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II in Zucker obese rats compromises oxygen and flow-mediated regulation of nitric oxide formation
Department of Cellular and Integrative Physiology, Indiana University Medical School, Indianapolis, Indiana 46202
Submitted 25 August 2003 ; accepted in final form 3 October 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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II to determine whether flow or oxygen-related NO regulation was improved. Blood flow velocity was increased by forcing arterioles to perfuse 
50% larger tissue areas by occlusion of nearby arterioles, and oxygen tension in the bath was lowered to create a modest oxygen depletion. When compared with lean Zucker rats, the periarteriolar NO concentration ([NO]) for obese rats was 30% below normal. At elevated shear rates, the [NO] for arterioles of obese animals was 20–30% below those in the arterioles of lean rats, and the NO response to decreased oxygen was about half normal in obese rats. All of these regulatory problems were essentially corrected in obese rats by PKC blockade with only minor changes in the microvascular behavior in lean rats. Therefore, activation of PKC-II in endothelial cells during obesity suppressed NO regulation both at rest and in response to increased flow velocity and decreased oxygen availability.
shear rate; ruboxistaurin; arteriole; intestine
II inhibitor (13, 22). Vascular smooth relaxation in response to exogenous sources of NO was essentially normal in obese animals and was unchanged by the blockade of PKC-II. These various observations raise the distinct possibility that obesity caused more of an endothelial than vascular smooth muscle compromise related to NO physiology.
With the assumption that the PKC-dependent abnormality of endothelial cells in obese rats extends to blood flow and oxygen-dependent vasodilation, the hypothesis that both processes are suppressed and amenable to improvement with PKC-II blockade was evaluated. Although blood flow-mediated vasodilation has not been studied in the microvasculature of obese humans, flow-dependent regulation of the brachial artery has been evaluated. In obese humans, the NO component of brachial artery dilation to increased flow velocity has been found to be compromised (9, 12), as has been extensively documented in obese diabetic humans (28). The microvasculature is likely to have even greater problems or at least have greater effects on the regulation of vascular resistance, assuming flow-mediated mechanisms are suppressed. The intestinal microvasculature was chosen for studies of flow-dependent vasodilation, because in the normal state, blood flow needs of the intestinal mucosa are transmitted through changes in blood flow velocity and shear rate to the intermediate and large arterioles that actually dominate vascular resistance (5). Studies of this phenomena indicated that 40% of the total resting vascular resistance was regulated by NO formation in the large and intermediate diameter arterioles in response to shear rate. Furthermore, shear-dependent regulation at higher and lower than normal blood flows was found to be NO dependent by selective manipulation of blood flow in single arterioles of the small intestine (6). As to oxygen regulation, recent studies from this laboratory (6, 23) found a major link of increased NO formation by intestinal arterioles during even mild reductions in perivascular oxygen tension. In fact, if the endothelial cells were prevented from making additional NO, microvascular dilation during reduced oxygen availability was all but eliminated.
For these studies, the Zucker fatty rat was used as an obese animal model that mimics severe obesity in humans. For the vast majority of severely obese humans, as well as the majority of obese humans with Type II diabetes mellitus who are receiving treatment, the person is hyperinsulinemic due to obesity but relatively normoglycemic. We used male Zucker fatty rats that were about 20% heavier than lean litter mates but not hyperglycemic even during the morning hours after overnight feeding. Measurements of intestinal blood flow and the perivascular NO concentration were used to test the hypothesis that obesity caused a PKC-dependent abnormality of endothelial cells that compromised blood flow and oxygen-dependent vasodilation. To acutely suppress PKC activity in endothelial cells, ruboxistaurin was used as a specific blocker of PKC-II.
