Biomedical subjects

P Rotwein

Publications and source records attributed to P Rotwein.

At least 73 records · Page 4Linked to original sources

PubMed1991

Expression of insulin-like growth factor-I during chicken development.

We have investigated the expression of insulin-like growth factor-I (IGF-I) during ontogeny in the chick. IGF-I mRNA was first detectable in whole embryos on day 6, while serum IGF-I could be measured on day 9, the earliest time point examined. Serum IGF-I values rose 10-fold from about 3 ng/ml on day 10 to a prehatch peak of 30-35 ng/ml during days 15-17, and then declined to about 10 ng/ml at the time of hatching. On days 17 and 20 of incubation, IGF-I mRNA was detected in eye, skeletal muscle, and brain, but could not be found in liver or heart until after hatching. During the posthatch period, serum IGF-I rose from 10 ng/ml in the first week to 35-40 ng/ml during weeks 3-6, and liver IGF-I mRNA increased nearly 5-fold from weeks 1-7. The increases observed during weeks 1-3 correlated with a posthatch rise in serum GH from 21 to 37 ng/ml, although GH levels declined over the subsequent 4 weeks without an appreciable change in serum IGF-I values. By contrast, before embryonic day 12 no GH could be detected in the circulation, while IGF-I was 19 ng/ml on day 11. These observations suggest that there is both GH-dependent and GH-independent regulation of IGF-I gene expression in the chick, as has been found in mammals, and support the idea that IGF-I plays a role in chicken embryonic development.

Animals↗

PubMed1991

Structure and function of a human insulin-like growth factor-I gene promoter.

We have identified and characterized a promoter regulatory region for the human insulin-like growth factor-I (IGF-I) gene. The 5'-ends of IGF-I mRNAs were first mapped to a series of sites within a 158-nucleotide portion of exon 1 that was found to be structurally similar to the recently delineated transcription start region of the chicken IGF-I gene. In both species multiple initiation sites are probably a reflection of the absence of typical transcriptional regulatory signals, such as a TATA or CCAAT box, in the proximal promoter, although neither gene sequence resembles a GC-rich housekeeping promoter, which also controls a dispersed initiation region. To test promoter function, hybrid genes were constructed linking human IGF-I DNA to a promoterless reporter plasmid. Fusion genes containing from 385-4300 nucleotides of the IGF-I 5'-flanking region enhanced luciferase activity after transfection into the IGF-I-producing SK-N-MC cell line. A plasmid with 1630 nucleotides of 5'-DNA gave maximal activity and directed accurate initiation of the hybrid gene while the same promoter fragment inserted in the reverse orientation was inert. Although cognate recognition sequences were identified for several transcription factors in the 1630 nucleotides 5' to the transcription start region, no sites were found that resembled the putative GH response element recently mapped to the proximal promoter of the rat Spi2.1 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1991

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.

Amino Acid Sequence↗

PubMed1990

Evolution of insulin-like growth factor II: characterization of the mouse IGF-II gene and identification of two pseudo-exons.

We have cloned the mouse insulin-like growth factor II (IGF-II) gene as a series of overlapping cosmid and lambda recombinants and have characterized its six exons. The gene extends over approximately 12 kb of mouse chromosome 7 and is located 18 kb 3' to the insulin 2 gene and in the same transcriptional polarity. Exons 1-3 encode distinct 5' untranslated regions and are transcribed by three different promoters, P1, P2, and P3, into three IGF-II mRNAs sharing common coding and 3' untranslated sequences. Promoters P2 and P3 each contain a TATA box and appear to direct transcription from single initiation sites. By contrast, exon 1 has three major transcriptional start sites distributed over 556 nucleotides, and P1 lacks a TATA region and other typical transcriptional control sequences. Exons 4-6 code for the 180-amino-acid IGF-II precursor, and exon 6 also contains a 3,045-nucleotide 3' untranslated region which ends at a single polyadenylation site. In addition to six functional IGF-II exons, we identified two 5' "pseudo-exons," which appear to be evolutionarily retained remnants of an alternative promoter-exon cassette that is active in human IGF-II. Loss of the homolog of this promoter, which directs "adult-specific" expression of the IGF-II gene in some human tissues, may explain the disappearance of this growth factor from most murine tissues in the early postnatal period.

Amino Acid Sequence↗

PubMed1990

Activation of insulin-like growth factor gene expression during work-induced skeletal muscle growth.

