Differential effects on proliferation of GH and IGFs in sea bream (Sparus aurata) cultured myocytes

Primary culture of gilthead sea bream skeletal muscle cells was used to examine the effects of growth hormone (GH) and insulin-like growth factors (IGFs) in fish muscle proliferation and growth. Proliferation was measured as the percentage of positive cells expressing the proliferating cell nuclear antigen (PCNA) analyzed by immunocytochemistry. First, the effects of GH from two different origins (mammals and fish) were tested. GH from human (hGH) did not stimulate proliferation except at 3 h at the dose of 1 nM. On the other hand, sea bream GH (sbGH) significantly stimulated proliferation, without differences between the three incubation times studied (3, 6, and 18 h), at the dose of 10 nM, demonstrating that the homologous hormone has a more potent effect. In addition, the results with the IGFs indicated that both peptides, IGF-I and IGF-II significantly stimulated proliferation of sea bream myocytes, but IGF-II showed higher effects than IGF-I, and even than those of sbGH. Finally, the combinations of peptide treatments (GHs with IGFs) indicated that IGF-I has higher effects on proliferation when it is combined with GHs compared with IGF-I alone, while IGF-II has similar effects alone or combined with either GH. These results indicate that IGF-II may have an important role on muscle proliferation that appears to be independent of GH. On the contrary, IGF-I seems to play a synergistic action with GH stimulating myocyte proliferation.     

Differential regulation of insulin-like growth factor-I (IGF-I) and IGF-II release from cultured neonatal mouse calvaria by parathyroid hormone, transforming growth factor-beta, and 1,25-dihydroxyvitamin D3

In a previous study we found that PTH stimulated bone resorption and release of insulin-like growth factor-I (IGF-I) and IGF-II from cultured neonatal mouse calvaria. Since IGF-I and IGF-II stimulate osteoblast proliferation and collagen synthesis, these results suggested that increased release of IGFs during resorption could mediate in part coupling of bone formation to bone resorption. In the present study two other osteolytic agents, transforming growth factor-beta (TGF beta) and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3 were examined for effects on IGF release from neonatal mouse calvaria. Like PTH, TGF beta stimulated resorption and increased release of IGF-I and IGF-II. 1,25-(OH)2D3, however, stimulated resorption and IGF-II release comparable to PTH, but inhibited release of IGF-I. 1,25-(OH)2D3 (0.1-100 nM) inhibited basal release of IGF-I, and 10 nM 1,25-(OH)2D3 inhibited release of IGF-I induced by PTH or TGF beta. The effects of 1,25-(OH)2D3 were specific to this vitamin D metabolite and did not occur with 25-hydroxyvitamin D3 or 24,25-(OH)2D3 at the same concentration. Calcitonin (50 mU/ml) decreased 1,25-(OH)2D3 stimulation of resorption, but did not affect 1,25-(OH)2D3 stimulation of IGF-II release and inhibition of IGF-I release. This evidence that effects of 1,25-(OH)2D3 on release of the IGFs were independent of bone resorption supports the conclusion that 1,25-(OH)2D3 modulated the production and secretion of IGF-I and IGF-II in calvarial cells. The results of this and the previous study suggest that PTH, TGF beta, and 1,25-(OH)2D3 differentially regulate mouse calvarial cell IGF-I and IGF-II production.     

Tissue-specific regulation of the growth hormone/insulin-like growth factor axis during fasting and re-feeding: Importance of muscle expression of IGF-I and IGF-II mRNA in the tilapia

