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Release of Glutamate Decarboxylase-65 into the Circulation by Injured Pancreatic Islet ß-Cells

Department of Medicine (M.A.W., A.T.S., S.D.C.), and Biomedical Sciences Graduate Program (A.T.S.), University of California, San Diego, La Jolla, California 92093-0726; and Northwest Lipid Metabolism and Diabetes Research Laboratories (S.M.M.), University of Washington, Seattle, Washington 98109

Address all correspondence and requests for reprints to: Steven D. Chessler, University of California, San Diego, Leichtag Biomedical Research Building, 9500 Gilman Drive, MC 0726, La Jolla, California 92093-0726. E-mail: schessler{at}ucsd.edu.

	Abstract

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The enzyme glutamate decarboxylase-65 (GAD65) is a major autoantigen in autoimmune diabetes. The mechanism whereby autoreactivity to GAD65, an intracellular protein, is triggered is unknown, and it is possible that immunoreactive GAD65 is released by injured pancreatic islet ß-cells. There is a great need for methods by which to detect and monitor ongoing islet injury. If GAD65 were released and, furthermore, were able to reach the circulation, it could function as a marker of ß-cell injury. Here, a novel GAD65 plasma immunoassay is used to test the hypotheses that ß-cell injury induces GAD65 discharge in vivo and that discharged GAD65 reaches the bloodstream. Plasma GAD65 levels were determined in rats treated with alloxan, and with diabetogenic and low, subdiabetogenic doses of streptozotocin. ß-Cell injury resulted in GAD65 release into the circulation in a dose-dependent manner, and low-dose streptozotocin resulted in a more gradual increase in plasma GAD65 levels than did diabetogenic doses. Plasma GAD65 levels were reduced in rats that had undergone partial pancreatectomy and remained undetectable in mice. Together, these data demonstrate that GAD65 can be released into the circulation by injured ß-cells. Autoantigen shedding may contribute to the pathogenesis of islet autoimmunity in the multiple low-dose streptozocin model and perhaps, more generally, in other forms of autoimmune diabetes. These results demonstrate that, as is true with other tissues, islet injury, at least in some circumstances, can be monitored by use of discharged, circulating proteins. GAD65 is the first such confirmed protein marker of islet injury.

	Introduction

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THE ENZYME GLUTAMATE decarboxylase-65 (GAD65) is a major autoantigen in autoimmune diabetes (1). GAD65 does not traffic to the plasma membrane, suggesting that, at least at certain times during the development of diabetes, it is released from within islet ß-cells and is able to come into contact with immune-effector cells (2). The detection of glutamate decarboxylase enzymatic activity in the media of cultured, injured rat ß-cells supports the hypothesis that GAD65 is susceptible to discharge (3). Whether discharge of diabetes-associated autoantigens, including GAD65, occurs in vivo is unknown.

There is currently no method for detecting and monitoring ongoing islet ß-cell injury. Metabolic measurements can reveal ß-cell loss, but only after extensive islet damage has occurred (4). The lack of a good biomarker for islet injury complicates the testing of therapies meant to prevent ß-cell loss, including loss after transplantation, and impedes research into the pathogenesis of diabetes (4, 5). Although there are no markers of ongoing islet injury, tests based on the measurement of circulating, discharged intracellular enzymes to monitor tissue injury have become an integral part of clinical care and research. For example, pathogenic processes affecting the liver can be assessed well before the onset of symptoms by monitoring discharged enzymes such as alanine aminotransferase, and myocardial injury can be detected by measuring serum troponin levels (6, 7). Measurement of lactate dehydrogenase in body fluids and cell culture media allows the monitoring of cell damage caused by a variety of processes (8). ß-Cell content of lactate dehydrogenase is minimal, however, further illustrating the need for markers of ß-cell damage for both in vivo and ex vivo work (9). GAD65, if discharged by injured ß-cells and capable of reaching the circulation, may function as a marker of islet injury. Expression of GAD65 is primarily restricted to inhibitory synapses in the central nervous system and to pancreatic islets (1, 10). Rat and human islets both express GAD65 in a ß-cell-specific manner. The limited tissue distribution of GAD65 contributes to its potential usefulness as a marker of ß-cell damage.