We have investigated the hypothesis that there is local regulation of insulin-like growth factor (IGF) gene expression during skeletal muscle growth. Compensatory hypertrophy was induced in the soleus, a predominantly slow-twitch muscle, and plantaris, a fast-twitch muscle, in 11- to 12-wk-old female Wistar rats by unilateral cutting of the distal gastrocnemius tendon. Animals were killed 2, 4, or 8 days later, and muscles of the nonoperated leg served as controls. Muscle weight increased throughout the experimental period, reaching 127% (soleus) or 122% (plantaris) of control values by day 8. In both growing muscles, IGF-I mRNA, quantitated by a solution-hybridization nuclease-protection assay, rose by nearly threefold on day 2 and remained elevated throughout the experimental period. IGF-II mRNA levels also increased over controls. A more dramatic response was seen in hypophysectomized rats, where IGF-I mRNA levels rose by 8- to 13-fold, IGF-II values by 3- to 7-fold, and muscle mass increased on day 8 to 149% (soleus) or 133% (plantaris) of the control contralateral limb. These results indicate that signals propagated during muscle hypertrophy enhance the expression of both IGF genes, that modulation of IGF-I mRNA levels can occur in the absence of growth hormone, and that locally produced IGF-I and IGF-II may play a role in skeletal muscle growth.

Animals↗

PubMed1990

Renal expression of IGF I in hypersomatotropic states.

We examined insulin-like growth factor I (IGF I) gene expression in kidney in two models of hypersomatotropism, rats implanted with GH3 pituitary tumors, and rats administered exogenous growth hormone (GH). Both GH3 tumor-bearing rats and rats administered GH gained weight more rapidly than control animals, and had kidneys that were larger than those of controls. Tumor-bearing rats had increased levels of circulating IGF I. Glomeruli from tumor-implanted rats were sclerotic. Immunostainable IGF I was increased in medullary collecting ducts from tumor-bearing and GH-injected rats compared with kidneys from control animals. Levels of IGF I mRNA in kidneys of rats implanted with GH3 tumors and GH-injected rats were elevated compared with levels in kidneys from controls. Our findings demonstrate enhanced renal IGF I gene expression in hypersomatotropism. Stimulation of renal IGF I synthesis by GH could be causative of changes in renal function and renal size that occur in states of GH excess such as acromegaly.

Animals↗

PubMed1990

Divergent effects of insulin on insulin-like growth factor-II gene expression in the rat hypothalamus.

The effect of peripheral insulin treatment on brain content and synthesis of insulin-like growth factor-II (IGF-II) was studied in acute and chronic conditions. After a 3-h hyperinsulinemic/euglycemic clamp, rats were killed, and brains were removed for IGF-II analysis. Insulin infusion elevated IGF-II concentrations in the dorsomedial hypothalamus (DMH) and suprachiasmatic nucleus, but decreased IGF-II in the paraventricular nucleus, lateral hypothalamus (LtH), and supraoptic nucleus compared to values in control animals. In separate parallel studies, the effects of 4 days of insulin injection (1 U insulin/rat, sc, twice daily) on brain IGF-II mRNA and peptide were determined. For RNA analysis the hypothalamus was divided into three regions (lateral, ventral, and dorsal). IGF-II mRNA content was shown to vary by an order of magnitude within the adult rat hypothalamus under control conditions, and there appeared to be a differential region-dependent response to insulin treatment. Relative abundance of IGF-II mRNA in control hypothalami was VH much greater than cortex greater than or equal to LtH greater than DMH. IGF-II mRNA content declined after insulin treatment in the VH, but rose in all other regions. Insulin increased IGF-II peptide content in the paraventricular nucleus, LtH, and VMH but decreased IGF-II in the DMH and suprachiasmatic nucleus. These results indicate that peripheral insulin status may be an important factor in the synthesis and secretion of IGF-II in the brain.

Animals↗

PubMed1990

Low prevalence of insulin-like growth factor-I gene mutations in human growth disorders.

In an attempt to identify genetic lesions contributing to human growth disorders, we evaluated a prospectively recruited group of children with growth failure for mutations in the insulin-like growth factor-I (IGF-I) gene. Two complementary approaches were used: Southern blot analysis to examine the large scale organization of the gene, and a solution hybridization, nuclease protection assay to identify small alterations, such as point mutations. From a total of 61 subjects studied, 52 had no organic basis for their short stature. Analysis of chromosomal DNA from these individuals failed to reveal any variation in the IGF-I gene except for a HindIII site polymorphism which maps near the 3' end of the last IGF-I exon. No single nucleotide substitutions were found within IGF-I-coding regions. Since the frequency of the length polymorphism was the same for both normal-sized and short individuals, it is unlikely to be associated with growth abnormalities. Our results suggest that there is minimal DNA sequence variability in the human IGF-I gene and that mutations in IGF-I exons are infrequent causes of growth failure.

