Specific Insulin-like Growth factor-I Receptors Research Articles

Expression of insulin receptor (IR) A and B isoforms, IGF-IR, and IR/IGF-IR hybrid receptors in vascular smooth muscle cells and their role in cell migration in atherosclerosis.

BackgroundAbnormal proliferation and migration of vascular smooth muscle cells (VSMCs) is a major contributor to the development of atherosclerotic process. In a previous work, we demonstrated that the insulin receptor isoform A (IRA) and its association with the insulin-like growth factor-I receptor (IGF-IR) confer a proliferative advantage to VSMCs. However, the role of IR and IGF-IR in VSMC migration remains poorly understood.MethodsWound healing assays were performed in VSMCs bearing IR (IRLoxP+/+ VSMCs), or not (IR−/− VSMCs), expressing IRA (IRA VSMCs) or expressing IRB (IRB VSMCs). To study the role of IR isoforms and IGF-IR in experimental atherosclerosis, we used ApoE−/− mice at 8, 12, 18 and 24 weeks of age. Finally, we analyzed the mRNA expression of total IR, IRB isoform, IGF-IR and IGFs by qRT-PCR in the medial layer of human aortas.ResultsIGF-I strongly induced migration of the four cell lines through IGF-IR. In contrast, insulin and IGF-II only caused a significant increase of IRA VSMC migration which might be favored by the formation of IRA/IGF-IR receptors. Additionally, a specific IGF-IR inhibitor, picropodophyllin, completely abolished insulin- and IGF-II-induced migration in IRB, but not in IRA VSMCs. A significant increase of IRA and IGF-IR, and VSMC migration were observed in fibrous plaques from 24-week-old ApoE−/− mice. Finally, we observed a marked increase of IGF-IR, IGF-I and IGF-II in media from fatty streaks as compared with both healthy aortas and fibrolipidic lesions, favoring the ability of medial VSMCs to migrate into the intima.ConclusionsOur data suggest that overexpression of IGF-IR or IRA isoform, as homodimers or as part of IRA/IGF-IR hybrid receptors, confers a stronger migratory capability to VSMCs as might occur in early stages of atherosclerotic process.Electronic supplementary materialThe online version of this article (doi:10.1186/s12933-016-0477-3) contains supplementary material, which is available to authorized users.

D-chiro-inositol phosphoglycan expression in human placenta at term in diabetes

We present a histological assessment of diabetic placenta for the localization of some insulin mediators that were reported to play a major role for lipid/glycogen metabolism in human placenta. Placental metabolism is fundamental to fetal wellbeing. In fact, all maternal metabolic alterations have a profound effect on placental development with a subsequent definite impact on pregnancy outcome and fetal growth [1]. We have previously reported abnormalities in expression and activation of specific insulin-like growth factor-I receptor signaling molecules and multiple members of the MAP kinase family in human placentas of fetuses with intra-uterine growth restriction [2]. Subsequent studies on placental metabolism have focused on a family of insulin mediators, namely inositol phosphoglycans that were found to be highly expressed in diabetic and preeclamptic placentas [3]. These molecules play a major role in the regulation of the disposal of glucose by oxidative and non-oxidative routes of storage by glycogen synthesis [4]. In fact, the complexity and specificity of insulin action are determined by an integrated signaling network of independent intracellular pathways with several downstream effectors that have been shown to modulate the signal amplitude and frequency for both shortand longterm effects. Specific acquired alterations in activation of proteins involved in insulin signaling in preeclamptic placenta were shown to be associated with the accumulation of D-chiro-inositol phosphoglycan and to play a role in the insulin-resistant state that occurs in preeclampsia [5]. A disturbed insulin signaling was demonstrated in these cases and associated to low activation of insulin receptor effectors through multiple serine phosphorylations in both IRS-1 and IRS-2 [5]. In this study, placental specimens from six healthy and diabetic women at term were assessed for chiro-inositol using antibodies (mouse anti-D-chiro-inositol phosphoglycan), kindly provided by Professor Eduardo Martinez (Pamplona, Spain). A standard procedure was used for the immunohistochemical staining. The primary anti-D-chiroinositol phosphoglycan antibody was diluted 1:100 in antibody diluent (Dako, USA) and incubated for 60 min at room temperature. The percentage of positive stained controls was used as a reference for comparison. In control cases (Fig. 1a), a clear immunostaining of the syncytiotrophoblast was found (p = 0.11) with a little activity within the villous stroma (p = 0.43). The endothelium of placental vessels shows a localization of D-chiro-inositol phosphoglycan in normal placenta while in diabetic samples is weakly stained (p \ 0.01, Fig. 1b). The extravillous trophoblast in the basal plate of diabetic cases reveals also staining (p = 0.23, Fig. 1c, d). In both cases, a weak staining was found in maternal plasma, while a strong staining was found in fetal plasma. According to these findings, we may hypothesize that the placenta uptakes D-chiro inositol phosphoglycan from M. Scioscia (&) Department of Obstetrics and Gynecology, Sacro Cuore Don Calabria General Hospital, Via Don A. Sempreboni, 5, 37024 Negrar, VR, Italy e-mail: marco.scioscia@sacrocuore.it

