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• W2077417063 abstract "Recent studies have identified a β-cell insulin receptor that functions in the regulation of protein translation and mitogenic signaling similar to that described for insulin-sensitive cells. These findings have raised the novel possibility that β-cells may exhibit insulin resistance similar to skeletal muscle, liver, and fat. To test this hypothesis, the effects of tumor necrosis factor-α (TNFα), a cytokine proposed to mediate insulin resistance by interfering with insulin signaling at the level of the insulin receptor and its substrates, was evaluated. TNFα inhibited p70s6k activation by glucose-stimulated β-cells of the islets of Langerhans in a dose- and time-dependent manner, with maximal inhibition observed at ∼20–50 ng/ml, detected after 24 and 48 h of exposure. Exogenous insulin failed to prevent TNFα-induced inhibition of p70s6k, suggesting a defect in the insulin signaling pathway. To further define mechanisms responsible for this inhibition and also to exclude cytokine-induced nitric oxide (NO) as a mediator, the ability of exogenous or endogenous insulin ± inhibitors of nitric-oxide synthase (NOS) activity, aminoguanidine or N-monomethyl-l-arginine, was evaluated. Unexpectedly, TNFα and also interleukin 1 (IL-1)-induced inhibition of p70s6k was completely prevented by inhibitors that block NO production. Western blot analysis verified inducible NOS (iNOS) expression after TNFα exposure. Furthermore, the ability of IL-1 receptor antagonist protein, IRAP, to block TNFα-induced inhibition of p70s6k indicated that activation of intra-islet macrophages and the release of IL-1 that induces iNOS expression in β-cells was responsible for the inhibitory effects of TNFα. This mechanism was confirmed by the ability of the peroxisome proliferator-activated receptor-γ agonist 15-deoxy-Δ12,14-prostaglandin J2 to attenuate TNFα-induced insulin resistance by down-regulating iNOS expression and/or blocking IL-1 release from activated macrophages. Overall, TNFα-mediated insulin resistance in β-cells is characterized by a global inhibition of metabolism mediated by NO differing from that proposed for this proinflammatory cytokine in insulin-sensitive cells. Recent studies have identified a β-cell insulin receptor that functions in the regulation of protein translation and mitogenic signaling similar to that described for insulin-sensitive cells. These findings have raised the novel possibility that β-cells may exhibit insulin resistance similar to skeletal muscle, liver, and fat. To test this hypothesis, the effects of tumor necrosis factor-α (TNFα), a cytokine proposed to mediate insulin resistance by interfering with insulin signaling at the level of the insulin receptor and its substrates, was evaluated. TNFα inhibited p70s6k activation by glucose-stimulated β-cells of the islets of Langerhans in a dose- and time-dependent manner, with maximal inhibition observed at ∼20–50 ng/ml, detected after 24 and 48 h of exposure. Exogenous insulin failed to prevent TNFα-induced inhibition of p70s6k, suggesting a defect in the insulin signaling pathway. To further define mechanisms responsible for this inhibition and also to exclude cytokine-induced nitric oxide (NO) as a mediator, the ability of exogenous or endogenous insulin ± inhibitors of nitric-oxide synthase (NOS) activity, aminoguanidine or N-monomethyl-l-arginine, was evaluated. Unexpectedly, TNFα and also interleukin 1 (IL-1)-induced inhibition of p70s6k was completely prevented by inhibitors that block NO production. Western blot analysis verified inducible NOS (iNOS) expression after TNFα exposure. Furthermore, the ability of IL-1 receptor antagonist protein, IRAP, to