ABSTRACT
Several lines of evidence have recently underscored the significance of fatty acids or fatty acid-derived metabolites as signaling molecules in adipocyte differentiation. The acyl-CoA-binding protein (ACBP), which functions as an intracellular acyl-CoA pool former and transporter, is induced during adipocyte differentiation. In this report we describe the effects of expression of high levels of ACBP antisense RNA on the differentiation of 3T3-L1 cells. Pools of 3T3-L1 cells transfected with vectors expressing ACBP antisense RNA showed significantly less lipid accumulation as compared with cells transfected with the control vector. When individual clones were analyzed the degree of differentiation at day 10 was inversely correlated with the level of ACBP antisense RNA expression at day 0. Furthermore, in the clones with the highest levels of ACBP antisense expression, the induction of expression of the adipogenic transcription factors peroxisome proliferator-activated receptor gamma and CCAAT/enhancer-binding protein alpha as well as several adipocyte-specific genes was significantly delayed and reduced. The adipogenic potential of antisense-expressing cells was partially restored by transfection with a vector expressing high levels of ACBP. Taken together, these results are strong evidence that inhibition of differentiation is causally related to the decreased expression of ACBP, indicating that ACBP plays an important role during adipocyte differentiation.
Subject(s)
Adipocytes/cytology , Adipocytes/metabolism , Carrier Proteins/genetics , RNA, Antisense/metabolism , Thiazolidinediones , 1-Methyl-3-isobutylxanthine/pharmacology , 3T3 Cells , Animals , CCAAT-Enhancer-Binding Proteins , Carrier Proteins/biosynthesis , Cell Differentiation , Clone Cells , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Dexamethasone/pharmacology , Diazepam Binding Inhibitor , Gene Expression Regulation/drug effects , Hypoglycemic Agents/pharmacology , Insulin/pharmacology , Mice , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , RNA, Messenger/genetics , Receptors, Cytoplasmic and Nuclear/biosynthesis , Receptors, Cytoplasmic and Nuclear/genetics , Recombinant Proteins/biosynthesis , Rosiglitazone , Thiazoles/pharmacology , Transcription Factors/biosynthesis , Transcription Factors/genetics , Transcription, Genetic , TransfectionABSTRACT
In previous work (Brooker, R. J., and Wilson, T. H. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3959-3963), lactose permease mutants were isolated which possessed an enhanced recognition for maltose. In some of these mutants, the wild-type alanine residue at position 177 was changed to valine or threonine. To gain further insight into the side chain requirement at position 177 that confers maltose recognition, further substitutions of isoleucine, leucine, phenylalanine, proline, and serine have been made via site-directed mutagenesis. Permeases containing alanine or serine exhibited poor maltose recognition whereas those containing isoleucine, leucine, phenylalanine, proline, or valine showed moderate or good recognition. As far as galactosides are concerned, the Val-177, Pro-177, and Ser-177 mutants were able to transport lactose as well as, or slightly better than, the wild-type strain. The other mutants displayed moderately reduced levels of lactose transport. For example, the Phe-177 mutant, which was the most defective, showed a level of downhill transport which was approximately 20% that of the wild-type strain. In uphill transport assays, all of the position 177 mutants were markedly defective in their ability to accumulate beta-D-thiomethylgalactopyranoside against a concentration gradient. Finally, the position 177 mutants were analyzed for their ability to catalyze an H+ leak. Interestingly, even though the wild-type permease does not leak H+ across the bacterial membrane, all of the position 177 mutants were shown to transport H+ in the absence of sugars. For most of the mutants, this H+ leak was blocked by the addition of beta-D-thiodigalactoside. Overall, these results are discussed with regard to the effects of position 177 substitutions on the sugar recognition site and H+ transport.
