Download Peptides that regulate food intake: orexin gene expression is increased during states of hypertriglyceridemia

Am J Physiol Regul Integr Comp Physiol 284: R1454–R1465, 2003.
First published January 30, 2003; 10.1152/ajpregu.00286.2002.
special topic
Peptides that Regulate Food Intake
Orexin gene expression is increased during states
of hypertriglyceridemia
Katherine E. Wortley,* Guo-Qing Chang,* Zoya Davydova, and Sarah F. Leibowitz
The Rockefeller University, New York, New York 10021
Submitted 20 May 2002; accepted in final form 17 January 2003
Wortley, Katherine E., Guo-Qing Chang, Zoya Davydova, and Sarah F. Leibowitz. Orexin gene expression is
increased during states of hypertriglyceridemia. Am J
Physiol Regul Integr Comp Physiol 284: R1454–R1465, 2003.
First published January 30, 2003; 10.1152/ajpregu.00286.
2002.—Previous reports implicate the orexins in eating and
body weight regulation. This study investigated possible
functional relationships between hypothalamic orexins and
circulating hormones or metabolites. In situ hybridization
and quantitative PCR were used to examine orexin expression in the perifornical hypothalamus (PF) of rats and mice
on diets varying in fat content and with differential propensity toward obesity. The results showed that orexin gene
expression was stimulated by a high-fat diet in close association with elevated triglyceride levels, suggesting a functional relationship between these measures. Results obtained in obesity-prone rats and mice revealed a similar
increase in orexin in close relation to triglycerides. A direct
test of this orexin-triglyceride link was performed with Intralipid, which increased PF orexin expression along with
circulating triglycerides. Whereas PF galanin is similarly
stimulated by dietary fat, double-labeling immunofluorescence studies showed that orexin and galanin neurons are
anatomically distinct. This evidence suggests that the orexins, like galanin, are “fat-responsive” peptides that respond
to circulating lipids.
(hypocretin) peptides A and B derive from a
common precursor protein, prepro-orexin, which is expressed specifically in the lateral hypothalamus (LH),
most densely within the perifornical region (PF) (15,
16). In addition to their role in the control of arousal
and sleep-wake cycle (34), there is evidence that the
orexins are involved in body weight regulation. Food
deprivation stimulates orexin gene expression (11, 48),
and injection of orexin A causes a small enhancement
of food intake in rats (32, 48, 52). Conversely, administration of an orexin receptor 1 antagonist (30, 31) or
anti-orexin antibody (61) inhibits feeding behavior.
That these pharmacological results reflect a physiological function is supported by a recent study showing
that genetic ablation of orexin neurons in mice causes
hypophagia (29). However, these mice also develop
late-onset obesity, indicating that the orexins may
have additional effects beyond the stimulation of food
intake. Indeed, other studies indicate that orexin peptides can increase metabolic rate and sympathetic nervous system (SNS) activity (3, 19, 49, 51) and have
variable effects on lipid utilization depending on time
of day (38).
Orexin neurons express leptin receptor immunoreactivity (27), and leptin administration downregulates
orexin A levels in this area (6, 37). Whereas these
findings suggest an inhibitory influence of leptin on the
orexins, other studies show that orexin gene expression
is downregulated in ob/ob mice and fa/fa rats, both of
which have disturbed leptin signaling (7, 20, 51, 62).
There are few studies that have actually tested the
relationship between leptin and orexin peptides under
physiological conditions. Two reports that exist fail to
reveal a change in orexin expression in feeding paradigms associated with elevated leptin (11, 53), suggesting that this hormone may not be a key physiological
regulator of the orexins.
Other evidence implicates glucose and insulin in the
control of the orexins. Because the LH contains neurons that are stimulated by a decline in glucose levels
(44), it is not surprising that a subset of orexin neurons
concentrated in this area is also affected by this change
in glucose (41). In addition, orexin expression increases
in response to insulin-induced hypoglycemia and also
to food deprivation, which reduces glucose as well as
insulin (11). Further studies are needed to characterize
a possible relationship between the orexins and this
metabolite or hormone under physiological conditions.
Circulating lipids may also influence peptide gene
expression in the hypothalamus. This has been demonstrated by studies with galanin (36) and dynorphin
(58), which are stimulated by a high-fat diet. Circulating lipid levels rise considerably with dietary obesity
(4, 17), and there is evidence that circulating fatty
acids derived from triglycerides can cross the bloodbrain barrier and gain access to the brain (35, 57).
Although not yet directly tested, the possibility that
lipids modulate the orexin system is suggested by evi-
* K. E. Wortley and G.-Q. Chang contributed equally to this work.
Address for reprint requests and other correspondence: S. F. Leibowitz, The Rockefeller Univ., 1230 York Ave., New York, NY 10021
(E-mail: [email protected]).
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. Section 1734
solely to indicate this fact.
perifornical lateral hypothalamus; triglycerides; high-fat
diet; obesity
THE OREXIN
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OREXINS AND CIRCULATING TRIGLYCERIDES
dence that orexin-synthesizing neurons may coexpress
dynorphin (5, 60) and galanin (27).
To further understand the various signals that modulate the orexin system, this study used different animal models of dietary obesity to measure orexin gene
expression in relation to shifts in circulating levels of
leptin, insulin, triglycerides, and glucose.
METHODS
Animals
Adult male Sprague-Dawley rats (275–285 g; Charles
River Breeding Labs, Kingston, NY) or 8-wk-old male
inbred C57L/J and AKR/J mice (Jackson Laboratories, Bar
Harbor, ME) were used. They were individually housed in
a fully accredited American Association for the Accreditation of Laboratory Animal Care facility (22°C, with lights
off at 3:30 PM for 12 h), according to institutionally approved protocols as specified in the NIH Guide to the Use
and Care of Animals and also with the approval of the
Rockefeller University Animal Care Committee. All animals were given 1 wk to acclimate to lab conditions, and all
protocols fully conformed to the “Guiding Principles for
Research Involving Animals and Human Beings” of the
American Physiological Society (2).
Diets
All diets were supplemented with vitamins and minerals,
as described previously (50, 55). In experiments 1 and 4, rats
were maintained on a single diet varying in fat composition.
