- Email: [email protected]
Differential regulation of metabolic parameters by energy deficit and hunger
Differential regulation of metabolic parameters by energy deficit and hunger Tam´as Kitka, Sebesty´en Tuza, Bal´azs Varga, Csilla Horv´ath, P´eter Kov´acs PII: DOI: Reference:
S0026-0495(15)00178-X doi: 10.1016/j.metabol.2015.06.017 YMETA 53237
To appear in:
Metabolism
Received date: Revised date: Accepted date:
20 February 2015 10 June 2015 22 June 2015
Please cite this article as: Kitka Tam´as, Tuza Sebesty´en, Varga Bal´azs, Horv´ath Csilla, Kov´acs P´eter, Differential regulation of metabolic parameters by energy deficit and hunger, Metabolism (2015), doi: 10.1016/j.metabol.2015.06.017
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Differential regulation of metabolic parameters by energy deficit and hunger Authors and affiliations:
RI P
T
Tamás Kitka; Gedeon Richter Plc., Division of Pharmacological and Drug Safety Research Sebestyén Tuza; Gedeon Richter Plc., Division of Pharmacological and Drug Safety Research
SC
Balázs Varga; Gedeon Richter Plc., Division of Pharmacological and Drug Safety Research
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Csilla Horváth; Gedeon Richter Plc., Division of Pharmacological and Drug Safety Research Péter Kovács; Gedeon Richter Plc., Division of Pharmacological and Drug Safety Research Corresponding author: Tamás Kitka PhD
ED
Gedeon Richter Plc., Division of Pharmacological and Drug Safety Research H-1103, Gyomroi ut 19-21, Budapest, Hungary
PT
Phone: +36-30/317-99-62
CE
Email: [email protected]
AC
Disclosure statement: The authors declare that they have no conflicts of interest.
1
ACCEPTED MANUSCRIPT Abstract Aims: Hypocaloric diet decreases both energy expenditure (EE) and respiratory exchange rate (RER), affecting the efficacy of dieting inversely. Energy deficit and hunger may be modulated separately
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T
both in human and animal studies by drug treatment or food restriction. Thus it is important to separate the effects of energy deficit and hunger on EE and RER.
SC
Methods: Three parallel and analogous experiments were performed using three pharmacologically distinct anorectic drugs: rimonabant, sibutramine and tramadol. Metabolic parameters of vehicle-,
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drug-treated and pair-fed diet-induced obese mice from the three experiments underwent common statistical analysis to identify effects independent of the mechanisms of action. Diet-induced obesity (DIO) test of tramadol was also performed to examine its anti-obesity efficacy.
ED
Results: RER was decreased similarly by drug treatments and paired feeding throughout the experiment irrespective of the cause of reduced food intake. Contrarily, during the passive phase, EE
PT
was decreased more by paired feeding than by both vehicle- and drug-treatment irrespective of the
CE
drug used. In the active phase, EE was influenced by the pharmacological mechanisms of action. Tramadol decreased body weight in the DIO test.
AC
Conclusions: Our results suggest that RER is mainly affected by the actual state of energy balance; conversely, EE is rather influenced by hunger. Therefore, pharmacological medications that decrease hunger may enhance the efficacy of a hypocaloric diet by maintaining metabolic rate. Furthermore, our results yield the proposal that effects of anorectic drugs on EE and RER should be determined compared to vehicle and pair-fed groups, respectively, in animal models.
2
ACCEPTED MANUSCRIPT 1
Introduction
Metabolic effects of various anti-obesity drugs compared to either ad libitum or pair-fed controls are
T
widely described [1,2]. Although both drug-treated and pair-fed groups experience energy deficit,
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only paired-feeding involves food restriction, i.e. prolonged or increased hunger. Human data suggest a possible relation between hunger and adaptive reduction of thermogenesis during a
SC
hypocaloric diet [1,3]. This leaves open the possibility of there being differences between the metabolic consequences of drug- and restriction-induced underfeeding.
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Low-calorie diet decreases both energy expenditure (EE) and respiratory exchange rate (RER) [4-6]. These processes affect the outcome of a hypocaloric diet targeting weight loss oppositely: decreased RER is a marker of increased fat catabolism [7,8], while reduced EE decreases the efficacy of dieting
ED
[9,10].
