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Objective: The aim of this study was to determine the effects of empagliflozin on glycerol-derived hepatic gluconeogenesis in adults with obesity without type 2 diabetes mellitus (T2DM) using oral carbon 13 ( 13 C)-labeled glycerol., Methods: A randomized, double-blind, placebo-controlled trial was performed in participants with magnetic resonance imaging assessment of body fat and measurement of glycerol-derived 13 C enrichment in plasma glucose by nuclear magnetic resonance spectroscopy following ingestion of [U- 13 C 3 ]glycerol. Participants were randomized to oral empagliflozin 10 mg once daily or placebo for 3 months. Glycerol-derived 13 C enrichment studies were repeated, and treatment differences in the mean percentage of 13 C glycerol enrichment in glucose were compared using mixed linear models., Results: Thirty-five participants completed the study. Empagliflozin increased glycerol-derived 13 C enrichment between baseline and follow-up by 6.5% (P = 0.005), consistent with less glycerol from visceral adipose tissue (VAT). No difference was found with placebo. Glycerol-derived 13 C enrichment was lower in participants with high VAT compared with low VAT by 12.6% (P = 0.04), but there was no heterogeneity of the treatment effect by baseline VAT. Glycerol-derived 13 C enrichment was inversely correlated with VAT but was not correlated with weight loss., Conclusions: VAT is associated with endogenous glycerol-derived hepatic gluconeogenesis, and empagliflozin reduces endogenous glycerol gluconeogenesis in adults with obesity without T2DM. These findings suggest a mechanism by which sodium-glucose cotransporter 2 inhibitors may prevent T2DM in obesity., (© 2020 The Obesity Society.)

Background: Therapies targeting altered activity of pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) have been proposed for hepatomas. However, the activities of these pathways in hepatomas in vivo have not been distinguished. Here we examined pyruvate entry into the tricarboxylic acid (TCA) cycle through PDH versus PC in vivo using hepatoma-bearing rats., Methods: Hepatoma-bearing rats were generated by intrahepatic injection of H4IIE cells. Metabolism of 13 C-labeled glycerol, a physiological substrate for both gluconeogenesis and energy production, was measured with 13 C NMR analysis. The concentration of key metabolites and the expression of relevant enzymes were measured in hepatoma, surrounding liver, and normal liver., Results: In orthotopic hepatomas, pyruvate entry into the TCA cycle occurred exclusively through PDH and the excess PDH activity compared to normal liver was attributed to downregulated pyruvate dehydrogenase kinase (PDK) 2/4. However, pyruvate carboxylation via PC and gluconeogenesis were minimal, which was linked to downregulated forkhead box O1 (FoxO1) by Akt activity. In contrast to many studies of cancer metabolism, lactate production in hepatomas was not increased which corresponded to reduced expression of lactate dehydrogenase. The production of serine and glycine in hepatomas was enhanced, but glycine decarboxylase was downregulated., Conclusions: The combination of [U- 13 C 3 ]glycerol and NMR analysis enabled investigation of multiple biochemical processes in hepatomas and surrounding liver. We demonstrated active PDH and other related metabolic alterations in orthotopic hepatomas that differed substantially not only from the host organ but also from many earlier studies with cancer cells., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Hepatic energy metabolism is a key element in many metabolic diseases. Hepatic anaplerosis provides carbons for gluconeogenesis (GNG) and triglyceride (TG) synthesis. We aimed to optimize a protocol that measures hepatic anaplerotic contribution for GNG, TG synthesis, and hepatic pentose phosphate pathway (PPP) activity using a single dose of oral [U -13 C 3 ]glycerol paired with an oral sugar tolerance test (OSTT) in a population with significant insulin resistance. The OSTT (75 g glucose + 25 g fructose) was administered to eight obese adolescents with polycystic ovarian syndrome (PCOS) followed by ingestion of [U- 13 C 3 ]glycerol at t = 180 or t = 210 min. 13 C-labeling patterns of serum glucose and TG-glycerol were determined by nuclear magnetic resonance. 13 C enrichment in plasma TG-glycerol was detectable and stable from 240 to 390 min with the [U- 13 C 3 ]glycerol drink at t = 180 min(3.65 ± 2.3 to 4.47 ± 1.4%; P > 0.4), but the enrichment was undetectable at 240 min with the glycerol drink at t = 210 min. The relative contribution from anaplerosis was determined at the end of the OSTT [18.5 ±3.4% ( t = 180 min) vs. 16.0 ± 3.5% ( t = 210 min); P = 0.27]. [U- 13 C 3 ]glycerol was incorporated into GNG 390 min after the OSTT with an enrichment of 7.5-12.5%. Glucose derived from TCA cycle activity was 0.3-1%, and the PPP activity was 2.8-4.7%. In conclusion, it is possible to obtain relative measurements of hepatic anaplerotic contribution to both GNG and TG esterification following an OSTT in a highly insulin-resistant population using a minimally invasive technique. Tracer administration should be timed to allow enough de novo TG esterification and endogenous glucose release after the sugar drink.

