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Increase of Neuronal Histamine in Obese Rats Is Associated with Decreases in Body Weight and Plasma Triglycerides Kjell Malmlo¨f,* Valeria Golozoubova,* Bernd Peschke,† Birgitte S. Wulff,‡ Hanne H.F. Refsgaard,§ Peter B. Johansen,¶ Thomas Cremers,储 and Karin Rimvall‡

Abstract ¨ F, KJELL, VALERIA GOLOZOUBOVA, MALMLO BERND PESCHKE, BIRGITTE S. WULFF, HANNE H.F. REFSGAARD, PETER B. JOHANSEN, THOMAS CREMERS, AND KARIN RIMVALL. Increase of neuronal histamine in obese rats is associated with decreases in body weight and plasma triglycerides. Obesity. 2006;14:2154 –2162. Objective: The purpose of the present study was to examine the metabolic effects of a specific histamine H3 receptor antagonist, the cinnamic amide NNC 0038-0000-1202 (NNC 38-1202). Research Methods and Procedures: Effects of NNC 381202 on paraventricular levels of histamine and acute effects on food intake were followed in normal rats, whereas effects on body weight homeostasis and lipid metabolism were studied in a rat model of diet-induced obesity (DIO). Results: NNC 38-1202, administered as single oral doses of 15 and 30 mg/kg, significantly (p ⬍ 0.01) increased paraventricular histamine by 339 ⫾ 54% and 403 ⫾ 105%, respectively, compared with basal levels. The same doses produced significant (p ⬍ 0.01) reductions in food intake. In DIO rats receiving NNC 38-1202 in a daily dose of 5 mg/kg for 22 days, a decrease in food intake was associated with a significant (p ⬍ 0.001) net loss of body weight (⫺11.0 ⫾

Received for review March 31, 2006. Accepted in final form August 31, 2006. The costs of publication of this article were defrayed, in part, by the payment of page charges. This article must, therefore, be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Departments of *Diabetes Pharmacology, †Protein and Peptide Chemistry, ‡Molecular Pharmacology, §Exploratory Absorption, Distribution, Metabolism and Excretion, Diabetes, and ¶Hemostasis Pharmacology, Novo Nordisk A/S, Måløv, Denmark; and 储Department of Bimonitoring and Sensoring (Brains on line), University Centre for Pharmacy, Groningen, The Netherlands. Address correspondence to Kjell Malmlo¨f, Department of Diabetes Pharmacology, Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark. E-mail: [email protected] Copyright © 2006 NAASO

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4.8 grams), compared with rats receiving vehicle, which gained 13.6 ⫾ 3.0 grams. Also, NNC 38-1202 significantly (p ⬍ 0.05) reduced plasma triglycerides by ⬃42%, in parallel with increases in plasma free fatty acids and ␤-hydroxybutyrate levels. Despite reductions in food intake and body weight following administration of NNC 38-1202, no sign of a decrease in energy expenditure was observed, and whole-body lipid oxidation was significantly (p ⬍ 0.05) increased in the period after dosing. Discussion: The present study suggests that antagonistic targeting of the histamine H3 receptor decreases food intake, body weight, and plasma TG levels and, thus, represents an interesting approach to treatment of obesity and associated hyperlipidemia. Key words: histamine H3 receptor antagonist, food intake, body weight, blood lipids, energy expenditure

Introduction Obesity is known to increase the risk of development of type 2 diabetes and cardiovascular disease (1). Although it has been shown that even a modest weight loss reduces the risk of acquiring these complications (2), very few effective therapies that can facilitate a successful treatment are presently available. Recent reviews (3–5) verify that even though weight loss can be achieved through dieting and other lifestyle changes, the long-term outcome is uncertain due to loss of compliance (6). For this reason, the possibility of finding an effective pharmacological treatment of obesity has stimulated intensive research during recent years (7). Although an impressive body of knowledge has been accumulating with regard to the appetite-regulating properties of molecules originating in the gastrointestinal tract (8,9) or in adipose tissue (10), relatively less attention has been paid to the involvement of neuronal histamine in regulation of energy homeostasis. Despite this, it is now established that neuronal histamine, through activation of

