Central Infusion of Histamine Reduces Fat Accumulation and Upregulates UCP Family in Leptin-Resistant Obese Mice
Takayuki Masaki,
Hironobu Yoshimatsu,
Seiichi Chiba,
Takeshi Watanabe, and
Toshiie Sakata
From the Department of Internal Medicine (T.M, H.Y., S.C., T.S.), School
of Medicine, Oita Medical University, Hasama, Oita; and the Department of
Molecular Immunology (T.W.), Medical Institute of Bioregulation, Kyushu
University, Fukuoka, Japan.
Address correspondence and reprint requests to Toshiie Sakata, MD, PhD,
Department of Internal Medicine I, School of Medicine, Oita Medical
University, 1-1 Idaigaoka, Hasama, Oita, 879-5593 Japan. E-mail:
sakata{at}oita-med.ac.jp
.
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ABSTRACT
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Leptin resistance has recently been confirmed not only in animal obese
models but in human obesity. Evidence is rapidly emerging that suggests that
activation of histamine signaling in the hypothalamus may have substantial
anti-obesity and antidiabetic actions, particularly in leptin-resistant
states. To address this issue, effects of central, chronic treatment with
histamine on food intake, adiposity, and energy expenditure were examined
using leptin-resistant obese and diabetic mice. Infusion of histamine (0.05
µmol · g body wt-1 · day-1) into the
lateral cerebroventricle (i.c.v.) for 7 successive days reduced food intake
and body weight significantly in both diet-induced obesity (DIO) and
db/db mice. Histamine treatment reduced body fat weight, ob
gene expression, and serum leptin concentration more in the model mice than in
pair-fed controls. The suppressive effect on fat deposition was significant in
visceral fat but not in subcutaneous fat. Serum concentrations of glucose
and/or insulin were reduced, and tests for glucose and insulin tolerance
showed improvement of insulin sensitivity in those mice treated with histamine
compared with pair-fed controls. On the other hand, gene expression of
uncoupling protein (UCP)-1 in brown adipose tissue and UCP-3 expression in
white adipose tissue were upregulated more in mice with i.c.v. histamine
infusion than in the pair-fed controls. These upregulating effects of
histamine were attenuated by targeted disruption of the H1-receptor in DIO and
db/db mice. Sustained i.c.v. treatment with histamine thus makes it
possible to partially restore the distorted energy intake and expenditure in
leptin-resistant mice. Together, i.c.v. treatment with histamine contributes
to improvement of energy balance even in leptin-resistant DIO and
db/db mice.
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INTRODUCTION
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Obesity, a common metabolic disorder characterized by chronic imbalance in
energy intake and energy expenditure, is a serious risk factor for type 2
diabetes, coronary artery disease, hypertension, hyperlipidemia, and other
common diseases (1). The
pathophysiological basis of obesity, however, is poorly understood. Since
discovery of the ob gene and its encoded protein leptin
(2), it has been understood
that leptin acts as a hormone at the level of the hypothalamus to inhibit food
intake and favor energy expenditure
(3,4,5).
The uncoupling protein (UCP) family, consisting of inner mitochondrial
proteins
(6,7,8,9),
is known to contribute to improvement of energy imbalance resulting from
energy insufficiency or excess
(7,10).
Gene expression of the UCP family is highly responsive to neural and humoral
factors
(10,11,12,13,14),
particularly leptin (14).
Serum leptin thus reflects energy stores in adipose tissue and serves to
signal the brain (15). To
improve understanding of the leptin signaling pathway, a number of approaches
have been tried to clarify the roles of leptin-modulated hypothalamic
neuropeptides in the regulation of feeding behavior and energy homeostasis
(16,17,18).
Leptin negatively regulates orexigenic neuropeptide Y
(16) and agouti gene-related
protein (17) and positively
regulates anorexigenic proopio-melanocortin-derived peptide through leptin
receptors on neurons in the hypothalamic arcuate nucleus
(18). Anorexigenic
corticotropin-releasing hormone in the paraventricular nucleus, which
negatively affects neuropeptide Y neurons in the arcuate nucleus
(19), is also positively
regulated by leptin (20). The
signals from such sites in the mediobasal hypothalamus thus communicate the
neural underpinning of hunger and satiety with the orexigenic mediators orexin
(21) and melanocortin
concentrating hormone (22),
both of which originate in the lateral hypothalamus.
