Molecular forms of serum insulin-like growth factor (IGF)-binding proteins in man: relationships with growth hormone and IGFs and physiological significance.

Insulin-like growth factor-I (IGF-I) and IGF-II are associated in the blood with specific binding proteins (BPs), forming complexes that elute in gel filtration with estimated mol wt around 40 and 150 kD. The latter appears to be under GH control. Five molecular forms of BP (41.5, 38.5, 34, 30, and 24 kD) have been identified by Western blotting using 125I-labeled IGF. All five forms are present in the smaller complexes, but only the 41.5- and 38.5-kD forms are found in the larger complexes. In this study immunoblotting showed that the 41.5- and 38.5-kD forms were recognized by antibodies directed against the GH-dependent BP purified from human plasma, and the 30-kD form was recognized by antibodies directed against the BP purified from amniotic fluid. The 34- and 24-kD forms proved to be immunologically unrelated to the other three. In sera with large quantities of the 41.5- and 38.5-kD forms, an additional band was often observed immediately ahead of the migration front of the 30 kD band. This was recognized by the anti-GH-dependent BP antibody and probably corresponds to a degradation product of the 41.5- and 38.5-kD BPs. Serum 41.5- and 38.5-kD BPs have been found to be elevated in acromegaly, where GH hypersecretion causes increased IGF-I levels, and diminished in cases of genetic or idiopathic GH deficiency and defects of the GH receptor (Laron's syndrome), where both IGF-I and IGF-II are decreased, as well as in Pygmy adults and children who have isolated IGF-I deficiency. In all of these conditions, the proportions of the 34- and 30-kD forms were inversely related to those of the 41.5- and 38.5-forms. Under treatment, the BP profiles tended to return to normal. In cases of GH deficiency caused by a tumor, the BP profiles resembled those of hypopituitary or normal serum, depending on whether IGF levels were diminished or normal. It, therefore, seems that BP synthesis is coordinated with IGF-I synthesis and may not be directly GH dependent. The results of neutral pH gel filtration analysis of hypopituitary (idiopathic and tumoral) and normal sera point to a relationship between the levels of circulating IGFs and those of the 150-kD IGF-BP complex whose binding units are the 41.5- and 38.5-kD BPs. It, therefore, seems that the 150-kD complex controls the bioavailability of IGF-I and IGF-II.(ABSTRACT TRUNCATED AT 400 WORDS)

Structure, specificity, and regulation of the insulin-like growth factor-binding proteins in adult rat serum.

Insulin-like growth factor-I (IGF-I), the principal IGF in adult rat serum, occurs complexed to specific binding proteins. After fractionation of serum on Sephadex G-200 at neutral pH, 62% of the immunoreactive IGF-I is recovered in the 150K region, 38% in the 40K region, and none is present as free 7.5K IGF-I. Adult rat serum also contains unoccupied binding sites for IGFs that also are predominantly (77%) located in the 150K region and have preferential binding affinity for IGF-II. IGF-binding protein components in the 150K and 40K regions were evaluated by affinity cross-linking to 125I-labeled IGFs and by ligand blotting (i.e. incubation of nitrocellulose blots of sodium dodecyl sulfate (SDS)-gels with [125I]IGFs). Affinity cross-linking of the 150K region revealed a major 43K binding protein complex and several minor covalent complexes of 97-210K that are formed during the cross-linking reaction. The 40K region of the gel filtration column contains a predominant 32K complex and smaller amounts of the 43K complex. Ligand blotting of the 150K region identifies a predominant cluster of binding components of about 40K and a smaller 29K protein. The apparent molecular masses of the 40K and 29K proteins are decreased by incubation with N-glycanase, indicating that they contain N-linked oligosaccharides. These glycoprotein components, designated gp40 and gp29, presumably combine with an acid-labile nonbinding subunit of about 100K to generate the 150K complex. The gp40 cluster represents glycosylation variants of a 34K protein; gp29 has been shown to correspond to an amino-terminal fragment of gp40. Ligand blotting of the 40K region indicates that it contains smaller amounts of gp40 and gp29, possibly representing free subunits not combined with the nonbinding subunit, as well as two proteins of apparent molecular mass 24K and 30K (p24 and p30) that are not glycosylated. Although p30 is similar in size to the binding protein from BRL-3A cells (BP-3A) that is present in fetal rat serum, immunoprecipitation and immunoblotting of whole and fractionated adult serum with an antiserum to BP-3A indicate that p30 in adult rat serum is an antigenically distinct protein. Serum levels of gp40 and gp29 are decreased by hypophysectomy and are restored by GH treatment; p24 and p30 show similar but smaller changes.

