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.     

Endometrial expression of the insulin-like growth factor system during uterine involution in the postpartum dairy cow

Rapid uterine involution in the postpartum period of dairy cows is important to achieve a short interval to conception. Expression patterns for members of the insulin-like growth factor (IGF) family were determined by in situ hybridisation at day 14 ± 0.4 postpartum (n = 12 cows) to investigate a potential role for IGFs in modulating uterine involution. Expression in each uterine tissue region was measured as optical density units and data were analysed according to region and horn. IGF-I mRNA was localized to the sub-epithelial stroma (SES) of inter-caruncular and caruncular endometrium. Both IGF-II and IGF-1R expression was detected in the deep endometrial stroma (DES), the caruncular stroma and myometrium. IGFBP-2, IGFBP-4 and IGFBP-6 mRNAs were all localised to the SES of inter-caruncular and caruncular uterine tissue, and in the DES and caruncular stroma, with IGFBP-4 mRNA additionally expressed in myometrium. IGFBP-3 mRNA was only detectable in luminal epithelium. IGFBP-5 mRNA was found in myometrium, inter-caruncular and caruncular SES and caruncular stroma. These data support a role for IGF-I and IGF-II in the extensive tissue remodelling and repair which the postpartum uterus undergoes to return to its non-pregnant state. The differential expression of binding proteins between tissues (IGFBP-3 in epithelium, IGFBP-2, -4, -5 and -6 in stroma and IGFBP-4 and -5 in myometrium) suggest tight control of IGF activity within each compartment. Differential expression of many members of the IGF family between the significantly larger previously gravid horn and the previously non-gravid horn may relate to differences in their rate of tissue remodelling.     

Effects of L-carnitine on fetal growth and the IGF system in pigs

The effects of L-carnitine on porcine fetal growth traits and the IGF system were determined. Fourth-parity sows were fed a gestation diet with either a 50-g top dress containing 0 (control, n = 6) or 100 mg of L-carnitine (n = 6). At midgestation, fetuses were removed for growth measurements, and porcine embryonic myoblasts (PEM) were isolated from semitendinosus. Real-time quantitative PCR was used to measure growth factor messenger RNA (mRNA) levels in the uterus, placenta, muscle, hepatic tissue, and cultured PEM. A treatment x day interaction (P = 0.02) was observed for maternal circulating total carnitine. Sows fed L-carnitine had a greater (P = 0.01) concentration of total carnitine at d 57 than control sows. Circulating IGF-I was not affected (P = 0.55) by treatment. Supplementing sows with L-carnitine resulted in larger (P = 0.02) litters (15.5 vs. 10.8 fetuses) without affecting litter weight (P = 0.07; 1,449.6 vs. 989.4 g) or individual fetal weight (P = 0.88) compared with controls. No treatment effect was found for muscle IGF-I (P = 0.36), IGF-II (P = 0.51), IGFBP-3 (P = 0.70), or IGFBP-5 (P = 0.51) mRNA abundance. The abundance of IGF-I (P = 0.72), IGF-II (P = 0.34), and IGFBP-3 (P = 0.99) in hepatic tissue was not influenced by treatment. Uterine IGF-I (P = 0.46), IGF-II (P = 0.40), IGFBP-3 (P = 0.29), and IGFBP-5 (P = 0.35) mRNA abundance did not differ between treatments. Placental IGF-I (P = 0.30), IGF-II (P = 0.18), IGFBP-3 (P = 0.94), and IGFBP-5 (P = 0.42) mRNA abundance did not differ between treatments. There was an effect of side of the uterus for IGF-I (P = 0.04) and IGF-II (P = 0.007) mRNA abundance; IGF-I mRNA abundance was greater in the left uterine horn than in the right uterine horn (0.14 and 0.07 relative units, respectively). Placental IGF-II mRNA abundance was greater (P = 0.007) in the left than in the right uterine horn (483.5 and 219.59, respectively). The abundance of IGFBP-3 was not affected by uterine horns in either uterine (P = 0.66) or placental (P = 0.13) tissue. There was no treatment difference for IGF-I (P = 0.31) or IGFBP-5 (P = 0.13) in PEM. The PEM isolated from sows fed L-carnitine had decreased IGF-II (P = 0.02), IGFBP-3 (P = 0.03), and myogenin (P = 0.04; 61, 59, and 67%, respectively) mRNA abundance compared with controls. These data suggest that L-carnitine supplemented to gestating sows altered the IGF system and may affect fetal growth and development.     

