31P and 13C NMR studies of isolated perfused hematopoietic cells from leukemic mice

The myeloproliferative leukemic virus (MPLV) induces within 2-3 weeks a massive infiltration of the adult mouse liver by hematopoietic leukemic cells. Since the metabolism of the infiltrated organ might be correlated with an interaction of two cell populations, it was decided to study the isolated hematopoietic cells separately. The metabolism of these cells embedded in an agarose gel and perfused with labeled substrates was investigated using 31P and 13C NMR. Using [1-13C]glucose as precursor, sequential 13C NMR spectra showed that the hematopoietic cells were able to store glucose as [1-13C]glycogen and to metabolize it through the glycolytic pathway to give [3-13C]lactate as sole end-product. The liver neoglucogenic substrates: [2-13C]pyruvate and [3-13C]alanine are not metabolized by these cells. This suggests that the tricarboxylic acid cycle was not efficient. To investigate further the glycolytic properties of the cells, 10 mM sodium azide was added to the medium containing [1-13C]glucose. When compared to the aerobic conditions, a 40% decrease of nucleotides (0.10 vs 0.17 mumole NTP/10(9) cells), a degradation of [1-13C]glycogen and an increase of ca 35% of the glycolytic rate were observed. The analysis of 13C NMR spectra of the perfusates at the end of the perfusion indicates a total conversion of [1-13C]glucose into [3-13C]lactate and [3-13C]pyruvate under anaerobic conditions. These results permit a better understanding of the metabolism of the perfused leukemic livers which are extensively infiltrated by these hematopoietic cells.     

Ketone bodies effectively compete with glucose for neuronal acetyl‐CoA generation in rat hippocampal slices

Ketone bodies can be used for cerebral energy generation in situ, when their availability is increased as during fasting or ingestion of a ketogenic diet. However, it is not known how effectively ketone bodies compete with glucose, lactate, and pyruvate for energy generation in the brain parenchyma. Hence, the contributions of exogenous 5.0 mM [1‐¹³C]glucose and 1.0 mM [2‐¹³C]lactate + 0.1 mM pyruvate (combined [2‐¹³C]lactate + [2‐¹³C]pyruvate) to acetyl‐CoA production were measured both without and with 5.0 mM [U‐¹³C]3‐hydroxybutyrate in superfused rat hippocampal slices by ¹³C NMR non‐steady‐state isotopomer analysis of tissue glutamate and GABA. Without [U‐¹³C]3‐hydroxybutyrate, glucose, combined lactate + pyruvate, and unlabeled endogenous sources contributed (mean ± SEM) 70 ± 7%, 10 ± 2%, and 20 ± 8% of acetyl‐CoA, respectively. With [U‐¹³C]3‐hydroxybutyrate, glucose contributions significantly fell from 70 ± 7% to 21 ± 3% (p < 0.0001), combined lactate + pyruvate and endogenous contributions were unchanged, and [U‐¹³C]3‐hydroxybutyrate became the major acetyl‐CoA contributor (68 ± 3%) – about three‐times higher than glucose. A direct analysis of the GABA carbon 2 multiplet revealed that [U‐¹³C]3‐hydroxybutyrate contributed approximately the same acetyl‐CoA fraction as glucose, indicating that it was less avidly oxidized by GABAergic than glutamatergic neurons. The appearance of superfusate lactate derived from glycolysis of [1‐¹³C]glucose did not decrease significantly in the presence of 3‐hydroxybutyrate, hence total glycolytic flux (Krebs cycle inflow + exogenous lactate formation) was attenuated by 3‐hydroxybutyrate. This indicates that, under these conditions, 3‐hydroxybutyrate inhibited glycolytic flux upstream of pyruvate kinase. Copyright © 2015 John Wiley & Sons, Ltd.     

