Porcine leptin inhibits protein breakdown and stimulates fatty acid oxidation in C2C12 myotubes

This study evaluated the potential mechanism(s) by which leptin treatment inhibits loss of muscle mass with fasting. Cultures of C2C12 myoblasts were differentiated into myotubes with 5% (vol/vol) horse serum in Dulbecco's modified Eagle's medium/F12. These myotubes were used to assess 3H-tyrosine incorporation and release following incubation with recombinant porcine leptin (0 to 500 ng/mL). Protein synthesis in myotubes, as measured by 3H-tyrosine incorporation, was not affected by leptin treatment (P > 0.05). Protein breakdown in C2C12 myotubes, as measured by 3H-tyrosine release, was inhibited by leptin treatment. A leptin concentration of 0.5 ng/mL was sufficient to inhibit 3H-tyrosine release by 3.5% (P < 0.05); 50 ng/mL produced a maximal inhibition of 10.2% (P < 0.05). Dexamethasone (1 microM) was used to maximally stimulate protein breakdown. Leptin (50 ng/mL leptin) decreased dexamethasone-induced 3H-tyrosine release by 32% (P < 0.05). The inhibition of 3H-tyrosine release in C2C12 myotubes suggests that leptin produces a protein-sparing effect in vitro by inhibiting protein breakdown. Fatty acid metabolism also was investigated because fatty acids are a major energy source for muscle during periods of reduced intake, as occurs with leptin treatment. Acute (4 h) and chronic (24 h) exposures to porcine leptin (0 to 500 ng/mL) were used to evaluate 14C-palmitate oxidation. Acute leptin treatment had no effect (P > 0.05) on palmitate metabolism. Chronic leptin exposure resulted in up to a 26% increase in palmitate oxidation (P < 0.05). The stimulation of fatty acid oxidation with chronic leptin treatment suggests that leptin spares other energy sources in muscle from oxidation during periods of a leptin-induced decrease in feed intake.     

Relationship of thyroid status to growth hormone and insulin-like growth factor-I (IGF-I) in plasma and IGF-I mRNA in liver and skeletal muscle of cattle

Steers were made hyperthyroid or hypothyroid to study the effects of physiological alterations in thyroid hormone status on plasma growth hormone (GH) profiles, plasma insulin-like growth factor-I (IGF-I) concentrations, and relative abundance of IGF-I mRNA in skeletal muscle and liver. Eighteen yearling crossbred steers (360 to 420 kg) were randomly allotted to hyperthyroid (subcutaneous injection 0.6 μg/kg BW L-thyroxine for 10 d), hypothyroid (oral thiouracil; 0.25% diet plus 12.5 g capsule/d for 17 d), or control (subcutaneous injection 0.9% NaCl) treatment groups. Blood samples were taken for measurement of GH, IGF-I, thyroxine (T₄) and triiodothyronine (T₃) by RIA. Samples of liver and skeletal muscle were taken by biopsy for measurement of IGF-I mRNA by solution hybridization. Steers receiving thiouracil had 57 and 53% (P<.05) lower T₄ and T₃, respectively, than control steers (84.1 and 1.7 ng/ml). The hyperthyroid steers had 228 and 65% greater (P<.05) T₄ and T₃ than control steers. Neither increased nor decreased thyroid status had any significant effects on plasma GH profiles, liver IGF-I mRNA, or plasma concentration of IGF-I. There was no effect of thyroid hormone alteration on skeletal muscle IGF-I mRNA concentrations. The results of this study suggest that short-term changes in thyroid status of cattle had no major impact on the GH-IGF-I axis or skeletal muscle IGF-I mRNA.     

Genetic variation in stimulated GH release and in IGF-I of young dairy cattle and their associations with the leucine/valine polymorphism in the GH gene

