Maternal protein restriction induce skeletal muscle changes without altering the MRFs MyoD and myogenin expression in offspring

Stimuli during pregnancy, such as protein restriction, can affect morphophysiological parameters in the offspring with consequences in adulthood. The phenomenon known as fetal programming can cause short- and long-term changes in the skeletal muscle phenotype. We investigated the morphology and the myogenic regulatory factors (MRFs) MyoD and myogenin expression in soleus, SOL; oxidative and slow twitching and in extensor digitorum longus, EDL; glycolytic and fast twitching muscles in the offspring of dams subjected to protein restriction during pregnancy. Four groups of male Wistar offspring rats were studied. Offspring from dams fed a low-protein diet (6?% protein, LP) and normal protein diet (17?% protein, NP) were euthanized at 30 and 112?days old, and their muscles were removed and kept at ?80?°C. Muscles histological sections (8?μm) were submitted to a myofibrillar adenosine triphosphatase histochemistry reaction for morphometric analysis. Gene and protein expression levels of MyoD and myogenin were determined by RT-qPCR and western blotting. The major findings observed were distinct patterns of morphological changes in SOL and EDL muscles in LP offspring at 30 and 112?days old without changes in MRFs MyoD and myogenin expression. Our results indicate that maternal protein restriction followed by normal diet after birth induced morphological changes in muscles with distinct morphofunctional characteristics over the long term, but did not alter the MRFs MyoD and myogenin expression. Further studies are necessary to better understand the mechanisms underlying the maternal protein restriction response on skeletal muscle.     

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.     

Effect of nitric oxide synthase inhibition on mitochondrial biogenesis in rat skeletal muscle.

The purpose of this study was to determine whether nitric oxide synthase (NOS) inhibition decreased basal and exercise-induced skeletal muscle mitochondrial biogenesis. Male Sprague-Dawley rats were assigned to one of four treatment groups: NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME, ingested for 2 days in drinking water, 1 mg/ml) followed by acute exercise, no l-NAME ingestion and acute exercise, rest plus l-NAME, and rest without l-NAME. The exercised rats ran on a treadmill for 53 +/- 2 min and were then killed 4 h later. NOS inhibition significantly (P < 0.05; main effect) decreased basal peroxisome proliferator-activated receptor-gamma coactivator 1beta (PGC-1beta) mRNA levels and tended (P = 0.08) to decrease mtTFA mRNA levels in the soleus, but not the extensor digitorum longus (EDL) muscle. This coincided with significantly reduced basal levels of cytochrome c oxidase (COX) I and COX IV mRNA, COX IV protein and COX enzyme activity following NOS inhibition in the soleus, but not the EDL muscle. NOS inhibition had no effect on citrate synthase or beta-hydroxyacyl CoA dehydrogenase activity, or cytochrome c protein abundance in the soleus or EDL. NOS inhibition did not reduce the exercise-induced increase in peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha) mRNA in the soleus or EDL. In conclusion, inhibition of NOS appears to decrease some aspects of the mitochondrial respiratory chain in the soleus under basal conditions, but does not attenuate exercise-induced mitochondrial biogenesis in the soleus or in the EDL.     

TNF‐α‐mediated reduction in PGC‐1α may impair skeletal muscle function after cigarette smoke exposure

