Effect of interleukin 1alpha and interleukin 2 over glucose metabolism in isolated uterus of restricted diet rats. Influence of NO and COX-2

A 25-day dietary restriction (50% of the normal diet) produce a fall in the production of 14CO2 from 14C-glucose in rats isolated uteri. The addition of 10 or 20 ngml(-1) interleukin 1alpha (IL-1alpha) or interleukin 2(IL-2) to the Krebs-Ringer bicarbonate solution medium stimulates glucose metabolism in uteri from underfed rats. Such concentrations are not effective in control rats. The addition of Nomega-nitro-L arginine methyl ester--an inhibitor of both the constitutive and inducible forms of nitric oxide synthase (NOS)--and of aminoguadinine--a preferential inhibitor of the inducible form of NOS--block such stimulation. In other experiments, the addition to the medium of arginine-a substrate for the formation of nitric oxide-increases interleukin stimulation of glucose metabolism, which is blocked by NOS inhibitor. At the same time, NS-398--a selective inhibitor of inducible cyclooxygenase (COX)--eliminates the interleukin metabolism stimulation. We conclude that IL-1alpha and IL-2 produce an increase of glucose metabolism in uteri isolated from underfed rats. Nitric oxide produced by the inducible form of NOS mediates the interleukins-induced glucose metabolism stimulation with the participation of inducible COX.     

Opioid effects on glucose and eicosanoid metabolism in isolated uterus of ovariectomized and non-ovariectomized restricted diet rats.

The effect of a 25-day restricted diet (50% of the normal food intake) on uterine glucose metabolism of ovariectomized (25 days) and non-ovariectomized rats, was studied. Underfeeding reduces (14)CO(2) production from U(14)C-glucose in intact animal. However, in spayed rats, results are the opposite. In intact rats receiving a low food intake, the effect of the addition to the KRB medium of various agonist opioids, was studied. Dinorphin A did not bring about any change. On the other hand, beta endorphin increased glucose metabolism. Also, the addition of Dago and Dadle increased (14)CO(2) production, while their corresponding specific blockers, beta-FNA and Naltrindole, reversed it. Ovariectomized rats subjected to food restriction are not affected by opioid agonists. In vitro morphine, like endogenous opioids, increased (14)CO(2) in intact restricted diet rats. Arachidonic acid metabolism in these rats show that underfeeding brings about a decrease in PGF(2 alpha) and PGE(2), but the addition of morphine does not alter this situation, for which eicosanoids metabolites are not related to the effect of morphine. The morphine effect was not altered by naloxone. The subcutaneous injection of morphine increased glucose metabolism in intact underfed animals, while naloxone reduced (14)CO(2) in spayed rats subjected to underfeeding. It can be concluded that uteri from ovariectomized rats receiving a restricted diet are influenced by a mechanism of upregulation related to endogenous opioids. These likely originate in other tissues, and so prevent us from seeing the morphine effect.     

Stimulation of arachidonic acid metabolism: differences in potencies of recombinant human interleukin-1 alpha and interleukin-1 beta on two cell types

Recombinant human interleukin-l (rIL-1) alpha and beta, which have 26% homology in their amino acid sequence, stimulated arachidonic acid metabolism by squirrel monkey smooth muscle cells and rat liver cells; their relative effectiveness, however, varied with the two cells. Recombinant IL-1 alpha was 3 times more effective than rIL-1 beta at stimulating arachidonic acid metabolism by the primate smooth muscle cells. Recombinant IL-1 alpha was 3 times less effective than rIL-1 beta when measured by their capacity to synergistically stimulate arachidonic acid metabolism of rat liver cells in the presence of palytoxin and anti-diuretic hormone (ADH). The rIL-1 alpha and rIL-1 beta also stimulated the release of radiolabelled arachidonic acid from the smooth muscle cells prelabelled with [3H]arachidonic acid. The two recombinant IL-1s have different heat stabilities, again when measured by their capacity to stimulate arachidonic acid metabolism; IL-1 alpha was more heat stable than IL-1 beta.     