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Figure 1: Molecular structure of the histamine H3 receptor antagonist NNC 38-1202.

central histamine H1 receptor signaling (11,12), plays a central role in regulation of food intake and body weight homeostasis (13). However, due to the widespread distribution of the histamine H1 receptor in extracerebral tissue, direct targeting of this receptor would not result in a specific effect on food intake. Instead, specific targeting of the histamine H3 receptor, which regulates intrasynaptic histamine levels (14) and has a pattern of expression that is essentially confined to the central nervous system (15,16), represents a more promising approach to achieving the desired degree of specificity. We recently published data demonstrating that administration of a selective histamine H3 receptor antagonist to rats was associated with an increase in paraventricular histamine levels, suppression of food intake, and a decrease in body weight in rats (17). Further evidence that histamine H3 receptor antagonists are potent regulators of body weight homeostasis is provided by data from another laboratory (18). In our previous study (17), a histamine H3 antagonist with a relatively short plasma half-life was observed to decrease plasma triglyceride (TG)1 levels in parallel with body weight. An increase in whole-body lipid oxidation was also indicated. We find these observations interesting because a malfunction in lipid metabolism is one of the main features of obesity and associated comorbidities, and a concomitant normalization of body weight and hyperlipidemia would represent a clear clinical advantage. The present study was undertaken with the main purpose of investigating this possibility in more detail. We report here the effects of a novel specific histamine H3 receptor antagonist, NNC 381202, with an improved pharmacokinetic profile.

Research Methods and Procedures Test Compound NNC 0038-0000-1202 (NNC 38-1202) (Figure 1), is a cinnamic amide and has a longer plasma half-life than our

1 Nonstandard abbreviations: TG, triglyceride; NNC 38-1202, NNC 0038-0000-1202; PVN, paraventricular nucleus; DIO, diet-induced obesity; EE, energy expenditure; RER, respiratory exchange ratio; SE, standard error; FFA, free fatty acid; BHBT, ␤-hydroxybutyrate.

previously described compound NNC 38-1049 (17,19), namely, 226 vs. 27 minutes after intravenous administration. Oral availability is also significantly improved, namely, 100% vs. 22%. For these reasons and from an efficacy point of view, NNC 38-1202 represents an interesting model compound, suitable for oral dosing in both acute and subchronic experimental studies. At higher doses (15 mg/kg) than used in the subchronic studies described below, the compound has been found to slightly stimulate locomotor activity, although to a significantly lesser degree than that observed with, for instance, sibutramine. It has also been determined that the compound achieves good penetration of the hypothalamic tissue. The synthesis and chemical properties of NNC 38-1202 were recently described in a separate article (20). In Vitro Characteristics of NNC 38-1202 The in vitro characteristics (20) of NNC 38-1202, and the methods (21) used to investigate them, have been described in previous publications and will not be repeated in this context other than to reiterate that NNC 38-1202 is a potent and selective antagonist at rat and human histamine H3 receptors. General Experimental Procedures and Animal Ethics Rats were acclimatized to the experimental environment for 2 to 4 weeks before start of experiments. On arrival, rats were placed in conventional rat cages harboring two to three animals each. Approximately 7 to 10 days before recording of food intake was initiated, animals were moved to single cages. Unless otherwise stated, rats were subjected to a 12-/12-hour light/dark regime, with lights on between 6 AM and 6 PM. Room temperature was maintained between 20 and 23 °C. Food was either freely available at all times or during a certain time period of 5 hours, i.e., schedule feeding (see below). Water was freely available at all times, and rats were weighed regularly. Diets were purchased from a local feed supplier (Brogaarden, Gentofte, Denmark). All experiments were concordant with the Declaration of Helsinki and were approved by the local ethics committees, Animal Experiment Inspectorate (Dyreforsøgstilsynet, Copenhagen, Denmark) and Animal Care Committee of the Faculty of Mathematics and Natural Science (University of Groningen, Groningen, The Netherlands). Microdialysis of Hypothalamic Histamine after Single Dose Effects of NNC 38-1202 on hypothalamic histamine release were studied in male albino Wistar rats from Harlan (Zeist, The Netherlands). On experimental days, animals were first anesthetized and fitted with microdialysis probes in the paraventricular nucleus (PVN) according to procedures described previously (17). OBESITY Vol. 14 No. 12 December 2006

