Regulation of contraction-induced FA uptake and oxidation by AMPK and ERK1/2 is intensity dependent in rodent muscle

Muscle contraction activates AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK1/2), two signaling molecules involved in the regulation of muscle metabolism. The purpose of this study was to determine whether activation of AMPK and/or ERK1/2 contributes to the regulation of muscle fatty acid (FA) uptake and oxidation in contracting muscle. Rat hindquarters were perfused during rest (R) or electrical stimulation (E) of increasing intensity by manipulating train duration (E1 = 25 ms, E2 = 50 ms, E3 = 100 ms, E4 = 200 ms). For matched FA delivery, FA uptake was significantly greater than R during E1, E2, and E3 (7.8 +/- 0.7 vs. 14.4 +/- 0.3, 16.9 +/- 0.8, 15.2 +/- 0.5 nmol.min(-1).g(-1), respectively, P < 0.05), but not during E4 (8.3 +/- 0.3 nmol.min(-1).g(-1), P > 0.05). FA oxidation was significantly greater than R during E1 and E2 (1.5 +/- 0.1 vs. 2.3 +/- 0.2, 2.5 +/- 0.2 nmol.min(-1).g(-1), P < 0.05) before returning to resting levels for E3 and E4 (1.8 +/- 0.1 and 1.5 +/- 0.2 nmol.min(-1).g(-1), P > 0.05). A positive correlation was found between FA uptake and ERK1/2 phosphorylation from R to E3 (R(2) = 0.55, P < 0.05) and between FA oxidation and ERK1/2 phosphorylation from R to E2 (R(2) = 0.76, P < 0.05), correlations that were not maintained when the data for E4 and E3 and E4, respectively, were included in the analysis (R(2) = 0.04 and R(2) = 0.03, P > 0.05). A positive correlation was also found between FA uptake and FA oxidation and AMPK activity for all exercise intensities (R(2) = 0.57, R(2) = 0.65 respectively, P < 0.05). These results, in combination with previous data from our laboratory, suggest that ERK1/2 and AMPK are the predominant signaling molecules regulating FA uptake and oxidation during low- to moderate-intensity muscle contraction and during moderate- to high-intensity muscle contraction, respectively.     

Insulin fails to alter plasma LCFA metabolism in muscle perfused at similar glucose uptake

Insulin has been shown to alter long-chain fatty acid (LCFA) metabolism and malonyl-CoA production in muscle. However, these alterations may have been induced, in part, by the accompanying insulin-induced changes in glucose uptake. Thus, to determine the effects of insulin on LCFA metabolism independently of changes in glucose uptake, rat hindquarters were perfused with 600 microM palmitate and [1-(14)C]palmitate and with either 20 mM glucose and no insulin (G) or 6 mM glucose and 250 microU/ml of insulin (I). As dictated by our protocol, glucose uptake was not significantly different between the G and I groups (10.3 +/- 0.6 vs. 11.0 +/- 0.5 micromol x g(-1) x h(-1); P > 0.05). Total palmitate uptake and oxidation were not significantly different (P > 0.05) between the G (10.1 +/- 1.0 and 0.8 +/- 0.1 nmol x min(-1) x g(-1)) and I (10.2 +/- 0.6 and 1.1 +/- 0.2 nmol. min(-1) x g(-1)) groups. Preperfusion muscle triglyceride and malonyl-CoA levels were not significantly different between the G and I groups and did not change significantly during the perfusion (P > 0.05). Similarly, muscle triglyceride synthesis was not significantly different between groups (P > 0.05). These results demonstrate that the presence of insulin under conditions of similar glucose uptake does not alter LCFA metabolism and suggest that cellular mechanisms induced by carbohydrate availability, but independent of insulin, may be important in the regulation of muscle LCFA metabolism.     

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.     

