Coupled Glucose Transport and Metabolism in Cultured Neuronal Cells: Determination of the Rate-Limiting Step

Whitesell, Richard R.; Ward, Michael P.; McCall, Anthony L.; Granner, Daryl K.; May, James M.

doi:10.1038/jcbfm.1995.102

Cited by 32 publications

(21 citation statements)

References 58 publications

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“…It is widely accepted that the major rate-limiting step in neuronal glucose metabolism is its phosphorylation by hexokinase, the first ATP-driven reaction of glycolysis (44). Insulin appears to upregulate hexokinase activity, localization, and expression, thus modulating glucose metabolism to meet the energy demands of sensory neurons (45).…”

Section: Discussionmentioning

confidence: 99%

Insulin Restores Metabolic Function in Cultured Cortical Neurons Subjected to Oxidative Stress

et al. 2006

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We previously demonstrated that insulin has a neuroprotective role against oxidative stress, a deleterious condition associated with diabetes, ischemia, and age-related neurodegenerative diseases. In this study, we investigated the effect of insulin on neuronal glucose uptake and metabolism after oxidative stress in rat primary cortical neurons. On oxidative stress, insulin stimulates neuronal glucose uptake and subsequent metabolism into pyruvate, restoring intracellular ATP and phosphocreatine. Insulin also increases intracellular and decreases extracellular adenosine, counteracting the effect of oxidative stress. Insulin effects are apparently mediated by phosphatidylinositol 3-K and extracellular signal-regulated kinase signaling pathways. Extracellular adenosine under oxidative stress is largely inhibited after blockade of ecto-5-nucleotidase, suggesting that extracellular adenosine results preferentially from ATP release and catabolism. Moreover, insulin appears to interfere with the ATP release induced by oxidative stress, regulating extracellular adenosine levels. In conclusion, insulin neuroprotection against oxidative stress-mediated damage involves 1) stimulation of glucose uptake and metabolism, increasing energy levels and intracellular adenosine and, ultimately, uric acid formation and 2) a decrease in extracellular adenosine, which may reduce the facilitatory activity of adenosine receptors.

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Section: Discussionmentioning

confidence: 99%

Insulin Restores Metabolic Function in Cultured Cortical Neurons Subjected to Oxidative Stress

et al. 2006

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“…Garfinkel et al derived this formula according to the data of Grossbard and Schimke (20) and assumed a random order ternary complex mechanism and assumed that G6P is a competitive inhibitor of ATP and a noncompetitive inhibitor of glucose. A sensitive and precise tracer technique allowed us to measure small amounts of enzyme activity at low substrate and high inhibitor concentrations (21). To use the glucose tracer data directly in the determination of the kinetic parameters, we modified the hexokinase rate law by dividing both sides of the equation by the concentration of glucose to correct for the specific activity of the tracer.…”

Section: Methodsmentioning

confidence: 99%

Functional Interaction between the N- and C-terminal Halves of Human Hexokinase II

Ardehali

Printz

Whitesell

et al. 1999

Journal of Biological Chemistry

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Mammalian hexokinases (HKs) I-III are composed of two highly homologous ϳ50-kDa halves. Studies of HKI indicate that the C-terminal half of the molecule is active and is sensitive to inhibition by glucose 6-phosphate (G6P), whereas the N-terminal half binds G6P but is devoid of catalytic activity. In contrast, both the N-and C-terminal halves of HKII (N-HKII and C-HKII, respectively) are catalytically active, and when expressed as discrete proteins both are inhibited by G6P. However, C-HKII has a significantly higher K i for G6P (K iG6P ) than N-HKII. We here address the question of whether the high K iG6P of the C-terminal half (C-half) of HKII is decreased by interaction with the N-terminal half (N-half) in the context of the intact enzyme. A chimeric protein consisting of the N-half of HKI and the C-half of HKII was prepared. Because the N-half of HKI is unable to phosphorylate glucose, the catalytic activity of this chimeric enzyme depends entirely on the C-HKII component. The K iG6P of this chimeric enzyme is similar to that of HKI and is significantly lower than that of C-HKII. When a conserved amino acid (Asp 209 ) required for glucose binding is mutated in the N-half of this chimeric protein, a significantly higher K iG6P (similar to that of C-HKII) is observed. However, mutation of a second conserved amino acid (Ser 155 ), also involved in catalysis but not required for glucose binding, does not increase the K iG6P of the chimeric enzyme. This resembles the behavior of HKII, in which a D209A mutation results in an increase in the K iG6P of the enzyme, whereas a S155A mutation does not. These results suggest an interaction in which glucose binding by the N-half causes the activity of the C-half to be regulated by significantly lower concentrations of G6P.Hexokinases (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1; HK) 1 catalyze the phosphorylation of glucose to glucose 6-phosphate (G6P). Type I, II, and III isozymes of mammalian HKs are ϳ100 kDa in size and are inhibited by the reaction product, G6P. Type IV HK (also known as glucokinase) is similar to yeast HK in that it has a relative mass of ϳ50 kDa and is insensitive to inhibition by physiologic concentrations of G6P. The deduced amino acid sequences of HKI-III reveal internal similarities between their N-and C-terminal halves and between each of these and yeast HK and glucokinase (1). This observation supports the hypothesis that the 100-kDa mammalian HKs evolved from the duplication and fusion of an ancestral 50-kDa HK (1, 2). The N-and C-terminal halves of HKI show a marked functional difference despite the similarity of their amino acid sequences. The C-terminal half of HKI is catalytically active, whereas the N-terminal half is inactive (3-5). Whereas G6P binds to both halves of HKI (3, 5), the G6P regulatory site of HKI is thought to be in the N-half of the intact enzyme, and the C-half binding site is latent (1, 6). In contrast, we have shown that both the N-and C-terminal halves of human and rat HKII (N-HKII and C-HKII, respectively) have cat...

