Cdkn2a/p16Ink4a Regulates Fasting-Induced Hepatic Gluconeogenesis Through the PKA-CREB-PGC1 ...

1

Cdkn2a/p16Ink4a

regulates fasting-induced hepatic gluconeogenesis through the PKA-

CREB-PGC1αααα pathway.

Kadiombo Bantubungi1,2,3,4*

, Sarah Hannou1,2,3,4*

, Sandrine Caron-Houde1,2,3,4

, Emmanuelle

Vallez1,2,3,4

, Morgane Baron1,2,3,4

, Anthony Lucas1,2,3,4

, Emmanuel Bouchaert1,2,3,4

, Réjane

Paumelle1,2,3,4+

, Anne Tailleux1,2,3,4+

, Bart Staels1,2,3,4+

.

* contributed equally

+ Senior authors

1 Univ Lille 2, F-59000, Lille, France

2 Inserm, U1011, F-59000, Lille, France

3 European Genomic Institut for Diabetes (EGID), FR 3508, F-59000 Lille, France

4 Institut Pasteur de Lille, F-59019, Lille, France

Running title: p16Ink4a

controls fasting-induced gluconeogenesis

Keywords: CDKI, liver, glucose homeostasis, PKA

Word count: 3509

Number of figures: 8

Number of Supplemental figures: 3

Number of supplementary table: 1

Correspondence to: Bart Staels. Inserm UMR 1011, Institut Pasteur de Lille, 1 rue du

Professeur Calmette, BP 245, Lille 59019, France, Tel: +33 3 20 87 73 88, Fax: +33 3 20 87

73 60, E-mail: bart.staels@pasteur-lille.

The authors declare no conflict of interest

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Abstract

Type 2 diabetes (T2D) is hallmarked by insulin resistance, impaired insulin secretion and

increased hepatic glucose production. The worldwide increasing prevalence of T2D calls for

efforts to understand its pathogenesis in order to improve disease prevention and

management. Recent genome wide association studies (GWAS) have revealed strong

associations between the CDKN2A/B locus and T2D risk. The CDKN2A/B locus contains

genes encoding cell cycle inhibitors, including p16Ink4a

, which have not yet been implicated in

the control of hepatic glucose homeostasis. Here we show that p16Ink4a

-deficiency enhances

fasting-induced hepatic glucose production in vivo by increasing the expression of key

gluconeogenic genes. p16Ink4a

down-regulation leads to an activation of PKA-CREB-PGC1α

signalling through increased phosphorylation of PKA regulatory subunits (PKAR2). Taken

together, these results provide evidence that p16Ink4a

controls fasting glucose homeostasis and

could as such modulate in T2D development.

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Introduction

Type 2 diabetes (T2D) is a complex metabolic disorder involving a combination of insulin

resistance, impaired insulin secretion and increased hepatic glucose production (1,2). The

pathogenesis of T2D is multifactorial, involving both genetic and environmental susceptibility

factors (3). These last few years, the search for genetic determinants of T2D greatly

progressed, identifying new loci contributing to T2D. A better understanding of the function

of the gene products of these loci is required to identify new strategies for the prevention and

treatment of T2D (3,4). Hence, recent human genome-wide association studies (GWAS) have

identified a polymorphism on chromosome 9p21 (rs 10811661), located ≈125kb upstream of

the CDKN2B and CDKN2A genes, which is strongly and reproducibly linked to T2D (5–7),

establishing genes on the CDKN2A/B locus among the strongest candidates conferring

susceptibility to T2D across different ethnicities (4).

The gene products are the cyclin-dependent kinase (CDK) inhibitors p16Ink4a

and p14ARF

for

the CDKN2A locus and p15Ink4b

for the CDKN2B locus, which are tumor suppressors acting

as cell cycle inhibitors (8,9). The p15Ink4b

and p16Ink4a

proteins bind to either CDK4 or CDK6,

thus inhibiting the action of cyclin D and preventing retinoblastoma protein phosphorylation

and subsequent release of the E2F1 transcription factor. As a consequence, the transcription of

genes required for cell cycle progression to the S phase is restrained.

However, how the CDKN2A/B gene products modulate glucose metabolism is less clear. In

murine models, increased expression of p15Ink4b

in pancreatic islets is associated with islet

hypoplasia and impaired glucose-induced insulin secretion (10). Moreover, p16Ink4a

plays a

crucial role in senescence and aging. p16Ink4a

expression increases with age in pancreatic ß

cells and promotes an age-dependent decline in islet regenerative potential (11). Additionally,

other cell cycle regulators, like CDK4, E2F1 and cyclin D, also play roles in glucose

homeostasis through actions in the pancreas, muscle and/or adipose tissue (12–16). However,

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whether the CDKN2A/B gene products modulate hepatic glucose production is unknown

(17).

Glucose homeostasis is determined by the balance of its production and utilization. Impaired

postprandial glucose control as well as the persistence of fasting hyperglycaemia are

hallmarks of T2D (18). Increased rates of hepatic glucose production are a major cause of

fasting hyperglycaemia in T2D patients (1). In physiological conditions, during prolonged

fasting, hepatic gluconeogenesis is a major pathway to maintain normal plasma glucose levels

(19) due to the action of different hormones, amongst which are glucagon and

glucocorticoids, like cortisol. During starvation, low blood glucose levels induce pancreatic α-

cell glucagon secretion and hypothalamic-pituitary-adrenal (HPA) axis activation. In the liver,

glucagon binds to its receptor, which then causes a GDP/GTP exchange, hence stimulating

adenylate-cyclase activity which converts ATP in cAMP (20). The rise in intracellular cAMP

levels stimulates the dissociation of the catalytic and regulatory subunits of protein kinase A

(PKA) (21). The catalytic PKA subunit then enters the nucleus where it phosphorylates the

cAMP-responsive binding protein (CREB) at Ser 133 converting it into its transcriptionally

active form which induces gluconeogenic gene expression (22–24). In concert,

glucocorticoids activate the glucocorticoid receptor which binds to glucocorticoid-responsive-

elements (GREs) in the promoters of gluconeogenic genes (25,26).

