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Mice lacking melanin-concentrating hormone receptor 1 demonstrate increased heart rate associated with altered autonomic activity

Annika Åstrand, Mohammad Bohlooly-Y, Sara Larsdotter, Margit Mahlapuu, Harriet Andersén, Jan Tornell, Claes Ohlsson, Mike Snaith and David G. A. Morgan

Am J Physiol Regul Integr Comp Physiol 287:R749-R758, 2004. First published 6 May 2004;

doi:10.1152/ajpregu.00134.2004

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Am J Physiol Regul Integr Comp Physiol 287: R749–R758, 2004. First published May 6, 2004; 10.1152/ajpregu.00134.2004.

Mice lacking melanin-concentrating hormone receptor 1 demonstrate increased heart rate associated with altered autonomic activity

Annika A ˚ strand, 1 Mohammad Bohlooly-Y, 2 Sara Larsdotter, 1 Margit Mahlapuu, 2 Harriet Anderse´n, 2 Jan Tornell, 2 Claes Ohlsson, 3 Mike Snaith, 2 and David G. A. Morgan 1

1 Department of Integrative Pharmacology and 2 AstraZeneca Transgenic and Comparative Genomics Centre, AstraZeneca Research and Development, SE-431 83 Mo¨lndal; and 3 Department of Internal Medicine, Sahlgrenska University Hospital, SE-413 45 Gothenburg Sweden

Submitted 27 February 2004; accepted in final form 22 April 2004

A ˚ strand, Annika, Mohammad Bohlooly-Y, Sara Larsdotter, Margit Mahlapuu, Harriet Anderse´n, Jan Tornell, Claes Ohlsson,

Mike Snaith, and David G. A. Morgan. Mice lacking melanin- concentrating hormone receptor 1 demonstrate increased heart rate associated with altered autonomic activity. Am J Physiol Regul Integr Comp Physiol 287: R749–R758, 2004. First published May 6, 2004; 10.1152/ajpregu.00134.2004.—Melanin-concentrating hormone (MCH) plays an important role in energy balance. The current studies were carried out on a new line of mice lacking the rodent MCH receptor

(MCHR1 / mice). These mice confirmed the previously reported lean phenotype characterized by increased energy expenditure and modestly increased caloric intake. Because MCH is expressed in the lateral hypo- thalamic area, which also has an important role in the regulation of the autonomic nervous system, heart rate and blood pressure were measured by a telemetric method to investigate whether the increased energy expenditure in these mice might be due to altered autonomic nervous

system activity. Male MCHR1 / mice demonstrated a significantly increased heart rate [24-h period: wild type 495 4 vs. MCHR1 /

  • 561 8 beats/min (P 0.001); dark phase: wild type 506 8 vs.

MCHR1 / 582 9 beats/min (P 0.001); light phase: wild type

  • 484 13 vs. MCHR1 / 539 9 beats/min (P 0.005)] with no

significant difference in mean arterial pressure [wild type 110 0.3 vs. MCHR1 / 113 0.4 mmHg (P 0.05)]. Locomotor activity and core body temperature were higher in the MCHR1 / mice during the dark phase only and thus temporally dissociated from heart rate differences. On fasting, wild-type animals rapidly downregulated body temperature and heart rate. MCHR1 / mice displayed a distinct delay in the onset of this downregulation. To investigate the mechanism underlying these differences, autonomic blockade experiments were carried out. Admin- istration of the adrenergic antagonist metoprolol completely reversed the

tachycardia seen in MCHR1 / mice, suggesting an increased sympa- thetic tone.

melanin-concentrating hormone; sympathetic; fasting

MELANIN-CONCENTRATING HORMONE (MCH) is a peptide synthe-

sized exclusively in the lateral hypothalamic area and zona incerta (6, 40) that has been implicated in the hypothalamic regulation of energy balance. Levels of MCH mRNA are regulated by altered energy balance in animal models such as fasting and in models with impaired leptin signaling (30). Intracerebroventricular (30) and intrahypothalamic (1) injec- tion of MCH increases food intake, and mice overexpressing MCH are hyperphagic and obese (22). Conversely, mice lack- ing a functioning MCH gene are hypophagic and lean (37).

Two receptors for MCH have been identified. The first, MCHR1, was originally characterized as an orphan receptor, SLC-1 (15). MCH was subsequently identified as the cognate ligand by a number of groups (5, 10, 21, 35, 38). It is distributed extensively in the central nervous system, with a distribution consistent with a role in energy balance (34). It demonstrates high sequence homology among species investi- gated so far. The second receptor, MCHR2, was identified by a number of groups using low-stringency homology searching or 5 /3 -rapid amplification of cDNA ends (3, 15, 24, 31, 33, 44). This receptor is expressed as a functional receptor in dogs, nonhuman primates, and humans but is expressed as a pseu- dogene in rabbits and guinea pigs and is absent in rodent species (42). Recently two groups have reported the phenotype of mice lacking the only known rodent receptor for MCH (11, 23). These have a similar phenotype to those lacking MCH itself. However, one striking difference is the lack of hypophagia in MCHR1 null mice; instead these mice eat slightly more than their wild-type littermates, suggesting that the lean phenotype of these animals is, to a large degree, brought about by an increase in energy expenditure. Indeed, a significantly raised 24-h energy expenditure has been recorded in these animals using indirect calorimetry. Increasing circumstantial evidence suggests that MCH may alter energy balance not only by increasing food intake but also by decreasing energy expenditure consistent with an altered level of autonomic nervous system activity. Thus a recent report suggests that intracerebroventricular MCH might de- crease body temperature and brown fat uncoupling protein (UCP-1) (16), whereas ob/ob mice lacking MCH showed an increased level of brown fat UCP-1 and an increased body temperature compared with control ob/ob animals (36). In addition, sympathetic projections to a number of organs have been retrogradely traced to the lateral hypothalamic area, and projections to the brown fat have even been traced to MCH- containing cells (26). In this study we investigate diurnal variations in energy expenditure of MCHR1 / mice and compare it to variations in heart rate and blood pressure activity. We have also inves- tigated the autonomic mechanisms underlying differences be- tween MCHR1 / mice and wild-type controls, using phar- macological blockade of sympathetic and parasympathetic ner- vous systems.

