Vol. 275, Issue 6, R2081-R2084, December 1998
RAPID COMMUNICATION
Leptin acts in the rat hypothalamic paraventricular nucleus to
induce gastric mucosal damage
Pauline M.
Smith,
Veronique
Mollaret, and
Alastair V.
Ferguson
Department of Physiology, Queen's University, Kingston, Ontario,
Canada K7L 3N6
 |
ABSTRACT |
Leptin is produced and secreted by
adipocytes to regulate body weight homeostasis. Leptin acts centrally
to reduce weight by decreasing food intake and increasing energy
expenditure. The paraventricular nucleus (PVN) is a central nervous
system structure suggested as a site at which leptin acts to exert its
central effects. Leptin microinjection
(10
6 M, 0.5 µl) into the
PVN of urethan-anesthetized male Sprague-Dawley rats (150-300 g)
resulted in significant gastric damage (mean score = 1.75, n = 16). Damage scores were
significantly different than those observed after saline microinjection
into the PVN (mean score = 0.00, n = 5, P < 0.05), or leptin
microinjection into non-PVN sites (mean score = 0.33, n = 6, P < 0.05). There were no changes in
blood pressure (mean area under curve = 401.9 ± 224.2 mmHg * s,
n = 11, P > 0.05) or heart rate (mean area
under curve = 40.9 ± 25.9 beats, n = 10, P > 0.05) in response to
leptin microinjection into PVN. These results suggest that leptin acts on a functionally specific population of PVN neurons involved in the
control of gastrointestinal function.
gastric mucosa; ulceration
| |
INTRODUCTION |
LEPTIN, THE PROTEIN PRODUCT of the
ob/ob
gene (40), is produced and secreted by adipocytes (9) to regulate body
weight homeostasis. Leptin induces weight reduction by decreasing food intake and increasing energy expenditure (5, 13, 27, 36). It has been demonstrated that circulating leptin levels correlate with
total body lipid content (8) and also that chronic leptin infusion in
conscious animals results in an increase in mean arterial blood
pressure (BP) and heart rate (HR) (33).
Leptin receptor mutations (see Ref. 14) and modifications in the
ob/ob
gene which result in a lack of leptin production (40) have both been
shown to result in obesity, hyperinsulinemia, and
hypercorticosteronemia. In addition, leptin injections into the third
ventricle reduce body weight (32, 38), suggesting that leptin acts in
the central nervous system (CNS) to exert its effects.
One CNS structure which may be the site at which leptin acts to exert
its effects is the hypothalamic paraventricular nucleus (PVN), which
has been implicated in the regulation of body fluid homeostasis, stress
responses, thyroid hormone secretion, and feeding behavior (37).
Electrolytic lesion of the PVN has been shown to produce hyperphagia
and obesity in the rat (4). Administration of neuropeptide Y (35),
morphine (39), norepinephrine (22), and GABA receptor agonists (21)
stimulate feeding, whereas many gut peptides such as cholecystokinin,
somatostatin, oxytocin, and monoamines act to inhibit feeding (23).
Neurons in the PVN have also been implicated in controlling the
integrity of the gastric mucosa through a combined modulation of
pituitary hormones (16), acid secretion (17, 30), and gastric motility
(31). In accordance with such observations, electrical stimulation of
PVN has been shown to produce acute (within 60 min) gastric ulceration
(7) through activation of cholinergic fibers of the vagus nerves (18).
Support for the PVN as the central site of leptin action comes from in
situ hybridization studies, which have revealed leptin receptor mRNA in
regions of the hypothalamus, including the PVN (12, 19, 24, 25).
Immunohistochemistry has demonstrated leptin receptor immunoreactive
neurons in this region (11). In addition, leptin administration into
the third ventricle elevates c-Fos immunoreactivity within the PVN
(38), whereas intravenous leptin administration also induces c-Fos
activity in the PVN (6). In accordance with such observations, we
recently used in vitro whole cell patch-clamp techniques to show that
leptin application to rat PVN slices results in dose-related
depolarizations in the majority of PVN neurons tested (28). These data
suggest that leptin acts as a satiety signal to inhibit feeding as a
result of its ability to influence the excitability of PVN neurons (3).