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.     

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.     

Expression of inhibins, activins, insulin-like growth factor-I and steroidogenic enzymes in the equine placenta

In this study, the expression patterns of inhibins, activins, insulin-like growth factor-I (IGF-I) and steroidogenic enzymes in equine placentae recovered during the latter two-thirds of gestation were examined. Concentrations of inhibin A and inhibin pro-alphaC in endometrial and fetal placental tissue homogenates were very low during the period examined, whereas these tissues contained high concentrations of activin A. In both maternal endometrial and fetal placental tissues, activin A levels decreased as pregnancy progressed. Expression of inhibin alpha-subunit was not observed in the placenta using either immunohistochemistry or in situ hybridization. Inhibin/activin betaA-subunit and its mRNA were confined to maternal endometrial glands, whereas immunopositive betaB-subunit was not detected in either endometrial glands or microcotyledons. Cytochrome P450 side chain cleavage enzyme was detected by immunohistochemistry in both endometrial glands and microcotyledons, whereas cytochrome P450 17alpha-hydroxylase/lyase was absent in these tissues. Immunopositive signals for 3beta-hydroxysteroid dehydrogenase and cytochrome P450 aromatase were localized in microcotyledons but not in endometrial glands. Immunohistochemistry revealed that IGF-I was highly expressed in microcotyledons around Day 130, and decreased as pregnancy progressed. Changes in the expression of IGF-I were correlated with the number of PCNA positive cells in the placenta. The present study demonstrated the presence and localized the site of expression of activin, IGF-I and steroidogenic enzymes in equine placental tissues during the latter two-thirds of gestation; the results suggest that activin and IGF-I may be involved in the regulation of placental development.     

Alteration of the gene and protein levels of insulin-like growth factors in streptozotocin-induced diabetic male rats

Insulin-like growth factor (IGFs: IGF-I and IGF-II) systems have been reported to be associated with the onset of diabetic mellitus. Therefore, we investigated the effect of diabetes on regulation of the IGF system in the liver, kidneys and heart, which are important organs in the pathogenesis of diabetes. The experimental groups were subdivided into three groups: 1) controls, 2) streptozotocin (STZ)-induced untreated diabetic group, and 3) an insulin-treated group (plus diabetic rats). In the present study, starting on the second day after STZ treatment, the diabetic group exhibited hyperglycemia, polyuria, and polydipsia, which are characteristic of diabetes melittus. Serum levels of IGF-I were decreased, but those of IGF-II were increased in the diabetic group compared with the controls. The expression levels of IGF-I and IGF-II protein in the livers of the diabetic group had a similar pattern to the serum. In addition, the expression levels of liver IGF-I mRNA and IGF-II mRNA were decreased in the diabetic groups. In the heart, IGF-I levels were decreased, but IGF-II levels were increased in the untreated diabetic groups, which was consistent with the expression levels of their mRNA. However, both the IGF-I and IGF-II levels in the kidneys were increased in the untreated diabetic groups, but the mRNA levels were decreased. Insulin treatment ameliorated the changes of IGF system in the serum, liver, kidneys, and heart. In conclusion, diabetes induced alteration of the IGF system tissue-specifically, and this was blocked by insulin treatment.     

