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Clinical Neurocardiology Section (J.N.P., D.S.G., G.E.), National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892; St. Bartholomew’s and The Royal London School of Medicine (S.W.C.), London E1 1BB, United Kingdom; and Endocrine Research Unit (J.M.M.), Mayo Clinic, Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Simon W. Coppack, M.D., Department of Diabetes and Metabolic Medicine, 5th Floor Alexandra Wing, The Royal London Hospital, Whitechapel, London E1 1BB, United Kingdom. E-mail: . s.w.coppack{at}mds.qmw.ac.uk
Abstract
Adipose tissue lipolysis is at least in part stimulated by the sympathetic nervous system (SNS). Although there is a generalized decrease in SNS activity with fasting, the rate of lipolysis during fasting increases. The aim of this study was to determine whether there is an association between activation of sympathetic nerves innervating adipose tissue and the increase in lipolysis seen during fasting in humans. We used the isotope dilution technique to measure regional norepinephrine spillover from abdominal sc adipose tissue from seven healthy subjects before and after a 72-h fast. Our results showed a significant increase in adipose tissue spillover of norepinephrine (mean ± SEM, 0.40 ± 0.09 vs. 1.08 ± 0.18 pmol·100 g-1·min-1, P < 0.05) and arterial norepinephrine concentrations (0.92 ± 0.10 vs. 1.23 ± 0.08 nmol·liter-1, P < 0.05) after the fast with no significant change in total body norepinephrine spillover, forearm norepinephrine spillover, epinephrine concentrations, or energy expenditure. We show for the first time, in humans, a selective regional increase in adipose tissue norepinephrine spillover in response to a 72-h fast and suggest that the SNS may play a greater role in the regulation of lipid metabolism during fasting than previously thought.
ADIPOSE TISSUE IS the major depot of lipid fuel within the body. Release of nonesterified fatty acids (NEFA) from adipose tissue is regulated by hormone-sensitive lipase (HSL), which in turn is under both humoral and neural control (1). In the postprandial state, insulin is a powerful inhibitor of HSL (2). During fasting, as insulin concentration decreases, circulating concentrations of catabolic hormones (especially growth hormone and cortisol) and catecholamines increase to promote lipolysis, resulting in the release of NEFA and glycerol into the circulation (3, 4). The changes in local sympathetic nervous activity during fasting are poorly understood and may differ among different tissues. For example, Young and Landsberg (5, 6) showed suppression during fasting of norepinephrine (NE) turnover in the heart, pancreas, and liver of rats. In contrast, NE turnover in rat adipose tissue increased with fasting (7).
Because catecholamines stimulate lipolysis and lipolysis is increased during fasting, we hypothesized that sympathetic nervous system (SNS) activity in adipose tissue increases during fasting to facilitate the mobilization of energy stores. To test this hypothesis, we used an isotope dilution technique with arteriovenous sampling to determine regional NE spillover from sc adipose tissue after an overnight (12–14 h) fast and again after 72 h of fasting.
Subjects and Methods
Experimental subjects
Seven healthy, lean Caucasian subjects (Table 1
) were recruited. The protocol was approved by the Institutional Review Board, and written, informed consent was obtained from all volunteers. The subjects were at a stable weight and taking no medication other than occasional acetaminophen. They were asked to refrain from smoking and caffeine for 24 h before the study.
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At 1700 h on the day before the study, subjects reported to the General Clinical Research Center. Body composition was determined by dual energy x-ray absorptiometry (Lunar Corp. Instruments, Madison, WI), and a blood sample was collected to serve as a blank for the NE assay. Subjects were then given a standard meal providing 15 kcal/kg lean body mass (LBM), with a 50% carbohydrate, 30% fat, and 20% protein caloric distribution. At 2200 h, subjects were given a fat-free snack (5 kcal/kg LBM).
At 0700 h the next morning (d 1), using local anesthesia, blood sampling cannulae were inserted into a superficial abdominal vein, an antecubital vein (usually the median cubital vein) draining deep forearm tissues, and the contralateral radial artery (8). The superficial abdominal vein cannula was positioned so that its tip was just inferior to the inguinal ligament (8). An infusion cannula was inserted in a forearm vein ipsilateral to the cannulated artery. All cannulae were kept clear with constant infusions of sterile 150 mM NaCl.
