UPTAKE OF GLUCOSE ANALOGUES BY RAT BRAIN CORTEX SLICES: MEMBRANE TRANSPORT VERSUS METABOLISM OF 2-DEOXY-d-GLUCOSE

Abstract— —The uptake of the glucose analogue 2-deoxy- d -glucose by rat brain cortex slices was studied in order to compare the rate of membrane transport with the rate of phosphorylation in the concentration range 5–12 mM-glucose plus 0.5–15 mM-2-deoxy-glucose. The comparison was carried out by fitting a model of the brain slice to uptake data and by determination of 2-deoxy-glucose and 2-deoxy-glucose-6-phosphate by ion exchange chromatography.
The rate of membrane transport exceeded the rate of phosphorylation by at least one order of magnitude. The membrane transport was so rapid that the extracellular diffusion became rate limiting for the uptake. The membrane transport could therefore only be determined as a minimum value and it was not possible to determine unidirectional flux across the cell membranes (initial rate). Accordingly, characterization of the membrane tranport with respect to maximal transport rate and affinity was not possible. The phosphorylation reaction, however, was so slow that it was accessible for exact determination and only the phosphorylation reaction was responsible for the fact that the cellular uptake of 2-deoxy-glucose was of the Michaelis-Menten type, thus emphasizing the importance of dissociation between membrane transport and metabolism when transport is studied of a substance which can undergo metabolism.
The data indicate that glucose transport across glial and neuronal membranes is not rate limiting for glucose metabolism of brain tissue in vitro.     

Microcalorimetric Study of Glucose Permeation in Microbial Cells

A microcalorimetric method for measuring the influence of extracellular glucose concentration on the rate of catabolism is described. This method has been applied to anaerobically growing cultures of Zymomonas mobilis and of a respiratory-deficient ("petite") mutant of Saccharomyces cerevisiae (strain YFa). The Michaelian kinetics recorded with both organisms were apparently related to glucose transport. With Z. mobilis, it was found that, in the range of glucose concentrations at which this organism was growing exponentially, cell activity was limited by the maximal rate of the catabolic enzymes; at lower concentrations, glucose transport was the rate controlling step. The metabolic activity of yeast always depended on external glucose concentration; when this was lowered under a threshold, a change of kinetics took place. The microcalorimetric method described seems to be widely applicable to kinetic studies of the permeation of metabolizable substrates in microorganisms.     

Growth and Glucose Repression Are Controlled by Glucose Transport in Saccharomyces cerevisiae Cells Containing Only One Glucose Transporter

A set of Saccharomyces cerevisiae strains with variable expression of only the high-affinity Hxt7 glucose transporter was constructed by partial deletion of the HXT7 promoter in vitro and integration of the gene at various copy numbers into the genome of an hxt1-7 gal2 deletion strain. The glucose transport capacity increased in strains with higher levels of HXT7 expression. The consequences for various physiological properties of varying the glucose transport capacity were examined. The control coefficient of glucose transport with respect to growth rate was 0.54. At high extracellular glucose concentrations, both invertase activity and the rate of oxidative glucose metabolism increased manyfold with decreasing glucose transport capacity, which is indicative of release from glucose repression. These results suggest that the intracellular glucose concentration produces the signal for glucose repression.     

Substrate regulation of the glucose transport system in rat skeletal muscle. Characterization and kinetic analysis in isolated soleus muscle and skeletal muscle cells in culture

