Insulin-like Growth factor-I Production Research Articles

Effect of Bovine Somatotropin on the Distribution of Immunoreactive Insulin-Like Growth Factor-I in Lactating Bovine Mammary Tissue

The distribution pattern of immunoreactive insulin-like growth factor-I in normal lactating bovine mammary tissue and in tissue obtained after bovine somatotropin treatment was determined by indirect immunofluorescence. In normal tissue, insulin-like growth factor-I immunoreactivity was observed almost exclusively associated with stromal elements. Intralobular stromal cells, small blood vessels, and capillaries all expressed moderate to high immunoreactivity. In contrast, mammary epithelial cells displayed only sparse cytoplasmic immunoreactivity. Immunoreactive material was also present in the periductular connective tissue area, possibly associated with the basal plasma membrane of epithelial cells. Somatotropin treatment of animals resulted in elevated serum insulin-like growth factor-I concentrations and altered the distribution of insulin-like growth factor-I-stainable material in mammary tissue. After somatotropin treatment, immunoreactivity was still detected in mammary stroma; however, prominent staining was also observed in the cytoplasm of mammary epithelial cells. Given the possible role of insulin-like growth factor-I in the regulation of bovine mammary epithelial cell growth and function, our findings raise the possibility that somatotropin may induce insulin-like growth factor-I production in mammary tissue, or other tissues, to influence indirectly the growth or function of the epithelial cells. This offers a possible mechanism for bovine somatotropin stimulation of lactation.

The role of growth hormone in diabetes mellitus.

The insulin and growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis are two endocrine systems that are interlinked at many levels. GH is one of the glucose counter-regulatory hormones, rising in response to hypoglycaemia, it has both intrinsic hyperglycaemic actions and causes insulin resistance. Both IGF-I and its receptor have high structural and functional homology to insulin and its receptor. Insulin can regulate IGF-I production, acting on the GH receptor or at a post-receptor site. Conversely IGF-I is thought to have a permissive effect on the pancreatic insulin response to glucose. Growth is compromised in poorly controlled diabetic children; however, a causal link with altered GH/IGF-I levels has not been proven. Insulin-dependent diabetes clearly causes derangements in the GH/IGF-I axis. In poorly controlled diabetics GH levels are invariably raised whilst normal or low levels of IGF-I are found, indicating a dissociation between the two factors. Altered IGF-binding protein levels are also found, with high levels of small binding protein and low levels of large binding protein. These derangements are probably the result of interactions at many levels although the exact mechanisms are not fully understood. Raised GH levels could result from altered hypothalamic/pituitary control or reduced feedback inhibition. The latter could, in turn, result from low IGF-I levels, reduced availability of IGF-I to relevant receptors or increased levels of inhibitors (possibly the small binding protein). Low IGF-I levels could be directly due to deficient insulin levels or simply to lack of available circulating binding protein. Alternative or altered molecular forms of circulating GH in diabetes seem unlikely on present evidence. That GH has an effect on glycaemic control is most evident from the abnormal glucose tolerance seen in acromegalics, but is also seen with physiological GH variations such as during the pubertal growth spurt. In diabetics the derangements to the GH/IGF-I axis, caused by poor metabolic control, leads to aggravation of the metabolic problems. Altered GH/IGF-I levels have been implicated in the long-term complications associated with diabetes, and whilst GH/IGF-I are not essential for the early changes involved in these complications they may still play an important role in their development, especially proliferative retinopathy.

Porcine insulin-like growth factor-I (pIGF-I): complementary deoxyribonucleic acid cloning and uterine expression of messenger ribonucleic acid encoding evolutionarily conserved IGF-I peptides.

In order to facilitate studies of insulin-like growth factor-I (IGF-I) expression during the pregnancy-associated development of uterus and mammary gland in the pig model, we have isolated several cDNA clones corresponding to porcine IGF-I (pIGF-I) mRNA. Sequence analysis of two cDNA fragments (sigf. 2 and sigf. 3) revealed an open reading frame encoding in order a putative 25 amino acid (aa) hydrophobic leader peptide, the mature (processed) 70 aa pIGF-I peptide and a 35 aa carboxy-terminal extension (E) peptide. The deduced aa sequence of the pIGF-I peptide is identical to human and bovine IGF-I but differs from that of rat and mouse at three and four residues, respectively. The sequences of the amino- and carboxy-terminal IGF extension peptides are also highly conserved among these species. Northern analysis using sigf. 3 as a probe revealed multiple IGF-I mRNAs (including species of 8000, 2300, and 1200 nucleotides in length) in uteri of pregnant pigs. Highest levels of the uterine IGF-I mRNAs were found at early pregnancy, when increased levels of immunoreactive tissue IGF-I were also observed. Mammary levels of IGF-I mRNAs and protein were considerably lower than that observed for uterus at the same time period. Thus, uterine production of IGF-I appears to be especially significant during early pregnancy in the pig when uterine growth, elevated IGF-I in uterine fluids, and rapid embryonic development are observed.