Overexpression of insulin-like growth factor-I receptor as a pertinent biomarker for hepatocytes malignant transformation

To investigate the dynamic features of insulin-like growth factor-I receptor (IGF-IR) expression in rat hepatocarcinogenesis, and the relationship between IGF-IR and hepatocytes malignant transformation at mRNA or protein level. Hepatoma models were made by inducing with 2-fluorenylacetamide (2-FAA) on male Sprague-Dawley rats. Morphological changes of hepatocytes were observed by pathological Hematoxylin and eosin staining, the dynamic expressions of liver and serum IGF-IR were quantitatively analyzed by an enzyme-linked immunosorbent assay. The distribution of hepatic IGF-IR was located by immunohistochemistry. The fragments of IGF-IR gene were amplified by reverse transcription-polymerase chain reaction, and confirmed by sequencing. Rat hepatocytes after induced by 2-FAA were changed dynamically from granule-like degeneration, precancerous to hepatoma formation with the progressing increasing of hepatic mRNA or IGF-IR expression. The incidences of liver IGF-IR, IGF-IR mRNA, specific IGF-IR concentration (ng/mg wet liver), and serum IGF-IR level (ng/mL) were 0.0%, 0.0%, 0.63 ± 0.17, and 1.33 ± 0.47 in the control; 50.0%, 61.1%, 0.65 ± 0.2, and 1.51 ± 0.46 in the degeneration; 88.9%, 100%, 0.66 ± 0.14, and 1.92 ± 0.29 in the precancerosis; and 100%, 100%, 0.96 ± 0.09, and 2.43 ± 0.57 in the cancerous group, respectively. IGF-IR expression in the cancerous group was significantly higher (P < 0.01) than that in any of other groups at mRNA or protein level. The closely positive IGF-IR relationship was found between livers and sera (r = 0.91, t = 14.222, P < 0.01), respectively. IGF-IR expression may participate in rat hepatocarcinogenesis and its abnormality should be an early marker for hepatocytes malignant transformation.

Insulin-like growth factor-I receptor (IGF-IR) targeting with monoclonal antibody cixutumumab (IMC-A12) inhibits IGF-I action in endometrial cancer cells

Specific insulin-like growth factor-I receptor (IGF-IR) targeting emerged in recent years as a promising therapeutic strategy in cancer. Endometrial cancer is the most common gynaecological cancer in the Western world. The aim of this study was to evaluate the potential of cixutumumab (IMC-A12, ImClone Systems), a fully human monoclonal antibody against the IGF-IR, to inhibit IGF-I-mediated biological actions and cell signalling events in four endometrial carcinoma-derived cell lines (ECC-1, Ishikawa, USPC-1 and USPC-2). Our results demonstrate that cixutumumab was able to block the IGF-I-induced autophosphorylation of the IGF-IR. In addition, the PI3K and MAPK downstream signalling pathways were also inactivated by cixutumumab in part of the cell lines. Prolonged (24 h and 48 h) exposures to cixutumumab reduced IGF-IR expression. Furthermore, confocal microscopy of GFP-tagged receptors shows that cixutumumab treatment led to IGF-IR redistribution from the cell membrane to the cytoplasm. Antiapoptotic effects were evaluated by cleavage of caspase 3 and PARP, and mitogenicity and transformation by proliferation and cell cycle assays. Results obtained showed that cixutumumab abrogated the IGF-I-stimulated increase in proliferation rate, and increased caspase-3 and PARP cleavage, two markers of apoptosis. Of importance, cixutumumab had no effect neither on insulin receptor (IR) expression nor on IGF-I activation of IR. In summary, in a cellular model of endometrial cancer cixutumumab was able to inhibit the IGF-I-induced activation of intracellular cascades, apoptosis and proliferation.

