Interaction of insulin-like growth factors (IGF) with isolated sheep hepatocytes. I. Binding and degradation of IGF-I

The binding and degradation of 125I-hIGF-I by isolated sheep hepatocytes have been examined. Hepatocytes were isolated by collagenase perfusion of 32-55 kg wether lambs and were incubated at 20 or 37 C at pH 7.4 in a 95% O2/5% CO2 atmosphere. Maximal binding was obtained at 60 min and declined slightly over the following 60-min period at both 20 and 37 C. Degradation of 125I-hIGF-I by the hepatocytes was minimal with 10-12% degradation over a 120-min period at 37 C. The lysosomal inhibitors chloroquine (0.2 mM), leupeptin and ammonium chloride had no significant effects on 125I-hIGF-I degradation or binding. At 20 C (60-min incubation), half maximal inhibition of 125I-hIGF-I binding was obtained with 8.4 +/- 1.1 nM hIGF-II, 16 +/- 2.4 nM hIGF-I, 36 +/- 6.2 nM oIGF-II, and 60 +/- 5.9 nM oIGF-I. Ovine insulin (0.01-10 uM) had no effect on 125I-hIGF-I binding. These observations suggest that IGF-I binds to the type II IGF receptor. The low molecular weight sheep serum IGF binding protein inhibited binding of 125I-hIGF-I to hepatocytes in a dose-dependent manner with half maximal inhibition occurring at 16.5 micrograms/ml, but did not affect IGF-I degradation. The current studies show that IGF-I interacts with ruminant hepatocytes via type II IGF receptors. The liver is not a major site of IGF-I degradation and the observed degradation is nonlysosomal and independent of receptor interaction.     

Insulin-like growth factor binding protein-3: its biological effect on bovine granulosa cells

This study was aimed at testing the hypothesis that insulin-like growth factor binding protein (IGFBP)-3 can modulate hormone-dependent differentiation of granulosa cells in vitro. Granulosa cells from small (1 to 5 mm) follicles were collected from cattle, cultured for 2 d in medium containing 10% fetal calf serum, washed, and then treated for an additional 2 d in serum-free medium with follicle-stimulating hormone (FSH) (50 ng/ml), recombinant human IGF-I (0, 1.3, 4.0, or 13.3 nM), or recombinant human IGFBP-3 (0 to 4.26 nM). In one series of experiments, IGFBP-3 (0.53 and 2.13 nM) inhibited (51% to 92% decreases; P < 0.05) progesterone and estradiol production induced by 1.3 nM of IGF-I, but did not influence (P > 0.10) granulosa cell numbers or steroidogenesis in the absence of IGF-I. Only 4.26 nM of IGFBP-3 inhibited (by 35%) the increase in granulosa cell numbers induced by 1.3 nM of IGF-I. In another series of experiments, 13.3 nM of IGF-I, but not 4.0 nM of IGF-I, was able to completely overcome the inhibitory effect of 4.26 nM of IGFBP-3 on estradiol production. The increase in cell numbers induced by 4.0 and 13.3 nM of IGF-I was attenuated (P < 0.001) by 4.26 nM of IGFBP-3. In a third series of experiments, IGFBP-3 inhibited 125I-IGF-I binding to granulosa cells. These results indicate that IGFBP-3 has a pronounced inhibitory effect on IGF-I action in cultured bovine granulosa cells, and that this inhibitory effect is likely attributable to IGFBP-3 binding/sequestering IGF-I. Thus, IGFBP-3 may play a significant role in regulating granulosa cell proliferation and steroidogenesis during follicular development in cattle.     