Diet composition, calculated as percentage of total kilocalories, ranged from a low-fat diet (LFD: 10% fat, 65% carbohydrate, 25% protein) with 3.75 kcal/g, to a moderate-fat diet
(MFD: 25% fat, 50% carbohydrate, 25% protein) with 3.98
kcal/g, to a high-fat diet (HFD: 50% fat, 25% carbohydrate,
25% protein) with 4.7 kcal/g. In experiments 2 and 5, rats
were maintained on an HFD. In experiment 3, inbred mice
were allowed to choose from separate sources of protein
(casein, 3.7 kcal/g), carbohydrate (dextrin, sucrose, cornstarch, 3.7 kcal/g), and fat (lard, 7.7 kcal/g) (55). In experiment 6, rats were maintained on laboratory chow.
Test Procedures
All rats were maintained ad libitum on food and water for
the duration of the experiment. At the end of the experiment,
food was removed 1 h before death (except in experiment 6),
and the animals were killed by rapid decapitation 1–2 h
before dark onset. Trunk blood was collected, and body fat
pads from four regions (inguinal, retroperitoneal, gonadal,
and mesenteric) were collected and weighed.
In experiments 1–4, the rats or mice were maintained on
their respective diets for 3 wk, and food intake was measured
three times per week and body weight one time per week for
the duration of the experiment. In experiment 2, the rats
were killed after 3 wk on an HFD and were designated lean
or obese based on the combined weight of their four fat pads.
The rats were ranked, with the lightest 33% designated lean
and the heaviest 33% designated obese, as described before
(36). Rats with a combined fat pad weight that fell in the
middle range were not used in this study.
In experiment 5, rats were killed after 5 days on an HFD
and were designated obesity resistant or obesity prone based
on their weight gain during this 5-day period. Preliminary
studies from this lab, consistent with the work of Hill and
colleagues (13), show this weight gain measure on a HFD to
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be a strong and reliable correlate of long-term body fat
accrual, permitting one to characterize obesity-prone rats
during the very early stages in their development of obesity.
The identification of obesity-prone rats with this measure
can occur even before a significant rise in fat pad weights,
with the elevated 5-day weight gain presumably reflecting
increased weight of nonadipose tissues, extra food in the gut,
and greater water retention. Based on this, a daily weight
gain score across the 5 days was recorded and averaged for
each rat. The subjects were then rank ordered and designated obesity resistant (lowest 33%) or obesity prone (highest
33%), with the middle range eliminated from the study.
In experiment 6, rats in the absence of food were given an
intraperitoneal injection of saline or 20% Intralipid (5 ml/rat,
Clintech Nutrition). This mode of administration was employed to minimize trauma to the rat and avoid the anesthesia required for intravenous injections, which can interfere
with triglyceride metabolism (8, 40). The rats were killed 4 h
after injection, which in pilot studies revealed the largest
increase in circulating triglycerides.
Hormone and Metabolite Assays
Trunk blood was assayed for insulin, leptin, glucose, and
triglycerides. Insulin (RIR-1685) and leptin (RLR-1143) were
assayed with radioimmunoassay kits from Linco Research.
Serum levels of glucose and triglycerides were analyzed using a commercially available serum chemistry analyzer at
Amgen.
In Situ Hybridization
In situ hybridization with digoxigenin-labeled probes was
used to quantify orexin gene expression. This technique measures specifically the density of neurons expressing the
orexin gene above threshold levels, rather than the level of
mRNA expressed per cell. This technique was chosen because
it is sensitive, due in part to our procedure of using freefloating sections, and it has a high signal-to-noise ratio, with
almost no background, making quantification possible (12,
36). It is also time efficient, allowing analyses of large groups
of animals, and it maintains high-quality morphology, permitting examination and quantification of cells in specific
hypothalamic nuclei. Particularly important, it also avoids
problems of isotope contamination and decay. Results obtained with this technique are generally consistent with
those obtained, in this and other labs, using such techniques
as immunocytochemistry, radioimmunoassay, RNase protection assay, and isotopic in situ hybridization. In the present
study, they are further validated by measurements of mRNA
using quantitative PCR, as described below.
After rapid decapitation, brains were removed, fixed, and
stored, as described previously (36). On the day of use, brains
were cut into 30-␮m-thick sections. An anti-sense cRNA
probe (final concentration, 5 ␮g/ml), labeled with digoxigenin, was prepared by in vitro transcription. Briefly, a pBC SK
plasmid, containing a 565-bp rat prepro-orexin cDNA (a
generous gift of Dr. L. de Lecea, The Scripps Research Institute), was linearized with EcoRI and NotI and was transcribed in vitro with T3 or T7 RNA polymerase for the
anti-sense and sense probes, respectively. Free-floating coronal sections were processed, as described previously (36).
They were incubated with the cRNA probe at 55°C for 18 h,
and, based on pilot studies, a 6-h period was chosen for the
orexin color reaction. The sense probe control was performed
in the same tissue, and no signal was found.
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Quantification of In Situ Hybridization
A digital imaging system, with the help of a rat brain atlas
(45), was used for quantification of cell density (density of
cells/mm2). The hypothalamic areas examined were the PF
and the LH (bregma ⫺2.8 to ⫺3.6 mm). To obtain a total of
six separate readings per subject at a given level, the density
of cells in three to four sections at the same level was
routinely analyzed, with a fourth section read if an area was
unreadable due to tissue damage. A Leitz microscope was
used with a ⫻4 illumination objective, and a Leitz volts
monitor (model 050260) kept the light voltage at 12 V to
maintain light intensity. The digital image was collected by a
video camera connected to an IBM computer with WScan
Array Software (Galai Lat, Migdal Haemek, Israel). The area
of interest was outlined using the Drawing Tool available in
the software.
The defined area was always at the same level on all
sections chosen for control and experimental animals. To
count the number of black pixels above background level
within the areas of interest, a threshold value was first
established, as described in the instruction manual. Briefly,
using 10 randomly selected sections from the experiment,
this threshold was set by matching the number of objects
counted by the software in a defined area of each section with
the number of objects counted manually in that same area.
