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Iowa Department of Veterans Affairs, Iowa City, Iowa; and Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa
Submitted 8 December 2005 ; accepted in final form 18 May 2006
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ABSTRACT |
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metabolic syndrome; Type 2 diabetes; oxidative stress; acetylcholine
The ZDF rat was derived from the Zucker obese rat by inbreeding for the hyperglycemia phenotype, which occurred in a subpopulation of the Zucker rat when fed a high-fat diet. Therefore, the genetic backgrounds for these rats are very similar. In the ZDF rat model, all fatty males become hyperglycemic by 10 wk of age when fed a high-fat diet, and glucose remains elevated throughout their life span (18). Initially, at 10–13 wk of age, ZDF rats are hyperinsulinemic. However, by 22–42 wk of age, serum insulin levels decline to below levels of insulin in age-matched lean control rats (18). A similar decrease in insulin levels is observed in human Type 2 diabetes, which is thought to be caused by pancreas 
-cell exhaustion. Throughout their life span, free fatty acids, triglycerides, and cholesterol levels are significantly higher in the euglycemic Zucker obese and hyperglycemic ZDF rats compared with lean littermate controls (18).
Previous studies have shown vascular dysfunction associated with the metabolic syndrome and Type 2 diabetes using these rat models. Stepp and colleagues showed augmented adrenergic vasoconstriction (23) and enhanced myogenic responses (7) in arteries from the gracilis muscle in the Zucker obese rat. Attenuated acetylcholine (ACh) responses have been shown in mesenteric (2) and cerebral (19) arteries of Zucker obese rats. Furthermore, Coppey et al. (5) have shown ACh-mediated responses are attenuated in epineurial arterioles of the sciatic nerve from ZDF rats. There has also been considerable evidence demonstrating enhanced reactive oxygen species as pathological factors responsible for the impaired vasomotor function in these rats (6, 9, 13, 20). However, these studies did not examine the development and progression of vascular dysfunction. To examine progression and development of vascular dysfunction associated with the metabolic syndrome and Type 2 diabetes, we designed a longitudinal study that was performed every 4 wk in rats from 8 to 40 wk of age. We measured indices of oxidative stress in serum and vascular reactivity in aorta and coronary and mesenteric small arteries.
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METHODS |
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Zucker obese, ZDF, and lean littermate rats were obtained from Charles River Laboratories (Wilmington, MA) at 6 wk of age. Food (Harlan Teklad, Madison, WI), no. 7001 (4% fat content, normal chow) for Zucker obese and lean rats or no. 7013 (6% fat content) for ZDF rats, and water were provided ad libitum.
On the day of the experiment, rats were weighed and anesthetized with pentobarbital sodium (Nembutal, 50 mg/kg ip; Abbott Laboratories, North Chicago, IL), and a catheter was inserted into the carotid artery to measure blood pressure. A blood sample was collected for determining nonfast serum glucose (Lifescan, Milpitas, CA), and serum samples were collected for determination of insulin levels (RIA kit; LINCO Research, St. Charles, MO). The rat was euthanized by exsanguination, and heart, aorta, and mesenteric tissues were harvested and placed in cold oxygenated Krebs solution maintained at 4°C.
Determination of oxidative stress. As an index of reactive oxygen species production in the serum, thiobarbituric acid reactive substance (TBARS) level was determined by the method of Mihara et al. (10) as modified by Siman and Eriksson (4, 21). TBARS measures a family of lipid peroxidation products (mostly lipid hydroperoxides) and is an indirect marker of oxidative stress.
Isolated aortic rings. Thoracic aorta was studied by using a standard isometric ring technique (14, 16). Segments of aorta were cut into ring segments, mounted on wire stirrups, and placed in a 10-ml- jacketed organ bath containing Krebs buffer (see Solutions and drugs). Isometric contractions and relaxations were measured on a data acquisition system. Rings were individually stretched to 2 g of resting tension. Response to 80 mM KCl confirmed vessel viability.
