1 Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia
2 Division of Biochemistry, Medical School, University of Tasmania, Hobart, Australia
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ABSTRACT |
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INTRODUCTION |
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While exploring insulins vascular actions, we observed that high-dose insulin infusion (10 mU · min-1 · kg-1) increased rat hindlimb blood flow in vivo and also enhanced the conversion of exogenously infused 1-methylxanthine (1-MX) to 1-methylurate (1-MU) by endothelial xanthine oxidase (8). Because this enzyme is located on the capillary endothelial surface (9) these data indicated that insulin enhances the exposure of 1-MX to xanthine oxidase (8). To our knowledge, this was the first report providing experimental evidence for an action of insulin on muscle microvascular recruitment. We also observed that epinephrine infusions, which enhanced total hindlimb blood flow to a similar degree as insulin infusions, did not affect microvascular recruitment as measured by 1-MX metabolism (8). This suggested that total flow and microvascular recruitment could be dissociated. Subsequently, we reported that high-dose (10 mU · min-1 · kg-1) insulin enhanced the laser Doppler signal in rat muscle obtained using either surface scanning or an implantable laser Doppler probe (10). Again, epinephrine infusion produced a similar increase in hindlimb blood flow but did not alter laser Doppler signal. These data (like the 1-MX result) could be explained on the basis of insulin-induced microvascular recruitment.
Most recently, we have used ultrasound during albumin microbubble infusion to estimate microvascular volume (MV) in rat skeletal muscle in vivo (11). Using contrast-enhanced ultrasound (CEU), we observed that exercise, a known stimulus for microvascular recruitment in muscle, enhanced MV. This technique is well suited to repeated measurement of microvascular perfusion in small animals and humans.
In the current study, we measured 1-MX metabolism and imaged microvascular perfusion (using CEU) in rat hindlimb muscle in vivo to ascertain whether physiological hyperinsulinemia exerts a hemodynamic action to increase microvascular perfusion. We then used CEU to examine whether insulin-induced changes in microvascular recruitment and total limb blood flow are temporally coupled.
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RESEARCH DESIGN AND METHODS |
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Surgery.
The hyperinsulinemic clamp was used as described previously (8). Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (55 mg/kg body wt). Polyethylene (PE50; Intramedic) cannulas were inserted into the carotid artery for arterial blood sampling and measurement of arterial blood pressure (Transpac IV pressure transducer; Abbott Critical Systems) and into both jugular veins for intravenous infusions. A tracheotomy tube was placed, and the animals breathed spontaneously throughout the experiment. The femoral vessels in the left leg were exposed by an 1.5-cm incision through the skin overlaying the vessels. The femoral artery was carefully separated from the femoral vein and saphenous nerve. The epigastric vessels were ligated, and a time transit flow probe that measured blood flow continuously by ultrasound (VB series 0.5 mm; Transonic Systems) was positioned over the femoral artery. The flow probe was interfaced through a flow meter to an IBM-compatible computer. Femoral blood flow, arterial blood pressure, and heart rate were continually measured using Windaq data acquisition software (Dataq Instruments). The animal was maintained under anesthesia for the duration of the experiment with aqueous sodium pentobarbital (0.6 mg · min-1 · kg-1) via the carotid artery. A heat lamp positioned above the rat maintained the rats body temperature.
Experimental protocols.
After a 60-min stabilization period following completion of the surgical procedure, rats were divided into two groups. Group 1 animals received either normal saline (10 µl/min, n = 6) or insulin (3.0 mU · min-1 · kg-1, n = 9) intravenously while maintaining blood glucose at baseline via a variable-rate infusion of 30% dextrose.
For the current study, MV and flow velocity were assessed with CEU at baseline and after 120 min of infusion. In addition, 1-MX metabolism was determined at the end of the 120-min infusion. In group 2 animals (n = 5), the time course of insulins action on microvascular blood volume and velocity were assessed by CEU. Measurements were performed at baseline and at 30 and 90 min after initiating insulin infusion. These time points were selected to reflect times at which plasma insulin concentration increases most markedly after a meal (12,13).
