Cellobiose uptake by the cellulolytic ruminal anaerobe Fibrobacter (Bacteroides) succinogenes

Cellobiose transport by the cellulolytic ruminal anaerobe Fibrobacter (Bacteroides) succinogenes was measured using randomly tritiated cellobiose. When assayed at the same concentration (1 mM), total cellobiose uptake was one-fourth to one-third that of total glucose uptake. The abilities of F. succinogenes to transport cellobiose or glucose were not affected by the sugar on which the cells were grown. Aspects of the simultaneous transport of [14C(U)]glucose and [3H(G)]cellobiose, the failure of high concentrations of cold glucose to compete with hypothetical [3H(G)]glucose (derived externally from [3H(G)]cellobiose), and differential metal-ion stimulation of cellobiose transport indicate a cellobiose permease, rather than cellobiase plus glucose permease, was responsible for cellobiose transport. Glucose (10-fold molar excess) partially inhibited cellobiose transport. This was enhanced by prior incubation of the cells with glucose, suggesting subsequent metabolism of the glucose was responsible for the inhibition. Compounds interfering with electron transport or maintenance of transmembrane ion gradients inhibited cellobiose uptake, indicating that active transport rather than a phosphoenolpyruvate:phosphotransferase system catalyzed cellobiose transport. Na+, but not Li+, stimulated cellobiose transport.     

Metabolism of [U-14C, 2-3H] glucose in rat liver during non-recirculating perfusion: effects of insulin

Livers from fed male rats were perfused in a non-recirculating manner with undiluted blood containing either 6 or 13 mM [U-14C,2-3H] glucose. At the lower concentration there was a small output of glucose which was unaffected by insulin whereas at the high concentration there was a substantial uptake of glucose which was significantly increased by the hormone. The rate of metabolism of [2-3H] glucose was greater than that of [U-14C] glucose in all experiments indicating an active substrate cycle between glucose:glucose 6-phosphate. Cycling was unaffected by insulin at the lower glucose concentration but was increased by perfusion with 13 mM glucose, the latter increase being abolished by insulin. These data show that although the perfused liver acts to autoregulate blood glucose, this is not achieved solely at the substrate cycle glucose:glucose 6-phosphate.     

Granulocyte-macrophage colony-stimulating factor signals for increased glucose transport via phosphatidylinositol 3-kinase- and hydrogen peroxide-dependent mechanisms

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates cellular glucose uptake by decreasing the apparent K(m) for substrate transport through facilitative glucose transporters on the plasma membrane. Little is known about this signal transduction pathway and the role of the alpha subunit of the GM-CSF receptor (alpha GMR) in modulating transporter activity. We examined the function of phosphatidylinositol 3-kinase (PI 3-kinase) in GM-CSF-stimulated glucose uptake and found that PI 3-kinase inhibitors, wortmannin and LY294002, completely blocked the GM-CSF-dependent increase of glucose uptake in Xenopus oocytes expressing the low affinity alpha GMR and in human cells expressing the high affinity alpha beta GMR complex. We identified a Src homology 3 domain-binding motif in alpha GMR at residues 358-361 as a potential interaction site for the PI 3-kinase regulatory subunit, p85. Physical evidence for p85 binding to alpha GMR was obtained by co-immunoprecipitation with antibodies to alpha GMR and p85, and an alpha GMR mutant with alteration of the Src homology 3 binding domain lost the ability to bind p85. Experiments with a construct eliminating most of the intracellular portion of alpha GMR showed a 50% reduction in GM-CSF-stimulated glucose uptake with residual activity blocked by wortmannin. Searching for a proximally generated diffusible factor capable of activating PI 3-kinase, we identified hydrogen peroxide (H(2)O(2)), generated by ligand or antibody binding to alpha GMR, as the initiating factor. Catalase treatment abrogated GM-CSF- or anti-alpha GMR antibody-stimulated glucose uptake in alpha GMR-expressing oocytes, and H(2)O(2) activated PI 3-kinase and led to some stimulation of glucose uptake in uninjected oocytes. Human myeloid cell lines and primary explant human lymphocytes expressing high affinity GM-CSF receptors responded to alpha GMR antibody with increased glucose uptake. These results identify the early events in the stimulation of glucose uptake by GM-CSF as involving local H(2)O(2) generation and requiring PI 3-kinase activation. Our findings also provide a mechanistic explanation for signaling through the isolated alpha subunit of the GM-CSF receptor.     