The aorta segments were equilibrated 30 min before study. To assess constriction, phenylephrine (3 nM–10 µM) concentration-response curves were performed. To evaluate vascular relaxation, the vessels were preconstricted to 50–70% of resting tension with the use of phenylephrine before cumulative concentration-response relationships were evaluated for ACh (3 nM–10 µM) and sodium nitroprusside (SNP) (3 nM–10 µM). Inclusion criteria included 1) development of >1 g of tension to phenylephrine and 2) dilation by >80% to SNP.
Isolated microvessels.
A standard in vitro pressurized microvessel preparation was used to study coronary and mesenteric vessels (15–17). Hearts and mesenteric tissues were removed and immediately placed in cold (4°C), oxygenated Krebs bicarbonate buffer solution (see Solutions and drugs). Coronary microvessels (75- to 175-µm intraluminal diameter and 
1 mm in length) and fourth-order mesenteric vessels of similar size were isolated and cannulated onto glass micropipettes with 10-0 ophthalmic suture. The organ chamber was placed on the stage of an inverted microscope, and oxygenated Krebs buffer (37°C) was circulated through the chamber. Intraluminal diameters were measured by using a video camera and video micrometer. The resolution of the system allowed measurement of small (1–2 µm) changes in vessel diameter.
Microvessels were allowed to equilibrate for 30 min at a hydrostatic distending pressure of 40 mmHg under no-flow conditions. KCl (75 mM) was added to the bath to test vessel viability. To evaluate relaxation responses to ACh (10–10–10–4 M) or SNP (10–10–10–4 M), vessels were preconstricted with the thromboxane analog U-46619 (0.3–3 µM; averages: 1.6 ± 0.1 µM for coronary vessels and 0.48 ± 0.03 µM for mesenteric vessels). Although coronary vessels required more U-46619 than mesenteric vessels, they were constricted to 30–60% of their baseline value. Coronary arteries were preconstricted to 42, 43, and 40% of their resting diameter in 28- to 36-wk-old lean, Zucker obese, and ZDF rats, respectively [P = not significant (NS)]. Mesenteric arteries were preconstricted with U-46619 to 51, 54, and 52% in 28- to 40-wk-old lean, Zucker obese, and ZDF rats, respectively (P = NS). Various vascular beds have altered sensitivity to many vasoactive agents. A single dose of papaverine (10–4 M) was given at the end of each experiment to determine maximal diameter. To evaluate the role of vascular superoxide, some vessels were pretreated with 1 mM tiron for 30 min before constriction with U-46619. The following criteria were required for an acceptable microvessel experiment: 1) microvessels had to demonstrate no leaks and maintain tone under 40 mmHg; 2) microvessels had to constrict >30% to 75 mM KCl and >30% to U-46619; and 3) microvessels had to dilate by >80% to 10–4 M papaverine.
Solutions and drugs. Krebs-Henseleit solution contained (in mM) 120.0 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 23.0 NaHCO3, 1.2 KH3PO4, 11 glucose, and 0.025 EDTA. Solutions were aerated with 20% O2-5% CO2-75% N2 and maintained at 37°C with pH at 7.4. U-46619 was obtained from BioMol (Plymouth Meeting, PA). All other salts, chemicals, and vasoactive agents were purchased from Sigma Chemical (St. Louis, MO). All solutions and vasoactive agents were prepared fresh on the day of the experiment.
Statistical analysis. Data are expressed as means ± SE. Vascular responses were grouped according to age of the rats. Results are expressed as 8–12 wk, 16–24 wk, and 28–40 wk of age. Analysis revealed no significant differences within these age groups. All concentration-response curves were evaluated for differences by using two-way repeated-measures ANOVA followed by the Fisher least significant difference correction for multiple comparisons using SigmaStat software (Jandel Scientific). Significance of differences among mean values of body weight, blood glucose, and plasma insulin levels was assessed with a one-way ANOVA. Differences with P < 0.05 were considered significant.
