Regulation of CPT I activity in intermyofibrillar and subsarcolemmal mitochondria from human and rat skeletal muscle

Carnitine palmitoyltransferase I (CPT I) is considered the rate-limiting enzyme in the transfer of long-chain fatty acids (LCFA) into the mitochondria and is reversibly inhibited by malonyl-CoA (M-CoA) in vitro. In rat skeletal muscle, M-CoA levels decrease during exercise, releasing the inhibition of CPT I and increasing LCFA oxidation. However, in human skeletal muscle, M-CoA levels do not change during moderate-intensity exercise despite large increases in fat oxidation, suggesting that M-CoA is not the sole regulator of increased CPT I activity during exercise. In the present study, we measured CPT I activity in intermyofibrillar (IMF) and subsarcolemmal (SS) mitochondria isolated from human vastus lateralis (VL), rat soleus (Sol), and red gastrocnemius (RG) muscles. We tested whether exercise-related levels ( approximately 65% maximal O2 uptake) of calcium and adenylate charge metabolites (free AMP, ADP, and Pi) could override the M-CoA-induced inhibition of CPT I activity and explain the increased CPT I flux during exercise. Protein content was approximately 25-40% higher in IMF than in SS mitochondria in all muscles. Maximal CPT I activity was similar in IMF and SS mitochondria in all muscles (VL: 282 +/- 46 vs. 280 +/- 51; Sol: 390 +/- 81 vs. 368 +/- 82; RG: 252 +/- 71 vs. 278 +/- 44 nmol.min-1.mg protein-1). Sensitivity to M-CoA did not differ between IMF and SS mitochondria in all muscles (25-31% inhibition in VL, 52-70% in Sol and RG). Calcium and adenylate charge metabolites did not override the M-CoA-induced inhibition of CPT I activity in mitochondria isolated from VL, Sol, and RG muscles. Decreasing pH from 7.1 to 6.8 reduced CPT I activity by approximately 34-40% in both VL mitochondrial fractions. In summary, this study reports no differences in CPT I activity or sensitivity to M-CoA between IMF and SS mitochondria isolated from human and rat skeletal muscles. Exercise-induced increases in calcium and adenylate charge metabolites do not appear responsible for upregulating CPT I activity in human or rat skeletal muscle during moderate aerobic exercise.     

Glycogenesis in muscle and liver during exercise

We hypothesized that glycogenesis increases in muscle during exercise before significant glycogen depletion occurs. Therefore, rats ran for 15 or 90 min at speeds of 8-22 m/min. D-[5-3H]glucose (10 microCi/100 g body wt) was administered 10 min before the end of exercise. Hindlimb muscles [soleus (SOL), plantaris (PL), extensor digitorum longus (EDL), and red (RG) and white gastrocnemius (WG)] and a portion of liver were analyzed for glycogen concentrations and rates of glycogen synthesis (i.e., D-[3H]glucose incorporated into glycogen). At rest, marked differences were observed among muscles in their rates of glucose incorporation into glycogen: i.e., SOL = 24.3 +/- 3.1, RG = 5.4 +/- 1.9, PL = 2.8 +/- 1.1, EDL = 0.54 +/- 0.10, WG = 0.12 +/- 0.02 (SE) dpm.micrograms glycogen-1.10 min-1 (P less than 0.05 between respective muscles). Compared with the glucose incorporation into glycogen at rest, increments in the PL (272%), RG (189%), WG (400%), EDL (274%), and liver (175%) were observed after 90 min of exercise (P less than 0.05, all data). In contrast, a decrease in glucose incorporation into glycogen (-62%) occurred in the SOL at min 15 (P less than 0.05), but this returned to the rates observed at rest after 90 min of exercise. This measure for rates of net glycogen synthesis (dpm.microgram glycogen-1.10 min-1) was weakly related to the ambient glycogen levels in most muscles; the exception was the SOL (r = -0.79; P less than 0.05). There was up to a 50-fold difference in glycogen synthesis among muscles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coordinately regulated expression of FAT/CD36 and FACS1 in rat skeletal muscle

