Effects of insulin-like growth factor-I and insulin on the in-vitro uptake of sulphate by eel branchial cartilage: evidence for the presence of independent hepatic and pancreatic sulphation factors.

The possible roles of insulin-like growth factor-I (IGF-I) and insulin in regulating cartilage growth were studied in the teleost Anguilla japonica. Significant sulphation activity was found in the extracts of pancreas, liver and muscle, but not in those of kidney, intestine or spleen. The hepatic sulphation activity was significantly decreased by hypophysectomy or by fasting for 14 days, suggesting that this activity is regulated by pituitary function and nutritional status. Northern blot analysis revealed that the hepatic IGF-I mRNA in the eel consists of a major 4.0 kb band. This mRNA was GH-dependent and was significantly decreased by fasting for 14 days. On the other hand, fasting for 14 days had no significant effect on pancreatic sulphation activity. Pancreatic extracts from both intact and hypophysectomized eels exhibited equally significant stimulating activity. Addition of bovine or human insulin (1-250 ng/ml) to the culture medium significantly stimulated sulphate uptake in a dose-dependent manner. Teleost (coho salmon) insulin was as effective as bovine insulin. Bovine insulin was more effective than IGF-I at lower concentrations (1-4 ng/ml) but less effective at higher concentrations (10-250 ng/ml). These results indicate that not only IGF-I but also insulin are likely to be involved in the regulation of cartilage growth in the eel.     

Effects of estrogens in vitro and in vivo on cartilage growth in the tilapia (Oreochromis mossambicus)

To study the effects of estrogens on cartilage growth in the tilapia Oreochromis mossambicus, an epiceratobranchial cartilage radioisotope incorporation assay was employed to measure proteoglycan synthesis and prechondrocyte proliferation by incorporation of radiolabeled sulfate and thymidine, respectively. Cartilage explants were cultured with estrogens with or without recombinant bovine insulin-like growth factor-I (IGF-I). In vitro experiments using the natural teleost estrogen, 17beta-estradiol (E2), showed a trend toward inhibition of sulfate incorporation and an inhibition of thymidine incorporation at higher doses (10 micrograms/ml), but not at physiological levels. E2 also showed a trend toward inhibition of sulfate and thymidine incorporation in the presence of IGF-I. Similar results were found with other estrogenic compounds in vitro: ethinylestradiol, diethylstilbestrol (DES), genistein, and nonylphenol. Ethinylestradiol inhibited sulfate and thymidine incorporation at 1000 ng/ml in the presence of IGF-I. DES inhibited thymidine incorporation at 1000 ng/ml in untreated or IGF-I-exposed cartilage. Genistein inhibited sulfate incorporation at 100 micrograms/ml in IGF-I-exposed cartilage and inhibited thymidine uptake at 1, 10, and 100 micrograms/ml in untreated and IGF-I-exposed cartilage. Nonylphenol inhibited sulfate uptake at 100 microM in untreated and IGF-I-exposed cartilage. Nonylphenol alone at 10 and 100 microM inhibited thymidine uptake. In IGF-I-exposed cartilage nonylphenol inhibited thymidine uptake at 100 microM. Fish receiving estrogen injections (E2 or DES) in vivo at a concentration of 2 micrograms/g body weight showed increased sulfate incorporation by cartilage in vitro. Stimulation in vivo by estrogens, in contrast to the inhibition by high doses in vitro, may be a result of the influence of estrogen on pituitary growth hormone release.     

