Sheep Skeletal Muscle Research Articles

Niacin supplementation induces type II to type I muscle fiber transition in skeletal muscle of sheep

BackgroundIt was recently shown that niacin supplementation counteracts the obesity-induced muscle fiber transition from oxidative type I to glycolytic type II and increases the number of type I fibers in skeletal muscle of obese Zucker rats. These effects were likely mediated by the induction of key regulators of fiber transition, PPARδ (encoded by PPARD), PGC-1α (encoded by PPARGC1A) and PGC-1β (encoded by PPARGC1B), leading to type II to type I fiber transition and upregulation of genes involved in oxidative metabolism. The aim of the present study was to investigate whether niacin administration also influences fiber distribution and the metabolic phenotype of different muscles [M. longissimus dorsi (LD), M. semimembranosus (SM), M. semitendinosus (ST)] in sheep as a model for ruminants. For this purpose, 16 male, 11 wk old Rhoen sheep were randomly allocated to two groups of 8 sheep each administered either no (control group) or 1 g niacin per day (niacin group) for 4 wk.ResultsAfter 4 wk, the percentage number of type I fibers in LD, SM and ST muscles was greater in the niacin group, whereas the percentage number of type II fibers was less in niacin group than in the control group (P < 0.05). The mRNA levels of PPARGC1A, PPARGC1B, and PPARD and the relative mRNA levels of genes involved in mitochondrial fatty acid uptake (CPT1B, SLC25A20), tricarboxylic acid cycle (SDHA), mitochondrial respiratory chain (COX5A, COX6A1), and angiogenesis (VEGFA) in LD, SM and ST muscles were greater (P < 0.05) or tended to be greater (P < 0.15) in the niacin group than in the control group.ConclusionsThe study shows that niacin supplementation induces muscle fiber transition from type II to type I, and thereby an oxidative metabolic phenotype of skeletal muscle in sheep as a model for ruminants. The enhanced capacity of skeletal muscle to utilize fatty acids in ruminants might be particularly useful during metabolic states in which fatty acids are excessively mobilized from adipose tissue, such as during the early lactating period in high producing cows.

Characterization of the Molecular Architecture of Human Caveolin-3 and Interaction with the Skeletal Muscle Ryanodine Receptor

Caveolin-3 facilitates both caveolae formation and a range of cell signaling pathways, including Ca(2+) homeostasis. Caveolin-3 forms a disc-shaped nonamer that binds the Ca(2+)-release channel, RyR1. Multiple caveolin-3 nonamers bind to a single RyR1 homotetramer. First three-dimensional structural insights into caveolin-3 assembly, interactions with RyR1 suggest a novel role in muscle contraction and/or for channel localization within the membrane. Caveolin-3 (cav-3), an integral membrane protein, is a building block of caveolae as well as a regulator of a number of physiological processes by facilitating the formation of multiprotein signaling complexes. We report that the expression of cav-3 in insect (Sf9) cells induces caveola formation, comparable in size with those observed in native tissue. We have also purified the recombinant cav-3 determining that it forms an oligomer of ∼220 kDa. We present the first three-dimensional structure for cav-3 (using transmission electron microscopy and single particle analysis methods) and show that nine cav-3 monomers assemble to form a complex that is toroidal in shape, ~16.5 nm in diameter and ~ 5.5 nm in height. Labeling experiments and reconstitution of the purified cav-3 into liposomes have allowed a proposal for the orientation of the protein with respect to the membrane. We have identified multiple caveolin-binding motifs within the ryanodine receptor (RyR1) sequence employing a bioinformatic analysis. We have then shown experimentally that there is a direct interaction between recombinant cav-3 nonamers and purified RyR1 homotetramers that would imply that at least one of the predicted cav-3-binding sites is exposed within the fully assembled RyR1 structure. The cav-3 three-dimensional model provides new insights as to how a cav-3 oligomer can bind multiple partners in close proximity to form signaling complexes. Furthermore, a direct interaction with RyR1 suggests a possible role for cav-3 as a modifier of muscle excitation-contraction coupling and/or for localization of the receptor to regions of the sarcoplasmic reticulum.