The effects of prolonged nutrient restriction (fasting) and subsequent restoration (re-feeding) on the growth hormone (GH)/insulin-like growth factor (IGF) axis were investigated in the tilapia (Oreochromis mossambicus). Mean weight and specific growth rate declined within 1 week in fasted fish, and remained lower than controls throughout 4 weeks of fasting. Plasma levels of IGF-I were lower than fed controls during 4 weeks of fasting, suggesting a significant catabolic state. Following re-feeding, fasted fish gained weight continuously, but did not attain the weight of fed controls at 8 weeks after re-feeding. Specific growth rate increased above the continuously-fed controls during the first 6 weeks of re-feeding, clearly indicating a compensatory response. Plasma IGF-I levels increased after 1 week of re-feeding and levels were not otherwise different from fed controls. Plasma GH levels were unaffected by either fasting or re-feeding. No consistent effect of fasting or re-feeding was observed on liver expression of GH receptor (GH-R), somatolactin (SL) receptor (SL-R), IGF-I or IGF-II. In contrast, muscle expression of GH-R increased markedly during 4 weeks of fasting, and then declined below control levels upon re-feeding for weeks 1 and 2. Similarly, muscle expression of SL-R increased after 4 weeks of fasting, and reduced below control levels after 1 and 2 weeks of re-feeding. On the other hand, muscle expression of IGF-I was strongly reduced throughout the fasting period, and levels recovered 2 weeks after re-feeding. Muscle expression of IGF-II was not affected by fasting, but was reduced after 1 and 2 weeks of re-feeding. These results indicate that GH/IGF axis, particularly muscle expression of GH-R, SL-R and IGF-I and -II, is sensitive to nutritional status in the tilapia.     

Parathyroid hormone stimulates release of insulin-like growth factor-I (IGF-I) and IGF-II from neonatal mouse calvaria in organ culture

Effects of increased bone resorption on release of insulin-like growth factor-I (IGF-I) and IGF-II into the osteoblast microenvironment were investigated using neonatal mouse calvaria organ cultures. Release of these growth factors from calvaria into serum-free medium was quantitated using a human IGF-I RIA and human IGF-II RRA. Untreated calvaria released several-fold more IGF-II than IGF-I. PTH (from 1-12 nM) stimulated a dose-dependent increase in the release of both growth factors that correlated with increased calcium release and was sustained for up to 6 days. IGF-I and IGF-II release were maximally stimulated 5- to 10-fold and 1.5- to 2-fold, respectively, compared to untreated control values. Calcitonin inhibited PTH-stimulated resorption, but had no effect on PTH stimulation of IGF-I and IGF-II release, suggesting that PTH effects on IGF-I and IGF-II release were not dependent on resorption. Furthermore, the amounts of IGF-I and IGF-II released from calvaria during 6 days of culture were 5-fold more than the amounts of IGF-I and IGF-II present in the calvaria (bone plus cells) at the beginning of culture, suggesting that much of the IGF-I and IGF-II released was newly produced by calvaria cells. The results suggest that PTH directly stimulated calvarial osteoblasts to release IGF-I and IGF-II. Since IGF-I and IGF-II stimulate osteoblastic cell proliferation, the effect of PTH on the release of these and other growth factors may mediate coupling of bone formation to bone resorption.     

Differential effects of IGF-I, IGF-II and insulin in human preadipocytes and adipocytes--role of insulin and IGF-I receptors

We compared insulin and IGF effects in adipocytes expressing IR (insulin receptors), and preadipocytes expressing IR and IGF-IR (IGF-I receptors). Treatment of adipocytes with insulin, IGF-II or IGF-I resulted in phosphorylation of IR. Order of potency was insulin>IGF-II>IGF-I. In preadipocytes IR, IGF-IR and insulin/IGF-I hybrid receptors (HR) were detected. Treatment of preadipocytes with IGF-I and IGF-II 10(-8)M resulted in activation of IGF-IR and IR whereas insulin was more potent in activating IR, with no effect on IGF-IR. In adipocytes glucose transport was 100-fold more sensitive to insulin than to IGFs and the maximal effect was higher with insulin. In preadipocytes glucose accumulation and DNA synthesis was equally sensitive to insulin and IGFs but the maximal effect was higher with IGF-I. In conclusion, insulin and IGF-I activate their cognate receptors and IGF-I also HR. IGF-II activates IR, IGF-IR and HR. Insulin and IGF-I are partial agonists to each other's receptors.     