87 POSTER Correlation analysis utilizing measurements of tumor biomarkers and antibody efficacy against EGFR, IGF1R and VEGFR2 receptors in multiple tumor models

Specific insulin-like growth factor-I receptor (IGF-IR) targeting emerged in recent years as a promising therapeutic strategy in cancer. Endometrial cancer is the most common gynaecological cancer in the Western world. The aim of this study was to evaluate the potential of cixutumumab (IMC-A12, ImClone Systems), a fully human monoclonal antibody against the IGF-IR, to inhibit IGF-I-mediated biological actions and cell signalling events in four endometrial carcinoma-derived cell lines (ECC-1, Ishikawa, USPC-1 and USPC-2). Our results demonstrate that cixutumumab was able to block the IGF-I-induced autophosphorylation of the IGF-IR. In addition, the PI3K and MAPK downstream signalling pathways were also inactivated by cixutumumab in part of the cell lines. Prolonged (24 h and 48 h) exposures to cixutumumab reduced IGF-IR expression. Furthermore, confocal microscopy of GFP-tagged receptors shows that cixutumumab treatment led to IGF-IR redistribution from the cell membrane to the cytoplasm. Antiapoptotic effects were evaluated by cleavage of caspase 3 and PARP, and mitogenicity and transformation by proliferation and cell cycle assays. Results obtained showed that cixutumumab abrogated the IGF-I-stimulated increase in proliferation rate, and increased caspase-3 and PARP cleavage, two markers of apoptosis. Of importance, cixutumumab had no effect neither on insulin receptor (IR) expression nor on IGF-I activation of IR. In summary, in a cellular model of endometrial cancer cixutumumab was able to inhibit the IGF-I-induced activation of intracellular cascades, apoptosis and proliferation.

Insulin and IGF-I receptors in trout adipose tissue are physiologically regulated by circulating hormone levels.

In fish, insulin is believed to act on adipose tissue to promote lipid accumulation, but a direct role for insulin in fish adipose tissue lipogenesis has yet to be demonstrated. To investigate the role of insulin and insulin-like growth factor I (IGF-I) in fish adipose tissue function, we have investigated the presence and the regulation of insulin and IGF-I receptors in adipose tissue of brown trout (Salmo trutta). Receptors for insulin and IGF-I were detected in trout adipose tissue, with IGF-I receptors being more abundant (two- to tenfold) and having a higher affinity (twofold) than insulin receptors. In contrast to the situation in mammals, arginine treatment, which elevates the levels of insulin and IGF-I in plasma, increased the number of insulin receptors 1.7-fold and the number of IGF-I receptors 2.3-fold. When plasma levels of insulin and IGF-I were decreased by fasting, insulin receptor numbers fell 3.6-fold and IGF-I receptor numbers fell 2.2-fold. These results demonstrate for the first time the presence of specific insulin and IGF-I receptors in adipose tissue of ectothermic vertebrates and suggest that adipose tissue may be a target for the actions of insulin and IGF-I in fish.

Insulin-Like Growth Factor I (IGF-I) Binding in Skeletal Muscle of Lamprey Lampetra fluviatilis Predominates over Insulin Binding and Increase in the Course of Pre-Spawning Migration

Binding of 125I-insulin and 125I-IGF-I to partially purified receptors of lamprey skeletal muscles was studied during pre-pawning migration. It has been shown that throughout this whole period the IGF-I binding to skeletal muscle predominates over the insulin binding. Besides, a certain time dynamics was observed: the insulin binding rose since October to reach maximum in February–March, then it decreased to a minimum level in May; the IGF-I binding also increased: it rose statistically significantly in March compared to October, became maximal in April, and then decreased to a minimum. The dynamics of the receptor IGF-I binding has been shown to depend on changes of receptor affinity, whereas the change of the insulin binding was determined by binding capacity (the number of binding sites). Highly specific IGF-I receptors of the lamprey skeletal muscle bound insulin with an affinity about 1% from that of IGF-I, while insulin receptors had identical affinity for the insulin and IGF-I binding. Both peptides, insulin and IGF-I, activated autophosphorylation of beta-subunits in their receptors. The increase of the IGF-I binding from October to April could be a factor that maintains a high functional activity of lamprey skeletal muscles in the course of the pre-pawning migration. It is suggested that IGF-I promotes maintaining this activity due to its property of inhibiting apoptosis.

