consumption was measured weekly (Fig. 1A and B). There was no significant difference in the mean amount of food intake and water consumption of individual mice between the water and GABA- treated groups of mice, consistent with our previous observations in a RA-prone stain of mice [18]. Although the body weights of individual mice in both groups of mice gradually increased following HFD-feeding, the mean body weights in the GABA- treated mice at 10 weeks post GABA treatment were significantly less than that of the water-fed mice (Fig. 1C) and remained significantly less than that of controls throughout the subsequent observation period. These data indicated that oral treatment with GABA did not significantly alter the food and water consumption, but reduced the HFD-associated gain in mouse body weight. Oral treatment with GABA improves glucose tolerance and insulin sensitivity in HFD-fed mice During the study period, we measured the concentrations of fasting blood glucose, glucose tolerance and insulin sensitivity in the water-fed control and GABA-fed mice longitudinally. We found that the concentrations of fasting blood glucose gradually increased in control mice and some mice at 20 weeks post HFD developed T2DM. In contrast, the concentrations of fasting blood glucose in mice that received GABA remained at a similar level throughout the observation period (Fig. 2A). As a result, the concentrations of fasting blood glucose in the GABA-fed mice were significantly lower than that of controls at 9 weeks post HFD (p,0.05). Similarly, oral feeding with GABA inhibited the development of glucose intolerance in the HFD-fed mice (Fig. 2B). Furthermore, while challenge of the control mice with insulin only resulted in moderate reduction in the concentrations of blood glucose, an indication of insulin resistance, challenge with insulin dramatically reduced the concentrations of blood glucose in the GABA-fed mice. Together, these data clearly demonstrated that oral treatment with GABA inhibited the HFD-induced glucose intolerance and insulin resistance in mice. Treatment with GABA improves glucose tolerance and insulin sensitivity in mice after the onset of obesity and T2DM To test whether treatment with GABA could modulate 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 glucose, glucose tolerance, and insulin sensitivity were measured. Individual mice with obesity (body weight . 48 g) and GABA Treatment for High Fat Diet-Fed Mice T2DM (abnormal fasting blood glucose and impaired glucose tolerance and insulin sensitivity, see Materials and Methods) were randomized and fed with water as controls or with water containing GABA (2 mg/ml) for another 12 weeks. Their body weights, fasting blood glucose levels, glucose tolerance, and insulin sensitivity were measured longitudinally. As shown in Fig. 3, mice that had been continually fed with HFD and plain water gradually increased their body weights while the mice fed with HFD and water containing GABA gained little or no body weight (Fig. 3A). More importantly, following treatment with GABA, all of diabetic mice became euglycemic and the mean concentrations of fasting blood glucose in the GABA-treated mice were significantly lower than that of controls at 32 weeks post HFD (p,0.05, Fig. 3B). In addition, treatment with GABA improved glucose intolerance and insulin sensitivity in the HFD-fed mice (Fig. 3C and D). While both groups of mice displayed similar levels of peak blood glucose after glucose challenge, the GABA-treated mice exhibited near normal levels of blood glucose at 2 hour post glucose challenge. In contrast, the concentrations of blood glucose at 2-hour post glucose challenge in the plain water-fed mice remained hypergly- cemic. Moreover, while insulin challenge reduced the concentra- tions of blood glucose by less than 25% in the plain water-fed mice the same treatment dramatically reduced the concentrations of blood glucose by nearly 50% during the early stages of responding to insulin in the GABA-treated mice. Therefore, treatment with GABA improved glucose intolerance and insulin sensitivity in the mice with established obesity and T2DM. Treatment with GABA reduces adipocyte mass and macrophage infiltration in the adipose tissues of HFD-fed mice Previous studies have shown that macrophage-related chronic inflammation in adipose tissues is crucial for the development of insulin resistance, obesity, and T2DM [1,22]. Given that GABAA- R subunits are expressed by macrophages and adipose tissues [13,15], we further tested whether GABA could modulate macrophage-associated inflammation in adipose tissues, or alter adipose tissue mass in the HFD-fed mice. Following HFD feeding for 20 weeks, control and GABA-treated mice were sacrificed and their VAT was dissected out and weighed. The total amount of VAT in the GABA-treated mice was significantly less than that of the controls (Fig. 4A), consistent with reduced body weights. Further analysis of VAT revealed that the size of adipocytes in the GABA-treated mice was also significantly less than that of the Figure 1. Oral treatment with GABA does not alter the amounts of water and food consumed, but reduces the gain of body weights in the HFD-fed mice. Groups of C57BL/6 mice were fed with HFD and provided with plain water or water containing 2 mg/ml of GABA for 20 weeks. The amounts of water and food consumed by individual mice and their body weights were measured longitudinally. Data are expressed as mean values of water and food consumed by individual mice per day and mean values of body weights of each group of mice (n = 20 per group) at the indicated time points post-HFD. Intragroup variations were less than 10% for the amounts of water and food consumption and less than 15% for the values of body weights. (A) The amounts of water consumed; (B) The amounts of food consumed; and (C) The body weights. *p, 0.05; ** p,0.01 vs. the GABA-fed mice. doi:10.1371/journal.pone.0025338.g001 PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e25338