Dapagliflozin impedes endothelial cell senescence by activating the SIRT1 signaling pathway in type 2 diabetes

Abstract

Background: Sodium-glucose cotransporter 2 inhibitors (SGLT2i) clinically reduce atherosclerosis and lower blood pressure. However, their impact on endothelial dysfunction in type 2 diabetes (T2D) remains unclear. In this study, we investigated the protective effect and underlying mechanism of the SGLT2 inhibitor dapagliflozin in diabetes.

Methods: Vascular reactivity was measured to assess the vasoprotective effect of dapagliflozin in a mouse model of high glucose (HG)-induced T2D. Pulse wave velocity was measured to quantify arterial stiffness. Protein expression was assessed by western blotting and immunofluorescence, oxidative stress was evaluated using dihydroethidium, nitric oxide was evaluated using the Griess reaction, and cellular senescence was assessed based on senescence-associated beta-galactosidase (SA-β-gal) activity and the expression of senescence markers. Furthermore, the endothelial nitric oxide synthase (eNOS) acetylation status was determined and eNOS interactions with SIRT1 were evaluated by coimmunoprecipitation assays.

Results: Dapagliflozin protected against impaired endothelium-dependent vasorelaxation and improved arterial stiffness in the mouse model of T2D; mouse aortas had significantly reduced levels of senescence activity and senescence-associated inflammatory factors. HG-induced increases in senescence activity, protein marker levels, and oxidative stress in vitro were all ameliorated by dapagliflozin. The decreases in eNOS phosphorylation and nitric oxide (NO) production in senescent endothelial cells were restored by dapagliflozin. SIRT1 expression was reduced in HG-induced senescent endothelial cells, and dapagliflozin restored SIRT1 expression. SIRT1 inhibition diminished the antisenescence effects of dapagliflozin. Coimmunoprecipitation showed that SIRT1 was physically associated with eNOS, suggesting that the effects of dapagliflozin are dependent on SIRT1 activation.

Conclusion: These findings indicate that dapagliflozin protects against endothelial cell senescence by regulating SIRT1 signaling in diabetic mice.

Keywords: Dapagliflozin; Diabetes; Endothelial cells; SGLT2 inhibitor; SIRT1; Senescence.

Fig. 1

Dapagliflozin protects against endothelium-dependent vasorelaxation in murine type 2 diabetes (T2D). (a) Strategy for dapagliflozin treatment and measurement of metabolic parameters (left). Scheme for blood collection, tissue extraction, and organ morphometry (right). Eight-week-old control and diabetic mice were randomly divided into four groups: (1) control receiving saline (db/m + saline, n = 17), (2) control receiving dapagliflozin (db/m + dapa, n = 14), (3) diabetic receiving saline (db/db + saline, n = 13), (4) diabetic receiving dapagliflozin (db/db + dapa, n = 15). (b, c, and d) Glucose and lipid metabolic measurements showing that T2D db/db mice had significantly higher fasting and non-fasting blood glucose and total triglyceride levels than those of heterozygous control mice. Treatment with dapagliflozin (8 weeks, 1.0 mg/kg/day) reversed these metabolic effects. (e and f) Impaired glucose tolerance test (IGTT) and homeostasis model assessment for insulin resistance (HOMA-IR) showing that T2D db/db mice had impaired glucose handling and significant insulin resistance, and these changes were attenuated by dapagliflozin treatment. (g) Pulse wave velocity (PWV) measurements to detect arterial stiffness. Dapagliflozin decreased PWV. (h) Vascular reactivity assessed to measure the vasoprotective effect of dapagliflozin. Data are presented as means ± SD. *P < 0.05 versus db/m; ^#P < 0.05 versus db/db. Dapa, dapagliflozin; FI, food intake; BW, body weight; BG, blood glucose.