block TNFα-induced inhibition of p70s6k indicated that activation of intra-islet macrophages and the release of IL-1 that induces iNOS expression in β-cells was responsible for the inhibitory effects of TNFα. This mechanism was confirmed by the ability of the peroxisome proliferator-activated receptor-γ agonist 15-deoxy-Δ12,14-prostaglandin J2 to attenuate TNFα-induced insulin resistance by down-regulating iNOS expression and/or blocking IL-1 release from activated macrophages. Overall, TNFα-mediated insulin resistance in β-cells is characterized by a global inhibition of metabolism mediated by NO differing from that proposed for this proinflammatory cytokine in insulin-sensitive cells. A hallmark of human type 2 diabetes is an initial attenuated responsiveness of cells to insulin (a term designated as insulin resistance) that is countered or compensated by the ability of pancreatic β-cells to secrete more insulin than normally required to achieve euglycemia (1Khan B.B. Cell. 1998; 92: 593-596Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 2Khan C.R. Cell. 1997; 88: 561-572Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar, 3Porte Jr., D. Diabetes. 1991; 40: 166-180Crossref PubMed Scopus (0) Google Scholar). Sustained hyperglycemia, however, occurs when the ability of β-cells to over-secrete insulin fails, resulting in overt diabetes. This sequence of events has generated the concept that a specific defect in the insulin-signaling pathway of insulin-sensitive cells such as skeletal muscle, fat, and liver is a primary cause for insulin resistance. As a secondary effect of insulin resistance, β-cell failure or the loss of the ability of β-cells to compensate has been proposed to be mediated by glucose toxicity, cellular exhaustion, and/or other undefined cellular mechanisms (4McClain D.A. Crook E.D. Diabetes. 1996; 45: 1003-1009Crossref PubMed Google Scholar, 5DeFronzo R.A. Diabetes Rev. 1997; 5: 177-269Google Scholar, 6Leahy J.L. Cooper H.W. Deal D.A. Weir C.G. J. Clin. Invest. 1986; 77: 908-915Crossref PubMed Scopus (295) Google Scholar). More recent findings using gene knockout approaches have shown that disruption of insulin receptor substrate protein-2 (IRS-2) 1The abbreviations used are: IRS-2, insulin receptor substrate 2; TNFα, tumor necrosis factor α; IL-1β, interleukin 1β; KRBB, Krebs-Ringer bicarbonate buffer; BSA, bovine serum albumin; iNOS, inducible nitric oxide synthase; NMMA, N-monomethyl-l-arginine; PPARγ, peroxisome proliferator-activated receptor γ; IRAP, IL-1 receptor antagonist protein; PHAS-I, phosphorylated heat- and acid-stable protein regulated by insulin; 4EBP-1, eukaryotic initiation factor 4E-binding protein 1; p70s6k, p70 S6 kinase; 15d-PGJ2, 15-deoxy-Δ12,14-prostaglandin J2; PAGE, polyacrylamide gel electrophoresis.1The abbreviations used are: IRS-2, insulin receptor substrate 2; TNFα, tumor necrosis factor α; IL-1β, interleukin 1β; KRBB, Krebs-Ringer bicarbonate buffer; BSA, bovine serum albumin; iNOS, inducible nitric oxide synthase; NMMA, N-monomethyl-l-arginine; PPARγ, peroxisome proliferator-activated receptor γ; IRAP, IL-1 receptor antagonist protein; PHAS-I, phosphorylated heat- and acid-stable protein regulated by insulin; 4EBP-1, eukaryotic initiation factor 4E-binding protein 1; p70s6k, p70 S6 kinase; 15d-PGJ2, 15-deoxy-Δ12,14-prostaglandin J2; PAGE, polyacrylamide gel electrophoresis. in mice results in defects in both insulin action and insulin secretion by β-cells, which closely mimics the development of human type 2 diabetes (7Withers 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 (1319) Google Scholar). Of particular significance, IRS-2 knockout mice exhibit a reduced mass of β-cells compared with wild-type mice, suggesting