The Effect Of OXPAT On The Morphology Of Lipid Droplet In Primary Cultured Mouse Hepatocytes

The disorders of lipid metabolism, such as obesity and hyperlipidemia, are becoming the serious diseases which damage the public health. Lipid is one of the major forms of energy storage. In the cells, neutral lipid, such as triglyceride (TG) and cholesterol ester (CE), is mainly stored as lipid droplets (LDs). LDs are found in many kinds of cells, including adipocytes, hepatocytes, adrenal cortical and muscle cells. Their surface is composed of a phospholipid monolayer which is coated with LD associated proteins. LD associated proteins are very important in the lipolysis, synthesis and fusion of LDs, and PAT family includes several proteins which play key roles in lipid metabolism. Perilipin, the first LD associated protein, was found on 1990, and ADRP, TIP47, S3-12, Vimentin, Caveolin-1, and CGI-58 were also found on the surface of lipid droplets. Recently, proteomics was introduced into analyzing LD associated proteins, which promote the identification of new LD associated proteins. Base on the proteomics, the LD associated proteins are divided into three categories: one includes the structural proteins specifically binding to LDs; one includes enzymes and transfer proteins which take part in the lipid metabolism; others are unidentified proteins without enough data. Some research found that perilipin, S3-12, Vimintin and ADRP always stayed on the surface of LDs under basic and activated status, but TIP47, Caveolin-1 and Tubulin are recruited on the LDs only under activated condition. The turnover of LD associated proteins implied that the surface was important platform for the regulation of LD metabolism. OXPAT is the homologue of Perilipin and ADRP, but mainly distributes in the liver, brown adipose tissue and muscle which have large capacities for fatty acid oxidation. OXPAT is a PPAR-induced lipid droplet associated protein that promotes fatty acid utilization.1. In this research, OXPAT gene was cloned from the mouse liver cDNA library with OXPAT specific primers, and the sequence was identical with that reported in Genebank. The plasmid pEGFP-OXPAT expressing the fusion protein of OXPAT and HA-tagged was constructed. The plasmid was introduced into the COS7 cells, and the LDs were stained with Bodipy 493/503. Under the fluorescent microscopy, we found OXPAT localized on the surface of LDs. This result is consistent with the published paper.2. We constructed adenovirus containing OXPAT using AdEasy-1 system. First, the plasmid pShuttle-HA-OXPAT was constructed by subcloning. The linearized pShuttle-HA-OXPAT with Pme I was recombinated with pAdEasy-1 in E coli. BJ5183, and the recombinant adenovirus vector pAd-HA-OXPAT was constructed. The linearized pAd-HA-OXPAT with Pac I was transfected into AD193 cells with Lipofecatmine 2000, and the cytopathologic effect (CPE) occurred on day 9. The package cells were harvested and cracked by frozen-thawed methods, and the adenovirus containing OXPAT was harvested from the lysate. The titer of adenovirus was measured using 293T cells, and the results showed the titer of adenovirus is more than 2×109/ml, and OXPAT could be expressed in 293T cells.3. Our result showed that OXPAT could reduce the content of lipid and the size of LDs in primary cultured hepatocytes. The mouse hepatocytes were isolated by liver perfusion and collagenase digestion, and cultured in vitro. The surviving cells were no less than 95%. The primary cultured hepatocytes were infectected by adenovirus expressing OXPAT, and the expression of OXPAT was detected by Western Blot, and the content of lipid and morphology of lipid droplets were observed. We found that OXPAT could reduce the content of lipid and the sized of LDs in primary mouse hepatocytes.In summary, OXPAT was cloned from the mouse liver cDNA library in the study, and the subcellular localization was identified. The adenovirus expressing OXPAT was constructed, and the primary hepatocytes were isolated and cultured successfully. The effect of OXPAT on the content of lipid and size of lipid droplets were studied. This study makes an experimental foundation for the role and mechanism of OXPAT in lipid metabolism.