Download Cocaine- and amphetamine-regulated transcript in the arcuate nucleus stimulates lipid metabolism to control body fat accrual on a high-fat diet

Regulatory Peptides 117 (2004) 89 – 99
www.elsevier.com/locate/regpep
Cocaine- and amphetamine-regulated transcript in the arcuate
nucleus stimulates lipid metabolism to control body fat
accrual on a high-fat diet
Katherine E. Wortley a, Guo-Qing Chang a, Zoya Davydova a,
Susan K. Fried b, Sarah F. Leibowitz a,*
a
The Rockefeller University, 1230 York Avenue, New York, N.Y. 10021, USA
b
Rutgers University, New Brunswick, New Jersey, N.J. 08901, USA
Received 21 May 2003; received in revised form 30 July 2003; accepted 10 August 2003
Abstract
Previous studies have indicated a relationship between cocaine- and amphetamine-related transcript (CART) and leptin. The present study
used quantitative PCR and in situ hybridization to examine this CART – leptin relationship in different animal models. With CART injection,
the function of this pathway was also investigated. The results demonstrate that CART mRNA in the arcuate nucleus (ARC) was significantly
increased in subjects fed a high-fat diet (HFD) compared to low-fat diet (LFD). It was also elevated in obese vs. lean rats and in normalweight obesity-prone vs. obesity-resistant rats. In each group tested, CART mRNA in the ARC was positively correlated specifically with
circulating levels of leptin. Its close association specifically with leptin was further supported by a stimulatory effect of this hormone on
CART expression. This leptin – CART relationship in the ARC, in contrast, was less consistent or undetectable in the paraventricular nucleus
and lateral hypothalamus. Central injection of CART peptide (55 – 102) increased circulating non-esterified fatty acid levels and decreased
lipoprotein lipase activity in adipose tissue. These results suggest that, on a fat-rich diet, this leptin – CART pathway originating in the ARC
inhibits excessive body fat accrual by causing a shift from lipid storage toward lipid mobilization.
D 2003 Elsevier B.V. All rights reserved.
Keywords: Leptin; Hypothalamus; Non-esterified fatty acids; Obesity
1. Introduction
Cocaine- and amphetamine-regulated transcript (CART)
is a neuropeptide with a possible role in energy homeostasis
[1]. Consistent with this idea, CART mRNA is heavily
expressed in the hypothalamus, with dense expression in the
arcuate and paraventricular (PVN) nuclei, as well as the
lateral hypothalamic area (LHA) [2]. Central injection of
recombinant CART peptide reduces food intake [1,3],
increases lipid substrate utilization [4] and stimulates the
expression of uncoupling proteins [5]. Further, antibodies
against CART peptides increase feeding [1,3], and CARTdeficient mice on a high-fat diet (HFD) exhibit elevated
food intake and body weight compared to wild-type mice
* Corresponding author. Tel.: +1-212-327-8378; fax: +1-212-3278447.
E-mail address: [email protected] (S.F. Leibowitz).
0167-0115/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.regpep.2003.08.005
[6]. Together, these findings indicate that CART is a satietyproducing peptide and, consistent with changes observed
with other satiety-producing peptides [7,8], hypothalamic
CART mRNA level is reduced by food deprivation [1].
There is evidence that the expression of CART in the
hypothalamus is linked to the satiety hormone, leptin, which
is synthesized in adipose tissue [9]. In animal models with
disrupted leptin signaling, CART mRNA in the ARC is
virtually absent and is restored by leptin injection [1], with
CART mRNA in the LHA less affected [1]. Both virusinduced hyperleptinemia and a high-fat diet, which
increases leptin levels, stimulate hypothalamic CART expression in rodents [4,10]. Further, in the ARC, where the
long-form leptin receptor (OBRb) is most heavily expressed
[11,12], CART neurons colocalize OBRb mRNA, in addition to SOCS-3 mRNA, a leptin-induced protein that inhibits leptin signal transduction [13]. In the PVN and LHA, the
leptin receptor is less concentrated [11,14] and SOCS-3
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mRNA in the PVN is less responsive to leptin’s stimulatory
effect [13], suggesting that the positive relationship between
leptin and CART neurons is weaker in these two areas than
in the ARC.
Further studies suggest that insulin may act similarly to
leptin in its effects on hypothalamic peptides. Whereas
insulin inhibits the expression of orexigenic peptides
[15,16], it potentiates mRNA levels of satiety-producing
peptides, as illustrated by analyses of proopoimelanocortin
mRNA in the ARC and a-melanin-concentrating hormone
peptide in the PVN [17]. Since CART colocalizes with
POMC in the ARC [18], insulin may be similar to leptin in
its relation to CART gene expression. Besides evidence
showing reduced CART mRNA in food-deprived animals
with low insulin [1], there are no reports that have implicated this hormone in the control of CART neurons.
