The Effects of High Fat Diet and Resveratrol on Mitochondrial Activity of Brown Adipocytes

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1 Originl Article Endocrinol Metb 216;31: pissn X eissn The Effects of High Ft Diet nd Resvertrol on Mitochondril Activity of Brown Adipocytes Cheol Ryong Ku 1, Yoon Hee Cho 1, Zhen-Yu Hong 2, H Lee 1, Sue Ji Lee 1, Seung-soo Hong 1, Eun Jig Lee 1 1 Division of Endocrinology, Deprtment of Internl Medicine, Yonsei University College of Medicine, Seoul, Kore; 2 Deprtment of Medicl Oncology, The First Affilited Hospitl, Xinxing Medicl University, Weihui, Chin Bckground: Resvertrol (RSV) is polyphenolic phytolexin tht hs mny effects on metbolic diseses such s dibetes nd obesity. Given the importnce of brown dipose tissue (BAT) for energy expenditure, we investigted the effects of RSV on brown dipocytes. Methods: For the in vitro study, interscpulr BAT ws isolted from 7-week-old mle Sprgue Dwley rts. For the in vivo study, 7-week-old mle Otsuk Long Evns Tokushim Ftty (OLETF) rts were divided into four groups nd treted for 27 weeks with: stndrd diet (); +RSV (1 mg/kg body weight, dily); high ft diet (HFD); HFD+RSV. RSV ws provided vi orl gvge once dily during the in vivo experiments. Results: RSV tretment of primry cultured brown predipocytes promoted mitochondril ctivity, long with over-expression of estrogen receptor α (ER-α). In OLETF rts, both HFD nd RSV tretment incresed the weight of BAT nd the differentition of BAT. However, only RSV incresed the mitochondril ctivity nd ER-α expression of BAT in the HFD-fed group. Finlly, RSV improved the insulin sensitivity of OLETF rts by incresing the mitochondril ctivity of BAT, despite hving no effects on white dipocytes nd muscles in either diet group. Conclusion: RSV could improve insulin resistnce, which might be ssocited with mitochondril ctivity of brown dipocyte. Further studies evluting the ctivity of RSV for both the differentition nd mitochondril ctivity of BAT could be helpful in investigting the effects of RSV on metbolic prmeters. Keywords: Adipocytes, brown; Estrogen receptor lph; Diet, high-ft; Mitochondri; Resvertrol INTRODUCTION Brown dipose tissue (BAT) is profoundly involved in the regultion of energy blnce nd the control of body weight, which re medited by non-shivering thermogenesis in mmmls [1,2]. As min meditor of dptive thermogenesis, BAT is highly dependent on the ctivity of uncoupling protein 1 (UCP-1), which represents the mitochondril ctivity [3]. Received: 1 April 215, Revised: 26 December 215, Accepted: 13 Jnury 216 Corresponding uthor: Eun Jig Lee Division of Endocrinology, Deprtment of Internl Medicine, Yonsei University College of Medicine, 5-1 Yonsei-ro, Seodemun-gu, Seoul 3722, Kore Tel: , Fx: , E-mil: ejlee423@yuhs.c Previous studies hve demonstrted tht BAT is lso importnt for humn metbolism [4,5]. Resvertrol (RSV), which hs biologicl ctivities similr to those of estrogen [6], is polyphenolic phytolexin tht is known to hve severl effects on metbolic diseses, crdiovsculr disese, nd mlignncies. With respect to energy metbolism, RSV incresed het production in mice, which ws ccompnied by prolonged life Copyright 216 Koren Endocrine Society This is n Open Access rticle distributed under the terms of the Cretive Commons Attribution Non-Commercil License ( licenses/by-nc/4./) which permits unrestricted non-commercil use, distribution, nd reproduction in ny medium, provided the originl work is properly cited

