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• W2040982134 abstract "Insulin receptor substrate-2 (IRS-2) plays a critical role in the survival and function of pancreatic β-cells. Gene disruption of IRS-2 results in failure of the β-cell compensatory mechanism and diabetes. Nonetheless, the regulation of IRS-2 protein expression in β-cells remains largely unknown. Inducible nitric-oxide synthase (iNOS), a major mediator of inflammation, has been implicated in β-cell damage in type 1 and type 2 diabetes. The effects of iNOS on IRS-2 expression have not yet been investigated in β-cells. Here, we show that iNOS and NO donor decreased IRS-2 protein expression in INS-1/832 insulinoma cells and mouse islets, whereas IRS-2 mRNA levels were not altered. Interleukin-1β (IL-1β), alone or in combination with interferon-γ (IFN-γ), reduced IRS-2 protein expression in an iNOS-dependent manner without altering IRS-2 mRNA levels. Proteasome inhibitors, MG132 and lactacystin, blocked the NO donor-induced reduction in IRS-2 protein expression. Treatment with NO donor led to activation of glycogen synthase kinase-3β (GSK-3β) and c-Jun N-terminal kinase (JNK/SAPK) in β-cells. Inhibition of GSK-3β by pharmacological inhibitors or siRNA-mediated knockdown significantly prevented NO donor-induced reduction in IRS-2 expression in β-cells. In contrast, a JNK inhibitor, SP600125, did not effectively block reduced IRS-2 expression in NO donor-treated β-cells. These data indicate that iNOS-derived NO reduces IRS-2 expression by promoting protein degradation, at least in part, through a GSK-3β-dependent mechanism. Our findings suggest that iNOS-mediated decreased IRS-2 expresssion may contribute to the progression and/or exacerbation of β-cell failure in diabetes. Insulin receptor substrate-2 (IRS-2) plays a critical role in the survival and function of pancreatic β-cells. Gene disruption of IRS-2 results in failure of the β-cell compensatory mechanism and diabetes. Nonetheless, the regulation of IRS-2 protein expression in β-cells remains largely unknown. Inducible nitric-oxide synthase (iNOS), a major mediator of inflammation, has been implicated in β-cell damage in type 1 and type 2 diabetes. The effects of iNOS on IRS-2 expression have not yet been investigated in β-cells. Here, we show that iNOS and NO donor decreased IRS-2 protein expression in INS-1/832 insulinoma cells and mouse islets, whereas IRS-2 mRNA levels were not altered. Interleukin-1β (IL-1β), alone or in combination with interferon-γ (IFN-γ), reduced IRS-2 protein expression in an iNOS-dependent manner without altering IRS-2 mRNA levels. Proteasome inhibitors, MG132 and lactacystin, blocked the NO donor-induced reduction in IRS-2 protein expression. Treatment with NO donor led to activation of glycogen synthase kinase-3β (GSK-3β) and c-Jun N-terminal kinase (JNK/SAPK) in β-cells. Inhibition of GSK-3β by pharmacological inhibitors or siRNA-mediated knockdown significantly prevented NO donor-induced reduction in IRS-2 expression in β-cells. In contrast, a JNK inhibitor, SP600125, did not effectively block reduced IRS-2 expression in NO donor-treated β-cells. These data indicate that iNOS-derived NO reduces IRS-2 expression by promoting protein degradation, at least in part, through a GSK-3β-dependent mechanism. Our findings suggest that iNOS-mediated decreased IRS-2 expresssion may contribute to the progression and/or exacerbation of β-cell failure in diabetes. All forms of diabetes develop when pancreatic β-cells can no longer secret a sufficient amount of insulin to maintain normal blood glucose levels. Hence, β-cell failure is the key event in the development of diabetes. Damage, death, and growth arrest of β-cells are, therefore, major contributors to the progression of diabetes. Insulin receptor substrate-2 (IRS-2) plays a crucial role in functional β-cell mass (1Withers D.J. Gutierrez J.S. Towery H. Burks D.J. Ren J.M. Previs S. Zhang Y. Bernal D. Pons S. Shulman G.I. Bonner-Weir S. White M.F. Nature. 