beta 3-Adrenergic agonist induces a functionally active uncoupling protein in fat and slow-twitch muscle fibers

Toshihide Yoshida1, Tsunekazu Umekawa1, Kenzo Kumamoto4, Naoki Sakane1, Akinori Kogure1, Motoharu Kondo1, Yasuo Wakabayashi2, Teruo Kawada3, Itsuro Nagase5, and Masayuki Saito5

1 First Department of Internal Medicine and 2 Department of Biochemistry, Kyoto Prefectural University of Medicine, Kyoto 602; 3 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-01; 4 Department of Anatomy, Meiji College of Oriental Medicine, Hiyoshi, Kyoto 629-03; and 5 Laboratory of Biochemistry, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The mitochondrial uncoupling protein (UCP) has usually been found only in brown adipose tissue. We recently observed that a chronic administration of the beta 3-adrenergic agonist CL-316,243 (CL) induced the ectopic expression of UCP in white fat and skeletal muscle in genetic obese yellow KK mice. The aim of the present study was to examine whether UCP could be induced in nongenetic obese animals produced by neonatal injections of monosodium L-glutamate (MSG). The daily subcutaneous injection of CL (0.1 mg/kg) to MSG-induced obese mice for 2 wk caused significant reductions of body weight (15%) and white fat pad weight (58%). Northern and Western blot analyses showed that CL induced significant expressions of UCP in the white fat and muscle, as well as in brown fat. Immunohistochemical observations revealed that the UCP stains in white fat were localized on multilocular cells and that those in muscle were localized on slow-twitch fibers rich in mitochondria. Immunoelectron microscopy confirmed the mitochondrial localization of UCP in the myocytes. The guanosine 5'-diphosphate (GDP) binding to mitochondria in brown fat doubled after the CL treatment. Moreover, significant GDP binding was detected in the white fat and muscle of the CL-treated mice, at about one-fourth and one-thirteenth the activity of brown fat, respectively, suggesting that ectopically expressed UCP is functionally active. We concluded that the beta 3-adrenergic agonist CL can induce functionally active UCP in white fat and slow-twitch muscle fibers of obese mice.

CL-316,243; guanosine 5'-diphosphate binding; monosodium L-glutamate-induced obese mice; white fat; gastrocnemius muscle

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THERMOGENESIS in brown adipose tissue (BAT) is regulated by sympathetic nerves distributed to this tissue principally through the beta -adrenergic action of norepinephrine (12). There are three isoforms of the beta -adrenergic receptor (AR) in brown adipocytes: beta 1-, beta 2-, and beta 3-AR (16). Because the beta 3-AR is expressed primarily, but not exclusively, in brown and white adipocytes, it has been expected that an agonist to the beta 3-AR, which would be a selective stimulant of lipolysis and BAT thermogenesis, might be useful as an antiobesity drug (1, 10, 20). In fact, various types of beta 3-AR agonists with different selectivities and potencies have been developed and confirmed to be effective in stimulating lipolysis in adipocytes in vitro and in reducing adipocity in vivo in experimental animals (3, 26, 27, 30). It has also been shown that beta 3-AR agonists are capable of inducing mitochondrial uncoupling protein (UCP), a key molecule in thermogenesis, in rodent (13, 27) and canine (6) BAT. We (21) recently demonstrated that the chronic treatment of genetically obese yellow KK mice with CL-316,243 (CL), a highly selective beta 3-AR agonist, induces UCP expression not only in BAT but also in various fat pads usually considered white adipose tissue (WAT), and even in skeletal muscle. These results, in contrast with the well-accepted view of the specific expression of UCP in BAT (12), suggest that such an ectopic expression of UCP in tissues other than typical BAT may also contribute to some degree to the potent antiobese effect of the beta 3-AR agonists. However, in human studies, especially, it has previously been shown that sympathomimetic stimulation results not in BAT thermogenesis but rather in a significant increase in skeletal muscle thermogenesis (2), and a significant part of this thermogenic response has subsequently been shown to be mediated by beta 3-adrenoceptors (18). Therefore, it remains to be determined whether or not the ectopically expressed UCP is an exceptional phenomenon found only in this genetically obese strain and whether or not the ectopically expressed UCP is functionally active. In the present study, we investigated 1) the effects of chronic CL treatment on the UCP expression in various fat tissues and skeletal muscle in nongenetic hypothalamic obese mice produced by monosodium L-glutamate (MSG) injection, and 2) the activity of UCP in the fat pads and skeletal muscle estimated by guanosine 5'-diphosphate (GDP) binding (14, 15, 22) to mitochondria, a reliable biochemical index of the thermogenic activity of UCP. We also examined the immunohistochemical localization of UCP, particularly focusing on UCP in skeletal muscle.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Chemicals. CL-316,243, disodium (R,R)-5{[2-(3-chlorophenyl)-2-hydroxyethyl]-aminopropyl}-1,3-benzodioxole-2,2-dicarboxylate (3), was provided by American Cyanamid (Pearl River, NY).

