Search

Objective: Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism, insulin resistance, and hepatic steatosis (HS). Because dietary essential amino acid (EAA) supplementation has been shown to decrease HS in various populations, this study's objective was to determine whether supplementation would decrease HS in PCOS., Methods: A randomized, double-blind, crossover, placebo-controlled trial was conducted in 21 adolescents with PCOS (BMI 37.3 ± 6.5 kg/m 2 , age 15.6 ± 1.3 years). Liver fat, very low-density lipoprotein (VLDL) lipogenesis, and triacylglycerol (TG) metabolism were measured following each 28-day phase of placebo or EAA., Results: Compared to placebo, EAA was associated with no difference in body weight (p = 0.673). Two markers of liver health improved: HS was lower (-0.8% absolute, -7.5% relative reduction, p = 0.013), as was plasma aspartate aminotransferase (AST) (-8%, p = 0.004). Plasma TG (-9%, p = 0.015) and VLDL-TG (-21%, p = 0.031) were reduced as well. VLDL-TG palmitate derived from lipogenesis was not different between the phases, nor was insulin sensitivity (p > 0.400 for both). Surprisingly, during the EAA phase, participants reported consuming fewer carbohydrates (p = 0.038) and total sugars (p = 0.046)., Conclusions: Similar to studies in older adults, short-term EAA supplementation in adolescents resulted in significantly lower liver fat, AST, and plasma lipids and thus may prove to be an effective treatment in this population. Additional research is needed to elucidate the mechanisms for these effects., (© 2024 The Authors. Obesity published by Wiley Periodicals LLC on behalf of The Obesity Society.)

Stable isotopes are powerful tools to assess metabolism. 13 C labeling is detected using nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry (MS). MS has excellent sensitivity but generally cannot discriminate among different 13 C positions (isotopomers), whereas NMR is less sensitive but reports some isotopomers. Here, we develop an MS method that reports all 16 aspartate and 32 glutamate isotopomers while requiring less than 1% of the sample used for NMR. This method discriminates between pathways that result in the same number of 13 C labels in aspartate and glutamate, providing enhanced specificity over conventional MS. We demonstrate regional metabolic heterogeneity within human tumors, document the impact of fumarate hydratase (FH) deficiency in human renal cancers, and investigate the contributions of tricarboxylic acid (TCA) cycle turnover and CO 2 recycling to isotope labeling in vivo. This method can accompany NMR or standard MS to provide outstanding sensitivity in isotope-labeling experiments, particularly in vivo., Competing Interests: Declaration of interests R.J.D. is a founder and advisor at Atavistik Bio and serves on the Scientific Advisory Boards of Agios Pharmaceuticals, Vida Ventures, Droia Ventures, and Nirogy Therapeutics., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Renal metabolism is essential for kidney functions and energy homeostasis in the body. The TCA cycle is the hub of metabolism, but the metabolic activities of the cycle in the kidney have rarely been investigated. This study is to assess metabolic processes at the level of the TCA cycle in the kidney based on isotopomer distributions in multiple metabolites. Isolated rat kidneys were perfused with media containing common substrates including lactate and alanine for an hour. One group of kidneys received [U- 13 C 3 ]lactate instead of natural abundance lactate while the other group received [U- 13 C 3 ]alanine instead of natural abundance alanine. Perfused kidneys and effluent were prepared for analysis using NMR spectroscopy. 13 C-labeling patterns in glutamate, fumarate, aspartate and succinate from the kidney extracts showed that pyruvate carboxylase and oxidative metabolism through the TCA cycle were comparably very active, but pyruvate cycling and pyruvate dehydrogenase were relatively less active. Isotopomer analyses with fumarate and malate from effluent, however, indicated that pyruvate carboxylase was much more active than the TCA cycle and other metabolic processes. The reverse equilibrium of oxaloacetate with four-carbon intermediates of the cycle was nearly complete (92%), based on the ratio of [2,3,4- 13 C 3 ]/[1,2,3- 13 C 3 ] in aspartate or malate. 13 C enrichment in glucose with 13 C-lactate supply was higher than that with 13 C-alanine. Isotopomer analyses with multiple metabolites (i.e., glutamate, fumarate, aspartate, succinate and malate) allowed us to assess relative metabolic processes in the TCA cycle in the kidney supplied with [U- 13 C 3 ]lactate. Data from the analytes were generally consistent, indicating highly active pyruvate carboxylase and oxidative metabolism through the TCA cycle. Different 13 C-labeling patterns in analytes from the kidney extracts versus effluent suggested metabolic compartmentalization., (© 2023 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)