1998; 391: 900-904Crossref PubMed Scopus (1347) Google Scholar, 2Kubota N. Terauchi Y. Tobe K. Yano W. Suzuki R. Ueki K. Takamoto I. Satoh H. Maki T. Kubota T. Moroi M. Okada-Iwabu M. Ezaki O. Nagai R. Ueta Y. Kadowaki T. Noda T. J. Clin. Invest. 2004; 114: 917-927Crossref PubMed Scopus (210) Google Scholar, 3Choudhury A.I. Heffron H. Smith M.A. Al-Qassab H. Xu A.W. Selman C. Simmgen M. Clements M. Claret M. Maccoll G. Bedford D.C. Hisadome K. Diakonov I. Moosajee V. Bell J.D. Speakman J.R. Batterham R.L. Barsh G.S. Ashford M.L. Withers D.J. J. Clin. Invest. 2005; 115: 940-950Crossref PubMed Scopus (209) Google Scholar, 4Lin X. Taguchi A. Park S. Kushner J.A. Li F. Li Y. White M.F. J. Clin. Invest. 2004; 114: 908-916Crossref PubMed Scopus (262) Google Scholar, 5Terauchi Y. Takamoto I. Kubota N. Matsui J. Suzuki R. Komeda K. Hara A. Toyoda Y. Miwa I. Aizawa S. Tsutsumi S. Tsubamoto Y. Hashimoto S. Eto K. Nakamura A. Noda M. Tobe K. Aburatani H. Nagai R. Kadowaki T. J. Clin. Invest. 2007; 117: 246-257Crossref PubMed Scopus (278) Google Scholar, 6Kubota N. Tobe K. Terauchi Y. Eto K. Yamauchi T. Suzuki R. Tsubamoto Y. Komeda K. Nakano R. Miki H. Satoh S. Sekihara H. Sciacchitano S. Lesniak M. Aizawa S. Nagai R. Kimura S. Akanuma Y. Taylor S.I. Kadowaki T. Diabetes. 2000; 49: 1880-1889Crossref PubMed Scopus (432) Google Scholar, 7Withers D.J. Burks D.J. Towery H.H. Altamuro S.L. Flint C.L. White M.F. Nat. Genet. 1999; 23: 32-40Crossref PubMed Scopus (485) Google Scholar). IRS-2 is essential for proliferation and insulin-stimulated activation of phosphatidylinositol 3-kinase and Akt in β-cells (8Assmann A. Ueki K. Winnay J.N. Kadowaki T. Kulkarni R.N. Mol. Cell Biol. 2009; 29: 3219-3228Crossref PubMed Scopus (119) Google Scholar). Global gene disruption of IRS-2 results in overt diabetes along with β-cell compensation failure (1Withers D.J. Gutierrez J.S. Towery H. Burks D.J. Ren J.M. Previs S. Zhang Y. Bernal D. Pons S. Shulman G.I. Bonner-Weir S. White M.F. Nature. 1998; 391: 900-904Crossref PubMed Scopus (1347) Google Scholar). IRS-2 is required for adaptive expansion of β-cell mass in response to high fat diet feeding (5Terauchi Y. Takamoto I. Kubota N. Matsui J. Suzuki R. Komeda K. Hara A. Toyoda Y. Miwa I. Aizawa S. Tsutsumi S. Tsubamoto Y. Hashimoto S. Eto K. Nakamura A. Noda M. Tobe K. Aburatani H. Nagai R. Kadowaki T. J. Clin. Invest. 2007; 117: 246-257Crossref PubMed Scopus (278) Google Scholar). In contrast, IRS-1 knock-out mice are insulin-resistant but exhibit normoglycemia because of compensatory hyperinsulinemia associated with adaptive expansion of β-cell mass (9Tamemoto H. Kadowaki T. Tobe K. Yagi T. Sakura H. Hayakawa T. Terauchi Y. Ueki K. Kaburagi Y. Satoh S. Nature. 1994; 372: 182-186Crossref PubMed Scopus (906) Google Scholar). Moreover, β-cell- or pancreas-specific gene disruption of IRS-2 results in reduced β-cell mass, glucose intolerance, and attenuated glucose-stimulated insulin secretion in mice (2Kubota N. Terauchi Y. Tobe K. Yano W. Suzuki R. Ueki K. Takamoto I. Satoh H. Maki T. Kubota T. Moroi M. Okada-Iwabu M. Ezaki O. Nagai R. Ueta Y. Kadowaki T. Noda T. J. Clin. Invest. 2004; 114: 917-927Crossref PubMed Scopus (210) Google Scholar, 10Cantley J. Choudhury A.I. Asare-Anane H. Selman C. Lingard S. Heffron H. Herrera P. Persaud S.J. Withers D.J. Diabetologia. 