Significance of abnormal serum binding of insulin-like growth factor II in the development of hypoglycemia in patients with non-islet-cell tumors.

We reported that serum and tumor from a hypoglycemic patient with a fibrosarcoma contained insulin-like growth factor II (IGF-II), mostly in a large molecular form designated "big IGF-II." We now describe two additional patients with non-islet-cell tumor with hypoglycemia (NICTH) whose sera contained big IGF-II. Removal of the tumor eliminated most of the big IGF-II from the sera of two patients. Because specific IGF-binding proteins modify the bioactivity of IGFs, the sizes of the endogenous IGF-binding protein complexes were determined after neutral gel filtration through Saphadex G-200. Normally about 75% of IGFs are carried as a ternary complex of 150 kDa consisting of IGF, a growth hormone (GH)-dependent IGF-binding protein, and an acid-labile complexing component. The three patients with NICTH completely lacked the 150-kDa complex. IGF-II was present as a 60-kDa complex with variable contributions of smaller complexes. In the immediate postoperative period, a 110-kDa complex appeared rather than the expected 150-kDa complex. Abnormal IGF-II binding may be important in NICTH because the 150-kDa complexes cross the capillary membrane poorly. The smaller complexes present in our patients' sera would be expected to enter interstitial fluid readily, and a 4- to 5-fold increase in the fraction of IGFs reaching the target cells would result.

Caloric restriction and 7,12-dimethylbenz(a)anthracene-induced mammary tumor growth in rats: alterations in circulating insulin, insulin-like growth factors I and II, and epidermal growth factor.

Caloric restriction (CR) inhibits many neoplastic diseases in rodents, yet the biochemical mechanism(s) for these effects are poorly understood. We have examined the effects of ad libitum (AL) feeding with 25 or 40% CR on the promotion of 7,12-dimethylbenz(a)anthracene-induced mammary tumorigenesis in virgin female Sprague-Dawley rats. Further, we have also studied the influence of chronic CR on temporal alterations in circulating insulin, insulin-like growth factor I/somatomedin C, insulin-like growth factor II/multiplication-stimulating activity, and epidermal growth factor levels at 0, 1, 3, 5, 11, and 20 weeks in carcinogen- and vehicle-treated animals. Tumor incidence and multiplicity were markedly inhibited (P less than 0.05) with increasing CR. Fasting serum insulin-like growth factor I/somatomedin C levels exhibited a significant acute decline with CR at 1 and 3 weeks, but were comparable to AL-fed controls throughout the remainder of the 5-month study, despite continued differences in weight gain between AL and CR rats. Levels of insulin-like growth factor II/multiplication-stimulating activity exhibited no discernible pattern in relation to CR. Serum insulin levels showed age-dependent increases, but were affected by increasing CR at all time points. Insulin levels were significantly (P less than 0.05) reduced in 40% CR rats from 3 weeks onward compared to controls, while 25% CR resulted in nonsignificant (P less than 0.07) reductions throughout the study. No significant differences in growth factor levels were observed between 7,12-dimethylbenz(a)anthracene- and vehicle-treated rats. Circulating epidermal growth factor was not detectable in any treatment group regardless of the nature or duration of the dietary regimen, time of blood collection, or subsequent tumor-bearing status. These data suggest that decreased serum insulin-like growth factor I/somatomedin C and insulin levels with CR and their complex interactions in vivo may play a role in the inhibition of mammary tumor promotion by CR.