Insulin-like growth factors act synergistically with basic fibroblast growth factor and nerve growth factor to promote chromaffin cell proliferation.

We have investigated the effects of insulin-likegrowth factors (IGFs), basic fibroblast growth factor (bFGF), and nerve growthfactor (NGF) on DNA synthesis in cultured chromaffin cells from fetal, neonatal,and adult rats by using 5-bromo-2'-deoxyuridine (BrdUrd) pulse labeling for24 or 48 h and immunocytochemical staining of cell nuclei. After 6 days inculture in the absence of growth factors, nuclear BrdUrd incorporation wasdetected in 30% of fetal chromaffin cells, 1.5% of neonatal cells, and 0.1% ofadult cells. Addition of 10 nM IGF-I or IGF-II increased the fraction ofBrdUrd-labeled nuclei to 50% of fetal, 20% of neonatal, and 2% of adultchromaffin cells. The ED50 value of IGF-I- and IGF-II-stimulated BrdUrd labelingin neonatal chromaffin cells was 0.3 nM and 0.8 nM, respectively. In neonataland adult chromaffin cells, addition of 1 nM bFGF or 2 nM NGF stimulated nuclearBrdUrd incorporation to approximately the same level as 10 nM IGF-I or IGF-II.However, the response to bFGF or NGF in combination with either IGF-I or IGF-IIwas more than additive, indicating that the combined effect of the IGFs and bFGFor NGF is synergistic. The degree of synergism was 2- to 4-fold in neonatalchromaffin cells and 10- to 20-fold in adult chromaffin cells compared with theeffect of each growth factor alone. In contrast, the action of bFGF and NGFadded together in the absence of IGFs was not synergistic or additive. IGF-IIacted also as a survival factor on neonatal chromaffin cells and the cellsurvival was further improved when bFGF or NGF was added together with IGF-II.In conclusion, we propose that IGF-I and IGF-II act in synergy with bFGF and NGFto stimulate proliferation and survival of chromaffin cells during neonatalgrowth and adult maintenance of the adrenal medulla. Our findings may haveimplications for improving the survival of chromaffin cell implants in diseasedhuman brain.     

Developmental expression of receptors for insulin, insulin-like growth factor I (IGF-I), and IGF-II in rat skeletal muscle

Insulin and insulin-like growth factors (IGFs) stimulate responses in skeletal muscle that include effects on carbohydrate and fat metabolism, protein turnover, growth, and differentiation. To gain insight into the relative importance of insulin and IGFs at different stages of development, the expression of their specific receptors was evaluated in skeletal muscle of rats from the late fetal period through 40 weeks of age. Distinct receptors for insulin. IGF-I and IGF-II are present in crude membrane preparations and wheat germ agglutinin-purified extracts of hindlimb muscle from rats at all ages, but each of the three receptors follows a different pattern of expression during development. There is a marked predominance of IGF-II receptors in fetal muscle (80- and 55-fold more abundant than insulin and IGF-I receptors, respectively) and a rapid decline in IGF-II receptors in early postnatal life. IGF-I receptors are more abundant than insulin receptors in the term fetus, remain constant in number until approximately 4 weeks of age, and then gradually decline to adult levels. Insulin receptor number rises 2- to 3-fold postnatally, peaks at approximately 4 weeks, and decreases to levels in the adult that are slightly lower than those in the term fetus. Although binding affinities and receptor specificity did not change during development, the relatively large number of IGF-II receptors in the fetus resulted in significant binding of IGF-I to receptors for both IGF-II and IGF-I. There was a modest increase in apparent mol wt of all three receptor types during development, suggesting a change in a common pathway, such as posttranslational glycosylation. The marked changes in number and distinct patterns of expression of the insulin, IGF-I, and IGF-II receptors in muscle during development are consistent with evolving functions of the three hormones determined by alterations in both receptor number and hormone concentrations.     

Identification of a second insulin-like growth factor in a fish species.

