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Am J Physiol Endocrinol Metab 290: E1014-E1021, 2006. First published December 20, 2005; doi:10.1152/ajpendo.00105.2005
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Indispensable role of mitochondrial UCP1 for antiobesity effect of beta3-adrenergic stimulation

Ken-ichi Inokuma,1 Yuko Okamatsu-Ogura,1 Asako Omachi,1 Yukiko Matsushita,1 Kazuhiro Kimura,1 Hitoshi Yamashita,2 and Masayuki Saito1

1Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo; 2Department of Molecular Genetics, National Institute for Longevity Sciences, Obu, Japan

Submitted 10 March 2005 ; accepted in final form 16 December 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mitochondrial uncoupling protein-1 (UCP1) has been thought to be a key molecule for thermogenesis during cold exposure and spontaneous hyperphagia and thereby in the autonomic regulation of energy expenditure and adiposity. However, UCP1 knockout (KO) mice were reported to be cold intolerant but unexpectedly did not get obese even after hyperphagia, implying that UCP1 may not be involved in the regulation of adiposity. Treatment of obese animals with beta3-adrenergic agonists is known to increase lipid mobilization, induce UCP1, and, finally, reduce body fat content. To obtain direct evidence for the role of UCP1 in the anti-obesity effect of beta3-adrenergic stimulation, in the present study, UCP1-KO and wild-type (WT) mice were fed on cafeteria diets for 8 wk and then given a beta3-adrenergic agonist, CL-316,243 (CL), or saline for 2 wk. A single injection of CL increased whole body oxygen consumption and brown fat temperature in WT mice but not in KO mice, and it elicited almost the same plasma free fatty acid response in WT and KO mice. WT and KO mice increased similarly their body and white fat pad weights on cafeteria diets compared with those on laboratory chow. Daily treatment with CL resulted in a marked reduction of white fat pad weight and the size of adipocytes in WT mice, but not in KO mice. Compared with WT mice, KO mice expressed increased levels of UCP2 in brown fat but decreased levels in white fat and comparable levels of UCP3. It was concluded that the anti-obesity effect of beta3-adrenergic stimulation is largely attributable to UCP1, but less to UCP2 and UCP3, and thereby to UCP1-dependent degradation of fatty acids released from white adipose tissue.

uncoupling protein-1; adiposity; CL-316,243; energy expenditure; hyperphagia

UNCOUPLING PROTEIN (UCP) IS A MOLECULE, as its name suggests, that uncouples mitochondrial oxidative phosphorylation by bypassing the electrochemical gradient across the inner membrane from the F1-ATPase and thereby dissipates energy as heat. Among several isoforms of the UCP family so far reported in mammals, UCP1 is the only one whose physiological importance has been firmly established; that is, UCP1 is present exclusively in brown adipose tissue (BAT), an organ specified for nonshivering thermogenesis during cold acclimation, arousal from hibernation, and recovery from anesthetic hypothermia (2). UCP1 has also been proposed to be involved in diet-induced thermogenesis, as well as cold-induced nonshivering thermogenesis, and play a significant role in the control of energy expenditure and whole body energy balance. This is supported by the observations, for example, that spontaneous overfeeding of highly palatable diets and/or high-fat diets gives rise to increased energy expenditure (oxygen consumption) in association with BAT hyperplasia and increased UCP1 contents in the same way as seen after cold acclimation (1, 2, 11, 20). It is also known that the thermogenic activity and UCP1 expression of BAT are decreased in most genetic and hypothalamic obese animals (18, 19, 21). Thus it seems likely that UCP1 is one of the key molecules for adaptive thermogenesis and energy expenditure in response to changes in energy intake, and thereby its dysfunction contributes to the development and maintenance of obesity.

However, the above-mentioned view was challenged by the findings of Enerback et al. (5) and Liu et al. (13) that transgenic mice with complete absence of UCP1 are not obese, but rather lean compared with wild-type control (WT) mice, both on normal and on high-fat diets, although they are apparently cold intolerant. This unexpected finding may suggest the existence of some critical molecules, other than, or in addition to, UCP1, that dissipate excess amounts of energy. Other UCP isoforms, such as UCP2 and UCP3, which are expressed ubiquitously in many tissues and abundantly in skeletal muscle, respectively, may be possible candidates because UCP2 expression is upregulated in BAT of UCP1-deficient mice (5). This possibility, however, has not been evidenced yet. Thus the precise role and significance of energy expenditure by UCP1 in body fat regulation are still to be debated.

