Glycolysis–respiration relationships in a neuroblastoma cell line

Background

Although some reciprocal glycolysis–respiration relationships are well recognized, the relationship between reduced glycolysis flux and mitochondrial respiration has not been critically characterized.

Methods

We concomitantly measured the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of SH-SY5Y neuroblastoma cells under free and restricted glycolysis flux conditions.

Results

Under conditions of fixed energy demand ECAR and OCR values showed a reciprocal relationship. In addition to observing an expected Crabtree effect in which increasing glucose availability raised the ECAR and reduced the OCR, a novel reciprocal relationship was documented in which reducing the ECAR via glucose deprivation or glycolysis inhibition increased the OCR. Substituting galactose for glucose, which reduces net glycolysis ATP yield without blocking glycolysis flux, similarly reduced the ECAR and increased the OCR. We further determined how reduced ECAR conditions affect proteins that associate with energy sensing and energy response pathways. ERK phosphorylation, SIRT1, and HIF1a decreased while AKT, p38, and AMPK phosphorylation increased.

Conclusions

These data document a novel intracellular glycolysis–respiration effect in which restricting glycolysis flux increases mitochondrial respiration.

General significance

Since this effect can be used to manipulate cell bioenergetic infrastructures, this particular glycolysis–respiration effect can practically inform the development of new mitochondrial medicine approaches.     

Excess exogenous pyruvate inhibits lactate dehydrogenase activity in live cells in an MCT1-dependent manner

Cellular pyruvate is an essential metabolite at the crossroads of glycolysis and oxidative phosphorylation, capable of supporting fermentative glycolysis by reduction to lactate mediated by lactate dehydrogenase (LDH) among other functions. Several inherited diseases of mitochondrial metabolism impact extracellular (plasma) pyruvate concentrations, and [1-¹³C]pyruvate infusion is used in isotope-labeled metabolic tracing studies, including hyperpolarized magnetic resonance spectroscopic imaging. However, how these extracellular pyruvate sources impact intracellular metabolism is not clear. Herein, we examined the effects of excess exogenous pyruvate on intracellular LDH activity, extracellular acidification rates (ECARs) as a measure of lactate production, and hyperpolarized [1-¹³C]pyruvate-to-[1-¹³C]lactate conversion rates across a panel of tumor and normal cells. Combined LDH activity and LDHB/LDHA expression analysis intimated various heterotetrameric isoforms comprising LDHA and LDHB in tumor cells, not only canonical LDHA. Millimolar concentrations of exogenous pyruvate induced substrate inhibition of LDH activity in both enzymatic assays ex vivo and in live cells, abrogated glycolytic ECAR, and inhibited hyperpolarized [1-¹³C]pyruvate-to-[1-¹³C]lactate conversion rates in cellulo. Of importance, the extent of exogenous pyruvate-induced inhibition of LDH and glycolytic ECAR in live cells was highly dependent on pyruvate influx, functionally mediated by monocarboxylate transporter-1 localized to the plasma membrane. These data provided evidence that highly concentrated bolus injections of pyruvate in vivo may transiently inhibit LDH activity in a tissue type- and monocarboxylate transporter-1–dependent manner. Maintaining plasma pyruvate at submillimolar concentrations could potentially minimize transient metabolic perturbations, improve pyruvate therapy, and enhance quantification of metabolic studies, including hyperpolarized [1-¹³C]pyruvate magnetic resonance spectroscopic imaging and stable isotope tracer experiments.     

