Changes In Intermediary Metabolites Research Articles

β-cell–selective inhibition of DNA damage response signaling by nitric oxide is associated with an attenuation in glucose uptake

Nitric oxide (NO) plays a dual role in regulating DNA damage response (DDR) signaling in pancreatic β-cells. As a genotoxic agent, NO activates two types of DDR signaling; however, when produced at micromolar levels by the inducible isoform of NO synthase, NO inhibits DDR signaling and DDR-induced apoptosis in a β-cell-selective manner. DDR signaling inhibition by NO correlates with mitochondrial oxidative metabolism inhibition and decreases in ATP and NAD+. Unlike most cell types, β-cells do not compensate for impaired mitochondrial oxidation by increasing glycolytic flux, and this metabolic inflexibility leads to a decrease in ATP and NAD+. Here, we used multiple analytical approaches to determine changes in intermediary metabolites in β-cells and non-β-cells treated with NO or complex I inhibitor rotenone. In addition to ATP and NAD+, glycolytic and tricarboxylic acid cycle intermediates as well as NADPH are significantly decreased in β-cells treated with NO or rotenone. Consistent with glucose-6-phosphate residing at the metabolic branchpoint for glycolysis and the pentose phosphate pathway (NADPH), we show that mitochondrial oxidation inhibitors limit glucose uptake in a β-cell-selective manner. Our findings indicate that the β-cell-selective inhibition of DDR signaling by NO is associated with a decrease in ATP to levels that fall significantly below the KM for ATP of glucokinase (glucose uptake) and suggest that this action places the β-cell in a state of suspended animation where it is metabolically inert until NO is removed, and metabolic function can be restored.

Metabolic Shifts Induced by Fatty Acid Synthase Inhibitor Orlistat in Non-small Cell Lung Carcinoma Cells Provide Novel Pharmacodynamic Biomarkers for Positron Emission Tomography and Magnetic Resonance Spectroscopy

PurposeAbnormal fatty acid (FA) synthesis is one of the common features of cancer. Fatty acid synthase (FASN), a multifunctional enzyme playing a key role in biosynthesis of FA, is up-regulated in prostate, breast, and lung carcinomas. Orlistat is a FDA-approved anti-obesity drug that inhibits the thioesterase domain of FASN, interferes with cellular FA synthesis, can arrest tumor cell proliferation, and induces tumor cell apoptosis. The current study was aimed to investigate the metabolic changes associated with FASN inhibition by orlistat and to understand the molecular mechanisms behind the observed metabolic changes in non-small cell lung carcinoma (NSCLC) cell lines.ProceduresChanges in metabolite pools in four NSCLC cell lines (H441, H1975, H3255, and PC14) with different mutational profiles were studied using NMR spectroscopy before and after in vitro incubation with sub-toxic concentration of orlistat and [1-13C]d-glucose or [1,2-13C2]choline. In vitro radiotracer accumulation assays in cells were performed with [3H]acetate, [14C]fluoroacetate, and 2-deoxy-2-[18F]fluoro-d-glucose. In parallel, microarray profiling of genes involved in the regulation of carbohydrate and lipid metabolism was performed.ResultsIn orlistat-treated NSCLC cells, FASN inhibition results in characteristic changes in intermediary metabolites (FAs, choline, phospholipids, and TCA cycle metabolites) as observed by magnetic resonance spectroscopy. Further, FASN inhibition by orlistat induces multiple adaptive changes in FA synthetic pathway and associated metabolic pathways, including induction of ketone metabolism and glutaminolysis, as well as the up-regulation of 5' adenosine monophosphate-activated protein kinase.ConclusionsThese observed changes in metabolic pools in orlistat-treated cells demonstrate the critical role of fatty acid de novo synthesis and metabolism for cellular energy production, especially in tumor cells with low glycolytic activity, which goes beyond the widely accepted concept that FA synthesis is important for cell membrane biosynthesis in rapidly proliferating tumor cells.Electronic supplementary materialThe online version of this article (doi:10.1007/s11307-012-0587-6) contains supplementary material, which is available to authorized users.