Effects of glucocorticoids on cartilage growth and response to IGF-I in the tilapia (Oreochromis mossambicus)

To study the effects of glucocorticoids and IGF-I on the modulation of growth in the tilapia Oreochromis mossambicus, we employed an epiceratobranchial cartilage radioisotope incorporation assay, wherein radiolabeled sulfate and thymidine uptakes are measured in vitro to indicate proteoglycan synthesis and cell proliferation, respectively. Cartilage explants were cultured with cortisol or dexamethasone with or without recombinant bovine insulin-like growth factor-I. Cortisol directly inhibited sulfate uptake at 100 and 1000 ng/mL concentrations in a concentration-dependent manner but inhibited thymidine uptake significantly only at the 1000 ng/mL concentration. Dexamethasone inhibited sulfate and thymidine uptake at concentrations similar to the effective concentrations of cortisol. Cortisol did not inhibit IGF-I stimulation of sulfate uptake at any of the concentrations tested. Furthermore, cortisol did not inhibit thymidine uptake when IGF-I was present in the medium. Cortisol appears to act directly on cartilage and not by interacting with the IGF-I system. However, the physiologically significant role of cortisol is mainly an inhibitory one on cartilage metabolism. The data generally indicate an inhibitory role for glucocorticoids on cartilage growth but an inability to counter the stimulation of sulfate uptake by IGF-I.     

Stimulatory effects of insulin and insulin-like growth factor I on migration and tube formation by vascular endothelial cells.

The effects of insulin and insulin-like growth factor I (IGF-I) on migration, proliferation and tube-forming activity of endothelial cells were investigated, by using bovine carotid artery endothelial cells. Migration was assayed by a filter membrane technique and tube formation was assayed by a quantitative angiogenesis in vitro model which we have recently developed. In this model, endothelial cells are cultured between two layers of type I collagen gel and become organized into tube-like structures which mimic capillaries in vivo ultrastructurally. Insulin (50-1000 microunits/ml) and IGF-I (10-200 ng/ml) significantly stimulated migration of endothelial cells in a dose-dependent manner with a maximal stimulation of 3.0-fold at 1000 microunits/ml for insulin and 3.8-fold at 200 ng/ml for IGF-I (P less than 0.01). Insulin at concentrations up to 1000 microunits/ml and IGF-I up to 100 ng/ml did not affect proliferation of endothelial cells. When insulin or IGF-I was added in culture medium on collagen gels, tube-forming activity of endothelial cells was markedly stimulated. The specific lengths of tubes significantly increased with the increase in insulin concentration from 25 to 100 microunits/ml (P less than 0.01). At 100 microunits/ml, the stimulation was 1.77-fold (P less than 0.01). IGF-I (1-100 ng/ml) also stimulated the elongation of tubes dose-dependently with a maximal stimulation of 1.96-fold at 100 ng/ml (P less than 0.01). Thus, insulin and IGF-I at pathophysiological concentrations stimulate migration and tube-forming activity of endothelial cells, suggesting that these polypeptides may stimulate repair of endothelial injury in cases such as atherosclerosis and may act as a stimulator of angiogenesis.     

Insulin-like growth factor I and insulin potentiate luteinizing hormone-induced androgen synthesis by rat ovarian thecal-interstitial cells

