Development and mechanism of barbiturate tolerance in glial cell cultures

The effects of exposure of glial cells in primary culture and in continuous line (clone NN) to pentobarbital over various periods of time on cellular respiration and activities of enzymes involved in carbohydrate metabolism were studied. The results obtained in glial cells in primary culture were qualitatively identical to those obtained in glial cells in clonal line (NN). Both types of glial cells were shown to develop biochemical tolerance to pentobarbital as defined by an attenuated response to the depressant effects of a challenging dose of pentobarbital on cellular respiration in barbiturate-cultivated cells compared to those grown in drug-free medium. The biochemical tolerance was evident in the presence of glucose and succinate but not malate as substrate. This tolerance to pentobarbital was accompanied by increased activities of hexokinase, glucose-6-phosphate dehydrogenase, succinate dehydrogenase, and glutamate dehydrogenase and by a marked increase in the number of glial cell mitochondria as observed in electron micrographs. The results are interpreted to indicate a compensation of glial cells to the continuous presence of PB by an accelerated glucose uptake and metabolism, an accelerated metabolism of succinate, and an increased mitochondrial activity.     

2-Deoxy-D-glucose uptake in cultured human muscle cells

Hexose uptake was studied with cultured human muscle cells using 2-deoxy-D-[1-3H]glucose. At a concentration of 0.25 and 4 mM, phosphorylation rather than transport was the rate-limiting step in the uptake of 2-deoxy-D-glucose. This was not due to inhibition of the hexokinase activity by either ATP depletion or 2-deoxyglucose 6-phosphate accumulation. In cellular homogenates, hexokinase showed a lower Km value for glucose as compared to 2-deoxyglucose. Intact cells preferentially phosphorylated glucose instead of 2-deoxyglucose. Therefore, transport instead of phosphorylation may be rate limiting in the uptake of glucose by cultured human muscle cells. These data suggest caution in using 2-deoxyglucose for measuring glucose transport.     

Factors controlling the metabolic response to glucose in isolated rat lung cells

Glucose decreases the oxygen utilization by isolated rat lung cells. Its effect displays saturation type kinetics with a “Ki” of 2.2. mM. The similarity of this value with the reported “Km” of 2.4 mM described for glucose uptake by these cells, suggests that both processes may be intimately related and both of them are under the control of the same rate limiting step. Several arguments point to glucose transport into these cells as the most important rate limiting step for its utilization: 1) Phloridzin prevented glucose inhibition of oxygen uptake while mannoheptulose did not; 2) The activity of hexokinase which is the least active glycolytic enzyme in these cells far exceeded the observed rates of glucose utilization and a decrease of 45 per cent in its activity in starved animals did not affect the rate of glucose uptake; 3) The “Km” of hexokinase for glucose is two orders of magnitude below the observed “Km” for glucose uptake and the “Ki” for glucose inhibition of respiration.     

Endothelin-1 stimulates the translocation and upregulation of both glucose transporter and hexokinase in astrocytes: relationship with gap junctional communication

We have previously shown that endothelin-1 increases glucose uptake in astrocytes. In the present work we investigate the mechanism through which endothelin-1 (ET-1) increases glucose uptake. Our results show that ET-1 activates a short-term and a long-term mechanism. Thus, ET-1 induced a rapid change in the localization of both GLUT-1 and type I hexokinase. These changes are probably aimed at rapidly increasing the entry and phosphorylation of glucose. In addition, ET-1 upregulated GLUT-1 and type I hexokinase and induced the expression of isoforms not normally expressed in astrocytes, such as GLUT-3 and type II hexokinase. These changes provide astrocytes with the machinery required to sustain a high rate of glucose uptake for a longer period of time. Our previous work had suggested that the effect of ET-1 on glucose uptake was associated with the inhibition of gap junctions. In this work, we compare the effect of ET-1 with that of carbenoxolone, a classical inhibitor of gap junction communication. Carbenoxolone increased glucose uptake to the same extent as ET-1 following the same mechanisms. Thus, carbenoxolone induced a rapid change in the localization of both GLUT-1 and type I hexokinase, upregulated GLUT-1 and type I hexokinase and induced the expression of GLUT-3 and type II hexokinase. When the inhibition of gap junction was prevented by tolbutamide, neither ET-1 nor carbenoxolone were able to increase the levels of GLUT-1, GLUT-3, type I hexokinase or type II hexokinase, indicating that these events are closely related to gap junctions.     

