1Division of Metabolism, Endocrinology and Nutrition, Department of Medicine, Veterans Affairs Puget Sound Health Care System and University of Washington; 2Hope Heart Institute and Department of Pathology, University of Washington, Seattle, Washington 98108; and 3British Columbia Research Institute for Children's and Women's Health, Vancouver British Columbia V5Z 4H4, Canada
Submitted 31 March 2003 ; accepted in final form 4 November 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
amylin; euglycemic hyperinsulinemic clamp; insulin sensitivity
IAPP is a 37-amino acid peptide localized to secretory granules and cosecreted with insulin in a molar ratio of 1:100 (25). The mechanism of IAPP deposition as islet amyloid is not known, although an amyloidogenic sequence between amino acids 20-29 such as that present in the primate and cat peptides seems to be essential (32). This region has the propensity to form
-pleated sheets, a secondary structure necessary for amyloid formation (45). The lack of the amyloidogenic sequence in rodent IAPP is thought to be the reason that rats and mice do not develop islet amyloid. However, the sequence alone is not sufficient for amyloidogenesis, since humans without type 2 diabetes or insulinomas rarely develop islet amyloid (8).
To study mechanisms responsible for islet amyloid formation, we generated transgenic mice that express human IAPP (hIAPP) in the -cells of their pancreata (13) and develop islet amyloid deposits and hyperglycemia when fed a high-fat diet for 12-16 mo (39). The length of time (12-16 mo) required for islet amyloid to be deposited in most of these transgenic mice is compatible with the middle-age onset of the syndrome in humans. However, such an approach is time consuming and expensive. Therefore, mechanisms to accelerate this process were deemed to be potentially useful so that intervention studies could be pursued in a shorter time frame. We hypothesized that, because insulinomas, like type 2 diabetes, are associated with islet amyloid deposition (33), hIAPP overproduction by such islet endocrine tumors may lead to more rapid islet amyloid deposition.
The RIP-Tag-transgenic mouse is an animal model of insulinomas. These mice express the large T-antigen of the SV-40 virus driven by the rat insulin promoter, so that transgene expression is targeted to the -cells of the pancreas (18). These mice develop islet hyperplasia and hyperinsulinemia, with 1-2% of these islets becoming solid, vascularized tumors (7). As a result, these mice die at
12 wk of age from hypoglycemia. Tumors from these mice have been excised and cultured in vitro to develop immortal cell lines (
TC-3,
TC-6), which have been extensively used to study synthesis and secretion of
-cell hormones (2, 15, 27).
To study islet amyloid formation in insulinomas, we therefore crossbred our hIAPP transgenic mice with RIP-Tag transgenic mice to produce a new, double-transgenic line (hIAPPxRIP-Tag) and characterized these new transgenic mice, which develop islet tumors and secrete hIAPP.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To produce the new line of transgenic mice, hemizygous RIP-Tag transgenic male mice (C57BL/6) were mated with hemizygous hIAPP transgenic females (C57BL/6 x DBA/2), and the resulting offspring (75% C57BL/6 x 25% DBA/2) were genotyped using the polymerase chain reaction of tail DNA (14). hIAPP and RIP-Tag were detected using specific primers, with the internal control being the 2-microglobulin gene. All of the studies were approved by the Animal Care Committee at the VA Puget Sound Health Care System.
Breeding of hIAPP and RIP-Tag hemizygous transgenic mice resulted in offspring containing no, one, or both transgenes. Only mice containing no (wild type), the RIP-Tag, or both hIAPP and RIP-Tag transgenes were studied. Thus all mice used in this study were littermates. Both hIAPPxRIP-Tag and RIP-Tag mice developed islet tumors. All animals were kept in a specific pathogen-free environment with a 12:12-h light-dark cycle and had free access to water and a high-fat diet containing 24.4% (wt/wt) fat (Research Diets, Brunswick, NJ). Both male and female mice were used in this study. All the mice were weighed and bled at 6, 9, and 12 wk of age for determination of body weight, plasma glucose, and immunoreactive insulin (IRI). They were then followed until natural death. The dates of birth and death were recorded so that life span (in days) could be calculated.
Plasma measurements and assays. Mice were fasted for 4 h and anesthetized with an intraperitoneal injection of pentobarbital sodium (100 mg/kg) before blood sampling. Blood was obtained from the retroorbital sinus by use of heparinized hematocrit capillary tubes, and the animals were allowed to recover. Plasma samples were stored at -20°C until assayed. Mice from which pancreas samples were obtained were killed at 12 wk of age after the final blood sample was collected. After cervical dislocation, a laparotomy was performed and the pancreas excised for histological analysis.