Genetic variations in plasma GH concentrations before and following thyrotropin-releasing hormone (TRH) stimulation and in IGF-I concentrations were studied in 11-mo-old Polish Friesian cattle (104 heifers and 110 bulls). A possible association between stimulated GH release, IGF-I, and the polymorphism in the GH gene causing substitution of leucine-Leu to valine-Val at amino acid position 127 of the protein was also investigated. The GH concentrations were determined in serial plasma samples collected every 15 min from 15 min before to 135 min after intravenous administration of 0.15 microg TRH/kg live weight. The analysis was performed on three variables: baseline (mean of samples at -15 and 0 min), peak (sample at 15 min after injection) and rate (peak minus sample at 60 min, divided by 45 min). The IGF-I concentrations were measured in plasma samples taken before the TRH stimulation. Additionally, first lactation records from the 75 cows earlier tested for GH release and IGF-I were used to study a possible association of milk production traits with GH genotypes. The data were analyzed by multivariate mixed linear models. The heritability of IGF-I reached a higher value (0.35) than variables baseline, peak, and rate (0.02, 0.14, and 0.14, respectively). The GH variables were positively genetically correlated with each other (0.22 to 0.93), whereas they had negative genetic correlations with IGF-I (-0.26). The Val/Val genotypes reached the highest peak value compared with other GH genotypes (P > 0.01), whereas the Leu/Leu genotypes had the highest IGF-I concentrations (P < or = 0.05). Moreover, the Leu/Val heterozygotes were superior to others in milk and protein yields, whereas the Leu/Leu homozygotes reached the highest fat yield (P > or = 0.01). We conclude that GH peak, GH rate, and IGF-I are heritable traits in young dairy cattle and are affected by the Leu/Val polymorphism in the GH gene.     

Low doses of estradiol partly inhibit release of GH in sheep without affecting basal levels

Estradiol increases basal growth hormone (GH) concentrations in sheep and cattle. This study sought to determine the effects of estradiol on GH-releasing hormone (GRH)-stimulated GH release in sheep. Growth hormone secretory characteristics, the GH response to GRH, and steady-state GH mRNA concentrations were determined in castrated male lambs treated with 2 different doses of estradiol 17-β for a 28-d experimental period. Although no differences between treatments in mean GH, basal GH, or GH pulse number were observed after 28 d of estradiol treatment, GH pulse amplitude was greater (P < 0.05) in the 2.00-cm implant-treated animals than in the control and 0.75-cm implant group. The effect of estradiol treatment on GRH-stimulated GH release revealed differences between the control and estradiol-treated animals (P < 0.05). The 15-min GH responses to 0.075 μg/kg hGRH in the control, 0.75-cm, and 2.00-cm implant groups, respectively, were 76 ± 10, 22.6 ± 2.1, and 43.6 ± 15.0 ng/mL. Growth hormone mRNA content was determined for pituitary glands from the different treatment groups, and no differences in steady-state GH mRNA levels were observed. There were no differences in the mean plasma concentrations of IGF-I, cortisol, T₃, or T₄ from weekly samples. Growth hormone release from cultured ovine pituitary cells from control sheep was not affected by estradiol after 72 h or in a subsequent 3-h incubation with estradiol combined with GRH. These data suggest that estradiol has differing actions on basal and GRH-stimulated GH concentrations in plasma, but the increase in pulse amplitude does not represent an increased pituitary sensitivity to GRH.     

Effect of growth hormone administration to mature miniature Brahman cattle treated with or without insulin on circulating concentrations of insulin-like growth factor-I and other metabolic hormones and metabolites

Previously, we determined that a primary cause of proportional stunted growth in a line of Brahman cattle was related to an apparent refractoriness in metabolic response to GH in young animals. The objective of this study was to determine the effect of administration of GH, insulin (INS), and GH plus INS to mature miniature Brahman cows (n = 6; 9.7 ± 2.06 y; 391 ± 48.6 kg) and bulls (n = 8; 9.4 ± 2.00 y; 441 ± 54.0 kg) on circulating concentrations of metabolic hormones and metabolites, primarily IGF-I and IGF-I binding proteins. We hypothesized that IGF-I secretion could be enhanced by concomitant administration of exogenous GH and INS, and neither alone would be effective. Animals were allotted to a modified crossover design that included four treatments: control (CON), GH, INS, and GH + INS. At the start of the study, one-half of the cattle were administered GH (Posilac; 14-d slow release) and the other one-half served as CON for 7 d. Beginning on day 8, and for 7 d, INS (Novolin L) was administered (0.125 IU/kg BW) twice daily (7:00 AM and 7:00 PM) to all animals; hence, the INS and GH + INS treatments. Cattle were rested for 14 d and then were switched to the reciprocal crossover treatments. Blood samples were collected at 12-hour intervals during the study. Compared with CON, GH treatment increased (P < 0.01) mean plasma concentrations of GH (11.1 vs 15.7 ± 0.94 ng/mL), INS (0.48 vs 1.00 ± 0.081 ng/mL), IGF-I (191.3 vs 319.3 ± 29.59 ng/mL), and glucose (73.9 vs 83.4 ± 2.12 mg/dL) but decreased (P < 0.05) plasma urea nitrogen (14.2 vs 11.5 ± 0.75 mg/dL). Compared with INS, GH + INS treatment increased (P < 0.05) mean plasma concentration of INS (0.71 vs 0.96 ± 0.081 ng/mL), IGF-I (228.7 vs 392.3 ± 29.74 ng/mL), and glucose (48.1 vs 66.7 ± 2.12 mg/dL), decreased (P < 0.01) plasma urea nitrogen (13.6 vs 10.4 ± 0.76 mg/dL), and did not affect GH (13.5 vs 12.7 ± 0.95 ng/mL). In the miniature Brahman model, both the GH and GH + INS treatments dramatically increased circulating concentrations of IGF-I in mature cattle, suggesting that this line of Brahman cattle is capable of responding to bioactive GH.     

