Islet Amyloid Develops Diffusely Throughout the Pancreas Before Becoming Severe and Replacing Endocrine Cells

Feng Wang 0 Rebecca L. Hull 0 Josep Vidal 0 Miriam Cnop 0 Steven E. Kahn 0 0 From the Division of Metabolism, Endocrinology and Nutrition, Department of Medicine, Veterans Affairs Puget Sound Health Care System and University of Washington, Seattle, Washington. Ch.B., Veterans Affairs Puget Sound Health Care System (151) , 1660 S. Columbian Way, Seattle, WA 98108 Islet amyloid occurs in >90% of type 2 diabetic patients and may play a role in the pathogenesis of this disease. To determine whether islet amyloid occurs diffusely throughout the pancreas, whether it affects islets equally, and whether it decreases islet endocrine cells, we characterized islet amyloidosis by computerized fluorescence microscopy in transgenic mice that develop typical islet amyloid. These mice produce the unique amyloidogenic component of human islet amyloid, human islet amyloid polypeptide (hIAPP). The prevalence of amyloid (number of islets containing amyloid/total number of islets 100) and the severity of amyloid (amyloid area/islet area 100) were found to be uniform throughout the pancreas. Furthermore, a high prevalence of amyloid was observed in islets when the severity of amyloid was only 1.5% of the islet area, suggesting a diffuse distribution of amyloid from the very early stages of islet amyloidosis. In 12 hIAPP transgenic mice with an amyloid severity of 9.6 3.4%, the proportion of islets composed of - and -cells was reduced in the transgenic mice compared with 6 nontransgenic mice that do not develop amyloid (-cells: 62.9 3.1% vs. 75.5 0.9%, P 0.02; -cells: 2.8 0.5% vs. 4.4 0.4%, P 0.05), whereas the proportion of islets composed of -cells did not significantly differ between the two groups of mice. In the individual islets in these transgenic mice, amyloid severity was inversely correlated with -cell, (r 0.59, P < 0.0001), -cell (r 0.32, P < 0.0001), and -cell (r 0.25, P < 0.0001) areas. In conclusion, islet amyloidosis occurs uniformly throughout the pancreas, affecting all islets before becoming severe. A reduction in islet endocrine mass starts at this early stage of islet amyloid development and progresses as amyloid mass increases. Diabetes 50:2514 -2520, 2001 - Imic cats and nonhuman primates (1 6). The amyloid slet amyloid deposits occur in 90% of type 2 diabetic patients and are also present in hyperglycedeposits are formed of unbranching fibrils in which islet amyloid polypeptide (IAPP, or amylin) is the unique protein component (7,8). IAPP is normally produced in islet -cells and coreleased with insulin (9). Islet amyloid has been proposed to play a role in the insufficient insulin output observed in type 2 diabetes, because amyloid deposits may decrease islet -cell mass and thereby reduce the capacity for insulin release. This hypothesis is consistent with several lines of evidence. In the monkey, glucose tolerance deteriorates as the amount of islet amyloid increases (4). Additionally, islet amyloidosis appears to be associated with a reduction in islet -cell mass, especially when marked islet amyloidosis is present (5,6). In vitro, IAPP-derived amyloid fibrils have been demonstrated to be cytotoxic, causing death of insulin-secreting -cells (10,11). Despite these observations, a number of questions remain to be answered to more fully elucidate the potential role of islet amyloid in the development of type 2 diabetes. For instance, it is important to know how islet amyloid is distributed in the pancreas, because it is unclear whether islet amyloid and the cellular composition observed in one pancreatic region represent the composition in the whole endocrine pancreas. At this point in time, it is also uncertain how early in the process of islet amyloidosis -cell mass starts to decrease. Finally, it would be of interest to know whether non-cell mass is also changed in amyloid-containing islets. Because rodent IAPP is nonamyloidogenic, islet amyloid never occurs in normal rats and mice. Therefore, to investigate the mechanism of islet amyloidosis in a mouse model, we and others have generated transgenic mice expressing the human IAPP (hIAPP) gene in their pancreatic -cells (1215). These transgenic mice do not develop islet amyloid spontaneously (12,13), in accordance with the fact that only small amounts of islet amyloid are observed in a few isolated islets in old nondiabetic primates and cats (3,4). However, islet amyloid occurs when these transgenic mice are exposed to other diabetogenic factors, including increased dietary fat (16), insulin resistance induced by growth hormone and dexamethasone (14), or obesity caused by crossbreeding with genetically obese mice (15,17). In the present study, we developed a method to precisely quantify islet amyloid and cellular components. Using this method, we addressed the following questions in our hIAPP transgenic mice: 1) How is islet amyloid distributed in different pancreatic regions? 2) Is the prevalence of amyloid in all islets related to the amount of amyloid in individual islets? and 3) Does the endocrine cell composition change in islets containing moderate amounts of amyloid? RESEARCH DESIGN AND METHODS hIAPP transgenic mice. Mice expressing the hIAPP gene in their pancreatic -cells were bred at the University of Washington (13). By breeding male hemizygous hIAPP transgenic mice (C57BL/6xDBA/2) with female nontransgenic mice (C57BL/6xDBA/2; Jackson Laboratories, Bar Harbor, ME), we produced offspring that did or did not express the hIAPP transgene. Genotyping was performed by polymerase chain reaction using primers specific for hIAPP (18). Because islet amyloid occurs more frequently in male than in female hIAPP transgenic mice (16), only male mice were studied. Male nontransgenic littermates were used when control mice were necessary. All of the studies were approved by the Animal Care Committee at the VA Puget Sound Health Care System. Preparation of pancreatic sections. To investigate the distribution of islet amyloid, three pancreases, designated as pancreases A, B, and C, were obtained from hIAPP transgenic mice. These mice were 15 months old and fed a diet containing 9% fat wt/wt (Mouse Diet 5021; Purina Mills, St. Louis, MO). Each pancreas was fixed in 4% paraformaldehyde in 0.1 mol/l phosphate buffer (pH 7.2) and embedded in paraffin with the pancreatic head oriented toward the top of the paraffin block. Each pancreas was cut completely, yielding 2,700 3,500 sections (5 m thick). We selected 20 sections at a fixed interval (750 m) throughout each pancreas. The sections were stained with thioflavin-S to visualize islet amyloid. To determine whether the distribution of -cells in the pancreas differs between hIAPP transgenic and nontransgenic mice, sections were taken from the head, body, and tail of three hIAPP transgenic pancreases (pancreases A, B, and C) and three nontransgenic pancreases (pancreases D, E, and F), the latter obtained from mice of the same age and genetic background. None of the six (...truncated)