Brief food restriction increases FA oxidation and glycogen synthesis under insulin-stimulated conditions

To determine the effects of brief food restriction on fatty acid (FA) metabolism, hindlimbs of F344/BN rats fed either ad libitum (AL) or food restricted (FR) to 60% of baseline food intake for 28 days were perfused under hyperglycemic-hyperinsulinemic conditions (20 mM glucose, 1 mM palmitate, 1,000 microU/ml insulin, [3-(3)H]glucose, and [1-(14)C]palmitate). Basal glucose and insulin levels were significantly lower (P < 0.05) in FR vs. AL rats. Palmitate uptake (34.3 +/- 2.7 vs. 24.5 +/- 3.1 nmol/g/min) and oxidation (3.8 +/- 0.2 vs. 2.7 +/- 0.3 nmol.g(-1).min(-1)) were significantly higher (P < 0.05) in FR vs. AL rats, respectively. Glucose uptake was increased in FR rats and was accompanied by significant increases in red and white gastrocnemius glycogen synthesis, indicating an improvement in insulin sensitivity. Although muscle triglyceride (TG) levels were not significantly different between groups, glucose uptake and total preperfusion TG concentration were negatively correlated (r(2) = 0.27, P < 0.05). In conclusion, our results show that under hyperglycemic-hyperinsulinemic conditions, brief FR resulted in an increase in FA oxidative disposal that may contribute to the improvement in insulin sensitivity.     

Impact of in vivo fatty acid oxidation blockade on glucose turnover and muscle glucose metabolism during low-dose AICAR infusion

AMPK plays a central role in influencing fuel usage and selection. The aim of this study was to analyze the impact of low-dose AMP analog 5-aminoimidazole-4-carboxamide-1-beta-d-ribosyl monophosphate (ZMP) on whole body glucose turnover and skeletal muscle (SkM) glucose metabolism. Dogs were restudied after prior 48-h fatty acid oxidation (FA(OX)) blockade by methylpalmoxirate (MP; 5 x 12 hourly 10 mg/kg doses). During the basal equilibrium period (0-150 min), fasting dogs (n = 8) were infused with [3-(3)H]glucose followed by either 2-h saline or AICAR (1.5-2.0 mg x kg(-1) x min(-1)) infusions. SkM was biopsied at completion of each study. On a separate day, the same protocol was undertaken after 48-h in vivo FA(OX) blockade. The AICAR and AICAR + MP studies were repeated in three chronic alloxan-diabetic dogs. AICAR produced a transient fall in plasma glucose and increase in insulin and a small decline in free fatty acid (FFA). Parallel increases in hepatic glucose production (HGP), glucose disappearance (R(d tissue)), and glycolytic flux (GF) occurred, whereas metabolic clearance rate of glucose (MCR(g)) did not change significantly. Intracellular SkM glucose, glucose 6-phosphate, and glycogen were unchanged. Acetyl-CoA carboxylase (ACC approximately pSer(221)) increased by 50%. In the AICAR + MP studies, the metabolic responses were modified: the glucose was lower over 120 min, only minor changes occurred with insulin and FFA, and HGP and R(d tissue) responses were markedly attenuated, but MCR(g) and GF increased significantly. SkM substrates were unchanged, but ACC approximately pSer(221) rose by 80%. Thus low-dose AICAR leads to increases in HGP and SkM glucose uptake, which are modified by prior FA(ox) blockade.     

Insulin stimulation of glucose uptake fails to decrease palmitate oxidation in muscle if AMPK is activated.

Fatty acid oxidation in muscle has been reported to be diminished when insulin and glucose levels are elevated. This study was designed to determine whether activation of AMP-activated protein kinase (AMPK) will prevent inhibitory effects of insulin and glucose on the rate of fatty acid oxidation. Rat hindlimbs were perfused with medium containing 0, 0.3, or 60 nM insulin with or without 2 mM 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). Glucose uptake was stimulated four- to fivefold by inclusion of insulin in the medium. Insulin attenuated the increase in AMPK caused by AICAR both in perfused hindlimbs and in isolated epitrochlearis muscles. The activation constant for citrate activation of acetyl-CoA carboxylase (ACC) was significantly increased in response to AICAR, and the increase was slightly attenuated if insulin was present in the perfusion medium. Insulin stimulated an increase in malonyl-CoA content of the muscles in the absence of AICAR. Malonyl-CoA was decreased to approximately the same value in AICAR-perfused muscle, regardless of insulin concentration. Muscle glucose 6-phosphate and citrate were significantly increased in response to AICAR and insulin. The rate of palmitate oxidation tended to decrease in response to insulin and in the absence of AICAR. AICAR increased palmitate oxidation to approximately the same level regardless of the insulin concentration or the rate of glucose uptake into the muscle. The rate of palmitate oxidation showed a curvilinear relationship as a function of muscle malonyl-CoA content, with half-maximal inhibition at approximately 0.6 nmol/g. We conclude that AMPK activation can prevent high rates of glucose uptake and glycolytic flux from inhibiting palmitate oxidation in predominantly fast-twitch muscle under these conditions.     

