Metabolic priming by multiple enzyme systems supports glycolysis, HIF1α stabilisation, and human cancer cell survival in early hypoxia.

Adaptation to chronic hypoxia occurs through changes in protein expression, which are controlled by hypoxia-inducible factor 1α (HIF1α) and are necessary for cancer cell survival. However, the mechanisms that enable cancer cells to adapt in early hypoxia, before the HIF1α-mediated transcription programme is fully established, remain poorly understood. Here we show in human breast cancer cells, that within 3 h of hypoxia exposure, glycolytic flux increases in a HIF1α-independent manner but is limited by NAD+ availability. Glycolytic ATP maintenance and cell survival in early hypoxia rely on reserve lactate dehydrogenase A capacity as well as the activity of glutamate-oxoglutarate transaminase 1 (GOT1), an enzyme that fuels malate dehydrogenase 1 (MDH1)-derived NAD+. In addition, GOT1 maintains low α-ketoglutarate levels, thereby limiting prolyl hydroxylase activity to promote HIF1α stabilisation in early hypoxia and enable robust HIF1α target gene expression in later hypoxia. Our findings reveal that, in normoxia, multiple enzyme systems maintain cells in a primed state ready to support increased glycolysis and HIF1α stabilisation upon oxygen limitation, until other adaptive processes that require more time are fully established.

Systematic diet composition swap in a mouse genome-scale metabolic model reveals determinants of obesogenic diet metabolism in liver cancer.

Dietary nutrient availability and gene expression, together, influence tissue metabolic activity. Here, we explore whether altering dietary nutrient composition in the context of mouse liver cancer suffices to overcome chronic gene expression changes that arise from tumorigenesis and western-style diet (WD). We construct a mouse genome-scale metabolic model and estimate metabolic fluxes in liver tumors and non-tumoral tissue after computationally varying the composition of input diet. This approach, called Systematic Diet Composition Swap (SyDiCoS), revealed that, compared to a control diet, WD increases production of glycerol and succinate irrespective of specific tissue gene expression patterns. Conversely, differences in fatty acid utilization pathways between tumor and non-tumor liver are amplified with WD by both dietary carbohydrates and lipids together. Our data suggest that combined dietary component modifications may be required to normalize the distinctive metabolic patterns that underlie selective targeting of tumor metabolism.

MOG analogues to explore the MCT2 pharmacophore, α-ketoglutarate biology and cellular effects of N-oxalylglycine.

α-ketoglutarate (αKG) is a central metabolic node with a broad influence on cellular physiology. The αKG analogue N-oxalylglycine (NOG) and its membrane-permeable pro-drug derivative dimethyl-oxalylglycine (DMOG) have been extensively used as tools to study prolyl hydroxylases (PHDs) and other αKG-dependent processes. In cell culture media, DMOG is rapidly converted to MOG, which enters cells through monocarboxylate transporter MCT2, leading to intracellular NOG concentrations that are sufficiently high to inhibit glutaminolysis enzymes and cause cytotoxicity. Therefore, the degree of (D)MOG instability together with MCT2 expression levels determine the intracellular targets NOG engages with and, ultimately, its effects on cell viability. Here we designed and characterised a series of MOG analogues with the aims of improving compound stability and exploring the functional requirements for interaction with MCT2, a relatively understudied member of the SLC16 family. We report MOG analogues that maintain ability to enter cells via MCT2, and identify compounds that do not inhibit glutaminolysis or cause cytotoxicity but can still inhibit PHDs. We use these analogues to show that, under our experimental conditions, glutaminolysis-induced activation of mTORC1 can be uncoupled from PHD activity. Therefore, these new compounds can help deconvolute cellular effects that result from the polypharmacological action of NOG.

Waste soybean frying oil for the production, extraction, and characterization of cell-wall-associated lipases from Yarrowia lipolytica.

The lipolytic yeast Yarrowia lipolytica produces cell-wall-associated lipases, namely Lip7p and Lip8p, that could have interesting properties as catalyst either in free (released lipase fraction-RLF) or cell-associated (cell-bound lipase fraction-CBLF) forms. Herein, a mixture of waste soybean frying oil, yeast extract and bactopeptone was found to favor the enzyme production. Best parameters for lipase activation and release from the cell wall by means of acoustic wave treatment were defined as: 26 W/cm2 for 1 min for CBLF and 52 W/cm2 for 2 min for RLF. Optimal pH and temperature values for lipase activity together with storage conditions were similar for both the free enzyme and cell-associated one: pH 7.0; T = 37 °C; and > 70% residual activity for 60 days at 4, - 4 °C and for 15 days at 30 °C.

