White adipose tissue sympathetic nervous system
denervation increases fat pad mass and fat cell number
TIMOTHY G. YOUNGSTROM1,2 AND TIMOTHY J. BARTNESS1,2,3,4
Departments of 1Psychology and 3Biology; and 2Neuropsychology and Behavioral Neurosciences and
4Neurobiology Programs, Georgia State University, Atlanta, Georgia 30303
Siberian hamsters; photoperiod; axotomy; innervation; body
weight; food intake; autonomic nervous system; proliferation
THE LARGEST COMPARTMENT for energy storage in mammals is white adipose tissue (WAT). Fluctuations in the
size of lipid energy stores occur through alterations in
energy intake, as well as through changes in energy
expenditure. Contraction of the lipid stores is reflected
in WAT cellularity through decreases in fat cell size
(FCS), but not fat cell number (FCN). Decreases in FCS
can occur through increases in the breakdown of stored
triacylglycerols (i.e., lipolysis) and/or through decreases in the synthesis or uptake of free fatty acids
into white adipocytes (9). To expand lipid stores, however, WAT pad growth occurs through increases in the
accumulation of lipid in existing mature adipocytes
(i.e., hypertrophy) and/or through the genesis of new fat
cells (i.e., hyperplasia). We have a better understanding of the contraction of lipid stores through lipolysis
and the expansion of lipid stores through adipocyte
hypertrophy than we do for mechanisms involved in
increasing FCN. Identification of the factors that control fat cell proliferation in vivo is a fundamental
problem in the regulatory biology of energy balance and
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R1488
the pathophysiology of obesity. If adipocyte precursor
cells are harvested from intact WAT and grown in cell
culture, stimulation or inhibition of the proliferation
and differentiation of these cells by single or multiple
factors can be studied (e.g., Refs. 16, 17, 31, 32).
One factor that has received little attention in the
study of WAT proliferation is the catecholamines. Catecholamines could reach the adipocyte precursor cells in
vivo through the vasculature originating from the
adrenal medulla or, alternatively, from the sympathetic
nervous system (SNS) innervation of WAT (33); for
review see Ref. 1. Norepinephrine (NE), the catecholaminergic postganglionic neurotransmitter for the SNS
(20), has been shown to inhibit preadipocyte proliferation in stromal-vascular cells harvested from the inguinal WAT of laboratory rats and grown in cell culture
(15). Moreover, this inhibition of preadipocyte proliferation is blocked by the general b-adrenergic receptor
blocker propranolol, but not the a-adrenergic receptor
blocker phenoxybenzamine (15). This effect does not
appear to be a pharmacological curiosity in that WAT
adipocytes possess b-adrenergic receptors (for reviews
see Ref. 18) and, moreover, that surgical denervation of
the retroperitoneal WAT pads in laboratory rats triggers increases in FCN in vivo (7). These denervationinduced increases in FCN are detectable as early as 1
wk postaxotomy and are reflected as increases in DNA;
however, increases in the number of mature adipocytes
are seen 1 mo after denervation (7).
To test this possible relation between the SNS innervation of WAT and fat cell proliferation, we chose to
study Siberian hamsters (Phodopus sungorus sungorus). Siberian hamsters adjust the level of their body
fat stores with changes in the photoperiod (3, 30). Body
fat is at its natural peak following exposure to long
‘‘summerlike’’ days (LDs) and at its nadir following
exposure to short ‘‘winterlike’’ days (SDs). The SDinduced decreases in body fat are not uniform, with the
more internally located WAT pads [e.g., epididymal
WAT (EWAT)] showing a greater relative initial depletion of lipid than the more externally located subcutaneous inguinal WAT (IWAT) pads (3). This fat pad-specific
pattern of decreased WAT mass appears to be related to
differential SNS drives on the fat pads causing differential rates of lipid depletion from the adipocytes. Specifically, we previously demonstrated that there are relatively separate neurologies associated with the
postganglionic SNS innervation of these pads, as revealed through anterograde and retrograde tracttracing studies (33). In addition, NE turnover was
greater in the EWAT compared with the IWAT pad
during this period of rapidly declining EWAT pad mass
(33). In subsequent weeks of SD exposure the elevated
0363-6119/98 $5.00 Copyright r 1998 the American Physiological Society
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Youngstrom, Timothy G., and Timothy J. Bartness.
White adipose tissue sympathetic nervous system denervation increases fat pad mass and fat cell number. Am. J.
