CCAAT/enhancer binding protein alpha is sufficient to initiate the 3T3-L1 adipocyte differentiation program.

Previous studies showed that CCAAT/enhancer binding protein alpha (C/EBP alpha) is required for differentiation of 3T3-L1 preadipocytes induced by exogenous hormonal agents. It was not possible to ascertain, however, whether C/EBP alpha alone is sufficient to induce differentiation because its antimitogenic activity precluded propagating 3T3-L1 cell lines that constitutively express C/EBP alpha at high levels. This problem was circumvented by using 3T3-L1 preadipocytes stably transfected with an isopropyl beta-D-thiogalactoside (IPTG)-inducible p42 C/EBP alpha expression vector system. IPTG-induced expression of the 42-kDa isoform of C/EBP alpha in preadipocytes caused expression of several endogenous adipocyte-specific genes (genes encoding the 422 adipose P2 protein, glucose transporter 4, and C/EBP alpha) and the accumulation of cytoplasmic triglyceride. Thus, C/EBP alpha is not only necessary but also is sufficient to trigger differentiation of growth-arrested 3T3-L1 preadipocytes without use of exogenous hormonal agents.     

Evidence for an insulin receptor substrate 1 independent insulin signaling pathway that mediates insulin-responsive glucose transporter (GLUT4) translocation.

Interaction of the activated insulin receptor (IR) with its substrate, insulin receptor substrate 1 (IRS-1), via the phosphotyrosine binding domain of IRS-1 and the NPXY motif centered at phosphotyrosine 960 of the IR, is important for IRS-1 phosphorylation. We investigated the role of this interaction in the insulin signaling pathway that stimulates glucose transport. Utilizing microinjection of competitive inhibitory reagents in 3T3-L1 adipocytes, we have found that disruption of the IR/IRS-1 interaction has no effect upon translocation of the insulin-responsive glucose transporter (GLUT4). The activity of these reagents was demonstrated by their ability to block insulin stimulation of two distinct insulin bioeffects, membrane ruffling and mitogenesis, in 3T3-L1 adipocytes and insulin-responsive rat 1 fibroblasts. These data suggest that phosphorylated IRS-1 is not an essential component of the metabolic insulin signaling pathway that leads to GLUT4 translocation, yet it appears to be required for other insulin bioeffects.     

Participation of the transcription factor C/EBP delta in the acute-phase regulation of the human gene for complement component C3.

C3, the third component of complement, is critical in the host immune response in that it is involved in both the classical and alternative pathways of complement activation. We have previously shown that a region (bp -127 to -70) within the C3 promoter is indispensable for conferring interleukin 1 (IL-1) responsiveness to this gene. A sequence comparison reveals two CCAAT/enhancer binding protein (C/EBP) consensus sequences, basic DNA binding region and leucine zippers 1 and 2 (bZIP1 and bZIP2), within this region. Site-directed mutagenesis of the more 3' C/EBP site (bZIP1) in the C3 promoter significantly reduced the basal level of expression and the IL-1 responsiveness of the reporter gene, whereas mutation in the second, more 5', C/EBP consensus sequence (bZIP2) had a minimal effect on basal expression and IL-1 inducibility. Electrophoretic-mobility-shift assays, with and without antibodies to the different C/EBP proteins that "supershift" protein-DNA complexes, demonstrated that proteins binding at the 3' C/EBP site formed several complexes. Antibodies to C/EBP alpha supershifted the majority of complexes formed with extracts from control cells. Antibodies directed against C/EBP delta supershifted the major IL-1-inducible complexes. Western immunoblot analyses showed that the level of C/EBP delta protein was increased dramatically in the nuclei of Hep 3B2 cells after 4 h of IL-1 treatment. When Hep 3B2 cells were cotransfected with a C/EBP delta expression vector and a construct with a C3 promoter and a reporter gene, C/EBP delta was able to trans-activate the C3 promoter in an IL-1-responsive manner. The data strongly suggest that C/EBP delta is the major protein responsible for regulating the acute-phase expression of the human C3 gene.