UCP1 thermogenesis in BAT is under direct control of sympathetic nerves abundantly entering into this tissue; that is, norepinephrine released from the sympathetic nerve endings stimulates the beta-adrenergic receptor (AR)-adenylate cyclase-protein kinase A signaling pathway and activates hormone-sensitive lipase. The released fatty acids activate UCP1 and are oxidized as a major substrate for thermogenesis. Because the same signaling pathway is also present in white adipose tissue (WAT), where UCP1 is absent, the activation of this pathway is expected to lead to UCP1-mediated energy dissipation of fatty acids released from WATs. In fact, it has been demonstrated that various agonists specific to the beta3-AR expressed predominantly in white and brown adipocytes effectively activate the pathway noted above, and chronic treatment of obese animals with beta3-AR agonists, as expected, reduces body fat content (10, 12, 16). Thus the anti-obesity effect of beta3-AR agonists has been believed to be due to the activation of UCP1. However, to our knowledge, there has been no direct evidence for this idea because almost all previous reports (10, 12) have shown only a parallel relationship between UCP1 activation and reduced adiposity but not the relation of cause and effect. Moreover, the observation of obesity resistance of UCP1-deficient mice noted above seems to not support the critical role of UCP1 in the long-term regulation of adiposity. Accordingly, to determine whether UCP1 is indispensable for the anti-obesity effect of beta3-AR agonists, in the present study we examined both short- and long-term effects of a selective beta3-AR agonist, CL-316,243 (CL), on the thermogenic and lipomobilizing activities and adiposity of UCP1 knockout (KO) mice, comparing them with those of WT control mice. Our results clearly indicate that the fat-reducing effect of beta3-AR stimulation is largely attributable to the activation of UCP1 thermogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. UCP1-KO (UCP1–/–) mice on a congenic C57BL/6J background were generated by backcross mating of heterozygous (UCP1+/–) mice on a mixed 129/SvPas and C57BL/6J background with C57BL/6J mice 15 times [mice were kindly given by Dr. L. Kozak (Pennington Biomedical Research Center, Baton Rouge, LA)]. All WT (UCP1+/+) mice were C57BL/6J. They were housed in plastic cages placed in an air-conditioned room at 26°C with a 12:12-h light-dark cycle (lights on at 0700–1900) and given free access to laboratory chow (Oriental Yeast, Tokyo, Japan) and tap water. The experimental procedures and care of animals were approved by the Animal Care and Use Committee of Hokkaido University.

Acute responses of thermogenesis and lipolysis to CL. BAT thermogenesis in response to a single injection of the beta3-AR agonist CL (American Cyanamid, Pearl River, NY) was assessed by measuring the temperature changes in BAT and the rectum, as described previously (22). Briefly, male mice (10–30 wk old) fasted overnight were anesthetized with pentobarbital sodium (50 mg/kg ip), a small incision was made above the scapula, and the interscapular brown fat pads were partially separated from the muscle below, with the vasculature and nerve supplies to the pads being left intact. Then, mice were placed on a heat plate, and a plastic-coated thermistor with a diameter of 1 mm was placed under the fat pads. Another thermistor was also inserted into the rectum, and the plate was heated gently. After the rectal temperature reached a steady level at about 37°C, CL (0.1 mg/kg) or saline was injected intraperitoneally, and the temperature changes were monitored for 20 min.

To assess lipolytic response to CL in WAT, CL (0.1 mg/kg) or saline was injected intraperitoneally into overnight-fasted conscious mice, and blood (20 µl) was taken from the tail vein 0–4 h after the injection. Plasma free fatty acid concentrations were measured enzymatically using a kit (NEFA C test; WAKO Pure Chemical, Tokyo, Japan).

Whole body oxygen consumption was measured for 2 h after intraperitoneal injection of CL (0.1 mg/kg) or saline into conscious mice by the use of an open-circuit-type metabolic chamber (MK-5000; Muromachi Kikai, Tokyo, Japan) in a room kept at 26°C.