Contribution of malonyl-CoA decarboxylase to the high fatty acid oxidation rates seen in the diabetic heart

Myocardial glucose oxidation is markedly reduced in the uncontrolled diabetic. We determined whether this was due to direct biochemical changes in the heart or whether this was due to altered circulating levels of insulin and substrates that can be seen in the diabetic. Isolated working hearts from control or diabetic rats (streptozotocin, 55 mg/kg iv administered 6 wk before study) were aerobically perfused with either 5 mM [(14)C]glucose and 0.4 mM [(3)H]palmitate (low-fat/low-glucose buffer) or 20 mM [(14)C]glucose and 1.2 mM [(3)H]palmitate (high-fat/high-glucose buffer) +/-100 microU/ml insulin. The presence of insulin increased glucose oxidation in control hearts perfused with low-fat/low-glucose buffer from 553 +/- 85 to 1,150 +/- 147 nmol x g dry wt(-1) x min(-1) (P < 0. 05). If control hearts were perfused with high-fat/high-glucose buffer, palmitate oxidation was significantly increased by 112% (P < 0.05), but glucose oxidation decreased to 55% of values seen in the low-fat/low-glucose group (P < 0.05). In diabetic hearts, glucose oxidation was very low in hearts perfused with low-fat/low-glucose buffer (9 +/- 1 nmol x g dry wt(-1) x min(-1)) and was not altered by insulin or high-fat/high-glucose buffer. These results suggest that neither circulating levels of substrates nor insulin was responsible for the reduced glucose oxidation in diabetic hearts. To determine if subcellular changes in the control of fatty acid oxidation contribute to these changes, we measured the activity of three enzymes involved in the control of fatty acid oxidation; AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), and malonyl-CoA decarboxylase (MCD). Although AMPK and ACC activity in control and diabetic hearts was not different, MCD activity and expression in all diabetic rat heart perfusion groups were significantly higher than that seen in corresponding control hearts. These results suggest that an increased MCD activity contributes to the high fatty acid oxidation rates and reduced glucose oxidation rates seen in diabetic rat hearts.     

Insulin increases FA uptake and esterification but reduces lipid utilization in isolated contracting muscle

We examined the effect of insulin on the synthesis and degradation of muscle lipid pools [phospholipid (PL), diacylglycerol (DG), triacylglycerol (TG)] and palmitate oxidation in isolated resting and contracting (20 tetani/min) soleus muscles. Lipid metabolism was monitored using the previously defined pulse-chase procedure. At rest, insulin significantly increased total palmitate uptake into soleus muscle (+49%, P < 0.05), corresponding to enhanced DG (+60%, P < 0.05) and TG (+61%, P < 0.05) esterification, but blunted palmitate oxidation (-38%, P < 0.05) and TG hydrolysis (-34%, P < 0.05). During muscle contraction, when total palmitate uptake was increased, insulin further enhanced uptake (+21%, P < 0.05) and esterification of fatty acids (FA) to PL (+73%, P < 0.05), DG (+19%, P < 0.05), and TG (+161%, P < 0.01). Despite a profound shift in the relative partitioning of FA away from esterification and toward oxidation during contraction, the increase in palmitate oxidation and TG hydrolysis was significantly blunted by insulin [oxidation, -24% (P = 0.05); hydrolysis, -83% (P < 0.01)]. The effects of insulin on FA esterification (stimulation) and oxidation (inhibition) during contraction were reduced in the presence of the phosphatidylinositol 3-kinase inhibitor LY-294002. In summary, the effects of insulin and contraction on palmitate uptake and esterification are additive, while insulin opposes the stimulatory effect of contraction on FA oxidation and TG hydrolysis. Insulin's modulatory effects on muscle FA metabolism during contraction are mediated at least in part through phosphatidylinositol 3-kinase.     

