Friday, July 25, 2014

Flux through hepatic pyruvate carboxylase and phosphoenolpyruvate carboxykinase detected by hyperpolarized 13C magnetic resonance


Merritt, M.E., et al., Flux through hepatic pyruvate carboxylase and phosphoenolpyruvate carboxykinase detected by hyperpolarized 13C magnetic resonance. Proc. Nat. Aca. Sci. USA, 2011. 108(47): p. 19084-19089.


In the heart, detection of hyperpolarized [13C]bicarbonate and 13CO2 by magnetic resonance (MR) after administration of hyperpolarized [1-13C]pyruvate is caused exclusively by oxidative decarboxylation of pyruvate via the pyruvate dehydrogenase complex (PDH). However, liver mitochondria possess alternative anabolic pathways accessible by [1-13C]pyruvate, which may allow a wider diagnostic range for hyperpolarized MR compared with other tissue. Metabolism of hyperpolarized [1-13C]pyruvate in the tricarboxylic acid (TCA) cycle was monitored in the isolated perfused liver from fed and fasted mice. Hyperpolarized [1-13C]pyruvate was rapidly converted to [1-13C]lactate, [1-13C]alanine, [1-13C]malate, [4-13C]malate, [1-13C]aspartate, [4-13C]aspartate, and [13C]bicarbonate. Livers from fasted animals had increased lactate:alanine, consistent with elevated NADH:NAD+. The appearance of asymmetrically enriched malate and aspartate indicated high rates of anaplerotic pyruvate carboxylase activity and incomplete equilibration with fumarate. Hyperpolarized [13C]bicarbonate was also detected, consistent with multiple mechanisms, including cataplerotic decarboxylation of [4-13C]oxaloacetate via phosphoenolpyruvate carboxykinase (PEPCK), forward TCA cycle flux of [4-13C]oxaloacetate to generate 13CO2 at isocitrate dehydrogenase, or decarboxylation of [1-13C]pyruvate by PDH. Isotopomer analysis of liver glutamate confirmed that anaplerosis was sevenfold greater than flux through PDH. In addition, signal from [4-13C]malate and [4-13C]aspartate was markedly blunted and signal from [13C]bicarbonate was completely abolished in livers from PEPCK KO mice, indicating that the major pathway for entry of hyperpolarized [1-13C]pyruvate into the hepatic TCA cycle is via pyruvate carboxylase, and that cataplerotic flux through PEPCK is the primary source of [13C]bicarbonate. We conclude that MR detection of hyperpolarized TCA intermediates and bicarbonate is diagnostic of pyruvate carboxylase and PEPCK flux in the liver.