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Regulatory Peptides 104 (2002) 153 – 159 www.elsevier.com/locate/regpep
Anorectic, thermogenic and anti-obesity activity of a selective orexin-1 receptor antagonist in ob/ob mice Andrea C. Haynes a,*, Helen Chapman a, Colleen Taylor a, Gary B.T. Moore a, Michael A. Cawthorne b, Mohammad Tadayyon a, John C. Clapham a, Jonathan R.S. Arch a a
GlaxoSmithKline, Third Avenue, Harlow, Essex, CM19 5AW, UK b University of Buckingham, Buckingham, MK18 1EG, UK
Abstract A single dose of the orexin-1 (OX1) receptor antagonist 1-(2-methylbenzoxazol-6-yl)-3-[1,5] naphthyridin-4-yl urea hydrochloride (SB334867-A) reduces orexin-A-induced feeding and natural feeding in Sprague Dawley rats. In this study, the anti-obesity effects of SB334867-A were determined in genetically obese (ob/ob) mice dosed with SB-334867-A (30 mg/kg, i.p.) once daily for 7 days, and then twice daily for a further 7 days. SB-334867-A reduced cumulative food intake and body weight gain over 14 days. Total fat mass gain, determined by Dual Emission X-ray Absorptiometry, was reduced, while gain in fat-free mass was unchanged. Fasting (5 h) blood glucose was also reduced at the end of the study, with a trend to reduced plasma insulin. Interscapular brown adipose tissue (BAT) weight was reduced, the tissue was noticeably darker in colour and quantitative PCR (TaqMan) analysis of this tissue showed a trend to an increase in uncoupling protein-1 mRNA expression, suggesting that SB-334867-A might stimulate thermogenesis. This was confirmed in a separate study in which a single dose of SB-334867-A (30 mg/kg, i.p.) increased metabolic rate over 4 h in ob/ob mice. OX1 receptor mRNA was detected in BAT, and its expression was increased by 58% by treatment with SB-334867-A. This is the first demonstration that OX1 receptor antagonists have potential as both anti-obesity and anti-diabetic agents. D 2002 Elsevier Science B.V. All rights reserved. Keywords: SB-334867-A; Food intake; Body weight; Energy expenditure
1. Introduction Orexins-A (OXA) and -B (OXB) are C-terminally amidated peptides of 33 and 28 amino acids, respectively, derived from the same precursor peptide [1]. They activate two Gprotein-coupled receptors: the orexin-1 (OX1) receptor, which has approximately 10-fold greater affinity for OXA than OXB, and the OX2 receptor, which has similar affinity for the two peptides [2,3]. Intracerebroventricular (i.c.v.) injection of the orexins, especially of OXA, stimulates feeding, giving rise to their name [1,4]. However, whether the orexins are physiologically important in the regulation of feeding or in the development of obesity is open to question because in some studies they have had only weak effects on food intake, and obese (fa/fa) Zucker rats have shown no difference in brain OXA or hypothalamic prepro –orexin mRNA levels from lean rats
*
Corresponding author. Tel.: +44-1279-627046; fax: +44-1279627049. E-mail address: Andrea [email protected] (A.C. Haynes).
[5 – 7]. An alternative role is thought to be in the regulation of sleep and arousal [6,8]. Other studies, in contrast, have shown reductions in hypothalamic prepro – orexin mRNA levels not only in fa/fa rats, but also in ob/ob and db/db mice [9,10]. Moreover, there was an elevated level of hypothalamic prepro –orexin mRNA in rats prone to obesity on a highfat diet that increases further when the rats are administered such a diet [11]. Overall, the evidence for OXA having a role in the regulation of feeding can be compared favourably with that for two other orexigenic peptides, melanin-concentrating hormone and galanin [12,13]. This does not preclude roles for orexins in the regulation of sleep and other behaviours, or in neurohormonal regulation. Indeed, it seems possible that orexins may mediate a range of responses that support food-seeking behaviour when there is a deficiency of fuel for the brain [13,14]. A key criterion supporting a role for a neuropeptide in the regulation of feeding is that blockade of its activity should alter natural feeding. This evidence is available for the orexin system since central administration of antibodies to both OXA and the OX1 receptor reduces food intake [15,16]. In addition, we have shown that SB-334867-A, an OX1 receptor
0167-0115/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 0 11 5 ( 0 1 ) 0 0 3 5 8 - 5
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Table 1 Oligonucleotide sequences for TaqMan primers and probes Target RNA
GenBank/EMBL accession no. (amplicon position)
Primer or probe
Primer or probe sequence
Rat OX1 receptor
AF041244 (1129 – 1213)
Mouse UCP1
U63419 (11 – 109)
Mouse b-actin
X03672 (412 – 497)
Mouse cyclophilin
X52803 (294 – 400)
Mouse HPRT
J00423 (579 – 672)
forward (sense) reverse (antisense) fluorogenic probe forward (sense) reverse (antisense) fluorogenic probe forward (sense) reverse (antisense) fluorogenic probe forward (sense) reverse (antisense) fluorogenic probe forward (sense) reverse (antisense) fluorogenic probe
5V-TCTGCCCGAGCTAGCCAAT-3V 5V-CCAGCGCTCATCACAGACAGAGAAGA-3V FAM-5V-GTGGTAGATCTTAGGGTAGAGTTCATCTG-3V-TAMRA 5V-CTGGGCTTAACGGGTCCTC-3V 5V-CTGGGCTAGGTAGTGCCAGTG-3V FAM-5V-AGCTCAAAGGTGAGCGTCCCTTCCTC-3V-TAMRA 5V-ACCCTAAGGCCAACCGTGAA-3V 5V-CACAGCCTGGATGGCTACGT-3V FAM-5V-CCCAGATCATGTTTGAGACCTTCAACACCC-3V-TAMRA 5V-GATGAGAACTTCATCCTAAAGCATACA-3V 5V-TCAGTCTTGGCAGTGCAGATAAA-3V FAM-5V-CCTGGCATCTTGTCCATGGCAAATG-3V-TAMRA 5V-GGTGAAAAGGACCTCTCGAAGTG-3V 5V-ATAGTCAAGGGCATATCCAACAACA-3V FAM-5V-CCAGACTTTGTTGGATTTGAAATTCCAGACAA-3V-TAMRA
Each TaqMan probe had the fluorescent reporter dye FAM (6-caboxyfluorescein) covalently linked to the 5V-end and the quencher dye TAMRA (6carboxytetramethylrhodamine) attached via a linker group to the 3V-end.
