Identification of a new calcitonin gene in the salmon Oncorhynchus gorbuscha.

Three isoforms of calcitonin (CT) exist in salmonids. Isohormones I and II are expressed in the pink salmon Oncorhynchus gorbuscha. We report here the existence in this species of a CT gene and of its transcripts, which encode for a fourth isohormone, the salmon CT (sCT) IV. This new CT gene was identified by PCR from genomic DNA and by sequencing the amplified DNA. The expression of this CT gene was established in ultimobranchial body and brain, by reverse transcription-PCR, hybridization and sequencing. The sCT IV gene, like the sCT I gene, is a complex transcription unit, containing exons encoding for a CT as a calcitonin gene-related peptide (CGRP) molecule. The predicted peptide, sCT IV, has a greater homology with the eel CT and the sCT II than with the sCT I. Alignment of the sCT IV with other fish and chicken CT showed amino acid modifications in similar positions as those found during evolution. The predicted salmon CGRP IV peptide is highly homologous to the known CGRP molecules in other species, confirming the high conservation of the molecule during evolution. This identification of a new salmon CT gene is interesting both for the therapeutic potential represented by the new molecules encoded by this gene and for phylogenetic studies.

Molecular physiology of the islet amyloid polypeptide (IAPP)/amylin gene in man, rat, and transgenic mice.

Islet amyloid polypeptide ("amylin") is the major protein component of amyloid deposits in pancreatic islets of type 2 (non-insulin-dependent) diabetic patients. Islet amyloid polypeptide consists of 37 amino acids, is co-produced and co-secreted with insulin from islet beta-cells, can act as a hormone in regulation of carbohydrate metabolism, and is implicated in the pathogenesis of islet amyloid formation and of type 2 diabetes mellitus. Rat islet amyloid polypeptide differs from human islet amyloid polypeptide particularly in the region of amino acids 25-28, which is important for amyloid fibril formation. In rat and mouse, diabetes-associated islet amyloid does not develop. To study the genetic organization and biosynthesis of islet amyloid polypeptide, we have isolated and analyzed the human and rat islet amyloid polypeptide gene and corresponding cDNAs. Both genes contain 3 exons, encoding precursor proteins of 89 amino acids and 93 amino acids, respectively. Apart from a putative signal sequence, these precursors contain amino- and carboxy-terminal flanking peptides in addition to the mature islet amyloid polypeptide. To understand regulation of islet amyloid polypeptide gene expression, we have identified several potential cis-acting transcriptional control elements that influence beta-cell-specific islet amyloid polypeptide gene expression. Using antisera raised against synthetic human islet amyloid polypeptide we developed a specific and sensitive radioimmunoassay to measure levels of islet amyloid polypeptide in plasma and tissue extracts. Also antisera raised against the flanking peptides will be used in studying human islet amyloid polypeptide biosynthesis. Elevated plasma islet amyloid polypeptide levels have been demonstrated in some diabetic, glucose-intolerant, and obese individuals, as well as in rodent models of diabetes and obesity. To examine the potential role of islet amyloid polypeptide overproduction in the pathogenesis of islet amyloid formation and type 2 diabetes, we generated transgenic mice that overproduce either the amyloidogenic human islet amyloid polypeptide or the nonamyloidogenic rat islet amyloid polypeptide in their islet beta-cells. Despite moderately to highly (up to 15-fold) elevated plasma islet amyloid polypeptide levels, no marked hyperglycemia, hyperinsulinemia or obesity was observed. This suggests that chronic overproduction of islet amyloid polypeptide "per se" does not cause insulin resistance. No islet amyloid deposits were detected in mice up to 63 weeks of age, but in every mouse producing human islet amyloid polypeptide (as in man), accumulation of islet amyloid polypeptide was observed in beta-cell lysosomal bodies. This may represent an initial phase in intracellular amyloid fibril formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of the human islet amyloid polypeptide/amylin gene transcripts: identification of a new polyadenylation site.

Islet amyloid polypeptide (IAPP) or Amylin is synthesized by the pancreatic beta-cells. IAPP is the major component of islet amyloid in the pancreas of patients with non-insulin-dependent diabetes mellitus. We report the composition and complete nucleotide sequence of the two human IAPP mRNAs of 1.6 and 2.1 kb. A new polyadenylation site was identified and shown to be used in generation of the 2.1 kb RNA. A previously identified polyadenylation signal is assigned to the 1.6 kb RNA. We exactly determined the major transcription start site, which is used in generation of these mRNAs. Lower abundance RNAs containing sequences located further upstream in the IAPP gene were also detected.