A self-regulatory mechanism of the glucose transport in rat skeletal muscle cells is described. In isolated rat soleus muscles and rat skeletal myocytes and myotubes in culture, pre-exposure to varying glucose concentrations modulated the rate of 2-deoxyglucose uptake. Maximal uptake was observed at glucose concentrations below 3 mM. Between 2.5 and 4.0 mM glucose it was reduced by 25-35%; further elevation of the glucose concentration resulted in a gradual decrease of the transport rate by approximately 2% for each millimolar glucose. The effect of glucose was time-dependent and fully reversible. Insulin rapidly increased the 2-deoxyglucose uptake in the soleus muscle; however, the insulin effect depended on the glucose concentration of the preincubation. Insulin was totally ineffective in muscles pre-exposed to 1.0-3.0 mM glucose, whereas its stimulatory action increased with increasing glucose concentrations above 4 mM. The effect of low glucose and insulin were not additive, and the maximal 2-deoxyglucose uptake rates induced by both conditions were of identical magnitude. It is postulated that glucose may "up- and down-regulate" its transport by affecting the number of active glucose transporters in the plasma membrane, and that insulin exerts its stimulatory effect only when the extracellular glucose reaches a threshold concentration.     

An alternative procedure for calculating glucose consumption from 2-deoxyglucose uptake

In the commonly used model (Sokoloff) for the transport and metabolism of glucose and 2-deoxyglucose in brain tissue a novel choice of constant parameters is proposed. In particular, the maximal transport capacity for glucose is assumed proportional to the rate of glucose consumption. The proportionality factor, the “transport factor”, may be calculated from the lumped constant and is more likely than the latter to remain constant under varying conditions. Calculations founded on these considerations should yield results similar to the Sokoloff procedure in many situations, but differences appear when the arterial glucose concentration changes. The model is flexible and allows changes.     

Glucose metabolism in murine fetal cortical brain cells: lack of insulin effects

Glucose uptake and oxidation were markedly higher in cultured than in freshly isolated neural cells, prepared from murine fetal brain cortices. The hexose transport process--measured as 3-O-methyl-D-glucose uptake--appeared comparable in both conditions, and proceeded proportionally to the extracellular sugar concentration up to 6 mM. In contrast, glucose oxidation occurred independently of the prevailing glucose concentration from 1.4 mM on. Acute or chronic exposure to insulin exerted no effect upon cellular glucose uptake or oxidation. These results suggest that glucose handling by maturing fetal cortical cells is mainly determined by the rate of cellular glucose breakdown rather than by the rate of glucose transport into the cell; the marked rise in cellular glucose metabolism during culture might result from the synthesis and/or activation of a key enzyme in glucose catabolism. Our observations also indicate that the previously described neurotrophic effects of insulin are not mediated via enhanced glucose handling.     

Pathways of reducing equivalents in hepatocytes from rats. Estimation of cytosolic fluxes by means of 3H-labelled substrates for either A- or B-specific dehydrogenases.

The rat insulinoma-derived RINm5F cell line retains many differentiated functions of islet beta-cells. However, it fails to recognize glucose as an insulin secretagogue in the physiological concentration range. With this cell line, glucose-transport kinetics were investigated, by using a double-label technique with the non-metabolizable glucose analogue 3-O-methylglucose (OMG). RINm5F cells possess a passive glucose-transport system with high capacity and low affinity. Equilibration across the plasma membrane of extracellular OMG concentrations up to at least 20 mM is achieved within 2 min at 37 degrees C. The half-saturation of OMG uptake occurs at 32 mM. At lower temperatures OMG uptake is markedly retarded, with a temperature coefficient (Q10) of 2.9. As indicated by efflux measurements, transport is symmetrical. Cytochalasin B at micromolar concentrations and phlorrhizin in millimolar concentrations are potent inhibitors of OMG uptake. Neutralization of the secreted insulin with antibodies does not alter OMG uptake kinetics. The glucose metabolism of RINm5F cells is much exaggerated compared with that of islet beta-cells. Nonetheless, when measured in parallel to uptake, transport exceeds by far the rate of metabolism at glucose concentrations above 3 mM. Measurements of intracellular D-glucose reveal a lower intracellular glucose concentration relative to the extracellular in RINm5F cells. This seems to be due to abnormalities in the subsequent steps of glucose metabolism, rather than to abnormalities in hexose uptake. The loss of glucose-induced insulin release in RINm5F cells cannot be explained by alterations in hexose transport.     