2007; 50: 1248-1256Crossref PubMed Scopus (66) Google Scholar). Conversely, β-cell-specific ectopic expression of IRS-2 prevents high fat diet-induced diabetes (11Hennige A.M. Burks D.J. Ozcan U. Kulkarni R.N. Ye J. Park S. Schubert M. Fisher T.L. Dow M.A. Leshan R. Zakaria M. Mossa-Basha M. White M.F. J. Clin. Invest. 2003; 112: 1521-1532Crossref PubMed Scopus (227) Google Scholar) and alleviates and/or delays β-cell destruction and diabetes development in nonobese diabetic mice (12Norquay L.D. D'Aquino K.E. Opare-Addo L.M. Kuznetsova A. Haas M. Bluestone J.A. White M.F. Endocrinology. 2009; 150: 4531-4540Crossref PubMed Scopus (18) Google Scholar) and streptozotocin-treated mice (11Hennige A.M. Burks D.J. Ozcan U. Kulkarni R.N. Ye J. Park S. Schubert M. Fisher T.L. Dow M.A. Leshan R. Zakaria M. Mossa-Basha M. White M.F. J. Clin. Invest. 2003; 112: 1521-1532Crossref PubMed Scopus (227) Google Scholar). Limited knowledge is, however, available about IRS-2 expression in islets in diabetes. Of interest, high fat diet reduced IRS-2 protein expression in islets of 90% pancreatectomized rats without alteration in IRS-2 mRNA expression (13Park S. Hong S.M. Lee J.E. Sung S.R. J. Appl. Physiol. 2007; 103: 1764-1771Crossref PubMed Scopus (52) Google Scholar). These data raise the possibility that high fat diet feeding might reduce IRS-2 protein expression by enhancing protein degradation. Interleukin-1β (IL-1β) has been proposed as a common important player in the pathogenesis of type 1 and type 2 diabetes (14Cnop M. Welsh N. Jonas J.C. Jörns A. Lenzen S. Eizirik D.L. Diabetes. 2005; 54: S97-S107Crossref PubMed Scopus (1200) Google Scholar). The blockade of IL-1β improves glycemic control and β-cell function in patients with type 2 diabetes (15Larsen C.M. Faulenbach M. Vaag A. Vølund A. Ehses J.A. Seifert B. Mandrup-Poulsen T. Donath M.Y. N. Engl. J. Med. 2007; 356: 1517-1526Crossref PubMed Scopus (1445) Google Scholar). A previous study has shown that circulating levels of IL-1 receptor antagonist, an endogenous antagonist of IL-1β, is associated with β-cell capacity in patients with type 1 diabetes (16Pfleger C. Mortensen H.B. Hansen L. Herder C. Roep B.O. Hoey H. Aanstoot H.J. Kocova M. Schloot N.C. Diabetes. 2008; 57: 929-937Crossref PubMed Scopus (69) Google Scholar). In nonobese diabetic mice, gene disruption of IL-1β receptor delays the development of diabetes (17Thomas H.E. Irawaty W. Darwiche R. Brodnicki T.C. Santamaria P. Allison J. Kay T.W. Diabetes. 2004; 53: 113-121Crossref PubMed Scopus (114) Google Scholar). Nitric oxide (NO) produced by inducible nitric-oxide synthase (iNOS, 2The abbreviations used are: iNOSinducible nitric-oxide synthasemTORmammalian target of rapamycinp70S6Kp70 ribosomal S6 kinaseGSNOS-nitroso-l-glutathioneSNAPS-nitroso-N-acetyl-dl-penicillamineL-NILN6-(1-iminoethyl)-l-lysinePTIO2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide. also known as NOS2) has been implicated in β-cell damage and death in both type 1 and type 2 diabetes (18Corbett J.A. Mikhael A. Shimizu J. Frederick K. Misko T.P. McDaniel M.L. Kanagawa O. Unanue E.R. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 8992-8995Crossref PubMed Scopus (151) Google Scholar, 19Corbett J.A. McDaniel M.L. J. Exp. Med. 1995; 181: 559-568Crossref PubMed Scopus (235) Google Scholar, 20Thomas H.E. Darwiche R. Corbett J.A. Kay T.W. Diabetes. 2002; 51: 311-316Crossref PubMed Scopus (152) Google Scholar, 21Steer S.A. Scarim A.L. Chambers K.T. Corbett J.A. PLoS Med. 2006; 3: e17Crossref PubMed Scopus (124) Google Scholar) and during islet transplantation (22Montolio M. Biarnés M. Téllez N. Escoriza J. Soler J. Montanya E. J. Endocrinol. 2007; 192: 169-177Crossref PubMed Scopus (56) Google Scholar, 23Chen Y.T. Fu S.H. Chen J.P. Hsu B.R. Transplant. Proc. 2009; 41: 1786-1788Crossref PubMed Scopus (1) Google Scholar, 24Brandhorst D. Brandhorst H. Zwolinski A. Nahidi F. Bretzel R.G. Transplantation. 