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Prolonged Treatment of Primary Hepatocytes with Oleate Induces Insulin Resistance through p38 Mitogen-activated Protein Kinase

2007; Elsevier BV; Volume: 282; Issue: 19 Linguagem: Inglês

10.1074/jbc.m609701200

ISSN

1083-351X

Autores

Hui-Yu Liu, Qu Fan Collins, Yan Xiong, Fatiha Moukdar, Edgar Lupo, Zhenqi Liu, Wenhong Cao,

Tópico(s)

Protein Kinase Regulation and GTPase Signaling

Resumo

Free fatty acid (FFA) is believed to be a major environmental factor linking obesity to Type II diabetes. We have recently reported that FFA can induce gluconeogenesis in hepatocytes through p38 mitogen-activated protein kinase (p38). In this study, we have investigated the role of p38 in oleate-induced hepatic insulin resistance. Our results show that a prolonged treatment of primary hepatocytes with oleate blunted insulin suppression of hepatic gluconeogenesis, and decreased insulin-induced phosphorylation of Akt in a p38-dependent manner. Reduction of the insulin-induced Akt phosphorylation by oleate correlated with activation of p38. In the presence of p38 inhibition, prolonged exposure of hepatocytes to oleate failed to reduce insulin-stimulated phosphorylation of Akt. An siRNA against p38α prevented oleate suppression of the insulin-induced phosphorylation of Akt. Furthermore, a prolonged exposure of hepatocytes to oleate decreased insulin-induced tyrosine phosphorylation of IRS1/2, while slightly increasing serine phosphorylation of IRS. The decrease of insulin-stimulated tyrosine phosphorylation of IRS1/2 in hepatocytes by oleate was reversed by the inhibition of p38. We further show that a prolonged exposure of primary hepatocytes to oleate elevated the protein level of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene in a p38-dependent manner, but had no effect on the mRNA level of PTEN. Knocking down the PTEN gene prevented oleate to inhibit insulin activation of Akt and insulin suppression of gluconeogenesis. Together, results from this study demonstrate a critical role for p38 in oleate-induced hepatic insulin resistance. Free fatty acid (FFA) is believed to be a major environmental factor linking obesity to Type II diabetes. We have recently reported that FFA can induce gluconeogenesis in hepatocytes through p38 mitogen-activated protein kinase (p38). In this study, we have investigated the role of p38 in oleate-induced hepatic insulin resistance. Our results show that a prolonged treatment of primary hepatocytes with oleate blunted insulin suppression of hepatic gluconeogenesis, and decreased insulin-induced phosphorylation of Akt in a p38-dependent manner. Reduction of the insulin-induced Akt phosphorylation by oleate correlated with activation of p38. In the presence of p38 inhibition, prolonged exposure of hepatocytes to oleate failed to reduce insulin-stimulated phosphorylation of Akt. An siRNA against p38α prevented oleate suppression of the insulin-induced phosphorylation of Akt. Furthermore, a prolonged exposure of hepatocytes to oleate decreased insulin-induced tyrosine phosphorylation of IRS1/2, while slightly increasing serine phosphorylation of IRS. The decrease of insulin-stimulated tyrosine phosphorylation of IRS1/2 in hepatocytes by oleate was reversed by the inhibition of p38. We further show that a prolonged exposure of primary hepatocytes to oleate elevated the protein level of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene in a p38-dependent manner, but had no effect on the mRNA level of PTEN. Knocking down the PTEN gene prevented oleate to inhibit insulin activation of Akt and insulin suppression of gluconeogenesis. Together, results from this study demonstrate a critical role for p38 in oleate-induced