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The Vollum Institute for Advanced Biomedical Research Oregon Health and Science University Portland, Oregon 97239
Address all correspondence and requests for reprints to: Simon A. Hinke, The Vollum Institute, Oregon Health and Science University (MRB322/L-474), 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97239. E-mail: hinkes{at}ohsu.edu.
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Introduction |
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 Top Introduction GAD65 and the ß-CellGAD65 as a Marker...Potential Impact of Early...References |
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Regrettably, the diagnosis of diabetes is often made at a late stage of disease progression, once the complications of chronic hyperglycemia have emerged and well after the initial ß-cell injury has taken place. Methods to detect early markers of ß-cell death have clear applications for the diagnosis and prevention of diabetes as well as monitoring the engraftment or rejection of islet transplants. In this issue of Endocrinology, Waldrop et al. (3) describe the use of an assay to detect circulating glutamic acid decarboxylase 65 (GAD65) to successfully track ß-cell death resulting from cell-type-specific toxins. This report marks the first time an assay of this kind has been applied in vivo using rats, overcoming obstacles preventing similar approaches in the past. The most remarkable finding of the study was the ability to detect ß-cell death at a time point well before the onset of hyperglycemia. It is hoped that this assay, and perhaps others like it, will facilitate a better understanding of the earlier stages of diabetes as well as permitting intervention strategies to prevent or limit the severity of the disease.
The primary thrust of the paper by Waldrop and colleagues (3) was to provide proof-of-principle under controlled conditions for the application of their sensitive GAD65 assay (4) to in vivo experiments with selective cytotoxic compounds. Two ß-cell-specific toxic compounds, alloxan and streptozotocin (STZ), were used at high doses to induce severe ß-cell destruction in Wistar rats, and markers of ß-cell function and integrity were monitored over time. Both toxins caused acute release of GAD65 into the peripheral circulation and concomitant induction of hyperglycemia; in the same animals, circulating insulin and C-peptide (a cosecreted peptide from insulin processing that remains in the circulation for a longer duration) were significantly depressed (3). To assess whether GAD65 release was proportional to the degree of injury, graded doses of STZ were administered to rats, and serum glycemia and GAD65 were measured at 6 and 24 h after injection. At 6 h, STZ caused a clear concentration-dependent shedding of GAD65 into the circulation; however, at the same time, it did not produce consistent effects on glycemia, which remained relatively close to basal values. By 24 h, overt hyperglycemia (>16 mM) was observed for all STZ doses 40 mg/kg and above, whereas variable but significantly elevated serum GAD65 was observed (3).
At the low sub-diabetogenic dose of 20 mg STZ/kg, significantly elevated GAD65 was measured in the absence of hyperglycemia. This observation implies that use of GAD65 as a ß-cell injury marker may be applied to detect mild insults to insulin-secreting cells. A series of experiments examining apoptosis in pancreatic sections from STZ-treated rats was used to correlate the severity of cell death to GAD65 release. Low-dose STZ was found to neither alter islet morphology nor induce programmed cell death, yet circulating GAD65 was significantly raised in these animals at 6 and 24 h. As expected, diabetogenic doses of STZ caused massive ß-cell loss and rearrangement of islet architecture (3). These promising results lend support for use of this assay to study the initiation of diabetes in animal models and human subjects, potentially allowing examination of triggering events before onset of hyperglycemia.
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GAD65 and the ß-Cell |
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TopIntroductionGAD65 and the ß-CellGAD65 as a Marker...Potential Impact of Early...References |
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-aminobutyric acid (GABA) from glutamate in neurons and islet endocrine cells (5). GABA is a well-known inhibitory neurotransmitter in the central nervous system, but less is known regarding its function in islets. Current research suggests GABA is localized to synaptic-like microvesicles, which may have differences in stimuli for regulated release, compared with insulin-containing large dense-core vesicles (6). Given the identification of both GABAA and GABAB receptors in islet endocrine cells (7, 8), it is largely believed that GABA functions as an autocrine and/or paracrine modulator of islet hormone release.
There are two main forms of GAD encoded by unique genes in mammals, GAD65 and GAD67; only trace amounts of either enzyme are found in adult human tissues other than brain and endocrine pancreas (9). Expression of GAD in islets of Langerhans exhibits species differences for the two isoforms; human 
-, ß-, and 
-cells express only GAD65, rat islets produce both GAD65 and GAD67, whereas mouse islets synthesize GAD67 almost exclusively (9, 10). In rodent islet tissue, the GAD65 is loosely membrane associated via acylation, whereas GAD67 appears to be cytosolic (11).
