Glutamine Oxidation Research Articles

Abstract 994: Monitoring glucose, glutamine and fatty acid metabolism to determine metabolic vulnerability of tumor cells in vitro

Abstract Tumor cells display a metabolism that is strikingly different from that of the normal tissue from which they originated. This metabolic alteration results from activation of oncogenes and/or loss of tumor suppressor genes controlling cellular metabolic pathways as well as adaptation to their microenvironments and provides energy and cellular building blocks, allowing tumor cells to proliferate and survive. The altered metabolism of glucose and glutamine are becoming one of the hallmarks of tumor cells. We present a method that enables rapid assessment of the ability of tumor cells to catabolize the three major nutrient substrates, glucose, glutamine and fatty acids in a microplate using the XF Extracellular Flux Analyzer. Cellular oxygen consumption rate is used as an indicator of substrate oxidation by the mitochondrial respiratory chain upon substrate addition and extracellular acidification rate upon glucose addition is used as an indicator of glycolysis. Furthermore, the maximal capacity of glycolysis and of mitochondrial substrate oxidation is determined by forcing one of the two energy-producing pathways to operate at maximum. In addition, pharmacological inhibition of distinct metabolic pathways allows us to discern the biochemical pathways used, i.e., whether glutamine is metabolized through aminotransferase or glutamate dehydrogenase. Using this approach, we have demonstrated that the nutrient levels in culture medium can markedly shift the ability of cancer cells to use glucose for glycolysis versus oxidation of glutamine and fatty acids and is accompanied by modulation of cell proliferation rate. For example, excessive nutrients induced activation of fatty acid oxidation and enhanced glutamine oxidation in myc-expressing SF188 glioblastoma cells. This shift in substrate use also sensitized these cells to inhibition of fatty acid oxidation. We observed a concomitant increase in the maximal capacity of the mitochondrial respiratory chain with elevated substrate oxidation. In contrast, the apparent glycolysis rate of SF188 cells was much reduced but the glycolytic capacity was retained, which implies these cells have the flexibility to channel glucose to either energy or building block production required for cell mass duplication. We suggest that this type of dynamic analysis provides a rapid means to determine the ability of various types of tumor cells to use glucose, glutamine and fatty acids as well as the maximal capacity of the machineries of glycolysis and substrate oxidation, which may be indicative of metabolic vulnerability and therefore targets of intervention of a particular tumor cell type. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 994. doi:10.1158/1538-7445.AM2011-994

Effects of α-ketoglutarate on energy status in the intestinal mucosa of weaned piglets chronically challenged with lipopolysaccharide

The present study determined whether α-ketoglutarate (AKG) might affect the expression of AMP-activated protein kinase (AMPK) and energy status in the intestinal mucosa of piglets challenged with Escherichia coli lipopolysaccharide (LPS). A total of eighteen piglets (weaned at 21d of age) were allocated to one of three treatments: (1) non-challenged (control); (2) LPS-challenged (LPS); (3) LPS+1% AKG (LPS+AKG). Piglets in the control and LPS groups were fed a maize- and soyabean meal-based diet, and the LPS+AKG group was fed the basal diet supplemented with 1% AKG. On days 10, 12, 14 and 16 of the trial, piglets in the LPS and LPS+AKG groups were challenged with LPS (80μg/kg body weight), whereas piglets in the control group received the same volume of sterile saline. Pigs were euthanised 24h after the last administration of LPS or saline to obtain intestinal mucosae for biochemical analysis. Compared with the control group, LPS administration decreased (P<0·05) the oxidation of AKG, oleic acid, glutamine and glucose in enterocytes, decreased concentrations of ATP in the duodenal and jejunal mucosae and decreased adenylate energy charge (AMP:ATP ratio) in the jejunal and ileal mucosae. Additionally, LPS treatment reduced (P<0·05) mucosal concentrations of phosphorylated AMPK in the jejunum and ileum as well as acetyl-CoA carboxylase in all segments of the small intestine. The adverse effects of LPS were reversed by AKG. Collectively, these results indicate that dietary supplementation with 1% AKG beneficially modulates the AMPK signalling pathway to improve energy status in the small intestine of LPS-challenged piglets.

