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Cooper, G. J. S., Willis, A. C., Clark, A., Turner, R. C., Sim, R. B.. & Reid, K. B. M. (1987) Proc. Natl. Acad. Sci. USA 84, 8628-. 8632. 7. Lukinius, A., Wilander, E., ...

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 3492-3496, April 1996 Medical Sciences

Islet amyloid formation associated with hyperglycemia in transgenic mice with pancreatic beta cell expression of human islet amyloid polypeptide mellitus/insulin/glucose) C. BRUCE VERCHERE*tt, DAVID A. D'ALESSIO*, RICHARD D. PALMITER§, GORDON C. WEIR¶, (non-insulin-dependent diabetes


*Division of Metabolism, Endocrinology and Nutrition, Department of Medicine, §Howard Hughes Medical Institute, and IlDepartment of Biological Structures, University of Washington, Seattle, WA 98195; tVeterans Affairs Medical Center, 1660 South Columbian Way, Seattle, WA 98108; and ¶Joslin Diabetes Center and Harvard Medical School, 1 Joslin Place, Boston, MA 02215

Contributed by Richard D. Palmiter, December 27, 1995

Several groups, including our own, have attempted to develop transgenic mouse model of islet amyloid formation by overexpressing hIAPP in pancreatic /3 cells (14-16). Islet amyloid deposits have not been reported in any of these transgenic lines even though they overproduce the human form of the peptide by up to 9-fold and have been followed up to 18 months of age (14-16). Thus, the available data suggest that simple overproduction of an amyloidogenic form of IAPP is insufficient for the formation of IAPP-derived amyloid within the lifespan of a mouse. Following a recent change in housing location and diet, we observed hyperglycemia in two transgenic male mice during routine screening of the colony. These hyperglycemic mice did not develop the acute syndrome typically associated with absolute insulin deficiency, suggesting that they had an NIDDM-like syndrome. Histopathological analysis of pancreatic tissue obtained from these two mice revealed the presence of islet amyloid, suggesting a relationship between the development of hyperglycemia and islet amyloid in these animals. To investigate this possibility, we undertook a complete analysis of the incidence of hyperglycemia and islet amyloid in our colony of hIAPP-transgenic and nontransgenic mice.

ABSTRACT 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 f3 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 -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 3 cells and islet capillaries. Strikingly, approximately half of the mice with islet amyloid deposits were hypeFglycemic (plasma glucose > 11 mM). In younger (6to 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 t3 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.


MATERIALS AND METHODS Transgenic Mice. Transgenic mice were generated at the University of Washington, as described (14). The /3 cells of

Amyloid deposits

are found in the pancreatic islets of most individuals with non-insulin-dependent diabetes mellitus (NIDDM) (1-3). The major component of islet amyloid is a 37-amino acid peptide, islet amyloid polypeptide (IAPP; amylin) (4-6), which is a normal secretory product of the pancreatic f3 cell (7, 8). Formation of islet amyloid deposits from IAPP may contribute to the progressive deterioration of ,3-cell function observed in this disease (9), since IAPP-derived amyloid has been shown to be toxic to islet cells in vitro (10) and the degree of amyloid deposition is associated with the severity of hyperglycemia in monkeys (11). The mechanism leading to IAPP deposition as islet amyloid is unknown. The sequence of human IAPP (hIAPP) contains an amyloidogenic region (amino acids 20-29) that is thought to be essential for fibril formation (12, 13); however, this sequence does not appear to be the sole prerequisite since nondiabetic individuals do not usually develop islet amyloid (1-3). Thus, some additional unrecognized factor(s) must be present in NIDDM that potentiates islet amyloid formation. IAPP-derived islet amyloid has never been observed in rodents, presumably because rodent IAPP, unlike hIAPP, does not contain the necessary amyloidogenic sequence (13).

