A null mutation in skeletal muscle FAT/CD36 reveals its essential role in insulin- and AICAR-stimulated fatty acid metabolism

Fatty acid translocase (FAT)/CD36 is involved in regulating the uptake of long-chain fatty acids into muscle cells. However, the contribution of FAT/CD36 to fatty acid metabolism remains unknown. We examined the role of FAT/CD36 on fatty acid metabolism in perfused muscles (soleus and red and white gastrocnemius) of wild-type (WT) and FAT/CD36 null (KO) mice. In general, in muscles of KO mice, 1) insulin sensitivity and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) sensitivity were normal, 2) key enzymes involved in fatty acid oxidation were altered minimally or not at all, and 3) except for an increase in soleus muscle FATP1 and FATP4, these fatty acid transporters were not altered in red and white gastrocnemius muscles, whereas plasma membrane-bound fatty acid binding protein was not altered in any muscle. In KO muscles perfused under basal conditions (i.e., no insulin, no AICAR), rates of hindquarter fatty acid oxidation were reduced by 26%. Similarly, in oxidative but not glycolytic muscles, the basal rates of triacylglycerol esterification were reduced by 40%. When muscles were perfused with insulin, the net increase in fatty acid esterification was threefold greater in the oxidative muscles of WT mice compared with the oxidative muscles in KO mice. With AICAR-stimulation, the net increase in fatty acid oxidation by hindquarter muscles was 3.7-fold greater in WT compared with KO mice. In conclusion, the present studies demonstrate that FAT/CD36 has a critical role in regulating fatty acid esterification and oxidation, particularly during stimulation with insulin or AICAR.     

Electrical stimulation alters fatty acid metabolism in isolated skeletal muscle

Little is known about the contribution of plasma free fatty acid (FFA) and intramuscular triacylglycerol (TG) as substrates for energy production during prolonged electrical stimulation of skeletal muscle. The purpose of this study was to investigate the effects of continuous and intermittent electrical stimulation protocols of different intensities on exogenous FFA oxidation, exogenous FFA incorporation into intracellular TG, and intracellular TG content in the isolated in vitro rat flexor digitorum brevis muscle preparation. Muscles were electrically stimulated for 0.5 h continuously at 0.2 Hz or intermittently (30 s on, 60 s off) at 0.2, 0.4, 0.8, and 5.0 Hz while incubated at 37 degrees C in 0.5 mM palmitate-3% bovine serum albumin medium (pH 7.4) in the presence of insulin (100 microU/ml) and glucose (11 mM). Control muscles were frozen immediately after excision or incubated for 0.5 h. At similar frequencies, less exogenous FFA esterification and more exogenous FFA oxidation occurred during continuous than during intermittent stimulation. As the frequency of intermittent stimulation increased, the amount of exogenous FFA esterified decreased and the amount of exogenous FFA oxidized increased. The data also indicate that at least a portion of TG was constantly being hydrolyzed during electrical stimulation. Under stimulation conditions in which exogenous FFA esterification was below the control (resting muscle) level, intramuscular TG content was significantly decreased compared with control TG content values. Thus both plasma FFA and intramuscular TG are substrates for energy production during electrical stimulation. However, the stimulation parameters employed affect the quantities utilized.     

Subsarcolemmal and intermyofibrillar mitochondria play distinct roles in regulating skeletal muscle fatty acid metabolism

