Effects Of Nucleoside Reverse Transcriptase Inhibitors On The Proliferation, Differentiation And Secretion Of Cytokines In 3T3-L1 Preadipocytes

Objective:1,To study the effects of D4T,AZT and DDI on proliferation and differentiation in 3T3-L1 preadipocytes.2,To study the effects of D4T,AZT and DDI on IGF-1 production by cultured proliferating preadipocytes and differentiating adipocytes.To study the effects of D4T,AZT and DDI on adiponectin secretion by differentiated adipocytes.To investigate the pathogenesis of highly active antiretroviral therapy(HAART) associated lipoatrophy based on above studies.Methods:1,3T3-L1 preadipocytes dying with CFSE were cultured in DMEM/F12 growth medium containing D4T,AZT or DDI at different concentrations(0.1μg/ml,1μg/ml,10μg/ml).Flow cytometry was used to measure the proliferation index of preadipocytes in treated and control groups.2,3T3-L1 preadipocytes were cultured with D4T(1μg/ml,4μg/ml), AZT(0.1μg/ml,1μg/ml) or DDI(1μg/ml,10μg/ml).MTT assay was applied to evaluate growth of preadipocytes.3,3T3-L1 preadipocytes were induced to differentiate in both treated groups containing D4T,AZT or DDI at different concentrations(0.1μg/ml,1μg/ml,10μg/ml) and control groups.Oilred O staining was used to assess lipid accumulation.4,ELISA was designed to measure IGF-1 and adiponectin levels in cell culture supernatants during the course of proliferation and differentiation.Results:1,It was shown by CFSE with flow cytometry that D4T in different concentrations(0.1μg/ml,1μg/ml,10μg/ml) could make proliferation index decrease 19.1%,39.8%,50.8%respectively compared with control group.The decreased levels by AZT interfering groups(0.1μg/ml,1μg/ml,10μg/ml) were 6.7%,18.1%,30.9%respectively.The fact that D4T and AZT were able to attenuate the proliferation ability of 3T3-L1 preadipocyte was also confirmed by MTT method.2,D4T and AZT in high concentration could also inhibit the differentiation ability of 3T3-L1 preadipocyte,then make less mature adipocyte.3,It was also discovered by ELISA testing that D4T in different concentrations(0.1μg/ml,1μg/ml,10μg/ml) could make the concentration of IGF-1 decrease 21.5%,31.7%,48.7%respectively in proliferation phrase compared with control group.D4T could also low down the IGF-1 secretion by mature adipocyte in the 8^th day of differentiation.The secretion decrease, which was concentration-dependent,were 10.5%,30.7%,42.6%respectively. AZT in different concentrations(0.1μg/ml,1μg/ml,10μg/ml) could only make the concentration of IGF-1 decrease 8.6%,21.7%,29.5%respectively in proliferation phrase compared with control group.4,Adiponectin secreted by mature adipocyte was also decreased in D4T in 10μg/ml and AZT in 10μg/ml interfering groups compared with control group.5,DDI had no effect on proliferation,differentiation and production of IGF-1 and APN in 3T3-L1 preadipocytes.Conclusion:1,Our study discovers that D4T and AZT impair the proliferative activity of 3T3-L1 preadipocytes dose-dependently by flow cytometry and MTT assay.2,We confirm that D4T and AZT induce the less number of mature adipocytes by impairing the differentiation of 3T3-L1 preadipocytes.3,To our knowledge,in vitro experiments our study is the first to discover that D4T and AZT impair the production of IGF-1 in proliferating preadipocytes,and D4T impairs the production of IGF-1 in mature adipocytes.4,It is confirmed that D4T and AZT can lead to the reduction of adiponectin in supernanants of differentiated adipocytes.So,D4T and AZT might induce lipoatrophy by bothering the proliferation, differentiation of preadipocytes and the secretion of adipokines.