An internal portion of insulin-like growth factor (IGF) amplified from the total cDNA of rainbow trout (Oncorhynchus mykiss) liver by a PCR was used to screen a rainbow trout liver cDNA library, and recombinant clones encoding two distinct IGFs were isolated. On the basis of a 98.7% nucleotide and 98.3% predicted amino acid identity to coho salmon IGF-I, one cDNA sequence was identified as rainbow trout preproinsulin-like growth factor I (rtIGF-I). The second cDNA sequence shared 46.1% and 43.3% identity with rtIGF-I at the nucleotide and predicted amino acid levels, respectively, and was identified as rainbow trout preproinsulin-like growth factor II (rtIGF-II). Predicted amino acid sequence comparisons of rtIGFs with those of human IGFs indicate that rtIGF-I is more similar to human IGF-I than to human IGF-II, and that rtIGF-II is more similar to human IGF-II than to human IGF-I. Southern blot analysis of rainbow trout genomic DNA probed with rtIGF-I and -II cDNA suggests that these two forms of IGF originate from separate genes. The presence of a teleost IGF-II suggests that the divergence of IGFs occurred early in vertebrate evolution.     

Cloning,characterization, and comparative activity of turbot IGF-I and IGF-II

IGF peptides belong to a complex system that is known to play a major role in the control of growth and development in mammals. Even if studies performed in nonmammalian species tend to demonstrate an important function of these molecules, use of heterologous ligands, especially in fish, partly limit our knowledge of the physiological role(s) of IGFs. We report in this study the cloning, production, and characterization in an evolved fish, the turbot Psetta maxima, of mature IGF-I and IGF-II. The deduced 68-amino-acid IGF-I and 70-amino-acid IGF-II show 75% and 74% sequence identity with their mammalian counterparts, respectively, confirming the high sequence homology observed in other species. The development of a simple and efficient system for the production and purification of both IGF-I and IGF-II in Escherichia coli was used to investigate the in vitro regulation of GH release in the turbot. Our results demonstrated for the first time in a Euteleost species that both peptides specifically inhibited GH release. Both hormones were equally potent in inhibiting GH release from dispersed pituitary cells, with maximal inhibitory effects of 92% and 91% at 1 nM doses after 12 days of culture, respectively. The biologically active recombinant turbot IGFs that we obtained will allow us to further investigate potential and perhaps the specific role(s) of these hormones in turbot as, in contrast with mammals, growth in fish is potentially continued during "adult" life.     

The effects of insulin-like growth factor-I (IGF-I), IGF-II and des(1-3)IGF-I, a potent IGF analogue, on growth hormone and IGF-binding protein secretion from cultured rat anterior pituitary cells.

The effects of insulin-like growth factor-I (IGF-I), IGF-II and des(1-3)IGF-I, a potent IGF-I analogue, on the secretion of GH and IGF-binding protein (IGFBP) from cultured rat anterior pituitary cells were measured. IGF-I and des(1-3)IGF-I stimulated GH secretion at low concentrations (maximally effective at 1 and 0.1 micrograms/l respectively) and inhibited GH secretion at higher concentrations. The half-maximal inhibitory concentrations (IC50) were approximately 20 micrograms/l and 1 microgram/l for IGF-I and des(1-3)IGF-I respectively. Thus des(1-3)IGF-I was more potent than IGF-I in these effects on GH secretion. We postulate that the increased potency of des(1-3)IGF-I in affecting GH secretion is due to decreased binding of this peptide by pituitary IGFBP compared with IGF-I. In contrast with IGF-I and des(1-3)IGF-I, IGF-II did not stimulate GH secretion at low concentrations, but did inhibit GH secretion from pituitary cells with an IC50 of approximately 20 micrograms/l. Several IGFBPs ranging in molecular mass from 22,000 to 52,000 were detected in medium conditioned by cultured anterior pituitary cells. When measured by Western-ligand blotting and competitive ligand-binding techniques, these IGFBPs exhibited decreased binding of des(1-3)IGF-I compared with IGF-I and IGF-II. The production of IGFBP by anterior pituitary cells was stimulated by the addition of IGFs to the culture medium.     

