Accumulation of Intrahepatic Islet Amyloid in a Nonhuman Primate Transplant Model

Abstract

Islet amyloid is hypothesized to play a role in nonimmunologic transplanted islet graft loss. We performed a quantitative histologic analysis of liver biopsies from intrahepatic islet grafts transplanted in streptozotocin-induced diabetic cynomolgus macaques. Seven animals treated with antithymocyte globulin (ATG) and rapamycin or ATG and rituximab experienced islet graft rejection with lymphocytic infiltrates present on islet graft biopsies. Except for one case involving the oldest and largest donor where amyloid was present on initial biopsy 1 month after transplant, none of the six other cases with rejection contained amyloid, including one case biopsied serially to 25 months. In contrast, four out of six animals treated with ATG and rituximab and rapamycin had no evidence of rejection at the time of biopsy (two animals that discontinued rapamycin had mild periislet lymphocytes), and all four cases followed more than 4 months demonstrated amyloid deposition at subsequent time points. Amyloid severity increased with time after transplant (r = 0.68; P < 0.05) and with decreasing islet β-cell area (r = −0.68; P < 0.05). In two islet recipients with no evidence of rejection and still normoglycemic and insulin independent at the first detection of amyloid, β-cell secretory capacity declined over time coincident with increasing amyloid severity and decreasing β-cell area, with both animals eventually becoming hyperglycemic and insulin dependent. Transplanted islet amyloid also developed in autologous islets placed sc. These results indicate that in cynomolgus macaques, transplanted islets may accumulate amyloid over time associated with subsequent decline in β-cell mass and function and support the development of intrahepatic islet amyloid as a potential mechanism for nonimmunologic islet graft loss.

Islet amyloid is a pathognomonic feature of type 2 diabetes (1) and is hypothesized to play a role in nonimmunologic islet graft loss in type 1 diabetic transplant recipients (2). Islet amyloid is composed of islet amyloid polypeptide (IAPP) (or amylin) fibrils deposited within and surrounding β-cells, where they exhibit direct toxicity (3). IAPP is cosecreted from β-cells with insulin (4, 5) but normally is inhibited from forming amyloid by appropriate proportions of insulin and other factors in the β-cell (6–8). Recently, a case report documented amyloid deposition in intrahepatic islets on postmortem examination of a type 1 diabetic recipient 5 yr after transplant (9). We subsequently demonstrated disproportionately increased IAPP-to-insulin secretion during glucose-potentiated arginine testing in insulin-independent type 1 diabetic recipients of intrahepatic islet transplants (10), suggesting disturbed regulation of insulin and IAPP within the transplanted islet β-cells as a possible mechanism for the reported deposition of amyloid in the intrahepatic islet graft.

Islet engraftment in the liver has been essential for the success of clinical islet transplantation to date. As a site, the liver provides transplanted islets with oxygenation via the portal circulation until revascularization by the hepatic arterial systems occurs (11, 12) and, in the case of allogeneic grafts, may represent an immunologically privileged site (13, 14). Indeed, the liver is the only site that has enabled sufficient survival of transplanted islets to consistently reverse diabetes and achieve insulin independence in large animal models (15). Nevertheless, although 70% of clinical islet transplant recipients may achieve insulin independence, only approximately 25% maintain insulin independence at 3 yr after transplant (16). Notwithstanding immunologic mechanisms such as alloimmune rejection (17, 18) and recurrent autoimmunity (19, 20) that can contribute to islet graft loss, there has long been speculated a nonimmunologic mechanism of islet “stress” believed to account for cases of recurrent hyperglycemia in the absence of evidence of immune system activation (21).

To investigate the deposition of intrahepatic islet amyloid as a possible mechanism for islet stress, we sought to establish a time course to amyloid deposition in intrahepatic transplanted islets and to evaluate for a relationship between islet graft dysfunction and the accumulation of islet amyloid by performing a quantitative histologic analysis of intrahepatic islet grafts transplanted in streptozotocin-induced diabetic cynomolgus macaques during a preclinical trial.

