Metabolism of glucose by unicellular blue-green algae

Summary A facultative photo- and chemoheterotroph, the unicellular bluegreen alga Aphanocapsa 6714, dissimilates glucose with formation of CO₂ as the only major product. A substantial fraction of the glucose consumed is assimilated and stored as polyglucose (probably glycogen). The oxidation of glucose proceeds through the pentose phosphate pathway. The first enzyme of this pathway, glucose-6-phosphate dehydrogenase, is partly inducible. In addition, the rate of glucose oxidation is controlled, at the level of glucose-6-phosphate dehydrogenase function, by the intracellular level of an intermediate of the Calvin cycle, ribulose-1,5-diphosphate, which is a specific allosteric inhibitor of this enzyme. As a consequence, the rate of glucose oxidation is greatly reduced by illumination, an effect reversed by the presence of DCMU, an inhibitor of photosystem II.Two obligate photoautotrophs, Synechococcus 6301 and Aphanocapsa 6308, produce CO₂ from glucose at extremely low rates, although their levels of pentose pathway enzymes and of hexokinase are similar to those in Aphanocapsa 6714. Failure to grow with glucose appears to reflect the absence of an effective glucose permease. A general hypothesis concerning the primary pathways of carbon metabolism in blue-green algae is presented.Abbreviations A (U)DPG ADP-glucose or UDP-glucose - G-1-P glucose-1-phosphate - G-6-P glucose-6-phosphate - G_(int.) intracellular glucose - F-6-P fructose-6-phosphate - 6-PG 6-phosphogluconate - Ru-5-P ribulose-5-phosphate - RUDP ribulose-1,5-diphosphate - PGA 3-phosphoglycerate - GAP glyceraldehyde-3-phosphate     

Continuous production of glucose-6-phosphate by immobilized Achromobacter butyri cells

Summary Whole cells of Achromobacter butyri OUT 8004 having polyphosphate glucokinase activity were immobilized in polyacrylamide gel. The immobilized cells were activated by organic solvents, especially acetone. The immobilization resulted in increased stability of polyphosphate glucokinase. Continuous high yield production of G-6-P from glucose and metaphosphate was performed with an immobilized cell column, which had a half-life of approximately 20 days.Abbreviations G-6-P glucose-6-phosphate - G-1-P glucose-1-phosphate - Cation-S stearyl trimethyl ammonium chloride - SDS sodium dodecyl sulfate - Tris tris(hydroxymethyl)-aminomethane; p-NPP, p-nitrophenyl phosphate - S.V. space velocity     

Muscle metabolite accumulation following maximal exercise. A comparison between short-term and prolonged kayak performance

Five elite flatwater kayak paddlers were studied during indoor simulated 500 and 10,000-m races, with performance times of 2 and 45 min, respectively. Muscle biopsies were obtained from the midportion of m. deltoideus immediately pre and post exercise. Concentrations of adenosine triphosphate (ATP), creatine phosphate (CP), glucose, glucose-6-phosphate (G-6-P), glycogen, and lactate were subsequently determined. Short term exercise resulted in statistically significant increases in glucose (P less than 0.001), G-6-P (P less than 0.05) and lactate (P less than 0.01) concentration concomitant with decreased CP (P less than 0.05) and glycogen (P less than 0.01). Following prolonged exercise, a non-significant elevation in glucose and a reduction (P less than 0.01) in glycogen were demonstrated. Evidently the metabolic demands for kayak competitions at 500 and 10,000 m are different. Thus, the energy contribution from glycolytic precursors and the anaerobic component is of greater relative importance in short distances than in exercise of long duration. A generalization of the findings to other athletic events of varying distances is proposed. The present data on arm-exercise is consistent with previous findings obtained in connection with leg exercises.     

Flux control in the rat gastrocnemius glycogen synthesis pathway by in vivo 13C/31P NMR spectroscopy

To determine the relative contributions of glucose transport/hexokinase, glycogen synthase (GSase), and glycolysis to the control of insulin-stimulated muscle glycogen synthesis, we combined 13C and 31P NMR to quantitate the glycogen synthesis rate and glucose 6-phosphate (G-6-P) levels in rat (Sprague-Dawley) gastrocnemius muscle during hyperinsulinemia at euglycemic (E) and hyperglycemic (H) glucose concentrations under thiopental anesthesia. Flux control was calculated using metabolic control analysis. The combined control coefficient of glucose transport/hexokinase (GT/Hk) for glycogen synthesis was 1.1 +/- 0.03 (direct measure) and 1.14-1.16 (calculated for a range of glycolytic fluxes), whereas the control coefficient for GSase was much lower (0.011-0.448). We also observed that the increase in in vivo [G-6-P] from E to H (0.22 +/- 0.03 to 0.40 +/- 0.03 mM) effects a supralinear increase in the in vitro velocity of GSase, from 14.6 to 26.1 mU. kg(-1). min(-1) (1.8-fold). All measurements suggest that the majority of the flux control of muscle glycogen synthesis is at the GT/Hk step.     