In order to separate the specific effects of certain drugs from their general anorectic effect, we have
PT
chosen three compounds with fundamentally different mechanisms of action (MoA-s): rimonabant
CE
(cannabinoid CB1 receptor antagonist), sibutramine (an amphetamine derivate, serotonin and noradrenaline reuptake inhibitor [11]) and tramadol (µ-opioid receptor agonist [12]).
AC
Both sibutramine and rimonabant decrease appetite in low doses and affect energy expenditure in higher doses [2,13-16]. Tramadol was recently reported to decrease food intake after surgery, but no vehicle group was used in that experiment [17]. Our aim was to test the hypothesis that there are fundamental differences between the metabolic effects of food restriction and drug-induced reduction in food intake, regardless of the exact pharmacological effect of a certain drug.
3
ACCEPTED MANUSCRIPT 2 2.1
Methods Drugs
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Sibutramine hydrochloride (Jiangyin Eastern Medical Raw Materials Co, Jiangyin, China) and tramadol
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hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in distilled water; rimonabant (Bosche Scientific, New Brunswick, NJ, USA) was dissolved in 5% Tween 80 in distilled water because
Animals
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2.2
SC
of solubility issues.
Male C57Bl6 mice (Harlan, Udine, Italy, 4 animals per cage) were kept on a 12:12 h light-dark cycle and fed with a high-fat diet (D12492, 60% of energy content from fat, Research Diets, New Brunswick, NJ, USA) throughout the whole process. After twelve weeks of fatting, 20-week-old
ED
animals were isolated and habituated to experimental circumstances. Oral treatments were performed before lights off. In case of tramadol, a second treatment was also performed six hours
PT
later in order to maintain the effective plasma level. Body weights were measured each day before
CE
the first treatment. All procedures had been approved by the local ethical committee and conformed to the national guidelines (decree No. 40/2013. (II. 14) of the Hungarian Government) and the
2.3
AC
directive 2010/63/EU of the European Parliament. Indirect calorimetry
Indirect calorimetry was performed using a 16-cage PhenoMaster system (TSE Systems GmbH, Bad Homburg, Germany). Three analogous experiments were performed dedicated to study the effects of sibutramine (3 mg/kg), tramadol (30 mg/kg per treatment) and rimonabant (3 mg/kg). Each experiment consisted of three separate experimental days (one day biweekly during the first, third and fifth week of the same 5-week-long period). Baseline measurements were performed before each experimental day. During each experimental day, each mouse was assigned to one of the three 4
ACCEPTED MANUSCRIPT groups (vehicle, drug-treated, pair-fed) using the same randomized crossover design for the three experiments. Paired-feeding was performed on an hourly basis. Every measurement started at lights
Statistical analysis
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2.4
T
off and lasted for 23 hours.
Regarding the DIO test, body weights have been analyzed with one-way ANOVA (n=8 per group).
SC
For the indirect calorimetry (n=16 per experiment), data were collected separately for the dark and
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light phases because we didn’t intend to compare the two phases by any means. RER and EE data for every 30-minute period were averaged for the given phases. Repeated measures ANOVA was performed with factors “group” (within-subject design) and “experiment” (between-subject design). Significant “group” effect suggested that there is a difference between vehicle, drug-treated and
ED
pair-fed groups, while significant “experiment” x “group” interaction suggested that this effect was influenced by which experiment they participated in. Contrarily, the absence of interaction suggested
PT
that the effects of drugs were identical, i.e. that effects differed between each drug. In case of
CE
significant “group” effect or “experiment” x “group” interaction, a one- or two-factor Tukey's HSD post hoc test was used, respectively.
AC
Mauchly's sphericity test was used to validate repeated measures ANOVA tests. All statistical analyses have been carried out using STATISTICA 10 software (StatSoft, Inc., Tulsa, OK, USA). 3 3.1
Results Diet-induced obesity test
Tramadol, administered 30 mg/kg twice daily decreased body weight significantly in dietary obese C57Bl/6 mice from day nine (n=8, F(1,14)>4.71, p<0.05, Figure 1).
5
ACCEPTED MANUSCRIPT 3.2
Indirect calorimetry
After exclusions based on baseline data, animal numbers per experiment were n=13 (rimonabant and
Food intake
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3.2.1
T
sibutramine) or n=12 (tramadol).
