Expression Of IAPP Research Articles

1730-P: In Vitro and In Vivo Effects of Increased IAPP Expression on Human Islet Function

1730-P: In Vitro and In Vivo Effects of Increased IAPP Expression on Human Islet Function

Abstract B049: Pancreatic beta cell stress pathways drive pancreatic ductal adenocarcinoma development in obesity

Abstract Epidemiologic studies have shown that obesity is a risk factor for more than a dozen cancers, including pancreatic ductal adenocarcinoma (PDAC). As PDAC is one of the leading causes of cancer-related death and obesity rates are rapidly increasing worldwide, it is imperative that we understand the mechanisms by which obesity promotes PDAC progression. To address this gap in knowledge, our lab combined a well-established genetic model for obesity (O: Lepob/ob) with a Kras-driven pancreatic cancer model (KC: Pdx1-Cre; KrasLSL-G12D/+) that mimics human PDAC progression. Resultant KCO mice had a drastic increase in tumor development compared to their lean counterparts, whereas induced weight loss intercepted tumor progression, showing that the tumor-promoting effects of obesity can be reversed. Bulk RNA sequencing of tumors from these mice revealed aberrantly increased expression of multiple peptide hormones expressed in insulin-producing beta (b) cells of pancreatic islets, including cholecystokinin (CCK) and amylin (Iapp). Strikingly, transgenic expression of CCK and Iapp in b-cells was sufficient to promote Kras-driven pancreatic tumorigenesis in non-obese mice. Single-cell RNA sequencing of b cells showed a trajectory of changes in gene expression with increasing obesity from wildtype to high-fat diet-fed to Lepob/ob mice. Further in silico analysis combining archetypal (AANet) with pseudotime (TrajectoryNet) methods demonstrated that b cells misexpressing hormones (CCK and Iapp) arose from a specific subtype of preexisting b cells, specifically virgin b cells, a rare, immature, regenerative population. Preliminary in vivo lineage tracing analyses in MIP-CreERT;LSL-TdTomato;Lepob/ob mice support these findings. Finally, we investigated the transcriptional networks that lead to aberrant CCK and Iapp expression in b cells. By combining Grainger causality analysis with known gene-gene relationships in the Transcriptional Regulatory Relationship Unraveled by Sentence-based Text mining (TRRUST) v2 database, we discovered a stress-induced mitogen-activated protein kinase (MAPK8)-cJun pathway that promotes pro-tumorigenic hormone expression in b cells. These combined experimental and computational methods nominate novel b cell targets to subvert the development of PDAC, an exocrine tumor. Citation Format: Cathy C. Garcia, Aarthi Venkat, Alex Tong, Sherry Agabiti, Lauren Lawres, Rebecca Cardone, Richard Kibbey, Smita Krishnaswamy, Mandar D Muzumdar. Pancreatic beta cell stress pathways drive pancreatic ductal adenocarcinoma development in obesity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B049.

Abstract C050: Elucidating mechanisms of endocrine-exocrine signaling in obesity-driven pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States. Various epidemiologic studies have shown that obesity increases the risk of PDAC. With rising rates of obesity worldwide, it is imperative that we understand the mechanisms by which obesity promotes PDAC progression. Therefore, to study the effect of obesity on PDAC, we combined a well-established genetic model of obesity (Lepob/ob) with a faithful oncogenic Kras-driven pancreatic cancer model (KC: Pdx1-Cre; KrasLSL-G12D/+) and showed that obese mice had both increased tumor burden and shortened survival compared to their lean counterparts. Strikingly, weight loss (via AAV-leptin rescue or caloric restriction), decreased tumor development, demonstrating that the tumor-promoting effects of obesity can be reversed. Bulk-RNA sequencing of tumors from obese mice exhibited aberrant expression of islet beta-cell genes, including those coding for the peptide hormones cholecystokinin (CCK) and amylin (Iapp). Single-cell RNA sequencing of islet beta-cells from wild-type, high fat diet-fed, and Lepob/ob mice revealed a trajectory of beta-cell adaptation to obesity with dysregulated hormone expression, including downregulation of insulin expression and upregulation of CCK and Iapp, concordant with loss of beta-cell identity transcription factors and beta-cell dysfunction. In silico analyses using dynamic optimal transport (TrajectoryNet) demonstrate that CCK-expressing beta-cells arise from pre-existing insulin low-expressing beta-cells, arguing for baseline beta-cell heterogeneity as a determinant of aberrant hormone expression. Lastly, transgenic expression of CCK or Iapp in beta-cells was sufficient to promote oncogenic Kras-driven pancreatic tumorigenesis, while converse beta-cell ablation impeded tumor progression in mice. Together, these results uncover a novel mechanism of obesity driven PDAC by local hormonal signaling between endocrine islets and exocrine acinar cells and reveal pancreatic endocrine adaptations that could be targeted to halt exocrine tumorigenesis. Citation Format: Cathy C. Garcia, Lauren Lawres, Sherry Agabiti, Jaffarguriqbal Singh, Alex Tong, Aarthi Venkat, Daniel B. Burkhardt, Rebecca Cardone, Richard G. Kibbey, Smita Krishnaswamy, Mandar D. Muzumdar. Elucidating mechanisms of endocrine-exocrine signaling in obesity-driven pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C050.