The effects of altered nutritional status upon insulin-like growth factors and their binding proteins in neonatal rats.

To investigate the role of nutrition in the regulation of IGFs during the perinatal period, 10-d-old rats were infused intravenously with various concentrations of nutrients for 24 h. Breast-fed litter mates served as controls. The effect of caloric intake on concentrations of IGF-I and IGF-II as well as IGF-binding proteins in serum, liver, and brain of neonatal rats was studied. A total of 45 rats from 10 litters was infused with solutions ranging from a caloric intake of 0 (saline) to 75% (glucose, amino acids, and lipids) of the estimated intake of control rats. In serum, both IGF-I and -II concentrations fell markedly in response to fasting. Serum IGF-II levels were linearly related to caloric intake in the pooled data from all groups. Concentrations of IGF-II, but not IGF-I, in liver and brain were depressed by caloric restriction. In contrast to the fall in IGF concentrations, activity of IGF-associated binding proteins rose in serum and in liver cytosol 2- to 4-fold in response to decreased nutrient intake. In serum, but not liver, the rise in binding protein activity was inversely related to to caloric intake. In liver, cytosol, but not serum, the rise in binding protein activity was inversely related to total serum amino acid concentration. Thus, IGF concentrations in preweanling rats change in response to alterations of nutrient intake. The fasting induced decrements in IGF levels, as well as the elevations in IGF-associated binding protein activity, may serve as a protective mechanism to depress growth in times of caloric restriction.

Simultaneous isolation of insulin-like growth factors I and II from adult sheep serum.

Ovine insulin-like growth factors I and II (oIGF-I and oIGF-II) have been purified from adult sheep serum. oIGF-II-like receptor-binding activity and IGF-I-like immunoactivity were enriched on SP-Sephadex C-25, then purified using HPLC in the presence of a variety of counter ions. IGF-I- and IGF-II-like activities were separated using HPLC in the presence of 0.2% tetrabutylammonium phosphate at pH 7.0. The final recovery of oIGF-I was 82.6 micrograms from 3.2 litres of adult sheep serum (a yield of 17.6%), and the recovery of oIGF-II was 388 micrograms (a yield of 13.3%). Both IGF preparations were considered to be homogeneous as judged by single sharp peaks during analytical HPLC, and unique N-terminal amino acid sequences. Purified ovine IGFs had molecular weights similar to that of other IGFs (approximately 7000), and the first 30 N-terminal amino acids of both peptides were identical to their human counterparts. The isoelectric points of oIGF-I (pI approximately 8.2) and oIGF-II (pI approximately 6.8) were similar to those of human (h) IGFs (hIGF-I pI approximately 8.2; hIGF-II pI approximately 6.5), and the overall amino acid content of the ovine IGFs was also similar to that of IGFs from other species. oIGF-II preparations from fetal sheep and from adult sheep appeared to be identical. The isolation procedure represents one of general utility that can be easily modified to facilitate the isolation of recombinant IGFs from culture fluid.

Insulinlike growth factors in patients with active nephrotic syndrome.

The serum concentrations of insulinlike growth factors 1 and 2 (IGF-1 and IGF-2) were measured by specific radioimmunoassays in 25 nephrotic patients. The serum concentration of IGF-1 in nephrotic patients (mean +/- SEM, 169 +/- 17 ng/mL) was significantly lower than that observed in 20 control subjects matched for sex and age (338 +/- 36 ng/mL). The IGF-2 serum concentration was significantly lower in nephrotic patients (343 +/- 22 ng/mL) than in control subjects (898 +/- 80 ng/mL). The IGF-1 and IGF-2 150-kd and 45-kd carrier protein complexes were found in the urine of nephrotic patients, whereas there was no binding of radiolabeled IGF-1 or IGF-2 to IGF carrier proteins in the urine of control subjects. The low serum IGF-1 and IGF-2 levels observed in nephrotic patients could be partially due to the increased urinary losses of the IGF carrier proteins.