Calcium as modulator of the hormonal-receptors-biological-response coupling system. Effects of Ca2+ ions on the insulin activated 2-deoxyglucose transport in rat fat cells.

Activation of 2-deoxyglucose transport in isolated rat fat cells by insulin is dependent upon the presence of Ca2+ in the external medium. When calcium concentration is kept below 100 micron, insulin acts like a partial agonist, giving only half of the maximal activation obtained normally with a millimolar concentration of this ion. Oxytocin, whose insulin-like action on adipocytes activates glucose oxidation by these cells, was found to be unable to affect the rate of 2-deoxyglucose transport. This, together with previous observations, suggests that calcium ions play a role in the mechanism of insulin action possibly by binding selectively to membrane sites involved in the transmission of the hormonal message to the glucose carrier. Oxytocin seems to trigger only intracellular glucose metabolism and it appears that there is an absolute requirement for calcium ions in the activation of a still unknown membrane signal.     

Transport and metabolism of D-glucose in human adipocytes. Studies of the dependence on medium glucose and insulin concentrations

Uptake and metabolism of the physiologically labelled D-glucose (D-[U-14C]glucose) has been characterized in human adipocytes at several unlabelled D-glucose concentrations in the absence and presence of insulin. Following a 90 min incubation, about 80% of the intracellular radioactivity was incorporated into total lipids at tracer glucose concentration, as well as at higher glucose concentrations in basal and insulin-stimulated cells, whereas 20% was recovered as hydrophilic metabolites. The only 14C-labelled metabolite escaping the cells in detectable amounts was CO2, which accounted about 4%. At trace glucose concentrations (5 mumol/l), the rate of glucose uptake was linear with time. Comparative studies of initial glucose uptake after 10 s and tracer D-glucose conversion to total lipids after 90 min showed high coefficients of correlation between basal rates (r = 0.87), maximal response above basal level to insulin (r = 0.92) and insulin sensitivity (r = 0.78). Thus, under these conditions glucose transport is rate-limiting for net glucose uptake, and measurements over long time intervals of rates for total cell-associated radioactivity or lipogenesis may serve as reliable estimates of initial glucose influx rates. However, the conversion rate of tracer glucose to metabolites decreased progressively with the glucose concentration and with an apparent Km of about 0.2 mmol/l. The three metabolic pathways exhibited similar percentage decreases in their activities, suggesting that a common enzymatic step is rate-limiting. In comparison, the Km for initial D-glucose uptake rate was about 7 mmol/l. Hence, the capacity for total glucose metabolism comprised only a small fraction of the glucose transport capacity at medium glucose concentrations above tracer concentrations. Both basal, half-maximal and maximal insulin-stimulated rates of adipocyte glucose utilization were dependent on the glucose concentration. Thus, comparing lipogenesis at tracer and at 0.5 mmol/l medium glucose concentration, it was shown that the higher medium glucose concentration was associated with a 60% lowering of the basal rate, a 35% reduction in the percentage response above baseline to maximal insulin stimulation and a 4-fold increase in the insulin sensitivity. Obviously, these findings reflect some intracellular step(s) being rate-limiting at medium glucose levels above tracer values.     

Mechanism of insulin resistance in the post receptor events in sheep: 3-O-methylglucose transport in ovine adipocytes

A severe resistance to the stimulatory action of insulin on glucose metabolism has been shown in ruminant adipose tissue or isolated adipocytes as compared to that of rats. To elucidate the mechanism of insulin resistance in ruminants, we measured the stimulatory effect of insulin on 3-O-methylgulose transport and on intracellular glucose metabolism in isolated adipocytes from sheep and rats. At a glucose concentration (0.1 mM) where transport is thought to be rate-limiting for metabolism, lipogenesis from [U-14C]glucose by ovine adipocytes was markedly less than by rat adipocytes in both the basal state and at all insulin concentrations. The responsiveness to insulin assessed by percent increase above basal was reduced to about 15% of that in rat adipocytes, but the insulin sensitivity was similar, because the insulin concentration giving half-maximal stimulation, ED50, did not differ significantly between ovine and rat adipocytes. The maximal insulin-stimulated 3-O-methylglucose transport in ovine adipocytes per cell was less than 20% of that in rat adipocytes, with a significant lowering in basal rates of transport. However, when data was expressed per 3-O-methylglucose equilibrium space no significant differences were found between ovine and rat in the basal transport rates, but a lowered ability of insulin to stimulate glucose transport was still seen in ovine adipocytes. The dose-response curve for glucose transport was slightly shifted to the right in ovine adipocytes compared to rat adipocytes, indicating a small decrease in insulin sensitivity. The decrease in glucose transport was due to 60% reduction in the maximum velocity in the insulin--stimulated state, with no change in the Km.     