1277-P: Spatial Whole Transcriptome Profiling of the Human Pancreas Uncovers Unique Insights into Histological Structures

Understanding the physiology of the pancreas and pathology that occurs during diabetes requires knowing transcriptional patterns driving biological activity within the functional structures of the tissue. Previous technologies have not enabled spatially resolved high plex profiling to elucidate these biological pathways. Using the unique ability of GeoMx® Digital Spatial Profiler to resolve functional units within FFPE tissue in situ, we provide whole transcriptome expression data from islets, acini, and ducts from four pancreata of nondiabetes patients. On average, 6000-8000 genes were detected from functional groups within each histological structure. Data were validated against the independent Human Protein Atlas, with ∼90% concordance between RNA expression and known protein localization. Deconvolution based on single-cell RNASeq enabled calculation of the proportion of distinct cell types within each structure, confirming cell composition. For example, while ductal components consist of small islands punctuating the acini, over 3000 genes were identified to be highly specific to each structure (FDR < 0.05) , and enriched for pathways related to focal adhesion, ECM structure, and diabetic disease processes. Within islets, we identified >300 genes with differences between α- and β-cells (FDR < 0.2) . Functional characterization confirmed enrichment of insulin biosynthesis and secretion pathways, with genes such as insulin, glucagon, and IAPP expression captured accurately with a single assay. Determination of expression in four individual pancreas samples identified variation between tissues, which may be central to health and disease processes. In conclusion, whole transcriptome data for spatial gene expression profiles of structures of the non-diseased pancreas are publicly available and can be used as standards to inform future profiling studies in normal and diseased tissue. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Disclosure W.Yang: None. M.F.Hogan: Employee; NanoString. Y.Liang: None. J.M.Beechem: None.

Type 2 Diabetes is a Beta Cell Protein Misfolding Disease

Islets in humans with T2D, and the brain in neurodegenerative disease, share many changes including protein misfolding of amyloidogenic proteins, inflammation, activation of vascular endothelium and dedifferentiation. Islet amyloid characteristic of T2D is derived from IAPP, the most aggregate prone and toxic of known amyloidogenic proteins. To address the question, does IAPP misfolding-induced stress initiate the wide-ranging changes in islets in T2D, we compared the transcriptome of islets from human IAPP transgenic mice before diabetes onset with islets from humans with T2D. Beta cell IAPP toxicity reproduces the transcriptome in islets from humans with T2D with prominent activation of inflammation and vascular endothelium, and beta cell dedifferentiation. In common with T2D and neurodegenerative diseases, these changes were mediated through activation of key injury response transcription factors NF-kB, STAT3, CTNNB1 and ESR1. We conclude that suppression of IAPP expression is a potentially disease modifying therapeutic intervention in T2D.

