Aerobic Exercise Restores Aging-Associated Reductions in Arterial Adropin Levels and Improves Adropin-Induced Nitric Oxide-Dependent Vasorelaxation

Abstract

Background Adropin is a peptide hormone that promotes nitric oxide (NO) production via activation of endothelial NO synthase (eNOS) in endothelial cells. Its circulating levels are reduced with aging and increased with aerobic exercise training (AT). Using a mouse model, we hypothesized that AT restores aging-associated reductions in arterial and circulating adropin and improves adropin-induced NO-dependent vasorelaxation. Further, we hypothesized these findings would be consistent with data obtained in elderly humans. Methods and Results In the animal study, 50-week-old SAMP1 male mice that underwent 12 weeks of voluntary wheel running, or kept sedentary, were studied. A separate cohort of 25-week-old SAMP1 male mice were used as a mature adult sedentary group. In the human study, 14 healthy elderly subjects completed an 8-week AT program consisting of 45 minutes of cycling 3 days/week. In mice, we show that advanced age is associated with a decline in arterial and circulating levels of adropin along with deterioration of endothelial function, arterial NO production, and adropin-induced vasodilation. All these defects were restored by AT. Moreover, AT-induced increases in arterial adropin were correlated with increases in arterial eNOS phosphorylation and NO production. Consistently with these findings in mice, AT in elderly subjects enhanced circulating adropin levels and these effects were correlated with increases in circulating nitrite/nitrate (NOx) and endothelial function. Conclusions Changes in arterial adropin that occur with age or AT relate to alterations in endothelial function and NO production, supporting the notion that adropin should be considered a therapeutic target for vascular aging. Registration URL: https://www.umin.ac.jp; Unique identifier: UMIN000035520.

Keywords: adropin; aging; exercise training; nitric oxide; vasodilation.

Figure 1. Aortic endothelium‐dependent relaxation responses are diminished with age and increased by aerobic exercise training (AT).

Aortic rings were isolated from mature adult (25‐week‐old), aged control (50‐week‐old, Aged‐Con), and aged with AT (50‐week‐old that underwent 12 weeks AT, Aged‐AT) mice. Rings were hung in wire myography systems and exposed to increasing concentrations of the endothelium‐dependent vasodilator acetylcholine (ACh, A) or the endothelium‐independent vasodilator sodium nitroprusside (SNP, B). Adropin‐induced aortic relaxation responses are nitric oxide synthase (NOS)‐dependent, diminished with age and increased by aerobic exercise training (AT). Aortic rings were isolated from mature adult (25‐week‐old), aged control (50‐week‐old, Aged‐Con), and aged with AT (50‐week‐old that underwent 12 weeks AT, Aged‐AT) mice. Rings were hung in wire myography systems and exposed to increasing concentrations of adropin with or without the NOS inhibitor, Nω‐nitro‐L‐arginine methyl ester (L‐NAME). C, Comparison of adropin‐induced relaxation between the Mature adult, Aged‐Con and Aged‐AT groups. D, Comparison of adropin‐induced relaxation with and without L‐NAME among the Mature adult group. E, Comparison of adropin‐induced relaxation with and without L‐NAME among the Aged‐Con group. F, Comparison of adropin‐induced relaxation with and without L‐NAME among the Aged‐AT group. Data are percent aortic relaxation responses from a preconstruction (Pre) induced with 30nM U‐46619 and expressed as means±SEM, n=7 to 10/group, *P<0.05.

Figure 2. Aortic and plasma adropin levels are diminished with age and increased by aerobic exercise training (AT).

Adropin levels were measured using a commercial ELISA kit in Aortic tissue (A) and plasma (B) obtained from mature adult (25‐week‐old), aged control (50‐week‐old, Aged‐Con), and aged with AT (50‐week‐old that underwent 12 weeks AT, Aged‐AT) mice. Aortic endothelial growth factor receptor‐2 (VEGFR2), phosphorylated Akt (p‐Akt), phosphorylated endothelial nitric oxide synthase (p‐eNOS) as well as aortic and plasma nitrate and nitrite (NOx) are diminished with age and increased by aerobic exercise training (AT). Aortic tissue and plasma were obtained from mature adult (25‐week‐old), aged control (50‐week‐old, Aged‐Con) and aged with AT (50‐week‐old that underwent 12 weeks AT, Aged‐AT) mice and subjected to Western blot analyses or NOx measurement. C, Representative blot and comparison of aortic VEGFR2 levels normalized to GAPDH between the three groups of mice. D, Representative blot and comparison of aortic p‐Akt to total Akt ratios between the three groups of mice. E, Representative blot and comparison of aortic p‐ERK1/2 to total ERK1/2 ratios between the three groups of mice. F, Representative blot and comparison of aortic p‐eNOS to total eNOS ratios between the three groups of mice. G, Comparison of aortic NOx levels between the three groups of mice. H) Comparison of plasma NOx levels between the three groups of mice. Data are expressed as means±SEM, n=7 to 10/group.

Figure 3. Aortic adropin mRNA is diminished with age and increased by aerobic exercise training (AT).

Adropin/β‐actin mRNA expression was determined in brown adipose tissue (BAT), liver, aorta, small intestine, heart, kidney, skeletal muscle, brain, lung, white adipose tissue (WAT), and spleen and compared between samples obtained from mature adult (25‐week‐old), aged control (50‐week‐old, Aged‐Con) and aged with AT (50‐week‐old that underwent 12 weeks AT, Aged‐AT) mice. A.U., arbitrary units. Data are expressed as means±SEM, n=7 to 10, *P<0.05 ​vs Aged‐Con.

Figure 4. Aortic adropin levels are positively correlated with plasma adropin, phosphorylated endothelial nitric oxide synthase (p‐eNOS) or aortic nitrate and nitrite (NOx).

A, Pearson’s correlation coefficient and linear fit between arterial adropin and plasma adropin levels. B, Pearson’s correlation coefficient and linear fit between arterial adropin and arterial p‐eNOS/eNOS. C, Pearson’s correlation coefficient and linear fit between arterial adropin and nitrite/nitrate (NOx) levels. Aortic tissues and plasma were obtained from mature adult (25‐week‐old), aged control (50‐week‐old, Aged‐Con), and aged with AT (50‐week‐old that underwent 12 weeks AT, Aged‐AT) mice. A.U., arbitrary units.

Figure 5. Serum adropin levels are positively correlated with plasma nitrate and nitrite (NOx) or brachial artery flow‐mediated dilation (FMD), as plasma NOx is also correlated with brachial FMD in human elderly subjects.

A, Pearson’s correlation coefficient and linear fit between serum adropin and plasma NOx levels. B, Pearson’s correlation coefficient and linear fit between serum adropin and brachial artery FMD. C, Pearson’s correlation coefficient and linear fit between plasma NOx levels and brachial artery FMD. Measurements were made before and after an 8‐week aerobic exercise training intervention.