Muscle Insulin-like Growth factor-I Research Articles

Regulation of protein synthesis after acute resistance exercise in diabetic rats.

These studies determined whether insulin-like growth factor-I (IGF-I) involvement in exercise-stimulated anabolic processes becomes more evident during hypoinsulinemia. Male Sprague-Dawley rats (n = 6-12/group) were made diabetic (blood glucose congruent with 300 mg/dl) by partial pancreatectomy (PPX) or remained nondiabetic (glucose congruent with 144 mg/dl). Rats performed acute resistance exercise by repetitive standing on the hindlimbs with weighted backpacks (ex), or they remained sedentary (sed). Resistance exercise caused increases in rates of protein synthesis (nmol Phe incorporated. g muscle-1. h-1, measured for gastrocnemius muscle in vivo 16 h after exercise) for both nondiabetic [sed = 154 +/- 6 (SE) vs. ex = 189 +/- 7] and diabetic rats (PPXsed = 152 +/- 11 vs. PPXex = 202 +/- 14, P < 0.05). Arterial plasma insulin concentrations in diabetic rats, congruent with180 pM, were less than one-half those found in nondiabetic rats, congruent with444 pM, (P < 0.05). The activity of eukaryotic initiation factor 2B (eIF2B; pmol GDP exchanged/min) was higher (P < 0.05) in ex rats (sed = 0.028 +/- 0.006 vs. ex = 0.053 +/- 0.015; PPXsed = 0.033 +/- 0.013 vs. PPXex = 0.047 +/- 0.009) regardless of diabetic status. Plasma IGF-I concentrations were higher in ex compared with sed diabetic rats (P < 0.05). In contrast, plasma IGF-I was not different in nondiabetic ex or sed rats. Muscle IGF-I (ng/g wet wt) was similar in ex and sed nondiabetic rats, but in diabetic rats was 2- to 3-fold higher in ex (P < 0.05) than in sed rats. In conclusion, moderate hypoinsulinemia that is sufficient to alter glucose homeostasis does not inhibit an increase in rates of protein synthesis after acute moderate-intensity resistance exercise. This preserved response may be due to a compensatory increase in muscle IGF-I content and a maintained ability to activate eIF2B.

Maturation of the myogenic program is induced by postmitotic expression of insulin-like growth factor I.

The molecular mechanisms underlying myogenic induction by insulin-like growth factor I (IGF-I) are distinct from its proliferative effects on myoblasts. To determine the postmitotic role of IGF-I on muscle cell differentiation, we derived L6E9 muscle cell lines carrying a stably transfected rat IGF-I gene under the control of a myosin light chain (MLC) promoter-enhancer cassette. Expression of MLC-IGF-I exclusively in differentiated L6E9 myotubes, which express the embryonic form of myosin heavy chain (MyHC) and no endogenous IGF-I, resulted in pronounced myotube hypertrophy, accompanied by activation of the neonatal MyHC isoform. The hypertrophic myotubes dramatically increased expression of myogenin, muscle creatine kinase, beta-enolase, and IGF binding protein 5 and activated the myocyte enhancer factor 2C gene which is normally silent in this cell line. MLC-IGF-I induction in differentiated L6E9 cells also increased the expression of a transiently transfected LacZ reporter driven by the myogenin promoter, demonstrating activation of the differentiation program at the transcriptional level. Nuclear reorganization, accumulation of skeletal actin protein, and an increased expression of beta1D integrin were also observed. Inhibition of the phosphatidyl inositol (PI) 3-kinase intermediate in IGF-I-mediated signal transduction confirmed that the PI 3-kinase pathway is required only at early stages for IGF-I-mediated hypertrophy and neonatal MyHC induction in these cells. Expression of IGF-I in postmitotic muscle may therefore play an important role in the maturation of the myogenic program.

