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Modest hepatic overexpression of Notch1 protein increased fasted and refed glucose and insulin levels, suggestive of insulin resistance. With HFD, L Rbpj mice showed normal weight gain and body composition, Everolimus but lower insulin levels in the face of similar serum glucose, and improved glucose tolerance as compared to controls. Insulin tolerance tests were unaltered in L Rbpj animals. L Rbpj livers showed increased Akt1 phosphorylation and lower fasted G6pc protein levels, indicative of increased hepatic insulin sensitivity. These data indicate that ablation of hepatic Notch signaling protects from diet induced insulin resistance. Notch1 induces G6pc expression in a FoxO1 dependent manner We transduced primary mouse hepatocytes with adenoviruses expressing N1 IC 18, FoxO1 ADA 19 or GFP, and analyzed gluconeogenic gene expression. Consistent with previous studies, transduction with FoxO1 ADA increased G6pc expression19, whereas transduction with N1 IC did not. The combination of N1 IC and FoxO1 ADA synergistically induced G6pc as compared to FoxO1 ADA Plastid alone and increased glucose release into culture medium. We also saw synergistic induction of canonical Notch targets, but not traditional FoxO1 targets such as Pck1 or Igfbp1, a finding recapitulated in luciferase assays using promoters containing either Rbp Jk or FoxO1 binding sites. When co transduced with FoxO1 ADA DBD 16, N1 IC was unable to induce G6pc, indicating that Notch1 requires FoxO1 DNA binding to regulate G6pc. To delineate the requirement of FoxO1 in Notch1 induced expression of G6pc, we performed luciferase assays using G6pc promoter reporter constructs. The G6pc promoter contains a conserved Rbp Cathepsin Inhibitor 1 J binding element 1. 1kb upstream of the transcriptional start site. N1 IC was able to induce luciferase activity only when we used constructs containing both Rbp Jk as well as functional FoxO1 binding sites. We saw similar using recombinant DLL4 that activates endogenous Notch signaling. Rbp J binds to the G6pc promoter during fasting Based on our luciferase data, we hypothesized that Rbp J directly binds to the G6pc promoter. Chromatin immunoprecipitation experiments showed a four fold enrichment of Rbp J binding to a G6pc promoter sequence containing the putative Rbp Jk element in control and L Foxo1, but not L Rbpj mice. No binding was seen in other regions of the G6pc promoter. Consistent with increased hepatic Notch1 activation in the fasted state, this binding was seen only during fasting. Gain of hepatic Notch1 function leads to insulin resistance As adenovirus mediated gene delivery leads to hepatocyte predominant expression, we employed this technique to determine effects of N1 IC in liver20. We noted increased G6pc expression in livers of mice transduced with N1 IC, as well as some but not all FoxO1 targets, providing further evidence that Notch1 regulates hepatic gluconeogenesis by inducing G6pc.