Interaction of insulin-like growth factor I with turkey satellite cells and satellite cell-derived myotubes

Satellite cells, isolated from the superficial pectoralis muscle of growing Nicholas tom turkeys, were cloned to obtain a pure population of myogenic cells. These cells proliferated rapidly and differentiated (fused) into myotubes typically containing 92-98% fused nuclei. Competitive binding assays were performed on near-confluent satellite cell or myotube cultures in 35 mm diameter wells by adding [125I]IGF-I along with increasing concentrations of unlabeled IGF-I, IGF-II, or insulin. Following incubation, the cultures were washed to remove the unbound hormones, solubilized with 0.5 N NaOH, and the radioactivity specifically bound was determined. Total and fused nuclei number as well as total protein were determined in parallel cultures. Our results indicate that turkey satellite cell and myotube cultures possess specific binding sites for IGF-I. Displacement of [125I]IGF-I was in the order of IGF-I greater than IGF-II greater than or equal to insulin. Although the [125I]IGF-I association constants were similar for turkey satellite cells and myotubes, a 2.8-fold decrease in the number of receptors per nuclei was observed as satellite cells differentiated into myotubes. The 50% inhibition constants for IGF-I, IGF-II, and insulin were 3.7 X 10(-9) M, 7.5 X 10(-8) M, and 8.7 X 10(-8) M for satellite cells and 3.1 X 10(-9) M, 7.5 X 10(-8) M, and 9.6 X 10(-8) M for myotubes, respectively. Receptor cross-linking analysis using disuccinimidyl suberate was performed on near-confluent satellite cell cultures incubated with [125I]IGF-I in the presence or absence of 1 X 10(-7) M IGF-I, IGF-II, or insulin. Receptor subunit species of Mr 130 kDa and 98 kDa were observed under reducing conditions (100 mM dithiothreitol) and at a Mr greater than 300 kDa (native receptor tetramer) under non-reduced conditions. Autoradiographic bands were displaced with IGF-I but not with equimolar levels of IGF-II or insulin. The results suggest that turkey satellite cells possess a type I IGF receptor.     

Porcine insulin-like growth factor (IGF)-binding protein-3 elicits bi-phasic effects on IGF-I stimulated DNA synthesis in neonatal porcine skin fibroblasts

The present study was undertaken to investigate the effects of porcine IGFBP-3 on IGF-I stimulated DNA synthesis in neonatal porcine skin fibroblasts. IGF-1 stimulated DNA synthesis in skin fibroblasts in a concentration dependent manner. DNA synthesis was maximally stimulated by 5 to 20 fold at 5 nM IGF-I; half-maximal stimulation was observed at approximately 1 nM IGF-I. Co-incubation of IGFBP-3 with a maximally effective dose of IGF-I (10 nM) did not inhibit the stimulatory effects of IGF-I on DNA synthesis. In contrast, when IGFBP-3 at concentrations of 0 to 20 nM was co-incubated with 1 nM IGF-I, a bi-phasic dose response was observed with IGFBP-3 being inhibitory only at a 10 to 20 fold molar excess to IGF-I. Based on the approximately equal molar ratio of IGFBP-3:IGF-I present in the circulation of control and pST-treated pigs our results suggest that IGFBP-3 does not inhibit the mitogenic effects of IGF-I. In summary, these results indicate that the combination of IGFBP-3 with IGF-I optimizes mitogenic signalling via the type I IGF receptor and suggest that IGFBP-3 does not inhibit the effects of ST that are mediated by IGF-I.     

Characterization of type II insulin-like growth factor (IGF) receptors in sheep liver plasma membranes

Interactions of insulin-like growth factors (IGFs) from recombinant human and natural ovine sources with sheep liver plasma membranes have been studied. Total specific binding of 125I-hIGF-II (40%) to liver plasma membranes greatly exceeded that of 125I-hIGF-I (1.5%) after incubation at 20 C for 90 min. Binding of 125I-hIGF-II to the plasma membranes was dependent upon time, temperature and membrane concentration of the incubation. Binding of 125I-hIGF-II was only partially reversed by addition of 100 nM IGF-II (18%) or by dilution with excess buffer (36%). Competitive inhibition studies of 125I-hIGF-II binding demonstrated that IGF-II from ovine or recombinant human sources was more effective at inhibiting binding than ovine or human IGF-I. Insulin did not affect binding of 125I-hIGF-II. Plasma membranes were affinity cross-linked to 125I-IGF-II followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence and absence of the reducing agent dithiothreitol. Following autoradiography, radioactive bands were localized at 274,000 Mr and 210,000-215,000 Mr in the presence and absence of reducing agent, respectively. This pattern was unaffected by 100 nM human or ovine IGF-I or 1,000 nM insulin, but coincubation with 100 nM human or ovine IGF-II eliminated the radioactive band. These data indicate that an IGF-II specific receptor is present in sheep liver plasma membranes which has characteristics similar to those of nonruminant Type II receptors.     

