Division of Biochemistry, School of Medicine, University of Tasmania, Hobart, Tasmania 7001, Australia
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ABSTRACT |
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In the rat muscle vascular bed,
vasoconstrictors either increase or decrease oxygen consumption
(O2). The present study compared the effects of norepinephrine (NE), angiotensin II (ANG II),
and 5-hydroxytryptamine (5-HT) on vasoconstriction-associated metabolism in the constant-flow perfused hindlimb of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) in the absence of
insulin. Basal perfusion pressure,
O2, glucose uptake, and lactate production were increased by 21.4, 11.9, 46.4, and 44.9% (P < 0.05 for all), respectively, in
SHR, which also had higher blood pressure and metabolic rate
(P < 0.05) in vivo. Dose-response curves for NE-induced perfusion pressure,
O2, and lactate production in SHR were shifted to the left compared with WKY. Associated with the
increased perfusion pressure, NE-induced
O2 and glucose uptake were
both decreased (P < 0.01),
particularly at high concentrations. These differences were unaffected
by 10 µM propranolol but were all diminished by further addition of
prazosin (2.5 nM). ANG II stimulated
O2, glucose uptake, and
lactate production in both strains, but the increased lactate
production was smaller in SHR (P < 0.05) with a proportional decrease (P < 0.05) in glucose uptake. Conversely, 5-HT decreased
O2 in both strains
(P < 0.01), and this effect was
greater in SHR (P < 0.01). These
data suggest that SHR muscle thermogenesis and glucose uptake are
impaired during vasoconstriction, especially in response to NE.
hypertension; oxygen consumption; glucose uptake; lactate production; norepinephrine; angiotensin II; 5-hydroxytryptamine; hindlimb
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INTRODUCTION |
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STUDIES IN BOTH HUMANS and animals have revealed a close correlation between hypertension and obesity as well as hyperinsulinemia, hyperglycemia, and hyperlipidemia, now often referred to as syndrome X (11, 18, 22, 24, 36). Although obesity is an important risk factor for the pathogenesis of hypertension, the effect of hypertension on metabolic disorders such as energy balance and glucose disposal is presently of great interest (22, 26-28, 31-35).
Skeletal muscle is a major thermogenic (or oxygen-consuming) source (4,
7, 9) and the primary tissue for glucose disposal (18, 33). Muscle
insulin resistance is a prerequisite for hyperglycemia seen in obesity
and hypertension (18, 23, 36). Both metabolic alterations in muscle
are, at least in part, controlled by the vascular system (7, 10, 19,
33, 35, 40). Compared with other organs such as gut and kidney, both vasoconstriction and oxygen consumption
(O2) in perfused rat hindlimbs are far more sensitive to norepinephrine (NE) and vasopressin (VP; see Ref. 41), implying that the muscle vascular bed is one of the
first organs affected by vasoconstrictors in vivo.
In the constant-flow perfused rat hindlimb, a muscle bed with many
characteristics similar to those in vivo (4, 7), all vasoconstrictive
hormones tested so far either increase or decrease
O2 and lactate production,
indicators for muscle metabolism. Furthermore, according to
their metabolic effects, vasoconstrictors may be classified as type
A, which induces a positive stimulation of both
O2 and lactate production,
and type B, which inhibits
O2 and lactate production
thermogenesis (Ref. 7 and references therein). Type A vasoconstrictors
include low concentration NE (LNE, <1 µM) and other
1-adrenoceptor agonists at low
doses, angiotensin II (ANG II), VP, and low-frequency sympathetic nerve stimulation (<4 Hz), whereas type B vasoconstrictors include high concentration NE (HNE, >1 µM) and
1-adrenoceptor agonists at high
doses, 5-hydroxytryptamine (5-HT), and high-frequency sympathetic nerve
stimulation (>4 Hz). Despite the disparity of their metabolic effects, vasoconstriction appears crucial because both metabolic and
pressure changes are reversed by infusion of vasodilators such as
nitroprusside for both type A and type B vasoconstrictors (7, 10, 13,
19, 42).
