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Divisions of Endocrine Research (M.A.A., D.P.S., M.L.H., H.M.H.), Research Technologies and Proteins (D.D.Y., N.F., A.K.), and Statistical and Mathematical Sciences (Y.-F.C.), Eli Lilly & Co., Indianapolis, Indiana 46285
Address all correspondence and requests for reprints to: Dr. Hansen M. Hsiung, Division of Endocrine Research, Lilly Research Laboratories, Eli Lilly & Co., Indianapolis, Indiana 46285. E-mail: hsiung_hansen_m{at}lilly.com
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The wide distribution of Cart mRNA and its encoded peptide in the brain and other endocrine tissues, such as adrenal gland and lung, suggests that CART is involved in diverse and perhaps very important neuroendocrine functions. Cart mRNA and peptide were found in the nucleus accumbens and basolateral amygdala, suggesting CART’s role in drug-induced reinforcement and reward (4). CART peptide is present in the hypothalamus, pituitary, and adrenal glands, suggesting CART’s roles in the hypothalamus-pituitary-adrenal axis (5). Furthermore, neurons in retina, olfactory bulb, sensory barrels, and the dorsal horn of the spinal cord contain high concentrations of CART peptide, indicating that it may also be involved in sensory processing (4, 6).
Cart mRNA species were found to be one of the most abundant mRNAs in rat hypothalamus (7). In some obese or hyperphagic rodent animal models, leptin administration increased the Cart and POMC gene expression in hypothalamus (8, 9, 10). Cart and POMC expression has been colocalized in arcuate and retrochiasmatic neurons, suggesting an important role of both peptides in feeding (11). CART’s role in feeding has been further examined by its expression in response to fasting states in animals. Cart mRNA expression in the arcuate nucleus and dorsomedial nucleus of the hypothalamus was decreased in normal fasted rats, Zucker (fa/fa) rats, and ob/ob mice (10). In addition, Cart expression in the arcuate can be restored by ip administration of leptin to fasted mice (10). That CART is an anorexigenic signal was further supported by the result that intracerebroventricular injection of the peptide completely blocks the NPY-induced feeding response (10). Conversely, when CART antiserum was injected intracerebroventricularly into rats, a significant increase in nighttime feeding, especially at the end of the feeding cycle, was observed (10).
To define further the physiological role of CART, we generated mice carrying a targeted deletion of the Cart gene. In this study, we investigated the effects of CART deficiency on feeding and on energy homeostasis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Construction of Cart targeting vector and generation of a targeted
mutant Cart mouse strain
A 300-bp mouse Cart cDNA fragment (Fig. 1A
) spanning
all three exons of the Cart gene was generated by RT-PCR
using a mouse brain cDNA library (CLONTECH Laboratories, Inc., Palo Alto, CA) as a template. This 300-bp fragment was
generated using two oligonucleotide primers (CART1F,
5'-TGCTACCTTTGCTGGGTGCCCGTGC-3'; and CART2R,
5'-CCCTTCACAAGCACTTCAAGAGGAA-3') designed from the homologous regions
of the rat and human Cart cDNA sequences. This 300-bp cDNA
probe was used to screen a mouse ES-129/SvJ I bacterial artificial
chromosome (BAC) genomic library (Genome Systems, Inc.,
St. Louis, MO) to obtain a BAC clone that contains the full length
Cart gene. This BAC clone was then mapped with multiple
restriction endonucleases, and the restriction fragments containing the
Cart gene were subcloned into pBluescript
SK+ (Stratagene, La Jolla, CA) or
pZErO-1 (Invitrogen, Carlsbad, CA). The final targeting
vector in pZErO-1 contained a 2.8-kb 5' Cart genomic
fragment (HindIII–Asp718) and a 3.5-kb 3'
genomic fragment (ScaI–EcoRI); 650 bp of the 5'
untranslated region and exons 1 and 2 of the Cart gene were
deleted (Fig. 1A). This targeting vector was linearized with
HindIII and introduced into E14 mouse embryonic stem (ES)
cells. G418 (350 µg/ml) was added 24 h after transfection, and
the antibiotic concentration was maintained for 8 d before stably
transfected clones were selected. A total of 240 G418-resistant clones
were picked and expanded for Southern hybridization analysis, and 3
correctly targeted ES cell clones were identified. Two of the clones
were injected into C57BL/6 blastocysts resulting in 12 male chimeric
offspring, which were bred to female C57BL/6 mice. However, only
chimeras from 1 of the injected ES cell clones transmitted the
disrupted Cart allele through its germline (as indicated by
agouti coat color) when bred to female C57BL/6 mice.