Extracellular Glucose Concentrations in the Rat Hippocampus Measured by Zero-Net-Flux

Abstract : The concentration of glucose in the brain's extracellular fluid remains controversial, with recent estimates and measurements ranging from 0.35 to 3.3 m M . In the present experiments, we used the method of zero-net-flux microdialysis to determine glucose concentration in the hippocampal extracellular fluid of awake, freely moving rats. In addition, the point of zero-net-flux was measured across variations in flow rate to confirm that the results for glucose measurement were robust to such variations. In 3-month-old male Sprague-Dawley rats, the concentration of glucose in the hippocampal extracellular fluid was found to be 1.00 ± 0.05 m M , which did not vary with changes in flow rate. Three-month-old and 24-month-old Fischer-344 rats both showed a significantly higher hippocampal extracellular fluid glucose concentration, at 1.24 ± 0.07 and 1.21 ± 0.04 m M , respectively ; there was no significant difference between the two age groups. The present data demonstrate variation in extracellular brain glucose concentration between rat strains. When taken together with previous data showing a striatal extracellular glucose concentration on the order of 0.5 m M , the data also demonstrate variation in extracellular glucose between brain regions. Traditional models of brain glucose transport and distribution, in which extracellular concentration is assumed to be constant, may require revision.     

D-Glucose Transport in Cultured Cells of Neural Origin: The Membrane as Possible Control Point of Glucose Utilization

Abstract: The function of plasma membrane as control point of glucose metabolism has been studied in confluent monolayer of C1300 neuroblastoma (N2A) and glioma (C6) cells. In neuroblastoma, steady state intracellular glucose concentration reached the extracellular levels, while intracellular contents in C6 glioma cells remained very low. In C6 glial cells the amount of glycogen as source of energy was much higher than that found in C1300 neuroblastoma cells. Influx rates of D-glucose in C6 glioma cells were only half those found in neuroblastoma cells. During the influx period (0-40 s) the transport of glucose in these cells did not exceed the phosphorylation rate, whereas a steady, time-dependent increase in glucose content was observed in neuroblastoma cells. While glucose uptake in neuroblastoma cells seems to be regulated at the level of phosphorylating enzymes, the control point in C6 glioma is believed to be membrane transport.     

Coupling of insulin receptors to glucose transport: a temperature-dependent time lag in activation of glucose transport.

We have studied the ability of occupied insulin receptors to activate (or couple to) the glucose transport system in isolated rat adipocytes. Maximal insulin action is seen when only a small proportion (<10%) of the receptors is occupied, and this fraction can be rapidly filled (<5 s) at an insulin concentration of 100 ng/ml. Additionally, control studies show that when the extracellular glucose concentration is tripled, the rate of transport triples within 10 s, indicating that changes in transport activity can be observed nearly instantaneously. Therefore, when cells are exposed to a high insulin concentration (100 ng/ml), any delay in the onset of insulin action beyond this time must be due to the time required for coupling of occupied insulin receptors to the glucose transport system. At 24 °C there is a lag of at least 200 s after insulin addition before a significant stimulation of 2-deoxyglucose transport is seen. The length of this lag phase is temperature dependent, decreasing to 45 s at 37 °C. An Arrhenius plot of the coupling lag is linear, with an activation energy of 25 kcal/mol. After the delay in the onset of initial transport activation the full response appears in a gradual manner, requiring 20 min at 24 °C to attain maximal stimulation. The time required for the full insulin response to appear is also temperature dependent, decreasing to 5 min at 37 °C. Similar results were obtained for the kinetics of insulin activation of 3-O-methyl glucose transport. Thus, the coupling of insulin receptors to the glucose transport system can be divided into two components: an initial absolute time lag followed by a gradual incremental process before the maximal, or full, effect of insulin is achieved. In conclusion, (1) there is an absolute delay in the onset of the insulin's initial action on glucose transport, (2) after an initial delay, activation of transport proceeds in a gradual manner, and (3) the coupling process between insulin receptors and the glucose transport system is temperature dependent and can be described by a linear Arrhenius plot. This suggests that the rate of activation is not limited by membrane fluidity.     

