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.     

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.     

Insulin and insulin-like growth factor-I stimulate DNA synthesis in bovine mammary tissue in vitro

Effects of insulin and insulin-like growth factor I (IGF-I) on [3H]thymidine incorporation, in vitro, by mammary tissue slices obtained from prepartum and lactating cows were investigated. Both insulin and IGF-I induced up to a 10-fold increase in [3H]thymidine incorporation in the mammary slices cultured in serum-free media. The effect of insulin-stimulated [3H]thymidine incorporation occurred at a threshold of greater than 1.75 pmol/ml and appeared to reach maximum at greater than 8.8 nmol/ml. The response to IGF-I occurred at greater than 6.5 pmol/ml and reached the equivalent of maximal insulin-stimulated incorporation at 39 pmol/ml. No synergistic or additive effects were observed between these two factors. The in vitro response took 3 to 4 d to reach maximum and was inhibited by cytarabine. Mammary tissue obtained from lactating cows incorporated more [3H]thymidine per microgram DNA in response to insulin (175 pmol/ml) than mammary tissue from pregnant cows. Culture of mammary tissue slices with growth hormone, cortisol, prolactin, or triiodothyronine showed no stimulation of [3H]thymidine incorporation over control. Autoradiography of the cultured lactating tissue showed incorporation of [3H]thymidine by 51, 24 and 29% of the ductal epithelial, secretory alveolar epithelial and myoepithelial cells, respectively. All alveolar epithelial cells that incorporated [3H]thymidine contained secretory products. Among nonsecretory cells, 25 and 28% of the fibroblasts and white blood cells, respectively, were labeled. Insulin-like growth factor I, but not bovine somatotropin, stimulated [3H]thymidine uptake into DNA in lactating bovine mammary tissue. Thus, our data support the concept that bovine somatotropin acts through IGF-I to increase DNA synthesis in mammary cells.     

The effects of bovine recombinant growth hormone administration on insulin-like growth factor-I and the haemopoietic system in thoroughbred geldings

The effect of intramuscularly administered recombinant bovine growth hormone (rbGH) on insulin-like growth factor-I (IGF-I) and white and red blood cell indices was studied in Thoroughbred geldings. An insulin-like growth factor binding protein (IGFBP)-blocked radioimmunoassay was modified and validated for the measurement of IGF-I in equine blood plasma. Baseline values of IGF-I and blood indices were determined over a 48 h period and then a single dose of 5 microg/kg, 10 microg/kg or 50 microg/kg of rbGH was administered. Insulin-like growth factor-I levels increased in a dose-dependent manner, with the highest values between 12 h and 24 h. The highest dose (50 microg/kg) yielded the greatest IGF-I response with a 90.2+/-10.8% increase at 24 h. White blood cell count increased following the three doses of rbGH with the highest white blood cell count at 12 h after the 50 microg/kg dose. Haemoglobin was significantly increased at 24 h (P< 0.05), when values following doses of 10 microg/kg and 50 microg/kg were significantly greater than after the vehicle or the dose of 5 microg/kg. Red blood cell count was not affected by any of the rbGH doses. These results indicated that rbGH is biologically active in the horse and that rbGH at a dose rate of 10 microg/kg or more could be used therapeutically.     

Growth hormone and lactogenic hormones can reduce the leptin mRNA expression in bovine mammary epithelial cells

Leptin mRNA is expressed in not only adipocytes but also mammary epithelial cells and leptin protein is present in milk. Although milk leptin is thought to influence metabolism or the immune system in neonates, there is little information about the regulation of leptin expression in mammary epithelial cells. We examined the effect of growth hormone (GH) and/or lactogenic hormone complex (DIP; dexamethasone, insulin and prolactin) on leptin mRNA expression in mammary epithelial cells. We used a bovine mammary epithelial cell (BMEC) clonal line, which was established from a 26-day pregnant Holstein heifer. We confirmed that the mRNA was expressed in BMECs and the expression was significantly reduced by GH and/or DIP, when the cells were cultured on both plastic plates and cell culture inserts at days 2 and 7 after stimulation with lactogenic hormones. GH and/or DIP significantly increased level of alpha-casein mRNA in BMECs after 7 days on the cell culture inserts, but no mRNA expression was detected at day 2. GH and DIP significantly stimulated the secretion of alpha-casein from BMEC on cell culture inserts at 3.5 and 7 days. However, neither alpha-casein mRNA expression nor secretion was observed in the BMECs cultured on plastic dishes, even in the presence of GH or/and DIP. These results indicate that GH and DIP can directly reduce leptin mRNA expression in both undifferentiated and functionally differentiated bovine mammary epithelial cell.     