Author Correction: MCT2 mediates concentration-dependent inhibition of glutamine metabolism by MOG.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

MCT2 mediates concentration-dependent inhibition of glutamine metabolism by MOG.

α-Ketoglutarate (αKG) is a key node in many important metabolic pathways. The αKG analog N-oxalylglycine (NOG) and its cell-permeable prodrug dimethyloxalylglycine (DMOG) are extensively used to inhibit αKG-dependent dioxygenases. However, whether NOG interference with other αKG-dependent processes contributes to its mode of action remains poorly understood. Here we show that, in aqueous solutions, DMOG is rapidly hydrolyzed, yielding methyloxalylglycine (MOG). MOG elicits cytotoxicity in a manner that depends on its transport by monocarboxylate transporter 2 (MCT2) and is associated with decreased glutamine-derived tricarboxylic acid-cycle flux, suppressed mitochondrial respiration and decreased ATP production. MCT2-facilitated entry of MOG into cells leads to sufficiently high concentrations of NOG to inhibit multiple enzymes in glutamine metabolism, including glutamate dehydrogenase. These findings reveal that MCT2 dictates the mode of action of NOG by determining its intracellular concentration and have important implications for the use of (D)MOG in studying αKG-dependent signaling and metabolism.

Effects of high-fat feeding on ectopic fat storage and postprandial lipid metabolism in mouse offspring.

OBJECTIVE: Parental high-fat feeding was proposed to negatively impact metabolic health in offspring. Here, the ectopic fat storage in heart and liver in offspring was investigated, and the effects on mitochondrial function, de novo lipogenesis, and postprandial lipid metabolism were explored in detail. METHODS: Male and female mice received either a high-fat (HF) or standard chow (LF) diet during mating, gestation and lactation. All offspring animals received the HF diet. RESULTS: Abdominal visceral adipose tissue tended to be higher in HF/HF mice. Cardiac lipid content was also higher in the HF/HF mice (LF/HF vs. HF/HF: 1.03% ± 0.08% vs. 1.33% ± 0.07% of water signal, P = 0.01). In contrast, hepatic lipid content tended to be lower in HF/HF mice compared to LF/HF mice. A severely disturbed postprandial lipid clearance was revealed in HF/HF mice by the results from the triglyceride (TG) tolerance tests (LF/HF vs. HF/HF: 6,753 ± 2,213 vs. 14,367 ± 1,978 mmol l(-1)  min(-1) , P = 0.01) and (13) C-fatty acid retention test (LF/HF vs. HF/HF: 2.73% ± 0.85% vs. 0.89% ± 0.26% retention from bolus, P = 0.04), which may underlie the lower hepatic lipid content. CONCLUSIONS: Here it is shown that HF diet negatively impacts postprandial TG clearance in offspring and results in an overall metabolic unfavorable phenotype and ectopic lipid deposition in the heart and in visceral storage sites.

Dietary lipids do not contribute to the higher hepatic triglyceride levels of fructose- compared to glucose-fed mice.

Fructose consumption has been associated with the surge in obesity and dyslipidemia. This may be mediated by the fructose effects on hepatic lipids and ATP levels. Fructose metabolism provides carbons for de novo lipogenesis (DNL) and stimulates enterocyte secretion of apoB48. Thus, fructose-induced hepatic triglyceride (HTG) accumulation can be attributed to both DNL stimulation and dietary lipid absorption. The aim of this study was to assess the effects of fructose diet on HTG and ATP content and the contributions of dietary lipids and DNL to HTG. Measurements were performed in vivo in mice by magnetic resonance imaging (MRI) and novel magnetic resonance spectroscopy (MRS) approaches. Abdominal adipose tissue volume and intramyocellular lipid levels were comparable between 8-wk fructose- and glucose-fed mice. HTG levels were ∼1.5-fold higher in fructose-fed than in glucose-fed mice (P<0.05). Metabolic flux analysis by (13)C and (2)H MRS showed that this was not due to dietary lipid absorption, but due to DNL stimulation. The contribution of oral lipids to HTG was, after 5 h, 1.60 ± 0.23% for fructose and 2.16 ± 0.35% for glucose diets (P=0.26), whereas that of DNL was higher in fructose than in glucose diets (2.55±0.51 vs.1.13±0.24%, P=0.01). Hepatic energy status, assessed by (31)P MRS, was similar for fructose- and glucose-fed mice. Fructose-induced HTG accumulation is better explained by DNL and not by dietary lipid uptake, while not compromising ATP homeostasis.

Resolving futile glucose cycling and glycogenolytic contributions to plasma glucose levels following a glucose load.