In vitro culture and induced differentiation of sheep skeletal muscle satellite cells

Skeletal muscle satellite cells are adult muscle-derived stem cells receiving increasing attention. Sheep satellite cells have a greater similarity to human satellite cells with regard to metabolism, life span, proliferation and differentiation, than satellite cells of the rat and mouse. We have used 2-step enzymatic digestion and differential adhesion methods to isolate and purify sheep skeletal muscle satellite cells, identified the cells and induced differentiation to examine their pluripotency. The most efficient method for the isolation of sheep skeletal muscle satellite cells was the type I collagenase and trypsin 2-step digestion method, with the best conditions for in vitro culture being in medium containing 20% FBS+10% horse serum. Immunofluorescence staining showed that satellite cells expressed Desmin, α-Sarcomeric Actinin, MyoD1, Myf5 and PAX7. After myogenic induction, multinucleated myotubes formed, as indicated by the expression of MyoG and fast muscle myosin. After osteogenic induction, cells expressed Osteocalcin, with Alizarin Red and ALP (alkaline phosphatase) staining results both being positive. After adipogenic induction, cells expressed PPARγ2 (peroxisome-proliferator-activated receptor γ2) and clear lipid droplets were present around the cells, with Oil Red-O staining giving a positive result. In summary, a successful system has been established for the isolation, purification and identification of sheep skeletal muscle satellite cells.

Central Leptin Activates Mitochondrial Function and Increases Heat Production in Skeletal Muscle

Leptin acts on the brain to increase postprandial heat production in skeletal muscle of sheep. To determine a mechanism for this effect, we examined the role of mitochondrial uncoupling and AMP-activated protein kinase (AMPK). Ovariectomized ewes (n=4/group) received infusion lines into the lateral cerebral ventricle, and leptin (10 μg/h) was infused to increase heat production in skeletal muscle. In animals that were program fed (1100-1600 h), skeletal muscle biopsies were taken after either central infusion of leptin or vehicle to measure the expression of uncoupling protein (UCP) mRNA and the phosphorylation status of AMPK. Respiratory function was also quantified in mitochondria isolated from skeletal muscle. Leptin infusion increased the expression of UCP2 and UCP3 mRNA as well as UCP3 protein but not UCP1 mRNA in muscle. Leptin also increased substrate-driven, coupled (ADP-driven), and uncoupled (oligomycin) respiration but had no effect on the total respiratory capacity. The respiratory control ratio was lower in leptin-treated (vs. vehicle-treated) animals, indicating a predominant effect on uncoupled respiration. There was no effect of central leptin treatment on AMPK phosphorylation. We then infused 5-aminoimidazole-4-carboxamide-1β-riboside (AICAR) (10 mg/h for 6 h) directly into the femoral artery and measured skeletal muscle temperature; muscle was also collected for isolated mitochondria studies. AICAR had no effect on heat production or substrate-driven, uncoupled, or total respiratory states in skeletal muscle mitochondria. However, AICAR increased ADP-driven (coupled) respiration in mitochondria. In conclusion, leptin acts at the brain to increase heat production in muscle through altered mitochondrial function, indicative of adaptive thermogenesis.

Central infusion of leptin does not increase AMPK signaling in skeletal muscle of sheep