Protein-mediated fatty acid uptake and intracellular fatty acid activation are key steps in fatty acid metabolism in muscle.We have examined (a) the abundance of fatty acid translocase (FAT/CD36) mRNA (a fatty acid transporter) and long-chain acyl CoA synthetase (FACS1) mRNA in metabolically heterogeneous muscles (soleus (SOL), red (RG) and white gastrocnemius (WG)), and (b) whether FAT/CD36 and FACS1 mRNAs were coordinately upregulated in red (RTA) and white tibialis muscles (WTA) that had been chronically stimulated for varying periods of time (0.25, 1, 6 and 24 h/day) for 7 days. FAT/CD36 mRNA and FACS1 mRNA abundance were scaled with (a) the oxidative capacity of muscle (SOL > RG > WG) (p < 0.05), (b) the rates of fatty acid oxidation in red and white muscles, and (c) fatty acid uptake by sarcolemmal vesicles, derived from red and white muscles. In chronically stimulated muscles (RTA and WTA), FAT/CD36 mRNA and FACS1 mRNA were up-regulated in relation to the quantity of muscle contractile activity (p < 0.05). FAT/CD36 mRNA and FACS1 mRNA up-regulation was highly correlated (r = 0.98). The coordinated expression of FAT/CD36 and FACS is likely a functional adaptive response to facilitate a greater rate of fatty acid activation in response to a greater rate of fatty acid transport, either among different types of muscles or in muscles in which capacity for fatty acid metabolism has been enhanced.     

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     

Overexpression of carnitine palmitoyltransferase I in skeletal muscle in vivo increases fatty acid oxidation and reduces triacylglycerol esterification

A key regulatory point in the control of fatty acid (FA) oxidation is thought to be transport of FAs across the mitochondrial membrane by carnitine palmitoyltransferase I (CPT I). To investigate the role of CPT I in FA metabolism, we used in vivo electrotransfer (IVE) to locally overexpress CPT I in muscle of rodents. A vector expressing the human muscle isoform of CPT I was electrotransferred into the right lateral muscles of the distal hindlimb [tibialis cranialis (TC) and extensor digitorum longus (EDL)] of rats, and a control vector expressing GFP was electrotransferred into the left muscles. Initial studies showed that CPT I protein expression peaked 7 days after IVE (+104%, P<0.01). This was associated with an increase in maximal CPT I activity (+30%, P < 0.001) and a similar increase in palmitoyl-CoA oxidation (+24%; P<0.001) in isolated mitochondria from the TC. Importantly, oxidation of the medium-chain FA octanoyl-CoA and CPT I sensitivity to inhibition by malonyl-CoA were not altered by CPT I overexpression. FA oxidation in isolated EDL muscle strips was increased with CPT I overexpression (+28%, P<0.01), whereas FA incorporation into the muscle triacylglycerol (TAG) pool was reduced (-17%, P<0.01). As a result, intramyocellular TAG content was decreased with CPT I overexpression in both the TC (-25%, P<0.05) and the EDL (-45%, P<0.05). These studies demonstrate that acute overexpression of CPT I in muscle leads to a repartitioning of FAs away from esterification and toward oxidation and highlight the importance of CPT I in regulating muscle FA metabolism.     

Regulation of carnitine palmitoyltransferase (CPT) I during fasting in rainbow trout (Oncorhynchus mykiss) promotes increased mitochondrial fatty acid oxidation