Blotting, Southern↗

PubMed1990

A novel human insulin-like growth factor I messenger RNA is expressed in normal and tumor cells.

We have identified and characterized a novel human insulin-like growth factor I (IGF-I) precursor from the transplantable T61 human breast cancer xenograft and from normal liver. The mRNA encoding this precursor contains a 5'-untranslated region that is 83% identical to the corresponding region of a previously described variant rat IGF-I. The nucleotide sequence of the cloned cDNA predicts an IGF-IA protein precursor of 137 amino acids, including a 32 residue signal peptide, 70 amino acid IGF-I, and a 35 residue COOH-terminal extension or E peptide. The exon encoding this variant maps in the genome between IGF-I exons 1 and 2, in a similar location to the homologous rat exon 1a. The rat and human exons 1a are 59% identical over 1443 nucleotides, with DNA sequence conservation occurring in a mosaic pattern. Human IGF-I mRNAs encoding this novel exon are expressed in liver, T61 tumor cells, and in an ovarian carcinoma cell line, NIH OVCAR3. These studies demonstrate that as in the rat, the human IGF-I gene contains six exons that are variably processed into multiple IGF-I mRNAs. The mechanisms responsible for generating different IGF-I mRNAs thus appear to be conserved among mammalian species.

Amino Acid Sequence↗

PubMed1990

Evolution of insulin-like growth factor I (IGF-I): structure and expression of an IGF-I precursor from Xenopus laevis.

By means of a cloning strategy employing the polymerase chain reaction, we have isolated and characterized cDNAs for Xenopus laevis insulin-like growth factor I (IGF-I). These cDNAs encode a primary IGF-I translation product of 153 residues that demonstrates considerable amino acid sequence similarity with IGF-IA peptides from other species. Fifty-seven of 70 residues of the mature protein are identical among human, rat, chicken, and Xenopus IGF-I, while less amino acid conservation is found at the COOH-terminus (25/35 identities) or at the NH2-terminus (24/48 identities) of the precursor protein. Despite the lower degree of structural similarity at the NH2-terminus, in vitro studies of IGF-I biosynthesis and proteolytic processing support a conserved function for the atypically long 48 residue NH2-terminal signal sequence in directing the nascent IGF-I peptide through the secretory pathway. The 5'-untranslated region of Xenopus IGF-I mRNA matches the human, rat, and chicken sequences in greater than 90% of 279 nucleotides. IGF-I mRNAs from all four species encode a conserved upstream open reading frame of 14 amino acids starting 240-250 nucleotides 5' to the translation start site, suggesting a possible role for this region in modulating IGF-I gene expression. The X. laevis IGF-I gene is transcribed and processed into three mRNAs of 1.6, 2.1, and 3.0 kilobases in liver, and IGF-I mRNAs can be detected in liver, lung, heart, kidney, and peritoneal fat of adult animals. These studies demonstrate that both the IGF-I protein precursor and potential regulatory regions of IGF-I mRNA have been conserved during vertebrate evolution, and indicate that like several other polypeptide growth factors, IGF-I may be of fundamental importance in regulating specific aspects of growth and development in all vertebrates.

Amino Acid Sequence↗

PubMed1989

Insulin-like growth factors (IGF) in muscle development. Expression of IGF-I, the IGF-I receptor, and an IGF binding protein during myoblast differentiation.