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Figure 2: Mean concentration of histamine in paraventricular extracellular fluid during a period of 200 minutes after oral administration of NNC 38-1202 in escalated single doses. Data are expressed as percentage of the basal histamine level before administration and represent means ⫾ SE (n ⫽ 8 to 11). p ⬎ 0.05 (ns) and p ⬍ 0.01 (**) compared with controls receiving 0 mg/kg (i.e., vehicle).

Acute Effects on Food Intake after Single Dosing in Rats Experiments were performed with 8- to 9-week-old male Sprague-Dawley rats purchased from Taconic M&B (Lille Skensved, Denmark). After being adapted to a 5-hour feeding schedule for at least 7 days, within the light period, groups (n ⫽ 8 to 10) of animals received single oral administrations of NNC 38-1202 in doses ranging from 0 to 30 mg/kg just before presentation of food. After this, food consumption was recorded for 5 hours. This was done either manually or automatically (FeedWin; Ellegård Systems, Faaborg, Denmark). Data are presented as cumulative food intake. Metabolic Effect of NNC 38-1202 in Rats with DietInduced Obesity (DIO) General Model of DIO. Female Wistar rats were obtained from Taconic M&B. DIO commenced at the age of 14 months and was continued until the age of 17 months, essentially according to procedures described previously (22). In this experiment, however, a slightly modified highfat (283 g/kg) diet containing sucrose (198 g/kg) was employed to elevate plasma TG. The diet contained 19.3 kJ/ gram (4.6 cal/g) of metabolizable energy, of which 55%, 30%, and 15% were derived from fat, carbohydrates, and protein, respectively. At the end of the fattening period, DIO rats were moved to single cages that permitted eye and olfactory contact with neighbor rats and free access to the same high-fat food. At this time, a reversed day cycle with 2156

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Figure 3: (A) Effects of NNC 38-1202 on 5-hour cumulative food intake in schedule-fed normal rats (n ⫽ 8 to 10) after administration of escalated single oral doses. p ⬎ 0.05 (ns) and p ⬍ 0.01 (**) compared with controls receiving 0 mg/kg (i.e., vehicle). (B) Representation of the pattern of 5-hour cumulative food intake after oral administration of either vehicle (▫, n ⫽ 25) or NNC 38-1202 (Œ, n ⫽ 10) given in a dose of 15 mg/kg. Data are means ⫾ SE.

a dark period between 9 AM and 9 PM was introduced. Rats were allowed a period of ⬃4 weeks to acclimatize to this transition. The same general procedures were followed in Experiments I and II. Experiment I: Effects on Body and Organ Weights and Plasma Variables. To study the effects of NNC 38-1202 on body weight and selected organ weights, rats were randomly assigned to one of two groups (n ⫽ 21). Two days later, one group received an oral dose of vehicle, and the other group

