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• W2106678312 abstract "The farnesoid X receptor (FXR) is a bile acid (BA)-activated nuclear receptor that plays a major role in the regulation of BA and lipid metabolism. Recently, several studies have suggested a potential role of FXR in the control of hepatic carbohydrate metabolism, but its contribution to the maintenance of peripheral glucose homeostasis remains to be established. FXR-deficient mice display decreased adipose tissue mass, lower serum leptin concentrations, and elevated plasma free fatty acid levels. Glucose and insulin tolerance tests revealed that FXR deficiency is associated with impaired glucose tolerance and insulin resistance. Moreover, whole-body glucose disposal during a hyperinsulinemic euglycemic clamp is decreased in FXR-deficient mice. In parallel, FXR deficiency alters distal insulin signaling, as reflected by decreased insulin-dependent Akt phosphorylation in both white adipose tissue and skeletal muscle. Whereas FXR is not expressed in skeletal muscle, it was detected at a low level in white adipose tissue in vivo and induced during adipocyte differentiation in vitro. Moreover, mouse embryonic fibroblasts derived from FXR-deficient mice displayed impaired adipocyte differentiation, identifying a direct role for FXR in adipocyte function. Treatment of differentiated 3T3-L1 adipocytes with the FXR-specific synthetic agonist GW4064 enhanced insulin signaling and insulin-stimulated glucose uptake. Finally, treatment with GW4064 improved insulin resistance in genetically obese ob/ob mice in vivo. Although the underlying molecular mechanisms remain to be unraveled, these results clearly identify a novel role of FXR in the regulation of peripheral insulin sensitivity and adipocyte function. This unexpected function of FXR opens new perspectives for the treatment of type 2 diabetes. The farnesoid X receptor (FXR) is a bile acid (BA)-activated nuclear receptor that plays a major role in the regulation of BA and lipid metabolism. Recently, several studies have suggested a potential role of FXR in the control of hepatic carbohydrate metabolism, but its contribution to the maintenance of peripheral glucose homeostasis remains to be established. FXR-deficient mice display decreased adipose tissue mass, lower serum leptin concentrations, and elevated plasma free fatty acid levels. Glucose and insulin tolerance tests revealed that FXR deficiency is associated with impaired glucose tolerance and insulin resistance. Moreover, whole-body glucose disposal during a hyperinsulinemic euglycemic clamp is decreased in FXR-deficient mice. In parallel, FXR deficiency alters distal insulin signaling, as reflected by decreased insulin-dependent Akt phosphorylation in both white adipose tissue and skeletal muscle. Whereas FXR is not expressed in skeletal muscle, it was detected at a low level in white adipose tissue in vivo and induced during adipocyte differentiation in vitro. Moreover, mouse embryonic fibroblasts derived from FXR-deficient mice displayed impaired adipocyte differentiation, identifying a direct role for FXR in adipocyte function. Treatment of differentiated 3T3-L1 adipocytes with the FXR-specific synthetic agonist GW4064 enhanced insulin signaling and insulin-stimulated glucose uptake. Finally, treatment with GW4064 improved insulin resistance in genetically obese ob/ob mice in vivo. Although the underlying molecular mechanisms remain to be unraveled, these results clearly identify a novel role of FXR in the regulation of peripheral insulin sensitivity and adipocyte function. This unexpected function of FXR opens new perspectives