these transgenic mice produce and secrete hIAPP in amounts 2-3 times greater than the native (mouse) peptide (14, 17, 18). The transgenic lineage has been maintained by breeding heterozygous transgenic mice with C57BL/6 x DBA/2 mates. Following publication of our initial characterization of the mice in which we reported that no islet amyloid was present (14), the colony was moved from the University of Washington Vivarium, where the mice were reared on Purina Laboratory Rodent Diet (5001; Purina) to the Animal Research Facility at the Seattle Veterans Affairs Medical Center, where the diet was changed to Purina Autoclavable Mouse Diet (5021). All data reported in the present study were obtained using mice bred and housed in the Seattle Veterans Affairs Medical Center colony and fed Purina diet 5021. Plasma Measurements. Plasma concentrations of glucose and immunoreactive insulin (IRI) were determined on multiple occasions during an 8-month period in mice aged 6-22 months. Prior to blood sampling, mice were fasted for 4-6 h Abbreviations: IAPP, islet amyloid polypeptide; hIAPP, human IAPP; NIDDM, non-insulin-dependent diabetes mellitus; IRI, immunoreactive insulin. 4To whom reprint requests should be addressed at: Veterans Affairs Medical Center, Mail Stop 151, 1660 South Columbian Way, Seattle, WA 98108.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.


M~edical Sciences: Verchere et al.

Proc. Natl. Acad. Sci. USA 93

and anesthetized with sodium pentobarbital (80 mg/kg) administered i.p. Blood samples were drawn by retroorbital bleed into heparinized microhematocrit capillary tubes. Plasma was stored at -20°C until assayed. Glucose was measured by the glucose oxidase technique. A mouse was considered hyperglycemic if plasma glucose measured >11 mM (200 mg/dl). IRI was measured by a specific radioimmunoassay (19). Histological Analysis. Human pancreas was obtained at autopsy from a 74-year-old male with NIDDM. Mouse pancreas was obtained at time of sacrifice (age 13-22 months). Samples of pancreas were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, rinsed in 70% ethanol, and embedded in paraffin. Sections (5 ,im) were deparaffinized and rehydrated prior to staining. For thioflavin S staining (20), sections were incubated with 0.5% thioflavin S for 1 min, rinsed briefly 3 times in 70% ethanol, washed for 5 min in water, mounted in polyvinyl alcohol, and viewed by fluorescence microscopy with UV light (365 nm) and a 420-nm barrier filter. A mouse was considered amyloid-positive if thioflavin S-positive islet staining was present. Congo Red staining of amyloid (21) was performed using a kit purchased from Sigma. Analysis of pancreatic sections from mice revealed no evidence of inflammatory response. For ultrastructural analysis, minced pieces of mouse pancreas (splenic lobe) were fixed in 4% formaldehyde/1% gluteraldehyde, 0.1 M phosphate buffer (pH 7.4), rinsed in 50 mM glycine and stored in the same phosphate buffer at 4°C. Hand-dissected islets were processed without osmication for Araldite embedding. Immunolocalization on ultrathin sections used 1:10-1:30 dilutions of primary antibody (hIAPP antiserum 5436) (14) and protein A-gold (Auroprobe EM protein

A-G10, Amersham). Data Analysis. Comparisons of weight and plasma insulin levels between groups of mice (transgenic vs. nontransgenic or male vs. female) were made by Mann-Whitney U test with Bonferroni adjustment for multiple comparisons. The effect of gender and transgenic status on the frequency of hyperglycemia and islet amyloid was assessed by log-linear analysis. The Fisher exact test was used to test for an association between hyperglycemia and the presence of islet amyloid; P < 0.05 was considered significant.




