Direct evidence for tonic sympathetic support of resting metabolic rate in healthy adult humans

Mary Beth Monroe1, Douglas R. Seals1,2, Linda F. Shapiro1,2, Christopher Bell1, David Johnson3, and Pamela Parker Jones1

1 Department of Kinesiology and Applied Physiology, University of Colorado at Boulder, Boulder 80309; 2 Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262; and 3 Department of Medicine, University of Arizona Health Sciences Center, Tucson, Arizona 85724


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

The sympathetic nervous system (SNS) plays an important role in the regulation of energy expenditure. However, whether tonic SNS activity contributes to resting metabolic rate (RMR) in healthy adult humans is controversial, with the majority of studies showing no effect. We hypothesized that an intravenous propranolol infusion designed to achieve complete beta -adrenergic blockade would result in a significant acute decrease in RMR in healthy adults. RMR (ventilated hood, indirect calorimetry) was measured in 29 healthy adults (15 males, 14 females) before and during complete beta -adrenergic blockade documented by plasma propranolol concentrations >= 100 ng/ml, lack of heart rate response to isoproterenol, and a plateau in RMR with increased doses of propranolol. Propranolol infusion evoked an acute decrease in RMR (-71 ± 11 kcal/day; -5 ± 0.7%, P < 0.0001), whereas RMR was unchanged from baseline levels during a saline control infusion (P > 0.05). The response to propranolol differed from the response to saline control (P < 0.01). The absolute and percent decreases in RMR with propranolol were modestly related to baseline plasma concentration of norepinephrine (r = 0.38, P = 0.05; r = 0.44, P = 0.02, respectively). These findings provide direct evidence for the concept of tonic sympathetic beta -adrenergic support of RMR in healthy nonobese adults.

sympathetic nervous system; resting energy metabolism; beta -adrenergic blockade


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE SYMPATHETIC NERVOUS SYSTEM (SNS) plays an important role in the regulation of energy expenditure. Sympathetic beta -adrenergic stimulation evokes an increase in metabolic rate (i.e., thermogenesis) under basal fasted conditions (4, 13, 14). Similarly, it has been shown that the SNS is largely responsible for the facultative component of the thermic effect of acute energy intake in humans (1, 2, 7, 15).

Although it is clear that stimulation of the SNS has a thermogenic effect, the possible role that tonic SNS activity plays in the support of resting metabolic rate (RMR) in healthy adult humans remains controversial. The results of studies to date are inconclusive, with most (2, 3, 7, 9, 12, 15, 16, 19), albeit not all (5, 8, 21, 22), failing to provide evidence for a significant contribution of the SNS. In all cases, RMR was measured before and after nonselective beta -adrenergic receptor blockade by use of either oral or intravenous propranolol. A significant reduction in RMR with propranolol has been interpreted as evidence for tonic sympathetic beta -adrenergic support of RMR, whereas lack of change in RMR has been interpreted as lack of tonic SNS support.

Careful review of the literature cited above, however, reveals at least two potential methodological explanations for the inconsistent findings. First, there was substantial variation in the dose of propranolol used, such that failure to achieve complete beta -adrenergic blockade could account for a negative result. The effective blocking dose, both oral and intravenous, and respective plasma concentrations of propranolol required to achieve complete beta -adrenergic blockade have been established (6). A plasma concentration of >= 100 ng/ml with intravenous infusion or 40 ng/ml with oral administration is necessary. These are typically achieved with a minimum dose of 20 (infusion) or 80 mg (oral). Most studies did not document complete beta -adrenergic receptor blockade. Second, the number of normal healthy subjects studied has generally been small (mean n = 8 per investigation). This leaves open the possibility of insufficient power to demonstrate a statistically significant effect should one be present (type II error).

