Peter Rotwein

Synthesis and Secretion of Insulin-like Growth Factor II by a Leiomyosarcoma with Associated Hypoglycemia

We describe a case of recurrent hypoglycemia apparently caused by secretion of insulin-like growth factor II (IGF-II) by a leiomyosarcoma. A 67-year-old woman presented with recurrent severe hypoglycemia and a large mass in the thorax. During hypoglycemia, plasma cortisol was elevated, but insulin and growth hormone levels were low. After resection of a large leiomyosarcoma, the hypoglycemia resolved. After an eight-year remission, both the tumor and symptomatic hypoglycemia recurred. During a second operation a second large tumor was removed, with relief of the patients hypoglycemia. The tumor contained high concentrations of IGF-II mRNA and 2100 ng of IGF-II immunoreactive peptide per gram. Filtration through a BioGel P-60 gel column established that 77 percent of the IGF-II was present as a larger molecule, demonstrating incomplete processing of the pro-IGF-II peptides. A similar fraction of high-molecular-weight IGF-II was present in the serum, indicating that the tumor was the chief source of IGF-II. The high-molecular-weight IGF-II found in both the tumor and serum was fully reactive with the IGF-II receptor. Radioimmunoassay showed that the concentrations of insulin-like growth factor I (IGF-I) in tumor and serum were low, suggesting feedback inhibition of growth hormone secretion by IGF-II. Eight months after reoperation, plasma concentrations of IGF-I and IGF-II were normal, and high-molecular-weight IGF-II was virtually undetectable. We conclude that the most likely cause of this patients recurrent hypoglycemia was IGF-II produced by the leiomyosarcoma.

Structure, Evolution, Expression and Regulation of Insulin-Like Growth Factors I and II

Insulin-like growth factors (IGF) I and II are chemically-related single-chain peptides with diverse actions on cellular growth and metabolism. This review will focus on recent information pertinent to the biochemical and molecular biological aspects of these peptides. Three areas will be examined: The structure of the two IGF molecules and their precursors will be analyzed; the complicated anatomy of the IGF genes and their mRNAs will be described; and the multiple ways in which the expression of IGF-I and IGF-II can be regulated will be discussed. Gaps in our understanding of these peptides will be highlighted in the context of opportunities for further investigation in this field.

Overexpression of insulin-like growth factor-II induces accelerated myoblast differentiation

Previous studies have shown that exogenous insulin‐like growth factors (IGFs) can stimulate the terminal differentiation of skeletal myoblasts in culture and have established a correlation between the rate and the extent of IGF‐II secretion by muscle cell lines and the rate of biochemical and morphological differentiation. To investigate the hypothesis that autocrine secretion of IGF‐II plays a critical role in stimulating spontaneous myogenic differentiation in vitro, we have established C2 muscle cell lines that stably express a mouse IGF‐II cDNA under control of the strong, constitutively active Moloney sarcoma virus promoter, enabling us to study directly the effects of IGF‐II overproduction. Similar to observations with other muscle cell lines, IGF‐II overexpressing myoblasts proliferated normally in growth medium containing 20% fetal serum, but they underwent enhanced differentiation compared with controls when incubated in low‐serum differentiation medium. Accelerated differentiation of IGF‐II overexpressing C2 cells was preceded by the rapid induction of myogenin mRNA and protein expression (within 1 h, compared with 24–48 h in controls) and was accompanied by an enhanced proportion of the retinoblastoma protein in an underphosphrylated and potentially active form, by a marked increase in activity of the muscle‐specific enzyme, creatine phosphokinase, by extensive myotube formation by 48 h, and by elevated secretion of IGF binding protein‐5 when compared with controls. These results confirm a role for IGF‐II as an autocrine/paracrine differentiation factor for skeletal myoblasts, and they define a model cell system that will be useful in determining the biochemical mechanisms of IGF action in cellular differentiation.

Growth hormone rapidly activates rat serine protease inhibitor 2.1 gene transcription and induces a DNA-binding activity distinct from those of Stat1, -3, and -4.

