GABA Treatment for High Fat Diet-Fed Mice Figure 2. Oral treatment with GABA improves glucose tolerance and insulin sensitivity in HFD-fed mice. Groups of C57BL/6 mice were fed with HFD and given plain water or water containing 2 mg/ml of GABA for 20 weeks. The concentrations of fasting blood glucose, intraperitoneal glucose tolerance (IPGT), and insulin sensitivity of individual mice were tested longitudinally, as described in the Materials and Methods. Data are expressed as mean values of the concentrations of fasting blood glucose (A), the AUC of IPGT (B), and the percentages of blood glucose changes following insulin challenge (C) in different groups of mice (n = 20 for each group). *p, 0.05; ** p,0.01 vs. the GABA-fed mice. doi:10.1371/journal.pone.0025338.g002 controls (Fig. 4B). In addition, characterization of F4/80+ macrophages in the adipose tissues indicated that the percentage of macrophages infiltrated in the adipose tissues of the GABA- treated mice was significantly less than that of the controls (Fig. 4C). Collectively, these data clearly indicated that treatment with GABA reduced the adipose tissue mass and macrophage infiltrates in the adipose tissues of HFD-fed mice. Oral treatment with GABA increases the frequency of splenic Tregs in vivo The Foxp3+ Tregs are negative regulators of inflammatory responses and recent studies have indicated that Tregs can inhibit obesity-related inflammation and insulin resistance in mice [12,23]. To further understand the mechanisms underlying the actions of GABA in regulating the HFD-induced obesity and insulin resistance, groups of C57BL/6 mice were fed with plain water or water containing GABA (2 mg/ml) for 4 weeks and their splenic CD4+Foxp3+ Tregs were characterized by flow cytometry analysis. As shown in Fig. 5, there was no significant difference in the frequency of CD4+ T cells between the plain water-fed controls and GABA-treated mice. However, the frequency of CD4+Foxp3+ Tregs in the GABA-treated mice was significantly higher than that of the controls (p,0.01). Therefore, treatment with GABA increased the frequency of Tregs in mice. Figure 3. Oral treatment with GABA improves glucose tolerance and insulin sensitivity in mice after the onset of obesity and T2DM. C57BL/6 mice were fed with HFD for 20 weeks and their fasting blood glucose and IPTG were measured. Individual mice with body weight .48g, fasting glucose level of .145 mg/dL and abnormal IPTG (blood glucose .220 mg/dL at 2 hours post-IPGT) were considered to have obesity and T2DM, and randomly treated orally with plain water (control) or water containing 2 mg/ml of GABA for another 12 weeks. Their body weights, fasting glucose concentrations, IPGT and insulin sensitivity were measured longitudinally. (A) The body weights; (B) The concentrations of fasting blood glucose; (C) The dynamic changes following glucose challenge; and (D) The percentages of blood glucose concentrations following insulin challenge. Data are expressed as mean values of each group (n = 4 per group) at the indicated time points and the concentrations of blood glucose before insulin challenge in individual mice were used as 100%. *p, 0.05; ** p,0.01 vs. the GABA-fed mice. doi:10.1371/journal.pone.0025338.g003 PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e25338