In sheep, central leptin infusion reduces food intake and increases energy expenditure in adipose tissue and skeletal muscle. The mechanisms for these peripheral effects of central leptin in sheep are not known but, on the basis of rodent studies, may involve AMPK. In mice, central leptin acutely increases both skeletal muscle AMPK activation and glucose uptake. Thus, to investigate whether these effects exist in higher-order mammals, ovariectomized Corriedale ewes (n = 4 per group) received a continuous lateral ventricular infusion (60 μl/h) of either leptin (50 μg/h) or artificial cerebrospinal fluid (aCSF; CON) for 8 days. Tritiated glucose (3-(3)H-glucose) was infused intravenously for calculation of whole body glucose turnover during both acute (6 h) and chronic (7-8 days) leptin/aCSF infusion. Muscle biopsies were also obtained. Leptin infusion reduced (P < 0.05) food intake and body weight, and it also increased plasma epinephrine concentration at 6 h and 7 days, suggesting increased sympathetic nerve activity. Despite this, and in contrast to rodent studies, central leptin infusion did not increase skeletal muscle AMPKα Thr(172) phosphorylation or ACCβ Ser(221) phosphorylation. Surprisingly, the glucose rate of appearance (glucose Ra) and rate of disappearance (glucose Rd) were reduced by both acute and chronic leptin infusion. Direct infusion of the AMPK activator 5-aminoimidazole-4-carboxyamide-ribonucleoside (AICAR) into the femoral artery increased skeletal muscle AMPK phosphorylation. In conclusion, although central leptin infusion in sheep caused the predicted reduction in food intake and increases plasma epinephrine concentration, it had no effect on AMPK activation in skeletal muscle and actually reduced glucose disposal. This suggests that there are species differences in the peripheral responses to central leptin infusion.

The Effect of Nutritional Supplements on Muscle-Derived Stem Cells in vitro.

Postnatal muscle stem cells, recognized as myogenic satellite cells, were isolated from sheep skeletal muscle and used in these experiments. Forty-one different metabolic compounds that are commonly found in commercially-available oral supplements were exposed to primary muscle stem cell cultures, in an effort to ascertain whether any one compound could alter satellite cell proliferation or differentiation (a first step towards elucidating the metabolomics or nutrigenomics of these stem cells). These compounds included energetic moieties, amino acid analogs, fatty acids and analogs including different forms of conjugated linoleic acid, minerals and mineral conjugates, insect hormones, caffeine, plant extracts, and extracts from over-the-counter supplements, and were obtained by key manufacturers in a form that would be commercially available. The compounds were sterilized and then exposed to myogenic satellite cell cultures at different levels (ranging from toxic to physiologic) to ascertain if there would be an effect. The results suggested that exposure of satellite cells to only a few compounds resulted in any measurable effect(s). Ten compounds elicited increases in proliferation, and four compounds promoted increases in differentiation. These results suggest avenues for the exploration of enhancing muscle stem cell activity of interest for muscle wasting disorders, sarcopenia of aging and physical performance.

Characterization of microRNAs from sheep (Ovis aries) using computational and experimental analyses

Ovis aries is one of the most important agricultural livestock for meat production, and also is an ideal model organism for biological and comparative genomics studies. Many miRNAs have been reported for their important roles in developmental processes in various animals, but there is limited information about O. aries miRNAs. In this study, combining a computational method based on expressed sequence tag (EST) analysis with experimental identification based on small RNA cDNA library, we identified 31 miRNAs belong to 24 families in sheep, 2 of which were novel miRNAs which had never been previously identified in any species. Especially, we cloned 12 miRNAs from the sheep skeletal muscle, which were good candidate miRNAs to be studied about the miRNA-dependant regulated process of muscle development, and we identified four pairs of miRNA/miRNA and one pair of miRNA-3p/miRNA-5p from sheep EST sequences. Expression analysis indicated that some miRNAs were expressed in a specific tissue, and the pair of miRNA-3p/miRNA-5p and one pair of miRNA/miRNA had a similar relative expression pattern in some tissues, respectively. Further, we predicted 120 potential target genes of 31 oar-miRNAs on the 3'UTR of O. aries genes. Gene ontology analysis showed that most of these genes took part in the cellular process and metabolic process. Our results enriched the O. aries miRNA database and provided useful information for investigating biological functions of miRNAs and miRNA in sheep.