Glucose (30% wt/vol solution) was infused at a variable rate into all insulin-treated animals to maintain euglycemia, and arterial blood glucose was monitored every 10 min for the first hour and then every 15 min for the rest of the study, using an Accu-Chek blood glucose monitoring system (Roche, Indianapolis, IN).
CEU.
CEU has been used extensively in the past to measure microvascular flow in the myocardium (14,15,16). Estimates of microvascular flow using CEU have been validated using 11-µm radiolabeled microspheres to show that there is a strong correlation between the two techniques (r = 0.96, P < 0.001) (14). The technique was modified for its use for the rat as follows: a linear-array transducer interfaced with an ultrasound system (HDI-5000; ATL Ultrasound) was positioned over the right leg of the rat and secured for the course of the experiment. The adductor magnus and semimembranosus muscles of the hindlimb were imaged in short-axis with intermittent harmonic imaging at a transmission frequency of 3.3 MHz. The mechanical index ([peak negative acoustic pressure] x [frequency]-1/2), a measure of acoustic power, was set at 0.9. The acoustic focus was set at the mid-portion of the muscle group. Gain settings were optimized and held constant. Data were recorded on 1.25-cm videotape using a S-VHS recorder (Panasonic MD830; Matsushita Electric). Albumin microbubbles containing octafluoropropane gas (Optison; Mallinckrodt Medical) were infused intravenously at 120 µl/min for the duration of data acquisition. The acoustic signal that is generated from the microbubbles exposed to ultrasound is proportional to the concentration of microbubbles within the volume of tissue being imaged. Essentially, all microbubbles within the ultrasound beam are simultaneously imaged and destroyed in response to a single pulse of high-energy ultrasound (14). As the time between successive pulses is prolonged, the beam becomes progressively replenished with microbubbles (14). Eventually, the beam will be fully replenished, and further increases in the time between each pulse will not affect microbubble signals in tissue (Fig. 1). The rate of microbubble reappearance within the ultrasound beam provides an indication of microvascular flow velocity (ß), and the plateau video intensity reached at long pulsing intervals provides a measurement of MV (14).
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To determine the appropriate microbubble infusion dose for in vivo imaging and to ensure linearity in the microbubble concentration versus video intensity relation, we performed experiments to define a microbubble dose-response curve both in vivo and in vitro. For the in vitro experiment, the ultrasound probe was immersed in a beaker of water, and video intensity was measured after injection of incremental numbers of microbubbles during continuous mixing with a stir bar. For the in vivo experiment, microbubbles were infused into the jugular vein of the rat, and the ultrasound probe was positioned over the thigh of the rat as described above. Background-subtracted video intensity in resting skeletal muscle was determined during step-wise increases in microbubble concentration. A microbubble infusion rate of 120 µl/min was selected for the in vivo study because this dose resulted in video intensity measurements that were well within the linear range of the microbubble concentration and video intensity indicated by both the in vitro and in vivo dose-range experiments, thus making it possible to accurately detect an increase in MV.
1-MX metabolism.
To date, the best indicator that we have that 1-MX metabolism reflects microvascular perfusion comes from studies of the effect of electrical stimulation of muscle (11,17). We have shown that 1-MX metabolism increases in response to electrical stimulation (a known stimulus for capillary recruitment) in rat hindlimb both in vivo (11) and in a perfused preparation (17). In the perfused rat hindlimb preparation, total blood flow is held constant (17), yet 1-MX conversion to 1-MU is increased, suggesting that microvascular perfusion can be affected independently of total flow.
In group 1 rats, 1-MX (0.4 mg · min-1 · kg-1) was infused intravenously at a constant rate during the last 60 min of each study. It was shown in previous experiments (8) that 1-MX is rapidly metabolized to 1-MU and that it is necessary to partially inhibit the xanthine oxidase before infusing 1-MX to slow its whole-body clearance and to allow for measurable concentrations in arterial and venous blood. We have performed allopurinol dose-response curves in the rat in vivo (data not shown) and found that 10 µmol/kg partially inhibited the xanthine oxidase and allowed steady-state systemic levels of 1-MX to be obtained.