for the treatment of type 2 diabetes. The farnesoid X receptor (FXR) 4The abbreviations used are: FXR, farnesoid X receptor; BA, bile acid; FFA, free fatty acid; MEF, mouse embryonic fibroblast; ITT, insulin tolerance test; GTT, glucose tolerance test; IR, insulin receptor; IRS, insulin receptor substrate; WAT, white adipose tissue; PDK1, phosphoinositide-dependent protein kinase 1; PPARγ, peroxisome proliferator-activated receptor γ; HDL, high density lipoprotein. (NR1H4) is a nuclear receptor that is activated by bile acids (BAs) (1Kuipers F. Claudel T. Sturm E. Staels B. Rev. Endocr. Metab. Disord. 2004; 5: 319-326Crossref PubMed Scopus (55) Google Scholar). A major physiological role of FXR is to protect liver cells from the deleterious effect of BA overload by decreasing their endogenous production and by accelerating BA biotransformation and excretion (1Kuipers F. Claudel T. Sturm E. Staels B. Rev. Endocr. Metab. Disord. 2004; 5: 319-326Crossref PubMed Scopus (55) Google Scholar). In addition, the generation and characterization of FXR-deficient (FXR-/-) mice has also established a critical role of FXR in lipid metabolism, since these mice display elevated serum levels of triglycerides and high density lipoprotein cholesterol (2Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1426) Google Scholar). Recently, several studies have suggested that FXR might also regulate hepatic carbohydrate metabolism (3Cariou B. Duran-Sandoval D. Kuipers F. Staels B. Endocrinology. 2005; 146: 981-983Crossref PubMed Scopus (36) Google Scholar). The first indication came from the observation that hepatic FXR expression is reduced in several rodent models of diabetes (4Duran-Sandoval D. Mautino G. Martin G. Percevault F. Barbier O. Fruchart J.C. Kuipers F. Staels B. Diabetes. 2004; 53: 890-898Crossref PubMed Scopus (204) Google Scholar). FXR expression also varies in mouse liver during nutritional changes, being increased during fasting and decreased upon refeeding (5Zhang Y. Castellani L.W. Sinal C.J. Gonzalez F.J. Edwards P.A. Genes Dev. 2004; 18: 157-169Crossref PubMed Scopus (296) Google Scholar, 6Duran-Sandoval D. Cariou B. Percevault F. Hennuyer N. Grefhorst A. van Dijk T.H. Gonzalez F.J. Fruchart J.C. Kuipers F. Staels B. J. Biol. Chem. 2005; 280: 29971-29979Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Moreover, FXR activation by BAs or the synthetic nonsteroidal specific agonist GW4064 (7Maloney P.R. Parks D.J. Haffner C.D. Fivush A.M. Chandra G. Plunket K.D. Creech K.L. Moore L.B. Wilson J.G. Lewis M.C. Jones S.A. Willson T.M. J. Med. Chem. 2000; 43: 2971-2974Crossref PubMed Scopus (458) Google Scholar) modulates the expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (3Cariou B. Duran-Sandoval D. Kuipers F. Staels B. Endocrinology. 2005; 146: 981-983Crossref PubMed Scopus (36) Google Scholar). However, conflicting data report either a positive (8Stayrook K.R. Bramlett K.S. Savkur R.S. Ficorilli J. Cook T. Christe M.E. Michael L.F. Burris T.P. Endocrinology. 2005; 146: 984-991Crossref PubMed Scopus (235) Google Scholar) or a negative effect (9De Fabiani E. Mitro N. Gilardi F. Caruso D. Galli G. Crestani M. J. Biol. Chem. 2003; 278: 39124-39132Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 10Yamagata K. Daitoku H. Shimamoto Y. Matsuzaki H. Hirota K. Ishida J. Fukamizu A. J. Biol. Chem. 2004; 279: 23158-23165Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar) of BA and/or GW4064 on phosphoenolpyruvate carboxykinase gene expression. A recent study, using FXR-/- mice, highlighted also a role of FXR in regulating the kinetics of hepatic carbohydrate metabolism during the