Identification and characterization of insulin-like growth factor receptors on adult rat cardiac myocytes: linkage to inositol 1,4,5-trisphosphate formation.

Cultured cardiac myocytes from adult Sprague-Dawley rats express both insulin-like growth factor-I (IGF-I) receptors and insulin-like growth factor-II/mannose 6-phosphate (IGF-II/Man6P) receptors and respond to IGF-I with a dose-dependent accumulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,4-bisphosphate [Ins(1,4)P2]. Specific binding of [125I]IGF-I to isolated membranes from cultured cardiac myocytes amounted to 1-1.2%. Binding of [125I]IGF-I was inhibited by unlabeled IGF-I at nanomolar concentrations and insulin at much higher concentrations. These data suggest that IGF-I binds to its own receptor on rat cardiac myocytes. Competitive binding studies using isolated membranes from cardiac myocytes and [125I]IGF-II showed 2-4% specific binding. Binding of [125I]IGF-II was inhibited by IGF-II and much less potently by IGF-I and insulin. Immunoglobulin G (IgG) 3637 (an IgG directed against the IGF-II/Man6P receptor) partially inhibited binding of [125I]IGF-II whereas nonimmune IgG did not. Affinity cross-linking studies with [125I]IGF-II and cardiac myocyte membranes and subsequent analysis of the ligand-receptor complex using SDS-PAGE and autoradiography showed a radiolabeled band of approximately 250 kilodalton (kDa). The formation of the [125I]IGF-II-receptor complex was inhibited by incubation with IGF-II and IgG 3637 but not by insulin or nonimmune IgG. Western blotting of protein extracts from cultured cardiac myocytes was performed using IgG 3637 and an immunoperoxidase technique for the visualization of the IGF-II/Man6P receptor protein. A specific band at 220 kDa under nonreducing conditions was detected on the blots, providing further evidence for the expression of the IGF-II/Man6P receptor by cardiac myocytes. The effect of IGFs on the accumulation of inositol phosphates was measured by HPLC analysis of perchloric acid extracts from myo-[3H]inositol-labeled cultured cardiac myocytes. IGF-I (50 ng/ml) stimulated the accumulation both of Ins(1,4,5)P3 and Ins(1,4)P2 after 30 sec by 43% and 63%. IGF-II (up to 500 ng/ml) had no significant effect on inositol phosphate accumulation under the same conditions. However, in the presence of millimolar concentrations of Man6P, IGF-II (500 ng/ml) also increased Ins(1,4,5)P3 accumulation by 59%. We conclude that cardiac myocytes from adult rats express IGF receptors and respond to IGFs with the accumulation of Ins(1,4,5)P3 and Ins(1,4)P2. This effect seems to be mediated by an IGF-I receptor-specific pathway.     

Expression of the insulin-like growth factor system in skeletal muscle during embryonic and postnatal development in the first filial generation pigs from Erhualian and Yorkshire reciprocal crosses

In this study, we detected the expression of IGF-I, IGF-II, IGF-IR, IGF-IIR, and IGFBP-3 mRNA at 50 (E50), 70 (E70), and 90 (E90) days of gestation, and 1 (D1), 20 (D20), 70 (D70), 120 (D120), and 180 (D180) days of age in the longissimus dorsi (LD) and the semitendinosus (ST) of pigs from a Yorkshire boar × Erhualian sow (YE) cross as well as a Erhualian boar × Yorkshire sow (EY) cross. We found that the expression of IGF-I and IGF-II mRNA in skeletal muscle tissues differed based on developmental age and reciprocal cross type (P < 0.05). The expression of IGF-I mRNA exhibited a fluctuant ascending trend. In contrast, IGF-II showed a fluctuant descending trend after birth. The levels of IGF-IR mRNA were higher before birth compared with after birth except for the ST of EY pigs at D120 (P < 0.05). The expression of IGF-IIR and IGFBP-3 mRNA remarkably changed with age and reciprocal cross type (P < 0.05). IGF-I, IGF-II, and IGFBP-3 mRNA were positively correlated with IGF-IR from 50E to 180D. These data suggest that the expression of IGF-system genes exhibits specific developmental patterns in the skeletal muscle tissues of pigs from reciprocal crosses at different developmental stages and may show linked expression during certain periods of development. Our results may provide a valuable resource for the molecular breeding of pigs.     