Hepatic carbohydrate metabolism in the spontaneously diabetic Bio-Breeding Worcester rat

The effects of diabetes on hepatic carbohydrate metabolism were investigated in spontaneously diabetic Bio-Breeding Worcester (BB/W) rats. The juvenile-onset-type syndrome displayed by these animals is characterized by beta-cell destruction with subsequent ketosis-prone insulinopenia. Livers from diabetic animals demonstrated increased adenosine 3',5'-cyclic monophosphate levels but subnormal total protein and glycogen content. Isolated perfused livers of diabetic BB/W rats demonstrated an increased rate of glucose production from [14C]lactate and an impaired rate of glycogen synthesis. These data were consonant with hepatic enzyme studies demonstrating markedly increased activities of component gluconeogenic (glucose-6-phosphatase, fructose-1,6-diphosphatase, phosphoenolpyruvate carboxykinase) and glycogenolytic (glycogen phosphorylase) enzymes with decreased activities of glycolytic (hexokinase, pyruvate kinase) and glycogenic (glycogen synthase) enzymes. These findings agree with previous studies using alloxan- and streptozotocin-induced diabetic animals and suggest that accelerated hepatic gluconeogenesis and impaired glucose utilization are pathognomonic of all insulin-deficient diabetic syndromes.     

Robust glycogen shunt activity in astrocytes: Effects of glutamatergic and adrenergic agents

The significance and functional roles of glycogen shunt activity in the brain are largely unknown. It represents the fraction of metabolized glucose that passes through glycogen molecules prior to entering the glycolytic pathway. The present study was aimed at elucidating this pathway in cultured astrocytes from mouse exposed to agents such as a high [K⁺], d-aspartate and norepinephrine (NE) known to affect energy metabolism in response to neurotransmission. Glycogen shunt activity was assessed employing [1,6-¹³C]glucose, and the glycogen phosphorylase inhibitor 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) to block glycogen degradation. The label intensity in lactate, reflecting glycolytic activity, was determined by mass spectrometry. In the presence of NE a substantial glycogen shunt activity was observed, accounting for almost 40% of overall glucose metabolism. Moreover, when no metabolic stimulant was applied, a compensatory increase in glycolytic activity was seen when the shunt was inhibited by DAB. Actually the labeling in lactate exceeded that obtained when glycolysis and glycogen shunt both were operational, i.e. supercompensation. A similar phenomenon was seen when astrocytes were exposed to d-aspartate. In addition to glycolysis, tricarboxylic acid (TCA) cycle activity was monitored, analyzing labeling by mass spectrometry in glutamate which equilibrates with α-ketoglutarate. Both an elevated [K⁺] and d-aspartate induced an increased TCA cycle activity, which was altered when glycogen degradation was inhibited. Thus, the present study provides evidence that manipulation of glycogen metabolism affects both glycolysis and TCA cycle metabolism. Altogether, the results reveal a highly complex interaction between glycogenolysis and glycolysis, with the glycogen shunt playing a significant role in astrocytic energy metabolism.     

Application of (13)C-filtered (1)H NMR to evaluate drug action on gluconeogenesis and glycogenolysis simultaneously in isolated rat hepatocytes

The effects of two inhibitors of hepatic glucose production, AICAR (5-aminoimidazole-4-carboxamide riboside) and metformin, whose precise mechanisms of action are a matter of some controversy, have been investigated in isolated rat hepatocytes by application of a novel NMR-based method whereby effects on metabolic flow from the two glucose-producing pathways, glycogenolysis and gluconeogenesis, and also lactate production, can be studied simultaneously. Hepatocytes were pre-incubated for 24 h with 15 mM 1-(13)C-glucose to load the cells with labeled glycogen, which under subsequent glycogenolytic conditions would yield predominantly 1-(13)C glucose and 3-(13)C-lactate, followed (after washing) by incubation in media with 2-(13)C-glycerol, which under subsequent gluconeogenic conditions would yield 2,5-(13)C-glucose, or if metabolized to lactate, 2-(13)C-lactate. Glucose production was then stimulated by glucagon for 3 h in the absence or presence of the inhibitors and then incubation media were analyzed by (13)C-HSQC (heteronuclear single quantum coherence)-filtered (1)H NMR spectra. The results show that metformin only inhibits glucose production by inhibition of gluconeogenesis, but also that it increases lactate production from both glycogenolysis and from glycerol, whereas, and contrary to expectations, AICAR inhibits glucose production by inhibiting both gluconeogenesis and glycogenolysis, and also increases lactate production from glycerol. The data show that application of this methodology can be used to answer important questions about drug action on hepatic metabolism that are not readily accessible by alternative means.     