Here, we have used a sensitive GAD65 assay to test the hypotheses that ß-cell injury induces GAD65 release in vivo and that discharged, immunoreactive GAD65 can reach the circulation (11). ß-Cell injury was induced using the selective ß-cell cytotoxins alloxan and streptozotocin (STZ) (Sigma, St. Louis, MO) (12). STZ accumulates specifically in ß-cells, is rapidly degraded into inactive metabolites, and does not cross the blood-brain barrier (12, 13, 14). In addition to inducing ß-cell necrosis at higher doses, repeated administration of STZ to immunologically susceptible rodents at doses too low to cause directly ß-cell destruction induces autoimmune diabetes (12, 15). The mechanism by which low-dose STZ triggers autoimmune diabetes is unknown. Here, along with testing the use of plasma GAD65 as a marker of ß-cell injury, we have begun to determine whether autoantigen discharge by injured ß-cells is a potential trigger of antiislet autoimmunity. The goals of the studies described here were to: 1) determine whether GAD65 can be detected in the serum of animals that have sustained ß-cell injury, and, more generally; 2) test whether, as is the case with other tissues, protein discharge into the circulation can provide a method by which to detect and monitor ß-cell damage.

	Materials and Methods

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Animals
Adult male Wistar rats weighing 150–250 g were purchased from Harlan (Indianapolis, IN). Pancreatectomized Wistar rats were purchased from Charles River Laboratories (Wilmington, MA); pancreatectomies were reported to be approximately 90% complete. Mice were adult male C57B6. All procedures were approved by the University of California, San Diego, Institutional Animal Care and Use Committee.

STZ and alloxan administration
Animals were fasted 16 h and anesthetized with isoflurane before injection. STZ was dissolved in sterile sodium citrate buffer (pH 4.5) and administered ip to normal rats (Px–) (n = 4–7 rats per time point) or rats that had undergone partial (90%) pancreatectomy (Px+) (n = 3–6). Alloxan (Sigma) was administered in the same manner (n = 3–4). Mice received a diabetogenic dose of 200 mg/kg STZ (n = 4) or vehicle alone (n = 4). Control rats or mice were injected with an equal volume of vehicle (n = 4–9 animals per time point). Blood glucose levels were monitored using a Freestyle Glucometer (Abbot, Alameda, CA). All glucose levels, except those of pancreatectomized rats at treatment (t = 0), were drawn nonfasting.

Plasma GAD65 analysis
Animals were anesthetized with 150 mg/kg Nembutal (Ovation Pharmaceuticals, Deerfield, IL), and blood was collected via cardiac puncture. Plasma samples (250 µl) were assayed in duplicate for GAD65 using a previously described immunoassay (11). Briefly, GAD65 was captured on antibody coated beads, eluted with 6 M guanidine HCl, transferred to a 96-well filter-bottom plate, and detected using anti-GAD65 antibodies and chemiluminescence. Plasma samples were diluted 1:2 in 1% BSA containing 20 mM Tris, 0.17% (vol/vol) Tween 20, and 4 mM EDTA. Samples with greater than 1.5 ng/ml GAD65 were further diluted to keep results within the range of the standard curve. Mouse plasma samples were diluted 1:7 due to a smaller sample volume. The interassay and intraassay coefficients of variance averaged 16 and 14%, respectively. The limit of the blank of the assay (the mean of the zero standard plus 2.5 times the SD) was used to determine the lower limit of detection, 56 pg/ml. Plasma GAD65 levels lower than 56 pg/ml were assigned a value of 28 pg/ml, half the detection limit (16).

Insulin and C-peptide determination
Plasma samples from rats treated with vehicle (n = 4 rats per time point) or from Px– (n = 4–6) and Px+ (n = 3–5) rats treated with 80 mg/kg STZ were submitted to the University of Cincinnati Mouse Metabolic Phenotyping Center for analysis by rat insulin and C-peptide ELISA (Linco Research, St. Charles, MO, and Wako Chemicals, Richmond, VA, respectively).