2001; 71: 179-184Crossref PubMed Scopus (34) Google Scholar). Previous studies have shown that iNOS expression plays an important role in IL-1β-induced dysfunction and death of cultured β-cells (20Thomas H.E. Darwiche R. Corbett J.A. Kay T.W. Diabetes. 2002; 51: 311-316Crossref PubMed Scopus (152) Google Scholar, 25Corbett J.A. Sweetland M.A. Wang J.L. Lancaster Jr., J.R. McDaniel M.L. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 1731-1735Crossref PubMed Scopus (405) Google Scholar, 26McCabe C. O'Brien T. Biochem. Biophys. Res. Commun. 2007; 357: 75-80Crossref PubMed Scopus (11) Google Scholar, 27Kato Y. Miura Y. Yamamoto N. Ozaki N. Oiso Y. Diabetologia. 2003; 46: 1228-1233Crossref PubMed Scopus (19) Google Scholar, 28Rydgren T. Sandler S. Eur. J. Endocrinol. 2002; 147: 543-551Crossref PubMed Scopus (31) Google Scholar), although other studies have concluded that cytokine induces β-cell damage through both NO-dependent and -independent pathways (29Zumsteg U. Frigerio S. Holländer G.A. Diabetes. 2000; 49: 39-47Crossref PubMed Scopus (90) Google Scholar, 30Andersson A.K. Börjesson A. Sandgren J. Sandler S. Mol. Cell. Endocrinol. 2005; 240: 50-57Crossref PubMed Scopus (17) Google Scholar). The expression of iNOS is increased in β-cells and intraislet macrophages of rodent models of type 1 and type 2 diabetes (18Corbett J.A. Mikhael A. Shimizu J. Frederick K. Misko T.P. McDaniel M.L. Kanagawa O. Unanue E.R. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 8992-8995Crossref PubMed Scopus (151) Google Scholar, 31Rabinovitch A. Suarez-Pinzon W.L. Sorensen O. Bleackley R.C. Endocrinology. 1996; 137: 2093-2099Crossref PubMed Scopus (124) Google Scholar, 32Kleemann R. Rothe H. Kolb-Bachofen V. Xie Q.W. Nathan C. Martin S. Kolb H. FEBS Lett. 1993; 328: 9-12Crossref PubMed Scopus (132) Google Scholar, 33Salehi A. Meidute Abaraviciene S. Jimenez-Feltstrom J. Ostenson C.G. Efendic S. Lundquist I. PLoS One. 2008; 3: e2165Crossref PubMed Scopus (43) Google Scholar, 34Reddy S. Young M. Ginn S. Histochem. J. 2001; 33: 317-327Crossref PubMed Scopus (26) Google Scholar, 35Reddy S. Yip S. Karanam M. Poole C.A. Ross J.M. Histochem. J. 1999; 31: 303-314Crossref PubMed Scopus (20) Google Scholar). Aminoguanidine, an iNOS inhibitor, prevents the development of obesity-induced diabetes in Zucker fa/fa rats (36Shimabukuro M. Ohneda M. Lee Y. Unger R.H. J. Clin. Invest. 1997; 100: 290-295Crossref PubMed Scopus (255) Google Scholar). iNOS depletion and iNOS inhibitor have been shown to block or ameliorate diabetes development in multiple low dose streptozotocin-treated mice and nonobese diabetic mice, murine models of type 1 diabetes (18Corbett J.A. Mikhael A. Shimizu J. Frederick K. Misko T.P. McDaniel M.L. Kanagawa O. Unanue E.R. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 8992-8995Crossref PubMed Scopus (151) Google Scholar, 27Kato Y. Miura Y. Yamamoto N. Ozaki N. Oiso Y. Diabetologia. 2003; 46: 1228-1233Crossref PubMed Scopus (19) Google Scholar, 28Rydgren T. Sandler S. Eur. J. Endocrinol. 2002; 147: 543-551Crossref PubMed Scopus (31) Google Scholar, 37Flodström M. Tyrberg B. Eizirik D.L. Sandler S. Diabetes. 1999; 48: 706-713Crossref PubMed Scopus (146) Google Scholar), although controversial results have been also reported (38Papaccio G. Pisanti F.A. Latronico M.V. Ammendola E. Galdieri M. J. Cell Biochem. 2000; 77: 82-91Crossref PubMed Scopus (66) Google Scholar, 39Yasuda H. Jin Z. Nakayama M. Yamada K. Kishi M. Okumachi Y. Arai T. Moriyama H. Yokono K. Nagata M. Diabetes Res. Clin. Pract. 