IGF-I mRNA Abundance Research Articles

Differential role of glucocorticoids in mediating endotoxin-induced changes in IGF-I and IGFBP-1.

The purpose of the present study was to determine whether endogenous elevations in glucocorticoids mediate the changes in insulin-like growth factor (IGF) 1 and IGF binding protein (IGFBP) 1 levels in plasma and tissues observed after in vivo administration of lipopolysaccharide (LPS). In overnight-fasted male rats LPS injected via the tail vein decreased the IGF-I concentration in plasma, liver, and skeletal muscle (30-45%) and increased IGF-I content in kidney (approximately 3-fold). LPS also decreased IGF-I mRNA abundance in liver and muscle and increased gene expression in kidney. Concomitantly, IGFBP-1 levels in plasma, liver, and muscle were markedly elevated by LPS. All these changes were associated with a greater than fourfold elevation in plasma corticosterone. Pretreatment of rats with the glucocorticoid receptor antagonist RU-486 completely prevented or blunted the LPS-induced changes in IGF-I content in plasma, liver, muscle, and kidney. In liver and muscle RU-486 significantly attenuated the reduction in IGF-I mRNA abundance produced by LPS, but in kidney the LPS-induced increase in IGF-I mRNA was still evident. In contrast, pretreatment with RU-486 did not prevent or attenuate the LPS-induced increase in IGFBP-1 levels in plasma, liver, or muscle. These data suggest that glucocorticoids play a major role in regulating IGF-I mRNA and peptide content in tissues in response to LPS, but the increased IGFBP-1 in blood and tissues induced by LPS appears largely glucocorticoid independent.

Intravenous infusion of insulin-like growth factor I in fetal sheep reduces hepatic IGF-I and IGF-II mRNAs

Liver contains the highest concentrations of insulin-like growth factor (IGF) I mRNA in adult rats and sheep and is a major source of circulating IGF-I. In rats, inhibition of hepatic IGF-I production by exogenous IGF-I has been reported. In fetal sheep, skeletal muscle and liver are major sites of IGF-I synthesis and potential sources of circulating IGF-I. To determine whether feedback inhibition of IGF gene expression in fetal liver or muscle by IGF-I occurs, IGF-I and IGF-II mRNAs were measured in these tissues after intravenous infusion of recombinant human IGF-I into fetal sheep. Infusion of IGF-I (26 +/- 4 micrograms.h-1.kg-1; n = 6) or saline (n = 6) commenced on day 120 of pregnancy (term = 150 days) and continued for 10 days. Plasma concentrations of IGF-I were threefold higher in infused fetuses at 130 days of gestation (P < 0.0003), whereas those of IGF-II were unchanged. IGF-I infusion reduced the relative abundance of IGF-I mRNA (P < 0.0002) and IGF-II mRNA (P < 0.01) in fetal liver by approximately 50% but did not alter IGF-I or IGF-II mRNA in skeletal muscle. These results indicate that IGF-I inhibits the expression of both IGF-I and IGF-II genes in fetal liver and that IGF gene expression in fetal liver and muscle is differentially regulated by IGF-I.

Ontogeny of insulin-like growth factor-I and -II gene expression in ovine fetal heart

Insulin-like growth factors (IGF)-I and -II have been implicated in growth and differentiation during embryonic and fetal development. To examine the role of the IGFs in growth of the fetal heart, we determined the gene expression of IGF-I and IGF-II in the four cardiac chambers of the ovine fetus from 58 to 146 days' gestation (term = 147 days). Total RNA was obtained from the cardiac chambers, analyzed by Northern blot, and hybridized to ovine specific cDNA probes for IGF-I and IGF-II. The resulting autoradiograms were subjected to light densitometry, and the intensity of the IGF signals was normalized to the respective 28S ribosomal RNA signals. In the atria and ventricles, IGF-I mRNA abundance was very low throughout the gestational period studied, whereas IGF-II mRNA levels were higher and readily detectable. In the atria, IGF-I mRNA was very low at 60 days' gestation and appeared to increase gradually toward term. Abundance of IGF-II mRNA was high at 60 days, increased further until 120 days, and decreased slightly toward term. In contrast, in the ventricles, IGF-I mRNA increased from 60 to 100 days and then declined moderately at term. Levels of IGF-II mRNA in the ventricles were high at 60 days and decreased progressively to low levels at term. No difference in IGF-I or IGF-II mRNA levels was noted between the right and left atria or right and left ventricles. These results suggest that developmental patterns for IGF-I and IGF-II gene expression exist in the ovine fetal heart, and the patterns differ between the atria and ventricles. Further, these gestational trends differ from those for atrial natriuretic factor (ANF) found in our previous studies, indicating that expression of the ANF gene in the fetal heart may not be associated with cardiac growth and differentiation.

