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The purpose of this study was to test mitochondrial functionality under conditions simulating postmortem metabolism. Isolated mitochondria from porcine longissimus lumborum (LLM) and masseter (MM) muscles were incorporated into an in vitro model that mimics postmortem metabolism. pH and 13 C-enrichment of glycolytic and tricarboxylic acid (TCA) cycle intermediates were evaluated at 0, 15, 30, 120, 240, and 1440 min. Addition of mitochondria to the in vitro model lowered its pH at 240 min compared with control. Reactions containing mitochondria had lower pyruvate and lactate [M + 2] and [M + 3] isotopomers at 240 and 1440 min than controls. Furthermore, LLM lowered the enrichment of [M + 2], [M + 3], and [M + 4]α-ketoglutarate at 1440 min compared with MM and control. Succinate [M + 2] and [M + 3] were greater in MM than the control and LLM. [M + 3]fumarate was greater in control at 240 and 1440 min than LLM and MM treatments. Our data indicated that mitochondria are capable of mobilizing pyruvate generated though glycolysis under conditions simulating muscle postmortem metabolism., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Acute activation of AMP-activated protein kinase (AMPK) increases monocarboxylate transporter (MCT) expression in skeletal muscle. However, the impact of chronic activation of AMPK on MCT expression in skeletal muscle is unknown. To investigate, MCT1, MCT2, and MCT4 mRNA expression and protein abundance were measured in the longissimus lumborum (glycolytic), masseter (oxidative), and heart from wild-type (control) and AMPK γ3 pigs. The AMPK γ3 gain in function mutation results in AMPK being constitutively active in glycolytic skeletal muscle and increases energy producing pathways. The MCT1 and MCT2 mRNA expression in muscle was lower ( < 0.05) from both wild-type and AMPK γ3 animals compared to other tissues. However, in both genotypes, MCT1 and MCT2 mRNA expression was greater ( < 0.05) in the masseter than the longissimus lumborum. The MCT1 protein was not detected in skeletal muscle, but MCT2 was greater ( < 0.05) in muscles with an oxidative muscle phenotype. Monocarboxylate transporter 2 was also detected in muscle mitochondria and may explain the differences between muscles. The MCT4 mRNA expression was intermediate among all tissues tested and greater ( < 0.05) in the longissimus lumborum than the masseter. Furthermore, MCT4 protein expression in the longissimus lumborum from AMPK γ3 animals was greater ( < 0.05) than in the longissimus lumborum from wild-type animals. In totality, these data indicate that chronic AMPK activation simultaneously increases MCT2 and MCT4 expression in skeletal muscle.

The Rendement Napole mutation (RN-), which is well known to influence pork quality, also has a profound impact on metabolic characteristics of muscle. Pigs with RN- possess a SNP in the γ3 subunit of adenosine monophosphate (AMP)-activated protein kinase (AMPK); AMPK, a key energy sensor in skeletal muscle, modulates energy producing and energy consuming pathways to maintain cellular homeostasis. Importantly, AMPK regulates not only acute response to energy stress but also facilitates long-term adaptation via changes in gene and protein expression. The RN- allele increases AMPK activity, which alters the metabolic phenotype of skeletal muscle by increasing mitochondrial content and oxidative capacity. Fibers with greater oxidative capacity typically exhibit increased protein turnover and smaller fiber size, which indicates that RN- pigs may exhibit decreased efficiency and growth potential. However, whole body and muscle growth of RN- pigs appear similar to that of wild-type pigs and despite increased oxidative capacity, fibers maintain the capacity for hypertrophic growth. This indicates that compensatory mechanisms may allow RN- pigs to achieve rates of muscle growth similar to those of wild-type pigs. Intriguingly, lipid oxidation and mitochondria function are enhanced in RN- pig muscle. Thus far, characteristics of RN- muscle are largely based on animals near market weight. To better understand interaction between energy signaling and protein accretion in muscle, further work is needed to define age-dependent relationships between AMPK signaling, metabolism, and muscle growth.