Phasic synaptic remodeling of the rat arcuate nucleus during the estrous cycle depends on insulin-like growth factor-I receptor activation.

Insulin-like growth factor-I (IGF-I) has trophic and plastic effects on neurons and glial cells and modulates neuroendocrine events by acting at the level of the hypothalamus. IGF-I and estrogen signaling interact to regulate in vitro hypothalamic neuronal survival and differentiation. In vivo, IGF-I levels fluctuate in the rat hypothalamic arcuate nucleus during the estrous cycle in parallel with a phasic remodeling of synaptic contacts and glial cell processes. Both the fluctuation of IGF-I levels and the synaptic and glial changes are induced by estrogen. The possible role of IGF-I in the regulation of arcuate nucleus synaptic plasticity has been assessed in the present study by intracerebroventricular administration to cycling female rats of a specific IGF-I receptor antagonist. In agreement with previous findings, the number of synaptic inputs to arcuate neuronal somas in control rats showed a significant decrease between the morning of proestrus and the morning of estrus. This decline in synaptic inputs and the accompanying increase in glial ensheathing of neuronal somas were blocked by the IGF-I receptor antagonist. In contrast, the IGF-I receptor antagonist did not affect the basal number of synapses or the morphology of synaptic terminals or length of the synaptic contacts. These findings indicate that IGF-I receptor activation may be involved in the phasic remodeling of arcuate nucleus synapses during the estrous cycle. Res.

Dynamics of insulin and insulin-like growth factor-I (IGF-I) ovarian receptors during maturation in the brown trout (Salmo trutta)

In salmonid fishes, the role of insulin and insulin-like growth factor-I (IGF-I) in the regulation of ovarian function is not well understood. Recently, we reported that isolated follicular layers of the preovulatory ovarian follicle of coho salmon (Oncorhynchus kisutch) have specific receptors for insulin and IGF-I and that IGF-I modulates steroid production in the follicular layers. In the present study we have investigated the structural and functional characteristics of insulin and IGF-I receptors in the ovary of brown trout (Salmo trutta) and the changes in insulin and IGF-I binding throughout the reproductive cycle of this species. The specific binding for IGF-I was 8- to 15-fold higher than the specific binding for insulin. IGF-I receptors were also more specific than insulin receptors because unlabeled insulin displaced bound radiolabeled insulin at concentrations 40- to 80-fold lower than unlabeled IGF-I; whereas, unlabeled IGF-I displaced bound radiolabeled IGF-I at concentrations 4000- to 8000-fold lower than unlabeled insulin. Insulin and IGF-I receptors from the brown trout ovary were composed of 120 kDa α-subunits and 90 kDa β-subunits, which underwent autophosphorylation in a concentration-dependent manner. Receptor tyrosine kinase activity was also stimulated in a concentration-dependent manner by insulin and IGF-I. When ovarian insulin and IGF-I binding was determined from mid-vitellogenesis (March) until ovulation (November), maximal binding for both peptides was detected in mid-vitellogenesis and gradually decreased until the end of vitellogenesis (August). In the preovulatory period (October), a small increase of insulin and IGF-I binding was observed. After ovulation, insulin binding was no longer detectable and IGF-I binding was very low. These results suggest that insulin and IGF-I receptors in the salmonid ovary follow the structural pattern described in other vertebrate species and that insulin and IGF-I could be involved in the regulation of ovarian function during reproductive stages other than the preovulatory period.

Upregulation of human chorionic gonadotrophin-induced steroidogenic acute regulatory protein by insulin-like growth factor-I in rat Leydig cells.

