SMHS1 is involved in oxidative/glycolytic-energy metabolism balance of muscle fibers

With the aim of finding important mediators of muscle atrophy, we cloned SMHS1, a novel gene that was found to be upregulated in rat soleus muscle atrophied by restriction of activity. The SMHS1 amino acid sequence shares 65% similarity with RTP801-which is a cellular stress response protein regulated by HIF-1-but SMHS1 expression was demonstrated to be independent of HIF-1. SMHS1 was found to be mainly expressed in skeletal muscle, and comparisons of its expression in atrophied versus hypertrophied muscles and in oxidative versus glycolytic muscles suggested that SMHS1 contributes to the muscle energy metabolism phenotypes.     

Hypoxia-Inducible Factor in ringed seal (Phoca hispida) tissues

Tissue hypoxia and ischemia-reperfusion pose a dangerous situation for oxidative stress. However, diving mammals and birds show pronounced resistance to oxidative injury under such conditions, which are a consequence of selective vasoconstriction during a dive. As the function of Hypoxia-Inducible Factor-1alpha (HIF-1alpha) in protection against and adaptation to hypoxia has been recognized in terrestrial animals, we have investigated the genomics and expression of this protein in ringed seal (Phoca hispida) in order to determine if it may play a protective role in this diving mammal. PCR studies using primers based on sequences from mouse HIF-1alpha exons 3, 4, 5, 6, 9, 10, 11, 12 and 15 showed that DNA from seal lung generated PCR products similar to those from mouse DNA. These studies have established that a putative HIF-1alpha gene exists in the seal genome that appears to have a similar but not identical sequence to the mouse gene. Seal lung and skeletal muscle tissues showed the highest relative levels of HIF-1alpha protein expression, with heart muscle showing significantly lower levels, and levels of HIF-1beta protein expression paralleled this situation. Analysis of oxidized cellular protein levels indicated that seal lung and heart muscle had the lowest levels of oxidized proteins. Thus, as seal lung tissue had the highest level of HIF-1alpha protein expression and the second lowest level of protein oxidation, this suggests that HIF-1alpha expression may have an important protective effect in this tissue in diving mammals. Our results support the hypothesis that HIF-1alpha expression is dependent on both tissue-specific energy requirements and adequate metabolic supply-to-demand ratio. Combined, the evidence available suggests that diving mammals have an overall anticipatory response to avoid the ill effects of dive-associated ischemia-reperfusion which may involve the HIF-1 system.     

Muscle oxidative capacity during IL-6-dependent cancer cachexia

Many diseases are associated with catabolic conditions that induce skeletal muscle wasting. These various catabolic states may have similar and distinct mechanisms for inducing muscle protein loss. Mechanisms related to muscle wasting may also be related to muscle metabolism since glycolytic muscle fibers have greater wasting susceptibility with several diseases. The purpose of this study was to determine the relationship between muscle oxidative capacity and muscle mass loss in red and white hindlimb muscles during cancer cachexia development in the Apc(Min/+) mouse. Gastrocnemius and soleus muscles were excised from Apc(Min/+) mice at 20 wk of age. The gastrocnemius muscle was partitioned into red and white portions. Body mass (-20%), gastrocnemius muscle mass (-41%), soleus muscle mass (-34%), and epididymal fat pad (-100%) were significantly reduced in severely cachectic mice (n = 8) compared with mildly cachectic mice (n = 6). Circulating IL-6 was fivefold higher in severely cachectic mice. Cachexia significantly reduced the mitochondrial DNA-to-nuclear DNA ratio in both red and white portions of the gastrocnemius. Cytochrome c and cytochrome-c oxidase complex subunit IV (Cox IV) protein were reduced in all three muscles with severe cachexia. Changes in muscle oxidative capacity were not associated with altered myosin heavy chain expression. PGC-1α expression was suppressed by cachexia in the red and white gastrocnemius and soleus muscles. Cachexia reduced Mfn1 and Mfn2 mRNA expression and markers of oxidative stress, while Fis1 mRNA was increased by cachexia in all muscle types. Muscle oxidative capacity, mitochondria dynamics, and markers of oxidative stress are reduced in both oxidative and glycolytic muscle with severe wasting that is associated with increased circulating IL-6 levels.     

