Decrease In Insulin-like Growth factor-I mRNA Research Articles

Hypoenergetic High-Carbohydrate or High-Fat Parenteral Nutrition Induces a Similar Metabolic Response with Differential Effects on Hepatic IGF-I mRNA in Dexamethasone-Treated Rats

The optimal level of energy for critically ill patients who require parenteral nutrition (PN) is unclear. Our objective was to determine whether 50% energy (50%E) restriction due to a reduction in carbohydrate or fat, with provision of adequate protein and micronutrients, ameliorates the detrimental effects of dexamethasone (Dex) on body protein catabolism, insulin resistance, and insulin-like growth factor-I (IGF-I) responses in rats administered PN. The experiment included 6 PN groups, adequate energy (AE) +/- Dex, 50% AE with high carbohydrate (50%E CHO) +/- Dex and 50% AE with high fat (50%E FAT) +/- Dex. There was a significant interaction between energy level and Dex such that the increase in body catabolism due to 50%E from CHO or FAT was reduced by approximately 50%, although the amount of body weight and nitrogen lost over 7 d was significantly greater with 50%E than with AE. AE+Dex induced a 60% increase in liver mass, whereas 50%E+Dex reduced the increase to 26%. AE+Dex induced a 5-fold increase in serum insulin level, whereas 50%E+Dex normalized the insulin to glucose ratio. Serum IGF-I levels were reduced 14-18% by Dex and 30% by 50%E. Hepatic immunoreactive IGF-I was significantly correlated with serum IGF-I and nitrogen balance. 50%E CHO and 50%E FAT had differential effects on hepatic IGF-I mRNA with a 40% decrease in IGF-I mRNA due to 50%E FAT+Dex. In summary,CHO or FAT hypoenergetic PN with adequate protein had similar effects in normalizing hyperinsulinemia, attenuating hepatomegaly, and reducing the increment, but not the total amount of body protein catabolism, induced by glucocorticoid excess.

Progesterone down-regulates insulin-like growth factor-I expression in cultured human uterine leiomyoma cells.

Insulin-like growth factor-I (IGF-I) plays crucial roles in uterine leiomyoma cell growth through stimulating proliferation and inhibiting apoptosis. The present study was conducted to elucidate whether progesterone affects IGF-I and its receptor expression in cultured leiomyoma cells. Isolated leiomyoma cells were subcultured in Phenol Red-free Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 120 h and then stepped down to serum-free conditions for an additional 48 h in the presence or absence of 17beta-estradiol (E(2)) (10 ng/ml) or progesterone (100 ng/ml). IGF-I and its receptor mRNA and immunoreactive IGF-I in the cultured cells were assessed by quantitative RT-PCR with Southern blot analysis and by radioimmunoassay with Seppak C18 chromatography, respectively. The presence of estrogen receptor (ER) and progesterone receptor (PR) in cultured leiomyoma cells was immunocytochemically examined. Both treatment with progesterone alone and treatment with E(2) and progesterone combined significantly decreased IGF-I mRNA and protein expression in cultured leiomyoma cells compared with that in untreated cultures, but treatment with E(2) alone did not. IGF-I receptor mRNA expression in those cells was not affected by treatment with either E(2) or progesterone. Immunocytochemical analysis revealed that PR protein expression in cultured leiomyoma cells maintained in a serum-free condition for 48 h whereas ER protein expression in the cells remarkably decreased after 24 h culture under the serum-free condition. The present study provided evidence for the first time that progesterone down-regulates IGF-I mRNA and protein expression in cultured leiomyoma cells without affecting IGF-I receptor mRNA expression.

Reduced Insulin-Like Growth Factor-I after Acute Feed Restriction in Lactating Dairy Cows is Independent of Changes in Growth Hormone Receptor 1A mRNA

Growth hormone receptor (GHR) and insulin-like growth factor-I (IGF-I) mRNA decrease in the liver of dairy cows at parturition. Epinephrine is released and feed intake is decreased at the same time. The objective of this study was to determine whether feed restriction and (or) administration of epinephrine could recapitulate the changes in the hepatic GHR 1A and IGF-I mRNA that occur at parturition. Eight lactating cows were randomly assigned to one of two rations (ad libitum or restricted). The cows assigned to the restricted ration were fed 75, 60, 60, and 25% of feed consumed by the ad libitum cows on successive days to mimic the changes in feed intake around parturition. Liver samples were collected by needle biopsy before and after feed restriction. Cows received either 0.02 mg/kg of epinephrine or saline approximately 24h before the second liver biopsy so that a 2 × 2 factorial arrangement of treatments was created. Feed restriction increased plasma nonesterified fatty acids and liver triglyceride percentages and decreased plasma IGF-I concentrations. The decrease in plasma IGF-I after feed restriction was associated with a decrease in IGF-I mRNA in feed-restricted cows. The amount of GHR 1A mRNA did not change after feed restriction. Epinephrine treatment did not affect the amount of GHR 1A or IGF-I mRNA. We conclude that the decrease in feed intake at parturition may be partially responsible for the decrease in IGF-I but may not cause the decrease in GHR 1A.