Identification and partial characterization of the salmon calcitonin/CGRP gene by polymerase chain reaction.

The polymerase chain reaction was used to amplify sequences encoding calcitonin and CGRP in the genomic DNA of salmon. Amplification products of the expected length were cloned and sequenced. In this way a new CGRP-coding sequence was identified. The new sequence and the known salmon calcitonin-coding sequence were shown to be part of one gene, implying that alternative gene expression takes place in fish.

Amylin-induced in vivo insulin resistance in conscious rats: the liver is more sensitive to amylin than peripheral tissues.

UNLABELLED: Amylin is a polypeptide of 37 amino acids, predominantly synthesized in pancreatic Beta cells. The peptide was suggested to be dysregulated in Type 2 (non-insulin-dependent) diabetes mellitus and it antagonized certain actions of insulin in vitro in rat muscle. This led to speculation that amylin is involved in the pathogenesis of Type 2 diabetes. We have examined the in vivo effects of rat amylin, amidated at the carboxy-terminus, on insulin-mediated carbohydrate metabolism in conscious rats, using the hyperinsulinaemic (+/- 1 nmol/l) euglycaemic (6 mmol/l) clamp technique combined with [3-3H]-glucose infusion. Basal plasma amylin levels were less than or equal to 75 pmol/l. Applied amylin levels of 220 +/- 75 pmol/l (infusion rate of 12.5 pmol/min) antagonized only the insulin action on liver, resulting in a 100% increase of hepatic glucose output. Amylin levels of 4750 +/- 750 pmol/l (infusion rate of 125 pmol/min) induced a 250% increase of insulin-inhibited hepatic glucose output and, in addition, a 30% decrease of insulin-stimulated peripheral glucose up-take. Amylin did not affect: 1) the metabolic clearance rate of insulin, 2) the levels of plasma glucagon, epinephrine, norepinephrine, and corticosterone, 3) in vitro insulin binding and insulin-stimulated receptor autophosphorylation. This suggests that amylin antagonizes insulin action via binding to a yet unknown receptor. IN CONCLUSION: amylin causes in vivo insulin resistance and the liver seems the predominant organ regulated by this hormone. The in vivo effects of amylin mimic the pathophysiological abnormalities of insulin action in Type 2 diabetes.

Islet amyloid polypeptide: structure and upstream sequences of the IAPP gene in rat and man.

Islet amyloid polypeptide (IAPP) or amylin is a pancreatic islet hormone which was first found in amyloid in insulinomas and in pancreases of patients with type 2 diabetes. In rat a similar polypeptide occurs; however, pancreatic amyloid in this species has not been described. Here we report the structure of the rat and human IAPP gene. Both consist of three exons and two introns which are very similar. The upstream sequence of the rat IAPP gene contains a TATA-box, a CCAAT-sequence and a GT-element, whereas the upstream sequence of the human IAPP gene contains a TATA-box and a rat insulin enhancer-like sequence. This suggests that the rat and human IAPP gene may be controlled differently at the transcriptional level.

IAPP/amylin gene transcriptional control region: evidence for negative regulation.

Aberrant expression of the islet amyloid polypeptide (IAPP) gene might be involved in the pathogenesis of non-insulin-dependent diabetes mellitus (NIDDM). Here, we report that IAPP promoter-luciferase constructs revealed tissue-specific activity. This activity was not mediated by cAMP. Sequential 5' deletions of the IAPP promoter caused a progressive derepression of the IAPP gene promoter in IAPP-producing cells. Comparison of the nucleotide sequence of the IAPP promoter with that of the insulin promoter (both active in pancreatic beta-cells) reveals two sequence elements of putative importance: an insulin enhancer-like sequence and an element which corresponds to a protected domain in rat insulin I gene promoter footprint experiments.

Detection of calcitonin-encoding mRNA by radioactive and non-radioactive in situ hybridization: improved colorimetric detection and cellular localization of mRNA in thyroid sections.