Functional proteasome complex is required for turnover of islet amyloid polypeptide in pancreatic β-cells

Human islet amyloid polypeptide (hIAPP) is the principal constituent of amyloid deposits and toxic oligomers in the pancreatic islets. Together with hyperglycemia, hIAPP-derived oligomers and aggregates are important culprits in type 2 diabetes mellitus (T2DM). Here, we explored the role of the cell's main proteolytic complex, the proteasome, in hIAPP turnover in normal and stressed β-cells evoked by chronic hyperglycemia. Moderate inhibition (10-35%) of proteasome activity/function in cultured human islets by the proteasome inhibitor lactacystin enhanced intracellular accumulation of hIAPP. Unexpectedly, prolonged (>1 h) and marked (>50%) impairment of proteasome activity/function had a strong inhibitory effect on hIAPP transcription and secretion from normal and stressed β-cells. This negative compensatory feedback mechanism for controlling IAPP turnover was also observed in the lactacystin-treated rat insulinoma β-cell line (INS 832/13), demonstrating the presence of an evolutionarily conserved mechanism for IAPP production. In line with these in situ studies, our current ex vivo data showed that proteasome activity and hIAPP expression are also down-regulated in islets isolated from T2DM subjects. Gene expression and promoter activity studies demonstrated that the functional proteasome complex is required for efficient activation of the hIAPP promoter and for full expression of IAPP's essential transcription factor, FOXA2. ChIP studies revealed that promoter occupancy of FoxA2 at the rat IAPP promoter region is an important and limiting factor for amylin expression in proteasome-impaired murine cells. This study suggests a novel regulatory pathway in β-cells involving proteasome, FOXA2, and IAPP, which can be possibly targeted to regulate hIAPP levels and islet amyloidosis in T2DM.

A Study of Short- and Long-term mRNA Levels of the Retn, Iapp, and Drd5 Genes in Obese Mice Induced with High-fat Diet.

Adipocyte gene expression is altered in obese individuals through multiple metabolic and biochemical pathways. In this study, we aimed to examine the expression of resistin (Retn), amylin (Iapp), and dopamine receptor domain 5 (Drd5) genes previously suggested to contribute to the pathogenesis of obesity, albeit controversially. We also aimed to determine the effects on short and long-term mRNA levels of these genes in obese mice, induced with high-fat diet (HFD). Two obesity models were created in our study: group T1 (20 mice) was fed with HFD (60% fat) for 3 months, and group T2 (20 mice) was fed with HFD (60% fat) for 6 months. The control group T0 (20 mice) was fed with a diet of 10% kcal fat supplement for 6 months. At the end of the experiment, their adipose tissues were dissected surgically. Tissue samples of each group were pooled for RNA isolation, cDNA synthesis was carried out and the mRNA levels were examined by quantitative real-time polymerase chain reaction. Serum resistin levels were measured using multiplex bead (luminex) technology for validation. In T2 mice, the mRNA expression of Retn showed a moderate up-regulation (fold change=8.32; p=0.0019) in the adipose tissues. Iapp expression was also significantly up-regulated (fold change=9.78; p=0.012). Moreover, a 6.36-fold up-regulation for Drd5 was observed in the adipose tissues of T2 mice (p<0.001). At the same time, serum levels of resistin were found to be high in T1 and T2 mice compared to the control group (p<0.001 and p=0.024, respectively). Our study demonstrated that the mRNA levels of the genetic markers considered to play a role in adipogenesis were different in short- and long-term obesity models formed in C57BL/6J mice using HFD.

Upregulation of β-cell genes and improved function in rodent islets following chronic glucokinase activation

Glucokinase (GK) plays a critical role in controlling blood glucose; GK activators have been shown to stimulate insulin secretion acutely both in vitro and in vivo. Sustained stimulation of insulin secretion could potentially lead to β-cell exhaustion; this study examines the effect of chronic GK activation on β-cells. Gene expression and insulin secretion were measured in rodent islets treated in vitro with GKA71 for 72 h. Key β-cell gene expression was measured in rat, mouse and global GK heterozygous knockout mouse islets (gk(del/wt)). Insulin secretion, after chronic exposure to GKA71, was measured in perifused rat islets. GKA71 acutely increased insulin secretion in rat islets in a glucose-dependent manner. Chronic culture of mouse islets with GKA71 in 5 mmol/l glucose significantly increased the expression of insulin, IAPP, GLUT2, PDX1 and PC1 and decreased the expression of C/EBPβ compared with 5 mmol/l glucose alone. Similar increases were shown for insulin, GLUT2, IAPP and PC1 in chronically treated rat islets. Insulin mRNA was also increased in GKA71-treated gk(del/wt) islets. No changes in GK mRNA were observed. Glucose-stimulated insulin secretion was improved in perifused rat islets following chronic treatment with GKA71. This was associated with a greater insulin content and GK protein level. Chronic treatment of rodent islets with GKA71 showed an upregulation of key β-cell genes including insulin and an increase in insulin content and GK protein compared with glucose alone.