Insulin-like growth factor-I (IGF-I) plays an essential role in reproductive function. Leydig cells express specific IGF-I receptors, and IGF-I enhances human chorionic gonadorphin (hCG)-induced testosterone formation. In the present study, we evaluate the effect of IGF-I on the gene expression and protein levels of steroidogenic acute regulatory protein (StAR), the rate-limiting step in steroidogenesis. StAR mRNA is expressed in rat Leydig cells as two major transcripts of 3.8 and 1.7 kb. StAR mRNA levels (both 3.8 and 1.7 kb) were markedly induced about 20-fold by hCG (10 ng/mL). Concomitant addition of IGF-I (50 or 100 ng/mL) and hCG (10 ng/mL) resulted in significant increases in StAR and cytochrome P450 side-chain cleavage (P450scc) mRNA levels, whereas lower doses of IGF-I (1 or 10 ng/ mL) had small effects. Synergistic effects of IGF-I and hCG on StAR mRNA levels were confirmed by ribonuclease protection assay (RPA). IGF-I (100 ng/mL) enhanced hCG- and 20 OH-cholesterol + hCG-induced testosterone formation, whereas the conversions of pregnenolone, 17-OH pregnenolone, dehydroepiandrosterone, and androstenedione to testosterone were not affected. This suggests that the major effect of IGF-I is at the steps of StAR and P450scc, whereas other steroidogenic enzymes are not affected. To evaluate whether increased StAR mRNA levels induced by IGF-I and hCG are associated with increased StAR protein levels, we carried out Western blot analyses. Basal StAR protein levels were low after 24 h in culture. hCG (10 ng/mL) increased StAR protein by 4.5-fold. In the presence of IGF-I (100 ng/mL), hCG-induced StAR protein levels were further increased. In conclusion, our present study demonstrated that IGF-I enhances Leydig cell steroidogenesis by upregulating hCG-induced StAR gene expression and protein production.

Ovarian Receptors for Insulin and Insulin-like Growth Factor I (IGF-I) and Effects of IGF-I on Steroid Production by Isolated Follicular Layers of the Preovulatory Coho Salmon Ovarian Follicle

In this study, receptors for insulin and insulin-like growth factor I (IGF-I) in isolated theca-interstitial layers and granulosa cells of the coho salmon preovulatory ovary were characterized, and the effects of IGF-I on ovarian steroidogenesis were examined. Specific receptors for insulin and IGF-I were found in granulosa and theca-interstitial layers. In both follicular layers, IGF-I receptors were greater in number and higher in affinity than insulin receptors. The effects of IGF-I onin vitroproduction of testosterone (T) and 17α-hydroxyprogesterone (17OH-P) by theca-interstitial layers and of 17β-estradiol (E2) and 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) by granulosa cell layers were evaluated during the preovulatory period. Both human and salmon recombinant IGF-I inhibited the basal and GTH II-stimulated T and 17OH-P production by theca-interstitial layers throughout the preovulatory period. In contrast, IGF-I stimulated the production of both E2and 17,20β-P by granulosa cell layers prior to germinal vesicle breakdown (GVBD) but only stimulated the production of 17,20β-P by granulosa cell layers after GVBD. The inhibitory effects of IGF-I on steroid production by the theca-interstitial layer and the opposite stimulatory effects on steroid production by the granulosa cell layer, coupled by the presence of specific IGF-I receptors in both follicular layers, suggest that IGF-I may play a role in the regulation of steroidogenesis in the preovulatory coho salmon ovary.

Luminal and basolateral uptake and receptor binding of IGF-I in rabbit renal proximal tubules.

The aim of the present study was to quantify and compare the luminal and basolateral binding and uptake of 125I-labeled insulin-like growth factor I (IGF-I) by means of 1) isolated, perfused, proximal tubules combined with electron microscope autoradiography and 2) luminal and basolateral membrane vesicles from rabbit proximal tubules. 125I-IGF-I was added to isolated perfused proximal tubules for 30 min in concentrations of 1.6-3.9 micrograms/l to either the perfusate or the bath. The luminal and basolateral uptake in 30 min averaged 447 and 410 fg/mm, respectively. About 20% of the luminally absorbed IGF-I was digested. Addition of excess unlabeled IGF-I (10(-7) M) to the bath produced complete inhibition of the basolateral binding/uptake, whereas no inhibition of the luminal uptake was seen. Electron microscope autoradiography showed that IGF-I after luminal endocytic uptake to a large extent was transported into lysosomes. After basolateral exposure the major portion of the grains was found over the basolateral cell membrane; however, a significant amount was located over endocytic vacuoles and lysosomes in both apical and basal parts of the cells. In both luminal and basolateral membrane vesicles, single-class, high-affinity binding sites for IGF-I were found with dissociation constants of 6.3 and 5.7 nM, respectively. Specific binding capacities averaged 2.7 and 25.7 pmol IGF-I/mg protein in luminal and basolateral vesicles. The biochemical data suggest an asymmetric distribution of specific IGF-I receptors in the luminal and basolateral membranes, with a greater abundance of receptors in the latter. The extensive basolateral endocytic binding/uptake of IGF-I compared with that of the luminal in isolated perfused tubules differs considerably from the processing of other peptide hormones.

