Expression and function of insulin/insulin-like growth factor I hybrid receptors during differentiation of 3T3-L1 preadipocytes.

During the assembly of cell surface receptors, insulin proreceptors are sometimes joined to insulin-like growth factor (IGF) receptor precursors to form covalently linked hybrid receptors. To address the biological consequences of hybrid receptor formation, we studied 3T3-L1 cells known to undergo a 50-70-fold increase in insulin binding while maintaining nearly constant levels of IGF-I binding during differentiation from preadipocytes into adipocytes. The presence of insulin/IGF receptor hybrids in 3T3-L1 adipocytes was demonstrated by the immunoprecipitation of phosphorylated receptors and a novel enzyme-linked immunoassay. Hybrid receptor levels were very low in the early stages of differentiation and increased rapidly between days 4 and 6, reaching a level about 100-fold higher in the mature adipocyte. Coincident with the hybrid assembly, the formation of archetypal (alpha2,beta2) IGF receptors decreased. In fully differentiated adipocytes, virtually all of the IGF receptors were in hybrid form. Stimulation by IGF-I of receptors isolated from mature adipocytes caused autophosphorylation of IGF receptor beta subunits in hybrid complexes, whereas autophosphorylated IGF holoreceptors were not demonstrable. Insulin and IGF-I were equipotent in stimulating glucose uptake in the differentiated adipocytes, leading to the conclusion that hybrid insulin/IGF receptors can transduce a transmembrane signal when activated by IGF-I. We conclude that hybrid formation constitutes a novel post-translational mechanism whereby increased synthesis of insulin receptors limits the cell surface expression of the homologous IGF receptor. Furthermore, biological actions in 3T3-L1 adipocytes, previously attributed to archetypal IGF receptors, are in fact mediated through hybrid receptors.

Failure of insulin-like growth factor type I to suppress HIV type 1 in adult or umbilical cord mononuclear blood cells.

Insulin-like growth factor type I (IGF-I) has been used as a treatment for cachexia in adults with AIDS and has been reported to show inhibitory activity against HIV-1IIIB in cord blood mononuclear cells (CBMCs) in vitro at low-concentration (1%) fetal bovine serum (FCS). We evaluated the effect of IGF-I on MN, IIIB, and BaL strains, as well as on a patient isolate of HIV-1 in CBMCs and adult peripheral blood mononuclear cells (PBMCs). IGF-I failed to show any inhibitory effect on HIV replication in CBMCs or adult PBMCs under various culture conditions. In contrast to an earlier report of an antiviral effect, IGF-I augmented HIV-1 replication in PHA-stimulated PBMCs maintained in a low concentration of FCS.

Insulin-like growth factor I receptor expression and function in fibroblasts from two patients with deletion of the distal long arm of chromosome 15.

Most patients with deletion of the distal long arm of chromosome 15 have intrauterine growth retardation and postnatal growth deficiency in addition to developmental abnormalities. It has been proposed that the absence of one copy of the insulin-like growth factor I (IGF-I) receptor gene may play a role in the growth deficiency seen in this syndrome. To address this question we examined IGF-I receptor expression and function in fibroblasts from two patients with deletion of the distal long arm of chromosome 15 (15q26.1-->qter). Quantitative Southern blot analysis of the IGF-I receptor gene was performed on HindIII digests of fibroblast DNA. Radioactivity in the 1.7-kilobase receptor fragment in the two patients was 55% and 51% of the values in controls, consistent with the absence of one copy of the IGF-I receptor gene. IGF-I receptor messenger ribonucleic acid levels were quantitated by a solution hybridization/nuclease protection assay. Receptor messenger ribonucleic acid levels in the two patients were 45% and 52% of the values in controls. Northern blotting demonstrated normal size IGF-I receptor transcripts and affinity crosslinking of [125I]IGF-I to Triton X-100-solubilized fibroblasts demonstrated a normal size receptor in the patients. Analysis of placental membranes prepared from one patient revealed no difference in [125I]IGF-I binding. In the patients' fibroblasts, however, binding of [125I]long [R3]-IGF-I to the IGF-I receptor was significantly reduced, as assessed by the amount of radioactivity competed by the monoclonal antibody alpha IR-3 or insulin and Scatchard analysis of binding data. To assess IGF-I receptor function, stimulation of [alpha-1-14C]-methylaminoisobutyric acid transport and stimulation of [methyl-3H]thymidine incorporation into DNA by a full range of IGF-I concentrations was examined in patient and control fibroblasts. There was a significant decrease in the maximal response to IGF-I in both assays for one of the two patients when data were expressed as fold response over the basal value. However, there was no evidence for impairment of response to IGF-I in either patient's fibroblasts when data were expressed as net stimulation (maximal response minus basal). In conclusion, although IGF-I receptor expression was decreased in fibroblasts from two patients with deletion of the distal long arm of chromosome 15, we were unable to provide conclusive evidence for impairment of the biological response to IGF-I.

