Role of the Glucagon and Glucagon-like Peptides in … · thank my brothers Mr. Arshed Ali and Mr....

Role of the Glucagon and Glucagon-like Peptides in pancreatic β-cell and cardiovascular function

By

Safina Ali

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Laboratory Medicine and Pathobiology University of Toronto

©Copyright by Safina Ali 2014

ii

Role of Glucagon and Glucagon-like peptides in pancreatic β-cell and

cardiovascular function

Safina Ali

Doctor of Philosophy

Laboratory Medicine and Pathobiology

University of Toronto

2014

ABSTRACT

Glucagon is inappropriately elevated in diabetes, and inhibition of glucagon receptor (Gcgr) signaling is

beneficial for glycemic control. However, the mechanism through which inhibition of Gcgr signaling leads to

improved glycemic control is unknown. Additionally, Gcgr is expressed in the cardiovascular system; however

the role of Gcgr signaling in the cardiovascular system is unexplored. In order to develop therapies targeting

Gcgr signaling for the treatment of type 2 diabetes (T2D) it is vital to understand Gcgr’ s role in regulation of

glucose homeostasis and cardiovascular function.

In mice, genetic deletion of the glucagon receptor results in increased levels of the insulinotropic hormone,

glucagon-like peptide-1 (GLP-1). I hypothesized Glp1r signaling contributed substantially to the improved

glucose tolerance observed in Gcgr-/- mice. I have generated and characterized the Gcgr and Glp-1 receptor

double knockout (Gcgr-/-:Glp1r-/-) mice. My studies demonstrate that Glp1r substantially contributes to the

delayed gastric emptying and improved intraperitoneal glucose tolerance in Gcgr-/- mice, but it did not

contribute to the improved oral glucose tolerance seen in the Gcgr-/- mice. Interestingly, expression of non-

classical incretin receptors and sensitivity to their exogenous agonists were increased in Gcgr-/-:Glp1r-/- mice,

suggesting that in the absence of the classical incretin receptors the non-classical incretin receptors compensate

to maintain the enteroinsular axis.

iii

I explored the role of Gcgr signaling in the cardiovascular system under normal and ischemic

conditions. My studies showed that exogenous glucagon increased mortality from myocardial infarction in WT

mice in a p38 MAPK-dependent manner. Conversely, Gcgr+/- and cardiac specific Gcgr-/- mice (GcgrCM-/-

)

had improved survival following myocardial infarction. Gene expression profiling of hearts from GcgrCM-/-

mice

showed reduced expression of fatty acid oxidation gene, consistent with a reduction in long-chain acylcarnitines

observed upon metabolic profiling of GcgrCM-/-

hearts. Therefore, partial or cardiomyocyte specific loss of Gcgr

signaling enhanced protection to ischemic injury by regulating fatty acid oxidation.

In conclusion, my studies suggest Gcgr signaling is essential for β-cell nutrient sensing and fuel metabolism

in cardiomyocytes and therapies aiming at modulating Gcgr signaling for the treatment of T2D require careful

assessment of cardiovascular outcomes.

iv

ACKNOWLEDGMENTS

I am forever grateful to my supervisor Dr. Daniel Drucker for his unconditional support, guidance,

encouragement, motivation and optimism during difficult situations. He has been a great mentor and has always

encouraged me to do great science and motivated me to stay on track in my career. Without Dr. Drucker’s

guidance, support and mentorship it would have been impossible for me to reach my career goals.

I would like to extend sincere thanks and appreciation to my program advisory committee members, Dr.

Amira Klip and Dr. Patricia Brubaker for their guidance and thoughtful advice throughout the years. I am also

thankful to my thesis defense examiners, for their review of this work.

I have been fortunate to share my PhD experience with a group of very talented and kind colleagues. Dr.

Ben Lamont, Dr. Christine Longuet and Dr. John Ussher were invaluable scientific collaborators and mentors.

The knowledge and tremendous generosity of Dr. Laurie Baggio will always be valued and appreciated. I would

like to thank Xiemin Cao for her skilled assistance and for rescuing me from the woes of islet isolation. Special

thanks are in order to the many other Drucker laboratory past and present members including Dr. Irene

Hadjiyanni, Dr. Jackie Koehler, Dr. Adriano Maida, Dr. Bernardo Yusta, Dr. Grace Flock, Dr. Jon Campbell,

Dr. Erin Muller, Dr. Holly Bates, Dr. Min Suk Kim, Kabir Gholam and Dianne Holland.

I am thankful to my family for their support and continued encouragement. I am most grateful to my

father late Mr. Kamran Rashed Ali who passed away during my doctoral studies for being my inspiration to do

research in diabetes and heart disease. I would like to thank my mother Mrs. Shamim Ali and my sisters Ms.

Sumaira Ali and Ms. Saima Naz, for their unconditional support, encouragement and assistance during my hard

times, without their love and support my doctorate journey would not have been possible. I would like to also

thank my brothers Mr. Arshed Ali and Mr. Murshed Ali for being a father figure to me and providing me with

their moral support.

v

TABLE OF CONTENTS

ABSTRACT ...................................................................................................................................................... ii

TABLE OF CONTENTS ................................................................................................................................ v

LIST OF FIGURES ........................................................................................................................................ ix

LIST OF TABLES ......................................................................................................................................... xii

ABREVIATIONS .......................................................................................................................................... xiii

CHAPTER 1: Introduction ............................................................................................................................. 1

1.1. Proglucagon .......................................................................................................................................... 2

1.2. Glucagon ............................................................................................................................................... 3

1.2.1. Glucagon synthesis and secretion ........................................................................................... 3

1.2.2. Glucagon metabolism and clearance ...................................................................................... 5

1.2.3. Glucagon action and the Gcgr ................................................................................................. 5

1.2.4. Glucagon and the Pathophysiology of Type 1 and 2 Diabetes ........................................... 15

1.2.5. Therapeutic potential of glucagon ........................................................................................ 16

1.3. Glucagon and the Cardiovascular System ....................................................................................... 20

1.3.1. Glucagon and blood vessels ................................................................................................... 21

1.3.2. Glucagon and calcium ions ................................................................................................... 22

1.3.3. Glucagon and blood pressure ............................................................................................... 22

1.3.4. Glucagon and cardiac ischemia ............................................................................................ 23

1.3.5. Glucagon and cardiac fuel metabolism ................................................................................ 25

1.3.6. Glucagon and myocardial oxygen consumption ................................................................. 26

1.4. Introduction to the incretins ............................................................................................................. 27

1.4.1. Incretin secretion and synthesis ............................................................................................ 27

vi

1.4.2. Incretin action in the pancreas ............................................................................................. 27

1.4.3. Incretin action in the adipose tissue ..................................................................................... 30

1.4.4. Incretin action in the liver ..................................................................................................... 31

1.4.5. GLP-1 and gastric emptying ................................................................................................. 31

1.4.6. Incretin action in the heart.................................................................................................... 32

1.4.7. Insight from incretin receptor knockout mice .................................................................... 34

1.5. Rationale & Hypotheses .................................................................................................................... 35

CHAPTER 2: Dual elimination of the glucagon and GLP-1receptors in mice reveals plasticity in the

incretin axis ................................................................................................................................................ 38

2.1 Research Summary ............................................................................................................................ 39

2.2 Introduction ........................................................................................................................................ 40

2.3 Materials and Methods ...................................................................................................................... 41

2.3.1 Animal studies ........................................................................................................................ 41

2.3.2 Peptides & drugs .................................................................................................................... 41

2.3.3 Assessment of food intake and energy expenditure ............................................................ 42

2.3.4 Tissue isolation and histological analysis ............................................................................. 42

2.3.5 Glucose, insulin tolerance test and measurement of plasma metabolites ......................... 43

2.3.6 Solid and liquid phase gastric emptying .............................................................................. 44

2.3.7 Islet isolation ........................................................................................................................... 44

2.3.8 Real-time qRT-PCR............................................................................................................... 45

2.3.9 Statistical Analysis ................................................................................................................. 45

2.4 Results ................................................................................................................................................. 45

2.4.1 Glp1r is not required for pancreas enlargement or α-cell hyperplasia in Gcgr-/-mice. ... 45

2.4.2 Disruption of Glp1r leads to increased fasting glycaemia in Gcgr-/-mice. ........................ 46

2.4.3 Elimination of Glp1r reverses improvements in i.p. glucose tolerance in Gcgr-/-mice. ... 51

vii

2.4.4 The GLP-1 receptor mediates reduced gastric emptying; however, oral glucose tolerance

remains improved independent of Glp1r in Gcgr-/-mice. ................................................... 51

2.4.5 Islets from Gcgr-/-:Glp1r-/-mice display increased sensitivity to GIP. .............................. 51

2.4.6 Plasticity of the incretin axis revealed through reduction of Gcgr action in Glp1r-/-

:Gipr-/-mice. ............................................................................................................................ 55

2.4.7 Gcgr-/-:Glp1r-/-mice display increased sensitivity to Gpr119 and Cckar agonists. ......... 68

2.5 Discussion............................................................................................................................................ 68

CHAPTER 3: Disruption of cardiomyocyte glucagon receptor signaling decreases flux through fatty

acid oxidation and enhances survival following ischemic injury .......................................................... 73

3.1 Research Summary ............................................................................................................................ 74

3.2 Introduction ........................................................................................................................................ 75

3.3 Materials and Methods ...................................................................................................................... 77

3.3.1 Animal studies ........................................................................................................................ 77

3.3.2 Peptide and drug injections: ................................................................................................. 77

3.3.3 Coronary artery ligation ....................................................................................................... 77

3.3.4 Ischemia reperfusion protocol .............................................................................................. 78

3.3.5 Blood pressure and heart rate measurements ..................................................................... 78

3.3.6 Myocardium metabolic profiling .......................................................................................... 78

3.3.7 Heart histology ....................................................................................................................... 78

3.3.8 Glucose tolerance and measurement of plasma insulin...................................................... 79

3.3.9 Western blotting ..................................................................................................................... 79

3.3.10 Heart RNA analyses ............................................................................................................... 80

3.3.11 PPARα Nuclear Translocation Immunoblotting in Primary Atrial Cardiomyocytes..... 80

3.3.12 Culture of HL-1 Atrial Cardiac Myocytes........................................................................... 80

3.3.13 PPARα Nuclear versus cytoplasmic expression .................................................................. 81

3.3.14 In vitro HL-1 cellular injury model ...................................................................................... 81

3.3.15 Statistical Analysis ................................................................................................................. 81

viii

3.4 Results: Glucagon impairs outcomes after myocardial infarction in a p38 MAPK-dependent

manner. ............................................................................................................................................... 82

3.4.2 Exogenous glucagon increases fatty acid oxidation in the ischemic heart ........................ 91

3.4.3 Partial deletion of whole body Gcgr signaling impairs survival following MI and

exacerbates LV remodeling ................................................................................................... 91

3.4.4 Loss of Gcgr signaling protects and glucagon perfusion worsens outcome from

ischemia/reperfusion injury in isolated mouse hearts. ....................................................... 91

3.4.5 Generation of cardiomyocyte-specific glucagon receptor knockout mice ........................ 94

3.4.6 Inactivation of Gcgr expression in cardiomyocytes increases survival after myocardial

infarction ................................................................................................................................. 94

3.4.7 Deletion of the cardiomyocyte Gcgr leads to reduced expression of genes and proteins

regulating fatty acid oxidation ............................................................................................ 105

3.4.8 Targeted metabolomics illustrates reduced fatty acid oxidation in normal and insulin-

resistant hearts with selective loss of Gcgr signaling ........................................................ 105

3.5 Discussion.......................................................................................................................................... 106

CHAPTER 4: General Discussion and Future Direction ......................................................................... 112

REFERENCES ............................................................................................................................................. 127

ix

LIST OF FIGURES

Figure 1.1. Structures of proglucagon ............................................................................................................ 2

Figure 1.2. Glucagon action in hepatic and extrahepatic tissues. .................................................................... 7

Figure 2.1. Body weight, food intake and energy expenditure…………………………………………...…47

Figure 2.2. Plasma levels of total GIP, active GLP-1, total GLP-2 and glucagon……………………..……48

Figure 2.3. Glp1r is not required for development of increased pancreas weight or α

Gcgr-/-mice. .......................................................................................................................................................... 49

Figure 2.4. Glp1r controls fasting and fed glycemia in Gcgr-/-mice. ........................................................... 50

Figure 2.5. Loss of Glp1r reverses improvements in i.p. glucose tolerance without altering insulin

sensitivity in Gcgr-/-mice. .................................................................................................................................... 53

Figure 2.6. Glp1r mediates reduced gastric emptying but not improved oral glucose tolerance in Gcgr-/-

mice. ...................................................................................................................................................................... 54

Figure 2.7. Function of GPCRs in isolated islets. .......................................................................................... 56

Figure 2.8. Gcgr-/-:Glp1r-/-mice exhibit enhanced sensitivity to [D-Ala2]GIP…………………...…. 57

Figure 2.9. Enteroinsular axis is maintained in DIRKO mice treated with Gcgr ASOs. ............................... 58

Figure 2.10. Expression of insulinotropic GPCRs in islets. ........................................................................... 59

Figure 2.11. GRP action in WT and knockout mice…………………………………………...……………60

Figure 2.12. PACAP action in WT and knockout mice……………………………………………….….....61

Figure 2.13. Gcgr-/-:Glp1r-/-mice exhibit enhanced sensitivity to the GPR119 agonist AR231453

(5mg/kg). ............................................................................................................................................................... 62

Figure 2.14. Gcgr-/-:Glp1r-/-mice exhibit enhanced sensitivity to the GPR119 agonist AR231453

(20mg/kg). ........................................................................................................................................................... 623

x

Figure 2.15. Gcgr-/-:Glp1r-/-mice exhibit increased sensitivity to CCK (9ug/kg)………………….... 64

Figure 2.16. Enhanced sensitivity to CCKAr ligand CCK (18ug/kg)………………………..………..65

Figure 2.17. Gut peptide gene expression in re-fed mice……………………………………………..66

Figure 2.18. Schematic of proposed model.................................................................................................... 67

Figure 3.1. Blood glucose and body weight before and after glucagon administration and LAD ligation. .. 84

Figure 3.2. Glucagon impairs survival after myocardial infarction in a p38 MAPK-dependent manner ...... 85

Figure 3.3. Glucagon impairs survival after myocardial infarction in a cardiac Gcgr-dependent manner ... 86

Figure 3.4. Glucagon has no effects onAd-βgal transfected HL-1 cell lines. ................................................ 87

Figure 3.5. Glucagon increases PPAR-αnuclear translocation, levels of cleaved caspase-3, and PDH

phosphorylation..................................................................................................................................................... 88

Figure 3.6. Glucagon increases long and medium chain fatty acid content in the heart. ............................... 90

Figure 3.7. Whole body Gcgr+/- mice have significantly improved survival following MI ........................ 92

Figure 3.8. Loss of Gcgr signaling protects whereas glucagon impairs recovery of ventricular developed

pressure after ischemia-reperfusion injury in the isolated heart ex vivo .............................................................. 93

Figure 3.9. Generation of mice and analysis of Gcgrexpression ................................................................... 95

Figure 3.10. Body weight, glucose tolerance and heart weight in mice with cardiac -specific inactivation of

the Gcgr ................................................................................................................................................................ 97

Figure 3.11. (A-C) Heart rate, systolic and diastolic blood pressure in αMHCCre

and GcgrCM-/-

mice were

measured. .............................................................................................................................................................. 98

Figure 3.12. Loss of cardiac Gcgr signaling enhances survival following MI and attenuates adverse LV

remodeling .......................................................................................................................................................... 100

xi

Figure 3.13. Selective loss of Gcgr signaling in cardiomyocytes lead to reduced expression of fatty

acid oxidation genes and proteins ....................................................................................................................... 100

Figure 3.14. Targeted metabolomics reveals reduced fatty acid oxidation in GcgrCM-/-

hearts ................... 102

Figure 3.15. Targeted metabolomics reveals reduced fatty acid oxidation in hearts from high fat fed mice

with loss of cardiac Gcgr signaling .................................................................................................................... 103

Figure 3.16. Schematic of proposed model .................................................................................................. 104

xii

LIST OF TABLES

Table 1. Heart rate and blood pressure measurements in GcgrCM-/-

mice.. ..................................................... 99

xiii

ABREVIATIONS

αIRKO α insulin receptor knockout

AMPK Adenosine monophosphate kinase

ANOVA Analysis of variance

ATP Adenosine triphosphate

ADP Adenosine diphosphate

AMP Adenosine monophosphate

AUC Area under the curve

ASOs Anti-sense oligonucleotides

N-glycosylation Asn-linked glycosylation

BAT Brown adipose tissue

BrDU Bromodeoxyuridine

Ca2+ Calcium

cAMP Cyclic adenosine monophosphate

CCK-8 Cholecystokinin

CHIP Chromatin immunoprecipitation

cDNA Complimentary DNA

CNS Central nervous system

CRE Cre recombinase

CREB Cyclic AMP response element-binding protein

C-terminal Carboxyl-terminus

db/db Leptin receptor knockout mice

DIRKO Double incretin knockout mice

DMEM Dulbecco's Modified Eagle Medium

DNA Deoxyribonucleic acid

DPPIV Dipeptidylpeptidase IV

EDTA Ethylenediaminetetraacetic acid

EGF Epidermal growth factor

EGFR Epidermal growth factor receptor

ELISA The enzyme-linked immunosorbent assay

Epac Exchange protein directly activated by cAMP

Ex (9-39) Exendin 9-39

Ex-4 Exendin-4

FBS Fetal Bovine Serum

FDA Federal drug administration

FGF21-/- Fibroblast growth factor 21 knockout mice

GABA Gamma aminobuteric acid

G protein G-protein coupled receptor 119

Gcgr ASO Glucagon receptor antisense oligonucleotide

GPCR Gprotein coupled receptor

GPR119 Gprotein coupled receptor 119

Gcg Glucagon

Gcgr Glucagon receptor

GI Gastrointestinal

GIP Gastric inhibitory peptide

xiv

GLP-1 Glucagon like-peptide 1

GLP-2 Glucagon like-peptide 2

Gcgr-/- Glucagon receptor knockout mice

Glp1r-/- Glucagon-like-peptide-1 receptor knockout mice

Gipr-/- Gastric inhibitory peptide receptor knockout mice

Gcgr-/-:Glp1r-/- Glucagon receptor and Glucagon-like peptide-1 receptor double knockout mice

Gsα Guanine nucleotide binding protein α

Gsα:Glp1r-/- Guanine nucleotide binding protein α and Glucagon-like peptide-1 receptor double

knockout mice

Gq Guanine nucleotide binding protein

GRP Gastrin releasing peptide

HFD High fat diet

IP-1 Intervening peptide-1

IP-2 Intervening peptide-2

IGF-1 Insulin growth factor-1

Isl-1 Islet-1

MBH Medial basal hypothalamus

MPGF Major proglucagon fragment

NEP24.11 Neutral endopeptidase 24.11

OXM Oxyntomodulin

PKA Protein kinase A

PKC Protein kinase C α

PI3k Protein inhibitory 3 kinase

PC2 Prohormone convertase 2

PC1/3 Prohormone convertase 1/3

K+

Potassium ion

RNA Ribonucleic acid

Rfx6 Regulatory Factor X 6

Sst Somatostatin

T1D Type 1 Diabetes

T2D Type 2 Diabetes

TCF7L2 Transcription factor 7-like 2

UCP2 Uncoupling protein 2

WAT White Adipose Tissue

xv

Methodological abbreviations

% Percent

BP Blood pressure

°C Degrees Celsius

Da Dalton

g Gram

h Hour(s)

l Litres

LVDP Left ventricular diastolic pressure

M Molar (moles/l)

min Minute(s)

mol Moles

sec Second(s)

U Units

wk Week

wt Weight

vol Volume

i.v. Intravenous

ip Intraperitoneal

s.c. Subcutaneous

Prefixes

k kilo- (x 103)

c centi- (x 10-2

)

m milli- (x 10-3

)

μ micro- (x 10-6

)

n nano- (x 10-9

)

p pico- (x 10-12

)

1

CHAPTER 1: Introduction

CHAPTER 1: Introduction

2

1.1. Proglucagon

The proglucagon gene is located on human chromosome 2 and comprises 6 exons and 5

introns [1]. The proglucagon gene is expressed in α-cells of the endocrine pancreas, in the L cells

of the intestine and in the brain, in the caudal brainstem and hypothalamic neurons [2]. The

proglucagon precursor contains the amino acid sequences for glucagon, glucagon-like peptide-1

(GLP-1), glucagon-like peptide-2 (GLP-2), oxyntomodulin, and glicentin (Figure 1.1). Tissue-

specific posttranslational processing of proglucagon in α-cells is mediated by prohormone

convertase (PC)2, which leaves proglucagon to liberate glucagon and leaves unprocessed

carboxy-terminal, major proglucagon fragment. In contrast, PC1/3 leaves proglucagon in the

intestinal L cells and the brain to liberate GLP-1, GLP-2, oxyntomodulin, and glicentin [3]. In

the pancreas, proglucagon gene expression is activated by hypoglycemia and fasting and is

inhibited by insulin. In the intestine, proglucagon gene is activated by nutrient ingestion and

inhibited by fasting [4].

Figure 1.1. (Adapted from [5]).(A): Structures of proglucagon (B): Tissue-specific

posttranslational processing of proglucagon in the pancreas gives rise to glicentin-related

pancreatic polypeptide (GRPP), glucagon, intervening peptide-1 (IP-1), and major

proglucagon fragment (MPGF), whereas glicentin, oxyntomodulin (OXM), intervening

peptide (IP-2), glucagon-like peptide-1 (GLP-1), and GLP-2 are liberated from the brain and

the intestine. S, signal peptide.

3

1.2. Glucagon

1.2.1. Glucagon synthesis and secretion

Glucagon is a 29-amino acid peptide hormone encoded within a single proglucagon

precursor. Studies with PC2 knockout mice showed how essential PC2 was for glucagon as

deletion of PC2 led to hypoglycemia and α-cell hyperplasia. These phenotypes were solely the

result of lack of glucagon as replacing glucagon by osmotic mini-pump normalized the

hypoglycemia and reduced the α-cell hyperplasia [6].

Glucagon secretion by α-cells is highly regulated and is achieved by specific electrical

machinery in the α-cells (ion channels). Multiple stimuli activate ion channels in the α-cells but

the most important stimuli are glucose and insulin [7]. Low glucose levels activate specific

channels in the pancreatic alpha cells, in particular the ATP-sensitive K+ (KATP) channel [8], to

generate action potentials of sodium and calcium currents, leading to glucagon secretion.

However, whether the modulating effect of glucose on glucagon secretion is predominantly

direct or indirect remains uncertain. Studies conducted with mouse and human α-cells show that

glucose can directly inhibit glucagon secretion. In contrast, studies with rat α-cells show that

glucose inhibits glucagon secretion in a paracrine manner [7]. High glucose mediated glucagon

suppression has been shown to be dependent on glucose stimulated somatostatin secretion.

Global deletion of somatostatin gene (Sst) in mice has been shown to increase basal glucagon

and insulin levels [9, 10]. Furthermore, high glucose failed to inhibit glucagon levels in islets

from Sst deleted mice suggesting the notion that somatostatin might be involved in high glucose

mediated inhibition of glucagon secretion [10]. Moreover, β-cell-derived products such as

insulin, GABA, and zinc can also inhibit glucagon secretion [11]. Many in vivo and ex-vivo

studies have demonstrated that insulin plays a pivotal role in regulation of glucagon secretion

[12-15]. Exogenous insulin administration led to suppression of glucagon secretion in

insulinopenic animal models, and infusion of anti-insulin antibody increased glucagon release.

The first direct genetic evidence that shows the significance of physiological insulin signaling in

inhibition of glucagon secretion in α-cells comes from studies conducted with the α-cell specific

insulin receptor knockout (αIRKO) mice. Deletion of insulin signaling in the αIRKO mice leads

to glucose intolerance, hyperglycemia, and increased plasma glucagon levels in the fed state.

Additionally, increased glucagon secretion is also observed following induction with L-arginine

4

in vivo and ex-vivo in whole pancreas perfusion suggesting deletion of insulin receptor from α-

cells leads to elevated glucagon levels. Furthermore, streptozotocin (STZ) treatment of αIRKO

mice leads to even higher plasma glucagon levels compared to glucagon levels in littermate

control mice. This suggests insulin receptor signaling on α-cells prevents glucagon secretion

during hyperglycemia and deletion of insulin receptor signaling from α-cells leads to elevated

glucagon secretion [16]. The mechanisms responsible for insulin-mediated inhibition of α-cell

glucagon secretion may involve insulin-mediated activation of GABA receptor and translocation

to the cell surface in an Akt-dependent manner [17]. Similarly, secretion of zinc from β-cells

appears to be important for suppression of glucagon secretion, and reduced zinc secretion

promotes enhanced glucagon secretion in response to hypoglycemia [18]. Nevertheless,

experiments using rat and human islets demonstrate that glucose-mediated suppression of

glucagon secretion may occur independently of GABA or zinc and requires functional KATP

channels [19]. Somatostatin inhibits glucagon secretion by inhibition of adenylate cyclase and

cAMP production, and genetic deletion of the somatostatin receptor subtype 2 is associated with

mild hyperglucagonemia and defective glucose- and somatostatin-mediated suppression of

glucagon secretion in isolated mice islets in vitro [20]. Similarly, the incretin hormone GLP-1

inhibits glucagon secretion in a glucose-dependent manner through mechanisms requiring the

somatostatin receptor subtype 2 [21]. Unlike GLP-1, the incretin hormone, Gastric inhibitory

polypeptide (GIP) has been shown to stimulate glucagon secretion under hyperglycemic

conditions [22]. In human subjects, infusion of GIP (20 ng/kg/min) led to elevation of glucagon

in plasma levels, and this resulted in hyperglycemia during a meal test. GIP mediated glucagon

secretion in α-cells can be direct as GIP receptors have been detected in human and rodent α-

cells [22].

Recent studies suggest uncoupling protein 2 (Ucp2) involvements in regulation of

glucagon secretion. UCP2 expression is increased in nutrient deprived human islets.

Additionally, uncoupling protein 2 knockout (Ucp2-/-) mice show a blunted glucagon response

in vivo 30 minutes after an insulin tolerance test. Furthermore, islets from Ucp2-/- mice have

impaired glucagon secretion under high to low glucose conditions compared to islets from

control mice [23]. Therefore, these studies suggest Ucp-2 plays an important role in normal

glucagon secretion.

5

1.2.2. Glucagon metabolism and clearance

Glucagon is metabolized by the membrane-bound zinc metallopeptidase, neutral

endopeptidase 24.11 (NEP 24.11) in vitro. NEP24.11 not only metabolizes glucagon but also

GLP-1 (7-36amide) [24]. Studies have shown that inhibition of NEP24.11 leads to an increase in

circulating levels of endogenously and exogenously administered glucagon in anesthetized pigs

[25]. This suggests NEP 24.11 plays an important role in glucagon metabolism. The mechanism

of glucagon clearance is not well understood in vivo, however, studies have found most cleaved

glucagon product in the kidney and very little in the liver [26]. Glucagon is cleaved by enzymes

at the glomerular brush border membrane of the proximal tubule prior to filtration out of the

kidney [27, 28]. Dipeptidyl peptidase-4 (DPPIV) is highly expressed in the circulation, kidney,

and to a lesser extent in the liver. Incubation of glucagon with purified porcine kidney DPPIV

leads to hydrolysis of glucagon (1-29) to glucagon (3-29), and glucagon (5-29) in vitro and in

human serum; these different processed glucagon fragments have been shown to be inactive

forms of glucagon as they fail to cause hyperglycemia in Wister rats upon intraperitoneal

injection [29]. Additionally, glucagon is processed by an endopeptidase in the circulation and by

target tissues such as the pancreas, the liver or the heart to liberate mini-glucagon (19-29) [30,

31]. A separate receptor for mini-glucagon has not been identified thus far, however mini-

glucagon has been shown to have biological function in the liver, the heart, and the pancreas [31,

32].

1.2.3. Glucagon action and the Gcgr

Glucagon receptor signaling

The major biological action of glucagon is to counteract the actions of insulin and

maintain normoglycemia during the fasting state by inducing hepatic glucose production.

Glucagon exerts its action on target tissues through activation of the glucagon receptor (Gcgr), a

G protein-coupled receptor, member of the class II G protein-coupled receptor superfamily [33].

Gcgr activation leads to signal transduction by G proteins (Gsα and Gq), whereby Gsα activates

adenylate cyclase, which causes cAMP production, resulting in an increase in levels of protein

kinase A. Gq activation leads to phospholipase, C-mediated increases in intracellular calcium

levels. Gcgr signaling in the liver results in increased hepatic glucose production by induction of

6

glycogenolysis and gluconeogenesis along with inhibition of glycogenesis [34]. The actions of

glucagon to promote increased hepatic glucose production are extremely rapid and reflect

changes in the activity of enzymes regulating gluconeogenesis and glycogenolysis. Glucagon-

stimulated increases in cAMP lead to activation of glycogen phosphorylase and inhibition of

glycogen synthase. The actions of glucagon to control gluconeogenesis are mediated through

coordinate regulation of the cAMP-regulated binding protein, regulated transcription coactivator

2, histone acetyltransferase p300, and the nutrient-sensing deacetylase sirtuin 1, resulting in

increased expression of genes regulating gluconeogenesis [35]. The Gcgr is also expressed in

extrahepatic tissues, which includes the heart, the intestinal smooth muscle, the kidney, the brain,

and the adipose tissue [36] (Figure 1.2).