Production of insulin-like growth factor-I by granulosa cells but not thecal cells is hormonally responsive in cattle

To determine whether the hormonal regulation of IGF-I production differs between granulosa and thecal cells in cattle, granulosa and thecal cells from bovine follicles were collected, cultured for 2 d in medium containing 10% fetal calf serum, washed, and then treated for an additional 24 h in serum-free medium with various hormones. In Exp. 1, granulosa cells were treated with 0 or 100 ng/mL of insulin and(or) 50 ng/mL of follicle-stimulating hormone (FSH), insulin plus 10 ng/mL of epidermal growth factor, or insulin plus 10 ng/mL of basic fibroblast growth factor. In Exp. 2, thecal cells were treated as described in Exp. 1 except that 100 ng/mL of luteinizing hormone (LH) was used instead of 50 ng/mL of FSH. In Exp. 3, granulosa and thecal cells were treated with 0 or 30 ng/mL of cortisol with or without 100 ng/mL of insulin, 300 pg/mL of glucagon, or glucagon plus insulin. In Exp. 4, granulosa and thecal cells were treated with 0 or 300 ng/mL of estradiol with or without 100 ng/mL of insulin and(or) 100 ng/mL of LH. At the end of treatment, medium was collected, concentrated with Centricon-3 concentrators, and assayed for IGF-I by radioimmunoassay. Cell numbers were determined by Coulter counting at the end of culture. Thecal cells produced low amounts of IGFI (0.48 +/- 0.04, 0.63 +/- 0.03, and 0.82 +/- 0.03 ng per 100,000 cells per 24 h in Exp. 2, 3, and 4, respectively), and this production was not influenced (P > 0.05) by the various treatments. In contrast, IGF-I production by granulosa cells (2.0 to 6.2 ng per 100,000 cells per 24 h) was influenced by treatment in Exp. 1, 3, and 4 and was greater than IGF-I production by thecal cells (Exp. 2, 3, and 4). Alone, insulin, FSH, LH, and cortisol (but not estradiol) each decreased (P < 0.05) granulosa-cell IGF-I production by 20 to 57%; combined treatments of insulin plus FSH or insulin plus cortisol decreased IGF-I production to levels seen with insulin alone. Glucagon had no effect (P > 0.10) on IGF-I production in the absence or presence of insulin. In the presence of insulin, epidermal growth factor, basic fibroblast growth factor, and estradiol decreased (P < 0.05) IGF-I production below that observed for insulin alone. These results indicate that, during follicular development in cattle, changes in intrafollicular levels of IGF-I may be due to hormonally-induced changes in granulosa-cell, but not thecal-cell, IGF-I production.     

Effects of growth factors on insulin-like growth factor binding protein (IGFBP) secretion by primary porcine satellite cell cultures.

Insulin-like growth factor-binding proteins (IGFBP) regulate the biological functions of insulin-like growth factors (IGF) and may affect cell growth through IGF-independent actions. Growth factors and hormones have been shown to alter IGFBP production by target cells suggesting that the effects of these factors may be partially mediated by the local production of IGFBP. Growth factors, including IGF-I, transforming growth factor-beta1 (TGF-beta1), and basic fibroblast growth factor (bFGF) have potent effects on satellite cell proliferation and differentiation, and some of these factors have been shown to alter IGFBP production in various cell types. Consequently, some of their actions on muscle satellite cells may be mediated by the local production of IGFBP. In this study, we measured the effects of IGF-I, bFGF, and TGF-beta1 on IGFBP production by primary porcine satellite cell (PSC) cultures after first determining physiologically active concentrations of these growth factors to use according to [3H]thymidine incorporation dose responses. There is little information on the effects of these growth factors on IGFBP production in primary porcine myogenic cells due to the confounding affects of contaminating nonmuscle fibroblasts. Comparative studies show that primary porcine satellite cells produce IGFBP-3 and -5 whereas porcine muscle-derived nonfusing cells (FIB) produce IGFBP-2 and -4 but not IGFBP-3 or -5. Because of this, our investigations have focused on growth factor-induced production of IGFBP-3 and -5 in primary porcine satellite cells cultures. Both IGF-I and bFGF exhibited dose-dependent increases in [3H]thymidine incorporation with increasing concentration from 1 to 50 ng/mL (P < 0.05), whereas TGF-beta1 caused a dose-dependent decrease from 0.01 to 0.5 ng/mL (P < 0.05). When 20 ng/ mL of IGF-I was added to the media, IGFBP-3 was increased approximately 65% (P < 0.05) and IGFBP-5 was increased approximately twofold (P < 0.05). The addition of 0.5 ng/mL TGF-beta1 caused more than a two-fold increase in IGFBP-3 (P < 0.05) and approximately an 80% increase in IGFBP-5 (P < 0.05), whereas 50 ng/ mL of bFGF caused approximately 40% (P < 0.05) and 70% (P < 0.05) increases in IGFBP-3 and -5, respectively. Neither IGFBP-3 nor -5 was detectable in the conditioned media from fibroblasts whether or not IGF-I, TGF- beta1 or bFGF were present. These data suggest that the effects of IGF-I, TGF- beta1 and bFGF on porcine satellite cells may in part be through the autocrine/ paracrine production of IGFBP-3 and -5 by porcine satellite cells.     

