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 I receptor analysis of satellite cell-derived myotube membranes established from two lines of Targhee rams selected for growth rate

Ovine-derived fibroblasts were used to validate an insulin-like growth factor I (IGF-I) membrane-receptor binding assay system. Competitive binding using fibroblasts revealed that half-maximal inhibition of 125I-IGF-I binding by IGF-I was 2.3 nM. SDS-polyacrylamide gel electrophoresis analysis of specific protein-associated 125I-IGF-I was consistent with the migration of 125I-IGF-I-labeled Type I IGF receptor alpha-subunits at Mr 133,000 daltons. Further, the efficiency of two cell solubilization methods was examined and time-dependent binding equilibrium was determined for the membrane assay system. Satellite cell-derived myotubes were subsequently isolated from primary satellite cell cultures established from the semimembranosus muscles of high and low efficiency-of-gain (EOG) Targhee rams, and IGF-I receptor dynamics were measured. A membrane competitive binding study revealed that half-maximal inhibition of 125I-IGF-I binding was achieved by 1-ng IGF-I for low, and 10-ng IGF-I for high, EOG myotube membrane preparations. Kd values were similar between the high EOG (4.78 nM) and low EOG (2.95 nM) groups; however, receptor concentrations (Bmax) appeared to differ between groups. High EOG membrane receptor Bmax was 3.88 pmole/micrograms protein (19.87 pmole/micrograms DNA), whereas low EOG membrane receptor Bmax was 1.22 pmole/micrograms protein (9.28 pmole/micrograms DNA). These preliminary findings support the hypothesis that genetic selection for EOG results in altered satellite cell responsiveness to IGF-I.     

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.     

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.     

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.     

Plasma IGF-I binding proteins in sheep: effect of recombinant growth hormone treatment and nutritional status.

In humans the IGF binding proteins (BP) are closely related to metabolic status. In this paper we have examined the influence of controlled feed intake and GH treatment on IGF binding proteins in growing lambs. Analyses were performed on plasma samples from animals maintained on two levels of feed intake (1.75% body weight as lucerne pellets or 3% body weight which is approximately equivalent to an ad libitum intake) either with or without recombinant bovine growth hormone (BST; 0.25 mg/kg body weight/day) administration. Samples used for the analyses reported in this paper were collected at 9.00 hr following 41 d of treatment. Total plasma IGF-I was increased on the higher plane of nutrition (P less than .01) and by BST (P less than .001) but only on high feed intake. IGF is associated with BP of 150 kDa and 40-50 kDa in sheep plasma. 150 kDa bound IGF-I was increased on the higher plane of nutrition (P less than .05) and by BST treatment (P less than .001) but only on the higher feed intake. By contrast no change in 40-50 kDa bound IGF-I was observed with treatment. Unbound IGF-I was also found in sheep plasma (2-5% of total) but demonstrated only minor changes in relation to treatment. Saturation analysis gave estimates of total binding capacity and saturation of the IGF-BP. In ovine plasma the binding capacity of the 150 kDa species is in excess of bound IGF (P less than .001). Saturation did not change with treatment despite the observed differences in 150 kDa bound IGF-I. Thus BP(s) contained in the 150 kDa fraction were responsive to treatment. By contrast large differences in saturation of the 40-50 kDa species were observed (P less than .001) despite little treatment dependent change in bound IGF-I. IGF-BP(s) in the 40-50 kDa fraction were elevated in the low nutrition group and suppressed on the higher feed intake resulting in near saturation. These data strongly suggest that the IGF BP are modulated according to metabolic status in the sheep.     

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.     

Effect of active immunization against growth hormone releasing factor on concentrations of somatotropin and insulin-like growth factor I in lactating beef cows.

