Irisin Alleviates Metabolic-associated Fatty Liver Disease And Underlying Mechanism

Background:Metabolic-associated fatty liver disease(MAFLD),which is characterized by an increase of intrahepatic fat,has become a serious public health issue because of its extremely high prevalence and potential progression to severe liver disease,such as cirrhosis.Approved pharmacological treatments for MAFLD are not available due to a lack of evidence-based recommendations.Exercise interventions are the non-pharmacological approaches with strong interventional efficiency,but the underlying mechanism of exercise for alleviating MAFLD is not clear.Exercise could induce irisin expression and secretion into circulation to exert its effects on browning of subcutaneous adipose tissue,suggesting that irisin may contribute to the regulation of energy homeostasis and thus is the potential target for therapy of MAFLD;however,the role of irisin in the alleviation of MAFLD by exercise is not clear.Therefore,further investigation is necessary to define the role of exercise-induced irisin in MAFLD,which will be beneficial to develop the complimentary agents in the prevention and treatment of MAFLD.Objective:The aims of this study are to explore the effect of aerobic exercise on the expression of irisin in the liver of db/db mice and the alleviation and improvement of MAFLD,and to provide a new potential therapeutic target or candidate intervention strategy and drug for the prevention and treatment of MAFLD.we also evaluated the expression levels of hepatic irisin during MAFLD progression and liver injury in HFD-induced fatty liver of rats and investigated its potential regulatory pathways in the development of insulin-resistance and lipid accumulation in hepatocytes as well as corresponding cell death.Methods:Here we investigated the effect of treadmill running in the regulation of irisin using a mouse model of db/db.Totally ten 8-week-old male m/m mice were selected as normal control group(NC group),and twenty db/db mice were randomly divided into two groups: MAFLD model group(MC group,n = 10)and MAFLD exercise group(ME group,n = 10).The mice from ME group were subjected to treadmill running for 8 consecutive weeks.At the end of the experimental period,all mice were sacrificed.The serum and liver tissues were collected to detect the expression of irisin and the contents of TG,TC,ALT and AST.HE and ORO staining of liver tissues were performed to measure the hepatic lipid content.Western blotting was performed to evaluate the protein expression of AMPK,p-AMPK,PGC-1α and irisin in hepatocytes.In order to further verify the effect and potential mechanism of exercise-induced irisin on the alleviation of MAFLD,a rat model of HFD-induced MAFLD was established,and intraperitoneal injection of exogenous recominbant irisin was conducted for rats to explore the effect of irisin on MAFLD and its regulatory mechanism,and to provide a new scientific basis for clinical prevention and treatment of MAFLD.Totally 30 8-week-old male SD rats were selected and randomly divided into 3 groups: normal control group(NC group,n = 10),and other twenty rats were fed with an HFD for 9 weeks to establish an HFD-induced fatty liver model.The fatty liver model mice were then randomly divided into two sub-groups: fatty liver model group(FL group,n = 10)and fatty liver model coupled with exogenous irisin intraperitoneal injection(FI group,n = 10)at the daily dose of 1 μg/kg irisin for 5consecutive weeks.At the end of the corresponding challenge,blood samples were collected,and all mice were immediately sacrificed and the liver tissues were harvested.The serum and liver tissue samples were collected to determine the expression levels of irisin and the contents of TG,TC,ALT,AST,SOD and MDA.HE and Oil Red staining of the liver tissue was performed to measure the hepatic lipid content.Western blotting was performed to evaluate the expression of lipogenesis-associated proteins(SREBP1 and ACC1),lipolysis-associated proteins(HSL and ATGL),fatty acid oxidation-associated proteins(PGC-1α,PPARα and PPARγ)and SIRT1/AMPK signal pathway,insulin signal pathway(PI3K and Akt),autophagy-related proteins(LC3,Beclin1 and p62),apoptosis-related proteins(Bax and Bcl2)and inflammatory cytokines(NF-κB,TNF-α,and IL-6)in hepatocytes.Results:1.Exercise-induced irisin alleviates MAFLD:The hepatic expression levels of irisin in MC group were significantly lower than that of the mice in NC group(P < 0.05).However,treadmill running could induce the expression of irisin in serum and liver tissues of db/db mice(P < 0.001,P < 0.001),which could reverse the low-level irisin in db/db mice caused by obesity.After 8weeks of treadmill running,the body weight(P < 0.05),the levels of TG(P < 0.001),TC(P < 0.01),AST(P < 0.01)and ALT(P < 0.05)in liver tissues were significantly increased