The insulin-like growth factors (IGFs) I and II exert pleiotropic effects on diverse cell types through interaction with specific high affinity cell surface receptors and with locally produced binding proteins. In skeletal muscle and in myoblast cell lines, the functions of IGF-I and -II are complex. Both growth factors appear capable of stimulating cellular proliferation and differentiation, as well as exerting insulin-like effects on intermediary metabolism. We have demonstrated recently that the expression of IGF-II and its receptor is induced during the terminal differentiation of the myoblast cell line, C2, and have suggested that IGF-II may be an autocrine growth factor in these cells (Tollefsen, S.E., Sadow, J.L., and Rotwein, P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 1543-1547). We now have examined this cell line for expression of other components involved in IGF signaling. The synthesis of IGF-I is low during myoblast proliferation; IGF-I mRNA can be detected only through use of a sensitive solution hybridization assay. Typical IGF-I receptors can be measured in myoblasts, whereas IGF binding proteins cannot be detected in proliferating cells or in conditioned culture medium. During myogenic differentiation, IGF-I mRNA levels increase transiently by 6-10-fold within 48-72 h. The expression of IGF-I mRNA is accompanied by a 2.5-fold accumulation of IGF-I in the culture medium. IGF-I receptors also increase transiently, doubling by 48 h after the onset of differentiation. By contrast, secretion of a Mr 29,000 IGF binding protein is induced 30-fold to 100 ng/ml within 16 h and continues to increase throughout differentiation. These studies demonstrate that several components critical to IGF action are produced in a fusing skeletal muscle cell line in a differentiation-dependent manner and suggest that both IGF-I and IGF-II may be autocrine factors for muscle.

Amino Acid Sequence↗

PubMed1989

Coordinate expression of insulin-like growth factor II and its receptor during muscle differentiation.

The role of polypeptide growth factors in promoting muscle differentiation is uncharacterized. We have used a fusing skeletal muscle cell line, C2, to examine the endogenous expression of one peptide, insulin-like growth factor II (IGF-II), and its receptor during differentiation. The synthesis of IGF-II is low during proliferation of myoblasts; IGF-II mRNA can be detected only through use of a highly sensitive solution-hybridization assay. Competition binding studies reveal that the IGF-II receptor is similarly nonabundant in myoblasts. During differentiation IGF-II mRNA rises rapidly. A nearly 4-fold increase is seen within 16 hr of onset of the differentiation process, and levels are 25 times higher than those in myoblasts by 96 hr, when myotubes have formed and muscle-specific alpha-actin mRNAs are synthesized. IGF-II accumulates in conditioned culture medium with similar kinetics. The expression of IGF-II receptors on the cell surface increases almost 6-fold 24 hr after the onset of differentiation and remains high. These studies suggest that IGF-II and its receptor are coordinately regulated during myogenic differentiation in C2 cells and that IGF-II may be an autocrine factor for skeletal muscle.

Amino Acid Sequence↗

PubMed1989

Complete thrombospondin mRNA sequence includes potential regulatory sites in the 3' untranslated region.

The nucleotide sequence of human thrombospondin (TS) mRNA has been determined from human fibroblast and endothelial cDNAs. The sequence of 5802 bp begins 110 bp upstream from the initiator codon and includes the entire 3' untranslated region (UTR) of the mRNA. The coding region (3510 bp) specifies a protein of 1170 amino acids with all of the known features of the TS subunit (Frazier, W. A. 1987. J. Cell Biol. 105:625-632). The long 3' UTR of 2166 nucleotides is extremely A/T-rich, particularly in the latter half. It contains 37 TATT or ATTT(A) sequences that have been suggested as mediators of the stability of mRNAs for cytokines, lymphokines, and oncogenes (Shaw, G., and R. Kamen. 1986. Cell. 46:659-667). Another unusual feature of the 3' UTR of TS mRNA is a stretch of 42 nucleotides of which 40 are thymidines (uridine in the mRNA) including an uninterrupted sequence of 26 thymidines. This region is flanked by two sets of direct repeats suggesting that it may be an insertion element of retrotranscriptional origin. Comparison of the 3' untranslated region of TS mRNA with the GenBank data base indicates the greatest degree of similarity with an alpha-interferon gene which contains a number of the TATT/ATTT consensus sites. The degree of similarity between the TS and interferon sequences is the same in regions of the interferon gene corresponding to its coding and noncoding regions suggesting that most of the TS 3' UTR may be derived from an interferon gene or pseudogene. The features of the TS mRNA 3' UTR provide a potential explanation for the rapid regulation of TS message observed in cultured cells in response to PDGF and suggest that TS is a member of a group of proteins which are intimately involved in the control of cell growth and differentiation.

Amino Acid Sequence↗

PubMed1989

Dual regulation of insulin-like growth factor I expression during renal hypertrophy.

