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Endocrinology, doi:10.1210/en.2003-0222
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Endocrinology Vol. 144, No. 10 4306-4314
Copyright © 2003 by The Endocrine Society

Hyperleptinemia, Visceral Adiposity, and Decreased Glucose Tolerance in Mice with a Targeted Disruption of the Histidine Decarboxylase Gene

András K. Fülöp, Anna Földes, Edit Buzás, Krisztina Hegyi, Ildikó H. Miklós, László Romics, Monika Kleiber, András Nagy, András Falus and Krisztina J. Kovács

Laboratory of Molecular Neuroendocrinology (A.Fö., I.H.M., K.J.K.), Institute of Experimental Medicine, H-1083 Budapest, Hungary; Department of Genetics, Cell, and Immunobiology (A.K.F., E.B., K.H., A.Fa.) and 3rd Department of Internal Medicine (L.R., M.K.), Semmelweis University, H-1089 Budapest, Hungary; Samuel Lunenfeld Research Institute (A.N.), Mount Sinai Hospital, Toronto, Canada M5G 1X5; and Molecular Immunology Research Group (A.Fa.), Hungarian Academy of Sciences, H-1089 Budapest, Hungary

Address all correspondence and requests for reprints to: Krisztina J. Kovács, Ph.D., Laboratory of Molecular Neuroendocrinology, Institute of Experimental Medicine, Szigony u. 43, H-1083 Budapest, Hungary. E-mail: kovacs{at}koki.hu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Histamine has been referred to as an anorexic factor that decreases appetite and fat accumulation and affects feeding behavior. Tuberomammillary histaminergic neurons have been implicated in central mediation of peripheral metabolic signals such as leptin, and centrally released histamine inhibits ob gene expression. Here we have characterized the metabolic phenotype of mice that completely lack the ability to produce histamine because of targeted disruption of the key enzyme in histamine biosynthesis (histidine decarboxylase, HDC). Histochemical analyses confirmed the lack of HDC mRNA, histamine immunoreactivity, and histaminergic innervation throughout the brain of gene knockout mouse. Aged histamine-deficient (HDC-/-) mice are characterized by visceral adiposity, increased amount of brown adipose tissue, impaired glucose tolerance, hyperinsulinemia, and hyperleptinemia. Histamine-deficient animals are not hyperphagic but gain more weight and are calorically more efficient than wild-type controls. These metabolic changes presumably are due to the impaired regulatory loop between leptin and hypothalamic histamine that results in orexigenic dominance through decreased energy expenditure, attenuated ability to induce uncoupling protein-1 mRNA in the brown adipose tissue and defect in mobilizing energy stores. Our results further support the role of histamine in regulation of energy homeostasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FIVE DISTINCT SUBPOPULATIONS (E1–E5) of histaminergic neurons in the tuberomammillary region provide histaminergic innervation throughout the brain (1). In addition to its pivotal role in regulation of sleep-wakefulness and arousal (2), histamine is an important anorexic factor that suppresses food intake via histamine H1 receptors in the hypothalamus (3, 4) and increases energy expenditure by stimulating lipolysis in adipose tissue via activation of the sympathetic nerve (5).

Leptin, the product of ob gene, secreted primarily by white adipose tissue is a pleiotropic molecule that signals to the brain about the overall metabolic state of the animal (6, 7). Leptin suppresses food intake and increases sympathetic tone by regulating the activity of two mutually antagonistic sets of hypothalamic neuropeptides, the anorexigenic [proopiomelanocortin (POMC), cocaine- and amphetamine-induced transcript (CART), and CRH] and orexigenic [neuropeptide Y (NPY), melanin-concentrating hormone, agouti-related peptide, orexin)] cell population (8, 9, 10, 11, 12, 13). In addition, leptin receptors were found in the caudal brain stem (14) and reward-related brain structures (15). Leptin also has effects in the periphery, influencing processes as diverse as immune regulation, inflammation, hematopoesis (16, 17), and ß-oxidation in muscle cells (18).

Data are accumulating to reveal a bidirectional regulatory loop between neuronal histamine and leptin. Although hypothalamic histaminergic neurons do not express leptin receptors and are not directly sensitive to leptin, their involvement in mediation of the central effects of leptin on food intake and feeding behavior is very likely. Intracerebroventricular infusion of leptin increases the histamine turnover in the hypothalamus (19) and pharmacological depletion of brain histamine levels by {alpha}-fluoromethylhistidine attenuates leptin-induced suppression of feeding (20). Histamine H1 receptor knockout (H1R-/-) animals display an attenuated response to leptin-induced feeding suppression, directly implicating histamine neurons in the regulation of feeding behavior as a downstream signal of leptin. H1R-/- mice display diet-induced fat deposition and increased serum leptin levels (21). On the other hand, histamine reduces ob gene expression (22) and induces lipolysis (5, 23), and central infusion of histamine reduces fat accumulation in obese mice (24).