After the cannulae were in place, a constant infusion of [9,10-3H] palmitate (0.4 Ci/min) was started. Thirty minutes later, an infusion of levo-[ring 2,5,6-3H] NE at 0.8 µCi/min (NEN Life Science Products, Boston, MA) was started, and both infusions were continued until all blood sampling and blood flow measurements were complete. At the same time, 150 µCi 133Xe (Malinkrodt, Northampton, UK) in approximately 0.2 ml normal saline was injected into the sc abdominal adipose tissue in the drainage of the abdominal vein being sampled.
Resting energy expenditure and respiratory exchange ratio were measured by indirect calorimetry (Deltatrac, Datex, Finland).
Sixty minutes after administration of the 133Xe isotope, adipose tissue blood flow was calculated from the rate of decline of activity in the 133Xe depot as previously described (8, 9). Forearm blood flow was measured by mercury strain-gauge plethysmography (10).
Four sets of blood samples (d 1 postabsorptive samples) were taken from the three sampling sites (two venous and one arterial) at 10-min intervals for measurement of palmitate, NE, epinephrine, [3H] NE, and [3H] palmitate. Samples from forearm veins and forearm blood flow measurements were always taken after exclusion of the hand circulation for 2 min by inflating a sphygmomanometer cuff at the wrist to 60–100 mm Hg above arterial pressure. Samples were also taken from the arterial site for measurement of insulin, glucagon, growth hormone, glucose, and ketone bodies.
After completing the postabsorptive sampling, subjects ate a standard meal (providing 15 kcal/kg LBM, with a 50% carbohydrate, 30% fat, and 20% protein caloric distribution) at about 1000 h. Postprandial samples were then taken 60–120 min after the meal.
The subjects then remained in the General Clinical Research Center and fasted for 3 d. They were allowed to drink water; caffeine-free diet drinks; and caffeine-free isotonic, glucose free, sports drinks. The study protocol was then repeated (d 4). In one subject, no anatomically suitable antecubital deep vein could be cannulated on d 4.
Analytical methods
Blood samples were taken and immediately transferred to prechilled tubes containing lithium heparin and kept on ice until centrifuged at 4 C, which was done within 30 min of sample collection. Plasma was stored at -70 C. Plasma insulin concentrations were determined by RIA as previously described (11) and hematocrit by centrifugation. Plasma epinephrine concentrations, together with NE concentrations and associated tritium specific activity, were determined as previously published (12). For plasma palmitate, concentration and specific activity were determined by HPLC as previously described (13).
Calculations
Total body NE spillover (TBS) was calculated according to the equation (14)
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Regional spillover of NE in adipose tissue or the forearm (Rs) was calculated according to the equation (14),
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Because previous work showed no net clearance of isotopically labeled palmitate by adipose tissue (15), local palmitate balance was calculated as the product of net arteriovenous difference in plasma palmitate concentration and local plasma flow.
Data are presented at mean ± SEM. Data were log transformed where necessary and paired t tests used to examine differences between the before fasting (d 1) and postfasting (d 4) values. For presentation purposes, the means of the four postabsorptive samples and four postprandial samples are given.
Results
Metabolic changes before and after the 72-h fast
There was no significant change in postabsorptive resting energy expenditure over the period of the fast (d 1 vs. d 4) (Table 2). Postabsorptive concentrations of ketone bodies (ß hydroxybutyrate and acetoacetate) increased significantly (P < 0.001), but respiratory exchange ratio (P < 0.01) and glucose and insulin concentrations (P < 0.01) decreased significantly from d 1 to d 4. Arterial glucagon concentrations rose significantly (P < 0.01), but the change in growth hormone was not significant.
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. In the postabsorptive state, deep forearm muscle took up palmitate on both d 1 and d 4, but there was no significant difference between the two days.
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shows the arterial insulin and palmitate concentrations before and after the meal on d 1 and d 4. As expected, the meal also produced an increase in glucose and reduction in NEFA concentrations (data not shown).
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, the postprandial palmitate concentration was greater (P < 0.01) on d 4 than on d 1 despite higher mean insulin concentrations. The postprandial whole body and adipose tissue palmitate flux were similarly increased on d 4, compared with d 1 (data not shown). Catecholamines
Arterial NE concentrations increased with the 3-d fast (e.g. postabsorptive, mean ±SEM, d, 0.9 ± 0.1 vs. d 4, 1.2 ± 0.1 nmol/l-1, P < 0.05) (Fig. 2). However, there was no significant change in arterial epinephrine concentrations following the 3-d fast. The NE concentration increased after the meal (P < 0.05) on both d 1 and d 4 after the meal, but epinephrine concentrations declined significantly (P < 0.05). These responses to the meal were not significantly different on d 1 and d 4.