Estimation of intracellular fluxes in cerebellar neurons after hypoglycemia: Importance of the pyruvate recycling pathway and glutamine oxidation

Although glucose is the primary cerebral fuel, the brain is able to metabolize other substrates in hypoglycemia. Nevertheless, the metabolic consequences of this pathology at the cellular level remain largely unknown. Taking advantage of the metabolic flux analysis (MFA) methodology, this work was aimed at investigating and quantifying the effects of hypoglycemia on cerebellar neurons. After 12 hr without glucose, primary cultures were incubated with medium containing [1,6-(13)C]glucose and unlabeled glutamine, and metabolism was monitored for 30 hr. Metabolic rates of glucose, lactate, and amino acids were determined based on cell supernatant analysis and used to estimate metabolic fluxes with MFA. Percent (13)C enrichment time profiles of different keto and amino acids were measured by mass spectrometry in cell extracts and compared with the MFA results. Hypoglycemia decreased the glucose uptake rate and glycolytic metabolism by 35% whereas glutamine uptake was increased fourfold. Flux estimations fit well with data from (13)C labeling dynamics, indicating a significant activation of the pyruvate recycling (PR) pathway, accounting for 43% of the total pyruvate synthesized under control conditions and up to 71% after hypoglycemia. Increased PR appeared to be due mainly to increased glutamine oxidation given the higher label dilution observed in the hypoglycemia group. In summary, this work provides new evidence for PR as an important pathway for glutamine oxidation in cerebellar neurons, particularly after glucose deprivation.

Brain Glutamine Synthesis Requires Neuronal Aspartate: A Commentary

Inspired by the paper, 'Brain glutamine synthesis requires neuronal-born aspartate as amino donor for glial glutamate formation' by Pardo et al, a modified model of oxidation-reduction, transamination, and mitochondrial carrier reactions involved in aspartate-dependent astrocytic glutamine synthesis and oxidation is proposed. The alternative model retains the need for cytosolic aspartate for transamination of α-ketoglutarate, but the 'missing' aspartate molecule is generated within astrocytes during subsequent glutamate oxidation. Oxaloacetate formed during glutamate formation is used during glutamate degradation, and all transmitochondrial reactions, oxidations-reductions, and cytosolic and mitochondrial transaminations are stoichiometrically balanced. The model is consistent with experimental observations made by Pardo et al.

Mechanism of Hyperinsulinism in Short-chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency Involves Activation of Glutamate Dehydrogenase

The mechanism of insulin dysregulation in children with hyperinsulinism associated with inactivating mutations of short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) was examined in mice with a knock-out of the hadh gene (hadh(-/-)). The hadh(-/-) mice had reduced levels of plasma glucose and elevated plasma insulin levels, similar to children with SCHAD deficiency. hadh(-/-) mice were hypersensitive to oral amino acid with decrease of glucose level and elevation of insulin. Hypersensitivity to oral amino acid in hadh(-/-) mice can be explained by abnormal insulin responses to a physiological mixture of amino acids and increased sensitivity to leucine stimulation in isolated perifused islets. Measurement of cytosolic calcium showed normal basal levels and abnormal responses to amino acids in hadh(-/-) islets. Leucine, glutamine, and alanine are responsible for amino acid hypersensitivity in islets. hadh(-/-) islets have lower intracellular glutamate and aspartate levels, and this decrease can be prevented by high glucose. hadh(-/-) islets also have increased [U-(14)C]glutamine oxidation. In contrast, hadh(-/-) mice have similar glucose tolerance and insulin sensitivity compared with controls. Perifused hadh(-/-) islets showed no differences from controls in response to glucose-stimulated insulin secretion, even with addition of either a medium-chain fatty acid (octanoate) or a long-chain fatty acid (palmitate). Pull-down experiments with SCHAD, anti-SCHAD, or anti-GDH antibodies showed protein-protein interactions between SCHAD and GDH. GDH enzyme kinetics of hadh(-/-) islets showed an increase in GDH affinity for its substrate, α-ketoglutarate. These studies indicate that SCHAD deficiency causes hyperinsulinism by activation of GDH via loss of inhibitory regulation of GDH by SCHAD.