Interactions of amino acids and hormones regulate the balance between growth and milk protein synthesis in lactating rats fed diets differing in protein content

Insulin-like growth factor-I (IGF-I), growth hormone (GH), and prolactin (PRL) play important roles in milk protein synthesis, and their plasma concentrations were reported to be affected by dietary protein intake. To investigate the relationship between circulating amino acid (AA) and concentrations of these hormones, 18 Wistar rats aged 14 wk were assigned to a low (LP; 9% protein), standard (SP; 21% protein), or high-protein (HP; 35% protein) diet from parturition through day 15 of lactation. Plasma, liver, pituitary gland, skeletal muscle, and mammary gland samples were collected at the end of treatment. Circulating and hepatic IGF-I concentrations increased linearly with elevated dietary protein concentrations (P < 0.0001). Rats receiving the HP diet had higher circulating GH (P < 0.01) and pituitary PRL concentrations (P < 0.0001) but lower pituitary GH concentration (P < 0.0001) relative to those in rats receiving the LP and SP diets. Pearson correlation test performed on composed data across treatments showed that several circulating AAs were correlated with circulating and tissue concentrations of IGF-I, GH, and PRL. Multiple linear regression analyses identified Leu, Gln, Ala, Gly, and Arg as the main AAs associated with hormone responses (R² = 0.37 ~ 0.80; P < 0.05). Rats fed the LP and HP diets had greater Igf1 and Ghr gene expression in skeletal muscle than those fed the SP diets (P < 0.01). However, LP treatment decreased Prlr mRNA abundance in mammary glands as compared with the SP and HP treatments (P < 0.05). The HP diets increased AA transporter expression (P < 0.01) but decreased mammalian target of rapamycin (P < 0.05) and 70 kDa ribosomal protein S6 kinase 1 (P < 0.01) phosphorylation in mammary glands as compared with the LP and SP diets. The results of the present study suggested that several circulating AAs mediated the effects of dietary protein supply on concentrations of IGF-I, GH, and PRL, which in turn altered the metabolism status in peripheral tissues including the lactating mammary glands.     

Effects of growth hormone and feeding level on endocrine measurements,hormone receptors,muscle growth and performance of prepubertal heifers