Periods of fasting, in most animals, are fueled principally by fatty acids, and changes in the regulation of fatty acid oxidation must exist to meet this change in metabolic substrate use. We examined the regulation of carnitine palmitoyltransferase (CPT) I, to help explain changes in mitochondrial fatty acid oxidation with fasting. After fasting rainbow trout (Oncorhynchus mykiss) for 5 wk, the mitochondria were isolated from red muscle and liver to determine (1) mitochondrial fatty acid oxidation rate, (2) CPT I activity and the concentration of malonyl-CoA needed to inhibit this activity by 50% (IC(50)), (3) mitochondrial membrane fluidity, and (4) CPT I (all five known isoforms) and peroxisome proliferator-activated receptor (PPARα and PPARβ) mRNA levels. Fatty acid oxidation in isolated mitochondria increased during fasting by 2.5- and 1.75-fold in liver and red muscle, respectively. Fasting also decreased sensitivity of CPT I to malonyl-CoA (increased IC(50)), by two and eight times in red muscle and liver, respectively, suggesting it facilitates the rate of fatty acid oxidation. In the liver, there was also a significant increase CPT I activity per milligram mitochondrial protein and in whole-tissue PPARα and PPARβ mRNA levels. However, there were no changes in mitochondrial membrane fluidity in either tissue, indicating that the decrease in CPT I sensitivity to malonyl-CoA is not due to bulk fluidity changes in the membrane. However, there were significant differences in CPT I mRNA levels during fasting. Overall, these data indicate some important changes in the regulation of CPT I that promote the increased mitochondrial fatty acid oxidation that occurs during fasting in trout.     

Differential effects of repetitive activity on sarcoplasmic reticulum responses in rat muscles of different oxidative potential

We investigated the hypothesis that muscles of different oxidative potential would display differences in sarcoplasmic reticulum (SR) Ca2+ handling responses to repetitive contractile activity and recovery. Repetitive activity was induced in two muscles of high oxidative potential, namely, soleus (SOL) and red gastrocnemius (RG), and in white gastrocnemius (WG), a muscle of low oxidative potential, by stimulation in adult male rats. Measurements of SR properties, performed in crude homogenates, were made on control and stimulated muscles at the start of recovery (R0) and at 25 min of recovery (R25). Maximal Ca2+-ATPase activity (Vmax, micromol x g protein(-1) x min(-1)) at R0 was lower in stimulated SOL (105 +/- 9 vs. 135 +/- 7) and RG (269 +/- 22 vs. 317 +/- 26) and higher (P < 0.05) in WG (795 +/- 32 vs. 708 +/- 34). At R25, Vmax remained lower (P < 0.05) in SOL and RG but recovered in WG. Ca2+ uptake, measured at 2,000 nM, was depressed (P < 0.05) in SOL and RG by 34 and 13%, respectively, in stimulated muscles at R0 and remained depressed (P < 0.05) at R25. In contrast, Ca2+ uptake was elevated (P < 0.05) in stimulated WG at R0 by 9% and remained elevated (P < 0.05) at R25. Ca2+ release, unaltered in SOL and RG at both R0 and R25, was increased (P < 0.05) in stimulated WG at both R0 and R25. We conclude that SR Ca2+-handling responses to repetitive contractile activity and recovery are related to the oxidative potential of muscle.     

Expression of genes participating in regulation of fatty acid and glucose utilization and energy metabolism in developing rat hearts

The heart is a unique organ that can use several fuels for energy production. During development, the heart undergoes changes in fuel supply, and it must be able to respond to these changes. We have examined changes in the expression of several genes that regulate fuel transport and metabolism in rat hearts during early development. At birth, there was increased expression of fatty acid transporters and enzymes of fatty acid metabolism that allow fatty acids to become the major source of energy for cardiac muscle during the first 2 wk of life. At the same time, expression of genes that control glucose transport and oxidation was downregulated. After 2 wk, expression of genes for glucose uptake and oxidation was increased, and expression of genes for fatty acid uptake and utilization was decreased. Expression of carnitine palmitoyltransferase I (CPT I) isoforms during development was different from published data obtained from rabbit hearts. CPT Ialpha and Ibeta isoforms were both highly expressed in hearts before birth, and both increased further at birth. Only after the second week did CPT Ialpha expression decrease appreciably below the level of CPT Ibeta expression. These results represent another example of different expression patterns of CPT I isoforms among various mammalian species. In rats, changes in gene expression followed nutrient availability during development and may render cardiac fatty acid oxidation less sensitive to factors that influence malonyl-CoA content (e.g., fluctuations in glucose concentration) and thereby favor fatty acid oxidation as an energy source for cardiomyocytes in early development.     

