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

Figures

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  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.

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  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…

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  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.

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  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.

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  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.