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Research has been conducted on the histidine-containing phosphotransfer (HPt) domain. The effect of the HPt YPD1 protein on the phosphostability of the response regulator domains has been investigated and the results are discussed.

Glucose import down its concentration gradient via the glucose transporter GLUT4 is one of the primary physiologic roles of the insulin-signaling cascade. Insulin sensitivity is therefore highly correlated with the levels of GLUT4 protein in adipose and muscle tissues. GLUT4 expression is known to be regulated by diverse tissue-specific, hormonal, and/or metabolic controls, and a loss of GLUT4 protein in diabetes mellitus has been correlated to a decrease in the transcriptional rate. Elucidating the molecular mechanisms that control the GLUT4 promoter could therefore aid in the search for new interventions for insulin resistance and diabetes. Our work has previously identified two of the cis-acting sites within the smallest functional GLUT4 promoter as well as the transcription factors binding those sites, namely the MEF2-family members MEF2A and MEF2D, and the GLUT4 Enhancer Factor (GEF). We have also recently identified a post-translational modification of MEF2A that correlates with a model of decreased GLUT4 promoter activity. We are examining whether an observed hyperphosphorylation event occurs within insulin-responsive tissues under the streptozotocin-induced diabetic phenotype. Interestingly, hyperphosphorylation correlates with increased cytoplasmic localization of MEF2A in transfected cells, suggesting that hyperphosphorylation regulates nuclear MEF2A concentration. Previously, we observed that MEF2A and GEF function cooperatively to regulate GLUT4 gene expression. Using a biotinylated oligonucleotide corresponding to a truncated GLUT4 promoter that MEF2A and GEF interact with their cognate binding sites cooperatively, thus providing a molecular mechanism to account for the functional cooperativity. Further examination of the regulatory elements governing GLUT4 expression in vivo and in vitro will lead to the development of an system that recapitulates all the essential molecular mechanisms that control GLUT4 promoter activity, aiding in further study of potential therapeutics. ADA-Funded Research [ABSTRACT FROM AUTHOR]

Objective: As the obesity pandemic continues to expand, novel molecular targets to reduce obesity-related insulin resistance and Type 2 Diabetes (T2D) continue to be needed. We have recently shown that obesity is associated with reactivated liver Notch signaling, which, in turn, increases hepatic insulin resistance, opening up therapeutic avenues for Notch inhibitors to be repurposed for T2D. Herein, we tested the systemic effects of γ-secretase inhibitors (GSIs), which prevent endogenous Notch activation, and confirmed these effects through creation and characterization of two different adipocyte-specific Notch loss-of-function mouse models through genetic ablation of the Notch transcriptional effector Rbp-Jk (A-Rbpj) and the obligate γ-secretase component Nicastrin (A-Nicastrin)., Methods: Glucose homeostasis and both local adipose and systemic insulin sensitivity were examined in GSI-treated, A-Rbpj and A-Nicastrin mice, as well as vehicle-treated or control littermates, with complementary in vitro studies in primary hepatocytes and 3T3-L1 adipocytes., Results: GSI-treatment increases hepatic insulin sensitivity in obese mice but leads to reciprocal lowering of adipose glucose disposal. While A-Rbpj mice show normal body weight, adipose development and mass and unchanged adipose insulin sensitivity as control littermates, A-Nicastrin mice are relatively insulin-resistant, mirroring the GSI effect on adipose insulin action., Conclusions: Notch signaling is dispensable for normal adipocyte function, but adipocyte-specific γ-secretase blockade reduces adipose insulin sensitivity, suggesting that specific Notch inhibitors would be preferable to GSIs for application in T2D.

GLUT4 promoter activity is regulated by hormonal, metabolic, and tissue-specific controls. This complicates the study of GLUT4 gene transcription, as no cell culture model adequately recapitulates these extracellular regulators. While investigating cultured primary adipocytes as a model system for GLUT4 transcription, we observed that GLUT4 mRNA was specifically and rapidly downregulated upon tissue dispersal. Downregulation of GLUT4 mRNA was mediated in part by loss of regulatory control by the trans-acting factors that control GLUT4 transcriptional activity [the myocyte enhancer factor 2 (MEF2) transcription factor family and the GLUT4 enhancer factor] and their cognate DNA binding sites in transgenic mice. The differences in GLUT4 transcription when whole adipose tissue and cell culture model systems are compared can be correlated to a posttranslational phosphorylation of the transcription factor MEF2A. The difference in the MEF2A phosphorylation state in whole tissue vs. isolated cells may provide a further basis for the development of an in vitro system that could recapitulate fully regulated GLUT4 promoter activity. Development of an in vitro system to reconstitute GLUT4 transcriptional regulation will further efforts to discern the molecular mechanisms that underlie GLUT4 expression.

As the primary glucose transporter in skeletal muscle, GLUT4 is an important factor in the regulation of blood glucose. We previously reported that stimulation of AMP-activated protein kinase (AMPK) with 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) increased GLUT4 expression in muscle. GLUT4 enhancer factor (GEF) and myocyte enhancer factor 2 (MEF2) have been shown to be important for normal GLUT4 expression because deletion or truncation of the consensus sequences on the promoter causes depressed GLUT4 mRNA expression. This led to the current study to investigate possible roles for GEF and MEF2 in mediating the activation of GLUT4 gene transcription in response to AMPK. Here we show that, although AMPK does not appear to phosphorylate MEF2A, AMPK directly phosphorylates the GEF protein in vitro. MEF2 and GEF are activated in response to AMPK as we observed translocation of both to the nucleus after AICAR treatment. Nuclear MEF2 protein content was increased after 2 h, and GEF protein was increased in the nucleus 1 and 2 h post-AICAR treatment. Last, GEF and MEF2 increase in binding to the GLUT4 promoter within 2 h after AICAR treatment. Thus we conclude that GEF and MEF2 mediate the AMPK-induced increase in transcription of skeletal muscle GLUT4. AMPK can phosphorylate GEF and in response to AICAR, GEF, and MEF2 translocate to the nucleus and have increased binding to the GLUT4 promoter.