Islet amyloid polypeptide/amylin messenger RNA and protein expression in human insulinomas in relation to amyloid formation.

OBJECTIVE: Islet amyloid polypeptide (IAPP), also named amylin, is the predominant protein component of amyloid deposits in human islet beta cell tumours of the pancreas (insulinomas). IAPP is co-produced with insulin by islet beta cells. We investigated IAPP expression in relation to insulin expression and to amyloid formation in eleven insulinomas. DESIGN AND METHODS: RNA and protein extracts were prepared from the same pieces of tumour tissue, and from specimens of two normal human pancreata. IAPP and insulin mRNA and peptide content were quantified using Northern blot analysis and radioimmunoassay (RIA) respectively. Molecular forms of IAPP immunoreactivity were analysed by reversed-phase high-performance liquid chromatography (HPLC). The presence of islet hormones and of amyloid was assessed by (immuno)histochemical staining of paraffin sections. Plasma levels of IAPP and insulin prior to tumour resection were determined by RIA. RESULTS: IAPP and insulin mRNA and peptide content varied widely between the tumour specimens, and there was considerable intratumour heterogeneity of peptide content. HPLC analysis indicated correct proteolytic processing of the IAPP precursor protein. Amyloid deposits were detected only in the three tumours with the highest IAPP content. In contrast to insulin, plasma levels of IAPP were not elevated in the insulinoma patients. CONCLUSIONS: The spectrum of hormone production by insulinomas cannot be inferred from only a few tissue sections due to intratumour heterogeneity. Expression of the IAPP and insulin genes is not coupled in insulinomas, which produce properly processed mature IAPP. In addition to IAPP overproduction, additional factors such as intracellular accumulation of IAPP are involved in amyloidogenesis in insulinomas.

Pancreatic cancer in rats and hamsters does not induce IAPP-related hyperglycaemia.

Many patients with exocrine pancreatic cancer develop diabetes mellitus due to insulin resistance. This may relate to concurrent over-production of islet amyloid polypeptide (IAPP) by the pancreatic beta cells. We investigated the effects of pancreatic cancer on circulating IAPP and glucose homeostasis in azaserine-treated rats (developing acinar pancreatic tumours) and BOP-treated hamsters (developing ductular pancreatic tumours). Glucose, insulin and IAPP levels in plasma were neither affected in azaserine-only treated rats nor in animals with enhanced carcinogenesis after chronic caerulein treatment. Azaserine-treated rats on a high-fat diet had decreased insulin levels and enhanced IAPP/insulin ratios in plasma, without hyperglycaemia. All BOP-treated hamsters showed pancreatic carcinogenesis at 6 months post-treatment. Supranormal plasma glucose levels in animals on a low-fat diet were the only change observed. After a second 6-month period, subnormal plasma glucose levels, at least 4-fold decreased plasma insulin and up to 2-fold decreased plasma IAPP levels were present in all hamsters. Remarkably, both in azaserine-treated rats on high-fat and in BOP-treated hamsters, decreased insulin levels and elevated IAPP/insulin ratios are not associated with hyperglycaemia. In contrast to humans with pancreatic cancer, IAPP over-production and hyperglycaemia do not develop in rats and hamsters with (pre-)neoplastic pancreatic lesions.

Biologically active human islet amyloid polypeptide/amylin in transgenic mice.

OBJECTIVE: Human islet amyloid polypeptide (hIAPP), also named amylin, is a pancreatic beta cell protein implicated in the pathogenesis of pancreatic islet amyloid formation and type 2 diabetes mellitus. To study the (patho)physiological roles of hIAPP, we have generated transgenic mice that overexpress hIAPP mRNA, in relation to endogenous mouse IAPP (mIAPP) mRNA, in pancreatic beta cells. The biological activity of human and mouse IAPP derived from pancreatic extracts was determined. METHODS: Pancreatic and plasma extracts of transgenic and control mice were analyzed by reversed-phase high-performance liquid chromatography (HPLC) and radioimmunoassay, yielding a separation of hIAPP from mIAPP. Biological activity of immunoreactive human and mouse IAPP components derived from pancreatic extracts was assessed by calcitonin receptor-mediated stimulation of cyclic AMP accumulation in T47D human breast carcinoma cells. RESULTS: The predominant immunoreactive human and mouse IAPP gene products had the retention times on HPLC analysis of the corresponding synthetic peptides. The ratio of bioactive over immunoreactive hIAPP and mIAPP was 0.93 +/- 0.18 and 1.19 +/- 0.56 respectively. In extracts of two plasma pools from 4 transgenic animals, hIAPP was 4.6- to 7-fold more abundant than mIAPP. CONCLUSION: This study has shown that correctly processed hIAPP produced in transgenic mouse pancreatic beta cells exhibits full biological activity. The results validate these transgenic mice for the study of (patho)physiological roles of hIAPP in vivo.