that this defect in the insulin signaling pathway also produces a functional defect in mitogenic signaling, resulting in decreased β-cell replication and growth. These novel results have altered this previous concept for the development of type 2 diabetes by demonstrating that disruption of a single gene product, i.e. IRS-2, involved in insulin signaling may be responsible for insulin resistance in insulin-sensitive tissues and also in pancreatic β-cells. Thus, β-cell failure may be because of insulin resistance rather than β-cell toxicity or exhaustion that occurs in parallel with the development of peripheral insulin resistance in type 2 diabetes. Evidence in support of a functional β-cell insulin receptor has been reported recently by several groups. Studies by Rothenberg et al. (8Rothenberg P.L. Willison L.D. Simon J. Wolf B.A. Diabetes. 1995; 44: 802-809Crossref PubMed Google Scholar) characterize an insulin-activated cell surface receptor tyrosine kinase and IRS-1 that associates with the 85-kDa α subunit of phosphoinositide 3-kinase in the β-cell line βTC3. Overexpression of the human insulin receptor in the β-cell line βTC6-F7 also regulates insulin gene expression and insulin content (9Xu G.G. Rothenberg P.L. Diabetes. 1998; 47: 1243-1252PubMed Google Scholar). Harbeck et al. (10Harbeck M.C. Louie D.C. Howland J. Wolf B.A. Rothenberg P.L. Diabetes. 1996; 45: 711-717Crossref PubMed Google Scholar) also describe expression of insulin receptor mRNA and IRS-1 in single primary rat β-cells. Furthermore, Leibiger et al. (11Leibiger I.B. Leibiger B. Moede T. Berggren P.O. Mol. Cell. 1998; 1: 933-938Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar) show that endogenous insulin secreted from the β-cell line HIT promotes insulin biosynthesis by enhancing insulin gene transcription in an autocrine manner. In addition, our laboratory presented findings suggesting that glucose-stimulated phosphorylation of the translational factors PHAS-I (also known as 4EBP-1) and p70s6k by β-cells of the islet is mediated via insulin interacting in an autocrine manner with its own insulin receptor (12Xu G. Marshall C.A. Lin T.-A. Kwon G. Munivenkatappa R.B. Hill J.R. Lawrence Jr., J.C. McDaniel M.L. J. Biol. Chem. 1998; 273: 4485-4491Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Our more recent studies show that amino acids, in particular branched-chain amino acids, stimulate phosphorylation of PHAS-I and p70s6k and are also essential for insulin and other growth factors to activate these same translational regulators (13Xu G. Kwon G. Marshall C.A. Lin T.-A. Lawrence Jr., J.C. McDaniel M.L. J. Biol. Chem. 1998; 273: 28178-28184Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). An understanding of causes responsible for the development of insulin resistance in insulin-sensitive cells has focused on the close relationship between obesity and defects in insulin signaling. A large number of studies have identified the proinflammatory cytokine tumor necrosis factor (TNFα) as a potential mediator of insulin resistance associated with obesity. Overexpression of TNFα by fat tissue in both obese animals and humans has been correlated with the development of peripheral insulin resistance (14Spiegelman B.M. Diabetes. 1998; 47: 507-514Crossref PubMed Scopus (1620) Google Scholar, 15Hotamisligil G.S. Murray D.L. Choy L.N. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4854-4858Crossref PubMed Scopus (1022) Google Scholar, 16Hotamisiligil G.S. Shargill N.S. Spiegelman B.M. Science. 1993; 259: 87-90Crossref PubMed Scopus (5970) Google Scholar, 17Hotamisiligil G.S. Peraldi P. Budavari A. Ellis R. White M.F. Spiegelman B.M. Science. 