Postabsorptive adipose tissue NE spillover increased significantly following the fast (d 1, 0.45 ± 0.11 vs. d 4, 0.94 ± 0.20 pmol/100 g-1 per min-1, P < 0.05), but there was no significant change in deep forearm muscle or whole-body NE spillover. The responses of adipose tissue, deep forearm muscle, and whole-body spillover to eating was not significantly different on d 1 and d 4 (data not shown).
Discussion
This study shows for the first time a divergent effect of prolonged fasting on SNS activity in adipose tissue, compared with its effect on total body SNS activity in humans. Local NE spillover from sc adipose tissue increased with fasting without a significant change in total body or forearm skeletal muscle NE spillover.
The metabolic and hormonal changes (Table 2) seen in this study were as expected and confirm adherence to the fast in all subjects (4, 16, 17, 18). The relative importance of humoral and neuronal control of metabolism in prolonged fasting (> 48 h) is not known. Adipose tissue is known to have adrenergic receptor systems (19, 20), and in vivo studies have demonstrated a role for catecholamines in regulating the key enzymes in the hydrolysis of triglycerides, namely lipoprotein lipase and HSL (21, 22). Adipose tissue NE spillover increases after eating, but the antilipolytic effects of insulin dominate over the prolipolytic effects of catecholamines following meal ingestion (23). Similarly, a decrease in plasma insulin is thought to mediate an increase in HSL activity in the early stages of fasting (24). Although a 3-d fast in rats produces a state of insulin unresponsiveness to glucose (25), the changes, if any, that occur in adipocyte insulin sensitivity as fasting progresses are not well understood.
It has been suggested that the increase in lipolysis seen during prolonged fasting is predominantly mediated by noncatecholaminergic mechanisms (26, 27). This view has been supported by findings from ß-adrenergic receptor blockade studies in both animals and humans (28, 29). However, interpretation of blockade studies is problematic because adrenergic blocking agents may affect lipolysis directly or alter blood flow (30). These effects may explain the failure, in some studies, of ß-blockade to cause a fall in circulating NEFA during fasting. Furthermore, when Klein et al. (31) measured whole-body rate of Ra rather than circulating concentrations of NEFA, increased lipolysis appeared to become more dependent on ß-adrenergic stimulation as fasting progressed. This suggests that the role of the SNS in lipolytic regulation during prolonged fasting may be more significant than previously suggested.
It is well established that regional activation of the SNS in response to certain stimuli can occur (32). In humans plasma concentrations of NE have been shown to increase following a 72-h fast (16), suggesting activation of the SNS in at least some tissues. However, the only previous study of sympathetic activity in human adipose tissue showed no significant change with fasting of a short (14–22 h) duration (33). In contrast, we have shown a significant increase in local NE spillover from adipose tissue after a 72-h fast. Although the work of Young and Landsberg (5, 6) suggested that fasting is associated with a decrease in SNS activity, they did not study adipose tissue. Our results in humans are consistent with the findings of Migliorini et al. (7), who demonstrated an increase in sympathetic nerve activity in the adipose tissue of rats during fasting.
Although the change in arterial plasma epinephrine concentration was not statistically significant, there was a tendency for an increase. This is in agreement with findings from previous studies (34) in which plasma epinephrine concentrations have been noted to rise in normal subjects fasted for 36 h.
We show for the first time that there is a selective regional increase in NE spillover from sc adipose tissue following a prolonged fast in healthy human subjects. Although a decrease in SNS activity in many tissues makes teleologic sense as part of an adaptive response to conserve energy during starvation, the tissue-specific increase observed in this study may play an important part in the mobilization of energy stores seen in response to starvation and be part of the same adaptive response.
Footnotes
This work was supported by The Wellcome Trust, The Mayo Foundation, and the U.S. Public Health Service (DK38092).
J.N.P. was a Sir Jules Thorn Charitable Trust Travelling Fellow.
Abbreviations: HSL, Hormone-sensitive lipase; LBM, lean body mass; NE, norepinephrine; NEFA, nonesterified fatty acids; Ra, rate of appearance; SNS, sympathetic nervous system.
Received November 15, 2001.
Accepted April 4, 2002.
References
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, Day 1, before fast; 
, day 4, after fast