Reduced expression of the LRP16 gene in mouse insulinoma (MIN6) cells exerts multiple effects on insulin content, proliferation and apoptosis

Abstract

This study assessed the effects of leukemia-related protein 16 (LRP16) on the regulation of pancreatic functions in mouse insulinoma (MIN6) cells. Cells with down-regulated expression of LRP16 were obtained by a shRNA interference strategy. Insulin content and glucose-stimulated insulin secretion (GSIS) were examined by radioimmunoassay. Western blotting was applied to detect protein expression. Glucose-stimulated sub-cellular localization of PDX-1 was immunocytochemically determined. Cell proliferation and apoptosis were detected by flow cytometry. Our results showed that LRP16 regulated insulin content in MIN6 cells by controlling expression of insulin and insulin transcription factors. LRP16 gene silence in MIN6 cells led to reduced cell proliferation and increased apoptosis. The observation of phosphorylation of serine-473 Akt and the localization of PDX-1 to the nucleus under glucose-stimulation exhibited that LRP16 was a component mediating Akt signaling in MIN6 cells. These results suggest that LRP16 plays a key role in maintaining pancreatic β-cell functions and may help us to understand the protective effects of estrogen on the functions of pancreatic β-cells.

Keywords

LRP16 / MIN6 / insulin / Akt signaling / proliferation / apoptosis

References

[1] SachdevaM., StoffersD.. Minireview: Meeting the demand for insulin: molecular mechanisms of adaptive postnatal beta-cell mass expansion. Mol Endocrinol, 2009, 23(6): 747-758

[2] CampbellR.. Type 2 diabetes: where we are today: an overview of disease burden, current treatments, and treatment strategies. J Am Pharm Asso, 2009, 49(Suppl1): S3-S9

[3] Le MayC., ChuK., HuM., et al.. Estrogens protect pancreatic beta-cells from apoptosis and prevent insulin-deficient diabetes mellitus in mice. Proc Natl Acad Sci USA, 2006, 103(24): 9232-9237

[4] RochiraV., BalestrieriA., MadeoB., et al.. Congenital estrogen deficiency in men: a new syndrome with different phenotypes; clinical and therapeutic implications in men. Mol Cell Endocrinol, 2002, 93(1–2): 19-28

[5] GodslandI.F.. Oestrogens and insulin secretion. Diabetologia, 2005, 48(11): 2213-2220

[6] LiuS., Mauvais-JarvisF.. Minireview: Estrogenic protection of beta-cell failure in metabolic diseases. Endocri nology, 2010, 151(3): 859-864

[7] KooptiwutS., SemprasertN., ChearskulS.. Estrogen increases glucose-induced insulin secretion from mouse pancreatic islets cultured in a prolonged high glucose condition. J Med Assoc Thai, 2007, 90(5): 956-961

[8] RenZ., ZouC., JiH., et al.. Oestrogen regulates proliferation and differentiation of human islet-derived precursor cells through oestrogen receptor alpha. Cell Biol Int, 2010, 34(5): 523-530

[9] SorianoS., RoperoA.B., Alonso-MagdalenaP., et al.. Rapid regulation of K(ATP) channel activity by 17 beta-estradiol in pancreatic beta-cells involves the estrogen receptor beta and the atrial natriuretic peptide receptor. Mol Endocrinol, 2009, 23(12): 1973-1982

[10] Alonso-MagdalenaP., RoperoA., CarreraM., et al.. Pancreatic insulin content regulation by the estrogen receptor ER alpha. PLoS One, 2008, 3(4): e2069

[11] HanW., YuL., LouF., et al.. The Application of RACE Technique to Clone the Full-Length cDNA of A Novel Leukemia Associated Gene LRP16. Zhongguo Shi Yan Xue Ye Xue Za Zhi (Chinese), 2001, 9(1): 18-21

[12] HanW.D., ZhaoY.L., MengY.G., et al.. Estrogenically regulated LRP16 interacts with estrogen receptor alpha and enhances the receptor’s transcriptional activity. Endocr Relat Cancer, 2007, 14(3): 741-753

[13] GloverL.E., NewtonK., KrishnanG., et al.. Dysferlin overexpression in skeletal muscle produces a progressive myopathy. Ann Neurol, 2010, 67(3): 384-393

[14] WangZ., GleichmannH.. GLUT2 in pancreatic islets: crucial target molecule in diabetes induced with multiple low doses of streptozotocin in mice. Diabete, 1998, 47(1): 50-56

[15] PalM.. Medicinal chemistry approaches for glucokinase activation to treat type 2 diabetes. Curr Med Chem, 2009, 16(29): 3858-3874

[16] Van SchaftingenE., DetheuxM., Veiga da CunhaM.. Short-term control of glucokinase activity: role of a regulatory protein. FASEB J, 1994, 8(6): 414-419

