Endocrinology Vol. 141, No. 6 1951-1954
Copyright © 2000 by The Endocrine Society
Blood to Brain Transfer of Leptin in Normal and Interleukin-1ß-Treated Male Rats1
Seymour Reichlin,
GuanJie Chen and
Margery Nicolson
Department of Medicine, University of Arizona College of Medicine
(S.R., G.C.), Tucson, Arizona 85724-5099; and Amgen, Inc.
(M.N.), Thousand Oaks, California 91320-1799
Address all correspondence and requests for reprints to: Dr. Seymour Reichlin, Department of Medicine, University of Arizona College of Medicine, 1501 North Campbell Avenue, P.O. Box 245099, Tucson, Arizona 85724-5099. E-mail: reichlin{at}arizona.edu
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Abstract
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To test the hypothesis that leptin was secreted from the brain into the
blood of the rat, its concentration was measured in the superior
sagittal sinus (SSS; which drains the cerebral cortex) and aortic blood
of normal fasting male rats and rats that had been treated with iv or
intracerebroventricular (icv) injections of interleukin-1ß (IL-1ß;
100 ng), a cytokine previously shown to induce peripheral leptin
secretion. Plasma levels of leptin in SSS were slightly, but
significantly, less than those in the aorta in control, saline-injected
rats (0.99 ± 0.07 vs. 1.19 ± 0.10 ng/ml;
n = 15; P = 0.03) and in rats injected with
human IL-1ß iv (1.56 ± 0.12 vs. 1.92 ±
0.15 ng/ml; n = 23; P = 0.004) or icv
(1.38 ± 0.11 vs. 1.57 ± 0.12 ng/ml; n =
23; P = 0.008). IL-1ß by either the iv or icv
route significantly increased leptin levels in the aorta [1.19 ±
0.10 vs. 1.92 ± 0.15 ng/ml (P
= 0.0002) and 1.19 ± 0.10 vs. 1.57 ± 0.12
ng/ml (P = 0.022), respectively]. SSS levels of
leptin were also raised after iv or icv injection
(P = 0.0002 and P = 0.0053,
respectively). These findings demonstrate a net uptake of leptin by the
cerebral cortex from peripheral blood in both normal and
IL-1ß-treated animals and show that peripheral blood levels of leptin
are increased by IL-1ß whether administered icv or iv.
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Introduction
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ESLER AND COLLEAGUES have reported that in
a group of fasting obese men the concentration of immunoreactive leptin
was significantly greater in internal jugular blood than in brachial
artery blood (1), a finding that was confirmed in lean and obese women
and obese men, but not in lean men (2). These observations led them to
hypothesize that the brain is a source of leptin circulating in the
blood. At the time that this work was performed, the idea that leptin
could be synthesized in the brain was unanticipated (3). In the first
report in which leptin was identified in mouse adipose tissue by
cloning methods, a survey of other potential tissue sites of synthesis
detected no leptin messenger RNA (mRNA) in brain (4), an observation
confirmed in the rat (5, 6) and sheep (7). However, a relatively small
amount of leptin mRNA could well escape detection when diluted with a
large amount of whole brain mRNA; no studies of leptin mRNA in human
brain have been published. More recently, Li and collaborators reported
that a population of neurons in the rat hypothalamus and brainstem
contained immunoreactive leptin (8), and Morash (9, 10) and Wilkinson
(11) and their respective collaborators found a number of sites in
brain and anterior pituitary that contained immunoreactive leptin and
leptin mRNA.
To test the hypothesis that the brain secretes leptin, we measured
levels of this peptide in the superior sagittal sinus (SSS) and
abdominal aorta of rats using methods previously applied to the study
of brain secretion of inflammatory cytokines (12, 13, 14, 15). The SSS is the
main drainage route of the cerebral cortex (16), receiving
approximately 30% of total brain venous outflow (17) and somewhat more
than half of the cerebrospinal fluid drainage (18). Sagittal sinus
blood, unlike jugular venous blood, does not drain the pituitary
vascular bed, an important difference for this study because the
anterior pituitary of rats (10) and humans (19) have recently been
shown to express leptin in several cell types. In addition to measuring
leptin levels in the basal, nonfasting state, measurements were made in
rats that had been injected with interleukin-1ß (IL-1ß) by the iv
or intracerebroventricular (icv) route. IL-1ß was used as a possible
inducer of brain leptin, because several proinflammatory cytokines,
including IL-1ß and bacterial endotoxin, have been reported to
increase blood levels of leptin in laboratory animals (20, 21, 22) and to
increase the production of leptin by adipose tissue (20, 21). Moreover,
leptin levels are reported to be elevated in patients with sepsis (23)
and to be induced by administration of IL-1
in normal individuals
(24). We postulated that if leptin was secreted by the brain, then
IL-1ß might induce its synthesis and cause an increased SSS to aortic
plasma gradient of leptin.