Immunohistochemical and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining
TUNEL staining was performed by the University of California, San Diego, Moores Cancer Center Histology and Immunohistochemistry Shared Resource using the ApopTag Peroxidase In Situ Apoptosis Detection Kit (Chemicon Int., Temecula, CA). Pancreata (n = 3–6) were collected 6 h after injection of STZ or of vehicle alone, and one to six frozen sections per pancreas were stained. For rats receiving 20 mg/kg STZ, pancreata were collected 6 or 24 h after injection (n = 3–6 per time point), and two sections were analyzed per pancreas. At higher STZ doses, islet destruction was too extensive at 24 h to allow TUNEL staining. Adjacent sections were stained using an antiglucagon monoclonal antibody (Sigma) and the Vectastain ABC Elite system (Vector Laboratories, Burlingame, CA) to confirm identification of islets. Sections were also stained with Harris’ modified hematoxylin and eosin (Fisher Scientific, Fairlawn, NJ). Pancreas sections (5 µm) were examined both frozen or after overnight fixation in Pen-Fix (Richard Allan Scientific, Kalamazoo, MI), followed by paraffin embedding. Results did not vary between frozen and paraffin-embedded sections. Sections were photographed at x200 magnification.

Elimination half-life
Rats were injected via the tail vein with 330 ng (n = 3) or 100 ng (n = 5) recombinant human GAD65 in PBS (rhGAD65) (produced in Spodoptera frugiperda cells; a gift from Diamyd Medical, Stockholm, Sweden). Blood was collected via the saphenous vein at 20 min and 3 h, and via cardiac puncture at 6 and 9 h after injection.

Statistical analysis and calculations
Data are expressed as means ± SEM. Plasma GAD65, insulin, and C-peptide levels were analyzed using the nonparametric Mann-Whitney U test. A P value < 0.05 was considered significant. For analysis that includes data points below the limit of detection, substitution of half the limit of detection is the optimal approach with sample numbers (n) in the range used here; therefore, this approach was used (16). GAD65 clearance was consistent with a one-compartment model with first-order elimination, and GAD65 concentrations after rhGAD65 injection were fitted to a first-order clearance equation (C = C_o · e^–kt), where C was the plasma concentration, C_o was the initial concentration after injection, and t was the time after determination of the initial (baseline) GAD65 concentration (17). The elimination time constant (k) was obtained from the resulting regression line. Regression lines were compared by analysis of covariance. Half-time of elimination was calculated as t_1/2 = ln 2/k (17).

	Results

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Circulating GAD65, insulin, and C-peptide levels after treatment with alloxan or STZ
Single ip doses of STZ 50 mg/kg body weight or greater as well as high doses of alloxan are sufficient to induce diabetes in rats (12, 18). We collected plasma samples at various time points after administration of 80 mg/kg STZ or 150 mg/kg alloxan to rats. After STZ treatment, blood glucose levels increased to more than 300 mg/dl within 24 h (Fig. 1B). Mean plasma GAD65 concentration increased from undetectable levels to 6811 ± 814 pg/ml 6 h after injection and decreased to 759 ± 195 pg/ml at 24 h (Fig. 1A). By 48 h, circulating GAD65 was no longer detectable. After alloxan treatment, mean plasma GAD65 levels increased to 3810 ± 1183 pg/ml by 6 h and decreased to 270 ± 41 pg/ml at 24 h. Rats injected with vehicle alone exhibited no increase in circulating GAD65, with plasma GAD65 remaining below detectable levels.

View larger version (18K): [in this window] [in a new window]   FIG. 1. Effect of STZ and alloxan treatments on circulating levels of GAD65 and on markers of ß-cell function. A, Rats were injected ip with STZ at a dose of 80 mg/kg body weight (STZ-tx), and circulating GAD65 was measured at 0, 3, 6, 10, 12, 24, and 48 h after injection. Control rats (Vehicle-tx) were injected ip with a corresponding volume of vehicle, and circulating GAD65 was measured at 0, 6, 24, and 48 h postinjection. Alloxan-treated (Alloxan-tx) rats were injected ip with alloxan at a dose of 150 mg/kg, and circulating GAD65 was measured at 0, 6, 24, and 48 h after injection. B, Blood glucose concentrations in vehicle-treated and toxin-treated rats. C and D, Plasma insulin (C) and C-peptide (D) in STZ-treated and vehicle-treated rats (all nonfasting). Data are shown as means ± SEM (n = 3–7 rats per time point). *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001 vs. 0 time point.