Lactate metabolism in the perfused rat hindlimb.

A preparation of isolated rat hindleg was perfused with a medium consisting of bicarbonate buffer containing Ficoll and fluorocarbon, containing glucose and/or lactate. The leg was electrically prestimulated to deplete partially muscle glycogen. The glucose was labelled uniformly with 14C and with 3H in positions 2, 5 or 6, and lactate uniformly with 14C and with 3H in positions 2 or 3. Glucose carbon was predominantly recovered in glycogen, and to a lesser extent in lactate. The 3H/14C ration in glycogen from [5-3H,U-14C]- and [6-3H,U-14C]-glucose was the same as in glucose. Nearly all the utilized 3H from [2-3H]glucose was recovered as water. Insulin increased glucose uptake and glycogen synthesis 3-fold. When the muscle was perfused with a medium containing 10 mM-glucose and 2 mM-lactate, there was little change in lactate concentration. 14C from lactate was incorporated into glycogen. There was a marked exponential decrease in lactate specific radioactivity, much greater with [3H]- than with [14C]-lactate. The 'apparent turnover' of [U-14C]lactate was 0.28 mumol/min per g of muscle, and those of [2-3H]- and [3-3H]-lactate were both about 0.7 mumol/min per g. With 10 mM-lactate as sole substrate, there was a net uptake of lactate, at a rate of about 0.15 mumol/min per g, and the apparent turnover of [U-14C]lactate was 0.3 mumol/min per g. The apparent turnover of [3H]lactate was 3-5 times greater. When glycogen synthesis was low (no prestimulation, no insulin), the incorporation of lactate carbon into glycogen exceeded that from glucose, but at high rates of glycogen deposition the incorporation of lactate carbon was much less than that of glucose. Lactate incorporation into glycogen was similar in fast-twitch white and fast-twitch red muscle, but was very low in slow-twitch red fibres. We find that (a) pyruvate in muscle is incorporated into glycogen without randomization of carbon, and synthesis is not inhibited by mercaptopicolinate or cycloserine; (b) there is extensive lactate turnover in the absence of net lactate uptake, and there is a large dilution of 14C-labelled lactate from endogenous supply; (c) there is extensive detritiation of [2-3H]- and [3-3H]-lactate in excess of 14C utilization.     

Kinetic advantage for transport into hamster intestine of glucose generated from phlorizin by brush border beta-glucosidase

Phlorizin, labeled with tritium only in the glucose moiety, was used as substrate for the beta-glucosidase present in brush border membranes from hamster intestine in order to study, simultaneously, the kinetics of hydrolysis and the fate of the [3H]glucose liberated by the enzyme. The [3H]glucose seems to experience the same hydrolase related transport into the intestinal villi as the hexoses liberated from the common disaccharides byu their respective hydrolases. The released [3H]glucose accumulation rate is only partially inhibited by unlabelled glucose added to the medium as either the free sugar or as the precursors sucrose, lactose or glucose 1-phosphate, and then only when these sugars are present at very high levels. Furthermore, glucose oxidase, added to the medium as a glucose scavenger, has no effect on the uptake rate of the phlorizin hydrolase-liberated sugar. These and other findings are presented as evidence that, under conditions where the Na+-dependent glucose carrier is more than 97% inhibited by phlorizin, the glucose derived from the inhibitor, like the hexoses from disaccharides, has a kinetic advantage for transfer into the intestinal tissue.     

Cardiac metabolism in mice: tracer method developments and in vivo application revealing profound metabolic inflexibility in diabetes