When these numbers of objects were found to match in all 10
sections, this threshold value was kept constant and used for
all sections counted within a given experiment. By setting
the threshold, the software then permitted the experimenter
to count the objects above background level.
Double-Labeling Studies
Sprague-Dawley rats (350–450 g) were injected intracerebroventricularly with colchicine (120 ␮g in 20 ␮l NaCl;
Sigma, St. Louis, MO) and killed 48 h later by rapid decapitation. The brains were removed and treated, as described
before (36). Thirty-micrometer free-floating cryostat sections
were blocked in 1% normal serum with 0.1% Triton X-100PBS for 30 min, followed by incubation in rabbit anti-orexin
serum (1:200, supplied by Dr. L. de Lecea, The Scripps
Research Institute) and guinea pig anti-GAL serum (1:100,
Peninsula Laboratories) overnight. After three washes in
PBS, 5 min each, the tissue was incubated in FITC-conjugated donkey anti-rabbit (1:50, JacksonImmuno Research,
PA) and Cy3-conjugated donkey anti-guinea pig serum (1:
100, JacksonImmuno Research, PA) for 30 min. Finally, the
tissue was washed in PBS, mounted onto glass slides, and
covered with Vectashield mounting medium (Vector Laboratories). Sections were then examined, and double-labeling
images were scanned using a Zeiss 510 laser confocal microscope.
RNA Extraction and cDNA Synthesis
The whole hypothalamus or PF, using the fornix and third
ventricle as landmarks, was rapidly microdissected. Total
RNA from individual and pooled samples was extracted with
TRIzol reagent (Invitrogen) and filtered with Qiagen
RNeasy, according to the manufacturer’s instructions. One
microgram of RNA was reverse transcribed into cDNA using
the Reverse Transcription System (Invitrogen) with oligo dT
primer and SuperScript Reverse Transcriptase. Before RT,
the RNA was treated with RNase-free DNase I (Invitrogen)
to remove any contaminating genomic DNA. The RT reaction
was carried out at 42°C for 50 min and terminated by heating
at 70°C for 15 min. Samples in which RT was omitted were
also forwarded for PCR as negative control samples, to confirm that no genomic DNA contamination occurred.
Quantitative PCR
For quantitative PCR, the SYBR Green PCR core reagents
kit (Applied Biosystems) was used, and the real-time PCR
was performed in MicroAmp Optic 96-well reaction plates
(Applied Biosystems) on an ABI PRISM 7700 Sequence Detection system (Applied Biosystems). The reaction mixture
(20 ␮l) was composed of 1⫻ SYBR Green buffer, 3.5 mM
MgCl2, 200 ␮M dNTP mixed with dUTP, 0.625 U AmpliTaq
Gold, 0.25 U AmpErase UNG, 400 nM ␤-actin primers, 800
nM orexin primers, 0.5 ␮l RT product, and RNase-, DNasefree water. The primers for rat ␤-actin were 5⬘-GGCCAACCGTGAAAAGATGA-3⬘ (forward) and 5⬘-CACAGCCTGGATGGCTACGT-3⬘ (reverse). The primers for rat orexin were
5⬘-AGATACCATCTCTCCGGATTGC-3⬘ (forward) and 5⬘-ACCAGGGAACCTTTGTAGAAGG-3⬘ (reverse). These primers
were designed with ABI Primer Express v.1.5a software. The
reaction conditions were an initial 2 min at 50°C, followed by
10 min at 95°C, then 40 cycles of 15 s at 95°C and 1 min at
60°C. The whole study consisted of four independent runs of
RT-PCR in triplicate. In addition to samples, each run included a standard curve, a nontemplate control, and a negative RT control. The levels of orexin mRNA expression were
quantified relative to the level of the housekeeping gene
␤-actin, using a standard curve method.
Data Analysis
Caloric intake, presented as kilocalories per 24 h, was
averaged from values taken in the last week of a given
experiment, unless otherwise stated. Body weight, body
weight gain, and fat pad weights are calculated for the final
week of measurements. Hypotheses regarding diet groups,
brain areas, and endocrine and behavioral measures were
tested using either a one-way or two-way ANOVA, followed
by a Bonferroni post hoc test for multiple comparisons between groups, when appropriate, or using unpaired or paired
t-tests. For quantitative PCR, the ratio of the relative concentration of orexin to ␤-actin in the test vs. control samples
was analyzed by a paired t-test. Within-group measures of fat
pad weights, hormones, metabolites, and orexin cell density
were related using a Pearson’s product-moment correlation.
The criterion for the use of the term “significant” in the text
is that the probability value (P) for a given test is ⬍0.05.
RESULTS
Experiment 1: Impact of an HFD on Orexin
Expression in Rats
This experiment tested the relationship between the
orexin system and dietary fat. Orexin mRNA was examined in three groups of rats (n ⫽ 7/group) maintained for 3 wk on either an LFD, MFD, or HFD.
Behavioral, hormonal, and metabolite measures.
Rats maintained on an HFD compared with an LFD
had greater body weight and fat pad weights (Table 1).
This increase in body weight was accompanied by, and
presumably attributed to, hyperphagia seen during the
first week on the HFD (87 ⫾ 2.9 kcal) vs. LFD (73 ⫾ 2.0
kcal, P ⬍ 0.05), but not during week 2 (HFD 109 ⫾ 1.9
vs. LFD 104 ⫾ 2.3) or week 3 (Table 1). The HFD rats
had higher levels of leptin but lower insulin levels
compared with the LFD rats (Table 1). In addition,
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Table 1. Measurements in rats fed an LFD, MFD,
or HFD for 3 wk in experiment 1
Final body weight, g
Body fat, g
Caloric intake, kcal/day
Leptin, ng/ml
Insulin, ng/ml
Triglycerides, mg/dl
Glucose, mg/dl
LFD
MFD
HFD
476 ⫾ 5
16 ⫾ 1.2
96 ⫾ 9.4
15 ⫾ 2.5
2.8 ⫾ 0.20
87 ⫾ 15
143 ⫾ 15
483 ⫾ 13
17 ⫾ 0.5
101 ⫾ 10.1
16 ⫾ 0.9
1.6 ⫾ 0.36†
189 ⫾ 49†
138 ⫾ 6
519 ⫾ 13†§
23 ⫾ 0.2†§
85 ⫾ 3.1
21 ⫾ 1.0†§
1.5 ⫾ 0.32†
207 ⫾ 32†
177 ⫾ 17*‡
Data are means ⫾ SE; n ⫽ 7/group. LFD, low-fat diet; MFD,
moderate-fat diet; HFD, high-fat diet. * P ⬍ 0.05, † P ⬍ 0.01 vs.