Single Point Mutations in Either Gene Encoding the Subunits of the Heterooctameric Yeast Phosphofructokinase Abolish Allosteric Inhibition by ATP

Yeast phosphofructokinase is a heterooctameric enzyme subject to a complex allosteric regulation. A mutation in the PFK1 gene, encoding the larger -subunits, rendering the enzyme insensitive to allosteric inhibition by ATP was found to be caused by an exchange of proline 728 for a leucine residue. By in vitro mutagenesis, we introduced this mutation in either PFK1 or PFK2 and found that the exchange in either subunit drastically reduced the sensitivity of the holoenzyme to ATP inhibition. This was accompanied by a lack of allosteric activation by AMP, fructose 2,6-bisphosphate, or ammonium and an increased resistance to heat inactivation. Yeast cells carrying either one mutation or both in conjunction did not display a strong phenotype when grown on fermentable carbon sources and did not show any significant changes in intermediary metabolites. Growth on non-fermentable carbon sources was clearly impaired. The strain carrying both mutant alleles was more sensitive to Congo Red than the wild-type strain or the single mutants indicating differences in cell wall composition. In addition, we found single pfk null mutants to be less viable than wild type at different storage temperatures and a pfk2 null mutant to be temperature-sensitive for growth at 37 degrees C. The latter mutant was shown to be respiration-dependent for growth on glucose.

Hemodialysate composition and intradialytic metabolic, acid–base and potassium changes

We compared the effects of dialysate composition on changes in intermediary metabolites, acid-base balance, and potassium removal during hemodialysis. Patients were dialyzed against dialysates containing acetate or bicarbonate, each with or without glucose, in a four-way cross-over study. Dialysates containing acetate were associated with significant perturbations in intermediary metabolism, including increases in blood citrate, acetoacetate and beta-hydroxybutyrate and a decrease in pyruvate. In contrast, bicarbonate-containing dialysates caused minimal perturbations in intermediary metabolism. Addition of glucose to the dialysate decreased the changes in intermediary metabolites; however, the magnitude of this effect was less than that observed for the change from acetate to bicarbonate. Use of acetate also resulted in lower post-dialysis blood-concentrations of base equivalents than obtained with bicarbonate; this difference was unaffected by the presence or absence of glucose. Although pre- and post-dialysis potassium concentrations were unaffected by the dialysate formulation, total potassium removal was significantly greater when glucose was omitted from the dialysate. Our results suggest that both bicarbonate and glucose should be included in the dialysate, particularly for those patients whose capacity for metabolism may be limited because of highly efficient dialysis, intercurrent illness, or starvation. However, addition of glucose to the dialysate may require a reduction in dialysate potassium to maintain proper potassium homeostasis.

Portacaval anastomosis: brain and plasma metabolite abnormalities and the effect of nutritional therapy.

Rats with portacaval shunts were used as a model of hepatic encephalopathy and compared to shamoperated controls. First, the changes in intermediary metabolites and amino acids in blood and whole brain were characterized and found to be similar at 4 and 7 weeks after shunting. Second, the effects of nutritional therapy on selected metabolites and tryptophan transport into brain were assessed in rats 5 weeks after surgery. Ordinary food was removed and the rats were treated with glucose given either by mouth or intravenously, or intravenous glucose plus branched chain amino acids. Several abnormalities in plasma amino acid concentrations were reversed by treatment. The abnormally high brain uptake index of tryptophan, a consequence of portacaval shunting, was not lowered by any of the treatment regimens; it was even higher in the groups given glucose by mouth and glucose plus amino acids. Calculated competition for entry of tryptophan, phenylalanine, and tyrosine into brain was unchanged (glucose plus amino acids), or reduced (glucose alone). Brain glutamine content was brought to near normal by all treatments. Infusion of glucose plus branched chain amino acids normalized brain content of tryptophan, phenylalanine, and tyrosine, even though the brain uptake index of tryptophan was higher in this group. Thus, partial or complete reversal of several abnormalities found after portacaval shunting was achieved by removal of oral food and administration of glucose. The addition of branched chain amino acids to the glucose infusion restored brain content of three aromatic amino acids to near normal, by a mechanism which appeared to be unrelated to transport across the blood-brain barrier.

Metabolic and endocrine consequences of depriving preterm infants of enteral nutrition.

Plasma enteroglucagon, pancreatic polypeptide, gastrin, motilin, neurotensin, gastric inhibitory polypeptide, secretin, vasoactive intestinal peptide and blood glucose, alanine, ketone bodies, lactate and pyruvate were measured on the sixth postnatal day in (a) a group of 10 preterm infants who on account of hyaline membrane disease had not received enteral feeding since birth and (b) before and at 55, 90, and 120 minutes after feeding in a group of healthy preterm infants fed three-hourly on human milk. Gut hormones were also measured in umbilical venous cord blood. The infants receiving regular boluses of milk from birth demonstrated postnatal surges in preprandial concentrations of gut hormones together with cyclical hormonal responses to feeding. None of these changes were seen in infants receiving intravenous fluids. The latter infants also had lower concentrations of blood alanine, glycerol and hydroxybutyrate and lacked the phasic changes in intermediary metabolites seen in the infants receiving enteral boluses of milk. Thus deprivation of enteral feeding results in a profound alteration of the metabolic and endocrine milieu which may have important effect on the process of adaptation to postnatal life.

