Roles And Underlying Mechanism Of FNDC5/irisin In Attenuating Hepatic Glucose And Lipid Metabolic Disorders

Irisin is a recently identifed myokine which induces uncouping protein-1(UCP1)expression and white adipose tissue(WAT)browning.Type III domain-containing protein 5(FNDC5)is consist of two fiber protein domains,a signal peptide and a transmembrane protein composed with embedded hydrophobic domains.Irisin is a cleaved and secreted fragment of FNDC5;it enhances energy expenditure and may be involved in the beneficial effects of exercise that have been noted independent of weight loss.Adenoviral vectors that express full-length FNDC5 in mice improve glucose tolerance and reduce insulin resistance.Serum irisin concentrations had an inverse association with the triacylglycerol content in the liver of obese adults.The thesis is divided into two parts,respectively.(1)Effects of irisin on hepatic gluconeogenesis and glycogen synthesis in type 2 diabetes.(2)Effects of FNDC5 on hepatic autophagy,fatty acid oxidation,lipogenesis and hepatosteatosis in mice.Part 1 Role and molecular mechanism of irisin in reducing hepatic gluconeogenesis,increasing glycogen synthesis in diabetic model1,BackgroundType 2 diabetes is characterized by hyperglycaemia and insulin resistance in target tissues.The liver plays a central role in the maintenance of blood glucose;it is of great significant in the regulation of glucose metabolism,energy storage and distribution.Increased synthesis of glucose into glycogen mainly in the liver responses to postprandial blood glucose rush,surplus glucose can also be converted into fat or reinforce pentose phosphate cycle,thus reducing postprandial blood glucose.On the contrary,increased gluconeogenesis in the liver transforms lactose,glycerin and glycogenic amino acid into glucose responses to low blood glucose or hepatic glycogen shortage.Strenuous exercise or hunger is major contributor to gluconeogenesis.Type 2 diabetes is mainly manifested as excessive hepatic gluconeogenesis and robustly down-regulated glycogen synthesis.The disorder of hepatic gluconeogenesis and glycogen synthesis further aggravated the glucose disturbance and insulin resistance in type 2 diabetes,thus reflecting a vicious circle.Therefore reversing the excessive hepatic gluconeogenesis,increasing hepatic glycogen synthesis has become one of the important strategies for prevention and treatment of type 2 diabetes.However,the role of irisin in hepatic gluconeogenesis and glycogen synthesis remains elusive.In this study,we determined the effects of irisin on hepatic gluconeogenesis and glycogen synthesis and their downstream signal pathway in type 2 diabetic mice and hepatocytes.2.ObjectiveThe study was to investigate the role and molecular mechanism of irisin on hepatic gluconeogenesis and glycogen synthesis and to evaluate the effects of persistently administration of irisin on glucose homeostasis and insulin resistance.3.MethodsHepG2 cells were maintained in Dulbecco's modified Eagle's medium(DMEM)with 25 mM glucose,10%FBS,penicillin(100 units/ml)and streptomycia(100(?g/ml)at 37 ? in a humidified atmosphere of 95%air and 5%CO2.The hepatocytes were maintained in special hepatocyte culture medium containing 10%FBS,with penicillin and streptomycin.Male C57BL/6J mice aged 6 weeks were used for inducing type 2 diabetes.After a week of adaptation in the living environment,the mice were randomly divided into three groups(n = 7 for each group).One group of mice(Ctrl)received an intraperitoneal injection of vehicle and was fed with a normal diet(14.7 kJ/g,13%of energy as fat)throughout the experiment.Another two groups of mice were combined as one group(STZ/HFD),to induce type 2 diabetes mellitus.These mice were subjected to 4-h fasting followed by intraperitoneal injection of low-dose STZ(120 mg/kg body weight in 10 mmol/1 citrate buffer,pH 4.0).After 3 weeks,the mice were fed with a HFD(21.8 kJ/g,60%of energy as fat)instead of the previously used normal diet.The mice were re-divided into two groups,8 weeks after the injection of STZ,and received,respectively,subcutaneous perfusion of either saline or irisin via micro-osmotic pumps for 2 weeks,and were maintained on HFD feeding.Acute experiments were carried out at the end of the 12th week after diet.The serum insulin and irisin levels were determined by an enzyme immunoassay kit.Glucose dehydrogenase coupling reaction and lactate dehydrogenase coupling reaction were used to evaluate glucose-6-phosphatase(G6Pase)and phosphoenolpyruvate carboxykinase(PEPCK)activity.PEPCK,G6Pase,glycogen synthase