Inverse relationship between PGC-1alpha protein expression and triacylglycerol accumulation in rodent skeletal muscle.

PGC-1alpha is a key regulator of tissue metabolism, including skeletal muscle. Because it has been shown that PGC-1alpha alters the capacity for lipid metabolism, it is possible that PGC-1alpha expression is regulated by the intramuscular lipid milieu. Therefore, we have examined the relationship between PGC-1alpha protein expression and the intramuscular fatty acid accumulation in hindlimb muscles of animals in which the capacity for fatty acid accumulation in muscle is increased (Zucker obese rat) or reduced [FAT/CD36 null (KO) mice]. Rates of palmitate incorporation into triacylglycerols were determined in perfused red (RG) and white gastrocnemius (WG) muscles of lean and obese Zucker rats and in perfused RG and WG muscles of FAT/CD36 KO and wild-type (WT) mice. In obese Zucker rats, the rate of palmitate incorporation into triacylglycerol depots in RG and WG muscles were 28 and 24% greater than in lean rats (P < 0.05). In FAT/CD36 KO mice, the rates of palmitate incorporation into triacylglycerol depots were lower in RG (-50%) and WG muscle (-24%) compared with the respective muscles in WT mice (P < 0.05). In the obese animals, PGC-1alpha protein content was reduced in both RG (-13%) and WG muscles (-15%) (P < 0.05). In FAT/CD36 KO mice, PGC-1alpha protein content was upregulated in both RG (+32%, P < 0.05) and WG muscles (+50%, P < 0.05). In conclusion, from studies in these two animal models, it appears that PGC-1alpha protein expression is inversely related to components of intramuscular lipid metabolism, because 1) PGC-1alpha protein expression is downregulated when triacylglycerol synthesis rates, an index of intramuscular lipid metabolism, are increased, and 2) PGC-1alpha protein expression is upregulated when triacylglycerol synthesis rates are reduced. Therefore, we speculate that the intramuscular lipid sensing may be involved in regulating the protein expression of PGC-1alpha in skeletal muscle.     

Acute hyperammonemia activates branched-chain amino acid catabolism and decreases their extracellular concentrations: different sensitivity of red and white muscle

Hyperammonemia is considered to be the main cause of decreased levels of the branched-chain amino acids (BCAA), valine, leucine, and isoleucine, in liver cirrhosis. In this study we investigated whether the decrease in BCAA is caused by the direct effect of ammonia on BCAA metabolism and the effect of ammonia on BCAA and protein metabolism in different types of skeletal muscle. M. soleus (SOL, slow-twitch, red muscle) and m. extensor digitorum longus (EDL, fast-twitch, white muscle) of white rat were isolated and incubated in a medium with or without 500 μM ammonia. We measured the exchange of amino acids between the muscle and the medium, amino acid concentrations in the muscle, release of branched-chain keto acids (BCKA), leucine oxidation, total and myofibrillar proteolysis, and protein synthesis. Hyperammonemia inhibited the BCAA release (81% in SOL and 60% in EDL vs. controls), increased the release of BCKA (133% in SOL and 161% in EDL vs. controls) and glutamine (138% in SOL and 145% in EDL vs. controls), and increased the leucine oxidation in EDL (174% of controls). Ammonia also induced a significant increase in glutamine concentration in skeletal muscle. The effect of ammonia on intracellular BCAA concentration, protein synthesis and on total and myofibrillar proteolysis was insignificant. The data indicates that hyperammonemia directly affects the BCAA metabolism in skeletal muscle which results in decreased levels of BCAA in the extracellular fluid. The effect is associated with activated synthesis of glutamine, increased BCAA oxidation, decreased release of BCAA, and enhanced release of BCKA. These metabolic changes are not directly associated with marked changes in protein turnover. The effect of ammonia is more pronounced in muscles with high content of white fibres.     

