The Mechanisms By Which Resveratrol Up-regulates Adiponectin Expression And Promotes The Multimerization Of Adiponectin

Part 1 Effect of RSV on Adiponectin expression and Multimerization in Adipocytes and the Role of Sirtl in the Regulating MechanismObjective:To explore the effect of RSV on adiponectin multimerization and cellular levels in Adipocytes and in vivo and the role of Sirtl in the regulating effect of RSV on adiponectin multimerization and cellular levels.Methods:(A) 3T3-L1 adipocytes were treated with or without resveratrol (RSV) at the different concentrations seperately for 24 hours. (B) 3T3-L1 adipocytes were treated with 50μM RSV for different times as indicated. (C) Adiponectin multimers in lysates from 3T3-L1 adipocytes treated with or without 50μM RSV were separated by gel-filtration chromatography. The expression levels of adiponectin were determined by Western blot. (D) The protein levels of DsbA-L and adiponectin in adipose tissues of the mice fed with normal chow or the RSV-containing diet were determined by Western blot using specific antibodies. (E) 3T3-L1 adipocytes were pre-treated with or without 10μM Sirtinol for 60 minutes, followed by 50μM RSV treatment for 24 hours. The acetylation and protein levels of immunoprecipitated PGC-1αwere determined by Western blot with specific antibodies. (F) The differentiated Sirtl-KO/WT adipocytes were treated with RSV at the indicated concentration for 24 hours. For (A), (B), (E), (F), the expression levels of adiponectin and DsbA-L in cell lysates were determined by Western blot. Data was representatives of 3 independent experiments with similar results.Results:(1) Treating 3T3-L1 adipocytes with RSV led to a time-and dose-dependent increase in the cellular levels of adiponectin and DsbA-L. Gel filtration experiments revealed that RSV treatment significantly increased the HMW form of adiponectin (P<0.05). (2) Consistent with the findings in vitro, RSV treatment significantly enhanced the protein levels of DsbA-L and adiponectin in mouse adipose tissue (P<0.05). (3) Inhibition of Sirt1 by sirtinol, as demonstrated by a marked increase in the acetylation levels of PGC-1a, had no significant effect on RSV-stimulated DsbA-L and adiponectin expression in 3T3-L1 adipocytes. On the other hand, RSV treatment also significantly enhanced the expression levels of DsbA-L and adiponectin in both wild-type and the Sirtl-deficient adipocytes.Conclusion:RSV up-regulates DsbA-L and adiponectin and promotes adiponectin multimerization in 3T3-L1 adipocytes and in vivo. Also, Sirtl is dispensable for RSV-stimulated up-regulation of DsbA-L and adiponectin in adipocytes. Part 2 Role of DsbA-L in RSV-induced Adiponectin Multimerization and Expression in AdipocytesObjective:To explore the role of DsbA-L in the regulating effect of RSV on adiponectin multimerization and cellular levels and the effect of RSV on mRNA levels of adiponectin and DsbA-L in adipocytes.Methods:(A) DsbA-L-suppressed or scramble 3T3-L1 adipocytes were treated with or without 50μM RSV for 24 hours. (B) Differentiated 3T3-CAR adipocytes were infected with adenoviruses encoding GFP plus DsbA-L or GFP alone at MOI=20. After Twenty-four hrs infection, the cells were treated with or without 50μM RSV for 24 hours. For (A) and (B), the expression levels of adiponectin in cell lysates were determined by Western blot using specific antibodies as indicated. (C) The cell lysates from DsbA-L-suppressed or scramble cells treated with or without 50μM RSV were separated by gel-filtration chromatography and the distribution of adiponectin complex was determined by Western blot using an antibody to adiponectin. Quantification of the relative abundance of adiponectin oligomers in cell lysates was performed by analyzing Western blots by using the National Institutes of Health Scion Image software. The data were shown as changes in the ratio of HMW/total adiponectin. (D) The mRNA levels of DsbA-L and adiponectin in 3T3-L1 adipocytes treated with or without 50μM RSV for 24 hours were determined by quantitative real-time PCR. All the data (A, C, D) are representatives of three independent experiments with similar results and were quantified by the NIH Scion-Image program. Differences between groups were tested for statistical significance using analysis of variance (ANOVA).