antagonist with 50-fold lower affinity for the OX2 receptor [17 – 19], reduces food consumption in male and female rats [20]. The structure of feeding behaviour (the behavioural satiety sequence) is well-preserved: SB-334867-A advanced the transition from feeding to resting in the manner of other satiety agents, and there was no disruption in the behavioural satiety sequence which known sedative agents elicit [21]. We now report that in genetically obese (ob/ob) mice, SB334867-A not only reduced food intake, but also increased energy expenditure, and on repeated administration, reduced body weight via a reduction in body fat content while preserving lean mass. Furthermore, fasting blood glucose was lowered despite a trend to reduced plasma insulin, indicating increased insulin sensitivity. These findings provide further evidence that orexins play a role in the regulation of energy balance, and suggest that OX1 receptor antagonists have potential in the treatment of obesity and Type 2 diabetes.
ethylamino]propyl]-phenoxyacetate hydrobromide (BRL35135) [22] were dissolved in 10% (w/v) 2-hydroxypropyl-b-cyclodextrin (Sigma) in sterile water to target concentrations of 3 and 0.05 mg/ml, respectively; 2-hydroxypropyl-b-cyclodextrin was added to the vehicle to increase the solubility of SB-334867-A [23]. Both compounds were dosed in a volume of 10 ml/kg by intraperitoneal (i.p.) administration. 2.3. Repeat administration of SB-334867-A The mice were housed in pairs (n = 6) on grid floors, and assigned to parallel treatment groups according to body weight. Either vehicle or SB-334867-A was administered once daily (late light phase) for 7 days, and then twice daily (early and late light phase) for a further 7 days; dosing was
2. Materials and methods 2.1. Animals Female ob/ob mice were obtained from Harlan Olac at 6 – 7 weeks of age. On arrival, they were housed under controlled temperature (24 F 2 C) and lighting conditions (12 h light –dark cycle), and were allowed 5 days habituation prior to the study. Food (chow pellets) and water were available ad libitum. All procedures carried out were in accordance with Home Office Guidelines. 2.2. Compound preparation and administration SB-334867-A [17 –19] and the b3-adrenoceptor agonist (R*,R*)-( F )-methyl-4-[2-[2-hydroxy-2-(3-chlorophenyl)-
Fig. 1. Effect of SB-334867-A (30 mg/kg, i.p.) on cumulative daily food intake over a 14 day period in ob/ob mice dosed once daily for 7 days and then twice daily for a further 7 days. Each point represents mean F SEM (n = 6 pairs); * P < 0.05, * * P < 0.01 vs. vehicle.
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Fig. 2. Effect of SB-334867-A (30 mg/kg, i.p.) on body weight gain in ob/ ob mice after once daily dosing for 7 days and twice-daily dosing for a further 7 days. Mean initial body weight were 36.1 F 0.6 g and 36.0 F 0.7 g for the vehicle and SB-334867-A groups, respectively. Each bar represents mean F SEM (n = 12); * P < 0.05, * * P < 0.01 vs. vehicle.
continued on days 15 and 16, while the second body composition measurement was made. 2.3.1. Food consumption and body weight Daily 24 h food intake measurements were carried out for 14 days; measurements were also made 4 h after dosing on days 1, 6 and 13 of the study. On each day, the mice were provided with a pre-weighed amount of chow pellets in hoppers when either the vehicle or SB-334867-A was administered during the late light phase. Food intake was then measured manually by weighing the remaining pellets in the hoppers and the residue from the tray positioned beneath the grid floor. Body weights were measured daily for the duration of the study.
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2.3.4. RNA isolation and analysis A PCR-based 5V nuclease assay with gene-specific fluorogenic TaqMan hydrolysis probes was used to quantify mRNA transcripts [25]. Total RNA, isolated from BAT using Trizol Reagent (Life Technologies), was treated with deoxyribonuclease I (Life Technologies), and reverse transcribed using random primers (Stratagene) and MMLV reverse transcriptase (Life Technologies). For each RNA sample, a negative control reaction without the reverse transcriptase (NoRT) was carried out. Transcript cDNAs were measured using TaqMan assay oligonucleotide primers (Sigma-Genosys) and fluorogenic probes (Applied Biosystems) designed for the rat OX1 receptor, the mouse uncoupling protein-1 (UCPl) and the following mouse reference genes: hypoxanthine phosphoribosyl-transferase (HPRT), b-actin, cyclophilin (Table 1). TaqMan analysis was performed using the ABI PRISMk 7700 Sequence Detection System for thermal cycling and real-time fluorescence measurements (Applied Biosystems). Each reaction contained a known amount of cDNA (equivalent to either 50 or 5 ng total RNA depending on the transcript), 1X TaqMan PCR Master Mix (Applied Biosystems), 300 nM forward primer, 300 nM reverse primer and 100 nM TaqMan probe in a total volume of 25 ml. The reaction conditions were as follows: 50 C for 2 min, 95 C for 10 min then 40 –50 cycles of 95 C for 15 min and 60 C for 1 min. For the standard curve, a 2-fold serial dilution of mouse brown adipose tissue cDNA (equivalent to 150 –0.3 ng total RNA) was assayed for each transcript. The NoRT values were subtracted from the corresponding cDNA measurement. 2.4. Single administration of SB-334867-A and energy expenditure Mice, housed in pairs according to body weight and in their home cages, were placed in a purpose-built 10-chamber
2.3.2. Body composition Body composition, determined by Dual Emission X-ray Absorptiometry (DEXA) densitometry (PIXImus, Lunar) [24], was measured before and after 14 days of dosing. The instrument was calibrated using a QC phantom supplied by the manufacturer. The mice were anaesthetised using isoflurane (1.5%) gas during data acquisition, which took no longer than 5 min per mouse. For the analysis, the heads of the mice were excluded by placing an exclusion region of interest over the head as described in the manufacturer’s instructions. The bone mineral density was determined using the femur of the left hind limb for each mouse. 2.3.3. Terminal blood analytes On the day after the last dose of SB-334867-A, mice were fasted for 5 h (starting approximately 4 h into the light phase) and then blood glucose was measured spectrophotometrically using a Cobas Mira plus Clinical Chemistry Analyser (Roche Diagnostics). Plasma insulin was measured in the same samples using a commercially available ELISA (Crystal Chem).