LFD; ‡ P ⬍ 0.05, § P ⬍ 0.01 vs. MFD.
they exhibited elevated triglycerides on the MFD and
HFD, with glucose levels significantly rising only on
the HFD.
Orexin gene expression. An increase in dietary fat
from 10 to 50% produced a rise in the density of
neurons with detectable levels of orexin mRNA, as
revealed by in situ hybridization using digoxigeninlabeled riboprobes. This effect reached statistical significance in the PF, as shown in Fig. 1, A and B, but not
in the LH (LFD 490 ⫾ 105; MFD 715 ⫾ 72; HFD 739 ⫾
65 cells/mm2), an area immediately lateral to the PF.
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To validate this diet-induced increase in orexin expression, two additional sets of rats, maintained on an
HFD vs. LFD, were tested using quantitative PCR.
Orexin mRNA, expressed as a ratio to ␤-actin mRNA,
was measured in the PF dissection of the first set of
rats with pooled tissue samples (n ⫽ 4/group) and the
second set of rats with individual samples (n ⫽
7/group). Consistent with the in situ hybridization results, the HFD subjects exhibited a clear increase in
the ratio of orexin to ␤-actin mRNA in the PF dissection (Fig. 2). This occurred in both sets of tissues,
whether pooled or examined individually.
Relation of orexins to circulating hormones and metabolites. The density of cells expressing the orexin
gene in the PF was strongly positively correlated with
circulating triglycerides. This was evident in rats on
the MFD (r ⫽ ⫹0.67, P ⬍ 0.05) or HFD (r ⫽ ⫹0.81, P ⬍
0.05), with a similar but statistically insignificant correlation seen in the LFD rats (r ⫽ ⫹0.57, P ⬎ 0.10).
The specificity of this relationship to circulating lipids
was indicated by the lack of significant correlations
obtained on an HFD, for example, between orexin cell
density and the measures of kilocalorie intake (r ⫽
⫹0.29), fat pad weight (r ⫽ ⫺0.31), leptin (r ⫽ ⫺0.26),
insulin (r ⫽ ⫺0.04), and glucose (r ⫽ ⫹0.30), measures
that are altered by the HFD.
Experiment 2: Orexin Expression in Relation
to Obesity in Rats on a HFD
With evidence from experiment 1 indicating a relationship between dietary fat, blood lipids, and orexin
neurons in the PF, this experiment tested an additional animal model, comparing obese vs. lean groups
on an HFD, which also involves elevated triglycerides.
Behavioral, hormonal, and metabolite measures.
Rats that became obese on an HFD accumulated a total
of 28 ⫾ 1.1 g of fat pad weight, compared with only
18 ⫾ 1.2 g for the lean rats on the same HFD. Similar
Fig. 1. Density of orexin-expressing cells in the perifornical hypothalamus (PF) of rats maintained on a low-fat (LFD), moderate-fat
(MFD), or high-fat diet (HFD) in experiment 1 (n ⫽ 7/group), as
measured by in situ hybridization. A: bar graph shows an increase in
orexin cell density in the PF as dietary fat content increases from 10
to 50%. Data are means ⫾ SE. * P ⬍ 0.05 vs. LFD. B: photomicrographs illustrate the increase in orexin cell density in the dorsal
region of the PF of rats fed HFD vs. LFD. F, fornix.
Fig. 2. Orexin mRNA in the PF of rats maintained on LFD or HFD
in experiment 1, as measured by quantitative PCR. Data (means ⫾
SE) show a significant increase in orexin mRNA levels, expressed as
a ratio to ␤-actin mRNA, in both pooled (n ⫽ 4/group) and individual
(n ⫽ 7/group) PF samples from HFD rats (* P ⬍ 0.05 vs. LFD rats).
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to the group differences in experiment 1 (Table 1), the
obese (vs. lean) rats (Table 2) had greater body weight
and exhibited higher levels of triglycerides as well as
leptin, with glucose levels tending to be elevated. These
two animal models differed, however, in their measures of insulin and total caloric intake. Both measures
were elevated in the obese (vs. lean) rats of this experiment (Table 2), while insulin levels were lower and
caloric intake unchanged in the final week in the HFD
(vs. LFD) rats of experiment 1.
Orexin gene expression. Despite these differences,
the density of orexin-expressing cells was increased in
the PF of obese compared with lean rats on the HFD
(Fig. 3A). No significant change in orexin cell density
was seen in the LH.
To validate this obesity-induced increase in orexin
expression, two additional sets of obese vs. lean rats on
an HFD were examined using quantitative PCR.
Orexin mRNA, as a ratio to ␤-actin mRNA, was measured in the whole hypothalamus of rats with pooled
tissue (n ⫽ 4/group) or individual samples (n ⫽
8/group). The obese rats of these groups showed, respectively, a 35 and 45% increase in the ratio of orexin
to ␤-actin mRNA in the hypothalamus compared with
lean rats (Fig. 3B), confirming the increase seen with
in situ hybridization.
Relation of orexins to circulating hormones and metabolites. The density of cells with detectable orexin
mRNA in the PF, as revealed by in situ hybridization,
was positively correlated with triglyceride levels across
individual subjects within the whole group (r ⫽ ⫹0.70,
P ⬍ 0.05). Orexin cell density was additionally related
to fat pad weight (r ⫽ ⫹0.69, P ⬍ 0.05) and to strong
correlates of body fat, namely, leptin (r ⫽ ⫹0.65, P ⬍
0.05) and insulin (r ⫽ ⫹0.70, P ⬍ 0.05). It showed little
relation, however, to glucose levels (r ⫽ ⫺0.27, P ⬎
0.10) or to total kilocalorie intake (r ⫽ ⫹0.48, P ⬎ 0.10).
