Effects of leptin on energy metabolism in b-less mice

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1 (2008) 32, & 2008 Nature Publishing Group All rights reserved /08 $30 ORIGINAL ARTICLE Effects of leptin on energy metabolism in mice CS Asensio 1,2, D Arsenijevic 2, L Lehr 3, J-P Giacobino 3, P Muzzin 1 and F Rohner-Jeanrenaud 2 1 Department of Cell Physiology and Metabolism, Medical University Centre, Geneva, Switzerland; 2 Division of Endocrinology, Diabetology and Nutrition, Department of Internal Medicine, Medical University Centre, Geneva, Switzerland and 3 Department of Medical Biochemistry, Medical University Centre, Geneva, Switzerland Objective: To investigate the impact of b-adrenoceptor deficiency on the metabolic effects of leptin. Measurements: was infused subcutaneously through an osmotic minipump in wild-type () and b 1 /b 2 /b 3 - adrenoceptor knockout () mice and its effects on food intake, energy expenditure, carbohydrate and lipid utilization as well as on the levels of expression of the brown adipose tissue (BAT), thermogenic marker uncoupling protein-1 (UCP1) and type II deiodinase (D2) mrnas were compared. Results: treatment decreased food intake by 23% in both the and the mice. In pair-fed animals being used as controls, leptin treatment was found to increase energy expenditure in, but not in mice. No difference was observed in carbohydrate or fat utilization between leptin-treated and mice. increased UCP1 and D2 mrna levels in mouse BAT 1.7- and 3-fold, respectively, but had no effect on the expression of these genes in mouse BAT. Conclusion: The stimulatory effects of leptin on oxygen consumption, BAT UCP1 and D2 expression require functional b-adrenoceptors, but its inhibitory effect on food intake and its stimulatory effect on fat utilization is independent of the b-adrenoceptor signalling. (2008) 32, ; doi: /ijo ; published online 19 February 2008 Keywords: leptin; b-adrenoceptor knockout mice; energy expenditure; brown adipose tissue; thermogenic markers Introduction The control of body weight and composition depends on the balance between caloric intake and energy expenditure., which is secreted by the adipocyte, functions as an afferent signal in a negative feedback loop that maintains body weight within a relatively narrow range by acting on both components of the balance. 1 administration, indeed, inhibits food intake and increases energy expenditure in rodents. 2 4 was first proposed to act essentially through the central nervous system. Both its inhibition of food intake and its stimulation of thermogenesis represent effects that are exerted at the level of the hypothalamus. 4 The energy-dissipating effects of leptin have two main targets: brown adipose tissue (BAT) thermogenesis and whole body oxidative substrate utilization. Brown adipose tissue thermogenesis is mainly regulated by the sympathetic nervous system activity. Stimulation of the latter leads to the local release of catecholamines by the Correspondence: Dr CS Asensio, Department of Neurology, University of California, San Francisco, th Street, Genentech Hall, N274, San Francisco, CA 94158, USA. Cedric.Asensio@ucsf.edu Received 22 July 2007; revised 7 January 2008; accepted 15 January 2008; published online 19 February 2008 sympathetic nerve terminals, and to an increase in circulating catecholamines by the activation of the adrenal medulla. Catecholamines then mediate their effects through a- and b-adrenoceptors. The b-adrenoceptor family comprises of the b 1 -, b 2 -, b 3 -subtypes and the three b-subtypes coexist in BAT. Numerous studies showed that leptin increases thermogenesis by acting centrally through the hypothalamus to activate the sympathetic neurons innervating the BAT, and thereby increase the thermogenic activity of this tissue. It has indeed been found that the administration of leptin in vivo to mice resulted in an increase in noradrenaline turnover in BAT. 5 An intracerebroventricular administration of leptin was also found to increase the thermogenic marker, uncoupling protein-1 (UCP1) mrna expression in the rodent BAT. 6 8 Furthermore, the stimulatory effect of peripheral or intracerebroventricular administration of leptin on rat BAT UCP1 mrna expression was suppressed by surgical denervation of the tissue. 9,10 might also modulate energy dissipation by activating glucose or fat catabolic pathways in various tissues. A peripheral administration of leptin was found to increase insulin sensivity and insulin-stimulated glucose uptake in skeletal muscles. 14 Intravenous or intracerebroventricular administration of leptin increased the uptake of glucose in skeletal muscles and in BAT 10,15 and the rate of glycolysis