Fig. 3. Effects of SB-334867-A (30 mg/kg, i.p.) on fat and lean mass in ob/ ob mice after a 14 day treatment period. Initial fat mass was 18.7 F 0.3 g, while lean mass was 18.2 F 0.2 g. Each bar represents mean F SEM (n = 12); * P < 0.05 vs. vehicle.
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Table 2 The effects of SB-334867-A (30 mg/kg, i.p.) on terminal blood/plasma analytes and tissue weight in ob/ob mice after a 14 day treatment period
Vehicle SB-334867-A P
Blood glucose (mmol/l)
Plasma insulin (ng/ml)
BAT weight (mg)
16.1 F 1.4 8.6 F 0.7 < 0.0001
26.7 F 4.9 16.5 F 2.5 0.0747
612 F 32 468 F 18 < 0.001
Data expressed as mean F SEM (n = 12).
open circuit, indirect calorimeter. The animals were dosed during the early light phase, with either vehicle, BRL-35135 or SB-334867-A, and food was removed (for approximately 8 h) until the onset of the dark phase; this was repeated over 4 consecutive days according to a random crossover design. Over a 20 h period, starting approximately 4 h into the light phase, samples from each chamber were monitored for 60 s every 20 min, with room air as reference, using a Servomax (model 1440C) oxygen/carbon dioxide gas analyser. Oxygen values (%) leaving the chamber were used to calculate VO2 (oxygen consumption), incorporating the Haldane correction for respiratory quotient, according to Eqs. (6) and (7) of Even et al. [26]. Energy expenditure was calculated from VO2 and VCO2 (carbon dioxide production) and expressed as kJ/min/ mouse. 2.5. Statistical analysis Statistical comparisons were made by analysis of variance (ANOVA) using the software package Statistica, version 5.1 (StatSoft). Relative TaqMan transcript data were log10-transformed prior to ANOVA, and expressed as a ratio to the vehicle unadjusted geometric mean with 95% confidence intervals. For statistical analysis of the energy expenditure data, the sum of the values over 4 h (2– 6 h post-dose) was used. Data in the figures are represented as mean F SEM.
effect on food intake in the period immediately following each injection than at later times during each 24 h period, food intake was also measured at 4 h post-dose on three occasions. At 4 h post-dose, SB-334867-A reduced food intake on days 1 (by 33 F 15%, P = 0.06), 6 (by 28 F 9%, P = 0.17) and 13 (by 29 F 22%, P = 0.24); these changes were not significantly different from the vehicle control groups. Body weight gain was significantly reduced after 7 days of once daily dosing, and also after a further 7 days of twice daily dosing (Fig. 2). 3.1.2. Body composition Total fat mass gain was significantly reduced (by 28%), while gain in fat-free mass was unchanged (Fig. 3). There were no changes in bone mineral density (data not shown). 3.1.3. Terminal blood analytes and brown adipose tissue weight At the end of the study, terminal blood glucose was significantly reduced by almost 50% in SB-334867-A-treated mice. Plasma insulin was also reduced (by 38%), but this was not significantly different from the vehicle control group ( P = 0.07). SB-334867-A caused a significant reduction in BAT weight, and the tissue was also noticeably darker in colour, suggestive of an increased density of mitochondrial cytochromes, the feature which gives rise to the term brown adipose tissue (Table 2). 3.1.4. mRNA quantification In SB-334867-A-treated mice, the OX1 receptor mRNA was significantly up-regulated (58%, P = 0.03) in BAT, and there was a trend to an increase in UCP1 (29%, P = 0.11) expression (Table 3). There was no significant change in expression of the reference genes (HPRT, b-actin and cyclophilin) in either the SB-334867-A or the vehicle-treated groups. There was a 30-fold lower expression of the OX1 receptor in BAT than in hypothalamus (data not shown). 3.2. Effects of single administration of SB-334867-A on energy expenditure
3. Results 3.1. Effects of repeat administration of SB-334867-A 3.1.1. Food consumption and body weight SB-334867-A significantly reduced cumulative 24 h food intakes between days 3 and 14 (last measurement) of the study (Fig. 1). Since SB-334867-A might have a greater
Over a 4-h period (2– 6 h post-dose) during the light phase, SB-334867-A caused a significant increase in energy expenditure. BRL-35135, the b3-adrenoceptor agonist included as a positive control, had a greater effect on energy expenditure than SB-334867-A (Fig. 4(a) and (b)).
Table 3 Effects of SB-334867-A (30 mg/kg, i.p.), after a 14-day treatment period, on BAT mRNA expression in ob/ob mice (n = 12)
Ratio to vehicle 95% CI for ratio Change from vehicle (%) P
OX1 receptor
UCP1
HPRT
b-actin
Cyclophilin
1.58 1.19 – 2.11 58 0.003
1.29 0.94 – 1.77 29 0.112
0.95 0.80 – 1.13 5 0.578
0.94 0.78 – 1.13 6 0.494
1.09 0.92 – 1.3 9 0.297
Data expressed as a ratio to vehicle geometric mean with 95% confidence intervals (CI), and the change from vehicle geometric mean (%).
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Fig. 4. Effects of a single dose of SB-334867-A (30 mg/kg, i.p.) and BRL35135 (0.5 mg/kg, i.p.) on energy expenditure in ob/ob mice over 4 h. Each point/bar represents mean F SEM (n = 5 – 7); * P < 0.05. (a) Energy expenditure at 20 min intervals over a 4 h period (2 – 6 h post- dose). (b) Cumulative 4 h energy expenditure for data shown in (a).
