Identification of divergent effects of FEN in fully differentiated adipocytes. Identification of divergent effects of FEN in fully differentiated adipocytes.

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Identification of divergent effects of FEN in fully differentiated adipocytes. Identification of divergent effects of FEN in fully differentiated adipocytes. A: Differentiated 3T3-L1 adipocytes were used to determine if FEN could reduce lipid content of mature adipocyte cells. 3T3-L1 cultures were differentiated with MDI alone for 8 days and then were subjected to the indicated treatments for an additional 8 days (FEN, RA, and ROSI at 1 μmol/L). Oil-red-O staining and lipid accumulation quantification of 3T3-L1 adipocytes revealed FEN significantly reduced triglyceride levels, unlike RA, which did not significantly change. MDI plus VEH had equivalent DMSO added as treated cells. Middle panels show 40× images of representative cell morphology/staining from six-well plate (left panel). Total lipid was quantified by eluting the stain and measuring absorbance at 520 nm (right panel). All graphs represent mean ± SEM of three separate experiments (performed on different plates of cells). Significance (P < 0.05 by one-way ANOVA followed by Tukey post hoc test or t test if indicated) is shown for *ROSI, FEN, or RA compared with vehicle (VEH) or for #FEN vs. RA. B: Relative expression levels of adipocyte genes observed from cultures described in (A) (quantitative PCR analysis, left panel). Immunoblots of HSL and ATGL protein levels (right panel). The t test was used to calculate significant difference for ATGL FEN or ROSI compared with VEH. HSL FEN vs. VEH trended to be significantly different by t test (P = 0.06). N = 2 different plates of cells. Otherwise significance was calculated by ANOVA. C: Retinoid metabolism gene expression levels observed in 3T3-L1 cultures shown in (A). The t test was used to calculate significant difference for RARγ FEN or RA vs. VEH, for CRBP1 FEN vs. VEH, RALDH1 ROSI or FEN vs. VEH, and CYP26A1 ROSI vs. VEH. Otherwise significance was calculated by ANOVA. D: Differentiated 3T3-L1 adipocytes were stimulated with 10 nmol/L insulin (for 15 min after overnight serum-free media) or saline (S) to determine if Fenretinide could increase insulin sensitivity of mature adipocyte cells. Phosphorylation of Akt/PKB at Ser473 and total Akt/PKB protein expression were determined by immunoblotting and the signal quantified. n = 2 different plates of cells. (A high-quality digital representation of this figure is available in the online issue.)‏ George D. Mcilroy et al. Diabetes 2013;62:825-836 ©2013 by American Diabetes Association