Glp 1

Mouse C2C12 myoblasts (ATCC, USA) were kindly given by Dr. Konhilas (University of Arizona, USA), and maintained in DMEM supplemented with 9% foetal calf serum, 5 mM d-glucose, 50 U/ml penicillin, and 50 μg/ml streptomycin. Before confluency, the medium was replaced to differentiation medium containing DMEM and 2% horse serum. After 4 additional days, the differentiated C2C12 cells fused into myotubes. Then, cells were switched to serum-free quiescent medium overnight before stimulation. The hyperlipidemic or hyperglycemic conditions were mimicked by 6–12 h incubation with high concentrations of a common saturated free FA (FFA) [Na⁺-palmitate (16:0), 0.12 mM], or glucose (d-glucose, 25 mM), respectively (Sigma). These concentrations were not lethal after 12 h incubation [15 (link)]. Palmitate was previously conjugated with BSA in a 3:1 molar ratio as published earlier [15 (link)]. In control cells, BSA was added as described but in the absence of palmitate. Some cells were pre-treated with GLP-1 (1 nM) or GLP-1(9-36) (0.3 nM) (Sigma) 30 min before stimulation. Wortmannin (50 nM) was added 1 h before stimulation.

Differentiation was performed according to a protocol previously reported by Tayaramma et al. [21 (link)]. Initially, the cells were cultured for 3 days in serum-free DMEM supplemented with trichostatin-a (TSA) at a concentration of 55 nanomoles (Sigma). Then, the cells were cultured for an additional 7 days in high-glucose (25 millimoles) medium containing a 1 : 1 ratio of DMEM : DMEM/F12 (Sigma). This mixture was supplemented with 10% fetal bovine serum and 10 nanomoles glucagon- (GCG-) like peptide-1 (GLP-1, Sigma).

The regulation of DPP-4 expression by metabolic effectors was evaluated by incubating human osteoblasts with five different compounds and/or combination of compounds for 2 and 24 h. Effectors used were glucose (5 mM, Sigma-Aldrich, Oakville, ON, Canada), insulin (0.3 nM, Humulin R, Eli Lilly, Indianapolis, IN, USA), glucose + insulin (5 mM and 0.3 nM), GLP-1 (10 nM, Sigma-Aldrich) and butyric acid (10 mM, Sigma-Aldrich). At confluence, after a 16 h pre-incubation with serum-free α-MEM media, effectors were mixed into serum-free α-MEM media before being added to the wells and incubated at 37 °C. For each sample and time point, the negative control consisted of cells incubated only with serum-free α-MEM media.

GLP-1, SB203580 and KG-501 were purchased from Sigma-Aldrich. Liraglutide was purchased from Novo Nordisk (Tianjin, China). LY294002 and U0126 were purchased from Cell Signaling Technology. H89 was from Adipogen Life Science. Anti-LC3A/B, anti-pERK1/2, anti-ERK1/2, anti-pAKT, and anti-AKT antibodies were purchased from Cell Signaling Technology. Anti-FNDC5 (ab174833) and anti-ATGL were from Abcam, while anti-UCP-1 and anti-β-actin antibodies were purchased from Sigma-Aldrich. HRP-conjugated secondary antibodies for western blotting were purchased from Cell Signaling Technology. Irisin-competitive ELISA kit (AG-45A-0046YEK-KI01) was from Adipogen Life Science.

Glucagon-like peptide-1 (GLP-1) was obtained from Sigma (St. Louis, MO, USA). Pegylation on free amino groups of the peptide was carried out using succinimide pegylating agent Sunbright ME-120 TS (NOF America Corporation, San Mateo, CA, USA), for which 1 mg of lyophilizate was dissolved in 2 mL of 20 mm Na-phosphate buffer, pH 7, containing 0.01% TWEEN 20 (Sigma, St. Louis, MO, USA), then 20 mg of pegylating agent (NOF America Corporation, San Mateo, CA, USA) was added to the solution. The reaction was stopped by the application of 200 µL of 0.1 M glycine solution. The molecular weight of GLP-1 was assessed by electrophoresis in polyacrylamide gel with SDS (Sodium dodecyl sulfate, Sigma, St. Louis, MO, USA) using a standard technique. Prior to pegylation, the samples contained 100% GLP-1. After pegylation, the samples contained 7% GLP- 1, 78% of monopeg-GLP-1, and 15% double peg-GLP-1.
GLP-1 and pegGLP-1 were daily administered intraperitoneally in the region of the pancreas at a dose of 3 mmol/kg on p149, 156, 157, 173, 184, 186, and 188 (Figure 10).