To give a perspective on this result, we included a b3adrenoceptor agonist in the study, since such compounds are well established and highly effective stimulants of energy expenditure that have potential in the treatment of obesity and Type 2 diabetes [27].
4. Discussion These findings in ob/ob mice demonstrate an anti-obesity effect with a selective OX1 receptor antagonist for the first time. The inhibitory effects of SB-334867-A on food intake are consistent with our previous work, which was carried out in Sprague Dawley [20] or Lister hooded [21] rats. Cumulative food intake in ob/ob mice was reduced over the entire 14 days of the study, without any signs of desensitisation. SB-334867-A also reduced body weight gain, with the greatest reduction being observed after the period of twice daily dosing. After 14 days of treatment, total fat mass gain and BAT weight were both reduced, while gain in fat-free mass was not significantly different from control, indicating that the change in weight gain was due to reduced adiposity. In
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addition, the BAT was noticeably darker in colour, which is a feature of exposure to cold (which raises sympathetic activity), or treatment with b3-adrenoceptor agonists and other sympathomimetic agents that increase metabolic rate [28 – 30]. Further analysis of this tissue revealed overexpression of OX1 receptor mRNA, and a trend to up-regulation of UCP1 mRNA expression — the latter also indicative of increased sympathetic activation of BAT thermogenesis [31]. The thermogenic activity of SB-334867-A was confirmed by indirect calorimetry in which a single dose increased energy expenditure in mice. This increase was less than that evoked by the b3-adrenoceptor agonist BRL35135, which acts directly on BAT and probably other thermogenic sites in the periphery such as skeletal muscle [27]. The stimulation of thermogenesis by SB-334867-A may be important in eliciting fat loss, since reduction in food intake, by itself, has relatively little effect on body lipid content in ob/ob mice [32]. Contrary to this indication that OX1 receptor antagonists are thermogenic agents, a single injection of OXA (3 nmol) directly into the third ventricle has been reported to increase metabolic rate [33]. A possible explanation for this discrepancy is that normal C57BL/6J mice were used to determine the effects of OXA, while we used ob/ob mice, which have diminished cold-induced sympathetic nervous system activity and therefore defective thermoregulation [34]. Antagonism of the OX1 receptor may increase sympathetic activity only when baseline activity is low. The report that OXA increases metabolic rate [33] also contrasts with our previous finding; that OXA does not affect BAT temperature when infused centrally in rats for 8 days [4]. The presence of OX1 receptor mRNA in mouse BAT, and significant blood levels of SB-334867-A detected up to 2 h after dosing in rats [20], raises the possibility that SB334867-A could have a direct effect on BAT. The OX1 receptor would have to be active tonically to reduce BAT thermogenesis for the antagonist to increase it. There is no evidence for OXA release in BAT, but the presence of picomolar concentrations of immunoreactive OXA in plasma raises the possibility that it is released in the periphery [35]. Moreover, the up-regulation of OX1 receptor expression in BAT by SB-334867-A treatment is consistent with the BAT OX1 receptor being tonically active. At the end of the study, fasting (5 h) blood glucose levels were almost halved and plasma insulin levels possibly slightly reduced (P = 0.07), suggesting that SB-334867-A improved insulin sensitivity in the ob/ob mouse. Based on the evidence we have so far for SB-334867-A as a thermogenic agent, it is likely that increased sympathetic activity and fat oxidation, with consequent lowering of intracellular fatty acyl CoA levels, is responsible for the increase in insulin sensitivity [36]. Alternatively, SB-334867-A could directly inhibit glucose output from liver or stimulate glucose uptake by muscle, possibly coupled with direct inhibition of insulin secretion. Thus, OXA has been reported to stimulate insulin secretion by the perfused rat pancreas, but OXA caused an
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increase in both insulin and glucose levels in rats [37]. Again, this explanation requires that OX1 receptors are present and tonically active in peripheral tissues. Moreover, it seems unlikely that this mechanism can explain the lowering of blood glucose at a time when (on the basis of pharmacokinetic studies in rats) blood levels of SB-334867-A were probably negligible. Sustained b-adrenoceptor activation, by contrast, is known to improve insulin sensitivity beyond the period when the activator is present in the circulation [38]. Although we cannot be certain that all the effects we describe are due to antagonism of OX1 receptors by SB334867-A, extensive screening has not revealed a significant interaction with other receptors [18], except for a 50-fold lower affinity for the OX2 receptor. The data here provide firm evidence for the involvement of the orexin system in the central regulation of feeding and energy balance, and for the first time, a potential role for OX1 receptor antagonists in the treatment of obesity with further beneficial effects on blood glucose and insulin sensitivity. Although we do not rule out a direct effect on BAT, we suggest that the thermogenic effect of SB-334867-A in the ob/ob mouse results primarily from central stimulation of the sympathetic nervous system, though the ability of OX1 receptor antagonists to produce a clinically relevant thermogenic (and consequential insulin sensitising) response in humans may depend on prevailing sympathetic tone.
