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 [Lipids]

July 18th, 2016 by Jin, E. S., Sherry, A. D., Malloy, C. R.

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-13C3]glycerol and blood was dawn at multiple times. Each subject completed three visits under differing nutritional states. High resolution 13C 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-13C3]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.
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Lactate contributes to glyceroneogenesis and glyconeogenesis in skeletal muscle by reversal of pyruvate kinase [Glycobiology and Extracellular Matrices]

October 21st, 2015 by Jin, E. S., Sherry, A. D., Malloy, C. R.

Phosphoenolpyruvate (PEP) generated from pyruvate is required for de novo synthesis of glycerol and glycogen in skeletal muscle. One possible pathway involves synthesis of PEP from the citric acid cycle intermediates via PEP carboxykinase while another could involve reversal of pyruvate kinase (PK). Earlier studies have reported that reverse flux through PK can contribute carbon precursors for glycogen synthesis in muscle but the physiological importance of this pathway remains uncertain especially in the setting of high plasma glucose. In addition, although PEP is a common intermediate for both glyconeogenesis and glyceroneogenesis, the importance of reverse PK in de novo glycerol synthesis has not been examined. Here we studied the contribution of reverse PK to synthesis of glycogen and the glycerol moiety of acylglycerols in skeletal muscle of animals with high plasma glucose. Rats received a single intraperitoneal bolus of glucose, glycerol and lactate under a fed or fasted state. Only one of the three substrates was 13C-labeled in each experiment. After 3 hours of normal awake activity, the animals were sacrificed and the contribution from each substrate to glycogen and the glycerol moiety of acylglycerols was evaluated. The fraction of 13C labeling in glycogen and the glycerol moiety exceeded the possible contribution from either plasma glucose or muscle oxaloacetate. The reverse PK served as a common route for both glyconeogenesis and glyceroneogenesis in skeletal muscle of rats with high plasma glucose. The activity of pyruvate carboxylase was low in muscle, and no PEP carboxykinase activity was detected.
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Interaction between the Pentose Phosphate Pathway and Gluconeogenesis from Glycerol in the Liver [Glycobiology and Extracellular Matrices]

October 6th, 2014 by Jin, E. S., Sherry, A. D., Malloy, C. R.

After exposure to [U-13C3]glycerol, the liver produces primarily [1,2,3-13C3]- and [4,5,6-13C3]glucose in equal proportions through gluconeogenesis from the level of trioses. Other 13C labeling patterns occur as a consequence of alternative pathways for glucose production. The pentose phosphate pathway (PPP), metabolism in the citric acid cycle, incomplete equilibration by triose phosphate isomerase, or the transaldolase reaction all interact to produce complex 13C labeling patterns in exported glucose. Here we investigated 13C labeling in plasma glucose in rats given [U-13C3]glycerol under various nutritional conditions. Blood was drawn at multiple time points to extract glucose for NMR analysis. Since the transaldolase reaction and incomplete equilibrium by triose phosphate isomerase cannot break a 13C-13C bond within the trioses contributing to glucose, the appearance of [1,2-13C2]-, [2,3-13C2]-, [5,6-13C2]- and [4,5-13C2]glucose provides direct evidence for metabolism of glycerol in the citric acid cycle or the PPP but not an influence of either triose phosphate isomerase or the transaldolase reaction. In all animals, [1,2-13C2]glucose / [2,3-13C2]glucose was significantly greater than [5,6-13C2]glucose / [4,5-13C2]glucose, a relationship that can only arise from gluconeogenesis followed by passage of substrates through the PPP. In summary, the hepatic PPP in vivo can be detected by 13C distribution in blood glucose after [U-13C3]glycerol administration.