Skeletal muscle dysfunction contributes to exercise limitation in COPD. In this study cigarette smoke exposure was hypothesized to increase expression of the inflammatory cytokine, TNF‐α, thereby suppressing PGC‐1α, and hence affecting down stream molecules that regulate oxygen transport and muscle function. Furthermore, we hypothesized that highly vascularized oxidative skeletal muscle would be more susceptible to cigarette smoke than less well‐vascularized glycolytic muscle. To test these hypotheses, mice were exposed to cigarette smoke daily for 8 or 16 weeks, resulting in 157% (8 weeks) and 174% (16 weeks) increases in serum TNF‐α. Separately, TNF‐α administered to C2C12 myoblasts was found to dose‐dependently reduce PGC‐1α mRNA. In the smoke‐exposed mice, PGC‐1α mRNA was decreased, by 48% in soleus and 23% in EDL. The vascular PGC‐1α target molecule, VEGF, was also down‐regulated, but only in the soleus, which exhibited capillary regression and an oxidative to glycolytic fiber type transition. The apoptosis PGC‐1α target genes, atrogin‐1 and MuRF1, were up‐regulated, and to a greater extent in the soleus than EDL. Citrate synthase (soleus—19%, EDL—17%) and β‐hydroxyacyl CoA dehydrogenase (β‐HAD) (soleus—22%, EDL—19%) decreased similarly in both muscle types. There was loss of body and gastrocnemius complex mass, with rapid soleus but not EDL fatigue and diminished exercise endurance. These data suggest that in response to smoke exposure, TNF‐α‐mediated down‐regulation of PGC‐1α may be a key step leading to vascular and myocyte dysfunction, effects that are more evident in oxidative than glycolytic skeletal muscles. J. Cell. Physiol. 222: 320–327, 2010. © 2009 Wiley‐Liss, Inc.     

Maternal postnatal high-fat diet,rather than gestational diet,affects morphology and mTOR pathway in skeletal muscle of weaning rat

The positive regulation of insulin pathway in skeletal muscle results in increased activity of the mammalian target of rapamycin (mTOR), a positive effector of mRNA translation rate and protein synthesis. Studies that assess the activity of this protein in response to chronic high-fat diet (HFD) are scarce and controversial, and to date, there are no studies evaluating the mTOR pathway in infants exposed to gestational and postgestational HFD. This study investigated the effect of maternal HFD on skeletal muscle morphology and on phosphorylation of proteins that comprise the intracellular mTOR signaling pathway in soleus muscle of offspring at weaning. For this purpose, 10 days prior to conception, 39 female Wistar rats were randomly assigned to either control diet (CTL) or HFD. Later, rats were distributed into four groups according to gestational and postpregnancy diet: CTL/CTL (n=10), CTL/HF (n=11), HF/HF (n=10) and HF/CTL (n=8). After 21 days of lactation, pups were killed, and blood samples and soleus and gastrocnemius skeletal muscle were collected for analysis. We observed an influence of maternal postgestational diet, rather than gestational diet, in promoting an obese phenotype, characterized by body fat accumulation, insulin resistance and high serum leptin, glucose, triglycerides and cholesterol levels (P<.05). We have also detected alterations on skeletal muscle morphology — with reduced myofiber density — and impairment on S6 kinase 1 and 4E binding protein-1 phosphorylation (P<.05). These results emphasize the importance of maternal diet during lactation on muscle morphology and on physiological adaptations of infant rats.     

Plausible role of naringenin against cerebrally implanted C6 glioma cells in rats

Sarcoplasmic and t-tubule membrane proteins regulating sarcoplasmic Ca²⁺ concentration exhibit fibre-type-dependent isoform expression, and play central roles in muscle contraction and relaxation. The purpose of this study was to evaluate the effects of in vitro electrical stimulation on the mRNA expression of components involved in Ca²⁺ regulation in oxidative and glycolytic skeletal muscle. The mRNA level of Ca²⁺-ATPase (SERCA1, 2), calsequestrin (CASQ1, 2), ryanodine receptor (RyR1), and dihydropyridine receptor (Cacna1) was assessed in rat extensor digitorum longus (EDL) and soleus (SOL) muscles at 4 h of recovery following in vitro stimulations (either short intensive (SHO) 60 Hz, 5 min, or prolonged moderate (PRO) 20 Hz, 40 min). Stimulation induced acute regulation of the mRNA level of Ca²⁺-regulating proteins in a manner that does not follow typical fibre-type-specific transitions. In general, stimulation decreased mRNA content of all proteins studied. Most prominent down-regulation was observed for Cacna1 (26 and 32 % after SHO and PRO, respectively, in SOL; 19 % after SHO in EDL). SERCA1, SERCA2, CASQ1, CASQ2, and RyR1 mRNA content also decreased significantly in both muscles relative to resting control. Of notice is that hexokinase II mRNA content was increased in EDL and unchanged in SOL underlining the specificity of the down-regulation of mRNA of Ca²⁺ regulatory proteins. The results demonstrate contraction-induced down-regulation of mRNAs for the main components of Ca²⁺-regulating system in skeletal muscle. The down-regulation of both isoforms of SERCA and CASQ after a single electrical stimulation session suggests that adaptations to repeated stimulation involve further regulatory mechanisms in addition to acute mRNA responses.     

