Central Infusion of Histamine Reduces Fat Accumulation and Upregulates UCP Family in Leptin-Resistant Obese Mice

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-3gene. 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 20× sodium chloride—sodium 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.

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 (0.05μmol · 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).

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).

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).

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/dbcontrols and mice combined with H1KO after i.c.v. infusion of histamine (0.05μmol · 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).

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/dbmice 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/dbmice with targeted disruption of the histamine H₁-R gene(Fig. 5B).

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 β₃agonists, 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.

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.