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Section: R Grue1ter Et Almentioning

confidence: 99%

“…Inside the brain, a different transporter may be responsible for glucose transport across the cell membranes. However, several studies have suggested that the large surface area of brain cells may result in rapid equilibration inside the brain's aqueous phase leading to similar intra-and extracellular glucose concentrations (LundAndersen, 1979;Gjedde and Diemer, 1983;Holden et al, 1991;Gjedde, 1992; Silver and Erecitiska, 1994;Whitesell et a!., 1995).Because the transporter isoform at the blood-brain barrier and that at the erythrocyte membrane are very similar, the kinetic constant for half-maximal transport, K,, should in principle be of similar magnitude. However, when reviewing the literature on animal brain glucose transport, a noticeable distribution of the measured brain K, was observed, ranging from 2 to 14 mM, as previously reviewed (Gjedde, 1992;Mason et a!., 1992).…”

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Steady‐State Cerebral Glucose Concentrations and Transport in the Human Brain

Gruetter¹,

Uğurbil

Seaquist³

1998

Journal of Neurochemistry

233

280

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Understanding the mechanism of brain glucose transport across the blood-brain barrier is of importance to understanding brain energy metabolism. The specific kinetics of glucose transport have been generally described using standard Michaelis-Menten kinetics. These models predict that the steady-state glucose concentration approaches an upper limit in the human brain when the plasma glucose level is well above the Michaelis-Menten constant for half-maximal transport, K~. In experiments where steady-state plasma glucose content was varied from 4 to 30 mM, the brain glucose level was a linear function of plasma glucose concentration. At plasma concentrations nearing 30 mM, the brain glucose level approached 9 mM, which was significantly higher than predicted from the previously reported Kõf --'4 mM (p < 0.05). The high brain glucose concentration measured in the human brain suggests that ablumenal brain glucose may compete with lumenal glucose for transport. We developed a model based on a reversible MichaelisMenten kinetic formulation of unidirectional transport rates. Fitting this model to brain glucose level as a function of plasma glucose level gave a substantially lower Kõ f 0.6 ±2.0 mM, which was consistent with the previously reported millimolar Km of GLUT-i in erythrocyte model systems. Previously reported and reanalyzed quantification provided consistent kinetic parameters. We conclude that cerebral glucose transport is most consistently described when using reversible Michaelis-Men±en kinetics. Key Words: NMR-Glucose transport-ln vivo studies-Spectroscopy-Human. J. Neurochem. 70, 397-408 (1998).Knowledge of the specific mechanism of glucose transport across the blood-brain barrier is important for understanding cerebral carbohydrate metabolism, which is the major source of energy for ATP production. Glucose transport across the blood-brain barrier occurs by facilitated diffusion mediated by specific transporter proteins. Glucose extraction is a saturable process in the brain/ (Crone, 1965) and erythrocytes (Le Fevre, 1961)atid is mediated by facilitated diffusion that has been well-established to be stereospecific and substrate-specific. Standard Michaelis-Menten models for glucose transport kinetics have since been used almost exclusively in most studies of the brain (for reviews, see, e.g., Lund-Andersen, 1979;Pardridge, 1983;Gjedde, 1992), with one exception .Many elegant techniques have been applied to study brain glucose uptake. The indicator-dilution techniques sample tracer extraction with a single pass across the capillary bed at a very high time resolution but with a somewhat limited spatial resolution (Knudsen et al., 1990). On the other hand, state-of-the art tracer techniques, such as positron emission tomography (PET), can provide a high spatial resolution (Svarer et al., 1996). However, the information on cerebral glucose content is indirect because the active metabolism of labeled glucose implies substantial label accumulation in metabolic products that cannot be distinguished fr...

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Coupled Glucose Transport and Metabolism in Cultured Neuronal Cells: Determination of the Rate-Limiting Step

Cited by 32 publications

References 58 publications

Insulin Restores Metabolic Function in Cultured Cortical Neurons Subjected to Oxidative Stress

Insulin Restores Metabolic Function in Cultured Cortical Neurons Subjected to Oxidative Stress

Functional Interaction between the N- and C-terminal Halves of Human Hexokinase II

Steady‐State Cerebral Glucose Concentrations and Transport in the Human Brain

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