We examined the regulation of insulin-like growth factor I (IGF-I) in kidney during the renal hypertrophy produced by two different experimental models: growth hormone treatment of hypophysectomized rats and compensatory hypertrophy subsequent to unilateral nephrectomy. Immunostaining for IGF-I in collecting ducts was enhanced in kidneys from growth hormone-repleted hypophysectomized rats, and the levels of IGF-I mRNAs were increased. In compensatory hypertrophy, no enhancement of the intensity of immunostaining was observed in kidneys of nephrectomized rats until 5 days postnephrectomy, at which time immunostainable IGF-I was increased markedly in medullary collecting ducts of hypertrophied kidneys compared with kidneys from sham-operated animals. No difference in steady-state levels of any IGF-I mRNA species was detected in whole kidneys or in collecting ducts from nephrectomized or sham-operated rats at any time postnephrectomy. Our findings demonstrate an increase in both IGF-I mRNA and in immunostainable IGF-I in collecting duct in the setting of growth hormone-induced renal hypertrophy but suggest that other, possibly translational, mechanisms underlie the induction of IGF-I synthesis during compensatory hypertrophy.

Animals↗

PubMed1989

Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations.

There is currently widespread interest in the IGFs (IGF-I and IGF-II) and their roles in the regulation of growth and differentiation of an ever increasing number of tissues are being reported. This selective review focused on the current state of our knowledge about the structure of mammalian IGFs and the multiple forms of mRNAs which arise from alternative splicing and promoter sites which arise from gene transcription. Current progress in the immunological measurement of the IGF is reviewed including different strategies for avoiding binding protein interference. The results of measurements of serum IGF-I and IGF-II in fetus and mother and at various stages of postnatal life are described. Existing knowledge of the concentration of these peptides in body fluids and tissues are considered. Last, an attempt is made to indicate circumstances in which the IGFs are exerting their actions in an autocrine/paracrine mode and when endocrine actions predominate. In the latter context it was concluded that an important role for GH action on skeletal tissues via hepatic production of IGF-I and endocrine action of IGF-I on growth cartilage is likely.

Amino Acid Sequence↗

PubMed1989

Structure and expression of a chicken insulin-like growth factor I precursor.

Insulin-like growth factor I (IGF-I) is a 70 amino acid growth-promoting polypeptide whose sequence and functions have been highly conserved among mammals. As an initial step in defining the role of IGF-I in other vertebrate species, we have isolated and characterized an IGF-I cDNA from the chicken. This cDNA encodes a 153 amino acid primary translation product which resembles in structure and sequence the IGF-IA protein of mammals. There is strong amino acid conservation between chicken and mammalian IGF-I throughout the entire protein. Sixty of 70 amino acids are identical in mature IGF-I among the chicken, rat, and human peptides, with five differences being localized to the C domain, and two to the D region. A comparable degree of amino acid identity is found in the COOH-terminal extension peptide (28/35 residues). At the NH2-terminus, where there is more amino acid divergence (32/48 identities), the most 5'-AUG codon is the only methionine residue conserved among all three species, suggesting that it functions as the authentic translation initiation site, an observation supported by cell-free studies of biosynthesis and cotranslational proteolytic processing. The pattern of IGF-I gene expression appears to be simpler in chickens than in mammals, since a single predominant mRNA of 2.6 kilobases can be detected in liver polyadenylated RNA on Northern blots. In the chicken, as in rats and humans, IGF-I mRNA is synthesized in multiple tissues, including liver, brain, skeletal muscle, and heart.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1988

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 patient's 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 patient's recurrent hypoglycemia was IGF-II produced by the leiomyosarcoma.

Aged↗

PubMed1988

Human tyrosine hydroxylase and insulin genes are contiguous on chromosome 11.

The gene for the catecholamine biosynthetic enzyme, tyrosine hydroxylase (TH), has been previously mapped to human chromosome 11 p15.5 in the vicinity of the loci for insulin (INS) and for the oncogene Harvey Ras 1 (HRAS). Here we show that gene probes derived from recombinant clones containing either human TH or INS cross-hybridize with each other. Direct DNA sequencing demonstrates that these genes are physically linked on chromosome 11. The TH gene is 5' to INS and is separated by only 2.7 kb of flanking DNA. Both genes have the same transcriptional polarity and form a head-to-tail linkage group with insulin-like growth factor 2 (IGF-2) in the order: 5' - TH - INS - IGF-2 - 3'. Because of the close physical proximity of these genes, previously described polymorphisms for INS are identical to those observed with TH. The localization of TH to the highly polymorphic INS locus provides four new restriction fragment length polymorphisms which should help determine rapidly whether defects in TH are responsible for bipolar affective disorder in the Old Order Amish and other populations.

Base Sequence↗