Comparison of insulin-like growth factor I serum binding proteins in sheep and cattle: species differences in size and endogenous binding capacities

Binding proteins (BP) for insulin-like growth factor I (IGF-I) were characterized in sheep and beef cattle serum for molecular weight (Mr) and binding characteristics. Serum was incubated with [125I] IGF-I at 37 degrees C before chromatography over a 1.6-cm X 94.0-cm column of Sephacryl S-300 (pH 7.4, 4 degrees C). Beef serum exhibited a 145 k Mr (mol. wt X 1,000) and a 35 to 39 k Mr BP. Sheep serum possessed a 170 to 190 k and a 35 to 38 k Mr protein. Binding of [125I] IGF-I was inhibited in the presence of excess unlabeled ovine somatomedin, demonstrating specific binding for each BP of both species. The high Mr component was pituitary-dependent in sheep, as evidenced by binding patterns from serum of hypophysectomized sheep. Direct binding studies of the Sephacryl-separated BP demonstrated that the native BP of high molecular weight of both species bound only minor amounts of [125I] IGF-I in a manner unrelated to BP concentration. The BP of low molecular weight of beef cows displayed a bell-shaped dose-response binding curve with maximum binding at 250 micrograms/ml BP, whereas binding to sheep BP of low molecular weight was independent of BP concentration. After chromatography on Sephadex G50 at pH 2.8, both BP from both species exhibited concentration-dependent binding that plateaued at 250 to 500 micrograms/ml of BP of low molecular weight but was curvilinear for the BP of high molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of somatotropin binding sites in pig skeletal muscle.

The present study was conducted to determine whether somatotropin (ST) binding sites are present in crude membrane preparations containing sarcolemma of pig skeletal muscle. Initial characterization experiments indicated that binding of bovine ST (bST) was time- and temperature-dependent and that binding was reversible. At 23 degrees C, binding was maximized between 36 and 48 h, whereas at 4 degrees C binding had not reached a maximum by 96 h. Somatotropin binding was stable between pH 5.5 and 8.5 and increased linearly between 100 and 600 micrograms of membrane protein. Addition of unlabeled bST decreased specific binding of [125I]bST in a dose-dependent manner (ED50: 1 to 1.6 ng/mL). The binding sites for bST were specific because porcine prolactin poorly inhibited bST binding. Scatchard analysis revealed the presence of a single class of binding sites (Ka: 9 to 15 x 10(9)M-1; Bmax: 5 to 6 fmol/mg of protein). In summary, the present report is the first to demonstrate that specific ST receptors are present in pig skeletal muscle. The role that ST plays in directly stimulating muscle growth and(or) muscle synthesis of insulin-like growth factor I (IGF-I) in pST-treated pigs as opposed to changes that occur as the result of an increase in plasma IGF-I concentration remains to be resolved.     

Interaction of insulin-like growth factors (IGF) with isolated sheep hepatocytes. II. Binding, internalization and degradation of IGF-II

