The transcriptional corepressor RIP140 regulates oxidative metabolism in skeletal muscle

Nuclear receptor signaling plays an important role in energy metabolism. In this study we demonstrate that the nuclear receptor corepressor RIP140 is a key regulator of metabolism in skeletal muscle. RIP140 is expressed in a fiber type-specific manner, and manipulation of its levels in null, heterozygous, and transgenic mice demonstrate that low levels promote while increased expression suppresses the formation of oxidative fibers. Expression profiling reveals global changes in the expression of genes implicated in both myofiber phenotype and metabolic functions. Genes involved in fatty-acid oxidation, oxidative phosphorylation, and mitochondrial biogenesis are upregulated in the absence of RIP140. Analysis of cultured myofibers demonstrates that the changes in expression are intrinsic to muscle cells and that nuclear receptor-regulated genes are direct targets for repression by RIP140. Therefore RIP140 is an important signaling factor in the regulation of skeletal muscle function and physiology.     

PGC-1alpha is not mandatory for exercise- and training-induced adaptive gene responses in mouse skeletal muscle

The aim of the present study was to test the hypothesis that peroxisome proliferator activated receptor-gamma coactivator (PGC) 1alpha is required for exercise-induced adaptive gene responses in skeletal muscle. Whole body PGC-1alpha knockout (KO) and littermate wild-type (WT) mice performed a single treadmill-running exercise bout. Soleus and white gastrocnemius (WG) were obtained immediately, 2 h, or 6 h after exercise. Another group of PGC-1alpha KO and WT mice performed 5-wk exercise training. Soleus, WG, and quadriceps were obtained approximately 37 h after the last training session. Resting muscles of the PGC-1alpha KO mice had lower ( approximately 20%) cytochrome c (cyt c), cytochrome oxidase (COX) I, and aminolevulinate synthase (ALAS) 1 mRNA and protein levels than WT, but similar levels of AMP-activated protein kinase (AMPK) alpha1, AMPKalpha2, and hexokinase (HK) II compared with WT mice. A single exercise bout increased phosphorylation of AMPK and acetyl-CoA carboxylase-beta and the level of HKII mRNA similarly in WG of KO and WT. In contrast, cyt c mRNA in soleus was upregulated in WT muscles only. Exercise training increased cyt c, COXI, ALAS1, and HKII mRNA and protein levels equally in WT and KO animals, but cyt c, COXI, and ALAS1 expression remained approximately 20% lower in KO animals. In conclusion, lack of PGC-1alpha reduced resting expression of cyt c, COXI, and ALAS1 and exercise-induced cyt c mRNA expression. However, PGC-1alpha is not mandatory for training-induced increases in ALAS1, COXI, and cyt c expression, showing that factors other than PGC-1alpha can exert these adaptations.     

Skeletal muscle mitochondrial FAT/CD36 content and palmitate oxidation are not decreased in obese women

A reduction in fatty acid oxidation has been associated with lipid accumulation and insulin resistance in the skeletal muscle of obese individuals. We examined whether this decrease in fatty acid oxidation was attributable to a reduction in muscle mitochondrial content and/or a dysfunction in fatty acid oxidation within mitochondria obtained from skeletal muscle of age-matched, lean [body mass index (BMI) = 23.3 +/- 0.7 kg/m2] and obese women (BMI = 37.6 +/- 2.2 kg/m2). The mitochondrial marker enzymes citrate synthase (-34%), beta-hydroxyacyl-CoA dehydrogenase (-17%), and cytochrome c oxidase (-32%) were reduced (P < 0.05) in obese participants, indicating that mitochondrial content was diminished. Obesity did not alter the ability of isolated mitochondria to oxidize palmitate; however, fatty acid oxidation was reduced at the whole muscle level by 28% (P < 0.05) in the obese. Mitochondrial fatty acid translocase (FAT/CD36) did not differ in lean and obese individuals, but mitochondrial FAT/CD36 was correlated with mitochondrial fatty acid oxidation (r = 0.67, P < 0.05). We conclude that the reduction in fatty acid oxidation in obese individuals is attributable to a decrease in mitochondrial content, not to an intrinsic defect in the mitochondria obtained from skeletal muscle of obese individuals. In addition, it appears that mitochondrial FAT/CD36 may be involved in regulating fatty acid oxidation in human skeletal muscle.     