Prepubertal Friesian heifer calves (n = 24, initial BW = 195 +/- 5 kg) were assigned to a 2 x 2 factorial block design and used to evaluate the effects of daily GH treatment (0 or 15 mg/d) at either a low or a high feeding level in a 5-wk treatment period on endocrine measurements, hormone receptors, muscle growth, and overall performance. In the pretreatment period, a low feeding level was employed for all calves. During the treatment period, animals at the low feeding level had free access to a roughage-based mixture, whereas animals at the high feeding level had free access to a concentrate mixture and were offered 2 kg/d of the roughage-based mixture. Blood samples were collected weekly starting 3 wk before treatment. Longissimus (LM) and supraspinatus (SS) muscles were obtained at slaughter. Metabolizable energy intake was 81% higher, digestible CP intake was 140% higher, and ADG was 115% higher (all P < 0.001) at the high vs. low feeding level. Feed (DMI, ME, and protein) intake was not affected by GH treatment, but ADG was 18% higher (P < 0.13) in GH-treated than in control heifers at both feeding levels. Although of different magnitudes, the muscle anabolic effects of GH treatment and high vs. low feeding level were additive, and both treatments increased carcass weights (P < 0.02 and P < 0.001, respectively), LM (P < 0.05 and P < 0.001), and SS (P < 0.06 and P < 0.003). The anabolic effect of GH treatment was similar in both muscles, whereas the effect of feeding level was most pronounced in LM. Overall, GH treatment increased plasma GH, IGF-I (both P < 0.001), and IGFBP-3 (P < 0.02); however, GH treatment increased total IGF-I, free IGF-I, and IGFBP-3, and decreased IGFBP-2 mainly at the high feeding level (GH x feeding level interaction; P < 0.02, 0.01, 0.03, and 0.10, respectively). The high feeding level increased insulin, free and total IGF-I, and IGFBP-3 (all P < 0.001), but decreased GH and IGFBP-2 (both P < 0.001). High feeding increased type-1 IGF receptor density (P < 0.02), mainly in LM, in accordance with the largest anabolic response in this muscle, whereas GH treatment had no effect on type-1 IGF receptors. The results suggest that in skeletal muscle, the anabolic effects of exogenous GH are related to endocrine changes in the GH-IGF axis, whereas the effects of feeding level also seem to rely on IGF receptor density in the muscles.     

The influence of level of feeding on growth and serum insulin-like growth factor I and insulin-like growth factor-binding proteins in growing beef cattle supplemented with somatotropin

The objective of this study was to determine the effects of level of feeding on growth, feed efficiency (gain:feed; G:F), body composition (BC), and serum concentrations of somatotropin (ST), IGF-I, and IGF-binding proteins (BP) in growing beef cattle supplemented with bovine (b) ST. In each of two consecutive years, 40 growing beef cattle were blocked by weight (average BW: yr 1 = 316 kg, yr 2 = 305 kg) and used in a 2 x 2 factorial arrangement with main effects of bST (0 or 33 microg x kg BW(-1) x d(-1)) and level of feed intake (ad libitum [AL] or 0.75 AL). Relative to uninjected cattle, treatment with bST increased ADG 9.6% (1.14 vs 1.25 kg/d; P < 0.05) and increased G:F 8.1% (12.3 vs 13.3 gain [g]:feed [kg]; P < 0.05), whereas ADG in AL animals was 39% greater than that in 0.75 AL animals (1.39 vs 1.00 kg/d; P < 0.05). There was a tendency (P = 0.10) for a bST x level of feeding interaction, such that the increase in ADG with bST was greater in AL cattle than in 0.75 AL cattle (10.6 vs 7.8%; P = 0.10). Serum concentrations of ST were greater in 0.75 AL cattle than in AL cattle (13.0 vs 8.6 ng/mL; P < 0.05) and in bST-treated cattle than in uninjected cattle (16.3 vs 5.2 ng/mL; P < 0.05). Due to a bST x level of feeding interaction (P < 0.01), the magnitude of the increase in serum ST to exogenous bST was greater (P < 0.01) in 0.75 AL cattle than in AL cattle. Relative to uninjected cattle, treatment with bST increased (P < 0.05) serum concentrations of IGF-I and IGFBP-3 and reduced (P < 0.05) concentrations of IGFBP-2. Similarly, AL cattle had greater (P < 0.05) serum concentrations of IGF-I and IGFBP-3 and reduced (P < 0.05) IGFBP-2 compared with 0.75 AL cattle. In summary, treatment with bST increased growth rate and G:F and stimulated serum IGF-I and IGFBP-3 while reducing IGFBP-2. Feeding at 0.75 ad libitum intake reduced the magnitude of response for each of these variables. Thus, limit-feeding may reduce the effect of exogenous bST on growth rate by blunting bST-induced increases in IGF-I and IGFBP-3 and bST-induced decreases in IGFBP-2.     

Growth hormone response to growth hormone-releasing hormone in beef cows divergently selected for milk production