Growth Hormone Promotes Somatic and Skeletal Muscle Growth Recovery in Rats Following Chronic Protein-Energy Malnutrition

The efficacy of recombinant human growth hormone (GH) and/or a diet enriched in protein and energy to improve growth recovery following prolonged malnutrition was examined in male rats food-restricted from birth until 120 d of age. At d 121, restricted rats were randomly assigned to recovery groups receiving either a control or enriched diet with or without daily subcutaneous injections of GH. Rats were killed after 16 or 47 d of recovery. At d 16, GH treatment stimulated liver, heart, plantaris, soleus, carcass and body weight gain and inhibited fat gain when compared to recovery controls. Rats receiving GH also exhibited the highest serum insulin-like growth factor-I (IGF-I) concentrations and total muscle protein. At d 47, GH effects on body and muscle recovery were minimal, and differences among recovery groups in serum IGF-I concentration and total muscle protein were no longer present. Consumption of an enriched diet increased fat pad and liver mass, but did not promote muscle recovery. There were no differences among treatment groups in skeletal muscle IGF-I mRNA levels at d 16 or 47. In summary, GH had positive effects on somatic and skeletal muscle growth early in the recovery process, possibly via endocrine IGF-I-stimulated protein accretion. In contrast, the enriched diet promoted fat deposition with no impact on skeletal muscle growth recovery.

Insulin-like growth factor-I, but not growth hormone, is dependent on a high protein intake to increase nitrogen balance in the rat

The aim of the present study was to investigate the influence of dietary protein level on the protein anabolic effects of growth hormone (GH) and insulin-like growth factor-I (IGF-I). Female growing rats were fed on either a high- or a low-protein diet with crude protein contents of 222 and 83 g/kg respectively. The diets contained the same amount of metabolizable energy (15.1 MJ/kg) and were given during a 14 d period. During the same time, three groups of rats (n 8) on each diet received subcutaneous infusions of either saline, recombinant human GH (rhGH) or recombinant human IGF-I (rhIGF-I). rhGH and rhIGF-I were given in doses of 360 and 500 micrograms/d respectively. The low-protein diet alone reduced significantly (P < 0.05) IGF-I concentrations in serum and in tissue taken from the gastrocnemius muscle as well as IGF-I mRNA from the same muscle. The responses to rhGH and rhIGF-I in terms of muscle IGF-I and its mRNA were variable. However, when rhIGF-I was infused into rats on the high-protein diet, significantly elevated levels of IGF-I in muscle tissues could be observed. This was associated with a significantly (P < 0.05) increased N balance, whereas rhGH significantly (P < 0.05) enhanced the N balance in rats on the low-protein diet. Thus, it can be concluded that the level of dietary protein ingested regulates not only the effect of IGF-I on whole-body N economy but also the regulation of IGF-I gene expression in muscles. The exact mechanism by which GH exerts its protein anabolic effect, however, remains to be elucidated.

Islet amyloid polypeptide decreases the effects of insulin-like growth factor-I on glucose transport and glycogen synthesis in skeletal muscle

Previous studies have shown that islet amyloid polypeptide (IAPP) is co-secreted with insulin from the β-cell. IAPP reduces insulin-stimulated rates of glycogen synthesis in skeletal muscle but the mechanisms are unclear. Insulin-like growth factor I (IGF-I) is an important regulator of glucose metabolism in skeletal muscle and acts through its own receptor, which has many structural and functional similarities with the insulin receptor. Despite this, the effects of IGF-I on glucose utilization are not identical to those of insulin. The aim of the study was to determine the effects of IAPP on IGF-I-stimulated rates of glucose transport and metabolism (measured by 3- O-methyl[ 3H]glucose and [U- 14C]glucose, respectively) in rat soleus muscle, and compare them with those stimulated by insulin. IAPP (10 nM) decreased the sensitivity of 3- O-methylglucose transport, the flux of glucose to hexosemonophosphate and the sensitivity of glycogen synthesis to IGF-I. In contrast, IAPP had no effect on IGF-I-stimulated rates of lactate formation (i.e. glycolysis). IAPP decreased the sensitivity of 3- O-methylglucose transport and glycogen synthesis to insulin. It is concluded that IAPP blunts the stimulation of glucose uptake and deposition by IGF-I or insulin in skeletal muscle. These observations expand those made initially for IAPP and insulin and suggest that IAPP affects IGF-I- or insulin-stimulated glucose metabolism in muscle by a mechanism which is common for both hormones. These experiments may serve as a framework for future studies in order to clarify the mechanisms by which IAPP affects glucose metabolism in skeletal muscle.