Histidine decarboxylase (HDC) is the key enzyme involved in histamine biosynthesis by decarboxylating L-histidine (25). The lack of HDC enzyme activity leads therefore to the complete absence of histamine in HDC-/- animals. The phenotypic features identified so far in HDC knockout animals include impaired allergic skin reactions, major reduction, and poor granulation of mast cells (26) weakened acute phase response (27) and blunted inducibility of IL-6 (28). Life-long deficit of HDC gene impairs cortical electroencephalogram, affects sleep-wakefulness states, and results in inability to remain awake and vigilant in response to environmental challenges (29).

The availability of a HDC-/- mouse (30) provides comprehensive model to study the role of histamine in regulation of metabolic balance and the regulatory connection between histaminergic neurons and leptin because all of the relevant histamine receptors expressed in the brain (H1R, H2R, and H3R) (2, 31) lack an appropriate agonist. The present study was therefore designed to compare the brain histaminergic system and characterize changes in feeding behavior, body weight regulation, and energy balance in wild-type and HDC-/- mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Generation of HDC gene targeted mice using R1 embryonic stem cells has been described (30, 32). Both homozygous HDC-deficient (HDC-/-) and wild-type (WT) animals were littermates in a segregating F2 population (32). Male wild-type and HDC-/- mice (CD1 background) were kept under controlled temperature and lighting conditions (lights on between 0600 and 1800 h). Mice had free access to standard laboratory chow diet (19% protein; 3.5% fat; Ssniff Spezialdiäten GmbH, Soest, Germany) for the first 3 months. Subsequently, they were maintained on histamine-free diet (<0.6 nmol histamine/g food; 17.5% protein; 5.15% fat; Altromin, Lage, Germany) for 7 d before experiments were performed. All experimental procedures were carried out in accordance with the European Communities Council Directive (86/609 EEC). Experiments were approved by the Animal Care and Use Committee at Semmelweis University.

To confirm the genotype of the mice, genomic DNA was obtained from tail samples and analyzed by PCR using primers (HDC forward: 5'-AGT GAG GGA CTG TGG CTC CAC GTC GAT GCT-3' and HDC reverse 5'-TAC AGT CAA AGT GTA CCA TCA TCC ACT TGG-3'; neor forward: 5'-AAA CAT CGC ATC GAG CGA GCA CGT ACT CGG-3' and neor reverse 5'-ATG TCC TGA TAG CGG TCC GCC ACA CCC AGC-3').

Procedures
All metabolic experiments were carried out on mice older than 12 wk. Body weights, and food intake was monitored on 7 consecutive days in WT and HDC-/- mice. Caloric efficiency [gram of body weight gained/(gram food ingested x caloric equivalent of the food)] was calculated. After anesthesia and killing by cervical dislocation, the thickness of sc fat tissue was determined by measuring the thickness of the skin fold on the animal’s back with a caliper (Oditest, Kroeplin Längenmesstechnik, Germany). The epididymal white fat pad and interscapular brown adipose tissue were dissected and weighed.

To assess metabolic responses in a challenge situation (33), WT and HDC-/- mice were housed individually in cages with wire bottom and were fasted for 5 h. Their body weights were measured immediately before and after fast. At this time, rectal temperature was measured with a digital thermometer, and then animals were transported to a cold room and kept at 4 C for 90 min. Their temperature was registered at 30, 60, and 90 min in cold, and their body weight was measured again at the end of the cold challenge.

Blood samples were obtained in the morning hours (between 0900 and 1100 h) from fed and overnight-fasted animals by puncture of retrobulbar venous plexus. Leptin was measured using Quantikine M mouse leptin immunoassay kit (R&D Systems, Minneapolis, MN), and insulin plasma concentration was determined by Mouse Insulin Ultrasensitive ELISA kit (DRG Instruments GmbH, Marburg, Germany), respectively, according to the manufacturer’s instructions. Plasma corticosterone levels were determined by Octeia ELISA kit (Immunodiagnostic Systems Ltd., Boldon, UK). Triglyceride and cholesterol levels were measured by enzymatic colorimetry (GPOL-PAP and CHOD-PAP, Roche, Basel, Switzerland). High-density lipoprotein (HDL) levels were assayed with a direct HDL-Chol test kit (Randox, Crumlin, UK).

To determine glucose tolerance, mice were fasted for 18 h and given an ip injection of glucose (1 mg/g body weight). Blood samples were obtained from the retrobulbar plexus immediately before and 30, 60, and 120 min after glucose injection.

To assess the glucose response to insulin injection, mice received an ip injection of insulin (Actrapid HMge, Novo Nordisk, Bagsværd, Denmark) at a dose of 1 U/kg body weight immediately after obtaining blood sample to determine basal glucose level. Further samples were withdrawn 15, 30, and 60 min after insulin challenge. Blood glucose levels were determined by One Touch glucose monitoring system (Lifescan, Milpitas, CA).