In this issue

AbstractDo rafts jump ship?pp. 4321–4328A raft, as most of us picture it, is a collection of floating material tied together to keep some kind of load afloat. A domain, on the other hand, is the area ruled by an individual, often based on inheritance. The cholesterol‐rich plasma membrane patches found on cell surfaces seem to fit the description of a domain reasonably well ‐ floating on the cell surface, they participate in cell signaling and endocytosis. And, presumably, when the host cell divides, its progeny receive a startup supply of domains (by foul means or fair). Kim et al. explored this question of distribution using synthetic glycosphingolipids (C8De and C8Lt). C8De stimulates endocytosis and rearrangement of domain proteins. Using lipid‐free methods, domains were isolated and analyzed by 2DGE, MALDI‐TOFMS and immunofluorescent microscopy. Proteins transferred to the surface after treatment were involved in ubiquitination, lipid transfer, integrin, small GTPases and several unknown functions. magnified imageFresh frozen versus formalin fixed ‐ and the winner is…pp. 4329–4339…biomedical research. The existence of a reservoir of millions of samples, many paired or containing areas of both normal and affected cells, has attracted many proteome researchers. These formalin‐fixed paraffin embedded (FFPE) samples, however, have proven very difficult to exploit. Addis et al, report here a protocol that yields full‐length protein products, suitable for 2D PAGE, Western (immuno) blots, LC‐MS/MS and MALDI‐MS, from FFPE sections. Protein extractions were performed using detergents and reducing agents and followed by TCA/acetone precipitation to remove interfering material. As many as 250 spots could be identified from FFPE sheep skeletal muscle, whereas 390 were found in fresh frozen samples. Not all 2D spots matched perfectly, but patterns were very reproducible and easily matched. magnified imagePeas porridge hot, peas porridge cold…pp. 4406–4415Agricultural applications of genetic engineering usually have regulatory requirements that GM offspring be essentially equivalent to the parental line with the exception of the target gene. This can be hard to do or hard to prove. Take the case of field peas, Mendel's favorite. To give the crop resistance to pea weevil, the α‐amylase inhibitor (α‐AI1) of the common bean was inserted into peas. Unfortunately, the engineers got more than they bargained for: the GM peas caused undesirable immunological effects in feeding tests. Upon proteomic analysis, Chen, et al., found a problem ‐ transfected genetic material included 33 genes that were over‐ or under‐expressed in five different patterns. Most of the problems were associated with Agrobacterium‐mediated events. Of the 16 proteins identified by 2D GE and MALDI‐TOF/TOF MS, many were potentially allergenic food storage proteins. A 2D gel is a cheap way to check the temperature of the soup. magnified image

Cover Picture: Proteomics 18'09

AbstractDo rafts jump ship?A raft, as most of us picture it, is a collection of floating material tied together to keep some kind of load afloat. A domain, on the other hand, is the area ruled by an individual, often based on inheritance. The cholesterol‐rich plasma membrane patches found on cell surfaces seem to fit the description of a domain reasonably well ‐ floating on the cell surface, they participate in cell signaling and endocytosis. And, presumably, when the host cell divides, its progeny receive a startup supply of domains (by foul means or fair). Kim et al. explored this question of distribution using synthetic glycosphingolipids (C8De and C8Lt). C8De stimulates endocytosis and rearrangement of domain proteins. Using lipid‐free methods, domains were isolated and analyzed by 2DGE, MALDI‐TOFMS and immunofluorescent microscopy. Proteins transferred to the surface after treatment were involved in ubiquitination, lipid transfer, integrin, small GTPases and several unknown functions.Kim, S.‐Y. et al., Proteomics 2009, 9, 4321–4328.Fresh frozen versus formalin fixed ‐ and the winner is……biomedical research. The existence of a reservoir of millions of samples, many paired or containing areas of both normal and affected cells, has attracted many proteome researchers. These formalin‐fixed paraffin embedded (FFPE) samples, however, have proven very difficult to exploit. Addis et al, report here a protocol that yields full‐length protein products, suitable for 2D PAGE, Western (immuno) blots, LC‐MS/MS and MALDI‐MS, from FFPE sections. Protein extractions were performed using detergents and reducing agents and followed by TCA/acetone precipitation to remove interfering material. As many as 250 spots could be identified from FFPE sheep skeletal muscle, whereas 390 were found in fresh frozen samples. Not all 2D spots matched perfectly, but patterns were very reproducible and easily matched.Addis, M. et al., Proteomics 2009, 9, 4329–4339.Peas porridge hot, peas porridge cold…Agricultural applications of genetic engineering usually have regulatory requirements that GM offspring be essentially equivalent to the parental line with the exception of the target gene. This can be hard to do or hard to prove. Take the case of field peas, Mendel's favorite. To give the crop resistance to pea weevil, the α‐amylase inhibitor (α‐AI1) of the common bean was inserted into peas. Unfortunately, the engineers got more than they bargained for: the GM peas caused undesirable immunological effects in feeding tests. Upon proteomic analysis, Chen, et al., found a problem ‐ transfected genetic material included 33 genes that were over‐ or under‐expressed in five different patterns. Most of the problems were associated with Agrobacterium‐mediated events. Of the 16 proteins identified by 2D GE and MALDI‐TOF/TOF MS, many were potentially allergenic food storage proteins. A 2D gel is a cheap way to check the temperature of the soup.Chen, H. et al., Proteomics 2009, 9, 4406–4415.

