Impaired Tricarboxylic Acid Cycle Activity in Mouse Livers Lacking Cytosolic Phosphoenolpyruvate Carboxykinase

Abstract

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....

Effects of insulin and cytosolic redox state on glucose production pathways in the isolated perfused mouse liver measured by integrated 2H and 13C NMR

“…More recently we used the isolated perfused mouse liver in conjunction with NMR spectroscopy to study glucose and energy metabolism in the isolated perfused mouse liver from liver specific PEPCK-null mice [24]. The primary goal of the present study was to evaluate the influence of variable redox state, substrate supply and insulin on glucose production, sources of glucose, O 2 consumption and energy homoeostasis in livers from freely fed mice using a combination of stable isotope tracers and NMR spectroscopy [24,27,28,32,42].…”

“…Absolute fluxes through the tricarboxylic acid cycle and associated pathways have been measured by an elegant combination of mass-isotopomer-distribution analysis ('MIDA') and substrate balance across the perfused rat liver [40] or by using a rigorous stoichiometric analysis of metabolite uptake and production [41]. More recently we used the isolated perfused mouse liver in conjunction with NMR spectroscopy to study glucose and energy metabolism in the isolated perfused mouse liver from liver specific PEPCK-null mice [24]. The primary goal of the present study was to evaluate the influence of variable redox state, substrate supply and insulin on glucose production, sources of glucose, O 2 consumption and energy homoeostasis in livers from freely fed mice using a combination of stable isotope tracers and NMR spectroscopy [24,27,28,32,42].…”

“…Each mouse was injected with heparin (0.05 ml, intraperitoneal) 15 min prior to a 0.1 ml intraperitoneal injection of anesthetic [ketamine (Fort Dodge Animal Health)/ xylazine (Boehringer Ingelheim), 85:15, w/w]. Once anesthetized, a midline laparotomy was performed to expose the hepatic portal vein and the livers were isolated and dissected using a previously described technique [24]. Briefly, the portal vein was cannulated and perfused with a buffered medium described below.…”

“…Once perfusion was initiated, the livers were dissected and suspended in a perfusion apparatus where temperature was maintained at 37 • C with a temperature-controlled water jacket. Efferent and afferent pO 2 was measured using a blood gas analyser (Instrumentation Laboratory, Lexington, MA, U.S.A.) to determine oxygen consumption [24]. Hepatic viability was evaluated by oxygen consumption and visual inspection.…”

Born to run; the story of the PEPCK-Cmus mouse

“…The importance of PEPCK-C for citric acid cycle flux has been well demonstrated in studies using mice in which the gene for PEPCK-C has been deleted, either in the whole animal [13] [14] or in the liver [14] [15] [16] [17]. Of course, removing citric acid cycle intermediates as PEP (via PEPCK-C) implies that the PEP itself will be further metabolized.…”