Skeletal muscle contains two populations of mitochondria that appear to be differentially affected by disease and exercise training. It remains unclear how these mitochondrial subpopulations contribute to fiber type-related and/or training-induced changes in fatty acid oxidation and regulation of carnitine palmitoyltransferase-1 (CPT1), the enzyme that controls mitochondrial fatty acid uptake in skeletal muscle. To this end, we found that fatty acid oxidation rates were 8.9-fold higher in subsarcolemmal mitochondria (SS) and 5.3-fold higher in intermyofibrillar mitochondria (IMF) that were isolated from red gastrocnemius (RG) compared with white gastrocnemius (WG) muscle, respectively. Malonyl-CoA (10 µM), a potent inhibitor of CPT1, completely abolished fatty acid oxidation in SS and IMF mitochondria from WG, whereas oxidation rates in the corresponding fractions from RG were inhibited only 89% and 60%, respectively. Endurance training also elicited mitochondrial adaptations that resulted in enhanced fatty acid oxidation capacity. Ten weeks of treadmill running differentially increased palmitate oxidation rates 100% and 46% in SS and IMF mitochondria, respectively. In SS mitochondria, elevated fatty acid oxidation rates were accompanied by a 48% increase in citrate synthase activity but no change in CPT1 activity. Nonlinear regression analyses of mitochondrial fatty acid oxidation rates in the presence of 0–100 µM malonyl-CoA indicated that IC₅₀ values were neither dependent on mitochondrial subpopulation nor affected by exercise training. However, in IMF mitochondria, training reduced the Hill coefficient (P < 0.05), suggesting altered CPT1 kinetics. These results demonstrate that endurance exercise provokes subpopulation-specific changes in mitochondrial function that are characterized by enhanced fatty acid oxidation and modified CPT1-malonyl-CoA dynamics. endurance exercise training; CPT-1; fiber type; rat; mitochondrial subpopulations     

Impaired fatty acid metabolism in type 2 diabetic skeletal muscle cells is reversed by PPARgamma agonists

The impact of type 2 diabetes on the ability of muscle to accumulate and dispose of fatty acids and triglycerides was evaluated in cultured muscle cells from nondiabetic (ND) and type 2 diabetic (T2D) subjects. In the presence of 5 microM palmitate, T2D muscle cells accumulated less lipid than ND cells (11.5 +/- 1.2 vs. 15.1 +/- 1.4 nmol/mg protein, P < 0.05). Chronic treatment (4 days) with the peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist troglitazone increased palmitate accumulation, normalizing uptake in T2D cells. There were no significant differences between groups with regard to the relative incorporation of palmitate into neutral lipid species. This distribution was also unaffected by troglitazone treatment. beta-Oxidation of both long-chain (palmitate) and medium-chain (octanoate) fatty acids in T2D muscle cells was reduced by approximately 40% compared with ND cells. Palmitate oxidation occurred primarily in mitochondrial ( approximately 40-50% of total) and peroxisomal (20-30%) compartments. The diabetes-related defect in palmitate oxidation was localized to the mitochondrial component. Both palmitate and octanoate oxidation were stimulated by a series of thiazolidinediones. Oxidation in T2D muscle cells was normalized after treatment. Troglitazone increased the mitochondrial component of palmitate oxidation. Skeletal muscle cells from T2D subjects express defects in free fatty acid metabolism that are retained in vitro, most importantly defects in beta-oxidation. These defects can be corrected by treatment with PPARgamma agonists. Augmentation of fatty acid disposal in skeletal muscle, potentially reducing intramyocellular triglyceride content, may represent one mechanism for the lipid-lowering and insulin-sensitizing effects of thiazolidinediones.     

Chronic leptin administration decreases fatty acid uptake and fatty acid transporters in rat skeletal muscle.

Chronic leptin administration reduces triacylglycerol content in skeletal muscle. We hypothesized that chronic leptin treatment, within physiologic limits, would reduce the fatty acid uptake capacity of red and white skeletal muscle due to a reduction in transport protein expression (fatty acid translocase (FAT/CD36) and plasma membrane-associated fatty acid-binding protein (FABPpm)) at the plasma membrane. Female Sprague-Dawley rats were infused for 2 weeks with leptin (0.5 mg/kg/day) using subcutaneously implanted miniosmotic pumps. Control and pair-fed animals received saline-filled implants. Leptin levels were significantly elevated (approximately 4-fold; p < 0.001) in treated animals, whereas pair-fed treated animals had reduced serum leptin levels (approximately -2-fold; p < 0.01) relative to controls. Palmitate transport rates into giant sarcolemmal vesicles were reduced following leptin treatment in both red (-45%) and white (-84%) skeletal muscle compared with control and pair-fed animals (p < 0.05). Leptin treatment reduced FAT mRNA (red, -70%, p < 0.001; white, -48%, p < 0.01) and FAT/CD36 protein expression (red, -32%; p < 0.05) in whole muscle homogenates, whereas FABPpm mRNA and protein expression were unaltered. However, in leptin-treated animals plasma membrane fractions of both FAT/CD36 and FABPpm protein expression were significantly reduced in red (-28 and -34%, respectively) and white (-44 and -56%, respectively) muscles (p < 0.05). Across all experimental treatments and muscles, palmitate uptake by giant sarcolemmal vesicles was highly correlated with the plasma membrane FAT/CD36 protein (r = 0.88, p < 0.01) and plasma membrane FABPpm protein (r = 0.94, p < 0.01). These studies provide the first evidence that protein-mediated long chain fatty acid transport is subject to long term regulation by leptin.     