The GAD65 isoform has long interested immunologists, because autoantibodies to GAD65 are detected in up to 80% of T1DM patients and can be considered predictive of the disease (12). The triggering events leading to GAD65 autoantibody formation in T1DM are still not entirely clear. One plausible hypothesis proposes an initial insult to ß-cells causing the release of GAD65 into the extracellular space, thus activating immune T cells; however, molecular mimicry of viral epitopes has also been put forth as a mechanism of autoimmunity (13).
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GAD65 as a Marker of ß-Cell Death |
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TopIntroductionGAD65 and the ß-CellGAD65 as a Marker...Potential Impact of Early...References |
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Two main issues have hampered the use of GAD65 assays in vivo to learn about early stages of islet damage: 1) sensitivity of the assays available and 2) assay interference by autoantibodies. The GAD65 RIA has reached a lower limit of detection of 1–3 ng/ml (17), and time-resolved fluorescent immunoassay improved this limit 10-fold to 0.1–0.33 ng/ml (20). Waldrop et al. (4) recently developed a novel magnetic bead-based assay to capture GAD65 with a monoclonal antibody directed at a region of the molecule distant from known autoantibody epitopes. This assay obtained a detection limit of 31–56 pg/ml and was equally effective in plasma from GAD65 autoantibody-positive or -negative patients (3, 4).
The question that everyone is asking is whether GAD65 can be used in vivo to detect early islet damage. In preclinical studies, use of a GAD enzymatic assay as a marker of islet auto- and allotransplantation rejection was performed in canine models with temporary immunosuppression. Using a direct 14CO2-release GAD65 assay, Shapiro and colleagues (21) achieved limited success in detecting islet graft rejection but were convinced of the potential of serological detection of islet-specific proteins before the onset of hyperglycemia. Now with a much more sensitive approach to measure GAD65, Chessler’s group has completed preliminary proof-of-concept experiments that seem to imply that the answer to the question is yes (3). Acute diabetogenic doses of STZ or alloxan produced rapid spikes in plasma GAD65 within 24 h; low-dose STZ, a mild insult model that can trigger progressive islet destruction, caused a gradual increase in plasma GAD65 over the course of a day. A concentration-dependent shedding of GAD65 was observed with STZ treatment at a time point before onset of hyperglycemia (3).
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Potential Impact of Early ß-Cell Injury Detection on Diabetes Therapy |
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TopIntroductionGAD65 and the ß-CellGAD65 as a Marker...Potential Impact of Early...References |
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Little is known regarding the earliest stages of TIDM initiation; however, genetic predisposition and extrinsic influences both have roles. Insulitis, islet infiltration by leukocytes, is commonly clinically observed in histopathological tissue analysis of autopsy and biopsy specimens from young recent-onset T1DM pancreata (25). It is theorized that cytokines, chemokines, and/or reactive oxygen species from the macrophages or damaged islet cells themselves precipitate the induction of the apoptotic program (14, 15). The destruction of the ß-cell mass is accelerated by the generation of autoimmune antibodies to insulin, GAD65, IA-2, and phogrin; worsening glucose tolerance may also contribute to ß-cell stress and possibly direct glucose toxicity at later stages (26, 27). At the time of diagnosis, it is common for over 70% of ß-cells to have been already lost (14).
Prevention of T1DM has been the focus of several large randomized multicenter trials. For T1DM, it has been shown that intervention with immunosuppressants at the time of disease diagnosis prolonged ß-cell function; however, benefits did not persist after cessation of treatment (28). Nicotinamide administration also demonstrated beneficial results toward halting progression of islet destruction in animal models of diabetes and human T1DM, but in the large-scale European Nicotinamide Diabetes Intervention Trial (ENDIT), subjects of the study showed identical disease development, regardless of treatment group (29). Preservation of residual ß-cell function during intensive insulin treatment (22, 23) was the basis for a similar diabetes prevention trial (DPT-1) examining the effect of oral and parenteral insulin. Unfortunately, progression of diabetes was not able to be prevented (30). Additional interventional strategies have shown promise but have not yet been tested in larger populations (28). The scale of these prevention trials is the primary logistic concern; however, the timing of the treatment is also certainly one of the largest obstacles. Inclusion criteria based on autoantibody positivity postulates disease progression is preventable after the initial insult has occurred. Clearly, the ability to detect the initial injury would allow the earliest possible intervention and likely the greatest chance for successful prevention.
Typically, T1DM requires lifelong insulin injections for survival; the quality of life of T1DM patients is reduced because of the need for painful injections and secondary complications from inferior glycemic control. Improvements in clinical islet transplantation have offered the promise of insulin independence, if only temporarily (31). However, the secondary effects of continuous immunosuppression necessary to prevent graft rejection may outweigh any benefit in quality of life for patients. The widespread use of this treatment is impeded not only by the availability of donor tissue but also by the high degree of apoptotic tissue loss during the isolation and engraftment of islet tissue (32). Currently, islet graft function is assessed by glycemic control and C-peptide secretion, but these parameters can be used to detect only the late stages of graft rejection (31). Here, a rapid cell viability marker specific to ß-cells would allow systematic optimization of the islet isolation and transplantation procedure, focusing on preserving the amount of functional secretory tissue.
Heredity also has a prominent impact on T2DM etiology, together with environmental factors, such as those brought about by a sedentary lifestyle. In health, islet mass is tightly controlled by expansion and involution to reach an equilibrium of ß-cell neogenesis, replication, and apoptosis to maintain normoglycemia (33). Glucose intolerance progresses to T2DM when insulin action fails to adequately reduce blood glucose to the normal range, usually the result of defective glucose sensing by the ß-cells and resistance of target tissues due to chronic hyperinsulinemia. As the disease worsens in severity, prolonged exposure to elevated circulating glucose and lipids can result in glucotoxicity and/or lipotoxicity to ß-cells (33). Postmortem analysis of ß-cell mass in T2DM can be as much as 60% lower than matched nondiabetic control patients (34). Although the risk of developing T2DM can be greatly diminished when diet and exercise routines are implemented (35, 36), intervention trials to prevent disease development in high-risk patients through the use of pharmaceutical drugs have also been considered (37, 38). Adequate glycemic control in T2DM can frequently be achieved with current oral drugs; however, patients eventually become refractory to these treatments, ultimately requiring insulin injections. The ability to detect islet apoptosis with a serum marker would allow tailoring the treatment regimen with the goal of preventing late-stage loss of ß-cell mass in individuals diagnosed with T2DM.
Although this new sensitive GAD65 assay shows great promise, whether it will be applicable to human disease prediction and prevention remains to be seen. The circulating half-life of GAD65 will have a great impact on the potential utility of it as an early marker. Waldrop et al. (3) estimated a half-time of elimination of just under 3 h for recombinant GAD65, and high-dose STZ resulting in near complete ß-cell destruction caused a spike within 6 h and a rapid return toward baseline but remaining statistically elevated for over 18 h. Asynchrony of ß-cell death in humans is suggested by the long duration over which it occurs in T1DM and the persistence of a small number of ß-cells in later stages of diagnosis (39). Mild acute STZ injury of ß-cells caused a progressive appearance of circulating GAD65 in rats (3); however, it is unclear whether the prolonged shedding of GAD65 in the progression of human T1DM will be detectable.
Unfortunately, because of species differences in GAD65 expression (10), this assay cannot be used directly in all murine models of spontaneous diabetes (e.g. the NOD mouse) or transgenic mice with abnormal glucose homeostasis. However, the next phase of preclinical experiments using this assay will provide the greatest insight into the applicability of this assay to the human state: what is the feasibility of measuring plasma GAD65 in spontaneous models of ß-cell death? The BB rat model spontaneously develops a type-I like diabetic phenotype due to autoimmune islet destruction, and the ZDF Fatty Zucker rat is a genetic model of severe obesity leading to a type-II-like diabetic phenotype, with late stage gluco- and lipotoxicity (40). If the technical challenges of such a longitudinal study can be met, and the GAD65 assay proves to offer an advantage over traditional glucose tolerance, C-peptide, and insulin measurements, it will be a unique means to study the first stages of disease progression.
In conclusion, the development of an assay for an early marker of islet injury and death has clear direct applications in clinical medicine. The GAD65 assay, or others like it, may be used to better optimize islet isolation and handling during transplantation and selection of appropriate immunosuppressive drugs to maximize engraftment and minimize graft rejection. Assays of this kind could be applied to detect and limit ß-cell destruction in T2DM patients as they become refractory to existing treatment regimens. This type of assay may be used to test diabetes-prone first-degree relatives of T1DM and T2DM patients to permit prevention or to allow the earliest detection and treatment of diabetes. In basic science, assay of early islet destruction could be used for selecting experimental treatments to selectively block ß-cell death in diabetic models and to better understand the first stages of spontaneous diabetes in appropriate rodent strains. Although it is premature to conclude the GAD65 assay can be used directly for these studies, it represents a leap in the right direction, and very well may.
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Footnotes |
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Disclosure Statement: The author has nothing to disclose.
Abbreviations: GABA, -Aminobutyric acid; GAD, glutamic acid decarboxylase; STZ, streptozotocin; T1DM, type I diabetes mellitus; T2DM, type II diabetes mellitus.
Received June 27, 2007.
Accepted for publication July 11, 2007.
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References |
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TopIntroductionGAD65 and the ß-CellGAD65 as a Marker...Potential Impact of Early...References |
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