Given the strong association of the CDKN2A/B locus with T2D risk, which in large

population studies is mainly established by the measurement of fasting hyperglycemia (5), we

set out to study whether p16Ink4a

plays a role in hepatic glucose homeostasis using p16Ink4a

-

deficient mice (p16-/-

), mouse primary hepatocytes and mouse hepatic cell line. Our results

identify p16Ink4a

as a modulator of the PKA-CREB-PGC1α signalling pathway and hence as a

regulator of fasting hepatic glucose homeostasis, independent of its function as cell cycle

regulator.

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Research Design & Methods

Animal experiments. p16Ink4a

-deficient (p16-/-

) and littermate control (p16+/+

) mice on a

C57Bl6 background (>97%) were housed under standard conditions in conventional cages

with free access to water and food unless indicated otherwise. 12-week-old male mice were

sacrificed by cervical dislocation at 9am after a 24h fasting. Experimental procedures were

conducted with the approval of the Ethics committee for animal experimentation of the Nord

Pas de Calais Region (CEEAA022008R).

Pyruvate test. Overnight fasted mice (5pm-9am) were injected intraperitoneally with sodium

pyruvate (P4562, Sigma) (2 g/kg body weight). Blood glucose levels were measured from the

tail vein at the indicated time points using an automatic glucose monitor (One Touch,

Lifescan).

Mouse primary hepatocyte isolation, culture and treatments. Mice were anesthetized with a

mixture of ketamine (100mg/kg), xylasine (20mg/kg) administered intraperitoneally. Livers

were perfused in situ through the inferior cava vein, with Hank Buffer Sodium Salt HBSS

(H9394, Sigma) containing 0.5mM EGTA and 50mM Hepes followed by HBSS containing

0.025% collagenase (C5138, Sigma) until loss of its firm texture. The soft liver was removed

and cut into pieces, the homogenate filtered and centrifuged for 2min. The pellet was washed

3x and resuspended in Williams medium supplemented with 0.1% bovine serum albumin

(BSA), 1% glutamine, 1% gentamycine, 100nM insulin, and 100nM dexamethasone. Cell

number and viability were assessed using trypan blue. Cells were plated on 6 well plates

during 2h for hepatocyte selection and then incubated in deprivation medium (DMEM, 1%

penicilline-streptomycine, 1% glutamine, with distinct concentrations of glucagon (0, 1, 10,

100 nM) during 6-8h (for RNA measurements) or 30 min (for protein analysis).

Mouse hepatocyte cell line culture and treatments. AML12 (Alpha Mouse Liver 12/ATCC-

CRL2254) cells were cultured in DMEM-Ham's F-12 supplemented with 10% fetal bovine

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serum (Invitrogen), 5g/ml insulin (Sigma), 5g/ml transferrin (Sigma), 5ng/ml selenium

(Sigma), 1% glutamine, 1% penicilline-streptomycine, and maintained at 37°C under 5%

CO2. AML12 cells were transfected with Small Interference RNA (siRNA) for CDKN2A

(043107-00-005, ThermoScientific; ON-TARGET plus SMART pool siRNA) or CDK4 (ON-

L-040106-00-0005, ThermoScientific, ON-TARGET plus SMARTpool siRNA) or Control

(D-001810-10-20, ThermoScientific, ON-TARGET plus non targeting pool siRNA) using the

Dharmafect1 reagent (Thermo Scientific) according to the manufacturer’s instructions.

AML12 cells were treated for the indicated times points with 10µM of forskolin.

Glucose production assay. Primary hepatocytes were cultured in six-well plates in Williams

medium with 0.1% BSA, 100nM dexamethasone, 1% penicilline-streptomycine and 1%

glutamine. After 2h, the medium was replaced with 1ml of glucose production buffer

consisting of glucose–free Krebs-ringer buffer (115mM NaCl, 5.9mM KCI, 1.2mM MgCl2,

1.2mM NaH2PO4, and 2.5mM NaHCO3 pH7.4) without phenol red, supplemented with

15mM sodium lactate and 1mM sodium pyruvate. Glucose concentrations were measured at

different time points with a colorimetric glucose assay kit (Sigma). The values were then

normalized to total protein content determined on whole-cell lysates.

Gene expression analysis. Liver total RNA was isolated using the guanidinium

isothiocyanate (GSCN) phenol/chloroform extraction method and total RNA from cultured

cells was extracted using the TRIzol reagent (Eurobio). One microgram of total RNA was

reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kits

(Applied Biosystems) according to the manufacturer's instructions. Reverse transcribed

cDNAs were quantified by Brilliant III Ultra-Fast SYBR green-based real-time PCR using

specific oligonucleotides (Table S1) on a Stratagene Mx3005P (AgilentTechnologies)

apparatus. mRNA levels were normalized to cyclophilinA expression as internal control and

mRNA fold induction was calculated using the comparative Ct ( 2-∆∆Ct

) method.