hepatic insulin resistance. Plasma levels of free fatty acids (FFAs) 2The abbreviations used are: FFA, free fatty acid; BSA, bovine serum albumin; ANOVA, analysis of variance; JNK, c-Jun N-terminal kinase; PKC, protein kinase C. 2The abbreviations used are: FFA, free fatty acid; BSA, bovine serum albumin; ANOVA, analysis of variance; JNK, c-Jun N-terminal kinase; PKC, protein kinase C. are usually increased in obese subjects, and FFAs are considered as a causative link between obesity and Type II diabetes (1Baldeweg S.E. Golay A. Natali A. Balkau B. Prato S. Del Coppack S.W. Eur. J. Clin. Investig. 2000; 30: 45-52Crossref PubMed Scopus (102) Google Scholar, 2Bolinder J. Kerckhoffs D.A. Moberg E. Hagstrom-Toft E. Arner P. Diabetes. 2000; 49: 797-802Crossref PubMed Scopus (78) Google Scholar, 3Boden G. Diabetes. 1997; 46: 3-10Crossref PubMed Scopus (0) Google Scholar, 4Kelley D.E. Mandarino L.J. Diabetes. 2000; 49: 677-683Crossref PubMed Scopus (767) Google Scholar, 5Lewis G.F. Carpentier A. Adeli K. Giacca A. Endocr. Rev. 2002; 23: 201-229Crossref PubMed Scopus (829) Google Scholar). A consequence of increased levels of FFA is insulin resistance. Insulin resistance is not only a strong risk factor for cardiovascular diseases (4Kelley D.E. Mandarino L.J. Diabetes. 2000; 49: 677-683Crossref PubMed Scopus (767) Google Scholar, 6Boden G. Life Sci. 2003; 72: 977-988Crossref PubMed Scopus (40) Google Scholar), but may also eventually lead to Type II diabetes (7Boden G. Curr. Diab. Rep. 2006; 6: 177-181Crossref PubMed Scopus (201) Google Scholar), which is currently a major health problem of industrialized countries and tends to increase in its incidence and economic cost because of the increasing trend of obesity. Insulin resistance in liver, adipose tissue, and skeletal muscles plays a central role in the development of Type II diabetes. Specifically, insulin resistance in liver leads to an increased glucose production from hepatic gluconeogenesis, while insulin resistance in adipose tissue and skeletal muscles decreases uptake, utilization, and storage of glucose in these tissues (8Lam T.K. Yoshii H. Haber C.A. Bogdanovic E. Lam L. Fantus I.G. Giacca A. Am. J. Physiol. Endocrinol. Metab. 2002; 283: E682-E691Crossref PubMed Scopus (168) Google Scholar, 9Lam T.K. Carpentier A. Lewis G.F. van de Werve G. Fantus I.G. Giacca A. Am. J. Physiol. Endocrinol. Metab. 2003; 284: E863-E873Crossref PubMed Scopus (202) Google Scholar). When insulin production from pancreatic islets fails to overcome the insulin resistance, hyperglycemia ensues to announce the presence of frank Type II diabetes. Hepatic insulin resistance is a powerful promoter of glucose production from liver through gluconeogenesis. The unrestrained hepatic gluconeogenesis is a major contributor to hyperglycemia in both Types I and II diabetes (10Accili D. Diabetes. 2004; 53: 1633-1642Crossref PubMed Scopus (149) Google Scholar). Hyperglycemia can damage every organ system by inflaming and clogging blood vessels, which consequently results in heart attacks, strokes, renal failure, blindness, and amputations (11Brownlee M. Metabolism. 2000; 49: 9-13Abstract Full Text PDF PubMed Scopus (269) Google Scholar, 12Hirsch I.B. Brownlee M. J. Diabetes Complications. 2005; 19: 178-181Crossref PubMed Scopus (346) Google Scholar), etc. Equally important, hepatic insulin resistance causes hyperlipidemia, which can further lead to or aggravate global insulin resistance and directly contributes to the development of cardiovascular disorders and other complications related to diabetes (8Lam T.K. Yoshii H. Haber C.A. Bogdanovic E. Lam L. Fantus I.G. Giacca A. Am. J. Physiol. Endocrinol. Metab. 2002; 283: E682-E691Crossref PubMed Scopus (168) Google Scholar, 10Accili D. Diabetes. 