Serum concentration of leptin is known to be increased in the great
majority of obese humans as well as in most rodent models, indicating that
most obesity is leptin resistant
(23,24).
Although the details and molecular basis of the mechanisms are unknown,
important factors are indicated by the following findings. First,
hyperleptinemia commonly develops along with the progress of obesity
(25,26).
Second, the high concentration of serum leptin in obesity is not paralleled by
a proportional rise in cerebrospinal fluid leptin
(23,24).
Third, exogenous application of leptin is relatively ineffective for weight
reduction of obese subjects
(27). Ob/ob mice that
either lack the ability to produce leptin or produce a truncated inactive form
are highly sensitive to leptin, and treatment with leptin markedly decreases
food intake and increases energy expenditure
(3,4,5).
In contrast, db/db mice, an obese model with a hypothalamic leptin
long-form receptor mutation
(28), are severely leptin
resistant
(3,4,5).
The leptin receptor mutation, although present in humans
(29), occurs rarely.
Diet-induced obesity (DIO) mice, in which obesity is acquired by the
environmental factor of excessive energy intake, are mildly leptin resistant
(30). In these contexts, such
mice are useful models for analysis of human obesity.
In parallel with neuropeptides regulated by leptin, it has been found that
histamine neurons are involved in leptin-induced feeding suppression as a
target in the hypothalamus
(31). Histamine neurons
originating from the tuberomammillary nucleus of the posterior hypothalamus
project diffusely to almost all the brain areas that contribute to maintenance
of energy homeostasis (32).
Histaminergic neurons have been particularly implicated in the neural
regulation of appetite through the postsynaptic histamine
H1-receptor (H1-R)
(33,34).
Indeed, histamine neuron activation suppresses food consumption in rats
(34). Thermoregulation, a
major factor involved in energy homeostasis, is mediated in part by brain
histamine neurons (35). Energy
deficiency in the brain, i.e., neural glucoprivation, activates histamine
neurons in the hypothalamus
(36) and augments
glycogenolysis in the brain
(37). Histamine neurons also
accelerate lipolysis in adipose tissues to supply energy to the brain through
activation of the sympathetic nervous system
(38). These findings regarding
functional roles of histamine neurons show that such systems are related to
nutritional status and energy storage across a broad range, from starvation to
hyperglycemia (32). Evidence
is thus rapidly emerging to suggest that hypothalamic histamine neurons may
play essential roles in the regulation of feeding, fat accumulation, energy
expenditure, and metabolism.
The aim of the present study was to examine central effects of chronic
histamine infusion on regulation of food intake, fat accumulation, energy
expenditure, and metabolism in leptin-resistant mice. To address this issue
more precisely, targeted disruption of the H1-R was introduced in
leptin-resistant mice.
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RESEARCH DESIGN AND METHODS
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Subjects. Mature male C57Bl/6J (C57Bl6),
C57Bl/KsJ-misty/misty (C57Ksj), C57Bl/KsJ-db/db (db/db)
obese (Seac Yoshitomi, Fukuoka, Japan) and histamine H1-R knockout
(H1KO) mice were used at 12-14 weeks of age. They were housed in a room
illuminated daily from 0700 to 1900 (a 12:12 h light-dark cycle) at a
temperature of 21 ± 1°C and humidity at 55 ± 5%. The mice
were allowed free access to standard mouse powder diet (CLEA Japan, Tokyo) and
tap water. In each experiment, mice were housed individually in an accustomed
cage at least 2 weeks before the start of each measurement. The animals used
were treated in accordance with the Oita Medical University Guidelines for the
Care and Use of Laboratory Animals.
Production and supply of H1KO mice. Male and female H1KO mice were
produced at the Medical Institute of Bioregulation (Kyushu University,
Fukuoka, Japan). The methods used to produce these mice are reported in detail
elsewhere (39). Backcrossing
H1-R-/- mice to the C57Bl6 strain for five generations resulted in
incipient congenic N4 mice of three genotypes (H1-R +/+,
H1-R +/-, and H1-R -/-) used here. All genotypes were
confirmed by Southern blotting.