IGF-binding protein-1 inhibits IGF effects on adipocyte function: implications for insulin-like actions at the adipocyte

IGF action in vivo is acutely regulated by IGF-binding protein-1 (IGFBP-1) and its phosphorylation state is implicated in modulating these effects. Since IGFs have an important regulatory role in adipocyte function, we investigated the effects of phosphorylated IGFBP-1 (pIGFBP-1) and non-phosphorylated IGFBP-1 (npIGF BP-1) on 3T3-L1 preadipocyte proliferation and adipocyte metabolism. IGFs stimulated clonal expansion of 3T3-L1 cells (IGF-I more potently than IGF-II (EC(50): 30 nM and 50 nM)). npIGFBP-1 inhibited IGF-I (50 nM) clonal expansion at a 5:1 molar ratio (P<0.01), whereas pIGFBP-1 (purified from HepG2 cell medium) abolished clonal expansion at a 1:1 molar ratio (P<0.005). In contrast, IGF-II-induced clonal expansion was inhibited 100% at a 1:1 molar ratio of npIGFBP-1. In mature adipocytes, IGF-I was equipotent with insulin in stimulating glucose uptake (EC(50): 10 nM) and inhibiting isoproterenol-induced lipolysis (EC(50): 15 nM). npIGFBP-1 completely reversed IGF-I effects at a 1:1 molar ratio (P<0.01). In summary, IGFs rather than insulin are potent regulators of clonal expansion in 3T3-L1 preadipocytes. Importantly, IGFs are equipotent with insulin in regulating adipocyte metabolism. IGFBP-1 inhibits IGF effects on preadipocyte proliferation and adipocyte metabolism, with pIGFBP-1 being more potent than npIGFBP-1 at inhibiting mitogenic actions. Since IGFBP-1 is acutely regulated by insulin, this could have important consequences in hyperinsulinaemic and insulin-resistant states.     

Effects of insulin-like growth factors (IGF-I and -II) on growth hormone and prolactin release and gene expression in euryhaline tilapia,Oreochromis mossambicus

We investigated in vitro effects of insulin-like growth factors (IGF-I and -II) on growth hormone (GH) and prolactin (PRL) release and gene expression in euryhaline tilapia, Oreochromis mossambicus. Pituitaries were removed from freshwater-acclimated adult males and incubated for 2-24h in the presence of human IGF-I or -II at doses ranging from 1-1000 ng/ml (0.13-130 nM). IGF-I at concentrations higher than 10 ng/ml and IGF-II higher than 100 ng/ml significantly inhibited GH release after 8, 16, and 24h. No effect of IGFs was seen during the first 4h of incubation. IGFs at the same concentrations also significantly attenuated GH gene expression after 24h, although no effect was seen at 2h. By contrast, PRL(188) release was stimulated significantly and in a dose-related manner by IGF-I at concentrations higher than 10 ng/ml and by IGF-II at concentrations higher than 100 ng/ml within 2h. No stimulation was observed after 4h. Similarly, both IGFs at concentrations higher than 10 ng/ml increased PRL(177) release within 2h. However, no significant effect of IGF-I or -II was observed on mRNA levels of both PRLs after 2 and 24h at all concentrations examined. These results clearly indicate differential regulation of GH and PRL release and synthesis by IGFs in the tilapia pituitary, i.e., rapid-acting, stimulatory effects of IGFs on PRL release and slow-acting, inhibitory effects on GH release and synthesis.     