[17] EfratS., TalM., LodishH.F.. The pancreatic beta-cell glucose sensor. Trends Biochem Sci, 1994, 19(12): 535-538

[18] ShaoS., FangZ., YuX., et al.. Transcription factors involved in glucose-stimulated insulin secretion of pancreatic beta cells. Biochem Biophys Res Commun, 2009, 384(4): 401-404

[19] CerfM.E.. Transcription factors regulating beta-cell function. Eur J Endocrinol, 2006, 155(5): 671-679

[20] BernardoA.S., HayC.W., DochertyK.. Pancreatic transcription factors and their role in the birth, life and survival of the pancreatic beta cell. Mol Cell Endocrinol, 2008, 294(1‐2): 1-9

[21] ElghaziL., Bernal-MizrachiE.. Akt and PTEN: beta-cell mass and pancreas plasticity. Trends Endocrinol Metab, 2009, 20(5): 243-251

[22] ElghaziL., RachdiL., WeissA.J., et al.. Regulation of beta-cell mass and function by the Akt/protein kinase B signalling pathway. Diabetes Obes Metab, 2007, 9(Suppl2): 147-157

[23] LawrenceM.C., McGlynnK., ParkB.H., et al.. ERK1/2-dependent activation of transcription factors required for acute and chronic effects of glucose on the insulin gene promoter. J Biol Chem, 2005, 280(29): 26 751-26 759

[24] LawrenceM., ShaoC., DuanL., et al.. The protein kinases ERK1/2 and their roles in pancreatic beta cells. Acta Physiol, 2008, 192(1): 11-17

[25] Le LayJ., SteinR.. Involvement of PDX-1 in activation of human insulin gene transcription. J Endocrinol, 2006, 188(2): 287-294

[26] FinegoodD.T., ScagliaL., Bonner-WeirS.. Dynamics of beta-cell mass in the growing rat pancreas. Estimation with a simple mathematical model. Diabetes, 1995, 44(3): 249-256

[27] HagmanD.K., HaysL.B., ParazzoliS.D., et al.. Palmitate inhibits insulin gene expression by altering PDX-1 nuclear localization and reducing MafA expression in isolated rat islets of Langerhans. J Biol Chem, 2005, 280(37): 32 413-32 418

[28] MacfarlaneW.M., McKinnonC.M., Felton-EdkinsZ.A., et al.. Glucose stimulates translocation of the homeodomain transcription factor PDX1 from the cytoplasm to the nucleus in pancreatic beta-cells. J Biol Chem, 1999, 274(2): 1011-1016

[29] KushnerJ.A., CiemerychM.A., SicinskaE., et al.. Cyclins D2 and D1 are essential for postnatal pancreatic beta-cell growth. Mol Cell Biol, 2005, 25(9): 3752-3762

[30] Cozar-CastellanoI., Fiaschi-TaeschN., BigatelT., et al.. Molecular control of cell cycle progression in the pancreatic beta-cell. Endocr Rev, 2006, 27(4): 356-370

[31] RaneS.G., DubusP., MettusR.V., et al.. Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in beta-islet cell hyperplasia. Nat Genet, 1999, 22(1): 44-52

[32] TsutsuiT., HesabiB., MoonsD.S., et al.. Targeted disruption of CDK4 delays cell cycle entry with enhanced p27(Kip1) activity. Mol Cell Biol, 1999, 19(10): 7011-7019

[33] WangJ., ShenL., NajafiH., et al.. Regulation of insulin preRNA splicing by glucose. Proc Natl Acad Sci USA, 1997, 94(9): 4360-4365

[34] IypeT., FrancisJ., GarmeyJ.C., et al.. 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, 2005, 280(17): 16 798-16 807

[35] GlickE., LeshkowitzD., WalkerM.D.. Transcription factor BETA2 acts cooperatively with E2A and PDX1 to activate the insulin gene promoter. J Biol Chem, 2000, 275(3): 2199-2204

[36] VanderfordN., AndraliS., OzcanS.. Glucose induces MafA expression in pancreatic beta cell lines via the hexosamine biosynthetic pathway. J Biol Chem, 2007, 282(3): 1577-1584

[37] NayaF., StellrechtC., TsaiM.. Tissue-specific regulation of the insulin gene by a novel basic helix-loop-helix transcription factor. Genes, 1995, 9(8): 1009-1019

[38] KitamuraT., NakaeJ., KitamuraY., et al.. The forkhead transcription factor Foxo1 links insulin signaling to Pdx1 regulation of pancreatic beta cell growth. J Clin Invest, 2002, 110(12): 1839-1847

[39] Bernal-MizrachiE., WenW., StahlhutS., et al.. Islet beta cell expression of constitutively active Akt1/PKB alpha induces striking hypertrophy, hyperplasia, and hyperinsulinemia. J Clin Invest, 2001, 108(11): 1631-1638