Molecular cloning of rat insulin-like growth factor I complementary deoxyribonucleic acids: differential messenger ribonucleic acid processing and regulation by growth hormone in extrahepatic tissues

Two classes of insulin-like growth factor I (IGF-I) cDNAs were isolated from an adult rat liver library using a human IGF-I cDNA probe. The two types of rat IGF-I cDNA differed by the presence or absence of a 52-base pair insert which altered the derived C-terminal amino acid sequence of the E peptide, but not the 3'-untranslated region or the sequence coding for the mature IGF-I protein. When probes derived from these cDNA clones were hybridized to Northern blots of rat mRNA, specific bands of 8.6, 2.1, and 1.0-1.4 kilobases were seen. Hybridization to poly(A)+ RNA from various tissues from GH-treated and control rats demonstrated an increase in IGF-I mRNA due to GH treatment in all tissues examined.     

Glucose starvation reduces IGF-I mRNA in tumor cells: evidence for an effect on mRNA stability

The purpose of this study was to characterize the mechanisms by which glucose regulates IGF-I gene expression in rat C6 glioma cells and in rat GH3 pituitary adenoma cells. Glucose starvation for periods of 12 to 48 h decreased IGF-I mRNA levels. In contrast, there was no stimulation of IGF-I mRNA by medium glucose between 1 and 25 mM over a 24-h period. Studies with hexoses and glycolytic metabolites suggested that glucose metabolism was required to maintain IGF-I mRNA. Glucose starvation lowered IGF-I mRNA half-life in both C6 and GH3 cells. Protein synthesis inhibition lowered IGF-I mRNA by about 20% in glucose-fed C6 and GH3 cells, while potently increasing IGF-I mRNA in glucose-starved C6 cells and not altering IGF-I mRNA in glucose-starved GH3 cells. Our results suggest that in these tumor cells, IGF-I mRNA stability is reduced by glucose starvation, secondary to a deficiency in intracellular glucose metabolism. Ongoing protein synthesis is not required for this mRNA de-stabilizing effect in GH3 cells. Rather, in glucose-starved C6 cells, decreased IGF-I mRNA stability may result from the action of a labile protein.     

The size heterogeneity of rat insulin-like growth factor-I mRNAs is due primarily to differences in the length of 3'-untranslated sequence

Previous studies have revealed multiple size classes of rat insulin-like growth factor-I (IGF-I) of estimated size 7.5-7.0, 1.9-1.5, and 1.2-0.9 kilobases (kb). Available sequence information accounts for only 2.1 kb of the 7.5-7.0 kb IGF-I mRNAs. We used oligomer directed ribonuclease H (RNase H) mapping to define the extent to which the unknown sequence in the large molecular weight mRNAs lies 5' or 3' to known sequence. Rat liver polyadenylated RNAs were incubated with oligomer probes complementary to internal rat IGF-I precursor (E domain) coding sequences. RNase H was used to hydrolyze IGF-I mRNAs at the point of annealment with the oligomers. Resultant 5' and 3'-IGF-I mRNA fragments were analyzed on Northern blots. A probe specific for type 1 (class C) 5'-sequences (the most predominant of multiple 5'-sequence types found on rat IGF-I mRNAs) identifies intact IGF-I mRNAs of 7.5-7.0, 1.9-1.5 and 1.2-0.9 kb but, after oligomer directed RNase cleavage of these mRNAs, identified only a single IGF-I mRNA 5'-fragment. Major differences in the length of sequence 5' to the IGF-I coding sequence therefore, do not account for the multiple size classes of type 1 (class C) IGF-I mRNAs. The size of the 5'-fragment suggests that the extent of sequence 5' to the IGF-I coding sequence is 0.4-0.7 kb in type 1 (class C) IGF-I mRNAs. Identification of multiple 3'-fragments of IGF-I mRNAs demonstrated heterogeneity in the 3'-ends of rat IGF-I mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell-specific localization of insulin-like growth factor binding protein mRNAs in rat liver

