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Vascular Biology Center, Medical College of Georgia, Augusta, Georgia
Submitted 4 March 2005 ; accepted in final form 1 June 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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microcirculation; adrenergic; nitric oxide; superoxide
Obesity also induces whole body insulin resistance, resulting in impaired control of plasma glucose, hyperinsulinemia, and chronic triglyceride dyslipidemia. Insulin resistance is considered an emerging risk factor for vascular disease and has been documented to impair nitric oxide (NO)-dependent mesenteric vasodilation in animal models of insulin resistance (20, 22–24). Given that NO is a major contributor to absorptive functional hyperemia (1, 9, 12, 17) and flow-mediated regulation (2, 12, 19), it raises the question of how the mesenteric circulation compensates for the impaired dilator response caused by insulin resistance against the elevated metabolic demands associated with increased ingestion of food in obese or hyperphagic individuals. Potential mechanisms include reduced responses to vasoconstriction, augmented responses to alternative vasodilator pathways, and increases in the vascularity of the gut.
Perfusion of the gut is a balance between the constrictor tone that maintains tonic resistance and the metabolic hyperemia that accounts for elevated flow during absorption. In the current study, we tested the hypothesis that NO-mediated dilation is impaired in an animal model of obesity and constrictor reactivity is altered to compensate for this impaired NO-mediated dilation. We used the obese Zucker rat (OZR), a well-established model of obesity and insulin resistance, as our model (26). Relative to lean controls (LZR), the OZR display intestinal organomegaly, elevated total mesenteric blood flow, and similar blood flow normalized to tissue mass (14). In vitro vasodilation to the NO-dependent agonist acetylcholine was examined in mesenteric resistance arteries [<175 µm inner diameter] to determine the effects of obesity on NO-mediated dilation. Because superoxide has been implicated in vascular dysfunction in other beds, the effects of the SOD mimetic tempol was used to examine a potential role for superoxide. Because metabolic dilation is balanced against extrinsic sympathetic tone, reactivity to the primary sympathetic neurotransmitter norepinephrine was examined. Specificity of constrictor reactivity changes was assessed by determining reactivity to endothelin and potassium chloride. To determine the potential contribution of insulin resistance independent of obesity, vasodilator reactivity and constrictor reactivity were examined in fructose-fed lean Zucker rats (FF-LZR), a model of insulin resistance without obesity.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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A moderate insulin resistance independent of obesity was induced by feeding a diet in which 66% of the calories were derived from fructose. The fructose diet was purchased from Harlan Teklad (89247 fructose diet formula TD pellets), and the diet regimen was derived from that described by Miller et al. (10). LZR were used in the fructose-feeding protocol to avoid variance in genetic background between groups. Fructose feeding was begun ad libitum at 8–9 wk of age and continued for 8 wk.
In vivo hemodynamics. Hemodynamic parameters were assessed in anesthetized rats under isoflurane anesthesia as described previously (14). Arterial pressure was measured by the advancement of a fluid-filled catheter into the thoracic aorta, and the heart rate was derived from the pressure pulse with the use of a cardiotachometer (CSI). The mesenteric blood flow was measured with a Transonic 1R flow probe around the superior mesenteric artery. The total perfused territory was determined by the infusion of crystal violet into the mesenteric circulation, and the dyed mass was dissected, cleaned of intestinal contents, and weighed. The blood flows were normalized to mesenteric tissue mass, and the vascular resistance was calculated as pressure divided by flow (in mmHg·ml–1·min·g). To assess whether the increased blood flow and mesenteric mass were simply due to fat accumulation, the omental mass and vascular mass were dissected free of the intestine and the change in the intestinal mass was quantified. As shown in Table 1, the intestinal mass increased in parallel to the mesenteric mass in the obese rats.