F1 agouti-coated mice were screened by PCR to detect
those with the heterozygous genotype. These heterozygotes were bred
together to obtain F2 mice. Genotypes of
F2 mice were determined by PCR and confirmed by
Southern hybridization. All phenotype testing was performed on
F2 hybrid mice (B6 129F2). Five
male or female mice with each genotype (wild-type [WT],
heterozygous [Cart+/-], and
homozygous Cart deficient
[Cart-/-]) were selected for
phenotypic studies.
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) was used as a 3' external probe to
detect correctly targeted ES cells and to confirm mouse genotypes. This
probe was prepared based on a segment of the mouse genomic sequence
downstream of the Cart gene sequence used for the
construction of the targeting vector. Targeted disruption of the
Cart gene with the PGK neomycin phosphotransferase
gene (pGKneo) cassette introduced two additional PvuII
recognition sites into the Cart gene loci. Consequently, the
probe detected a PvuII fragment of greater than 12 kb in
length with the wild-type allele(s) and a 6-kb PvuII
fragment (Fig. 1B) with the correctly targeted, mutant Cart
allele(s).
Mouse genotyping by PCR
Genotype analysis of large numbers of mice was facilitated by
PCR (Fig. 1C). Multiplex PCR was performed using a common 5' primer
(CARTcF, 5'-TATGTGTACACGAGTGCAGG-3') with both of the two different 3'
primers to amplify regions of DNA for genotyping. The sequence of one
of the 3' primers was derived from a segment of wild-type
Cart DNA that was replaced by the pGKneo cassette in the
targeted allele (CARTwtR, 5'-AAGGTAGCAGTAGCAGCAGG-3'). The sequence of
the second 3' primer was derived from a segment of the pGKneo cassette
gene sequence (CARTmutR, 5'-GAAAATGGCCGCTTTTCTGG-3'). Using the PCR
protocol suggested by the manufacturer (TaKaRa Biomedicals, Shiga,
Japan), the wild-type allele produced an 880-bp DNA fragment and the
mutant allele produced a 658-bp fragment (Fig. 1C).
Detection of CART expression in brain by RNA (Northern)
hybridization
Whole brains were removed from euthanized 2-month-old WT,
Cart+/-, and
Cart-/- mice of both sexes. These
specimens were immediately frozen in liquid nitrogen until total brain
poly(A+) RNAs were extracted using an mRNA
isolation kit (Roche Diagnostics, Indianapolis, IN). Two
micrograms of each poly(A+) RNA sample was loaded
onto a 1.1% formaldehyde agarose gel and subjected to electrophoresis
at 100 V for 4 h. RNAs were transferred by capillary action in
20x sodium chloride-sodium citrate (3 M NaCl,
0.3 M sodium citrate) to Hybond-XL
(Amersham Pharmacia Biotech, Zurich, Switzerland)
overnight. The mRNA was cross-linked to the membrane by UV light in a
UV Stratalinker (Stratagene, La Jolla, CA). The resulting
blot was dried and then incubated in hybridization solution (5x sodium
chloride-sodium phosphate-EDTA, 0.2% SDS, 5x Denhardt’s reagent,
50% formamide, 10% dextran sulfate, and 100 µg/ml denatured salmon
sperm DNA) overnight at 42 C. The same 300-bp mouse Cart
cDNA probe (Fig. 2A) used for library
screening was used to detect the Cart transcript. The
32P-labeled probe was then stripped from the
membrane, and the membrane was further hybridized to a ß-actin probe
to confirm that all RNA samples were not degraded and that comparable
quantities were loaded onto the gel (Fig. 2B).