Materials and Methods

Islet graft donors and recipients

Islet donors were normoglycemic (blood glucose, <100 mg/dl) healthy cynomolgus macaques with body weights ranging 4.4–7.9 kg except for one older animal that weighed 9.4 kg (Table 1). Islet recipients weighed 1.3–3.0 kg and were rendered diabetic by injection of 150 mg/kg streptozotocin (22). C-peptide negative diabetes was confirmed by blood glucose more than 300 mg/dl and serum C-peptide less than 0.5 ng/ml. In select animals (01C0266, 211050, and CR14) undergoing posttransplant liver biopsies, a native pancreas biopsy was also performed that in all cases demonstrated a complete absence of islet β-cells by immunohistochemistry, supporting that our biochemical criteria for diabetes was associated with complete destruction of native islet β-cells. Islet transplantation involved portal vein delivery of 2–3 × 10⁴ islet equivalents/kg recipient body weight. Groups of animals received immunosuppression with either antithymocyte globulin (ATG) and rapamycin, ATG and rituximab, ATG and rituximab and rapamycin, or ATG and rituximab and rapamycin where the rapamycin was discontinued 200 d after transplant. The immunosuppression regimens were designed to determine whether B lymphocyte directed immunotherapy with rituximab would promote long-term islet allograft survival. We previously reported significantly prolonged islet graft survival in animals that received all three drugs, including evidence of long-term immunologic tolerance after discontinuation of rapamycin (23). Liver biopsies were performed at the time of recurrent hyperglycemia or after at least 2 months of normoglycemia and periodically thereafter. A final group of animals was evaluated who had undergone subtotal pancreatectomy followed 24 h later by autologous transplantation of their islets (1.2–2.4 × 10⁴ islet equivalents/kg recipient body weight) in the sc compartment of the abdominal wall. All animal care and handling were performed in accordance with guidelines established by the Department of Health and Human Services for care and use of nonhuman primates, as well as guidelines established by the Institutional Animal Care and Use Committee of the University of Pennsylvania that approved the study protocol.

Table 1.

Islet recipient and donor ages and body weights at transplant

Islet recipients	Age (yr)	Body weight (kg)	Donor age (yr)	Donor weight (kg)
01C0008	2.5	2.8	5	5.7
T3973	1.5	1.4	10	9.4
00C0759	1.5	1.8	6	6.3
00C0087	2	2.1	5	4.6
01C0266	2	2.3	8	7.0
01C0298	2	2.2	7	7.0
T3967	1	1.3	9	7.7
211050	2.5	2.4	6	5.6
T4536	1.5	1.5	6	5.9
01C0246	3	2.9	7	7.9
93-100	3	3.2	9	7.0
3845	1.5	1.5	5	4.4
CR14	2	1.8	6	6.4
210069 (sc)	3.5	3.9	n.a.	n.a.
212027 (sc)	3.5	3.5	n.a.	n.a.

n.a., Not applicable (autologous).

Islet biopsy assessment

Liver or native pancreatic tissue biopsy specimens were fixed in 10% buffered formalin. Formalin fixed tissues were processed for routine histology and stained with hematoxylin and eosin. For immunohistochemical analysis, adjacent paraffin sections were cut (5 μm) and stained using antibodies against porcine insulin and human glucagon to detect islet β- and α-cells, or against human CD3 and human CD20cy to detect T and B lymphocytes (Dako Cytomation, Carpinteria, CA). Antibodies conjugated with Cy3 IgG (Jackson ImmunoResearch, West Grove, PA) were used as the secondary antibody. Control experiments were performed omitting the primary antibody. Islet amyloid was detected by staining with thioflavin S (5 g/1000 ml solution; Sigma-Aldrich, St. Louis, MO) as previously described (24) using normal cynomolgus and human pancreata as negative controls and type 2 diabetic human pancreata as positive controls (Table 2). Representative biopsies deemed positive and negative for amyloid by thioflavin S staining, and control samples were stained with rabbit antibodies against rat IAPP (1:750–1:2000; a gift from Per Westermark) that has 100% cross-reactivity with human IAPP (9) and with rabbit antibodies against human IAPP (1:400–1:800) that has 100% cross-reactivity with rat and cat IAPP (Bachem Peninsula Laboratories, San Carlos, CA).

Table 2.