Extracellular matrix is required for skeletal muscle differentiation but not myogenin expression

Skeletal muscle cells are a useful model for studying cell differentiation. Muscle cell differentiation is marked by myoblast proliferation followed by progressive fusion to form large multinucleated myotubes that synthesize muscle-specific proteins and contract spontaneously. The molecular analysis of myogenesis has advanced with the identification of several myogenic regulatory factors, including myod1, myd, and myogenin. These factors regulate each other's expression and that of muscle-specific proteins such as the acetylcholine receptor and acetylcholinesterase (AChE). In order to investigate the role of extracellular matrix (ECM) in myogenesis we have cultured myoblasts (C₂C₁₂) in the presence or absence of an exogenous ECM (Matrigel). In addition, we have induced differentiation of myoblasts in the presence or absence of Matrigel and/or chlorate, a specific inhibitor of proteoglycan sulfation. Our results indicated that the formation of fused myotubes and expression of AChE was stimulated by Matrigel. Treatment of myoblasts induced to differentiate with chlorate resulted in an inhibition of cell fusion and AChE activity. Chlorate treatment was also found to inhibit the deposition and assembly of ECM components such fibronectin and laminin. The expression of myogenin mRNA was observed when myoblasts were induced to differentiate, but was unaffected by the presence of Matrigel or by culture of the cells in the presence of chlorate. These results suggest that the expression of myogenin is independent of the presence of ECM, but that the presence of ECM is essential for the formation of myotubes and the expression of later muscle-specific gene products. © 1996 Wiley-Liss, Inc.     

Mast cells are not required for anaphylatoxin-induced ileal smooth muscle contraction

The complement-derived anaphylatoxin peptides, C3a and C5a, have long been considered to manifest their spasmogenic activities primarily through stimulation of mast cells. Although mast cells represent the major non-circulating repository for histamine, these cells also elaborate a number of additional, highly potent spasmogenic mediators derived from arachidonic acid. The same lipid mediators can be released by many other cell types. As a result, evaluation of the role of mast cells in anaphylatoxin-dependent responses cannot be based exclusively upon an analysis of the mediators released. We evaluated the role of mast cells in anaphylatoxin-induced ileal smooth muscle contractions by testing isolated segments of ileal tissues derived from genetically mast cell-deficient mice and their congenic normal (+/+) littermates. Isolated tissues from either congenic normal (+/+) or mast cell-deficient Sl/Sld mice responded similarly to acetylcholine, histamine, serotonin, prostaglandin E2, and the thromboxane A2 analog, U-46619. At 1 microgram/ml, histamine induced contractions of greater magnitude in tissues from mast cell-deficient animals; however, this mediator also desensitized the tissues to repeat challenge with histamine at the same concentration. C5a at 1 nM resulted in contractions equivalent to approximately 50% of the maximal KCl response; normal and mast cell-deficient tissues responded in a similar manner. C5a also released histamine from the normal mouse ileum, in addition to causing contraction of the tissues. C3a at 200 nM also produced similar contractile responses in both +/+ and S1/S1d tissues. These studies show that the anaphylatoxin peptides C3a and C5a are capable of contracting smooth muscle-containing tissues by a mechanism completely independent of mast cells. In addition, we also demonstrated that mast cell degranulation does not necessarily provoke ileal contraction. Thus compound 48/80, a mast cell degranulating agent unrelated to the anaphylatoxins, did not induce contractions in ileal tissues, even when used at concentrations as high as 100 micrograms/ml. Compound 48/80 did release histamine from the +/+ ileum, however, indicating that the agent was able to cause degranulation of ileal mast cells. Taken together, these data indicate that spasmogenic responses to anaphylatoxins (and possibly other agents) that are associated with mast cell degranulation need not necessarily require mast cell mediator release for their expression.     

