An Oral Load of [13C3]Glycerol and Blood NMR Analysis Detect Fatty Acid Esterification, Pentose Phosphate Pathway, and Glycerol Metabolism through the Tricarboxylic Acid Cycle in Human Liver

Abstract

Drugs and other interventions for high impact hepatic diseases often target biochemical pathways such as gluconeogenesis, lipogenesis, or the metabolic response to oxidative stress. However, traditional liver function tests do not provide quantitative data about these pathways. In this study, we developed a simple method to evaluate these processes by NMR analysis of plasma metabolites. Healthy subjects ingested [U-13 C 3 ]glycerol, and blood was drawn at multiple times. Each subject completed three visits under differing nutritional states. High resolution 13 C NMR spectra of plasma triacylglycerols and glucose provided new insights into a number of hepatic processes including fatty acid esterification, the pentose phosphate pathway, and gluconeogenesis through the tricarboxylic acid cycle. Fasting stimulated pentose phosphate pathway activity and metabolism of [U-13 C 3 ]glycerol in the tricarboxylic acid cycle prior to gluconeogenesis or glyceroneogenesis. Fatty acid esterification was transient in the fasted state but continuous under fed conditions. We conclude that a simple NMR analysis of blood metabolites provides an important biomarker of pentose phosphate pathway activity, triacylglycerol synthesis, and flux through anaplerotic pathways in mitochondria of human liver.

Effects of visceral adiposity on glycerol pathways in gluconeogenesis

“…Total 13 C enrichment in plasma glucose is measured by the sum of all glucose isotopomers with excess 13 C. Additional information about specific pathways is derived from specific glucose isotopomers [17, 18]. Initially, glycerol is phosphorylated in the liver by glycerol kinase and is converted to DHAP and GA3P.…”

“…Metabolism of [U- 13 C 3 ] glycerol to [U- 13 C 3 ] pyruvate followed by either decarboxylation to [U- 13 C 2 ] acetyl-CoA or carboxylation to [1,2,3- 13 C 3 ] oxaloacetate allows rearrangement of 13 C in the TCA cycle. Two possible phosphoenolpyruvate (PEP) isotopomers through glycerol metabolism in the TCA cycle are [1,2- 13 C 2 ]- and [2,3- 13 C 2 ]PEP [18]. The PEP metabolism to glucose will produce [4,5- 13 C 2 ]- or [5,6- 13 C 2 ] glucose (Figure 2).…”

“…Previs and colleagues found that a significant fraction of glucose derived from [U- 13 C] glycerol in rodents and non-human primates passed through the oxaloacetate pool in the TCA cycle prior to gluconeogenesis [16]. More recent studies in rodents [17] and healthy humans [18] demonstrated that most glycerol is metabolized directly to glucose but that a modest fraction enters the TCA cycle prior to re-synthesis to glucose and a small fraction of glycerol carbons are redistributed in the oxidative arm of the PPP. These observations are consistent with reports of bidirectional metabolism in the gluconeogenic and glycolytic pathways [19, 20].…”

Assessing the pentose phosphate pathway using [2, 3‐13C2]glucose

“…With additional measurements corrections are possible but require the assumption that the lactate pool is fully mixed. [U‐ 13 C 3 ]glycerol was used to detect hepatic PPP activity by analyzing 13 C‐labeling patterns in glucose because the PPP shares intermediates with gluconeogenesis . However, this approach requires active gluconeogenesis.…”

“…C-labeling patterns in glucose because the PPP shares intermediates with gluconeogenesis. 9,10 However, this approach requires active gluconeogenesis. Hyperpolarized [1-13 C 1 ]gluconolactone detects the PPP by production of hyperpolarized 13 C bicarbonate released in the 6phosphogluconate dehydrogenase reaction.…”