Essential hypertension is characterized by increased peripheral
vascular resistance. Spontaneously hypertensive rats (SHR) have been
widely used to delineate the mechanisms for abnormalities in the
cardiovascular system (1, 5, 6, 38) and metabolic disorders in
hypertension (3, 8, 25, 27, 28, 31-33). In SHR, there is an
elevated peripheral vascular resistance in muscle caused by either
increased vascular tension due to a higher sensitivity of resistance
blood vessels or/and elevated vasoconstrictor levels (1, 3, 5, 6, 25,
39). At the level of microcirculation, vascular rarefaction has been
found in skeletal muscle (3, 26, 38, 39). These alterations are likely
to affect muscle metabolism controlled by vasoconstrictors. To test
this idea, we compared the effects of NE, ANG II, and 5-HT on perfusion
pressure and associated changes in
O2, glucose uptake, and
lactate production in the perfused hindlimbs of SHR and their
normotensive counterparts, Wistar-Kyoto rats (WKY).
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MATERIALS AND METHODS |
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Animals. Age-matched (11 wk) male SHR and WKY used for the experiments were purchased from the Animal Resources Center of Western Australia. Twenty SHR and 20 WKY were used for this study. Hypertension has been shown by others (1) to be well developed at this age in the SHR from the same source. The animals were housed at 20°C with a 12:12-h light-dark cycle and allowed free access to water. The diet consisted of 20.4% protein, 4.6% lipid, 69% carbohydrate, and 6% crude fiber with added vitamins and minerals (Gibson, Hobart, Australia). All experiments were approved by the Ethics Committee of the University of Tasmania under the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.
Whole body metabolic rate. Metabolic
rates were measured in pairs (SHR-WKY, or WKY-SHR) each day between
10:00 AM and 6:00 PM by an indirect calorimetry system under conscious
conditions at 25 ± 0.5°C as previously described (42). To
minimize stress, the animals had been exposed to human handling for 2 wk before the experiment. The respiratory chamber was covered with
black cloth to prevent any visual disturbance from the environment. The
flow rate of air through the chamber was adjusted to 3 l/min to keep
the expired CO2 below 0.3% and
monitored by a mass flowmeter (Hartings, Hampton, VA). The expired air
was dried by passing it through a column (50 × 5 cm) filled with
CaSO4 placed before a modified
O2/CO2
gas analyzer (Datex; Labtech, Helsinki, Finland) and continuously
monitored throughout the experiment. The inlet air was measured before
and after each experiment for the calculation of
O2 and
CO2 emission on the basis of the
difference between the inlet and expired airs as previously described
(42). The measurement for each rat lasted 3 h, and the resting
metabolic rate was taken from the average of minimal readings for a
period of 10 min.
Blood pressure and interscapular brown adipose tissue. Eight animals from each strain were used for these measurements using the method described by Sexton et al. (39) under anesthesia with pentobarbital sodium (60 mg/kg body weight ip). Blood pressure was determined in the anesthetized state from a cannulated carotid artery by the use of a sphygmomanometer (ALPK2) placed at the level of the heart. Three consecutive steady-state blood pressure recordings for each rat were obtained and averaged. After the measurement of blood pressure, the carotid artery was tied off, and the interscapular brown adipose tissue (IBAT) was removed and weighed.
Hindlimb perfusion. The surgical procedures were similar to those described before (10) under anesthesia (mentioned above) with modifications of additional occlusions of abdominal vessels to improve the leg muscle perfusion (17) and restrict flow distribution to the contralateral trunk region. In brief, a midline abdominal incision was made to expose the abdominal cavity. The lower part of the anterior abdominal wall was removed, and the cut edges were ligated with sutures. Gut, seminal vesicles, and testes were removed after appropriate ligations. The right common iliac artery and vein were ligated, and a string was firmly placed around the tail at its root. To prevent any perfusate spillover when perfusion pressure increased, blood vessels connected with tissues other than the left hindlimb were carefully tied off. These included the right hypogastric vessels, the left inferior and superficial epigastric vessels, the inferior mesenteric vessels and superior vesicle vessels, and the iliolumbar and spermatic vessels in both sides. The perfused areas (as confirmed by perfusion with Evan's blue) included both the left leg, the whole left trunk, and a small part (usually 0.3-0.5 cm) of the right trunk along the spine. This flow distribution pattern was more comparable to the single hindquarter perfusion preparation with hypogastric occlusion as described by Gorski et al. (17). After heparin (0.5 ml, 1,000 U/ml) was injected into the vena cava, two cannulas (Ohmeda) were inserted caudally into the abdominal aorta (16-gauge) and vena cava (18-gauge) between the left renal and iliolumbar vessels. The animal was then immediately placed on a perspex platform to begin the perfusion. The animal was killed with an overdose of the anesthetic. Ligatures were placed firmly around the lumbar trunk approximately between L3-L4 vertebrate, the right thigh (near the inguinal ligament), and the genitalia (above the penis).