Physiol. 275 (Regulatory Integrative Comp. Physiol. 44):
R1488–R1493, 1998.—The sympathetic nervous system (SNS)
drive on white adipose tissue (WAT) was varied to test its
effects on fat cell number (FCN) under conditions in which
lipolysis would be minimized and therefore partially separable from SNS trophic effects. The inguinal subcutaneous
WAT (IWAT) pad of Siberian hamsters was chosen because 1)
it is innervated by the SNS, 2) short day (SD) exposure
increases its SNS drive (,250%) without proportionately
increasing lipolysis, and 3) surgical denervation eliminates
its SNS innervation. IWAT was either unilaterally surgically
or sham denervated, while the contralateral pad was left
intact. In long day- or SD-exposed hamsters (11 wk), IWAT
denervation decreased norepinephrine content (,80%) and
increased fat pad mass (,200%) and FCN (,250 and ,180%,
respectively) compared with the contralateral intact pads,
but did not affect fat cell size (FCS). The denervation-induced
increased FCN in SDs occurred despite naturally occurring
decreased food intake. SDs decreased IWAT FCS regardless of
the surgical treatment. These results support an important
role of WAT SNS innervation in the control of FCN in vivo.
DENERVATION OF WHITE ADIPOSE TISSUE
NE turnover rate in EWAT declines while the rate in
IWAT increases slightly corresponding to the decline in
IWAT mass. Therefore, the purpose of the present
investigation was to test the role of varied SNS drive on
the consequent changes in IWAT adipose tissue cellularity, especially alterations in FCN. This was accomplished by varying SNS drive (NE turnover) through
housing Siberian hamsters in LDs (relatively low NE
turnover) or SDs (relatively higher NE turnover) and
by attempting to eliminate SNS drive through surgical
denervation of IWAT.
METHODS
associated nerves and blood vessels. After surgery the sham
or denervated pad was replaced against the outside abdominal wall and rinsed with physiological saline, the incision was
closed with wound clips, and nitrofurozone powder was
applied to the wound surface.
Body mass and food intake. Body mass and food intakes
were measured weekly to the nearest 0.01 g, the latter
corrected for spillage and pouching.
Testes mass. Paired testes mass was measured as an
indicator of the responsiveness of the SD-housed hamsters to
the winterlike photoperiod. SD-housed hamsters with paired
testes masses greater than 0.3 g were considered photoinsensitive (24), their previous data were discarded, and they were
dropped from further measurements and analysis.
WAT measurements. Animals were killed by decapitation
and the IWAT pads were removed, chilled on wet ice, blotted
dry, and weighed. The samples were then prepared for
measurements of cellularity and NE content.
WAT cellularity. Single samples of the finely minced and
mixed IWAT pads were processed for automated cell number
determination according to the osmium fixation method of
Hirsch and Gallian (13). Triplicate samples were processed
for lipid content (ng triglyceride/adipocyte) using the procedure of Folch et al. (12).
WAT NE content. The SNS denervation was verified by
measuring NE content by HPLC with electrochemical detection. An additional sample of the IWAT pad was snap frozen
in liquid nitrogen and subsequently stored at 280°C until
assayed for NE content. NE content was measured according
to our previously described method (33). Briefly, samples were
thawed, weighed, sonicated in 350 µl 0.2 M perchloric acid
containing 1 µg/ml ascorbic acid (PCA/AA) and 350 µl dihydroxybenzoacetic acid [1028 (10 pg/ml of PCA/AA)] as an
internal standard and then centrifuged. A sample of the
infranatant was removed and processed for protein content
according to the method of Lowry et al. (21). Catecholamines
were extracted from the remainder of the infranatant using
alumina. The extracted samples were assayed using an ESA
(Bedford, MA) HPLC system with electrochemical detection
(guard cell: 135 mV; cell 1: 110 mV; cell 2: 230 mV). The
mobile phase consisted of 0.05 M citric acid (pH 3.0) containing 2% acetonitrile (wt/vol), EDTA-disodium salt (0.025 mM),
and sodium duodecylsulfate (0.07 mM). Standard solutions
[1022 (10 mg/ml)] containing known concentrations of catecholamines and their metabolites were prepared and aliquots
diluted to 1028 and 1029 in PCA/AA and run on the HPLC
before and after sets of the unknowns. The results were
analyzed offline and are expressed as micrograms NE per
gram protein.