A possible genetic defect in 25-hydroxylation as a cause of rickets.

We examined two siblings who had severe rickets at ages 2 and 7 years, respectively, despite a history of adequate vitamin D intake. The patients' sera had calcium concentrations at the lower limits of normal, low phosphate concentrations, elevated alkaline phosphatase activity, and low levels of 25-hydroxyvitamin D. Treatment with high doses of vitamin D2 resulted in resolution of the biochemical abnormalities and radiographic deformities; pharmacologic doses of vitamin D2 were required to maintain normal concentrations of 25-hydroxyvitamin D in the serum even though vitamin D absorption was normal. These children may have a genetic defect of the 25-hydroxylation step in vitamin D activation.

The 5'-untranslated region of the IGF-I receptor gene modulates reporter gene expression by both pre- and post-transcriptional mechanisms.

The insulin-like growth factor I (IGF-I) receptor gene has a large, complex 5'-untranslated region (UTR). In order to examine the role that this region plays in regulating IGF-I receptor expression, we isolated fragments of the human IGF-I receptor 5'-UTR and interposed them between the SV40 early promoter and the chloramphenicol acetyl transferase reporter gene. Fragments of the IGF-I receptor gene 5'-UTR were found to enhance reporter gene expression by increasing gene transcription. In addition, the increased transcription and mRNA levels were in excess of the increase in enzyme activity, providing indirect evidence that these fragments inhibit translation, consistent with predictions.

Accuracy and precision of low-dose insulin administration.

OBJECTIVE: To determine the lowest dose of concentrated (U100) insulin that can be reproducibly delivered. METHODS: A telephone survey was used to determine current practices in major pediatric hospitals regarding the administration of low doses of concentrated insulin. A sensitive gravitometric technique was used to determine the error in measurement of low doses of U100 insulin by pediatric nurses and parents of diabetic children. RESULTS: A telephone survey revealed that doses as low as 0.5 or 1.0 U (5 to 10 microL) are routinely administered in pediatric hospitals. In our study of pediatric nurses, attempts to deliver 0.5, 1.0, and 2.0 U resulted in delivered doses of 0.975 +/- 0.315, 1.638 +/- 0.376, and 2.153 +/- 0.435 U (mean +/- standard deviation of the mean), respectively. The use of 0.3-mL syringes compared to 0.5-mL syringes did not improve accuracy or precision. Taken as a group, parents of children with diabetes were more accurate (mean delivered dose of 1.063 +/- 0.276 for the 1-U dose), but the individual means ranged from 0.641 to 1.300 and coefficients of variation ranged from 5% to 33%. Only three of the seven parents could deliver 1.0 U with acceptable precision and accuracy. CONCLUSIONS: When currently available syringes are used, insulin injections of less than 20 microL (2 U of U100) have an unacceptably large error. It is recommended that, in the inpatient setting, diluted insulin be used if the prescribed dose is less than 2.0 U.

Effects of insulin-like growth factor-I on androgen production by highly purified pubertal and adult rat Leydig cells.