A temperature-regulated cabinet (25°C) contained the perfusion system, which consisted of a perfusion pump (Masterflex, Chicago, IL), microinfusion infusion pump (model 11; Harvard Apparatus), an in-line Clark-type oxygen electrode, sphygmomanometer, pressure transducer, artificial lung, heat exchange, and polyethylene tubing. The oxygen electrode and pressure transducer were connected to a dual channel chart recorder (OmniScribe, series D5000; Houston Instrument). The oxygen electrode was calibrated against 100% oxygen, room air, and 99.9% nitrogen. The perfusate consisting of a cell-free Krebs-Ringer bicarbonate buffer (pH 7.4) containing 8.3 mM glucose, 1.27 mM CaCl2, and 2% bovine serum albumin was equilibrated by the artificial lung with a mixture of 95% O2 and 5% CO2. The perfusion flow was set at 6 ml/min by adjusting the perfusion pump speed and confirmed by intermittent collection of the venous efflux from the hindlimb in a measuring cylinder. The hindlimb was perfused in a nonrecirculating manner at 25°C. The hindlimb perfused under these conditions gives qualitatively similar results to those perfused with erythrocyte-containing media at 37°C in its metabolic responses to various vasoconstrictors (4, 7, 33). The venous oxygen partial pressure was always >150 mmHg even when a maximal oxygen extraction took place while the arterial oxygen measured was 684 ± 3.3 mmHg (n = 58). Adequate oxygen delivery at this flow rate had been confirmed in our earlier studies (10). The perfusion was completed within 180 min, and our previous experiments under similar conditions have shown that this preparation was stable for at least 180 min with similar muscle metabolic characteristics as those in vivo (10). The heart was weighed after perfusion.
Perfusion pressure was monitored via the pressure transducer from a
side arm of the arterial line immediately before the arterial cannula.
Oxygen partial pressure of the perfusate was measured by the oxygen
electrode, which was calibrated before and after each perfusion with
oxygen and air. The oxygen content in the perfusate was calculated
according to the partial pressures using a Bunsen coefficient at
25°C for plasma as described previously (10).
O2 by the perfused hindlimb
was then calculated from the arteriovenous difference of oxygen
contents multiplied by flow rate and divided by the mass of perfused
muscle. The perfused muscle mass was measured by weighing
dye-containing muscle dissected from hindlimbs (11 SHR and 11 WKY) that
had been infused with Evan's blue (1% wt/vol) under the same
perfusion conditions. The average values were used for the calculation
of the rest of the rats for each strain of rat. The gastrocnemius,
plantaris, and soleus muscle groups in the right hindlimb were
dissected from eight animals of each strain for the measurement of
weight, assuming that the weight of both hindlimbs was equal. Both
glucose and lactate were measured with a glucose analyzer (YSI 2300 STAT plus). The venous perfusate was sampled in the steady state as
indicated by a constant venous partial oxygen pressure record for each
dose of the vasoconstrictors. Arterial perfusate was also taken before and after each perfusion to calculate glucose uptake and lactate production in the same way as for
O2.
Experimental protocols. After the
perfusion began, an equilibration period of 30 min was allowed to
elapse during which the partial pressure of venous oxygen reached the
same constant value. The basal values for perfusion pressure,
O2, glucose uptake, and
lactate production were obtained between 30 and 40 min. The experiments
in each strain were assigned to three equal groups with four rats in
each group scheduled to receive either NE, ANG II, or 5-HT infusion.
With the effects of NE, the involvement of
- and
1-adrenoceptors was assessed in
the presence of 10 µM propranolol as well as a combination of
propranolol with 2.5 nM prazosin. The antagonists were infused 20 min
before NE and then were coinfused with NE throughout the perfusion.