Insulin secretory response to glucose challenge was determined in fasted mice. Blood samples were withdrawn immediately before and 30, 60, and 120 min after a single ip injection of 1 mg/g body weight D-glucose.

For histidine loading, to increase hypothalamic neuronal histamine concentration and release, L-histidine (Sigma-Aldrich Corp., St. Louis, MO), the precursor of histamine was injected ip (dose: 0.1 mg/g body weight in 0.2 ml). The animals were then left undisturbed in their individual cages until perfusion 2.5 h later.

Another set of WT and histamine-deficient mice was used to assess uncoupling protein (UCP)-1 mRNA levels in the brown adipose tissue (BAT). Animals were decapitated, the interscapular BAT dissected, cleared from white adipose tissue (WAT), and frozen immediately. For Northern blots, total RNA from BAT was purified by the method of Chomczynski and Sacchi (33), separated in a 1.2% agarose gel containing 8% formaldehyde, and transferred onto Hybond-N membranes (Amersham, Freiburg, Germany) using the capillary transfer method. DNA probe for UCP-1 (kindly provided by Barbara Cannon, Stockholm University, Stockholm, Sweden) and the ß-actin riboprobe were labeled by the random primer method (HexaLabel DNA labeling kit, MBI Fermentas, Szeged, Hungary) using 32P-dCTP (Izinta, Budapest, Hungary). Prehybridizations (42 C, 4 h) and hybridizations (42 C, overnight) were carried out in solution containing 50% (vol/vol) formamide, 6x sodium chloride/sodium citrate (SSC), 5x Denhardt’s solution, 0.5% sodium dodecyl sulfate (SDS), 50 mM Na-phosphate buffer, 100 µg/ml tRNA, 10 µg/ml polyU-homopolymer, and 7.5 µg/ml denatured salmon-sperm DNA. The labeled probes were added to the hybridization solution at 1 x 106 cpm/ml. Filters were washed at high-stringency conditions (room temperature in 2x SSC/0.1% SDS for 5 min, 68 C, 2x SSC/0.1% SDS for 30 min, 68 C in 0.2x SSC/0.1% SDS for 30 min). Between hybridizations, filters were washed in a solution containing 5 mM Na-phosphate/0.1% SDS at 100 C for 30 min to remove the labeled probe. Blots were detected by phosphor imager (Sigma) and exposed to x-ray films (XAR, Kodak, Rochester, NY) for 1–2 d at -70 C using intensifying screens; the data were analyzed by ImageJ software (http://rsb.info.nih.gov/ij). All comparisons were made from RNA samples hybridized on the same filter and normalized to the content of ß-actin RNA detected in each individual sample.

Histological procedures
Brown and epididymal fat tissues were dissected and fixed in Bouin’s fixative, dehydrated, and embedded into paraffin. Ten-micrometer sections were cut and stained with hematoxylin-eosin.

Perfusion and tissue processing
Mice were deeply anesthetized with pentobarbital and perfused through the heart with saline and then with 100 ml ice-cold fixative [4% paraformaldehyde in 0.1 M borate buffer (pH 9)]. Serial sections in the frontal plane were cut on freezing microtome, collected into antifreeze solution, and stored at -20 C.

Rabbit antibody raised against a synthetic N-terminal fragment (residues 4–17) of human Fos (sc-52, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used to identify L-histidine-activated neuronal profiles within the mouse brain. Sections were incubated in normal goat serum (1:10) for 1 h at room temperature and then in the primary antibody (dilution 1:5000) at 4 C for 72 h. Further incubation steps included 1 h in biotinylated antirabbit antibody (1:1000) and 1 h in avidin-biotin-horseradish peroxidase complex (1:500) (Vectastain Elite kit, Vector, Burlingame, CA). The peroxidase reaction was completed by diaminobenzidine tetrahydrochloride, 0.5 mg/ml) and nickel-ammonium-sulfate (1.5%) with 0.03% H2O2.

Histamine immunohistochemistry
To intensify histamine staining, male mice were injected with 200 mg L-histidine 24 h before perfusion. Fixation was performed according to Wang and Nakai (34) with some modifications. Brains were quickly flushed with saline followed by 4% 1-ethyl-3-(3 dimethylaminopropyl)-carbodiimide (Sigma-Aldrich) in 0.1 M phosphate buffer (pH 7.2). Then animals were consecutively perfused with 2% paraformaldehyde, 4% 1-ethyl-3-(3 dimethylaminopropyl)-carbodiimide, and 4% paraformaldehyde, respectively. A rabbit antiserum raised against hemocyanin-conjugated histamine (generous gift from Dr. P. Panula, Turku, Finland) was applied in 1:50,000 dilution and visualized by standard avidin-biotin horseradish peroxidase method.