Insulin resistance in soleus muscle from obese Zucker rats. Involvement of several defective sites

1. The effect of insulin upon glucose transport and metabolism in soleus muscles of genetically obese (fa/fa) and heterozygote lean Zucker rats was investigated at 5–6 weeks and 10–11 weeks of age. Weight-standardized strips of soleus muscles were used rather than the intact muscle in order to circumvent problems of diffusion of substrates. 2. In younger obese rats (5–6 weeks), plasma concentrations of immunoreactive insulin were twice those of controls, whereas their circulating triacylglycerol concentrations were normal. Insulin effects upon 2-deoxyglucose uptake and glucose metabolism by soleus muscles of these rats were characterized by both a decreased sensitivity and a decrease in the maximal response of this tissue to the hormone. 3. In older obese rats (10–11 weeks), circulating concentrations of insulin and triacylglycerols were both abnormally elevated. A decrease of 25–35% in insulin-binding capacity to muscles of obese rats was observed. The soleus muscles from the older obese animals also displayed decreased sensitivity and maximal response to insulin. However, at a low insulin concentration (0.1m-i.u./ml), 2-deoxyglucose uptake by muscles of older obese rats was stimulated, but such a concentration was ineffective in stimulating glucose incorporation into glycogen, and glucose metabolism by glycolysis. 4. Endogenous lipid utilization by muscle was calculated from the measurements of O₂ consumption, and glucose oxidation to CO₂. The rate of utilization of fatty acids was normal in muscles of younger obese animals, but increased in those of the older obese rats. Increased basal concentrations of citrate, glucose 6-phosphate and glycogen were found in muscles of older obese rats and may reflect intracellular inhibition of glucose metabolism as a result of increased lipid utilization. 5. Thus several abnormalities are responsible for insulin resistance of muscles from obese Zucker rats among which we have observed decreased insulin binding, decreased glucose transport and increased utilization of endogenous fatty acid which could inhibit glucose utilization.     

Regulation of Glycogen Metabolism in Primary and Transformed Astrocytes In Vitro

Glycogen metabolism was studied in primary and Herpesvirus-transformed cultures of neonatal rat brain astrocytes. A small fraction of the glucose consumed was conserved in glycogen in both the primary and the transformed astrocytic cell cultures. After addition of culture medium containing 5.5 mM glucose, glycogen increased to maximal levels within 2.5 h, the approximate time at which half of the medium glucose was consumed, and rapidly declined thereafter in both the primary and transformed astrocytic cultures. Maximum levels of glycogen were apparently related to the cell density of the Herpesvirus-transformed cultures, but primary cultures did not show this behavior. At any given cell density, maximal levels of glycogen were dependent on the concentration of extracellular glucose. Administration of glucose caused a transient activation of glycogen synthase alpha and a rapid inactivation of glycogen phosphorylase alpha.     