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Western blot analysis. AML12 cells and mouse primary hepatocytes were lysed with cell

lysis buffer (50mM Tris-HCl, pH 8, 137mM NaCl, 5mM Na2EDTA, 2mM EGTA, 1%

Triton, 20mM sodium pyrophosphate, 10mM beta-glycerophosphate, 1mM Na3VO4, 10µM

leupeptin, 5µM PepestatinA) (Sigma-Aldrich) on ice. Cells were scraped and transferred to

1.5ml Eppendorf tubes and rotated for 30min at 4°C, followed by centrifugation at 13000g for

10min at 4°C. The resulting supernatants were stored in aliquots at -80°C until required.

Protein concentration in the cell lysates was determined using BCA protein assay kit (Pierce).

The cell lysates were mixed with 4X-SDS sample buffer NOVEX (Life technologies).

Samples were heated at 100°C for 10min before loading and being separated on precasted 4–

12% or 3-8% SDS-polyacrylamide gels (Invitrogen). Proteins were electrotransfered to a

nitrocellulose membrane (Millipore, Bedford) in 1X transfer buffer (Invitrogen) using the

Nupage Systeme for 1h at 30V. Non specific binding to the membrane was blocked for 1h at

room temperature with 5% non fat milk in TTBS buffer (20mM Tris, 500mM sodium NaCl,

and 0.1% Tween 20). Membranes were then incubated O/N at 4°C with various primary

antibodies in blocking buffer containing 5% non fat milk at the dilution specified by the

manufacturers. The following primary antibodies were used: phospho-CREB (Ser133) (9198,

Cell Signaling Technology), CREB (9197, Cell Signaling Technology), phospho-(S/T)-PKA

substrates (9621, Cell Signaling Technology), phospho-pRb (3590, Cell Signaling

Technology), pRb (9313, Cell Signaling Technology), PGC1α (sc-13067, Santa Cruz

Biotechnology), GAPDH (sc-25778, Santa Cruz Biotechnology) p16ink4a

(sc-1207, Santa Cruz

Biotechnology), phospho-PKAR2 (ab32390, Abcam), PKAR2 (ab-38949, Abcam).

Membranes were then incubated with the secondary antibody conjugated with the enzyme

horseradish peroxidase. The visualization of immunoreactive bands was performed using the

ECL plus Western blotting detection system (GE Healthcare). Quantification of phospho-

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CREB level in mouse primary hepatocytes and AML12 cells was performed by volume

densitometry using the Image J 1.47t (NIH) software.

Cyclic AMP and PKA assay. Intracellular cAMP concentrations were measured using a ready

to use competitive enzyme immunoassay kit (R&D Systems). Briefly, cells were lysed

according to the manufacturer protocol and 100µl of sample were mixed with 50µl of cAMP

conjugated and then added to cAMP-specific antibody pre-coated microplate. After 2h of

incubation at room temperature, substrate solution was added for 20min. Colour development

was stopped and the absorbance at 450nm was measured using Dynex MRX TC Revelation

Microplate Reader. PKA activity was measured by the signaTECT® cAMP-Dependent

Protein Kinase (PKA) assay System by using the Kemptide (LRRASLG) as a peptide

substrate.

Co-immunoprecipitation assay. Co-immunoprecipitation of CDK4 from whole AML12 cell

extracts was performed using the Thermo ScientificTM

Pierce TM

Crosslink Magnetic IP/Co-IP

Kit. Briefly, 48h after siRNA transfection, cells were lysed and 500µg of total protein extract

incubated with 3µg of the CDK4 antibody (Santa Cruz Biotechnology sc-260) according to

the manufacturer's protocol. The eluate was then subjected to western blot analysis using

PKAR2 (ab-38949, Abcam) and CDK4 (Santa Cruz Biotechnology sc-260).

Immunofluorescence assay in AML12 cells. Cells were grown on coverslips. 48h after

siRNA transfection, cells were washed with PBS and fixed with 4% paraformaldehyde during

20 min. After fixation and permeabilization with 0.1% TRITON, cells were incubated

over/night with antibodies against p16ink4a

(Santa Cruz Biotechnology M-156, sc-1207

California) and phospho-PKAR2 (Ab-32390, Abcam) and subsequently incubated with a

combination of Texas red-conjugated anti-rabbit IgG and FITC-conjugated anti-mouse IgG. A

Nuclear DAPI Counterstain was also performed.

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Statistics. Data are expressed as means +/- SEM. Results were analyzed by unpaired 2-tailed

Student's t-test or 1-way ANOVA with LSD Fisher post-hoc test or 2-way ANOVA with LSD

Fisher post-hoc test as appropriate using GraphPad Prism software. A p value of < 0.05 was

considered statistically significant.

Results

p16Ink4a

-deficiency results in fasting hyperglycemia and increased gluconeogenesis.

