The major findings of this study are that 6 weeks of high sucrose or high fat feeding results in increased body mass and adiposity (Table 2) concomitant with increased oxidative stress and impaired vasodilation, although the mechanism of impaired vasodilation differed for each group. Vasodilation of arteries from Chow fed rats is dependent on NO as well as H2O2 (Figures 3 and 6). Feeding rats either HSD or HFD results in diminished vascular smooth muscle sensitivity to NO (Figure 7). Vasodilation is further impaired in rats fed HSD as a result of increases in H2O2 which acts to oppose vasodilation (Figures 1, 4 & 6). In contrast, the findings of this study implicate O2˙ˉ-mediated scavenging of NO to form ONOO- in arteries from rats fed HFD (Figures 1 & 5).
Feeding rats a HSD diet alone elicited hypertriglyceridemia (Table 2), impaired glucose tolerance (Figure 1A), hyperleptinemia (Table 2), enhanced oxidative stress (Table 2 & Figure 1B) and impaired acetylcholine-mediated vasodilation (Figure 2), which are characteristics of metabolic syndrome and pre-diabetes. Animals on the HFD gained more adiposity compared to the HSD rats (Table 2) and demonstrated further characteristics of metabolic syndrome as they developed fasting hyperglycemia (Figure 1A) in addition to hyperleptinemia (Table 2), oxidative stress (Table 2 & Figure 1B), and impaired acetylcholine-induced vasodilation (Figure 2). These significant alterations in oxidative stress and vascular reactivity demonstrate that vascular dysfunction occurs well before the development of obesity and that different diets can have varying effects on biochemical parameters and vascular reactivity in rats.
Elevated plasma glucose, free fatty acids and triglycerides contribute to increased ROS production [9–11]. Previous studies have shown that feeding obesity-prone Sprague-Dawley rats a moderately high fat diet (32% kcal% as fat) for 16 weeks results in elevated TBARS and O2˙ˉ in aorta . Similarly, our studies demonstrate increased fasting glucose in the HFD rats (Figure 1A) and plasma triglycerides in the HSD rats (Table 2) with both groups of rats developing elevated TBARS and DCF fluorescence, indicative of vascular ROS (Table 2 & Figure 1B).
Multiple oxidative stress pathways can potentially lead to decreases in NO bioavailability and therefore, a reduction in vasodilatory responses. A direct pathway occurs by O2˙ˉ-induced scavenging of endothelial-derived NO to form peroxynitrite (ONOO-) [6, 7]. Another mechanism involves the uncoupling of eNOS as a result of diminished levels of the essential eNOS cofactor, tetrahydrobiopterin, resulting in enhanced O2˙ˉ formation and further decreases in NO levels [6–8, 11]. Furthermore, ONOO- itself can oxidize and deactivate tetrahydrobiopterin . Moreover, H2O2, produced from the breakdown of O2˙ˉ, is reported to exhibit vasodilatory or vasoconstrictor effects depending on the concentration or tissue examined [29–31]. Since the observed increase in oxidative stress in HSD and HFD rats may directly affect blood vessel reactivity or impact NO bioavailability, the effects of oxidative stress on endothelium-mediated vasodilation were measured (Figures 3, 4, 5, 6).
Data from the present study demonstrate that high sucrose and high fat feeding result in significantly attenuated endothelium-dependent vasodilation compared to Chow-fed controls (Figure 2). In Chow-fed rats, ACh-mediated vasodilation appears to rely on NO since the NOS inhibitor LNNA nearly abolished the vasodilatory response (Figure 3). In contrast, inhibition of NOS caused no further impairment of vasodilation in HSD and HFD fed rats (Figures 4 & 5). These data suggest that ROS may be involved in the scavenging of NO in these animals as eNOS protein expression levels were not significantly different in the experimental groups compared to Chow fed rats (Figure 8). Further studies demonstrated that arteries from HSD and HFD rats have impaired vascular smooth muscle sensitivity to NO contributing to the impaired ACh-mediated vasodilation (Figure 7). Therefore, it is evident that the residual response to ACh following high calorie feeding is NO-independent and likely involves other endothelium-dependent vasodilatory pathways.