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METHODS |
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Measurement techniques. The perivascular [NO] was monitored with glass micropipettes that contained a 7-mm carbon fiber sealed into the glass tip during pipette pulling (7). The carbon fiber was cut near the end of the glass tip and polished on an alumina grit (0.005 µm) surface until the carbon fiber was surrounded by glass. This yields a pipette tip polished to a sharp point with about a 25–30° angle. The tip was then coated with three dips (5 s) of Nafion (Sigma Chemical; St. Louis, MO) and heated at 110°C for 10–15 min between brief dips. The total outer diameter of the glass and thin layers of Nafion was 8–9 µm when the electrode was dry. Nafion decreased the responsiveness of the electrodes to nitrite by at least 10-fold, and for practical purposes, the electrodes cannot respond to biological concentrations of nitrite ions. Nitrate ions are not a problem as they are fully oxidized and stable. The electrodes were calibrated at 37.5°C using 100% nitrogen for 0 nM NO and 600 and 1,200 nM NO (NO gas in nitrogen gas) using a commercially produced tonometer (Diamond General; Ann Arbor, MI). If the electrodes did not return their resting N2 current after exposure to 600 nM NO, they were recoated with Nafion because they were sensitive to nitrite formed in the tonometer fluids. Typical currents in 0 nM NO are 3–8 pA, and at 1,200 nM, the current increases 1.5–2 pA. In terms of voltage output of the Keithley model 6517A Electrometer polarizing the electrodes at +0.9 V, the typical calibration was 1 mV equaled 3–5 nM NO, and changes in voltage of 0.5 mV could be followed with the PowerLab analog-to-digital recording system used to collect data. Because microelectrode current drift with time was a routine concern, the virtual baseline current during tissue measurements was calculated from the current at 0 nM NO before and after measurements at a location 200 µm above the tissue surface. In general, drift rates were sufficiently low that 30-min recordings were possible with minor (±5%) corrections.
To measure the perivascular NO, the equivalent "0" nM NO concentration was determined at 200 µm above the tissue. For practical purposes, any point from 100 to 300 µm above the tissue would be adequate if no large arterioles and venules were directly beneath the pipette tip location. The micropipette tip was advanced through the thin muscle layers of the bowel to the side of the submucosal arteriole to be studied using Narishige (Tokyo, Japan) hydraulic micromanipulators. The goal was to have the microelectrode tip appear to touch the outer side of the arteriole yet not mechanically stimulate the arteriole to release additional NO. As the vessel changed diameter, the microelectrode tip was moved relative to the vessel wall to maintain the pipette-vessel association. To minimize intestinal motility, 10–8 M isoproterenol (Sigma Chemical) was added to all bathing fluids to take advantage of the sensitivity of visceral smooth muscle to -adrenergic relaxation: arterioles rarely responded to the low concentration of isoproterenol, but a 2–3% dilation was tolerated.
Vessel diameter was recorded from digital images collected with an Image 1 Image Analysis System (Universal Imaging; West Chester, PA) calibrated to a 10- and 100-µm marked stage micrometer. The vessel diameter was measured with a virtual caliper system superimposed on the inner diameter of the vessel image. Blood flow velocity was determined with a Doppler velocitmeter from the Department of Medical Physiology at Texas A&M University (8) and calibrated against red blood cells on the surface of a rotating disk spun by a 1-rpm synchronous motor. The system was calibrated at 2–6 mm/s and found to have a linear velocity and voltage output relationship with a correlation coefficient of 0.99. During actual experiments, the transilluminating light intensity was increased well above the minimum light intensity required to record a maximum velocity output for a given situation. This precaution was used because the output signal is intensity related up to some threshold light level and independent as the light intensity was increased above threshold. The dynamic output signal of the system was measured with an oscilloscope to ensure maximum systolic-diastolic velocity amplitude of the signal when the vessel was slightly defocused. A slight defocus was necessary even with infinity-corrected optics because the focal points in the optical path for the velocimeter and video camera were not identical. In general, the maximum auditory signal of the velocimeter and ideal oscilloscopic measurement can be deduced by an operator after literally a few minutes of experience.