Statistics. Weekly body mass and food intakes were compared using the analysis of variance with repeated measures
program [BMDP program 2V (Los Angeles, CA)]. Testes
masses were compared using paired t-tests. Left and right
measurements obtained in the same animals were treated as
paired samples and analyzed using program 2V of BMDP.
Comparisons between groups were made using Duncan’s new
multiple-range test. All differences were considered statistically different at P , 0.05. Exact probability and test values
were omitted for simplification and clarity of the presentation
of the results.
RESULTS
There were no lateralization effects of the denervation or of the sham surgery on IWAT mass or cellularity.
Therefore, the left and right side groups were collapsed
within a surgical condition for all measures, resulting
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Animals and housing. Adult male Siberian hamsters (n 5
175) were obtained from our breeding colony established in
1988 with stock donated by Dr. Bruce Goldman (University of
Connecticut). Second-generation wild-trapped hamsters donated by Dr. Katherine Wynne-Edwards (Queen’s University,
Kingston, ON) were introduced into the colony in 1990.
Animals were born and raised initially in LDs (16:8-h lightdark cycle; lights on at 0200) and group housed 10–12/cage in
the vivarium until they were 2–3 mo of age. The vivarium
was kept at a constant room temperature (23 6 1°C) throughout the experiment, and the animals had ad libitum access to
Purina Rodent Chow (no. 5001) and tap water. Hamsters
were individually housed with corn cob bedding and had
nestlets available.
Experimental design. Animals were randomly divided into
eight groups so that the mean body masses and variance were
the same among the groups. Because we have noticed that the
left IWAT pad tends to be larger than the right (unpublished
observations), we divided each surgical group into left and
right sham or denervated groups. Thus the experimental
design was a 2 3 2 3 2 design (photoperiod 3 surgery 3 side
of surgery), with the following numbers of hamsters in each
group at the end of the experiment: LD-sham-left side, n 5 12;
LD-sham-right side, n 5 12; LD-denervated-left side, n 5 15;
LD-denervated-right side, n 5 15; SD-sham-left side, n 5 13;
SD-sham-right side, n 5 15; SD-denervated-left side, n 5 19;
SD-denervated-right side, n 5 15. Care, housing and experimental treatments were approved by the Georgia State
University Institutional Animal Care and Use Committee
and conducted in accordance with Public Health Service and
US Department of Agriculture guidelines.
Surgical procedures. Animals were anesthetized with pentobarbital sodium (,50 mg/kg), the fur was removed from the
selected hindquarter of the animal, and the area was wiped
with 95% ethanol-soaked gauze. An incision was made dorsally on the skin from a point near the tail and lateral to the
spinal column. The incision continued rostrally along the
dorsum adjacent to the spinal column to a point just rostral to
the hind limb, then laterally and ventrally to a point ,2 cm
from the ventral midline. Finally, the incision extended
caudally to a point near the tail. Care was taken with the
depth of the incision to avoid the underlying blood vessels and
musculature. The IWAT pad was separated from the abdominal wall and overlying skin by blunt dissection, keeping
intact the major blood vessels leading into or through the pad.
Nerves identified at 34 magnification as terminating in the
pad were cut in two or more locations. Throughout the
surgery, the pad was kept moist with 0.15 M NaCl-soaked
gauze. The sham surgery was as above except the skin was
spread so that the lateral edges of the target IWAT could be
seen. The edges of the fat pad were then lifted with forceps,
taking care to avoid stretching or tearing of the pad and
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R1490
DENERVATION OF WHITE ADIPOSE TISSUE
Fig. 1. Weekly body mass (mean 6 SE). Presurgical body masses
(Pre-Surg) were balanced for mass and variance at the beginning of
the experiment. Long day (LD)-housed groups were significantly
heavier than short day (SD)-housed groups after 4 wk. Den, denervated; Sham, sham denervated.
Fig. 2. Weekly food intake (mean 6 SE).
photoperiod, there was no significant difference in food
intake regardless of surgical condition.
IWAT NE content. NE content only was significantly
decreased in denervated pads (P values , 0.05; Fig. 3),
although a small, but not statistically significant, decrease was apparent in IWAT pads from both shamdenervated groups (Fig. 3). There were no effects of
photoperiod on NE content.