Insulin-like growth factor (IGF-I) mRNA and IGF-I receptor in trout testis and in isolated spermatogenic and Sertoli cells.

Few data exist concerning the occurrence and potential role of an insulin-like growth factor (IGF) system in fish gonads. Using Northern and slot blot hybridization with a specific salmon IGF-I cDNA, we confirmed that IGF-I transcription occurs in trout testis. Testicular IGF-I mRNA abundance may be increased by long-term GH treatment in juvenile fish, while shorter treatment with growth hormone (GH) or a gonadotropin (GTH-II) in maturing males had no statistically significant effect. Radiolabelled recombinant human IGF-I binds with high affinity to crude trout testis preparation, to cultured isolated testicular cells, and to a membrane fraction of these cells (Ka = 0.2 to 0.7 x 10(10) M-1; Bmax = 10 to 20 fmol/10(7) cells, and 68 fmol/mg protein of membrane). The binding site was identified as type 1 IGF receptor by its binding specificity (IGF-I > IGF-II >>> insulin) and the molecular size of its alpha-subunit labelled with 125I-IGF-I (M(r)125-140 kDa). 125I-IGF-II also bound to the type 1 receptor whereas IGF-II/ mannose 6 phosphate receptors could not be detected. Separation of isolated testicular cells by Percoll gradient and centrifugal elutriation provided populations enriched in different types of intratubular cells. IGF-I mRNA (detected by reverse transcription + polymerase chain reaction [PCR]) and IGF-I receptors (measured by competitive binding) were observed to a greater extent in Sertoli cell-enriched populations and in spermatogonia with primary spermatocytes. Therefore, IGF-I is a potential paracrine/autocrine regulator inside the spermatogenic compartment and appears as a possible mediator of GH action at the gonadal level in fish.

Long-term effects of gestational protein malnutrition on postnatal growth, insulin-like growth factor (IGF)-I, and IGF-binding proteins in rat progeny.

We examined the long-term effects of dietary protein restriction during rat pregnancy on serum IGF-I, serum IGF binding proteins, and liver IGF-I gene expression during postnatal development. Pregnant Wistar rats were fed ad libitum throughout gestation a normal (20% casein diet; P20 controls) or a low (5% casein; P5) protein diet. At birth, the pups from both P20 and P5 dams were cross-fostered to well nourished lactating dams, and litters (n = 5/dietary group) were reduced in size to 6 pups. After weaning (d 22), the pups were fed the control diet ad libitum. The pups were killed at 8, 22, and 63 d of age. Gestational protein restriction caused significant growth retardation and mortality in newborn pups. Despite food rehabilitation during the suckling period (d 0-22), body weight, tail length, and the weight of liver, heart, kidney, and brain in the P5 pups remained significantly reduced at 8 and 22 d (-17 to -35%) compared with control pups. At the same time, serum and liver IGF-I concentrations in the P5 pups (on d 8: 100 +/- 9 ng/mL and 11 +/- 1 ng/g, respectively; on d 22: 340 +/- 20 ng/mL and 42 +/- 3 ng/g) were lower than in age-matched controls (on d 8: 170 +/- 12 ng/mL and 26 +/- 2 ng/g; on d 22: 470 +/- 30 ng/mL and 73 +/- 5 ng/g), although liver IGF-I mRNA abundance was not affected. After long-term food rehabilitation (d 63), tail length and organ weight recovered, and serum and liver IGF-I concentrations were normalized. However, although the P5 rats had resumed a normal growth rate, their body weight remained lower than in the controls. There were no differences in serum IGF binding proteins 1-4, insulin, and GH concentrations between the groups at any age studied. These results suggest that reduction in serum IGF-I may contribute to the reduced somatic and organ growth observed in rats after gestational protein malnutrition, and further support a role for IGF-I in the control of catch-up growth.

