S-1 cells, in a dynamin-dependent manner. Pharmacokinetic studies in CD1 mice show that after a single intraperitoneal injection, the fusion protein rapidly appears in circulation and remained at a high level for more than a week. The fusion protein has anti-diabetic effects, which is exemplified by its capacity in reducing the incidence of diabetes in multiple-low-dose streptozotocin-induced type 1 diabetes mouse model. Our results suggest that GLP-1/hIgG2 may serve as an alternative potent GLP-1 receptor agonist for the treatment of diabetes. itoneal glucose tolerance testing, water consumption, food intake and urine volume were measured and a fasting blood sample was taken from the saphenous vein for serum insulin and glucagon measurement. For diabetes induction, 50 mg/kg body weight of STZ was dissolved in 0.01 M cold citrate buffer immediately before intraperitoneal injection. The development of diabetes was monitored by measuring blood glucose from the tail vein using Ascensia ELITE XL glucometer and Ascensia Elite blood glucose test strips. Cell culture Rat insulinoma INS-1 cells were maintained in RPMI 1640 medium containing fetal bovine serum, 100 Units/ml penicillin G sodium, 100 mg/ml streptomycin sulphate, 55 mg/500 ml sodium pyruvate, 1.14 g/500 ml HEPES, and 1.7 ml/500 ml bmercaptoethanol at 37uC in an atmosphere of humidified air and CO2. In studies involving serum-starvation, serum was replaced by 0.1% BSA in RPMI 1640 without glucose. Materials and Methods Animals 7-week-old CD1 mice were housed under controlled temperature conditions and a 12-h light/12-h dark cycle in the St Michael’s Hospital MedChemExpress Degarelix Animal facility with free access to normal rodent chow and water except where noted. All procedures were conducted according to protocols and guidelines approved by the Canadian Council of Animal Care and the St Michael’s Hospital Animal Care committee. Before intervention, body weight, feeding blood glucose, intraper- Expression and purification of GLP-1/hIgG2 A schematic shows that the cDNA encoding the fusion protein hGHRH/hGLP-1 was chemically synthesized, ligated 2 September 2010 | Volume 5 | Issue 9 | e12734 GLP-1-Human IgG Fusion Protein to a PCR-amplified cDNA fragment encoding human IgG2 FC and inserted into the NcoI and Hind III sites of the pAV0243 vector to generate GLP-1/hIgG-Fc/pAV0243. GLP-1/ hIgG2 encoding DNA fragment was then amplified using pfu DNA polymerase. The GLP-1/ hIgG2 gene was then double digested by restriction enzyme BamH I and sub-cloned into a mammalian expression vector pMPGCR5. For stable expression, CHO cells were expanded in CD-CHO complete medium containing with 16HT, 4 mM Lglutamine, then transfected with GLP-1/hIgG2-pMPGCR5 constructs. Stable expressing clones were selected by 600 mg/ml Hygromycin B screen and finally amplified into 500 ml CD-CHO complete medium in suspension culture at 225 rpm at 37uC until the cells density reached at 76106 cells/ml. The cultured CD-CHO medium was harvested, filtered and the GLP-1/hIgG2 fusion protein was purified by using Protein A Ceramic HyperDH F sorbent and Immunopure Gentle Ag/Ab binding and elution buffers. Selected fractions were pooled, dialyzed into 16PBS, pH 7.4 and stored 
at 280uC. detected by HRP-conjugated donkey anti-goat IgG. Ortho-Phenylenediamine substrate was added for enzymatic reaction and the colorimetric change was analyzed by reading absorbance at 490 nm in a Beckman microplate reader. Internalization Studies Internalization of rec
