Control of beta-cell function by  PHIP: Implications for Type I and

II Diabetes

Laboratory Medicine and PathobiologyFaculty of Medicine

University of Toronto

IGF-1 Signaling Pathways Play Crucial Role in Pancreatic β-cell Function

β-Cell

PH

Introduction

PH PTB

NPXpY?

Receptor interaction

1 1243

IR

IRS1pY

pYpY

pY

SHP-2NCK

GRB2PI3K

IRS-1 PH

1 902 aa

PBR 150

PHIP

•PHIP (Pleckstrin Homology domain Interacting Protein) was originally identified as a 902 amino acid protein that interacts with the PH domain of insulin receptor substrate 1 (IRS1)

•PHIP was shown to regulate insulin-dependent mitogenic and metabolic responses in skeletal muscle cells (Farhang-Fallah J, et al. MCB 2002)

Objective 1

Given the apparent role of PHIP in IRS signaling pathways and the importance of IRS proteins in proliferation and survival in β-cells we sought to investigate the functional role of PHIP in β-cells

Q: Is PHIP expressed in beta-cells?

PHIP is Highly Expressed In Mouse Islets(QPCR)

Rel

ativ

e P

HIP

mR

NA

exp

ress

ion

MIN6Isl

ets

Acinar

Who

le

WAT

Brain

Pancreas

7.3 Fold

10.0

0.2

0.4

0.6

0.8

1.0

Muscle

Min6 INS-1

a-PHIP

DAPI

PHIP Is Localized to Nuclei of Insulinoma Cell Lines (IHC)

PHIP Predominantly Expressed in Nuclei of Islet Cells (IHC)

a-PHIP a-Insulin a-PDX1

DAPI a-Glucagon H&E

x100

x100

x200

a-PHIP

DAPI

PHIP Structure

•WD40 domains - protein-protein interaction modules implicated in transcriptional regulation, vesicular formation and trafficking and control of various aspects of cell division.

•BromoDomains - found predominantly in proteins that regulate chromatin remodeling such as nuclear histone acetyltransferases and transcriptional coactivators

1821 a.a (206kDa)

1821 a.a (206kDa)

1498 a.a (165kDa)

1266 a.a (143kDa)

902 a.a (105kDa)

BD

isoform 9

Q: What isoform of PHIP1 predominantly expressed in beta-

cells?

PHIP1 – a 206 kDa PHIP Isoform is Predominant Species in Mouse Islets and Insulinoma Cell Lines

ISLETS

INS1

MIN

6

230 -

95 -

a-PHIP 130 -

206 kDa

230 -

95 -

130 -

PHIP1

PHIP

Cont.

Short Isoform

Long Isoform

Transfection

a-PHIP

Western Blotting

Q: Does overexpression of PHIP1 influence the growth of pancreatic β-cells ?

24 48 72 96

100

200

300

400

500

600

700

800

Adenoviral-mediated Expression of PHIP1 Enhances Proliferation in INS-1 cells (MTT Assay)

Hours after Infection

Nu

mb

er

of

Via

ble

Ce

lls(O

D45

0)

(10%FBS)

*

IRS2PHIP1GFP

Q: Does PHIP1 over expression influence the IGF-1 dependent growth of β-cells ?

10.0

0.5

1.0

1.5

2.0

2.5

3.0

Adenoviral-mediated overexpression of PHIP1 enhances IGF1-dependent mitogenesis in INS-1 Cells (BRDU Assay)

BR

DU

In

corp

ora

tio

n(F

old

Ch

ang

es)

IGF-1 - + - + - +

AdGFP AdPHIP1 AdIRS210nM

1.8 Fold

1.3 Fold

Q: What possible mechanisms mediate increase in proliferation rate of PHIP1

overexpressing cells ?

