Exendin-4 Improves Reversal of Diabetes in NOD Mice Treated with ...

0013-7227/07/$15.00/0 Printed in U.S.A.

Endocrinology 148(11):5136 –5144 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2007-0358

Exendin-4 Improves Reversal of Diabetes in NOD Mice Treated with Anti-CD3 Monoclonal Antibody by Enhancing Recovery of ␤-Cells Nicole A. Sherry,* Wei Chen,* Jake A. Kushner, Mariela Glandt, Qizhi Tang, Sue Tsai, Pere Santamaria, Jeffrey A. Bluestone, Anne-Marie B. Brillantes, and Kevan C. Herold Departments of Immunobiology and Medicine (W.C., M.G., A.-M.B.B., K.C.H.), Yale University, New Haven, Connecticut 06520; Department of Pediatrics and the Naomi Berrie Diabetes Center (N.A.S., K.C.H.), College of Physicians and Surgeons, Columbia University, New York, New York 10032; Children’s Hospital of Philadelphia (J.A.K., A.-M.B.B.), Division of Endocrinology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19019; Diabetes Center (Q.T., J.A.B.), Department of Medicine, University of California at San Francisco, San Francisco, California 94143; and Julia McFarlane Diabetes Research Centre and Department of Microbiology and Infectious Diseases (S.T., P.S.), University of Calgary, Calgary, Alberta, Canada T2N 1N4 Immune modulators can arrest loss of insulin secretion in type 1 diabetes mellitus (T1DM), but they have not caused permanent disease remission or restored normal insulin secretion. We tested whether exendin-4, a glucagon-like peptide-1 receptor agonist, would enhance remission of T1DM in NOD mice treated with anti-CD3 monoclonal antibody (mAb) and studied the effects of exendin-4 treatment on cellular and metabolic responses of ␤-cells. Diabetic NOD mice treated with anti-CD3 mAb and exendin-4 had a higher rate of remission (44%) than mice treated with anti-CD3 mAb alone (37%) or exendin-4 (0%) or insulin or IgG alone (0%) (P < 0.01). The effect of exendin-4 on reversal of diabetes after anti-CD3 mAb was greatest in mice with a glucose level of less than 350 mg/dl at diagnosis (63 vs. 39%, P < 0.05). Exendin-4 did not affect

R

ECENT CLINICAL TRIALS have shown that treatment of patients with new onset type 1 diabetes mellitus (TIDM) with anti-CD3 monoclonal antibody (mAb) can prevent the loss of insulin production for at least 1 yr after diagnosis (1–3). However, the long-term clinical impact of the mAb treatment is uncertain because both published studies showed deterioration of C-peptide responses beyond the first year after the single course of treatment. In addition, not all patients responded to the drug treatment. In the European study, the clinical response to drug treatment was most apparent in those subjects in whom the magnitude of the Cpeptide response to a glucose clamp at diagnosis was in the upper half of C-peptide responses of all subjects at diagnosis (3). This is reminiscent of the experience with cyclosporine A where the C-peptide level at entry was a predictor of clinical response to treatment (4). This combined experience suggests that improved ␤-cell function at the time of diagnosis is an

First Published Online August 2, 2007 * N.A.S. and W.C. contributed equally to this publication. Abbreviations: ALS, Antilymphocyte serum; DAPI, 4⬘,6-diamidino2-phenylindole; GLP-1, glucagon-like peptide-1; GLUT-2, glucose transporter-2; IPGTT, ip glucose tolerance test; mAb, monoclonal antibody; NRP-V7, NOD-relevant V7 peptide; T1DM, type 1 diabetes mellitus. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

␤-cell area, replication, or apoptosis or reduce the frequency of diabetogenic or regulatory T cells or modulate the antigenicity of islet cells. Reversal of T1DM with anti-CD3 mAb was associated with recovery of insulin in glucose transporter-2ⴙ/insulinⴚ islet cells that were identified at diagnosis. Glucose tolerance and insulin responses improved in mice treated with combination therapy, and exendin-4 increased insulin content and insulin release from ␤-cells. We conclude that treatment with glucagon-like peptide-1 receptor agonist enhances remission of T1DM in NOD mice treated with anti-CD3 mAb by enhancing the recovery of the residual islets. This combinatorial approach may be useful in treatment of patients with new-onset T1DM. (Endocrinology 148: 5136 –5144, 2007)

important determinant of response to treatment with immune therapy, and therefore strategies that improve ␤-cell mass and/or function may have a positive effect on response to immune treatment. Glucagon-like peptide-1 (GLP-1) and its related peptide exendin-4 have been shown to augment glucose-stimulated insulin release, decrease levels of glucagon, and delay gastric emptying (5, 6). In addition, it has been shown to increase ␤-cell replication, decrease ␤-cell apoptosis, stimulate ␤-cell neogenesis, and induce expansion of ␤-cell mass in rodent models (7–12). These cellular effects of exendin-4 and other GLP-1 receptor agonists may be useful to expand ␤-cell mass in the setting of immune tolerance (13–16). Indeed, it is conceivable that after induction of immune tolerance, a GLP-1 receptor agonist might promote recovery of ␤-cell mass. Ogawa et al. (17) found that the addition of exendin-4 to antilymphocyte serum (ALS) enhanced the reversal of diabetes in hyperglycemic NOD mice compared with treatment with ALS alone. In these studies, it was suggested that the combination therapy enhanced ␤-cell replication because the insulin content of pancreases from the mice treated with both agents was increased compared with diabetic mice, but the rates of ␤-cell replication and ␤-cell mass were not directly measured. To determine whether a GLP-1 receptor agonist will enhance the effects of anti-CD3 mAb in autoimmune diabetes,

5136

Sherry et al. • Reversal of T1DM with Exendin-4 and Anti-CD3 mAb

we studied the combination of exendin-4 and anti-CD3 mAb treatment in NOD mice with new-onset T1DM. Previous studies of anti-CD3 mAb in this setting have shown that diabetes can be reversed and immunological tolerance to the autoimmune disease can be induced (18, 19). We have therefore tested whether the addition of exendin-4 to the treatment regimen enhances reversal of diabetes and determined the mechanisms that are responsible for this effect. Our studies show that the combination of anti-CD3 mAb and exendin-4 induces remission in a higher proportion of mice than treatment with either agent alone. However, we found that the effect of exendin-4 is to enhance revival of degranulated ␤-cells and to increase total ␤-cell insulin rather than to stimulate ␤-cell regeneration, decrease ␤-cell apoptosis, or cause immune modulation. Materials and Methods

Endocrinology, November 2007, 148(11):5136 –5144

5137

in liquid nitrogen, and then kept at ⫺70 C. Insulin was extracted by mechanical homogenization in 2 ml iced acid ethanol (0.7 m HCl/ethanol 25:75 ⫹ 0.1% Triton X-100). After 24 h at 4 C, samples were centrifuged, and the supernatants were collected. Insulin concentration of samples was measured by mouse ultrasensitive insulin ELISA (Alpco Diagnostics, Salem, NH), and total protein content was determined with the 2D Quant kit (GE Healthcare, Piscataway, NJ).