2004; 53: 1633-1642Crossref PubMed Scopus (149) Google Scholar). Hepatic insulin resistance is tightly correlated with plasma levels of FFA. For example, chronic elevation of plasma FFA in obesity is almost always accompanied by hepatic insulin resistance (7Boden G. Curr. Diab. Rep. 2006; 6: 177-181Crossref PubMed Scopus (201) Google Scholar, 8Lam T.K. Yoshii H. Haber C.A. Bogdanovic E. Lam L. Fantus I.G. Giacca A. Am. J. Physiol. Endocrinol. Metab. 2002; 283: E682-E691Crossref PubMed Scopus (168) Google Scholar). Acute increase of plasma levels of FFA via lipid infusion can also lead to hepatic insulin resistance (8Lam T.K. Yoshii H. Haber C.A. Bogdanovic E. Lam L. Fantus I.G. Giacca A. Am. J. Physiol. Endocrinol. Metab. 2002; 283: E682-E691Crossref PubMed Scopus (168) Google Scholar, 13Boden G. She P. Mozzoli M. Cheung P. Gumireddy K. Reddy P. Xiang X. Luo Z. Ruderman N. Diabetes. 2005; 54: 3458-3465Crossref PubMed Scopus (419) Google Scholar). The exact mechanism by which FFA induces hepatic insulin resistance has been intensively studied, but much remains undetermined. Elevation of plasma FFA levels is known to activate a series of stress- and inflammation-related kinases, including at least JNK, IKKβ/NF-κB, PKC, and p38 (14Evans J.L. Goldfine I.D. Maddux B.A. Grodsky G.M. Endocr. Rev. 2002; 23: 599-622Crossref PubMed Scopus (1759) Google Scholar). All these kinase systems have been shown to be able to mediate fat-induced insulin resistance in various cellular and tissue types (14Evans J.L. Goldfine I.D. Maddux B.A. Grodsky G.M. Endocr. Rev. 2002; 23: 599-622Crossref PubMed Scopus (1759) Google Scholar, 15Muoio D.M. Newgard C.B. Annu. Rev. Biochem. 2006; 75: 367-401Crossref PubMed Scopus (289) Google Scholar). For example, deletion of the JNK1 gene in mouse models (JNK-/-) can slow down high fat diet-induced global insulin resistance (16Hirosumi 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 (2629) Google Scholar). The predominant alteration in JNK1-/- mice is the reduced adipose depot, indicating that the primary tissue affected by JNK is adipose tissue, but not liver (16Hirosumi 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 (2629) Google Scholar). Deletion of the IKKβ gene from liver can also prevent high fat diet-induced global and hepatic insulin resistance, while liver-specific expression of the IKKβ gene can promote high fat diet-induced hepatic and global insulin resistance (17Cai D. Yuan M. Frantz D.F. Melendez P.A. Hansen L. Lee J. Shoelson S.E. Nat. Med. 2005; 11: 183-190Crossref PubMed Scopus (1795) Google Scholar, 18Arkan M.C. Hevener A.L. Greten F.R. Maeda S. Li Z.W. Long J.M. Wynshaw-Boris A. Poli G. Olefsky J. Karin M. Nat. Med. 2005; 11: 191-198Crossref PubMed Scopus (1478) Google Scholar). Therefore, it is clear that the IKKβ/NF-κB signaling pathway can play a role in fat-induced hepatic insulin resistance. Some PKC isoforms such as PKCɛ has been shown to be able to mediate FFA-induced insulin resistance in adipocytes (19Gao Z. Zhang X. Zuberi A. Hwang D. Quon M.J. Lefevre M. Ye J. Mol. Endocrinol. 2004; 18: 2024-2034Crossref PubMed Scopus (266) Google Scholar). FFA can activate PKCδ in isolated hepatocytes and liver (13Boden G. She P. Mozzoli M. Cheung P. Gumireddy K. Reddy P. Xiang X. Luo Z. Ruderman N. Diabetes. 2005; 54: 3458-3465Crossref PubMed Scopus (419) Google Scholar, 20Collins Q.F. Xiong Y. Lupo J.E.J. Liu H.Y. Cao W. J. Biol. Chem. 2006; 281: 24336-24344Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Furthermore, PKCδ is probably involved in FFA-induced insulin resistance (13Boden G. She P. Mozzoli M. Cheung P. Gumireddy K. Reddy P. Xiang X. Luo Z. Ruderman N. Diabetes. 