CLINICAL USE OF NUCLEAR MAGNETIC RESONANCE IN THE INVESTIGATION OF MYOPATHY

Topical nuclear magnetic resonance allows non-invasive investigation of the metabolism of skeletal muscle and other tissues. Energy exchange can be followed by measurement of 31P NMR spectra which characterise changes in muscle content of inorganic phosphorus (Pi) and phosphorylcreatine. Muscle pH is determined from the chemical shift of Pi with respect to phosphorylcreatine. Topical NMR can be used to study changes in intermediary metabolites during exercise, and the clinical value is illustrated in a patient with phosphofructokinase deficiency who would otherwise have required multiple biopsies of muscle. In diseases such as Duchenne muscular dystrophy, where muscle may be partly replaced by fat or other tissue, the findings with 31P NMR can be related to independent estimates of tissue composition obtained from 1H and 13C NMR spectra and from X-ray computerised tomography.

The effects of acute carbon monoxide intoxication on the cerebral energy metabolism of the rat.

The cerebal metabolic effects of 60 min exposure to 0.5, 1.0, 1.5, and 2.0% carbon monoxide (CO) and 60 min exposure to 1.0% CO were studied in lightly anesthetized rats by measurement of brain tissue contents of glycolytic and citric acid cycle intermediates, as well as tissue energy phosphates. The results indicate that cerebral energy homeostasis is maintained until advanced levels of CO intoxication (2.0%) are reached. Animals exposed to 2.0% CO developed significant decreases in systemic blood pressure, with attendent decreases in cerebral ATP, increases in ADP and AMP, plus early depletions of tissue citrate and alpha-oxyglutarate. The similarity of this pattern to that previously documented for various cerebral oligemic states suggests a possible modifying role for altered cerebral production in its production. A correlation between conscious behavior and cerebral energy state was suggested by the observation that unanesthetized animals exposed to 1.0% CO for 30 and 60 min retained consciousness, whereas animals exposed to 2.0% CO for 30 min became unresponsive late on in the exposure. A comparison of CO induced changes in intermediary metabolites, energy phosphates, intracellular pH, and cytoplasmic redox state with those seen in hypoxemia indicate no basic qualitative or quantiative differences in the metabolic response of brain tissue to the two conditions.

The role of cyclic AMP in the control of liver metabolism

1. 1.|Gluconeogenesis from lactate in the perfused liver is stimulated by blood levels of glucagon and high levels of catecholamines. This effect of the hormones appears to be mediated by cyclic AMP. The changes in intermediary metabolites produced by the hormones or cyclic AMP with lactate or amino acids as substrate are consistent with the activation of a reaction or reactions between pyruvate and P-pyruvate in the gluconeogenic pathway. The absence of any effect of glucagon on glucose or lactate formation from fructose suggests that cyclic AMP does not produce any significant effect on reactions between triose-P and glucose or on the conversion of P-pyruvate to pyruvate. 2. 2.|Gluconeogenesis appears to be as sensitive as glycogenolysis to stimulation by glucagon, catecholamines and cyclic AMP. The nucleotide mimics the actions of the hormones on urea production and K + release by the perfused liver. 3. 3.|The accumulation of cyclic AMP in the liver is promoted by much lower levels of glucagon than catecholamines and the maximum cyclic AMP concentration with glucagon is 20 to 30 fold higher than that with epinephrine. Isopropyl norepinephrine stimulates glycogenolysis and cyclic AMP formation in the liver and the effects of epinephrine are blocked by pronethalol. The liver receptors therefore appear to resemble β-adrenergic receptors more closely than α-receptors. 4. 4.|Insulin deficiency produced by anti-insulin serum or alloxan causes an elevation of the liver cyclic AMP level in vivo which can be reversed by insulin treatment. Perfused livers from antiserum-treated rats also show an increase in cyclic AMP and this probably accounts for the increased rates of glycogenolysis, gluconeogenesis, urea production and K + release. 5. 5.|Insulin added to the perfusion medium reverses the effects of antiserum treatment. It also inhibits many processes in normal livers which are stimulated by cyclic AMP and it antagonizes the actions of glucagon and epinephrine on glucose output. It lowers the level of cyclic AMP in normal livers and in those treated with glucagon or obtained from rats injected with antiserum. 6. 6.|Adrenal glucocorticoids are required for a normal effect of glucagon on lactate gluconeogenesis. They appear to exert a permissive action by increasing the sensitivity of a reaction or reactions in gluconeogenesis to activation by cyclic AMP.