Two experiments were conducted to determine the effects of immunoneutralization of growth hormone-releasing factor [GRF(1-29)-NH2] on concentrations of somatotropin (ST) and insulin-like growth factor I (IGF-I) in lactating beef cows. In Experiment 1, multiparous Hereford cows were immunized against 2 mg GRF(1-29)-(Gly)4-Cys-NH2 conjugated to human serum albumin (GRFi, n = 3) or 2 mg human serum albumin (HSAi, n = 3) at 52 +/- 1 d prior to parturition. Boosters (1 mg) were administered on days 12, 40 and 114 postpartum (pp). Serum samples were collected at 15-min intervals for 5 hr on days 18, 46 and 120 pp, followed by administration (IV) of an opioid agonist (FK33-824; 10 micrograms/kg) and an antagonist (naloxone; .5 mg/kg) at hours 5 and 7, respectively. A GRF-analog ([desamino-Tyr1, D-Ala2, Ala15] GRF (1-29)-NH2; 3.5 micrograms/kg) and arginine (.5 g/kg) were administered at hour 10 on days 47 and 121, respectively. Percentage binding of [125I]GRF (1:100 dilution of serum) 28 d after primary immunization was greater in GRFi (14.3 +/- 4.9) than in HSAi (.7 +/- .3) cows. Binding increased to 29.3 +/- 6.5% after first booster in GRFi cows. Episodic release of ST was abolished by immunization against GRF; concentration and frequency of release of ST were lower (P less than .05) in GRFi than in HSAi cows on all days pp. Concentrations of IGF-I were lower in GRFi than in HSAi cows throughout lactation. Serum ST failed to increase following FK33-824 or arginine in GRFi; however, ST increased after both compounds in HSAi cows. Concentrations of ST following GRF-analog were greater (P less than .05) in HSAi than in GRFi cows. Experiment 2 was conducted to determine if a lower dose of antigen and a single booster would be sufficient to lower ST and IGF-I in lactating cows. Multiparous Hereford and Angus cows were assigned to GRFi (n = 6) or HSAi (n = 6). Primary (1.2 mg) and booster (.5 mg) immunizations were administered -14 and 8 d from calving, respectively. Cows were restricted to 60% of recommended intake of energy during lactation in order to elevate concentrations of ST. Serum samples were collected at 15-min intervals for 6 hr on days 26, 50, 73, 90 and 109 pp. Two of six GRFi cows had binding less than 10% (1:1,000 dilution of serum) and were omitted from further analyses.(ABSTRACT TRUNCATED AT 400 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.     

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.     

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.     

Identification and partial purification of serum growth hormone binding protein in domestic animal species.

The chemical nature and variations in serum concentrations of growth hormone binding protein (GHBP) from humans, rabbits, and rodents have been reported. To date little is known about the GHBP of domestic animals. Therefore, we initiated these studies to determine whether a serum GHBP was present in domestic animals and to purify the binding protein (BP) from serum of selected species. Using a dextran-coated charcoal separation assay, specific growth hormone (GH) binding was demonstrated in ovine, bovine, chicken, human, goose, porcine, and equine serum (listed in sequence from lowest to highest binding). Variation in BP activity was relatively high, both within and between species. Yearling ewes had higher serum GHBP than either prepubertal (4 mo) or older (5 yr) ewes. The GHBP was partially purified from chicken, ovine, and porcine serum using GH affinity chromatography. These BP had high affinity (Ka = 2 x 10(8) to 2 x 10(9) L/mol, depending on species) and low capacity (2 x 10(-10) to 5 x 10(-11) mol/unit of protein) for human GH but showed lower binding affinity for homologous GH (Ka = 2 x 10(7) L/mol). The porcine GHBP had the highest and ovine GHBP the lowest affinity for human GH. Other heterologous somatotropic hormones, ovine placental lactogen, and ovine GH displayed higher binding affinity to chicken and pig BP than the respective homologous hormones. Further chromatographic purification of the porcine GHBP resulted in an additional 1,000-fold purification. The estimated molecular weight of porcine GHBP is 50,000 to 60,000 Da. These results demonstrate that the serum from all domestic species tested contains a specific GH-binding moiety and that under the conditions described here human GH is a more efficient ligand than the homologous hormone.     

