Effect of maternal nutrient restriction in sheep on the development of fetal skeletal muscle

The effect of maternal nutrient restriction on mTOR (mammalian target of rapamyosin) signaling and the ubiquitin system as well as their possible relation to growth of fetal muscle was determined. Ewes were fed to 50% (nutrient-restricted) or 100% (control-fed) of total digestible nutrients (National Research Council requirement) from Days 28 to 78 of gestation. Ewes were killed at Day 78 of gestation, and the fetal longissimus dorsi muscle was sampled for the measurement of mTOR, ribosomal protein S6, AMP-activated protein kinase (AMPK), calpastatin, and protein ubiquitylation. No difference was observed in the content of mTOR and ribosomal protein S6, but the phosphorylation of mTOR at Ser2448 and ribosomal protein S6 at Ser235/336 were reduced (P <0.05) in muscle from nutrient-restricted fetuses. Because phosphorylation of mTOR and ribosomal protein S6 up-regulates protein translation, these results show that nutrient restriction down-regulates protein synthesis in fetal muscle. No difference in AMPK activity was detected. The lack of difference in calpastatin and ubiquitylized protein content shows that nutrient restriction did not affect degradation of myofibrillar proteins in fetal muscle. Fetuses of nutrient-restricted ewes showed retarded development of muscles and skeleton. Muscle from nutrient-restricted fetuses contained fewer secondary myofibers than muscle from control fetuses, and the average area of fasciculi was smaller (P <0.05). The decreased number of secondary myofibers in nutrient-restricted fetuses may result from the decreased mTOR signaling. Lower activation of mTOR signaling in nutrient-restricted fetuses may reduce the proliferation of myoblasts and, thus, reduce the formation of secondary myofibers. This decrease in secondary myofibers in fetuses may predispose fetuses to metabolic diseases, such as diabetes and obesity, in their postnatal lives.     

Acute ethanol exposure in pregnancy alters the insulin-like growth factor axis of fetal and maternal sheep

Maternal ethanol intake during pregnancy impairs fetal growth, but mechanisms are not clearly defined. Reduced IGF abundance or bioavailability in the fetus and/or mother may contribute to this growth restriction. We hypothesized that an episode of acute ethanol exposure, mimicking binge drinking would restrict fetal growth and perturb the maternal and fetal IGF axes. Pregnant sheep were infused intravenously with saline or ethanol (1 g/kg maternal wt) over 1 h, on days 116, 117, and 118 of gestation (start of 1st infusion = time 0, term is 147 days). Maternal and fetal plasma IGF and IGF-binding protein (IGFBP) concentrations were measured before and after each infusion. Compared with controls, ethanol exposure reduced fetal weight at day 120 by 19%, transiently reduced maternal plasma IGF-I (-35%) at 30 h, and decreased fetal plasma IGF-II (-28%) from 24 to 54 h after the first infusion. Ethanol exposure did not alter maternal or fetal plasma concentrations of IGFBP-2 and IGFBP-3, measured by Western ligand blotting. We conclude that suppression of maternal and fetal IGF abundance may contribute to fetal growth restriction induced by acute or binge ethanol exposure.     

Isocaloric maternal low-protein diet alters IGF-I,IGFBPs, and hepatocyte proliferation in the fetal rat

We investigated the effect of an isocaloric maternal low-protein diet during pregnancy in rats on the proliferative capacity of cultured fetal hepatocytes. The potential roles of these changes on the IGF-IGF-binding protein (IGFBP) axis, and the role of insulin and glucocorticoids in liver growth retardation, were also evaluated. Pregnant Wistar rats were fed a control (C) diet (20% protein) or a low-protein (LP) diet (8%) throughout gestation. In primary culture, the DNA synthesis of hepatocytes derived from LP fetuses was decreased by approximately 30% compared with control hepatocytes (P < 0.05). In parallel, in vivo moderate protein restriction in the dam reduced the fetal liver weight and IGF-I level in fetal plasma (P < 0.01) and augmented the abundance of 29- to 32-kDa IGFBPs in fetal plasma (P < 0.01) and fetal liver (P < 0.01). By contrast, the abundance of IGF-II mRNA in liver of LP fetuses was unaffected by the LP diet. In vitro, the LP-derived hepatocytes produced less IGF-I (P < 0.01) and more 29- to 32-kDa IGFBPs (P < 0.01) than hepatocytes derived from control fetuses. These alterations still appeared after 3-4 days of culture, indicating some persistence in programming. Dexamethasone treatment of control-derived hepatocytes decreased cell proliferation (54 +/- 2.3%, P < 0.01) and stimulated 29- to 32-kDa IGFBPs, whereas insulin promoted fetal hepatocyte growth (127 +/- 5.5%, P < 0.01) and inhibited 29- to 32-kDa IGFBPs. These results show that liver growth and cell proliferation in association with IGF-I and IGFBP levels are affected in utero by fetal undernutrition. It also suggests that glucocorticoids and insulin may modulate these effects.     