Growth characteristics, blood metabolites, and insulin-like growth factor system components in maternal tissues of gilts fed L-carnitine through day seventy of gestation

A total of 59 gilts (BW = 137.7 kg) from 3 breeding groups were used to assess the effects of feeding l-carnitine during gestation on gilt growth characteristics, blood metabolites, and uterine and chorioallantoic expression of IGF axis components at d 40, 55, and 70 of gestation. Experimental treatments were arranged in a 2 x 3 factorial, with main effects of added l-carnitine (0 or 50 ppm) and day after initial breeding (d 40, 55, or 70 of gestation). All gilts received a constant feed allowance of 1.75 kg/d and a top-dress containing 0 or 50 ppm of l-carnitine beginning on the first day of breeding through the assigned day of gestation. No dietary treatment differences were observed for gilt BW, backfat, or estimated protein or fat mass at any day of gestation. No differences were observed in circulating total and free carnitine at breeding, but concentrations increased (P < 0.01) as day of gestation increased for gilts fed diets containing l-carnitine compared with those fed the control diet. Maternal IGF-I concentration decreased (P < 0.01) from d 0 to 70 for all gilts, with no differences between treatments. Insulin-like growth factor binding protein-3 mRNA (P = 0.05) and IGFBP-5 mRNA (P = 0.01) increased in the endometrium of gilts supplemented with l-carnitine. These data demonstrate that l-carnitine supplementation and day of gestation alter the expression of the IGF axis by changing the expression of IGFBP at the fetal-maternal interface in swine. These changes in the IGF axis at the fetal maternal interface may aid in determining the reasons for the effects of l-carnitine on reproductive traits.     

The insulin-like growth factor-II/mannose-6-phosphate receptor is present in fetal bovine tissues throughout gestation

The insulin-like growth factor-II (IGF-II) and the IGF-II/mannose-6-phosphate (M6P) receptor are thought to play an important role in fetal growth and development. We have studied the expression of the IGF-II/M6P receptor in fetal bovine tissues from 5 through 36 weeks' gestation. Tissues from bovine fetuses were extracted in buffer containing 2% Triton-X-100 and 2% sodium dodecyl sulfate (SDS). Aliquots of the protein extracts were analyzed by SDS polyacrylamide gel electrophoresis and the protein bands were transferred onto nitrocellulose. Immunoblotting was performed with anti-bovine IGF-II/M6P receptor antiserum. In a subset of experiments, ligand blotting was carried out with radiolabeled IGF-II and subsequent autoradiography. IGF-II/M6P receptors were expressed in all tissues examined, with the highest amount of receptor being present in fetal lung and liver. Low amounts of receptors were measured in fetal brain. The amount of receptor was developmentally regulated throughout fetal life. The developmental regulation of receptor expression varied among the different tissues. In conclusion, the IGF-II/M6P receptor is present in all fetal bovine tissues examined. The presence of the IGF-II/M6P receptor seems to be developmentally regulated during bovine fetal life. We hypothesize that this receptor exerts important biologic effects during fetal growth and tissue and organ development.     

Insulin-like growth factor (IGF)-I and -II and IGF binding proteins in serum and mammary secretions during the dry period and early lactation in dairy cows.

Concentrations of IGF-I and IGF-II, and IGF binding proteins (IGFBP) in serum and mammary gland secretions were surveyed during the dry period and early lactation of 30 Holstein cows. Although there was a threefold drop in the concentration of IGF-I in serum from the last week of the dry period to parturition (81 +/- 7 to 24 +/- 3 ng/ml, P less than .01), there was no significant change in serum IGF-II concentration during this period (150 +/- 17 vs 173 +/- 13 ng/ml, P greater than .05). Furthermore, a 57% increase in serum IGF-I was observed from the last week of lactation to the second week of drying off (100 +/- 5 to 157 +/- 8 ng/ml, P less than .05). Changes in serum IGF-II were not observed (126 +/- 11 vs 150 +/- 10 ng/ml, respectively; P greater than .05). Although IGF-I, IGF-II, and IGFBP concentrations in mammary secretions peaked 2 wk before parturition (2.95 +/- 1.1, 1.83 +/- .6, and 7.27 +/- .76 micrograms/ml, respectively), total output/quarter was highest in colostrum (394 +/- 119, 295 +/- 132, and 2,680 +/- 1,967 micrograms/quarter, respectively). Weekly milking of two individual quarters during the dry period did not affect (P greater than .05) IGF-I or IGF-II concentration (ng/ml) or total output (microgram/quarter) and milk yield in colostrum and milk (2 wk and 7 wk) compared with the ipsilateral quarter. The data support the hypothesis that IGF-I may be transported by the mammary gland epithelium. Furthermore, the secretion mechanisms of IGF-I, IGF-II, and IGFBP by the gland may be related to each other.     