At the end of each experiment in rats from group 1, blood from the carotid artery and femoral vein was sampled and immediately centrifuged, and 100 µl of plasma was added to 20 µl of 2 mol/l perchloric acid. The plasma was immediately neutralized with 12 µl of 2.5 mol/l K2CO3 and stored at -20°C until analysis of 1-MX.
Plasma 1-MX, 1-MU, allopurinol, and oxypurinol were analyzed using reverse-phase high-performance liquid chromatography from a modified version of the protocol used by Wynants et al. (18). The metabolites were separated and analyzed using an Ultrasphere ODS column (25 cm, 5-mm particles; Beckman) at 1.0 ml/min. Two buffers were used for the aqueous mobile phase. Buffer A consisted of 4.35 mmol/l acetic acid, and buffer B consisted of 87 mmol/l acetic acid/20% methanol/5% acetonitrile/0.2% tetrahydrofuran, pH 4.0. Buffer A was used during 010 min, and at that time buffer B was introduced and buffer A was decreased by using a linear gradient from 10 to 30 min. Buffer B was used from 30 to 40 min. Detection between 0 and 20.5 min was at a wavelength of 250 nm, and between 20.5 and 40 min it was at 272 nm.
Statistical analysis.
For animals studied in group 1, an unpaired Students t test was used to test differences between saline- and insulin-infused rats for mean arterial blood pressure, heart rate, arterial blood glucose level, vascular resistance, and 1-MX metabolism after 120 min of infusion. A paired Students t test was used to compare the effects of insulin or saline versus baseline on MV, ß, and femoral artery blood flow after 120 min of infusion. For group 2, where each animal served as its own control, repeated-measures analysis of variance was used to test differences between baseline and 30 and 90 min of insulin infusion on MV, ß, and femoral artery flow. When a significant difference was found, pairwise comparisons by Student-Newman-Keuls test were used to determine at which individual times the differences were significant. For all comparisons, significance was recognized at P < 0.05.
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RESULTS |
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The effect of insulin and saline on arterial 1-MX concentrations and hindleg 1-MX metabolism is shown in Fig. 3. At the end of the 120-min experimental period, arterial 1-MX concentration was similar between saline and insulin (18.3 ± 3.6 vs. 16.8 ± 2.7 µmol/l). Hindleg 1-MX extraction during saline infusion was 7.1 ± 0.8 µmol/l, and this was not significantly different from the insulin-treated group (7.7 ± 1.2 µmol/l). However, because of differences in total blood flow, 1-MX metabolism was 47% higher (P < 0.01) for insulin than saline, consistent with the conclusion that insulin had enhanced the exposure of 1-MX to xanthine oxidase by recruiting capillaries.
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DISCUSSION |
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The relative changes in 1-MX metabolism and CEU were not identical; indeed, within the CEU, results seen in groups 1 and 2 were not quantitatively identical. There are a number of things that might contribute to both the 1-MX and CEU measurements and thus influence the quantitative nature of the responses. First, the 1-MX measurement is made across the whole hindlimb (arteriovenous difference x flow), whereas the CEU measurement is made in one area and exclusively in muscle. Therefore, if insulin had no effect to increase flow in nonmuscle tissue (bone and skin), the proportionate rise in MV based on the CEU measurement would be greater than for the 1-MX measurement. Likewise, it is known that there are significant differences in insulin-induced increases in microsphere perfusion among different types of muscle (i.e., red versus white fibers). Beyond that, it should be recognized that 1-MX metabolism and CEU are fundamentally different techniques for estimating microvascular recruitment. Microbubbles are entirely intravascular and produce a signal that we can measure as video intensity. The background subtraction procedure that we use allows us to remove the contribution from larger vessels, in which flow is rapid; this cannot be done with 1-MX, although it is thought that xanthine oxidase is predominantly a microvascular endothelial marker.