S-nitrosation of monocarboxylate transporter 1: Inhibition of pyruvate-fueled respiration and proliferation of breast cancer cells

Energy substrates metabolized through mitochondria (e.g., pyruvate, glutamine) are required for biosynthesis of macromolecules in proliferating cells. Because several mitochondrial proteins are known to be targets of S-nitrosation, we determined whether bioenergetics are modulated by S-nitrosation and defined the subsequent effects on proliferation. The nitrosating agent S-nitroso-l-cysteine (L-CysNO) was used to initiate intracellular S-nitrosation, and treatment decreased mitochondrial function and inhibited proliferation of MCF7 mammary adenocarcinoma cells. Surprisingly, the d-isomer of CysNO (D-CysNO), which is not transported into cells, also caused mitochondrial dysfunction and limited proliferation. Both L- and D-CysNO also inhibited cellular pyruvate uptake and caused S-nitrosation of thiol groups on monocarboxylate transporter 1, a proton-linked pyruvate transporter. These data demonstrate the importance of mitochondrial metabolism in proliferative responses in breast cancer and highlight a novel role for inhibition of metabolic substrate uptake through S-nitrosation of exofacial protein thiols in cellular responses to nitrosative stress.     

Reaction between nitric oxide,glutathione, and oxygen in the presence and absence of protein: How are S-nitrosothiols formed?

The reaction between NO, thiols, and oxygen has been studied in some detail in vitro due to its perceived importance in the mechanism of NO-dependent signal transduction. The formation of S-nitrosothiols and thiol disulfides from this chemistry has been suggested to be an important component of the biological chemistry of NO, and such subsequent thiol modifications may result in changes in cellular function and phenotype. In this study we have reinvestigated this reaction using both experiment and simulation and conclude that: (i) S-nitrosation through radical and nonradical pathways is occurring simultaneously, (ii) S-nitrosation through direct addition of NO to thiol does not occur to any meaningful extent, and (iii) protein hydrophobic environments do not catalyze or enhance S-nitrosation of either themselves or of glutathione. We conclude that S-nitrosation and disulfide formation in this system occur only after the initial reaction between NO and oxygen to form nitrogen dioxide, and that hydrophobic protein environments are unlikely to play any role in enhancing and targeting S-nitrosothiol formation.     

Epigallocatechin gallate, a green tea polyphenol, mediates NO-dependent vasodilation using signaling pathways in vascular endothelium requiring reactive oxygen species and Fyn

Green tea consumption is associated with reduced cardiovascular mortality in some epidemiological studies. Epigallocatechin gallate (EGCG), a bioactive polyphenol in green tea, mimics metabolic actions of insulin to inhibit gluconeogenesis in hepatocytes. Because signaling pathways regulating metabolic and vasodilator actions of insulin are shared in common, we hypothesized that EGCG may also have vasodilator actions to stimulate production of nitric oxide (NO) from endothelial cells. Acute intra-arterial administration of EGCG to mesenteric vascular beds isolated ex vivo from WKY rats caused dose-dependent vasorelaxation. This was inhibitable by L-NAME (NO synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), or PP2 (Src family kinase inhibitor). Treatment of bovine aortic endothelial cells (BAEC) with EGCG (50 microm) acutely stimulated production of NO (assessed with NO-specific fluorescent dye DAF-2) that was inhibitable by l-NAME, wortmannin, or PP2. Stimulation of BAEC with EGCG also resulted in dose- and time-dependent phosphorylation of eNOS that was inhibitable by wortmannin or PP2 (but not by MEK inhibitor PD98059). Specific knockdown of Fyn (but not Src) with small interfering RNA inhibited both EGCG-stimulated phosphorylation of Akt and eNOS as well as production of NO in BAEC. Treatment of BAEC with EGCG generated intracellular H(2)O(2) (assessed with H(2)O(2)-specific fluorescent dye CM-H(2)DCF-DA), whereas treatment with N-acetylcysteine inhibited EGCG-stimulated phosphorylation of Fyn, Akt, and eNOS. We conclude that EGCG has endothelial-dependent vasodilator actions mediated by intracellular signaling pathways requiring reactive oxygen species and Fyn that lead to activation of phosphatidylinositol 3-kinase, Akt, and eNOS. This mechanism may explain, in part, beneficial vascular and metabolic health effects of green tea consumption.     