Balancing biosynthesis and bioenergetics: metabolic programs in oncogenesis

Cancer biologists' search for new chemotherapy targets is reinvigorating the study of how cancer cell metabolism determines both oncogenic potential and chemotherapeutic responses. Oncogenic metabolic programs support the bioenergetics associated with resistance to programed cell death and provide biosynthetic building blocks for cell growth and mitogenesis. Both signal transduction pathway activation and direct mutations in key metabolic enzymes can activate the metabolic programs that support cancer cell growth. Cancer-associated metabolic programs include glycolysis, glutamine oxidation, and fatty acid metabolism. Recent observations are revealing the regulatory mechanisms that activate cancer-associated metabolism, and the competitive advantages provided to transformed cells by their metabolic programs. In this study, we review recent results illustrating the mechanisms and functional impact of each of these oncogenic metabolic programs in cancer cell growth and survival.

The Mitochondrial 2-Oxoglutarate Carrier Is Part of a Metabolic Pathway That Mediates Glucose- and Glutamine-stimulated Insulin Secretion

Glucose-stimulated insulin secretion from pancreatic islet beta-cells is dependent in part on pyruvate cycling through the pyruvate/isocitrate pathway, which generates cytosolic alpha-ketoglutarate, also known as 2-oxoglutarate (2OG). Here, we have investigated if mitochondrial transport of 2OG through the 2-oxoglutarate carrier (OGC) participates in control of nutrient-stimulated insulin secretion. Suppression of OGC in clonal pancreatic beta-cells (832/13 cells) and isolated rat islets by adenovirus-mediated delivery of small interfering RNA significantly decreased glucose-stimulated insulin secretion. OGC suppression also reduced insulin secretion in response to glutamine plus the glutamate dehydrogenase activator 2-amino-2-norbornane carboxylic acid. Nutrient-stimulated increases in glucose usage, glucose oxidation, glutamine oxidation, or ATP:ADP ratio were not affected by OGC knockdown, whereas suppression of OGC resulted in a significant decrease in the NADPH:NADP(+) ratio during stimulation with glucose but not glutamine + 2-amino-2-norbornane carboxylic acid. Finally, OGC suppression reduced insulin secretion in response to a membrane-permeant 2OG analog, dimethyl-2OG. These data reveal that the OGC is part of a mechanism of fuel-stimulated insulin secretion that is common to glucose, amino acid, and organic acid secretagogues, involving flux through the pyruvate/isocitrate cycling pathway. Although the components of this pathway must remain intact for appropriate stimulus-secretion coupling, production of NADPH does not appear to be the universal second messenger signal generated by these reactions.

Glutamine: precursor or nitrogen donor for citrulline synthesis?

Although glutamine is considered the main precursor for citrulline synthesis, the current literature does not differentiate between the contribution of glutamine carbon skeleton vs. nonspecific nitrogen (i.e., ammonia) and carbon derived from glutamine oxidation. To elucidate the role of glutamine and nonspecific nitrogen in the synthesis of citrulline, l-[2-(15)N]- and l-[5-(15)N]glutamine and (15)N-ammonium acetate were infused intragastrically in mice. The amino group of glutamine labeled the three nitrogen groups of citrulline almost equally. The amido group and ammonium acetate labeled the ureido and amino groups of citrulline, but not the delta-nitrogen. D(5)-glutamine also infused in this arm of the study, which traces the carbon skeleton of glutamine, was utilized poorly, accounting for only 0.2-0.4% of the circulating citrulline. Dietary glutamine nitrogen (both N groups) incorporation was 25-fold higher than the incorporation of its carbon skeleton into citrulline. To investigate the relative contributions of the carbon skeleton and nonspecific carbon of glutamine, arginine, and proline to citrulline synthesis, U-(13)C(n) tracers of these amino acids were infused intragastrically. Dietary arginine was the main precursor for citrulline synthesis, accounting for approximately 40% of the circulating citrulline. Proline contribution was minor (3.4%), and glutamine was negligible (0.4%). However, the glutamine tracer resulted in a higher enrichment in the ureido group, indicating incorporation of nonspecific carbon from glutamine oxidation into carbamylphosphate used for citrulline synthesis. In conclusion, dietary glutamine is a poor carbon skeleton precursor for the synthesis of citrulline, although it contributes both nonspecific nitrogen and carbon to citrulline synthesis.

