Acute effects of cycloheximide on the translocation of glucose transporters in rat adipose cells

Insulin's rapid action to increase glucose transport is believed to occur primarily through the translocation of glucose transporters from an intracellular pool to the plasma membrane. To better understand the mechanism involved, we studied the role of protein synthesis in glucose transporter translocation by using the protein synthesis inhibitor, cycloheximide. Isolated rat epididymal adipose cells were incubated in the presence or absence of cycloheximide (10 micrograms/ml) for a total of 120 min. Insulin (7 nM) was added to half of the cells from both groups for the final 30 min. Protein synthesis was inhibited by approximately 90%, as measured by [14C]leucine incorporation, in the cells exposed to cycloheximide. The 3-O-methylglucose uptake in intact cells was slightly increased in the basal state with cycloheximide treatment, but the insulin-stimulated 3-O-methylglucose uptake was unchanged by cycloheximide. The distribution of glucose transporters in the different subcellular membrane fractions, as measured by the cytochalasin B binding assay, was unchanged by cycloheximide. These results suggest that insulin's stimulation of glucose transport and translocation of glucose transporters can occur without acute protein synthesis.     

Stimulation of 3-O-methylglucose transport by anaerobiosis in rat thymocytes.

Transport of 3-O-methylglucose by rat thymocytes occurs by facilitated diffusion and follows a biphasic time course. The half-times of the two phases of uptake are 0.8 min and 20 to 30 min; the rapid phase contributes 10 to 20% of the total 3-O-methylglucose taken up at equilibrium. Cells incubated under anaerobic conditions for 1 hour undergo a 3- to 4-fold increase in the initial rate of 3-O-methylglucose uptake. The relative contribution of the rapid phase of uptake increases nearly 4-fold in anaerobically incubated cells, although the half-time of the rapid phase remains the same. Anaerobiosis also reduces the half-time of the slow phase of uptake by a factor of three. In the absence of exogenous glucose, anaerobiosis reduces cellular ATP by 97% after 1 hour at 37 degrees. However, full stimulation of transport activity does not occur in cells with such low levels of ATP. When anaerobically incubated cells are re-exposed to oxygen, ATP synthesis proceeds and transport activity increases by 100% within 5 to 10 min. Adding 1 mM 2,4-dinitrophenol at the time the anaerobic cells are reexposed to oxygen completely blocks the subsequent ATP synthesis and the associated increase in transport activity. Cells incubated aerobically in the presence of 1 mM 2,4-dinitrophenol show a 90% reduction in ATP levels and a 2-fold increase in the rate of 3-O-methylglucose uptake. An additional 70% increase in transport activity is observed when the cells are washed free of uncoupler and incubated an additional 10 min. The results suggest that transport activity is stimulated when cellular ATP levels decline but that the stimulation process requires some minimal level of ATP for full expression.     

Interactions of insulin, catecholamines and adenosine in the regulation of glucose transport in isolated rat cardiac myocytes

The regulation of the glucose transport system by catecholamines and insulin has been studied in isolated rat cardiomyocytes. In the basal state, 1-isoproterenol exhibited a biphasic concentration-dependent regulation of 3-O-methylglucose transport. At low concentrations (less than 10 nM), isoproterenol induced a maximal inhibition of 65-70% of the basal rates, while at higher concentrations (greater than 10 nM) a 25-70% stimulation of transport was observed. In the presence of adenosine deaminase, the inhibition of isoproterenol at low doses was attenuated. No effect of adenosine deaminase was observed on the stimulation of transport at high doses of isoproterenol. The inhibitory effect of isoproterenol returned when N6-phenylisopropyladenosine (a non-metabolizable analog of adenosine) was included along with adenosine deaminase. Dibutyryl cAMP and forskolin both inhibited basal transport rates. In the presence of maximally stimulating concentrations of insulin, cardiomyocyte 3-O-methylglucose transport was generally elevated 200-300% above basal levels. In the presence of isoproterenol, insulin stimulation was inhibited at both high and low concentrations of catecholamine, with maximum inhibition occurring at the lowest concentrations tested. When cells were incubated with both adenosine deaminase and isoproterenol, the inhibition of the insulin response was greater at all concentrations of catecholamine and was almost completely blocked at isoproterenol concentrations of 10 nM or less. Dibutyryl cAMP inhibited the insulin response to within 10% of basal transport levels, while forskolin completely inhibited all transport activity in the presence of insulin. These results suggest that catecholamines regulate basal and insulin-stimulated glucose transport via both cAMP-dependent and cAMP-independent mechanisms and that this regulation is modulated in the presence of extracellular adenosine.     