The liver is a major source of circulating insulin-like growth factor I (IGF-I), and it also synthesizes several classes of IGF binding proteins (IGFBPs). Synthesis of IGF-I and IGFBPs is regulated by hormones, growth factors, and cytokines. They are nutritionally regulated and expressed in developmentally specific patterns. To gain insight into cellular regulatory mechanisms that determine hepatic synthesis of IGF-I and IGFBPs and to identify potential target cells for IGF-I within the liver, we studied the cellular sites of synthesis of IGF-I, IGF receptor, growth hormone (GH) receptor, and IGFBPs in freshly isolated rat hepatocytes, endothelial cells, and Kupffer cells. We also localized cellular sites of IGFBP synthesis by in situ hybridization histochemistry. Western ligand and immunoblot analyses were used to determine IGFBP secretion by isolated cells. Two IGF-I mRNA subtypes with different 5' ends (class 1 and class 2) were detected in all isolated liver cell preparations. Type 1 IGF receptor mRNA was detected in endothelial cells, indicating that these cells are a local target for IGF actions in liver. GH receptor was expressed in all cell preparations, consistent with GH regulation of IGF-I and IGFBP synthesis in multiple liver cell types. The IGFBPs expressed striking cell-specific expression. IGFBP-1 was synthesized only in hepatocytes, and IGFBP-3 was expressed in Kupffer and endothelial cells. IGFBP-4 was expressed at high levels in hepatocytes and at low levels in Kupffer and endothelial cells. Cell-specific expression of distinct IGFBPs in the liver provides the potential for cell-specific regulation of hepatic and endocrine actions of IGF-I.     

Tissue and development specific regulation of a complex family of rat insulin-like growth factor I messenger ribonucleic acids

To obtain information about the functional significance of the structural heterogeneity that has been described for rat insulin-like growth factor I (IGF-I) cDNAs, we hybridized polyadenylated RNAs from rat tissues at different developmental stages with probes specific for two variant 5'-sequences (designated here as type 1 and type 2), with a probe specific for IB type E domain coding sequences and with a probe for E domain sequences common to IA and IB type IGF-I cDNAs. Northern blot analyses revealed that previously reported rat liver IGF-I mRNAs of estimated size 7.5-7.0, 1.9-1.5, and 1.2-0.9 kilobases each are comprised of multiple closely migrating IGF-I mRNA species containing either of two 5'-sequences and either IA or IB type E domain coding sequences. In liver, each of these detected IGF-I mRNA species showed postnatal increases in abundance. The mRNAs detected with the probe for type 2 5'-sequences were detected exclusively in postnatal liver and also showed a different pattern of postnatal increase in abundance than other IGF-I mRNA types. IGF-I mRNAs detected with the probe for IB type E domain coding sequences likewise were highly liver specific and were undetectable or barely detectable in other fetal or adult rat tissues. In contrast, IGF-I mRNAs that hybridized with probes for type 1 5'-sequences or for E domain coding sequences common to IA and IB type IGF-I mRNAs were detected in all fetal and adult rat tissues tested. These findings suggest development and tissue specific regulation of the expression of different rat IGF-I mRNA types, and also suggest a possible role of different precursor sequences encoded by the various mRNAs in targeting of IGF-I to a local site of action.     

Distribution and regulation of rat insulin-like growth factor I messenger ribonucleic acids encoding alternative carboxyterminal E-peptides: evidence for differential processing and regulation in liver

Alternative splicing of insulin-like growth factor I (IGF-I)/somatomedin C mRNAs generates two IGF-I mRNAs coding for IGF-I peptides with different sequences in the E domain of the IGF-I prohormone. These two mRNAs encode alternative E peptides due to the presence (IGF-Ib) or absence (IGF-Ia) of a 52-base insert in the region coding for the E domain. We have used a solution hybridization/RNase protection assay to determine the tissue distribution and regulation by GH of the expression of these alternative IGF-I mRNAs. IGF-Ib mRNAs are present in low abundance (representing approximately 2.5% of the total IGF-I mRNA) in heart, lung, muscle, testes, stomach, kidney, and brain, but represent approximately 13% of the IGF-I mRNA in liver. GH treatment of hypophysectomized rats increased steady-state IGF-I mRNA levels in liver, kidney, lung, and heart. In kidney, lung, and heart, IGF-Ia and IGF-Ib mRNA levels were coordinately regulated by GH, but, in liver, the fold increase in IGF-Ib mRNA levels was approximately three times greater than the fold increase in IGF-Ia mRNA levels. These data suggest that the processing of IGF-I mRNA in liver is different than in nonhepatic tissues. These results also further elucidate the organization of the rat IGF-I gene as well as the generation of multiple IGF-I mRNAs by alternative splicing.     