Effects of chronic AICAR treatment on fiber composition, enzyme activity, UCP3, and PGC-1 in rat muscles.

This study was designed to determine the histological and metabolic effects of the administration of 5'-AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) for 14 successive days. AICAR treatment caused a significant decrease in the percentage of type IIB fibers and the concomitant increase in the percentage of type IIX fibers in extensor digitorum longus (EDL) muscle. The capillary density and the capillary-to-fiber ratio were not altered by AICAR. AICAR treatment increased the glycolytic and oxidative enzyme activities but not the antioxidant enzyme activities. The AICAR treatment increased the uncoupling protein 3 (UCP3) level in EDL and the peroxisome proliferator-activated receptor-gamma coactivator-1alpha protein level in the soleus and EDL muscles, whereas the myogenin level was not altered by AICAR. These results seem to imply that the chronic activation of AMPK alters such muscle histochemical and metabolic characteristics.     

Calcium signalling in the regulation of PGC-1alpha, PDK4 and HKII mRNA expression

The role of calcium signalling and specific intracellular calcium signalling pathways in regulating skeletal muscle tissue peroxisome proliferator-activated receptor gamma co-activator (PGC)-1alpha, hexokinase (HK)II and pyruvate dehydrogenase kinase (PDK)4 mRNA was examined. Cultured primary rat skeletal muscle cells were incubated for 6 h in caffeine or ionomycin. Because PGC-1alpha mRNA clearly showed greater induction with ionomycin, the latter was chosen for the main experiments, whereby cells were incubated for 6 h with either ionomycin alone or in combination with either cyclosporin A or KN-62. The PGC-1alpha mRNA level was increased (p<0.05) approximately six-fold and HKII mRNA content approximately two-fold by ionomycin relative to the corresponding controls, whereas the PDK4 mRNA content remained unaffected. Cyclosporin A abolished (p<0.05) and KN-62 reduced (p<0.1) the ionomycin-induced increase in PGC-1alpha mRNA. Electrical stimulation of in vitro incubated rat EDL muscle increased (p<0.05) PGC-1alpha mRNA by 2.2-fold after 4 h of recovery relative to a resting control, and this increase was absent when muscles were incubated with KN-62 or cyclosporin A. The present data strongly suggest that calcium signalling is involved in regulating the PGC-1alpha and HKII genes, but not PDK4. Both calcineurin and CaMK signalling seem to be involved in the calcium- and contraction-mediated PGC-1alpha up-regulation in skeletal muscle.     

Lpaatδ/Agpat4 deficiency impairs maximal force contractility in soleus and alters fibre type in extensor digitorum longus muscle

