Palmitate Inhibits Insulin Gene Expression by Altering PDX-1 Nuclear Localization and Reducing MafA Expression in Isolated Rat Islets of Langerhans

Abnormalities in lipid metabolism have been proposed as contributing factors to both defective insulin secretion from the pancreatic beta cell and peripheral insulin resistance in type 2 diabetes. Previously, we have shown that prolonged exposure of isolated rat islets of Langerhans to excessive fatty acid levels impairs insulin gene transcription. This study was designed to assess whether palmitate alters the expression and binding activity of the key regulatory factors pancreas-duodenum homeobox-1 (PDX-1), MafA, and Beta2, which respectively bind to the A3, C1, and E1 elements in the proximal region of the insulin promoter. Nuclear extracts of isolated rat islets cultured with 0.5 mm palmitate exhibited reduced binding activity to the A3 and C1 elements but not the E1 element. Palmitate did not affect the overall expression of PDX-1 but reduced its nuclear localization. In contrast, palmitate blocked the stimulation of MafA mRNA and protein expression by glucose. Combined adenovirus-mediated overexpression of PDX-1 and MafA in islets completely prevented the inhibition of insulin gene expression by palmitate. These results demonstrate that prolonged exposure of islets to palmitate inhibits insulin gene transcription by impairing nuclear localization of PDX-1 and cellular expression of MafA. Abnormalities in lipid metabolism have been proposed as contributing factors to both defective insulin secretion from the pancreatic beta cell and peripheral insulin resistance in type 2 diabetes. Previously, we have shown that prolonged exposure of isolated rat islets of Langerhans to excessive fatty acid levels impairs insulin gene transcription. This study was designed to assess whether palmitate alters the expression and binding activity of the key regulatory factors pancreas-duodenum homeobox-1 (PDX-1), MafA, and Beta2, which respectively bind to the A3, C1, and E1 elements in the proximal region of the insulin promoter. Nuclear extracts of isolated rat islets cultured with 0.5 mm palmitate exhibited reduced binding activity to the A3 and C1 elements but not the E1 element. Palmitate did not affect the overall expression of PDX-1 but reduced its nuclear localization. In contrast, palmitate blocked the stimulation of MafA mRNA and protein expression by glucose. Combined adenovirus-mediated overexpression of PDX-1 and MafA in islets completely prevented the inhibition of insulin gene expression by palmitate. These results demonstrate that prolonged exposure of islets to palmitate inhibits insulin gene transcription by impairing nuclear localization of PDX-1 and cellular expression of MafA. The prevalence of diabetes mellitus is increasing dramatically in Western countries, in part because of the increase in obesity. Type 2 diabetes mellitus, the most frequent form of the disease, is characterized by defective insulin secretion from the pancreatic beta cells and peripheral insulin resistance. 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Previous studies in our laboratory have shown that deleterious effects of fatty acids appear mediated by distinct mechanisms; whereas inhibition of insulin secretion is observed after culture with palmitate, oleate, and other fatty acids (7Moore P.C. Ugas M.A. Hagman D.K. Parazzoli S.D. Poitout V. Diabetes. 2004; 53: 2610-2616Crossref PubMed Scopus (74) Google Scholar), insulin gene expression is only affected by palmitate and is mediated via de novo synthesis of ceramide (11Kelpe C.L. Moore P.C. Parazzoli S.D. Wicksteed B. Rhodes C.J. Poitout V. J. Biol. Chem. 2003; 278: 30015-30021Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). In isolated rat islets, we have shown that palmitate markedly blunts the activation by glucose of an insulin promoter reporter construct, indicating a transcriptional mode of action (11Kelpe C.L. Moore P.C. Parazzoli S.D. Wicksteed B. Rhodes C.J. Poitout V. J. Biol. Chem. 2003; 278: 30015-30021Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). However, the mechanisms by which palmitate affects the insulin promoter are unknown. Both beta cell-specific expression and metabolic regulation of the insulin gene are conferred by a highly conserved region lying ∼340 bp upstream of the transcription initiation site that constitutes the promoter/enhancer region (12Stein R. Cherrington A. Jefferson J. Handbook of Physiology. Section 7: The Endocrine System. II. American Physiology Society, Washington, D. C.2001: 25-78Google Scholar, 13Ohneda K. Ee H. German M. Semin. Cell Dev. Biol. 2000; 11: 227-233Crossref PubMed Scopus (106) Google Scholar, 14Melloul D. Marshak S. Cerasi E. Diabetologia. 2002; 45: 309-326Crossref PubMed Scopus (269) Google Scholar). The main glucose-responsive elements on the insulin promoter are the highly conserved A3 (15Melloul D. Ben-Neriah Y. Cerasi E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3865-3869Crossref PubMed Scopus (159) Google Scholar), C1 (16Sharma A. Fusco-DeMane D. Henderson E. Efrat S. Stein R. Mol. Endocrinol. 