Since the observed increase in oxidative stress in HSD and HFD rats may impact NO bioavailability, the effects of oxidative stress on endothelium-mediated vasodilation were measured (Figures 3, 4, 5, 6). ACh-mediated vasodilation responses in arteries from HFD rats were greatly attenuated across a broad range of ACh doses and were normalized by the inhibition of ROS using either tiron and catalase or EUK-134 (Figure 5). In contrast, vasodilation of arteries from HSD rats was impaired at only high doses of ACh which likewise demonstrated improved vasodilation in the presence of tiron and catalase but not EUK-134 (Figure 4). These data illustrate that oxidative stress plays a role in the limitation of ACh-mediated vasodilation in HSD and HFD rats with only modest increases in adiposity. Oxidative stress is similarly involved in the impaired vasodilatory responses of aortic rings and renal arterioles from alloxan-induced diabetic rabbits . Impaired endothelium-dependent vasodilation, as occurs in coronary artery diseases, has likewise been linked with increased oxidative stress since administration of the antioxidant vitamin C improved the response in humans . Similarly, Zucker obese rats exhibit attenuated responses to ACh in isolated coronary as well as middle cerebral arteries [34, 35]. Moreover, diminished ACh-mediated vasorelaxation in thoracic aorta from insulin resistant rats has been observed .
To test whether impaired vasodilation in arteries from HSD and HFD is due to an interaction of ROS and NO, vessels from each group were incubated in the presence of both the NOS inhibitor LNNA and the ROS scavengers tiron and catalase or EUK-134. Data from these studies demonstrate that impaired vasodilatory responses recorded in arteries from HSD rats is at least in part mediated by reduced NO bioavailability as the combined treatment nearly abolished vasodilation (Figure 4). Similar results were observed in arteries from HFD rats treated with LNNA and tiron and catalase, suggesting that ROS may also reduce the bioavailability of NO in these vessels (Figure 5). However, superfusion of arteries with LNNA and EUK-134 did not normalize vasodilation in arteries from these rats (Figure 5). This apparent discrepancy may be due to the different mechanisms of action of each of these ROS scavengers.
Since H2O2 is known to exert both vasodilatory and vasoconstrictor properties, we examined the role of H2O2 by exposing arteries to catalase in the absence of superoxide dismutase mimetics. In arteries from HFD rats, this exposure did not improve vasodilation (Figure 6C) whereas the combined superoxide dismutase mimetics and catalase were successful at normalizing vasodilation (Figure 5). This suggests that in HFD vessels, elevated O2˙ˉ may be responsible for scavenging of NO resulting in the production of ONOOˉ. In contrast, arteries from HSD fed rats demonstrate only a mild improvement in ACh-mediated vasodilation in the presence of tiron and catalase but no effect of EUK-134 (Figure 4). Since inhibition of H2O2 alone normalized vasodilation in this group (Figure 6B), this supports a role for H2O2 as a vasoconstrictor following HSD feeding. DCF can be oxidized by both ONOOˉ and H2O2 resulting in increased fluorescence as we observed in arteries from HSD and HFD rats (Figure 1B). Our data also demonstrate a physiological role of H2O2 in vasodilatory responses of arteries from Chow rats (Figure 6A), that has been described by others [29–31].
We also observed a differential role of COX products between the different feeding regimens. In Chow rats, COX inhibition blunted ACh-mediated vasodilation (Figure 6A). In contrast, indomethacin normalized vasodilation in arteries from HSD rats suggesting a switch to vasoconstrictor COX products following high sucrose feeding (Figure 6B) that was only minimally present in the HFD group (Figure 6C). Thus, the profile of COX-derived vasoactive products may be altered by diet.
In summary, our data demonstrate that feeding rats either a high fat or high sucrose diet results in the development of oxidative stress as well as impaired vasodilation. The data highlight the importance of the type of diet as it can produce divergent effects on vascular reactivity pathways despite both groups developing increased body mass, adiposity and oxidative stress. Although rats in the HSD fed group develop similar levels of oxidative stress as observed in the HFD rats, the impaired vasodilation is not as severe and the mechanisms of impaired vasodilation are divergent. In the HFD group, the impaired vasodilation appears to be mediated in part by O2˙ˉ scavenging of NO. In contrast, H2O2 is implicated in the impaired vasodilatory responses in vessels from HSD rats. In conclusion, the impaired vasodilatory responses to acetylcholine in rats fed either HSD or HFD are mediated by ROS scavenging of NO, impaired smooth muscle sensitivity to NO as well as by inflammatory factors.