Address for reprint requests and other correspondence: D. G. A. Morgan, Dept. of Integrative Pharmacology, AstraZeneca R&D Mo¨lndal, S-431 83 Mo¨lndal, Sweden (E-mail david.ga.morgan@astrazeneca.com).

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.

http://www.ajpregu.org 0363-6119/04 $5.00 Copyright © 2004 the American Physiological Society R749

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INCREASED SYMPATHETIC ACTIVITY IN MCHR1 / MICE

Fasting at an ambient temperature below thermoneutrality results in large transient depressions in heart rate, mean arterial pressure, and oxygen consumption in mice (46). This response is thought to be mediated in part via a reduction in sympathetic nervous system activity, and so changes in this response can indicate changes in sympathetic nervous system output. To investigate this we have compared the cardiovascular re- sponses to fasting in MCHR1 / mice and wild-type controls.

MATERIALS AND METHODS

Animal care. Knockout animals were generated using R1 ES cells

(derived from 129/SvJ). All animals used in these studies had been

backcrossed for a minimum of three generations to C57Bl/6. Control

animals used in the experiments were always the wild-type littermates

of the MCHR1 / mice. Mice were maintained under a standard 12-h

light cycle regime, the relative humidity was between 45 and 55%, the

temperature was kept at 20°C, and the animals had ad libitum access

to chow and water, with environmental enrichment (in the form of egg

cartons, wooden tongue depressors, and cotton nesting pads) present

in home cages unless otherwise stated. The study was performed after

approval from the local ethical committee for animal experimentation.

Generation of MCHR1 null mice. The mouse MCHR1 locus was

subcloned from a positive BAC identied by screening a 129/SvJ

genomic library. The targeting construct was generated by replacing

the entire MCHR1 coding sequence with a neomycin phosphotrans-

ferase (Neo) cassette driven off the phosphoglycerate kinase (PGK)

promoter. After linearization, the targeting construct was electropo-

rated into R1 ES cells and neomycin-resistant clones were selected in

G-418-containing media (300 g/ml). Of 250 G418-resistant clones

screened, four targeted clones were identied using a Southern screen-

ing strategy with an EcoR1-BamHI fragment 5 to the targeted

sequence as a probe. One positive clone was expanded and injected

into C57Bl/6 blastocysts to generate chimeric mice. Chimeric males

were crossed to C57Bl/6 females, and genotyping of the offspring was

performed from tail biopsies by triplex PCR using a common (for-

ward) primer located at the 3 -end of the short arm (5 -cattgagaacag-

tacccctggctt-3 ), a reverse primer specic for the exon 2 of the

wild-type allele (5 -ctagatccacctgttactcagagga-3 ), and a reverse

primer in the PGK promoter specic for the targeted allele (5 -

agcgcatgctccagactgcctt-3 ).

To verify that the deletion of MCHR1 coding sequences resulted in

a null mutation, total RNA was prepared from the brains of 4-wk-old

homozygous, heterozygous, and control mice using the RNA

STAT-60 Kit according to the manufacturers instructions (Tel-Test,

Friendswood, TX). cDNA was synthesized using Superscript TM II

RNase H-Reverse Transcriptase and random hexamer primers (Life

Technologies, Frederick, MD). Real-time semiquantitative PCR was

performed in the ABI PRISM 7700 Sequence Detector System (Ap-

plied Biosystems, Warrington, UK) using SYBR Green. All samples

were run in triplicate, and data were normalized using the mouse

acidic ribosomal phosphoprotein PO (M36B4) as an internal control.

The primers for real-time PCR specic for the MCHR1 were derived

from rat SLC-1 sequences (20): 5 -tcagcttgggctatgctaacag-3 (for-

ward) and 5 -caacaccaagcgttttcgaa-3 (reverse).

Measurement of body weight and food intake. Mice used for

weight-gain studies were housed three to four per cage and given free

access to standard mouse chow containing 4% of total energy as fat

(R-34; Lactamin AB, Stockholm, Sweden) or to cafeteria diet, an ad

libitum choice of full-fat cheese (38% fat), almond paste, milk

chocolate, and pastry (based on vegetarian butter, sugar, rolled oats,

cocoa, and coconut shreds). From 4 wk of age, mice were weighed

weekly to the nearest 0.1 g on an electronic scale.

Food intake was measured in 6-wk-old single-housed mice for 6

consecutive days. The mice were housed individually 3 wk before

food consumption was measured. Mice were fed standard mouse

chow (see above), which had a total energy content of 3.0 kcal/g or a

high-fat diet. Cafeteria diet consisted of a number of human food-

stuffs, which rapidly became contaminated and dispersed in the cages

of animals fed this diet. Therefore, we turned to an alternative high-fat

diet for measurement of food intake. This contained 45% of total

energy as fat (D12451; Research Diets, New Brunswick, NJ; a total

energy content of 4.7 kcal/g). Cages were carefully monitored for

spillage of food, which was undetectable.

Measurements of the body composition. The total fat content of

16-wk-old mice fed standard chow diet was measured by dual-energy

X-ray absorptiometry (DEXA) as previously described (39) using the

Norland pDEXA Sabre (Fort Atkinson, WI).

Indirect calorimetry. Oxygen consumption (V ˙ O 2 ), carbon dioxide

production (V ˙ CO 2 ), and activity were measured using an open-circuit

calorimetry system (Oxymax, Columbus Instruments International,

Columbus, OH). The animals were placed in calorimeter chambers

with ad libitum access to normal lab chow and water for 72 h.