This study examines the binding and degradation of IGF-II by the ovine liver. Binding and degradation of 125I-IGF-II to isolated hepatocytes was time, temperature and cell number dependent. Ovine and human IGF-II were 2-5 times more effective in inhibiting 125I-hIGF-II binding than were the IGF-I preparations. Insulin did not affect binding. Autoradiographs of 125I-hIGF-II affinity cross-linked to hepatocytes showed a major band of molecular weight 271,000 under reduced conditions. This band was eliminated by 100 nM hIGF-II or oIGF-II but not by excess hIGF-I, oIGF-I or insulin. The internalization of IGF-II was examined by treating the cells with trypsin or sodium acetate to remove surface-bound IGF-II. Both treatments showed that 20-25% of 125I-hIGF-II was internalized. Mannose-6-phosphate at 1, 2 and 4 mM enhanced the binding of 125I-hIGF-II to hepatocytes 3.5, 12.8 and 16.4%, respectively. The lysosomal inhibitors ammonium chloride, chloroquine and leupeptin had no effect on 125I-hIGF-II degradation or cell-associated radioactivity indicating a nonlysosomal pathway of degradation for 125I-hIGF-II in the ovine hepatocyte. The low molecular weight sheep serum IGF binding protein inhibited binding of 125I-hIGF-II in a dose-dependent manner but had no effect on degradation, which suggests that degradation of 125I-hIGF-II is independent of receptor interaction. These studies demonstrate that IGF-II binds to specific high affinity sites in sheep hepatocytes which display the characteristics of type II IGF receptors. A significant fraction of the receptor bound IGF-II is internalized but not degraded by these cells, which suggests that the biological actions of IGF-II may be exerted by an intracellular pathway in sheep hepatocytes.     

Characterization of leptin binding in bovine kidney membranes

The interactions of leptin with its receptor and other leptin binding sites is not well described or understood. We have used Scatchard analysis of saturation binding data to characterize the affinity of leptin for binding sites in bovine kidney membranes. 125I-Leptin was used in saturation studies, over a range of concentrations from 50 pM to 9 nM. 125I-Leptin differentiated a high affinity binding site from an abundant low affinity site. The high affinity/low density binding site (putative leptin receptor) had K(d)=0.098 nM and B(max)=46.2f mol/mg protein. An additional class of low affinity, highly abundant sites with an apparent K(d)=175 nM, and B(max)=574 fmol/mg protein was characterized. The association and dissociation kinetics for 125I-leptin binding were also studied. Dissociation of the leptin-receptor complex was very rapid, and this necessitated the use of a specially developed separation method for radioligand binding studies (precipitation with PEG and filtration). Competitive displacement of 125I-leptin by mouse and human leptin and polyclonal anti-bovine leptin antibodies was dose-dependent. Specificity of binding was shown as bound 125I-leptin was not displaced by insulin or control antibodies. These data indicate that leptin binds the bovine leptin receptor with high affinity and that a pool of leptin is bound to abundant cell membrane-associated proteins. These observations are consistent with the plasma concentration range for leptin and imply that free leptin concentration in the tissues may be partially buffered by cell-associated and bound forms in plasma. Thus, acute changes in leptin secretion may have little effect at the leptin receptor. The development of leptin agonists/antagonists should facilitate further characterization of leptin binding and clarify the role of abundant low affinity binding sites at the leptin axis.     

Potential role of G-protein-coupled receptor 30 (GPR30) in estradiol-17beta-stimulated IGF-I mRNA expression in bovine satellite cell cultures

Androgenic and estrogenic steroids enhance muscle growth in animals and humans. Estradiol-17beta (E2) and trenbolone acetate (TBA) (a synthetic testosterone analog) increased IGF-I mRNA expression in bovine muscle satellite cell (BSC) cultures. The goal of this study was to evaluate the mechanisms responsible for this increase by evaluating the effects of ICI 182 780 (an E2 receptor antagonist), flutamide (an androgen receptor inhibitor), G1 (a GPR30 agonist), and BSA-conjugated E2 on E2 and/or TBA-stimulated IGF-I mRNA expression in BSC cultures. Flutamide completely suppressed TBA-stimulated IGF-I mRNA expression in BSC cultures. ICI 182 780 did not suppress E2-stimulated IGF-I mRNA expression and 100nM ICI 182 780 enhanced (93%, p<0.05) IGF-I mRNA levels in BSC cultures. G1 (100nM) stimulated IGF-I mRNA expression (100%, p<0.05) but had no effect on proliferation in BSC cultures. E2-BSA, which cannot cross the cell membrane, stimulated IGF-I mRNA expression (approximately 100%, p<0.05) in BSC but even at extremely high concentrations had no effect on proliferation. In summary, our data indicate the E2-stimulation of proliferation and E2-stimulation of IGF-I mRNA expression in BSC cultures occur via different mechanisms. Our previous results showing that ICI 182 780 inhibited BSC proliferation and results of the current study showing lack of response to E2-BSA or G1 suggest that E2-stimulated proliferation in BSC cultures is mediated through classical estrogen receptors. Stimulation by ICI 182 780, G1 and E2-BSA suggests the E2-stimulated IGF-I mRNA expression in BSC cultures is mediated through the GPR30 receptor.     