Exercise training increases branched-chain oxoacid dehydrogenase kinase content in human skeletal muscle

The branched-chain oxoacid dehydrogenase complex (BCOAD) is rate determining for the oxidation of branched-chain amino acids (BCAAs) in skeletal muscle. Exercise training blunts the acute exercise-induced activation of BCOAD (BCOADa) in human skeletal muscle (McKenzie S, Phillips SM, Carter SL, Lowther S, Gibala MJ, Tarnopolsky MA. Am J Physiol Endocrinol Metab 278: E580-E587, 2000); however, the mechanism is unknown. We hypothesized that training would increase the muscle protein content of BCOAD kinase, the enzyme responsible for inactivation of BCOAD by phosphorylation. Twenty subjects [23 +/- 1 yr; peak oxygen uptake (.VO(2peak)) = 41 +/- 2 ml.kg(-1).min(-1)] performed 6 wk of either high-intensity interval or continuous moderate-intensity training on a cycle ergometer (n = 10/group). Before and after training, subjects performed 60 min of cycling at 65% of pretraining .VO(2peak), and needle biopsy samples (vastus lateralis) were obtained before and immediately after exercise. The effect of training was demonstrated by an increased .VO(2peak), increased citrate synthase maximal activity, and reduced muscle glycogenolysis during exercise, with no difference between groups (main effects, P < 0.05). BCOADa was lower after training (main effect, P < 0.05), and this was associated with a approximately 30% increase in BCOAD kinase protein content (main effect, P < 0.05). We conclude that the increased protein content of BCOAD kinase may be involved in the mechanism for reduced BCOADa after exercise training in human skeletal muscle. These data also highlight differences in models used to study the regulation of skeletal muscle BCAA metabolism, since exercise training was previously reported to increase BCOADa during exercise and decrease BCOAD kinase content in rats (Fujii H, Shimomura Y, Murakami T, Nakai N, Sato T, Suzuki M, Harris RA. Biochem Mol Biol Int 44: 1211-1216, 1998).     

Nuclear SIRT1 activity, but not protein content, regulates mitochondrial biogenesis in rat and human skeletal muscle

The domestic cat (Felis catus), a carnivore, naturally eats a very low carbohydrate diet. In contrast, the dog (Canis familiaris), a carno-omnivore, has a varied diet. This study was performed to determine the expression of the intestinal brush border membrane sodium/glucose cotransporter, SGLT1, sweet receptor, T1R2/T1R3, and disaccharidases in these species adapted to contrasting diets. The expression (this includes function) of SGLT1, sucrase, maltase and lactase were determined using purified brush border membrane vesicles and by quantitative immunohistochemistry of fixed tissues. The pattern of expression of subunits of the sweet receptor T1R2 and T1R3 was assessed using fluorescent immunohistochemistry. In proximal, middle, and distal small intestine, SGLT1 function in dogs was 1.9- to 2.3-fold higher than in cats (P = 0.037, P = 0.0011, P = 0.027, respectively), and SGLT1 protein abundance followed an identical pattern. Both cats and dogs express T1R3 in a subset of intestinal epithelial cells, and dogs, but not cats, express T1R2. In proximal and middle regions, there were 3.1- and 1.6-fold higher lactase (P = 0.006 and P = 0.019), 4.4- and 2.9-fold higher sucrase (both P < 0.0001), and 4.6- and 3.1-fold higher maltase activity (P = 0.0026 and P = 0.0005), respectively, in the intestine of dogs compared with cats. Dogs have a potential higher capacity to digest and absorb carbohydrates than cats. Cats may suffer from carbohydrate malabsorption following ingestion of high-carbohydrate meals. However, dogs have a digestive ability to cope with diets containing significant levels of carbohydrate.     

Progesterone increases skeletal muscle mitochondrial H2O2 emission in nonmenopausal women

