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.     

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.     

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.     

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.     

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)     

Feeding different amounts of colostrum or only milk replacer modify receptors of intestinal insulin-like growth factors and insulin in neonatal calves

Colostrum intake influences growth and development of the gastrointestinal tract (GIT) in several species and colostral insulin-like growth factors I and II (IGF-I and IGF-II), and insulin are involved in neonatal intestinal tissue growth. We have studied IGF type 1, IGF type 2, and insulin receptors in the intestine of 8-day-old calves fed different amounts of colostrum or only milk replacer. Calves were fed colostrum of the first six milkings on the first 3 days and then milk replacer (GrC(6)) or colostrum only once and then milk replacer (GrC(1)) or they were fed only milk replacer from the beginning, i.e., no colostrum (GrM). Competitive binding studies and ligand blots confirmed the presence of IGF type 1, IGF type 2, and insulin receptors in mucosal cell membranes of duodenum, jejunum, ileum, and colon. The IGF type 1 receptor number in ileum and total intestine in GrC(6) was greater (P<0.05) than in GrC(1) and in GrM, and IGF type 2 receptor number in total intestine was greater (P<0.05) in GrC(6) than in GrM. Insulin binding was best fitted by a model with two binding sites. High affinity insulin receptor numbers in duodenum, ileum, and total intestine were greater (P<0.05) in GrC(6) than in GrM. The data demonstrate that IGF type 1, IGF type 2, and insulin receptors in intestinal mucosa of neonatal calves are influenced by feeding.     

An association between lifespan and variation in insulin-like growth factor I receptor in sheep

Longevity in livestock is a valuable trait. When productive animals live longer, fewer replacement animals need to be raised. However, selection for longevity is not commonly the focus of breeding programs as direct selection for long-lived breeding stock is virtually impossible until late in the reproductive life of the animal. Additionally the underlying genetic factors or genes associated with longevity are either not known, or not well understood. In humans, there is evidence that IGF 1 receptor (IGF1R) is involved in longevity. Polymorphism in the IGF1R gene has been associated with longevity in a number of species. Recently, 3 alleles of ovine IGF1R were identified, but no analysis of the effect of IGF1R variation on sheep longevity has been reported. In this study, associations between ovine IGF1R variation, longevity and fertility were investigated. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) was used to type IGF1R variation in 1,716 New Zealand sheep belonging to 6 breeds and 36 flocks. Ovine IGF1R C was associated with age when adjusting for flock (present 5.5 ± 0.2 yr, absent 5.0 ± 0.1 yr, P = 0.02). A general linear mixed effects model suggested an association (P = 0.06) between age and genotype, when correcting for flock. Pairwise comparison (least significant difference) of specific genotypes revealed the difference to be between AA (5.0 ± 0.1 yr) and AC (5.6 ± 0.2 yr, P = 0.02). A weak negative Pearson correlation between fertility and longevity traits was observed (r = -0.25, P < 0.01). The finding of an association between variation in IGF1R and lifespan in sheep may be useful in prolonging the lifespan of sheep.     

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.     

Effects of EGF receptor ligands on fetal ovine myoblasts

Epidermal growth factor (EGF) receptors are widely distributed in mammalian tissues, including muscle. One ligand of these receptors, heparin-binding epidermal growth factor-like growth factor (HB-EGF) is also strongly expressed in adult muscle. However, in vitro studies of EGF action in cultured muscle cells of different species have yielded conflicting results. The purpose of this study was to investigate the potential role of EGF and related factors in the growth and development of fetal ovine muscle. High affinity EGF receptors were detected on clonally purified ovine fetal myoblasts, using [(125)I] human EGF as a ligand (K(d) values of 47 and 54 pM in separate experiments). Competitive binding studies in mixed secondary cultures showed that EGF had the highest affinity for the fetal ovine receptor, followed by HB-EGF and transforming growth factor alpha (TGF-alpha). These ligands all stimulated DNA synthesis in clonally purified ovine myoblasts, with their relative potencies at 0.1 nM reflecting their receptor binding affinities. Maximal effects were seen at 1-10 nM. EGF (10 nM) did not significantly inhibit the differentiation of clonally purified fetal ovine myoblasts, although there was increased proliferation of nondifferentiating cells. Hence a variety of EGF receptor ligands have the potential to influence the proliferation ovine muscle cell precursors in utero, but it is unlikely that they promote differentiation.     

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.     