7

Figure1.2.(Adapted from [5]).Glucagon action in hepatic and extrahepatic tissues. The

actions of glucagon in the liver, kidney, gastrointestinal tract, pancreas, adipose tissue,

cardiovascular system, and the central nervous system.

Glucagon, the Kidney, and the Gastrointestinal Tract

Glucagon stimulates adenylate cyclase and cAMP production in nephrons and in cell-free

preparations of human renal medullas [37, 38]. Although the role of glucagon in the control of

renal glucose output remains uncertain, glucagon regulates the rate of kidney filtration, urea

excretion, and water reabsorption by the kidney [36] via direct and indirect mechanisms [39].

8

Paradoxically, long-term infusion of glucagon in mice leads to kidney injury through the

development of hypertension, hypertrophy, and increased proliferation of mesangial cells [40].

Although the Gcgr is expressed in the gut, where it regulates motility, very little is known about

the physiological role of glucagon in the gut [41, 42]. However, pharmacological doses of

glucagon infusion in men have been shown to delay gastric emptying following a radiolabelled

meal in double-blind placebo-controlled study. Gastric release of nutrition was suppressed

during the first 60 min of glucagon infusion, and an increased insulin release was also observed

[43]. The mechanism through which glucagon inhibits gastric emptying is unknown.

Glucagon and the Endocrine Pancreas

Gcgr immunoreactivity and mRNA expression have been detected predominantly in β-

cells from rodent pancreas; however, subsets of α- and δ-cells also express the Gcgr [44].

Additionally, glucagon has been shown to regulate cAMP production in β-cells. However,

glucagon-mediated cAMP production in β-cells is less potent than that induced by the incretin

hormones GLP-1 and gastric inhibitory polypeptide (GIP) [45]. Nevertheless, glucagon induces

insulin secretion in human subjects. Moreover, insulin secretion is increased from perfused

pancreas and isolated β-cells in the presence of glucagon [44, 45]. The stimulatory actions of

glucagon on the islet’s β-cell may be mediated through dual activation of both the Gcgr and the

GLP-1 receptor (Glp1r) [46]. The research group headed by Gelling et al generated transgenic

mice overexpressing the Gcgr in pancreatic β-cells using the rat insulin promoter to understand

the role of the Gcgr in the β-cells and found these mice to have increased insulin secretion,

pancreatic insulin content, and β-cell mass. After high fat feeding, these mice were partially

protected against hyperglycemia and impaired glucose tolerance [47]. However, the molecular

mechanism(s) and physiological importance of glucagon-stimulated insulin secretion require

further explanation. Even less is known about the role of the Gcgr in α-cells; yet, several studies

have demonstrated Gcgr expression in at least a subset of rodent α-cells [44, 48]. Glucagon

stimulates cAMP production in a dose-dependent manner from rat and mouse α-cells and

increases α-cell exocytosis in a PKA-dependent manner, suggesting that it may regulate its own

secretion [44, 48]. Furthermore, glucagon is also important for α-cell proliferation and survival

as the PC2 knockout mice that lack glucagon action develop α-cell hyperplasia, and the

replacment of glucagon using mini-pump reduces α-cell hyperplasia. Similarly, Gcgr whole body

9

knockout mice develop α-cell hyperplasia. The effects of glucagon on α-cell proliferation may

not be direct as Longuet et al has elegantly showed that disruption of hepatic Gcgr signaling

leads to α-cell hyperplasia [49]. Furthermore, transplanting islets from wild-type mice into either

Gcgr-/- recipients, or liver specific Gcgr knockout recipients, results in proliferation of islet α-

cells underneath the kidney capsule. These findings imply that interruption of the liver glucagon

receptor pathways regulates islet α-cell proliferation independent of the normal islet localization

and pancreatic location. Nonetheless, the importance of direct glucagon action on α-cells is

uncertain.

Glucagon Action in the Brain

The proglucagon gene is expressed in the brainstem and, to a lesser extent, in the

hypothalamus, and different projections distribute proglucagon-derived peptides to diverse brain

regions [50, 51]. Glucagon binds to the brain membranes and to the mouse astrocytes and

stimulates adenylate cyclase and cAMP production respectively [52, 53]. Intracerebral

administration of pharmacological levels of glucagon in the brain produces dose-dependent

hyperglycemia in rodents through mechanisms requiring cholinergic and α-adrenergic neural

pathways [54, 55]. Glucagon infusion in the central nervous system also inhibits food intake, and

the anorectic actions of glucagon require functional vagal afferents [56, 57]. Moreover,

neutralization of endogenous glucagon via intraportal infusion of glucagon antibodies increases

meal size in normal rats, effects that are abolished in rats with selective hepatic vagotomy [58].

The satiety-promoting effects of glucagon may also involve suppression of ghrelin secretion,

actions that require an intact hypothalamic-pituitary axis, and ghrelin has been shown to regulate

feeding behavior, suggesting that the satiety effect of glucagon can be mediated through ghrelin

[59].

Recently, the research group headed by Mighiu et al explored the central role of glucagon

signaling in regulation of hepatic glucose production in mice and rats using mediobasal

hypothalamus (MBH) infusion of glucagon intravenously during a pancreatic euglycemic clamp.

The glucose was maintained at euglycemic level by continuous infusion, and insulinemia was

maintained, leading to inhibition of hepatic glucose production [60]. Furthermore, the inhibitory

effects of glucagon infusion on hepatic glucose production were dependent on brain PKA

activation [60]. Moreover, these effects of glucagon were absent in Gcgr-/- mice with infusion of

10

central glucagon and in rats treated with inhibitors of PKA signaling in the MBH or in rats after

hepatic vagotomy. These findings suggest central glucagon-mediated regulation of hepatic

glucose production requires central PKA signaling and neuronal signaling between the brain and

the liver. In addition, rats fed a high fat diet had higher plasma glucose concentrations and had

impaired MBH glucagon signaling. Therefore, this study suggests that hypothalamic glucagon

signaling is essential for inhibition of hepatic glucose production [60].

Glucagon and food intake

During a mixed meal glucagon levels increase higher than basal levels but lower than

fasting levels, and injecting glucagon antiserum before a meal increases food intake suggesting

elevated levels of endogenous glucagon during a meal may contribute to satiety [61-64]. A

substantial number of studies in rodents and humans have shown that pharmacological doses of

glucagon lead to reduced feeling of hunger and therefore reduced food intake [65-68]. Reduction

in food intake has been postulated to be solely a result of glucagon-mediated satiety and not

alteration in food taste as studies where rats are given glucagon before a meal show significant

reduction in overall meal size without alteration of inter-meal interval [69]. Additionally,

grooming and exploratory behavior is unaltered in glucagon-treated rats suggesting glucagon is

not causing toxicity which is resulting in reduced food intake [69]. Based on these studies

glucagon has been postulated to be a potent satiety factor.

Glucagon-mediated satiety may be mediated by vagal afferent fibers located in the

hepatic branch that transmits signal to the CNS [70]. This finding emerges from studies where

glucagon is administered via the hepatic portal vein in rats with sham-vagotomy leading to

reduced food intake. However, in rats with bilateral subdiaphagmatic truncal vagotomy glucagon

fails to reduce food intake [70-72]. Additionally, complete vagotomy ameliorates glucagon’s

suppression on food intake and sparing the vagal hepatic branch preserves glucagon-mediated

satiety effects [71].

The role of central glucagon receptor signaling in regulation of food intake is unknown.

However, high levels of glucagon immunoreactivity have been found in the hypothalamus, and

administration of glucagon into the third ventricle region of the brain in rats results in more

potent reduction in food intake compared to peripherally administered glucagon [56, 73, 74]. The

importance of central Gcgr signaling in food intake is not well understood and requires further

11

studies.

Glucagon and energy expenditure

Glucagon-mediated increases in oxygen consumption were measured in several species

including rats, mice, quail, and human subjects. Glucagon increased oxygen consumption in rats,

quails and humans, but not in mice and dogs [75, 76]. However, hyperinsulinemia has been

shown to blunt glucagon’s effects on oxygen consumption. [77]. Plasma glucagon levels are

elevated under low temperatures, and one of the proposed mechanisms for glucagon-induced

oxygen consumption is through glucagon action on brown adipose tissue (BAT). In vitro studies

showed that glucagon increased blood flow, body temperature, DNA, protein content, and

mitochondrial mass in BAT [78-80]. Additionally, a study in rats showed glucagon increased

BAT mass, therefore glucagon could increase the thermogenic capacity in rats by activating BAT

[81]. The sympathetic nervous system also plays an important role in glucagon-mediated

thermogenesis in BAT as glucagon-mediated increases in oxygen consumption and

thermogenesis were inhibited by adrenergic and ganglionic blocking agents [75, 76, 80, 82].

Furthermore, glucagon’s effects on oxygen consumption and thermogenesis were diminished by

chemical sympathectomy, which blocked the release of catecholamines [75, 76, 80, 82]. These

findings suggest that glucagon-mediated induction of oxygen consumption may involve

glucagon-mediated secretion of catecholamines from adrenal medulla. Glucagon has also been

reported by some studies to be important in white adipose tissue (WAT) lipolysis, and glucagon

action on WAT is dependent on the nerves surrounding the WAT [83-86]. Following denervation

of WAT, glucagon-mediated free fatty acid release from WAT was reduced but not completely

abolished, and therefore the sympathetic nervous system was involved in glucagon action in both

BAT and WAT [86].

Another mechanism through which glucagon has recently been shown to regulate energy

expenditure is through inducing the secretion of Fibroblast Growth Factor 21 (FGF21) from

hepatocytes in rodents and humans [87]. Gcgr agonists have been shown to reduce body weight

and increase energy expenditure. A recent study reported that glucagon-mediated increases in

energy expenditure, locomotors activity, and reduction in body weight gain were dependent on

FGF21 expression as these effects of Gcgr agonists were abolished in FGF21 knockout (FGF21-

12

/-) mice. This study suggested that FGF21 signaling was essential in regulation of energy

expenditure by glucagon [87].

Glucagon and body weight regulation

In human studies, glucagon administered at a dose of 1mg prior to each meal for a period

of 5 weeks showed significant reduction in body weight and food intake [65]. Furthermore,

chronic infusion of glucagon via a mini-pump over seven days resulted in reduced body weight

in rats with no effects on food intake [65]. Additionally, another study where glucagon was

injected 3 times a day at 0.25mg per injection for 3 days showed significant reduction in body

weight following induction of diabetes with STZ, and no changes in food intake were observed

[65, 81, 88]. In Zucker diabetes rats, long-term glucagon administration reduced body weight

without having any changes in food intake [89]. It is not clear from these studies whether the

effects of glucagon on reduction of body weight are the result of direct glucagon’s effect on

energy expenditure, adipogenesis or both as these studies do not assess fat mass or locomotion

activity.

Glucagon and lipids

In humans, canines, and rodents following 30 minutes of intravenous glucagon

administration, total plasma cholesterol and lipid levels are significantly reduced [67, 90-92]. A

number of different mechanisms have been proposed for the glucagon-mediated reduction in

plasma lipids and cholesterol. One of the mechanisms includes glucagon-mediated reduction in

amino acid incorporation in hepatic apolipoprotein production, therefore resulting in reduced

hepatic lipoprotein production [93]. In a rat model of hyperlipidemia, pharmacological glucagon

administered over 4 days decreased the synthesis of liver lipoprotein apoproteins. Reduced liver

lipoprotein was also associated with reduced plasma VLDL, triglyceride, and serum lipoprotein

[93]. However, this study did not utilize physiological glucagon levels; therefore, it was not clear

if physiological glucagon levels lower liver triglyceride production.

A second proposed mechanism through which glucagon reduces plasma cholesterol is by

increasing urinary secretion of cholesterol and by directing cholesterol towards bile acid

generation [94]. Supporting this concept a study looked at the influence of twice-daily glucagon

injections for 3 weeks in Wistar rats on urinary secretion of cholesterol and bile acid generation

[94]. This study reported reduced plasma cholesterol, phospholipids, and triglyceride levels in

13

addition to reduced plasma glucose and insulin levels following chronic glucagon administration.

Interestingly, chronic glucagon administration in the study did not lead to hyperglycemia or

reduced liver triglyceride production. However, this study reported increased bile acid synthesis

in the liver and increased urinary secretion of cholesterol. Elevated bile acids following

glucagon treatment may be preventing hyperglycemia as studies have previously reported that

bile acids can improve glucose homeostasis [94]([95].

A third mechanism is through glucagon increasing the binding ability of Low-density

lipoprotein (LDL) to its receptor[96]. Studies conducted in rats supported this hypothesis. In rats

glucagon administration led to increased binding of LDL to its receptor and a reduction in

plasma cholesterol (apoB and apoE) [96]. The mRNA expression of LDL receptor was

unchanged suggesting glucagon-mediated increase in LDL binding to its receptor could be a

posttranscriptional or posttranslational modification change in LDL receptor by glucagon [96].

A fourth proposed mechanism through which glucagon has been suggested to reduce

plasma lipids and cholesterol is by increasing lipid catabolism [97]. Wistar rats fed a diet rich in

sucrose and chronically treated with glucagon had reduced triglycerides in chylomicron and

VLDL and no difference in triglyceride secretion [97]. Furthermore, upon an intravenous fat

tolerance test glucagon significantly improved fat tolerance in Wistar rats. Therefore, overall the

studies concluded that glucagon increased triglyceride clearance through glucagon-mediated

enhancement in lipid catabolism and had no effect on triglyceride secretion [97].

Physiological and pharmacological doses of glucagon have been shown to promote

lipolysis in white adipose tissue in rodents and human subjects [84, 86, 98]. Glucagon-mediated

lipolysis in adipose tissue seems to involve the sympathetic nervous system as denervation of

adipose tissue reduces glucagon-mediated decreases in release of nonesterified fatty acids,

however the release of glycerol from adipose tissue is not completely blocked suggesting other

mechanisms may be involved in glucagon-mediated lipolysis other than the sympathetic nervous

system [86]. Recent studies in humans and rodents suggest involvement of FGF21 in glucagon-

mediated lipolysis [99]. In healthy and T1D human subjects glucagon treatment increased

lipolysis. Similarly, glucagon increased lipolysis in healthy and STZ induced diabetic rodents in

vivo and in isolated adipocytes [99]. Glucagon treatment increased plasma FGF-21 in healthy

and diabetic human subjects and rodents [99]. Glucagon also stimulated FGF21 from isolated

14

rodent adipocytes and hepatocytes. Immunoneutralization or reduction of FGF21 mRNA

expression using si-RNA attenuated glucagon-mediated lipolysis. These studies suggest that the

glucagon effect on lipolysis is dependent on FGF21 [99].

Several studies of rodents and humans have shown that glucagon is essential for ketone-

body production [100-102]. In human subjects with T1D blocking glucagon secretion with

somatostatin led to suppression of ketoacidosis [101]. Conversely, in rabbit hepatocytes,

treatment with glucagon led to an increased ketone-body production [102]. Therefore, these

studies show that glucagon is necessary for ketoacidosis.

Lipid metabolism is regulated by glucagon and studies have also shown that lipids can

regulate glucagon receptor signaling [103-105]. HFD induced fatty liver was improved in

exercised rats in accordance with increased plasma glucagon levels [103]. Furthermore, reduced

glucagon tolerance was observed in rats fed HFD with hepatic steatosis [103]. Additionally,

Gcgr expression was reduced in hepatocytes from HFD fed rats with hepatic steatosis. Gcgr

degradation into amino acids is one of the mechanisms that have been proposed for HFD-

induced hepatic steatosis-mediated decrease in Gcgr expression [104, 105]. However, this is not

proven by any study as of yet. A marked increase in plasma membrane endosomal and lysosomal

compartments was observed in HFD-fed rat hepatocytes in addition to reduced Gcgr expression

suggesting Gcgr internalization on the hepatocyte plasma membrane may be modulated by HFD

feeding [104, 105]. Moreover, increased expression of protein kinase C (PKC) is also observed,

and it is known that PKC inhibits receptor internalization by phosphorylating G-protein related

kinases [106]. Therefore, HFD feeding may lead to inhibition of Gcgr internalization and

subsequently lead to Gcgr desensitization.

Glucagon and bile acid metabolism

Bile acids are important for lipid homeostasis, and glucagon has been shown to regulate

lipid metabolism in the liver [107]. Nevertheless, it is not clear if glucagon-mediated actions on

lipid homeostasis involve bile acid metabolism by glucagon. There are studies showing that

glucagon can decrease the expression of cholesterol 7α-hydroxylase (CYP7A1), which is a key

enzyme involved in the synthesis of bile acid from cholesterol in rodent and human hepatocytes

[108, 109]. Glucagon represses CYP7A1 transcription via promoting PKA phosphorylation of

HNF4, which prevents binding of HNF4 to the transcription site in CYP7A1 and is required for

15

the transcriptional activation of CYP7A1 [109]. However, these studies did not measure plasma

or intestinal level of bile acids; therefore it is not clear if glucagon-mediated reduction of

CYP7A1 has any impact on plasma bile acid levels.

1.2.4. Glucagon and the Pathophysiology of Type 1 and 2 Diabetes

Glucagon and Type 1 Diabetes

Studies have reported perfusion of glucose in rat pancreas with anti-insulin serum or

normal guinea pig serum results in significant rise in glucagon levels. Terminating the perfusion

of anti-insulin serum results in glucagon levels return them back to baseline values. This

suggests that intraislet insulin levels are important for regulation of α-cell glucagon secretion

[15]. In subjects with Type 1 Diabetes (T1D) it has been observed that in the absence of insulin

signaling α-cell numbers increase; this has been suggested to contribute to the inappropriate

hyperglucagonemia in T1D subjects [110]. A study conducted by Lee et al[111] showed Gcgr

knockout mice did not develop T1D and diabetes complications, such as hyperglycemia, ketosis,

and cachexia, despite complete depletion of β-cells by streptozotocin treatment. These studies

suggest that without glucagon signaling some of the metabolic complications of T1D do not

occur. Therefore, the hepatic actions of glucagon such as glycogenolysis, gluconeogenesis,

cytogenesis, and hypercatabolism are enhanced in the absence of insulin, which may be

contributing to the ketoacidosis, cachexia, coma, and death observed in T1D [111].

Glucagon and Type 2 Diabetes

Type 2 Diabetes is characterized by impaired insulin secretion and/or action, and many

subjects also exhibit inappropriate levels of circulating glucagon in the fasting and postprandial

state. An increase in the glucagon/insulin ratio is likely an important determinant of the

hyperglycemia seen in Type 2 Diabetes patients [112-114]. Consistent with the importance of

glucagon for fasting hyperglycemia, infusion of low doses of glucagon leads to the development

of hyperglycemia, whereas suppression of glucagon secretion in the fasting state by somatostatin

infusion significantly reduces hepatic glucose production [112, 115]. Lack of suppression of

postprandial glucagon secretion in subjects with T2D also plays an important role in the

pathogenesis of postprandial hyperglycemia [101, 116, 117]. The molecular mechanisms

16

responsible for dysregulation of α-cell glucagon secretion in diabetic subjects remain unclear but

may include impaired glucose sensing by α-cells and/or resistance of α-cells to the inhibitory

actions of insulin or other β-cell secretory products such as zinc or GABA.

1.2.5. Therapeutic potential of glucagon

Reduction of Gcgr Signaling for the Treatment of Diabetes

Considerable preclinical evidence supports targeting of glucagon action as an effective

approach to reduction of hyperglycemia. Immunoneutralization of glucagon with a monoclonal

antibody produces significant improvements in plasma glucose in rats with streptozotocin-

induced diabetes [118]. Similarly, glucagon antibodies markedly reduce hepatic glucose

production and reduce the extent of hyperglycemia in normal and diabetic rabbits [119].

Additionally, immunoneutralization of plasma glucagon decreases hepatic glucose output and

reduces glucose and HbA1c in ob/ob mice, providing further evidence for the role of glucagon in

the pathogenesis of diabetic hyperglycemia [120]. Both peptide and nonpeptide glucagon

receptor antagonists have been generated for use as experimental tools to block glucagon action

[34]. Consistent with data from glucagon immunoneutralization studies, Gcgr antagonists lower

blood glucose in response to exogenous glucagon administration in nondiabetic rodents and

block the actions of endogenously elevated levels of glucagon, leading to reduction of

hyperglycemia in diabetic rodents [121-123]. Several different classes of small molecule-based,

orally available Gcgr antagonists have been identified, including trisubstituted ureas,

benzimidazole, alkylidene hydrazides, and β-alanine derivatives. These molecules are actively

following oral administration in dogs, rhesus monkeys, and nondiabetic and diabetic rodents

[124-127]. Furthermore, BAY27-995, a small-molecule Gcgr antagonist, successfully blocks

exogenous glucagon-stimulated glucose production in human subjects [128]. Complementary

strategies for reduction of hepatic Gcgr signaling have utilized antisense oligonucleotide (ASO)

to target hepatic Gcgr expression. Twice weekly intraperitoneal administration of Gcgr ASOs to

db/db mice significantly reduced plasma levels of glucose, triglycerides, and free fatty acids

without associated hypoglycemia [129]. Similarly, Gcgr ASOs reduced hyperglycemia in ob/ob

and db/db mice and Zucker diabetic fatty rats together with a reduction in plasma and hepatic

triglyceride content. Peculiarly, plasma levels of glucagon and GLP-1 were markedly elevated in

17

rodents treated with Gcgr ASOs, in association with the development of α-cell hyperplasia and

hypertrophy, findings that were reversible following discontinuation of ASO therapy [130].

Taken together, these studies demonstrate that transient inhibition of Gcgr expression and/or

glucagon action can inhibit hepatic glucose production, leading to improved glucose homeostasis

in rodents.

Elimination of Gcgr Signaling: Insights from Gcgr−/− Mice

Studies of mice with targeted disruption of the Gcgr gene (Gcgr-/-) have demonstrated

that Gcgr-/-mice are viable, exhibit mild fasting hypoglycemia and have relatively low blood

glucose throughout the day compared to control littermates [131, 132]. Gcgr-/- mice have

significantly improved intraperitoneal and oral glucose tolerance with a significant increase in

plasma insulin levels following glucose challenge compared to littermate control mice.

Additionally, insulin tolerance was also improved, and gastric emptying rates were delayed in

Gcgr-/- mice [120, 131]. Despite having improved glucose tolerance and increased plasma

insulin levels following glucose challenge in vivo, islets from Gcgr-/- mice displayed blunted

responses to glucose and various insulin secretagogues including GLP-1, GIP, carbachol,

arginine, and CCK-8. Moreover, Gcgr-/- mice islets also showed an impaired glucose oxidation

rate. Reduced response to various insulin secretagogues can be a result of impaired glucose

oxidation in the Gcgr-/- mice islets [120]. Therefore, Gcgr signaling seems essential for normal

islet function in the whole body of Gcgr-/- mice. However, in this study Gcgr is deleted in all

germline tissues, and hence it is not clear if Gcgr signaling is essential for normal glucose

oxidation under physiological conditions. Future studies are required with Gcgr β-cell specific

knockouts where Gcgr is deleted at a postnatal stage to understand the physiological importance

of Gcgr signaling in the regulation of glucose and insulin signaling in the islets.

Despite normal body weight, food intake, and energy expenditure the Gcgr-/- mice had

significantly reduced whole body adiposity, reduced plasma leptin levels, and increased lean

mass [131]. Gcgr-/- mice also had differential expression of compensatory hormones including a

decrease in plasma levels of insulin growth factor-1 (IGF-1) and a two-fold increase in fasting

corticosterone levels. In addition, Gcgr-/- mice exhibited increased cAMP responsiveness to

epinephrine in liver membranes suggesting that in the absence of Gcgr signaling there was

compensation from other counter-regulatory hormones [131]. Gcgr-/- mice exhibit α-cell

18

hyperplasia, an enlarged pancreas and enhanced somatostatin staining of islets, suggesting a

possible increase in delta cell numbers. Although similar random fed and fasted insulin levels

were observed, the plasma ambient and fasting glucagon levels were increased by 56 to 280-fold

in the Gcgr-/- mice, which could be a result of increased α-cell numbers in the pancreas [131]. In

the Gcgr-/- mice the pancreatic and plasma total and amidated GLP-1 levels were also increased

10-25 fold. It is not clear whether the elevated plasma GLP-1 levels in the Gcgr-/- mice are

solely a result of increased GLP-1 synthesis from the pancreas, and/or the gastrointestinal L cells

are also involved in amplifying the plasma GLP-1 levels in Gcgr-/- mice [131, 133].

Pancreas from 1 day old Gcgr-/- pups had normal weight suggesting the enlarged

pancreas phenotype in the Gcgr-/- mice was a postnatal event [131]. Gcgr signaling seems to be

vital to maintain normal glycaemia in the new born as some Gcgr-/- pups from Gcgr-/- mothers

die 24 hours after birth due to severe hypoglycemia [133]. Gcgr signaling is also essential for

normal fetal development and normal pregnancy as ablation of Gcgr leads to intra-uterine

growth retardation (IUGR), which is characterized by reduced fetal body weight and placental

nutrient deficiency. Gcgr-/- placentas have more edema, vessel necrosis, and narrowing of

vessels and downregulation of gene expression associated with growth, oxidation stress,

adrenergic signaling, and upregulation of apoptotic gene expression. Therefore, like insulin

signaling, Gcgr is essential for normal female reproductive function [134].

Deletion of Gcgr signaling in the whole body also resulted in delayed differentiation of β-

cells as the insulin positive cells appeared later in the Gcgr-/- embryos compared to littermate

control mice [133]. Additionally, Gcgr-/- embryos had increased α-, β-, and delta-cell

proliferation [133]. Although total β-cell area was not altered in the Gcgr-/- mice, the total

number of α-, delta- cells in islets were increased in pancreata from Gcgr-/- mice [133]. Islet

cells from the adult Gcgr-/-mice had embryonic traits; normally β-cells only express Glut2

however, α-cells from the Gcgr-/- mice also expressed Glut2 [133]. Additionally, in Gcgr+/+

pancreas, islet cells co-expressing insulin and glucagon were present early during development;

however Gcgr-/- pancreata had cells co-expressing insulin and glucagon at later stages of

development in adults. β cells from Gcgr-/- mice had reduced expression of Glut2, PC3/1, Ins-1,

Maf-A, and Pdx1 [133]. These findings suggest Gcgr expression is essential for islet cell

development and differentiation.

19

Under a high fat diet (HFD), Gcgr-/- mice gained 30 percent less body weight and were

significantly leaner than the Gcgr+/+ littermate controls. Food intake was also significantly

lower in the Gcgr-/- mice compared to littermate controls on HFD suggesting reduced body

weight gain under HFD in the Gcgr-/- mice could be a result of differences in food intake.

Additionally, Gcgr-/- mice also had significantly reduced white and brown fat compared to

littermate control on HFD. Gcgr-/- mice had improved oral and intraperitoneal glucose tolerance

and increased plasma insulin levels following glucose challenge compared to littermate controls

on HFD. Furthermore, Gcgr-/- mice on HFD were resistant to STZ induced hyperglycemia and

β-cell injury. Overall, Gcgr-/- mice are resistant to HFD-induced obesity and STZ-induced

diabetes [135].

Gcgr signaling is essential for maintaining normal glycaemia, and numerous independent

studies have shown deletion of Gcgr signaling leads to prolonged hypoglycemia in the Gcgr-/-

mice [134]. Metabolic stress from prolonged hypoglycemia in the Gcgr-/- mice has been

reported to cause loss of vision and eventual death of retinal cells in the Gcgr-/- mice [136].

Therefore, long-term hypoglycemia in diabetes may increase the likelihood of vision loss and/or

retinal complications as suggested by Gcgr-/- mice studies.

Gcgr-/- mice have been a useful tool to study Gcgr’s physiological role in hepatocyte

survival and lipid oxidation. Studies with the Gcgr-/- mice have suggested that Gcgr signaling is

essential for hepatocyte survival. Gcgr-/- mice exhibited an increased susceptibility to liver

injury, and that was reversed by partially restoring Gcgr expression in the liver. Gcgr-/- mice had

elevated plasma levels of triglycerides (TGs) and free fatty acids (FFA) following an overnight

fast. Fasting increased fatty acid (FA) oxidation in the hepatocytes from the Gcgr+/+ mice,

however, hepatocytes from Gcgr-/- mice fail to increase FA oxidation. Accordingly, fasting

failed to increase expression of hepatic FA oxidation genes in Gcgr-/- mice compared to

Gcgr+/+ mice. Gcgr signaling-mediated regulation of hepatocyte FA oxidation was dependent

on PPAR-α activation and translocation to the nucleus in a p38 MAPK signaling-dependent

manner [107]. This study suggests that Gcgr signaling is essential for fasting induced fatty acid

oxidation in murine hepatocytes.

Exercise has been shown to reduce the development of fatty liver and to increase

glucagon action in the liver, which enhances liver fatty acid oxidation. Gcgr-/- mice are also

20

more susceptible to developing liver steatosis on a HFD [107]. A study investigated whether

exercise mediated reduction in fatty liver development was associated with Gcgr signaling.