Animals. Immediately after birth and for 5 consecutive days, seventy-two ICR female mice (Charles River Japan, Tokyo) received daily subcutaneous injections of MSG (Wako Pure Chemical, Osaka, Japan) dissolved in physiological saline at a concentration of 2 mg/g (28-30), which made these mice obese. Another 72 control female animals received physiological saline in a similar manner (lean control mice). The mice were kept in a controlled temperature room (22 ± 2°C) with artificial light for 12 h a day, and they received commercial powdered chow (Charles River Japan) and tap water ad libitum. At 6 wk after treatment, the MSG obese group and the lean control group were each further divided into two groups; some mice were given a daily subcutaneous injection of CL (0.1 mg/kg) (30) for 2 wk, and the other mice were given a daily subcutaneous injection of distilled water (vehicle) for 2 wk. Thus four groups were examined: 1) 36 CL-treated MSG obese mice, 2) 36 distilled water-treated MSG obese mice, 3) 36 CL-treated lean control mice, and 4) 36 distilled water-treated lean control mice. At the end of 2 wk, body weights of the mice were measured. These mice were then killed by cervical dislocation, and the skeletal muscle (gastrocnemius), interscapular BAT, and inguinal and retroperitoneal WATs were rapidly removed in their entirety. Some of these tissues were frozen in liquid nitrogen for Western blot and RNA analyses. Other sets of these tissues were obtained and treated by the methods described below for immunohistochemistry and electron-microscopic observations, and some parts of the gastrocnemius muscles, interscapular BATs, and inguinal and retroperitoneal WATs were prepared for the assay of GDP binding by the method described below. The animal care and experimental procedures were approved by the Animal Care and Use Committee of the Kyoto Prefectural University of Medicine.

Western blot analysis. Each tissue (6 individual samples in each group) was homogenized in 5-10 volumes of a solution containing 10 mM tris(hydroxymethyl)aminomethane · HCl and 1 mM EDTA (pH 7.4) for 30 s with a polytron. After centrifugation at 1,500 g for 5 min, the fat cake was discarded, and the infranant (fat-free extract) was used for the Western blot analysis of UCP, as described previously (24, 25). Briefly, the fat-free extract (10-40 µg protein) was solubilized, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to a nitrocellulose filter. After the filter was blocked with 6% nonfat dry milk, it was incubated with a rabbit antiserum (×1,000 diluted) against rat UCP purified from interscapular BAT (16). The filter was then incubated with 125I-labeled protein A (ICN, Irvine, CA). The dry blot was exposed to an X-ray film for autoradiography or an imaging plate of the BAS1000 system (Fuji Film, Tokyo).

RNA analysis. Total RNA (6 individual samples in each group) was extracted from 0.1-1 g tissue using TRIzol (GIBCO-BRL, Tokyo). For Northern blot analysis, 20 or 40 µg total RNA were electrophoresed on a 1.0% agarose-formaldehyde gel and transferred to and fixed on a nylon membrane. A 488-bp cDNA probe corresponding to the coding region of rat UCP was prepared by digestion of whole UCP cDNA (a gift from Dr. D. Ricquier, Centre National de la Recherche Scientifique, Meudon, France) by BamH I and labeling by random priming with [alpha -32P]dCTP (ICN). The blots were hybridized to the labeled probe at 42°C for 20 h in the presence of 500 µg/ml salmon sperm DNA and were exposed to an X-ray film.