Reduced growth hormone receptor (GHR) messenger ribonucleic acid in liver of periparturient cattle is caused by a specific down-regulation of GHR 1A that is associated with decreased insulin-like growth factor I.

GH receptor (GHR) messenger RNA (mRNA) is transcribed from at least three different promoters within the liver of cattle. The first promoter (P1) is liver specific and alternatively splices exon 1A onto the GHR mRNA (GHR 1A mRNA). The second and third promoters (P2 and P3) have constitutive activity in many tissues and alternatively splice exons 1B and 1C onto the GHR mRNA (GHR 1B and GHR 1C mRNA). The total amount of GHR in the liver partially determines liver insulin-like growth factor I (IGF-I) synthesis in response to GH. Two studies were conducted to characterize the changes in GHR 1A mRNA, alternatively spliced GHR mRNA, and IGF-I mRNA during late pregnancy and early lactation in dairy cattle. Liver RNA was isolated from pregnant Holstein cattle (Bos taurus) on days -14, 0, and 21 relative to parturition (study 1) or days -14, 0, 15, 30, 60, and 90 relative to parturition (study 2). Ribonuclease protection assays were used to quantify total GHR (all GHR variants) as well as liver-specific GHR 1A and alternatively spliced GHR mRNA. Likewise, total IGF-I as well as alternatively spliced IGF-I mRNA (class 1 and class 2 transcripts) were measured. A decrease in total GHR mRNA at parturition (P < 0.01) was associated with a specific decrease in GHR 1A mRNA (P < 0.001). The amount of alternatively spliced GHR mRNA (including GHR 1B and GHR 1C mRNA) did not change at parturition (P > 0.10). Total liver IGF-I mRNA and blood IGF-I concentrations were also decreased at parturition (P < 0.05 and P < 0.01, respectively). However, a decrease in IGF-I mRNA was observed for both class 1 and class 2 IGF-I transcripts (P < 0.01 and P < 0.05, respectively). We conclude that the reduced amount of GHR mRNA during early lactation is caused by a specific down-regulation of GHR 1A mRNA that was associated with decreased liver IGF-I mRNA and decreased blood IGF-I concentrations. These data provide evidence for independent regulation of GHR mRNA by mechanisms that discriminate between GHR P1 (transcribes GHR 1A) and alternative promoters that transcribe constitutive GHR mRNA.

Prostaglandin A2 Specifically Represses Insulin-Like Growth Factor-I Gene Expression in C6 Rat Glioma Cells

The cyclopentenone PGs (PGA and PGJ series) inhibit tumor cell proliferation in vitro and tumorigenesis in vivo via mechanisms that are at present poorly understood. The C6 rat glioma cell line synthesizes and secretes insulin-like growth factor-I (IGF-I), which is believed to act as an autocrine factor for these cells. PGA2 inhibits the proliferation of the C6 cells and causes an increase in the fraction of cells in the G1 phase of the cell cycle. The inhibition of cell proliferation by PGA2 is accompanied by a decrease in the abundance of IGF-I messenger RNA (mRNA). This regulation of IGF-I gene expression is specific, as the abundance of hypoxanthine-guanine phosphoribosyl transferase (HPRT) and ubiquitin mRNA is not significantly affected by PGA2. The repression of IGF-I gene expression is observed at PGA2 concentrations as low as 10 μm and is evident within 4 h after treatment of the C6 cells with PGA2. In addition to specifically regulating the expression of the IGF-I gene, PGA2 also decreases the abundance of cyclin D1 mRNA and increases the abundance of Waf1 mRNA. The inhibition of cell proliferation by PGA2 is partially reversed by coaddition of IGF-I, indicating partial dominance of IGF-I action over PGA2 action. To investigate the molecular basis for the regulation of IGF-I gene expression by PGA2, we developed a sensitive RT-PCR assay for IGF-I nuclear transcripts. A similar assay was developed for quantifying HPRT transcripts, which were used as a control. Treatment of the C6 cells with 20 μm PGA2 resulted in approximately a 6-fold decrease in IGF-I mRNA and IGF-I nuclear transcripts. In contrast, HPRT mRNA and nuclear transcript levels were not significantly affected by PGA2. These results indicate that the decrease in IGF-I mRNA abundance that occurs in response to PGA2 is caused largely by a decrease in IGF-I nuclear transcript levels. To identify the cis-acting element that mediates the effect of PGA2 on IGF-I transcription, C6 cells were transiently transfected with IGF-I/luciferase expression constructs in which luciferase transcription is driven by IGF-I P1 promoter fragments extending from −1711 to +328 or from −1114 to +328 relative to the beginning of exon 1. Treatment of cells with PGA2 in these transient transfection assays did not decrease luciferase activity. These results suggest that the cis-acting regulatory element required for the response to PGA2 is located outside the −1711 to +328 promoter interval.