It is a challenge to assess metabolic dysregulation in fatty liver of human patients prior to clinical manifestations. Here, we recruited obese, but otherwise healthy, subjects to examine biochemical processes in the liver with simple triglyceride accumulation using stable isotopes and NMR analysis of metabolic products in blood. Intrahepatic triglycerides were measured using 1 H magnetic resonance spectroscopy, and volunteers received 2 H 2 O and [U- 13 C 3 ]glycerol orally, followed by a series of blood draws. NMR analysis of plasma triglycerides and glucose provided detailed information about metabolic pathways in patients with simple hepatic steatosis. Compared with subjects with low hepatic fat, patients with hepatic steatosis were characterized by the following: lower 13 C enrichments in the glycerol backbones of triglycerides (i.e., TG-[ 13 C]glycerol), higher [U- 13 C 3 ]glycerol metabolism through the tricarboxylic acid (TCA) cycle, delayed gluconeogenesis from [U- 13 C 3 ]glycerol, and less flexibility in adjusting supporting fluxes of glucose production upon an oral load of glycerol. In summary, simple hepatic steatosis was associated with enhanced [U- 13 C 3 ]glycerol metabolism through pathways that intersect the TCA cycle and delayed gluconeogenesis from glycerol.

Objective: To determine the feasibility of using oral 13 C labeled glycerol to assess effects of visceral adiposity on gluconeogenic pathways in obese humans., Research Design and Methods: Obese (BMI ≥30kg/m 2 ) participants without type 2 diabetes underwent visceral adipose tissue (VAT) assessment and stratification by median VAT into high VAT-fasting (n=3), low VAT-fasting (n=4), and high VAT-refed (n=2) groups. Participants ingested [U- 13 C 3 ] glycerol and blood samples were subsequently analyzed at multiple time points over 3h by NMR spectroscopy. The fractions of plasma glucose (enrichment) derived from [U- 13 C 3 ] glycerol via hepatic gluconeogenesis, pentose phosphate pathway (PPP), and tricarboxylic acid (TCA) cycle were assessed using 13 C NMR analysis of glucose. Mixed linear models were used to compare 13 C enrichment in glucose between groups., Results: Mean age, BMI, and baseline glucose were 49years, 40.1kg/m 2 , and 98mg/dl, respectively. Up to 20% of glycerol was metabolized in the TCA cycle prior to gluconeogenesis and PPP activity was minor (<1% of total glucose) in all participants. There was a 21% decrease in 13 C enrichment in plasma glucose in the high VAT-fasting compared with low VAT-fasting group (p=0.03), suggesting dilution by endogenous glycerol. High VAT-refed participants had 37% less 13 C enrichment in glucose compared with high VAT-fasting (p=0.02). There was a trend toward lower [1,2- 13 C 2 ] (via PPP) and [5,6- 13 C 2 ]/[4,5,6- 13 C 3 ] (via TCA cycle) glucose in high VAT versus low VAT groups., Conclusions: We applied a simple method to detect gluconeogenesis from glycerol in obese humans. Our findings provide preliminary evidence that excess visceral fat disrupts multiple pathways in hepatic gluconeogenesis from glycerol., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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, whereas 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 (13)C-labeled in each experiment. After 3 h 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 (13)C 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 the skeletal muscle of rats with high plasma glucose. The activity of pyruvate carboxylase was low in muscle, and no PEP carboxykinase activity was detected., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Context: The ability of insulin to suppress hepatic glucose production is impaired among subjects with increased intrahepatic triglycerides (IHTG). However, little is known about the roles of insulin on the supporting fluxes of glucose production among patients with fatty liver., Objective: To evaluate the effects of insulin on fluxes through the three potential sources of plasma glucose (glycerol, the citric acid cycle, and glycogen) among patients with fatty liver. Design, Settings, Participants, and Intervention: Nineteen men with a range of IHTG (∼0.5% to 23%) were studied after an overnight fast and during hyperinsulinemia using magnetic resonance spectroscopy and stable isotope tracers., Main Outcome Measures: IHTG, gluconeogenesis from glycerol, gluconeogenesis from the citric acid cycle, glycogenolysis, and (13)C-labeled glucose produced from the citric acid cycle during hyperinsulinemia were measured., Results: Men with high IHTG had higher fluxes through all pathways contributing to glucose production during hyperinsulinemia, compared to men with low IHTG, but they had similar fluxes after the fast. Consequently, men with fatty liver had impaired insulin efficiency in suppressing total glucose production as well as fluxes through all three biochemical pathways contributing to glucose. The detection of glucose isotopomers with (13)C arising from [U-(13)C3]propionate ingested during hyperinsulinemia demonstrated continuous gluconeogenesis from the citric acid cycle in all subjects., Conclusions: These findings challenge the concept that individual glucose production pathways are selectively dysregulated during hepatic insulin resistance. Overproduction of glucose during hyperinsulinemia in men with fatty liver results from inadequate suppression of all the supporting fluxes of glucose production in response to insulin.

