UCP3 gene expression does not correlate with muscle oxidation rates in troglitazone-treated Zucker fatty rats

Uncoupling protein-3 (UCP3), a mitochondrial carrier protein predominantly expressed in muscle, has been suggested to release stored energy as heat. The insulin-sensitizing thiazolidinediones enhance glucose disposal in skeletal muscle and have been reported to increase the expression of uncoupling proteins in various experimental systems. We therefore studied the effect of troglitazone treatment on UCP3 gene expression in muscles from lean and obese Zucker rats. In comparison with obese littermates, basal UCP3 mRNA levels in lean Zucker rats tended to be higher in white and red gastrocnemius muscles, but were lower in soleus (P<0.001) muscle and heart (P<0.01). In lean rats, troglitazone significantly increased UCP3 gene expression in white and red gastrocnemius and heart muscles (all P<0.01). In contrast, the drug reduced UCP3 mRNA expression in red gastrocnemius and soleus muscles of obese littermates (all P<0.001). The troglitazone-dependent decrease in UCP3 gene expression was accompanied by an increased weight gain in obese rats, while no such effect was observed in lean rats. In obese rats, improvement of insulin resistance by troglitazone was associated with increased rates of basal and insulin-stimulated CO(2) production from glucose measured in soleus muscle. These studies demonstrate that effects of troglitazone on UCP3 gene expression depend on the phenotype of Zucker rats and that troglitazone-induced metabolic improvements are not related to increased uncoupling resulting from upregulation of UCP3 mRNA expression in muscle.     

Effects of chronic exercise during aging on muscle and end-plate morphology in rats

Terminal sprouting, myofiber atrophy, and fiber type changes were studied in soleus and distal gastrocnemius muscles of 21- and 26-mo-old rats and in rats who performed treadmill running from 21 to 26 mo. End-plate structure and muscle fiber types were demonstrated by staining for acetylcholinesterase and myosin ATPase activity. Terminal sprouting was expressed as the percentage of end plates with growth configurations. Fiber atrophy was assessed as percentage of small-diameter fibers. In all three groups, the percentage of small-diameter fibers was significantly smaller and the percentage of growth configurations significantly larger in the soleus than in the gastrocnemius. The exercised-soleus group had a significantly higher percentage of growth configurations than the 26-mo controls, which had a higher percentage than the 21-mo controls. Percentages among gastrocnemius groups were not different. Fiber type was similar among gastrocnemius groups. However, the exercised-soleus had significantly more slow-twitch fibers than the controls. These data suggest that the soleus responds differently to chronic exercise during aging than does the gastrocnemius.     

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

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

Effects of aging and denervation on the expression of uncoupling proteins in slow- and fast-twitch muscles of rats

We investigated the effects of aging and denervation on the gene expression of uncoupling proteins (UCPs) in slow-twitch soleus and fast-twitch gastrocnemius muscles. In a comparison between the control limbs of 6- and 24-month-old rats, the mRNA levels of UCP3, heart-type fatty acid binding protein (HFABP), and glucose transporter-4 (GLUT4) were considerably lower in the gastrocnemius muscles of the older rats, whereas no significant differences in the mRNA levels of those genes as well as UCP2 and cytochrome oxidase subunit IV (COX-IV) were observed in the soleus muscles of young and old rats. The UCP3 and COX-IV protein levels were also reduced considerably in the aged gastrocnemius muscles with atrophy. Denervation of the sciatic nerve caused an increase in UCP3 mRNA levels in both muscles, but the regulation of other genes contrasted between the two types of skeletal muscles. In spite of the increased mRNA level, a remarkable reduction in UCP3 protein was found in the denervated gastrocnemius muscles. These results indicate that the effects of aging and denervation on the gene expression of UCPs, HFABP, GLUT4, and COX-IV are different between the muscle types. The reduction in the mitochondrial UCP3 and COX proteins in aged fast-twitch muscles may have a negative effect on energy metabolism and thermogenesis in old animals.     