Environmental enrichment was removed. An air sample was with-

drawn for 75 s every 20 min, and the O 2 and CO 2 content were

measured by a paramagnetic oxygen sensor and a spectrophotometric

CO 2 sensor. These values were used to calculate V ˙ O 2 and V ˙ CO 2 . Data

from the rst 24 h were not used in analysis of results to allow

acclimatization to the novel environment. Data from corresponding

hours during the second and third 24-h periods were combined in 1-h

bins. After the 3-day period in the Oxymax system, the body compo-

sition of each animal was measured by DEXA, and the lean body mass

was calculated. Metabolic rate (kcal/h) was calculated using a rear-

rangement of the Weir equation (45) as supplied by Columbus

Instruments: (3.815 1.232RER) V ˙ O 2 , where RER is the respira-

tory exchange ratio [volume of CO 2 produced per volume of O 2

consumed

(both

ml kg 1 min 1 )]

and

V ˙ O 2

is the volume

of

O 2

consumed per hour. Data for the last two 24-h periods of the exper-

iment were combined and analyzed by two-way ANOVA. An eight-

channel activity monitor, forming part of the Oxymax system, pro-

vided ambulatory counts for each channel.

Remote measurement of temperature, heart rate, and blood pres-

sure. Core body temperature, heart rate, mean arterial pressure, and

locomotor activity were continuously monitored using remote telem-

etry transmitters (DSI, St. Paul, MN) on individually housed 8-wk-old

male mice at room temperature. Transmitters were implanted intra-

abdominally under isourane (Forene) anesthesia. For measurement

of blood pressure, a catheter was inserted in the carotid artery. Mice

were allowed to recover for at least 14 days postsurgery. All mea-

surements were carried out in the home-cage environment, and envi-

ronmental enrichment was available in all studies except those involv-

ing fasting.

In one experiment, knockout (n 7) or wild-type (n 8) male

mice were implanted with PA-C20 transmitters (DSI). Data on heart

rate and mean arterial pressure were collected every 30 s and averaged

in 10-min bins over a 72-h period. The average for each bin from the

same point over 3 days was calculated, and these values were used to

estimate 24-h diurnal rhythms. A further experiment was carried out

using knockout (n 8) or wild-type (n 8) male mice implanted

with ETA-F20 transmitters (DSI) to monitor baseline body tempera-

ture. As mean arterial pressure showed only small variations, ETA-

F20 transmitters, which deliver ECG-data as well as body tempera-

ture, were used for further experiments described below.

Another set of experiments was designed to compare autonomic

control of heart rate in MCHR1 / and wild-type mice. Animals were

implanted with ETA-F20 telemetry transmitters and allowed to re-

cover as above before undergoing four treatments, each separated by

a recovery period of at least 3 days. On each treatment day animals

were randomly assigned to one of four treatment groups and received

intraperitoneal administration of either 0.9% saline vehicle (10 ml/

kg), methylscopolamine (0.5 mg/kg), metoprolol (5 mg/kg), or a

combination of methylscopolamine (0.5 mg/kg) and metoprolol (5

mg/kg), so that each animal received all four treatments in random

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INCREASED SYMPATHETIC ACTIVITY IN MCHR1

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order. The doses of metoprolol and methylscopolamine were chosen

after consultation with the literature (e.g., Refs. 1719) and in-house

dose-nding experiments in wild-type mice from the same supplier.

Data were collected from each animal for a 2-h baseline period and

then for a further 2 h after intraperitoneal injection of the autonomic

blocker. Data were averaged over 15-min bins, and the data for each

bin were compared by one-way ANOVA and post hoc Tukeys test.

In the nal experiment, data were collected for 72 h from wild-type

(n 7) and MCHR1 / (n 8) mice implanted with ETA-F20

telemetry transmitters. Recording was started at 1600 on day 1, and

baseline measurements were recorded for 24 h, during which time

animals had ad libitum access to food and water. Food was removed

at 1600 on the second day, and animals were fasted for 24 h. Access

to water was ad libitum during this period. Their recovery from the

fast was monitored for 24 h, starting at 1600 on day 3. Average heart

rate for each animal was compared under fasted and nonfasted

conditions. Episodes of reduced heart rate and body temperature were

dened as 5-min bins in which the average value was 3 SDs from

the mean value seen during the dark period on the previous nonfasted

day. The total number of such bins and the time to onset of the rst

episode were calculated for each animal, and the average values for

wild-type and knockout animals were compared by t-test.

RESULTS

Generation and characterization of MCHR1 / mice. The targeting construct for generation of MCHR1 / mice is shown in Fig. 1A. R1 ES cell clones carrying the truncated allele were selected by Southern analysis (Fig. 1B) and used to generate MCHR1-null mice. Real-time PCR with MCHR1 specic primers on the brains from homozygous mice showed that a null mutation had been generated (Fig. 1C), with MCHR1 expression undetectable in the homozygous MCHR1 / animals. In the brains of the heterozygous mice, the expression of MCHR1 was reduced by approximately one-half, indicating that loss of one allele is not compensated by an increase in expression from the other (Fig. 1C). Homozy- gous MCHR1 mutant mice were represented in the litters from heterozygous crosses at the normal Mendelian frequency. MCHR1 / mice were viable into adulthood, fertile, and appeared normal by gross inspection. As previously demonstrated for alternative lines (11, 23), male MCHR1 / mice maintained with ad libitum access to

INCREASED SYMPATHETIC ACTIVITY IN MCHR1 MICE R751 order. The doses of metoprolol and methylscopolamine were chosen