Exendin‐4 protects pancreatic beta cells from human islet amyloid polypeptide‐induced cell damage: potential involvement of AKT and mitochondria biogenesis

Glucagon-like peptide-1 (GLP-1) stimulates beta-cell proliferation and enhances beta-cell survival, whereas oligomerization of human islet amyloid polypeptide (hIAPP) may induce beta-cell apoptosis and reduce beta-cell mass. Type 2 diabetes is associated with increased expression of IAPP. As GLP-1-based therapy is currently developed as a novel antidiabetic therapy, we examined the potential protective action of the GLP-1 receptor agonist exendin-4 on hIAPP-induced beta-cell apoptosis. The study was performed in clonal insulinoma (INS-1E) cells. Both method of transcriptional and translational and sulphorhodamine B (SRB) assays were used to evaluate cell viability and cell mass. Western blot analysis was applied to detect protein expression. Transfection of constitutively active protein kinase B (PKB/AKT) was performed to examine the role of AKT. Mitochondrial biogenesis was quantified by mitogreen staining and RT-PCR. First, we confirmed that hIAPP induced cell apoptosis and growth inhibition in INS-1E cells. These effects were partially protected by exendin-4 in association with partial recovery of the hIAPP-mediated AKT inhibition. Furthermore, AKT constitutive activation attenuated hIAPP-induced apoptosis, whereas PI3K/AKT inhibition abrogated exendin-4-mediated effects. These findings suggest that the antiapoptotic and proliferative effects of exendin-4 in hIAPP-treated INS-1E cells were partially mediated through AKT pathway. Moreover, hIAPP induced FOXO1 but inhibited pdx-1 nucleus translocation. These effects were restored by exendin-4. Finally, mitogreen staining and RT-PCR revealed enhanced mitochondrial biogenesis by exendin-4 treatment. Collectively, these results suggest that GLP-1 receptor agonist protects beta cells from hIAPP-induced cell death partially through the activation of AKT pathway and improved mitochondrial function.

Selective effect of INGAP-PP upon mouse embryonic stem cell differentiation toward islet cells

We evaluated the effect of islet neogenesis-associated protein pentadecapeptide (INGAP-PP) upon islet β- and non-β cell differentiation from mouse embryonic stem (mES) cells. ES-D3 cell lines were cultured following Lumelsky's protocol with or without INGAP-PP (5 µg/ml) at different stages. Gene expression was quantified using qPCR. mES cells were fixed and immunostained using anti insulin-, somatostatin-, glucagon-, Pdx-1-, Ngn-3-, Nkx-6.1 and PGP9.5 specific antibodies. PCNA was used to measure replication rate. Bcl 2 (immunostaining) and caspase-3 (enzyme activity and gene expression) were determined as apoptosis markers. INGAP-PP increased IAPP, Glut-2, Kir-6.2, SUR-1 and insulin gene expression, and the percentage of insulin-immunostained cells. Conversely, INGAP-PP reduced significantly glucagon and somatostatin gene expression and immunopositivity. While nestin gene expression was not affected, there was a significant reduction in the percentage of PGP9.5-immunostained cells. Pdx-1 gene expression increased by 115% in INGAP-PP treated cells, as well as the percentage of Pdx-1, Ngn-3 and Nkx-6.1 immunopositive cells. Neither caspase-3 (expression and activity) nor Bcl 2 positively immunostained cells were affected by INGAP-PP. Accordingly, INGAP-PP would promote stem cell differentiation into a β-like cell phenotype, simultaneously decreasing its differentiation toward non-β-cell precursors. Therefore, INGAP-PP would be potentially useful to obtain β-cells from stem cells for replacement therapy.

