Solubilization of brain mitochondrial hexokinase by thiopental

Summary The control of hexokinase activity probably is accomplished by regulating the partitioning of the enzyme between soluble and particulate forms, the latter being more active. In the present investigation we have examined the thiopental effect on the cerebral hexokinase distribution. In anesthesia, after administration of thiopental to male Sprague Dawley rats, the increase of the soluble fraction of hexokinase was dose dependent.The change in the intracellular hexokinase distribution was reversible and lasted as long as general anesthesia existed.Also in experiments in vitro a solubilization of the mitochondrial hexokinase by thiopental (0.1–1 mM) occurred; it was depending on drug concentration. An inhibition of hexokinase was found neither in the total brain extract, nor in the soluble or the particulate fraction. The results suggest that phosphorylation of glucose in brain may be suppressed in anesthesia by shifting hexokinase activity from a more active mitochondrial form to its less active soluble form.This effect seems to be caused by a direct action of thiopental and is obviously correlated with anesthesia.The results have been briefly reported at the 17th spring meeting of the Deutsche Pharmakologische Gesellschaft at Mainz (Krieglstein and Bielicki, 1976)     

Activity of enzymes of glucose and triglyceride metabolism in adipose and muscle tissue from normal pregnant women at term

In order to further investigate insulin insensitivity in pregnancy, the activities of key enzymes in glycolysis and lipid metabolism were measured in adipose and muscle tissue biopsies from 20 normal pregnant women undergoing caesarean section at term, and 23 non-pregnant women of similar age and body weight undergoing gynaecological surgery. The activity of pyruvate kinase was decreased in pregnant women in both adipose tissue (0.015 (0.009-0.024) (median and range) vs 0.020 (0.009-0.038) Ug-1 wet weight, p less than 0.05) and muscle tissue (6.7 (3.6-10.9) vs 12.0 (2.8-16.2) U g-1 wet weight, p less than 0.001). The activity of hexokinase was decreased in adipose tissue only (0.045 (0.022-0.085) vs 0.057 (0.025-0.097) U g-1 wet weight, p less than 0.05), while the activity of phosphofructokinase was decreased in muscle tissue only (1.3 (0.7-2.6) vs 2.1 (0.3-4.5) U g-1 wet weight, p less than 0.01). Glucose-6-phosphate dehydrogenase activity was increased in muscle tissue (0.30 (0.11-0.59) vs 0.17 (0.09-0.48) U g-1 wet weight, p less than 0.05), while the activity of hydroxyacyl-CoA dehydrogenase was decreased in adipose tissue (0.5 (0.3-1.1) vs 1.0 (0.5-2.3) U g-1 wet weight p less than 0.001) from the pregnant women. Similar results were found when enzyme activities were calculated per gram of protein, but with poorer reproducibility.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bioenergetic dysfunction in Huntington's disease human cybrids

In this work we studied the mitochondrial-associated metabolic pathways in Huntington's disease (HD) versus control (CTR) cybrids, a cell model in which the contribution of mitochondrial defects from patients is isolated. HD cybrids exhibited an interesting increase in ATP levels, when compared to CTR cybrids. Concomitantly, we observed increased glycolytic rate in HD cybrids, as revealed by increased lactate/pyruvate ratio, which was reverted after inhibition of glycolysis. A decrease in glucose-6-phosphate dehydrogenase activity in HD cybrids further indicated decreased rate of the pentose-phosphate pathway. ATP levels of HD cybrids were significantly decreased under glycolysis inhibition, which was accompanied by a decrease in phosphocreatine. Nevertheless, pyruvate supplementation could not recover HD cybrids' ATP or phosphocreatine levels, suggesting a dysfunction in mitochondrial use of that substrate. Oligomycin also caused a decrease in ATP levels, suggesting a partial support of ATP generation by the mitochondria. Nevertheless, mitochondrial NADH/NAD_t levels were decreased in HD cybrids, which was correlated with a decrease in pyruvate dehydrogenase activity and protein expression, suggesting decreased tricarboxylic acid cycle (TCA) input from glycolysis. Interestingly, the activity of alpha-ketoglutarate dehydrogenase, a critical enzyme complex that links the TCA to amino acid synthesis and degradation, was increased in HD cybrids. In accordance, mitochondrial levels of glutamate were increased and alanine was decreased, whereas aspartate and glutamine levels were unchanged in HD cybrids. Conversely, malate dehydrogenase activity from total cell extracts was unchanged in HD cybrids. Our results suggest that inherent dysfunction of mitochondria from HD patients affects cellular bioenergetics in an otherwise functional nuclear background.     