Fig. 1. Targeting the melanin-concentrating hormone receptor 1 (MCHR1) gene. A: neomycin phosphotransferase (Neo) cassette driven off the phosphoglycerate kinase (PGK) promoter replaced the coding region (hatched area) of the 2-exon MCHR1 gene in the targeting construct. The latter contained a total of 11 kb from the MCHR1 locus, 2 kb on the short arm and 9 kb on the long. B: G-418-resistant colonies obtained by electroporation of the targeting construct into R1 ES cells were screened by Southern blotting of EcoRV-digested DNA hybridized with a probe (EcoRV/BamHI) located outside of the short arm. wt, wild type. C: for routine genotyping a triplex PCR was used with a common sense primer located in the short arm, an antisense primer specic for the targeted allele in the PGK promoter, and a wild-type-specic antisense primer in the exon 2 of MCHR1 (all designated by black arrowheads in A). D: real-time PCR was used to analyze the relative MCHR1 expression in the brains of wild-type, MCHR1 / , and MCHR1 / mice (% of wild type, y-axis). Each bar represents mean SE. **** P 0.001 as determined by 2-tailed unpaired Students t-tests.

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INCREASED SYMPATHETIC ACTIVITY IN MCHR1 / MICE

chow diet gained less body weight compared with their wild- type littermates. By 14 wk of age, male MCHR1 / mice were 10% lighter than wild-type controls (P 0.05; Fig. 2A). Male heterozygous mice of the same age also had a signicant reduction ( 7%) in body weight compared with wild-type animals (P 0.01, data not shown). MCHR1-decient females showed a similar pattern of reduction in weight gain compared with wild-type littermates, but this difference did not reach statistical signicance (Fig. 2B). Wild-type male mice fed a cafeteria diet showed dramati- cally increased body weight compared with mice fed normal chow. MCHR1 deciency imparted dramatic resistance to this diet-induced obesity (Fig. 2, A and B). At the age of 14 wk, knockout mice were 20% lighter than wild-type littermates (P 0.001 for males, P 0.05 for females). The weight of the MCHR1 / mice fed a cafeteria diet did not differ signi- cantly from that of wild-type males fed a normal chow diet. MCHR1-decient females demonstrated a similar resistance to diet-induced obesity (data not shown). To determine the total body fat content of MCHR1-decient mice, we assessed adiposity by DEXA. At the age of 16 wk, in animals fed a standard chow diet, total body fat was decreased by 45% in MCHR1 / male mice compared with wild-type control male mice (P 0.05; Fig. 2C). Daily food intake of 6-wk-old mice was measured over a 6-day period. As demonstrated in previous studies, daily food intake of MCHR1 / mice on normal lab chow was increased compared with the wild-type controls. Male homozygous mice ingested 8.3 2.5% more calories than wild-type siblings over a 24-h period (P 0.005). Measurement of high-fat food intake was carried out using a commercially available pelleted high-fat diet. Although the magnitude of the diet-induced obesity in wild-type mice was not so great as that seen with the cafeteria diet, similar resistance to diet-induced obesity was seen in MCHR1 /

mice (at 14 wk MCHR1 / 26.4 1.0 vs. wild type 31.5 1.6 g, P 0.05, n 10). In contrast to intake of normal chow, the caloric intake of male MCHR1 / fed a high-fat diet did not differ from that of controls (MCHR1 / 12.3 1.9 vs. wild type 12.0 0.6 kcal mouse 1 day 1 ). Energy expenditure, locomotor activity, heart rate, body temperature, and mean arterial pressure. Data detailing dif- ferences between wild-type and MCHR1 / mice are summa- rized in Table 1. Energy expenditure was calculated both per kilogram body mass and per kilogram lean mass. In these experiments, MCHR1 / mice showed a signicantly in- creased caloric expenditure per kilogram body mass compared with wild-type animals as analyzed by two-way ANOVA (Fig. 2D) but not by t-test comparison of mean caloric expenditure (Table 1). When analyzed per kilogram lean body mass, similar results were seen, although this did not reach statistical signif- icance (data not shown). Locomotor activity as measured in the Oxymax system was increased during the dark phase but not during the light phase in MCHR1 / mice [locomotor activity:

24-h period, wild type 294 33 vs. MCHR1 / 419 48 beam breaks (P 0.05); dark phase, wild type 405 36 vs. MCHR1 / 579 53 beam breaks (P 0.05); light phase, wild type 173 21 vs. MCHR1 / 245 35 beam breaks (P 0.1)] (Fig. 3A). We used implanted telemetry chips to measure the 24-h prole of locomotor activity, heart rate, temperature, and mean arterial pressure in 8-wk-old MCHR1 / and wild-type male mice (Fig. 3, BD). The MCHR1-decient mice showed in- creases in all these parameters compared with controls by two-way ANOVA, but these increases were not uniform throughout the light/dark cycle. Of the parameters measured, only heart rate differed signicantly between MCHR1 / mice during both dark and light phases, as well as over the entire 24-h period [24-h period: wild type 495 4 vs. MCHR1 / 561 8 beats/min (P 0.001); dark phase: wild type 506

Fig. 2. Decreased body weight and fat mass in MCHR1 / mice. Growth curves of group- housed age-matched cohorts of male (A) and female (B) wild-type and MCHR1 / mice. Mice were fed standard lab chow (F, MCHR1 / ; , wild type) or cafeteria (caf) diet (beginning at the age of 4 wk; E, MCHR1 / ; , wild type); n 715 ani- mals/group. C: total body fat, as measured by dual-energy X-ray absorptiometry (DEXA), in wild-type (open bars) and MCHR1 / (lled bars) male mice maintained on normal laboratory chow (n 8 animals/group). Each bar represents mean SE. * P 0.05 as determined by 2-tailed unpaired Students t- tests. D: energy expenditure (cal min 1 kg body wt 1 ) of MCHR1 / (, n 4) and wild-type (, n 4) male mice as measured by open-circuit calorimetry. The data are the average SE of data collected at 3 time points in each hour from 4 mice over 2 con- secutive 24-h periods. Genotype has a signif- icant effect as measured by 2-way ANOVA (P 0.0001).

