Metabolic effects of an essential amino acid supplement in adolescents with PCOS and obesity.

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/m2, 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.

Comprehensive isotopomer analysis of glutamate and aspartate in small tissue samples.

Stable isotopes are powerful tools to assess metabolism. 13C labeling is detected using nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry (MS). MS has excellent sensitivity but generally cannot discriminate among different 13C 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 13C 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 CO2 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.

Isotopomer analyses with the tricarboxylic acid cycle intermediates and exchanging metabolites from the rat kidney.

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 C3 ]lactate instead of natural abundance lactate while the other group received [U-13 C3 ]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 C3 ]/[1,2,3-13 C3 ] 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 C3 ]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.

Glycerol as a precursor for hepatic de novo glutathione synthesis in human liver.

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-13C3]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/m2 [range 41.3-63.3]). The concentrations of free glutamate, cysteine, and glycine were similar among participants, and so were the fractions of 13C-labeled glutamate and glycine derived from [U-13C3]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 [13C2]glycine or [13C2]glutamate derived from the [U-13C3]glycerol drink were readily detected, and 13C-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-13C3]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.

Recent progress in analysis of intermediary metabolism by ex vivo 13 C NMR.

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.

A randomized clinical trial evaluating the effect of empagliflozin on triglycerides in obese adults: Role of visceral fat.

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/m2) 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-13C3]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-13C3]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.

13 C NMR of glutamate for monitoring the pentose phosphate pathway in myocardium.

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 C2 ]glucose and the tissue extracts were analyzed. Metabolism of [2,3-13 C2 ]glucose yields [1,2-13 C2 ]pyruvate via glycolysis and [2,3-13 C2 ]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 C2 ]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 C2 ]lactate was minimal. Benfotiamine, known to induce the PPP, caused an increase in production of [4,5-13 C2 ]glutamate. In rats receiving [2,3-13 C2 ]glucose, brain extracts showed well-resolved signals from both [2,3-13 C2 ]lactate and [4,5-13 C2 ]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 C2 ]glucose.

The presence of 3-hydroxypropionate and 1,3-propanediol suggests an alternative path for conversion of glycerol to Acetyl-CoA.

BACKGROUND: In our recent study using [U-13C3]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-13C3]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-13C3]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-13C3]glycerol was metabolized to acetyl-CoA as evidenced with downstream products detected in glutamate and ß-hydroxybutyrate, yet 13C labeling in pyruvate and glucose was minimal (p < 0.001, 13C 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.

Insulin resistance is mechanistically linked to hepatic mitochondrial remodeling in non-alcoholic fatty liver disease.

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.

Divergent effects of glutathione depletion on isocitrate dehydrogenase 1 and the pentose phosphate pathway in hamster liver.

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 C3 ]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.

Effects of Empagliflozin Treatment on Glycerol-Derived Hepatic Gluconeogenesis in Adults with Obesity: A Randomized Clinical Trial.

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 C3 ]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.

Advances in stable isotope tracer methodology part 1: hepatic metabolism via isotopomer analysis and postprandial lipolysis modeling.

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-13C3]-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 [2H5]-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.

Active pyruvate dehydrogenase and impaired gluconeogenesis in orthotopic hepatomas of rats.

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 13C-labeled glycerol, a physiological substrate for both gluconeogenesis and energy production, was measured with 13C 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-13C3]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.

A simple method to monitor hepatic gluconeogenesis and triglyceride synthesis following oral sugar tolerance test in obese adolescents.

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-13C3]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-13C3]glycerol at t = 180 or t = 210 min. 13C-labeling patterns of serum glucose and TG-glycerol were determined by nuclear magnetic resonance. 13C enrichment in plasma TG-glycerol was detectable and stable from 240 to 390 min with the [U-13C3]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-13C3]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.

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

The pentose phosphate pathway (PPP) is essential for reductive biosynthesis, antioxidant processes and nucleotide production. Common tracers such as [1,2-13 C2 ]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 C2 ]glucose. 13 C NMR analysis of the resulting isotopomers is informative because [1,2-13 C2 ]lactate arises from glycolysis and [2,3-13 C2 ]lactate arises exclusively through the PPP. A correction for natural abundance is unnecessary. In rats receiving [2,3-13 C2 ]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 C2 ]glucose and 13 C NMR simplify assessment of the PPP.

Fatty liver disrupts glycerol metabolism in gluconeogenic and lipogenic pathways in humans.

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 1H magnetic resonance spectroscopy, and volunteers received 2H2O and [U-13C3]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 13C enrichments in the glycerol backbones of triglycerides (i.e., TG-[13C]glycerol), higher [U-13C3]glycerol metabolism through the tricarboxylic acid (TCA) cycle, delayed gluconeogenesis from [U-13C3]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-13C3]glycerol metabolism through pathways that intersect the TCA cycle and delayed gluconeogenesis from glycerol.

Pentose phosphate pathway activity parallels lipogenesis but not antioxidant processes in rat liver.

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-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.

Effects of visceral adiposity on glycerol pathways in gluconeogenesis.

OBJECTIVE: To determine the feasibility of using oral 13C labeled glycerol to assess effects of visceral adiposity on gluconeogenic pathways in obese humans. RESEARCH DESIGN AND METHODS: Obese (BMI ≥30kg/m2) 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-13C3] 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-13C3] glycerol via hepatic gluconeogenesis, pentose phosphate pathway (PPP), and tricarboxylic acid (TCA) cycle were assessed using 13C NMR analysis of glucose. Mixed linear models were used to compare 13C enrichment in glucose between groups. RESULTS: Mean age, BMI, and baseline glucose were 49years, 40.1kg/m2, 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 13C 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 13C enrichment in glucose compared with high VAT-fasting (p=0.02). There was a trend toward lower [1,2-13C2] (via PPP) and [5,6-13C2]/[4,5,6-13C3] (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.

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.

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.