The fundamental chemistry of life : An attempt to define and identify the basic reaction responsible for life''s creation and evolution

All natural growth follows exponential characteristics which vary from a simple exponential equation (non-solid cancer, bacteria) to complex Gompertzian functions describing solid cancer and multicellular organisms. Like all chemical processes the reagents (energy sources of food) react during life to produce vital energy, but in addition also create the next generation of life. This latter reaction is unique in that a simple proportional increase in the reagents creates an exponential increase in products: it is the sole invariable criterion of all life. The target of combined ionising and non-ionising radiations in cancer cells appears to be identical with this fundamental exponential chemical reaction. Identification of this target as a system of anaerobic glycolysis suggests that life's first reaction is a unique one whereby a simple proportional increase in available glucose causes an exponential proportional increase in energy which is available solely for reproduction.     

Glucose avidity of carcinomas

The cancer cell phenotype has been summarized in six hallmarks [D. Hanahan, R.A. Weinberg, The hallmarks of cancer, Cell 100 (1) (2000) 57-70]. Following the conceptual trait established in that review towards the comprehension of cancer, herein we summarize the basis of an underlying principle that is fulfilled by cancer cells and tumors: its avidity for glucose. Our purpose is to push forward that the metabolic reprogramming that operates in the cancer cell represents a seventh hallmark of the phenotype that offers a vast array of possibilities for the future treatment of the disease. We summarize the metabolic pathways that extract matter and energy from glucose, paying special attention to the concerted regulation of these pathways by the ATP mass-action ratio. The molecular and functional evidences that support the high glucose uptake and the "abnormal" aerobic glycolysis of the carcinomas are detailed discussing also the role that some oncogenes and tumor suppressors have in these pathways. We overview past and present evidences that sustain that mitochondria of the cancer cell are impaired, supporting the original Warburg's formulation that ascribed the high glucose uptake of cancer cells to a defective mitochondria. A simple proteomic approach designed to assess the metabolic phenotype of cancer, i.e., its bioenergetic signature, molecularly and functionally supports Warburg's hypothesis. Furthermore, we discuss the clinical utility that the bioenergetic signature might provide. Glycolysis is presented as the "selfish" pathway used for cellular proliferation, providing both the metabolic precursors and the energy required for biosynthetic purposes, in the context of a plethora of substrates. The glucose avidity of carcinomas is thus presented as the result of both the installment of glycolysis for cellular proliferation and of the impairment of mitochondrial activity in the cancer cell. At the end, the repression of mitochondrial activity affords the cancer cell with a cell-death resistant phenotype making them prone to malignant growth.     

Glucose deprivation produces a prolonged increase in sensitivity to glutamate in cultured rat cortical neurons

In this study we investigated whether the link between mitochondrial dysfunction and deregulation of Ca(2+) homeostasis preceding excitotoxic cell death is mediated by cellular deenergization. Glycolytic and/or mitochondrial ATP synthesis was inhibited with 2-deoxy-D-glucose (2DG) and oligomycin, respectively. Changes in cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) and mitochondrial membrane potential were simultaneously measured in response to low (10 microM) glutamate concentrations, using the fluorescence dyes fura-2FF and rhodamine 123. 2DG, which blocks glycolysis and also inhibits mitochondrial respiration due to depletion of pyruvate, greatly increased and accelerated glutamate-induced elevation of [Ca(2+)](c) and mitochondrial depolarization. The 2DG-induced hypersensitivity to glutamate was observed even after 150-min washout of 2DG with glucose-containing medium, suggesting a permanent deterioration of mitochondrial function. Prior blockade of only glycolytic (2DG with pyruvate) or only mitochondrial (oligomycin) ATP synthesis did not affect neuronal sensitivity to glutamate. Collectively, these studies show that to maintain the sensitivity of neurons to glutamate at control levels at least one of the cellular sources of ATP production must be intact. Either glycolysis or oxidative phosphorylation can effectively support Ca(2+) homeostasis in cultured forebrain neurons.     

Nitric oxide accounts for an increased glycolytic rate in activated astrocytes through a glycogenolysis-independent mechanism

The possible interference of nitric oxide (NO) in glucose metabolism was studied in activated astrocytes. Lipopolysaccharide (LPS) treatment triggered a NO-mediated increase in glucose consumption and lactate production, suggesting an enhanced rate of glycolysis. Active glycogen synthesis was also observed after LPS treatment, but NO synthase inhibition was unable to prevent this effect. These results strongly suggest that endogenously-formed NO stimulates glycolysis through a glycogenolysis-independent mechanism in astrocytes.     

