The Role And Preliminary Mechanism Of LSDP5in Mice Lipotoxic Liver Injury

Current studies have suggested that lipotoxicity may play a key role in theprogression of the nonalcoholic steatohepatitis (NASH) generated from simple fatty liver.Abnormalities of non-esterified fatty acids (NEFAs) and the relative derivatives have beenreported to be the crucial factors inducing intracellular lipotoxic injury. Normally, NEFAsare stored in lipid droplets (LD) in the form of triglyceride (TG), therefore, closerelationship exists between abnormalities of LD metabolism and lipotoxic liver injury. Asis well accepted, LDs are metabolic-active “subcellular organelles” and play critical rolesin the process of lipid storage and metabolism, which is regulated by lipiddroplet-associated proteins. LSDP5, the fifth member of the PAT family, which is the mostimportant class of lipid droplet-associated proteins, is primarily expressed in oxidativetissues, including the liver, heart, skeletal muscle and BAT. Thus, LSDP5is also known as perilipin5, OXPAT or MLDP. However, the definite physiological function andmechanism of LSDP5in liver still remains unknown.In this study, LSDP5knock-out mice were generated as the animal model. The mainchanges of phenotypes, liver morphology and physiological function of the LSDP5knock-out mice were observed. Moreover, the effects of LSDP5deficiency on theexpression of LD-and mitochondria-associated molecules were also examined. Finally,the role and preliminary mechanism of LSDP5in lipotoxic liver injury were discussed.The results of this study could show us a deeper understanding about the relationshipbetween the metabolic process of lipid droplets and lipotoxic liver injury, and they alsohave great significance to the clinical diagnosis and treatment of fatty liver and NASH.1. LSDP5knock-out mice exhibited liver injury.Through analyzing the growth and reproduction conditions and genotypes of LSDP5knock-out mice, we found no changes in the growth and reproduction processes.Meanwhile, we neither found any obvious changes of the body weight, insulin sensitivity(GTT and ITT) and the weight of important organs in LSDP5knock-out mice. In addition,no obvious changes of the serum level of glucose, lipid, total protein and albumin werefound between normal diet (ND) and high-fat diet (HFD) groups in LSDP5knock-outmice. However, as shown in the results of the blood biochemistry analysis, LSDP5knock-out mice presented an apparent increase in the serum level of ALT, AST, DB andTB, which indicated that LSDP5deficiency could induce liver injury.2. LSDP5knock-out mice exhibited decreased TG content yet increased mitochondriaproliferation in the liver.To determine the main performance of LSDP5deficiency-induced liver injury inmice, histological methods were applied to analyze the hepatic morphological changes inLSDP5knock-out mice. The results of HE staining showed that LSDP5knock-out miceexhibited injurious changes, such as the disorder of hepatic plates and non-uniform nucleus size in the liver tissue, which were more obvious in LSDP5knock-out mice withHFD. The results of Oil Red O staining showed that, compared with wild-type ones,hepatic TG content of LSDP5knock-out mice with ND or HFD were both significantlyreduced. Meanwhile, the quantity and size of the LDs in the cultured LSDP5-deficiencyhepatocytes were both decreased. The results of electron microscope showed that themitochondria in the liver tissue of LSDP5knock-out mice were increased in quantity yetdecreased in size, which was confirmed by MitoTracker staining in LSDP5-deficiencyhepatocytes. Moreover, the expression of mitochondria markers, Cox IV and CytochromeC, were also increased. These results indicated that LSDP5deficiency could suppress lipidaccumulation and promote mitochondria proliferation in hepatocytes.3. LSDP5knock-out mice exhibited increased fatty acid oxidation and subsequentlylipid peroxidation injury in hepatocytes.To determine the changes of hepatic lipid metabolism in LSDP5knock-out mice,3Hlabeled oleic acid (Oleic acid-[9,10-3H]) was used to detect the rate of fatty acid oxidation,TG synthesis and secretion in LSDP5-deficiency hepatocytes. We found that the rate offatty acid β-oxidation was increased and TG storage and secretion were both decreased.Furthermore, we found that, in LSDP5knock-out mice with ND and HFD, the levels ofMDA were both elevated significantly, but those of the anti-oxidant SOD were bothreduced. JC-1staining revealed a reduction in mitochondrial membrane potential ofLSDP5-deficiency hepatocytes. To sum up, we assumed that LSDP5deficiency could leadto mitochondria proliferation and accelerated fatty acid oxidation, and finally result inlipid peroxidation, which may be an important explanation for liver injury in LSDP5knock-out mice.4. LSDP5was localized to the surface of LDs, and LSDP5knock-out mice exhibitedincreased exprssion of PPARα and fatty acid decomposition-ralated molecules butdeclined exprssion of lipid biosynthesis-ralated molecules.To further confirm the LDs localization characteristics of LSDP5, we used the techniques of subcellular organelles isolation and GFP fusion protein, and found thatLSDP5was mainly located on the surface of LDs and partially exited in cytosol.Meanwhile, the N-terminal region (1-128aa) of LSDP5was determined as the LDtargeting domain. Based on the results of microarry analysis, Real-time PCR was used toanalyze the expression of the hepatic lipid metabolism-ralated molecules in LSDP5knock-out mice, and the results suggested that the expression of ADRP was significantlydecreased while that of TIP47was increased. Moreover, the expression of themitochondria-related molecules, including Cox IV, Cytochrome C and CPT1a, were allsignificantly increased, while that of the key lipid synthesis-related molecules, includingACC1, ACC2, FAS, exhibited different degrees of decline. More importantly, we foundthat, PPARα, the key regulatory molecule in lipid metabolism of hepatocytes, wassignificantly overexpressed at both mRNA and protein levels. These results indicated thatLSDP5knock-out mice showed suppressed lipid synthesis but enhanced lipolysis process,and the increased expression of PPARα may be the crucial regulatory mechanism resultingin the changes of hepatic lipid metabolism in LSDP5knock-out mice.In this study, we found that LSDP5deficiency could result in lipotoxic liver injury byusing LSDP5knock-out mice model. We concluded that, LSDP5, as an important memberof the PAT family, was located both on the surface of LDs and in cytosol, and couldsuppress TG lipolysis in LDs. In LSDP5-deficiency hepatocytes, lipolysis of LDs wasaccelerated, which could reduce the hepatocellular lipid storage, increase the intracellularNEFA content and finally activate PPARα. Eventually, activated PPARα could promotemitochondria proliferation and accelerate fatty acid oxidation rate, resulting in excessiveROS in hepatocytes and subsequently lipotoxic liver injury. In the further studies, we willcontinue to explore the specific role and molecular mechanism of LSDP5in the process ofLDs metabolic regulation.