Insulin-like Growth factor-I In Medium Research Articles

The release of insulin-like growth factor-I by luteinized human granulosa cells in vitro: regulation by growth hormone, oxytocin, steroids and cAMP-dependent intracellular mechanisms.

The aim of the present experiments was to demonstrate the release of insulin-like growth factor-I (IGF-I) by human granulosa cells, and to examine the role of growth hormone (GH), oxytocin, steroids and cAMP-dependent intracellular mechanism in its control. A significant accumulation of IGF-I in a serum-supplemented medium in which the human granulosa cells were cultured for 4 days was observed. The concentration of IGF-I in the medium was particularly high at 3 and 4 days of culture. The addition of GH (1-10,000 ng/ml) to the medium increased IGF-I secretion by the cells. A higher GH dose (100,000 ng/ml) was inhibitory. Oxytocin stimulated IGF-I release at doses of 10-10,000 ng/ml. Dibutyryl-cAMP, isobutyl-methyl-xanthine (inhibitor of cAMP catabolism) or forskolin (stimulator of cAMP production) inhibited IGF-I output at these doses. Additions of progesterone (1-1,000 ng/ml) did not affect IGF-I release, whilst adrostenedione and estradiol were stimulatory at doses of 1, 10, 100, 1,000 ng/ml and 10, 100 and 1,000 ng/ml respectively. Testosterone inhibited IGF-I at a dose of 1,000 ng/ml but not at lower doses (1, 10 or 100 ng/ml). Blockade of estradiol (but not of testosterone) in the medium by specific antisera (1 or 10%) significantly reduced IGF-I output. The same effect was observed with an antiserum to progesterone when added at 0.1%, whilst higher doses (1 or 10%) stimulated IGF-I secretion. The present observations demonstrate the involvement of peptide, steroid hormones and cAMP in the regulation of IGF-I secretion by luteinized human granulosa cells. In particular, both GH and oxytocin are stimulators of IGF-I release. Estradiol and androstenedione, but not testosterone, may also be stimulators of IGF-I output. The involvement of progesterone in this process can also not be excluded. A cAMP-dependent intracellular mechanism appears to play an inhibitory role in the regulation of IGF-I secretion by luteinized human granulosa cells.

A quantitative real-time RT-PCR assay for salmon IGF-I mRNA, and its application in the study of GH regulation of IGF-I gene expression in primary culture of salmon hepatocytes

The hormone insulin-like growth factor-I (IGF-I) regulates vertebrate growth. The liver produces most circulating IGF-I, under the control of pituitary growth hormone (GH) and nutritional status. To study the regulation of liver IGF-I production in salmon, we established a primary hepatocyte culture system and developed a TaqMan quantitative real-time RT-PCR assay for salmon IGF-I gene expression. A portion of the coho salmon acidic ribosomal phosphoprotein P0 (ARP) cDNA was sequenced for use as a reference gene. A systematic bias across the 96 well PCR plate was discovered in an initial IGF-I assay, which was corrected when the assay was redesigned. IGF-I mRNA levels measured with the validated assay correlated well with levels measured with an RNase protection assay, and were highest in liver compared with other tissues. We examined the time course of hepatocyte IGF-I gene expression over 48 h in culture, the response to a range of GH concentrations in hepatocytes from fed and fasted fish, and potential effects of variation in IGF-I in the medium. IGF-I gene expression decreased over time in culture in hepatocytes in plain medium, and in cells treated with 5 nM GH with or without a combination of metabolic hormones (1 μM insulin, 100 nM triiodothyronine, and 0.1 nM dexamethasone). GH stimulated IGF-I gene expression at all time points. In cells treated with GH plus metabolic hormones, IGF-I gene expression was intermediate between the controls and GH alone. Increasing concentrations of GH resulted in biphasic IGF-I gene expression response curves in cells from fed and fasted fish, with the threshold for stimulation from 0.5 to 2.5 nM GH, maximal response from 5 to 50 nM, and a reduced response at 500 nM. Medium IGF-I (5 nM) did not affect basal or GH stimulated IGF-I gene expression. This study shows that primary hepatocyte culture and the TaqMan IGF-I assay can be used to study the regulation of hepatic IGF-I gene expression in salmon, and provides the first evidence of a biphasic response to GH concentration in fish hepatocyte culture.