Late diagnosis of a McArdle disease's case (type V glycogenosis)

McArdle's disease is a metabolic myopathy characterized by a myophosphorylase deficiency resulting in an inability to degrade glycogen stores. We report the case of a 48 years old patient who complained since adolescence of rest and exercise myalgias and presented a chronic increased plasma creatine kinase activity. First, a maximal exercise test was performed. This test demonstrated a quasi lack of rise of respiratory exchange ratio and of blood lactate, possibly due to a glycogenolytic/glycolytic pathway deficiency. Second, a biopsy of vastus lateralis muscle was performed using Bergstr?m needle. As expected, the analysis of mitochondrial function was normal. The in vitro screening test of the glycogenolysis/glycolysis pathway showed a lack of lactate production in presence of glycogen substrate. The study of muscular metabolism of glycogen revealed a glycogen accumulation and a decrease of active and total phosphorylase activities. These data allowed us to diagnose a type V glycogenosis, or McArdle's disease. The patient appeared heterozygous for the most frequent mutation (p.R50X).     

Magnetic resonance spectroscopic investigation of mitochondrial fuel metabolism and energetics in cultured human fibroblasts: effects of pyruvate dehydrogenase complex deficiency and dichloroacetate

The pyruvate dehydrogenase complex (PDC) is integral to metabolism and energetics. Congenital PDC deficiency leads to lactic acidosis, neurological degeneration and early death. An investigational compound for such defects is dichloroacetate (DCA), which activates the PDC (inhibiting reversible phosphorylation of the E1alpha subunit) and decreases its turnover. Here, primary human fibroblast cultures from five healthy subjects and six patients with mutations in the PDC-E1 component were grown in media+/-DCA, exposed to media containing (13)C-labeled glucose, and studied (as cell extracts) by nuclear magnetic resonance (NMR) spectroscopy. Computer modeling of NMR-derived (13)C-glutamate isotopomeric patterns estimated relative carbon flow through TCA cycle-associated pathways and characterized effects of PDC deficiency on metabolism and energetics. Rates of glucose consumption (GCR) and lactate production (LPR) were measured. With the exception of one patient cell line expressing an unusual splicing mutation, PDC-deficient cells had significantly higher GCR, LPR and label-derived acetyl-CoA, indicative of increased glycolysis vs. controls. In all cells, DCA caused a major shift (40% decrease) from anaplerotic-related pathways (e.g., pyruvate carboxylase) toward flux through PDC. Ignoring the patient with the splicing mutation, DCA decreased average glycolysis (29%) in patient cells, but had no significant effect on control cells, and did not change LPR or the nucleoside triphosphate to diphosphate ratio (NTP/NDP) in either cell type. Maintenance of NTP despite reduced glycolysis indicates that DCA improves metabolic efficiency by increasing glucose oxidation. This study demonstrates that NMR spectroscopy provides insight into biochemical consequences of PDC deficiency and the mechanism of putative therapeutic agents.     

Hepatic metabolism of genetically diabetic (db/db) mice. I. Carbohydrate metabolism.

Hepatic carbohydrate metabolism in genetically diabetic mice (db/db) and their normal littermates has been studied. In db/db mice, body water was below normal and declined with age. The liver of db/db mice was abnormally large in relation to the metabolic mass of the body at all ages studied. In db/db mice, hepatic glycogenolysis, glycogen synthesis, glycogen synthetase, and phosphorylase were markedly increased. Gluconeogenesis from alanine or lactate in perfused livers of db/db mice was greater than normal per 100 g body water. Activities of fructose-1, 6-biophosphatase, glucose-6-phosphatase, glucokinase + hexokinase, and pyruvate kinase were elevated in livers of db/db mice. Diabetic mouse livers perfused with lactate showed a markedly reduced concentration of P-enolpyruvate and clear "forward crossover" between fructose-1, 6-P2 and fructose-6-P. In vivo glucose clearance, measured with [3-3H]glucose, in db/db mice was 170% that of normal mice. Data presented indicate that in livers of db/db mice: 1) glucose production is elevated prior to hyperglycemia, 2) glycogen turns over more rapidly, and 3) glycolytic and gluconeogenic enzymes are elevated paradoxically. These abnormalities are discussed from the viewpoint of their etiology.     

