0021-972X/99/$03.00/0
The Journal of Clinical Endocrinology & Metabolism
Copyright © 1999 by The Endocrine Society
Vol. 84, No. 8
Printed in U.S.A.
Norepinephrine Spillover in Forearm and Subcutaneous
Adipose Tissue before and after Eating*
J. N. PATEL, G. EISENHOFER, S. W. COPPACK,
AND
J. M. MILES
Royal London Hospital Medical College, London E1 18B, United Kingdom; Clinical Neuroscience
Branch, National Institutes of Health, Bethesda, Maryland 20892; and St. Luke’s Hospital,
Kansas City, Missouri 64110
ABSTRACT
The sympathetic nervous system regulates lipolysis. There are
regional differences in the sensitivity of lipolysis to adrenergic regulation. Little is known about regional sympathetic activity in response to eating in humans. We studied the effect of feeding on
systemic and local sympathetic nervous system activity and lipolysis
in lean healthy subjects (three women and five men; age, 27.0 6 2.0;
body mass index, 23.4 6 1.2 kg/m22) using isotope dilution methodology and arterio-venous sampling. Feeding increased arterial norepinephrine (NE) concentration (mean premeal, 0.96 6 0.12 nmol/LzL;
mean postmeal, 1.28 6 0.14 nmol/LzL; P , 0.02) and total body NE
spillover (mean premeal, 2.11 6 0.30 nmol/minzL; mean postmeal,
2.76 6 0.31 nmol/minzL; P , 0.02), whereas the arterial epinephrine
concentration decreased (mean premeal, 289 6 61 pmol/L; mean post-
T
HE SYMPATHETIC nervous system (SNS) regulates numerous metabolic processes. The adrenergic regulation
of lipolysis is particularly complex, as it involves both catecholamines delivered to adipose tissue via the circulation
(1–3) and norepinephrine (NE) that is released locally in
adipose tissue from sympathetic neurons (4). Moreover, adrenergic regulation of lipolysis depends on the balance between the stimulatory effects mediated through b-receptors
and the inhibitory effects that are mediated through a2-receptors (4, 5). To make matters more complicated, in vivo
studies in humans have demonstrated regional differences in
the lipolytic activity of adipose tissue (6 – 8). However, such
studies of regional adipose tissue metabolism are difficult to
interpret in the absence of data on local sympathetic activity.
Little is known from human in vivo studies about changes
in NE spillover in adipose tissue in response to such activities
as eating, fasting, and exercise. This is principally because no
convenient method for measuring exocytotic release of NE in
adipose tissue has heretofore been available. NE is synthesized and stored in sympathetic nerve endings and is the
neurotransmitter involved in SNS signal transmission. Although most of the NE released from sympathetic postganglionic neurons is cleared locally by neuronal and extraReceived November 23, 1998. Revision received April 16, 1999. Accepted April 21, 1999.
Address all correspondence and requests for reprints to: Dr. Jigisha
Patel, M.R.C.P., National Institute of Neurological Disorders and Stroke,
National Institutes of Health, 10 Center Drive, MSC-1424, Building 10,
Room 6N252, Bethesda, Maryland 20892-1424. E-mail: jnpatel@box-j.
nih.gov.
* This work was supported in part by NIH Grants DK-38092 and
RR-00585, The Wellcome Trust, and the British Diabetic Association.
meal, 170 6 5 pmol/L; P , 0.02). Palmitate concentration and total
body systemic rate of appearance of palmitate declined postprandially
(mean premeal, 117 6 15 mmol/min; mean postmeal, 38 6 4 mmol/min;
P , 0.01). NE spillover increased by the same proportion in both
forearm and adipose tissue [in forearm, mean premeal and postmeal,
1.02 6 0.11 and 2.41 6 0.44. nmol/100 mLzmin, respectively (P , 0.02);
in adipose tissue, mean premeal and postmeal, 0.41 6 0.12 and 0.73 6
0.17 nmol/100 gzmin, respectively (P , 0.02)]. The results show that
a meal caused differential changes in systemic sympatho-adrenal
activity and an increase in sympathetic activity in adipose tissue
postprandially, However, this increase in postprandial sympathetic
activity was not enough to overcome the inhibition of lipolysis by
insulin (J Clin Endocrinol Metab 84: 2815–2819, 1999)
neuronal reuptake, a portion of released NE spills over into
the bloodstream. Measurements of the rates of spillover of
NE to the systemic circulation provides a better reflection of
the activity of the SNS than simple measurements of plasma
NE concentration (9).
