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

In the heart, detection of hyperpolarized (13)Cbicarbonate and (13)CO(2) by magnetic resonance (MR) after administration of hyperpolarized 1-(13)Cpyruvate is caused exclusively by oxidative decarboxylation of pyruvate via the pyruvate dehydrogenase complex (PDH). However, liver mitochondria possess alternative anabolic pathways accessible by 1-(13)Cpyruvate, which may allow a wider diagnostic range for hyperpolarized MR compared with other tissue. Metabolism of hyperpolarized 1-(13)Cpyruvate in the tricarboxylic acid (TCA) cycle was monitored in the isolated perfused liver from fed and fasted mice. Hyperpolarized 1-(13)Cpyruvate was rapidly converted to 1-(13)Clactate, 1-(13)Calanine, 1-(13)Cmalate, 4-(13)Cmalate, 1-(13)Caspartate, 4-(13)Caspartate, and (13)Cbicarbonate. 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 (13)Cbicarbonate was also detected, consistent with multiple mechanisms, including cataplerotic decarboxylation of 4-(13)Coxaloacetate via phosphoenolpyruvate carboxykinase (PEPCK), forward TCA cycle flux of 4-(13)Coxaloacetate to generate (13)CO(2) at isocitrate dehydrogenase, or decarboxylation of 1-(13)Cpyruvate by PDH. Isotopomer analysis of liver glutamate confirmed that anaplerosis was sevenfold greater than flux through PDH. In addition, signal from 4-(13)Cmalate and 4-(13)Caspartate was markedly blunted and signal from (13)Cbicarbonate was completely abolished in livers from PEPCK KO mice, indicating that the major pathway for entry of hyperpolarized 1-(13)Cpyruvate into the hepatic TCA cycle is via pyruvate carboxylase, and that cataplerotic flux through PEPCK is the primary source of (13)Cbicarbonate. We conclude that MR detection of hyperpolarized TCA intermediates and bicarbonate is diagnostic of pyruvate carboxylase and PEPCK flux in the liver.