Plasma glucose was measured by an automated glucose oxidase method (Beckman Glucose Analyzer II). Plasma IRI was measured by a modification of the double-antibody radioimmunoassay method of Morgan and Lazarow (31), using rat insulin as the standard. Plasma human IAPP-like immunoreactivity (hIAPP-LI) was measured by an enzyme immunoabsorbance assay by using F024 as the capture and F002 as the detection antibody [kind gift of Amylin Pharmaceuticals, La Jolla, CA (34)]. Plasma mouse IAPP-like immunoreactivity (mIAPP-LI) was measured as previously described (3).
Euglycemic hyperinsulinemic clamp procedure. Twelve-week-old mice were anesthetized as described above. Catheters were inserted into the left jugular vein for the infusion of insulin and glucose and the right carotid artery for blood sampling. Insulin was infused at a rate of 25 mU·kg-1·min-1, and plasma glucose concentrations were kept at euglycemia with the infusion of a 5% glucose solution. Stable plasma glucose concentrations were achieved after 60-90 min of insulin infusion, at which time the glucose infusion rate was determined and used as a measure of insulin sensitivity. To account for differences in the steady-state plasma glucose and IRI levels, insulin sensitivity was calculated as glucose infusion rate/(clamped insulin level x clamped glucose level) (6).
Immunocytochemical analysis. For light microscopy, pancreata were fixed in 0.1 M phosphate buffer (pH 7.4) containing 4% paraformaldehyde, rinsed in 0.1 M phosphate buffer (pH 7.4), and embedded in paraffin. Sections (5 µm) were deparaffinized and rehydrated before staining.
Immunodetection of pancreatic hormones was performed with the following antibodies diluted in 0.1 M phosphate buffer (pH 7.4)/0.5% BSA: insulin antibody (Sigma, St. Louis, MO) at a dilution of 1:2,000; rodent IAPP antibody (8342, which recognizes both mouse and rat but not human IAPP) at a dilution of 1:500; somatostatin antibody (AS-10, kind gift from Dr. John Ensinck) at a dilution of 1:2,000; and glucagon antibody [14C, gift from Dr. Robert McEvoy (5)] at a dilution of 1:1,000. Slides were rinsed and treated with the Vector ABC kit (Vectastain ABC Kit Elite PK-6100 Standard) and then treated with the Vector DAB Peroxidase Substrate Kit. Slides were counterstained with hematoxylin and dehydrated, and coverslips were mounted with permount.
Western blotting analysis. Tissue was powdered in liquid nitrogen and lysed in lysis buffer containing 50 mM Tris·HCl (pH 8), 150 mM NaCl, 0.02% sodium azide, 0.1% sodium dodecyl sulfate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 20 µg/ml leupeptin, 1 µM pepstatin A, 1% Nonidet P-40, and 0.5% sodium deoxycolate for 25 min on ice. Samples were centrifuged at 15,000 g, and the supernatant was used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Ten milligrams of protein were electrophoresed on a Tris-tricine gel, transferred to a polyvinylidene difluoride membrane, and blocked with 5% skim milk for 2 h at room temperature. The membrane was incubated with 1:1,000 rabbit anti-rat IAPP antibody (cat. no. RIN7323; Peninsula Laboratories, San Carlos, CA) for 1 h followed by 1:5,000 horseradish peroxidase-conjugated anti-rabbit antibody for 1 h at room temperature. Detection was achieved using enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ).
Statistical analysis. The Mann-Whitney U-test was used to compare data from the two groups of mice. The Wilcoxon signed rank test was used when data were paired. Analysis of life span was performed using Kaplan-Meier survival statistics. A P value of 0.05 was considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Plasma hIAPP-LI was measured with a two-site immunoabsorbance assay in all mice at 12 wk of age. The capture antibody binds to the amidated COOH terminus of IAPP, and the detection antibody binds to the 20- to 29-amino acid region of human IAPP, so that the assay measures only hIAPP-LI. Thus, in mice that did not express the hIAPP gene (i.e., wild-type and RIP-Tag), plasma hIAPP-LI was not detected. In hIAPPxRIP-Tag mice, the mean plasma hIAPP-LI concentration was 24.6 ± 7.0 pmol/l, which is 2.5 times higher than in hIAPP transgenic littermates (9.3 ± 2.6 pmol/l, P < 0.05) that had been fed the same diet and had blood sampled at 12 wk of age.