IGF-I and serine protease inhibitor 2·1 nuclear transcript abundance in rat liver during protein restriction

Restriction of dietary protein consumption of young male rats results in decreased growth velocity and a reduction in the abundance of hepatic IGF-I mRNA. It is not known whether the reduction of IGF-I mRNA abundance in the liver of protein-restricted rats results from a decrease in IGF-I gene transcription. In the present study, three experiments were performed with 4-week-old male rats to examine the effect of protein restriction on IGF-I gene transcription in liver. In these experiments, we monitored IGF-I nuclear transcripts (pre-mRNA) within total cellular RNA using a ribonuclease protection assay. In the first experiment, a consistent decrease in IGF-I mRNA from animals fed isocaloric diets containing 20% (control), 12%, 8% and 4% protein (dietary effect, P < 0.001) was not paralleled by a decrease (P > 0.50) in IGF-I pre-mRNA. Two additional experiments examining the effect of 4% vs 20% protein diets yielded comparable results. Pooled results from these two studies (n = 12/treatment) demonstrated that a 64% reduction (P < 0.0001) in IGF-I mRNA abundance was not accompanied by a decrease in IGF-I pre-mRNA (1.17 vs 1.31 +/- 0.21 image density units for 4% and 20% protein treatments). Unlike IGF-I, the abundance of carbamyl phosphate synthetase-I (CPS-I) pre-mRNA and mRNA was comparably reduced (approximately 70%, P < 0.001), indicating that the decrease in mRNA of this urea cycle enzyme during protein restriction occurs predominantly by a transcriptional mechanism. A common feature of all experiments was a pronounced variability in the expression of hepatic IGF-I pre-mRNA among animals, which was not diet specific. To test whether the variability in IGF-I gene transcription was correlated with variability in the transcription of another gene that is regulated by GH, we quantified the abundance of nuclear transcripts for the serine protease inhibitor 2.1 (SPI 2.1) gene. A positive association (r = 0.81, P < 0.0001) between SPI 2.1 and IGF-I nuclear transcripts was demonstrated. The correlation between IGF-I and SPI 2.1 transcripts was specific, because the quantity of IGF-I and CPS-I nuclear transcripts was not correlated in this study. Although transcription of the IGF-I and SPI 2.1 genes was similar, the abundance of SPI 2.1 mRNA was not altered by protein deprivation.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of Dietary Energy Restriction on the Expression of Insulin-like Growth Factor-I in Liver and Corpus Luteum of Heifers

The effects of energy balance on the corpus luteum may be mediated by IGFI-I. Our objective was to determine whether negative energy balance decreases expression of IGF-I mRNA in the liver or corpus luteum of heifers. For four consecutive estrous cycles, 14 Holstein heifers were maintained in negative or positive energy balance to lose 510 g/d of BW or gain 560 g/d of BW, respectively. The liver was biopsied and the corpus luteum was collected 7 d after fourth estrus. Heifers fed below maintenance had a smaller corpus luteum than did control heifers. Compared with that of controls, negative energy balance increased growth hormone in serum but decreased IGF-I in serum and the abundance of mRNA for growth hormone receptor and IGF-I in liver. In contrast, diet did not affect the abundance of mRNA for the growth hormone receptor or IGF-I in luteal tissue. Negative energy balance increased IGF binding protein-2 in serum but did not affect IGF binding protein-3. We conclude that negative energy balance of cattle decreases IGF-I mRNA abundance in the liver but not in the 7-d corpus luteum. If decreased IGF-I mediates the adverse effect of negative energy balance on luteal growth, the mode of action is likely endocrine, not autocrine or paracrine.

Effects of maternal protein malnutrition on fetal growth, plasma insulin-like growth factors, insulin-like growth factor binding proteins, and liver insulin-like growth factor gene expression in the rat.