Characterization of Lipolytic Inhibitor G(0)/G(1) Switch Gene-2 Protein (G0S2) Expression in Male Sprague-Dawley Rat Skeletal Muscle Compared to Relative Content of Adipose Triglyceride Lipase (ATGL) and Comparitive Gene Identification-58 (CGI-58)

The rate-limiting enzyme in lipolysis, adipose triglyceride lipase (ATGL), is activated by comparative gene identification-58 (CGI-58) and inhibited by the G(0)/G(1) switch gene-2 (G0S2) protein. It is speculated that inhibition of ATGL is through a dose dependent manner of relative G0S2 protein content. There is little work examining G0S2 expression in lipolytic tissues, and the relative expression across oxidative tissues such as skeletal muscle has not yet been described. Three muscles, soleus (SOL), red gastrocnemius (RG), and white gastrocnemius (WG) were excised from 57-day old male Sprague-Dawley rats (n = 9). QRT-PCR was used for mRNA analysis, and western blotting was conducted to determine protein content. ATGL and G0S2 protein content were both greatest in the lipolytic SOL, with the least amount of both ATGL and G0S2 protein content found in the WG. CGI-58 protein content however did not mirror ATGL and G0S2 protein content, since the RG had the greatest CGI-58 protein content when compared to the SOL and WG. When comparing our tissues based on CGI-58-to-ATGL ratio and G0S2-to-ATGL ratio, it was discovered that contrary to oxidative demand, the glycolytic WG had the greatest activator CGI-58-to-ATGL ratio with the oxidative SOL having the least, and no differences in G0S2-to-ATGL across the three muscle types. These data suggest that the content of G0S2 relative to the lipase in skeletal muscle would not predict lipolytic potential.     

Na+-K+-ATPase in rat skeletal muscle: content, isoform, and activity characteristics.

The purpose of this study was to investigate the hypothesis that muscle Na+-K+-ATPase activity is directly related to Na+-K+-ATPase content and the content of the alpha2-catalytic isoform in muscles of different fiber-type composition. To investigate this hypothesis, tissue was sampled from soleus (Sol), red gastrocnemius (RG), white gastrocnemius (WG), and extensor digitorum longus (EDL) muscles at rest from 38 male Wistar rats weighing 413 +/- 6.0 g (mean +/- SE). Na+-K+-ATPase activity was determined in homogenates (Hom) and isolated crude membranes (CM) by the regenerating ouabain-inhibitable hydrolytic activity assay (ATPase) and the 3-O-methylfluorescein K+-stimulated phosphatase (3-O-MFPase) assay in vitro. In addition, Na+-K+-ATPase content (Bmax) and the distribution of alpha1-, alpha2-, beta1-, and beta2-isoforms were determined by [3H]ouabain binding and Western blot, respectively. For the ATPase assay, differences (P < 0.05) in enzyme activity between muscles were observed in Hom (EDL > WG) and in CM (Sol > EDL = WG). For the 3-O-MFPase assay, differences (P < 0.05) were also found for Hom (Sol > RG = EDL > WG) and CM (Sol = WG > RG). For Bmax, differences in the order of RG = EDL > Sol = WG (P < 0.05) were observed. Isoform distribution was similar between Hom and CM and indicated in CM, a greater density (P < 0.05) of alpha1 in Sol than WG and EDL (P < 0.05), but more equal distribution of alpha2 between muscles. The beta1 was greater (P < 0.05) in Sol and RG, and the beta2 was greater in EDL and WG (P < 0.05). Over all muscles, the correlation (r) between Hom 3-O-MFPase and Bmax was 0.45 (P < 0.05) and between Hom alpha2 and Bmax, 0.59 (P < 0.05). The alpha1 distribution correlated to Hom 3-O-MFPase (r = 0.79, P < 0.05) CM ATPase (r = 0.69, P < 0.005) and CM 3-O-MFPase activity (r = 0.32, P < 0.05). The alpha2 distribution was not correlated with any of the Na+-K+-ATPase activity measurements. The results indicate generally poor relationships between activity and total pump content and alpha2 isoform content of the Na+-K+-ATPase. Several factors, including the type of preparation and the type of assay, appear important in this regard.     