We tested the hypothesis that insulin-like growth factor I (IGF-I) and insulin play a role in androgen production by rat ovarian thecal-interstitial cells. Collagenase/DNase-dispersed rat ovarian thecal-interstitial cells obtained from immature hypophysectomized Sprague-Dawley rats were cultured at a concentration of 10(6) cells/ml in serum-free medium in the presence of increasing concentrations of LH, IGF-I, or insulin. The medium was replaced every 48 h, and the androsterone concentration in the culture supernatants was used as an index of androgen production. In the absence of added hormones (control) androsterone levels were consistently less than 0.1 ng/ml. Increasing concentrations of LH stimulated androsterone synthesis in a dose-dependent manner. IGF-I, in the absence of LH, did not significantly increase androsterone levels above control values. However, when combined with 10 ng/ml LH, IGF-I increased androsterone synthesis above levels seen with LH alone in a dose-related fashion: for example, the peak androsterone levels seen with LH and 100 ng/ml (13 nM) IGF-I at 96 h of culture were significantly greater than the peak level seen with 10 ng/ml LH alone (302 +/- 71 vs. 17 +/- 7 ng/ml; P less than 0.0125). Similarly, while insulin alone did not increase androsterone synthesis above control values, androsterone concentrations were increased by insulin in combination with 10 ng/ml LH; a peak value of 240 +/- 67.7 ng/ml was observed at 96 h of culture with 100 ng/ml (18 mM) insulin (P less than 0.025 vs. LH alone) Androsterone levels were slightly less with insulin than with IGF-I, but this difference was not significant. The combination of IGF-I and insulin did not increase levels of androsterone synthesis above those observed with each hormone alone. IGF-I bound to a high affinity binding site on ovarian cell monolayer cultures with an apparent binding affinity of 1.3 x 10(-9) M. Insulin also competed for binding with radiolabeled IGF-I in a dose-dependent manner, but the affinity of insulin was approximately 500-fold less; half-maximal inhibition of [125I] IGF-I binding occurred with an insulin concentration of approximately 300 nM (or approximately 1700 ng/ml). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of thecal-interstitial cell monolayers affinity labeled with radiolabeled IGF-I in the absence and presence of unlabeled hormone revealed proteins with characteristics of type I IGF receptors. Affinity labeling to a protein of a relative molecular mass of approximately 45,000 was also noted, probably representing IGF carrier proteins synthesized by thecal-interstitial cell monolayers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Epidermal growth factor stimulates glycogen synthesis in fetal rat hepatocytes: comparison with the glycogenic effects of insulin-like growth factor I and insulin

The effects of epidermal growth factor (EGF) on glycogen metabolism and the binding of [125I]iodo-EGF to receptors in fetal rat hepatocytes have been examined. The actions of EGF have been compared with those of insulin-like growth factor I (IGF-I) and insulin. EGF (0.1-45 nM) stimulated dose-dependent increases in [14C]glucose incorporation into glycogen (8.8-31.1%, P less than 0.01) and total cellular glycogen content (5.6-21.4%, P less than 0.05). The concentration of EGF causing half-maximal stimulation of glycogen synthesis was 2 ng/ml, and maximal stimulation occurred at 1 h of incubation. EGF had no effect on the uptake of the nonmetabolizable monosaccharide [14C]O-methyl-D-glucose, suggesting that the glycogenic effect of EGF was not mediated through stimulation of glucose transport. Although IGF-I (1-100 nM) and insulin (14 nM to 10 microM) also stimulated glycogen synthesis in fetal liver, the maximal effects of these hormones occurred at 2 h incubation, and the dose-response curves of IGF-I and insulin were not parallel to that of EGF. In addition, the maximal glycogenic effect of EGF was only 40% that of insulin or IGF-I, and the effects of EGF and insulin on [14C]glucose incorporation were additive. These findings suggest that EGF stimulates glycogen synthesis through a mechanism distinct from that of IGF-I or insulin. The binding of [125I]iodo-EGF to fetal hepatocytes was specific, saturable, and time- and temperature-dependent. Maximal specific binding occurred at 1 h of incubation at 37 C or at 24 h of incubation at 4 C. Unlabeled EGF (0.05-250 ng/ml) caused a dose-dependent inhibition of the binding of [125I]iodo-EGF to fetal hepatocytes, with half-maximal displacement of [125I]iodo-EGF by 1.7 ng unlabeled EGF/ml. The specific binding of [125I] iodo-EGF was not inhibited by high concentrations of insulin or IGF-I, suggesting that the differences in the mechanisms by which EGF, insulin, and IGF-I stimulate glycogenesis may be explained in part by differences in the binding of these hormones to fetal liver receptors. In addition to having mitogenic effects in fetal tissue, EGF or other EGF-like growth factors may have acute effects on fetal hepatic intermediary metabolism and may contribute to the accumulation of liver glycogen in the mammalian fetus during late gestation.     