Stimulating Effect of Recombinant Human Erythropoietin (rhEPO) on Insulin-like Growth Factor-I (IGF-I) Production by Hepatocytes in Rats with or without Subtotal Nephrectomy

S We investigated the effect of recombinant human erythropoietin (rhEPO) on insulin-like growth factor-I (IGF-I) production in primary cultured rat hepatocytes. Hepatocytes were obtained by in situ collagenase perfusion from adult male Wistar rats with sham-operation (control rats) or 5/6 nephrectomy (chRF rats), in which renal function was impaired and associated with renal anemia 4 weeks after the surgery. The hepatocytes were cultured at a density of 5 X 10 5 cells/mL in Williams' medium E with different doses of rhEPO for 48 h. IGF-I concentrations were measured by specific radioimmunoassay after acid-ethanol extraction. Plasma EPO levels were lower in chRF rats than in control rats at 4 weeks after the surgery (1.8′0.1 vs 2.6′0.3 mU/mL, P<0.05), whereas plasma IGF-I levels were rather higher in chRF rats (778.3 ′53.5 vs 636.0′70.0, P<0.05). Medium IGF-I levels were not different between the two groups (control rats 9.8′1.7 ng/mL vs chRF rats 10.1′ 1.8 ng/mL) and were dose-dependently increased by rhEPO (10 - 1 1 -10 - 8 M) in both groups of animals. An increment of medium IGF-I after the incubation with 10 - 8 M rhEPO for 48 h was 5 fold greater in control rats than in chRF rats (Δ13.6′3.9 vs Δ2.7′1.5 ng/mL, P<0.05). These findings suggest that EPO directly stimulates IGF-I release from rat hepatocytes and that the stimulating effect of EPO is blunted in chRF rats.

Stimulatory effect of zinc on insulin-like growth factor-I and transforming growth factor-beta1 production with bone growth of newborn rats.

The effect of zinc, an essential trace element, on insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 production was investigated to determine the role of this metal in bone growth of newborn rats. Femoral-diaphyseal and metaphyseal tissues were obtained between 1 and 28 days after birth of newborn rats, and cultured for 24 h in a serum-free Dulbecco's modified Eagle's medium containing either vehicle or zinc sulfate (10(-6) - 10(-4) M). Protein concentration in the medium was significantly increased by culture with bone tissues of newborn rats with increasing age (14 and 21 days). Medium IGF-I and TGF-beta1 concentration was gradually reduced with increasing age after birth. The presence of zinc (10(-5) and 10(-4) M) caused a significant increase in protein, IGF-I, and TGF-beta1 concentrations in the medium cultured with the diaphyseal or metaphyseal tissues obtained at 7 and 14 days after birth. The expression of IGF-I and TGF-beta1 mRNA was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) analysis in the diaphyseal and metaphyseal tissues cultured for 24 h using rat IGF-I or TGF-beta1-specific primers. These expressions were significantly raised in the presence of zinc (10(-4) M) in culture medium. The present study demonstrates that zinc has a stimulatory effect on IGF-I and TGF-beta1 production in the femoral tissues with bone growth of newborn rats.

Direct adenovirus-mediated insulin-like growth factor I gene transfer enhances transplant chondrocyte function.