Wild-type mice on HFD exercised on treadmills had increased fat oxidation in the liver and

reduced liver fat composition, however in the Gcgr-/- mice, exercise had no effect on liver FA

oxidation or fat composition. Effects of exercise on reduction of liver fat composition were

independent of body weight and were solely due to changes in fuel metabolism by the liver

[137]. Therefore, exercise-induced increase in liver fat oxidation and elimination of liver fat

composition requires Gcgr signaling.

1.3. Glucagon and the Cardiovascular System

Glucagon receptor and cardiac contractility

Pharmacological doses of glucagon increase cardiac contractility [138-140]. The

mechanism(s) responsible for the contractility effects of glucagon in the heart is not dependent

on β-adrenergic receptor signaling. Glucagon-mediated increase in cAMP was also thought to be

a result of an increase in endogenous catecholamine levels. However, studies with reserpine to

deplete endogenous catecholamine levels failed to prevent glucagon induced increases in cardiac

contractility suggesting glucagon induced positive chronotropic effects were not dependent on

endogenous catecholamine levels [141, 142]. The glucagon-mediated increase in cardiac heart

rate was also independent of hyperglycemia induced by glucagon treatment. Maintenance of

normal glucose levels by insulin treatment failed to prevent glucagon-mediated increase in

cardiac contractility [143]. Positive chronotropic effects of glucagon were dependent on the

stimulation of Gcgr associated with Gs protein, which caused adenylase cyclase activation and

the consequent increase of cAMP production in the myocardium [144]. Glucagon-mediated

increases in cardiac contractility occur 6-8 minutes after glucagon administration and last up to

25 minutes after the administration [145]. Glucagon-mediated increases in cAMP in the

myocardium are hydrolyzed by cyclic nucleotide phosphodiesterase (PDE) enzymes 3 and 4;

therefore the contractile effects of glucagon are short-lived [146, 147]. The inotropic effects

mediated by glucagon in the cardiovascular system may be preferentially localized to the

ventricular myocardium. A study by Gonzalez-Munoz et al [148] showed higher expression of

Gcgr in the ventricle versus atria in the rat heart [148]. Although direct assessment of Gcgr

21

expression has not been examined in fetal hearts, glucagon-mediated induction of cAMP is

absent in fetal hearts of mice, rats, and sheep [149]. However, a cAMP response to glucagon is

detected in the hearts of rats and mice on days 13-22 post gestation, suggesting Gcgr expression

is delayed during development in the myocardium until weaning [149, 150]. Interestingly,

glucagon-mediated increases in cAMP and contractile force are dependent on the health of the

heart. Accordingly, glucagon failed to increase cardiac contractility in heart tissue obtained from

human heart failure patients, but was able to increase cAMP content in tissues from non-failing

hearts [151, 152]. Due to glucagon’s ability to increase cardiac contractility of the heart in

humans, canines, and rodents, glucagon is occasionally used for the treatment of poisoning

caused by cardio depressant drugs such as β-blockers or calcium channel blockers [144].

Recently a study investigated whether glucagon and oxyntomodulin-mediated increases

in heart rate were dependent on a functional Gcgr. 15 μg of oxyntomodulin and 1.5 μg of

glucagon were singly dosed through an i.p injection in mice. Both glucagon and oxyntomodulin

failed to increase heart rate in Gcgr-/- mice. Furthermore, Gcgr-/- mice had higher resting heart

rate than control mice (including Gcgr+/+ and Gcgr+/- mice) at thermal neutral temperature

(30°C). The authors speculated the elevated heart rate in the Gcgr-/- mice could be the result of

low parasympathetic system activity [153].

1.3.1. Glucagon and blood vessels

Glucagon-mediated relaxation and cAMP production were measured in strips of different

arteries or veins isolated from dogs. Glucagon-mediated cAMP production was more potent in

renal compared to mesenteric or coronary arteries. Similarly, glucagon had the highest effect on

relaxation of renal compared to mesenteric or coronary arteries. Furthermore, removal of

endothelium from renal artery did not alter glucagon-mediated relaxation suggesting glucagon

might have a direct effect on the renal artery or the smooth muscle cell. This study suggests

differential impact of glucagon on relaxation and cAMP production on different arteries, which

can be a result of heterogeneous expression of Gcgr in smooth muscle cells and blood vessels

[154]. Glucagon is a potent vasodilator, and a large number of studies in canines have shown that

a single injection of glucagon increased hepatic blood flow by 100% [155]. Glucagon had the

highest impact on superior mesenteric artery (SMA) blood flow, that increased by 190% [155]

22

compared to its impact on other arteries. As Gcgr expression was not assessed in different blood

vessels or cell types of blood vessels it was not clear whether these effects of glucagon on SMA

artery were the result of higher Gcgr expression in the smooth muscle cells in the SMA artery.

1.3.2. Glucagon and calcium ions

Glucagon has been reported to increase cAMP in cardiac tissues and cells. In turn cAMP

is known to upregulate calcium uptake and releases it from the sarcoplasmic reticulum leading to

cardiac action potentials and ultimately increases in cardiac contractility [139, 156, 157]. A

number of studies have shown that glucagon can increase both intracellular calcium

accumulation and mitochondrial calcium uptake in cardiac microsomal fraction and in isolated

cardiac cells [139, 156, 157]. Glucagon-mediated uptake of calcium in cardiac microsomal

fraction is not dependent on β-adrenergic receptor signaling as these effects of glucagon on

calcium signaling are not blocked by β-adrenergic blocking agents [139].

1.3.3. Glucagon and blood pressure

Gcgr signaling activation has been shown to increase blood pressure in dogs and rodents.

A study reported administration of pharmacological dose of glucagon into the pulmonary artery

increased heart rate, cardiac output, stroke volume aortic mean, and left ventricular end-diastolic

pressure [158]. Myocardial oxygen consumption was also increased with glucagon

administration, which was also associated with an increase in coronary flow. These effects of

glucagon lasted from 3 to 20 minutes following glucagon injection [158]. It has been

demonstrated that the induction of systemic hyperglucagonemia is also associated with increased

blood pressure. A study conducted by Li et al [159] showed glucagon administration in mice via

osmotic minipump resulted in elevation of serum glucagon levels by 129% and a raise in systolic

blood pressure [159]. An increase in kidney weight/body weight ratio was observed, and the 24

hour urinary albumin excretion augmented by 108%. Concurrent administration of a Gcgr

antagonist, [Des-His1-Glu9] glucagon with glucagon significantly attenuated glucagon-mediated

increase in systolic blood pressure, kidney weight/body weight ratio, and 24 hour urinary

albumin excretion [159]. The study suggested chronic glucagon administration led to systemic

hypertension [159], however whether the increased blood pressure was due to direct kidney

damage, vascular effects, and/or direct or indirect glucagon action on the heart, remained unclear

[40]. Conversely, studies have also reported down-regulation of cardiac Gcgr expression in

23

rodents with experimental hypertension, in association with the development of cardiac

hypertrophy [160], but the significance of this correlative observation is uncertain [160].

Additionally, G-->A/GT (Gly40Ser) polymorphism of the Gcgr gene has been associated with

hypertension in human subjects. G-->A/GT (Gly40Ser) polymorphism of the Gcgr has been

reported to lead to less responsiveness of the receptor to its ligand. An association study in

humans investigated the correlation between Gcgr polymorphism and the risk to develop

hypertension by measuring serum uric acid, fractional excretion of uric acid, and exogenous

lithium levels in individuals with or without hypertension. The study suggested that the Gly40Ser

polymorphism of the Gcgr gene was associated with higher risk of hypertension, and it could be

due to an enhanced renal sodium reabsorption associated with the polymorphism of the Gcgr

gene. [161]. Therefore, it is unclear if activation or inhibition of the Gcgr signaling is beneficial

or detrimental for the development of hypertension and requires further investigation.

1.3.4. Glucagon and cardiac ischemia

Following an acute myocardial infarction an increased incidence of hyperglycemia and

impaired glucose tolerance was observed in non-diabetic patients. These patients also had

elevated levels of plasma glucagon. This suggests myocardial infarction may induce secretion of

glucagon. However, the significance of the increase in glucagon levels following ischemia was

not explored [162]. A number of studies have examined the effects of physiological and

pharmacological dose of glucagon in ischemia; the majority of those were conducted in canines,

rats, and some in patients with myocardial infarction or chronic rheumatic heart disease [142,

158, 163-166]. Following myocardial infarction the contractility of the heart is depressed; left

ventricular performance is estimated by the relationship between left ventricular end-diastolic

pressure and left ventricular minute work. The left ventricular performance declines with cardiac

ischemia leading to reduced cardiac output and increased peripheral vascular resistance.

Glucagon has been shown to be a cardiac inotrophic and chronotropic agent in human subjects,

rodents, and canines, and hence, glucagon has been proposed to be an effective therapy to

prevent depression of cardiac contractility following myocardial infarction. A single dose of

glucagon (1-3 mg) directly injected into pulmonary artery of patients with acute myocardial

infarction resulted in a significant rise in blood pressure, slight rise in chronotropic effect and

temporary improvements in cardiac output [142, 158, 163-166]. Accordingly, glucagon’s effect

24

on patients with acute myocardial infarction or chronic rheumatic heart disease was investigated

following a single 2-5 mg injection or continuous infusion of glucagon (20 mg) into the

pulmonary artery. Cardiac parameters were measured 2, 5, 10, 20, 30 minutes following

glucagon injection or 24 or 48 hours following glucagon infusion. After a single injection of

glucagon a significant increase in cardiac inotropic action, cardiac output, and an enhanced

cardiac performance were observed in the acute phase of myocardial infarction. Those effects of

glucagon were noticeable 10 minutes following glucagon injection and lasted until 30 minutes

following injection. In the chronic rheumatic heart disease setting of mitral stenosis, glucagon

increased left atrial pressure, an undesirable effect as this might worsen the outcome [164].

Overall, in human studies glucagon administration in acute versus chronic disease had different

effects, and single versus continuous infusion also gave different results. Similar to human

studies, in dogs, a pharmacological dose of glucagon administered intravenously either as a bolus

or as an infusion following an acute myocardial infarction resulted in augmentation of the

contractile state of the non-infarcted portion of the left ventricle. Left ventricular performance

and overall cardiac output was increased following glucagon treatment suggesting the

dysfunctional impact of myocardial infarction on the left ventricle could be reversed with

pharmacological doses of glucagon [142]. In contrast to glucagon’s effect in chronic rheumatic

heart disease in humans, an improved outcome was noted in dogs with chronic myocardial

infarction after glucagon administration. In studies with dogs, 50 minutes following

pharmacological dose of glucagon infusion recovery from myocardial infarction was monitored.

The infusion of glucagon reduced left ventricular failure, which led to an increase in cardiac

performance in dogs. There were no significant changes in coronary flow nor myocardial oxygen

consumption with glucagon 1 hour or 1 week after myocardial ischemia suggesting glucagon

might be improving cardiac outcome from acute ischemia by increasing cardiac efficiency [158].

This study also suggested that the improvements in outcome from ischemia by glucagon could be

due in part to glucagon action to increase heart rate, which was similar to the effects of external

pacing of hearts in patients going through myocardial infarction. Additionally, another study

reported i.v. injection of 100ug/kg of glucagon led to increased heart rate, decreased blood

pressure, and an induction of positive chronotropic effect in anaesthetized dogs, but not in un-

anaesthetized dogs with ligation of the anterior descending branch of the left coronary artery

[165]. Although none of the studies have explored how glucagon protects the heart from

25

myocardial infarction mechanistically, one proposed mechanism through which glucagon can be

protecting the myocardium from ischemia is by increasing potassium ions (K+) flux into the

heart. Studies have shown that infusion of glucose, insulin, and potassium, or potassium alone in

the heart leads to improved outcome from myocardial infarction through a shift in cardiac fuel

metabolism from fatty acid to glucose utilization. Studies have also shown that depletion of K+ in

the heart leads to development of arrhythmia (abnormal heart rate) and impairs cardiac outcomes

from myocardial ischemia, and inhibition of K+

depletion can correct the arrhythmia and

improves outcomes from myocardial infarction. [142, 166]. In contrast, there are studies that

show that glucagon can impair outcome from myocardial infarction. In one study,

pharmacological levels of glucagon were perfused (1ug/ml) in the isolated working rat heart

model before ischemia, and recovery from ischemia was monitored. Glucagon impaired recovery

from ischemia and cardiac glycogen levels in those hearts were also depleted. The authors of this

study conclude that glucagon-mediated depletion of glycogen levels in the heart can be a

mechanism through which glucagon worsens outcomes from ischemia [167]. So, the role of

glucagon receptor signaling in the setting of cardiac ischemia is not clear and requires further

investigation.

1.3.5. Glucagon and cardiac fuel metabolism

Glucagon has been shown to play an important role in lipid metabolism in the liver;

however the role of glucagon in cardiac fuel metabolism is not fully explained. Perfusion of

pharmacological dose of glucagon in working, normal, and insulin-resistant rat hearts increased

inotropic and chronotropic activity of the heart followed by an increase in cardiac glucose and

palmitate oxidation [168]. Furthermore, glucagon caused an increase in fat oxidation in normal

hearts through a malonyl CoA-independent mechanism [168]. Similarly, infusion of glucagon in

perfused rat hearts at levels designed to achieve physiological concentrations led to induction of

glycolysis and glucose oxidation, similar to insulin actions in the heart that was mediated via

phosphatidylinositol 3-kinase-dependent and adenylate cyclase- and cAMP-independent

pathways [169]. Hence, unlike the effects of glucagon in the liver that generally oppose insulin

action, glucagon and insulin action in the heart may overlap in regards to stimulation of fuel

metabolism.

26

1.3.6. Glucagon and myocardial oxygen consumption

Infusion of pharmacological doses of glucagon stimulated cardiac contractility, increased

blood flow and caused elevation in myocardial oxygen consumption in humans and dogs [170].

In one study glucagon was infused in hearts of dogs, and left ventricular work per minute was

recorded. Glucagon increased the left ventricular work per minute; however, index for minute to

work efficiency was significantly lower in glucagon-treated hearts without any changes in ATP

levels suggesting the generation and utilization of energy was unchanged with glucagon. These

findings indicate that glucagon reduces the cardiac efficiency by increasing myocardial oxygen

consumption [145]. Yet in a different study pharmacological doses of glucagon in conscious

dogs with intact myocardial infarction did not produce any changes in myocardial oxygen

consumption. It is postulated that during myocardial infarction there are reciprocal changes in

factors that increase myocardial infarction such as elevations in circulating levels of glucagon

(increases cardiac inotrophy) whereas factors that decrease oxygen consumption (ventricular end

diastolic wall stress) may balance out leading to no overall changes in myocardial oxygen

consumption [171].

Mini glucagon and the heart:

Glucagon has been reported to be processed by target tissues locally such as the pancreas,

the liver, and the heart as well as in the circulation by a specific ectoendopeptidase to liberate

COOH-terminal (19-29) fragment, mini-glucagon [172]. Mini-glucagon has been shown to

contribute significantly to the positive cardiac contractile response of glucagon as the

unprocessed form of glucagon has minimal effects on cardiomyocyte contraction [173, 174].

However, both mini-glucagon and glucagon synergistically cause accumulation of Ca2+

into the

cardiac sarcoplasmic reticulum stores [173, 174]. Mini-glucagon does not act through the known

glucagon receptor. Mini-glucagon has no effect on cAMP, cGMP, or inositol 1,4,5-triphosphate

production in cardiac cells or tissues [174]. Therefore, the actions of mini-glucagon are not

dependent on the classical glucagon transduction pathways. Nonetheless, in the cardiac cells

mini-glucagon mediates release of arachadonic acid from ventricular chick cardiomyocytes, and

adding arachadonic acid to cardiomyocytes has similar effects as mini-glucagon on Ca2+

homeostasis and cell contraction. Hence, mini-glucagon actions on Ca2+ homeostasis and

cardiomyocyte contraction may be dependent on arachidonic acid release [174].

27

1.4. Introduction to the incretins

Incretins are gut hormones that are secreted from the gut during meal ingestion; they

induce insulin secretion by the pancreatic β-cells in a glucose-dependent manner. Two classical

incretins that have been the most studied thus far are glucagon-like peptide-1 (GLP-1) and

gastric inhibitory polypeptide (GIP). GLP-1 and GIP exert their actions on target tissues through

distinct G-protein coupled receptors (GPCRs): Glucagon-like peptide 1 receptor (Glp1r) and

Gastric inhibitory peptide receptor (Gipr) respectively. In addition to GLP-1 and GIP there are

numerous other peptide hormones, nutrients, bile acids, and lipid amides that are also secreted

following nutrient ingestion and many of these molecules also bind to receptors on islet β-cells

and stimulate insulin secretion.

1.4.1. Incretin secretion and synthesis

GLP-1 is derived from proglucagon, which is processed by prohormone convertase 1/3

(PC1/3) (Figure 1.1). GLP-1 is predominantly made in the L cells of the gut, circumvallate

papillae and adjacent salivary gland, and the central nervous system (mostly in the brainstem

from which it is delivered to other parts of the brain) [175]. GLP-1 expression is also detected in

a subset of α-cells and is stimulated by exogenous IL-6 or exercise [176]. Ingestion of nutrients

including carbohydrates, fats, and proteins can stimulate GLP-1 secretion through secretion of

other hormones, neural signals, and luminal nutrient interactions with the gut [177, 178].

Unlike GLP-1, GIP is synthesized and secreted from the K cells located in the intestinal

epithelium. GIP biosynthesis in gut K cells is not well understood. Studies have identified

regulatory factor X 6 (Rfx6) as an important factor in GIP secretion from K cells and for GIP

synthesis in K cells [179]. GIP has been detected in neurons, Schwann cells, and

oligodendrocytes in the CNS [180]. GIP is also detected in pancreatic α-cells and in islets from

glucagon knockout mice, GIP is detected in pancreatic β-cells [181, 182]. It is not clear if

detection of GIP from pancreatic cells suggests a possibility for intra-islet paracrine GIP action.

GIP secretion is stimulated by nutrients including fat [183].

1.4.2. Incretin action in the pancreas

One of the most important functions of GLP-1 and GIP is to stimulate insulin secretion

from the pancreatic β-cells in a glucose-dependent manner. GLP-1 and GIP stimulate insulin

28

secretion via numerous different mechanisms including 1) enhancement of glucose sensing

mechanisms by increasing β-cell expression of glucokinase and GLUT2; 2) direct inhibition of

KATP channels which leads to β-cell membrane depolarization; 3) influx of extracellular

Ca2+

through opening voltage-dependent Ca2+

channels, mobilization of intracellular Ca2+

through activation of nonselective cation channels; 4) membrane depolarization by increase in

mitochondrial ATP synthesis; 5) reduction in Kv currents which prevent β-cell repolarization by

closing voltage dependent Kv channels; 6) increase β-cell insulin storage granule exocytosis

[184]. In addition to stimulating insulin from β-cells, GLP-1 has also been shown to promote β-

cell proliferation, neogenesis, and glucose sensitivity to glucose-resistant β-cells and reduce β-

cell apoptosis[185]. However, these effects of GLP-1 are secluded to rodent islets as Glp1r

agonists do not have proliferative effects on human islets. One possible reason for the species

difference could be because the rates and capacity for islet cell turnover and growth in humans

are different from rodents [186]. Furthermore, it is difficult to assess beta cell mass in human

subjects noninvasively [186].

β-cell Glp1r signaling is dependent on β-arrestin-1 as GLP-1 mediated activation of cAMP,

CREB, and ERK1/2 activation is reduced following knock down of β-arrestin-1 with siRNA in

INS-1 cells [187]. Furthermore, GLP-1 mediated insulin secretion, and prevention of β-cell

apoptosis was ameliorated following knockdown of β-arrestin-1 with siRNA in MIN-6 cell lines

[187]. Therefore, β-arrestin-1 is essential for Glucagon-like peptide-1 receptor (GLP-1R)

signaling in β-cells. Additionally, another key transcription factor activated by GLP-1 is

TCF7L2, and is a downstream target of the Wnt/ β -catenin pathway [188]. In INS-1 cells and

primary islets in culture the GLP-1R agonist, exendin-4 (Ex-4) has been shown to activate the

canonical Wnt signaling pathway, enhance β-catenin/Tcf7l2 mediated cyclin D1 gene expression

and β-cell proliferation in a cAMP/PKA, AKT, and ERK1/2 dependent manner [188]. These

studies are consistent with the loss of function studies with Tcf7l2 pancreas-specific knockout

mice; GLP-1-stimulated insulin release from pancreas-specific Tcf7l2 knockout mice islets is

compromised [189]. Therefore, Tcf7l2 is an essential transcription factor for GLP-1’s action in

β-cells. GLP-1’s proliferative and anti-apoptotic actions on β-cells are also dependent on Insulin-

like growth factor 1/2 (IGF) signaling. GLP-1 fails to increase β-cell proliferation in islets from

Igf1r-/- mice. Furthermore, GLP-1 mediated activation of IGF pathway stimulates β-cell

29

proliferation in islets [190]. GLP-1 failed to prevent apoptosis and promote phosphorylation of

AKT and BAD in cytokine treated MIN-6 cells and mouse islets following knockdown of the

Igf-1/2 receptor [191]. Thus, IGF signaling is essential for GLP-1’s action regulating

proliferation and survival of β-cells.

Like GLP-1, GIP also stimulates insulin secretion in a glucose-dependent manner in islets

and INS-1 cells [192-196]. However, unlike GLP-1, which has been consistently shown to

promote β-cell proliferation and survival in multiple in vivo studies, GIP’s action on β-cell

proliferation and survival are inconsistent in in vivo studies. The research group headed by

Weidenmeir et al has shown that the GIP analog, [D-Ala2]GI treatment in vivo preserved β-cell

mass and prevented apoptosis in multiple rat models of T2D and in INS-1 cells [197]. Similarly,

in ZDF rats, iv injections of [D-Ala2]GIP for two weeks led to decreased Bax and upregulation

of Bcl-2 in β-cells. Conversely, Maida et al demonstrated that chronic treatment with [D-

Ala2]GIP had a modest effect in preventing apoptosis in β-cells and no effect on β-cell mass,

whereas chronic treatment with Ex-4 significantly increased β-cell mass and prevented β-cell

apoptosis in STZ treated diabetic mice [198]. Furthermore, deletion of Glp1r increased

susceptibility to STZ induced diabetes, however, deletion of Gipr did not increase susceptibility

to STZ-induced diabetes in mice [198]. Therefore, GIP’s anti-apoptosis and proliferative actions

on β-cells are not clearly understood and may require further investigation. GIP mediates its

insulinotropic actions on β-cells through cAMP/PKA and cAMP/Epac2 pathways, similar to

GLP-1 [192]. GIP and GLP-1 have both been shown to activate AKT independent of PI3K or

Thr (308) and Ser (473) phosphorylation [199]. GIP has been proposed by Widenmaier et al to

activate the AKT pathway through a non-canonical pathway [199]. Furthermore, in INS-1 cells

and mouse islets, GIP has been shown to increase antiapoptotic protein expression, Bcl-2,

through PKA-dependent phosphorylation of CREB [199]. Gipr signaling is not heavily studied

like the Glpr1r as downregulation of Gipr expression is observed in experimental and clinical

diabetes. A possible mechanism through which Gipr expression is downregulated in diabetes can

be linked to PPAR-γ; under glucotoxic conditions reduced PPAR-γ activity, and Gipr expression

has been observed [200]. Downregulation of Gipr is also associated with ubiquitination and

degradation of the Gipr during hyperglycemia in rodent and human islets [201]. In the majority

of the hyperglycemic subjects with T2D, GIP also fails to stimulate insulin secretion, and this

has been associated with SNP (rs10423928) in the GIPR gene. T2D subjects with this SNP

30

showed lower GIPR expression and had lower GIP-induced insulin response [202]. Therefore,

genetic and environmental stimuli may be involved in downregulation of Gipr during

hyperglycemia and diabetes.

1.4.3. Incretin action in the adipose tissue

Glp1r expression is controversial in white adipose tissues (WAT). Glp1r expression in

human or rodent WAT was not detectable even though GLP-1 bindings sites were detected in

human and rat WAT membranes using radiology and binding assays [203]. Studies suggest

GLP-1 mediates lipolysis in human primary adipocytes and in 3T3-L1 cell lines in vitro [204].

However, the lipolytic actions of GLP-1 on WAT in vivo were not clear and might be indirect

through the sympathetic nervous system as studies suggested direct infusion of GLP-1 into

abdominal subcutaneous adipose tissue failed to reduce WAT in human subjects[205].

Additionally, intracerebroventicular (icv) infusion of GLP-1 led to decreased WAT mass and

inhibition of Glp1r signaling using Exendin 9-39 (Ex-9) infusion icv led to increased fat mass.

The effect of central Glp1r signaling on WAT mass was independent of food intake and body

weight loss [206]. Glp1r expression was not detected in brown adipose tissue (BAT). A single

icv injection of GLP-1 led to an increase in BAT thermogenesis and increase in sympathetic

nerve activity in BAT in rodents [207]. Therefore, GLP-1 mediated BAT thermogenesis is

indirect via the central nervous system.

Gain and loss of function studies with GIP illustrates an essential role of Gipr signaling in

WAT lipolysis in rodents and humans [208-213]. Gipr signaling activation leads to an increase

in lipid uptake, secretion of adipokine, weight gain, and insulin resistance [208]. Conversely,

Gipr-/- mice or ablation of GIP action using immunoneutralizing antisera leads to protection

from diet-induced insulin resistance. Additionally, rescuing Gipr expression in the adipocyte in

whole body Gipr-/- mice leads to normal body weight gain, and these mice are no longer

resistant to diet induced obesity [213]. It is not clear if these effects of GIP on adipocytes are

direct or indirect via insulin. Asmar et al showed that in human subjects, in the absence of

hyperinsulinemia, GIP’s effects on lipid metabolism and free fatty acids were significantly

reduced. GIP infusion under hyperinsulinimic conditions led to increased adipose tissue blood

flow [214]. Therefore, this study suggests GIP’s action on adipose tissue may be insulin-

dependent. Gogebakan et al demonstrated that GIP infusion alone led to increased plasma insulin

31

levels, and lowered plasma free fatty acids in healthy nondiabetic subjects. However, it was not

clear if GIP infusion alone led to changes in free fatty acid or that was the result of increased

plasma insulin following GIP infusion [215]. Human subjects with a Gipr SNP (rs10423928)

had reduced adipogenesis. It is not clear if this SNP is associated with lower Gipr expression in

adipocyte or lower plasma insulin levels [216]. Therefore further investigation is required to

understand if GIP has direct effects in adipocyte or if the effects are indirect via insulin.

1.4.4. Incretin action in the liver

Glp1r agonists have been shown to lower liver lipid content, hepatic steatosis, liver weights, and

inhibit hepatic glucose output [217, 218]. Similarly, during a hyperinsulinemic-euglycemic

clamp, Glp1r-/- mice showed reduced phosphorylation of liver AKT and GSK-3β in the liver

and defective hepatic glucose output [219]. To date no full length GLP-1 receptor expression

has been detected in murine hepatocytes nor was a cAMP response detected in murine

hepatocytes following GLP-1 or Glp1r agonist treatment [220, 221]. However, in rat and human

hepatocytes, Glp1r mRNA expression was detected. Additionally, Glp1r binding sites were not

detected in human liver sections [222, 223]. Thus, Glp1r expression in the liver is controversial.

Glp1r mediated actions in the liver could be indirect through the brain as central administration

of GLP-1 led to inhibition of endogenous glucose production in rodent studies [224, 225].

Another possibility can be that GLP-1 promotes insulin secretion and inhibits glucagon

secretion, and this can result in reduction of hepatic glucose production indirectly by GLP-1.

However, the majority of the studies that examined GLP-1 mediated liver glucose output did not

control insulin nor glucagon levels. Therefore, it is difficult to make conclusions whether GLP-1

mediated hepatic glucose production is a result of changes in plasma insulin or glucagon levels.

1.4.5. GLP-1 and gastric emptying

GLP-1 has been shown universally to be a potent inhibitor of gastric emptying in rodents,

pigs and human studies[226], [227] [228] [229] [230].. GLP-1 mediated inhibition of gastric

emptying contributes significantly to the reduced glycemic excursion as such infusion of low

dose of GLP-1 inhibited gastric emptying and lowered glycemic excursion without changes in

insulin secretion [228]. Although GLP-1 has been shown to directly stimulate cAMP formation

in rat gastric gland preparations, GLP-1 mediated inhibition of gastric emptying is mediated

32

through vagal afferents as vagotomy prevented GLP-1 mediated inhibition of gastric emptying in

rats [229] [230].

1.4.6. Incretin action in the heart

Glp1r expression in the heart:

GLP-1 has been shown to have direct effects on the myocardium [231-234]. However,

numerous studies have shown reduced Glp1r expression in ventricular cardiomyocytes relative

to atria in multiple species [235-237]. Recent studies in mice by Kim et al[235] showed that the

level of Glp1r expression in the atrial cardiomyocyte is significantly higher compared to the

expression in ventricular cardiomyocyte [235]. In neonatal and adult cardiomyocytes from mice

and rats Glp1r activation is coupled to increased cAMP production [231, 238]. In whole

cardiomyocytes cell patch clamp studies from adult dogs, GLP-1 treatment increased cAMP

production and induced Ca2+ channel current in a PKA-dependent manner [239]. In neonatal

rodent cardiomyocytes and mouse atrial cardiomyocyte cell lines HL-1, activation of the Glp1r

led to increase in AKT and ERK phosphorylation and also reduced apoptosis in a PI3K and

ERK-dependent manner [240, 241] Therefore, these studies suggest GLP-1 has protective effect

on cardiomyocytes and activates cardioprotective signaling pathways in primary cardiomyocytes

in culture.