ANOVA test revealed that the difference between treatment groups (active: F2,70=51.173, p<0.001,
SC
passive: F2,70=9.709, p<0.001) was identical between the experiments only in the active phase (active: F4,70=0.942, p=0.445, passive: F4,70=3.056, p=0.022), when drug treatments and paired feedings
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similarly decreased food intake. In the passive phase, only sibutramine and its pair-fed group showed decreased food intake compared to vehicle (Figure 2/A). 3.2.2
Respiratory Exchange Rate
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For both phases, ANOVA tests revealed that the difference between treatment groups (active: F2,70=38.759, p<0.001, passive: F2,70=33.798, p<0.001) were identical between the experiments in
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both phases (active: F4,70=0.247, p=0.911, passive: F4,70=0.665, p=0.619). Drug treatment and paired
3.2.3
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feeding decreased RER in both phases (Figure 2/B). Energy Expenditure
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The difference between treatment groups (active: F2,70=20.287, p<0.001, passive: F2,70=20.949, p<0.001), was identical between the experiments only in the passive phase (active: F4,70=9.510, p<0.001, passive: F4,70=0.443, p=0.777). Post hoc tests revealed the followings: ●
None of the drug treatments decreased EE, in fact, tramadol increased EE in the dark phase compared to corresponding vehicle-treated and pair-fed animals;
6
ACCEPTED MANUSCRIPT ●
decrease of EE in pair-fed groups did not reach statistical significance in the active phase compared to corresponding vehicle-treated groups (rimonabant, sibutramine and tramadol:
paired feeding decreased EE in the passive phase compared to both vehicle- and drug-
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●
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p=0.064, p=0.103 and p=0.195, respectively);
treatment while the latter two groups were identical (Figure 2/C). Discussion
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4
The common statistical evaluation of three parallel and analogous experiments with three
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fundamentally different drugs allowed the separation of their common anorectic effects from the effects characteristic for a certain drug. This separation was successful and reliable: „group x experiment” interaction was either highly significant (p<0.001) or very far from the level of
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significance (p>0.6) for the studied metabolic parameters.
The decreased RER in case of groups with energy deficit compared to the groups in neutral energy
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balance throughout the whole experiment and absence of “group x experiment” interaction suggest
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that RER depends only on the actual state of energy balance and independent of certain pharmacological actions of drugs applied.
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Regarding EE, MoA-independent unanimous changes among experiments were seen only in the passive phase, where paired-feeding decreased energy expenditure compared to both vehicle and drug-treated groups. It suggests that adaptive decrease of thermogenesis may not depend on energy deficit per se, only can be observed during exogenous food restriction, implying that hunger plays a role. It is also supported by literature data, for instance the reduction of food intake by increasing satiety using dietary fibers also decreases RER without affecting EE [18]. Furthermore, it provides a possible explanation of the unexpected finding that preserved fat free mass does not fully prevent metabolic slowing during a hypocaloric diet [19]. 7
ACCEPTED MANUSCRIPT The effect of tramadol on palatable food intake and body weight was not reported previously. Since this effect is not characteristic for μ-opiate agonists, other neurochemical processes may also be
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involved, for example tramadol is known to inhibit noradrenaline and serotonin reuptake [20].
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The aim of this study was to describe general physiological phenomena observed at the level of the organism independent of a certain MoA. Regarding obesity pharmacotherapy, our data suggest that
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an anorectic drug may increase the efficacy of a hypocaloric diet by alleviating the adaptive decrease of thermogenesis. In terms of animal experiments, MoA-specific effects of anorectic drugs on EE
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should be determined compared to an ad libitum fed vehicle group, while RER should be determined compared to a pair-fed group. Other comparisons may involve confounding effects of energy deficit or hunger.
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However, this approach also implies that further studies are needed to decipher the neurophysiological connection between hunger and energy expenditure. Furthermore, studies about
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these phenomena on the long run may also be of interest.
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In conclusion, our results suggest fundamental differences in the physiological regulation of the two main indirect calorimetric parameters during a hypocaloric diet. RER seems to be driven only by the
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actual state of energy balance. Contrarily, the adaptive decrease of thermogenesis may basically occur during exogenous food restriction, implying a role of hunger. 5
Funding
The study was partially supported by ERNYŐ 2013 of the Hungarian Government. 6
Disclosure statement
The authors declare that they have no conflicts of interest.