Indinavir alters regulators of protein anabolism and catabolism in skeletal muscle

The HIV protease inhibitor indinavir adversely impairs carbohydrate and lipid metabolism, whereas its influence on protein metabolism under in vivo conditions remains unknown. The present study tested the hypothesis that indinavir also decreases basal protein synthesis and impairs the anabolic response to insulin in skeletal muscle. Indinavir was infused intravenously for 4 h into conscious rats, at which time the homeostasis model assessment of insulin resistance was increased. Indinavir decreased muscle protein synthesis by 30%, and this reduction was due to impaired translational efficiency. To identify potential mechanisms responsible for regulating mRNA translation, several eukaryotic initiation factors (eIFs) were examined. Under basal fasted conditions, there was a redistribution of eIF4E from the active eIF4E.eIF4G complex to the inactive eIF4E.4E-BP1 complex, and this change was associated with a marked decrease in the phosphorylation of 4E-BP1 in muscle. Likewise, indinavir decreased constitutive phosphorylation of eIF4G and mTOR in muscle, but not S6K1 or the ribosomal protein S6. In contrast, the ability of a maximally stimulating dose of insulin to increase the phosphorylation of PKB, 4E-BP1, S6K1, or mTOR was not altered 20 min after intravenous injection. Indinavir increased mRNA expression of the ubiquitin ligase MuRF1, but the plasma concentration of 3-methylhistidine remained unaltered. These indinavir-induced changes were associated with a marked reduction in the plasma testosterone concentration but were independent of changes in plasma levels of IGF-I, corticosterone, TNF-alpha, or IL-6. In conclusion, indinavir acutely impairs basal protein synthesis and translation initiation in skeletal muscle but, in contrast to muscle glucose uptake, does not impair insulin-stimulated signaling of protein synthetic pathways.     

Detecting metabolic differences in fetal and adult sheep adipose and skeletal muscle tissues

The primary metabolic pathway required to produce ATP differs as a result of tissue type, developmental stage and substrate availability. We utilized molecular and histological techniques to define the metabolic status in foetal and adult, adipose and skeletal muscle tissues. Redox ratios of these tissues were also determined optically by two‐photon microscopy. Adult perirenal adipose tissue had a higher optical redox ratio than fetal perirenal adipose tissue, which aligned with glycolysis being used for ATP production; whereas adult skeletal muscle had a lower optical redox ratio than fetal skeletal muscle, which aligned with oxygen demanding oxidative phosphorylation activity being utilized for ATP production. We have compared traditional molecular and microscopy techniques of metabolic tissue characterization with optical redox ratios to provide a more comprehensive report on the dynamics of tissue metabolism.     

Relationship between accelerations and decelerations in heart rate and skeletal muscle activity in fetal sheep

Accelerations in fetal heart rate have been shown to be closely related to fetal body movements and are indicative of well-being in the human fetus. We have examined the association of accelerations and decelerations in heart rate with skeletal muscle activity in 8 fetal sheep between 125 and 145 days' gestation. Accelerations/decelerations were defined as transient increases/decreases in fetal heart rate of greater than or equal to 10 beats/min. lasting for 5 s or longer. For accelerations (n = 1180), the mean duration was 18.8 +/- 1.5 s (SEM) and the mean amplitude was 25.3 +/- 1.2 beats/min; for decelerations (n = 237), the mean duration was 17.4 +/- 1.6 s and the mean amplitude was 18.7 +/- 1.0 beats/min. Electromyograms were recorded from the nuchal muscles and antagonistic muscle groups of the fetal forelimb and hindlimb. Electromyogram activity occurred during 88.4 +/- 2.8% of accelerations and 60.6 +/- 7.7% of decelerations. There was a 36.6% reduction in the number of accelerations following fetal paralysis with gallamine, but no change in their amplitude or duration. It is concluded that accelerations in heart rate are highly associated with skeletal muscle activity in fetal sheep. The majority of these occur as a result of central neuronal output rather than as a consequence of fetal movement.     