There is evidence that lipids circulating in the blood may
also have impact on hypothalamic peptides. Triglyceride
levels rise as dietary fat or body fat increases [19,20]. A
possible effect of lipids on hypothalamic peptides is suggested by studies of the feeding-stimulatory peptides, galanin and orexin, showing a significant increase in the
expression and production of these peptide under all conditions associated with elevated lipids [21,22]. Thus, whereas the stimulation of CART mRNA by a high-fat diet [10]
may reflect the rise in leptin, it may also occur in response to
an elevation in circulating lipids.
To advance our understanding of the signals that modulate hypothalamic CART, the present report performed
several experiments to determine whether this peptide
under different conditions is related to circulating levels
of insulin and lipids in addition to leptin. An additional
experiment involving CART injections was conducted to
analyze this peptide’s effects on endocrine and metabolic
processes.
2. Methods
2.1. Animals
Adult, male Sprague –Dawley rats (275 – 285 g; Charles
River Breeding Labs, Kingston, NY) were individually
housed in a fully accredited American Association for the
Accreditation of Laboratory Animal Care facility (22 jC,
with lights off at 3:30 p.m. for 12 h), according to
institutionally approved protocols as specified in the NIH
Guide to the Use and Care of Animals and with the approval
of the Rockefeller University Animal Care Committee. All
animals were given 1 week to acclimate to laboratory
conditions.
2.2. Diets
All diets were supplemented with vitamins and minerals,
as described previously [23]. Diet composition, calculated
as percent of total kilocalories, ranged from a low-fat diet
(LFD: 25% protein, 65% carbohydrate, 10% fat) with 3.75
kcal/g, to a moderate-fat diet (MFD: 25% protein, 50%
carbohydrate, 25% fat) with 3.98 kcal/g and to a HFD (25%
protein, 25% carbohydrate, 50% fat) with 4.7 kcal/g. In
Experiments 1 and 3, the rats were maintained on a HFD,
whereas Experiment 2 had three groups of rats maintained
on a LFD, MFD or HFD. In Experiment 4, the rats were
maintained on standard laboratory chow.
2.3. Test procedures
All rats were maintained ad libitum on food and water for
the duration of the experiment. For all experiments, food
was removed 1 h before sacrifice and rats were killed by
rapid decapitation within 1 –2 h before dark onset. Trunk
blood was collected and fat pads from three regions (inguinal, retroperitoneal and gonadal), as well as the mesenteric
fat pad, were collected and weighed. Total fat pad weights
were recorded.
In Experiment 1, rats were sacrificed following 3 weeks
on a HFD and were designated lean or obese based on their
body fat accrual as described before [21,22]. In Experiment
2, rats were maintained on their respective diets for 3 weeks.
In both Experiments 1 and 2, food intake and body weight
were measured four times or once a week, respectively, for
the duration of the experiment. In Experiment 3, kcal intake
and body weight on a HFD were measured every day over a
5-day period, and the rats were sacrificed on the fifth day.
Rats were designated obesity-prone or obesity-resistant
based on their body weight gain over the 5 days on the
diet, as described before [22].
In Experiment 4, rats were implanted with a steel
cannula aimed at the third ventricle and allowed 1 week
to recover, as described previously [24]. After the 1-week
recovery period, the rats were prepared for a test to
determine whether they were responsive to the inhibitory
effect of CART on food intake, to verify that their cannula
was patent and the site of implantation was on target.
Baseline food intake over the first 2 h following lights out
was established for each rat over 3 days and the three
values were averaged to obtain a mean value for food
intake. Then, on the fourth day, the rats were injected with
CART (55 – 102, Phoenix Pharmaceuticals, CA) (two i.c.v.
injections of 500 pmol each, at 11:.30 a.m. and 1:30 p.m.).
Food intake was then measured 2 h following lights out.
Approximately 10% of the rats failed to show a feeding
suppression in response to CART and were, therefore,
eliminated from the experiment. The other rats responded
to CART with a marked, 60% suppression of feeding
compared to baseline food intake, decreasing intake from
9.2 F 2.3 to 3.8 F 1.2 kcal ( P < 0.01). These rats were,
then, randomly assigned to a saline or CART group for
the final experiment. On the test day, they were each
given two injections, one at 10 a.m. and at 12 noon, of
either saline (3 Al) or CART 55 –102 (500 pmol/3 Al).
K.E. Wortley et al. / Regulatory Peptides 117 (2004) 89–99
Food was removed following the first injection, to rule out
any effect of CART that was secondary to a reduction in
food intake, and the rats were sacrificed 2 h after the
second injection.
2.4. Hormone and metabolite assays
Trunk blood was analyzed for insulin, leptin, triglycerides and non-esterified fatty acids (NEFA). Insulin and
leptin were assayed with commercially available kits from
Linco Research, MO. Plasma levels of triglycerides and
NEFA were analyzed with a commercial serum chemistry
analyzer at Amgen, CA.
