Cellular retinol-binding protein type III is a PPARgamma target gene and plays a role in lipid metabolism.

Cellular retinol-binding protein (CRBP) type III (CRBP-III) belongs to the family of intracellular lipid-binding proteins, which includes the adipocyte-binding protein aP2. In the cytosol, CRBP-III binds retinol, the precursor of retinyl ester and the active metabolite retinoic acid. The goal of the present work is to understand the regulation of CRBP-III expression and its role in lipid metabolism. Using EMSAs, luciferase reporter assays, and chromatin immunoprecipitation assays, we found that CRBP-III is a direct target of peroxisome proliferator-activated receptor-gamma (PPARgamma). Moreover, CRBP-III expression was induced in adipose tissue of mice after treatment with the PPARgamma agonist rosiglitazone. To examine a potential role of CRBP-III in regulating lipid metabolism in vivo, CRBP-III-deficient (C-III-KO) mice were maintained on a high-fat diet (HFD). Hepatic steatosis was decreased in HFD-fed C-III-KO compared with HFD-fed wild-type mice. These differences were partly explained by decreased serum free fatty acid levels and decreased free fatty acid efflux from adipose tissue of C-III-KO mice. In addition, the lack of CRBP-III was associated with reduced food intake, increased respiratory energy ratio, and altered body composition, with decreased adiposity and increased lean body mass. Furthermore, expression of genes involved in mitochondrial fatty acid oxidation in brown adipose tissue was increased in C-III-KO mice, and C-III-KO mice were more cold tolerant than wild-type mice fed an HFD. In summary, we demonstrate that CRBP-III is a PPARgamma target gene and plays a role in lipid and whole body energy metabolism.

Role of versican and hyaluronan in the differentiation of 3T3-L1 cells into preadipocytes and mature adipocytes.

We have previously shown that during the adipose conversion of these cells the culture medium changed its viscoelastic properties due to the presence of hyaluronan and a chondroitin sulfate proteoglycan [Calvo, J.C., Rodbard, D., Katki, A., Chernick, S., and Yanagishita, M., 1991. Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans. J. Biol. Chem. 266, 11237-11244., Calvo, J.C., Gandjbakhche, A.H., Nossal, R., Hascall, V.C., and Yanagishita, M., 1993. Rheological effects of the presence of hyaluronic acid in the extracellular media of differentiated 3T3-L1 preadipocyte cultures. Arch. Biochem. Biophys. 302, 468-475]. Here, we analyze the time course for the appearance of these molecules during drug-induced cell differentiation. The synthesis of both hyaluronan and the proteoglycan, was maximal at 48 h in the presence of isobutylmethylxanthine and dexamethasone, but while hyaluronan remained high after changing the culture medium, the proteoglycan dropped to almost basal levels after a few days. Northern analysis revealed the presence of message for a "versican-like" molecule as well as the possibility of alternative splicing. Three major bands of 9.39, 8.48, and 7.69 kb appeared in the analysis. These bands showed a dramatic increase in intensity when RNA from non-differentiated cells was compared to differentiating 3T3-L1 cells. In addition, when the time course of appearance for this message was analyzed, it perfectly correlated the metabolic labeling of the glycosaminoglycans during cell culture. The nucleotide sequencing of two exons revealed between a 100-94% homology with proteoglycan PG-M from murine endothelial cells. At least 13% of the proteoglycan was able to bind hyaluronan. Disruption of the synthesis of the proteoglycan molecule by exogenous addition of xyloside, did not prevent triglyceride accumulation but was inhibitory to preconfluent 3T3-L1 cell proliferation. Coating of plastic culture dishes with conditioned medium from differentiating 3T3-L1 cells, resulted in decreased cell adhesion. Cell adhesion was partially recovered after degradation of hyaluronan and chondroitin sulfate by enzymatic treatment. All these results indicate a possible role of these molecules in the observed changes in the viscoelastic properties of the culture medium, as well as open the field for a more thorough study of their role in 3T3-L1 cell proliferation and/or differentiation.

Changes in IGF-I receptor and IGF-I mRNA during differentiation of 3T3-L1 preadipocytes.

Insulin-like growth factor-1 (IGF-I) is an essential factor for the differentiation of preadipocytes into adipocytes. We investigated the expression of IGF-I receptor and IGF-I RNA messenger during 3T3-L1 preadipocyte differentiation. Levels of IGF-I receptor decreased in the mature adipocytes compared to cells before the initiation of differentiation. In addition, cultures not induced to differentiate showed a decrease on the receptor levels after 4 days in the presence of insulin compared to cultures without treatment. The levels of the IGF-I RNA messenger were shown to be higher in mature adipocytes compared to preadipocytes. We propose an autocrine and/or paracrine action of IGF-I in this adipocyte differentiation model, where IGF-I produced by the differentiating preadipocytes acts over their adjacent cells and, in this way, diminishes the expression of IGF-I receptor.