Effects of ATP depletion on the mechanism of hexose transport in intact human erythrocytes

Depletion of ATP is known to inhibit glucose transport in human erythrocytes, but the kinetic mechanism of this effect is controversial. Selective ATP depletion of human erythrocytes by 10 micrograms/ml A23187 in the presence of extracellular calcium inhibited 3-O-methylglucose influx noncompetitively and efflux competitively. ATP depletion also decreased the ability of either equilibrated 3-O-methylglucose or extracellular maltose to inhibit cytochalasin B binding in intact cells, whereas neither total high-affinity cytochalasin B binding nor its Kd was affected. Under the one-site model of hexose transport these data indicate that ATP depletion decreases both the affinity of the inward-facing glucose carrier for substrate and its ability to reorient outwardly in intact cells.     

Counter-regulation of insulin-stimulated glucose transport by catecholamines in the isolated rat adipose cell

The interaction between catecholamines and insulin in regulating glucose transport in isolated rat adipose cells has been evaluated. In the absence of insulin, 1 microM isoproterenol stimulates 3-O-methylglucose transport approximately 2-fold. However, isoproterenol in combination with adenosine deaminase inhibits glucose transport activity approximately 60%. N6-Phenylisopropyladenosine, a nonmetabolizable adenosine analogue, substantially reverses this inhibitory effect and actually stimulates glucose transport activity approximately 2-fold in the absence of isoproterenol. Dibutyryl cAMP inhibits glucose transport activity approximately 75% regardless of adenosine deaminase. While none of these agents significantly influences the basal concentration of plasma membrane glucose transporters, as assessed by specific D-glucose-inhibitable cytochalasin B binding, isoproterenol or dibutyryl cAMP in combination with adenosine deaminase reduces that in the low density microsomes 19 and 58%, respectively. In the presence of insulin, both isoproterenol and adenosine deaminase alone inhibit glucose transport activity approximately 25%. However, only the latter is accompanied by a corresponding decrease in the insulin-stimulated concentration of plasma membrane glucose transporters. Together, isoproterenol and adenosine deaminase inhibit insulin-stimulated glucose transport activity approximately 75%, even in the presence of 5 mM glucose to maintain cellular ATP levels. A similar inhibition is observed with dibutyryl cAMP. However, these agents decrease the insulin-stimulated concentration of plasma membrane glucose transporters only approximately 45%. Nevertheless, all of these inhibitory effects occur through decreases in the transport Vmax. In addition, N6-phenylisopropyladenosine partially reverses the inhibitory effects induced by the presence of adenosine deaminase. These results suggest that catecholamines counter-regulate basal and insulin-stimulated glucose transport in rat adipose cells through a cAMP-mediated mechanism, but only in part by modulating the translocation of glucose transporters.     

G-protein-mediated regulation of the insulin-responsive glucose transporter in isolated cardiac myocytes.

Isolated muscle cells from adult rat heart were used to study the involvement of G-proteins in the regulation of the glucose transporter by insulin and isoprenaline. Efficient modification of G-protein functions was established by measuring isoprenaline-stimulated cyclic AMP production, viability and ATP content after treating the cells with cholera toxin and pertussis toxin for 2 h. Under these conditions cholera toxin decreased the stimulatory action of insulin on 3-O-methylglucose transport by 56%, but pertussis toxin had no effect. Basal transport was not affected by toxin treatment. Isoprenaline increased 3-O-methylglucose transport by 63%. This effect was not mimicked by dibutyryl cyclic AMP, but was completely blocked by cholera toxin. Streptozotocin-diabetes abolished isoprenaline action and decreased stimulation of transport by 64%. Concomitantly, cholera-toxin sensitivity of glucose transport was lost in cells from diabetic animals. This was paralleled by a large decrease (87 +/- 4%) in mRNA expression of the insulin-regulatable glucose transporter, as shown by Northern-blot analysis of RNA isolated from cardiomyocytes of diabetic rats. These data suggest a functional association between the insulin-responsive glucose transporter and a cholera-toxin-sensitive G-protein mediating stimulation by insulin and isoprenaline.     