Protocols. Blood flow in large and intermediate diameter arterioles were increased by selective occlusion of arterioles arranged naturally in parallel to force the observed vessel to perfuse more tissue. To reduce blood flow in the vessel of choice, distal downstream occlusion was used. For distal occlusion, there are at least four branched vessels from the large arteriole from the site of measurement to the area of occlusion. Past analysis indicated that microvascular pressure changed very little with this protocol (6). In both downstream and collateral vessel occlusion protocols, the tissues in the vicinity of the NO measurements were normally perfused and not subject to oxygen deprivation. The details of this protocol have been published (6). In each situation, the vasculature was allowed to equilibrate to the new status of flow for at least 5 min before the data collection began. In general, steady-state responses to flow perturbations were reached within 2–3 min and remained stable thereafter. Because simultaneous measurements of the [NO] and vessel responses during vessel occlusions in the same tissue area were exceedingly difficult, only one or two vessels per animal were studied under natural conditions and 30 min after ruboxistaurin had been used to locally suppress PKC-II. The exact area of the vessel studied at rest was reevaluated after ruboxistaurin had been added to the preparation so that dimensions of the vessels, flows, and NO measurements were at equivalent locations. A bath concentration of 20 nM ruboxistaurin was used because it was effective to restore the resting [NO] of intestinal arterioles in obese Zucker rats, based on a prior study from this laboratory (7). Of equal importance, IIshi et al. (13) found that plasma concentrations of 20 µM ruboxistaurin in insulin-deficient diabetic rats ameliorated microvascular complications.
The responses of the vasculature at a bath oxygen tension of 40–45 and 5–10 mmHg were compared before and after 20 nM ruboxistaurin was exposed to just the intestinal preparation. The low oxygen tension was not meant to cause hypoxia. In fact, it was not possible to decrease perivascular oxygen tension more than 10 mmHg with this protocol, although "tissue" oxygen tensions away from arterioles can be reduced by much larger amounts. We have previously shown that the dilation of arterioles exposed to lowered oxygen tension in normal rats was highly dependent on NO and not related to adenosine released from the tissue or hyperpolarizing factors likely to activate calcium-dependent potassium channels (23). That the NO primarily was derived from endothelial cells was shown in the earlier study by oxygen embolization to stun the endothelial cells. For several hours after embolization, a reduction in bath oxygen tension in lean animals did not increase the [NO] or cause significant arteriolar dilation.
Two-way analysis of variance for repeated measures (rest and perturbation versus natural state and postruboxistaurin PKC inhibition) was used to detect significant effects, and individual changes were evaluated with a post hoc Tukey's test using SigmaStat Software (Jandel). Significance was accepted at P 
0.05.
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RESULTS |
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The data in Fig. 1A present the acute effects of PKC-II blockade on the percentage of control blood flow, and in Fig. 1B, the resting [NO] at low and normal (5%) oxygen concentrations in the bath media are presented. The data set are based on observations in six lean and five obese rats. The periarteriolar [NO] was measured on the first-order arterioles, the largest arterioles to enter the intestinal wall. The averaged resting diameter of the arterioles studied in lean rats was 97.7 ± 8.2 µm compared with 102.2+7.1 µm in obese rats. The calculated flow in arterioles of all lean animals studied was 0.037 ± 0.007 and 0.048+0.008 mm3/s in the entire group of obese rats. The intestine is larger in obese than lean rats, which may explain the higher flow per vessel than normal. Because arterioles were chosen on the basis of clarity for observation and a strong signal for optical flow velocity measurements, the flow data per vessel may not be representative of flows in larger arterioles in general. Therefore, the results of flow changes will be given in percentage of control resting flow to allow a comparison of reactivity independent of variations in absolute flow between animals. Prior studies of both diabetic rats and the effects of acute hyperglycemia on the normal in vivo intestinal microvasculature (3, 4, 15, 19, 20) have shown the larger arterioles to be a good barometer of NO physiological problems. The resting periarteriolar [NO] in obese rats of 311 ± 29 nM was consistently about 100 µm below that in lean rats of 447 ± 54 nM. The resolution of the NO microelectrodes in the calibration system was of the order of ±5 µm, therefore there was no question that a lower than normal periarteriolar [NO] existed in obese rats. After blockade of PKC-II the [NO] and blood flow in lean rats did not change for either superfusion oxygen concentration relative to the untreated state. However, blockade increased [NO] and blood flow both at what is considered a normal oxygen environment for the small intestine, 40–50 mmHg oxygen, and at a low oxygen tension of 5–10 mmHg in the bathing media. PKC blockade in obese rats also increased both the [NO] and blood flow responses to low oxygen tension. The improvement in obese rats after PKC blockade was so great during reduced oxygen availability that both the blood flow increase and [NO] absolute and relative responses rivaled or surpassed those in lean rats.