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received NNC 38-1202 (5 mg/kg). These administrations were maintained once daily for a period of 22 days and were performed 5 to 30 minutes before lights out. Body weights and food consumptions were recorded once daily. The amount of food consumed was estimated by subtracting the residual food recovered from each cage from the total amount presented. On the last day of treatment (Day 22), unanesthetized animals were killed by decapitation 2 to 4 hours post-dosing. They had free access to food and water until the moment of decapitation. After bleeding, plasma was prepared and stored frozen at ⫺20 °C until analyzed for various metabolites. Selected organs were also dissected out, weighed, and frozen. Experiment II: Energy Expenditure (EE) and Substrate Oxidation. The main purpose of this experiment was to investigate the effects of NNC 38-1202 on EE and lipid/ carbohydrate oxidation. Oxygen (O2) consumption was used as a measure of EE, and respiratory exchange ratio (RER), i.e., the ratio of carbon dioxide (CO2) production to O2 consumption, was employed as a measure of the type of substrate being oxidized. Exchange of gases was studied using an Oxymax equal flow system (Columbus Instruments, Columbus, OH). The system was calibrated daily before the start of measurements. Groups of DIO rats, comprising nine to 10 animals, received once-daily oral doses of either vehicle alone or NNC 38-1202 (5 mg/kg). After 1, 2, and 3 weeks of dosing, animals were transferred to the Oxymax chambers, where they had access to water only. This transfer was carried out in the afternoon about halfway into the dark period, and measurements were started after a 2-hour adaptation period and were continued for a total period of 22 hours. Rats were dosed 16 hours into this period (i.e., the next morning) and ⬃15 minutes before lights went out again. At this time, they were also provided with food. O2 and CO2 concentrations in the reference air and in the chambers were measured every 20.7 minutes. Oxygen consumption was calculated per metabolic weight (kilograms of live weight0.75). Data for each individual rat are the average of three repeated measurements performed in the 1st, 2nd, and 3rd weeks of dosing. Results are expressed as mean values (n ⫽ 9 to 10) together with standard error (SE). In addition, mean levels of RER in the 5-hour period directly after dosing and presentation of food were calculated, on an individual basis, in the two groups and were subsequently tested for statistical differences. Analyses Histamine from microdialysis samples was separated on a reversed-phase high-performance liquid chromatography column, essentially as described previously (17). Plasma concentrations of metabolites were determined using a Synchron CX5 autoanalyzer system (Beckman Instruments, Fullerton, CA), employing standard methods. Plasma insulin was determined using an enzyme-linked immunosorbent

assay (23). An enzyme-linked immunosorbent assay was also employed for determination of serum leptin (Assay Design Inc., Ann Arbor, MI), with inter- and intra-assay variations below 5%. Statistics Statistical calculations were performed using GraphPad Prism version 4.0 (GraphPad Software, San Diego, CA) or SAS version 9.1 (SAS Institute Inc., Cary, NC). Potential differences between treatment groups exhibiting normally distributed data were tested by one-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons. In other cases, data were analyzed by non-parametric one-way ANOVA (Kruskal-Wallis test) using Dunn’s post hoc test for multiple comparisons. Body weight data were analyzed by repeated measures ANOVA within the frames of mixed linear models using the baseline body weight as covariate and a structure that allows for unequal variances (heterogeneous compound symmetry). Comparisons having p ⬍ 0.05 were considered to be statistically significant. Data are presented as means ⫾ SE.

Results Hypothalamic Histamine Levels When given in doses of 15 and 30 mg/kg, NNC 38-1202 produced a highly significant (p ⬍ 0.01) rise in the mean concentration of histamine in dialysates recovered during a period of 200 minutes post-dosing from the paraventricular region of rat brains, whereas a dose of 7.5 mg/kg produced insignificant elevation (Figure 2). Acute Effects on Food Intake In schedule-fed rats, oral doses of NNC 38-1202 ranging from 15 to 30 mg/kg produced significant (p ⬍ 0.01) reductions in the accumulated food intake during a 5-hour period after presentation of food (Figure 3A). As can be seen in Figure 3B, this effect was established already in the 1st hour post-dosing and was not compensated for later on. Metabolic Effects in DIO Rats Experiment I: Effects on Body and Organ Weight, and Plasma Variables. In DIO rats with free access to food, daily administration of NNC 38-1202 (5 mg/kg) resulted in a sustained reduction in food intake. At the end of the 22-day study, control rats that received vehicle had consumed a total of 251.3 ⫾ 9.2 grams of food, whereas the equivalent number for rats that had received NNC 38-1202 was significantly (p ⬍ 0.05) lower, namely, 216.8 ⫾ 9.4 grams (Figure 4). This was associated with a highly significant (p ⬍ 0.001) change (⫺11.0 ⫾ 4.8 grams) in body weight compared with control rats, which had gained 13.6 ⫾ 3.0 grams. Also, the profiles of change in body weight (Figure 5A) differed, as the interaction term (treatOBESITY Vol. 14 No. 12 December 2006