Effect of chemotactic factors on hexose transport in polymorphonuclear leucocytes

Transport of the nonmetabolizable glucose analogue, 3-O-methylglucose, was assessed in human polymorphonuclear leucocytes with or without the chemotactic peptide N-formylmethionylleucylphenylalanine (fMet-Leu-Phe). The peptide increased entry of labelled 3-O-methylglucose about 5-fold and the intracellular distribution space about 70%. The half-time of equilibration was 3 s in the treated cells. Similar effects were observed with zymosan-treated serum (containing the chemotactic factor C5a), with arachidonic acid, calcium ionophore A23187 and phorbol myristate acetate. However, the chemotactic protein, thrombin, had no effect, even though binding to high-affinity receptors was demonstrated. Km for zero-trans entry of 3-O-methylglucose was about 1 mM and fMet-Leu-Phe increased Vmax from 5 to about 25 amol.s-1.cell-1. Similar values were obtained from incubations for a few seconds with glucose and 2-deoxyglucose. The rate of 2-deoxyglucose uptake (8 min incubations) was limited by the transport step at substrate concentrations lower than approx. 0.1 mM, whereas the phosphorylation step became rate-limiting at higher concentrations. Thus, 2-deoxyglucose uptake can only be taken as a measure of transport at a tracer concentration. It is concluded that chemotactic factors can, but do not necessarily, increase the maximal transport velocity of hexoses entering the polymorphonuclear leucocyte via the glucose transporter.     

Cycloheximide decreases glucose transporters in rat adipocyte plasma membranes without affecting insulin-stimulated glucose transport.

This study examines the relationship between insulin-stimulated glucose transport and insulin-induced translocation of glucose transporters in isolated rat adipocytes. Adipose cells were incubated with or without cycloheximide, a potent inhibitor of protein synthesis, for 60 min and then for an additional 30 min with or without insulin. After the incubation we measured 3-O-methylglucose transport in the adipose cells, and subcellular membrane fractions were prepared. The numbers of glucose transporters in the various membrane fractions were determined by the cytochalasin B binding assay. Basal and insulin-stimulated 3-O-methylglucose uptakes were not affected by cycloheximide. Furthermore, cycloheximide affected neither Vmax. nor Km of insulin-stimulated 3-O-methylglucose transport. In contrast, the number of glucose transporters in plasma membranes derived from cells preincubated with cycloheximide and insulin was markedly decreased compared with those from cells incubated with insulin alone (10.5 +/- 0.8 and 22.2 +/- 1.8 pmol/mg of protein respectively; P less than 0.005). The number of glucose transporters in cells incubated with cycloheximide alone was not significantly different compared with control cells. SDS/polyacrylamide-gel-electrophoretic analysis of [3H]cytochalasin-B-photolabelled plasma-membrane fractions revealed that cycloheximide decreases the amount of labelled glucose transporters in insulin-stimulated membranes. However, the apparent molecular mass of the protein was not changed by cycloheximide treatment. The effect of cycloheximide on the two-dimensional electrophoretic profile of the glucose transporter in insulin-stimulated low-density microsomal membranes revealed a decrease in the pI-6.4 glucose-transporter isoform, whereas the insulin-translocatable isoform (pI 5.6) was decreased. Thus the observed discrepancy between insulin-stimulated glucose transport and insulin-induced translocation of glucose transporters strongly suggests that a still unknown protein-synthesis-dependent mechanism is involved in insulin activation of glucose transport.     

