Mouse models designed to examine hepatic metabolism are critical to diabetes and obesity research. Thus, a microscale method to quantitatively assess hepatic glucose and intermediary metabolism in conscious, unrestrained mice was developed. [(13)C3]propionate, [(2)H2]water, and [6,6-(2)H2]glucose isotopes were delivered intravenously in short- (9 h) and long-term-fasted (19 h) C57BL/6J mice. GC-MS and mass isotopomer distribution (MID) analysis were performed on three 40-μl arterial plasma glucose samples obtained during the euglycemic isotopic steady state. Model-based regression of hepatic glucose and citric acid cycle (CAC)-related fluxes was performed using a comprehensive isotopomer model to track carbon and hydrogen atom transitions through the network and thereby simulate the MIDs of measured fragment ions. Glucose-6-phosphate production from glycogen diminished, and endogenous glucose production was exclusively gluconeogenic with prolonged fasting. Gluconeogenic flux from phosphoenolpyruvate (PEP) remained stable, whereas that from glycerol modestly increased from short- to long-term fasting. CAC flux [i.e., citrate synthase (VCS)] was reduced with long-term fasting. Interestingly, anaplerosis and cataplerosis increased with fast duration; accordingly, pyruvate carboxylation and the conversion of oxaloacetate to PEP were severalfold higher than VCS in long-term fasted mice. This method utilizes state-of-the-art in vivo methodology and comprehensive isotopomer modeling to quantify hepatic glucose and intermediary fluxes during physiological stress in mice. The small plasma requirements permit serial sampling without stress and the affirmation of steady-state glucose kinetics. Furthermore, the approach can accommodate a broad range of modeling assumptions, isotope tracers, and measurement inputs without the need to introduce ad hoc mathematical approximations.

A polymorphism located in the G6PC2 gene, which encodes an islet-specific glucose-6-phosphatase catalytic subunit, is the most important common determinant of variations in fasting blood glucose (FBG) levels in humans. Studies of G6pc2 knockout (KO) mice suggest that G6pc2 represents a negative regulator of basal glucose-stimulated insulin secretion (GSIS) that acts by hydrolyzing glucose-6-phosphate (G6P), thereby reducing glycolytic flux. However, this conclusion conflicts with the very low estimates for the rate of glucose cycling in pancreatic islets, as assessed using radioisotopes. We have reassessed the rate of glucose cycling in pancreatic islets using a novel stable isotope method. The data show much higher levels of glucose cycling than previously reported. In 5 mmol/L glucose, islets from C57BL/6J chow-fed mice cycled ∼16% of net glucose uptake. The cycling rate was further increased at 11 mmol/L glucose. Similar cycling rates were observed using islets from high fat–fed mice. Importantly, glucose cycling was abolished in G6pc2 KO mouse islets, confirming that G6pc2 opposes the action of the glucose sensor glucokinase by hydrolyzing G6P. The demonstration of high rates of glucose cycling in pancreatic islets explains why G6pc2 deletion enhances GSIS and why variants in G6PC2 affect FBG in humans.

In order to provide timely treatment for organ damage initiated by therapeutic drugs or exposure to environmental toxicants, we first need to identify markers that provide an early diagnosis of potential adverse effects before permanent damage occurs. Specifically, the liver, as a primary organ prone to toxicants-induced injuries, lacks diagnostic markers that are specific and sensitive to the early onset of injury. Here, to identify plasma metabolites as markers of early toxicant-induced injury, we used a constraint-based modeling approach with a genome-scale network reconstruction of rat liver metabolism to incorporate perturbations of gene expression induced by acetaminophen, a known hepatotoxicant. A comparison of the model results against the global metabolic profiling data revealed that our approach satisfactorily predicted altered plasma metabolite levels as early as 5 h after exposure to 2 g/kg of acetaminophen, and that 10 h after treatment the predictions significantly improved when we integrated measured central carbon fluxes. Our approach is solely driven by gene expression and physiological boundary conditions, and does not rely on any toxicant-specific model component. As such, it provides a mechanistic model that serves as a first step in identifying a list of putative plasma metabolites that could change due to toxicant-induced perturbations.

The G6PC1, G6PC2 and G6PC3 genes encode distinct glucose-6-phosphatase catalytic subunit (G6PC) isoforms. In mice, germline deletion of G6pc2 lowers fasting blood glucose (FBG) without affecting fasting plasma insulin (FPI) while, in isolated islets, glucose-6-phosphatase activity and glucose cycling are abolished and glucose-stimulated insulin secretion (GSIS) is enhanced at submaximal but not high glucose. These observations are all consistent with a model in which G6PC2 regulates the sensitivity of GSIS to glucose by opposing the action of glucokinase. G6PC2 is highly expressed in human and mouse islet beta cells however, various studies have shown trace G6PC2 expression in multiple tissues raising the possibility that G6PC2 also affects FBG through non-islet cell actions. Using real-time PCR we show here that expression of G6pc1 and/or G6pc3 are much greater than G6pc2 in peripheral tissues, whereas G6pc2 expression is much higher than G6pc3 in both pancreas and islets with G6pc1 expression not detected. In adult mice, beta cell-specific deletion of G6pc2 was sufficient to reduce FBG without changing FPI. In addition, electronic health record-derived phenotype analyses showed no association between G6PC2 expression and phenotypes clearly unrelated to islet function in humans. Finally, we show that germline G6pc2 deletion enhances glycolysis in mouse islets and that glucose cycling can also be detected in human islets. These observations are all consistent with a mechanism by which G6PC2 action in islets is sufficient to regulate the sensitivity of GSIS to glucose and hence influence FBG without affecting FPI.

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