Identification of cold-shock protein RBM3 as a possible regulator of skeletal muscle size through expression profiling

Changes in gene expression associated with skeletal muscle atrophy due to aging are distinct from those due to disuse, suggesting that the response of old muscle to inactivity may be altered. The goal of this study was to identify changes in muscle gene expression that may contribute to loss of adaptability of old muscle. Muscle atrophy was induced in young adult (6-mo) and old (32-mo) male Brown Norway/F344 rats by 2 wk of hindlimb suspension (HS), and soleus muscles were analyzed by cDNA microarrays. Overall, similar changes in gene expression with HS were observed in young and old muscles for genes encoding proteins involved in protein folding (heat shock proteins), muscle structure, and contraction, extracellular matrix, and nucleic acid binding. More genes encoding transport and receptor proteins were differentially expressed in the soleus muscle from young rats, while in soleus muscle from old rats more genes that encoded ribosomal proteins were upregulated. The gene encoding the cold-shock protein RNA-binding motif protein-3 (RBM3) was induced most highly with HS in muscle from old rats, verified by real-time RT-PCR, while no difference with age was observed. The cold-inducible RNA-binding protein (Cirp) gene was also overexpressed with HS, whereas cold-shock protein Y-box-binding protein-1 was not. A time course analysis of RBM3 mRNA abundance during HS showed that upregulation occurred after apoptotic nuclei and markers of protein degradation increased. We conclude that a cold-shock response may be part of a compensatory mechanism in muscles undergoing atrophy to preserve remaining muscle mass and that RBM3 may be a therapeutic target to prevent muscle loss.     

No limiting role for glycogenin in determining maximal attainable glycogen levels in rat skeletal muscle

We examined whether the protein level and/or activity of glycogenin, the protein core upon which glycogen is synthesized, is limiting for maximal attainable glycogen levels in rat skeletal muscle. Glycogenin activity was 27.5 +/- 1.4, 34.7 +/- 1.7, and 39.7 +/- 1.3 mU/mg protein in white gastrocnemius, red gastrocnemius, and soleus muscles, respectively. A similar fiber type dependency of glycogenin protein levels was seen. Neither glycogenin protein level nor the activity of glycogenin correlated with previously determined maximal attainable glycogen levels, which were 69.3 +/- 5.8, 137.4 +/- 10.1, and 80.0 +/- 5.4 micromol/g wet wt in white gastrocnemius, red gastrocnemius, and soleus muscles, respectively. In additional experiments, rats were exercise trained by swimming, which resulted in a significant increase in the maximal attainable glycogen levels in soleus muscles ( approximately 25%). This increase in maximal glycogen levels was not accompanied by an increase in glycogenin protein level or activity. Furthermore, even in the presence of very high glycogen levels ( approximately 170 micromol/g wet wt), approximately 30% of the total glycogen pool continued to be present as unsaturated glycogen molecules (proglycogen). Therefore, it is concluded that glycogenin plays no limiting role for maximal attainable glycogen levels in rat skeletal muscle.     

Estrogen effects on skeletal muscle insulin-like growth factor 1 and myostatin in ovariectomized rats

Previous work showed that estrogen replacement attenuates muscle growth in immature rats. The present study examined muscle insulin-like growth factor-1 (IGF-1) and myostatin expression to determine whether these growth regulators might be involved in mediating estrogen's effects on muscle growth. IGF-1 and myostatin message and protein expression in selected skeletal muscles from 7-week-old sham-ovariectomized (SHAM) and ovariectomized rats that received continuous estrogen (OVX/E2) or solvent vehicle (OVX/CO) from an implant for 1 week or 5 weeks was measured. In the 1-week study, ovariectomy increased IGF-1 mRNA expression in fast extensor digitorum longus and gastrocnemius muscles; the increase was reversed by estrogen replacement. A similar trend was observed in the slow soleus muscle, although the change was not statistically significant. In contrast to mRNA, muscle IGF-1 protein expression was not different between SHAM and OVX/ CO animals in the 1-week study. One week of estrogen replacement significantly decreased IGF-1 protein level in all muscles examined. Myostatin mRNA expression was not different among the 1-week treatment groups. One week of estrogen replacement significantly increased myostatin protein in the slow soleus muscle but not the fast extensor digitorum longus and gastrocnemius muscles. There was no treatment effect on IGF-1 and myostatin expression in the 5-week study; this finding suggested a transient estrogen effect or upregulation of a compensatory mechanism to counteract the estrogen effect observed at the earlier time point. This investigation is the first to explore ovariectomy and estrogen effects on skeletal muscle IGF-1 and myostatin expression. Results suggest that reduced levels of muscle IGF-1 protein may mediate estrogen's effect on growth in immature, ovariectomized rats. Increased levels of muscle myostatin protein may also have a role in mediating estrogen's effects on growth in slow but not fast skeletal muscle.     