Preparation of mice with diet-induced obesity. For preparation of
DIO-C57Bl6 and DIO-H1KO mice, the mice were fed a high-energy diet. Matched on
the basis of body weight at 8 weeks of age, H1KO and C57Bl6 mice were placed
on a high-fat diet (n = 6 for each subgroup). The high-fat diet
consisted of 45% fat, 35% carbohydrate, and 20% protein with an energy density
of 4.73 kcal/g. The standard diet consisted of 10% fat, 70% carbohydrate, and
20% protein, with an energy density of 3.85 kcal/g. DIO mice were fed a
high-energy diet for 6 weeks. DIO H1-R null (DIO:-/-) and DIO wild
(DIO:+/+) mice were used in the experiment.
Cross-breeding of H1KO and db/db mice. H1-R
heterozygous gene (+/-) and db/db breeder pairs were cross-bred to
create db/db H1KO models (db/+:+/-). H1-R gene
carriers were identified by Southern blotting analysis with genomic DNA
followed by allele-specific hybridization to identify presence or absence of
the H1-R mutation. db/db H1-R null
(db/db:-/-) and db/db (db/db: +/+) mice were used
in the experiment.
Measurement of body composition and food intake. DIO,
db/db, and their corresponding controls of C57Bl6 and C57Ksj mice (12
in each) were equally divided into histamine-treatment and phosphate-buffered
saline (PBS)-treatment groups, respectively. To evaluate parameters regarding
adipose tissues, DIO and db/db mice (18 in each) were equally divided
into histamine, PBS, and pair-fed control groups. DIO and db/db mice
with and without H1KO (12 in each) were equally divided into the histamine and
PBS control groups. These groupings at the start of experiment were made to
avoid any difference in body weight between the groups. After the mice were
killed, total fat pads were surgically removed and separated into brown
adipose tissue (BAT), subcutaneous white adipose tissue (WAT), and visceral
WAT, including mesenteric (Mes), retroperitoneal (Ret) and epididymal (Epi)
fat. These samples were immediately weighed, and all the tissues were then
frozen in liquid nitrogen and stored at -80°C. Epi WAT and BAT were
thawed, and RNA was extracted for measurement of gene expression of
ob and UCP families. To exclude differences in food consumption
between the histamine and control groups, the mice used in each histamine
infusion study were also pair-fed using standard powdered mouse food (pair-fed
groups were restricted to histamine-treated levels). In addition, evaluation
of regional fat accumulation was assessed by analytical balance for small
animals (Mettler. Tolado, Osaka, Japan).
Chronic implantation of a cannula and infusion methods. Before
surgery, mice were anesthetized with nembutal (1 mg/kg) intraperitoneally
(i.p.). Mice used were placed in a stereotactic device and implanted with a
29-gauge stainless steel cannula into the left lateral cerebroventricle (0.5
mm posterior, 1.0 mm lateral, and 2.0 mm ventral to the bregma). To prevent
the cannula from being blocked by blood coagulation, a 30-gauge wire plug
remained inserted into each cannula until use. Subjects were allowed 1 week of
postoperative recovery, during which they were handled daily to equilibrate
their arousal levels. Cannula placement was verified on each brain slice at
the end of each experiment by injecting 1.0 µl of 1% Indiana green. The
test solution was infused by a 30-gauge stainless steel injector that
projected 1.0 mm below the tip of the cannula. Histamine (Sigma, St. Louis,
MO) was freshly dissolved in PBS on the infusion day. Histamine was infused
intracerebroventricularly (i.c.v.) at a dose of 0.05 µmol/g body wt daily
for 7 successive days. Intracerebroventricular infusion volume of histamine
and PBS solutions was limited to a total volume of 1.0 µl.
Blood sampling procedures and methods for assay. Blood samples were
collected from timed treatment mice at 1000-1030. The samples were separated
into serum, immediately frozen at -20°C, and briefly stored until
measurement. For serum glucose, insulin, and free fatty acid (FFA)
measurement, DIO, db/db, and their appropriate controls of C57Bl6 and
C57Ksj mice (18 in each) were equally divided into three groups, i.e.,
histamine-treated, pair-fed, and PBS control. To measure serum leptin, DIO and
db/db mice (18 in each) were equally divided into histamine, PBS, and
pair-fed control groups. Serum glucose, insulin, FFA (Eiken Chemical, Tokyo,
Japan), and leptin (sandwich enzyme immunoassay; Immune Biological Laboratory,
Gunma, Japan) were measured by commercially available kits. To test glucose
tolerance, each mouse was injected i.p. with glucose at a dose of 1.0 mg/g
body wt after an 8-h fast. For the insulin tolerance test, each mouse was
injected with human regular insulin (Nobolin R; Novo Nordisk, Bagsvaerd,
Denmark) at a dose of 0.5 mU/g body wt after a 2-h fast. Procedures for
histamine treatment were the same as those in the forgoing methods. Blood
sampling for these tolerance tests was carried out succeeding the 7-day
treatment with histamine.