Dietary coconut oil affects more lipoprotein lipase activity than the mitochondria oxidative capacities in muscles of preruminant calves

The presence of coconut oil in a milk replacer stimulates the growth rate of calves, suggesting a better oxidation of fatty acid in muscles. Because dietary fatty acid composition influences carnitine palmitoyltransferase I (CPT I) activity in rat muscles, this study was designed to examine the effects of a milk replacer containing either tallow (TA) or coconut oil (CO) on fatty acid utilization and oxidation and on the characteristics of intermyofibrillar (IM) and subsarcolemmal (SS) mitochondria in the heart and skeletal muscles of preruminant calves. Feeding CO did not affect palmitate oxidation rate by whole homogenates, but induced higher palmitate oxidation by IM mitochondria (+37%, P < 0.05). CPT I activity did not significantly differ between the two groups of calves. Heart and longissimus thoracis muscle of calves fed CO had higher lipoprotein lipase activity (+27% and 58%, respectively; P < 0.05) but showed no differences in fatty acid binding protein content or activity of oxidative enzymes. Whatever the muscle and the diet, IM mitochondria had higher respiration rates and enzyme activities than those of SS mitochondria (P < 0.05). Furthermore, CPT I activity of the heart was 28-fold less sensitive to malonyl-coenzyme A inhibition in IM mitochondria than in SS mitochondria. In conclusion, dietary CO marginally affected the activity of the two mitochondrial populations and the oxidative activity of muscles in the preruminant calf. In addition, this study showed that differences between IM and SS mitochondria in the heart and muscles were higher in calves than in other species studied so far.     

Effect of hindlimb unweighting on anaerobic metabolism in rat skeletal muscle.

Female Sprague-Dawley rats (250 g) were hindlimb suspended for 14 days, and the effects of hindlimb unweighting (HU) on skeletal muscle anaerobic metabolism were investigated and compared with nonsuspended controls (C). Soleus (SOL), plantaris (PL), and red and white portions of the gastrocnemius (RG, WG) were sampled from resting and stimulated limbs. Muscle atrophy after HU was 46% in SOL, 22% in PL, and 24% in the gastrocnemius compared with nonsuspended C animals. The muscles innervated by the sciatic nerve were stimulated to contract with an occluded circulation for 60 s with trains of supramaximal impulses (100 ms, 80 Hz) at a train rate of 1.0 Hz. Peak tension development by the gastrocnemius-PL-SOL muscle group was similar in HU and C animals (13.0 +/- 1.2, 12.2 +/- 0.8 N/g wet muscle). Occlusion of the circulation before stimulation created a predominantly anaerobic environment, and in situ glycogenolysis and glycolysis were estimated from accumulations of glycolytic intermediates. Total glycogenolysis and glycolysis were higher in the RG muscle of HU animals (74.6 +/- 3.3, 58.1 +/- 1.1) relative to C (57.1 +/- 4.6, 46.1 +/- 2.9 mumol glucosyl units/g dry muscle). Consequently, total anaerobic ATP production was also increased (HU, 251.3 +/- 1.1; C, 204.6 +/- 8.9 mumol ATP/g dry muscle). Total ATP production, glycogenolysis, and glycolysis were unaffected by HU in SOL, PL, and WG muscles. The enhanced glycolytic activity in RG after HU may be attributed to a shift in the metabolic profile from oxidative to glycolytic in the fast oxidative-glycolytic fiber population.     