After exposure to [U-(13)C3]glycerol, the liver produces primarily [1,2,3-(13)C3]- and [4,5,6-(13)C3]glucose in equal proportions through gluconeogenesis from the level of trioses. Other (13)C-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 (13)C-labeling patterns in exported glucose. Here, we investigated (13)C labeling in plasma glucose in rats given [U-(13)C3]glycerol under various nutritional conditions. Blood was drawn at multiple time points to extract glucose for NMR analysis. Because the transaldolase reaction and incomplete equilibrium by triose phosphate isomerase cannot break a (13)C-(13)C bond within the trioses contributing to glucose, the appearance of [1,2-(13)C2]-, [2,3-(13)C2]-, [5,6-(13)C2]-, and [4,5-(13)C2]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-(13)C2]glucose/[2,3-(13)C2]glucose was significantly greater than [5,6-(13)C2]glucose/[4,5-(13)C2]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 (13)C distribution in blood glucose after [U-(13)C3]glycerol administration., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Obesity is a risk factor for type 2 diabetes in cats. The risk of developing diabetes is severalfold greater for male cats than for females, even after having been neutered early in life. The purpose of this study was to investigate the role of different metabolic pathways in the regulation of endogenous glucose production (EGP) during the fasted state considering these risk factors. A triple tracer protocol using (2)H(2)O, [U-(13)C(3)]propionate, and [3,4-(13)C(2)]glucose was applied in overnight-fasted cats (12 lean and 12 obese; equal sex distribution) fed three different diets. Compared with lean cats, obese cats had higher insulin (P < 0.001) but similar blood glucose concentrations. EGP was lower in obese cats (P < 0.001) due to lower glycogenolysis and gluconeogenesis (GNG; P < 0.03). Insulin, body mass index, and girth correlated negatively with EGP (P < 0.003). Female obese cats had approximately 1.5 times higher fluxes through phosphoenolpyruvate carboxykinase (P < 0.02) and citrate synthase (P < 0.05) than male obese cats. However, GNG was not higher because pyruvate cycling was increased 1.5-fold (P < 0.03). These results support the notion that fasted obese cats have lower hepatic EGP compared with lean cats and are still capable of maintaining fasting euglycemia, despite the well-documented existence of peripheral insulin resistance in obese cats. Our data further suggest that sex-related differences exist in the regulation of hepatic glucose metabolism in obese cats, suggesting that pyruvate cycling acts as a controlling mechanism to modulate EGP. Increased pyruvate cycling could therefore be an important factor in modulating the diabetes risk in female cats.

A recently introduced tracer, [3,4-(13)C(2)]glucose, was compared to the widely used tracer, [6,6-(2)H(2)]glucose, for measurement of whole-body glucose turnover. The rate of glucose production (GP) was measured in rats after primed infusions of [3,4-(13)C(2)]glucose, [6,6-(2)H(2)]glucose, or both tracers simultaneously followed by a constant infusion of tracer(s) over 90 min. Blood glucose was purified and converted into monoacetone glucose for analysis by (13)C NMR (for [3,4-(13)C(2)]glucose) or (1)H and (2)H NMR (for [6,6-(2)H(2)]glucose). The values of GP measured during infusion of each single tracer were not significantly different. In rats infused with both tracers simultaneously, GP was identical as reported by each tracer, 42 +/- 4 micromol/kg/min. Since (2)H and (13)C enrichment in glucose is typically much less than 2% for in vivo studies, [3,4-(13)C(2)]glucose does not interfere with measurements of (13)C or (2)H enrichment patterns and therefore is valuable when multiple metabolic pathways are being evaluated simultaneously.