IWAT mass. IWAT pad mass was increased for both
denervated and sham-denervated groups compared
with their contralateral nonoperated pads in both
photoperiods (P values , 0.05; Fig. 4). The increased
mass of the denervated pads in both photoperiods,
however, was greater than the IWAT mass of the
sham-denervated pads in the same photoperiod (LDexposed denervated group ,118% of their sham counterparts; SD-exposed denervated group ,127% of their
Fig. 3. Norepinephrine (NE) content (mean 6 SE) was generally
greater in unoperated pads, intermediate in sham-operated pads,
and significantly less in denervated pads from animals in either
photoperiod. Bars with the same letter are not significantly different
from one another (P values , 0.05).
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in a 2 3 2 (photoperiod 3 surgery) design with one
repeated measure (surgical condition). All further statistical analysis was based on this model.
Testes and body mass and food intake. Testes masses
were not significantly different for groups housed in the
same photoperiod, but paired testes masses for each
LD-housed group were significantly larger than for
each SD-housed group (mean 6 SE: LD-denervated,
0.9377 6 0.0422 g; LD-sham, 0.8753 6 0.0403 g;
SD-denervated, 0.0776 6 0.0080 g; SD-sham, 0.0677 6
0.0066 g).
LD-housed hamsters showed typical body mass increases following surgery, regardless of the surgical
condition (Fig. 1). Among SD-housed groups, body mass
declined from their presurgery baseline. There were no
significant differences in body mass within each photoperiod when the side or type of surgical procedure was
considered. Compared with their LD-housed counterparts, SD-housed hamsters weighed significantly less
beginning with week 4 and throughout the remainder of
the experiment.
As expected, food intake was decreased in SDs compared with LDs overall (P , 0.05; Fig. 2), but only was
significantly decreased consistently from week 3 to
the end of the experiment for the SD-sham group (P
values , 0.05; Fig. 2). Although SD-denervated hamsters generally had significantly decreased food intakes
compared with both LD-housed groups, there were
occasionally weeks subsequent to week 3 where food
intakes were not different among either the LD group
or the SD-denervated group (Fig. 2). Within each
DENERVATION OF WHITE ADIPOSE TISSUE
sham counterparts; P values , 0.05; Fig. 4). In addition, the denervated and sham-denervated IWAT pads
from LD-housed hamsters were larger than their respective same-surgery pads from their SD-housed counterparts (P values , 0.05; Fig. 4). As expected, nonoperated IWAT pads were smaller in SD- than in LD-housed
hamsters (P values , 0.05; Fig. 4). There was no
significant surgery 3 photoperiod interaction on IWAT
mass.
IWAT cellularity. FCN was significantly greater from
fat pads that were either denervated or sham denervated compared with their nonoperated contralateral
IWAT pads in both photoperiods (P values , 0.05; Fig.
5). However, denervation produced a greater hyperplasia than did sham denervation in LDs, but not in SDs
(P , 0.05; Fig. 5).
FCS (i.e., volume) was larger for all LD pads (except
for the LD-denervated pads) compared with the pads
from all SD-housed groups (P values , 0.05; Fig. 6).
Within a photoperiod, there was no difference in FCS
for all groups (Fig. 6).
Fig. 5. Fat cell number (mean 6 SE) was always greater in operated
pad compared with contralateral pad and was greatest in LD-housed
denervated animals. There was no difference in unoperated pads
from LD- or SD-housed animals. Bars with the same letter are not
significantly different from one another (P values , 0.05).
because application of NE to stromal-vascular cells
harvested from WAT inhibits their unprovoked proliferation in vitro (15). This assertion also is supported by
the intermediate increase in fat pad mass and FCN of
sham-denervated IWAT pads from hamsters in both
photoperiods in the present experiment. Specifically,
these changes in fat pad mass and cellularity are
understandable in sham-denervated pads given that
the NE content of the sham-operated pads was intermediate between that of the neurally intact and denervated pads (,40% decrease in NE content). These
latter data suggest that we may have been too rigorous
DISCUSSION
The results of the present experiment support the
view that the SNS innervation of WAT plays a role in
fat pad mass and FCN. Specifically, surgical denervation of IWAT resulted in increased fat pad mass in both
LDs and SDs compared with neurally intact pads and
did not affect FCS. Increases in FCN after surgical
denervation of retroperitoneal WAT in laboratory rats
was reported while the present study was in progress
(7). Thus it appears that the SNS inhibits fat cell
proliferation in vivo (present results and Ref. 33) and,
moreover, that it is NE that is the inhibiting stimulus
Fig. 6. IWAT fat cell size (FCS; volume in pl; mean 6 SE) was
generally greater in LD-housed animals compared with SD-housed
animals. FCS in the denervated pad of LD-housed animals was not
significantly different from any group. Bars with the same letter are
not significantly different from one another (P values , 0.05).