Studies of cardiac fuel metabolism in mice have been almost exclusively conducted ex vivo. The major aim of this study was to assess in vivo plasma FFA and glucose utilization by the hearts of healthy control (db/+) and diabetic (db/db) mice, based on cardiac uptake of (R)-2-[9,10-(3)H]bromopalmitate ([3H]R-BrP) and 2-deoxy-D-[U-14C]glucose tracers. To obtain quantitative information about the evaluation of cardiac FFA utilization with [3H]R-BrP, simultaneous comparisons of [3H]R-BrP and [14C]palmitate ([14C]P) uptake were first made in isolated perfused working hearts from db/+ mice. It was found that [3H]R-BrP uptake was closely correlated with [14C]P oxidation (r2 = 0.94, P < 0.001). Then, methods for in vivo application of [3H]R-BrP and [14C]2-DG previously developed for application in the rat were specially adapted for use in the mouse. The method yields indexes of cardiac FFA utilization (R(f)*) and clearance (K(f)*), as well as glucose utilization (R(g)'). Finally, in the main part of the study, the ability of the heart to switch between FFA and glucose fuels (metabolic flexibility) was investigated by studying anesthetized, 8-h-fasted control and db/db mice in either the basal state or during glucose infusion. In control mice, glucose infusion raised plasma levels of glucose and insulin, raised R(g)' (+58%), and lowered plasma FFA level (-48%), K(f)* (-45%), and R(f)* (-70%). This apparent reciprocal regulation of glucose and FFA utilization by control hearts illustrates metabolic flexibility for substrate use. By contrast, in the db/db mice, glucose infusion raised glucose levels with no apparent influence on cardiac FFA or glucose utilization. In conclusion, tracer methodology for assessing in vivo tissue-specific plasma FFA and glucose utilization has been adapted for use in mice and reveals a profound loss of metabolic flexibility in the diabetic db/db heart, suggesting a fixed level of FFA oxidation in fasted and glucose-infused states.     

Hexose transport by brain slices: Further studies on energy dependence

We studied the uptake of [3H]2-deoxyglucose [( 3H]2DG) by slices of rat cerebral cortex in vitro as a model of glucose transport by brain. Slices were incubated with [3H]2DG, or with L-[3H]glucose as a marker for diffusion; the difference between [3H]2DG uptake and L-[3H]glucose uptake was defined as net [3H]2DG transport. Net [3H]2DG transport was a function of incubation temperature, with an estimated temperature coefficient of 1.87 from 15 degrees C to 25 degrees C. The net uptake of [3H]2DG was not inhibited by phlorizin or phloretin in concentrations well above the reported Ki of these inhibitors for hexose uptake in other systems. To examine the hypothesis that [3H]2DG transport by brain slices is dependent on mitochondrial energy, we studied net [3H]2DG uptake by slices which had been preincubated in media designed to alter intracellular ATP stores. The transport process was very sensitive to inhibition by DNP, but the correlation between [3H]2DG transport and ATP levels was unclear. In contrast to our published hypothesis that the transport process required mitochondrial energy, these data indicate that dependence on energy is not absolute.     

Glucose uptake in vivo in skeletal muscles of insulin-injected chicks

Glucose uptake across the plasma membrane in animal cells plays a crucial role in whole-body glucose homeostasis. Insulin-stimulated glucose transport activity in vivo in several tissues was estimated using the 2-deoxy-D-[1-(3)H]glucose ([(3)H]2DG) uptake determination method. A tracer dose of [(3)H]2DG was injected intravenously into 8-day-old chicks (Gallus gallus) administered simultaneously or previously with porcine insulin (40 microg/kg BW). After 10 or 20 min, several major tissues, including skeletal and cardiac muscle, were sampled and their 2-deoxy-D-[1-(3)H]glucose 6-phosphate content analyzed. Plasma glucose concentration and [(3)H]2DG radioactivity were lowered by insulin within 20 min of [(3)H]2DG administration, while the plasma [(3)H]2DG/glucose ratio was not significantly different between chicks injected with insulin and their control counterparts. A marked uptake of 2DG was observed in cardiac tissue and brain, followed by kidney and skeletal muscles. In skeletal muscles, insulin increased the 2DG uptake in soleus, extensor digitorum longus and pectoralis superficialis muscles. On the other hand, no significant increases in insulin-induced 2DG uptake were detected in cardiac muscle or adipose tissue compared to controls. The results show that glucose transport across the plasma membrane in vivo in most skeletal muscles tested, but not cardiac muscle, was increased by insulin administration to chicks. These findings suggest that an insulin-responsive glucose transport mechanism is present in chickens, even though they intrinsically lack GLUT4 homologous gene, the insulin-responsive glucose transporter in mammals.     