Stimulation of glucose transport in skeletal muscle by hypoxia

Hypoxia caused a progressive cytochalasin B-inhibitable increase in the rate of 3-O-methylglucose transport in rat epitrochlearis muscles to a level approximately six-fold above basal. Muscle ATP concentration was well maintained during hypoxia, and increased glucose transport activity was still present after 15 min of reoxygenation despite repletion of phosphocreatine. However, the increase in glucose transport activity completely reversed during a 180-min-long recovery in oxygenated medium. In perfused rat hindlimb muscles, hypoxia caused an increase in glucose transporters in the plasma membrane, suggesting that glucose transporter translocation plays a role in the stimulation of glucose transport by hypoxia. The maximal effects of hypoxia and insulin on glucose transport activity were additive, whereas the effects of exercise and hypoxia were not, providing evidence suggesting that hypoxia and exercise stimulate glucose transport by the same mechanism. Caffeine, at a concentration too low to cause muscle contraction or an increase in glucose transport by itself, markedly potentiated the effect of a submaximal hypoxic stimulus on sugar transport. Dantrolene significantly inhibited the hypoxia-induced increase in 3-O-methylglucose transport. These effects of caffeine and dantrolene suggest that Ca2+ plays a role in the stimulation of glucose transport by hypoxia.     

CA(2+) mobilization in the human submandibular duct cell line A253

Ca(2+)mobilization induced by ATP, isoproterenol and the Ca(2+)-ATPase inhibitor thapsigargin in the human submandibular duct cell line A253 was investigated using the Ca(2+)-sensitive fluorescent indicator fura-2. ATP and isoproterenol increased cytosolic free Ca(2+)([Ca(2+)](i)) and subsequent exposure to thapsigargin after ATP or isoproterenol stimulation caused a further increase in [Ca(2+)](i). However, ATP and isoproterenol were not able to elicit a further increase in [Ca(2+)](i)after exposure of the cells to thapsigargin. Relatively few cells reacted to isoproterenol stimulation, but nearly all cells reacted to isoproterenol if ATP was added together with, or prior to isoproterenol stimulation. Moreover, the effect of ATP was potentiated by prior or simultaneous addition of isoproterenol. Furthermore, ATP decreased [Ca(2+)](i)in the presence of thapsigargin probably due to agonist-induced export of intracellular calcium. The results may suggest the existence of three thapsigargin sensitive pools; one opened by ATP acting through P(2)-purinergic receptors and IP(3), one opened by isoproterenol acting through beta2-adrenergic receptors, and a third pool not sensitive to ATP or isoproterenol.     

Effect of glucocorticoids on the glucose transport system of isolated fat cells.

Glucocorticoids inhibit glucose utilization by fat cells. The possibility that this effect results from altered glucose transport was investigated using an oil-centrifugation technique which allows a rapid (within 45 s) estimation of glucose or 3-O-methylglucose uptake by isolated fat cells. At high concentration (greater than 25 muM), dexamethasone inhibited glucose uptake within 1 min of its addition to fat cells. Efflux of 3-O-methylglucose was also impaired by 0.1 mM dexamethasone. However, diminished glucose uptake was not a specific effect of glucocorticoids; high concentrations (0.1 mM) of 17beta-estradiol, progesterone, and deoxycorticosterone produced a similar response in adipocytes. At a more physiologic steroid concentration (0.1 muM), glucocorticoids inhibited glucose uptake in a time-dependent manner (maximum effect in 1 to 2 hours). This effect was specific for glucocorticoids since, under these conditions, glucose uptake was not changed by the non-glucocorticoid steroids. Lineweaver-Burk analysis showed that 0.1 muM dexamethasone treatment produced a decrease in Vmax for glucose uptake but did not change the Ku. Hexokinase activity and ATP levels were not altered by this treatment, suggesting that processes involved in glucose phosphorylation were not affected. Dexamethasone treatment also caused a reduction in uptake of 3-O-methylglucose when assayed using a low sugar concentration (0.1 mM). At a high concentration (10 mM), uptake of the methyl sugar was only slightly less than normal in treated cells. Stimulation by insulin markedly enhanced uptake of glucose and 3-O-methylglucose by both treated and untreated cells. At a low hexose concentration (0.1 mM) and in the presence of insulin, sugar uptake by dexamethasone-treated cells was slightly less than control cells. Stimulation by insulin did however completely overcome the alteration in hexose uptake when larger concentrations of sugars (greater than 5 mM) were used. There was no detectable change in total protein synthesis during incubation of fat cells with dexamethasone. However, actinomycin C blocked the inhibitory effect of dexamethasone on glucose uptake. Cycloheximide, which caused a small inhibition in glucose uptake, prevented the full expression of the inhibitory effect of dexamethasone on glucose transport. These results indicate that dexamethasone alters the facilitated transport of glucose and, secondly, suggest that synthesis of RNA and protein is needed for glucocorticoid action.     

