Nesfatin 1

Seven days after surgery, intra-DRN injections were administered in accordance with the Paxinos and Watson atlas¹⁹ . Rats in the MS model were microinjected with anti-nesfatin-1/NUCB2 (1 µL, 10 µg; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or vehicle (1 µL, sterile water) into the DRN; then, abdominal withdrawal reflex (AWR) scores and magnitude of electromyogram (EMG) were recorded 2 h later. While normal rats were microinjected with nesfatin-1 (1 µL, 50 pmol; Phoenix Pharmaceuticals, Burlingame, CA, USA) or vehicle (1 µL, sterile water) into the DRN, nesfatin-1 was injected daily for 7 consecutive days^{15 (link)} then visceral sensitivity was detected at the 7th day.

Blood samples were collected into EDTA and serum tubes and were centrifuged for 15 min at 1000g at 4°C and 21°C, respectively. Aliquots were immediately frozen in liquid nitrogen and subsequently stored at −80°C until analysis. Plasma glucose, FFA, TAG and total cholesterol were analyzed with standard enzymatic methods (ABX Pentra 400 autoanalyzer, Horiba ABX, Montpellier, France). Plasma glycerol was analyzed with an enzymatic assay (Enzytec Glycerol, Roche Biopharm, Basel, Switzerland) automated on a Cobas Fara spectrophotometric autoanalyzer. Plasma insulin, leptin, and adiponectin concentrations were analyzed with commercially available radioimmunoassay (RIA) kits (Human insulin specific RIA, human leptin RIA, human adiponectin RIA, Millipore Corporation, Billerica, MA). Plasma concentrations of IL‐6 (Meso Scale Discovery, Gaithersburg, MD), apelin‐12 (Phoenix pharmaceuticals Inc., Burlingame, CA), RBP4 (R&D systems, Minneapolis, MN) and Vaspin (Adipogen, San Diego, IL) were determined by ELISA. The apelin‐12 ELISA has 100% cross‐reactivity with human apelin‐12, apelin‐13, and apelin‐36. Serum ACE activity was measured with a standard enzymatic assay (Bühlmann, Basel, Switzerland) while serum nesfatin‐1 (Phoenix pharmaceuticals Inc., Burlingame, CA) concentrations were measured by ELISA.

Unless otherwise stated, all chemicals were purchased from Sigma Chemical Co. (St. Louis. MO, USA). MES23.5 cells were provided by Professor Wei-Dong Le (Baylor College of Medicine, TX, USA). Nesfatin-1 was obtained from Phoenix Pharmaceuticals Inc. (Burlingame, CA, USA). Dulbecco’s modified Eagle’s medium nutrient mixture-F12 (DMEM/F12) was purchased from Gibco (Grand Island, NY, USA). The PE-conjugated monoclonal active-caspase-3 antibody apoptosis kit was from BD Bioscience (San Diego, CA, USA). PD98059 and the BCA kit was from Beyotime (Shanghai, CN). KT5720 and anti-rabbit secondary antibody conjugated to horseradish peroxidase were from Santa Cruz (Santa Cruz, CA). The cell fractionation kit was from Clontech (Mountain View, CA, USA). Anti-Cox IV monoclonal antibody was from Clontech (CA, USA). The primary antibody against TH was from Millipore (Darmstadt, Germany). Primary antibodies against ERK1/2 and phospho-ERK1/2 were from cell signaling technology (MA, USA). Alexa Fluor 555 donkey anti-rabbit secondary antibody was from Eugene (OR, USA). GW5074 was from Selleck (TX, USA).