Exogenous somatotropin alters IGF axis in porcine endometrium and placenta

The aim of this study was to examine whether exogenous somatotropin (ST) can alter the insulin-like growth factor (IGF) axis in the porcine epitheliochorial placenta. Crossbred gilts were injected either 6 mg of recombinant porcine ST or vehicle from days 10 to 27 after artificial insemination (term day 116). Control and ST-treated gilts were euthanized on day 28 (8 control/5 treated), day 37 (4 control/6 treated), and day 62 (4 control/6 treated) of gestation. Endometrium and placental tissue samples were collected and subjected to mRNA analyses. In control gilts, somatotropin receptor (STR) and IGF-I mRNA abundance in the endometrium decreased with gestation. Conversely, the amounts of IGF-II mRNA and of IGF binding protein (BP)-2 and -3 mRNA, which were analyzed in endometrium and placental chorion, increased with gestation. The endometrium contained less IGF-II mRNA but more IGFBP-2 and-3 mRNA than the placental chorion. In response to pST treatment, the amounts of endometrial STR and IGF-I mRNA were lower at days 28 and 37, but higher at day 62 of gestation. The content of IGF-II mRNA was higher in the endometrium of pST-treated than control gilts on day 37. The amount of IGFBP-2 mRNA was increased on day 37 in endometrium and placenta of pST-treated gilts, whereas no changes in IGFBP-3 mRNA were observed. The IGF-II/IGFBP-2 ratio was higher in the placenta in response to pST on day 28 of gestation. Results show that pST treatment of pregnant gilts during early gestation alters IGF axis in maternal and fetal placental tissues and suggest pST may exert an effect on fetal growth by altering the relative amount of IGFBPs and IGFs at the fetal-maternal interface.     

Genetic models for the study of insulin-like growth factors (IGF) and muscle development in birds compared to mammals.

IGFs are important positive modulators of overall body and muscle growth in different species. Genetic variation in IGF-I gene expression exists as shown by the possibility of genetic selection for high or low circulating IGF-I concentrations in mice and the associated variations in growth potential. Targeted over-expression of IGF-I in transgenic mice results in muscle hypertrophy, but it is yet unknown whether genetic variability in muscle IGF-I gene expression exists. Much less data are available in birds. This review is focussed on the potential role of IGFs on chicken muscle development. Apart from the absence of a type 2 IGF receptor (IGF-II receptor), the general characteristics of the chicken IGF system seem to be similar to mammalian species. In different genetic models with altered growth rates or body composition, differences in the IGF system are observed suggesting its importance in regulating body growth in those species. The components for a paracrine action of IGF are also present in chicken muscle but it has not yet been demonstrated if they contribute to differences in muscle development.     

Periparturient endocrine and metabolic changes in healthy cows and in cows affected by mastitis

Transition from pregnancy to lactation in dairy cows involves considerable metabolic adaptation. Additional stress is incurred during infections such as periparturient mastitis. Multiparous Holstein-Friesian cows kept under normal production conditions (n = 15) were used to evaluate changes in circulating metabolite and hormone concentrations from 5 days before to 5 days after calving. Insulin-like growth factor binding protein (IGFBP) profiles were also monitored. Marked time-related changes were observed for plasma thyroid hormone, IGF, cortisol, insulin, beta-hydroxybutyrate (BHB) and non-esterified fatty acid (NEFA) concentrations but not for plasma leptin. A decrease in IGF-II concentration and maximal intensity of the putative IGFBP-1 band occurred at parturition. When compared with the five healthy cows,low IGF-II levels were prolonged to day 2 post-partum in five cows with Escherichia coli-associated mastitis. However, marked decreases in IGFBP-2 band intensity were evident only in two of the four cases examined. Individual total ligand (IGF-I + IGF-II) concentration and IGFBP pattern prepartum were largely regained 5 days post-partum in all cows. Hormone and metabolite concentrations in the two cows with Staphylococcus aureus-associated mastitis were very similar to those in the five healthy cows. Plasma thyroxine (T4) was lower 2 days prepartum in the cows, which later developed Gram-negative mastitis. Multiregression analysis showed that variance in T4 concentration was significantly and independently associated with triiodothyronine (T3) and IGF-I positively and with cortisol negatively (R2 = 0.648). This study confirms the close inter-relationship between the thyroid hormone and IGF axes in cattle and indicates possible effects of Gram-negative mastitis infection on IGF-II metabolism.     

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.     

Hypothyroid goitre in a ram: chemical analysis gives indirect evidence for a structurally altered type of ovine thyroglobulin

The thyroglobulin of a ram of the East Friesian milk sheep breed suffering from goitre was investigated by physico- and immunochemical methods. The respective ram was the only animal amongst the other sheep of the flock, that exhibited severe goitre, additionally showing depressed behaviour. Results of the thyroid-stimulating hormone response test were indicative of hypothyroidism. The dysfunction of the thyroid gland could be treated by additional iodine supplementation quite successfully, although all sheep had been given iodinated cattle salt throughout the course of the history. Without reducing conditions sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of isolated thyroglobulin molecules of the ram and control sheep did not reveal different band patterns, but under reducing conditions different band patterns were evident for the respective animals: the ram's thyroglobulin displayed two main bands, those of healthy reference sheep only one. Both bands reacted equally with anti-thyroglobulin antibodies, even with those produced by immunizing rabbits with single bands. The reduced single thyroglobulin band of healthy sheep corresponded to a truncated form of that molecule, whereas the additional main band of the ram was a more resistant, intact thyroglobulin subunit, as was shown by mass spectrometry. In conclusion, results of physico- and immunochemical investigations gave evidence of a modification of thyroglobulin with suspected different iodine binding properties in the ram. The latter finding may have clinical relevance in similar cases in other species, as it is an example of the impact that a minor change in a protein molecule may have on a complete metabolic pathway. Additionally, it could be shown, that in the ovine species the generally found single main band of thyroglobulin after reduction is a truncated form and not an intact subunit. This truncation seems to be induced in vitro by the reductive sample pretreatment prior to SDS-PAGE.     