Involvement of brain lactate in neuronal metabolism

The involvement of brain lactate in neuronal metabolism was analyzed by ex vivo NMR spectroscopy with rats under the effects of pentobarbital, alphachloralose or morphine, which were infused with a solution of either [1-(13)C]glucose+lactate or glucose+[3-(13)C]lactate for 20 min. Electroencephalogram recordings indicated different brain electrical activity levels under the three drugs with a clear distinction between pentobarbital, on the one hand, and alphachloralose and morphine on the other. Labeling of metabolites in brain perchloric acid extracts and of blood glucose and lactate was determined by (13)C- and/or (1)H-observed/(13)C-edited-NMR spectroscopy. The following were found: (i) the ratio between glutamate C3 and C4 (13)C-enrichments increased from pentobarbital to alphachloralose and morphine whatever the labeled precursor, indicating a link between metabolic and electrical activity; (ii) under glucose+[3-(13)C]lactate infusion, alanine C3 and acetyl-CoA C2 enrichments were higher than that of lactate C3, revealing the occurrence of an isotopic dilution of the brain exogenous lactate (arising from blood) by lactate from brain (endogenous lactate); the latter was synthesized from glycolysis in a compartment other than the neurons; (iii) the contributions of labeled glucose and lactate to acetyl-CoA C2 enrichment indicated that the involvement of blood glucose relative to that of blood lactate to brain metabolism was correlated with brain activity. It can therefore be concluded that the brain electrical activity-dependent increase in the contribution of blood glucose relative to that of blood lactate to brain metabolism occurred partly via the increase in the metabolism of lactate generated from astrocytic glycolysis. This conclusion supports the hypothesis of an astrocyte-neuron lactate shuttle component in the coupling mechanism between cerebral activity and energy metabolism.     

The action of glibenclamide on glycogen catabolism and related parameters in the isolated perfused rat liver

Inhibitory effects on glycogenolysis have been reported for glibenclamide in the presence of insulin after stimulation of glycogenolysis by glucagon. Inhibition of oxidative phosphorylation, which has been equally reported for this drug, however, should stimulate glycogenolysis. The present work aimed to find an answer to the question of how glibenclamide affects glycogen catabolism in the liver of fed rats undergoing substrate- and hormone-free perfusion. The experimental system was the isolated perfused liver of ad libitum fed rats. Metabolites in the outflowing perfusate were assayed enzymatically. Oxygen uptake was measured polarographically. Glibenclamide (25-500 microM) stimulated glucose production and lactate release, with a clear correlation between concentrations and effects. Maximal stimulations were 132 and 127% for lactate production and glucose release, respectively. At low glibenclamide concentrations (up to 100 microM) both oxygen uptake and pyruvate production were stimulated, but at higher concentrations inhibition took place. Uric acid production was stimulated by glibenclamide. All effects of glibenclamide are probably due to decreases in oxidative phosphorylation. Stimulation of glucose release is the opposite of what should be expected for a hypoglycemic drug and it also contrasts with some reports of diminishing effects in the presence of glucagon plus insulin. This means that the stimulatory action on glycogenolysis that was seen as a net effect under the specific conditions of the present work could be counterbalancing inhibitory effects in vivo. This combination of events could eventually diminish the effectiveness of the drug as a hypoglycemic agent in the fed state.     

Two-compartment model for determination of glycolytic rates of solid tumors by in vivo 13C NMR spectroscopy

Carbon-13 NMR spectroscopy of 13C enriched substrates is useful for non-invasively determining metabolic fluxes of cells and tissues. Our study demonstrates that for RIF-1 tumor cells, examined under monolayer culture with continuous perfusion and also grown as solid subcutaneously (sc) implanted tumors in vivo, the levels of intracellular glucose and intermediates of the glycolytic pathway are below the level of detection by NMR spectroscopy. For these tumors, glucose transport into the cell is the most probable rate limiting step of the glycolytic pathway. Under these limiting conditions a simple two-compartment model of glycolysis applies. This model yields two parameters: the average rate of glycolysis and the rate of lactate clearance through the vasculature. For the RIF-1 tumor these parameters were 0.022 +/- 0.01 and 0.034 +/- 0.006 min(-1), respectively.     