Experiment 3: Orexin in Relation to a Propensity
Toward Obesity in Inbred Mice
To cross-validate these findings observed in rats, we
examined two strains of inbred mice. Orexin expression in AKR/J mice, which are susceptible to obesity on
a HFD, was compared with that of C57L/J mice, which
are less responsive to the obesity-promoting effects of a
HFD (59). All mice were maintained ad libitum for 3
wk with a choice of three macronutrient diets: fat,
carbohydrate, and protein (see METHODS).
Table 2. Measurements in lean and obese rats
on HFD in experiment 2
Final body weight, g
Caloric intake, kcal/day
Leptin, ng/ml
Insulin, ng/ml
Triglycerides, mg/dl
Glucose, mg/dl
Fig. 3. Density of orexin-expressing cells in the hypothalamus of
obese vs. lean rats on an HFD in experiment 2. A: data (means ⫾ SE)
reveal an increase in orexin cell density in the perifornical hypothalamus (PF) but not the lateral hypothalamus (LH) of obese rats, as
measured by in situ hybridization (n ⫽ 6/group). B: orexin mRNA
(ratio to ␤-actin mRNA) in the hypothalamus of lean and obese rats
in experiment 2, as measured by quantitative PCR, shows a significant increase in orexin expression in both pooled (n ⫽ 4/group) and
individual samples (n ⫽ 8/group) from obese rats. (* P ⬍ 0.05 vs. lean
rats).
Behavioral, hormonal, and metabolite measures. The
lean C57L/J mice on these macronutrient diets exhibited a strong preference for dietary carbohydrate over
fat, consuming 62% of their daily kilocalorie intake in
the form of carbohydrate and only 13% as fat (Table 3).
The obese AKR/J mice, in contrast, consumed 33% of
their daily caloric intake in the form of fat. They also
Table 3. Measurements in lean C57L/J and obese
AKR/J mouse strains given 3 macronutrient
diets in experiment 3
C57L/J
Lean
Obese
463 ⫾ 28
99 ⫾ 2.3
12 ⫾ 0.8
0.7 ⫾ 0.06
86 ⫾ 9
104 ⫾ 7.4
525 ⫾ 5*
107 ⫾ 2.7*
22 ⫾ 0.7†
1.0 ⫾ 0.04†
127 ⫾ 21*
117 ⫾ 5.3
Data are means ⫾ SE; n ⫽ 6/group. * P ⬍ 0.05, † P ⬍ 0.01 vs.
lean.
Final body weight, g
Fat intake, %caloric
intake
Carbohydrate intake,
%caloric intake
Protein intake,
%caloric intake
Body fat, g
Leptin, ng/ml
Triglycerides, mg/dl
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24 ⫾ 0.49
32 ⫾ 0.81*
13 ⫾ 2.2
33 ⫾ 4*
62 ⫾ 1.7
42 ⫾ 3.7*
17 ⫾ 1.9
1.0 ⫾ 0.18
2.4 ⫾ 0.8
66 ⫾ 9.0
26 ⫾ 2.2*
1.9 ⫾ 0.17*
8.1 ⫾ 1.1*
101 ⫾ 6.6*
Data are means ⫾ SE; n ⫽ 5/group. * P ⬍ 0.01 vs. C57L/J.
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had significantly greater fat pad weights, in addition to
higher levels of triglycerides and leptin compared with
the C57L/J mice (Table 3). Levels of insulin and glucose were not measured, due to the low volume of blood
collected from these mice.
Orexin gene expression. Together with elevated triglycerides and body weight, the obese AKR/J mice
exhibited a greater density of cells expressing the
orexin gene in the PF compared with the lean C57L/J
mice (Fig. 4). These results are very similar to those
obtained in the obese rats of experiment 2.
Experiment 4: Relationship of the Orexins to Galanin
in the PF
This experiment performed two tests to determine
whether galanin-expressing cells in the PF are responsive to dietary fat and, if so, whether the orexin and
galanin peptides actually colocalize in neurons of the
PF, as suggested by a preliminary report (27). In two
sets of rats maintained on either an LFD or HFD, this
experiment examined diet-induced changes in the density of PF galanin-expressing cells in one set (n ⫽
5/group) and analyzed peptide immunoreactivity (peptide-ir) for orexin and galanin together in the second
set (n ⫽ 3/group), using double-labeling immunocytochemistry.
Galanin gene expression in PF. Similar to the orexins
(experiment 1), a rise in dietary fat from 10 to 50%
produced a small, but significant, increase in the density of galanin-expressing cells in the PF (Fig. 5). This
change was detected immediately dorsal to the fornix,
in the same area where orexin neurons are expressed.
Galanin and orexin peptide-ir in PF. With singlelabeling immunocytochemistry, a dense concentration
of orexin-positive neurons and terminals was observed
in the PF, both the dorsal and ventromedial areas
around the fornix (Fig. 6, A and B). Galanin-positive
neurons and terminals were also evident in these same
areas of the PF (Fig. 6, C and D). While exhibiting
Fig. 5. Density of galanin-expressing cells in the PF of rats maintained on LFD, MFD, or HFD in experiment 4 (n ⫽ 5/group). Data
(means ⫾ SE) show a significant increase in galanin cell density in
the PF as dietary fat content increases from 10 to 50%. * P ⬍ 0.05 vs.
LFD; † P ⬍ 0.01 vs. LFD; ‡ P ⬍ 0.05 vs. MFD.
anatomic overlap, the orexin-positive neurons were
considerably larger than the galanin-containing neurons. They also had larger processes, suggesting that
these peptides are expressed in separate neurons.
Consistent with this, the double-labeling immunocytochemistry failed to reveal any coexistence of these
two peptides in neurons of the PF. Whereas there was
a suggestion at low magnification (⫻25) that these
peptides may colocalize in a few neurons, a higher
magnification (⫻63 or ⫻100) using laser confocal microscopy failed to reveal any double-labeled neurons, in
either the dorsal or ventromedial areas of the PF (Fig.