*p< 0.05,**p<0.01.Results:(1) RSV treatment greatly stimulated the cellular levels of DsbA-L and adiponectin in the scramble 3T3-L1 adipocytes. The stimulatory effect of RSV on adiponectin expression, however, was markedly diminished in the DsbA-L-suppressed cells (P<0.01). Gel filtration experiments revealed that RSV promoted adiponectin multimerization in the scramble 3T3-L1 adipocytes and the stimulatory effect of RSV was blocked in DsbA-L-suppressed cells (P<0.01). (2) On the other hand, overexpression of DsbA-L greatly increased adiponectin expression in 3T3-L1 adipocytes and RSV had no further stimulatory effect of RSV in the DsbA-L-overexpressed cells. (3) RSV significantly increased the mRNA levels of DsbA-L, but had no significant effect on the mRNA levels of adiponectin (P<0.05).Conclusion:DsbA-L plays a critical role in RSV-induced adiponectin multimerization and expression and the promoting effect of RSV on adiponectin up-regulation is predominantly mediated by enhanced expression levels of DsbA-L Part 3 RSV promotes DsbA-L and Adiponectin Expression in Adipocytes partly dependent on PDK1/AKT/FOXO1 Signaling PathwayObjective:To explore the role of PDK1/Akt/Foxol signaling pathway in the stimulatory effect of RSV on DsbA-L and adiponectin expression in adipocytes.Methods:(A) Serum-starved 3T3-L1 adipocytes were pre-treated with or without 50μM RSV for 20 minutes and then treated with or without 10 nM insulin for 10 minutes. The phosphorylation of Akt at Thr308, Ser473 and Foxol at Ser236 and the protein levels of these kinases in cell lysates were determined by Western blot using antibodies as indicated. (B) PDK1-KO/WT MEF cells were treated with or without 50μM RSV for 24 hours. (C) 3T3-L1 adipocytes were pre-treated with or without 10 pM Akt inhibitor VIII for 60 minutes, followed by treatment of 50μM RSV for 24 hours. (D) Foxo1-suppressed or scramble cells were treated with or without 50μM RSV for 24 hours. For (B-D), the expression of DsbA-L, adiponectin, PDK1, Akt and Foxo1, and phosphorylated Akt (Thr-308) and Foxo1 (Ser 256) in cell lysates were determined by Western blot using antibodies as indicated. The data was representatives of three independent experiments with similar results and were quantified by the NIH Scion-Image program. Differences between groups were tested for statistical significance using analysis of variance (ANOVA).*p< 0.05,**p<0.01.Results:(1) Treating 3T3-L1 adipocytes with RSV remarkably reduced insulin-stimulated Akt phosphorylation at Thr308 and Ser473, concurrently with a decrease in FOXO1 phosphorylation at Ser2D6, an Akt-phosphorylation site, in DsbA-L-suppressed cells. (2) The expression levels of DsbA-L is greatly enhanced in the PDK1-KO MEFs compared with wild-type cells (P<0.01). Interestingly, RSV treatment led to a further increase in the expression levels of DsbA-L (P<0.05). (3) AKTi VIII treatment inhibited the phosphorylation of Akt and FOXO1, concurrently with an increase in the expression levels of DsbA-L and adiponectin (P<0.01). The expression levels of DsbA-L and adiponectin were further increased in cells co-treated with both AKTi VIII and RSV (P<0.05). (4) The expression levels of DsbA-L and adiponectin were greatly