Cell-based cartilage-resurfacing procedures may be enhanced by the addition of insulin-like growth factor I (IGF-I) to the transplant biomatrix. Given the relatively short half-life of IGF-I in biological systems, however, maintenance of effective concentrations of this peptide necessitates either high initial doses, or repeated treatment. This study investigated IGF-I delivery via adenoviral gene therapy, targeting graftable articular chondrocytes. Cultured articular chondrocytes were infected with an E1-deleted adenoviral vector containing IGF-I-coding sequence under CMV promoter control. Increased adenovirus-IGF-I concentrations resulted in coordinate increase in IGF-I mRNA and ligand expression; however, chondrocyte matrix synthesis was maximized by the lower adenovirus-IGF-I concentration (100 MOI) without additional increase at 200 or 500 MOI. Using 100 MOI, infected monolayers produced medium IGF-I content of at least 10 ng/ml in each 48-hr period for 28 days, reaching a day 4 peak concentration of 66 +/- 4.0 ng/ml. These concentrations were sufficient to produce significant stimulation of normal cartilage matrix gene expression. The concentration of secreted matrix products in medium from infected monolayers was increased up to 8-fold over uninfected control cultures. Moreover, compared with uninfected cultures, cells in infected cultures were more resistant to de-differentiation over time under serum-starved conditions, maintaining a normal chondrocyte molecular phenotype for at least 28 days. These data indicate that cultured chondrocytes are readily transduced by recombinant adenoviral vectors. The adenoviral-IGF transgene is abundantly expressed and its product secreted at therapeutic concentrations for at least 28 days, resulting in increased matrix biosynthesis and maintenance of the chondrocytic phenotype. Combined, this information suggests that there may be significant value in preimplantation adenoviral-IGF gene therapy for chondrocytes destined for cartilage resurfacing.

Effect of Transforming Growth Factor-β on the Insulin-like Growth Factor-I Autocrine/Paracrine Axis in Cultured Rat Articular Chondrocytes

Transforming growth factor-β (TGF-β) and insulin-like growth factor-I (IGF-I) are essential anabolic factors in articular cartilage. In this study, we concentrated on the elucidation of TGF-β interaction with IGF-I on cell growth and differentiation in monolayer articular chondrocytes obtained from 5-week-old rats. TGF-β (1 ng/ml) and IGF-I (25 ng/ml) stimulated DNA synthesis about 6.5- and 2.1-fold over control values, respectively. When TGF-β and IGF-I were added in combination, DNA synthesis was enhanced about 10.4-fold, indicating that the two peptides act in synergism. This synergistic action was also present in the expression of aggrecan mRNA. To study the mechanism of synergistic action, the effect of TGF-β on the IGF-I autocrine/paracrine axis was investigated. Administration of increasing concentrations of TGF-β (0.1-10 ng/ml) resulted in a dose-dependent decrease in medium IGF-I concentration that was reflected by decreased levels of IGF-I mRNA. TGF-β also inhibited the production of a 41-kDa IGF-binding protein into the culture medium. Pretreatment with TGF-β (1 ng/ml) for 12 h increased the binding of [125I]IGF-I to 140% of control by increasing the number of receptors without changes of affinity. Immunoprecipitation against phosphorylated tyrosine indicated that IGF-I-dependent autophosphorylation of IGF-I receptor β-subunit was inhibited by simultaneous TGF-β stimulation. These observations demonstrate that TGF-β acts synergistically with IGF-I and regulates the IGF-I autocrine/paracrine axis via a complex regulatory mechanism with decreased production of IGF-I and IGFBPs and dephosphorylation of IGF-I receptor, whereas there is an apparent up-regulation of the binding of [125I]IGF-I.

Anabolic effects of 3,3',5-triiodothyronine and triiodothyroacetic acid in cultured neonatal mouse parietal bones.

