ERK1/2 inhibition prevents contraction-induced increase in plasma membrane FAT/CD36 content and FA uptake in rodent muscle.

AIM: The purpose of this experiment was to investigate the role of extracellular signal-regulated kinase 1/2 (ERK1/2) signalling in the contraction-induced increase in muscle FA uptake. METHODS: Male Wistar rats (n = 41) were randomly assigned to either a resting or stimulated group. Within each group, animals were randomly assigned to receive PD-98059, an inhibitor of MAP/ERK kinase 1/2 (MEK1/2), a kinase upstream of ERK1/2 and perfused with 550 microM palmitate, [(14)C]palmitate, 7 mM glucose, and no insulin. In the stimulated group, electrical stimulation (ES) of supramaximal trains of 100 ms was delivered every 2 s for 20 min. RESULTS: ERK1/2 phosphorylation was increased by 50% (P < 0.05) during ES but the contraction-induced increase was prevented by the addition of PD-98059. Glucose uptake increased by 3.6-fold (P < 0.05) from rest to ES in muscle perfused without PD-98059 and was not affected by the addition of PD-98059 either at rest (P > 0.05) or during ES (P > 0.05). For a matched palmitate delivery, ES increased palmitate uptake by 35% (P < 0.05). PD-98059 had no effect on palmitate uptake at rest but completely abolished the increase in palmitate uptake during ES. Plasma membrane FAT/CD36 protein content was increased by 38% during ES (P < 0.05) but the contraction-induced increase was prevented by the addition of PD-98059. AMPK activity was increased by ES (P < 0.05) but was unaffected by PD-98059. CONCLUSION: These results show for the first time that the increase in FA uptake and in plasma membrane FAT/CD36 protein content is mediated, at least in part, by the ERK1/2 signalling pathway during muscle contraction.

Increased rates of fatty acid uptake and plasmalemmal fatty acid transporters in obese Zucker rats.

Giant vesicles were used to study the rates of uptake of long-chain fatty acids by heart, skeletal muscle, and adipose tissue of obese and lean Zucker rats. With obesity there was an increase in vesicular fatty acid uptake of 1.8-fold in heart, muscle and adipose tissue. In some tissues only fatty acid translocase (FAT) mRNA (heart, +37%; adipose, +80%) and fatty acid-binding protein (FABPpm) mRNA (heart, +148%; adipose, +196%) were increased. At the protein level FABPpm expression was not changed in any tissues except muscle (+14%), and FAT/CD36 protein content was altered slightly in adipose tissue (+26%). In marked contrast, the plasma membrane FAT/CD36 protein was increased in heart (+60%), muscle (+80%), and adipose tissue (+50%). The plasma membrane FABPpm was altered only in heart (+50%) and adipose tissues (+70%). Thus, in obesity, alterations in fatty acid transport in metabolically important tissues are not associated with changes in fatty acid transporter mRNAs or altered fatty acid transport protein expression but with their increased abundance at the plasma membrane. We speculate that in obesity fatty acid transporters are relocated from an intracellular pool to the plasma membrane in heart, muscle, and adipose tissues.

Increased fatty acid uptake and altered fatty acid metabolism in insulin-resistant muscle of obese Zucker rats.

Altered muscle fatty acid (FA) metabolism may contribute to the presence of muscle insulin resistance in the genetically obese Zucker rat. To determine whether FA uptake and disposal are altered in insulin-resistant muscle, we measured palmitate uptake, oxidation, and incorporation into di- and triglycerides in isolated rat hindquarters, as well as muscle plasma membrane fatty acid-binding protein (FABP(PM)) content of lean (n = 16, fa/+) and obese (n = 15, fa/fa) Zucker rats (12 weeks of age). Hindquarters were perfused with 7 mmol/l glucose, 1,000 micromol/l albumin-bound palmitate, and albumin-bound [1-(14)C]palmitate at rest (no insulin). Glucose uptake was 42% lower in the obese than in the lean rats and indicated the presence of muscle insulin resistance. Fractional and total rates of palmitate uptake were 42 and 74% higher in the obese than in the lean rats and were associated with higher muscle FABP(PM) content (r(2) = 0.69, P < 0.05). The percentage of palmitate oxidized was not significantly different between groups. FA disposal to storage was altered according to fiber type. When compared with lean rats, the rate of triglyceride synthesis in red muscle was 158% higher in obese rats, and the rate of palmitate incorporation into diglycerides in white muscle was 93% higher in obese rats. Pre- and postperfusion muscle triglyceride levels were higher in both red and white muscles of the obese rats. These results show that increased FA uptake and altered FA disposal to storage may contribute to the development of muscle insulin resistance in obese Zucker rats.