Control specimens used for amyloid and IAPP immunohistochemistry

Specimen	Source	Age (yr)	Body weight (kg)	BMI (kg/m2)
8889	Normal monkeya	5	4.2
B0134	Normal monkeya	6.5	7.0
F195	Normal monkeya	3	2.7
CITH30	Normal human	48	103	31.7
CITH43	Normal human	36	108	42.1
CITH56	Normal human	40	88	35.8
CITH86	Normal human	10	32	18.3
ICRH8	T2D human	45	120	37.0
ICRH11	T2D human	53	125	28.3
ICRH14	T2D human	58	90	29.3

T2D, Type 2 diabetic.

^aCynomolgus macaque.

When stained with Cy3, islet area positive for insulin fluoresced red at excitation 554 nm and emission 568 nm, and when stained with thioflavin S, islet area positive for amyloid fluoresced green at excitation 430 nm and emission 550 nm. The biopsy slides were imaged with an automated multispectral microscope (Vectra, Caliper Life Sciences). The multispectral capability was used to isolate label signal from background fluorescence, thus significantly increasing the contrast in component images of insulin label (Cy3) and of amyloid label (thioflavin S). In some cases, spectral unmixing was required to reveal amyloid deposits that were obscured by background fluorescence. Insulin and amyloid positive areas of each biopsy were determined using image analysis software developed to process multispectral imagery (inForm, Caliper Life Sciences). The inForm software was trained by M.D.F. to identify islet tissue as distinct from pancreatic exocrine or hepatic parenchymal tissue through pattern recognition that included accounting for the other endocrine cell types as well as vascular and neural tissue contained within an islet to determine the total islet area.

Total islet, β-cell, and amyloid areas determined by the multispectral imaging software were compared with those determined by manual segmentation performed by K.L. in a validation sample (n = 5) consisting of biopsies of a normal human pancreas, two monkey livers with amyloid affected intrahepatic islets, and two type 2 diabetic human pancreata. Although it is expected that the automated method using multispectral image analysis may underestimate β-cell area relative to manual segmentation, because it excludes vascular, neural, and non-β-cell endocrine tissue that interdigitates between β-cells and cannot be excluded with manual segmentation, better agreements between the two methods are expected for total islet and amyloid areas. Indeed, there was a shallower slope for β-cell area measured by the manual vs. automated methods relative to total islet and amyloid areas, with the two approaches nonetheless highly correlated for islet area (r² = 0.98; P = 0.001), β-cell area (r² = 0.88; P = 0.02), and amyloid area (r² = 0.99; P = 0.0001) (Supplemental Fig. 1, published on The Endocrine Society's Journals Online web site at http://endo.endojournals.org). Furthermore, both the manual and automated segmentation methods were found to be highly reproducible. Average interobserver r² values, based upon three operators segmenting the same images, were calculated to be 0.989 and 0.998 for manual and automated segmentation, respectively. Average intraobserver r² values were also calculated, based upon one operator repeating the segmentation three times on the same images, and found to be 0.996 and 0.999 for manual and automated segmentation, respectively. Average automated r² values for images from intrahepatic islet biopsy sites and from native pancreas, when analyzed separately, were both 0.999.

The degree of amyloidosis affecting all islets of a given biopsy was quantitated as amyloid severity calculated as the proportion of total islet area occupied by amyloid deposits (Σamyloid area/Σislet area × 100), and β-cell area was calculated similarly (Σinsulin area/Σislet area × 100) (25). Statistical comparisons of islet amyloid quantification parameters were performed by least squares linear regression using Origin software (Northampton, MA).