Abstract LB-108: Glutamine addition and glucose addiction - linking metabolic abnormalities to genetic alterations in glioblastoma SF188 and hereditary leiomyomatosis renal carcinoma UOK262

Abstract Metabolic transformations of malignant cells are essential for tumor development and progression. Oncogene Akt and c-myc stimulate glucose metabolism and glutamine metabolism, respectively, and render the cancers dependent on glucose or glutamine for growth and survival (otherwise known as glucose addiction and glutamine addiction). However, many other oncogenes, tumor suppressor genes and signaling molecules also regulate glucose and glutamine metabolism, which results in various metabolic abnormalities in different cancers. The question we want to ask is how one or more genetic mutations can lead to the ultimate metabolic abnormalities of a given cancer. Toward this end, we report here the metabolic abnormalities of two human cancer cell lines, glioblastoma SF188, which is c-myc-dependent and is addicted to glutamine, and hereditary leiomyomatosis renal carcinoma (HLRCC) UOK262, which is fumarate hydratase-deficient and is addicted to glucose. We performed our metabolic analysis using the XF24 Extracellular Flux Analyzer and pharmacological modulators of cellular metabolism. The XF24 Analyzer simultaneously monitors oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), which are indicators of mitochondrial respiration and aerobic glycolysis respectively. Oxygen consumption rate is also used as an indicator for the oxidation of glucose and glutamine when they are added to the cells after baseline measurement. We found that SF188 cells showed a small increase in acidification rate upon glucose addition, suggesting a relatively low basal glycolysis rate. We estimated the glycolytic capacity of the cells by determining the acidification rate after the addition of mitochondrial ATP syntase inhibitor oligomycin. The glycoltic capacity remained high although basal glycolysis rate was low. Glucose was not oxidized at all by these cells. Most importantly, we observed significant increases in oxygen consumption rate upon addition of glutamine indicating active uptake and oxidation of glutamine. Associated with active glutamine oxidation, SF188 cells exhibited high basal reparation rate and high respiratory capacity. Finally, SF188 cells grew and survived in medium containing glutamine but no glucose; however, they started to die in the medium containing glucose but no glutamine. In contrast with glioblastoma SF188, we found that HLRCC UOK262 cells exhibit high basal glycolysis rate, which is close to its maximal capacity. Interestingly, basal mitochondrial respiration and respiratory capacity are extremely low, which is associated with reported defective mitochondrial complex I. The growth and survival of UOK262 is dependent on a high concentration of glucose in the culture medium. Collectively, we established metabolic abnormalities of glioblastama SF188 and HLRCC UOK262, which contain genetic alterations in c-myc and fumarate hydratase, respectively, that makes them addicted to glutamine and glucose. The metabolic abnormalities we found include the preferential use of glucose and glutamine, the activation or inactivation of relevant enzymatic pathways that catabolize glucose and glutamine, and the modulation of mitochondrial bioenergetic activities in the two cancer cell lines. Most of cancer cell lines that we have studied showed metabolic abnormalities falling between the two extremes. Therefore, this type of metabolic analysis coupled with the analysis of genetic abnormalities in cancers will provides a rapid means to gain insight into the aberrant metabolism of each type of cancer and improve the efficacy of cancer therapy for a cancer with a particular set of metabolic abnormalities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-108.