1996; 27: 665-668Crossref Scopus (2164) Google Scholar, 18Uysal K.T. Wiesbrock S.M. Marin M.W. Hotamisiligil G.S. Nature. 1997; 389: 610-614Crossref PubMed Scopus (1869) Google Scholar, 19Peraldi P. Spiegelman B.M. Mol. Cell. Biochem. 1998; 182: 169-175Crossref PubMed Scopus (236) Google Scholar). A key observation indicating that TNFα may exert a role in the development of insulin resistance is that neutralization of TNFα in obese rats increases insulin sensitivity (16Hotamisiligil G.S. Shargill N.S. Spiegelman B.M. Science. 1993; 259: 87-90Crossref PubMed Scopus (5970) Google Scholar). In addition, TNFα also interferes with insulin signaling in vitro by inhibiting insulin receptor tyrosine kinase activity and tyrosine phosphorylation of insulin receptor substrates (17Hotamisiligil G.S. Peraldi P. Budavari A. Ellis R. White M.F. Spiegelman B.M. Science. 1996; 27: 665-668Crossref Scopus (2164) Google Scholar). In this latter case, TNFα induces serine phosphorylation of IRS-1, which then is believed to function as an inhibitor of insulin receptor tyrosine kinase activity. Another important feature of insulin resistance is the ability of the PPARγ agonists troglitazone and a derivative of prostaglandin D2, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), to correct this defect (20Spiegelman B.M. Cell. 1998; 93: 153-155Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 21Zhang B. Berger J. Hu E. Szalkowski D. White-Carrington S. Spiegelman B.M. Moller D.E. Mol. Endocrinol. 1996; 10: 1457-1466Crossref PubMed Scopus (306) Google Scholar). The antidiabetic agent troglitazone also ameliorates both insulin resistance and hyperinsulinemia in diabetic animals and type 2 diabetics (22Lee M.-K. Miles P.D.G. Khoursheed M. Gao K.-M. Moossa A.R. Olefsky J.M. Diabetes. 1994; 43: 1435-1439Crossref PubMed Scopus (231) Google Scholar, 23Nolan J.J. Ludvik B. Beerdsen P. Joyce M. Olefsky J. N. Eng. J. Med. 1994; 331: 1188-1193Crossref PubMed Scopus (914) Google Scholar). Furthermore, troglitazone normalizes TNFα-induced inhibition of insulin-stimulated glucose disposal in rodents (24Miles P.D.G. Romeo O.M. Higo K. Cohen A. Rafaat K. Olefsky J.M. Diabetes. 1997; 46: 1678-1683Crossref PubMed Google Scholar). Troglitazone and 15d-PGJ2 also prevent TNFα-induced inhibition of the most proximal steps in insulin signalingi.e. tyrosine phosphorylation of the insulin receptor and its substrate IRS-1 and activation of phosphoinositide 3-kinasein vitro (25Peraldi P. Xu M. Spiegelman B.M. J. Clin. Invest. 1997; 100: 1863-1869Crossref PubMed Scopus (296) Google Scholar). Of particular importance, troglitazone also improves the reduced response of β-cells to glucose observed in subjects with impaired glucose tolerance (26Cavaghan M.K. Ehrmann D.A. Byrne M.M. Polonsky K.S. J. Clin. Invest. 1997; 100: 530-537Crossref PubMed Scopus (192) Google Scholar) and decreases the ratio of plasma proinsulin to immunoreactive insulin in type 2 diabetics (27Prigeon R.L. Kahn S.E. Porte Jr., D. J. Clin. Endocrinol. Metab. 1998; 83: 819-823Crossref PubMed Scopus (78) Google Scholar). Overall, these findings suggest that troglitazone may improve β-cell function in addition to insulin sensitivity. To further assess if defects in insulin signaling may negatively impact β-cells in a manner analogous to that described for insulin-sensitive cells, we have examined whether TNFα, an important mediator of insulin resistance in insulin-sensitive cells, also mediates insulin resistance in pancreatic β-cells. As a measure of β-cell insulin resistance, we have examined the phosphorylation level of p70s6k in response to stimulation by glucose or amino acids. This parameter has been shown to be a reliable measure of insulin action (28Avruch J. Mol. Cell. Biochem. 