Potentiation of Acetylcholine-Induced Relaxation of Aorta in Male UC Davis Type 2 Diabetes Mellitus (UCD-T2DM) Rats: Sex-Specific Responses

Abstract

Previous reports suggest that diabetes may differentially affect the vascular beds of females and males. The objectives of this study were to examine whether there were (1) sex differences in aortic function and (2) alterations in the relative contribution of endothelium-derived relaxing factors in modulating aortic reactivity in UC Davis Type 2 Diabetes Mellitus (UCD-T2DM) rats. Endothelium-dependent vasorelaxation (EDV) in response to acetylcholine (ACh) was measured in aortic rings before and after exposure to pharmacological inhibitors. Relaxation responses to sodium nitroprusside were assessed in endothelium-denuded rings. Moreover, contractile responses to phenylephrine (PE) were measured before and after incubation of aortic rings with a nitric oxide synthase (NOS) inhibitor in the presence of indomethacin. Metabolic parameters and expression of molecules associated with vascular and insulin signaling as well as reactive oxygen species generation were determined. Diabetes slightly but significantly impaired EDV in response to ACh in aortas from females but potentiated the relaxation response in males. The potentiation of EDV in diabetic male aortas was accompanied by a traces of nitric oxide (NO)- and prostanoid-independent relaxation and elevated aortic expression of small- and intermediate conductance Ca²⁺-activated K⁺ channels in this group. The smooth muscle sensitivity to NO was not altered, whereas the responsiveness to PE was significantly enhanced in aortas of diabetic groups in both sexes. Endothelium-derived NO during smooth muscle contraction, as assessed by the potentiation of the response to PE after NOS inhibition, was reduced in aortas of diabetic rats regardless of sex. Accordingly, decreases in pAkt and peNOS were observed in aortas from diabetic rats in both sexes compared with controls. Our data suggest that a decrease in insulin sensitivity via pAkt-peNOS-dependent signaling and an increase in oxidative stress may contribute to the elevated contractile responses observed in diabetic aortas in both sexes. This study demonstrates that aortic function in UCD-T2DM rats is altered in both sexes. Here, we provide the first evidence of sexual dimorphism in aortic relaxation in UCD-T2DM rats.

Keywords: aorta; insulin resistance; nitric oxide; sex differences; type-2 diabetes.

FIGURE 1

Western blot analysis of IRS1 and IRS2 expression in control and UCD-T2DM rat aorta. Protein levels of (A) aortic insulin receptor substrate-I (IRS1) and (B) insulin receptor substrate-2 (IRS2) from the samples of male and female control and diabetic rats. IRS1 (A) and IRS2 (B) were quantified by densitometry and normalized to corresponding beta actin. Values are represented as mean ± SEM. Each bar represents the values obtained from n = 4–5 animal s per group. To show representative bands, images from different parts of the same gel have been juxtaposed, indicated by white dividing lines. Capped lines indicate significant differences between two groups (p < 0.05), as analyzed by unpaired Student’s t-test.

FIGURE 2

Concentration-response curves to acetylcholine (ACh) in control and UCD-T2DM rat aorta. Relaxation responses to cumulative concentrations of ACh (10^–8 to 10^–5 M) in intact aortic rings pre-contracted with phenylephrine (PE, 2 μM) from male (A) and female (B) control and diabetic rats. Data are expressed as mean ± SEM. n = 6–12 animals per group. *p < 0.05 between two groups analyzed using two-way ANOVA followed by Bonferroni’s post hoc test.

FIGURE 3

Endothelium-derived relaxing factors (EDRF) contribution to acetylcholine (ACh)− induced relaxation responses in control and UCD-T2DM rat aorta. Effects of inhibiting cyclooxygenase, soluble guanylyl cyclase and nitric oxide synthase on ACh-induced vasorelaxation in aortic rings taken from (A) male control and (B) male diabetic, (C) female control and (D) female diabetic rats. ACh relaxation was measured in the presence of indomethacin (Indo, 10 μM), followed by addition of ODQ (10 μM), and then with N-nitro-L-arginine (L-NNA; 100 μM). Data are expressed as mean ± SEM. *p < 0.05 vs. no drug; ^#P < 0.05 vs. Indo; analyzed using two-way ANOVA with repeated measures followed by Bonferroni post hoc test (n = 5–8 per group). Light gray shaded area: contribution of endothelium-derived hyperpolarizing factor (EDHF)-type to endothelium-dependent vasorelaxation (EDV).

FIGURE 4

Western blot analysis IK_Ca and SK_Ca expression in control and UCD-T2DM rat aorta. Protein levels of (A) aortic intermediate conductance calcium activated potassium channel (IK_Ca) and (B) small conductance calcium activated potassium channels (SK_Ca) were measured from the samples of male control and diabetic rats. IK_Ca (A) and SK_Ca (B) were quantified by densitometry and normalized to corresponding GAPDH. Values are represented as mean ± SEM. Each bar represents the values obtained from n = 4–5 per group. To show representative bands, images from different parts of the same gel have been juxtaposed, indicated by white dividing lines. Capped lines indicate significant differences between two groups (p < 0.05), as analyzed by unpaired Student’s t-test. MC, male control; MD, male diabetic.

