Recent studies in mice reported that alteration of the gut microbiota changes host bile acid composition, notably alteration of taurine-conjugated bile acids that can antagonize the intestinal FXR, and could give rise to metabolic dysfunction including obesity and insulin resistance. Bile acids could also influence NAFLD through activation of the hepatic FXR and the G-protein coupled receptor TGR5 expressed in non-parenchymal cells. However, questions remain about the role of the gut microbiota, bile acids, and intestinal and hepatic FXR signaling in the pathogenesis of hepatic steatosis. In the current study, mice were fed a high-fat diet (HFD) to induce NAFLD. A combination of bacitracin, neomycin, and streptomycin (BNS) to kill certain members of the gut microbiota, or tempol treatment to specifically modulate the gut microbiota were employed to determine the role of the gut microbiota in NAFLD pathogenesis. Intestine-specific Fxr-null (FxrdIE) mice were used to elucidate the mechanism by which the gut microbiota contributes to NAFLD. A critical role for FXR in NAFLD was established by use of intestine-specific Fxr-null mice that had reduced HFD-induced hepatic triglyceride accumulation as compared to wild-type mice, due to decreased circulating ceramides that result in part from repressed of expression of ceramide synthesis genes. Tempol- and antibiotic- treatment of HFD-fed wild-type mice also reduced the levels of ileum and serum ceramides. The lower serum ceramides downregulated hepatic SREBP1C and CIDEA expression leading to decreased de novo lipogenesis. Administration of C16:0 ceramide to HFD-fed mice reversed the suppression of hepatic SREBP1C-CIDEA signaling and steatosis found with antibiotic treatment. These studies demonstrate that inhibition of an intestinal FXR-ceramide axis mediates the gut microbiota-associated development of NAFLD and reveals a mechanistic link between the microbiome, nuclear receptor signaling, and NAFLD. This work suggests that inhibition of intestinal FXR is a therapeutic target for treatment of NAFLD. Role of FGF 21 in the early stage of NASH induced by methionine- and choline-deficient diet: FGF21 is a modulator of energy homeostasis and is increased in human NAFLD and after feeding of methionine- and choline-deficient diet (MCD), a conventional inducer of murine nonalcoholic steatohepatitis (NASH). However, the significance of FGF21 induction in the occurrence of MCD-induced NASH remains undetermined. C57BL/6J Fgf21-null and wild-type mice were treated with MCD for 1week. Hepatic Fgf21 mRNA was increased early after commencing MCD treatment independent of PPARalpha and FXR. While no significant differences in white adipose lipolysis were seen in both genotypes, hepatic triglyceride (TG) contents were increased in Fgf21-null mice, likely due to the up-regulation of genes encoding CD36 and phosphatidic acid phosphatase 2a/2c, involved in fatty acid uptake and diacylglycerol synthesis, respectively, and suppression of increased mRNAs encoding carnitine palmitoyl-CoA transferase 1alpha, PPARgamma coactivator 1alpha, and adipose TG lipase, which are associated with lipid clearance in the liver. The MCD-treated Fgf21-null mice showed increased hepatic endoplasmic reticulum (ER) stress. Exposure of primary hepatocytes to palmitic acid elevated the mRNA levels encoding DNA damage-inducible transcript 3, an indicator of ER stress, and FGF21 in a PPARalpha-independent manner, suggesting that lipid-induced ER stress can enhance hepatic FGF21 expression. Collectively, FGF21 is elevated in the early stage of MCD-induced NASH likely to minimize hepatic lipid accumulation and ensuing ER stress. These results provide a possible mechanism on how FGF21 is increased in NAFLD/NASH Role of FGF 21 in the early stage of NASH induced by methionine- and choline-deficient diet: Due to excess calorie intake and sedentary lifestyle, surplus lipids are stored in hepatocytes, a condition designated as hepatic steatosis or NAFLD as described above. NASH may also develop from NAFLD through increasing hepatic inflammation and/or hepatocyte damage to steatotic liver, leading to hepatic fibrosis, hepatocellular carcinoma, and eventually death. The MCD model of NASH was used to investigate the mechanism of NASH and the role of FGF21. FGF21 is a modulator of energy homeostasis and is increased in human NAFLD. Fgf21 mRNA and serum FGF21 are elevated in human NAFLD and correlated with the degree of steatosis, but the mechanism by which FGF21 is induced in steatotic hepatocytes and its role in hepatic steatosis are not known. C57BL/6J Fgf21-null and wild-type mice were treated with MCD for 1week. Hepatic Fgf21 mRNA was increased early after commencing MCD treatment independent of PPARalpha and FXR. While no significant differences in white adipose lipolysis were seen in both genotypes, hepatic triglyceride (TG) contents were increased in Fgf21-null mice, likely due to the up-regulation of genes encoding CD36 and phosphatidic acid phosphatase 2a/2c, involved in fatty acid (FA) uptake and diacylglycerol synthesis, respectively, and suppression of increased mRNAs encoding carnitine palmitoyl-CoA transferase 1alpha, PPARgamma coactivator 1alpha, and adipose TG lipase, which are associated with lipid clearance in the liver. The MCD-treated Fgf21-null mice showed increased hepatic endoplasmic reticulum (ER) stress. Exposure of primary hepatocytes to palmitic acid elevated the mRNA levels encoding DNA damage-inducible transcript 3, an indicator of ER stress, and FGF21 in a PPARalpha-independent manner, suggesting that lipid-induced ER stress can enhance hepatic FGF21 expression. Collectively, FGF21 is elevated in the early stage of MCD-induced NASH likely to minimize hepatic lipid accumulation and ensuing ER stress. These results provide a possible mechanism on how FGF21 is increased in NAFLD/NASH. Adipocyte-specific disruption of fat-specific protein 27 causes hepatosteatosis and insulin resistance in high-fat diet-fed mice: White adipose tissue (WAT) functions as an energy reservoir where excess circulating fatty acids are transported to WAT, converted to triglycerides, and stored as unilocular lipid droplets. Fat-specific protein 27 (FSP27, CIDEC in humans) is a lipid-coating protein highly expressed in mature white adipocytes that contributes to unilocular lipid droplet formation. However, the influence of FSP27 in adipose tissue on whole-body energy homeostasis remains unclear. Mice with adipocyte-specific disruption of the Fsp27 gene (Fsp27dAd) were generated using an aP2-Cre transgene with the Cre/LoxP system. Upon high-fat diet feeding, Fsp27dAd mice were resistant to weight gain. In the small WAT of these mice, small adipocytes containing multilocular lipid droplets were dispersed. The expression levels of the genes associated with mitochondrial abundance and brown adipocyte identity were increased, and basal lipolytic activities were significantly augmented in adipocytes isolated from Fsp27dAd mice compared with the Fsp27(F/F) counterparts. The impaired fat-storing function in Fsp27dAd adipocytes and the resultant lipid overflow from WAT led to marked hepatosteatosis, dyslipidemia, and systemic insulin resistance in high-fat diet-treated Fsp27dAd mice. These results demonstrate a critical role for FSP27 in the storage of excess fat in WAT with minimizing ectopic fat accumulation that causes insulin-resistant diabetes and non-alcoholic fatty liver disease. This mouse model may be useful for understanding the significance of fat-storing properties of white adipocytes and the role of local FSP27 in whole-body metabolism and estimating the pathogenesis of human partial lipodystrophy caused by CIDEC mutations.