Carbohydrate metabolism in perfused livers of adrenalectomized and steroid-replaced rats.

In livers from fasted rats perfused with bicarbonate buffer containing bovine albumin and erythrocytes, adrenalectomy decreased glycogen levels and glucose production, impaired the incorporation of 14C from [14C]lactate into glucose or glycogen, and decreased the activity of the active (I) form of glycogen synthase. Cortisol treatment restored gluconeogenesis after 1 h and glycogen synthesis after 2 h. Adrenalectomy did not alter the production of glucose or lactate or the levels of gluconeogenic intermediates in livers from fasted rats perfused with fructose, but reduced the formation of glycogen from this substrate. Adrenalectomy increased the levels of lactate and decreased the levels of P-pyruvate and subsequent intermediates in the gluconeogenic pathway. These changes were reversed by cortisol treatment. It is concluded that glucocorticoids support gluconeogenesis and glycogen synthesis in livers from fasted rats primarily by facilitating a reaction(s) located between pyruvate and P-pyruvate in the gluconeogenic pathway and by promoting the conversion of inactive to active glycogen synthase.     

Metabolic remodeling triggered by salivation and diabetes in major salivary glands

The metabolic profile of major salivary glands was evaluated by ¹³C nuclear magnetic resonance isotopomer analysis (¹³C NMR‐IA) following the infusion of [U‐¹³C]glucose in order to define the true metabolic character of submandibular (SM) and parotid (PA) glands at rest and during salivary stimulation, and to determine the metabolic remodeling driven by diabetes. In healthy conditions, the SM gland is characterized at rest by a glycolytic metabolic profile and extensive pyruvate cycling. On the contrary, the PA gland, although also dominated by glycolysis, also possesses significant Krebs’ cycle activity and does not sustain extensive pyruvate cycling. Under stimulation, both glands increase their glycolytic and Krebs’ cycle fluxes, but the metabolic coupling between the two pathways is further compromised to account for the much increased biosynthetic anaplerotic fluxes. In diabetes, the responsiveness of the PA gland to a salivary stimulus is seriously hindered, mostly as a result of the incapacity to burst glycolytic activity and also an inability to improve the Krebs’ cycle flux to compensate. The Krebs’ cycle activity in the SM gland is also consistently compromised, but the glycolytic flux in this gland is more resilient. This diabetes‐induced metabolic remodeling in SM and PA salivary glands illustrates the metabolic need to sustain adequate saliva production, and identifies glycolytic and oxidative pathways as key players in the metabolic dynamism of salivary glands.     

Integration of [U-13C]glucose and 2H2O for quantification of hepatic glucose production and gluconeogenesis

Glucose metabolism in five healthy subjects fasted for 16 h was measured with a combination of [U-13C]glucose and 2H2O tracers. Phenylbutyric acid was also provided to sample hepatic glutamine for the presence of 13C-isotopomers derived from the incorporation of [U-13C]glucose products into the hepatic Krebs cycle. Glucose production (GP) was quantified by 13C NMR analysis of the monoacetone derivative of plasma glucose following a primed infusion of [U-13C]glucose and provided reasonable estimates (1.90 +/- 0.19 mg/kg/min with a range of 1.60-2.15 mg/kg/min). The same derivative yielded measurements of plasma glucose 2H-enrichment from 2H2O by 2H NMR from which the contribution of glycogenolytic and gluconeogenic fluxes to GP was obtained (0.87 +/- 0.14 and 1.03 +/- 0.10 mg/kg/min, respectively). Hepatic glutamine 13C-isotopomers representing multiply-enriched oxaloacetate and [U-13C]acetyl-CoA were identified as multiplets in the 13C NMR signals of the glutamine moiety of urinary phenylacetylglutamine, demonstrating entry of the [U-13C]glucose tracer into both oxidative and anaplerotic pathways of the hepatic Krebs cycle. These isotopomers contributed 0.1-0.2% excess enrichment to carbons 2 and 3 and approximately 0.05% to carbon 4 of glutamine.     