Immunohistochemical analysis Pancreata were resected, weighed, fixed, and embedded in paraffin. Noncontiguous 5-␮m pancreatic sections were stained with antibodies to insulin, 4⬘,6-diamidino-2-phenylindole (DAPI), Ki67, or active caspase-3 and counterstained with hematoxylin. The bound antibodies were detected by immunofluorescent secondary antibodies (Vectastain; Vector Laboratories, Burlingame, CA). The anti-Ki67 and anti-cleaved caspase-3 antibodies were purchased from Novocastra, Inc. (Newcastle upon Tyne, UK) and Trevigen, Inc. (Gaithersburg, MD), respectively. To improve the detection of ␤-cells before and after treatment, additional immunohistochemical staining was performed involving detection of insulin and glucose transporter-2 (GLUT-2) as described (21).

Animals Female NOD/LtJ mice and NOD/scid mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and maintained under pathogenfree conditions. NOD mice were screened for glycosuria three times per week beginning at 12 wk of age and diagnosed with diabetes when two random glucose levels were more than 200 mg/dl (20). Glucose levels were measured in whole blood from the tail vein with the Glucometer Elite XL (Bayer, Elkhart, IN). Animal use was approved by the Columbia and Yale University Animal Use Committees.

Treatment with anti-CD3 mAb and exendin-4 Diabetic NOD mice were treated with mAb 145-2C11 (antimouse CD3), 10 ␮g/d iv for 5 d, and/or exendin-4 (Bachem, King of Prussia, PA), 0.075 ␮g ip for 10 d, beginning at the onset of hyperglycemia (9, 10, 19). Control diabetic mice received hamster IgG and/or exendin-4. Reversal of diabetes was considered to have occurred when random glucose levels were less than 200 mg/dl 3 wk after diagnosis. Some mice were followed for longer periods of time to establish the duration of treatment effects. To determine the effects of increased insulin levels on the reversal of diabetes, certain diabetic mice received an insulin pellet (Linsen, Toronto, Canada) implanted sc at the time of diagnosis of diabetes and at the time of initiation of treatment with anti-CD3 mAb. The insulin pellet was removed after 2 wk, and the glucose levels were followed.

Adoptive transfer of diabetes into NOD/scid mice To determine the effect of exendin-4 treatment on islet cells in vivo, a single-cell suspension was prepared of splenocytes from diabetic NOD mice, and 10 ⫻ 106 cells were administered iv to NOD/scid recipients. Recipient mice were treated with exendin-4 0.075 ␮g ip for 10 d beginning 3 d before the transfer of splenocytes, and the rate of diabetes (glucose ⬎ 200 mg/dl) was compared with cell recipients not treated with exendin-4.

Glucose tolerance tests After a 3-wk observation period after diagnosis and treatment, a subset of mice, selected at random, underwent an ip glucose tolerance test (IPGTT) after a 14- to 16-h fast. One milligram dextrose per gram body weight was injected ip, and glucose levels were measured in whole blood from the tail vein taken before and 15, 30, and 60 min after the injection. Murine glucagon and insulin levels were measured by Luminex bead assay and ELISA, respectively (Linco, St. Louis, MO).

Pancreatic insulin content To determine the effects of exendin-4 on pancreatic insulin content, pancreases were harvested from diabetic NOD mice 2 wk after treatment with anti-CD3 mAb or anti-CD3 mAb and exendin-4. Pancreata were collected after overnight fasting, immediately frozen

Measurement of ␤-cell area, ␤-cell replication, and ␤-cell apoptosis Previously published methods were used to determine ␤-cell area (20). Five to 16 NOD mice from each treatment group, selected at random, were studied. Insulin-positive and total pancreatic areas were calculated from eight to 10 noncontiguous sections from pancreas using a Nikon light microscope, Spot Insight digital imaging camera, and Image-Pro Plus software. ␤-Cell area was determined by calculating the total insulin-positive area/average area of a single insulin⫹ cell/insulinnegative area in the pancreatic section and reported as the number of ␤-cells per square millimeter of pancreatic area. To determine the percentage of replicating and apoptotic ␤-cells, images were taken of pancreatic sections stained with antibody to insulin, DAPI, Ki67, or active caspase-3. The percent replication or apoptosis was defined as the number of Ki67⫹/insulin⫹ cells or caspase⫹/insulin⫹ cells/total insulin⫹ cells ⫻ 100. All of the nuclei in at least four pancreatic sections or the first 1000 ␤-cells were counted.

Immunological studies Identification of regulatory T cells. Two to five islets from mice treated with anti-CD3 mAb alone or in combination with exendin-4 that did and did not correct diabetes were stained with rabbit antimouse Foxp3 (kindly provided by Roli Khattri) followed by biotin-labeled secondary antibody and peroxidase-labeled streptavidin (Vectastain ABC kit). The sections were counterstained with hematoxylin and analyzed using Spot Insight digital imaging camera and Image-Pro Plus software for the percentage of total intra- and peri-islet lymphocytes expressing Foxp3, a marker of regulatory T cells. Enumeration of diabetogenic CD8⫹ T cells. Splenocytes from new-onset diabetic NOD mice treated with either anti-CD3 mAb alone or anti-CD3 mAb and exendin-4 were isolated and stained with NOD-relevant V7 peptide (NRP-V7) or control TUM tetramer and antibody to CD8 (22). The percentage of CD8⫹/NRP-V7⫹ cells was measured by FACS analysis.

Studies of exendin-4-treated ␤-cells in vitro The effects of exendin-4 on ␤-cell functions, insulin release, and insulin content were evaluated in islets from adult NOD/scid mice. Islets were isolated from 10- to 14-wk-old NOD/scid mice. Twenty islets were placed on a culture plate insert (Millicell-CM; Millipore, Billerica, MA) and kept in culture medium RPMI 1640 supplemented with 10% fetal bovine serum, 10 mm nicotinamide, l-glutamine, and antibiotics at 37 C and 5% CO2 (23). After overnight rest, the islets were treated with or without exendin-4 (1 ␮g/ml) for 24 h. Subsequently, the islets were washed twice with culture medium and placed back into culture. At the indicated time, insulin release was measured in culture supernatants after 1 h incubation with Krebs-Ringer solution [129 mm NaCl, 4.8 mm


Anti-CD3 mAb Anti-CD3 mAb

A

10

20

30

40

50

60

70

80

650 600 550 500 450 400 350 300 250 200 150 100 50 0

0

20

10

30

Day after diagnosis

D

Anti-CD3 mAb + Ex-4

Anti-CD3 mAb + Ex-4

650 600 550 500 450 400 350 300 250 200 150 100 50 0

Glucose (mg/dl) 0

10

20

30

40

80

50

60

70

70

80

Anti-CD3 mAb + Ex-4

80

0

10

60

50

40

30

20

Day after diagnosis

Day after afterdiagnosis diagnosis Day

E

F

Anti-CD3 mAb + insulin

*

75

550 500 450 400 350 300 250 200 150 100 50 0

R ate of rem is s ion

60 45

All mice

30

Gluc < 350mg/dl

15

n-

4

G

4

di en

Ab m

CD an

ti-

CD

3

tian

Ex

40

/I g

30

Day after diagnosis

x-

20

+E

10

3

0

Ab

0 m

Glucose (mg/dl)