IGF-I protects human oral buccal mucosal epithelial cells from sodium nitroprusside-induced apoptosis via PI3-kinase

OBJECTIVE: Cancers of the head and neck account for the vast majority of all malignancies of the oral cavity. The insulin-like growth factor (IGF) family of proteins is well documented to have an important role in rescuing cells from apoptosis. While it is known the IGF proteins are present in normal oral epithelial and cancer cells its role is not fully understood. Our aim was to study the ability of IGFs to rescue sodium nitroprusside (SNP)-induced apoptotic normal oral epithelial cells in vitro. DESIGN: Cultured normal human oral keratinocytes (NOKs) or epithelial cells were used. Apoptosis was induced by SNP then cells were exposed to IGF-I or IGF-II to rescue them. Cell viability was assessed by ELISA (for cell death and caspase 3) and FACS analysis; post receptor effects of IGF-I or IGF-II were assessed by [(3)H] thymidine incorporation. Cell signaling events were measured by western blotting using antibodies against phosphorylated Akt or p42/p44 MAPK, and measuring PI3-K activity by ELISA. RESULTS: SNP induced apoptosis of NOKs and activated the PI3-K/Akt survival pathway. Exposing cells to IGF proteins prevented their apoptosis. IGF-I and -II caused significant increases in PI3-K, but not MAPK, activity. SNP and LY294002, a PI3-K inhibitor, both caused a significant rise in caspase 3 release from NOKs which was reduced in the presence of IGFs. CONCLUSIONS: The data establishes the importance of IGF-activated PI3-K in rescuing cells from apoptosis. It lends further evidence to the significance of IGF proteins in the possible development of oral cancer.     

Effects of insulin-like growth factor II on the generation of inositol trisphosphate,diacylglycerol and cAMP in human fibroblasts

In human fibroblasts, exogenous insulin-like growth factor-II (IGF-II) induce a rapid redistribution of mannose 6-phosphate/IGF-II receptors. To analyze the mechanism transducing the IGF-II signal the phosphoinositide hydrolysis, 1,2-diacylglycerol and cAMP formation were studied after incubation with IGFs. While IGF-I (10 nM, 30 s) increased the inositol trisphosphate formation IGF-II (10 nM, up to 10 min) failed to affect phosphoinositide hydrolysis and had neither an effect on basal concentrations of diacylglycerol containing arachidonic acid or myristic acid nor on intracellular cAMP. On the contrary, pretreatment with IGF-II for 10 min enhanced the cAMP production stimulated by bradykinin (10 nM, 3 min) by 2.5-fold whereas no additive effects of IGF-II on the increased ligand binding to the mannose 6-phosphate/IGF-II receptor in response to bradykinin were observed. These results indicate that in fibroblasts the rapid IGF-II-induced redistribution of mannose 6-phosphate/IGF-II receptors is not mediated by inositol trisphosphate, diacylglycerol or cAMP, but that IGF-II may modulate permissively other agonist-generated signals.     

Characterization of the receptors for insulin and the insulin-like growth factors on micro- and macrovascular tissues

Insulin and insulin-like growth factors (IGFs) have been implicated in the pathogenesis of diabetic retinopathy and peripheral vascular complications. Previously, we have shown that retinal capillary endothelial cells responded to insulin and IGFs for metabolic and growth effects, whereas aortic endothelial cells were not responsive. In contrast, vascular supporting cells from both retinal capillaries (i.e. pericytes) and aorta (i.e. smooth muscle cells) responded equally to insulin, IGF-I, and IGF-II. The structure and ligand specificities of the receptor for these peptides were studied by covalently cross-linking 125I-labeled peptide hormones to their respective receptors using disuccinimidyl suberate, followed by polyacrylamide gel electrophoresis and autoradiography. The binding subunit of the insulin receptor, alpha-subunit, for all cell types was found to have a mol wt 145,000 under reduced conditions. Labeling of this band was inhibited by 10(-9) M insulin, antiinsulin receptor antibodies, and 10(-8) M IGF-I, but not by multiplication-stimulating activity (IGF-II). The beta-subunit of the insulin receptor in endothelial cells was identified by its ability to be autophosphorylated when stimulated by insulin and was found to have a mol wt of 99,000. Covalent cross-linking of IGF-I to its receptor revealed a mol wt of 145,000, similar to that of insulin receptor, except that IGF-I was 100-fold more potent than insulin in competing with [125I]IGF-I for binding. [125I]IGF-II in all cells was cross-linked to receptor with mol wt of 260,000 and 230,000 under reduced and nonreduced conditions, respectively. IGF-I competed weakly with [125I]IGF-II, whereas insulin was ineffective. [125I]IGF-II also bound to the band with alpha mol wt of 135,000, which was inhibited by insulin, IGF-I, and IGF-II. In summary, receptors for insulin, IGF-I, and IGF-II on cells from micro- and macrovessels are biochemically similar to those in other cells. Interestingly, the finding of large numbers of IGF-I and IGF-II receptors on endothelial cells suggests that these growth factors play a physiological role and are involved in vascular complications associated with diabetes.     

