receptors may modulate the HFD-induced adipocyte dysfunction and inflammation, as well as associated obesity and glucose metabolic disorder. To test this hypothesis, we employed the HFD-induced obesity and T2DM model and treated orally with GABA to test whether activation of GABA receptors could prevent the HFD-induced obesity and T2DM, and improve glucose tolerance and insulin sensitivity after the onset of T2DM. We found that oral administration of GABA did not affect the amount of water and food consumption by animals, but reduced the HFD-induced gain in body weight and epididymal fat mass, accompanied by reducing the numbers of infiltrated macrophages in adipose tissues. Furthermore, treatment with GABA improved glucose tolerance and insulin sensitivity in mice, even after the onset of obesity and T2DM. Finally, we observed that treatment with GABA significantly increased the frequency of peripheral Tregs in mice, which are known to negatively regulate inflammation. Materials and Methods Mice and treatments All experiments were approved by the Animal Research Committee of University of California, Los Angeles (protocol number 1993-211). Male C57BL/6 mice at 4 weeks of age were from Jackson Laboratories (Bar Harbor, ME, USA) and were housed in a specific pathogen free facility. The mice were fed with HFD beginning at 5 weeks of age (60% fat of caloric intake at 5.32 kcal/g, Research Diets, New Brunswick, USA) and provided with plain water (control) or water containing 2 mg/ml of GABA (Sigma, St. Louis, USA). The water bottles were changed weekly with fresh material. Their food intake and water consumption were measured weekly and their body weights, fasting blood glucose, glucose tolerance, and insulin sensitivity were measured longitudinally. In addition, we tested whether treatment with GABA could modulate glucose intolerance and insulin resistance in mice after the onset of obesity and T2DM. C57BL/6 mice were fed with HFD and plain water for 20 weeks and tested for fasting blood glucose, glucose tolerance, and insulin sensitivity. Individual mice with obesity (body weights . 48 g) and T2DM (fasting blood glucose .145 mg/dL; blood glucose at 2 hours post-intraperito- neal glucose challenge .220 mg/dL; blood glucose at 30 min post insulin injection , 28% before insulin injection) were randomly divided and fed with HFD and plain water or water containing 2 mg of GABA for another 12 weeks, respectively. Their food intake and water consumption were measured weekly and their body weights, fasting blood glucose, glucose tolerance, and insulin sensitivity were measured longitudinally. Measurement of fasting blood glucose, glucose tolerance, and insulin sensitivity The mice were fasted for 16 hours and the concentrations of blood glucose in individual mice were measured using an OneTouch ultra blood glucose meter (LifeScan, Milpitas, USA). Glucose tolerance in individual mice was injected intraperitoneally with 2 g/kg glucose (Sigma) and their blood glucose concentra- tions were measured at 15, 30, 60, 90, 120 and 180 min post challenge and the areas of under the curve (AUC) of blood glucose levels were calculated. For insulin tolerance tests, non-fasted individual mice were measured for the concentrations of blood glucose as 0 time point and injected intraperitoneally with 0.75 units/kg of human regular insulin (Eli Lilly, Indianapolis, USA). Subsequently, the concentrations of blood glucose were measured at 15, 30, 60, 90, 120 and 150 min post insulin injection. The GABA Treatment for High Fat Diet-Fed Mice changes in the percentage of blood glucose relative to the values at 0 time point for individual mice were calculated. Histological analysis of visceral adipose tissue At the end of the experiments, mice were sacrificed and their visceral adipose tissue (VAT) were dissected and weighed. 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. A total 5 sections from individual mice of each group (n = 8) were examined in a blinded fashion using the Lplab image analysis software. Another portion of the VAT was fixed with the Bouin buffer for 48 hours and then paraffin-embedded. Their sections at 5 mm of 100 mm intervals were rehydrated, treated with 3% of H202 in methanol, and subjected to antigen retrieval with 0.01 M sodium citrate buffer (pH 6.0) in a high pressure-steamer. Subsequently, these sections were blocked sequentially with a biotin blocking system (Dako) and 20% of FBS in PBS. The sections were probed with biotinylated anti-F4/80 (PharMigen, San Diego, USA) overnight at 4uC. After washing, the immunocomplex was visualized using HRP-striptoavidin (Dako) and the peroxidase substrate diaminobenzidine (DAB, Dako), followed by counter- staining with hematoxylin and examination under a light microscope. A total of 400 nuclei from 5 fields of 5 sections of each mouse were counted in a blinded manner and the percentage of macrophage infiltrates was calculated. Flow cytometry analysis Groups of C57BL/6 mice were fed with plain water or water containing 2 mg/ml of GABA for 4 weeks. Their splenic mononuclear cells were prepared and the frequency of CD4+Foxp3+ Tregs was characterized by flow cytometry analysis. Briefly, splenic mononuclear cells (106/tube) were treated in duplicate with anti-CD16/32 on ice for 20 minutes. After washing, the cells were stained with FITC-anti-CD4 (BD PharMigen, San Diego, USA) for 25 minutes, fixed, permeabilized and stained with PE-anti-Foxp3 or isotype control (Biolegend, San Diego. USA), respectively. After washing, the cells were characterized by flow cytometry analysis and the percentage of CD4+Foxp3+ cells in total CD4+ T cells was calculated. Statistical analysis Data are expressed as mean or mean 6 SD of each group of mice. The difference between groups was analyzed by Students’ t- test and X2 test. The potential correlation between measures was analyzed by Sperman’s correlation analysis. A p value of less than 0.05 was considered statistically significant. Results Oral treatment with GABA does not change the amounts of water and food consumed, but significantly reduces the gain of body weight in HFD-fed mice GABAA-R subunits are expressed by immunocompetent cells, including T cells and macrophages and are also expressed by adipose tissues [13,14,15,17]. Our previous studies and those of others have shown that treatment with GABA or a GABAA receptor agonist inhibits inflammation in different mouse models of autoimmune diseases [16,17,18,20,21]. To determine whether GABA could modulate HFD-induced inflammation, obesity, and glucose intolerance, groups of 5-week-old male C57BL/6 mice were fed with HFD and provided with plain water or water containing 2 mg/ml of GABA for 20 weeks. Their food and water PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e25338