GLUT4 repression in response to oxidative stress is associated with reciprocal alterations in C/EBP alpha and delta isoforms in 3T3-L1 adipocytes

Insulin responsiveness of adipocytes is acquired during normal adipogenesis, and is essential for maintaining whole-body insulin sensitivity. Differentiated adipocytes exposed to oxidative stress become insulin resistant, exhibiting decreased expression of genes like the insulin-responsive glucose transporter GLUT4. Here we assessed the effect of oxidative stress on DNA binding capacity of C/EBP isoforms known to participate in adipocyte differentiation, and determine the relevance for GLUT4 gene regulation. By electrophoretic mobility shift assay, nuclear proteins from oxidized adipocytes exhibited decreased binding of C/EBPalpha-containing dimers to a DNA oligonucleotide harboring the C/EBP binding sequence from the murine GLUT4 promoter. C/EBPdelta-containing dimers were increased, while C/EBPbeta-dimers were unchanged. These alterations were mirrored by a 50% decrease and a 2-fold increase in the protein content of C/EBPalpha and C/EBPdelta, respectively. In oxidized cells, GLUT4 protein and mRNA levels were decreased, and a GLUT4 promoter segment containing the C/EBP binding site partially mediated oxidative stress-induced repression of a reported gene. The antioxidant lipoic acid protected against oxidation-induced decrease in GLUT4 and C/EBPalpha mRNA, but did not prevent the increase in C/EBPdelta mRNA. We propose that oxidative stress induces adipocyte insulin resistance partially by affecting the expression of C/EBPalpha and delta, resulting in altered C/EBP-dimer composition potentially occupying the GLUT4 promoter.     

Hepatocyte nuclear factor 4α regulates the expression of the murine pyruvate carboxylase gene through the HNF4-specific binding motif in its proximal promoter

Pyruvate carboxylase (PC) is the first regulatory enzyme of gluconeogenesis. Here we report that the proximal promoter of the murine PC gene contains three binding sites for hepatocyte nuclear factor 4α (HNF4α). These sites include the classical direct repeat 1 (DR1) (− 386/− 374), non-perfect DR1 (− 118/− 106) and HNF4α-specific binding motif (H4-SBM) (− 26/− 14). Under basal conditions, mutation of the non-perfect DR1 decreased promoter activity by 50%, whereas mutation of neither the DR1 nor the H4-SBM had any effect. In marked contrast, only mutation of the H4-SBM decreased HNF4α-transactivation of the promoter activity by 65%. EMSA revealed that HNF4α binds to the DR1site and H4-SBM with similar affinity while it binds poorly to the non-perfect DR1. Interestingly, this non-perfect DR1 also coincides with two E-boxes. Mutation of the non-perfect DR1 together with the nearby E-box reduced USF1- but not USF2-transactivation of promoter activity, suggesting that USF1 partly contributes to the basal activity of the promoter. Substitution of the H4-SBM with the DR1 marginally reduced the basal promoter activity but did not eliminate HNF4α-transactivation, suggesting that HNF4α can exert its effect via DR1 within this promoter context. ChIP-assay confirmed that HNF4α is associated with the H4-SBM. Suppression of HNF4α expression in AML12 cells down-regulated PC mRNA and PC protein by 60% and 50%, respectively, confirming that PC is a target of HNF4α. We also propose a model for differential regulation of P1 promoter of PC gene in adipose tissue and liver.     

Binding of USF to a non-canonical E-box following stress results in a cell-specific derepression of the lama3 gene

Expression of the lama3 gene, encoding the laminin α3A chain, is restricted to specialized epithelia. We previously showed that lama3 gene expression is controlled by an epithelial enhancer through the cooperative effect of AP-1 binding sites. In fibroblasts, there is no lama3 expression because of the recruitment of a repressor complex absent or inactive in epithelial cells. In this paper, we show evidence that this repression of the lama3 gene is relieved by exogenous and UV-induced USF-1 through its interaction with a non-canonical E-box site. Using a chromatin immunoprecipitation assay, we find that UV stress induces USF to bind to the lama3 promoter in vivo. We further demonstrate that this loss of cell specificity is directly related to the accessibility of the E-box, resulting in a strong induction in fibroblasts, while expression remains constitutively high in keratinocytes. This accessibility appears to be dependent upon the recruitment of a fibroblastic repressor complex. Therefore, we speculate that anchorage of this repressor complex in fibroblasts modifies the enhancer geometry, allowing USF to interact under stress-inducing conditions with its heptameric binding site.