8
ACCEPTED MANUSCRIPT 7
Author contributions
T.K. determined the scientific question, designed the study and wrote the manuscript. S.T. and B.V.
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performed the measurements and contributed to manuscript sections. C.H. researched data. P.K.
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contributed to study design, statistical analysis and the „results” section. C.H and P.K. revised the manuscript.
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References
[1] Tremblay A., Royer M.M., Chaput J.P., Doucet E. Adaptive thermogenesis can make a
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difference in the ability of obese individuals to lose body weight. Int J Obes (Lond) 2013; 37:759-764.
[2] Zhang L.N., Gamo Y., Sinclair R. et al. Effects of chronic oral rimonabant administration on
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energy budgets of diet-induced obese C57BL/6 mice. Obesity (Silver Spring) 2012; 20:954-
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962.
[3] Caudwell P., Finlayson G., Gibbons C. et al. Resting metabolic rate is associated with hunger,
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self-determined meal size, and daily energy intake and may represent a marker for appetite. Am J Clin Nutr 2013; 97:7-14.
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[4] Thearle MS., Pannacciulli N., Bonfiglio S., Pacak K., Krakoff J. Extent and determinants of thermogenic responses to 24 hours of fasting, energy balance, and five different overfeeding diets in humans. J Clin Endocrinol Metab 2013; 98(7):2791-9. [5] Westbrook R., Bonkowski M.S., Arum O., Strader A.D., Bartke A. Metabolic alterations due to caloric restriction and every other day feeding in normal and growth hormone receptor knockout mice. J Gerontol A Biol Sci Med Sci 2014; 69:25-33.
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ACCEPTED MANUSCRIPT [6] Wueest S., Item F., Boyle C.N. et al. Interleukin-6 contributes to early fasting-induced free fatty acid mobilization in mice. Am J Physiol Regul Integr Comp Physiol 2014; 306:R861-R867.
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[7] Ellis A.C., Hyatt T.C., Hunter G.R., Gower B.A. Respiratory quotient predicts fat mass gain in
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premenopausal women. Obesity (Silver Spring) 2010; 18:2255-2259.
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[8] Longo K.A., Charoenthongtrakul S., Giuliana D.J. et al. The 24-hour respiratory quotient predicts energy intake and changes in body mass. Am J Physiol Regul Integr Comp Physiol
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2010; 298:R747-R754.
[9] Bosy-Westphal A., Schautz B., Lagerpusch M. et al. Effect of weight loss and regain on adipose tissue distribution, composition of lean mass and resting energy expenditure in
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young overweight and obese adults. Int J Obes (Lond) 2013; 37(10):1371-7 [10] Siervo M., Faber P., Laraa J. et al. Imposed rate and extent of weight loss in obese men and
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adaptive changes in resting and total energy expenditure. Metabolism 2015; 64(8): 896–904
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[11] Ryan D.H., Kaiser P., Bray G.A. Sibutramine: a novel new agent for obesity treatment. Obes Res 1995; 3 Suppl 4:553S-559S.
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[12] Gillen C., Haurand M., Kobelt D.J., Wnendt S. Affinity, potency and efficacy of tramadol and its metabolites at the cloned human mu-opioid receptor. Naunyn Schmiedebergs Arch Pharmacol 2000; 362:116-121. [13] Boozer C.N., Leibel R.L., Love R.J., Cha M.C., Aronne L.J. Synergy of sibutramine and low-dose leptin in treatment of diet-induced obesity in rats. Metabolism 2001; 50:889-893. [14] Seagle H.M., Bessesen D.H., Hill J.O. Effects of sibutramine on resting metabolic rate and weight loss in overweight women. Obes Res 1998; 6:115-121. 10
ACCEPTED MANUSCRIPT [15] Thornton-Jones Z.D., Kennett G.A., Benwell K.R. et al. The cannabinoid CB1 receptor inverse agonist, rimonabant, modifies body weight and adiponectin function in diet-induced obese
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rats as a consequence of reduced food intake. Pharmacol Biochem Behav 2006; 84:353-359.