In the present study we determined the effect of the administration of
leptin directly into PVN on both cardiovascular and gastric function.
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MATERIALS AND METHODS |
Urethan-anesthetized male Sprague-Dawley rats (150-300 g; fasted
for a minimum of 18 h before experiment) were fitted with femoral
arterial catheters (Intramedic PE-50) for the measurement of BP and HR
and placed on a feedback-controlled heating blanket to maintain body
temperature at 37°C. Animals were placed in a stereotaxic frame,
and the skull was exposed so that a small burr hole could be drilled
and a cannula electrode (tip diameter 150 µm) could be advanced into
the region of PVN according to the coordinates of Paxinos and Watson
(26).
The arterial catheter was connected to a pressure transducer, and the
signal was sent to a computer programmed for online data acquisition
and later offline analysis (Spike-2; Cambridge Electronic Design). Once
a stable baseline was achieved (minimum 5 min), a 0.5-µl bolus
microinjection of biologically active leptin fragment 22
56
[106 M, shown to
exert maximum single cell effects in PVN neurons (28)] or saline
(vehicle control) was delivered into the region of PVN and the effect
on BP and HR was assessed. The effects of each microinjection on BP and
HR were analyzed for 60 s before microinjection (baseline) until 300 s
after microinjection. The area under the curve (AUC; area between
baseline and each individual BP and HR response) was calculated for
normalized BP and HR changes for the 300 s after microinjection. At the
conclusion of the experiment, the animal was overdosed with anesthetic,
the abdominal cavity was opened, and the stomach was removed. The
stomach was cut along the greater curvature such that an observer,
unaware of the experimental protocol, could macroscopically assign a
gastric damage score according to a five-point scale [0 = normal
stomach lining (no visible damage), 1 = redness of gastric epithelial
tissue, 2 = 1-3 pin-sized dark red dots on gastric tissue, 3 = 4 or more small red dots of superficial epithelial damage, and 4 = extensive gastric damage, including regions of hemorrhage]
modified from Hierlihy et al. (15). The rats were then perfused through
the left ventricle of the heart with saline followed by 10% Formalin,
and the brain was removed and placed in Formalin overnight. The
following day, 100-µm coronal sections were cut through the region of
PVN using a vibratome. The sections were mounted and stained with
cresyl violet, and the anatomic location of the microinjection sites was verified at the light microscopic level by an observer unaware of
the experimental protocol.
Statistical analysis.
Animals were divided according to the anatomic location of the
microinjection site and the substance microinjected into PVN (leptin or
saline) and non-PVN (leptin) groups. Gastric damage scores were
statistically evaluated using a Kruskal-Wallis test for nonparametric
data followed by Dunn's multiple-comparison post hoc analysis. The
mean AUC was calculated for each group, and effects of microinjections
on BP and HR were assessed using one-sample
t-tests.
| |
RESULTS |
The animals used in the present study
(n = 36) were assigned to PVN (leptin,
n = 16; saline,
n = 5) and non-PVN (leptin,
n = 6) groups (see Fig.
1). The remaining animals were excluded from the present analysis because they could not be precisely classified as either PVN or non-PVN sites.
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Fig. 1.
A: anatomic location of a typical
paraventricular nucleus (PVN) microinjection site. V, ventricle.
B: location of microinjection sites
for all animals included in present study.  , PVN (leptin);  , PVN
(saline);  , non-PVN (leptin). Scale bar represents 500 µm.
|
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The majority of stomachs removed from animals in which leptin was
microinjected into PVN were assigned damage scores of 
1 (modal damage
score = 3). In contrast, leptin microinjection into non-PVN sites
resulted primarily in stomach damage scores of 0, (4 out of 5 stomachs)
with only one stomach receiving a damage score of 2. Saline
microinjection into the PVN did not induce any macroscopic damage in
any of the stomachs in this group (see Fig.