Postmortem lactate accumulation in skeletal muscle is linearly associated with the extent of pH decline. Yet, pigs harboring the AMPKγ3(R200Q) mutation produce meat with similar lactate levels to that of wild-type pigs but have a lower ultimate pH. We hypothesized that lower initial lactate levels and (or) lower buffering capacity in muscle of these pigs may help explain this discrepancy. Longissimus lumborum muscle samples were harvested at 0 and 1440 min postmortem from AMPKγ3(R200Q) and wild-type pigs. As expected, AMPKγ3(R200Q) muscle exhibited a lower ultimate pH but similar lactate levels to that of wild-type pigs at 1440 min postmortem. However, the total net lactate produced postmortem was greater in the AMPKγ3(R200Q) muscle due to lower initial lactate levels at 0 min postmortem. Buffering capacity measured over the pH range of 5.5-7.0 was also lower in AMPKγ3(R200Q) muscle. Greater net lactate accumulation postmortem (i.e., glycolytic flux) coupled with a lower buffering capacity explains the lower ultimate pH of meat from AMPKγ3(R200Q) pigs., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Muscles in most domestic animals differ in function and growth potential based largely on muscle fiber type composition. Though much is known about satellite cells (SC), information is limited regarding how populations of SC differ with muscle fiber type, especially in pigs. Therefore, the objective of this study was to isolate and culture SC from red (RST) and white (WST) portions of the semitendinosus muscle of neonatal and adult pigs and determine their capacity to proliferate, differentiate, and express various myosin heavy chain (MyHC) isoforms in vitro. Porcine satellite cells were isolated from RST and WST muscles of 6-wk-old and adult (>6-mo-old) pigs and cultured under standard conditions. Muscle from neonatal pigs yielded nearly 10 times more (P < 0.001) presumptive satellite cells as those from adult pigs, with fusion percentages close to 60% for the former. The RST yielded more (P < 0.001) SC per gram muscle compared to WST, 8.1 ± 0.2 × 10(4) cells versus 6.7 ± 0.1 × 10(4) cells/gram muscle in young pigs, and 9.7 ± 0.4 × 10(3) cells versus 5.5 ± 0.4 × 10(3) cells/gram muscle in adult pigs, respectively. Likewise, satellite cells from RST proliferated faster (P < 0.001) than those from WST across both ages, as indicated by a shorter cell doubling time, 18.6 ± 0.8 h versus 21.3 ± 0.9 h in young pigs, and 23.2 ± 0.7 h versus 26.7 ± 0.9 h in adult pigs, respectively. As a result of shorter times to confluence, satellite cells from RST also formed myotubes earlier than those SC originating from WST. Once induced, however, SC from WST differentiated and fused faster (P < 0.05) as evidenced by fusion percentage within the first 24 h, 41.6% versus 34.3%, respectively; but reached similar ultimate fusion percentages similar to WST by 48 h. Over 90% of MyHC expressed in maximally fused SC cultures from both RST and WST was restricted to the embryonic isoform. Type IIX MyHC mRNA was not detected in any culture. Myotube cultures from RST expressed more (P < 0.01) Type I MyHC isoform mRNA than those from WST, whereas those cultures from WST expressed more (P < 0.05) Type II (including Types IIA and IIB) MyHC transcripts. These data show SC cultures from porcine fast and slow muscles express MyHC profiles largely reflective of their muscle of origin and suggest satellite cells are partially restricted to a particular muscle phenotype in which they are juxtapositioned. Understanding the molecular nature of these intrinsic control mechanisms may lead to improved strategies for augmenting meat animal growth or muscle regeneration.