2 High Ft Diet nd Resvertrol on Brown Adipocytes spn nd improved insulin sensitivity [7]. Furthermore, RSV hd inhibitory effects on dipokine expression nd secretion in humn white dipose tissue [8]. However, there hve been few reports demonstrting the exct role of RSV on BAT. Although Mirnd et l. [9] reported the poptotic role of RSV on BAT, Andrde et l. [1] demonstrted tht RSV promoted the biologicl function of BAT through incresing SIRT1 nd energy expenditure. Although white dipose tissue hs mjor functions in the development of obesity, recent studies hve demonstrted tht BAT hs n importnt role s modultor of metbolic disese. However, there re few reports on gents which could modulte the ctivity of BAT, especilly in ptients with type 2 dibetes. With this bckground, we evluted the role of RSV on the mitochondri of BAT, both in vivo nd in vitro. The protein levels of UCP-1, which represents the mitochondril ctivity of BAT, were evluted in this study. METHODS Ethics sttement This study ws conducted in ccordnce with the Institutionl Animl Cre nd Use Committee of Yonsei University Helth System bsed on the Lbortory Animl Mnul nd the Guide for the Cre nd Use of Lbortory Animls edited by the Ntionl Reserch Council of the Ntionl Acdemies. All niml studies were performed in fcilities pproved by the Assocition for Assessment nd Accredittion of Lbortory Animl Cre. Experimentl protocols were reviewed nd pproved by the Institutionl Animl Cre nd Use Committee of the Yonsei Lbortory Animl Reserch Center (permit no.: ). Primry cell isoltion nd culture Brown predipocytes were isolted from the interscpulr BAT of 4-week-old mle Sprgue Dwley rts (Orient Bio Inc., Seongnm, Kore) nd differentited s described previously [9,11]. Isolted brown predipocytes were incubted in high glucose Dulbecco s Modified Egle s Medium contining 1% ntibiotic solution nd 1% fetl bovine serum (Invitrogen, Crlsbd, CA, USA) t 37 C in humidified tmosphere with 5% CO2. For differentition, the immortlized brown dipocytes were grown in Dulbecco s Modified Egle s Medium contining 1% fetl bovine serum, 1 nm T3, nd 2 nm insulin (Boehringer-Mnnheim, Mnnheim, Germny) until 7% confluent; therefter, it ws clled differentition medium (DM). Then, the cells were cultured in DM supplemented with.5 mm isobutylmethylxnthine,.125 μm indomethcin (Sigm-Aldrich Inc., St. Louis, MO, USA), nd.5 μm dexmethsone (Sigm-Aldrich Inc.) for 2 dys; therefter, it ws clled induction medium (IM). Next, the cells were cultured in DM until they exhibited fully differentited phenotype with multiple multilobulr lipid droplets in the cytoplsm. For the in vitro study, RSV (Sigm-Aldrich Inc.) ws provided during the IM supplementtion period nd mintined therefter. RSV ws dissolved in dimethyl sulfoxide (Sigm-Aldrich Inc.) for stock solution with concentrtion of 1 mm. Animls nd tretment mngement Mle Otsuk Long Evns Tokushim Ftty (OLETF) rts (Otsuk Phrmceuticl Co. Ltd., Tokushim, Jpn), 9 weeks of ge nd weighing 29 to 32 g, were housed in n niml room controlled t 23 C±2 C nd 55%±5% room humidity under 12-hour light/12-hour drk cycle, nd given tp wter d libitum. Animls were seprted into four groups: (1) stndrd chow (stndrd diet []) diet-fed with sline tretment (n=7); (2) diet-fed with RSV (Federl Lbortories Corp., Alden, NY, USA) tretment (n=8); (3) high ft diet (HFD)-fed with sline tretment (n=7); nd (4) HFD-fed with RSV tretment (n=8). RSV t dose of 1 mg/kg dissolved in 1.5 ml sline, or the sme volume of sline, ws dministered vi orl gvge once dily for 27 weeks. diet ws consisted of 24% protein, 65% crbohydrte, nd 12% ft (PicoLb Rodent Diet 2, Lb- Diet, St. Louis, MO, USA). The cloric content of the HFD ws 17% protein, 43% crbohydrte, nd 41% ft (RD Western Diet, D1279B, Reserch Diets Inc., New Brunswick, NJ, USA). Body weights nd fsting glucose were checked weekly. Insulin tolernce tests were conducted every month. After 27 weeks of tretment with RSV or sline, OLETF rts were scrificed nd their plsm ws collected. Furthermore, ech orgn ws weighed, including the hert, liver, kidney, epididyml ft, nd BAT. BAT ws isolted from ech rt nd prepred for Western blotting nlysis nd histopthologicl evlution s described previously [12]. Plsm leptin nd diponectin levels were mesured with n enzyme immunossy (Mouse/Rt Adiponectin enzyme-linked immunossy kit, BioTrder Inc., Seoul, Kore). Immunoblotting Cell lystes were prepred nd subjected to Western blot nlysis. Membrnes were immunoblotted with primry ntibodies for UCP-1 (Snt Cruz Biotechnology, Snt Cruz, CA, USA), AMP-ctivted protein kinse (AMPK, Cell Signling Tech- Copyright 216 Koren Endocrine Society 329