Impaired Energy Metabolism in Airway Epithelial Cells Exposed to the Industrial Toxicant Phosgene

Phosgene is a common, highly toxic industrial gas that causes latent non‐cardiogenic pulmonary edema and is a model for chemically induced lung injury. Its toxicity arises from HCl release and acylation of proteins in pulmonary alveoli leading to breakdown of the blood:air barrier. We have developed an in vitro, air‐liquid interface exposure model in which airway epithelial cultures on Transwell inserts are combined with a Vitrocell exposure apparatus. Exposure to 0–64 ppm phosgene for 8 min caused dose‐dependent decreases in transepithelial electrical resistance, a measure of epithelial barrier integrity, and cell viability. Neither recovered at &gt; 8 ppm. In view of the loss of cell viability, we examined the effects on energy metabolism. At 64 ppm phosgene, the rate of [14C]glutamine oxidation to [14C]O2 was inhibited by 72 ± 8% at 6 hrs and 68 ± 8% at 24 hrs after exposure (p &lt; 0.05). However, 8 ppm only inhibited the rate by 23 ± 17% at 6 hrs and 16 ± 7% at 24 hrs (NS). Data for 6 vs 24 hrs did not differ. Preliminary assays of glycolysis showed no inhibition by phosgene. These data suggest that inhibition of aerobic metabolism may play a key role in the loss of cell viability and barrier integrity following phosgene exposure, in vitro. Supported by an Interagency Agreement from NIAID. The opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the US Army or the DoD.

Effects of glyoxal or methylglyoxal on the metabolism of amino acids, lactate, glucose and acetate in the cerebral cortex of young and adult rats

The in vitro effects of glyoxal and methylglyoxal on the metabolism of glycine, alanine, leucine, glutamate, glutamine, glucose, lactate and acetate were evaluated in cortico-cerebral slices from young (10-day-old) or adult (3-month-old) rats. In a first set of experiments with cortico-cerebral slices from young animals, the compounds glyoxal or methylglyoxal at 400 μM, increased the oxidation of alanine, leucine and glycine to CO 2 and decreased the protein synthesis from these amino acids. Lipid synthesis from alanine, leucine and glycine was not changed in the cortico-cerebral slices from young rats after glyoxals exposure. Moreover, glutamine oxidation to CO 2 decreased by glyoxals exposure, but glutamate oxidation was not affected. In a second set of experiments with brain slices from adult animals, glycine metabolism (oxidation to CO 2, conversion to lipids or incorporation into proteins) was not changed by glyoxals exposure. In addition, the oxidation rates of glucose, lactate, acetate, glutamine and glutamate to CO 2 were also not modified. Taken together, these results indicate that glyoxal disrupts the energetic metabolism of the rat cerebral cortex in vitro. However, only young animals were susceptible to such events, suggesting that the immature cerebral cortex is less capable of dealing with glyoxal than the mature one.

128. A ROLE FOR SIRTUIN 3 IN THE DEVELOPING MAMMALIAN EMBRYO

Pre-implantation embryo development relies critically on the balance between cytoplasmic and mitochondrial metabolism for the generation of metabolic intermediates such as NAD+. SIRT3 is a mitochondrial sirtuin with NAD+-dependant deacetylase activity that, targets glutamate dehydrogenase (GDH). In this study we characterised SIRT3 mRNA, protein and activity through pre-implantation development and determined whether modulation of SIRT3 activity influenced GDH activity. Embryos (zygotes, 2-cell, 8-cell and blastocyst stages) were recovered from female CBA/C57Bl6 mice following ovarian stimulation and mating with CBA/C57Bl6 males. Expression of SIRT3 mRNA was measured using real-time RTPCR, protein localisation examined using immunohistochemistry and SIRT3 activity measured using a Fluor-de-Lys SIRT3 fluorescentassay. Functional GDH activity was assessed in 2-cell embryos indirectly by measuring glutamine oxidation, following culture from zygote to 2-cell in the presence of nicotinamide, (a sirtuin inhibitor), G1.2 media, or simpleG1 media, compared to in vivo controls. SIRT3 mRNA was detected at all stages of development, with significantly greater levels expressed in the blastocyst. SIRT3 protein was localised predominantly around the nucleus of zygote and 2-cell embryos, and was mainly cytoplasmic in 8-cell embryos and blastocysts. SIRT3 activity remained constant throughout pre-implantation development, and tended to increase at the blastocyst stage. Glutamine oxidation was reduced for embryos cultured in G1.2 media relative to in vivo controls (0.14 pmol/e/hr vs 0.21pmol/e/hr), and this was further reduced by the addition of nicotinamide (0.07pmol/e/hr). Embryo culture in perturbing simpleG1 increased glutamine metabolism (0.33pmol/e/hr). In conclusion, SIRT3 mRNA, protein and activity was detected throughout pre-implantation development. Modulation of sirtuins by nicotinamide decreased glutamine metabolism, likely as a result of decreased deacetylation, thus decreased activity of GDH. SIRT3 can translocate to the mitochondria during cellular stress, thus the increased glutamine metabolism in simpleG1 conditions may be caused by translocation of SIRT3 to mitochondria, potentially increasing GDH deacetylation and enzymatic activity.