Uptake and phosphorylation of glucose and fructose in Daucus carota cell suspensions are differently regulated

Cell suspensions of Daucus carota L. were grown in batch culture on 50 mM sucrose, 100 mM glucose or 100 mM fructose. Sucrose was rapidly converted extra-cellularly into equimolar amounts of glucose and fructose, and glucose was then taken up preferentially. This impaired uptake of fructose could partially be explained by the eight-fold lower affinity of the hexose carrier in the plasmamembrane for fructose compared to glucose. However, cells grown on fructose as the sole carbon source showed a shorter lag phase and showed more biomass production compared to glucose-grown cells, indicating that conversion of glucose and fructose were also differently regulated. Ninety-five % of the glucose phosphorylating activity was membrane-associated and most probably confined to mitochondria; therefore, it might be present in a respiratory ‘compartment’ making glucose a better substrate for respiration than fructose. The soluble fraction contained the majority of the fructokinase activity. This activity was hypothesized to be more or less randomly distributed through the cytosol; in this soluble ‘compartment’ a pool of fructose-6-phosphate is formed. Concomitantly, via glucose-6-phosphate (G-6-P) and glucose-1-phosphate (G-1-P), it is converted into UDPG-glucose, resulting in structural cell components. The observed transient obstruction of the conversion of G-1-P into UDP-glucose in fructose-grown cells, leading to G-1-P accumulation, might be a result of both an altered equilibrium maintained by phosphoglucomutase, interconverting G-6-P and G-1-P and low levels of nucleotide triphosphates. Low nucleotide triphosphate production, connected with a low initial respiration rate, might be caused by the ten-fold lower affinity of the membrane-associated phosphorylating enzymes for fructose compared to glucose. Our results were taken to indicate that two separate pools of glycolytic intermediates exist in D. carota cells: one distributed throughout the cytosol and one surrounding the mitochondria.     

Kinetic analysis of glucose-6-phosphatase: an investigative approach to carbohydrate metabolism and kinetics

The enzyme glucose-6-phosphatase (G-6-Pase) catalyzes the hydrolysis of glucose-6-phosphate (G-6-P) to glucose. This is one of the key steps in gluconeogenesis and is critically important in maintaining stable blood glucose levels in most mammals. G-6-Pase is primarily found in the endoplasmic reticulum (ER) of hepatocytes and can easily be studied using isolated microsomes prepared from liver ER. A three-part undergraduate laboratory exercise uses rat liver microsomes to focus on the enzymatic analysis of G-6-Pase. The assessment of G-6-Pase activity is conducted using a stopped assay protocol combined with a colorimetric determination of inorganic phosphate (P_i) levels. The laboratory exercise was designed to carry out an independent inhibition investigation using orthovanadate, a competitive inhibitor of G-6-Pase with potential clinical importance. The format of the three-part investigation provides a useful mechanism for demonstrating enzyme kinetics and competitive inhibition using an enzyme that is important for carbohydrate metabolism and glycogen storage disease.     