Chemical methods to detect S-nitrosation

Nitric oxide (NO) is a cell-signaling molecule involved in a number of physiological and pathophysiological processes. Modification of cysteine residues by NO (or NO metabolites), that is S-nitrosation, changes the function of a broad spectrum of proteins. This reaction represents an important post-translational modification that transduces NO-dependent signals. However, the detection and quantification of S-nitrosation in biological samples remain a challenge mainly because of the lability of S-nitrosation products: S-nitrosothiols (SNO). In this review we summarize recent developments of the methods to detect S-nitrosation. Our focus is on the methods which can be used to directly conjugate the site(s) of S-nitrosation.     

Soluble guanylyl cyclase is a target of angiotensin II-induced nitrosative stress in a hypertensive rat model

Nitric oxide (NO) by activating soluble guanylyl cyclase (sGC) is involved in vascular homeostasis via induction of smooth muscle relaxation. In cardiovascular diseases (CVDs), endothelial dysfunction with altered vascular reactivity is mostly attributed to decreased NO bioavailability via oxidative stress. However, in several studies, relaxation to NO is only partially restored by exogenous NO donors, suggesting sGC impairment. Conflicting results have been reported regarding the nature of this impairment, ranging from decreased expression of one or both subunits of sGC to heme oxidation. We showed that sGC activity is impaired by thiol S-nitrosation. Recently, angiotensin II (ANG II) chronic treatment, which induces hypertension, was shown to generate nitrosative stress in addition to oxidative stress. We hypothesized that S-nitrosation of sGC occurs in ANG II-induced hypertension, thereby leading to desensitization of sGC to NO hence vascular dysfunction. As expected, ANG II infusion increases blood pressure, aorta remodeling, and protein S-nitrosation. Intravital microscopy indicated that cremaster arterioles are resistant to NO-induced vasodilation in vivo in anesthetized ANG II-treated rats. Concomitantly, NO-induced cGMP production decreases, which correlated with S-nitrosation of sGC in hypertensive rats. This study suggests that S-nitrosation of sGC by ANG II contributes to vascular dysfunction. This was confirmed in vitro by using A7r5 smooth muscle cells infected with adenoviruses expressing sGC or cysteine mutants: ANG II decreases NO-stimulated activity in the wild-type but not in one mutant, C516A. This result indicates that cysteine 516 of sGC mediates ANG II-induced desensitization to NO in cells.     

Rapid Analysis of Glycolytic and Oxidative Substrate Flux of Cancer Cells in a Microplate

Cancer cells exhibit remarkable alterations in cellular metabolism, particularly in their nutrient substrate preference. We have devised several experimental methods that rapidly analyze the metabolic substrate flux in cancer cells: glycolysis and the oxidation of major fuel substrates glucose, glutamine, and fatty acids. Using the XF Extracellular Flux analyzer, these methods measure, in real-time, the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of living cells in a microplate as they respond to substrates and metabolic perturbation agents. In proof-of-principle experiments, we analyzed substrate flux and mitochondrial bioenergetics of two human glioblastoma cell lines, SF188s and SF188f, which were derived from the same parental cell line but proliferate at slow and fast rates, respectively. These analyses led to three interesting observations: 1) both cell lines respired effectively with substantial endogenous substrate respiration; 2) SF188f cells underwent a significant shift from glycolytic to oxidative metabolism, along with a high rate of glutamine oxidation relative to SF188s cells; and 3) the mitochondrial proton leak-linked respiration of SF188f cells increased significantly compared to SF188s cells. It is plausible that the proton leak of SF188f cells may play a role in allowing continuous glutamine-fueled anaplerotic TCA cycle flux by partially uncoupling the TCA cycle from oxidative phosphorylation. Taken together, these rapid, sensitive and high-throughput substrate flux analysis methods introduce highly valuable approaches for developing a greater understanding of genetic and epigenetic pathways that regulate cellular metabolism, and the development of therapies that target cancer metabolism.     