Muscle palmitate uptake and binding are saturable and inhibited by antibodies to FABP(PM).

Studies show that uptake of long-chain fatty acids (LCFA) across the plasma membranes (PM) may occur partly via a carrier-mediated process and that the plasma membrane fatty acid-binding protein (FABP(PM)) may be a component of this system. To test the hypothesis that FABP(PM) is involved in transsarcolemmal transport of LCFA in muscle, we measured palmitate uptake in giant sarcolemmal vesicles and palmitate binding to PM proteins in rat muscles, (1) in the presence of increasing amounts of unbound palmitate and (2) in the absence or presence of antibody to FABP(PM). Both palmitate uptake and binding were found to be saturable functions of the unbound palmitate concentration with calculated Vmax values of 10.5 +/- 1.2 pmol/mg protein/15 sec and 45.6 +/- 2.9 nmol/mg protein/15 min and Km values of 12.8 +/- 3.8 and 18.4 +/- 1.8 nmol/L, respectively. The Vmax values for both palmitate uptake and binding were significantly decreased by 75-79% in the presence of a polyclonal antibody to the rat hepatic FABP(PM). Antibody inhibition was found to be dose-dependent and specific to LCFA. Glucose uptake was not affected by the presence of the antibody to FABP(PM). Palmitate uptake and binding were also inhibited in the presence of trypsin and phloretin. These results support the hypothesis that transsarcolemmal LCFA transport occurs in part by a carrier-mediated process and that FABP(PM) is a component of this process in muscle.

Lower calorie intake enhances muscle insulin action and reduces hexosamine levels.

Previous studies have demonstrated enhanced insulin sensitivity in calorie-restricted [CR, fed 60% ad libitum (AL) one time daily] compared with AL-fed rats. To evaluate the effects of reduced food intake, independent of temporal differences in consumption, we studied AL (unlimited food access)-fed and CR (fed one time daily) rats along with groups temporally matched for feeding [fed 3 meals (M) daily]: MAL and MCR, eating 100 and 60% of AL intake, respectively. Insulin-stimulated glucose transport by isolated muscle was increased in MCR and CR vs. AL and MAL; there was no significant difference for MCR vs. CR or MAL vs. AL. Intramuscular triglyceride concentration, which is inversely related to insulin sensitivity in some conditions, did not differ among groups. Muscle concentration of UDP-N-acetylhexosamines [end products of the hexosamine biosynthetic pathway (HBP)] was lower in MCR vs. MAL despite unaltered glutamine-fructose-6-phosphate aminotransferase activity (rate-limiting enzyme for HBP). These results indicate that the CR-induced increase in insulin-stimulated glucose transport in muscle is attributable to an altered amount, not timing, of food intake and is independent of lower triglyceride concentration. They further suggest that enhanced insulin action might involve changes in HBP.

Muscle fatty acid uptake during exercise: possible mechanisms.

Muscle long-chain fatty acid (LCFA) utilization may be regulated by the ability of the muscle cell to carry LCFA across the plasma membrane. The presence of saturable LCFA uptake kinetics and the identification of putative LCFA transporter proteins whose expression can be modulated by acute and chronic exercise adaptations provide evidence for the existence of a carrier-mediated transport system in muscle.

Markers of capacity to utilize fatty acids in human skeletal muscle: relation to insulin resistance and obesity and effects of weight loss.