Because of the limited blood volume of mice, mIAPP-LI in plasma was measured by pooling plasma samples from 3-4 animals obtained at death (12 wk of age). The results suggest an increased amount of mIAPP-LI in hIAPPxRIP-Tag (47.1 ± 11.8 pmol/l, n = 2) and RIP-Tag mice (28.6 ± 4.1 pmol/l, n = 2) compared with wild-type mice (5.7 ± 0.3, n = 4).
Pancreatic histology. Results of islet morphology and immunostaining of serial sections of pancreas for insulin, IAPP, glucagon, and somatostatin are shown in Fig. 1. Sections through tumorous and normal-sized islets from hIAPPxRIP-Tag mouse pancreata revealed immunolabeling for insulin and IAPP throughout. Glucagon and somatostatin immunoreactivity in tumors was rare, confirming previous reports that the islet tumors in RIP-Tag mice were composed almost exclusively of -cells (18, 35). In normal-sized islets from hIAPPxRIP-Tag mice, glucagon and somatostatin immunoreactivity was located in the periphery, where
- and
-cells were expected to reside. The pattern of immunostaining in RIP-Tag mouse pancreata was essentially the same as in hIAPPxRIP-Tag pancreata (data not shown).
|
Wild-type mice displayed insulin and IAPP immunoreactivity throughout regularly shaped islets, whereas glucagon and somatostatin labeling was primarily located in the periphery of the islets (Fig. 1).
Western blotting. Impaired proteolytic processing of proinsulin to mature insulin is a feature of insulinomas. Western blotting was performed to determine whether processing of IAPP from its precursor proIAPP was impaired in tumor-bearing mice and to ascertain whether differences existed in IAPP processing between RIP-Tag and hIAPPxRIP-Tag tumors. Figure 2 shows that, although the major band corresponded to mature IAPP (4 kDa), both RIP-Tag and hIAPPxRIP-Tag tumors had more higher molecular mass bands corresponding to unprocessed proIAPP (8 kDa) than wild-type islets. There was no large difference in the intensity of these higher molecular mass bands between RIP-Tag and hIAPPxRIP-Tag tumors (Fig. 2).
|
Microscopy for identification of amyloid and amyloid fibrils. Light microscopy of thioflavin S-stained tumors and islets from hIAPPxRIP-Tag and RIP-Tag transgenic mice showed no evidence of amyloid at 12 wk of age.
Because the presence of amyloid deposits visible by light microscopy is preceded by the development of fibrils visible only by electron microscopy, we examined four hIAPPxRIP-Tag mice at 12 wk of age and two hIAPPxRIP-Tag mice at 22 wk of age for the presence of fibrils. Despite extensive examination, amyloid fibrils were not found in hIAPPxRIP-Tag mice. As expected, assessment of tumors from RIP-Tag mice (n = 6) did not display any fibrils, as they produce and secrete rodent IAPP, which is nonamyloidogenic.
Life span assessment of RIP-Tag vs. hIAPPxRIP-Tag transgenic mice. During follow-up of these transgenic mice, we observed that hIAPPxRIP-Tag had higher body weights and plasma glucose concentrations at 12 wk of age compared with RIP-Tag mice. We also observed that, in our initial cohort, the decrement in plasma glucose between 6 and 12 wk of age was smaller in hIAPPxRIP-Tag compared with RIP-Tag mice (-2.6 ± 0.5 vs. -4.2 ± 0.6 mmol/l, respectively, P = 0.05).
We thus hypothesized that hIAPPxRIP-Tag mice may live longer than RIP-Tag mice due to a slower decrease in plasma glucose levels. A group of hIAPPxRIP-Tag and RIP-Tag mice were thus bled at 6 and 12 wk of age and then followed until natural death. In this group, weight, plasma glucose, and IRI were not different between hIAPPxRIP-Tag and RIP-Tag mice at 6 wk of age (Table 2). However, as we observed in the original cohort, body weight and plasma glucose levels were higher in hIAPPxRIP-Tag mice compared with RIP-Tag mice at 12 wk of age (P < 0.05), whereas plasma IRI concentrations were similar in both groups of mice.