The insulin-like growth factor II/mannose-6-phosphate receptor is present in fetal and maternal sheep serum.

A large mol wt binding protein for insulin-like growth factor II (IGF-II) has been described in fetal sheep serum. We now provide evidence to demonstrate that this binding protein is the IGF-II/mannose-6-phosphate (Man-6-P) receptor. Serum and plasma were gel filtered on Sephadex G-200, and the column fractions were assayed for binding of radiolabeled IGF-II. There was significant binding of [125I]IGF-II to the void volume fractions in addition to binding to the 150K and 40K carrier proteins. Binding to the void volume fractions was increased in fetal serum as well as maternal serum and dramatically decreased in the nonpregnant adult. Competitive binding studies with [125I]IGF-II and the void volume pools from fetal and maternal sheep serum demonstrated that IGF-I competed less potently than IGF-II, and insulin did not compete. There was no specific binding of [125I]IGF-I to the void volume pools of either fetal or maternal samples. Chemical cross-linking of [125I]IGF-II to aliquots of the void volume pools from fetal and maternal sheep serum samples and analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of dithiothreitol demonstrated a specific band at about 240K. Western blotting using a specific antiserum (no. 3637) against rat IGF-II/Man-6-P receptor was performed on aliquots of the Sephadex G-200 void volume pools of fetal, maternal, uterine vein, and adult sheep serum; a band of approximately 210K (without dithiothreitol) was seen. The IGF-II/Man-6-P receptor band was more intense in fetal serum than in either maternal or adult nonpregnant sheep serum. There was also increased binding of [125I]IGF-II in the 40K region of the Sephadex G-200 column fractions in the maternal serum compared to that in serum from nonpregnant adult ewes. When fetal, maternal, and adult nonpregnant sheep serum Sephadex G-200 pools were gel filtered on Sephadex G-50 in 1 mol/liter acetic acid to separate bound from free IGF, and IGF-II was measured by RRA, approximately 50% of the circulating IGF-II was associated with this IGF-II/Man-6-P receptor in fetal serum compared to 7% in maternal serum and 3% in adult nonpregnant serum. These data demonstrate that the IGF-II/Man-6-P receptor circulates in fetal sheep serum as well as maternal serum and appears to be a significant carrier for IGF-II in fetal sheep.

Growth hormone and testosterone can independently stimulate the growth of hypophysectomized prepubertal lambs without any alteration in circulating concentrations of insulin-like growth factors.

Castrated prepubertal lambs were hypophysectomized and then treated with GH and testosterone either alone or in combination over a series of 3-week treatment periods. Hypophysectomy resulted in a rapid reduction in skeletal growth rate which could be reversed by the administration of either GH (4 IU three times a week for 3 weeks) or testosterone propionate (10 mg daily for 3 weeks). When GH or testosterone treatment was withdrawn, skeletal growth fell to the post-operative rate. Combined treatment with both GH and testosterone was no more or less effective than either hormone given singly. The order of administration did not have any effect on the growth rate. Circulating concentrations of insulin-like growth factor-I (IGF-I) were reduced by hypophysectomy, but neither GH nor testosterone treatment, alone or in combination, had any effect on IGF-I concentrations. Concentrations of IGF-II rose following hypophysectomy, and again were not affected by any of the hormonal replacement treatments. In conclusion, both GH and testosterone could stimulate skeletal growth in the hypophysectomized lamb without any alteration of circulating IGF concentrations, and testosterone can clearly stimulate skeletal growth in the complete absence of GH.

The study of insulin-like growth factors in Tilapia, Oreochromus mossambicus.