2.5. Lipoprotein lipase assay
Lipoprotein lipase activity (expressed as Amol of fatty
acids released/g tissue/h) was measured as total extractable
lipoprotein lipase activity in samples of retroperitoneal
adipose tissue by Dr. Susan Fried (Rutgers University), as
described previously [25].
2.6. Fatty acid synthase (FAS) assay
Fatty acid synthase activity (expressed as the amount of
enzyme needed to catalyze the oxidation of 1 nM NADPH),
in samples of retroperitoneal fat pads, was assayed using a
precision microplate reader at 340 nm, according to methods
previously described by Halestrap and Denton [26].
2.7. RNA extraction and cDNA synthesis
The whole hypothalamus or the ARC, PVN or LHA,
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 jC for 50 min and terminated by heating at
70 jC for 15 min. Samples in which reverse transcriptase
was omitted were also forwarded for PCR as negative
control samples, to confirm that no genomic DNA contamination occurred.
2.8. Quantitative PCR
For quantitative PCR, the SYBR Green PCR core
reagents kit (Applied Biosystems, CA) 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).
91
The reaction mixture (20 Al) was composed of 1 SYBR
Green buffer, 3.0 mM MgCl2, 200 AM dNTP mixed with
dUTP, 0.3 U AmpliTaq Gold, 0.12 U AmpErase UNG, 200
nM h-actin primers, 400 nM CART primers, 0.5 Al RT
product and RNase-, DNase-free water. The primers for rat
h-actin were: 5V-GGCCAACCGTGAAAAGATGA-3V (forward) and 5V-CACAGCCTGGATGGCTACGT-3V(reverse).
The primers for rat CART were: 5V-GGATGATGCGTCCCATGAG-3V (forward) and 5V-CAGCGCTTCAATCTGCAACA-3V (reverse). These primers were designed with
ABI Primer Express v.1.5a software. The reaction conditions were an initial 2 min at 50 jC, followed by 10 min at
95 jC, then 40 cycles of 15 s at 95 jC and 1 min at 60 jC.
The whole study consisted of 4 independent runs of RTPCR in triplicate. In addition to samples, each run included
a standard curve, a non-template control and a negative RT
control. The levels of CART mRNA expression were
quantified relative to the level of the housekeeping gene
h-actin, using a standard curve method.
2.9. In situ hybridization
In situ hybridization with digoxigenin-labeled probes
was used to examine CART gene expression, as described
previously [21,22]. This technique measures specifically the
density of neurons expressing the CART gene above threshold levels, rather than the level of mRNA expressed per cell.
Following rapid decapitation, brains were removed, fixed
and stored [21]. On the day of use, brains were cut into 30
Am thick sections. An anti-sense cRNA probe (final concentration, 5 Ag/ml) labeled with digoxigenin was prepared
by in vitro transcription. Briefly, an 866-bp fragment of the
long alternatively spliced rat CART cDNA (a generous gift
of Dr. Mike Kuhar, Emory University, GA) was subcloned
into pBluescript SK () and linearized with Not1. Freefloating coronal sections were processed as described previously [21,22]. They were incubated with the cRNA probe
at 55 jC for 18 h and, based on pilot studies, a 6-h period
was chosen for the CART color reaction. The sense probe
control was performed in the same tissue and no signal was
found.
2.10. Quantification of in situ hybridization
A digital imaging system, with the help of a rat brain
atlas [27], was used for quantification of cell density
(density of cells/mm2) in a specific area of the brain,
according to procedures described previously [22]. The
hypothalamic areas examined were the arcuate nucleus
(ARC) and LHA (bregma 2.56 to 2.80 mm) and the
paraventricular nucleus (PVN) (bregma 1.80 mm). To
obtain a total of six separate readings per subject at a given
level, the observer, who was experimentally blinded, routinely analyzed the density of cells in three to four sections
at the same level, with a fourth section read if an area was
unreadable due to tissue damage.
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2.11. Data analysis
Caloric intake, presented as kcal/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.
With a standard statistical package (Statview), hypotheses
regarding diet groups, brain areas, 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. Within-group measures of
fat pad weights, hormones, metabolites and hypothalamic
CART cell density were related using a Pearson’s product
moment correlation. Correlations were performed on measures that showed significant changes compared to control
values under the various conditions in each of the experiments. All values are expressed as mean F S.E.M. 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.
Fig. 1. CART mRNA in the ARC and PVN nuclei of lean and obese rats in
Experiment 1, as measured by quantitative PCR. The data (mean F S.E.M.)
show a significant increase in CART mRNA levels, expressed as a ratio to
h-actin mRNA, in both pooled (Pool) and individual (Ind) samples from
obese rats (*P < 0.05 vs. lean rats).
3. Results
3.1. Experiment 1: relationship between CART, hormones
and body fat on a fat-rich diet
This experiment tested whether a change in CART
expression can be associated with shifts in hormones and
metabolites that are affected by an increase in body fat.