2009; 83: 200-207Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Moreover, β-cell-specific iNOS expression per se leads to insulin-dependent diabetes and loss of β-cells without insulitis in mice (41Takamura T. Kato I. Kimura N. Nakazawa T. Yonekura H. Takasawa S. Okamoto H. J. Biol. Chem. 1998; 273: 2493-2496Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). However, it is not fully understood how NO and iNOS induce and/or exacerbate β-cell damage and loss of functional β-cell mass in diabetes. Here, we show that iNOS and NO donor reduce the protein expression of IRS-2 by promoting proteasome-dependent degradation of IRS-2 in cultured insulinoma cells and mouse islets. inducible nitric-oxide synthase mammalian target of rapamycin p70 ribosomal S6 kinase S-nitroso-l-glutathione S-nitroso-N-acetyl-dl-penicillamine N6-(1-iminoethyl)-l-lysine 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide. S-Nitroso-l-glutathione (GSNO), S-nitroso-N-acetyl-dl-penicillamine (SNAP), l-NIL, and carboxy-PTIO (Cayman Chemical, Ann Arbor, MI); MG132, reduced glutathione (GSH), and oxidized glutathione (GSSG) (Sigma); cycloheximide (Calbiochem); lactacystin (Boston Biochem, Cambridge, MA); SB216763 and SB415268 (Tocris Bioscience, Ellisville, MO); recombinant mouse IFN-γ (R&D Systems, Minneapolis, MN); mouse IL-1β (Roche Applied Science); human tumor necrosis factor-α (TNF-α; Cell Signaling, Beverly, MA); anti-IRS-2, anti-iNOS, and anti-phosphotyrosine (Millipore, Billerica, MA); anti-ubiquitin (Affiniti Research Products, Golden, CO); anti-phospho-glycogen synthase (Novus Biologicals, Littleton, CO); anti-GSK-3β (BD Transduction Laboratories); anti-IRS-2 (EMD Chemicals, Gibbstown, NJ); anti-GAPDH (Trevigen, Gaithersburg, MD); anti-Akt, anti-phospho-Akt (Ser-473); and anti-phospho-GSK-3β (Ser-9), anti-glycogen synthase, anti-c-Jun, anti-phospho-c-Jun (Ser-63), anti-p70 S6 kinase, and anti-phospho-p70 S6 kinase (Thr-389) antibodies (Cell Signaling) were purchased commercially. siRNA oligonucleotides for rat iNOS and GSK-3β were purchased from Invitrogen (iNOS: RSS302394, RSS302395, and RSS351448; GSK-3β: r(CGAUUACACGUCUAGUAUA)dTdT (sense) and r(UAUACUAGACGUGUAAUCG)dTdT (antisense)). Rat INS-1/832 insulinoma cells, a kind gift of Dr. C. B. Newgard (Duke University) (42Hohmeier H.E. Mulder H. Chen G. Henkel-Rieger R. Prentki M. Newgard C.B. Diabetes. 2000; 49: 424-430Crossref PubMed Scopus (717) Google Scholar), were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 10 mm HEPES, 0.05 mm 2-mercaptoethanol, 1 mm sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin. The mouse βTC-6 insulinoma cell line was obtained from ATCC (Manassas, VA). βTC-6 cells were cultured in Dulbecco's modified Eagle's medium with 15% heat-inactivated FBS, 4 mm l-glutamine, 1.5 g/liter sodium bicarbonate, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were treated with GSNO (30–300 μm), SNAP (300 μm), or cytokine (IL-1β (2.5–10 units/ml), IFN-γ (100 ng/ml)) in the presence or absence of insulin (100 nm), l-NIL (200 μm), carboxy-PTIO (300 μm), cycloheximide (1 μg/ml), MG132 (1 μm), lactacystin (1 μm), SB216763 (10 μm), SB415268 (10 μm), SP600125 (10 μm), or rapamycin (1 μm), as indicated in the figure legends. INS-1/832 cells were transfected with siRNA for iNOS, GSK-3β, or control siRNA (43Ota H. Tokunaga E. Chang K. Hikasa M. Iijima K. Eto M. Kozaki K. Akishita M. Ouchi Y. Kaneki M. Oncogene. 