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It is well established that heat stress (HS) negatively impacts growth rate in swine. Although reduced feed intake undoubtedly plays a significant role in this reduction, studies in laboratory animals and other non-swine species indicate muscle growth also is affected by HS-related alterations in muscle physiology. There is now emerging evidence that heat shock proteins (Hsp), produced in response to HS and other types of cellular stress, may play an important role in regulating rate and efficiency of muscle growth. Because muscle satellite cells play a crucial role in postnatal muscle growth, the effects of HS on rates of satellite cell proliferation, protein synthesis, and protein degradation play an important role in determining the rate and extent of muscle growth. Consequently, in the current study we have examined the effects of mild HS (40.5°C for 48 h) on the rates of proliferation, protein synthesis, and protein degradation and on levels of Hsp90, Hsp70, and Hsp25/27 mRNA and protein in cultured porcine muscle satellite cells (PSC). Mild HS of PSC cultures resulted in 2.5-, 1.4-, and 6.5-fold increases (P < 0.05) in the levels of Hsp90, Hsp70, and Hsp25/27 mRNA, respectively, relative to the levels in control cultures. Levels of Hsp 90, 70, and 25/27 proteins were also increased in HS PSC cultures compared to those in control cultures. Proliferation rates in HS PSC cultures were 35% less (P < 0.05) than those in control cultures. Protein synthesis rates in HS fused PSC cultures were 85% greater (P < 0.05) than in control cultures, and protein degradation rates in HS fused PSC were 23% less (P < 0.05) than in control cultures. In light of the crucial role satellite cells play in postnatal muscle growth, the HS-induced changes we have observed in rates of proliferation, protein turnover, and in levels of Hsp mRNA and protein in PSC cultures indicate that mild HS affects the physiology of PSC in ways that could affect muscle growth in swine. [ABSTRACT FROM AUTHOR]

We previously showed that a combined trenbolone acetate (TBA)/estradiol-17β (E2) implant significantly increases IGF-I mRNA levels in the LM of feedlot steers by 28 d after implantation. Here we compare the effects of E2 (25.7 mg), TBA (120 mg), and combined TBA (120 mg)/E2 (24 mg) implants on IGF-I, ICF-I receptor (IGFR-1), estrogen receptor (ER)-α and androgen receptor (AR) mRNA levels in the LM of steers. Twenty yearling crossbred steers with an average initial BW of 421.1 ± 3.6 kg were stratified by BW and randomly assigned to 1 of 4 treatments: 1) nonimplanted, control; 2) implanted with TBA and E2; 3) implanted with E2; or 4) implanted with TBA. Steers were weighed weekly starting on d 0, and muscle biopsy samples were taken from each steer on d 0 (before implantation), 7, 14, and 28. Ribonucleic acid was prepared from each sample and real-time reverse transcription-PCR was used to determine the levels of IGF-I, IGFR-1, ER-α, and AR mRNA. Body weight of implanted steers, adjusted by using d-0 BW as a covariant, tended (P = 0.09) to be greater than that of control steers. On d 7 and 28, IGF-I mRNA levels were greater (58 and 78%, respectively; P < 0.009) in E2-implanted animals than in control steers. Similarly, on d 28 the LM IGF-I mRNA level was 65% greater (P = 0.017) in TBA/E2-implanted steers than in control animals. In contrast, the TBA implant did not increase (P = 0.99) LM IGF-I mRNA levels after 28 d of implantation. Muscle IGFR-1, AR, and ER-α mRNA levels were not different (P > 0.47) in any of the treated groups compared with the control group. These data suggest that E2 is responsible for the increased muscle ICF-I mRNA level observed in steers implanted with a combined TBA/E2 implant. [ABSTRACT FROM AUTHOR]

Both transforming growth factor (TGF-β) and growth and development factor (GDF)-8 (myostatin) affect muscle differentiation by suppressing proliferation and differentiation of myogenic cells. In contrast, insulin-like growth factors (IGFs) stimulate both proliferation and differentiation of myogenic cells. In vivo, IGFs are found in association with a family of high-affinity insulin-like growth factor binding proteins (IGFBP 1-6) that affect their biological activity. Treatment of porcine embryonic myogenic cell (PEMC) cultures with either TGF-β1 or GDF-8 suppressed proliferation and increased production of IGFBP-3 protein and mRNA (P<0.005). An anti-IGFBP-3 antibody that neutralizes the biological activity of IGFBP-3 reduced the ability of either TGF-β1 or GDF-8 to suppress PEMC proliferation (P<0.005). However, this antibody did not affect proliferation rate in the presence of both TGFβ1 and GDF-8. These data show that IGFBP-3 plays a role in mediating the activity of either TGF-β1 or GDF-8 alone but not when both TGF-β1 and GDF-8 are present. In contrast to findings in T47D breast cancer cells, treatment of PEMC cultures with IGFBP-3 did not result in increased levels of phosphosmad-2. Since TGF-β and GDF-8 are believed to play a significant role in regulating proliferation and differentiation of myogenic cells, our current data showing that IGFBP-3 plays a role in mediating the activity of these growth factors in muscle cell cultures strongly suggest that IGFBP-3 also may be involved in regulating these processes in myogenic cells. [ABSTRACT FROM AUTHOR]