We examined the effects of maternal dietary protein restriction on fetal growth and expression of IGF-I and -II, and IGF-binding proteins (IGFBP). We sought to dissociate the respective effects of maternal protein versus calorie restriction on growth indices and IGF synthesis by the neonates of protein-restricted dams. Pregnant Wistar rats (six to eight per group) fed a low (5%) protein diet throughout gestation had impaired body weight gain compared with controls fed a normal (20%) protein diet (by 45%, p < 0.001). Their serum and liver IGF-I concentrations and liver IGF-I mRNA concentrations were also reduced by 60, 80, and 50%, respectively. Serum IGFBP-3 was reduced by 60% in protein-restricted dams within 1 to 2 h after delivery (p < 0.001 versus controls), although IGFBP-1, -2, and -4 were not significantly affected by the dietary protein intake. In pups of protein-restricted dams, the mean body and liver weight at birth was 15-20% less than that observed in the progeny from normal protein-fed dams (p < 0.01). Their plasma and liver IGF-I concentrations were 30 and 60% lower, respectively, whereas liver IGF-I mRNA abundance was reduced by 50% (p < 0.01). In contrast, neonatal plasma IGF-II and liver IGF-II mRNA concentrations were not significantly affected by the maternal protein malnutrition. Also, the plasma levels of IGFBP were not altered in the growth-retarded pups. Maternal protein restriction did not affect fetal and placental growth, plasma and liver IGF-I levels, and liver IGF-I mRNA abundance in 20-d-old fetuses.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional variation of insulin-like growth factor-I gene expression in mature rat bone and cartilage

Regulation of long bone growth by growth hormone and other endocrine factors is mediated by the local synthesis of IGF-I in the growth plate. Recent evidence suggests that different regions of the growth plate exhibit variable growth rates. To investigate whether IGF-I gene expression in the growth plate differs in relation to growth, we examined the distribution of IGF-I mRNA and peptide using in situ hybridization and immunohistochemistry, respectively, in the tibiae of 18-week-old rats ( n = 6). Osteoblasts were identified by osteocalcin immunoreactivity, and osteoclasts by tartrateresistant acid phosphatase (TRAP) histochemistry. The abundance of IGF-I mRNA in growth plate chondrocytes was quantified by counting the autoradiographic signal associated with each cell. IGF-I mRNA was identified in chondrocytes of both the proliferative and hypertrophic zones of the growth plate. Cells in the marginal regions of both zones contained significantly more IGF-I mRNA than those in the central region ( p < 0.05). In addition, IGF-I mRNA levels were greater in the periphery of the growth plate on the medial side of the tibia ( p < 0.05) in which there was more active growth than the lateral side. IGF-I immunoreactivity was present predominantly in the hypertrophic zone chondrocytes and no regional differences in its distribution were observed. IGF-I mRNA and peptide were also identified in periosteal fibroblasts, notably at sites of muscle attachment to bone, and in osteoblasts at active sites of bone remodelling in the periosteal, endocortical, and endosteal bone envelopes. In the TRAP-positive osteoclasts, IGF-I immunoreactivity, but not IGF-I mRNA, was detected. In addition, both IGF-I mRNA and peptide were identified in the hemopoietic cells of the metaphyseal bone marrow, whereas only IGF-I immunoreactivity was detectable in the diaphysis. We conclude that, in the tibiae of mature rats: (i) IGF-I gene expression in the growth plate is related to its growth and/or synthetic activity; and (ii) the presence of IGF-I in osteoblasts and osteoclasts suggests its involvement in active bone growth and remodeling.

Expression of Insulin-like Growth Factor-I in Cows at Different Stages of Lactation and in Late Lactation Cows Treated with Somatotropin

Relative amounts of IGF-I mRNA were measured in livers of Holstein cows at different stages of lactation (6 early, 6 mid, 6 late lactation, 6 dry) and 6 late lactation cows treated with bST for 1 wk. Milk yield was greater for early lactation cows than for mid and late lactation controls. All cows except those in early lactation were in positive energy balance. Serum IGF-I increased as lactation progressed and was greatest during the dry period. Liver IGF-I mRNA was less in cows in early than in late lactation but greater in lactating than in dry cows. Treatment with bST increased milk yield and concentrations of serum IGF-I, hepatic IGF-I mRNA, and serum IGF-binding protein-3, decreased concentration of serum IGF-binding protein-2, and did not alter abundance of mammary IGF-I mRNA. These parallel changes in serum IGF-I and hepatic IGF-I mRNA suggest that exogenous bST increases IGF-I synthesis in liver of cows during late lactation and that IGF-I synthesis is depressed during early lactation when cows are in negative energy balance. We conclude that IGF-I may play an endocrine role in mediating galactopoietic effects of exogenous bST during late lactation. However, the role of IGF-I during early lactation remains unclear.