Mechanism of muscle glycogen autoregulation in humans

To examine the mechanism by which muscle glycogen limits its own synthesis, muscle glycogen and glucose 6-phosphate (G-6-P) concentrations were measured in seven healthy volunteers during a euglycemic ( approximately 5.5 mM)-hyperinsulinemic ( approximately 450 pM) clamp using (13)C/(31)P nuclear magnetic resonance spectroscopy before and after a muscle glycogen loading protocol. Rates of glycogen synthase (V(syn)) and phosphorylase (V(phos)) flux were estimated during a [1-(13)C]glucose (pulse)-unlabeled glucose (chase) infusion. The muscle glycogen loading protocol resulted in a 65% increase in muscle glycogen content that was associated with a twofold increase in fasting plasma lactate concentrations (P < 0.05 vs. basal) and an approximately 30% decrease in plasma free fatty acid concentrations (P < 0.001 vs. basal). Muscle glycogen loading resulted in an approximately 30% decrease in the insulin-stimulated rate of net muscle glycogen synthesis (P < 0.05 vs. basal), which was associated with a twofold increase in intramuscular G-6-P concentration (P < 0.05 vs. basal). Muscle glycogen loading also resulted in an approximately 30% increase in whole body glucose oxidation rates (P < 0.05 vs. basal), whereas there was no effect on insulin-stimulated rates of whole body glucose uptake ( approximately 10.5 mg. kg body wt(-1). min(-1) for both clamps) or glycogen turnover (V(syn)/V(phos) was approximately 23% for both clamps). In conclusion, these data are consistent with the hypothesis that glycogen limits its own synthesis through feedback inhibition of glycogen synthase activity, as reflected by an accumulation of intramuscular G-6-P, which is then shunted into aerobic and anaerobic glycolysis.     

Control of glycogen content in retina: allosteric regulation of glycogen synthase

Retinal tissue is exceptional because it shows a high level of energy metabolism. Glycogen content represents the only energy reserve in retina, but its levels are limited. Therefore, elucidation of the mechanisms controlling glycogen content in retina will allow us to understand retina response under local energy demands that can occur under normal and pathological conditions. Thus, we studied retina glycogen levels under different experimental conditions and correlated them with glucose-6-phosphate (G-6-P) content and glycogen synthase (GS) activity. Glycogen and G-6-P content were studied in ex vivo retinas from normal, fasted, streptozotocin-treated, and insulin-induced hypoglycemic rats. Expression levels of GS and its phosphorylated form were also analyzed. Ex vivo retina from normal rats showed low G-6-P (14±2 pmol/mg protein) and glycogen levels (43±3 nmol glycosyl residues/mg protein), which were increased 6 and 3 times, respectively, in streptozotocin diabetic rats. While no changes in phosphorylated GS levels were observed in any condition tested, a positive correlation was found between G-6-P levels with GS activity and glycogen content. The results indicated that in vivo, retina glycogen may act as an immediately accessible energy reserve and that its content was controlled primarily by G-6-P allosteric activation of GS. Therefore, under hypoglycemic situations retina energy supply is strongly compromised and could lead to the alterations observed in type 1 diabetes.     

Regulation of pyruvate kinase from Propionibacterium shermanii

Pyruvate kinase from Propionibacterium shermanii was shown to be activated by glucose-6-phosphate (G-6-P) at non-saturating phosphoenol pyruvate (PEP) concentrations but other glycolytic and hexose monophosphate pathway intermediates and AMP were without effect. Half-maximal activation was obtained at 1 mM G-6-P. The presence of G-6-P decreased both the PEP_0.5V and ADP_0.5V values and the slope of the Hill plots for both substrates. The enzyme was strongly inhibited by ATP and inorganic phosphate (P_i) at all PEP concentrations. At non-saturating (0.5 mM) PEP, half-maximal inhibition was obtained at 1.8 mM ATP or 1.4 mM P_i. The inhibition by both P_i and ATP was largely overcome by 4 mM G-6-P. The specific activity of pyruvate kinase was considerably higher in lactate-, glucose- and glycerol-grown cultures than that of the enzyme catalysing the reverse reaction, pyruvate, phosphate dikinase. It is suggested that the activity of pyruvate kinase in vivo is determined by the balance between activators and inhibitors such that it is inhibited during gluconeogenesis while, during glycolysis, the inhibition is relieved by G-6-P.Abbreviations PEP phosphoenolpyruvate - G-6-P glucose-6-phosphate - P_i inorganic phosphate     

The effects of exogenous glucose, uracil, and inorganic phosphate on differentiation in Dictyostelium discoideum.

