Cellular localization of somatomedin (insulin-like growth factor) messenger RNA in the human fetus.

The somatomedins or insulin-like growth factors (IGFs) are synthesized in many organs and tissues, but the specific cells that synthesize them in vivo have not been defined. By in situ hybridization histochemistry, IGF I (somatomedin C) and IGF II messenger RNAs were localized to connective tissues or cells of mesenchymal origin in 14 organs and tissues from human fetuses. IGF messenger RNAs were localized to perisinusoidal cells of liver, to perichondrium of cartilage, to sclera of eye, and to connective tissue layers, sheaths, septa, and capsules of each organ and tissue. All of the hybridizing regions are comprised predominantly of fibroblasts or other cells of mesenchymal origin. Because these cells are widely distributed and anatomically integrated into tissues and organs, they are ideally located for production of IGFs, which may exert paracrine effects on nearby target cells.

Insulin-like growth factors as intraovarian regulators of granulosa cell growth and function.

A relatively large body of evidence now appears to support the existence of the essential ingredients for novel intraovarian IGF-driven control mechanisms. Indeed, evidence presented in this communication is in keeping with the possibility that the granulosa cell may be the site of IGF production, reception, and action. Although the relevance of IGFs to ovarian cell types other than the granulosa cell is largely unknown, one cannot at the present time exclude the possibility of nongranulosa cell contributions to intraovarian IGF production, reception, and action. Indeed, preliminary affinity cross-linking studies (Adashi, Resnick, Svoboda, Van Wyk and D'Ercole; unpublished data) suggest the existence of type-I and type-II receptors in nongranulosa cell compartments. The above notwithstanding, IGFs of granulosa (and possibly circulatory) origins may interact with granulosa cell autoreceptors either independently or in synergy with other granulosa cell agonists. According to this view, IGFs may act in the autocrine mode to stimulate granulosa cell replication on the one hand and promote granulosa cell differentiation on the other. Although proliferation and terminal differentiation may prove mutually exclusive under some circumstances, coexistence of the two processes is being increasingly recognized. In this context, some studies of porcine granulosa cells support a dual role for IGFs in granulosa cell ontogeny. As such, the IGFs can be added to a growing list of growth factors known to modulate granulosa cell growth and function, including EGF, PDGF, and FGF. Our findings indicate that Sm-C/IGF-I synergizes with FSH in the induction of rat granulosa cell aromatase activity at nanomolar concentrations compatible with its granulosa cell receptor binding affinity (thus far studied only in porcine cells. A role for Sm-C/IGF-I in the regulation of this key granulosa cell function would be in keeping with the possibility that Sm-C/IGF-I may partake in the assertion and maintenance of dominance by the selected follicle(s) or in promoting juvenile and early follicular development. Moreover, the ability of Sm-C/IGF-I to potentiate this and other FSH-driven ovarian functions may also account, at least in part, for the puberty-promoting effect of growth hormone. This permissive action of growth hormone has been initially suggested by observation in growth hormone-deficient rats, mice (dwarf mutants, and humans (sporadic, hereditary or acquired growth hormone deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)

Affinity-labeled plasma somatomedin-C/insulinlike growth factor I binding proteins. Evidence of growth hormone dependence and subunit structure.

By using disuccinimidyl suberate, we have covalently cross-linked 125I-labeled somatomedin-C (Sm-C)/insulinlike growth factor I to specific binding proteins in human plasma. In unfractionated plasma samples from normal and acromegalic donors, 125I-Sm-C binding-protein complexes with relative molecular weights (Mr) of 160,000, 135,000, 110,000, 80,000, 50,000, 43,000-35,000, and 28,000-24,000 were consistently observed. In contrast, the 43,000-35,000-mol wt species were frequently the only specific complexes observed in hypopituitary plasma and were consistently more intensely labeled in such samples. Reduction of samples with beta-mercaptoethanol did not alter the electrophoretic pattern of these 125I-Sm-C binding-protein complexes. All Sm-C binding proteins, with the exception of the 43,000-35,000-mol wt complex, were adsorbed by concanavalin A-Sepharose. When acromegalic or normal plasma was fractionated on a Sephadex G-200 column and affinity labeled, the same complexes that were adsorbed by concanavalin A were found in fractions that eluted near the gamma-globulin peak. On the other hand, the 43,000-35,000-mol wt complex consistently eluted in size-appropriate fractions near the albumin peak. These data suggest that the growth hormone (GH)-dependent Sm-C binding protein, represented by the 160,000-mol wt complex, is in some way composed of smaller species, i.e., the 135,000-, 110,000-, 80,000-, 50,000-, and 28,000-24,000-mol wt complexes. Acid incubation of plasma prior to Sephadex G-200 chromatography results in the elimination of specific 125I-Sm-C binding-protein complexes which elute near gamma-globulin and a concurrent increase in the labeling intensity of the 28,000-24,000-mol wt complexes. We speculate, therefore, that each of the GH-dependent Sm-C binding-protein complexes represents an oligomer composed of 28,000-24,000-mol wt protomers. The 43,000-35,000-mol wt species is not dependent upon GH and appears to represent a different type of Sm-C binding protein.

