GABA Treatment for High Fat Diet-Fed Mice Figure 4. Oral treatment with GABA reduces adipocyte mass and macrophage infiltration in the adipose tissues of 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 mice were sacrificed and the amounts of VAT in individual mice were measured. One portion of the VAT was fixed with 4% paraformaldehyde for 24 hours and the VAT sections at 5 mm were stained with Toluidine blue O, followed by examination under a light microscope. The sizes of adipocytes in 5 sections of individual mice from each group (n = 8) were examined in a blinded fashion. Another portion of VAT was fixed with the Bouin buffer for 48 hours and the VAT sections were subjected to immunohistochemistry analysis of infiltrated macrophages using anti-F4/80 antibodies and DAB substrate (brown), and the percentages of macrophages in 400 nuclear cells from 5 sections of each mouse in individual groups of mice were quantified in a blinded manner. Data are representative images of the adipocytes, macrophages stained and expressed as mean 6 SEM from each group of mice (n = 8 for immunohistological examination and n = 12 for measuring the amounts of VAT per group). *p, 0.05; ** p,0.01 vs. the GABA-fed mice. doi:10.1371/journal.pone.0025338.g004 Discussion Macrophage-related chronic inflammation in adipose tissues is associated with the development of insulin resistance and glucose intolerance, leading to the development of obesity and T2DM. Our previous studies, and those of others, have shown that GABA, through the activation of GABAA-R, inhibits inflammation in mouse models of autoimmune diseases [16,17,18,19,20]. In this study, we examined the impact of oral treatment with GABA on the development of HFD-induced glucose intolerance and insulin resistance as well as obesity in mice. We found that oral treatment with GABA did not modulate the amount of food intake and water consumption by mice, consistent with our previous findings [18]. Given that GABA has little or no ability to pass through the blood brain barrier, it is not surprising that activation of peripheral GABA receptors did not alter calorie intake in mice. Furthermore, while most of the HFD-fed control mice displayed a significant increase in body weights, the mice that had been fed PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e25338