High glucose-induced inhibition of alpha-methyl-D-glucopyranoside uptake is mediated by protein kinase C-dependent activation of arachidonic acid release in primary cultured rabbit renal proximal tubule cells

Abnormal glucose handling in the proximal tubule may play an important role in the development of diabetic nephropathy. Thus, the present study was designed to examine the effect of high glucose on alpha-methyl-D-glucopyranoside (alpha-MG) uptake and its signaling pathways in the primary cultured rabbit renal proximal tubule cells (PTCs). When PTCs were preincubated with 25 or 50 mM glucose for 4 h, 25 or 50 mM glucose significantly inhibited alpha-MG uptake, while 25 or 50 mM mannitol and L-glucose did not affect. Actinomycin D and cycloheximide did not block the effect of high glucose on alpha-MG uptake. Twenty-five millimoles glucose-induced inhibition of alpha-MG uptake was blocked by mepacrine and AACOCF(3), phospholipase A(2) (PLA(2)) inhibitors. Twenty-five millimoles of glucose, not mannitol or L-glucose, significantly increased the [(3)H]-arachidonic acid (AA) release compared to control. In addition, the 25 mM glucose-induced [(3)H]-AA release was completely blocked by mepacrine or AACOCF(3). Indomethacin, a cyclooxygenase inhibitor, blocked the high glucose-induced inhibition of alpha-MG uptake, although econazole, cytochrome P-450 a epoxygenase inhibitor, and nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, did not. On the other hand, staurosporine and bisindolylmaleimide I, protein kinase C (PKC) inhibitors, blocked 25 mM glucose-induced increase of [(3)H]-AA release and inhibition of alpha-MG uptake. However, neomycin, U 73122, and phospholipase c(PLC) inhibitors did not block the effect of 25 mM glucose on [(3)H]-AA release and alpha-MG uptake. Pretreatment of methoxyverapamil, an L-type Ca(2+) channel blocker, abolished 25 mM glucose-induced increase of [(3)H]-AA release. Indeed, 25 mM glucose increased translocation of cPLA(2) from cytosolic fraction to membrane fraction. In conclusion, the present results demonstrate that high glucose inhibits alpha-MG uptake by the increase of AA release via the activation of PKC.     

Characteristics of Glucose Transport in Neuronal Cells and Astrocytes from Rat Brain in Primary Culture

Glucose transport systems in cultured neuronal cells and astrocytes of rats were characterized by measuring the uptake of 2-deoxy-D-[3H]glucose ([3H]2-DG) into the cells. Various sugars inhibited 2-DG uptake by neuronal cells and astrocytes similarly, a finding indicating that the substrate specificities of the transporters in the two types of cells were almost the same. However, the Km values for 2-DG of neuronal cells and astrocytes were 1.7 and 0.36 mM, respectively. The uptake of 2-DG was strongly inhibited by cytochalasin B. Nucleosides, such as adenosine, inosine, and uridine, inhibited 2-DG uptake competitively in both neuronal cells and astrocytes. The uptake by both types of cells were also inhibited by forskolin, but not by cyclic AMP, an observation suggesting that forskolin bound directly to the transporters to cause inhibition. Its inhibition was competitive in astrocytes and noncompetitive in neuronal cells. Astrocytes contained a glucose transporter with a subunit molecular weight of 45K, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after photoaffinity labeling using [3H]cytochalasin B as a probe.     

Inhibition of mitochondrial respiratory chain in the brain of rats after hepatic failure induced by acetaminophen

To explore the effect of LYRM1 over-expression on basal and insulin-stimulated glucose uptake in rat skeletal muscle cells, and to understand the underlying mechanisms, Rat myoblasts (L6) transfected with either an empty expression vector (pcDNA3.1Myc/His B) or a LYRM1 expression vector were differentiated into myotubes. Glucose uptake was determined by measuring 2-deoxy-D-[(3)H] glucose uptake into L6 myotubes. Western blotting was performed to assess the translocation of insulin-sensitive glucose transporter 4 (GLUT4). It was also used to measure the phosphorylation and total protein contents of insulin-signaling proteins, such as the insulin receptor (IR), insulin receptor substrate (IRS)-1, phosphatidylinositol-3-kinase (PI3K) p85, Akt, ERK1/2, P38, and JNK. LYRM1 over-expression in L6 myotubes reduced insulin-stimulated glucose uptake and impaired insulin-stimulated GLUT4 translocation. It also diminished insulin-stimulated tyrosine phosphorylation of IRS-1, PI3K (p85), and serine phosphorylation of Akt without affecting the phosphorylation of IR, ERK1/2, P38, and JNK. LYRM1 regulates the function of IRS-1, PI3K, and Akt, and decreases GLUT4 translocation and glucose uptake in response to insulin. These observations highlight the potential role of LYRM1 in glucose homeostasis and possibly in the pathophysiology of type 2 diabetes related to obesity.     