Lysophosphatidic acid acyltransferase (LPAAT) δ/acylglycerophosphate acyltransferase 4 is a mitochondrial enzyme and one of five homologues that catalyze the acyl-CoA-dependent synthesis of phosphatidic acid (PA) from lysophosphatidic acid. We studied skeletal muscle LPAATδ and found highest levels in soleus, a red oxidative fibre-type that is rich in mitochondria, and lower levels in extensor digitorum longus (EDL) (white glycolytic) and gastrocnemius (mixed fibre-type). Using Lpaatδ-deficient mice, we found no change in soleus or EDL mass, or in treadmill time-to-exhaustion compared to wildtype littermates. There was, however, a significant reduction in the proportion of type I and type IIA fibres in EDL but, surprisingly, not soleus, where these fibre-types predominate. Also unexpectedly, there was no impairment in force generation by EDL, but a significant reduction by soleus. Oxidative phosphorylation and activity of complexes I, I?+?II, III, and IV in soleus mitochondria was unchanged and therefore could not explain this effect. However, pyruvate dehydrogenase activity was significantly reduced in Lpaatδ^?/? soleus and EDL. Analysis of cellular lipids indicated no difference in soleus triacylglycerol, but specific elevations in soleus PA and phosphatidylethanolamine levels, likely due to a compensatory upregulation of Lpaatβ and Lpaatε in Lpaatδ^?/? mice. An anabolic effect for PA as an activator of skeletal muscle mTOR has been reported, but we found no change in serine 2448 phosphorylation, indicating reduced soleus force generation is unlikely due to the loss of mTOR activation by a specific pool of LPAATδ-derived PA. Our results identify an important role for LPAATδ in soleus and EDL.     

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

Acute effects of free fatty acids (FFA) were investigated on: (1) glucose oxidation, and UCP-2 and -3 mRNA and protein levels in 1 h incubated rat soleus and extensor digitorium longus (EDL) muscles, (2) mitochondrial membrane potential in cultured skeletal muscle cells, (3) respiratory activity and transmembrane electrical potential in mitochondria isolated from rat skeletal muscle, and (4) oxygen consumption by anesthetized rats. Long-chain FFA increased both basal and insulin-stimulated glucose oxidation in incubated rat soleus and EDL muscles and reduced mitochondrial membrane potential in C2C12 myotubes and rat skeletal muscle cells. Caprylic, palmitic, oleic, and linoleic acid increased O₂ consumption and decreased electrical membrane potential in isolated mitochondria from rat skeletal muscles. FFA did not alter UCP-2 and -3 mRNA and protein levels in rat soleus and EDL muscles. Palmitic acid increased oxygen consumption by anesthetized rats. These results suggest that long-chain FFA acutely lead to mitochondrial uncoupling in skeletal muscle.     

Catalase-positive microperoxisomes in rat soleus and extensor digitorum longus muscle fiber types

The size, distribution, and content of catalase-reactive microperoxisomes were studied cytochemically in slow-twitch oxidative (SO), fast-twitch oxidative glycolytic (FOG), and fast-twitch glycolytic (FG) fibers of soleus and extensor digitorum longus (EDL) rat muscles. Fiber types were classified on the basis of mitochondrial content and distribution, Z-band widths, and myofibril size and shape. Microperoxisomes were generally located between myofibrils at the I-bands. The absence of crystalloid inclusions prevented positive identification of microperoxisomes in nonreacted and aminotriazole-inhibited muscles. EDL and soleus SO fibers possessed the largest microperoxisomes, whereas FOG and FG fibers of the EDL contained small- to medium-sized microperoxisomes. Comparing either microperoxisome number per muscle fiber area or microperoxisome area per fiber area revealed significant differences between fiber types with this ranking: soleus SO greater than EDL SO greater than EDL FOG greater than EDL FG. The present observations demonstrate that the content of catalase-positive microperoxisomes is greatest in the oxidative muscle fiber types. These cytochemical findings account for the higher catalase activity in homogenates of soleus muscles as compared to that of EDL muscles, because the soleus contains more oxidative fibers than EDL.     

Biochemical and histochemical changes in energy supply enzyme pattern of muscles of the rat during old age.

