Identification of rat cell lines that preferentially express insulin-like growth factor binding proteins rlGFBP-1, 2, or 3.

The bioavailability and action of the insulin-like growth factors (IGFs) are determined by specific IGF-binding proteins (IGFBP) to which they are complexed. Complementary DNA clones have been isolated that encode three related IGFBPs: human IGFBP-1 (hIGFBP-1), human IGFBP-3 (hIGFBP-3), and rat IGFBP-2 (rIGFBP-2). IGFBP-1 and IGFBP-3 are regulated differently in human plasma, suggesting that they have different functions. In order to study the molecular basis of the regulation of the different IGFBPs, we have identified a panel of rat cell lines that express a single predominant binding protein and developed an assay strategy to distinguish the different binding proteins. Proteins in conditioned medium were examined by ligand blotting, and by immunoprecipitation and immunoblotting using antibodies to rIGFBP-2 and hIGFBP-1; RNAs were hybridized to cDNA probes for rIGFBP-2 and hIGFBP-1. 1) C6 glial cells and B104 neuroblastoma cells express an approximately 40 kilodalton (kDa) glycosylated binding protein that most likely represents rIGFBP-3, the binding subunit of the 150 kDa IGF: binding protein complex in adult rat serum. The C6 and B104 binding proteins do not react with antibodies to rIGFBP-2, and RNAs from C6 and B104 cells do not hybridize to cDNA probes for rIGFBP-2 or hIGFBP-1. 2) BRL-3A, Clone 9, and TRL 12-15 cell lines derived from normal rat liver express rIGFBP-2, a 30 kDa nonglycosylated IGF-binding protein that is recognized by antibodies to rIGFBP-2 but not by antibodies to hIGFBP-1. RNAs from these cells hybridize to a rIGFBP-2 cDNA probe, but not to a hIGFBP-1 probe. 3) H35 rat hepatoma cells express a 30 kDa nonglycosylated IGFBP that is presumptively identified as rIGFBP-1. It does not react with antibodies to rIGFBP-2, but is recognized by polyclonal and monoclonal antibodies to hIGFBP-1. RNA from H35 cells hybridizes to a hIGFBP-1 cDNA probe, but not to a rIGFBP-2 probe. Expression of rIGFBP-1 by the H35 cell line has enabled us to establish and validate specific assays for this protein that allow us to study its regulation in intact rats. Identification of a panel of rat cell lines expressing specific IGFBPs should be useful in elucidating the molecular mechanisms of IGFBP regulation.

The fetal rat binding protein for insulin-like growth factors is expressed in the choroid plexus and cerebrospinal fluid of adult rats.

The biological effects of the insulin-like growth factors, IGF-I and IGF-II, on their receptors are modulated by IGF-binding proteins. Recently, we isolated a cDNA clone for one member of the family of IGF-binding proteins, BP-3A, a 30 kilodalton (kDa) protein synthesized by the BRL-3A rat liver cell line. BP-3A is related to but distinct from two other cloned IGF-binding proteins, the human amniotic fluid binding protein and the glycosylated binding subunit of the 150 kDa IGF-binding protein complex in serum. It is expressed in multiple nonneural tissues and in serum in the fetal rat and decreases after birth, similar to the developmental pattern of IGF-II expression. IGF-I, IGF-II, and their receptors are expressed in brain. The present study examines the expression of BP-3A in the rat central nervous system. By Northern blot analysis, BP-3A mRNA is present at high levels in brain stem, cerebral cortex, and hypothalamus from 21-day gestation rats and, like IGF-II mRNA, persists in adult rat brain. The site of BP-3A mRNA synthesis was localized by in situ hybridization to coronal sections of adult rat brain using 35S-labeled oligonucleotides, 48 bases in length, complementary and anticomplementary to the coding region of BP-3A. Specific hybridization of the BP-3A probe was observed exclusively to the choroid plexus extending from the level of the medial preoptic nucleus to the arcuate nucleus of the hypothalamus, similar to the previously reported preferential localization of IGF-II mRNA to the choroid plexus. Synthesis of BP-3A mRNA by choroid plexus suggested that BP-3A might be secreted into the cerebrospinal fluid. A 30 kDa IGF-binding protein was demonstrated in rat cerebrospinal fluid that is recognized by antibodies to BP-3A and, like purified BP-3A, has equal affinity for IGF-I and IGF-II. By analogy with other transport proteins synthesized by the choroid plexus, BP-3A may facilitate the secretion of IGF-II to the cerebrospinal fluid and modulate its biological actions at distant sites within the brain.

