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. 2014 Sep;63(9):3128-34.
doi: 10.2337/db13-1385.

Combined Therapy With GABA and Proinsulin/Alum Acts Synergistically to Restore Long-Term Normoglycemia by Modulating T-cell Autoimmunity and Promoting β-Cell Replication in Newly Diabetic NOD Mice

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Free PMC article

Combined Therapy With GABA and Proinsulin/Alum Acts Synergistically to Restore Long-Term Normoglycemia by Modulating T-cell Autoimmunity and Promoting β-Cell Replication in Newly Diabetic NOD Mice

Jide Tian et al. Diabetes. .
Free PMC article

Abstract

Antigen-based therapies (ABTs) fail to restore normoglycemia in newly diabetic NOD mice, perhaps because too few β-cells remain by the time that ABT-induced regulatory responses arise and spread. We hypothesized that combining a fast-acting anti-inflammatory agent with an ABT could limit pathogenic responses while ABT-induced regulatory responses arose and spread. γ-Aminobutyric acid (GABA) administration can inhibit inflammation, enhance regulatory T-cell (Treg) responses, and promote β-cell replication in mice. We examined the effect of combining a prototypic ABT, proinsulin/alum, with GABA treatment in newly diabetic NOD mice. Proinsulin/alum monotherapy failed to correct hyperglycemia, while GABA monotherapy restored normoglycemia for a short period. Combined treatment restored normoglycemia in the long term with apparent permanent remission in some mice. Proinsulin/alum monotherapy induced interleukin (IL)-4- and IL-10-secreting T-cell responses that spread to other β-cell autoantigens. GABA monotherapy induced moderate IL-10 (but not IL-4) responses to β-cell autoantigens. Combined treatment synergistically reduced spontaneous type 1 T-helper cell responses to autoantigens, ABT-induced IL-4 and humoral responses, and insulitis, but enhanced IL-10 and Treg responses and promoted β-cell replication in the islets. Thus, combining ABT with GABA can inhibit pathogenic T-cell responses, induce Treg responses, promote β-cell replication, and effectively restore normoglycemia in newly diabetic NOD mice. Since these treatments appear safe for humans, they hold promise for type 1 diabetes intervention.

Figures

Figure 1
Figure 1
Longitudinal blood glucose levels in newly diabetic NOD given GABA or proinsulin/alum monotherapy or combined treatment. Newly diabetic NOD mice (with two blood glucose levels >250 mg/dL recorded on consecutive days) were untreated (n = 6) (A) or were continually given GABA, 6 mg/mL (n = 7) (B), or GABA, 20 mg/mL (n = 10) (C), through their drinking water. Other groups of mice received proinsulin/alum monotherapy (n = 7) (D) or combined proinsulin/alum + GABA, 20 mg/mL (n = 9) (E). Four of the mice shown in E remained normoglycemic at 20, 30, 45, and 50 weeks after initiating treatment (at the time of manuscript proofing). Data shown are longitudinal blood glucose levels for individual mice. Note the change of scale in C and E. The dashed line indicates a blood glucose level of 250 mg/dL. Combined therapy significantly prolonged the period of normoglycemia compared with GABA (20 mg/mL) monotherapy (P = 0.001 by the log-rank test).
Figure 2
Figure 2
Effect of monotherapy and combined therapy on T-cell immunity and humoral responses. NOD mice at 15 weeks of age were treated with alum, proinsulin/alum, GABA, or proinsulin/alum + GABA, as described in Research Design and Methods. A control group of mice received no treatment. Ten days after completing treatment, their splenic T cells were isolated, and the frequency of T cells secreting IFN-γ (A), IL-4 (B), or IL-10 (C) in response to MSA, proinsulin, GAD65, or HSPp277 was determined by ELISPOT. Responses to control MSA were at background levels in all mice (data not shown). Data are expressed as the mean number of spot-forming cells (SFCs) ±SEM per million splenic mononuclear cells. D: The levels of proinsulin-specific IgG, IgG1, and IgG2a antibodies, as determined by ELISA. Data are expressed as the mean optical density (O.D.) value ± SEM for each group. E: Effect of monotherapy and combined therapies on Treg responses, as determined by flow cytometry. Data are expressed as the mean percentage of Tregs ± SEM. The experimental and control groups of mice (n = 5 per group) were tested simultaneously in two separate experiments. *P < 0.05, **P < 0.01 vs. the control. #P < 0.05, ##P < 0.01 vs. the proinsulin/alum group. †P < 0.05, ††P < 0.01 vs. the GABA group.
Figure 3
Figure 3
Effect of monotherapy and combined therapy on insulitis, β-cell replication, and the percentage of insulin+ cells per islet in newly diabetic NOD mice. Newly diabetic NOD mice were randomized to receive water without GABA (untreated) or water containing GABA (20 mg/mL) and/or intraperitoneal proinsulin/alum (n = 6 mice per group). Additionally, their water contained BrdU. After 10 days, their pancreata were analyzed for insulitis or β-cell replication. At least 12–15 islets from two sections (with an interval of 150 µm) of each pancreatic tissue were examined in a blinded manner. A: Insulitis scores. *P < 0.05 vs. untreated, †P < 0.05 vs. the GABA monotherapy group. B: Representative image of anti-Ki67 (red) and anti-insulin (green) staining cells in an islet from a mouse that had been treated with combined therapy (original magnification ×400). Arrows indicate Ki67+insulin+ cells. Scale bar = 50 μm. C: The percentages of Ki67+insulin+ β-cells in total insulin+ β-cells. Data are expressed as the mean ± SEM of the percentages of Ki67+insulin+ islet cells in different groups of mice. Similar results were observed using BrdU/insulin immunostaining (data not shown). D: The number of insulin+ cells per islet. †P < 0.05 vs. the GABA monotherapy group.

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