Type 2 Diabetes Mellitus (T2D) is a disease strongly characterized by the loss of functional pancreatic beta cell mass. The Carbohydrate Response Element Binding Protein (ChREBP) is a glucosesensing transcription factor necessary for glucosestimulated proliferation in pancreatic beta cells, a process that drives adaptive beta cell mass expansion. Understanding the regulation of ChREBP is therefore critical for developing therapeutic strategies geared towards the controlled expansion of functional pancreatic beta cell mass. Glycolytic metabolites, in particular, have been previously demonstrated to play a role in ChREBP activity, although none have been definitively confirmed. Consistent with the stimulus-secretory nature of pancreatic beta cells, we hypothesized that alternate metabolites may partake in ChREBP activation. In this study, four independent model systems were developed to assess different metabolic pathways for their ability to activate ChREBP by the biochemical and/or transgenic rewiring of metabolism. Here we will explore the contribution of lower glycolytic and mitochondrial metabolites as candidates. Using adenoviruses, we overexpressed either cytosolic Phosphoenolpyruvate Carboxykinase (PCK1) or Glycerol Kinase (GLYK) along with their respective substrates dimethyl malate (DMM) or glycerol. Overexpression of PCK1 with DMM resulted in an additive increase in glucose-stimulated mRNA levels. Furthermore, recruitment of ChREBP to target gene carbohydrate response element (ChoRE) sites was additively increased using a Chromatin Immunoprecipitation (ChIP) assay. BrdU incorporation and insulin release was augmented in response to these treatments. Overexpression of GLYK with glycerol dose-dependently increased mRNA levels of ChREBP target genes in low glucose, both in INS-1 832/13 cells and in primary rat and human islet beta cells. In addition, ChREBP was recruited to target gene ChoREs equally by Ad-GLYK plus glycerol and control adenoviruses plus high glucose in ChIP assays. Remarkably, GLYK plus glycerol (cultured in low glucose) stimulated proliferation in INS-1 832/13 cells and human islet beta cells to the same extent as high glucose. In order to specifically modulate rates of mitochondrial phosphoenolpyruvate (PEP) turnover, either the GDP or ADP-binding isoform of Succinyl CoA Synthetase (SCS) were stably overexpressed in INS-1 832/13 cells. The SCS-ATP cells exhibited attenuated glucose-stimulated gene expression, promoter activity, and proliferation in response to reduced mitochondrial PEP metabolism. Treatment of INS-1 832/13 cells with succinyl acid methyl ester (SAME), a mitochondrial intermediate, augmented ChREBP-β and L-PK mRNA levels. Mass spectrometry was subsequently performed in all model systems to correlate ChREBP-β mRNA levels to metabolite concentrations. While previously established metabolic activators of ChREBP partially correlated with activation, stronger correlations were measured with downstream glycolytic intermediates as well as Tricarboxylic Acid (TCA) Cycle metabolites. In conclusion, we have provided evidence that alternate metabolites may contribute to the activation of ChREBP. These effects are associated with functional enhancements in pancreatic beta cells and may provide insight for more targeted strategies for T2D therapeutics.
|Advisor:||Scott, Donald K.|
|Commitee:||Germain, Doris, Sobie, Eric A., Walsh, Martin J., Yang, Fajun|
|School:||Icahn School of Medicine at Mount Sinai|
|School Location:||United States -- New York|
|Source:||DAI-B 79/04(E), Dissertation Abstracts International|
|Keywords:||Beta cells, Chrebp, Glucose metabolism, Pancreas|
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