Circulating GAD65 levels in mice and pancreatectomized rats
To confirm that GAD65 of pancreatic origin was released, we administered STZ to pancreatectomized rats and to mice. Because of the diffuse nature of the rat pancreas and other aspects of its anatomy, removing more than approximately 90–95% the pancreas in the rat is highly challenging and, in general, not possible in rats more than approximately 150 g (20). For this reason, rats that had undergone approximately 90% Px were used to test whether partial Px had an effect on the pattern of increase in plasma GAD65 levels after STZ treatment. Total pancreatic GAD65 content of normal rats was 76 ± 4 µg; GAD65 content of the pancreas remnants of Px+ rats was 13 ± 7 µg (see supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Mice exhibit a high level of GAD65 expression in the brain but have very low levels of islet GAD65, detectable only by highly sensitive methods (10, 11). Thus, mice present an ideal model in which to test whether circulating GAD65 levels increase after high-dose STZ treatment, despite a very minimal pancreatic GAD65 content.

Increasing the functional activity of ß-cells renders them more sensitive to the toxic effects of STZ (21). Because the remaining ß-cells in rats that had undergone Px would be relatively hyperfunctional, it was likely that they would be more susceptible to STZ-induced cytotoxicity. Indeed, in the Px+ rats, there was significant insulin and C-peptide discharge into the circulation 6 h after STZ treatment, consistent with acute ß-cell damage (19). Despite the reduced size of their pancreata, these rats had markedly higher plasma insulin and C-peptide levels than Px– rats at this time (Fig. 2C). Both Px+ and Px– rats exhibited elevated plasma GAD65 protein levels at the 6-h point (Fig. 2A). However, after the initial period of acute ß-cell injury, plasma GAD65 concentrations in the Px+ rats were observed to be markedly lower than in the Px– rats and declined more rapidly with time (Fig. 2B). Mice became hyperglycemic after STZ administration (Fig. 2D) but did not exhibit elevated plasma GAD65 levels: there was no detectable plasma GAD65 after either 6 or 24 h.

View larger version (20K): [in this window] [in a new window]   FIG. 2. Effect of STZ on plasma GAD65 in mice, and on discharge of GAD65, insulin, and C-peptide in 90% pancreatectomized rats. A, Circulating GAD65 was measured 6 h after STZ treatment (80 mg/kg) of approximately 90% Px+ and Px– rats. Control rats (left-hand-most bar on chart) received vehicle only. Mice, which have very minimal levels of islet GAD65 but a high level of expression in the brain, were injected ip with vehicle or a diabetogenic dose of STZ (200 mg/kg). In mice, plasma GAD65 was, as expected, not detectable 6 or 24 h after injection of STZ or of vehicle alone, and consequently mouse results are not depicted in A or B. ***, P < 0.005; ****, P < 0.001 vs. control (vehicle only). B, Circulating GAD65 was measured 12 or 24 h after STZ injection. Px– and Px+ rats received 80 mg/kg STZ. Mice were treated as described in A. ***, P < 0.005 (Px– vs. Px+). C, Circulating insulin and C-peptide were measured in Px– and Px+ rats at 0, 6, and 24 h after STZ injection (+ below the x-axis identify the bars on the chart showing data from Px+ rats). Probably due to its enhanced toxicity in hyperfunctional ß-cells, in Px+ rats, STZ induced acute C-peptide and insulin discharge (seen at 6 h) and, as seen in A, acute GAD65 discharge. *, P < 0.05, Px– vs. Px+. (C-peptide concentrations divided by 10 to enhance clarity of bar chart.) D, Blood glucose levels after STZ treatment in Px– and Px+ rats and in mice. Glucose levels in vehicle-treated mice (data not shown) remained 100–150 mg/dl (n = 3–7 animals per time point). All data are shown as means ± SEM.