3 Ku CR, et l. nologies, Dnvers, MA, USA), nd estrogen receptor α (ER-α, Snt Cruz Biotechnology). Peroxidse-conjugted nti-rbbit or nti-mouse ntibodies were used s secondry ntibodies (Thermo Fisher Scientific Inc., Wlthm, MA, USA). Oil-Red-O stining Dishes were wshed with phosphte-buffered sline nd fixed with 1% buffered formlin for 16 hours t 4 C. Then, cells were stined for 4 hours t room temperture with Oil-Red-O solution (5 g/l in isopropyl lcohol), wshed five times with wter, nd visulized. Sttisticl nlysis Dt were nlyzed using Mnn-Whitney tests. All sttisticl nlyses were performed using SPSS version 18. (SPSS Inc., Chicgo, IL, USA). All sttisticl tests were two-tiled, nd P<.5 were considered significnt. RESULTS Resvertrol promotes the differentition of brown predipocytes in the erly phse To evlute the effect of RSV on the differentition of brown predipocytes, RSV ws provided together with the IM nd mintined therefter. Oil-Red-O stining reveled tht RSV promoted the differentition of predipocytes in dose-dependent mnner (Fig. 1A). In seprte experiment to evlute the role of RSV in brown dipogenesis, RSV tretment ws performed t ech step promoting the differentition of brown predipocytes t fixed concentrtion of 1 μm. However, there ws no effect of RSV on brown dipogenesis when RSV tretment ws performed fter the period of IM supplementtion. When RSV ws dministered simultneously with IM nd mintined therefter, Western blotting reveled tht the expression of UCP-1, phospho-ampk, nd ER-α hd incresed (Fig. 1B). Similr to the results of the Oil-Red-O stining, no effect of RSV tretment on the expression of these proteins ws found fter IM supplementtion. Resvertrol improves insulin sensitivity in OLETF rts, without ffecting white dipocytes OLETF rts were treted with RSV nd fed the or HF diet for 27 weeks. Although significnt reduction in body weight ws observed fter 12 weeks of RSV tretment, the difference diminished therefter (Fig. 2A). However, insulin tolernce tests reveled tht RSV tretment improved insulin sensitivity in both diet groups (Fig. 2B). There were no differences in the histologies nd orgn weights of the hert, liver, epididyml ft, retroperitonel ft, gstrocnemius muscle, nd soleus muscle fter the RSV tretment (Fig. 2C, dt not shown). Further- RSV ( ) 1 μm 5 μm 1 μm UCP-1 Control 1 4 p-ampk AMPK RSV 5 μm 1 4 ER-α β-actin RSV 1 μm 1 4 A B Fig. 1. Effects of resvertrol (RSV) on the differentition of brown dipocytes. (A) Oil-red-O stining of primry cultured brown predipocytes. RSV tretment ws performed with induction medium t doses of 5 nd 1 μm nd mintined therefter. Adipogenesis ws significntly incresed with RSV tretment. (B) Western blotting for mitochondril ctivity nd estrogen receptor α (ER-α) in brown dipocytes. Brown predipocytes were cultured nd differentited with or without RSV tretment. The protein lystes were collected fter the differentition of brown dipocytes ws confirmed. UCP-1, uncoupling protein 1; p-ampk, phospho-amp-ctivted protein kinse; AMPK, AMP-ctivted protein kinse Copyright 216 Koren Endocrine Society

4 High Ft Diet nd Resvertrol on Brown Adipocytes 9 +RSV HF HF+RSV 8 Body weight (g) Weeks of RSV tretment since 9 weeks old A 3 +RSV HF HF+RSV Blood glucose (mg/dl) Time (min) Glucose (mg/dl/min) RSV HF HF+RSV AUC (mg/dl/min) 18, 12, 6, +RSV HF HF+RSV B Weight (g) RSV HF HF+RSV +RSV HF HF+RSV Hert Kidney Epididyml ft Liver C Leptin (pg/ml) Adiponcetin (μg/ml) D Fig. 2. Effects of resvertrol (RSV) on metbolic prmeters in Otsuk Long Evns Tokushim Ftty (OLETF) rts. (A) Body weight chnges fter RSV tretment of OLETF rts. RSV hd been provided since the ge of 9 weeks nd mintined for 27 weeks. (B) Insulin tolernce test of OLETF rts t 35 weeks of ge. (C) Orgn weights t 36 weeks of ge. (D) Chnges in leptin nd diponectin levels. Serum smples from 36-week-old rts were collected nd evluted with n enzyme-linked immunossy kit., stndrd diet; HF, high ft; AUC, re under the curve. P vlue below.5 with tretment of RSV in ech diet group (Mnn-Whitney U test). Copyright 216 Koren Endocrine Society 331