Background: Glycerol is a substrate for gluconeogenesis and fatty acid esterification in the liver, processes which are upregulated in obesity and may contribute to excess fat accumulation. Glycine and glutamate, in addition to cysteine, are components of glutathione, the major antioxidant in the liver. In principle, glycerol could be incorporated into glutathione via the TCA cycle or 3-phosphoglycerate, but it is unknown whether glycerol contributes to hepatic de novo glutathione biosynthesis., Methods: Glycerol metabolism to hepatic metabolic products including glutathione was examined in the liver from adolescents undergoing bariatric surgery. Participants received oral [U- 13 C 3 ]glycerol (50 mg/kg) prior to surgery and liver tissue (0.2-0.7g) was obtained during surgery. Glutathione, amino acids, and other water-soluble metabolites were extracted from the liver tissue and isotopomers were quantified with nuclear magnetic resonance spectroscopy., Results: Data were collected from 8 participants (2 male, 6 female; age 17.1 years [range 14-19]; BMI 47.4 kg/m 2 [range 41.3-63.3]). The concentrations of free glutamate, cysteine, and glycine were similar among participants, and so were the fractions of 13 C-labeled glutamate and glycine derived from [U- 13 C 3 ]glycerol. The signals from all component amino acids of glutathione - glutamate, cysteine and glycine - were strong and analyzed to obtain the relative concentrations of the antioxidant in the liver. The signals from glutathione containing [ 13 C 2 ]glycine or [ 13 C 2 ]glutamate derived from the [U- 13 C 3 ]glycerol drink were readily detected, and 13 C-labelling patterns in the moieties were consistent with the patterns in corresponding free amino acids from the de novo glutathione synthesis pathway. The newly synthesized glutathione with [U- 13 C 3 ]glycerol trended to be lower in obese adolescents with liver pathology., Conclusions: This is the first report of glycerol incorporation into glutathione through glycine or glutamate metabolism in human liver. This could represent a compensatory mechanism to increase glutathione in the setting of excess glycerol delivery to the liver., Competing Interests: Declaration of competing interest None., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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 13 C NMR of blood or tissue biopsies. Simple molecules such as 13 C-labeled glucose are readily administered to patients prior to surgical biopsies, and 13 C-labeled glycerol is easily administered orally to outpatients. Here, we review recent progress in practical applications of 13 C 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., (© 2022 John Wiley & Sons, Ltd.)