3.1.1. Behavioral, hormonal and metabolite measures
As shown in Table 1, the obese rats, subgrouped based
on their greater body fat pad weights (28 F 0.6 g) compared
to the lean rats (17 F 0.70 g), were heavier in body weight
and were also hyperphagic. Although leptin levels were
high in both groups, the obese rats had two-fold higher
levels of circulating leptin than the lean rats. Their levels of
insulin and triglycerides were also significantly increased,
with no change in NEFA (Table 1).
3.1.2. CART gene expression
Two sets of obese vs. lean rats on a HFD were examined
using quantitative PCR. CART mRNA, as a ratio to h-actin
Table 1
Measurements in lean and obese rats on a high-fat diet in Experiment 1
Body weight (g)
kcal intake (kcal/day)
Leptin (ng/ml)
Insulin (ng/ml)
Triglycerides (mg/dl)
NEFA (mEq/l)
Lean
Obese
428 F 7.3
101 F 3.5
8.9 F 1.7
1.2 F 0.13
64 F 5
0.70 F 0.11
500 F 7.2*
134 F 5.0*
21 F 2.5y
2.1 F 0.16*
83 F 8*
0.64 F 0.08
Data are mean F S.E.M. n = 6/group.
* P < 0.05 vs. lean.
y
P < 0.01 vs. lean.
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 a
60– 65% increase in the ratio of CART/h-actin mRNA in
the hypothalamus compared to lean rats (Fig. 1).
In addition to quantifying CART mRNA expression, in
situ hybridization using digoxigenin-labeled riboprobes
was used to investigate the density of cells expressing
CART mRNA in different hypothalamic nuclei in an
additional set of rats. Fig. 2 shows that the density of cells
expressing detectable levels of CART mRNA was significantly enhanced in the obese rats compared to lean subjects. This effect was strongest in the ARC, as illustrated in
Fig. 2A. This rise in CART expression in the ARC is
illustrated in the photomicrographs of Fig. 2B. Together
with the quantitative PCR data, these results demonstrate
that obese rats have significantly greater mRNA level in
CART neurons and higher density of CART-expressing
cells in hypothalamic nuclei involved in eating and body
weight regulation.
3.1.3. Relation of CART to leptin
Analyses revealed strong, positive correlations between
leptin and the density of cells expressing CART mRNA
across the whole group. This is evident in the ARC
(r = + 0.72, P < 0.01) and PVN (r= + 0.77, P < 0.01) across
the group, with no relationship seen in the LHA
(r = 0.07). A similar pattern was also observed between
body fat pad weights and CART mRNA in the ARC
(r = + 0.66, P < 0.05) and PVN (r = + 0.77, P < 0.01), but
not in the LHA (r = 0.50, P > 0.05). A weaker but still
significant relationship was also observed between triglyceride levels and CART cell density in the ARC (r= + 0.59,
K.E. Wortley et al. / Regulatory Peptides 117 (2004) 89–99
93
Fig. 2. CART cell density in the ARC, PVN and LHA of lean and obese rats on a HFD in Experiment 1 (N = 6/group), as measured by in situ hybridization. (A)
Bar graph shows an increase in CART cell density specifically in the ARC and LHA of obese rats (*P < 0.05 vs. lean). (B) Photomicrographs illustrate the
increase in CART cell density in the ARC of obese vs. lean rats.
P < 0.05), while no consistent relationship was evident
between insulin and CART in any area.
3.2. Experiment 2: relationship between CART, hormones
and dietary fat
This experiment tested CART expression in a model of
obesity associated with decreased insulin levels.
3.2.1. Behavioral, hormonal and metabolite measures
Subjects fed a MFD or HFD for 3 weeks were heavier and
had greater body fat compared to rats fed a LFD (Table 2).
Table 2
Measurements in rats fed a LFD, MFD or HFD in Experiment 2
Body weight (g)
Fat pad weight (g)
kcal intake (kcal/day)
Leptin (ng/ml)
Insulin (ng/ml)
Triglycerides (mg/dl)
NEFA (mEq/l)
LFD
MFD
HFD
478 F 5
15 F 0.8
107 F 4
14 F 1.1
2.4 F 0.2
85 F 10
0.61 F 0.05
480 F 8
19 F 0.9y
103 F 3
16 F 0.8*
1.7 F 0.24y
142 F 16y
1.7 F 0.16y
512 F 7y,§
23 F 1.0y,§
104 F 2
20 F 1.1y,§
2.0 F 0.28y
156 F 8y
1.6 F 0.09y
Data are mean F S.E.M. n = 7/group.
* P < 0.05 vs. LFD.
y
P < 0.05 vs. LFD.
§
P < 0.01 vs. MFD.