Delayed fetal pulmonary maturation in a rabbit model of the diabetic pregnancy.

A rabbit model for the diabetic pregnancy was used to investigate the etiology of delayed pulmonary maturation observed in infants of diabetic mothers. Pregnant rabbit does were made glucose intolerant and insulinopenic by injection of alloxan, a pancreatic beta-cell cytotoxin. At 28 d (term approximately 31 d) fetuses of these animals were hyperglycemic, but were not hyperinsulinemic and did not demonstrate tissue overgrowth. Fetal pulmonary maturity was assessed by measurement of pressure-volume relationships on the fetal lungs. Fetuses of glucose-intolerant does demonstrated less retention of air on deflation. Phospholipid components of pulmonary surfactant were assayed on fluid obtained from lavage of the fetal lungs. Levels of disaturated phosphatidylcholine (per-cent total-lavage phospholipids) were diminished in fetuses of glucose-intolerant does compared to control fetuses (20.5 +/- 4.2 vs. 38.0 +/ 4.3%; P less than 0.01). Lecithin/sphingomyelin ratios were similar in both groups and phosphatidylglycerol was not detected in either group. There was a direct correlation between the percentage of alveolar disaturated phosphatidylcholine and retention of air on lung deflation. These findings suggest that in this model pulmonary instability was a result of diminished alveolar disaturated phosphatidylcholine, and this diminution did not result from fetal hyperinsulinemia.

Estimation of somatomedin-C levels in normals and patients with pituitary disease by radioimmunoassay.

The development of a radioimmunoassay for somatomedin-C has for the first time made it possible to discriminate between serum concentrations of a single peptide or closely related group of peptides and the net somatomedin activity measured by less specific bioassay and radioreceptor techniques. Antibodies to human somatomedin-C were raised in rabbits using a somatomedin-C ovalbumin complex as the antigen. A variety of peptide hormones at concentrations up to 1 muM are not recognized by the antibody. Insulin at concentrations >0.1 muM cross reacts in a non-parallel fashion; purified somatomedin-A is only 3% as active as somatomedin-C; and radiolabeled cloned rat liver multiplication stimulating activity does not bind to the antibody. Immunoreactive somatomedin-C can also be quantitated in the sera of a variety of subhuman species. Unusual assay kinetics, which are manifest when reactants are incubated under classic "equilibrium" assay conditions, appear to result from the failure of (125)I-somatomedin-C to readily equilibrate with the somatomedin-C serum binding protein complex. It is, therefore, necessary to use nonequilibrium assay conditions to quantitate somatomedin-C in serum. With this assay it is possible to detect somatomedin-C in normal subjects using as little as 0.25 mul of unextracted serum. Serum somatomedin-C concentrations in normal subjects were lowest in cord blood and rose rapidly during the first 4 yr of life to near adult levels. In 23 normal adult volunteers, the mean serum somatomedin-C concentration was 1.50+/-0.10 U/ml (SEM) when compared to a pooled adult serum standard. 19 children with hypopituitary dwarfism had concentrations below 0.20 U/ml. 17 of these were below 0.1 U/ml, the lower limit of sensitivity of the assay. The mean concentration in 14 adults with active acromegaly was 6.28+/-0.37 U/ml (SEM), five times greater than the normal volunteers. Significant increases in serum somatomedin-C concentrations were observed in 8 of 10 hypopituitary children within 72 h after the parenteral administration of human growth hormone. Three patients with Cushing's disease had elevated serum somatomedin-C concentrations (2.61+/-0.14 U/ml [SEM]). Three patients with hyperprolactinemia had normal concentrations (1.74+/-0.11 U/ml [SEM]).The important new discovery brought to light by quantitation of immunoassayable somatomedin in patient sera is that all previously used assays detect, in addition to somatomedin-C, serum substances that are not under as stringent growth hormone control.

