Role of adropin in arterial stiffening associated with obesity and type 2 diabetes

Abstract

Adropin is a peptide largely secreted by the liver and known to regulate energy homeostasis; however, it also exerts cardiovascular effects. Herein, we tested the hypothesis that low circulating levels of adropin in obesity and type 2 diabetes (T2D) contribute to arterial stiffening. In support of this hypothesis, we report that obesity and T2D are associated with reduced levels of adropin (in liver and plasma) and increased arterial stiffness in mice and humans. Establishing causation, we show that mesenteric arteries from adropin knockout mice are also stiffer, relative to arteries from wild-type counterparts, thus recapitulating the stiffening phenotype observed in T2D db/db mice. Given the above, we performed a set of follow-up experiments, in which we found that 1) exposure of endothelial cells or isolated mesenteric arteries from db/db mice to adropin reduces filamentous actin (F-actin) stress fibers and stiffness, 2) adropin-induced reduction of F-actin and stiffness in endothelial cells and db/db mesenteric arteries is abrogated by inhibition of nitric oxide (NO) synthase, and 3) stimulation of smooth muscle cells or db/db mesenteric arteries with a NO mimetic reduces stiffness. Lastly, we demonstrated that in vivo treatment of db/db mice with adropin for 4 wk reduces stiffness in mesenteric arteries. Collectively, these findings indicate that adropin can regulate arterial stiffness, likely via endothelium-derived NO, and thus support the notion that "hypoadropinemia" should be considered as a putative target for the prevention and treatment of arterial stiffening in obesity and T2D.NEW & NOTEWORTHY Arterial stiffening, a characteristic feature of obesity and type 2 diabetes (T2D), contributes to the development and progression of cardiovascular diseases. Herein we establish that adropin is decreased in obese and T2D models and furthermore provide evidence that reduced adropin may directly contribute to arterial stiffening. Collectively, findings from this work support the notion that "hypoadropinemia" should be considered as a putative target for the prevention and treatment of arterial stiffening in obesity and T2D.

Keywords: adropin; endothelial cells; mesenteric arteries; nitric oxide; smooth muscle cellss.

Figure 1.

Decreased adropin is associated with increased arterial stiffness. A: human hepatic adropin mRNA expression is inversely associated with glycosylated hemoglobin (HbA1c) and body mass index (BMI) in subjects that underwent a liver biopsy during bariatric surgery (n = 45, F = 36, M = 9, n = 3–24/cluster). B: plasma adropin concentrations of healthy subjects (n = 33, F = 20, M = 13) and subjects with type 2 diabetes (T2D; n = 42, F = 21, M = 21). Carotid-to-femoral pulse wave velocity in healthy subjects (n = 33, F = 20, M = 13) and subjects with T2D (n = 36, F = 19, M = 17). Pearson correlation between plasma adropin concentration and pulse wave velocity in healthy subjects (n = 33, F = 20, M = 13) and subjects with T2D (n = 36, F = 19, M = 17). C: hepatic adropin mRNA expression in db/db and db/+ male mice (n = 9/genotype). Plasma adropin concentrations in db/db and db/+ male mice (n = 9–10/genotype). Incremental modulus of elasticity (E_inc) of mesenteric arteries of db/db and db/+ male mice (n = 9–10/genotype). D: adropin mRNA expression in various tissues harvested from adropin knockout and wild-type littermate male mice (n = 5–14/genotype). Final body weight of adropin knockout and wild-type littermate male mice (n = 9–14/genotype). Blood glucose concentration during a glucose tolerance test in adropin knockout and wild-type littermate male mice (n = 10–14/genotype). E_inc of mesenteric arteries from adropin knockout and wild-type littermate male mice (n = 9–14/genotype). Student’s unpaired t tests were performed in A–D. E_inc data (C and D) are represented using simple linear regression. Mann-Whitney test was performed for the comparison of adropin mRNA expression in the brain (D). A two-way, repeated-measures ANOVA was performed to assess glucose tolerance (D). *P < 0.05 compared with control.

Figure 2.

