GABA exerts protective and regenerative effects on islet beta cells and reverses diabetes

Abstract

Type 1 diabetes (T1D) is an autoimmune disease characterized by insulitis and islet β-cell loss. Thus, an effective therapy may require β-cell restoration and immune suppression. Currently, there is no treatment that can achieve both goals efficiently. We report here that GABA exerts antidiabetic effects by acting on both the islet β-cells and immune system. Unlike in adult brain or islet α-cells in which GABA exerts hyperpolarizing effects, in islet β-cells, GABA produces membrane depolarization and Ca(2+) influx, leading to the activation of PI3-K/Akt-dependent growth and survival pathways. This provides a potential mechanism underlying our in vivo findings that GABA therapy preserves β-cell mass and prevents the development of T1D. Remarkably, in severely diabetic mice, GABA restores β-cell mass and reverses the disease. Furthermore, GABA suppresses insulitis and systemic inflammatory cytokine production. The β-cell regenerative and immunoinhibitory effects of GABA provide insights into the role of GABA in regulating islet cell function and glucose homeostasis, which may find clinical application.

Fig. 1.

GABA activates Ca²⁺–PI3K/Akt pathway in the β-cells. (A) [³H]thymidine incorporation in INS-1 cells (n = 4). (B) BrdU assay (brown) in the islet β-cells (pink) of CD1 mice injected with GABA. Two i.p. injections of GABA (20 μmol per mouse) were made within 48 h, and BrdU was injected i.p. (100 mg/kg) 6 h before the animal was killed (150–200 islets were examined; n = 5). (C) Apoptosis assay in the INS-1 cells challenged with STZ (15 mM, 24 h) in the presence of different concentrations of GABA (n = 5). (D) TUNEL (red) and insulin (green) dual staining of the isolated islets from CD1 mice, pretreated with or without GABA (100 μM, 16 h), and challenged with a mixture of cytokines (10 ng/mL IL-1β, 50 ng/mL TNF-α, 50 ng/mL IFN-γ) for 20 h (n = 5). (E) Immunoblot of p-Akt and Akt in INS-1 cells treated with GABA (100 μM) for various time intervals, in the presence or absence of bicuculline (Bic, 20 μM) or the PI3K inhibitor LY294002 (LY, 20 μM; n = 8). (F) Immunoblot of p-Akt and Akt in INS-1 cells treated with GABA (100 μM) in the presence or absence of Bic or the Ca²⁺ channel blocker nifedipine (Nif, 10 nM) for 2 h (n = 4). (*P < 0.05, **P < 0.01 vs. control groups.)

Fig. 2.

GABA produces membrane depolarization in β-cells. Representative traces show GABA-evoked currents (I_GABA, A) and GABA-induced depolarization (intracellular sharp recordings) (B) in INS-1 cells. (C) Current-clamp recording (at membrane potential of −60 mV) of INS-1 cells in the presence of GABA (100 μM) with or without Bic (100 μM). Intracellular Ca²⁺ measurements in INS-1 cells (D) and isolated islet β-cells (E) in the presence of GABA or GABA_AR agonist muscimol (10 μM) (E) with or without Bic. (*P < 0.05 and **P < 0.01; n = 3–5.)

Fig. 3.

