Enhancing effect of troglitazone on porcine adipocyte differentiation in primary culture: a comparison with dexamethasone.

Abstract

OBJECTIVE This study compares the effects of the thiazolidinedione, troglitazone (TGZ), dexamethasone (DEX), and DEX plus TGZ on preadipocyte differentiation and the expression of CCAAT/enhancer-binding protein-alpha (C/ EBPalpha) and peroxisome proliferator-activated receptor-gamma (PPARgamma). RESEARCH METHODS AND PROCEDURES Adipose tissue was obtained from postnatal pigs to isolate stromal-vascular cells. First, we applied 1, 5, or 10 microM TGZ and 10% fetal bovine serum for 3 days and counted the number of recruited preadipocytes. Next, we used either 10 microM TGZ, 80 nM DEX, or DEX plus TGZ with 10% fetal bovine serum for 3 days and then switched to serum-free medium with insulin for 6 days. On day 3 of culture, we counted preadipocytes, and on days 3 and 6 of culture, we performed immunostaining and Western blot analysis to determine the expression of C/EBPalpha and PPARgamma proteins. On day 9 of culture, we stained for lipids with oil red-O and measured the activity of glycerol phosphate dehydrogenase. RESULTS DEX and TGZ equally enhanced recruitment of preadipocytes and late differentiation, but these effects were not additive with DEX plus TGZ treatment. However, TGZ and DEX had a differential effect on morphogenesis; DEX-treated adipocytes were larger and organized in loose clusters, whereas TGZ-treated cells were smaller and formed compact clusters. Both agents increased C/EBPalpha expression but in a temporally distinct manner. DEX was a better inducer than TGZ, and its effect was early and temporary. However, treatment with either TGZ or DEX did not change PPARgamma protein expression as evaluated by a Western blotting, but immunocytochemistry showed a tendency for increased numbers of PPARgamma positive cells. DISCUSSION TGZ and DEX equally enhance early and late adipocyte differentiation, possibly by using some common pathways for preadipocyte recruitment. The differential effect on morphogenesis implies a potential differential effect on the expression of extracellular matrix components. C/EBPalpha may be the critical transcription factor involved in TGZ- and DEX-induced adipogenesis.

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