Diet and chronic treatment with CL. When mice became 10–12 wk old, they were divided into three groups consisting of four males and four females in each group. One group was fed on laboratory chow as previously described, whereas the other two groups were kept on cafeteria feeding for 10 wk, during which they were allowed free access to, in addition to laboratory chow, two kinds of snacks with various tastes (ChocoCrisp; Nisshin Cisco and Mire-Fry; Watayoshi Seika, Nagoya, Japan) that contained 68.1–70.5 g carbohydrate, 18.8–19.8 g fat (mixture of rapeseed, sunflower, and coconut oil), 7.7–7.8 g protein, and 481–483 kcal in 100 g. Laboratory chow contained 54 g carbohydrate, 5.1 g fat, 23.8 g protein, and 357 kcal in 100 g. During the last 2 wk, the cafeteria-feeding groups were subcutaneously given either CL (0.1 mg/kg) or saline once a day at 1300–1400. The laboratory chow-fed mice were similarly injected with saline. Body weight and the amount of food intake were measured every day. For calculation of total energy intake, the amount of actual intake of individual foods (laboratory chow and snacks) was measured after those lying scattered in the cages were corrected for. At week 10, all mice were killed by cervical dislocation at 1000–1300, and fat pads of various regions (interscapular BAT and inguinal, perigonadal, retroperitoneal, and mesenteric adipose tissues) and some other tissues were quickly removed and weighed. Tissue specimens were transferred into RNALater (Invitrogen, Carlsbad, CA) for RNA analysis, 10% buffered formalin for conventional histological examinations, or liquid nitrogen for Western blot analysis.

UCP mRNA analysis. Total RNA was extracted with the use of RNALater according to the manufacturer's protocol, and mRNA levels of UCP1, UCP2, and UCP3 were measured semiquantitatively by real-time RT-PCR using respective cDNA fragment as a standard and expressed as relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels. Briefly, 2 µg of total RNA were reverse transcribed with an oligo(dT) 15-adaptor primer and MMLV reverse transcriptase (Invitrogen). Real-time PCR was performed on a fluorescence thermal cycler (LightCycler system; Roche Diagnostics, Mannheim, Germany), using SYBR Green I as a double-strand DNA-specific dye according to the manufacturer's protocol. Primers used were 5'-GTG AAG GTC AGA ATG CAA GC-3' and 5'-AGG GCC CCC TTC ATG AGG TC-3' for mouse UCP1, 5'-GGC TGG TGG TGG TCG GAG AT-3' and 5'-CCG AAG GCA GAA GTG AAG TG-3' for mouse UCP2, 5'-GAG CGG ACC ACT CCA GCG TC-3' and 5'-TGA GAC TCC AGC AAC TTC TC-3' for mouse UCP3, and 5'-GAA GGT CGG TGT GAA CGG ATT-3' and 5'-GAA GAC ACC AGT AGA CTC CAC GAC ATA-3' for mouse GAPDH.

Histological examinations. Sections of formalin-fixed tissue specimens were stained with hematoxylin-eosin and examined light microscopically. The average diameter of adipocytes of each mouse was calculated from those of 100–200 cells at three different sections.

Data analysis. All values were presented as means ± SE, unless otherwise specified, and analyzed by analysis of variance with post hoc testing by the Scheffé's multiple range test.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Acute responses to CL of lipomobilization, BAT thermogenesis, and oxygen consumption. To confirm the impaired responses of thermogenic activity of BAT of UCP1-KO mice, we first monitored temperature changes in the interscapular BAT and the rectum after a single injection of CL. Although the basal temperatures of the BAT and rectum did not differ between UCP1-KO and WT mice, BAT temperature was slightly lower than rectal temperature in all mice. In WT mice, CL injection elicited a rapid rise in BAT temperature in couples of minute followed by a gradual rise in rectal temperature (Fig. 1, A and B). In contrast, in UCP1-KO mice, the temperature responses were much less. Saline injection elicited no temperature change in either type of mice. Being consistent with these results, CL increased the whole body oxygen consumption by 46% in WT mice, whereas it did not change in UCP1-KO mice (Fig. 1C). To assess lipolytic response in WAT, changes in plasma free fatty acid concentration were also monitored after CL injection. As shown in Fig. 1D, CL produced remarkable and sustained rises in the fatty acid concentrations for ≥4 h. There was no notable difference in the fatty acid responses between UCP1-KO and WT mice. It was thus confirmed that UCP1-KO mice are incapable of increasing BAT thermogenesis and energy expenditure in response to acute beta3-AR stimulation, although they can respond normally in lipid mobilization from WAT.