reduced in FOXO1-suppressed 3T3-L1 adipocytes compared with the scramble cells (P<0.05). Suppressing the cellular levels of FOXO1 greatly reduced basal and RSV-induced expression levels of DsbA-L and adiponectin (P<0.05). Consistent with the view that there is an Akt/FOXO1-independent mechanism by which RSV enhances DsbA-L and adiponectin cellular levels, RSV was still able to stimulate DsbA-L and adiponectin expression in the FOXO1-supppressed cells, although the stimulatory effect was much smaller compared to that observed in the scramble cells (P<0.05). Conclusion:RSV inhibits the PDK1/Akt/Foxo1 signaling pathway and the stimulatory effect of RSV on DsbA-L and adiponectin expression is partially mediated by suppression of the PDK1/Akt signaling pathway. Besides, there might exist a PDK1/Akt/FOXO1-independent mechanism by which RSV enhances DsbA-L and adiponectin cellular levels. Part 4 Role of AMPK Signaling Pathway in the Stimulatory Effect of RSV on DsbA-L and Adiponectin Expression in AdipocytesObjective:To explore the role of AMPK signaling pathway in the stimulatory effect of RSV on DsbA-L and adiponectin expression in adipocytes, and to determine whether both PDK1/Akt/FOXO1 and AMPK are the two major pathways mediating the stimulatory effect of RSV on DsbA-L and adiponectin expression.Methods:(A) 3T3-L1 adipocytes were pre-treated with or without 10μM Compound C for 60 minutes, followed with or without 50μM RSV treatment for 24 hours. The expression levels of DsbA-L, adiponectin and AMPK, and the phosphorylation levels of AMPK (Thr-172) in cell lysates were determined by Western blot using specific antibodies as indicated. (B) AMPK-suppressed or scramble cells were treated with or without 50μM RSV for 24 hours. The expression levels of DsbA-L and AMPK in cell lysates were determined by Western blot using antibodies as indicated. (C) 3T3-L1 adipocytes were treated with or without 500μM AICAR in the presence or absence of 50μM RSV for 24 hours. The expression levels of DsbA-L, adiponectin and AMPK, and the phosphorylation levels of AMPK (Thr-172) in cell lysates were determined by Western blot using specific antibodies as indicated. (D) Differentiated 3T3-CAR adipocytes were infected with adenoviruses encoding Foxo1-siRNA or scramble RNA at MOI=100 for 24 hours. Cells were then pre-treated with or without 10μM Compound (Comp.) C for 60 minutes, followed with or without 50μM RSV for 24 hours. The expression levels of DsbA-L, adiponectin and Foxo1 in cell lysates were determined by Western blot using antibodies as indicated. Data was representatives of three independent experiments with similar results and were quantified by the NIH Scion-Image program. Differences between groups were analyzed by ANOVA.*p<0.05.Results:(1) Treating 3T3-L1 adipocytes with Compound C significantly suppressed RSV-stimulated DsbA-L and adiponectin expression (P<0.05). (2) The inhibition of AMPK suppressed RSV stimulated DsbA-L expression compared with scramble C2C12 cells (P<0.05). (3) Activation of AMPK by AICAR markedly enhanced the protein levels of DsbA-L and adiponectin in 3T3-L1 adipocytes. However, AICAR co-treatment had no further effect on RSV-stimulated DsbA-L and adiponectin up-regulation. (4) Compound C treatment partially suppressed the stimulatory effect of RSV on the expression levels of DsbA-L and adiponectin in the scramble 3T3-L1 adipocytes. Interestingly, the promoting effect of RSV was completely diminished in the FOXO1-RNAi cells treated with Compound C.Conclusion:The AMPK signaling pathway is involved in RSV-stimulated up-regulation of DsbA-L and adiponectin. Both the PDK1/Akt/FOXO1 and the AMPK signaling pathways might be the two major pathways mediating the stimulatory effect of RSV on DsbA-L and adiponectin expression.