Stimulation of insulin secretion by GLP-1/<i>h</i>IgG2 in INS- cells.

<p>(A) Cells grown in 24-well plates were incubated with fresh KRB buffer devoid of glucose for 2×60 min. The cells were then treated with 2.8 or 16.8 mM glucose and various concentrations of purified GLP-1/<i>h</i>IgG2 fusion proteins in KRB buffer for 2 h. The insulin levels in the conditioned KRB buffer were measured using a rat insulin RIA kit. (B) Active GLP-1 was measured by active GLP-1 ELISA kit in the mouse serum samples incubated with recombinant GLP-1 (75 pM) or GLP-1/<i>h</i>IgG2 fusion proteins (75 pM) at 37°C for indicated time period. The reactions were terminated by adding excessive DPP-IV inhibitors and aprotinin.</p

Pharmacokinetic study of GLP-1/<i>h</i>IgG2 fusion protein in CD1 mice.

<p>CD1 mice were i.p. injected by a single-dose of GLP-1/<i>h</i>IgG2 (1 µg/mouse). Blood samples were taken from tail vein at different time points and serum GLP-1 levels were measured by GLP-1 RIA kit.</p

Intraperitoneal Glucose Tolerance Test (IPGTT) shows that GLP-1/<i>h</i>IgG2 improves glucose tolerance in CD-1 mice.

<p>After 16 h fasting, CD1 mice were i.p. injected with GLP-1/<i>h</i>IgG2 (1 µg/mouse). 30 min after the injection, IPGTT were conducted by i.p. injection of 1.5 g/kg of glucose and blood glucose levels were measured by a glucometer at 0, 10, 20, 30, 60 minutes after glucose administration. (B) 192-h after a single-dose injection of GLP-1/<i>h</i>IgG2 in CD1 mice, the mice were fasted for 16 h and IPGTT were conducted as described in (A).</p

GLP-1/<i>h</i>IgG2 improves glucose regulation in MDSD mice.

<p>(A) The intraperitoneal injections of GLP-1/<i>h</i>IgG2 (1 µg/mouse) were made every three days during the course of experiment and the first drug injection was made 3 days prior to the multiple low dose streptozotozin treatment (50 mg/kg/day for 4 consecutive i.p. injections). Glucose levels were measured by a glucometer at indicated times. (B) The glucose levels were expressed as the area under the curve (AUC). (C) Insulin tolerance test (ITT) was conducted by injecting insulin (2.0 U/kg, i.p.) and blood glucose levels were measured at indicated times. (D) IPGTT was performed in MDSD mice treated with PBS or GLP-1/<i>h</i>IgG2-Fc at the end of the experiment as shown in (A).</p

Endocytosis of GLP-1/<i>h</i>IgG2 is via a dynamin dependent manner.

<p>(A) Internalization of GLP-1/hIgG2-Fc in INS-1 cells (A) Cells were incubated with 1 µM GLP-1/<i>h</i>IgG2 at 4°C for 30 minutes, then switched to 37°C for 0, 10, 20 and 60 minutes. After fixation and blocking for non-specific binding, cells were then incubated with goat-anti-human IgG antibody (1∶500) followed by secondary Cy3-conjugated anti-goat antibody (1∶500) and Top3 dye (1∶20,000) for nuclei staining. The images were then visualized by a confocal microscope. (B) The internalization experiments were performed in cells co-transfected with either wild type dynamin or dominant negative dynamin and green fluorescent protein (GFP). 24 hours after transfection, internalization of GLP-1/<i>h</i>IgG2-Fc was conducted as described in (A). DAPI (1∶10,000) was used for nuclei staining and images were taken by a Nikon fluorescent microscope.</p

Construction of GLP-1/<i>h</i>IgG2 fusion protein.

<p>(A) Illustration shows that the cDNA encoding the fusion protein hGLP-1 chemically synthesized was ligated to a PCR-amplified cDNA fragment coding human IgG2 FC (hinge-ch2-ch3) and inserted into the NcoI and Hind III sites of a modified mammalian expression vector. (B) The secretable GLP-1/<i>h</i>IgG2-Fc fusion proteins are homodimeric. (C) Protein production efficiency was evaluated by the Dot blot with anti-human IgG antibodies using conditional medium from the CHO cells stably expressing the fusion expression. (D) Western blot shows that the GLP-1 fusion protein form stable dimer of molecular weight ∼70 kDa and monomer formation under reducing conditions.</p

Assessment of the binding affinity of GLP-1/<i>h</i>IgG2.

<p>(A) Cellular ELISA based ligand-receptor binding assay shows the maximum binding of GLP-1/hIgG2 to INS-1 cells is ∼1 µM with Kd of 13.90±1.52 nM. (B). Competitive binding assay was performed in a reaction mixture containing 10 µM GLP-and its competitors Ex-4, GLP-1 and glucagon of varying concentration (1×10⁻⁵ to 1×10⁻¹² M).</p