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[16] Kunz I., Meier M.K., Bourson A., Fisseha M., Schilling W. Effects of rimonabant, a cannabinoid
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CB1 receptor ligand, on energy expenditure in lean rats. Int J Obes (Lond) 2008; 32:863-870. [17] Ratsep M.T., Barrette V.F., Winterborn A., Adams M.A., Croy B.A. Hemodynamic and
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behavioral differences after administration of meloxicam, buprenorphine, or tramadol as analgesics for telemeter implantation in mice. J Am Assoc Lab Anim Sci 2013; 52:560-566. [18] Rasoamanana R., Even P.C., Darcel N., Tome D., Fromentin G. Dietary fibers reduce food intake by satiation without conditioned taste aversion in mice. Physiol Behav 2013; 110-
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111:13-19.
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[19] Johannsen D.L., Knuth N.D., Huizenga R., Rood J.C., Ravussin E., Hall K.D. Metabolic slowing with massive weight loss despite preservation of fat-free mass. J Clin Endocrinol Metab 2012;
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97:2489-2496.
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[20] Munro G., Baek C.A., Erichsen H.K. et al. The novel compound (+/-)-1-[10-((E)-3-Phenyl-allyl)3,10-diaza-bicyclo[4.3.1]dec-3-yl]-propan -1-one (NS7051) attenuates nociceptive transmission in animal models of experimental pain; a pharmacological comparison with the combined mu-opioid receptor agonist and monoamine reuptake inhibitor tramadol. Neuropharmacology 2008; 54:331-343.
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ACCEPTED MANUSCRIPT Figure legends Figure 1 Body weights of DIO mice receiving tramadol (30 mg/kg, po, bid) or vehicle. H1-H3:
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habituation days, D0-D14: experimental days, n=8 per group, means ± SEM, *: significant
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difference compared to vehicle, p<0.05.
Figure 2 Food intake (A), respiratory exchange rate (B) and energy expenditure (C) during the dark
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and light phases, n=13 (rimonabant and sibutramine) or n=12 (tramadol) per group, means ± SEM. black columns: dark (active) phase, white columns: light (passive) phase; R:
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rimonabant (3 mg/kg, po, qd), S: sibutramine (3 mg/kg, po, qd), T: tramadol (30 mg/kg, po, bid). Statistically significant differences compared to vehicle groups are indicated by ** (p<0.01) or *** (p<0.001). Statistically significant differences between drug-treated and
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CE
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ED
pair-fed groups are indicated by ### (P<0.001).
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ACCEPTED MANUSCRIPT
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Figure 1
13
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PT
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ACCEPTED MANUSCRIPT
Figure 2
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Highlights Respiratory exchange rate (RER) is affected by the actual state of energy balance. Energy expenditure (EE) is rather influenced by subjective feelings of hunger. Tramadol causes weight loss in diet-induced obese mice.
15
S0026-0495(15)00178-X doi: 10.1016/j.metabol.2015.06.017 YMETA 53237
To appear in:
Metabolism
Received date: Revised date: Accepted date:
20 February 2015 10 June 2015 22 June 2015
Please cite this article as: Kitka Tam´as, Tuza Sebesty´en, Varga Bal´azs, Horv´ath Csilla, Kov´acs P´eter, Differential regulation of metabolic parameters by energy deficit and hunger, Metabolism (2015), doi: 10.1016/j.metabol.2015.06.017
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Differential regulation of metabolic parameters by energy deficit and hunger Authors and affiliations:
RI P
T
Tamás Kitka; Gedeon Richter Plc., Division of Pharmacological and Drug Safety Research Sebestyén Tuza; Gedeon Richter Plc., Division of Pharmacological and Drug Safety Research
SC
Balázs Varga; Gedeon Richter Plc., Division of Pharmacological and Drug Safety Research
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Csilla Horváth; Gedeon Richter Plc., Division of Pharmacological and Drug Safety Research Péter Kovács; Gedeon Richter Plc., Division of Pharmacological and Drug Safety Research Corresponding author: Tamás Kitka PhD
ED
Gedeon Richter Plc., Division of Pharmacological and Drug Safety Research H-1103, Gyomroi ut 19-21, Budapest, Hungary
PT
Phone: +36-30/317-99-62
CE
Email: [email protected]
AC
Disclosure statement: The authors declare that they have no conflicts of interest.