2006; 25: 176-185Crossref PubMed Scopus (386) Google Scholar) using Lipofectamine RNAi MAX (Invitrogen) according to the manufacturers' instructions. Islets were isolated from male wild-type C57BL/6 mice, iNOS knock-out (−/−) mice on C57BL/6 background, wild-type BKS mice, and obese, diabetic (db/db) mice on BKS background (Jackson Laboratory, Bar Harbor, ME) at 9–12 weeks of age by collagenase digestion followed by centrifugation over a Histopaque (Sigma) gradient, as described previously (44Kitamura T. Kido Y. Nef S. Merenmies J. Parada L.F. Accili D. Mol. Cell Biol. 2001; 21: 5624-5630Crossref PubMed Scopus (91) Google Scholar). Upper laparotomy was performed by midline incision, and after clamping the common bile duct at its entrance to the duodenum, 3 ml of M199 medium containing collagenase P (1 mg/ml; Roche Applied Science) was injected into the duct. The swollen pancreas was surgically removed. Human islets from two non-diabetic subjects (male aged 39 years; female aged 45 years) and two type 2 diabetes patients (male aged 63 years on anti-diabetic medication for 5 years; male age 42 years on anti-diabetic medication for 4 years) were provided by the National Disease Research Interchange (Philadelphia, PA). Islets isolated from wild-type C57BL/6 mice and from non-diabetic subjects were cultured in RPMI 1640 supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. The islets were treated with and without GSNO (400 or 500 μm) for 5 h in the presence or absence of SB216763 (10 μm) or lactacystin (1 μm) or with and without IL-1β (10 units/ml) and IFN-γ (100 ng/ml) or IL-1β (10 units/ml), IFN-γ (100 ng/ml), TNF-α (10 ng/ml), and lipopolysaccharide (LPS, 3 μg/ml; Sigma) as indicated in the figure legends for 20 h in the presence or absence of l-NIL (200 μm). Cells or islets were lysed on ice for 30 min in lysis buffer (10 mm Tris-HCl, pH 7.4, 5 mm EDTA, 1% Nonidet P-40, 0.4% sodium deoxycholate, 10 mm sodium pyrophosphate, 10 mm sodium fluoride, 2 mm sodium vanadate, 1 mm phenylmethylsulfonyl fluoride, protease inhibitor mixture (Sigma)), as described previously (45Yasukawa T. Tokunaga E. Ota H. Sugita H. Martyn J.A. Kaneki M. J. Biol. Chem. 2005; 280: 7511-7518Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). The protein samples were denatured by boiling for 5 min and separated in a 7.5 or 10% polyacrylamide gel and electrophoretically transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked in 2% ECL advance blocking agent (GE Healthcare) for 1 h at room temperature and incubated with primary antibody for 2 h at room temperature or overnight at 4 °C. This was followed by incubation with secondary antibody conjugated with horseradish peroxidase for 1 h at room temperature. The blots were visualized by an enhanced chemiluminescence method using an ECL advance Western blotting detection kit (GE Healthcare). Bands of interest were scanned using an HP Scanjet 4850 and were quantified by NIH ImageJ 1.410 software (National Institutes of Health, Bethesda, MD). Lysates were preincubated with 20 μl of protein A/G-agarose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 2 h at 4 °C to minimize nonspecific absorption. The supernatants were then incubated with 2 μg of anti-IRS-2 antibody (Santa Cruz Biotechnology, Inc.) and 20 μl of protein A/G-agarose beads at 4 °C overnight. After centrifugation at 1,000 × g for 5 min, the pellets were washed five times with Tris-buffered saline (10 mm Tris-HCl, pH 7.4, 150 mm NaCl) and dissolved in 30 μl of SDS-sample buffer. Total RNA was purified using TRIzol reagent (Invitrogen). The first-strand cDNA was synthesized from 1 μg of total RNA using a high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). Real-time PCR reactions were performed using 10 ng of cDNA and TaqMan probes (Applied Biosystems) for IRS-2 (Rn01482270_s1 or Hs0065185_m1) and 18 S ribosomal RNA (Hs99999901_s1), conducted with Mastercycler® ep realplex (Eppendorf, Westbury, NY). Results were normalized to 18 S ribosomal RNA as an endogenous reference gene, and the relative amount of each mRNA was calculated by the comparative Ct (threshold cycle) method. iNOS mRNA content in the islets was evaluated by RT-PCR, as described previously (46Bellenger J. Bellenger S. Bataille A. Massey K.A. Nicolaou A. Rialland M. Tessier C. Kang J.X. Narce M. Diabetes. 