Overexpression of IGF-I in skeletal muscle of transgenic mice does not prevent unloading-induced atrophy.

This study examined the association between local insulin-like growth factor I (IGF-I) overexpression and atrophy in skeletal muscle. We hypothesized that endogenous skeletal muscle IGF-I mRNA expression would decrease with hindlimb unloading (HU) in mice, and that transgenic mice overexpressing human IGF-I (hIGF-I) specifically in skeletal muscle would exhibit less atrophy after HU. Male transgenic mice and nontransgenic mice from the parent strain (FVB) were divided into four groups (n = 10/group): 1) transgenic, weight-bearing (IGF-I/WB); 2) transgenic, hindlimb unloaded (IGF-I/HU); 3) nontransgenic, weight-bearing (FVB/WB); and 4) nontransgenic, hindlimb unloaded (FVB/HU). HU groups were hindlimb unloaded for 14 days. Body mass was reduced (P < 0.05) after HU in both IGF-I (-9%) and FVB mice (-13%). Contrary to our hypothesis, we found that the relative abundance of mRNA for the endogenous rodent IGF-I (rIGF-I) was unaltered by HU in the gastrocnemius (GAST) muscle of wild-type FVB mice. High-level expression of hIGF-I peptide and mRNA was confirmed in the GAST and tibialis anterior (TA) muscles of the transgenic mice. Nevertheless, masses of the GAST and TA muscles were reduced (P < 0.05) in both FVB/HU and IGF-I/HU groups compared with FVB/WB and IGF-I/WB groups, respectively, and the percent atrophy in mass of these muscles did not differ between FVB and IGF-I mice. Therefore, skeletal muscle atrophy may not be associated with a reduction of endogenous rIGF-I mRNA level in 14-day HU mice. We conclude that high local expression of hIGF-I mRNA and peptide in skeletal muscle alone cannot attenuate unloading-induced atrophy of fast-twitch muscle in mice.

Muscle insulin-like growth factor-I (IGF-I) receptors in chickens with high or low body weight: effects of age and muscle fibre type

IGF-I receptors were characterized in leg and breast muscles from 1- and 7-week-old chickens selected for high (HG) or low (LG) growth rate. Following whole muscle solubilization, receptors were partially purified by wheat germ agglutinin (WGA) chromatography. IGF-I and insulin binding did not differ between genotypes, but significantly decreased with age, without any change in receptor affinity. In the older birds, insulin binding was lower in breast than in leg muscle, whereas IGF-I binding was similar. Using the artificial substrate poly(Glu-Tyr) 4:1, the IGF-I-stimulated tyrosine kinase activity of the receptors was measured and compared between genotypes (at 1 week), and in HG chickens between muscle types (1 and 7 weeks) and ages (in leg muscle). It was not modified by any of these factors. We conclude that: (1) IGF-I and insulin receptor number do not differ between genotypes, (2) the number of both receptors decreases with age, and (3) in 7-week-old birds, breast muscle could become less responsive to insulin than leg muscles, following a decrease in receptor number.

Cytokines block the effects of insulin-like growth factor-I (IGF-I) on glucose uptake and lactate production in skeletal muscle but do not influence IGF-I-induced changes in protein turnover.