Porcine somatotropin (pST) increases IGF-I mRNA abundance in liver and subcutaneous adipose tissue but not in skeletal muscle of growing pigs

The present study was undertaken to determine the effects of exogenous porcine somatotropin (pST) on IGF-I gene expression in liver, skeletal muscle (longissimus dorsi), and s.c. adipose tissue of growing pigs. Twenty prepubertal gilts (approximately 60 kg BW) were allotted to four treatment groups (n = 5) and treated with either 0, 35, 70, or 140 micrograms/kg BW of recombinantly derived pST by daily i.m. injection for 7 d. Serum concentrations of IGF-I were determined by RIA and IGF-I mRNA levels were determined by direct counting of individual samples on slot blots. Administration of pST increased IGF-I concentration in serum. This was accompanied by significant increases (P < .05) in IGF-I mRNA abundance in liver and s.c. adipose tissue; the effects were maximal at the lowest dose of pST. Insulin-like growth factor I mRNA levels were increased 2.5- and 4.5-fold, respectively. Levels of IGF-I mRNA were very low in longissimus muscle and were unaffected by administration of pST. When expressed as picograms of mRNA/10 micrograms of total RNA, IGF-I mRNA levels were highest in s.c. adipose tissue. Levels of IGF-I mRNA were 1.9-fold higher in s.c. adipose tissue than in liver of control animals, and pST administration increased this difference to 3.2-fold. Our results suggest that 1) the effects of pST administered by daily i.m. injection on IGF-I gene expression in pigs are tissue-specific and 2) the stimulatory effects of pST administered in this manner on muscle growth in pigs are not associated with increased expression of the IGF-I gene in skeletal muscle.

Differential regulation of IGF-I leader exon transcription.

We have shown that there is differential expression of the two IGF-I leader exons during physiological changes in GH and nutritional status in liver, with exon 2 being more responsive than exon 1. It may be that this exon is responsible for endocrine IGF-I synthesised in response to specific anabolic stimuli inducing subsequent whole body and skeletal muscle growth.

Effects of glucocorticoids on circulating levels and hepatic expression of insulin-like growth factor (IGF)-binding proteins and IGF-I in the adrenalectomized streptozotocin-diabetic rat

Circulating levels and hepatic expression of insulin-like growth factor-binding protein-1 (IGFBP-1) are increased in insulin-deficient streptozotocin (STZ)-diabetic rats. Glucocorticoids stimulate and insulin suppresses hepatocellular expression of IGFBP-1 in vitro. We asked whether increased IGFBP-1 expression in STZ-diabetic animals is due to an effect of insulin deficiency per se or whether insulin deficiency represents a permissive state where glucocorticoids may play an important role in the regulation of IGFBP-1 and other circulating peptides involved in the modulation of IGF bioactivity. Intact female Sprague-Dawley-derived rats and rats undergoing bilateral adrenalectomy (ADNX) were injected with STZ (140 mg/kg) or buffer. Corticosterone acetate (50 mg/kg) or vehicle was administered to diabetic and nondiabetic animals immediately after ADNX and 24 h later. All rats were killed 48 h after surgery and/or STZ administration. Serum [125I]IGF-I-binding activity was increased 4-fold (P < 0.01), and Western ligand and immunoblotting demonstrated that levels of IGFBP-1 were high in intact STZ-diabetic animals. ADNX prevented these effects of STZ-diabetes, and corticosterone treatment restored serum IGF-binding activity and IGFBP-1 to intact diabetic levels. Similarly, Northern analysis demonstrated that the abundance of hepatic IGFBP-1 mRNA was increased 6-fold in intact STZ-diabetic animals (P < 0.01), but not in adrenalectomized diabetic animals. Corticosterone treatment restored hepatic IGFBP-1 mRNA to intact diabetic levels, and serum concentrations of corticosterone correlated with the abundance of IGFBP-1 mRNA (r = 0.475; P < 0.01), indicating that glucocorticoids play an important role in the regulation of expression of IGFBP-1 in insulin-deficient animals. In contrast, neither ADNX nor corticosterone altered the abundance of hepatic IGFBP-1 mRNA levels in nondiabetic animals. This pattern of regulation appeared to be specific; serum levels of immunoreactive IGFBP-2 and -4 tended to rise in adrenalectomized animals, and levels of IGFBP-3 were not affected by either ADNX or corticosterone treatment. Of note, serum levels of IGF-I by RIA were reduced in STZ-diabetic animals compared to control values (168 +/- 16 vs. 587 +/- 55 ng/ml, respectively; P < 0.01), were partially restored toward control values with ADNX (320 +/- 22 ng/ml), and were reduced again by corticosterone treatment (195 +/- 26 ng/ml), indicating that glucocorticoids also contribute to the regulation of IGF-I levels in insulin-deficient animals. The abundance of IGF-I mRNA was reduced in STZ-diabetic animals, and ADNX also partially prevented this effect of diabetes.