Impaired fatty acid oxidation in muscle of aging rats perfused under basal conditions

The purpose of the present study was to examine the utilization of fatty acids (FA) and muscle substrates by skeletal muscle in young, middle-aged, and old adult rats under conditions of euglycemia with low insulin levels. Male Fischer 344 x Brown Norway rats aged 5, 15, or 24 mo underwent hindlimb perfusion with a medium of 8 mM glucose, 1 mM palmitate, 25 microU/ml insulin, [1-(14)C]palmitate, and [3-(3)H]glucose. Glucose and palmitate uptake were similar among age groups. The percent and total palmitate oxidized (nmol.min(-1).g(-1)) were 30-36 and 41-49% lower (P < 0.05) in 15-mo- and 24-mo-old than in 5-mo-old animals. Compared with 5-mo- and 15-mo-old animals, pre- and postperfusion muscle triglyceride (TG) levels were significantly (P < 0.05) elevated 91-305% in red and 118-219% in white muscles of 24-mo-old animals. Fatty acid-binding protein content was 40-64% higher (P < 0.05) in 24-mo- than in 5-mo- or 15-mo-old animals. In red muscle, hormone-sensitive lipase (HSL) content was 28% lower (P < 0.05) in 24-mo- than in 5-mo-old animals. These results indicate that, under euglycemic conditions in the presence of low insulin levels, the reduction in FA disposal to oxidation and the decrease in HSL content may contribute to the accumulation of TG in muscle of old animals.     

Fatty acid oxidation and triacylglycerol hydrolysis are enhanced after chronic leptin treatment in rats

Leptin acutely increases fatty acid (FA) oxidation and triacylglycerol (TG) hydrolysis and decreases TG esterification in oxidative rodent muscle. However, the effects of chronic leptin administration on FA metabolism in skeletal muscle have not been examined. We hypothesized that chronic leptin treatment would enhance TG hydrolysis as well as the capacity to oxidize FA in soleus (SOL) muscle. Female Sprague-Dawley rats were infused for 2 wk with leptin (LEPT; 0.5 mg x kg(-1) x day(-1)) by use of subcutaneously implanted miniosmotic pumps. Control (AD-S) and pair-fed (PF-S) animals received saline-filled implants. Subsequently, FA metabolism was monitored for 45 min in isolated, resting, and contracting (20 tetani/min) SOL muscles by means of pulse-chase procedures. Food intake (-33 +/- 2%, P < 0.01) and body mass (-12.5 +/- 4%, P = 0.01) were reduced in both LEPT and PF-S animals. Leptin levels were elevated (+418 +/- 7%, P < 0.001) in treated animals but reduced in PF-S animals (-73 +/- 8%, P < 0.05) relative to controls. At rest, TG hydrolysis was increased in leptin-treated rats (1.8 +/- 2.2, AD-S vs. 23.5 +/- 8.1 nmol/g wet wt, LEPT; P < 0.001). In contracting SOL muscles, TG hydrolysis (1.5 +/- 0.6, AD-S vs. 3.6 +/- 1.0 micromol/g wet wt, LEPT; P = 0.02) and palmitate oxidation (18.3 +/- 6.7, AD-S vs. 45.7 +/- 9.9 nmol/g wet wt, LEPT; P < 0.05) were both significantly increased by leptin treatment. Chronic leptin treatment had no effect on TG esterification either at rest or during contraction. Markers of overall (citrate synthase) and FA (hydroxyacyl-CoA dehydrogenase) oxidative capacity were unchanged with leptin treatment. Protein expression of hormone-sensitive lipase (HSL) was also unaltered following leptin treatment. Thus leptin-induced increases in lipolysis are likely due to HSL activation (i.e., phosphorylation). Increased FA oxidation secondary to chronic leptin treatment is not due to an enhanced oxidative capacity and may be a result of enhanced flux into the mitochondrion (i.e., carnitine palmitoyltransferase I regulation) or electron transport uncoupling (i.e., uncoupling protein-3 expression).     