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Fig. 4. Inguinal white adipose tissue (IWAT) pad mass (mean 6 SE)
was consistently and significantly greater on operated side compared
with its contralateral counterpart. IWAT of LD-housed animals was
heavier than that of the same group housed in SDs. Bars with the
same letter are not significantly different from one another (P
values , 0.05).
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R1492
DENERVATION OF WHITE ADIPOSE TISSUE
Perspectives
Factors affecting hyperplasia of adipocytes in WAT
pads have received considerable speculation, but surprisingly little empirical elucidation. Several factors
have been shown to increase WAT cell proliferation in
vitro; however, a problem associated with such preparations is the ready ability of plated stromal-vascular
cells to proliferate and then differentiate into mature
adipocytes in serum-free media. The results of the
present experiment, and those of others (7), suggest
that the SNS can inhibit fat cell proliferation in WAT.
This finding, and the ability of NE to inhibit WAT
proliferation in vitro (15), suggests that the SNS can
inhibit proliferation in vivo, most likely in response to
the level of SNS drive on the fat pads. Therefore,
although paracrine, autocrine, or humoral factors may
stimulate WAT cell proliferation, it may be that a
predominant regulator of FCN may be the SNS drive on
WAT. This view is supported by the findings and
hypotheses of Bray (6) that suggest that most obesities
are associated with deficits in the SNS activity. Although this view has focused on the diminished SNS
drive on brown adipose tissue in the obese state, rather
than on WAT, it may be that the essence of this
hypothesis is supported for the effects of SNS drive on
WAT.
Using a transneuronal viral retrograde tract-tracer
(Bartha’s K strain of the pseudorabies virus) to define
the SNS outflow from brain to WAT, we found that one
of the most heavily infected central nervous system
areas was the parvicellular region of the paraventricular nucleus of the hypothalamus (PVN). This area is
thought to be part of descending autonomic nervous
system projections to the spinal cord preganglionic
neurons of the SNS (14, 22, 27, 28). We have found in
Siberian hamsters (25), and others have found in
laboratory rats (10), that bilateral disruption of these
descending projections from the PVN result in increases in WAT FCN. These data suggest that some of
the origins of the SNS inhibition of WAT FCN include
the PVN and implicate this response in the developing
obesity produced by lesions of the PVN (e.g., Refs. 2, 10,
19) in addition to the effects of these lesions on food
intake (e.g., Refs. 10, 19, 29). Indeed, laboratory rats
with microknife cuts that sever these descending projections from the PVN and that also are pair body mass
matched to controls show increases in retroperitoneal
WAT pad FCN (10). Thus the obesity-promoting effects
of PVN lesions may have disinhibition of the SNS drive
on WAT as part of their underlying mechanism.
Finally, there appears to be an interesting relation
between the density of the innervation of WAT by the
SNS and the proliferation seen in the pads in vivo. For
example, in a preliminary report (26), it was found
using a combination of histofluorescence and confocal
microscopy that, in addition to observations of catecholaminergic neural fluorescence in direct contact with
white adipocytes, the mesenteric WAT had the densest
NE innervation, whereas IWAT had the least dense NE
innervation (26). In a model of spontaneous obesity
(aging Wistar rats; Ref. 8), the increases in mesenteric
WAT pad mass that occur with aging are almost
exclusively due to increases in FCS, whereas the increases in IWAT pad mass with age are largely accountable by the increases in FCN. Therefore, it may be that
the SNS innervation of WAT plays an important role in
the regulation of FCN in this tissue.
This research was supported in part by National Institute of
Mental Health Research Scientist Development Award KO2-MH00841 and National Institute of Diabetes and Digestive and Kidney
Diseases Grant RO1-DK-35254 to T. J. Bartness.
Address for reprint requests: T. J. Bartness, Departments of
Psychology and Biology, and Neuropsychology and Behavioral Neurosciences and Neurobiology Programs, Georgia State Univ, Atlanta,
GA 30303.
Received 26 May 1998; accepted in final form 21 July 1998.
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DENERVATION OF WHITE ADIPOSE TISSUE
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