Effects of glucocorticoids on circulating levels and hepatic expression of insulin-like growth factor (IGF)-binding proteins and IGF-I in the adrenalectomized streptozotocin-diabetic rat.

Circulating levels and hepatic expression of insulin-like growth factor-binding protein-1 (IGFBP-1) are increased in insulin-deficient streptozotocin (STZ)-diabetic rats. Glucocorticoids stimulate and insulin suppresses hepatocellular expression of IGFBP-1 in vitro. We asked whether increased IGFBP-1 expression in STZ-diabetic animals is due to an effect of insulin deficiency per se or whether insulin deficiency represents a permissive state where glucocorticoids may play an important role in the regulation of IGFBP-1 and other circulating peptides involved in the modulation of IGF bioactivity. Intact female Sprague-Dawley-derived rats and rats undergoing bilateral adrenalectomy (ADNX) were injected with STZ (140 mg/kg) or buffer. Corticosterone acetate (50 mg/kg) or vehicle was administered to diabetic and nondiabetic animals immediately after ADNX and 24 h later. All rats were killed 48 h after surgery and/or STZ administration. Serum [125I]IGF-I-binding activity was increased 4-fold (P < 0.01), and Western ligand and immunoblotting demonstrated that levels of IGFBP-1 were high in intact STZ-diabetic animals. ADNX prevented these effects of STZ-diabetes, and corticosterone treatment restored serum IGF-binding activity and IGFBP-1 to intact diabetic levels. Similarly, Northern analysis demonstrated that the abundance of hepatic IGFBP-1 mRNA was increased 6-fold in intact STZ-diabetic animals (P < 0.01), but not in adrenalectomized diabetic animals. Corticosterone treatment restored hepatic IGFBP-1 mRNA to intact diabetic levels, and serum concentrations of corticosterone correlated with the abundance of IGFBP-1 mRNA (r = 0.475; P < 0.01), indicating that glucocorticoids play an important role in the regulation of expression of IGFBP-1 in insulin-deficient animals. In contrast, neither ADNX nor corticosterone altered the abundance of hepatic IGFBP-1 mRNA levels in nondiabetic animals. This pattern of regulation appeared to be specific; serum levels of immunoreactive IGFBP-2 and -4 tended to rise in adrenalectomized animals, and levels of IGFBP-3 were not affected by either ADNX or corticosterone treatment. Of note, serum levels of IGF-I by RIA were reduced in STZ-diabetic animals compared to control values (168 +/- 16 vs. 587 +/- 55 ng/ml, respectively; P < 0.01), were partially restored toward control values with ADNX (320 +/- 22 ng/ml), and were reduced again by corticosterone treatment (195 +/- 26 ng/ml), indicating that glucocorticoids also contribute to the regulation of IGF-I levels in insulin-deficient animals. The abundance of IGF-I mRNA was reduced in STZ-diabetic animals, and ADNX also partially prevented this effect of diabetes.(ABSTRACT TRUNCATED AT 400 WORDS)

Skeletal muscle growth and expression of skeletal muscle αs-actin mRNA and insulin-like growth factor I mRNA in pigs during feeding and withdrawal of ractopamine