Accordingly, the aim of the present study was to determine whether there is a tonic SNS beta -adrenergic contribution to RMR in healthy adult humans. We hypothesized that an intravenous propranolol infusion designed to achieve complete beta -adrenergic blockade would result in a statistically significant acute decrease in RMR in an appropriately sized group of healthy men and women.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Subjects. Twenty-nine healthy young (27 ± 1 yr, mean ± SE) men (n = 15) and women (n = 14) were studied. All subjects were nondiabetic, normotensive (systolic/diastolic blood pressure <140/90 mmHg), free of known cardiovascular and metabolic disease, nonobese (body mass index <28), and otherwise healthy as assessed by medical history and focused physical examination. Subjects were nonsmokers and were not taking any regular medications. The nature, purpose, and risks of the study were explained to each subject before written informed consent was obtained. The experimental protocol was approved by the Human Research Committee at the University of Colorado at Boulder and by the Colorado Multiple Institutional Review Board.

Protocol. Subjects reported to the laboratory on three separate occasions. The first visit involved a physical exam, health history screening, measurement of resting heart rate and blood pressure, and body composition analysis. The second and third visits were the experimental and control sessions for determination of SNS beta -adrenergic support of RMR. The order of these latter two sessions was randomized. For the experimental session, RMR was measured before and during beta -adrenergic receptor blockade (iv infusion of propranolol: 0.25 mg/kg bolus followed by continuous infusion at 0.004 mg · kg-1 · min-1). For the control condition, saline was infused at the same volume rate as for propranolol. All measurements were made in the morning after a 12-h fast. Subjects were studied under quiet resting conditions in the semirecumbent position. Females were tested during the follicular phase of their menstrual cycle (days 1-10). Measurements were performed between 0600 and 0900 in a dimly lit room at a comfortable temperature (~23°C).

Measurements. For the experimental and control sessions, subjects were instrumented for measurement of heart rate (ECG) and blood pressure (finger photoplethysmography, Finapres BP monitor, model 2300, Ohmeda, Englewood, CO). A catheter was introduced into an antecubital vein that was kept patent with a slow saline drip for intravenous infusions and blood sampling. After a 30-min rest period, a 45-min measurement of baseline RMR was begun. The first 15 min were considered an habituation period, after which oxygen consumption (VO2) and carbon dioxide production (VCO2) were averaged each minute for 30 min using a ventilated hood, indirect calorimetry system as described previously (18) (DeltaTrac Metabolic Monitor, SensorMedics, Yorba Linda, CA). RMR was then calculated from the average of the 30-min period using the Weir formula (20). The hood was then removed, while an intravenous bolus was given of either propranolol or saline (0.25 mg/kg). After the bolus, continuous infusion of propranolol or saline (0.004 mg · kg-1 · min-1) was given, during which the second RMR measurement was performed. For this second measurement, there was a 5-min habituation period, followed by a 30-min sampling period from which average RMR was calculated.

Blood was sampled at three time points for determination of plasma propranolol concentrations: immediately after the bolus and at minutes 15 and 30 during the RMR measurement. Propranolol concentrations were determined by high performance liquid chromatography (Associated Regional and University Pathologists, Salt Lake City, UT). In a subset of subjects (n = 15), the effectiveness of the beta -blockade was documented independently by use of an isoproterenol challenge test. Isoproterenol was infused in stepwise increments (sequentially for 6 min each at 0.05, 0.1, 0.2, and 0.3 µg/min) until an increase in heart rate of >= 25 beats/min above resting baseline was observed. The infusion was then stopped, followed by a 30-min washout period. The dose of isoproterenol that induced a heart rate increase of >= 25 beats/min was recorded as the challenge dose. Immediately after the second measurement of RMR, the isoproterenol challenge was repeated while the subject was still receiving propranolol. The protocol is presented in Fig. 1. In another subset of subjects (n = 10), a dose-response study was performed using three incremental doses of propranolol (0.004, 0.005, 0.006 µg · kg-1 · min-1). This was done to assure that beta 3-adrenergic receptors were blocked, because the other criteria of plasma propranolol concentrations and lack of heart rate response to isoproterenol document blockade of beta 1- and beta 2-receptors only.


