Present addresses Y. Kontani, Department of Microbiology, Kanazawa Medical University, 1–1 Uchinada, Ishikawa 920–0293, Japan. Y. Wang, Department of Physiology, UT South-western Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA. Z. Wang, Division of Neurodegenerative Disorders, St. Boniface Hospital Research Centre, R4048–351 Tache Avenue, Winnipeg, MB R2H 2A6 Canada. N. Mori, Department of Neuroanatomy and Molecular Neurobiology, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki 852–8523, Japan.
UCP1 deficiency increases susceptibility to diet-induced obesity with age
Article first published online: 9 MAY 2005
Volume 4, Issue 3, pages 147–155, June 2005
How to Cite
Kontani, Y., Wang, Y., Kimura, K., Inokuma, K.-I., Saito, M., Suzuki-Miura, T., Wang, Z., Sato, Y., Mori, N. and Yamashita, H. (2005), UCP1 deficiency increases susceptibility to diet-induced obesity with age. Aging Cell, 4: 147–155. doi: 10.1111/j.1474-9726.2005.00157.x
- Issue published online: 9 MAY 2005
- Article first published online: 9 MAY 2005
- Accepted for publication 04 April 2005
- adrenergic receptor;
- brown adipose tissue;
- diet-induced thermogenesis;
- uncoupling protein
Loss of nonshivering thermogenesis in mice by inactivation of the mitochondrial uncoupling protein gene (Ucp1−/– mice) causes increased sensitivity to cold and unexpected resistance to diet-induced obesity at a young age. To clarify the role of UCP1 in body weight regulation throughout life and influence of UCP1 deficiency on longevity, we longitudinally analyzed the phenotypes of Ucp1−/– mice maintained in a room at 23 °C. There was no difference in body weight and lifespan between genotypes under the standard chow diet condition, whereas the mutant mice developed obesity with age under the high-fat (HF) diet condition. Compared with Ucp1+/+ mice, Ucp1−/– mice showed increased expression of genes related to thermogenesis and fatty acid metabolism, such as β3-adrenergic receptor, in adipose tissues of the 3-month-old mutants; however, the augmented expression was reduced in Ucp1+/+ mice in 11-month-old Ucp1−/– mice fed the HF diet. Likewise, the increased levels of UCP3 and cAMP-dependent protein kinase in the brown adipose tissue of Ucp1−/– mice given the standard diet were decreased significantly in that of Ucp1−/– mice fed the HF diet, which animals showed impaired norepinephrine-induced lipolysis in their adipose tissues. These results suggest profound attenuation of β-adrenergic responsiveness and fatty acid utilization in Ucp1−/– mice fed the HF diet, bringing them to late-onset obesity. Our findings provide evidence that UCP1 is neither essential for body weight regulation nor for longevity under conditions of standard diet and normal housing temperature, but deficiency increases susceptibility to obesity with age in combination with HF diet.
At present, obesity is a global health problem especially in Western countries and Japan, as it is associated profoundly with type 2 diabetes mellitus and cardiovascular disease and also decreases longevity. In modern society obesity is basically late-onset and its incidence apparently increases with age, being prominent after the age of 20–30 years, likely due to a combination of genetic and environmental factors (Kopelman, 2000). In the etiology of obesity, the contribution of the thermoregulatory mechanisms to body weight regulation appears to be critical in homeothermic animals (Kozak & Harper, 2000; Lowell & Spiegelman, 2000; Ricquier & Bouillaud, 2000). To date, a number of genes related to thermogenesis, such as those encoding adrenergic receptors (AR) and uncoupling proteins (UCP), have been considered as candidate genes for human obesity (Oppert et al., 1994; Clement et al., 1995; and 1996; Kogure et al., 1998). Age-related declines in β-adrenergic responsiveness (Schwartz et al., 1990) and the virtual absence of UCP1 in adult humans (Lean, 1992; Lowell & Spiegelman, 2000) also suggest a relationship between the reduced thermogenic ability and increased susceptibility to obesity with age. Indeed, a role for UCP1 in resistance against diet-induced obesity was postulated from the results of studies on transgenic mice (Lowell et al., 1993; Kopecky et al., 1995; Li et al., 2000). Recently, Bachman et al. showed that the efferent action via a β-adrenergic signaling pathway plays a crucial role in the defense against diet-induced obesity by studying mice lacking the 3 β-AR genes (β-less mice), in which UCP1 was scarcely induced in the brown adipose tissue (BAT) and cold sensitivity and extreme obesity were phenotypic (Bachman et al., 2002). In contrast, the targeted disruption of genes such as those for β3-AR or UCPs did not enhance obesity (Susulic et al., 1995; Enerback et al., 1997; Arsenijevic et al., 2000; Gong et al., 2000; Vidal-Puig et al., 2000; Liu et al., 2003). Two-month-old UCP1-deficient (Ucp1−/–) mice were previously reported to be resistant to diet-induced obesity, suggesting the importance of alternative or additional mechanisms compensating the loss of UCP1 function in energy dissipation (Enerback et al., 1997; Liu et al., 2003). Mice deficient in dopamine β-hydroxylase, thus lacking norepinephrine and epinephrine, showed an impaired induction of the UCP1 gene but were rather lean compared with control mice (Thomas & Palmiter, 1997). Thus, the thermoregulatory mechanisms associated with the regulation of body weight in UCP1-deficient animals are complex. However, in almost all of these previous studies, young animals were used, despite increased susceptibility to obesity with age in both rodents and humans. Because body weight changes greatly in the developmental phase and is affected by various environmental factors, the importance of UCP1 in body weight regulation remains to be clarified throughout life. Furthermore, the impact of UCP1 deficiency on longevity was totally unknown, although Ucp1−/– mice cannot survive in the cold at 4 °C (Golozoubova et al., 2001). In the present study therefore we investigated the effects of age and diet condition on body weight regulation in UCP1-deficient mice as well as the effect of UCP1 deficiency on lifespan under an ambient temperature of 23 °C.