1998; 182: 31-48Crossref PubMed Scopus (319) Google Scholar). Male Sprague-Dawley rats were purchased from Harlan Sprague-Dawley (Indianapolis, IN). CMRL-1066 and RPMI 1640 tissue culture media, penicillin, streptomycin, Hanks' balanced salt solution, l-glutamine, minimum essential medium amino acid solution, and nonessential minimum essential medium solution were obtained from Life Technologies, Inc.. Fetal bovine serum was from Hyclone (Logan, UT). Human recombinant interleukin-1β (IL-1β) was from Cistron Biotechnology (Pine Brook, NJ). Collagenase type P and human recombinant TNFα were from Roche Molecular Biochemicals. Porcine insulin was from ICN (Aurora, OH). Ficoll and aminoguanidine were from Sigma. NMMA was from Calbiochem. IRAP was from R&D Systems (Minneapolis, MN). Mouse macrophage nitric oxide synthase (iNOS) antibody and 15d-PGJ2 were from Cayman Chemical (Ann Arbor, MI). The antibody for p70s6k was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The secondary antibody was peroxidase-conjugated donkey anti-rabbit IgG, Jackson Immunoresearch Laboratories (West Grove, PA). Troglitazone was a kind gift from John Johnson, Parke-Davis (Ann Arbor, MI). All other chemicals were from commercially available sources. Islets were isolated from male Sprague-Dawley rats (200–250 g) by collagenase digestion as described previously (12Xu G. Marshall C.A. Lin T.-A. Kwon G. Munivenkatappa R.B. Hill J.R. Lawrence Jr., J.C. McDaniel M.L. J. Biol. Chem. 1998; 273: 4485-4491Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 13Xu G. Kwon G. Marshall C.A. Lin T.-A. Lawrence Jr., J.C. McDaniel M.L. J. Biol. Chem. 1998; 273: 28178-28184Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Briefly, pancreases were inflated with Hanks' balanced salt solution, and the tissue was isolated, minced, and digested with 7 mg of collagenase/pancreas for 7 min at 38 °C. Islets were separated on a Ficoll step density gradient and then selected with a stereomicroscope to exclude any contaminating tissues. Islets were cultured overnight in an atmosphere of 95% air, 5% CO2 in “complete” CMRL-1066 tissue culture medium containing 5.5 mm glucose, 2 mm l-glutamine, 10% (v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Where CMRL is stated, the culture medium does not contain fetal bovine serum. RINm5F cells, an insulin secreting β-cell line (29Bhathena S.J. Oie H.K. Gazdar A.F. Voyles N.R. Wilkins S.D. Recant L. Diabetes. 1982; 31: 521-531Crossref PubMed Google Scholar), were cultured by the Washington University Tissue Culture Support Center in RPMI 1640 containing 10% (v/v) heat-inactivated fetal bovine serum, 2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin, 0.1 mm nonessential amino acids, 16.8 mm glucose, 1 mm sodium pyruvate, and 10 mm HEPES. Cells were subcultured in complete CMRL in Petri dishes (60 × 15 mm) at a concentration of 3 × 106 cells/3 ml and incubated at 37 °C under an atmosphere of 95% air, 5% CO2 for 24 h before initiating experiments. Islets (200/1 ml) and RINm5F cells (3 × 106 cells/3 ml) were washed free of fetal bovine serum and incubated in CMRL, 3 mm glucose, 0.1% BSA at 37 °C, 95% air, 5% CO2 for 0 to 48 h ± TNFα (50 ng/ml ∼ 2.9 nm), IL-1β (5 units/ml ∼ 5.7 pm), aminoguanidine, NMMA, 15d-PGJ2, actinomycin D, or IRAP. IRAP, troglitazone, and 15d-PGJ2 were added 30 min before TNFα or IL-1β. Culture media was collected for accumulated nitrite determinations. Islets were then incubated for 30 min with either 3 or 20 mm glucose ± cytokines, insulin, and inhibitors. RINm5F cells were incubated for 2 h in Krebs-Ringer bicarbonate buffer (KRBB) in the absence of glucose and amino acids, followed by a 30-min incubation ±1× amino acid mixture (13Xu G. Kwon G. Marshall C.A. Lin T.