Feeding Aspergillus awamori reduces skeletal muscle protein breakdown and stimulates growth in broilers

This study was conducted to show that dietary supplementation of a fungus, Aspergillus awamori called Koji in Japan, reduces skeletal muscle protein breakdown and stimulates growth in broiler chickens. A total of 30 chicks at 15 days of age was divided into control and two treatment groups (10 birds per treatment). Control group was fed basal diet and treatment groups were fed the basal diets supplemented with A. awamori at levels of 0.05% and 0.2%. The birds were raised for 12 days from 15 to 27 days of age and then the effect on growth, organ weights and plasma 3‐methylhistidine concentration and digestibilities of protein and energy was evaluated. The messenger RNAs (mRNAs) of atrogin‐1, ubiquitin, proteasome, m‐calpain, µ‐calpain, β‐actin, myosin and pax‐7 in the breast muscle were also measured. Body weight gain and breast muscle weight were increased, although feed intake was decreased by the fungus and thus feed efficiency was increased. Protein and energy digestibilities were increased. Furthermore, plasma 3‐methylhistidine concentration was decreased by the fungus. The mRNAs of atrogin‐1, ubiquitin, proteasome, m‐calpain and µ‐calpain were all decreased. The mRNA of β‐actin but not myosin and pax‐7 was slightly increased by the fungus. In conclusion, feeding A. awamori improves growth performance because skeletal muscle proteolytic activity is reduced and digestibilities of energy and protein are increased.     

Plasma concentrations of growth hormone and insulin-like growth factor-I in prepuberal quarter horses and ponies

The hypotheses were tested that among types of horses with phenotypically different mature sizes, a difference in pattern of secretion of 1) GH and 2) insulin-like growth factor-I (IGF-I) would exist prepuberally. To test these hypotheses, plasma was collected each 20 min for 8 hr from three types of horses [Quarter Horses (n=5), ponies (n=4), and Quarter Horse-pony F₁ crosses (n=5)] at 2, 4, and 10 months of age. Plasma concentrations of GH and IGF-I were determined by RIA and the patterns of secretion were quantified. Type of horse had no effect on tonic patterns of secretion of GH (P=0.92) or IGF-I (P=0.39), so the hypotheses were rejected and the data were pooled across types within age. Mean plasma concentrations of GH did not differ (P=0.74) with respect to age of horse. In contrast, number of pulses of GH per 8 hour (2 months = 2.3±0.4; 4 months = 2.2±0.5; 10 months = 2.8±0.9) and the interval between pulses (2 months = 87.1±23.1; 4 months = 121.7±25; 10 months = 111.5±15 min) changed quadratically (P=0.03 and P=0.02). Plasma concentrations of IGF-I decreased quadratically (P=0.01) from 2 months through 10 months of age. These data provide evidence to suggest that tonic secretion of GH and IGF-I may differ among prepuberal Quarter Horses and ponies with respect to age of horse but not type of horse.     