2 in the whole animal. 15 Peripheral administration of leptin was also found to increase ex vivo lipolysis and fatty acid b-oxidation in skeletal muscles 16,17 and lipolysis in white adipose fat pads. 18 It has been demonstrated that the increase of fatty acid b-oxidation by leptin in skeletal muscles comprises two components: an early activation of the 5 0 -AMP-activated protein kinase elicited directly by leptin and a later activation mediated by the sympathetic nervous system through the a-adrenoceptor. 17 Whereas, as mentioned above, it seems clear that the effects of leptin on BAT thermogenesis are mediated by the sympathetic nervous system, it would be important to determine in vivo the part played by the b-adrenergic system in the effects of leptin on energy expenditure and on glucose or fat catabolic pathways. In 2002, two b 1 /b 2 /b 3 -adrenoceptor knockout () mouse models were generated. The mice exhibited normophagic obesity and a defective cold-induced thermogenesis. 19,20 Furthermore, they did not respond to a high-fat diet by an increase in oxygen consumption, suggesting a failure in their diet-induced thermogenesis. 19 The mouse model would be an invaluable tool to test in vivo the respective contributions of the b-adrenergic system and of putative direct peripheral effects of leptin on thermogenesis and oxidative substrate utilization. Therefore, leptin was infused subcutaneously through osmotic minipumps in wild-type () and mice and its effects on food intake, body weight, energy expenditure, glucose and lipid utilization, as well as BAT thermogenic gene expression were determined. Materials and methods Materials All organic and inorganic chemicals of analytical or molecular biology grade were purchased from Merck (Darmstadt, Germany), Boehringer Mannheim (Mannheim, Germany), Sigma Chemical Co. (St Louis, MI, USA) and Fluka (Buchs, Switzerland). (Ala-100) hleptin, a human leptin analog was provided by Eli Lilly and Co. (Indianapolis, IN, USA). Mice 3- to 4-month-old male and mice, obtained as previously described, 20 were housed individually and kept at 24±1 1C on a 12 h light dark cycle ( hours). They were allowed ad libitum access to water and to a standard laboratory chow (Nordos, Cergy, France) unless otherwise stated. Osmotic minipumps (Alzet, model 2001; Cupertino, CA, USA) delivering 24 mg of leptin per day for 6 days or its vehicle alone were implanted subcutaneously on the back of the mice under isofluorane (Abbott, Abbott Park, IL, USA) anesthesia. Food intake was measured daily during a 1-week pretreatment period and during the 6 days of leptin infusion and b-adrenoceptors and corrected for spillage. Pair-fed animals were provided at the beginning of each dark cycle with the same amount of food as ingested by leptin-treated animals on a daily basis. At the end of the experiment, the mice were killed by cervical dislocation. The mice were treated in accordance with our institutional guidelines. Indirect calorimetry Oxygen consumption and carbon dioxide production of and mice were measured by indirect calorimetry, using the OXYMAX system (Columbus instruments, Columbus, OH, USA). Mice were caged individually in four 1-l chambers at 24 1C with free access to food and water, unless otherwise stated. Mice were adapted to the chambers for 3 days before surgery. Settling and measuring times were both of 120 s. Room air served as reference air. Data were recorded from 24 to 11 hours on day 2 6 after surgery. Values were collected every 12 min. The VO 2 was expressed as oxygen consumption (ml h 1 ) and the VCO 2 as carbon dioxide production (ml h 1 ). The respiratory quotient (RQ) is defined as the ratio of VCO 2 over VO 2. Energy expenditure (EE) in kcal