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ABSTRACT

Messenger RNA for rat islet amyloid polypeptide (IAPP) has been identified not only in the pancreas but also, in lesser amounts, in preparations from the stomach and dorsal root ganglia. In the stomach, insulin mRNA was not detectable, ruling out possible contamination by pancreatic tissue. Because IAPP and calcitonin gene-related peptide (CGRP) are related and CGRP is present in both stomach and dorsal root ganglia, it was possible that 'IAPP' signals were in fact due to cross-hybridization with CGRP mRNA. A second IAPP probe was constructed which does not cross-react. This probe also detected mRNA in both tissues, confirming the expression of IAPP in both tissues. The regional distribution of IAPP mRNA in the stomach did not parallel that of gastrin mRNA. IAPP mRNA was present in the antrum, centrum and pylorus and, like gastrin, the highest amounts were in the pylorus. However, the ratio between the pylorus and centrum was 3·6:1 for IAPP and 156:1 for gastrin. The effects of dietary manipulation were determined; a period of 48 h of starvation reduced pancreatic IAPP mRNA by approximately 60%, whereas in the stomach there was no significant reduction. If the action of IAPP was hormonal, pancreas and stomach would not be acting in concert. A paracrine role for gastric IAPP therefore seems more likely.

ABSTRACT

Gastric inhibitory peptide (GIP) is a 42 amino acid gastrointestinal peptide which inhibits gastric acid secretion and stimulates pancreatic insulin secretion in the presence of glucose. Here we report the sequence of the cDNA encoding the rat GIP precursor. PreproGIP was 144 amino acids in length and comprised the GIP peptide itself, N- and C-terminal flanking peptides of 22 and 59 amino acids respectively and a typical hydrophobic signal peptide. The sequence indicated that GIP is released from its precursor by cleavage at single arginine residues. The C-terminal flanking peptide may have an important function since it was well conserved and contained a region of 16 amino acids with only a single, conservative replacement. Rat GIP mRNA was found in the duodenum and jejunum. Levels of GIP mRNA in the duodenum were increased twofold after a period of 2 days of starvation. There was no detectable expression of the GIP gene in other parts of the gastrointestinal tract or in other endocrine tissues. However, in pancreatic mRNA preparations, a larger mRNA was detected after low stringency hybridization. This could represent a further member of this gene family.

ABSTRACT

Islet amyloid polypeptide (IAPP) in the pancreas of the spontaneously diabetic (BB) Wistar rat was examined by radioimmunoassay, and IAPP mRNA levels were determined by Northern blotting. IAPP-like immunoreactivity in the diabetic rat pancreas was found to be significantly depleted compared with control (non-diabetic) BB rats (85·9±5 pmol/g in control rats, n = 8, vs 8·97 ± 0·9 pmol/g in diabetic rats, n=5; mean ± s.e.m.). A similar change in insulin concentrations was found, although insulin was present in approximately 100-fold greater amounts than IAPP. Chromatography of the IAPP immunoreactivity revealed a single molecular form, corresponding to synthetic IAPP. Northern blot analysis of pancreatic RNA (n = 4) revealed that IAPP mRNA in the diabetic group was depleted to 22% of the signal intensity in the control group. Insulin mRNA was dramatically reduced to only 4% of the control group and, in contrast, somatostatin was relatively unaffected, with the diabetic group retaining 86% of signal compared with the controls.

This animal model of insulin-dependent diabetes results from severe autoimmune destruction of the β cell. The extremely low levels of both insulin and its messenger RNA are in agreement with this. These results demonstrate that this pathological state is also associated with a loss of IAPP from the pancreas. Insulin-dependent diabetes is associated with a range of metabolic disturbances. It is possible that the concomitant depletion of IAPP may be a contributory factor in exacerbating the condition.

ABSTRACT

We have used the polymerase chain reaction with mixed sequence primers to generate a probe for rat amylin and have used this to detect expression in various rat tissues. Amylin mRNA is found in greatest concentrations in the pancreas where a single mRNA species can be detected giving a hybridisation signal intensity approximately 10% that of insulin mRNA. When the beta cell population was depleted with streptozotocin, both amylin and insulin mRNAs were reduced to a similar extent. Consistent with its supposed role in the control of carbohydrate metabolism, amylin mRNA was also found in the stomach. Unlike the related peptide, CGRP, amylin mRNA is not present in the thyroid and is not widely distributed in the central nervous system. The only nervous tissue in which it could be detected was the dorsal root ganglion. Surprisingly, amylin mRNA was also found in the lung though only at very low levels.

ABSTRACT

An abundant, seven trans-membrane domain receptor related to the calcitonin receptor has been studied by a number of groups without identification of its ligand. A recent report claimed that the receptor was a type 1 CGRP receptor (Aiyar et al J. Biol. Chem. 271 11325-11329 (1996)). We have studied the equivalent rat sequence in transfected cells. When expressed in 293 cells the receptor interacts with CGRP and adrenomedullin with KD values of 1.2 nM for CGRP and 11 nM for adrenomedullin. Both ligands cause an elevation of intracellular cAMP with EC₅₀ values of 4 nM and 20 nM respectively and these effects are inhibited by the antagonist CGRP_8-37. The receptor is expressed at high levels in the pulmonary vascular endothelium. Both the pharmacological data and the localisation are consistent with the conclusion that the orphan receptor is a type 1 CGRP receptor. However, when expressed in COS-7 cells, no receptor activity could be demonstrated suggesting that 293 cells contain a factor necessary for functional receptor expression.

ABSTRACT

This study has quantified, for the first time, the relative levels of neuromedin U (NmU) mRNA in the rat gastrointestinal tract using Northern blot analysis. NmU message was detected in all regions of the gastrointestinal tract from the oesophagus to the rectum. The greatest levels were found in the duodenum and jejunum, the principal sites for absorption, which were 2·5- and 3-fold respectively above ileal levels.

Quantification of NmU mRNA and peptide contents in the duodenum, jejunum and ileum during postnatal development of the rat showed message and peptide levels to be greater in the maturing rat than in neonates. Message levels in the duodenum, jejunum and ileum showed 14-, 7- and 4-fold increases respectively between 1 and 56 days after birth, whilst the corresponding peptide levels in the duodenum, jejunum and ileum showed 33-, 14- and 25-fold increases respectively.

Food deprivation caused a small, but significant, decrease in message levels in the jejunum and colon, but there was no change in the duodenum or ileum. This shows that the presence of food has little effect on NmU mRNA levels in the gut.