A number of biochemical defects have been identified in glucose metabolism within skeletal muscle in obesity, and positive effects of weight loss on insulin resistance are also well established. Less is known about the capacity of skeletal muscle for the metabolism of fatty acids in obesity-related insulin resistance and of the effects of weight loss, though it is evident that muscle contains increased triglyceride. The current study was therefore undertaken to profile markers of human skeletal muscle for fatty acid metabolism in relation to obesity, in relation to the phenotype of insulin-resistant glucose metabolism, and to examine the effects of weight loss. Fifty-five men and women, lean and obese, with normal glucose tolerance underwent percutaneous biopsy of vastus lateralis skeletal muscle for determination of HADH, CPT, heparin-releasable (Hr) and tissue-extractable (Ext) LPL, CS, COX, PFK, and GAPDH enzyme activities, and content of cytosolic and plasma membrane FABP. Insulin sensitivity was measured using the euglycemic clamp method. DEXA was used to measure FM and FFM. In skeletal muscle of obese individuals, CPT, CS, and COX activities were lower while, conversely, they had a higher or similar content of FABP(C) and FABP(PM) than in lean individuals. Hr and Ext LPL activities were similar in both groups. In multivariate and simple regression analyses, there were significant correlations between insulin resistance and several markers of FA metabolism, notably, CPT and FABP(PM). These data suggest that in obesity-related insulin resistance, the metabolic capacity of skeletal muscle appears to be organized toward fat esterification rather than oxidation and that dietary-induced weight loss does not correct this disposition.

Training-induced elevation in FABP(PM) is associated with increased palmitate use in contracting muscle.

To evaluate the effects of endurance training in rats on fatty acid metabolism, we measured the uptake and oxidation of palmitate in isolated rat hindquarters as well as the content of fatty acid-binding proteins in the plasma membranes (FABP(PM)) of red and white muscles from 16 trained (T) and 18 untrained (UT) rats. Hindquarters were perfused with 6 mM glucose, 1,800 microM palmitate, and [1-(14)C]palmitate at rest and during electrical stimulation (ES) for 25 min. FABP(PM) content was 43-226% higher in red than in white muscles and was increased by 55% in red muscles after training. A positive correlation was found to exist between succinate dehydrogenase activity and FABP(PM) content in muscle. Palmitate uptake increased by 64-73% from rest to ES in both T and UT and was 48-57% higher in T than UT both at rest (39.8 +/- 3.5 vs. 26.9 +/- 4. 4 nmol. min(-1). g(-1), T and UT, respectively) and during ES (69.0 +/- 6.1 vs. 43.9 +/- 4.4 nmol. min(-1). g(-1), T and UT, respectively). While the rats were resting, palmitate oxidation was not affected by training; palmitate oxidation during ES was higher in T than UT rats (14.8 +/- 1.3 vs. 9.3 +/- 1.9 nmol. min(-1). g(-1), T and UT, respectively). In conclusion, endurance training increases 1) plasma free fatty acid (FFA) uptake in resting and contracting perfused muscle, 2) plasma FFA oxidation in contracting perfused muscle, and 3) FABP(PM) content in red muscles. These results suggest that an increased number of these putative plasma membrane fatty acid transporters may be available in the trained muscle and may be implicated in the regulation of plasma FFA metabolism in skeletal muscle.

Role of fats in exercise. Types and quality.

Plasma TGs, FFAs, and muscle TG are oxidizable lipid fuel sources for skeletal muscle metabolism during prolonged exercise. Plasma FFAs are a major fuel oxidized by skeletal muscle, and their rate of use by muscle depends on several factors, including plasma FFA availability, transport from plasma to the mitochondria, and intracellular metabolism. Mobilization of FFAs from adipose tissue is the first committed step in FFA metabolism, and it depends on the rate of adipose tissue lipolysis. Adipose tissue lipolysis increases with exercise duration and exercise intensity up to intensities of approximately 60% to 65%. Evidence suggests that FFAs are transported from plasma to the mitochondria by FFA transporter proteins that include the plasma membrane and cytosolic FABPPM and FABPC. Plasma FFA use can also be regulated at the mitochondrial transport step by changing the activity of carnitine palmitoyltransferase (CPT-1). Although results from biopsy and tracer studies indicate that muscle TG contribute to skeletal muscle oxidative metabolism during exercise, their exact contribution is difficult to ascertain. Evidence shows that muscle TG use depends on exercise intensity, duration, and mode. The contribution of plasma TG to skeletal muscle metabolism is small. The rate of use of plasma TG is dependent on lipoprotein lipase activity, which is correlated with the oxidative capacity of the muscle fibers. Dietary manipulations can modulate substrate use during exercise and can potentially affect exercise performance. High carbohydrate availability before exercise is associated with an increase in blood glucose and plasma insulin concentrations, which can ultimately decrease the rate of adipose tissue lipolysis and the availability of plasma FFAs. Increased glucose flux has also been shown to decrease lipid oxidation by directly inhibiting the transport of FFAs across the mitochondrial membranes. High lipid availability can be changed by short-term or long-term exposure to high-fat diets. Because carbohydrate reserves are diminished with exposure to high-fat diets, improvements in exercise performance have been difficult to measure under these conditions.