R752 INCREASED SYMPATHETIC ACTIVITY IN MCHR1 MICE chow diet gained less body weight compared with their
R752 INCREASED SYMPATHETIC ACTIVITY IN MCHR1 MICE chow diet gained less body weight compared with their
R752 INCREASED SYMPATHETIC ACTIVITY IN MCHR1 MICE chow diet gained less body weight compared with their

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Table 1. Phenotypic analysis of energy balance parameters in MCHR1 / mice and wild type littermates

 

Wild Type

 

MCHR1 /

 

P

n

Locomotor activity, beam breaks/10 min Light

173

21

245

35

0.1

8

Dark

405

36

579

53

0.05

8

24 h Core temperature, °C

294

33

419

4

0.05

8

Light

36.4

0.1

36.5

0.1

NS

8

Dark

37.3

0.1

37.6

0.1

0.005

8

24 h

36.8

0.1

37.0

0.1

0.05

8

Heart rate, beats/min Light

484

13

539

9

0.005

8

Dark

506

8

582

9

0.001

8

24 h Mean arterial pressure, mmHg

495

4

561

8

0.001

8

Light

107

2.4

108

2.3

NS

8

Dark

113

2.7

117

3.4

NS

8

24 h

110

0.3

113

0.4

NS

8

Body weight (15 wk), g Male chow

31.4

0.7

27.9

0.4

0.0005

15

Male cafeteria

44.5

1.2

35.6

2.0

0.001

8

Female chow

24.1

0.6

23.4

0.3

NS

16

Female cafeteria

36.4

3.1

26.1

1.5

0.05

6

Energy expenditure, kcal min 1 kg total body wt 1 Light

221.1

10.9

230.0

6.0

0.5

4

Dark

242.0

12.7

272.0

13.8

0.16

4

24 h Energy expenditure, kcal min 1 kg lean body wt 1

231.3

11.7

251.7

6

0.19

4

Light

248.8

12.4

245.1

6.4

NS

4

Dark

273.5

14.1

292.1

14.4

NS

4

24 h

261.2

13.2

268.6

7.8

NS

4

Body composition, g Fat

4.3

0.8

2.0

0.2

0.01

8

Lean

32.9 1.2

29.8 1.2

NS

8

Values are means SE. MCHR1 / , mice lacking melanin-concentrating hormone receptor 1.

INCREASED SYMPATHETIC ACTIVITY IN MCHR1 MICE R753 Table 1. Phenotypic analysis of energy balance parameters in
INCREASED SYMPATHETIC ACTIVITY IN MCHR1 MICE R753 Table 1. Phenotypic analysis of energy balance parameters in
INCREASED SYMPATHETIC ACTIVITY IN MCHR1 MICE R753 Table 1. Phenotypic analysis of energy balance parameters in

Fig. 3. Mechanisms underlying decreased fat

mass in MCHR1 / mice. A: 24-h locomotor

activity in 8-wk-old MCHR1 / (open sym-

bols) and wild-type (lled symbols) male

mice maintained on normal laboratory chow,

as measured by beam breaks in the Oxymax

open-circuit calorimetry system. Data are the

average SE of data collected at 3 time

points in each hour from 4 male 8-wk-old

mice over 2 consecutive 24-h periods. Geno-

type has a signicant difference as measured

  • by 2-way ANOVA (n 4, P 0.0001). A very similar pattern of locomotor activity was recorded using implanted telemetry chips

INCREASED SYMPATHETIC ACTIVITY IN MCHR1 MICE R753 Table 1. Phenotypic analysis of energy balance parameters in

(data not shown). Implanted telemetry chips

were also used to record 24-h temperature

(B), heart rate (C), and mean arterial pressure

proles (D) in 8-wk-old MCHR1 / and

wild-type male mice maintained on normal

laboratory chow. Data presented are aver-

age SE of data collected from 8 mice in

1-h time bins over a 24-h period. Genotype

has a signicant effect on all parameters

except mean arterial pressure, as measured

INCREASED SYMPATHETIC ACTIVITY IN MCHR1 MICE R753 Table 1. Phenotypic analysis of energy balance parameters in
INCREASED SYMPATHETIC ACTIVITY IN MCHR1 MICE R753 Table 1. Phenotypic analysis of energy balance parameters in
INCREASED SYMPATHETIC ACTIVITY IN MCHR1 MICE R753 Table 1. Phenotypic analysis of energy balance parameters in

by two-way ANOVA (P 0.01).

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INCREASED SYMPATHETIC ACTIVITY IN MCHR1 / MICE

8 vs. MCHR1 / 582 9 beats/min (P 0.001); light phase:

wild type 484 13 vs. MCHR1 / 539 9 beats/min (P 0.005)]. The telemetry chip system was a much less sensitive measure of locomotor activity than the Oxymax system, with a number of bins during the light phase recording no locomotor activity and no signicant difference (NS) in the average number of movements between genotypes [24-h period: wild type 3.57 0.18 vs. MCHR1 / 5.06 0.22 (P NS); dark

phase: wild type 5.33 0.28 vs. MCHR1 / 7.82 0.34 (P NS); light phase: wild type 1.83 0.20 vs. MCHR1 / 2.33

  • 0.25 (P NS)]. Nevertheless, a very similar overall pattern

was recorded, with genotype giving a signicant effect by two-way ANOVA, and with locomotor activity increased dur- ing the dark phase to a greater extent than during the light

phase in MCHR1 / mice. Body temperature was increased during the dark phase but not the light phase in MCHR1 / mice [24-h period: wild type 36.8 0.1 vs. MCHR1 /

  • 37.0 0.1°C (P 0.05); dark phase: wild type 37.3 0.1 vs.