Figure 1
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  		Fig. 1. Acute responses of brown adipose tissue (BAT) thermogenesis, lipolysis, and energy expenditure to CL-316,243 (CL). Wild-type (WT) and uncoupling protein (UCP)1 knockout (KO) male mice were anesthetized, and temperature changes of interscapular BAT (A) and rectum (B) were monitored after ip injection of CL (0.1 mg/kg) or saline. Values are means ± SE for 4 mice. Difference between values of WT and UCP1-KO mice was significant (P < 0.05) at 10 min and thereafter. C: whole body oxygen consumption was measured for 2 h after ip injection of CL (0.1 mg/kg) or saline to free-moving male mice. Values are means ± SE for 6 mice. *P < 0.05 vs. saline. D: to assess lipolytic response of white adipose tissue (WAT) to CL, male mice were fasted overnight and injected ip with saline or CL (0.1 mg/kg). Changes in plasma free fatty acid concentration were monitored for 4 h. Values are means ± SE for 4 mice. Difference between values of CL- and saline-injected mice was significant (P < 0.05) at all time points except 0 h.

 
Effects of cafeteria feeding and CL treatment on body and tissue weights. Mice were kept in a cafeteria-feeding condition for 10 wk, during which they were allowed free access to palatable foods, in addition to laboratory chows, for 10 wk. Female WT mice kept under such a cafeteria-feeding condition, compared with those kept on laboratory chow, took 43% more energy due to a large intake of snacks despite decreased intake of laboratory chow (Fig. 2C). In parallel, these mice gained their body weight more rapidly, and after 8 wk they weighed more (26.4 ± 1.5 vs. 23.1 ± 0.7 g). Similar to WT mice, female UCP1-KO mice were also hyperphagic (+37%) and weighed slightly more (24.8 ± 0.8 vs. 22.4 ± 0.5 g) when kept under the cafeteria-feeding condition. Because quite similar effects of cafeteria feeding were also found in male mice, the data obtained from male and female mice were combined and are summarized in Fig. 2. After the 8-wk period, cafeteria-fed mice were injected with either CL or saline every day for 2 wk while they were kept under the same feeding condition. As shown in Fig. 2, A and B, the CL treatment stopped the weight gain in WT mice but not in UCP1-KO mice, which gained more weight, similarly to those treated with saline. The total energy intake during the 2-wk period was not affected by CL treatment (Fig. 2C).


Figure 2
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  		Fig. 2. Body weight and energy intake during cafeteria feeding and CL treatment. WT and UCP1-KO mice of both sexes were fed on laboratory chow or cafeteria diets for 8 wk and then injected sc with CL (0.1 mg·kg–1·day–1) or saline for an additional 2 wk. Mean body weights of female WT and UCP1-KO mice at week 0 were 21.1 ± 0.5 (n = 12) and 20.3 ± 0.3 g (n = 12), respectively, and those of males were 24.4 ± 0.2 (n = 12) and 24.5 ± 0.6 g (n = 12), respectively. A and B: mean body weight changes from week 0 were calculated for each group, which consisted of 4 males and 4 females. Difference between values of cafeteria- and laboratory chow-fed WT mice was significant (P < 0.05) at week 5 and thereafter, as well as the difference between values of CL- and saline-treated WT mice at week 9 and thereafter. There was no significant difference between the groups of UCP1-KO mice. C: energy intake during the last 2-wk period was calculated from the amount of intake of laboratory chow and snacks (Mire-Fry and ChocoCrisp). All values are means ± SE for 8 mice. *P < 0.05 vs. laboratory chow group.