Protein-mediated palmitate uptake and expression of fatty acid transport proteins in heart giant vesicles.

Giant sarcolemmal vesicles were isolated from rat heart and hindlimb muscles for a) characterization of long-chain fatty acid transport in the absence of metabolism and b) comparison of fatty acid transport protein expression with fatty acid transport. Giant vesicles contained cytosolic fatty acid binding protein. Palmitate uptake was completely divorced from its metabolism. All palmitate taken up was recovered in the intravesicular cytosol as unesterified FA. Palmitate uptake by heart vesicles exhibited a K m of 9.7 nm, similar to that of muscle (K m = 9.7 nm). Vmax (2.7 pmol/mg protein/s) in heart was 8-fold higher than in muscle (0.34 pmol/mg protein/s). Palmitate uptake was inhibited in heart (55-80%) and muscle (31-50%) by trypsin, phloretin, sulfo-N-succinimidyloleate (SSO), or a polyclonal antiserum against the 40 kDa plasma membrane fatty acid binding protein (FABPpm). Palmitate uptake by heart and by red and white muscle vesicles correlated well with the expression of fatty acid translocase (FAT/CD36) and fatty acid binding protein FABPpm, which may act in concert. The expression of fatty acid transport protein (FATP), was 10-fold lower in heart vesicles than in white muscle vesicles. It is concluded that long-chain fatty acid uptake by heart and muscle vesicles is largely protein-mediated, involving FAT/CD36 and FABPpm. The role of FATP in muscle and heart remains uncertain.

Effect of extracellular palmitate on 2-deoxy-d-glucose uptake in muscle from Ad libitum fed and calorie restricted rats.

We studied the effect of a high physiologic concentration of palmitate (1mM) on in vitro 2-deoxy-D-glucose (2DG) uptake by flexor digitorum brevis (FDB) muscle from ad libitum fed rats (AL) and rats fed 60% of ad libitum intake (CR) for 20 days. CR did not alter muscle 2DG uptake in the absence of insulin, but relative to AL, CR significantly (p<0.01) increased 2DG uptake in the presence of 20,000 microU/ml insulin. This effect of CR persisted in the presence of 1mM palmitate. The presence of 1mM palmitate significantly (p<0.01) impaired 2DG glucose uptake, both in the presence and absence of insulin, to the same extent in AL and CR muscle, despite an 18% decrease in FABPpm expression with CR. Thus, although CR profoundly affects insulin-mediated muscle glucose uptake, it does not alter the ability of extracellular fatty acid to modulate glucose utilization by skeletal muscle.

Palmitate transport and fatty acid transporters in red and white muscles.