PURPOSE: After a glucose load, futile glucose/glucose-6-phosphate (G6P) cycling (FGC) generates [2-(2) H]glucose from (2)H(2)O thereby mimicking a paradoxical glycogenolytic contribution to plasma glucose levels. Contributions of load and G6P derived from gluconeogenesis, FGC, and glycogenolysis to plasma glucose levels need resolution. A simple methodology is proposed integrating the administration of (2)H(2)O with a glucose load containing [1-(2)H, 1-(13)C]glucose and [2-(2)H, 2-(13)C]glucose. METHODS: Mice fasted for 6 (n = 7) or 24 h (n = 5) were intraperitoneally injected with 2 mg/g 10% enriched glucose in 35 µL/g (2)H(2)O. Plasma glucose enrichment was analyzed by (2)H NMR after 30 min. RESULTS: For 6-h fasted mice, 12.3 ± 1.5% of plasma glucose was pre-existing, 44.3 ± 2.7% was load derived, and 43.4 ± 1.8% G6P derived. G6P origins were 26.0 ± 2.0% gluconeogenesis, 10.9 ± 2.6% FGC, and 6.5 ± 3.4% glycogenolysis. For 24-h fasted mice, 18.2 ± 8.5% was pre-existing, 41.1 ± 5.0 % was load derived, and 40.8 ± 4.3% G6P derived. G6P origins were 27.1 ± 3.3% gluconeogenesis, 13.1 ± 2.8% FGC, and 0.6 ± 2.4% glycogenolysis. CONCLUSION: After a glucose load, glycogenolytic contribution to plasma glucose was negligible, whereas FGC was significant for both 6- and 24-h fasted mice.

Effect of cyclosporine A on hepatic carbohydrate metabolism and hepatic gene expression in rat.

OBJECTIVE: Cyclosporine A (CsA)-based therapy has been implicated in the development of diabetes. Hence, its effects on hepatic carbohydrate metabolism and gene expression will be investigated. METHODS: Sprague-Dawley rats given 15 mg/kg body weight/day of CsA for 20 days, as well as healthy untreated animals, received a glucose load enriched with [U-(13)C]glucose and deuterated water to resolve load and endogenous contributions to plasma glucose. Blood glucose and plasma insulin levels were assayed and at 60-min post-load, plasma glucose (13)C and (2)H-enrichments were analyzed by nuclear magnetic resonance spectroscopy and liver tissue analyzed for hepatic gene expression. RESULTS: CsA-treated rats were glucose intolerant relative to controls (AUC(glucose) = 21,297 ± 857 versus 14,183 ± 1094, p < 0.01). Contributions from endogenous glucose production (EGP) were significantly elevated in CsA-treated rats (179 ± 16 versus 123 ± 13 mg/dl, p < 0.05). The increased endogenous contributions were attributable to glycogenolysis or glucose-G6P cycling and not to gluconeogenesis. Significantly higher expressions of fatty acid synthase and acetyl-CoA carboxylase 1 and 2 genes were observed in CsA-treated rats. CONCLUSIONS: CsA-altered glucose metabolism and gene expression could reflect increased hepatic insulin resistance. In the liver of CsA-treated animals, EGP suppression is impaired whereas hepatic de novo lipogenesis is enhanced contributing to dysregulated glucose and lipid metabolism.

Resolving the sources of plasma glucose excursions following a glucose tolerance test in the rat with deuterated water and [U-13C]glucose.

Sources of plasma glucose excursions (PGE) following a glucose tolerance test enriched with [U-(13)C]glucose and deuterated water were directly resolved by (13)C and (2)H Nuclear Magnetic Resonance spectroscopy analysis of plasma glucose and water enrichments in rat. Plasma water (2)H-enrichment attained isotopic steady-state within 2-4 minutes following the load. The fraction of PGE derived from endogenous sources was determined from the ratio of plasma glucose position 2 and plasma water (2)H-enrichments. The fractional gluconeogenic contributions to PGE were obtained from plasma glucose positions 2 and 5 (2)H-positional enrichment ratios and load contributions were estimated from plasma [U-(13)C]glucose enrichments. At 15 minutes, the load contributed 26±5% of PGE while 14±2% originated from gluconeogenesis in healthy control rats. Between 15 and 120 minutes, the load contribution fell whereas the gluconeogenic contribution remained constant. High-fat fed animals had significant higher 120-minute blood glucose (173±6 mg/dL vs. 139±10 mg/dL, p<0.05) and gluconeogenic contributions to PGE (59±5 mg/dL vs. 38±3 mg/dL, p<0.01) relative to standard chow-fed controls. In summary, the endogenous and load components of PGE can be resolved during a glucose tolerance test and these measurements revealed that plasma glucose synthesis via gluconeogenesis remained active during the period immediately following a glucose load. In rats that were placed on high-fat diet, the development of glucose intolerance was associated with a significantly higher gluconeogenic contribution to plasma glucose levels after the load.