There is evidence that proinflammatory cytokines are involved in the regulation of muscle protein breakdown in various catabolic conditions but the mechanisms are not fully understood. Previous studies suggest that cytokines reduce circulating and tissue levels of insulin-like growth factor-I (IGF-I) and may block the anabolic effects of the hormone in certain cell types and tissues. We tested the hypothesis that a mixture of tumor necrosis factor alpha, interleukin-1 alpha, and interferon-gamma block the anabolic effects of IGF-I in skeletal muscle. Muscles from burned or unburned rats were incubated in the absence or presence of 1 microgram/mL of IGF-I with or without the addition of the cytokines. As expected, IGF-I stimulated protein synthesis and inhibited protein breakdown in incubated muscles. The cytokines did not influence protein turnover rates in muscles incubated with or without IGF-I. In additional experiments, the effects of IGF-I on glucose uptake and lactate production were tested. IGF-I increased glucose uptake approximately 2.5-fold and stimulated lactate production approximately 5-fold. These effects of the hormone were significantly inhibited by the cytokine mixture. The results suggest that cytokines do not induce protein catabolism by directly inhibiting the anabolic effects of IGF-I in muscle tissue. The inhibitory effects of the cytokines on IGF-I-stimulated glucose transport and lactate production suggest that the lack of effect of cytokines on protein metabolism was not due to a metabolic unresponsiveness of the incubated muscles to the cytokines.

Developmental changes in embryonic and extra-embryonic insulin-like growth factor-I tissue concentrations in the turkey embryo

The ontogeny of insulin-like growth factor-I (IGF-I) embryonic and extra-embryonic tissue concentrations were determined in the developing turkey embryo. At 2-d intervals, starting on Day 6 of incubation, individual tissues (n = 8 for each stage of incubation) were removed, weighed, pulverized, and extracted in 1 M acetic acid for IGF-I determination. Amniotic and allantoic fluid were collected starting on Day 8, serum on Day 12, and analyzed for IGF-I levels. Serum IGF-I levels were the lowest on Days 12 and 28 of incubation (5.9 and 9.5 ng/mL), respectively, and the highest on Day 20 (16.2 ng/mL). Allantoic and amniotic fluid IGF-I concentrations were essentially unchanged during incubation. Extra-embryonic tissue IGF-I levels increased in both the yolk sac and chorioallantoic membranes as incubation advanced with concentrations being 8- to 10-fold greater in the chorioallantois. Embryo tissue IGF-I concentrations varied greatly with regard to tissue and stage of development. Brain IGF-I levels were the highest on Day 8 and lowest on Day 26 (423 vs 35 pg/mg protein, respectively). Tissue IGF-I pattern in the heart mirrored that of brain. Liver IGF-I was low (< 40 pg/mg protein) from Day 10 to 20 and undetectable on Days 22 to 28. Muscle IGF-I levels were similar in the final days of development to those observed in early incubation. Bone IGF-I levels were highest in mid-incubation and lowest on Day 26. Peptide levels in the gastrointestinal tract (GI) tract and gizzard were dissimilar in that IGF-I was elevated in the GI tract in early incubation and declined with advancing incubation, whereas gizzard IGF-I levels peaked in late incubation. It is apparent that tissue synthesis of IGF-I is differentially regulated within a given tissue and stage of incubation during embryogenesis in the turkey embryo.