Relative roles of polysomes and cytoplasmic enzymes in regulating bovine skeletal muscle protein synthesis

Activities of cytoplasmic enzyme or polysomal fractions, both prepared from longissimus muscle of bovine animals differing markedly in muscle growth were measured in cell-free, protein synthesis assays. Two groups of animals were used: 1) eight animals comprising two "double-muscled" animals fed a high-energy diet, two "double-muscled" animals fed a low-energy diet, two "normal" animals fed a high-energy diet and two "normal" animals fed a low-energy diet and 2) six animals differing in age and sex and including two bull calves, 87 and 145 kg; two 18- to 24-mo-old animals, 411 and 596 kg and two cows, 594 and 681 kg. Ability of cell-free, protein-synthesis systems prepared from muscle of different animals to incorporate amino acids into TCA-precipitable protein differed significantly and was in direct correlation with phenotypic muscle growth. Mixing experiments using cytoplasmic enzymes from one set of animals with polysomes from another set showed that source of cytoplasmic enzymes had a greater effect on rate of in vitro protein synthesis than source of polysomes. Cytoplasmic enzymes and polysomes from double-muscled animals and animals fed a high-energy diet supported more rapid in vitro protein synthesis than the same components from normal animals or animals fed low-energy diets. The "double-muscling" characteristic had a greater effect on rate of in vitro protein synthesis than did high or low-energy diets. Cytoplasmic enzymes and polysomes from calves supported a greater rate of in vitro protein synthesis than the same components from 18- to 24-mo-old animals or cows. Based on these limited results in one species and with two groups of animals in that species, cytoplasmic enzymes may have a greater role in limiting rate of muscle protein synthesis in the current population of domestic animals than polysomes. Composition of the crude cytoplasmic enzyme fraction used in this study is unknown, but it likely contains elongation and termination factors in addition to aminoacyl-tRNA synthases and regulatory RNA.     

胰岛素、胰高血糖素对体外单层培养的肝细胞胰岛素样生长因子-Ⅰ mRNA丰度的影响

以持家基因β肌动蛋白(β-actin)作为内参照,通过竞争PCR法检测不同浓度的胰岛素和胰高血糖素对犊牛肝细胞中胰岛素样生长因子-Ⅰ mRNA丰度的影响.结果表明,胰岛素和胰高血糖素都能直接调控胰岛素样生长因子-Ⅰ mRNA的表达,胰岛素促进胰岛素样生长因子-Ⅰ mRNA的表达,而胰高血糖素抑制胰岛素样生长因子-ⅠmRNA的表达,存在量变关系.     

The effect of glycerol on mRNA expression of growth hormone,insulin-like growth factor,and mitochondrial breast muscle genes of Japanese quail

1. A study was carried out to evaluate the expression of growth hormone (GH), insulin-like growth factor I (IGF-I), mitochondrial adenine nucleotide translocase (ANT), cytochrome oxidase III (COX III) and avian uncoupling protein (avUCP) genes in 14-d-old Japanese quail that were fed different levels of glycerol (0%, 4%, and 12% dietary glycerol) which replaced maize as an energy source.

2. Total RNA was extracted from the breast muscle, and cDNA was amplified using real-time PCR with primers specific to the examined genes.

3. Quail fed the diet with 12% glycerol supplementation presented higher growth hormone (GH) mRNA expression than did those fed 0% glycerol. Supplementation with 12% glycerol negatively influenced IGF-I mRNA expression and reduced ANT mRNA expression in comparison with the treatment with no glycerol. COX III mRNA expression in the pectoralis superficialis muscle was decreased by 26% in quail fed 12% glycerol compared with those fed 0 and 4% glycerol.

4. There was no difference in UCP mRNA expression between quail fed 0 and 4% glycerol; however, UCP expression was reduced (73%) in birds fed the 12% glycerol diet compared with the level in those fed the 4% glycerol diet.

5. The inclusion of 4% glycerol in the diet produced results similar to those of the diet with no glycerol. Based on quail performance and the expression of the GH, IGF-I, ANT, COX III and UCP genes, 4% glycerol can be used in quail feeding without any harmful effects.     

The role of endocrine insulin-like growth factor-I (IGF-I) in female bovine reproduction

Insulin-like growth factor-I (IGF-I) plays a pivotal role in cattle fertility, acting as a monitoring signal that allows reproductive events to occur when nutritional conditions for successful reproduction are reached. However, endocrine IGF-I is not a predictor of reproductive events, but rather an indirect estimator of the suitability of the animal to achieve the reproductive event in question. Although measuring circulating IGF-I concentrations might not have any clinical application in the cattle industry, endocrine IGF-I screening will continue to be important for the study of interactions between nutrition and reproduction. In addition, endocrine IGF-I screening could be used as an ancillary test for the selection of cattle for high reproductive potential, especially in herds of high genetic merit for milk production, in which a decline in fertility has been identified.     