P/16 Uncoupling Protein 2: Physiology data and biochemistry questions

The mitochondrial Uncoupling Protein 2 UCP2 is a member of the mitochondrial carrier family and belongs to the UCP subfamily. It is widely expressed in tissues. In immune cells, UCP2 has a regulatory function through its effect on the production of reactive oxygen species and MAPK signalling. Ucp2 mice are resistant to infection by parasites and intracellular bacteria but are more sensitive to chronic inflammation and experimental neurodegeneration. We found that autoimmune diabetes was strongly accelerated in Ucp2 mice compared to Ucp2 mice with increased intra-islet lymphocytic infiltration. These data highlight UCP2 as a new player in autoimmune diabetes. In addition, in agreement with the known inhibitory role of UCP2 on insulin secretion, loss of function of UCP2 contributes to congenital hyperinsulinism in patients. The question of whether UCP2 fully uncouples respiration from ATP synthesis is still debated. Ucp2 cells display enhanced proliferation associated with a metabolic switch from fatty acid oxidation to glucose metabolism. This metabolic switch requires the unrestricted availability of glucose, and Ucp2 cells more readily activate autophagy than wild-type cells when deprived of glucose. Altogether, these results suggest that UCP2 promotes mitochondrial fatty acid oxidation while limiting mitochondrial catabolism of pyruvate. UCP2 expression is also required for efficient oxidation of glutamine in macrophages. This role of UCP2 in glutamine metabolism appears independent from its uncoupling activity.

Elimination of KATP Channels in Mouse Islets Results in Elevated [U-13C]Glucose Metabolism, Glutaminolysis, and Pyruvate Cycling but a Decreased γ-Aminobutyric Acid Shunt

Pancreatic beta cells are hyper-responsive to amino acids but have decreased glucose sensitivity after deletion of the sulfonylurea receptor 1 (SUR1) both in man and mouse. It was hypothesized that these defects are the consequence of impaired integration of amino acid, glucose, and energy metabolism in beta cells. We used gas chromatography-mass spectrometry methodology to study intermediary metabolism of SUR1 knock-out (SUR1(-/-)) and control mouse islets with d-[U-(13)C]glucose as substrate and related the results to insulin secretion. The levels and isotope labeling of alanine, aspartate, glutamate, glutamine, and gamma-aminobutyric acid (GABA) served as indicators of intermediary metabolism. We found that the GABA shunt of SUR1(-/-) islets is blocked by about 75% and showed that this defect is due to decreased glutamate decarboxylase synthesis, probably caused by elevated free intracellular calcium. Glutaminolysis stimulated by the leucine analogue d,l-beta-2-amino-2-norbornane-carboxylic acid was, however, enhanced in SUR1(-/-) and glyburide-treated SUR1(+/+) islets. Glucose oxidation and pyruvate cycling was increased in SUR1(-/-) islets at low glucose but was the same as in controls at high glucose. Malic enzyme isoforms 1, 2, and 3, involved in pyruvate cycling, were all expressed in islets. High glucose lowered aspartate and stimulated glutamine synthesis similarly in controls and SUR1(-/-) islets. The data suggest that the interruption of the GABA shunt and the lack of glucose regulation of pyruvate cycling may cause the glucose insensitivity of the SUR1(-/-) islets but that enhanced basal pyruvate cycling, lowered GABA shunt flux, and enhanced glutaminolytic capacity may sensitize the beta cells to amino acid stimulation.