Findings from ex-vivo studies with GLP-1 on cardiac contractility are not consistent. In

one study perfusion of GLP-1 in the rat heart led to reduced left ventricular developed pressure,

and in another study a similar dose of GLP-1 increased left ventricular diastolic pressure (LVDP)

in mouse hearts [232, 233]. However, in both studies, GLP-1 perfusion led to increased glucose

uptake by the heart, and this was associated with increased activity of p38 MAPK, and increased

GLUT1 protein expression at the cardiac plasma membrane [232, 233]. Similar findings were

observed in isolated dog hearts, where GLP-1 infusion increased glucose uptake in a p38

MAPK-dependent manner [242]. GLP-1-mediated changes in heart rate (HR) and blood

pressure (BP) are complex depending on the species. GLP-1 administration in rodents increases

HR, and BP via a -adrenergic receptor signaling-independent, Glp1r-dependent manner. The

effects of GLP-1 on HR and BP disappear in the presence of Ex-9-39 and not with a -

adrenergic receptor antagonist [243-245]. In rodents the effects of GLP-1 on HR and BP involve

both CNS and peripheral mechanism. In vagotomized rats, icv GLP-1 failed to increase HR or

33

BP. Therefore this suggests GLP-1 mediated increases in BP and HR involves intact vagal

nerves and the central nervous system [246]. In rodents, central GLP-1 mediated regulation of

BP also involves vasopressin, which is a posterior pituitary gland hormone [247]. Vasopressin

receptor antagonist injected in the artery-prevented icv GLP-1 induced rise in BP in mice and

rats [247]. Therefore, GLP-1 mediated increases in HR and BP in rodents involves a complex

pathway from the CNS. Although Glp1r activation in healthy animals leads to increased HR and

BP, in obese/diabetic (db/db mice) mice that are fed salt to induce hypertension sustained Glp1r

activation leads to a reduction in BP [248, 249]. One of the potential mechanism via which

GLP-1 is mediating antihypertensive effects can be through induction of nitric oxide (NO)

secretion by endothelial cells, which has antihypertensive properties [250].

Studies on human subjects with T2D or heart failure reported inconsistent findings. In

some studies infusion of native GLP-1 or GLP-1R agonists led to an increase, and in other

studies it led to a reduction in HR and BP [251, 252]. These differences can possibly be

attributed to structural difference in GLP-1R agonists as these studies have utilized different

GLP-1R agonists. However, in the majority of the clinical trial studies with long-term GLP-

1Ragonists’ including exenatide and liraglutide, BP reduction was detected in non-diabetic obese

and T2D subjects [253-255]. Furthermore, the majority of the BP lowering effect of GLP-1R

agonists in clinical trials could attribute to reduced body weight as body weight was reduced in

most of the clinical trial studies [256, 257]. Therefore, it is not entirely clear if long term

treatments with GLP-1Ragonists leads to lower BP and HR as a result of reduction in body

weight or these effects of GLP-1R agonists are independent of changes in body weight and

require future investigation.

The majority of the studies have reported that native GLP-1 and Glp1r agonists have

cardioprotective effects in ex-vivo and in vivo models of ischemia [232, 233, 238, 258]. Direct

perfusion of GLP-1 or Ex-4 during reperfusion after ischemia led to improvement in LVDP in rat

and mice hearts [233, 238, 259]. Similarly, reduced infarct sizes were observed in studies with

temporary or permanent left anterior descending (LAD) ligations following iv or sc infusion/

injections of GLP-1 and Glp1r agonists in rats, mice, and pigs [258, 260-262]. Multiple

mechanisms for the beneficial effects of GLP-1 in cardiac ischemia have been proposed.

Albiglutide, which is a Glp1r agonist, has been shown to cause cardioprotective effects in the

34

heart through improving cardiac energetics. Injection of albiglutide for 3 days followed by

temporary LAD coronary artery occlusion in rats led to reduced infarct size, and this was

associated with reduced fatty acid oxidation and increased glucose oxidation rates [263].

Therefore, albiglutide has been suggested to improve recovery from ischemia through improving

fuel metabolism in the heart. Another proposed mechanism for Glp1r activation leading to

cardioprotection is in part through mechanisms requiring the heart to receive signals from the

vasculature, or neurons. Direct administration of liraglutide in coronary arteries at the onset of

reperfusion injury failed to protect the heart. However, liraglutide injection into mice in vivo

before ischemia/reperfusion led to improved LVDP [238]. Not all studies with GLP-1 or Glp1r

agonists have shown to be protective against cardiac ischemia suggesting that universal

activation of Glp1r does not lead to cardioprotection. Therefore, the exact mechanism involved

in GLP-1 mediated cardiac protection from ischemia is not clear. Furthermore, it is not clear if in

the setting of diabetes, GLP-1 is effective in protecting the heart against ischemia, and this

requires further investigation.

In human studies, GLP-1 infusion for 72h improved left ventricular (LV) ejection

fraction (LVEF) and wall motion of the infarcted area of the heart [242]. Furthermore, in

patients with coronary artery disease, iv infusion of GLP-1 protected against LV dysfunction and

prevented myocardial contractile abnormality following ischemia [264]. Furthermore, exenatide

reduced ischemic area following infusion for 6 hours in patients undergoing primary angioplasty

[265]. However, the majority of the studies in humans with Glp1r agonists were with small

patient numbers, were not double blinded, or Glp1r agonists were not given long term in these

studies. Hence, future studies need to investigate whether long term Glp1r agonist treatment is

cardioprotective in larger patient populations and subjects with T2D.

1.4.7. Insight from incretin receptor knockout mice

Genetic deletion of Glp1r led to a mild impairment in glucose-stimulated insulin

secretion and glucose homeostasis in vivo [192, 266]. Surprisingly, despite inhibitory effects of

GLP-1 on food intake, disruption of Glp1r did not impact food intake, or body weight and

paradoxically increased susceptibility to diet-induced obesity [266, 267]. Despite Glp1r’s action

in the cardiovascular system in reducing ischemia and blood pressure, Glp1r-/- mice in C57bL/6

background had mostly normal cardiac morphometric parameters including normal LV function

35

and heart rate basally [232, 233, 238, 258]. However, Glp1r-/- mice in the CD-1 background

had thicker ventricular walls and had significantly increased baseline LVDP during Langendorff

perfusion of the hearts under aerobic conditions suggesting Glp1r signaling might be important

for development of the heart in some mouse strains [231, 232].

Like Glp1r-/- mice, genetic deletion of the Gipr also led to mild impairment in glucose

stimulated insulin secretion. Gipr-/- mice are resistant to diet-induced obesity and are more

insulin sensitive than wild-type littermate control mice [268, 269]. Additionally, genetic

variation in the GIPR in humans has also been shown to be associated with increased body mass

index and elevated fasting glucose levels [270, 271].

Double incretin knockout (DIRKO) mice were first characterized by Hansotia et al [272];

despite loss of both classical incretin receptors, the DIRKO mice had normal body weight and

food intake but had impaired glucose tolerance during oral glucose challenge and not during an

ip glucose tolerance. Surprisingly, glucose-stimulated insulin secretion in perifused DIRKO mice

islets was preserved despite loss of both Gipr and Glp1r [272]. Furthermore, studies with

DIRKO mice have shown that DPPIV inhibitors work to reduce glucose excursion solely,

through elevation of GLP-1 and GIP levels as the effect of the DPPIV inhibitors

valinepyrrolidide (Val-Pyr) and SYR106124 are diminished in the DIRKO mice [272]. The most

surprising finding from the DIRKO studies was that despite the loss of both the incretins those

mice on an HFD had preservation of insulin sensitivity and elevated energy expenditure which

was associated with increased locomotor activity [267].

1.5. Rationale & Hypotheses

Glucagon secretion has been observed to be inappropriately increased in many T2D

subjects, which has fostered great interest in reduction of glucagon action for the treatment of

T2D [111, 112, 117, 273]. Genetic or pharmacological inhibition of Gcgr signaling leads to

improved glucose homeostasis, reduced fasting glycaemia, delayed gastric emptying, and

increased pancreas weight [118, 119, 128-130, 274, 275]. In addition to these phenotypes

antagonism or genetic deletion of Gcgr also leads to elevated plasma active and total GLP-1

levels [131]. It is unknown whether direct loss of Gcgr signaling is responsible for the

36

phenotypes observed in the Gcgr-/- mice or if the elevated GLP-1 levels contribute to the

phenotypes observed in the Gcgr-/- mice.

Cardiovascular disease is the most common complication in individuals with diabetes and

is the number one cause of death in diabetes [276-279]. Glucagon levels are elevated in diabetes,

and hyperglucagonemia is fundamental to the pathophysiology of hyperglycemia, ketoacidosis,

and impaired counter-regulation [111, 112, 117, 273]. Glucagon receptor antagonists and

Gcgr/Glp1rco-agonists show promising results to treat diabetes and obesity [118, 119, 128-130,

274, 280-282]. The Gcgr is expressed in the heart and promotes both cAMP production and

enhances cardiac contraction [146, 283]. However, the consequence of hyperglucagonemia in

the development of cardiovascular complications has not been explored in diabetes and obesity.

Furthermore, the physiological role of the Gcgr in the cardiovascular system is unknown.

The investigations began with the following research objective/question and hypotheses,

respectively:

Pharmacological levels of GLP-1 contribute to the improved glucose homeostasis, β-

cell function and delayed gastric emptying in the Gcgr-/- mice

The extent to which the improved metabolic phenotype of Gcgr−/− mice reflects the

direct loss of Gcgr signaling in liver vs. the contribution of enhanced GLP-1 action remains to be

determined. Multiple phenotypes are consistent with enhanced GLP-1 action, including

improved glucose homeostasis, improved β-cell function, increased insulin sensitivity, reduction

in fasting glycaemia, and delayed gastric emptying are observed in the Gcgr-/-mice (Table 1)

[131, 133, 135, 274, 280]. This has led me to hypothesize that GLP-1 receptor signalling

substantially determines the improved glucose tolerance, increased insulin sensitivity,

reduction in fasting glycaemia and delayed gastric emptying in the Gcgr-/-mice. I have

aimed to delineate the role of GLP-1 receptor signalling to the overall phenotype of the Gcgr-/-

mice using a newly generated double knockout mouse with a disruption of the known Glp1 and

Gcg receptors.

What is the physiological role of the glucagon receptor in the cardiovascular system

under normal and cardiac ischemic conditions?

A substantial body of literature supports the acute transient use of glucagon to reverse the

37

effects of β-adrenergic agonists, or to increase heart rate, blood pressure, and cardiac output in

subjects with circulatory collapse refractory to adrenergic stimulation [143, 284, 285]. In

contrast, more sustained administration of glucagon may increase the severity of ischemic

cardiac injury, and this has led me to hypothesize that enhancing Gcgr signalling may increase

and reducing Gcgr signalling may reduce susceptibility to cardiac ischemia [167, 286]. I

have aimed to identify the role of Gcgr signalling in cardiac ischemia by treating mice in vivo,

and using ex-vivo hearts and in vitro primary cardiomyocyte and atrial cardiac cell lines with

glucagon and by utilizing Gcgr+/- mice and by generating cardiac specific Gcgr-/- mice.

38

CHAPTER 2: Dual elimination of the glucagon and GLP-1receptors in mice reveals plasticity in the incretin axis

CHAPTER 2

Dual elimination of the glucagon and GLP-1receptors in mice reveals plasticity in the incretin axis

The work presented in this chapter corresponds to the following publication: Ali S., Lamont

B., Charron M., Drucker D.J. Journal of Clinical Investigation (2011) 121(5): 1917-29

Author contributions:

B. Lamont provided assistance in islet isolation and insulin secretion experiments (Figure

2.6).

39

2.1 Research Summary

Disordered glucagon secretion contributes to the symptoms of diabetes, and reduced

glucagon action is known to improve glucose homeostasis. In mice, genetic deletion of the

glucagon receptor (Gcgr) results in increased levels of the insulinotropic hormone glucagon-like

peptide 1 (GLP-1), which may contribute to the alterations in glucose homeostasis observed in

Gcgr-/-mice. Here, we assessed the contribution of GLP-1 receptor (Glp1r) signaling to the

phenotype of Gcgr-/- mice by generating Gcgr-/-:Glp1r-/- mice. Although insulin sensitivity was

similar in all genotypes, fasting glucose was increased in Gcgr-/-:Glp1r-/- mice. Elimination of

the Glp1r normalized gastric emptying and impaired intraperitoneal glucose tolerance in Gcgr-/-

mice. Unexpectedly, deletion of Glp1r in Gcgr-/-mice did not alter the improved oral glucose

tolerance and increased insulin secretion characteristic of that genotype. Although Gcgr-/-

:Glp1r-/ -islets exhibited increased sensitivity to the incretin glucose-dependent insulinotropic

polypeptide (GIP), mice lacking both Glp1r and the GIP receptor (Gipr) maintained preservation

of the enteroinsular axis following reduction of Gcgr signaling. Moreover, Gcgr-/-:Glp1r-/-

islets expressed increased levels of the cholecystokinin A receptor (Cckar) and G protein–

coupled receptor 119 (Gpr119) mRNA transcripts, and Gcgr-/-:Glp1r-/- mice exhibited

increased sensitivity to exogenous CCK and the GPR119 agonist AR231453. Our data reveal

extensive functional plasticity in the enteroinsular axis via induction of compensatory

mechanisms that control nutrient-dependent regulation of insulin secretion.

40

2.2 Introduction

Glucagon, a 29-amino-acid peptide hormone, is produced in pancreatic α-cells and,

together with insulin, plays a key role in regulating hepatic glucose production, serving as the

first line of defense against hypoglycemia [287]. Disordered control of glucagon secretion is

observed in Type 2 Diabetes (T2D) [288-290] and together with insulin deficiency or resistance

results in metabolic derangements characteristic of T2D [291]. Glucagon exerts its actions

through a class B family G protein–coupled receptor (GPCR), the glucagon receptor (Gcgr), that

is expressed not only in the liver, but in a broad range of tissues, including the heart, the kidney,

the endocrine pancreas, and the central nervous system [292-296]. The observations that

inappropriately increased levels of glucagon promote increased hepatic glucose production and

hyperglycemia [289, 297-300] had fostered efforts targeting suppression of glucagon action for

the treatment of T2D [34]. Reduction of glucagon activity using glucagon antagonists,

immunoneutralizing antisera, or antisense oligonucleotides (ASOs) directed against Gcgr

attenuates hyperglycemia in experimental models of diabetes [118, 121, 122, 129, 130, 301-303].

The importance of glucagon action has also been examined via generation and characterization

of the Gcgr-/- mouse. Surprisingly, mice with complete germline disruption of Gcgr are viable

and grow normally [131, 274]. Gcgr-/- mice exhibited normal body weight, food intake, and

energy expenditure, yet display improved glucose tolerance, enhanced β-cell function and

insulin sensitivity, resistance to streptozotocin-induced (STZ- induced) diabetes, and delayed

gastric emptying [131, 135, 280]. These favorable phenotypes support reduction of glucagon

action for the therapy of T2D.

Interpretation of metabolic derangements arising from loss of Gcgr signaling is

complicated by the concomitant finding of markedly increased circulating levels of glucagon-

like peptide 1 (GLP-1) in Gcgr-/- mice [131]. GLP-1, a related proglucagon-derived peptide

normally secreted from enteroendocrine L cells, produces many of the actions described in Gcgr-

/- mice. The increased circulating levels of GLP-1 in Gcgr-/- mice likely reflect increased

production of bioactive GLP-1 in the Gcgr-/- pancreas [131]. As GLP-1 improves β-cell

function and glucose tolerance, reduces gastric emptying, and promotes islet cell proliferation

and resistance to STZ-induced diabetes, attribution of precise mechanisms responsible for the

improved metabolic phenotype in Gcgr-/- mice is challenging. We now demonstrate that a

41

functional GLP-1 receptor (Glp1r) is essential for control of fasting and ambient glycaemia,

enhancement of glucose-stimulated insulin secretion following i.p. glucose challenge, and

inhibition of gastric emptying in Gcgr-/- mice. Unexpectedly, Gcgr-/-:Glp1r-/- mice continued

to exhibit improved oral glucose tolerance and enhanced β-cell function despite loss of two key

insulinotropic β-cell receptors. Although loss of GLP-1 action is classically compensated for by

upregulation of the other major incretin, glucose-dependent insulinotropic polypeptide (GIP)

[304], we demonstrate that reduction of Gcgr expression in mice with combined inactivation of

both Glp1r and the GIP receptor (Gipr) genes continues to be associated with preservation of

oral glucose tolerance and enhanced insulin secretion. Disruption of the classical (Glp1r and

Gipr) entero-insular axis was associated with enhanced expression and activity of the

cholecystokinin A receptor (Cckar) and G protein–coupled receptor 119 (Gpr119), functionally

related insulinotropic receptors that respond to nutrient-sensitive signals. These findings reveal

considerable plasticity in the incretin-related mechanisms regulating β-cell function and glucose

homeostasis.

2.3 Materials and Methods

2.3.1 Animal studies

Gcgr-/-:Glp1r-/- double knockout (DKO) mice were generated by crossing double

heterozygote Glp1r+/-and Gcgr+/- mice to obtain littermate Gcgr+/+:Glp1r+/+(WT), Gcgr-/-

andGlp1r-/- single knockouts, and Gcgr-/-:Glp1r-/- DKO mice. Gipr-/-mice were provided by

Y. Seino (Kansai Electric Power Hospital, Osaka, Japan) and used to generate DIRKO mice as

previously described [192]. All mice used in these studies were male and were housed up to 5

per cage under a light/dark cycle of 12 hours in the Toronto Centre for Phenogenomics animal

facility, with free access to food and water except where noted. All procedures were conducted

according to protocols and guidelines approved by the Toronto Centre for Phenogenomics

Animal Care Committee. For confirmation of genotypes, genomic DNA prepared from tail snips

was analyzed by PCR and Southern blotting as described previously [131, 266].

2.3.2 Peptides & drugs

Treatment of mice with Gcgr ASOs. DIRKO and WT mice (11–13 weeks old) were given

42

subcutaneous injections of Gcgr ASOs (Sigma-Aldrich, ISIS 180475, hybridizes to bases 1348–

1367 of mouse Gcgr sequence NM 008101.1 and bases 1398–1417 of rat Gcgr sequence

M96674.1) at a dose of 25 mg/kg every 3.5 days for a maximum of 6 injections (22).

Intraperitoneal glucose tolerance tests (IPGTTs) were performed after administration of 3

injections of the ASOs, and oral glucose tolerance tests (OGTTs) were performed after

administration of 4 injections of the ASOs. Mice were euthanized after administration of 6

injections of the ASOs, and islets and livers were obtained and examined for gene expression.

DIRKO and WT mice treated with the Gcgr ASOs were fasted for 6 hours prior to the glucose

tolerance tests.

Treatment of mice with peptides and agonists. Gcgr-/-:Glp1r-/- mice and littermate

controls were treated orally 30 minutes prior to IPGTT with vehicle (80% polyethylene glycol

(PEG) 400, 10% Tween 80, and 10% ethanol) or the Gpr119 agonist AR231453 (5 mg/kg and 20

mg/kg, Arena Pharmaceuticals) [305]. Gcgr-/-:Glp1r-/- mice and littermate controls were

injected i.p. with PBS vehicle, [D-Ala2]GIP (1 or 2 nmol/kg) (CHI Scientific), GRP (20

nmol/kg) (Bachem), CCK-8 (9 μg/kg or 18 μg/kg), or PACAP-38 (1.3 nmol/kg), immediately

prior to IPGTT (Sigma-Aldrich).

2.3.3 Assessment of food intake and energy expenditure

Assessment of food intake. 16-18 week-old mice were fasted overnight (16–18 hours),

weighed, and then placed in individual cages containing pre-weighed rodent chow, with free

access to water. Food was reweighed after 1, 2, 4, 8, and 24 hours, and food intake was

expressed as grams consumed per gram of body weight.

Indirect calorimetry. 8-10-week-old mice were placed in individual metabolic chambers,

with free access to food and water. Oxygen consumption, CO2 production, and total and

ambulatory activity were determined by indirect calorimetry using an Oxymax System

(Columbus Instruments) as described previously [267].

2.3.4 Tissue isolation and histological analysis

Pancreata from 20- 24-week-old male mice were weighed, fixed in 10% neutral buffered

43

formalin solution for 48 hours, and then embedded in paraffin. For assessment of islet area and

histology, pancreatic sections were immunostained for insulin and/or glucagon as previously

described in [198], followed by scanning using the ScanScope CS system (Aperio Technologies)

at ×20 magnification[198]. Digital images were analyzed with ScanScope software (Aperio

Technologies). Percent islet area was calculated as the sum of the total cross-sectional area of β

and α-cells/total pancreas area multiplied by 100.

For gut mRNA analyses, 16- to 18-week-old mice were fasted in individual cages

overnight (16 hours) and allowed access to a premeasured amount of rodent chow for 1 hour.

Subsequently, duodenums were isolated and snap frozen.

2.3.5 Glucose, insulin tolerance test and measurement of plasma metabolites

Glucose tolerance and measurement of plasma metabolites. Eight- to 11-week-old male

mice were fasted overnight (16–18 hours), and glucose (1.5 mg/g body weight) was administered

orally (through a gavage tube) or via injection into the peritoneal cavity (IPGTT). Blood samples

were drawn from the tail vein at 0, 15, 30, 60, 90, and 120 minutes after glucose administration,

and blood glucose levels were measured using a Glucometer Elite blood glucose meter

(Ascensia; Bayer HealthCare). For plasma insulin determinations, blood samples (100 μl) were

drawn from the tail vein during the 0- and 15-minute time periods following glucose

administration in a heparinized tube. Plasma was separated by centrifugation at 4°C and stored at

–80°C until assayed. Plasma was assayed for insulin using a mouse insulin ELISA kit (Alpco).

Plasma GLP-1 levels were measured using a mouse/rat total GLP-1 assay kit (Meso Scale

Discovery). Plasma GIP levels were assessed using a mouse/rat total GIP ELISA kit (Linco).

Plasma GLP-2 levels were assessed using a mouse/rat total GLP-2 assay kit (Alpco). Plasma

levels of active GLP-1 and glucagon were measured using a mouse Milliplex endocrine assay

(Millipore).

Insulin tolerance test. Twelve- to 13-week-old male mice were fasted for 5 hours and

given 0.7 U/kg insulin (Humulin R, 100 U/ml; Lilly) by i.p. injection. Blood samples for blood

glucose determination were drawn from the tail vein at 0, 20, 40, 60, 90, 120, and 180 minutes

following insulin administration.

44

2.3.6 Solid and liquid phase gastric emptying

Gastric emptying. Gastric emptying was assessed using two protocols. For measurement

of solid-phase gastric emptying, 12-week-old mice were fasted in individual cages overnight (16

hours) and allowed access to a pre-measured amount of rodent chow for 1 hour. Food intake was

determined by reweighing the rodent chow after 1 hour of refeeding. The stomach was isolated

and gastric contents retrieved and weighed. The gastric emptying was determined using the

following calculation: gastric emptying (%) = (1 – [stomach content wet weight/food intake]) ×

100. In the second protocol, liquid-phase gastric emptying was assessed using the acetaminophen

absorption test [306]. Ten- to 11-week-old mice were fasted overnight and administered a

solution containing acetaminophen at a dose of 100 mg/kg by gavage. Tail vein blood (50 μl)

was collected into heparinized tubes at 0 and 15 minutes after acetaminophen administration.

Plasma was separated by centrifugation at 4°C and stored at –20°C until measurement of

acetaminophen levels using an enzymatic- spectrophotometric assay (Diagnostic Chemicals

Ltd.).

2.3.7 Islet isolation

After CO2 euthanasia, pancreata from mice were inflated via the pancreatic duct with

collagenase type V (0.7 mg/ml in HBSS), excised, and digested at 37°C for 10–15 minutes. The

resulting digest was washed twice with cold HBSS (containing 0.25% wt/vol BSA), and islets

were separated using a Histopaque density gradient (Sigma-Aldrich). The interface containing

islets was removed and washed with HBSS plus BSA, and islets were resuspended in RPMI

medium containing 10% FBS, 2 mM l-glutamine, 11 mM glucose, 100 U/ml penicillin, and 100

μg/ml streptomycin. After 4 hours of incubation at 37°C, islets were handpicked into fresh

RPMI medium (containing 5.6 mM glucose) and allowed to recover overnight. Islets with

preserved architectural integrity were either used for insulin secretion experiments or washed

twice in PBS before being lysed by QIAshredder columns for RNA extraction using the RNeasy

Micro Kit (QIAGEN). For insulin secretion, islets were preincubated in Krebs-Ringer buffer

(KRB) containing 0.1% BSA, 10 mM HEPES (pH 7.4), and 2.8 mM glucose for 60 minutes.

Batches of 10 islets were distributed into wells containing 0.5 ml KRB with either 2.8 or 16.7

mM glucose, with or without [D-Ala2]GIP, L-arginine, PACAP, tolbutamide, or Ex-4 at the

45

indicated concentrations. After incubation for 1 hour at 37°C, secretion medium was collected

and stored at –20°C for assessment of insulin secretion. Islet insulin was extracted by transfer of

islets to cold acid-ethanol solution (70% ethanol, 0.18 M HCl) and brief sonication (10 seconds).

Insulin levels in secretion media and islet extracts were measured by RIA (Millipore). For cAMP

studies, batches of 10 islets were distributed into wells containing 0.5 ml KRB with 16.7 mM

glucose, with or without 1, 3, or 10 nM [D-Ala2]GIP for 1 hour at 37°C. Reactions were

terminated by the addition of ice-cold absolute ethanol, and cell extracts were collected and

stored at –80°C until measurement of cAMP using a cAMP RIA kit according to the

manufacturer’s instructions (Biomedical Technologies). CAMP levels were normalized to insulin

content.

2.3.8 Real-time qRT-PCR

RNA analyses. Following RNA isolation, first-strand complementary DNA was

synthesized from total RNA using the SuperScript III reverse transcriptase synthesis system

(Invitrogen) and random hexamers. Real-time PCR was performed with the ABI Prism 7900

Sequence Detection System using TaqMan Gene Expression Assays and TaqMan Universal PCR

Master Mix (Applied Biosystems). Levels of RNA transcripts were quantified using the 2–ΔΔCt

method normalized to peptidyl-propyl isomerase A (cyclophilin).

2.3.9 Statistical Analysis

Statistics. Results are presented as mean ± SEM. Statistical significance was determined

using 1-way or 2-way ANOVA with Bonferroni post-hoc tests (as appropriate) using GraphPad

Prism 4.0 (GraphPad Software Inc.). Statistical significance was noted when P values were less

than 0.05.

2.4 Results

2.4.1 Glp1r is not required for pancreas enlargement or α-cell hyperplasia in Gcgr-/-mice.

Body weight, food intake, physical activity, and energy expenditure were comparable in

WT, single incretin receptor knockout, and Gcgr-/-:Glp1r-/- mice (Figure 2.1). As Gcgr-/-mice

exhibit increased pancreatic mass, marked islet, and α-cell hyperplasia [307], and Glp1r

46

activation promotes expansion of islet and pancreatic mass [308-310], we assessed the

contribution of Glp1r to the development of these abnormalities in Gcgr-/- mice. Consistent with

previous findings, Gcgr-/- mice exhibited very high circulating levels of GLP-1 (Figure 2.2A),

significantly increased pancreas weight, and an approximately 4-fold increase in islet area

(Figure 2.3); however, islet area and pancreatic mass remained significantly increased to a

similar extent in Gcgr-/-:Glp1r-/- mice (Figure 2.3, A and B). Immunohistochemical analysis

revealed that the increased islet area was predominantly due to α-cell hyperplasia, with most

Gcgr-/- and Gcgr-/-:Glp1r-/- islets containing a core of β-cells surrounded by an expanded

mantle of hyperplastic α-cells (Figure 2.3C). Hence, Glp1r is not required for development of

increased pancreatic mass and islet hyperplasia following loss of Gcgr action.

2.4.2 Disruption of Glp1r leads to increased fasting glycaemia in Gcgr-/-mice.

Both fasting and random glucose were reduced in Gcgr-/- mice (Figure 2.4, A and B),

consistent with the central role of glucagon in the maintenance of euglycemia [131]. Basal GLP-

1R signaling also regulates fasting glycaemia [266] classically through suppression of glucagon

secretion [311]. Surprisingly, a significant increase in fasting glucose was observed in Gcgr-/-

:Glp1r-/- compared with Gcgr-/- mice (Figure 2.4A) and elimination of the Glp1r normalized

random-fed glycaemia in Gcgr-/-:Glp1r-/- mice (Figure 2.4B). Hence, loss of Glp1r substantially

attenuates improvements in both ambient and fasting glycaemia in Gcgr-/- mice [131].

47

Figure 2.1. Body weight, food intake and energy expenditure.

(A) Bodyweight from 12 week old mice (n = 4-7). (B) Food intake was determined 1, 2, 4, 8 and

24 hours following an overnight fast (n = 4-16). (C) Energy expenditure was determined in 8-10

week old mice. Oxymax measurements were starting at 12pm (n = 6 mice per genotype). Values

are expressed as mean ± SEM.