Changes in the local regulation of insulin-like growth factors I and II and insulin-like growth factor-binding proteins in osteoarthritis of the canine stifle joint secondary to cruciate ligament rupture

OBJECTIVES: To investigate changes in concentrations of insulin-like growth factors I (IGF-I) and II (IGF-II) and the expression of IGF-binding proteins (IGFBP) in synovial fluids from dogs with naturally occurring osteoarthritis (OA) of the canine stifle joint secondary to cranial cruciate ligament (CCL) rupture. STUDY DESIGN: Prospective study with synovial fluid sampling from diseased and contralateral unaffected joints at 0, 1.5, and 5 months. SAMPLE POPULATION: Eleven dogs with unilateral CCL deficiency, with unaffected contralateral joints. METHODS: IGF-I and IGF-II concentrations in synovial fluids were estimated by radioimmunoassay at 0, 1.5, and 5 months; Western ligand blotting was performed for intact IGFBPs at 0, 1.5, 5, and 9 months. Both stifle joints were radiographed at 0, 7, and 13 months. RESULTS: The IGF system is altered after CCL rupture and during development of early OA. Mean IGF-I and IGF-II concentrations in index stifle joints at study entry were 201.6 microg/mL and 345.7 microg/mL, respectively, compared with 57.7 microg/mL and 79.4 microg/mL, respectively, for contralateral joints. Index joint IGF concentrations increased after surgical treatment and then declined, although they remained higher than contralateral joints. Index joints had increases in IGFBP-3 and -4, and a decrease in IGFBP-2 expression compared with contralateral joints. CONCLUSIONS: Although IGF concentrations are increased in canine OA, alterations in IGFBP profiles may limit the tissue availability of IGF. CLINICAL RELEVANCE: Manipulation of the IGF system may provide an opportunity for novel treatments of OA in dogs.     

Peripheral circulating insulin-like growth factor-I and -II in cattle

Interrelationships between circulating concentrations of the insulin-like growth factors (IGF-I and IGF-II) were investigated in 235 blood samples taken from 145 healthy beef or dairy calves, bulls and cows of different breeds and ages. Autoradiography of Western ligand blots indicated different IGF binding protein (IGFBP) profiles between sera from different categories of cattle. Each IGF radioimmunoassay was validated by determining the effects of IGFBPs, ligand and contraligand, as well as serial dilution and comparison with results obtained after molecular sieve chromatography in acid. In female cattle mean values for IGF-I varied from 5.1 nmol/l in postparturient Holstein cows to 18.5-20.5 nmol/l in growing beef heifers, while mean IGF-II concentrations ranged from 30.0 nmol/l in the cows to 14.7-15.7 nmol/l in the beef heifer calves. In male cattle mean serum IGF-I ranged widely from 8.2 nmol/l in 1-day-old Holstein calves to 67.4 nmol/l in 16-month-old Simmental-type bulls. Mean IGF-II concentrations decreased from 22.9 nmol/l in 1-day-old Holstein bull calves to 11.9 nmol/l in 12-month-old beef bulls. Thus, total molar IGF concentrations were fairly stable in female cattle (24.7-35.1 nmol/l) but extended from 27.3 nmol/l to 81.8 nmol/l in the male cattle. The tendency for a reciprocal relationship between serum concentrations of these growth factors was most obvious in the periparturient cows.     