Small Interfering RNA–Mediated Suppression of Proislet Amyloid Polypeptide Expression Inhibits Islet Amyloid Formation and Enhances Survival of Human Islets in Culture

OBJECTIVE—Islet amyloid, formed by aggregation of the β-cell peptide islet amyloid polypeptide (IAPP; amylin), is a pathological characteristic of pancreatic islets in type 2 diabetes. Toxic IAPP aggregates likely contribute to the progressive loss of β-cells in this disease. We used cultured human islets as an ex vivo model of amyloid formation to investigate whether suppression of proIAPP expression would inhibit islet amyloid formation and enhance β-cell survival and function.RESEARCH DESIGN AND METHODS—Islets from cadaveric organ donors were transduced with a recombinant adenovirus expressing a short interfering RNA (siRNA) designed to suppress human proIAPP (Ad-hProIAPP-siRNA), cultured for 10 days, and then assessed for the presence of islet amyloid, β-cell apoptosis, and β-cell function.RESULTS—Thioflavine S–positive amyloid deposits were clearly present after 10 days of culture. Transduction with Ad-hProIAPP-siRNA reduced proIAPP expression by 75% compared with nontransduced islets as assessed by Western blot analysis of islet lysates 4 days after transduction. siRNA-mediated inhibition of IAPP expression decreased islet amyloid area by 63% compared with nontransduced cultured islets. Cell death assessed by transferase-mediated dUTP nick-end labeling staining was decreased by 50% in transduced cultured human islets, associated with a significant increase in islet insulin content (control, 100 ± 4 vs. +Ad-siRNA, 153 ± 22%, P < 0.01) and glucose-stimulated insulin secretion (control, 222 ± 33 vs. +Ad-siRNA, 285 ± 21 percent basal, P < 0.05).CONCLUSIONS—These findings demonstrate that inhibition of IAPP synthesis prevents amyloid formation and β-cell death in cultured human islets. Inhibitors of IAPP synthesis may have therapeutic value in type 2 diabetes.

In vitro derivation of functional insulin-producing cells from human embryonic stem cells

The capacity for self-renewal and differentiation of human embryonic stem (ES) cells makes them a potential source for generation of pancreatic beta cells for treating type I diabetes mellitus. Here, we report a newly developed and effective method, carried out in a serum-free system, which induced human ES cells to differentiate into insulin-producing cells. Activin A was used in the initial stage to induce definitive endoderm differentiation from human ES cells, as detected by the expression of the definitive endoderm markers Sox17 and Brachyury. Further, all-trans retinoic acid (RA) was used to promote pancreatic differentiation, as indicated by the expression of the early pancreatic transcription factors pdx1 and hlxb9. After maturation in DMEM/F12 serum-free medium with bFGF and nicotinamide, the differentiated cells expressed islet specific markers such as C-peptide, insulin, glucagon and glut2. The percentage of C-peptide-positive cells exceeded 15%. The secretion of insulin and C-peptide by these cells corresponded to the variations in glucose levels. When transplanted into renal capsules of Streptozotocin (STZ)-treated nude mice, these differentiated human ES cells survived and maintained the expression of beta cell marker genes, including C-peptide, pdx1, glucokinase, nkx6.1, IAPP, pax6 and Tcf1. Thirty percent of the transplanted nude mice exhibited apparent restoration of stable euglycemia; and the corrected phenotype was sustained for more than six weeks. Our new method provides a promising in vitro differentiation model for studying the mechanisms of human pancreas development and illustrates the potential of using human ES cells for the treatment of type I diabetes mellitus.

Beta-Amyloid peptide induces formation of actin stress fibers through p38 mitogen-activated protein kinase.

Based on the critical role of actin in the maintenance of synaptic function, we examined whether expression of familial beta-amyloid precursor protein APP-V642I (IAPP) or mutant presenilin-1 L286V (mPS1) affects actin polymerization in rat septal neuronal cells. Expression of either IAPP or mPS1 but not wild-type amyloid precursor protein or presenilin-1induced formation of actin stress fibers in SN1 cells, a septal neuronal cell line. Treatment with beta-amyloid (Abeta) peptide also caused formation of actin stress fibers in SN1 cells and primary cultured hippocampal neurons. Treatment with a gamma-secretase inhibitor completely blocked formation of actin stress fibers, indicating that overproduction of Abeta peptide induces actin stress fibers. Because activation of the p38 mitogen-activated protein kinase (p38MAPK)-mitogen-associated protein kinase-associated protein kinase (MAPKAPK)-2-heat-shock protein 27 signaling pathway mediates actin polymerization, we explored whether Abeta peptide activates p38MAPK and MAPKAPK-2. Expression of IAPP or mPS1 induced activation of p38MAPK and MAPKAPK-2. Treatment with a p38MAPK inhibitor completely inhibited formation of actin stress fibers mediated by Abeta peptide, IAPP or mPS1. Moreover, treatment with a gamma-secretase inhibitor completely blocked activation of p38MAPK and MAPKAPK-2. In summary, our data suggest that overproduction of Abeta peptide induces formation of actin stress fibers through activation of the p38MAPK signaling pathway in septal neuronal cells.