Temporal pattern of insulin-like growth factor-I response to exogenous bovine somatotropin in lactating cows

The effect of exogenous bovine somatotropin (bST) treatment on the temporal pattern of insulin-like growth factor-I (IGF-I) in serum of four multiparous Holstein cows was examined. Cows (190 +/- 24 days postpartum) were treated with daily subcutaneous injections of recombinant bST (40 mg) or excipient for 12-day periods in a crossover experimental design. During excipient treatment, concentrations of IGF-I in serum were relatively constant throughout the day and averaged 70 ng/ml. Following the first bST injection, serum IGF-I began increasing after a lag of 5 to 7 hr and progressively increased over the first 2 days of treatment. Serum IGF-I levels were approximately 2-fold greater than control values at the end of day 1 of bST treatment, with a 3-fold elevation observed at the end of day 2. Concentrations of IGF-I in serum plateaued by day 3 of bST treatment. Serum concentrations of IGF-I did not follow the oscillating pattern of bST in serum resulting from daily bST injections. Milk yield (3.5% fat-corrected) plateaued after 6 days of bST treatment and was increased 61% (+15.3 kg). Both IGF-I and milk yield remained essentially constant across days for the remainder of treatment. Following cessation of treatment, serum IGF-I and milk yield gradually declined, returning to control values after approximately 4 days. The temporal pattern of circulating concentrations of IGF-I is consistent with a role for IGF-I in mediating a portion of the effects of exogenous bST in lactating cows.     

Injection of porcine growth hormone releasing hormone gene plasmid in skeletal muscle increases piglets' growth and whole body protein turnover

The aim of the current study was to investigate the effects of a porcine growth hormone releasing hormone (pGHRH) gene plasmid injection in piglets on growth performance and whole body protein turnover. Sixty male Canadian Landrace × Chinese Taihu piglets were assigned to an intramuscular injection of 0 (control), 0.25, 0.5, 1 and 2 mg. All pigs were fed with the same diet (crude protein: 239.8 g/kg, digestible energy: 14.28 MJ/kg) at ad libitum intake. Protein turnover was determined on the 22nd day with a three-pool model by using a single-dosage, end-product analysis method with ¹⁵ N-glycine as a tracer. Injection of the pGHRH gene plasmid increased the piglets' growth rate, altered feed intake and decreased feed conversion ratio. It increased plasma growth hormone releasing hormone (GHRH), growth hormone (GH), insulin-like growth factor-I (IGF-I) and somatostatin but reduced serum urea and triglyceride. It reduced the urinary nitrogen excretion and led to higher nitrogen retention as well as the efficiencies of nitrogen retention and digestible N utilization. It increased the rates of protein synthesis, protein breakdown and net protein gain. Excretion of endogenous urinary nitrogen was reduced and nitrogen reutilization rate was improved. Conclusions: Injection of the pGHRH gene plasmid in skeletal muscle stimulated GHRH, GH and IGF-I excretion in piglets. Protein deposition was increased by an increase in protein synthesis and a smaller increase in protein breakdown, which was accompanied by reducing amino acid oxidation and increasing nitrogen reutilization.     

Effects of flax supplementation and a combined trenbolone acetate and estradiol implant on circulating insulin-like growth factor-I and muscle insulin-like growth factor-I messenger RNA levels in beef cattle