Six hours after STZ administration, plasma GAD65 levels were found to be higher with increasing STZ dosage, plateauing at doses above 80 mg/kg (Fig. 3A). These elevations in plasma GAD65 preceded the onset of hyperglycemia (Fig. 3C). At 24 h, as at 6 h, all STZ doses 20 mg/kg and above resulted in significant elevations in plasma GAD65 levels (Fig. 3B).

View larger version (16K): [in this window] [in a new window]   FIG. 3. Circulating GAD65 in response to varying doses of STZ. Rats were injected ip with STZ doses ranging from 0–160 mg/kg body weight. A, Concentration of plasma GAD65 (n = 4–10) 6 h postinjection. B, Plasma GAD65 (n = 4–6) 24 h after injection. C, Blood glucose levels at 6 and 24 h postinjection after administration of different doses of STZ. The result shown for 60 mg/kg STZ, 6 h was skewed by one rat that exhibited a blood glucose level of 500 mg/dl. Without this outlier, the mean blood glucose at this point was 135 ± 11 mg/dl. D, Circulating GAD65 at 6 and 24 h after injection of low, subdiabetogenic doses of STZ. Data are shown as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001 vs. GAD65 concentrations at time 0.

STZ-induced ß-cell death is preceded by DNA strand breakage, enabling detection of the severe ß-cell injury by TUNEL assay (23). Consistent with prior reports, low-dose STZ (20 mg/kg) did not cause hyperglycemia or change histochemical islet appearance (Figs. 3C and 4) (18, 22). Low-dose STZ also did not induce detectable ß-cell DNA strand breakage (Fig. 4).

View larger version (49K): [in this window] [in a new window]   FIG. 4. Examination of rat pancreas sections by TUNEL, histological, and immunohistochemical staining. A–E, TUNEL staining of pancreas sections to identify DNA strand breakage. Sections from rats treated with STZ doses of 0 (A), 20 (B and C), 40 (D), or 80 mg/kg (E). Pancreata were from 6 h after STZ treatment except C, which was from 24 h. Representative islets are shown and surrounded by arrowheads in each panel. Islet TUNEL staining was only observed in sections from rats treated with the two higher doses of STZ (D and E). There were no TUNEL-positive cells observed in sections from rats treated with vehicle alone (A) or, after 6 or 24 h, in rats treated with 20 mg/kg (B and C). F–H, Sections from rats treated for 6 h with vehicle alone (F) or 20 mg/kg STZ for 6 h (G) or 24 h (H) stained for glucagon to identify islet periphery (mantle). Consistent with past reports, low-dose STZ did not alter islet morphology (for review, see Refs. 18 and 22 ). I and J, Hematoxylin and eosin staining of sections from rats treated 24 h with 20 mg/kg STZ (I) or 80 mg/kg (J). Consistent with past reports, high-dose STZ, as is seen in panel J, disrupts islet architecture and causes extensive depletion of islet ß-cells. (A–J, Magnification, x 200).

	Discussion

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We have found that STZ- and alloxan-induced ß-cell injury results in GAD65 discharge into the circulation. Discharge after STZ treatment occurs in a dose-dependent fashion, and, therefore, plasma GAD65 levels likely reflect the degree of ongoing islet injury. Both high- and low-dose treatments resulted in increased blood GAD65 levels. After high-dose treatment, circulating GAD65 became detectable and reached high levels before the onset of hyperglycemia. In partially (90%) pancreatectomized rats, the increased functional state of the remaining ß-cells led to a more acute course of STZ-induced toxicity, as manifested by the significant C-peptide and insulin discharge observed at 6 h, and the time course of GAD65 discharge was correspondingly more compressed (21). At later time points, plasma GAD65 was markedly lower in the pancreatectomized rats. Plasma GAD65 remained undetectable after high-dose STZ treatment of mice, which have only very minimal islet GAD65 content (10, 11). GAD65, at least in the case of STZ- and alloxan-induced injury, is the first confirmed circulating marker of ongoing ß-cell injury. More generally, the results presented here provide proof-of-principle that monitoring of ß-cell enzymes discharged into the blood can provide a method for detecting and monitoring islet injury.