Islet function assessment

β-Cell secretory capacity was determined from glucose-potentiated arginine tests (26) performed serially in the two islet recipients with the longest duration of insulin-independent graft function and compared with results obtained from one insulin-independent recipient of autologous islets in the sc compartment and four normal control animals. Testing was conducted after an 18-h overnight fast under ketamine sedation. Bilateral saphenous vein catheters were placed for the infusion of test substances and blood sampling, respectively. After baseline blood samples at −5 and −1 min, arginine (0.07 g/kg) was injected over 1 min followed by blood sampling at 2, 3, 4, and 5 min. Then, at t = 10 min, a hyperglycemic clamp was conducted using a variable rate infusion of 20% dextrose solution administered via a syringe pump to achieve a blood glucose of approximately 250 mg/dl. The blood glucose concentration was determined every 5–10 min with an Elite XL glucometer (Bayer, Elkhart, IN) to adjust the infusion rate and achieve the desired glucose concentration. After 45 min of the dextrose infusion (at t = 55 min), arginine (0.07 g/kg) was injected again with identical blood sampling. All samples were collected on ice into tubes containing EDTA and protease inhibitor (Sigma, St. Louis, MO), centrifuged at 4 C, separated, and frozen at 80 C for subsequent analysis. Plasma glucose was determined on prestimulus samples in duplicate using the glucose oxidase method on an automated glucose analyzer (YSI 2300; Yellow Springs Instruments, Yellow Springs, OH). Plasma immunoreactive insulin was measured on pre- and poststimulus samples in duplicate by an ELISA that has been validated with an intraassay coefficient of variation 1.5% and an interassay coefficient of variation 2.3% in cynomolgus macaques (Mercodia, Uppsala, Sweden, previously distributed by ALPCO Diagnostics, Windham, NH) (27). Functional β-cell mass was estimated from the β-cell secretory capacity measured as the acute insulin response to glucose-potentiated arginine (AIR_pot) calculated as the mean of the three highest postinjection insulin levels minus the mean of the preinjection insulin levels under the hyperglycemic clamp condition (26).

Results

Islet transplant biopsies

As we have previously reported (23), immunologic rejection as evidenced by islet infiltration with T and/or B lymphocytes occurred in animals that received either ATG and rapamycin or ATG and rituximab and not in animals that received the combination of ATG, rituximab, and rapamycin (Table 3). Of the seven animals studied under double agent immunosuppression with early histologic evidence of islet lymphocytic infiltration, only one (T3973) exhibited islet amyloid deposition, this despite all the animals being hyperglycemic. Although one of these animals (01C0266) underwent biopsy out to 26 months, the remaining biopsies associated with immunologic rejection were performed within the first 4 months after transplant. In the one case (T3973) where islet amyloid was detected at 1 month after transplant in conjunction with lymphocytic infiltration, we speculate that amyloid may have already been present in the islets pretransplant, because they were derived from the oldest and largest donor (Table 1) (28). Unfortunately, donor pancreatic tissue was not obtained before islet isolation to evaluate for the presence of pretransplant amyloid.

Table 3.

Islet recipient characteristics at each time of biopsy assessment and islet biopsy results

Islet recipient	Immunosuppression	Months after transplant	Body weight (kg)	Glucose (mg/dl)	Insulin use (U/d)	Islet lymphocytes	Islet amyloid
01C008	ATG + rapa	1	2.3	370	5	Present	Absent
T3973	ATG + rapa	1	1.3	340	4.5	Present	Present
00C0759	ATG + rapa	1	1.6	150	1	Present	Absent
00C0087	ATG + rapa	2	2.7	350	5	Present	Absent
01C0266	ATG + ritux	9	4.8	52	0	Present	Absent
		15	4.5	168	2	Present	Absent
		25	4.0	378	5.5	Present	Absent
01C0298	ATG + ritux	1	1.5	207	2	Present	Absent
T3967	ATG + ritux	4	1.5	200	2	Present	Absent
211050	ATG + ritux + rapa	4	1.6	100	0	Absent	Absent
T4536	ATG + ritux + rapa	18	1.8	59	0	Absent	Present
		40	2.7	66	0	Absent	Present
		46	3.2	93	3.4	Absent	Present
01C0246	ATG + ritux + rapaa	2	2.7	65	0	Absent	Absent
93–100	ATG + ritux + rapaa	10	3.1	355	5	Presentb	Present
3845	ATG + ritux + rapaa	43	2.2	383	5.5	Presentb	Present
CR14	ATG + ritux + rapaa	14	2.4	74	0	Absent	Absent
		48	4.8	92	0	Absent	Present
		66	5.7	384	5.5	Absent	Present
210069 (sc)	None (autologous)	2	4.0	234	2	Absent	Absent
		8	4.7	280	5	Absent	Present
212027 (sc)	None (autologous)	2	3.7	48	0	Absent	Absent

rapa, Rapamycin; ritux, rituximab.

^aRapamycin discontinued at 200 d after transplant.

^bMild periislet lymphocytic infiltrates were present in these two animals after discontinuation of rapamycin.