fasting-refeeding transition phase. Indeed, FXR appears to modulate glycolytic and lipogenic pathways by interfering directly with the transcription of glucose-regulated genes, such as liver pyruvate kinase (6Duran-Sandoval D. Cariou B. Percevault F. Hennuyer N. Grefhorst A. van Dijk T.H. Gonzalez F.J. Fruchart J.C. Kuipers F. Staels B. J. Biol. Chem. 2005; 280: 29971-29979Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Furthermore, FXR also controls the adaptive response to fasting, since FXR-/- mice exhibit transient hypoglycemia upon fasting (11Cariou B. van Harmelen K. Duran-Sandoval D. van Dijk T. Grefhorst A. Bouchaert E. Fruchart J.C. Gonzalez F.J. Kuipers F. Staels B. FEBS Lett. 2005; 579: 4076-4080Crossref PubMed Scopus (73) Google Scholar). In the present study, we investigated the role of FXR in whole-body glucose homeostasis and insulin sensitivity by performing glucose tolerance tests and hyperinsulinemic-euglycemic clamp studies in FXR-/- mice. Interestingly, a decreased peripheral insulin sensitivity was observed in these mice. In vitro studies indicated that FXR directly modulates adipocyte function. Furthermore, we found that treatment with the specific FXR agonist GW4064 improved insulin sensitivity in ob/ob mice. These findings provide new evidence to support an important role of FXR in the maintenance of normal glucose homeostasis. Materials—Chemicals were obtained from Sigma France, GW4064 from Genfit SA (Loos, France). Rabbit polyclonal anti-IRβ antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); mouse monoclonal anti-phosphotyrosine (4G10), rabbit polyclonal anti-phosphatidylinositol 3-kinase p85, and anti-IRS-1 antibodies were from Upstate Biotechnology, Inc. (Lake Placid, NY); anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-PDK1 (Ser241), and anti-PDK1 antibodies were from Cell Signaling. Different kits were used to determine plasma concentrations of various metabolites: insulin (Mercodia AB), leptin and adiponectin (R&D systems), triglycerides, total, and HDL cholesterol (Roche Applied Science), and FFAs (WAKO). Fat content in feces was measured as previously described (12Kok T. Hulzebos C.V. Wolters H. Havinga R. Agellon L.B. Stellaard F. Shan B. Schwarz M. Kuipers F. J. Biol. Chem. 2003; 278: 41930-41937Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Animals—Homozygous FXR-/- male mice of 18-20 weeks of age and sex- and age-matched wild type mice (FXR+/+) bred on the C57BL/6N genetic background (2Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1426) Google Scholar) were housed with a 12-h light/12-h dark cycle with free access to water and a standard laboratory chow diet (UAR A03, Villemoison/Orge, France). Ob/Ob mice of 20 weeks of age were treated with the FXR agonist GW4064 (30 mg/kg of body weight/day) or with its vehicle (corn oil) for 10 days via intraperitoneal injection. Glucose and Insulin Tolerance Tests—Mice were fasted for 6 h with free access to water. For the glucose tolerance test (GTT), glucose (1 g/kg) was administered intraperitoneally, and blood glucose was measured with the Accu-Check active® (Roche Applied Science) at 15, 30, 60, 90, and 120 min. For ITT, recombinant human insulin (Actrapid®, Novo Nordisk) was administered intraperitoneally (FXR+/+ and FXR-/- mice, 0.75 units/kg; ob/ob mice, 2 units/kg), and blood glucose was measured at 0, 30, 60, 90, and 120 min after insulin injection. Hyperinsulinemic-Euglycemic Clamp Study—These experiments were performed in chronically catheterized, freely moving animals, exactly as described before (13van Dijk T.H. Boer T.S. Havinga R. Stellaard F. Kuipers F. Reijngoud D.J. Anal. Biochem. 