Construction Of Recombinant Adenoviral Vector Of SOCS2 And Its Effect On Lipid Metabolism Induced By GH In Porcine Primary Adipocyte

Lipid metabolism is closely related with some human diseases, so it is significant to human health. GH act through a complex signaling network to regulate diverse biological processes which control growth, development and metabolism. Therefore, it is important to reveal the molecular mechanisms of GH regulating lipid metabolism. SOCS2 is a member of suppressor of cytokine signaling (SOCS) family, it mainly participates in the negative regulation of JAK/STAT signaling induced by GH. Porcine is an ideal model of human disease, because porcine is remarkably similar to human biological, metabolic and genetic levels.In this research, to investigate the role of SOCS2 on adipocyte differentiation and lipid metabolism in porcine adipocytes induced by GH, we firstly tested the effect of 500 ng/mL GH on adipocytes differentiation, TG accumulation; and tested the expression of PPARγ, FAS, ATGL, HSL, SOCS2 and SOCS3 mRNA by Real-Time PCR. Then, we constructed a recombinant adenovirus encoding SOCS2 gene (Ad-SOCS2) which was used to infect differentiated adipocytes. The expression and phosphorylation of GH signaling components were determined by Real-Time PCR and Western blotting. The main results were summarized as following:1. 500 ng/mL GH significantly decreased the process of porcine preadipocytes differentiation into mature adipocytes. And TG accumulation enhanced at GH- stimulated 0.5 h (P<0.05), reduced from 8 h. Expression of PPARγ, FAS, ATGL and HSL mRNA incresed during earlier stage of GH treatment. With induced time prolonged, expression of PPARγ, FAS, ATGL and HSL mRNA decreased. However, expressions of ATGL and HSL mRNA are always significantly higher than those in control group. SOCS2 expression increases steadily with time after GH stimulation while the expression of SOCS3 is rapid but transient.2. Restriction enzyme and sequencing demonstrated that the recombinant adenovirus vector encoding SOCS2 gene (Ad-SOCS2) was constructed correctly, after packaging and purifying, the virus titer reached 1.0×10¹⁰ PFU/mL. Recombinant adenovirus encoding SOCS2 gene (Ad-SOCS2) could effectively infect porcine adipocytes and notably enhance SOCS3 expression of porcine adipocytes.3. Overexpression of SOCS2 in porcine adipocytes significantly decreased the GH-induced enhance of PPARγ, FAS, ATGL and HSL mRNA.4. Overexpression of SOCS2 in porcine adipocytes significantly decreased STAT3 and STAT5 mRNA level, GH-stimulated STAT3 and STAT5 tyrosine phosphorylation. In Ad-SOCS2 infected adipocytes SOCS3 mRNA keep in a lower level. So overexpression of SOCS2 inhibited GH signaling in porcine adipocytes.In summary, our research indicated that SOCS2 expression increases steadily with time after GH stimulation while the expression of SOCS3 is rapid but transient. And expression of PPARγ, FAS, ATGL and HSL mRNA increases in porcine adipocytes induced with GH. But overexpression of SOCS2 significantly inhibits expression of PPARγ, FAS, ATGL, HSL and SOCS3 mRNA, GH-stimulated STAT3 and STAT5 tyrosine phosphorylation. Therefore, SOCS2 is an important negative regulator of GH signaling in porcine adipocytes, it may be a newly recognized molecular target for obesity.