Vasoactive prostanoids are generated from arachidonic acid by COX-1 and COX-2 in the mouse

Generation of vasoactive prostanoids from arachidonic acid by cyclooxygenase (COX)-1 and COX-2 was investigated in anesthetized mice. Intravenous injections of the prostanoid precursor arachidonic acid increased pulmonary arterial pressure and decreased systemic arterial pressure. Pulmonary pressor and systemic depressor responses were attenuated by SC-560 and nimesulide, inhibitors of COX-1 and COX-2, in doses that did not alter responses to injected prostanoids. Pulmonary pressor responses to arachidonic acid were blocked and a depressor response was unmasked, whereas systemic depressor responses were not altered, by a thromboxane receptor antagonist. Pulmonary and systemic pressor responses to angiotensin II injections and systemic pressor responses to angiotensin II infusion were not modified by COX-1 or COX-2 inhibitors but were attenuated by losartan. Systemic depressor responses to arachidonic acid were smaller in COX-1 and COX-2 knockout mice, whereas responses to angiotensin II, norepinephrine, U-46619, endothelin-1, and PGE(1) were not different in COX-1 and COX-2 knockout and wild-type control mice. These results suggest that vasoactive prostanoids with pulmonary pressor and systemic vasodepressor activity are formed by COX-1 and COX-2 and are consistent with Western blot analysis and immunostaining showing the presence of COX-1 and COX-2. These data suggest that thromboxane A(2) (TxA(2)) is formed from the precursor by COX-1 and COX-2 in the lung and are in agreement with immunofluorescence studies showing thromboxane synthase. The present data suggest that COX-1- or COX-2-derived prostanoids do not modulate responses to angiotensin II or other vasoactive agents and that prostanoid responses are similar in CD-1 and C57BL/6 and in male and female mice.     

Influence of arachidonic acid metabolites on cardiac alpha 1-adrenoceptor responses in diabetic rats.

There is evidence that one or more metabolites of arachidonic acid can produce positive inotropic effects and may also be implicated in the enhanced alpha 1-adrenoceptor responses in hearts from diabetic rats. We therefore carried out a study to investigate the possibility that arachidonic acid metabolites could be involved in the altered cardiac alpha 1-effect of norepinephrine (in the presence of propranolol) in chronic streptozotocin-diabetic rats. Our results have shown that in the presence of the cyclooxygenase inhibitor indomethacin or the thromboxane synthetase inhibitor imidazole, the norepinephrine-stimulated positive inotropic effect and the formation of inositol 1,4,5-trisphosphate were significantly increased in control hearts but were unaltered in hearts from diabetic rats. The addition of the prostacyclin synthetase inhibitor tranylcypromine reduced the norepinephrine-stimulated positive inotropic effect and inositol 1,4,5-trisphosphate formation only in diabetic hearts and had no effect in the controls. The nature and physiological significance of the enhanced positive inotropic effect and inositol 1,4,5-trisphosphate formation in the control heart with the addition of indomethacin and imidazole are still unclear. The effect of tranylcypromine may indicate the participation of prostacyclin in mediating the enhanced alpha 1-inotropic effect of norepinephrine in the chronic diabetic heart.     

Effects of arachidonic acid and prostaglandin F2 alpha in isolated rat lungs

Isolated rat lungs were ventilated and perfused by saline-Ficoll perfusate at a constant flow. The baseline perfusion pressure (PAP) correlated with the concentration of 6-keto-PGF1 alpha the stable metabolite of PGI2 (r = 0.83) and with the 6-keto-PGF1 alpha/TXB2 ratio (r = 0.82). A bolus of 10 micrograms exogenous arachidonic acid (AA) injected into the arterial cannula of the isolated lungs caused significant decrease in pulmonary vascular resistance (PVR) which was followed by a progressive increase of PVR and edema formation. Changes in perfusion pressure induced by AA injection also correlated with concentrations of the stable metabolites (6-keto-PGF1 alpha: r = -0.77, TxB2: -0.76), and their ratio: (6-keto-PGF1 alpha/TXB2: r = -0.73). Injection of 10 and 100 micrograms of PGF2 alpha into the pulmonary artery stimulated the dose-dependent production of TXB2 and 6-keto-PGF1 alpha. No significant correlations were found between the perfusion pressure (PAP) which was increased by the PGF2 alpha and the concentrations of the former stable metabolites. The results show that AA has a biphasic effect on the isolated lung vasculature even in low dose. The most potent vasoactive metabolites of cyclooxygenase, prostacyclin and thromboxane A2 influence substantially not only the basal but also the increased tone of the pulmonary vessels.     

