Acquired resistance to the action of insulin to stimulate glucose transport in skeletal muscle is associated with obesity and promotes the development of type 2 diabetes. In skeletal muscle, insulin resistance can result from high levels of circulating fatty acids that disrupt insulin signalling pathways. However, the severity of insulin resistance varies greatly among obese people. Here we postulate that this variability might reflect differences in levels of lipid-droplet proteins that promote the sequestration of fatty acids within adipocytes in the form of triglycerides, thereby lowering exposure of skeletal muscle to the inhibitory effects of fatty acids.

Mitochondrial transcription factor A (mtTFA), the product of a nuclear gene, stimulates cription from the two divergent mitochondrial promoters and is likely the principal activator of mitochondrial gene expression in vertebrates. Here we establish that the proximal promoter of the human mtTFA gene is highly dependent upon recognition sites for the nuclear respiratory factors, NRF-1 and NRF-2, for activity. These factors have been previously implied in the activation of numerous nuclear genes that contribute to mitochondrial respiratory function. The affinity-purified factors from HeLa cells specifically bind to the mtTFA NRF-1 and NRF-2 sites through guanine nudeotide contacts that are characteristic for each site. Mutations in these contacts eliminate NRF-1 and NRF-2 binding and also dramatically reduce promoter activity in transfected cells. Although both factors contribute, NRF-1 binding appears to be the major determinant of promoter function. This dependence on NRF-1 activation is confirmed by in vitro ranscription using highly purified recombinant proteins that display the same binding specificities as the HeLa cell factors. The activation of the mtTFA promoter by both NRF-1 and NRF-2 therefore provides a link between the expression of nuclear and mitochondrial genes and suggests a mechanism for their coordinate regulation during organelle biogenesis.

Signal transmission by many cell surface receptors results in the activation of phosphoinositide (PI) 3-kinases that phosphorylate the 3' position of polyphosphoinositides. From a screen for mouse proteins that bind phosphoinositides, the protein GRP1was identified. GRP1 binds phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4, 5)P3] through a pleckstrin homology (PH) domain and displays a region of high sequence similarity to the yeast Sec7 protein. The PH domain of the closely related protein cytohesin-1, which, through its Sec7 homology domain, regulates integrin beta2 and catalyzes guanine nucleotide exchange of the small guanine nucleotide-binding protein ARF1, was also found to specifically bind PtdIns(3,4,5)P3. GRP1 and cytohesin-1 appear to connect receptor-activated PI 3-kinase signaling pathways with proteins that mediate biological responses such as cell adhesion and membrane trafficking.

Nuclear respiratory factor 1 (NRF-1) was first discovered as an activator of the cytochrome c gene and was subsequently found to play a broader role in nuclear-mitochondrial interactions. We have now cloned a HeLa cDNA encoding NRF-1 using degenerate oligomers derived from tryptic peptide sequences for PCR amplification. The cDNA-encoded protein was indistinguishable from the authentic HeLa cell factor on denaturing gels, displayed the expected NRF-1 DNA-binding specificity, and made the same guanine nucleotide contacts as HeLa NRF-1 on binding known NRF-1 recognition sites. Antiserum raised against the highly purified recombinant protein recognized the identical DNA-protein complex formed using either a crude nuclear fraction or nearly homogeneous HeLa NRF-1. Recombinant NRF-1 also activated transcription through specific sites from several NRF-l-responsive promoters, confirming both the transcriptional activity and specificity of the cDNA product. Portions of NRF-1 are closely related to sea urchin P3A2 and the erect wing (EWG) protein of Drosophila. Both are recently identified developmental regulatory factors. The region of highest sequence identity with P3A2 and EWG was in the amino-terminal half of the molecule, which was found by deletion mapping to contain the DNA-binding domain, whereas the carboxy-terminal half of NRF-1 was highly divergent from both proteins. The DNA-binding domain in these molecules is unrelated to motifs found commonly in DNA-binding proteins; thus, NRF-1, P3A2, and EWG represent the founding members of a new class of highly conserved sequence-specific regulatory factors.