High glucose concentrations inhibit glucose phosphorylation, but not glucose transport, in human endothelial cells.

Glucose uptake is autoregulated in a variety of cell types and it is thought that glucose transport is the major step that is subjected to control by sugar availability. Here, we examined the effect of high glucose concentrations on the rate of glucose uptake by human ECV-304 umbilical vein-derived endothelial cells. A rise in the glucose concentration in the medium led a dose-dependent decrease in the rate of 2-deoxyglucose uptake. The effect of high glucose was independent of protein synthesis and the time-course analysis indicated that it was relatively slow. The effect was not due to inhibition of glucose transport since neither the expression nor the subcellular distribution of the major glucose transporter GLUT1, nor the rate of 3-O-methylglucose uptake was affected. The total in vitro assayed hexokinase activity and the expression of hexokinase-I were similar in cells treated or not with high concentrations of glucose. In contrast, exposure of cells to a high glucose concentration caused a marked decrease in phosphorylated 2-deoxyglucose/free 2-deoxyglucose ratio. This suggests the existence of alterations in the rate of in vivo glucose phosphorylation in response to high glucose. In summary, we conclude that ECV304 human endothelial cells reduce glucose utilization in response to enhanced levels of glucose in the medium by inhibiting the rate of glucose phosphorylation, rather than by blocking glucose transport. This suggests a novel metabolic effect of high glucose on cellular glucose utilization.     

Glucose uptake and metabolism by cultured human skeletal muscle cells: rate-limiting steps

To use primary cultures of human skeletal muscle cells to establish defects in glucose metabolism that underlie clinical insulin resistance, it is necessary to define the rate-determining steps in glucose metabolism and to improve the insulin response attained in previous studies. We modified experimental conditions to achieve an insulin effect on 3-O-methylglucose transport that was more than twofold over basal. Glucose phosphorylation by hexokinase limits glucose metabolism in these cells, because the apparent Michaelis-Menten constant of coupled glucose transport and phosphorylation is intermediate between that of transport and that of the hexokinase and because rates of 2-deoxyglucose uptake and phosphorylation are less than those of glucose. The latter reflects a preference of hexokinase for glucose over 2-deoxyglucose. Cellular NAD(P)H autofluorescence, measured using two-photon excitation microscopy, is both sensitive to insulin and indicative of additional distal control steps in glucose metabolism. Whereas the predominant effect of insulin in human skeletal muscle cells is to enhance glucose transport, phosphorylation, and steps beyond, it also determines the overall rate of glucose metabolism.     

Is glucose transport enhanced in virus-transformed mammalian cells? A dissenting view

Much of the literature on the uptake of glucose by untransformed and transformed animal cells is based on experiments carried out with 2-deoxy-D-glucose (2-DOG). Results obtained with this analog can be ambiguous, since 2-DOG can be phosphorylated by hexokinases of animal cells. An intracellular trapping mechanism is thus provided. Therefore, the total flux of 2-DOG into the cell is a resultant of both transport and hexokinase action, and the measurement of total 2-DOG incorporation is a valid measurement of transport only if 2-DOG is phosphorylated as rapidly as it enters the cell. Evidence is presented here that this is not necessarily the case, significant levels of free intracellular 2-DOG approaching external concentrations were found in untransformed and transformed mouse 3T3 cells even at early times during uptake. Differences in total intracellular 2-DOG between untransformed and transformed cells were accounted for entirely by 2-deoxyglucose phosphate. Thus, it appears the apparent increase of 2-DOG uptake accompanying transformation in these cell lines is not due to an effect on the transport process, but on enhanced phosphorylation, which is a reflection of an alteration in the regulation of glycolysis. The ambiguity introduced by phosphorylation can be oviated by the use of an analog that cannot be phosphorylated, such as 3-O-methyl-D-glucose. The rate of transport and efflux of this sugar was not found to be different in untransformed versus transformed 3T3 cells. Moreover, deficiencies of this analog as a substrate for the glucose transport system are pointed out.     