Preparation of the probes and Northern blotting analysis. Polymerase
chain reaction (PCR) primers of 5'-AGTGCCACTGTTGTCTTCAG-3' and
5'-TTCTCCAAGTCGCCTATGTG-3' were designed for the coding region of
the mouse UCP-1 gene, and primers of
5'-GTTACCTTTCCACTGGACAC-3' and
5'-CCGTTTCAGCTGCTCATAGG-3' were designed for the UCP-3
gene. Reverse transcription of 10 µg total RNA from C57Bl6 mice was
performed using Moloney murine leukemia virus reverse transcriptase (Life
Technologies, Gaithersburg, MD). PCR was carried out with Taq DNA polymerase
(Amersham International, Buckinghamshire, England) and 20 pmol of each primer.
The reaction profiles were as follows: denaturation at 94°C for 1 min,
annealing at 50°C for 1 min, and extension at 72°C for 1 min, for 30
cycles. The PCR fragment was subcloned into pCRTM2.1 vector (TA cloning kit;
Invitrogen, San Diego, CA), and the nucleotide sequence of amplified cDNA was
confirmed by sequencing. The nucleotide sequences were determined by the
dideoxynucleotide chain termination method, using synthetic oligonucleotide
primers, which were complementary to the vector sequence and ABI373A,
automated DNA Sequencing System (Perkin-Elmer, Norwalk, CT). All DNA sequences
were confirmed by reading both DNA strands. The ob probe was
generated in an analogous fashion (Genbank accession No. U18812). Total
cellular RNA was prepared from various rat tissues with the use of Isogen
(Nippon gene, Toyama, Japan) according to the manufacturer's protocol. Total
RNA (20 µg) was electrophoresed on 1.2% formaldehyde-agarose gel. The
separated RNA was transferred onto a Biodyne B membrane (Pall Canada, Toronto,
ON, Canada) in 20x sodium chloridesodium citrate by capillary
blotting and immobilized by exposure to ultraviolet light (0.80 J).
Prehybridization and hybridization were carried out according to the
manufacturer's protocol. Membranes were washed under high-stringency
conditions. After washing the membranes, the hybridization signals were
analyzed with the BIO-image analyzer BAS 2000 (Fuji Film Institution, Tokyo).
The membranes were stripped by exposure to boiling 0.1% SDS, and ethidium
bromide staining was used to quantify the amounts of RNA species on the
blots.
Evaluation of data and statistical analysis. All the data were
expressed as means ± SE. Values of parameters excluding food intake,
body weight, and humoral factors were expressed as percentage of the values in
normally fed controls with PBS. Unpaired t test or two-way analysis
of variance with repeated measures assessed the statistical analysis of
difference between mean values.
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RESULTS
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Effects of histamine treatment on food intake and body weight.
Figure 1A and
C show time-course changes in food intake and body weight
of DIO and C57B16 control mice after i.c.v. infusion of histamine (0.05
µmol · g body wt-1 · day-1) for 7
successive days. Histamine infusion into DIO mice for 7 days induced 25.5 and
11.2% decreases in cumulative food intake and body weight, respectively
[F(1,11) = 12.10, P < 0.01; F(1,11) = 16.94, P <
0.01]. In the mice fed a normal diet, the suppressive effect of histamine was
18.8 and 6.8% decreases of food intake and body weight, respectively, compared
with vehicle-treated C57B16 controls [F(1,11) = 7.84, P < 0.01;
F(1,11) = 2.19 P < 0.05] (Fig.
1A and C). Histamine infusion with the same dose
and same period as described in DIO mice caused 31.7 and 13.3% decreases of
cumulative food intake and body weight, respectively, in db/db mice
[F(1,11) = 36.03, P < 0.01; F(1,11) = 29.81, P <
0.01]. The decrease in cumulative food intake for C57Ksj was 16.8% after
histamine treatment [F(1,11) = 2.04, P < 0.05]
(Fig. 1B and
D). The body weight decrease in db/db and DIO
mice was greater than in the pair-fed controls [F(1,11) = 2.53, P
< 0.01; F(1,11) = 6.15, P < 0.01]
(Fig. 1C and
D).