Peri‐implantation and late gestation maternal undernutrition differentially affect fetal sheep skeletal muscle development

Poor prenatal nutrition is associated with a greater risk of adult glucose intolerance and insulin insensitivity in the offspring. Skeletal muscle is the primary tissue for glucose utilization, and insulin resistance in muscle is the earliest identifiable abnormality in the pre-diabetic patient. We investigated the effect of early and late gestation undernutrition on structure and markers of growth and glucose metabolism regulation in the fetal triceps brachii (TB, slow- and fast-twitch myofibres) and soleus (slow-twitch myofibres) muscles. Pregnant sheep were fed 100% nutrient requirements (C, n = 8) or a restricted diet peri-implantation (PI, n = 9; 40%, 1-31 days gestation (dGA) (term approximately 147)) or in late gestation (L, n = 6; 50%, 104-127 dGA). At 127 +/- 1 dGA we measured myofibre and capillary density in the fetal TB and soleus muscles, and mRNA levels in the TB of insulin receptor (InsR), glucose transporter-4 (GLUT-4) and type 1 insulin-like growth factor receptor (IGF-1R). Total myofibre and capillary densities were lower in the TB, but not the soleus, of PI and L fetuses. The predominant effect in the L group was on slow-twitch myofibres. In TB, InsR, GLUT-4 and IGF-1R mRNA levels were greater in L group fetuses. Our finding of reduced myofibre density is consistent with a redistribution of resources at the expense of specific peripheral tissues by early and late gestation undernutrition which may be mediated by a decrease in capillary density. The increase in key regulatory components of glucose uptake following late gestation undernutrition may constitute a short-term compensation to maintain glucose homeostasis in the face of fewer type I (insulin-sensitive) myofibres. However, together these adaptations may influence the risk of later metabolic disease and thus our findings have implications for future strategies aimed at improving maternal diet.

Mesenchymal Stem Cells: Isolation, Characterisation and In Vivo Fluorescent Dye Tracking

Cell therapies have been used to regenerate the heart by direct myocardial delivery, by coronary infusion and by surface attached scaffolds. Multipotent mesenchymal stem cells (MSC) with capacity to differentiate into cardiomyocytes and other cell lines have been predominantly trialled in rodents. However, large animal models are increasingly needed to translate basic research into new, safe regenerative therapies. Understanding the mode of action of cell therapies in the mammalian heart has been limited by cell tracking capability. This study examined the ability to track the fate of allogeneic MSC in sheep using various fluorescent dyes. MSC isolated from sheep bone marrow were grown in culture following extraction and flow cytometric characterisation. After labelling with fluorescent tracking dyes (e.g. CFSE and DiI) cells were tested for in vitro and in vivo signal up to six weeks. Labelling effect on cell division and differentiation was studied. Several dyes lost fluorescence and slowed cell division. However, the thiol reactive dye CM-DiI showed detectable in vivo fluorescence in labelled MSC six weeks after injection into sheep skeletal muscle and two weeks after implantation of an MSC coated biomaterial scaffold. CM-DiI labelled MSC differentiated in vitro showed label retention over four weeks. The fluorescent membrane dye CM-DiI tracks implanted sheep MSC and provides an alternative to traditional cell markers such as gene modified GFP.