In rats that received low-dose STZ (20 mg/kg), plasma GAD65 concentrations continued to increase between 6 and 24 h. STZ is completely degraded within 4 h at pH 7.4 and breaks down even more rapidly in plasma (13). Therefore, prolonged GAD65 release cannot be attributed to slow clearance of the drug. Low-dose STZ may have resulted in a milder form of injury that caused prolonged GAD65 shedding. That low-dose STZ induces mild ß-cell injury different from the fatal damage caused by high-dose STZ is consistent with past morphological analysis of islets from STZ-treated rats and with our finding that low-dose STZ, in contrast to high-dose, does not cause detectable DNA strand breakage (18, 22).

In immunologically susceptible strains of rodents, repeated administration of low-dose STZ results in autoimmune diabetes (12, 15). Our results are consistent with the hypothesis that ß-cell autoantigen release promotes antiislet autoimmunity in this model. Diabetes-associated autoantigens, perhaps due to their characteristic vesicle association, may be especially susceptible to release (24). In the case of GAD65, which becomes bound to the surface of synaptic-like microvesicles, high susceptibility to release may help account for the occurrence of STZ- and diabetes-associated GAD65 autoreactivity in mice, despite the minimal GAD65 content of mouse islets (10, 12, 25). The diabetes-prone BioBreeding rat does not exhibit autoreactivity to GAD65; whether GAD65 functions as an autoantigen in the rat low-dose STZ model remains to be determined (26). In humans, possible instigators of anti-ß-cell autoreactivity include ß-cell injury by dietary or other environmental toxins (1, 4). Such initial triggering ß-cell insults may, like STZ, induce autoantigen shedding. Our results provide the first in vivo evidence of ß-cell autoantigen discharge.

Twenty minutes after injection of 330 ng rhGAD65, the plasma GAD65 concentration was 22 ± 2 ng/ml, suggesting that (based on a half-life of 2.9 h) the concentration was approximately 26 ng/ml just after injection. Based on the approximation that 330 ng GAD65 yields a plasma concentration of approximately 26 ng/ml, the levels of plasma GAD65 attained 6 h after STZ administration (6.8 ng/ml) may have been due to release of approximately 86 ng GAD65 into the circulation. This obviously simplified estimation assumes that the GAD65 is discharged by ß-cells shortly before the 6 h point. The rats used here have a total pancreatic GAD65 content of 76 ± 4 µg, and the GAD65 content of the pancreas remnant in Px+ rats, in which increased ß-cell functional state may have increased islet GAD65 levels, was 13 ± 7 µg (27, 28) (see supplemental data). Thus, the levels of GAD65 that we observed in both Px– and Px+ rats are of a magnitude that could be reasonably expected to result from islet injury.

We have determined the half-life of elimination of rhGAD65 in the rat circulation to be approximately 3 h. It is uncertain whether this reflects the half-life of GAD65 in humans. The half-life of GAD65 in rats lies between that of lipase (7–14 h) and troponin T (2 h), which are discharged protein markers of acute and chronic pancreatic and myocardial injury, respectively (7, 29). Given the small size of the ß-cell mass, use of GAD65 as a biomarker may require the use of a very highly sensitive, nucleic acid-based immunodetection methodology, several of which have recently been described (see Ref.30).

It remains to be determined whether measurement of discharged ß-cell proteins will provide a method for monitoring immune-, autoimmune-, or virus-mediated islet injury. Blood assays for enzymes discharged from other tissues serve extremely well for monitoring a variety of different disease processes, and this is a cause for optimism that this approach will work in the case of islets as well. Preliminary evidence from a canine islet transplant model suggests that measurement of plasma GAD65 may provide a method to test for ongoing rejection of transplanted islets (5). New technologies are enabling the development of ever more sensitive assays (30). Continued development of circulating biomarkers of islet damage should greatly facilitate research into the pathogenesis and treatment of diabetes and improve clinical care of individuals with the disease.

	Footnotes

 
This work was supported by the Juvenile Diabetes Research Foundation. S.D.C. was also supported by Grants DK002944 and DK077466 from the National Institutes of Health. A.T.S. is supported by a Graduate Research Fellowship from the National Science Foundation. Insulin and C-peptide assays and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining were performed by the University of Cincinnati Mouse Metabolic Phenotyping Center (DK59630), and the University of California, San Diego Moores Cancer Center Histology and Immunohistochemistry Shared Resource, respectively. Recombinant glutamate decarboxylase-65 protein was a kind gift from Diamyd Medical, A.B.