The cellular slime mold was exposed to exogenous glucose, uracil, and inorganic phosphate for either 900 or 90 min to determine their effects on the cellular levels of glucose 6-phosphate (glucose-6-P), UDP-glucose, glycogen, trehalose, and cellulose. Glucose, and phosphate to a lesser extent, increase the levels of glucose-6-P and trehalose, whereas glycogen levels are increased only by glucose. Uracil inhibits glucose-6-P and trehalose accumulation, and this inhibition is reversed by glucose or phosphate. Uracil, especially in the presence of glucose, stimulates the accumulation of UDP-glucose and cellulose. In an attempt to understand the dynamics of the biochemical mechanisms underlying these experimental observations, fluxes of the same metabolites were imposed on a kinetic model of this system. The effects of glucose, uracil, and phosphate either singly or in various combinations on the accumulation of glycogen and trehalose can be predicted quantitatively by applying the appropriate external flux(es) of these additives to the model; the predicted effects on glucose-6-P levels are qualitatively consistent with the observations, but are greater in magnitude, suggesting compartmentation of glucose-6-P. Matching the observed and simulated results requires a lower level of additive in the simulated system than in the actual experiment, which is consistent with earlier studies on the cellular permeability of these metabolites.It is concluded that the complex of flux changes induced in the model by the perturbing metabolites may also occur in vivo, and that endogenous glucose availability is a critical variable controlling the rate and cessation of differentiation as well as the relative amounts of the saccharide end products of differentiation.     

Inactivation of the phosphoglucomutase gene pgm in Corynebacterium glutamicum affects cell shape and glycogen metabolism

In Corynebacterium glutamicum formation of glc-1-P (α-glucose-1-phosphate) from glc-6-P (glucose-6-phosphate) by α-Pgm (phosphoglucomutase) is supposed to be crucial for synthesis of glycogen and the cell wall precursors trehalose and rhamnose. Furthermore, Pgm is probably necessary for glycogen degradation and maltose utilization as glucan phosphorylases of both pathways form glc-1-P. We here show that C. glutamicum possesses at least two Pgm isoenzymes, the cg2800 (pgm) encoded enzyme contributing most to total Pgm activity. By inactivation of pgm we created C. glutamicum IMpgm showing only about 12% Pgm activity when compared to the parental strain. We characterized both strains during cultivation with either glucose or maltose as substrate and observed that (i) the glc-1-P content in the WT (wild-type) and the mutant remained constant independent of the carbon source used, (ii) the glycogen levels in the pgm mutant were lower during growth on glucose and higher during growth on maltose, and (iii) the morphology of the mutant was altered with maltose as a substrate. We conclude that C. glutamicum employs glycogen as carbon capacitor to perform glc-1-P homeostasis in the exponential growth phase and is therefore able to counteract limited Pgm activity for both anabolic and catabolic metabolic pathways.     

Effects of hyperglycemia on hepatic gluconeogenic flux during glycogen phosphorylase inhibition in the conscious dog

The aim of these studies was to investigate the effect of hyperglycemia with or without hyperinsulinemia on hepatic gluconeogenic flux, with the hypothesis that inhibition would be greatest with combined hyperglycemia/hyperinsulinemia. A glycogen phosphorylase inhibitor (BAY R3401) was used to inhibit glycogen breakdown in the conscious overnight-fasted dog, and the effects of a twofold rise in plasma glucose level (HI group) accompanied by 1) euinsulinemia (HG group) or 2) a fourfold rise in plasma insulin were assessed over a 5-h experimental period. Hormone levels were controlled using somatostatin with portal insulin and glucagon infusion. In the HG group, net hepatic glucose uptake and net hepatic lactate output substantially increased. There was little or no effect on the net hepatic uptake of gluconeogenic precursors other than lactate (amino acids and glycerol) or on the net hepatic uptake of free fatty acids compared with the control group. Consequently, whereas hyperglycemia had little effect on gluconeogenic flux to glucose 6-phosphate (G-6-P), net hepatic gluconeogenic flux was reduced because of increased hepatic glycolytic flux during hyperglycemia. Net hepatic glycogen synthesis was increased by hyperglycemia. The effect of hyperglycemia on gluconeogenic flux to G-6-P and net hepatic gluconeogenic flux was similar. We conclude that, in the absence of appreciable glycogen breakdown, the increase in glycolytic flux that accompanies hyperglycemia results in decreased net carbon flux to G-6-P but no effect on gluconeogenic flux to G-6-P.     