Romina: A powerful strawberry with in vitro efficacy against uterine leiomyoma cells

Uterine leiom yomas are benign tumors highly prevalent in reproductive women. In thecurrent study, initially, we aimed to screen five different strawberry cultivars (Alba, Clery, Portola, Tecla, and Romina) to identify efficient cultivars in terms of phytochemical characterization and biological properties by measuring phenolic and anthocyanin content as well as antioxidant capacity, and by measuring apoptotic rate and reactive oxygen species (ROS) production in uterine leiomyoma cells. Next, we focused on the most efficient ones, cultivar Alba (A) and Romina (R) as well as Romina anthocyanin (RA) fraction for their ability to regulate oxidative phosphorylation (oxygen consumption rate [OCR]) glycolysis (extracellular acidification rate [ECAR]), and also fibrosis. Leiomyoma and myometrial cells were treated with a methanolic extract of A and R (250 μg/ml) or with RA (50 μg/ml) for 48 hr to measure OCR and ECAR, as well as gene expression associated with fibrosis. In the leiomyoma cells, RA was more effective in inducing apoptosis and increasing intracellular ROS levels, followed by R and A. In myometrial cells, all strawberry treatments increased the cellular viability and decreased ROS concentrations. Leiomyoma cells showed also a significant decrease in ECAR, especially after RA treatment, while OCR was slightly increased in both myometrial and leiomyoma cells. R and RA treatment significantly decreased collagen 1A1, fibronectin, versican, and activin A messenger RNA expression in leiomyoma cells. In conclusion, this study suggests that Romina, or its anthocyanin fraction, can be developed as a therapeutic and/or preventive agent for uterine leiomyomas, confirming the healthy effects exerted by these fruits and their bioactive compounds.     

Phosphorus nuclear magnetic resonance studies of phosphorus metabolites in frog muscle.

31P-nuclear magnetic resonance was applied to living muscles of bullfrogs, and the time courses of metabolic changes of ATP, creatine phosphate, inorganic phosphate, and sugar phosphates were studied under anaerobic and aerobic conditions. A decrease in creatine phosphate was observed in the resting muscle under anaerobic conditions with a concomitant decrease in the intracellular pH, while the ATP level remained constant. With the use of 2,4-dinitro-1-fluorobenzene and iodoacetic acid, ATP disappeared quickly. When the resting muscle was perfused with oxygen-saturated glucose-Ringer's solution, the amount of creatine phosphate increased gradually. These findings indicate that anaerobic glycolysis is insufficient for even the resting energy consumption whereas oxidative phosphorylation is sufficient. The effects of tetanic stimulation on living muscles were also studied. When glycolysis and oxidative phosphorylation were suppressed, the intracellular energy store was depleted by the tetanic contraction. Anaerobic glycolysis produced rapid recovery of the energy store level, although it was insufficient to reach the initial level. Aerobic oxidative phosphorylation produced sufficient energy to reach the initial level, and this level was never exceeded. This finding suggests the existence of a regulatory mechanism for the energy store level.     

Regulation of lipolysis and cyclic AMP synthesis through energy supply in isolated human fat cells.

The effects of glucose and of various inhibitors of glycolysis or of oxidative phosphorylation on stimulated lipolysis and on intracellular cyclic AMP and ATP levels were investigated in isolated human fat cells. The glycolysis inhibitors, NaF and monoiodoacetate, inhibited epinephrine or theophylline-stimulated lipolysis and parallely reduced the intracellular cyclic AMP and ATP levels; however, neither NaF nor monoidoacetate significantly affected dibutyryl cyclic AMP-induced lipolysis. Removal of glucose from the medium also reduced the rate of epinephrine-stimulated lipolysis and the intracellular cyclic AMP and ATP levels but failed to modify the lipolytic activity of dibutyryl cyclic AMP. The oxidative phosphorylation inhibitors, antimycin A and, under fixed conditions, 2,4-dinitrophenol also strongly decreased the adipocyte cyclic AMP and ATP levels but inhibited as well the rate of epinephrine- and of dibutyryl cyclic AMP-induced lipolysis. N-Ethylmaleimide, a mixed glycolysis and oxidative phosphorylation inhibitor, not only reduced the intracellular cyclic AMP and ATP levels and epinephrine- or theophylline-induced lipolysis, but also that stimulated by dibutyryl cyclic AMP. When glycolysis was almost fully inhibited, human fat cells were insensitive to epinephrine but remained fully responsive to dibutyryl cyclic AMP. These results, showing a relationship between ATP availability, cyclic AMP synthesis and lipolysis, suggest a different ATP requirement for cyclic AMP synthesis and triacylglycerol lipase activation, a difference which could explain why ATP issued from glucose breakdown appears to be a determinant factor for cyclic AMP synthesis, but not for triacylglycerol lipase activation in human fat cells.     