Ad-mediated Overexpression of PHIP1 Promotes IGF1-Dependent Increase of Cyclin D2 Expression in INS-1 cells

Cyclin D2-

Cyclin D1-

Actin-

Western Blotting

IGF-1 - + - + - +

AdGFP AdPHIP1 AdIRS2

Luciferase Assay

IGF-1 - + - + AdGFP AdPHIP1

0.5 0.6 0.7 0.8 0.9 1.00.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

2.1 Fold

2.5 Fold

Cyclin D2 promoter Luc

Lu

cife

rase

Un

its

(Fo

ld C

ha

ng

es

)

Cyclin D2reporter

Q: Does PHIP1 is necessary component of IGF-1 and IRS2 signaling pathways leading to

mitogenic responses?

siRNA-mediated Knockdown of PHIP1 Blocks IGF-1 and IRS2-induced Mitogenesis and Expression of Cyclin D2, but

not AKT phosphorylation.

H3 -

Th

ymid

ine

Inco

rpo

rati

on

(Fo

ld C

han

ges

)

ScrGFP

SiPHIPGFP

ScrIRS2

siPHIPIRS2

*

- + - + - + - +IGF-1 (10nM)

- + - + - + - +IGF-1 (10nM)

PHIP1-

IRS2-

Cyclin D2-

H3Thymidine Incorporation Assay

Western Blotting

ScrGFP

SiPHIPGFP

ScrIRS2

siPHIPIRS2

pSer473AKT-

*

Summary of PHIP Role in β-cell Proliferation:

• Long Isoform of PHIP – PHIP1 is predominantly expressed in nuclei of β-cells

• PHIP1 promotes proliferation and potentiates IGF1-stimulated mitogenesis in β-cells

• PHIP1 drives transcriptional induction of Cyclin D2 promoter

• PHIP1 expression is essential for IGF1 and IRS2-induced β-cell replication

Q: Does PHIP1 plays any role in β-cell apoptosis?

•Chronic elevation of free fatty acids (FFAs) leads to the generation of reactive oxygen species, inhibition of insulin biosynthesis and induction of pancreatic -cell apoptosis both in vivo and in vitro (β-cell lipotoxicity)

•Inflammatory cytokines such as TNF-α , IL-1ß, and Interferon-γ are cytotoxic to ß-cells and may contribute to ß-cell death in obesity which has been described as a state of low-level chronic inflammation

•TNF-α , IL-1ß, and Interferon-γ are major mediators of β-cell apoptosis in Type 1 diabetes

•Increased IRS2 expression or exposure to IGF1 inhibits FFA and cytokine-induced apoptosis in β-cells

Role of β-cell apoptosis in diabetes

To evaluate the effect of PHIP1 overexpression on FFA-induced apoptosis

Objective 2

- IGF1 + IGF1

Adenoviral-mediated Overexpression of PHIP1 Inhibits FFA-induced INS-1 Cell Apoptosis

2.6

4.1

Q: What molecular mechanisms could mediate the inhibitory effect of PHIP1 overexpression on FFA-induced apoptosis?

• Prevent AKT translocation to membrane

• Activation of PP2A

• Increased Ser/Thr Phosphorylation of IRS2 by activated PKC

10.0

0.5

1.0

1.5

2.0

pS

er47

3A

kt

/AK

T r

ati

o

GFPBSA

GFPOA

PHIP1 OA

IRS2OA

Akt-

pSer473 AKT-

IRS2-

PHIP-

IGF-1 (10ng/ml)

Casp9-(cleaved)

Adenoviral-mediated Expression of PHIP1 Induces Activation of pAKT and Inhibition of pro-Caspase-9 and -3 Cleavage

Casp3-(cleaved)

GFPBSA

IGF-1

GFP OA

PHIP1OA

IRS2OA

- -+ + - -+ +

- + - + - + - +

10.0

0.5

1.0

1.5

2.0

Ca

sp

as

e9

/A

cti

n C

as

pa

se

3 /

Ac

tin

8.6 fold

1.75 fold

2.7 folds

*

*

5.8 Folds

Apoptosis

Apoptosis

Q: Does AKT activation is crucial for PHIP1 antiapoptotic effect ?

•KD-AKT -“Kinase Dead” AKT due to mutation in ATP-binding site

Apoptosis

Overexpression of “kinase-dead” AKT Blocks Protective Effect of PHIP1 on FFA-induced apoptosis

•AKT plays crucial role in antiapoptotic effect of PHIP1

Q: How does PHIP1 increase activity of AKT?

PHIP1

?

PHIP1

?

?

?

??