Insulin activation of glycogen synthase in cultured human fibroblasts is not mediated solely via the insulin receptor

Insulin binds to its specific cell surface receptor in cultured human fibroblasts and also stimulates the conversion of glycogen synthase from the glucose-6-phosphate (G-6-P) dependent to the G-6-P independent form. Although these two processes are tightly coupled in most target tissues for insulin action, in the fibroblast a variety of findings question the relationship of these two events to one another. In human fibroblasts the amount of insulin required to displace half of the 125I-insulin bound to the insulin receptor is 4 ng/ml (6.6 X 10(-10)M), but the activation of glycogen synthase is not maximal until 1-10 micrograms/ml with an ED50 of 30 ng/ml insulin. Antibodies directed against the insulin receptor, which activate glycogen synthase in both fat and muscle, do not stimulate the activation of glycogen synthase in the fibroblast. Fab fragments from anti-insulin receptor antibody compete for insulin binding, but do not inhibit the insulin-stimulated rise in independent activity. The insulin-like growth factor, MSA, which is 1% as potent as insulin in stimulating glucose oxidation in rat fat cells and in inhibiting 125I-insulin binding to human fibroblasts, is 25% as potent as insulin in stimulating glycogen synthase. Proinsulin is 2-10% as potent as insulin, but behaves as a "partial agonist" of insulin action in the fibroblast, i.e. proinsulin is able to elicit only 60% of the maximal response of insulin in the glycogen synthase assay, even at high concentrations. Finally, cell lines from patients with clearly defective insulin receptors exhibit normal insulin dose response curves for the activation of glycogen synthase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Brain contains a functional glucose-6-phosphatase complex capable of endogenous glucose production

Glucose is absolutely essential for the survival and function of the brain. In our current understanding, there is no endogenous glucose production in the brain, and it is totally dependent upon blood glucose. This glucose is generated between meals by the hydrolysis of glucose-6-phosphate (Glc-6-P) in the liver and the kidney. Recently, we reported a ubiquitously expressed Glc-6-P hydrolase, glucose-6-phosphatase-beta (Glc-6-Pase-beta), that can couple with the Glc-6-P transporter to hydrolyze Glc-6-P to glucose in the terminal stages of glycogenolysis and gluconeogenesis. Here we show that astrocytes, the main reservoir of brain glycogen, express both the Glc-6-Pase-beta and Glc-6-P transporter activities and that these activities can couple to form an active Glc-6-Pase complex, suggesting that astrocytes may provide an endogenous source of brain glucose.     

The role of acetate in alcohol-induced alterations of uterine glucose metabolism in the mouse during pregnancy

The acute exposure of mice to ethanol during post-implantation pregnancy has been reported to cause alterations in the levels of several glycolytic intermediates in the uterus, suggesting a possible indirect mechanism of alcohol embryo-toxicity. The present study was undertaken to assess whether the ethanol metabolite, acetate is implicated in this phenomenon. Blood and uterine alcohol concentrations in day 9--pregnant Quackenbush Swiss mice were maximal 15 minutes after the intraperitoneal injection of ethanol (3.5 g/kg body weight), and fell to almost negligible levels 6 hours later. In response to this treatment, the levels of blood and uterine acetate increased, liver glycogen decreased, plasma glucose increased, and uterine glucose, glucose-6-phosphate (G-6-P), fructose-6-phosphate (F-6-P), and citrate increased. When acetate was administered to pregnant mice in amounts approximating those generated by exposure to alcohol, the levels of uterine F-6-P and citrate increased while other metabolic parameters remained unaffected. The administration of 4-methylpyrazole to mice subsequently treated with alcohol produced conditions of alcohol exposure in the absence of ethanol-derived acetate and depressed the ethanol-induced rise in uterine G-6-P and citrate. The results support the notion that acetate contributes to the alcohol-induced alterations in metabolism, at least as far as the regulation of uterine citrate and hexose monophosphates are concerned. This, together with stress responses induced by exposure to the acute dose of alcohol, may present mechanisms underlying the fetal alcohol syndrome associated in particular with "binge" drinking.     

Loss of the major isoform of phosphoglucomutase results in altered calcium homeostasis in Saccharomyces cerevisiae