In dairy cattle, increased circulating growth hormone has been associated with selection for greater milk yield. This study tested the hypothesis that beef cows divergently selected for milk production would have differing GH responses to a challenge dose of GHRH. Growth hormone response to a challenge of GHRH was measured in 36 Angus-sired cows ranging from 6 to 10 yr of age. The cows were classified as high milking (n = 16) or low milking (n = 20), on the basis of their sires' milk EPD. Mean milk EPD (in kilograms) were 16.6 and -14.4 for high and low milking cows, respectively. Milk production was estimated by the weigh-suckle-weigh procedure. Blood samples were taken immediately before and 10 min after a clearance dose of 4.5 microg of GHRH/100 kg BW (injected i.v.) and, 3 h later, immediately before and 10 min after a challenge dose of either 1.5 or 4.5 microg of GHRH/100 kg BW. Each animal received both challenge doses, and the doses were randomly assigned across 2 d of blood collection. Serum concentrations of GH and IGF-I were measured by RIA. Serum IGF-I was measured in the baseline blood sample on d 1 of blood collection. A positive relationship (r = 0.35; P = 0.03) was observed between the cows' rankings for each dose of GHRH; that is, high responders to the low dose were high responders to the high dose. Growth hormone response to the 4.5 microg/100 kg BW challenge dose of GHRH was positively related to sire milk EPD (R2 = 0.09; P = 0.03). Response of GH to the 1.5 microg GHRH/100 kg BW challenge dose also tended to be related (P = 0.08) to sire milk EPD of high milking cows. In addition, IGF-I concentrations of high milking cows were inversely related (R2 = 0.24; P = 0.04) to sire milk EPD. Growth hormone response to GHRH challenge may have potential as an additional tool in the evaluation of milk production in beef cattle.     

Epitope-tagged insulin-like growth factor-I expression in muscle

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.     

Effects of fish oil supplementation on the performance and the immunological, adrenal, and somatotropic responses of weaned pigs after an Escherichia coli lipopolysaccharide challenge

Seventy-two crossbred pigs (7.58 +/- 0.30 kg BW) weaned at 28 +/- 3 d of age were used to investigate the effects of fish oil supplementation on pig performance and on immunological, adrenal, and somatotropic responses following an Escherichia coli lipopolysaccharide (LPS) challenge in a 2 x 2 factorial design. The main factors consisted of diet (7% corn oil [CO] or 7% fish oil [FO]) and immunological challenge (LPS or saline). On d 14 and 21, pigs were injected intraperitoneally with either 200 microg/kg BW of LPS or an equivalent amount of sterile saline. Blood samples were collected 3 h after injection for analysis of interleukin-1beta (IL-1beta), prostaglandin E2 (PGE2), cortisol, growth hormone (GH), and insulin-like growth factor (IGF)-I. On d 2 after LPS challenge, peripheral blood lymphocyte proliferation (PBLP) was determined. Lipopolysaccharide challenge decreased ADG (487 vs. 586 g; P < 0.05) and ADFI (as-fed, 776 vs. 920 g; P < 0.05) from d 14 to 21 and ADG (587 vs. 652 g; P < 0.10) from d 21 to 28. Fish oil improved ADG (554 vs. 520 g; P < 0.10) and ADFI (891 vs. 805 g; P < 0.10) from d 14 to 21. On d 14, LPS challenge x diet interactions were observed for IL-1beta (P < 0.10), PGE2 (P < 0.001), and cortisol (P < 0.05) such that these measurements responded to the LPS challenge to a lesser extent (IL-1beta: 93 vs. 114 pg/mL, P < 0.05; PGE2: 536 vs. 1,285 pg/mL, P < 0.001; cortisol: 143 vs. 206 ng/mL, P < 0.05) in pigs receiving the FO diet than in pigs fed the CO diet. In contrast, among LPS-treated pigs, pigs fed the FO diet had higher IGF-I (155 vs. 101 ng/mL; P < 0.10) than those fed the CO diet. On d 21 among LPS-treated pigs, pigs fed FO had lower IL-1beta (70 vs. 84 pg/mL; P < 0.10) and cortisol (153 vs. 205 ng/mL; P < 0.05) than those fed CO. Pigs fed FO had lower PGE2 (331 vs. 444 pg/mL; P < 0.05) and higher IGF-I (202 vs. 171 ng/mL; P < 0.10) compared with those fed CO. Lipopolysaccharide challenge decreased GH (0.27 vs. 0.33 ng/mL; P < 0.05) on d 14, whereas it had no effect on GH on d 21. During both LPS challenge periods, the challenge increased PBLP when these cells were incubated with 8 (1.46 vs. 1.32; P < 0.10) or 16 microg/mL (1.46 vs. 1.30; P < 0.05) of concanavalin A. Fish oil had no effect on PBLP. These results suggest that FO alters the release of proinflammatory cytokines, which might lead to improved pig performance during an immunological challenge.     

