FIGURE 2.

Apelin-36(L28A) exhibits impaired APJ activation and blood pressure regulation, but retains full metabolic activity. A, apelin-36 potently activates APJ, but apelin-36(L28A) is >100-fold less potent. A U2OS cell line stably expressing human APJ was incubated with apelin peptides at the indicated concentrations. (n = 4 replicates/concentration, error bars indicate standard deviation; data are from one experiment that is representative of three independent experiments). B, apelin-36(L28A) retains full activity in protecting mice from the metabolic consequences of a high-fat diet. Chow refers to BDF mice on chow diet, Secreted GFP refers to BDF mice that were injected with 1 × 10¹² genome copies (“1E+12 GC”) of rAAV expressing secreted GFP and placed on a high-fat diet, Apelin-36 refers to mice injected with 1E+12 GC of rAAV expressing apelin-36 and placed on a high-fat diet, and Apelin-36(L28A) refers to mice injected with 1E+12 GC of rAAV expressing apelin-36(L28A) and placed on a high-fat diet (n = 7 mice per group; data are from one experiment that is representative of two independent experiments). Body weight was measured weekly beginning 2 weeks following AAV delivery. Recombinant AAV expressing murine apelin-36 or apelin-36(L28A) significantly reduced body weight in mice on a high-fat diet. C, fasting blood glucose (4-h fast) was measured weekly beginning 2 weeks following AAV delivery. Recombinant AAV expressing murine apelin-36 or apelin-36(L28A) significantly reduced blood glucose in mice on a high-fat diet. (n = 7/group). D, i.p. glucose tolerance was measured 4 weeks following AAV delivery. Following a 4-h fast, mice were administered 2 g/kg of glucose. Blood glucose levels were measured before glucose delivery (0 min) and 15, 30, 60, and 120 min following delivery. Apelin-36 or apelin-36(L28A) significantly improved glucose tolerance. Inset, glucose area under the curve shown in column graph. (n = 7/group). E, fasting serum insulin (4-h fast) was measured 3 weeks following AAV delivery. (n = 7/group). F, G, and H, serum total cholesterol (cholesterol) (F), LDL cholesterol (LDL) (G), and triacylglycerides (TAG) (H) were measured 6 weeks following AAV delivery. (n = 4/group). *, p < 0.05; **, p < 0.01; ***, p < 0.001 apelin-36 versus secreted GFP. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 apelin-36(L28A) versus secreted GFP. ∧∧, p < 0.01; ∧∧∧, p < 0.001 Chow versus secreted GFP. I, apelin-36 lowers mean blood pressure (MBP) in SHR, whereas apelin-36(L28A) does not. Blood pressure was measured using a non-invasive tail cuff blood pressure monitor. *, p < 0.05; **, p < 0.01 apelin-36 versus apelin-36(L28A). Statistics were analyzed using Student's t test with two-tailed distribution.