Effective treatment of steatosis and steatohepatitis by fibroblast growth factor 1 in mouse models of nonalcoholic fatty liver disease.
ABSTRACT: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder and is strongly associated with obesity and type 2 diabetes. Currently, there is no approved pharmacological treatment for this disease, but improvement of insulin resistance using peroxisome proliferator-activated receptor-γ (PPARγ) agonists, such as thiazolidinediones (TZDs), has been shown to reduce steatosis and steatohepatitis effectively and to improve liver function in patients with obesity-related NAFLD. However, this approach is limited by adverse effects of TZDs. Recently, we have identified fibroblast growth factor 1 (FGF1) as a target of nuclear receptor PPARγ in visceral adipose tissue and as a critical factor in adipose remodeling. Because FGF1 is situated downstream of PPARγ, it is likely that therapeutic targeting of the FGF1 pathway will eliminate some of the serious adverse effects associated with TZDs. Here we show that pharmacological administration of recombinant FGF1 (rFGF1) effectively improves hepatic inflammation and damage in leptin-deficient ob/ob mice and in choline-deficient mice, two etiologically different models of NAFLD. Hepatic steatosis was effectively reduced only in ob/ob mice, suggesting that rFGF1 stimulates hepatic lipid catabolism. Potentially adverse effects such as fibrosis or proliferation were not observed in these models. Because the anti-inflammatory effects were observed in both the presence and absence of the antisteatotic effects, our findings further suggest that the anti-inflammatory property of rFGF1 is independent of its effect on lipid catabolism. Our current findings indicate that, in addition to its potent glucose-lowering and insulin-sensitizing effects, rFGF1 could be therapeutically effective in the treatment of NAFLD.
Project description:Therapeutic approach for NAFLD is limited and there are no approved drugs. Pioglitazone (PGZ), a thiazolidinedione (TZD) that acts via peroxisome proliferator activated receptor gamma (PPARγ) is the only agent that has shown consistent benefit and efficacy in clinical trials. However, the mechanism of its therapeutic effect on NAFLD remains unclear. The poor understanding may be due to problems with mouse, a species most used for animal experiments. TZDs exacerbate fatty liver in mouse models while they improve it in rat models like in human patients. Therefore, we compared the effects of TZDs including PGZ and rosiglitazone (RGZ) in ob/ob mice and Lep<sup>mkyo</sup>/Lep<sup>mkyo</sup> rats, models of leptin-deficient obesity, and A-ZIP/F-1 mice and seipin knockout (SKO) rats, models of generalized lipodystrophy. Pparg mRNA expression was markedly upregulated in fatty livers of mouse models while it was unchanged in rat models. TZDs exacerbated fatty liver in ob/ob and A-ZIP/F-1 mice, improved it in Lep<sup>mkyo</sup>/Lep<sup>mkyo</sup> rats and showed no effect in SKO rats. Gene expression analyses of Pparg and its target gene, Fsp27 revealed that PPARγ in the adipose tissue is the exclusive therapeutic target of TZDs in rats but PPARγ in the liver in addition to the adipose tissue is also a major site of actions for TZDs in mice. Although the response to TZDs in mice is the complete opposite of that in human patients, no report has pointed out the problem with TZD studies using mouse models so far. The present study might provide useful suggestions in research on TZDs.
Project description:OBJECTIVE:Glucagon is well known to regulate blood glucose but may be equally important for amino acid metabolism. Plasma levels of amino acids are regulated by glucagon-dependent mechanism(s), while amino acids stimulate glucagon secretion from alpha cells, completing the recently described liver-alpha cell axis. The mechanisms underlying the cycle and the possible impact of hepatic steatosis are unclear. METHODS:We assessed amino acid clearance in vivo in mice treated with a glucagon receptor antagonist (GRA), transgenic mice with 95% reduction in alpha cells, and mice with hepatic steatosis. In addition, we evaluated urea formation in primary hepatocytes from ob/ob mice and humans, and we studied acute metabolic effects of glucagon in perfused rat livers. We also performed RNA sequencing on livers from glucagon receptor knock-out mice and mice with hepatic steatosis. Finally, we measured individual plasma amino acids and glucagon in healthy controls and in two independent cohorts of patients with biopsy-verified non-alcoholic fatty liver disease (NAFLD). RESULTS:Amino acid clearance was reduced in mice treated with GRA and mice lacking endogenous glucagon (loss of alpha cells) concomitantly with reduced production of urea. Glucagon administration markedly changed the secretion of rat liver metabolites and within minutes increased urea formation in mice, in perfused rat liver, and in primary human hepatocytes. Transcriptomic analyses revealed that three genes responsible for amino acid catabolism (Cps1, Slc7a2, and Slc38a2) were downregulated both in mice with hepatic steatosis and in mice with deletion of the glucagon receptor. Cultured ob/ob hepatocytes produced less urea upon stimulation with mixed amino acids, and amino acid clearance was lower in mice with hepatic steatosis. Glucagon-induced ureagenesis was impaired in perfused rat livers with hepatic steatosis. Patients with NAFLD had hyperglucagonemia and increased levels of glucagonotropic amino acids, including alanine in particular. Both glucagon and alanine levels were reduced after diet-induced reduction in Homeostatic Model Assessment for Insulin Resistance (HOMA-IR, a marker of hepatic steatosis). CONCLUSIONS:Glucagon regulates amino acid metabolism both non-transcriptionally and transcriptionally. Hepatic steatosis may impair glucagon-dependent enhancement of amino acid catabolism.