A case of carnitine palmitoyltransferase II deficiency in human skeletal muscle

A 20-year-old man was shown to have a deficiency of carnitine palmitoyltransferase (CPT) II in skeletal muscle. The evidence was: (i) there was no significant oxidation of [9,10-3H]-palmitate or of [1-14C]palmitate in mitochondrial fractions from fresh skeletal muscle from the patient; (ii) all the CPT activity in a homogenate of fresh muscle from the patient was overt (CPT I) with no increase in activity after the inner membrane was disrupted; (iii) all the CPT activity in the patient's muscle was inhibited by malonyl-CoA; and (iv) an immunoreactive peptide of 67 kDa corresponding to CPT II, present in mitochondria from controls, was absent in those from the patient.     

Glycogen content has no effect on skeletal muscle glycogenolysis during short-term tetanic stimulation

The effect of skeletal muscle glycogen content on in situ glycogenolysis during short-term tetanic electrical stimulation was examined. Rats were randomly assigned to one of three conditions: normal (N, stimulated only), supercompensated (S, stimulated 21 h after a 3-h swim), and fasted (F, stimulated after a 20-h fast). Before stimulation, glycogen contents in the white (WG) and red gastrocnemius (RG) and soleus (SOL) muscles were increased by 13-25% in S and decreased by 15-27% in F compared with N. Hindlimb blood flow was occluded 60 s before stimulation to produce a predominantly anaerobic environment. Muscles were stimulated with trains of supramaximal impulses (100 ms at 80 Hz) at a rate of 1 Hz for 60 s. Muscle glycogenolysis was measured from the decrease in glycogen content and estimated from the accumulation of glycolytic intermediates in the closed system. The resting glycogen content had no effect on measured or estimated glycogenolysis in all muscles studied. Average glycogenolysis in the WG, RG, and SOL muscles was 98.4 +/- 4.3, 60.9 +/- 4.0, and 11.2 +/- 3.6 mumol glucosyl U/g dry muscle, respectively. Hindlimb tension production was similar across conditions. The results suggest that in vivo glycogen phosphorylase activity in skeletal muscle is not regulated by the content of its substrate glycogen (range 80-165 mumol/g) during short-term tetanic stimulation in an anaerobic environment.     

Differential apoptosis-related protein expression, mitochondrial properties, proteolytic enzyme activity, and DNA fragmentation between skeletal muscles

Increased skeletal muscle apoptosis has been associated with a number of conditions including aging, disuse, and cardiovascular disease. Skeletal muscle is a complex tissue comprised of several fiber types with unique properties. To date, no report has specifically examined apoptotic differences across muscles or fiber types. Therefore, we measured several apoptotic indices in healthy rat red (RG) and white gastrocnemius (WG) muscle, as well as examined the expression of several key proteins across fiber types in a mixed muscle (mixed gastrocnemius). The protein content of apoptosis-inducing factor (AIF), apoptosis repressor with caspase recruitment domain (ARC), Bax, Bcl-2, cytochrome c, heat shock protein 70 (Hsp70), and second mitochondria-derived activator of caspases (Smac) were significantly (P < 0.05) higher in RG vs. WG muscle. Cytosolic AIF, cytochrome c, and Smac as well as nuclear AIF were also significantly (P < 0.05) higher in RG compared with WG muscle. In addition, ARC protein expression was related to muscle fiber type and found to be highest (P < 0.001) in type I fibers. Similarly, AIF protein expression was differentially expressed across fibers; however, AIF was correlated to oxidative potential (P < 0.001). Caspase-3, -8, and -9 activity, calpain activity, and DNA fragmentation (a hallmark of apoptosis) were also significantly higher (P < 0.05) in RG compared with WG muscle. Furthermore, total muscle reactive oxygen species generation, as well as Ca(2+)-induced permeability transition pore opening and loss of membrane potential in isolated mitochondria were greater in RG muscle. Collectively, these data suggest that a number of apoptosis-related indices differ between muscles and fiber types. Given these findings, muscle and fiber-type differences in apoptotic protein expression, signaling, and susceptibility should be considered when studying cell death processes in skeletal muscle.