Growth factors regulate immunoreactive insulin-like growth factor-I production by cultured porcine granulosa cells.

The effects of various growth factors on the production of immunoreactive insulin-like growth factor I (iIGF-I) in short term (3-day) cultures of porcine granulosa cells was investigated. Epidermal growth factor (EGF) was shown to be a potent dose-dependent stimulator of iIGF-I production, achieving a 3.6-fold stimulation at a dose of 10 ng/ml. Transforming growth factor-alpha (10 ng EGF equivalents/ml) was also stimulatory. Platelet-derived growth factor (10 ng/ml) had no effect of its own, but enhanced EGF-stimulated iIGF-I production. The acidic and basic fibroblast growth factors (100 ng/ml) had no effect alone or in combination with EGF. Transforming growth factor-beta (10 ng/ml) had no effect of its own, but inhibited EGF-stimulated iIGF-I production. The interactive effects of EGF and FSH (200 ng/ml) on iIGF-I production were investigated in short term and longer term (7-day) cultures. In short term cultures under conditions optimized for EGF-dependent iIGF-I production, FSH had no effect of its own and inhibited EGF action. Conversely, in longer term cultures optimized for FSH-dependent iIGF-I production, EGF had no effect of its own and inhibited FSH action. Thus IGF production by cultured porcine granulosa cells is regulated in a complex manner and is highly dependent on the culture conditions. Our results suggest that IGF production in the ovary may also be regulated in a complex manner which is dependent on the developmental state of the follicle.

Insulin-like growth factor-I as an amplifier of follicle-stimulating hormone action: studies on mechanism(s) and site(s) of action in cultured rat granulosa cells.

The ovarian granulosa cell has recently been shown to be a site of insulin-like growth factor-I (IGF-I) production, reception, and action. In large measure, IGF-I action (in the rat) appears contingent upon its ability to synergize with FSH, a major promoter of granulosa cell differentiation. It is the objective of the in vitro studies reported herein to elucidate the cellular mechanism(s) whereby IGF-I amplifies FSH hormonal action, placing special emphasis on the potential role of the putative intracellular second messenger cAMP in this regard. Basal FSH binding (115.7 +/- 2.1 fmol/mg cell protein) to rat granulosa cells cultured under serum-free conditions remained unchanged after 72 h of treatment with IGF-I (50 ng/ml) by itself (107.1 +/- 1.0 fmol/mg cell protein). In contrast, treatment with FSH (20 ng/ml) resulted in a significant (P less than 0.05) decrease in FSH binding capacity (but not affinity) relative to controls in either the absence or presence of IGF-I. Whereas treatment with FSH resulted in a substantial increase in forskolin-stimulatable adenylate cyclase activity (10 +/- 1.7% conversion of [3H] ATP to [3H]cAMP), concurrent treatment with IGF-I resulted in 2.2-fold enhancement of FSH action. This IGF-I effect proved dose dependent with an apparent median effective dose of 3.6 +/- 0.8 ng/ml, a concentration in keeping with its granulosa cell receptor binding affinity. Significantly, however, IGF-I proved capable of enhancing Bt2cAMP-stimulated progesterone accumulation suggesting that IGF-I may be also acting at site(s) distal to cAMP generation. Taken together, these and previous studies indicate that nanomolar concentrations of exogenously added IGF-I may be interacting with the FSH transduction signal at multiple cellular site(s) to effect amplification of FSH action.

Mitogenic effects of growth hormone in cultured human fibroblasts. Evidence for action via local insulin-like growth factor I production.