1995; 9: 1468-1476PubMed Google Scholar), and E1 (16Sharma A. Fusco-DeMane D. Henderson E. Efrat S. Stein R. Mol. Endocrinol. 1995; 9: 1468-1476PubMed Google Scholar) sites, which respectively bind the homeodomain protein pancreas-duodenum homeobox-1 (PDX-1) 2The abbreviations used are: PDX-1, pancreas-duodenum homeobox-1; BSA, bovine serum albumin; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; RT, reverse transcription; RPA, ribonuclease protection assay; Ad, adenovirus; pfu, plaque-forming units.2The abbreviations used are: PDX-1, pancreas-duodenum homeobox-1; BSA, bovine serum albumin; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; RT, reverse transcription; RPA, ribonuclease protection assay; Ad, adenovirus; pfu, plaque-forming units. (17Marshak S. Totary H. Cerasi E. Melloul D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15057-15062Crossref PubMed Scopus (151) Google Scholar), the basic region leucine zipper MafA (18Olbrot M. Rud J. Moss L.G. Sharma A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6737-6742Crossref PubMed Scopus (257) Google Scholar, 19Kataoka K. Han S.I. Shioda S. Hirai M. Nishizawa M. Handa H. J. Biol. Chem. 2002; 277: 49903-49910Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 20Matsuoka T.A. Zhao L. Artner I. Jarrett H.W. Friedman D. Means A. Stein R. Mol. Cell. Biol. 2003; 23: 6049-6062Crossref PubMed Scopus (254) Google Scholar), and a heterodimeric complex of basic helix-loop-helix proteins consisting of the ubiquitous class A (E12/E47 and E2/5) and beta cell-restricted class B (Beta2/NeuroD) proteins (21Shieh S.-Y. Tsai M.-J. J. Biol. Chem. 1991; 266: 16708-16714Abstract Full Text PDF PubMed Google Scholar). The aims of this study were 1) to examine whether prolonged exposure of isolated rat islets to palmitate alters the binding activity of MafA, PDX-1, and Beta2 to their cognate sequences on the insulin promoter and, if so, 2) to ascertain whether this effect is because of reduced expression or post-translational modifications of the transcription factors and 3) whether overexpression of the proteins prevents palmitate inhibition of insulin gene expression. Reagents—RPMI 1640 was obtained from Invitrogen. α-32PUTP and α-32PdCTP were from PerkinElmer Life Sciences. Fatty acid-free bovine serum albumin (BSA) was from Serologicals Corporation (Norcross, GA). Palmitic acid (sodium salt) and all other reagents (analytical grade) were from Sigma. Animals—Normal 225–275-g male Wistar rats were purchased from Charles River Laboratories (Wilmington, MA). Animals were housed on a 12-h light/dark cycle with free access to standard laboratory chow and water. All procedures using animals were approved by the Pacific Northwest Research Institute Animal Care and Use Committee. Rat Islet Isolation and Culture—Rat islets were isolated by collagenase digestion and dextran density gradient centrifugation as described previously (11Kelpe C.L. Moore P.C. Parazzoli S.D. Wicksteed B. Rhodes C.J. Poitout V. J. Biol. Chem. 2003; 278: 30015-30021Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) and then allowed to recover overnight in RPMI 1640 supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 and mm glucose. culture were as described The of culture palmitate was as described previously (11Kelpe C.L. Moore P.C. Parazzoli S.D. Wicksteed B. Rhodes C.J. Poitout V. J. Biol. Chem. 2003; 278: 30015-30021Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) with a of of All the of and as with palmitate. of and Nuclear cellular proteins were from as described previously Briaud I. Rhodes C.J. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). nuclear from of islets were in phosphate-buffered in of mm mm mm 0.5 mm mm mm 0.5 mm 0.5 and allowed to on the of of 10% the were by centrifugation and in of mm mm mm mm mm 0.5 and All protein were was using the were by 10% and to were blocked in in mm mm and and with MafA Beta2 PDX-1 by C. or transcription overnight was using a and Life on were using and islets were with PBS, with on with PBS, and in of the was in on standard and were on and was using mm to and then allowed to to The were with blocked with serum in PBS, and then overnight with an in PBS, to was used in PBS, of the islets were by the of to with were using a insulin A3 C1 and E1 elements were by with the and α-32PdCTP Life In we used an to the of the insulin promoter M. J. German Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Nuclear extracts were with of with or in a to with binding mm mm mm 2 mm mm and 100 of were on in mm mm mm 2 and by The of the protein in the binding was by using PDX-1 or MafA. The were using Isolation and was from of 100 islets as described previously P. N. PubMed Scopus Google Scholar). and the rat MafA, PDX-1, and were designed using the MafA reverse PDX-1 reverse and reverse was using the Life and an as described previously Robertson R.P. Diabetes. 