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The data in Fig. 2 present the blood flow behaviors of intestinal arterioles to forced increases and decreases in blood flow under conditions that could not cause a localized deficit in oxygen to the vessels. Simultaneous measurements of NO, diameter, and flow velocity measurements were continuously made during this protocol. These animals were a separate group of seven lean and obese Zucker rats from those used to obtain the data in Fig. 1. The resting [NO] and diameter was 621 ± 70 nM and 97.2 ± 4.3 µm in lean rats, respectively, compared with 565 ± 55 nM and 112.4 ± 7.2 µm in obese rats, respectively. Again note that PKC inhibition increased blood flow in obese rats and elevated the resting [NO] but had no significant effects on either blood flow or [NO] in lean rats at reduced or normal blood flow conditions. However, PKC inhibition in lean rats did increase the [NO] during collateral flow but not the actual blood flow. The data in Fig. 3 represent the relationship of shear rate and [NO] for the conditions presented in Fig. 2. By comparing the conditions in both graphs, the nature of how obesity interfered with the [NO] versus shear rate relationship, how PKC-II substantially corrected the problem, and the consequences for blood flow regulation can be better appreciated.
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Note in Fig. 3 that in the natural state, arterioles of obese animals had a lower [NO] for a given shear rate than that occurred in normal vessels of lean rats. The consequences in obese rats were less of an increase in blood flow during forced collateral blood, less of an absolute increase in [NO] at elevated shear rates, but surprisingly as shown in Fig. 2, similar percentage of control increases in [NO] as in lean animals. After ruboxistaurin acute administration in obese rats, the PKC blockade increased the [NO] for a given shear rate to nearly the status found in lean rats during the natural state. In lean rats after PKC-II inhibition, the [NO] versus shear rate relationship shifted upward at high shear rates but was not influenced at low and normal shear rates.
It is important to point out in Figs. 2 and 3 that when blood flow was forced to be decreased by downstream arteriolar occlusion, normal rats demonstrated a large decrease in [NO] as the shear rate was decreased, and this behavior remained after PKC-II inhibition. Obese rats under natural conditions have little or no ability to reduce [NO] when shear rate was decreased. However, after PKC inhibition this behavior improved essentially to normal in that the [NO] decreased as much or more than in lean rats for a given drop in shear rate.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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II activation with ruboxistaurin. Furthermore, the intestinal arterioles in obese animals were constricted at rest and demonstrated both dilation and increased [NO] with blockade of PKC-II (Figs. 1 and 2). In prior studies of obese Zucker rats, Bohlen and Lash (5) found their arteriolar dilation to exogenous NO to be normal, as has been a consistent finding in obese humans and rats (21, 24–27). These current and past observations raise the distinct possibility that severe obesity in part increases vascular resistance by limiting NO availability due to a negative effect of PKC on endothelial cell NO generation rather than a deficit in vascular smooth muscle responsiveness to NO.
One of the hallmarks of obesity in animals and humans is that damage of host organs by impaired microvascular regulation and structural abnormalities is very rare even with many years of severe obesity. However, this lack of pathology does not indicate a lack of impaired regulation, such as impaired flow-dependent regulation or elevated vascular resistance leading to mild to moderate hypertension. The intestinal vasculature was chosen as a model for flow-dependent regulation because it is highly dependent on flow-dependent vasodilation to communicate the flow needs of the small arterioles of the mucosa to the larger arterioles some distance away in the superficial submucosa (5). Because the metabolic needs of the mucosa are altered by the presence or absence of food molecules, the smaller arterioles appropriately respond, and in doing so, change the blood flow and shear rates in the larger arterioles. As shown in Fig. 3, at resting, elevated, and increased flow states, the arterioles of lean animals generated a higher [NO] for a given shear rate than existed in obese rats. Whereas obese rats exhibited an increased [NO] at elevated shear rate, the response was substantially below that in lean rats. In practical terms, as shown in Fig. 2, a depressed ability to respond to increased shear rates generated half the increase blood flow in obese than lean rats during a need to improve collateral blood flow. Instead of blood flow nearly doubling, as in lean rats, the blood flow only increased 50% in obese rats. In this same context of some form of NO impairment related to flow-mediated dilation, in obese normoglycemic men, (16), the NO component of brachial artery dilation to increased flow velocity is compromised, and this abnormality was even more compromised once hyperglycemia was present (28). The absence of a robust blood flow response in obese rats is believed to be purely because of too little NO generation rather than some deficit of response by vascular muscle cells to the [NO]. Blockade of PKC-II in obese rats dramatically improved the blood flow response to essentially normal, and the absolute [NO] and increase in [NO] in response to elevated shear rate was improved to the normal ranges (Figs. 2 and 3). These impressive improvements occurred after 30 min of treatment at a drug concentration equivalent to that in blood plasma during chronic treatment to improve microvascular status in diabetic rats (14). The blockade in lean rats slightly improved the [NO]-shear rate responses in lean animals at the high shear rates during collateral flow. The benefits of PKC-II blockade were so great in obese animals that one can only conclude that PKC-II activation during severe obesity substantially and negatively altered endothelial NO regulatory physiology.