Phosphatidylinositol 3-kinase and xanthine oxidase regulate nitric oxide and reactive oxygen species productions by apoptotic lymphocyte microparticles in endothelial cells

Microparticles (MPs) are membrane vesicles released during cell activation and apoptosis. We have previously shown that MPs from apoptotic T cells induce endothelial dysfunction, but the mechanisms implicated are not completely elucidated. In this study, we dissect the pathways involved in endothelial cells with respect to both NO and reactive oxygen species (ROS). Incubation of endothelial cells with MPs decreased NO production that was associated with overexpression and phosphorylation of endothelial NO synthase (eNOS). Also, MPs enhanced expression of caveolin-1 and decreased its phosphorylation. Microparticles enhanced ROS by a mechanism sensitive to xanthine oxidase and P-IkappaBalpha inhibitors. PI3K inhibition reduced the effects of MPs on eNOS, but not on caveolin-1, whereas it enhanced the effects of MPs on ROS production. Microparticles stimulated ERK1/2 phosphorylation via a PI3K-depedent mechanism. Inhibition of MEK reversed eNOS phosphorylation but had no effect on ROS production induced by MPs. In vivo injection of MPs in mice impaired endothelial function. In summary, MPs activate pathways related to NO and ROS productions through PI3K, xanthine oxidase, and NF-kappaB pathways. These data underscore the pleiotropic effects of MPs on NO and ROS, leading to an increase oxidative stress that may account for the deleterious effects of MPs on endothelial function.     

Combining p53 stabilizers with metformin induces synergistic apoptosis through regulation of energy metabolism in castration-resistant prostate cancer

Since altered energy metabolism is a hallmark of cancer, many drugs targeting metabolic pathways are in active clinical trials. The tumor suppressor p53 is often inactivated in cancer, either through downregulation of protein or loss-of-function mutations. As such, stabilization of p53 is considered as one promising approach to treat those cancers carrying wild type (WT) p53. Herein, SIRT1 inhibitor Tenovin-1 and polo-like kinase 1 (Plk1) inhibitor BI2536 were used to stabilize p53. We found that both Tennovin-1 and BI2536 increased the anti-neoplastic activity of metformin, an inhibitor of oxidative phosphorylation, in a p53 dependent manner. Since p53 has also been shown to regulate metabolic pathways, we further analyzed glycolysis and oxidative phosphorylation upon drug treatments. We showed that both Tennovin-1 and BI2536 rescued metformin-induced glycolysis and that both Tennovin-1 and BI2536 potentiated metformin-associated inhibition of oxidative phosphorylation. Of significance, castration-resistant prostate cancer (CRPC) C4-2 cells show a much more robust response to the combination treatment than the parental androgen-dependent prostate cancer LNCaP cells, indicating that targeting energy metabolism with metformin plus p53 stabilizers might be a valid approach to treat CRPC carrying WT p53.     