Background: Empagliflozin, a sodium glucose cotransporter 2 inhibitor, is a medication to treat type 2 diabetes. The effect of empagliflozin in persons without diabetes has received less attention. Here we conducted a randomized, double-blind placebo-controlled clinical trial to examine the effect of empagliflozin on plasma triglycerides in obese non-diabetic adults., Methods: Participants (n = 35; BMI ≥ 30 kg/m 2 ) underwent body composition assessments using MRI, and were randomly assigned to either placebo or empagliflozin (10 mg/d) for three months. At the baseline and post-treatment visit, after an overnight fast, blood was drawn for biochemical analysis. Participants received [U- 13 C 3 ]glycerol orally followed by multiple blood draws over 3 h to examine glycerol incorporation into triglycerides using NMR spectroscopy., Results: The changes in blood triglyceride concentration with empagliflozin therapy related to the mass of baseline visceral adipose tissue (VAT; r = 0.53, p = 0.04). Empagliflozin slightly lowered triglycerides in obese subjects with low VAT, but increased triglycerides in the subjects with high VAT. Consistently, empagliflozin effectively suppressed triglyceride synthesis following [U- 13 C 3 ]glycerol administration in the subjects with low VAT (p < 0.05), but not in the subjects with high VAT. The subjects with high VAT lost body weight after three months of empagliflozin treatment. In all subjects, about 20% of the triglyceride backbone originated from mitochondrial metabolism of glycerol., Conclusions: The effect of empagliflozin on triglycerides in obese adults differed depending on VAT. Empagliflozin suppressed triglyceride synthesis in the subjects with low VAT, but tended to increase triglycerides in those with high VAT., Competing Interests: All authors, except Dr. Neeland, declare no competing interests. Dr. Neeland is a former member of the scientific advisory board of AMRA Medical, and he has received honoraria, consulting, and speaker's bureau fees, and travel support from Boehringer-Ingelheim/Lilly Alliance, and a research grant from Novo Nordisk., (© 2021 The Authors.)

After administration of 13 C-labeled glucose, the activity of the pentose phosphate pathway (PPP) is often assessed by the distribution of 13 C in lactate. However, in some tissues, such as the well-oxygenated heart, the concentration of lactate may be too low for convenient analysis by NMR. Here, we examined 13 C-labeled glutamate as an alternative biomarker of the PPP in the heart. Isolated rat hearts were perfused with media containing [2,3- 13 C 2 ]glucose and the tissue extracts were analyzed. Metabolism of [2,3- 13 C 2 ]glucose yields [1,2- 13 C 2 ]pyruvate via glycolysis and [2,3- 13 C 2 ]pyruvate via the PPP. Pyruvate is in exchange with lactate or is further metabolized to glutamate through pyruvate dehydrogenase and the TCA cycle. A doublet from [4,5- 13 C 2 ]glutamate, indicating flux through the PPP, was readily detected in 13 C NMR of heart extracts even when the corresponding doublet from [2,3- 13 C 2 ]lactate was minimal. Benfotiamine, known to induce the PPP, caused an increase in production of [4,5- 13 C 2 ]glutamate. In rats receiving [2,3- 13 C 2 ]glucose, brain extracts showed well-resolved signals from both [2,3- 13 C 2 ]lactate and [4,5- 13 C 2 ]glutamate in 13 C NMR spectra. Assessment of the PPP in the brain based on glutamate had a strong linear correlation with lactate-based assessment. In summary, 13 C NMR analysis of glutamate enabled detection of the low PPP activity in isolated hearts. This analyte is an alternative to lactate for monitoring the PPP with the use of [2,3- 13 C 2 ]glucose., (© 2021 John Wiley & Sons, Ltd.)