The localization of mRNA encoding calcitonin was studied by in situ hybridization using 35S-labeled RNA probes and biotin-labeled DNA probes. Radiolabeled probes were detected by autoradiography and biotin-labeled probes by streptavidin-biotin-peroxidase. To intensify the colorimetric signal, the indirect avidin-biotin complex (ABC) method was performed. However, the results were often variable. To improve the sensitivity, the peroxidase reaction signal was enhanced with a gold-silver deposit intensification reaction. To shorten the incubation times and to enhance the colorimetric reaction, several reaction steps were performed in a microwave oven. The localization of calcitonin mRNA in thyroid tissue, as detected with in situ hybridization, was confirmed by immunohistochemical localization of the calcitonin polypeptide. The results of in situ hybridization using biotinylated probes were compared to in situ hybridization using radioactive probes. Our data show that the results of in situ hybridization applied on frozen and paraffin-embedded sections using biotinylated DNA probes, detected with an indirect streptavidin-biotin-peroxidase reaction and intensified by silver-gold enhancement, were comparable to those obtained with radioactive probes. The localization of calcitonin encoding mRNA was in agreement with the localization of the calcitonin polypeptide.

In vitro splicing analysis of mini-gene constructs of the alternatively processed human calcitonin/CGRP-I pre-mRNA.

The human calcitonin/CGRP-I (CALC-I) gene can be alternatively expressed into calcitonin mRNA in thyroid C-cells and into CGRP-I mRNA in particular nerve cells. Formation of calcitonin mRNA requires splicing of exons 1, 2, 3 and 4 and addition of poly(A) at exon 4, whereas splicing of exons 1, 2, 3, 5 and 6 and addition of poly(A) at exon 6 yields CGRP-I mRNA. The calcitonin and CGRP-I mRNA-specific splicing reactions were investigated in vitro, in nuclear extracts of HeLa cells, using model precursor RNAs containing the exon 3 to exon 5 region of the gene. A precursor RNA containing the full-length exon 3 to exon 5 region was only poorly spliced in vitro. Therefore, a systematic analysis was performed of the effect of deletions introduced in the intron 3, exon 4 and intron 4 of this precursor RNA on calcitonin/CGRP mRNA-specific splicing. The deletions increased the efficiency of splicing considerably. In all cases CGRP mRNA-specific splicing is strongly favoured over calcitonin mRNA-specific splicing. In addition, splicing reactions using cryptic 5' splice sites were detected which interfered with the usage of processing signals for calcitonin and CGRP mRNA-specific splicing. The results imply a major regulatory role for the exon 4 poly(A) addition reaction in the generation of calcitonin mRNA.

The complete islet amyloid polypeptide precursor is encoded by two exons.

Islet amyloid polypeptide (IAPP) is the 37-amino acid peptide subunit of amyloid found in pancreatic islets of type 2 diabetic patients and in insulinomas. Recently, we isolated the human gene encoding IAPP [(1988) FEBS Lett. 239, 227-232]. We now report the nucleotide sequences of a human insulinoma cDNA encoding a complete IAPP precursor, and of the corresponding parts of the IAPP gene. Two exons, which are approx. 5 kb apart in the human genome, encode the 89-amino acid pre-pro-IAPP. At least one additional exon is present further upstream in the IAPP gene. A putative signal sequence at the amino-terminus of the precursor suggests that IAPP is a secreted protein.

Islet amyloid polypeptide: identification and chromosomal localization of the human gene.

Islet or insulinoma amyloid polypeptide (IAPP) is a 37 amino acid polypeptide isolated from pancreatic amyloid. Here, we describe the isolation and partial characterization of the human gene encoding IAPP. The DNA sequence predicts that IAPP is excised from a larger precursor protein and that its carboxy-terminus is probably amidated. The predicted normally occurring IAPP is identical to the reported polypeptides isolated from pancreatic amyloid, except for the amidated carboxy-terminus. IAPP specific polyadenylated RNAs of 1.6 kb and 2.1 kb are present in human insulinoma RNA. The human IAPP gene is located on chromosome 12.

Unusual branch point selection involved in splicing of the alternatively processed Calcitonin/CGRP-I pre-mRNA.

To study splice site selection in alternative RNA processing we used the human Calcitonin/CGRP-I (CALC-I) gene. Expression of the CALC-I gene in thyroid C-cells results predominantly in calcitonin (CT) mRNA (containing exons 1 to 4) whereas CGRP-I mRNA (containing exons 1,2,3,5 and 6) is the exclusive product in particular nerve cells. We previously reported that a model precursor RNA containing the exon 3 to exon 5 region is predominantly processed into CGRP-I mRNA in vitro using nuclear extracts of three different cell types. To study CT specific processing in Hela cell nuclear extracts we have used precursor RNAs corresponding to the exon 3 to exon 4 region containing only CT specific processing signals. The results revealed the usage of a uridine residue 23 nucleotides upstream of the 3' splice site as the major site of lariat formation in CT specific splicing. The implications of this finding for the alternative, tissue specific processing of the CALC-I pre-mRNA and for branch point selection in general are discussed.