We examined human growth hormone's (hGH) effect on mitogenesis in cultured human fibroblasts, and the role of local insulin-like growth factor I (IGF-I). With 0.5% human hypopituitary serum (HPS), hGH increased thymidine incorporation (TI) over serum-free medium dose responsively, with half-maximal effect at 10 ng/ml (0.5 nM) (hGH 127 +/- 8.8%; IGF-I 107 +/- 1.7% [SEM]) (n = 10). Similarly, with 0.5% HPS, hGH and IGF-I increased cell replication by 172 +/- 8.2% and 169 +/- 25%, respectively (n = 4). Specific IGF-I monoclonal antibody (Sm1.2) dose dependently blunted TI stimulated by 10 ng/ml hGH or IGF-I (at 1:1000, 38 +/- 6.5% and 30 +/- 14% reduction, respectively). Sm1.2 also reduced cell replication by both 10 ng/ml hGH and IGF-I, respectively, to 32% and 42% of stimulated values. Dexamethasone (0.1 microM) synergistically enhanced TI by both IGF-I and hGH. A 28-h time course for TI showed that hGH stimulated a similar peak to IGF-I, lagging in its effect by 4-10 h. We have provided further evidence that hGH stimulates growth of cultured human fibroblasts via local IGF-I production, consistent with IGF-I's paracrine-autocrine role.

Stimulatory effects of insulin-like growth factors on DNA synthesis in the human embryonic cornea

10- to 12-week-old human embryonic eye globes were microdissected so that a passage was opened between the outer environment and the anterior chamber which rendered free access of tissue culture medium to the endothelial cell monolayer. The dissected eye globes were maintained in organ culture for 24 h in the continuous presence of tritiated thymidine. Cross sections were cut through the whole eye globes and subjected to autoradiographic analysis in order to estimate the mitogenic response of human embryonic corneal endothelial cells to externally supplied growth factors and hormones. It was found that the corneal endothelial cells could be stimulated to initiate DNA synthesis by exposure to insulin-like growth factor I (IGF-I). The thymidine-labelling index doubled after IGF-I supplementation. Northern blot analysis revealed the abundant presence of IGF-II transcripts in the posterior eye. In contrast, the anterior portion of the eye, including the cornea, contains barely detectable levels of IGF-II transcripts. IGF-I transcripts were detected in both parts of the eye at much lower concentrations than those for IGF-II. No insulin transcripts were found. These results demonstrate that mRNA for both IGF-I and IGF-II is present in the late first trimester eye. The observed stimulatory effects of IGF-I in organ culture suggest that local production of IGF-I and IGF-II may stimulate cell proliferation in vivo.

Identification of insulin-like growth factor-I and its receptors in the rat testis.

As part of a study of the testicular production and action of insulin-like growth factor-I (IGF-I), adult rat testes were extracted with acidified methanol, yielding an immunoreactive IGF-I fraction corresponding in size to human IGF-I. The mean IGF-I content (+/- SEM) of testes weighing approximately 1.1 g was 51.5 +/- 5.6 ng/testis, and was not due to serum contamination. After a 3-day fast testicular IGF-I decreased by 80%, whereas serum IGF-I levels declined by 90%. Testicular homogenates and isolated Leydig cells were shown to contain specific IGF-I receptors, Ka = 2 X 10(9) M-1, with 10% IGF-II cross-reactivity. The concentration of these receptors was 2 pmol binding sites per testis, or 3.3 fmol per 10(6) Leydig cells. However IGF-I at 250 ng/ml had no effect on basal or hCG-stimulated testosterone production by isolated Leydig cells, measured over 3 h. Although an effect of IGF-I over longer incubation periods cannot be excluded, it is also possible that testicular IGF-I has a mitogenic role, rather than acting on differentiated testicular functions.

Omeprazole, a specific inhibitor of H +-K +-ATPase, inhibits prostaglandin stimulated bone resorption in vitro