Prostaglandin A2 specifically represses insulin-like growth factor-I gene expression in C6 rat glioma cells.

The cyclopentenone PGs (PGA and PGJ series) inhibit tumor cell proliferation in vitro and tumorigenesis in vivo via mechanisms that are at present poorly understood. The C6 rat glioma cell line synthesizes and secretes insulin-like growth factor-I (IGF-I), which is believed to act as an autocrine factor for these cells. PGA2 inhibits the proliferation of the C6 cells and causes an increase in the fraction of cells in the G1 phase of the cell cycle. The inhibition of cell proliferation by PGA2 is accompanied by a decrease in the abundance of IGF-I messenger RNA (mRNA). This regulation of IGF-I gene expression is specific, as the abundance of hypoxanthine-guanine phosphoribosyl transferase (HPRT) and ubiquitin mRNA is not significantly affected by PGA2. The repression of IGF-I gene expression is observed at PGA2 concentrations as low as 10 microM and is evident within 4 h after treatment of the C6 cells with PGA2. In addition to specifically regulating the expression of the IGF-I gene, PGA2 also decreases the abundance of cyclin D1 mRNA and increases the abundance of Waf1 mRNA. The inhibition of cell proliferation by PGA2 is partially reversed by coaddition of IGF-I, indicating partial dominance of IGF-I action over PGA2 action. To investigate the molecular basis for the regulation of IGF-I gene expression by PGA2, we developed a sensitive RT-PCR assay for IGF-I nuclear transcripts. A similar assay was developed for quantifying HPRT transcripts, which were used as a control. Treatment of the C6 cells with 20 microM PGA2 resulted in approximately a 6-fold decrease in IGF-I mRNA and IGF-I nuclear transcripts. In contrast, HPRT mRNA and nuclear transcript levels were not significantly affected by PGA2. These results indicate that the decrease in IGF-I mRNA abundance that occurs in response to PGA2 is caused largely by a decrease in IGF-I nuclear transcript levels. To identify the cis-acting element that mediates the effect of PGA2 on IGF-I transcription, C6 cells were transiently transfected with IGF-I/luciferase expression constructs in which luciferase transcription is driven by IGF-I P1 promoter fragments extending from -1711 to -328 or from -1114 to +328 relative to the beginning of exon 1. Treatment of cells with PGA2 in these transient transfection assays did not decrease luciferase activity. These results suggest that the cis-acting regulatory element required for the response to PGA2 is located outside the -1711 to +328 promoter interval.

Decreased IGF-I gene expression during the apoptosis of Purkinje cells in pcd mice

Insulin-like growth factor I (IGF-I) plays a potential functional role in cerebellar development in the rat, as indicated by its spatio-temporally coordinated expression with the IGF-I receptor (IGFR-I), IGF binding protein (IGFBP) 2 and 5 during the postnatal critical growth period. Although IGF-I promotes the survival of cultured cerebellar neurons, its role during cerebellar development in vivo is unclear. Growth factor deprivation has been shown to trigger apoptosis, the developmental cell death which, if abnormal, may lead to various pathological states. To understand the involvement of IGF-I in Purkinje cell survival, we examined mRNA expression of IGF-I, IGFR-I, IGFBP 2 and 5 in the Purkinje cell degeneration ( pcd) mice. During pcd cerebellar development, Purkinje cells rapidly degenerate leading to their almost complete depletion by adult life. IGF system mRNA expression was studied during Purkinje cell death in the pcd mutants ( pcd pcd ) at postnatal day (D) 11, 17, 24 and adult. At D11 and D17, no significant difference of the IGF-I system mRNA expression was observed between the normal and pcd pcd cerebellum. At D24, a significant decrease of IGF-I mRNA was found in the apoptotic Purkinje cells in the pcd pcd cerebellum, which was accompanied by a severe astrogliosis and activation of astrocytic IGF-I expression. In the adult pcd pcd cerebellum, with few Purkinje cells remaining, many granule cells underwent apoptosis. In conclusion, decreased IGF-I mRNA expression was correlated with Purkinje cell apoptosis in pcd cerebellum. Whether the decrease of IGF-I mRNA expression in the cause or result of the Purkinje cell degeneration needs to be further elucidated.

