Control of beta-cell function by PHIP: Implications for Type I and II Diabetes Laboratory Medicine and Pathobiology Faculty of Medicine University of Toronto

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

Introduction PHPTB NPXpY ? Receptor interaction IR IRS1 pY SHP-2 NCK GRB2 PI3K IRS-1 PH 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) Relative PHIP mRNA expression MIN6 Islets Acinar Whole WATBrain Pancreas 7.3 Fold 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 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) 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 a-PHIP kDa PHIP1 PHIP Cont. Short Isoform Long Isoform Transfection a-PHIP Western Blotting

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

Adenoviral-mediated Expression of PHIP1 Enhances Proliferation in INS-1 cells (MTT Assay ) Hours after Infection Number of Viable Cells (OD450) (10%FBS) * IRS2 PHIP1 GFP

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

Adenoviral-mediated overexpression of PHIP1 enhances IGF1-dependent mitogenesis in INS-1 Cells (BRDU Assay) BRDU Incorporation (Fold Changes) IGF AdGFP AdPHIP1 AdIRS2 10nM 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 AdGFP AdPHIP1 AdIRS2 Luciferase Assay IGF AdGFP AdPHIP1 2.1 Fold 2.5 Fold Cyclin D2 promoter Luc Luciferase Units (Fold Changes) Cyclin D2 reporter

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. H 3 -Thymidine Incorporation (Fold Changes) Scr GFP SiPHIP GFP Scr IRS2 siPHIP IRS2 * IGF-1 (10nM) IGF-1 (10nM) PHIP1- IRS2- Cyclin D2- H 3 Thymidine Incorporation Assay Western Blotting Scr GFP SiPHIP GFP Scr IRS2 siPHIP IRS2 pSer 473 AKT- *

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

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

pSer 473 Akt /AKT ratio GFP BSA GFP OA PHIP1 OA IRS2 OA Akt- pSer 473 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) GFP BSA IGF-1 GFP OA PHIP1 OA IRS2 OA Caspase9 /Actin Caspase3 /Actin 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 and IRS2 have different subcellular localization in β-cells IF (Min6) Anti-PHIP Anti-IRS2 DAPI PHIP1 role in regulation AKT signalling PTEN mRNA SHIP2 mRNA mTOR mRNA 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 Hyperlipidemia Obesity Increased Peripheral Resistance 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 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 Body Weight, g (M+SEM) WT (n=19-25) TG-PHIP (n=14-18) 10,2% 25% Fed Blood Glucose, mM (M+SEM) Day 020weeks Normal Chow 20weeks HFDiet Day 020weeks Normal Chow 20weeks HFDiet 43% 27%

TG-PHIP Mice Have Improved Glucose Tolerance After 12 and 20 Weeks on High Fat Diet (GTTest) Blood Glucose mM (M+SEM) * * * * * (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?

Insulin (ng/ml*min/Cells DNA) Insulin (ng/ml/min/50 islets) 16.7mM Glucose GLP1 Phase I 16.7mM Glucose GLP1 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)

* Insulin Content (ng/ml/min/ 50 islets) Insulin Content (ng/ml*min/ DNA Content) INSULIN 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 2.5 fold Cont PHIP1 Cytokines (24hrs) - + Apoptosis (fold changes) Cont PHIP1 - + Cytokines (24hrs) PHIP1- Caspase3- Tubulin- MDM2- PSerAKT- Cytokines mix: Interferon-γ 50ng/ml IL-1β 10ng/ml

WT +cytokine mix TG-PHIP +cytokine mix INSULIN TUNEL DAPI PHIP1 Inhibits Induction of Cytokine-induced Apoptosis in Dispersed Islets of RIP7-PHIP1 mice (48hrs exposure; TUNEL assay) Wild Type Tg-PHIP1 Cytokines (24hrs) fold Merge INSULIN Merge TUNEL DAPI % of TUNEL positive β-cells

Q: Does PHIP1 knockdown enhances cytokine- induced apoptosis?

siRNAi-mediated PHIP1 Knockdown Enhances Cytokine- induced Apoptosis in INS1 Cells Apoptosis (fold changes) Cont siPHIP1 Cytokines (24hrs) fold Cytokines (24hrs) PHIP1- Caspase3- Tubulin- MDM2- PSerAKT- Cont siPHIP1 - +

PHIP1 Enhances Cytokine-induced Expression of STAT- dependent Antiapoptotic Genes IRF1, SOCS1 and SOCS3 (qPCR) Relative mRNA level * * * STAT1 IRF1 Relative mRNA level Cytokine Mix * * * * * SOCS1SOCS3 AdPHIP1 AdGFP AdPHIP1 AdGFP 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. Wheeler Dr. Adria Giacca Dr. H. Gaisano Special Thanks to BBDC for providing financial support of this study