We evaluated effects of a 5% (dry matter basis) ground flaxseed supplement (flax) and a trenbolone acetate and estradiol-17beta implant, Revalor-S, on circulating IGF-I and muscle IGF-I messenger RNA (mRNA). Sixteen crossbred yearling steers (initial BW = 397 kg) were assigned randomly to one of four treatments: 1) flax/implant; 2) nonflax/implant; 3) flax/nonimplant; and 4) nonflax/nonimplant. Serum was harvested from blood collected on d 0 (before implant or flax addition), 14, and 28, and used in subsequent analyses of circulating IGF-I. Biopsy samples (0.5 g) were obtained from the longissimus muscle on d 0, 14, and 28. Total RNA was isolated from the muscle samples, and real-time quantitative-PCR was used to assess relative differences in IGF-I mRNA. Flax supplementation had no effect (P > 0.10) on circulating IGF-I concentrations. Following implantation, sera from implanted steers had 52 and 84% greater (P < 0.05) IGF-I concentrations than sera from nonimplanted steers on d 14 and 28, respectively. On d 28, local muscle IGF-I mRNA levels increased 2.4-fold (P < 0.01) in biopsy samples obtained from implanted compared with nonimplanted steers. Muscle biopsy samples from nonflax cattle had 4.4-fold higher (P < 0.01) levels of IGF-I mRNA than those from flax cattle on d 28. To determine whether a component of flax, alpha-linolenic acid (alphaLA), was directly responsible for IGF-I mRNA down-regulation, we incubated primary cultures of bovine satellite cells, from implanted and nonimplanted steers, in two concentrations of alphaLA (10 nM and 1 microM). An implant x dose interaction (P < 0.05) was observed for IGF-I mRNA concentrations in bovine satellite cells cultured for 72 h with alphaLA. Satellite cells from nonimplanted steers had similar (P > 0.10) IGF-I mRNA concentration regardless of the level of alphaLA exposure; however, satellite cells from implanted steers exposed to 10 nM and 1 microM alphaLA had 2.5- and 2.0-fold greater IGF-I mRNA levels, respectively, than cells from implanted steers that were not exposed to alphaLA (P < 0.05). Administration of a Revalor-S implant increased circulating IGF-I and local muscle IGF-I mRNA concentrations in finishing cattle. However, muscle IGF-I mRNA levels were decreased by flax supplementation. Muscle cell culture experiments suggested that alphaLA was not responsible for the IGF-I mRNA down-regulation.     

Influence of gonadotropins on insulin- and insulin-like growth factor-I (IGF-I)-induced steroid production by bovine granulosa cells

To determine the effect of gonadotropins on insulin- and insulin-like growth factor (IGF-I)-induced bovine granulosa cell functions, granulosa cells from bovine ovarian follicles were cultured for 2 days in the presence of 10% fetal calf serum (FCS), and then cultured for an additional 2 days in serum-free medium with added hormones. In the presence of 0 or 1 ng/mL of insulin or IGF-I, FSH had little or no effect (P>0.05) on estradiol production by granulosa cells from both small (1–5 mm) and large (≥8 mm) follicles. However, in the presence of ≥3 ng/mL of insulin, FSH increased (P<0.05) estradiol production by granulosa cells from small and large follicles such that the estimated dose (ED₅₀) of insulin necessary to stimulate 50% of the maximum estradiol production was decreased by 2- to 3-fold from 22 to 28 ng/mL in the absence of FSH to 7–14 ng/mL in the presence of FSH. Similarly, in the presence of ≥3 ng/mL of IGF-I, FSH increased (P<0.05) estradiol production by granulosa cells from small and large follicles such that the ED₅₀ of IGF-I for estradiol production was decreased by 4- to 5-fold from 25 to 36 ng/mL in the absence of FSH to 5–6 ng/mL in the presence of FSH. In the presence of FSH, the maximal effect of insulin on estradiol production was much greater than that of IGF-I (137- versus 12-fold increase) and were not additive; when combined, 100 ng/mL of IGF-I completely blocked the stimulatory effect of 100 ng/mL of insulin. In the absence of FSH, the maximal effect of insulin and IGF-I on estradiol production was similar. Concomitant treatment with 30 ng/mL of LH reduced (P<0.05) insulin-stimulated estradiol production by 52% on day 1 and 19% on day 2 of treatment. Insulin, IGF-I and FSH also increased (P<0.05) granulosa cell numbers and progesterone production but their maximal effects were less (i.e., <4-fold increase) than their effects on estradiol production. In conclusion, insulin and IGF-I synergize with FSH to directly regulate ovarian follicular function in cattle, particularly granulosa cell aromatase activity.