Defective P2Y purinergic receptor function: A possible novel mechanism for impaired glucose transport

Extracellular ATP is an ubiquitous mediator that regulates several cellular functions via specific P2 plasma membrane receptors (P2Rs), for which a role in modulating intracellular glucose metabolism has been recently suggested. We have investigated glucose uptake in response to P2Rs stimulation in fibroblasts from type 2 diabetic (T2D) patients and control subjects. P2Rs expression was evaluated by RT-PCR; intracellular calcium release by fluorometry; glucose transporter (GLUT1) translocation by immunoblotting and chemiluminescence; glucose uptake was measured with 2-deoxy-D-[1-(3)H]glucose (2-DOG) and ATP by luminometry. Cells from T2D patients, in contrast to those from healthy controls, showed no increase in glucose uptake after ATP stimulation; extracellular ATP caused, however, a similar GLUT1 recruitment to the plasma membrane in both groups. P2Rs expression did not differ between fibroblasts from diabetic and healthy subjects, but while plasma membrane depolarization, a P2X-mediated response was similar in both groups, no evident intracellular calcium increase was detectable in the cells from the former group. The calcium response in fibroblasts from diabetics was restored by co-incubation with apyrase or hexokinase, suggesting that P2YRs in those cells were normally expressed but chronically desensitised. In support to this finding, fibroblasts from T2D subjects secreted a two-fold larger amount of ATP compared to controls. Pre-treatment with apyrase or hexokinase also restored ATP stimulated glucose uptake in fibroblasts from diabetic subjects. These results suggest that extracellular ATP plays a role in the modulation of glucose transport via GLUT1, and that the P2Y-dependent GLUT1 activation is deficient in fibroblasts from T2D individuals. Our observations may point to additional therapeutic targets for improving glucose utilization in diabetes.     

Inhibition of glucose metabolism by n-hexadecane in Cladosporium (Amorphotheca) resinae.

When Cladosporium resinae is provided with n-hexadecane and glucose, n-hexadecane is used preferentially. Studies using [14C]glucose indicated that n-hexadecane did not inhibit glucose uptake but did retard oxidation of glucose to CO2 and assimilation of glucose carbon into trichloroacetic acid-insoluble material. Glucose could be recovered quantitatively from hydrocarbon-grown cells that had been transferred to glucose. Four enzymes that may be involved in glucose metabolism, hexokinase, glucose-6-phosphate dehydrogenase, glucose-phosphate isomerase, and succinate dehydrogenase, were not detected in cells grown on hexadecane but were present in cells grown on glucose. Addition of hexadecane to extracts of glucose-grown cells resulted in immediate loss of activity for each of the four enzymes, but two other enzymes did not directly involved in glucose metabolism, adenosine triphosphatase and alanine-ketoacid aminotransferase, were not inhibited by hexadecane in vitro. Cells grown on hexadecane and transferred to glucose metabolize intracellular hexadecane; after 1 day, activity of hexokinase, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, and succinate dehydrogenase could be detected and 22% of the intracellular hydrocarbon had been metabolized. Hexadecane-grown cells transferred to glucose plus cycloheximide showed the same level of activity of all the four enzymes as cells transferred to glucose alone. Thus, intracellular n-hexadecane or a metabolite of hexadecane can inthesis of those enzymes is not inhibited.     

Sugar induced cell death in yeast is dependent on the rate of sugar phosphorylation as determined by Arabidopsis thaliana hexokinase

Sugars like glucose and fructose induce death of yeast cells within a few hours, in the absence of additional nutrients to support growth, while cells incubated in water remain viable for weeks. This sugar-induced cell death (SICD) by glucose and fructose required glucose or fructose phosphorylation since yeast cells deficient in hexose phosphorylation did not die. However, when hexose phosphorylation is restored by complementation with Arabidopsis thaliana hexokinase, the cells died. The affinity of A. thaliana hexokinase is about 400 times higher for glucose than for fructose, therefore, A. thaliana hexokinase was further utilized to study the role of hexose phosphorylation in SICD. The rate of SICD of hexokinase-deficient yeast cells expressing A. thaliana hexokinase was significantly slower in fructose than in glucose, indicating that SICD is determined by the rate of hexose phosphorylation. The significance of hexose phosphorylation and its role in SICD is discussed.     