Inasmuch as CEU is a noninvasive method, it permitted repeated measurement of insulins vascular action in individual animals. We observed that insulin increased MV well before it affected hindlimb total blood flow. Indeed, the changes in steady-state video intensity (MV) at 30 and 90 min during the insulin infusion was not different from those seen at 120 min, by which time total hindlimb flow had begun to rise. This suggests that microvascular recruitment occurred early and remained stable for the duration of the infusion period, and that it occurred independently of changes in total flow. Therefore, insulin-mediated microvascular recruitment and total limb flow follow a different time course. This result is reminiscent of our previous findings using pharmacological doses of insulin (10 mU · min-1 · kg-1). In that study, insulin infusion caused an increase in laser Doppler signal in muscle within 20 min that reached a maximum by 50 min. Femoral artery flow measured continuously for the duration of the experiment was not increased until at least the 60 min time point (10). Thus, increases in laser Doppler signal preceded by 3040 min any changes in total muscle blood flow. The marked increase in femoral artery flow seen at 60 min in the laser Doppler study was likely caused by the higher dose of insulin (10 mU · min-1 · kg-1) used for the former study. As noted earlier, we have also reported that epinephrine infusion increases rat hindlimb blood flow without recruiting capillaries, measured by either hindleg 1-MX metabolism or laser Doppler flowmetry (8,10), again providing experimental evidence that changes in microvascular flow can be dissociated from changes in total limb flow. Kuznetsova et al. (19) have also shown dissociation between total muscle flow and microvascular flow, as measured by microspheres and laser Doppler flowmetry, respectively. In those studies, angiotensin II or phenylephrine infusion into the anesthetized rat during ganglionic blockade resulted in an increase in laser Doppler signal but no change in total flow. In contrast, isoproterenol markedly enhanced total muscle flow, despite a reduction in laser Doppler signal.
In addition to microvascular blood volume, CEU provides an estimate of ß. The current results showed that ß did not change significantly during the first 90 min of insulin infusion but may have increased at 120 min, a time when hindlimb flow had likewise risen. This is not surprising because the product of ß x MV should provide an index of flow rate at a microvascular level. However, because this product increased without accompanying changes in femoral artery blood flow at 30 and 90 min of insulin infusion, the possibility should be considered that in addition to microvascular recruitment, insulin redistributed flow at the microvascular level within the rat hindleg. Femoral artery blood flow is a measurement of total hindleg flow that feeds muscle, bone, skin, and fat, whereas our measurement of MV and ß is determined within a discrete area of the hindleg that is exclusively in muscle. Thus, it is possible to find a mismatch between total hindleg blood flow and MV x ß within this small area of muscle. For example, we have found in the constant-flow perfused rat hindlimb that microvascular recruitment can increase in muscle without any change in total hindleg flow, and that this is most likely caused by flow redistribution within the hindlimb. Also, as noted previously, muscle blood flow in the rat hindlimb is very heterogeneous, based on the distribution of the different fiber types (20). For instance, in the anesthetized rat, blood flow (measured by 15-µm microspheres) to the red quadricep is 9 ml · 100 g-1 · min-1 but is only 4 ml · 100 g-1 · min-1 in the white quadricep and the extensor digitorum longus. Thus, based on the distinct differences in total muscle blood flow for each individual muscle of the rat hindlimb, it is highly likely that there could be a mismatch between total hindlimb flow and microvascular flow within a discrete area of muscle.
The albumin microbubbles used as the contrast-enhancing agent averaged 4 µm in diameter, had similar rheology to red blood cells, and were contained entirely within the vascular space (21). As a result, they were an excellent perfusion tracer. Furthermore, over the range of video intensity studied here, there was a linear correlation between the concentration of microbubbles within the vasculature and video intensity. CEU is particularly useful because it allows background subtraction of the tissue image and thus elimination of signal from large vessels, which fill very rapidly, allowing microvascular flow (which is slower) to be imaged selectively. The CEU method has the additional appeal that it allows imaging of specific regions within muscle and, unlike 1-MX metabolism or measures of total hindlimb flow, is not influenced by contributions from nonmuscle tissue (skin, bone, and adipose tissue). Furthermore, compared with laser Doppler flowmetry, CEU samples a relatively large volume of muscle and does not require surgical exposure of the muscle surface. The laser Doppler method applied to muscle has the potential for producing artifacts, given that it only samples the immediate region around an implanted or surface probe.