Carbohydrate metabolism of the rat C6 glioma. An in vivo 13C and in vitro 1H magnetic resonance spectroscopy study

Surface coil 13C nuclear magnetic resonance (NMR) spectroscopy was used to investigate the in vivo carbohydrate metabolism of rat C6 gliomas during and after infusion with [1-13C] glucose. In vivo 1H-decoupled 13C NMR spectra of the glioma following infusion with [1-13C]glucose revealed the direct production of [3-13C]lactic acid, [1-13C]glycogen, and [4-13C], [3-13C], and [2-13C]glutamate/glutamine. Lactate levels of in vivo gliomas increased and reached steady state levels during [1-13C]glucose infusion, and decreased following termination of infusion. Complementary in vitro studies using supernatant media collected from C6 glioma cells incubated with media containing [1-13C] or [6-13C]glucose and glutamine were examined by 1H NMR spectroscopy. The [3-(13C/12C)]lactate ratios obtained from 1H spectra of supernatant media containing [1-13C]glucose revealed the percentage of glucose metabolized through the hexose monophosphate shunt to be 10.01 +/- 0.85% (n = 3), while similar measurements of media containing [6-13C]glucose and glutamine showed that glutaminolysis contributed 9.0 +/- 1.0% of total lactate production under these conditions. Enzymatic analysis of media determined lactate production to be 139 +/- 9 nmol per 10(6) cells per h (n = 4). These measurements demonstrate the ability of NMR to monitor brain tumor carbohydrate metabolism both in vitro and in vivo.     

Effect of perfusate glucose concentration on rat lung glycolysis.

We investigated the relationship between perfusate concentration of glucose and its utilization and lactate production derived from exogenous glucose and from metabolism of endogenous substrates. Isolated rat lungs were ventilated with 5% CO2 in air and perfused for 100 min with Krebs-Ringer bicarbonate buffer containing 3% bovine serum albumin, 10(-2) U/ml insulin, [U-14C]glucose and [5-3H]glucose. Glucose utilization, total lactate production, [14C]lactate production, and 3H2O production were measured. The apparent Km and Vmax for glucose utilization were 3.4 mM and 72.5 mumol/g dry wt per h, respectively. Lactate production from endogenous substrates, calculated as the difference between total and [14C]lactate, was 37.6 +/- 2.2 mumol/g dry wt (n = 36); it was unaffected by perfusate glucose concentration and by omission of insulin, but increased threefold with anoxia. Lactate production from 1.5 mM glucose was significantly less (P less than 0.02) with insulin omitted. Glycogen content was unchanged during perfusion without glucose. These results suggest that: 1) protein catabolism contributes to lung lactate production; 2) glucose utilization by lung is not maximal at resting physiological glucose concentrations; and 3) insulin is required at low glucose concentrations for maximal glycolytic rates.     

Hyperpolarized 13C NMR studies of glucose metabolism in living breast cancer cell cultures

The recent development of dissolution dynamic nuclear polarization (DNP) gives NMR the sensitivity to follow metabolic processes in living systems with high temporal resolution. In this article, we apply dissolution DNP to study the metabolism of hyperpolarized U‐¹³C,²H₇‐glucose in living, perfused human breast cancer cells. Spectrally selective pulses were used to maximize the signal of the main product, lactate, whilst preserving the glucose polarization; in this way, both C₁‐lactate and C₃‐lactate could be observed with high temporal resolution. The production of lactate by T47D breast cancer cells can be characterized by Michaelis–Menten‐like kinetics, with K_m = 3.5 ± 1.5 mm and V_max = 34 ± 4 fmol/cell/min. The high sensitivity of this method also allowed us to observe and quantify the glycolytic intermediates dihydroxyacetone phosphate and 3‐phosphoglycerate. Even with the enhanced DNP signal, many other glycolytic intermediates could not be detected directly. Nevertheless, by applying saturation transfer methods, the glycolytic intermediates glucose‐6‐phosphate, fructose‐6‐phosphate, fructose‐1,6‐bisphosphate, glyceraldehyde‐3‐phosphate, phosphoenolpyruvate and pyruvate could be observed indirectly. This method shows great promise for the elucidation of the distinctive metabolism and metabolic control of cancer cells, suggesting multiple ways whereby hyperpolarized U‐¹³C,²H₇‐glucose NMR could aid in the diagnosis and characterization of cancer in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

Mechanistic Model of Myocardial Energy Metabolism Under Normal and Ischemic Conditions