Drugs were infused from a port in the arterial line at a rate <1% of
the perfusion flow rate and were mixed by a magnetic stirrer in a small
bubble trap immediately before entering the hindlimb. Dose-response curves were constructed for each vasoconstrictor in a cumulative fashion. Data were collected at the steady-state levels for each dose
of vasoconstrictors, which usually took 5-10 min with weak vasoconstriction and 20-30 min with strong vasoconstriction, based on the continuously recorded perfusion pressure and
O2. Earlier studies using
the same preparation showed sustained changes in vasoconstriction and
metabolism in response to different vasoconstrictors (7, 10, 13). The
preliminary experiments in this study showed no differences in the
development between SHR and WKY of the shape of either perfusion
pressure or
O2.
Venous perfusate was collected throughout the last 5 min at the steady
state. The perfusion flow rate was monitored and readjusted to 6 ml/min
if changes occurred due to changing the dose of vasoconstrictors. The
experiments on SHR and WKY were conducted under the same conditions and
were interspersed randomly.
Chemicals. ()-NE bitartrate,
ANG II, 5-HT-HCl, DL-propranolol-HCl, and prazosin-HCl were
obtained from Sigma. Both glucose and lactate standards were purchased
from YSI. Bovine serum albumin (fraction V) was obtained from
Boehringer Mannheim. Other chemicals were analytical grade from Ajax Chemicals.
Calculation and statistical analysis.
EC50 and
IC50 (designated here as the
stimulatory and inhibitory effects of NE, respectively) were calculated
individually from the best-fit dose-response regression curves by Sigma
Plot for Windows on the basis of observed maximal changes in perfusion
pressure or O2. The
regression coefficient closest to one was used to determine the best
fitness of a curve. Negative log values of
EC50 and
IC50 (M) were calculated and
expressed as pEC50 and
pIC50, respectively, according to
the recommendation by the International Union of Pharmacology Committee
(21). Data are presented as means ± SE. Dose-response curves were
determined to differ (P < 0.05)
using ANOVA (Startview SE; Abacus Concept, Berkeley, CA) with dose as a
repeated measure to test differences within subjects (effects of a
vasoconstrictor), differences between subjects (SHR vs. WKY), and the
interactions of these two factors. Student's
t-tests were used for the comparison
between two mean values, with P < 0.05 as statistically significant.
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RESULTS |
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Blood pressure, heart weight, and whole body metabolic
rate. Table 1 shows the
body weight, blood pressure, whole body
O2, and weight of the heart
and IBAT in SHR and WKY. Compared with the age-matched WKY rat, SHR
showed significantly higher mean blood pressure under anesthesia (94%,
P < 0.01) and increased heart weight
(20%, P < 0.01). The measured
resting metabolic rate (expressed as
O2) was also increased by
11.6% (P < 0.05) in SHR. However,
there were no significant differences in body weight and IBAT mass
between these two strains (P > 0.05).
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Basal values of perfusion pressure,
O2, glucose uptake, and
lactate production. The basal values of the perfused hindlimb in the absence of vasoconstrictor are summarized in Table 2. At similar perfusion flow rates,
perfusion pressure,
O2, glucose uptake, and lactate production were increased by 21.4% (P < 0.01), 11.9%
(P < 0.01), 46.4%
(P < 0.05), and 44.9%
(P < 0.01), respectively, in SHR
when compared with WKY. However, no significant differences were found
in the basal perfused muscle mass or weight of the individual muscle
groups measured (P > 0.05).
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Effects of NE on perfusion pressure,
O2, glucose uptake, and
lactate production. Infusion of NE led to a
dose-dependent sigmoidal increase in perfusion pressure in both strains
of rats (P < 0.001). In WKY,
NE-induced increases in perfusion pressure started at 33 nM and reached
a plateau at 10 µM with a maximal increment of 199.3 ± 3.1 mmHg.
In SHR, the NE-induced increases in the dose-perfusion pressure curve
ranged between 1 nM and 1 µM, with a general upshift in magnitude
(Fig. 1). The maximal increase at 3.3 µM
was 235.7 ± 7.5 mmHg, significantly higher than that of WKY
(P < 0.001). Comparisons of the
pEC50 values (Table
3) indicate that the NE-induced perfusion
pressure curve in SHR was shifted to the left for more than twofold
(P < 0.05) compared with WKY.