5 Ku CR, et l. RSV ( ) RSV (+) 8 HFD No. of brown ipocytes A +RSV HF HF+RSV B 1.8 RSV UCP-1 p-ampk ER-α HFD ( ) (+) ( ) (+) Reltive expression of UCP GAPDH C.2 +RSV HF HF+RSV D Fig. 3. Effects of resvertrol (RSV) on the differentition nd ctivtion of brown dipocytes in Otsuk Long Evns Tokushim Ftty (OLETF) rts. (A) Hemtoxylin nd eosin stining for brown dipocytes. Brown dipose tissue ws collected from OLETF rts t 36 weeks of ge. With the tretment of RSV nd feeding of the high ft diet (HFD), the differentition of brown dipocytes incresed. (B) The number of brown dipocytes visible in the high-power field incresed significntly with RSV tretment nd HFD feeding. The verge number of brown dipocytes ws clculted t four sites of ech section. The number of rts in ech group is described in the Methods. (C) Western blotting for mitochondril ctivity nd estrogen receptor α (ER-α). (D) Reltive expression in Western blotting, determined using densitometer. All protein lystes were evluted nd compred with those of rts on the stndrd diet., stndrd diet; HF, high ft; UCP-1, uncoupling protein 1; p-ampk, phospho-amp-ctivted protein kinse; GAPDH, glycerldehyde 3-phosphte dehydrogense. P vlue below.5 with tretment of RSV in ech diet group (Mnn-Whitney U test). more, RSV tretment did not lter the serum levels of leptin nd diponectin in either diet group (Fig. 2D). Resvertrol ccelertes the ctivity of brown dipocytes in obese rts Histologicl nlysis reveled tht RSV prevented the deposit formtion of lipid droplets in ll of the spces between the brown dipocytes of OLETF rts. The OLEFT rts fed the HFD hd more brown dipocytes thn those fed the diet. Furthermore, RSV tretment of OLETF rts fed the HF diet incresed the number of brown dipocytes, s confirmed by H&E stin (Fig. 3A, B). However, nlysis of protein lystes from the BAT of OLETF rts indicted tht RSV incresed the expression of UCP-1 nd ER-α only in the HFD-fed group (Fig. 3C). Although the histology of BAT reveled n incresed number of brown dipocytes in HFD-fed OLETF rts, the protein expression of UCP-1 ws reduced, wheres it ws restored in HFD-fed rts treted with RSV (Fig. 3D). DISCUSSION Adipose tissues, both white nd brown, ply pivotl roles in energy homeostsis. While white dipocytes store energy nd cuse obesity, BAT induces non-shivering thermogenesis nd Copyright 216 Koren Endocrine Society

6 High Ft Diet nd Resvertrol on Brown Adipocytes consumes excess clories [13]. Given the role of BAT in energy expenditure, severl studies hve suggested BAT s new therpeutic trget for obesity. For thermogenesis, BAT utilizes unique protein, UCP-1, which is locted in the inner mitochondril membrne nd uncouples mitochondril respirtion, leding to the relese of energy s het [14]. Together with UCP-1, peroxisome prolifertor-ctivted receptor coctivtor 1α (PGC-1α) hs been show to express multiple physiologic roles for mitochondril biogenesis nd ctivity [15]. Severl studies hve demonstrted tht PGC-1α were expressed in BAT nd were dysregulted in obesity, dibetes nd neurodegenertion. There re severl physiologic regultors of the differentition nd ctivity of BAT, including cold exposure, the sympthetic nervous system, the β-drenergic system, ctecholmines, thyroid hormones, nutrition, nd sex hormones [13]. In this study, we confirmed tht over-nutrition nd RSV ply importnt roles in the mitochondril differentition of brown dipocytes. The HFD itself ugmented the number of brown dipocytes in OLETF rts. There hve been debtes bout the chnges in brown dipocytes during the development of