Muscle fiber type comparison of PDH kinase activity and isoform expression in fed and fasted rats

Fiber type specificity for expression of all three rat skeletal muscle pyruvate dehydrogenase kinase (PDK) isoforms (PDK1, 2, and 4) was determined in fed and 24-h fasted rats. PDK activity and isoform protein and mRNA contents were determined in white gastrocnemius (WG; fast-twitch glycolytic), red gastrocnemius (RG; fast-twitch oxidative), and soleus (Sol; slow-twitch oxidative) muscles. PDK activity was lower in WG compared with oxidative muscles (RG, Sol) in both fed and fasted rats. PDK activities from fed muscles were 0.12 +/- 0.04, 0.30 +/- 0.01, and 0.36 +/- 0.08 min(-1) in WG, Sol, and RG, respectively, and increased in fasted muscles (0.36 +/- 0.09, 0.68 +/- 0.18, and 0.80 +/- 0.14 min(-1)). This correlated with increased PDK4 protein and to a lesser extent with PDK4 mRNA. PDK2 protein was not different between fiber types in fed or fasted rats, but PDK2 mRNA content was twofold greater in RG from fasted rats compared with fed rats. PDK1 was unaltered by fasting in all muscle types at both the protein and mRNA level, but in both fed and fasted rats had much greater protein and mRNA content in the oxidative vs. glycolytic muscles. In conclusion, PDK activity and PDK1 and 4 protein and mRNA were lower in glycolytic vs. oxidative muscles from fed and fasted rats. Fasting for 24 h induced a two- to threefold increase in PDK activity that was mainly due to increases in PDK4 protein and mRNA. PDK1 and 2 protein and mRNA were generally unaltered by fasting in all fiber types, except for increased PDK2 mRNA in the fast oxidative fibers. Because the PDK isoforms vary greatly in their kinetic properties, their relative proportions in the three fiber types at any given time during fasting could significantly alter the acute regulation of the pyruvate dehydrogenase complex.     

Lack of AMPKalpha2 enhances pyruvate dehydrogenase activity during exercise

5'-AMP-activated protein kinase (AMPK) was recently suggested to regulate pyruvate dehydrogenase (PDH) activity and thus pyruvate entry into the mitochondrion. We aimed to provide evidence for a direct link between AMPK and PDH in resting and metabolically challenged (exercised) skeletal muscle. Compared with rest, treadmill running increased AMPKalpha1 activity in alpha(2)KO mice (90%, P < 0.01) and increased AMPKalpha2 activity in wild-type (WT) mice (110%, P < 0.05), leading to increased AMPKalpha Thr(172) (WT: 40%, alpha(2)KO: 100%, P < 0.01) and ACCbeta Ser(227) phosphorylation (WT: 70%, alpha(2)KO: 210%, P < 0.01). Compared with rest, exercise significantly induced PDH-E(1)alpha site 1 (WT: 20%, alpha(2)KO: 62%, P < 0.01) and site 2 (only alpha(2)KO: 83%, P < 0.01) dephosphorylation and PDH(a) [ approximately 200% in both genotypes (P < 0.01)]. Compared with WT, PDH dephosphorylation and activation was markedly enhanced in the alpha(2)KO mice both at rest and during exercise. The increased PDH(a) activity during exercise was associated with elevated glycolytic flux, and muscles from the alpha(2)KO mice displayed marked lactate accumulation and deranged energy homeostasis. Whereas mitochondrial DNA content was normal, the expression of several mitochondrial proteins was significantly decreased in muscle of alpha(2)KO mice. In isolated resting EDL muscles, activation of AMPK signaling by AICAR did not change PDH-E(1)alpha phosphorylation in either genotype. PDH is activated in mouse skeletal muscle in response to exercise and is independent of AMPKalpha2 expression. During exercise, alpha(2)KO muscles display deranged energy homeostasis despite enhanced glycolytic flux and PDH(a) activity. This may be linked to decreased mitochondrial oxidative capacity.     