Leydig cells were isolated and purified from adult and midpubertal rats to study the effects of insulin-like growth factor-I (IGF-I) on steroidogenesis. Androgen production, as measured in Leydig cell conditioned culture media, from four different treatment groups (1 = no hormone; 2 = 70 ng/ml IGF-I; 3 = 0.1 ng/ml LH; 4 = 70 ng/ml IGF-I + 0.1 ng/ml LH) were compared daily. After 3 days in culture, the cells were treated with a maximally stimulating dose of luteinizing hormone (LH) (100 ng/ml) for 3 hours. Androgen production was highest in the cells treated with both IGF-I and low concentrations of LH. In the presence of IGF-I, regardless of LH, cells derived from pubertal animals had a greater increase in steroidogenesis during the culture period than did cells from adult animals. Pretreatment with IGF-I prior to maximal LH stimulation induced a greater increase in androgen production in cells from pubertal rats than in cells from adult animals. It is concluded that IGF-I has a direct effect on Leydig cells and may act synergistically with LH to promote androgen synthesis. The greater response in pubertal cells raises the possibility that IGF-I is important in the maturing process of the testis.

Expression of a type I insulin-like growth factor receptor with low affinity for insulin-like growth factor II.

We investigated the binding properties of the type I insulin-like growth factor (IGF) receptor expressed in NIH-3T3 fibroblasts transfected with a human type I receptor cDNA. Cell surface receptors bound IGF-I with KD = 1 nM as predicted. Although recent studies have suggested that IGF-I and IGF-II bind to type I receptors with near-equal affinity, the receptors in this system bound IGF-II with much lower affinity (KD = 15-20 nM). When type I receptors from the transfected cells were solubilized and immunopurified, however, both 125I-IGF-I and 125I-IGF-II bound to the purified receptors with extremely high and relatively similar affinities (KD = 8 and 17 pM respectively). Thus the immunopurified receptors had higher affinity but lower specificity for the two ligands. The monoclonal antibody alpha IR-3 effectively inhibited IGF-I binding to cell surface receptors (75 +/- 10%), but did not inhibit IGF-II binding. In the purified receptor assay, alpha IR-3 also inhibited IGF-I binding more effectively than IGF-II binding (38 +/- 7% versus 10 +/- 4%). We conclude that the products of this cDNA can account for the binding patterns that we previously observed in receptors immunopurified from human placenta. The differential effect of alpha IR-3 on IGF-I versus IGF-II raises the possibility that these homologous growth factors bind to immunologically distinct epitopes on the type I receptor.

Analysis of the human type I insulin-like growth factor receptor promoter region.

We isolated genomic fragments containing the 5' region of the human type I insulin-like growth factor receptor gene. A unique transcription start site was identified, defining a 1038 bp 5'-untranslated region. No TATA or CCAAT elements were identified in the proximal 480 nucleotides of 5'-flanking region. The region surrounding the transcription start site was similar to a recently described "initiator" sequence. The 5'-flanking and 5'-untranslated regions were highly GC-rich, with numerous potential Sp1 binding sites. A potential AP-2 binding site was identified in the 5'-flanking region and a potential thyroid response element was identified in the 5'-untranslated region. The 5' region of the human gene was very similar to that of the rat gene, with conservation of many of the potential regulatory elements.

Isolation and characterization of cDNA encoding the alpha subunit of Cap Z(36/32), an actin-capping protein from the Z line of skeletal muscle.

cDNA encoding the alpha chain of Cap Z has been isolated by screening a lambda gt11 library with affinity-purified antibodies. A single cDNA insert (designated CE2) of 2153 base pairs (bp) contains an open reading frame of 836 bp, which is incomplete at its 5' end. The technique of "rapid amplification of cDNA ends" has been used to extend the 5' end of this open reading frame to a potential transcription initiation site that is preceded by 320 bp of an apparently untranslated region. The protein predicted by the resulting nucleotide sequence has a Mr of 32,960 and contains four regions that show close homology with four alpha-chymotryptic digestion fragments of the alpha chain. The amino acid composition of the alpha chain of Cap Z and the predicted protein are also similar. Northern blot analysis of whole chicken embryos shows two mRNA species of 1.9 and 2.4 kilobases, respectively, that hybridize with CE2. Three potential polyadenylylation signals in two regions of CE2 460 bp apart are identified, suggesting that the two messages may result from the use of alternative polyadenylylation sites. Comparison of the sequence data with that of other known actin-capping and severing proteins shows no significant homologies, suggesting that Cap Z may be a member of a unique group of capping, nonsevering proteins.

cDNAs encoding the beta subunit of cap Z, the actin-capping protein of the Z line of muscle.