Rat somatotroph insulin-like growth factor-II (IGF-II) signaling: role of the IGF-I receptor.

The anterior pituitary contains insulin-like growth factor-II (IGF-II) and expresses abundant IGF-II-binding sites, but the IGF-II signaling pathway in the pituitary has not been defined. We, therefore, tested the effects of recombinant human IGF-II on pituitary function by assessing the GH responsiveness of the primary rat somatotroph to IGF-II. IGF-II (3.3 nM) suppressed GH secretion by 50%, similar to the effect elicited by equimolar doses of IGF-I. In contrast, a low concentration of IGF-II (0.2 nM) did not attenuate GH secretion, while a similar IGF-I dose was sufficient to produce 50% inhibition of basal GH secretion. Fifty percent competition for [125I]IGF-I binding by IGF-I and IGF-II in GC rat pituitary cells demonstrated a 14-fold lesser affinity of the IGF-II ligand for the IGF-I receptor compared to IGF-I; therefore, the binding affinity of IGF-II for the IGF-I receptor correlates with the concentration of IGF-II required for 50% GH inhibition. Transfected GH-secreting cell lines derived from GC cells overexpressing intact human IGF-I receptors exhibited enhanced responsiveness to IGF-II. In contrast, cells transfected with a truncated IGF-I receptor cDNA lacking the cytoplasmic receptor beta-subunit (952STOP) failed to transduce the IGF-II signal, indicating that functional IGF-I receptors are required for IGF-II signaling. In addition, a mutant IGF ligand, [Leu27]IGF-II, which selectively exhibits high affinity for the type II receptor, but only minimal binding to the IGF-I receptor, did not attenuate GH secretion. However, the analog [Arg54,Arg55]IGF-II, which exhibits high affinity to the IGF-I receptor, but no binding to the type II receptor, appropriately suppressed GH secretion. This unique model of somatotroph signaling provides evidence for IGF-II regulation of polypeptide hormone secretion mediated by the IGF-I receptor.     

Maternal nutrition affects the ability of treatment with IGF-I and IGF-II to increase growth of the placenta and fetus, in guinea pigs

The aim of this study was to investigate how administration of IGF-I and IGF-II, during early to mid pregnancy, affects maternal growth and body composition as well as fetal and placental growth, in ad libitum fed, and in moderately, chronically food restricted guinea pigs. From day 20 of gestation, mothers (3-4 months old) were infused with IGF-I, IGF-II (565 microg/day) or vehicle for 17 days and then killed on day 40 of gestation. Maternal organ weights, fetal and placental weights were assessed. Treatment with IGFs did not alter body weight gain and had small effects on body composition in the mothers. Both IGF-I and IGF-II increased fetal and placental weights in ad libitum fed dams and IGF-I increased placental weight in food restricted dams. In conclusion, treatment with IGF-I during the first half of pregnancy stimulates placental growth in both ad libitum fed and food restricted guinea pigs without affecting maternal growth while fetal growth is stimulated by IGF treatment only in ad libitum fed animals.