Effects Of Zinc Alpha2 Glycoprotein On Adipocyte Mitochondrial Synthesis In Ob / Ob Mice Liver

Objective: To examine the expression of ZAG and lipid metabolism-related genes in the liver in ob/ob mice and explore the relationship between ZAG and development of NAFLD.Methods:All of 36 male wild type C57BL/6 mice and 22 ob/ob mice aged 8 weeks were fed with standard diet, 2-4 mice in each cage. During the period of feeding, mice bedding material was replaced every other day, fodder and water were added every day. The general states, such as body size, hair, activities, food intake, water intake and urine volume were observed. 2 weeks later, according to their fasting weight, 8 mice were randomly chosen from wild type as WT group and the same number mice were randomly chosen from the ob/ob mice as ob/ob group. The serum and liver of mice were taken for detection. The weight and liver index of two mice groups were detected and the content of TG in liver, the content of ALT/AST in serum were detected; Histopathological changes of liver tissue were observed by H&E staining; ZAG m RNA expression level of liver was assessed by RT-q PCR; Furthermore, The protein expression of ZAG and lipid metabolism-related genes of liver were detected by Western blotting.Results:Compared with WT group, ob/ob mice displayed larger body size. The level of weight, liver index were significantly increased in ob/ob mice(P?0.01); The liver volume of obese subjects, which owned the histologic appearance of hepatic fatty change, were overtly bigger than these lean subjects; Moreover, The content of TG in liver and the content of ALT/AST in serum were significantly increased in ob/ob mice(P?0.01). ZAG m RNA synthesis in the liver was downregulated(P?0.05), as were its protein levels(P?0.01), in ob/ob mice. The protein expression of liver FXR and CPT1, which were involved in lipolysis, were decreased(P?0.05), and the PPARa were decreased obviously in ob/ob mice; However, lipid synthesis-related genes, such as LXRa, SREBP-1c, FAS and ACCa were all strongly increased on protein level(P?0.01). Interestingly, protein expression of GAPDH, which has long been known as one of the important glycolytic enzymes, was much higher in obese subjects with NAFLD than subjects without NAFLD(P?0.01).Conclusion:The expression of ZAG and lipolysis-related genes, such as FXR, PPARα and CPT1, were decreased in the liver in ob/ob mice; However, lipogenesis-related genes, such as LXRα, SREBP-1c, FAS and ACCα, have the opposite tendencies.Objective: To observe the effects of recombinant adeno-associated virus-2 mediated overexpression of Zinc-alpha2 glycoprotein on relevant factors of mitochondria biogenesis in adipocyte.Methods:1.Culture and differentiafion of 3T3-Ll preadipocytes: The 3T3-L1 cells were incubated in high glucose medium containing 10% FBS under an atmosphere with 5%CO2, at 37℃, and inoculated into 6-well plate. The cells were incubated for another two days when they reached 100% confluence. The induced differentiation, named “Cocktail”, followed the protocol of medium I for 3 days,medium II for 3 days and the fresh medium II for 2 days.Then, the accumulation of TG in adipocytes were assayed by oil red O staining, and morphological changes of the adipocytes were determined by the inverted microscope. 2.Determined the time of infection and the multiplicity of infection(MOI):r AAV2-EGFP were used to infect adipocytes under the conditions of various MOI. After infection, observed the number of fluorescent cells by the fluorescence microscope every 24 hours. 3.Adipocytes were respectively re-infected with the r AAV2-ZAG and r AAV2-EGFP for 72 hours under the best conditions, and were assayed by oil red O staining. What’s more, the protein expression of ZAG, UCP-1 and relevant factors of mitochondria biogenesis in adipocytes were detected by Western blotting.Results:1. On the 4th day, a small amount of lipid droplets were observed in the cytoplasm under inverted microscope. On the 6th day, the cells changed to round gradually, and it was clear that the lipid droplets merged with each other inside the cells. On the 10 th day, more than 80% of 3T3-L1 cells had differentiated into adipocyte cells totally, which were round or ellipse in shape and a large amount of lipid droplets were observed, gathered around the nucleus in the cytoplasm, formed a typical "ring ring" structure. Oil red O staining indicated that lipid droplets were stained orange clearly. 2. It demonstrated that the number of green fluorescence-labeled cells were the most when took MOI 5 x 105 v.g./cell and infected 72 h. Further studies conclusively showed the expression of ZAG gene was at the peak under this condition.3. Oil red O staining indicated that compared with r AAV2-EGFP-infected cell, large lipid drops were replaced by the small one in the r AAV2-ZAG group. The interest protein was expressed successfully in adipocytes(P?0.05). The results of western blotting exhibited that protein expression of UCP-1 was increased(P?0.01), and similar results were obtained for the relevant factors of mitochondria biogenesis such as PGC-1a(P?0.01), NRF-1(P?0.01), NRF-2(P?0.05)and mt TFA(P?0.05).Conclusion:The lipid accumulation in adipocytes could be rescued by overexpression of the relevant factors of mitochondria biogenesis, and this could be attributable to the role of ZAG.