A triple-tracer method was developed to provide absolute fluxes contributing to endogenous glucose production and hepatic tricarboxylic acid (TCA) cycle fluxes in 24-h-fasted rats by (2)H and (13)C nuclear magnetic resonance (NMR) analysis of a single glucose derivative. A primed, intravenous [3,4-(13)C(2)]glucose infusion was used to measure endogenous glucose production; intraperitoneal (2)H(2)O (to enrich total body water) was used to quantify sources of glucose (TCA cycle, glycerol, and glycogen), and intraperitoneal [U-(13)C(3)] propionate was used to quantify hepatic anaplerosis, pyruvate cycling, and TCA cycle flux. Plasma glucose was converted to monoacetone glucose (MAG), and a single (2)H and (13)C NMR spectrum of MAG provided the following metabolic data (all in units of micromol/kg/min; n = 6): endogenous glucose production (40.4+/-2.9), gluconeogenesis from glycerol (11.5+/-3.5), gluconeogenesis from the TCA cycle (67.3+/-5.6), glycogenolysis (1.0+/-0.8), pyruvate cycling (154.4+/-43.4), PEPCK flux (221.7+/-47.6), and TCA cycle flux (49.1+/-16.8). In a separate group of rats, glucose production was not different in the absence of (2)H(2)O and [U-(13)C]propionate, demonstrating that these tracers do not alter the measurement of glucose turnover.

The generally accepted metabolic concept that fructose 2,6-bisphosphate (Fru-2,6-P2) inhibits gluconeogenesis by directly inhibiting fructose 1,6-bisphosphatase is based entirely on in vitro observations. To establish whether gluconeogenesis is indeed inhibited by Fru-2,6-P2 in intact animals, a novel NMR method was developed using [U-13C]glucose and 2H2O as tracers. The method was used to estimate the sources of plasma glucose from gastric absorption of oral [U-13C]glucose, from gluconeogenesis, and from glycogen in 24-h fasted rats. Liver Fru-2,6-P2 increased approximately 10-fold shortly after the glucose load, reached a maximum at 60 min, and then dropped to base-line levels by 150 min. The gastric contribution to plasma glucose reached approximately 50% at 30 min after the glucose load and gradually decreased thereafter. Although the contribution of glycogen to plasma glucose was small, glucose formed from gluconeogenesis was substantial throughout the study period even when liver Fru-2,6-P2 was high. Liver glycogen repletion was also brisk throughout the study period, reaching approximately 30 micromol/g at 3 h. These data demonstrate that Fru-2,6-P2 does not inhibit gluconeogenesis significantly in vivo.

Advanced imaging technologies, large‐scale metabolomics, and the measurement of gene transcripts or enzyme expression all enable investigations of intermediary metabolism in human patients. Complementary information about fluxes in individual metabolic pathways may be obtained by ex vivo 13C NMR of blood or tissue biopsies. Simple molecules such as 13C‐labeled glucose are readily administered to patients prior to surgical biopsies, and 13C‐labeled glycerol is easily administered orally to outpatients. Here, we review recent progress in practical applications of 13C NMR to study cancer biology, the response to oxidative stress, gluconeogenesis, triglyceride synthesis in patients, as well as new insights into compartmentation of metabolism in the cytosol. The technical aspects of obtaining the sample, preparing material for analysis, and acquiring the spectra are relatively simple. This approach enables convenient, valuable, and quantitative insights into intermediary metabolism in patients. [ABSTRACT FROM AUTHOR]

The pentose phosphate pathway (PPP) is widely assumed to play a key role in both reductive biosynthesis and protection from oxidative stress because it is the major source of NADPH. However, little is known about the activity of the PPP in fatty liver, which is characterized by both oxidative stress and lipogenesis. This study was designed to test whether the PPP is active in parallel with lipogenesis and antioxidant processes in the fatty liver of whole animals. Eightand 16-wk-old obese Zucker diabetic fatty rats and their lean littermates received [U-13C3]glycerol, and 13C labeling patterns of glucose and triglycerides were analyzed for the assessment of hepatic PPP activity and the potentially related processes simultaneously. Oxidative stress, antioxidant activity, and NADPH-producing enzymes in the liver were further examined. Both PPP activity and lipogenesis increased in the fatty liver of young obese Zucker rats but decreased together in older obese Zucker rats. As expected, lipid peroxidation measured by malondialdehyde increased in the fatty liver of obese Zucker rats at both ages. However, evidence for antioxidant processes such as [glutathione] or activities of glutathione reductase, glutathione peroxidase, and catalase was not altered. Hepatic PPP activity paralleled lipogenesis but was dissociated from biomarkers of oxidative stress or antioxidant processes. In summary, NADPH from the PPP was presumably consumed for reductive biosynthesis rather than antioxidant defense in the fatty liver. [ABSTRACT FROM AUTHOR]