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Fig. 1.   The experimental protocol. RMR, resting metabolic rate.

Total body mass and composition were determined as described previously by our laboratory (17). Body mass was measured on a physician's scale. Fat mass and fat-free mass were measured using dual-energy X-ray absorptiometry (DXA-IQ; Lunar Radiation, Madison, WI, software version 4.1).

Data analysis and statistics. A two-way repeated-measures analysis of variance (ANOVA) was used to identify changes in RMR with the infusion of propranolol or saline and differences between the control and experimental conditions. To determine whether effective beta -blockade was achieved, 1) propranolol concentrations at each time point and the overall average were assessed to ensure that levels were >= 100 ng/ml; 2) differences between heart rate responses to isoproterenol before compared with during beta -adrenergic receptor blockade also were identified using a two-way repeated-measures ANOVA; and 3) linear regression analysis of dose-response gain and repeated-measures ANOVA were performed to assure that RMR did not decrease with each incremental dose of propranolol. Univariate correlation analysis was performed to determine the relation between baseline plasma catecholamine concentrations and sympathetic support of RMR. The level of statistical significance was set at P < 0.05. Data are expressed as means ± SE.


    RESULTS
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INTRODUCTION
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Selected subject characteristics are shown in Table 1. Table 2 shows documentation that complete beta -adrenergic receptor blockade was achieved, as indicated by serum propranolol concentrations of >= 100 ng/ml and a lack of heart rate response to the intravenous isoproterenol infusion. The results of incremental dose-rate experiments revealed no consistent or significant dose response as assessed by linear regression analysis (P = 0.64). Furthermore, the magnitude of decrease in RMR from baseline was similar regardless of the dose (P = 0.26).

                              
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Table 1.   Subject characteristics


                              
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Table 2.   Evidence that complete beta -adrenergic blockade was achieved

SNS beta -adrenergic support of RMR. Figure 2 shows the change in RMR with propranolol and saline infusions. Propranolol infusion evoked an acute decrease in RMR (1,555 ± 26 vs. 1,484 ± 26 kcal/day; -5%, P < 0.0001), whereas RMR was unchanged from baseline levels during saline infusion (1,559 ± 29 vs. 1,544 ± 29 kcal/day; P > 0.05). The response to propranolol differed from the response to saline (P < 0.01). The respiratory exchange ratio (RER) was unchanged with propranolol (mean change = -0.0072 ± 0.0082; r = -0.06 to 0.08). Figure 3 presents the minute-by-minute values for RMR, VO2, and VCO2 during baseline and propranolol infusion.


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Fig. 2.   Change in RMR during saline () and propranolol () infusions. *P < 0.0001 vs. baseline; dagger P < 0.01 vs. saline control.



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Fig. 3.   Mean values, minute by minute, of RMR (kcal/day), O2 consumption (VO2), and CO2 production (VCO2) during the 30-min baseline and 30-min propranolol conditions.

Relations between SNS beta -adrenergic support of RMR and plasma catecholamine concentrations. The absolute and percent decreases in RMR with propranolol were modestly related to baseline plasma concentration of norepinephrine (r = -0.38, P = 0.05; r = -0.44, P = 0.02, respectively). The change in RMR was not significantly related to baseline plasma epinephrine concentration (r = -0.32, P = 0.13; r = -0.30, P = 0.14).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The primary finding from the present study is that beta -adrenergic receptor blockade induces an acute reduction in RMR. This finding provides direct evidence for the concept of tonic sympathetic beta -adrenergic support of RMR in healthy, nonobese adults. To obtain this evidence, we developed a propranolol infusion protocol that was designed specifically to achieve complete beta -adrenergic blockade. Complete blockade was documented using three approaches: plasma concentrations of propranolol, the absence of a heart rate response to the beta -adrenergic receptor agonist isoproterenol, and lack of dose response with incremental infusion dose rates of propranolol.