2003; 322: 1-13Crossref PubMed Scopus (55) Google Scholar). In Vivo Insulin Stimulation—After an overnight fast, insulin (0.1 IU/kg) or saline was injected into the inferior venae cavae of anesthetized mice. After 5 min, epididymal fat pads and quadriceps muscle were dissected, frozen in liquid nitrogen, and stored at -80 °C until further analysis. Real Time Quantitative Reverse Transcription-PCR—Total RNA was isolated from white adipose tissue (WAT) and muscle using the acid guanidinium thiocyanate/phenol/chloroform method and isolated from 3T3-L1 cells and MEFs using the Trizol reagent (Invitrogen), as previously described (4Duran-Sandoval D. Mautino G. Martin G. Percevault F. Barbier O. Fruchart J.C. Kuipers F. Staels B. Diabetes. 2004; 53: 890-898Crossref PubMed Scopus (204) Google Scholar). The complete table of primers can be found in the supplemental data. Western Blot Analysis—Tissue or cell proteins were extracted in lysis buffer, as described previously (6Duran-Sandoval D. Cariou B. Percevault F. Hennuyer N. Grefhorst A. van Dijk T.H. Gonzalez F.J. Fruchart J.C. Kuipers F. Staels B. J. Biol. Chem. 2005; 280: 29971-29979Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). For coimmunoprecipitation experiments, proteins were incubated overnight at 4 °C with the indicated antibodies in the presence of protein A-agarose. The immunoprecipitates were washed in lysis buffer, subjected to SDS-PAGE, and transferred to a polyvinylidene difluoride membrane. Immunoreactive bands were revealed using an ECL detection kit (Amersham Biosciences). Adipocyte Size Determination—The samples from epididymal adipose tissue were fixed in 4% neutral buffered paraformaldehyde, embedded in paraffin, cut into 7-μm sections, and stained with hematoxylin. Cell size was determined using the computer-assisted Quips image analysis system (Leica Mikroskopic und System GmbH, Wetzlar, Germany). Cell Culture—3T3-L1 cells were cultured in growth medium containing Dulbecco's modified Eagle's medium and 10% calf serum. The cells were differentiated by the method of Bernlohr et al. (14Bernlohr D.A. Angus C.W. Lane M.D. Bolanowski M.A. Kelly Jr., T.J. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 5468-5472Crossref PubMed Scopus (243) Google Scholar). The fully differentiated adipocytes were cultured in postdifferentiation medium (Dulbecco's modified Eagle's medium, 10% fetal calf serum, 5 μm insulin) with or without GW4064 (5 μm) before experiments. Mouse embryonic fibroblasts (MEFs) were derived from 13.5-day wild-type and FXR-/- embryos. Adipocyte differentiation was initiated after 2 days at confluence. MEFs were differentiated with AmnioMAX-C100 medium (Invitrogen), 7.5% AmnioMAX-C100 supplement, 7.5% fetal calf serum, 0.5 mm 3-isobutyl-1-methylxanthine, 1 μm dexamethasone, 5 μm insulin. From day 3 to 8, cells were incubated with the same AmnioMAX-C100 medium with 5 μm insulin. At days 0, 4, and 8, cells were lysed and homogenized for RNA isolation or fixed in 4% paraformaldehyde and stained with Oil Red O. All experiments were performed in triplicate. Glucose Uptake—Fully differentiated 3T3-L1 adipocytes were starved overnight and thereafter incubated in PBS with or without human recombinant insulin for 30 min. During the last 6 min, 0.5 μCi/ml 2-deoxy-d-[2,6-3H]glucose (Amersham Biosciences) and 0.1 mm 2-deoxy-d-glucose were added. The reaction was ended by adding ice-cold phosphate-buffered saline containing 20 μg/ml cytochalasin B. Cells were lysed with ice-cold 1 m NaOH, the radioactivity was measured using a scintillation counter, and the data were expressed in dpm/min/mg of protein. Results were corrected for the