Hexose transporters GLUT1 and GLUT3 are colocalized with hexokinase I in caveolae microdomains of rat spermatogenic cells

Postmeiotic spermatogenic cells, but not meiotic spermatogenic cells respond differentially with glucose-induced changes in [Ca2+]i indicating a differential transport of glucose via facilitative hexose transporters (GLUTs) specifically distributed in the plasma membrane. Several studies have indicated that plasma membrane in mammalian cells is not homogeneously organized, but contains specific microdomains known as detergent-resistant membrane domains (DRMDs), lipid rafts or caveolae. The association of these domains and GLUTs isoforms has not been characterized in spermatogenic cells. We analyzed the expression and function of GLUT1 and GLUT3 in isolated spermatocytes and spermatids. The results showed that spermatogenic cells express both glucose transporters, with spermatids exhibiting a higher affinity glucose transport system. In addition, spermatogenic cells express caveolin-1, and glucose transporters colocalize with caveolin-1 in caveolin-enriched membrane fractions. Experiments in which the integrity of caveolae was disrupted by pretreatment with methyl-beta-cyclodextrin, indicated that the involvement of cholesterol-enriched plasma membrane microdomains were involved in the localization of GLUTs and uptake of 2-deoxyglucose. We also observed cofractionation of GLUT3 and caveolin-1 in low-buoyant density membranes together with their shift to higher densities after methyl-beta-cyclodextrin treatment. GLUT1 was found in all fractions isolated. Immunofluorescent studies indicated that caveolin-1, GLUT1, and hexokinase I colocalize in spermatocytes while caveolin-1, GLUT3, and hexokinase I colocalize in spermatids. These findings suggest the presence of hexose transporters in DRMDs, and further support a role for intact caveolae or cholesterol-enriched membrane microdomains in relation to glucose uptake and glucose phosphorylation. The results would also explain the different glucose-induced changes in [Ca2+]i in both cells.     

The inactivation of hexokinase activity does not prevent glucose repression in Candida utilis

Abstract High hexokinase activity was not related to glucose repression in Candida utilis IGC 3092. The addition of Cibacron Blue 3G-A to growing cells in batch culture led to a permanent in vivo hexokinase inactivation, decreased growth rate and inhibited alcohol dehydrogenase. Hexokinase inactivation up to 90% did not alleviate glucose repression of α-glucosidase, as has been described for Saccharomyces cerevisiae and other yeasts. Moreover, when cells were physiologically derepressed by growing them in a chemostat at low glucose concentrations, the highest hexokinase activity was shown by the derepressed cells, and decreased as repression increased. Thus, in our strain of C. utilis , hexokinase activity was inversely proportional to glucose repression.     

Insulin stimulation of glucose transport and metabolism in a human Wilms' tumor-derived myoblast-like cell Line: Modulation of hormone effects by glucose deprivation