Inhibition of p38 MAPK and AMPK restores adenosine-induced cardioprotection in hearts stressed by antecedent ischemia by altering glucose utilization

p38 mitogen-activated protein kinase (MAPK) and 5'-AMP-activated protein kinase (AMPK) are activated by metabolic stresses and are implicated in the regulation of glucose utilization and ischemia-reperfusion (IR) injury. This study tested the hypothesis that inhibition of p38 MAPK restores the cardioprotective effects of adenosine in stressed hearts by preventing activation of AMPK and the uncoupling of glycolysis from glucose oxidation. Working rat hearts were perfused with Krebs solution (1.2 mM palmitate, 11 mM [(3)H/(14)C]glucose, and 100 mU/l insulin). Hearts were stressed by transient antecedent IR (2 x 10 min I/5 min R) before severe IR (30 min I/30 min R). Hearts were treated with vehicle, p38 MAPK inhibitor (SB-202190, 10 microM), adenosine (500 microM), or their combination before severe IR. After severe IR, the phosphorylation (arbitrary density units) of p38 MAPK and AMPK, rates of glucose metabolism (micromol x g dry wt(-1) x min(-1)), and recovery of left ventricular (LV) work (Joules) were similar in vehicle-, SB-202190- and adenosine-treated hearts. Treatment with SB-202190 + adenosine versus adenosine alone decreased p38 MAPK (0.03 +/- 0.01, n = 3 vs. 0.48 +/- 0.10, n = 3, P < 0.05) and AMPK (0.00 +/- 0.00, n = 3 vs. 0.26 +/- 0.08, n = 3 P < 0.05) phosphorylation. This was accompanied by attenuated rates of glycolysis (1.51 +/- 0.40, n = 7 vs. 3.95 +/- 0.65, n = 7, P < 0.05) and H(+) production (2.12 +/- 0.76, n = 7 vs. 6.96 +/- 1.48, n = 7, P < 0.05), and increased glycogen synthesis (1.91 +/- 0.25, n = 6 vs. 0.27 +/- 0.28, n = 6, P < 0.05) and improved recovery of LV work (0.81 +/- 0.08, n = 7 vs. 0.30 +/- 0.15, n = 8, P < 0.05). These data indicate that inhibition of p38 MAPK abolishes subsequent phosphorylation of AMPK and improves the coupling of glucose metabolism, thereby restoring adenosine-induced cardioprotection.     

AMPKα2 deficiency uncovers time dependency in the regulation of contraction-induced palmitate and glucose uptake in mouse muscle

AMP-activated protein kinase (AMPK) is a fuel sensor in skeletal muscle with multiple downstream signaling targets that may be triggered by increases in intracellular Ca(2+) concentration ([Ca(2+)]). The purpose of this study was to determine whether increases in intracellular [Ca(2+)] induced by caffeine act solely via AMPKα(2) and whether AMPKα(2) is essential to increase glucose uptake, fatty acid (FA) uptake, and FA oxidation in contracting skeletal muscle. Hindlimbs from wild-type (WT) or AMPKα(2) dominant-negative (DN) transgene mice were perfused during rest (n = 11), treatment with 3 mM caffeine (n = 10), or muscle contraction (n = 11). Time-dependent effects on glucose and FA uptake were uncovered throughout the 20-min muscle contraction perfusion period (P < 0.05). Glucose uptake rates did not increase in DN mice during muscle contraction until the last 5 min of the protocol (P < 0.05). FA uptake rates were elevated at the onset of muscle contraction and diminished by the end of the protocol in DN mice (P < 0.05). FA oxidation rates were abolished in the DN mice during muscle contraction (P < 0.05). The DN transgene had no effect on caffeine-induced FA uptake and oxidation (P > 0.05). Glucose uptake rates were blunted in caffeine-treated DN mice (P < 0.05). The DN transgene resulted in a greater use of intramuscular triglycerides as a fuel source during muscle contraction. The DN transgene did not alter caffeine- or contraction-mediated changes in the phosphorylation of Ca(2+)/calmodulin-dependent protein kinase I or ERK1/2 (P > 0.05). These data suggest that AMPKα(2) is involved in the regulation of substrate uptake in a time-dependent manner in contracting muscle but is not necessary for regulation of FA uptake and oxidation during caffeine treatment.     