48

Figure 2.2. Plasma levels of total GIP, active GLP-1, total GLP-2 and total Glucagon.

(A) Random fed active GLP-1 levels in plasma. (B) Plasma levels of total GIP at 0,15 and 30

min following oral glucose administration. (C) Random fed total glucagon levels in plasma. (D)

Random fed total GLP-2 levels in plasma. n=4-10 per genotype. Values are expressed as mean ±

SEM.

49

Figure 2.3. Glp1r is not required for development of increased pancreas weight or αcell

hyperplasia in Gcgr-/-mice.

(A) Pancreas weight of 20- to 24-week-old mice shown as percentage of the final body

weight (n = 7–20 mice per group). (B) Islet area shown as a percentage of total pancreas area

(n = 4–12 mice per group) (C) Representative histological sections of pancreas stained for

insulin or glucagon alone. . Values are expressed as mean ± SEM. *P < 0.05, Gcgr-/-mice

versus WT littermate controls; #P < 0.05, Gcgr-/-:Glp1r-/-mice versus WT littermate control

mice; †P < 0.05, Glp1r-/-versus Gcgr-/-:Glp1r-/-mice.

50

Figure 2.4. Glp1r controls fasting and fed glycaemia in Gcgr-/-mice.

(A) Blood glucose following 5 or 16 hours of fasting in 8- to 12-week-old WT, Gcgr-/-,

Glp1r-/-and Gcgr-/-:Glp1r-/-mice (n = 5–30 mice per genotype). (B) Weekly random-fed

blood glucose levels in 8- to 20-week-old Gcgr-/-:Glp1r-/-, Gcgr-/-, Glp1r-/-, and littermate

control WT mice (n = 3–20 mice per group). Values are expressed as mean ± SEM. *P <

0.05, Gcgr-/-versus Gcgr-/-:Glp1r-/-mice; #P < 0.05, Gcgr-/-versus WT mice; ‡P < 0.05,

Glp1r-/-versus WT mice; †P < 0.05, Glp1r-/-versus Gcgr-/-:Glp1r-/-mice.

51

2.4.3 Elimination of Glp1r reverses improvements in i.p. glucose tolerance in Gcgr-/-mice.

To clarify the contribution of enhanced GLP-1 receptor signaling to improved β-cell

function and glucose tolerance in Gcgr-/- mice [131], we first assessed clearance of i.p. glucose

in mice of different genotypes. Intraperitoneal glucose tolerance was significantly enhanced and

plasma insulin levels increased in Gcgr-/- mice; conversely, i.p. glucose tolerance was impaired

in Glp1r–/– mice (Figure 2.5A), consistent with previous studies [131, 266]. Furthermore,

disruption of Glp1r in Gcgr-/- mice reversed the improvements in i.p. glucose tolerance and

normalized plasma insulin levels in Gcgr-/-:Glp1r-/- mice (Figure 2.5, A and B), whereas insulin

sensitivity, approximated by insulin tolerance, was comparable among all genotypes (Figure

2.5C). Consequently, the elevated levels of GLP-1 leading to increased Glp1r signaling is

primarily responsible for enhanced β-cell function and improved glucose clearance after i.p.

glucose challenge in Gcgr-/- mice.

2.4.4 The GLP-1 receptor mediates reduced gastric emptying; however, oral glucose

tolerance remains improved independent of Glp1r in Gcgr-/-mice.

As GLP-1–mediated reduction in gastric emptying may substantially account for the

improved oral glucose tolerance in Gcgr-/- mice [131, 135], we quantified gastric emptying with

two complementary methods. Both liquid-phase gastric emptying, assessed via measurement of

plasma acetaminophen levels, and solid-phase gastric emptying were significantly reduced in

Gcgr-/- mice and normalized in Gcgr-/-:Glp1r-/- mice (Figure 2.6, A and B). We hypothesized

that normalization of gastric emptying would be associated with deterioration of oral glucose

tolerance in Gcgr-/-:Glp1r-/- mice [131]. Unexpectedly, oral glucose tolerance remained

significantly improved in Gcgr-/-:Glp1r-/- mice to an extent comparable to that in Gcgr-/- mice

alone (Figure 2.6, C and D). Furthermore, in contrast to the normalization of plasma insulin

levels seen following i.p. glucose challenge in Gcgr-/-:Glp1r-/- versus Gcgr-/- mice (Figure

2.5B), Gcgr-/-:Glp1r-/- mice continued to exhibit significantly increased levels of plasma insulin

following oral glucose challenge (Figure 2.6D).

2.4.5 Islets from Gcgr-/-:Glp1r-/-mice display increased sensitivity to GIP.

As Glp1r-/- mice exhibit enhanced GIP secretion and increased sensitivity to GIP [304],

52

we explored whether GIP-related mechanisms underlie the enhanced enteral glucose-stimulated

insulin secretion in Gcgr-/-:Glp1r-/- mice. Although levels of GIP were modestly elevated in

Glp1r-/- mice, GIP levels were not significantly increased in Gcgr-/-:Glp1r-/- mice (Figure 2.2

B). We then examined control of insulin secretion from WT, Glp1r-/-, Gcgr-/-, and Gcgr-/-

:Glp1r-/- islets. No significant differences across genotypes were detected in response to 16.7

mM glucose with a modest but non-significant reduction in insulin secretion observed with

Gcgr-/-:Glp1r-/- islets (Figure 2.7A). Consistent with the loss of the Glp1r, the insulinotropic

response to Ex-4 was absent in Glp1r-/- and Gcgr-/-:Glp1r-/- islets (Figure 2.7A). Although GIP

sensitivity was not enhanced in Glp1r-/- or Gcgr-/- islets, the insulinotropic response to GIP was

significantly increased in Gcgr-/-:Glp1r-/- islets (Figure 2.7A). To explore the selectivity of the

enhanced response to GIP, we tested a range of other insulin secretagogues. In contrast to the

enhanced response to GIP, Gcgr-/-:Glp1r-/- islets exhibited a normal response to pituitary

adenylate cyclase–activating peptide (PACAP) but significantly reduced insulin secretory

responses to tolbutamide and l-arginine (Figure 2.7B). Consistent with the increased GIP

sensitivity demonstrated for insulin secretion (Figure 2.7A), Gcgr-/-:Glp1r-/- islets also

exhibited enhanced cAMP accumulation in response to GIP (Figure 2.7C). To evaluate GIP

sensitivity in vivo, we administered i.p. glucose in the presence or absence of submaximal doses

of exogenous GIP to WT, Glp1r-/-, Gcgr-/-, and Gcgr-/-:Glp1r-/- mice. Both Glp1r-/- and to a

greater extent Gcgr-/-:Glp1r-/- mice exhibited enhanced sensitivity to GIP, as revealed by

reduced glycemic excursions and increased circulating levels of plasma insulin in response to

exogenous GIP administration (Figure 2.8, A–D).

53

Figure 2.5. Loss of Glp1r reverses improvements in i.p. glucose tolerance without altering insulin

sensitivity in Gcgr-/-mice.

(A) IPGTT in 8- to 10-week- old WT, Gcgr-/-, Glp1r-/-, and Gcgr-/-:Glp1r-/-mice (n = 9–24 mice

per group). (B) Area under the curve and plasma insulin levels at 0 and 15 minutes following i.p.

glucose challenge (n = 4–8 mice per group). (C) Insulin tolerance test in 12- to 14-week-old mice;

values are normalized to basal glucose, with right graph showing area under the glucose curve (n = 5–

20 mice per group). Values are expressed as mean ± SEM. *P < 0.05, Gcgr-/-versus Gcgr-/-:Glp1r-/-

mice; #P < 0.05, Gcgr-/-versus WT mice; ‡P < 0.05, Glp1r-/-versus WT mice; †P < 0.05, Glp1r-/-

versus Gcgr-/-:Glp1r-/-mice.

54

Figure 2.6. Glp1r mediates reduced gastric emptying but not improved oral glucose tolerance in

Gcgr-/-mice.

(A) Liquid-phase gastric emptying (as determined by the appearance of acetaminophen in the

circulation after 15 minutes) in 10- to 11-week-old mice (n = 4–14 mice per group). (B) Solid-phase

gastric emptying in 20-week-old mice (n = 4–10 mice per group). Values are expressed as mean ±

SEM. *P < 0.05, Gcgr-/-versus Gcgr-/-:Glp1r-/-mice; #P < 0.05, Gcgr-/-versus WT mice. (C) Blood

glucose levels during an OGTT in 10- to 11-week-old mice (n = 11–22 mice per group). (D) Area

under the glucose curve and plasma insulin levels 0 and 15 minutes following oral glucose challenge

(n = 4–9 mice per group). Values are expressed as mean ± SEM. In C and D: *P < 0.05, Gcgr-/-

versus WT littermate control mice; #P < 0.05, Gcgr-/-:Glp1r-/-versus WT mice; †P < 0.05, Glp1r-/-

versus Gcgr-/-:Glp1r-/- littermate control mice; ‡P < 0.05, Glp1r-/-versus WT littermate control

mice.

55

2.4.6 Plasticity of the incretin axis revealed through reduction of Gcgr action in Glp1r-/-

:Gipr-/-mice.

The available data strongly suggests that preservation of improved glucose tolerance and

enhanced insulin secretion despite loss of GLP-1 action reflects increased GIP sensitivity in

Gcgr-/-:Glp1r-/- islets. To more rigorously test this hypothesis, we reduced Gcgr expression

using ASOs in mice lacking both functional incretin receptors, i.e., Glp1r-/-:Gipr-/- (double

incretin receptor knockout [DIRKO]) mice [267]. Hepatic Gcgr mRNA transcripts were

markedly decreased, and plasma levels of GLP-1 progressively increased in WT and DIRKO

mice following ASO treatment (Figure 2.9, A and B, respectively). WT mice treated with Gcgr

ASOs showed improved i.p. glucose tolerance and increased insulin levels following i.p. glucose

challenge (Figure 2.9C). In contrast, DIRKO mice treated with Gcgr ASOs showed no

improvement in glucose tolerance or insulin levels following i.p. glucose challenge (Figure

2.9D), consistent with the importance of the Glp1r for improved β-cell function following

reduction of Gcgr expression [129, 130, 311]. WT mice treated with Gcgr ASOs also showed

improved oral glucose tolerance and significantly higher plasma insulin levels than vehicle-

treated controls (Figure 2.9E). Remarkably, despite loss of both classical incretin receptors,

DIRKO mice treated with Gcgr ASOs also exhibited improved oral glucose tolerance and

significantly increased plasma insulin levels (Figure 2.9F). Hence, preferential improvement of

glucose tolerance and enhanced β-cell function following glucose administration in the gut can

be achieved through loss of the Gcgr despite loss of both incretin receptors.

To identify mechanisms responsible for improvement of oral glucose tolerance and β-cell

function despite absence of both GLP-1 and GIP receptors, we assessed the expression of

insulinotropic receptors in islets from (a) DIRKO mice treated with Gcgr ASOs and (b) Gcgr-/-

:Glp1r-/- mice. Remarkably, levels of mRNA transcripts for the insulinotropic receptors gastrin-

releasing peptide receptor (Grpr), Cckar, and Gpr119 were significantly increased in islet RNA

from Gcgr-/-:Glp1r-/- mice (Figure 2.10A). Similarly, Gpr119 and Cckar mRNA transcripts

were also significantly increased in islet RNA from DIRKO mice following Gcgr ASO

administration (Figure 2.10B). These findings raised the possibility that increased activity of

related insulinotropic receptors may compensate for the loss of GLP-1 and GIP action on islet β-

56

cells.

Figure 2.7. Function of GPCRs in isolated islets. Islet insulin secretion was assessed by preincubation of islets in KRB for 60 minutes at 2.8 mM

glucose at 37°C before distribution in batches of 10 islets per condition into wells containing 16.7

mM glucose with or without (A) exendin-4 (Ex-4, 10 nM), [D-Ala2]GIP (GIP, 10 nM), (B) PACAP

(10 nM), tolbutamide (Toll, 100 M), or l-arginine (L-art, 10 mM) for 1 hour at 37°C. Levels of

insulin in the secretion medium were normalized to levels of islet insulin content and are expressed as

a fold change in insulin secretion relative to WT high-glucose treatment. Insulin content values

averaged approximately 30–40 ng/islet for Glp1r-/-and WT mice and 15–25 ng/islet for Gcgr-/-and

Gcgr-/-:Glp1r-/-mice (n = 3 mice per group). Data shown are representative of 2–3 independent

experiments, each with 3 replicates per condition. (C) Total cellular and media cAMP in islets from

WT, Glp1r–/–, Gcgr-/-, and Gcgr-/-:Glp1r-/-mice were quantified following treatment of the islets

57

with 0, 1, 3, or 10 nM [D-Ala2]GIP. Levels of cAMP in the secretion medium were normalized to

levels of islet insulin content and are expressed as a fold change in islet cAMP levels relative to WT

high-glucose treatment (n = 3 mice per group). Values are expressed as mean ± SEM. §P < 0.05,

Glp1r-/-versus Gcgr-/-:Glp1r-/- mice; #P < 0.05, Gcgr-/-versus Gcgr-/-:Glp1r-/- mice; ‡P < 0.05,

Gcgr-/-:Glp1r-/- versus WT mice; †P < 0.05, Glp1r-/-versus WT mice; *P < 0.05, Gcgr-/- versus WT

mice.

Figure 2.8. Gcgr-/-:Glp1r-/-mice exhibit enhanced sensitivity to [D-Ala2]GIP.

An IPGTT was performed in 20- to 22-week-old (A) WT, (B) Gcgr-/-, (C) Glp1r–/–, and (D)

Gcgr-/-:Glp1r-/-mice following treatment with 1 nmol/kg [D-Ala2]GIP or saline (vehicle

[Veh]). Insets depict the area under the glucose excursion curve (AUC) in mM×min and

plasma insulin levels at 0 and 15 minutes following glucose challenge (n = 5–8). Values are

expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, [D-Ala2]GIP–treated

versus saline-treated group.

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Figure 2.9. Enteroinsular axis is maintained in DIRKO mice treated with Gcgr ASOs. (A) mRNA expression of Gcgr in the liver (n = 3 per group) following treatment with 6 injections of

25 mg/kg Gcgr ASOs. (B) Total plasma GLP-1 levels following 2, 4, or 6 injections of 25 mg/kg

saline or Gcgr ASOs (n = 5 per group). (C and D) An i.p. glucose challenge was performed on 13- to

14-week-old male (C) WT mice and (D) DIRKO mice that had been treated with 3 injections of

vehicle or 25 mg/kg Gcgr ASOs (n = 5 per group). (E and F) An OGTT was performed on 15- to 16-

week-old (E) WT mice and (F) DIRKO mice that had been treated with 4 injections of vehicle or 25

mg/kg Gcgr ASOs (n = 5 per group). Insets depict plasma insulin levels at 0 and 15 minutes

following glucose challenge for saline- or Gcgr ASO–treated mice (n = 5 per treatment group).

Values are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, vehicle- versus Gcgr

ASO–treated WT or DIRKO mice.

59

Figure 2.10. Expression of insulinotropic GPCRs in islets.

(A) Islets were isolated from WT, Gcgr-/-, Glp1r-/- and Gcgr-/-:Glp1r-/-mice, followed by

isolation of mRNA for real-time PCR of basal levels of transcripts encoding Gipr, Pacapr,

Gpr40, Grpr, Cckar, and Gpr119. (B) Islets were isolated from WT or DIRKO mice

following 6 injections of vehicle or 25 mg/kg Gcgr ASOs, and mRNA levels of Grpr,

Gpr119, and Cckar were determined. Levels of transcripts were normalized to levels of

cyclophilin for each RNA sample. n= 4 mice per genotype. Values are expressed as mean ±

SEM. §P < 0.05, Glp1r-/- versus Gcgr-/-:Glp1r-/- mice; #P < 0.05, Gcgr-/- versus Gcgr-/-

:Glp1r-/-mice; ‡P < 0.05, Gcgr-/-:Glp1r-/- versus WT mice; ¶P < 0.01, WT Gcgr ASO– vs

DIRKO Gcgr ASO–treated mice; *P < 0.01, WT saline- versus DIRKO Gcgr ASO–treated

mice; †P < 0.01, DIRKO saline- versus DIRKO Gcgr ASO–treated mice.

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Figure 2.11. GRP action in WT and knockout mice. Intraperitoneal glucose tolerance test was performed in 22-24 week old (A) WT, (B) Gcgr-/-, (C) Glp1r-/- ;and (D) Gcgr-/-:Glp1r-/- mice immediately following treatment with 20 nmol/kg of GRP or with saline. Insets depict plasma insulin levels at 0 and 15 min following glucose challenge (n = 5-8). Values are expressed as mean ± SEM; *P < 0.05, **P <0.01, ***P<.001 GRP- vs. vehicle treated mice.

61

Figure 2.12. PACAP action in WT and knockout mice. Intraperitoneal glucose tolerance test was performed in 22-24 week old mice A) WT, (B) Gcgr-/-, (C) Glp1r-/- ; and (D) Gcgr-/-:Glp1r-/- mice immediately following treatment with 1.3 nmol/kg of PACAP-38 or saline. Insets depict plasma insulin levels at 0 and 15 min following glucose challenge (n = 5-8). Values are expressed as mean ± SEM; *P < 0.05 for PACAP-38- vs. vehicle- treated mice.

62

Figure 2.13. Gcgr-/-:Glp1r-/-mice exhibit enhanced sensitivity to the GPR119 agonist

AR231453 (5mg/kg). An IPGTT was performed in 22- to 24-week-old (A) WT (B), Gcgr-/-

(C), Glp1r-/-, and (D) Gcgr-/-:Glp1r-/- mice 30 minutes following treatment with 5 mg/kg

AR231453 or vehicle. Insets depict the area under the glucose excursion curve in mM.min

and plasma insulin levels at 0 and 15 minutes following glucose challenge (n = 5–8). Values

are expressed as mean ± SEM. **P < 0.01, AR231453- versus vehicle-treated mice.

63

Figure 2.14. Enhanced sensitivity to the GPR119 agonist AR231453 (20mg/kg). An Intraperitoneal glucose tolerance test was performed in 22-24 week old (A) WT, (B) Gcgr-/-, (C) Glp1r-/- ; and (D) Gcgr-/:Glp1r-/- mice 30 min following treatment with 20 mg/kg of AR231453 or vehicle. Insets depict plasma insulin levels at 0 and 15 min following glucose challenge (n = 5-8). Values are expressed as mean ± SEM; *P <0.05, **P <0.01, *** P<.001 AR231453- vs. vehicle- treated mice.

64

Figure 2.16. Gcgr-/-:Glp1r-/-mice exhibit increased sensitivity to CCK (9ug/kg). An IPGTT was performed in 22- to 24-week-old (A) WT, (B) Gcgr-/-, (C) Glp1r-/-, and (D) Gcgr-/-

:Glp1r-/- mice following treatment with 9g/kg of CCK-8 or vehicle. Insets depict the area under the

glucose excursion curve (AUC) in mM.min and plasma insulin levels at 0 and 15 minutes following

glucose challenge (n = 5–8). Values are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P

<0.001, CCK-8– versus saline-treated mice.

65

Figure 2.15. Enhanced sensitivity to CCKAr ligand CCK (18ug/kg). Intraperitoneal glucose tolerance test was performed in 22-24 week old (A) WT, (B) Gcgr-/-, (C) Glp1r-/- ;and (D) Gcgr-/-:Glp1r-/- mice immediately following treatment with 18 ug/kg of CCK-8 or saline. Insets depict plasma insulin levels at 0 and 15 min following glucose challenge (n = 5-8). Values are expressed as mean ± SEM; *P < 0.05, **P <0.01, ***P<.001 CCK-8- vs. vehicle- treated mice.

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Figure 2.17. Gut peptide gene expression in re-fed mice.

Duodenums were isolated from Gcgr-/-:Glp1r-/-, Gcgr-/-, Glp1r-/- and WT mice following an

overnight fast and refed for 1 hour followed by assessment of basal levels of transcripts encoding

FAAH, GIP, CCK, GRP, PACAP and VIP. Levels of transcripts were normalized to levels for

cyclophilin for each RNA sample. n = 4 mice per genotype. Values are expressed as mean ±

SEM.

67

Figure 2.18. Schematic of proposed model. (A) Loss of Gcgr in the liver markedly elevates plasma GLP-1 levels, which promotes glucose-

stimulated insulin secretion during i.p. glucose challenge and inhibits gastric emptying in Gcgr-/-

mice. (B) Loss of Glp1r in the Gcgr-/-mice results in preservation of the enteroinsular axis via β-cell

compensatory mechanisms such as upregulation of Gpr119 and Cckar action in βcells, independent

of the increase in GIP sensitivity.

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2.4.7 Gcgr-/-:Glp1r-/-mice display increased sensitivity to Gpr119 and Cckar agonists.

To assess the functional significance of increased islet receptor expression, we carried out

glucose tolerance tests in the presence or absence of exogenous GRP, CCK, PACAP, and the

GPR119 agonist AR231453 [305] in WT, Gcgr-/-,Glp1r-/-, and Gcgr-/-:Glp1r-/- mice. We did

not detect enhanced sensitivity to exogenous GRP or PACAP (Figure 2.11 and Figure 2.12, A–

D). However, although AR231453 failed to improve i.p. glucose tolerance in WT, Gcgr-/-, or

Glp1r-/-mice, a robust improvement in glucose tolerance and marked stimulation of plasma

insulin levels were observed following AR231453 administration in Gcgr-/-:Glp1r-/- mice

(Figure 2.13, A–D, and Figure 2.14, A–D). Similarly, doses of CCK that failed to improve i.p.

glucose tolerance in WT or single Gcgr-/- or Glp1r–/– mice produced a significant reduction in

glycemic excursion and significantly elevated plasma insulin levels in Gcgr-/-:Glp1r-/- mice,

consistent with increased sensitivity to CCK (Figure 2.15, A–D, and Figure 2.16, A–D).

2.5 Discussion

The central importance of glucagon action for the maintenance of euglycemia, together

with observations that glucagon secretion may be inappropriately increased in many subjects

with T2D, has fostered great interest in reduction of glucagon action for the treatment of T2D.

Indeed, multiple therapeutic modalities, including insulin, amylin analogs, Glp1r agonists, and

dipeptidyl peptidase–4 inhibitors, exert their anti-diabetic actions in part through suppression of

glucagon secretion [5]. The Gcgr-/-mice are healthy mice with reduced fasting and postprandial

glycaemia [307], resistance to diet-induced obesity, and enhanced β-cell function and survival

[135] further support the concept of reducing glucagon receptor signaling for treatment of T2D.

Our studies clearly show that a substantial component of the improved metabolic phenotype of

Gcgr-/- mice, including control of fasting and fed glycaemia, reduced gastric emptying,

improved i.p. glucose tolerance, and enhanced β-cell function, reflects concomitant upregulation

of GLP-1 action in Gcgr-/- mice (Figure 2.18).

Although incretin action classically controls postprandial glucose excursion, considerable

evidence supports a role for GLP-1 in the regulation of fasting glucose. GLP-1 receptor agonists

69

reduce fasting glucose in human subjects, in association with reduced levels of circulating

glucagon [185]. Moreover, the Glp1r antagonist Ex-9-39 increases fasting glucose and glucagon

levels in baboons [312], and genetic disruption of the Glp1r is associated with fasting

hyperglycemia in Glp1r-/- mice, without detectable changes in levels of fasting glucagon [266].

Surprisingly, elimination of GLP-1 action significantly increased fasting glycaemia in Gcgr-/-

:Glp1r-/- mice. These observations further demonstrate that Glp1r dependent pathways may

regulate fasting glucose independent of glucagon action, perhaps through incompletely

understood neural mechanisms or through reductions in basal cAMP levels in the β-cell s [313].

Reduction or loss of Gcgr signaling [274, 307] or resistance to glucagon action [314] lead to

hyperplasia of the exocrine and endocrine pancreas. Selective liver-specific deletion of the gene

encoding the G protein Gsα generates a similar phenotype characterized by markedly increased

levels of glucagon and GLP-1, increased pancreatic mass, and islet hyperplasia [314]. Moreover,

this constellation of pancreatic abnormalities is reversible, as glucagon replacement in glucagon-

deficient PC2-knockout mice normalizes a substantial proportion of these pancreatic phenotypes

[315]. Although Glp1r activation promotes expansion of islet and pancreatic mass [308, 310], α-

cell hyperplasia is not generally observed following administration of Glp1r agonists. Consistent

with these findings, our data demonstrate that the Glp1r does not mediate pancreatic or islet α-

cell proliferation in Gcgr-/- mice. As Gcgr-/- mice are born with normal pancreatic mass and

only modest α-cell hyperplasia [131, 133], the precise mechanisms linking loss of the Gcgr to

marked postnatal expansion of the exocrine and endocrine islet compartments remain unclear.

The finding of reduced gastric emptying in Gcgr-/- mice [135] might be explained by

enhanced action of multiple proglucagon-derived peptides. GLP-1, oxyntomodulin, and, to a

lesser extent, GLP-2 inhibit gastric emptying [316, 317], and GLP-1, and oxyntomodulin exert

their actions on gastric emptying through the GLP-1 receptor. Unexpectedly, however, despite

normalization of gastric emptying in Gcgr-/-:Glp1r-/- mice, oral glucose tolerance remained

substantially improved in Gcgr-/-:Glp1r-/- mice, despite elimination of Glp1r action. Further to

this, the levels of circulating insulin remained significantly increased following oral glucose

challenge despite loss of Glp1r action in Gcgr-/-:Glp1r-/- mice. These findings implicate

sustained enhancement of β-cell function as a mechanism underlying persistent improvement in

oral glucose tolerance despite loss of Glp1r action in Gcgr-/-:Glp1r-/- mice.

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The demonstration that the Gcgr-/-:Glp1r-/- β-cell maintains enhanced insulin secretion

in response to oral glucose was unexpected given the previous demonstration of selectively

impaired β-cell function in Gcgr-/- mice compared with age- and sex-matched WT C57BL/6

mice [120], and the multiple lines of evidence supporting important roles for both GLP-1 and

glucagon in the control of glucose-stimulated insulin secretion. Glucagon directly stimulates

insulin secretion in the rat pancreas independent of GLP-1 receptor action [318], and transgenic

Gcgr expression in murine β-cell s significantly augment glucose-stimulated insulin secretion

and reduce random glycaemia in RIP-Gcgr transgenic mice [47]. Moreover, the glucagon

antagonist des-His1-[Glu9]-glucagon-amide significantly reduced glucose-stimulated insulin

release in human islets [319]. Similarly, GLP-1 induces β-cell glucose competence, upregulates

insulin biosynthesis and secretion, and markedly enhances glucose sensitivity and glucose-

stimulated insulin secretion even in poorly responsive diabetic β-cells [320]. Hence, given the

results of previous observations reporting impaired β-cell function in Gcgr-/- islets [120], we

initially predicted that a further deterioration in β-cell function would ensue following removal

of the Glp1r signaling system in Gcgr-/- mice.

Surprisingly, however, elimination of Glp1r in Gcgr-/- mice did not lead to deterioration

in glycemic excursion after oral glucose loading in Gcgr-/-:Glp1r-/- mice. The preferential

preservation of improved oral glucose tolerance and enhanced insulin secretion following

glucose administration via the gastrointestinal tract, despite functional elimination of both the

Gcgr and Glp1r, strongly implicates the existence of one or more gut-derived compensatory

factors that augment β-cell function in an “incretin-like” manner in Gcgr-/-:Glp1r-/- mice. As

compensatory upregulation of GIP action has been described in Glp1r-/- mice, we postulated that

upregulation of the GIP-Gipr axis might similarly explain preservation of improved oral glucose

tolerance in mice following loss of glucagon and GLP-1 action. Although circulating levels of

GIP and levels of Gipr mRNA transcripts were not increased in islets from Gcgr-/-:Glp1r-/-

mice, we detected increased insulin secretion and enhanced cAMP accumulation following

treatment of Gcgr-/-:Glp1r-/- islets with GIP, consistent with increased GIP sensitivity.

Furthermore, glucose tolerance was significantly improved, and plasma insulin levels were

markedly increased following administration of GIP in vivo, further supporting enhanced

sensitivity to GIP as a compensatory mechanism augmenting β-cell function in Gcgr-/-:Glp1r-/-

71

mice.

Unexpectedly, however, we continued to observe preferential improvement of oral

glucose tolerance and β-cell function in DIRKO mice treated with ASOs to reduce Gcgr

expression, implying the existence of additional incretin-like mechanisms compensating for the

lack of insulinotropic activity normally subserved by the glucagon, GLP-1, and GIP receptors.

Our data demonstrating increased islet expression and functional activity of Cckar and Gpr119

mRNA transcripts from Gcgr-/-:Glp1r-/- mice, and in islets from DIRKO mice treated with Gcgr

ASOs, reveal a mechanism compensating for the loss of insulinotropic incretin receptors (Figure

11B). The finding of greatly enhanced sensitivity in vivo (improved glucose tolerance and

plasma insulin levels) to exogenous ligands for both the Cckar and Gpr119 receptors is

consistent with an important role for these receptors in maintaining an incretin response to

enteral glucose administration despite loss of GLP-1 and GIP action. It is notable that the

putative ligands for both Cckar, namely CCK, and Gpr119, principally lipid-derived amides such

as oleoylethanolamide and N-oleoyldopamine, would be expected to increase significantly

following oral, but not i.p., glucose administration, consistent with our findings of enhanced oral,

but not i.p., glucose clearance in Gcgr-/-:Glp1r-/- mice. Interestingly, β-cells from DIRKO mice

appeared to compensate for loss of incretin receptor and Gcgr expression by similar induction of

Cckar and Gpr119 expression, extending the findings of islet incretin receptor plasticity to a

second related, yet genetically distinct model.