Whole and acid-separated serum samples from fed, starved, and refed Tilapia were analyzed for insulin-like growth factors 1 (IGF-1) and 2 (IGF-2) using human fetal brain radioreceptorassay (RRA-IGF-1), rat liver membrane radioreceptorassay (RRA-IGF-2), and radioimmunoassay (RIA-IGF-1). Triidothyronine (T3) and thyroxine (T4) levels were measured by commercial kits for RIA. For serum separation, acid Sephadex G-50 and G-100 and neutral Sephadex G-200 columns were used. Whole serum and separated serum cross-reacted in RRA-IGF-1, but only slightly in RRA-IGF-2. IGF activity eluted in two peaks after acid G-50 chromatography. Peak I eluted at the void volume, and peak II eluted with an apparent molecular weight of approximately 7 kDa. The 7 kDa activity did not cross-react in RIA-IGF-1 excluding identity with human intact or truncated IGF-1, but did suggest the presence of an IGF-1 variant form. Whole serum was separated over a neutral G-200 column, and all activity eluted at the void volume indicated an apparent molecular weight equal to or greater than 250 kDa. No IGF-binding activity was displayed by either whole serum or peak I after acid G-50 chromatography. Despite significant changes in body weight, an influence of starvation and refeeding on serum IGF activity could not be established. No correlation was seen between serum IGF and T3 and T4 levels.

Pulsatile growth hormone, insulin-like growth factors and antler development in red deer (Cervus elaphus scoticus) stags.

Plasma samples taken every 30 min over a 26-h period each month from six 4- to 15-month-old red deer stags were analysed for GH. In addition, two samples taken at 10.00 and 22.00 h were analysed for insulin-like growth factor-I (IGF-I) and insulin-like growth factor-II (IGF-II). A concentrate diet was available ad libitum. Food intake, body weight and antler status were recorded. Concentrations of GH were analysed using the PULSAR peak detection routine. Secretion of GH was pulsatile in every month of sampling, but the pattern of pulsatility differed seasonally. During the autumn and early winter (April-June in the Southern hemisphere) GH pulses were frequent and of low amplitude. In contrast, GH pulses in spring (August-September) were of high amplitude and high frequency resulting in a high mean level of GH circulating in the plasma. In early summer (November) the GH pulse amplitude was much lower and pulse frequency fell. There was a rise in GH pulse frequency not accompanied by an increase in GH pulse amplitude in summer (December-January). GH pulse amplitude seemed to be the main determinant of mean GH plasma level. Secretion of IGF-I was raised 1 month after peak monthly mean GH secretion. There was little consistent relationship between concentrations of IGF-II and mean daily GH. Concentrations of GH correlated positively and significantly with liveweight gain and antler growth rate with a delay of 1 month. Significantly positive correlations between concentrations of IGF-I, liveweight gain and antler growth rate were observed. It is considered that the spring and summer (September-December) seasonal acceleration of liveweight gain and antler development in stags could be a consequence of high winter/early spring (August-September) GH pulse frequency and amplitude resulting in increased concentrations of IGF-I, particularly in October.

Serum levels of insulin-like growth factor (IGF) I, II and IGF binding protein in diabetic adolescents treated with continuous subcutaneous insulin infusion.

IGF-I and IGF-II as well as the low molecular type of IGF binding protein (IGFPB) were determined in serum from 11 adolescents with insulin-dependent diabetes mellitus (IDDM) during a cross-over study with conventional and continuous subcutaneous insulin infusion (CIT and CSII) therapy. At the onset of the study the mean IGF-I level, 127 +/- 15 ng ml-1, was significantly decreased (P less than 0.001) in comparison with age-matched controls, whereas the mean IGF-II level, 1024 +/- 48 ng ml-1, was increased. A significant correlation (r = 0.70, P less than 0.05) was found between IGF-II and HbA1c levels. The mean morning level of IGFBP, 75 +/- 17 ng ml-1, at the onset of the study, was increased threefold above that in age-matched controls (P less than 0.01). There was a significant correlation between IGFBP and blood glucose values (r = 0.66, P less than 0.05). During CSII therapy a significant decrease (P less than 0.05) of the IGFBP levels was seen in subjects with a decrease in glucose levels, whereas no change was observed in IGF levels. The findings of elevated IGF-II and IGFBP levels and correlations between IGFBP and blood glucose concentration as well as IGF-II and HbA1c levels in adolescents with IDDM indicate that both IGF-II and IGFBP reflect a deranged metabolism caused by inadequate insulin administration.