Anatomic capillarization is maintained in relative excess of fiber oxidative capacity in some skeletal muscles of late middle-aged rats.

The anatomic size of the capillary-to-fiber (C/F) interface plays an important role in O(2) flux from blood to tissue by determining the surface area available for diffusion and is maintained in relative proportion to fiber mitochondrial volume across a wide range of muscle aerobic capacity. In the present study, we examined an estimate of the anatomic size of the C/F interface [the quotient of the individual C/F ratio and fiber perimeter, C/F perimeter exchange (CFPE) index] and fiber oxidative capacity in different skeletal muscles, or muscle regions, to test the hypothesis that capillarization would be maintained in relative excess of reduced fiber oxidative capacity in aged muscles. The right gastrocnemius, plantaris, and soleus muscles from young adult (8 mo old) and late middle-aged (28-30 mo old) Fischer 344 x Brown Norway F1 hybrid rats were excised for evaluation of flux through electron transport chain complexes I-III and/or morphometric estimation of capillarization. Muscle mass was lower in the gastrocnemius muscles of the older animals (2,076 +/- 32 vs. 1,825 +/- 47 mg in young adult vs. late middle-aged, respectively; mean +/- SE) but not the plantaris or soleus muscles. Fibers were smaller in the white region of gastrocnemius muscles but larger in the red region of gastrocnemius muscles of the older animals. There was no difference in the number of capillaries around a fiber, the individual C/F ratio, or the CFPE index between groups for any muscle/region, whereas flux through complexes I-III was reduced by 29-43% in late middle-aged animals. Thus the greater quotient of indexes of anatomic capillarity (individual C/F ratio or CFPE index) and fiber oxidative capacity in soleus and the white region of gastrocnemius muscles, but not in the red region of gastrocnemius muscles of the older animals, shows that anatomic capillarity is maintained in relative excess of oxidative capacity in some muscle regions in late middle-aged rats.     

Aging is associated with elevated muscle triglyceride content and increased insulin-stimulated fatty acid uptake

The purpose of the present study was to examine the utilization of fatty acids (FA) and muscle substrates by skeletal muscle in young, middle-aged, and old adult rats under hyperglycemic and hyperinsulinemic conditions. Male Fischer 344 x Brown Norway rats aged 5, 15, or 24 mo underwent hindlimb perfusion with a medium of 20 mM glucose, 1 mM palmitate, 1,000 microU/ml insulin, [1-14C]palmitate, and [3-3H]glucose. Glucose uptake and palmitate delivery were similar among age groups. Palmitate uptake and oxidation as well as muscle protein concentration of fatty acid translocase (FAT/CD36) and plasma membrane fatty acid-binding protein (FABPPM) were significantly increased (P < or = 0.05) in 24- vs. 5- and 15-mo-old animals. Compared with 5- and 15-mo-old animals, pre- and postperfusion muscle triglyceride (TG) levels were significantly (P < 0.05) elevated 72-145% in red and 112-129% in white muscles of 24-mo-old animals. Palmitate uptake was associated with total preperfusion TG concentration (r2 = 0.27, P < 0.05) and total TG synthesis rate (r2 = 0.68, P < 0.05). These results indicate that, under insulin-stimulated conditions, FA uptake is significantly increased in old animals, which is associated with increased rates of TG synthesis and may contribute to the accumulation of TG in muscle of old animals.     

Effect of acute exercise on the content of free sphinganine and sphingosine in different skeletal muscle types of the rat.