Effects of strenuous maternal exercise on fetal organ weights and skeletal muscle development in rats

The purpose of the present study was to observe the effects of strenuous maternal aerobic exercise throughout gestation on fetal outcome in the rat. The strenuous exercise intensity consisted of a treadmill speed of 30 m.min-1 on a 10 degrees incline, for 120 min.day-1, 5 days.week-1. The rats were conditioned to run on a motor-driven treadmill by following a progressive two-week exercise program, so that by the end of the two weeks the rats were capable of running comfortably at this strenuous intensity in the non-pregnant state. Following the two-week running programme, the rats were paired by weight and randomly assigned to either a pregnant group that continued the running program throughout gestation (pregnant runner), or a pregnant group that did not continue the running program throughout pregnancy (pregnant control). At birth the neonates born to the pregnant running group did not differ in average neonatal body weight values, number per litter or total litter weight values when compared to controls, nor were superficial gross abnormalities observed in neonates born to the pregnant control or pregnant running groups. The strenuous maternal exercise intensity did not alter neonatal organ weight values (brain, heart, liver, lung, kidney), nor neonatal skeletal muscle (gastrocnemius, sternomastoid, diaphragm) when compared to control values. It is suggested that maternal exercise of this intensity throughout gestation does not affect fetal outcome in the rat, and may be due to the animals accustomization to the strenuous exercise protocol prior to pregnancy.     

MicroRNA transcriptome profiles during swine skeletal muscle development

Background

Dietary polyunsaturated fatty acids (PUFA), in particular the long chain marine fatty acids docosahexaenoic (DHA) and eicosapentaenoic (EPA), are linked to many health benefits in humans and in animal models. Little is known of the molecular response to DHA and EPA of the small intestine, and the potential contribution of this organ to the beneficial effects of these fatty acids. Here, we assessed gene expression changes induced by DHA and EPA in the wildtype C57BL/6J murine small intestine using whole genome microarrays and functionally characterized the most prominent biological process.

Results

The main biological process affected based on gene expression analysis was lipid metabolism. Fatty acid uptake, peroxisomal and mitochondrial beta-oxidation, and omega-oxidation of fatty acids were all increased. Quantitative real time PCR, and -in a second animal experiment- intestinal fatty acid oxidation measurements confirmed significant gene expression differences and showed in a dose-dependent manner significant changes at biological functional level. Furthermore, no major changes in the expression of lipid metabolism genes were observed in the colon.

Conclusion

We show that marine n-3 fatty acids regulate small intestinal gene expression and increase fatty acid oxidation. Since this organ contributes significantly to whole organism energy use, this effect on the small intestine may well contribute to the beneficial physiological effects of marine PUFAs under conditions that will normally lead to development of obesity, insulin resistance and diabetes.     

Maternal overnutrition suppresses the phosphorylation of 5'-AMP-activated protein kinase in liver, but not skeletal muscle, in the fetal and neonatal sheep

Epidemiological studies have shown that infants exposed to an increased supply of nutrients before birth are at increased risk of type 2 diabetes in later life. We have investigated the hypothesis that fetal overnutrition results in reduced expression and phosphorylation of the cellular fuel sensor, AMP-activated kinase (AMPK) in liver and skeletal muscle before and after birth. From 115 days gestation, ewes were fed either at or approximately 55% above maintenance energy requirements. Postmortem was performed on lamb fetuses at 139-141 days gestation (n = 14) and lambs at 30 days of postnatal age (n = 21), and liver and quadriceps muscle were collected at each time point. The expression of AMPKalpha1 and AMPKalpha2 mRNA was determined by quantitative RT-PCR (qRT-PCR). The abundance of AMPKalpha and phospho-AMPKalpha (P-AMPKalpha) was determined by Western blot analysis, and the proportion of the total AMPKalpha pool that was phosphorylated in each sample (%P-AMPKalpha) was determined. The ratio of AMPKalpha2 to AMPKalpha1 mRNA expression was lower in fetuses compared with lambs in both liver and muscle, independent of maternal nutrition. Hepatic %P-AMPKalpha was lower in both fetuses and lambs in the Overfed group and %P-AMPKalpha in the lamb liver was inversely related to plasma glucose concentrations in the first 24 h after birth (r = 0.73, P < 0.025). There was no effect of maternal overnutrition on total AMPKalpha or P-AMPKalpha abundance in liver or skeletal muscle. We have, therefore, demonstrated that AMPKalpha responds to signals of increased nutrient availability in the fetal liver. Suppression of hepatic AMPK phosphorylation may contribute to increased glucose production, and basal hyperglycemia, present in lambs of overfed ewes in early postnatal life.     