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Materials and Methods
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Male Sprague Dawley rats (Taconic Farms, Inc.,
Germantown, NY), 250–360 g BW, were maintained at a room temperature
of 20.0–23.8 C, fed rat chow (Harlan Teklad Diet 700, 4% protein,
Harlan Bioproducts for Science, Inc., Indianapolis,
IN) ad libitum, were not fasted, and were tested between
0800–1400 h on the experimental day. They were prepared 5–7 days
before intraventricular injection by placing cerebral guide cannulas
under stereotaxic control using coordinates and methods previously
described (12, 13, 14, 15). On the day of the experiment, rats were
anaesthetized with sodium pentobarbital (35–50 mg/kg, ip) and placed
in a stereotaxic headholder, and an inner cannula was inserted into the
lateral ventricle. Rats were given either normal pyrogen-free saline or
human IL-1ß (catalogue no. 40042, Collaborative Biochemical Products,
Bedford, MA; 100 ng in a total volume of 10 µl) over a 2-min period.
To minimize leakage, the tubing through which the test substance was
administered was sealed by compression with a hemostat, and the inner
cannula was not removed until the end of the experiment, 2 h
later. This time interval was chosen because it is the time of peak
elevation of aortic IL-6 after icv injection of IL-1 (12, 15). Animals
were allowed to recover from anesthesia. At the end of 2 h, they
were reanesthetized and replaced in the stereotaxic headholder, the
superior sagittal sinus was exposed using a dental drill, and 1 ml
blood was taken from the sinus with a 26-gauge hypodermic needle and
syringe. Blood was then drawn from the abdominal aorta, collected in
heparinized tubes, spun down at 5000 rpm, separated, and frozen until
assayed for leptin by a previously reported method (25). Each set of
paired samples was measured in the same assay. In addition, the effects
of iv injection of IL-1ß on leptin levels were compared with
responses to icv injection. For parenteral injection, IL-1ß (100 ng)
in 100 µl pyrogen-free saline was injected under pentobarbital
anesthesia into the right atrium by way of a catheter threaded into the
external jugular vein.
Statistical analysis of SSS and aortic plasma levels was performed
using Fisher’s t test for paired samples, and comparison of
control and IL-1ß-treated animals was made using Fisher’s two-tailed
t test, assuming equal variance.
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Results
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Paired sagittal sinus and aortic plasma levels were measured in
groups of rats given saline iv (n = 8) and icv (n = 7; Fig 1
). The mean sagittal sinus plasma value
was 0.99 ± 0.07 compared with 1.19 ± 0.15 ng/ml for aortic
blood (by Fisher’s paired t test, P =
0.03). For 23 paired samples of rats injected iv with IL-1ß, the mean
sagittal sinus plasma value was also less than that for aorta
(1.56 ± 0.12 vs. 1.92 ± 0.15 ng/ml;
P = 0.004). In 23 paired samples from rats that had
been injected icv with IL-1ß, mean leptin levels in SSS were also
slightly, but significantly, lower than those in aortic blood
(1.38 ± 0.11 vs. 1.57 ± 0.12 ng/ml;
P = 0.008).
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Figure 1. Plasma leptin concentration in SSS and aorta in
normal rats injected with normal saline either iv or icv and in rats
injected with human IL-1ß (100 ng) either iv or icv. Although the
differences between aortic and SSS blood are small ( 18%), when
analyzed by pair comparison, they are significant: saline control,
0.99 ± 0.07 vs. 1.19 ± 0.15 ng/ml
(P = 0.03); iv IL-1ß, 1.56 ± 0.12
vs. 1.92 ± 0.015 ng/ml (P =
0.004); and icv IL-1ß, 1.38 ± 0.11 vs. 1.57
± 0.12 ng/ml (P = 0.008). The effects of IL-1ß
to raise aortic levels of leptin are also significant whether
administered icv or iv: after iv administration, 1.19 ± 0.10
vs. 1.92 ± 0.15 (P = 0.0002);
and after icv injection, 1.19 ± 0.10 vs. 1.57
± 0.12 (P = 0.022). Levels in SSS are also
significantly increased by IL-1ß either iv (P =
0.0002) or icv (P = 0.005).
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IL-1ß induced a statistically significant increase in aortic plasma
leptin values in rats injected either iv or icv [iv, 1.19 ± 0.10
vs. 1.92 ± 0.15 ng/ml (P = 0.0002);
icv, 1.19 ± 0.10 vs. 1.57 ± 0.12 ng/ml
(P = 0.022)]. Leptin-induced differences in SSS plasma
were also observed [control vs. iv IL-1ß, 0.99 ±
0.07 vs. 1.56 ± 0.12 ng ng/ml (P =
0.0002); control vs. icv IL-1ß, 0.99 ± 0.07
vs. 1.38 ± 0.11 ng/ml (P =
0.005)].