We previously reported that both T3 and triiodothyroacetic acid (Triac) stimulate bone resorption in organ culture, with Triac somewhat more potent than T3. In this study we compared the effects of T3 and Triac on DNA and protein synthesis in cultured neonatal mouse calvariae. After 24 h of preculture, with 72 h of treatment, but not 24 or 48 h, these hormones stimulated [3H]thymidine incorporation. After 72 h of preculture, stimulation was observed with only 24 h of treatment. These effects were significant at 10(-10) M and maximal at 10(-9) M for both T3 and Triac (treated/control ratios, 2.2 and 2.0, respectively). In contrast to their effects on bone resorption, T3 was more potent than Triac in stimulating [3H]thymidine incorporation. Insulin-like growth factor-binding protein-3 did not decrease the stimulation of DNA synthesis by T3 or Triac. The medium insulin-like growth factor-I concentration was less than 10(-10) M and was not regulated by these hormones. T3 and Triac stimulated [3H]proline incorporation into both collagen and noncollagen protein to a similar extent (approximately 30% at 10(-8) M). Indomethacin did not alter the effects of T3 and Triac on DNA or collagen synthesis. Aphidicolin, which blocked DNA synthesis completely, partially decreased the effects of thyroid hormones on collagen synthesis. The effect of aphidicolin on bone formation was less than that observed previously on bone resorption. We speculate that the effects of thyroid hormones on bone formation as well as bone resorption may be partially dependent on their mitogenic effects. As Triac is more potent in stimulating resorption and less potent in stimulating formation, Triac may be more catabolic than T3.

Regulation of Growth Hormone (GH) Secretion by GH-Releasing Factor, Somatostatin, and Insulin-Like Growth Factor I in Ovine Fetal and Neonatal Pituitary Cellsin Vitro*

Serum GH concentrations in the ovine fetus are much higher than those in the neonate, and the maximal GH response induced by GRF is 5-fold greater in the fetus than in the neonate. To clarify these in vivo observations further, we studied the effects of GRF, somatostatin (SRIF), and insulin-like growth factor I (IGF-I) on primary cultures of fetal and neonatal ovine pituitary cells. GH secretion from fetal ovine pituitary cells increased from 148 +/- 34 to 950 +/- 130 ng/10(5) cells.3 h in response to 1 nM GRF, whereas GH secretion from neonatal pituitary cells rose from 113 +/- 26 to 1221 +/- 129 ng/10(5) cells.3 h, a significantly greater response (P less than 0.001). This greater GRF-induced GH response in neonatal than fetal cells differs from the response in vivo and suggests that the increased in vivo response in the fetus is not due to inherently increased sensitivity of pituitary cells to GRF. SRIF (10 nM) decreased maximal GRF-induced GH secretion by 37 +/- 3% in fetal cells compared with 59 +/- 8% in neonatal cells (P less than 0.01). This may explain in part the decreased in vivo sensitivity to SRIF in the ovine fetus compared to that in the neonatal lamb. In fetal pituitary cells, 10 nM GRF increased ovine (o) GH mRNA from 100 +/- 14% to 145 +/- 40%, SRIF decreased oGH mRNA to 84 +/- 3%, and GRF and SRIF in combination increased fetal oGH mRNA to 126 +/- 24%. Values in neonatal pituitary cell cultures were similar (control, 100 +/- 17%; GRF, 132 +/- 6%, SRIF, 85 +/- 15%; GRF plus SRIF, 105 +/- 26%). Pretreating fetal cells with 100 nM IGF-I for 3 days reduced GRF-stimulated GH secretion from 1049 +/- 38 to 232 +/- 8 ng/10(5) cells.3 h (P less than 0.001). Similarly, IGF-I pretreatment of neonatal cells reduced GRF-stimulated GH secretion from 810 +/- 18 to 419 +/- 16 ng/10(5) cells.3 h (P less than 0.001). The mean secreted IGF-I was 0.58 U/ml (36 nM) in culture medium from neonatal cells and was unchanged by incubation for 3 days with 5 micrograms/ml hGH. Secreted IGF-I in medium from fetal cells was 0.87 U/ml (54 nM) without GH and 0.81 U/ml (51 nM) after incubation with human GH. IGF-I mRNA was present in neonatal pituitary and brain.(ABSTRACT TRUNCATED AT 400 WORDS)