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A.C. Haynes et al. / Regulatory Peptides 104 (2002) 153–159 [31] Ricquier D, Bouillard F, Toumelin P, Mory G, Bazin R, Arch JRS, et al. Expression of uncoupling protein mRNA in thermogenic or weakly thermogenic brown adipose tissue. J Biol Chem 1986;261:13905 – 10. [32] Arch JR. The contribution of increased thermogenesis to the effect of anorectic drugs on body composition in mice. Am J Clin Nutr 1981; 34:2763 – 9. [33] Lubkin M, Stricker-Krongrad A. Independent feeding and metabolic actions of orexins in mice. Biochem Biophys Res Commun 1998; 253:241 – 5. [34] Young JB, Landsberg L. Diminished sympathetic nervous system activity in genetically obese (ob/ob) mouse. Am J Physiol 1983;245: E148 – 54. [35] Arihara Z, Takahashi K, Murakami O, Totsune K, Sone M, Satoh F, et
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al. Immunoreactive orexin-A in human plasma. Peptides 2001;22: 139 – 42. [36] Laybutt DR, Schmitz-Peiffer C, Saha AK, Ruderman NB, Biden TJ, Kraegen EW. Muscle lipid accumulation and protein kinase C activation in the insulin-resistant chronically glucose-infused rat. Am J Physiol 1999;277:E1070 – 6. [37] Nowak M, Hochol A, Tortorella C, Jedrzejczak N, Ziolkowska A, Nussdorfer GG, et al. Modulatory effects of orexins on the function of rat pituitary – adrenocortical axis under basal and stressful conditions. Biomed Res (Tokyo) 2000;21:89 – 93. [38] Cawthorne MA, Sennitt MV, Arch JRS, Smith SA. BRL 35135, a potent and selective atypical b-adrenoceptor agonist. Am J Clin Nutr 1992;55:S252 – 7.
Anorectic, thermogenic and anti-obesity activity of a selective orexin-1 receptor antagonist in ob/ob mice Andrea C. Haynes a,*, Helen Chapman a, Colleen Taylor a, Gary B.T. Moore a, Michael A. Cawthorne b, Mohammad Tadayyon a, John C. Clapham a, Jonathan R.S. Arch a a
GlaxoSmithKline, Third Avenue, Harlow, Essex, CM19 5AW, UK b University of Buckingham, Buckingham, MK18 1EG, UK
Abstract A single dose of the orexin-1 (OX1) receptor antagonist 1-(2-methylbenzoxazol-6-yl)-3-[1,5] naphthyridin-4-yl urea hydrochloride (SB334867-A) reduces orexin-A-induced feeding and natural feeding in Sprague Dawley rats. In this study, the anti-obesity effects of SB334867-A were determined in genetically obese (ob/ob) mice dosed with SB-334867-A (30 mg/kg, i.p.) once daily for 7 days, and then twice daily for a further 7 days. SB-334867-A reduced cumulative food intake and body weight gain over 14 days. Total fat mass gain, determined by Dual Emission X-ray Absorptiometry, was reduced, while gain in fat-free mass was unchanged. Fasting (5 h) blood glucose was also reduced at the end of the study, with a trend to reduced plasma insulin. Interscapular brown adipose tissue (BAT) weight was reduced, the tissue was noticeably darker in colour and quantitative PCR (TaqMan) analysis of this tissue showed a trend to an increase in uncoupling protein-1 mRNA expression, suggesting that SB-334867-A might stimulate thermogenesis. This was confirmed in a separate study in which a single dose of SB-334867-A (30 mg/kg, i.p.) increased metabolic rate over 4 h in ob/ob mice. OX1 receptor mRNA was detected in BAT, and its expression was increased by 58% by treatment with SB-334867-A. This is the first demonstration that OX1 receptor antagonists have potential as both anti-obesity and anti-diabetic agents. D 2002 Elsevier Science B.V. All rights reserved. Keywords: SB-334867-A; Food intake; Body weight; Energy expenditure
1. Introduction Orexins-A (OXA) and -B (OXB) are C-terminally amidated peptides of 33 and 28 amino acids, respectively, derived from the same precursor peptide [1]. They activate two Gprotein-coupled receptors: the orexin-1 (OX1) receptor, which has approximately 10-fold greater affinity for OXA than OXB, and the OX2 receptor, which has similar affinity for the two peptides [2,3]. Intracerebroventricular (i.c.v.) injection of the orexins, especially of OXA, stimulates feeding, giving rise to their name [1,4]. However, whether the orexins are physiologically important in the regulation of feeding or in the development of obesity is open to question because in some studies they have had only weak effects on food intake, and obese (fa/fa) Zucker rats have shown no difference in brain OXA or hypothalamic prepro –orexin mRNA levels from lean rats
*
Corresponding author. Tel.: +44-1279-627046; fax: +44-1279627049. E-mail address: Andrea [email protected] (A.C. Haynes).
[5 – 7]. An alternative role is thought to be in the regulation of sleep and arousal [6,8]. Other studies, in contrast, have shown reductions in hypothalamic prepro – orexin mRNA levels not only in fa/fa rats, but also in ob/ob and db/db mice [9,10]. Moreover, there was an elevated level of hypothalamic prepro –orexin mRNA in rats prone to obesity on a highfat diet that increases further when the rats are administered such a diet [11]. Overall, the evidence for OXA having a role in the regulation of feeding can be compared favourably with that for two other orexigenic peptides, melanin-concentrating hormone and galanin [12,13]. This does not preclude roles for orexins in the regulation of sleep and other behaviours, or in neurohormonal regulation. Indeed, it seems possible that orexins may mediate a range of responses that support food-seeking behaviour when there is a deficiency of fuel for the brain [13,14]. A key criterion supporting a role for a neuropeptide in the regulation of feeding is that blockade of its activity should alter natural feeding. This evidence is available for the orexin system since central administration of antibodies to both OXA and the OX1 receptor reduces food intake [15,16]. In addition, we have shown that SB-334867-A, an OX1 receptor
0167-0115/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 0 11 5 ( 0 1 ) 0 0 3 5 8 - 5
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Table 1 Oligonucleotide sequences for TaqMan primers and probes Target RNA
GenBank/EMBL accession no. (amplicon position)
Primer or probe
Primer or probe sequence
Rat OX1 receptor
AF041244 (1129 – 1213)
Mouse UCP1
U63419 (11 – 109)
Mouse b-actin
X03672 (412 – 497)
Mouse cyclophilin
X52803 (294 – 400)
Mouse HPRT
J00423 (579 – 672)
forward (sense) reverse (antisense) fluorogenic probe forward (sense) reverse (antisense) fluorogenic probe forward (sense) reverse (antisense) fluorogenic probe forward (sense) reverse (antisense) fluorogenic probe forward (sense) reverse (antisense) fluorogenic probe
5V-TCTGCCCGAGCTAGCCAAT-3V 5V-CCAGCGCTCATCACAGACAGAGAAGA-3V FAM-5V-GTGGTAGATCTTAGGGTAGAGTTCATCTG-3V-TAMRA 5V-CTGGGCTTAACGGGTCCTC-3V 5V-CTGGGCTAGGTAGTGCCAGTG-3V FAM-5V-AGCTCAAAGGTGAGCGTCCCTTCCTC-3V-TAMRA 5V-ACCCTAAGGCCAACCGTGAA-3V 5V-CACAGCCTGGATGGCTACGT-3V FAM-5V-CCCAGATCATGTTTGAGACCTTCAACACCC-3V-TAMRA 5V-GATGAGAACTTCATCCTAAAGCATACA-3V 5V-TCAGTCTTGGCAGTGCAGATAAA-3V FAM-5V-CCTGGCATCTTGTCCATGGCAAATG-3V-TAMRA 5V-GGTGAAAAGGACCTCTCGAAGTG-3V 5V-ATAGTCAAGGGCATATCCAACAACA-3V FAM-5V-CCAGACTTTGTTGGATTTGAAATTCCAGACAA-3V-TAMRA
Each TaqMan probe had the fluorescent reporter dye FAM (6-caboxyfluorescein) covalently linked to the 5V-end and the quencher dye TAMRA (6carboxytetramethylrhodamine) attached via a linker group to the 3V-end.