Model for tissue specific Calcitonin/CGRP-I RNA processing from in vitro experiments.

The Calcitonin/CGRP-I (CALC-I) gene is known to be expressed in a tissue specific fashion resulting in the production of Calcitonin mRNA in thyroid C-cells and CGRP-I mRNA in particular nerve cells. The alternative RNA processing reactions include splicing of exons 1, 2 and 3 to exon 4 and poly (A) addition at exon 4 (Calcitonin mRNA) or splicing of exons 1, 2 and 3 to exons 5 and 6 and poly (A) addition at exon 6 (CGRP-I mRNA). Using a model precursor RNA containing the exon 3 to exon 5 region of the human CALC-I gene we have investigated the Calcitonin- and CGRP-I mRNA-specific processing reactions in vitro, in nuclear extracts of Hela, PC12 and Ewing-1B cells, respectively. Extracts of PC12- and Ewing-1B cells were expected to perform CGRP mRNA-specific splicing, whereas Calcitonin mRNA specific processing was expected to occur in Hela cell extracts. Surprisingly, CGRP mRNA-specific splicing of exon 3 to exon 5 was the predominant reaction in all three extracts. Significant Calcitonin mRNA-specific splicing of exon 3 to exon 4 only took place upon elimination of the dominant downstream 3' splice site used in CGRP mRNA-specific splicing. This elimination occurs most definitively by cleavage at the Calcitonin mRNA specific poly (A) site at exon 4 which may then be the major regulatory mechanism for tissue-specific expression of the CALC-I gene.

A third human CALC (pseudo)gene on chromosome 11.

A genomic locus in man (CALC-III) containing nucleotide sequences highly homologous to both exon 2 and exon 3 of the CALC-I and -II genes, is described in this paper. The CALC-I gene produces calcitonin (CT) (encoded by exon 4) or calcitonin gene-related peptide (CGRP) (encoded by exon 5) in a tissue-specific fashion. The CALC-II gene produces a second human CGRP, but probably not a second CT. The CALC-III gene does not seem to encode a CT- or CGRP-related polypeptide hormone and is probably a pseudogene. Like the other two CALC genes, the CALC-III gene is located on human chromosome 11.

Evolutionary pathways of the calcitonin genes.

Since the development of molecular biology, knowledge about polypeptide hormones has increased rapidly. Recombinant DNA techniques have made it possible to establish the structure of genes encoding polypeptide hormones. The results have provided insight into the mechanisms underlying the increasing diversity of polypeptide hormones. Comparison of nucleotide sequences of various genes has revealed surprising similarities and variations. The calcitonin (CT) genes offered an opportunity for speculation about the evolutionary origin on one hand and relationships between these genes on the other.

Expression of insulin-like growth factor-I and -II genes in human smooth muscle tumours.

The insulin-like growth factors I and II (IGF-I and -II) are polypeptides which play an important role in growth and development of the organism. In the present report we describe the detection of human IGF-I RNAs (both type Ia and type Ib) and IGF-II RNAs in benign (leiomyoma) and malignant (leiomyosarcoma) tumours from smooth muscle origin, using Northern blot hybridization analysis. In normal smooth muscle tissue of the uterus we found low levels of IGF-I RNAs only. In the tumours the same IGF-I RNA species were detected as in adult non-tumour tissues (uterus, liver). For transcription of the IGF-II gene in these tumours, two promoters are used which are expressed in fetal liver, but not in adult liver. The presence of IGF-I and IGF-II RNAs was also established by nucleotide sequence analysis of recombinant DNA clones isolated from cDNA libraries derived from two leiomyosarcomas. The nucleotide sequences of these cDNA clones, together covering the entire coding regions of IGF-Ia and IGF-II var RNA, predict that IGFs encoded by the tumour RNAs do not differ in amino acid sequence from the corresponding polypeptides isolated from serum. In those tissues containing IGF-I RNAs, IGF-I immunoreactivity was also demonstrated.