FIGURE 5

Concentration-response curves to phenylephrine (PE) in control and UCD-T2DM rat aorta. Contractile responses to cumulative concentrations of PE (10^–8 to 10^–5 M) in intact aortic rings of (A) male and (B) female control and diabetic rats. Data are expressed as mean ± SEM. n = 5–6 per group. *p < 0.05 between two groups analyzed using two-way ANOVA followed by Bonferroni’s post hoc test.

FIGURE 6

Concentration-response curves to phenylephrine (PE) in intact aortic rings from (A) male control, (B) male diabetic, (C) female control, and (D) female diabetic rats. Contraction to PE was measured in absence of any drug (ND) or in presence of indomethacin (Indo, 10 μM) followed by addition of N-Nitro-L-arginine methyl ester (Indo + L-NAME, 200 μM). Results are expressed as a percent of the maximal response to PE (10 μM) obtained in the absence of any drug. Data are expressed as mean ± SEM, analyzed using two-way ANOVA with repeated measures followed by Tukey’s post hoc test: ^∗p < 0.05 vs. ND; ^#p < 0.05 vs. Indo, n = 6–8 per group.

FIGURE 7

Western blot analysis of eNOS and peNOS expression in control and UCD-T2DM rat aorta. Protein levels of (A) total endothelial nitric oxide synthase (eNOS) and (B) phosphorylated eNOS (peNOS) in aortic samples from male and female control and diabetic rats. eNOS (A) and peNOS (B) were quantified by densitometry and normalized to corresponding beta actin. Each bar represents the mean ± SEM of values obtained from n = 4–5 animals per group. To show representative bands, images from different parts of the same gel have been juxtaposed, indicated by white dividing lines. Capped lines indicate significant differences between two groups (p < 0.05), as analyzed by unpaired Student’s t-test.

FIGURE 8

Western blot analysis of Akt and pAkt expression in control and UCD-T2DM rat aorta. Protein levels of aortic (A) total V-akt murine thymoma viral oncogene homolog-2 (Akt) and (B) phosphorylated V-akt murine thymoma viral oncogene homolog-2 (pAkt) from the samples of male and female control and diabetic rats. Akt (A) and pAkt (B) were quantified by densitometry and normalized to corresponding GAPDH. Each bar represents the mean ± SEM of values obtained from n = 4–5 animals per group. To show representative bands, images from different parts of the same gel have been juxtaposed, indicated by white dividing lines. Capped lines indicate significant differences between two groups (p < 0.05), as analyzed by unpaired Student’s t-test.

FIGURE 9

Western blot analysis of NOX1 and NOX4 expression in intact aorta, and oxidant generation in primary aortic endothelial cells isolated from control and UCD-T2DM rats. Protein levels of aortic NADPH oxidases (NOX1) (A) and NOX4 (B) from the samples of male and female control and diabetic rats. NOX1 and NOX4 were quantified by densitometry and normalized to corresponding GAPDH. To show representative bands, images from different parts of the same gel have been juxtaposed, indicated by white dividing lines. Hydrogen peroxide (H₂0₂) (C) and intracellular oxidant (CellROX oxidation) (D) generation in primary aortic endothelial cells isolated from male and female control and diabetic rats. Values are presented as mean ± SEM. Each bar represents the values obtained from n = 4–5 animals per group for NOX expression and n = 3–4 animals per group for oxidant generation studies. Capped lines indicate significant differences between two groups (p < 0.05), analyzed by unpaired Student’s t-test.

FIGURE 10

Proposed drivers of an elevated contractile response in UCD-T2DM rat aortas. (A green one) Impaired insulin signaling, elevated ROS generation, and decreased basal NO activity may drive elevated contractile responses in aortic rings of both male and female UCD-T2DM rats. (B blue one) Male UCD-T2DM rat aortas display enhanced EDV (despite elevated contractile responses) along with elevated IK_Ca and SK_Ca channel expression and traces of NO-independent responses. (C red one) Female UCD-T2DM rat aortas display impaired EDV, possibly due to decreased NO activity and enhanced contractile responses. IRS, insulin receptor substrate; pAkt, phosphorylated V-Akt murine thymoma viral oncogene homolog-2; peNOS, phosphorylated endothelial nitric oxide synthase; NOX, NADPH oxidase; ROS, reactive oxygen species; NO, nitric oxide; IKCa, intermediate-conductance calcium- activated potassium channel; SKca, small-conductance calcium-activated potassium channel; EDV, endothelium-dependent vasorelaxation.