We performed studies 1) to investigate the kinetics of palmitate transport into giant sarcolemmal vesicles, 2) to determine whether the transport capacity is greater in red muscles than in white muscles, and 3) to determine whether putative long-chain fatty acid (LCFA) transporters are more abundant in red than in white muscles. For these studies we used giant sarcolemmal vesicles, which contained cytoplasmic fatty acid binding protein (FABPc), an intravesicular fatty acid sink. Intravesicular FABPc concentrations were sufficiently high so as not to limit the uptake of palmitate under conditions of maximal palmitate uptake (i.e., 4.5-fold excess in white and 31.3-fold excess in red muscle vesicles). All of the palmitate taken up was recovered as unesterified palmitate. Palmitate uptake was reduced by phloretin (-50%), sulfo-N-succinimidyl oleate (-43%), anti-plasma membrane-bound FABP (FABPpm, -30%), trypsin (-45%), and when incubation temperature was lowered to 0 degrees C (-70%). Palmitate uptake was also reduced by excess oleate (-65%), but not by excess octanoate or by glucose. Kinetic studies showed that maximal transport was 1.8-fold greater in red vesicles than in white vesicles. The Michaelis-Menten constant in both types of vesicles was approximately 6 nM. Fatty acid transport protein mRNA and fatty acid translocase (FAT) mRNA were about fivefold greater in red muscles than in white muscles. FAT/CD36 and FABPpm proteins in red vesicles or in homogenates were greater than in white vesicles or homogenates (P < 0.05). These studies provide the first evidence of a protein-mediated LCFA transport system in skeletal muscle. In this tissue, palmitate transport rates are greater in red than in white muscles because more LCFA transporters are available.

Contraction-induced increase in Vmax of palmitate uptake and oxidation in perfused skeletal muscle.

To evaluate the effects of contractions on the kinetics of uptake and oxidation of palmitate in a physiological muscle preparation, rat hindquarters were perfused with glucose (6 mmol/l), albumin-bound [1-14C]palmitate, and varying amounts of albumin-bound palmitate (200-2,200 micro mol/l) at rest and during muscle contractions. When plotted against the unbound palmitate concentration, palmitate uptake and oxidation displayed simple Michaelis-Menten kinetics with estimated maximal velocity (Vmax) and Michaelis-Menten constant (Km) values of 42.8 +/- 3.8 (SE) nmol . min-1 . g-1 and 13.4 +/- 3.4 nmol/l for palmitate uptake and 3.8 +/- 0.4 nmol . min-1 . g-1 and 8.1 +/- 2.9 nmol/l for palmitate oxidation, respectively, at rest. Whereas muscle contractions increased the Vmax for both palmitate uptake and oxidation to 91.6 +/- 10.1 and 16.5 +/- 2.3 nmol . min-1 . g-1, respectively, the Km remained unchanged. Vmax and Km estimates obtained from Hanes-Woolf plots (substrate concentration/velocity vs. substrate concentration) were not significantly different. In the resting perfused hindquarter, an increase in palmitate delivery from 31.9 +/- 0.9 to 48.7 +/- 1.2 micro mol . g-1 . h-1 by increasing perfusate flow was associated with a decrease in the fractional uptake of palmitate so that the rates of uptake and oxidation of palmitate remained unchanged. It is concluded that the rates of uptake and oxidation of long-chain fatty acids (LCFA) saturate with an increase in the concentration of unbound LCFA in perfused skeletal muscle and that muscle contractions, but not an increase in plasma flow, increase the Vmax for LCFA uptake and oxidation. The data are consistent with the notion that uptake of LCFA in muscle may be mediated in part by a transport system.

Effect of endurance training on ammonia and amino acid metabolism in humans.

Few studies examine ammonia and amino acid metabolism in response to endurance training. Trained humans generally experience less increase in plasma ammonia during either prolonged or intense exercise. This is probably a reflection of reduced ammonia production and release from the active muscle; it could be a reflection of decreased AMP deaminase activity, decreased glutamate dehydrogenase activity, and/or increased alanine and glutamine formation. Little is known regarding the associated enzyme systems in humans, but in experiments with animal models, aerobic training decreases AMP deaminase and increases the enzymes of amino acid transamination and oxidation.

Membrane associated fatty acid binding protein (FABPpm) in human skeletal muscle is increased by endurance training.

Endurance training increases the capacity for utilization of fatty acids. Since fatty acids are believed to enter cells via facilitated diffusion a possible mechanism behind this adaptation to training might be a training-induced increase in membrane content of putative fatty acid transporters. We investigated whether the expression of the 40 KD membrane associated fatty acid binding protein (FABPpm) in skeletal muscle is increased with endurance training in man. The FABPpm was detectable in a crude membrane preparation from human skeletal muscle. Three weeks of intense one-legged endurance training increased (p < 0.05) the content of FABPpm by 49% whereas in the untrained control muscle no change was observed. In addition, the activity of citrate synthase was increased (p < 0.05) by 20% in the trained compared with the untrained muscle. It is concluded that expression of FABPpm in human skeletal muscle is increased with endurance training consistent with a role of FABPpm as a sarcolemmal fatty acid transporter.