Glucose Metabolism in Sediments of a Eutrophic Lake: Tracer Analysis of Uptake and Product Formation

The uptake of glucose and the formation of end products from glucose catabolism have been measured for sediments of eutrophic Wintergreen Lake with a combination of tritiated and ¹⁴C-labeled tracers. Time course analyses of the loss of [³H]glucose from sediments were used to establish rate constants for glucose uptake at natural substrate concentrations. Turnover times from these analyses were about 1 min for littoral and profundal sediments. No seasonal or site differences were noted in turnover times. Time course analyses of [U-¹⁴C]glucose uptake and ¹⁴C-labeled end product formation indicated that glucose mass flow could not be calculated from end product formation since the specific activity of added [¹⁴C]glucose was significantly diluted by pools of intracellular glucose and glucose metabolites. Mass flow could only be accurately estimated by use of rates of uptake from tracer studies. Intermediate fermentation end products included acetate (71%), propionate (15%), lactate (9%), and only minor amounts of butyrates or valerates. Addition of H₂ to sediments resulted in greater production of lactate (28%) and decreased formation of acetate (50%), but did not affect glucose turnover. Depth profiles of glucose uptake indicated that rates of uptake decreased with depth over the 0- to 18-cm interval and that glucose uptake accounted for 30 to 40% of methanogenesis in profundal sediments.     

Plasma glucose and insulin responses to oral and intravenous glucose in cold-exposed humans

Although glucose tolerance and skeletal muscle glucose uptake are markedly improved by cold exposure in animals, little is known about such responses in humans. This study used two variations of a glucose tolerance test (GTT) to investigate changes in carbohydrate metabolism in healthy males during nude exposure to cold. In experiment 1, an oral GTT was performed in the cold and in the warm (3 h at 10 or 29 degrees C). To bypass the gastrointestinal tract, and to suppress hepatic glucose output, a second experiment was carried out as described above, using an intravenous GTT. Even though cold exposure raised metabolic rate greater than 2.5 times, plasma glucose and insulin responses to an oral GTT remained unaltered. In contrast, cold exposure reduced the entire plasma glucose profile as a function of time during the intravenous GTT (P less than 0.05), as plasma glucose was returned to basal levels within 1 h in comparison to a full 2 h in the warm, despite low insulin levels. The results of the intravenous GTT demonstrate that even with low insulin levels, carbohydrate metabolism is increased in cold-exposed males. This effect could be masked in the oral GTT by gastrointestinal factors and a high hepatic glucose output. Cold exposure may enhance insulin sensitivity and/or responsiveness for glucose uptake, mainly in shivering skeletal muscles.     

Dehydroepiandrosterone (DHEA) stimulates glucose uptake in rat adipocytes: activation of phospholipase D

We examined the effect of dehydroepiandrosterone (DHEA) on glucose uptake and phospholipase D (PLD) activation in rat adipocytes. DHEA (1 microM) provoked a twofold increase in [3H]2-deoxyglucose (DG) uptake for 30 min. Incorporation of [3H]glycerol into diacylglycerol was increased 150% above basal level for 20 min after stimulation with 1 microM DHEA. DHEA increased PLD activity, measured by the incorporation into [3H]phosphatidylethanol in [3H]palmitate labelled rat adipocytes, or by [3H]choline release in [methyl-(3)H]choline labeled rat adipocytes. Our results suggest that DHEA stimulates glucose uptake with activation of PLD in rat adipocytes.     

Accumulation of 2-deoxy-D-glucose-6-phosphate as a measure of glucose uptake in the isolated perfused heart: a 31P NMR study