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Body composition by dual energy x-ray absorptiometry (DEXA)
scan
We used a pDEXA Forearm Bone Densitometer (Norland Medical Systems, Inc., White Plains, NY) to perform DEXA scans on
each animal to determine their bone, lean, and fat masses. This
densitometer uses an x-ray generator, a "K-edge" filter, two solid
state detectors, and proprietary energy discrimination software to
determine the attenuation of high and low energy photon counts as they
pass through the animal. Measurements were performed at a scan rate of
10 mm/sec and a resolution of 0.5 x 0.5 mm. The animals were
sedated in a Plexiglas container by inhalation of isoflurane and
positioned for the scan with all limbs and tail tucked under the
animal, and the container and animal were placed on the
spectrophotometer. The attenuation of the photon energies was
proportional to the density of the tissue that they were passing
through. The software transformed this count data into bone, lean, and
fat mass. Body composition analyses by DEXA scan were performed during
the 1st, 7th, 14th, 22nd, and 29th wk of the feeding study.
Indirect calorimetry
Energy expenditure and respiratory quotient measurements were
obtained using an Oxymax (Columbus Instruments International Corp.,
Columbus, OH) open circuit indirect calorimetry system. These
measurements were taken when the animals were 5 wk of age, after 2 wk
of acclimation to the high fat diet, and before any difference in body
weight was noted. After the system was calibrated against standard gas
mixtures, the animals were individually placed into acrylic calorimeter
chambers with food and water. To measure O2 and
CO2 by paramagnetic and spectrophotometric
sensors, respectively, the system automatically withdrew gas samples
from each chamber hourly for approximately 24 h. The system then
calculated the volumes of O2 consumed (ml/kg body
weight) and CO2 generated (ml/kg body weight) by
each animal in 1 h. The respiratory quotient was the ratio of the
volumes of CO2 generated to
O2 consumed. Energy expenditure was calculated as
the product of the calorific value of oxygen (=3.815 + 1.232 x
respiratory quotient) and the volume of O2
consumed. Daily fuel use was determined by calculating the total
calories expended in 1 d. We used Flatt’s proposal
(11A ) and assumed that protein oxidation and intake were
equivalent in adult stable animals to calculate the proportions of
protein, fat, and carbohydrate used during the 24-h period. Caloric
intake in a 24-h period was the product of the mass (g) of food
consumed in 1 d and the nutritional content of the diet (kcal/g).
Ambulatory and fine locomotion of each animal were also detected during
the 24-h period. Ambulatory movement was measured by counting the
number of times an animal broke adjacent light beams during the
calorimetry measurements. Fine movement was calculated by subtracting
the ambulatory movement from the total number of beam breaks (same or
adjacent) that occurred during the same period. The first 2 h of
measurements was used as a period of adaptation for the animals, and
the data obtained during this period were excluded from analysis.
Serum leptin and free T3 measurements
Blood was collected by cardiac puncture from mice on a regular
chow or a high fat diet. Serum leptin and free T3 concentrations were
measured by RIA (Linco Research, Inc., Reference
Laboratory Division, St. Charles, MO).
Statistical method
Measurements were taken over time for each animal. The mixed
models were applied to test the difference in various genotypes with
respect to body weight and fat mass. For each animal, measurements were
serially correlated. Therefore, in SAS mixed models (12),
we adopted the time series covariance structure AR1, in which the
measurements taken at adjacent times are assumed to be more highly
correlated than two measurements taken several time points apart. Based
on standard errors derived from this model with respect to body weight,
food consumption, and fat mass, statistical comparisons were made
between gender and genotype groups at each time point. Body weight on d
1 was considered the baseline for making adjustments of heterogeneity
between animals.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Using
Southern hybridization analysis to screen 240 ES cell clones, we
detected 3 ES clones (E11 and G1-8 are shown in Fig. 1B) that carried a
correctly targeted Cart allele, as shown by the presence of
both 6- and 12-kb fragments in the hybridization result (Fig. 1B).
Chimeras of only one of the ES cell clones transmitted the mutation
through the germline when mated with C57BL/6 females.
F1 heterozygous mice were intercrossed to generate
F2 WT, Cart+/-, or
Cart-/- mice (Fig. 1C).
The Cart-/- mice were born at the
expected Mendelian frequency, suggesting no embryonic lethality.