kinase-3(GSK3),glycogen synthase(GS),Forkhead box O1(FOXO1)and the phosphorylation of GSK,GS,Akt,FOXO1 expression were measured by Western blot.The mRNA of PEPCK and G6Pase levles were anlyzed by real-time quantitative PCR.Liver glycogen content was measured by Periodic Acid-Schiff(PAS)staining.Glucose tolerence test(GTT)and insulin tolerence test(ITT)were uased for analyzing mice glucose homeostasis and inuslin resistance.Glycogen levels were tested using a glycogen assay kit;hepatocytes glucose prodution was tested using a glucose oxidase-peroxidase assay kit.4.Results(1)Irisin rectified the enhanced gluconegenesis and glucose production in hepatocytes with insulin resistance induced by glucosamine or palmitate.(2)Irisin promoted the phosphorylation of GSK3,inhibted the phosphorylation of GS,thus preventing reduced glycogen synthesis content in hepatocytes with insulin resistance.(3)Glucosamine pretreatment prevented both PI3K P100? and PI3K P100? ptotein expression.Irisin promoted hepatocytes PI3K P100? protein expression but not PI3K P100? ptotein expression.Irisin increaed the phosphorylation of Akt and FOXO1,thereby inhibiting FOXO1 activity.Irisin showed similar phosphorylation effects on Akt and FOXO1 to insulin in hepatocytes.(4)Irisin prevented the excessive glucose production of hepatocytes caused by glucosamine or palmitate,which was abolished by PI3K inhibitor LY294002 or Akt inhibitor MK2206.The effects of irisin on promoting the phosphorylation of GSK3,inhibiting the phosphorylation of GS,and subsequently increasing glycogen synthesis were prevented by LY294002 or MK2206.(5)Serum and liver irisin levels were lower in STZ/HFD induced type 2 diabetic mice than control mice.Irisin administration through micro-osmotic pump increased serum and liver irisin levels in STZ/HFD mice,but had no significant effect on mice body weight,food intake and systolic blood pressure.(6)Persistent administration of irisin reduces blood glucose levels,increases liver glycogen,promoted glucose and insulin tolerance,but had no significant effect on serum insulin levels.(7)Persistent administration of irisin prevented the enhanced gluconeogenesis and decreased glycogen synthesis via the Akt-mediated regulation of key enzymes of gluconeogenesis and glycogen synthesis in type 2 diabetes.5.ConclusionIrisin decreases gluconeogenesis by the PI3K/Akt/FOXO 1-mediated down-regulation of PEPCK and G6Pase,and increases glycogenesis by the PI3K/Akt/GSK3-mediated GS activation in hepatocytes and mouse models of type 2 diabetes.Long-term subcutaneous administration of irisin attenuates hyperglycaemia and insulin resistance in mice with type 2 diabetes.Part 2 Role and molecular mechanism of FNDC5 in enhancing hepatic autophagy,fatty acid oxidation,and deducing lipogenesis in mice.1.BackgroundNonalcoholic fatty liver disease(NAFLD)is characterized by triacylglycerol(TG)accumulation within hepatocytes.Fatty acid oxidation(FAO)in mitochondria is a process to shorten the fatty acids into acetyl-CoA,which can be converted into ketone bodies or incorporated into the tricarboxylic acid cycle for full oxidation.Autophagy is another programmed cell death in addition to apoptosis;it is a highly conserved mechanism of self-preservation in the process of evolution.Autophagy is a mechanism involved in cellular homeostasis delivering cytoplasmic content to the lysosomes for degradation to macronutrients.Impaired FAO or autophagy and excess de novo lipogenesis are critical to hepatic steatosis.Therefore,it is necessary to find an important target molecule for FAO,autophagy and lipogenesis,which is one of the important strategies for the treatment of hepatic steatosis.Fibronectin type III domain containing 5(FNDC5)is a type I membrane protein that has 209 amino acid residues.Adenoviral vectors that express full-length FNDC5 reduce body weight in mice,FNDC5 inducesbrowning of subcutaneous adipocytes and mediates the beneficial effect of exercise on metabolism.Our recent studies have shown that FNDC5 overexpression ameliorates hyperlipemia and enhances lipolysis in adipose tissues in obese mice.However,it is not clear whether FNDC5 played a role in FAO,autophagy or lipogenesis.2.ObjectiveThe study was to investigate the effect of FNDC5 on FAO,autophagy,lipogenesis and hepatic steatosis and its underlying molecular mechanism,and to determine the therapeutic effects of FNDC5 on hepatosteatosis.3.Methods4-8 weeks old male C57BL/6 wild-type(WT)and FNDC5-/-mice on a C57BL/6 background were used in the experiments.The primary culture of hepatocyte from WT and FNDC5-/-mice