Fig. 2

Dapagliflozin prevents endothelial cell senescence in T2D mice. (a and b) Senescence-associated β-galactosidase (SA-β-gal) staining of aortas (β-galactosidase staining is blue); n = 3–5 showing senescence. (c and d) Immunofluorescent detection of senescence markers in aortas. CD31 was used to identify endothelial cells and p21 and p53 protein levels were detected to assess senescence. n = 6, Scale bar = 100 μm. (e, f, and g) Flow-based fluorescence immune-microbead assay to measure levels of circulating inflammatory cytokines IL-8, IL-17A, and IL-17F. n = 4–8. Data are presented as means ± SD. *P < 0.05, versus db/m; ^#P < 0.05 versus db/db. Dapa, dapagliflozin.

Fig. 3

Dapagliflozin restores endothelial nitric oxide synthase (eNOS) activity and mitigates oxidative stress in senescent endothelial cells. (a and b) Senescence-associated β-galactosidase (SA-β-gal) staining to detect senescence in endothelial cells stimulated with HG (30 mM). Mannitol was used as a control to rule out osmotic effects. n = 6–8, scale bar = 50 μm. Data are presented as means ± SD. *P < 0.05 versus normal glucose; ^#P < 0.05 versus high glucose. (c, d, and e) Immunoblotting to measure p53 and p21 protein levels in HG-stimulated endothelial cells. β-Actin was used for normalization. n = 3. Data are presented as means ± SD. *P < 0.05, versus normal glucose; ^#P < 0.05 versus high glucose. (f) Griess reaction to detect endothelial nitric oxide (NO) production in the plasma of db/db mice. NO levels decreased in the plasma of db/db mice, and this effect was reversed by the addition of dapagliflozin. n = 3. Data are presented as means ± SD. *P < 0.05, versus db/m; ^#P < 0.05 versus db/db. (g and h) The fluorescent probe dihydroethidium (DHE) used to detect intracellular reactive oxygen species production. n = 3, scale bar = 50 μm. (i and j) Immunoblotting to measure total eNOS expression and eNOS phosphorylation status. n = 3. Data are presented as mean s± SD. *P < 0.05 versus normal glucose; ^#P < 0.05 versus high glucose. NG, normal glucose; D, dapagliflozin; HG: high glucose; M, mannitol.

Fig. 4

Dapagliflozin rescues decreased SIRT1 expression in senescent endothelial cells. (a and b) SIRT1 expression in HG-stimulated endothelial cells normalized to β-actin levels. Mannitol was used as a control to rule out osmotic effects. n = 3. (c) NAD⁺/NADH ratios to evaluate SIRT1 activity in HG-stimulated endothelial cells. n = 3. (d and e) Nicotinamide (NAM), a specific and potent pharmacological inhibitor of SIRT1, was used to inhibit SIRT1 activity. Cells were then treated with dapagliflozin and SA-β-gal staining was used to identify senescent cells. n = 6–8, Scale bar = 50 μm. (f, g, and h) p53 and p21 protein levels were assessed using immunoblotting, with β-actin as a loading control. n = 3. Data are presented as means ± SD. *P < 0.05, versus normal glucose; ^#P < 0.05 versus high glucose; ⁺P < 0.05 versus high glucose + dapa. NG, normal glucose; D, dapagliflozin; HG, high glucose; M, mannitol; N, nicotinamide.

Fig. 5

SIRT1 is required for the antisenescence effects of dapagliflozin. (a and b) The fluorescence probe DHE was used to detect intracellular reactive oxygen species (ROS) production following the inhibition of SIRT1 expression. n = 3, scale bar = 50 μm. (c and d) Western blot analyses of total endothelial nitric oxide synthase (eNOS) expression and eNOS phosphorylation status following SIRT1 inhibition. n = 3. (e) NAD⁺/NADH ratios determined following SIRT1 inhibition. n = 3. (f) Coimmunoprecipitation and immunoblotting to measure eNOS acetylation and SIRT1 levels. Data are presented as means ± SD. *P < 0.05 versus normal glucose; ^#P < 0.05 versus high glucose; ⁺P < 0.05 versus high glucose + dapa. NG, normal glucose; D, dapagliflozin; HG, high glucose; M, mannitol; N, nicotinamide; Ac-Lys, acetyl-lysine; ROS, reactive oxygen species; DHE, dihydroethidium.