Immunohistochemistry. Six mice, each treated with CL or distilled water from both the MSG and saline control groups, were anesthetized with diethyl ether and perfused transcardially with 4% paraformaldehyde in phosphate-buffered saline (PBS), and the fat pads and gastrocnemius muscles were removed and fixed in Bouin solution. The fixed tissues were dehydrated in ethanol, paraffin embedded, and cut into 7-mm-thick sections. The dewaxed sections were incubated in 0.3% hydrogen peroxide in methanol to inhibit endogenous peroxidase activity, and then with 10% normal goat serum, the rabbit antiserum against rat UCP (×500 diluted), goat anti-rabbit immunoglobulin G (IgG, ×400 diluted; Vector, Burlingame, CA), and finally with avidin-biotin-peroxidase complex (Vector) according to the conventional avitin-biotin complex method. The sections were also counterstained with hematoxylin and examined by a light microscope. To determine whether the increased expression of UCP in muscle is found in slow-twitch fibers, a monoclonal antibody (MEDAC Diagnostika, Hamburg, Germany), which specifically recognizes slow-type myosin heavy chain (8), was used and stained by the methods described elsewhere (8, 9) with the muscles (n = 6 each) of mice treated with CL or distilled water.

For the electron-microscopic observations, specimens of gastrocnemius muscle fixed in 1% glutaraldehyde-0.1 M phosphate buffer (pH 7.4) from six mice of each of the four groups were dehydrated in dimethyl formamide and embedded in Lowicry K4M (Chemische Werke Lowi, Waldkaiburg, Germany) in gelatin capsules polymerized by ultraviolet irradiation. Ultrathin sections were mounted on nickel grids, immersed in 2% normal goat serum in PBS for 1 h, and treated with the rabbit antiserum against rat UCP (×100 diluted) in PBS containing 1% bovine serum albumin (BSA) overnight in a moist chamber at 4°C. After being washed well with PBS, the sections were treated with colloidal gold-labeled anti-rabbit IgG F(ab')2 (10 nm; Bio Cell, Cardiff, UK) diluted at 1:15 in PBS containing 1% BSA for 2 h at room temperature, postfixed with 2.5% glutaraldehyde for 10 min, and stained with uranyl acetate for 15 min and lead citrate for 1 min. In the controls, nonimmune rabbit serum was substituted for the primary antiserum.

GDP binding. Interscapular BAT, inguinal and retroperitoneal WATs, and gastrocnemius muscle samples (6 individual samples from each group) were rapidly removed after cervical dislocation, weighed, and placed in ice-cold sucrose buffer. For the preparation of mitochondria, samples were homogenized in an ice-cold medium (pH 7.2) containing 250 mM sucrose and 5 mM N-tris (hydroxy-methyl)-2-aminoethanesulfonic acid (TES). The mitochondria in the sample were isolated by differential centrifugation according to the procedure described by Cannon and Lindberg (5). The mitochondrial protein content was estimated by the method of Lowry and Lindberg (19). Mitochondrial GDP binding was determined by the method of Nicholls (22). The mitochondria were incubated at 20°C in 0.5 ml of medium containing 48,100 Bq of [3H]GDP, 4,551 Bq of [14C]sucrose, 100 mM potassium atractyloside, 20 mM TES (pH 7.1), 10 mM choline chloride, and 5 mM rotenone. After a 7-min incubation, 0.4 ml aliquots containing 0.26 mg of mitochondrial protein were withdrawn and filtered through a nitrocellulose membrane filter with a pore size of 0.45 mm (Sartorius, Gottingen, Germany). The 3H and 14C radioactivities of the filters were measured by scintillation spectrometry (Packard, Downers Grove, IL). To calculate the volume of the medium trapped on the filter, [14C]sucrose was included.

Data analysis. Values are expressed as means ± SE. Data were analyzed by one-way or two-way analysis of variance (ANOVA). After justification of the ANOVA, the Bonferroni t-test was performed. Representative results are shown in Figs. 1-6.

    RESULTS
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Abstract
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Methods
Results
Discussion
References

MSG obese mice and lean control mice were given CL (0.1 mg · kg-1 · day-1) or distilled water once a day. After 2 wk, the MSG obese mice treated with CL weighed significantly less than those given distilled water and had smaller white fat pads, as shown in Table 1. The CL treatment also produced a hypertrophy of the interscapular BAT, with an ~1.3-fold increase in the mitochondrial protein content (P < 0.05), but had no effects on the tissue weight or protein content of the gastrocnemius muscle. Similar effects of CL were also observed in the lean control mice, but to a lesser extent.