Lung injury-induced skeletal muscle wasting in aged mice is linked to alterations in long chain fatty acid metabolism

Introduction

Older patients are more likely to acquire and die from acute respiratory distress syndrome (ARDS) and muscle weakness may be more clinically significant in older persons. Recent data implicate muscle ring finger protein 1 (MuRF1) in lung injury-induced skeletal muscle atrophy in young mice and identify an alternative role for MuRF1 in cardiac metabolism regulation through inhibition of fatty acid oxidation.

Objectives

To develop a model of lung injury-induced muscle wasting in old mice and to evaluate the skeletal muscle metabolomic profile of adult and old acute lung injury (ALI) mice.

Methods

Young (2 month), adult (6 month) and old (20 month) male C57Bl6 J mice underwent Sham (intratracheal H₂O) or ALI [intratracheal E. coli lipopolysaccharide (i.t. LPS)] conditions and muscle functional testing. Metabolomic analysis on gastrocnemius muscle was performed using gas chromatography-mass spectrometry (GC–MS).

Results

Old ALI mice had increased mortality and failed to recover skeletal muscle function compared to adult ALI mice. Muscle MuRF1 expression was increased in old ALI mice at day 3. Non-targeted muscle metabolomics revealed alterations in amino acid biosynthesis and fatty acid metabolism in old ALI mice. Targeted metabolomics of fatty acid intermediates (acyl-carnitines) and amino acids revealed a reduction in long chain acyl-carnitines in old ALI mice.

Conclusion

This study demonstrates age-associated susceptibility to ALI-induced muscle wasting which parallels a metabolomic profile suggestive of altered muscle fatty acid metabolism. MuRF1 activation may contribute to both atrophy and impaired fatty acid oxidation, which may synergistically impair muscle function in old ALI mice.

     

A potent PPARalpha agonist stimulates mitochondrial fatty acid beta-oxidation in liver and skeletal muscle

The proposed mechanism for the triglyceride (TG) lowering by fibrate drugs is via activation of the peroxisome proliferator-activated receptor-alpha (PPARalpha). Here we show that a PPARalpha agonist, ureido-fibrate-5 (UF-5), approximately 200-fold more potent than fenofibric acid, exerts TG-lowering effects (37%) in fat-fed hamsters after 3 days at 30 mg/kg. In addition to lowering hepatic apolipoprotein C-III (apoC-III) gene expression by approximately 60%, UF-5 induces hepatic mitochondrial carnitine palmitoyltransferase I (CPT I) expression. A 3-wk rising-dose treatment results in a greater TG-lowering effect (70%) at 15 mg/kg and a 2.3-fold elevation of muscle CPT I mRNA levels, as well as effects on hepatic gene expression. UF-5 also stimulated mitochondrial [3H]palmitate beta-oxidation in vitro in human hepatic and skeletal muscle cells 2.7- and 1.6-fold, respectively, in a dose-related manner. These results suggest that, in addition to previously described effects of fibrates on apoC-III expression and on peroxisomal fatty acid (FA) beta-oxidation, PPARalpha agonists stimulate mitochondrial FA beta-oxidation in vivo in both liver and muscle. These observations suggest an important mechanism for the biological effects of PPARalpha agonists.     