Studies of islet adaptation classically carried out in the context of β-cell injury and

regeneration, pregnancy, or high-fat feeding have revealed numerous changes in islet gene

expression thought to be linked to the need to expand β-cell mass and/or enhance β-cell

function in response to insulin resistance [321-323]. Consequently, the concept of β-cell

plasticity is reasonably well established under physiological conditions requiring enhanced

functional β-cell mass. There is considerably less information concerning the potential plasticity

of the gut-islet axis in circumstances associated with impairment or disruption of classic incretin

receptor signals normally emanating from the GLP-1 and GIP receptors. Our observations made

using two distinct genetic models, the Gcgr-/-:Glp1r-/- mouse and the DIRKO mouse treated

with Gcgr ASOs, reveal a heretofore unrecognized capacity for β-cell adaptation to loss of

insulinotropic receptor signaling (Figure 11). It was fascinating how upregulation of Cckar and

72

Gpr119 expression was also observed in murine islets from pregnant mice, although the

functional importance of these findings was not ascertained [323]. Our findings may explain

why the metabolic phenotype arising from loss of one or both incretin receptors is comparatively

mild [192, 267, 311] and identify new models for exploring the expanding importance of

incretin-related mechanisms that potentiate β-cell function following oral nutrient ingestion.

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CHAPTER 3: Disruption of cardiomyocyte glucagon receptor signaling decreases flux through fatty acid oxidation and enhances

survival following ischemic injury

CHAPTER 3

Disruption of cardiomyocyte glucagon receptor signaling decreases flux through fatty acid oxidation and enhances

survival following ischemic injury

Author contributions:

J.R. Ussher provided assistance in designing experiments, HL-1 cell line and cardiac

acylcarnitine profiling experiments (Figures 2.6). Min Suk Kim provided assistance in culturing

primary cardiomyocytes and ischemia reperfusion experiments ex-vivo (Figures 3.5 and 3.8). I

would like to acknowledge Dr. Christopher Newgard and his lab for their expertise in providing

assistance with acylcarnitine profile assessment (Figures 3.2, 3.4 and 3.5).

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3.1 Research Summary

Glucagon maintains normoglycemia during the fasting state by inducing hepatic glucose

production. Although modulation of glucagon receptor (Gcgr) signaling is being explored for the

treatment of diabetes, the cardiovascular consequences of manipulating glucagon action are

poorly understood. We show that exogenous glucagon administration impairs recovery of

ventricular pressure in ischemic mouse hearts ex vivo, and increases mortality from myocardial

infarction after left anterior descending artery ligation in a p38 mitogen activated protein kinase

(MAPK)-dependent manner. In contrast, whole body reduction of glucagon action in Gcgr+/-

mice, or cardiac-specific reduction of glucagon action in GcgrCM-/-

mice, significantly improved

survival, reduced heart weights and infarct size following myocardial infarction. Metabolic

profiling of hearts from GcgrCM-/-

mice revealed a marked reduction in long chain acylcarnitines

in both aerobic ischemic hearts and following high fat diet feeding, consistent with an essential

role for Gcgr signaling in the control of cardiac fatty acid utilization. These findings may have

implications for strategies designed to augment or inhibit Gcgr signaling for the treatment of

metabolic disorders.

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3.2 Introduction

Glucagon is a 29 amino acid peptide hormone secreted from islet -cells that plays a

critical role in maintenance of euglycemia, predominantly by increasing hepatic glucose output.

Activation of glucagon receptor (Gcgr) signaling promotes glycogenolysis and enhanced

gluconeogenesis, and regulates pathways controlling hepatic lipid oxidation and lipid secretion

[5, 324]. More recent evidence demonstrates that Gcgr signaling also controls cell survival

pathways in hepatocytes, as genetic interruption of hepatic Gcgr signaling increases the

susceptibility to hepatic injury [325].

The Gcgr is widely expressed in extrahepatic tissues including the central and peripheral

nervous system, pancreatic islets, adipose tissue, kidney, blood vessels, and heart [5, 154]. In the

pancreas, glucagon action potentiates glucose-dependent insulin secretion, whereas activation of

Gcgr signaling in the brain regulates hepatic glucose production, control of appetite, and body

weight [60, 318, 319, 326]. Glucagon actions in adipose tissue and kidney are less well

understood, but have been linked to control of fatty acid and glucose metabolism, respectively.

The central importance of glucagon action for the pathophysiology of hyperglycemia was

suggested by observations that glucagon levels are inappropriately increased in many subjects

with type 2 diabetes (T2D) [117, 327, 328]. Furthermore, studies attenuating glucagon action

using glucagon immunoneutralizing antisera, Gcgr antagonists, antisense Gcgr oligonucleotides

and Gcgr-/- mice, demonstrate amelioration of hyperglycemia in experimental models of

diabetes [118, 121-123, 129, 130]. Collectively, these findings have fostered enthusiasm in

reduction of glucagon action as a potential treatment for T2D.

Paradoxically, complementary efforts are exploring whether partial enhancement of

glucagon action, together with agonism of the glucagon-like peptide-1 receptor (Glp1r), may be

useful for the treatment of diabetes and/or obesity. Oxyntomodulin, a naturally occurring

proglucagon-derived peptide, contains the 29 amino acid sequence of glucagon plus a

carboxyterminal extension. Oxyntomodulin exerts potent glucoregulatory and anorectic actions

in humans and rodents through activation of the GLP-1 and glucagon receptors [329]. More

recent studies have demonstrated that simultaneous activation of the glucagon and GLP-1

76

receptors using synthetic balanced co-agonists produces potent glucoregulatory activity and

greater weight loss than observed with Glp1r agonists alone [282]. Hence, there is ongoing

interest in understanding the metabolic consequences arising from partial selective activation of

Gcgr signaling.

The development of drugs that reduce or activate Gcgr signaling raises important

questions about the cardiovascular actions and safety of such actions. A substantial body of

literature supports the acute transient use of glucagon to reverse the effects of -adrenergic

agonists, or to increase heart rate, blood pressure, and cardiac output in subjects with circulatory

collapse refractory to adrenergic stimulation [143, 284]. In contrast, more sustained

administration of glucagon may increase the severity of ischemic cardiac injury [167, 286]. We

have now examined the consequences of manipulating Gcgr signaling in the ischemic mouse

heart. We demonstrate that exogenous glucagon impairs survival following ligation of the left

anterior descending (LAD) coronary artery, actions requiring p38 MAP kinase. In contrast,

partial reduction of Gcgr expression and activity in Gcgr+/-

mice produces a cardioprotective

response to ischemic injury, a phenotype recapitulated in mice with cardiac-specific inactivation

of the Gcgr (GcgrCM-/-

mice). Furthermore, expression of fatty acid oxidation genes and proteins

was reduced, and levels of total long and medium chain fatty acids were decreased in hearts from

GcgrCM-/-

mice. Our studies demonstrate that cardiac Gcgr signaling plays an essential role in fat

oxidation under normal, ischemic, and insulin resistant conditions, findings with implications for

pharmaceutical efforts to manipulate Gcgr signaling for the treatment of human disease.

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3.3 Materials and Methods

3.3.1 Animal studies

Gcgr+/- mice were generated by heterozygous-heterozygous breeding in the C57BL/6

background and were maintained as previously described [131]. Inducible αMHCcre

(stock

005657) [330] and FLPe (stock 005703) transgenic mice in the C57BL/6 background were

purchased from Jackson Laboratory. GcgrFlox

mice in the C57BL/6 background were described

previously [49]. GcgrCM-/-

mice were generated by crossing αMHCCre

mice with Gcgr

Flox mice

and were maintained in the C57BL/6 background. In the αMHCCre

mice, Cre gene was expressed

under the αMHC promoter and inducible construct was utilized [331]. All mice were housed 5

per cage under a light/dark cycle of 12 hours in the Toronto Centre for Phenogenomics (TCP)

animal facility, with free access to food and water except where noted. All procedures were

conducted according to protocols and guidelines approved by the TCP Animal Care Committee.

For confirmation of genotypes, genomic DNA prepared from tail snips was analyzed by

polymerase chain reaction (PCR). Tamoxifen (Tam, Sigma Aldrich 20mg/kg) was injected for 5

days (2 consecutive days). Tamoxifen was dissolved in corn oil (Mazola) and was aliquoted and

stored in -20C until injected in mice. The day of injection tamoxifen solution was thawed on a

shaker at 55C for 30 min.

3.3.2 Peptide and drug injections:

30 ng/g/body weight glucagon (Sigma) or saline in 10% gelatin was administered in

C57BL/6 mice 3 injections daily with or without 2 injections daily of 1 mol/body weight

SB290386 (p38 MAPK inhibitor, Sigma) for 7 days.

3.3.3 Coronary artery ligation

Left anterior descending (LAD) artery ligation was used to induce myocardial infarction

in 12-14 week old male mice. Mice were anesthetized using 1% isoflurane, intubated, and

ventilated with room air using positive-pressure respirator (moel 680; Harvard South Natick,

MA). The pericardium was opened following retraction of the lungs to expose the heart, and left

thoracotomy was performed via the fourth intercostal space. The LAD was ligated with a 7-0

78

silk suture. In the shams group the 7-0 silk suture was passed under the coronary artery and then

removed. When the anterior wall of the left ventricle (LV) turned pale it was considered

successful induction of acute myocardial ischemia. Animals were ventilated until awake.

3.3.4 Ischemia reperfusion protocol

Global ischemia in ex-vivo hearts were performed as described previously [233]. Hearts

underwent a 30 minute perfusion phase with Kreb buffer alone, or 1ug/mL of glucagon (20

minutes) was perfused for by 30 minute ischemia, and 50 minute reperfusion during which left

ventricular developed pressure (LVDP) was recorded using Biopac instruments.

3.3.5 Blood pressure and heart rate measurements

Blood pressure and heart rates were measured using a telemetry system (DSI technology)

as described previously [235]. Blood pressure and heart rates were monitored for 1 week after

surgery for 24 hours.

3.3.6 Myocardium metabolic profiling

Mass spectrometry-based metabolic profiling was performed to determine myocardial

levels of acylcarnitines and organic acids. Triglyceride was extracted from frozen myocardial

tissue (~20 mg) with a 2:1 chloroform-methanol solution and quantified with a commercially

available enzymatic assay kit (Wako Pure Chemical Industry) as previously described [332].

3.3.7 Heart histology

Animals were anesthetized using avertin (10ul/g of body weight). 1 mol/l of KCl was

apically injected into the heart to arrest the heart in diastole. Perfusion of saline to flush out

blood from the heart was followed by perifusion of 4% buffered formalin at physiological

pressure to fix the hearts. Hearts were post fixed in formalin, embedded in paraffin, sectioned at

6 μm, and stained with Masson’s Trichrome, or were used for Tunnel staining. Cardiac images

were obtained from mid-ventricular cross-sections, and morphometry was performed with

LEICA QWin V3 software (2003). The infarcted area was calculated as a % of total LV area.

79

TUNEL staining was performed using the ApopTag peroxidase kit for apoptosis (EMD

millipore).

3.3.8 Glucose tolerance and measurement of plasma insulin

12-14 week old male mice were fasted overnight (16–18 hours), and glucose (1.5 mg/g

body weight) was administered orally (through a gavage tube) or via injection into the peritoneal

cavity (intraperitoneal glucose tolerance test). Blood samples were drawn from the tail vein at 0,

15, 30, 60, 90, and 120 minutes post glucose administration, and blood glucose levels were

measured using a Glucometer Elite blood glucose meter (Ascensia; Bayer HealthCare). For

plasma insulin determinations, blood samples (100 μl) were drawn from the tail vein during the 0

and 15 minute time periods following glucose administration in a heparinized tube. Plasma was

separated by centrifugation at 4°C and stored at –80°C until assayed. Plasma was assayed for

insulin using a mouse insulin ELISA kit (Linco).

3.3.9 Western blotting

Hearts were collected from 5 hour fasted mice 30 minutes following ischemia or without

ischemia and were washed in Krebs buffer containing 11mM glucose and frozen. Frozen hearts

were powdered using mortar and pestle and were subjected to homogenization for 30s in buffer

containing 50mM Tris HCL, pH to 8, 1mM EDTA, 10% glycerol, 0.02% Brij-35. The

homogenate was kept on ice for 10 minutes before centrifugation at 10,000 x g for 20 minutes.

Immunoblotting was performed on the resultant supernatant. The protein concentration of

homogenates was determined via BCA assay (Thermo Scientific). Samples (20 ug protein each)

were resolved via 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

and transferred onto a 0.45 μm nitrocellulose membrane. 10% fat free milk was used to block for

2 hours, and membranes were probed with either primary antibodies (Cell signaling) in 5% fatty

acid free of BSA. 1x phosphate buffered saline was used to wash the membrane, which was

subsequently probed with goat anti-rabbit (GE Healthcare UK limited NA934V 1/2000 dilution)

secondary antibody in 1% fat-free milk. Immunoblots were visualized with the Kodak imager

Station 4000MM PRO and quantified with Carestream Molecular Imaging software.

80

3.3.10 Heart RNA analyses

Hearts were collected from 5 hour fasted mice 30 minutes following ischemia or without

ischemia and were washed in Krebs buffer containing 11mM glucose and frozen. Frozen hearts

were powdered using mortar and pestle and were subjected to homogenization for 30s in buffer

containing Trizol (Sigma), and RNA was isolated using the Trizol method (Sigma). Following

RNA isolation, first-strand complementary DNA was synthesized from total RNA using the

SuperScript III reverse transcriptase synthesis system (Invitrogen) and random hexamers. Real-

time polymerase chain reaction was performed with the ABI Prism 7900 Sequence Detection

System using TaqMan Gene Expression Assays and TaqMan Universal PCR Master Mix

(Applied Biosystems). Levels of RNA transcripts were normalized to levels of peptidyl-propyl

isomerase A (cyclophilin) RNA.

3.3.11 PPARα Nuclear Translocation Immunoblotting in Primary Atrial Cardiomyocytes

Atrial cardiomyocytes were isolated as described previously [235]. Briefly, 3 to 4 month

old mice were anesthetized using avertin and perfused using a Langendorff system following

opening of the chest cavity. After perfusion for 2 minutes, the heart was digested with 2 mg/ml

collagenase II (Cellutron) for 15 minutes. The atrium was isolated from the ventricle and the rest

of the heart and agitated gently for 10 minutes. Tissues were resuspended and dissociated using

stopping buffer (20% FBS in minimum essential medium (MEM); Sigma Aldrich) by repeated

gentle pipetting followed by protein isolation for Western blot analysis.

3.3.12 Culture of HL-1 Atrial Cardiac Myocytes

HL-1 atrial cardiac myocytes were kindly provided by Dr. William Claycomb (Harvard

medical school), and cultured in supplemented Claycomb Media (Sigma-Aldrich) with 10%

FBS, 1% penicillin/streptomycin, 0.1 mM norepinephrine, and 2 mM L-glutamine. For

experiments, cells were seeded onto 6-well plates (BD Falcon), coated in 0.02% gelatin / 0.5%

fibronectin. Upon reaching confluence, cells were serum starved and supplemented with

Claycomb Media without FBS and norepinephrine prior to infection with AdβGal or AdGcgr at

10x multiplicity of infection. 24 hours following infection, cells were treated with either 1x PBS

or glucagon for 3 hours in supplemented Claycomb Media without FBS and norepinephrine.

81

3.3.13 PPARα Nuclear versus cytoplasmic expression

Confluent HL-1 cells were grown in supplemented Claycomb Media without FBS and

norepinephrine prior to infection with AdβGal or AdGcgr at 10x multiplicity of infection. 24

hours following infection, cells were treated with either 1x PBS or glucagon for 3 hours in

supplemented Claycomb Media without FBS and norepinephrine. Cells were then extracted in

nuclear fractionation buffer to obtain nuclear and cytoplasmic fractions. Protein concentrations

of each fraction were determined via Bradford protein assay, and 20 μg of cytoplasmic and 8 μg

of nuclear fractions were resolved on a 10% SDS gel to determine PPARα nuclear translocation.

β-actin was used as a positive control for cytoplasmic fractions, whereas lamin A/C was used as

a positive control for nuclear fractions.

3.3.14 In vitro HL-1 cellular injury model

HL-1 cells were subjected to cellular injury via treatment with H2O2 as previously

described [333]. In brief, confluent HL-1 cells were grown in supplemented Claycomb Media

without FBS and norepinephrine prior to infection with AdβGal or AdGcgr at 10x multiplicity of

infection. 24 hours following infection, cells were treated with either 1x PBS or 20 nM glucagon

concurrently with 100 μM H2O2 for 24 hours in supplemented Claycomb Media without FBS

and norepinephrine.

3.3.15 Statistical Analysis

Results are presented as mean ± SEM. Statistical significance was determined using 1-

way or 2-way analysis of variance with Bonferroni post hoc tests (as appropriate) using

GraphPad Prism 4.0 (GraphPad Software Inc). Statistical significance was noted when P< 0.05.

82

3.4 Results: Glucagon impairs outcomes after myocardial infarction in a

p38 MAPK-dependent manner.

To assess the impact of enhanced Gcgr signaling in the setting of acute cardiac ischemia,

I injected wild-type mice with glucagon 2 days prior to and 5 days after ligation of the left

anterior descending artery. Exogenous glucagon markedly reduced survival (Fig. 3.2B) without

changing levels of blood glucose or body weight (Fig. 3.1 A-C). Infarct sizes assessed at day 15

were similar in glucagon versus saline treated mice (Fig 3.2B-D). As p38 MAPK links G protein

coupled receptor signaling to cardiomyocyte cell survival pathways [334]. I assessed whether

glucagon activates p38 MAPK pathway in aerobic and ischemic hearts from wild-type mice.

Glucagon increased p38 MAPK phosphorylation in aerobic and ischemic hearts from wild-type

mice (Fig 3.2A). Next we assessed whether the increased mortality following glucagon treatment

is dependent on the p38 MAPK pathway; we treated separate groups of mice with glucagon, with

and without the p38 MAPK inhibitor SB203580, prior to and after LAD ligation and induction of

myocardial infarction. Although SB203580 alone had no effect, it completely eliminated the

glucagon-mediated impairment of survival after myocardial infarction (Fig 3.2B). Consistent

with these findings, cardiomyocyte apoptosis was increased in glucagon-treated mice, and

attenuated by co-administration of SB203580 (Fig. 3.2D). To determine whether the deleterious

effects of glucagon were mediated by the cardiomyocyte Gcgr, I generated mice with

cardiomyocyte-specific inactivation of the Gcgr gene (Fig. 3.3). Survival was reduced, and

infarct size was greater in glucagon-treated αMHCCre

control relative to outcomes in similarly-

treated GcgrCM-/-

mice (Fig 3.3). These findings indicate that glucagon reduces survival after

myocardial infarction in mice through actions requiring a cardiomyocyte Gcgr.

We next explored signaling pathways activated by glucagon in cardiac cell lines. As we

did not detect endogenous Gcgr expression in several immortalized cell lines, we transduced HL-

1 cells with an adenovirus encoding the rat Gcgr (AdGcgr). As Gcgr signaling regulates fatty

acid oxidation in the liver via PPAR-α, we assessed the PPAR-α pathway in the HL-1 cell line

[324]. Transduction of AdGcgr alone in HL-1 cells significantly increased PPAR-α expression

compared to HL-1 cells infected with βgal control virus (Adβgal) (Fig. 3.4A). Furthermore,

glucagon (20 nM for 3 hours) treatment increased PPAR-α target gene expression in AdGcgr

83

infected, but not in Adβgal control virus-infected cells (Fig. 3.5A and 3.4B), and increased the

activity of a PPAR-α luciferase reporter plasmid (Fig. 3.4B). This glucagon-dependent induction

of PPAR-α promoter activity was abrogated by pretreatment with the p38 MAPK inhibitor (10

μM SB203580) but not by the PKA inhibitor, H89 (Fig. 3.5C).

We next examined whether glucagon regulates PPAR-α translocation in cardiomyocytes.

PPAR-α nuclear translocation was enhanced by treatment with 20 nM glucagon for 3 hours in

primary cultures of atrial cardiomyocytes and in AdGcgr infected HL-1 cells, actions requiring

p38 MAPK activity (Fig. 3.5 D&E).

In HL-1 cells glucagon upregulated PPAR-α and downstream targets of PPAR-α

suggesting glucagon can upregulate fatty acid oxidation gene expression. Next we explored

whether glucagon can regulate the protein expression of key enzyme involved in glucose

oxidation; pyruvate dehydrogenase (PDH) [332]. Exogenous treatment with glucagon

upregulated P-PDH expression in aerobic and ischemic hearts from wild type mice (Fig. 3.5F).

Furthermore, a 3-hour treatment with 20 nM glucagon increased PDH phosphorylation in

AdGcgr-transduced HL-1 cells but not in cells infected with AdβGal (Fig 3.5G).

To identify whether glucagon directly regulated cardiomyocyte survival, we induced

apoptosis in HL-1 cardiomyocytes with H2O2. Glucagon increased caspase-3 cleavage in HL-1

cells, actions attenuated by treatment with 1.5mM DCA, an inhibitor of the PPAR-α downstream

target gene, pyruvate dehydrogenase kinase 4 (PDK4), which phosphorylated and inhibited the

activity of PDH (Fig. 3.5H).

84

Figure 3.1.Blood glucose and body weight before and after glucagon administration and

LAD ligation.

(A) Blood glucose levels before and after left anterior descending coronary artery (LAD)

ligation surgeries in 11-14 week old C57BL/6 mice injected with 30ng/g subcutaneous

glucagon or saline (B) with p38 MAPK inhibitor (SB203586, 1umol/kg)(C) Body weight in

11-14 week old C57BL/6 mice with 30ng/g subcutaneous glucagon injections or saline (D)

with p38 MAPK inhibitor (SB203586) Data are mean ± S.E.M (n = 5-8 mice per group).

85

Figure 3.2.Glucagon impairs survival after myocardial infarction in a p38 MAPK-dependent

manner

Left coronary artery descending ligation (LAD) was performed in 11-14 week old C57BL/6

mice treated with saline or 30ng/g subcutaneous glucagon injections (every 8 hours for 7

days) with or without co-administration of the p38 MAPK inhibitor (SB203580 1umol/kg)

and survival was monitored for 15 days following surgery. * = p<0.05 WT saline vs. WT

glucagon group. Data are mean ± S.E.M (LAD n = 13-15 per treatment) (B) Infarct size 15

days following LAD ligation. Data are mean ± S.E.M (LAD n = 5-6 per treatment) (C)

C57BL/6J mice treated with saline or glucagon (30ng/g body weight) were subjected to LAD

coronary artery ligation for 48 hour, following which hearts were isolated for analysis of

apoptosis by TUNEL staining. * = p<0.05 WT saline vs. WT glucagon group. Data are mean

± S.E.M (LAD n=7-8 mice per treatment). (D-E) C57BL/6J mice treated with saline or

glucagon (30ng/g body weight) with or without SB203580 were subjected to LAD coronary

86

artery ligation for 48 hours, hearts were isolated and apoptosis assessed by TUNEL staining.

* = p<0.05 WT saline vs. WT glucagon group. Data are mean ± S.E.M (LAD n =3-5).

Figure 3.3.Glucagon impairs survival after myocardial infarction in a cardiac Gcgr-

dependent manner

Left anterior descending coronary artery ligation (LAD) surgeries were performed in 11-14

week old MHCCre

WT and GcgrCM-/-

mice treated with 30ng/g subcutaneous glucagon

injections (every 8 hours for 7 days) and survival was monitored for 15 days following

surgery. * = p<0.05 MHCCre

WT vs. GcgrCM-/-

mice. Data are mean ± S.E.M (LAD n = 12-

13 per genotype). Infarct size 15 days following LAD ligation surgeries.

87

Figure 3.4.Glucagon has no effects on Ad-βgal transfected HL-1 cell lines.

(A)HL-1 cell lines transfected with Adenovirus encoding βgal or rat Gcgr were treated with

saline or 20nM Glucagon for 3 hours following which RNA was isolated and analyzed ** =

p<0.01 Adβgal vs AdGcgr adenovirus infected cells. Data are mean ± S.E.M (n =5).

(B)HL-1 cell lines were transfected with Adenovirus encoding βgal for 24 hour followed by

transfection with PPAR-α reported gene-luciferase construct for another 24 hours and

luciferase expression was assessed. Data are mean ± S.E.M (n =5).

(C) HL-1 cell lines were infected with Adβgal or AdGcgr for 24 hours followed by a 3 hour

treatment with 20 nM glucagon. Cell lysates were analyzed by Western blotting for

expression of p-PDH and total PDH. Data are mean ± S.E.M (n = 5).

88

Figure 3.5.Glucagon increases PPAR-αnuclear translocation, levels of cleaved caspase-3,

and PDH phosphorylation

(A)HL-1 cells infected with adenovirus expressing the Gcgr were treated with saline or

20nM glucagon for 3 hours following which cells were harvested for RNA isolation and

analysis.

(B-C)HL-1 cells were infected with adenovirus containing β-gal or Gcgr for 24 hour

followed by transfection with PPARα gene promoter -luciferase construct for 24 hours. Cells

were treated with saline or 20nM glucagon for three hours with or without PKA inhibitor

(H89) or p38 MAPK inhibitor (SB203580) and luciferase expression was assessed as

described in Methods.

(D)Primary cultures of atrial cardiac myocytes were treated with saline or 20nM glucagon for

3 hours and cells were harvested for cytoplasmic and nuclear protein expression using

Western blot analysis

(E) HL-1 cell lines infected with AdGcgr were treated with saline or 20 nM glucagon with

and without SB203580 for three hours and cells were harvested for nuclear and cytoplasmic

protein content followed by Western blotting and quantitation of protein expression.

89

(F) 11-13 week old mice were treated with exogenous glucagon every 8-hour for 24 hours.

Mice were fasted for 5 hours and hearts were collected 30 min following LAD ligation

surgeries for protein analysis as described in methods (n = 6 per treatment).

(G) HL-1 cell lines were infected with Adenovirus encoding β-gal or Gcgr for 24 hours

followed by 3 hour treatment with glucagon followed by protein isolation and western

blotting for expression of p-PDH and total PDH.

(H) HL-1 cell lines were infected with AdGcgr for 24 hours followed by 24 hour treatment

with 100 µM H2O2 and 3 hours of exposure to 20nM glucagon. Western blot analysis of

cleaved caspase-3 from cells treated with or without 1.5 mM DCA, an inhibitor of the PPAR-

α target gene, PDK4.

90

Figure 3.6.Glucagon increases long and medium chain fatty acid content in the heart.

(A) Acylcarnitine levels in aerobic hearts were assessed following glucagon or saline

subcutaneous injections (every 8 hours for 24 hours) and hearts were collected following a 5

hour fast (for ischemic samples: hearts were collected 30 minutes following left coronary

artery descending ligation (LAD) surgeries) (n = 5 per genotype). Data is expressed as

percent of levels in aerobic mouse hearts.* = p<0.05 Saline vs. glucagon-treated group. Data

are mean ± S.E.M (LAD n = 13-15 per genotype). (B) Citric acid cycle intermediate levels in

aerobic or ischemic hearts harvested 30 min following LAD ligation from mice injected with

glucagon or saline (every 8 hours for 24 hours). Values are expressed as percent of C-

Cre vs. WT mice values (n = 5 per genotype).

91

3.4.2 Exogenous glucagon increases fatty acid oxidation in the ischemic heart

Since exogenous glucagon increased expression of genes and proteins regulating fatty

acid oxidation, we assessed cardiac lipid content in hearts from fasted wild-type mice treated

with glucagon or saline for 2 days. No difference in lipid content was detected in aerobic hearts

from mice treated with glucagon vs. saline. However, levels of total long and medium chain fatty

acids (acylcarnitines) were significantly increased in ischemic hearts from glucagon-treated mice

(Fig. 3.6A). In contrast, we did not observe differences in citric acid cycle intermediates,

including succinate, fumarate, malate, and citrate (Fig. 3.6B) in hearts from glucagon- vs. saline-

treated mice.

3.4.3 Partial deletion of whole body Gcgr signaling impairs survival following MI and

exacerbates LV remodeling

Next we assessed the susceptibility of 12-week-old global Gcgr+/-and Gcgr+/+ mice in

response to ischemia 28 days following induction of experimental myocardial infarction. This

operation is described in methods and in articles by Ohta in 2004 and Fazel in 2005 [335, 336].

We observed improved survival in Gcgr+/- mice compared to +/+ control mice following

experimental myocardial infarction (Fig. 3.7B). To assess infarct size in the heart following

myocardial infarction, immunostaining for collagen fibers were performed 28 days following

induction of myocardial infarction in the heart of Gcgr-/-, +/- and +/+ mice. Our results show

comparable infarct sizes in the global Gcgr+/- mice compared to Gcgr+/+ mice (Fig. 3.7E).

3.4.4 Loss of Gcgr signaling protects and glucagon perfusion worsens outcome from

ischemia/reperfusion injury in isolated mouse hearts.