Dissociation of insulin-stimulated glucose transport from the translocation of glucose carriers in rat adipose cells

Cycloheximide, a potent inhibitor of protein synthesis, has been used to examine the relationship between recruitment of hexose carriers and the activation of glucose transport by insulin in rat adipocytes. Adipocytes were preincubated +/- cycloheximide for 90 min then +/- insulin for a further 30 min. We measured 3-O-methylglucose uptake in intact cells and in isolated plasma membrane vesicles. The concentration of glucose transporters in plasma membranes and low density microsomes was measured using a cytochalasin B binding assay. Cycloheximide had no affect on basal or insulin-stimulated 3-O-methylglucose uptake in intact cells or in plasma membrane vesicles. However, the number of glucose carriers in plasma membranes prepared from cells incubated with cycloheximide and insulin was markedly reduced compared to that from cells incubated with insulin alone (14 and 34 pmol/mg protein, respectively). Incubation of cells with cycloheximide alone did not change the concentration of glucose carriers in either plasma membranes or in low density microsomes compared to control cells. When isolated membranes were analyzed with an antiserum prepared against human erythrocyte glucose transporter, decreased cross-reactivity was observed in plasma membranes prepared from cycloheximide/insulin-treated cells compared to those from insulin cells. The present findings indicate that incubation of adipocytes with cycloheximide greatly reduces the number of hexose carriers in the plasma membrane of insulin-stimulated cells. Despite this reduction, insulin is still able to maximally stimulate glucose uptake. Thus, these data suggest an apparent dissociation between insulin stimulation of glucose transport activity and the recruitment of glucose carriers by the hormone.     

Effects of cellular ATP depletion on glucose transport and insulin signaling in 3T3-L1 adipocytes

Glucosamine induced insulin resistance in 3T3-L1 adipocytes, which was associated with a 15% decrease in cellular ATP content. To study the role of ATP depletion in insulin resistance, we employed sodium azide (NaN3) and dinitrophenol (DNP), which affect mitochondrial oxidative phosphorylation, to achieve a similar 15% ATP depletion. Unlike glucosamine, NaN3 and DNP markedly increased basal glucose transport, and the increased basal glucose transport was associated with increased GLUT-1 content in the plasma membrane without changes in total GLUT-1 content. These agents, like glucosamine, did not affect the early insulin signaling that is implicated in insulin stimulation of glucose transport. In cells with a severe 40% ATP depletion, basal glucose transport was similarly elevated, and insulin-stimulated glucose transport was similar in cells with 15% ATP depletion. In these cells, however, early insulin signaling was severely diminished. These data suggest that cellular ATP depletion by glucosamine, NaN3, and DNP exerts differential effects on basal and insulin-stimulated glucose transport and that ATP depletion per se does not induce insulin resistance in 3T3-L1 adipocytes.     