Autonomous growth of a human neuroblastoma cell line is mediated by insulin-like growth factor II.

Insulin-like growth factor II (IGF-II) mRNA was increased in two of eight neuroblastomas and in eight of eight pheochromocytomas, tumors of the adrenal medulla that occur in childhood and adulthood, respectively. RNA encoding the type I IGF receptor, the receptor thought to mediate the mitogenic effects of IGF-I and IGF-II, also was uniformly expressed in these cells. To assess the role of IGF-II in the growth of these tumor cells, we have used the SK-N-AS cultured neuroblastoma cell line, which can be continuously propagated in mitogen-free medium, as a model system. Our results strongly suggest that IGF-II, synthesized by SK-N-AS cells and acting through type I IGF receptors, contributes to the autonomous growth of this tumor cell line. (a) SK-N-AS cells synthesized large amounts of IGF-II RNA and secreted greater than 50 ng/ml of IGF-II (as determined by specific radioimmuno- and radioreceptor assays). Little, if any, IGF-I RNA or immunoreactive IGF-I were detected. (b) SK-N-AS cells possess type I IGF receptors. (c) Exogenous IGF-I and IGF-II stimulated DNA synthesis in SK-N-AS cells, and this stimulation was abolished by a blocking antibody to the type I IGF receptor. (d) This anti-receptor antibody also abolished the multiplication of SK-N-AS cells in the absence of added mitogens. We conclude that IGF-II is an autocrine growth factor for SK-N-AS cells and suggest that this mechanism may contribute to the growth of some adrenal medullary tumors.

The 34 kilodalton insulin-like growth factor binding proteins in human cerebrospinal fluid and the A673 rhabdomyosarcoma cell line are human homologues of the rat BRL-3A binding protein.

Three members of a family of insulin-like growth factor binding proteins have been identified by nucleotide sequencing of cDNA clones: the binding subunit of the 150 kDa IGF-binding protein complex in human serum, the 30 kDa IGF binding protein in human amniotic fluid, and a 30 kDa binding protein (BP-3A) isolated from the rat BRL-3A cell line. The present study demonstrates by molecular hybridization and immunoreactivity that the human counterpart of rat BP-3A is a 34 kDa IGF binding protein that is present in human cerebrospinal fluid and is synthesized and secreted by the A673 human rhabdomyosarcoma cell line.

Synthesis of insulin-like growth factor II (IGF-II) in fetal rat tissues: translation of IGF-II ribonucleic acid and processing of pre-pro-IGF-II.

The single insulin-like growth factor II (IGF-II) gene is transcribed into multiple RNA species in most fetal and neonatal rat tissues. For IGF-II to serve as a local growth factor in fetal tissues, IGF-II RNA must be translated into pre-pro-rat (r) IGF-II, and the biosynthetic precursor processed to smaller biologically active forms. IGF-II RNA extracted from fetal rat liver, muscle, intestine, lung, and stomach, from rat placenta, and from fetal or neonatal mouse liver and lung directed the synthesis of 22,000 mol wt pre-pro-IGF-II in a reticulocyte lysate cell-free translation system. A biosynthetic precursor of this size had been observed previously in translation of RNA from BRL-3A rat liver cells and is predicted by the nucleotide sequence of cDNA clones encoding rIGF-II. Consistent with the developmental pattern of expression of IGF-II RNA observed in hybridization studies, RNA from adult rat liver, muscle, and intestine did not direct the synthesis of pre-pro-rIGF-II. To determine whether the IGF-II biosynthetic precursor was processed to smaller biologically active IGF-II, term fetal rat tissues were extracted with acid-ethanol, the extracts were fractionated by acid gel filtration, and the IGF pools were examined in a RIA specific for IGF-II. Levels of 1-2 micrograms/g were observed in liver, limb, lung, intestine, and brain; lower levels were observed in heart and kidney. In general, the levels of immunoreactive IGF-II corresponded to the levels of IGF-II mRNA. These results suggest that IGF-II mRNA is translated, and pre-pro-IGF-II processed to mature IGF-II in different fetal rat tissues. In contrast to IGF-I, in which alternative RNA splicing generates possible precursor molecules containing different COOH-terminal propeptide segments, we find no evidence for an IGF-II precursor in rat tissues other than 22,000 mol wt pre-pro-rIGF-II.