2005; 54: 3458-3465Crossref PubMed Scopus (419) Google Scholar) and FFA-induced decrease of insulin binding in hepatocytes (21Chen S. Lam T.K. Park E. Burdett E. Wang P.Y. Wiesenthal S.R. Lam L. Tchipashvili V. Fantus I.G. Giacca A. Biochem. Biophys. Res. Commun. 2006; 346: 931-937Crossref PubMed Scopus (8) Google Scholar). Finally, p38 has been shown to mediate FFA-induced insulin resistance in various cell types (22Fujishiro M. Gotoh Y. Katagiri H. Sakoda H. Ogihara T. Anai M. Onishi Y. Ono H. Abe M. Shojima N. Fukushima Y. Kikuchi M. Oka Y. Asano T. Mol. Endocrinol. 2003; 17: 487-497Crossref PubMed Scopus (163) Google Scholar, 23Greene M.W. Sakaue H. Wang L. Alessi D.R. Roth R.A. J. Biol. Chem. 2003; 278: 8199-8211Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 24Bloch-Damti A. Bashan N. Antioxid. Redox Signal. 2005; 7: 1553-1567Crossref PubMed Scopus (307) Google Scholar, 25Wang X.L. Zhang L. Youker K. Zhang M.X. Wang J. Lemaire S.A. Coselli J.S. Shen Y.H. Diabetes. 2006; 55: 2301-2310Crossref PubMed Scopus (197) Google Scholar), but its role in hepatic insulin resistance is unclear. Because desaturation of saturated FFAs such as stearate into monounsaturated oleate by stearoyl-CoA desaturase-1 (SCD1) is necessary for the onset of high fat diet-induced hepatic insulin resistance (26Gutierrez-Juarez R. Pocai A. Mulas C. Ono H. Bhanot S. Monia B.P. Rossetti L. J. Clin. Investig. 2006; 116: 1686-1695Crossref PubMed Scopus (248) Google Scholar), and oleate is the most abundant FFA in the plasma (27de Almeida I.T. Cortez-Pinto H. Fidalgo G. Rodrigues D. Camilo M.E. Clin. Nutr. 2002; 21: 219-223Abstract Full Text PDF PubMed Scopus (191) Google Scholar, 28Bysted A. Holmer G. Lund P. Sandstrom B. Tholstrup T. Eur. J. Clin. Nutr. 2005; 59: 24-34Crossref PubMed Scopus (27) Google Scholar), we have investigated the role of p38 in oleate-induced insulin resistance in isolated hepatocytes in this study. Our results show that a prolonged exposure of primary hepatocytes to oleate can blunt insulin suppression of hepatic glucose production via gluconeogenesis through p38. We further show that oleate can activate p38 in isolated hepatocytes, and activation of p38 is required for oleate to reduce insulin-stimulated phosphorylation of Akt and tyrosine phosphorylation of IRS1/2. Finally, we have observed that PTEN plays a critical role in oleate-induced hepatic insulin resistance. Reagents—Oleate and palmitate, fatty acid-free albumin from bovine serum, antibody against phospho-IRS-1(Tyr896), and the ascites fluid containing the anti-β-actin monoclonal antibody were from Sigma-Aldrich. Antibodies against total/phospho-p38, total/phospho-Akt, total/phospho-PTEN, and phosphotyrosine (p-Tyr-100) were from Cell Signaling Technology (Danvers, MA). Antibodies against insulin receptor substrate-1 (IRS-1) (C-20) and IRS-2 (H-205) and the siRNA against p38α and the scrambled siRNA were from Santa Cruz Biotechnology. The antibody against phosphoserine/threonine (clone 22a) was from BD Biosciences (San Jose, CA). SB202190 and SB203580 were purchased from Calbiochem. The siRNA against PTEN (cat. no. 15034) and related scrambled siRNA (cat. no. 4635) were from Ambion (Austin, TX). Lipofectamine™ 2000 Transfection Reagents were from Invitrogen. Both lactate dehydrogenase (LDH) assay kit and Cell Death Detection ELISAplus kit were from Roche Applied Science (Indianapolis, IN). Other materials were all obtained commercially and are of analytical quality. Isolation of Hepatocytes—Primary hepatocytes were isolated from C57BL/6 mice as previously described (20Collins Q.F. Xiong Y. Lupo J.E.J. Liu H.Y. Cao W. J. Biol. Chem. 2006; 281: 24336-24344Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 29Cao W.H. Collins Q.F. Becker T.C. Robidoux J. Lupo J.E.G. Xiong