h 1 was calculated by the OXYMAX system as follows: EE ¼ (3.815 þ RQ) VO 2 Under our experimental conditions, protein oxidation was assumed to be equivalent to daily protein intake. Knowing that the chow diet contained 20% of protein, the adjusted RQ and the carbohydrate utilization rate could be calculated as follows: 21,22 Carbohydrate utilization rate (kcal h 1 ) ¼ % carbohydrate EE (kcal h 1 ) %carbohydrate ¼percentage of substrate used as carbohydrate ¼ðð8021:12ÞðRQ adj 0:71ÞÞ=ð21:12 ðrq adj 0:71Þþ19:61ð1 RQ adj ÞÞ RQ adj ¼RQ adjusted for protein utilized ¼ðVCO 2 4:76NÞ=ðVO 2 5:94NÞ N ¼protein intake=6:25: Real-time PCR The interscapular BAT was used for total RNA preparation by the technique of Chomczynski and Sacchi. 23 Oligo-dTprimed first strand cdna was synthesized using the Superscriptt II RNase H Reverse Transcription kit (Invitrogen, Carlsbad, CA, USA) and real-time PCR was performed using ABI rapid thermal cycler system, and a SYBR Green PCR master mix according to the manufacturer s instructions. Cyclophilin A was used as a control to account for any variations due to the efficiencies of the reverse transcription. UCP1 oligonucleotide primers used were upstream 5 0 -CGATGTCCATGTACACCAAGGA-3 0 and downstream 5 0 -TTGTGGCTTCTTTTCTGCGA-3 0, covering the nucleotides of UCP1 cdna (GenBank accession number NM_009463). Type II deiodinase (D2) oligonucleotide 937

3 938 primers used were upstream 5 0 -CGCCCAGTGTCAAGTTGT-3 0 and downstream 5 0 -CACGATGCACACACGTTCAA, covering the nucleotides of D2 cdna (GenBank accession number NM_010050). Cyclophilin A oligonucleotide primers used were upstream 5 0 -CAAATGCTGGACCAAACACAA-3 0 and downstream 5 0 -CCATCCAGCCATTCAGTCTTG-3 0, covering the nucleotides of cyclophilin A (GenBank accession number NM_008907). The conditions of PCR were a step at 50 1C for 2 min followed by a denaturing step at 95 1C for 10 min and by 50 cycles at 95 1C for 15 s and 60 1C for 1 min. The upstream and downstream oligonucleotide primers were chosen on both sides of an intron to prevent amplification of possible contaminating genomic DNA. Statistical analysis The results are given as mean±s.e.m. Statistical analysis was performed using one-way analysis of variance followed by Student Newman Keuls procedure or using Kruskal- Wallis one-way analysis of variance on ranks for multiple comparisons. The calculations were performed using the Sigma STAT software (SPSS Inc., Chicago, IL, USA). A P-value o5 was considered statistically significant. Results and b-adrenoceptors The effects of leptin on food intake in and mice were measured. was administered using osmotic minipumps implanted subcutaneously on the back of the mice and delivering 1 mgh 1 of the hormone in NaCl 0.9%. The untreated control group consisted in mice implanted with osmotic minipumps delivering the vehicle alone. As shown in Figure 1a, leptin treatment decreased food intake in both and mice. The magnitude of the decreases was similar in both genotypes, that is, 23% in both and mice. To avoid confounding effects of the decrease in food intake on the various parameters tested in this study, it was necessary to introduce a pair-fed group as a control for the leptin-treated group. As a consequence, the leptin effects referred to below will be analysed and compared to the pairfed values. As shown in Figure 1b, in mice, 6 days of pair feeding or leptin treatment induced similar body weight losses (3.1 and 2.5 g, respectively). In mice, pair feeding or leptin treatment also induced similar body weight losses (2.6 and 2.4 g, respectively). Therefore, under the conditions of our experiment, leptin per se did not affect body weight. The lack of effect of leptin on body weight other than by reducing food intake does not exclude a fine-tuning by leptin of energy expenditure. We, therefore, performed a series of experiments in which the latter was measured by indirect calorimetry in or mice treated or without leptin. The oxygen consumption was recorded during five consecutive days and energy expenditure values were derived from oxygen consumption and CO 2 production, as described in Materials and methods. The average energy expenditure per group from day 2 to 6 is presented in Figure 