The accumulation of 2-deoxy-D-glucose-6-phosphate (2DG6P), detected using 31P NMR spectroscopy, has been used as a measure of the rate of glucose uptake, yet the accuracy of this measurement has not been verified. In this study, isolated rat hearts were perfused with different substrates or isoproterenol for 30 min before measurement of either 2DG6P accumulation or [2-3H]glucose uptake, without and with insulin. Basal contractile function and metabolite concentrations were the same for all hearts. The basal rates of 2DG6P accumulation differed significantly, depending on the preceding perfusion protocol, and were 38-60% of the [2-3H]glucose uptake rates, whereas insulin-stimulated 2DG6P accumulation was the same or 71% higher than the [2-3H]glucose uptake rates. Therefore the ratio of 2DG6P accumulation/[2-3H]glucose uptake rates varied from 0.38 to 1.71, depending on the prior perfusion conditions or the presence of insulin. The rates of 2DG6P hydrolysis were found to be proportional to the intracellular 2DG6P concentrations, with a K(m) of 17.5mM and V(max) of 1.4 micromol/g dry weight/min. We conclude that the rates of 2DG6P accumulation do not accurately reflect glucose uptake rates under all physiological conditions in the isolated heart and should be used with caution.     

The kinetic advantage for transport into hamster intestine of glucose generated from phlorizin by brush border β-glucosidase

Phlorizin, labeled with tritium only in the glucose moiety, was used as substrate for the β-glucosidase present in brush border membranes from hamster intestine in order to study, simultaneously, the kinetics of hydrolysis and the fate of the [³H]glucose liberated by the enzyme. The [³H]glucose seems to experience the same hydrolase related transport into the intestinal villi as the hexoses liberated from the common disaccharides by their respective hydrolases. The released [³H]glucose accumulation rate is only partially inhibited by unlabelled glucose added to the medium as either the free sugar or as the precursors sucrose, lactose or glucose 1-phosphate, and then only when these sugars are present at very high levels. Furthermore, glucose oxidase, added to the medium as a glucose scavenger, has no effect on the uptake rate of the phlorizin hydrolase-liberated sugar. These and other findings are presented as evidence that, under conditions where the Na⁺-dependent glucose carrier is more than 97% inhibited by phlorizin, the glucose derived from the inhibitor, like the hexoses from disaccharides, has a kinetic advantage for transfer into the intestinal tissue.     

Hexose uptake in primary cultures of bovine brain microvessel endothelial cells. II. Effects of conditioned media from astroglial and glioma cells.

Regulation of glucose uptake by an astroglial cell secreted factor(s) was studied in primary cultures of brain microvessel endothelial cells (BMECs). Uptake of a non-metabolizable glucose analog, 3-O-[3H]methyl-D-glucose ([3H]3MG), was measured after the BMECs were treated with media conditioned by primary cultures of rat astrocytes (Astrocyte Conditioned Media: ACM) or rat C6 glioma cells (Glioma Cell Conditioned Media: GCM). Uptake of [3H]3MG was significantly increased by ACM (30-50%) and GCM (60-200%) treatments, whereas conditioned medium from 3T3 fibroblasts (3T3) caused no significant effect. The elevation in [3H]3MG uptake increased with increasing time of exposure of BMECs to these conditioned media (CM), and the effect was shown to be reversible. Glucose depletion of CM was shown not to be a factor. The presence of cycloheximide, a protein synthesis inhibitor, during treatment of the BMECs with ACM and GCM blocked the increase in [3H]3MG uptake by the cells. These results suggested that ACM or GCM treatment elevated de novo synthesis of brain-type glucose transporter (GLUT1). Indeed, enhanced GLUT1 expression by these treatments in BMECs was demonstrated directly by enzyme-linked immunosorbent assay (ELISA) using antibodies against human GLUT1. After trypsinization of ACM and GCM, both conditioned media still induced significant stimulation of [3H]3MG uptake by BMECs. A significant increase in [3H]3MG uptake was also observed when ACM or GCM was exposed to BMECs through a dialysis membrane with a molecular weight cutoff of 1000. To examine whether the effects were specific to brain endothelial cells, [3H]3MG uptake experiments were performed employing aortic endothelial cells (AECs), pulmonary microvessel endothelial cells (PMECs), and 3T3 cells. ACM treatment did not alter 3MG uptake by these cells, suggesting that the ACM effect was specific to BMECs. On the other hand, [3H]3MG uptake by AECs and PMECs treated with GCM was significantly enhanced. The present study demonstrated that some factor(s) of relatively small molecular weight, which was released from astrocytes or glioma cells, stimulated glucose uptake by enhancing GLUT1 synthesis in BMECs.     

Sugar transport in rat liver lysosomes. Direct demonstration by using labelled sugars.