1
ACCEPTED MANUSCRIPT Abstract Aims: Hypocaloric diet decreases both energy expenditure (EE) and respiratory exchange rate (RER), affecting the efficacy of dieting inversely. Energy deficit and hunger may be modulated separately
RI P
T
both in human and animal studies by drug treatment or food restriction. Thus it is important to separate the effects of energy deficit and hunger on EE and RER.
SC
Methods: Three parallel and analogous experiments were performed using three pharmacologically distinct anorectic drugs: rimonabant, sibutramine and tramadol. Metabolic parameters of vehicle-,
MA NU
drug-treated and pair-fed diet-induced obese mice from the three experiments underwent common statistical analysis to identify effects independent of the mechanisms of action. Diet-induced obesity (DIO) test of tramadol was also performed to examine its anti-obesity efficacy.
ED
Results: RER was decreased similarly by drug treatments and paired feeding throughout the experiment irrespective of the cause of reduced food intake. Contrarily, during the passive phase, EE
PT
was decreased more by paired feeding than by both vehicle- and drug-treatment irrespective of the
CE
drug used. In the active phase, EE was influenced by the pharmacological mechanisms of action. Tramadol decreased body weight in the DIO test.
AC
Conclusions: Our results suggest that RER is mainly affected by the actual state of energy balance; conversely, EE is rather influenced by hunger. Therefore, pharmacological medications that decrease hunger may enhance the efficacy of a hypocaloric diet by maintaining metabolic rate. Furthermore, our results yield the proposal that effects of anorectic drugs on EE and RER should be determined compared to vehicle and pair-fed groups, respectively, in animal models.
2
ACCEPTED MANUSCRIPT 1
Introduction
Metabolic effects of various anti-obesity drugs compared to either ad libitum or pair-fed controls are
T
widely described [1,2]. Although both drug-treated and pair-fed groups experience energy deficit,
RI P
only paired-feeding involves food restriction, i.e. prolonged or increased hunger. Human data suggest a possible relation between hunger and adaptive reduction of thermogenesis during a
SC
hypocaloric diet [1,3]. This leaves open the possibility of there being differences between the metabolic consequences of drug- and restriction-induced underfeeding.
MA NU
Low-calorie diet decreases both energy expenditure (EE) and respiratory exchange rate (RER) [4-6]. These processes affect the outcome of a hypocaloric diet targeting weight loss oppositely: decreased RER is a marker of increased fat catabolism [7,8], while reduced EE decreases the efficacy of dieting
ED
[9,10].
In order to separate the specific effects of certain drugs from their general anorectic effect, we have
PT
chosen three compounds with fundamentally different mechanisms of action (MoA-s): rimonabant
CE
(cannabinoid CB1 receptor antagonist), sibutramine (an amphetamine derivate, serotonin and noradrenaline reuptake inhibitor [11]) and tramadol (µ-opioid receptor agonist [12]).
AC
Both sibutramine and rimonabant decrease appetite in low doses and affect energy expenditure in higher doses [2,13-16]. Tramadol was recently reported to decrease food intake after surgery, but no vehicle group was used in that experiment [17]. Our aim was to test the hypothesis that there are fundamental differences between the metabolic effects of food restriction and drug-induced reduction in food intake, regardless of the exact pharmacological effect of a certain drug.
3
ACCEPTED MANUSCRIPT 2 2.1
Methods Drugs
T
Sibutramine hydrochloride (Jiangyin Eastern Medical Raw Materials Co, Jiangyin, China) and tramadol
RI P
hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in distilled water; rimonabant (Bosche Scientific, New Brunswick, NJ, USA) was dissolved in 5% Tween 80 in distilled water because
Animals
MA NU
2.2
SC
of solubility issues.