Effects of beta-adrenergic receptor stimulation and blockade on substrate metabolism during submaximal exercise

We used beta-adrenergic receptor stimulation and blockade as a tool to study substrate metabolism during exercise. Eight moderately trained subjects cycled for 60 min at 45% of VO(2 peak) 1) during a control trial (CON); 2) while epinephrine was intravenously infused at 0.015 microg. kg(-1) x min(-1) (beta-STIM); 3) after ingesting 80 mg of propranolol (beta-BLOCK); and 4) combining beta-BLOCK with intravenous infusion of Intralipid-heparin to restore plasma fatty acid (FFA) levels (beta-BLOCK+LIPID). beta-BLOCK suppressed lipolysis (i.e., glycerol rate of appearance) and fat oxidation while elevating carbohydrate oxidation above CON (135 +/- 11 vs. 113 +/- 10 micromol x kg(-1) x min(-1); P < 0.05) primarily by increasing rate of disappearance (R(d)) of glucose (36 +/- 2 vs. 22 +/- 2 micromol x kg(-1) x min(-1); P < 0.05). Plasma FFA restoration (beta-BLOCK+LIPID) attenuated the increase in R(d) glucose by more than one-half (28 +/- 3 micromol x kg(-1) x min(-1); P < 0.05), suggesting that part of the compensatory increase in muscle glucose uptake is due to reduced energy from fatty acids. On the other hand, beta-STIM markedly increased glycogen oxidation and reduced glucose clearance and fat oxidation despite elevating plasma FFA. Therefore, reduced plasma FFA availability with beta-BLOCK increased R(d) glucose, whereas beta-STIM increased glycogen oxidation, which reduced fat oxidation and glucose clearance. In summary, compared with control exercise at 45% VO(2 peak) (CON), both beta-BLOCK and beta-STIM reduced fat and increased carbohydrate oxidation, albeit through different mechanisms.     

Glucose kinetics and substrate oxidation during exercise in the follicular and luteal phases.

The purpose of this investigation was to determine whether plasma glucose kinetics and substrate oxidation during exercise are dependent on the phase of the menstrual cycle. Once during the follicular (F) and luteal (L) phases, moderately trained subjects [peak O(2) uptake (V(O(2))) = 48.2 +/- 1.1 ml. min(-1). kg(-1); n = 6] cycled for 25 min at approximately 70% of the V(O(2)) at their respective lactate threshold (70%LT), followed immediately by 25 min at 90%LT. Rates of plasma glucose appearance (R(a)) and disappearance (R(d)) were determined with a primed constant infusion of [6,6-(2)H]glucose, and total carbohydrate (CHO) and fat oxidation were determined with indirect calorimetry. At rest and during exercise at 70%LT, there were no differences in glucose R(a) or R(d) between phases. CHO and fat oxidation were not different between phases at 70%LT. At 90%LT, glucose R(a) (28.8 +/- 4.8 vs. 33.7 +/- 4.5 micromol. min(-1). kg(-1); P < 0.05) and R(d) (28.4 +/- 4.8 vs. 34.0 +/- 4.1 micromol. min(-1). kg(-1); P < 0.05) were lower during the L phase. In addition, at 90%LT, CHO oxidation was lower during the L compared with the F phase (82.0 +/- 12.3 vs. 93.8 +/- 9.7 micromol. min(-1) .kg(-1); P < 0.05). Conversely, total fat oxidation was greater during the L phase at 90%LT (7.46 +/- 1.01 vs. 6.05 +/- 0.89 micromol. min(-1). kg(-1); P < 0.05). Plasma lactate concentration was also lower during the L phase at 90%LT concentrations (2.48 +/- 0.41 vs. 3.08 +/- 0.39 mmol/l; P < 0.05). The lower CHO utilization during the L phase was associated with an elevated resting estradiol (P < 0.05). These results indicate that plasma glucose kinetics and CHO oxidation during moderate-intensity exercise are lower during the L compared with the F phase in women. These differences may have been due to differences in circulating estradiol.     

Increased submaximal insulin-stimulated glucose uptake in mouse skeletal muscle after treadmill exercise.