This increase in body weight was accompanied by, and
presumably attributed to, hyperphagia seen during the first
week on the HFD (112 F 2 kcal) vs. LFD (98 F 3 kcal,
P < 0.05), but not during week 3 (Table 2). Further, both the
MFD and HFD produced an increase in circulating levels of
leptin, as well as triglycerides and NEFA. Insulin levels,
however, were lower in the MFD and HFD rats compared to
the LFD rats. Whereas a HFD generally causes insulin levels
to rise with body fat over the long-term [29,30], their levels
during the first few weeks on a HFD, in association with a
relatively small increase in body fat, remain low presumably
due to the reduced amount of carbohydrate in the diet. These
endocrine changes differ from those observed in obese rats
(Experiment 1), which compared to lean rats displayed
elevated insulin levels, in addition to hyperphagia and no
change in NEFA.
3.2.2. CART gene expression
Two sets of rats, maintained on a HFD vs. LFD, were
tested using quantitative PCR. Since the results of the in situ
hybridization study in Experiment 1 revealed differences in
the density of CART-expressing cells in the different hypothalamic nuclei, CART mRNA in this experiment was
measured in ARC, PVN and LHA dissections. In the first
set of rats, CART mRNA was analyzed in pooled tissue
samples (n = 4/group) and in the second set of rats it was
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K.E. Wortley et al. / Regulatory Peptides 117 (2004) 89–99
3.2.3. Relation of CART to leptin
Within-group analyses of the MFD and HFD groups
showed that CART cell density in the ARC and PVN was
consistently and closely related to circulating leptin, with
correlations ranging from r= + 0.76 to r= + 0.91 ( P < 0.05).
The density of cells expressing CART in the PVN, but not
the ARC, was also related to body fat, in both the MFD
(r= + 0.81, P>0.05) and HFD (r= + 0.91, P < 0.05) groups.
These relationships were not apparent in the LFD animals,
nor were they seen in the LHA of any of the groups. No
significant correlations were detected between CART cell
density and the measures of insulin or triglycerides, in
addition to NEFA.
Fig. 3. CART mRNA (ratio to h-actin mRNA) in the ARC, PVN and LHA
of rats maintained on a LFD or HFD in Experiment 2, as measured by
quantitative PCR. The data (mean F S.E.M.) exhibit a significant increase
in CART expression in both pooled (Pool) and individual (Ind) samples
from HFD rats (*P < 0.05 vs. LFD).
analyzed in individual samples (n = 7/group). Fig. 3 shows
that the HFD subjects exhibited a clear increase in the ratio
of CART/h-actin mRNA in the ARC dissection of the
pooled ( + 74%) and individual ( + 60%) tissue samples. A
somewhat smaller effect ( + 50%, p < 0.05) was evident in
the PVN of both sets, whereas the LHA exhibited a 56%
increase ( p < 0.05) only in the pooled tissue, with little
change evident in the individual samples (Fig. 3).
Again, in situ hybridization was used to measure the
density of CART-expressing cells in the different hypothalamic nuclei. As illustrated in Fig. 4, a rise in dietary fat
from 10% to 50% caused a significant increase in the
density of cells expressing detectable levels of CART
mRNA in the ARC and PVN, but not the LHA.
3.3. Experiment 3: relationship between CART and
propensity toward obesity on a HFD
This experiment tested whether a change in CART
expression can be associated with a rise in leptin and
triglycerides in the absence of a change in body fat,
specifically in rats in a pre-obese state. In this experiment,
rats maintained for only 5 days on a HFD were examined
with respect to their weight gain during this 5-day period
(see Section 2).
3.3.1. Behavioral, hormonal and metabolite measures
During the 5-day period on a HFD, the obesity-prone
subjects exhibited a weight gain of 11.1 F 0.5 g/day, compared to 6.7 F 0.1 g/day for the obesity-resistant subjects.
With only a small elevation of body weight ( + 26 g), the
obesity-prone rats showed no difference from the obesityresistant rats in the weight of their dissected body fat pads
(Table 3). Despite their similar body fat, the obesity-prone
compared to obesity-resistant subjects exhibited changes in
the endocrine and metabolic parameters that were similar to
those seen in already obese rats in Experiment 1. These
included a significant increase in kcal intake, leptin, insulin
and triglycerides, with no change in NEFA levels (Table 3).
3.3.2. CART gene expression
In situ hybridization revealed a significant change in
CART cell density that was localized to the ARC. As
Table 3
Measurements in obesity-resistant and obesity-prone rats in Experiment 3
Fig. 4. CART cell density in the ARC, PVN and LHA of rats maintained on
a LFD, MFD or HFD in Experiment 2 (N = 7/group), as measured by in situ
hybridization. The data (mean F S.E.M.) reveal an increase in CART cell
density specifically in the ARC and PVN (*P < 0.05 vs. LFD, zP < 0.05 vs.
MFD).