Biosynthesis of the low molecular weight carrier protein for insulin-like growth factors in rat liver and fibroblasts.

Biosynthesis of the low mol wt (Mr) carrier protein for insulin-like growth factors (IGFs) was studied in the BRL-3A rat liver cell line, rat embryo fibroblasts (REFs), and fetal rat liver by biosynthetic labeling of intact cells and cell-free translation of extracted RNA. [35S]Cysteine-labeled carrier protein precursors were immunoprecipitated using antibodies raised to the approximately 33,000 Mr carrier protein from BRL-3A cells that recognize the IGF carrier protein present in fetal and neonatal rat serum, but not in adult rat serum. The IGF carrier protein is synthesized as a 35,000 Mr precursor in a reticulocyte lysate translation system directed by RNA from BRL-3A cells or REFs. Supplementation of the translation incubation with microsomal membranes decreases the size of the precursor to 33,000 Mr, presumably by removal of a signal peptide. In continuous labeling or pulse-chase experiments of intact BRL-3A cells or REFs, the 33,000 Mr protein is labeled within 10 min intracellularly, appears in the medium after 40 min, and persists in the medium for 24 h without a change in size. The intracellular carrier protein was biosynthetically labeled in BRL-3A cells with [3H]leucine, [3H]phenylalanine, [3H]arginine, or [35S]cysteine and purified, and its NH2-terminal amino acid sequence was determined. Eleven of 34 residues were identified and correspond to those of mature unlabeled carrier protein purified from conditioned medium, indicating that after removal of the signal peptide, the carrier protein undergoes no detectable further processing at its NH2-terminus. These results establish that although they are regulated coordinately, IGF-II and the fetal IGF carrier protein are synthesized as separate proteins. Finally, RNA extracted from fetal, but not adult, rat liver directs the synthesis of the 35,000 Mr carrier protein precursor, suggesting that the developmental regulation of the carrier protein may occur at the level of RNA abundance.

Coordinate developmental regulation of high and low molecular weight mRNAs for rat insulin-like growth factor II.

Insulin-like growth factor II (IGF-II) is a mitogenic polypeptide that is thought to play a role in fetal growth and development. To study the hormonal and developmental regulation of IGF-II gene expression, we have isolated a cDNA clone for rat IGF-II (rIGF-II) from a 12S [1.2-kilobase-pair (kbp)] fraction of mRNA from a rat liver cell line (BRL-3A) that directs the cell-free synthesis of pre-pro-rIGF-II. In the present study, the rIGF-II probe was used to determine the size of IGF-II RNA. Surprisingly, in BRL-3A cells and in neonatal liver, the probe hybridized under stringent conditions 10-20 times more strongly to a larger (4 kbp) RNA than to 1.2-kbp RNA. The 4-kbp RNA is almost exclusively cytoplasmic and is colinear with a 551-base fragment of the rIGF-II cDNA insert containing coding and 3' noncoding regions. The 4-kbp and 1.2-kbp RNA species are regulated coordinately with developmental age, being high in liver from neonatal rats but not detectable in liver from older animals, suggesting that both IGF-II mRNA species arise from a single primary transcript by alternative RNA processing. Although oligodeoxynucleotide hybridization and S1 nuclease protection experiments suggest that the 4-kbp RNA contains an intact protein-coding region, fractions enriched in 4-kbp RNA do not direct the translation of pre-pro-rIGF-II in vitro. This may indicate that the 4-kbp RNA specifies an altered protein product that has not yet been recognized, or alternatively that it contains a normal protein-coding region but requires further RNA processing to be activated for translation.

Insulin-like growth factor carrier proteins in neonatal and adult rat serum are immunologically different: demonstration using a new radioimmunoassay for the carrier protein from BRL-3A rat liver cells.