Y. Daniel K. Floering L. Collins S. J. Biol. Chem. 2005; 280: 42731-42737Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 30Xiong Y. Collins Q.F. Lupo J.G. Liu H.Y. Jie A. Liu D. Cao W. J. Biol. Chem. 2006; 282: 4975-4982Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). All mice used in the present study for isolation of hepatocytes were fed normal chow diet under a regular schedule unless otherwise noted. Briefly, under anesthesia with pentobarbital (IP, 50 mg/kg body weight), livers were perfused with Ca2+-free Hanks’ Balanced Solution (Invitrogen) at 5 ml/min for 8 min, followed by continuous perfusion with serum-free Williams’ Medium E containing collagenase (Worthington, Type II, 50 units/ml) (Invitrogen) supplemented with 10 mm HEPES for 12 min. Hepatocytes were harvested and purified with Percoll. The viability of hepatocytes was examined with trypan blue exclusion. Only cell isolates with viability over 95% were used. Hepatocytes were inoculated into collagen-coated plates (5 × 105 cells/well in 6-well plates and 1.25 × 105 cell per well in 24-well plates) in Williams’ Medium E with 10% FBS, and were incubated for 24 h before experimentation. All studies were approved by The Hamner Institutes for Health Sciences Animal Care and Use Committee and complied with guidelines from the United States National Institutes of Health. Oleate Preparation and Treatment—Oleate was freshly prepared from a 50 mm stock solution in methanol. Specifically, 0.5 mm oleate and 0.1 mm BSA were added to the pre-warmed culture media (37 °C), and mixed by vortexing for 1 min. The media with both oleate and BSA was then used to replace the media in the cell culture immediately. Lower concentrations of oleate/BSA were prepared by adding an equal volume of prewarmed media. BSA was prepared from a 10× stock solution. Equal amounts of methanol were added into the control cells in each experiment. Measurement of Glucose Production in Primary Hepatocytes—Primary hepatocytes were isolated from mice, which had been fasted for 24 h to deplete glycogen in liver. Glucose production from primary hepatocytes were measured as previously described (20Collins Q.F. Xiong Y. Lupo J.E.J. Liu H.Y. Cao W. J. Biol. Chem. 2006; 281: 24336-24344Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 31Zhou H. Song X. Briggs M. Violand B. Salsgiver W. Gulve E.A. Luo Y. Biochem. Biophys. Res. Commun. 2005; 338: 793-799Crossref PubMed Scopus (58) Google Scholar). Briefly, cells were incubated in Williams’ Medium E supplemented with 10% fetal bovine serum and 0.5 mm oleate for 16 h, and were then washed three times with warm PBS to remove glucose. Subsequently, cells were pretreated with insulin, followed by stimulation with cAMP/dexamethasome in glucose-free Dulbecco’s modified Eagle’s medium containing gluconeogenic substrates (2 mm sodium pyruvate). Glucose concentrations were determined with a glucose assay kit from Roche Applied Science (cat. no. 0716251) and normalized to protein concentrations. The total glucose production was derived from both glycogenolysis and gluconeogenesis. Glucose production from glycogenolysis was measured in the absence of a gluconeogenic substrate. The amount of glucose production by gluconeogenesis is defined as the difference between total glucose production and glycogenolysis. Apoptosis Assay—Using a Cell Death Detection ELISAplus kit, levels of cellular apoptosis were quantified by measuring cytoplasmic DNA-histone nucleosome complexes generated during apoptotic DNA fragmentation. In brief, hepatocytes were incubated with oleate or palmitate for 16 h as noted, and then were lysed with the lysis buffer provided by the assay kit. The supernatants of the lysates were incubated with biotinylated