Corticosteroids increase glutamine utilization in human splanchnic bed

Glutamine is the most abundant amino acid in the body and is extensively taken up in gut and liver in healthy humans. To determine whether glucocorticosteroids alter splanchnic glutamine metabolism, the effect of prednisone was assessed in healthy volunteers using isotope tracer methods. Two groups of healthy adults received 5-h intravenous infusions of l-[1-(14)C]leucine and l-[(2)H(5)]glutamine, along with q. 20 min oral sips of tracer doses of l-[1-(13)C]glutamine in the fasting state, either 1) at baseline (control group; n = 6) or 2) after a 6-day course of 0.8 mg.kg(-1).day(-1) prednisone (prednisone group; n = 8). Leucine and glutamine appearance rates (Ra) were determined from plasma [1-(14)C]ketoisocaproate and [(2)H(5)]glutamine, respectively, and leucine and glutamine oxidation from breath (14)CO(2) and (13)CO(2), respectively. Splanchnic glutamine extraction was estimated by the fraction of orally administered [(13)C]glutamine that failed to appear into systemic blood. Prednisone treatment 1) did not affect leucine Ra or leucine oxidation; 2) increased plasma glutamine Ra, mostly owing to enhanced glutamine de novo synthesis (medians +/- interquartiles, 412 +/- 61 vs. 280 +/- 190 mumol.kg(-1).h(-1), P = 0.003); and 3) increased the fraction of orally administered glutamine undergoing extraction in the splanchnic territory (means +/- SE 64 +/- 6 vs. 42 +/- 12%, P < 0.05), without any change in the fraction of glutamine oxidized (means +/- SE, 75 +/- 4 vs. 77 +/- 4%, not significant). We conclude that high-dose glucocorticosteroids increase in splanchnic bed the glutamine requirements. The role of such changes in patients receiving chronic corticoid treatment for inflammatory diseases or suffering from severe illness remains to be determined.

Modified glutamine catabolism in macrophages of Ucp2 knock-out mice

Uncoupling protein 2 (UCP2) belongs to a family of transporters of the mitochondrial inner membrane and is reported to uncouple respiration from ATP synthesis. Our observation that the amino acid glutamine specifically induces UCP2 protein expression prompted us to investigate metabolic consequences of a UCP2 knockdown ( Ucp2-KO) when glutamine is offered as a substrate. We found that Ucp2-KO macrophages incubated in the presence of glutamine exhibit a lower ammonium release, a decreased respiratory rate, and an intracellular accumulation of aspartate. Therefore, we conclude that UCP2 expression is required for efficient oxidation of glutamine in macrophages. This role of UCP2 in glutamine metabolism appears independent from the uncoupling activity of UCP2.

Requirement for, and patterns of, pyruvate and glutamine metabolism in the domestic dog oocyte in vitro

Supplementation of energy substrates to culture medium is essential for resumption and completion of meiosis in vitro for many mammalian species. Objectives were to study the dog oocyte, specifically the influences of pyruvate and glutamine on maturation and the utilization of these two substrates at various developmental stages and incubation times. Ovarian oocytes (n=681) were obtained from spayed bitches and cultured for 48 hr in TCM 199 medium containing various concentrations of pyruvate (0-2.5 mM) and glutamine (0-4 mM) before being assessed for nuclear status. For analyzing metabolic activity, 259 dog oocytes were cultured for 0, 12, 24, 36, or 48 hr, assessed for pyruvate and glutamine metabolism using the hanging drop method and then evaluated for nuclear status. Neither pyruvate nor glutamine had influence (P > 0.05) on oocyte maturation in vitro (IVM). However, both culture interval and meiotic status influenced pyruvate uptake (P < 0.05). Specifically, pyruvate uptake declined as the oocyte progressed from the germinal vesicle (GV) to metaphase II (MII) stage. Glutamine oxidation decreased as culture duration progressed (P < 0.05). In summary, pyruvate or glutamine is not required to promote successful IVM of dog oocytes. But, both substrates are being metabolized, and in patterns different to the domestic cat, another carnivore species. Pyruvate played an important role earlier in the maturational process, and less glutamine was oxidized as the oocyte neared nuclear maturation. These variations emphasize the importance of defining species specificities in carnivores before expecting consistently successful IVM/IVF.