Senile muscles of the rat (28-36 months) show loss of overall activity of glycolytic and aerobic enzymes. However, there is a differential loss and shift of enzyme activity pattern in the three types of muscles. The extensor digitorum longus (EDL) and diaphragm show a decrease of ratios of glycolytic to aerobic-oxidative enzymes. This shift to a more oxidative type of metabolism is not observed in the soleus muscle. Decrease of enzyme activities is least marked in the diaphragm muscle. Biochemical analysis shows a trend to levelling out of metabolic differences between the different muscle types. This trend of 'dedifferentiation' is most marked when comparing EDL and soleus, least marked when comparing EDL and diaphragm muscle. The histochemical analysis shows a shift from the original mixed to a more uniform pattern of muscle fibres in the EDL and soleus muscle; this levelling-out of differences between enzymatic activities of different muscle fibres is not observed in the diaphragm muscle. Preferential atrophy and loss of ATPase activity in II muscle fibres in the soleus muscle and the occurrence of 'type grouping' are further characteristic features of senile muscle change. The findings show general (i.e. loss of enzyme activities) and differential trends of biochemical and histochemical enzyme changes in different types of muscles.     

Regulation of skeletal muscle dihydropyridine receptor gene expression by biomechanical unloading.

Biomechanical unloading of the rat soleus by hindlimb unweighting is known to induce atrophy and a slow- to fast-twitch transition of skeletal muscle contractile properties, particularly in slow-twitch muscles such as the soleus. The purpose of this study was to determine whether the expression of the dihydropyridine (DHP) receptor gene is upregulated in unloaded slow-twitch soleus muscles. A rat DHP receptor cDNA was isolated by screening a random-primed cDNA lambda gt10 library from denervated rat skeletal muscle with oligonucleotide probes complementary to the coding region of the rabbit DHP receptor cDNA. Muscle mass and DHP receptor mRNA expression were assessed 1, 4, 7, 14, and 28 days after hindlimb unweighting in rats by tail suspension. Isometric twitch contraction times of soleus muscles were measured at 28 days of unweighting. Northern blot analysis showed that tissue distribution of DHP receptor mRNA was specific for skeletal muscle and expression was 200% greater in control fast-twitch extensor digitorum longus (EDL) than in control soleus muscles. A significant stimulation (80%) in receptor message of the soleus was induced as early as 24 h of unloading without changes in muscle mass. Unloading for 28 days induced marked atrophy (control = 133 +/- 3 vs. unweighted = 62.4 +/- 1.8 mg), and expression of the DHP receptor mRNA in the soleus was indistinguishable from levels normally expressed in EDL muscles. These changes in mRNA expression are in the same direction as the 37% reduction in time to peak tension and 28% decrease in half-relaxation time 28 days after unweighting. Our results suggest that muscle loading necessary for weight support modulates the expression of the DHP receptor gene in the soleus muscle.     

Fucoxanthin promotes translocation and induction of glucose transporter 4 in skeletal muscles of diabetic/obese KK-A(y) mice

Fucoxanthin (Fx) isolated from Undaria pinnatifida suppresses the development of hyperglycemia and hyperinsulinemia of diabetic/obese KK-A(y) mice after 2 weeks of feeding 0.2% Fx-containing diet. In the soleus muscle of KK-A(y) mice that were fed Fx, glucose transporter 4 (GLUT4) translocation to plasma membranes from cytosol was promoted. On the other hand, Fx increased GLUT4 expression levels in the extensor digitorum longus (EDL) muscle, although GLUT4 translocation tended to increase. The expression levels of insulin receptor (IR) mRNA and phosphorylation of Akt, which are in upstream of the insulin signaling pathway regulating GLUT4 translocation, were also enhanced in the soleus and EDL muscles of the mice fed Fx. Furthermore, Fx induced peroxisome proliferator activated receptor γ coactivator-1α (PGC-1α), which has been reported to increase GLUT4 expression, in both soleus and EDL muscles. These results suggest that in diabetic/obese KK-A(y) mice, Fx improves hyperglycemia by activating the insulin signaling pathway, including GLUT4 translocation, and inducing GLUT4 expression in the soleus and EDL muscles, respectively, of diabetic/obese KK-A(y) mice.     

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

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