Newborn rabbit gastric smooth muscle cell culture: EGF and TGF-alpha are potent mitogens.

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) are two in a family of growth-promoting peptides for many gastrointestinal epithelia. This study was designed to assess their mitogenic effect on cultured gastric myocytes and to characterize specific EGF receptors on these cells. Single myocytes were isolated from newborn rabbit gastric fundus and placed into tissue culture. The composition of the culture at confluence as assessed by immunostaining with smooth muscle actin-specific monoclonal antibody (CGA7) was > 95% myocytes. To assess the effect of putative growth factors, freshly isolated myocytes were incubated in Dulbecco's modified Eagle's (DME) medium containing 1% fetal bovine serum in the presence or absence of growth factors. After 6 days, cells were incubated in serum-free medium with [3H]thymidine (1 microCi/ml) in the continued presence or absence of growth factors. After 24 h, EGF and TGF-alpha but not insulin-like growth factor I (IGF-I) induced a dose-dependent increase in [3H]thymidine incorporation. Ten nanomolar EGF or TGF-alpha increased [3H]thymidine incorporation more than sixfold over control. EGF was more potent than was TGF-alpha, with apparent median effective dose (ED50) values of 64 +/- 14 pM and 166 +/- 62 pM (p < 0.05), respectively. EGF bound to cultured myocytes with Kd = 7.6 +/- 1.8 nM and Bmax = 27 +/- 11 pmol/mg DNA or 440,000 receptors/cell. TGF-alpha competed for binding at these receptors. Although IGF-I did not stimulate thymidine incorporation, specific high-affinity receptors for IGF-I were detected on gastric myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgen induction of steroid 5 alpha-reductase may be mediated via insulin-like growth factor-I.

The action of added insulin-like growth factor-I (IGF-I) and dihydrotestosterone (DHT) on steroid 5 alpha-reductase (5 alpha R) activity was studied using primary cultures of rat or human scrotal skin fibroblasts. Agents were added to cultured cells (2 x 10(5) cells) for 2 days, and enzyme activity was measured by the percent conversion of [3H] testosterone to DHT over a 4-h period in the absence of fetal calf serum or other growth factors. DHT, but not testosterone, at 10(-7) M significantly increased 5 alpha R activity (rat, 1.5 +/- 0.3% to 3.0 +/- 0.4%; human, 7.6 +/- 1.7% to 11.4 +/- 2.9%; P < 0.01). IGF-I (10(-9)-6.4 x 10(-9) M), but not IGF-II (10(-9)-10(-8) M) or insulin (10(-9)-10(-7) M), increased enzyme activity in a dose-related fashion [i.e. 1.5 +/- 0.5 to 10 +/- 2 in rat and 6.0 +/- 1.1 to 9.8 +/- 1.6% (P < 0.01) in human cells]. No change in cell numbers was observed in any experiment. Since the effect of IGF-I was about 100 times that of androgen, we studied the possibility that androgen induction of the enzyme activity could be via IGF-I production. Addition of a monoclonal antibody against IGF-I significantly reduced the effect of DHT, and simultaneous addition of a specific IGF-I receptor antibody blocked the expected induction of 5 alpha R activity (control, 4.9 +/- 0.5; DHT, 8.0 +/- 1.9; DHT plus IGF-I receptor antibody, 3.7 +/- 0.4%). No effect on 3 alpha-reduction of [3H]DHT to 3 alpha-androstanediol was detected in separate experiments. These studies indicate that IGF-I may be an important regulator of skin 5 alpha R activity and, thus, may influence DHT formation. The previously known androgen induction of this peripheral steroidogenic enzyme may be via paracrine/autocrine production of an IGF-I-type growth factor.

Insulin and IGF-I receptors and tyrosine kinase activity in carp ovaries: changes with reproductive cycle

Insulin and insulin-like growth factor I (IGF-I) receptors from carp ovaries were semipurified with wheat germ agglutinin at different moments of the reproductive cycle and their binding characteristics and tyrosine kinase activity were studied. Specific receptors for insulin and IGF-I were found. IGF-I receptors presented higher binding (23.8 ± 1.5%), number of receptors (965 ± 20fm/mg) and affinity (KD 0.24 ± 0.03nM) than those shown for insulin receptors (4.1 ± 1%, 530 ± 85fm/mg and 0.85 ± 0.1nM, respectively). Insulin and IGF-I receptors have a tyrosine kinase activity which is not different from that found in muscle of the same species. Seasonal changes were found in binding, with maximum values for insulin and IGF-I reached at the end of pre-spawning period (June). However, while IGF-I binding was observed in all stages, insulin binding decreased in autumn and disappeared in winter, which suggests a different role for the two peptides in ovarian physiology.