Since GWAS revealed an association between the CDKN2A/B locus and T2D risk,

primarily based on the fasting plasma glucose trait, we first measured fed and fasted blood

glucose levels in 12-week-old mice. p16-/-

mice displayed a less pronounced hypoglycemia

after 24h of fasting compared to p16+/+

mice (Figure 1A). This effect was not due to

differences in plasma glucagon levels between fasted p16+/+

and p16-/-

mice (Supplemental

Figure 1). To evaluate whether gluconeogenesis was influenced, a pyruvate tolerance test

(PTT) was performed in fasted p16-/-

and p16+/+

mice. Interestingly, p16-/-

mice produced

higher blood glucose levels, upon pyruvate administration, suggesting an increased hepatic

glucose production (Figure 1B-C). Consistent with this, hepatic mRNA levels of

gluconeogenic genes, such as G6pase and Pepck, were significantly higher in livers of fasted

p16-/-

versus p16+/+

mice (Figure 1D, F), while Fbp1 mRNA was not different between the

genotypes (Figure 1E). Conversely, genes involved in other metabolic pathways regulated

during fasting, such as glycolysis (Gk, Lpk) and beta-oxidation (Cpt1a, Lcad), were not

differently expressed between both genotypes upon fasting (Figure 1G-H, J-K), although

mRNA levels of Pdk4, which blocks glycolysis at the level of PDH, tended to be higher in

fasted p16-/-

livers (Figure 1I). Altogether, these data indicate that among the different hepatic

metabolic pathways regulated by fasting, gluconeogenesis is the only one modulated in p16-/-

mice.

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Since p16Ink4a

is a tumor suppressor and a cell cycle regulator, and since hepatic

proliferation and tumor growth may perturb glucose homeostasis, we investigated whether

p16Ink4a

-deficiency is associated with spontaneous liver tumor growth or altered hepatocyte

proliferation in our experimental conditions. At the age of 12 weeks, p16-/-

mice did not

display macroscopic liver abnormalities nor differences in liver weight compared to p16+/+

mice (Supplemental Figure 2A-B). Moreover, immunohistochemical Ki-67 staining of liver

sections showed no differences between p16-/-

mice and their littermate controls under fasting

conditions (Supplemental Figure 2C-H), indicating that hepatocyte proliferation is not

different. These data indicate that p16Ink4a

-deficiency increases fasting-induced hepatic

gluconeogenesis in vivo, independent of any action on hepatocyte proliferation.

p16Ink4a

-deficiency increases gluconeogenic gene expression and glucose production in

vitro in hepatocytes.

To analyse whether the altered regulation of hepatic gluconeogenic gene expression in p16-/-

mice is a cell-autonomous phenomenon, primary hepatocytes from p16-/-

mice and their

littermate controls were isolated and incubated with increasing concentrations of glucagon to

mimic the fasting conditions. Basal levels of gluconeogenic gene expression were 1.5 fold

higher for G6Pase (±0.15 *p<0.05), 4.4 fold higher for Pepck (±0.96 **p<0.01) and 2.3 fold

higher for Fbp1 (0.17 ***p<0.001) in p16-/-

compared to p16+/+

primary hepatocytes (Figure

2A-C). Moreover, glucagon, which activates the PKA-CREB signalling pathway, more

pronouncedly induced G6pase, Pepck and Fbp1 (Figure 2A-C) mRNA levels in p16-/-

vs

p16+/+

primary hepatocytes. Further, hepatic glucose production was higher in primary

hepatocytes of p16-/-

than of p16

+/+ mice (Figure 2D). Next, p16

Ink4a was silenced using a

CDKN2A siRNA (which affects both p16Ink4a

and p19ARF

expression) in AML12 cells (Figure

3 A-B), a mouse hepatocyte cell line which expresses very high levels of p16Ink4a

compared to

liver and primary hepatocytes (Figure 3C). Incubation with forskolin, to activate the PKA-

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CREB pathway, resulted in a more pronounced increase of G6pase and Fbp1 gene expression

when p16Ink4a

was silenced, while no effect was observed on Pepck gene expression (Figure

3D-F). Moreover, although G6pase and Fbp1 gene expression only marginally increased upon

forskolin treatment in p16Ink4a

-expressing AML12 cells, p16Ink4a

silencing resulted in the

restoration of a strong response (Figure 3D-E).

Altogether, these results indicate that p16Ink4a

expression levels influence the response to

fasting-induced stimuli both in vivo and in vitro.

p16Ink4a

levels modulate PGC1αααα expression in vivo and in vitro.

To study the mechanism by which p16Ink4a

regulates gluconeogenic gene expression, mRNA

and protein levels of peroxisome proliferator-activated receptor γ coactivator 1 alpha

(PGC1α), a master regulator of the fasting adaptation process (24), were measured. The

fasting response of Pgc1a mRNA was significantly more pronounced in livers of p16-/-

compared to p16+/+

mice (Figure 4A). In line, p16-/-

primary hepatocytes displayed a 3.4 fold

increased Pgc1a mRNA level (±0.83 **p<0.01, 2-tailed Student’s t test) and a stronger

induction by glucagon compared to p16+/+

hepatocytes (Figure 4B). p16Ink4a

silencing in

AML12 cells significantly increased Pgc1a expression at both mRNA and protein levels upon

forskolin treatment (Figure 4C-D).

p16Ink4a

-deficiency increases the PKA-CREB signalling pathway.

To gain insight into how PGC1α is induced upon p16Ink4a

-deficiency, we first analyzed the

phosphorylation status of CREB, a transcription factor inducing PGC1α expression. p-Ser133-

CREB was markedly higher in p16-/-

compared to p16+/+

hepatocytes, at the basal level as well

as following glucagon exposure (Figure 5A,B). Similar results were obtained upon forskolin

treatment (data not shown). Likewise, p16Ink4a

silencing in AML12 cells resulted in a stronger

CREB phosphorylation, both at the basal level as upon forskolin treatment (Figure 5C,D).