DECREASE IN IGF-I mRNA CORRELATES WITH INCREASED APOPTOTIC CELL DEATH IN NEONATAL HYPOXIA-ISCHEMIA. ▴ 1196

Perinatal hypoxia-ischemia remains the leading cause of severe brain damage in newborns. Hypoxic-ischemic brain injury includes apoptotic and necrotic cell death resulting from persistent alterations in cellular energy homeostasis. Insulin-like growth factor I (IGF-I) is an anabolic pleiotrophic factor required by all cell types in the developing brain for optimal proliferation, differentiation, and survival. To determine how cell death and changes in IGF-I gene expression relate to the events of varying times of hypoxia-ischemia, we evaluated the time course of apoptosis in a neonatal hypoxic-ischemic injury model in relationship to IGF-I mRNA levels. One hour after an hypoxic-ischemic insult to the newborn rat brain mRNA levels of IGF-I decreased in the whole hemisphere ipsilateral to the ligated carotid artery. This decrease is more pronounced at 24 hours, especially in areas vulnerable to hypoxic-ischemic injury, such as the thalamus and hippocampus. At 72 hours post-injury, gene expression of IGF-I was activated in reactive astrocytes. The decrease in IGF-I mRNA was concurrent with an increase in apoptosis. The immediate decrease in IGF-I gene expression may be partially responsible for the impending neuronal death and selective vulnerability of myelinogenesis during the perinatal period. The temporal relationship between the decrease in IGF-I and apoptosis may provide clues of prognostic or therapeutic value, especially as it relates to delineation of a “therapeutic window,” where IGF-I gene therapy could theoretically prevent devastating neuronal injury.

Dexamethasone attenuates the estradiol-induced increase of IGF-I mRNA in the rat uterus

In recent years growth factors, e.g. insulin-like growth factor-I (IGF-I), transforming growth factor-α (TGF-α) and epidermal growth factor (EGF), have been considered as mediators of estradiol-stimulated growth in the uterus. In the liver, dexamethasone (Dex) has been shown to inhibit the IGF-I mRNA increase induced by growth hormone (GH). In the present study the influence of Dex on estradiol-stimulated induction of uterine IGF-I mRNA was examined. The concentration of IGF-I mRNA in the uterus and liver was monitored, as well as the levels of ER mRNA and estrogen receptor (ER). Since it has been previously shown that the maximal induction of uterine IGF-I mRNA after estradiol (E 2) stimulation occurs after 21–24 h, Dex was administered to ovariectomized (ovx) rats 3 h before an E 2 injection and 24 h before sacrifice. There was a significant decrease in IGF-I mRNA in the Dex + E 2 treated rats compared to the rats given E 2 only. In both groups an increase was seen compared to the level in the ovx control group. The uterine ER mRNA levels in E 2 and Dex + E 2 treated animals were significantly elevated compared to the ovx control. There were no significant changes in uterine ER content after hormone treatment compared to the level in ovx control rats. In the liver no effects on IGF-I mRNA were detected. Hepatic ER mRNA was significantly increased in the E 2 treated group, compared to both the ovx control group and the animals that received Dex + E 2. The hepatic ER level was also increased in the E 2 treated group compared to the ovx control and the group which received Dex + E 2. In conclusion, Dex does attenuate the estrogen-induced uterine IGF-I mRNA increase in ovx rats. In addition to this, Dex was found to inhibit the estrogen-induced increase in ER and ER mRNA in the liver of ovx rats.

The effect of fasting on insulin-like growth factor-I nuclear transcript abundance in rat liver.

The abundance of insulin-like growth factor I (IGF-I) messenger RNA (mRNA) is decreased in the liver of fasting, protein-restricted, and energy-restricted rats. The extent to which this decrease in steady state mRNA abundance may be attributed to a decrease in IGF-I gene transcription remains unresolved. In the present study, we used an RNase protection assay to quantify IGF-I nuclear transcript (pre-mRNA) and mRNA abundance in whole cellular RNA isolated from liver of fasted and nonfasted male rats (4-6 weeks of age). The results of the RNase protection assay of IGF-I nuclear transcripts were strongly correlated with the results of nuclear transcription elongation (run-on) assays (r > 0.90; P < 0.001). In addition, the RNase protection assay allows for a greater capability for sensitively monitoring gene transcription in a large number of samples. In four different experiments, a consistent decrease in the quantity of IGF-I nuclear transcripts was observed in liver of animals fasted for 72 h, whereas IGF-I pre-mRNA abundance in animals fed ad libitum was highly variable (average intraassay coefficient of variation = 74% vs. 34% for nonfasted and fasted groups). When data from the four experiments were pooled, fasting reduced IGF-I pre-mRNA and mRNA levels by 78% and 70% (P < 0.001), respectively. Fasting also caused a significant decrease in mRNA and nuclear transcript abundance for another nutritionally sensitive gene, the gene encoding transthyretin (TTR). To determine whether the decrease in IGF-I and TTR nuclear transcripts was gene specific, levels of nuclear transcripts for serum albumin, H-ferritin, and ribosomal RNA were also quantified. The results indicated that serum albumin, H-ferritin, and ribosomal RNA nuclear transcripts were not decreased by fasting, demonstrating that the negative effect of fasting was specific for IGF-I and TTR. In summary, these results indicate that IGF-I and TTR nuclear transcripts are specifically decreased by fasting. The decrease in IGF-I mRNA is matched by a similar decrease in IGF-I nuclear transcripts, suggesting that fasting controls IGF-I gene expression primarily at the transcriptional level.