antibodies against histone and peroxidase (POD)-labeled antibodies against DNA in streptavidin-coated microplates for 2 h. Plates were washed to remove unbound lysates and antibodies. The 2,2′-azino-di[3-ethylbenzthia-zoline sulfonate diammonium salt was then added. POD activity (apoptosis) was quantified at 405 nm against A490 (blank). Immunoprecipitation and Western Blotting—Cells were lysed in Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mm NaCl, 10% glycerol, 2 mm EDTA, 20 mm Tris (pH 8.0), 1 mm dithiothreitol, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, and 10 μg/ml aprotinin (32Liu H.Y. MacDonald J.I. Hryciw T. Li C. Meakin S.O. J. Biol. Chem. 2005; 280: 19461-19471Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Cell lysates (15 μg per lane) were resolved in 10% Tris-glycine gels (Invitrogen) and transferred to nitrocellulose membranes (Bio-Rad). For immunoprecipitation, cell lysates were mixed with the indicated antibody and protein G-agarose at 4 °C overnight, followed by extensive washes with lysis buffer twice and PBS once. Precipitated proteins were released by heating the protein sample buffer at 75 °C for 5 min (32Liu H.Y. MacDonald J.I. Hryciw T. Li C. Meakin S.O. J. Biol. Chem. 2005; 280: 19461-19471Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 33Liu H.Y. Meakin S.O. J. Biol. Chem. 2002; 277: 26046-26056Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Proteins were subsequently resolved in 4–20% Tris glycine gels, and detected by immunoblotting with primary antibodies as indicated and alkaline phosphatase-conjugated secondary antisera. The fluorescent bands were visualized with Typhoon 9410 variable mode Imager from GE Healthcare (Piscataway, NJ), and then quantified by densitometry analysis using ImageQuant 5.2 software from Molecular Dynamics (Piscataway, NJ). Gene Silencing with siRNA—The cognate siRNA against p38α or PTEN and scrambled siRNA were purchased from Santa Cruz Biotechnology or Ambion, and were introduced into primary mouse hepatocytes by reverse transfection with Lipofectamine™ 2000 Transfection Reagents. Briefly, the siRNA transfection mixture was applied to collagen-coated 6-well plates right before the plating of primary hepatocytes in culture media without antibiotics. After 24–48 h, cells were treated with oleate together with BSA for 16 h prior to the treatment with insulin as noted. Real-time PCR—Total RNA was extracted from hepatocytes with an RNeasy Mini Kit (Qiagen), and reverse-transcribed into cDNA. The cDNA was quantified by TaqMan® Real-time PCR with specific probes and primers from Applied Biosciences, and normalized to levels of GAPDH. Statistical Analysis—Data are presented as mean ± S.E. of at least three independent experiments. Data were compared by Student’s t test or one-way ANOVA analysis using GraphPad Prism version 4.0 for Windows (San Diego, CA). Differences at values of p < 0.05 were considered significant. Prolonged Exposure of Hepatocytes to Oleate Blunts Insulin Suppression of Gluconeogenesis in a p38-dependent Manner—Insulin is the predominant suppressor of hepatic gluconeogenesis, and this suppression is blunted in type II diabetes (reviewed in Refs. 10Accili D. Diabetes. 2004; 53: 1633-1642Crossref PubMed Scopus (149) Google Scholar and 34Kahn S.E. Hull R.L. Utzschneider K.M. Nature. 2006; 444: 840-846Crossref PubMed Scopus (3457) Google Scholar). It is known that plasma levels of FFA in diabetes are almost always increased, and FFA can induce insulin resistance in various cell types (22Fujishiro M. Gotoh Y. Katagiri H. Sakoda H. Ogihara T. Anai M. Onishi Y. Ono H. Abe M. Shojima N. Fukushima Y. Kikuchi M. Oka Y. Asano T. Mol. Endocrinol. 