α-Tocopherol Improves Microcirculatory Dysfunction on Fructose Fed Hamsters

Abstract

Fructose, an everyday component of western diet associated to chronic hyperglycemia and enhanced free radical production, impairs endothelial function and supplementation with antioxidants might improve it. In this study we investigated if vitamin E could reverse the microvascular damage elicited by fructose. Male Syrian golden hamsters drank either 10% fructose solution (F) or filtered water (C), combined with three concentrations of vitamin E in their chows [zero, normal (VE) or 5X (5XVE)] during 60 days. Microvascular reactivity in response to topical application of acetylcholine (Ach; endothelium-dependent vasodilator) or sodium nitroprusside (SNP; endothelium-independent vasodilator) and macromolecular permeability increase induced by either 30 min ischemia followed by reperfusion (I/R) or topical application of histamine (5 μM) were assessed using the cheek pouch preparation. Compared to controls (drinking filtered water), fructose-drinking animals showed decreased vasodilatation to acetylcholine in all concentrations tested (-56.2% for 10-9M, -53.9% for 10-7M and -43.7% for 10-5M). On the other hand, vitamin E supplementation resulted in increased responses for both water and fructose drinking groups (177.4% for F vs. F/5XVE and 241.6% for C vs. C/5XVE for 10-5M Ach). Endothelial-independent vasodilatation explored by topical application of SNP was restored and even enhanced with the supplementation of 5X vitamin E in both groups (80.1% for F vs. F/5XVE; 144.2% for C vs. C/5XVE; 3.4% of difference for C/5XVE vs. F/5XVE on 10-5M SNP). The number of leaky sites after I/R and histamine stimuli in vitamin E supplemented animals decreased (-25.1% and -15.3% for F vs. F/5XVE; and -21.7% and -16% of leaky sites comparing C vs. C/5XVE, respectively for I/R and histamine stimuli) pointing to tightening of the endothelial barrier for macromolecular permeability. Our results strongly suggest that vitamin E could improve the endothelial function and permeability barrier and also reverse impairments elicited by sugar overload.

Fig 1. Experimental design and protocol.

(1A) Eight weeks-old hamsters were treated during 8 weeks as described: animals were divided into two major groups, substitution of the drinking water by 10% fructose solution or kept drinking filtered water. Each major group had the formulated chow associated to three different concentrations of vitamin E: zero vitamin E (groups F and C), 75U/kg (normal concentration of vitamin E—groups F/VE and C/VE) and 375U/kg (5 times the normal concentration of vitamin E—groups F/5XVE and C/5XVE). After the 8^th week of treatment, the hamster cheek pouch microcirculation was evaluated by intravital microscopy and animals were euthanized for blood collection. (1B) Microcirculatory function was evaluated in two fronts: endothelial function by topical application of either acetylcholine or sodium nitroprusside, both in three different concentrations (10⁻⁹, 10⁻⁷ and 10^-5M), in a cumulative dose-response curve and macromolecular permeability increase induced by either ischemia/reperfusion (30 min local ischemia followed by reperfusion) or topical application of histamine (5 μM during 5 min).

Fig 2. Mean arteriolar diameters after topical application of Ach–Endothelial- dependent evaluation.

Data are shown as changes of average diameter, expressed as mean ± SD and plotted in superimposed symbols. (A) Overall mean arteriolar diameters after topical application of three concentrations of acetylcholine (10⁻⁹, 10⁻⁷ and 10⁻⁵ M) in the 2 treated groups. (B) Endothelial-dependent responses of groups treated with filtered water, concomitantly associated with chows without vitamin E (C), with normal concentration of vitamin E (75U/kg–C/VE) and supplemented concentrations of vitamin E (375U/kg–C/5XVE). (C) Endothelial-dependent responses of groups that had the filtered water substituted by 10% fructose solution, concomitantly associated with chows without vitamin E (F), with normal concentration of vitamin E (75U/kg–F/VE) and supplemented concentrations of vitamin E (375U/kg–F/5XVE). ⁺Significantly different in relation to control; ⁺ p<0.05 [10^-7M] and both control and control with normal concentration of vitamin E; ⁺ p<0.01 [10^-5M]. ^#Significantly different from fructose-drinking without vitamin E in chow (^# p<0.05).

Fig 3. Mean arteriolar diameters after topical application of SNP–Endothelial- independent evaluation.

Data are shown as changes of average diameter, expressed as mean ± SD and plotted in superimposed symbols. (A) Overall mean arteriolar diameters after topical application of three concentrations of sodium nitroprusside (10⁻⁹, 10⁻⁷ and 10⁻⁵ M) in the 2 treated groups. (B) Endothelial-independent responses of groups treated with filtered water, concomitantly associated with chows without vitamin E (C), with normal concentration of vitamin E (75U/kg–C/VE) and supplemented concentrations of vitamin E (375U/kg–C/5XVE). (C) Responses of groups that had the filtered water substituted by 10% fructose solution, concomitantly associated with chows without vitamin E (F), with normal concentration of vitamin E (75U/kg–F/VE) and supplemented concentrations of vitamin E (375U/kg–F/5XVE). ⁺ p<0.05 [10^-9M and 10^-7M] and p<0.01 [10^-5M]. Significantly different from Control without vitamin E and ^#p<0.01 significantly different from fructose-drinking solution without vitamin E.

Fig 4. Microvascular permeability measurements after I/R procedure and topical application of histamine.

Data are shown as number of leaks, expressed as mean ± SD and plotted in column bars. (A) Number of extravasations after 30 min ischemia followed by reperfusion, 10 minutes after the onset of reperfusion. Significant differences were found between *C vs. F (p<0.01), + C vs. C/VE and C/5XVE (p<0.001), and F vs. F/VE and F/5XVE (p<0.01). (B) Histamine-mediated microvascular permeability. Animals treated with 10% fructose solution without vitamin E in the chow presented higher number of leaky sites, when compared to controls (C—filtered water without vitamin E in the chow) (^* p<0.01- F vs. C). Supplementation with vitamin E restored microcirculatory function, as improvements may be seen on both fructose (^# p<0.01 –F vs. F/5XVE) and control responses (⁺ p<0.01 –C vs. C/5XVE).