The Molecular Mechanisms Of Perilipin 5 In Regulating Hepatic Lipid Droplet Metabolism

Non-alcoholic fatty liver disease(NAFLD),which is mainly characterized as diffuse hepatic steatosis,is recognized as the liver manifestation of metabolic syndrome.Despite the close relationship between the excess hepatic lipid accumulation and the development of NAFLD,current studies have suggested that neutral lipids in hepatocytes are not the direct reason for non-alcoholic steatohepatitis(NASH),while lipotoxicity induced by excess free fatty acids(FFAs)in hepatocytes is the key mechanism underlying the occurrence of NASH.The excess FFAs are physically stored in lipid droplets(LDs)in the form of triglyceride(TG)in hepatocytes.Therefore,close relationship exists between abnormalities of LDs metabolism and lipotoxic liver injury.Once perceived as relatively simple storage particles for neutral lipids,LDs are now widely recognized as multifunctional dynamic organelles.LDs play crucial roles in cellular lipid accumulation,energy supply,membrane formation,viral replication and protein degradation.LDs are composed of neutral lipids as hydrophobic core,which is surrounded with a phospholipid monolayer and a variety of proteins named as lipid droplet-associated proteins(LDPs).Perilipin family members are the most important LDPs,and play critical roles in regulating lipid metabolism and maintaining intracellular lipid homeostasis.As the latest discovered member of Perilipin family,Perilipin 5(Plin5)is primarily abundant in oxidative tissues,including liver,heart,skeletal muscle and brown adipose tissue(BAT).Currently,a number of studies have investigated the role of Plin5 in heart and skeletal muscle.Using knockout mice,our previous studies showed no significant changes in the general physiological parameters in Plin5-null mice,including growth and reproduction processes,body weight,serum levels of glucose,TG and cholesterol,as well as insulin sensitivity,whereas Plin5-null mice exhibited reduced hepatic lipid contents and liver injury.However,the specific role of Plin5 in hepatic lipid metabolism and possible mechanisms still remains unrevealed.In this study,human liver specimens and fatty liver mouse models were used to determine the relationship between the expression levels of Plin5 and the development of fatty liver.Then,Plin5-null mice and in vitro overexpression models were established to further determine the effects of Plin5 deficiency on liver morphology and hepatic lipid metabolism,and to clarify the molecular mechanism of Plin5 in regulating the hydrolysis of hepatic LD.Finally,we investigated the interaction between Plin5 and Cideb,as well as their effects on LDs morphology in hepatocytes.1)The expression of Plin5 was increased in steatotic liversGenerally,Plin5 in liver is mainly distributed around the central veins of hepatic lobule,which is a common site for hepatic steatosis induced by metabolic abnormality.Using immunohistochemistry,we found that Plin5 expression was significantly elevated in the liver tissues of simple steatosis and NASH patients,which was closely related to the development of NAFLD.Moreover,Plin5 expression was also significantly elevated in the livers of ob/ob mice and steatotic livers induced by high-fat diet(HFD)or fasting,further indicating that the expression of Plin5 was closely related to the development of fatty liver.Although Plin2 expression was also elevated in steatotic livers,we found that the increasing extent of Plin5 expression were significantly higher than that of Plin2 in LDs derived from steatotic livers induced by fasting,suggesting that Plin5 plays a more important role in hepatic LD accumulation.2)Plin5 deficiency induced lipotoxic liver injuryTo clarify the specific role of Plin5 in hepatic lipid metabolism,Plin5-null mice and primary hepatocytes were used to analyze the effects of Plin5 deficiency on oxidative stress,endoplasmic reticulum(ER)stress,inflammation,fibrosis,and mitochondrial function in mice livers.We found that Plin5 deficiency resulted in significantly increased rate of lipolysis in hepatocytes and elevated levels of intracellular FFA and serum ketone bodies.Plin5 deficiency also resulted in significantly elevated levels of reactive oxygen species(ROS)in mice livers and subsequently led to lipid peroxidation,oxidative stress,ER stress and significant inflammatory responses,suggesting Plin5 deficiency induced lipotoxic liver injury.In addition,PPAR? was activated in Plin5 deficient livers,and the expression levels of key molecules involved in mitochondrial respiratory chain complex were increased as well,indicating that Plin5 deficiency enhanced mitochondrial oxidative capacity.Together,these