Cultured fibroblast monolayers secrete a protein that alters the cellular binding of somatomedin-C/insulinlike growth factor I.

We studied somatomedin-C/insulinlike growth factor (Sm-C/IGF-I) binding to human fibroblasts in both adherent monolayers and in suspension cultures. The addition of Sm-C/IGF-I in concentrations between 0.5 and 10 ng/ml to monolayers cultures resulted in a paradoxical increase in 125I-Sm-C/IGF-I binding and concentrations between 25 and 300 ng/ml were required to displace the labeled peptide. The addition of unlabeled insulin resulted in no displacement of labeled Sm-C/IGF-I from the adherent cells. When fibroblast suspensions were used Sm-C/IGF-I concentrations between 1 and 10 ng/ml caused displacement, the paradoxical increase in 125I-Sm-C/IGF-I binding was not detected, and insulin displaced 60% of the labeled peptide. Affinity cross-linking to fibroblast monolayers revealed a 43,000-mol wt 125I-Sm-C-binding-protein complex that was not detected after cross-linking to suspended cells. The 43,000-mol wt complex was not detected after cross-linking to smooth muscle cell monolayers, and binding studies showed that 125I-Sm-C/IGF-I was displaced greater than 90% by Sm-C/IGF-I using concentrations between 0.5 and 10 ng/ml. Because fibroblast-conditioned medium contains the 43,000-mol wt complex, smooth muscle cells were incubated with conditioned medium for 24 h prior to initiation of the binding studies. 125I-Sm-C/IGF-I-binding increased 1.6-fold compared to control cultures and after cross-linking the 43,000-mol wt complex could be detected on the smooth muscle cell surface. Human fibroblast monolayers secrete a protein that binds 125I-Sm-C/IGF-I which can be transferred to the smooth muscle cell surface and alters 125I-Sm-C/IGF-I binding.

Cloning and characterization of a novel RNA involved in cellular growth regulation.

During the course of antisense oligodeoxynucleotide (oligo) inhibition experiments investigating the role of insulin-like growth factor I (IGF-I) in the WI-38 cell cycle, we found that a sense-strand oligo (S oligo), used as a control, inhibited DNA synthesis 90 to 95%. S1 nuclease protection assays demonstrated that this S oligo formed intracellular duplexes with WI-38 RNA, and Northern (RNA) hybridization analyses demonstrated specific hybridization of this 32P-labeled S oligo to 1.8-, 2.3-, and 3.2-kb RNAs. We have cloned and sequenced a 2,251-bp cDNA, designated BB1, corresponding to the 2.3-kb RNA. Decoding of the BB1 cDNA sequence reveals several open reading frames arranged in a motif similar to that seen in proteins subject to translational control mechanisms. Homology searches of nucleic acid and protein data bases reveal no significant homology of BB1 with known sequences other than a 234-bp region in the BB1 5' untranslated region that shared 97% homology with a region in the 3' untranslated region of the human cdc42 mRNA. S1 nuclease protection analyses performed with IGF-I gene fragments and computer homology searches demonstrated that the BB1 RNA does not derive from transcription from the opposite strand of the IGF-I gene. Northern hybridization analyses of RNA extracted from serum-starved HeLa S3 cells demonstrated that steady-state BB1 RNA levels increased upon serum growth stimulation, with steady-state levels peaking 4 h after release from the block induced by serum starvation. Antisense oligo inhibition experiments using specific BB1 antisense oligos targeted to the putative open reading frames of the BB1 RNA reduce DNA synthesis of HeLa S3 cells to 15% of control levels, indicating that the BB1 RNA is essential for cell cycle traversal and, as such, encodes a growth-reguLating gene product.

Insulin-like growth factor-I promotes neurogenesis and synaptogenesis in the hippocampal dentate gyrus during postnatal development.

The in vivo actions of insulin-like growth factor-I (IGF-I) on the growth and development of the hippocampal dentate gyrus were investigated in transgenic mice that overexpress IGF-I postnatally in the brain and in normal nontransgenic littermate controls. Stereological analyses of the dentate gyrus were performed by light and electron microscopy on days 7, 14, 21, 28, 35, and 130 to determine postnatal changes in the numerical density and total number of neurons and synapses. The volumes of both the granule cell layer and the molecular layer of the dentate gyrus were significantly increased by 27-69% in transgenic mice after day 7, with the greatest relative increases occurring by day 35. Although the numerical density of neurons in the granule cell layer did not differ significantly between transgenic and control mice at any age studied, the total number of neurons was significantly greater in transgenic mice by 29-61% beginning on day 14. The total number of synapses in the molecular layer was significantly increased by 42-105% in transgenic mice from day 14 to day 130. A transient increase in the synapse-to-neuron ratio was found in transgenic mice at postnatal days 28 and 35 but not at day 130. This finding indicates a disproportionate increase in synaptogenesis, exceeding that expected for the observed increase in neuron number. Our results demonstrate that IGF-I overexpression produces persistent increases in the total number of neurons and synapses in the dentate gyrus, indicating that IGF-I promotes both neurogenesis and synaptogenesis in the developing hippocampus in vivo.