|
The life span of hIAPPxRIP-Tag mice (120.9 ± 7.5 days, n = 13) was significantly longer compared with their RIP-Tag littermates (101.5 ± 5.3 days, n = 10, P < 0.05). This mean difference of 19.4 days represents a 19% increase in life span for the hIAPPxRIP-Tag mice. Analysis of the data with Kaplan-Meier survival statistics confirmed this and showed marked divergence of the two survival curves with hIAPPxRIP-Tag mice living longer than RIP-Tag mice (P < 0.05; Fig. 3). Life span correlated significantly with body weight at 12 wk of age (r = 0.48, P < 0.05; Fig. 4A) but did not correlate with plasma glucose (r = 0.33, P = 0.13) or plasma IRI (r = 0.17, P = 0.42) concentrations.
|
|
Insulin sensitivity of RIP-Tag vs. hIAPPxRIP-Tag transgenic mice. Euglycemic hyperinsulinemic clamps were performed to determine whether the higher body weight and plasma glucose levels and consequent increased life span in hIAPPxRIP-Tag mice were due to reduced insulin sensitivity. Plasma glucose levels were significantly higher and plasma IRI concentrations tended to be higher in hIAPPxRIP-Tag compared with RIP-Tag transgenic mice (Table 3). Although the glucose infusion rate was not different, when corrected for differences in the plasma glucose and IRI levels at steady state, this parameter was significantly lower in hIAPPxRIP-Tag compared with RIP-Tag transgenic mice (Table 3). Furthermore, there was a significant inverse correlation between body weight at 12 wk of age and insulin sensitivity (r = 0.70, P < 0.05; Fig. 4B). This finding is compatible with the heavier hIAPPxRIP-Tag mice being more insulin resistant compared with RIP-Tag transgenic mice.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Close examination of islet tumors from hIAPPxRIP-Tag mice at both the light and electron microscopic level did not reveal any evidence of amyloid deposits or fibrils. This is despite increased plasma hIAPP-LI levels in hIAPPxRIP-Tag mice compared with age-matched hIAPP transgenic littermates. The most probable reason for the absence of amyloid is the fact that hIAPPxRIP-Tag mice have a considerably shortened life span compared with normal hIAPP transgenic or wild-type littermates. Although we examined islet tumors from 12- and 22-wk-old (longest surviving) high-fat-fed hIAPPxRIP-Tag mice, this may not be sufficient time for amyloid deposits or fibrils to form. We (39) have previously shown that islet amyloid forms after hIAPP transgenic mice are fed a high-fat diet for at least 12 mo. Furthermore, islet amyloid deposition was reduced in 12-mo-old mice that were hyperglycemic and had impaired hIAPP release due to disruption of the -cell glucokinase gene (3). These studies and the present one suggest that, although a certain level of secretion needs to be maintained, increased hIAPP production and secretion alone are not associated with an acceleration of islet amyloid deposition.
Another possible reason for the lack of amyloid formation in the islet tumors may be that the increased production of insulin and its precursors may impair fibril formation in vivo, as has been demonstrated to occur in vitro (22, 28, 47). Because insulinomas secrete increased amounts of proinsulin, insulin, and C-peptide (10), it is possible that these peptides may act to impair fibril formation. Furthermore, it has been shown that insulin can form heteromolecular complexes with hIAPP in vitro that suppress amyloid fibril formation (21), and mouse IAPP can also antagonize and dose-dependently inhibit fibril formation by hIAPP (43). It is also likely that these tumors release increased amounts of proIAPP, as this propeptide and proinsulin are proteolytically cleaved by the same processing enzymes (19, 41) and, as mentioned above, insulinomas are known to produce and release increased amounts of proinsulin (10, 36). We have now been able to demonstrate by Western blotting that the tumors do in fact contain increased amounts of a high molecular mass band that, based on its molecular mass, is consistent with it being proIAPP. Although the tumors contained increased amounts of this larger IAPP-like peptide, the similar intensities of the bands did not allow us to determine whether there was a difference in the amount of higher molecular mass IAPP produced by the RIP-Tag and hIAPPxRIP-Tag tumors. However, although proIAPP output may be increased, we do not believe that proIAPP is likely to be inhibitory to the amyloidogenic process in hIAPPxRIP-Tag for the following reasons. 1) As stated earlier, pancreatic islet tumors are known to contain islet amyloid (1, 38, 48) and to be associated with increased propeptide secretion (10); 2) islet amyloid and increased proinsulin release occur in patients with type 2 diabetes (42), and disproportionate proinsulin levels predict the development of this disease (26); and 3) histological evidence has shown that the NH2-terminal portion of the proIAPP molecule is associated with islet amyloid in humans with type 2 diabetes (44, 46).