Opiates have potent antidiarrheal actions that are mediated in part by delta-opioid receptors (DOR). We examined DOR localization within subregions of porcine ileum, a tissue analogous to human small bowel. A partial cDNA sequence for porcine DOR was obtained after reverse transcription-polymerase chain reaction cloning of forebrain RNA; it encoded the end of transmembrane domain 1 through the beginning of transmembrane domain 7 and exhibited 93% nucleotide identity with human DOR. Positive signals for DOR mRNA were found in all subregions of the porcine ileal wall. With an antiserum recognizing an N-terminal epitope in murine DOR, DOR-like immunoreactivity was localized in neurons within myenteric and submucous ganglia, longitudinal and circular smooth muscle, and villous lamina propria. The DOR agonist [D-Ser2, Leu5, Thr6]enkephalin (DSLET) attenuated circular smooth muscle contractions in porcine ileum that were evoked by electrical stimulation of myenteric cholinergic neurons. These results are consistent with previous reports of the DOR-mediated neuromodulation that underlies the antipropulsive and antisecretory effects of opioids in the intestinal tract. [ABSTRACT FROM AUTHOR]

Insulin-like growth factor binding proteins (IGFBP)-3 and -5 play a significant role in the mechanism by which TGF-beta and myostatin suppress proliferation of porcine embryonic myogenic cells (PEMC) and porcine muscle satellite cells (PMSC). RNA interference (RNAi) utilizing small inhibitory RNA (siRNA) is currently extensively used to silence specific genes in mammalian cells. Consequently, we have cloned a small hairpin (sh) IGFBP-3 RNA sequence (complementary 19mer siRNA sequences separated by a hairpin loop) into the pSilencer 2.1 (Ambion) siRNA expression vector. Electroporation was used to transiently transfect this construct into PEMC cells (IGFBP-3-silenced PEMC). As a control, the same electroporation procedure was used to transfect the vector containing a nonsense sequence supplied by the manufacturer into PEMC (mock-silenced cells). As compared to mock-silenced or non-transfected control cells, IGFBP-3-silenced PEMC showed a 90% reduction (p<0.01) in IGFBP-3 protein and mRNA levels. Neither IGFBP-2 nor IGFBP-5 mRNA or protein levels were significantly affected in the IGFBP-3-silenced cell population, indicating that the suppression of IGFBP-3 production was specific. Suppression of IGFBP-3 production in IGFBP-3-silenced PEMC persisted for at least 120 h after transfection, providing ample time to accomplish experiments assessing the effects of IGFBP-3 knock-down on proliferation and cell signaling. Additionally, immunohistochemical studies show that intracellular IGFBP-3 levels were dramatically reduced in IGFBP-3-silenced cells as compared to mock-silenced cells. We also have identified an shRNA that reduces IGFBP-5 mRNA in PEMC by 95% (as compared to mock-silenced control PEMC) (p<0.01) without significantly altering the levels of IGFBP-2 or -3 mRNA or protein. Silencing IGFBP-3 and IGFBP-5 production in PEMC cells will provide a valuable research tool for use in assessing the role of these IGFBPs in mediating the anti-proliferative effects of myostatin and TGF-beta on these cells. [ABSTRACT FROM AUTHOR]

The proliferation-suppressing actions of myostatin and TGF-beta in porcine embryonic myogenic cell (PEMC) cultures are mediated, at least in part, by insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5. Consequently, understanding the mechanism of action of these IGFBPs in myogenic cells is important to understanding how TGF-beta and myostatin regulate growth of muscle. We have used anti-rpIGFBP-3 (anti-BP3), anti-rpIGFBP-5 (anti-BP5) and anti-desmin antibodies to localize IGFBP-3, IGFBP-5 and desmin, respectively, in PEMC. IGFBP-3 was detected in the cytoplasm and nuclei of desmin-positive, mononucleated cells in proliferating PEMC cultures; thereby, establishing that IGFBP-3 is present in PEMC (controls using non-specific IgG show no staining). Similarly, IGFBP-3 was detected in cultured PEMC myotubes. In proliferating PEMC cultures, treatment for 24 h with 20 ng TGF-beta/ml medium resulted in an 80% increase (p<0.01) in the number of nuclei containing IGFBP-3. Myogenic cells pre-treated with anti-BP3 for 24 h prior to immunohistochemical localization showed dramatically reduced intracellular levels of IGFBP-3 as compared to control cells that received no pre-treatment. This confirms reports in other cell types that a significant portion, if not all, of the intracellular IGFBP-3 represents uptake of secreted IGFBP-3. Additionally, these results establish that anti-BP-3 interferes with the transport of IGFBP-3 into myogenic cells. IGFBP-5 was detected in the cytoplasm and nuclei in proliferating and fused PEMC cultures (controls with non-specific IgY show no staining). Localization of IGFBP-3 and IGFBP-5 in PEMC at different stages of differentiation or after treatment with specific growth factors should lead to a greater understanding of the roles of these IGFBPs in muscle growth and differentiation. [ABSTRACT FROM AUTHOR]