Objective: Insulin resistance and altered hepatic mitochondrial function are central features of type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD), but the etiological role of these processes in disease progression remains unclear. Here we investigated the molecular links between insulin resistance, mitochondrial remodeling, and hepatic lipid accumulation., Methods: Hepatic insulin sensitivity, endogenous glucose production, and mitochondrial metabolic fluxes were determined in wild-type, obese (ob/ob) and pioglitazone-treatment obese mice using a combination of radiolabeled tracer and stable isotope NMR approaches. Mechanistic studies of pioglitazone action were performed in isolated primary hepatocytes, whilst molecular hepatic lipid species were profiled using shotgun lipidomics., Results: Livers from obese, insulin-resistant mice displayed augmented mitochondrial content and increased tricarboxylic acid cycle (TCA) cycle and pyruvate dehydrogenase (PDH) activities. Insulin sensitization with pioglitazone mitigated pyruvate-driven TCA cycle activity and PDH activation via both allosteric (intracellular pyruvate availability) and covalent (PDK4 and PDP2) mechanisms that were dependent on PPARγ activity in isolated primary hepatocytes. Improved mitochondrial function following pioglitazone treatment was entirely dissociated from changes in hepatic triglycerides, diacylglycerides, or fatty acids. Instead, we highlight a role for the mitochondrial phospholipid cardiolipin, which underwent pathological remodeling in livers from obese mice that was reversed by insulin sensitization., Conclusion: Our findings identify targetable mitochondrial features of T2D and NAFLD and highlight the benefit of insulin sensitization in managing the clinical burden of obesity-associated disease., (Copyright © 2020 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Background: In our recent study using [U- 13 C 3 ]glycerol, a small subset of hamsters showed an unusual profile of glycerol metabolism: negligible gluconeogenesis from glycerol plus conversion of glycerol to 1,3-propanediol (1,3PDO) and 3-hydroxypropionate (3HP) which were detected in the liver and blood. The purpose of the current study is to evaluate the association of these unusual glycerol products with other biochemical processes in the liver., Methods: Fasted hamsters received acetaminophen (400 mg/kg; n = 16) or saline (n = 10) intraperitoneally. After waiting 2 h, all the animals received [U- 13 C 3 ]glycerol intraperitoneally. Liver and blood were harvested 1 h after the glycerol injection for NMR analysis and gene expression assays., Results: 1,3PDO and 3HP derived from [U- 13 C 3 ]glycerol were detected in the liver and plasma of eight hamsters (two controls and six hamsters with acetaminophen treatment). Glycerol metabolism in the liver of these animals differed substantially from conventional metabolic pathways. [U- 13 C 3 ]glycerol was metabolized to acetyl-CoA as evidenced with downstream products detected in glutamate and β-hydroxybutyrate, yet 13 C labeling in pyruvate and glucose was minimal (p < 0.001, 13 C labeling difference in each metabolite). Expression of aldehyde dehydrogenases was enhanced in hamster livers with 1,3PDO and 3HP (p < 0.05)., Conclusion: Detection of 1,3PDO and 3HP in the hamster liver was associated with unorthodox metabolism of glycerol characterized by conversion of 3HP to acetyl-CoA followed by ketogenesis and oxidative metabolism through the TCA cycle. Additional mechanistic studies are needed to determine the causes of unusual glycerol metabolism in a subset of these hamsters., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

The liver regenerates NADPH via multiple pathways to maintain redox balance and reductive biosynthesis. The pentose phosphate pathway (PPP) contributes to hepatic lipogenesis by supplying NADPH, and it is thought to play a major role in response to oxidative stress. This study determined the significance of the PPP and related NADPH-regenerating enzymes in the liver under oxidative stress. Fasted hamsters received acetaminophen (400 mg/kg) to deplete glutathione in the liver and [U- 13 C 3 ]glycerol to measure the PPP activity by analysis of 13 C distribution in plasma glucose. Blood and liver were harvested to assess NADPH-producing enzymes, antioxidant defense, PPP, and other relevant biochemical processes. Acetaminophen caused glutathione depletion and decreased activities of glutathione peroxidase and catalase in the liver, but it did not change triglyceride synthesis. Although the PPP is potentially an abundant source of NADPH, its activity was decreased and the expression of glucose 6-phosphate dehydrogenase remained unchanged after acetaminophen treatment. The effects of acetaminophen on other NADPH-producing enzymes were complex. Isocitrate dehydrogenase 1 was overexpressed, both isocitrate dehydrogenase 2 and malic enzyme 1 were underexpressed, and methylenetetrahydrofolate dehydrogenase 1 remained unchanged. In summary, isocitrate dehydrogenase 1 was most sensitive to glutathione depletion caused by acetaminophen, but glucose 6-phosphate dehydrogenase, the regulatory enzyme of PPP, was not., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

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.)