Altogether, these data demonstrate that p16Ink4a

knock-down increases CREB

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phosphorylation. It is well known that the cAMP-PKA signalling pathway regulates fasting-

induced CREB phosphorylation (22,27). To test whether alterations in PKA activity may

explain the increased CREB phosphorylation upon p16Ink4a

-deficiency, p16Ink4a

-silenced

AML12 cells were treated with H89, a specific PKA inhibitor. H89 treatment prevented

CREB phosphorylation induced by p16Ink4a

silencing (Figure 6A). Accordingly, PKA activity

in p16Ink4a

-silenced AML12 cells was 1.5 fold higher compared to control (Figure 6B). This

increase was substantiated by the increase in total PKA substrate phosphorylation profiles

upon p16Ink4a

silencing (Figure 6C). Likewise, several PKA substrates were more

phosphorylated in p16-/-

than in p16+/+

primary hepatocytes both under basal conditions as

following glucagon stimulation (Figure 6D). Since PKA activity is controlled at least in part

by the phosphorylation of its regulatory subunits (PKAR2), the expression and

phosphorylation of PKAR2 were measured in p16Ink4a

-silenced AML12 cells and in p16-/-

primary hepatocytes. p16Ink4a

silencing or deficiency resulted in increased PKAR2

phosphorylation in AML12 cells (Figure 7A), and in p16-/-

primary hepatocytes both at the

basal state and upon glucagon stimulation (Figure 7B). This result was confirmed by the

enhanced P-PKAR2 immunostaining in p16Ink4a

-silenced AML12 cells (Figure 7C).

Noteworthy, the increased PKA activity was not due to an increase in cAMP levels (Figure

7D). Collectively, these data demonstrate that p16Ink4a

-deficiency activates the PKA-CREB-

PGC1α signalling pathway independent of changes in intracellular cAMP levels. To

understand the underlying mechanism by which p16Ink4a

increases phosphorylation of PKAR2

and thereby the increase of gluconeogenic genes, we investigated the involvement of CDK4, a

well-known target of p16Ink4a

. Silencing of CDK4 in p16Ink4a

-silenced AML12 cells (Figure

8A-B) abrogated the induction of Ppc1α and Fbp1 mRNA levels by p16Ink4a

-silencing (Figure

8 C-D). Moreover, co-immunoprecipitation experiments in AML12 cells after p16Ink4a

-knock

down demonstrates a physical interaction between CDK4 and PKAR2 (Figure 8E).

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Discussion

In recent years, a growing body of evidence supports the emerging notion that cell cycle

regulatory proteins contribute to metabolic processes in addition to, or linked with their role in

cell growth (17,28). Today, these proteins are perceived as sensors of external signals that

require a particular adapted metabolic response. The CDK-Rb-E2F1 pathway, which is

inhibited by p16Ink4a

, has already been shown to control adipogenesis by modulating the

expression of the nuclear receptor PPARγ (15,29), a master regulator of adipogenesis as well

as by controlling oxidative metabolism in adipose tissue (30). The CDK-Rb-E2F1 pathway is

also a negative regulator of energy expenditure through repression of mitochondrial oxidative

metabolism in muscle (16). Disruption of CDK inhibitors (CKI) genes in the mouse has not

revealed profound cell cycle abnormalities, but does result in a specific metabolic phenotype.

Mice lacking either p18Ink4c

(31,32), p21cip1

or p27Kip1

display growth abnormalities and

adipocyte hyperplasia (33). Double knockout mice (p21-/-

; p27-/-

) develop

hypercholesterolemia, glucose intolerance and insulin insensitivity (33). Surprisingly, and

until today, the role of these cell cycle regulators in the liver, one of the main metabolic

organs controlling glucose homeostasis, has not been yet demonstrated.

It is well known that an increased rate of hepatic gluconeogenesis contributes to fasting

hyperglycemia observed in T2D patients. Genetic analysis in genome-wide studies identified

an association of the CDKN2A/B locus with T2D risk (5,34,35). The association is based on

the measurement of fasting glycemia and confers to the CDKN2A/B locus a high

susceptibility to T2D across different ethnicities. In this study, we tried to elucidate the

mechanism by which a product of CDKN2A/B, ie p16Ink4a

, may influence the hepatic

gluconeogenic program and thereby be implicated in T2D pathogenesis. We found that

p16Ink4a

-deficiency raises PKAR2 phosphorylation leading to an increased PKA activity. The

increased PKA activity enhances CREB-PGC1α signalling, regulating gluconeogenic gene

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expression program. Since the p16Ink4a

protein shares several ankyrin repeat domains, which

are involved in protein-protein interaction, we assessed whether p16Ink4a

may associate with

the PKA complex. Immunoprecipitation of endogenous p16Ink4a

in AML12 cells failed to

demonstrate an interaction of p16Ink4a

with the PKA regulatory subunit or the PKA catalytic

subunit (data not shown), suggesting the existence of other proteins able to connect p16Ink4a

to

PKA complex. One good candidate bridging p16Ink4a

to the PKA complex was CDK4, a well-

known interaction partner of p16Ink4a

. Indeed, siRNA knock-down of CDK4 in AML12 cells

abrogates the effect of p16Ink4a

deficiency on the expression of gluconeogenic gene, like

Pgc1α and Fbp1, suggesting a direct role of CDK4 in the regulation of PKA activity by

p16Ink4a

. Moreover, it has already been shown that CDK4 can displace the interaction between

cyclin D and the PKAR2 - A-kinase anchoring protein /AKAP95 complex when CDK4 is

activated (36). Other studies demonstrated that CDK1 also phophorylates PKAR2 (37)

suggesting that other CDKs than CDK4 can have the same activity on PKAR2 (38). All these

data support the existence of a dynamic complex including p16Ink4a

-(CDK4/Cyclin D)-PKA-

AKAP95 involved in the control of hepatic glucose production (Figure 8F).