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 drawn 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. [ABSTRACT FROM AUTHOR]

The source of hyperpolarized (HP) [13C]bicarbonate in the liver during metabolism of HP [1-13C]pyruvate is uncertain and likely changes with physiology. Multiple processes including decarboxylation through pyruvate dehydrogenase or pyruvate carboxylase followed by subsequent decarboxylation via phosphoenolpyruvate carboxykinase (gluconeogenesis) could play a role. Here we tested which metabolic fate of pyruvate contributed to the appearance of HP [13C]bicarbonate during metabolism of HP [1-13C]pyruvate by the liver in rats after 21 h of fasting compared to rats with free access to food. The 13C NMR of HP [13C]bicarbonate was observed in the liver of fed rats, but not in fasted rats where pyruvate carboxylation and gluconeogenesis was active. To further explore the relative fluxes through pyruvate carboxylase versus pyruvate dehydrogenase in the liver under typical conditions of hyperpolarization studies, separate parallel experiments were performed with rats given non-hyperpolarized [2,3-13C]pyruvate. 13C NMR analysis of glutamate isolated from the liver of rats revealed that flux from injected pyruvate through pyruvate dehydrogenase was dominant under fed conditions whereas flux through pyruvate carboxylase dominated under fasted conditions. The NMR signal of HP [13C]bicarbonate does not parallel pyruvate carboxylase activity followed by subsequent decarboxylation reaction leading to glucose production. In the liver of healthy well-fed rats, the appearance of HP [13C]bicarbonate exclusively reflects decarboxylation of HP [1-13C]pyruvate via pyruvate dehydrogenase. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd. [ABSTRACT FROM AUTHOR]

Abstract: Objective: A three-day high-fat diet induces hepatic steatosis and hepatic insulin resistance in rats without altering fasting plasma glucose concentration or the rate of glucose production. However, as the nutrient profile available to the liver is substantially altered by a high-fat diet, we hypothesized that the relative fluxes supporting hepatic glucose production would be altered. Materials/Methods: To test this hypothesis, we used multiple tracers ([3,4-13C2]glucose, 2H2O, and [U-13C3]propionate) followed by NMR analysis of blood glucose to quantify net glucose production and the contributions of glycogen and key gluconeogenesis precursors in 4–5-h fasted rats. Results: NMR analysis demonstrated that the majority of blood glucose was derived from glycogen and the citric acid cycle, while a smaller fraction of glucose was derived from glycerol in both controls and high-fat-fed animals. High-fat feeding was associated with a two-fold increase in plasma glycerol concentration and an increase in the contribution (both fractional and absolute) of glycerol-gluconeogenesis. The increase in gluconeogenesis from glycerol tended to be balanced by a decrease in glycogenolysis. The absolute fluxes associated with the citric acid cycle including gluconeogenesis from the cycle intermediates, pyruvate cycling and the citric acid cycle flux itself, were not altered by this short high-fat diet. Conclusions: A short term high-fat diet altered the specific pathways for hepatic glucose production without influencing the overall rate of glucose production or flux in the citric acid cycle. [Copyright &y& Elsevier]

The metabolic mechanism of hepatic glucose overproduction was investigated in 3,3′-5-triiodo-L-thyronine (T3)-treated rats and Zucker diabetic fatty (ZDF) rats (fa/fa) after a 24-h fast. ²H2O and [U-13C3]propionate were administered intraperitoneally, and [3,4-13C2]glucose was administered as a primed infusion for 90 min under ketamine-xylazine anesthesia. 13C NMR analysis of monoacetone glucose derived from plasma glucose indicated that hepatic glucose production was twofold higher in both T3-treated rats and ZDF rats compared with controls, yet the sources of glucose overproduction differed significantly in the two models by ²H NMR analysis. In T3-treated rats, the hepatic glycogen content and hence the contribution of glycogenolysis to glucose production was essentially zero; in this case, excess glucose production was due to a dramatic increase in gluconeogenesis from TCA cycle intermediates. 13C NMR analysis also revealed increased phosphoenolpyruvate carboxykinase flux (4x), increased pyruvate cycling flux (4x), and increased TCA flux (5x) in T3-treated animals. ZDF rats had substantial glycogen stores after a 24-h fast, and consequently nearly 50% of plasma glucose originated from glycogenolysis; other fluxes related to the TCA cycle were not different from controls. The differing mechanisms of excess glucose production in these models were easily distinguished by integrated ²H and 13C NMR analysis of plasma glucose. [ABSTRACT FROM AUTHOR]