Ursodeoxycholic acid treatment of hepatic steatosis: a (13)C NMR metabolic study.

Ursodeoxycholic acid (UDCA) is commonly used for the treatment of hepatobiliary disorders. In this study, we tested whether a 4-week treatment with this bile acid (12-15 mg/kg/day) could improve hepatic fatty acid oxidation in obese Zucker rats - a model for nonalcoholic fatty liver disease and steatosis. After 24 h of fasting, livers were perfused with physiological concentrations of [U-(13) C]nonesterified fatty acids and [3-(13) C]lactate/[3-(13) C]pyruvate. Steatosis was associated with abundant intracellular glucose, lactate, alanine and methionine, and low concentrations of choline and betaine. Steatotic livers also showed the highest output of glucose and lactate. Glucose and glycolytic products were mostly unlabeled, indicating active glycogenolysis and glycolysis after 24 h of fasting. UDCA treatment resulted in a general amelioration of liver metabolic abnormalities with a decrease in intracellular glucose and lactate, as well as their output. Hepatic betaine and methionine were also normalized after UDCA treatment, suggesting the amelioration of anti-oxidative defenses. Choline levels were not affected by the bile acid, which may indicate a deficient synthesis of very-low-density lipoproteins. The percentage contribution of [U-(13) C]nonesterified fatty acids to acetyl-coenzyme A entering the tricarboxylic acid (TCA) cycle was significantly lower in livers from Zucker obese rats relative to control rats: 23.1 ± 4.9% versus 44.1 ± 2.7% (p < 0.01). UDCA treatment did not alter significantly fatty acid oxidation in control rats, but improved significantly oxidation in Zucker obese rats to 46.0 ± 6.1% (p > 0.05), comparable with control group values. The TCA cycle activity subsequent to fatty acid oxidation was reduced in steatotic livers and improved when UDCA was administered (0.24 ± 0.04 versus 0.37 ± 0.05, p = 0.05). We further suggest that the mechanism of action of UDCA is either related to the activity of the farnesoid receptor, or to the amelioration of the anti-oxidative defenses and cell nicotinamide adenine dinucleotide (NAD(+) /NADH) ratio, favoring TCA cycle activity and ß-oxidation.

Quantifying endogenous glucose production and contributing source fluxes from a single 2H NMR spectrum.

Endogenous glucose production (EGP), gluconeogenic and glycogenolytic fluxes by analysis of a single (2)H-NMR spectrum is demonstrated with 6-hr and 24-hr fasted rats. Animals were administered [1-(2)H, 1-(13)C]glucose, a novel tracer of glucose turnover, and (2)H(2)O. Plasma glucose enrichment from both tracers was quantified by (2)H-NMR analysis of monoacetone glucose. The 6-hr fasted group (n = 7) had EGP rates of 95.6 +/- 13.3 micromol/kg/min, where 56.2 +/- 7.9 micromol/kg/min were derived from PEP; 12.1 +/- 2.1 micromol/kg/min from glycerol, and 32.1 +/- 4.9 micromol/kg/min from glycogen. The 24-hr fasted group (n = 7) had significantly lower EGP rates (52.8 +/- 7.2 micromol/kg/min, P = 0.004 vs. 6 hr) mediated by a significantly reduced contribution from glycogen (4.7 +/- 5.9 micromol/kg/min, P = 0.02 vs. 6 hr) while PEP and glycerol contributions were not significantly different (39.5 +/- 3.9 and 8.5 +/- 1.2 micromol/kg/min, respectively). These estimates agree with previous assays of EGP fluxes in fasted rats obtained by multinuclear NMR analyses of plasma glucose enrichment from (2)H(2)O and (13)C-glucose tracers.

Elimination of glucose contamination from adipocyte glycogen extracts.

Glycogen was quantified in rat adipocytes by isolation using conventional KOH digestion and ethanol precipitation, followed by hydrolysis and spectrophotometric assay of the glucose product. A concentration of 0.193+/-0.020 micromol glucosyl units/10(6)cells was recorded. When this procedure was modified by including a 4h incubation with glucose oxidase prior to glycogen hydrolysis, the glycogen concentration was found to be 0.055+/-0.008 micromol glucosyl units/10(6) cells. Therefore in adipocytes, conventional glycogen assays give substantial overestimates due to incomplete removal of glucose during glycogen isolation. Contaminant glucose can be scavenged in a simple manner by incubation with glucose oxidase prior to glycogen hydrolysis.