Differential serotonin responses in the skeletal muscle microcirculation

Closed circuit television microscopy was used to quantitate in vivo responses of small vessels in the rat cremaster muscle to topically applied serotonin. Sprague-Dawley rats were anesthetized with a combination of urethane (800 mg/kg) and alpha-chloralose (60 mg/kg). The cremaster muscle with intact circulation and innervation was suspended in a bath which had controlled pH, pCO2, and pO2. Microvascular diameters of first order arterioles and venules and fourth-order arterioles were measured from the television monitor while serotonin (10(-9)M-10(-4)M) was added to the bath. Fourth-order arterioles (3-11 micron diameter) dilated to a maximum of 267% of their control value with a serotonin concentration of 10(-6)M. Serotonin (10(-4)M) constricted first-order arterioles (78-121 micron) to 61% of their control value. The threshold concentration (10(-8)M) for a serotonin-induced dilation of fourth-order arterioles was 1000 fold less than the threshold concentration (10(-5)M) for serotonin-induced constriction of first-order arterioles. Serotonin (10(-8)M - 10(-4)M) did not alter the diameter of first-order venules (115-195 micron) from the control value. The dose-dependent constriction of first-order arterioles and dose-dependent dilation of fourth-order arterioles by serotonin appear to be independent of each other. In addition, the lack of constriction of first-order venules suggests a heterogenous distribution of serotonin receptors and that the predominate control mechanisms are different at different levels of the arteriolar and venous microcirculation of rat skeletal muscle.     

Dystrophin-related protein, utrophin, in normal and dystrophic human fetal skeletal muscle

Summary Dystrophin is the product of the Duchenne muscular dystrophy (DMD) gene. Dystrophin-related protein (utrophin), an autosomal homologue of dystrophin, was studied in skeletal muscle from normal fetuses aged 9–26 weeks and one stillbirth of 41 weeks' gestation, and compared with low- and high-risk DMD fetuses aged 9–20 weeks. Utrophin was present at the sarcolemma from before 9 weeks' gestation, although there was variability in intensity both within and between myotubes. Sarcolemmal immunolabelling became more uniform, and levels of utrophin increased to a maximum at approximately 17–18 weeks. Levels then declined, until by 26 weeks sarcolemmal labelling was negligible and levels were similar to adult control muscle. By 41 weeks there was virtually no sarcolemmal labelling, although immunolabelling of capillaries was bright. Similar results were obtained with normal and DMD fetal muscle. Utrophin is therefore expressed in the presence and absence of dystrophin and down-regulated before birth in normal fetal muscle fibres. Samples were not available to determine whether or when, utrophin levels decline in DMD fetal muscle. On Western blots, utrophin was shown to have a smaller relative molecular mass than adult dystrophin, but similar to the fetal isoform. Blood vessels were brightly immunolabelled at all ages, although utrophin immunolabelling of peripheral nerves increased with gestational age.     

Matching of maternal and fetal flow ratios in the sheep placenta

Local interaction of maternal and fetal placental blood flows was studied in two groups of unanaesthetized near-term sheep. Five sheep were exposed to a simulated dive to 100 feet of seawater (4.03 atmospheres) for 25 min. Six fetuses received an infusion of noradrenaline (6.8 micrograms/[kg x min]). Radioactive microspheres were administered simultaneously to mother and fetus before (control) and after (test) the experimental manipulation. Maternal and fetal relative activities, defined as % of total placental radioactivity divided by % of total placental weight, were calculated for 1-g pieces of cotyledonary tissue under control and test conditions. Pieces of cotyledons were defined as matched if the direction of change in relative activity from control to test was the same for mother and fetus. In the absence of an interaction between the maternal and fetal placental circulations, the probability of a piece of cotyledon being matched is 0.5. In each series of experiments the proportion of all cotyledon pieces having maternal and fetal relative activities that changed in the same direction was significantly greater than 0.5. Thus, the majority of the placental mass responds to a physical or chemical perturbation of the fetus in such a way that changes in relative perfusion are qualitatively matched in the adjacent maternal and fetal placental circulations.     