 
At week 10, all mice were killed and their individual tissues were weighed. There was no significant difference in the weights of liver and some other organs such as skeletal muscle, brain, heart, and kidney among the three groups of both WT and UCP1-KO mice. In contrast, the weight of fat pads, particularly those of WAT, was markedly influenced by cafeteria feeding and CL treatment; that is, WAT of cafeteria-fed mice was apparently heavier (+99% in WT and +75% in UCP1-KO mice) than that of mice fed on laboratory chow. WATs of the four regions of UCP1-KO mice were similar to those of WT mice, except for perigonadal WAT, which was smaller. The effects of cafeteria feeding were almost abolished by the CL treatment in WT mice but not in UCP1-KO mice (Fig. 3A). In WT mice, the weight-reducing effect of CL treatment was similarly observed in all WAT regions (Fig. 3B). The weight of interscapular BAT was not changed by cafeteria feeding or CL treatment in WT mice. The BAT weight of UCP1-KO mice was heavier than that of WT mice and increased by cafeteria feeding, but little affected by CL treatment.


Figure 3
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  		Fig. 3. Effects of cafeteria feeding and CL treatment on tissue weight. WT and UCP1-KO mice were treated as in Fig. 2 and decapitated on week 10. The weight of interscapular BAT, liver, and WAT of 4 different regions was measured and expressed as relative to body weight. Values are means ± SE for 8 mice. *P < 0.05 vs. laboratory chow group; {dagger}P < 0.05 vs. saline-treated cafeteria feeding group; §P < 0.05 vs. WT mice.

 
Histology of WAT and BAT. Figure 4 shows the histology of BAT and WAT. Interscapular BAT consisted of multilocular adipocytes in both WT and UCP1-KO mice but with apparently larger lipid droplets in UCP1-KO mice, particularly in those kept under the cafeteria feeding condition (Fig. 4A). In perigonadal WAT, almost all adipocytes were unilocular (Fig. 4C), and cafeteria feeding increased their size similarly in WT (48 ± 4 vs. 36 ± 3 µm in diameter, P < 0.05) and UCP1-KO mice (45 ± 6 vs. 34 ± 2 µm, P < 0.05). When treated with CL, the adipocytes became smaller, to 35 ± 3 µm (P < 0.05), in WT mice but not in UCP1-KO mice (42 ± 2 µm). In inguinal WAT of WT mice, most adipocytes were unilocular, and multilocular cells were observed to be abundant after CL treatment (Fig. 4B). In inguinal WAT of UCP1-KO mice, however, abundant multilocular adipocytes were observed regardless of whether they were treated with CL or not.


Figure 4
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  		Fig. 4. Histological features of BAT and WAT. Interscapular BAT (A), inguinal WAT (B), and perigonadal WAT (C) of female mice treated as in Fig. 2 were stained with hematoxylin and eosin.

 
UCP expression. The mRNA expression levels of UCP1, UCP2, and UCP3 were examined by real-time PCR. As shown in Fig. 5, the effects of cafeteria feeding and CL treatment were considerably different among the UCP isoforms and also among the tissues. In inguinal WAT of WT mice, UCP1 mRNA was increased slightly by cafeteria feeding and markedly by CL treatment, keeping with the appearance of multilocular adipocytes in this depot (Fig. 4B). Such changes were not seen in perigonadal WAT (Fig. 4C). These results confirm the previously reported results of the beta3-AR agonist-induced ectopic expression of UCP1 in WAT (10, 12, 16). As expected, no UCP1 mRNA was detected in any tissue of UCP1-KO mice. Nevertheless, abundant multilocular adipocytes were present in inguinal WAT of UCP1-KO mice, as also seen in Liu et al. (13), regardless of whether they were treated with CL or not.


Figure 5
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  		Fig. 5. mRNA levels of 3 UCP isoforms in adipose tissues. mRNA of UCP1, UCP2, and UCP3 was measured by real-time PCR and expressed as relative to GAPDH mRNA. There was no significant difference in the GAPDH mRNA levels between the groups. Values are means ± SE for 4 male mice. *P < 0.05 vs. laboratory chow group; {dagger}P < 0.05 vs. saline-treated cafeteria-feeding group; §P < 0.05 vs. WT mice.

