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An siRNA Screen in Pancreatic Beta Cells Reveals a Role for in Insulin Production


The prevalence of type 2 diabetes in the United States is projected to double or triple by 2050. We reasoned that the genes that modulate insulin production might be new targets for diabetes therapeutics. Therefore, we developed an siRNA screening system to identify genes important for the activity of the insulin promoter in beta cells. We created a subclone of the MIN6 mouse pancreatic beta cell line that expresses destabilized GFP under the control of a 362 base pair fragment of the human insulin promoter and the mCherry red fluorescent protein under the control of the constitutively active rous sarcoma virus promoter. The ratio of the GFP to mCherry fluorescence of a cell indicates its insulin promoter activity. As G protein coupled receptors (GPCRs) have emerged as novel targets for diabetes therapies, we used this cell line to screen an siRNA library targeting all known mouse GPCRs. We identified several known GPCR regulators of insulin secretion as regulators of the insulin promoter. One of the top positive regulators was Gpr27, an orphan GPCR with no known role in beta cell function. We show that knockdown of Gpr27 reduces endogenous mouse insulin promoter activity and glucose stimulated insulin secretion. Furthermore, we show that Pdx1 is important for Gpr27's effect on the insulin promoter and insulin secretion. Finally, the over-expression of Gpr27 in 293T cells increases inositol phosphate levels, while knockdown of Gpr27 in MIN6 cells reduces inositol phosphate levels, suggesting this orphan GPCR might couple to Gq/11. In summary, we demonstrate a MIN6-based siRNA screening system that allows rapid identification of novel positive and negative regulators of the insulin promoter. Using this system, we identify Gpr27 as a positive regulator of insulin production.


Vyšlo v časopise: An siRNA Screen in Pancreatic Beta Cells Reveals a Role for in Insulin Production. PLoS Genet 8(1): e32767. doi:10.1371/journal.pgen.1002449
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1002449

Souhrn

The prevalence of type 2 diabetes in the United States is projected to double or triple by 2050. We reasoned that the genes that modulate insulin production might be new targets for diabetes therapeutics. Therefore, we developed an siRNA screening system to identify genes important for the activity of the insulin promoter in beta cells. We created a subclone of the MIN6 mouse pancreatic beta cell line that expresses destabilized GFP under the control of a 362 base pair fragment of the human insulin promoter and the mCherry red fluorescent protein under the control of the constitutively active rous sarcoma virus promoter. The ratio of the GFP to mCherry fluorescence of a cell indicates its insulin promoter activity. As G protein coupled receptors (GPCRs) have emerged as novel targets for diabetes therapies, we used this cell line to screen an siRNA library targeting all known mouse GPCRs. We identified several known GPCR regulators of insulin secretion as regulators of the insulin promoter. One of the top positive regulators was Gpr27, an orphan GPCR with no known role in beta cell function. We show that knockdown of Gpr27 reduces endogenous mouse insulin promoter activity and glucose stimulated insulin secretion. Furthermore, we show that Pdx1 is important for Gpr27's effect on the insulin promoter and insulin secretion. Finally, the over-expression of Gpr27 in 293T cells increases inositol phosphate levels, while knockdown of Gpr27 in MIN6 cells reduces inositol phosphate levels, suggesting this orphan GPCR might couple to Gq/11. In summary, we demonstrate a MIN6-based siRNA screening system that allows rapid identification of novel positive and negative regulators of the insulin promoter. Using this system, we identify Gpr27 as a positive regulator of insulin production.


Zdroje

1. NarayanKMBoyleJPThompsonTJSorensenSWWilliamsonDF 2003 Lifetime risk for diabetes mellitus in the United States. JAMA 290 1884 1890

2. CowieCCRustKFFordESEberhardtMSByrd-HoltDD 2009 Full accounting of diabetes and pre-diabetes in the U.S. population in 1988–1994 and 2005–2006. Diabetes Care 32 287 294

3. UKPDS 1998 Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). UK Prospective Diabetes Study (UKPDS) Group. Lancet 352 837 853

4. MiyazakiJArakiKYamatoEIkegamiHAsanoT 1990 Establishment of a pancreatic beta cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms. Endocrinology 127 126 132

5. OdagiriHWangJGermanMS 1996 Function of the human insulin promoter in primary cultured islet cells. J Biol Chem 271 1909 1915

6. IypeTFrancisJGarmeyJCSchislerJCNesherR 2005 Mechanism of insulin gene regulation by the pancreatic transcription factor Pdx-1: application of pre-mRNA analysis and chromatin immunoprecipitation to assess formation of functional transcriptional complexes. J Biol Chem 280 16798 16807

7. KonigRChiangCYTuBPYanSFDeJesusPD 2007 A probability-based approach for the analysis of large-scale RNAi screens. Nat Methods 4 847 849

8. KimHToyofukuYLynnFCChakEUchidaT Serotonin regulates pancreatic beta cell mass during pregnancy. Nat Med 16 804 808

9. GriffithMGriffithOLMwenifumboJGoyaRMorrissyAS Alternative expression analysis by RNA sequencing. Nat Methods 7 843 847

10. GaultonKJNammoTPasqualiLSimonJMGiresiPG A map of open chromatin in human pancreatic islets. Nat Genet 42 255 259

11. RegardJBKataokaHCanoDACamererEYinL 2007 Probing cell type-specific functions of Gi in vivo identifies GPCR regulators of insulin secretion. J Clin Invest 117 4034 4043