Several other investigators have attempted to determine the effect of insulin on muscle microvascular flow. Raitakari et al. (22) used positron emission tomography during inhalation of [15O]carbon monoxide. The labeled carbon monoxide binds to erythrocytes, thereby providing a vascular tracer. High-dose insulin concentrations augmented leg blood flow and increased muscle blood volume by 9%. The high dose of insulin used limited any effort to separately assess effects on total flow from those on microvascular recruitment. Moreover, the positron emission tomography method measures blood within both the larger vessels and the microvasculature; thus, changes in MV may not be accurately quantified.
In a subsequent study, Bonadonna et al. (23) used a very different approach and injected a bolus of nonmetabolizable radiolabeled L-glucose into the brachial artery to measure forearm transit time and to estimate extracellular volume. Pharmacological concentrations of insulin enhanced total forearm blood flow, as measured by the dilution of intra-arterially infused indocyanine green dye. The mean transit time of L-glucose lengthened in response to insulin because of an expansion of the extracellular volume by 33%. This measure includes more than the vascular space, and would not report separately on large and small vessels.
More recently, Baron et al. (24) modeled the effects of insulin on microvascular flow by raising and lowering blood flow in the human leg during a steady-state insulin clamp and compared the results to the predicted values of the noncapillary recruitment model of Renkin (25). It was concluded that because glucose uptake was greater than predicted when leg blood flow was increased and smaller than predicted when leg blood flow was decreased, this suggested that the number of perfused capillaries changed considerably under insulin-stimulated conditions, assuming that glucose permeability remained constant. Although it was concluded that insulin-mediated increments in muscle perfusion are accompanied by microvascular recruitment, no direct experimental measure of microvascular recruitment was made.
The relation between insulins action on total muscle blood flow and its metabolic actions remains embedded in controversy. Yki-Jarvinen and Utriainen (2) have concluded that insulins effect on total limb flow occurs after its metabolic effects on glucose uptake. It should be noted that this temporal discordance is based on the assumption that insulins vascular action is restricted to increases in total blood flow. The present study indicated that insulin produces an early hemodynamic action at the microvascular level during a time that insulin is producing a metabolic action. Interestingly, microvascular recruitment (as measured by laser Doppler flowmetry) closely follows changes in glucose infusion rate and not total muscle flow (10). This relation also held for the current study because the glucose infusion rate at 30 min of insulin infusion was 90% of the glucose infusion rate used at 90 min (the plateau for insulins metabolic action).
Insulin-stimulated total blood flow in many insulin-resistant disease states, such as type 2 diabetes, hypertension, and obesity, is reported to be blunted (1,4,26,27), providing further evidence that the hemodynamic involvement of insulin may be important for its action. However, controversies regarding the vascular actions of physiological doses of insulin persist because some investigators have failed to see an increase in blood flow within healthy humans (28) or an impairment during insulin-resistant states (29,30). None of these studies address separately the actions of insulin on microvascular recruitment and total flow. This is unfortunate because, based on the theoretical models of tissue perfusion developed by Renkin (25), microvascular recruitment might be expected to be a more potent determinant of tissue glucose exchange than total flow. For instance, we have recently reported that when insulin-mediated microvascular recruitment is prevented with the vasoconstrictor -methyl-5-HT, an acute insulin-resistant state is induced (31).
In summary, these results indicate that insulin at physiological concentrations recruits flow to the microvasculature in muscle, and at the insulin concentration used here, this effect precedes by at least 60 min any changes in total muscle blood flow. Changes in total muscle blood flow have later onset and do not involve further changes in the microvasculature. Therefore, a full assessment of the role of insulins vascular actions in healthy and insulin-resistant states may require focusing on the effects on both total blood flow and microvascular flow, which could be the key to insulins vascular action.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication 12 March 2001 and accepted in revised form 18 October 2001.
ß, microvascular flow velocity; CEU, contrast-enhanced ultrasound; 1-MU, 1-methylurate; MV, microvascular volume; 1-MX, 1-methylxanthine.
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REFERENCES |
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