Lack of islet amyloid polypeptide/amylin-immunoreactivity in urine collected from healthy volunteers after ingestion of a carbohydrate-rich meal.

Islet amyloid polypeptide (IAPP), or amylin, is synthesized by beta cells in the islets of Langerhans of the pancreas. Plasma IAPP levels are highly elevated in patients with advanced renal failure. To investigate the involvement of the kidney in the clearance of IAPP, the response of plasma and urinary IAPP to a carbohydrate-rich meal was investigated in 14 healthy volunteers. Although plasma IAPP levels increased severalfold after the meal, no IAPP-immunoreactivity was detected in the urine samples up to 4 hours after the meal. This might be due to the fact that urinary IAPP levels are under the detection limit of the assay or, assuming the presence of IAPP in the primary urine, immunoreactive IAPP molecules may be processed by renal mechanisms in such a way that they are no longer recognized by the antibodies used in the radioimmunoassay. Processed IAPP molecules may be reabsorbed in the proximal tubules of the kidney and/or excreted.

Pancreatic islet amyloid formation in patients with noninsulin-dependent diabetes mellitus. Implication for therapeutic strategy.

Islet amyloid polypeptide (IAPP or amylin) is the main component of pancreatic islet amyloid found in the vast majority of patients with noninsulin-dependent (Type-2) diabetes mellitus (NIDDM). IAPP may also act as a hormone that antagonizes the effects of insulin on peripheral tissues, but the results with IAPP overproducing transgenic mice and other recent findings indicate that IAPP overproduction is unlikely to induce peripheral insulin resistance in NIDDM. However, IAPP may contribute to the progression of NIDDM by impairing beta-cell function via islet amyloid formation. This may be initiated by locally elevated IAPP concentrations, induced by insulin-resistance-associated beta-cell hyperactivity. In order to improve therapeutic results, we propose strategies to inhibit IAPP production and islet amyloid formation during the pathogenesis of NIDDM.

Human islet amyloid polypeptide accumulates at similar sites in islets of transgenic mice and humans.

The cellular mechanisms responsible for conversion of islet amyloid polypeptide (IAPP) into insoluble amyloid deposits in non-insulin-dependent diabetes mellitus (NIDDM) are not clear. Overexpression of IAPP and the amino acid sequence of human IAPP (hIAPP) have both been implicated. To examine factors involved in amyloid formation, transgenic mice expressing the hIAPP or rat IAPP (rIAPP) gene were generated. These mice had elevated plasma IAPP concentrations, and they were normoglycemic and normoinsulinemic. No amyloid deposits were detected by light microscopy. To examine the ultrastructure of islets, pancreatic tissue was studied from hIAPP and rIAPP transgenic mice and from age-matched control mice by immunoelectron microscopy. IAPP was immunolocalized in beta-cell secretory granules of all mice, and the COOH- and NH2-terminal flanking peptides of hIAPP were localized in beta-cell granules of hIAPP mice. Accumulations of nonfibrillar perivascular IAPP-immunoreactive material were found between capillaries and beta-cells in hIAPP transgenic mice but not in rIAPP transgenic or control mice. Similar nonfibrillar masses were identified in islets of an NIDDM patient. Secondary lysosomes in beta-cells and macrophages of hIAPP transgenic mice showed dense labeling for IAPP. We suggest that hIAPP is degraded more slowly than rIAPP or mouse IAPP by beta-cell lysosomes. Accumulations of IAPP in islet perivascular spaces may represent the early stages of islet amyloid formation.

An improved method for the determination of islet amyloid polypeptide levels in plasma.