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RESULTS |
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100 mg/dl). Glucose values in lean rats remained around 100 mg/dl throughout the study (Fig. 1B). Glucose values in Zucker obese rats were similar to lean animals for the first 36 wk but were increased slightly at 40 wk, which was preceded by a decrease in serum insulin levels (Fig. 1C). ZDF rats were hyperglycemic at 8 wk, and glucose values remained elevated throughout the 40 wk. Insulin levels in lean rats remained steady throughout the study (Fig. 1C). Plasma insulin levels in Zucker obese rats were markedly elevated early in the study but consistently declined after 16 wk of age, as the rats became progressively insulinopenic. At 36 wk of age, insulin levels in Zucker obese rats were no longer elevated compared with lean rats. Insulin levels in ZDF rats decreased at 28 wk of age, which is the same time point that these rats began to lose weight.
Mean arterial blood pressure was determined by using a catheter inserted in the carotid artery. At 8–12 wk of age and 36–40 wk of age, the mean arterial blood pressure was 120 ± 4 and 117 ± 6 in lean animals, 129 ± 4 and 146 ± 5 (P < 0.05 vs. lean; P < 0.05 vs. 8- to 12-wk-old rats) in Zucker obese rats, and 129 ± 3 and 139 ± 4 mmHg (P < 0.05 vs. lean) in ZDF rats, respectively.
TBARS are indicative of changes in lipid peroxidation. Serum TBARS level was increased in Zucker obese rats by 16 wk (Fig. 2) and remained elevated throughout the 40 wk. TBARS levels were increased in ZDF rats at an earlier time point compared with lean rats (8–12 wk) and remained elevated throughout the study. These data indicate that oxidant stress is increased in serum in both models of the metabolic syndrome but is increased at an earlier age when diabetes is superimposed.
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SNP concentration-response curves were performed to evaluate smooth muscle-dependent relaxation in segments of thoracic aorta. SNP produced complete relaxation of the phenylephrine-induced constriction in aorta from all groups of rats. In the 28–40 wk groups 10–7 M SNP produced 93.3 ± 1.2, 98.4 ± 0.4, and 92.2 ± 1.2% relaxation in aorta segments from lean, Zucker obese, and ZDF rats, respectively (P = NS).
Vascular responses in small coronary and mesenteric arteries. Intraluminal pressurized diameter was 167 ± 16, 141 ± 12, and 127 ± 10 µm in coronary arteries from lean, Zucker obese, and ZDF rats, respectively, in 28- to 36-wk-old animals (P = NS). Vasomotor responses in coronary arteries from 40-wk-old rats are not included due to decreased ability of the vessels to constrict to KCl or maintain a stable constriction to U-46619. In mesenteric arteries, intraluminal pressurized diameter was 161 ± 11, 151 ± 8, and 159 ± 6 µm from 28- to 40-wk-old lean, Zucker obese, and ZDF rats, respectively; P = NS. ACh induced endothelium-dependent relaxation in rat coronary arteries. ACh-induced relaxation was not altered in coronary arteries from lean rats (Fig. 4A). In coronary arteries from Zucker obese rats (Fig. 4B), relaxation to ACh was not altered at 16–24 wk; however, it was nearly abolished in the 28- to 36-wk age group. In coronary arteries from the ZDF rats (Fig. 4C), ACh-induced dilation was attenuated by 16–24 wk of age and remained decreased throughout the study. Thus diabetic rats demonstrated endothelial dysfunction in coronary arteries before Zucker obese rats without hyperglycemia.
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In a comparison of data from the same aged animals, ACh responses were not altered in lean versus Zucker obese and ZDF rats in coronary vessels in the 8- to 12-wk age groups. At 16–24 wk, ACh responses in coronary vessels were attenuated in the ZDF group compared with responses from lean animals (Fig. 4, A and C). In coronary vessels from 28- to 36-wk-old rats, ACh-induced dilator responses were nearly abolished in vessels from Zucker obese and ZDF rats (Fig. 5A).