Sixty crossbred barrows were used to study the effect of ractopamine (a phenethanolamine/beta-adrenergic agonist) treatment and its withdrawal on muscle growth and on the relative abundance of skeletal muscle alpha-actin (sk-alpha-actin) mRNA and of liver and longissimus muscle IGF-I mRNA at 4 wk. Ractopamine was fed (20 ppm) for periods of 2, 4, and 6 wk (six pigs per group). Additional pigs (four per group) were fed ractopamine (20 ppm) for 6 wk and then slaughtered 1, 3, and 7 d after withdrawal of ractopamine. Ractopamine increased (P < .05) longisimus muscle weight and protein content, although protein concentrations were not different. The increased muscle weight and protein content attained by feeding ractopamine for 6 wk was retained when ractopamine was withdrawn. The RNA and DNA concentrations did not change, whereas total DNA and RNA content per muscle was 18 and 26.7% greater, respectively, in ractopamine-treated pigs at 4 wk, but there were no differences at 2 or 6 wk or among the withdrawal groups. The relative abundance of sk-alpha-actin mRNA in the longissimus muscle was 41 and 62% greater (P < .05) in treated animals at 2 and 4 wk but was similar to that in controls at 6 wk and during the withdrawal period. The relative abundance of IGF-I mRNA in liver and longissimus muscle was not altered with ractopamine treatment for 4 wk. These results indicate that the ractopamine-enhanced muscle growth may result from increased myofibrillar gene expression at the pretranslational level, which is maximal with short-term treatment of ractopamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Ontogeny of the porcine insulin-like growth factor system

The developmental expression of the individual components of the insulin-like growth factor (IGF) system in pigs was examined. Serum IGF-I and IGF-binding protein-3 (IGFBP-3) levels were low during fetal life and increased during postnatal development. Levels of mRNAs encoding these proteins were not greater for liver than for nonhepatic tissues (skeletal muscle, lung, kidney) and did not increase during the postnatal period, whereas hepatic growth hormone (GH) receptor mRNA expression was increased postnatally. Serum IGF-II levels exceeded IGF-I levels at all developmental stages examined and both exhibited postnatal increases. IGF-II mRNA abundance, in contrast, was high in the fetal tissues with the exception of lung and declined during the perinatal transition. Hepatic IGFBP-2 mRNA and serum IGFBP-2 levels increased during the latter half of gestation and then declined postnatally. The levels in muscle and liver of type I IGF receptors and the corresponding mRNAs also exhibited postnatal decreases. The discordance of changes in hepatic IGF-I and IGF-II mRNA abundance with serum IGF levels during the postnatal period does not support the concept that liver is the primary endocrine source of IGFs in the young pig. The postnatal increases in serum IGF levels may reflect decreased plasma clearance rates of these peptides which may be related to the transition in IGFBP type from IGFBP-2 to IGFBP-3 in blood and the reduced tissue expression of IGF receptors.

Messenger ribonucleic acid expression of the insulin-like growth factors and their binding proteins in uterine fibroids and myometrium.

Fibroids (leiomyomata) are the most common tumors in women, but their etiology is unknown. The insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) may be important in the growth of these benign neoplasms. We have examined the presence of mRNA encoding both IGF-I and IGF-II and IGFBP-1, -2, and -3 in fibroids and corresponding myometrium from 20 women undergoing hysterectomy for symptomatic uterine fibroids. Northern blots of total cellular RNA were probed with oligonucleotides for IGF-I, IGF-II, IGFBP-2, and IGFBP-3 and a human IGFBP-1 cDNA. Western ligand blotting was also used to detect the presence of IGFBP proteins in both fibroid and myometrium. The data showed that in fibroids compared to myometrium, 1) the relative abundance of IGF-I mRNA was not different, but there was an increase in the relative abundance of IGF-II mRNA (P < 0.001); 2) IGFBP-1 mRNA was undetectable in fibroids and detectable in only 1 specimen of myometrium; 3) there was no difference in the relative abundance of IGFBP-2 mRNA, but there was an increase in the relative abundance of IGFBP-3 mRNA in myometrium (P < 0.05). By Western ligand blotting, both IGFBP-2 and -3 proteins were present. Our data show that the mRNAs encoding IGF-I, IGF-II, IGFBP-2, and IGFBP-3 are expressed in both fibroids and myometrium and that fibroids express more IGF-II and less IGFBP-3 mRNA than myometrium. We postulate that the net effect of the changes seen is to increase the bioavailability of free (bioactive) IGF, which may then play a major role in promoting fibroid tumor growth.

Influence of somatotropin on lipid metabolism and IGF gene expression in porcine adipose tissue.