antagonist with 50-fold lower affinity for the OX2 receptor [17 – 19], reduces food consumption in male and female rats [20]. The structure of feeding behaviour (the behavioural satiety sequence) is well-preserved: SB-334867-A advanced the transition from feeding to resting in the manner of other satiety agents, and there was no disruption in the behavioural satiety sequence which known sedative agents elicit [21]. We now report that in genetically obese (ob/ob) mice, SB334867-A not only reduced food intake, but also increased energy expenditure, and on repeated administration, reduced body weight via a reduction in body fat content while preserving lean mass. Furthermore, fasting blood glucose was lowered despite a trend to reduced plasma insulin, indicating increased insulin sensitivity. These findings provide further evidence that orexins play a role in the regulation of energy balance, and suggest that OX1 receptor antagonists have potential in the treatment of obesity and Type 2 diabetes.
ethylamino]propyl]-phenoxyacetate hydrobromide (BRL35135) [22] were dissolved in 10% (w/v) 2-hydroxypropyl-b-cyclodextrin (Sigma) in sterile water to target concentrations of 3 and 0.05 mg/ml, respectively; 2-hydroxypropyl-b-cyclodextrin was added to the vehicle to increase the solubility of SB-334867-A [23]. Both compounds were dosed in a volume of 10 ml/kg by intraperitoneal (i.p.) administration. 2.3. Repeat administration of SB-334867-A The mice were housed in pairs (n = 6) on grid floors, and assigned to parallel treatment groups according to body weight. Either vehicle or SB-334867-A was administered once daily (late light phase) for 7 days, and then twice daily (early and late light phase) for a further 7 days; dosing was
2. Materials and methods 2.1. Animals Female ob/ob mice were obtained from Harlan Olac at 6 – 7 weeks of age. On arrival, they were housed under controlled temperature (24 F 2 C) and lighting conditions (12 h light –dark cycle), and were allowed 5 days habituation prior to the study. Food (chow pellets) and water were available ad libitum. All procedures carried out were in accordance with Home Office Guidelines. 2.2. Compound preparation and administration SB-334867-A [17 –19] and the b3-adrenoceptor agonist (R*,R*)-( F )-methyl-4-[2-[2-hydroxy-2-(3-chlorophenyl)-
Fig. 1. Effect of SB-334867-A (30 mg/kg, i.p.) on cumulative daily food intake over a 14 day period in ob/ob mice dosed once daily for 7 days and then twice daily for a further 7 days. Each point represents mean F SEM (n = 6 pairs); * P < 0.05, * * P < 0.01 vs. vehicle.
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Fig. 2. Effect of SB-334867-A (30 mg/kg, i.p.) on body weight gain in ob/ ob mice after once daily dosing for 7 days and twice-daily dosing for a further 7 days. Mean initial body weight were 36.1 F 0.6 g and 36.0 F 0.7 g for the vehicle and SB-334867-A groups, respectively. Each bar represents mean F SEM (n = 12); * P < 0.05, * * P < 0.01 vs. vehicle.
continued on days 15 and 16, while the second body composition measurement was made. 2.3.1. Food consumption and body weight Daily 24 h food intake measurements were carried out for 14 days; measurements were also made 4 h after dosing on days 1, 6 and 13 of the study. On each day, the mice were provided with a pre-weighed amount of chow pellets in hoppers when either the vehicle or SB-334867-A was administered during the late light phase. Food intake was then measured manually by weighing the remaining pellets in the hoppers and the residue from the tray positioned beneath the grid floor. Body weights were measured daily for the duration of the study.
