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

Abstract

In the heart, detection of hyperpolarized [ 13 H epatic metabolism is comprised of a network of exergonic reactions that facilitate energy capture, such as fatty acid oxidation, carbohydrate oxidation and flux through the tricarboxylic acid (TCA) cycle, and endergonic reactions required for gluconeogenesis, lipogenesis, and amino acid biosynthesis. The dysregulation of these pathways underlie the pathology of a variety of diseases, including diabetes, cancer, and inborn errors of metabolism, making their diagnostic evaluation critically important. In particular, the hepatic TCA cycle is an important diagnostic target because its intermediates integrate lipid, carbohydrate, and amino acid metabolism during normal physiology. As examples, oxaloacetate is the common intermediate by which all noncarbohydrate sources enter gluconeogenesis; citrate is required to shuttle acetyl-CoA from the mitochondria for cytosolic lipogenesis; and the ketoacids of the TCA cycle serve as intermediates of amino acid synthesis and catabolism. The interconnectivity of these hepatic pathways makes their examination difficult but worthwhile because technologies that detect these fluxes provide important advances for basic and clinical investigation of mechanisms of disease.Dynamic nuclear polarization (DNP) of 13 C is a powerful molecular imaging technology that uses principles of magnetic resonance (MR) (1). Prepolarization of 13 C-labeled tracers enhances the sensitivity of MR by 10,000-fold or more and enables studies of cancer (2), hepatic metabolism (3, 4), and cardiac metabolism (5-7). In the heart, metabolism of hyperpolarized [1-13 C]pyruvate to 13 CO 2 and [ 13 C]bicarbonate is caused exclusively by flux through the pyruvate dehydrogenase complex (PDH). Consequently, 13 CO 2 detection is not necessarily indicative of flux in the TCA cycle, because non-PDH sources of acetyl-CoA (e.g., fat oxidation) dominate the metabolism of certain tissues, like the liver. Recently, DNP was used to observe altered cytosolic redox state in liver following ethanol administration (3), and hyperpolarized bicarbonate was observed in vivo after administration of [1-13 C]lactate, but analysis of oxidative versus biosynthetic metabolism in the liver has not been reported. In contrast to the heart, hepatic anaplerosis is four-to sixfold higher than acetyl-CoA oxidation (8)(9)(10) C]pyruvate, followed by rapid but incomplete "backwards" equilibration with the symmetric intermediate, fumarate. The polarization profiles of these hepatic metabolites changed as anticipated during feeding and fasting. Signal from hyperpolarized [ 13 C]bicarbonate was also observed. A 13 C isotopomer analysis of glutamate isolated from separate livers demonstrated that flux through PEPCK is ∼sevenfold higher than flux through PDH, indicating that flux through PEPCK may be a significant source of the The authors declare no conflict of interest.

In Vivo Hyperpolarized Carbon-13 Magnetic Resonance Spectroscopy Reveals Increased Pyruvate Carboxylase Flux in an Insulin-Resistant Mouse Model

“…The anaplerotic role of pyruvate was observed by its conversion into OAA, a vital intermediate metabolite involved in gluconeogenesis and oxidative phosphorylation. [1- 13 4,12 A representative time course displaying the progression of metabolite signals is shown in Fig. 2B.…”

“…From the 1 Singapore Bioimaging Consortium, Singapore; 2 Clinical Imaging Research Centre, National University of Singapore, Center for Translation Medicine, Singapore; 3 Metabolism in Human Diseases, Institute of Molecular and Cell Biology, Singapore; and 4 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.…”

“…The recent development of hyperpolarized 13 C MRS addresses this problem by improving the signal-to-noise ratio (SNR) by more than 10,000-fold, 3 making it possible to visualize uptake of 13 C labeled pyruvate in the liver and its subsequent metabolic conversion catalyzed by specific enzymes in real time. 4,5 In gluconeogenesis, the conversion of pyruvate into phosphoenolpyruvate (PEP) in the liver is accomplished in two enzyme-mediated steps: anaplerosis of pyruvate into oxaloacetate (OAA) catalyzed by pyruvate carboxylase (PC), followed by conversion of OAA into PEP mediated by PEP carboxykinase (PEPCK). PEPCK is commonly considered the control point for liver gluconeogenesis and its overexpression leads to hyperglycemia.…”

Simultaneous Steady-state and Dynamic 13C NMR Can Differentiate Alternative Routes of Pyruvate Metabolism in Living Cancer Cells

“…These experiments are an extension of combining oxygen consumption measurements with isotopomer analysis data in perfused hearts and livers (45,48). In those cases, isotopomer analysis was used to evaluate the amount of H[…”

“…2), the lack of observable [ 13 C]citrate suggests that forward cycling does not contribute significantly to the H[ 13 C]O 3 Ϫ signal. Even the perfused liver, which contains abundant PC activity, produces only minimal amounts of observable hyperpolarized [6-13 C]citrate (45). Pyruvate cycling may also contribute to decarboxylation of pyruvate at C1.…”