Effects of recombinant insulin-like growth factor I on insulin secretion and renal function in normal human subjects.

Insulin-like growth factor I (IGF-I) is an important mediator of growth hormone (GH) action and it appeared tempting to evaluate possible clinical applications. Recombinant IGF-I was infused s.c. at a dose of 20 micrograms/kg of body weight per hour during 6 days in two healthy adult subjects. Blood glucose and fasting insulin levels remained within normal limits and IGF-II levels were suppressed. In contrast to insulin, fasting C peptide levels were decreased. GH secretion was also suppressed by IGF-I. Our preliminary data allow us to distinguish between the effects of GH per se and those of IGF-I: GH causes hyperinsulinism, whereas IGF-I leads to decreased insulin secretion. Glomerular filtration rate, as estimated by creatinine clearance, increased to 130% of preinfusion values during the IGF-I infusion. Total creatinine and urea excretion remained unchanged. We conclude that IGF-I influences kidney function and, in contrast to GH, exerts an insulin-sparing effect. It may be speculated that the therapeutic spectrum of IGF-I is quite different from that of GH.

Serum insulin-like growth factor II in chronic liver disease.

Insulin-like growth factor II is secreted primarily by the liver and is reported to be transcribed in many primary hepatocellular carcinoma (PHC) cell lines. We have studied diagnostic significance of serum IGF-II in chronic liver diseases using specific enzyme immunoassay. Serum IGF-II levels (mean +/- SE) were decreased in chronic hepatitis (538 +/- 51 ng/ml; N = 29), liver cirrhosis (427 +/- 45; 50) and PHC (260 +/- 41; 17) compared to controls (830 +/- 49; 57). Serum IGF-II was not different from controls in any of nonhepatic diseases such as diabetes (1032 +/- 97; 19) pancreatic cancer (1413 +/- 282; 8), chronic pancreatitis (999 +/- 126; 17), peptic ulcer (1186 +/- 43; 11), irritable bowel syndrome (1002 +/- 109; 12), gastrointestinal tract cancer (1250 +/- 216; 21) and chronic renal failure (733 +/- 135; 14). In liver diseases serum IGF-II showed a significant correlation with liver function test (negative with retention of indocyanine green and total bile acids; positive with albumin, thrombo-test, and cholinesterase). These results suggest that serum IGF-II reflects a reduced production of IGF-II in the liver and that it can be an index for the residual capacity of liver function.

Sheep insulin-like growth factors I and II: sequences, activities and assays.

This report describes the purification, sequences, and activities of insulin-like growth factors (IGFs) from adult and fetal sheep plasma. IGF-1 from adult sheep is identical to human and bovine IGF-I, except for substitution in the sheep of Ala at residue 66 for Pro in the human and bovine polypeptides. IGF-II from adult sheep differs from bovine IGF-II also by a single amino acid, with residue 62 being Ala in ovine and Thr in bovine IGF-2. The first 10 amino-terminal residues of fetal sheep plasma IGF-I and 92% of the amino acids of fetal IGF-II were identified and found to be the same as those of the corresponding IGFs isolated from adult sheep. Ovine IGF-I was virtually equipotent with human IGF-I in growth-related bioassays and in a RIA for human and bovine IGF-I and inhibited the binding of radiolabeled human IGF-I to type I IGF receptors and to a pure IGF-binding protein. Ovine and bovine IGF-II were also found to be similar to each other in biological and immunochemical activities, and in their binding to type I and II IGF receptors and IGF-binding protein. As observed with human and bovine IGF-I and IGF-II, ovine IGF-I bound slightly better to type I IGF receptors than ovine IGF-II, but bound very poorly to type II IGF receptors. This study shows that IGFs from sheep are very similar to those of human and bovine in structure and activity and defines sensitive radioligand assays specific for ovine IGF-I and ovine IGF-II.