PTH ameliorates acidosis-induced adverse effects in skeletal growth centers: The PTH–IGF-I axis.Chronic metabolic acidosis (CMA) exerts profound adverse effects on bone metabolism thereby leading to impaired skeletal linear growth. We have recently shown that CMA in vitro causes distinct morphological changes in skeletal growth centers along with inhibition of endochondral differentiation. In addition, CMA causes an end organ resistance to the anabolic effects of growth hormone (GH) and locally produced insulin-like growth factor-I (IGF-I) in skeletal growth centers. Given the effects of parathyroid hormone (PTH) and PTH related protein (PTHrP) on the development of cartilaginous bone, we sought to determine whether PTH has any effects on the changes induced by CMA in skeletal growth centers. The interaction between PTH and IGF-I in growth centers during neutral or acidic conditions were studied specifically.An in vitro organ culture system using the murine mandibular condyle was employed as a model for endochondral active growth center. Condyles from six-day-old mice were cultured in BGJb medium of either neutral pH (pH ∼7.4) or acidic pH (pH ∼7.15) in the presence or absence of 10-10 mol/L1-34 PTH. After 24, 48, 72 and 96 hours of culture, the condyles were washed, fixed in formaldehyde, and processed for paraffin embedding. Histologic markers of the growth center were assessed. In addition, the protein level and mRNA expression for various markers of cartilage differentiation were evaluated by immunohistochemistry and in situ hybridization, respectively. The abundance and expression levels of IGF-I and IGF-I receptor (IGF-I-R) were assessed also.Following incubation for 72 hours in acidic conditions, there was a marked attenuation of the chondroblastic zone, suggesting a defect in the process of cellular differentiation. Acidosis also down-regulated endochondral differentiation markers (cartilage specific proteoglycans, collagen type II). This was accompanied by a reduction in the expression of IGF-1, IGF-1 receptor and PTH receptors. PTH (10-10 mol/L) added to acidic cultures prevented the adverse effects of CMA on endochondral differentiation and increased the overall condylar growth, when compared to acidic conditions without PTH. PTH also up-regulated its own receptor in control as well as during acidic conditions, and increased the expression levels of IGF-1 and IGF-1 receptor in the acidotic condyle. Acidosis increased the expression of IGF-I binding protein-4 (IGFBP-4, an inhibitor of IGF-I activity), whereas coincubation with PTH during acidic conditions abrogated the up-regulation of IGFBP-4. Addition of a neutralizing antibody to IGF-I-R during PTH treatment under acidic conditions resulted in the abrogation of the ameliorative effect of PTH on endochondral differentiation. The protein kinase C (PKC) signaling pathway was modulated negatively by CMA. However, PTH activated PKC-alpha under both control and acidic conditions. The phorbol ester, PMA (phorbol 12-myristate 13-acetate), a PKC activator, mimicked the effect of PTH on chondrocyte differentiation.Parathyroid hormone at low concentration stimulates the differentiation and proliferation of cartilage cells and prevents the suppressive effect of acidosis on endochondral bone differentiation and on the IGF-I/IGF-I-R system in skeletal growth centers. Increased local production of IGF-I by PTH, which takes place even during acidotic conditions, mediates, at least in part, the ameliorative effect of PTH. Protein kinase C is probably one of the signaling pathways mediating the salutary effects of PTH on chondrocyte differentiation in growth centers. This study lends further credence to the notion that under certain conditions, PTH or PTHrP can exert anabolic effects in the skeleton. These findings may be of clinical-therapeutic significance in children and patients with CMA.

Developmental patterns of insulin-like growth factor-I and -II synthesis and regulation in rat fibroblasts.

Insulin-like growth factor-I (IGF-I) and IGF-II are chemically similar polypeptides with related receptor specificity and in vitro biological activity1. IGF-I and IGF-II have a 62% amino acid sequence identity and are 38–48% homologous with the A and B domains of human proinsulin. They can be distinguished by specific radioimmunoassays, appear to have different patterns of hormonal regulation, and presumably serve different in vivo biological functions. Plasma IGF-I levels reflect circulating concentrations of pituitary growth hormone (GH), and exogenous IGF-I substitutes for GH in inducing somatic and skeletal growth in GH-deficient rats2. Several recent observations in rats have suggested a possible role for IGF-II in fetal growth. IGF-II levels are 20- to 100-fold higher in fetal rat serum than in maternal serum and decline within days after birth3. IGF-I exhibits the reciprocal developmental pattern—low in the early neonate, rising to adult levels by approximately 4 weeks of age4,5. We recently reported6 that cultured rat embryo fibroblasts synthesize large amounts of multiplication-stimulating activity (MSA or rIGF-II), the rat homologue of human IGF-II7. We now demonstrate that fibroblast cultures established from rats 2 to 50 days of age mimic the developmental switch from IGF-II to IGF-I production observed in serum. In addition, we show that placental lactogen (PL) but not GH stimulates IGF-II synthesis in fetal fibroblasts. By contrast, both GH and PL stimulate IGF-I synthesis in fibroblasts from older rats with no effect on IGF-II synthesis. Stimulation of IGF-II synthesis in fetal fibroblasts by PL provides a mechanism whereby PL may regulate fetal growth.