Effects of morphine and naloxone on glucose metabolism in uterine strips from ovariectomized and non-ovariectomized restricted diet rats

The effect of underfeeding over glucose metabolism in uteri isolated from ovariectomized and non-ovariectomized rats subjected to a restricted diet for 25 days (50% of the normal food intake), was studied. Underfeeding decreases (14)CO(2) formation from U(14) C-glucose in intact animal uteri. While in ovariectomized rats (25 days), the effect is the opposite. The addition of morphine 10(-6) M to the medium does not affect rats fed ad libitum. However, (14)CO(2) levels increase significantly in intact animals receiving a restricted diet. In ovariectomized rats morphine does not show any activity, regardless of the type of diet rats were subjected to. None of the rat groups seems to be sensitive to naloxone 10(-6) M. The s.c. injection of morphine (4 mg.kg (-1)) increases glucose metabolism only in intact rats provided with a restricted diet, while naloxone (2.5 mg.kg (-1) ) produces a decrease of ( 14)CO(2) in ovariectomized underfed animals. To conclude, morphine either 'in vivo' or 'in vitro' is active only in uteri from intact rats subjected to underfeeding. Naloxone produces a decrease in (14)CO(2) production, particularly when it is s.c. injected to ovariectomized rats undergoing a dietary restriction. Since the uterus does not react to naloxone, the effect of the opiod blocker may be the result of endogenous opioids originated in other tissues.     

Molecular dynamics simulations of arachidonic acid complexes with COX-1 and COX-2: insights into equilibrium behavior

The cyclooxygenase (COX) enzymes are responsible for the committed step in prostaglandin biosynthesis, the generation of prostaglandin H(2). As a result, these enzymes are pharmacologically important targets for nonsteroidal antiinflammatory drugs, such as aspirin and newer COX-2 selective inhibitors. The cyclooxygenases are functional homodimers, and each subunit contains both a cyclooxygenase and a peroxidase active site. These enzymes are quite interesting mechanistically, as the conversion of arachidonic acid to prostaglandin H(2) requires two oxygenation and two cyclization reactions, resulting in the formation of five new chiral centers with nearly absolute regio- and stereochemical fidelity. We have used molecular dynamics (MD) simulations to investigate the equilibrium behavior of both COX-1 and COX-2 enzyme isoforms with bound arachidonate. These simulations were compared with reference simulations of arachidonate in solution to explore the effect of enzyme on substrate conformation and positioning in the active site. The simulations suggest that the substrate has greater conformational freedom in the COX-2 active site, consistent with the larger COX-2 active site volume observed in X-ray crystal structures. The simulations reveal different conformational behavior for arachidonate in each subunit over the course of extended equilibrium MD simulations. The simulations also provide detailed information for several protein channels that might be important for oxygen and water transport to or from active sites or for intermediate trafficking between the cyclooxygenase and peroxidase active sites. The detailed comparisons for COX-1 versus COX-2 active site structural fluctuations may also provide useful information for design of new isozyme-selective inhibitors.     