A carrier protein for insulin-like growth factors (IGFs) has been purified from serum-free medium conditioned by the Buffalo rat liver (BRL)-3A cell line and used to immunize rabbits. Purified carrier protein was 125I labeled and affinity purified on IGF-Sepharose. The major labeled protein had a mol wt of about 33,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (appropriate for the IGF carrier protein subunit) and gave a single predominant peak of radioactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and acid-urea gel electrophoresis that was immunoprecipitated by immune serum and comigrated with unlabeled proteins that bind [125I] IGF. A RIA was developed using affinity purified [125I]carrier protein and immune serum. Tracer binding was inhibited only by preparations containing IGF carrier proteins, but not by unrelated proteins or by the IGFs themselves. Carrier proteins from BRL-3A cells gave equivalent strong reactivity either after dissociation of endogenous IGF or as an IGF-carrier protein complex. The antiserum effectively recognized the approximately 40,000 mol wt (Mr approximately 40,000) carrier protein from neonatal rat serum, both as a native complex and after acid stripping. It did not effectively recognize the Mr approximately 150,000 carrier protein from adult rat serum either as endogenous complex or after acid stripping. These results suggest that the Mr approximately 40,000 carrier protein of neonatal rat serum and the Mr approximately 40,000 binding subunit of the Mr approximately 150,000 carrier protein in adult rat serum are immunologically distinct. These antisera to the BRL-3A carrier protein should be useful tools with which to dissect the relationships between different carrier protein species and to study the regulation of IGF carrier protein gene expression.

Production of insulin-like growth factors by ovarian granulosa cells.

Evidence for ovarian secretion of somatomedins or insulin-like growth factors (IGF's) was generated by two approaches. First, porcine granulosa cells were shown to produce IGF's and an IGF-binding protein under serum-free conditions in vitro. The ovarian IGF's were recognized in two competitive binding assays specific for IGF's, a RIA using antibodies to human IGF-I and a radioreceptor assay using rat liver plasma membranes. IGF secretion was maintained for at least 10 days in culture. Second, ovarian production of IGF's in vivo was suggested by studies which showed that IGF levels in follicular fluid from preovulatory follicles were significantly greater than those in either serum or immature follicles. In contrast, similar low levels of insulin were observed in the follicles and serum. In conjunction with previous evidence of IGF action on granulosa cells, the present studies suggest the possibility of an autocrine role of IGF's in regulating follicular growth and development.

Neonatal rat islet cell cultures synthesize insulin-like growth factor I.

Monolayer cultures of islet B-cells were established from neonatal rat pancreas. Serum-free media conditioned by these cultures for 72 h were concentrated and fractionated on Sephadex G-50 at acid pH into a high-molecular-weight pool containing binding protein for insulin-like growth factors (IGFs) and a low-molecular-weight pool containing IGFs. IGF activity in the IGF pool was demonstrated by a specific radioreceptor assay using rat liver plasma membranes and 125I-labeled rat IGF-II. The IGF in islet cell media was characterized further by radioimmunoassays specific for human IGF-I and for rat IGF-II. Islet cell IGF was identified as predominantly IGF-I or a closely related species and not IGF-II. Levels of approximately 15-50 ng IGF-I (based on human IGF-I standard)/10(6) islet cells accumulated in media after 72 h, and presumably represented synthesis by the islet cells. Concentrations of IGF-I attained in culture media, approximately 0.1 ng/ml, were sufficient to stimulate [3H]thymidine incorporation into B-cells. Growth hormone did not consistently increase IGF-I synthesis, suggesting that the previously described effects of growth hormone on islet cell replication do not result from stimulation of IGF-I synthesis by islet cells. Thus, although the IGF-I synthesized by islet cells may be a physiologically relevant growth factor for these cells, the mitogenic effects of growth hormone in islet cells appear to be independent and not mediated by IGF-I.

Biosynthesis of rat insulin-like growth factor II. I. Immunochemical demonstration of a approximately 20-kilodalton biosynthetic precursor of rat insulin-like growth factor II in metabolically labeled BRL-3A rat liver cells.