Male C57Bl6 mice (Harlan, Udine, Italy, 4 animals per cage) were kept on a 12:12 h light-dark cycle and fed with a high-fat diet (D12492, 60% of energy content from fat, Research Diets, New Brunswick, NJ, USA) throughout the whole process. After twelve weeks of fatting, 20-week-old
ED
animals were isolated and habituated to experimental circumstances. Oral treatments were performed before lights off. In case of tramadol, a second treatment was also performed six hours
PT
later in order to maintain the effective plasma level. Body weights were measured each day before
CE
the first treatment. All procedures had been approved by the local ethical committee and conformed to the national guidelines (decree No. 40/2013. (II. 14) of the Hungarian Government) and the
2.3
AC
directive 2010/63/EU of the European Parliament. Indirect calorimetry
Indirect calorimetry was performed using a 16-cage PhenoMaster system (TSE Systems GmbH, Bad Homburg, Germany). Three analogous experiments were performed dedicated to study the effects of sibutramine (3 mg/kg), tramadol (30 mg/kg per treatment) and rimonabant (3 mg/kg). Each experiment consisted of three separate experimental days (one day biweekly during the first, third and fifth week of the same 5-week-long period). Baseline measurements were performed before each experimental day. During each experimental day, each mouse was assigned to one of the three 4
ACCEPTED MANUSCRIPT groups (vehicle, drug-treated, pair-fed) using the same randomized crossover design for the three experiments. Paired-feeding was performed on an hourly basis. Every measurement started at lights
Statistical analysis
RI P
2.4
T
off and lasted for 23 hours.
Regarding the DIO test, body weights have been analyzed with one-way ANOVA (n=8 per group).
SC
For the indirect calorimetry (n=16 per experiment), data were collected separately for the dark and
MA NU
light phases because we didn’t intend to compare the two phases by any means. RER and EE data for every 30-minute period were averaged for the given phases. Repeated measures ANOVA was performed with factors “group” (within-subject design) and “experiment” (between-subject design). Significant “group” effect suggested that there is a difference between vehicle, drug-treated and
ED
pair-fed groups, while significant “experiment” x “group” interaction suggested that this effect was influenced by which experiment they participated in. Contrarily, the absence of interaction suggested
PT
that the effects of drugs were identical, i.e. that effects differed between each drug. In case of
CE
significant “group” effect or “experiment” x “group” interaction, a one- or two-factor Tukey's HSD post hoc test was used, respectively.
AC
Mauchly's sphericity test was used to validate repeated measures ANOVA tests. All statistical analyses have been carried out using STATISTICA 10 software (StatSoft, Inc., Tulsa, OK, USA). 3 3.1
Results Diet-induced obesity test
Tramadol, administered 30 mg/kg twice daily decreased body weight significantly in dietary obese C57Bl/6 mice from day nine (n=8, F(1,14)>4.71, p<0.05, Figure 1).
5
ACCEPTED MANUSCRIPT 3.2
Indirect calorimetry
After exclusions based on baseline data, animal numbers per experiment were n=13 (rimonabant and
Food intake
RI P
3.2.1
T
sibutramine) or n=12 (tramadol).
ANOVA test revealed that the difference between treatment groups (active: F2,70=51.173, p<0.001,
SC
passive: F2,70=9.709, p<0.001) was identical between the experiments only in the active phase (active: F4,70=0.942, p=0.445, passive: F4,70=3.056, p=0.022), when drug treatments and paired feedings
MA NU
similarly decreased food intake. In the passive phase, only sibutramine and its pair-fed group showed decreased food intake compared to vehicle (Figure 2/A). 3.2.2
Respiratory Exchange Rate
ED
For both phases, ANOVA tests revealed that the difference between treatment groups (active: F2,70=38.759, p<0.001, passive: F2,70=33.798, p<0.001) were identical between the experiments in
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both phases (active: F4,70=0.247, p=0.911, passive: F4,70=0.665, p=0.619). Drug treatment and paired
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feeding decreased RER in both phases (Figure 2/B). Energy Expenditure
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The difference between treatment groups (active: F2,70=20.287, p<0.001, passive: F2,70=20.949, p<0.001), was identical between the experiments only in the passive phase (active: F4,70=9.510, p<0.001, passive: F4,70=0.443, p=0.777). Post hoc tests revealed the followings: ●
None of the drug treatments decreased EE, in fact, tramadol increased EE in the dark phase compared to corresponding vehicle-treated and pair-fed animals;
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decrease of EE in pair-fed groups did not reach statistical significance in the active phase compared to corresponding vehicle-treated groups (rimonabant, sibutramine and tramadol:
paired feeding decreased EE in the passive phase compared to both vehicle- and drug-
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p=0.064, p=0.103 and p=0.195, respectively);
treatment while the latter two groups were identical (Figure 2/C). Discussion
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The common statistical evaluation of three parallel and analogous experiments with three
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fundamentally different drugs allowed the separation of their common anorectic effects from the effects characteristic for a certain drug. This separation was successful and reliable: „group x experiment” interaction was either highly significant (p<0.001) or very far from the level of
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significance (p>0.6) for the studied metabolic parameters.