MCHR1 / 37.6 0.1°C (P 0.005); light phase: wild type

  • 36.4 0.1 vs. MCHR1 / 36.5 0.1°C (P NS)]. Average

mean arterial pressure showed no signicant difference be- tween MCHR1 / mice and wild-type mice [24-h period: wild type 110 0.3 vs. MCHR1 / 113 0.4 mmHg (P 0.05)]. Thus there appeared to be temporal differentiation between the effects on temperature, mean arterial pressure, and loco- motor activity, where the difference was more marked during the dark phase, and the effect on heart rate, which was apparent throughout the 24-h period. As the effect on mean arterial pressure was relatively minor, telemetry chips measuring heart rate but not mean arterial pressure were used for experiments on the intrinsic heart rate and fasting responses of MCHR1 / mice described below. Autonomic blockade and regulation of heart rate. The in- traperitoneal injection procedure caused a rapid, transient in- crease in heart rate, which returned to a steady level after 1 h. Treatment with sympathetic and parasympathetic blockade altered heart rate in both wild-type and MCHR1 / mice during the 3-h period after administration (Fig. 4A), but due to the transient injection effect, comparison of strains during the period after administration has been discounted, and statistical

Fig. 4. Increased sympathetic activity underlies increased heart rate in MCHR1 / mice. A: vari- ations in heart rate in 8-wk-old male MCHR1 / () and wild-type () mice after administration of vehicle, metoprolol, methylscopolamine, or metoprolol methylscopolamine. Due to the large transient increase in heart rate seen after injection, statistical comparisons of effects of sympathetic and vagal blockade were carried out on data collected between 1 and 1.25 h after administration (B). MCHR1 / mice (lled bars) showed a higher heart rate than wild-type animals (open bars). This appeared to be due to the dom- inance of sympathetic tone in these animals com- pared with dominant parasympathetic tone in wild-type animals. Data are the average SE from 78 animals of 3 time points in each 15-min bin. * P 0.05 vs. wild-type saline, P 0.05 vs. MCHR1 / saline as measured by 1-way ANOVA and post hoc Tukeys test at the indi- vidual time point.

R754 INCREASED SYMPATHETIC ACTIVITY IN MCHR1 MICE 8 vs. MCHR1 582 9 beats/min ( P 0.001);
R754 INCREASED SYMPATHETIC ACTIVITY IN MCHR1 MICE 8 vs. MCHR1 582 9 beats/min ( P 0.001);
R754 INCREASED SYMPATHETIC ACTIVITY IN MCHR1 MICE 8 vs. MCHR1 582 9 beats/min ( P 0.001);
R754 INCREASED SYMPATHETIC ACTIVITY IN MCHR1 MICE 8 vs. MCHR1 582 9 beats/min ( P 0.001);
R754 INCREASED SYMPATHETIC ACTIVITY IN MCHR1 MICE 8 vs. MCHR1 582 9 beats/min ( P 0.001);

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analysis was carried out on data gathered between 1 and 1.25 h postinjection (Fig. 4B). During this period, intraperitoneal vehicle-treated male MCHR1 / mice had a signicantly higher heart rate than wild-type animals receiving a similar treatment [wild type 355 30 vs. MCHR1 / 502 24 beats/min (P 0.001)]. Treatment with the sympathetic blocker metoprolol had no effect on the heart rate of wild-type animals but signicantly reduced the tachycardia seen in

MCHR1 / mice [wild type 385 30 beats/min (P 0.05 vs. wild type saline); MCHR1 / 417 15 beats/min (P 0.01 vs. knockout saline; P 0.05 vs wild type metoprolol)]. Treatment with methylscopolamine signicantly increased the heart rate of wild-type animals and also caused a signicant increase in the knockout animals [wild type 522 28 beats/ min (P 0.001 vs. wild type saline); MCHR1 / 550 22 beats/min (P 0.01 vs. MCHR1 / saline; P 0.05 vs. wild type methylscopolamine)]. Treatment with a combination of methylscopolamine and metoprolol showed that there was no signicant difference in intrinsic heart rate between wild-type and MCHR1 / mice and that this was intermediate between the basal heart rates of the two mouse strains [wild type 477

  • 23 beats/min (P 0.001 vs. wild type saline); MCHR1 /

451 21 beats/min (P 0.01 vs. MCHR1 / saline; P 0.05 vs. wild type methylscopolamine metoprolol)]. Effect of fasting on heart rate and body temperature. As

above, MCHR1 / mice showed a signicantly higher heart rate compared with wild-type mice, both during the fed and fasted periods (fed period: wild type 518 12 vs. MCHR1 / 561 10 beats/min; P 0.05; fasted period: wild type 447

  • 14 vs. MCHR1 / 536 23 beats/min; P 0.01). Transient

falls in heart rate were seen in both wild-type and MCHR1 /

mice (Fig. 5A); however, the overall average heart rate during the 12-h after removal of food decreased signicantly more in the wild-type mice than in the MCHR1 / mice (wild type 70 13 vs. MCHR1 / 25 14 beats/min, P 0.05). This difference in average heart rate represented both an increase in latency to the rst profound drop in heart rate, as assessed by a 3-SD difference from the mean value for the fed period (MCHR1 / 7.7 1.1 h after fasting vs. wild type 3.3 1.4 h after fasting, P 0.05) and in the overall time spent with depressed heart rate, as assessed by the number of 15-min bins where heart rate was 3 SDs below the average for the fed period (MCHR1 / 21 6.9 vs. wild type 44 5.4 bins, P 0.05). Body temperature also showed transient and profound falls during the fasting period (Fig. 5B), and there was a temporal relationship between drops in heart rate and body temperature. Again, MCHR1 / mice had a small but statistically signi- cant elevation in body temperature during both fed and fasted phases (fed period: wild type 36.7 0.1 vs. MCHR1 / 36.9 0.05°C, P 0.05; fasted period: wild type 35.1 0.2 vs. MCHR1 / 36.1 0.3°C, P 0.05). Similar transient falls in average body temperature during the 12-h after removal of food were seen in both wild-type and MCHR1 / mice (Fig. 5B). Although this did not reach signicant difference, there was a trend toward a greater decrease in average body temper- ature in the wild-type animals (wild type 1.5 0.24 vs. MCHR1 / 0.78 0.27°C, P 0.055). This difference represented both an increase in latency to the rst profound drop in temperature (wild type 2.7 1.0 vs. MCHR1 / 6.9 1.5 h after fasting, P 0.05) and in the overall time spent with depressed body temperature (wild type 63 10 vs. MCHR1 / 33 7 bins, P 0.05).