Effect of glucose on potassium contracture tension in rat heart

The effect of substrates on potassium contracture tension of the isolated rat ventricle strip was investigated. The contracture tension magnitude of ventricle strips exposed to potassium rich medium was markedly greater with medium containing pyruvate or acetate than with glucose as the substrate. The effect of substrates on contracture tension was not related to their ability to maintain the ATP stores of the heart, for there was not a significant difference in the ATP levels in ventricle strips incubated in medium containing pyruvate, pyruvate + glucose, or glucose. Glucose reduced the K-contracture tension magnitude of heart strips suspended in medium containing pyruvate or acetate as substrate; 3-0-methylglucose and 2-deoxy-D-glucose did not have this action on K-contracture tension. The reduction of K-contracture tension by glucose was inhibited by iodoacetate and fluoride. Under anaerobic conditions, 50 mM glucose significantly reduced the K-contracture tension of ventricle strips suspended in pyruvate medium. The findings of this study suggest that glucose metabolism has an action on K-contracture tension in cardiac muscle that is not shared by acetate or pyruvate.     

Subcellular distribution and phosphorylation state of insulin receptors from insulin- and isoproterenol-treated rat adipose cells

Rat adipose cells treated with insulin followed by isoproterenol exhibit a change in glucose transporter intrinsic activity (lowered maximal activity) and a decrease in insulin sensitivity (rightward shift of the concentration-response curve) when assayed for 3-O-methylglucose transport. To investigate the latter phenomenon, the distribution and phosphorylation state of insulin receptors was examined. Isoproterenol augmented the effect of insulin to reduce cell surface receptors by 20-30%. These receptors were recovered in microsomal fractions. Isoproterenol also markedly reduced insulin-stimulated [32P]phosphate incorporation into the plasma membrane receptor beta-subunit. These effects may account for the effect of isoproterenol to decrease the sensitivity of the glucose transport response to insulin.     

Ca2+ ions and the stimulation of 3-O-methylglucose transport by uncouplers in rat thymocytes.

The uptake of 3-O-methylglucose by rat thymocytes follows a biphasic time course. 2,4-Dinitrophenol (10-3 M), carbonyl cyanide m-chlorophenylhydrazone (10-5 M) and oligomycin (5 microgram/ml) each reduce ATP levels in rat thymocytes by 85% and bring about 3- to 4-fold stimulation of the slow phase of 3-O-methylglucose uptake. No consistent effect is observed on either the half-time of the rapid phase of uptake or the relative proportions of the two phases of uptake in the presence of these agents. Ca2+ ions do not appear to play a necessary role in the stimulation of transport activity since cells depleted of exchangeable Ca2+ by treatment with the Ca2+-Mg2+ ionophore, A23187, plus [ethylenebis(oxyethylenenitrilo)]tetraacetic acid respond to uncouplers in exactly the same manner as untreated cells. The effect of dinitrophenol on the slow phase of 3-O-methylglucose uptake is reversible after 10 min of incubation. After 60 min however, cells washed free of dinitrophenol and incubated at 37 degrees exhibited an additional acceleration in transport activity. This stimulation of transport is characterized by an increase in the proportion of the rapid phase of uptake with little or no change in its half-time. The results suggest that rat thymocytes regulate their 3-O-methylglucose transport activity in two distinct fashions.     

Alternate models for shared carriers or a single maturing carrier in hexose uptake into rabbit jejunum in vitro

The uptake (tissue accumulation) of three hexoses into rabbit jejunum was measured in a flux chamber in conditions of effective stirring. Glucose uptake was inhibited by galactose or 3-O-methylglucose: 1-40 mM galactose caused a progressive decline in glucose uptake; 1-5 mM 3-O-methylglucose inhibited glucose uptake but higher concentrations of 3-O-methylglucose had no further effect. When 1-40 mM 3-O-methylglucose was added to glucose plus galactose there was a further decrease in the uptake of glucose; adding 1-40 mM galactose to glucose plus 3-O-methylglucose also produced a decrease in glucose uptake. Both glucose and 3-O-methylglucose inhibited uptake of galactose but the pattern of inhibition varied between the two sugars. The uptake of 3-O-methylglucose was also inhibited by glucose and by galactose, but the uptake of 3-O-methylglucose in the presence of either galactose or glucose was no further reduced by adding the third hexose. Graphical analysis and analysis by non-linear regression both showed that neither the single Michaelis-Menten function, nor the single Michaelis-Menten-plus-competitive-inhibition function was appropriate for any of these data. The results are consistent with the hypothesis that either there are multiple (at least three) intestinal carriers for hexoses; alternatively that there is a single carrier whose transport properties for the three hexoses change differentially during cell maturation and migration up the villus.     