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.     

Doxycycline binding to plasma albumin of several species

The affinity of doxycycline for crystalline plasma albumin fraction V, originating from sheep, dogs, cats, cows, pigs and humans, was evaluated by means of double-reciprocal and Scatchard plots. Mathematical modelling and weighted least-squares non-linear regression analysis of each Scatchard plot identified one binding component characterized by one high affinity binding site, and a second component attributed to non-specific binding to albumin. Association constants for this binding site ranged from 38,471 +/- 13,369 (SEM) l/mol for the interaction of doxycycline with ovine albumin to 6405 +/- 2375 l/mol for the interaction of doxycycline with human albumin. Statistical evaluation of the results suggested slight species-related differences in the values of association constants. Diphenylhydantoin, phenobarbital or carbamazepine did not displace doxycycline from binding sites on bovine albumin.     

Regulation of protein and energy metabolism by the somatotropic axis.

The somatotropic axis plays a key role in the co-ordination of protein and energy metabolism during postnatal growth. This review discusses the complexity of the regulation of protein and energy metabolism by the somatotropic axis using three main examples: reduced nutrition, growth hormone (GH) treatment and insulin-like growth factor-1 (IGF-1) treatment. Decreased nutrition leads to elevated GH secretion, but it reduces hepatic GH receptor (GHR) number and plasma levels of IGF-1; it also changes the relative concentrations of IGF binding proteins (IGFBPs) in plasma. GH treatment improves the partitioning of nutrients by increasing protein synthesis and decreasing protein degradation and by modifying carbohydrate and lipid metabolism. However, these well-established metabolic responses to GH can change markedly in conditions of reduced nutritional supply or metabolic stress. Short-term infusion of IGF-1 in lambs reduces protein breakdown and increases protein synthesis. However, long-term IGF-1 administration in yearling sheep does not alter body weight gain or carcass composition. The lack of effect of IGF-1 treatment can be explained by activation of feedback mechanisms within the somatotropic axis, which lead to a reduction in GH secretion and hepatic GHR levels. The somatotropic axis has multiple levels of hormone action, with complex feedback and control mechanisms, from gene expression to regulation of mature peptide action. Given that GH has a much wider range of biologic functions than previously recognized, advances in research of the somatotropic axis will improve our understanding of the normal growth process and metabolic disorders.     

Sheep macrophages express at least two distinct receptors for IgG which have similar affinity for homologous IgG1 and IgG2.

Ovine bone marrow-derived macrophages (BMM) may express several IgG receptor (Fc gamma receptor; FcR) subsets. To study this, model particles (opsonized erythrocytes; EA), which are selectively handled by certain FcR subsets of human macrophages were used in cross-inhibition studies and found to react in a similar manner with FcR subsets of sheep macrophages. In experiments with monoclonal antibodies against subsets of human FcR, human erythrocytes (E) treated with human anti-D-IgG (anti-D-EAhu) and sheep E treated with bovine IgG1 (Bo1-EAs) were handled selectively by human macrophage FcRI and FcRII, respectively. Rabbit-IgG-coated sheep E (Rb-EAs) were recognized by FcRI, FcRII and possibly also by FcRIII of human macrophages. Anti-D-EAhu, Bo1-EAs and Rb-EAs were also ingested by sheep BMM. Competitive inhibition tests, using various homologous and heterologous IgG isotypes as fluid phase inhibitors and the particles used as FcR-specific tools in man (anti-D-EAhu and Bo1-EAs), revealed a heterogeneity of FcR also in sheep BMM. Thus, ingestion of anti-D-EAhu by ovine BMM was inhibited by low concentrations of competitor IgG from rabbit or man in the fluid phase, but not at all by bovine IgG1, whereas ingestion of Bo1-EAs was inhibited by bovine IgG1. This suggested that anti-D-EAhu were recognized by a FcR subset distinct from that recognizing bovine-IgG1. It was concluded that sheep BMM express functional analogs of human macrophage FcRI and FcRII and that Bo1-EAs and anti-D-EAhu are handled by distinct subsets of BMM FcR. All EAhu tested (EAhu treated with anti-D, sheep IgG1 or sheep IgG2) were ingested to a lower degree than EAs. This inefficient phagocytosis could be enhanced by treatment of EAhu with antiglobulin from the rabbit, suggesting that it is caused by a low degree of activity of opsonizing antibodies rather than special properties of the erythrocytes themselves. Several lines of evidence suggested that both FcR subsets of ovine BMM recognize both ovine IgG1 and IgG2. In contrast, bovine IgG1 reacts with one FcR subset and bovine IgG2 interacts inefficiently with all FcR of ovine BMM.