Four of the six animals treated with ATG, rituximab, and rapamycin had no evidence of islet lymphocytic infiltration at the time of biopsy, with two animals demonstrating a mild periislet lymphocytic infiltrate in biopsies conducted after rapamycin had been discontinued at 200 d after transplant. Importantly, all four cases biopsied beyond 4 months after transplant demonstrated islet amyloid deposition that was initially detected at 10, 18, 43, and 48 months after transplant (Fig. 1). Although the two animals with mild periislet lymphocytic infiltrates (93–100 and 3845) were already hyperglycemic and receiving insulin at the time of their biopsy demonstrating amyloid, the other two animals (T4536 and CR14) were normoglycemic and insulin independent (Table 3). One of these animals (CR14) that had discontinued rapamycin had a negative biopsy for amyloid at 14 months and subsequently evidenced islet amyloid deposits by 48 months. One of the animals that received sc transplantation of autologous islets also evidenced islet amyloid deposits by 8 months after transplant (Table 3).

Fig. 1.

Intrahepatic islet transplant biopsies stained for insulin (red) and thioflavin S (green) are shown from recipient T4536 at posttransplant months 18 (A), 40 (B), and 46 (C), recipient 211050 at month 4 (D), recipient 93–100 at month 10 (E), recipient 3845 at month 43 (F), and recipient CR14 at months 14 (G), 48 (H), and 66 (I). Apparent is an increased accumulation of islet amyloid and decrease in islet β-cells when examining the same animals (A–C and G–I) or different animals (D–F) over time. Magnification, ×40. mo, Month.

IAPP immunoreactivity that colocalized with insulin within β-cells was detectable in samples from normal monkey and human pancreata (Table 2), as well as in amyloid negative intrahepatic monkey islets (Fig. 2). In the type 2 diabetic human and intrahepatic monkey islet biopsies with amyloid present by thioflavin S, there was IAPP immunoreactivity both within β-cells and extracellularly immediately surrounding β-cells in the region of the amyloid deposits (Fig. 2).

Fig. 2.

Native pancreas biopsies stained for insulin (red) and thioflavin S (green) are demonstrated from normal monkey (A), normal human (B), and type 2 diabetic human (C). Native pancreas biopsies stained for insulin (red) and IAPP (green) are also demonstrated from normal monkey (D), normal human (E), and type 2 diabetic human (F), where intracellular colocalization of insulin and IAPP appears orange. Only the type 2 diabetic pancreas with amyloid present by thioflavin S evidences additional IAPP present extracellularly immediately surrounding β-cells. Intrahepatic islet biopsies stained for insulin (red) and IAPP (green) similarly show intracellular colocalization of insulin and IAPP in a biopsy without amyloid (G), and intra- and extracellular IAPP in a biopsy with amyloid (H). In I, separate images for insulin (red, 1), IAPP (green, 2), and their combination (3) are shown.

Quantification of islet amyloid

Amyloid quantification was performed in biopsy samples from animals who received ATG, rituximab, and rapamycin and so did not have lymphocytic islet infiltration. Biopsy specimens were freshly cut and stained for insulin and amyloid; however, material was not available for 93–100 and the first biopsy of CR14. The median (range) number of islets detected on the intrahepatic biopsies was 3.5 (1–9), and on biopsies with amyloid present, all islets were involved, so that the amyloid prevalence denoted by the frequency of islets containing amyloid (number of islets containing amyloid/total number of islets × 100) was either 0 or 100%. This result is consistent with studies of amyloid deposition in native monkey pancreata, where if amyloid is present, all islets are invariably involved (29, 30). Figure 3 depicts the segmentation of the multispectral imaging for islet area, β-cell area, and amyloid area. Multispectral imaging analysis of the intrahepatic islet biopsy specimens gave a median (range) islet β-cell area of 36.2% (5.0–45.6%) and a median (range) amyloid severity 18.6% (0.0–60.6%). Negative controls included three normal monkey pancreases and four normal human pancreases with islet β-cell area ranging 26.1–68.0% and no amyloid detectable. Positive controls included three type 2 diabetic human pancreata with islet β-cell area ranging 21.5–38.8% and amyloid severity 4.4–40.2%. All intrahepatic islet biopsies identified as having amyloid present by visual inspection at ×40 power had at least 2% amyloid severity. A few biopsy specimens identified visually as being absent for amyloid had less than 0.3% amyloid severity. Amyloid severity increased with time after transplant (r = 0.68; P < 0.05), with increasing blood glucose (r = 0.94; P < 0.001), and with decreasing islet β-cell area (r = −0.68; P < 0.05) (Fig. 4, A–C). Decreasing islet β-cell area also inversely correlated by trend to increasing blood glucose (r = −0.57; P = 0.08) (Fig. 4D).

