Recurrent Hypoglycemia Impaired Vascular Function in Advanced T2DM Rats by Inducing Pyroptosis

Abstract

Background: Hypoglycemia is a dangerous side effect of intensive glucose control in diabetes. Even though it leads to adverse cardiovascular events, the effects of hypoglycemia on vascular biology in diabetes have not been adequately studied.

Methods: Aged Sprague-Dawley rats were fed a high-fat diet and given streptozotocin to induce type 2 diabetes mellitus (T2DM). Acute and recurrent hypoglycemia were then induced by glucose via insulin administration. Vascular function, oxidative stress, and pyroptosis levels in aortic tissue were assessed by physiological and biochemical methods.

Results: Hypoglycemia was associated with a marked decrease in vascular function, elevated oxidative stress, and elevated pyroptosis levels in the thoracic aorta. The changes in oxidative stress and pyroptosis were greater in rats with recurrent hypoglycemia than in those with acute hypoglycemia.

Conclusions: Hypoglycemia impaired vascular function in aged rats with T2DM by inducing pyroptosis. The extent of injury increased with the duration of blood glucose fluctuation.

Figure 1

Glucose levels in T2DM model rats and insulin-induced acute or recurrent hypoglycemia; ^∗p < 0.05 pretreatment vs. posttreatment.

Figure 2

Vascular function, eNOS, and iNOS expression in aortas from aged T2DM rats in response to acute and recurrent hypoglycemia. (a) ACh- and (b) SNP-induced relaxation responsiveness and (c) PE-induced contraction responsiveness in controls, aged diabetes (DM), acute hypoglycemia (H-DM), and recurrent hypoglycemia (RH-DM) groups were determined by testing the reactivity of aorta rings. Vascular function was reported by EC₅₀, E_max, and the AUC. eNOS and iNOS protein expressions were assayed in (d) western blots and (e) by immunohistochemical staining. Aorta morphology was evaluated by hematoxylin and eosin staining; ^∗p < 0.05 DM vs. control; ^#p < 0.05 H-DM, RH-DM vs. DM; ^&p < 0.05 H-DM vs. RH-DM.

Figure 3

Oxidative stress in the aorta in response to acute and recurrent hypoglycemia in aged T2DM rats. NOX2/4 expression was assayed by (a) western blotting, (b) serum levels of SOD, (c) GSH-Px, and (d) MDA were tested. (e) 8-OHdG location and expression were determined by immunofluorescence; ^∗p < 0.05 DM vs. control; ^#p < 0.05 H-DM, RH-DM vs. DM; ^&p < 0.05 H-DM vs. RH-DM.

Figure 4

NF-κB, NLRP3, and cGAS–STING pathway activity of the aortas in response to acute and recurrent hypoglycemia in aged T2DM rats. (a) p-p65, NLRP3, ASC, (b) cleaved caspase-1, cGAS, and (e) STING expression were assayed by western blotting. The expression and location of NLRP3 were determined by (c) immunohistochemistry and (d) immunofluorescence; ^∗p < 0.05 DM vs. control; ^#p < 0.05 H-DM, RH-DM vs. DM; ^&p < 0.05 H-DM vs. RH-DM.

Figure 5

Pyroptosis in the aortas in response to acute and recurrent hypoglycemia in aged T2DM rats. (a) GSDMD-N, Bax, and (b) Bcl-2 expression were assayed by western blotting. Expression and location of GSDMD-N were identified by (b) immunohistochemistry and (c) immunofluorescence; ^∗p < 0.05 DM vs. control; ^#p < 0.05 H-DM, RH-DM vs. DM; ^&p < 0.05 H-DM vs. RH-DM.