Nutritional regulation of hormones and growth factors that control mammalian growth.

Juvenile animals stop growing if they are fed a diet containing an inadequate amount of energy or protein. The molecular basis for this growth arrest is not completely understood. The cessation of growth that occurs in nutritionally restricted animals is not generally explained by a decrease in circulating growth hormone (GH). In most species, plasma GH is increased rather than decreased under conditions of nutritional restriction. Current evidence suggests that the biosynthesis of insulin-like growth factor-I (IGF-I) is a key control point for nutritional regulation of growth. Plasma IGF-I peptide levels and hepatic IGF-I mRNA abundance are correlated with growth velocity and are consistently decreased when growth is arrested by nutritional deprivation. The decreased IGF-I mRNA abundance observed in the fasting rat appears to be caused primarily by a decrease in IGF-I gene transcription. In tissues and plasma, the insulin-like growth factors are complexed with high-affinity binding proteins, which are thought to modulate the tissue access and action of the IGFs. The hepatic mRNA abundance of two of the binding proteins (IGFBP-1 and -2) is increased in nutritionally restricted animals. This increase in mRNA abundance is caused primarily by an increase in transcription of the IGFBP-1 and IGFBP-2 genes. Current research is focused on molecular mechanisms for regulation of IGF-I and IGF-binding protein gene expression.

Intracellular calcium regulates insulin-like growth factor-I messenger ribonucleic acid levels

Insulin-like growth factor-I (IGF-I) is involved in repair and regeneration in tissues in which non-GH-mediated regulation of its production has been shown to be important. We have investigated the effects of a second messenger signaling pathway, intracellular calcium, on IGF-I mRNA levels in cultured rat dermal fibroblasts using a RNase protection assay. Intracellular calcium concentrations ([Ca2+]i) were increased using either the calcium ionophore A23187 or thapsigargin. The ability of these agents to increase [Ca2+]i was confirmed by spectrofluorimetry, using fluo-3 as the Ca2+ indicator. Treatment of cells in serum-free medium and 0.25% BSA [Minimum Essential Medium (MEM) + BSA] with 500 nM A23187 or 1 micron thapsigargin decreased IGF-I mRNA levels in a time-responsive manner over 4-8 h. A23187 and thapsigargin also decreased IGF-I mRNA levels to 36% and 47% of control levels, respectively, in a dose-responsive fashion. Basic fibroblast growth factor mRNA levels, which were simultaneously determined, were either unchanged or increased in cells treated with thapsigargin or A23187. Consistent with the change in IGF-I mRNA levels, immunoreactive IGF-I levels in medium conditioned for 48 h by A23187 or thapsigargin decreased to 25% and 14%, respectively, of control levels in cells maintained in MEM + BSA. To determine the role of protein synthesis in the effects of A23187 and thapsigargin, cells were treated with these agents in the presence or absence of cycloheximide. Cycloheximide had no effect on the decrease in IGF-I mRNA levels mediated by thapsigargin, but significantly attenuated the response to A23187. Given these differences in the role of protein synthesis in and the time course of the effects of A23187 and thapsigargin on IGF-I mRNA levels, additivity experiments were performed. Treatment of cells with the combination of A23187 and thapsigargin resulted in IGF-I mRNA levels that were approximately 70% of the levels present in cells treated with either agent alone. These data are consistent with a small additive effect, but suggest that the majority of the effect of A23187 and thapsigargin occurs via the same final pathway.

Intracellular calcium regulates insulin-like growth factor-I messenger ribonucleic acid levels.

Insulin-like growth factor-I (IGF-I) is involved in repair and regeneration in tissues in which non-GH-mediated regulation of its production has been shown to be important. We have investigated the effects of a second messenger signaling pathway, intracellular calcium, on IGF-I mRNA levels in cultured rat dermal fibroblasts using a RNase protection assay. Intracellular calcium concentrations ([Ca2+]i) were increased using either the calcium ionophore A23187 or thapsigargin. The ability of these agents to increase [Ca2+]i was confirmed by spectrofluorimetry, using fluo-3 as the Ca2+ indicator. Treatment of cells in serum-free medium and 0.25% BSA [Minimum Essential Medium (MEM) + BSA] with 500 nM A23187 or 1 micron thapsigargin decreased IGF-I mRNA levels in a time-responsive manner over 4-8 h. A23187 and thapsigargin also decreased IGF-I mRNA levels to 36% and 47% of control levels, respectively, in a dose-responsive fashion. Basic fibroblast growth factor mRNA levels, which were simultaneously determined, were either unchanged or increased in cells treated with thapsigargin or A23187. Consistent with the change in IGF-I mRNA levels, immunoreactive IGF-I levels in medium conditioned for 48 h by A23187 or thapsigargin decreased to 25% and 14%, respectively, of control levels in cells maintained in MEM + BSA. To determine the role of protein synthesis in the effects of A23187 and thapsigargin, cells were treated with these agents in the presence or absence of cycloheximide. Cycloheximide had no effect on the decrease in IGF-I mRNA levels mediated by thapsigargin, but significantly attenuated the response to A23187. Given these differences in the role of protein synthesis in and the time course of the effects of A23187 and thapsigargin on IGF-I mRNA levels, additivity experiments were performed. Treatment of cells with the combination of A23187 and thapsigargin resulted in IGF-I mRNA levels that were approximately 70% of the levels present in cells treated with either agent alone. These data are consistent with a small additive effect, but suggest that the majority of the effect of A23187 and thapsigargin occurs via the same final pathway.(ABSTRACT TRUNCATED AT 400 WORDS)