•PHIP1 and IRS2 have different subcellular localization in β-cells

IF (Min6)

Anti-PHIP Anti-IRS2

DAPI DAPI

PHIP1 role in regulation AKT signalling

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1 2

PTE

N m

RN

A

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

1 2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

1 2

SH

IP2 m

RN

Am

TO

R m

RN

A

•The precise mechanism by which PHIP1 converge on AKT signaling is still not clear

AdGFP

qPCR(INS1)

AdPHIP1

Summary on PHIP role in FFA-induced apoptosis:

•PHIP1 overexpression protects INS1 cells against FFA induced apoptosis in vitro

•Ectopic PHIP1 overexpression induces AKT phosphorylation and inhibits Caspase-9 and -3 activation

•AKT has essential role in the PHIP1 signaling pathway that promotes beta-cell survival

Q: Will PHIP1 exhibit similar effect on β-cells in vivo model of Type 2 Diabetes?

Rodent High Fat Diet (HFD) induced Experimental Diabetes as a Model of Type 2 Diabetes

•Prolonged (>3 months) HFD in C57BL/6J background mice results in overt diabetes (Mills E, et al., Am.J.Physiol, 1993)

Compensatory β-cell hyper-

plasia

Diabetes

HyperlipidemiaObesity

Increased PeripheralResistance to Insulin

Hyperinsulinemia

Failure of β -cells to adapt to

changes in metabolic demand

High Fat Diet 3-5 months

•Overexpression of IRS2, AKT, PDX1, Glucokinase and CA-STAT5 in β-cells blocks or alleviates development of HFD-induced Diabetes

Generation and Charachterization of Tg-PHIP1 mice

PHIP1 HA

DAPI DAPI

x200

WB from extracted Islets

RIP HA PHIP1

RIP-HA-PHIP1 construct

IHC

WT TG1 TG2 TG3

Body Weight and Fed Blood Glucose Levels in Wild Type (WT) or TG-PHIP Mice after 20 weeks on Standard or High Fat Diet

10

5

10

15

20

25

30

35

40

45

50

B

ody

Wei

ght,

g (

M+

SE

M)

WT (n=19-25) TG-PHIP (n=14-18)

10,2%

10

2

4

6

8

10

12

14

25%

Fed

Blo

od

Glu

cose

, mM

(M

+S

EM

)

Day 0 20weeksNormalChow

20weeksHFDiet

Day 0 20weeksNormalChow

20weeksHFDiet

43%27%

0 20 40 60 80 100 12002468

101214161820222426283032

0 20 40 60 80 100 12002468

101214161820222426283032

0 20 40 60 80 100 12002468

101214161820222426283032

0 20 40 60 80 100 12002468

10121416182022242628303234

TG-PHIP Mice Have Improved Glucose Tolerance After 12 and 20 Weeks on High Fat Diet (GTTest)

Blo

od

Glu

co

se

mM

(M

+S

EM

)

Blo

od

Glu

co

se

mM

(M

+S

EM

)

Blo

od

Glu

co

se

mM

(M

+S

EM

)

Blo

od

Glu

co

se

mM

(M

+S

EM

)

*

*

* *

*

(Min) (Min)

(Min) (Min)

Normal Chow 12 Weeks

Normal Chow 20 Weeks

HF Diet 12 Weeks

HF Diet 20 Weeks

WT (n=10-17) TG-PHIP (n=10-12) IP Glucose Injection 2g/kg

Q: How does PHIP1 overexpression improve glucose tolerance?

Ins

uli

n (

ng

/ml*

min

/Ce

lls

DN

A)

Ins

uli

n (

ng

/ml/

min

/50

is

lets

)

16.7mM Glucose

GLP1

Phase I Phase I

16.7mM Glucose

GLP1

Phase I Phase I

TG-PHIP Mice Have Improved Glucose Stimulated Insulin Secretion (Perfusion Assay) After 20 Weeks on HFD

Q: Is Insulin secretion from TG-PHIP islets higher due to increased number of β-cells per islet?