in MC group when compared with those in the NC group.Whereas,treadmill running significantly decreased the body weight(P < 0.0001),and the levels of TG(P < 0.05),TC(P < 0.05),AST(P < 0.05),and ALT(P < 0.05)in liver tissue when compared to those in the db/db mice.Notable steatosis of hepatocytes accompanied by hepatocytes ballooning and lobular inflammation was revealed by HE staining in the MC group(P < 0.01),while treadmill running improved the hepatic steatosis,hepatocytes ballooning,and lobular inflammation.Likewise,the NAS score was significantly higher in the MC group when compared with that in the NC group.HE staining in the ME groups showed a lower NAS score when compared with the MC group(P < 0.0001).Oil Red staining also confirmed hepatic steatosis and increased lipid deposition in the MC group,whereas the number of lipid-loaded hepatocytes was significantly decreased in mice upon treadmill running.In db/db mice,treadmill running significantly upregulated AMPK phosphorylation(P < 0.01)and PGC-1α expression(P < 0.001)in liver.2.Exogenous recombinant irisin improves MAFLD:Based on the observation from the appearance of liver tissues collected from the rats at the end of the experiments,HFD-induced fatty liver rats revealed the larger and fatter livers when compared with the normal rats.Interestingly,after 5 weeks of irisin treatment,the body weight(P < 0.01),fasting blood glucose(P < 0.01),the levels of TG(P < 0.01),TC(P < 0.01),AST(P < 0.01)and ALT(P < 0.01)in serum and liver tissues were significantly increased in the rats from HFD-induced fatty liver group when compared with those in the NC group.Whereas,irisin treatment significantly decreased the body weight(P < 0.01),fasting blood glucose,and the levels of TG(P< 0.01),TC(P < 0.01),AST(P < 0.01),and ALT(P < 0.01)in serum and liver tissues when compared to those in HFD-induced fatty live model rats.Notable steatosis of hepatocytes accompanied by hepatocyte ballooning and lobular inflammation was revealed by HE staining in liver tissues of the rats from the FL group,while irisin treatment rescued hepatic steatosis,hepatocyte ballooning,and lobular inflammation.Likewise,the NAS score was significantly higher in the FL group when compared with the NC group(P < 0.0001)and HE staining of the liver tissues in FI group showed a lower NAS score when compared with the FL group(P < 0.01).Oil Red staining also confirmed hepatic steatosis and increased lipid deposition in the FL group(P < 0.001),whereas the number of lipid-loaded hepatocytes were significantly decreased in irisin-treated rats(P < 0.01).3.Possible mechanism of exogenous irisin for treating MAFLD:Our results showed significantly lower activity of SOD and higher level of MDA in the MAFLD model rats when compared to the rats from the NC group(P < 0.01).Compared with the FL group,the activity of SOD(P < 0.01)was increased and the level of MDA was decreased in the FI group.In rats with HFD-induced fatty liver,irisin significantly suppressed the expression of SREBP1(P < 0.01)and ACC1(P <0.01),enhanced HSL(P < 0.01)and ATGL expression(P < 0.01),and up-regulated PGC1α,PPARα and PPARγ expression(P < 0.01).Irisin also significantly increased SIRT1 and phosphorylated AMPK(P < 0.01)and inhibited the phosphorylation of mTOR(P < 0.01)when compared with the FL group.Irisin could significantly increase LC3-Ⅱ/LC3-Ⅰ ratio and Beclin1 expression(P < 0.001)and downregulate p62(P < 0.05);in contrast,irisin significantly suppressed the expression of Bax,cleaved-Caspase-3 and Caspase-8 as well as Bax/Bcl2 ratio(P < 0.01),and increased the expression of Bcl2(P < 0.001).Irisin also upregulated PI3 K and Akt in hepatocytes.Furthermore,irisin significantly reduced the expression of TNF-α(P <0.01),IL-6(P < 0.01)and NF-κB(P < 0.01)in the liver of rats with HFD-induced fatty liver.Conclusion:1.Treadmill running can induce the expression of irisin in the liver tissues of db/db mice,and alleviate the fatty liver disease,which may be correlated to the upregulation of AMPK and PGC-1α in liver tissue,and enhanced energy metabolism and lipid homeostasis.2.Exercise-induced irisin is able to alleviate MAFLD in HFD-fed rats through suppressing metabolic regulators.The upregulation of hepatic irisin in MAFLD models could protect against lipid accumulation,insulin resistance and hepatic cell injury in hepatocytes.3.The underlying mechanisms associated with the alleviation of irisin-mediated MAFLD may be correlated with the activated AMPK in liver tissues,thereby resulting in the reduction of de novo lipogenesis,the promotion of fatty acid oxidation,the induction of autophagy and the suppression of apoptosis.However,the additional studies to better understand the regulatory role of secreted irisin are needed to provide the more solid evidence for irisin as the potential therapeutic target and drug candidate.