INCREASED SYMPATHETIC ACTIVITY IN MCHR1 MICE R755 analysis was carried out on data gathered between 1
INCREASED SYMPATHETIC ACTIVITY IN MCHR1 MICE R755 analysis was carried out on data gathered between 1
INCREASED SYMPATHETIC ACTIVITY IN MCHR1 MICE R755 analysis was carried out on data gathered between 1
INCREASED SYMPATHETIC ACTIVITY IN MCHR1 MICE R755 analysis was carried out on data gathered between 1
INCREASED SYMPATHETIC ACTIVITY IN MCHR1 MICE R755 analysis was carried out on data gathered between 1

Fig. 5. MCHR1 / mice show a reduced

cardiovascular response to fasting. Heart rate

(A and B) and core temperature recordings (C

  • and D) from 8-wk-old male wild-type (A and C) and MCHR1 / (B and D) mice during 24-h periods with ad libitum access to food (open symbols) or with food removed from cages (lled symbols). Data shown are from a representative animal because the timing of large, transient drops in heart rate and body temperature varied between individuals. Data are the average SE from a representative animal of 12 time points in each 1-h bin.

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DISCUSSION

In this series of studies we show that a third line of MCHR1 / mice has a similar phenotype to that of the MCHR1 / mice lines previously described by Marsh et al. (23) and Chen et al. (11). All three lines have a lean phenotype and are resistant to diet-induced obesity. In each case, in- creased energy expenditure appears to be an important factor (11, 23) because MCHR1 / mice show a slight increase in food consumption. We also demonstrate that the absence of a functional MCHR1 system leads to an increased basal heart rate independent of increased locomotor activity and that this appears to be caused by increased sympathetic and decreased parasympathetic activity compared with wild-type animals. We also demonstrate that under fasting conditions MCHR1 / mice maintain heart rate and body temperature for longer than wild-type mice before the initiation of transient but profound drops in these two parameters, a response normally involving reduced sympathetic activity. It thus appears that the MCHR1 system is involved in both the tonic regulation of autonomic activity and the fast-induced autonomic suppression of heart rate and body temperature. MCH has been considered an orexigenic peptide, with in- tracerebroventricular injection causing increased food intake (10, 32), and MCH antagonists causing acute (41) and chronic reductions (7) of food intake. Thus it was an unexpected result that mice lacking the MCHR1 receptor should be hyperphagic compared with wild-type animals. It is plausible that the increase in food intake is due to compensatory mechanisms acting to rectify the negative energy balance in these animals, and one might even speculate that the compensatory hyperpha- gia is somewhat impaired because MCHR1 / mice show only a modest increase in food intake, inadequate to compensate fully and maintain a normalwild-type body weight. Thus it is possible that the slight compensatory hyperphagia might be masking an effect on food intake responsible in part for the lean phenotype in these animals. This explanation would be in keeping with the reports of reduced food intake after admin- istration of MCHR1 antagonists (7, 41). It is interesting to note that in the semichronic study reported by Borowsky et al. (7), there appears to be a steady reduction in the effect of the MCH antagonist SNAP-7941 on food intake, while no such normal- ization of body weight loss is seen. MCHR1 / mice show an increase in dark-phase locomotor activity, accompanied by increased body temperature, in keep- ing with previous reports (11, 23). However, energy expendi- ture, as measured by open-circuit calorimetry, is increased during the light phase as well as the dark phase, whereas no increased locomotor activity is seen during the light phase. This suggests that, while increased locomotor activity might be responsible for part of the increased energy expenditure, an increase in basal metabolic rate, or involuntary energy expen- diture is also likely to be present. Such an increase in invol- untary energy expenditure is likely to be regulated by the autonomic nervous system, and in keeping with altered auto- nomic regulation we also observed an increase in heart rate in MCHR1 / mice. Like the increase in energy expenditure, this mild tachycardia was present throughout the light/dark cycle, and thus did not appear to be wholly caused by increased locomotor activity.