Enhanced mitochondrial metabolism may account for the adaptation to insulin resistance in islets from C57BL/6J mice fed a high-fat diet

Hyperinsulinaemia maintains euglycaemia in insulin-resistant states. The precise cellular mechanisms by which the beta cells adapt are still unresolved. A peripherally derived cue, such as increased circulating fatty acids, may instruct the beta cell to initiate an adaptive programme to maintain glucose homeostasis. When this fails, type 2 diabetes ensues. Because mitochondria play a key role in beta cell pathophysiology, we tested the hypothesis that mitochondrial metabolism is critical for beta cell adaptation to insulin resistance. C57BL/6J mice were given high-fat (HF) diet for 12 weeks. We then analysed islet hormone secretion, metabolism in vivo and in vitro, and beta cell morphology. HF diet resulted in insulin resistance and glucose intolerance but not frank diabetes. Basal insulin secretion was elevated in isolated islets from HF mice with almost no additional response provoked by high glucose. In contrast, a strong secretory response was seen when islets from HF mice were stimulated with fuels that require mitochondrial metabolism, such as glutamate, glutamine, alpha-ketoisocaproic acid and succinate. Moreover, while glucose oxidation was impaired in islets from HF mice, oxidation of glutamine and palmitate was enhanced. Ultrastructural analysis of islets in HF mice revealed an accumulation of lipid droplets in beta cells and a twofold increase in mitochondrial area. We propose that beta cells exposed to increased lipid flux in insulin resistance respond by increasing mitochondrial volume. This expansion is associated with enhanced mitochondrial metabolism as a means of beta cell compensation.

Green Tea Polyphenols Modulate Insulin Secretion by Inhibiting Glutamate Dehydrogenase

Insulin secretion by pancreatic beta-cells is stimulated by glucose, amino acids, and other metabolic fuels. Glutamate dehydrogenase (GDH) has been shown to play a regulatory role in this process. The importance of GDH was underscored by features of hyperinsulinemia/hyperammonemia syndrome, where a dominant mutation causes the loss of inhibition by GTP and ATP. Here we report the effects of green tea polyphenols on GDH and insulin secretion. Of the four compounds tested, epigallocatechin gallate (EGCG) and epicatechin gallate were found to inhibit GDH with nanomolar ED(50) values and were therefore found to be as potent as the physiologically important inhibitor GTP. Furthermore, we have demonstrated that EGCG inhibits BCH-stimulated insulin secretion, a process that is mediated by GDH, under conditions where GDH is no longer inhibited by high energy metabolites. EGCG does not affect glucose-stimulated insulin secretion under high energy conditions where GDH is probably fully inhibited. We have further shown that these compounds act in an allosteric manner independent of their antioxidant activity and that the beta-cell stimulatory effects are directly correlated with glutamine oxidation. These results demonstrate that EGCG, much like the activator of GDH (BCH), can facilitate dissecting the complex regulation of insulin secretion by pharmacologically modulating the effects of GDH.

Experimental study to show that growth hormone treatment before trauma increases glutamine uptake in the intestinal tract.

This study examined whether growth hormone treatment deprived the intestinal tract of glutamine after trauma. Piglets were treated with growth hormone 24 units daily 3 days before and at the start of the trauma (GH-3, n = 8) or at the start of the trauma only (GH-1, n = 8). Eight piglets acted as non-treated controls. The trauma consisted of a standardized abdominal surgical procedure. Primed constant infusions of U-14C-glutamine were given. Intestinal, hepatic, renal and hindleg glutamine fluxes were measured. Growth hormone treatment increased mean(s.e.m.) net intestinal glutamine uptake: GH-3, 39.7(9.4) and 48.7(12.7) mumol/min; GH-1, 33.2(5.5) and 25.7(12.3) mumol/min; controls, 19.5(10.3) and 2.0(15.3) mumol/min at 1 h and 5 h after trauma, respectively, (P = 0.02). The treatment increased glutamine oxidation (P = 0.025), and decreased hindleg glutamine net (P = 0.0052) and absolute release (P = 0.0063), glutamine rate of appearance (P = 0.01), and percentage of glucose coming from glutamine (P = 0.05). Growth hormone treatment before trauma increased intestinal glutamine uptake.