OVER-EXPRESSION OF IGF-I IN SKELETAL MUSCLE DOES NOT PREVENT UNLOADING-INDUCED ATROPHY 385

This study examined the hypothesis that high level local expression of insulin-like growth factor I (IGF-I) in skeletal muscle would prevent or minimize unloading-induced atrophy. We employed transgenic mice in which the-424 promoter, first intron and 3' untranslated region of the avian skeletal alpha actin gene drives expression of human IGF-I. Expression of the transgene results in -47 fold increase in IGF-I concentration specific to skeletal muscle (J. Biol. Chem. 270: 12109-12116, 1995). Male transgenic mice and non-transgenic mice from the parent strain (FVB) were divided into four groups (n = 10/group): 1) Transgenic, weight-bearing (IGF-I/WB); 2) transgenic, hindlimb unloaded (IGF-I/HU); 3) non-transgenic, weight-bearing(FVB/WB); and 4) non-transgenic, hindlimb unloaded (FVB/HU). HU groups were tail suspended for 14 days. Body mass was reduced (P<0.05) following HU in both IGF-I (-9%) and FVB mice (-13%). Mass of the gastrocnemius (GAST) and tibialis anterior (TA) muscles were reduced (p<0.05) in both FVB/HU and IGF-I/HU groups compared to FVB/WB and IGF-I/WB, respectively. The percent atrophy of mass in the GAST and TA following HU did not differ between IGF-I and FVB mice. Elevated expression of IGF-I mRNA was confirmed in the GAST and TA of the transgenic mice. However, HU resulted in a marked reduction in transgene expression in these muscles (although still much greater than FVB mice). Further, transgene expression was found to be undetectable in the soleus of transgenic mice. Therefore, high level local expression of IGF-I in skeletal muscle does not prevent unloading-induced skeletal muscle atrophy. Also, the avian skeletal alpha actin regulatory sequences used to drive this transgene confer fiber-type specific expression of human IGF-I and are sensitive to loading of the muscles.

Acute exercise attenuates age-associated resistance to insulin-like growth factor I.

This study examined the influence of acute exercise on the ability of insulin-like growth factor I (IGF-I) to stimulate protein synthesis in an isolated in vitro soleus muscle preparation in female C57Bl/6 mice aged 5, 12, and 24 mo. Gastrocnemius muscles were analyzed for IGF-I receptor mRNA levels, IGF-I receptor number, and IGF-I receptor binding capacities and affinity constants. IGF-I (20 nM) was unable to stimulate rates of protein synthesis in the 12- and 24-mo-old control animals. These animals demonstrated declines in IGF-I receptor protein. After acute exercise, IGF-I receptor mRNA was elevated, IGF-I receptor protein levels were unaffected, and rates of protein synthesis were stimulated by IGF-I in the 12- and 24-mo-old animals. Acute exercise significantly increased IGF-I receptor binding capacity and affinity constants in the 24- but not the 12-mo-old animals. We conclude that acute exercise can restore the action of IGF-I in aged muscle, potentially by increasing the IGF-I receptor binding capacity and affinity or as a result of factors downstream of the IGF-I receptor.

Insulin and insulin-like growth factor-I (IGF-I) binding in fish red muscle: regulation by high insulin levels

Insulin and IGF-I binding to semi-purified red muscle receptors was characterized in brown trout, Salmo trutta and the common carp, Cyprinus carpio. The yield of glycoprotein obtained after semipurification of receptors with WGA-agarose affinity chromatography in μg g −1 initial tissue was 210.6±21 μg g −1 in trout and 108.5±2.5 μg g −1 in carp. IGF-I specific binding (4.72±0.64%/10 μg glycoprotein) was 4–5-times higher than insulin binding (1.04±0.12%/10 μg glycoprotein) in trout red muscle. This difference in binding was due to a higher number and a greater affinity of the IGF-I ( K d, 0.21±0.03 nM) compared with the insulin ( K d, 0.67±0.06 nM) receptors in this tissue. Carp red muscle IGF-I binding (9.14±0.55%/10 μg glycoprotein) surpassed insulin binding (2.59±0.094%/10 μg glycoprotein) mainly because of a greater affinity of the IGF-I ( K d, 0.092±0.027 nM) compared with the insulin ( K d, 0.1515±0.0285 nM) receptor. IGF-I and insulin binding in carp red muscle were higher than in trout, as a consequence of a higher affinity of carp red muscle receptors. Arginine injection provoked acute hyperinsulinemia in both trout (23.3±1.01 ng ml −1) and carp (24.3±1.34 ng ml −1). Specific binding of insulin and IGF-I to the red muscle decreased 4 h after injection. In trout, a decrease of insulin and IGF-I binding of 47.0% and 63.3%, respectively was observed compared with controls; in carp, these values were 44.0% and 45.0%. The number of insulin and IGF-I receptors decreased (42–55%) but affinities did not change suggesting that receptor down-regulation is a consequence of high insulin levels.