Expression of uncoupling protein-3 in subsarcolemmal and intermyofibrillar mitochondria of various mouse muscle types and its modulation by fasting.

Uncoupling protein-3 (UCP3) is a mitochondrial inner-membrane protein abundantly expressed in rodent and human skeletal muscle which may be involved in energy dissipation. Many studies have been performed on the metabolic regulation of UCP3 mRNA level, but little is known about UCP3 expression at the protein level. Two populations of mitochondria have been described in skeletal muscle, subsarcolemmal (SS) and intermyofibrillar (IMF), which differ in their intracellular localization and possibly also their metabolic role. To examine if UCP3 is differentially expressed in these two populations and in different mouse muscle types, we developed a new protocol for isolation of SS and IMF mitochondria and carefully validated a new UCP3 antibody. The data show that the density of UCP3 is higher in the mitochondria of glycolytic muscles (tibialis anterior and gastrocnemius) than in those of oxidative muscle (soleus). They also show that SS mitochondria contain more UCP3 per mg of protein than IMF mitochondria. Taken together, these results suggest that oxidative muscle and the mitochondria most closely associated with myofibrils are most efficient at producing ATP. We then determined the effect of a 24-h fast, which greatly increases UCP3 mRNA (16.4-fold) in muscle, on UCP3 protein expression in gastrocnemius mitochondria. We found that fasting moderately increases (1.5-fold) or does not change UCP3 protein in gastrocnemius SS or IMF mitochondria, respectively. These results show that modulation of UCP3 expression at the mRNA level does not necessarily result in similar changes at the protein level and indicate that UCP3 density in SS and IMF mitochondria can be differently affected by metabolic changes.     

Mitochondrial gene expression in mammalian striated muscle. Evidence that variation in gene dosage is the major regulatory event

The oxidative capacity of mammalian striated muscles can vary markedly over a nearly 10-fold range, reflecting major differences in the expression of genes that encode enzymes of oxidative metabolism, including genes located exclusively within mitochondrial DNA. To clarify the regulatory events that govern expression of mitochondrial genes in striated muscle, nucleic acid hybridization procedures employing cloned segments of mitochondrial DNA as probes were utilized to determine the concentrations of mitochondrial DNA, mitochondrial ribosomal RNA, and cytochrome b mRNA (a mitochondrial gene product) in rabbit striated muscles of markedly different oxidative capacities. When cardiac muscle and Type I (red, oxidative) skeletal muscle were compared to Type II (white, glycolytic) skeletal muscle, mitochondrial DNA, mitochondrial ribosomal RNA, and cytochrome b mRNA, each increased in direct proportion to increases in oxidative capacity. Furthermore, when the phenotypic characteristics of Type II skeletal muscle were altered by electrical stimulation in vivo, mitochondrial DNA, mitochondrial rRNA, and cytochrome b mRNA also increased proportionately with increases in oxidative capacity. These results indicate that the expression of mitochondrial genes in mammalian striated muscle is proportionate to their copy number, and support the hypothesis that amplification of the mitochondrial genome relative to chromosomal DNA is an important feature underlying enhanced expression of mitochondrial genes in highly oxidative tissues.     