To ascertain whether improved survival of Gcgr+/- mice reflects the consequences of

reduced cardiac Gcgr expression, we assessed the response of isolated Gcgr-/- vs. Gcgr+/+

hearts to ischemia and reperfusion ex vivo [233]. The hearts from Gcgr-/- mice exhibited

significantly improved recovery following ischemia reperfusion injury compared to wild-type

littermate controls (Fig. 3.8A, C&E). In contrast, wild-type mouse hearts perfused with glucagon

exhibited significantly reduced recovery following ischemia (Fig. 3.8B, D&F). Hence, activation

92

of cardiac Gcgr signaling worsens whereas loss of Gcgr expression improves functional recovery

from ischemia.

Figure 3.7. Whole body Gcgr+/- mice has significantly improved survival following MI

(A)Heart weight to body weight ratio in 11-14 week old male Gcgr+/+ and Gcgr+/- mice (n

= 4 per genotype). (B)Left coronary artery descending ligation (LAD) surgeries were

performed in 11-14 week old male Gcgr+/+, and Gcgr+/- mice and survival was monitored

for 28 days following surgery. * = p<0.05 Gcgr+/+ vs Gcgr+/- Data are mean ± S.E.M

(SHAM n = 10 per genotype and LAD n = 22-25 per genotype) (C) Heart weight to body

weight ratio 28 days following LAD ligation surgery. * = p<0.05 Gcgr +/+ SHAM vs Gcgr

+/+ LAD, Gcgr +/- SHAM vs .Gcgr+/- LAD. Data are mean ± S.E.M (n = 5-11 per

genotype) (D) Masson trychome staining and infarct area quantification 28 days following

surgery. * = p<0.05 Gcgr +/+ vs Gcgr +/-. Data are mean ± S.E.M (n = 4-8 per genotype)

(E) Blood glucose before and after LAD surgery.

93

Figure 3.8. Loss of Gcgr signaling protects whereas glucagon impairs recovery of

ventricular developed pressure after ischemia-reperfusion injury in the isolated heart ex vivo

(A&B) Schematic depiction of peptide infusions, ischemia and reperfusion times and

representative LVDP recordings from isolated perfused hearts (C) Determination of LVDP in

isolated perfused WT and Gcgr-/- hearts subjected to ischemia-reperfusion. Data are shown

as the means ± S.E.M. of 3 mice per group. *P < 0.05, **P < 0.01 and ***P < 0.001

compared to the control group. (D) Determination of LVDP in isolated perfused WT hearts

with pre-treatment of PBS or glucagon (1 ug/ml for 20min) to ischemia/reperfusion. Data

shown are means ± S.E.M., n= 3 mice per group. *P < 0.05, **P < 0.01 and***P < 0.001

compared to the control group (E&F). Bar graphs depict the percentage recovery rate of

LVDP from ischemia. Data are shown as the means ± S.E.M. of 3–6 mice per group. *P

<0.05 compared to the control group, Statistical significance was determined by ANOVA

followed by Bonferroni’s post hoc test.

WT Kreb WT Glucagon0

5

10

15

20 *

Re

co

ve

ry(%

In

itia

l L

VD

P)

Ischemia

WT Gcgr-/-0

20

40

*

Recovery

(% in

itia

l L

VD

P)

0

min

30

min 60

min

Reperfusion

120

min

Ischemia

0

min 30

min 60

min

Reperfusion

110

min

E F

A B

Gcgr+/+

Gcgr-/-

WT Kreb

WT Glucagon

10 30 50 70 90 1100

20

40

60

80

100

120 Gcgr-/-

Gcgr+/+

******

******

*** ***

***************

Time (min)

LV

DP

(m

m H

g)

Perfusion Perfusion

Glucagon

(20 min)

C D

0 20 40 60 80 1000

20

40

60

80

100

KrebGlucagon

** * * *** ***

Time (min)

LV

DP

(m

mH

g)

94

3.4.5 Generation of cardiomyocyte-specific glucagon receptor knockout mice

To determine whether loss of Gcgr expression in cardiomyocytes promotes resistance to

ischemic cardiac injury, we generated inducible cardiac-specific Gcgr-/- (GcgrCM-/-)

mice (Fig.

3.9A). Gcgr expression was preserved in liver and kidney from GcgrCM-/

vs. wild-type, GcgrFlox

and αMHCCre

mice (Fig. 3.9B). To disrupt the Gcgr gene in the heart, mice were injected with

tamoxifen, 5-6 week following tamoxifen injections ~ 80% reduction in levels of Gcgr mRNA

transcripts were observed in RNA isolated from hearts of GcgrCM-/-

mice (Fig. 3.9B).

3.4.6 Inactivation of Gcgr expression in cardiomyocytes increases survival after

myocardial infarction

GcgrCM-/-

mice exhibited normal body weight, glucose tolerance, plasma insulin levels,

and basal heart weights compared to littermate controls (Fig. 3.10A-F). Additionally, heart rate

and blood pressure were comparable in GcgrCM-/-

vs. littermate control mice 5-6 weeks following

tamoxifen injections (Table 2). Survival was significantly greater, and heart weights, and infarct

size was reduced following LAD ligation in GcgrCM-/-

mice (Fig. 3.11A-C). Although induction

of Cre expression in αMHCCre

mice induces a transient cardiomyopathy [337], GcgrCM-/-

mice

had significantly lower mortality after tamoxifen administration compared to αMHCCre

mice

(Fig. 3.12) suggesting inactivation of the Gcgr in cardiomyocytes leads to protection from

cardiac injury.

95

Figure 3.9. Generation of mice and analysis of Gcgr expression

GcgrFlox

mice containing the neomycin cassette were mated with FLPe mice to remove the

neomycin cassette [49]. The resulting GcgrFlox

mice were then mated with αMHCCre

mice to

generate GcgrCM-/-

mice. (B-D) Relative Gcgr expression assessed in liver (B), kidney (C)

and heart (D) * = p<0.05 GcgrCM-/-

vs littermate control mice. Data are mean ± S.E.M (n = 5-

6 per genotype).

97

Figure 3.10. Body weight, glucose tolerance and heart weight in mice with cardiac -specific

inactivation of the Gcgr

(A) Body weight was monitored from 7-14 week before and after tamoxifen injections

GcgrCM-/-

and littermate control mice. (B) Blood glucose levels during an intraperitoneal

glucose tolerance test in 10 to 14 week-old mice (n = 11–22 mice per group). (C) Plasma

insulin levels 0 and 15 minutes following intraperitoneal glucose challenge (n = 4–9 mice per

group). (D) Blood glucose levels during an oral glucose tolerance test in 10- to 14-week-old

mice (n = 11–22 mice per group). (E) Plasma insulin levels 0 and 15 minutes following oral

glucose challenge (n = 4–9 mice per group). (F) Heart weight to body weight ratios in 12-14

week old GcgrCM-/-

and littermate control mice. Values are expressed as mean ± SEM.

98

Figure 3.11. (A-C) Heart rate, systolic and diastolic blood pressure in αMHCCre

and GcgrCM-/-

mice was measured. Data are mean ± S.E.M. (n = 4 mice in each group monitored for 20 hours

from Day 1: 5:30pm to Day 2: 1:30 pm).

Time (in 2 min, Day 1-5:30pm-Day 2-2:30pm)

99

Table 1. Heart rate and blood pressure measurements in GcgrCM-/-

mice were conducted using

telemetry system. Systolic and diastolic blood pressure and heart rates were measured in 12-14

week old GcgrCM-/-

and αMHCCre

mice from Day 1, 5:30 pm to Day 2, 1:30 pm. Values are

expressed as mean ± SEM.

Parameters αMHCCre

GcgrCM-/-

Systolic blood pressure, mm Hg 137.7±9.492 132.5±15.7

Diastolic blood pressure, mm Hg 104.7±11 108±14

Heart rate, bpm 506.6±23.6 487.5±12.9

100

Figure 3.12. Loss of cardiac Gcgr signaling enhances survival following MI and attenuates

adverse LV remodeling

(A) Left anterior descending coronary artery ligation (LAD) was performed in 11-14 week

old GcgrCM-/-

and littermate control mice and survival was monitored for 15 days following

surgery. * = p<0.05 GcgrCM-/-

vs per control group. Data are mean ± S.E.M (SHAM n = 10

per genotype and LAD n = 11-23 per genotype) (B) Heart weight: body weight ratio 15 days

following LAD ligation. * = p<0.05 GcgrCM-/-

vs GcgFlox

and wild-type mice. Data are mean ±

S.E.M (n = 8-11 per genotype) (C) Masson trychome staining of infarcted heart, black arrows

represent areas quantified for infarct size measurements. Infarct area quantification 15 days

following surgery using Aperio image Scanscope program. * = p<0.05 GcgrCM-/-

vs littermate

control mice. Data are mean ± S.E.M (n = 9-11 per genotype).

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Figure 3.13. Selective loss of Gcgr signaling in cardiomyocytes lead to reduced expression

of fatty acid oxidation genes and proteins

(A) Quantification of mRNA from aerobic hearts and 30 minutes following ischemia from 12

week old αMHCCre

WT vs GcgrCM-/-

mice fasted 5 hours prior to collection of hearts (n = 4).

* = p<0.05 GcgrCM-/-

vs αMHCCre

WT. Data are mean ± S.E.M. (B) 5 hour fasted 12 week

old αMHCCre

vs GcgrCM-/-

mice were euthanized, and hearts were frozen and processed

followed by Western blot analysis as described in Methods with and without 30 min

ischemia. * = p<0.05 for levels of proteins in αMHCCre

vs GcgrCM-/-

Data are mean ± S.E.M.

(n = 4 mice in each group).

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Figure 3.14. Targeted metabolomics reveals reduced fatty acid oxidation in GcgrCM-/-

hearts

(A) Acylcarnitine levels in aerobic hearts harvested from 5 hour fasted αMHCCre

WT vs

GcgrCM-/-

(n = 5 per genotype). Values are expressed as percent of αMHCCre

mice values.

(B) Aerobic myocardial total long, medium and short chain acylcarnitines.* = p<0.05

αMHCCre

vs GcgrCM-/-

(C) TG levels (D) Lactate levels (E-I) Citric acid cycle intermediate

levels in aerobic hearts. * = p<0.05 αMHCCre

vs GcgrCM-/-

(J) Acylcarnitine levels in hearts

harvested 30 min following ischemia from 5 hour fasted αMHCCre

vs GcgrCM-/-

mice, n = 5

per genotype). Values are expressed as percent of αMHCCre

mice values. (K) Levels of total

long, medium and short chain in ischemic hearts.* = p<0.05 αMHCCre

vs. GcgrCM-/-

. * =

p<0.05 αMHCCre

vs. GcgrCM-/-

(L) Ischemic TG levels. * = p<0.05 αMHCCre

vs. GcgrCM-/-

(M)

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Ischemic lactate levels. * = p<0.05 αMHCCre

vs. GcgrCM-/-

(N-R) Ischemic citric acid cycle

intermediate levels in aerobic hearts from 5 hour fasted αMHCCre

vs. GcgrCM-/-

mice. * =

p<0.05 αMHCCre

vs. GcgrCM-/-

.

Figure 3.15. Targeted metabolomics reveals reduced fatty acid oxidation in hearts from high

fat fed mice with loss of cardiac Gcgr signaling

(A) Acylcarnitine levels in aerobic hearts harvested from 5 hour fasted and fed high fat diet

for 24 weeks αMHCCre

vs GcgrCM-/-

(n = 5 per genotype). Values are expressed as percent of

αMHCCre

mice values. (B) Aerobic high fat diet fed myocardial total long, medium and short

chains. (C) Aerobic high fat diet fed TG levels (D) Aerobic high fat diet fed lactate levels (E-

I) Aerobic high fat diet fed citric acid cycle intermediate levels.

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Figure 3.16.Schematic of proposed model

(A) Exogenous glucagon promotes PPAR- translocation to the nucleus in a p38 MAPK-

dependent manner leading to upregulation of PPAR-α target genes including cpt1b and genes

involved in β oxidation. Subsequently fatty acid oxidation is increased and increased

mortality is increased following myocardial infarction. (B) Cardiac-specific or partial

reduction of Gcgr expression in cardiomyocytes leads to reduced expression of PPAR- and

genes involved in β oxidation leading to reduced fatty acid uptake into cardiomyocytes and

reduced uptake into mitochondria. Hence, flux through fatty acid metabolism is reduced,

leading to increased glucose oxidation and protection from cardiac ischemic injury.

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3.4.7 Deletion of the cardiomyocyte Gcgr leads to reduced expression of genes and

proteins regulating fatty acid oxidation

As hepatic Gcgr signaling controls fatty acid oxidation in the liver via PPARα, we

analyzed this pathway in GcgrCM-/-

mice [324]. The expression of PPARα and its target genes

was lower in aerobic and ischemic hearts from GcgrCM-/-

mice (Fig. 3.13). We also detected

lower levels of PPARα and PDK4, an inhibitor of PDH, in ischemic GcgrCM-/-

mice hearts (Fig.

3.13B-C). Furthermore, levels of cardioprotective proteins such as P-AKT and P-GSK3β were

increased in aerobic GcgrCM-/-

hearts (Fig. 3.13D-E). Hence, the presence or absence of cardiac

Gcgr signaling alters the expression of genes and proteins important for fatty acid and glucose

oxidation.

3.4.8 Targeted metabolomics illustrates reduced fatty acid oxidation in normal and

insulin-resistant hearts with selective loss of Gcgr signaling

Since lower expression of fatty acid oxidation genes and proteins was detected in

GcgrCM-/-

hearts, we assessed cardiac lipid content. Significantly lower levels of total long and

medium chain fatty acids (acylcarnitines) were present in aerobic hearts from GcgrCM-/-

mice

(Fig. 3.14 A&B). Although triglyceride content was similar, citric acid cycle intermediates

including succinate, fumarate, malate, and citrate trended lower in GcgrCM-/-

hearts (Fig.3.14 C-

I). Similarly, levels of long chain fatty acids were significantly lower and medium chain fatty

acids were reduced after induction of ischemia in GcgrCM-/-

hearts (Fig. 3.14 J-K). Unlike

findings in aerobic hearts, ischemic GcgrCM-/-

hearts had significantly higher triglyceride content

and lower levels of lactate (Fig. 3.14 L-M), whereas levels of citric acid cycle intermediates were

lower in both aerobic and ischemic GcgrCM-/-

hearts (Fig. 3.14 N-R). These findings suggest that

loss of basal cardiac Gcgr signaling leads to reduced fatty acid oxidation, increased triglyceride

pools and higher glucose oxidation resulting in lower levels of lactate.

To investigate whether similar changes in cardiac acylcarnitines arise under conditions of

insulin resistance, GcgrCM-/-

mice were maintained on a high fat diet for 24 weeks. Consistent

with findings in regular chow-fed mice, levels of total long and medium chain acylcarnitine

levels were lower in hearts after prolonged high fat feeding (Fig. 3.15 A&B). Although lactate

levels were similar, citric acid cycle intermediates were lower in GcgrCM-/-

hearts (Fig. 3.15 C-I),

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suggesting that under conditions of insulin resistance, loss of cardiac Gcgr signaling leads to

reduced fatty acid oxidation.

3.5 Discussion

Adults with type 2 diabetes are two to four times more likely to develop heart disease or a

stroke, findings attributable to associated risk factors such as hypertension, dyslipidemia,

obesity, and abnormalities in coagulation [276-279]. Nevertheless, diabetes itself is widely

viewed as an independent risk factor for the development of cardiovascular disease, although

mechanisms linking hyperglycemia to the pathogenesis of macro vascular disease are complex

and poorly understood [338, 339]. Although deregulated glucagon secretion is fundamental to

the pathophysiology of hyperglycemia, ketoacidosis, and impaired counter-regulation [340], a

role for glucagon in the development of cardiovascular complications has not been extensively

explored.

Acute administration of glucagon increases heart rate and cardiac output, providing the

rationale for use of glucagon as an inotropic or chronotropic agent in subjects with refractory

bradycardia or in the setting of cardiac failure not responsive to classical inotropic therapies

[341, 342]. Nevertheless, these beneficial actions of glucagon are transient, and efforts to use

chronic glucagon administration as a treatment for congestive heart failure were subsequently

abandoned. Our studies show that short-term activation of glucagon receptor signaling in mice

worsens outcomes from experimental myocardial infarction. The deleterious actions of glucagon

required the cardiac Gcgr and were mediated by the p38 MAPK-dependent pathway.

Conversely, partial reduction or cardiac-specific loss of Gcgr signaling improves outcome from

myocardial infarction, and is associated with reduced expression of genes and proteins regulating

fatty acid oxidation in the heart.

Our data linking cardiac Gcgr signaling to fatty acid oxidation is consistent with previous

findings linking Gcgr signaling to PPAR-dependent control of hepatic lipid oxidation [324].

Glucagon enhances PPARα translocation to the nucleus in cardiomyocytes, actions requiring p38

MAPK signalling. Consistent with the functional importance of a glucagon-PPAR pathway in

the heart, our loss of function studies in hearts from GcgrCM-/-

mice reveal significantly reduced

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cardiac expression of PPARα and downstream targets such ascd36, gpat1, dgat1, and dgat2.

To gain insight into the effects of glucagon on cardiac fuel metabolism we utilized

acylcarnitine profile as a tool to detect abnormalities in fatty acid metabolism. We observed

reduced levels of long and medium chain acylcarnitines strongly suggestive of overall reduced

fatty acid oxidation in GcgrCM-/-

hearts. However, short chain acylcarnitines (C2) levels were

normal in hearts from GcgrCM-/-

mice suggesting selective inhibition of fat oxidation and not

more widespread changes in oxidation of other substrates. It is important to emphasize that while

acylcarnitine profiling is often used as a tool to obtain information on changes in fatty acid

metabolism and substrate utilization, these changes may not always reveal actual overall rates of

fatty acid oxidation [343]. Acylcarnitine levels indicate which step of fatty acid oxidation might

be inhibited for instance; if long chain acylcarnitines are increased and medium chain

acylcarnitines are reduced, this would indicate an inhibition of one of the enzymes involved in

oxidation of medium chain acylcarnitines, and this may not be suggestive of an overall reduction

in fatty acid oxidation [343]. Therefore, assessment of the actual rate of fatty acid oxidation may

further solidify our findings.

Reduction in fat utilization in GcgrCM-/-

hearts leads to increase in Triacylglycerol (TAG)

pools and glucose oxidation, as suggested by reduced phosphorylation of PDH and decreased

lactate levels. Decreased TCA cycle activity in hearts from GcgrCM-/-

mice may indicate that less

energy (ATP) is being made, a hypothesis that can be further solidified by direction

measurement of nucleotide levels in the heart. The heart relies primarily on fatty acid oxidation

under conditions of hyperglycemia and insulin resistance [279, 344-347], and even during states

of low oxidative capacity such as ischemia, fatty acid oxidation predominates as a source of

residual oxidative metabolism [348, 349]. Increased fat oxidation decreases the heart’s ability to

metabolize glucose, and several lines of evidence suggest that utilization of fat over glucose as

substrate for cardiac fuel metabolism decreases cardiac efficiency [350-355]. Utilization of

glucose is more efficient as fatty acid oxidation requires approximately 10% more oxygen to

produce equivalent amounts of ATP than glucose [350]. Additionally, reduced ATP synthesis

may ensue as a result of fatty acid oxidation. ATP synthesis can occur through translocation of

protons from the mitochondrial matrix to the inner membrane space by F1/F0 ATPase; during

fatty acid oxidation expression of uncoupling proteins (UCP 2 and 3) are increased [351, 352].

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UCPs promote an alternate route for protons from ATP synthesis, thereby uncoupling ATP

synthesis, and leading to reduced cardiac efficiency [351-353]. Moreover, increased fatty acid

utilization can also promote futile cycling of fatty acid intermediates that results in consumption

of ATP for non-contractile functions versus contractile purposes leading to cardiac inefficiency

[354, 355]. Therefore, reduced fat and increased glucose utilization in GcgrCM-/-

hearts may

increase cardiac efficiency, which in turn may contribute to protection from myocardial ischemia

(Figure 3.16).

Previous studies have reported that glucagon can promote fatty acid oxidation in the heart

[356]. Consistent with these findings we observed increased long and medium chain

acylcarnitine levels in glucagon-treated ischemic hearts. These findings complement our loss of

function studies, and are consistent with the notion that glucagon promotes fat oxidation in hearts

and reduce glucose oxidation under ischemic conditions as suggested by the increased inhibition

of PDH by glucagon. Glucagon-mediated increases in fat and reduced glucose oxidation may

contribute to cardiac inefficiency and increased mortality following ischemic cardiac injury

(Figure 3.16).

Glucagon increases heart rate and contractility, and these actions of glucagon are opposite to

the effects of adrenergic β-blockers, which reduce heart rate and slow cardiac contractility,

reducing cardiac energy demand [341, 357-359]. β -blockers reduce myocardial oxygen

consumption and improve myocardial efficiency by shifting myocardial substrate utilization

from increased free fatty acid oxidation to increased glucose oxidation thereby producing

cardioprotection [358, 359]. The consequences of reduced signaling through the adrenergic β-

receptor overlap with the cardiac phenotype observed in GcgrCM-/-

mice. We suspect that GcgrCM-

/- hearts may produce energy more efficiently, which in turn may protect the ischemic heart and

enhance survival after myocardial infarction.

In the diabetic or insulin-resistant hearts it is unclear whether fatty acid oxidation is increased

or decreased as studies have reported inconsistent findings; results are variable depending on the

experimental design or genetic model utilized [279, 332, 360-365]. Impaired fatty acid oxidation

has been suggested to lead to an increase in accumulation of cardiac lipid intermediates including

DAG and ceramide levels. These lipid intermediates have been suggested to inhibit insulin

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sensitivity and possibly to lead to cardiac lipotoxicity [360, 361, 366]. This is further supported

by genetic models where adipose triglyceride lipase (ATGL) was deleted in mice which resulted

in reduced PPAR-α and PPAR-γ expression in the heart, and blunted fatty acid oxidation, leading

to excessive lipid accumulation, cardiac insufficiency, and lethal cardiomyopathy. Similarly,

another mouse model with overexpression of PPAR-γ in the heart resulted in dilated

cardiomyopathy and deletion of PPAR-α rescued the cardiomyopathy phenotype in the PPAR-γ

overexpressing mice. Surprisingly, deletion of PPAR-α in PPAR-γ overexpressing mice resulted

in upregulation of many PPAR-α targets involved in fatty acid uptake, oxidation and triglyceride

synthesis leading to increased fatty acid oxidation. Despite having increased fatty acid oxidation

these mice have improved cardiac efficiency and reduced cardiac apoptosis [363]. Alternatively,

increasing cardiac fatty acid oxidation has been suggested to be also protective in diabetic or

insulin-resistant hearts, and this is partly due to reduced levels of acylcarnitines, which, when

present at high levels, may disrupt biomembranes and increase the susceptibility to cardiac

apoptosis [364]. Based on these findings, stimulating β-oxidation can benefit the hearts in

diabetes or obesity by reducing the accumulation of intramyocardial lipids and increasing insulin

sensitivity and cardiac efficiency [360, 361, 363, 364, 366, 367].

Although previous studies demonstrate impaired fat oxidation in obese animal models, the

contribution of endogenous TAG turnover rates to these findings is not well defined [360, 361,

363, 364, 366]. In mouse cardiomyocytes, 50 to 70% of total fatty acid oxidation is from

endogenous TAG storage pools [368]. In studies where PPARwas overexpressed in the mouse

heart it resulted in higher TAG turnover and an increased reliance on the endogenous TAG as a

source for fatty acid oxidation was observed [368]. Therefore, by not measuring the TAG

turnover rates these studies are lacking a large portion of fatty acid oxidation measurements,

which may lead to an underestimation of the fatty acid oxidation rates in diabetes and obese

models. Therefore, it is vital to measure TAG turnover rates to make definite conclusions about

the overall fatty acid oxidation rates in diabetes/obesity and in genetic knockout models.

A limitation to enhancing fat oxidation for the treatment of cardiovascular disease is the

possibility of simultaneous reduction of carbohydrate oxidation, which may result in cardiac

inefficiency [279, 332, 367]. Moreover, increased fatty acid oxidation can reduce insulin-

stimulated glucose metabolism based on the Randle cycle [369-372]. Therefore, blocking

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cardiac fatty acid oxidation as a strategy for the treatment of cardiovascular complications in

diabetes and obesity may not be ideal.

Excessive fat oxidation is also observed in hearts from T2D animal models and in obese

human subjects [279, 332, 365]. Furthermore, in obese or diabetic human subjects with chronic

heart failure reduction of fatty acid oxidation using anti-anginal drugs that inhibit enzymes

involved in fatty acid oxidation or fatty acid transport into mitochondria showed an improved

cardiac function [373, 374]. However, these studies also report overall improved whole body

insulin sensitivity as well. Nonetheless, a number of genetic mouse models also suggest that

enhanced fatty acid oxidation is not always beneficial, as transgenic mice with cardiac over-

expression of PPAR- exhibited excessive fatty acid oxidation and features of diabetic

cardiomyopathy [375]. Furthermore, deletion of CD36, a fatty acid transporter, in PPAR-

transgenic mice led to reduction of fatty acid oxidation and rescued the excessive fatty acid

oxidation and diabetic cardiomyopathy phenotype [376]. Under ischemic conditions

manipulation of PPAR-α expression has also been shown to modulate outcomes from ischemia.

Genetic deletion of of PPAR-α gene in mice resulted in improved cardiac outcomes and

activation of PPAR-α during ischemia led to increase fatty oxidation, which resulted in reduced

cardiac efficiency and post-ischemic functional loss [377, 378]. Therefore, these studies suggest

blocking fatty acid oxidation in cardiac ischemia or diabetes and obesity can be beneficial.

Our results are consistent with the concept that reduced expression of PPAR-α in hearts of

GcgrCM-/-

mice may contribute to improved outcomes following ischemic injury as a result of

reduced fat oxidation and increased glucose oxidation. Conversely, activation of Gcgr signaling

activation results in increased PPAR-α translocation to the nucleus in a p38 MAPK-dependent

manner, resulting in increased expression of PPAR-α target genes in the heart. This in turn leads

to increased fat uptake in cardiomyocytes and mitochondria resulting in increased fatty acid

oxidation and decreased glucose oxidation, as suggested by PDH phosphorylation, which results

in cardiac inefficiency following glucagon treatment in the setting of cardiac ischemia. This is

further supported by our findings that glucagon-mediated increases in apoptosis in hydrogen

peroxide-treated HL-1 cells was dependent on PDH phosphorylation by PDK-4, suggesting that

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glucagon may also increase apoptosis by blocking glucose oxidation.

Under ischemia when the oxygen supply to the heart is minimal the substrate utilization by

the heart is minimized [379, 380]. Accordingly, fatty acid oxidation and glucose oxidation are

reduced under ischemic conditions. Reduction in fatty acid oxidation in the heart under ischemic

conditions results in lower cardiomyocyte levels of very long chain and long chain

acylcarnitines[379, 380]. Reduced levels of long chain acylcarnitines have been suggested to be

beneficial for cardiomyocyte health as increased levels of cardiomyocyte acylcarnitines

minimize oxygen consumption by cardiomyocytes, inhibit ATPase activity, and they also have

detergent-like properties that can disrupt membranes and increase lysosomal activity of the cells

leading to poor cardiomyocyte health[379, 380]. We propose here another mechanism through

which glucagon may be increasing cardiomyocyte injury - by increasing long chain acylcarnitine

levels in the heart under ischemic conditions. Similarly, in the GcgrCM-/-

mice reduced long chain

acylcarnitine levels can also be beneficial for the survival of the cardiomyocytes under ischemic

conditions.

In summary our studies demonstrate that enhanced glucagon action in the cardiovascular

system may be detrimental, while partial global reduction of Gcgr activity in Gcgr+/- mice or

cardiomyocyte-specific attenuation of Gcgr signaling may be beneficial in the setting of cardiac

ischemic injury. Our findings have implications for ongoing development of therapeutic

strategies designed to augment or inhibit Gcgr signaling [5, 282, 381, 382] for the treatment of

metabolic disorders.

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CHAPTER 4: General Discussion and Future Direction

CHAPTER 4

General Discussion and Future Directions

113

.

Diabetes consists of inappropriate increases in glucagon secretion and a higher risk of

cardiovascular disease. Reduced glucagon action is known to improve glucose homeostasis

however; the exact mechanism through which reduction of glucagon action leads to improved

glucose homeostasis is unknown. Current US Federal drug administration guidelines recommend

that manufacturers developing novel therapies for type 2 diabetes (T2D) provide evidence that

the therapy will not increase the risk of cardiovascular mortality. A functional Gcgr is expressed

in the heart. However, glucagon’s physiological role in the heart is unknown, and it is not clear if

therapies that target the inhibition or activation of glucagon receptor (Gcgr) signaling will have

detrimental effect in the cardiovascular system.