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FIG. 1. Central effects of chronic histamine (HA) infusion on food intake
(A, B) and body weight (C, D) in DIO (A, C) and
db/db (B, D) obese mice. Each pair-fed group in C
and D was pair-fed with the corresponding HA-treated mice. HA in this
and succeeding figures was infused intracerebroventricularly (i.c.v.) at a
dose of 0.05 µmol/g body wt daily for 7 successive days. Values and
vertical bars are means ± SE (n = 6 for each). Each horizontal
bar shows the infusion period of test solution i.c.v. C57B1/6J (C57B16)-HA,
C57B16 control mice with HA; C57B16-PBS, C57B16 control mice with PBS;
C57B1/KsJ (C57Ksj)-HA, C57Ksj control mice with HA; C57Ksj-PBS, C57Ksj control
mice with PBS; db/db-HA, db/db mice with HA;
db/db-PBS, db/db control mice with PBS; DIO-HA, DIO mice
treated with HA; DIO-PBS, DIO control mice with PBS. Statistical significance
marked in parentheses: *P < 0.05,
**P < 0.01 vs. the corresponding PBS controls;
P < 0.01 vs. the corresponding pair-fed PBS
controls.
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Effects on serum glucose, insulin, and FFA. Concentrations of serum
glucose and FFAs were not changed in C57B16 and C57Ksj mice after histamine
treatment (0.05 µmol · g body wt-1 ·
day-1 for 7 days), but serum insulin in C57B16 mice was
significantly different (P < 0.05 vs. ad libitum vehicle controls)
(Table 1). However, the histamine
treatment attenuated or abolished hyperglycemia, hyperinsulinemia, and
hyper-free fatty acidemia detectable in both DIO and db/db mice
(P < 0.05 and P < 0.01 vs. vehicle controls). In
addition, these moderating effects of histamine on serum glucose in
db/db mice and insulin in DIO mice were more potent than those in
pair-fed controls (P < 0.05 for each)
(Table 1).
Effects on visceral adiposity in DIO and db/db mice. To
examine net effects of histamine treatment (0.05 µmol · g body
wt-1 · day-1 for 7 days) on fat distribution in
DIO and db/db mice, corresponding pair-fed controls were used. As
shown in Fig. 2, both pair-fed
DIO and db/db mice reduced their visceral fat (P < 0.05
and P < 0.01 vs. the corresponding ad libitum controls). Although
each showed similar food reduction, histamine treatment caused a greater
decrease in visceral fat in the obese models than in the controls (P
< 0.05 for each vs. the corresponding pair-fed controls). Decreases in Mes,
Ret, and Epi fat in DIO mice were 21.8, 22.6, and 10.8%, respectively
(P < 0.05 for each vs. the corresponding pair-fed DIO controls)
(Fig. 2A). Similar
inhibitory effects of histamine were manifest in db/db mice (22.6%,
16.2%, and 11.1% decrease in Mes, Ret, and Epi fat, respectively; P
< 0.05 for each vs. the corresponding pair-fed db/db controls)
(Fig. 2B). Of note,
subcutaneous fat in either DIO or db/db mice was not affected by
histamine treatment (Fig.
2).

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FIG. 2. Central effects of chronic histamine (HA) infusion on fat weight (A,
B), ob gene expression (C), and serum leptin
concentration (D) in DIO and db/db obese mice. Each pair-fed
group was pair-fed with the corresponding HA-treated mice. HA treatment and
other procedures were the same as those in
Fig. 1, as applicable. Values
are means ± SE (n = 6 for each). Each value is expressed as
percentage of PBS controls. db/db-HA, db/db mice with HA;
db/db-PBS, db/db mice with PBS; DIO-HA, DIO mice treated
with HA; DIO-PBS, DIO mice with PBS; Sub, subcutaneous; r.a.u., relative
arbitrary unit. *P < 0.05 and **P
< 0.01 vs. the corresponding PBS controls; P < 0.05 vs.
the corresponding pair-fed PBS controls.