                              
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Table 1.   Body and mass weights, mitochondrial protein, and specific GDP binding in mitochondria of IBAT, WAT, and gastrocnemius muscle of MSG-induced obese mice and lean controls treated with CL-316,243 for 2 wk

The presence of UCP and its mRNA was detected by Western and Northern blot analyses of samples obtained from various regions of the fat pads and also from skeletal muscle. As shown in Fig. 1, the Northern blot analysis detected no UCP mRNA in any of the samples except the BAT in the two groups of mice given distilled water. In contrast, in the MSG obese mice treated with CL, a clear signal corresponding to UCP mRNA was detected not only in the BAT but also in the inguinal and retroperitoneal fat pads. In addition, a similar band was also detected in the gastrocnemius muscle. In the lean control mice given CL, a signal was detected in the inguinal fat pads in addition to the BAT. The Western blot analysis also revealed the presence of UCP in the inguinal and retroperitoneal fat pads and the gastrocnemius muscle of the MSG obese mice treated with CL (Fig. 2). The UCP protein in muscle was not detected in the lean control mice, regardless of whether they were given CL.


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Fig. 1.   Northern blot analysis of uncoupling protein (UCP) mRNA of fat pads and muscles. Obese monosodium L-glutamate (MSG) and lean control mice were given CL-316,243 (CL) or distilled water (Dist.) for 2 wk, and the total RNA [20 µg from brown adipose tissues (BAT), 40 µg from other tissues] was used for Northern blot analysis. Results are representative of 5 independent samples. WAT, white adipose tissue.


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Fig. 2.   Western blot analysis of UCP. Mice were treated as described in Fig. 1, and fat-free extracts containing 10 µg protein of BAT, 20 µg of WAT, and 40 µg of muscle were used for Western blot analysis. Results are representative of 3 independent samples.

The morphological characteristics of the fat pads expressing UCP were examined immunohistochemically. Macroscopically, the inguinal and retroperitoneal fat pads of the CL-treated MSG obese mice were less pale than those of the distilled water-treated mice. Histochemically, they contained many multilocular cells that were positive for UCP (Fig. 3). Unilocular cells were also present in the fat pads but were always negative for UCP, and their size was smaller than the typical unilocular cells found in the distilled water-treated mice. The fat pads of the distilled water-treated mice were occupied mostly by unilocular cells, filled with a single large lipid droplet, which were always negative for UCP. Thus the CL-treated mice had many typical brown adipocytes in the fat pads usually considered WAT. The localization of UCP in the gastrocnemius muscle of the CL-treated MSG obese mice was also examined at both the light- and electron-microscopic levels. As shown in Fig. 4, the muscle tissue was clearly stained with anti-UCP serum, the positive regions being in the myocytes. No stains were detected when a preimmune serum or the anti-UCP serum pretreated with purified UCP was used. The muscle tissues from the distilled water-treated MSG obese mice and lean control mice were always negative for UCP (data not shown). The mouse gastrocnemius muscle contains both slow-twitch oxidative and fast-twitch glycolytic muscle fibers, whose mitochondrion contents are high and low, respectively. To determine the type of fiber expressing UCP, the muscle tissue was stained with a monoclonal antibody that specifically recognizes the myosin heavy chain of the slow-twitch fiber. As shown in Fig. 5, the muscle fibers positive for UCP were always stained by this monoclonal antibody, suggesting that UCP is localized in the mitochondria in slow-twitch fibers. The intracellular localization of UCP in myocytes was also determined by electron microscopy by use of a gold-labeled anti-UCP antiserum. UCP-gold particles were localized predominantly on mitochondria of the myocytes of the CL-treated MSG obese mice (Fig. 6), whereas no gold particles were found in the other mice (data not shown).


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Fig. 3.   Immunohistochemical detection of UCP in white fat pads. Sections of an inguinal fat pad of an obese MSG mouse treated with CL (A) or distilled water (B) were stained by a rabbit antiserum against rat UCP (×80). Results are representative of 3 mice. Arrows show multilocular cells.


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Fig. 4.   Immunohistochemical detection of UCP in skeletal muscle. Gastrocnemius muscle of an obese MSG mouse treated with CL was stained by a rabbit antiserum against rat UCP (A). Sections of gastrocnemius muscle were also stained with hematoxylin (B). Arrows show typical UCP signal in myocytes (×80). Results are representative of 3 mice.