Insulin-like growth factor-I overexpression attenuates cerebellar apoptosis by altering the expression of Bcl family proteins in a developmentally specific manner.

In studies of transgenic (Tg) mice that overexpress insulin-like growth factor-I (IGF-I) exclusively in the CNS, we demonstrated a dramatic increase in cerebellar granule cell number that appeared to be attributable predominantly to enhanced survival. IGF-I anti-apoptotic actions are well established in cultured neurons, but comparable studies in vivo are few. Using the same Tg mice, therefore, we set out to document IGF-I anti-apoptotic effects during cerebellar development and to probe IGF-I signaling mechanisms. Compared with cerebella (CBs) of non-Tg littermates, those of Tg mice had fewer apoptotic cells at postnatal day 7 (P7) and showed a similar tendency at P14 and P21. At each age studied, procaspase-3 and caspase-3 were decreased in CBs of Tg mice. The caspase-3 decline was accompanied by decreases in the 85 kDa fragment of Poly(ADP-ribose) polymerase, a known product of caspase cleavage, suggesting decreased caspase activity. At P7 decreased apoptosis in Tg mice was associated with increased expression of the anti-apoptotic Bcl genes, Bcl-x(L) and Bcl-2. The mRNA expression of the proapoptotic Bcl genes, Bax and Bad, also was increased, but no changes were observed in the abundance of their proteins. At P14 Bcl-xL and Bcl-2 expression were similar in normal and Tg mice; Bax mRNA was unchanged in Tg mice, but its protein abundance was decreased, and both Bad mRNA and protein abundance were decreased. At P21 Bcl-xL and Bcl-2 expression were unchanged, but Bax and Bad expression were decreased. Our data show that IGF-I exerts anti-apoptotic actions during cerebellar development, and thereby alters the magnitude of naturally occurring apoptosis. IGF-I appears to affect multiple steps in the apoptotic pathway in a developmentally specific manner. IGF-I decreases caspase-3 availability and activity, increases the expression of anti-apoptotic Bcl-x(L) and Bcl-2 during early postnatal development, and decreases proapoptotic Bax and Bad expression at later developmental stages.

Identification of a new gene (rat TM6P1) encoding a fasting-inducible, integral membrane protein with six transmembrane domains.

We describe the isolation of a new gene that encodes a membrane-integrated protein with six transmembrane domains, termed TM6P1. A 403-bp expressed sequence tag was isolated from fasted rat liver subtracted cDNA library, and its full-length cDNA is 1482 bp long. It contains an open reading frame of 816 bp and is predicted to encode a 271-amino acid protein with a deduced mass of 29520 Da. A sequence homology search failed to show significant correspondence to any known protein in the databank. TM6P1 has six highly hydrophobic domains that are predicted to be transmembrane helices. Consistent with this prediction, the TM6P1-EGFP fusion protein was shown to localize to the plasma membrane. TM6P1 mRNA is widely expressed in rat tissues, with placenta and liver being the most abundant sites. Fasting increased TM6P1 mRNA nearly two-fold in liver. Taken together, our data suggest that TM6P1 is a unique new membrane integral protein that might have a function important during fasting-induced catabolism.

Physiological differences in insulin-like growth factor binding protein-1 (IGFBP-1) phosphorylation in IGFBP-1 transgenic mice.