It has previously been shown that prolonged exercise of moderate intensity reduces the content of ceramide in each type of skeletal muscle. This was accompanied by a reduction in the activity of neutral, Mg++-dependent sphingomyelinase (the major enzyme responsible for ceramide formation from sphingomyelin) in the soleus and red gastrocnemius, but not in the white gastrocnemius (A. Dobrzyń and J. Górski, Am. J. Physiol.: Endorcinol. Metab. 282: E277 - E285, 2002). No other data on regulation of ceramide metabolism in contracting muscles are available. The aim of the present study was to examine the content of sphinganine (a key precursor of ceramide on the de novo synthesis route) and the content of sphingosine (the main product of ceramide catabolism) in different skeletal muscle types after two kinds of acute exercise. The experiments were carried out on 30 male Wistar rats, 250 - 280 g of body weight. The rats were divided equally into three groups: 1 - control, 2 - run until exhaustion (1200 m/h, +10 degree incline), 3 - a group in which the sciatic nerve was stimulated 10 min with tetanic pulses (60 pulses/min). Samples were taken of the soleus and of the red and white section of the gastrocnemius. These muscles are composed mostly of the slow-twitch oxidative, fast-twitch oxidative-glycolytic and fast-twitch glycolytic fibers, respectively. Lipids were extracted with chloroform/methanol. Sphinganine and sphingosine were quantified by high-performance liquid chromatography. At rest, the content of sphinganine in the soleus was higher than in the red gastrocnemius (p < 0.05), and in the latter, it was higher than in the white gastrocnemius (p < 0.01). Prolonged exercise increased the content of sphinganine approximately 6-fold in each muscle. The resting content of sphingosine in the soleus and in the red gastrocnemius was similar--higher than in the white gastrocnemius (p < 0.001 and p < 0.01, respectively). The content of sphingosine increased over 3-fold in the soleus and nearly 2-fold in the red and white sections of the gastrocnemius. Stimulation of the sciatic nerve increased the content of both compounds approximately 2-fold in each muscle. We conclude that acute exercise increases both de novo synthesis and catabolism of ceramide in skeletal muscles. Accumulation of sphingosine in contracting muscles may contribute to the development of fatigue.     

Age-associated differential expression of Na(+)-K(+)-ATPase subunit isoforms in skeletal muscles of F-344/BN rats.

Skeletal muscle expresses multiple isoforms of the Na(+)-K(+)-ATPase. Their expression has been shown to be differentially regulated under pathophysiological conditions. In addition, previous studies suggest possible age-dependent alterations in Na(+)-K(+) pump function. The present study tests the hypothesis that advancing age is associated with altered Na(+)-K(+)-ATPase enzyme activity and isoform-specific changes in expression of the enzyme subunits. Red and white gastrocnemius (Gast) as well as soleus muscles of male Fischer 344/Brown Norway (F-344/BN) rats at 6, 18, and 30 mo of age were examined. Na(+)-K(+)-ATPase activity, measured by K(+)-stimulated 3-O-methylfluorescein phosphatase activity, increased by approximately 50% in a mixed Gast homogenate from 30-mo-old compared with 6- and 18-mo-old rats. Advancing age was associated with markedly increased alpha(1)- and beta(1)-subunit, and decreased alpha(2)- and beta(2)-subunit in red and white Gast. In soleus, there were similar changes in expression of alpha(1)- and alpha(2)-subunits, but levels of beta(1)-subunit were unchanged. Functional Na(+)-K(+)-ATPase units, measured by [(3)H]ouabain binding, undergo muscle-type specific changes. In red Gast, high-affinity ouabain-binding sites, which are a measure of alpha(2)-isozyme, increased in 30-mo-old rats despite decreased levels of alpha(2)-subunit. In white Gast, by contrast, decreased levels of alpha(2)-subunit were accompanied by decreased high-affinity ouabain-binding sites. Finally, patterns of expression of the four myosin heavy chain (MHC) isoforms (type I, IIA, IIX, and IIB) in these muscles were similar in the three age groups examined. We conclude that, in the skeletal muscles of F-344/BN rats, advancing age is associated with muscle type-specific alterations in Na(+)-K(+)-ATPase activity and patterns of expression of alpha- and beta-subunit isoforms. These changes apparently occurred without obvious shift in muscle fiber types, since expression of MHC isoforms remained unchanged. Some of the alterations occurred between middle-age (18 mo) and senescence (30 mo), and, therefore, may be attributed to aging of skeletal muscle.     

Sepsis and inflammatory insults downregulate IGFBP-5, but not IGFBP-4, in skeletal muscle via a TNF-dependent mechanism