The decreased RER in case of groups with energy deficit compared to the groups in neutral energy
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balance throughout the whole experiment and absence of “group x experiment” interaction suggest
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that RER depends only on the actual state of energy balance and independent of certain pharmacological actions of drugs applied.
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Regarding EE, MoA-independent unanimous changes among experiments were seen only in the passive phase, where paired-feeding decreased energy expenditure compared to both vehicle and drug-treated groups. It suggests that adaptive decrease of thermogenesis may not depend on energy deficit per se, only can be observed during exogenous food restriction, implying that hunger plays a role. It is also supported by literature data, for instance the reduction of food intake by increasing satiety using dietary fibers also decreases RER without affecting EE [18]. Furthermore, it provides a possible explanation of the unexpected finding that preserved fat free mass does not fully prevent metabolic slowing during a hypocaloric diet [19]. 7
ACCEPTED MANUSCRIPT The effect of tramadol on palatable food intake and body weight was not reported previously. Since this effect is not characteristic for μ-opiate agonists, other neurochemical processes may also be
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involved, for example tramadol is known to inhibit noradrenaline and serotonin reuptake [20].
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The aim of this study was to describe general physiological phenomena observed at the level of the organism independent of a certain MoA. Regarding obesity pharmacotherapy, our data suggest that
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an anorectic drug may increase the efficacy of a hypocaloric diet by alleviating the adaptive decrease of thermogenesis. In terms of animal experiments, MoA-specific effects of anorectic drugs on EE
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should be determined compared to an ad libitum fed vehicle group, while RER should be determined compared to a pair-fed group. Other comparisons may involve confounding effects of energy deficit or hunger.
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However, this approach also implies that further studies are needed to decipher the neurophysiological connection between hunger and energy expenditure. Furthermore, studies about
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these phenomena on the long run may also be of interest.
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In conclusion, our results suggest fundamental differences in the physiological regulation of the two main indirect calorimetric parameters during a hypocaloric diet. RER seems to be driven only by the
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actual state of energy balance. Contrarily, the adaptive decrease of thermogenesis may basically occur during exogenous food restriction, implying a role of hunger. 5
Funding
The study was partially supported by ERNYŐ 2013 of the Hungarian Government. 6
Disclosure statement
The authors declare that they have no conflicts of interest.
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Author contributions
T.K. determined the scientific question, designed the study and wrote the manuscript. S.T. and B.V.
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performed the measurements and contributed to manuscript sections. C.H. researched data. P.K.
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contributed to study design, statistical analysis and the „results” section. C.H and P.K. revised the manuscript.
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ACCEPTED MANUSCRIPT Figure legends Figure 1 Body weights of DIO mice receiving tramadol (30 mg/kg, po, bid) or vehicle. H1-H3:
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habituation days, D0-D14: experimental days, n=8 per group, means ± SEM, *: significant
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difference compared to vehicle, p<0.05.
Figure 2 Food intake (A), respiratory exchange rate (B) and energy expenditure (C) during the dark
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and light phases, n=13 (rimonabant and sibutramine) or n=12 (tramadol) per group, means ± SEM. black columns: dark (active) phase, white columns: light (passive) phase; R:
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rimonabant (3 mg/kg, po, qd), S: sibutramine (3 mg/kg, po, qd), T: tramadol (30 mg/kg, po, bid). Statistically significant differences compared to vehicle groups are indicated by ** (p<0.01) or *** (p<0.001). Statistically significant differences between drug-treated and
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pair-fed groups are indicated by ### (P<0.001).
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Highlights Respiratory exchange rate (RER) is affected by the actual state of energy balance. Energy expenditure (EE) is rather influenced by subjective feelings of hunger. Tramadol causes weight loss in diet-induced obese mice.
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