GLUT-nonspecific uptake by removing uptake values obtained in the presence of 10 μg/ml cytochalasin B. Statistical Analysis—Statistical significance was analyzed using the unpaired Student's t test or analysis of variance for clamp experiments. All values are reported as means ± S.E. Values of p < 0.05 were considered significant. FXR-deficient Mice Exhibit Reduced Adipose Tissue Mass, Hypoleptinemia, and Increased Circulating FFA Levels—Body and organ weights as well as several plasma parameters were determined in chow-fed male wild-type (FXR+/+) and FXR-deficient (FXR-/-) mice under basal conditions (Table 1). Whereas total body weights were similar, organ weights varied between the two genotypes. FXR-/- mice showed a significant decrease in both epididymal (65%) and inguinal (35%) fat pads, whereas liver weight was increased (45%). The weight variations appeared to be restricted to these organs, since heart and kidney weights did not differ between the two genotypes. The reduction in adipose tissue mass was associated with a 35% reduction in adipocyte volume in FXR-/- compared with FXR+/+ mice (Fig. 1). Total weight and fat content of feces, as well as food intake, were similar in FXR+/+ and FXR-/- mice (Table 1). Thus, FXR deficiency is not associated with impaired fat absorption under chow-fed conditions.TABLE 1Morphometric and metabolic parameters in FXR+/+ and FXR-/- mice 18-20-week-old male FXR+/+ (n = 22) and FXR-/- (n = 21) mice were sacrificed after 6 h of fasting, body and organs were weighed, and plasma samples were collected to measure metabolite parameters. Values are presented as means ± S.E.FXR+/+FXR–/–Body weight (g)29.6 ± 0.628.5 ± 0.5Epididymal fat mass (g)0.71 ± 0.060.24 ± 0.03ap < 0.001; significantly different from FXR+/+ miceInguinal fat mass (g)0.42 ± 0.040.28 ± 0.01bp < 0.01; significantly different from FXR+/+ miceLiver mass (g)1.35 ± 0.031.95 ± 0.14ap < 0.001; significantly different from FXR+/+ miceHeart mass (g)0.14 ± 0.010.16 ± 0.01Kidney mass (g)0.20 ± 0.030.21 ± 0.01Feces weight (mg/day)835 ± 27910 ± 37Total fat (μmol/mg feces)89.5 ± 3.881.0 ± 4.3Food consumption (g/day)5.9 ± 0.16.0 ± 0.2Glucose (mg/dl)184 ± 7145 ± 8ap < 0.001; significantly different from FXR+/+ miceInsulin (pg/ml)1349 ± 235593 ± 137bp < 0.01; significantly different from FXR+/+ miceLeptin (ng/ml)3.86 ± 0.910.70 ± 0.11bp < 0.01; significantly different from FXR+/+ miceAdiponectin (μg/ml)6.97 ± 0.277.46 ± 0.98Triglycerides (mg/dl)71 ± 5161 ± 27bp < 0.01; significantly different from FXR+/+ miceTotal cholesterol (mg/dl)83 ± 3162 ± 6ap < 0.001; significantly different from FXR+/+ miceHDL cholesterol (mg/dl)62 ± 2689 ± 7bp < 0.01; significantly different from FXR+/+ miceFFA (mmol/liter)0.51 ± 0.030.71 ± 0.04ap < 0.001; significantly different from FXR+/+ micea p < 0.001; significantly different from FXR+/+ miceb p < 0.01; significantly different from FXR+/+ mice Open table in a new tab Basal plasma glucose concentrations were significantly lower in FXR-/- mice, associated with an impaired hepatic glucose production during short term fasting (11Cariou B. van Harmelen K. Duran-Sandoval D. van Dijk T. Grefhorst A. Bouchaert E. Fruchart J.C. Gonzalez F.J. Kuipers F. Staels B. FEBS Lett. 2005; 579: 4076-4080Crossref PubMed Scopus (73) Google Scholar). Plasma insulin levels were decreased in FXR-/- mice, probably reflecting an adaptive response to the relative hypoglycemia. Whereas adiponectin concentrations did not differ between both genotypes, plasma leptin concentrations were drastically decreased in FXR-/- mice, presumably reflecting the decrease in adipose tissue mass. As expected (2Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1426) Google Scholar), cholesterol and triglyceride levels were increased in FXR-/- mice. Moreover, FFA plasma levels were also increased in FXR-/- mice. A similar phenotype was observed in female mice as well as in another FXR-deficient mouse model on a different genetic background (12Kok T. Hulzebos C.V. Wolters H. Havinga R. Agellon L.B. Stellaard F. Shan B. Schwarz M. Kuipers F. J. Biol. Chem. 2003; 278: 41930-41937Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar) (data not shown). FXR Deficiency Leads to Peripheral Insulin Resistance—The ability of FXR-/- mice to respond to a glucose challenge was investigated. After a single intraperitoneal bolus injection of glucose, FXR-/- mice displayed a more pronounced plasma glucose excursion than the FXR+/+ mice, with a significantly larger area under the curve (+263%, p < 0.001) (Fig. 2A). The decreased clearance of plasma glucose could result from either an impaired insulin secretion, a peripheral insulin resistance or an increased hepatic glucose production. In view of our earlier studies (6Duran-Sandoval D. Cariou B. Percevault F. Hennuyer N. Grefhorst A. van Dijk T.H. Gonzalez F.J. Fruchart J.C. Kuipers F. Staels B. J. Biol. Chem. 2005; 280: 29971-29979Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 11Cariou B. van Harmelen K. Duran-Sandoval D. van Dijk T. Grefhorst A. Bouchaert E. Fruchart J.C. Gonzalez F.J. Kuipers F. Staels B. FEBS Lett. 2005; 579: 4076-4080Crossref PubMed Scopus (73) Google Scholar), the latter possibility can be excluded. To address the remaining options, intraperitoneal insulin tolerance tests (ITTs) were performed. The insulin-mediated decrease of plasma glucose was less pronounced in FXR-/- than in FXR+/+ mice, as indicated by the lower integrated area under the curve (-65%, p < 0.01) (Fig. 2B), suggesting that FXR deficiency alters insulin sensitivity. To directly evaluate the effect of FXR deficiency on whole-body insulin sensitivity, a 6-h hyperinsulinemic-euglycemic clamp was performed in awake, 9-h-fasted, male FXR+/+ and FXR-/- mice. The glucose infusion rate required to maintain euglycemia was ∼22% lower in FXR-/- than in wild-type mice, indicative of the existence of whole-body insulin resistance. Endogenous blood glucose production rate, which mainly reflects hepatic glucose production, was suppressed to similar values by insulin infusion in both genotypes, indicating the absence of hepatic insulin resistance in FXR-/- mice. In contrast, the insulin-stimulated glucose disposal rate and metabolic clearance rate of glucose were reduced by ∼20% in FXR-/- mice (Fig. 2C). These results show that FXR deficiency results in peripheral insulin resistance. FXR Modulates Insulin Signaling at the Level of Akt in Peripheral Insulin-sensitive Tissues—To investigate whether alteration in insulin receptor (IR)-mediated signaling occurs in peripheral insulin-sensitive tissues, in vivo insulin stimulation was performed in FXR+/+ and FXR-/- mice. Both insulin-stimulated tyrosine phosphorylation of IR and insulin substrate receptor 1 (IRS-1), as well as the recruitment of the p85α regulatory subunit of phosphoinositide 3-kinase to IRS-1 were similar between both genotypes in skeletal muscle and WAT (Fig. 3A). However, FXR deficiency altered the level of insulin-stimulated phosphorylation of the serine/threonine kinase Akt/protein kinase B on serine 473 in WAT and to a lesser extent in skeletal muscle (Fig. 3B). The protein expression level of phosphoinositide-dependent protein kinase 1 (PDK1), which plays a central role in activating Akt (15Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar), was