The purpose of the present study was to determine whether catabolic stimuli that induce muscle atrophy alter the muscle mRNA abundance of insulin-like growth factor binding protein (IGFBP)-4 and -5, and if so determine the physiological mechanism for such a change. Catabolic insults produced by endotoxin (LPS) and sepsis decreased IGFBP-5 mRNA time- and dose-dependently in gastrocnemius muscle. This reduction did not result from muscle disuse because hindlimb immobilization increased IGFBP-5. Continuous infusion of a nonlethal dose of tumor necrosis factor-alpha (TNF-alpha) decreased IGFBP-5 mRNA 70%, whereas pretreatment of septic rats with a neutralizing TNF binding protein completely prevented the reduction in muscle IGFBP-5. The addition of LPS or TNF-alpha to cultured C(2)C(12) myoblasts also decreased IGFBP-5 expression. Although exogenously administered growth hormone (GH) increased IGFBP-5 mRNA 2-fold in muscle from control rats, muscle from septic animals was GH resistant and no such elevation was detected. In contrast, exogenous administration of IGF-I as part of a binary complex composed of IGF-I/IGFBP-3 produced comparable increases in IGFBP-5 mRNA in both control and septic muscle. Concomitant determinations of IGF-I mRNA content revealed a positive linear relationship between IGF-I and IGFBP-5 mRNA in the same muscle in response to LPS, sepsis, TNF-alpha, and GH treatment. Although dexamethasone decreased muscle IGFBP-5, pretreatment of rats with the glucocorticoid receptor antagonist RU486 did not prevent the sepsis-induced decrease in IGFBP-5 mRNA. In contrast, muscle IGFBP-4 mRNA abundance was not significantly altered by LPS, sepsis, or hindlimb immobilization. In summary, these data demonstrate that various inflammatory insults decrease muscle IGFBP-5 mRNA, without altering IGFBP-4, by a TNF-dependent glucocorticoid-independent mechanism. Finally, IGF-I appears to be a dominant positive regulator of IGFBP-5 gene expression in muscle under both normal and catabolic conditions.     

Reduced lipoprotein lipase activity in postural skeletal muscle during aging.

Lipoprotein lipase (LPL) is a key enzyme for fatty acid and lipoprotein metabolism in muscle. However, the effect of aging on LPL regulation in skeletal muscle is unknown. We report the effect of aging on LPL regulation in the soleus (red oxidative postural) muscle and the tibialis anterior (white glycolytic non-weight-bearing) muscle in 4- and 24-mo-old Fischer 344 rats and 18- and 31-mo-old Fischer 344 x Brown-Norway F1 (F-344 x BN F1) rats. Total and heparin-releasable LPL (HR-LPL) activities were decreased 38% (P < 0.01) and 52% (P < 0.05), respectively, in the soleus muscle of the older Fischer 344 rats. There was a 32% reduction (P < 0.05) of total LPL protein mass in the soleus muscle with aging. The results were confirmed in another strain. A decrease of total LPL activity (-50%, P < 0.05) was also found in the soleus muscle between 18- and 31-mo-old F-344 x BN F1 rats. LPL mRNA concentration in the soleus muscle was not different between ages. Total LPL protein mass was reduced by 46% (P < 0.05) in the soleus muscle of the 31-mo-old F-344 x BN F1 rats. In the tibialis anterior muscle, neither LPL activity nor mRNA concentration was affected by age in either strain. In conclusion, LPL regulation in a non-weight-bearing muscle was not affected by aging. However, there was a pronounced reduction in LPL activity and LPL protein mass in postural muscle with aging.     

Increased DNA fragmentation and altered apoptotic protein levels in skeletal muscle of spontaneously hypertensive rats.

Apoptosis is a highly conserved process that plays an important role in controlling tissue development, homeostasis, and architecture. Dysregulation of apoptosis is a hallmark of numerous human pathologies including hypertension. In the present work we studied the effect of hypertension on apoptosis and the expression of several apoptotic signaling and/or regulatory proteins in four functionally and metabolically distinct muscles. Specifically, we examined these markers in soleus, red gastrocnemius, white gastrocnemius, and left ventricle (LV) of 20-wk-old normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Compared with WKY rats SHR had a significantly greater heart weight, LV weight, and mean arterial pressure. In general, SHR skeletal muscle had increased Bax protein, procaspase-3 protein, caspase-3 activity, cleaved poly(ADP-ribose) polymerase protein, and DNA fragmentation as well as decreased Bcl-2 protein and a lower Bcl-2-to-Bax ratio. Subcellular distribution studies demonstrated increased levels of apoptosis-inducing factor protein in cytosolic or nuclear extracts as well as elevated nuclear Bax protein in SHR skeletal muscle. Moreover, heat shock protein 70 in red gastrocnemius and soleus was significantly correlated to several apoptotic factors. With the exception of lower heat shock protein 90 levels in SHR no additional differences in any apoptotic markers were observed in LV between groups. Collectively, this report provides the first evidence that apoptotic signaling is altered in skeletal muscle of hypertensive animals, an effect that may be mediated by both caspase-dependent and -independent mechanisms. This proapoptotic state may provide some understanding for the morphological and functional abnormalities observed in skeletal muscle of hypertensive animals.     

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.     