The luteal phase of the female menstrual cycle is associated with both 1) elevated serum progesterone (P4) and estradiol (E2), and 2) reduced insulin sensitivity. Recently, we demonstrated a link between skeletal muscle mitochondrial H(2)O(2) emission (mE(H2O2)) and insulin resistance. To determine whether serum levels of P4 and/or E(2) are related to mitochondrial function, mE(H2O2) and respiratory O(2) flux (Jo(2)) were measured in permeabilized myofibers from insulin-sensitive (IS, n = 24) and -resistant (IR, n = 8) nonmenopausal women (IR = HOMA-IR > 3.6). Succinate-supported mE(H2O2) was more than 50% greater in the IR vs. IS women (P < 0.05). Interestingly, serum P4 correlated positively with succinate-supported mE(H2O2) (r = 0. 53, P < 0.01). To determine whether P4 or E2 directly affect mitochondrial function, saponin-permeabilized vastus lateralis myofibers biopsied from five nonmenopausal women in the early follicular phase were incubated in P4 (60 nM), E2 (1.4 nM), or both. P4 alone inhibited state 3 Jo(2), supported by multisubstrate combination (P < 0.01). However, E2 alone or in combination with P4 had no effect on Jo(2). In contrast, during state 4 respiration, supported by succinate and glycerophosphate, mE(H2O2) was increased with P4 alone or in combination with E2 (P < 0.01). The results suggest that 1) P4 increases mE(H2O2) with or without E2; 2) P4 alone inhibits Jo(2) but not when E2 is present; and 3) P4 is related to the mE(H2O2) previously linked to skeletal muscle insulin resistance.     

Contraction-induced increases in Na+-K+-ATPase mRNA levels in human skeletal muscle are not amplified by activation of additional muscle mass

The present study tested the hypothesis that exercise with a large compared with a small active muscle mass results in a higher contraction-induced increase in Na(+)-K(+)-ATPase mRNA expression due to greater hormonal responses. Furthermore, the relative abundance of Na(+)-K(+)-ATPase subunit alpha(1), alpha(2), alpha(3), alpha(4), beta(1), beta(2), and beta(3) mRNA in human skeletal muscle was investigated. On two occasions, eight subjects performed one-legged knee extension exercise (L) or combined one-legged knee extension and bilateral arm cranking (AL) for 5.00, 4.25, 3.50, 2.75, and 2.00 min separated by 3 min of rest. Leg exercise power output was the same in AL and L, but heart rate at the end of each exercise interval was higher in AL compared with L. One minute after exercise, arm venous blood lactate was higher in AL than in L. A higher level of blood epinephrine and norepinephrine was evident 3 min after exercise in AL compared with L. Nevertheless, none of the exercise-induced increases in alpha(1), alpha(2), beta(1), and beta(3) mRNA expression levels were higher in AL compared with L. The most abundant Na(+)-K(+)-ATPase subunit at the mRNA level was beta(1), which was expressed 3.4 times than alpha(2). Expression of alpha(1), beta(2), and beta(3) was less than 5% of the alpha(2) expression, and no reliable detection of alpha(3) and alpha(4) was possible. In conclusion, activation of additional muscle mass does not result in a higher exercise-induced increase in Na(+)-K(+)-ATPase subunit-specific mRNA.     

Thin filaments are not of uniform length in rat skeletal muscle

The variation in thin filament length was investigated in slow and fast muscle from adult and neonatal rats. Soleus (slow) muscle from adult, 3- , 7-, and 9-d-old rats, and extensor digitorum longus (EDL; fast) muscle from adult rats were serially cross-sectioned. The number of thin filaments per 0.06 microns2 (TF#) was counted for individual myofibrils followed from the H zone of one sarcomere, through the I-Z-I region, to the H zone of an adjacent sarcomere TF# was pooled by distance from the Z band or AI junction. In both adult muscles, thin filament length varied from 0.18 to 1.20 microns, with approximately 25% of the thin filaments less than 0.7 microns in length. In 7- and 9- d soleus, thin filament length ranged from 0.18 to 1.08 microns; except for the longest (0.18 to 1.20 microns) filaments, the distribution of thin filament lengths was similar to that in adult muscle. In 3-d soleus, thin filament length was more uniform, with less than 5% of the filaments shorter than 0.7 microns. In all neonatal muscles, there were approximately 15% fewer thin filaments per unit area as compared to adult muscles. We conclude: (a) In rat skeletal muscle, thin filaments are not of uniform length, ranging in length from 0.18 to 1.20 microns. (b) There may be two stages of thin filament assembly in neonatal muscle: between 3 and 7 d when short thin filaments may be preferentially or synthesized or inserted near the Z-band, and between 9 d and adult when thin filaments of all lengths may be synthesized or inserted into the myofibril.     

