Pancreatic b-cell dysfunction is central to the pathogenesis of type 2 diabetes and may involve secretory failure through glucolipotoxity. The relative importance of the transcription factors carbohydrate-responsive element binding protein (ChREBP), sterol-responsive element binding protein-1c (SREBP-1c), and upstream stimulatory factor (USF) in the induction of lipogenic genes by glucose remains unclear. By confocal imaging, we show that ChREBP translocates to the nucleus in MIN6 b cells in response to glucose. Both ChREBP and SREBP-1c were required for the induction of the fatty acid synthase (FAS) promoter by glucose, and chromatin immunoprecipitation (ChIP) assay revealed that glucose induced the binding of both ChREBP and SREBP-1c to the FAS promoter without affecting USF2 binding. By contrast, ChIP assay revealed that high glucose prompted direct binding of ChREBP, but not SREBP-1c or USF2, to the liver-type pyruvate kinase (L-PK) promoter. This event was indispensable for the induction of the L-PK gene by glucose, as demonstrated by RNA silencing, single-cell promoter analysis, and quantitative real-

The process of epithelial mesenchymal transition, whereby cells acquire molecular alterations and fibroblastic features, is a fundamental process of embryogenesis and cancer invasion/metastasis. The mechanisms responsible for epithelial mesenchymal transition remain elusive. Human tumors frequently establish constitutively activated RAS signaling, which contributes to the malignant phenotype. In an effort to dissect distinct RAS isoform specific functions, we previously established human colon cell lines stably overexpressing activated Harvey-RAS (Ha-RAS) and Kirsten-RAS (Ki-RAS). Using these, we observed that only oncogenic Ha-RAS overexpression resulted in morphologic and molecular changes suggestive of epithelial to mesenchymal transition. We showed that vimentin, a key molecule of epithelial mesenchymal transition, was differentially regulated between Ha-RAS and Ki-RAS leading to a Ha-RAS specific induction of a migrative phenotype and eventually epithelial to mesenchymal transition. We demonstrated that the AP-1 sites in vimentin promoter could be involved in this regulation. A potential role of FRA-1 was suggested in the regulation of vimentin during the Ha-RAS-induced epithelial to mesenchymal transition, in association with colon cell migration. Our results therefore propose that in colon cells, the induction of epithelial mesenchymal transition by oncogenic Ha-RAS could occur through the overexpression of proteins like FRA-1 and vimentin. ' 2007 Wiley-Liss, Inc.Key words: Ras; vimentin; FRA-1; epithelial-mesenchymal transition RAS proteins (comprising Ha-RAS, Ki-RAS and N-RAS) are very important molecular switches for a wide variety of signal pathways that control proliferation, cell adhesion, apoptosis and cell migration. RAS proteins are often deregulated in cancer, leading to increased invasion and metastasis and decreased apoptosis. Mutations in the RAS family of proto-oncogenes are very common, found in 30% of all human tumors and in 50% of colon tumors in particular. 1 The most common mutations are found on residues 12 and 61. The glycine to valine mutation on residue 12 renders RAS insensitive to inactivation. RAS protein functions within a signal transducing cascade of reactions. Among them, the mitogen activated protein (MAP) kinases that transmit signals downstream to other protein kinases and gene regulatory proteins, are of leading importance. 2 Epithelial to mesenchymal transition (EMT) is a highly conserved and fundamental process that not only governs morphogenesis but also cancer invasion and metastasis in multicellular organisms. There is good evidence that EMT gives rise to the dissemination of single carcinoma cells from the site of primary tumors. In addition, increasing evidence suggests that EMT could play a specific role in the migration of cells from a primary tumor into the blood circulation. EMT can require the cooperation of oncogenic RAS or tyrosine kinase receptor, with endogenous signaling molecules. It involves the transition from an epithelial to a fibroblastic or mesench...

Acetyl-CoA carboxylase I (ACCI) is a key lipogenic enzyme whose induction in islet ␤-cells may contribute to glucolipotoxicity. Here, we provide evidence that enhanced insulin release plays an important role in the activation of this gene by glucose. Glucose (30 vs. 3 mmol/l) increased ACCI mRNA levels ϳ4-fold and stimulated ACCI (pII) promoter activity >30-fold in MIN6 cells. The latter effect was completely suppressed by blockade of insulin release or of insulin receptor signaling. However, added insulin substantially, but not completely, mimicked the effects of glucose, suggesting that intracellular metabolites of glucose may also contribute to transcriptional stimulation. Mutational analysis of the ACCI promoter, and antibody microinjection, revealed that the effect of glucose required sterol response element binding protein (SREBP)-1c. Moreover, adenoviral transduction with dominant-negative-acting SREBP1c blocked ACCI gene induction, whereas constitutively active SREBP1c increased ACCI mRNA levels. Finally, glucose also stimulated SREBP1c transcription, although this effect was independent of insulin release. These data suggest that glucose regulates ACCI gene expression in the ␤-cell by complex mechanisms that may involve the covalent modification of SREBP1c. However, overexpression of SREBP1c also decreased glucose-stimulated insulin release, implicating SREBP1c induction in ␤-cell lipotoxicity in some forms of type 2 diabetes.

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Protein Kinase B (PKB, also known as Akt) is an important signalling molecule which has been shown to become activated in response to many stimuli, including insulin, growth factors and a variety of survival promoting agents. The signalling pathway by which insulin activates PKB has been well characterised. Insulin receptor ligation results in Phosphatidylinositol3' kinase activation, which leads to phosphorylation of PKB on its activatory sites (Thr 308 and Ser 473) following its translocation to the membrane. PKB is also activated by p adrenergic agonists (such as isoproterenol) in fat cells by an as yet unidentified mechanism. This is particularly interesting since these agonists usually produce opposing physiological effects to insulin. In order to elucidate the mechanism by which p adrenergic agonists activate PKB, we stimulated primary rat adipocytes with isoproterenol and other agents such as CPT-CAMP and forskolin. We then measured the activity of PKB and one of its downstream effectors, Glycogen Synthase Kinase 3 (GSK-3), in the presence and absence of inhibitors such as wortmannin and the PKA inhibitor H-89. We present evidence for the involvement of several different pathways in the regulation of PKB and GSK-3 by isoproterenol.Agonists in Rat Epididymal Fat Cells.