Increased glucose oxidation in H-FABP null soleus muscle is associated with defective triacylglycerol accumulation and mobilization, but not with the defect of exogenous fatty acid oxidation

Heart-type fatty acid-binding protein (H-FABP) is a major fatty acid-binding factor in skeletal muscles. Genetic lack of H-FABP severely impairs the esterification and oxidation of exogenous fatty acids in soleus muscles isolated from chow-fed mice (CHOW-solei) and high fat diet-fed mice (HFD-solei), and prevents the HFD-induced accumulation of muscle triacylglycerols (TAGs). Here, we examined the impact of H-FABP deficiency on the relationship between fatty acid utilization and glucose oxidation. Glucose oxidation was measured in isolated soleus muscles in the presence or absence of 1 mM palmitate (simple protocol) or in the absence of fatty acid after preincubation with 1 mM palmitate (complex protocol). With the simple protocol, the mutation slightly reduced glucose oxidation in CHOW-muscles, but markedly increased it in HFD-muscles; unexpectedly, this pattern was not altered by the addition of palmitate, which reduced glucose oxidation in both CHOW- and HFD-solei irrespective of the mutation. In the complex protocol, the mutation first inhibited the synthesis and accumulation of TAGs and then their mobilization; with this protocol, the mutation increased glucose oxidation in both CHOW- and HFD-solei. We conclude: (i) H-FABP mediates a non-acute inhibition of muscle glucose oxidation by fatty acids, likely by enabling both the accumulation and mobilization of a critical mass of muscle TAGs; (ii) H-FABP does not mediate the acute inhibitory effect of extracellular fatty acids on muscle glucose oxidation; (iii) H-FABP affects muscle glucose oxidation in opposing ways, with inhibition prevailing at high muscle TAG contents.     

Cytokine regulation of skeletal muscle fatty acid metabolism: effect of interleukin-6 and tumor necrosis factor-alpha

IL-6 and TNF-alpha have been associated with insulin resistance and type 2 diabetes. Furthermore, abnormalities in muscle fatty acid (FA) metabolism are strongly associated with the development of insulin resistance. However, few studies have directly examined the effects of either IL-6 or TNF-alpha on skeletal muscle FA metabolism. Here, we used a pulse-chase technique to determine the effect of IL-6 (50-5,000 pg/ml) and TNF-alpha (50-5,000 pg/ml) on FA metabolism in isolated rat soleus muscle. IL-6 (5,000 pg/ml) increased exogenous and endogenous FA oxidation by approximately 50% (P < 0.05) but had no effect on FA uptake or incorporation of FA into endogenous lipid pools. In contrast, TNF-alpha had no effect on FA oxidation but increased FA incorporation into diacylglycerol (DAG) by 45% (P < 0.05). When both IL-6 (5,000 pg/ml) and insulin (10 mU/ml) were present, IL-6 attenuated insulin's suppressive effect on FA oxidation, increasing exogenous FA oxidation (+37%, P < 0.05). Furthermore, in the presence of insulin, IL-6 reduced the esterification of FA to triacylglycerol by 22% (P < 0.05). When added in combination with IL-6 or leptin (10 microg/ml), the TNF-alpha-induced increase in DAG synthesis was inhibited. In conclusion, the results demonstrate that IL-6 plays an important role in regulating fat metabolism in muscle, increasing rates of FA oxidation, and attenuating insulin's lipogenic effects. In contrast, TNF-alpha had no effect on FA oxidation but increased FA incorporation into DAG, which may be involved in the development of TNF-alpha-induced insulin resistance in skeletal muscle.     

Nonacute effects of H-FABP deficiency on skeletal muscle glucose uptake in vitro

Heart-type fatty acid-binding protein (H-FABP) is required for high rates of skeletal muscle long-chain fatty acid (LCFA) oxidation and esterification. Here we assessed whether H-FABP affects soleus muscle glucose uptake when measured in vitro in the absence of LCFA. Wild-type and H-FABP null mice were fed a standard chow or high-fat diet before muscle isolation. With the chow, the mutation increased insulin-dependent deoxyglucose uptake by 141% (P < 0.01) at 0.02 mU/ml of insulin but did not cause a significant effect at 2 mU/ml of insulin; skeletal muscle triglyceride and long-chain acyl-CoA (LCA-CoA) levels remained normal. With the high-fat diet, the mutation increased insulin-dependent deoxyglucose uptake by 190% (P < 0.01) at 2 mU/ml of insulin, thus partially preventing insulin resistance, and it completely prevented the threefold (P < 0.001) diet-induced increase of muscle triglyceride levels; however, muscle LCA-CoA levels showed little or no reduction. With both diets, the mutation reduced the basal (insulin-independent) soleus muscle deoxyglucose uptake by 28% (P < 0.05). These results establish a close relation between FABP-dependent lipid pools and insulin sensitivity and indicate the existence of a nonacute, antagonistic, and H-FABP-dependent fatty acid regulation of basal and insulin-dependent muscle glucose uptake.     