We describe an improved method for the determination of islet amyloid polypeptide (IAPP) levels in plasma. Plasma is first extracted with acid-acetone, followed by a specific and sensitive radioimmunoassay (RIA) for IAPP using rabbit-anti-human-IAPP serum. Recovery of synthetic IAPP from plasma was 82 +/- 6% (n = 16). Standard samples, prepared in 'hormone-free' serum, were also extracted with acid-acetone. Displacement curves of serially diluted acid-acetone extracted plasma samples were parallel to the standard curve. The lower detection limit of the RIA was 2.3 +/- 0.1 fmol/sample (n = 5). Intra-assay variations for IAPP concentrations of 4, 17 and 32 pM were 16.3% (n = 10), 9.2% (n = 10) and 6.2% (n = 10); interassay variations were 35.9% (n = 14), 19.9% (n = 15) and 15.4% (n = 15), respectively. Non-stimulated IAPP levels ranged from 2.4 to 12 pM (mean 6 +/- 4 pM, n = 10) in healthy control subjects. IAPP was not detectable in type 1 (insulin-dependent) diabetic patients before and after glucagon administration. In type 2 (non-insulin-dependent) diabetic patients basal levels ranged from 2.2 to 14.5 pM and glucagon-stimulated levels ranged from 2.2 to 38.9 pM. The increase in IAPP varied from 0 to 24.4 pM. The anti-human-IAPP serum had full cross-reactivity with rat IAPP (= mouse IAPP). Transgenic mice overexpressing the human IAPP gene showed elevated plasma IAPP levels as compared to (non-transgenic) control mice. It is concluded that the method presented for the determination of IAPP in plasma is reliable and easy to perform, yielding reproducible results.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Chronic overproduction of islet amyloid polypeptide/amylin in transgenic mice: lysosomal localization of human islet amyloid polypeptide and lack of marked hyperglycaemia or hyperinsulinaemia.

Type 2 (non-insulin-dependent) diabetes mellitus is characterised by hyperglycaemia, peripheral insulin resistance, impaired insulin secretion and pancreatic islet amyloid formation. The major constituent of islet amyloid is islet amyloid polypeptide (amylin). Islet amyloid polypeptide is synthesized by islet beta cells and co-secreted with insulin. The ability of islet amyloid polypeptide to form amyloid fibrils is related to its species-specific amino acid sequence. Islet amyloid associated with diabetes is only found in man, monkeys, cats and racoons. Pharmacological doses of islet amyloid polypeptide have been shown to inhibit insulin secretion as well as insulin action on peripheral tissues (insulin resistance). To examine the role of islet amyloid polypeptide in the pathogenesis of Type 2 diabetes, we have generated transgenic mice with the gene encoding either human islet amyloid polypeptide (which can form amyloid) or rat islet amyloid polypeptide, under control of an insulin promoter. Transgenic islet amyloid polypeptide mRNA was detected in the pancreas in all transgenic mice. Plasma islet amyloid polypeptide levels were significantly elevated (up to 15-fold) in three out of five transgenic lines, but elevated glucose levels, hyperinsulinaemia and obesity were not observed. This suggests that insulin resistance is not induced by chronic hypersecretion of islet amyloid polypeptide. Islet amyloid polypeptide immunoreactivity was localized to beta-cell secretory granules in all mice. Islet amyloid polypeptide immunoreactivity in beta-cell lysosomes was seen only in mice with the human islet amyloid polypeptide gene, as in human beta cells, and might represent an initial step in intracellular formation of amyloid fibrils.(ABSTRACT TRUNCATED AT 250 WORDS)

Uridine branch acceptor is a cis-acting element involved in regulation of the alternative processing of calcitonin/CGRP-l pre-mRNA.

The human calcitonin/CGRP-I (CALC-I) gene contains 6 exons and encodes two polypeptide precursors. In thyroid C-cells, calcitonin (CT) mRNA is produced by splicing of exons 1-2-3 to exon 4 (CT-encoding) and polyadenylation at exon 4. CGRP-I mRNA is produced in particular neural cells by splicing of exons 1-2-3 to exon 5 (CGRP-I-encoding) and the polyadenylated exon 6. We previously reported that model precursor RNAs containing the exon 3 to exon 5 region of the CALC-I gene are processed predominantly into CGRP-I mRNA in vitro, in nuclear extracts of several cell types (neural and non-neural). Using truncated precursor RNAs containing only the exon 3 to exon 4 region of the CALC-I gene it was shown that CT splicing is an inefficient reaction in which a uridine residue serves as the major site of lariat formation. Here we report that the low CT splicing efficiency and the dominance of CGRP-I splicing over CT splicing in vitro are primarily due to the usage of the CT-specific uridine branch acceptor. Mutation of this uridine residue into an adenosine residue resulted in a strong increase in CT splicing efficiency causing a reversal of the splicing pattern. In addition, it was shown that this point mutation also increased CT splicing efficiency in vivo. These results and data obtained from other experiments involving mutation of the CT splice acceptor site suggest that the uridine branch acceptor is a cis-acting element involved in regulation of the alternative processing of the CALC-I pre-mRNA.