2002; PubMed Scopus Google Scholar). are as a of gene to were using the and from a of the rat gene and a conserved of the were on as described previously (11Kelpe C.L. Moore P.C. Parazzoli S.D. Wicksteed B. Rhodes C.J. Poitout V. J. Biol. Chem. 2003; 278: 30015-30021Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). The were to in The were using the of and Islet PDX-1 and MafA the promoter were by the Pacific Northwest Research Institute as described previously (11Kelpe C.L. Moore P.C. Parazzoli S.D. Wicksteed B. Rhodes C.J. Poitout V. J. Biol. Chem. 2003; 278: 30015-30021Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) using a PDX-1 by M. Institute and a MafA by R. Stein A the of the promoter was used as a The was in by 100 islets with increasing concentrations to plaque-forming The islets were then in to are as were by or of with was Palmitate MafA and PDX-1 to on the in rat islets were cultured or with or mm glucose in the or of 0.5 mm palmitate. of nuclear extracts to the A3, C1, and was by a after a were observed with and in the of The of MafA and PDX-1, in the with the C1 and A3 was by the with The of mm glucose the of the binding complex to the C1 and, to a to the A3 and E1 The of 0.5 mm palmitate markedly reduced binding to the C1 and A3 elements in the of mm glucose to the E1 and was not affected by palmitate. The of binding to the A3 and C1 elements in the of 0.5 mm palmitate glucose was after of culture and after and These results demonstrate that palmitate impairs the binding activity of MafA and PDX-1 to their cognate sequences on the insulin promoter in the of glucose levels in beta with our that palmitate blunts glucose activation of an insulin (11Kelpe C.L. Moore P.C. Parazzoli S.D. Wicksteed B. Rhodes C.J. Poitout V. J. Biol. Chem. 2003; 278: 30015-30021Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Palmitate PDX-1 Nuclear and MafA whether the of binding activity observed in was because of protein expression or expression of PDX-1, MafA, and Beta2 in or protein extracts of rat islets cultured in or mm glucose in the or of 0.5 mm palmitate was by of PDX-1 in was not affected by glucose or palmitate mm PDX-1 was in the nuclear as shown by Western and A and In the of palmitate, the expression of PDX-1 in the was reduced These results that palmitate impairs the of PDX-1 from the to the that glucose stimulation I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. K. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). mm MafA expression was in by In contrast, mm glucose MafA protein was a stimulation of MafA protein expression by glucose. In the of palmitate, MafA protein expression was on by in A and MafA protein was in the and its levels in the nuclear were reduced by by palmitate A and in with its reduced expression in These results that palmitate inhibits MafA protein expression. Beta2 the protein transcription was affected by palmitate. the mechanisms by which palmitate affects protein MafA and PDX-1 mRNA levels were in islets after a culture in or mm glucose with or 0.5 mm palmitate MafA mRNA expression In the of palmitate, the effect of glucose was The course of this effect is shown in In contrast, glucose palmitate a effect on the expression of PDX-1 mRNA with the protein expression observed A and These results that palmitate affects PDX-1 and MafA binding by distinct PDX-1 is the post-translational in its nuclear whereas palmitate inhibits glucose stimulation of MafA mRNA of PDX-1 and MafA Palmitate of the of PDX-1 and MafA in palmitate inhibition of insulin gene we to whether overexpression of transcription factors insulin mRNA expression in the of palmitate. isolated islets were with PDX-1 or MafA a of and expression of the proteins was by after or shown in protein expression was to to whether the proteins were islets were with increasing of and the binding activity of nuclear extracts to the A3 and C1 elements was by shown in binding of PDX-1 and MafA to their sequences levels and with increasing the of was with PDX-1, MafA, or both were then cultured in the of mm glucose with or 0.5 mm palmitate. gene expression was by and shown previously I. Harmon J.S. Kelpe C.L. Segu V.B. Poitout V. Diabetes. 