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NE caused dose-dependent changes in
O2 and lactate production
(P < 0.001 for both) characterized
by stimulatory action at low doses and inhibitory action from the
maximal increase to values near or below basal levels at high NE doses
(Fig. 1). The dose-dependent biphasic change in
O2 was also shifted to the
left in SHR by more than twofold (P < 0.05), as indicated by the values of
pEC50 and
pIC50 (Table 3). Compared with
WKY, the bell-shaped
O2 curves in SHR were downshifted significantly with different NE doses
(P < 0.01).
Glucose uptake was also increased by NE during vasoconstriction (P < 0.001) in both strains, and the dose-response curves were distinctly different between SHR and WKY (P < 0.002, Fig. 1). In WKY, the increased glucose uptake reached a plateau at 0.1 µM and remained at that level with further increases in NE dose. In contrast, a bell-shaped dose-response curve of glucose uptake was observed in SHR, with the maximal increase in glucose uptake only one-half of that in WKY. Above 0.33 µM, NE-induced glucose uptake started to decline in SHR with further increases in NE dose.
Overall, there was a highly significant interaction
(P < 0.001) between SHR
and WKY in vasoconstriction,
O2, and glucose uptake in response to NE.
Effects of propranolol and prazosin on NE-induced
changes in perfusion pressure,
O2, glucose uptake, and
lactate production. Although appearing somewhat
smaller, a similar pattern of increased vasoconstriction
(P < 0.01) and reduced
O2
(P < 0.01) in SHR also occurred at
different doses of NE in the presence of 10 µM propranolol. Compared
with WKY, NE-induced glucose uptake in SHR was still lower at the high
dose range (P < 0.05). The interaction between NE doses and rat groups was also highly significant for NE-mediated dose-response curves of perfusion pressure,
O2, and glucose
uptake (P < 0.01 for all 3 parameters, Fig. 2).
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However, when the NE-elicited maximal increase in perfusion pressure in
SHR was normalized to a similar level as that of WKY by further
addition of the 1-adrenergic
antagonist prazosin, there were no significant differences between SHR
and WKY in the curves for
O2,
glucose uptake, and lactate production (2.5 nM, Fig.
3).
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Effects of ANG II on perfusion pressure,
O2, glucose uptake, and
lactate production. ANG II elicited increases in
O2, glucose uptake, and
lactate production during vasoconstriction in both strains at all doses
tested (P < 0.001 for all 4 parameters). Whereas ANG II-mediated
O2 dose-response curves were
similar between SHR and WKY (P > 0.5), the curves for glucose uptake and lactate production tended to be
lower in SHR (P < 0.12, Fig.
4). When analyzed as percentage changes
above the basal levels (Fig. 5), the maximal increments in
glucose uptake (P < 0.05) and
lactate (P < 0.01) in SHR
were less than one-half compared with WKY. The doses ranged between 3.3 and 10 nM with similar pEC50
values in both strains (Table 3).
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Effects of 5-HT on perfusion pressure,
O2, glucose uptake, and
lactate production. 5-HT caused dose-dependent
increases in perfusion pressure (P < 0.001) in both strains of rats within the range between 0.1 and 10 µM
(Fig. 6). 5-HT inhibited
O2 at all doses during
vasoconstriction in both SHR and WKY
(P < 0.001), and this inhibition was
greater in SHR (P < 0.01). However, there were no significant differences in the dose-response curves for
perfusion pressure (P > 0.5),
glucose uptake (P > 0.5), and lactate production (P > 0.2) between
SHR and WKY (Table 3).
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Maximal changes in perfusion pressure,
O2, glucose uptake, and
lactate production induced by NE, ANG II, and 5-HT. Because the measured basal metabolites (in the absence of
vasoconstrictors) were different between SHR and WKY (as shown in Table
2), we further evaluated the metabolic alterations during
vasoconstriction by expressing them as percentages of the basal values.
The data in Fig. 5 represent the maximal
O2 (%) over the basal values obtained from Figs. 1, 4, and 6. The results with NE clearly show marked proportional reductions in
O2, glucose uptake, and
lactate production in SHR during vasoconstriction in SHR. ANG
II-induced glucose uptake and lactate production were also
proportionally inhibited in SHR. An increased inhibition of
O2 was found when 5-HT
elicited vasoconstriction. However, there were no significant increases
in proportion to their basal values of perfusion pressure for NE and
5-HT, whereas the ANG II-elicited maximal perfusion pressure was in
fact proportionally smaller.