Sharing of biological effect and receptors between guinea pig insulin and platelet-derived growth factor.

Insulins from the hystricomorphs (guinea pig, porcupine, coypu, and casiragua) at high concentration stimulate DNA synthesis in human fibroblasts to a greater level than other mammalian insulins or insulin-like growth factors (IGFs). 125I-Labeled guinea pig insulin binds to a specific receptor and this binding is competed for by hystricomorph insulins but not by porcine insulin or IGFs. Fetal bovine serum also inhibits the binding of 125I-labeled guinea pig insulin and is more potent than fetal bovine plasma, in concordance with their relative potencies for growth stimulation in human fibroblasts. Of several other known growth factors tested, only platelet-derived growth factor (PDGF) inhibits binding of 125I-labeled guinea pig insulin. Four preparations of PDGF that vary in purity and potency for the stimulation of DNA synthesis in human fibroblasts over a 1,000-fold range compete with binding of 125I-labeled guinea pig insulin in proportion to their biological potencies. The purest preparation of PDGF is able to inhibit binding of 125I-labeled guinea pig insulin by 50% at 15 ng/ml (0.25 nM). Biologically, guinea pig insulin, like PDGF, exhibits a synergistic effect with plasma in initiating DNA synthesis in human fibroblasts; this effect is not observed with other mammalian insulins or IGFs. Thus, hystricomorph insulins appear to be mediating their growth-promoting effect through a different receptor and mechanism than other mammalian insulins or IGFs. Further, hystricomorph insulins may be sharing the mechanism of action for their growth effects with PDGF, perhaps suggesting some relationship between these peptides from very different sources.     

Effects of gonadotropins, insulin and insulin-like growth factor I on ovarian oxytocin and progesterone production.

Oxytocin is produced in the granulosa-derived cells of the ruminant corpus luteum where its gene is dramatically up-regulated within days of ovulation. Regulation of these processes is poorly understood but oxytocin release can be increased by insulin, insulin-like growth factor I (IGF-I), and gonadotropins. Here we have assessed interactions between these regulatory systems. Follicle-stimulating hormone (FSH), luteinizing hormone (LH) and human chorionic gonadotropin (hCG) caused dose-dependent release of oxytocin from bovine granulosa cells cultured in medium containing 100 ng/ml insulin. The gonadotropins also increased oxytocin mRNA levels and their effects were mimicked by forskolin. The effects of these stimuli on oxytocin and progesterone release were synergistically increased by insulin or IGF-I. Binding studies revealed separate binding sites with characteristics of insulin and IGF-I receptors. Insulin potentiated the effects of hCG and forskolin on oxytocin mRNA levels and release of oxytocin and progesterone in cells from follicles containing greater than 50 ng/ml estradiol. In cells from follicles containing less than 5 ng/ml estradiol these stimuli had little effect on oxytocin release although progesterone release was synergistically increased by insulin and forskolin. The data suggest that gonadotropins regulate oxytocin synthesis and release and that these effects are amplified by insulin or IGF-I acting via their own receptors. Changes associated with maturation of the target cells in vitro appear prerequisite for oxytocin production in response to increased cAMP levels in the presence of insulin or IGF-I.     

Growth regulation in the gobiid teleost, Gillichthys mirabilis: roles of growth hormone, hepatic growth hormone receptors and insulin-like growth factor-I.