Insulin-like growth factor binding protein (IGFBP)-1 has been shown to alter cellular responses to insulin-like growth factor 1 (IGF-1). Human IGFBP-1 undergoes serine phosphorylation, and this enhances both its affinity for IGF-1 by six- to eightfold and its capacity to inhibit IGF-1 actions. To investigate the physiological role of IGFBP-1 in vivo, transgenic mice have been generated using either the human IGFBP-1 or rat IGFBP-1 transgene. Both lines of mice expressed high concentrations of IGFBP-1 in serum and tissues; however, human IGFBP-1 transgenic mice did not show glucose intolerance and exhibited no significant intrauterine growth retardation, whereas rat IGFBP-1 transgenic mice showed fasting hyperglycemia and intrauterine growth restriction. The aim of this study was to investigate the physiological differences in the phosphorylation state of human IGFBP-1 and rat IGFBP-1 in these transgenic mice. The phosphorylation status of IGFBP-1 in transgenic mouse serum was analyzed by nondenaturing PAGE. Almost all of the IGFBP-1 in serum from the human IGFBP-1 transgenic mice was present as a nonphosphorylated form. Most of the rat IGFBP-1 in the serum of the mice expressing the rat IGFBP-1 was phosphorylated. Immunoprecipitation showed that mouse hepatoma (Hepa 1-6) cells (exposed to [32P]H3PO4) secrete 32P-labeled IGFBP-1. When the human IGFBP-1 transgene was transfected into Hepa 1-6 cells, all of the IGFBP-1 was secreted in the nonphosphorylated form. However, when the rat IGFBP-1 transgene was transfected into these cells, phosphorylated forms of IGFBP-1 were secreted. To confirm this result, the mouse hepatoma cell protein kinase was partially purified. This kinase activity phosphorylated mouse and rat IGFBP-1 in vitro, but it did not phosphorylate human IGFBP-1. Scatchard analysis showed that the affinity of phosphorylated rat IGFBP-1 for IGF-1 was 3.9-fold higher than that of nonphosphorylated human IGFBP-1. We conclude that the mouse IGFBP-1 kinase activity cannot phosphorylate human IGFBP-1, whereas it can phosphorylate rat IGFBP-1. The phosphorylation state of human IGFBP-1 may account for part of the phenotypic differences noted in the two studies of transgenic mice, and it is an important determinant of the capacity of human IGFBP-1 to inhibit IGF-1 actions in vivo.

Serum somatomedin-C concentrations in a rabbit model of diabetic pregnancy.

We have developed and validated a method for measuring immunoreactive somatomedin-C (Sm-C) in serum of rabbits, and have shown that during midgestation (11-26 days; gestation = 31 days) serum Sm-C concentrations are higher in normal pregnant animals than in pregnant diabetic animals. Sm-C concentrations in the serum of 28-day gestation fetuses of diabetic rabbits (3.14 +/- 0.25 U/ml) were significantly higher than in the fetuses of nondiabetic rabbits (2.31 +/- 0.23 U/ml; P less than 0.05). Fetuses from litters of the most severely hyperglycemic diabetic mothers (glucose greater than 250 mg/dl) had higher serum Sm-C (3.66 +/- 0.41 U/ml) than those of mothers who were mildly hyperglycemic (2.71 +/- 0.2 U/ml). Although these differences were not statistically significant, fetuses from the former litters accounted in great part for the difference between the fetuses of diabetic and normal pregnancy. The diabetes-related increment in Sm-C does not appear to be due to insulin, since fetal insulin concentrations were not different between the normal and diabetic litters (normal, 50.0 +/- 3.6 microU/ml versus diabetic, 49.6 +/- 7.6 microU/ml). Despite their elevation in serum Sm-C, fetuses from litters of diabetic rabbits were growth retarded in weight (26.8 +/- 6.9 g and 33.8 +/- 6.9 g, diabetic versus normal pregnancy; P less than 0.05) and in length (7.9 +/- 0.7 cm and 8.6 +/- 0.7 cm, diabetic versus normal pregnancy; P less than 0.025). We speculate that these discrepancies between growth and Sm-C might be secondary to the toxic effects of glucose on embryonic growth and that later in gestation, the excessive energy provided to the fetus might stimulate Sm-C synthesis.

Alterations in the synthesis of a fibroblast surface associated 35 K protein modulates the binding of somatomedin-C/insulin-like growth factor I.