P2-23 Peri-implantation and late gestation maternal undernutrition differentially alter pathways of insulin and IGF-1 action in fetal sheep skeletal muscle

P2-23 Peri-implantation and late gestation maternal undernutrition differentially alter pathways of insulin and IGF-1 action in fetal sheep skeletal muscle

Protein synthesis rates in skin components and skeletal muscle of sheep selected for divergent clean fleece weight in response to below- and above-maintenance nutrition

Protein metabolism in skin and muscle was studied in Merino wethers selected for high (F+, n = 10) or low (F–, n = 10) estimated breeding values for clean fleece weight, but with similar estimated breeding values for liveweight and fibre diameter, raised to 20 months of age under the same conditions, and then offered two levels of nutrition (0.8 or 1.8 × maintenance) for 37 days. Over 37 days, F+ sheep had greater rate of wool production, liveweight gain, and had greater eye-muscle and fat depth than F– sheep (P &lt; 0.05). Fractional synthesis rates of protein (%/day) in the epidermis, dermis, whole skin and muscle were affected by both feeding level (P &lt; 0.05) and genotype (P &lt; 0.05). The fractional synthesis rates of protein were greater (P &lt; 0.05) in F+ sheep at both levels of intake. There was an interaction (P &lt; 0.01) between genotype and feeding level for the protein fractional synthesis rate in muscle, where F+ sheep were more responsive to higher feed intake. Muscle of F– sheep responded to increased amino acid supply by reducing the rate of protein degradation without altering synthesis rate; whereas muscle of F+ sheep responded by increasing the rates of both protein synthesis and degradation. The overall muscle fractional synthesis rate (1.6%/day) was ~7-times lower than the skin fractional synthesis rate (10.8%/day) in these animals (P &lt; 0.01). F+ sheep had a higher rate of protein synthesis in dermis and whole skin to support their higher wool protein accretion at both levels of feed intake. Muscle protein synthesis rate was greater in F+ sheep offered above-maintenance metabolisable energy (ME) intake than those given below-maintenance ME intake but was unaffected by ME intake in F– sheep. The results indicate that selection for wool growth not only affects production of wool and the wool follicle, but also affects the rate of protein turnover in components of the skin and skeletal muscle.

PREPARATION AND EVALUATION OF CROSS LINKED CHITOSAN-POLYURETHANE MESH IN TISSUE REPAIR IN SHEEP

In the present study, an interpenetrating polymer network (cross-linked chitosan-polyurethane mix) was successfully prepared. It was found that the mixing of 70% chitosan and 30% polyurethane gave the best results as far as the mechanical properties of the network are concerned. Toxicological evaluation of the network was done in 15 rabbits, and it was found to be pathologically non-toxic. The possible effects of the network on the liver, kidneys, and skeletal muscles of sheep with clean or infected open wounds were studied through the measurement of plasma aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and acid phosphatase. In sheep with clean open wounds that were implanted with the network, the levels of the enzymes were 11.04  0.54; 30.83  1.20; 36.37  1.52; and 5.52  0.37, respectively. In sheep with contaminated wounds, the levels of the enzymes were 11.29  0.30; 38.58  1.16; 38.20  1.21, and 6.31  0.46, respectively. Statistically significant differences were not encountered in the values of alanine aminotransferase, alkaline phosphatase, and acid phosphatase in sheep with clean wounds and those with contaminated wounds. However, the level of aspartate aminotransferase was higher significantly (P

Postnatal changes in the expression of genes located in the callipyge region in sheep skeletal muscle