Transcriptional regulation by growth hormone (GH) represents the culmination of signal transduction pathways that are initiated by the cell surface GH receptor and are targeted to the nucleus. Recent studies have demonstrated that the activated GH receptor can stimulate Stat1, a cytoplasmic transcription factor that becomes tyrosine phosphorylated and translocates to the nucleus, where it can interact with specific DNA sequences to modulate gene expression. GH also has been found to induce protein binding to a portion of the rat serine protease inhibitor (Spi) 2.1 gene promoter that is required for GH-induced transcription of Spi 2.1. Using GH-deficient hypophysectomized rats as a model, we show that GH treatment rapidly and potently induces both nuclear Spi 2.1 mRNA expression in the liver and specific nuclear protein binding to a 45-bp segment of the Spi 2.1 gene promoter. A GH-inducible gel-shifted complex appears within 15 min of systemic hormone administration and can be inhibited by an antiphosphotyrosine monoclonal antibody but is not blocked by a polyclonal antiserum to Stat1, Stat3, or Stat4, even though the nucleotide sequence contains two gamma interferon-activated sequence-like elements that could interact with STAT proteins. By Southwestern (DNA-protein) blot analysis, approximately 41- and 35-kDa GH-inducible proteins were detected in hepatic nuclear extracts with the Spi 2.1 DNA probe. Thus, a GH-activated signaling pathway stimulates Spi 2.1 gene expression through a unique mechanism that does not appear to involve known members of the STAT family of transcription factors.

Rapid Nuclear Actions of Growth Hormone

The mechanisms by which growth hormone (GH) initiates its effect on growth are largely unknown. In this report we examine the acute actions of GH with a focus on the intracellular signaling pathways leading from the cell-surface GH receptor into the nucleus, and culminating in the activation of specific target genes. We show that in vivo GH treatment leads to the rapid appearance of tyrosine-phosphorylated nuclear proteins and the equally rapid induction of c-fos and insulin-like growth factor I gene transcription. A model is proposed for a GH-activated intracellular signal transduction pathway.

Islet β-Cell Function and Polymorphism in the 5′-Flanking Region of the Human Insulin Gene

The present study investigates the possible relationship between human β-cell secretory capacity and polymorphism in the 5′-flanking region of the human insulin gene. The glucose potentiation slope was measured in normal and non-insulin-dependent diabetic subjects (NIDDM). This slope, as reported previously (Ward, W. K., et al., Am. J. Physiol. 1984; 246:E405–11), is an index of the ability of hyperglycemia to potentiate the insulin response to arginine and as such is a measure of β-cell responsiveness to glucose. Restriction enzyme analysis using a human insulin gene probe was performed on leukocyte DNA isolated from the same individuals. We conclude that a 1.6 kb polymorphism in the 5′-flanking region of the human insulin gene in both normal and NIDDM subjects has no association with insulin secretory responses as defined here by the glucose potentiation Slope.

Human renin gene is on chromosome 1

DNA sequences encoding kidney renin were localized to region p21 → qter of human chromosome 1 by Southern blot analysis of mouse-human somatic cell hybrids with a cloned human renin DNA probe. The renin gene may be a member of a chromosome 1 linkage group which is conserved in mouse and man. Available evidence suggests this gene is present in one copy per haploid genome. Thus those renin-like molecules detected immunologically in tissues other than the kidney (such as brain, placenta, uterus, pituitary, vasculature, and adrenal) may be derived from this single gene. Since renin messenger RNA in human kidney is about 1550 nucleotides long, reported molecular weights in excess of 45,000 for circulating renin represent posttranslational or postsecretory modifications of the polypeptide.