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Figure 4: Cumulative daily food intake in DIO rats after daily oral administration of either vehicle (▫) or NNC 38-1202 (Œ) in a dose of 5 mg/kg. Data are means ⫾ SE (n ⫽ 21). A potential statistical difference between groups was tested on Day 22 and was found to be statistically significant (p ⬍ 0.05). For details, see “Results.”

ment ⫻ day) produced in the repeated measures ANOVA was highly significant (p ⬍ 0.001). In the NNC 38-1202 group, body weight was lost in a proportional fashion as judged by the consistency of relative weight (percentage of body weight) of selected organs and tissues between rats receiving NNC 38-1202 and those receiving vehicle (Table 1). Plasma glucose, high-density lipoprotein-cholesterol, insulin, and leptin remained unchanged after administration of NNC 38-1202 (Table 2, Figure 6), whereas plasma TGs were significantly (p ⬍ 0.05) decreased, namely, 2.3 ⫾ 0.3 mM, compared with the 4.0 ⫾ 0.6 mM observed for the vehicle group (Figure 6). A concomitant (p ⬍ 0.05) increase in ␤-hydroxybutyrate (BHBT) and free fatty acid (FFA) levels was also observed after administration of NNC 381202 (Figure 6). Experiment II: EE and Substrate Oxidation. EE remained unchanged after administration of NCC 38-1202 compared with vehicle (Figure 7A). This was true regardless of whether EE was calculated per rat, per kilogram of body weight, or per kilogram of body weight0.75. However, the RER was significantly (p ⬍ 0.05) attenuated in rats receiving NNC 38-1202 compared with vehicle in the 5-hour period after dosing and presentation of food, signifying an increase in lipid oxidation (Figure 7B). Lipid oxidation appeared to be slightly, but systematically, elevated in this group throughout the whole observation period, but this trend was not statistically significant.

Discussion In the present study, a novel histamine H3 receptor antagonist, NNC 38-1202 (20), was employed to increase 2158

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Figure 5: (A) Change in body weight and (B) total body weight in 17-month-old DIO rats after daily oral administration of either vehicle (▫) or NNC 38-1202 (Œ), in a dose of 5 mg/kg. Data represent means ⫾ SE (n ⫽ 21). The effect of NNC 38-1202 on both variables was tested with repeated measures ANOVA, and both the whole profile and the final change in body weight were found to be highly significant (p ⬍ 0.001). For details, see “Results.”

neural histamine activity and to examine subsequent effects on body weight, whole-body lipid oxidation, and plasma lipids. The compound NNC 38-1202 (20), which was used in the present study, has an improved pharmacokinetic profile compared with our previously described histamine H3 receptor antagonist, NNC 38-1049 (17,19). We (17) and others (18) have shown that an increase in neural histamine, evoked by specific targeting of central histamine H3 receptors, is associated with dose-dependent acute decreases in food intake in rats. In our previous study (17), an extensive series of experiments showed that the reduction of food intake was specific and without serious side effects. Here, we report that oral administration of the histamine H3 receptor antagonist NNC 38-1202 effectively