Human fibroblasts, a cell type that is used extensively to determine the pleiotypic effects of the insulin-like growth factors, have been shown to secrete a 35K protein that binds somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) but not insulin. This 35 K protein is associated with the fibroblast surface and following transfer to the surface of cell types that do not have this protein on their surfaces, it alters the binding of radiolabeled Sm-C/IGF-I. In this study human fibroblast monolayers that were incubated with cyclohexamide (50 micrograms/ml) for 14 h at 37 C had no detectable 35 K protein on their cell surface, but type I Sm-C/IGF-I receptors were still present. Loss of the 35 K protein was associated with 60-70% increase in binding of Sm-C/IGF-I to type I receptors. The relative affinity of the type I receptor for Sm-C/IGF-I was apparently increased because unlabeled Sm-C/IGF-I (12 ng/ml) competitively displaced 63% of radiolabeled Sm-C/IGF-I after cycloheximide exposure, whereas in cultures not exposed to cycloheximide [125I]Sm-C/IGF-I binding was increased by 11%. Coincubation of fibroblast conditioned media containing the 35 K protein with cycloheximide-treated fibroblast monolayers resulted in restoration of the paradoxical increase in Sm-C/IGF-I binding and loss of sensitivity to competition by unlabeled Sm-C/IGF-I. Exposure of suspended fibroblasts, which do not have 35 K on their cell surface, to media conditioned by fibroblast monolayers also induced both of these changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor-I (IGF-I) antisense oligodeoxynucleotide mediated inhibition of DNA synthesis by WI-38 cells: evidence for autocrine actions of IGF-I.

Insulin-like growth factor-I (IGF-I) is elaborated into culture medium by WI-38 cells, a human embryonic lung fibroblast cell line, and may participate in the autocrine stimulation of DNA synthesis. We have confirmed the expression of IGF-I by these cells and documented that they express the type 1 IGF receptor and a number of IGF-binding proteins. In situ hybridization histochemistry demonstrated relatively uniform expression of IGF-I and type 1 IGF receptor transcripts among WI-38 cells. To determine whether WI-38-synthesized IGF-I exerted mitogenic effects, a 15-base oligodeoxynucleotide complementary to the 5'IGF-I mRNA sequence (IGF-I AS-Oligo), including the translation start site, was incubated with cultured cells in an attempt to inhibit IGF-I synthesis. The IGF-I AS-Oligo was stable in cell culture, formed intracellular duplexes with IGF-I mRNA, and at 2 microM reduced IGF-I in conditioned medium by 83%. The IGF-I AS-Oligo also inhibited [3H]thymidine incorporation into DNA in a dose-dependent fashion (by 77% at 2 microM and by 95% at 20 microM). This reduction in DNA synthesis was prevented when the medium was supplemented with 100 ng/ml IGF-I. The oligomer also decreased the abundance of IGF-binding proteins in conditioned medium. The IGF-I AS-Oligo appears to exert its effects by blocking IGF-I mRNA translation, rather than blocking transcription or initiating RNase-H activity, because the abundance of IGF-I transcripts was not decreased in its presence. These findings confirm an essential role for IGF-I in WI-38 cell DNA synthesis and are consistent with autocrine actions by WI-38 cell IGF-I.

Growth hormone dependence of somatomedin-C/insulin-like growth factor-I and insulin-like growth factor-II messenger ribonucleic acids.

The GH dependence of somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) and insulin like growth factor II (IGF-II) mRNAs was investigated by Northern blot hybridizations of polyadenylated RNAs from liver, pancreas, and brain of normal rats, untreated hypophysectomized rats, and hypophysectomized rats 4 h or 8 h after an ip injection of human GH (hGH). Using a 32P-labeled human Sm-C/IGF-I cDNA as probe, four Sm-C/IGF-I mRNAs of 7.5, 4.7, 1.7, and 1.2 kilobases (kb) were detected in rat liver and pancreas but were not detectable in brain. In both liver and pancreas, the abundance of these Sm-C/IGF-I mRNAs was 8- to 10-fold lower in hypophysectomized rats than in normal rats. Within 4 h after injection of hGH into hypophysectomized animals, the abundance of liver and pancreatic Sm-C/IGF-I mRNAs was restored to normal. A human IGF-II cDNA was used as a probe for rat IGF-II mRNAs which were found to be very low in abundance in rat liver and showed no evidence of regulation by GH status. In pancreas, IGF-II mRNA abundance was below the detection limit of the hybridization procedures. The brain contained two IGF-II mRNAs of 4.7 and 3.9 kb that were 5-fold lower in abundance in hypophysectomized rats than in normal rats. These brain IGF-II mRNAs were not, however, restored to normal abundance at 4 or 8 h after ip hGH injection into hypophysectomized animals. To investigate further, the effect of GH status on abundance of Sm-C/IGF-I and IGF-II mRNAs in rat brain, a second experiment was performed that differed from the first in that hypophysectomized rats were given an injection of hGH into the lateral ventricle (intracerebroventricular injection) and a rat Sm-C/IGF-I genomic probe was used to analyze Sm-C/IGF-I mRNAs. In this experiment, a 7.5 kb Sm-C/IGF-I mRNA was detected in brain polyadenylated RNAs. The abundance of the 7.5 kb mRNA was 4-fold lower in hypophysectomized rats than in normal rats and was increased to 80% of normal within 4 h after icv administration of hGH to hypophysectomized animals. As in the first experiment, the abundance of the 4.7 and 3.9 kb brain IGF-II mRNAs was lower than normal in hypophysectomized rats. Brain IGF-II mRNAs were increased to 50% of normal in hypophysectomized rats given an icv injection of hGH but within 8 h after the injection rather than at 4 h as with Sm-C/IGF-I mRNAs.

