The Physiology of Calcitonin Gene-Related Peptide in the Islet Compared with That of Islet Amyloid Polypeptide (Amylin) D. BRETHERTON-WATT, M. A. GHATEI, H . JAMAL, S. G. GILBEY, P. M. JONES, AND S. R. BLOOM" Department o j Medicine Royal Postgraduate Medical School London W12 O N N , United Kingdom Calcitonin gene-related peptide (a-CGRP or CGRP-1) is a 37-amino acid peptide produced by alternative splicing of the primary transcript of the calcitonin gene.[ CGRP is widely distributed throughout the central nervous system and peripheral tissues, and has been shown to possess a number of biological actions, including potent effects on gastric acid secretion and food intake, pancreatic function, and on the cardiovascular system (for reviews, see Refs. 2 and 3). Subsequently, a second CGRP-encoding gene was discovered and predicted to encode a 37-amino acid ~ e p t i d e The . ~ production of this peptide, known as 0CGRP or CGRP-2, has been demonstrated to occur in uiuo.s P-CGRP possesses a very similar spectrum of biological actions to CK-CGRP.'.~ However, the peptides exhibit different tissue specificities of distribution, which probably reflects the preferential biological actions of that form,5 such that a-CGRP is the predominant form in central nervous tissue and sensory innervation, while P-CGRP forms the main component of the gut intrinsic innervation.' In 1986, a new peptide of the calcitonin/CGRP family was identified.' Islet amyloid polypeptide (IAPP or amylin) was isolated from the amyloid deposits of an insulinoma' and of a type I1 diabetic pancreas.8 IAPP has a strong familial resemblance to CGRP, with 43% and 46% amino acid sequence homology with human a- and p-CGRPs, respectively (for review, see Ref. 9). Furthermore, both peptides are 37 amino acids long, are C-terminally amidated, and have an N-terminal disulfide bridge linking cysteine residues in positions 2 and 7. The similarities between the peptides suggested that IAPP and CGRP are evolutionary homologues that arose as a result of a gene duplication event. The location of the IAPP encoding gene to chromosome 12, a n evolutionary homologue of chromosome 1 1 where a-and P-CGRPs are encoded,'(' agrees with this theory. The close structural similarity between IAPP and CGRP suggests they may bind to similar or even identical receptors. Indeed, IAPP has been shown to bind to and activate CGRP receptors in the 1iver.I' This structural homology has also caused problems with antisera and cast doubt upon the validity of earlier reports of CGRP in the pancreas, since it was shown that islet amyloid could react with anti-CGRP antisera."

a Address for correspondence: Professor S . R. Bloom, 2"d Floor Francis Fraser Labs., Department of Medicine, Royal Postgraduate Medical School, I50 DuCane Road, London W12 ONN, United Kingdom.

299

300

ANNALS NEW YORK ACADEMY OF SCIENCES

IAPP is deposited as amyloid only in certain animals. Comparison of the amino acid sequences of IAPP from different species have pinpointed the amino acids 20-29 as the amyloidogenic region of the m ~ l e c u l e . 'This ~ region has the least degree of homology to the CGRP m o l e ~ u l eIt. ~is thought that in certain pathological conditions-for example, non-insulin dependent diabetes (N1DDM)-IAPP molecules can polymerize to form the large insoluble P-pleated sheets characteristic of amyloid proteins.I4 In NIDDM, IAPP is depleted from the p-cell, accumulating as fibrils in the islet interstitium.Is Unlike CGRP, IAPP is not thought to be widespread throughout the body. The primary site of IAPP production is the p-cell of the pancreatic islet, where IAPP is co-localized with insulin to the p-cell secretory granule.16 However, there have been reports of IAPP in the human and rat gastrointestinal tracts.l7-I9 In addition, IAPP mRNA has been identified in rat stomach, lung, and dorsal root ganglia of the spinal cord."' IAPP has been shown to change in parallel with insulin in response to a range of physiological and pharmacological stimuli, both in the c i r ~ u l a t i o n ~ "and ~ in isolated pancreatic preparation^.'^-'^ IAPP may be of pathological significance in the development of NIDDM. Deposition of pancreatic amyloid is the most common pathological feature of NIDDM.I4 Furthermore, the disease is characterized by elevated glucose in the presence of normal insulin, suggesting that tissues are not able to respond to insulin and promote lowering of blood glucose-that is, they are insulin re~istant.~' IAPP and CGRP have both been shown to counteract insulin action in peripheral tissues.'8.2' In this paper, we will review some of the recent work relevant to the glucoregulatory actions of CGRP and IAPP, both in normal physiology and in the pathology of insulin resistance and NIDDM.

PRODUCTION OF CGRP AND IAPP IN THE PANCREAS IAPP is synthesized within the p-cell of the pancreatic islet and stored with insulin in the p-cell secretory granule.I6 Levels of IAPP immunoreactivity have been reported to be in the range of 1/3 to 1/100 insulin level^,?^.^^^^" while IAPP mRNA in rat pancreas has been reported as 1\10 insulin levels."' IAPP is released from isolated rat i ~ l e t s ,p~-' c e l l ~ . ~and " perfused whole rat p a n c r e a ~ , ? ~in- ?parallel ~ with insulin in response to glucose and arginine. CGRP is also found in the pancreas of several specie^,^'-^^ where it is thought to be present in the innervation around pancreatic blood vessels and islets. Within the islet, CGRP has been localized within cells around the islet periphery, colocalizing with the somatostatin-containing S cells in the rat.33-35In other species, however-for instance, the mouse-CGRP has been identified in the insulinsecreting p-celk.33 Capsaicin treatment in neonatal rats is known to deplete CGRP-like immunoreactivity (-LI)from spinal cord and peripheral sensory innervation, while the intrinsic innervation of the enteric nervous system is unaffected. Using two radioimmunoassays, one that was specific for a-CGRP and one that cross-reacted fully with both a- and p-CGRP, Mulderry et d.sestablished that the non-capsaicin sensitive population was due to P-CGRP innervation, while the capsaicin-sensitive innervation was a-CGRP. In the whole rat pancreas, a-and P-CGRP immunoreactivities are approximately equal. Following capsaicin treatment, there is a specific loss of a-CGRP immunoreactivity. Such a result is consistent with the existence of two separate populations of CGRP within the pancreas.

BRETHERTON-WATT et al.: CGRP IN THE ISLET TABLE 1.

301

Details of the CGRP and IAPP Radioimmunoassays

Antiserum

Specificity

Sensitivity

a-CGRP Assav

Total CGRP Assav

IAPP Assav

Synthetic rat a-CGRP (1-37) conjugated to BSA by glutaraldehyde (CG7) 100% cross-reactivity a-CGRP, 2%' crossreactivity p-CGRP

Synthetic rat p-CGRP (1-37) conjugated to BSA by carbodiimide (CG3) 100% cross-reactivity with both a- and pCGRP

1 fmoliassay tube

2 fmoliassay tube

Synthetic human IAPP (1-37) conjugated to BSA by bis-diazotisation 100% cross-reactivity with rat IAPP,