glycemia is increased by expression of the hIAPP transgene (P
13 months of age, were sacrificed for histopathologic evaluation of the pancreas. At the time of sacrifice, transgenic and nontransgenic animals of both genders were well matched for age and weight (Table 1). Samples of pancreas were examined for the presence of islet amyloid deposits using thioflavin S, a specific amyloid stain (20, 22). In 81% (13/16) of transgenic male mice, numerous thioflavin S-positive islet amyloid deposits were observed (Fig. 1A). Following Congo Red staining, these islet deposits were birifringent when viewed under polarized light, again consistent with amyloid. By contrast, in nontransgenic animals, islet amyloid deposits were never observed (0/17, Fig. 1B), even in three hyperglycemic animals (Table 1). Islet amyloid was rarely observed in female hIAPP-transgenic mice (1/9, 11%). Amyloid deposits were usually found in perivascular and peripheral regions of the islet and were never observed in the surrounding exocrine pancreas. In many of these mice, the amount of islet amyloid was extensive, occurring in the majority of islets and composing a major proportion of islet area. These deposits thus bear a striking resemblance to the islet amyloid seen in humans with NIDDM (Fig. 1C). Ultrastructural analysis using hIAPP antisera revealed immunogold particles localized over secretory granules of (3 cells and over the extracellular fibrillar material present in the pericapillary space adjacent to islet /3 cells, but not over granules of a or 5 cells (Fig. 2). Fibrillar material was not observed within cells nor near a or 8 cells. Further, fibrils were not observed in the islets of nontransgenic mice. As detailed in Table 1, every hyperglycemic male transgenic mouse analyzed had islet amyloid deposits. However, islet amyloid deposits were also observed in 67% (6/9) of normoglycemic transgenic males. Islet amyloid occurred in only two of eight male transgenic mice aged 6-9 months and, in these two mice, was associated with severe hyperglycemia (fasting plasma glucose concentrations of 27 mM and 28 mM). Thus, the incidence of islet amyloid deposition increased with aging and was associated with the development of hyperglycemia (P

Frequency of Hyperglycemia and Plasma Insulin ConcenhIAPP-Transgenic Mice. The frequency of hyperglycemia was evaluated in 99 mice of various ages over an < 0.001). 8-month period, as detailed in Materials and Methods. The mean plasma glucose level in normoglycemic mice was 6.8 0.1 mM (n 82). Spontaneous hyperglycemia (fasting plasma DISCUSSION glucose > 11 mM) was occasionally observed in younger mice We have developed a transgenic mouse model in which =80% (6-9 months), but increased in frequency with aging. Hyperof male mice with pancreatic (3-cell expression of an hIAPP glycemia was more common in male (10/32, 31%) than in female (2/19, 11%) transgenic mice. Hyperglycemia was also transgene spontaneously develop extensive islet amyloid demore frequently observed in nontransgenic male mice (5/35, posits by 1.5 years of age. These amyloid deposits share characteristics with the islet amyloid of human NIDDM, 14%) as it was never observed in nontransgenic female mice (0/13). Thus, male mice in our colony are prone to the including the presence of hIAPP-immunoreactive fibrils (23, development of hyperglycemia, and the frequency of hyper24), increasing deposition with age (25), and an association Table 1. Frequency of islet amyloid in hyperglycemic and normoglycemic 13- to 22-month-old hIAPP-transgenic and nontransgenic mice trations in



Hyperglycemic mice Normoglycemic mice Mean age, Mean Total amyloid/total amyloid/total amyloid/total N Sex months examined examined examined Genotype weight, g ± Male 16 16.1 0.5 53.5 ± 2.9 6/9 7/7 13/16 Transgenic Female 9 18.0 ± 0.4 56.3 ± 5.6 1/2 0/7 1/9 11 17.1 ± 0.5 59.9 + 4.2 0/3 0/8 0/11 Nontransgenic Male Female 6 17.0 + 0.4 58.3 ± 5.0 0/0 0/6 0/6 Data for age and weight at time of sacrifice are mean ± SEM. Mean age and mean weight were not significantly different between mice of different genotypes

or sexes.


Proc. Natl. Acad. Sci. USA 93 (1996)

Medical Sciences: Verchere et al.