12. WelshMNielsenDAMacKrellAJSteinerDF 1985 Control of insulin gene expression in pancreatic beta-cells and in an insulin-producing cell line, RIN-5F cells. II. Regulation of insulin mRNA stability. J Biol Chem 260 13590 13594

13. TakahashiRIshiharaHTakahashiKTamuraAYamaguchiS 2007 Efficient and controlled gene expression in mouse pancreatic islets by arterial delivery of tetracycline-inducible adenoviral vectors. J Mol Endocrinol 38 127 136

14. BrissovaMShiotaMNicholsonWEGannonMKnobelSM 2002 Reduction in pancreatic transcription factor PDX-1 impairs glucose-stimulated insulin secretion. J Biol Chem 277 11225 11232

15. GauthierBRWiederkehrABaquieMDaiCPowersAC 2009 PDX1 deficiency causes mitochondrial dysfunction and defective insulin secretion through TFAM suppression. Cell Metab 10 110 118

16. SgourakisNGBagosPGHamodrakasSJ 2005 Prediction of the coupling specificity of GPCRs to four families of G-proteins using hidden Markov models and artificial neural networks. Bioinformatics 21 4101 4106

17. GriffithMTangMJGriffithOLMorinRDChanSY 2008 ALEXA: a microarray design platform for alternative expression analysis. Nat Methods 5 118

18. YangCLeeBChenTHHsuWH 1997 Mechanisms of bradykinin-induced insulin secretion in clonal beta cell line RINm5F. J Pharmacol Exp Ther 282 1247 1252

19. YangCHsuWH 1995 Stimulatory effect of bradykinin on insulin release from the perfused rat pancreas. Am J Physiol 268 E1027 1030

20. ParandehFAbaravicieneSMAmistenSErlingeDSalehiA 2008 Uridine diphosphate (UDP) stimulates insulin secretion by activation of P2Y6 receptors. Biochem Biophys Res Commun 370 499 503

21. SassmannAGierBGroneHJDrewsGOffermannsS The Gq/G11-mediated signaling pathway is critical for autocrine potentiation of insulin secretion in mice. J Clin Invest 120 2184 2193

22. KebedeMAAlquierTLatourMGPoitoutV 2009 Lipid receptors and islet function: therapeutic implications? Diabetes Obes Metab 11 Suppl 4 10 20

23. MatsumotoMSaitoTTakasakiJKamoharaMSugimotoT 2000 An evolutionarily conserved G-protein coupled receptor family, SREB, expressed in the central nervous system. Biochem Biophys Res Commun 272 576 582

24. RegardJBSatoITCoughlinSR 2008 Anatomical profiling of G protein-coupled receptor expression. Cell 135 561 571

25. JuhlKSarkarSAWongRJensenJHuttonJC 2008 Mouse pancreatic endocrine cell transcriptome defined in the embryonic Ngn3-null mouse. Diabetes 57 2755 2761

26. GuGWellsJMDombkowskiDPrefferFAronowB 2004 Global expression analysis of gene regulatory pathways during endocrine pancreatic development. Development 131 165 179

27. GuettierJMGautamDScarselliMRuiz de AzuaILiJH 2009 A chemical-genetic approach to study G protein regulation of beta cell function in vivo. Proc Natl Acad Sci U S A 106 19197 19202

28. KongKCButcherAJMcWilliamsPJonesDWessJ M3-muscarinic receptor promotes insulin release via receptor phosphorylation/arrestin-dependent activation of protein kinase D1. Proc Natl Acad Sci U S A

29. MillarRPNewtonCL The year in G protein-coupled receptor research. Mol Endocrinol 24 261 274

30. WeiHAhnSShenoySKKarnikSSHunyadyL 2003 Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2. Proc Natl Acad Sci U S A 100 10782 10787

31. LevoyeADamJAyoubMAGuillaumeJLJockersR 2006 Do orphan G-protein-coupled receptors have ligand-independent functions? New insights from receptor heterodimers. EMBO Rep 7 1094 1098

32. OhnedaKEeHGermanM 2000 Regulation of insulin gene transcription. Semin Cell Dev Biol 11 227 233

33. JohnsonJDZhangWRudnickARutterWJGermanMS 1997 Transcriptional synergy between LIM-homeodomain proteins and basic helix-loop-helix proteins: the LIM2 domain determines specificity. Mol Cell Biol 17 3488 3496

34. SanderMGriffenSCHuangJGermanMS 1998 A novel glucose-responsive element in the human insulin gene functions uniquely in primary cultured islets. Proc Natl Acad Sci U S A 95 11572 11577

35. ChangWCNgJKNguyenTPellissierLClaeysenS 2007 Modifying ligand-induced and constitutive signaling of the human 5-HT4 receptor. PLoS ONE 2 e1317 doi:10.1371/journal.pone.0001317

36. VenturaAMeissnerADillonCPMcManusMSharpPA 2004 Cre-lox-regulated conditional RNA interference from transgenes. Proc Natl Acad Sci U S A 101 10380 10385

37. HeTCZhouSda CostaLTYuJKinzlerKW 1998 A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A 95 2509 2514

38. LuoJDengZLLuoXTangNSongWX 2007 A protocol for rapid generation of recombinant adenoviruses using the AdEasy system. Nat Protoc 2 1236 1247

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Genetika Reprodukčná medicína

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PLOS Genetics


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