Total body NE spillover, however, reflects the release of
NE from many tissues, including the adrenal medulla, and
thus does not provide information on SNS activity within
specific tissues. The combined use of isotope dilution and
arterio-venous sampling allows the investigator to measure
spillover from individual tissues (10). Radiolabeled NE has
thereby been used to determine regional NE clearance and
spillover in skeletal muscle (11), myocardium (12), kidney
(11), lung (11), and mesenteric organs and liver (13). There
are very little data on the sympathetic response to food in
adipose tissue, but sympathetic activity in brown adipose
tissue has been shown to decrease in response to food intake
in rats (14).
We combined the isotope dilution method with the arteriovenous sampling technique to estimate NE spillover in sc
abdominal adipose tissue in vivo in humans. We studied the
effect of feeding on adipose tissue and forearm norepinephrine kinetics in a group of lean healthy subjects. Postprandial
measurements were made during the time period that previous studies (15) suggested that metabolic responses to a
mixed meal would be maximal. We postulated that there
would be a heterogeneous regional sympathetic response to
eating so that sympathetic activity in adipose tissue would
decrease as lipolysis was inhibited postprandially, whereas
that in the forearm would increase as part of the thermogenetic response to eating (16).
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PATEL ET AL.
Experimental Subjects
Eight healthy lean Caucasian subjects (Table 1) were recruited. The
protocol was approved by the Mayo Clinic 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.
Materials and Methods
At 1700 h on day 0 of the study, subjects reported to the Mayo General
Clinical Research Center and on arrival were given a standard meal
containing 15 Cal/kg lean body mass (LBM), with a 50% carbohydrate,
30% fat, and 20% protein caloric distribution. Body composition was
determined by dual energy x-ray absorptiometry (Lunar Corp., Madison, WI). A blood sample was collected at that time to serve as a blank
for the NE assay. At 2200 h, the subject was given a fat-free snack (5
Cal/kg LBM). At 0630 h the next morning sampling catheters were
inserted under local anesthesia in a radial artery and in the superficial
abdominal and deep forearm veins as previously described (17). We
assumed that the deep forearm vein drained principally muscle, and the
sc adipose tissue vein drained principally adipose tissue (15, 17). An
infusion catheter was placed in a superficial midforearm vein contralateral to the sampling catheters. At 0800 h an infusion of levo-[ring-2,5,63
H]NE at 0.8 mCi/min and an infusion of [9,10-3H]palmitate (both from
New England Nuclear, Boston, MA) at 0.4 mCi/min were started and
continued to the end of the study. At the same time, 150 mCi 133Xe
(Malinkrodt, Northampton, UK) in approximately 0.5 mL normal saline
were injected into the sc abdominal adipose tissue in the drainage of the
abdominal vein being sampled, and tracings of 133Xe activity were
obtained 15 min before the meal, immediately before the meal, and 100
and 120 min after the meal (18). Forearm blood flow, with the wrist
occluded, was determined 30 and 10 min before and 60 and 100 min after
the meal using mercury strain gauge plethysmography (Hokanson, Inc.,
Bellevue, WA) (19). At 0 min, the subject was given a mixed meal
containing 15 Cal/kg LBM (50% carbohydrate, 30% fat, and 20% protein). Blood samples were taken from the three sampling sites at 30, 20,
and 10 min and immediately before the meal and again at 60, 80, 100,
and 120 min after the meal for measurement of NE, epinephrine, [3H]NE,
and palmitate. Samples were also taken from the arterial site for measurement of insulin.
Analytical methods
Blood samples were taken, immediately transferred to precooled
tubes containing ethylenediamine tetraacetate, and kept on ice until
centrifuged at 4 C, which was performed within 30 min of the sample
being drawn. Plasma was stored at 270 C.
Plasma insulin concentrations were determined by RIA as previously
described (1). Plasma epinephrine concentrations, together with NE
concentration and radioactivity, were determined as previously published (20).
Plasma palmitate concentrations and specific activity were determined by high performance liquid chromatography using [2H31]palmitate as an internal standard (21),
Calculations
Systemic rates of appearance (Ra) of palmitate in the postabsorptive
state were calculated using steady state equations (22, 23): Ra 5 tracer
infusion rate/SAa, where SAa is the arterial specific activity. In the
postprandial period nonsteady state equations were used (22, 23): Ra 5
{2F 2 [(C60 1 C120)(Vd)(SAa120 2 SAa60)}/(t120 2 t60)]/(SAa60 2 SAa120),
TABLE 1. Subject details
Subject details
27.0 6 2
5:3
1.7 6 0.02
71.8 6 5.1
23.4 6 1.2
Age (yr)
Sex (m:f)
Ht (m)
Wt (kg)
BMI (kg/m22)
Values are the mean 6
SE.
BMI, Body mass index.
where F is the isotope infusion rate in disintegrations per min (kilograms
per min), Vd is the estimated volume of distribution for palmitate (90
mL/kg) (22), C is the arterial plasma concentration, t is the time in
minutes, and subscripts 60 and 120 indicate values at 60 and 120 min.