Studies of the teleost Gillichthys mirabilis were undertaken to assess the role of GH in regulating hepatic GH receptors, insulin-like growth factor-I (IGF-I) activity and cartilage growth. Hypophysectomized G. mirabilis were injected with saline vehicle or with 10, 100 or 1000 ng tilapia GH (tGH)/g every other day for 2-3 weeks. Growth, judged by increased body weight and length, was inhibited by hypophysectomy and stimulated by increasing tGH doses. Hepatic GH receptors, measured by 125I-labelled tGH binding, were decreased 50% by hypophysectomy. An additional dose-dependent reduction in binding was observed 24 h after tGH injection, but MgCl2 stripping of membranes suggested receptor occupation by exogenous tGH. IGF-I activity, measured by 35SO4 incorporation into oral cartilage explants in vitro, was decreased 50% by hypophysectomy and increased to 200% of intact control levels after injection of 1000 ng tGH/g. In a second experiment, 35SO4 incorporation by oral cartilage from hypophysectomized fish injected with 10, 100 or 1000 ng tGH/g was stimulated to intact control levels. Effects of feeding and tGH injection on in-vitro responsiveness of oral cartilage to recombinant bovine IGF-I (rbIGF-I; 10-1000 ng/ml) were also assessed. Oral cartilage from fed fish showed a parabolic dose-response curve, whereas oral cartilage from starved fish had a lower basal rate of 35SO4 incorporation and a linear dose-response relationship. Oral cartilage from hypophysectomized G. mirabilis showed a significantly attenuated response to rbIGF-I which was restored by tGH injection, suggesting that the GH status of the animal is important for sensitivity of target tissue to IGF-I. Because of its similarity to other vertebrate systems, G. mirabilis presents a good teleost model of growth regulation and of the functions and interactions of GH and IGF-I.     

Characterization of a specific insulin-like growth factor-I/somatomedin-C receptor on high density, primary monolayer cultures of bovine articular chondrocytes: regulation of receptor concentration by somatomedin, insulin and growth hormone

In order to obtain a phenotypically stable cell population of chondrocytes, high density primary monolayer cultures of bovine articular chondrocytes were established. Using these cultures, a specific insulin-like growth factor-I/somatomedin-C (IGF-I/SM-C) receptor was demonstrated and characterized. At 15 degrees C steady-state binding was attained by 5 h, and averaged 25% per 2.2 X 10(6) cells. Fifty per cent displacement of 125I-labelled IGF-I/SM-C by unlabelled IGF-I/SM-C occurred at concentrations of only 2.3 ng/ml, whereas IGF-II and porcine insulin were approximately 15- and 1000-fold less potent respectively. Scatchard analysis gave a linear plot, with a calculated association constant of 2.26 X 10(9) l/mol and a receptor number of 15 400 sites per cell. Preincubation of chondrocyte monolayers with either IGF-I SM-C or porcine insulin at 37 degrees C for 20 h resulted in reduction of 125I-labelled IGF-I/SM-C binding in a dose-dependent manner, although higher concentrations were required with insulin. More than 40% down-regulation of the receptor occurred with IGF-I/SM-C at concentrations of 10 nmol/l and nearly 70% reduction at 50 nmol/l. Interestingly, after preincubation with either human (h) or bovine (b) GH, 40% down-regulation of 125I-labelled IGF-I/SM-C binding was observed at concentrations of 10 mumol/l. Local production of IGF-I/SM-C by chondrocytes in response to GH stimulation may have occurred, but, because only 120 pmol IGF-I/SM-C and less than 30 pmol IGF-I/SM-C per litre were recovered from serum-free conditioned media preincubated with bGH and hGH respectively, this was not established.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unprocessed insulin proreceptor in cultured fibroblasts from a patient with extreme insulin resistance

Insulin receptors and IGF-I receptors in cultured fibroblasts were investigated in a patient with extreme insulin resistance due to unprocessed insulin receptors. Insulin binding to cultured fibroblast monolayers and partially purified insulin receptors was extremely decreased to 27% and 18% of control value, respectively. Affinity cross-linking study revealed that molecular weight of the insulin receptor was 210 kDa and that it could not be dissociated to alpha- and beta-subunit with dithiothreitol treatment. Because IGF-I binding to the fibroblasts from the patient was normal and alpha-subunit of IGF-I receptor was 135 KDa, the defect was specific to the insulin receptor. Autophosphorylation of the 210 kDa unprocessed insulin proreceptor was stimulated by insulin in a dose-dependent manner. In the fibroblasts from the patient, insulin-stimulated alpha-aminoisobutyric acid uptake was fivefold shifted to the right in the dose-response curve (ED50 20 ng/mL for the patient v 3.5 ng/mL for the control subjects), but the maximally stimulated uptake was normal. With 0.025% trypsin treatment, insulin binding and alpha-aminoisobutyric acid uptake were normalized. These results suggested that (1) abnormal processing of insulin proreceptor also occurred in the cultured fibroblasts, (2) the postreceptor steps of insulin action were totally intact, and (3) IGF-I receptors were normally processed in this patient.     