not altered by FXR deficiency. Furthermore, the level of serine 241 phosphorylation of PDK1 that is esssential for its catalytic activity did not differ between the two genotypes (16Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (291) Google Scholar) (Fig. 3C). As previously described (16Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (291) Google Scholar, 17Wick M.J. Ramos F.J. Chen H. Quon M.J. Dong L.Q. Liu F. J. Biol. Chem. 2003; 278: 42913-42919Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), insulin stimulation did not increase Ser241-PDK1 phosphorylation. FXR deficiency did not alter the expression of several upstream negative regulators of insulin signaling, such as PTEN (18Tang X. Powelka A.M. Soriano N.A. Czech M.P. Guilherme A. J. Biol. Chem. 2005; 280: 22523-22529Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), protein-tyrosine phosphatase 1B (19Gum R.J. Gaede L.L. Koterski S.L. Heindel M. Clampit J.E. Zinker B.A. Trevillyan J.M. Ulrich R.G. Jirousek M.R. Rondinone C.M. Diabetes. 2003; 52: 21-28Crossref PubMed Scopus (183) Google Scholar), and SOCS-3 (20Emanuelli B. Peraldi P. Filloux C. Sawka-Verhelle D. Hilton D. Van Obberghen E. J. Biol. Chem. 2000; 275: 15985-15991Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar) (Table 2). Taken together, these results suggest that FXR may act directly at the level of Akt. Since Akt plays a key role in the regulation of the metabolic effects of insulin (21Whiteman E.L. Cho H. Birnbaum M.J. Trends Endocrinol. Metab. 2002; 13: 444-451Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar), defective Akt activation may contribute to the whole-body insulin resistance observed in FXR-/- mice.TABLE 2Expression of genes involved in glucose and lipid metabolism in skeletal muscle and white adipose tissue of FXR+/+ and FXR-/- mice The indicated mRNA levels were measured in muscle (top) and WAT (bottom) of FXR+/+ and FXR-/- mice after 6 h of fasting. Values are normalized relative to 28 S RNA (for skeletal muscle) and cyclophilin mRNA levels (for WAT) and are expressed (means ± S.E.) relative to those of FXR+/+ mice, which are arbitrarily set at 1 (n = 7 mice/group). Statistical analysis was performed using the unpaired Student's t test. NS, not significant.GeneFXR+/+FXR-/-Statistical significance%%Skeletal muscleInsulin signalingPTP-1B100 ± 14100 ± 17NSPTEN100 ± 1873 ± 18NSGlucose metabolismGLUT4100 ± 9116 ± 10NSGLUT1100 ± 8122 ± 7NSPDK4100 ± 12137 ± 11p = 0.05Fatty acid uptakeFATP1100 ± 16223 ± 43p < 0.05CD36100 ± 3122 ± 10NSLPL100 ± 4228 ± 36p < 0.05Fatty acid oxidationCPT1b100 ± 4140 ± 10p < 0.01UCP-2100 ± 16171 ± 15p < 0.05UCP-3100 ± 4159 ± 6p < 0.001Fatty acid synthesisDGAT-1100 ± 4210 ± 25p < 0.05SCD-1100 ± 18164 ± 48NSFAS100 ± 29146 ± 66NSPeroxisomal β-oxidationACO100 ± 7133 ± 10p < 0.05Transcription factorsPPARα100 ± 8181 ± 27p < 0.05PPARδ100 ± 7207 ± 13p < 0.05PGC1α100 ± 7152 ± 28NSSREBP-1C100 ± 7143 ± 17p < 0.05Epididymal adipose tissueInsulin signalingPTP-1B100 ± 10102 ± 11NSSOCS-3100 ± 26145 ± 30NSPTEN100 ± 6112 ± 12NSGlucose metabolismGLUT4100 ± 1389 ± 13NSGLUT1100 ± 8216 ± 56p = 0.05Fatty acid uptakeFATP1100 ± 9289 ± 87p = 0.07CD36100 ± 8109 ± 18NSLPL100 ± 7192 ± 32p < 0.05Fatty acid metabolismDGAT-1100 ± 6132 ± 17NSFAS100 ± 767 ± 17NSPEPCk100 ± 1986 ± 17NSHSL100 ± 1046 ± 8p < 0.001Adipocyte differentiationaP2100 ± 11102 ± 37NSPPARγ100 ± 978 ± 8NSc-EBPα100 ± 6113 ± 13NSc-EBPβ100 ± 14260 ± 37p < 0.01SREBP-1c100 ± 5153 ± 18p < 0.05AdipocytokinesLeptin100 ± 1818 ± 3p = 0.01Adiponectin100 ± 11130 ± 23NSResistin100 ± 7103 ± 19NSTNFα100 ± 1149 ± 11p < 0.05IL-6100 ± 2590 ± 14NS Open table in a new tab FXR Is