TGH Expression And Primary Study On Its Role In Lipolysis In Porcine Adipocyte

Triglyceride hydrolysis is important to energy metabolism. The balanced lipolysis can not only provide energy for organism, but also prevent obese, insulin resistance and other related diseases caused by excess adipose deposition. So the lipolysis regulation is important. Lipases are key factors in lipolysis. TGH is another key lipase involved in lipolysis after HSL. However, most previous studies on TGH focused on rodents , there are few reports on TGH expression and its role in lipolysis in mammals so far as now. It is unknown that whether its role in lipolysis is similar to HSL through stimulation by hormones, or playing its role in the basal condition . Pig is considered as the best model for studying obese, diabetes and other lipid metabolism related diseases. So our study investigated TGH mRNA and protein expression and its role in starvation–induced and Isoproterenol-stimulated lipolysis in porcine primary adipocyte by RT-PCR , Western Blot and lipolytic analysis. Comparing with HSL, we preliminarily learned the role of TGH in lipolysis. Besides, pSilencer-TGH vector was constructed in this study, which would be prepared for next research on TGH function. The main results were summarized as following:1 TGH mRNA and protein expression were detected during porcine primary preadipocyte differentiation. Both TGH mRNA and protein were not detected at day 0 and its expression was increased greatly during differentiation, its mRNA was highest expressed at day 4, whereas its protein was at day 6 ,and its expression became decreased then.2 TGH expression and lipolysis alteration were investigated in starvation-induced condition. TGH mRNA and protein expression were downregulated when the cells were cultured with serum-free midia, while lipolysis was inenhanced . TGH expression decreased while changed into complete media again ,but there found no significant decrease of lipolysis.3 The effect of Isoproterenol on lipolysis and TGH expression in porcine adipocyte were investigated, and analysized the role of TGH in lipolyis. Isoproterenol with 0.1 and 1μmol/L had not change the mass of glycerol release, and Isoproterenol with higher concentration (10,100,200μmol/L) increased it greatly. Beside 0.1μmol/L ,Isoproterenol decreased TG content significantly among groups by dose-dependent manner. Meanwhile, HSL mRNA and protein expression were changed with the Isoproterenol concentration and its time of treatment. But there was few changes of TGH expression during this process.4 Screened the best killing concentration of hygromycin and constructed pSilencer-TGH vector. 200μg/mL is the best killing concentration. After digestion,recovery,ligation, transformation, plasmid extraction, finally pSilencer-TGH vector was proved successfully constructed by sequencing.