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DISCUSSION |
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A close correlation between obesity, insulin resistance, and hypertension has been recognized in humans (11, 18, 22, 24, 36). These complex diseases are multifactorial traits with both environmental and genetic determinants. To delineate the mechanisms involved, genetic animal models have proved to be helpful. For example, most metabolic disorders seen in humans resemble those observed in genetically obese rodents, although hypertension is not as profound in these animals as in humans (25, 36). In terms of the cardiovascular abnormalities, the SHR is a well-characterized model. Metabolic disorders are also found in this model. For instance, insulin resistance has been indicated with the euglycemic-hyperinsulinemic clamp technique in vivo (33, 36). In the skeletal muscle, altered Na+-K+-ATP pump number and activity have been demonstrated (31). There are also reports showing a transition of muscle fiber from the slow to fast type (3). These changes could potentially affect the thermogenic capacity and glucose utilization rate. On the other hand, higher rates of glycogen synthesis and lactate formation have been observed at certain doses of insulin concentrations in incubated soleus muscle strips of SHR compared with that of WKY (27). These controversial reports on SHR metabolism and glucose utilization may be related to the experimental conditions employed. As elegantly demonstrated by Rao (33), interpretation of insulin resistance in SHR can be very different in glucose clamp studies depending on whether the assessment is based on insulin infusion rate or on plasma insulin levels. Likewise, the influence of vascular function on muscle thermogenesis and glucose uptake can not be ignored (33, 35). Nonetheless, the relationship between vascular function and muscle metabolism has not been emphasized in studies involving SHR.
Several important findings have emerged from the present experiment.
First, in the absence of vasoconstrictors, the basal perfusion
pressure, O2, glucose uptake,
and lactate production were all higher in the SHR hindlimb when
compared with WKY rats. These results appear to be consistent with the
increased blood pressure and increased resting metabolic rate of the
whole animal in vivo. Second and most important, our results clearly
showed impaired muscle thermogenesis and glucose uptake associated with vasoconstriction elicited by NE. Similar defects were also found with
5-HT or ANG II during vasoconstriction.
Vascular resistance and metabolism in the absence of vasoconstrictors. Whereas resting hindlimb perfusion pressures are at first sight low, arterial pressures will necessarily be lower than in vivo when employing a cell-free, low-viscosity perfusate. In addition, there is neither resting sympathetic tone nor circulating vasoconstrictors in the perfused hindlimb. Thus physiological pressures require high flow rates (40), or, alternatively, physiological flow rates generate subphysiological pressure (4, 10). In the present study, a compromise approach was adopted by using the same constant perfusion flow rather than constant pressure. Even so, the low vascular tone of the hindlimb is unlikely to have had significant bearing on the experimental observations, as a number of studies suggest that the vasodilatation is uniform across both nutritive and nonnutritive networks and does not alter the proportion of flow between the two routes (7). Over a range of perfusion pressures, total hindquarter flows are 76% lower in SHR than in WKY regardless of muscle fiber types (39).
Consistent with earlier reports by others (1, 3, 26, 39), the results in Table 2 clearly showed a marked increase in basal perfusion pressure in the SHR hindlimb. This change is probably due to morphological changes in resistance vessels, which could start as early as 4 wk of age in SHR (1, 12). Although it may be different from that of a conscious animal in terms of the absolute value, the blood pressure measured under anesthesia confirmed a fully developed hypertension at this age as reported by others (1).
It is of interest to note that the basal
O2, glucose uptake, and
lactate production were higher (11.5, 43, and 41.5%, respectively) in
the perfused SHR hindlimb. This appears puzzling at first because the
vascular changes resulting from the increased basal perfusion pressure
could be argued to restrict the exchange rate between capillaries and
muscle cells. However, it is possible that the capillary changes at
this stage have not developed to such an extent that basal metabolism
is inhibited at a constant flow rate. The observed higher basal
metabolism may relate to changes in cellular properties. For example,
an increased number of sarcolemmal Na+-K+-ATP
pumps and their overall activity have been noted in soleus muscle of
young SHR (up to 16 wk) in compensation for expelling increased
intracellular Na+ (31). These
changes could be expected to consume more ATP, leading to a rise in
muscle metabolism. Elevated vascular tone due to increased smooth
muscle layers (12) might also use more oxygen (9). Other changes such
as increased lactate dehydrogenase activity and slow-to-fast fiber type
transition (3) may also contribute to the increased lactate production.