Stable isotope tracers have been used to gain an understanding of integrative animal and human physiology. More commonly studied organ systems include hepatic glucose metabolism, lipolysis from adipose tissue, and whole body protein metabolism. Recent improvements in isotope methodology have included the use of novel physiologic methods/models and mathematical modeling of data during different physiologic states. Here we review some of the latest advancements in this field and highlight future research needs. First we discuss the use of an oral [U- 13 C 3 ]-glycerol tracer to determine the relative contribution of glycerol carbons to hepatic glucose production after first cycling through the tricarboxylic acid cycle, entry of glycerol into the pentose phosphate pathway or direct conversion of glycerol into the glucose. Second, we describe an adaptation of the established oral minimal model used to define postprandial glucose dynamics to include glycerol dynamics in an oral glucose tolerance test with a [ 2 H 5 ]-glycerol tracer to determine dynamic changes in lipolysis. Simulation results were optimized when parameters describing glycerol flux were determined with a hybrid approach using both tracer-based calculations and constrained parameter optimization. Both of these methodologies can be used to expand our knowledge of not only human physiology, but also the effects of various nutritional strategies and medications on metabolism., Competing Interests: Competing interests: Yes, there are competing interests for one or more authors and I have provided a Competing Interests statement in my manuscript., (© American Federation for Medical Research 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

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.

The pentose phosphate pathway (PPP) is essential for reductive biosynthesis, antioxidant processes and nucleotide production. Common tracers such as [1,2- 13 C 2 ]glucose rely on detection of 13 C in lactate and require assumptions to correct natural 13 C abundance. Here, we introduce a novel and specific tracer of the PPP, [2,3- 13 C 2 ]glucose. 13 C NMR analysis of the resulting isotopomers is informative because [1,2- 13 C 2 ]lactate arises from glycolysis and [2,3- 13 C 2 ]lactate arises exclusively through the PPP. A correction for natural abundance is unnecessary. In rats receiving [2,3- 13 C 2 ]glucose, the PPP was more active in the fed versus fasted state in the liver and the heart, consistent with increased expression of key enzymes in the PPP. Both the PPP and glycolysis were substantially increased in hepatoma compared with liver. In summary, [2,3- 13 C 2 ]glucose and 13 C NMR simplify assessment of the PPP., (© 2019 John Wiley & Sons, Ltd.)

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.

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. Eight- and 16-wk-old obese Zucker diabetic fatty rats and their lean littermates received [U- 13 C 3 ]glycerol, and 13 C 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.

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.)

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)C3]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)C3]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., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The source of hyperpolarized (HP) [(13)C]bicarbonate in the liver during metabolism of HP [1-(13)C]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 [(13)C]bicarbonate during metabolism of HP [1-(13)C]pyruvate by the liver in rats after 21 h of fasting compared to rats with free access to food. The (13)C NMR of HP [(13)C]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-(13)C]pyruvate. (13)C 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 [(13)C]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 [(13)C]bicarbonate exclusively reflects decarboxylation of HP [1-(13)C]pyruvate via pyruvate dehydrogenase., (© 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)

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.)

Metabolic reprogramming facilitates cancer cell growth, so quantitative metabolic flux measurements could produce useful biomarkers. However, current methods to analyze flux in vivo provide either a steady-state overview of relative activities (infusion of (13)C and analysis of extracted metabolites) or a dynamic view of a few reactions (hyperpolarized (13)C spectroscopy). Moreover, although hyperpolarization has successfully quantified pyruvate-lactate exchanges, its ability to assess mitochondrial pyruvate metabolism is unproven in cancer. Here, we combined (13)C hyperpolarization and isotopomer analysis to quantify multiple fates of pyruvate simultaneously. Two cancer cell lines with divergent pyruvate metabolism were incubated with thermally polarized [3-(13)C]pyruvate for several hours, then briefly exposed to hyperpolarized [1-(13)C]pyruvate during acquisition of NMR spectra using selective excitation to maximize detection of H[(13)C]O3(-) and [1-(13)C]lactate. Metabolites were then extracted and subjected to isotopomer analysis to determine relative rates of pathways involving [3-(13)C]pyruvate. Quantitation of hyperpolarized H[(13)C]O3(-) provided a single definitive metabolic rate, which was then used to convert relative rates derived from isotopomer analysis into quantitative fluxes. This revealed that H[(13)C]O3(-) appearance reflects activity of pyruvate dehydrogenase rather than pyruvate carboxylation followed by subsequent decarboxylation reactions. Glucose substantially altered [1-(13)C]pyruvate metabolism, enhancing exchanges with [1-(13)C]lactate and suppressing H[(13)C]O3(-) formation. Furthermore, inhibiting Akt, an oncogenic kinase that stimulates glycolysis, reversed these effects, indicating that metabolism of pyruvate by both LDH and pyruvate dehydrogenase is subject to the acute effects of oncogenic signaling on glycolysis. The data suggest that combining (13)C isotopomer analyses and dynamic hyperpolarized (13)C spectroscopy may enable quantitative flux measurements in living tumors.