IGF-I receptors in embryonic skeletal muscle of three strains of chickens selected for differences in growth capacity.

IGF-I receptors in embryonic skeletal muscle from three strains of chickens have been characterized. Specific receptors for IGF-I were present in breast and thigh skeletal muscle of 13 d chicken embryos. Dissociation rate constants (Kd) for breast muscle of a slow growing layer strain (Leghorn), an intermediate growth strain (Synthetic Whiterock), and a fast growing broiler strain (Cornish) were 3.24, 3.04, and 2.68 nM, respectively. Kd for the thigh muscle of the slow, intermediate and fast growing strains were 3.30, 2.90 and 3.29 nM, respectively. Kd values were not significantly different between strains or between tissues (p greater than .05). Receptor concentration (Bmax) in breast muscle of slow, intermediate and fast growing strains were 1.22, 1.08 and 0.92 pM/mg protein, respectively. Receptor concentration in thigh muscle of the slow, intermediate and fast growing strains were 0.95, 0.76, and 0.79 pM/mg protein, respectively. Bmax values were significantly different (p less than .05) between breast muscle (1.08 +/- .05 pM/mg protein) and thigh muscle (.84 +/- .05 pM/mg protein). IGF-I receptor number for breast muscle although not statistically significant (p greater than .05), indicated a trend towards differences between the slow growing Leghorn strain and the faster growing Cornish strain. The results suggest that strains of birds with different growth capacity may have different densities of IGF-I receptors in breast skeletal muscle plasma membranes.     

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.     

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.     

Evaluation of interference by insulin-like growth factor I (IGF-I) binding proteins in a radioimmunoassay for IGF-I in serum from dairy cows.

Insulin-like growth factor I (IGF-I) circulates in serum bound to a number of different binding proteins (BPs). With antibodies currently available, BPs must be dissociated and inactivated or removed from serum prior to measurement of IGF-I by radioimmunoassay (RIA). Serum samples which spanned a 13-fold range in IGF-I concentration were obtained from lactating dairy cows and used to develop conditions for assay of IGF-I with minimal interference from BPs. Removal of BPs from serum by acid-ethanol extraction resulted in interference in the RIA. Therefore, serum was incubated with 0.1 M glycyl-glycine HCl to inactivate BPs as suggested by Underwood et al. Time, temperature and pH were optimum when serum was incubated for 48 hr at 37 C, pH 3.7. Binding protein inactivation was evaluated by ability of glycyl-glycine incubated serum to reassociate with 125I-IGF-I. In addition, BPs isolated by gel filtration of glycyl-glycine incubated serum were tested for interference in the RIA. The concentration of IGF-I in serum where inactivated BPs were removed by acid gel filtration was compared to corresponding glycyl-glycine incubated serum. There was a 1:1 relationship which intersected at zero indicating that total IGF-I could be measured. Therefore, incubation of serum with glycyl-glycine is a reliable method for measuring total IGF-I in serum from dairy cows.     