Phosphoglucomutase (PGM) is a key enzyme in glucose metabolism, where it catalyzes the interconversion of glucose 1-phosphate (Glc-1-P) and glucose 6-phosphate (Glc-6-P). In this study, we make the novel observation that PGM is also involved in the regulation of cellular Ca(2+) homeostasis in Saccharomyces cerevisiae. When a strain lacking the major isoform of PGM (pgm2Delta) was grown on media containing galactose as sole carbon source, its rate of Ca(2+) uptake was 5-fold higher than an isogenic wild-type strain. This increased rate of Ca(2+) uptake resulted in a 9-fold increase in the steady-state total cellular Ca(2+) level. The fraction of cellular Ca(2+) located in the exchangeable pool in the pgm2Delta strain was found to be as large as the exchangeable fraction observed in wild-type cells, suggesting that the depletion of Golgi Ca(2+) stores is not responsible for the increased rate of Ca(2+) uptake. We also found that growth of the pgm2Delta strain on galactose media is inhibited by 10 microM cyclosporin A, suggesting that activation of the calmodulin/calcineurin signaling pathway is required to activate the Ca(2+) transporters that sequester the increased cytosolic Ca(2+) load caused by this high rate of Ca(2+) uptake. We propose that these Ca(2+)-related alterations are attributable to a reduced metabolic flux between Glc-1-P and Glc-6-P due to a limitation of PGM enzymatic activity in the pgm2Delta strain. Consistent with this hypothesis, we found that this "metabolic bottleneck" resulted in an 8-fold increase in the Glc-1-P level compared with the wild-type strain, while the Glc-6-P and ATP levels were normal. These results suggest that Glc-1-P (or a related metabolite) may participate in the control of Ca(2+) uptake from the environment.     

Glycogen Metabolism in Neonatal Rat Brain During Anoxia and Recovery

Abstract: Metabolic alterations in glycogen and in glycogen-related metabo lites were studied in neonatal rat brain during controlled anoxia and recovery. One-day postnatal rats were exposed to 100% N, at 37°C for up to 20 min; some rats were allowed to recover in air. Animals were frozen in liquid N, and the brains were prepared for fluorometric analysis of compounds involved in glycogen turnover. During anoxia, glycogen decreased by 29% and 42% at 10 and 20 min, respectively; the free (soluble) and bound (insoluble) components of glycogen decreased in nearly equal proportions. Brain glucose decreased by 72% at 10 min with little further change there after; G-6-P, G-1-P, and UDPG also declined. During recovery from anoxia, glucose and G-6-P increased above control levels for up to 60 min. G-1-P paralleled G-6-P levels, but UDPG remained low. Glycogen returned to control values by 4 h. The findings suggest that although glycogen is mobilized slowly in newborn rat brain, the metabolite contributes at least one-third of the cerebral energy supply during anoxia. Presumably, readily available stores of glycogen combined with low cerebral metabolic requirements underscore the known tolerence of immature animals to hypoxic stress. Glycogen accumulation during recovery appears to be facilitated at the synthetase step, since equilibrium measurements of the phosphoglucomutase and pyrophosphorylase systems indicate that these reactions are not rate-limiting for glycogen synthesis.     

Effects of high-fat diet and exercise training on intracellular glucose metabolism in rats

We examined the effects of high-fat diet (HFD) and exercise training on insulin-stimulated whole body glucose fluxes and several key steps of glucose metabolism in skeletal muscle. Rats were maintained for 3 wk on either low-fat (LFD) or high-fat diet with or without exercise training (swimming for 3 h per day). After the 3-wk diet/exercise treatments, animals underwent hyperinsulinemic euglycemic clamp experiments for measurements of insulin-stimulated whole body glucose fluxes. In addition, muscle samples were taken at the end of the clamps for measurements of glucose 6-phosphate (G-6-P) and GLUT-4 protein contents, hexokinase, and glycogen synthase (GS) activities. Insulin-stimulated glucose uptake was decreased by HFD and increased by exercise training (P < 0.01 for both). The opposite effects of HFD and exercise training on insulin-stimulated glucose uptake were associated with similar increases in muscle G-6-P levels (P < 0.05 for both). However, the increase in G-6-P level was accompanied by decreased GS activity without changes in GLUT-4 protein content and hexokinase activities in the HFD group. In contrast, the increase in G-6-P level in the exercise-trained group was accompanied by increased GLUT-4 protein content and hexokinase II (cytosolic) and GS activities. These results suggest that HFD and exercise training affect insulin sensitivity by acting predominantly on different steps of intracellular glucose metabolism. High-fat feeding appears to induce insulin resistance by affecting predominantly steps distal to G-6-P (e.g., glycolysis and glycogen synthesis). Exercise training affected multiple steps of glucose metabolism both proximal and distal to G-6-P. However, increased muscle G-6-P levels in the face of increased glucose metabolic fluxes suggest that the effect of exercise training is quantitatively more prominent on the steps proximal to G-6-P (i.e., glucose transport and phosphorylation).