2001; 50: 315-321Crossref PubMed Scopus (248) Google Scholar, 11Kelpe C.L. Moore P.C. Parazzoli S.D. Wicksteed B. Rhodes C.J. Poitout V. J. Biol. Chem. 2003; 278: 30015-30021Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), palmitate insulin gene expression after in islets of MafA insulin gene expression to that of the islets mm glucose. overexpression of PDX-1 reduced insulin mRNA In the of palmitate, MafA PDX-1 was to the in insulin gene expression However, overexpression of MafA and PDX-1 completely prevented the effect of palmitate on insulin gene expression and the key of transcription factors in the mechanisms of lipotoxicity on the insulin The aims of this study were to whether prolonged exposure of isolated rat islets to palmitate alters the binding activity of MafA, PDX-1, and Beta2 to the insulin to ascertain whether this effect was to reduced expression or post-translational modifications of transcription and to whether overexpression of proteins palmitate inhibition of insulin gene expression. results that exposure of rat islets to levels of palmitate inhibits binding of MafA and PDX-1 but not Beta2 to the insulin promoter. the mechanisms of this inhibition in that palmitate alters the cellular localization of PDX-1 by its to the whereas glucose-stimulated MafA mRNA expression is in reduced overall MafA protein palmitate inhibition of insulin gene expression is prevented by overexpression of MafA and S. Briaud I. Rouault C. Reach G. Poitout V. Metabolism. 2000; 49: 532-536Abstract Full Text PDF PubMed Scopus (123) Google Scholar, 10Briaud I. Harmon J.S. Kelpe C.L. Segu V.B. Poitout V. Diabetes. 2001; 50: 315-321Crossref PubMed Scopus (248) Google Scholar, 11Kelpe C.L. Moore P.C. Parazzoli S.D. Wicksteed B. Rhodes C.J. Poitout V. J. Biol. Chem. 2003; 278: 30015-30021Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) and (8Ritz-Laser B. Meda P. Constant I. Klages N. Charollais A. Morales A. Magnan C. Ktorza A. Philippe J. Endocrinology. 1999; 140: 4005-4014Crossref PubMed Scopus (81) Google Scholar, S. C. G. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. Y. Y. H. Metabolism. 2001; 50: Full Text PDF PubMed Scopus Google Scholar) have shown previously that exposure to levels of glucose and palmitate inhibits insulin gene expression. this effect of palmitate was observed to the transcriptional (11Kelpe C.L. Moore P.C. Parazzoli S.D. Wicksteed B. Rhodes C.J. Poitout V. J. Biol. Chem. 2003; 278: 30015-30021Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). demonstrate that the mechanisms of this transcriptional inhibition key transcription factors of the insulin PDX-1 and MafA. reduced binding of PDX-1 and MafA to the A3 and C1 was by the that palmitate nuclear localization of PDX-1 and mRNA expression of MafA that the site of action of palmitate upstream of the binding of the transcription gene expression is highly by which both the of transcription and mRNA D. Marshak S. Cerasi E. Diabetologia. 2002; 45: 309-326Crossref PubMed Scopus (269) Google Scholar). studies have shown that binding to the A3 and C1 elements is by glucose in 14Melloul D. Marshak S. Cerasi E. Diabetologia. 2002; 45: 309-326Crossref PubMed Scopus (269) Google Scholar). A site of glucose regulation of PDX-1 function is the post-translational D. Marshak S. Cerasi E. 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This MafA and PDX-1 as of lipotoxicity the of the insulin previously that palmitate inhibition of insulin gene expression de novo synthesis of ceramide (11Kelpe C.L. Moore P.C. Parazzoli S.D. Wicksteed B. Rhodes C.J. Poitout V. J. Biol. Chem. 2003; 278: 30015-30021Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). The results of the study the as to ceramide affect PDX-1 and MafA. the mechanisms of PDX-1 regulation by glucose are of the been H. K. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, I. G. S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, R. K. R. M. K. Diabetes. 2000; 49: PubMed Scopus Google Scholar, K. Diabetes. 2001; 50: PubMed Scopus Google Scholar, P. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). ceramide been shown to with this the of protein B in all cell Diabetes. PubMed Scopus Google Scholar), is to that ceramide a from glucose to PDX-1 via the ceramide in beta cells as in other cells S. J. PubMed Scopus Google Scholar, 2003; PubMed Scopus Google Scholar). In beta activation been shown to PDX-1 binding H. G. N. S. S. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar) and nuclear localization D. Y. H. Y. Y. H. Y. M. Diabetes. 2003; PubMed Scopus Google Scholar). These are in our The mode of regulation of MafA by glucose is unknown. results that palmitate inhibition of MafA expression is the transcriptional the mechanisms of effects of the promoter/enhancer region of the MafA In this study that the mechanisms by which palmitate impairs insulin gene transcription reduced binding of PDX-1 and MafA to the insulin promoter. both factors are affected levels by PDX-1 is affected in its nuclear whereas MafA is affected the transcriptional The of the mechanisms of lipotoxicity is by the of the in insulin gene expression in the of palmitate observed after overexpression of PDX-1 and MafA. the of fatty acid inhibition of insulin gene transcription to beta cell in type 2 diabetes to been shown in that regulation of insulin gene expression by glucose is to insulin in the of G. G. Cerasi E. N. Endocrinology. 2002; 143: PubMed Scopus Google Scholar). of the of fatty acids the of to the of beta cell function during the course of type 2 diabetes. the PDX-1 Stein the MafA and Institute the PDX-1 Robertson and Rhodes of the and Harmon