70

650 600 550 500 450 400 350 300 250 200 150 100 50 0

l in

Glucose (mg/dl)

C

60

50

40

Day after diagnosis

su

0

Anti-CD3 mAb+Ex-4 Anti-CD3 mAb + Ex-4

B

650 600 550 500 450 400 350 300 250 200 150 100 50 0

Glucose (mg/dl)

Glucose (mg/dl)


In

5138

FIG. 1. Remission of diabetes by treatment with insulin, exendin-4, and/or anti-CD3 mAb. At the time of diagnosis of diabetes, NOD mice were treated with exendin-4 (n ⫽ 8), anti-CD3 mAb (n ⫽ 71), anti-CD3 mAb and exendin-4 (n ⫽ 45), or insulin or hamster IgG (n ⫽ 7) or with anti-CD3 mAb and a sc implanted insulin pellet (n ⫽ 11). The glucose levels in the mice treated with anti-CD3 mAb with (B–D) or without (A and F) exendin-4 are shown. The glucose levels in all mice (A and B) and with glucose levels less than 350 mg/dl (C) or more than 350 mg/dl (D) at diagnosis are shown. E, The rate of correction of diabetes was similar in mice that received anti-CD3 mAb and an insulin pellet implant at the time of diagnosis compared with mice treated with anti-CD3 mAb alone. When the insulin pellet was removed (arrow), reversal of diabetes was found at 3 wk in three of 11 mice. F, The rates of remission (glucose level ⬍ 200 mg/dl) in the treatment groups 3 wk after diagnosis are shown. Overall, a significantly greater proportion of mice treated with anti-CD3 mAb alone or with exendin-4 showed reversal of diabetes (␹2, P ⬍ 0.01) compared with mice treated with insulin (sc or with an implant) or exendin-4. In addition, the remission rate of diabetes was greater in mice treated with anti-CD3 mAb with exendin-4 (n ⫽ 32) compared with those treated with anti-CD3 mAb alone (n ⫽ 57) in whom the glucose level was less than 350 mg/dl at diagnosis; *, ␹2, P ⬍ 0.05.


We determined the total ␤-cell area among the treatment groups and between mice that did and did not show correction of diabetes. The ␤-cell area was greater in mice that 10 *** **

**

1

all mice Diabetes remission

0.1

Persistent hyperglycemia

0.01

/s ci d O D

N

on

se t

in ew

N

In su l

-4

tiC

D3 +E x

m Ab

0.001

D3

The response to treatment with anti-CD3 mAb or antiCD3 mAb ⫹ exendin-4 was generally seen within 3 wk after onset of diabetes and treatment and did not change with longer follow-up (Fig. 1, A–D). In mice that showed a correction of diabetes (defined as a glucose ⬍ 200 mg/ dl), the time to correction of diabetes was statistically greater but not clinically meaningful in mice that were treated with anti-CD3 mAb ⫹ exendin-4 compared with mice treated with anti-CD3 mAb alone (15 ⫾ 0.8 days vs. 12 ⫾ 1.4 d, P ⬍ 0.05). We therefore compared the rates of correction between the treatment groups 3 wk after diagnosis of diabetes (Fig. 1E). A greater proportion of mice showed correction of diabetes after treatment with antiCD3 mAb with exendin-4 (20 of 45, 44%) or anti-CD3 mAb alone (26 of 71, 37%) compared with treatment with IgG (zero of seven), insulin (zero of five), or exendin-4 alone (zero of eight) (P ⬍ 0.01). When all of the mice were considered, the difference in the rate of correction of diabetes between mice treated with anti-CD3 mAb ⫹ exendin-4 did not reach statistical significance compared with mice treated with anti-CD3 mAb alone. This was due to mice with the highest levels of glucose (i.e. ⬎350 mg/dl) at diagnosis, which generally failed to respond to treatment with anti-CD3 mAb with or without exendin-4. In these mice (n ⫽ 31), the overall the rate of correction of diabetes was 19%, 29% in the mice treated with anti-CD3 mAb alone (n ⫽ 21) and 0% (n ⫽ 10) in the mice treated with anti-CD3 mAb ⫹ exendin-4. In these mice, ␤-cell function or mass may have been severely compromised, and recovery of ␤-cell function may not have been possible. Indeed, when mice with glucose levels less than 350 mg/dl at diagnosis were considered (n ⫽ 89), there was overall a higher rate of correction of diabetes (47%), and there was a significant effect of exendin-4 treatment on the correction of diabetes when added to anti-CD3

Effects of exendin-4 on ␤-cell dynamics

an

Results Correction of diabetes after treatment with anti-CD3 mAb and/or exendin-4

tiC

The data are expressed as the mean ⫾ sem. The glucose and insulin responses to the IPGTT were compared by taking the average of the fasting and maximal values for each test. Data from treatment groups of mice were compared with a Student’s t test. The frequency of the response to treatment between groups was compared by a ␹2 test or by Fisher’s exact test for comparison of two groups. A P value of ⬍0.05 was considered to be of statistical significance.

Beta cell area (mm2)

Statistical analysis

5139

mAb (63%, n ⫽ 32, vs. 39%, n ⫽ 57; P ⬍ 0.05) (Fig. 1E). This suggests that exendin-4 is most effective when there is residual ␤-cell mass or function at the time that therapy is initiated. It was possible that the improved response to the antiCD3 mAb treatment with exendin-4 was due to improved glucose levels and/or higher insulin levels as a result of the exendin-4 treatment rather than a direct effect of the drug on islet or immune cells. We therefore also studied the rate of correction of diabetes in hyperglycemic mice treated with anti-CD3 mAb that received an insulin pellet implant (Fig. 1F). The insulin pellet was implanted at the time of diagnosis of diabetes. Two weeks after diagnosis and after treatment with anti-CD3 mAb and the insulin pellet, the glucose level had fallen from 387 ⫾ 9.4 mg/dl (n ⫽ 11) to 171 ⫾ 39 mg/dl (n ⫽ 11; P ⬍ 0.001). The insulin pellet was removed, and when the glucose levels were measured 1 wk later, the average glucose level was 261 ⫾ 36 mg/dl. Similar to mice treated with anti-CD3 mAb alone, 27% (three of 11) of the mice showed reversal of diabetes.

an

KCl, 2.5 mm CaCl2, 1.2 mm KH2PO4, 1.2 mm MgSO4, 5.5 mm NaHCO3, 10 mm HEPES, 0.1% (wt/vol) BSA] containing either 1.7 mm glucose (basal level) or 17 mm glucose (stimulation level). For insulin content analysis, the islet extracts were prepared using an acid/ethanol method with some modifications (24). The insulin concentrations in culture supernatants or islet extracts were measured using the mouse insulin ELISA kit (Alpco Diagnostics). To correct for differences in islet size, the DNA content of islets was measured using the Quant-It PicoGreen dsDNA kit (Invitrogen, Carlsbad, CA), and the insulin concentrations in the supernatants and insulin content of the islets was expressed as nanograms of insulin per nanogram DNA. The stimulation index of insulin release in response to glucose was calculated by dividing the insulin concentration in stimulation supernatants by the insulin concentration in the basal supernatants.


