Induction of Pancreatic beta Cell Stress by Functional Anti-Voltage-Gated Calcium Channel Autoantibodies in Type 1 Diabetes

Author: Ahmed Alshehri

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Alshehri, Ahmed, 2019 Induction of Pancreatic beta Cell Stress by Functional Anti-Voltage-Gated Calcium Channel Autoantibodies in Type 1 Diabetes, Flinders University, College of Medicine and Public Health

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The production of autoantibodies (Abs) is a key feature of type 1 diabetes. Typically against internal components of the insulin response in beta (β) cells, as yet a direct role for humoral autoimmunity in the pathogenesis of β cell loss is yet to be established. Recently, a functional autoantibody (Ab) derived from the serum of patients with T1D and targeting L-type voltage gated calcium channels (VGCCs) has been demonstrated to disrupt autonomic regulation of visceral smooth muscle in whole organ assays of murine colon, bladder and vas deferens. The functional Ab was reported to act as an agonist at the dihydropyridine (DHP) site of VGCCs following both ex vivo addition and passive transfer to mice. Interestingly, positivity in serum for the functional Ab was reported to be correlated with symptoms in patients of diabetic autonomic dysfunction (DAN), suggesting these Abs may play a role in extraglandular symptoms of autonomic dysregulation in patients.

More recently, these Abs have been demonstrated to exert effects on β cells, in which VGCCs play a key role in the insulin response. Specifically, functional anti-VGCC Abs have been reported to induce intracellular calcium (Ca2+) dysregulation, altered mitochondrial membrane potential, and apoptosis in cultured murine β cells. As yet, a mechanism linking Ab-mediated Ca2+ dysregulation and apoptosis in β cells is yet to be established.

In this thesis, a novel cell-based assay of reactive oxygen species (ROS) stress induction in the rat insulinoma cell line, Rin A12, was established to investigate the role of anti-VGCC Abs in mediating β cell apoptosis. Using the 2’, 7’-Dichlorofluorescein diacetate (DCF-DA) fluorescence method for ROS detection, and a flow cytometry method for the selective detection of increased ROS in viable cells, it was demonstrated that incubation of Rin A12 cells with immunoglobulin G (IgG) containing anti-VGCC Abs from patients with T1D significantly increased the ROS, with subsequent induction of apoptosis, as determined by positivity of annexin V expression. Neither T1D patient-derived IgG lacking anti-VGCC Abs activity, nor IgG from healthy donors altered ROS or annexin V expression, indicating the new assay is specific to the detection of functional anti-VGCC Abs. Furthermore, the cell-based ROS assay was successfully used to screen a cohort of patients with T1D indicating a prevalence of anti-VGCC Abs of approximately 75 %. In addition, the effects of the functional Ab on ROS generation was blocked by co-incubation of cells with the DHP antagonist, nicardipine, and by pre-absorption of functional Ab using Ig from healthy individuals or patients negative for anti-VGCC Abs, presumably due to the presence of anti-idiotypic Abs. As such, the assay may offer a method for identification of small molecule blockers which might provide a method of treatment for patients

In summary, the cell-based ROS assay provides, for the first time, evidence supporting a plausible pathophysiological mechanism underlying anti-VGCC Ab-mediated apoptosis induction in β cells, which being ROS-mediated apoptosis secondary to increased Ca2+ flux. While the proposed hypothesis of this thesis was supported by the present results, other potential effects on β cell function, particularly insulin release, resulting from functional anti-VGCC warrant further exploration. Given that the assay provides both a method of detection and an indication of pathogenicity arising from the functional activity of the antibodies, the assay should facilitate a greatly improved understanding of the prevalence, timing and consequences of anti-VGCC Abs in patients with T1D.

Keywords: type 1 diabetes; autoantibodies, voltage-gated calcium channels, reactive oxygen species, beta cells, apoptosis

Subject: Clinical Immunology thesis

Thesis type: Doctor of Philosophy
Completed: 2019
School: College of Medicine and Public Health
Supervisor: Michael W Jackson