IGF-I- and EGF-dependent DNA synthesis of porcine myoblasts is influenced by the dietary isoflavones genistein and daidzein

Soy-derived isoflavones have been reported to be specific inhibitors of protein tyrosine kinases like the type 1 insulin-like growth factor receptor (IGF-1R) and the epidermal growth factor receptor (EGFR). This study was conducted to investigate, whether IGF-I and EGF stimulate porcine myoblast growth and whether the responses are influenced by isoflavones. Satellite cell-born myoblasts derived from the semimembranosus muscle of newborn piglets were treated for 26h with IGF-I or EGF alone and in combination with genistein or daidzein. The DNA amount was measured and DNA synthesis was recorded as 6 h-[(3)H]thymidine incorporation during exponential growth in serum-free basal medium. IGF-I and EGF synergistically stimulated DNA synthesis of porcine myoblast with EGF causing a greater response. Genistein (100mumol/l) effectively reduced the growth factor-mediated DNA synthesis, which was associated with an inhibition of growth factor receptor protein expression. In response to daidzein no reduction in growth factor-mediated DNA synthesis was found. Daidzein (1; 10mumol/l) combined with IGF-I caused even a slight increase in DNA amount compared with the untreated control. The expression of the IGF-1R precursor protein was reduced with 10 and 100mumol/l daidzein, whereas the EGFR expression remained unchanged with daidzein. The results suggest that dietary isoflavones may interact with growth factor-induced stimulation of pig skeletal muscle growth.     

Leucine stimulates mammalian target of rapamycin signaling in C2C12 myoblasts in part through inhibition of adenosine monophosphate-activated protein kinase

Mammalian target of rapamycin (mTOR) signaling is one of the main signaling pathways controlling protein synthesis. Leucine treatment upregulates mTOR signaling, which enhances protein synthesis; however, the mechanisms are not well understood. Herein, treatment of C2C12 myoblast cells with leucine enhanced the phosphorylation of mTOR and ribosomal protein S6 kinase. Leucine treatment also decreased the adenosine monophosphate/ATP ratio in myoblasts by 36.4 +/- 9.1% (P < 0.05) and reduced the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) alpha subunit at Thr172 (28.6 +/- 4.9% reduction, P < 0.05) and inhibited AMPK activity (43.6 +/- 3.5% reduction, P < 0.05). In addition, leucine increased the phosphorylation of mTOR at Ser2448 by 63.5 +/- 10.0% (P < 0.05) and protein synthesis by 30.6 +/- 6.1% (P < 0.05). Applying 5-aminoimidazole-4-carbox-amide 1-beta-d-ribonucleoside, an activator of AMPK, abolished the stimulation of mTOR signaling by leucine, showing that AMPK negatively controls mTOR signaling. To further show the role of AMPK in mTOR signaling, myoblasts expressing a dominant negative AMPKalpha subunit were employed. Negative myoblasts had very low AMPK activity. The activation of mTOR induced by leucine in these cells was abated, showing that AMPK contributed to mTOR activation. In conclusion, leucine stimulates mTOR signaling in part through AMPK inhibition. This study implicates AMPK as an important target for nutritional management to enhance mTOR signaling and protein synthesis in muscle cells, thereby increasing muscle growth.     

Relationships of metabolic hormones and serum glucose to growth and reproductive development in performance-tested Angus, Brangus, and Brahman bulls

Understanding mechanisms that regulate growth and reproduction are important for improving selection strategies in cattle. In this study, Angus, Brangus, and Brahman bulls (n = 7 per breed) of similar age were selected from a group of 65 weanlings. Bulls were evaluated after weaning (i.e., approximately 6 mo of age) for 112 d for serum concentrations of metabolic hormones and glucose, growth, and reproductive traits. Performance data and blood sera were collected on d 0, 28, 56, 84, and 112. Sera were also collected in periods from d 50 to 59 (56D) and 103 to 112 (112D). Angus bulls were heavier (P < 0.05) throughout the study than Brahman bulls and were heavier than Brangus bulls on d 56, 84, and 112. Initial and final BW for Angus, Brangus, and Brahman bulls were 292.7, 260.6, and 230.4 and 468.3, 435.6, and 350.7 +/- 12 kg, respectively. Conversely, Brahman bulls had greater hip height (P < 0.05) than Brangus, and Brangus were taller (P < 0.05) than Angus. Angus bulls had the greatest (P < 0.05) scrotal circumference (SC) and Brahman bulls the least. Mean SC across days was 31.5, 29.7, and 25.0 +/- 0.6 cm for the three respective breeds. Serum testosterone was greater (P < 0.01) in Angus and Brangus bulls (10.0 and 8.9 +/- 1.4 ng/mL) than in Brahman bulls (4.0 +/- 1.4 ng/mL) throughout the study. After d 112, 100, 86, and 57% of the Angus, Brangus, and Brahman bulls passed a breeding soundness exam (P = 0.51). Serum concentrations of IGF-I and leptin were greater (P < or = 0.06) in Angus bulls on d 56, 84, and 112 than in Brangus and Brahman bulls. Serum concentrations of GH (P < 0.08) and glucose (P < 0.03) were greater in Brangus bulls than in Angus or Brahman bulls throughout the study. Prediction analyses suggested that serum concentrations of leptin could be used to predict (P < or = 0.08) BW and SC (R2 > 0.82) in the 56D and 112D periods among these breeds. Leptin was also useful in predicting (P < or = 0.09) serum concentrations of GH and testosterone in the 112D period (R2 > 0.32). Residual correlation analyses with the effect of breed removed suggested that leptin was correlated (r > or => 0.53, P < 0.05) with both SC and serum testosterone. Angus and Brahman cattle differ in phenotype, level of adiposity, and rate of sexual development. Data herein suggest that these characteristics could be due to varying mechanisms by which metabolic hormones such as leptin, GH, and(or) IGF-I are regulated.     