The effects of insulin and glucose on parameters of metabolism were investigated in myoblast-like (MBL) cells, a human myoblast-like cell line derived from a Wilms' tumor. Insulin responses were studied after 4 hr pre-incubation in serum free media, with or without 5 mM glucose. Insulin was added during the last 2 hr. Glucose starvation markedly increased basal glucose transport (measured as 2-deoxyglucose uptake) as well as the net uptake of [¹⁴C]glucose and [¹⁴C]glucose incorporation into glycogen. Insulin stimulated net glucose uptake and incorporation into glycogen in a dose-dependent manner in glucose-fed and starved cells. These insulin responses were markedly enhanced in glucose-starved cells. Insulin accelerated 2-deoxyglucose transport in glucose-fed cells but did not further stimulate basal glucose transport in glucose-deprived cells. Insulin increased the incorporation of [³H]leucine into protein in glucose-fed or -starved MBL cells equally. The dose of insulin required for half-maximal insulin responses was similar for all parameters studied. Cycloheximide did not prevent the increased basal glucose incorporation in glucose-starved cells, but markedly inhibited the insulin response, while in glucose-fed cells, cycloheximide stimulated basal glucose incorporation. We conclude that MBL cells resemble fibroblasts in their insulin-independent stimulation of glucose transport in response to glucose-deprivation; when provided with glucose, they respond to insulin like fibroblasts. However, after brief glucose-starvation, the stimulated glucose transport system is no longer insulin-responsive in MBL cells, while pathways leading to the synthesis of macromolecules demonstrate preserved or enhanced stimulation by insulin, suggesting that these cells may serve as models to study the regulation of receptor-response coupling by the metabolic milieu.     

Altered metabolic states do not change the intracellular distribution of hexokinase in Zajdela hepatoma ascites cells.

The distribution of hexokinase between bound and soluble forms was studied by digitonin fractionation of Zajdela hepatoma ascites cells maintained under various metabolic conditions. Addition of glucose to Zajdela cells respiring on endogenous substrates induces an immediate inhibition of respiration by 50-60% ( Crabtree effect), and a production of acid due to glycolysis. Acid production decreases abruptly after 60s to 50% of the initial rate. The ATP/ADP ratio is not altered by the addition of glucose or by different rates of glycolysis. The uncoupling agent carbonyl cyanide m-chlorophenylhydrazone decreases the ATP/ADP ratio by 10-fold in cells respiring on endogenous substrate, but has little effect on cells oxidizing glucose. Rapid fractionation of the cells under these various metabolic conditions revealed no change in the distribution of hexokinase. Approx. 75% of hexokinase is bound in all cases, in contrast with lactate dehydrogenase, 95% of which was in the soluble form. Longer-term incubations (to 20 min) revealed only slight (10-15%) increases in soluble hexokinase in cells incubated with glucose. Various metabolic inhibitors had little additional affect on the subcellular distribution of hexokinase. Thus a rapid release of hexokinase from mitochondrial membrane is not a mechanism by which glycolysis is regulated in rapidly growing Zajdela hepatoma.     

Silybin and dehydrosilybin decrease glucose uptake by inhibiting GLUT proteins

Silybin, the major flavonoid of Silybum marianum, is widely used to treat liver diseases such as hepatocellular carcinoma and cirrhosis-associated insulin resistance. Research so far has focused on its anti-oxidant properties. Here, we demonstrate that silybin and its derivative dehydrosilybin inhibit glucose uptake in several model systems. Both flavonoids dose-dependently reduce basal and insulin-dependent glucose uptake of 3T3-L1 adipocytes, with dehydrosilybin showing significantly stronger inhibition. However, insulin signaling was not impaired, and immunofluorescence and subcellular fractionation showed that insulin-induced translocation of GLUT4 to the plasma membrane is also unchanged. Likewise, hexokinase activity was not affected suggesting that silybin and dehydrosilybin interfere directly with glucose transport across the PM. Expression of GLUT4 in CHO cells counteracted the inhibition of glucose uptake by both flavonoids. Moreover, treatment of CHO cells with silybin and dehydrosilybin reduced cell viability which was partially rescued by GLUT4 expression. Kinetic analysis revealed that silybin and dehydrosilybin inhibit GLUT4-mediated glucose transport in a competitive manner with K(i)=60 and 116 μM, respectively. We conclude that silybin and dehydrosilybin inhibit cellular glucose uptake by directly interacting with GLUT transporters. Glucose starvation offers a novel explanation for the anti-cancer effects of silybin.     

Effects of Different Substrates on Glucose Uptake and Hexokinase Activity in Euglena gracilis*

SYNOPSIS. Hexokinase levels and glucose uptake rates were measured in cells of Euglena gracilis adapted for growth on glucose, acetate, and ethanol. Glucose-grown cells had 3 times the hexokinase levels found in acetate- and ethanol-grown cells. However, glucose- and ethanol-grown cells had the same rates of glucose uptake, while acetate-grown cells accumulated only 1/8 as much glucose in the same interval.     