Final body weight (g)
Fat pad weight (g)
kcal intake (kcal/day)
Leptin (ng/ml)
Insulin (ng/ml)
Triglycerides (mg/dl)
NEFA (mEq/l)
Obesity-resistant
Obesity-prone
353 F 5
12 F 1.1
112 F 4.6
12 F 0.9
1.1 F 0.16
44 F 4.8
0.81 F 0.03
379 F 7y
13 F 0.9
138 F 8.6*
16 F 0.7y
1.8 F 0.18y
54 F 2.7*
0.70 F 0.03
Data are mean F S.E.M. n = 8/group.
* P < 0.05 vs. obesity-resistant.
y
P < 0.01 vs. obesity-resistant.
K.E. Wortley et al. / Regulatory Peptides 117 (2004) 89–99
95
illustrated in Fig. 5, a significant increase in the density
of cells expressing detectable levels of CART mRNA
was evident in the ARC of the obesity-prone compared
to obesity-resistant rats. This change in CART was
specific to the ARC, not seen in the PVN (1888 F 105
vs. 1686 F 115 objects/mm2) or LHA (992 F 35 vs.
1035 F 83 objects/mm2).
3.3.3. Relation of CART to leptin
Within-group analyses revealed a significant, positive
correlation between leptin levels and the density of cells
expressing CART mRNA in the ARC (r= + 0.56, P < 0.05)
but not in the other areas. No correlations between CART
and the measures of body fat, insulin or triglycerides were
evident.
3.4. Experiment 4: effect of CART on circulating hormones,
metabolites and enzyme activities
To examine the possible physiological consequences of
an increase in hypothalamic CART as shown in Experiments
1 –3, rats were tested, in the absence of food, following
intracerebroventricular injection of synthetic CART peptide.
3.4.1. Hormonal and metabolite measures
Compared to rats injected with saline, CART (two i.c.v.
injections of 500 pmol each) produced a 66% increase in
circulating levels of NEFA (Fig. 6A). This elevation in
NEFA levels was seen in the absence of a change in body
weight (336 F 12 vs. 342 F 8 g), body fat pad weights
(4.1 F 0.46 vs. 4.8 F 0.46 g), leptin (2.12 F 0.35 vs.
1.65 F 0.39 ng/ml), insulin (0.76 F 0.08 vs. 0.89 F 0.16
ng/ml) or triglycerides (72 F 3.5 vs. 77 F 4.1 mg/dl).
Fig. 6. (A) Serum NEFA levels 2 h after intracerebroventricular injection of
saline or CART (500 pmol/3 Al) in Experiment 4 (N = 8/group). The data
(mean F S.E.M.) demonstrate that CART (55 – 102) increases plasma levels
of NEFA. (B) aLPL in retroperitoneal adipose tissue following intracerebroventricular injection of saline or CART (500 pmol/3 Al in
Experiment 4 (N = 8/group)). The data (mean F S.E.M.) show that CART
(55 – 102) decreases aLPL activity (*P < 0.05 vs. saline, yP < 0.01 vs.
saline).
3.4.2. Metabolic enzyme measures
In this same set of rats, measurements of adipose
lipoprotein lipase activity (aLPL), an enzyme involved in
the hydrolysis of triglycerides in the vascular endothelium
and transport of fatty acids [31,32], were taken in the
dissected retroperitoneal fat pad. Central CART injection
caused a significant decrease in aLPL activity (Fig. 6B).
Measurements of the activity of FAS, an enzyme involved in
de novo lipogenesis [33], were also taken in the same fat
pad. Compared to the saline control, central administration
of CART peptide had no apparent impact on the activity of
this enzyme (101 F 5.4 vs. 98 F 7.8 AM).
4. Discussion
The results of these experiments revealed a strong
relationship, under physiological conditions, between endogenous leptin and CART in the ARC. They further
suggested a possible functional role for CART in the control
of fat deposition.
4.1. CART expression in the ARC is stimulated in close
association with leptin under physiological conditions
Fig. 5. CART cell density in the ARC of obesity-resistant and obesity-prone
rats in Experiment 3 (N = 8/group). The data (mean F S.E.M.) show an
increase in CART cell density in the ARC of obesity-prone rats, which have
elevated leptin levels (*P < 0.05 vs. obesity-resistant).
This study demonstrated that, under natural feeding
conditions, elevated CART expression in the ARC is
accompanied by a spontaneous rise in endogenous leptin.