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The role of superoxide in impaired endothelium-dependent vasodilation was examined in the presence of tiron (1 mM), a nonspecific free radical scavenger. Treatment of coronary arteries with tiron completely restored the attenuated vasodilatory response to ACh from Zucker obese (Fig. 6A) and ZDF (Fig. 6B) rats similar to dilation observed in vessels from lean rats. In mesenteric arteries, improved ACh-induced dilation was observed in Zucker obese (82.6 ± 8.5 and 52.0 ± 10.1%) and ZDF rats (58.7 ± 11.4 and 47.7 ± 4.7% relaxation) in tiron versus untreated vessels, respectively. These data indicate that superoxide produces significant injury to nitric oxide (NO)-mediated vasodilation in coronary and mesenteric arteries from Zucker obese and ZDF rats.
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DISCUSSION |
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The metabolic syndrome, as defined by National Heart, Lung, and Blood Institute and the American Heart Association, consists of six major components: abdominal obesity, atherogenic dyslipidemia (elevated triglycerides and low HDL cholesterol), hypertension, insulin resistance ± glucose intolerance, a proinflammatory state, and a prothombotic state (8). The metabolic syndrome encompasses Type 2 diabetes, and estimates are that there will be 200–300 million cases in Western society by 2010 (1). All characteristics of the metabolic syndrome are known to be significant risk factors for cardiovascular disease.
The Zucker obese and ZDF rat strains have been well characterized as models of the metabolic syndrome and Type 2 diabetes (7, 13, 18, 22). The lean rats in this study were normotensive and euglycemic, had normal insulin values, and gained small amounts of weight throughout the 40 wk of the study. Zucker obese animals were also euglycemic; however, they developed hypertension, were insulin resistant, and gained a substantial amount of weight throughout the study. ZDF rats were hypertensive, insulin resistant, and hyperglycemic and lost weight after 28 wk of age, corresponding with reduced serum insulin levels.
Increased oxidative stress has been implicated in several disease models, including the metabolic syndrome and diabetes. We found enhanced formation of lipid peroxide in serum from Zucker obese and ZDF rats. When compared with samples from lean rats, serum from ZDF rats showed increased lipid peroxide throughout the study, whereas values from Zucker obese rats showed increased formation of lipid peroxide at later stages of disease progression. Katakam et al. (9) showed increased reactive oxygen species in coronary arteries from Zucker obese rats. Phillips et al. (19) and Frisbee et al. (7) showed increased superoxide production in cerebral and femoral arteries from Zucker obese rats, respectively. Our studies suggest that hyperglycemia contributes to superoxide formation in ZDF rats. Oxidative stress, as measured by serum TBARS, increases with age, as does vascular dysfunction in the coronary arteries.
We observed an attenuated response to phenylephrine-induced constriction with increasing age in aorta from all three groups of rats (lean, Zucker obese, and ZDF). This appeared to be an age-related effect, as responses from lean rats were also attenuated. In our studies, we found an attenuated response to phenylephrine in Zucker obese and ZDF aorta compared with aorta from lean rats at 8–12 wk, suggesting early dysfunction in contractile properties. Potential mechanisms for the decreased phenylephrine response include receptor desensitization or signaling pathways or alteration in calcium sensitivity/handling mechanisms. In contrast, Subramanian et al. (24) showed no changes in norepinephrine- or endothelin-1-induced constriction in aorta from 20- and 32-wk-old Zucker obese rats. This difference could be due to the vasoconstrictors used between these studies.