The role of arachidonic acid metabolism in the activities of interleukin 1 and 2

Several investigations have suggested that products of arachidonic acid metabolism have modulatory effects on the development of cellular immunity. In this report we have studied the role of arachidonic acid metabolism in the specific effects of interleukin 1 (IL 1) induction of interleukin 2 (IL 2), and also IL 2 stimulation of proliferation and interferon-gamma (IFN-gamma) production. Utilizing cell lines that are specifically responsive to IL 1 or IL 2, it was found that both interleukins stimulate lipoxygenation of arachidonic acid in their respective target cell. The ability of each interleukin to induce monohydroxyeicosatetraenoic acid (HETE) correlated with the induction of secondary lymphokine secretion. Utilizing selective and partially selective pharmacologic inhibitors of arachidonic acid metabolism, the data suggest that the participation of lipoxygenase activity is required for both IL 1 induction of IL 2 production and IL 2 regulation of proliferation and IFN-gamma secretion. The same requirement for lipoxygenase activity was seen when phorbol myristate acetate (PMA) was used as a secretory stimulant, suggesting a similar mode of action for stimulation-secretory activity between PMA and interleukins. Studies performed with an endogenous inhibitor of 5-lipoxygenase (15-HETE) demonstrated the requirement of this enzyme system for IL 2-dependent proliferation and IFN-gamma production. Although leukotrienes could replace IL 2 for IFN-gamma secretion, they had no effect on IL 2 growth promotion. The results suggest that both IL 1 and IL 2, and PMA, may share the lipoxygenase pathway of arachidonic acid metabolism which is a component of the intracellular signal transduction process that regulates secretory activity and/or cellular proliferation.     

The metabolism of arachidonic acid in isolated perfused fetal and neonatal rabbit lungs

The developmental pattern of fetal and neonatal rabbit lungs to metabolize arachidonic acid (AA) to different cyclo-oxygenase products was studied in isolated rabbit lungs, which were perfused with Krebs bicarbonate buffer. 14C-AA (66 nmol) was injected into the pulmonary circulation and the nonrecirculating perfusion effluent was collected for four minutes. About ten per cent of the injected radioactivity was found in the 0-4 min perfusion effluent. The metabolites of AA in the effluent were analyzed by thin layer chromatography. The major metabolites of AA were PGE2 and its 15-keto-derivates, but also PGF2 alpha and its 15-keto-derivates, TXB2 and 6-keto-PGF1 alpha were found in the effluent. The most drastic developmental change was the increase in the amount of 15-keto-metabolites of PGE2 from late fetal period to the lungs of one day old rabbits (1.8 fold increase between birth and first postnatal day). Smaller changes were detected in the amounts of other cyclo-oxygenase products.     

The metabolism of arachidonic acid in isolated perfused fetal and neonatal rabbit lungs

The developmental pattern of fetal and neonatal rabbit lungs to metabolize arachidonic acid (AA) to different cyclo-oxygenase products was studied in isolated rabbit lungs, which were perfused with Krebs bicarbonate buffer. ¹⁴C-AA (66 nmol) was injected into the pulmonary circulation and the nonrecirculating perfusion effluent was collected for four minutes. About ten per cent of the injected radioactivity was found in the 0–4 min perfusion effluent. The metabolites of AA in the effluent were analyzed by thin layer chromatography. The major metabolites of AA were PGE₂ and its 15-keto-derivates, but also PGF_2α and its 15-keto-derivates, TXB₂ and 6-keto-PGF_1α were found in the effluent. The most drastic developmental change was the increase in the amount of 15-keto-metabolites of PGE₂ from late fetal period to the lungs of one day old rabbits (1.8 fold increase between birth and first postnatal day). Smaller changes were detected in the amounts of other cyclo-oxygenase products.     

Stimulation of arachidonic acid metabolism by CCl4 in isolated rat hepatocytes

Arachidonic acid metabolism was evaluated in isolated rat hepatocytes after CCl4 exposure. CCl4 induced dose-dependently the synthesis and release of prostacyclin (PGI2) and thromboxane (TXB2). Treatment with prostaglandin E2 (PGE2) 30 min after exposure to CCl4, significantly reduced the cell damage as well as the release of TXB2 from the cells.     

Aspirin inhibits arachidonic acid metabolism via lipoxygenase and cyclo-oxygenase in hamster isolated lungs