2. Pair feeding similarly decreased energy expenditure by 44 and 47% in and mice, respectively. Although two-way analysis of variance did not show any significant interaction between treatment and genotype, leptin treatment increased energy expenditure by 1.5-fold in mice, while it had no effect in mice. As a consequence, after leptin administration, the energy expenditure of the mice was significantly lower than that of mice. The measurement of oxygen consumption and carbon dioxide production allows for the calculation, through the RQ, of carbohydrate and lipid disappearance rates. As shown Food intake [g] BW changes [g] Figure 1 (a) Daily food intake of 3- to 4-month-old male or mice treated with saline (control), or with 1 mgh 1 of leptin (leptin), both delivered subcutaneously by minipumps. The values for each mouse were the average in grams day 1 of the food intake during the 6 days of leptin infusion. The results illustrated in the figure are the means±s.e.m. of the average values of 6 8 mice. Po05 vs corresponding controls. (b) Total body weight changes of or mice treated with saline (control), with saline but pair-fed with the leptin-treated group (), or with 1 mgh 1 of leptin (leptin). The results are the means±s.e.m. of 6 8 mice. Po05 vs corresponding controls., wild type. -4.0

4 Energy expenditure kcal h o in Figure 3a, in and mice, pair feeding decreased average carbohydrate utilization from day 2 to 6 by 35 and 28%, respectively. The pair-fed values of the and mice were not different from each other. had no effect on carbohydrate utilization in either genotype. As shown in Figure 3b, pair feeding did not significantly alter average lipid utilization from day 2 to 6 in either or in mice. The pair-fed values of the and mice were not different from each other. Compared to values of the pair-fed groups, leptin increased lipid utilization 1.4-fold in mice, but it had no significant effect in the group. However, after leptin administration, there was no difference in the lipid utilization rate between and mice. We further dissected the effects of pair feeding and of leptin by calculating the substrate utilization rates obtained during the dark phase, a phase during which the nutritional state of the various groups is the closest. As shown in Figure 3c, during this phase, pair feeding had no effect on carbohydrate utilization neither in nor in mice. The pair-fed values of the and mice were not different from each other. Compared to values of pair-fed mice, leptin decreased carbohydrate utilization in the group, but had no significant effect in animals, although a trend toward a decrease was observed (P ¼ 0.1). As shown in Figure 3d, pair feeding had no effect on fat utilization either in or in mice. markedly increased lipid utilization both in the and the group. No difference was observed, after leptin administration, between and mice. Figure 2 Average energy expenditure (kcal h 1 ) from day 2 to 6 of or mice treated with saline (control, n ¼ 6 and 7), with saline and pair-fed with the leptin-treated group (, n ¼ 8 and ¼ 7) or with 1 mgh 1 of leptin (leptin, n ¼ 8 and 6). The results are the means±s.e.m. Po01 vs corresponding controls; þ Po5 vs corresponding ; 1Po5 between genotypes., wild type. and b-adrenoceptors It has been proposed that the effects of leptin on energy expenditure are mediated by sympathetic activation of BAT UCP1 expression in rodents. 5 9 We therefore measured this parameter in BAT of or mice treated with or without leptin. As shown in Figure 4a, when considering BAT of control groups, UCP1 mrna level was 1.2-fold lower in than in mice (Po2). Pair feeding had no effect on UCP1 mrna level in either genotype. increased UCP1 mrna level 1.7-fold in BAT of the (Po05), but had no effect in BAT of the group. After leptin administration, UCP1 mrna level in BAT of mice was twofold lower than in BAT of animals (Po05). Stimulation of the sympathetic nervous system activity through the a and b 1 /b 2 /b 3 -adrenoceptors acting in a synergistic manner is known to stimulate D2 mrna expression and activity, thereby increasing BAT T3 concentration and UCP1 expression We therefore measured D2 mrna in the BAT of or mice treated with or without