6, E and F). This was true for each of the six rats
examined, whether on an LFD or an HFD. This lack of
colocalization agrees with the morphological differences of the orexin- and galanin-containing neurons in
suggesting that they are distinct populations.
Experiment 5: Orexin mRNA in Relation
to a Propensity Toward Obesity in Rats
Fig. 4. Density of orexin-expressing cells in the PF of lean C57L/J
mice and obese AKR/J mice (n ⫽ 5/group) in experiment 3. Data
(means ⫾ SE) show a reliable increase in orexin cell density in the
PF of AKR/J mice. * P ⬍ 0.05 vs. C57L/J control mice.
This next experiment provided a further test of the
observed orexin-triglyceride relationship, specifically
in a physiological state where triglyceride levels increase before significant changes in fat pad weights
and leptin.
Behavioral, hormonal, and metabolite measures.
Obesity-prone rats, defined by their weight gain of
10.2 ⫾ 0.2 g/day on an HFD compared with 6.8 ⫾ 0.2
g/day for the obesity-resistant rats, exhibited an 8% (28
g) increase in body weight and were hyperphagic during the 5-day period (Table 4). With this small increase
in body weight, the obesity-prone rats exhibited little
change in fat pad weight, only a 1-g (⫹10%) increase,
and they also had normal levels of leptin (Table 4).
These rats, however, showed a similar pattern to the
already-obese rats of experiment 2, in having elevated
levels of triglycerides compared with the obesity-
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OREXINS AND CIRCULATING TRIGLYCERIDES
Fig. 6. Photomicrographs of immunofluorescent orexin- and galanin-staining neurons in the PF in experiment 4.
A and B: immunofluorescent orexin cell
bodies and terminals are shown in the
dorsal and ventromedial areas around
the fornix. C and D: galanin-positive
neurons and terminals are shown in
the same dorsal and ventromedial areas of the PF. E and F: double-labeling
immunocytochemistry fails to reveal
any coexistence of orexin and galanin
in neurons of the PF. Magnification,
⫻25.
resistant rats, as well as higher insulin and glucose
(Table 4).
Orexin gene expression. This endocrine profile of obesity-prone rats was accompanied by a small but significant increase in the density of orexin-expressing cells
in the PF compared with obesity-resistant subjects
(Fig. 7). Again, no difference in cell density was seen in
the LH.
Relation of orexin to circulating hormones and metabolites. Within-group analyses revealed, once again,
a positive correlation between the density of orexin
cells in the PF and levels of triglycerides (r ⫽ ⫹0.54,
P ⬍ 0.05) across the group. Orexin mRNA showed no
relation, in contrast, to insulin (r ⫽ ⫺0.10), glucose
(r ⫽ ⫺0.12), or total kilocalorie intake (r ⫽ ⫹0.38).
triglycerides. Acute intraperitoneal administration of
this emulsion significantly increases circulating levels
of triglycerides (1, 39).
Behavioral, hormonal, and metabolite measures. Injection of Intralipid caused an ⬃29% increase in serum
triglyceride levels (Table 5). This effect was seen in the
absence of any change in leptin, insulin, or glucose, as
well as in body weight and fat pad weight (Table 5).
Orexin gene expression. Rats injected with Intralipid
compared with saline rats showed a significant increase in the density of cells with detectable levels of
orexin mRNA in the PF (Fig. 8). No change in orexin
expression was seen in the LH.
Experiment 6: Impact of Intralipid Injection
on Orexin Expression in Rats
The results of these experiments reveal a strong and
consistent relationship, under physiological conditions,
DISCUSSION
This final experiment was conducted to test the possibility that triglycerides elevated in the blood have
impact, direct or indirect, on orexin neurons in the PF.
This was examined using the compound Intralipid, a
commercially available lipid emulsion containing 20%
Table 4. Measurements in obesity-resistant and
obesity-prone rats on a HFD for 5 days
in experiment 5
Final body weight, g
Body fat, g
Caloric intake, kcal/day
Leptin, ng/ml
Insulin, ng/ml
Triglycerides, mg/dl
Glucose, mg/dl
Obesity
Resistant
Obesity
Prone
350 ⫾ 3
12 ⫾ 0.5
113 ⫾ 3.1
12 ⫾ 1.2
0.6 ⫾ 0.07
117 ⫾ 12
116 ⫾ 8.6
378 ⫾ 3†
13 ⫾ 0.4
132 ⫾ 3.4†
12 ⫾ 1.4
1.1 ⫾ 0.21*
168 ⫾ 14†
145 ⫾ 8.4*
Data are means ⫾ SE; n ⫽ 8/group. * P ⬍ 0.05, † P ⬍ 0.01 vs.
obesity resistant.
Fig. 7. Density of orexin-expressing cells in the PF or LH of obesityresistant and obesity-prone rats on HFD in experiment 5 (n ⫽
8/group). Data (means ⫾ SE) reveal an increase in orexin cell density
in the PF but not LH of obesity-prone rats. * P ⬍ 0.05 vs. obesityresistant rats.
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OREXINS AND CIRCULATING TRIGLYCERIDES
Table 5. Measurements in rats 4 h after
intraperitoneal injection of saline
or intralipid in experiment 6
Final body weight, g
Body fat, g
Leptin, ng/ml
Insulin, ng/ml
Triglycerides, mg/dl
Glucose, mg/dl
Saline
Intralipid
322 ⫾ 3
4.0 ⫾ 0.35
1.20 ⫾ 0.14
2.0 ⫾ 0.21
56 ⫾ 4.6
162 ⫾ 13
323 ⫾ 4
4.3 ⫾ 0.33
1.43 ⫾ 0.16
1.9 ⫾ 0.16
72 ⫾ 6.4*
162 ⫾ 19
Data are means ⫾ SE; n ⫽ 8/group. * P ⬍ 0.05 vs. saline.
between dietary triglycerides and orexin expression in
the PF. Although anatomically overlapping in the PF
and similarly responsive to dietary fat, the orexin and
galanin peptide systems are distinct and likely function separately as they contribute to the physiological
control and pathology of eating and body weight.