Fig. 3.

Images shown are multilayer composites created by recoloring and scaling spectrally unmixed label component planes. A, Mask overlay indicates where inForm pattern-recognition software detected islet tissue. B, Insulin segmentation mask indicates the detection of islet β-cells as green fill. C, Thioflavin S segmentation mask indicates the detection of islet amyloid as green fill, with the islet representing an intrahepatic islet affected with amyloid from monkey CR14 at 66 months after transplant. The corresponding panels D–F demonstrate the software-detected islet tissue, β-cells, and amyloid, respectively, from a normal human islet not affected with amyloid.

Fig. 4.

Relationship of time since transplantation to islet amyloid severity (A), the relationship of amyloid severity to blood glucose (B) and to insulin positive area (C), and the relationship of insulin positive area to blood glucose (D). Islet amyloid severity increased with further time from transplantation, and this increase in amyloid severity was associated with both increasing blood glucose and decreasing islet insulin positive area.

β-Cell secretory capacity

The AIR_pot in the two intrahepatic islet transplant recipients with the longest duration of insulin-independent graft function was initially close to the normal mean (159 ± 62 μU/ml, n = 4) but declined from 24 to 40 to 46 months in T4536 (from 139 to 44 to 24 μU/ml) (Fig. 5, A–C) and from 26 to 50 months in CR14 (from 86 to 25 μU/ml) (Fig. 5, D and E), coincident with increased accumulation of islet amyloid. Both animals became hyperglycemic and resumed insulin therapy either just before (T4536) or just after (CR14) their last functional islet assessment, suggesting an AIR_pot more than 25 μU/ml, which is to say an AIR_pot more than 16% of normal, may be required for insulin independence in macaques, a finding slightly lower than what has been reported for human intrahepatic islet transplant recipients (31, 32). Also, consistent with an AIR_pot threshold more than 25 μU/ml for insulin independence, the one sc autologous islet transplant recipient that was insulin independent at 18 months after transplant had an AIR_pot of 31 μU/ml (Fig. 5F).

Fig. 5.

Plasma insulin levels in response to the iv administration of 0.07 g/kg arginine under fasting and 250 mg/dl hyperglycemic clamp conditions during the glucose-potentiated arginine test, where the latter response gives AIR_pot. The shaded region gives the mean ± se range for insulin values obtained from four normal monkeys. A–C, Decline in AIR_pot in intrahepatic islet recipient T4536 from 24 to 40 to 46 months, by which time insulin therapy had been resumed for treatment of hyperglycemia. D and E, Decline in AIR_pot in intrahepatic islet recipient CR14 from 26 to 54 months, shortly after which time insulin therapy was also required. F, β-Cell secretory capacity of sc autologous islet recipient 212027 at 18 months after transplant while remaining insulin independent. mo, Month.

Discussion

These results indicate that in cynomolgus macaques, amyloid deposition occurs in intrahepatic transplanted islets, and its accumulation may be associated with subsequent decline in AIR_pot with eventual recurrence of hyperglycemia. Although the numbers of animals and biopsies studied are small, these results document a pattern of intrahepatic islet amyloid deposition that is shown to begin before the development of hyperglycemia in animals without islet graft rejection and progresses over time. The small number of islets available on each biopsy is another limitation to these data. However, unlike the situation in the diabetic human pancreas where the prevalence of amyloid positive islets can be quite variable (33, 34), when amyloid is present in native pancreata of nonhuman primates, all islets are invariably involved (29, 30), similar to our intrahepatic islet amyloid prevalence of either 0 or 100%. The severity of intrahepatic islet amyloid deposits was inversely correlated with the islet β-cell area, and in the two rejection-free animals with longitudinal assessments, intrahepatic islet amyloid was detected before a decrease in AIR_pot that predated the recurrence of hyperglycemia. This supports the development of intrahepatic islet amyloid as a mechanism for nonimmunologic islet graft loss.