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100 µm tip diameter) and secured to the inflow and outflow pipettes with the use of a 10-0 nylon suture. Any side branches were ligated with a single strand teased from 6-0 silk sutures. The inflow and outflow pipettes were connected to a reservoir system that allowed the intraluminal pressure to be controlled. Arteries were extended to their in situ length and equilibrated at 60 mmHg. After initial pressurization, the inflow and outflow pressures were equal and the vessel experienced no flow. The vessel diameter was measured with television microscopy and an on-screen video micrometer. Serial dose-response curves to vasoactive agents were generated over the following dose ranges: acetylcholine (1 x 10–8 to 3 x 10–6), norepinephrine (1 x 10–8 to 3 x 10–6), endothelin (1 x 10–11 to 1 x 10–9), and KCl (0–20 mM). Vasodilation was expressed as the percentage of the passive diameter in a Ca2+-free PSS. Because myogenic tone in extraparenchymal resistance arteries tends to be limited (10% in these studies), the tone was supplemented with norepinephrine (1 x 10–7 to 3 x 10–7) until 50% constricted in each group. No differences in myogenic activity were observed among groups. Vasoconstriction was expressed as the percentage of baseline diameter. One vessel for each dilator or constrictor curve was generated in each animal, and no curve was repeated in vessels from the same animal.
Passive characteristics of mesenteric vessels were determined in Ca2+-free PSS supplemented with 2 mM EGTA. The calculations of passive arteriolar wall mechanics were as follows: The vessel wall thickness (WT, in µm) was calculated as WT = (OD – ID)/2, where OD and ID represent arteriolar outer and inner diameter (in µm), respectively. For the calculation of circumferential stress (
), the intraluminal pressure (PIL) was converted from millimeters of mercury to Newtons per square meter, where 1 mmHg = 1.334 x 102 N/m2. The circumferential stress was then calculated as = (PIL x ID)/(2WT). Circumferential strain (
) was calculated as = (ID – ID5)/ID5, where ID5 represents the internal arteriolar diameter at the lowest intraluminal pressure (i.e., 5 mmHg). Each stress-strain relationship was fitted with the equation = 
x exp(
x), where is the intercept and is the "slope" of the exponential fit. The -coefficient is used as a relative measure of vascular stiffness.
Plasma chemistry. Blood glucose concentrations were assessed with a Precision Glucometer (Medisense, Bedford, MA). Plasma total cholesterol and triglycerides were assessed with colorimetric assays from Wako Chemicals. Lipid peroxides were assessed with a colorimetric assay from Calbiochem. Plasma insulin was determined with the Mercodia Rat Insulin ELISA assay from ALPCO Diagnostics.
Drugs and chemicals. All buffer components and experiment reagents were purchased from Sigma Chemical.
Statistics. All data are expressed as means ± SE. Comparisons between groups were performed by two-way ANOVA (reactivity curves with dose and condition as variables) with the use of the Fisher least significant difference post hoc test or Student’s t-test for baseline comparisons. All analyses were performed with SigmaStat software (Systat Software). Significance was accepted at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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10 mmHg) increase in arterial pressure was evident under isoflurane anesthesia in both insulin-resistant rat groups. Heart rates were similar among all groups. Mesenteric blood flow was significantly elevated in OZR compared with LZR. OZR displayed splanchnomegaly, explaining the increased blood flow because blood flow was similar in LZR and OZR when normalized to tissue mass. Normalized mesenteric blood flow was reduced in FF-LZR. Mesenteric vascular resistance was also significantly higher in FF-LZR compared with LZR. Resistance was similar in LZR and OZR. In the rats that were fasted overnight, blood glucose levels were similar in LZR, OZR, and FF-LZR, indicating no fasting hyperglycemia in any of the strains. Plasma insulin levels were markedly elevated in OZR and more moderately increased in FF-LZR compared with LZR. These data are consistent with the literature values (7, 16, 20) and indicate that fructose feeding induced a state of moderate insulin resistance, whereas obesity induces a more severe form. Plasma cholesterol and triglycerides were significantly elevated in both OZR and FF-LZR, consistent with insulin resistance. Lipid changes in OZR rats were more pronounced than those in FF-LZR, consistent with the greater severity of insulin resistance in OZR. Plasma lipid peroxides were assessed as an index of whole body oxidant stress. Lipid peroxides reflect the combined concentration of malondialdehydes and hydroxynonenals. Lipid peroxide levels were elevated in fructose-fed rats relative to controls and further elevated in OZR. These data indicate that oxidant stress is a component of vascular disease in both moderate and severe states of insulin resistance.