The primary purpose of this study was to determine the effect of prior exercise on insulin-stimulated glucose uptake with physiological insulin in isolated muscles of mice. Male C57BL/6 mice completed a 60-min treadmill exercise protocol or were sedentary. Paired epitrochlearis, soleus, and extensor digitorum longus (EDL) muscles were incubated with [3H]-2-deoxyglucose without or with insulin (60 microU/ml) to measure glucose uptake. Insulin-stimulated glucose uptake for paired muscles was calculated by subtracting glucose uptake without insulin from glucose uptake with insulin. Muscles from other mice were assessed for glycogen and AMPK Thr172 phosphorylation. Exercised vs. sedentary mice had decreased glycogen in epitrochlearis (48%, P < 0.001), soleus (51%, P < 0.001), and EDL (41%, P < 0.01) and increased AMPK Thr172 phosphorylation (P < 0.05) in epitrochlearis (1.7-fold), soleus (2.0-fold), and EDL (1.4-fold). Insulin-independent glucose uptake was increased 30 min postexercise vs. sedentary in the epitrochlearis (1.2-fold, P < 0.001), soleus (1.4-fold, P < 0.05), and EDL (1.3-fold, P < 0.01). Insulin-stimulated glucose uptake was increased (P < 0.05) approximately 85 min after exercise in the epitrochlearis (sedentary: 0.266 +/- 0.045 micromol x g(-1) x 15 min(-1); exercised: 0.414 +/- 0.051) and soleus (sedentary: 0.102 +/- 0.049; exercised: 0.347 +/- 0.098) but not in the EDL. Akt Ser473 and Akt Thr308 phosphorylation for insulin-stimulated muscles did not differ in exercised vs. sedentary. These results demonstrate enhanced submaximal insulin-stimulated glucose uptake in the epitrochlearis and soleus of mice 85 min postexercise and suggest that it will be feasible to probe the mechanism of enhanced postexercise insulin sensitivity by using genetically modified mice.     

Skeletal muscle lipid metabolism with obesity

The objectives of this study were to 1). examine skeletal muscle fatty acid oxidation in individuals with varying degrees of adiposity and 2). determine the relationship between skeletal muscle fatty acid oxidation and the accumulation of long-chain fatty acyl-CoAs. Muscle was obtained from normal-weight [n = 8; body mass index (BMI) 23.8 +/- 0.58 kg/m(2)], overweight/obese (n = 8; BMI 30.2 +/- 0.81 kg/m(2)), and extremely obese (n = 8; BMI 53.8 +/- 3.5 kg/m(2)) females undergoing abdominal surgery. Skeletal muscle fatty acid oxidation was assessed in intact muscle strips. Long-chain fatty acyl-CoA concentrations were measured in a separate portion of the same muscle tissue in which fatty acid oxidation was determined. Palmitate oxidation was 58 and 83% lower in skeletal muscle from extremely obese (44.9 +/- 5.2 nmol x g(-1) x h(-1)) patients compared with normal-weight (71.0 +/- 5.0 nmol x g(-1) x h(-1)) and overweight/obese (82.2 +/- 8.7 nmol x g(-1) x h(-1)) patients, respectively. Palmitate oxidation was negatively (R = -0.44, P = 0.003) associated with BMI. Long-chain fatty acyl-CoA content was higher in both the overweight/obese and extremely obese patients compared with normal-weight patients, despite significantly lower fatty acid oxidation only in the extremely obese. No associations were observed between long-chain fatty acyl-CoA content and palmitate oxidation. These data suggest that there is a defect in skeletal muscle fatty acid oxidation with extreme obesity but not overweight/obesity and that the accumulation of intramyocellular long-chain fatty acyl-CoAs is not solely a result of reduced fatty acid oxidation.     

Crucial role for LKB1 to AMPKα2 axis in the regulation of CD36-mediated long-chain fatty acid uptake into cardiomyocytes