Luteinizing hormone,growth hormone,insulin-like growth factor-i,insulin and metabolites before puberty in heifers fed to gain at two rates

Fall born Angus x Hereford heifers were allotted to treatments at 9 mo of age to achieve the following growth rates: 1) fed to gain 1.36 kg/d (n = 10; HGAIN); and 2) fed to gain 0.23 kg/d for 16 wk, then fed to gain 1.36 kg/d (n = 9; LHGAIN). Growth hormone (GH), insulin-like growth factor-1 (IGF-I), insulin, glucose, nonesterified fatty acids (NEFA), and progesterone were quantified in twice weekly blood samples until onset of puberty. Body weight, hip height, and pelvic area were recorded every 28 d. Frequent blood samples (n = 8 heifers/treatment) were collected every 14 d, commencing on day 29 of treatment until onset of puberty to evaluate secretion of luteinizing hormone (LH) and GH. The HGAIN heifers were younger (369 d; P < 0.001), were shorter at the hip (115 cm; P < 0.05) and had smaller pelvic area (140 cm²; P < 0.10), but body weight (321 kg) did not differ at puberty compared with LHGAIN heifers (460 d; 119 cm; 155 cm²; 347 kg, respectively). The HGAIN heifers had greater (P < 0.05) concentrations of LH, IGF-I, and insulin in serum and glucose in plasma during the first 84 d of treatment than LHGAIN heifers, whereas LHGAIN heifers had greater (P < 0.05) concentrations of GH in serum and NEFA in plasma than HGAIN heifers. On Day 68 of treatment, HGAIN heifers had less mean GH (P < 0.01) and greater (P < 0.05) LH pulse frequency than LHGAIN heifers, whereas LH pulse amplitude and mean LH did not differ (P > 0.10) between treatments. Treatment did not influence secretion of LH and GH at 1 and 3 wk before puberty. Mean GH concentrations in serum and GH pulse amplitude in all heifers were greater (P < 0.05) 2 to 9 d (12.9 and 40.7 ng/ml, respectively) than 16 to 23 d (10.4 and 20.0 ng/ml, respectively) before puberty. Nutrient restriction decreased LH pulse frequency and delayed puberty in beef heifers. Furthermore, dramatic changes in mean concentration and amplitude of GH pulses just before puberty in beef heifers may have a role in pubertal development.     

New insights into the mechanism and actions of growth hormone (GH) in poultry.

Despite well documented anabolic effects of GH in mammals, a clear demonstration of such responses in domestic poultry is lacking. Recently, comprehensive dose-response studies of GH have been conducted in broilers during late post-hatch development (8 to 9 weeks of age). GH reduced feed intake (FI) and body weight gain in a dose-dependent manner, whereas birds pair-fed to the level of voluntary FI of GH-infused birds did not differ from controls. The reduction in voluntary FI may involve centrally mediated mechanisms, as hypothalamic neuropeptide Y protein and mRNA were reduced with GH, coincident with the maximal depression in FI. Growth of breast muscle was also reduced in a dose-dependent manner. Circulating IGF-I was not enhanced by GH, despite evidence that early events in the GH signaling pathway were intact. A GH dose-dependent increase in circulating 3,3',5-triiodothyronine(T3) paralleled decreases in hepatic 5D-III monodeiodinase activity, whereas 5'D-I activity was not altered. This confirms that a marked hyperthyroid response to GH occurs in late posthatch chickens, resulting from a decrease in the degradative pathway of T3 metabolism. This secondary hyperthyroidism would account for the decreased skeletal muscle mass (52) and lack of enhanced IGF-I (53) in GH-treated birds. Based upon these studies, it is now evident that GH does in fact have significant effects in poultry, but metabolic responses may confound the anabolic potential of the hormone.