FIG. 2. Cell area in NOD mice in the treatment groups: ␤-Cell area was measured in NOD mice at the onset of diabetes (n ⫽ 5), in NOD/scid control mice (n ⫽ 4), and in diabetic NOD mice 3 wk after treatment with anti-CD3 mAb (n ⫽ 16), anti-CD3 mAb and exendin-4 (n ⫽ 12), or insulin (n ⫽ 5). There was a significant increase in ␤-cell area in diabetic NOD mice treated with anti-CD3 mAb with or without exendin-4 compared with mice treated with insulin (**, P ⫽ 0.002). The ␤-cell area in both of these treatment groups is less than in NOD/scid controls (***, P ⬍ 0.001). The mice treated with anti-CD3 mAb and anti-CD3 mAb ⫹ exendin-4 were classified according to their response to treatment (anti-CD3 mAb responders, n ⫽ 10, nonresponders, n ⫽ 6; anti-CD3 mAb with exendin-4 responders, n ⫽ 6, nonresponders, n ⫽ 6). There was a significant difference in the ␤-cell area in the mice treated with anti-CD3 mAb in responders vs. nonresponders, but the difference in the area between mice that were treated with anti-CD3 mAb and exendin-4 that did and did not correct diabetes did not reach statistical significance (P ⫽ 0.09). Among the mice with correction of diabetes, the cell area was greater in mice treated with anti-CD3 mAb alone compared with those treated with both anti-CD3 and exendin-4 (P ⫽ 0.03).


FIG. 3. Cell replication in NOD mice in the treatment groups: A, Costaining with Ki67⫹/ insulin and was used to identify proliferating ␤-cells. There was a significant increase in ␤-cell proliferation in mice with new-onset diabetes (n ⫽ 5) compared with NOD/scid control mice (n ⫽ 4; **, P ⱕ 0.02), as well as in mice treated with anti-CD3 mAb (n ⫽ 14) or anti-CD3 mAb ⫹ exendin-4 (n ⫽ 10). ␤-Cell proliferation was significantly higher in newonset mice compared with mice treated with anti-CD3 mAb alone or anti-CD3 mAb and exendin-4 (**, P ⫽ 0.02). ␤-Cell proliferation was significantly lower in mice treated with anti-CD3 mAb ⫹ exendin-4 compared with mice treated with anti-CD3 mAb alone (***, P ⬍ 0.01). ␤-Cell proliferation was similar in mice with remission of diabetes (gray bars) compared with those with persistent hyperglycemia (white bars) that had been treated with anti-CD3 mAb or anti-CD3 mAb ⫹ exendin-4, but was lower in mice treated with anti-CD3 mAb alone, with persistent hyperglycemia (*, P ⬍ 0.05).


6 **

P ercent K i67+ beta cells

5140

5 4

***

all mice Diabetes remission Persistent hyperglycemia

*

3 2 1 0

Anti-CD3 mAb

had received anti-CD3 mAb or anti-CD3 mAb with exendin-4 regardless of whether there was correction of hyperglycemia compared with mice treated with insulin and IgG (P ⫽ 0.002; Fig. 2). The ␤-cell area was also greater in mice that showed correction of hyperglycemia (0.29 ⫹ 0.05 cells/ mm2, n ⫽ 16 mice) compared with those that did not (0.08 ⫹ 0.016 cells/mm2, n ⫽ 12 mice; P ⫽ 0.001), whether they were treated with anti-CD3 mAb alone or with exendin-4, but the addition of exendin-4 did not significantly affect the ␤-cell area in mice treated with anti-CD3 mAb. In previous studies, we and others have reported that the rate of ␤-cell proliferation increases as NOD mice develop diabetes and is significantly greater than in NOD/ scid mice that do not develop insulitis (20, 25). Accordingly, we found that the rate of ␤-cell proliferation was increased in NOD mice with new-onset disease compared with NOD/scid control mice (P ⫽ 0.01; Fig. 3). After 3 wk, there was a significant reduction in the rate of ␤-cell proliferation in mice treated with anti-CD3 mAb or anti-CD3 mAb ⫹ exendin-4 when compared with mice with newonset disease (P ⬍ 0.01; Fig. 3). Overall, the rate of ␤-cell proliferation was significantly greater in mice treated with anti-CD3 mAb compared with those receiving anti-CD3 mAb with exendin-4 (P ⬍ 0.01), but in the mice that showed remission of diabetes, the rates of proliferation were not different between the mice treated with anti-CD3 mAb that did and did not receive exendin-4. In mice with persistent hyperglycemia, the rates of ␤-cell proliferation were higher in mice that were treated with anti-CD3 mAb compared with those treated with anti-CD3 mAb and exendin-4 (P ⬍ 0.03). The rates of ␤-cell apoptosis were similar in mice treated with anti-CD3 mAb and anti-CD3 mAb with exendin-4 (Fig. 4). Caspase-3 staining was greater in mice with newonset diabetes than in mice treated with anti-CD3 mAb or anti-CD3 mAb and exendin-4 (P ⬍ 0.001). When the mice that did and did not show remission of diabetes were considered together, there was not a significant difference in the proportion of ␤-cells that were caspase-3⫹ between

Anti-CD3 mAb+Ex-4

New onset

NOD/scid

the mice treated with anti-CD3 mAb ⫹ exendin-4 (54.7 ⫾ 7.6%) or anti-CD3 mAb alone (45.2 ⫾ 7.4%). There was a significantly reduced proportion of caspase-3 staining in sections from mice that did (35.9 ⫹ 5.7%, n ⫽ 12 mice) compared with those that did not (63.0 ⫹ 7.5%, n ⫽ 11 mice) correct diabetes (P ⬍ 0.01), but in the mice with diabetes remission, the rates were not significantly different between those treated with anti-CD3 mAb with exendin (48.8 ⫹ 10, n ⫽ 5 mice) compared with those treated with anti-CD3 mAb alone (28.6 ⫹ 4.64%). Effects of exendin-4 treatment on immune responses