Chicken liver and muscle carnitine palmitoyltransferase 1: nutritional regulation of messengers

In mammals, carnitine palmitoyltransferase 1 (CPT1) is a rate limiting enzyme of fatty acid oxidation. Two isoforms are present. We characterized a full-length cDNA sequence encoding chicken liver L-CPT1 isoform and a partial cDNA sequence encoding chicken muscle M-CPT1 isoform. CPT1 messengers showed the expected tissue specificity. M-CPT1 messenger and CPT1 activity were higher in oxidative than in glycolytic muscle. Expression of both isoforms was assessed in various tissues of genetically fat or lean chickens. Fasting considerably increased L-CPT1 mRNA expression and beta-hydroxyacyl CoA dehydrogenase (HAD) activity in the liver of fat or lean chickens. Unexpectedly, fasting did not increase M-CPT1 mRNA levels nor HAD activity in muscles of either chicken genotype. It however increased succinyl-CoA:3-ketoacid CoA transferase (SCOT) mRNA expression (an enzyme related to ketone body utilization) in oxidative muscle. SCOT messenger was slightly more abundant in oxidative muscle of lean chickens but not in glycolytic muscle. In conclusion, the regulation of fatty acid oxidation is probably not impaired in fat chicken. The absence of fasting stimulation of M-CPT1 mRNA expression, which is at variance with the situation observed in mammals, suggests that during fasting, chicken muscles preferentially use ketone bodies as fuel, at least in the short term.     

Molecular adaptations in human skeletal muscle to endurance training under simulated hypoxic conditions.

This study was performed to explore changes in gene expression as a consequence of exercise training at two levels of intensity under normoxic and normobaric hypoxic conditions (corresponding to an altitude of 3,850 m). Four groups of human subjects trained five times a week for a total of 6 wk on a bicycle ergometer. Muscle biopsies were taken, and performance tests were carried out before and after the training period. Similar increases in maximal O(2) uptake (8.3-13.1%) and maximal power output (11.4-20.8%) were found in all groups. RT-PCR revealed elevated mRNA concentrations of the alpha-subunit of hypoxia-inducible factor 1 (HIF-1) after both high- (+82.4%) and low (+78.4%)-intensity training under hypoxic conditions. The mRNA of HIF-1alpha(736), a splice variant of HIF-1alpha newly detected in human skeletal muscle, was shown to be changed in a similar pattern as HIF-1alpha. Increased mRNA contents of myoglobin (+72.2%) and vascular endothelial growth factor (+52.4%) were evoked only after high-intensity training in hypoxia. Augmented mRNA levels of oxidative enzymes, phosphofructokinase, and heat shock protein 70 were found after high-intensity training under both hypoxic and normoxic conditions. Our findings suggest that HIF-1 is specifically involved in the regulation of muscle adaptations after hypoxia training. Fine-tuning of the training response is recognized at the molecular level, and with less sensitivity also at the structural level, but not at global functional responses like maximal O(2) uptake or maximal power output.     

Leucine treatment enhances oxidative capacity through complete carbohydrate oxidation and increased mitochondrial density in skeletal muscle cells

Leucine has been largely implicated for increasing muscle protein synthesis in addition to stimulating mitochondrial biosynthesis. Limited evidence is currently available on the effects and potential benefits of leucine treatment on skeletal muscle cell glycolytic and oxidative metabolism. This work identified the effects of leucine treatment on oxidative and glycolytic metabolism as well as metabolic rate of human and murine skeletal muscle cells. Human rhabdomyosarcoma cells (RD) and mouse myoblast cells (C2C12) were treated with leucine at either 100 or 500 μM for 24 or 48 h. Glycolytic metabolism was quantified by measuring extracellular acidification rate (ECAR) and oxidative metabolism was quantified by measuring oxygen consumption rate. Peroxisome proliferator-activated receptor coactivator 1 alpha (PGC-1α), an important stimulator of mitochondrial biosynthesis, was quantified using flow cytometry and verified by immunofluorescent confocal microscopy. Mitochondrial content was quantified using mitochondrial and cytochrome C staining measured by flow cytometry and confirmed with confocal microscopy. Treatment with leucine significantly increased both basal and peak oxidative metabolism in both cell models. Leucine treated cells also exhibited significantly greater mitochondrial proton leak, which is associated with heightened energy expenditure. Basal ECAR was significantly reduced in both cell models following leucine treatment, evidence of reduced lactate export and more complete carbohydrate oxidation. In addition, both PGC-1α and cytochrome C expression were significantly elevated in addition to mitochondrial content following 48 h of leucine treatment. Our observations demonstrated few dose-dependent responses induced by leucine; however, leucine treatment did induce a significant dose-dependent expression of PGC-1α in both cell models. Interestingly, C2C12 cells treated with leucine exhibited dose-dependently reduced ATP content, while RD ATP content remain unchanged. Leucine presents a potent dietary constituent with low lethality with numerous beneficial effects for increasing oxidative preference and capacity in skeletal muscle. Our observations demonstrate that leucine can enhance oxidative capacity and carbohydrate oxidation efficiency, as well as verify previous observations of increased mitochondrial content.     