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 21, 2007

Abbreviations: GAD65, Glutamate decarboxylase-65; Px, pancreatectomy; Px+, pancreatectomized; Px–, nonpancreatectomized; rhGAD65, recombinant human glutamate decarboxylase-65 in PBS; STZ, streptozotocin; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling.

Received October 6, 2006.

Accepted for publication June 5, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Ellis TM, Atkinson MA 1996 The clinical significance of an autoimmune response against glutamic acid decarboxylase. Nat Med 2:148–153[CrossRef][Medline]
2. Kanaani J, Diacovo MJ, El-Husseini Ael D, Bredt DS, Baekkeskov S 2004 Palmitoylation controls trafficking of GAD65 from Golgi membranes to axon-specific endosomes and a Rab5a-dependent pathway to presynaptic clusters. J Cell Sci 117:2001–2013[Abstract/Free Full Text]
3. Smismans A, Ling Z, Pipeleers D 1996 Damaged rat beta cells discharge glutamate decarboxylase in the extracellular medium. Biochem Biophys Res Commun 228:293–297[CrossRef][Medline]
4. Atkinson MA, Eisenbarth GS 2001 Type 1 diabetes: new perspectives on disease pathogenesis and treatment. Lancet 358:221–229[CrossRef][Medline]
5. Shapiro AM, Hao EG, Lakey JR, Yakimets WJ, Churchill TA, Mitlianga PG, Papadopoulos GK, Elliott JF, Rajotte RV, Kneteman NM 2001 Novel approaches toward early diagnosis of islet allograft rejection. Transplantation 71:1709–1718[CrossRef][Medline]
6. Pratt DS, Kaplan MM 1999 Evaluation of the liver: laboratory tests. In: Schiff ER, Sorrell MF, Maddrey WC, eds. Schiff’s diseases of the liver. 8th ed. Philadelphia: Lippincott-Raven Publishers; 205–225
7. Zimmermann R, Baki S, Dengler TJ, Ring GH, Remppis A, Lange R, Hagl S, Kubler W, Katus HA 1993 Troponin T release after heart transplantation. Br Heart J 69:395–398[Abstract/Free Full Text]
8. Danpure CJ 1984 Lactate dehydrogenase and cell injury. Cell Biochem Funct 2:144–148[CrossRef][Medline]
9. Sekine N, Cirulli V, Regazzi R, Brown LJ, Gine E, Tamarit-Rodriguez J, Girotti M, Marie S, MacDonald MJ, Wollheim CB, Rutter GA 1994 Low lactate dehydrogenase and high mitochondrial glycerol phosphate dehydrogenase in pancreatic ß cells. Potential role in nutrient sensing. J Biol Chem 269:4895–4902[Abstract/Free Full Text]
10. Chessler SD, Lernmark A 2000 The role of glutamic acid decarboxylase and GABA in the pancreas and diabetes. In: Martin DL, Olson RW, eds. GABA in the nervous system: the view at fifty years. Philadelphia: Lippincott Williams, Wilkins; 471–484
11. Waldrop MA, Suckow AT, Hall TR, Hampe CS, Marcovina SM, Chessler SD 2006 A highly sensitive immunoassay resistant to autoantibody interference for detection of the diabetes-associated autoantigen glutamic acid decarboxylase 65 in blood and other biological samples. Diabetes Technol Ther 8:207–218[CrossRef][Medline]
12. Mordes JP, Greiner DL, Rossini AA 2000 Animal models of autoimmune diabetes mellitus. In: LeRoith D, Taylor SI, Olefsky JM, eds. Diabetes mellitus: a fundamental and clinical text. 2nd ed. Philadelphia: Lippincott Williams, Wilkins; 430–441
13. Lee JY, Kim MJ, Moon CK, Chung JH 1993 Degradation products of streptozotocin do not induce hyperglycemia in rats. Biochem Pharmacol 46:2111–2113[CrossRef][Medline]
14. Karunanayake EH, Baker JR, Christian RA, Hearse DJ, Mellows G 1976 Autoradiographic study of the distribution and cellular uptake of (14C)-streptozotocin in the rat. Diabetologia 12:123–128[CrossRef][Medline]