During hepatic lipogenesis, the glycerol backbone of acylglycerols originates from one of three sources: glucose, glycerol, or substrates passing through the citric acid cycle via glyceroneogenesis. The relative contribution of each substrate source to glycerol in rat liver acylglycerols was determined using (13)C-enriched substrates and NMR. Animals received a fixed mixture of glucose, glycerol, and lactate; one group received [U-(13)C6]glucose, another received [U-(13)C3]glycerol, and the third received [U-(13)C3]lactate. After 3 h, the livers were harvested to extract fats, and the glycerol moiety from hydrolyzed acylglycerols was analyzed by (13)C NMR. In either fed or fasted animals, glucose and glycerol provided the majority of the glycerol backbone carbons, whereas the contribution of lactate was small. In fed animals, glucose contributed >50% of the total newly synthesized glycerol backbone, and 35% of this contribution occurred after glucose had passed through the citric acid cycle. By comparison, the glycerol contribution was ~40%, and of this, 17% of the exogenous glycerol passed first through the cycle. In fasted animals, exogenous glycerol became the major contributor to acylglycerols. The contribution from exogenous lactate did increase in fasted animals, but its overall contribution remained small. The contributions of glucose and glycerol that had passed through the citric acid cycle first increased in fasted animals from 35 to 71% for glucose and from 17 to 24% for glycerol. Thus, a substantial fraction from both substrate sources passed through the cycle prior to incorporation into the glycerol moiety of acylglycerols in the liver.

Studies of glycerol metabolism in the heart have largely emphasized its role in triglyceride synthesis. However, glycerol may also be oxidized in the citric acid cycle, and glycogen synthesis from glycerol has been reported in the nonmammalian myocardium. The intent of this study was to test the hypothesis that glycerol may be metabolized to glycogen in mammalian heart. Isolated rat hearts were supplied with a mixture of substrates including glucose, lactate, pyruvate, octanoate, [U-(13)C(3)]glycerol, and (2)H(2)O to probe various metabolic pathways including glycerol oxidation, glycolysis, the pentose phosphate pathway, and carbon sources of stored glycogen. NMR analysis confirmed that glycogen production from the level of the citric acid cycle did not occur and that the glycerol contribution to oxidation in the citric acid cycle was negligible in the presence of alternative substrates. Quite unexpectedly, (13)C from [U-(13)C(3)]glycerol appeared in glycogen in carbon positions 4-6 of glucosyl units but none in positions 1-3. The extent of [4,5,6-(13)C(3)]glucosyl unit enrichment in glycogen was enhanced by insulin but decreased by H(2)O(2). Given that triose phosphate isomerase is generally assumed to fully equilibrate carbon tracers in the triose pool, the marked (13)C asymmetry in glycogen can only be attributed to conversion of [U-(13)C(3)]glycerol to [U-(13)C(3)]dihydroxyacetone phosphate and [U-(13)C(3)]glyceraldehyde 3-phosphate followed by rearrangements in the nonoxidative branch of the pentose phosphate pathway involving transaldolase that places this (13)C-enriched 3-carbon unit only in the bottom half of hexose phosphate molecules contributing to glycogen.