results indicated that the increased intracellular FFA induced by Plin5 deficiency resulted in activated of PPAR? and accelerated FFA oxidation in hepatocytes,in order to attenuate lipotoxic liver injury induced by excess FFA oxidation.3)Plin5 inhibited ATGL lipolysis activity through interaction with CGI-58To determine the molecular mechanism of Plin5 in regulating the hydrolysis of hepatic LD,we examined the expression of the Perilipin family proteins and the key molecules involved in lipid synthesis and lipolysis in Plin5-deficient mice livers.The results showed that the expression of Plin2 but not Plin3 was significantly reduced by Plin5 deficiency in livers.The expression of FAS and ACC,the key enzymes regulating FA synthesis,were significantly down-regulated in Plin5-deficient livers,but Plin5 deficiency didn't affect the expression of DGAT2,the key enzyme catalyzing TG synthesis,as well as the expression of lipolytic enzyme ATGL and its co-activator CGI-58.Further study showed that Plin5 interacted with CGI-58,and the C-terminal of Plin5(212-382aa)was essential for the interaction between Plin5 and CGI-58.Moreover,we found that interaction of Plin5 and CGI-58 disrupted the binding between CGI-58 and ATGL,and the lipolytic rate of ATGL was significantly increased in Plin5-deficient hepatocytes,especially in ATGL and/or CGI-58 overexpression cells.These results indicated that Plin5 could disrupt the binding between CGI-58 and ATGL through interaction with CGI-58 in hepatocytes,thereby inhibiting the lipolytic activity of ATGL.4)Plin5 interacted with Cideb,exerting differential effects in the process of hepatic LD formationIn addition to lipolysis,the formation and transport of LDs also play important roles in cellular lipid metabolism.Our previous study showed that Plin5 may interact with Cideb.As the most abundant CIDE family protein in liver,Cideb plays a crucial role in hepatic LD formation.To further investigate the specific roles of Plin5 and Cideb in hepatic LD formation,knock-out mice were used to determine the effects of Plin5 or Cideb deletion on the expression of LDPs in liver.Results showed that Plin5 expression was significantly increased in Cideb-deficient livers,while Cideb expression was also slightly increased in Plin5-deficient livers.Plin2 expression was significantly reduced or increased in Plin5-or Cideb-deficient livers,respectively,which was positively correlated with hepatic lipid content.Plin5 deficiency resulted in reduced hepatic lipid content,which is characterized as decreased LDs numbers,and overexpressed Cideb in Plin5-deficient hepatocytes could not recover the impaired lipid content.However,Plin5 overexpression in Cideb-deficient hepatocytes resulted in significantly increased LDs number rather than LDs size,exhibiting more small LDs storage.Using immunofluorescence,we confirmed the co-localization between Plin5 and Cideb.Moreover,Plin5 interacted with Cideb,and the C-terminal of Cideb(118-219aa)was essential for its interaction with Plin5.Thus,we deduce that,different from the promotion effects on the fusion and enlargement of LDs by Cideb,Plin5 could increase LDs number in hepatocytes,indicating the involvement of Plin5 in the generation of hepatic LDs.However,the specific role of interaction between Plin5 and Cideb in the generation of hepatic LDs remains to be further investigated.In summary,Plin5 expression is closely related to the development of NAFLD.Plin5 disrupts the binding between CGI-58 and ATGL through interacting with CGI-58 in hepatocytes,thereby inhibiting the lipolytic activity of ATGL and reducing LDs lipolysis.In Plin5-deficient hepatocytes,the lipolytic activity of ATGL could be increased by binding to CGI-58 on the surface of LDs,resulting in reduced LDs content and increased intracellular FFA content.Subsequently,increased intracellular FFA activates PPAR?,resulting in the increased expression of a series of key molecules involved in lipid metabolism.Excess intracellular FFA could be removed by mitochondrial proliferation,enhanced oxidative capacity and increased ? oxidation.Moreover,excess intracellular FFA oxidation induces ROS production,lipid peroxidation,oxidative stress,ER stress and inflammation,and eventually leads to lipotoxic liver injury.In terms of LDs formation,Plin5 or Cideb deficiency promotes mutual expression and Plin5 also interacts with Cideb.Moreover,Plin5 increases the number of LDs,while Cideb promotes the fusion and enlargement of LDs in hepatocytes,both of which participates in the process of hepatic LDs formation.However,the specific role of interaction between Plin5 and Cideb in the generation of hepatic LDs remains to be further investigated.