Action of multiplication-stimulating activity on [3H]thymidine incorporation in rabbit and human fetal chondrocytes in vitro

The mitogenic action of multiplication-stimulating activity (MSA) on normal mammalian chondrocytes has been examined. Addition of MSA (NIH, PkII-MSA, 2.5-500 ng/ml or Collaborative Research, CR-MSA, 50-250 ng/ml) to primary suspensions of chondrocytes prepared by enzymic digestion of costal and articular cartilage of rabbits (356-481 g body wt) resulted in a dose-dependent increase in [3H]thymidine incorporation into the trichloroacetic acid-precipitated cell contents. CR-MSA (50-250 ng/ml) also had a significant stimulatory effect on [3H]thymidine incorporation into human fetal chondrocytes (22 weeks of gestation) prepared by enzymic digestion. When PkII-MSA was added in the presence of 1.25% of a standard adult or cord plasma to either rabbit or human fetal (18 weeks) chondrocytes, the increase in [3H]thymidine incorporation appeared to be synergistic. The mitogenic action of MSA can thus be demonstrated on primary suspensions of mammalian chondrocytes. The action of MSA on human chondrocytes has not previously been reported.     

Effects of growth hormone-releasing factor and vasoactive intestinal peptide on proliferation and steroidogenesis of bovine granulosa cells.

Recent studies have suggested that growth hormone-releasing factor (GRF), like vasoactive intestinal peptide (VIP), may enhance follicle-stimulating hormone (FSH)-stimulated steroidogenesis in cultured rat granulosa cells (GC). Because effects of GRF or VIP on GC proliferation have not been reported, we evaluated and compared the effect of GRF to that of VIP using cultured bovine GC. Undifferentiated GC from 1-5 mm bovine follicles were established for 2 days in medium containing 10% fetal calf serum, washed and then cultured in chemically defined medium for an additional 2 days. Two-day treatment with 2.5-1000 ng/ml of VIP had no effect (P greater than 0.05) on proliferation or progesterone production of bovine GC in the presence or absence of 200 ng/ml FSH. In comparison, 100, 250, 500, 1000 or 2000 pg/ml of human [desNH2Tyr1,D-Ala2,Ala15]-GRF(1-29)-NH2 analog caused a dose-dependent stimulation (P less than 0.05) of GC proliferation in the absence and presence of 5 micrograms/ml insulin. However, the GRF analog had no effect (P greater than 0.05) on GC progesterone production (expressed as ng/10(5) cells/24 h) in the absence or presence of 5 micrograms/ml insulin. The effects of GRF analog on progesterone production and cell proliferation were not influenced by co-culture with 200 ng/ml FSH. GRF(1-44)-NH2 also stimulated cell proliferation but had no effect on basal or FSH-induced progesterone production. These results suggest that GRF may play a role in GC proliferation during follicular development in the bovine.     