In situ hybridization histochemistry
To assess HDC mRNA expression, sections were hybridized with 35S-uridine 5-triphosphate-labeled antisense riboprobes corresponding to a 530-bp fragment of the HDC gene. Hybridization and autoradiographic techniques were performed as described (35). Briefly, tissue sections were mounted onto poly-L-lysine-coated slides, postfixed with 4% paraformaldehyde, and digested with Proteinase K [10 mg/ml in 50 mM Tris (pH 8), and 5 mM EDTA at 37 C, 30 min], acetylated [0.25% acetic anhydride in 0.1 M triethanolamine (pH 8)], and dehydrated. Hybridization mixture [50% formamide, 0.3 M NaCl, 10 mM Tris (pH 8), 2 mM EDTA, 1x Denhardt’s, 10% dextran sulfate, 0.5 mg/ml yeast tRNA] was pipetted onto the slides (100 µl, containing probe at 107 dpm/ml), and hybridized overnight at 56 C. Sections were then rinsed in 4x SSC [1x SSC = 0.15 M NaCl and 15 mM trisodium-citrate buffer (pH 7)], digested with ribonuclease A (20 mg/ml in Tris-EDTA buffer with 0.5 M NaCl at 37 C for 30 min), gradually desalted, and washed in 0.1x SSC at 65–75 C for 30 min. Slides were exposed to x-ray film and dipped in NTB-2 nuclear emulsion (Kodak) and exposed for 3–5 d, developed in D-19 developer, and lightly counterstained with thionin.

Statistics
Statistical analysis was performed using STATISTICA 6.0 software (StatSoft Inc., Tulsa, OK). Results are expressed as the mean ± SEM; differences between the groups was demonstrated using ANOVA followed by Dunnett’s post hoc tests. When comparisons were restricted to two experimental groups, a t test was used. Plasma glucose concentrations after ip injections of glucose or insulin and insulin secretory response to ip glucose were analyzed by repeated-measures of ANOVA. In all cases, differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Body weight, fat pads, and energy balance
As shown in Fig. 1Go, up to 10–11 wk of age, the body weights of WT and HDC-/- mice were indistinguishable (32.54 ± 1.46 g WT vs. 32.54 ± 4.13 g HDC-/- n = 9; P = 0.480, at age 10 wk). However, the weights diverged after 10 wk and knockout mice weighed 13% more than WT control at the age of 16 wk (34.74 ± 2.16 g WT vs. 39.38 ± 5.71 g HDC-/-; n = 8, P = 0.022). This difference in body weight increased with age. Knockout mice older than 30 wk were dramatically overweight, compared with WT controls (39.10 ± 1.90 g WT vs. 46.40 ± 2.31 g HDC-/- n = 5, P = 0.0026).



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  		FIG. 1. Body weight curves for male WT and histamine-deficient HDC-/- mice kept on normal diet. Each point represents the mean of 5–10 animals.

 
Knockout animals exhibited selective increase of the epididymal WAT and interscapular BAT fat pads (Fig. 2Go) without significant differences in the thickness of sc tissue (1.18 ± 0.3 mm vs. 1.07 ± 0.2 mm in WT and HDC-/-, respectively). Average size of epididymal WAT adipocytes was significantly greater in histamine-deficient animals, compared with controls (551.15 ± 25.56 and 329.75 ± 25.26 µm2 in HDC-/- and WT, respectively). Significant histological alterations were not detected in BAT in HDC knockout animals, compared with WT control mice.



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  		FIG. 2. Fat deposition in HDC-/- mice. A, Weights of epididymal fat and BAT pads. Mean ± SEM values; n = 16; *, P < 0.01, compared with WT controls. B, Bright-field photomicrographs showing adipocytes from the epididymal WAT and in male, 3-month-old WT and HDC knockout animals. Note the reduced number of mast cells (arrows in WT) in HDC-/- mice.

 
During a 7-d test period, HDC knockout mice (age 13 wk) gained more weight (P = 0.014) and had higher caloric efficiency (P = 0.012) than age-matched controls (Fig. 3Go).



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  		FIG. 3. Comparison of metabolic parameters. Body weight gain (A), cumulative food intake (B), and caloric efficiency (C) in WT and HDC-/- mice. Mean ± SEM values, n = 10/group; *, P < 0.01.

 
To get a more dynamic, though indirect, measure of the metabolic rate, we followed the protocol described by Forbes et al. (36) by detecting the weight loss of mice during a 5-h fast. Although mice lost 6–9% of their initial weight, the differences in body weights of WT (-2.41 ± 0.25 g) and in HDC knockout animals (-2.95 ± 0.45 g) were not statistically significant.