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Table 1. Absolute and relative organ weights in DIO rats following 22 days of administration of either vehicle or NNC 38-1202 in a dose of 5 mg/kg; data are means ⫾ standard error (n ⫽ 21) Organ

Vehicle

NNC 38-1202

Liver (g) Liver (% body weight) Perirenal fat pad (g) Perirenal fat pad (% body weight) Thigh muscle (g) Thigh muscle (% body weight)

9.3 ⫾ 0.55 2.2 ⫾ 0.07 17.2 ⫾ 1.76

9.0 ⫾ 0.26 2.2 ⫾ 0.04 16.3 ⫾ 1.41

3.9 ⫾ 0.26 2.5 ⫾ 0.06

3.9 ⫾ 0.26 2.4 ⫾ 0.05

0.59 ⫾ 0.018

0.60 ⫾ 0.014

DIO, diet-induced obesity; NNC 38-1202, NNC 0038-0000-1202.

and dose-dependently inhibited food intake in rats and that this inhibition occurred in parallel with significant increases in extracellular concentration of histamine in the PVN of the hypothalamus. This is of particular interest because the PVN has been pointed out to be the main site for coordination of pathways regulating energy homeostasis (24). Even though our data clearly show that there is a tight covariation between histamine levels in the PVN and inhibition of food intake, other key transmitters could also possibly contribute to the complex neurobiological changes leading to the inhibition of food intake observed after administration of a histamine H3 receptor antagonist (25). In DIO rats receiving NNC 38-1202, the sustained reduction in food intake was associated with maintained (not decreased) EE, which, in essence, means that a negative energy balance was established and sustained. Obviously,

Table 2. Plasma concentrations of glucose and hormones in DIO rats following 22 days of administration of either vehicle or NNC 38-1202 in a dose of 5 mg/kg; data are means ⫾ SE (n ⫽ 21) Variables

Vehicle

NNC 38-1202

Insulin (pM) Glucose (mM) Leptin (␮g/L)

605 ⫾ 87.7 7.3 ⫾ 0.17 29.0 ⫾ 3.27

393 ⫾ 60.2 7.3 ⫾ 0.14 36.6 ⫾ 5.64

DIO, diet-induced obesity; NNC 38-1202, NNC 0038-0000-1202. A potential difference in insulin levels between rats receiving vehicle and rats receiving NNC 38-1202 with borderline p ⬍ 0.053 was observed.

Figure 6: Plasma levels of TGs, high-density lipoprotein-cholesterol, FFAs, and BHBT in DIO rats after 22 days of daily oral administration of either vehicle or NNC 38-1202 in a dose of 5 mg/kg. Data are means ⫾ SE (n ⫽ 21). p ⬎ 0.05 (ns) and p ⬍ 0.05 (*) compared with vehicle.

these are the two main reasons why a significant reduction of body weight was seen in the present study. The observation that NNC 38-1202 effectively produced a negative energy balance resulting in weight loss should eliminate any existing doubts (26) that selective histamine H3 receptor antagonists with good oral bioavailability represent an interesting group of compounds in relation to treatment of human obesity. In parallel with the observed changes in body weight homeostasis, administration of NNC 38-1202 was associated with a significant decrease in plasma TGs, confirming our previous findings (17). The decrease in plasma TG levels is most certainly, and predominantly, the result of the reduction in food intake because caloric restriction (27,28) or semi-starvation (29) has been shown to decrease plasma TG levels in humans also. The fall in TG levels seen after administration of NNC 38-1202 is, of course, encouraging, from at least two viewpoints. Obesity-related hyperlipidemia (30,31) and associated derangements of lipid metabolism (32,33) are known to increase the risk for development of type 2 diabetes (34) and cardiovascular disease (35). It appears evident that the fall in plasma TGs seen in this study constitutes an integrated part of a metabolic adaptation to an energy-deficient state created primarily by the reduction in food intake and in which circulating TGs are oxidized to maintain EE unchanged. The energy-rich FFAs, which constitute the quantitatively and metabolically (36,37) most important component of TGs, represent, in fact, the preferred energy source during fasting conditions (38). Accordingly, an up-regulated expression of a great number of genes associated with FFA transport and catabolism was recently observed in normal mice subjected to prolonged caloric restriction (39). OBESITY Vol. 14 No. 12 December 2006