2011; 60: 1090-1099Crossref PubMed Scopus (121) Google Scholar, 47Baylis S.A. Strijbos P.J. Sandra A. Russell R.J. Rijhsinghani A. Charles I.G. Weiner C.P. Mol. Hum. Reprod. 1999; 5: 277-286Crossref PubMed Scopus (25) Google Scholar), using specific primers for mouse and human iNOS (mouse, 5′-ACAGCCTCAGAGTCCTTCAT-3′ and 5′TTGTCACCACCAGCAGTAGT-3′; human, 5′-CAGTACGTTTGGCAATGGAGACTGC-3′ and 5′-GGTCACATTGGAGGTGTAGA GCTTG-3′). RT-PCR products were quantified using a densitometer and image analyzer (Bio-Rad) (46Bellenger J. Bellenger S. Bataille A. Massey K.A. Nicolaou A. Rialland M. Tessier C. Kang J.X. Narce M. Diabetes. 2011; 60: 1090-1099Crossref PubMed Scopus (121) Google Scholar). 36B4 gene expression was used as an internal control (48Sekiya M. Osuga J. Nagashima S. Ohshiro T. Igarashi M. Okazaki H. Takahashi M. Tazoe F. Wada T. Ohta K. Takanashi M. Kumagai M. Nishi M. Takase S. Yahagi N. Yagyu H. Ohashi K. Nagai R. Kadowaki T. Furukawa Y. Ishibashi S. Cell Metab. 2009; 10: 219-228Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Cell viability of INS-1/832 cells and islet cells was assessed using Sytox Green (Molecular Probes, Inc., Eugene, OR) and TOX-8 (Sigma) according to the manufacturers' instructions. For Sytox staining, cells were incubated with Sytox Green (1 μm) for 20 min in the dark and observed under a Nikon Eclipse TE2000-5 inverted fluorescence microscope. Nitrite accumulation in culture medium was determined by Griess reagent (Sigma). 50 μl of culture medium was mixed and incubated with 50 μl of Griess reagent for 15 min at room temperature, and absorbance at 540 nm was measured in a microplate reader. Serial dilutions of sodium nitrite were used as standards. The data were compared using one-way analysis of variance followed by Tukey's least significant difference test or unpaired Student's t test. A value of p < 0.05 was considered statistically significant. All data are expressed as mean ± S.E. Treatment with IL-1β or with IL-1β plus interferon-γ (IFN-γ) resulted in a time- and dose-dependent induction of iNOS expression and nitrite accumulation in the culture medium in INS-1/832 cells. The induction of iNOS paralleled the reduction in IRS-2 protein expression (FIGURE 1, FIGURE 2). Treatment with IL-1β for 24 h significantly decreased IRS-2 protein expression starting at a dose of 2.5 units/ml, and the maximum level of the inhibitory effect of IL-1β on IRS-2 was observed at 10 and 25 units/ml (Fig. 1C). The suppression of IRS-2 protein expression by IL-1β or IL-1β plus IFN-γ was prevented by a specific inhibitor of iNOS, l-NIL, in INS-1/832 cells, along with the reversal of accumulation of nitrite in the culture medium (FIGURE 2, FIGURE 3). The protein expression of GAPDH was not affected by IL-1β, IL-1β plus IFN-γ, or l-NIL. Likewise, siRNA-mediated knockdown of iNOS inhibited IL-1β-induced decreased protein expression of IRS-2 in INS-1/832 cells (Fig. 3F). In contrast to the decreased protein expression of IRS-2, IL-1β did not reduce mRNA expression of IRS-2 in INS-1/832 cells (IRS-2 mRNA level: control, 100 ± 11% (mean ± S.E.); IL-1β, 104 ± 5%. Treatment with neither IL-1β, IL-1β plus IFN-γ, nor l-NIL for up to 24 h increased cell death in INS-1/832 cells and mouse islets, as judged by Sytox staining (data not shown).FIGURE 2Time-dependent reduction in IRS-2 expression by cytokine and its reversal by iNOS inhibitor in β-cells. IL-1β (5 units/ml) alone and the combination