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2.3.4. RNA isolation and analysis A PCR-based 5V nuclease assay with gene-specific fluorogenic TaqMan hydrolysis probes was used to quantify mRNA transcripts [25]. Total RNA, isolated from BAT using Trizol Reagent (Life Technologies), was treated with deoxyribonuclease I (Life Technologies), and reverse transcribed using random primers (Stratagene) and MMLV reverse transcriptase (Life Technologies). For each RNA sample, a negative control reaction without the reverse transcriptase (NoRT) was carried out. Transcript cDNAs were measured using TaqMan assay oligonucleotide primers (Sigma-Genosys) and fluorogenic probes (Applied Biosystems) designed for the rat OX1 receptor, the mouse uncoupling protein-1 (UCPl) and the following mouse reference genes: hypoxanthine phosphoribosyl-transferase (HPRT), b-actin, cyclophilin (Table 1). TaqMan analysis was performed using the ABI PRISMk 7700 Sequence Detection System for thermal cycling and real-time fluorescence measurements (Applied Biosystems). Each reaction contained a known amount of cDNA (equivalent to either 50 or 5 ng total RNA depending on the transcript), 1X TaqMan PCR Master Mix (Applied Biosystems), 300 nM forward primer, 300 nM reverse primer and 100 nM TaqMan probe in a total volume of 25 ml. The reaction conditions were as follows: 50 C for 2 min, 95 C for 10 min then 40 –50 cycles of 95 C for 15 min and 60 C for 1 min. For the standard curve, a 2-fold serial dilution of mouse brown adipose tissue cDNA (equivalent to 150 –0.3 ng total RNA) was assayed for each transcript. The NoRT values were subtracted from the corresponding cDNA measurement. 2.4. Single administration of SB-334867-A and energy expenditure Mice, housed in pairs according to body weight and in their home cages, were placed in a purpose-built 10-chamber
2.3.2. Body composition Body composition, determined by Dual Emission X-ray Absorptiometry (DEXA) densitometry (PIXImus, Lunar) [24], was measured before and after 14 days of dosing. The instrument was calibrated using a QC phantom supplied by the manufacturer. The mice were anaesthetised using isoflurane (1.5%) gas during data acquisition, which took no longer than 5 min per mouse. For the analysis, the heads of the mice were excluded by placing an exclusion region of interest over the head as described in the manufacturer’s instructions. The bone mineral density was determined using the femur of the left hind limb for each mouse. 2.3.3. Terminal blood analytes On the day after the last dose of SB-334867-A, mice were fasted for 5 h (starting approximately 4 h into the light phase) and then blood glucose was measured spectrophotometrically using a Cobas Mira plus Clinical Chemistry Analyser (Roche Diagnostics). Plasma insulin was measured in the same samples using a commercially available ELISA (Crystal Chem).
Fig. 3. Effects of SB-334867-A (30 mg/kg, i.p.) on fat and lean mass in ob/ ob mice after a 14 day treatment period. Initial fat mass was 18.7 F 0.3 g, while lean mass was 18.2 F 0.2 g. Each bar represents mean F SEM (n = 12); * P < 0.05 vs. vehicle.
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Table 2 The effects of SB-334867-A (30 mg/kg, i.p.) on terminal blood/plasma analytes and tissue weight in ob/ob mice after a 14 day treatment period
Vehicle SB-334867-A P
Blood glucose (mmol/l)
Plasma insulin (ng/ml)
BAT weight (mg)
16.1 F 1.4 8.6 F 0.7 < 0.0001
26.7 F 4.9 16.5 F 2.5 0.0747
612 F 32 468 F 18 < 0.001
Data expressed as mean F SEM (n = 12).
open circuit, indirect calorimeter. The animals were dosed during the early light phase, with either vehicle, BRL-35135 or SB-334867-A, and food was removed (for approximately 8 h) until the onset of the dark phase; this was repeated over 4 consecutive days according to a random crossover design. Over a 20 h period, starting approximately 4 h into the light phase, samples from each chamber were monitored for 60 s every 20 min, with room air as reference, using a Servomax (model 1440C) oxygen/carbon dioxide gas analyser. Oxygen values (%) leaving the chamber were used to calculate VO2 (oxygen consumption), incorporating the Haldane correction for respiratory quotient, according to Eqs. (6) and (7) of Even et al. [26]. Energy expenditure was calculated from VO2 and VCO2 (carbon dioxide production) and expressed as kJ/min/ mouse. 2.5. Statistical analysis Statistical comparisons were made by analysis of variance (ANOVA) using the software package Statistica, version 5.1 (StatSoft). Relative TaqMan transcript data were log10-transformed prior to ANOVA, and expressed as a ratio to the vehicle unadjusted geometric mean with 95% confidence intervals. For statistical analysis of the energy expenditure data, the sum of the values over 4 h (2– 6 h post-dose) was used. Data in the figures are represented as mean F SEM.
effect on food intake in the period immediately following each injection than at later times during each 24 h period, food intake was also measured at 4 h post-dose on three occasions. At 4 h post-dose, SB-334867-A reduced food intake on days 1 (by 33 F 15%, P = 0.06), 6 (by 28 F 9%, P = 0.17) and 13 (by 29 F 22%, P = 0.24); these changes were not significantly different from the vehicle control groups. Body weight gain was significantly reduced after 7 days of once daily dosing, and also after a further 7 days of twice daily dosing (Fig. 2). 3.1.2. Body composition Total fat mass gain was significantly reduced (by 28%), while gain in fat-free mass was unchanged (Fig. 3). There were no changes in bone mineral density (data not shown). 3.1.3. Terminal blood analytes and brown adipose tissue weight At the end of the study, terminal blood glucose was significantly reduced by almost 50% in SB-334867-A-treated mice. Plasma insulin was also reduced (by 38%), but this was not significantly different from the vehicle control group ( P = 0.07). SB-334867-A caused a significant reduction in BAT weight, and the tissue was also noticeably darker in colour, suggestive of an increased density of mitochondrial cytochromes, the feature which gives rise to the term brown adipose tissue (Table 2). 3.1.4. mRNA quantification In SB-334867-A-treated mice, the OX1 receptor mRNA was significantly up-regulated (58%, P = 0.03) in BAT, and there was a trend to an increase in UCP1 (29%, P = 0.11) expression (Table 3). There was no significant change in expression of the reference genes (HPRT, b-actin and cyclophilin) in either the SB-334867-A or the vehicle-treated groups. There was a 30-fold lower expression of the OX1 receptor in BAT than in hypothalamus (data not shown). 3.2. Effects of single administration of SB-334867-A on energy expenditure
3. Results 3.1. Effects of repeat administration of SB-334867-A 3.1.1. Food consumption and body weight SB-334867-A significantly reduced cumulative 24 h food intakes between days 3 and 14 (last measurement) of the study (Fig. 1). Since SB-334867-A might have a greater
Over a 4-h period (2– 6 h post-dose) during the light phase, SB-334867-A caused a significant increase in energy expenditure. BRL-35135, the b3-adrenoceptor agonist included as a positive control, had a greater effect on energy expenditure than SB-334867-A (Fig. 4(a) and (b)).
