Reactive Oxygen Species in the Aorta and Perivascular Adipose Tissue Precedes Endothelial Dysfunction in the Aorta of Mice with a High-Fat High-Sucrose Diet and Additional Factors

Abstract

Metabolic syndrome (Mets) is the major contributor to the onset of metabolic complications, such as hypertension, type 2 diabetes mellitus (DM), dyslipidemia, and non-alcoholic fatty liver disease, resulting in cardiovascular diseases. C57BL/6 mice on a high-fat and high-sucrose diet (HFHSD) are a well-established model of Mets but have minor endothelial dysfunction in isolated aortas without perivascular adipose tissue (PVAT). The purpose of this study was to evaluate the effects of additional factors such as DM, dyslipidemia, and steatohepatitis on endothelial dysfunction in aortas without PVAT. Here, we employed eight-week-old male C57BL/6 mice fed with a normal diet (ND), HFHSD, steatohepatitis choline-deficient HFHSD (HFHSD-SH), and HFHSD containing 1% cholesterol and 0.1% deoxycholic acid (HFHSD-Chol) for 16 weeks. At week 20, some HFHSD-fed mice were treated with streptozocin to develop diabetes (HFHSD-DM). In PVAT-free aortas, the endothelial-dependent relaxation (EDR) did not differ between ND and HFHSD (p = 0.25), but in aortas with PVAT, the EDR of HFHSD-fed mice was impaired compared with ND-fed mice (p = 0.005). HFHSD-DM, HFHSD-SH, and HFHSD-Chol impaired the EDR in aortas without PVAT (p < 0.001, p = 0.019, and p = 0.009 vs. ND, respectively). Furthermore, tempol rescued the EDR in those models. In the Mets model, the EDR is compromised by PVAT, but with the addition of DM, dyslipidemia, and SH, the vessels themselves may result in impaired EDR.

Keywords: endothelial dysfunction; hypercholesterolemia; oxidative stress; perivascular adipose tissue; steatohepatitis; type 2 diabetes mellitus.

Figure 1

All the HFHSD groups revealed higher glucose levels and insulin resistance, and the HFHSD-DM group, in particular, showed the highest levels of glucose: (A–D) Oral glucose tolerance test (OGTT) time curve for glucose levels (n = 8); (E–H) insulin tolerance test (ITT) time curve for glucose levels (n = 8). OGTT and ITT are expressed as mean blood glucose levels ± standard error of the mean (SEM). ND, normal diet group; HFHSD, high-fat/high-sucrose (HFHSD) diet group; HFHSD-DM, HFHSD group that received streptozocin (STZ) (50 mg/kg/day for 2 days at 20 weeks old); HFHSD-SH, choline-deficient HFHSD group; HFHSD-Chol, 1% cholesterol and 0.1% deoxycholic acid-containing HFHSD group. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the ND group.

Figure 2

HFHSD-SH had the worst steatohepatitis and HFHSD-Chol had the worst steatohepatitis: (A) Hematoxylin and eosin staining of the liver for the ND, HFHSD, HFHSD-DM, HFHSD-SH, and HFHSD-Chol. Scale bars, 200 µm; (B) Masson’s trichrome staining of the liver for the ND, HFHSD, HFHSD-DM, HFHSD-SH, and HFHSD-Chol. Scale bars, 200 µm; (C) oil red O staining of the liver for the ND, HFHSD, HFHSD-DM, HFHSD-SH, and HFHSD-Chol. Scale bars, 200 µm; (D) serum ALT levels (n = 12–16); (E,F) hepatic cholesterol and triglyceride content (n = 8); (G,H) non-alcoholic fatty liver disease (NAFLD) activity score (n = 8) and staging of fibrosis (n = 8) were adapted from Brunt et al. ND, normal diet group; HFHSD, high-fat/high-sucrose (HFHSD) diet group; HFHSD-DM, HFHSD group that received streptozocin (STZ) (50 mg/kg/day for 2 days at 20 weeks old); HFHSD-SH, choline-deficient HFHSD group; HFHSD-Chol, 1% cholesterol and 0.1% deoxycholic acid-containing HFHSD group; ALT, alanine aminotransferase; NAFLD, non-alcoholic fatty liver disease. Error bars represent the standard error of the mean (SEM). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the ND group and † p < 0.05 and ††† p < 0.001 compared with the HFHSD group.

Figure 3

Endothelial-dependent relaxation in the HFHSD-DM, HFHSD-SH, and HFHSD-Chol groups was significantly impaired and accompanied by increased superoxide production from aortas without PVAT: (A) DHE staining of aortic rings and (B) aortic rings with tempol; (C) quantification of DHE fluorescence (n = 8 for each group). Vascular relaxation of aortic rings with acetylcholine in the HFHSD group (D), HFHSD-DM group (E), HFHSD-SH group (F), and HFHSD-Chol group (G) compared with the ND group (ND, n = 16; HFHSD, n = 12; HFHSD-DM, n = 12; HFHSD-SH, n = 16; HFHSD-Chol, n = 14). Acetylcholine indicates acetylcholine; DHE, dihydroethidium; ND, normal diet group; HFHSD, high-fat/high-sucrose (HFHSD) diet group; HFHSD-DM, HFHSD group that received streptozocin (STZ) (50 mg/kg/day for 2 days at 20 weeks old); HFHSD-SH, choline-deficient HFHSD group; HFHSD-Chol, 1% cholesterol, and 0.1% deoxycholic acid-containing HFHSD group. Error bars represent the standard error of the mean (SEM). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the ND group and † p < 0.05 and †† p < 0.01 compared with aorta without treatment of tempol.

Figure 4

PVAT increased EDR in the ND group but decreased EDR in the HFHSD group and was accompanied by increased superoxide production from PVAT: (A) DHE staining of aortic rings with PVAT and (B) with tempol; (C) quantification of DHE fluorescence (n = 8 for each group). Vascular relaxation of aortic rings with PVAT or without PVAT on acetylcholine in the HFHSD group (D), HFHSD-DM group (E), HFHSD-SH group (F), and HFHSD-Chol group (G) (n = 8 for each group). Acetylcholine indicates acetylcholine; PVAT, perivascular adipose tissue; DHE, dihydroethidium; ND, normal diet group; HFHSD, high-fat/high-sucrose (HFHSD) diet group; HFHSD-DM, HFHSD group that received streptozocin (STZ) (50 mg/kg/day for 2 days at 20 weeks old); HFHSD-SH, choline-deficient HFHSD group; HFHSD-Chol, 1% cholesterol and 0.1% deoxycholic acid-containing HFHSD group. Error bars represent the standard error of the mean (SEM). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the ND group and † p < 0.05 and ††† p < 0.001 compared with the HFHSD group.

Figure 5

Summary of experimental results. ACh, acetylcholine; EDR, endothelium-dependent relaxation; PVAT, perivascular adipose tissue; STZ, streptozocin; T2DM, type 2 diabetes mellitus; ND, normal diet group; HFHSD, high-fat/high-sucrose (HFHSD) diet group; HFHSD-DM, HFHSD group that received STZ (50 mg/kg/day for 2 days at 20 weeks old); HFHSD-SH, choline-deficient HFHSD group; HFHSD-Chol, 1% cholesterol and 0.1% deoxycholic acid-containing HFHSD group.