Baseline characteristics of mesenteric resistance arteries from LZR, OZR, and FF-LZR are shown in Table 2. Passive diameter at 60 mmHg was similar in all groups. The WT and wall-to-lumen ratio were unaltered by obesity or insulin resistance, indicating a lack of vascular hypertrophy in this segment of the mesenteric circulation. The -coefficent of the stress-strain curve was also similar among all groups, ruling out alteration in vascular distensibility or compliance as a contribution to altered vascular function.
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Dose-response curves to acetylcholine in control and insulin-resistant rats are shown in Fig. 1. In both OZR and FF-LZR rats, vasodilation induced by acetylcholine was significantly reduced. No statistical difference in reactivity was resolved between OZR and FF-LZR, suggesting that both moderate and severe insulin resistance impairs NO-mediated vasodilation to a similar degree.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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NO is a key mediator of tissue perfusion in the mesenteric circulation, in both the response to flow (2) and the absorption of nutrients (1, 17, 25). The effect of vascular risk factors on NO-mediated dilation has been the subject of intensive study, and recent evidence has documented that obesity also impairs NO-dependent dilation in both clinical (6, 21) and experimental animal populations (3, 5, 24). The aspects of obesity that contribute to impaired NO-mediated vasodilation remain incompletely understood, but the associated insulin resistance common in obesity has been implicated as causal. Indeed, even moderate insulin resistance induced by fructose feeding is sufficient to induce impaired NO-dependent dilation in the aorta (15), the coronary circulation (10), and the cerebral microcirculation (4). Our results (Fig. 1) are in good agreement with these previous results in that we also find impairment of NO-dependent vasodilation in the mesenteric microcirculation in both OZR and FF-LZR. Furthermore, with moderate insulin resistance induced by fructose feeding, similar impairment in NO-dependent dilation occurs in FF-LZR as observed in more profoundly insulin-resistant OZR. We interpret this result to indicate that moderate reductions in insulin sensitivity may be sufficient to induce the reduced endothelium-dependent relaxation observed in obese animals.
The mechanisms of impaired NO-dependent responses in obese and insulin-resistant animals remain controversial. An emerging concept is that elevated oxidant stress is a major mechanism impeding normal endothelium-dependent dilation. Laight et al. (8) found that exacerbation of oxidant stress impaired depressor responses to NO-mediated vasodilator in OZR, and previous studies from our laboratory have correlated superoxide production with impaired microvascular dilation in the skeletal muscle of OZR (5). Shinozaki et al. (15) reported that superoxide limited NO-dependent vasodilation in the aorta of fructose-fed rats, and Onuma et al. (11) have recently reported that scavenging of superoxide with tempol improves renal medullary blood flow in fructose-fed rats in vivo. Our results (Fig. 2) provide new evidence from isolated microvessels that vascular superoxide production limits NO-mediated vasodilation in insulin-resistant animals. In the mesenteric microcirculation, treatment with the SOD mimetic tempol improves NO-mediated dilation to acetylcholine in both OZR and FF-LZR. Furthermore, increases in lipid peroxidation (Table 1) were observed in both groups of insulin-resistant rats. These findings provide new support for the hypothesis that insulin resistance provokes a prooxidant state that impairs NO-mediated dilation. The extension of this observation into the mesenteric circulation, when combined with previous results, argues the pervasive nature of this defect. This ubiquity suggests that chronic exposure to altered plasma composition may be causal because plasma is the one component of the body that touches all vessels. The identity of the causal factor in the effect of insulin resistance on oxidant tone remains to be elucidated.