Phosphatidylinositol 3-kinase and p70 s6 kinase participate in the regulation of protein turnover in skeletal muscle by insulin and insulin-like growth factor I.

This study was designed to evaluate the role of phosphatidylinositol (PI3) kinase, p70 S6 kinase (p70S6K), and mitogen-activated protein (MAP) kinase in the regulation of muscle protein metabolism by insulin and insulin-like growth factor I (IGF-I). Wortmannin and LY294002 (inhibitors of P13 kinase) both abolished the stimulation of protein synthesis by insulin or IGF-I in epitrochlearis muscle incubated in vitro. LY294002 also totally reversed the antiproteolytic action of these hormones. Although p70S6K activation by insulin and IGF-I may be mediated by PI3 kinase in epitrochlearis muscle, the specific inhibition of this kinase by rapamycin caused only partial (25%) inhibition of the stimulation of protein synthesis by these two hormones. Rapamycin had no effect on proteolysis. Finally, insulin or IGF-I did not stimulate MAP kinase activity at any of the times tested (2-25 min), suggesting that this protein kinase was not directly involved in the regulation of muscle protein metabolism. These observations provide evidence that PI3 kinase and p70S6K, but not MAP kinase, play a role in the regulation of muscle protein turnover by insulin or IGF-I.

Phosphatidylinositol 3-kinase and p70 s6 kinase participate in the regulation of protein turnover in skeletal muscle by insulin and insulin-like growth factor I

This study was designed to evaluate the role of phosphatidylinositol (PI3) kinase, p70 S6 kinase (p70S6K), and mitogen-activated protein (MAP) kinase in the regulation of muscle protein metabolism by insulin and insulin-like growth factor I (IGF-I). Wortmannin and LY294002 (inhibitors of P13 kinase) both abolished the stimulation of protein synthesis by insulin or IGF-I in epitrochlearis muscle incubated in vitro. LY294002 also totally reversed the antiproteolytic action of these hormones. Although p70S6K activation by insulin and IGF-I may be mediated by PI3 kinase in epitrochlearis muscle, the specific inhibition of this kinase by rapamycin caused only partial (25%) inhibition of the stimulation of protein synthesis by these two hormones. Rapamycin had no effect on proteolysis. Finally, insulin or IGF-I did not stimulate MAP kinase activity at any of the times tested (2-25 min), suggesting that this protein kinase was not directly involved in the regulation of muscle protein metabolism. These observations provide evidence that PI3 kinase and p70S6K, but not MAP kinase, play a role in the regulation of muscle protein turnover by insulin or IGF-I.

Parallel insulin-like growth factor I and insulin resistance in muscles of rats fed a high fat diet.

The possibility of a similarity between insulin-like growth factor-I (IGF-I) and insulin resistance in epitrochlearis muscles of rats fed a high fat diet [20% (wt/wt) fat] vs. a low fat diet [5% (wt/wt) fat] was investigated. Half-maximally and maximally effective concentrations of IGF-I- and insulin-stimulated 3-O-methyl-glucose transport were 2.32 and 25 nM, respectively, for IGF-I, compared with 0.79 and 20 nM for insulin. The high fat diet reduced both responsiveness and sensitivity of IGF-I-stimulated 3-O-methyl-glucose transport in parallel with the impaired insulin-stimulated 3-O-methyl-glucose transport. IGF-I binding at 2.5 nM to incubated epitrochlearis muscle was also decreased by the high fat diet (P < 0.05). Thus, the high fat diet caused simultaneous IGF-I and insulin resistance in rat skeletal muscle. The IGF-I resistance induced by feeding the high fat diet seems at least partially due to a reduction in IGF-I binding.