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Effects on ob gene expression and serum leptin
concentration. Histamine treatment i.c.v. (0.05 µmol · g body
wt-1 · day-1 for 7 days) reduced Epi-fat
ob gene mRNA expression in both DIO and db/db mice compared
with pairfed controls or ad libitum controls
(Fig. 2C), consistent
with the results in Fig. 2. The
percent decrease in ob gene expression in DIO and db/db mice
was 34.4 and 22.3%, respectively, compared with ad libitum controls
(P < 0.01 for each), and 18.4 and 11.3%, respectively, compared
with pair-fed controls (P < 0.05 for each)
(Fig. 2C). Reflecting
the reduced mRNA, serum leptin concentration after histamine infusion was 15.4
and 11.2% less in DIO and db/db mice, respectively, than in pair-fed
controls (P < 0.05 for each), and 30.4 and 21.1% less,
respectively, than in ad libitum controls (P < 0.01 for each)
(Fig. 2D).
Glucose and insulin tolerance tests. We examined the effects of
histamine on glucose and insulin tolerance after administration of standard
intraperitoneal glucose or insulin after 7 days of histamine treatment. Serum
glucose concentrations during glucose tolerance tests were lowered in both
histamine-treated DIO and db/db mice compared with PBS-treated DIO
and pair-fed PBS-treated db/db mice [DIO: F(1,3) = 3.94, P
< 0.05 and F(1,3) = 3.76, P < 0.05; db/db: F(1,3) =
4.03, P < 0.05 and F(1,3) = 3.23, P < 0.05]
(Fig. 3). As with glucose
loading, the insulin tolerance test showed that hypoglycemic responses were
exaggerated more in histamine-treated DIO and db/db mice than in
PBS-treated DIO and pair-fed PBS-treated db/db mice [DIO: F(1,3) =
3.20, P < 0.05 and F(1,3) = 4.06, P < 0.05;
db/db: F(1,3) = 3.35, P < 0.05 and F(1,3) = 3.44,
P < 0.05] (Fig. 3). There were no significant differences between PBS and pair-fed PBS groups
(Fig. 3).

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FIG. 3. Effects of histamine (HA) on intraperitoneal glucose tolerance (A,
B) and insulin tolerance (C, D) in DIO and db/db obese
mice. Mice in A and B were injected i.p. with 1.0 mg/g body
wt glucose. Mice in C and D were injected i.p. with 0.5 mU/g
body wt insulin. Each pair-fed group was pair-fed with the corresponding
HA-treated mice. Values and vertical bars are means ± SE (n =
6 for each). db/db-HA, db/db mice after 7-day HA treatment;
db/db-PBS, db/db mice after 7-day PBS treatment; DIO-HA, DIO
mice after 7-day HA treatment; DIO-PBS, DIO mice after 7-day PBS treatment.
*P < 0.05 vs. the corresponding PBS controls;
P < 0.05 vs. the corresponding pair-fed control.
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Effects of histamine treatment on food intake and body weight in H1KO
mice. Figure 4 shows
time-course changes in food intake and body weight of DIO and db/db
controls and mice combined with H1KO after i.c.v. infusion of histamine (0.05
µmol · g body wt-1 · day-1) for 7 days.
Histamine infusion into DIO and db/db mice induced decreases in
cumulative food intake [F(1,11) = 17.65, P < 0.01; F(1,11) =
40.87, P < 0.01]. In the DIO- and db/db-H1KO mice, the
suppressive effects of histamine on food intake were decreased compared with
histamine-treated wild type (+/+) controls [F(1,11) = 4.51, P <
0.01; F(1,11) = 4.64, P < 0.01]
(Fig. 4A and
D). Histamine infusion at the same dose and for the same
period as described in DIO and db/db mice decreased body weight
[F(1,11) = 7.69, P < 0.01; F(1,11) = 12.08, P < 0.01].
In the DIO- and db/db-H1KO mice, however, the suppressive effects on
body weight were decreased compared with histamine-treated wild type (+/+)
controls [F(1,11) = 2.53, P < 0.01; F(1,11) = 4.37, P
< 0.01] (Fig. 4A, and
D).