Acute effect of fatty acids on metabolism and mitochondrial coupling in skeletal muscle

Acute effects of free fatty acids (FFA) were investigated on: (1) glucose oxidation, and UCP-2 and -3 mRNA and protein levels in 1 h incubated rat soleus and extensor digitorium longus (EDL) muscles, (2) mitochondrial membrane potential in cultured skeletal muscle cells, (3) respiratory activity and transmembrane electrical potential in mitochondria isolated from rat skeletal muscle, and (4) oxygen consumption by anesthetized rats. Long-chain FFA increased both basal and insulin-stimulated glucose oxidation in incubated rat soleus and EDL muscles and reduced mitochondrial membrane potential in C2C12 myotubes and rat skeletal muscle cells. Caprylic, palmitic, oleic, and linoleic acid increased O(2) consumption and decreased electrical membrane potential in isolated mitochondria from rat skeletal muscles. FFA did not alter UCP-2 and -3 mRNA and protein levels in rat soleus and EDL muscles. Palmitic acid increased oxygen consumption by anesthetized rats. These results suggest that long-chain FFA acutely lead to mitochondrial uncoupling in skeletal muscle.     

Liver fatty acid-binding protein gene ablation inhibits branched-chain fatty acid metabolism in cultured primary hepatocytes

Whereas the role of liver fatty acid-binding protein (L-FABP) in the uptake, transport, mitochondrial oxidation, and esterification of normal straight-chain fatty acids has been studied extensively, almost nothing is known regarding the function of L-FABP in peroxisomal oxidation and metabolism of branched-chain fatty acids. Therefore, phytanic acid (most common dietary branched-chain fatty acid) was chosen to address these issues in cultured primary hepatocytes isolated from livers of L-FABP gene-ablated (-/-) and wild type (+/+) mice. These studies provided three new insights: First, L-FABP gene ablation reduced maximal, but not initial, uptake of phytanic acid 3.2-fold. Initial uptake of phytanic acid uptake was unaltered apparently due to concomitant 5.3-, 1.6-, and 1.4-fold up-regulation of plasma membrane fatty acid transporter/translocase proteins (glutamic-oxaloacetic transaminase, fatty acid transport protein, and fatty acid translocase, respectively). Second, L-FABP gene ablation inhibited phytanic acid peroxisomal oxidation and microsomal esterification. These effects were consistent with reduced cytoplasmic fatty acid transport as evidenced by multiphoton fluorescence photobleaching recovery, where L-FABP gene ablation reduced the cytoplasmic, but not membrane, diffusional component of NBD-stearic acid movement 2-fold. Third, lipid analysis of the L-FABP gene-ablated hepatocytes revealed an altered fatty acid phenotype. Free fatty acid and triglyceride levels were decreased 1.9- and 1.6-fold, respectively. In summary, results with cultured primary hepatocytes isolated from L-FABP (+/+) and L-FABP (-/-) mice demonstrated for the first time a physiological role of L-FABP in the uptake and metabolism of branched-chain fatty acids.     

Peripheral effect of alpha-melanocyte-stimulating hormone on fatty acid oxidation in skeletal muscle