Northern hybridization analysis demonstrated that
Cart-/- mice did not express
Cart mRNA in the brain and
Cart+/- mice expressed
Cart mRNA at reduced levels (Fig. 2). Furthermore, we did
not detect differential expression of Cart mRNA as a result
of gender differences (Fig. 2).
The Cart-/- mice were about the same size or slightly smaller at weaning than their wild-type siblings (male WT, 13.5 g vs. male Cart-/-, 13.6 g; female WT, 13.5 g vs. female Cart-/-, 11.7 g). CART-deficient mice appeared to be healthy and were viable into adulthood. The gross anatomy and histology of the brains and other organs of the CART-deficient mice were unremarkable (data not shown). Both female and male mutant mice were fertile and raised normal sized litters.
After the mice were weaned, we initiated feeding studies using a high
fat diet to increase the likelihood of detecting phenotypic differences
between WT and mutant mice. To ensure that food intake was not affected
mainly by significant changes in body weight, we analyzed the weekly
food intake data in two time intervals (1–14 vs. 15–29 wk;
Fig. 3) or three intervals (1–10,
11–20, and 21–29 wk; data not shown) during the study. In general, we
did not observe any changes in food consumption pattern during the
study intervals (Fig. 3), suggesting that differences in food
consumption were not attributable to increases in body weight alone.
Fig. 3 shows that the statistically significant increases in weekly
food consumption were observed in female
Cart+/- mice (P <
0.001) or female Cart-/- mice
(P < 0.001) compared with their WT controls. However,
we only observed a significant increase in weekly food consumption in
male Cart-/- mice (0.001 
P < 0.05 for 1–14 wk and P < 0.001
for 15–29 wk) but not in male
Cart+/- mice compared with their WT
controls (Fig. 3). In fact, inexplicably, male
Cart+/- mice consumed significantly
less food than their WT controls (Fig. 3).
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1). Body weights in
male Cart+/- mice were not different
from those of their WT littermates. Table 1 also shows that significant
body weight differences were observed as early as the 7th wk of the
feeding study in male mice, whereas the body weight differences in
female Cart-/- and
Cart+/- mice became statistically
significant only after the 10th and 13th wk, respectively.
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and Table 1). Table 1 also shows
that fat mass in male Cart-/- mice
was significantly greater than that of their WT littermates beginning
at the 8th wk of the study, whereas fat mass of the female mutant mice
increased significantly after the 15th wk of the feeding study.
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). A slight but not statistically
significant decrease in ambulatory movement between male and female
mutant mice and WT controls was detected (data not shown).
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). However, we observed a significant
mean body weight increase (0.001 P < 0.05) in
Cart-/- or female
Cart+/- mice fed a high fat diet
compared with the mice of the same genotypes fed regular chow at this
age (Fig. 6).
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). We did not observe any differences in serum
leptin or free T3 levels for mice fed a regular chow diet (data not
shown). In mice fed a high fat diet, serum leptin levels correlated
with the fat mass but T3 levels were not changed (Table 2). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we generated CART-deficient mice and studied the effect
of CART deficiency on energy homeostasis. Strain C57BL/6 was used to
generate CART-deficient mice because mice of this strain are
susceptible to adiposity when fed a calorie-dense diet. In fact, WT
C57BL/6 mice are known to become leptin insensitive, hyperglycemic, and
obese over time on a high fat diet alone (15). The
contributions of the two different genetic backgrounds (C57BL/6 and
129/Ola) in the F2 hybrids probably resulted in some
variation within the study groups that we observed. To increase the
likelihood of detecting phenotypic differences between obese WT and
obese mutant mice, we challenged the mice with a high fat diet starting
from weaning. We showed that the food intake (Fig. 3) and body weight
(Fig. 4 and Table 1) of female
Cart-/- and
Cart+/- and male
Cart-/- mice (compared with WT) were
increased on a high fat diet. However, to our surprise, male
Cart+/- mice had decreased food
consumption compared with their WT or
Cart-/- littermates. The cause of
this aberration is largely unknown. Furthermore, female
Cart-/- and
Cart+/- and male
Cart-/- mice also had greater fat
mass than their WT littermates (Fig. 5 and Table 1). Finally, body
weights of mutant mice were not increased during 14 wk of feeding study
when animals were fed a regular chow diet, suggesting that CART
deficiency can predispose mice to obesity only in a calorically rich
environment (Fig. 6). It is surprising that body weights of WT (B6
129F2) and Cart+/- mice
were unaffected by the high fat diet (Fig. 6). It is likely that a
14-wk feeding study with a high fat diet is not long enough to affect
body weight in WT mice and male
Cart+/- mice but is definitely long
enough to affect body weight in female
Cart-/- and
Cart+/- and male
Cart-/- mice (Fig. 6).