Investigation of the insulin-like growth factor system in the avian epiphyseal growth plate

Components of the insulin-like growth factor (IGF) system were investigated in chondrocytes isolated from the avian growth plate. The genes for IGF-I, IGF-II, type 1 IGF receptor (IGF-R), IGF binding protein-2 (IGFBP-2), IGFBP-3, IGFBP-5 and IGFBP-7 were found to be expressed in both proliferative and hypertrophic chondrocytes. The expression of IGF-II in proliferative chondrocytes was extremely high relative to IGF-I. Although IGF-I expression was significantly increased in hypertrophic chondrocytes, the level was still low relative to IGF-II. In cell culture, IGF-I stimulated proteoglycan synthesis and increased the expression of Indian hedgehog (Ihh) and type X collagen, markers of chondrocyte differentiation. IGF-II was found to be equally efficacious in stimulating proteoglycan biosynthesis. These observations suggest that IGF-II may play a significant role in avian growth plate physiology, which is consistent with several reports on mammalian endochondral bone growth.     

Insulin binding to bovine mammary membranes: comparison of microsomes versus smooth membranes

The study was undertaken to determine if membrane preparations of bovine mammary tissue bound insulin. If binding occurred, it was also the intent to compare binding kinetics between microsomes and smooth membranes. Insulin binding to bovine mammary membranes attained equilibrium, was saturable and was specific for insulin. Additional studies showed binding to be pH sensitive and maximal at 10 mM calcium. Binding affinity of insulin to microsomes and smooth membranes was similar, with the exception that smooth membranes bound 1.8 times more insulin per unit of membrane protein than microsomes. Two different methods were used to generate data for kinetic analysis of the insulin-receptor interaction in microsomes. Competitive binding assays (.6 ng [125I]insulin plus 0 to 100 ng insulin) indicated the presence of two binding sites with dissociation constants (Kd) of .32 and 15.8 nM. Direct titration of microsomes with [125I]insulin (.02 to 10 ng/ml) revealed two binding sites with Kd of .017 and .31 nM. The affinity of the second binding site measured by the competitive binding assay method (Kd of 15.8 nM) is low and therefore may not be of physiological importance for insulin action. Insulin appears to bind to two high-affinity receptor sites in bovine mammary microsomes with Kds of .017 nM and .32 nM. These findings show that bovine mammary tissue contains receptors for insulin. In addition, isolation of smooth membranes from microsomes enriches the number of insulin receptors per unit of membrane protein without altering their binding characteristics.     

Expression of insulin-like growth factor binding protein (IGFBP)-3, and the effects of IGFBP-2 and -3 in the bovine corpus luteum.

The present study was conducted to gain insight into the insulin-like growth factor (IGF) system in the bovine corpus luteum (CL). Specific aims were to measure the levels of IGF binding protein-3 (IGFBP-3) and RNA encoding IGFBP-3 in the CL throughout diestrus, and to investigate the effects of IGFBP-2 and -3 on IGF-I-stimulated progesterone (P4) production and IGF-I-receptor binding. Bovine CL were collected from a local abattoir and classified according to stage of diestrus based on anatomical characteristics. Corpora lutea from early, mid and late diestrus were each analyzed for the presence of IGFBP-3 by ligand blot analysis, and for RNA encoding IGFBP-3 by Northern blot analysis. Dissociated cells from mid-cycle CL were treated with IGF-I, IGFBP-2 or -3, or a combination of IGF-I and IGFBP-2 or -3. The effect of IGFBP-2 and IGFBP-3 on [(125)I] IGF-I binding to its receptor on CL plasma membranes also was investigated. IGFBP-3 protein and RNA expression were higher in early CL, compared to mid or late CL (p < 0.05). IGF-I stimulated P4 production in a dose-dependant manner (p < 0.05). IGFBP-2 and -3 blocked the stimulatory effect of IGF-I on P4 production (p < 0.05). Both IGFBP-2 and -3 inhibited [(125)I]-IGF-I binding to its receptor in a dose-dependant manner. These results demonstrate that IGFBP-3 protein and RNA are expressed predominantly during early diestrus in the bovine CL. Moreover, both IGFBP-2 and -3 can modulate IGF-I actions in the CL by interfering with binding of IGF-I to its receptor.     