Whereas macro- and microvascular responses to changes in shear rate are undoubtedly important, perhaps the single greatest responsibility of the microvasculature is the supply of tissue oxygenation. For the sake of relevance to physiological events, the reduction in oxygen availability was 7–10 mmHg PO2 at the arteriolar wall of the vessels studied during maximum depletion of the bathing media oxygen content. The loss of oxygen from the tissue to the bathing media was not a trivial strain on the microvasculature. In normal rats, intestinal blood flow increased to 150–200% of control (Fig. 1). This increase in blood flow is as large or larger than the increase in intestinal blood flow during nutrient absorption (5). The increase in microvascular [NO] in obese animals during oxygen depletion was small compared with that in arterioles of lean animals, as shown in the Fig. 1B. However, the relative increase in blood flow was only slightly less than normal, as shown in Fig. 1A. At first approximation, these results seemed to indicate no particular problems with oxygen regulation in obese rats. However, after blockade of PKC-II, the resting blood flow was increased 20–40% with an average of 30%, and the blood flow response to lowered oxygen availability in obese animals was nearly doubled. Normal animals demonstrated no significant changes in microvascular behavior to reduced oxygen availability with PKC-II blockade. Whereas it is unlikely that the intestine of obese rats was at risk for oxygenation in natural conditions, blockade of PKC-II definitely improved the potential for oxygenation by restoring NO and increasing both resting and reactive blood flow. These data were interpreted to indicate that while the arterioles of obese rats did respond to lowered oxygen tension, the flow response was likely mediated to a large extent by means other than NO because the [NO] marginally increased. This may be similar to Frisbee's (10) findings in isolated skeletal muscle arterioles from obese Zucker rats. Frisbee found that the NO component of dilation to severe reduction in oxygen availability was quite weak in Zucker rats but well developed in arterioles of lean rats. This is essentially the same pattern of results found for in vivo intestinal arterioles of lean and obese Zucker rats during a reduction in oxygen tension that was too small to cause hypoxia. What is very important to stress is that suppression of PKC-II revealed a potent NO mechanism that was quickly restored in the arterioles of obese animals to assist in full regulation of microvascular responses to a reduction in oxygen availability. This would be consistent with suppression of endothelial NO synthase by activation of endothelial PKC in obese animals rather than some type of damage to the eNOS process.
The present study used obese rats that by human standards would be about 25% heavier due to obesity than their age-matched lean counterparts. Comparable increases in body mass in humans due to fatty tissue are generally considered as serious obesity. If the wide variety of altered physiological processes in obese humans mirror those in the obese Zucker rat, the current study would predict that endothelial regulation while adequate during obesity has been compromised. From the increase in resting intestinal blood flow in obese rats following PKC-II blockade and improved vascular responses to decreased oxygen availability and elevated shear rates, increased endothelial PKC activity during the multiple biochemical abnormalities of obesity compromised the endothelial NO-dependent aspects of microvascular regulation.
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ACKNOWLEDGMENTS |
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GRANTS
The study was supported by National Heart, Lung, and Blood Institute Grant HL-25628.
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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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