Project description:<h4>Background and aims</h4>Fibroblast growth factor (FGF) 1 demonstrated protection against nonalcoholic fatty liver disease (NAFLD) in type 2 diabetic and obese mice by an uncertain mechanism. This study investigated the therapeutic activity and mechanism of a nonmitogenic FGF1 variant carrying 3 substitutions of heparin-binding sites (FGF1<sup>△HBS</sup> ) against NAFLD.<h4>Approach and results</h4>FGF1<sup>△HBS</sup> administration was effective in 9-month-old diabetic mice carrying a homozygous mutation in the leptin receptor gene (db/db) with NAFLD; liver weight, lipid deposition, and inflammation declined and liver injury decreased. FGF1<sup>△HBS</sup> reduced oxidative stress by stimulating nuclear translocation of nuclear erythroid 2 p45-related factor 2 (Nrf2) and elevation of antioxidant protein expression. FGF1<sup>△HBS</sup> also inhibited activity and/or expression of lipogenic genes, coincident with phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and its substrates. Mechanistic studies on palmitate exposed hepatic cells demonstrated that NAFLD-like oxidative damage and lipid accumulation could be reversed by FGF1<sup>△HBS</sup> . In palmitate-treated hepatic cells, small interfering RNA (siRNA) knockdown of Nrf2 abolished only FGF1<sup>△HBS</sup> antioxidative actions but not improvement of lipid metabolism. In contrast, AMPK inhibition by pharmacological agent or siRNA abolished FGF1<sup>△HBS</sup> benefits on both oxidative stress and lipid metabolism that were FGF receptor (FGFR) 4 dependent. Further support of these in vitro findings is that liver-specific AMPK knockout abolished therapeutic effects of FGF1<sup>△HBS</sup> against high-fat/high-sucrose diet-induced hepatic steatosis. Moreover, FGF1<sup>△HBS</sup> improved high-fat/high-cholesterol diet-induced steatohepatitis and fibrosis in apolipoprotein E knockout mice.<h4>Conclusions</h4>These findings indicate that FGF1<sup>△HBS</sup> is effective for preventing and reversing liver steatosis and steatohepatitis and acts by activation of AMPK through hepatocyte FGFR4.
Project description:Impaired autophagy has been implicated in the pathogenesis of nonalcoholic fatty liver disease. Catalpol (CAT), a bioactive compound from Rehmannia (Di Huang) glutinosa, is known to ameliorate insulin resistance and the histological NAFLD spectrum in obese mice. Here, we investigated the effects of CAT on hepatic steatosis and autophagy in ob/ob and high-fat diet-induced obese mice, as well as in hepatocytes. In ob/ob mice, CAT reduced liver weight, liver triglyceride and cholesterol content, and hepatic lipogenic enzyme levels and increased fatty acid oxidase levels. In addition, CAT administration increased LC3-II levels and decreased SQSTM1/P62 levels in ob/ob mice. Similar effects on hepatic steatosis and autophagy were observed in high-fat diet-induced mice after administration of CAT. Additionally, we found that CAT stimulated AMPK and increased nuclear translocation of transcription factor EB (TFEB) in obese mice and hepatocytes. Inhibition of AMPK completely blocked the effects of CAT on TFEB nuclear localization, hepatic autophagy, and liver steatosis. These findings revealed that diminished AMPK/TFEB-dependent autophagy is involved in the pathogenesis of liver steatosis in obesity, and that CAT might be a novel therapeutic candidate for treatment of this condition.