Because HDC-deficient mice are not hyperphagic (Fig. 3Go), we hypothesized that increased fat deposition and weight gain in HDC-/- animals are due to decreased energy expenditure. To test this hypothesis, thermoregulatory responses to cold were followed and taken as an indirect measure of ability to mobilize energy (36). The baseline core temperature of WT and knockout animals were not different (37.50 ± 0.34 C and 37.49 ± 0.29 C). However, when fasted mice were challenged at 4 C, histamine-deficient animals have an impaired ability to metabolize energy stores, and their core temperature dropped by 3.82 ± 1.02 C, whereas WT animals fell by only 1.34 ± 0.40 C by the end of 90-min test period (Fig. 4Go).



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  		FIG. 4. Changes in body core temperature of WT and HDC-/- mice during cold challenge. Animals (age >12 wk) were individually caged and fasted for 5 h and then placed into a cold room at 4 C. Core temperatures were detected immediately before and 30, 60, and 90 min in cold using a digital thermometer. Mean ± SEM values, n = 10/group; *, P < 0.01.

 
Blood glucose levels and insulin secretion
To determine whether the increased adiposity in HDC-/- animals is accompanied with abnormalities in glucose metabolism, we performed glucose tolerance and insulin challenge tests. Basal glucose levels after 18 h fasting were not different between the groups (WT, 4.18 ± 0.18 mM; HDC-/-, 3.83 ± 0.24 mM). As shown in Fig. 5AGo, after ip injection of D-glucose (1 mg/g body weight), blood glucose levels in adult HDC-/- mice were significantly higher than in WT littermates, indicating glucose intolerance (P < 0.01, repeated measures of ANOVA). Maximal differences in blood glucose concentrations among the groups (32% and 30%; P = 0.037 and 0.039 by t test) were detected at 30 and 60 min post glucose time points, respectively.



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  		FIG. 5. Profile of blood glucose levels in response to ip glucose and insulin challenge. Tests were performed on 3-month-old male knockout mice (dashed line) and age-matched WT controls (solid line). Mean ± SEM values of blood glucose levels before and at different time points after challenge. A, Glucose tolerance test. Blood glucose levels of male mice that were fasted for 18 h to ip injection of 1 mg/g body weight glucose (n = 9). *, P < 0.05, compared with WT. B, Insulin test. Glucose response to insulin administration in HDC-deficient (HDC-KO) and WT control mice; n = 10). Mice received 1 U/kg insulin immediately after blood sampling at time 0. C, Insulin secretory response to glucose in WT and HDC-/- mice. Fasted animals were challenged with 1 mg/g body weight D-glucose. Mean ± SEM values (n = 6) of insulin levels.

 
To assess the effects of histamine deficiency on insulin action in vivo, we measured the changes in plasma glucose concentrations after a single ip injection of insulin (Fig. 5BGo). Although the basal glucose levels were slightly but not significantly higher in adult male nonfasted HDC-/- animals than in the WT mice, their glucose disappearance curves were comparable.

To examine insulin secretory response to glucose, mice from both genotypes were fasted for 18 h and injected ip with 1 mg/g body weight D-glucose. Plasma insulin levels were measured before and 30, 60, and 120 min after glucose challenge. Within 30 min plasma insulin levels reached peak and remain elevated up to 120 min. Plasma insulin levels rose 334% in WT but only 85.7% in HDC knockout animals; however, at the plateau of the insulin secretory curve, there was no significant differences between WT and histamine-deficient mice (Fig. 5CGo).

Hormone levels and other variables
Serum leptin levels were significantly higher in HDC-/- animals than in WT mice (0.64 ± 0.07 ng/ml in WT vs. 3.60 ± 0.80 in HDC-/-; n = 10; P < 0.001). Following overnight fast, serum leptin levels were reduced in WT controls (0.26 ± 0.17 ng/ml) but remained unchanged at high levels in knockout animals (3.48 ± 0.93 ng/ml) (Fig. 6AGo).



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  		FIG. 6. Hormone levels of WT and histamine-deficient male mice. Leptin (A), insulin (B), and corticosterone (C) levels in ad libitum-fed and fasted male mice (age > 12 wk). Mean ± SEM values, n = 6–12; *, P < 0.01, compared with WT; +, P < 0.01, compared with fed mice.

 
Steady-state plasma insulin levels were comparable in WT controls (345 ± 53 pM) and HDC-deficient mice (361 ± 48 pM). After fasting, however, insulin levels of HDC-knockout animals remained at higher levels (212.5 ± 46.3 pM) than in WT mice (11.6 ± 2.3 pM, n = 5, P = 0.024) (Fig. 6BGo).

Morning levels of plasma corticosterone were elevated in HDC-/- male mice (42.68 ± 6.88), compared with WT animals (23.03 ± 7.34 ng/ml), although the difference was not statistically significant (P = 0.068). After fasting, corticosterone levels were increased in both genotypes (Fig. 6CGo).

Serum triglyceride, cholesterol, and HDL levels were not different in HDC-/- mice, compared with wild-type controls (Table 1Go).