Close Relation Between Orexin-Expressing Neurons
and Circulating Triglycerides
A main result of this report, revealed by in situ
hybridization, is that consumption of an HFD in rats
increases the density of orexin-expressing cells and
that this effect occurs specifically in the PF. This dietinduced increase in orexin expression was confirmed
with measurements of orexin mRNA in PF dissections
or hypothalamus using quantitative PCR. This effect
was not seen, however, in the LH, an area lying immediately adjacent to the PF. This high degree of site
specificity, in addition to differences in experimental
conditions, may explain why measurements of orexin
mRNA or orexin A peptide in whole hypothalamus
failed to reveal a significant change on an HFD (53, 65).
Because orexin neurons are restricted spatially and
other neurons are unlikely to have the necessary transcription factors required to turn on orexin expression,
the rise in density of orexin-expressing cells in the PF
may reflect a rise in expression from basal levels undetectable by digoxigenin-labeled probes. This is particularly likely because the animals were killed at the
end of the light period, at a time when PF orexin is
generally at its nadir (22, 64).
Of particular note is the additional evidence that the
HFD-induced increase in PF orexin cell density is accompanied invariably by a rise in circulating triglyceride levels. These parallel changes in orexin expression
and metabolic fuels are characteristic of each of the rat
feeding models tested. These models are the 25% fat
MFD or 50% fat HFD rats, the already-obese rats on an
HFD, and the obesity-prone rats in a preobese state on
an HFD. A specific relationship between triglycerides
and the orexins is substantiated by strong, positive
correlations repeatedly detected between these measures in the different groups tested. Consistent with
these findings in rats, the density of cells expressing
orexin mRNA is also elevated in obese AKR/J mice,
which compared with C57L/J mice consume greater fat
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and have higher circulating triglycerides, as well as
body weight. These results confirm the basic finding
that orexin expression rises with elevated lipids and
obesity on a fat-rich diet, although it remains to be
determined, with basal measurements independent of
diet, whether the increased orexin mRNA in AKR/J
mice reflects an inherent strain difference. The specificity of an orexin relationship to triglycerides is underscored by the finding, in experiment 5, that animals
with a propensity toward obesity, but still with normal
fat pad weights equivalent to controls, have elevated
orexin cell density in the PF, together with higher
triglycerides. Thus triglycerides or closely related metabolic fuels appear to be a necessary component of the
enhanced orexin gene expression.
Distinct Populations of Fat-Responsive Galanin
and Orexin Neurons in the PF
The results of the present and published studies
reveal similarities between the orexins and galanin,
suggesting that these two peptides may function as
part of a common system regulating energy balance.
Like the orexins, galanin expression is stimulated by
an HFD in the PF, as shown here, and in the PVN (36),
and it is elevated in obese rats (36). Moreover, preliminary studies indicate a possible coexistence of galanin
and orexins in a few isolated neurons in the PF (27).
Despite these similarities, there is little support for
the possibility that these two peptides exist and function within the same neuronal population of the PF.
Whereas the orexins and galanin at low magnification
may appear to colocalize in a few PF neurons (27), this
was not evident here at higher magnification, even in
rats maintained on an HFD. Moreover, their neurons
exhibit clear morphological differences, with the
orexin-positive neurons and their processes considerably larger.
Fig. 8. Density of orexin-expressing cells in the PF or LH of salineor Intralipid-treated rats (n ⫽ 10/group). Data (means ⫾ SE) reveal
an increase in orexin cell density in the PF of Intralipid-treated rats
in experiment 6. * P ⬍ 0.01 vs. saline-treated rats.
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Little Relation of Orexin-Expressing Neurons to Total
Caloric Intake or Body Weight
The increased density of orexin-expressing cells in
the various models of dietary obesity raises the possibility that the orexins are responding to either a rise in
total caloric intake or body weight. The present study,
together with published results, however, is inconsistent with these possibilities. Although the HFD, obese,
and obesity-prone rats exhibit hyperphagia during
their initial exposure to the HFD, no significant correlations were seen between orexin cell density and caloric intake in any of these groups. These findings
agree with published evidence, showing that orexin
expression is actually inhibited by the presence of food
in the gut (9), and it rises in association with a deprivation-induced reduction in caloric intake (11, 48). It is
also unchanged in rats that overeat on a highly palatable diet compared with chow-fed rats (11) and is
actually decreased in hyperphagic ob/ob mice and fa/fa
rats (7, 20, 51, 62).
Although the increased orexin gene expression in the
three dietary models is accompanied by an elevation of
body weight, several pieces of evidence dissociate these
phenomena. In the present study, Intralipid stimulates
orexin cell density in the absence of a change in body
weight. Further, there were no significant correlations
between orexin cell density and fat pad weights in the
different groups tested, except the obese subjects of
experiment 2. Although further tests are needed to
provide direct evidence dissociating the orexins and
body weight, published studies indicate that excess
body weight per se does not increase and may, in fact,
decrease orexin expression. For example, orexin
mRNA is unchanged in obese rats fed a highly palatable diet (11), and it is actually reduced in ob/ob mice
and fa/fa rats, which are profoundly obese (7, 20, 51,
62). Thus orexin neurons appear to be responsive specifically to a rise in dietary or circulating lipids, such as
triglycerides, which occurs independently of changes in
caloric intake or body weight. The orexins are, therefore, referred to as “fat-responsive” peptides.
Little Association Between the Orexins and
Circulating Levels of Insulin, Glucose, and Leptin
In contrast to its association with triglycerides,
orexin expression in the PF on an HFD shows little
relation to circulating insulin and glucose, at least with
food available ad libitum. The results demonstrate an
increase in orexin expression in the PF of all models
showing a rise in triglycerides, despite variable shifts
in insulin and glucose. Consistent with these findings,
the expression and production of the orexins are not
affected by insulin-induced hypoglycemia in freely
feeding animals (9, 11) or in insulin-injected animals
given concurrent injections of glucose (25). This evidence argues against a functional link between orexin
neurons and physiological changes in insulin or glucose. Under conditions of food deprivation, however,
the orexins are shown to be stimulated in association
with hypoglycemia (9, 11).