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FIG. 4. Central effects of chronic histamine (HA) infusion on food intake
(A, B) and body weight (C, D) in DIO (A, C) and
db/db obese (B, D) mice with and without H1KO. Values and
vertical bars are means ± SE (n = 6 for each). Horizontal bars
show the i.c.v. infusion period of test solution.
db/db-HA-H1R(-), H1KO-db/db mice treated with HA;
db/db-HA-H1R(+), wild db/db mice with HA;
db/db-PBS-H1R(-), H1KO-db/db mice with PBS;
db/db-PBS-H1R(+), wild db/db mice with PBS;
DIO-HA-H1R(-), H1KO-DIO mice with HA; DIO-HA-H1R(+),
wild-type DIO mice with HA; DIO-PBS-H1R(-), H1KO-DIO mice with PBS;
DIO-PBS-H1R(+), wild DIO mice with PBS. *P <
0.01 vs. the corresponding PBS control; P < 0.01 vs. the
corresponding wild-type control.
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Effects of histamine to regulate adiposity and BAT and WAT UCP gene
expression are partially mediated by H1-R.
Intracerebroventricular infusion of histamine (0.05 µmol · g body
wt-1 · day-1 for 7 days) decreased visceral fat
in DIO and db/db mice compared with pair-fed controls (P
< 0.01 for each). In contrast to these mice, induced changes in such
adiposity were attenuated in H1-R knockout DIO and db/db
mice compared with pair-fed controls (P < 0.01 for each).
Intralateralventricular treatment with histamine (0.05 µmol · g body
wt-1 · day-1 for 7 days) remarkably increased BAT
UCP-1 mRNA expression by 172.4 and 154.5% in DIO and db/db mice,
respectively, compared with pair-fed controls. These effects of histamine were
attenuated in the DIO and db/db mice with targeted disruption of the
histamine H1-R gene (Fig.
5A). Central treatment with histamine (0.05 µmol
· g body wt-1 · day-1 for 7 days)
remarkably increased WAT UCP-3 expression by 173.2 and 165.9% in DIO and
db/db mice, respectively, compared with pair-fed controls. However,
these effects of histamine were also attenuated in the DIO and db/db
mice with targeted disruption of the histamine H1-R gene
(Fig. 5B).

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FIG. 5. Chronic central effects of histamine (HA) on gene expression of UCP-1 in
BAT (A) and UCP-3 in WAT (B) in PBS-treated wild-type (WT)
( ), PBS-treated H1KO ( ), HA-treated WT ( ), and HA-treated H1KO
( ) (n = 6) DIO and db/db obese mice. Values are means
± SE (n = 6 for each). Each value is expressed as percentage
of PBS controls. *P < 0.05, **P
< 0.01 vs. PBS control; P < 0.05 vs. HA-treated
control. C and D: Representative blots of BAT UCP-1 gene
expression in PBS-treated WT mice (PBS-WT), PBS-treated H1KO mice (PBS-H1KO),
HA-treated WT mice (HA-WT), and HA-treated H1KO mice (HA-H1KO) in DIO
(C) and db/db (D) mice. E and F:
Representative blots of WAT UCP-3 gene expression in PBS-WT, PBS-H1KO, HA-WT,
and HA-H1KO in DIO (E) and db/db (F) mice. r.a.u.,
relative arbitrary unit.
|
|
 |
DISCUSSION
|
---|
The present study shows that chronically central treatment with histamine
contributes to improvement of the abnormality in energy metabolism of DIO and
db/db mice. DIO mice are known to be an acquired leptin-resistant
model, whereas db/db mice are an inherited model with a
leptin-receptor mutation (28).
In the present study, i.c.v. treatment with histamine reduced food intake in
DIO mice similar to that observed in db/db mice. However, the
difference in leptin resistance between DIO and db/db mice leaves the
possibility that if leptin were actually infused i.c.v. in DIO mice, it would
cause responses similar to those observed after histamine treatment in DIO
mice. Histamine-treated DIO and db/db mice lost more body weight than
pair-fed PBS controls. The results suggest that the weight loss after
histamine treatment may be attributable not only to the decrease in food
intake but also, at least in part, to the histamine-derived increase in
lipolysis. In fact, the present data showed a greater decrease in fat pads in
histamine-treated mice than in the pair-fed PBS controls. In particular, the
histamine treatment was predominantly effective to reduce visceral fat,
leaving subcutaneous fat unaffected. Histamine has been shown to activate
peripheral lipolysis through H1- and/or H2- Rs
(40,41).
Previous studies showed that activation of histamine signaling promoted
lipolysis through sympathetic nerves
(38,41).