The present study was designed to evaluate the effects of porcine somatotropin (pST) treatment (2 mg/day) and dietary fat (10%) separately and in combination on the metabolic activity of subcutaneous adipose tissue, serum adipogenic activity, and insulin-like growth factor (IGF) gene expression within adipose tissue from growing 5- to 6-mo-old barrows. This study attempted to determine how these factors might contribute to the reported changes in adiposity of treated swine. Biopsies of adipose tissue were collected after 28 days of treatment following anesthesia with thiopental sodium (15 mg/kg iv). Somatotropin inhibited in vitro glucose oxidation and lipogenesis in adipose tissue but did not affect fatty acid esterification. Adipogenic activity of serum was not altered by pST treatment. Subcutaneous adipose tissue contained mRNA for IGF-I and -II, and pST administration increased the abundance of IGF-I mRNA. Dietary fat had no effect on these variables. Thus somatotropin reduces glucose metabolism in porcine subcutaneous adipose tissue. Preadipocyte proliferation and differentiation are not affected by somatotropin through its actions on systemic factors. Dietary fat provides no additional benefit in combination with pST administration to affect accretion of adipose tissue in growing swine.

Octreotide inhibits insulin-like growth factor-I hepatic gene expression in the hypophysectomized rat: evidence for a direct and indirect mechanism of action.

Somatostatin and somatostatin analogs are known to interact with the GH-insulin-like growth factor (IGF)-I axis by inhibiting GH secretion and consequently hepatic IGF-I production. Indirect evidence suggests that octreotide, a somatostatin analog, reduces serum IGF-I levels relatively more than expected from GH reduction, implying a GH-independent pathway of action. To study the role of octreotide in the regulation of IGF-I production, independently of endogenous GH, we used the hypophysectomized (hypox) rat to measure hepatic IGF-I expression and also employed cultured rat hepatocytes to examine whether octreotide has any direct effect on the production of IGF-I. Forty male hypox Sprague-Dawley rats were randomized into 4 groups to receive daily injections for 3 days of either saline, human GH (hGH) (100 g), octreotide (100 g twice), or both hGH (100 g) and octreotide (100 g twice). GH stimulated serum IGF-I levels to 104 +/- 10 micrograms/liter as compared to saline (26 +/- 2 micrograms/liter). Octreotide alone had no effect, but combining octreotide and hGH significantly reduced the hGH-induced rise in the IGF-I levels (52 +/- 6 micrograms/liter). The relative expression of hepatic IGF-I in the rats treated with hGH increased by 4-fold compared to that in the saline-treated rats. Octreotide administered simultaneously with hGH potently blocked the hGH-induced IGF-I expression to control levels. In cultured hepatocytes, IGF-I mRNA levels maximally stimulated by combining bGH and glucagon were significantly inhibited in the presence of octreotide at low concentrations (0.3 and 3 ng/ml) by 25% and 45%, respectively. In contrast, high concentrations of octreotide (30 and 300 ng/ml) had no significant effect on IGF-I mRNA abundance. We conclude that: 1) octreotide inhibits IGF-I serum levels and hepatic gene expression in the hypox rat; and 2) octreotide can inhibit partially the direct effects of GH and glucagon on hepatic IGF-I production.

Fibroblast growth factor inhibits insulin-like growth factor-II (IGF-II) gene expression and increases IGF-I receptor abundance in BC3H-1 muscle cells.

Muscle is an important target tissue for insulin-like growth factor (IGF) action. We have previously reported that muscle cell differentiation is associated with down-regulation of the IGF-I receptor at the level of gene expression that is concomitant with an increase in the expression and secretion of IGF-II. Furthermore, treatment of myoblasts with IGF-II resulted in a similar decrease in IGF-I receptor mRNA abundance, suggesting an autocrine role of IGF-II in IGF-I receptor regulation. To explore further the role of IGF-II in IGF-I receptor regulation, BC3H-1 mouse muscle cells were exposed to differentiation medium in the presence of basic fibroblast growth factor (FGF), a known inhibitor of myogenic differentiation. FGF treatment of cells resulted in a 50% inhibition of IGF-II gene expression compared to that in control myoblasts and markedly inhibited IGF-II secretion. Concomitantly, FGF resulted in a 60-70% increase in IGF-I binding compared to that in control myoblasts. Scatchard analyses and studies of gene expression demonstrated that the increased IGF-I binding induced by FGF reflected parallel increases in IGF-I receptor content and mRNA abundance. These studies indicate that FGF may up-regulate IGF-I receptor expression in muscle cells through inhibition of IGF-II peptide expression and further support the concept of an autocrine role of IGF-II in IGF-I receptor regulation. In addition, these studies suggest that one mechanism by which FGF inhibits muscle cell differentiation is through inhibition of IGF-II expression.