Table 1.
Effect of dietary treatment on body weight, adiposity and plasma parameters.
Figure 1.
Effect of PVAT on contractile responses to noradrenaline.
Cumulative concentration-response curves to noradrenaline (NA, 1 nM–10 µM) in MA with (+) and without (−) PVAT from control (C) [A] and high fat diet (HFD) animals [B]. Data are means ± S.E.M. (n≥5 animals per group). **p<0.01, compared to MA (-) PVAT. Cumulative concentration-response curves to NA (0.1–10 µM) in MB from C and HFD animals [C]. Data are means ± S.E.M. (n≥5 animals per group). **p<0.01, compared with C.
Figure 2.
Contribution of endothelial nitric oxide to Ach-mediated relaxant responses.
Cumulative concentration-response curves to acetylcholine (Ach, 1 nM–0.1 mM) in MB [A] and in MA [B] from control (C) and high fat diet (HFD) animals in absence/presence of L-NAME (0.1 mM). Data are means ± S.E.M. (n≥5 animals per group). ***p<0.001;**p<0.01, compared to C; ###p<0.001 compared to their corresponding matched control groups.
Table 2.
Emax and pD2 values of Ach-induced relaxation in mesenteric arteries (MA-PVAT) and in the perfused mesenteric bed (MB).
Figure 3.
Nitric oxide and superoxide anion availability in mesenteric arteries.
[A] Confocal projections showing in situ NO generation, determined with DAF2-DA (10−5 M), in MA. Adventitial cells are round; vascular smooth muscle cells are elongated and perpendicular to blood flow; endothelial cells are elongated and parallel to blood blow. [B] Fluorescence intensity in MA from control (C) and high fat diet (HFD) animals. Data are means ± S.E.M. (n≥5 animals per group). ***p<0.001 compared to C. [C] Confocal projections showing in situ superoxide generation determined with dihydroethidium (DHE, 3 µM) in MA and [D] quantification of DHE fluorescence intensity in MA from C and HFD. Data are means ± S.E.M. (n = 5 animals per group). ***p<0.001 compared to C.
Figure 4.
eNOS phosphorylation and NOX activity in mesenteric arteries.
[A] Representative immunoblots of p-eNOS in MA. Diagram bars show the result of densitometric analysis of p-eNOS immunoblots, expressed as percentage of p-eNOS/eNOS in the control (C) group. Data are means ± S.E.M. (n≥5 animals per group). *p<0.05 compared to C. [B] NOX activity in MA. Results are expressed as percentage of NADPH oxidase activity in C animals. Data are means ± S.E.M. (n≥5 animals per group). *p<0.05 compared to C.
Figure 5.
Contribution of pro-oxidant systems on contractile responses to noradrenaline in both mesenteric arteries and the mesenteric bed.
[A] Confocal projections showing in situ superoxide generation determined with dihydroethidium (DHE, 3 µM) in mesenteric PVAT from control (C) and high fat diet (HFD) animals. [B] NOX activity in mesenteric PVAT. Results are expressed as percentage of NOX activity in C. Data are means ± S.E.M. (n≥5 animals per group). *p<0.05 compared to C animals. [C] Cumulative concentration-response curves to noradrenaline (NA, 0.1–10 µM) in MB and [D] cumulative concentration-response curves to NA (1 nM–10 µM) in MA from C and HFD animals in absence/presence of apocynin (0.1 mM). Data are means ± S.E.M. (n≥5 animals per group). #p<0.05; ###p<0.001 compared to their corresponding matched control groups.
Figure 6.
Contribution of antioxidant systems on contractile responses to noradrenaline in both mesenteric arteries and the mesenteric bed.
Effect of HFD on ec-SOD protein expression in mesenteric PVAT [A] and in MA [B]. Diagram bars show the result of densitometric analysis of ec-SOD immunoblots, expressed as percentage of ec-SOD in the control (C) group. Data are means ± S.E.M. (n≥5 animals per group). Total SOD activity in mesenteric PVAT [C] and in MA [D]. Data are presented as means ± S.E.M. (n≥5 animals per group). *p<0.05 compared to C animals. [E] Cumulative concentration-response curves to noradrenaline (NA, 0.1–10 µM) in MB and [F] cumulative concentration-response curves to NA (0.1–10 µM) in MA from C and HFD animals in absence/presence of 3-amino-1,2,4-triazole (3-AT, 20 mM). Data are means ± S.E.M. (n≥5 animals per group). ##p<0.01; ###p<0.001 compared to their corresponding matched control groups.
Figure 7.
Representative diagram with a possible mechanism explaining the effect of long-term high fat diet (HFD) on endothelial dysfunction.
Adipokine dysregulation in PVAT (increase in leptin release together with reduced adiponectin levels) lead to an increase in NOX activity but a reduction in total SOD activity and ec-SOD expression. Therefore, obesity induces a huge increment of superoxide levels in PVAT. Circulating and PVAT-derived adipokines might also lead to an increased NOX activity and consequently, to enhance superoxide and H2O2 levels in the vascular wall. PVAT-derived adipokines might also contribute to a reduction in eNOS phosphorylation and, consequently to reduced NO availability that accounts for endothelial dysfunction aggravated by PVAT-derived superoxide.