IGF-I restores satellite cell proliferative potential in immobilized old skeletal muscle.

One of the key factors responsible for the age-associated reduction in muscle mass may be that satellite cell proliferation potential (number of doublings contained within each cell) could become rate limiting to old muscle regrowth. No studies have tested whether repeated cycles of atrophy-regrowth in aged animals deplete the remaining capacity of satellite cells to replicate or what measures can be taken to prevent this from happening. We hypothesized that there would be a pronounced loss of satellite cell proliferative potential in gastrocnemius muscles of aged rats (25- to 30-mo-old FBN rats) subjected to three cycles of atrophy by hindlimb immobilization (plaster casts) with intervening recovery periods. Our results indicated that there was a significant loss in gastrocnemius muscle mass and in the proliferative potential of the resident satellite cells after just one bout of immobilization. Neither the muscle mass nor the satellite cell proliferation potential recovered from their atrophied values after either the first 3-wk or later 9-wk recovery period. Remarkably, application of insulin-like growth factor I onto the atrophied gastrocnemius muscle for an additional 2 wk after this 9-wk recovery period rescued approximately 46% of the lost muscle mass and dramatically increased proliferation potential of the satellite cells from this muscle.     

Effect of acute streptozotocin diabetes on fatty acid content and composition in different lipid fractions of rat skeletal muscle.

The aim of the present study was to examine the effect of acute streptozotocin diabetes on long chain fatty acid content and composition in different lipid classes of particular muscle types in the rat. Two days after streptozotocin administration, rats were anesthetised, and the white and red sections of the gastrocnemius, the soleus and the blood were taken. Lipids were extracted with chloroform/methanol and separated into different fractions (phospholipids, free fatty acids, di- and triacylglycerols) by means of thin layer chromatography. Fatty acids of each fraction were identified and quantified by means of gas-liquid chromatography. The diabetes resulted in elevation of the concentration of blood glucose (over four-fold) and the plasma free fatty acid (over two-fold). Total free fatty acid content in the muscles of diabetic rats increased by 26% in the white, 24% in the red gastrocnemius and 21% in the soleus. There were also changes in the composition of that fraction in each muscle. Diacylglycerol fatty acid content was elevated in both parts of the gastrocnemius (the white part by 15%, the red part by 44%) and remained stable in the soleus of the diabetic rats. The content of triacylglycerol fatty acids was elevated only in the red gastrocnemius in the diabetic group (by 112%), but changes in fatty acid composition in this fraction occurred in each muscle. The content of phospholipid fatty acids was elevated in the white gastrocnemius (by 13%) and remained stable in other muscles. There were only minor changes in phospholipid fatty acid composition in the diabetic rats. We concluded that acute insulin deficiency changes fatty acid content and composition in skeletal muscle lipids. The changes depend both on lipid fraction and muscle type.     

Effect of prolonged intermittent hypoxia and exercise training on glucose tolerance and muscle GLUT4 protein expression in rats

We compared the chronic effect of intermittent hypoxia and endurance training on the glucose tolerance and GLUT4 protein expression in rat skeletal muscle. Thirty-two Sprague-Dawley rats were matched for weight and assigned to one of the following four groups: control, endurance training, hypoxia, or hypoxia followed by endurance training. Hypoxic treatment consisted of breathing 14% O₂ for 12 h/day under normobaric conditions, and the training protocol consisted of making animals swim 2 times for 3 h/day. At the end of the 3rd week, an oral glucose tolerance test (OGTT) was performed 16 h after treatments. At the end of the 4th week, GLUT4 protein, mRNA, and glycogen storage in skeletal muscle were determined. Endurance training significantly improved OGTT results. Glycogen content and GLUT4 protein expression in the plantaris and red gastrocnemius, but not in the soleus or white gastrocnemius muscles, were also elevated. Chronic intermittent hypoxia also improved OGTT results, but did not alter GLUT4 protein expression. Additionally, hypoxia followed by exercise training produced significant increases in GLUT4 protein and mRNA in a greater number of muscles compared to endurance training alone. Both exercise training and hypoxia significantly reduced body mass, and an additive effect of both treatments was found. In conclusion, chronic intermittent hypoxia improved glucose tolerance in the absence of increased GLUT4 protein expression. This treatment facilitated the exercise training effect on muscle GLUT4 expression and glycogen storage. These new findings open the possibility of utilizing intermittent hypoxia, with or without exercise training, for the prevention and clinical treatment of type 2 diabetes or insulin resistance.