Such an explanation is also consistent with an increased ATP turnover
in skeletal muscle found in patients with untreated primary
hypertension (37). Interestingly, the measured whole body
O2 at rest was also higher in
SHR (Table 1). This latter result agrees with earlier studies by others
showing a slightly higher metabolic rate (3) and body temperature (8).
The data taken together suggest that the muscle vascular bed may not
only contribute to the increased peripheral vascular resistance but
also to the elevated whole body metabolic rate during hypertension.
Because increased
O2 could
potentially improve glucose uptake and utilization, impaired muscle
metabolic capacity in SHR may not be readily demonstrable under
conditions in which the cardiovascular system is not challenged, such
as in incubated muscle strips (27). This interpretation would also
appear to be consistent with the observations by Rao (33). In the study
by Rao, insulin resistance was most convincingly revealed using
euglycemic and hyperinsulinemic clamp technique with a series
concentration doses of insulin even although neither hyperinsulinemia
nor hyperglycemia were present in SHR under basal conditions. This is true for the SHR and WKY from the same
source used in the present experiment [the fasted plasma insulin
levels were 188 ± 34 and 169 ± 22 pmol/l, respectively
(32)].
Changes in vasoconstrictor-controlled muscle
thermogenesis and glucose uptake. An increased vascular
sensitivity to vasoconstrictors has been shown in muscle vascular beds
of SHR (1, 5, 6, 26, 39). The present study also found an augmented
vasoconstriction in SHR hindlimb produced by NE (Fig. 1). NE-elicited
biphasic changes in O2 and
lactate production in the perfused rat hindlimb have been previously
observed in Wistar rats, and both phases are predominantly mediated by
1-adrenoceptors (7, 35)
presumably by different subtypes (13). In SHR, LNE-induced increases in
O2 were significantly
reduced, and the inhibitory phase of
O2 produced by HNE was much
greater compared with WKY. This accentuated inhibition of
O2 and glucose uptake still
remained in the presence of 10 µM propranolol despite a significant
leftward shift of the dose-response curve of the perfusion pressure of
WKY together with a slight inhibition of the maximal
O2. However, further addition
of 2.5 nM prazosin (which normalized SHR and WKY perfusion pressure
without full blockade of the NE effect) abolished the decreased
O2 produced by NE
in SHR when compared with WKY. Interestingly, these effects of prazosin
on
O2 were associated with a
much stronger inhibition of vasoconstriction in SHR than in WKY,
suggesting that the altered muscle thermogenesis in SHR is closely
associated with changes of vasoconstriction primarily attributed to
1-adrenoceptors.
A stimulatory effect of -adrenergic stimulation on glucose uptake by
the perfused rat hindlimb has been described by others when epinephrine
was administered, and this effect was associated with increased
O2 and perfusion pressure
(35). Of particular interest is the striking reduction of glucose
uptake in SHR induced by NE compared with that of WKY. In parallel with
changes in
O2, this impaired
glucose uptake in SHR appeared to be associated with vasoconstriction
mediated by
1- rather than
-adrenoceptors because it could only be reversed when the increased
vasoconstriction was abolished by prazosin. Coincidentally, Mondon et
al. (28) also suggested that the altered vascular system may be
responsible for the impaired insulin removal in SHR.
NE does not directly stimulate
O2 in muscle preparations in
which nutrients and oxygen are not delivered by the vascular system
(7). By contrast, in the muscle preparations in which nutrients and
oxygen are delivered via the vascular system, NE elicits changes in
O2, glucose uptake, and
lactate production, and these changes are all reversed when
vasoconstriction is blocked (7, 10, 35). Therefore, a hemodynamic
mechanism for NE-controlled
O2 in perfused muscle
preparations is highly probable (7, 34). Such an explanation is
supported by the vasoconstriction-associated metabolic changes produced
by ANG II and 5-HT, neither of which showed direct action on muscle
metabolism in superfused or incubated muscle preparations (7, 34).
However, ANG II-elicited glucose uptake in the perfused SHR hindlimb
was proportionally lower with a reduced lactate production, and 5-HT
inhibition of
O2 was much
greater compared with that in WKY. Hence, in general,
vasoconstrictor-controlled metabolism in SHR hindlimb muscle was
downregulated although not all three parameters measured were uniformly
altered by ANG II and 5-HT.