Expressed in White Adipose Tissue and Differentiating Adipocytes—Previous studies have suggested that FXR might be expressed in WAT (22Li J. Pircher P.C. Schulman I.G. Westin S.K. J. Biol. Chem. 2005; 280: 7427-7434Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 23Zhang Y. Kast-Woelbern H.R. Edwards P.A. J. Biol. Chem. 2003; 278: 104-110Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). In accordance with this hypothesis, both FXR mRNA and protein levels increased during 3T3-L1 adipocyte differentiation (Fig. 4A). Furthermore, FXR mRNA levels were also induced during adipogenesis of MEFs, with a peak 4 days after the induction of differentiation (Fig. 4B). FXR is expressed at low levels in WAT (∼50-fold lower than in liver), whereas FXR mRNA levels were undetectable in skeletal muscle in mice (Fig. 4C). Nevertheless, FXR gene expression was reduced in WAT of C57Bl/6J mice upon high fat feeding (Fig. 4D) as well as in WAT of ob/ob mice (Fig. 4E), strengthening the hypothesis of a functional role of FXR in this insulin-sensitive tissue. FXR Activation Improves Insulin Signaling in 3T3-L1 Adipocytes—The effect of FXR activation with the synthetic agonist GW4064 on insulin signaling was then measured in differentiated 3T3-L1 adipocytes. As observed in WAT of FXR-/- mice, alteration of FXR activity did not alter proximal insulin signaling (Fig. 5A). However, GW4064 treatment increased the level of serine 473 Akt/protein kinase B phosphorylation and enhanced glucose uptake in 3T3-L1 adipocytes upon insulin stimulation (Fig. 5, B and C). Taken together, these results suggest that FXR directly modulates insulin signaling in the adipocyte. FXR Deficiency Directly Alters Adipocyte Differentiation in Vitro—To further assess a direct role for FXR in the adipocyte, the adipogenic process in MEFs derived from FXR-/- mice was investigated in vitro. Interestingly, FXR deficiency altered the kinetics of the adipogenic program in MEFs. Indeed, the induction of several transcription factors acting as critical adipogenic regulators was delayed in FXR-/- mice (Fig. 6A). Notably, mRNA levels of both peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (c-EBPα) were significantly lower 4 days after the initiation of the differentiation in MEFs derived from FXR-/- compared with FXR+/+ mice. In addition, CCAAT/enhancer-binding protein β (c-EBPβ), a marker of early adipocyte differentiation, was less induced at day 2 in FXR-/- MEFs. The expression of the adipocyte-specific marker aP2 was also lower in MEFs from FXR-/- mice. Moreover, the expression of both leptin and the glucose transporter GLUT4 was significantly decreased at day 8 in MEFs from FXR-/- mice, reflecting an impaired adipogenic process. The effect of FXR deficiency on adipocyte differentiation was further investigated using Oil Red O staining to assess triglyceride accumulation in MEFs induced to differentiate (Fig. 6, B and C). In accordance with the gene expression results, there was a decrease in triglyceride accumulation in MEFs derived from FXR-/- mice, with a reduced number of lipid droplets at day 4. In addition, there was a striking difference in the morphology of the lipid droplets between both genotypes (see magnification × 40; Fig. 6C). Indeed, the size of lipid droplets was drastically reduced in MEFs from FXR-/- mice, indicating that FXR directly interferes with the lipid storage process. Altogether, these results suggest that FXR plays a role in adipocyte differentiation and function. The PPARα-β/δ Gene Regulatory Pathways Are Activated in Skeletal Muscle of FXR-deficient Mi" @default.
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