These data indicate that exendin-4 improved the remission of diabetes after anti-CD3 mAb treatment, but this effect could not be accounted for by an effect on ␤-cell area, death, or replication. We considered whether exendin-4 could modify the islets themselves, thereby affecting their antigenicity, or in some way affect the autoimmune response as has been recently suggested (26). NOD/scid mice that were treated with exendin-4 developed diabetes at the same rate as NOD/ scid control mice when they received splenocytes from diabetic NOD mice (Fig. 5). We also enumerated the frequency of a prevalent population of autoreactive CD8⫹ T cells that recognize residues 206 –214 of islet-specific glucose 6 phosphatase catalytic subunit-related protein (IGRP) in the spleen using NRP-V7Kd tetramers (22, 27–29). The number of tetramer-positive T cells was modestly higher in mice that did not show correction of diabetes compared with mice that did (14.1 ⫾ 3.04%, n ⫽ 12 mice, vs. 6.76 ⫾ 1.76%, n ⫽ 5 mice; P ⫽ 0.05) but the percentage of tetramer-positive cells was similar in mice treated with anti-CD3 mAb and exendin-4 compared with those treated with anti-CD3 mAb alone. Finally, we compared the number of Foxp3 regulatory T cells in the islets of mice treated with anti-CD3 mAb and anti-CD3 mAb ⫹ exendin-4 (30). Interestingly, there was a significantly greater percentage of Foxp3 in mice treated with anti-CD3 mAb alone compared with those treated with the combination, although the differences were modest [anti-CD3 mAb, 6.29 ⫾


** **

60

all mice Diabetes remission

40

Persistent hyperglycemia

20

0

anti-CD3 mAb

0.81% Foxp3⫹ lymphocytes per islet (n ⫽ 7), vs. anti-CD3 mAb ⫹ exendin-4, 4.18 ⫾ 0.6% Foxp3⫹ lymphocytes per islet (n ⫽ 11); P ⬍ 0.05]. Thus, these studies do not suggest that exendin-4 improves the remission of diabetes after anti-CD3 mAb treatment by affecting the immune response. Enhanced ␤-cell recovery and pancreatic insulin content accounts for the improvement in glucose tolerance and correction of diabetes when exendin-4 is added to anti-CD3 mAb

We previously reported that insulin-negative ␤-cells could be found in the islets of NOD mice at the time of diagnosis of diabetes by staining with anti-GLUT-2 mAb (20). We therefore examined islets after treatment with anti-CD3 mAb with or without exendin-4 to determine whether recovery of these cells could account for the improvement in ␤-cell area after treatment with anti-CD3 mAb. Similar to our previous observations, we identified cells in the islets of mice at the time of diagnosis of diabetes that did not stain for insulin but did stain for GLUT-2 (Fig. 6A). Three weeks later, after treatment with anti-CD3 mAb, we were unable locate GLUT-2Percent with diabetes

100

5141

80

Percent Caspase+ beta cells

FIG. 4. ␤-Cell apoptosis in NOD mice. A, Apoptotic ␤-cells were identified by staining for active caspase-3 and insulin. The percentage of apoptotic ␤-cells was increased in NOD mice with new-onset disease (n ⫽ 5) and mice treated with antiCD3 mAb (n ⫽ 14) or anti-CD3 mAb ⫹ exendin-4 (n ⫽ 9) compared with NOD/ scid control mice (n ⫽ 5; **, all P ⬍ 0.01). The frequency of apoptotic cells was lower in mice treated with anti-CD3 mAb with remission of diabetes (gray bars, n ⫽ 7) compared with those treated with antiCD3 mAb but with persistent hyperglycemia (white bars, n ⫽ 7; P ⬍ 0.01). In the mice treated with anti-CD3 mAb alone, there was a significantly reduced proportion of caspase-3⫹ ␤-cells in mice in which diabetes was corrected compared with those in which diabetes was not corrected (P ⬍ 0.01).


anti-CD3 mAb+Ex-4

New onset

NOD/scid

positive cells that were insulin negative. This was similar in mice treated with anti-CD3 mAb whether or not exendin-4 had been administered (Fig. 6, B and C). These observations suggest that degranulated ␤-cells become replete with insulin granules during recovery from diabetes with anti-CD3 mAb with or without exendin-4. We addressed the quantitative effects of exendin-4 on insulin recovery in vivo and in vitro. We performed IPGTTs in a randomly selected subset of mice to characterize the ␤-cell functional capacity of the treated mice 3 wk after the development of diabetes. The fasting glucose was significantly lower in the mice treated with anti-CD3 mAb ⫹ exendin-4 compared with those treated with anti-CD3 mAb alone (P ⬍ 0.01), and the glucose tolerance reflected by the average glucose or the area under the curve during an IPGTT were significantly improved (P ⬍ 0.05; Table 1 and Fig. 7). The mice treated with anti-CD3 mAb and exendin-4 showed a greater insulin release during the IPGTT than mice treated with anti-CD3 mAb alone (Table 1; P ⬍ 0.05). Even when mice with significant fasting hyperglycemia (i.e. ⬎ 350 mg/ dl) were excluded from the studies, the insulin responses were still found to be higher in the group treated with antiCD3 ⫹ exendin-4 (2.16 ⫾ 0.33 IU/ml) compared with those

Exendin-4 Control

75

New onset DM

A

50

Anti-CD3mAb

B

Anti-CD3 mAb+Ex-4

C

25

0

0

10

20

30

40

Day after adoptive transfer FIG. 5. Adoptive transfer of diabetes into NOD/scid control mice and exendin-4-treated NOD/scid recipients. NOD/scid mice were untreated (n ⫽ 14, 䡺) or treated with exendin-4 (n ⫽ 15, f) for 10 d. On d 2 of the exendin-4 treatment, all of the mice received 107 splenocytes from diabetic NOD donors. The plasma glucose levels were measured every 2– 4 d, and mice were considered diabetic if the glucose level was more than 200 mg/dl.

FIG. 6. Recovery of ␤-cells after treatment with anti-CD3 mAb. Pancreatic sections from NOD mice with new-onset diabetes (A) or 3 wk after treatment with anti-CD3 mAb with (C) or without (B) exendin-4 were stained for insulin (green), GLUT-2 (red), and DAPI (blue) as described in Materials and Methods. GLUT-2⫹/insulin⫺ cells could be identified in the islets of NOD mice with new-onset diabetes (arrows). Three weeks later, the GLUT-2⫹ cells are all insulin⫹ (B and C). Islets representative of 11 islets from eight mice are shown.