WT (n=3) TG-PHIP (n=3)

10

1

2

3

4

5

6

7

8

9

10

11

10.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

*In

sulin

Co

nte

nt

(ng

/ml/m

in/ 5

0 is

lets

)

Insu

lin C

on

ten

t(n

g/m

l*m

in/ D

NA

Co

nte

nt)

INS

ULIN

WT TG-PHIP WT TG-PHIP

TG-PHIP mice islets Wild Type mice islets

Tg-PHIP Mice Islets Have Increased Number of β-cells

Summary on PHIP role in HFD-induced Diabetes:

•Transgenic mice selectively overexpressing PHIP1 in β-cells have decreased incidence of HFD induced diabetes

•Protective effect of PHIP1 on HFD induced Diabetes mediated via increase of β-cell proliferation or inhibition of apoptosis

Objective 3

To evaluate role of PHIP1 in cytokine-induced β-cell apoptosis

A Model of Signaling Pathways Involved in β-cell Cytokine-induced Apoptosis

β-Cell

Q: Does PHIP1 overexpression inhibit cytokine-induced β-cell apoptosis?

Adenoviral-mediated PHIP1 over expression inhibits cytokine-induced (IL-1β+IFNγ) apoptosis in INS1 cells

10.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.5 fold

Cont PHIP1

Cytokines(24hrs)

- + - +

Ap

op

tosi

s(f

old

ch

ang

es)

Cont PHIP1

- + - +Cytokines(24hrs)

PHIP1-

Caspase3-

Tubulin-

MDM2-

PSerAKT-

Cytokines mix:Interferon-γ 50ng/mlIL-1β 10ng/ml

WT +cytokine mix

TG-PHIP +cytokine mix

INS

UL

IN

TU

NE

LD

AP

I

PHIP1 Inhibits Induction of Cytokine-induced Apoptosis in Dispersed Islets of RIP7-PHIP1 mice (48hrs exposure; TUNEL assay)

102468

1012141618202224262830323436

WildType

Tg-PHIP1

Cytokines(24hrs)

- + - +

3.7 fold

Mer

ge

INS

UL

INM

erg

e

TU

NE

LD

AP

I

% o

f T

UN

EL

po

siti

ve β

-cel

ls

Q: Does PHIP1 knockdown enhances cytokine-induced apoptosis?

siRNAi-mediated PHIP1 Knockdown Enhances Cytokine-induced Apoptosis in INS1 Cells

10

2

4

6

8

10

12

Ap

op

tosi

s(f

old

ch

ang

es)

Cont siPHIP1

Cytokines(24hrs)

- + - +

3.1 fold

Cytokines(24hrs)

PHIP1-

Caspase3-

Tubulin-

MDM2-

PSerAKT-

Cont siPHIP1

- + - +

0 4 8 12 16 20 24 2802468

1012141618202224262830

0 4 8 12 16 20 24 2805

10152025303540455055

PHIP1 Enhances Cytokine-induced Expression of STAT-dependent Antiapoptotic Genes IRF1, SOCS1 and SOCS3

(qPCR)R

ela

tive m

RN

A level

* *

*STAT1 IRF1

0 4 8 12 16 20 24 280

5

10

15

20

25

30

35

0 4 8 12 16 20 24 280.0

0.5

1.0

1.5

2.0

2.5

3.0

Rela

tive m

RN

A level

Rela

tive m

RN

A level

Rela

tive m

RN

A level

Cytokine Mix Cytokine Mix

*

*

*

*

*SOCS1 SOCS3

AdPHIP1

AdGFP

AdPHIP1

AdGFP

hrs. hrs.

1. We have identified a novel WD40 repeat-containing isoform of PHIP which expressing predominantly in the nuclei of beta-cells.

2. Overexpression of PHIP1 in β-cells results in enhanced levels of proliferation and decreased apoptosis.

3. The PHIP signaling network seems to regulate survival and apoptosis through a variety of intracellular mediators such as caspases, D-type cyclins, PKB and STAT1/SOCS1 signaling.

4. PHIP1 appears to be a new physiological regulator of IRS2 and STAT signaling in pancreatic -cells. This implies that a critical expression level of PHIP1 is required for general β-cell survival.

5.Approaches that promote PHIP1 expression in β-cells could provide important treatments for β-cell failure and diabetes.

Summary of Our Study

Acknowledgements

Collaborators:

Dr. Michael B. WheelerDr. Adria Giacca

Dr. H. Gaisano

Special Thanks to BBDC for providing financial support of this study