Ontogeny of hepatic bovine growth hormone receptors in cattle

A series of studies examined the binding characteristics and ontogeny of hepatic growth hormone binding sites in dairy bulls on d 2, 30, 180, and 365 of age. Binding of iodinated recombinant bovine growth hormone ([125I]rbGH) to liver membrane receptors was membrane protein-dependent. Receptors were considered growth hormone-specific, because physiological concentrations of bovine prolactin (bPRL) failed to displace [125I]rbGH from bovine hepatocyte membranes. Only 50% of [125I]rbGH was bound reversibly to hepatic microsomes. Addition of dithiothreitol (DTT) to the receptor-assay buffer increased the binding of [125I]rbGH to hepatic membranes in a time-dependent manner. Moderate concentrations of Ca++ and Mg++ in the receptor-assay buffer had no detectable effects on binding of [125I]rbGH to hepatic microsomes. In growing dairy bulls, specific binding of [125I]rbGH per milligram of membrane protein increased from 1.9 +/- 1.8% at d 2 to 14.1 +/- 1.8% at d 180 and then declined to 5.2 +/- 1.6% at d 365. Likewise, concentration of insulin-like growth factor (IGF)-I in serum was low during the 1st mo of age (d 2, 13.3 +/- 8.8 ng/ml; d 30, 9.7 +/- 8.8 ng/ml), but it became maximal at d 180 (151.0 +/- 8.8 ng/ml). Circulating concentrations of IGF-II increased linearly during the 1st yr of growth. Serum concentrations of GH, triiodothyronine, and thyroxine declined from 39.9 +/- 6.5, 2.7 +/- .2, and 75.4 +/- 4.6 ng/ml at d 2 to 16.5 +/- 6.5, 1.3 +/- .2, and 53.4 +/- 4.6 ng/ml at d 30, respectively, and remained low through 1 yr of age. Insulin concentration in serum did not change significantly with development. Results indicated that increasing concentrations of specific bGH receptors in the bovine liver may play a key role in regulating postnatal growth in cattle.     

Validation of a rapid solid-phase radioimmunoassay for canine, bovine, and equine insulin

A rapid and convenient commercial radioimmunoassay kit, developed for quantifying hormones in specimens from human beings, was validated for use in measuring insulin in serum of dogs, cattle, and horses. The procedure uses polypropylene assay tubes treated with rabbit anti-porcine insulin serum and porcine [125I]iodoinsulin. Specificity was proven by demonstrating that standard solutions of porcine insulin and serial dilutions of canine, bovine, and equine sera and pancreatic extracts inhibited binding of [125I]iodoinsulin to the antibody in a parallel manner. Gel-filtration chromatography of pancreatic extracts yielded a major peak of immunoreactive material that eluted identically with [125I]iodoinsulin. Immunoreactivity was not associated with fractions that contain larger and smaller molecular weight peptides (eg, proinsulin and C-peptide, respectively). Biological specificity of the assay was shown by demonstrating increased insulin in serum after injection of glucose into heifers and glucagon into dogs and horses. Purified insulin and insulin in pancreatic extracts could be quantitatively recovered from serum, thereby demonstrating accuracy of the assay. Interassay precision of 5 control specimens run in 20 consecutive assays ranged from 6.7% to 20.1% (coefficient of variation) and intra-assay precision of 6 control specimens each assayed 10 times ranged from 4.4% to 10.7% (coefficient of variation). Sensitivity of the assay was 3.2 microIU/ml. This radioimmunoassay for insulin is ideal for veterinary research and diagnosis, because a single set of reagents and procedures can be used for at least 3 species.     

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.     

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.     