In summary, GWAS identified SNPs near CDKN2A/B associate with fasting glycemia and

the risk of T2D development. Our study establishes that the p16Ink4a

gene product of this locus

modulates hepatic glucose production by increasing hepatic gluconeogenic gene expression.

Further, we provide evidence that p16Ink4a

acts via the PKA-CREB-PGC1α signalling

pathway. Although the functional role of several cell cycle regulators (CDK4, pRb, E2F,

p21cip1

, p27kip1

, p18Ink4c

) in metabolic control has been described in tissues such as adipose

tissue and the pancreas (28), this is the first study which demonstrates a role of a cell cycle

regulator, p16Ink4a

, in the liver, a master organ regulating glucose homeostasis in a manner

independent of its function in cell proliferation. Thus, altered p16Ink4a

activity may contribute

to the association between the GWAS locus and the risk to develop T2D.

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Acknowledgments

We thank P. Krimpenfort for providing the p16-deficient mice and J. Dumont for her

assistance. Bart Staels is a member of the Institut Universitaire de France. This work was

supported by grants from European Genomic Institute for Diabetes (EGID, ANR-10-LABX-

46). We thank also Cost Action (BM0602), the Conseil régional Nord Pas-de-Calais and the

Fonds Européens de Développement Régional (FEDER). K Bantubungi was supported by a

post-doctoral fellowship from Fondation pour la Recherche Médicale (FRM). S. Hannou was

supported by a doctoral fellowship from Université Lille2/ Conseil régional Nord Pas-de-

Calais and Fondation pour la Recherche Médicale (FRM). No potential conflicts of interest

relevant to this article were reported.

K.B., S.H., S.C-H, E.V., M.B., A.L. and E.B. performed experiments. K.B., S.H., R.P., A.T.

and B.S. designed experiments. K.B., S.H., R.P., A.T. and B.S. analyzed data. K.B., S.H.,

A.T, R.P. and B.S. wrote the paper. B.S. is the guarantor of this work and, as such, had full

access to all the data in the study and takes responsibility for the integrity of the data and the

accuracy of the data analysis.

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Legends

Figure 1. p16Ink4a

-deficiency increases glucose production and gluconeogenic gene

expression without modulating genes involved in glucose and lipid utilization in liver. (A) 12

week old p16-/-

mice (n=9) display higher blood glucose levels after 24h fasting than wild-

type p16+/+

mice (n=9). Unpaired Student t-test (*compared between the genotypes of the

same treatment group; #compared between the treatment groups of the same genotype; *or

#<0.05, **or ##<0.01). Data are means +/- SEM. (B) Pyruvate tolerance test (PTT) shows

increased glucose production in 12 week old p16-/-

compared to p16+/+

mice (n=6). 2 way

ANOVA and Newman-Keuls post-hoc test (*compares genotypes; *<0.05, **<0.01). (C)

Area under the curve (iAUC) of PTT is higher in p16-/-

vs p16+/+

mice. Student t-test (*<0.05).

Data are means ± SEM. (D-F) The expression of gluconeogenic genes (G6pase, Fbp1, Pepck)

is increased in p16-/-

vs p16+/+

mice (n=10) after 24h fasting. (G-I) mRNA level of genes

involved in glycolysis (Gk, Lpk, Pdk4) and (J-K) beta-oxydation (Cpt1a, Lcad) pathways are

unchanged in livers of p16 -/-

vs p16+/+

mice (n=10). 2 way ANOVA and LSD Fisher post-hoc

test (*compared between the genotypes of the same treatment group; #compared between the

treatment groups of the same genotype; *or #<0.05, **or ##<0.01, ***or ###<0.001, ****or

####<0,0001). Data are means ± SEM. Open bars/open squares, p16+/+

mice; black bars/black

circles, p16-/-

mice.

Figure 2. p16Ink4a

-deficiency increases gluconeogenic gene expression and glucose

production in primary hepatocytes.

The induction of gluconeogenic genes (G6pase (A), Fbp1 (B), Pepck (C)) by glucagon (8h) is

higher in primary hepatocytes isolated from p16-/-

vs p16+/+

mice. 2 way ANOVA and LSD

Fisher post-hoc test (*compared between the genotypes of the same treatment group;

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#compared between the treatment groups of the same genotype; *or #<0.05, **or ##<0.01,

***or ###<0.001, ****or ####<0,0001). Data are means ± SEM. Open bars, p16+/+

primary

hepatocytes; black bars, p16-/-

primary hepatocytes. Glucose production is higher in primary

hepatocytes isolated from p16-/-

vs p16+/+

mice (D). Student t-test (*compared between the

genotypes of the same treatment group; **<0.01). Data are means +/- SEM.

Figure 3. p16Ink4a

down-regulation increases gluconeogenic gene expression in AML12 cells.

SiRNA CDKN2A treatment (which affects both p16Ink4a

and p19ARF

expression) in AML12

strongly decreases (A) p16Ink4a

mRNA level measured by RT-qPCR and (B) p16Ink4a

protein

level measured by Western blot analysis. Student’s t-test (***<0.001). Data are means ±

SEM. (C) p16Ink4a

protein level is comparable in liver, primary hepatocytes and AML12 cells.