Ligands that activate protein kinase-C differ in their ability to regulate basic fibroblast growth factor and insulin-like growth factor- I messenger ribonucleic acid levels

In this study, rat dermal fibroblasts were used as a model system to examine the ability of ligands that are known to activate protein kinase-C to regulate the levels of the mRNAs encoding basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I), two growth factors that are thought to be important in processes such as tissue repair and regeneration and wound healing. Treatment of fibroblasts with the phorbol ester phorbol 12-myristate 13-acetate (PMA), thrombin, bradykinin, serotonin, angiotensin-II, or bombesin increased protein kinase-C activity to a similar degree. Treatment of fibroblasts with 1 microM serotonin transiently increased bFGF mRNA levels about 3-fold compared to the level in control cells maintained in serum-free medium with 0.25% BSA and decreased IGF-I mRNA levels by approximately 50% compared to the level in control cells. This is similar to the previously described changes induced by bradykinin in these cells, but different from the more marked and sustained changes induced by thrombin and PMA. In contrast, angiotensin-II and bombesin had no effect on bFGF or IGF-I mRNA levels. The effects of serotonin, bradykinin, and PMA on bFGF and IGF-I mRNA levels were abrogated by preincubation of cells in 250 nM PMA to down-regulate protein kinase-C. In contrast, the effect of thrombin on bFGF mRNA levels was only partially inhibited by down-regulation of protein kinase-C, while its effect on IGF-I mRNA levels was unaffected. The activation of signaling pathways by the different ligands was further investigated to begin to determine the mechanism for the differences in the effects of thrombin vs. serotonin and bradykinin and in the effects of these three ligands vs. angiotensin-II and bombesin. All of the ligands activated phospholipase-D to a similar degree, suggesting that activation of this enzyme was not responsible for the differential effects of the ligands. In contrast, thrombin, serotonin, and bradykinin had marked effects on the hydrolysis of phosphatidylcholine and phosphatidylinositol, whereas bombesin and angiotensin-II had a small effect on phosphatidylcholine hydrolysis and no effect on phosphatidylinositol hydrolysis.

Ligands that activate protein kinase-C differ in their ability to regulate basic fibroblast growth factor and insulin-like growth factor-I messenger ribonucleic acid levels.

In this study, rat dermal fibroblasts were used as a model system to examine the ability of ligands that are known to activate protein kinase-C to regulate the levels of the mRNAs encoding basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I), two growth factors that are thought to be important in processes such as tissue repair and regeneration and wound healing. Treatment of fibroblasts with the phorbol ester phorbol 12-myristate 13-acetate (PMA), thrombin, bradykinin, serotonin, angiotensin-II, or bombesin increased protein kinase-C activity to a similar degree. Treatment of fibroblasts with 1 microM serotonin transiently increased bFGF mRNA levels about 3-fold compared to the level in control cells maintained in serum-free medium with 0.25% BSA and decreased IGF-I mRNA levels by approximately 50% compared to the level in control cells. This is similar to the previously described changes induced by bradykinin in these cells, but different from the more marked and sustained changes induced by thrombin and PMA. In contrast, angiotensin-II and bombesin had no effect on bFGF or IGF-I mRNA levels. The effects of serotonin, bradykinin, and PMA on bFGF and IGF-I mRNA levels were abrogated by preincubation of cells in 250 nM PMA to down-regulate protein kinase-C. In contrast, the effect of thrombin on bFGF mRNA levels was only partially inhibited by down-regulation of protein kinase-C, while its effect on IGF-I mRNA levels was unaffected. The activation of signaling pathways by the different ligands was further investigated to begin to determine the mechanism for the differences in the effects of thrombin vs. serotonin and bradykinin and in the effects of these three ligands vs. angiotensin-II and bombesin. All of the ligands activated phospholipase-D to a similar degree, suggesting that activation of this enzyme was not responsible for the differential effects of the ligands. In contrast, thrombin, serotonin, and bradykinin had marked effects on the hydrolysis of phosphatidylcholine and phosphatidylinositol, whereas bombesin and angiotensin-II had a small effect on phosphatidylcholine hydrolysis and no effect on phosphatidylinositol hydrolysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of insulin-like growth factor I production in rat C6 glioma cells: possible role as an autocrine/paracrine growth factor.