The expression of five genes surrounding the callipyge (CLPG) mutation was analysed in skeletal muscles from lambs at one prenatal and two postnatal ages that coincide with the onset and establishment of muscle hypertrophy. Genotype-specific changes in transcript abundance were detected for paternal allele-specific DLK1 and PEG11 (the official symbol of the latter is RTL1) and the maternal allele-specific MEG3, PEG11AS and MEG8 when the mutation was inherited in cis. There were differences in the temporal and muscle-specific effects on expression between the maternal allele-specific genes and paternal allele-specific genes. Maternal inheritance of the CLPG allele had a significant effect on the expression of MEG3 and MEG8 at prenatal and postnatal ages, whereas paternal inheritance of DLK1 and PEG11 only affected postnatal expression. Genotype-specific changes in PEG11AS expression were detected only in prenatal muscle. Maternal inheritance of the mutation caused similar changes in MEG3 and MEG8 expression in the semimembranosus, which undergoes hypertrophy, and the supraspinatus, which does not hypertrophy. Paternal inheritance of the mutation caused changes in PEG11 expression in both muscles, although the magnitude of expression in semimembranosus was more than 100-fold greater than in supraspinatus. DLK1 expression was upregulated in callipyge animals at both postnatal ages in the semimembranosus, but there was no effect of genotype on DLK1 expression in the supraspinatus at any age. Increased DLK1 expression was likely the primary cause of muscle hypertrophy, but a contribution of PEG11 to the phenotype cannot be ruled out based on gene expression.

Effects of selective hyperglycemia and hyperinsulinemia on glucose transporters in fetal ovine skeletal muscle.

We measured net fetal glucose uptake rate from the placenta, shown previously to be equal to total fetal glucose utilization rate (GUR(f)) and proportional to fetal hindlimb skeletal muscle glucose utilization, under normal conditions and after 1, 2.5, and 24 h of selective hyperglycemia increasing G or selective hyperinsulinemia increasing I. We simultaneously measured the amount of Glut 1 and Glut 4 glucose transporter proteins in fetal sheep skeletal muscle. With increasing G , GUR(f) was increased approximately 40% at 1 and 2.5 h but returned to the control rate by 24 h. This transient increasing G -specific increasing GUR(f) was associated with increased plasma membrane-associated Glut 1 (4-fold) and intracellular Glut 4 (3-fold) protein beginning at 1 h. With increasing I, GUR(f) was increased approximately 70% at 1, 2.5, and 24 h. This more sustained increasing I-specific increasing GUR(f) was associated with a significant increase in Glut 4 protein (2-fold) at 2.5 h but no change in Glut 1 protein. These results show that increasing G and increasing I have independent effects on the amount of Glut 1 and Glut 4 glucose transporter proteins in ovine fetal skeletal muscle. These effects are time dependent and isoform specific and may contribute to increased glucose utilization in fetal skeletal muscle. The lack of a sustained temporal correlation between the increase in transporter proteins and glucose utilization rates indicates that subcellular localization and activity of a transporter or tissues other than the skeletal muscle contribute to net GUR(f).

Branched-Chain Amino Acid Aminotransferase Activity Decreases during Development in Skeletal Muscles of Sheep

The catabolism of branched-chain amino acid (BCAA) differs between sheep and monogastric animals. The transamination of BCAA seems to be affected by development of the sheep. We studied the developmental changes in the activity and expression of the BCAA aminotransferase (BCAT) isoenzymes in skeletal muscle of sheep. Five muscles were taken from fetus, newborn, preruminant and ruminant lambs. BCAT specific activity and the contribution of each BCAT isoenzyme [mitochondrial and cytosolic (BCATm and BCATc, respectively)] were quantified using radioenzymatic and immunoprecipitation assays. BCATm and BCATc mRNAs were assessed by real-time reverse transcription-polymerase chain reaction. BCAT specific activities were 62% (diaphragma) to 83% (longissimus dorsi) lower in the ruminant lamb than in the fetal sheep. BCATm and BCATc were both expressed in sheep skeletal muscle at all developmental stages. BCATc was mainly responsible for the developmental decrease in BCAT specific activity. BCATc specific activities were 77% (diaphragma) to 92% (longissimus dorsi) lower in the ruminant lamb than in the fetal sheep, whereas BCATm specific activities were only 36% (semimembranosus) to 56% (longissimus dorsi) lower. BCATc and BCATm mRNAs in the longissimus dorsi were not affected by development of the sheep. The developmental decrease in BCATc activity, and to a lesser extent in BCATm activity, probably involves posttranscriptional mechanisms in sheep. The present results are consistent with lower in vivo metabolism of BCAA in ruminant than in the fetal sheep.