To study the peripheral effects of melanocortin on fuel homeostasis in skeletal muscle, we assessed palmitate oxidation and AMP kinase activity in alpha-melanocyte-stimulating hormone (alpha-MSH)-treated muscle cells. After alpha-MSH treatment, carnitine palmitoyltransferase-1 and fatty acid oxidation (FAO) increased in a dose-dependent manner. A strong melanocortin agonist, NDP-MSH, also stimulated FAO in primary culture muscle cells and C2C12 cells. However, [Glu6]alpha-MSH-ND, which has ample MC4R and MC3R agonistic activity, stimulated FAO only at high concentrations (10(-5) M). JKC-363, a selective MC4R antagonist, did not suppress alpha-MSH-induced FAO. Meanwhile, SHU9119, which has both antagonistic activity on MC3R and MC4R and agonistic activity on both MC1R and MC5R, increased the effect of alpha-MSH on FAO in both C2C12 and primary muscle cells. Small interference RNA against MC5R suppressed the alpha-MSH-induced FAO effectively. cAMP analogues mimicked the effect of alpha-MSH on FAO, and the effects of both alpha-MSH and cAMP analogue-mediated FAO were antagonized by a protein kinase A inhibitor (H89) and a cAMP antagonist ((Rp)-cAMP). Acetyl-CoA carboxylase activity was suppressed by alpha-MSH and cAMP analogues by phosphorylation through AMP-activated protein kinase activation in C2C12 cells. Taken together, these results suggest that alpha-MSH increases FAO in skeletal muscle, in which MC5R may play a major role. Furthermore, these results suggest that alpha-MSH-induced FAO involves cAMP-protein kinase A-mediated AMP-activated protein kinase activation.     

Identification of fatty acid translocase on human skeletal muscle mitochondrial membranes: essential role in fatty acid oxidation

Fatty acid translocase (FAT/CD36) is a transport protein with a high affinity for long-chain fatty acids (LCFA). It was recently identified on rat skeletal muscle mitochondrial membranes and found to be required for palmitate uptake and oxidation. Our aim was to identify the presence and elucidate the role of FAT/CD36 on human skeletal muscle mitochondrial membranes. We demonstrate that FAT/CD36 is present in highly purified human skeletal mitochondria. Blocking of human muscle mitochondrial FAT/CD36 with the specific inhibitor sulfo-N-succimidyl-oleate (SSO) decreased palmitate oxidation in a dose-dependent manner. At maximal SSO concentrations (200 muM) palmitate oxidation was decreased by 95% (P<0.01), suggesting an important role for FAT/CD36 in LCFA transport across the mitochondrial membranes. SSO treatment of mitochondria did not affect mitochondrial octanoate oxidation and had no effect on maximal and submaximal carnitine palmitoyltransferase I (CPT I) activity. However, SSO treatment did inhibit palmitoylcarnitine oxidation by 92% (P<0.001), suggesting that FAT/CD36 may be playing a role downstream of CPT I activity, possibly in the transfer of palmitoylcarnitine from CPT I to carnitine-acylcarnitine translocase. These data provide new insight regarding human skeletal muscle mitochondrial fatty acid (FA) transport, and suggest that FAT/CD36 could be involved in the cellular and mitochondrial adaptations resulting in improved and/or impaired states of FA oxidation.     

Heparin inhibits skeletal muscle growth in vitro

Heparin or heparan sulfate proteoglycan (HeSPG), but not chondroitin sulfate or hyaluronic acid, exerts a pronounced inhibitory effect on muscle growth in vitro, as determined by total protein, myosin accumulation or synthesis, and [3H]thymidine incorporation studies. Primary muscle fibroblast culture growth is also inhibited by heparin but to a substantially lesser degree compared to muscle (30% and over 90% inhibition of growth, respectively). Heparin-induced inhibition of skeletal muscle growth is a consequence of its interaction with a growth factor(s) present in the media used to support myogenesis; heparin-Sepharose column absorbed horse serum can support muscle growth only in the presence of added heparin-binding growth factors like fibroblast growth factor (FGF) or chicken muscle growth factor (CMGF). Furthermore, heparin prevents the binding of iodinated FGF to the myoblast surface. We also show that the extent of muscle growth is a function of the relative amounts of heparin and FGF in culture. Finally, we provide evidence indicating that FGF can combine with endogenously occurring heparin-like components: immobilized FGF binds sodium-[35S]sulfate labeled components secreted in muscle culture conditioned medium, an interaction inhibited by anti-HeSPG antibodies or heparin, but not by other sulfated glycosaminoglycans. Since heparin binding growth factors not only stimulate myoblast proliferation but also actively inhibit the onset of muscle differentiation (G. Spitzz, D. Roman, and A. Strauss (1986). J. Biol. Chem. 261, 9483-9488), their interaction with naturally occurring heparin-like components may be an important physiological mechanism for modulating muscle growth and differentiation in development and regeneration.     