Insulin Gene Expression in the Developing Rat Pancreas

In the developing rat pancreas from about day 15 until birth there is a marked increase in the concentration of insulin. The present experiments were designed to determine whether enhanced insulin biosynthesis is accompanied by a coordinate increment in proinsulin mRNA. Using a sensitive RNA filter hybridization technique and a cloned rat proinsulin 32P-cDNA as a hybridizing probe, the concentration of proinsulin mRNA was measured. A greater than 900-fold accumulation of proinsulin mRNA was found, which closely paralleled that of immunoreactive insulin. Proinsulin mRNA and immunoreactive insulin were highest 1–2 days after birth, more than eightfold higher than that in adult pancreas. The patterns of digestion by a number of restriction endonucleases of the two nonallelic insulin genes were identical in adult and fetal DNA and in DNA from an insulinoma. We found no evidence of major gene rearrangement during embryogenesis as a prerequisite to insulin gene expression. Glucose stimulated insulin biosynthesis at the earliest time examined (day 15 of gestation). Insulin biosynthesis was enhanced almost fourfold while proinsulin mRNA differed by less than 20% during a 2-h incubation. This suggested that the short-term effect of glucose on insulin biosynthesis in the developing rat pancreas occurs at the level of translation of existing mRNA, similar to that noted in adult pancreatic islets. These data taken together suggest that the same controls for insulin biosynthesis in the adult exist at these early times in pancreatic development and that the controls for insulin biosynthesis appear coordinately with the appearance of increased insulin during endocrine pancreatic differentiation.

Contrasting acute in vivo nuclear actions of growth hormone and prolactin

Growth hormone (GH) and prolactin (PRL) exert long-term effects on cellular metabolism, growth, and development through changes in gene expression and protein biosynthesis that are initiated by hormone binding to specific cell-surface receptors. Recent studies have demonstrated that ligand-induced activation of both GH and PRL receptors leads to the tyrosine phosphorylation of multiple intracellular proteins by the identical non-receptor tyrosine kinase, JAK2. We have shown previously that in vivo administration of human recombinant GH rapidly stimulated the inducible transcription factors, Stats1, 3, and 5, and acutely altered gene transcription in the liver. Because human GH can bind to both lactogenic and somatogenic receptors with high affinity, in this study we have addressed the question of specificity of the hormonal response by examining the early nuclear events following a single injection of rat GH or rat PRL to hormone-deficient hypophysectomized female rats. We find that PRL stimulated tyrosine phosphorylation of Stat5, induced nuclear protein binding to the GH-responsive element of the serine protease inhibitor (Spi) 2.1 promoter, and activated Spi 2.1 gene expression. These acute actions of rat PRL were modest compared to the effects of rat GH. GH treatment induced tyrosine phosphorylation of several hepatic nuclear proteins, activated Stats1, 3, and 5, stimulated Spi 2.1 gene expression, and inhibited albumin gene transcription. All of the effects of rat GH paralleled responses to human GH that we have measured previously. Based on these results, it is likely that most of the actions of human GH in the liver are mediated by the GH receptor rather than by the PRL receptor. The diminished response to PRL may be secondary to the high density of short PRL receptor isoforms in the liver, which do not participate effectively in ligand-induced signal transmission.

Whole Body Nitrogen Kinetics and Their Relationship to Growth in Short Children Treated with Recombinant Human Growth Hormone

ABSTRACT: We studied the effects of growth hormone on retention of 15N-labeled amino acids in 34 short, prepubertal, growth hormone-sufficient children and three growth hormone-deficient subjects. All 34 non-growth hormone-deficient children had apparently normal circulating growth hormone molecules and no mutations were detected in the growth hormone or IGF-I genes of any subjects. Fibroblasts from 34 children responded normally when challenged with recombinant human IGF-I. During the last 72 h of a 4-d challenges with recombinant human growth hormone (16 μg/kg body wt), retention of a mixed 15N-amino acid dose varied between 5.7 and 50.5%. Whole body protein synthesis, breakdown, and net anabolism calculated from the 15N kinetics were all increased by the acute growth hormone challenge. However, no routine clinical feature or laboratory determination correlated with the nitrogen retention response. After subsequent treatment (75 μg/kg three times a week) with recombinant human growth hormone for 1 y, there was a significant increase in height velocity. but this increase was not related significantly to pretreatment variables other than inversely to pretreatment height velocity. There was a significant (p = 0.03) correlation between the change in height velocity Z score and the degree of nitrogen retention to acute challenge with growth hormone, but this correlation was too weak (r = 0.37) to be of practical value in predicting the treatment growth response in an individual child.