The increase in CART expression was apparent as a rise
in CART mRNA level, revealed by RT-PCR, and in the
density of CART-expressing neurons in the ARC,
revealed by in situ hybridization. The increase in the
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K.E. Wortley et al. / Regulatory Peptides 117 (2004) 89–99
density of CART-expressing cells most likely reflects a
rise in expression from basal levels undetectable by
digoxigenin probes. In published studies, a close link
between circulating leptin and CART-containing neurons
in the ARC has been suggested by the findings that
CART gene expression in this nucleus is virtually absent
in ob/ob mice, and it is restored to normal levels in this
strain by leptin administration [1]. Building on these
previous reports, the present results indicated that a rise
in endogenous leptin level is most strongly associated
with a rise in CART mRNA in the ARC, with a weaker
relation apparent in the PVN and no relation detected in
the LHA. This pattern, consistent with another study
examining CART expression in whole hypothalamus
[10], is evident in rats given free access to a fat-rich
diet, compared to a diet with moderate or low levels of
fat. Rats that become obese or obesity-prone on a fat-rich
diet, compared to lean rats, also exhibit elevated CART in
this nucleus, together with higher leptin levels. The
possibility of a direct interaction between leptin and
ARC CART neurons under these physiological conditions
is strengthened by the strong, positive correlations consistently detected between leptin levels and the density of
cells expressing CART mRNA in the ARC of the various
groups tested.
This site-specificity is consistent with investigations
showing that the expression of the leptin receptor, OBRb,
is particularly dense in the ARC [11,12] and that approximately 35% of CART-containing neurons in this nucleus
co-localize OBRb mRNA [13]. Also, leptin injection
produces a considerably stronger increase in c-Fos and
SOCS-3 mRNA expression in CART-containing neurons
of the ARC, compared to that observed in the PVN or
LHA [13,18]. Together, these findings provide strong
evidence that endogenous leptin in normal rats impacts
on hypothalamic CART, specifically in neurons of the
ARC.
4.2. CART neurons in the PVN are less responsive to leptin
Whereas a dense population of CART-expressing neurons also exists in the PVN [33], the relationship between
leptin and CART invariably seen in the ARC appears less
consistent and weaker in this nucleus. The density of cells
expressing CART mRNA in the PVN was unaltered in the
obesity-prone rats, despite an increase in leptin over the 5day test period. This weaker relationship in the PVN may be
related to the finding that OBRb mRNA is less dense in this
nucleus [34]. Also, c-Fos and SOCS-3 mRNA in PVN
neurons are less responsive than in ARC neurons to the
stimulatory effect of leptin [13].
Nevertheless, PVN CART neurons may respond to
leptin under conditions where this hormone is chronically
elevated, together with body fat. Both the obese and HFD
rats, maintained for 3 weeks on the fat-rich diet, exhibited
an increase in the density of cells expressing CART
mRNA in the PVN, along with elevated leptin and body
fat. They also showed strong, positive correlations between these measures. These relationships, in contrast,
were not evident in the PVN of obesity-prone rats, which
were maintained for only 5 days on a high-fat diet, had
normal body fat pad weights and showed only a small
increase in leptin.
4.3. CART neurons in the LHA show little relation to leptin
In the LHA, CART neurons exhibited different characteristics from those in the ARC and PVN. In this area,
CART neurons were relatively unresponsive to dietary
fat, exhibiting only a small change in the pooled tissue
samples assayed using the quantitative PCR. They were
also unaffected in obesity-prone rats and showed only a
small rise in CART mRNA in obese compared to lean
subjects. These results are consistent with the finding
that ob/ob mice and obese Zucker rats show a considerably smaller decline in CART mRNA in the LHA than
in the ARC compared to control animals [1]. Thus, the
subpopulations of CART neurons in the LHA, ARC and
PVN may have distinct functional roles in energy
homeostasis.
4.4. Positive association between leptin and ARC CART
neurons is independent of changes in body fat and caloric
intake
Analyses of obesity-prone rats provide insight into the
question as to whether endogenous CART is stimulated
by other signals, besides leptin, that are related to body
fat. There is evidence that rapid changes in leptin can
occur under conditions of stable body fat [35]. This is
also evident in Experiment 3, where leptin levels were
elevated in the obesity-prone subjects, along with higher
caloric intake, prior to any measurable increase in body
fat. This pattern was accompanied by a rise in the
density of cells expressing CART mRNA in the ARC,
indicating the greater importance of leptin, relative to
body fat itself, in modulating CART expression. A
positive correlation within this group, between CART
mRNA and leptin levels but not body fat, underscores
this relationship.
Although all three of the obesity models studied were
associated with some degree of hyperphagia, it is unlikely
that a simple rise in caloric intake was responsible for
stimulating CART expression. Ob/ob mice are markedly
hyperphagic yet lack CART gene expression in the ARC,
and leptin injection decreases caloric intake while stimulating CART expression. Further, no correlations were
detected in the present study between caloric intake and
CART expression. Although additional studies are needed
to substantiate this idea, the available evidence fails to
support a role for caloric intake in the regulation of
hypothalamic CART.