Animal models of obesity and metabolic dysregulation during growth (or childhood) are lacking. Our objective was to increase adiposity and induce metabolic syndrome in young, genetically lean pigs. Pre-pubertal female pigs, age 35 d, were fed a high-energy diet (HED; n = 12), containing 15% tallow, 35% refined sugars and 9.1-12.9% crude protein, or a control corn-based diet (n = 11) with 12.2-19.2% crude protein for 16 wk. Initially, HED pigs self-regulated energy intake similar to controls, but by wk 5, consumed more (P<0.001) energy per kg body weight. At wk 15, pigs were subjected to an oral glucose tolerance test (OGTT); blood glucose increased (P<0.05) in control pigs and returned to baseline levels within 60 min. HED pigs were hyperglycemic at time 0, and blood glucose did not return to baseline (P = 0.01), even 4 h post-challenge. During OGTT, glucose area under the curve (AUC) was higher and insulin AUC was lower in HED pigs compared to controls (P = 0.001). Chronic HED intake increased (P<0.05) subcutaneous, intramuscular, and perirenal fat deposition, and induced hyperglycemia, hypoinsulinemia, and low-density lipoprotein hypercholesterolemia. A subset of HED pigs (n = 7) was transitioned back to a control diet for an additional six weeks. These pigs were subjected to an additional OGTT at 22 wk. Glucose AUC and insulin AUC did not improve, supporting that dietary intervention was not sufficient to recover glucose tolerance or insulin production. These data suggest a HED may be used to increase adiposity and disrupt glucose homeostasis in young, growing pigs.

The purpose of this study was to determine the effect of 5'-AMP-activated protein kinase (AMPK) on energy metabolism and myosin heavy chain (MyHC) isoform expression in growing pigs using chronic treatment with 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) as a model. Four-week-old pigs were given daily injections of AICAR or 0.9% saline for 10 d. Treatment with AICAR increased (P < 0.05) AMPK activity in semitendinosus muscles (STM). Expression of skeletal muscle specific glucose transporter 4 (GLUT4) was also enhanced (P < 0.05) by AICAR treatment. Using real-time PCR, electrophoresis, and Western blot analyses, we confirmed that AICAR treatment caused a decrease (P < 0.05) in type IIa MyHC isoform mRNA and protein levels and a concomitant increase (P < 0.05) in type IIx MyHC containing fibers. Consistent with a MyHC isoform shift from IIa to IIx, muscles from pigs treated with AICAR had greater (P < 0.05) lactate dehydrogenase (LDH) activity. Moreover, muscle of treated pigs expressed greater (P < 0.05) message for LDH. Administration of AICAR, however, did not alter expression of PPAR-gamma coactivator-1alpha, fatty acid translocase, citrate synthase, or the activity of cytochrome c oxidase. Overall, these results indicate that activation of AMPK by AICAR causes muscle to assume a faster-contracting, more glycolytic nature. These data are in direct contrast to documented effects in rodent models, but these effects may be dependent on the time of administration and the overall growth status of the animal.

Genetic selection for improved growth and overall meatiness has resulted in the occurrence of 2 major mutations in pigs, the Rendement Napole (RN) and Halothane (Hal) gene mutations. At the tissue level, these mutations influence energy metabolism in skeletal muscle and muscle fiber type composition, yet also influence total body composition. The RN mutation affects the adenosine monophosphate-activated protein kinase gamma subunit and results in increased glycogen deposition in the muscle, whereas the Hal mutation alters sarcoplasmic calcium release mechanisms and results in altered energy metabolism. From a meat quality standpoint, these mutations independently influence the extent and rate of muscle energy metabolism postmortem, respectively. Even though these mutations alter overall muscle energy metabolism and histochemically derived muscle fiber type independently, their effects have not been yet fully elucidated in respect to myosin heavy chain (MyHC) isoform content and those enzymes responsible for defining energetics of the tissue. Therefore, the objective of this study was to determine the collective effects of the RN and Hal genes on genes and gene products associated with different muscle fiber types in pig skeletal muscle. To overcome potential pitfalls associated with traditional muscle fiber typing, real-time PCR, gel electrophoresis, and Western blotting were used to evaluate MyHC composition and several energy-related gene expressions in muscles from wild-type, RN, Hal, and Hal-RN mutant pigs. The MyHC mRNA levels displayed sequential transitions from IIb to IIx and IIa in pigs bearing the RN mutation. In addition, our results showed MyHC protein isoform abundance is correlated with mRNA level supporting the hypothesis that MyHC genes are transcriptionally controlled. However, transcript abundance of genes involved in energy metabolism, including lactate dehydrogenase, citrate synthase, glycogen synthase, and peroxisome proliferator-activated receptor alpha, was not different between genotypes. These data show that the RN and Hal gene mutations alter muscle fiber type composition and suggest that muscle fiber energy metabolism and speed of contraction, the 2 determinants of muscle fiber type, can be uncoupled.