Control of cellular proliferation by modulation of oxidative phosphorylation in human and rodent fast-growing tumor cells

The relationship between cell proliferation and the rates of glycolysis and oxidative phosphorylation in HeLa (human) and AS-30D (rodent) tumor cells was evaluated. In glutamine plus glucose medium, both tumor lines grew optimally. Mitochondria were the predominant source of ATP in both cell types (66-75%), despite an active glycolysis. In glucose-free medium with glutamine, proliferation of both lines diminished by 30% but oxidative phosphorylation and the cytosolic ATP level increased by 50%. In glutamine-free medium with glucose, proliferation, oxidative phosphorylation and ATP concentration diminished drastically, although the cells were viable. Oligomycin, in medium with glutamine plus glucose, abolished growth of both tumor lines, indicating an essential role of mitochondrial ATP for tumor progression. The presumed mitochondrial inhibitors rhodamines 123 and 6G, and casiopeina II-gly, inhibited tumor cell proliferation and oxidative phosphorylation, but also glycolysis. In contrast, gossypol, iodoacetate and arsenite strongly blocked glycolysis; however, they did not affect tumor proliferation or mitochondrial metabolism. Growth of both tumor lines was highly sensitive to rhodamines and casiopeina II-gly, with IC(50) values for HeLa cells lower than 0.5 microM, whereas viability and proliferation of human lymphocytes were not affected by these drugs (IC(50) > 30 microM). Moreover, rhodamine 6G and casiopeina II-gly, at micromolar doses, prolonged the survival of animals bearing i.p. implanted AS-30D hepatoma. It is concluded that fast-growing tumor cells have a predominantly oxidative type of metabolism, which might be a potential therapeutic target.     

Pyruvate protects against kainate-induced epileptic brain damage in rats

Although the majority of epileptic seizures can be effectively controlled with antiepileptic drugs and/or surgery, a significant number progress to status epilepticus of sufficient duration to cause permanent brain damage. Combined treatment with antiepileptic drugs and neuroprotective agents, however, may help protect these individuals from permanent brain damage. Since toxicity induced by endogenous zinc contributes to epileptic brain injury, and since pyruvate is effective in reducing zinc-triggered neuronal death in cortical culture as well as ischemic neuronal death in vivo, we examined whether systemic pyruvate administration reduces seizure-induced brain damage. Na pyruvate (500 mg/kg) or osmolarity-matched saline (265 mg/kg NaCl, i.p.) were given to adult SD rats 30 or 150 min after 10 mg/kg kainite injection (i.p.), and there was no significant difference in the time course or severity of seizures between these groups. Zinc accumulation in neuronal cell bodies in the hippocampus, however, was much lower in the pyruvate than in the saline group. There was a close correlation between zinc accumulation and cell death, as assessed by acid-fuchsin and TUNEL staining. Pyruvate treatment markedly reduced neuronal death in the hippocampus, neocortex and thalamus. Pyruvate increased HSP-70 expression in hippocampal neurons. These results suggest that pyruvate, a natural glucose metabolite, may be useful as adjunct treatment in status epilepticus to reduce permanent brain damage.     

Inhibition of phosphatidylinositol 3-kinase-mediated glucose metabolism coincides with resveratrol-induced cell cycle arrest in human diffuse large B-cell lymphomas

An abnormally high rate of aerobic glycolysis is characteristic of many transformed cells. Here we report the polyphenolic compound, resveratrol, inhibited phosphatidylinositol 3-kinase (PI-3K) signaling and glucose metabolism, coinciding with cell-cycle arrest, in germinal center (GC)-like LY1 and LY18 human diffuse large B-cell lymphomas (DLBCLs). Specifically, resveratrol inhibited the phosphorylation of Akt, p70 S6K, and S6 ribosomal protein on activation residues. Biochemical analyses and nuclear magnetic resonance spectroscopy identified glycolysis as the primary glucose catabolic pathway in LY18 cells. Treatment with the glycolytic inhibitor 2-deoxy-D-glucose, resulted in accumulation of LY18 cells in G0/G1 -phase, underscoring the biological significance of glycolysis in growth. Glycolytic flux was inhibited by the PI-3K inhibitor LY294002, suggesting a requirement for PI-3K activity in glucose catabolism. Importantly, resveratrol treatment resulted in inhibition of glycolysis. Decreased glycolytic flux corresponded to a parallel reduction in the expression of several mRNAs encoding rate-limiting glycolytic enzymes. These results are the first to identify as a mechanism underlying resveratrol-induced growth arrest, the inhibition of glucose catabolism by the glycolytic pathway. Taken together, these results raise the possibility that inhibition of signaling and metabolic pathways that control glycolysis might be effective in therapy of DLBCLs.