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

摘要

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.