Insulin-like growth factor I protects oligodendrocytes from tumor necrosis factor-alpha-induced injury.

Tumor necrosis factor-alpha (TNF-alpha) has been causally implicated in several demyelinating disorders, including multiple sclerosis. Because insulin-like growth factor I (IGF-I) is a potent stimulator of myelination, we investigated whether it can protect oligodendrocytes and myelination from TNF-alpha-induced damage using mouse glial cultures as a model. Compared with controls, TNF-alpha decreased oligodendrocyte number by approximately 40% and doubled the number of apoptotic oligodendrocytes and their precursors. Addition of Boc-aspartyl(Ome)-fluoromethyl ketone (BAF), an inhibitor of interleukin-1beta converting enzyme (ICE)/caspase proteases, blocked TNF-alpha-induced reductions in oligodendrocytes, indicating that the TNF-alpha-induced reduction in oligodendrocytes is, at least in part, due to apoptosis, and that ICE/caspases are one of TNF-alpha action mediators. Simultaneous addition of IGF-I to TNF-alpha-treated cultures negated these TNF-alpha effects nearly completely. Furthermore, IGF-I promoted oligodendrocyte precursor proliferation and/or differentiation in TNF-alpha-treated cultures. To analyze TNF-alpha and IGF-I actions on oligodendrocyte function, we measured the abundance of messenger RNAs (mRNAs) for two major myelin-specific proteins, myelin basic protein (MBP) and proteolipid protein (PLP). While TNF-alpha decreased MBP and PLP mRNA abundance by 5- to 6-fold, IGF-I abrogated TNF-alpha-induced reductions in a dose- and time-dependent manner. The changes in MBP and PLP mRNA abundance could not be completely explained by the changes in oligodendrocyte number, indicating that myelin protein gene expression is regulated by both TNF-alpha and IGF-I. These data support the hypothesis that TNF-alpha can mediate oligodendrocyte and myelin damage, and indicate that IGF-I protects oligodendrocytes from TNF-alpha insults by blocking TNF-alpha-induced apoptosis, and by promoting oligodendrocyte and precursor proliferation/differentiation and myelin protein gene expression.

Somatomedin-C stimulates glycogen synthesis in fetal rat hepatocytes.

The effects of somatomedin-C/insulin-like growth factor I (Sm-C) on glycogen metabolism in cultured hepatocytes from 20-day-old rat fetuses have been examined and compared with the effects of insulin. Sm-C (25-375 ng/ml; 3.25-50 nM) stimulated dose-dependent increases in [14C]glucose incorporation into glycogen (14.4-72.9%; P less than 0.001) and total cell glycogen content (10.6-34.3%; P less than 0.01). Maximal stimulation of glycogen synthesis by Sm-C occurred at 2-4 h of incubation. Insulin (10 nM to 10 microM) also stimulated [14C]glucose incorporation but its potency was only 1/20th that of Sm-C. The time course of stimulation of glucose incorporation by insulin was identical to that of Sm-C, the dose-response curves of the two hormones were parallel, and the maximal effects of insulin were not enhanced by simultaneous exposure of cells to Sm-C. These findings suggest that Sm-C and insulin stimulate glycogenesis in fetal liver through similar or identical mechanisms. Since the potency of Sm-C was 20 times greater than that of insulin, the glycogenic action of insulin in fetal liver may be mediated through binding to a hepatic receptor which also binds Sm-C. In addition to having mitogenic effects on fetal tissues, Sm-C may have direct anabolic effects on fetal carbohydrate metabolism.

Affinity labeled somatomedin-C-binding proteins in rat sera.