leptin. As shown in Figure 4b, D2 mrna level was similar in the BAT of control and mice. Pair feeding decreased D2 mrna level by twofold in the (Po05), but it had no effect in mice. increased D2 mrna by threefold in the (Po01), an effect that was not observed in the group. As a consequence, after leptin administration, D2 mrna levels in the BAT of mice were 1.9-fold lower than those in the BAT of leptin-treated mice (Po1). Discussion As expected, leptin infusion decreased food intake in mice. It also decreased in animals. The amplitude of the leptin effects was similar (23% decrease) in and mice. This suggests that the b-adrenoceptors do not play an essential role in driving the anorectic effects of leptin. Pair feeding in mice induced a decrease in energy expenditure, a decrease in carbohydrate disappearance rate and a trend toward an increase in fat disappearance rate. These results were expected since it is well known that food restriction induces a shift from glucose to fat utilization. It should be mentioned at this point that pair-fed mice received their food at the beginning of the dark phase. Thus, during the light phase, the pair-fed animals ran out of food and shifted their metabolism toward increased fat utilization, as compared to ad libitum-fed mice. When considering carbohydrate and fat utilization rates during the dark period only, no difference between control and food-restricted mice was observed. Pair- feeding did not change BAT UCP1 mrna expression in mice. The 23% decrease in food intake of this study is probably not sufficient to affect BAT UCP1 expression. Pair feeding, however, decreased D2 mrna expression in mice, suggesting that the expression of this gene is more 939

5 and b-adrenoceptors 940 Carbohydrate disappearance rate mg h 1 Carbohydrate disappearance rate mg h Dark period only * + Fat disappearance rate mg h 1 Fat disappearance rate mg h ** Dark period only Figure 3 (a) Average carbohydrate disappearance rate determined by indirect calorimetry from day 2 to 6 in or mice treated with saline (control, n ¼ 6 and 7), with saline and pair-fed with the leptin-treated group (, n ¼ 8 and 7) or with 1 mgh 1 of leptin (leptin, n ¼ 8 and 6). The results are expressed in milligram of carbohydrate used per hour. They are the means±s.e.m. (b) Average lipid disappearance rate determined by indirect calorimetry from day 2 to 6 in or mice treated with saline (control, n ¼ 6 and 7), with saline and pair-fed with the leptin-treated group (, n ¼ 8 and 7) or with 1 mgh 1 of leptin (leptin, n ¼ 8 and 6). The results are expressed in milligram of lipids used per hour. They are the means±s.e.m. (c) Average carbohydrate disappearance rate during the dark period determined as described above. (d) Average lipid disappearance rate during the dark period determined as described above. *Po5, **Po1 and Po05 vs corresponding controls; þ Po5 and þþþ Po05 vs corresponding., wild type. susceptible to food restriction than that of UCP1. The comparative analysis of the effects of pair feeding in and mice showed that the decreases in body weight, in energy expenditure and in 23 h (that is, hours) carbohydrate disappearance rate observed are b-adrenoceptor independent. It can therefore be concluded that the effects of food restriction on energy balance are not mediated by the b-adrenergic system. This does not exclude that the effects of a more severe food restriction than that of this study might depend on the b-adrenergic system. Concerning UCP1 in BAT, we observed that this parameter is lower in than in animals, an observation in keeping with previously reported results. 27 As was observed in mice, food restriction had no effect on BAT UCP1 expression in animals. The situation is different for BAT D2 expression, as we observed that food restriction failed to promote a decrease in this parameter in mice as it does in mice. These results indicate an involvement of the b-adrenergic system in the response of BAT D2 to caloric restriction. Concerning the effects of leptin, our study provides a series of new findings. The stimulatory effect of leptin in vivo on energy expenditure was abolished in the mice, suggesting that it is essentially b-adrenoceptor dependent.