Ractopamine (RAC) improves growth by increasing lean accretion and decreasing fat deposition through repartitioning nutrients from adipose tissue to skeletal muscle. Although the process is not completely understood, RAC alters the proportion of muscle fiber type composition toward a faster-contracting phenotype. Because one of the primary determinants of contractile speed is the relative abundance of myosin heavy chain (MyHC) isoforms and because the genes encoding these isoforms are transcriptionally regulated, RAC likely alters MyHC gene expression. Using real-time PCR, the relative abundance of transcripts of individual type I, IIA, IIX, and IIB, and total MyHC, as well as glycogen synthase, citrate synthase, lactate dehydrogenase, peroxisome proliferator activated receptor alpha, beta1-adrenergic receptor (AR), and beta2-AR were determined in the LM of 44 pigs fed RAC (20 mg/kg) for 0, 1, 2, or 4 wk. In addition, MyHC isoform expression was determined in the LM and red semitendinosus and white semitendinosus muscles of 48 pigs fed RAC (20 mg/kg) for shorter periods of 12, 24, 48, or 96 h. Type I MyHC expression was unaffected (P > 0.73) by RAC administration. Type IIA MyHC expression decreased (P < 0.0001) by 96 h, was lower (P < 0.0001) by 1 wk, and returned to normal by 4 wk. Type IIX MyHC mRNA decreased (P < 0.001) by 2 wk and continued to decrease (P < 0.0001) by 4 wk. Most interesting was an increase (P < 0.0001) in type IIB MyHC by 12 h, which was maintained at an elevated level throughout the 4-wk feeding period. Abundance of glycogen synthase transcript was increased (P < 0.05) by 12 h, but was not different from controls at 2 wk, and was lower (P < 0.01) at 4 wk. Gene expression of beta1-AR was not affected by feeding RAC, whereas beta2-AR gene expression was decreased (P < 0.05) by 2 wk. These data show MyHC genes are differentially regulated by RAC and suggest that the beta adrenergic agonist-induced repartitioning effect is, in part, mediated by changing muscle fiber type-specific gene expression, perhaps through the beta2-AR.

Muscle fiber types are classified based on contractile speed and type of metabolism. Fast-contracting fibers involve mainly glycolytic-based metabolism, whereas slow-contracting fibers involve a more oxidative type of energy metabolism. The relationship between expression of the genes controlling these functional characteristics and their relative protein abundance in porcine muscle is unknown. The objective of this study was to determine the expression of adult myosin heavy-chain (MyHC) genes and their corresponding protein content in various porcine muscles. Moreover, changes in expression of 2 genes involved in energy metabolism (glycogen synthase and citrate synthase) were determined on muscles varying in MyHC. Using real-time PCR, the relative transcript abundance was determined for the adult MyHC isoforms (types I, IIA, IIX, and IIB), glycogen synthase, and citrate synthase in the masseter (MAS), diaphragm, longissimus, cutaneous trunci, and red and white semitendinosus muscles of 7 pigs. Each muscle was subjected to SDS-PAGE analyses to determine the relative abundance of each MyHC. The relative transcript abundance of type IIB MyHC was greatest (P < 0.05) in the longissimus, white semitendinosus, and cutaneous trunci muscles, whereas type I MyHC expression was greatest (P < 0.05) in the MAS, diaphragm, and red semitendinosus muscles. Glycogen synthase gene expression was least in the MAS (P < 0.01) but exhibited a pattern similar to MyHC IIB expression across muscles. Citrate synthase transcript abundance, however, varied (P < 0.05) independently of MyHC gene expression. Expression of types I and IIB MyHC was correlated with their tissue protein content (R2 = 0.76 and 0.78, respectively), whereas type IIA and X MyHC expression did not correlate with the SDS-PAGE-determined protein content. These data show differences in MyHC gene expression across various porcine muscles and suggest that expression of these genes is reflective of the type of myosin contained within the muscle. Moreover, these data show that expression of energy-specific genes differs greatly across porcine muscles with different functions.