Hypophysectomy of the fetal lamb leads to a fall in the plasma concentration of insulin-like growth factor I (IGF-I), but not IGF-II.

The role of the pituitary gland in the regulation of the plasma concentrations of insulin-like growth factors (IGFs) in the late gestation sheep fetus has been examined. Singleton sheep fetuses were either hypophysectomized or sham-operated between days 110-120 of gestation. Blood samples were then collected via carotid cannulae at least three times weekly for the remainder of gestation. In some hypophysectomized fetuses T4 was administered (100 g/day) to overcome the hypothyroidism caused by hypophysectomy. Blood samples were also obtained from lambs during the perinatal period, neonatal lambs within 1-10 days after birth, and pregnant and nonpregnant adult ewes. All plasma samples were subjected to Sephadex G-50 gel filtration under acidic conditions (pH 2.3) to eliminate IGF-binding protein activity. The fractions containing the free IGF peptides were collected and assayed for IGF-I by heterologous RIA, and IGF-II by a homologous RRA. Plasma concentrations of IGF-I and IGF-II did not change with advancing gestational age in any fetal group and were not affected by the prolonged gestation that results from hypophysectomy. The mean plasma IGF-I and IGF-II concentrations in the sham fetuses were 112 +/- 8 and 1340 +/- 112 ng/ml, respectively. Hypophysectomy without thyroid hormone replacement resulted in a significant decrease in plasma IGF-I concentrations to 50 +/- 5 ng/ml, whereas IGF-II concentrations were not affected (1096 +/- 124 ng/ml). IGF-I concentrations in the hypophysectomized fetuses that received T4 were significantly increased (67 +/- 6.0 ng/ml) compared to those in the hypophysectomized fetuses that did not receive T4. The IGF-II concentrations in the hypophysectomized fetuses that received T4 were similar to those in the sham-operated fetuses (1120 +/- 112 ng/ml). At term IGF-I concentrations were increased (180 +/- 21 ng/ml) and IGF-II concentrations were decreased (264 +/- 25 ng/ml) compared to fetal values. Plasma IGF-I concentrations in the prepubertal lamb were similar to the fetal values. Pregnancy in the adult ewe was associated with a significant increase in IGF-II, but had no effect on IGF-I plasma concentrations. These data show that circulating IGF-I concentrations in the fetal lamb are under some pituitary and thyroid control, whereas IGF-II concentrations are independently of pituitary or thyroid status. We confirm, using a homologous assay, that fetal IGF-II concentrations are high and then decrease at term. These data also support the concept that a pregnancy-related factor may regulate plasma IGF-II concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

Serum form of the rat insulin-like growth factor II/mannose 6-phosphate receptor is truncated in the carboxyl-terminal domain.