-A. Lawrence Jr., J.C. McDaniel M.L. J. Biol. Chem. 1998; 273: 28178-28184Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar) and TNFα. Following experimental treatments, cells or islets were washed with phosphate-buffered saline and solubilized in 300 μl or 30 μl, respectively, of Laemmli sample buffer, heated at 100 °C for 5 min, and centrifuged at 10,000 × g for 15 min to remove insoluble materials. The supernatants were processed for SDS-PAGE and Western blotting of iNOS or p70s6k as described previously (13Xu G. Kwon G. Marshall C.A. Lin T.-A. Lawrence Jr., J.C. McDaniel M.L. J. Biol. Chem. 1998; 273: 28178-28184Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 30Kwon G. Corbett J.A. Hauser S. Hill J.R. Turk J. McDaniel M.L. Diabetes. 1998; 47: 583-591Crossref PubMed Scopus (74) Google Scholar). Detection was performed using ECL reagents from Amersham Pharmacia Biotech. Quantitation of Western blots was performed by densitometry using a Molecular Dynamics personal densitometer scanning instrument (Sunnyvale, CA). Data are expressed as percent of phosphorylated p70s6k over total. Isolated islets (150/1 ml) were cultured for 48 h at 37 °C in CMRL medium, 8 mm glucose, 0.1% BSA ± TNFα (50 ng/ml), ± aminoguanidine (0.5 mm). In the absence of fetal calf serum, the glucose concentration was raised to 8 mm to maintain the glucose-stimulated insulin secretory response of β-cells. Culture medium was collected for accumulated nitrite measurements, and islets were washed and incubated for 2 h in CMRL, 0.1% BSA, minus glucose. Following this period, islets were washed in KRBB, 3 mm glucose, 0.1% BSA, and groups of 20 islets were placed into 10 × 75-mm siliconized, borosilicate tubes. KRBB was replaced with 200 μl of fresh KRBB, 3 mmglucose, 0.1% BSA, and the islets were preincubated for 30 min at 37 °C under 95% air, 5% CO2 in a shaking water bath. Buffer was replaced with 200 μl of KRBB containing 3 or 20 mm glucose, and islets were incubated for 30 min. TNFα and aminoguanidine were present during the entire experiment. Insulin secretion was determined in the incubation buffer by insulin radioimmunoassay at the Radioimmunoassay Core Facility, Diabetes Research Training Center, Washington University Medical Center. Culture media was removed, and 50-μl aliquots were mixed with 50 μl of Griess reagent (31Green L.C. Wagner D.A. Glogowski J. Skipper P.L. Wishnok J.S. Tannenbaum S.R. Anal. Biochem. 1982; 126: 131-138Crossref PubMed Scopus (10586) Google Scholar). Nitrite production was determined at an absorbance of 540 nm using a Molecular Devices Thermomax platereader (Sunnyvale, CA). Our initial focus was to determine whether preincubation of pancreatic islets in the presence of TNFα would inhibit insulin signaling at concentrations and periods of exposure similar to that used previously with insulin-sensitive cells. As shown in Fig.1 A (lanes 1 and2), islets incubated under control conditions for 48 h and subsequently stimulated with an insulin stimulatory concentration of glucose (20 mm) showed enhanced phosphorylation of p70s6k compared with basal glucose (3 mm), as determined by gel shift analysis. Pretreatment of islets for 48 h with TNFα concentrations of 20 ng/ml or greater reduced phosphorylation of p70s6k to basal or lower levels. As shown in Fig. 1 B, the inhibitory effect of TNFα was detected at 24 h (lanes 5 and 6) and 48 h (lanes 7 and 8). Thus, both the dose and time dependence of TNFα-induced inhibition of p70s6k by pancreatic islets is quite similar to that described previously for TNFα on other aspects of insulin signaling in insulin-sensitive cells. To determine whether TNFα-induced inhibition of p70s6k represents a specific defect in insulin signaling by islets, the ability of exogenous insulin to