The hemodynamic mechanism underlying the altered muscle metabolism in
SHR may involve both functional and morphological changes in the muscle
microvasculature. Julius et al. (22) have proposed that
hypertension-related changes in the muscle microcirculation may
contribute to the impaired glucose uptake in hypertension by impairing
the delivery of insulin and glucose to muscle cells. As reviewed by us
earlier (Ref. 7 and references therein), muscle resting
O2 may be largely controlled
by the ratio of nutritive to nonnutritive flow. Type A vasoconstrictors
LNE and ANG II may preferentially constrict the precapillary arterioles before nonnutritive routes, thereby redirecting to nutritive routes. Accordingly,
O2, lactate
production, and glucose uptake are increased. Conversely, type B
vasoconstrictors HNE and 5-HT may redistribute flow to nonnutritive
routes by closing nutritive capillaries, causing an inhibition of
muscle metabolism. The muscle tendon vessels have been shown to be
possible nonnutritive routes for type B vasoconstrictors (29). In SHR
skeletal muscle, a decreased vascular flow capacity has been shown to
correlate with increased vascular resistance and alleviated capillary
exchange function (39) due to vascular remodeling such as capillary
rarefaction (3, 26). These changes are likely to reduce the nutritive route reserve and thus suppress vasoconstrictor-controlled muscle metabolic capacity in SHR. On the other hand, the transition of muscle
fiber types from the slow to fast type, which may be partly related to
capillary rarefaction (3), could also contribute to the overall
metabolic changes in SHR muscle. It is well known that the slow muscle
fiber has higher
O2 and
glucose utilization rate with denser capillaries (27).
Comparison of NE, ANG II, and 5-HT. A
comparison of the effects of NE, ANG II, and 5-HT in SHR indicates that
NE-induced changes in perfusion pressure and altered muscle metabolism
were most profound in the perfused SHR hindlimb. The reported plasma
ANG II level in SHR is ~100 pM in a low-salt diet and 57 pM in a
high-salt diet (5), whereas the plasma 5-HT level in normal humans is ~6 pM, and under some disease conditions such primary pulmonary hypertension it can be increased fivefold (20). These concentrations are below the range of the dose-response curves observed in Figs. 3 and
4. Although plasma NE ranges between ~1 and 3 nM in rats (1, 2) and
humans (2, 23) under normal physiological conditions, the measured NE
in rat muscle is ~0.12 mg/g wet tissue weight (1, 30), equivalent to
153 mg/l water (or 0.9 µM). Importantly, NE is a major
neurotransmitter of the peripheral sympathetic nerves that widely
innervate muscle resistance vessels. NE concentration in the
neuromuscular junction is estimated to be as high as 10-50 µM
(16). Such concentrations would cover the full dose-response range of
perfusion pressure and metabolism for NE seen in Fig. 1. Furthermore,
sympathetic nerve plexus density in skeletal muscle small arteries is
significantly higher (38), with a 40% increased NE turnover in muscle
tissue (1) in SHR than in WKY rats. Similarly, both plasma NE levels
and the recorded muscle sympathetic nerve activity are elevated in
young hypertensive subjects (2, 15). Thus NE-induced changes found in
this study may have significant implications in vivo. It has been shown
that chronic treatment of SHR with trandolapril, an
angiotensin-converting enzyme inhibitor, normalizes blood pressure and
improves the response of glycogen metabolism to insulin in isolated
soleus muscle (27). It would be interesting to see whether chronic
blockage of -adrenoceptors in SHR will also ameliorate the altered
metabolism during vasoconstriction.
Conclusion. Data from the present study have clearly shown an impaired potential of muscle thermogenesis and glucose uptake in SHR associated with vasoconstrictor action, although the resting metabolism in the absence of vasoconstrictors is increased. These data may imply, at least in part, a vascular role in some metabolic disorders such as obesity and glucose intolerance during hypertension.
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ACKNOWLEDGEMENTS |
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We thank Michael G. Clark for constructive criticism of the manuscript.
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FOOTNOTES |
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This study was supported by the National Health and Medical Research Council of Australia.
Address for reprint requests: J.-M. Ye, Diabetes Group, Garvan Institute of Medical Research, 384 Victoria St., Darlinghurst, Sydney NSW 2010, Australia.
Received 29 December 1997; accepted in final form 19 August 1998.
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