The initial studies with the Gcgr-/- mice revealed improved glucose homeostasis, delayed

gastric emptying, and improved β-cell function in addition to elevated GLP-1 levels in the Gcgr-

/- mice. However, initial studies did not thoroughly explore the mechanism(s) through which

deletion of the Gcgr led to improved glucose homeostasis, delayed gastric emptying, and

improved β-cell function, and whether elevated GLP-1 played a role in the improved metabolic

phenotype in the Gcgr-/- mice. In this thesis, I have addressed the role of the elevated GLP-1

levels in the improved metabolic phenotype of the Gcgr-/- mice by generation and

characterization of the Gcgr-/-:Glp1r-/- mice in Chapter 2. In this thesis, I have identified for

the first time that β-cells can sense signal from the gut and can compensate for the lack of

classical incretins. I made the observations that the non-classical incretin receptors could

compensate in the absence of classical incretin signaling as deletion of the classical incretin

receptors led to compensation from the non-classical incretins including GPR119 and CCKA

receptors.

The number one cause of death in diabetes is heart disease. Previous studies have

explored the pharmacological role of glucagon in cardiac ischemia. Some studies have reported

beneficial, while others have shown worsening outcomes from cardiac ischemia following

glucagon administration. In Chapter 3, I addressed the physiological role of Gcgr signaling in

cardiac ischemia. I aimed to delineate the physiological role of cardiac-specific Gcgr signaling in

myocardial ischemia by generating cardiac-specific Gcgr knockout mice. I identified for the first

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time that the physiological Gcgr signaling regulates cardiac fatty acylcarnitine levels and could

influence susceptibility to cardiac ischemia, and this might be dependent on p38 MAPK and

PPARα pathway. Together, these studies demonstrate for the first time the importance of

endogenous Gcgr signaling in cardiac ischemia.

How does the Glp1r contribute to the improved ipGTT, gastric emptying, and

reduced fasting glycaemia in Gcgr-/- mice? Is Glp1r signaling solely responsible for

these phenotypes in the Gcgr-/- mice?

Deletion of the Glp1r in the Gcgr-/- mice led to reversal of improved pit, reduced fasting

glycaemia, and delayed gastric emptying to levels observed in wild-type mice. Pharmacological

activation of the Glp1r using Glp1r agonists or native GLP-1 have universally been shown to

improve glucose tolerance, increase insulin secretion, reduce appetite, and delay gastric

emptying in rodents, pigs, and humans [184, 308, 383]. Consistent with the evidence that GLP-1

plays an important role in improving glucose homeostasis and regulation of fasting glucose

levels; Glp1r-/- mice exhibit impaired glucose tolerance and fasting hyperglycemia [384]. GLP-

1-mediated improvements in glucose tolerance are largely mediated through Glp1r signaling on

β-cells and mainly through cAMP activation. Furthermore, overexpression of Glp1r in a clonal

β-cell line leads to increased resting cAMP levels [385]. One possible mechanism through

which deletion of Glp1r could lead to increases in fasting glycaemia and impaired glucose

tolerance during an intraperitoneal glucose challenge could be due to defects in β-cell plasma

membrane depolarization. This is supported by studies with DIRKO mice islets that show 50%

reduction in the amplitude of insulin secretion as a result of defects in plasma membrane

depolarization in β-cells from DIRKO mice. I observed reduced insulin secretory responses to

tolbutamide and l-arginine in islets from the Gcgr-/- and Gcgr-/-:Glp1r-/- mice; suggesting

defect in membrane depolarization in islets from Gcgr-/- and Gcgr-/-:Glp1r-/- mice [386].

Additionally, GLP-1 mediated regulation of fasting glucose could also be through control of

glucagon and regulation of liver glucose production. Although no Glp1r expression has been

detected in the liver, it is possible that central Glp1r activation shuts down liver glucose

production during fasting [225]. Elevated GLP-1 levels in the Gcgr-/- mice could be inhibiting

gastric emptying via the vagal nerves. Consistent with our findings, previous studies have shown

that denervation/treatment with Ex-9-39 abolished glucose-induced inhibition of gastric

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emptying [387]. These studies concluded that endogenous GLP-1 released upon food ingestion

inhibits gastric emptying via the vagal nerves. Hence, elevated GLP-1 levels are responsible for

majority of the overall improved metabolic control observed in the Gcgr-/- mice.

Deletion of Glp1r in Gcgr-/- mice did not completely reverse the reduced fasting

glycaemia and improved ipGTT to levels observed in Glp1r-/- mice. Glp1r signaling may be

contributing to most of the improved ipGTT and reduced fasting glycaemia; however, loss of

Gcgr signaling may indirectly or directly affect the improved ipGTT and reduced fasting

glycaemia in addition to the elevated GLP-1 levels. Gcgr signaling in the liver has been shown

to be important for basal liver glucose production and gluconeogenesis [388]. Therefore, loss of

Gcgr signaling in the liver may be reducing the basal glucose production or gluconeogenesis by

the liver. Furthermore, Gcgr-/- mice have been reported to also have high plasma bile acid levels

[49]. Bile acids have been shown to improve glucose tolerance and improve β-cell function.

Another possibility could be that in addition to the elevated GLP-1 levels, elevated bile acids

could also contribute to the improved ipGTT and reduced fasting glycaemia in the Gcgr-/- mice.

Future studies should explore the role of liver Gcgr signaling and bile acid in contribution to the

improved ipGTT and reduced fasting glycaemia in the Gcgr-/- mice. To test if liver Gcgr

signaling is involved in the improved ipGTT and reduced random fed glycaemia in the Gcgr-/-

mice one can restore the expression of Gcgr in the liver of Gcgr-/-:Glp-1r-/- mice through

adenoviral transfer of Gcgr gene under Albumin promoter. If this restores the improved ipGTT

and reduced fasting glycaemia in the Gcgr-/-:Glp1r-/- mice to levels in Glp1r-/- mice then this

would imply involvement of liver Gcgr signaling. To test if bile acids are involved in the

improved ipGTT and reduced fasting glycaemia in the Gcgr-/- mice Gcgr-/-:Glp-1r-/- mice

should be mated with the G protein-coupled bile acid receptor 1 and membrane-type bile acid

receptor (TGR5) knockout mice (TGR5-/-) to make a triple Gcgr-/-:Glp1r-/-:TGR5-/- mice.

TGR5 receptor is present in brown adipose tissue and intestine, where its agonism increases

energy expenditure and lowers blood glucose [389]. If the triple knockout mice have similar or

worse ipGTT and fasting glucose levels compared to Glp1r-/- mice then this will imply

involvement of bile acids in improving ipGTT and fasting glycaemia in the Gcgr-/- mice in

addition to the elevated GLP-1 levels.

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Do the elevated GLP-1 levels in the Gcgr-/- mice contribute to increased insulin

sensitivity?

In Chapter 2, I show that the Gcgr-/-:Glp1r-/- DKO mice continue to exhibit increased

insulin sensitivity similar to Gcgr-/- mice following an insulin challenge. Similarly, liver-specific

Gcgr-/- mice also exhibit increased insulin sensitivity comparable to the whole body Gcgr-/-

mice [49]. Therefore, the study by Longuet et al and my findings from Chapter 2 suggests that

the impact of glucagon on insulin sensitivity can be largely mediated by hepatic Gcgr signaling

and is independent of Glp1r signaling. In the whole body Gcgr-/- mice, hepatic phospho-cAMP

response element binding protein (P-CREB), a major component of glucagon’s signal

transduction pathway, and the mRNA of phosphoenol pyruvate carboxykinase (PEPCK), a

glucagon-responsive gluconeogenic target enzyme studies have shown to be downregulated

compared to wild-type littermate control mice [390]. Previously studies have shown that

knocking down CREB using antisense oligonucleotide in the liver prevents hepatic insulin

resistance in ZDF rats and ob/ob mice diabetic models. Therefore, it can be possible that deletion

of Gcgr in the liver leads to low expression of CREB, which is driving the increased insulin

sensitivity by reducing hepatic glucose production and storage [391]. Future studies need to

address how loss of liver Gcgr signaling regulates insulin sensitivity and investigate if CREB,

PEPCK, and/or other signaling pathways are involved.

What mechanisms regulate α-cell hyperplasia and the enlarged pancreas in Gcgr-/-

mice if not Glp1r signaling?

. In Chapter 2, I investigated the role of the Glp1r in α-cell hyperplasia and enlarged

pancreas observed in Gcgr-/- mice by assessing insulin staining and weight of the pancreas in the

Gcgr-/-:Glp1r-/- mice . The pancreas in the Gcgr-/-:Glp1r-/- mice continued to be enlarged and

the islet structure remained perturbed as observed in the Gcgr-/- mice [131]. This suggests that

Glp1r is not involved in the α-cell hyperplasia or in the increased pancreas weight seen in the

Gcgr-/- mice. Related studies have shown that liver-specific knockout of the gene encoding for

the Gsα protein which is a G-protein involved downstream of the Gcgr signaling, leads to

enlargement of the pancreas and α-cell hyperplasia as observed in the Gcgr-/-mice [314]. The

enlarged pancreas and α-cell hyperplasia phenotype in the Gsα-/- mice were independent of the

117

Glp1r as the Gsα-/-:Glp1r-/- double knockout mice continued to show an enlarged pancreas

despite deletion of the Glp1r. Overall, the findings indicate that there may be other factors,

independent of Glp1r signaling, playing a role in non β-cell hyperplasia and enlarged pancreas

weight seen in Gcgr-/- mice.

Since islets account for approximately 2% of total pancreatic weight, increase in islet

number and size cannot account for the overall increase in pancreas weight in the Gcgr-/-. It is

not clear whether the increased pancreas weight represents increased cell size and/or cell

numbers and/or water content in the exocrine pancreas. Future studies need to investigate

whether the increased pancreas weight is a result of increased cell size/number or water content

in the exocrine pancreas.

What are the mechanisms by which eliminating the Gcgr and Glp1r lead to

compensation from other incretin receptors?

In Chapter 2, we show that deletion of both Gcgr and Glp1r or knocking down Gcgr

expression using Gcgr antisense oligonucleotides in the DIRKO mice results in upregulation of

GPR119 and CCKA receptor expression in islets. Additionally, sensitivity to GPR119 and

CCKA receptors are also upregulated as demonstrated by increased plasma insulin and lower

glucose excursion following treatment with GPR119 and CCKA receptor agonists or ligands in

the Gcgr-/-:Glp1r-/- DKO mice. Previous studies have shown that in the absence of Glp1r, there

is compensation by GIP through both upregulation of plasma GIP levels and through increased

Gipr expression in islets [392]. Therefore, in activation of the classical incretin receptors Gipr or

Glp1r only caused a mild impairment in glucose tolerance [386]. In this thesis, I speculate that

signals to β-cells are sent from the gut to increase expression of other incretin receptors in the

absence of the classical incretin signaling. Studies have previously shown that specialized cells

from the intestine can detect changes in glucose concentrations and hormone levels in the

circulations and can send signals to peripheral tissues such as the pancreatic β-cells through the

vagus nerve [393]. Therefore, this could be one of the mechanisms through which in our studies

the gut is communicating to β-cell s to upregulate the expression of incretin receptors in the

absence of the classical incretin receptors. The exact molecular mechanisms of how the gut

regulates β-cell function have not been defined clearly and require further investigation [393].

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Additionally, it is not excluded that the changes in expression of CCKa and GPR119 receptors in

β-cells could be mediated through the brain-β-cell axis. The β-cells from nervous system

specific deletion of GLUT-2 have been shown to have lower insulin secretion and lower β-cell

mass due to lower β-cell proliferation. Furthermore, electrophysiology revealed lower

parasympathetic activity in the CNS specific GLUT-2 knockout mice. This is suggesting brain-β-

cell axis could be regulating the β-cell changes observed in CNS specific GLUT-2 knockout mice

[394]. Therefore, brain-β-cell axis interaction has been reported previously, and this could be

one of the mechanisms through which β-cell s in the Gcgr-/-:Glp1r-/- mice could be mediating

upregulation of CCKa and GPR119 receptors. Direct loss of Gcgr and Glp1r in β-cells could

also be mediating the upregulation of CCKa and GPR119 receptors. Therefore, future studies

need to explore what tissue or cell type is involved in mediating these signals to beta cells.

Although little is known about the control of Glp1r and Gipr gene transcription and

posttranslational modifications, studies suggest glucose, transcription factors, kinase signaling,

and posttranslational modification including N-glycosylation can impact Glp1r and Gipr

expression [395, 396]. PPAR-α has been shown to be involved in metformin-induced increase in

islet cell incretin receptor expression [395]. In the PPARα-/- mice, metformin failed to increase

islet incretin receptor transcripts including CCKar expression [395]. Previously PPARα has

been shown to be involved in Gipr expression [397]. Furthermore, direct treatment of BRIN-

D11 cells or rat islets with PPARα agonist WY14643 for 8-24 hours significantly increased the

expression of Gipr[398]. Therefore, one possible mechanism through which genetic deletion of

one or more of the incretin receptors can lead to compensation from the other incretin receptors

can be through upregulation of PPARα leading to increased expression of other non-classical or

classical incretin receptors. Studies on human islets isolated from T2D subjects revealed

decreased expression of GIPR and GLP1RmRNA transcripts and this was associated with

decreased protein levels of TCF7L2 (T cell factor 7 L 2) [399]. TCF7L2 is a transcription factor

involved in the WNT signaling pathway. Knock down of TCF7L2 in human islets was

associated with reduced levels of incretin receptors [399]. Therefore, another possible

mechanism through which deletion of one or more of the incretin receptors can lead to

compensation from other incretin receptors may be through upregulation of TCF7L2

transcription factor activity or expression. Transcription factors involved in pancreatic

119

development including islet 1 (isl-1) and its co-regulator, LIM-domain–binding coregulators

(Ldb1) have also been shown to regulate Glp1r expression. Pancreas-specific deletion of isl-1 or

Ldb1 led to reduced levels of Glp1r mRNA transcripts and Glp1r promoter binding was detected

using CHIP experiments using antisera against either isl-1 or Ldb1 [400]. Therefore, another

mechanism through which loss of one or more incretin receptors can lead to induction of the

expression of other incretin receptors may be a result of upregulation of isl-1 and/or Ldb1 gene

expression or activity. AMP kinase has also been shown to be involved in the regulation of

glucose-induced incretin receptor expression. Treatment with AMPK inhibitor and double

negative-AMPK expression led to significant increase in incretin receptor expression under low

glucose, and inhibition of AMPK had no effect on incretin receptor expression under

hyperglycemic conditions [401]. Therefore, this could be one of the mechanisms through which

loss of one or more incretin receptors on islet β-cells can lead to compensation from other

incretin receptors can be through reduction of AMPK activity or expression. Future experiments

should investigate expression at the protein and mRNA levels of PPAR-α, TCF7L2, isl-1, Ldb1,

and AMPK in islets from Gcgr-/-:Glp1r-/- DKO and DIRKO mice treated with Gcgr antisense

oligonucleotides or in Ins-1 cells following knock down of Gcgr and Glp1r. Posttranslational

modification of GPCRs can also influence the plasma membrane expression of GPCRs. Most of

the GPCRs including Gipr and Glp1r possess N-terminus Asn-linked (N)-glycosylation [402].

Studies in Chinese hamster ovary cells and Ins-1 cells have shown that N-glycosylation can

enhance cell surface expression and function of Glp1r and Gipr [402]. N-glycosylation

lengthens receptor half-life by reducing degradation in the endoplasmic reticulum. Studies have

also shown that expression of Gipr mutant lacking N-glycosylation can be rescued by co-

expressing wild-type Glp1r [402]. Therefore, genetic deletion of the classical incretin receptors

can lead to compensation from other incretin receptors can be through modification on N-

glycosylation which may increase the half-life of the receptors on cell surface and/or increase

receptor function as well. Future studies should investigate whether N-glycosylation of Glp1r,

Gipr, CCKar, and GPR119 controls cell surface availability and receptor expression.

120

Are the elevated GLP-1 levels in the Gcgr-/- mice responsible for the improved

outcome from ischemia reperfusion?

In Chapter 3, I observed improved outcomes from ischemia reperfusion injury in hearts

from Gcgr-/- mice ex-vivo. Improved outcome from myocardial infarction can be a result of

elevated plasma GLP-1 levels in the Gcgr-/- mice. Glp1r activation in rodents, pigs, and human

studies has been shown to be cardioprotective in ex-vivo and in vivo [232, 233, 238, 258, 259].

However, we have prevented the influence of GLP-1 and other secreted factors in ischemia

reperfusion experiments by assessing outcomes in isolated hearts from Gcgr-/- mice in the

absence of elevated GLP-1 levels. However, it is not excluded that the hearts from Gcgr-/- mice

may have better outcomes as the hearts had chronic exposure to elevated GLP-1 levels during

development. To date none of the studies have assessed the outcome from ischemia ex-vivo or in

vivo following chronic exposure to GLP-1 during development. Nonetheless, in our gain of

function glucagon treated hearts in ex-vivo, glucagon worsened outcomes from myocardial

infarction suggesting direct activation of Gcgr signaling may not be ideal for the outcome from

ischemia reperfusion. Similarly, in the Gcgr+/- mice and cardiac specific Gcgr-/- mice which

lack elevated GLP-1 levels and have similar glucose tolerance to wild-type control, we found

improved outcomes from myocardial infarction suggesting the effects of Gcgr signaling in the

outcome from myocardial infarction are cardiac-specific and may not be influenced by the

elevated GLP-1 levels.

Does loss or enhanced Gcgr signaling control fat and/or glucose oxidation in the

heart?

In Chapter 3, we show that enhancing cardiac Gcgr signaling impairs outcome from

myocardial infarction and suppressing cardiac Gcgr signaling improves outcomes from

myocardial infarction. We speculate the differences in survival can be a result of differences in

fatty acid oxidation as suggested by changes in acylcarnitine profiles; however, our studies

require further investigation as measurement of acylcarnitine levels is not a direct assessment of

fatty acid flux in the heart. Accordingly, differences in acylcarnitine levels may not always

indicate changes in fatty acid flux. For instance if long chain acylcarnitines are increased and

medium chain acylcarnitines are decreased there may not be an overall change in fatty acid flux

121

but this may indicate there is a block in medium chain acylcarnitine generation [343]. Therefore,

direct assessment of fatty acid oxidation rates is required to make solid conclusions about fatty

acid flux rates. There are various methods for measuring fatty acid oxidation, and these include

both direct and indirect methods [403]. The most commonly used method in the heart is to

assess whole flux measurement. Whole flux measurement involves measuring the byproducts of

fatty acid oxidation. One of the byproducts of fatty acid oxidation is CO2, one can perfuse 14

C

radiolabelled fatty acids in the heart in an ex-vivo working heart model followed by measurement

of byproducts which will be radiolabelled using 14

C to quantify the rate of fatty acid oxidation

[403].

In Chapter 3, we also claim that the GcgrCM-/-

mice have increased glucose oxidation as seen

by a trend towards a reduction in PDH phosphorylation, and we also observed glucagon

increased PDH phosphorylation by PDK-4 in HL-1 cells and in hearts in vivo. This suggests that

glucagon can block glucose oxidation, and deletion of Gcgr in the heart can increase glucose

oxidation. However, we did not assess directly the glucose oxidation rates in hearts from

GcgrCM-/-

mice, nor in our gain of function studies did we assess glucose oxidation rates

following glucagon treatment in vivo. Therefore, our future studies need to explore the direct

rate of glucose oxidation in the heart. Glucose oxidation can be measured by perfusing

radiolabelled glucose into the hearts ex-vivo followed by measurement of by-products of glucose

oxidation, which is CO2 [403].

Is Gcgr signaling mediated changes in outcome from myocardial infarction

dependent on the rate of glucose and/or fat oxidation?

Our studies suggest that augmentation of Gcgr signaling leads to an increase and

inhibition of Gcgr signaling results in reduction of cardiac fatty acid oxidation, and this may be a

mechanism through which Gcgr signaling regulates susceptibility to myocardial infarction.

However, it is not clear if the reduced or increased cardiac fatty acid oxidation is directly

involved in protecting GcgrCM-/-

mice hearts from myocardial infarction. To address this, we

have treated GcgrCM-/-

mice with a PPAR-α agonist, fenofibfrate. Fenofibrate has been shown to

increase fatty acid oxidation in the heart following oral administration in mice. [404]. In our

study, we gavaged fenofibrate twice a day, from 24 hours prior to LAD ligations until 7 days

122

following LAD ligations and found treatment with fenofibrate failed to worsen outcomes from

myocardial infarction in the GcgrCM-/-

mice (data not shown). This could be due to fenofibrate’s

effect in the liver. Fenofibrate has been shown to increase fatty acid oxidation in the liver leading

to reduction of plasma lipids, resulting in reduced myocardial fatty acid supply and protection

from myocardial infarction. Studies have shown previously that administration of fenofibrate in

mice leads to protection from myocardial infarction, and this was associated with reduced fat

oxidation in the hear[404-406]. Therefore, administration of fenofibrate in vivo to the whole

animal may not be ideal as it may be increasing fatty acid oxidation in the liver of GcgrCM-/-

mice

and may cause further protection from myocardial infarction. Currently, there are no fatty acid

oxidation agonists that can be targeted only to the heart without impacting fatty acid oxidation

elsewhere. One way of eliminating the liver effect of increasing fatty acid oxidation is to isolate

the hearts in ex-vivo and perfuse fenofibrate into the heart. Our future experiments should

explore working heart models of ischemia reperfusion from GcgrCM-/-

mice ex-vivo with and

without perfusion of fenofibrate. Perfusion of fenofibrate in the GcgrCM-/-

hearts ex vivo should

result in an increase in fatty acid oxidation, allowing us to investigate if we can reverse the

improvements in survival from myocardial infarction. Similarly, in our gain of function studies,

glucagon should be perfused in the working heart model ex-vivo with or without fatty acid

oxidation inhibitors including trimetazidine or ranolazine or glucose oxidation activators using

dicholoroacetate [407]. If glucagon fails to worsen outcome from ischemia reperfusion ex-vivo in

the presence of fatty acid oxidation inhibitors or glucose oxidation activators, then this will

imply glucagon-mediated worsening of outcomes from myocardial infarction is dependent on fat

and/or glucose oxidation.

Does Gcgr signaling regulate susceptibility to myocardial infarction through p38

MAPK and/or PPAR-αpathway?

In Chapter 3, our data suggests that glucagon increases susceptibility to myocardial

infarction in a p38 MAPK-dependent manner and upregulated phosphorylation of p38 MAPK

expression in vivo. However, we did not assess the expression of p38 MAPK in our loss of

function model. Therefore, future studies need to look at expression of p38 MAPK in GcgrCM-/-

hearts before and after experimental myocardial infarction. Furthermore, we show upregulation

of PPAR-α regulated gene expression and increase PPAR-α translocation to the nucleus in HL-1

123

cell lines following glucagon treatment. However, our studies require further investigation; it is

not clear if glucagon’s detrimental effect on myocardial infarction is dependent on PPAR-α

activity in vivo. Future studies should investigate if glucagon treatment worsens outcomes from

myocardial infarction in the whole body or cardiac specific PPAR-α knockout mice. If glucagon

fails to impair outcomes from myocardial infarction in the PPAR-α knockout mice, this would

imply PPAR-α is involved in glucagon-mediated impairment of outcomes from myocardial

infarction. Furthermore, our studies have shown that PPAR-α translocation to the nucleus by

glucagon is dependent on p38 MAPK pathway and is independent of PKA pathway. Future

studies need to explore whether glucagon activates PPAR-α exclusively through p38 MAPK or if

it involves other kinases including PKC, PI3K, ERK, GSK3, or other signaling proteins which

have been shown previously to activate PPAR-α [408-410]. Additionally, future studies also

need to investigate if in addition to PPAR-α, there are other p38 MAPK mediated pathways

activated by glucagon including ATF2 and PGC-1α, which have been previously shown to be

activated by p38 MAPK [411].

Does Gcgr signaling in the heart have direct effects on myocardial infarction or are

the effects indirect?

In Chapter 3, my studies suggest a direct role of Gcgr signaling in the heart in modulating

susceptibility to myocardial infarction. I showed that perfusion of glucagon in hearts ex-vivo

caused protection from ischemia reperfusion. Similarly hearts from the whole body Gcgr-/- mice

show protection during ischemia reperfusion in ex-vivo experiments. Furthermore, treatment with

glucagon in mice led to increased susceptibility to myocardial infarction and these effects of

glucagon were dependent on cardiac Gcgr signaling. Conversely deletion of Gcgr signaling in

the heart led to protection from myocardial infarction. Altogether, both the in vivo and ex vivo

gain of function and loss of function studies suggest the effects of Gcgr signaling on myocardial

infarction are direct. However, future studies need to assess whether treatment with glucagon in

primary cardiomyocyte in culture leads to increased apoptosis and if so, does glucagon utilize

p38 MAPK and PPAR-α pathways to mediate pro-apoptotic pathways in primary

cardiomyocytes similar to the mechanisms we observed in the GcgrCM-/-

mice and HL-1 cell line.

Interestingly, none of the studies in primary cardiomyocyte in culture so far has looked at the

role of Gcgr signaling in primary cardiomyocytes.

124

Are there differences in physiological versus pharmacological activation of Gcgr

signaling in the heart and susceptibility to myocardial infarction?

In Chapter 3, for the first time I have investigated the role of the endogenous Gcgr

signaling in myocardial infarction. In loss of function studies with the GcgrCM-/-

mice, we

observed protection from myocardial infarction suggesting endogenous Gcgr signaling plays a

role in regulation of myocardial infarction. This also suggested that activation of Gcgr signaling

in the heart might not be beneficial for the outcomes from myocardial infarction. We utilized

pharmacological doses of glucagon for ischemia reperfusion and for our in vivo gain of function

studies (1ug/mL perfusion and 30ug/kg respectively). However, in our in vivo studies, we picked

a pharmacological dose that did not cause chronic hyperglycemia and/or reduced food intake

and/or body weight; as changes in these parameters can impact outcomes from myocardial

infarction. My studies in Chapter 3 and the majority of the studies conducted with glucagon

previously have looked at the cardiovascular outcomes following a pharmacological dose of

glucagon. None of the studies have looked at the outcome from myocardial infarction following

chronic activation of Gcgr signaling using physiological glucagon levels. Therefore, future

studies should investigate the role of physiological activation of Gcgr signaling in myocardial

infarction by administration of physiological dose of glucagon in the heart in ex-vivo and/or in

vivo and look at acylcarnitine levels in the heart and susceptibility to myocardial infarction.

Does glucagon protect the heart from myocardial infarction during diabetes or high

fat diet-induced obesity?

In Chapter 3, my studies illustrate that the levels of long and medium chain acylcarnitine

in diet-induced obese GcgrCM-/-

mice are lower than that observed in littermate control mice. This

is suggesting that the GcgrCM-/-

mice hearts have lower fatty acid flux even following diet

induced obesity. This implies under diet induced obesity, deletion of Gcgr signaling in the heart

may have beneficial outcomes from myocardial infarction by reduction in fatty acid flux.

Interestingly, recent studies in mice, rats, and humans have shown that fatty acid oxidation rates

are increased in obesity and insulin resistance. High levels of fatty acid oxidation blocks glucose

oxidation pathways leading to decrease in insulin-stimulated glucose oxidation and resulting in

125

insulin resistance in the heart and worsening of outcomes from myocardial infarction [344, 345,

362, 412-415]. Additionally, in our gain of function studies we observed an increase in

acylcarnitine levels with glucagon treatment following ischemia. This is suggesting that

glucagon may increase fatty acid flux in the heart and reduce glucose oxidation leading to

increase in cardiac insulin resistance and worsening outcome from myocardial infarction.

Therefore, my findings in Chapter 3 prompts future studies to explore the role of

hyperglucagonemia in T2D or obesity induced cardiovascular complications.

Hyperglucagonemia in obesity and T2D can be driving the increase in fatty acid oxidation and

blockage in glucose oxidation in the heart.

Potential therapeutic impact:

Recent data from mice studies suggests that repeated administration of a dual

glucagon/Glp-1 receptor agonist causes more weight loss, reduction in food intake, fat mass, and

induction of energy expenditure and improvement in plasma lipids relative to Glp1r agonism

alone in high fat diet fed mice [281, 282]. Dual agonism of GLP-1 and glucagon receptors does

not cause hyperglycemia despite glucagon action to promote glucose production by the liver in

part due to GLP-1’s anti-hyperglycemic actions, which counteract glucagon-mediated increase in

blood glucose levels. The lipolytic and thermogenic properties of glucagon in addition to the

satiation-inducing properties of GLP-1 make Gcgr-Glp1r co-agonist an ideal therapeutic for the

treatment of diabetes [282]. However, my studies reveal potential limitations to developing

Gcgr agonists for the treatment of T2D. Activation of Gcgr signaling in the heart may not be

ideal for cardiovascular outcomes as glucagon reduces survival following myocardial infarction

possibly through increasing fatty acid oxidation and decreasing glucose oxidation in the heart.

Conversely, partial reduction or cardiac specific inhibition of Gcgr signaling seems to improve

outcomes during myocardial infarction as Gcgr+/- and GcgrCM-/-

mice are protected from

myocardial infarction. Therefore, my studies support development of partial Gcgr antagonists

for the treatment of myocardial infarction. My findings also encourage future investigation

looking at the role of hyperglucagonemia in promoting cardiovascular

abnormalities/complications in T2D, and whether chronic hyperglucagonemia may be driving

the increased fatty acid oxidation in diabetic hearts.

126

Overall Summary:

My thesis reveals an important role of Gcgr signaling in regulating β-cell nutrient

sensing and cardiomyocyte fuel metabolism. My findings suggests careful assessment of

therapies that target modulation of Gcgr signaling for the treatment of diabetes as Gcgr

signaling seems to be important for cardiovascular outcomes in myocardial infarction.

127

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