Preservation of metabolic reserves and function after storage of myocytes in hypothermic UW solution

Isolated rat myocytes coldstored anaerobically up to 24 h in University of Wisconsinsolution lost 95% of their ATP and 100% of their glycogen. Theyunderwent contracture when rewarmed in a Krebs-Henseleit (KH) mediumthat contained Ca unless Ca addition was delayed. In the latter case,cell function, measured by stimulation-induced cell shortening, wassurprisingly well retained. Aerobically stored cells were resistant toCa on rewarming, although 96% of glycogen was still lost, along with46% of ATP. Cells that were incubated for 48 h aerobically withthe substrates glucose and pyruvate at pH 6.2 retained 77% of theirATP and 59% of their glycogen, with good cell morphology. At pH 6.2, the demand for ATP was only 55% of that at pH 7.4. However, afterrewarming, these cells functioned no better than anaerobically storedcells, although their inotropic response to isoproterenol was improved.We conclude that 1) aerobic conditions with substrates atlow pH preserve myocyte metabolic reserves well for 48 h, partlyby reducing the demand for ATP; 2) rewarming conditions arecritical for anaerobically stored cells with metabolic stores that areseverely depleted; and 3) unloaded cell function issurprisingly insensitive to a period of severe metabolic deprivation.

     

Role of glycolysis in the relaxation process in mammalian cardiac muscle: comparison of the influence of glucose and 2-deoxyglucose on maintenance of resting tension

This study explores the effect of glucose on resting tension in mammalian cardiac muscle. Exogenous glucose (5 mM) ameliorates the increase in resting tension seen in severely hypoxic cardiac muscle. This effect of glucose is independent of any influence on total adenosine triphosphate (ATP) concentration, and cannot be duplicated with 2-deoxyglucose. Resting tension remains stable in muscles exposed to 1 mM iodoacetate (IAA) under aerobic conditions; contracture occurs rapidly when IAA exposed preparations are made severely hypoxic. These observations suggest that glycolysis is sufficient but not necessary for the maintenance of stable resting tension under our experimental conditions. While it is not clear from these data that glycolytically produced ATP is used preferentially, the possibility that a small compartment of ATP is supplied to the relaxing system is clearly suggested.     

A simple indirect colorimetric assay for measuring mitochondrial energy metabolism based on uncoupling sensitivity

PurposeCancer cells rapidly adjust their balance between glycolytic and mitochondrial ATP production in response to changes in their microenvironment and to treatments like radiation and chemotherapy. Reliable, simple, high throughput assays that measure the levels of mitochondrial energy metabolism in cells are useful determinants of treatment effects. Mitochondrial metabolism is routinely determined by measuring the rate of oxygen consumption (OCR). We have previously shown that indirect inhibition of plasma membrane electron transport (PMET) by the mitochondrial uncoupler, FCCP, may also be a reliable measure of mitochondrial energy metabolism. Here, we aimed to validate these earlier findings by exploring the relationship between stimulation of oxygen consumption by FCCP and inhibition of PMET.MethodsWe measured PMET by reduction of the cell impermeable tetrazolium salt WST-1/PMS. We characterised the effect of different growth conditions on the extent of PMET inhibition by FCCP. Next, we compared FCCP-mediated PMET inhibition with FCCP-mediated stimulation of OCR using the Seahorse XF96e flux analyser, in a panel of cancer cell lines.ResultsWe found a strong inverse correlation between stimulation of OCR and PMET inhibition by FCCP. PMET and OCR were much more severely affected by FCCP in cells that rely on mitochondrial energy production than in cells with a more glycolytic phenotype.ConclusionIndirect inhibition of PMET by FCCP is a reliable, simple and inexpensive high throughput assay to determine the level of mitochondrial energy metabolism in cancer cells.