| Cytoplasmic lipid droplets(CLDs), consisting a hydrophobic core filled with neutrallipid inside and single layer of lipid molecules outside, are multifunctional dynamicorganelles resulted from the different functional proteins located in that single layermembrane.Lipid droplets are widely found in many types of cells and physiological disordersof lipid droplets maybe linked to some lipid metabolic diseases and cancers.Milk fat,normally in the form of milk lipid droplets, is one of the main components of milk. Milk lipiddroplets are composed of triacylglycerol mainly and small amount of cholesterol ester withthree layer of phospholipid molecules outside. The process that secretion of milk lipiddroplets from the apical plasma membrane of mammary epithelial cells are at least associatedwith the function of butyrophilin (BTN), xanthine oxidoreductase (XOR) and adiposedifferentiation-related protein (ADRP). ADRP, a member of PAT protein family, is widelyexpressed in many tissues and supposed to be related to process of lipid droplets formation,transshipment,secretion and even intake the long chain fatty acid as a kind of fatty acidbinding protein. ADRP gene is directly regulated by peroxisome proliferator activatedreceptors (PPARs). In this study, we have constructed over-expression adenovirus vectorsbased on goat ADRP gene. And with the help of successfully constructed RNAi recombinantadenovirus targeting goat ADRP gene, we studied the effects and analysed the molecularmechanisms of over-expression and knockdown of ADRP gene on lipid metabolism in goatmammary epithelial cells (GMEC). And we also explored the transcription regulationrelationship between peroxisome proliferator activated receptor gamma (PPARG) and ADRPgene. Main results obtained in this study as follows:1. We successfully constructed over-expression adenovirus vectors based on goatADRP gene (AdEasy-ADRP) and packaged and amplified adenovirus of high titer (109U/mL). And we also detected the over-expression and knockdown efficiency of ADRP genein mRNA, protein and cellular level with the use of Ad-ADRP and Ad-sh511in GMEC,respectively, which ensured the reliability of subsequent study of ADRP gene functionidentification.2. Based on over-expression and knockdown of goat ADRP gene, we found ADRP gene could significantly promote lipid accumulation in GMEC and increase the cellularcontent of triacylglycerol, cholesterol and non-esterified fatty acid. Meanwhile, ADRPfacilitated the intake of exogenous oleic acid and increased the proportion of long-chainunsaturated fatty acids. In addition, ADRP may protect lipid droplets from ATGL-inducedlipolysis as a kind of passive barrier. By the study of submicrostructure of GMEC, we foundADRP may promote the formation of lipid droplets ranged in0.25-0.5μm and the secretionof CLDs, and we also obtained more evidence to support that ADRP could inhibitATGL-induced lipolysis.3. Using Real-time PCR, we found that ADRP gene transcription level rised by severaltimes after over-expression of PPARG by which we speculated that goat ADRP gene maybetranscriptionally regualated by nuclear receptor PPARG. Through cloning of goat ADRP genepromoter2378bp and bioinformatic prediction of transcription factor binding sites, wediscovered a putative PPRE which was not conservative with that on humans and mice. Thenwe constructed luciferase reporter gene vectors including ADRP promoter we cloned andfound that ligand-activated PPARG could significantly enhance the transcriptional activity ofADRP promoter, however, this effect could be abolished after the potential PPRE wassite-directed mutated or deleted. We further designed oligonucleotide probes to conductEMSA to verify PPARG could interact with the PPRE in ADRP gene promoter in vitro，which demonstrated that PPARG could direct regulate transcription of ADRP gene.In conclusion, goat ADRP gene can promote lipid storage in GMEC through inhibitingATGL-induced lipolysis and accelerating intake of exogenous long chain fatty acid. Inaddition, ADRP gene can promote the process of formation and secretion of CLDs inGMEC and be transcriptionally regulated by PPARG. |
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