Insulin stimulation of aminoisobutyric acid transport in human skin fibroblasts is mediated through both insulin and type I insulin-like growth factor receptors

A monoclonal antibody to the human type I insulin-like growth factor (IGF-I) receptor (alpha IR-3) was used to distinguish actions of insulin and IGF-I that are mediated through insulin as opposed to IGF-I receptors on human skin fibroblasts. Both insulin and IGF-I stimulate uptake of the nonmetabolized alanine analog alpha-aminoisobutyric acid (AIB) in these cells. alpha IR-3 inhibited AIB uptake stimulated by both of these hormones in a dose-dependent manner. However, the pattern of hormone action in the presence of alpha IR-3 differed for the two hormones. In the case of IGF-I, alpha IR-3 potently inhibited AIB uptake at low hormone concentrations, but this inhibition was overcome by high hormone concentrations, consistent with impairment of IGF-I action through the IGF-I receptor. In the case of insulin, the action of low concentrations (i.e. 10 ng/ml) was not inhibited, but that of higher insulin concentrations was, suggesting a dual receptor mechanism of cell stimulation by insulin. alpha IR-3 will be an important tool in further studies of the biology of the IGF-I receptor in normal and abnormal human cells.     

Effect of growth hormone and insulin-like growth factor-I on DNA synthesis and matrix production in rat epiphyseal chondrocytes in monolayer culture.

The influence of various culture conditions was studied on the effect of GH and insulin-like growth factor-I (IGF-I) on DNA and matrix synthesis in epiphyseal rat chondrocytes in monolayer culture. Chondrocytes from enzymatically digested rat tibia epiphyseal growth plates were seeded in 48-well culture plates and precultured for 10 days in Ham's F-12 medium supplemented with 1% (v/v) newborn calf serum and 1% (v/v) of a serum substitute. After preculture, the medium was changed to Ham's F-12 medium supplemented with 1% serum from hypophysectomized rats, and the effect of GH and IGF-I on DNA synthesis ([3H]thymidine incorporation) and matrix production ([35S]sulphate uptake) was studied during an additional 96-h culture period. Isotopes were present during the last 24 h of culture. Both hGH and IGF-I stimulated DNA synthesis in a dose-dependent manner. A maximal effect of GH was seen at a concentration of 25 micrograms/l (60 +/- 11% stimulation over control) and for IGF-I at 10 micrograms/l (162 +/- 12%). The stimulatory effects of the same concentrations of human GH (hGH) and IGF-I on [35S]sulphate uptake were 135 +/- 25 and 320 +/- 42% respectively. In-vitro pulse labelling revealed that GH did not produce a response during the first 3 days of culture (after addition of GH) but was effective during days 4 and 5 of culture. In contrast, IGF-I was effective throughout the culture period. Pretreatment of cells with GH or IGF-I for 2.5 days showed that GH but not IGF-I produced a sustained effect on [3H]thymidine uptake. In order to study the influence of cell density on the effect of GH and IGF-I on DNA synthesis, the effect of added peptides was evaluated after different preculture periods (5-15 days). A maximal stimulatory effect of hGH was seen at a cell density of 150,000-300,000 cells/cm2. GH had no significant effect at a low (less than 100,000 cells/cm2) or a high (greater than 400,000 cells/cm2) cell density. The magnitude of the stimulatory effect of IGF-I was the same at densities between 10,000 and 250,000 cells/cm2, but was reduced at higher cell densities (over 250,000 cells/cm2). Chondrogenic properties of cells that had been cultured for 15 days were verified in vitro by positive alcian blue staining and identification of type II collagen, and in vivo by development of cartilage nodules in nude mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of insulin-like growth factor I on deoxyribonucleic acid synthesis and galactopoiesis in bovine undifferentiated and lactating mammary tissue in vitro