2003; 17: 487-497Crossref PubMed Scopus (163) Google Scholar, 23Greene M.W. Sakaue H. Wang L. Alessi D.R. Roth R.A. J. Biol. Chem. 2003; 278: 8199-8211Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 24Bloch-Damti A. Bashan N. Antioxid. Redox Signal. 2005; 7: 1553-1567Crossref PubMed Scopus (307) Google Scholar, 25Wang X.L. Zhang L. Youker K. Zhang M.X. Wang J. Lemaire S.A. Coselli J.S. Shen Y.H. Diabetes. 2006; 55: 2301-2310Crossref PubMed Scopus (197) Google Scholar). Lipid infusion in animal models and in humans can also induce hepatic insulin resistance (8Lam T.K. Yoshii H. Haber C.A. Bogdanovic E. Lam L. Fantus I.G. Giacca A. Am. J. Physiol. Endocrinol. Metab. 2002; 283: E682-E691Crossref PubMed Scopus (168) Google Scholar, 13Boden G. She P. Mozzoli M. Cheung P. Gumireddy K. Reddy P. Xiang X. Luo Z. Ruderman N. Diabetes. 2005; 54: 3458-3465Crossref PubMed Scopus (419) Google Scholar, 35Bevilacqua S. Bonadonna R. Buzzigoli G. Boni C. Ciociaro D. Maccari F. Giorico M.A. Ferrannini E. Metabolism. 1987; 36: 502-506Abstract Full Text PDF PubMed Scopus (195) Google Scholar, 36Boden G. Chen X. Ruiz J. White J.V. Rossetti L. J. Clin. Investig. 1994; 93: 2438-2446Crossref PubMed Scopus (851) Google Scholar, 37Lewis G.F. Vranic M. Harley P. Giacca A. Diabetes. 1997; 46: 1111-1119Crossref PubMed Google Scholar, 38Rebrin K. Steil G.M. Mittelman S.D. Bergman R.N. J. Clin. Investig. 1996; 98: 741-749Crossref PubMed Scopus (263) Google Scholar, 39Sindelar D.K. Chu C.A. Rohlie M. Neal D.W. Swift L.L. Cherrington A.D. Diabetes. 1997; 46: 187-196Crossref PubMed Google Scholar, 40Wiesenthal S.R. Sandhu H. McCall R.H. Tchipashvili V. Yoshii H. Polonsky K. Shi Z.Q. Lewis G.F. Mari A. Giacca A. Diabetes. 1999; 48: 766-774Crossref PubMed Scopus (146) Google Scholar). However, the direct role of FFA in the development of insulin resistance in isolated hepatocytes has not been defined. Oleate is the most abundant FFA in the plasma (27de Almeida I.T. Cortez-Pinto H. Fidalgo G. Rodrigues D. Camilo M.E. Clin. Nutr. 2002; 21: 219-223Abstract Full Text PDF PubMed Scopus (191) Google Scholar, 28Bysted A. Holmer G. Lund P. Sandstrom B. Tholstrup T. Eur. J. Clin. Nutr. 2005; 59: 24-34Crossref PubMed Scopus (27) Google Scholar), and conversion of the saturated FFA such as stearate into monounsaturated FFA (oleate) by SCD-1 is required for high fat diet to induce hepatic insulin resistance (26Gutierrez-Juarez R. Pocai A. Mulas C. Ono H. Bhanot S. Monia B.P. Rossetti L. J. Clin. Investig. 2006; 116: 1686-1695Crossref PubMed Scopus (248) Google Scholar). Therefore, we chose to study the role of oleate in the development of hepatic insulin resistance. As shown in Fig. 1A, insulin failed to efficiently inhibit the glucose production via gluconeogenesis in hepatocytes exposed to oleate for 16 h, while insulin completely inhibited the glucose production via gluconeogenesis stimulated by cAMP/dexamethasone in control cells. These results suggest that oleate can induce insulin resistance in isolated hepatocytes. Because previous studies have suggested a role for p38 in fat-induced insulin resistance in vivo (41Bouzakri K. Roques M. Gual P. Espinosa S. Guebre-Egziabher F. Riou J.P. Laville M. March Le -Brustel Y. Tanti J.F. Vidal H. Diabetes. 2003; 52: 1319-1325Crossref PubMed Scopus (239) Google Scholar, 42Carlson C.J. Koterski S. Sciotti R.J. Poccard G.B. Rondinone C.M. Diabetes. 