of IL-1β (10 units/ml) and IFN-γ (100 ng/ml) time-dependently decreased IRS-2 protein expression in parallel with iNOS induction and nitrite accumulation in the culture medium in INS-1/832 cells (A–D). iNOS inhibitor, l-NIL (200 μm), blocked the cytokine-induced decreases in IRS-2 expression and nitrite accumulation. Neither IL-1β, IL-1β plus IFN-γ, nor l-NIL altered GAPDH expression. IB, immunoblotting. Error bars, S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Inhibition of iNOS blocked cytokine-induced reduction in IRS-2 expression in β-cells. INS-1/832 cells were treated for 24 h with or without IL-1β (5 units/ml) (A–C and F) or IL-1β (10 units/ml) plus IFN-γ (100 ng/ml) (D and E) in the presence and absence of iNOS inhibitor, l-NIL (A–E), or siRNA to iNOS (F). IL-1β alone and IL-1β plus IFN-γ induced iNOS expression and reduced IRS-2 protein expression. l-NIL reverted decreased IRS-2 expression and nitrite accumulation in the culture medium in cytokine-treated cells. Likewise, siRNA-mediated knockdown prevented IL-1β-induced suppression of IRS-2 expression in β-cells in parallel with reversal of elevated nitrite concentration in the medium, as compared with control siRNA. Unless treated with cytokine, l-NIL and iNOS knockdown did not affect IRS-2 expression and nitrite concentrations. GAPDH expression was not altered by cytokine, l-NIL, or iNOS knockdown. IB, immunoblotting. ***, p < 0.001 versus the cells without cytokine and those with cytokine + l-NIL; §§§, p < 0.001 versus the cells without cytokine. Error bars, S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In accord with decreased IRS-2 expression by IL-1β, basal and insulin-stimulated phosphorylation of IRS-2 and Akt was attenuated by IL-1β, which was partially prevented by iNOS inhibitor, l-NIL, in INS-1/832 cells (Fig. 4). In the absence of IL-1β, l-NIL did not affect the phosphorylation status of IRS-2 and Akt. The protein expression of Akt was not altered by IL-1β or l-NIL. Similar to INS-1/832 cells, treatment with IL-1β alone or IL-1β plus IFN-γ decreased IRS-2 protein expression in cultured islets isolated from wild-type mice along with induction of iNOS expression (Fig. 5A), whereas mRNA content of IRS-2 was not decreased (mouse IRS-2 mRNA level: control, 100 ± 9%; IL-1β, 95 ± 14%) IL-1β plus IFN-γ induced more profound decrease in IRS-2 protein expression in association with greater increases in iNOS expression and nitrite accumulation in wild-type islets (Fig. 5B). In islets from iNOS-deficient mice, however, neither IL-1β alone nor IL-1β plus IFN-γ affected IRS-2 expression or increased nitrite concentration in the culture medium (Fig. 5). iNOS inhibitor, l-NIL (200 μm), blocked IL-1β plus IFN-γ-induced decrease in IRS-2 protein expression and nitrite accumulation in wild-type mouse islets (data not shown). NO donor, GSNO, decreased the protein expression of IRS-2 in a time-dependent manner in INS-1/832 cells (Fig. 6A). GSNO (300 μm) significantly decreased IRS-2 expression starting at 2 h after the addition of the NO donor. Treatment with GSNO for 5 h elicited the inhibitory effects on IRS-2 expression in a dose-dependent manner. A significant reduction in IRS-2 protein expression was observed starting at a dose of 100 μm (Fig. 6B). To assess cell viability, we evaluated cell membrane integrity and metabolic cell viability by Sytox staining and TOX" @default.
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• W2040982134 title "Inducible Nitric-oxide Synthase and Nitric Oxide Donor Decrease Insulin Receptor Substrate-2 Protein Expression by Promoting Proteasome-dependent Degradation in Pancreatic β-Cells" @default.
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