High plasma amylin/islet amyloid polypeptide levels in patients with residual medullary thyroid carcinoma after total thyroidectomy.

We have previously reported that amylin/islet amyloid polypeptide (IAPP) mRNA is detected in a substantial subset of medullary carcinomas of the thyroid (MTCs). The aim of this study was to determine if the amylin/IAPP gene is expressed as the IAPP peptide in MTC tissues. In 10 patients with a histological diagnosis of MTC and with persisting or recurrent disease (basal calcitonin levels >250 pg/ml), the fasting serum insulin and plasma glucose, IAPP and calcitonin levels were measured and compared with those of 18 normal control subjects matched for age and body mass index. IAPP expression was studied by immunohistochemistry in MTCs and lymph-node metastasis tissues. Seven of ten MTC patients had abnormally elevated IAPP levels. Plasma IAPP and serum insulin levels were correlated in both patients and controls, but the slope of the regression line was significantly higher for MTC patients. IAPP staining was detected in four out of 12 random MTC samples and in two out of five lymph-node metastases, using immunohistochemistry. These results indicate that MTC cells express IAPP at the peptide level and that this raises the peripheral plasma levels. Further studies may reveal whether this is a feature of malignant disease.

The Transplanted Fetal Endocrine Pancreas Undergoes an Inherent Sequential Differentiation Similar to That in the Native Pancreas

This study examines, at the ultrastructural level, whether the fetal porcine endocrine pancreas (insulin, glucagon, somatostatin, and pancreatic polypeptide [PP]- and islet amyloid polypeptide [IAPP]-containing cells) develops normally after transplantation under the kidney capsule in athymic mice. We have thus used an in vivo pig-to-mouse model for the differentiation of the endocrine pancreas removed from its normal milieu. Islet-like cell clusters (ICCs) were prepared from the fetal porcine pancreas as previously described and transplanted under the renal capsule of athymic mice. At various times after transplantation, the endocrine pancreas was removed and the level of differentiation was compared with the native pancreas of the same biological age. At the ultrastructural level, several sequential steps could be identified based on the morphology and hormone content of the secretory granules of the endocrine cell examined. Applying this approach, we could demonstrate that the ontogeny of the transplanted fetal pig pancreas follows the same sequential differentiation as the native pancreas. The process seems to be under stringent control, apparently directly related to the biological age of the tissue, and independent not only of the new environment under the kidney capsule but also of the adult and xenogeneic milieu provided after transplantation to the athymic nude mouse. Therefore, all four major hormone-producing cells seem to develop normally after transplantation when compared with the development in the native pancreas. IAPP was produced by the pluripotent fetal endocrine cells as well as the adult alpha-, beta-, and delta-cell granules in the native pancreas; however, in the transplanted pancreas, IAPP expression was demonstrated only in beta-cells, delta-cells, and PP cells. No IAPP was found in granules of the alpha-cell lineage. The results suggest a sequential differentiation of all four major types of islet cells from a common pluripotent progenitor cell, which seems to be located in the pancreatic ducts. Therefore, the results presented strongly suggest that the ontogeny of the four major endocrine islet cells is determined by genetic information carried by the progenitor cells and not by the systemic or local environment.