These data have at least three important implications. First, our results emphasize that it is necessary to achieve complete beta -adrenergic blockade to properly interpret the contribution of the SNS to metabolic rate. Only one of the previous studies using an intravenous propranolol infusion measured plasma propranolol concentration (9). The mean propranolol concentration in that study was 60 ng/ml, a lower concentration than what has been shown to be necessary to achieve complete beta -adrenergic blockade (100 ng/ml). As such, in that study, RMR did not change significantly. No other propranolol infusion studies reported any documentation of complete beta -adrenergic blockade. Furthermore, all but one study (7) used infusion rates that were less than one-half of what is necessary to achieve a plasma concentration of 100 ng/ml, according to previous pharmacology studies (6) and our pilot research. Only one of these studies found a reduction in RMR (5), which, although reported as statistically significant, was much less than that found in the present study with complete beta -adrenergic blockade (1.6 vs. 5% in the current study). In the studies involving long-term oral propranolol (ranging from 5 to 15 days), none documented complete beta -adrenergic blockade (8, 21, 22). However, based on previous pharmacology literature (6), in each case the dose was likely to have achieved complete blockade. Among these studies, each reported a significant decrease in RMR. Thus future studies in which intravenous propranolol infusion is used to quantify the SNS beta -adrenergic contribution to RMR should be designed with careful attention to the dose of propranolol given, with the goal of complete beta -adrenergic blockade.

Second, clarification that there is, in fact, significant beta -adrenergic support of RMR contributes to our understanding of energy balance in humans. Although many factors can influence RMR, and some are known to play a very important role (e.g., fat-free mass), there remains large unexplained interindividual variability in RMR. Because individuals with low RMR are predisposed to body weight gain and obesity (10), an understanding of potential factors that could be related to low RMR (i.e., low tonic SNS beta -adrenergic support) is important in developing strategies for reducing obesity.

In this context, we wish to emphasize the physiological importance of the contribution of this mechanism to overall energy expenditure. On average, SNS beta -adrenergic support of RMR in this population accounts for 71 kcal/day. Hence, in the absence of SNS beta -adrenergic support, one would have to compensate ~26,000 kcal/year through decreased energy intake or increased energy expenditure by use of non-SNS mechanisms to prevent weight gain. Thus this may be an important contributor to the maintenance of energy balance over time and the prevention of obesity.

Third, beta -adrenergic blockers are widely used in the treatment of cardiovascular diseases such as hypertension. Weight loss is often recommended as well in such patients. Thus clinicians should recognize that beta -blocker therapy might present a challenge to weight loss/maintenance by reducing energy expenditure. In fact, long-term propranolol treatment is associated with weight gain (11). Appropriate adjustments in energy intake and physical activity-related energy expenditure may be necessary to counteract the potentially adverse effects of beta -adrenergic blocker therapy on energy balance.

In conclusion, we have shown that there is tonic SNS beta -adrenergic support of RMR in healthy adult humans. We developed an appropriate methodology to achieve complete beta -adrenergic blockade to eliminate the interpretative confounding of partial (incomplete) blockade. These findings are important for our understanding of the role of the SNS in the regulation of metabolic rate and long-term energy balance, as well as in designing future studies using this methodology.


    ACKNOWLEDGEMENTS

We thank Mary Jo Reiling for administrative and technical assistance and Jason Lashbrook for technical assistance.


    FOOTNOTES

This work was supported by National Institutes of Health Grants HL-39966, AG-13038, AG-06537, AG-00828, and DK-07658 and by American Heart Association Grants 9920445Z and CWFW-0298.

Address for reprint requests and other correspondence: P. P. Jones, Dept. of Kinesiology and Applied Physiology, Campus Box 354, Univ. of Colorado, Boulder, CO 80309 (E-mail: pamela{at}spot.colorado.edu).

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.

Received 25 May 2000; accepted in final form 15 January 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
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DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 280(5):E740-E744
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