2003; 52: 634-641Crossref PubMed Scopus (189) Google Scholar, 43Shen Y.H. Zhang L. Gan Y. Wang X. Wang J. LeMaire S.A. Coselli J.S. Wang X.L. J. Biol. Chem. 2006; 281: 7727-7736Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), and we have previously described a role for p38 in FFA-induced hepatic gluconeogenesis (20Collins Q.F. Xiong Y. Lupo J.E.J. Liu H.Y. Cao W. J. Biol. Chem. 2006; 281: 24336-24344Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), we examined the role of p38 in oleate-induced hepatic insulin resistance. As shown in Fig. 1B, a preincubation of hepatocytes with a p38 inhibitor, SB203580, significantly reduced the capability of oleate to blunt the insulin suppression of hepatic gluconeogenesis. These results indicate that p38 plays a critical role in oleate induction of hepatic insulin resistance. To examine whether the exposure to oleate was toxic to hepatocytes, levels of LDH release and apoptosis in hepatocytes were measured. As shown in Fig. 1, C and D, oleate did not cause significant cytotoxicity and apoptosis at concentrations lower than 1 mm while palmitate caused obvious toxicity and apoptosis at concentrations higher than 0.2 mm. Prolonged Exposure of Primary Hepatocytes to Oleate Decreases Insulin-induced Activation of Akt—Insulin suppresses hepatic gluconeogenesis through activation of Akt (reviewed in Refs. 10Accili D. Diabetes. 2004; 53: 1633-1642Crossref PubMed Scopus (149) Google Scholar and 44Cohen P. Nat Rev Mol. Cell. Biol. 2006; 7: 867-873Crossref PubMed Scopus (178) Google Scholar). To determine the mechanism by which oleate blunted insulin suppression of hepatic gluconeogenesis, isolated primary hepatocytes were incubated with increasing amount of oleate for 16 h, followed by treatment with insulin for 15 min. Levels of Akt phosphorylation were then measured. As shown in Fig. 2, insulin stimulated phosphorylation of Akt as expected, and this stimulation was decreased by the prolonged incubation with oleate in a dose-dependent manner. Together, these results indicate that a prolonged exposure to oleate can decrease insulin-induced activation of Akt in hepatocytes. Oleate Stimulates p38 Phosphorylation in Primary Hepatocytes—Previous studies have shown that p38 mediates FFA-induced insulin resistance in several non-hepatocyte cell types (22Fujishiro M. Gotoh Y. Katagiri H. Sakoda H. Ogihara T. Anai M. Onishi Y. Ono H. Abe M. Shojima N. Fukushima Y. Kikuchi M. Oka Y. Asano T. Mol. Endocrinol. 2003; 17: 487-497Crossref PubMed Scopus (163) Google Scholar, 23Greene M.W. Sakaue H. Wang L. Alessi D.R. Roth R.A. J. Biol. Chem. 2003; 278: 8199-8211Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 24Bloch-Damti A. Bashan N. Antioxid. Redox Signal. 2005; 7: 1553-1567Crossref PubMed Scopus (307) Google Scholar, 25Wang X.L. Zhang L. Youker K. Zhang M.X. Wang J. Lemaire S.A. Coselli J.S. Shen Y.H. Diabetes. 2006; 55: 2301-2310Crossref PubMed Scopus (197) Google Scholar), and we have recently shown that FFA can activate p38 in primary hepatocytes (20Collins Q.F. Xiong Y. Lupo J.E.J. Liu H.Y. Cao W. J. Biol. Chem. 2006; 281: 24336-24344Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Therefore, we postulated that p38 might play a role in oleate-induced hepatic insulin resistance. To test this hypothesis, primary hepatocytes were treated with increasing amount of oleate in the presence or absence of insulin, followed by measurements of p38 phosphorylation. As shown in Fig. 3, p38 phosphorylation was increased by oleate, and the effect was not influenced by insulin. Insulin itself did not initiate detectable phosphorylation of p38. p38 Mediates Oleate Suppression of Insulin-induced Activation of Akt—To determine whether the function of oleate in blunting insulin-induced activation of Akt is linked to oleate activation of p38, we took the advantage of chemical inhibitors of p38. Both SB203580 and SB202190 are potent and cell-permeable inhibitors of p38, and they do not suppress other MAP kinases including ERK1/2 and JNK even at 100 μm (45Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D.

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