The objective of this study was to examine the effects of somatotropin (ST) on porcine reproductive and metabolic statuses during early pregnancy. Four pregnant crossbred gilts received 6 mg of recombinant porcine somatotropin (rpST) daily from days 10 to 27 after artificial insemination while six pregnant gilts served as controls. Blood samples were taken on days 8, 10, 12, 14, 18, 22, and 27 prior to rpST injections (8:00 h) and subsequently at 9:00, 10:00, 12:00, 14:00, 16:00, 18:00, and 20:00 h. On all remaining days of treatment, samples were taken once daily before injections (8:00 h). The samples were assayed for the metabolic hormones: ST, insulin-like growth factor I (IGF-I), insulin, thyroxine (T(4)), triiodothyronine (T(3)), and cortisol; for metabolites: free fatty acids (FFA) and glucose; and for the reproductive hormones: luteinizing hormone (LH), progesterone, estradiol-17beta, estrone sulfate, and prostaglandin F(2alpha). Delivery of rpST daily induced a 20- to 40-fold increase in plasma ST concentrations. Moreover, repeated administration of rpST resulted in a continuous increase in plasma IGF-I concentration (P < 0.001), from 191.0 +/- 22.3-340.0 +/- 15.3 ng/mL 24 h after initial injection to 591.3 +/- 46.8 ng/mL after final injections. Mean serum insulin tended to be greater in rpST-treated gilts. Blood concentrations of T(4) were reduced (P < 0.05) from day 14 of gestation in treated gilts while T(3) concentrations remained unchanged. Concentrations of both glucose and FFA were greater (P < 0.01) and cortisol concentrations were unchanged in treated gilts. Changes in reproductive steroid hormones were minimally affected. Circulating progesterone (P = 0.078), and estradiol-17beta (P = 0.087) concentrations tended to be lower in treated animals. These data show that treatment of pregnant gilts with rpST during early gestation mainly impacts metabolic rather than reproductive status., (Copyright 2002 Elsevier Science Inc.)

Feeding beta-adrenergic agonists promotes muscle growth. Early histological techniques failed to show precisely how feeding ractopamine-HCl (Paylean) alters muscle growth in pigs. To understand these effects, an indirect enzyme-linked immunosorbent assay (ELISA) was used to determine the abundance of each adult skeletal muscle myosin heavy chain isoform, one means of assigning muscle fiber type, in fast and slow muscles of pigs fed Paylean. Sixty growing pigs (-85 kg) were randomly assigned to three Paylean doses (0, 20, or 60 ppm). At 3, 7, 14, 28, and 42 d of treatment, four pigs per dose were harvested and white (WST) and red (RST) semitendinosus and longissimus (LM) muscles were removed and processed, and myosin heavy chain was quantified by ELISA. Feeding Paylean enhanced (P < 0.05) pigs' average daily gain. Muscle myosin heavy chain (slow, 2A, 2AX, and 2B) composition differed (P < 0.05) across muscles. Compared with LM, RST contained approximately five times more (P < 0.0001) slow and type 2A myosin heavy chain and three times more 2AX myosin heavy chain but nearly undetectable amounts of 2B myosin heavy chain. Myosin heavy chain composition of the WST closely resembled that of the LM (i.e., greater 2AX and 2B and less slow and 2A). After 42d of 60 ppm Paylean, the amount of slow, 2A, and 2AX myosin heavy chain decreased (P < 0.05) across the three muscles whereas the amount of 2B myosin heavy chain increased (P < 0.05). In contrast, relative amounts of 2A and 2AX myosin heavy chain increased (P < 0.05) in muscle of control pigs at 42d. Changes associated with the 20-ppm dose were intermediate to and different from (P < 0.05) control and 60 ppm treatments. Correlations (P < 0.05) among various myosin heavy chain within muscles suggest that slow, type 2A, and 2X decrease with increases in 2B myosin heavy chain. These data show that administration of Paylean affects myosin heavy chain isoform composition in a time- and dose-dependent manner and provides a mechanism of action for Paylean altering animal growth.