The growth of rat glioma C6 cells, which provide an in vitro model of glial cells, is inhibited by retinoic acid and glucocorticoids, two agents which are important in brain differentiation and growth. To determine whether the growth-inhibitory effects of these agents are mediated by alterations in insulin-like growth factor I (IGF-I) production, the effects of retinoic acid and dexamethasone on IGF-I production and messenger RNA levels in C6 cells were investigated. IGF-I mRNA levels were determined using a solution hybridization/RNase protection assay. Treatment of C6 cells with dexamethasone or retinoic acid decreased IGF-I mRNA levels in a time-dependent fashion. The time course of the effect of the two agents differed, with the peak effect of dexamethasone between 6 and 12 h and the peak effect of retinoic acid at 27 h. In dose-response studies, IGF-I mRNA levels decreased to 27% of control levels (cells maintained in serum-free media) after treatment with 5 ng/ml dexamethasone, while half-maximal inhibition was achieved with approximately 0.5 ng/ml (1.4 nM) dexamethasone. Treatment with 10 microM retinoic acid decreased IGF-I mRNA levels to 24% of control levels with half-maximal inhibition occurring with approximately 0.5 microM retinoic acid. Cycloheximide prevented the inhibitory effect of these agents on IGF-I mRNA levels, suggesting that their effect is at least partly dependent upon protein synthesis. Immunoreactive IGF-I levels in media conditioned for 48 h by cells treated with dexamethasone or retinoic acid decreased to 32% and 42% of control levels, respectively. Treatment of C6 cells with retinoic acid or dexamethasone decreased thymidine incorporation into DNA. Treatment of cells with IGF-I alone had no effect on thymidine incorporation into DNA, but addition of 10 or 50 ng/ml IGF-I to dexamethasone-treated cells stimulated a small, but significant (P less than 0.01), increase in thymidine incorporation into DNA. IGF-I was not, however, able to reverse the inhibitory effect of retinoic acid. Finally, treatment of cells with 150 ng/ml of IGF binding protein 1 significantly decreased (P less than 0.01) thymidine incorporation into DNA by 17% as compared to incorporation into control cells maintained in serum-free media.(ABSTRACT TRUNCATED AT 400 WORDS)

Growth hormone regulation of insulin-like growth factor-I mRNA in rat adipose tissue and isolated rat adipocytes

The effects of hypophysectomy and hormonal replacement therapy on insulin-like growth factor-I (IGF-I) mRNA in rat adipose tissue and adipocytes were studied. The effects of GH and IGF-I in vitro on IGF-I mRNA and IGF-I production were also studied in cultured rat adipocytes. Male rats were hypophysectomized at about 50 days of age and given replacement therapy with cortisol (400 micrograms/kg per day) and thyroxine (10 micrograms/kg per day). GH was given as a single i.v. or s.c. injection and also as a continuous s.c. infusion for 6 days. Epididymal fat pads were excised and used either for isolation of adipocytes or for determination of IGF-I mRNA in adipose tissue. A solution hybridization assay was used. The IGF-I mRNA content of adipocytes was analysed either immediately after isolation or after short-term (2-3 days) culture with or without GH or IGF-I. Hypophysectomy resulted in a marked decrease in IGF-I mRNA in both tissue and cells. Replacement therapy (in vivo) with cortisol and thyroxine alone had no effect, whereas additional treatment with GH caused a dose-dependent increase in IGF-I mRNA. IGF-I mRNA was also increased after a continuous s.c. infusion of GH. A single i.v. injection of GH (100 micrograms) resulted in an increase in IGF-I mRNA after approximately 2 h, with maximal levels around 6 h after the injection. In cultured adipocytes, addition of GH to the culture medium increased IGF-I mRNA in a dose-dependent manner and a marked increase was observed with a concentration of GH of 1 ng/ml. Addition of IGF-I (100 ng/ml) had no effect. The increase in IGF-I mRNA after addition of GH (100 ng/ml) was detectable after 3 h. The concentration of IGF-I in the culture medium was increased 24 h after the addition of GH. These results demonstrate that GH induces IGF-I mRNA in both adipose tissue and isolated fully differentiated adipocytes and that this increase in IGF-I mRNA results in increased IGF-I production.