Local rates of appearance of palmitate were calculated as previously
described (23).
Total body NE spillover (TBS) was calculated according to the equation (10): TBS 5 I/SAa, where I is the infusion rate of [3H]NE in arterial
plasma (disintegrations per min), and SAa is the specific activity of
[3H]NE in arterial plasma (disintegrations per min/pg).
Regional spillover of NE in adipose tissue or the forearm (Rs) was
calculated according to the equation: RS 5 [([3H]NEa 2 [3H]NEv)/
3
[ H]NEa] 3 [3H]NEa 1 (NEv 2 NEa) 3 PF, where [3H]NEa and [3H]NEv
are the arterial and venous plasma concentrations of [3H]NE (disintegrations per min/mL), NEa and NEv are the arterial and venous plasma
concentrations of NE (picograms per mL), respectively, and PF is the
plasma flow.
Adipose tissue and muscle densities were taken from the report by
Durnin and Wormersley (24).
Data was log transformed, and paired t tests were used to examine
differences between pre- and postprandial values.
Results
As expected, the insulin concentration increased (P ,
0.001) in response to the meal (Fig. 1, upper panel). After the
meal, the palmitate concentration declined (Fig. 1, lower panel). Arterio-venous differences across adipose tissue (Fig. 1),
systemic rates of appearance of palmitate, uptake by forearm
tissue of palmitate, and release by sc adipose tissue of palmitate all declined (Table 2).
Effect of meal on the systemic sympatho-adrenal
medullary system
The effect of feeding on plasma catecholamine concentrations and NE kinetics is shown in Table 3. There was a
significant postprandial increase in the arterial NE concentration, whereas the arterial epinephrine concentration decreased after the meal. (Fig. 2). Total body NE spillover
increased postprandially (Fig. 3).
Effect of meal on local NE spillover
Forearm plasma flow did not change significantly (1.40 6
0.30 vs. 1.78 6 0.3 mL/100 mLzmin), whereas adipose tissue
plasma flow increased significantly from 1.27 6 0.1 to 2.08 6
0.3 mL/100 mgzmin (P , 0.05) in response to the meal.
Feeding increased NE spillover in both forearm and adipose
tissue (Fig. 3).
Regional differences
Regional comparisons between forearm and adipose tissue were made after using the density of adipose tissue to
convert spillover from units of weight to units of volume (24).
NE spillover was higher in the forearm compared to adipose
tissue in both the baseline and postprandial states. The absolute increase in forearm spillover (1.39 6 0.47 pmol/100
mLzmin) was greater (P , 0.05) than the absolute increase in
adipose tissue spillover (0.32 6 0.12 pmol/100 gzmin), but the
proportional changes in each tissue in response to the meal
were not significantly different.
Discussion
We have combined novel measurements of NE spillover
from adipose tissue with measurements of total body and
REGIONAL NE SPILLOVER AFTER FOOD
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TABLE 2. Systemic and net local palmitate kinetics
Premeal
Systemic Ra (mmol/min)
Forearm tissue uptake
(nmol/100 mlzmin)
sc adipose tissue release
(nmol/100 gzmin)
Postmeal
117 6 15
31.7 6 2.2
38 6 4
3.0 6 0.4
85.2 6 24.4
0.7 6 3.2
Values are the mean 6 SE. All postmeal values are significantly
different from premeal values (P , 0.01).
TABLE 3. Catecholamine concentrations and norepinephrine
kinetics
Norepinephrine (nmol/L)
Arterial
Forearm vein
Abdominal vein
Epinephrine (pmol/L)
Arterial
Forearm vein
Abdominal vein
Specific activity (dpm/pmol)
Artery
Forearm vein
Abdominal vein
Norepinephrine spillovers
Total body (nmol/min)
Forearm tissue (pmol/100
mLzmin)
Adipose tissue (pmol/100
gzmin)
Premeal
Postmeal
0.96 6 0.12
1.13 6 0.13
0.86 6 0.13
1.28 6 0.14a
1.97 6 0.22a
1.17 6 0.18a
289 6 61
83 6 17
121 6 26
170 6 5a
90 6 22
110 6 10
683 6 30
210 6 28
334 6 35
441 6 45a
128 6 19b
289 6 31a
2.11 6 0.30
1.02 6 0.11
2.76 6 0.31a
2.41 6 0.44a
0.41 6 0.12
0.73 6 0.17a
Values are the mean 6 SE.
a
Postmeal values significantly different from premeal values, P ,
0.02.
b
Postmeal values significantly different from premeal values, P ,
0.05.
FIG. 1. Upper panel, Arterial concentration of insulin before and after a mixed meal eaten between 0 –15 min. Lower panel, Arterial
(black line, squares), sc abdominal venous (black line, diamonds), and
deep forearm venous (black filled squares) concentrations of palmitate
before and after a mixed meal eaten between 0 –15 min.
forearm NE spillover and plasma concentrations of epinephrine. The results show that ingestion of a meal causes differentiated changes in sympatho-adrenal medullary activity;
systemic epinephrine concentrations declined, whereas both
systemic and local NE spillover increased.