GABA preserves β-cell mass and prevents diabetes in MDSD and NOD mice. (A) Immunohistochemistry of islet β-cells (green) and α-cells (red) in the MDSD mice that received daily saline solution or GABA injections. Analysis of β-cell mass (B) and α-cell mass (C) in GABA- or saline solution-treated MDSD mice (in non–STZ-injected mice, β-cell mass was 1.78 ± 0.25; α-cell mass was 0.22 ± 0.03). (D) Daily i.p. injection of GABA (20 μmol per mouse) prevented STZ-induced (40 mg/kg for 4 d) diabetic hyperglycemia in CD1 mice. (E) i.p. glucose tolerance test (IPGTT) was performed in MDSD mice treated with or without GABA, or in these mice before the STZ injections (Ctrl). (F) Immunostaining for insulin (brown) and glucagon (black) in pancreatic sections of NOD mice (13 wk of age) that received injections of saline solution or GABA; the severity of insulitis was scored on H&E-stained slides. (G) Blood glucose measurement of the NOD mice during the feeding course. (H) Islet immunohistochemistry of insulin (red) and glucagon (black) in the NOD mice at 23 wk of age treated with PBS solution or GABA. (I) IPGTT performed in the NOD mice at 23 wk of age. IPGTT was performed at 7 wk of age before the onset of diabetes as control (Ctrl). (J) Blood glucose measurement of TCR-8.3 NOD mice during the feeding course (n = 10). (K) β-Cell mass measurement of TCR-8.3 NOD mice. (L) Diabetogenic TCR-8.3 NOD CD8⁺ T cells were cocultured with irradiated antigen-presenting cells as described in Experimental Procedures and stimulated with a peptide mimotope. T-cell proliferation was measured by MTT assay at 72 h. (*P < 0.05 and **P < 0.01; n = 5–8.)

Fig. 4.

GABA restores β-cell mass and reverses diabetes in MDSD and NOD mice. (A) Staining of insulin (brown) and glucagon (black) in pancreatic sections of hyperglycemic MDSD mice (n = 8) receiving daily injections of GABA or saline solution (red arrows indicate infiltrating lymphocytes). (B) β-Cell mass measurement of the MDSD mice. (C) Blood glucose measurement in established diabetic MDSD mice that received daily GABA or saline solution injections during the feeding course (n = 8). Circulating insulin (D) and glucagon (E) levels of the MDSD mice measured by RIA. (F) Blood glucose measurement in the course of administration of GABA or saline solution in hyperglycemic NOD mice (the injections started when the blood glucose level was >12 mM; n = 6–10). (G) IPGTT performed in the NOD mice before the animals were killed (n = 6). (H) β-Cell mass measurement in the NOD mice (n = 5) that received chronic GABA or saline solution injections. (*P < 0.05 and **P < 0.01.)

Fig. 5.

GABA exerts anti-inflammatory and immune regulatory effects in mice. Circulating levels of the inflammatory cytokines IL-1β (A), TNF-α (B), IFN-γ (C), IL-12 (D), IL-6 (E), and IL-10 (F) in the MDSD mice by using multiplex bead-based cytokine assay. IL-12 release (determined by ELISA) of LPS⁺IFN-γ–stimulated splenic adherent cells (macrophage and DCs), with or without GABA (G). IFN-γ release (by ELISA) of cultured splenic CD4⁺ (H) or CD8⁺ T cells (I) stimulated with anti-CD3 and anti-CD28 antibodies, with or without GABA. (J) Measurement of TGF-β1 in cultured splenic T cells stimulated with anti-CD3⁺anti-CD28 antibodies, or not stimulated (Ctrl), in the presence or absence of GABA. (K) Evaluation of TGFβRI expression by flow cytometry in activated CD4⁺ T cells in the presence or absence of GABA, with or without GABA_AR antagonist picrotoxin (Pic). (*P < 0.05; **P < 0.01, n = 5–8.)

Fig. 6.

Model shows GABA_AR–Ca²⁺–PI3K/Akt pathway in mediating GABA-induced tropic effects in β-cells (A). (1) GABA activates GABA_AR Cl⁻ channel in an autocrine fashion. (2) Cl⁻ efflux leads to membrane depolarization. (3) Activation of VDCCs and subsequent Ca²⁺ influx. (4) Activation of Ca²⁺-dependent PI3-K/Akt signaling. (5) Cl⁻-dependent insulin secretion. (6) Insulin-stimulated activation of PI3K/Akt signaling. The tropic effects mediated by GABA_BR (47) are not displayed. GABA dose-dependently enhances insulin secretion from INS-1 cells, which is blocked by bicuculline (Bic, 20 μM) and picrotoxin (Pic, 100 μM) (B). GABA enhances insulin-simulated Akt phosphorylation in INS-1 cells, which can be blocked by Bic and LY294002 (LY, 20 μM) (C). (*P < 0.05 and **P < 0.01, n = 5.)