FIG. 1. Demonstration of amyloid deposits in pancreatic islets of hIAPPtransgenic male mice. Thioflavin S staining of sections of pancreas from (A) a 16-month-old, hyperglycemic hIAPPtransgenic male mouse exhibiting severe amyloid deposition; (B) a 16-month-old, hyperglycemic, nontransgenic male mouse; and (C) a human with NIDDM. Thioflavin S-positive staining is present in the islets of the hyperglycemic transgenic mouse and the

diabetic human, but


in the islets of the

hyperglycemic nontransgenic mouse. (Bars 50 Am.) =

Proc. Natl. Acad. Sci. USA 93 (1996)

Medical Sciences: Verchere et al. .AVU.



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FIG. 2. Ultrastructural analysis of pancreatic islet amyloid in hIAPP-transgenic mice. (A) Abundant immunogold labeling with hIAPP antiserum is found over the fibrillar accumulation present in the enlarged extracellular space between ,3 cells and the islet capillaries of hIAPP-transgenic mice that had thioflavin S-positive islet amyloid. (B) Islets of nontransgenic and transgenic mice without thioflavin S-positive amyloid have less pericapillary space with no noticeable fibrils or immunogold particles. (C) A higher magnification view of an extracellular amyloid deposit illustrating the fibrillar nature of these accumulations. Fibrils are 10 nm in diameter, consistent with the reported size of IAPP-derived amyloid fibrils (12). In both A and B the lumen of the capillary (top of each panel) is clear of gold particles but some, albeit sparse, immunogold labeling is found over insulin secretory granules. (Bars = 0.5j/m.)

with hyperglycemia (1-3). Ultrastructural analysis of the pancreatic islets of these transgenic mice indicates that hIAPPimmunoreactive fibrils accumulate in the extracellular space between f3 cells and islet capillaries, suggesting that IAPP deposition as amyloid occurs following its secretion from the P3 cell. Thus, these mice provide the first rodent model to study islet amyloid formation in vivo and its role in the pathogenesis of NIDDM. Mice with islet amyloid frequently had fasting hyperglycemia in the absence of weight loss (Table 1) or absolute insulin deficiency. Since hyperglycemia was less commonly observed in female transgenic mice (in which islet amyloid was rarely observed) or in nontransgenic mice, it is possible that islet amyloid formation may play a causative role in the development of hyperglycemia in these mice. Indeed, IAPP-derived amyloid fibrils have been shown to be toxic to islet cells in vitro (10). However, since islet amyloid deposits were present in 67% of normoglycemic transgenic male mice (see Table 1), and since hyperglycemia was also observed without amyloid in nontransgenic male mice, our data suggest that islet amyloid alone is neither sufficient nor essential for the development of hyperglycemia in these animals. Rather, the increased frequency of hyperglycemia in mice with islet amyloid suggests that each increases the likelihood of the other in this model. Male gender clearly contributes to the development of both hyperglycemia [as seen in other rodent models of NIDDM (26-28)] and islet amyloid. This sex-specificity was independent of weight (Table 1), but may be related to decreased insulin sensitivity in males as indicated by their higher fasting plasma insulin levels (29). We (14) and others (15, 16) have previously reported that mice with P-cell expression of hIAPP did not develop islet amyloid or hyperglycemia. However, after moving our colony to a new facility that routinely uses a different diet, we began to observe hyperglycemia and islet amyloid deposits. These changes were associated with an increase in mean body weight (56.0 ± 2.0 g at the Seattle Veterans Affairs Medical Center vs. 36.5 ± 1.8 g at the University of Washington) at 1 year of age. Although there are other differences between the two diets, it is our hypothesis that the change in dietary fat (9.0% in Purina Diet 5021 vs. 4.5% in Purina Diet 5001) is the most notable difference and is responsible for the weight gain and