Selective agonists of the b3 adrenoceptor were found to accelerate
lipolysis of visceral fat more than that of subcutaneous fat
(42). Ultimately, the
suppressive and selective effect of histamine on visceral fat deposition
depends most likely on activation of the sympathetic nervous system. In this
context, it is understandable why the present histamine treatment reduced
ob gene expression in WAT and serum leptin concentration. In other
words, the downregulation of ob mRNA expression and the resultant
decrease in leptin production reflect reduction in body fat content by
histamine treatment because WAT ob mRNA level and serum concentration
of leptin are positively and tightly correlated with body fat mass
(25,26).
Elevation of circulating FFAs in obese animals has been regarded as a major
determinant of decreased insulin sensitivity because it increases hepatic
glucose output and decreases glucose disposal in muscle
(43). Treatment of DIO and
db/db obese mice with histamine lowered serum concentrations of
glucose and insulin in the present study. In addition, both intraperitoneal
glucose and insulin tolerance tests showed that histamine treatment improved
glucose tolerance and insulin sensitivity. The lowered serum FFA concentration
produced simply by virtue of histamine-induced reduction of visceral fat may
be a major factor that improved insulin sensitivity.
To clarify the possibility of reduction in body weight and adiposity
induced by increased energy expenditure, we investigated the effects of
histamine on UCP expression in peripheral tissues. Intracerebroventricular
infusion of histamine in DIO and db/db obese mice upregulated mRNA
expression of BAT UCP-1 and WAT UCP-3 in the present study. It is well known
that BAT is richly innervated by sympathetic nerves
(6). Expression of UCP-1 and
UCP-3 in BAT and WAT is known to be modulated, in part, by ß3
agonists, indicating sympathetic influence on UCP expression
(44). According to previous
studies, hypothalamic neuronal histamine affects peripheral lipid metabolism
and autonomic function
(38,41).
Such observations suggest that pharmacological potentials of enhanced
histamine signaling in the hypothalamus may regulate UCP expression through
the sympathetic nervous system.
Further experiments as to whether the H1-R per se may be
involved in energy intake and expenditure were carried out to examine the
effects of H1KO on both food intake and expression of BAT and WAT UCPs in DIO
and db/db mice. The present data from DIO and db/db mice
revealed that the effects of histamine treatment on food intake and gene
expression of BAT UCP-1 and WAT UCP-3 were attenuated in H1KO mice compared
with those in the histamine-treated wild controls, although H1KO mice were not
restored to the levels of the PBS controls. Intracerebroventricular infusion
of histamine thus suppresses energy intake and accelerates energy expenditure
through the H1-R in the hypothalamus. It is intriguing that the
partial but not complete abolishment of the effects on food intake and UCP
expression in DIO and db/db mice is confirmed by targeted disruption
of H1-R. In this regard, histamine receptors other than
H1-R may be involved in the control of feeding and UCP
expression.
In conclusion, activation of hypothalamic histamine signaling induced by
histamine treatment contributes to maintenance of energy balance even in
leptin-resistant DIO and db/db mice through reduction of food intake,
visceral adiposity, ob gene expression, and circulating leptin
concentration together with upregulation of BAT and WAT UCPs gene expression.
The improvement of adiposity results in recovery of insulin sensitivity in DIO
and db/db mice. Evidence that regulatory actions of histamine are
mediated at least in part through H1-R has been shown by targeted
disruption of H1-R.
 |
ACKNOWLEDGMENTS
|
---|
Supported by Grant-in-Aid 10470233 from the Japanese Ministry of Education,
Science, and Culture; by Research Grants for Intractable Diseases from the
Japanese Ministry of Health and Welfare, 1997-1998; and by Research Grants
from the Japanese Fisheries Agency for Research into Efficient Exploitation of
Marine Products for Promotion of Health, 1997-1998.
We thank Dr. Knight, DS, Department of Anatomy, Louisiana State University,
for help in preparation of the manuscript.
 |
FOOTNOTES
|
---|
BAT, brown adipose tissue; Epi, epididymal; FFA, free fatty acid;
H1-R, H1-receptor; i.c.v., intracerebroventricularly;
i.p., intraperitoneally; Mes, mesenteric; PBS, phosphate-buffered saline; PCR,
polymerase chain reaction; Ret, retroperitoneal; UCP, uncoupling protein; WAT,
white adipose tissue.
Received for publication February 15, 2000
and accepted in revised form October 11, 2000
 |
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