Whereas previous studies demonstrate an inhibitory
effect of leptin injection on orexin gene expression (6,
37), there is little evidence for a close association between this hormone and peptide system under natural
feeding conditions. Increased orexin gene expression,
while accompanied by a rise in leptin in HFD rats and
in obese rats or mice on an HFD, can be similarly seen
in the absence of a change in leptin, in the obesityprone rats. Further, investigations in genetically obese
mutants with disrupted leptin signaling generally
show no change (53) or a decrease in orexin peptides or
gene expression (10, 51, 62). Thus endogenous leptin
may not be important in the control of orexin neurons
under physiological conditions.
Direct Impact of Triglycerides on Orexin-Expressing
Neurons in the PF
The strong association between the orexins and triglycerides led us to test whether the orexin neurons in
the PF are, in fact, responsive to experimentally induced changes in triglyceride levels. Such changes in
triglycerides can be seen with acute injection of the
compound, Intralipid (39). In experiment 6, this agent
significantly raised circulating levels of triglycerides
within 4 h after injection, without affecting insulin,
glucose, or leptin levels. With this specific increase in
triglycerides, Intralipid stimulated orexin gene expression in the PF, in precisely the same site affected by an
HFD or dietary obesity. This finding supports the idea
that circulating triglycerides, elevated under natural
feeding conditions, contribute to the enhanced orexin
gene expression.
As for how triglycerides may impact on the brain,
evidence indicates that these lipids are rapidly hydrolyzed in capillary endothelium and that fatty acids
derived from triglycerides enter the brain from the
plasma, through as yet undefined mechanisms (57).
Although there is no direct evidence in the present
study that fatty acids stimulate orexin neurons, it has
long been known that they activate neurons in the LH
in vitro (43). This suggests that fatty acids, derived
from dietary triglycerides carried in chylomicrons, may
actually stimulate neurons in this area in vivo.
Whereas the mechanism of this response remains to be
determined, it may involve both a direct, stimulatory
effect of fatty acids on neuronal activity (43) or an
indirect, inhibitory effect of fatty acids on glucose
transport, in a manner similar to that observed in the
periphery (24). The idea that neurons in the PF are
responsive to a rise in lipids is supported by additional
evidence that exposure to an HFD stimulates c-Fos
expression in this area (54). Further experiments involving measurements of circulating fatty acids are
needed to test this idea.
Possible Function of PF Orexin Neurons
on a Fat-Rich Diet
Taken together, the above evidence suggests that
orexin neurons in the PF are stimulated by a rise in
circulating lipids during a state of positive energy
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OREXINS AND CIRCULATING TRIGLYCERIDES
balance, as well as by a decline in glucose levels during
a state of negative energy balance. This suggests that
the orexin peptides may have multiple functions depending on nutritional status, consistent with other
evidence distinguishing functional subsets of orexin
neurons (23). An interaction of the orexins with other
obesity-related systems may provide the mechanisms
for their multiple functions in energy balance. Among
other areas, orexin neurons project to the arcuate nucleus
(ARC) (42), where they innervate one set of neurons that
expresses NPY and another set that expresses proopiomelanocortin (POMC) together with cocaine- and amphetamine-related transcript (CART) (26).
Evidence showing a relation between orexin and
NPY neurons demonstrates that both are stimulated
by hypoglycemia (28, 47), and the feeding-stimulatory
effect of orexin A is blocked by NPY antagonists (18, 33,
63). Thus, in states of negative energy balance, PF
orexin neurons may be activated by a decline in glucose
and, through ARC NPY neurons, trigger counterregulatory responses that maintain glucose levels and protect the body during starvation. In states of positive
energy balance, in contrast, the orexin neurons in the
PF, projecting to POMC/CART neurons, may function
very differently. The orexins are similar to the POMC/
CART peptides in being stimulated by an HFD (46, 56)
and implicated in the stimulation of lipid metabolism
(46, 51) and SNS activity (19, 21, 49). Thus, with a
fat-rich diet freely available, PF orexin neurons may be
activated by the lipids and specifically target POMC/
CART neurons, which help the body deal with surplus
lipid calories.
This idea, relating the orexins to fat metabolism,
receives support from the finding that orexin A stimulates lipid utilization during the light period but not at
dark onset (38). At dark onset, NPY neurons are presumably the predominantly active neurons in the ARC,
which initiate food intake and suppress POMC/CART
neuronal activity (14). Thus injection of orexin A at this
time may further stimulate NPY neurons, while having little impact on POMC/CART neurons and, thus,
lipid utilization. In contrast, during the light period
when NPY neurons are presumably quiescent, orexin
A should be able to stimulate POMC/CART neurons
and, thereby, increase lipid metabolism. This may help
to explain why mice depleted of orexin neurons, which
lack this excitatory input to both orexigenic NPY neurons and metabolism-promoting POMC/CART neurons, exhibit hypophagia in addition to obesity (29).
In conclusion, the present results indicate that
orexin neurons in the PF are fat-responsive, stimulated in parallel with a rise in triglycerides under both
physiological and nonphysiological conditions. Although galanin in the same region also has properties
of a fat-responsive peptide, these peptides most likely
function as separate systems involved in lipid storage
and utilization. Based on this together with published
evidence, it is proposed that orexin neurons respond to
elevated levels of circulating lipids, as well as to a
decline in glucose. These neurons, in turn, project to
different sets of downstream neurons, to maintain en-
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ergy homeostasis in states of both positive and negative energy balance.
We thank Dr. L. de Lecea (The Scripps Research Institute) for the
generous supply of orexin cDNA and antibody, and Clintech Nutrition for the supply of Intralipid. We are grateful to Prof. J. Arch
(Univ. of Buckingham) for reading the manuscript and providing
helpful comments and to F. Li and Dr. Z. Han (Rockefeller Univ.) for
assistance with the quantitative PCR technique. We very much
appreciate the help of K. Sepiashvili in the preparation of this
manuscript. We thank Amgen for analysis of triglycerides and glucose levels.
This research was supported by grants from the Wellcome Trust
(058240) and the National Institutes of Health (MH-43422).
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