OZR face a conundrum in which mesenteric NO-mediated vasodilation is blunted, thus compromising the regulation of mesenteric blood flow and chronic hyperphagia in which the demand for absorptive hyperemia is increased. This study hypothesized that one resolution of this dilemma may involve reducing the degree of sensitivity to vasoconstrictor stimulation to help preserve perfusion of the gut when active vasodilator tone is impaired. To test this hypothesis, constrictor reactivity was assessed in vitro to remove the potential confounding variables of parenchymal factors, innervation, or differences in plasma composition. In the current studies, we found that reactivity to norepinephrine and endothelin (Figs. 3 and 4) was reduced in mesenteric resistance arteries from OZR relative to those from LZR. In nonobese FF-LZR, where NO-mediated dilation is impaired to the same degree as observed in OZR, reactivity to norepinephrine was unaffected. When one examines basal mesenteric vascular resistance (Table 1), one finds that mesenteric resistance is comparable between LZR and OZR, despite the impairment of NO-mediated vasodilation in OZR. Thus compensation may exist to normalize perfusion when an essential vasodilator is lost. In FF-LZR, where NO-mediated vasodilation is impaired but reactivity to norephinephrine is not altered, mesenteric resistance is significantly elevated. Thus our findings suggest that the reduced reactivity to vasoconstrictors may help preserve perfusion in the face of obesity and hyperphagia when NO-mediated dilation is compromised.
The precise mechanism by which vasoconstrictor reactivity is reduced in obese animals remains to be elucidated. Nevertheless, three findings in the current studies offer insight into potential mechanisms. First, the reduction in reactivity is relatively specific for G protein-coupled receptors. Reactivity to endothelin and norepinephrine is blunted but not to depolarization with potassium chloride (Fig. 4). This finding suggests that receptor expression or receptor signaling is the mechanistic site of reduced sensitivity to constrictors, rather than alterations in reactivity to calcium. These changes in receptor function may be generalized or may reflect desensitization secondary to changes in concentrations of these ligands in the mesenteric perivascular interstitium. Second, the reduction in constrictor reactivity does not reflect a simple reaction to impaired dilation to NO. Both OZR and FF-LZR show similar levels of impaired dilation to NO, yet only OZR show reduced reactivity to vasoconstrictors. This comparison also rules out oxidant stress as a factor in reduced reactivity to norepinephrine because oxidant stress (as assessed by lipid peroxides and impairment of NO-mediated vasodilation) was elevated in fructose-fed rats without changes in reactivity to norepinephrine. Finally, the reduced reactivity is specific to obesity or severely insulin-resistant states. In FF-LZR, a model of moderate insulin resistance, constrictor reactivity to norepinephrine was not different from LZR. This suggests that other aspects of obesity, including increased sympathetic nerve activity (3, 13), hyperphagia, or more severe insulin resistance (Table 1), may be causal in reducing vascular reactivity to noradrenergic stimulation. Because reactivity to norepinephrine is actually increased in the skeletal muscle circulation (18) of OZR, we speculate that hyperphagia superimposed on impaired vasodilation is a key mechanism for reduced vasoconstriction in the mesenteric circulation.
In summary, we find that metabolic dysfunction induces impairments of NO-mediated vasodilation secondarily to oxidant stress. We find that when obesity and hyperphagia occur concurrently with impaired vasodilation, vasoconstrictor reactivity is reduced. This reduced reactivity to constriction may provide an important compensation to maintain adequate perfusion in the mesenteric circulation of obese individuals. Moreover, because diversion of mesenteric blood flow by adrenergic constriction is a hemodynamic requirement of exercise or compensation for hemorrhage, the reduced vasoconstrictor reactivity present in the mesenteric circulation may represent a source of hemodynamic dysfunction when obese animals are subjected to physiological challenge.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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