BRL-3A rat liver cells synthesize mature 7484-dalton rat insulin-like growth factor II (rIGF-II) as a approximately 22-kDa precursor, presumably prepro-rIGF-II. In the present study, we have biosynthetically labeled intact BRL-3A cells with [35S]cysteine and immunoprecipitated cell lysates and media with antisera to rIGF-II. A approximately 20-kDa protein was identified in immunoprecipitates of cell lysates having properties consistent with pro-rIGF-II. The approximately 20-kDa protein is precipitated by immune sera but not by nonimmune serum. Its immunoprecipitation is specifically inhibited by unlabeled rIGF-II but not by insulin. It is not precipitated from labeled lysates of a subclone of BRL-3A cells (BRL-3A2) that does not synthesize rIGF-II. The approximately 20-kDa protein is rapidly labeled intracellularly (10 min) but is not detected in BRL-3A media. In pulse-chase experiments, radioactivity in the approximately 20-kDa protein disappears during the chase and appears, at later times, in specifically immunoprecipitated approximately 19-, approximately 10-, approximately 8-, and approximately 7-kDa proteins in media and, to a limited extent, intracellularly. A protein with electrophoretic mobility identical to that of the approximately 20-kDa protein observed in cell lysates is immunoprecipitated from 35S-proteins whose synthesis is directed by BRL-3A RNA in a reticulocyte lysate cell-free translation system supplemented with microsomal membranes, and presumably arises by cotranslational removal of the signal peptide from approximately 22-kDa prepro-rIGF-II. Processing of the approximately 20-kDa protein in intact BRL-3A cells to intermediate and mature rIGF-II species appears to occur at the time of secretion and/or shortly thereafter, with the different forms appearing at approximately the same time.

Interaction of insulin-like growth factors with a nonfusing mouse muscle cell line: binding, action, and receptor down-regulation.

Insulin and the insulin-like growth factors (IGFs) are chemically related polypeptides that interact with distinct receptors and elicit the same biological responses. We have sought a readily propagated cell line from a potential target tissue in which to probe the multiple and complex interrelationships among receptor and effector pathways for these polypeptides. We now report that the mouse muscle cell line BC3H-1 represents such a model system. BC3H-1 cells differentiate spontaneously at high density to form cells with muscle-specific properties, but do not fuse. Standaert et al. reported that differentiated BC3H-1 myocytes possess insulin receptors that mediate glucose and amino acid uptake and are down-regulated by prolonged incubation with insulin. The present report demonstrates that BC3H-1 myocytes also possess functional and regulated IGF receptors. Two subtypes of IGF receptors, types I and II, differing in structure and peptide specificity, were demonstrated by competitive binding and affinity cross-linking experiments. Low concentrations of IGFs stimulated glucose incorporation and alpha-aminoisobutyric acid uptake by BC3H-1 myocytes, suggesting that these effects were mediated primarily by IGF receptors rather than insulin receptors. Preincubation with IGFs (or high concentrations of insulin) selectively down-regulated type I IGF receptors without affecting type II IGF receptors. Since [125I]IGF-I binds to both type I and type II receptors in BC3H-1 cells, and since type I receptors have a higher affinity for IGF-I, the selective down-regulation of type I IGF receptors results in an apparent decrease in affinity for IGF-I. This difference in the regulation of type I and type II receptors in BC3H-1 myocytes is consistent with observations in other systems in which only one IGF receptor was present or examined. In their ability to be down-regulated by IGFs and insulin, type I IGF receptors are more similar to the structurally homologous insulin receptors than to the structurally dissimilar type II IGF receptors. These findings indicate that the BC3H-1 cell line provides an excellent model system in which to study the structure-function relationships of the receptor and effector pathways for insulin and the IGFs.

Insulin regulation of insulin-like growth factor action in rat hepatoma cells.

We have reported previously that insulin causes a complete but reversible desensitization to insulin action in rat hepatoma HTC cells in tissue culture, and that this insulin resistance is mediated by postbinding mechanisms rather than receptor down-regulation (Heaton, J. H., and Gelehrter, T. D. (1981) J. Biol. Chem. 256, 12257-12262). We report here that insulin causes a similar desensitization to the induction of tyrosine aminotransferase by the insulin-like growth factors IGF-I and IGF-II isolated from human plasma, and by multiplication-stimulating activity, the rat homologue of IGF-II. The results of both competition-binding studies and affinity cross-linking experiments indicate that insulin-like growth factors (IGFs) bind primarily to IGF receptors rather than to insulin receptors. The low concentrations at which these factors induce transaminase is consistent with their acting primarily via IGF receptors. This is confirmed by experiments utilizing anti-insulin receptor antibody which both inhibits 125I-insulin binding and shifts the concentration dependence of insulin induction of tyrosine aminotransferase to the right. This same immunoglobulin does not inhibit 125I-multiplication-stimulating activity binding and only minimally inhibits 125I-IGF-I binding. Anti-insulin receptor antibody also does not significantly shift the concentration dependence for the IGFs, suggesting that IGFs induce transaminase by acting via IGF receptors. Although insulin down regulates insulin receptors, it does not decrease IGF-I or IGF-II binding. We conclude that insulin causes desensitization of HTC cells to IGFs by affecting a postbinding step in IGF action, which may be common to the actions of both insulin and insulin-like growth factors.