Cell-mediated gene transfer is a potential tool for studying muscle growth, but efficient genetic manipulation and implantation strategies have not been developed for pigs. The objectives of the present study were to determine methods for transient and stable incorporation of reporter genes into porcine muscle cells and to investigate their use for cell-mediated gene transfer in pigs. Porcine myoblasts and fibroblasts were isolated from muscle of 2-wk-old male pigs. Myogenic cell lines were identified using muscle-specific monoclonal antibodies, myotube fusion assays, and the presence of muscle-specific markers (MyoD and desmin). Four commercial cationic liposomes (lipofectAMINE, lipofectin, cellFECTIN, and DMRIE-C) were tested at different DNA:lipid ratios for their ability to transfect myoblasts and fibroblasts transiently with a luciferase reporter plasmid. LipofectAMINE resulted in the greatest (P < .01) transient luciferase activity for both cell types. Electroporation of cells for transient transfection resulted in less luciferase activity than cationic transfection. Stable transfections were conducted using a green fluorescence protein (GFP) reporter plasmid containing the neomycin resistance gene. LipofectAMINE transfection resulted in stable GFP expression in 1:16,000 myoblasts and 1:33,000 fibroblasts. Stable electroporation resulted in efficiencies that were significantly lower than established with cationic liposomes. Porcine cells were transduced with GFP using vesicular stomatitis virus glycoprotein G pseudotyped retrovirus and resulted in efficiencies of 1:1.2 for myoblasts and 1:1.1 for fibroblasts. These results show that cationic liposomes are superior to electroporation for transfection, but retroviral transduction produced stable reporter gene expression in > 80% of porcine muscle cells. Transduced GFP-positive cells were separated from GFP-negative cells by fluorescence-activated cell sorting and implanted into 2-wk-old male pigs. On d 4, implanted muscles were removed and subjected to immunodetection of GFP protein. Fibroblast implantation resulted in limited GFP expression within muscle, whereas myoblast implantation resulted in GFP within muscle fibers. This suggests that cell-mediated gene transfer is possible in porcine muscle and may be useful as an approach for studying muscle growth in pigs.

Development of a recombinant insulin like growth factor I (IGF-I) that is distinguishable from its endogenous counterpart would provide a powerful tool for delineating the role of IGF in myogenesis. Therefore, the objective of this study was to create an epitope-tagged IGF-I that retains biological activity and determine whether expression of this construct is possible in muscle tissue following direct DNA injection. Expression vectors were created that encoded porcine IGF-I containing a T7 (11-amino acid) epitope-tag (TIGF). Immunoreactivity of the purified recombinant TIGF was confirmed using monoclonal antibodies. Biological activity was evaluated by examining differentiation of myoblasts cultured with TIGF or transfected with TIGF plasmid DNA. Addition of purified TIGF to myoblast cultures stimulated (P < 0.05) muscle creatine kinase levels similar to insulin (10(-5) M). Likewise, transfection of L6A1 with TIGF DNA hastened (P < 0.01) differentiation compared to control pcDNA-transfected myoblasts. The integrity of the recombinant protein was confirmed using a sandwich-configured enzyme linked immunosorbent assay. Finally, recombinant TIGF DNA was injected in porcine muscle and the ability to detect TIGF protein was evaluated. TIGF expression was detected in muscle fibers of injected porcine muscle. These data show that a T7 amino acid tag placed on the amino terminus of the IGF-I protein remains intact during processing and does not interfere with the biological activity of the molecule. Use of this DNA construct is an excellent tool for investigating the role of IGFs in control muscle development and provides a model to investigate other regulators of animal growth.

