Delineation of the insulin-responsive sequence in the rat cytosolic aspartate aminotransferase gene: binding sites for hepatocyte nuclear factor-3 and nuclear factor I.

Expression of the rat cytosolic aspartate aminotransferase gene is stimulated by glucocorticoids and repressed by insulin in the liver. The regulation by insulin and part of the glucocorticoid effect are mediated by a distal region in the promoter. A 142 bp fragment (-1844 to -1702) confers hormonal sensitivity to the heterologous thymidine kinase promoter in transient-transfection assays in H4IIEC3 hepatoma cells. Footprinting and gel-shift assays showed that several nuclear proteins bind to this region at conserved CCAAT-enhancer binding protein (C/EBP), activator protein (AP-1) and E-box sequences. Hepatocyte nuclear factor-3alpha (HNF-3)alpha and beta bind to sequences upstream of a glucocorticoid-responsive element (GRE) half-site as demonstrated by supershift experiments. Nuclear factor I (NFI)-like proteins bind downstream of the GRE half-site. These sites around the GRE motif overlap with five insulin responsive element (IRE) -like sequences (TG/ATTT). The effect of insulin was not prevented by any single mutation in the IRE-like sites. However, mutation of two IRE sites (namely IREc and d) prevented the insulin effect although only marginally affecting the glucocorticoid effect. The results suggest that the effect of insulin is due to a complex interplay of factors requiring the synergistic contribution of at least two sites and underline the contribution of HNF-3 and NFI-like proteins.

Transcriptional activation of CYP2C, MxA and Fas in sudden infant death syndrome.

The expression of cytochrome P4502C has been shown to be upregulated in sudden infant death syndrome (SIDS) and could be linked to viral infection through the release of interferon alpha and interleukins. MxA is a reliable marker of IFNalpha release and its level was significantly enhanced in SIDS reflecting the release of IFNalpha in response to viral infection. Similarly, the concentration of Fas protein was increased in SIDS (2.6x control) and indicated a stimulation of the Fas gene expression. Accumulation of MxA and Fas proteins were visible in liver and to a lesser extent in lung and kidney. The amount of RNA encoding CYP2C9 (4.4x control), 2C8 (2.5x) and 2C18 (2.3x) was markedly higher in SIDS than in age-matched children and would suggest a transcriptional activation of CYP2C gene expression. Finally, CYP2C genes were shown to be adjacent to two IFN-inducible genes (IFI54 and IFI56) on chromosome 10. We conclude that in SIDS a viral infection leads to the release of IFNalpha which could activate a battery of IFN-inducible genes. This might modify the chromatin structure and facilitate the accessibility to promoter/regulatory sequences of CYP2C and Fas genes close to IFN-inducible gene on chromosome 10.

Identification of an adipocyte-specific negative glucose response region in the cytosolic aspartate aminotransferase gene.

Cytosolic aspartate aminotransferase (cAspAT) participates in gluconeogenesis in the liver and is expected to exert a glyceroneogenic function in the adipose tissue when the supply of glucose is limited. Here we demonstrate that adipose cAspAT messenger RNA (mRNA) is increased when rats are fed a low carbohydrate diet. In the 3T3-F442A, BFC-1 adipocyte cell lines and differentiated adipocytes in primary culture, a 24 h glucose deprivation induces approximately a 4-fold increase in cytosolic AspAT (cAspAT) mRNA, whereas mitochondrial AspAT mRNA remains unchanged. cAspAT activity is also increased in a weaker but reproducible manner. Addition of glucose within a physiological range of concentrations reverses the increase of cAspAT mRNA in 8 h (EC50 = 1.25 g/liter). Such a regulation requires protein synthesis and is specific for adipocytes differentiated in culture. It does not occur in Fao or H4IIE hepatoma cells, in C2 muscle cells, or in 293 kidney cells. 2-deoxyglucose mimicks glucose, while 3-orthomethyl-glucose has no effect, suggesting that glucose-6-phosphate is the effector. cAspAT mRNA stability is not affected by glucose deprivation. To ascertain the transcriptional nature of the glucose effect, we have stably transfected 3T3-F442A adipoblasts with constructs containing the chloramphenicol acetyltransferase reporter gene under the control of either 5'-deletions of the cAspAT gene promoter or internal fragments in an heterologous context. We demonstrate that a glucose response element(s) is present in the region between -1838 and -1702 bp relative to the translation start site. In this region, three DNA sequences bind nuclear proteins from adipocytes as shown by footprinting experiments. Our results indicate that cAspAT gene transcription is repressed by glucose selectively in adipocytes.