Fasting increases plasma membrane fatty acid-binding protein (FABP(PM)) in red skeletal muscle.

The present study was designed to investigate the presence of the fatty acid-binding protein (FABP(PM)) in the plasma membranes of skeletal muscles with different oxidative capacities for free fatty acid (FFA) oxidation during conditions of normal (fed) or increased (fasted) FFA utilization in the rat. Female Sprague-Dawley rats were either fed or fasted for 12, 24, or 48 h and, plasma membranes (PM) fractions from red and white skeletal muscles were isolated. Short-term fasting significantly decreased body weight by 11% and blood glucose concentration by 42% (6.6+/-0.2-3.8+/-0.4 mmol/l) and increased plasma FFA concentration by 5-fold (133+/-14-793+/-81 micromol/l). Immunoblotting of PM fractions showed that FABP(PM) protein content was 83+/-18% higher in red than in white skeletal muscle and correlated with oxidative capacity as measured by succinate dehydrogenase activity (r = 0.78, p < 0.05). Short-term fasting significantly increased FABP(PM) protein content by 60+/-8% in red skeletal muscle but no change was measured in white skeletal muscle. These results show that FABP(PM) protein content in skeletal muscle is related to oxidative potential and can be increased during a physiological condition known to be associated with an increase in FFA utilization, suggesting that cellular expression of FABP(PM) may play a role in the regulation of FFA metabolism in skeletal muscle.

Lactate and H+ uptake in inactive muscles during intense exercise in man.

1. The present study examined how uptake of lactate and H+ in resting muscle is affected by blood flow, arterial lactate concentration and muscle metabolism. 2. Six males subjects performed intermittent arm exercise in two separate 32 min periods (Part I and Part II) and in one subsequent 20 min period in which one leg knee-extensor exercise was also performed (Part III). The exercise was performed at various intensities in order to obtain different steady-state arterial blood lactate concentrations. In the inactive leg, femoral venous blood flow (draining about 7.7 kg of muscles) was measured and femoral arterial and venous blood was collected frequently. Biopsies were taken from m. vastus lateralis of the inactive leg at rest and 10 and 30 min into both Part I and Part II as well as 10 min into recovery from Part II. 3. The arterial plasma lactate concentrations were 7, 9 and 16 mmol l-1 after 10 min of Parts I, II and III, respectively, and the corresponding arterial-venous difference (a-vdiff) for lactate in the resting leg was 1.3, 1.4 and 2.0 mmol l-1. The muscle lactate concentration was 2.8 mmol (kg wet wt)-1 after 10 min of Part I and remained constant throughout the experiment. During Parts I and II, a-vdiff lactate decreased although the arterial lactate concentration and plasma-muscle lactate gradient were unaltered throughout each period. Thus, membrane transport of lactate decreased during each period. 4. Blood flow in the inactive leg was about 2-fold higher during arm exercise compared to the rest periods, resulting in a 2-fold higher lactate uptake. Thus, lactate uptake by inactive muscles was closely related to blood flow. 5. Throughout the experiment a-vdiff for actual base excess and for lactate were of similar magnitude. Thus, in inactive muscles lactate uptake appears to be coupled to the transport of H+.

Circulating palmitate uptake and oxidation are not altered by glycogen depletion in contracting skeletal muscle.

