| Background:Non-alcoholic fatty liver disease(NAFLD)is a chronic liver disease ranged from simple hepatic steatosis to non-alcoholic steatohepatitis(NASH),and insulin resistance plays a key role on the pathogenesis of NAFLD.High-fat diet is a single risk factor leading to insulin resistance and type 2 diabetes,also named as "lipotoxicity",which is characterized by increased body weight,elevated plasma FFA levels and abnormal accumulation of fat in insulin target organs,such as skeletal muscles,liver and adipocytes.Although both epidemiological and experimental researches have demonstrated the effects of high-fat exposure on insulin resistance in several insulin target organs,including skeletal muscles,liver and adipocytes,and the roles of insulin resistance on NAFLD,but the precise mechanism has not been fully elucidated yet.To date,both in vivo and in vitro studies have demonstrated that high-fat induced insulin resistance is accompanied with impaired activity or expression of several insulin signal moleculars,such as insulin receptor,insulin receptor substrate and GluT4 etc,and this was proposed as possible contributors to high-fat induced insulin resistance. However,the upstream mechanism underlying such impaired insulin signaling caused by high-fat exposure remains unclear.AMPK,AMP-activated protein kinase,is a key regulator of intracellular fatty acid metabolism.Stresses such as exercise,hypoxia,or prolonged starvation could activate AMPK via increasing intracellular AMP/ATP ratio.Once activated,its classical effect is to phosphorylate and inactivate acetyl-CoA carboxylase(ACC),resulting in a decrease in malonyl-CoA,thus relieving inhibition of CPT1 and facilitating FFA entry into mitochondria forβ-oxidation.Besides regulating fatty acid oxidation,AMPK has been proved recently as a regulator of insulin signaling.AMPK overexpression exhibited enhanced insulin sensitivity.It is also known that metformin is an activator of AMPK.Our previous work and other studies have indicated that metformin may enhance AMPKαactivity and reduce lipotoxicity in both rat skeletal muscle and mature adipocytes.Whether AMPK also participates in the liver pathophysiology of high-fat induced insulin resistance and NAFLD is unclear.Peroxisome proliferator-activated receptors(PPARs),members of the nuclear receptor superfamily,regulate target gene expression in response to ligand binding.Of these,PPARαmainly regulates the expression of genes involved in fatty acid metabolism,particularly when coactivated by PPARγ,coactivator(PGC)-1.Thus, PPARαagonists,such as fenofibrate,are used clinically to treat patients with hypertriglyceridemia. PPARγ,is primarily involved in adipocyte differentiation and insulin sensitization,and its agonists have been used clinically to ameliorate IR and type diabetes.Furthermore,recent study found that a PPARγagonist,pioglitazone,reduced fasting plasma triacylglycerol and hepatic fat content in patients with type 2 diabetes.Recently,an association between AMPK and PPARαor PPARγ,has been paid more attentions.It is possible that AMPK activation has different effects on PPARαandγin the liver.It is known that the liver is a major player in lipid homeostasis.In order to investigate their characteristics and gain a better understanding of the roles of metformin on PPARs in NAFLD,it is necessary to evaluate the activity of PPARαandγ, in liver.Published studies,however,are currently lacking.Based on our animal trial above,we also made a half-one year clinical prospective study on patients with NAFLD and metabolic syndrome(MS)to investigate the therapeutic steps for NAFLD and lipotoxicity.In our clinical study,we observed the effects of metformin and fenofibrate on NAFLD and lipotoxicity.Objective: ①The effects of chronic high-fat feeding on insulin sensitivity and lipotoxicity of rat at body level and its effects on lipid content in rat liver,and the effects of metformin on the changes of them.②We investigated the effects of chronic high-fat feeding on the activity and expression of AMPKα,PPARαand PPARγin rat liver,and the effects of metformin treatment on the activity of AMPKα,PPARαand PPARγ,as well as whether these changes were accompanied by changes of lipid accumulation in rat liver and serum.③By clinical prospective study,we investigated the effects and salty of metformin and fenofibrate on lipotoxicity and NAFLD to detect the therapeutic steps for lipotoxicity and NAFLD.Methods:①Animal feeding:Thirty male Wistar rats were divided randomly into three groups according to their body weight:control group,high-fat group(HF)and metformin-treated group(HF+MET).In control group,the rat standard diet was provided.While in HF group and HF + MET group,rats received a high-fat diet,in which 59%carolie was provided by fatty acid(mainly lard).Metformin was administrated in HF+MET group with daily dose of 50rag·kg-1after fed with high-fat diet for four months.The total feeding time was five months.