An interesting consequence of hIAPP expression and production in hIAPPxRIP-Tag transgenic mice was the increased life span compared with RIP-Tag mice. This increased longevity of our new line of double-transgenic mice is of interest and implies that, besides islet amyloidogenesis, hIAPP may have a biological role in carbohydrate metabolism. It has previously been suggested that IAPP may cause insulin resistance by inhibiting glucose uptake and glycogen storage in muscle (29). Chronic systemic administration of IAPP has been shown to be associated with increased body weight gain and increased fat mass (12). We performed hyperinsulinemic clamps and observed that hIAPPxRIP-Tag mice had reduced insulin sensitivity compared with RIP-Tag mice. Thus the presence of the hIAPP transgene resulted in increased circulating hIAPP and reduced insulin sensitivity. Because the concentrations of both plasma human and mouse IAPP in hIAPPxRIP-Tag transgenic mice were in the physiological range (3, 20, 37), it is unlikely that the effects on insulin sensitivity were due to a pharmacological effect of these peptides. As indicated by the significant inverse correlation between insulin sensitivity and body weight (Fig. 4B), this reduction in insulin sensitivity may contribute to, or be the result of, the higher body weight and plasma glucose levels in the hIAPPxRIP-Tag mice, thereby increasing their life span. This reduction in insulin sensitivity in the hIAPPxRIP-Tag mice is also compatible with the trend for the steady-state plasma insulin levels during the clamp to be increased as a manifestation of a reduction in insulin clearance (16). IAPP may also affect insulin release, as suggested by a study of IAPP-null mice, which found that the absence of IAPP was associated with increased insulin release and improved glucose tolerance (17). Because hIAPP was present in the circulation of the hIAPPxRIP-Tag mice and therefore they had greater circulating total (human and mouse) IAPP levels than the RIP-Tag mice, we cannot exclude the possibility that increased release of IAPP in these double-transgenic mice may also have resulted in a reduction in insulin release and contributed to the increased life span of these mice. Because insulinomas are poorly responsive to glucose stimulation, it was not possible to perform a reliable assessment of stimulated insulin secretion. However, the possibility that insulin release was reduced in the hIAPPxRIP-Tag mice is supported indirectly by the fact that plasma insulin levels tended to be lower in these mice despite the fact that they were insulin resistant and had higher plasma glucose levels, two scenarios that would be expected to raise insulin levels.
Another possible reason for the increased life span in the double-transgenic mice is that hIAPP is killing -cells, leading to reduced insulin release. Human IAPP has been shown to be cytotoxic to and induce apoptosis in pancreatic islet cells (4, 23, 30). To determine whether hIAPP production in hIAPPxRIP-Tag mice may be killing tumorous
-cells, we performed propidium iodide staining of pancreatic sections but found no evidence of increased numbers of picnotic nuclei (a marker of apoptotic/necrotic cells) in hIAPPxRIP-Tag compared with RIP-Tag mice (data not shown). Furthermore, as stated earlier, we did not observe amyloid fibrils in islets or tumors from hIAPPxRIP-Tag mice, and the plasma IRI levels were comparable in hIAPPxRIP-Tag and RIP-Tag mice. Thus we do not believe that islet cell cytotoxicity is a likely explanation for the increased life span of the hIAPPxRIP-Tag mice.
In conclusion, we have generated a new line of transgenic mice that develop pancreatic islet tumors that produce and secrete hIAPP, resulting in elevated plasma hIAPP levels. Despite this increase in hIAPP release, these mice did not develop islet amyloid, suggesting that overproduction of hIAPP is not likely to greatly accelerate amyloid formation in vivo. Interestingly, these mice also develop insulin resistance and live significantly longer than mice that develop islet tumors but do not produce hIAPP, suggesting a possible role of IAPP to impair glucose metabolism.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
Present address of S. Andrikopoulos: Department of Medicine, Royal Melbourne Hospital, University of Melbourne, Parkville, Victoria 3050, Australia.
Present address of C. B. Verchere: B.C. Research Institute for Children's and Women's Health, Vancouver BC V5Z 4H4, Canada.
Present address of F. Wang: Department of Surgery, Karolinska Institute at Huddinge University Hospital, 171 76 Stockholm, Sweden.
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |