The Role of Glutamic Acid Decarboxylase in Insulin Dependent Diabetes Mellitus

By Amine Gebremichael

Type 1 diabetes, also referred as an insulin-dependent diabetes mellitus (IDDM) is a severe metabolic disorder which affects one in 300-400 children and whose incidence is steadily increasing throughout the world (4). Sixteen million people in the United States have diabetes mellitus. It is the leading cause of kidney failure, blindness in adults and amputations. It is a major risk factor for heart disease, stroke, and birth defects, shortens average life expectancy by up to 15 years. At present, there is no method to prevent or cure IDDM.

IDDM is an organ-specific autoimmune disease with aberrant immune response to specific beta cell autoantigens (1). IDDM is caused by T cell dependent immune mediated destruction of pancreatic beta cells and deficiency in the secretion of insulin. Insulin is essential for the cells of our body to metabolize glucose properly and function normally. The disease begins early in life and quickly becomes severe. Most people do not realize they are developing IDDM until the damage is complete. Characteristic symptoms of diabetes are excessive thirst and frequent urination that lead to the intake of large volumes of water and weight loss. The changes are due to the excretion of large amounts of glucose in the urine.

Again, IDDM is caused by progressive loss of cells in the pancreas and the beta cell death is caused by macrophages and followed by invasion of lymphocytes (7). The human immune system mistakenly targets non-foreign cells. When the immune system recognizes the presence of Glutamic acid decarboxylase (GAD), it acts by invading the beta cells of the pancreas. The presence of GAD autoantibodies has been shown to be strong predictive marker for eventual onset of IDDM.

Glutamic acid decarboxylase is an enzyme that is found in high concentrations in the pancreas, it catalyzes the conversion of L-glutamic acid into g aminobutyrate (GABA) and carbon dioxide via an irreversible reaction (Fig. 1) (2). GABA is an inhibitory neurotransmitter that is found in pancreas and other parts of the body and considered to have a primary role as a signaling molecule in the pancreatic islets (islets are a small scattered endocrine glands in the pancreas that secrete insulin) (3).

Figure 1. Catalyzes of L-glutamic Acid to g-Aminobutyrate

GAD is present in pancreatic beta cells and has two different isoforms, GAD65 and GAD67. Both isoforms depends on the co-factor pyridoxal phosphate (PLP) for full enzymatic activity but they differ in their molecular weights, their affinities for PLP and their postulated cellular function (4). Both forms of GAD require the cofactor PLP for activity and the interaction of GAD with PLP plays a central role in the regulation of GAD activity. Most of the GAD is present without a cofactor, which provides a reserve of inactive enzyme that can be activated when additional GABA synthesis is required. GAD65 is more responsive to PLP than GAD67 (9). GAD65 has a molecular mass of 65kDa and GAD67 has a molecular mass of 67kDa and GAD65 is predominant in pancreas of human than GAD67. Both isoforms have carboxyl and amino terminal ends and that are encoded by two different genes located on different chromosomes. The amino acid sequences of GAD65 and GAD67 have 65% homology and difference between both forms of GAD exists largely in the amino-terminal region (7).

GAD65 is a major autoantigen in IDDM and thought to be involved in the destruction of insulin-secreting cells that causes IDDM. The pancreas has more GAD65 and the production of GABA exhibit high level of GAD activity. Some experiments have shown that GAD expression is necessary for the autoimmune destruction of cells. A study was done on non-obese diabetic (NOD) mice similar to human IDDM and the mice were injected with GAD antigen (suppress GAD expression in the cells) and found that IDDM did not develop (5). The T cells did not destroy the beta cells because GAD was absent. In addition, a study was done on human with IDDM and no IDDM (control group) and T cells responded more (were more active) to GAD on IDDM patients than the control group.

The T cells activated by antigens found in pancreatic beta cells cause IDDM by destroying the beta cells. There are two types of T cells that are involved in the destruction of beta cells in the pancreas, CD4 (T helper) and CD8 (cytotoxic) T cells. Both T cells are required for the activation of the islet reactive autoimmunity in the NOD mice. Once activated, the CD8 T cells appear to carry out an early function required for developing of a abnormal CD4 T cells response that destroy islet beta cells (6). When CD4 and CD8 T cells were injected to non-diabetic mice and both T cells were capable of causing diabetes. To study the relationship of GAD in IDDM, researchers performed an experiment of T-cells response to GAD expressed as stimulation index (SI) (Fig. 2) (12). SI a method researchers use to analyze how T-cells respond to GAD level in the body in patients with IDDM and control group. The result was T cells responsiveness to GAD of newly diagnosed IDDM patients was higher than the control group.

Figure 2. Distribution of T-cell responses to GAD

There is evidence that CD8 T cells directly recognize beta cells via peptide bound to cell surface major histocompatibility complex (MHC) class I protein but CD4 T cells are unlikely to recognize beta cells directly because they do not express MHC class I proteins (11). MHC I protein provide the molecular basis for non-self recognition and are found embedded in the surface of the immune system cells. Recent research shows that the percent of CD4 T cells is significantly higher in the diagnosed patients than non-diabetic controls. The CD4 fraction was increased in recent onset of IDDM compared to IDDM patients who have had the disease for a longer period (8). Scientists suspect that there might be some cooperation between CD4 and CD8 before the damage of beta cells. To this day, the mechanism of beta cell death is unknown and researchers have been studying these systems in hoping to understand the main cause.

Although there is some controversy with regard to the central role of GAD in IDDM, there is evidence that strongly show that GAD does play an important role in the development of T cell-mediated autoimmune diabetes. Experiments were done on NOD mice (similar to human IDDM) and two different results were found. First, when NOD mice were injected with purified GAD65 protein at early age, the T cell-mediated immune response system did not attack pancreatic beta cells. As a result, the NOD mice were protected from developing early diabetes. Injection of pure GAD65 does not completely prevent diabetes, but only delays the development of the disease (4,12). Second, the beta cell-specific suppression of GAD resulted in the prevention of autoimmune diabetes in NOD mice. GAD suppression inhibited the generation of diabetogenic T cells and inhibited T cell proliferative immune responses to other beta-cell autoantigens as well as to GAD (7).

There are circulating antibodies against GAD, the immune system has two main branches, the cell mediated immune system (CMI) and the humoral immune system. Immune systems are stimulated when an antigen (foreign substance) enters the body. When antigens enter the body, the antigen-presenting cells (APCs) process the antigen and present it on their surface. After the antigen is presented, the branch of the immune system get activated via its specific T helper lymphocytes. The T helper 1 lymphocytes activates CMI, which develops natural killer cells and cytotoxic T-cells that attack and destroy infected cells. On the other hand, T helper 2 lymphocytes stimulates the humoral immune system, which produce B cells. The B cells produce cells that manufacture antibodies specific to the antigens presented by the APCs (12).

The detection of anti-GAD65 and anti-GAD67 antibodies using recombinant GAD suggested that the presence of anti-GAD antibodies is a predisposing factor for the development of diabetes. Anti-GAD antibody appears to be primary against the GAD65 and the anti-GAD was detected at early stage before antibodies to beta cell autoantigens developed (7). The CMI has low level of GAD antibody and high level of T cells against GAD and humoral response has high level of GAD antibody and low level of T cells against GAD. High level of T-cell response tends to cause more destruction of beta cells. Therefore, the cell mediated immune system is responsible for high incidence of IDDM. On the other hand, the humoral immune system has low T-cells against GAD and high number of antibodies.

In this research, GAD plays an important role in the onset of IDDM. When GAD catalyzes the conversion of L-glutamic acid into g-aminobutyric acid, T-cells recognize the presence of GAD as foreign antigens. The two types of T-cells, CD4 and CD8 destroy the beta cells in the pancreas. When T-cells attack and destroy insulin producing cells, IDDM progress occurs. Both forms of GAD play different roles in human and mice. Humans have more GAD65 than GAD67 and mice have more GAD67 than GAD65. Both forms of GAD have different level of reactivity and both are more active with the presence of PLP (cofactor). The suppression of both GAD in NOD mice showed less responses from T-cells and no sign of IDDM. Even though there is some controversy with regard to the central role of GAD in IDDM, researchers concluded that GAD plays an important role in the development of T-cell mediated autoimmune diabetes. Researchers hope to have a better understanding of how GAD functions with in the beta cells in the future.

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