K.E. Wortley et al. / Regulatory Peptides 117 (2004) 89–99
4.5. ARC CART mRNA rises in parallel with circulating
lipids
A rise in leptin levels is often accompanied by an
increase in circulating lipids [19]. Consistent with this,
serum triglyceride levels were elevated, together with leptin
and ARC CART mRNA, in each of the three animal models
tested in the present experiments. In fact, a significant
correlation between triglyceride levels and CART mRNA
was detected in Experiment 1, in rats on a high-fat diet.
Recent studies show that an increase in circulating lipids
stimulates the expression of orexin and galanin in the
hypothalamus [21,22]. Thus, a similar relation between
hypothalamic CART and circulating lipids, in addition to
leptin, needs to be considered.
4.6. CART neurons are unrelated to measures of insulin
under physiological conditions
Whereas an elevation in leptin levels together with
body fat is frequently associated with a rise in insulin
levels [28,29], the findings of the present investigation
dissociated insulin from changes in CART. Specifically,
while CART was elevated in all animal models that
showed an increase in leptin levels, this effect occurred
despite variable shifts in insulin, which were reduced in
normal rats on a high-fat diet, while elevated with in obese
rats. This dissociation was supported by the lack of
significant correlations detected between CART mRNA
and insulin.
97
effects of CART are similar to those reported for leptin.
Injection of this hormone decreases aLPL activity [38],
stimulates lipolysis [39] and increases fat oxidation
[40,41].
4.8. Leptin may stimulate CART specifically to control body
fat accrual on a fat-rich diet
These similarities between leptin and CART support the
idea that CART is a central mediator of leptin’s actions
under physiological conditions [1]. The evidence further
suggests that this occurs specifically under conditions of a
HFD, which stimulates both CART and leptin levels. On a
fat-rich diet, leptin may increase the activity of CART
neurons specifically to reduce lipid uptake and storage, by
minimizing aLPL activity, and to enhance fat oxidation,
effects that limit excessive body fat accrual on this diet.
Consistent with this proposal is the finding that a HFD [42],
similar to CART [5], increases markers of thermogenic
activity in brown adipose tissue. The importance of leptin
in activating the CART pathway is reflected in the additional finding that CART-deficient mice are heavier and
have greater fat mass than their wild-type littermates specifically on a HFD, when leptin levels rise, but not on a
LFD, when leptin remains low [6]. The evidence that central
CART injection inhibits food intake and causes weight loss
in obese fa/fa rats, which lack the leptin receptor and are
unresponsive to leptin [37], indicates that CART functions
downstream of the leptin receptor in mediating leptin’s
effects on lipid mobilization and oxidation.
4.7. CART has similar effects to leptin on lipid metabolism
5. Conclusion
The present findings lead one to consider the possibility
that the functional role of hypothalamic CART in body
weight regulation may be similar to the central effects of
exogenous leptin. The results of the CART injection test,
in the absence of food, demonstrated that this peptide
increases circulating levels of NEFA. This rise in NEFA
without food may reflect the mobilization and hydrolysis
of stored triglycerides by hormone-sensitive lipase in white
adipose tissue [36]. CART also reduced the activity of
aLPL, an enzyme in the vascular endothelium of adipose
tissue that releases fatty acids from triglyceride-rich lipoproteins for uptake into adipocytes [30]. In contrast to its
suppression of aLPL, CART had no apparent effect in
adipose tissue on the activity of fatty acid synthase, an
enzyme involved in de novo lipogenesis [32]. Thus,
together with recent studies showing that chronic CART
administration reduces body weight while decreasing respiratory quotient, reflecting enhanced lipid oxidation
[4,37], the present findings suggest that CART’s overall
effect on a fat-rich diet is to cause a shift from lipid
storage to lipid utilization. Whereas the technical limitations of central peptide injections leave open the possibility
of non-physiological changes, it is notable that these
The findings of this investigation focus attention on
leptin in terms of its relation to hypothalamic CART
function under physiological conditions. It is postulated that
CART neurons are activated on a HFD as a result of
elevated leptin secretion. The rise in leptin stimulates CART
neurons most strongly in the ARC, with weaker effects in
the PVN and little change in the LHA. The activation of
these CART neurons, then, stimulates pathways that decrease aLPL activity and increase plasma NEFA availability.
These actions, resulting in a reduction in lipid storage and
promotion of fat utilization, help to minimize excessive
body fat accrual under conditions of positive energy balance. This close relationship of CART to endogenous leptin
may also extend to circulating triglycerides, which are
known to affect leptin gene expression [43].
Acknowledgements
This study was supported by grants from the Wellcome
Trust (058240) and the National Institutes of Health
(MH43422). K.E.W. and G.Q.C. contributed equally to the
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K.E. Wortley et al. / Regulatory Peptides 117 (2004) 89–99
experimental work. We thank Dr. Michael Kuhar at Emory
University for supplying the CART plasmid. We also thank
Amgen (CA) for their analyses of serum triglyceride and
NEFA levels and their generous supply of leptin. We are
very grateful to Ms. Kate Sepiashvili for her help in the
preparation of this manuscript.
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