GABA Treatment for High Fat Diet-Fed Mice Figure 5. Oral treatment with GABA increases the frequency of splenic Tregs in vivo. C57BL/6 mice were fed with plain water or water containing GABA (2mg/ml) for four weeks. Their splenic mononuclear cells were prepared and treated with anti-CD16/32. Subsequently, splenic mononuclear cells (106/tube) were stained in duplicate with FITC-anti-CD4 and after washing, the cells were fixed, permeabilized and stained with PE- anti-Foxp3, followed by flow cytometry analysis. The cells were stained with single fluorescent-labeled antibodies or with isotype-matched IgG as controls. Data are representative charts and expressed as mean 6 SEM of the percentage of CD4+Foxp3+ Tregs in splenic CD4+ T cells of different groups of mice (n = 3–4 mice per group) from two separate experiments. *p,0.01 vs. the water-fed mice. doi:10.1371/journal.pone.0025338.g005 with HFD and GABA had significantly less gain in body weights over the observation period. Over-consumption of calories can cause obesity and lead to the development of insulin resistance and T2DM [22,24]. We found that oral treatment with GABA significantly improved glucose intolerance and insulin resistance in HFD-fed mice. Evidentially, the levels of fasting blood glucose, blood glucose at 2 hours post-glucose challenge, and the relative levels of blood glucose post-insulin treatment in the GABA-treated mice were significantly lower than that of the control mice. More importantly, after the establishment of obesity and T2DM, oral treatment with GABA significantly reduced the gain in body weight and improved glucose tolerance and insulin sensitivity in mice. These novel data demonstrated that oral treatment with GABA not only prevented the HFD-induced obesity and T2DM development, but also inhibited the progression of obesity and T2DM in mice. Given that GABA is safe for human consumption, GABA may be valuable for the prevention and treatment of obesity and T2DM in the clinic. Recent studies have shown that adipose tissue-related chronic inflammation contributes to the development of insulin resistance, a key component of metabolic syndrome, and leads to the development of obesity and T2DM as well as other metabolic disorder-related diseases [1,22]. To understand the potential mechanisms underlying the therapeutic effect of GABA in inhibiting the HFD-induced obesity and T2DM, we examined the mass of adipose tissues and inflammatory infiltrates. In comparison with that in the control mice, oral treatment with GABA significantly reduced the total amounts of epididymal fat tissues and the size of adipocytes in the HFD-fed mice. Because macrophages are predominant players in the development of chronic inflammation in adipose tissues [10,11,25,26,27], we further characterized the frequency of macrophage infiltrates in the epididymal fat tissues. We found that oral treatment with GABA significantly decreased the frequency of macrophage infiltrates in adipose tissues of the HFD-fed mice. More importantly, GABAA receptor subunits are expressed by adipo- cytes and macrophages, and GABAA receptor agonists can inhibit macrophage activation [13,15,17]. It is possible that GABA, through the GABAA receptors, inhibits macrophage activation and migration, reducing chronic inflammation in adipose tissues. Given that inflammatory adipocytes can produce adipokines that recruit inflammatory infiltrates, such as macrophages, GABA may alternatively inhibit adipogenesis and adipokine production, indirectly limiting the macrophage infiltration into adipose tissue in the HFD-fed mice. Recent studies have highlighted the importance of glucagon in regulating glucose homeostasis [28,29]. Physiologically, GABA acts synergistically with insulin to inhibit the secretion of glucagon by the pancreatic a-cells, and reverse insulin-deficiency-related hyperglycemia in mice [29,30]. Although we can not completely exclude the possible effect of GABA-mediated inhibition of glucagon secretion, our data from islet transplant suggest that treatment with GABA alone has little ability to reverse T1D in diabetic NOD mice [19]. Hence, the effect of GABA-mediated inhibition of glucagon secretion may be minor in inhibiting HFD-induced glucose intolerance, insulin resistance, and macrophage-related inflammation. Conceivably, GABA, through its receptors, on macrophages and adipocytes, improves glucose tolerance and insulin sensitivity in our experimental model. We are interested in further investigating how activation of GABAA receptors modulates adipogenesis and macrophage activation and migration as well as adipokine and cytokine production by adipocytes. Notably, Tregs are potent inhibitors for macrophage activation and function [23]. Tregs are negative regulators of obesity-related insulin resistance and T2DM in mice [12,31]. We found that oral treatment with GABA significantly increased the frequency of splenic Tregs in C57BL/6 mice, indicating that GABA promoted Treg proliferation and maturation in vivo. Our previous study has shown that GABA, through the GABAA receptors, induces effector T cell cycle arrest, consistent with the notion that engagement of GABAA receptors induces the hyperpolarization of membrane potentials, which may inhibit the TCR-related signaling [16]. The significantly increased frequency of splenic Tregs by GABA treatment suggests that GABA, through the GABAA receptors, may promote the depolarization of membrane potentials by opening the voltage-dependent calcium channel and enhancing the TCR-triggering calcium-dependent downstream survival and growth signaling [16,32]. Given that Tregs are potent inhibitors of inflammation and insulin resistance the GABA- induced increase in Tregs may also inhibit macrophage infiltration and related chronic inflammation, contributing to the therapeutic effect of GABA in inhibiting the HFD-induced obesity and T2DM. In summary, our data demonstrated that oral treatment with GABA inhibited the HFD-induced obesity and improved glucose intolerance and insulin sensitivity, even after the establishment of obesity and T2DM in mice. Furthermore, oral treatment with GABA reduced the HFD-induced adipocyte hypertrophy and PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e25338