Effect of feed intake on antimicrobially induced increases in porcine serum insulin-like growth factor I

This study was conducted to determine whether an antimicrobially induced (ASP-250) increase in serum IGF-I was the result of differences in feed intake. Serum IGF-I concentrations were measured in crossbred pigs that were fed a control diet or a diet supplemented with ASP-250 either for ad libitum consumption or limited to 85% of the control pigs' consumption. The pigs that consumed either diet ad libitum, control or ASP-250, consumed similar quantities of feed. The ASP-250 ad libitum-intake pigs had serum IGF-I concentrations that were greater (P<.01) than those of their ad libitum-intake control littermates. Similarly, the ASP-250 limit-fed pigs had serum IGF-I concentrations that were greater (P<.01) than those of the controls. Although the serum IGF-I concentrations of pigs fed the ASP-250-supplemented diet for ad libitum intake were greater than the serum IGF-I concentrations of the pigs limit-fed the ASP-250-supplemented diet, the differences were not significant (P<.08). The ASP-250-fed pigs had higher serum IGF binding protein (BP)-3 concentrations than did their control littermates (P<.003). A time course of antimicrobially induced alterations in serum IGF-I concentrations revealed that the effect of increased serum IGF-I levels in ASP-250-supplemented pigs (P<.02) was observed within 4 d and was maintained throughout the 4-wk study. These findings show that feed intake is not responsible for the increase in serum IGF-I observed with ASP-250 supplementation. Additionally, the antimicrobially induced increase in serum IGF-I concentrations occurs within a few days after initiation of the treatment.     

Impact of blood serum insulin-like growth factor I on platelet-activating factor in bull spermatozoa

The objective of this study was to examine differences in platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine; PAF] in spermatozoa between two lines of Angus beef cattle divergently selected for blood serum insulin-like growth factor I (IGF-I) concentration. Endogenous lipids were extracted from the spermatozoa and endogenous PAF content was determined by radioimmunoassay. The amount of PAF detected in spermatozoa obtained from high IGF-I bulls (n = 8) ranged from 0.145 to 3.571 pM/10(6) cells. The level of PAF extracted from spermatozoa obtained from low IGF-I- bulls (n = 5) ranged from 0.001 to 1.024 pM/10(6) cells. Polynomial regression analysis revealed a significant cubic relationship (R(2) = 0.374; F = 6.292; P < 0.05) between spermatozoa PAF content and blood serum IGF-I concentration. Spermatozoa-derived PAF levels (mean +/- SEM) were significantly higher (P < 0.05) in the high IGF-I group (1.90 +/- 0.39 pM/10(6) cells) than in the low IGF-I group (0.59 +/- 0.20 pM/10(6) cells). High IGF-I bulls have a greater than three-fold higher PAF content in their spermatozoa than low IGF-I bulls. The data demonstrate that not only is PAF present in bull spermatozoa but that levels are significantly higher in individuals with high serum IGF-I concentrations.     

Ontogeny of a specific high-affinity binding site for ovine placental lactogen in fetal and postnatal liver

Ovine placental lactogen (oPL) exerts actions in sheep and rodent fetal tissues that growth hormone (GH) does not. However, in postnatal tissues, both oPL and GH possess these activities. Although a high-affinity binding site for oPL in ovine fetal liver has been reported, some investigators believe this to be the GH receptor. It was our objective to discriminate between oPL and GH binding to fetal liver microsomes using competitive saturation analyses. Microsomal membranes from fetal liver (Days 60, 90, 105, 120, and 135 of gestation) and postnatal liver (1 wk of age) were incubated with increasing amounts of [¹²⁵I]oPL in the absence or presence of a 100-fold molar excess of unlabeled oPL. Saturable binding of [¹²⁵I]oPL was observed with fetal liver and postnatal liver microsomes. The K_d of the oPL-binding site in fetal liver was 122.1 ± 8.2 pM (mean ± standard error), and receptor concentrations remained relatively constant (9.8 ± 1.1 fmol/mg of membrane protein) across gestation. The highest concentration of oPL binding was detected in 1-wk postnatal liver microsomes (53.0 fmol/mg of membrane protein). Saturation analyses using [¹²⁵I]GH and [¹²⁵I] prolactin (PRL) were also conducted with fetal liver membrane preparations. Although specific binding for these two radiolabeled ligands was observed in control tissues, no specific binding was observed in fetal liver. These data are in agreement with earlier reports that a high-affinity binding site for oPL exists in fetal tissues. The fact that saturable binding could not be demonstrated for either GH or PRL with fetal liver microsomes contradicts recent suggestions that oPL is binding the GH receptor.