Macaques of a variety of species [e.g. Macaca nigra (29); Macaca mulatta, also known as rhesus (30); and Macaca fascicularis, also known as cynomolgus (28)] have been shown to develop islet amyloidosis and diabetes that resembles type 2 diabetes in humans (35). In Macaca nigra, normal glucose-tolerant animals had 0–3% islet amyloid, those with impaired glucose tolerance approximately 30% islet amyloid, and those with overt diabetes approximately 60% islet amyloid (29), which was inversely correlated with reductions in both islet β-cell area and first-phase insulin responses during iv glucose tolerance tests (36); progression from normal to impaired glucose tolerance or from impaired to diabetic glucose tolerance in the same animals occurred over an approximately 5-yr period. In Macaca mulatta, young animals had no islet amyloid, about half that were older and overweight had less than 45% islet amyloid, and all those that were older and diabetic had approximately 60% islet amyloid, with islet β-cell area significantly reduced in the diabetic group (30). And in Macaca fascicularis, young animals had no islet amyloid, about a quarter of older animals had less than 50% islet amyloid, and all older diabetic animals were overweight and had more than 50% islet amyloid (28). Thus, in macaques, increasing age and body weight are associated with the development of native islet amyloidosis that precedes the development of hyperglycemia and overt diabetes, similar to the findings reported here in transplanted intrahepatic islets in cynomolgus macaques.

Transplanted human (37) and human IAPP transgenic mouse (38) islets have previously been reported to develop amyloidosis in rodent models. In nude mice, transplanted human islets developed amyloid in most grafts after 4 wk whether placed under the kidney capsule, intrahepatically via the portal vein, or directly implanted into the splenic parenchyma (37). Because amyloid deposits were found in both normoglycemic and hyperglycemic animals, their role in islet graft failure could not be established. Importantly, rodent IAPP is not amyloidogenic, and so human IAPP transgenic mice have been developed that can accumulate amyloid deposits associated with β-cell loss (25). Syngeneic mice rendered diabetic by streptozotocin and transplanted with 100 human IAPP transgenic or nontransgenic islets under the kidney capsule were normoglycemic 1 wk after transplant, but by 6 wk, half of the mice receiving the human IAPP transgenic islets developed recurrent hyperglycemia associated with progressive accumulation of amyloid in their transplanted islets (38). Importantly, that amyloid was detectable in the human IAPP transgenic islet grafts at 1 wk, before the recurrence of hyperglycemia (38) is consistent with our findings in cynomolgus macaques, where amyloid could be detected in intrahepatic islet grafts, while the animals remained normoglycemic and subsequently accumulated over time coincident with a decline AIR_pot and eventual recurrence of hyperglycemia.

The role of IAPP in the development of intrahepatic islet amyloid lesions in this nonhuman primate model is further supported by our findings of IAPP immunoreactivity both within β-cells and, in those biopsies that were positive for amyloid by thioflavin S, extracellularly immediately surrounding β-cells. A limitation, however, is that although we obtained similar results using two antibodies against IAPP known to recognize rat, cat, and human IAPP, neither antibody has been previously shown to recognize monkey IAPP. Nonetheless, that evidence of extracellular IAPP immunostaining was only seen in amyloid positive biopsies from human type 2 diabetic pancreata and in amyloidogenic monkey intrahepatic islets supports a role for IAPP in transplanted islet amyloidosis in monkeys as has also been shown in humans (9).

In our study, we were able to conduct a longitudinal histologic assessment on three animals. Monkey 01C0266 that developed intrahepatic islet lymphocytic infiltration by 9 months and was hyperglycemic by 15 months lost body weight during the course of islet graft rejection and did not develop islet amyloidosis, similar to the absence of islet amyloid in human autoimmune insulitis of type 1 diabetes (39). In contrast, monkeys T4536 and CR14 with no evidence of rejection progressively gained weight, even after becoming hyperglycemic with amyloid accumulated in their intrahepatic islets. We speculate that the growth of the animals after transplant may have led to an increased secretory demand on the transplanted islets leading to inappropriate IAPP secretion. Our group has shown in type 1 diabetic human islet transplant recipients with an AIR_pot only approximately 22% of normal that intrahepatically transplanted islets secrete disproportionately more IAPP relative to insulin than is normal under hyperglycemic clamp conditions (10). Because appropriate proportions of insulin and other factors in the β-cell are required to inhibit IAPP fibril formation (6–8), disproportionately increased IAPP secretion in settings of increased β-cell secretory demand may lead to amyloid formation. Indeed, it has recently been shown in human IAPP transgenic mouse islets cultured under hyperglycemic conditions that increasing IAPP secretion by administering exendin-4 or potassium chloride increases amyloid deposition, whereas decreasing IAPP secretion with diazoxide or somatostatin has the opposite effect (40). In the present study, islets came from donor animals that were larger and so older than the recipients, which may explain their inability to compensate for growth of the animal and is consistent with the association of increasing age and body weight with native islet amyloidosis and diabetes in cynomolgus monkeys and other macaques (28). Longitudinal evaluation of additional animals will be required to establish that intrahepatic islet amyloid development can lead to transplanted islet graft failure.

