Palmitate Induces Insulin Resistance in H4IIEC3 Hepatocytes through Reactive Oxygen Species Produced by Mitochondria

Abstract

Visceral adiposity in obesity causes excessive free fatty acid (FFA) flux into the liver via the portal vein and may cause fatty liver disease and hepatic insulin resistance. However, because animal models of insulin resistance induced by lipid infusion or a high fat diet are complex and may be accompanied by alterations not restricted to the liver, it is difficult to determine the contribution of FFAs to hepatic insulin resistance. Therefore, we treated H4IIEC3 cells, a rat hepatocyte cell line, with a monounsaturated fatty acid (oleate) and a saturated fatty acid (palmitate) to investigate the direct and initial effects of FFAs on hepatocytes. We show that palmitate, but not oleate, inhibited insulin-stimulated tyrosine phosphorylation of insulin receptor substrate 2 and serine phosphorylation of Akt, through c-Jun NH2-terminal kinase (JNK) activation. Among the well established stimuli for JNK activation, reactive oxygen species (ROS) played a causal role in palmitate-induced JNK activation. In addition, etomoxir, an inhibitor of carnitine palmitoyltransferase-1, which is the rate-limiting enzyme in mitochondrial fatty acid β-oxidation, as well as inhibitors of the mitochondrial respiratory chain complex (thenoyltrifluoroacetone and carbonyl cyanide m-chlorophenylhydrazone) decreased palmitate-induced ROS production. Together, our findings in hepatocytes indicate that palmitate inhibited insulin signal transduction through JNK activation and that accelerated β-oxidation of palmitate caused excess electron flux in the mitochondrial respiratory chain, resulting in increased ROS generation. Thus, mitochondria-derived ROS induced by palmitate may be major contributors to JNK activation and cellular insulin resistance. Visceral adiposity in obesity causes excessive free fatty acid (FFA) flux into the liver via the portal vein and may cause fatty liver disease and hepatic insulin resistance. However, because animal models of insulin resistance induced by lipid infusion or a high fat diet are complex and may be accompanied by alterations not restricted to the liver, it is difficult to determine the contribution of FFAs to hepatic insulin resistance. Therefore, we treated H4IIEC3 cells, a rat hepatocyte cell line, with a monounsaturated fatty acid (oleate) and a saturated fatty acid (palmitate) to investigate the direct and initial effects of FFAs on hepatocytes. We show that palmitate, but not oleate, inhibited insulin-stimulated tyrosine phosphorylation of insulin receptor substrate 2 and serine phosphorylation of Akt, through c-Jun NH2-terminal kinase (JNK) activation. Among the well established stimuli for JNK activation, reactive oxygen species (ROS) played a causal role in palmitate-induced JNK activation. In addition, etomoxir, an inhibitor of carnitine palmitoyltransferase-1, which is the rate-limiting enzyme in mitochondrial fatty acid β-oxidation, as well as inhibitors of the mitochondrial respiratory chain complex (thenoyltrifluoroacetone and carbonyl cyanide m-chlorophenylhydrazone) decreased palmitate-induced ROS production. Together, our findings in hepatocytes indicate that palmitate inhibited insulin signal transduction through JNK activation and that accelerated β-oxidation of palmitate caused excess electron flux in the mitochondrial respiratory chain, resulting in increased ROS generation. Thus, mitochondria-derived ROS induced by palmitate may be major contributors to JNK activation and cellular insulin resistance. Insulin is the major hormone that inhibits gluconeogenesis in the liver. Visceral adiposity in obesity causes hepatic steatosis and insulin resistance. In an insulin-resistant state, impaired insulin action allows enhancement of glucose production in the liver, resulting in systemic hyperglycemia (1Saltiel A.R. Kahn C.R. Nature. 2001; 414: 799-806Crossref PubMed Scopus (3870) Google Scholar) and contributing to the development of type 2 diabetes. In addition, we have demonstrated experimentally that insulin resistance accelerated the pathology of steatohepatitis in genetically obese diabetic OLETF rats (2Ota T. Takamura T. Kurita S. Matsuzawa N. Kita Y. Uno M. Akahori H. Misu H. Sakurai M. Zen Y. Nakanuma Y. Kaneko S. Gastroenterology. 2007; 132: 282-293Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). In contrast, lipid-induced oxidative stress caused steatohepatitis and hepatic insulin resistance in mice (3Matsuzawa N. Takamura T. Kurita S. Misu H. Ota T. Ando H. Yokoyama M. Honda M. Zen Y. Nakanuma Y. Miyamoto K. Kaneko S. Hepatology. 2007; 46: 1392-1403Crossref PubMed Scopus (403) Google Scholar). In fact, steatosis of the liver is an independent predictor of insulin resistance in patients with nonalcoholic fatty liver disease (4Sakurai M. Takamura T. Ota T. Ando H. Akahori H. Kaji K. Sasaki M. Nakanuma Y. Miura K. Kaneko S. J. Gastroenterol. 2007; 42: 312-317Crossref PubMed Scopus (46) Google Scholar). It remains unclear whether hepatic steatosis causally contributes to insulin resistance or whether it is merely a resulting pathology. Excessive dietary free fatty acid (FFA) 2The abbreviations used are: FFA, free fatty acid; IRS, insulin receptor substrate; JNK, c-Jun NH2-terminal kinase; ER, endoplasmic reticulum; ROS, reactive oxygen species; NAC, N-acetyl-l-cysteine; H2DCFDA, 2′,7′-dichlorofluorescin diacetate; OXPHOS, oxidative phosphorylation; PVDF, polyvinylidene difluoride.2The abbreviations used are: FFA, free fatty acid; IRS, insulin receptor substrate; JNK, c-Jun NH2-terminal kinase; ER, endoplasmic reticulum; ROS, reactive oxygen species; NAC, N-acetyl-l-cysteine; H2DCFDA, 2′,7′-dichlorofluorescin diacetate; OXPHOS, oxidative phosphorylation; PVDF, polyvinylidene difluoride. flux into the liver via the portal vein may cause fatty liver disease and hepatic insulin resistance. Indeed, elevated plasma FFA concentrations correlate with obesity and decreased target tissue insulin sensitivity (5Boden G. Diabetes. 1997; 46: 3-10Crossref PubMed Scopus (0) Google Scholar). Experimentally, lipid infusion or a high fat diet that increases circulating FFA levels promotes insulin resistance in the liver. Candidate events linking FFA to insulin resistance in vivo are the up-regulation of SREBP-1c (6Ide T. Shimano H. Yahagi N. Matsuzaka T. Nakakuki M. Yamamoto T. Nakagawa Y. Takahashi A. Suzuki H. Sone H. Toyoshima H. Fukamizu A. Yamada N. Nat. Cell Biol. 2004; 6: 351-357Crossref PubMed Scopus (278) Google Scholar), inflammation caused by activation of c-Jun amino-terminal kinase (JNK) (7Hirosumi J. Tuncman G. Chang L. Gorgun C.Z. Uysal K.T. Maeda K. Karin M. Hotamisligil G.S. Nature. 2002; 420: 333-336Crossref PubMed Scopus (2612) Google Scholar) or IKKβ (8Boden G. She P. Mozzoli M. Cheung P. Gumireddy K. Reddy P. Xiang X. Luo Z. Ruderman N. Diabetes. 2005; 54: 3458-3465Crossref PubMed Scopus (413) Google Scholar), endoplasmic reticulum (ER) stress (9Ozcan U. Cao Q. Yilmaz E. Lee A.H. Iwakoshi N.N. Ozdelen E. Tuncman G. Gorgun C. Glimcher L.H. Hotamisligil G.S. Science. 2004; 306: 457-461Crossref PubMed Scopus (2950) Google Scholar), ceramide (10Kim J.K. Fillmore J.J. Chen Y. Yu C. Moore I.K. Pypaert M. Lutz E.P. Kako Y. Velez-Carrasco W. Goldberg I.J. Breslow J.L. Shulman G.I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7522-7527Crossref PubMed Scopus (585) Google Scholar, 11Turinsky J. O'Sullivan D.M. Bayly B.P. J. Biol. Chem. 1990; 265: 16880-16885Abstract Full Text PDF PubMed Google Scholar), and TRB3 (12Du K. Herzig S. Kulkarni R.N. Montminy M. Science. 2003; 300: 1574-1577Crossref PubMed Scopus (707) Google Scholar). However, which event is the direct and initial target of FFA in the liver is unclear. Insulin resistance induced by lipid infusion or a high fat diet is complex and may be accompanied by alterations not restricted to the liver, making it difficult to determine the contribution of FFAs to hepatic insulin resistance. For example, hyperinsulinemia and hyperglycemia secondary to the initial event also may contribute to the development of diet-induced insulin resistance in vivo (6Ide T. Shimano H. Yahagi N. Matsuzaka T. Nakakuki M. Yamamoto T. Nakagawa Y. Takahashi A. Suzuki H. Sone H. Toyoshima H. Fukamizu A. Yamada N. Nat. Cell Biol. 2004; 6: 351-357Crossref PubMed Scopus (278) Google Scholar). To address the early event(s) triggering the development of high fat diet- or obesity-induced insulin resistance, we investigated the molecular mechanism(s) underlying the direct action of FFA on hepatocytes to cause insulin resistance in vitro, using the rat hepatocyte cell line H4IIEC3. We found that mitochondria-derived reactive oxygen species (ROS) were a cause of palmitate-induced insulin resistance in hepatocytes. Materials—The antibody against IRS-2 was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Antibodies against phosphotyrosine, Akt, phospho-Akt (Ser473), stress-activated protein kinase/JNK, phospho-stress-activated protein kinase/JNK (Thr183/Tyr185), and phospho-GSK (glycogen synthase kinase)-3 (Ser21/9) were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against GSK-3 and phospho-c-Jun were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Insulin from porcine pancreas, sodium palmitate, sodium oleate, myriocin, N-acetyl-l-cysteine, rotenone, thenoyltrifluoroacetone, cyanide m-chlorophenylhydrazone, oxypurinol, etomoxir, and tunicamycin was obtained from Sigma. SP600125 and apocynin were from Calbiochem. dl-α-tocopherol and 2′,7′-dichlorofluorescin diacetate (H2DCFDA) were from Wako (Osaka, Japan). Cell Culture and Fatty Acid Treatment—Studies were performed in the rat hepatoma cell line H4IIEC3, purchased from the American Type Culture Collection (Manassas, VA). Cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), penicillin (100 units/ml), and streptomycin (0.1 mg/ml; Invitrogen). The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2, with medium changes three times a week. All studies were conducted using 80–90% confluent cells, which were treated with the indicated concentrations of FFAs in the presence of 2% FFA-free bovine serum albumin (Sigma). Cell Harvest and Western Blot Analysis—H4IIEC3 hepatocytes, grown to 80–90% confluence in 6-well plates, were treated with the indicated reagents for 16 h in Dulbecco's modified Eagle's medium. After treatment, the cells were stimulated with insulin (1 ng/ml) for 15 min. Then the cells were washed with ice-cold phosphate-buffered saline and lysed in buffer containing 20 mm Tris-HCl (pH 7.5), 5 mm EDTA, 1% Nonidet P-40, 2 mm Na3VO4, 100 mm NaF, and a protease inhibitor mixture (Sigma). After sonication with a Bioruptor (Cosmo Bio, Tokyo, Japan), the lysates were centrifuged to remove insoluble materials. The supernatants (10 μg/lane) were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). For detection of phosphotyrosine insulin receptor and phosphotyrosine IRS-2, the supernatants (400 μgof protein) were immunoprecipitated with a phosphotyrosine antibody and protein G beads for 2 h at 4 °C before SDS-PAGE. The membranes were blocked in a buffer containing 5% nonfat milk, 50 mm Tris (pH 7.6), 150 mm NaCl, and 0.1% Tween 20 (TBS-T) for 1 h at room temperature. They were then incubated with specific primary antibodies and subsequently with horseradish peroxidase-linked secondary antibodies. Signals were detected with a chemiluminescence detection system (ECL Plus Western blotting detection reagents; GE Healthcare). Densitometric analysis was conducted directly on the blotted membrane, using a CCD camera system (LAS-3000 Mini; Fujifilm, Tokyo, Japan) and Scion Image software. Quantitative Real Time PCR—Total RNA was extracted from cultured H4IIEC3 hepatocytes using an RNeasy mini kit (Qiagen, Germantown, MD), according to the manufacturer's protocol. The cDNA was synthesized from total RNA (100 ng) using random hexamer primers, N6, and a high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). Quantitative real time PCR was performed with an ABI Prism 7900HT (Applied Biosystems). The set of specific primers and TaqMan probes in the present study was obtained from Applied Biosystems. The PCR conditions were one cycle at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Analysis of XBP-1 RNA was extracted from H4IIEC3 hepatocytes, and cDNA was synthesized as The cDNA was with a of primers and to the rat XBP-1 The PCR conditions were initial at °C for min, followed by cycles of °C for °C for °C for and a at °C for min. The PCR were separated by of of ROS was detected using the H2DCFDA, according to a T. S. T. Y. M. Nature. PubMed Scopus Google Scholar). H4IIEC3 hepatocytes, grown to confluence in plates, were treated with the indicated reagents in Dulbecco's modified Eagle's medium for After treatment, the cells were washed with phosphate-buffered with 10 H2DCFDA, and incubated for min at 37 The was using a MA). of cellular of containing carbonyl that with to the was using a protein carbonyl kit according to the manufacturer's and as N. Takamura T. Ando H. S. Kurita S. Misu H. Ota T. Yokoyama M. Honda M. Miyamoto K. Kaneko S. Full Text Full Text PDF PubMed Scopus Google Scholar). are as were using were by analysis of All were performed with for Insulin chain fatty were for the palmitate, a saturated fatty and oleate, a monounsaturated fatty To whether FFAs impaired insulin signal transduction in H4IIEC3 hepatocytes, we the of FFAs on insulin-stimulated tyrosine phosphorylation of IRS-2 and serine phosphorylation of and with mm palmitate inhibited insulin-stimulated tyrosine phosphorylation of IRS-2 by in H4IIEC3 of IRS-2, insulin-stimulated serine phosphorylation of and were also inhibited by mm palmitate treatment, by and an insulin-resistant However, the protein levels of total IRS-2, Akt, and GSK-3 were by we that palmitate, but not oleate, impaired insulin-stimulated serine phosphorylation in the hepatoma cell line JNK by to Insulin a stress-activated protein to and at serine T. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, G. W. Lee Karin M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). phosphorylation of tyrosine to a in insulin studies have the role of JNK in insulin resistance in (7Hirosumi J. Tuncman G. Chang L. Gorgun C.Z. Uysal K.T. Maeda K. Karin M. Hotamisligil G.S. Nature. 2002; 420: 333-336Crossref PubMed Scopus (2612) Google Scholar, Y. H. M. T. Y. M. Y. M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, H. S. J.L. T. J. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, we the of FFAs on JNK activation and in insulin but not oleate, increased JNK and c-Jun and inhibitor of JNK, SP600125 Sasaki W. A. S. Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: PubMed Scopus Google Scholar), the palmitate-induced phosphorylation of c-Jun that palmitate To whether palmitate-induced JNK activation cellular insulin resistance, we inhibited the JNK with SP600125 insulin-stimulated serine phosphorylation of and GSK-3 in H4IIEC3 hepatocytes to palmitate that JNK activation by palmitate to palmitate-induced insulin of a JNK inhibitor on palmitate-induced alterations in insulin-stimulated phosphorylation of and GSK-3 in H4IIEC3 hepatocytes. H4IIEC3 cells were incubated in the presence or of palmitate and the JNK inhibitor SP600125 for 16 h to with insulin (1 15 cell lysates were by transferred to a membrane, and with the indicated antibodies. was by are the from of or independent were to the of total or GSK-3 and as the palmitate Image for SREBP-1c and in JNK and Insulin in H4IIEC3 SREBP-1c to a role in diet-induced insulin resistance in (6Ide T. Shimano H. Yahagi N. Matsuzaka T. Nakakuki M. Yamamoto T. Nakagawa Y. Takahashi A. Suzuki H. Sone H. Toyoshima H. Fukamizu A. Yamada N. Nat. Cell Biol. 2004; 6: 351-357Crossref PubMed Scopus (278) Google Scholar) found that high diet-induced hyperglycemia and hyperinsulinemia hepatic of to of IRS-2 at the However, in the present palmitate the of SREBP-1c in H4IIEC3 hepatocytes with the and protein levels of IRS-2 were by Thus, palmitate not to cause insulin resistance in hepatocytes via the SREBP-1c stress is induced in insulin-resistant as obesity and type 2 and in stress to to the of insulin through of JNK (9Ozcan U. Cao Q. Yilmaz E. Lee A.H. Iwakoshi N.N. Ozdelen E. Tuncman G. Gorgun C. Glimcher L.H. Hotamisligil G.S. Science. 2004; 306: 457-461Crossref PubMed Scopus (2950) Google Scholar). excessive FFAs have to stress in E. C. L. T. M. A. PubMed Scopus Google Scholar), we whether palmitate caused stress in H4IIEC3 hepatocytes. stress the of XBP-1 which the transcription of molecular protein) M. Cell Biol. 2002; PubMed Scopus Google Scholar). at mm not the of or the of an used to stress we the of palmitate and tunicamycin on insulin-stimulated signal transduction and JNK activation The of tunicamycin on insulin-stimulated serine phosphorylation of was and not with that of the in JNK by tunicamycin was and not with that of that stress played a role in palmitate-induced JNK activation and cellular insulin resistance in H4IIEC3 hepatocytes. ROS to increased cellular ROS levels are to phosphorylation of JNK H. Y. S. 2004; PubMed Scopus Google Scholar). Indeed, ROS levels are increased in conditions with insulin resistance, as and type 2 S. T. M. M. Yamada Y. Y. M. M. J. 2004; PubMed Scopus Google Scholar). FFAs have to ROS in cells, as cells C. N. PubMed Google Scholar), Y. Ruderman 2005; PubMed Scopus Google Scholar), and S. T. M. M. Yamada Y. Y. M. M. J. 2004; PubMed Scopus Google Scholar). Thus, we that palmitate increased ROS production and JNK, to the impaired insulin To H4IIEC3 hepatocytes were incubated with H2DCFDA, a to ROS, with or was elevated by with mm palmitate for and palmitate induced ROS production with the of protein a of oxidative increased in hepatocytes with cells that palmitate, cause ROS production and oxidative stress in H4IIEC3 hepatocytes. Insulin to whether palmitate-induced ROS a causal role in insulin resistance by whether and also as insulin and palmitate-induced ROS at 10 mm and at mm ROS production by 50 and In with decreased ROS the the insulin-stimulated phosphorylation impaired by at 10 mm and at mm the phosphorylation by 40 and palmitate-induced JNK phosphorylation; at 10 mm and at mm it by and that palmitate increased ROS levels in H4IIEC3 hepatocytes and JNK, resulting in insulin of on palmitate-induced alterations in insulin-stimulated serine phosphorylation of in H4IIEC3 hepatocytes. H4IIEC3 cells were incubated in the presence or of palmitate and for 16 h to with insulin (1 15 cell lysates were by transferred to a membrane, and with the indicated antibodies. was by are the from of or independent were to the of total protein and as the palmitate NAC, N-acetyl-l-cysteine; Image of on palmitate-induced JNK activation in H4IIEC3 hepatocytes. H4IIEC3 cells were incubated in the presence or of palmitate and for 16 cell lysates were by transferred to a membrane, and with the indicated antibodies. was by are the from of independent were to the of total JNK protein and as the palmitate Image ROS in the of ROS induced by palmitate in H4IIEC3 hepatocytes, we the cellular in ROS and ROS production was by rotenone, an inhibitor of mitochondrial respiratory chain complex thenoyltrifluoroacetone, an inhibitor of mitochondrial respiratory chain complex and carbonyl cyanide m-chlorophenylhydrazone, an of oxidative phosphorylation In contrast, ROS production in H4IIEC3 cells was not by an inhibitor of or oxypurinol, an inhibitor of that the mitochondrial respiratory chain is in palmitate-induced ROS in H4IIEC3 hepatocytes. ROS through the Fatty Acid are in the mitochondrial fatty acid β-oxidation which the mitochondrial respiratory chain with of the respiratory chain may cause of to ROS production. Thus, we whether palmitate-induced ROS production was on mitochondrial fatty acid is the rate-limiting enzyme in mitochondrial fatty acid etomoxir, a decreased palmitate-induced ROS by palmitate, but not oleate, increased of the up-regulation may contribute to palmitate-induced ROS because the accelerated β-oxidation cause excessive electron flux in the respiratory In the present we investigated the direct action of fatty on insulin in hepatocytes. The saturated fatty acid palmitate, but not the fatty acid oleate, impaired tyrosine phosphorylation of IRS-2, serine phosphorylation of Akt, and serine phosphorylation of of which are of insulin resistance in cultured H4IIEC3 hepatocytes in vivo findings (6Ide T. Shimano H. Yahagi N. Matsuzaka T. Nakakuki M. Yamamoto T. Nakagawa Y. Takahashi A. Suzuki H. Sone H. Toyoshima H. Fukamizu A. Yamada N. Nat. Cell Biol. 2004; 6: 351-357Crossref PubMed Scopus (278) Google Scholar), the of the SREBP-1c was by palmitate to cultured H4IIEC3 hepatocytes, which is a of a for fatty acid and IRS-2 protein levels were insulin resistance in cells, as H. S. J.L. T. J. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) and cells 2003; PubMed Scopus Google Scholar). with the present that FFA inhibits insulin at the of tyrosine phosphorylation of of cell to the findings in H. S. J.L. T. J. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) and primary hepatocytes and G. W. Lee Karin M. Proc. Natl. Acad. Sci. U. S. A. 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In our palmitate increased the of ceramide in H4IIEC3 hepatocytes at the the of ceramide was not blocked by a inhibitor of serine at the in ceramide ceramide was not blocked was used in with an inhibitor of ceramide synthase not Therefore, we the that ceramide contributes to palmitate-induced insulin resistance in H4IIEC3 hepatocytes. studies are to the role of the ceramide in palmitate-induced insulin resistance in hepatocytes. In the present etomoxir, an inhibitor of decreased palmitate-induced ROS production. palmitate, but not oleate, increased the of the which may for the in insulin action palmitate and it was that fatty acid may be a in insulin sensitivity C.R. Nat. 2007; PubMed Scopus Google Scholar, H. K. Hotamisligil G.S. Full Text Full Text PDF PubMed Scopus Google Scholar). In we investigated the of on insulin in hepatocytes. palmitate-induced ROS and JNK phosphorylation and palmitate-induced phosphorylation of and T. at the molecular underlying the and of FFAs are In study mitochondrial ROS as a in palmitate-induced hepatic insulin resistance. may excess for mitochondrial OXPHOS, and ROS then the insulin by JNK, tyrosine phosphorylation of IRS-2, and hepatic insulin resistance The that an initial event in high diet-induced or obesity-induced insulin resistance in the liver is mitochondrial ROS which be a In to JNK inhibitors or mitochondrial as cyanide m-chlorophenylhydrazone, may a for by ROS generation. We and and for on Western analysis of with