Autonomic control of heart rate. In many cases, factors altering food intake also have a reciprocal effect on energy expenditure (for review, see Ref. 8). In many cases the sym- pathetic nervous system plays a major role in such changes in energy expenditure. The sympathetic nervous system is made up of multiple differentially regulated segments, and heart rate in particular can be regulated by a number of these, including several that do not appear important in regulation of energy balance (see Ref. 25 for review). However, under a number of circumstances, heart rate and thermogenesis are under coordi- nate autonomic control, and specic areas of the brain, such as the raphe pallidus, have been shown to control both (9). Thus it appears that under some circumstances heart rate and brown fat thermogenesis might be controlled by the same single unit of the sympathetic nervous system (8). Because both parame- ters appear altered in MCHR1 / mice, it seems at least possible that the same mechanisms underlie the increased heart rate and a proportion of the increased energy expenditure in MCHR1 / mice. We therefore investigated the autonomic mechanisms underlying the increased heart rate. To do this we measured the effects of sympathetic and parasympathetic blockade on heart rate in MCHR1 / mice using metoprolol and methylscopolamine. This also allowed us to differentiate between autonomic regulation and intrinsic heart rate of MCHR1 / and wild-type mice. Directly after injection, we observed a transient stress- induced increase in heart rate. This transient increase in heart rate disappeared within 1 h of administration, and heart rate then returned to a steady level. We compared the effects of sympathetic and parasympathetic blockade during this later steady period. Intrinsic heart rate, as measured in the presence of both sympathetic and parasympathetic blockade, was the same for both MCHR1 / and wild-type mice and lay inter- mediate to the baseline levels for the two strains. In both MCHR1 / and wild-type mice, heart rate was controlled by a balance between sympathetic and parasympa- thetic inputs. In MCHR1 / mice, it appeared that the sympa- thetic nervous system was the predominant input. Thus admin- istration of the sympathetic blocker metoprolol reduced heart rate to the level seen in wild-type mice. However, when MCHR1 / mice were treated with both metoprolol and meth- ylscopolamine, they showed a heart rate signicantly higher than when treated with metoprolol alone, demonstrating that parasympathetic tone was present in these animals that had been unmasked by metoprolol treatment. Conversely, in wild-type mice, it appeared that parasympa- thetic inputs predominated. Thus treatment with methylscopol- amine alone increased heart rate in wild-type mice to the same level as seen in MCHR1 / mice. Again, it was clear that parasympathetic blockade unmasked a tonic sympathetic ac- tivity in wild-type mice because treatment with both metopro- lol and methylscopolamine induced a heart rate signicantly lower than that seen after treatment with methylscopolamine alone. In summary, in this paradigm, ablation of the MCHR1 gene leads to a state where sympathetic rather than parasym- pathetic control of heart rate predominates. This is in keeping with the changes expected if altered autonomic regulation were responsible for the changes in energy expenditure in these mice. It is often stated that sympathetic activity is the predominant autonomic controller of heart rate in mice (for review, see Ref.

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18). However, contrary to this nding, we see a predominance

of parasympathetic control of heart rate in wild-type mice in

our study. A careful review of the literature shows that this is

not a unique nding. Indeed there are studies showing a

spectrum of autonomic control from predominantly sympa-

thetic (12, 17) to a balance between sympathetic and parasym-

pathetic (43) to predominantly parasympathetic control (2).

Other than the differences in autonomic control, perhaps the

most pertinent observed difference between these studies is in

the resting heart rate. Not surprisingly, the higher the resting

heart rate is, the larger is the sympathetic component in the

control of heart rate. For example, whereas we observed a

resting heart rate of approximately 350 400 beats/min in

wild-type animals, Gehrmann et al. (12), using a similar

telemetry system, reported resting heart rates over 700 beats/

min. A number of variables might contribute to these variations

in resting heart rate. These include the different methods of

sampling heart rate data (telemetry vs. externalized catheters);

the length of time between operation and measurements; ad-

ministration, novel environment, and handling stress-induced

effects; strain, age, and gender effects; and effects of the

ambient temperature at which the experiments were carried

out. Of these factors, ambient temperature seems a possible

unreported variant between studies. Thus in our study although

the room temperature was 20°C, well below the thermoneutral

temperature for most mice strains, the animals did have access

to nesting material in the cages, in keeping with local ethical

guidelines and in an attempt to make the surroundings as

normal as possible. It seems quite likely that the mice were

thus able to maintain thermoneutrality and that this maintains

the parasympathetic balance of the autonomic inputs. Nesting

material was removed during fasting experiments, and this

perhaps explains the smaller differences in heart rate seen

between knockout and wild-type animals in these experiments.

Responses to fasting. Fasting induces a distinctive pattern of

responses in rodents. This includes reductions in heart rate,

arterial pressure, V ˙ O 2 , and body temperature, as well as an

initial hyperactivity followed by periods of hypoactivity (28,

46, 47). A large body of indirect evidence suggests that

decreased sympathetic activity and increased parasympathetic

activity underlie this process (4, 13, 14, 27). Given that we had

seen altered autonomic activity in MCHR1 / mice, we hy-

pothesized that the MCH system might play an important role

in mediating the autonomic changes leading to altered cardio-

vascular parameters during fasting. To test this we measured

heart rate and body temperature changes in MCHR1 / and

wild-type mice during a 24-h fast. In this model we observed

a reduced response to fasting in MCHR1 / mice.

The cardiovascular elements of the fasting response are not

constant but instead occur as transient bouts of profound

suppression of parameters such as heart rate and body temper-

ature (46). In MCHR1 / mice we observed that both the

latency to onset of these bouts and the overall time with

depressed heart rate and body temperature were reduced in

MCHR1 / mice. However, we did observe that the typical

cardiovascular response to fasting was eventually seen. Thus

the data suggest that the MCHR1 system is an important

regulator of the physiological responses to fasting (as its

removal slowed the response) but does not represent the one

and only mechanism (since the response still eventually oc-

curred).

A number of hypothalamic peptide systems that are known

to alter food intake also have reciprocal effects on sympathetic

nervous activity (see Ref. 8 for review), a coordinate energy-

conserving response to negative energy balance. From this

model, it would appear that MCH can be added to that number.

It appears that MCH may play an important role in the balance

of autonomic nervous system activity, as in its absence there

appears to be a switch from predominantly parasympathetic

control to sympathetic control. It could be also be speculated

that increases in MCH, which occur during fasting and food

restriction, play a role in the suppression of sympathetic

activity in these states. It remains to be investigated whether

direct intracerebroventricular injection of MCH causes a sup-

pression of sympathetic activity and heart rate in rodents,

although intracerebroventricular MCH has been shown to have

no effect on heart rate in sheep (29). It also remains unproven

whether the increased heart rate seen in MCHR1 / mice is

related to an increase in brown fat thermogenesis in these

animals.

In summary, it appears that the MCH system may be an

important tonic regulator of sympathetic nervous activity. This

may form part of a coordinated response to negative energy

balance and may be of importance when considering modula-

tion of this system as a target for body weight control.

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