(D-F) p16Ink4a

-silenced and p16Ink4a

-expressing AML12 were treated with 10µM forskolin

(FSK) for 16h. The expression of G6pase, Fbp1 genes (D-E) were increased but not Pepck

(F) in p16Ink4a

-silenced compared to p16Ink4a

-expressing AML12 cells. 2 way ANOVA and

LSD Fisher post-hoc test (*compared between the genotypes of the same treatment group; #

compared between the treatment groups of the same genotype; *or #<0.05, **or ##<0.01,

***or ###<0.001, ****or ####<0,0001). Data are means ± SEM. Open bars, p16Ink4a

-

expressing AML12; black bars, p16Ink4a

-silenced AML12.

Figure 4. p16Ink4a

down-regulation increases Pgc1a gene expression. Pgc1a mRNA levels

are higher in (A) livers of p16-/-

compared to p16+/+

mice, in (B) primary hepatocytes isolated

from p16-/-

compared to p16+/+

mice and treated with glucagon for 8h and in (C) p16Ink4a

-

silenced compared to p16Ink4a

-expressing AML12 cells treated with 10µM forskolin (FSK) for

16h. (D) Western blots show higher increase of PCG1α protein level in p16Ink4a

-silenced

compared to p16Ink4a

-expressing AML12 cells treated with 10µM forskolin (FSK) for 1h. 2

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way ANOVA and LSD Fisher post-hoc test (*compared between the genotypes of the same

treatment group; #compared between the treatment groups of the same genotype; *or #<0.05,

**or ##<0.01, ***or ###<0.001, ****or ####<0,0001). Data are means ± SEM. Open bars,

p16+/+

mice (A), p16+/+

primary hepatocytes (B) or p16Ink4a

-expressing AML12 (C); black

bars, p16-/-

mice (A), p16-/-

primary hepatocytes (B) or p16Ink4a

-silenced AML12 (C).

Figure 5. p16Ink4a

down-regulation increases the phosphorylation of CREB. Western blots

show higher increase of p-CREB in (A) primary hepatocytes isolated from p16-/-

compared to

p16+/+

mice and treated with glucagon for 1h and in (C) p16Ink4a

-silenced compared to

p16Ink4a

-expressing AML12 cells treated with 10µM forskolin (FSK) for 1h. (B, D) The bar

graphs are the quantification of p-CREB western blots in primary hepatocytes isolated from

p16-/-

and p16+/+

mice and treated with glucagon for 1h and p16Ink4a


-

expressing AML12 cells treated with 10µM forskolin (FSK) for 1h. 2 way ANOVA and LSD

Fisher post-hoc test (*compared between the genotypes of the same treatment group;

#compared between the treatment groups of the same genotype; *or #<0.05, **or ##<0.01,

***or ###<0.001, ****or ####<0,0001). Data are means ± SEM. Open bars, p16+/+

primary

hepatocytes (B) or p16Ink4a

-expressing AML12 (D); black bars, p16-/-

primary hepatocytes (B)

or p16Ink4a

-silenced AML12 (D).

Figure 6. p16Ink4a

down-regulation increases PKA activity. Western blots show (A) a stronger

decreased p-CREB in p16Ink4a

-silenced compared to p16Ink4a

-expressing AML12 cells treated

with 20µM H89 and (B) an increased PKA activity upon p16Ink4a

-silencing in AML12 cells.

Student t-test (*<0.05). Data are means ± SEM. Western blots show higher increase of global

profile of PKA phosphorylated substrates in (C) p16Ink4a


-expressing

AML12 under basal conditions and in (D) primary hepatocytes isolated from p16-/-

and p16+/+

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mice treated with glucagon for 1h. Open bars, p16Ink4a

-expressing AML12; black bars,

p16Ink4a

-silenced AML12.

Figure 7. p16Ink4a

down-regulation increases PKAR2 phosphorylation without affecting

intracellular cAMP levels. Western blots show higher p-PKAR2 in p16Ink4a

-silenced than in

p16Ink4a

-expressing AML12 cells (A) and in primary hepatocytes from p16-/-

than p16+/+

mice

treated or not with glucagon for 1h (B). (C) Immunofluorescent staining of p-PKAR2 in

p16Ink4a


-expressing AML12 cells. Original magnification X20. (D)

cAMP levels were measured in p16Ink4a


-expressing AML12 under basal

conditions. Student t test (ns). Data are means ± SEM. Open bars, p16Ink4a

-expressing

AML12; black bars, p16Ink4a

-silenced AML12.

Figure 8. p16Ink4a

down-regulation increases gluconeogenic gene expression in AML12 cells.

SiRNA CDK4 treatment in AML12 strongly decreases (A) CDK4 mRNA level measured by

RT-qPCR without affecting p16Ink4a

mRNA level. (B) The p16Ink4a

down-regulation-induced

increase of Pgc1a and Fbp1 mRNA expression (C-D) were abrograted by siRNA CDK4

treatment. 2 way ANOVA and LSD Fisher post-hoc test (*compared AML12 treated by

SiRNA CDKN2A or not; # compared AML12 treated by siRNA CDK4; *or #<0.05, **or

##<0.01, ***or ###<0.001, ****or ####<0,0001). Data are means ± SEM. Open bars,

p16Ink4a

-expressing AML12; black bars, p16Ink4a

-silenced AML12. (E) Co-

immunoprecipitation of CDK4 from whole AML12-cell extracts was performed. The eluate

was then subjected to western blot analysis against PKAR2. (F) Proposed pathway for the

control of hepatic gluconeogenesis through p16Ink4a

and CDK4. When p16Ink4a

is unable to

bind CDK4 in the nucleus, CDK4 translocates to the cytoplasm where it interacts with the

PKA complex through regulatory sub-unit 2 of PKA (PKAR2). This interaction leads to an

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increase of PKA activity, independently of changes in intracellular cAMP levels. Increased

PKA activity leads to the activation of the transcription factor CREB and expression of the

PGC1α co activator, which in turn drives the transcription of gluconeogenic enzymes such as

PEPCK and G6Pase.