In previous studies of SNS activity in the postprandial
state systemic, SNS activity has been shown to increase after
a meal (25), and this may contribute to the increase in energy
expenditure known as the thermic effect of food (26). Local
NE spillover has also been shown to increase postprandially
in forearm and renal tissues (27).
The current study measured the systemic, local forearm,
and local sc adipose tissue catecholamine responses to eating
a mixed meal. The postprandial period is known to be associated with an increase in energy expenditure (28), an
increase in cardiac output, and a redistribution of the circulating volume (29). The time course of the postprandial
changes in systemic and local fatty acid suggested that the
response of the sympatho-adrenal system was measured
around the time when such changes were maximal. In some
tissues, catecholamines and insulin have antagonistic effects,
and the response to food is the consequence of divergent
effects of insulin and catecholamines.
Plasma insulin concentrations increased after the mixed
meal, whereas the different indexes of sympatho-adrenal
system activity showed a more heterogeneous response.
Thus, eating caused plasma epinephrine to decrease, in line
with a similar trend previously seen using mixed meals (29,
30). The change in epinephrine concentration presumably
reflects a fall in adrenal gland output. In contrast, systemic
NE concentrations and systemic NE spillover increased postprandially. This suggests that sympathetic nerve terminal
activity increased. However, changes in NE spillover can
reflect either or both changes in local removal or neuronal
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PATEL ET AL.
FIG. 2. Upper panel, Arterial NE concentrations before and after a
mixed meal eaten between 0 –15 min. *, Postmeal values significantly
different from premeal values, P , 0.02. Lower panel, Arterial epinephrine concentrations before and after a mixed meal eaten between
0 –15 min. *, Postmeal values significantly different from premeal
values, P , 0.02.
release of NE. The design of the present study did not allow
these influences to be distinguished.
Previous local NE spillover studies suggest that spillover
from forearm tissue increases postprandially (27). This was
also seen in our data. Whether NE spillover in forearm principally mediates an effect of NE on vascular structures to
induce a vasomotor response, an effect on skeletal muscle
cells to induce a metabolic response, or both is a matter of
speculation.
NE kinetics have not previously been measured in an
adipose tissue depot after a meal. We expected sympathetic
activity to adipose tissue to decrease after the meal, as lipolysis is suppressed postprandially; however, NE spillover
to sc adipose tissue increased after the meal, suggesting
JCE & M • 1999
Vol 84 • No 8
FIG. 3. Upper panel, Total body NE spillover before and after eating
a mixed meal. *, Postmeal values significantly different from premeal
values, P , 0.02. Lower panel, Adipose tissue and forearm NE spillover before and after eating a mixed meal. *, Postmeal values significantly different from baseline, P , 0.02.
increased SNS activity in this tissue. Although there is uncertainty about the target cells of the increased NE release in
the forearm, the chief result of increased NE release in adipose tissue would presumably be increased lipolysis in fat
cells. However the net release of palmitate from the sc adipose tissue declined. The inhibition of local palmitate release
was practically complete in all subjects, thereby obscuring
any possible interindividual differences in relative sensitivity of palmitate release to insulin levels or to local NE spillover. The results would imply that any prolipolytic effect of
increased NE spillover within the adipose tissue was being
overridden by the increased insulinemia postprandially.
This is in agreement with recent work by Horowitz et al. (31),
who showed that in the fasting state, insulin concentration,
not local NE spillover, determines local NEFA release. Thus,
whereas adipose tissue sympathetic activity might be an
important modulator of lipolytic activity in other situations,
it would appear that during meal absorption in normal lean
subjects, an increase in the insulin concentration is sufficient
REGIONAL NE SPILLOVER AFTER FOOD
to override any effect of NE and completely inhibit lipolysis
in sc adipose tissue.
In this study we have examined the responses of the SNS
to a mixed meal in a group of healthy lean subjects. As
expected, there were increases in systemic NE concentrations
and spillover as well as an increase in forearm NE spillover.
We also found that epinephrine concentrations declined after
this meal. We report for the first time that the meal also
increased NE spillover in sc adipose tissue. However, this
increased NE spillover did not prevent the complete suppression of adipose tissue lipolysis in the tissue bed. This
observation affirms the primacy of insulin in lipolytic regulation during meal absorption in healthy individuals.
13.
14.
15.
16.
17.
18.
Acknowledgments
19.
We thank Dr. R. A. Rizza for providing insulin measurements, the
staff of the Mayo General Clinical Research Center for excellent assistance, and Douglas Hooper of the NIH for technical expertise.
20.
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