5142



TABLE 1. Metabolic responses during an IPGTT Treatment group

Fasting Fasting Fasting Fasting Fasting

blood glucose (mg/dl) ⫹ maximal glucose/2 during IPGTT (mg/dl) insulin level (␮IU/ml) ⫹ maximal insulin levels/2 (␮IU/ml) glucagon levels (pM)

Anti-CD3 mAb

Anti-CD3 mAb and exendin-4

243 ⫾ 37, n ⫽ 24 211 ⫹ 24, n ⫽ 22 1.35 ⫾ 0.129 1.48 ⫾ 0.12, n ⫽ 21 9.6 ⫾ 2.9, n ⫽ 4

110 ⫾ 14, n ⫽ 24a 147 ⫹ 9, n ⫽ 21a 1.87 ⫾ 0.37 2.16 ⫾ 0.33, n ⫽ 17b 28.2 ⫾ 4.4a, n ⫽ 5

A subset of mice, selected at random, underwent an IPGTT after a 14- to 16-h fast, 3 wk after diagnosis of diabetes and treatment with anti-CD3 mAb or anti-CD3 mAb and exendin-4 as described in Materials and Methods. Murine glucagon and insulin levels were measured by Luminex bead assay and ELISA, respectively (Linco). a P ⬍ 0.01. b P ⬍ 0.05

receiving anti-CD3 mAb alone (1.49 ⫾ 0.127 IU/ml, P ⫽ 0.06), and the glucose responses were lower (322 ⫾ 53.7 vs. 180 ⫾ 27.9 mg/dl, P ⫽ 0.03). The improvement in glucose tolerance could not be explained by suppression of glucagon by exendin-4 because the glucagon levels at the start of the IP GTT were actually higher in mice treated with anti-CD3 mAb and exendin-4 compared with those receiving just anti-CD3 mAb (P ⫽ 0.01; Table 1). We measured the total insulin content in pancreases from mice that were treated with anti-CD3 mAb alone or with exendin-4 2 wk after the initiation of treatment (Fig. 8). There were significantly greater levels of extracted insulin in pancreases from mice that were treated with anti-CD3 mAb and exendin-4 (182 ⫾ 113 ␮g/mg protein, n ⫽ 11, vs. 668 ⫾ 195 ␮g/mg protein, n ⫽ 8; P ⬍ 0.05; Fig. 8). To directly test the effects of exendin-4 on islet cells and determine how exendin-4 affected their function, we cultured islets from nondiabetic NOD/scid mice with exendin-4 and then measured glucose-stimulated insulin release and insulin content (Table 2). At 1 or 4 d after a 24-h exposure to exendin-4, the insulin concentrations in culture supernatants in 1.7 and 17 mm glucose were significantly higher than in control islets cultured for the same period of time (P ⬍ 0.01). However, the stimulation index defined as the insulin release

during culture with 17 mm glucose/insulin release during 1.7 mm glucose was similar in the islets that had and had not been cultured with exendin-4. The total insulin content of islets exposed to exendin-4 was higher than control islets even 6 d after exposure to exendin-4 for 24 h (2838 ⫾ 190 ng/ng DNA vs. 1883 ⫾ 260 ng/ng DNA, P ⬍ 0.05). These findings indicate that treatment with exendin-4 improves total insulin content of islets, insulin release, and glucose tolerance in vivo. Discussion

We have shown that treatment with the GLP-1 receptor agonist exendin-4 improves the rate of correction of diabetes in newly diabetic NOD mice treated with anti-CD3 mAb. Exendin-4 treatment did not enhance ␤-cell replication, decrease ␤-cell death, or increase the average ␤-cell area in the treated mice, nor did it have a detectable effect on the autoimmune response (7, 10, 12). Instead, we found that exendin-4 improved the revival of ␤-cells after treatment with anti-CD3 mAb, reflected by recovery of insulin staining in GLUT-2-positive/insulin-negative cells that were found at diagnosis and higher insulin content in the pancreases compared with mice treated with anti-CD3 mAb alone. Glucose tolerance, insulin release, and the insulin content of ␤-cells

*

Glucose (mg/dl)

400 300

anti-CD3 mAb Anti-CD3 mAb+Ex-4

200 100 0 0min

15

30

60

Time (min) FIG. 7. Glucose tolerance in mice after treatment. Mice treated with anti-CD3 mAb alone (n ⫽ 14) or with exendin-4 (n ⫽ 14) underwent a glucose tolerance test. The mean ⫾ SEM glucose level at each time point is plotted. The average AUC of the glucose curves was lower in mice that had been treated with anti-CD3 mAb ⫹ exendin-4 compared with mice treated with anti-CD3 mAb alone (P ⬍ 0.05).

ng insulin/mg protein

500

900 800 700 600 500 400 300 200 100 0

anti-CD3 mAb

anti-CD3 mAb+Ex-4

FIG. 8. Effects of exendin-4 treatment on pancreatic insulin content in mice treated with anti-CD3 mAb. The total insulin content of pancreases from mice treated with anti-CD3 mAb alone (black bar) or anti-CD3 mAb and exendin-4 (stippled bar) was measured 2 wk after diagnosis of diabetes (*, P ⬍ 0.05).



5143

TABLE 2. Effects of exendin-4 on islet responses in vitro d1 Insulin concentration Insulin concentration at 1.7 mM glucose at 17 mM glucose (ng/␮g DNA) (ng/␮g DNA)

Exendin-4 treated Control

573 ⫾ 95.8a 290 ⫾ 14.0

1642 ⫾ 163b 668 ⫾ 99.9

d4 Stimulation index

Insulin concentration Insulin concentration at 1.7 mM glucose at 17 mM glucose (ng/␮g DNA) (ng/␮g DNA)

2.97 ⫾ 0.24 2.30 ⫾ 0.0.31

775 ⫾ 109 506 ⫾ 84.1

2199 ⫾ 151b 740 ⫾ 86.1

Stimulation index

Insulin content

3.00 ⫾ 0.55 7856 ⫾ 270b 1.49 ⫾ 0.09 4809 ⫾ 232

Islets, isolated from NOD/scid mice, were placed in culture for 1 d with exendin-4 and then washed and placed into culture for the indicated number of days. They were washed with culture medium and incubated with Krebs-Ringer solutions containing either 1.7 mM or 17 mM glucose as described in Materials and Methods. The insulin concentration was measured in the culture supernatants. After the culture in 17 mM glucose on d 4, insulin was extracted from the islets and the concentration was measured. The insulin concentrations in the supernatants and the insulin content of islets were corrected for islet mass by dividing by the DNA content of the islets. a P ⬍ 0.05. b P ⬍ 0.01.

