Trysin

VIN was obtained from Harbin Sanlian Pharmaceutical Factory (Heilongjiang, China). Dulbecco’s Modified Eagle’s medium and Nonessential amino acids (DMEM) were purchased from GIBCO (USA). Solutol HS 15 was supplied by BASF (Ludwigshafen, Germany). Transcutol P was obtioned from Castris (Gattefosse, France). Ethyl oleate was purchased from Beijing Changcheng Chemical Ltd. (Beijing, China). Fetal bovine serum (FBS) was gained from Hycolone (USA). Penicillin, streptomycin, trysin and ethylenediaminetertraacetic acid (EDTA) were purchased from Sigma (USA). Transwell^® nunclone™ was supplied by COSTAR (Camgridge, MA, USA). Eudragit L30D55 and Eudragit FS30D were obtioned from Evonik Degussa. HPMC was gained from Colorcon. Sucrose, mannitol and sodium bicarbonate?were purchased from Bodi chemical co, LTD (Tianjin, China).
Other materials were Solutol HS 15 (BASF, Ludwigshafen, Germany), Transcutol P (Castris, Gattefosse, France), ethyl oleate (Beijing Changcheng Chemical Ltd., Beijing, China), fetal bovine serum (FBS) (Hyclone, USA), Penicillin, streptomycin, trypsin, ethylenediaminetetraacetic acid (EDTA) (Sigma, USA), Transwell^® nunclon™(COSTAR, Cambridge, MA, USA), Eudragit L30D55 and Eudragit FS30D (Evonik Degussa), HPMC (Colorcon), sucrose, mannitol, sodium bicarbonate (Bodi chemical co., LTD, Tianjin, China), All other reagents were of analytical grade.

Dexamethasone, insulin, protease inhibitor cocktail, rosiglita zone and 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]–glucose (2-NBDG), TBA, SRB and trysin were purchased from Sigma Aldrich, United States. Phenazine methosulphate (PMS), nitroblue tetrazolium (NBT), and nicotinamide adenine dinucleotide (NADH) were procured from Himedia labs, India. Hydrogen peroxide was purchased from MP biomedicals, United States. Kits for ALT, AST and ALP, albumin, BUN, CK analysis were purchased from ERBA (Germany). Anti-mouse insulin monoclonal antibody (Thermo Fischer Scientific, United States) and anti-rabbit glucagon receptor N-term polyclonal antibody (Abcam, United Kingdom) were used for the study. Analytical grade chemicals and solvents were used in the current study.

Details of isolations of DRGN and SciN were given in previous studies^{15 (link),34 (link)}. Briefly, the DRGN and SciN were minced with iridectomy scissors and incubated with enzymes including trysin (type III, Sigma) and 0.5 mg/ml collagenase (type XI, Sigma) in 5 ml DMEM at 37 °C in a shaking bath for 40 min after removing the attached nerves and surrounding connective tissues. To stop the enzymatic digestion 1.25 mg/ml soybean trypsin inhibitor (type II-S1, Sigma) was added. After dissociation with a sterile syringe, the DRGN and SciN suspensions of mediums were centrifuged at 1,500 g and the medium and high size neurons were removed for the analysis. The isolated neurons were transferred into a 35-mm culture dish and kept still for at least 30 min.
The brain was also taken as follows; the cortex was dissected out after the brain was split in the mid-sagittal plane. The gastrocnemius muscle samples from right legs were also taken. After preparing brain and muscle homogenates in ice-cold Tris-HCl buffer (50 mM, pH 7.4), they were stored at −85 °C for analyses of lipid peroxidation and antioxidants. Half of the muscle sample with DRGN and SciN was frozen at −85 °C for using Western blot analyses. Plasma and erythrocyte samples from anticoagulated blood (sodium EDTA) were obtained as described in a previous studies^{17 (link),18 (link)} and they were stored at −85 °C.