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-(13)C(2)]glucose, (2)H(2)O, and [U-(13)C(3)]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 © 2013 Elsevier Inc. All rights reserved.)

Mitochondrial metabolism provides precursors to build macromolecules in growing cancer cells. In normally functioning tumour cell mitochondria, oxidative metabolism of glucose- and glutamine-derived carbon produces citrate and acetyl-coenzyme A for lipid synthesis, which is required for tumorigenesis. Yet some tumours harbour mutations in the citric acid cycle (CAC) or electron transport chain (ETC) that disable normal oxidative mitochondrial function, and it is unknown how cells from such tumours generate precursors for macromolecular synthesis. Here we show that tumour cells with defective mitochondria use glutamine-dependent reductive carboxylation rather than oxidative metabolism as the major pathway of citrate formation. This pathway uses mitochondrial and cytosolic isoforms of NADP(+)/NADPH-dependent isocitrate dehydrogenase, and subsequent metabolism of glutamine-derived citrate provides both the acetyl-coenzyme A for lipid synthesis and the four-carbon intermediates needed to produce the remaining CAC metabolites and related macromolecular precursors. This reductive, glutamine-dependent pathway is the dominant mode of metabolism in rapidly growing malignant cells containing mutations in complex I or complex III of the ETC, in patient-derived renal carcinoma cells with mutations in fumarate hydratase, and in cells with normal mitochondria subjected to acute pharmacological ETC inhibition. Our findings reveal the novel induction of a versatile glutamine-dependent pathway that reverses many of the reactions of the canonical CAC, supports tumour cell growth, and explains how cells generate pools of CAC intermediates in the face of impaired mitochondrial metabolism.

Tumor cells require a constant supply of macromolecular precursors, and interrupting this supply has been proposed as a therapeutic strategy in cancer. Precursors for lipids, nucleic acids, and proteins are generated in the tricarboxylic acid (TCA) cycle and removed from the mitochondria to participate in biosynthetic reactions. Refilling the pool of precursor molecules (anaplerosis) is therefore crucial to maintain cell growth. Many tumor cells use glutamine to feed anaplerosis. Here we studied how "glutamine-addicted" cells react to interruptions of glutamine metabolism. Silencing of glutaminase (GLS), which catalyzes the first step in glutamine-dependent anaplerosis, suppressed but did not eliminate the growth of glioblastoma cells in culture and in vivo. Profiling metabolic fluxes in GLS-suppressed cells revealed induction of a compensatory anaplerotic mechanism catalyzed by pyruvate carboxylase (PC), allowing the cells to use glucose-derived pyruvate rather than glutamine for anaplerosis. Although PC was dispensable when glutamine was available, forcing cells to adapt to low-glutamine conditions rendered them absolutely dependent on PC for growth. Furthermore, in other cell lines, measuring PC activity in nutrient-replete conditions predicted dependence on specific anaplerotic enzymes. Cells with high PC activity were resistant to GLS silencing and did not require glutamine for survival or growth, but displayed suppressed growth when PC was silenced. Thus, PC-mediated, glucose-dependent anaplerosis allows cells to achieve glutamine independence. Induction of PC during chronic suppression of glutamine metabolism may prove to be a mechanism of resistance to therapies targeting glutaminolysis.