Table 3 Effects of SB-334867-A (30 mg/kg, i.p.), after a 14-day treatment period, on BAT mRNA expression in ob/ob mice (n = 12)
Ratio to vehicle 95% CI for ratio Change from vehicle (%) P
OX1 receptor
UCP1
HPRT
b-actin
Cyclophilin
1.58 1.19 – 2.11 58 0.003
1.29 0.94 – 1.77 29 0.112
0.95 0.80 – 1.13 5 0.578
0.94 0.78 – 1.13 6 0.494
1.09 0.92 – 1.3 9 0.297
Data expressed as a ratio to vehicle geometric mean with 95% confidence intervals (CI), and the change from vehicle geometric mean (%).
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Fig. 4. Effects of a single dose of SB-334867-A (30 mg/kg, i.p.) and BRL35135 (0.5 mg/kg, i.p.) on energy expenditure in ob/ob mice over 4 h. Each point/bar represents mean F SEM (n = 5 – 7); * P < 0.05. (a) Energy expenditure at 20 min intervals over a 4 h period (2 – 6 h post- dose). (b) Cumulative 4 h energy expenditure for data shown in (a).
To give a perspective on this result, we included a b3adrenoceptor agonist in the study, since such compounds are well established and highly effective stimulants of energy expenditure that have potential in the treatment of obesity and Type 2 diabetes [27].
4. Discussion These findings in ob/ob mice demonstrate an anti-obesity effect with a selective OX1 receptor antagonist for the first time. The inhibitory effects of SB-334867-A on food intake are consistent with our previous work, which was carried out in Sprague Dawley [20] or Lister hooded [21] rats. Cumulative food intake in ob/ob mice was reduced over the entire 14 days of the study, without any signs of desensitisation. SB-334867-A also reduced body weight gain, with the greatest reduction being observed after the period of twice daily dosing. After 14 days of treatment, total fat mass gain and BAT weight were both reduced, while gain in fat-free mass was not significantly different from control, indicating that the change in weight gain was due to reduced adiposity. In
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addition, the BAT was noticeably darker in colour, which is a feature of exposure to cold (which raises sympathetic activity), or treatment with b3-adrenoceptor agonists and other sympathomimetic agents that increase metabolic rate [28 – 30]. Further analysis of this tissue revealed overexpression of OX1 receptor mRNA, and a trend to up-regulation of UCP1 mRNA expression — the latter also indicative of increased sympathetic activation of BAT thermogenesis [31]. The thermogenic activity of SB-334867-A was confirmed by indirect calorimetry in which a single dose increased energy expenditure in mice. This increase was less than that evoked by the b3-adrenoceptor agonist BRL35135, which acts directly on BAT and probably other thermogenic sites in the periphery such as skeletal muscle [27]. The stimulation of thermogenesis by SB-334867-A may be important in eliciting fat loss, since reduction in food intake, by itself, has relatively little effect on body lipid content in ob/ob mice [32]. Contrary to this indication that OX1 receptor antagonists are thermogenic agents, a single injection of OXA (3 nmol) directly into the third ventricle has been reported to increase metabolic rate [33]. A possible explanation for this discrepancy is that normal C57BL/6J mice were used to determine the effects of OXA, while we used ob/ob mice, which have diminished cold-induced sympathetic nervous system activity and therefore defective thermoregulation [34]. Antagonism of the OX1 receptor may increase sympathetic activity only when baseline activity is low. The report that OXA increases metabolic rate [33] also contrasts with our previous finding; that OXA does not affect BAT temperature when infused centrally in rats for 8 days [4]. The presence of OX1 receptor mRNA in mouse BAT, and significant blood levels of SB-334867-A detected up to 2 h after dosing in rats [20], raises the possibility that SB334867-A could have a direct effect on BAT. The OX1 receptor would have to be active tonically to reduce BAT thermogenesis for the antagonist to increase it. There is no evidence for OXA release in BAT, but the presence of picomolar concentrations of immunoreactive OXA in plasma raises the possibility that it is released in the periphery [35]. Moreover, the up-regulation of OX1 receptor expression in BAT by SB-334867-A treatment is consistent with the BAT OX1 receptor being tonically active. At the end of the study, fasting (5 h) blood glucose levels were almost halved and plasma insulin levels possibly slightly reduced (P = 0.07), suggesting that SB-334867-A improved insulin sensitivity in the ob/ob mouse. Based on the evidence we have so far for SB-334867-A as a thermogenic agent, it is likely that increased sympathetic activity and fat oxidation, with consequent lowering of intracellular fatty acyl CoA levels, is responsible for the increase in insulin sensitivity [36]. Alternatively, SB-334867-A could directly inhibit glucose output from liver or stimulate glucose uptake by muscle, possibly coupled with direct inhibition of insulin secretion. Thus, OXA has been reported to stimulate insulin secretion by the perfused rat pancreas, but OXA caused an
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increase in both insulin and glucose levels in rats [37]. Again, this explanation requires that OX1 receptors are present and tonically active in peripheral tissues. Moreover, it seems unlikely that this mechanism can explain the lowering of blood glucose at a time when (on the basis of pharmacokinetic studies in rats) blood levels of SB-334867-A were probably negligible. Sustained b-adrenoceptor activation, by contrast, is known to improve insulin sensitivity beyond the period when the activator is present in the circulation [38]. Although we cannot be certain that all the effects we describe are due to antagonism of OX1 receptors by SB334867-A, extensive screening has not revealed a significant interaction with other receptors [18], except for a 50-fold lower affinity for the OX2 receptor. The data here provide firm evidence for the involvement of the orexin system in the central regulation of feeding and energy balance, and for the first time, a potential role for OX1 receptor antagonists in the treatment of obesity with further beneficial effects on blood glucose and insulin sensitivity. Although we do not rule out a direct effect on BAT, we suggest that the thermogenic effect of SB-334867-A in the ob/ob mouse results primarily from central stimulation of the sympathetic nervous system, though the ability of OX1 receptor antagonists to produce a clinically relevant thermogenic (and consequential insulin sensitising) response in humans may depend on prevailing sympathetic tone.
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