The extent to which muscle glycogen depletion affects plasma free fatty acid (FFA) metabolism in contracting skeletal muscle is not well characterized. To study this question, rats were glycogen depleted (GD) or supercompensated (SC) by swimming exercise and diet treatment 24 h before perfusion of their isolated hindquarters at rest and during electrically induced muscle contractions. After 20 min of equilibration with glucose (6 mM), palmitate (2,000 microM), and [1-14C]palmitate, palmitate uptake and oxidation were found to be similar between groups at rest and during electrical stimulation. Palmitate uptake increased by 55% during electrical stimulation and averaged 2.75 +/- 0.56 mumol.g-1.h-1. Resting palmitate oxidation averaged 0.14 +/- 0.03 mumol.g-1.h-1 and increased to 0.53 +/- 0.06 and 0.47 +/- 0.08 mumol.g-1.h-1 during electrical stimulation in GD and SC, respectively. Glucose uptake was significantly higher in GD than in SC at rest and during electrical stimulation and significantly increased in both groups during electrical stimulation to reach values of 11.8 +/- 1.2 and 7.6 +/- 1.4 mumol.g-1.h-1, respectively. Lactate release was lower in GD than in SC at rest and during electrical stimulation and was highest after 2 min of stimulation in both groups. Additional experiments at perfusate palmitate concentrations of 600-900 microM yielded similar results. These results show that, in contracting perfused skeletal muscle, muscle glycogen depletion increases glucose utilization but does not affect total plasma FFA oxidation, suggesting that regulation within pathways of carbohydrate metabolism takes precedence over regulation between pathways of lipid and carbohydrate metabolism.

Training and muscle ammonia and amino acid metabolism in humans during prolonged exercise.

We studied the responses of NH3 and amino acids (AA) to prolonged exercise (3 h) in trained (Tr; n = 6) and untrained (Utr; n = 6) men. Each subject exercised the knee extensor muscles of one leg at 60% of maximum capacity. Thigh blood flow and femoral arteriovenous differences (0, 30, 60, 120, 150, and 180 min) as well as muscle biopsies (0, 120, and 180 min) were taken for NH3 and AA measurements. In both groups, muscle Glu decreased (P < 0.05) and Asp increased (P < 0.05), but the intramuscular AA pool, including the essential AA, remained constant despite a total AA efflux of 22.4 +/- 8.3 and 24.4 +/- 6.8 mmol/kg wet wt in Tr and Utr, respectively. Tr had greater (P < 0.05) muscle Tau, Phe, Ala, and Glu. Both groups had a large Glu uptake and effluxes of NH3, Gln, and Ala as well as essential AA. The latter implies that there was a net protein catabolism. The efflux of NH3 and Gln was much greater than that expected from AMP deamination, suggesting that deamination of AA was occurring. Many of the AA responses use Glu, and Tr maintained the intramuscular Glu pool at a higher concentration (P < 0.05), implying that they derived more Glu from protein catabolism and/or AA transaminations. Under these conditions, prolonged dynamic knee extensor exercise is associated with a large release of alpha-amino moieties both as NH3 and as Gln as well as a net protein catabolism; these responses are similar in Tr and Utr.

Impaired plasma FFA oxidation imposed by extreme CHO deficiency in contracting rat skeletal muscle.

The extent to which carbohydrate (CHO) availability affects free fatty acid (FFA) metabolism in contracting skeletal muscle is not well characterized. To study this question, rats were depleted of glycogen by swimming exercise and lard feeding 24 h before perfusion of their isolated hindquarters. After 20 min of preperfusion with a medium containing no glucose, palmitate (600 or 2,000 microM), and [1-14C]palmitate, flow was restricted to one hindlimb, which was electrically stimulated for 2 min to further deplete muscles of glycogen. After 2 min of recovery, glucose was added to the perfusate at final concentrations of 0, 6, or 20 mM, and after another 3 min muscles were stimulated for 30 min. At 6 and 2,000 microM palmitate, glucose uptake after 30 min of stimulation averaged 23.5 +/- 9.3 and 45.9 +/- 10.6 mumol.g-1.h-1 with 6 and 20 mM glucose, respectively. At 6 and 2,000 microM palmitate, palmitate uptake was lower (30-37%, P < 0.05) with 0 than with 6 or 20 mM glucose. At 600 microM palmitate, percent palmitate oxidation was higher (27%, P < 0.05) with 0 than with 6 or 20 mM glucose, resulting in similar total palmitate oxidation with the three glucose concentrations (0.28 +/- 0.01 mumol.g-1.h-1). At 2,000 microM palmitate, percent palmitate oxidation was not significantly different among glucose concentrations, resulting in a significantly lower rate of palmitate oxidation with 0 (0.62 +/- 0.18 mumol.g-1.h-1) than with 6 or 20 mM glucose (0.77 +/- 0.25 and 0.78 +/- 0.20 mumol.g-1.h-1, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)