Expression of Islet Amyloid Polypeptide. Localization and regulation in the pancreatic islets, gastrointestinal tract and sensory nervous system

Islet amyloid polypeptide (IAPP; also named ”amylin”) is a member of the calcitonin superfamily of peptides, displaying the closest structural relationship to the neuropeptide calcitonin gene-related peptide (CGRP). It is expressed in islet beta-cells and forms islet amyloid in type 2 diabetic patients. IAPP may restrain release and peripheral actions of insulin. Thus, IAPP has been implicated in the pathogenesis of type 2 diabetes. By examinations of tissue-specific expression of IAPP and its regulation under normal and experimental conditions, we aimed to illuminate a possible role of IAPP. IAPP was expressed in islet beta-cells in nearly all species examined; in some species IAPP occurred also in delta-cells. IAPP expression was of early onset during embryonic islet development. In vivo, we found a parallel regulation of IAPP and insulin expression by administration of glucose. These findings agree with a role for IAPP in glucose homeostasis by autocrine/paracrine regulation of islet function as well as by endocrine secretion. Following dexamethasone treatment of rats, a model for type 2 diabetes, we found a non-parallel expression of IAPP and insulin; in relative terms, IAPP was over-expressed compared with insulin. In rodents given streptozotocin or alloxan, conferring beta-cell depletion reminiscent of type 1 diabetes, IAPP expression was consistently less impaired than that of insulin. These studies show that IAPP and insulin gene expression are differentially regulated. Apparently, beta-cell stress, regardless of ethiology, results in over-expression of IAPP. IAPP was also found to be expressed in the gastrointestinal tract in a region- and species-dependent manner; when present, IAPP expression was most abundant proximally, occurring in somatostatin and gastrin cells. Also gastrointestinal IAPP expression was of early embryonic onset. In vitro, IAPP relaxed pre-contracted gut musculature. In the sensory nervous system IAPP was expressed in CGRP neurons and displayed a distribution typical of sensory neuropeptides. Upon sciatic axotomy, IAPP and CGRP expression were co-ordinately downregulated. In contrast, following adjuvant-induced rat paw inflammation, IAPP expression was upregulated in innervating neurons, indicating that IAPP may play a role in neurogenic inflammation. In conclusion, our studies have expanded the view of IAPP as an exclusive islet hormone to become a ”classical” neurohormonal peptide, with several endocrine and neuronal expression sites. The studies on the regulation of islet IAPP and insulin expression in response to glucose and in experimental diabetes may form the basis for an understanding of the role of IAPP in diabetes. Provided that the amyloidogenic mechanisms and metabolic effects of IAPP are elucidated, an over-expression of IAPP may prove to be relevant for diabetes development.

Reduced nociceptive behavior in islet amyloid polypeptide (amylin) knockout mice

Islet amyloid polypeptide (IAPP or amylin) is predominantly expressed by insulin cells, but occurs also in primary sensory neurons in the rat. Here, using mice targeted for a null mutation in the IAPP gene, we establish murine expression of IAPP in sensory neurons; its distribution in a population of calcitonin gene-related peptide-containing neurons in the spinal cord and dorsal root ganglion is similar to that previously described in the rat. We also report the IAPP mutant mice display a reduced pain response in the paw formalin test. Adjuvant-induced joint inflammation was not altered in IAPP mutants, arguing against a peripheral inflammatory abnormality. These findings lead us to suggest that IAPP has a pro-nociceptive function in primary sensory neurons.

Islet amyloid formation associated with hyperglycemia in transgenic mice with pancreatic beta cell expression of human islet amyloid polypeptide.

Pancreatic islet amyloid deposits are a characteristic pathologic feature of non-insulin-dependent diabetes mellitus and contain islet amyloid polypeptide (IAPP; amylin). We used transgenic mice that express human IAPP in pancreatic beta cells to explore the potential role of islet amyloid in the pathogenesis of non-insulin-dependent diabetes mellitus. Extensive amyloid deposits were observed in the pancreatic islets of approximately 80% of male transgenic mice > 13 months of age. Islet amyloid deposits were rarely observed in female transgenic mice (11%) and were never seen in nontransgenic animals. Ultrastructural analysis revealed that these deposits were composed of human IAPP-immunoreactive fibrils that accumulated between beta cells and islet capillaries. Strikingly, approximately half of the mice with islet amyloid deposits were hyperglycemic (plasma glucose > 11 mM). In younger (6- to 9-month-old) male transgenic mice, islet amyloid deposits were less commonly observed but were always associated with severe hyperglycemia (plasma glucose > 22 mM). These data indicate that expression of human IAPP in beta cells predisposes male mice to the development of islet amyloid and hyperglycemia. The frequent concordance of islet amyloid with hyperglycemia in these mice suggests an interdependence of these two conditions and supports the hypothesis that islet amyloid may play a role in the development of hyperglycemia.