A moderate reduction in coronary blood flow results in decreased myocardial oxygen consumption, accelerated glycolysis, decreased pyruvate oxidation, and lactate accumulation. To quantitatively understand cardiac metabolism during ischemia, we have developed a mechanistic, mathematical model based on biochemical mass balances and reaction kinetics in cardiac cells. By numerical solution of model equations, computer simulations showed the dynamic responses in glucose, fatty acid, glucose-6-phosphate, glycogen, triglyceride, pyruvate, lactate, acetyl-CoA, and free-CoA as well as CO₂, O₂ phosphocreatine/creatine, nicotinamide adenine dinucleotide (reduced form)/nicotinamide adenine dinucleotide (oxidized form) NADH/NAD⁺, and adenosine diphosphate/adenosine triphosphate (ADP/ATP). When myocardial ischemia was simulated by a 60% reduction in coronary blood flow, the model generated myocardial concentrations, uptakes, and fluxes that were consistent with experimental data from in vivopig studies. After 60 min of ischemia the concentrations of glycogen, phosphocreatine, and ATP were decreased by 60%, 75%, and 50%, respectively. With the onset of ischemia, myocardial lactate concentration increased and the myocardium switched from net consumer to net producer of lactate. Our model predicted a rapid 13-fold increase in NADH/NAD but only a twofold increase in the ratio of acetyl-CoA to free-CoA. These findings are consistent with the concept that pyruvate oxidation is inhibited during ischemia partially by the rise in NADH/NAD⁺. © 2002 Biomedical Engineering Society. PAC2002: 8717Aa     

On the inhibition of hepatic glycogenolysis by fructose. A 31P-NMR study in perfused rat liver using the fructose analogue 2,5-anhydro-D-mannitol.

Inhibition of hormone-stimulated hepatic glycogenolysis by fructose (Fru) has been attributed to accumulation of the competitive inhibitor Fru1P and/or to the associated depletion of the substrate phosphate (Pi). To evaluate the relative importance of either factor, we used the Fru analogue 2,5-anhydro-D-mannitol (aHMol). This analogue is avidly phosphorylated, traps Pi, and inhibits hormone-stimulated glycogenolysis, but it is not a gluconeogenic substrate, and hence does not confound glycogenolytic glucose production. Livers were continuously perfused with dibutyryl-cAMP (100 microM) to clamp phosphorylase in its fully activated a form. We administered aHMol (3.8 mM), and studied changes in glycogenolysis (glucose, lactate and pyruvate output) and in cytosolic Pi and phosphomonoester (PME), using in situ 31P-NMR spectroscopy (n = 4). Lobes of seven livers perfused outside the magnet were extracted for evaluation, by high-resolution 31P-NMR, of the evolution of aHMol1P and of aHMol(1,6)P2. After addition of aHMol, both glycogenolysis and the NMR Pi signal dropped precipitously, while the PME signal rose continuously and was almost entirely composed of aHMol1P. Inhibition of glycogenolysis in excess of the drop in Pi could be explained by continuing accumulation of aHMol1P. A subsequent block of mitochondrial ATP synthesis by KCN (1 mM) caused a rapid increase of Pi. Despite recovery of Pi to values exceeding control levels, glycogenolysis only recovered partially, attesting to the Pi-dependence of glycogenolysis, but also to inhibition by aHMol phosphorylation products. However, KCN resulted in conversion of the major part of aHMol1P into aHMol(1,6)P2. Residual inhibition of glycogenolysis was due to aHMol1P. Indeed, the subsequent withdrawal of aHMol caused a further gradual decrease in the proportion of aHMol1P (being converted into aHMol(1,6)P2, in the absence of de novo aHMol1P synthesis), and this resulted in a gradual de-inhibition of glycogenolysis, in the absence of marked changes in Pi. Glycogenolytic rates were consistently predicted by a model assuming non-saturated Pi kinetics and competition by aHMol1P exclusively: In conclusion, limited Pi availability and the presence of competitive inhibitors are decisive factors in the control of the in situ catalytic potential of phosphorylase a.     

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‐¹³C]nonesterified fatty acids and [3‐¹³C]lactate/[3‐¹³C]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‐¹³C]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 β‐oxidation. Copyright © 2011 John Wiley & Sons, Ltd.