Three cDNAs encoding the beta chain of Cap Z have been isolated from lambda gt11 chicken libraries screened with antibodies. The coding region, which is identical among the cDNAs, contains 831 basepairs encoding a protein with 277 amino acid residues and Mr = 31,352. The predicted protein sequence contains eight regions that match perfectly the NH2-terminal sequence of eight peptides isolated from muscle Cap Z beta. The amino acid compositions of the protein predicted from the cDNA sequence and Cap Z beta are similar. Comparison of the cDNA sequence and the predicted protein sequence with those of other actin-binding proteins and all sequences in the GenBank and NBRF Protein databases shows no remarkable similarities. Two of the three cDNAs contain the complete coding region. Both coding regions begin with a consensus translation start site; however, their 5'-untranslated regions are different. Northern analysis of whole chicken embryos and adult chicken tissues shows two mRNA species. The embryo and non-muscle tissues have transcripts of 1.35 and 2.00 kilobases, and the muscle tissues have transcripts of 2.15 and 1.45 kilobases. Southern analysis of chicken genomic DNA shows that there are probably two related genes, one more similar to the cDNA than the other.

Isolation of rat testis cDNAs encoding an insulin-like growth factor I precursor.

We have characterized rat testis cDNAs encoding insulin-like growth factor I (IGF-I) precursor to facilitate studies of IGF-I expression in the male reproductive system. Two clones, P2 and P3, with inserts of 786 and 1200 bp, respectively, were isolated from a lambda gt11 library of rat testis cDNAs. The longest open reading frame of cDNA P2 predicts a 153-amino-acid residue IGF-I precursor that has only 11 amino acid substitutions compared with a human IGF-IA precursor encoded by a human liver mRNA. Three substitutions are within the predicted rat IGF-I sequence: a Pro for Asp in the B domain, an Ile for Ser in the C domain, and Thr for Ala in the D domain. Only two substitutions distinguish the predicted rat sequence from a mouse liver IGF-IA precursor: Thr for Ala in the signal peptide and Ala for Ser in the D domain. P2 hybridizes with poly(A)+ mRNAs of 7.5, 4.7, 1.7, and 1.2-0.9 kb in rat liver and testis. The other testis cDNA, P3, appears to represent a partially processed rat IGF-I mRNA precursor. By comparing the sequence of cDNA P2 with that of cDNA P3 and a 2.3-kb rat IGF-I genomic fragment, we predict exon splice sites within the codon for residue 26 and between residues 86-87 of the rat IGF-I precursor. Both of the predicted splice sites align with exon-intron junctions in the human IGF-I gene. We conclude, therefore, that IGF-I is synthesized as a precursor in the rat testis and that the structure of IGF-I genes, mRNAs, and precursors are highly conserved across species.

Insulin-like growth factor II binding to the type I somatomedin receptor. Evidence for two high affinity binding sites.

We have previously shown that the antireceptor antibody alpha IR-3 inhibits binding of 125I-somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) to the 130-kDa alpha subunit of the type I receptor in human placental membranes, but does not block 125I-insulin-like growth factor II (IGF-II) binding to a similar 130-kDa complex in these membranes. To determine whether the 130-kDa 125I-IGF-II binding complex represents a homologous receptor or whether 125I-IGF-II binds to the type I receptor at a site that is not blocked by alpha IR-3, type I receptors were purified by affinity chromatography on Sepharose linked alpha IR-3. The purified receptors bound both 125I-Sm-C/IGF-I and 125I-IGF-II avidly (KD = 2.0 X 10(-10) M and 3.0 X 10(-10) M, respectively). The maximal inhibition of 125I-Sm-C/IGF-I binding by the antibody, however, was 62% while only 15% of 125I-IGF-II binding was inhibited by alpha IR-3. In the presence of 500 nM alpha IR-3, Sm-C/IGF-I bound with lower affinity (KD = 6.5 X 10(-10) M) than IGF-II (KD = 4.5 X 10(-10) M) and IGF-II was the more potent inhibitor of 125I-Sm-C/IGF-I binding. These findings suggest that the type I receptor contains two different binding sites. The site designated IA has highest affinity for Sm-C/IGF-I and is blocked by alpha IR-3. Site IB has higher affinity for IGF-II than for Sm-C/IGF-I and is not blocked by alpha IR-3.

