Vascular hyperacetylation is associated with vascular smooth muscle dysfunction in a rat model of non-obese type 2 diabetes

Abstract

Background: Advanced type 2 diabetes mellitus (T2DM) accelerates vascular smooth muscle cell (VSMC) dysfunction which contributes to the development of vasculopathy, associated with the highest degree of morbidity of T2DM. Lysine acetylation, a post-translational modification (PTM), has been associated with metabolic diseases and its complications. Whether levels of global lysine acetylation are altered in vasculature from advanced T2DM remains undetermined. We hypothesized that VSMC undergoes dysregulation in advanced T2DM which is associated with vascular hyperacetylation.

Methods: Aged male Goto Kakizaki (GK) rats, a non-obese murine model of T2DM, and age-matched male Wistar rats (control group) were used in this study. Thoracic aortas were isolated and examined for measurement of global levels of lysine acetylation, and vascular reactivity studies were conducted using a wire myograph. Direct arterial blood pressure was assessed by carotid catheterization. Cultured human VSMCs were used to investigate whether lysine acetylation participates in high glucose-induced reactive oxygen species (ROS), a crucial factor triggering diabetic vascular dysfunction.

Results: The GK rats exhibited marked glucose intolerance as well as insulin resistance. Cardiovascular complications in GK rats were confirmed by elevated arterial blood pressure and reduced VSMC-dependent vasorelaxation. These complications were correlated with high levels of vascular global lysine acetylation. Human VSMC cultures incubated under high glucose conditions displayed elevated ROS levels and increased global lysine acetylation. Inhibition of hyperacetylation by garcinol, a lysine acetyltransferase and p300/CBP association factor (PCAF) inhibitor, reduced high glucose-induced ROS production in VSMC.

Conclusion: This study provides evidence that vascular hyperacetylation is associated with VSMC dysfunction in advanced T2DM. Understanding lysine acetylation regulation in blood vessels from diabetics may provide insight into the mechanisms of diabetic vascular dysfunction, and opportunities for novel therapeutic approaches to treat diabetic vascular complications.

Keywords: High glucose; Lysine acetylation; PCAF; ROS; Type 2 diabetes; Vascular dysfunction; Vascular smooth muscle cells.

Fig. 1

Altered glucose metabolism, insulin sensitivity and lipid profile from GK rats in advanced T2DM. a Fasting blood glucose (mg/dL), b HbA1C levels (%), c Oral glucose tolerance test (oGTT) performed during 120 min after administration of glucose (2 g/kg body weight), d Total blood glucose accumulation reported as area under the curve (AUC), e TyG Index (insulin sensitivity marker), f Fasting triglycerides (mg/dL), g Non-esterified fatty acids (NEFA) (mM) of male GK rats. *p < 0.05 vs. control, n = 5–7 per group. Values are means ± SEM

Fig. 2

Increased arterial blood pressure, and impaired VSMC-dependent relaxation from GK rats in advanced T2DM. a Representative tracing of direct arterial blood pressure (BP) measurement via carotid catheterization. b Systolic BP (mmHg) and c Diastolic BP (mmHg). d Cumulative concentration–response curves to Sodium Nitroprusside (SNP) in aortas from GK rats (gray circles) in comparison to aortas from age-matched male Wistar rats (black circles). Each point represents the maximal response to each concentration of SNP, e Maximal relaxation response (Emax) to SNP, f pD2 for SNP curve. *p < 0.05 vs. control, n = 6 per group. Values are means ± SEM

Fig. 3

Vascular remodeling in aortas from GK rats in advanced T2DM. a Representative photomicrographs of the aortic cross-sections from the control and GK groups stained with H&E. Scale bar: 500 μm. b Wall thickness (Wm) (μm), c Cross-Sectional Area (CSA) (mm²), d Lumen diameter (μm), and e Wall-to-lumen (W_m/L) ratio were obtained using Image J analysis software. *p < 0.05 vs. control, n = 6–10 per group. Values are means ± SEM

Fig. 4

Lysine acetylation and PCAF expression is increased in aortas from GK rats in advanced T2DM. a Global lysine acetylation was detected by using a specific antibody for acetylated lysine residue. β actin used as internal control. b Representative immunoblotting for PCAF (top panel). β actin used as internal control. Bar graphs are means ± SEM of four independent experiments as determined from densitometry relative to β actin. *p < 0.05 vs. control, n = 4–5 per group

Fig. 5

Lysine acetylation and PCAF expression is increased and ROS levels is elevated in aortic-VSMC from GK rats in advanced T2DM. Aortic VSMCs were isolated from Wistar and GK rats and maintained in primary cultures. a Morphology of aortic VSMCs cultures in passage 1. b Global lysine acetylation was detected by using a specific antibody for acetylated lysine residue. β actin used as internal control. c Representative immunoblotting for PCAF (top panel). β tubulin used as internal control. d ROS levels detected by DHE fluorescence in each experimental group. Quantification of DHE staining was determined through fluorescent intensity in each cell by pixel intensity of the cell. Bar graphs are means ± SEM of four independent experiments as determined from densitometry relative to internal control. *p < 0.05 vs. control

Fig. 6

High glucose increases levels of lysine acetylation and PCAF expression in primary human VSMCs cultures. Human VSMCs (hVSMC) were stimulated with 25 mM high glucose (HG) for 12 h. a Global lysine acetylation was detected by using a specific antibody for acetylated lysine residue. β actin used as internal control. b Bar graphs are means ± SEM for PCAF expression as determined from densitometry relative to internal control. β tubulin used as internal control. *p < 0.05 vs. control, n = 5 per group. Molecular weight (MW)

Fig. 7

Garcinol reduces HG-induced ROS production in hVSMC. Quiescent hVSMC were pretreated with 15 µM garcinol, followed by stimulation with 25 mM HG or 5 mM NG for 60 min. a ROS production was detected using DHE staining. Representative photomicrographs depicting DHE fluorescence in each experimental group. Quantification of DHE staining was determined through fluorescent intensity in each cell by pixel intensity of the cell. b Global lysine acetylation was detected by western blot. β actin used as internal control. Results represent means ± SEM. n = 4 per group, *p < 0.05 vs. controls, **p < 0.05 vs. HG group

Fig. 8

Schematic summary of the present study. Advanced T2DM leads to increased global lysine acetylation (AC) and PCAF expression in association with elevated ROS formation in VSMCs, which may be contributing to dysfunctional VSMC-induced impaired vasodilation. Created with BioRender.com