obesity. In some studies, both impired mitochondril ctivity nd reduced mount of brown dipocytes were observed in obese niml models nd humn ptients [4,16,17]. On the other hnd, other studies hve found tht the differentition of brown dipocytes correlted with incresed supplement of nutrition [18]. In this study, we used OLETF rts to confirm the role of brown dipocytes in type 2 dibetes combined with obesity. OLETF rts were generted with inctive cholecystokinin-1 receptors on the bckground of Long-Evns Tokushim Otsuk rts, leding to the phenotype of type 2 dibetes with mild obesity. To eliminte the genetic influence of OLETF rts on brown dipocyte, the brown predipocytes were isolted from Sprgue Dwley rts for in vitro study. As described in this study, the HFD led to significnt increse in body weight nd induced the differentition of brown dipocytes. However, the HFD itself did not ffect the mitochondril ctivity of brown dipocytes. Although we could not find the exct mechnism, impired biogenesis could hve reduced mitochondril ctivity, despite the incresed differentition of BAT fter HFD intke [19]. Mny studies hve reported the effects of RSV on obesity [2-22]. Although most of them hve focused on chnges in white dipocyte, few recent studies focused on the role of BAT or the browning of white dipocyte [23,24]. They evluted the development of brown-dipocyte-like chrcteristics in white dipocytes, through the min mechnism of SIRT1 ctivtor combined with phytoestrogen ctivity [25]. Furthermore, Mirnd et l. [9] reported tht RSV leded Akt/protein kinse B nd foxo1 dephosphoryltion/decetyltion nd induced poptosis of brown dipocyte. In the present study, RSV restored both the differentition nd ctivtion of BAT nd incresed the expression of ER-α, especilly in the HFD group. Considering tht sex-dependent difference in BAT expression ws reported even in humn subjects, nd RSV tretment of postmenopusl women hd fvourble effects on estrogen-ssocited metbolism [26-28], RSV might modulte the ctivity of brown dipocytes through the estrogen pthwy. However, RSV hd no effects on white dipocytes, which were suggested by previous studies to be the trget of the nti-obesity effects of RSV [29,3]. In our obesity-relted inflmmtory mrker nlysis, there were no differences in the levels of leptin nd diponectin following RSV tretment. Considering tht the histologies nd orgn weights of the hert, liver, epididyml ft, retroperitonel ft, gstrocnemius muscle, nd soleus muscle did not differ with the RSV tretment, improved insulin sensitivity my hve been chieved through the incresed differentition nd ctivtion of BAT. The differences in body weight were diminished fter 19 weeks of ge in the OLETF rts, possibly due to the reltively long study durtion, the high dose of the HFD tretment, nd differences in niml species for type 2 dibetes. Andrde et l. [1] demonstrted tht erly tretment of RSV on mice fed with incresed BAT thermogenesis mrkers by incresing SIRT1 nd energy expenditure. However, Mirnd et l. [9] reported tht RSV induced poptosis of poptosis. The differences might be resulted from the time-point of RSV tretment. In our study, the effect of RSV on brown dipogenesis ws confirmed t ech step during in vitro study. Although RSV tretment ws performed t different stges of dipogenesis, only the erly tretment before the ppliction of IM significntly ffected the differentition nd ctivity of BAT. This suggests tht RSV tretment might hve role in the erly development of brown dipocytes with more prominent mitochondril ctivities thn those induced by obesity. There re severl limittions of current study. Although mitochondril ctivities were evluted by UCP-1, the role of RSV on mitochondril biogenesis of BAT, such s PGC-1α, ws not demonstrted. Furthermore, the direct mechnism of RSV involving mitochondril ctivtion of BAT ws not clrified. Finlly, the effect of RSV on BAT thermogenesis ws not defined in in vivo condition using obesity niml model. However, we identified the reltionship between RSV nd mitochondril ctivities of BAT in current study, both from in vitro Copyright 216 Koren Endocrine Society 333