Hormonal Regulation of Leptin mRNA Expression and Preadipocyte Recruitment and Differentiation in Porcine Primary Cultures of S-V Cells

The hormonal regulation of leptin mRNA expression and the association between leptin expression and adipocyte differentiation were examined in primary cultures of porcine S-V cells with Northern blot and immunocytochemical analysis. Seeding for 3 days with fetal bovine serum (FBS) with varying levels of dexamethasone (Dex) increased levels of leptin mRNA in a dosedependent manner in parallel with increases in the proportion of preadipocytes (AD-3 positive cells; AD-3, a preadipocyte marker). Six-day treatment with 10 or 850 nM insulin after FBS+Dex treatment resulted in a similar increase in leptin mRNA expression and morphological differentiation. However, significantly lower levels of leptin mRNA and smaller fat cells were observed in cultures treated with 1 nM insulin or 10 nM insulin-like growth factor-I (IGF-I). Dex-induced increases in leptin mRNA levels and AD-3 cell numbers were blocked completely by the addition of transforming growth factor-β (TGF-β) to FBS+Dex-treated cultures. However TGF-β significantly increased fat cell size and leptin mRNA expression when added to ITS (insulin, 850 nM; transferrin, 5 μg/ml; and selenium, 5 ug/mL) treated cultures during the lipid-filling stage. When added with FBS+DEX for the first 3 days, growth hormone (GH) did not influence the Dex-induced increase in AD-3 cells and leptin mRNA expression, but GH reduced leptin mRNA levels when added with insulin for 6 days after FBS+Dex. These results demonstrated that regulation of leptin mRNA expression by Dex, insulin, IGF-I, TGF-β, and GH may be associated with changes in preadipocyte number and fat cell size.     

Organ-specific and age-dependent expression of insulin-like growth factor-I (IGF-I) mRNA variants: IGF-IA and IB mRNAs in the mouse

Insulin-like growth factor-I (IGF-I) gene generates several IGF-I mRNA variants by alternative splicing. Two promoters are present in mouse IGF-I gene. Each promoter encodes two IGF-I mRNA variants (IGF-IA and IGF-IB mRNAs). Variants differ by the presence (IGF-IB) or absence (IGF-IA) of a 52-bp insert in the E domain-coding region. Functional differences among IGF-I mRNAs, and regulatory mechanisms for alternative splicing of IGF-I mRNA are not yet known. We analyzed the expression of mouse IGF-IA and IGF-IB mRNAs using SYBR Green real-time RT-PCR. In the liver, IGF-I mRNA expression increased from 10 days of age to 45 days. In the uterus and ovary, IGF-I mRNA expression increased from 21 days of age, and then decreased at 45 days. In the kidney, IGF-I mRNA expression decreased from 10 days of age. IGF-IA mRNA levels were higher than IGF-IB mRNA levels in all organs examined. Estradiol-17beta (E2) treatment in ovariectomized mice increased uterine IGF-IA and IGF-IB mRNA levels from 3 hr after injection, and highest levels for both mRNAs were detected at 6 hr, and relative increase was greater for IGF-IB mRNA than for IGF-IA mRNA. These results suggest that expression of IGF-I mRNA variants is regulated in organ-specific and age-dependent manners, and estrogen is involved in the change of IGF-I mRNA variant expression.     

Expression of insulin-like growth factor-I and its receptor by SV40-transformed rat granulosa cells

Cellular proliferation is a dominant aspect of ovarian follicular development in the rat, and insulin-like growth factor I (IGF-I) has been proposed as a mediator of cellular growth and differentiation in the ovary. An SV40-transformed rat granulosa cell line (RGA-41S) has been established as a model for studies on dividing cells of granulosa origin. Granulosa cells from the ovaries of immature diethylstilbestrol-treated rats were infected with the tsA255 mutant of SV40, followed by cloning in serum-free medium to select transformed cell lines which were serum independent. At the permissive temperature (33 C), RGA-41S cells exhibited a transformed phenotype and rapidly formed high density multilayers of compact cells that readily overgrew nontransformed cells. At the nonpermissive temperature (40 C) cell replication declined and division ceased after 4 days. Furthermore, at 40 C the cells grew as a monolayer and assumed a tetrahedral shape with a high cytoplasm-to-nucleus ratio, and displayed reduced ability to overgrow nontransformed cells. The transformed ovarian cells did not express detectable gonadotropin receptors and steroidogenic activity but retained their epithelial phenotype as demonstrated by cytokeratin staining of the cytoskeleton, the presence of microvilli, and the formation of tight junctions between cells. In support of the proposed autocrine-paracrine actions of IGF-I in the ovary, assay of conditioned serum-free culture medium revealed secretion of IGF-I-immunoreactive material by RGA-41S cells. HPLC-purified IGF-I immunoreactivity from these cells eluted with the same retention time as recombinant human IGF-I. When hybridized with a 32P-labeled rat IGF-I cDNA probe, poly(A)+ mRNA prepared from RGA-41S cells grown at both temperatures showed the typical three size classes of IGF-I mRNA on Northern blots (7.5, 1.7, and 0.8-1.2 kilobase kb), although the levels were somewhat higher at 33 C. The presence of IGF-I receptors in transformed cells was demonstrated by specific 125I-IGF-I binding to intact cells. Scatchard analysis indicated a single class of high affinity receptors at a density of 10(5) binding sites per cell and a dissociation constant (Kd) = 0.52 x 10(-9) M. Furthermore, hybridization of a 32P-labeled IGF-I receptor probe to Northern blots of poly(A+) RNA prepared from cells grown at 33 C and 40 C revealed an 11-kilobase rat IGF-I receptor mRNA. Physiological concentrations of IGF-I increased [3H]aminoisobutyric acid uptake by RGA-41S cells grown at either temperature, attesting to the retention of responsiveness to IGF-I in these transformed granulosa cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