Project description:Nonalcoholic fatty liver disease (NAFLD) is strongly associated with insulin resistance. The peroxisome proliferator-activated receptor (PPAR) activators, thiazolidinediones, (TZDs), are insulin sensitizers used as a treatment for NAFLD. However, TZDs are a controversial treatment for NAFLD because of conflicting results regarding hepatic steatosis and fibrosis. To evaluate a possible effective drug for treatment of NAFLD, we investigated the effects of a newly developed TZD, lobeglitazone, with an emphasis on hepatic lipid metabolism. Lobeglitazone treatment for 4 weeks in high fat diet- (HFD-) induced obese mice (HL group) improved insulin resistance and glucose intolerance compared to HFD-induced obese mice (HU group). The gene levels related to hepatic gluconeogenesis also decreased after treatment by lobeglitazone. The livers of mice in the HL group showed histologically reduced lipid accumulation, with lowered total plasma cholesterol and triglyceride levels. In addition, the HL group significantly decreased the hepatic expression of genes associated with lipid synthesis, cholesterol biosynthesis, and lipid droplet development and increased the hepatic expression of genes associated with fatty acid <i>?</i>-oxidation, thus suggesting that lobeglitazone decreased hepatic steatosis and reversed hepatic lipid dysregulation. Livers with steatohepatitis contained increased levels of PPAR<i>?</i> and phosphorylated PPAR<i>?</i> at serine 273, leading to downregulation of expression of genes associated with insulin sensitivity. Notably, the treatment of lobeglitazone increased the protein levels of PPAR<i>?</i> and diminished levels of PPAR<i>?</i> phosphorylated at serine 273, which were increased by a HFD, suggesting that induction of PPAR<i>?</i> and posttranslational modification of PPAR<i>?</i> in livers by lobeglitazone might be an underlying mechanism of the improvement seen in NAFLD. Taken together, our data showed that lobeglitazone might be an effective treatment for NAFLD.
Project description:Ob/ob mice provide an animal model for non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH) in patients with obesity and type-2 diabetes. Low liver copper has been linked to hepatic lipid build-up (steatosis) in animals with systemic copper deficiency caused by low-copper diets. However, hepatic copper status in patients with NAFLD or NASH is uncertain, and a validated animal model useful for the study of hepatic copper regulation in common forms of metabolic liver disease is lacking. Here, we report parallel measurements of essential metal levels in whole-liver tissue and defatted-dried liver tissue from ob/ob and non-obese control mice. Measurements in whole-liver tissue from ob/ob mice at an age when they have developed NAFLD/NASH, provide compelling evidence for factitious lowering of copper and all other essential metals by steatosis, and so cannot be used to study hepatic metal regulation in this model. By marked contrast, metal measurements in defatted-dried liver samples reveal that most essential metals were actually normal and indicate specific lowering of copper in ob/ob mice, consistent with hepatic copper deficiency. Thus ob/ob mice can provide a model useful for the study of copper regulation in NAFLD and NASH, provided levels are measured in defatted-dried liver tissue.
Project description:Mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) is an integral component of the respiratory chain, and recent studies have suggested that it plays an important role in hepatic glucose homeostasis. However, its function in hepatic lipid metabolism is unclear. Here, we identified a role for mGPDH in nonalcoholic fatty liver disease (NAFLD). Specifically, mGPDH expression and activity were lower in fatty livers from patients and mice with NAFLD (ob/ob, high-fat diet [HFD] and db/db). Liver-specific depletion of mGPDH in mice or mGPDH knockdown in cultured hepatocytes exacerbated diet-induced triglyceride accumulation and steatosis through enhanced lipogenesis. RNA-sequencing revealed that mGPDH regulated endoplasmic reticulum (ER)-related proteins and processes. mGPDH deletion exacerbated tunicamycin (ER stress inducer)-induced hepatic steatosis, whereas tauroursodeoxycholic acid (ER stress inhibitor) rescued mGPDH depletion-induced steatosis on an HFD. Moreover, ER stress induced by mGPDH depletion could be abrogated by the intracellular Ca2+ chelator 1,2-bis (2-aminophenoxy) ethane N,N,N´,N´-tetraacetic acid acetoxymethyl ester, mitochondrial permeability transition pore (mPTP) inhibitor cyclosporine A, or cyclophilin-D (Cyp-D) knockdown. mGPDH promoting Cyp-D ubiquitination was also observed. Finally, liver-specific mGPDH overexpression attenuated hepatic steatosis in ob/ob and HFD mice. Conclusion: mGPDH is a pivotal regulator of hepatic lipid metabolism. Its deficiency induces ER stress by suppressing Cyp-D ubiquitination, a key regulator of the mitochondrial Ca2+ conductance channel mPTP, and results in hepatic steatosis. mGPDH may be a potential therapeutic target for the treatment of NAFLD.
Project description:Nonalcoholic fatty liver disease (NAFLD) is highly prevalent and associated with considerable morbidities. Unfortunately, there is no currently available drug established to treat NAFLD. It was recently reported that intraperitoneal administration of taurine-conjugated ursodeoxycholic acid (TUDCA) improved hepatic steatosis in ob/ob mice. We hereby examined the effect of oral TUDCA treatment on hepatic steatosis and associated changes in hepatic gene expression in ob/ob mice. We administered TUDCA to ob/ob mice at a dose of 500 mg/kg twice a day by gastric gavage for 3 weeks. Body weight, glucose homeostasis, endoplasmic reticulum (ER) stress, and hepatic gene expression were examined in comparison with control ob/ob mice and normal littermate C57BL/6J mice. Compared to the control ob/ob mice, TUDCA treated ob/ob mice revealed markedly reduced liver fat stained by oil red O (44.2±5.8% vs. 21.1±10.4%, P<0.05), whereas there was no difference in body weight, oral glucose tolerance, insulin sensitivity, and ER stress. Microarray analysis of hepatic gene expression demonstrated that oral TUDCA treatment mainly decreased the expression of genes involved in de novo lipogenesis among the components of lipid homeostasis. At pathway levels, oral TUDCA altered the genes regulating amino acid, carbohydrate, and drug metabolism in addition to lipid metabolism. In summary, oral TUDCA treatment decreased hepatic steatosis in ob/ob mice by cooperative regulation of multiple metabolic pathways, particularly by reducing the expression of genes known to regulate de novo lipogenesis.
Project description:Men with nonalcoholic fatty liver disease (NAFLD) are more exposed to nonalcoholic steatohepatitis (NASH) and liver fibrosis than women. However, the underlying molecular mechanisms of NALFD sex dimorphism are unclear. We combined gene expression, histological and lipidomic analyses to systematically compare male and female liver steatosis. We characterized hepatosteatosis in three independent mouse models of NAFLD, <i>ob/ob</i> and lipodystrophic fat-specific (<i>PpargF</i><sup>Δ/Δ</sup>) and whole-body PPARγ-null (<i>Pparg</i><sup>Δ/Δ</sup>) mice. We identified a clear sex dimorphism occurring only in <i>Pparg</i><sup>Δ/Δ</sup> mice, with females showing macro- and microvesicular hepatosteatosis throughout their entire life, while males had fewer lipid droplets starting from 20 weeks. This sex dimorphism in hepatosteatosis was lost in gonadectomized <i>Pparg</i><sup>Δ/Δ</sup> mice. Lipidomics revealed hepatic accumulation of short and highly saturated TGs in females, while TGs were enriched in long and unsaturated hydrocarbon chains in males. Strikingly, sex-biased genes were particularly perturbed in both sexes, affecting lipid metabolism, drug metabolism, inflammatory and cellular stress response pathways. Most importantly, we found that the expression of key sex-biased genes was severely affected in all the NAFLD models we tested. Thus, hepatosteatosis strongly affects hepatic sex-biased gene expression. With NAFLD increasing in prevalence, this emphasizes the urgent need to specifically address the consequences of this deregulation in humans.
Project description:Ochratoxin A(OTA) is considered to be one of the most important contaminants of food and feed worldwide. The liver is one of key target organs for OTA to exert its toxic effects. Due to current lifestyle and diet, nonalcoholic fatty liver disease (NAFLD) has been the most common liver disease. To examine the potential effect of OTA on hepatic lipid metabolism and NAFLD, C57BL/6 male mice received 1 mg/kg OTA by gavage daily. Compared with controls, OTA increased lipid deposition and TG accumulation in mouse livers. In vitro OTA treatment also promoted lipid droplets accumulation in primary hepatocytes and HepG2 cells. Mechanistically, OTA prevented PPARγ degradation by reducing the interaction between PPARγ and its E3 ligase SIAH2, which led to activation of PPARγ signaling pathway. Furthermore, downregulation or inhibition of CD36, a known of PPARγ, alleviated OTA-induced lipid droplets deposition and TG accumulation. Therefore, OTA induces hepatic steatosis via PPARγ-CD36 axis, suggesting that OTA has an impact on liver lipid metabolism and may contribute to the development of metabolic diseases.