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).
Ambulatory movement of CART-deficient mice was decreased slightly from
that of their WT littermates (data not shown). We observed a significant difference in food consumption between mutant and WT mice only after 4–6 wk of the feeding study (data not shown). However, we did observe a significant increase in food consumption before we observed a significant body weight increase in mice; therefore, we believe the effect of CART is more pronounced on food consumption than on metabolism. Our indirect calorimetry results also support this conclusion.
Our results demonstrate that the absence of Cart expression induces adiposity when the mice were consuming a calorie-rich diet. These data suggest that CART participates in the regulation of energy homeostasis. When animals are fed a calorie-rich diet and a net gain of energy is stored, many peripheral signals, including leptin and insulin, are secreted to inform the hypothalamus (the energy regulatory center) of energy abundance. In turn, physiological adjustments such as decreased food intake and increased energy expenditure occur. CART has been proposed to participate as an efferent signal to help limit positive caloric balance. Indeed, our data indicate that CART-deficient mice were more hyperphagic than WT mice as they gained body fat, suggesting an impaired ability to adjust. With time, this gradually led to obesity.
The phenotypic changes related to fat accretion in CART-deficient mice are distinct from those of MC3R-deficient, MC4R-deficient, or POMC-deficient mice (16, 17, 18, 19, 20). Up to 17 wk of age and at the 14th wk of the feeding studies, CART-deficient mice fed a regular chow diet do not gain body fat compared with their WT littermates. In contrast, MC4R-null mice (18), or POMC-null mice (20) exhibited dramatic increases in food intake and body weight compared with their WT siblings, whereas MC3R-null mice exhibited a trend toward increased fat mass without an increase in body weight (16, 17). In addition, MC3R- and MC4R-deficient mice sustained on regular chow showed significant increases in serum leptin and insulin levels, whereas CART deficiency did not cause a change in leptin or free T3 levels in mice fed regular chow. These observations suggest that CART peptide might play a different role in energy homeostasis than other anoretic peptides such as POMC peptides or leptin.
We observed a significant phenotype difference between male and female
Cart+/- mice. Male
Cart+/- mice had a phenotype similar
to that of male WT mice, whereas female
Cart+/- mice behaved like female
Cart-/- mice. Fagergren and Hurd
(21) showed that male rats express higher levels of
Cart mRNA in the mesolimbic region of the brain than female
rats. Therefore, a phenotype difference between male and female
Cart+/- mice may be attributable to
gender-based differential Cart expression in specific areas
of the brain. However, we did not detect any differential expression of
the mouse Cart mRNAs in the whole brain of either WT or
Cart+/- mice (Fig. 2).
In contrast to the expected phenotype of hyperphagia, our results show that CART deficiency in mice does not cause the dramatic changes in energy intake that are seen in leptin- or MC4R-deficient mice (18, 19). Recently, Kimmel et al. (22) showed that injection of CART peptide into the ventral tegmental area of the rat brain induced locomotor activity and promoted conditioned place preference, suggesting that the CART peptide may play an important role in other central nervous system functions, such as reward or motivation. The high abundance of CART in the brain and the lack of dramatic effects on energy homeostasis (particularly when mutant mice were fed regular chow) suggest that other activities of the CART peptide in the brain indirectly caused changes in some parameters of energy homeostasis. Our results, however, demonstrate that CART is likely a minor player among the numerous factors involved in energy homeostasis.
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Acknowledgments |
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Footnotes |
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Received February 16, 2001.
Accepted for publication June 5, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]dCTP. The RNA blot was subsequently hybridized
to an [