Heat stress inhibits skeletal muscle hypertrophy

Heat shock proteins (Hsps) are molecular chaperones that aid in protein synthesis and trafficking and have been shown to protect cells/tissues from various protein damaging stressors. To determine the extent to which a single heat stress and the concurrent accumulation of Hsps influences the early events of skeletal muscle hypertrophy, Sprague-Dawley rats were heat stressed (42 degrees C, 15 minutes) 24 hours prior to overloading 1 plantaris muscle by surgical removal of the gastrocnemius muscle. The contralateral plantaris muscles served as controls. Heat-stressed and/or overloaded plantaris muscles were assessed for muscle mass, total muscle protein, muscle protein concentration, Type I myosin heavy chain (Type I MHC) content, as well as Hsp72 and Hsp25 content over the course of 7 days following removal of the gastrocnemius muscle. As expected, in non-heat-stressed animals, muscle mass, total muscle protein and MHC I content were significantly increased (P < 0.05) following overload. In addition, Hsp25 and Hsp72 increased significantly after 2 and 3 days of overload, respectively. A prior heat stress-elevated Hsp25 content to levels similar to those measured following overload alone, but heat stress-induced Hsp72 content was increased significantly greater than was elicited by overload alone. Moreover, overloaded muscles from animals that experienced a prior heat stress showed a lower muscle mass increase at 5 and 7 days; a reduced total muscle protein elevation at 3, 5, and 7 days; reduced protein concentration; and a diminished Type I MHC content accumulation at 3, 5, and 7 days relative to nonheat-stressed animals. These data suggest that a prior heat stress and/or the consequent accumulation of Hsps may inhibit increases in muscle mass, total muscle protein content, and Type I MHC in muscles undergoing hypertrophy.     

Ammonia and amino acid metabolism in human skeletal muscle during exercise.

This review focuses on the ammonia and amino acid metabolic responses of active human skeletal muscle, with a particular emphasis on steady-state exercise. Ammonia production in skeletal muscle involves the purine nucleotide cycle and the amino acids glutamate, glutamine, and alanine and probably also includes the branched chain amino acids as well as aspartate. Ammonia production is greatest during prolonged, steady state exercise that requires 60-80% VO2max and is associated with glutamine and alanine metabolism. Under these circumstances it is unresolved whether the purine nucleotide cycle (AMP deamination) is active; if so, it must be cycling with no IMP accumulation. It is proposed that under these circumstances the ammonia is produced from slow twitch fibers by the deamination of the branched chain amino acids. The ammonia response can be suppressed by increasing the carbohydrate availability and this may be mediated by altering the availability of the branched chain amino acids. The fate of the ammonia released into the circulation is unresolved, but there is indirect evidence that a considerable portion may be excreted by the lung in expired air.     

Increased mitochondrial matrix-directed superoxide production by fatty acid hydroperoxides in skeletal muscle mitochondria

Previous studies have shown that muscle atrophy is associated with mitochondrial dysfunction and an increased rate of mitochondrial reactive oxygen species production. We recently demonstrated that fatty acid hydroperoxides (FA-OOHs) are significantly elevated in mitochondria isolated from atrophied muscles. The purpose of this study was to determine whether FA-OOHs can alter skeletal muscle mitochondrial function. We found that FA-OOHs (at low-micromolar concentrations) induce mitochondrial dysfunction assessed by a decrease in the rate of ATP production, oxygen consumption, and activity of respiratory chain complexes I and III. Using methods to distinguish superoxide release toward the matrix and toward the intermembrane space, we demonstrate that FA-OOHs significantly elevate oxidative stress in the mitochondrial matrix (and not the intermembrane space), with complex I as the major site of superoxide production (most probably from a site upstream of the ubiquinone binding site but downstream from the flavin binding site-the iron sulfur clusters). Our results are the first to indicate that FA-OOHs are important modulators of mitochondrial function and oxidative stress in skeletal muscle mitochondria and may play an important role in muscle atrophies that are associated with increased generation of FA-OOHs, e.g., denervation-induced muscle atrophy.