Myogenic Vector Expression of Insulin-like Growth Factor I Stimulates Muscle Cell Differentiation and Myofiber Hypertrophy in Transgenic Mice

The avian skeletal alpha-actin gene was used as a template for construction of a myogenic expression vector that was utilized to direct expression of a human IGF-I cDNA in cultured muscle cells and in striated muscle of transgenic mice. The proximal promoter region, together with the first intron and 1.8 kilobases of 3'-noncoding flanking sequence of the avian skeletal alpha-actin gene directed high level expression of human insulin-like growth factor I (IGF-I) in stably transfected C2C12 myoblasts and transgenic mice. Expression of the actin/IGF-I hybrid gene in C2C12 muscle cells increased levels of myogenic basic helix-loop-helix factor and contractile protein mRNAs and enhanced myotube formation. Expression of the actin/IGF-I hybrid gene in mice elevated IGF-I concentrations in skeletal muscle 47-fold resulting in myofiber hypertrophy. IGF-I concentrations in serum and body weight were not increased by transgene expression, suggesting that the effects of transgene expression were localized. These results indicate that sustained overexpression of IGF-I in skeletal muscle elicits myofiber hypertrophy and provides the basis for manipulation of muscle physiology utilizing skeletal alpha-actin-based vectors.

Effect of training and growth hormone suppression on insulin-like growth factor I mRNA in young rats

The growth hormone (GH)-insulin-like growth factor I (IGF-I) axis plays a role in the adaptation to exercise training, but IGF-I gene expression in response to exercise training and GH suppression has not been studied. Twenty female rates underwent a 4-wk treadmill training program begun in the prepubertal period (day 14 of life). In 10 of the training rats, GH production was suppressed by anti-GH-releasing hormone antibodies (GH suppressed). IGF-I mRNA and protein levels were measured in liver and hindlimb skeletal muscle. GH suppression reduced IGF-I mRNA expression in the liver to a much greater extent than in the muscle. In the GH control rats, training induced significant increases in hepatic exon 1-derived IGF-I mRNA (mean increase 30%; P < 0.05) and muscle exon 2-derived mRNA (mean increase 35%; P < 0.05). In the GH-suppressed rats, only muscle exon 1-derived transcripts were significantly increased by training (55%; P < 0.05) and this was associated with a significant increase in muscle IGF-I protein levels (P < 0.05). We speculate that the anabolic response to training may involve both GH-dependent increases in IGF-I mRNA in the liver and GH-independent increases in the muscle.

Modulation of IGF mRNA abundance during stretch-induced skeletal muscle hypertrophy and regression.

Increased load on a muscle (synergistic overload or stretch) results in muscle hypertrophy. The expression of insulin-like growth factor I (IGF-I) mRNA in rat skeletal muscle is increased during synergistic overload-induced hypertrophy. Although it has also been established that fasting animals lose muscle protein, it has been shown that compensatory muscle hypertrophy occurs in adult fasting rats that are undergoing a net loss of body weight. The purpose of this investigation was to determine whether a relationship exists between IGF-I mRNA levels and muscle growth and regression. This was accomplished by examining whether IGF-I mRNA levels were altered during muscle hypertrophy after stretch and regression and the effect of fasting on IGF-I mRNA levels during stretch-induced hypertrophy. Patagialis (PAT) muscle weights increased 13 and 44% at 2 and 11 days of stretch, respectively. However, after removal of the stretch stimulus on day 11, PAT weights began to decrease, reaching control weights by 18 days. During the first time point (2 days), PAT muscle IGF-I mRNA remained constant. IGF-I mRNA abundance was threefold greater than contralateral control levels by 11 days of stretch. IGF-I mRNA levels decreased but remained significantly above control levels throughout the regression of hypertrophy (13, 18, and 25 days). Fasting did not alter PAT muscle response to stretch. After 11 days of stretch, PAT muscle weight increased 60% compared with contralateral control muscles and IGF-I mRNA levels increased three-fold. This study supports a role for IGF-I in muscle hypertrophy but not muscle atrophy.