Insulin-like growth factors are associated with myogenesis in vivo, and their actions are mediated by IGF binding proteins (IGFBP). Sites of IGFBP production and their location during early development are not clear. The objective of this research was to examine the developmental expression and location of IGFBP-2, -4, and -5 mRNA and peptides in developing porcine skeletal muscle and liver. Pregnant pigs were euthanatized at various times postconception (pc). Developmental expression of IGFBP was evaluated using total RNA extracted from skeletal muscle and liver of 30-, 44-, 59-, 68-, 75-, 89-, and 109-d pc fetuses and from adult and neonatal pigs. Localization of IGFBP-2, -4, and -5 mRNA and peptides was examined by in situ hybridization and immunocytochemistry of muscle samples from contralateral pelvic limbs of each pig. Overall muscle IGFBP gene expression decreased (P < .05) with increasing age. Moreover, expression of liver IGFBP-2 and -5, but not of IGFBP-4, was greater (P < .05) during prenatal than during postnatal periods. The majority of immunoreactive IGFBP was located in developing muscle cells, with little localized to connective tissue, except at later stages of development. These data show that IGFBP-2, -4, and -5 expression is time- and tissue-dependent in fetal liver and muscle.

Three main fiber types (one slow [type I] and two fast [type IIA and IIB] can be distinguished using conventional actomyosin ATPase (AM-ATPase) histochemistry after acidic pretreatment in mature pig skeletal muscle. We report the isolation, characterization, and identification of four adult 3'-untranslated regions corresponding to types I, IIA, IIB, and IIX myosin heavy chains (MyHC) from a cDNA library. Identification of different type II clones was based on sequence homology, in situ hybridizations (ISH), AM-ATPase histochemistry, and immunocytochemistry. Enzyme histochemistry, immunocytochemistry, and ISH were performed on serial transverse sections of longissimus and red portion of semitendinosus muscle. Results showed that all three fast MyHC transcripts were expressed in the longissimus, whereas only type IIA and IIX transcripts were present in deep red semitendinosus muscle. Type I and IIA fibers contained mostly type I and IIA transcripts, respectively, whereas type IIB fibers contained a heterogeneous population of transcripts. In longissimus muscle, 18, 31, and 51% of conventional IIB fibers were pure IIX, hybrid IIX/IIB, and pure IIB fibers, respectively. Conversely, conventional IIB fibers were actually IIX in deep red semitendinosus muscle. Expression of the three fast adult MyHC isoforms in longissimus was spatially regulated around the typical islets of type I fibers encountered in pig skeletal muscle. Thus, IIA fibers were contiguous to type I fibers, pure IIX fibers were in the direct vicinity of type I and IIA fibers, and hybrid IIX/IIB fibers were located mostly within primary fascicles between the islets of type I fibers; however, pure IIB fibers were located mainly at the periphery of the rosettes near the edges of primary fascicles. In light of the present study, conventional IIB fibers, as defined with AM-ATPase staining, are a heterogeneous population that should be split into pure IIX, hybrid IIX/ IIB, and pure IIB fibers for a more accurate fiber typing.