UCP-mediated energy depletion in skeletal muscle increases glucose transport despite lipid accumulation and mitochondrial dysfunction

To address the potential role of lipotoxicity and mitochondrial function in insulin resistance, we studied mice with high-level expression of uncoupling protein-1 in skeletal muscle (UCP-H mice). Body weight, body length, and bone mineral density were decreased in UCP-H mice compared with wild-type littermates. Forelimb grip strength and muscle mass were strikingly decreased, whereas muscle triglyceride content was increased fivefold in UCP-H mice. Electron microscopy demonstrated lipid accumulation and large mitochondria with abnormal architecture in UCP-H skeletal muscle. ATP content and key mitochondrial proteins were decreased in UCP-H muscle. Despite mitochondrial dysfunction and increased intramyocellular fat, fasting serum glucose was 22% lower and insulin-stimulated glucose transport 80% higher in UCP-H animals. These beneficial effects on glucose metabolism were associated with increased AMP kinase and hexokinase activities, as well as elevated levels of GLUT4 and myocyte enhancer factor-2 proteins A and D in skeletal muscle. These results suggest that UCP-H mice have a mitochondrial myopathy due to depleted energy stores sufficient to compromise growth and impair muscle function. Enhanced skeletal muscle glucose transport in this setting suggests that excess intramyocellular lipid and mitochondrial dysfunction are not sufficient to cause insulin resistance in mice.     

The urokinase-type plasminogen activator receptor is not required for skeletal muscle inflammation or regeneration

The hypothesis of this study was the urokinase-type plasminogen activator receptor (uPAR) is required for accumulation of inflammatory cells in injured skeletal muscle and for efficient muscle regeneration. Expression of uPAR was elevated at 1 and 3 days after cardiotoxin-induced muscle injury in wild-type mice before returning to baseline levels. Neutrophil accumulation peaked 1 day postinjury in muscle from both wild-type (WT) and uPAR null mice, while macrophage accumulation peaked between 3 and 5 days postinjury, with no differences between strains. Histological analyses confirmed efficient muscle regeneration in both wild-type and uPAR null mice, with no difference between strains in the formation or growth of regenerating fibers, or recovery of normal morphology. Furthermore, in vitro experiments demonstrated that chemotaxis is not different between WT and uPAR null macrophages. Finally, fusion of cultured satellite cells into multinucleated myotubes was not different between cells isolated from WT and uPAR null mice. These results demonstrate that uPAR is not required for the accumulation of inflammatory cells or the regeneration of skeletal muscle following injury, suggesting uPA can act independently of uPAR to regulate events critical for muscle regeneration.     

Creatine but not betaine supplementation increases muscle phosphorylcreatine content and strength performance

We aimed to investigate the role of betaine supplementation on muscle phosphorylcreatine (PCr) content and strength performance in untrained subjects. Additionally, we compared the ergogenic and physiological responses to betaine versus creatine supplementation. Finally, we also tested the possible additive effects of creatine and betaine supplementation. This was a double-blind, randomized, placebo-controlled study. Subjects were assigned to receive betaine (BET; 2?g/day), creatine (CR; 20?g/day), betaine plus creatine (BET?+?CR; 2?+?20?g/day, respectively) or placebo (PL). At baseline and after 10?days of supplementation, we assessed muscle strength and power, muscle PCr content, and body composition. The CR and BET?+?CR groups presented greater increase in muscle PCr content than PL (p?=?0.004 and p?=?0.006, respectively). PCr content was comparable between BET versus PL (p?=?0.78) and CR versus BET?+?CR (p?=?0.99). CR and BET?+?CR presented greater muscle power output than PL in the squat exercise following supplementation (p?=?0.003 and p?=?0.041, respectively). Similarly, bench press average power was significantly greater for the CR-supplemented groups. CR and BET?+?CR groups also showed significant pre- to post-test increase in 1-RM squat and bench press (CR: p?=?0.027 and p?<?0.0001; BET?+?CR: p?=?0.03 and p?<?0.0001 for upper- and lower-body assessments, respectively) No significant differences for 1-RM strength and power were observed between BET versus PL and CR versus BET?+?CR. Body composition did not differ between the groups. In conclusion, we reported that betaine supplementation does not augment muscle PCr content. Furthermore, we showed that betaine supplementation combined or not with creatine supplementation does not affect strength and power performance in untrained subjects.     