6 UCP-1 mrna [% of control] ** οο οο οοο D2 mrna [% of control] and b-adrenoceptors Figure 4 UCP1 (a) and D2 (b) mrna expression in the BAT of or mice treated with saline (control), with saline but pair-fed with the leptin-treated group () or with 1 mgh 1 of leptin delivered subcutaneously by minipumps (leptin). Quantitative RT-PCR determinations were performed as described under Materials and methods. The results are expressed as percentage of the control mice. They are the mean±s.e.m. of arbitrary values normalized using the corresponding cyclophilin values of six mice per group. *Po5, **Po2 and Po05 vs corresponding controls; þþþ Po05 vs corresponding ; 11Po2 and 111Po05 between genotypes. BAT, brown adipose tissue; D2, type II deiodinase; UCP1, uncoupling protein-1; RT-PCR, reverse transcription PCR;, wild type * οοο οο 941 This effect might be, for a large part, mediated by an activation of BAT thermogenic genes, such as UCP1 and D2, which were also confirmed in this study to be stimulated by leptin through a mechanism involving the b-adrenergic system. Since leptin increases energy expenditure in but not in mice, it should have influenced carbohydrate or fat utilization differently in the two genotypes. However, no significant difference in the effects of leptin on either 23 h carbohydrate or fat utilization was observed between and mice. This is probably due to variance in the data and insufficient power of the technical approach used. The lack of an effect of leptin on average fat utilization in mice is surprising, but can be explained by our experimental conditions. As mentioned above, pair-fed mice were essentially food-deprived during the light phase, which was not the case for leptin-treated animals. These different nutritional patterns could have a significant impact on substrate utilization rates during the light phase and could have masked genuine effects of leptin. This is the reason why we analyzed the results obtained during the dark phase, during which all animals have free access to food. This revealed that, in mice, leptin had no effect on carbohydrate utilization, an observation which appears to be in contrast with results showing that an acute leptin administration increases glucose metabolism. 15 This discrepancy could be explained by the conditions of our experiment that consists in a long-lasting increase in circulating leptin associated with a decrease in food intake. It is not in contradiction with the observation that glucose turnover (glycogen to glucose and back to glycogen) was increased by leptin, 15 since the latter was not measured in this study. When considering the data obtained during the dark phase, we observed that leptin stimulates fat utilization and this effect seems to be b-adrenoceptor independent since, after leptin administration, fat utilization was similar in and mice. It has been shown that leptin increases fatty acid b-oxidation in skeletal muscle by an early direct activation of AMP-activated protein kinase, which is followed by a later activation of the enzyme by the sympathetic nervous system through the a-adrenoceptor. 17 The results of our study allow extrapolating from the muscle to the whole organism in vivo, showing an effect of leptin on fat utilization, that is, b-adrenoceptor independent. An explanation for this effect of leptin in the mice would be that it is either direct or mediated by the a-adrenoceptor. In conclusion, our study shows that the stimulatory effects of leptin on energy expenditure as well as on BAT UCP1 and D2 expression require functional b-adrenoceptors, but that its inhibitory effect on food intake and its stimulatory effect on fat utilization is independent of the b-adrenoceptor signaling. Acknowledgements This study was funded by the Swiss National Science Foundation Grants no. 3100AO (to FRJ), the Fondation Boninchi (to PM), the Fondation du Centenaire de la Société suisse d Assurances générales sur la vie humaine pour la santé publique et les recherches médicales (to PM) and the European Community (EC) FP6 funding (to FRJ), contract no. LSHM-CT This study was part of the Geneva Programme for Metabolic Disorders. We are very grateful to Dr Kobilka for providing us with the double KO mice. Finally, we thank Ms Marcella Klein for her excellent technical assistance.

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