In the adult heart, regulation of fatty acid oxidation and mitochondrial genes is controlled by the PPARgamma coactivator-1 (PGC-1) family of transcriptional coactivators. However, in response to pathological stressors such as hemodynamic load or ischemia, cardiac myocytes downregulate PGC-1 activity and fatty acid oxidation genes in preference for glucose metabolism pathways. Interestingly, despite the reduced PGC-1 activity, these pathological stressors are associated with mitochondrial biogenesis, at least initially. The transcription factors that regulate these changes in the setting of reduced PGC-1 are unknown, but Myc can regulate glucose metabolism and mitochondrial biogenesis during cell proliferation and tumorigenesis in cancer cells. Here we have demonstrated that Myc activation in the myocardium of adult mice increases glucose uptake and utilization, downregulates fatty acid oxidation by reducing PGC-1alpha levels, and induces mitochondrial biogenesis. Inactivation of Myc in the adult myocardium attenuated hypertrophic growth and decreased the expression of glycolytic and mitochondrial biogenesis genes in response to hemodynamic load. Surprisingly, the Myc-orchestrated metabolic alterations were associated with preserved cardiac function and improved recovery from ischemia. Our data suggest that Myc directly regulates glucose metabolism and mitochondrial biogenesis in cardiac myocytes and is an important regulator of energy metabolism in the heart in response to pathologic stress.

Conversion of lactate to glucose was examined in myotubes, minced muscle tissue, and rats exposed to 2H2O or 13C-enriched substrates. Myotubes or minced skeletal muscle incubated with [U-(13)C3]lactate released small amounts of [1,2,3-(13)C3]- or [4,5,6-(13)C3]glucose. This labeling pattern is consistent with direct transfer from lactate to glucose without randomization in the tricarboxylic acid (TCA) cycle. After exposure of incubated muscle to 2H2O, [U-(13)C3]lactate, glucose, and glutamine, there was minimal release of synthesized glucose to the medium based on a low level of 2H enrichment in medium glucose but 50- to 100-fold greater 2H enrichment in glucosyl units from glycogen. The 13C enrichment pattern in glycogen from incubated skeletal muscle was consistent only with direct transfer of lactate to glucose without exchange in TCA cycle intermediates. 13C nuclear magnetic resonance (NMR) spectra of glutamate from the same tissue showed flux from lactate through pyruvate dehydrogenase but not flux through pyruvate carboxylase into the TCA cycle. Carbon from an alternative substrate for glucose production that requires metabolism through the TCA cycle, propionate, did not enter glycogen, suggesting that TCA cycle intermediates do not exchange with phosphoenolpyruvate. In vivo, the 13C labeling patterns in hepatic glycogen and plasma glucose after administration of [U-(13)C3]lactate did not differ significantly. However, skeletal muscle glycogen was substantially enriched in [1,2,3-(13)C3]- and [4,5,6-(13)C3]glucose units that could only occur through skeletal muscle glyconeogenesis rather than glycogenesis. Lactate serves as a substrate for glyconeogenesis in vivo without exchange into symmetric intermediates of the TCA cycle.

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.

Sources of plasma glucose and glucose turnover were investigated in 8-week-old (pre-diabetic) and 13-week-old (diabetic) Zucker (fa/fa) rats after a 24-h fast. Intraperitoneal (2)H(2)O was administered and [3,4-(13)C(2)]glucose and [U-(13)C(3)]propionate were infused into conscious active rats. (13)C nuclear magnetic resonance analysis of monoacetone glucose derived from blood glucose indicated that glucose production was increased significantly in 8- and 13-week-old fa/fa rats compared with age-matched Zucker (+/+) rats, and hepatic glycogen was dramatically higher among fa/fa animals regardless of age. Glycogenolysis, essentially 0 in +/+ rats after a 24-h fast, was significant in fa/fa rats (11 +/- 6 and 17 +/- 7% of glucose production in 8- and 13-week-old rats, respectively), even after a 24-h fast. Tricarboxylic acid (TCA) cycle flux and efflux of carbon skeletons from the cycle (cataplerosis) were both significantly higher in fa/fa rats compared with controls, but net gluconeogenesis from the TCA cycle was not higher because products leaving the cycle were returned to the cycle via a pyruvate cycling pathway. Thus, pyruvate cycling flux increased in proportion to TCA cycle flux, leaving net gluconeogenesis unchanged in fa/fa animals compared with control animals. The distribution of (2)H in skeletal muscle glycogen suggested that at least a fraction of glucose molecules entering glycogen pass through phosphomannose isomerase.

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.

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.

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.