ACh-induced relaxation was attenuated in aorta from 28- to 40-wk-old ZDF rats. Subramanian et al. (24) found an increase in ACh response in aorta from 32-wk-old Zucker obese rats, which was not observed in the current study. Zhang et al. (25) showed decreasing endothelial function over time (3–9 mo) in ZDF rat aorta. Smooth muscle responses as determined by SNP were not altered in aorta from Zucker obese or ZDF rats in our study. We found limited impairment in the vasoactive properties of aorta from Zucker obese and ZDF rats. Thus conduit vessels, such as the aorta, appear more resistant to vascular damage than do the smaller arteries from coronary and mesenteric beds in Zucker obese and ZDF rats.
Progression of endothelium-dependent vascular dysfunction in coronary and mesenteric arteries was examined by performing ACh concentration-response curves in vessels from lean, Zucker obese, and ZDF rats. ACh-induced dilation was not altered in coronary and mesenteric vessels from 8- to 40-wk-old lean rats. Thus there was not an effect of age on ACh-induced dilation in these rats. ACh-induced relaxation in coronary arteries from 8- to 12-wk-old and 16- to 24-wk-old Zucker obese rats was normal; however, vasodilation to ACh by vessels from 28- to 36-wk-old Zucker obese rats was severely impaired. Data from the younger Zucker obese rats in this study are consistent with Katakam et al. (9), who showed that ACh responses were not altered in larger (225 um) coronary arteries from 12-wk-old Zucker obese rats. Normal relaxation was observed in mesenteric vessels from the same rats, which suggests that endothelial dysfunction occurs earlier in the coronary circulation than in some other microvascular beds in Zucker obese rats. Thus vessels from the coronary bed may be more susceptible to the effects of the metabolic syndrome or diabetes.
In coronary arteries from ZDF rats, ACh-induced responses were attenuated in 16- to 24-wk-old and 28- to 36-wk-old rats compared with 8- to 12-wk-old rats. The attenuated ACh response was also observed in mesenteric arteries from 28- to 40-wk-old ZDF rats, again suggesting that endothelium-dependent responses were altered in the coronary vascular bed before dysfunction in vessels from the mesenteric bed in ZDF rats. It is not surprising that endothelial dysfunction occurs at different times during the progression of the disease states; because these vessels are subjected to different stresses, they may have diverse receptor and ion channel populations and the prominence of NO and endothelium-derived hyperpolarizing factor pathways may be different in coronary and mesenteric vascular beds.
The mechanism of attenuated ACh response in Zucker obese and ZDF rats is controversial. A leading hypothesis in several vascular beds is that endothelium-dependent dilation is altered by increases in oxidative stress (7, 9, 20). Our data support this concept, because endothelial dysfunction observed in coronary vessels follows observed increased oxidative stress in the serum. Also, our data with a free radical scavenger show that oxidant stress is involved in the attenuated ACh response observed in coronary and mesenteric arteries from Zucker obese and ZDF rats.
SNP responses were not altered by age or disease state in coronary arteries from lean, Zucker obese, or ZDF rats. This has also been observed in larger coronary arteries (225 µm) from Zucker lean and Zucker obese rats (9). This suggests that the effect of the metabolic state of these animals does not alter smooth muscle relaxation in the coronary bed. SNP responses were not altered in mesenteric arteries from lean or ZDF rats. In mesenteric arteries from the Zucker obese animals, we found an enhanced SNP response. This may be due to an altered NO response, which could increase guanylate cyclase activity (3, 11).
In summary, vascular function was evaluated in aorta, coronary, and mesenteric arteries of rats by using isolated vessel techniques. These various vessels react differently to a variety of stimuli, as they have different functions and diverse receptor and ion channel populations. Our results suggest that vascular dysfunction in Zucker obese and ZDF rats progresses at a different rate of time in coronary and mesenteric arteries and precedes changes in conduit (aorta) arteries. An option for decreasing the late-stage complications of the metabolic syndrome and diabetes may be intervening earlier in the disease process before vascular dysfunction occurs.
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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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0.05 vs. lean animals.