②Evaluation of lipotoxicity:Rat' body weights(W)and fasting plasma glucose (FPG)levels were monitored during the feeding stage.After five months of feeding,the levels of fasting serum insulin(FINS),FPG,fasting serum triglyceride(TG)and total cholesterol(TC)as well as contents of hepatic TG and TC were measured.③Measurements of hepatic AMPKα,PPARαand PPARγactivitites,gene and protein expressions in rat liver:mRNA levels of alpha 1 and alpha 2 subunit of AMPK as well as PPARαwere measured using RT-Real time PCR.Protein levels of total AMPKαsubunit and PPARαwere detected using Western blot and activities of AMPK were evaluated by measuring the protein levels of phosphorlated AMPKα(P-AMPKα). Activity of PPARαand PPARγwere detected using ELISA.To further explore the role of AMPK in lipotoxicity,the effects of metformin on insulin resistance induced by high-fat feeding,and associations between AMPK and PPARαor PPARγactivity in rat liver were evaluated.④Methods for our clinical study:178 cases of patient with NAFLD and MS were divided into four groups:group D given dietary treatment alone,group A given metformin and dietary treatment,group B given fenofibrate and dietary treatment and group C without any steps.By comparing the differences of physical characteristics, including W,BMI,WC and BP,and biochemical parameters,including FBG,2hPG, FINS,2hINS,HOMA-IR,TG,TC,HDL,LDL,LA,ALT and AST,before and after treatment for 6 months.Glucose metabolism was evaluated using oral glucose tolerance test and NAFLD was evaluated with color doppler sonography.to evaluate the effects and salty of metformin and fenofibrate on lipotoxicity and NAFLD.Results:①Evaluation of lipotoxicity.Compared with control,high-fat fed rats showed significantly increased levels of body weight(51.1%,P＜0.01),fast plasma glucose (21.5%,P＜0.05)and serum insulin(17%,P＜0.05).Plasma TG and TC levels(both P＜0.01)as well as hepatic content of TG and TC(both P＜0.05)also obviously elevated after high-fat feeding.These changes indicated onset of insulin resistance, lipotoxicity and hepatic steatosis,and metformin apparently reversed these changes.②Changes of hepatic AMPKα,PPARαand PPARγactivity,and their changes in the levels of gene and protein expression in rat liver.A high fat diet decreased the mRNA expression and protein content of both AMPKαand PPARαin rat livers (respectively,P＜0.05),while metformin obviously increased the mRNA expression and protein content of AMPKα(P＜0.05),and increased that of PPARαin a certain extent. High fat diet also obviously decreased the AMPKαactivity(P-AMPKαcontent) (P＜0.05),PPARαand PPARγactivity decreased 21.6%and 16.7%(respectively,P＞0.05) in rat liver.Metformin significantly increased AMPKαactivity and enhanced PPARαactivity in rat liver(respectively,P＜0.05),while the PPARγ,activity only increased 5.7% (P＞0.05).Compared with high-fat fed rats,enhancements of AMPK and PPARαactivity induced by metformin obviously ameliorated insulin resistance,lipotoxicity and hepatic steatosis induced by high-fat feeding,thrther suggesting the possible roles of AMPK and PPARαon lipotoxicity and NAFLD,and effects of metformin on ameliorating lipotoxicity and NAFLD.③Effects of metformin and fenofibrate on lipotoxicity and NAFLD in our clinical study.Treatment with dietary treatment alone could apparently decrease W,BMI,WC, and levels of 2hPG,2hINS,TG and TC(all P＜0.05),but HOMA-IR did not decrease significantly.Treatment with metformin accompanied with dietary control was more effective on ameliorating lipotoxicity and NAFLD than B and D groups,and no side effects on patients,although the effect of metformin on decreasing serum TG was less than that of fenofibrate.Fenofibrate accompanied with dietary treatment can obviously decrease serum TG and TC,and also ameliorate lipotoxicity and NAFLD,but the effects of fenofibrate on ameliorating lipotoxicity and NAFLD were less than that of metformin.Patients treated without any steps deteriorated in all aspects after 6 months.Conclusions:①Chronic high-fat feeding could induce insulin resistance and NAFLD,and metformin could ameliorate them.②High-fat feeding impaired both the expressions and activities of AMPKαand PPARαin rat liver,while metformin could obviously activate AMPKαand enhance PPARαactivity in rat liver.These findings indicated that the enhancement of PPARαactivity was consistent with metformin-induced AMPK activation in rat liver.AMPKαand PPARαall should be partly responsible for reducing insulin resistance and lipid accumulation in rat liver and serum.③Dietary treatment is the fundmental step for ameliorating lipotoxicity and NAFLD.Not only metformin can obviously decrease the levels of blood glucose, severity of abdominal obesity and insulin resistance,but also effectively and safely ameliorate NAFLD and decrease the levels of TG and TC in patients with NAFLD and MS.Fenofibrate can obviously decrease the levels of TG and TC,and also effectively and safely ameliorate lipotoxicity and NAFLD in patients with NAFLD and MS.
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