Neither the liver as an engraftment site nor immunosuppression drugs can be held responsible for the development of transplanted islet amyloid. As discussed above, human islets transplanted under the kidney capsule or in the spleen of nude mice (not treated with any immunotherapy) deposit just as much amyloid as those placed intrahepatically (37). We also demonstrated amyloid accumulation in autologous islets transplanted in the sc space of an animal after pancreatectomy that also did not receive immunosuppression. And in monkey CR14 that discontinued rapamycin therapy and remained rejection free (and so immunologically tolerant to its islet graft), no amyloid was detected 8 months after stopping rapamycin but developed subsequently in the absence of any ongoing immunotherapy. Thus, an increased β-cell secretory demand on the transplanted islets leading to inappropriate IAPP secretion remains the most likely mechanism to explain the development of transplanted islet amyloid. To evaluate this hypothesis further, future studies of rodent and nonhuman primate models of islet transplantation and amyloidosis should determine whether increasing the islet dose transplanted may alleviate increased β-cell secretory demand and ameliorate the development of transplanted islet amyloid.

Other approaches to combat the development of islet amyloid relevant to both transplantation and type 2 diabetes have involved ex vivo suppression of IAPP expression (41) and in vitro inhibition of IAPP fibril formation (42). Translation of these approaches to the transplantation of islets for type 1 diabetes or the prevention of type 2 diabetes in humans will require in vivo investigation using animal models starting with human IAPP transgenic mice followed by preclinical testing in a macaque model such as that reported here. An alternative solution to the problem of transplanted islet amyloidosis may be the use of islets whose IAPP is not amyloidogenic, such as porcine islets (43, 44). Porcine IAPP, like rodent IAPP, contains sequence substitutions in the region corresponding to residues 20–29 that render it resistant to IAPP fibril formation (44, 45). Intrahepatically transplanted porcine islets in an autograft model maintained normoglycemia in young pigs that experienced a 50% increase in body weight over an 18-month period without the development of intrahepatic islet amyloid (43). Importantly, advances in immunosuppression agents have protected intrahepatically transplanted porcine islets from rejection in streptozotocin-induced diabetic cynomolgus macaques that have demonstrated prolonged diabetes reversal (46, 47).

In conclusion, intrahepatic transplanted islets in cynomolgus macaques accumulate amyloid deposits in the absence of rejection coincident with animal growth that increases the β-cell secretory demand placed on the initially engrafted islet β-cell mass. The presence of amyloid predates a decline in AIR_pot, accumulates over time with decreasing islet β-cell area, and is associated with subsequent recurrence of hyperglycemia. Although the IAPP fibrils of amyloid deposits have been shown to exhibit direct β-cell toxicity (3), it remains possible that additional mechanisms resulting from islet β-cell overstimulation, such as endoplasmic reticulum stress or the generation of reactive oxygen species, may also contribute to islet stress and graft loss after transplant. Nonetheless, the identification of transplanted islet amyloid can serve as a marker of islet stress, and future studies will need to determine whether the prevention of IAPP fibrillogenesis itself is sufficient to prolong normoglycemia in islet transplant recipients, or whether reducing metabolic demand through the provision of exogenous insulin or increasing the engrafted islet β-cell mass will be necessary. The development of amyloid in transplanted islets over a period of months rather than years in native monkey pancreases (29) makes a nonhuman primate transplant model such as that reported here attractive for preclinical testing of new therapeutic approaches to prevent the development of islet amyloid in both transplantation and type 2 diabetes.