was performed with enzymatic digestion.4 weeks old WT and FNDC5-/-mice were subject to high fat diets(HFD)(21.8 kJ/g,60%of energy as fat)for 12 weeks to induce obesity models.ELISA,Western blot and Real-time PCR were employed to measure the efficiency of FNDC5 gene knock out.Enzymatic assays were conducted with standard protocols and kits to evaluate serum and tissue contents of TG(triglyceride),total cholesterol(CHO),non-esterified fatty acid(NEFA),Alanine aminotransferase(ALT),Glutamic-oxalacetic transaminase(AST).Real-time PCR was used to determine the mRNA expression of FAO related genes(Hmgcs2,Cptl,Acox1,Ehhadh,Acsll,Peci,Cyp4a10,and Cyp4a12),autophagy genes(UNC51-like kinase 1(Ulk1),Ulk2,Atg5,Atg8,Atg10)and lipogenesis genes(Srebplc,Dgatl,Fasn,and Scdl).The protein levels of AMPK/mTORC1 signal pathway(AMPK,S6 and raptor)and autophagy related protein(p62,LC3B,ULK1)were assessed by Western blot and immunohistochemistry.Oil red O staining was used to detect lipid accumulation in hepatocytes and liver.14C radioactivity labeling method was performed to evaluate FAO rate.The tandem GFP-RFP-LC3 adenovirus was transfected into hepatocytes to monitor autophagy flux.4.Results(1)Moderate lipid accumulation developed in FNDC5-/-mice liver at baseline at the age of 8 weeks,and became much more sever after fasting for 16h.Serum TG,CHO and NEFA were obviously increased in FNDC5-/-mice in response to fasting overnight.(2)The FAO related genes expression was relatively low in FNDC5-/-mice liver compared with that in WT mice both in fed and fasted state.AMPK agonist AICAR and PPARa agonist WY14643 greatly enhanced the reduced FAO genes expression in FNDC5-/-mice,and significantly attenuated the increased lipid accumulation of FNDC5-/-mice liver.Importantly,knockdown of AMPK with siRNA promoted lipid accumulation both in WT and FNDC5-/-mice.These findings indicate that FAO is reduced in FNDC5-/-mice livers and that FNDC5 is important for fasting-induced FAO gene expressions,which are mediated by the AMPK pathway.FNDC5 deficiency causes impairment in AMPK/PPAR?-mediated FAO.(3)Treatment with mTORC1 inhibitor rapamycin greatly enhanced FAO related genes in FNDC5-/-mice livers.Moreover,inhibition of mTORC1 suppressed the increased expression of lipogenesis genes and lipid accumulation in FNDC5-/-mice livers.(4)Starvation or amino acid deprivation treatment obviously prevented p62 expression,increased LC3B expression in WT hepatocytes,but had no significant effect in FNDC5-/-hepatocytes.Similar change was observed by autophagy flux detection.Amino acid deprivation or metformin incubation promoted the phosphorylation of AMPK,raptor and ULK1 in WT hepatocytes in a time dependent manner,but had much weaker effects in FNDC5-/-hepatocytes.Stimulation with AICAR significantly increased autophagy development both in WT and FNDC5-/-hepatocytes.Correspondingly,autophagy was reduced by AMPK siRNA both in WT and FNDC5-/-hepatocytes.(5)Treatment with rapamycin promoted the expression of LC3B,reduced p62 expression and AST,ALT concentration in FNDC5-/-mice.Similarly,autophagy flux response to rapamycin greatly enhanced both in WT and FNDC5-/-hepatocytes.Importantly,palmitate induced lipid accumulation was prevented by rapamycin,which was predominantly depend on Atg5 gene.(6)Liver weight and the ratio of liver weight to body weight were higher in FNDC5-/-/HFD mice compared with WT/HFD mice,but no significant difference in body weight and food intake was observed between the two groups.FNDC5 deficiency aggravates hepatosteatosis,lipogenesis,liver injury,as well as FAO and autophagy impairment.(7)Exogenous FNDC5 dose and time-dependently stimulated the expression of FAO genes,almost reaching its maximal effects at concentration of 100nmol/1 and time of 24h.FNDC5 abolished the increased TG content induced by palmitate in FNDC5-/-hepatocytes.Moreover,treatment with FNDC5 prevented p62 protein,increased LC3B protein expression via AMPK/mTORC1 signaling pathway.(8)FNDC5 overexpression prevents hepatosteatosis and attenuates the FAO and autophagy impairment via AMPK/mTORC1 signaling pathway in HFD-induced obese mice.5.ConclusionOur data provide a novel view that FNDC5 gene deficiency aggravates hepatosteatosis,lipogenesis,liver injury,as well as FAO and autophagy impairment.FNDC5 promotes FAO and autophagy,attenuates lipogenesis by positively regulates AMPK activity and subsequent mTORC1 suppression,thus prevents hepatosteatosis and liver injury.Our findings provide new insight that intervention of FNDC5 maybe a promising therapeutic strategy for hepatosteatosis.