Receptor binding and growth-promoting activity of insulin-like growth factor-I in a bovine mammary epithelial cell line (MAC-T3).

Insulin-like growth factor-I (IGF-I) has been known to be mitogenic to a variety of cell types, although a growth-regulatory role for IGF-I on bovine mammary epithelial cells has not been fully investigated. In the present study, we examined the receptor binding of IGF-I and its effect on growth in a bovine mammary epithelial cell line (MAC-T3). Specific receptors for IGF-I were detected on cultured bovine mammary epithelial cells. Competitive binding revealed that half-maximal inhibition of 125I-labelled IGF-I binding by IGF-I was approximately 3 micrograms/l. Dissociation rate constant of the IGF-I receptor was 3.10 +/- 0.06 nmol/l (S.E.M.) with a receptor site concentration of 366 +/- 8 fmol/mg protein for the average of three experiments. IGF-I exerted a positive mitogenic effect on MAC-T3 cells according to both direct DNA assay and thymidine incorporation assay. Moreover, the mitogenic effect of IGF-I on MAC-T3 cells was enhanced by the addition of fetal calf serum in the culture media. The present results suggest that the bovine mammary epithelial cell line (MAC-T3) provides a useful model system with which to study the biological actions of insulin-like growth factors on the bovine mammary secretory tissue in vitro.

Regulation of insulin-like growth factor-binding protein expression by thyroid hormone in rat GH3 pituitary tumor cells.

Rat pituitary GH3 tumor cells express GH and insulin-like growth factor-I (IGF-I) mRNAs and possess specific IGF-I and IGF-II receptors which mediate the inhibitory effect of IGF on GH secretion. T3 increases the rate of cell replication and GH gene transcription, and causes an increase in IGF-I binding to cell membranes. Since the IGFs circulate in association with specific binding proteins (IGFBPs) that appear to modulate the access of IGFs to their receptors, we have investigated the effect of T3 treatment on the expression of IGFBPs in GH3 cells. Cells were grown in serum-free defined medium, and IGFBP secretion was determined by Western ligand blotting of conditioned medium after the addition of T3 and/or various peptides for 48-72 h. The conditioned medium of GH3 cells revealed a complex of bands migrating at 40-45 Mr, a pattern typical of rat (r) IGFBP-3. T3 treatment resulted in an increase in rIGFBP-3. IGF-I, IGF-II, and insulin did not alter rIGFBP-3 levels. After concentrating (10-fold) conditioned medium samples, two additional bands at 24K and 28K mol wt were also seen. These bands corresponded in size to rIGFBP-4 (24K) and its glycosylated form (28K). The mRNAs for both rIGFBP-3 and rIGFBP-4 were present in GH3 cells; T3 treatment increased steady state levels of rIGFBP-3 mRNA, but did not affect BP-4 mRNA levels. To learn whether the increased expression of IGFBPs could be responsible for the increased IGF-I binding seen after T3 treatment, [125I]IGF-I was cross-linked to GH3 membranes, and the proteins were separated on a 5-15% gradient sodium dodecyl sulfate-polyacrylamide gel under reducing conditions. T3 treatment induced a large increase in the intensity of bands migrating at 135K and 280K, representing the alpha-subunit of the IGF-I receptor and an incompletely reduced alpha-alpha-dimer, respectively. No membrane-associated IGFBPs were detected. In conclusion, GH3 cells express two IGFBPs, rIGFBP-3 and rIGFBP-4, which are differentially regulated by T3. The increased binding of IGF-I to GH3 cell membranes after T3 treatment indicates that thyroid hormone induces an up-regulation of IGF-I receptors and that the increased IGF-I binding to GH3 membranes is not due to increased expression of membrane-associated IGFBPs.