Ectopic overexpression of porcine DGAT1 increases intramuscular fat content in mouse skeletal muscle

The microsomal enzyme 1, 2-acyl CoA: diacylglyceroltransferase-1 (DGAT1) plays an important role in triglyceride storage in adipose tissue and expresses in skeletal muscle as well. The primary goal of the present study was to investigate the effect of porcine DGAT1 on intramuscular fat (IMF) content of transgenic mice produced by pronuclear microinjection with muscle specific promoter of porcine muscle creatine kinase (MCK). In normal chow-fed diet, 4 month-old male transgenic mice expressed more DGAT1, ACC1, UCP1, and FABP4 mRNAs and proteins in skeletal muscle than control mice by real-time PCR and western blot. No significant changes were detected for ACC2, CD36, ADRP, PPAR gamma and LPL. Triacylglycerol assay and soleus muscle sections showed overexpression of porcine DGAT1 in skeletal muscle increased intramyocellular triglyceride and percent of the total cell surface covered by lipid droplets. Thus, upregulation of porcine DGAT1 in skeletal muscle increases IMF content. The present study may further serve to develop transgenic pigs with higher IMF content and improved meat quality.     

AMP kinase is not required for the GLUT4 response to exercise and denervation in skeletal muscle

An acute bout of exercise increases muscle GLUT4 mRNA in mice, and denervation decreases GLUT4 mRNA. AMP-activated protein kinase (AMPK) activity in skeletal muscle is also increased by exercise, and GLUT4 mRNA is increased in mouse skeletal muscle after treatment with AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside(AICAR). These findings suggest that AMPK activation might be responsible for the increase in GLUT4 mRNA expression in response to exercise. To investigate the role of AMPK in GLUT4 regulation in response to exercise and denervation, transgenic mice with a mutated AMPK alpha-subunit (dominant negative; AMPK-DN) were studied. GLUT4 did not increase in AMPK-DN mice that were treated with AICAR, demonstrating that muscle AMPK is inactive. Exercise (two 3-h bouts of treadmill running separated by 1 h of rest) increased GLUT4 mRNA in both wild-type and AMPK-DN mice. Likewise, denervation decreased GLUT4 mRNA in both wild-type and AMPK-DN mice. GLUT4 mRNA was also increased by AICAR treatment in both the innervated and denervated muscles. These data demonstrate that AMPK is not required for the response of GLUT4 mRNA to exercise and denervation.     

Kinase-specific responsiveness to incremental contractile activity in skeletal muscle with low and high mitochondrial content

Muscle contractions activate protein kinases, leading to signal transduction. We hypothesized that kinase activation would be influenced by mitochondrial content, as well as by contractile activity-induced increases in muscle O(2) consumption (Vo(2)). Kinase phosphorylation in high-oxidative red and low-oxidative white tibialis anterior (TA) muscle (RTA and WTA, respectively) with 2.5-fold differences in mitochondrial content were compared. Stimulation of the TA muscle elicited large increases in Vo(2) (3- to 6-fold and 4- to 60-fold above resting levels in WTA and RTA, respectively). At rest, AMP-activated protein kinase (AMPK), p38, p42, and p44 activation were nearly twofold greater in WTA than in RTA, suggesting an inverse relationship between mitochondrial content and kinase activation in resting muscle. During contractions, similar degrees of phosphorylation in RTA and WTA were evident as a function of Vo(2) for p38 and p42. During increases in Vo(2) up to sixfold above rest, greater responses were observed in RTA than in WTA for AMPK and p44, whereas Akt activation was greater in WTA. In RTA, elevations in Vo(2) elicited increases in AMPK and p44 activation, whereas Akt, p38, and p42 were less sensitive to increments in Vo(2). Reactive oxygen species (ROS) production was greater in mitochondria from white muscle, but when it was calculated in the context of the whole muscle, ROS production was twofold greater in red than in white myofibers. Thus mitochondrial content influences ROS production and is inversely related to kinase activation in resting muscle. During contractions, kinases are differentially sensitive to contraction-induced increments in Vo(2), suggesting that muscle mitochondrial content is important, but it is not the sole determinant of kinase activation during exercise of different intensities.