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  		TABLE 1. Some metabolic parameters in WT and HDC-/- mice kept in normal or histamine (HA)-free diet

 
UCP-1 mRNA in BAT
Northern blot analysis of UCP-1 mRNA levels in the BAT did not reveal any significant difference in UCP-1 expression between WT and HDC-/- animals under normal conditions (fed animals kept in room temperature). When placed into a cold room (4 C) for 18 h, WT animals displayed a 4.5-fold induction of UCP-1 mRNA levels. Histamine-deficient mice were much less efficient in up-regulating BAT UCP-1 expression in response to cold challenge than WT controls (Fig. 7Go).



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  		FIG. 7. UCP-1 mRNA expression in the BAT. A, Representative Northern blots of UCP-1 mRNA in the BAT of wild-type (+/+) and HDC knockout (-/-) animals. ß-Actin hybridization shows that equal amount of samples were loaded. B, Effect of cold challenge on UCP-1 mRNA levels in the BAT. Mean ± SEM values. Each value is expressed as percentage of WT control in normal (room) temperature (RT). +, P < 0.01, compared with values from animals kept in normal temperature. *, P < 0.01, compared with WT, cold-challenged animals.

 
Histological analysis of HDC-/- animals
Distribution of HDC mRNA and histamine-immunoreactive (ir) profiles in the brain of WT mice was essentially similar to that observed in rats (1). Cell clusters in the tuberomammilary region corresponding to E1-E5 cell groups were identified by HDC in situ hybridization, using a cRNA probe corresponding to bases 121–650 of the mouse HDC sequence. No hybridization signal in the tuberomammilary region of HDC knockout animals could be detected (Fig. 8Go). Immunocytochemistry also confirmed the absence of histamine-containing neurons and fibers throughout the brain of HDC-/- animals (Fig. 8Go). Histidine loading, a process that usually leads to a strong increase of HDC activity and histamine release as well as enhanced histamine in normal animals did not elicit any histamine-ir signal in HDC-/- mice (not shown).



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  		FIG. 8. Lack of histamine-synthesizing neurons in HDC-/- mice. Dark-field photomicrographs (top row) demonstrate distribution of HDC mRNA in the tuberomammillary region of a WT animal. Corresponding bright-field images (middle) show clusters of histamine-ir neurons. Histamine-containing profiles are completely missing from the HDC-/- mice. Dense histaminergic innervation of arcuate nucleus was revealed in WT mice (bottom row). This area contains neuropeptides (POMC, NPY) regulating food intake. Note the disappearance of histaminergic fibers from the arcuate nucleus in HDC-/- animal. Arc, Arcuate nucleus; DM, dorsomedial nucleus; f, fornix; Mtu, medial tuberal nucleus; PMv, ventral premammillary nucleus; V3, third ventricle; VM, ventromedial nucleus. Bars, 200 µm.

 
Neuronal activation following histidine challenge
L-Histidine, a precursor of neuronal histamine, has been shown to increase the activity of HDC and concentration of histamine in the hypothalamus (37). We take the advantage of this finding to reveal histamine-responsive profiles in the hypothalamus using c-fos as an immediate-early gene marker of neuronal activation. Immunohistochemical staining patterns for c-Fos on ip L-histidine administration was compared in the hypothalamus of WT and HDC knockout animals. Both WT and HDC-/- animals injected with PBS displayed weak to moderate c-Fos immunoreactivity throughout the hypothalamus, limited to scattered cell clusters in the anterior hypothalamic area, lateral hypothalamus, and suprachiasmatic-, periventricular-, lateroanterior hypothalamic-, and ventromedial nuclei. A significant increase of c-Fos signal was detected in the arcuate nucleus and lateral hypothalamic area of L-histidine-injected WT mice 2 h after challenge. In contrast, HDC-/- animals failed to display any c-Fos induction in these cell clusters (Fig. 9Go).



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  		FIG. 9. Histamine-responsive neurons in the arcuate nucleus. Bright-field photomicrographs showing the medial basal hypothalamus at the level of arcuate nucleus. In vehicle-injected (PBS) WT mice, scattered c-Fos-positive profiles are seen in the ventromedial nucleus and lateral hypothalamus. L-Histidine administration in WT controls resulted in significant c-Fos-induction in the arcuate nucleus and neurons of the lateral hypothalamic area. The same treatment was ineffective to induce c-Fos immunoreactivity in the hypothalamus of HDC knockout mice. ARC, Arcuate nucleus; LHA, lateral hypothalamic area; V3, third ventricle; VM, ventromedial nucleus. Bar, 250 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Histamine has been implicated as an important modulator of central autonomic mechanisms that govern food intake, feeding behavior, metabolism, and energy expenditure. Here we provide evidence that histamine deficiency in HDC gene targeted mice results in a phenotype characterized by increased weight gain, epididymal fat deposition, and increased amount of BAT. The body weights of HDC-/- mice kept on normal or histamine-free diet are not significantly different from WT controls in young animals; however, the weight difference is increasing with aging. The mechanisms, however, that lead to obese phenotype in adult animals despite life long histamine deficiency remain to be elucidated.