Sheep cytosolic branched-chain amino acid aminotransferase: cDNA cloning, primary structure and molecular modelling and its unique expression in muscles

This paper presents the cloning and the molecular modelling of the cytosolic branched-chain amino acid aminotransferase (BCATc) from sheep brain. The sheep BCATc cDNA (3 kb) encodes a mature polypeptide of 385 amino acids with a calculated molecular mass of 43 072.93 Da. The sequence of the sheep BCATc cDNA is more similar to other mammalian BCATc cDNAs (53–87% identical) than to the sheep mitochondrial branched-chain amino acid aminotransferase (52%). Sheep BCATc belongs to the IV Folding class of pyridoxal-5′-phosphate-depending enzymes. Based on the known structure of the branched-chain amino acid aminotransferase (BCAT) from Escherichia coli, a molecular model of sheep BCATc (amino acid residues 62–385) was built. This is the first three-dimensional model of any mammalian BCAT. It suggests that the enzymatic mechanism of sheep BCATc and likely of all mammalian BCAT is very similar to the mechanism of the E. coli BCAT and confirms the hypotheses regarding to the substrate binding sites of E. coli BCAT. Sheep skeletal muscle, which is the main in vivo site for transamination of branched-chain amino acids, exhibits an unique expression of BCATc.

Purification and cloning of the mitochondrial branched-chain amino acid aminotransferase from sheep placenta.

This paper presents the first purification of the mitochondrial branched-chain amino acid aminotransferase (BCATm) from sheep placenta. It is a homodimer with an apparent subunit molecular mass of 41 kDa. The enzyme differs from those of the rat and human as it appears to form at least one intermolecular disulfide bond. The sheep BCATm cDNA (1.4 kb) encodes a mature polypeptide of 366 amino acids with a calculated molecular mass of 41 329 Da and a partial mitochondrial targeting sequence of seven amino acids. The sheep BCATm sequence shares higher identity with other mammalian BCATm isoenzymes (82-85%) than with the cytosolic isoenzymes (60%). By Northern blot analysis, a message of 1.7 kb was detected in sheep placenta and skeletal muscle. Measurements of BCAT activity, mRNA and BCATm protein in sheep placenta and skeletal muscle revealed that BCATm is the sole BCAT isoenzyme expressed in placenta, whereas it contributes 57 and 71% of the BCAT activity in tensor fascia latae and masseter muscles from weaned lambs respectively. Skeletal muscle, the main site of branched-chain amino acid transamination, exhibits significantly lower BCAT activity in sheep than in rat. Our results suggest that the low BCATm mRNA level probably accounts for the low BCAT activity in sheep skeletal muscle, and that the metabolic scheme for branched-chain amino acid catabolism is specific to each species.

Two alternative procedures for isolating adipofibroblasts from sheep skeletal muscle

Methods to isolate cells used in the study of adipocyte differentiation are lengthy, potentially damaging to the cells collected, and usually result in a mixed group of cells which are difficult to define clearly. Additionally, much work done on the differentiation of fibroblasts to preadipocytes or preadipocytes to adipocytes has relied on the use of populations of cells which are either embryonic in origin or from genetically unique animals. We therefore report two simple and rapid protocols for obtaining relatively pure populations of preadipocytes from perimuscular fat and from intramuscular fat depots of normal sheep skeletal muscle. In the first procedure, a finely minced preparation of perimuscular adipose tissue is placed directly into flasks for ceiling culture. During the second procedure, free-floating adipocytes, resulting from the enzymatic preparation of satellite cells from skeletal muscle, are placed into ceiling culture. Cells from both isolation procedures attach, grow, and are later harvested for use. These cells demonstrate proliferation and differentiation abilities of normal preadipocytes. Cell populations may be expanded for use in cloning pure populations of adipocytes or used directly in studies to identify mechanisms of adipocyte development and intercellular communication with myogenic cells.