S-nitrosothiols inhibit uterine smooth muscle cell proliferation independent of metabolism to NO and cGMP formation

S-nitrosothiols (RSNOs) are important mediators of nitric oxide (NO) biology. The two mechanisms that appear to dominate in their biological effects are metabolism leading to the formation of NO and S-nitrosation of protein thiols. In this study we demonstrate that RSNOs inhibit uterine smooth muscle cell proliferation independent of NO. The antiproliferative effects of NO on vascular smooth muscle are well defined, with the classic NO-dependent production of cGMP being demonstrated as the active pathway. However, less is known on the role of NO in mediating uterine smooth muscle cell function, a process that is important during menstruation and pregnancy. The RSNOs S-nitrosoglutathione and S-nitroso-N-acetyl pencillamine inhibited growth factor-dependent proliferation of human and rat uterine smooth muscle cells (ELT-3). Interestingly, these cells reduced RSNOs to generate NO. However, use of NO donors and other activators of the cGMP pathway failed to inhibit proliferation. These findings demonstrate the tissue-specific nature of responses to NO and demonstrate the presence of a RSNO-dependent but NO-independent pathway of inhibiting DNA synthesis in uterine smooth muscle cells.     

Localization of endothelial nitric-oxide synthase phosphorylated on serine 1179 and nitric oxide in Golgi and plasma membrane defines the existence of two pools of active enzyme.

The subcellular localization of endothelial nitric-oxide synthase (eNOS) is critical for optimal coupling of extracellular stimulation to nitric oxide production. Because eNOS is activated by Akt-dependent phosphorylation to produce nitric oxide (NO), we determined the subcellular distribution of eNOS phosphorylated on serine 1179 using a variety of methodologies. Based on sucrose gradient fractionation, phosphorylated-eNOS (P-eNOS) was found in both caveolin-1-enriched membranes and intracellular domains. Co-transfection of eNOS with Akt and stimulation of endothelial cells with vascular endothelial growth factor (VEGF) increased the ratio of P-eNOS to total eNOS but did not change the relative intracellular distribution between these domains. The proper localization of eNOS to intracellular membranes was required for agonist-dependent phosphorylation on serine 1179, since VEGF did not increase eNOS phosphorylation in cells transfected with a non-acylated, mistargeted form of eNOS. Confocal imaging of P-eNOS and total eNOS pools demonstrated co-localization in the Golgi region and plasmalemma of transfected cells and native endothelial cells. Finally, VEGF stimulated a large increase in NO localized in both the perinuclear region and the plasma membrane of endothelial cells. Thus, activated, phosphorylated eNOS resides in two cellular compartments and both pools are VEGF-regulated to produce NO.     

Assessment of drug-induced mitochondrial dysfunction via altered cellular respiration and acidification measured in a 96-well platform

High-throughput applicable screens for identifying drug-induced mitochondrial impairment are necessary in the pharmaceutical industry. Hence, we evaluated the XF96 Extracellular Flux Analyzer, a 96-well platform that measures changes in the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of cells. The sensitivity of the platform was bench-marked with known modulators of oxidative phosphorylation and glycolysis. Sixteen therapeutic agents were screened in HepG2 cells for mitochondrial effects. Four of these compounds, thiazolidinediones, were also tested in primary feline cardiomyocytes for cell-type specific effects. We show that the XF96 platform is a robust, sensitive system for analyzing drug-induced mitochondrial impairment in whole cells. We identified changes in cellular respiration and acidification upon addition of therapeutic agents reported to have a mitochondrial effect. Furthermore, we show that respiration and acidification changes upon addition of the thiazoldinediones were cell-type specific, with the rank order of mitochondrial impairment in whole cells being in accord with the known adverse effects of these drugs.     

Effects of metabolic inhibition on conduction, Ca transients, and arrhythmia vulnerability in embryonic mouse hearts