reverse this inhibition was next evaluated. As shown in Fig. 2,lane 3, pretreatment of islets for 48 h with TNFα (50 ng/ml) resulted in marked inhibition of p70s6k following exposure of islets to an insulin stimulatory concentration of glucose (20 mm) compared with the control in the absence of TNFα (lane 2). Furthermore, this inhibition was not reversed by exogenous insulin over a concentration range of 10–200 nm(lanes 4–6). These findings suggest that TNFα induces insulin resistance in islets similar to insulin-sensitive cells. A possible mechanism to explain these results is that TNFα may mediate serine phosphorylation of IRS proteins, which then functions as an inhibitor of insulin receptor tyrosine kinase activity (17Hotamisiligil G.S. Peraldi P. Budavari A. Ellis R. White M.F. Spiegelman B.M. Science. 1996; 27: 665-668Crossref Scopus (2164) Google Scholar). Alternatively, our previous studies have demonstrated that proinflammatory cytokines including TNFα, either alone or in synergy with other cytokines, stimulate expression of the inducible isoform of iNOS and the overproduction of NO (32Corbett J.A. McDaniel M.L. J. Exp. Med. 1995; 181: 559-568Crossref PubMed Scopus (232) Google Scholar). NO potently inhibits cellular metabolism by inactivating iron-sulfur-containing enzymes localized primarily in the mitochondria, which results in reduced levels of ATP. Thus, an alternate mechanism to explain the ability of TNFα to inhibit p70s6k activation by islets is that NO mediates a global inhibition of cellular metabolism.Figure 2TNFα-induced inhibition of p70 s6k phosphorylation is not reversed by exogenous insulin. Islets (200 in 1 ml CMRL supplemented with 3 mm glucose and 0.1% BSA) were incubated for 48 h in the presence or absence of 50 ng/ml TNFα as indicated. Islets were then stimulated with either 3 or 20 mm glucose for 30 min. For lanes 4 through 5, increasing concentrations of insulin were added in addition to 20 mm glucose. Islets were then processed for SDS-PAGE, followed by Western blot for p70s6k as described under “Experimental Procedures.” Results are representative of three individual experiments.View Large Image Figure ViewerDownload (PPT) Studies were performed next to evaluate this possibility. As shown in Fig. 3 A (lanes 3and 4), co-incubation of aminoguanidine (0.5 mm) with TNFα (50 ng/ml) for 48 h completely prevented the inhibition of glucose-stimulated activation of p70s6k. Also, aminoguanidine (0.05, 0.1, and 0.5 mm) in the absence of TNFα at noninsulin stimulatory levels of glucose (3 mm) exerted no effect on basal levels of p70s6k(lanes 6–8). As shown in Fig. 3 B, exposure of islets for 48 h to TNFα (50 ng/ml) also inhibited glucose-stimulated insulin secretion, and this inhibition was completely reversed by co-incubation with aminoguanidine. Based on these new findings, the ability of exogenous insulin to reverse TNFα-mediated inhibition of p70s6k as described in Fig. 2 was re-evaluated but now in the presence of aminoguanidine, which prevents formation of NO and inhibition of cellular metabolism. As shown in Fig. 4, pretreatment of islets with TNFα (50 ng/ml) for 48 h inhibited p70s6k phosphorylation (lanes 2 and3). Furthermore, exogenous insulin (200 nm) at basal glucose levels (3 mm) in the presence of an inhibitor of iNOS activity, aminoguanidine, completely reversed this inhibition compared with the absence of aminoguanidine (lanes 4 and5). Also shown in Fig. 4 (lane 6), exogenous insulin (200 nm) at basal glucose (3 mm) enhanced phosphorylation of p70s6k similar to elevated glucose (20 mm). These results indicate that although TNFα-induced NO formation inhibits endogenous insulin secretion (Fig.3 B), β-cell insulin resistance is reversed by exogenous