Purified rat liver lysosomes ('tritosomes') were prepared from rats injected with Triton WR-1339. 2. The water space of tritosomes, measured by using [3H]water and [14C]sucrose, was 2.15 +/- 0.72 microliter/mg of protein (mean +/- S.E.M., n = 12). 3. Tritosomes, when compared with a crude preparation of normal lysosomes by an indirect method of study, showed sugar specificity but decreased stereospecificity of sugar uptake. 4. At 125 mM the relative rates of net uptake of D-[14C]ribose, D-[14C]- or D-[3H]glucose and 2-deoxy-D-[3H]glucose were the same as that inferred from the indirect study. 5. The entry of D-[3H]glucose into tritosomes showed concentration-dependence suggestive of saturation, with a Km of 48 +/- 18 mM (4). 6. D- and L-glucose, D-ribose, 2-deoxy-D-glucose and D-mannose competed with D-[14C]glucose or D-[14C]ribose for uptake. 7. Cytochalasin B inhibited D-[3H]glucose uptake. 8. Uptake of 1 mM-L-[14C]glucose was slower than for 1 mM-D-[14C]glucose. 9. It is concluded that a facilitated-diffusion transport system is present in purified rat liver lysosomes.     

Propranolol prevents epinephrine from limiting insulin-stimulated muscle glucose uptake during contraction.

Beta-blockade results in rapid glucose clearance and premature fatigue during exercise. To investigate the cause of this increased glucose clearance, we studied the acute effects of propranolol on insulin-stimulated muscle glucose uptake during contraction in the presence of epinephrine with an isolated rat muscle preparation. Glucose uptake increased in both fast- (epitrochlearis) and slow-twitch (soleus) muscle during insulin or contraction stimulation. In the presence of 24 nM epinephrine, glucose uptake during contraction was completely suppressed when insulin was present. This suppression of glucose uptake by epinephrine was accompanied by a decrease in insulin receptor substrate (IRS)-1-phosphatidylinositol 3 (PI3)-kinase activity. Propranolol had no direct effect on insulin-stimulated glucose uptake during contraction. However, epinephrine was ineffective in attenuating insulin-stimulated glucose uptake during contraction in the presence of propranolol. This ineffectiveness of epinephrine to suppress insulin-stimulated glucose uptake during contraction occurred in conjunction with its inability to completely suppress IRS-1-PI3-kinase activity. Results of this study indicate that the effectiveness of epinephrine to inhibit insulin-stimulated glucose uptake during contraction is severely diminished in muscle exposed to propranolol. Thus the increase in glucose clearance and premature fatigue associated with beta-blockade could result from the inability of epinephrine to attenuate insulin-stimulated muscle glucose uptake.     

Oxidative stress stimulates skeletal muscle glucose uptake through a phosphatidylinositol 3-kinase-dependent pathway

We determined the acute effects of oxidative stress on glucose uptake and intracellular signaling in skeletal muscle by incubating muscles with reactive oxygen species (ROS). Xanthine oxidase (XO) is a superoxide-generating enzyme that increases ROS. Exposure of isolated rat extensor digitorum longus (EDL) muscles to Hx/XO (Hx/XO) for 20 min resulted in a dose-dependent increase in glucose uptake. To determine whether the mechanism leading to Hx/XO-stimulated glucose uptake is associated with the production of H2O2, EDL muscles from rats were preincubated with the H2O2 scavenger catalase or the superoxide scavenger superoxide dismutase (SOD) prior to incubation with Hx/XO. Catalase treatment, but not SOD, completely inhibited the increase in Hx/XO-stimulated 2-deoxyglucose (2-DG) uptake, suggesting that H2O2 is an intermediary leading to Hx/XO-stimulated glucose uptake with incubation. Direct H2O2 also resulted in a dose-dependent increase in 2-DG uptake in isolated EDL muscles, and the maximal increase was threefold over basal levels at a concentration of 600 micromol/l H2O2. H2O2-stimulated 2-DG uptake was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, but not the nitric oxide inhibitor NG-monomethyl-l-arginine. H2O2 stimulated the phosphorylation of Akt Ser473 (7-fold) and Thr308 (2-fold) in isolated EDL muscles. H2O2 at 600 micromol/l had no effect on ATP concentrations and did not increase the activities of either the alpha1 or alpha2 catalytic isoforms of AMP-activated protein kinase. These results demonstrate that acute exposure of muscle to ROS is a potent stimulator of skeletal muscle glucose uptake and that this occurs through a PI3K-dependent mechanism.