the appearance of islet amyloid in our mice. Considering that infusion of a fat emulsion impairs islet function in rodents (30), it is possible that increased dietary fat had a direct effect on the /3 cell, which may have contributed to islet amyloid formation. Alternatively, it is possible that the increase in body weight (and presumably fat stores and insulin resistance) may have contributed to islet amyloid deposition by enhancing hIAPP synthesis and/or secretion, as suggested by in vitro studies using isolated islets from hIAPP-transgenic mice (31). However, simply increasing transgene expression in our original colony (by generation of homozygotes or chemical induction of insulin resistance) was not associated with islet amyloid formation (18), and other groups have failed to observe islet amyloid deposits in vivo despite an up to 9-fold increase in hIAPP production (15, 16). Moreover, our preliminary data suggest that hIAPP production, as assessed by plasma and pancreatic IAPP-like immunoreactivity levels, was not substantially increased by either dietary fat or male gender. Finally, background strain may have played a permissive role as the hIAPP transgene was bred onto a hybrid background of two strains (C57BL/6 and DBA/2) that show different susceptibilities to diet-induced obesity and diabetes (32-34). In conclusion, we have observed the spontaneous development of pancreatic islet amyloid deposits containing human IAPP in mice bearing an hIAPP transgene. The development of these deposits was associated with an increase in the fat content of the diet and an increased frequency of hyperglycemia. Thus, these mice provide a rodent model that may allow us to study the mechanism(s) underlying the formation of the islet amyloid that is characteristic of human NIDDM. We thank Stein Wang, Maggie Abrahamson, Richard Chan, Ruth Hollingsworth, Vicki Hoagland, Chare Vathanaprida, and Christopher Cahill for expert technical assistance; Dr. David Thorning for human NIDDM pancreas; Dr. Ronald Prigeon for statistical advice; and Drs. Daniel Porte, Jr., John Ensinck, and Renee LeBoeuf for helpful comments and advice. This work was supported by the Medical Research Service of the Department of Veterans Affairs and National

Institutes of Health Grants DK-12829, DK-17047, DK-35816, and DK-36836. C.B.V. was supported by a Medical Research Council of Canada Postdoctoral Fellowship and S.E.K. by a Young Investigator Award from the Diabetes Endocrinology Research Center of the University of Washington.


Medical Sciences: Verchere et al.

1. Westermark, P. (1972) Uppsala J. Med. Sci. 77, 91-94. 2. Clark, A., Cooper, G. J. S., Lewis, C. E., Morris, J. F., Willis, A. C., Reid, K. B. M. & Turner, R. C. (1987) Lancet ii, 231-234. 3. Clark, A., Saad, M. F., Nezzer, T., Uren, C., Knowler, W. C., Bennett, P. H. & Turner, R. C. (1990) Diabetologia 33, 285-289. 4. Westermark, P., Wernstedt, C., Wilander, E. & Sletten, K. (1986) Biochem. Biophys. Res. Commun. 140, 827-831. 5. Westermark, P., Wernstedt, C., Wilander, E., Hayden, D. W., O'Brien, T. D. & Johnson, K. H. (1987) Proc. Natl. Acad. Sci. USA 84, 3881-3885. 6. Cooper, G. J. S., Willis, A. C., Clark, A., Turner, R. C., Sim, R. B. & Reid, K. B. M. (1987) Proc. Natl. Acad. Sci. USA 84, 86288632. 7. Lukinius, A., Wilander, E., Westermark, G. T., Engstrom, U. & Westermark, P. (1989) Diabetologia 32, 240-244. 8. Kahn, S. E., D'Alessio, D. A., Schwartz, M. W., Fujimoto, W. Y., Ensinck, J. W., Taborsky, G. J., Jr., & Porte, D., Jr. (1990) Diabetes 39, 634-638. 9. Porte, D., Jr. (1991) Diabetes 40, 166-180. 10. Lorenzo, A., Razzaboni, B., Weir, G. C. & Yankner, B. A. (1994) Nature (London) 368, 756-760. 11. Howard, C. F., Jr. (1986) Diabetologia 29, 301-306. 12. Glenner, G. G., Eanes, E. D. & Wiley, C. A. (1988) Biochem. Biophys. Res. Commun. 155, 608-614. 13. Betsholtz, C., Christmanson, L., Engstrom, U., Rorsman, F., Jordan, K., O'Brien, T. D., Murtaugh, M., Johnson, K. H. & Westermark, P. (1990) Diabetes 39, 118-122. 14. D'Alessio, D. A., Verchere, C. B., Kahn, S. E., Hoagland, V., Baskin, D. G., Palmiter, R. D. & Ensinck, J. W. (1994) Diabetes 43, 1457-1461. 15. Fox, N., Schrementi, J., Nishi, M., Ohagi, S., Chan, S. J., Heisserman, J., Westermark, G. T., Leckstrom, A., Westermark, P. & Steiner, D. F. (1993) FEBS Lett. 323, 40-44. 16. Hoppener, J. W. M., Verbeek, J. S., de Koning, E. J. P., Oosterwijk, C., van Hulst, K. L., Visser-Vernooy, H. J., Hofhuis, F. M. A., van Gaalen, S., Berends, M. J. H., Hackeng, W. H. L., Jansz, H. S., Morris, J. F., Clark, A., Capel, P. J. A. & Lips, C. J. M. (1993) Diabetologia 36, 1258-1265.