Developing myocardium is more dependent on glycolysis than adult myocardium, yet the effects of selectively inhibiting glycolysis versus oxidative phosphorylation on embryonic heart function have not been well characterized. Accordingly, we investigated how selective metabolic inhibition affects membrane voltage and intracellular Ca (Ca(i)) transients in embryonic mouse hearts, including their susceptibility to arrhythmias. A total of 136 isolated embryonic mouse hearts were exposed to either 1) 2-deoxyglucose (2DG; 10 mM) or iodoacetate (IAA; 0.1 mM) with 10 mM pyruvate in place of glucose to selectively inhibit glycolysis or 2) the mitochondrial uncoupler protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP; 500 nM) with 10 mM glucose present to selectively inhibit oxidative phosphorylation. Using confocal imaging, we found that mitochondrial membrane potential monitored with tetramethylrhodamine methyl ester (200 nM) remained stable with 2DG or IAA but depolarized within 5 min after exposure to FCCP. IAA and FCCP decreased heart rate, inhibited Ca(i) transient amplitude, shortened action potential duration at 80% repolarization (APD(80)), and prolonged atrioventricular conduction time to similar extents. Although 2DG decreased heart rate and Ca(i) transient amplitude, it did not significantly affect APD(80) and AV conduction time. In addition, spontaneous arrhythmias occurred in 77 of 136 embryonic hearts (57%) after exposure to IAA (28/53) or FCCP (49/83). There were no significant differences in the types or incidence of arrhythmias induced by IAA and FCCP. These data support the idea that both glycolysis and oxidative phosphorylation play critical metabolic roles in regulating cardiac function in the embryonic mouse heart.     

Cell energy metabolism: An update

In living cells, growth is the result of coupling between substrate catabolism and multiple metabolic processes that take place during net biomass formation and maintenance processes. During growth, both ATP/ADP and NADH/NAD⁺ molecules play a key role. Cell energy metabolism hence refers to metabolic pathways involved in ATP synthesis linked to NADH turnover. Two main pathways are thus involved in cell energy metabolism: glycolysis/fermentation and oxidative phosphorylation. Glycolysis and mitochondrial oxidative phosphorylation are intertwined through thermodynamic and kinetic constraints that are reviewed herein. Further, our current knowledge of short-term and long term regulation of cell energy metabolism will be reviewed using examples such as the Crabtree and the Warburg effect.     

Impairment of human ether-à-go-go-related gene (HERG) K+ channel function by hypoglycemia and hyperglycemia. Similar phenotypes but different mechanisms

Hyperglycemia and hypoglycemia both can cause prolongation of the Q-T interval and ventricular arrhythmias. Here we studied modulation of human ether-à-go-go-related gene (HERG) K(+) channel, the major molecular component of delayed rectifier K(+) current responsible for cardiac repolarization, by glucose in HEK293 cells using whole-cell patch clamp techniques. We found that both hyperglycemia (extracellular glucose concentration [Glu](o) = 10 or 20 mm) and hypoglycemia ([Glu](o) = 2.5, 1, or 0 mm) impaired HERG function by reducing HERG current (I(HERG)) density, as compared with normoglycemia ([Glu](o) = 5 mm). Complete inhibition of glucose metabolism (glycolysis and oxidative phosphorylation) by 2-deoxy-d-glucose mimicked the effects of hypoglycemia, but inhibition of glycolysis or oxidative phosphorylation alone did not cause I(HERG) depression. Depletion of intracellular ATP mimicked the effects of hypoglycemia, and replacement of ATP by GTP or non-hydrolysable ATP failed to prevent the effects. Inhibition of oxidative phosphorylation by NaCN or application of antioxidants vitamin E or superoxide dismutase mimetic (Mn(III) tetrakis(4-benzoic acid) porphyrin chloride) abrogated and incubation with xanthine/xanthine oxidase mimicked the effects of hyperglycemia. Hyperglycemia or xanthine/xanthine oxidase markedly increased intracellular levels of reactive oxygen species, as measured by 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA) fluorescence dye, and this increase was prevented by NaCN, vitamin E, or Mn(III) tetrakis(4-benzoic acid) porphyrin chloride. We conclude that ATP, derived from either glycolysis or oxidative phosphorylation, is critical for normal HERG function; depression of I(HERG) in hypoglycemia results from underproduction of ATP and in hyperglycemia from overproduction of reactive oxygen species. Impairment of HERG function might contribute to Q-T prolongation caused by hypoglycemia and hyperglycemia.