One possibility for the excess body weight gain of HDC-/- animals is the altered sleep-wake cycle (29). Histamine-deficient mice are more somnolent in the phase of light-dark transition and in response to environmental challenges that might effect their feeding behavior. However, no correlation was found between body weight and the daily amount of sleep-wake stages in either genotype (29).

The increased body weight in HDC-/- animals is due to the increased fat deposition to visceral (epididymal) stores, but sc fat stores are not affected significantly. The role of neuronal histamine in regulation of WAT depots is supported by the following facts: 1) recent reports by Tsuda et al. (5) and Yoshimatsu et al. (23) show that activation of central histaminergic neurons by histamine H3 autofeedback receptor antagonist or by histidine accelerates lipolysis in WAT (2). In line with this observation, infusion of histamine into the third cerebral ventricle dose-dependently increased glycerol concentration in the perfusate from the epididymal adipose tissue and results in activation of sympathetic nerve (2). Chronic, central administration of histamine reduces body fat weight in db/db mice or in mice with diet-induced obesity (24). On the contrary, accelerated fat deposition was detected in histamine H1 receptor knockout mice kept on a high-fat diet (21). These results offer an explanation for the relatively selective increase of epididymal fat deposition seen in histamine-deficient HDC-/- mice. Slight excess of glucocorticoids at the circadian trough (38) and elevated insulin levels (39) may also contribute to the selective increase of visceral fat stores seen in HDC-/- mice.

Because HDC-/- mice consume slightly less food than their WT counterparts, the increased weight gain and caloric efficiency probably is due to the impairment of mobilization of fat/energy stores. Consistent with this hypothesis, HDC-/- mice show defect in thermoregulation and decreased accessibility of fat stores when fasted animals are exposed to cold, although these measures are indirect indices of energy expenditure.

In addition to the lipolytic action in WAT, sympathetic nervous system activates BAT, hence increasing diet-induced thermogenesis and dissipating excess energy as heat. This effect is mediated via ß3-adrenoceptors and activation of UCPs in the BAT. Lack of histaminergic stimulation of sympathetic nerve activity and/or decreased energy expenditure may account for the increased interscapular BAT weight seen in histamine-deficient mice. Consistent with this hypothesis histidine decarboxylase gene targeted mice are defective to up-regulate BAT UCP-1 transcription in response to cold stimulation. At this stage of the analysis, however, it is not known whether defects of sympathetic outflow or impaired ß-adrenergic receptor function is responsible for the lack of up-regulation of UCP-1 mRNA in HDC-/- animals. It is interesting to note that H1R-/- animals display a significant reduction of the leptin-induced up-regulation of UCP-1 in BAT (21), and icv histamine injection up-regulated UCP-1 mRNA levels in leptin-resistant and diabetic mice (24).

A large body of evidence supports the hypothesis that histaminergic neurons are key players of the central mediation of peripheral signals as well as of the translation of central, hypothalamic-signaling events to the periphery. Leptin administration increases the release and turnover of histamine in the hypothalamus (19, 20); leptin-induced suppression of food intake is attenuated in mice treated with {alpha}-fluoromethylhistidine, a specific inhibitor of HDC (40). Furthermore, leptin is unable to reduce food intake in mutant mice lacking histamine H1 receptors (21, 40). On the other hand, icv infusion of histamine reduces ob gene expression, suppresses fat deposition, and decreases serum leptin concentrations in both diet-induced obesity and diabetic db/db mice (24).

HDC-deficient mice provide an excellent tool to study the interrelationship between histamine and leptin in a situation of chronic histamine insufficiency. Leptin may directly or indirectly target histaminergic neurons. Although some cells in the histaminergic premammillary nucleus express leptin receptors (41) and display c-fos induction in response to systemic leptin administration (42), the major histaminergic cell clusters in the tuberomammilary region are not directly sensitive to leptin.

Leptin receptors were identified on anorexigenic POMC-, CART-, and CRH-synthesizing neurons as well as on orexigenic NPY, melanin-concentrating hormone, and orexin neurons (10) in the rat hypothalamus. Previous studies have revealed that leptin inhibits the expression of orexigenic neuropeptides and stimulates the expression of anorexigenic peptides in the hypothalamus (8). These leptin-sensitive neurons are in a position to relay the hormonal influence of leptin to histaminergic system. In support this hypothesis, {alpha}MSH-containing axons were shown to innervate histaminergic neurons (43). This innervation seems to be reciprocal because histaminergic fibers densely innervate hypothalamic nuclei that synthesize anorexigenic and orexigenic neuropeptides (44). We have identified histaminergic fibers in close apposition with NPY-containing profiles in the arcuate nucleus (Miklós, I. H., unpublished observation). There is also a reciprocal connection between histamine- and orexin-containing neurons in the hypothalamus. Histamine-synthesizing neurons innervate leptin-sensitive orexin neurons in the lateral hypothalamus, but orexin-A and -B depolarize tuberomammilary histamine neurons (45).