We have developed an affinity labeling technique that uses disuccinimidyl suberate to covalently cross-link [125I]somatomedin-C (Sm-C) to specific binding proteins in rat serum. Normal rat serum contains four major classes of intensely labeled [125I]Sm-C-binding protein complexes which are sensitive to competition with unlabeled Sm-C with relative molecular masses of 95, 49, 36-33, and 26-23 K. In addition, less intensely labeled complexes are observed migrating between 175 and 115 K. Of the Sm-C binding complexes observed in normal serum, hypophysectomized (hypox) serum contains only an intensely labeled 36-33-K complex and a faint 49-K complex. Chronic administration of ovine (100 micrograms, ip, daily) to hypox rats induces the 95-K complex and possibly complexes between 175-115 K. With increasing duration of treatment, these complexes as well as the 49-K complex appear to increase in intensity. Binding proteins in both hypox and normal sera do not appear to distinguish between Sms, since both unlabeled Sm-C and multiplication-stimulating activity were equally potent in competing with [125I]Sm-C for binding. This affinity labeling technique appears to be a useful investigative tool to study the physiology and structure of Sm-binding proteins.

Effect of maternal fasting on fetal growth, serum insulin-like growth factors (IGFs), and tissue IGF messenger ribonucleic acids.

Pregnant rats were fasted or allowed access to ad libitum feeds for the last 3 days of gestation to determine if the fetal growth retardation that results from maternal nutrient deprivation correlates with reductions in serum insulin-like growth factor-I (IGF-I) and IGF-II. In addition, IGF-I and IGF-II mRNA concentrations in liver and lung were measured by specific solution hybridization assays to determine if changes in steady state levels of mRNA correlate with changes in serum values. Fetal serum IGF-I concentrations were 30% lower in fasted than in control fetuses, although fasting did not significantly reduce the abundance of IGF-I mRNA in their livers or lungs. Serum IGF-I concentrations in the fasted dams were 34% lower than those in controls. IGF-I mRNA concentrations in the livers and lungs of the fasted dams were also lower than those in controls and correlated with serum IGF-I values (liver: r = 0.833; P less than 0.001; lung: r = 0.610; P less than 0.05). Therefore, whereas IGF-I appears to be transcriptionally regulated by fasting in dams, regulation of circulating IGF-I in fetuses may occur at a post-transcriptional step. Serum IGF-II and liver IGF-II mRNA concentrations were much higher than IGF-I levels in the fetuses and were not influenced by maternal fasting. Dam serum IGF-II concentrations were low compared to those in fetal serum and also were not reduced by fasting. We conclude that one mechanism by which maternal malnutrition causes intrauterine growth retardation is through decreased expression of IGF-I. On the other hand, short term nutrient restriction does not appear to be a regulator of IGF-II during either fetal or adult life.

Regulation of insulin-like growth factor (IGF) binding proteins in transgenic mice with altered expression of growth hormone and IGF-I.

The insulin-like growth factors (IGFs) are present in extracellular fluids bound to specific, high affinity IGF binding proteins (IGFBPs). IGFBPs are believed to mediate IGF transport to tissues and to modulate their actions on target cells. To determine whether IGF-I can modulate IGFBP concentrations in blood and to distinguish the effects of IGF-I from those of GH, we assessed serum IGFBP concentrations in four genotypically distinct groups of sibling transgenic (Tg) mice that differed in respect to their expression of IGF-I and GH. This unique physiological situation was created by crossing IGF-I Tg mice to GH-deficient, dwarf mice in whom somatotrophs were genetically ablated by the expression of a diphtheria toxin transgene in the somatotrophs. Because both Tg mouse lines are hemizygous for their respective transgene, progeny of the cross differ genotypically, according to whether or not they carry one or both transgenes, and phenotypically in regard to their relative expression of IGF-I and GH. GH-deficient mice showed a 15.7-fold decrease in serum IGF-I and a 5.5-fold decrease in serum IGFBP-3, but no change in a serum doublet band of 29,000 to 34,000 Mr, as assessed by ligand blotting. When IGF-I was expressed in the GH-deficient mice, serum levels of IGF-I and IGFBP-3 were 69% and 64% of those in normal sera, respectively. The 29,000 to 34,000 Mr doublet bands also increased. The ternary 150 kilodalton IGF-IGFBP complex, however, was not restored, presumably because IGF-I has no influence on the expression of the acid-labile subunit in this complex. In mice with IGF-I overexpression, serum IGFBP-3 was increased 2.1-fold and the sum of the 29,000 to 34,000 doublet bands was increased 2.9-fold. Immunoblotting showed that the changes in the 29,000 to 34,000 Mr forms observed by ligand blotting appeared to be predominantly due to changes in IGFBP-2. The results show that IGF-I can induce IGFBP-3 and IGFBP-2 independently of GH and that IGF-I is a major controller of these binding proteins.