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Discussion
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In this study net secretion of leptin into the blood from the
cerebral cortex could not be demonstrated, a finding that differs from
observations reported previously for jugular venous-arterial
differences in obese men and lean and obese women (1, 2). These studies
cannot be compared directly because there are a number of important
methodological differences between the work reported here and that
conducted previously in humans. Work in humans used internal jugular
vein sampling, which would include blood from the whole brain and the
pituitary gland, a known site of leptin expression (10, 19). Jugular
venous blood, even if obtained from the base of the skull, conceivably
could contain an uncertain amount of blood refluxed from nonbrain
structures in the head. In the present studies, the venous drainage
source is almost completely from the cerebral cortex and includes a
component derived from cerebrospinal fluid that enters the SSS from the
subarachnoid space. Other differences between the current studies, on
the one hand, and those of Esler et al. (1) and Wiesner
et al. (2), on the other, are that theirs were of humans in
a fasting state (not ad libitum-fed rats) and that they
noted differences only in women and obese men. They found no increase
in jugular venous-aortic gradient in lean men (who may be more
comparable to the normal weight male rats used in this experiment).
In contrast to the human studies, the mean concentration of leptin in
SSS was lower that that in aorta. The differences between SSS and
aortic blood were relatively small in the three experimental states
studied (18.0%, 18.8%, and 12.1%, respectively) and would not have
been statistically significant unless paired comparisons had been made
in a large number of test animals. Nevertheless, this finding is
evidence for a net uptake of leptin from blood to brain. Specific,
carrier-mediated leptin uptake into the brain and its bulk transfer
from brain to blood have previously been established using
radioiodinated tracer in the mouse (26), but this approach does not
show the difference in the concentration of endogenous leptin entering
and leaving the brain under steady state conditions. Transport of
leptin from blood to brain is attributed to receptors in the choroid
plexus (4) and in brain microvessels (27, 28).
The finding that the concentration of leptin in blood leaving the
cerebral cortex is less than that entering the brain from blood does
not exclude the possibility that leptin may arise from within the
brain, as reported by several workers (8, 9, 10, 11), and be secreted by
brain. However, the absolute amount of leptin that could be synthesized
in the brain is likely to be small, because leptin mRNA was not
detected in mouse (4), rat (5, 6), or sheep (7) brain by hybridization
analysis, and the successful demonstration of leptin mRNA by Morash
et al. (10) used PCR amplification. Further evidence that
appreciable amounts of leptin are not synthesized in the brain is the
demonstration by several groups that the leptin concentration in the
cerebrospinal fluid is much lower than that in peripheral blood and
that in lean individuals, cerebrospinal fluid leptin correlates
significantly with the serum leptin concentration. In nonobese humans
the ratio of cerebrospinal fluid to serum leptin concentration ranged
between 0.023–0.0068, depending on blood levels (29, 30, 31, 32). In normal
rats the mean cerebrospinal fluid/serum leptin ratio was 0.031, and in
obese rats lacking the leptin receptor ratios were 1/10th of those in
normal animals (0.0029) (25).
Injection of IL-1ß by either iv or icv routes induced a significant
increase in leptin levels in peripheral blood obtained from either the
aorta or the SSS. Stimulation of leptin secretion by IL-1 or LPS has
been well documented in several species of laboratory animals and in
humans. For example, the blood leptin concentration reportedly is
increased after parenteral injection of LPS and/or IL-1ß or tumor
necrosis factor- in hamsters (20) and mice (21, 22). In humans, iv
IL-1 induces the appearance in blood of leptin (23), and leptin
levels are elevated in septic patients who also have elevated blood
levels of IL-6 (24).
Mechanisms by which icv injection of IL-1ß induced an increased
concentration of leptin in aortic and SSS plasma should be considered.
That icv IL-1ß induced a peripheral response does not necessarily
mean that leptin has been induced in the brain. On the basis of studies
of the effects of IL-1ß on IL-6 in peripheral blood of the rat (15),
it appears to be more likely that the injected IL-1ß has passed from
the brain into peripheral blood and that the effect on leptin secretion
is due to its action on peripheral fat depots. As demonstrated
previously (15), radioiodinated IL-1ß appears in peripheral blood
within 5 min after icv injection of the labeled cytokine, and 70% of
the dose injected icv enters peripheral blood within 2 h of
injection. Blood levels of IL-1ß after icv injection exceed those
observed after iv injection by 60 min after injection.
It is also possible that the elevation of leptin levels induced by
injection of IL-1ß icv (or iv) is due not only to a direct action of
IL-1ß on fat tissue, but also to the well documented cytokine-induced
pituitary-adrenal activation that is observed after IL-1ß
administration (33). That glucocorticoids can induce leptin is shown by
the findings that leptin levels are elevated in Cushing’s disease (34, 35), that administered glucocorticoids raise blood levels of leptin in
the human (35), and that adipocytes treated with glucocorticoids
secrete larger amounts of leptin and express greater amounts of leptin
mRNA (36, 37).
Taken together, the data presented here show a net cerebral cortical
uptake of leptin from the blood, and that peripheral plasma levels of
leptin are increased by either iv or icv injection of IL-1ß.
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Footnotes
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1 This work was supported by NIH Grant 16894. 
Received October 27, 1999.
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Abstract
Introduction