Differential regulation of myofilament protein isoforms underlying the contractility changes in skeletal muscle unloading

Weight-bearing skeletal muscles change phenotype in response to unloading. Using the hindlimb suspension rat model, we investigated the regulation of myofilament protein isoforms in correlation to contractility. Four weeks of continuous hindlimb unloading produced progressive atrophy and contractility changes in soleus but not extensor digitorum longus muscle. The unloaded soleus muscle also had decreased fatigue resistance. Along with the decrease of myosin heavy chain isoform I and IIa and increase of IIb and IIx, coordinated regulation of thin filament regulatory protein isoforms were observed: - and -tropomyosin decreased and -tropomyosin increased, resulting in an / ratio similar to that in normal fast twitch skeletal muscle; troponin I and troponin T (TnT) both showed decrease in the slow isoform and increases in the fast isoform. The TnT isoform switching began after 7 days of unloading and TnI isoform showed detectable changes at 14 days while other protein isoform changes were not significant until 28 days of treatment. Correlating to the early changes in contractility, especially the resistance to fatigue, the early response of TnT isoform regulation may play a unique role in the adaptation of skeletal muscle to unloading. When the fast TnT gene expression was upregulated in the unloaded soleus muscle, alternative RNA splicing switched to produce more high molecular weight acidic isoforms, reflecting a potential compensation for the decrease of slow TnT that is critical to skeletal muscle function. The results demonstrate that differential regulation of TnT isoforms is a sensitive mechanism in muscle adaptation to functional demands. troponin T; fatigue resistance; troponin I; tropomyosin; myosin; hindlimb-suspended rat; Western blot protein quantification     

Downhill exercise alters immunoproteasome content in mouse skeletal muscle

Content of the immunoproteasome, the inducible form of the standard proteasome, increases in atrophic muscle suggesting it may be associated with skeletal muscle remodeling. However, it remains unknown if the immunoproteasome responds to stressful situations that do not promote large perturbations in skeletal muscle proteolysis. The purpose of this study was to determine how an acute bout of muscular stress influences immunoproteasome content. To accomplish this, wild-type (WT) and immunoproteasome knockout lmp7 ^?/? /mecl1 ^?/? (L7M1) mice were run downhill on a motorized treadmill. Soleus muscles were excised 1 and 3 days post-exercise and compared to unexercised muscle (control). Ex vivo physiology, histology and biochemical analyses were used to assess the effects of immunoproteasome knockout and unaccustomed exercise. Besides L7M1 muscle being LMP7/MECL1 deficient, no other major biochemical, histological or functional differences were observed between the control muscles. In both strains, the downhill run shifted the force-frequency curve to the right and reduced twitch force; however, it did not alter tetanic force or inflammatory markers. In the days post-exercise, several of the proteasome’s catalytic subunits were upregulated. Specifically, WT muscle increased LMP7 while L7M1 muscle instead increased β5. These findings indicate that running mice downhill results in subtle contractile characteristics that correspond to skeletal muscle injury, yet it does not appear to induce a significant inflammatory response. Interestingly, this minor stress activated the production of specific immunoproteasome subunits that if knocked out were replaced by components of the standard proteasome. These data suggest that the immunoproteasome may be involved in maintaining cellular homeostasis.     

Sepsis alters pyruvate dehydrogenase kinase activity in skeletal muscle

Chronic sepsis promotes a stable increase in pyruvate dehydrogenase kinase (PDHK) activity in skeletal muscle. PDHK is found tightly bound to the pyruvate dehydrogenase (PDH) complex and as free kinase. We investigated the ability of sepsis to modify the activity of the PDHK intrinsic to the PDH and free PDHK. Sepsis was induced by the intraabdominal introduction of a fecal-agar pellet infected with E. coli and B. fragilis. Five days later, mitochondria were isolated from skeletal muscle and PDHK measured in mitochondrial extracts. Sepsis caused an approximate 2-fold stimulation of PDHK. The mitochondrial extracts from control and septic rats were fractionated by gel chromatography on Sephacryl S-300 to separate PDHK intrinsic to PDH complex and free PDHK. PDH complex eluted at void volume and was assayed for PDHK intrinsic to the complex. The activity of PDHK intrinsic to PDH complex was a significantly increased 3 fold during sepsis. Free PDHK activity eluted after the PDH complex and its activity was enhanced by 70% during sepsis. Incubation of PDHK intrinsic to PDH with dichloroactate, an uncompetitive inhibitor of PDHK, showed the PDHK from septic rats relatively less sensitive to inhibition than controls. These results indicate that sepsis induces stable changes in PDHK in skeletal muscle.