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Figure 7: Oxygen consumption (A) and RER (B) as measures of whole-body EE and substrate oxidation. DIO rats received daily oral doses of either vehicle (▫) or NNC 38-1202 (Œ), given in a dose of 5 mg/kg, for 21 days. Data for each individual rat are based on three repeated measurements performed once weekly during the study period. Each data point represents means ⫾ SE of n ⫽ 9 to 10. In addition, 5-hour mean RER levels in the post-dosing period were calculated on an individual basis (n ⫽ 9 to 10) in the two groups (dotted lines). The difference between these mean levels was found to be statistically significant (*, p ⬍ 0.05).

Following administration of NNC 38-1202, several indications of mobilization and catabolism of FFAs could be observed, notably elevation of plasma FFA and BHBT levels. The fact that these variables were concomitantly increased implies that the liver had plentiful access to FFAs and was oxidizing them at an increased rate (40). It should be mentioned that blood samples reflecting these metabolic events were obtained 2 to 4 hours post-dosing in the early phase of the feeding period. Curves of RER dynamics (Figure 7) from the same period showed that administration of NNC 38-1202 attenuated the postprandial rise in this 2160

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variable seen in the vehicle control group. Instead, comparatively low RER values were maintained, suggesting a higher level of lipid oxidation. Although the immediate origin of these lipids is unknown, it must be anticipated that they were of both endogenous and dietary origin. In view of this, it might seem somewhat surprising that the relative size of the perirenal fat pad remained unchanged after administration of NNC 38-1202. However, it must be remembered that this does not exclude the possibility that other fat pads, not examined in this study, were more affected. Regional differences in body fat breakdown during weight loss have been reported (41,42). This interesting point will be addressed in future studies with H3 antagonists. There are, of course, a number of questions arising from the present study that merit further attention and need to be investigated more thoroughly. One of the more pertinent of these issues is the consequence of the slightly elevated FFA levels for the glucoregulatory capacity of insulin. Increased plasma FFA levels are commonly seen as a metabolic risk factor responsible for development of insulin resistance/ type 2 diabetes (41,42). Insulin sensitivity was not directly assessed in the present study. However, no indications of a decrease in this parameter could be observed, in that nonfasting blood glucose levels were normal and insulin levels tended to be decreased. In view of this, it could be speculated that an increase in circulating FFAs in a metabolic context of energy deficiency and increased lipid oxidation does not have the same detrimental effects on insulinmediated glucose uptake as when high FFA levels are the result of lipid-heparin infusions (43) or prolonged overnutrition. In future studies, it would certainly be of interest to investigate whether the pool of intramyocellular lipids is reduced under experimental conditions similar to those prevailing in the present study. An accumulation of intramyocellular lipids has been suggested to be one of the main reasons for the decrease in insulin-stimulated transport of glucose into skeletal muscle such as that seen in obese states (33). Another issue of interest for further investigation is whether the changes seen in lipid metabolism following administration of a histamine H3 receptor antagonist are due, in part, to specific stimulation of sympathetic neural activity, thereby actively promoting lipolysis (44), or whether the changes seen are secondary to the effect of reduced food intake alone. In summary, the present study showed that an increase in central histamine levels induced by oral administration of a selective histamine H3 receptor antagonist not only decreased food intake and body weight but also resulted in significant reductions in plasma TG levels. It may, therefore, be concluded that administration of histamine H3 receptor antagonists represents an interesting approach to treatment of obesity and one of its associated comorbidities, hyperlipidemia.

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