7 Ku CR, et l. nd in vivo conditions. In conclusion, we confirmed tht RSV might hve role on promotion of mitochondril ctivity in BAT, both in vitro nd in vivo. In the in vivo study, RSV restored the impired mitochondril ctivity induced by the HFD, incresed the expression of ER-α, nd improved insulin sensitivity. Further studies evluting the reltionship between mitochondril ctivity nd ER-α could be helpful in understnding the nti-obesity effects of RSV. CONFLICTS OF INTEREST No potentil conflict of interest relevnt to this rticle ws reported. ACKNOWLEDGMENTS This work ws supported by grnt of the Kore Helth Technology R&D Project through the Kore Helth Industry Development Institute (KHIDI), funded by the Ministry of Helth nd Welfre, Republic of Kore (grnt number: HI13C423 [to EJL]). ORCID Cheol Ryong Ku REFERENCES 1. Nicholls DG, Locke RM. Thermogenic mechnisms in brown ft. Physiol Rev 1984;64: Tryhurn P, Milner RE. A commentry on the interprettion of in vitro biochemicl mesures of brown dipose tissue thermogenesis. Cn J Physiol Phrmcol 1989;67: Nedergrd J, Golozoubov V, Mtthis A, Asdi A, Jcobsson A, Cnnon B. UCP1: the only protein ble to medite dptive non-shivering thermogenesis nd metbolic inefficiency. Biochim Biophys Act 21;154: Cypess AM, Lehmn S, Willims G, Tl I, Rodmn D, Goldfine AB, et l. Identifiction nd importnce of brown dipose tissue in dult humns. N Engl J Med 29;36: Sito M, Okmtsu-Ogur Y, Mtsushit M, Wtnbe K, Yoneshiro T, Nio-Kobyshi J, et l. High incidence of metboliclly ctive brown dipose tissue in helthy dult humns: effects of cold exposure nd diposity. Dibetes 29; 58: Cos P, De Bruyne T, Apers S, Vnden Berghe D, Pieters L, Vlietinck AJ. Phytoestrogens: recent developments. Plnt Med 23;69: Bur JA, Person KJ, Price NL, Jmieson HA, Lerin C, Klr A, et l. Resvertrol improves helth nd survivl of mice on high-clorie diet. Nture 26;444: Olholm J, Pulsen SK, Cullberg KB, Richelsen B, Pedersen SB. Anti-inflmmtory effect of resvertrol on dipokine expression nd secretion in humn dipose tissue explnts. Int J Obes (Lond) 21;34: Mirnd S, Gonzlez-Rodriguez A, Revuelt-Cervntes J, Rondinone CM, Vlverde AM. Beneficil effects of PTP1B deficiency on brown dipocyte differentition nd protection ginst poptosis induced by pro- nd nti-inflmmtory stimuli. Cell Signl 21;22: Andrde JM, Frde AC, Guimres JB, Freits KM, Lopes MT, Guimres AL, et l. Resvertrol increses brown dipose tissue thermogenesis mrkers by incresing SIRT1 nd energy expenditure nd decresing ft ccumultion in dipose tissue of mice fed stndrd diet. Eur J Nutr 214;53: Klein J, Fsshuer M, Ito M, Lowell BB, Benito M, Khn CR. Bet(3)-drenergic stimultion differentilly inhibits insulin signling nd decreses insulin-induced glucose uptke in brown dipocytes. J Biol Chem 1999;274: Kim WK, Choi HR, Prk SG, Ko Y, Be KH, Lee SC. Myosttin inhibits brown dipocyte differentition vi regultion of Smd3-medited bet-ctenin stbiliztion. Int J Biochem Cell Biol 212;44: Lee P, Swrbrick MM, Ho KK. Brown dipose tissue in dult humns: metbolic renissnce. Endocr Rev 213; 34: Cnnon B, Nedergrd J. Brown dipose tissue: function nd physiologicl significnce. Physiol Rev 24;84: Hndschin C, Spiegelmn BM. Peroxisome prolifertor-ctivted receptor gmm coctivtor 1 coctivtors, energy homeostsis, nd metbolism. Endocr Rev 26;27: Vegiopoulos A, Muller-Decker K, Strzod D, Schmitt I, Chichelnitskiy E, Ostertg A, et l. Cyclooxygense-2 controls energy homeostsis in mice by de novo recruitment of brown dipocytes. Science 21;328: Lowell BB, S-Susulic V, Hmnn A, Lwitts JA, Himms-Hgen J, Boyer BB, et l. Development of obesity in trnsgenic mice fter genetic bltion of brown dipose tissue. Nture Copyright 216 Koren Endocrine Society

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