Intracellular Glucose Concentration in Derepressed Yeast Cells Consuming Glucose Is High Enough To Reduce the Glucose Transport Rate by 50%

In Saccharomyces cerevisiae cells exhibiting high-affinity glucose transport, the glucose consumption rate at extracellular concentrations above 10 mM was only half of the zero trans-influx rate. To determine if this regulation of glucose transport might be a consequence of intracellular free glucose we developed a new method to measure intracellular glucose concentrations in cells metabolizing glucose, which compares glucose stereoisomers to correct for adhering glucose. The intracellular glucose concentration was 1.5 mM, much higher than in most earlier reports. We show that for the simplest model of a glucose carrier, this concentration is sufficient to reduce the glucose influx by 50%. We conclude that intracellular glucose is the most likely candidate for the observed regulation of glucose import and hence glycolysis. We discuss the possibility that intracellular glucose functions as a primary signal molecule in these cells.     

Long-term effects of glucose on insulin release and glucose oxidation by mouse pancreatic islets maintained in tissue culture

Rates of glucose oxidation and insulin release in response to a wide range of glucose concentrations were studied in short-term experiments in isolated mouse pancreatic islets maintained in tissue culture for 6 days at either a physiological glucose concentration (6.7mm) or at a high glucose concentration (28mm). The curves relating glucose oxidation or insulin release to the extracellular glucose concentration obtained with islets cultured in 6.7mm-glucose displayed a sigmoid shape similar to that observed for freshly isolated non-cultured islets. By contrast islets that had been cultured in 28mm-glucose showed a linear relationship between the rate of glucose oxidation and the extracellular glucose concentration up to about 8mm-glucose. The maximal oxidative rate was twice that of the non-cultured islets and the glucose concentration associated with the half-maximal rate considerably decreased. In islets cultured at 28mm-glucose there was only a small increase in the insulin release in response to glucose, probably due to a depletion of stored insulin in those B cells that had been cultured in a high-glucose medium. It is concluded that exposure of B cells for 6 days to a glucose concentration comparable with that found in diabetic individuals causes adaptive metabolic alterations rather than degeneration of these cells.     

Effect of Sorbinil on myo-Inositol Metabolism in Cultured Neuroblastoma Cells Exposed to Increased Glucose Levels

Neuroblastoma cells were used to determine the effect of sorbinil on myo-inositol metabolism in cells exposed to elevated levels of glucose in culture. Exposing cells to elevated levels of glucose led to an increase in levels of intracellular sorbitol. The increase in sorbitol levels was dependent on the extracellular glucose concentration. In contrast, the myo-inositol content of cells was decreased in the presence of increasing concentrations of extracellular glucose. Increasing the concentration of glucose in the culture medium caused a decrease in myo-inositol uptake and in the incorporation of extracellular myo-inositol into phospholipid. The effect of elevated glucose levels on myo-inositol metabolism and sorbitol accumulation was blocked by addition of 0.4 mM sorbinil. The ability of sorbinil to block the decrease in myo-inositol metabolism and sorbitol accumulation caused by 30 mM extracellular glucose was dependent on its concentration. Maximal effects were obtained with 0.4 mM sorbinil. However, there was some variation in the degree of effectiveness among batches of sorbinil. These results at the cellular level suggest that the intracellular accumulation of sorbitol is responsible for the alteration of myo-inositol metabolism observed in neuroblastoma cells exposed to elevated glucose concentrations.     

Impaired glucose transporter-1 degradation and increased glucose transport and oxidative stress in response to high glucose in chondrocytes from osteoarthritic versus normal human cartilage

Introduction  

Disorders that affect glucose metabolism, namely diabetes mellitus (DM), may favor the development and/or progression of osteoarthritis (OA). Thus far, little is known regarding the ability of chondrocytes to adjust to variations in the extracellular glucose concentration, resulting from hypoglycemia and hyperglycemia episodes, and so, to avoid deleterious effects resulting from deprivation or intracellular accumulation of glucose. The aim of this study was to compare the ability of normal and OA chondrocytes to regulate their glucose transport capacity in conditions of insufficient or excessive extracellular glucose and to identify the mechanisms involved and eventual deleterious consequences, namely the production of reactive oxygen species (ROS).