were greater in mice and islets exposed to exendin-4 compared with those that were not. Our findings confirm a previous study by Ogawa et al. (17) in which exendin-4 was used with ALS for treatment of NOD mice with new-onset diabetes but clarify the mechanism to account for synergistic effect of exendin-4 with immune modulators. The rate of correction of diabetes that we found after treatment with anti-CD3 mAb was lower than that originally reported by Chatenoud et al. (19) but similar to that found by that group in their initial report and by Turvey et al. (32) and recently reported by our group (18, 31). The differences with our results may be related to the glucose levels in the mice at diagnosis and/or the time to the start of treatment with anti-CD3 mAb. We have recently shown that the reversal of diabetes after treatment with anti-CD3 mAb is primarily due to the functional recovery of ␤-cells in the islets rather than the growth of new cells. Indeed, these new observations, indicating that exendin-4 improves the functional recovery rather than stimulating ␤-cell replication or decreasing ␤-cell death are consistent with that mechanism. Likewise, the improvement with exendin-4 treatment with anti-CD3 mAb was most evident in mice that had moderate hyperglycemia (200 –350 mg/dl at diagnosis), suggesting that residual ␤-cells need to be present for the effects of exendin-4. In other studies, we have found that when exendin-4 treatment was delayed for 6 wk after diagnosis, reversal of diabetes did not occur even when the mice were pretreated with anti-CD3 mAb (data not shown). In a similar manner, studies in NOD mice using anti-CD3 mAb and intranasal insulin peptide (von Herrath, M., personal communication) and in patients indicate that residual ␤-cell mass is needed for optimal immune therapy effects. In a clinical trial of anti-CD3 mAb, in the upper half of C-peptide responses at diagnosis were better responders than those with the lower half of C-peptide responses (3). In settings where ␤-cell mass is not marginal, the effects of the exendin-4 may be less clinically apparent. In preliminary studies, we have found that prediabetic NOD mice with impaired glucose intolerance treated with anti-CD3 mAb alone (n ⫽ 3) or anti-CD3 mAb ⫹ exendin-4 were completely protected from progression to diabetes, which occurred in four fifths of untreated mice followed over the same period of time (P ⬍ 0.05). Our studies in vivo and in vitro show that the total insulin content of islets and response to glucose is enhanced by exposure to exendin-4. Interestingly, the relative proportion

of insulin released in response to glucose, the stimulation index, was not affected, indicating that the effect of the exendin-4 treatment was to augment the total insulin content rather than the secretory response to glucose per se. The effect of exendin-4 on insulin responses of islets in vitro were persistent for at least 10 d after exposure to the exendin-4 for 24 h (Table 2 and data not shown). Similarly, the improved responses that we found in vivo occurred approximately 2 wk after the last dose of exendin-4. When given over weeks, exendin-4 has been shown to increase insulin sensitivity (33). However, a difference in insulin sensitivity possibly due to a lower body weight of the exendin-4-treated mice was not a likely explanation for the results because the weights of the mice that received anti-CD3 mAb (23.2 ⫾ 0.25 g) were similar to those treated with the combination (22.8 ⫾ 0.12 g). In our studies, exendin-4 did not enhance ␤-cell proliferation. This is in concordance with the findings by Baggio et al. (34), who did not find an increase in ␤-cell replication or mass in mice with chronic long-term exposure to exendin-4 expressed as a transgene under control of the mouse metallothionein I promoter. It is possible that with longer exposure to exendin-4, there might be improved rates of ␤-cell replication, increased ␤-cell area, or reduced ␤-cell apoptosis, as has been suggested by previous studies (7, 12), but this is not suggested by our data in mice followed for 70 d after diabetes onset (Fig. 1), and our earlier studies indicate that functional rather than anatomic recovery of ␤-cells accounts for the improvement after anti-CD3 mAb. In summary, we found that the addition of exendin-4 to treatment with anti-CD3 mAb enhanced the remission rate of T1DM in diabetic NOD mice with moderate hyperglycemia. The basis for the improvement involves the recovery of ␤-cells and an increase in insulin content of islets. These effects are seen primarily in mice with residual ␤-cells at diagnosis and are not extremely hyperglycemic. In the time course of our studies that involve the relatively acute responses to anti-CD3 mAb, we did not find evidence for enhanced ␤-cell proliferation, decreased ␤-cell apoptosis, or a change in the autoimmune response with treatment with exendin-4. These studies have implications for the design of clinical trials in which immune therapy alone or with incretins are considered. Because there are also substantial residual ␤-cell responses in patients at the time of diagnosis of type 1 diabetes, the addition of a GLP-1 receptor agonist may improve responses to immune therapies by improving the functional recovery of ␤-cells.

5144



Acknowledgments We thank Youping Liu and Michelle Nowroozi for their technical assistance. Received March 15, 2007. Accepted July 20, 2007. Address all correspondence and requests for reprints to: Kevan C. Herold, M.D., Department of Immunobiology, Yale University, 300 Cedar Street, S155B, New Haven, Connecticut 06520. E-mail: [email protected]. This work was supported by National Institutes of Health Grants DK068678, AI-98-010, P30 DK063608, and DK57846, the Juvenile Diabetes Research Foundation Grant 2007-234, the Russell Berrie Foundation, and a Pediatric Endocrine Fellowship award from the Lawson Wilkins Pediatric Endocrine Society. P.S. is supported by the Canadian Institutes of Health Research and is a scientist of the Alberta Heritage Foundation for Medical Research (AHFMR). S.T. is supported by an AHFMR studentship. Author Disclosure Summary: The authors state that they have nothing to declare.

References 1. Herold KC, Gitelman SE, Masharani U, Hagopian W, Bisikirska B, Donaldson D, Rother K, Diamond B, Harlan DM, Bluestone JA 2005 A single course of anti-CD3 monoclonal antibody hOKT3␥1(Ala-Ala) results in improvement in C-peptide responses and clinical parameters for at least 2 years after onset of type 1 diabetes. Diabetes 54:1763–1769 2. Herold KC, Hagopian W, Auger JA, Poumian-Ruiz E, Taylor L, Donaldson D, Gitelman SE, Harlan DM, Xu D, Zivin RA, Bluestone JA 2002 Anti-CD3 monoclonal antibody in new-onset type 1 diabetes mellitus. N Engl J Med 346:1692–1698 3. Keymeulen B, Vandemeulebroucke E, Ziegler AG, Mathieu C, Kaufman L, Hale G, Gorus F, Goldman M, Walter M, Candon S, Schandene L, Crenier L, De Block C, Seigneurin JM, De Pauw P, Pierard D, Weets I, Rebello P, Bird P, Berrie E, Frewin M, Waldmann H, Bach JF, Pipeleers D, Chatenoud L 2005 Insulin needs after CD3-antibody therapy in new-onset type 1 diabetes. N Engl J Med 352:2598 –2608 4. Bougneres PF, Carel JC, Castano L, Boitard C, Gardin JP, Landais P, Hors J, Mihatsch MJ, Paillard M, Chaussain JL, Bach JF 1988 Factors associated with early remission of type I diabetes in children treated with cyclosporine. N Engl J Med 318:663– 670 5. Buse JB, Henry RR, Han J, Kim DD, Fineman MS, Baron AD 2004 Effects of exenatide (exendin-4) on glycemic control over 30 weeks in sulfonylureatreated patients with type 2 diabetes. Diabetes Care 27:2628 –2635 6. Kolterman OG, Buse JB, Fineman MS, Gaines E, Heintz S, Bicsak TA, Taylor K, Kim D, Aisporna M, Wang Y, Baron AD 2003 Synthetic exendin-4 (exenatide) significantly reduces postprandial and fasting plasma glucose in subjects with type 2 diabetes. J Clin Endocrinol Metab 88:3082–3089 7. Li Y, Hansotia T, Yusta B, Ris F, Halban PA, Drucker DJ 2003 Glucagon-like peptide-1 receptor signaling modulates ␤-cell apoptosis. J Biol Chem 278:471– 478 8. Farilla L, Bulotta A, Hirshberg B, Li Calzi S, Khoury N, Noushmehr H, Bertolotto C, Di Mario U, Harlan DM, Perfetti R 2003 GLP-1 inhibits cell apoptosis and improves glucose responsiveness of freshly isolated human islets. Endocrinology 144:5149 –5158 9. Xu G, Stoffers DA, Habener JF, Bonner-Weir S 1999 Exendin-4 stimulates both ␤-cell replication and neogenesis, resulting in increased ␤-cell mass and improved glucose tolerance in diabetic rats. Diabetes 48:2270 –2276 10. Stoffers DA, Kieffer TJ, Hussain MA, Drucker DJ, Bonner-Weir S, Habener JF, Egan JM 2000 Insulinotropic glucagon-like peptide 1 agonists stimulate expression of homeodomain protein IDX-1 and increase islet size in mouse pancreas. Diabetes 49:741–748 11. Stoffers DA, Desai BM, DeLeon DD, Simmons RA 2003 Neonatal exendin-4 prevents the development of diabetes in the intrauterine growth retarded rat. Diabetes 52:734 –740 12. De Leon DD, Deng S, Madani R, Ahima RS, Drucker DJ, Stoffers DA 2003

