Resveratrol up-regulates DsbA-L and adiponectin in 3T3-L1 adipocytes and in vivo. A, 3T3-L1 adipocytes were treated with or without resveratrol (RSV) at the indicated concentrations for 24 h. B, 3T3-L1 adipocytes were treated with 50 μm RSV for different times as indicated. For A and B, the expression levels of adiponectin and DsbA-L in cell lysates were determined by Western blot. Data are representative of three independent experiments with similar results. C, adiponectin multimers in lysates from 3T3-L1 adipocytes treated with or without 50 μm 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. Quantification of the relative abundance of DsbA-L or adiponectin in the tissue homogenates was performed by analyzing the Western blots with National Institutes of Health Scion Image software. *, p < 0.05 (n = 3). Data are presented as mean ± S.E.

DsbA-L plays a critical role in RSV-stimulated up-regulation of adiponectin. A, DsbA-L-suppressed or scramble 3T3-L1 adipocytes were treated with or without 50 μm RSV for 24 h. B, differentiated 3T3-CAR adipocytes were infected with adenoviruses encoding GFP plus DsbA-L or GFP alone at multiplicity of infection = 20. Twenty-four h after infection, the cells were treated with or without 50 μm RSV for 24 h. 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 50 μm 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 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 50 μm RSV for 24 h were determined by quantitative real time PCR. All data (A, C, and D) are representatives of three independent experiments with similar results and were quantified by the NIH Scion Image program. Data are presented as mean ± S.E. Differences between groups were analyzed using analysis of variance. **, p < 0.01; *, p < 0.05.

Resveratrol up-regulates DsbA-L and adiponectin expression is independent of Sirt1. A, 3T3-L1 adipocytes were pre-treated with or without 10 μm sirtinol for 60 min, followed with or without 50 μm RSV for 24 h. The acetylation and protein levels of immunoprecipitated PGC-1α were determined by Western blot with specific antibodies. B, the differentiated Sirt1-KO/WT adipocytes were treated with RSV at the indicated concentrations for 24 h. For A and B, the expression levels of adiponectin and DsbA-L in cell lysates were determined by Western blot using specific antibodies.

The stimulatory effects of RSV on DsbA-L and adiponectin expression are partially through suppression of the PI3K pathway. A, serum-starved 3T3-L1 adipocytes were pre-treated with or without 50 μm RSV for 20 min and then with or without 10 nm insulin for 10 min. The phosphorylation of Akt at Thr³⁰⁸, Ser⁴⁷³, and Foxo1 at Ser²⁵⁶ 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 50 μm RSV for 24 h. C, 3T3-L1 adipocytes were pre-treated with or without 10 μm Akt inhibitor VIII for 60 min, followed with or without 50 μm RSV for 24 h. D, Foxo1-suppressed or scramble cells were treated with or without 50 μm RSV for 24 h. For B–D, the expression levels of DsbA-L, adiponectin, PDK1, Akt, Foxo1, and phosphorylation levels of Akt (Thr³⁰⁸) and Foxo1 (Ser²⁵⁶) in cell lysates were determined by Western blot using antibodies as indicated. The data are representative of three independent experiments with similar results and quantified by the NIH Scion Image program. Data are presented as mean ± S.E. Differences between groups were tested for statistical significance using analysis of variance. *, p < 0.05; **, p < 0.01.

The AMPK signaling pathway is involved in RSV-stimulated up-regulation of DsbA-L and adiponectin expression. A, 3T3-L1 adipocytes were pre-treated with or without 10 μm Compound C for 60 min, followed with or without 50 μm RSV for 24 h. The expression levels of DsbA-L, adiponectin, and AMPK, and the phosphorylation levels of AMPK (Thr¹⁷²) in cell lysates were determined by Western blot using specific antibodies as indicated. B, AMPK-suppressed or scramble cells were treated with or without 50 μm RSV for 24 h. 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 500 μm AICAR in the presence or absence of 50 μm RSV for 24 h. The expression levels of DsbA-L, adiponectin, and AMPK, and the phosphorylation levels of AMPK (Thr¹⁷²) 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 multiplicity of infection = 100 for 24 h. Cells were then pre-treated with or without 10 μm Compound (Comp.) C for 60 min, followed with or without 50 μm RSV for 24 h. The expression levels of DsbA-L, adiponectin, and Foxo1 in cell lysates were determined by Western blot using antibodies as indicated. Data are representative of three independent experiments with similar results and quantified by the NIH Scion Image program. Data are presented as mean ± S.E. Differences between groups were analyzed by analysis of variance. *, p < 0.05.