IGF-I causes an ultrasensitive reduction in GH mRNA levels via an extracellular mechanism involving IGF binding proteins

IGF-I-dependent decreases in endogenous GH mRNA expression were studied in individual rat MtT/S somatotroph cells using in situ hybridization. It was first shown that increasing IGF-I concentrations (0-90 nM) decreased GH mRNA levels in a ultrasensitive manner when averaged over the entire population, such that the decrease occurred over a narrow range of IGF-I concentration with an EC50 of 7.1 nM. The degree of ultrasensitivity of the population average was expressed by calculating the Hill coefficient (nA), which had a value of -2.0. GH mRNA levels in individual dispersed cells from these cultures were then measured. These results were first summed for all cells to show that the average response of the population remained ultrasensitive (nA = -2.6, EC50 = 8.1 nM). Then, parameters for individual cells of the population were calculated using mathematical modeling of the distribution of individual cell GH mRNA levels after treatment with 0-90 nM IGF-I. Solution of the data from the individual cells yielded a Hill coefficient (nI = -0.65) and a heterogeneity coefficient (mI = -1.2) indicative of individual cell responsiveness to IGF-I that was not ultrasensitive and very heterogeneous. These results suggested that ultrasensitivity in the population may likely be caused by an extracellular mechanism regulating IGF-I concentrations, such as IGF binding proteins. Increasing concentrations of long (Arg)3IGF-1, an analog that binds the IGF type-1 receptor but not IGF binding proteins, showed a linear inhibition of GH mRNA levels. Treatment with IGF binding protein ligand inhibitor, an IGF-I analog that binds to IGF binding proteins but not the IGF type-1 receptor, decreased GH mRNA levels in the absence of exogenous IGF-I. Thus, IGF binding proteins provide the extracellular sequestration of IGF-I necessary for the precise and ultrasensitive regulation of GH mRNA levels in the entire cell population, although expression within individual cells is regulated in a graded fashion.     

Nucleotide sequence and growth hormone-regulated expression of salmon insulin-like growth factor I mRNA

Protein and cDNA sequence analysis have revealed that the insulin-like growth factor (IGF-I) has been highly conserved among several mammalian species. Using the combined techniques of polymerase chain reaction and molecular cloning, we have now obtained the cDNA sequence encoding preproIGF-I from a teleost species, Oncorhynchus kisutch (coho salmon). The 2020 nucleotide (nt) cloned cDNA sequence contains a 528 nt open reading frame encoding 176 amino acids in preproIGF-I and 175 nt and 1317 nt of flanking 5'- and 3'-untranslated regions, respectively. The deduced amino acid sequence of salmon IGF-I is highly conserved relative to its mammalian homologues and there are only 14 amino acid differences out of 70 between salmon and human IGF-I. Interestingly, the C-terminal E domain of salmon proIGF-I, which is presumed to be proteolytically cleaved during biosynthesis, also shows striking amino acid sequence homology with its mammalian counterpart, except for an internal 27 residue segment that is unique to salmon proIGF-I. Northern analysis revealed that salmon preproIGF-I mRNA consists predominantly of a single 3900 nt sized band although minor bands were also observed after prolonged autoradiographic exposure. The RNA analysis also revealed that the level of preproIGF-I mRNA is increased 6-fold in liver RNA isolated from salmon injected with bovine GH, as compared to untreated controls. These results demonstrate that the primary structure and regulated expression of IGF-I by GH have been conserved in teleosts.