We have demonstrated that insulin-like growth factor I (IGF-I), at physiological concentrations, is a potent mitogen of bovine undifferentiated mammary epithelial cells cultured in collagen in serum-free medium. Its activity is independent of insulin, although at pharmacological concentrations insulin may substitute for IGF-I. The maximal [3H]thymidine incorporation stimulated by either IGF-I or insulin was only 25-40% of that in medium supplemented with 10% fetal calf serum (FCS) only. Epidermal growth factor (EGF) exhibited low mitogenic activity which was not synergistic with IGF-I in serum-free medium. IGF-I and EGF had low synergistic activity when added separately to 10% FCS-supplemented medium. Strong synergism (100% or more) was observed, however, when both factors were added simultaneously, indicating that their maximum mitogenic effect is dependent on a simultaneous presence of other factors existing in FCS. The galactopoietic effect of IGF-I was tested in organ culture of bovine lactating mammary gland. Neither fatty acid synthesis nor alpha-lactalbumin secretion was stimulated by IGF-I, even at 2000 ng/ml. These results indicate that, at least in our in vitro system, galactopoiesis is not affected by IGF-I.     

Calf serum modifies the mitogenic activity of epidermal growth factor in WRT thyroid cells

It has already been shown that Wistar rat thyroid (WRT) cells in low concentrations of calf serum (0.5%) are under the influence of both thyrotropin (TSH) and insulin as regards growth. The present data show that epidermal growth factor (EGF), in concentrations up to 10 micrograms/ml, is not able to modify DNA synthesis in WRT cells. On the other hand, insulin-like growth factor I (IGF-I) stimulates DNA synthesis from a dose which is 10-fold lower than that of insulin alone. Combined stimulation of EGF and TSH in WRT cells is equal to that of TSH alone in relation to DNA synthesis, while the combined presence of TSH and IGF-I, or TSH and insulin, in the same medium results in an effect which is greatly superior to the theoretical sum of activities. Repetition of the same experiments using the original clone of WRT cells, but in high concentrations of calf serum (5%), shows that EGF stimulates DNA synthesis in a dose-dependent way from 0.1 to 100 ng/ml. Under these conditions, combined stimulation of EGF with TSH shows that DNA synthesis is equal to the predicted theoretical sum. No other differences in WRT cell sensitivity to either IGF-I or insulin, or IGF-I and TSH and insulin and TSH, can be noted. This finding is confirmed by the demonstration of specific and sensitive binding sites for EGF on WRT cells cultured in 5% calf serum; these binding sites are not present on WRT cells adapted to grow in 0.5% calf serum. Present data support the hypothesis that EGF and serum growth actions are mediated through the same analogous pathway, which is, however, different from those of TSH and/or IGF-I and/or insulin.     

The effects of insulin and growth hormone on the release of somatomedin by the isolated rat liver.

Livers from hypophysectomized (hypox) rats were perfused with oxygenated Waymouth's medium in a system which permitted continuous recirculation for separate 30 minute periods after which fresh medium was supplied. In most experiments 6 changes of medium were carried out over a 3 hour period. The somatomedin activity of each perfusate was determined by measuring its ability to stimulate sulfate uptake in hypox rat cartilage in vitro. For comparison between experiments the results are expressed as the per cent stimulation of sulfate uptake by the perfusate compared with the unperfused buffer. Without hormonal additions there was a progressive fall in the release of somatomedin activity during the 6 periods of study. When compared with the results without hormone, the addition of 1000 muU/ml of insulin per ml of medium during the 2nd to 6th period led to a significant increase in perfusate somatomedin activity at all periods. The addition of 100 muU/ml of insulin was without significant effect. The possible inter-relationship between insulin and growth hormone in the regulation of somatomedin release was studied with a dose of bGH of 250 ng/ml which had previously been shown to be insufficient by itsel to stimulate somatomedin release. When added to a medium containing 1000 muU/ml of insulin, this dose of bGH did not significantly stimulate somatomedin release beyond that obtained with insulin alone. However, when 250 ng/ml was added to a medium containing 100 muU/ml insulin, a significant stimulation of somatomedin release was observed while the addition of each hormone separately was without significant effect. These results support the hypothesis that insulin shares with GH the regulation of somatomedin release by the liver. Differences in insulin concentration may explain some clinical situations in which somatomedin concentrations cannot be correlated with GH levels.