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Author contributions: N.S., G.J.P., and Q.W. designed research; N.S., H.Q., M.A., Y.G., F.Z.,
R.L., N.Z., R.C., T.N., H.Z., Z.-P.F., and Q.W. performed research; N.S., H.Q., M.A., Y.G., F.Z.,
R.L., Y.L., N.Z., R.C., T.N., T.J., H.Z., W.-Y.L., Z.-P.F., G.J.P., and Q.W. analyzed data; and N.S.,
G.J.P., and Q.W. wrote the paper.
Conict of interest statement: A patent application authored by N.S and Q.W. has been
submitted for an invention related to this study.
*This Direct Submission article had a prearranged editor.
1
Present address: First Teaching Hospital of Xinjiang Medical University, Urumqi 830054,
China.
PHYSIOLOGY
Edited* by Roger H. Unger, Touchstone Center for Diabetes Research, Dallas, TX, and approved June 2, 2011 (received for review February 23, 2011)
Fig. 1. GABA activates Ca2+PI3K/Akt pathway in the -cells. (A) [3H]thymidine incorporation in INS-1 cells (n = 4). (B) BrdU assay (brown) in the islet
-cells (pink) of CD1 mice injected with GABA. Two i.p. injections of GABA
(20 mol per mouse) were made within 48 h, and BrdU was injected i.p. (100
mg/kg) 6 h before the animal was killed (150200 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 M, 16 h), and challenged with a mixture
of cytokines (10 ng/mL IL-1, 50 ng/mL TNF-, 50 ng/mL IFN-) for 20 h (n = 5).
(E) Immunoblot of p-Akt and Akt in INS-1 cells treated with GABA (100 M)
for various time intervals, in the presence or absence of bicuculline (Bic, 20
M) or the PI3K inhibitor LY294002 (LY, 20 M; n = 8). (F ) Immunoblot of
p-Akt and Akt in INS-1 cells treated with GABA (100 M) 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.)
To assess whether GABA has an inhibitory effect on diabetogenic, islet cell-reactive CD8+ CTLs, we investigated its
effects in T-cell receptor transgenic NOD mice (TCR-8.3 NOD).
We found that GABA was highly effective at preventing hyperglycemia in these mice (Fig. 3J). It markedly suppressed insulitis
(Fig. S3 A and B), preserved -cell mass (Fig. 3K), enhanced Cpeptide levels, and reduced glucagon levels (Fig. S3 C and E).
Because this disease is dependent on diabetogenic CTLs, we
examined whether GABA could suppress these cells. Indeed,
in vitro, we observed that GABA inhibited the proliferation of
the TCR-transgenic CD8+ T cells induced by a peptide mimotope (Fig. 3L). Although this suppression was partial, it was
signicant.
Soltani et al.
Fig. 4. GABA restores -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 inltrating lymphocytes). (B)
-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 = 610). (G) IPGTT performed in the NOD mice before
the animals were killed (n = 6). (H) -Cell mass measurement in the NOD mice
(n = 5) that received chronic GABA or saline solution injections. (*P < 0.05
and **P < 0.01.)
PHYSIOLOGY
Fig. 6. Model shows GABAARCa2+PI3K/Akt pathway in mediating GABAinduced tropic effects in -cells (A). (1) GABA activates GABAAR Cl channel in
an autocrine fashion. (2) Cl efux leads to membrane depolarization. (3)
Activation of VDCCs and subsequent Ca2+ inux. (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 GABABR (47) are not displayed. GABA dose-dependently enhances insulin
secretion from INS-1 cells, which is blocked by bicuculline (Bic, 20 M) and
picrotoxin (Pic, 100 M) (B). GABA enhances insulin-simulated Akt phosphorylation in INS-1 cells, which can be blocked by Bic and LY294002 (LY,
20 M) (C). (*P < 0.05 and **P < 0.01, n = 5.)
Soltani et al.
Soltani et al.
PHYSIOLOGY
Immunoblotting, Immunostaining, and - and -Cell Mass Measurement. Immunoblotting, tissue process, and islet cell mass analysis were performed as
we described previously (27). Information on the antibodies used is provided
in SI Experimental Procedures.
Lymphocytic Stimulation in Vitro Flow Cytometry Analysis. Spleen cell isolation, in vitro culture, and ow cytometry analysis were performed as described previously (39). The antibodies used and the lymphocytic stimulation
in vitro assays were performed as described in SI Experimental Procedures.
Statistical Analysis. All data are presented as mean SEM. Statistical analysis
was done by a Student t test or ANOVA with Tukey post-hoc test as appropriate. Signicance was assumed at a P value of less than 0.05.
Experimental Procedures
Animal Handling and Tissue Processing. Male CD1 mice, female NOD/Lt mice,
and female TCR-8.3 NOD mice (Jackson Laboratory) were housed under
a light/dark cycle of 12 h with free access to food and water. All procedures
were approved by the institutional animal care committee. Animal handling
and drug injections are described in SI Experimental Procedures.
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