insulin providing aminoguanidine is present to prevent inhibition of cellular metabolism (Fig. 4, lanes 4 and 5). Thus, β-cell insulin resistance is not because of a decrease in secreted insulin but a defect in insulin signaling by β-cells.Figure 5TNFα and IL-1-induced inhibition of p70 s6k is reversed by aminoguanidine and NMMA . Islets were treated with ±50 ng/ml TNFα, ±5 unit/ml IL-1, ± 0.5 mm aminoguanidine (AG), ± 0.5 mm NMMA for 48 h as indicated. After incubation, islets were stimulated with either 3 or 20 mm glucose for 30 min. Islets were then processed for SDS-PAGE, followed by Western blot for p70s6k as described under “Experimental Procedures.” Results are representative of three individual experiments.View Large Image Figure ViewerDownload (PPT) Previous studies by Spiegelman et al. (15Hotamisligil G.S. Murray D.L. Choy L.N. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4854-4858Crossref PubMed Scopus (1022) Google Scholar) demonstrate that other proinflammatory cytokines, in particular IL-1, interfered with insulin signaling in cultured 3T3-F442A adipocytes, similar to the effects produced by TNFα (15Hotamisligil G.S. Murray D.L. Choy L.N. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4854-4858Crossref PubMed Scopus (1022) Google Scholar). Similar comparisons with pancreatic islets (Fig. 5) indicated that IL-1 also markedly inhibited glucose-stimulated activation of p70s6k(lanes 2 and 6). Furthermore, the inhibitory effects produced by both TNFα and IL-1 were completely prevented by inhibitors of iNOS activity, aminoguanidine and NMMA. To further establish a role for NO as a mediator of TNFα-induced β-cell insulin resistance, Western blot analysis of iNOS expression was performed. As shown in Fig. 6, TNFα (1–50 ng/ml) dose-dependently induced iNOS expression by islets following a 48-h pretreatment. TNFα-induced iNOS expression was blocked by the transcription inhibitor actinomycin D and IRAP (lanes 7 and 8). These latter findings suggest that TNFα mediates iNOS expression by activating intra-islet macrophages, resulting in the release of IL-1 that then acts directly on the β-cell. This mode of action for TNFα was further supported by findings shown in Fig. 7 A, indicating that IRAP reversed in a dose-dependent manner TNFα-induced inhibition of p70s6k (lanes 3 and4–6). As expected, IRAP also prevented IL-1-induced inhibition of p70s6k (lanes 7 and8).Figure 7IRAP reverses TNFα-induced inhibition of p70 s6k .Islets were incubated for 48 h in the presence and absence of 50 ng/ml TNFα ± increasing concentrations of IRAP as indicated. For lanes 7 and8, 5 units/ml IL-1 ± 1 μg/ml IRAP was added as a control. After incubation, islets were stimulated with either 3 or 20 mm glucose for 30 min. Islets were then processed for SDS-PAGE, followed by Western blot for p70s6k as described under “Experimental Procedures.” B, TNFα has no effect on amino acid-stimulated p70s6k phosphorylation by RINm5F cells. RINm5F cells (3 × 106/3 ml) were incubated with increasing concentrations of TNFα for 48 h in CMRL supplemented with 3 mm glucose and 0.1% BSA. After incubation, cells were washed 2× with KRBB in the absence of glucose and amino acids, followed by a 2-h incubation in the same buffer. After a 2-h incubation in KRBB, cells were stimulated" @default.
• W2077417063 created "2016-06-24" @default.
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• W2077417063 date "1999-06-01" @default.
• W2077417063 modified "2023-10-17" @default.
• W2077417063 title "Tumor Necrosis Factor α-induced Pancreatic β-Cell Insulin Resistance Is Mediated by Nitric Oxide and Prevented by 15-Deoxy-Δ12,14-prostaglandin J2 and Aminoguanidine" @default.
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