Enhanced contractile activity increases cardiac long-chain fatty acid (LCFA) uptake via translocation of CD36 to the sarcolemma, similarly to increase in glucose uptake via GLUT4 translocation. AMP-activated protein kinase (AMPK) is assumed to mediate contraction-induced LCFA utilization. However, which catalytic isoform (AMPKα1 versus AMPKα2) is involved, is unknown. Furthermore, no studies have been performed on the role of LKB1, a kinase with AMPKK activity, on the regulation of cardiac LCFA utilization. Using different mouse models (AMPKα2-kinase-dead, AMPKα2-knockout and LKB1-knockout mice), we tested whether LKB1 and/or AMPK are required for stimulation of LCFA and glucose utilization upon treatment of cardiomyocytes with compounds (oligomycin/AICAR/dipyridamole) which induce CD36 translocation similar to that seen upon contraction. In AMPKα2- kinase-dead cardiomyocytes, the stimulating effects of oligomycin and AICAR on palmitate and deoxyglucose uptake and palmitate oxidation were almost completely lost. Moreover, in AMPKα2- and LKB1-knockout cardiomyocytes, oligomycin-induced LCFA and deoxyglucose uptake were completely abolished. However, the stimulatory effect of dipyridamole on palmitate uptake and oxidation was preserved in AMPKα2-kinase-dead cardiomyocytes. In conclusion, in the heart there is a signaling axis consisting of LKB1 and AMPKα2 which activation results in enhanced LCFA utilization, similarly to enhanced glucose uptake. In addition, an unknown dipyridamole-activated pathway can stimulate cardiac LCFA utilization by activating signaling components downstream of AMPK.     

Preexercise medium-chain triglyceride ingestion does not alter muscle glycogen use during exercise.

This investigation determined whether ingestion of a tolerable amount of medium-chain triglycerides (MCT; approximately 25 g) reduces the rate of muscle glycogen use during high-intensity exercise. On two occasions, seven well-trained men cycled for 30 min at 84% maximal O(2) uptake. Exactly 1 h before exercise, they ingested either 1) carbohydrate (CHO; 0.72 g sucrose/kg) or 2) MCT+CHO [0.36 g tricaprin (C10:0)/kg plus 0.72 g sucrose/kg]. The change in glycogen concentration was measured in biopsies taken from the vastus lateralis before and after exercise. Additionally, glycogen oxidation was calculated as the difference between total carbohydrate oxidation and the rate of glucose disappearance from plasma (R(d) glucose), as measured by stable isotope dilution techniques. The change in muscle glycogen concentration was not different during MCT+CHO and CHO (42.0 +/- 4.6 vs. 38.8 +/- 4.0 micromol glucosyl units/g wet wt). Furthermore, calculated glycogen oxidation was also similar (331 +/- 18 vs. 329 +/- 15 micromol. kg(-1). min(-1)). The coingestion of MCT+CHO did increase (P < 0.05) R(d) glucose at rest compared with CHO (26.9 +/- 1.5 vs. 20.7 +/- 0. 7 micromol.kg(-1). min(-1)), yet during exercise R(d) glucose was not different during the two trials. Therefore, the addition of a small amount of MCT to a preexercise CHO meal did not reduce muscle glycogen oxidation during high-intensity exercise, but it did increase glucose uptake at rest.     

Effect of acute activation of 5'-AMP-activated protein kinase on glycogen regulation in isolated rat skeletal muscle.

5'-AMP-activated protein kinase (AMPK) has been implicated in glycogen metabolism in skeletal muscle. However, the physiological relevance of increased AMPK activity during exercise has not been fully clarified. This study was performed to determine the direct effects of acute AMPK activation on muscle glycogen regulation. For this purpose, we used an isolated rat muscle preparation and pharmacologically activated AMPK with 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR). Tetanic contraction in vitro markedly activated the alpha(1)- and alpha(2)-isoforms of AMPK, with a corresponding increase in the rate of 3-O-methylglucose uptake. Incubation with AICAR elicited similar enhancement of AMPK activity and 3-O-methylglucose uptake in rat epitrochlearis muscle. In contrast, whereas contraction stimulated glycogen synthase (GS), AICAR treatment decreased GS activity. Insulin-stimulated GS activity also decreased after AICAR treatment. Whereas contraction activated glycogen phosphorylase (GP), AICAR did not alter GP activity. The muscle glycogen content decreased in response to contraction but was unchanged by AICAR. Lactate release was markedly increased when muscles were stimulated with AICAR in buffer containing glucose, indicating that the glucose taken up into the muscle was catabolized via glycolysis. Our results suggest that AMPK does not mediate contraction-stimulated glycogen synthesis or glycogenolysis in skeletal muscle and also that acute AMPK activation leads to an increased glycolytic flux by antagonizing contraction-stimulated glycogen synthesis.