Liver-specific phosphoenolpyruvate carboxykinase (PEPCK) null mice, when fasted, maintain normal whole body glucose kinetics but develop dramatic hepatic steatosis. To identify the abnormalities of hepatic energy generation that lead to steatosis during fasting, we studied metabolic fluxes in livers lacking hepatic cytosolic PEPCK by NMR using 2 H and 13 C tracers. After a 4-h fast, glucose production from glycogenolysis and conversion of glycerol to glucose remains normal, whereas gluconeogenesis from tricarboxylic acid (TCA) cycle intermediates was nearly absent. Upon an extended 24-h fast, livers that lack PEPCK exhibit both 2-fold lower glucose production and oxygen consumption, compared with the controls, with all glucose production being derived only from glycerol. The mitochondrial reductionoxidation (red-ox) state, as indicated by the NADH/ NAD ؉ ratio, is 5-fold higher, and hepatic TCA cycle intermediate concentrations are dramatically increased in the PEPCK null livers. Consistent with this, flux through the TCA cycle and pyruvate cycling pathways is 10-and 40-fold lower, respectively. Disruption of hepatic cataplerosis due to loss of PEPCK leads to the accumulation of TCA cycle intermediates and a nearly complete blockage of gluconeogenesis from amino acids and lactate (an energy demanding process) but intact gluconeogenesis from glycerol (which contributes to net NADH production). Inhibition of the TCA cycle and fatty acid oxidation due to increased TCA cycle intermediate concentrations and reduced mitochondrial red-ox state lead to the development of steatosis. Hepatic phosphoenolpyruvate carboxykinase (PEPCK)1 is a major control point for gluconeogenesis (1). Excess PEPCK expression in mice causes hyperglycemia (2), hyperinsulinemia, and increased glucose turnover (3). Inhibition of PEPCK by pharmaceutical interventions causes hypoglycemia (4) and, as expected, the global ablation of the cytosolic isoform of PEPCK in mice by genetic manipulation results in nonviable offspring (5). Most surprisingly, the liver-specific deletion of cytosolic PEPCK yielded a phenotype that, except during fasting and exercise, was virtually indistinguishable from control mice (5, 6). Even after a 24-h fast, when liver glycogen is depleted and flux through liver PEPCK should be essential to maintain plasma glucose, these animals are euglycemic and glucose turnover is normal. By using NMR spectroscopy and stable isotope tracers, we demonstrated that approximately ϳ60% of whole body glucose production in liver-specific PEPCK knock-out animals is derived from lactate and alanine (6). This suggests that either an alternative route to glucose production that bypasses PEPCK in these livers exists or that the majority of whole body gluconeogenesis is extrahepatic (6). In marked contrast to the minimal impact that the absence of hepatic PEPCK has on systemic glucose kinetics, these mice develop dramatic hepatic steatosis after fasting (6) even though enzymes of the TCA cycle and -oxidation are up-regulated (5) in liver tissue....
. Effect of murine strain on metabolic pathways of glucose production after brief or prolonged fasting. -Background strain is known to influence the way a genetic manipulation affects mouse phenotypes. Despite data that demonstrate variations in the primary phenotype of basic inbred strains of mice, there is limited data available about specific metabolic fluxes in vivo that may be responsible for the differences in strain phenotypes. In this study, a simple stable isotope tracer/NMR spectroscopic protocol has been used to compare metabolic fluxes in ICR, FVB/N (FVB), C57BL/6J (B6), and 129S1/SvImJ (129) mouse strains. After a short-term fast in these mice, there were no detectable differences in the pathway fluxes that contribute to glucose synthesis. However, after a 24-h fast, B6 mice retain some residual glycogenolysis compared with other strains. FVB mice also had a 30% higher in vivo phosphoenolpyruvate carboxykinase flux and total glucose production from the level of the TCA cycle compared with B6 and 129 strains, while total body glucose production in the 129 strain was ϳ30% lower than in either FVB or B6 mice. These data indicate that there are inherent differences in several pathways involving glucose metabolism of inbred strains of mice that may contribute to a phenotype after genetic manipulation in these animals. The techniques used here are amenable to use as a secondary or tertiary tool for studying mouse models with disruptions of intermediary metabolism. nuclear magnetic resonance; tricarboxylic acid cycle; phosphoenolpyruvate carboxykinase; metabolic flux; stable isotope tracers; deuterium; mouse phenotype THE MOUSE HAS BEEN EMPHASIZED in studies of type 2 diabetes and obesity because of genetic homologies with humans and the relative simplicity of generating targeted mutations. Because these disorders result from a complex interaction among multiple genes and the environment, it is attractive in principle to probe individual genes to determine the magnitude of their contribution to the disease phenotype. However, the potential for a single mutation to produce diabetes or obesity is exquisitely sensitive to the inbred strain background (8). For example, mutations have been described in both the leptin receptor gene and the leptin gene. In a B6 background, either mutation produces obesity and transient diabetes, but the same mutations produce a severe diabetes phenotype in the C57/BlKSJ strain (12). More recently, Colombo et al. (9) found that the depletion of adipose tissue and hyperinsulinemia in the A-ZIP/F-1 lipoatrophic mouse was similar in both the FVB and B6 inbred backgrounds, but on the B6 background, the hyperglycemia, hypertriglyceridemia, and mortality were all attenuated. It was suggested that the B6 strain has inherently higher triglyceride clearance in the liver, which may protect against adverse effects of circulating triglycerides. A related problem is the observation that disruption of critical features of glucose metabolism may fail to yield the expected result. For example, ...
A great deal is known about hepatic glucose production and its response to a variety of factors such as redox state, substrate supply and hormonal control, but the effects of these parameters on the flux through biochemical pathways which integrate to control glucose production are less clear. A combination of 13 C and [ 2 H]water tracers and NMR isotopomer analysis were used to investigate metabolic fluxes in response to altered cytosolic redox state and insulin. In livers isolated from fed mice and perfused with a mixture of substrates including lactate/pyruvate (10:1, w/w), hepatic glucose production had substantial contributions from glycogen, PEP (phosphoenolpyruvate) and glycerol. Inversion of the lactate/pyruvate ratio (1:10, w/w) resulted in a surprising decrease in the contribution from glycogen and an increase in that from PEP to glucose production. A change in the lactate/pyruvate ratio from 10:1 to 1:10 also stimulated flux through the tricarboxylic acid cycle (2-fold), while leaving oxygen consumption and overall glucose output unchanged. When lactate and pyruvate were eliminated from the perfusion medium, both gluconeogenesis and tricarboxylic-acid-cycle flux were dramatically lower. Insulin lowered glucose production by inhibiting glycogenolysis at both low and high doses, but only at high levels of insulin did gluconeogenesis or tricarboxylic-acid-cycle flux tend towards lower values (P < 0.1). Our data demonstrate that, in the isolated mouse liver, substrate availability and cellular redox state have a dramatic impact on liver metabolism in both the tricarboxylic acid cycle and gluconeogenesis. The tight correlation of these two pathways under multiple conditions suggest that interventions which increase or decrease hepatic tricarboxylic-acid-cycle flux will have a concomitant effect on gluconeogenesis and vice versa.
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