Hepatic And Cardiac Beneficial Effects Of A Long-acting Fc-apelin-13 Fusion Protein In Diet-induced Obese Mice

Aim Apelin is a peptide ligand of the G-protein-coupled receptor APJ.It is widely expressed in many tissues and exhibits anti-diabetes and anti-heart failure activities.However,short serum half-life of the apelin peptide limits in vivo biological studies.In this study,we attempted to engineer a long-acting Fc-apelin-13 fusion protein and measure the biological activity of the resulting fusion protein.Materials and Methods First,a DNA primer containing the codon for C-terminal 13 amino acids of human apelin was synthesized with a linker sequence and used to generate a DNA fragment encoding a fusion protein of ht PA-Fc-GGGS-linkerapelin13 by PCR using a plasmid containing ht PA-Fc-GGS-linker as template.The resulting PCR fragment was subcloned into a Gateway p Entra vector by Infusion cloning.To make the lentiviral destination vector p SMPUW-CMV-EGFP,a fragment of CMV promoter–CCD-IRES-EGFP was cloned into the universal lentiviral vector p SMPUW.Standard Gateway LR cloning protocol was used to generate Fc-GGGS-linker-apelin13-EGFP by using LR Clonase II.All lentiviral vector plasmids were amplified in STBL3 bacteria.The resulting fusion protein was expressed in HEK293 cells for purification by Protein A affinity chromatography.Next,we conducted a pharmacokinetics study of the purified fusion protein in mice by measuring blood protein levels after a single subcutaneous injection of recombinant Fc-apelin-13 at 5mg/kg body weight,and determined its biological activity in suppressing c AMP and activating ERK signaling by luciferase reporter assay.Finally,we investigated the effects of Fc-apelin13 on food intake,body weight,glucose levels,insulin sensitivity,hepatic steatosis and cardiac function and fibrosis by subcutaneous administration of Fc-apelin-13 injection at a dose of 3 mg/kg/d in diet-induced obese mice.Results With protein A affinity chromatography,we were able to purify the Fc-apelin-13 fusion protein in one step at a purity of > 95%.The yield of Fc-fusion proteins was approximately 20-40 mg/L in culture medium.Pharmacokinetic analysis showed the estimated half-life of Fc-apelin-13 in blood is approximately 35 hours.Reporter assay showed that Fc-apelin-13 was nearly as active as the apelin-13 peptide in suppressing CRE-luciferase activity and in activating SRE-luciferase activity.A four-weeks treatment of Fc-apelin-13 in diet-induced obese mice led to a 14% decrease of fasting glucose and a significant improvement of glucose tolerance.In addition,the treatment reduced hepatic steatosis,triglyceride content and serum ALT levels.Moreover,cardiac stroke volume and output was increased and cardiac fibrosis was decreased in the treated mice.Conclusions Fc-apelin-13 fusion protein has an extended in vivo half-life and exerts multiple benefits on obese mice with respect to the improvement of glucose disposal,amelioration of liver steatosis and heart fibrosis,and increase of cardiac output.Hence,Fc-apelin-13 is potentially a therapeutic for obesity-associated disease conditions.