Biological activity of insulin-like growth factor-I purified from chicken serum

This study describes a rapid purification of insulin-like growth factor-I from chicken serum and the immunological, biological and receptor binding activity of the peptide. It was purified after initial extraction, by cation exchange chromatography, hydrophobic interaction chromatography and reverse phase chromatography up to 1.4 x 10(6)-fold with an overall yield ranging from 10-30%. The N-terminal amino acid sequence was the same as predicted from the nucleotide sequence of a chicken IGF-I cDNA and the partial sequence obtained from a previously reported purification. The material was both immunologically and biologically active. It had a 50% potency compared to human IGF-I in a radioimmunoassay using an antiserum raised against human IGF-I, stimulated the incorporation of [3H]-thymidine into DNA in cultured chick embryo myoblasts with a half-maximum effective dose of 5 ng/ml and displaced [125I]-labelled human IGF-I and IGF-II from binding sites in microsomal membranes prepared from both the chicken liver and the lactating rabbit mammary gland in a dose dependent manner.     

Evaluation of serum insulin-like growth factor binding proteins (IGFBP) in Angus cattle divergently selected for serum IGF-I concentration

Postweaning serum insulin-like growth factor-I (IGF-I) concentrations and serum IGF binding proteins (IGFBP) were investigated in 68 (1992 Fall-born) and 84 (1999 Fall-born) Angus cattle selected for either high or low serum IGF-I concentrations since 1989. Relative serum levels of IGFBP were determined by [¹²⁵I]IGF-I Western ligand blotting. IGFBP species of 38–42, 34, 30, and 24 kDa were identified. The 34 kDa species was identified as IGFBP-2 by immunoblot analysis. No significant line effects were observed for any of the IGFBP. In both 1992 and 1999, heifers had higher IGFBP-2 levels than bulls (P<0.0005). In 1992 calves, relative levels of the 38–42 and 24 kDa species were significantly correlated with serum IGF-I concentration. In 1999 calves, none of the IGFBP were correlated with serum IGF-I, although IGFBP-2 was negatively correlated with several measures of body weight. No significant line effects were observed for growth or serum IGF-I traits in 1992 calves. However, 1999 high line calves had higher serum IGF-I concentrations and body weights than low line calves (P<0.05). In both 1992 and 1999 calves, bulls had higher serum IGF-I concentrations and body weights than heifers (P<0.05). Thus, while selection for high versus low serum IGF-I concentrations has resulted in divergence between the selection lines and also in changes in body weights, it has not resulted in changes in serum IGFBP levels. Furthermore, circulating IGFBP-2 appears to be higher in heifers than in bulls, and also appears to be negatively correlated with body weights.     

Basic principles of muscle development and growth in meat-producing mammals as affected by the insulin-like growth factor (IGF) system

This presentation aims to describe how the basic events in prenatal muscle development and postnatal muscle growth are controlled by the insulin-like growth factor system (IGF). The prenatal events (myogenesis) cover the rate of proliferation, the rate and extent of fusion, and the differentiation of three myoblast populations, giving rise to primary fibers, secondary fibers, and a satellite cell population, respectively. The number of muscle fibers, a key determinant of the postnatal growth rate, is fixed late in gestation. The postnatal events contributing to myofiber hypertrophy comprise satellite cell proliferation and differentiation, and protein turnover. Muscle cell cultures produce IGFs and IGF binding proteins (IGFBPs) in various degrees depending on the origin (species, muscle type) and state of development of these cells, suggesting an autocrine/paracrine mode of action of IGF-related factors. In vivo studies and results based on cell lines or primary cell cultures show that IGF-I and IGF-II stimulate both proliferation and differentiation of myoblasts and satellite cells in a time and concentration-dependent way, via interaction with type I IGF receptors. However, IGF binding proteins (IGFBP) may either inhibit or potentiate the stimulating effects of IGFs on proliferation or differentiation. During postnatal growth in vivo or in fully differentiated muscle cells in culture, IGF-I stimulates the rate of protein synthesis and inhibits the rate of protein degradation, thereby enhancing myofiber hypertrophy. The possible roles and actions of the IGF system in regulating and determining muscle growth as affected by developmental stage and age, muscle type, feeding levels, treatment with growth hormone and selection for growth performance are discussed.