Pr~c. Natl. Acad. Sci. USA 93 (1996) 17. Verchere, C. B., D'Alessio, D. A., Palmiter, R. D. & Kahn, S. E. (1994) Diabetologia 37, 725-728. 18. Verchere, C. B., D'Alessio, D. A. & Kahn, S. E. (1996) in Lessons from Animal Diabetes VI, ed. Shrafrir, E. (Birkhauser, Boston, MA), in press. 19. Morgan, D. R. & Lazarow, A. (1963) Diabetes 12, 115-126. 20. Schwartz, P. (1970) Amyloidosis: Cause and Manifestation of Senile Dementia (Thomas, Springfield, IL). 21. Puchtler, H., Sweat, F. & Levine, M. (1962) J. Histochem. Cytochem. 10, 355-364. 22. Games, D., Adams, D., Alessandrini, R., Barbour, R., Berthelette, P., et al. (1995) Nature (London) 373, 523-527. 23. Johnson, K. H., O'Brien, T. D., Hayden, D. W., Jordan, K., Ghobrial, H. K. G., Mahoney, W. C. & Westermark, P. (1988) Am. J. Pathol. 130, 1-8. 24. Clark, A., Edwards, C. A., Ostle, L. R., Sutton, R., Rothbard, J. B., Morris, J. F. & Turner, R. C. (1989) Cell Tissue Res. 257, 179-185. 25. Bell, E. T. (1959) Am. J. Pathol. 35, 801-805. 26. Kava, R., West, D. B., Lukasik, V. A. & Greenwood, M. R. C. (1989) Diabetes 38, 159-163. 27. Leiter, E. H., Chapman, H. D. & Coleman, D. L. (1989) Endocrinology 124, 912-922. 28. Lowell, B. B., S-Susulic, V., Hamann, A., Lawitts, J. A., HimmsHagen, J., Boyer, B. B., Kozak, L. P. & Flier, J. S. (1993) Nature (London) 366, 740-742. 29. Kahn, S. E., Prigeon, R. L., McCulloch, D. K., Boyko, E. J., Bergman, R. N., Schwartz, M. W., Neifing, J. L., Ward, W. K., Beard, J. C., Palmer, J. P. & Porte, D., Jr. (1993) Diabetes 42, 1663-1672. 30. Sako, Y. & Grill, V. E. (1990) Endocrinology 127, 1580-1589. 31. de Koning, E. J. P., Morris, E. R., Hofhuis, F. M. A., Posthuma, G., Hoppener, J. W. M., Morris, J. F., Capel, P. J. A., Clark, A. & Verbeek, J. S. (1994) Proc. Natl. Acad. Sci. USA 91, 8467-8471. 32. Kaku, K., Fiedorek, F. T. J., Province, M. & Permutt, M.A. (1988) Diabetes 37, 707-713. 33. Surwit, R. S., Kuhn, C. M., Cochrane, C., McCubbin, J. A. & Feinglos, M. N. (1988) Diabetes 37, 1163-1167. 34. Leiter, E. H. (1989) FASEB J. 3, 2231-2241.

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