Adropin reduces F-actin stress fibers and stiffness in endothelial cells (EC) and isolated mesenteric arteries of db/db male mice: role of NO. A: phosphorylation of eNOS Ser1177 relative to total eNOS in human aortic EC treated with vehicle vs. adropin (10 ng/mL) for 30 min (n = 8–9/condition); representative Western blot images are also included. Only the top band was responsive to adropin and selected for quantification and analysis. Nitrite concentrations in the supernatant of human aortic EC treated with vehicle vs. adropin (10 ng/mL) for 24 h (n = 11/condition). B: volume of F-actin stress fibers in human aortic EC treated with vehicle vs. adropin (10 ng/mL) for 24 h (n = 20–24/condition); representative confocal microscope images of F-actin (yellow) and nuclei (blue) are also included (scale bar = 30 mm). Cortical stiffness of human aortic EC treated with vehicle vs. adropin (10 ng/mL) for 24 h (n = 18/condition). Below panels are repeat experiments in the presence of N^G-nitro-l-arginine methyl ester (l-NAME; added 30 min before adropin; n = 17–18/condition). C: volume of F-actin content and incremental modulus of elasticity (E_inc) of isolated mesenteric arteries from db/db male mice treated with vehicle vs. adropin (10 ng/mL) for 24 h (n = 8/condition); representative images of confocal microscope images of F-actin (yellow) and nuclei (blue) are also included (scale = 30 mm). Below panels are repeat experiments in the presence of l-NAME (added 30 min before adropin; n = 7–8/group). Student’s unpaired (A and B) and paired t tests (C) were performed, according to experimental design. E_inc data (C) are represented using simple linear regression. *P < 0.05 compared with control.

Figure 3.

Stimulation of vascular smooth muscle cells (VSMCs) with sodium nitroprusside (SNP), an NO mimetic, reduces F-actin stress fibers and stiffness. A: volume of F-actin stress fibers in human coronary artery VSMCs treated with vehicle vs. SNP (10 µM) for 1 h (n = 28–32/condition); representative fluorescent images of F-actin (yellow) and nuclei (blue) are also included (scale bar = 30 mm). Below panels are repeat experiments in the presence of LIMKi or jasplakinolide (added 30 min before SNP; n = 14–16/condition). B: cortical stiffness of human coronary artery VSMCs treated with vehicle vs. SNP (10 µM) for 1 h (n = 6–7/condition). Incremental modulus of elasticity (E_inc) of mesenteric arteries from db/db male mice treated with vehicle vs. SNP (10 µM) for 4 h (n = 14–16/condition). Student’s unpaired (A and B) and paired (B) t tests were performed according to experimental design. E_inc data are represented using simple linear regression. *P < 0.05 compared with control.

Figure 4.

In vivo treatment of db/db male mice with adropin for 4 wk reduces mesenteric arterial stiffness. Plasma adropin concentration in db/db male mice implanted with osmotic minipumps containing either vehicle or adropin (63 mg/kg/h, n = 5–11/group). Final body weight of db/db male mice after 4 wk of vehicle or adropin administration (n = 7–12/group). Incremental modulus of elasticity (E_inc) of mesenteric arteries after 4 wk of vehicle or adropin administration (n = 6–12/group). Student’s unpaired and paired t tests were performed, as appropriate. E_inc data are represented using simple linear regression. *P < 0.05 compared with control.

Figure 5.

Proteomic analysis of endothelial cells treated with vehicle vs. adropin. Upregulation of pathways linked to organization of the cytoskeleton in human aortic endothelial cells treated with vehicle vs. adropin (10 ng/mL) for 24 h (n = 10/condition). Data expressed as average log2 ratio relative to vehicle. Student’s unpaired t test was performed for all protein comparisons (see Supplemental material for further description of the statistical analysis and individual proteins within figure).

Figure 6.

Schematic illustrating the interpretation of the results. Adropin-induced nitric oxide (NO) production in endothelial cells (EC) promotes vascular smooth muscle cell (VSMC) actin depolymerization and reduced stiffness. Destiffening of VSMCs likely contributes to reduced whole artery stiffness. Not depicted here, adropin also causes actin depolymerization and reduced stiffness in EC. Although endothelial rigidity does not physically contribute to whole artery stiffness, it is possible that decreased EC stiffness augments endothelial nitric oxide synthase (eNOS) activation and, consequently, increased NO signaling in VSMCs.