15. Lukic ML, Ejdus L, Shahin A, Pravica V, Stosic-Grujicic S, Stojkovic MM, Kolarevic S, Liew E, Ramic Z, Badovinac V 1997 Down-regulation of Th1 mediated autoimmune pathology. In: Lukic ML, Colic M, Stojkovic MM, Cuperlovic K, eds. Immunoregulation in health and disease: experimental and clinical aspects. New York: Academic Press; 265–278
16. Clarke JU 1998 Evaluation of censored data methods to allow statistical comparisons among very small samples with below detection limit observations. Environ Sci Technol 32:177–183
17. Gibaldi M 1991 Biopharmaceutics and clinical pharmacokinetics. 4th ed. Philadelphia: Lea & Febiger
18. Mythili MD, Vyas R, Akila G, Gunasekaran S 2004 Effect of streptozotocin on the ultrastructure of rat pancreatic islets. Microsc Res Tech 63:274–281[CrossRef][Medline]
19. Nagasao J, Yoshioka K, Amasaki H, Tsujio M, Ogawa M, Taniguchi K, Mutoh K 2005 Morphological changes in the rat endocrine pancreas within 12 h of intravenous streptozotocin administration. Anat Histol Embryol 34:42–47[Medline]
20. Scow RO 1957 Total pancreatectomy in the rat: operation, effects, and postoperative care. Endocrinology 60:359–367[Medline]
21. Eizirik DL, Strandell E, Sandler S 1988 Culture of mouse pancreatic islets in different glucose concentrations modifies ß cell sensitivity to streptozotocin. Diabetologia 31:168–174[CrossRef][Medline]
22. Kohnert KD, Falt K, Ziegler B, Odselius R, Ziegler M, Falkmer S 1990 Histopathological lesions in the pancreas of a rat model of diabetes induced with complete Freund’s adjuvant and low-dose streptozotocin. Exp Clin Endocrinol 95:47–56[Medline]
23. Shibata S, Asanuma Y, Koyama K, Saito K 1995 Detection of beta cell-specific DNA damage in streptozotocin-treated rats by in situ nick translation with immunostaining of alpha cells. Pancreas 10:354–359[Medline]
24. Solimena M 1998 Vesicular autoantigens of type 1 diabetes. Diabetes Metab Rev 14:227–240[CrossRef][Medline]
25. Ziegler B, Augstein P, Luhder F, Northemann W, Hamann J, Schlosser M, Kloting I, Michaelis D, Ziegler M 1994 Monoclonal antibodies specific to the glutamic acid decarboxylase 65 kDa isoform derived from a non-obese diabetic (NOD) mouse. Diabetes Res 25:47–64[Medline]
26. Bieg S, Hanlon C, Hampe CS, Benjamin D, Mahoney CP 1999 GAD65 and insulin B chain peptide (9–23) are not primary autoantigens in the type 1 diabetes syndrome of the BB rat. Autoimmunity 31:15–24[Medline]
27. Bjork E, Kampe O, Andersson A, Karlsson FA 1992 Expression of the 64 kDa/glutamic acid decarboxylase rat islet cell autoantigen is influenced by the rate of insulin secretion. Diabetologia 35:490–493[CrossRef][Medline]
28. Inuwa IM, El Mardi AS 2005 Correlation between volume fraction and volume-weighted mean volume, and between total number and total mass of islets in post-weaning and young Wistar rats. J Anat 206:185–192[CrossRef][Medline]
29. World Health Organization 2002 Use of anticoagulants in diagnostic laboratory investigations, stability of blood, plasma and serum samples. (WHO/DIL/LAB/99.1 Rev. 2) Geneva: World Health Organization
30. Zhang H, Cheng X, Richter M, Greene MI 2006 A sensitive and high-throughput assay to detect low-abundance proteins in serum. Nat Med 12:473–477[CrossRef][Medline]

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