 
UCP2 mRNA in BAT of UCP1-KO mice was upregulated by cafeteria feeding and CL treatment, being much higher than that of WT mice. On the contrary, UCP2 mRNA in WAT seemed to be downregulated. UCP3 mRNA was expressed at comparable levels in BAT and WAT, being increased in BAT and perigonadal WAT of WT mice by cafeteria feeding and CL treatment, respectively.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The purposes of the present study were to determine whether UCP1 is involved in the long-term regulation of adiposity and the anti-obesity effect of beta3-adrenergic stimulation. For this, we examined the effects of hyperphagia induced by feeding cafeteria diets and treatment with the selective beta3-adrenergic agonist CL on the adiposity in UCP1-KO mice and compared them with those of WT mice. The principal findings of the present study were as follows: 1) prolonged hyperphagia by cafeteria feeding produced obesity in both WT and UCP1-KO mice, and 2) daily treatment with CL ameliorated the diet-induced obesity in WT mice but not in UCP1-KO mice.

UCP1-KO mice have been proved to lack beta3-adrenergically activated thermogenesis and to be cold intolerant (5). In fact, we confirmed that oxygen consumption and the temperature of BAT and rectum were increased in response to a single injection of CL in WT mice, but these responses were absent or greatly blunted in UCP1-KO mice. CL injection increased oxygen consumption by 46% in WT mice but not in UCP1-KO mice. These results seem to be a little different from some previous reports (5–8) of substantial increases in oxygen consumption in WT mice (+90–110%) and even in UCP1-KO mice (+30–50%). Although the precise reason for these differences is not clear at present, it may be due to the different doses of CL (0.1 and 1 mg/kg in the present and previous studies, respectively). Moreover, mice had been acclimated at 26°C and underwent the measurement of oxygen consumption at the same temperature in our study, whereas mice had been acclimated at 24–25°C and underwent the measurement of oxygen consumption at higher temperatures (28–30°C) in the previous studies. It is likely that mice acclimated at lower temperatures have more UCP1 and higher activities of some other thermogenic mechanisms, if any, and thereby greater responsiveness to acute adrenergic stimulation. In contrast, the plasma free fatty acid concentration was increased similarly in UCP1-KO and WT mice. These results indicate that UCP1-KO mice are incapable of increasing BAT thermogenesis and energy expenditure in response to acute beta3-adrenergic stimulation, although they can respond normally in lipid mobilization from WAT.

The anti-obesity effect of beta3-AR stimulation has been well documented in various animal models of obesity, including those induced by cafeteria feeding (10, 12, 15, 16). In this study, we also confirmed the marked fat-reducing effect of the highly selective beta3-adrenergic agonist CL in obesity induced by feeding cafeteria diets; that is, WT mice fed on cafeteria diets gained more body and WAT weights than those on laboratory chow, but a 2-wk treatment with CL completely prevented the obesity induced by cafeteria feeding. In contrast with WT mice, the fat-reducing effect of CL was absent in UCP1-KO mice. Because the total energy intake during the 2-wk period was not affected by CL treatment in either WT or UCP1-KO mice, the different effects of CL treatment are due not to the difference in energy intake but to the impaired response of energy expenditure to CL in UCP1-KO mice. Histological examinations revealed that CL treatment seemed to reduce the lipid droplets remarkably in brown adipocytes of WT mice but little in those of UCP1-KO mice. Moreover, CL treatment reduced the adipocyte size of WAT remarkably in WT mice but little in UCP1-KO mice. Thus chronic treatment with CL reduces WAT in WT mice but not in UCP1-KO mice, although CL can stimulate lipid mobilization from WAT similarly in the two types of mouse. All these results suggest that the fat-reducing effect of CL is largely attributable to UCP1-dependent thermogenesis and degradation of fatty acids released from WAT.