Follicular Dominance in Cattle Is Associated With Divergent Patterns of Ovarian Gene Expression for Insulin-Like Growth Factor (IGF)-I,IGF-II,and IGF Binding Protein-2 in Dominant and Subordinate Follicles

A decrease in insulin-like growth factor (IGF) binding protein (BP) amount occurs within the follicular fluid of dominant ovarian follicles. At the same time, concentrations of follicular fluid IGF-I do not change. The mRNA for IGF-I, IGF-II, IGFBP-2, and IGFBP-3 in dominant and subordinate follicles were measured to determine if changes in IGF or IGFBP gene expression are associated with follicular dominance. Heifers were ovariectomized during a follicular wave, either during early-dominance (emerging dominant follicle, 9 mm diameter) or mid-dominance (established dominant follicle, 14–16 mm diameter). Follicles were classified as either dominant (DF), subordinate (SF), or not-recruited (NRF; small antral follicles). mRNA was localized by in situ hybridization and measured by image analyses. The IGF-I mRNA (granulosa cells) was greatest in DF and increased in DF, SF, and NRF from early- to mid-dominance. Likewise, IGF-II mRNA (theca cells) was greatest in DF compared with SF or NRF. The IGFBP-2 mRNA (granulosa cells), however, was nearly undetectable in DF, whereas adjacent SF expressed abundant IGFBP-2 mRNA. The NRF were not uniform in their IGFBP-2 expression because only 5 of 13 NRF had IGFBP-2 mRNA. The IGFBP-3 mRNA (granulosa cells) was found only in two NRF, suggesting that local synthesis is not a predominant source of follicular fluid IGFBP-3. These data show that changes in gene expression for IGFBP-2 are opposite to those for IGF-I or IGF-II. Increased IGF-I and IGF-II mRNA and decreased IGFBP-2 mRNA within the DF may be one mechanism leading to follicular dominance. The opposite pattern of IGFBP-2 gene expression in SF and some NRF may lead to follicular atresia.     

Effect of recombinant porcine somatotropin on fetal and placental growth in gilts with reduced uterine capacity

Crowded uterine conditions were induced by unilateral hysterectomy-ovariectomy (UHO) in 42 gilts to determine the effect of recombinant porcine somatotropin on fetal and placental growth. Gilts were randomly assigned across three replicates to one of three treatments: Control (C; n = 14), daily injections of 1 mL saline from d 0 to 64 of gestation, Early (E; n = 12), 5 mg of rpST/d from d 0 to 30, followed by 1 mL saline from d 31 to 64, and Late (L; n = 16), 1 mL saline/d from d 0 to 29, followed by 5 mg of rpST/d from d 30 to 64 of gestation. Blood was collected from each gilt via jugular venipuncture at d 0 and every 15 d thereafter. Gilts were hysterectomized on d 65 of gestation. Length of placental attachment and fetal crown-rump length were measured. Placentas and fetuses were weighed. Placental length, wet weight, and dry weight were recorded. Treatment with rpST (either E or L) increased (P < 0.0001) maternal plasma IGF-I concentrations relative to controls. Treatment with rpST did not affect placental wet weight or placental DNA content. However, E and L treatments increased the percentage of placental protein (P = 0.01) and placental dry matter (P = 0.10) and increased contact area of uterine-placental interface (P = 0.01). Despite changes in placental composition and morphology, weights of fetuses collected from L-treated gilts did not differ from controls, whereas weights of fetuses collected from E-treated gilts tended to be less than controls (P < 0.06). Administration of rpST increased maternal IGF-I concentrations and placental surface area but failed to increase fetal growth in the UHO model. Therefore, mechanisms that are independent of maternal IGF-I or placental contact area may control early fetal growth under crowded uterine conditions.     

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.     

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.