Glucose metabolism in Neurospora is altered by heat shock and by disruption of HSP30

We compared the metabolism of [1-13C]glucose by wild type cells of Neurospora crassa at normal growth temperature and at heat shock temperatures, using nuclear magnetic resonance analysis of cell extracts. High temperature led to increased incorporation of 13C into trehalose, relative to all other metabolites, and there was undetectable synthesis of glycerol, which was a prominent metabolite of glucose at normal temperature (30 degrees C). Heat shock strongly reduced formation of tricarboxylic acid cycle intermediates, approximately 10-fold, and mannitol synthesis was severely depressed at 46 degrees C, but only moderately reduced at 45 degrees C. A mutant strain of N. crassa that lacks the small alpha-crystallin-related heat shock protein, Hsp30, shows poor survival during heat shock on a nutrient medium with restricted glucose. An analysis of glucose metabolism of this strain showed that, unlike the wild type strain, Hsp30-deficient cells may accumulate unphosphorylated glucose at high temperature. This suggestion that glucose-phosphorylating hexokinase activity might be depressed in mutant cells led us to compare hexokinase activity in the two strains at high temperature. Hexokinase was reduced more than 35% in the mutant cell extracts, relative to wild type extracts. alpha-Crystallin and an Hsp30-enriched preparation protected purified hexokinase from thermal inactivation in vitro, supporting the proposal that Hsp30 may directly stabilize hexokinase in vivo during heat shock.     

Hexokinase II-deficient mice. Prenatal death of homozygotes without disturbances in glucose tolerance in heterozygotes.

Type 2 diabetes is characterized by decreased rates of insulin-stimulated glucose uptake and utilization, reduced hexokinase II mRNA and enzyme production, and low basal levels of glucose 6-phosphate in insulin-sensitive skeletal muscle and adipose tissues. Hexokinase II is primarily expressed in muscle and adipose tissues where it catalyzes the phosphorylation of glucose to glucose 6-phosphate, a possible rate-limiting step for glucose disposal. To investigate the role of hexokinase II in insulin action and in glucose homeostasis as well as in mouse development, we generated a hexokinase II knock-out mouse. Mice homozygous for hexokinase II deficiency (HKII(-/-)) died at approximately 7.5 days post-fertilization, indicating that hexokinase II is vital for mouse embryogenesis after implantation and before organogenesis. HKII(+/-) mice were viable, fertile, and grew normally. Surprisingly, even though HKII(+/-) mice had significantly reduced (by 50%) hexokinase II mRNA and activity levels in skeletal muscle, heart, and adipose tissue, they did not exhibit impaired insulin action or glucose tolerance even when challenged with a high-fat diet.     

Polyamines stimulate the binding of hexokinase type II to mitochondria

Spermine and spermidine enhanced the binding of hexokinase isoenzyme type II to mitochondria, both of which were prepared from Ehrlich-Lettre hyperdiploid ascites tumor cells, at much lower concentrations than Mg2+. Chymotrypsin-treated hexokinase II could not bind to the mitochondrial membrane in the presence of either spermine or Mg2+, indicating that the effect of spermine is not a nonspecific action, since the treatment of chymotrypsin cleaves only the region essential for the binding without any significant effect of the catalytic activity. Both spermine and Mg2+ antagonized the glucose 6-phosphate-induced release of mitochondria-bound hexokinase, and promoted the binding of the solubilized hexokinase II even in the presence of glucose 6-phosphate. However, inhibition of the activity of soluble hexokinase by glucose 6-phosphate was not reversed by spermine and Mg2+. Hexokinase II rebound to mitochondria with spermine and Mg2+ produced glucose 6-phosphate using ATP generated inside the mitochondria, and no difference was observed between the spermine- and Mg2+-rebound systems. Significance of the binding of hexokinase to mitochondria, especially with polyamines, is discussed with reference to high glycolytic rate in tumor cells.