The effect of aspirin on the fate of exogenous arachidonic acid (AA) was investigated in isolated perfused lungs of female hamsters. During pulmonary infusion of aspirin (10 μM, 100 μM or 1 mM) 45 nmol of ¹⁴C-AA was infused in two minutes into the pulmonary circulation. The nonrecirculating perfusion effluent was collected for 6 minutes after the beginning of the AA infusion. Arachidonate infusion increased the perfusion pressure. This pressor response was completely abolished by 1 mM aspirin. When aspirin was infused into the pulmonary circulation, the amount of radioactivity was increased in the perfused lungs and decreased dose dependently in the nonrecirculating perfusion effluent. The amount of unmetabolized free arachidonate was not changed significantly by aspirin in the perfused lungs or in the perfusion effluent. In the effluent the amounts of all arachidonate metabolites, which were extracted with ethyl acetate first at pH 7.4 and then at pH 3.5 and analysed by thin layer chromatography, were decreased quite similarly by aspirin. The formation of arachidonate metabolites was completely inhibited by 1 mM aspirin. In the perfused lung tissue the amount of ¹⁴C-AA was increased by aspirin in phospholipids and neutral lipids. The present study indicates that the metabolism of arachidonic acid is inhibited by aspirin in hamster lungs not only via cyclo-oxygenase but also via other lipoxygenases.     

The role of nitric oxide in the metabolism of labeled glucose in isolated rat uterus. Influence of 17β-estradiol

The influence of nitric oxide (NO) on the production of ¹⁴CO₂ from labeled glucose in uteri isolated from ovariectomized-estrogenized rats was studied. Nitroprusside, an NO donor (NP), 200 μM increased the formation of labeled CO₂ from [U-¹⁴C]glucose. This effect was blunted by hemoglobin (Hb) 20 μg/mL, an NO scavenger. The addition of N-monomethyl arginine (NMMA), an inhibitor of NO synthase decreased the stimulatory action of NP at 400 mM. Incubation of uterine strips in the presence of NP plus acetylsalicylic acid (ASA) 10⁻⁴ M (a cyclooxygenase inhibitor), inhibited the stimulatory action of NP on glucose metabolism. PGE₂ (10⁻⁷ M) added to the incubation medium containing NP and ASA reversed the effect of the inhibitor. Neither NP nor Hb nor NMMA modified the ¹⁴CO₂ production from labeled glucose in uterine strips from ovariectomized rats. The addition of NP to the incubating medium increased PGE accumulation by uterine strips from rats treated with estradiol, but not in ovariectomized animals. These results suggest that NO exerts a positive influence on glucose metabolism and PGE synthesis in isolated rat uteri from estrogenized animals.     

Influence of insulin on the metabolism of glucose in uteri isolated from ovariectomized and non ovariectomized underfed rats

The effects of insulin on the metabolism of U14C-glucose in uteri isolated from ovariectomized and non-ovariectomized rats receiving a restricted diet (50% of the normal food intake) for 25 days, were studied. As a result of food restriction, the production of 14CO2 diminishes in intact rats, while results are reversed in ovariectomized ones. Various concentrations of insulin were added to the medium, but only 0.50 IU. ml(-1)was effective in increasing glucose metabolism in intact rats receiving a restricted diet; neither underfed castrated animals nor control ones receiving a normal diet, reacted to this concentration. The increase of 14CO2 produced by insulin is not affected by acetyl salicylic acid. Insulin does not alter the effect of underfeeding over arachidonic acid metabolism. On the contrary, the increase in glucose metabolism was blocked by N(G)methyl-L-arginine or by hemoglobin, increased with the addition of L arginine and is not affected by acetyl salicylic acid. Hemoglobin and L-arginine show no effects without insulin. We can conclude that the stimulating effect of insulin on glucose metabolism in uteri isolated from intact rats subjected to dietary restriction, is nitric oxide dependent.     

Mechanism of arachidonic acid modulation of the T-type Ca2+ channel alpha1G

Arachidonic acid (AA) modulates T-type Ca(2+) channels and is therefore a potential regulator of diverse cell functions, including neuronal and cardiac excitability. The underlying mechanism of modulation is unknown. Here we analyze the effects of AA on the T-type Ca(2+) channel alpha(1G) heterologously expressed in HEK-293 cells. AA inhibited alpha(1G) currents within a few minutes, regardless of preceding exposure to inhibitors of AA metabolism (ETYA and 17-ODYA). Current inhibition was also observed in cell-free inside-out patches, indicating a membrane-delimited interaction of AA with the channel. AA action was consistent with a decrease of the open probability without changes in the size of unitary currents. AA shifted the inactivation curve to more negative potentials, increased the speed of macroscopic inactivation, and decreased the extent of recovery from inactivation at -80 mV but not at -110 mV. AA induced a slight increase of activation near the threshold and did not significantly change the deactivation kinetics or the rectification pattern. We observed a tonic current inhibition, regardless of whether the channels were held in resting or inactivated states during AA perfusion, suggesting a state-independent interaction with the channel. Model simulations indicate that AA inhibits T-type currents by switching the channels into a nonavailable conformation and by affecting transitions between inactivated states, which results in the negative shift of the inactivation curve. Slow-inactivating alpha(1G) mutants showed an increased affinity for AA with respect to the wild type, indicating that the structural determinants of fast inactivation are involved in the AA-channel interaction.     

