The Roles And Mechanism Of Lipid Droplet Protein Plin5 In Inhibiting Hepatic Lipid Accumulation By Atorvastatin

Non-alcoholic fatty liver disease(NAFLD)is characterized by diffuse fatty degeneration and lipid accumulation in liver cells due to causes other than excessive alcohol use.Excessive lipid accumulation in liver cells is closely related to the development of NAFLD,which has affected about 1/3 of the population in developed countries and is becomig the most common cause of chronic liver diseases.With the improving of our living,the incidence of NAFLD has also increased rapidly.NAFLD will become the leading cause of cirrhosis and hepatocellular carcinoma,with the extensive use of hepatitis vaccination and the improvement of treatments.When abnormal lipid accumulation exceeds the compensatory capacity of liver cells,the excessive lipids and their metabolites produce a series of damages on hepatocytes,which is called lipotoxic injuries,including oxidative stress,inflammation and endoplasmic reticulum stress.,which could promote NAFLD progression from simple fatty liver to non-alcoholic steatohepatitis,hepatic fibrosis,cirrhosis,and even malignant transformation.In mammalian cells,excessive lipids are usually stored in lipid droplets(LDs)in the form of neutral lipids [mainly including cholesteryl esters(CE)and triglycerides(TG)].Lipid droplets are an important “buffer system” in the lipid metabolism of liver cells and play crucial roles in maintaining the intracellular lipid homeostasis.Furthermore,the enlarged and redundant lipid droplets are the important pathological hallmark of NAFLD,and the number and size of hepatic lipid droplets are closely related to the progression of NAFLD.For a long time,lipid droplets are considered as a simple structure for storing neutral lipids.In recent years,more and more researches shows that lipid droplets are multifunctional metabolically active organelles that participate not only in the accumulation of intracellular lipids,but also in various metabolic processes,such as energy supply and cell membrane biosynthesis.The multifunctions of lipid droplets are regulated by the proteins archored on their surfaces,of which Perilipins are the important markers of lipid droplets.As a key lipid droplet protein,Plin5 plays a very important role in regulating of the metabolism of lipid droplets and maintaining the balance of lipid metabolism in cell.Plin5 is abundantly expressed in the oxidative tissues,such as liver,heart,skeletal muscle and brown adipose tissue(BAT),in both mice and humans.Our previous studies showed the hepatic lipid contents in Plin5 knockout mice was significantly reduced,indicating that Plin5 was envolved in regulating hepatic lipid metabolism.Studies have also shown that Plin5 could inhibit lipolysis of triglycerides in lipid droplets and/or reduce the fatty acid oxidation,and promote intracellular lipid accumulation.However,their specific regulatory functions and mechanisms in the liver diseases should be further clarified.Statins are the representative lipid-regulating drugs,are most widely used in clinical practice.Statins can inhibit the activity of hydroxymethylglutaryl coenzyme A reductase(HMGCR)and are mainly used to decrease blood cholesterol levels.Statins are widely used in the treatment of dyslipidemia,such as hypercholesterolemia and atherosclerosis.With a wide range of applications,the pleiotropic effects of statins have also been gradually observed,such as reducing oxidative stress,inflammation,and improving endothelial function,in addition to lipid-lowering effects.Recently,more and more studies suggest that statins could inhibit liver inflammation,fibrosis,and even have protective effects on the development of hepatocellular carcinoma.Recently,it was reported that statins could inhibited the expression of Plin5 in liver,resulting in elevating the lipolysis of LDs and reducing the intracellular lipid accmulation,but the detail functions and mechanism of Plin5 should be further investigated.Aims:1.To investigate the effect of atorvastatin on liver lipid accumulation in patients with NAFLD.2.To investigate the roles of lipid droplet protein Plin5 in regulating hepatic lipid contents with atorvastatin in Plin5 knockout mice.3.To clarify the molecular mechanism of Plin5 phosphorylation in the hepatic lipid-lowering effects of atorvastatin.Methods;1.According to inclusion and exclusion criteria,we collected and analyzed 101 patients with fatty liver who received atorvastatin calcium treatment for 6 months.The patients were divided into normal weight group(18.5≤BMI<24,n=48)and overweight group(BMI≥24,n=53),according to the body mass index.Then we analyzed the effect of atorvastatin on hepatic lipid accumulation in the patients with fatty liver.2.To further study the role of Plin5 in lowering hepatic lipid accumulation by atorvastatin,high-fat diet(HFD)was used to stimulate liver lipid accumulation in mice.These mice were then administered with atorvastatin by gavage,and the effects of atorvastatin on hepatic lipid accumulation and Plin5 expression were observed.Moreover,the wild-type and Plin5-deficient mice and hepatocytes were used to investigate the roles of Plin5 in the hepatic lipid-lowering effects of atorvastatin in vivo and in vitro.3.By separating the lipid droplets and the cytosolic fraction,the changes of Plin5 in lipid droplets and cytoplasm were analyzed after treatment with atorvastatin.To clarify the relationship of atorvastatin and Plin5,we further analyzed the specific effects of atorvastatin on the lipid droplets hydrolysis