Oestradiol treatment increases the sensitivity of MCF-7 cells for the growth stimulatory effect of IGF-I

MCF-7 cells were grown in serum free medium (Dulbecco MEM without phenol red, supplemented with Costar SF-1 without insulin). Insulin was added as required and gave dose dependent growth stimulation at concentrations between 5 and 10,000 nM. Identical growth response curves were obtained for thymidine uptake and cell number. Oestradiol and insulin-like growth factor I (IGF-I) added individually both gave a dose dependent stimulation of cell growth in serum free medium containing 50 nM insulin. The growth stimulatory effect of oestradiol was to a large extent inhibited with suramine, a general inhibitor of growth factors, indicating that the effect of oestradiol was mediated through stimulating autocrine secretion of a growth factor. To investigate a possible link between the effects of oestradiol and IGF-I, a specific IGF-I receptor antibody (αIR-3), 10 μg/ml was used. These experiments were carried out with 2.5 nM insulin in the medium, a concentration at which insulin had no growth stimulatory effect. Stimulation was carried out for 18 h before assay of thymidine uptake. The effect of oestradiol was not significantly reduced by αIR-3, indicating that IGF-I was not an autocrine mediator of oestradiol stimulation of cell growth under these conditions, whereas αIR-3 extensively reduced growth stimulation by IGF-I. On long term stimulation (5 days) oestradiol had a marked stimulatory effect on cell number and αIR-3 almost totally abrogated this effect. When oestradiol (1 nM) and IFG-I (2.5 nM) were added together, the combined effect on thymidine incorporation and cell number was significantly greater than additive. This synergistic effect on the IGF-I growth response was totally abolished by the IGF-I receptor antibody. The results suggest a cooperative interaction of oestradiol and IGF-I. It is concluded that growth stimulation of MCF-7 cells by long term treatment with oestradiol may be mediated through autocrine secretion of IGF-I. The effect of short term stimulation of thymidine incorporation suggest that the growth response of oestradiol is more complex, and indicate that a cooperative interaction with IGF-I is involved, which is unrelated to stimulated autocrine secretion.

Evidence for an Insulin‐Like Growth Factor Autocrine‐Paracrine System in the Retinal Photoreceptor‐Pigment Epithelial Cell Complex

The interphotoreceptor matrix (IPM), lying between retinal photoreceptor and pigment epithelial (RPE) cells, contains insulin-like growth factor I (IGF-I) immunoreactivity that co-elutes with authentic human IGF-I in HPLC analyses. Cultured human RPE cells synthesize and release IGF-I, raising the possibility that the RPE serves as a source of IPM IGF-I in vivo. Photoreceptor rod outer segments and cultured monkey RPE cells express specific IGF-I receptors with alpha-subunits of 120 and 138 kDa, respectively. They thus appear to be of the "brain" (in photoreceptors) and "peripheral" (in RPE cells) receptor subtypes. Additionally, the IPM contains high levels of an IGF binding protein (IGF-BP) that specifically binds IGF-I and IGF-II. The IPM-BP is visualized as a single radiographic band by both ligand blot and affinity cross-linking procedures. With enzymes specific for removing N- and O-linked oligosaccharides, the IPM-BP was found to contain O- but not N-linked glycosylated side chains. The distinctive size and glycosylation pattern of the IPM-BP indicate that it is not derived from the vitreous or serum but instead is synthesized locally. The presence of IGF-I and IGF-BP in the IPM, together with the presence of IGF-I receptors on both photoreceptor and RPE cells, suggests the presence of an outer retina autocrine-paracrine system.

Insulin-Like Growth Factor-I Binds Selectively to Human Peripheral Blood Monocytes and B-Lymphocytes*

Insulin-like growth factor-I (IGF-I) is a potential modulator of responses to a variety of immunological challenges. Previous studies have suggested that specific IGF-I receptors are present on peripheral blood leukocytes, including polymorphonuclear leukocytes, lymphocytes, and monocytes. We sought to determine what type of IGF receptor was present on peripheral blood mononuclear cells and which types of cells possessed these receptors. Binding of [125I]IGF-I to mononuclear cells was inhibited by both unlabeled IGF-I and insulin, insulin being 200-fold less potent than IGF-I. Covalent affinity labeling with [125I]IGF-I, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, revealed a specific binding species with an apparent mol wt of 130 kDa. Two color flow cytometric analysis of mononuclear cells stained with mouse monoclonal antibodies specific for the human IGF-I receptor, the human insulin receptor, and monoclonal antibodies directed against specific monocyte and lymphocyte subset cell surface antigens revealed that both IGF-I receptors and insulin receptors were present on nearly all monocytes and B-lymphocytes, but were present on only 2% of T-lymphocytes. We conclude from these data that among human peripheral blood nonactivated mononuclear cells, IGF-I binds to specific type I IGF receptors found predominantly on monocytes and B-lymphocytes.