Control of beta-cell function by PHIP: Implications for Type I and II Diabetes Laboratory Medicine...

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Transcript of Control of beta-cell function by PHIP: Implications for Type I and II Diabetes Laboratory Medicine...

  • Slide 1
  • Control of beta-cell function by PHIP: Implications for Type I and II Diabetes Laboratory Medicine and Pathobiology Faculty of Medicine University of Toronto
  • Slide 2
  • IGF-1 Signaling Pathways Play Crucial Role in Pancreatic -cell Function -Cell PH
  • Slide 3
  • Introduction PHPTB NPXpY ? Receptor interaction 11243 IR IRS1 pY SHP-2 NCK GRB2 PI3K 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)
  • Slide 4
  • Objective 1 Given the apparent role of PHIP in IRS signaling pathways and the importance of IRS proteins in proliferation and survival in -cells we sought to investigate the functional role of PHIP in -cells
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  • Q: Is PHIP expressed in beta-cells?
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  • PHIP is Highly Expressed In Mouse Islets (QPCR) Relative PHIP mRNA expression MIN6 Islets Acinar Whole WATBrain Pancreas 7.3 Fold Muscle
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  • Min6 INS-1 a-PHIP DAPI PHIP Is Localized to Nuclei of Insulinoma Cell Lines (IHC)
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  • 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
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  • 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
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  • Q: What isoform of PHIP1 predominantly expressed in beta-cells?
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  • PHIP1 a 206 kDa PHIP Isoform is Predominant Species in Mouse Islets and Insulinoma Cell Lines ISLETS INS1 MIN6 230 - 95 - a-PHIP 130 - 206 kDa 230 - 95 - 130 - PHIP1 PHIP Cont. Short Isoform Long Isoform Transfection a-PHIP Western Blotting
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  • Q: Does overexpression of PHIP1 influence the growth of pancreatic -cells ?
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  • 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
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  • Q: Does PHIP1 over expression influence the IGF-1 dependent growth of -cells ?
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  • Adenoviral-mediated overexpression of PHIP1 enhances IGF1-dependent mitogenesis in INS-1 Cells (BRDU Assay) BRDU Incorporation (Fold Changes) IGF-1 - + - + - + AdGFP AdPHIP1 AdIRS2 10nM 1.8 Fold 1.3 Fold
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  • Q: What possible mechanisms mediate increase in proliferation rate of PHIP1 overexpressing cells ?
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  • 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 2.1 Fold 2.5 Fold Cyclin D2 promoter Luc Luciferase Units (Fold Changes) Cyclin D2 reporter
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  • Q: Does PHIP1 is necessary component of IGF-1 and IRS2 signaling pathways leading to mitogenic responses?
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  • 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- *
  • Slide 20
  • Summary of PHIP Role in -cell Proliferation: Long Isoform of PHIP PHIP1 is predominantly expressed in nuclei of -cells PHIP1 promotes proliferation and potentiates IGF1- stimulated mitogenesis in -cells PHIP1 drives transcriptional induction of Cyclin D2 promoter PHIP1 expression is essential for IGF1 and IRS2-induced -cell replication
  • Slide 21
  • Q: Does PHIP1 plays any role in -cell apoptosis?
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  • 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 (-cell lipotoxicity) Inflammatory cytokines such as TNF-, IL-1, and Interferon- are cytotoxic to -cells and may contribute to -cell death in obesity which has been described as a state of low-level chronic inflammation TNF-, IL-1, and Interferon- are major mediators of -cell apoptosis in Type 1 diabetes Increased IRS2 expression or exposure to IGF1 inhibits FFA and cytokine-induced apoptosis in -cells Role of -cell apoptosis in diabetes
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  • To evaluate the effect of PHIP1 overexpression on FFA-induced apoptosis Objective 2
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  • - IGF1+ IGF1 Adenoviral-mediated Overexpression of PHIP1 Inhibits FFA-induced INS-1 Cell Apoptosis 2.6 4.1
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  • 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
  • Slide 26
  • 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
  • Slide 27
  • 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
  • Slide 28
  • Overexpression of kinase-dead AKT Blocks Protective Effect of PHIP1 on FFA-induced apoptosis AKT plays crucial role in antiapoptotic effect of PHIP1
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  • Q: How does PHIP1 increase activity of AKT? PHIP1 ? ? ? ? ? ?
  • Slide 30
  • PHIP1 and IRS2 have different subcellular localization in -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
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  • 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 - cells in vivo model of Type 2 Diabetes?
  • Slide 32
  • 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 - cell hyper- plasia Diabetes Hyperlipidemia Obesity Increased Peripheral Resistance to Insulin Hyperinsulinemia Failure of -cells to adapt to changes in metabolic demand High Fat Diet 3-5 months Overexpression of IRS2, AKT, PDX1, Glucokinase and CA-STAT5 in - cells blocks or alleviates development of HFD-induced Diabetes
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  • 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
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  • 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%
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  • 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
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  • Q: How does PHIP1 overexpression improve glucose tolerance?
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  • 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 -cells per islet? WT (n=3) TG-PHIP (n=3)
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  • * 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 -cells
  • Slide 39
  • Summary on PHIP role in HFD-induced Diabetes: Transgenic mice selectively ov