Glycerol Regulation of Gluconeogenesis and Fatty Acid Metabolism

[Fasting hyperglycemia in diabetes mellitus (DM) is caused by unregulated hepatic gluconeogenesis (GNG), and the associated hyperinsulinemia promotes fatty acid (FA) and triglyceride (TAG) synthesis. Importantly the more common, insulin-resistant form of DM (T2DM) is less prevalent in women, even after controlling for obesity. Increased glucagon signaling, insulin resistance, and altered substrate availability have been offered as explanations, but the lack of correlation between hepatic gene expression and metabolic pathway function has hindered mechanistic progress. Understanding these limitations, our laboratories are uniquely positioned to study and integrate sex-specific differences in transcriptional regulation of GNG and lipogenic genes with direct measurement of metabolic pathways using 13C tracing and flux analyses. The overall hypothesis: glycerol metabolism is sex-dependent and crucial for both hepatic GNG and lipid metabolism. Several recent or new observations support this hypothesis: 1) Glycerol supplies the majority of the carbons for glucose and lactate synthesis in vivo. 2) Glycerol promotes its own conversion to glucose by simultaneously activating G6PC and inhibiting PCK1 expression. 3) E2 dramatically suppresses GK expression and glucose production from glycerol in primary hepatocytes (PHs) derived from ovariectomized mice. 4) Hepatic G3-P levels are low in liver- specific insulin receptor (Ir) and glycerol kinase (Gk) KO mice, demonstrating GK’s critical role in generating G3- P for GNG and TAG synthesis. 5) Liver-specific Gk KO eliminates glycerol as a GNG substrate, dramatically reducing total glucose production, hepatic expression of GNG and lipogenic genes and HFD-induced steatosis. 6) Glycerol metabolism increases the level of P300, a transcriptional co-activator and key regulator of hepatic function. Three specific aims are proposed: Aim 1 To characterize sex-dependent utilization of glycerol in obesity and T2DM mouse models. Employing obesity and T2DM models, estradiol (E2) depletion and replacement models, and a liver estrogen receptor KO model, we will study GNG substrate use in female and male mice using 13C labeled substrate infusions. Hypothesis: Glycerol use in GNG is sex-dependent and increased in obesity and T2DM. Aim 2 To determine the mechanism(s) by which glycerol regulates glucagon-stimulated GNG. Glycerol’s critical importance for glucose production in PHs suggests that mechanisms based on data obtained from PHs cultured in the absence of glycerol may be incorrect. Hypothesis: Glycerol augments glucagon- stimulated GNG by increasing G6pc gene expression while limiting PCK1 expression and substrates entering the lower GNG pathway. Aim 3 To determine how GK expression regulates hepatic GNG and lipid metabolism. We will study how insulin stimulates, and E2 suppresses GK expression, the former exacerbating and the latter mitigating hyperglycemia, elevated GNG, and hepatic steatosis. Hypothesis: Insulin and E2 regulate glycerol use as both a substrate for GNG and triglyceride synthesis and as a nutritional factor stimulating co-activator (P300) expression and regulating both GNG and lipogenesis transcription.]