The insulin-like growth factor (IGF)-II/mannose 6-phosphate (Man-6-P) receptor present in mammalian tissues as an apparent molecular mass = 250 kDa glycoprotein has recently been detected in fetal rat serum in a lower molecular mass form (240 kDa). In the present studies the serum receptor was affinity labeled with 125I-IGF-II after its adsorption onto pentamannosyl 6-phosphate-Sepharose, demonstrating that it can also bind both ligands simultaneously. The receptors in both serum and fresh plasma exhibited the lower molecular mass compared to tissue receptors, indicating this form circulates in vivo. In order to probe the structural basis of the serum receptor's lower mass, we raised antipeptide antibodies against cytoplasmic and extracellular domains of the tissue form of the rat receptor deduced from complementary DNA clones (MacDonald, R. G., Pfeffer, S. R., Coussens, L., Tepper, M. A., Brocklebank, C. M., Mole, J. E., Anderson, J. K., Chen, E., Czech, M. P., and Ullrich, A. (1988) Science 239, 1134-1137). Peptide 22C, Glu-Glu-Glu-Thr-Asp-Glu-Asn-Glu-Thr-Glu-Trp-Leu-Met-Glu-Glu-Ile-Gln-Val- Pro-Ala - Pro-Arg, located in the cytoplasmic domain 32 residues carboxyl-terminal to the transmembrane region, and peptide 13D, Tyr-Tyr-Leu-Asn-Val-Cys-Arg-Pro-Leu-Asn-Pro-Val-Pro-Gly-Cys-Asp, located 1476 residues amino-terminal to the transmembrane domain were synthesized and used as immunogens in rabbits. IGF-II/Man-6-P receptors were first immunoprecipitated from either rat serum or a Triton X-100 extract of rat placental plasma membranes using a polyclonal antireceptor antibody. The immunoadsorbed receptors were then reduced, alkylated, electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto nitrocellulose, and probed with antipeptide antibodies. Anti-13D revealed the major receptor band in all the membrane and serum samples tested as well as several minor species of lower apparent mass in serum. Fetal and neonatal rat sera contained 3-4 times as much of the receptor as adult serum. In contrast, anti-22C recognized the membrane IGF-II/Man-6-P receptor but failed to recognize any of the serum receptor species. These results indicate that the serum IGF-II/Man-6-P receptor is truncated or altered in its cytoplasmic domain, consistent with the hypothesis that it is derived from cells by proteolytic cleavage.

Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations.

There is currently widespread interest in the IGFs (IGF-I and IGF-II) and their roles in the regulation of growth and differentiation of an ever increasing number of tissues are being reported. This selective review focused on the current state of our knowledge about the structure of mammalian IGFs and the multiple forms of mRNAs which arise from alternative splicing and promoter sites which arise from gene transcription. Current progress in the immunological measurement of the IGF is reviewed including different strategies for avoiding binding protein interference. The results of measurements of serum IGF-I and IGF-II in fetus and mother and at various stages of postnatal life are described. Existing knowledge of the concentration of these peptides in body fluids and tissues are considered. Last, an attempt is made to indicate circumstances in which the IGFs are exerting their actions in an autocrine/paracrine mode and when endocrine actions predominate. In the latter context it was concluded that an important role for GH action on skeletal tissues via hepatic production of IGF-I and endocrine action of IGF-I on growth cartilage is likely.

Degradation of insulin and insulin-like growth factors by enzyme purified from human erythrocytes. Comparison of degradation products observed with A14- and B26-[125I]monoiodoinsulin.

An insulin-degrading enzyme has been purified from human erythrocytes. This enzyme degraded 125I-labeled insulin-like growth factor I (IGF-I) more slowly than 125I-IGF-II and degraded IGF-II more slowly than 125I-insulin. The time course of 125I-insulin degradation suggested the presence of intermediates, each of which was itself shown to be a substrate for the enzyme. One of these intermediates appeared to be made up entirely of B-chain residues and had HisB10 as its NH2-terminal. The final major radiolabeled degradation product of A14-[125I]monoiodoinsulin was a peptide with TyrA14 at the A-chain NH2 terminal. This peptide could be reduced with dithiothreitol, suggesting that it contained amino acid residues from both A- and B-chains. It was partially precipitated by trichloroacetic acid and anti-insulin antibody but bound poorly to IM-9 lymphocytes. The final major degradation product of B26-[125I]monoiodoinsulin was a peptide whose NH2-terminal was TyrB26 and could not be reduced by dithiothreitol. It was partially precipitated by anti-insulin antibody but was precipitated poorly, if at all, by trichloroacetic acid and bound poorly to IM-9 lymphocytes. The results show that this enzyme degraded insulin by sequential cleavage of peptide bonds on both A- and B-chains. We identified LeuA13-TyrA14, SerB9-HisB10, and PheB25-TyrB26 as three of the bonds that are cleaved.