Here we identified dense plexus of histaminergic fibers in the arcuate nucleus, which contain POMC ({alpha}MSH), CART, and NPY neurons. Following L-histidine injection, a subset of these arcuate neurons expresses c-Fos, suggesting an impact of histaminergic innervation on arcuate neurons. Further anatomical studies are required, however, to identify elements of the neuronal circuit underlying leptin-histamine interaction.

Based on the data presented, we propose that the lack of histamine in HDC-/- mice blocks leptin signaling through the central histaminergic neurons. As a consequence, the histaminergic stimulation of anorexigenic effector mechanisms in the hypothalamus is disrupted, resulting in an orexigenic dominance, leading to increased fat deposition and hyperleptinemia. Importantly, however, the increase in serum leptin levels is disproportionate to the increased fat mass and can be explained by a lack of feedback inhibition of leptin expression and/or leptin resistance.

Like leptin, the pancreatic hormone insulin is also an important metabolic signal to the brain (46). The secretion of insulin and its level in the blood and brain is directly proportional to the adipose mass. It has recently been shown that central anorexic effects of insulin is mediated by POMC-expressing cells in the arcuate nucleus (47). In HDC-/- mice, the elevated insulin levels suggest that anorexigenic insulin signaling is also disturbed. Based on the glucose disappearance curves, HDC knockout animals retain their insulin sensitivity; however, insulin-secretory responses to fasting and glucose challenge are affected.

The role of histaminergic mechanisms in regulation of food intake and metabolism was also addressed in histamine H1 receptor knockout mice (21). These mice, when kept on normal diet, do not show any significant metabolic phenotype, compared with WT controls; however, they became obese, accumulate visceral fat, and up-regulate ob gene when loaded with a high-fat diet. Thus, lifelong, complete absence of histamine in HDC-/- mice has a significantly more profound impact on metabolism than anticipated from the H1R-/- mice. To paint a more complex picture of the relevant receptors involved in the metabolic aspects of histamine action, it has been recently shown that mice with targeted disruption of H3 presynaptic autoreceptors are mildly obese and leptin and insulin resistant (48).

Our results point to the regulatory role of histamine in mediating anorectic (leptin and/or insulin) signals to the central nervous system. Importantly, because this is a model that lacks histamine systemically, we cannot exclude the possibility that the lack of histamine may also have a direct effect on a target in the periphery. It should be noted, that histamine weakly stimulates lipolysis in human subcutaneous fat cells (49).

In contrast to most animal models of obesity such as ob/ob mice, obesity in humans is often associated with metabolic X syndrome, which includes hyperleptinemia, glucose intolerance, and visceral fat deposition. Here we show that the HDC-/- mice display a metabolic phenotype characterized by leptin resistance, hyperinsulinemia, impaired glucose tolerance, and increased epididymal white and brown fat depots, supporting the important role that histamine plays in regulation of energy metabolism.


   Acknowledgments
 
We thank Dr. Pretti Panula (Department of Biology, Abo Akademi University, Finland) for generously providing histamine antisera; Dr. Barbara Cannon (Stockholm University, Stockholm, Sweden) for providing UCP-1 plasmid; and Drs. Attila Sándor and Péter Jakus (Department of Biochemistry, Medical University, Pécs, Hungary) for their valuable help in preparation of BAT samples. Assistance of Dr. B. Bali (graphical), Ms. O. Szalay, and V. Tökési (technical) is acknowledged.


   Footnotes
 
This work was supported by the Hungarian Ministry of Health, ETT 159, and 300/2000 (to A.Fa.), 473/2003 (to K.J.K.); Hungarian Research Fdn. OTKA T031887 (to A.Fa.), T043056 (to K.J.K.) and T032134 (to B.E.).

A.K.F. and A.Fö. contributed equally to the manuscript.

Abbreviations: BAT, Brown adipose tissue; CART, cocaine- and amphetamine-regulated transcript; HDC, histidine decarboxylase; HDL, high-density lipoprotein; H1R, histamine receptor, type I; ir, immunoreactive; {alpha}MSH, {alpha}-melanocyte-stimulating hormone; NPY, neuropeptide Y; POMC, proopiomelanocortin; SDS, sodium dodecyl sulfate; SSC, sodium chloride/sodium citrate; UCP, uncoupling protein; WAT, white adipose tissue; WT, wild-type.

Received February 18, 2003.

Accepted for publication June 19, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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