Conversion of arachidonic acid to prostanoids in the avian uterus

In an effort to obtain information on the possible source of prostaglandins which have been shown to play an important role in oviposition we examined the metabolism of arachidonic acid in microsomal preparations of both the muscular and the glandular tissue of the hen uterus. We found that adrenaline and tryptophan (but not hydroquinone) were effective stimulators of prostanoid synthesis. On incubation with [3H]arachidonic acid we identified, using TLC radiochromatography and several solvent systems, prostaglandins F2 alpha and E2 and, predominantly, thromboxane B2 which could not be attributed to platelet contamination. Addition of reduced glutathione increased prostaglandin E2 formation at the expense of thromboxane B2 and at 1 mM concentration suppressed adrenaline-promoted prostanoid synthesis. While the former effect has been documented in many other systems and could be ascribed to the activation of prostaglandin H2 to prostaglandin E2 isomerase, the latter effect is postulated to be due to an inhibition of cyclooxygenase. Interestingly, this inhibitory effect was shared by a number of reducing agents. Although the subcellular preparations were derived from structurally and functionally different tissues, there was no qualitative difference with respect to prostanoid synthesis. Our data support the role of locally produced primary prostaglandins in the regulation of oviposition and raise the question of a potential role for thromboxane in this process.     

Potent inhibition of gastric acid secretion by intravenous interleukin-1 beta and -1 alpha in rats.

The influence of human and rat recombinant interleukin-1 (hIL-1 beta and -1 alpha and rIL-1 beta) on acid secretion was investigated in conscious pylorus-ligated rats. Intravenous injection of either hIL-1 beta, hIL-1 alpha or rIL-1 beta dose dependently inhibited gastric acid output with an ED50 of 0.05 microgram, 0.5 microgram and 2.2 micrograms, respectively. The antisecretory action of IL-1 beta was associated with an increase in circulating levels of gastrin. hIL-1 beta-induced inhibition of acid secretion was dose dependently reversed by peripheral injection of the IL-1 receptor antagonist, IL-RA, with a dose ratio of 1:10(3) for complete reversal. The inhibitory effect of hIL-1 beta was blocked by indomethacin and was not modified by IV injections of the CRF receptor antagonist, alpha-helical CRF(9-41), or the monoclonal somatostatin antibody CURE.S6, or by systemic capsaicin pretreatment. These results show that systemic hIL-1 beta-induced inhibition of gastric acid secretion is mediated through IL-1 receptors and prostaglandin pathways, and does not involves CRF receptors, afferent fibers, or changes in circulating gastrin or somatostatin levels.     

The effect of cigarette smoke on the metabolism of arachidonic acid in isolated hamster lungs

The effects of cigarette smoke on the metabolism of exogenous arachidonic acid (AA) were investigated in isolated hamster lungs. Arachidonate was injected into the pulmonary circulation and the metabolites were analysed from the nonrecirculating perfusion effluent by thin layer chromatography. After the pulmonary injection of 66 nmol of ¹⁴C-AA about 20 % of the injected radioactivity appreated in the perfusion effluent mostly as metabolites in six minutes. When isolated lungs were ventilated with cigarette smoke during the perfusion, the amounts of PGF_2α, PGE₂ and two unidentified metabolite groups increased in the lung effluent. In two other experimental series hamsters were exposed to cigarette smoke before the lung perfusion either once for 30 min or during one hour daily for ten consecutive days. Neither pre-exposures caused any changes in the amounts of arachidonate metabolites in the lung effluent.     

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).