13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

23. 24. 25. 26. 27. 28.

29. 30. 31.

32. 33.

34.

Role of endogenous glucagon-like peptide-1 in islet regeneration after partial pancreatectomy. Diabetes 52:365–371 Greenbaum CJ, Prigeon RL, D’Alessio DA 2002 Impaired ␤-cell function, incretin effect, and glucagon suppression in patients with type 1 diabetes who have normal fasting glucose. Diabetes 51:951–957 Dupre J 2005 Glycaemic effects of incretins in type 1 diabetes mellitus: a concise review, with emphasis on studies in humans. Regul Pept 128:149 –157 Behme MT, Dupre J, McDonald TJ 2003 Glucagon-like peptide 1 improved glycemic control in type 1 diabetes. BMC Endocr Disord 3:3 Dupre J, Behme MT, Hramiak IM, McFarlane P, Williamson MP, Zabel P, McDonald TJ 1995 Glucagon-like peptide I reduces postprandial glycemic excursions in IDDM. Diabetes 44:626 – 630 Ogawa N, List JF, Habener JF, Maki T 2004 Cure of overt diabetes in NOD mice by transient treatment with anti-lymphocyte serum and exendin-4. Diabetes 53:1700 –1705 Chatenoud L, Thervet E, Primo J, Bach JF 1994 Anti-CD3 antibody induces long-term remission of overt autoimmunity in nonobese diabetic mice. Proc Natl Acad Sci USA 91:123–127 Chatenoud L, Primo J, Bach JF 1997 CD3 antibody-induced dominant self tolerance in overtly diabetic NOD mice. J Immunol 158:2947–2954 Sherry NA, Kushner JA, Glandt M, Kitamura T, Brillantes AM, Herold KC 2006 Effects of autoimmunity and immune therapy on ␤-cell turnover in type 1 diabetes. Diabetes 55:3238 –3245 Tomita T 1999 Immunocytochemical localization of glucose transporter-2 (GLUT-2) in pancreatic islets and islet cell tumors. Endocr Pathol 10:213–221 Trudeau JD, Kelly-Smith C, Verchere CB, Elliott JF, Dutz JP, Finegood DT, Santamaria P, Tan R 2003 Prediction of spontaneous autoimmune diabetes in NOD mice by quantification of autoreactive T cells in peripheral blood. J Clin Invest 111:217–223 Hober C, Benhamou PY, Watt PC, Watanabe Y, Nomura Y, Stein E, Brunicardi FC, Mullen Y 1997 A new culture method for human pancreatic islets using a biopore membrane insert. Pancreas 14:199 –204 Davalli AM, Ogawa Y, Scaglia L, Wu YJ, Hollister J, Bonner-Weir S, Weir GC 1995 Function, mass, and replication of porcine and rat islets transplanted into diabetic nude mice. Diabetes 44:104 –111 Sreenan S, Pick AJ, Levisetti M, Baldwin AC, Pugh W, Polonsky KS 1999 Increased ␤-cell proliferation and reduced mass before diabetes onset in the nonobese diabetic mouse. Diabetes 48:989 –996 Baggio LL, Holland D, Wither J, Drucker DJ 2006 Lymphocytic infiltration and immune activation in metallothionein promoter-exendin-4 (MT-exendin) transgenic mice. Diabetes 55:1562–1570 Amrani A, Verdaguer J, Serra P, Tafuro S, Tan R, Santamaria P 2000 Progression of autoimmune diabetes driven by avidity maturation of a T-cell population. Nature 406:739 –742 Lieberman SM, Evans AM, Han B, Takaki T, Vinnitskaya Y, Caldwell JA, Serreze DV, Shabanowitz J, Hunt DF, Nathenson SG, Santamaria P, DiLorenzo TP 2003 Identification of the ␤-cell antigen targeted by a prevalent population of pathogenic CD8⫹ T cells in autoimmune diabetes. Proc Natl Acad Sci USA 100:8384 – 8388 Anderson B, Park BJ, Verdaguer J, Amrani A, Santamaria P 1999 Prevalent CD8⫹ T cell response against one peptide/MHC complex in autoimmune diabetes. Proc Natl Acad Sci USA 96:9311–9316 Belghith M, Bluestone JA, Barriot S, Megret J, Bach JF, Chatenoud L 2003 TGF-␤-dependent mechanisms mediate restoration of self-tolerance induced by antibodies to CD3 in overt autoimmune diabetes. Nat Med 9:1202–1208 Bresson D, Togher L, Rodrigo E, Chen Y, Bluestone JA, Herold KC, von Herrath M 2006 Anti-CD3 and nasal proinsulin combination therapy enhances remission from recent-onset autoimmune diabetes by inducing Tregs. J Clin Invest 116:1371–1381 Turvey SE, Swart E, Denis MC, Mahmood U, Benoist C, Weissleder R, Mathis D 2005 Noninvasive imaging of pancreatic inflammation and its reversal in type 1 diabetes. J Clin Invest 115:2454 –2461 Young AA, Gedulin BR, Bhavsar S, Bodkin N, Jodka C, Hansen B, Denaro M 1999 Glucose-lowering and insulin-sensitizing actions of exendin-4: studies in obese diabetic (ob/ob, db/db) mice, diabetic fatty Zucker rats, and diabetic rhesus monkeys (Macaca mulatta). Diabetes 48:1026 –1034 Baggio L, Adatia F, Bock T, Brubaker PL, Drucker DJ 2000 Sustained expression of exendin-4 does not perturb glucose homeostasis, ␤-cell mass, or food intake in metallothionein-preproexendin transgenic mice. J Biol Chem 275:34471–34477

Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.