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Fig. 5.   Detection of slow-twitch fibers in gastrocnemius muscle of an obese MSG mouse treated with CL using anti-myosin heavy chain. Results are representative of 3 mice. A: anti-UCP stain; B: anti-myosin heavy chain. Bar, 20 µm.


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Fig. 6.   Immunoelectron microscopy of UCP in skeletal muscle. An ultrathin section of gastrocnemius muscle of an obese MSG mouse treated with CL was stained by a rabbit antiserum against rat UCP and colloidal gold-labeled anti-rabbit immunoglobulin G F(ab')2 (10 nm). Results are representative of 3 mice. M, mitochondria; MF, myofibril; Z, Z line; G, glycogen particles. Bar, 0.1 mm.

The activity of UCP in the fat pads and muscle of the MSG obese mice was estimated from the specific GDP binding to the mitochondrial fractions prepared from these tissues. As shown in Table 1, the GDP binding to the mitochondrial fraction of the interscapular BAT was 114.5 ± 8.9 pmol/mg mitoprotein in the distilled water-treated MSG obese mice; it increased by 2.3-fold in the CL-treated obese mice (266.3 ± 10.3 pmol/mg). The mitochondrial preparations from the inguinal and retroperitoneal fat pads and those from the gastrocnemius muscle showed undetectable GDP binding (<10 pmol/mg mitoprotein) in the distilled water-treated mice, but in the CL-treated MSG obese mice, significant binding values of 66.9 ± 7.1, 55.0 ± 9.3, and 20.4 ± 3.8 pmol/mg were observed in the inguinal and retroperitoneal WATs and gastrocnemius muscle, respectively, and in the CL-treated lean mice at 50.8 ± 7.6, 32.7 ± 7.5, and 12.1 ± 3.1, respectively (Table 1).

    DISCUSSION
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Introduction
Methods
Results
Discussion
References

The present results showed that the chronic treatment of nongenetic hypothalamic MSG obese mice with a selective beta 3-AR agonist, CL, resulted in the significant expression of UCP in the inguinal and retroperitoneal fat pads (which are conventionally considered white adipose tissue) and also in skeletal muscle. These results are similar to those obtained with genetic obese yellow KK mice (21), confirming the ectopic expression of UCP after chronic treatment with a beta 3-AR agonist, regardless of the type of obesity. A similar occurrence of UCP in various fat pads other than typical BAT was reported in rats exposed to cold (7) or treated with CL (13, 27), and also in dogs treated with another beta 3-AR agonist, ICI-D7114 (6). The present immunohistochemical examinations showed that the UCP-expressing fat pads contained numerous multilocular cells indistinguishable from typical brown adipocytes. The fat pads also contained many typical unilocular "white" fat cells, but they were always negative for UCP signals. Thus the beta 3-AR agonist treatment resulted in the appearance of "brown" adipocytes expressing UCP in adipose deposits usually considered white fat.

As in yellow KK mice (21), the CL-induced ectopic expression of UCP was more obvious in the MSG obese mice than in the lean controls. The effect of the beta 3-AR agonist on body adiposity was also more remarkable in the obese animals. Although the reason for these differences is not clear at present, it may be related to the different sympathetic nerve activity: that is, both MSG-treated (28, 29) and yellow KK obese mice (23) had reduced sympathetic nerve activity compared with their respective lean controls. Thus the obese mice could be much more sensitive to adrenergic stimulation.

Several groups (4, 11) have recently cloned genes that code for the novel uncoupling proteins UCP-2 and UCP-3, which have 59 and 57% amino acid identity to the conventional UCP (UCP-1), respectively. UCP-2 is expressed in a wide variety of tissues, including adipose tissues, lung, and skeletal muscle, whereas UCP-3 is specific to BAT and skeletal muscle. Thus it may be possible that the UCP signals detected in the tissues other than typical BAT are those of UCP-2 and UCP-3 rather than UCP-1. However, this is unlikely, because 1) in the present study the signals were not detected in any tissues except BAT in the lean control animals without CL treatment, and 2) the ectopic signals were found only after the CL treatment. The UCP-2 expression was reported not to be influenced by beta 3-AR agonist treatments (11), although the relationship between UCP-3 and the beta 3-AR agonist is unclear. The relative importance of UCP-1, UCP-2, and UCP-3 in regulatory thermogenesis remains to be clarified in further studies.