Receptors for insulinlike growth factor I are defective in fibroblasts cultured from a patient with leprechaunism.

We previously have demonstrated that fibroblasts from a patient with leprechaunism exhibited markedly decreased insulin binding to insulin receptors and that the ability of insulin to stimulate glucose incorporation in the patient's cells was greatly impaired. In addition, the insulinlike growth factor, multiplication-stimulating activity (MSA), also exhibited an impaired ability to stimulate glucose incorporation in the patient's fibroblasts, although in normal fibroblasts this response appears to be mediated by an insulinlike growth factor receptor. The present study examines 125I-labeled insulinlike growth factor I (IGF-I) binding to patient's and control fibroblasts. 125I-labeled IGF-I binds to a specific IGF-I receptor in normal fibroblasts. At steady state, binding was inhibited by unlabeled IGF-I, IGF-II, MSA III-2, MSA II, insulin, and proinsulin, in order of potency, but not by high concentrations of epidermal growth factor and human growth hormone, chemically unrelated polypeptides 125I-labeled IGF-I binding to patient's cells was decreased by approximately 75%, whereas binding of epidermal growth factor to its cell surface receptors was unaffected. Computer curve-fitting of untransformed equilibrium binding data suggests that the decreased binding resulted from a decreased Ka for IGF-I. The ability of the patient's IGF-I receptor to recognize insulin also appears to be altered. Impaired IGF-I binding by the leprechaun patient's fibroblasts may contribute to the abnormal biological response to insulinlike growth factors observed in vitro and to the in utero growth retardation.

Stimulation of glucose incorporation and amino acid transport by insulin and an insulin-like growth factor in fibroblasts with defective insulin receptors cultured from a patient with leprechaunism.

Fibroblasts cultured from an infant with leprechaunism and insulin resistance have been reported to exhibit profound, selective defect in insulin binding. We now examine the effect of this defect on two acute metabolic actions of insulin thought to be mediated by the insulin receptor, glucose incorporation and N-methyl-alpha-aminoisobutyric acid (Me-AiBu) uptake. In the patient's fibroblasts, maximal insulin-stimulated glucose incorporation was less than 25% of that in control fibroblasts, whereas stimulation by hydrogen peroxide, an insulinomimetic agent that acts distal to the insulin receptor, was normal. By contrast, insulin stimulated Me-AiBu uptake to the same extent in patient's and control fibroblasts. Impaired glucose incorporation and relatively normal Me-AiBu uptake also were observed in the patient's cells with multiplication-stimulating activity, an insulin-like growth factor, despite the fact that multiplication-stimulating activity appeared to stimulate both responses in normal fibroblasts via an insulin-like growth factor receptor. The divergent effects on two hormone-stimulated functions in the patient's cells suggests differences in the coupling of a receptor to different effectors. The same coupling mechanisms appear to be used by insulin receptors and receptors for insulin-like growth factors.

Multiplication stimulating activity (MSA) from the BRL 3A rat liver cell line: relation to human somatomedins and insulin.

The properties of multiplication stimulating activity (MSA), an insulin-like growth factor (somatomedin) purified from culture medium conditioned by the BRL 3A rat liver cell line are summarized. The relationship of MSA to somatomedins purified from human and rat plasma are considered. MSA appears to be the predominant somatomedin in fetal rat serum, but a minor component of adult rat somatomedin. In vitro biological effects of MSA and insulin in adipocytes, fibroblasts and chondrocytes are examined to determine whether they are mediated by insulin receptors or insulin-like growth factor receptors. The possible relationship of a primary defect of insulin receptors observed in fibroblasts from a patient with the rare genetic disorder, leprechaunism, to intrauterine growth retardation is discussed.