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Supplemental Legend

Supplemental Figure 1. p16Ink4a

-deficiency does not influence plasma glucagon level. 12

week old p16-/-

mice (n=9) and wild-type p16+/+

mice (n=9) were fed or fasted and plasma

glucagon concentration measured. 2 way ANOVA and LSD Fisher post-hoc test (*compared

between the genotypes of the same treatment group; #compared between the treatment groups

of the same genotype; *or #<0.05). Data are means ± SEM. Open bars/open squares, p16+/+

mice; black bars/black circles, p16-/-

mice.

Supplemental Figure 2. (A) Macroscopic pictures of livers from p16+/+

and p16-/-

mice. (B)

Liver weights of p16+/+

and p16-/-

mice. (C) Immunohistochemical Ki-67 staining of liver

sections of p16+/+

treated by CCL4 (C-F), p16+/+

(D-G) and p16

-/- (E-H) mice under fed or

fasting condition, indicating no differences between p16-/-

mice and their littermate controls

under fasting conditions.

Supplemental Figure 3. mRNA levels of p15Ink4b

(A-E), p18Ink4c

(B-F), p19Ink4d

(C-G) and

p19ARF

(D-H) in livers of (A-D) fed or (E-H) fasted wild-type and p16ink4a

-deficient mice.

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Supplemental Table 1

Supplemental Table 1: primer pairs to quantitative RT-PCR

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Bantubungi et al. Figure 1

0

200

400

Pe

pc

k m

RN

A lev

el

*

fed fasted

###

#

0

50

100

150

200

Gk

mR

NA

le

ve

l

fed fasted

####

####

0

500

1000

1500

Pd

k4

mR

NA

le

ve

l

fed fasted

*###

0,08

0

50

100

150

200

Lp

k m

RN

A le

vel

fed fasted

###

####

G H I

0

400

800

1200

G6

Pa

se

mR

NA

le

ve

l

*

fed fasted

#

###

E F

0

200

400

600

800

Lc

ad

mR

NA

le

ve

l

fed fasted

########

J K

A B C

D

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Figure 3 Bantubungi et al.

p16 Ink4a

siRNA CDKN2A - +

GAPDH

B

A

C

p16 Ink4a

GAPDH

E F D

Fb

p1

mR

NA

le

ve

l

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A

p-CREB

CREB

Glucagon (nM)

GAPDH

p16+/+

0 10 100

p16-/-

0 10 100

p-CREB

CREB

GAPDH

siRNA CDKN2A - + + -

- - + + FSK (10 μM)

C

B

D

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Bantubungi et al. Figure 6 Bantubungi et al.

A

siRNA CDKN2A - - + +

- + + - H89

p-CREB

CREB

p16 Ink4a

GAPDH

B

D

P-s

ub

stra

tes

of

PK

A

GAPDH

siRNA CDKN2A - +

P-s

ub

stra

tes

of

PK

A

GAPDH

p16+/+

0 10 100

p16-/-

0 10 100

C Glucagon (nM)

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Figure 7 Bantubungi et al. Bantubungi et al.

C

p16 Ink4a p-PKAR2 Merge

SiR

NA

C

DK

N2

A

Scra

mb

le

A B

T-PKAR2

GAPDH

p-PKAR2

p16 Ink4a

- + siRNA CDKN2A

p-PKAR2

Actin

T-PKAR2

p16+/+

0 10 100

p16-/-

0 10 100 Glucagon (nM)

D

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Supplemental Figures

Supplemental Figure 1. p16Ink4a

-deficiency does not influence plasma glucagon level. 12

week old p16-/- mice (n=9) and wild-type p16

+/+ mice (n=9) were fed or fasted and plasma

glucagon concentration measured. 2 way ANOVA and LSD Fisher post-hoc test (*compared

between the genotypes of the same treatment group; #compared between the treatment groups

of the same genotype; *or #<0.05). Data are means ± SEM. Open bars/open squares, p16+/+

mice; black bars/black circles, p16-/- mice.

Supplemental Figure 2. (A) Macroscopic pictures of livers from p16+/+

and p16-/- mice. (B)

Liver weights of p16+/+

and p16-/- mice. (C) Immunohistochemical Ki-67 staining of liver

sections of p16+/+

treated by CCL4 (C-F), p16+/+

(D-G) and p16

-/- (E-H) mice under fed or

fasting condition, indicating no differences between p16-/- mice and their littermate controls

under fasting conditions.

Supplemental Figure 3. mRNA levels of p15Ink4b

(A-E), p18Ink4c

(B-F), p19Ink4d

(C-G) and

p19ARF

(D-H) in livers of (A-D) fed or (E-H) fasted wild-type and p16ink4a

-deficient mice.

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Cdkn2a/p16Ink4a Regulates Fasting-Induced Hepatic Gluconeogenesis Through the PKA-CREB-PGC1 ...

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