Cultured fibroblast monolayers secrete a protein that alters the cellular binding of somatomedin-C/insulinlike growth factor I.

We studied somatomedin-C/insulinlike growth factor (Sm-C/IGF-I) binding to human fibroblasts in both adherent monolayers and in suspension cultures. The addition of Sm-C/IGF-I in concentrations between 0.5 and 10 ng/ml to monolayers cultures resulted in a paradoxical increase in 125I-Sm-C/IGF-I binding and concentrations between 25 and 300 ng/ml were required to displace the labeled peptide. The addition of unlabeled insulin resulted in no displacement of labeled Sm-C/IGF-I from the adherent cells. When fibroblast suspensions were used Sm-C/IGF-I concentrations between 1 and 10 ng/ml caused displacement, the paradoxical increase in 125I-Sm-C/IGF-I binding was not detected, and insulin displaced 60% of the labeled peptide. Affinity cross-linking to fibroblast monolayers revealed a 43,000-mol wt 125I-Sm-C-binding-protein complex that was not detected after cross-linking to suspended cells. The 43,000-mol wt complex was not detected after cross-linking to smooth muscle cell monolayers, and binding studies showed that 125I-Sm-C/IGF-I was displaced greater than 90% by Sm-C/IGF-I using concentrations between 0.5 and 10 ng/ml. Because fibroblast-conditioned medium contains the 43,000-mol wt complex, smooth muscle cells were incubated with conditioned medium for 24 h prior to initiation of the binding studies. 125I-Sm-C/IGF-I-binding increased 1.6-fold compared to control cultures and after cross-linking the 43,000-mol wt complex could be detected on the smooth muscle cell surface. Human fibroblast monolayers secrete a protein that binds 125I-Sm-C/IGF-I which can be transferred to the smooth muscle cell surface and alters 125I-Sm-C/IGF-I binding.

Evidence from monoclonal antibody studies that insulin stimulates deoxyribonucleic acid synthesis through the type I somatomedin receptor.

Somatomedin-C/insulin-like growth factor-I (Sm-C/IGF-I) and insulin stimulate DNA synthesis and cell replication in cultured human fibroblasts. It has been postulated that the growth-promoting actions of both peptides are mediated through the type I Sm-C/IGF-I receptor. This study tests this hypothesis using two recently developed monoclonal antibodies. The antibody designated sm 1.2 is directed to Sm-C, whereas the antibody designated alpha IR-3 is directed against the type I receptor for Sm-C/IGF-I. Radiolabeled monoclonal antibody alpha IR-3 was bound to human foreskin fibroblasts in a reversible time-dependent fashion, with 90% of the specific binding complete after 6 h of incubation at 15 C. Binding of [125I]alpha IR-3 was completely inhibited by excess unlabeled antibody, but not by 50 nM Sm-C or 1000 nM insulin. Specific binding of [125I]Sm-C fell to 27% of the control value in the presence of 50 nM alpha IR-3, and this concentration of antibody significantly reduced the mitogenic response to both Sm-C and insulin. Antibody sm 1.2 blocked the mitogenic response to exogenous Sm-C, but did not block the response to insulin; indeed, in some experiments, sm 1.2 enhanced the response to insulin. We postulate that this enhancement is the result of neutralizing endogenously produced Sm-like substances. This study provides further evidence that the growth-promoting effects of insulin in this cell type are the result of interaction with the Sm-C/IGF-I receptor.