Reduced serum concentrations of insulin-like growth factor-I (IGF-I) in protein-restricted growing rats are accompanied by reduced IGF-I mRNA levels in liver and skeletal muscle

The serum concentration of insulin-like growth factor-I (IGF-I) is reduced in growing rats fed a low-protein diet, and this decrease is age-dependent, being more pronounced in younger animals. To determine whether this decrease in serum IGF-I is related to a decrease in IGF-I mRNA, growing female rats were given free access to either a 15% protein-sufficient or a 5% protein-deficient diet for 1 week. Protein restriction in 4-week-old rats decreased body weight gain by 44% (P less than 0.001 compared with 4-week controls), serum IGF-I concentration by 67% (P less than 0.001) and liver IGF-I mRNA abundance by 51% (P less than 0.001). During week 6, protein restriction for 1 week resulted in a 20% increase in food intake with no change in weight gain, a 38% reduction in serum IGF-I (P less than 0.001 compared with 6-week controls) and a 39% decrease in liver IGF-I mRNA (P less than 0.001). The serum IGF-I concentration was highly correlated (r = 0.80; P less than 0.001) with the hepatic IGF-I mRNA concentration. Skeletal muscle IGF-I mRNA abundance was also decreased significantly by protein restriction (37% at week 4, P less than 0.001, and 24% at week 6, P less than 0.01) and was closely correlated (r = 0.71; P less than 0.001) with body weight gain. Liver GH-binding protein and GH receptor mRNA abundance were reduced by 1 week of protein deprivation at week 6 but not at week 4.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential expression of insulin-like growth factor-I and insulin-like growth factor binding protein-1 in the diabetic rat

Multiple factors contribute to the growth retardation which is a characteristic feature of uncontrolled diabetes. In this report we have examined the effects of streptozotocin-induced (STZ) diabetes on expression of insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-1 (IGFBP-1) in various tissues. As early as 7 days after STZ administration there was a modest reduction in IGF-I mRNA abundance. The reduction (10-30%) was of similar magnitude in each of the 7 tissues examined; liver, kidney, lung, diaphragm, quadraceps, heart and adipose tissue. However, the reduction achieved statistical significance only in the lung (p less than 0.05) and diaphragm (p less than 0.01). A further reduction in IGF-I mRNA abundance was seen in many tissues, 32 and 91 days after STZ administration. In contrast to the decrease in IGF-I mRNA, IGFBP-1 mRNA was significantly increased in the liver and kidney of diabetic rats. IGFBP-1 mRNA was detectable at only very low levels in other tissues but was increased in diabetic rats compared non-diabetic rats. In diabetic rats, a highly significant correlation (R = 0.75, p less than 0.001) between hepatic IGFBP-1 mRNA and glucose was observed whereas there was no significant correlation between serum glucose and hepatic IGF-I mRNA abundance (R = 0.24, p = NS). Treatment of diabetic rats with insulin resulted in a small, non significant increase in hepatic and renal IGF-I mRNA and a significant decrease in renal IGFBP-1 mRNA abundance. The observations reported here are consistent with the hypothesis that diminished IGF-I expression and inhibition of available IGF-I by increased levels of IGFBP-1 may explain the impaired growth seen in diabetic animals.

Regulation of Insulin-Like Growth Factor I Messenger Ribonucleic Acid Levels by Serum in Cultured Rat Fibroblasts*

Fibroblasts represent one of the in vivo sites of extrahepatic insulin-like growth factor I (IGF-I) production. In this study, cultured fibroblasts prepared from the skin of neonatal rats were used as a model to assess the role of serum in regulating IGF-I messenger RNA (mRNA) levels. IGF-I mRNA, as demonstrated by Northern blot analysis, was present in the cultured fibroblasts, and serum free media which was conditioned by fibroblasts for 20 h contained 108 pg/ml of immunoreactive IGF-I. Fetal calf serum (FCS) decreased steady state IGF-I mRNA levels, as measured by solution hybridization/RNase protection assay, in fibroblasts in a time- and dose-dependent fashion. Incubation of fibroblasts for 18 h in the presence of 0.3%, 0.6%, or 1% FCS decreased IGF-I mRNA levels to 76%, 56%, and 46% of the levels present in control cells which were maintained in serum free media with 0.25% BSA. Maximal inhibition to approximately 20% of control levels was seen with 4-10% FCS. In contrast, basic fibroblast growth factor and beta-actin mRNA levels increased 2- and 4-fold, respectively, with increasing concentrations of FCS. Treatment of the cells with 10 micrograms/ml cycloheximide resulted in partial abrogation of the inhibitory effect of FCS while protein synthesis in the cells was decreased to 6% of control levels. The addition of 2 micrograms/ml of insulin or 15-100 ng/ml of IGF-I to the fibroblasts did not reproduce the inhibitory effect of FCS. Finally, the inhibitory factor(s) present in the FCS was partially removed/inactivated by charcoal stripping or heat inactivating the serum, but delipidation of the FCS by chloroform extraction had no effect on the inhibitory effect of FCS. In summary, FCS contains a factor(s) that decreases IGF-I mRNA levels in cultured fibroblasts in a time- and dose-dependent fashion. The partial abrogation of the inhibitory effect of FCS with cycloheximide treatment suggests that this effect is at least partially dependent upon new protein synthesis. Furthermore, the studies using delipidated, heat-inactivated, and charcoal-stripped serum suggest that the inhibitory factor(s) is a peptide.