A new recombinant pituitary adenylate cyclase-activating peptide-derived peptide efficiently promotes glucose uptake and glucose-dependent insulin secretion

Abstract

The recombinant peptide, DBAYL, a promising therapeutic peptide for type 2 diabetes, is a new, potent, and highly selective agonist for VPAC2 generated through sitedirected mutagenesis based on sequence alignments of pituitary adenylate cyclase-activating peptide (PACAP), vasoactive intestinal peptide (VIP), and related analogs. The recombinant DBAYL was used to evaluate its effect and mechanism in blood glucose metabolism and utilization. As much as 28.9 mg recombinant DBAYL peptide with purity over 98% can be obtained from 1 l of LuriaBertani medium culture by the method established in this study and the prepared DBAYL with four mutations (N10Q, V18L, N29Q, and M added to the N-terminal) were much more stable than BAY55-9837. The half-life of recombinant DBAYL was about 25 folds compared with that of BAY55-9837 in vitro. The bioactivity assay of DBAYL showed that it displaced [ 125 I]PACAP38 and [ 125 I]VIP from VPAC2 with a half-maximal inhibitory concentration of 48.4 + + + + + 6.9 and 47.1 + + + + + 4.9 nM, respectively, which were significantly lower than that of BAY55-9837, one established VPAC2 agonists. DBAYL enhances the cAMP accumulation in CHO cells expressing human VPAC2 with a half-maximal stimulatory concentration (EC 50 ) of 0.68 nM, whereas the receptor potency of DBAYL at human VPAC1 (EC 50 of 737 nM) was only 1/1083 of that at human VPAC2, and DBAYL had no activity toward human PAC1 receptor. Western blot analysis of the key proteins of insulin receptor signaling pathway: insulin receptor substrate 1 (IRS-1) and glucose transporter 4 (GLUT4) indicated that the DBAYL could significantly induce the insulin-stimulated IRS-1 and GLUT4 expression more efficiently than BAY55-9837 and VIP in adipocytes. Compared with BAY55-9837 and PACAP38, the recombinant peptide DBAYL can more efficiently promote insulin release and decrease plasma glucose level in Institute of Cancer Research (ICR) mice. These results suggested that DBAYL could efficiently improve glucose uptake and glucose-dependent insulin secretion by VPAC2-mediated effect.

A novel selective VPAC2 agonist peptide-conjugated chitosan modified selenium nanoparticles with enhanced anti-type 2 diabetes synergy effects

“…Thus, PACAP derivatives as VPAC2-specific agonists can effectively promote glucose-dependent insulin secretion, control circulating glucose and protect islet beta cells without causing glucagon secretion and glycogenolysis, and have been proposed as potential T2D therapeutics. [12][13][14] BAY55-9837 is a reported specific VPAC2 agonist constructed through site-directed mutagenesis, and it can increase plasma insulin levels in a dose-dependent manner, but not leading to any hypoglycemia in rats. 15,16 Nevertheless, its therapeutic application has been hampered by its short half-life (~5 min) and limited bioavailability in vivo.…”

“…12,18,19 Pan et al explored the coupling of dipeptidyl peptidase IV-resistant VPAC2 analog peptides with 22 or 44 kDa polyethylene glycol (PEG) through specific cysteine moieties to improve the pharmacokinetics of these peptides in vivo. 13 But the large polymers formed by PEG or other modifications often decrease the biological activity of the peptides or interfere with the biological function of the peptides.…”

“…Sixty microliters of the sample was taken out from Eppendorf tube at appropriate intervals (0, 3,6,12,18,24,30,36,42 and 48 h). The quantity of DBAYL was measured by HPLC method at 280 nm.…”

A New Recombinant PACAP-Derived Peptide Efficiently Promotes Corneal Wound Repairing and Lacrimal Secretion

“…The vector pKY-MPAPO was transformed into E. coli ER2566 and the fusion proteins were expressed and purified by the optimized procedure previously described. 23 After chitin bead (NEB) affinity chromatography purification for the cell lysate, MPAPO was purified and prepared by reverse-phase high-performance liquid chromatography (HPLC) using 4.6-3 150-mm 300 SB-C18 Sep-Pak column (Agilent Technologies, Beijing, China) through gradient elution of acetonitrile from 3% to 60% for 20 minutes at 0.5 mL/min. The eluate containing MPAPO was dried by lyophilization.…”

“…23 The half-maximal inhibitory concentration (IC50) of MPAPO, PACAP27, PACAP38, and VIP was detected. [K15,R16,L27]VIP(1-7)/GRF(8-27), a VPAC1-specific agonist, was used as the negative control in the receptor binding assay.…”