The ETS domain proteins are a diverse family of transcriptional activators that have been implicated recently in the expression of a number of cell-specific and viral promoters. Nuclear respiratory factor 2 (NRF-2) is a nuclear transcription factor that activates the proximal promoter of the rat cytochrome c oxidase subunit IV (RCO4) gene through tandem sequence elements. These elements conform to the consensus for high-affinity ETS domain recognition sites. We have now purified NRF-2 to homogeneity from HeLa cells and find that it consists of five polypeptides, only one of which has intrinsic DNA-binding ability. The others participate in the formation of heteromeric complexes with distinct binding properties. NRF-2 also specifically recognizes multiple binding sites in the mouse cytochrome c oxidase subunit Vb (MCO5b) gene. As in the functionally related RCO4 gene, tandemly arranged NRF-2 sites are essential for the activity of the proximal MCO5b promoter, further substantiating a role for NRF-2 in respiratory chain expression. Determination of peptide sequences from the various subunits of HeLa NRF-2 reveals a high degree of sequence identity with mouse GA-binding protein (GABP), a multisubunit ETS domain activator of herpes simplex virus immediate early genes. A cellular role in the activation of nuclear genes specifying mitochondrial respiratory function is thus assigned to an ETS domain activator of viral promoters.

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Phosphatidylinositol 3-kinases (PI 3-kinases) have been implicated in membrane trafficking in the secretory and endocytic pathways of yeast and mammalian cells, but the molecular mechanisms by which these lipid kinases operate are not known. Here we identify a protein of 170 kDa that is rapidly released from cell membranes in response to wortmannin, a potent inhibitor of mammalian PI 3-kinases. The amino acid sequence of peptides from p170 reveal its identity to early endosomal antigen (EEA) 1, an endosomal antigen with homology to several yeast proteins genetically implicated in membrane trafficking. Immunof luorescence analysis of 3T3-L1 adipocytes with antisera against p170͞EEA1 reveal a punctate peripheral pattern that becomes diffuse in response to wortmannin. In vitro, p170͞EEA1 binds specifically to liposomes containing PIns(3)P, suggesting that the effect of wortmannin on cells is due to inhibition of PIns(3)P production. Thus, p170͞EEA1 may define a family of proteins that mediate the regulatory effects of 3-phosphoinositides on membrane trafficking in yeast and mammalian cells.

The insulin-regulated glucose transporter GLUT4 is a key modulator of whole body glucose homeostasis, and its selective loss in adipose tissue or skeletal muscle causes insulin resistance and diabetes. Here we report an RNA interference-based screen of protein kinases expressed in adipocytes and identify four negative regulators of insulin-responsive glucose transport: the protein kinases PCTAIRE-1 (PCTK1), PFTAIRE-1 (PFTK1), I B kinase ␣, and MAP4K4͞NIK. Integrin-linked protein kinase was identified as a positive regulator of this process. We characterized one of these hits, MAP4K4͞NIK, and found that it is unique among mitogenactivated protein (MAP) kinases expressed in cultured adipocytes in attenuating hexose transport. Remarkably, MAP4K4͞NIK suppresses expression of the adipogenic transcription factors C͞EBP␣, C͞EBP␤, and PPAR␥ and of GLUT4 itself in these cells. RNA interference-mediated depletion of MAP4K4͞NIK early in differentiation enhances adipogenesis and triglyceride deposition, and even in fully differentiated adipocytes its loss up-regulates GLUT4. Conversely, conditions that inhibit adipogenesis such as TNF-␣ treatment or depletion of PPAR␥ markedly up-regulate MAP4K4͞ NIK expression in cultured adipocytes. Furthermore, TNF-␣ signaling to down-regulate GLUT4 is impaired in the absence of MAP4K4͞NIK, indicating that MAP4K4 expression is required for optimal TNF-␣ action. These results reveal a MAP4K4͞NIK-dependent signaling pathway that potently inhibits PPAR␥-responsive gene expression, adipogenesis, and insulin-stimulated glucose transport.GLUT4 function ͉ adipocyte differentiation ͉ protein kinase screening