and fatty acid oxidation.Finally,we systematically analyzed the phosphorylation domains in Plin5,including protein kinase A(PKA),protein kinase B(PKB)/AKT and AMP-activated protein kinase(AMPK),to clarify the relationship between atorvastatin and Plin5 phosphorylation.Results:(1)Atorvastatin reduces hepatic lipid accumulation in the patients with fatty liver.To determine the effect of atorvastatin on fatty liver,we collected and analyzed 101 patients with fatty livers from the patients who received atorvastatin calcium treatment for 6 months.The results showed that after 6 months of treatment with atorvastatin calcium,the levels of blood glutamic-pyruvic transaminase,aspartate transaminase,total bilirubin,direct bilirubin and indirect bilirubin were unchanged,but the levels of plasm triglyceride and total cholesterol were decreased compared to these before treatment,with a more significant effect in overweight patients(BMI ≥24).We further analyzed the effects of atorvastatin on severity of fatty liver,and the results showed that atorvastatin calcium could significantly reduce the severity of fatty liver in patients.Meanwhile,atorvastatin calcium had a better therapeutic effect on fatty liver in overweight patients.In summary,atorvastatin calcium could significantly improve liver lipid accumulation in patients with fatty liver.(2)Atorvastatin reduces hepatic lipid accumulation by inhibiting Plin5 expressionIt was proven that Plin5 could promote lipid accumulation in liver,and Plin5 deficiency could stimulate the lipolysis of LDs and induce the hepatic lipotoxicity.To further study the role of Plin5 in lowering hepatic lipid accumulation by atorvastatin,high-fat diet(HFD)was used to stimulate liver lipid accumulation in mice.These mice were then administered with atorvastatin by gavage,and the effects of atorvastatin on hepatic lipid accumulation and Plin5 expression were observed.Moreover,the wild-type and Plin5-deficient mice and hepatocytes were used to investigate the roles of Plin5 in the hepatic lipid-lowering effects of atorvastatin in vivo and in vitro.The results showed that atorvastatin could effectively reduce the liver triglyceride(TG)and cholesterol contents.In addition to lipid-lowering effects,atorvastatin also reduce the expression of hepatic Plin5,but not the other lipid droplet proteins,including adipose differentiation-related proteins(ADRP).More importantly,the lipid-lowering effects of atorvastatin were dimished in Plin5-deficient mice and hepatocyts,although it could still effectively decrease cholesterol levels in liver.These results suggestted that the Plin5 might play an important role in the hepatic lipid-lowering effects of atrovastatin.(3)Atorvastatin increases the lipolysis of hepatic lipid droplets by stimulating the phosphorylation of Plin5To clarify the roles and mechanism of lipid droplet protein Plin5 in hepatic lipid accumulation inhibited by atorvastatin,we first analyzed the distribution of Plin5 in lipid droplets and cytoplasm.The results showed that atorvastatin could significantly reduce the expression of Plin5 in cytoplasm,without directly affecting the contents of Plin5 protein on lipid droplet fractions.We further analyzed the specific effects of atorvastatin on the TG hydrolysis of lipid droplets and fatty acid oxidation.The result indicated that atorvastatin could promote the lipolysis of lipid droplets and promotes the oxidation of fatty acidswithout affecting the Plin5 protein levels on the lipid droplet.These results suggestted that atorvastatin might inhibit the protective effects of Plin5 on LDs through an alterative pathway other than the protein levels.Phosphorylation is the common regulation of perilipins,and it was reported that Plin5 could be phosphorylated at serine-155.Here,we systematically analyzed the phosphorylation domains in Plin5,including protein kinase A(PKA),protein kinase B(PKB)/AKT and AMP-activated protein kinase(AMPK).The result showed that atorvastatin increased PKA-dependent Plin5 phosphorylation in the livers of high-fat diet(HFD)-induced mice.Taking a further step,it showed that atorvastatin could activated PKA in liver,resulting in the phosphorylation of Plin5 atserine-155.The phosphorylation of Plin5 affected its interaction with CGI-58,and then increased activity of adipose triglyceride lipase(ATGL)on the surface of lipid droplets,thereby reduced lipid accumulation in hepatocytes.The results is not only helpful for us to deeply understand the molecular mechanism of atorvastatin in lowering hepatic lipid accumulation,but also provide a basis for exploring the regulation of Plin5.Conclusion:In summary,we analyzed the clinical data of patients and found that atorvastatin could reduce hepatic lipid contents in patients with fatty liver,and this effect was more obvious in overweight patients.Using Plin5 knockout mice and primary hepatocytes,we found that Plin5 played a crucial roles in lowering the hepatic lipid contents by atorvastatin.Moreover,we investigated the roles and mechanisms of atorvastatin in regulating the lipolysis of hepatic lipid droplets.The results showed that atorvastatin could promote PKA-dependent phosphorylation of Plin5,thereby promoting the hydrolysis of hepatic lipid droplet and reducing lipid accumulation in the liver.This research systematically studied the roles and molecular mechanism of atorvastatin in inhibiting hepatic lipid accumulation.It not only helps to understand the regulatory mechanisms of hepatic lipid metabolism,but also provides experimental basis for the prevention and treatment of NAFLD.