Role of mitochondria–cytoskeleton interactions in respiration regulation and mitochondrial organization in striated muscles

The aim of this work was to study the regulation of respiration and energy fluxes in permeabilized oxidative and glycolytic skeletal muscle fibers, focusing also on the role of cytoskeletal protein tubulin βII isotype in mitochondrial metabolism and organization. By analyzing accessibility of mitochondrial ADP, using respirometry and pyruvate kinase–phosphoenolpyruvate trapping system for ADP, we show that the apparent affinity of respiration for ADP can be directly linked to the permeability of the mitochondrial outer membrane (MOM). Previous studies have shown that MOM permeability in cardiomyocytes can be regulated by VDAC interaction with cytoskeletal protein, βII tubulin. We found that in oxidative soleus skeletal muscle the high apparent K_m for ADP is associated with low MOM permeability and high expression of non-polymerized βII tubulin. Very low expression of non-polymerized form of βII tubulin in glycolytic muscles is associated with high MOM permeability for adenine nucleotides (low apparent K_m for ADP).     

Differential expression of metabolic genes essential for glucose and lipid metabolism in skeletal muscle from spinal cord injured subjects

Skeletal muscle plays an important role in the regulation of energy homeostasis; therefore, the ability of skeletal muscle to adapt and alter metabolic gene expression in response to changes in physiological demands is critical for energy balance. Individuals with cervical spinal cord lesions are characterized by tetraplegia, impaired thermoregulation, and altered skeletal muscle morphology. We characterized skeletal muscle metabolic gene expression patterns, as well as protein content, in these individuals to assess the impact of spinal cord injury on critical determinants of skeletal muscle metabolism. Our results demonstrate that mRNA levels and protein expression of skeletal muscle genes essential for glucose storage are reduced, whereas expression of glycolytic genes is reciprocally increased in individuals with spinal cord injury. Furthermore, expression of genes essential for lipid oxidation is coordinately reduced in spinal cord injured subjects, consistent with a marked reduction of mitochondrial proteins. Thus spinal cord injury resulted in a profound and tightly coordinated change in skeletal muscle metabolic gene expression program that is associated with the aberrant metabolic features of the tissue.     

Expression of the Na(+)/Ca(2+) exchanger in skeletal muscle

The expression of the Na(+)/Ca(2+) exchanger was studied in differentiating muscle fibers in rats. NCX1 and NCX3 isoform (Na(+)/Ca(2+) exchanger isoform) expression was found to be developmentally regulated. NCX1 mRNA and protein levels peaked shortly after birth. Conversely, NCX3 isoform expression was very low in muscles of newborn rats but increased dramatically during the first 2 wk of postnatal life. Immunocytochemical analysis showed that NCX1 was uniformly distributed along the sarcolemmal membrane of undifferentiated rat muscle fibers but formed clusters in T-tubular membranes and sarcolemma of adult muscle. NCX3 appeared to be more uniformly distributed along the sarcolemma and inside myoplasm. In the adult, NCX1 was predominantly expressed in oxidative (type 1 and 2A) fibers of both slow- and fast-twitch muscles, whereas NCX3 was highly expressed in fast glycolytic (2B) fibers. NCX2 was expressed in rat brain but not in skeletal muscle. Developmental changes in NCX1 and NCX3 as well as the distribution of these isoforms at the cellular level and in different fiber types suggest that they may have different physiological roles.