To investigate the role of IGF in muscle development in vivo, developmental expression and location of IGF-I and -II protein and mRNA were examined in fetal, postnatal, and adult skeletal muscle. Muscle tissue was collected from 30-, 44-, 59-, 68-, 75-, 89-, and 109-d porcine fetuses, 21-d neonatal pigs, and 6-mo-old (adult) pigs. Relative amounts of IGF-II mRNA peaked (P < .05) in 59-d fetal muscle and decreased thereafter. Inversely, muscle IGF-I expression increased (P < .05) to maximal levels around birth. For in situ hybridization, frozen muscle tissue sections (10 microm) were hybridized with a hydrolyzed form of the same riboprobes or incubated with polyclonal or monoclonal antibodies to IGF-I or -II, respectively. The majority of IGF-I and IGF-II mRNA was localized to developing muscle fibers, whereas little signal was found in the surrounding connective tissues. Immunofluorescent localization of IGF-I and -II confirmed that muscle IGF are present in developing muscle fibers. Collectively, these data show that IGF-I and -II are expressed and produced primarily in muscle cells within developing muscle tissue and support the hypothesis that IGF-I and -II modulate fetal muscle development.

Transcriptional regulation of the porcine alpha-skeletal actin gene was investigated by comparative transient transfection assays in cultured mammalian cells and by direct DNA injection in skeletal muscle. Intron I sequences were necessary to direct high-level, cell-specific porcine alpha-skeletal actin expression in C2C12 myotubes, but they inhibited transcription in skeletal muscle. A 5' distal sequence (-1929 to -550), had enhancer-like activity in C2C12 myotubes and directly injected muscle, and inhibited transcription in Hela cells. In contrast, a central region (-550 to -388) enhanced basal transcription in directly injected muscle, but not in C2C12 myotubes. A distal regulatory element localized to the 3' untranslated region modulated SV40 promoter activity only in cell culture studies. These results suggest that the intragenic and 3' distal regulatory element may be differentially utilized during differentiation and maturation of skeletal muscle. All three regions decreased SV40 promoter activity in Hela cells, suggesting that they play a role in defining tissue-specific expression of porcine alpha-skeletal actin. Furthermore, different regulatory programs of alpha-skeletal actin expression appear to exist in these two experimental systems.

The objective of this study was to determine the effects of recombinant porcine somatotropin (rpST) on placental size, fetal growth, and maternal and fetal IGF-I and IGF-II concentrations. Twenty-four pregnant gilts received daily injections of either 1 mL of saline (control) (n = 12) or 5 mg of rpST (n = 12) from d 30 to 43 of gestation. Gilts were slaughtered on d 44 of gestation, reproductive tracts were removed, and fetal weight and length, placental weight, and implantation length were recorded. There was no effect of rpST on fetal or implantation length. Placental weight increased with rpST administration (71.20 +/- 3.52 vs 58.35 +/- 3.41 g; P < .02), as did fetal weight (18.06 +/- .55 vs 16.44 +/- .53 g; rpST vs control, respectively; P < .05). Implantation lengths were partitioned into quartiles to determine the effect of rpST on fetuses with different implantation lengths. The effect of rpST of fetal weight was greater in the first quartile ( < 137.5 mm) than in the fourth quartile ( > 240 mm) (16.04 vs 13.86 g compared with 19.47 vs 18.21 g, respectively). Analysis using a modified Brody curve suggests that the effect of rpST treatment on fetal weight is equivalent to the effect of increasing implantation length by 58.8 mm. Administration of rpST numerically raised IGF-I (P = .07) and IGF-II (P = .12) concentrations in fetal serum. Although maternal serum IGF-I concentrations were similar at d 30, treatment with rpST increased these concentrations over time (77.76, 247.75, 267.85 vs 82.59, 79.59, 77.97 ng/mL on d 30, 37, 43, respectively; P < .001, SE = 14.09). Maternal IGF-II concentrations were also similar at d 30 but decreased over time with rpST treatment (265.78, 219.61, 191.05 vs 285.44, 284.72, 283.05 ng/mL; P < .03, SE = 14.03). Increased maternal IGF-I concentrations may exhibit negative feedback on maternal IGF-II concentrations. The more pronounced effect of rpST on growth in fetuses with shorter implantation lengths suggests that rpST may increase uptake or utilization of nutrients by fetuses. In addition, nutrient transfer across placental membranes may be enhanced by rpST.