Supplementary Materialsnutrients-10-01757-s001. periphery. These effects were due to increases in sirtuin 1 activity in the liver, leptin receptors in muscle and protection against endoplasmic reticulum (ER)-stress in adipose tissue. In general, the resveratrol metabolites associated with these beneficial effects were derived from both phase II and microbiota metabolism, although only those derived from microbiota increased proportionally with the administered dose of resveratrol. In conclusion, resveratrol reversed leptin resistance caused by diet-induced obesity in peripheral organs using tissue-specific mechanisms. = 30; 200 50 g body weight) were purchased from Charles River Laboratories (Barcelona, Spain). The animals were housed in pairs under a 12 h light-dark cycle at 22 C, fed a standard chow diet (Panlab A04, Barcelona, Spain) ad libitum, and were provided access to tap water during the adaptation week. Then, the animals were distributed into equal groups composed of 6 rats. One group was fed a standard chow diet (STD group) with a calorie breakdown of 14% protein, 8% fat and 73% carbohydrates, while the others were fed an STD plus cafeteria diet (CAF group). The CAF diet WIN 55,212-2 mesylate small molecule kinase inhibitor was composed of 14% protein, 35% fat and 51% carbohydrates and consisted of bacon, carrots, cookies, foie gras, cupcakes, cheese and sugary milk. Nine weeks later, an oral treatment with RSV (Fagron Iberica, Barcelona, Spain) was administered together with the CAF diet for 22 days. The treatment groups were supplemented daily with 50, 100 or 200 mg/kg WIN 55,212-2 mesylate small molecule kinase inhibitor body weight of RSV dissolved in low-fat sugary milk diluted 1:1 in water. The STD and CAF diet groups were supplemented with the same quantity of vehicle (750 L) (Figure S1, Supplemental Data). Before supplementation, all rats were trained to voluntarily lick the milk to avoid oral gavage. On the day of sacrifice, the rats received vehicle or RSV and then were fasted for 3 h before sacrifice by decapitation. Blood was collected, and the serum was obtained by centrifugation (1500 for 20?min at 4 C. The supernatant was placed in fresh tubes and used to determine total protein and for immunoblotting analyses. The total protein content of the supernatant was measured using the Pierce bicinchoninic acid assay (BCA) protein assay kit (Thermo Scientific, Madrid, Spain). Samples were denatured by mixing with loading buffer solution (Tris-HCl 0.5 M pH 6.8, glycerol, sodium dodecyl sulfate (SDS), -mercaptoethanol and Bromophenol Blue) and then heated at 99 C for 5 min in a thermocycler (Multigen Labnet, Barcelona, Spain). Acrylamide gels were prepared using TGX Fast Cast Acrylamide Kit(Bio-Rad, Barcelona, Spain), and 25 g of protein was subjected to SDS-polyacrylamide Rabbit Polyclonal to BLNK (phospho-Tyr84) gel electrophoresis (PAGE) in WIN 55,212-2 mesylate small molecule kinase inhibitor electrophoresis buffer (glycine 192 mM, Tris base 25 mM and 1% SDS). Proteins were electrotransferred onto supported polyvinylidene difluoride (PVDF) membranes (Trans-Blot Turbo Mini PVDF Transfer Packs, Bio-Rad). After blocking with 5% non-fat dried milk, the membranes were incubated with gentle agitation overnight at 4 C with a specific antibody for ObRb (ab177469, Abcam, Cambridge, UK) diluted 1:1000. For -actin analysis as a loading control, membranes were incubated with a rabbit anti-actin primary antibody (A2066, Sigma-Aldrich, Madrid, Spain) diluted 1:1000. Finally, membranes were incubated with anti-rabbit horseradish peroxidase secondary antibody (NA9344, GE Healthcare, Barcelona, Spain) diluted 1:10,000. Protein levels were detected with the chemiluminescent detection reagent ECL Select (GE Healthcare, Barcelona, Spain) and GeneSys image acquisition software (G:Box series, WIN 55,212-2 mesylate small molecule kinase inhibitor Syngene, Barcelona, Spain). The protein bands were quantitated by densitometry using ImageJ software (W.S Rasband, Bethesda, MD, USA), and each band was normalized by the corresponding -actin band, and finally, the treatment groups were normalized by the STD group. 2.9. SIRT1 Activity Assay The SIRT1 activity in liver, calf skeletal muscles and.