2).
Statistical analysis (Kruskal-Wallis test for nonparametric parametric
data) demonstrated that a significant difference existed between the gastric damage scores of the different groups
(P < 0.01). Post hoc analysis
(Dunn's multiple-comparison test) revealed that stomach damage scores
in response to leptin microinjection into PVN were significantly
greater than both saline microinjection into PVN
(P < 0.05) and leptin microinjection
into non-PVN sites (P < 0.05).
In contrast to the observed effects of leptin on the gastric mucosa,
there were no significant changes in BP (mean AUC = 401.9 ± 224.2 mmHg * s, n = 11, P > 0.05) or HR (mean AUC = 40.9 ± 25.9 beats, n = 10, P > 0.05) in response to
leptin microinjection into PVN (Fig. 3).
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Fig. 3.
Mean normalized blood pressure (BP;
top) and heart rate (HR,
bottom) response to a single
0.5-µl injection of 106
leptin into PVN. Arrow, time of microinjection; bpm, beats/min; AUC,
area under curve.
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DISCUSSION |
The results of the present study demonstrate that leptin administration
into the PVN of anesthetized rats causes significant gastric mucosal
damage. The magnitude of this effect is similar to the acute gastric
mucosal damage seen in response to 1 h of electrical stimulation within
the PVN (7). Although the specific mechanism(s) by which leptin acts to
induce gastric damage was not determined in this study, we speculate
that this ulceration may have occurred as a result of activation of
cholinergic fibers of the vagus nerves, as demonstrated previously in
the stimulation studies (18). PVN stimulation-induced damage requires
the integrity of the vagus nerves (7), whereas vagally induced gastric
mucosal damage requires simultaneous activation of both efferent and
afferent vagal fibers (15). It is clear, from previous studies, that gastric damage induced by vagal and PVN stimulation utilizes similar pathways which do not include projections to the pituitary (16) or
projections to sympathetic preganglionic neurons of the
intermediolateral cell column (18).
Our results showing that microinjection of leptin into the PVN is
without effect on BP or HR are in agreement with previous studies
demonstrating acute leptin infusion to be without effect on BP or HR
(20, 29, 34). In contrast, these cardiovascular parameters are elevated
after chronic leptin administration (33). The fact that leptin
microinjection was without effect on BP or HR suggests that the leptin
acts on a specific population of neurons within the PVN responsible for
control of the gastrointestinal system (rather than an overall
stimulatory effect in this area).
Similar microinjections of saline into the PVN and injections of leptin
into areas adjacent to the PVN did not produce significant acute
gastric mucosal damage. These findings lend support to the specificity
of action of leptin within the PVN. Further evidence that leptin acts
at specific leptin-sensitive PVN neurons comes from in vitro work
demonstrating dose-related depolarizations of PVN neurons to bath
application of leptin (28). The results of the present study clearly
demonstrate that leptin acts in the PVN to cause gastric mucosal
damage. Therefore, in addition to the demonstrated roles of leptin in
reproductive (1, 2), neuroendocrine (2), and hemopoietic (10)
functions, it may also act as a humoral factor involved in gastric ulceration.
Perspectives
Current literature implies that the anorexic consequences of leptin are
behavioral and specific. The results of the present study suggest an
alternative explanation. Our demonstration that leptin administration
into the PVN results in profound gastrointestinal damage suggests a
more general cause for malaise. Perhaps the anorexic effects of leptin
administration into the brain occur as a consequence of its damaging
effects on the gastrointestinal mucosa. The lack of appetite, exhibited
by animals after such treatment, may be due to the broad-based
pathological consequences caused by gastric ulceration.
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FOOTNOTES |
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. §1734 solely to indicate this fact.
Address reprint requests to A. V. Ferguson.
Received 30 June 1998; accepted in final form 17 September 1998.
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Abstract
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