The thermogenic activity of UCP can be estimated by measuring the GDP-binding activity of mitochondrial preparation in vitro, although the GDP binding is not always parallel to the amount of UCP because of the "unmasking" phenomenon (12, 22). In the present study, we found that the GDP binding of the mitochondrial preparation of interscapular BAT was increased more than twofold by the CL treatment, in parallel with the increase in immunoreactive UCP. In the control untreated mice, the mitochondrial fractions prepared from the inguinal and retroperitoneal fat pads showed negligible GDP-binding activity, below the limit of detection (10 pmol/mg mitochondrial protein). However, in the obese mice treated with CL, the mitochondrial preparations from the fat pads showed the GDP binding at about one-fourth the activity of BAT. These results suggest that the mitochondria of the inguinal and retroperitoneal fat pads of CL-treated mice are also thermogenically active because of the significant expressions of UCP. A low but significant GDP binding was also detected in the fat pads of lean control mice treated with CL, although Western and Northern blot analyses detected no UCP signals in the retroperitoneal fat pad. A possible reason for this discrepancy may be the difference in sensitivity of these two methods. Alternatively, part of the GDP binding estimated in the present study may be due to the GDP binding by UCP-2 and/or UCP-3. Further studies are necessary to determine the relative contribution of the individual UCP isoforms to the total mitochondrial GDP-binding activity.

The immunohistochemical examination of the skeletal muscles of the present CL-treated mice revealed that UCP signals are localized in slow-twitch fibers, which are known to be rich in mitochondria (8, 9). The mitochondrial localization of UCP in the myocytes was also confirmed by immunoelectron microscopy. These results suggest that the UCP in muscles, as well as that in adipocytes, is also functionally active in mitochondrial uncoupling. In fact, a very low but significant GDP-binding activity was detected in the mitochondrial fractions of the gastrocnemius muscles of the CL-treated obese mice. It is difficult to evaluate the contribution of the ectopically expressed UCP in white fat and skeletal muscle to whole body thermogenesis, but a rough estimation is possible on the basis of data shown in Table 1: in the CL-treated obese mice, provided that the total weights of BAT, white fat, and skeletal muscle are ~0.3, 10, and 15 g, respectively, the total GDP bindings could be expected to be estimated as ~2,200, 731, and 343 pmol. Such a rough estimation suggests a significant contribution of the ectopically expressed UCP to increased energy expediture and thereby to the anti-obese effect of CL.

In conclusion, the chronic CL treatment of MSG-treated hypothalamic obese mice induced the increased expression of UCP in typical BAT but also the ectopic expression of UCP in fat pads usually considered WAT and also in skeletal muscle, similar to the results obtained in genetically obese yellow KK mice. Because the ectopically expressed UCP is functionally active in mitochondrial GDP binding, this UCP, in addition to the UCP in BAT, may also contribute to the increased thermogenesis and antiobese effect of the beta 3-AR agonist CL. Unlike the findings in rodents, the contribution of BAT has been considered to be negligible in humans because of the apparent lack of BAT and UCP in adults. However, the present findings imply that the expression of the UCP gene can be activated in the white fat and even in skeletal muscle by beta 3-adrenergic stimulation. Although it remains to be examined whether this is also the case in humans, our results undoubtedly suggest that beta 3-AR and UCP are important targets for the treatment of human obesity, as for rodent obesity. This is supported further by recent findings of the presence of UCP isoforms UCP-2 (11) and UCP-3 (4) in humans, and also by those demonstrating a role of beta 3-AR in human obesity (18).

    ACKNOWLEDGEMENTS

We are grateful to American Cyanamid Co. for providing the CL-316,243, and to Dr. D. Ricquier (Centre National de la Recherche Scientifique, Meudon, France) for the kind gift of the UCP cDNA.

    FOOTNOTES

This work was supported in part by Grants-in-Aid 09671067 and 07671149 for scientific research from the Ministry of Education, Science, Culture and Sports of Japan, and by The Smoking Research Foundation, Japan.

Address for reprint requests: T. Yoshida, First Dept. of Internal Medicine, Kyoto Prefectural Univ. of Medicine, 465-Kajiicho, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602, Japan.

Received 25 June 1997; accepted in final form 26 November 1997.

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Abstract
Introduction
Methods
Results
Discussion
References

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AJP Endocrinol Metab 274(3):E469-E475
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