CL treatment induced considerable expression of UCP1 in inguinal fat pad, which was usually thought of as WAT. Such ectopic expression of UCP1 has been demonstrated in animals that were acclimated to cold environments and those treated with beta3-adrenergic agonists and/or leptin (3, 10, 12, 15–17, 23). In contrast to UCP1, the effect of CL treatment on UCP2 and UCP3 was rather complicated; that is, UCP2 mRNA was upregulated in BAT but downregulated in WAT of UCP1-KO mice. UCP3 expression was not changed in BAT and inguinal WAT and was increased in perigonadal WAT of WT mice. Collectively, no consistent relationship between the UCP2 and UCP3 expression levels and the fat-reducing effect of CL was found. UCP2 and UCP3 have been suggested to be involved in the regulation of cellular energy levels and/or oxidation of fatty acids (4). However, our results indicate that most of the fat-reducing effect of CL disappeared in the UCP1-KO mice despite the considerable expression levels of UCP2 and UCP3, suggesting the minor role of these UCP isoforms for the effect of CL. This view is quite consistent with a report (14) that adrenergically induced thermogenesis in brown fat cells is fully UCP1 dependent and not substituted by UCP2 or UCP3.

Granneman et al. (8) reported that acute activation of beta3-AR with CL significantly increased oxygen consumption and body temperature, even in UCP1-KO mice, and that chronic activation increased basal and CL-stimulated metabolic rates in WAT, suggesting the presence of beta3-AR-mediated but UCP1-independent thermogenesis in WAT. However, we could not detect any significant increase in oxygen consumption in UCP1-KO mice after a single injection of CL. In contrast to oxygen consumption, body and BAT temperatures were slightly but apparently increased after CL injection in UCP1-KO mice. Because the temperature response is a consequence of changes in heat loss as well as heat production, our results may suggest some CL-induced changes in heat loss, such as vasoconstriction and piloerection.

The present study clearly indicates the indispensable role of UCP1 for the anti-obesity effect of beta3-AR stimulation. Enerback et al. (5) and Liu et al. (13) demonstrated that UCP1-KO mice are cold intolerant but do not become any more obese than WT mice on either normal or high-fat diets. In the present study, we also found that hyperphagia induced by cafeteria feeding produced obesity in both WT and UCP1-KO mice. UCP1-KO mice were originally predicted to be more susceptible to diet-induced obesity than WT mice because of their lack of ability to use increased UCP1-mediated thermogenesis, but, as shown here, they are only equally susceptible. One of the likely explanations is, as proposed by Liu et al. (13), that in the absence of UCP1, some alternative, calorically more costly pathways of metabolism are used for thermogenesis to maintain body temperature and that these pathways also contribute to the regulation of adiposity. UCP1-KO mice are known to be able to acclimate to mild cold and to maintain their body temperature by using shivering thermogenesis (6). Thus it is conceivable that the importance of UCP1 in the development of hyperphagia-induced obesity is dependent on the environmental temperature and the possibility that compensatory shivering is occurring. In fact, when UCP1-KO mice are kept at a thermoneutral temperature when they do not need to activate the shivering thermogenesis to any major extent, they become obese under certain experimental regimens (Jan Nedergaard, personal communication). This would be in keeping with the very similar development of obesity in cafeteria-fed UCP1-KO and WT mice kept at 26°C in our experiments. Indeed, the previous study showed a relative resistance to high-fat diet-induced obesity in UCP1-KO mice compared with WT mice at 20°C but similar development of obesity at 27°C (13). In this previous study (13), abundant multilocular adipocytes appeared in inguinal WAT of UCP1-KO mice given a high-fat diet at 20°C but not when they lived at 27°C. A similar increase was seen in our mice living at 26°C whether they were treated with CL or not. However, in the absence of UCP1, these multilocular adipocytes do not appear to contribute to UCP1-independent thermogenesis evoked by beta3-AR stimulation, as judged by the complete absence of thermogenic response to CL as assessed by indirect calorimetry. Thus our results do not support the concept (8) of UCP1-independent beta3-AR-mediated thermogenesis in WAT.

In conclusion, the anti-obesity effect of beta3-adrenergic stimulation is largely attributable to UCP1 in BAT and ectopically expressed in WAT, but less to UCP2 and UCP3 and thereby to UCP1-dependent degradation of fatty acids released from WAT.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.


   ACKNOWLEDGMENTS
 
We thank Dr. L. Kozak (Pennington Biomedical Research Center) for providing us with UCP1-KO mice.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Saito, Dept. of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060–0818, Japan (e-mail: saito{at}vetmed.hokudai.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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