An Enzyme Immunoreceptor Assay for the Quantitation of Insulin-like Growth Factor-I and Insulin Receptors in Bovine Muscle Tissue

To investigate the regulation of insulin-like growth factor-I (IGF-I) and insulin receptor in muscle, a sensitive enzyme immunoreceptor assay (EIRA), which allows for the determination of both affinity and capacity of the receptors, was developed. After solubilization with Triton X-100, receptors were immobilized in microtiter plates using receptor specific monoclonal antibodies that recognize the intracellular β-domain of the respective receptors (clone 17A3 and clone 29B4). The immobilized receptors were labeled with either biotinylated IGF-I or insulin. The bound hormones were detected with a streptavidin-horseradish peroxidase technique. The assay had a detection limit of 1 fmol receptor/well. The intraassay variation was 9% (n = 22) for the IGF-I receptor concentration and 12% (n = 22) for the insulin receptor. The interassay variation was 5% (n = 4) for the IGF-I receptor and 10% (n = 4) for the insulin receptor. The coefficient of variation of the dissociation constants (Kd) was 26% (n = 7) for the IGF-I receptor and 24% (n = 7) for the insulin receptor. The assay system was used to study the effect of growth hormone treatment upon IGF-I and insulin receptors in bovine skeletal muscle. Three groups of 12 heifers (13 months old) were treated with either 320 or 640 mg recombinant bovine somatotropin (slow release preparation) every fortnight for 3 months. When samples of m. splenius were assayed for IGF-I and insulin receptors, there was no difference between groups for receptor concentration or affinity. The amount of receptor (average of all groups) was 49 ± 11 fmol/mg protein (insulin) and 97 ± 18 fmol/mg protein (IGF-I). The average Kd obtained was 0.76 ± 0.22 nM (insulin) and 0.66 ± 0.18 nM (IGF-I).

Differential regulation of transcription initiation from insulin-like growth factor-I (IGF-I) leader exons and of tissue IGF-I expression in response to changed growth hormone and nutritional status in sheep.

In the mammalian insulin-like growth factor-I (IGF-I) gene, exons 1 and 2 are differentially spliced to exon 3 producing alternate class 1 and class 2 transcripts. The aim of this study was to investigate the tissue expression of these leader exons in lambs growing at different rates as a result of chronic manipulation of nutritional and GH status. Riboprobes were developed so that leader exon-specific and total IGF-I gene expression could be determined using a single RNase protection assay. Lambs were fed a diet containing high or low protein content either ad libitum or at a restricted intake; within these dietary groups they were treated with either saline or GH for 10 weeks. Total hepatic IGF-I messenger RNA (mRNA) transcripts were significantly increased by GH (P = 0.004), protein (P = 0.002), and energy (P < 0.001) status as were circulating IGF-I concentrations (GH, P < 0.001; protein, P = 0.026; energy, P < 0.001). Exons 1 and 2 were expressed in liver but to a variable extent. Increased dietary energy and protein induced increased expression from both class 1 and 2 transcripts, but the percent increases was at least 5-fold greater for class 2 than for class 1 mRNA. GH treatment only stimulated significant increases in expression from class 2 transcripts. In the low protein, energy-restricted, saline-treated lambs exon 1 transcripts accounted for approximately 70% of total class 1 and 2 transcripts, and this proportion declined significantly as class 1 and 2 transcripts, and this proportion declined significantly as GH and nutritional status increased to only 30% in the high protein, ad libitum-fed, GH-treated animals; class 2 transcripts therefore displayed the opposite pattern of expression. These data indicate that exon 2 may be far more sensitive than exon 1 in intact animals which have been stimulated within normal physiological limits. Muscle IGF-I gene expression was at least 20-fold less than that for the liver and consisted mainly of class 1 transcripts. Muscle total IGF-I mRNA was insensitive to changed nutritional status or to GH treatment, even though significant increases in muscle growth occurred in response to ad libitum intake and GH, indicating that hepatically derived endocrine IGF-I could have a role in the regulation of muscle growth.