Late–onset obesity in UCP1-deficient mice
We analyzed the phenotypes of congenic B6.Ucp1−/– mice given a standard chow or high-fat (HF) diet and the effect on body weight regulation, at room temperature. We used mice from 3-months-old through their life, because there was no difference in size or in various biochemical parameters between the genotypes at 3 months of age (Table 1). In the longitudinal analyses using the first cohort of mice, there was no apparent difference in body weight between genotypes even under the HF diet condition by around 5–6 months of age (Fig. 1A), which is consistent with previous results (Enerback et al., 1997; Liu et al., 2003). However, the Ucp1−/– mice tended to become more obese gradually from the age of young adults compared with Ucp1+/+ mice. The Ucp1−/– males similarly developed diet-induced obesity with age (data not shown), but the age-associated development of obesity in Ucp1−/– mice was more remarkable in female mice given the HF diet (Fig. 1A), despite no significant difference in energy intake between the genotypes in either diet group (Fig. 1B). An age-associated increase in obesity was also observed in Ucp1−/– mice fed another type of HF diet containing 42% kcal from fat, TD88137 (Harlan TEKLAD; data not shown). Regression analysis using data from the second cohort of female mice fed the HF diet confirmed the age-associated development of diet-induced obesity in Ucp1−/– mice (Fig. 1C). The data showed increases in tissue mass paralleled those in body weight with age, but the slopes of correlation in many parameters such as BAT, white adipose tissue (WAT), heart, and liver were significantly greater in Ucp1−/– mice than in Ucp1+/+ mice (Fig. 1D–G), suggesting the progress of complication such as cardiac hypertrophy and fatty liver. On the contrary, there was no difference in the increase in skeletal muscle mass between the genotypes, which reached a plateau at the age of approximately 6 months, meaning the maturity of muscle development (Fig. 1H).
|Genotype||Body weight (g)||Glucose (mg/dL)||Insulin (ng/dL)||Leptin (ng/dL)||Free fatty acid (mEq/L)||Cholesterol (mg/dL)|
|Male +/+||26.7 ± 0.4 (19)||150 ± 6 (19)||0.49 ± 0.10 (12)||4.94 ± 1.76 (10)||1.57 ± 0.10 (17)||94.0 ± 4.2 (17)|
|–/–||27.0 ± 0.4 (18)||166 ± 6 (18)||0.43 ± 0.06 (10)||4.22 ± 1.16 (10)||1.56 ± 0.09 (18)||99.0 ± 3.3 (18)|
|Female +/+||20.6 ± 0.4 (15)||141 ± 6 (15)||0.45 ± 0.09 (12)||2.90 ± 0.75 (10)||1.24 ± 0.08 (15)||75.0 ± 4.9 (15)|
|–/–||21.0 ± 0.2 (22)||134 ± 4 (22)||0.47 ± 0.09 (15)||2.85 ± 0.20 (14)||1.22 ± 0.08 (21)||74.2 ± 4.3 (22)|
Despite the age-associated increase in susceptibility to diet-induced obesity in UCP1-deficient mice, there was no difference in the lifespan under the standard diet condition between genotypes (Fig. 2). The 50% survivals were 27.4 and 28.6 months old in Ucp1+/+ and Ucp1−/–, respectively. The maximum lifespan was 34.0 and 36.2 months old in Ucp1+/+ and Ucp1−/–, respectively.
Pathogenesis of diet-induced obesity in UCP1-deficient mice
To verify the pathogenesis of late-onset obesity in Ucp1−/– mice, we analyzed the second cohort of mice fed the HF diet for 8 months in comparison with those fed the standard chow diet. We first examined the glucose tolerance of 6-month-old mice, at which age the mice fed the HF diet were already obese compared with the littermates fed the standard diet; and the severity was significantly higher in Ucp1−/– mice than in Ucp1+/+ mice (data not shown). The data from the intraperitoneal glucose tolerance test (IPGTT) indicated that the impairment of glucose tolerance was concomitant with obesity, which impairment was more serious in the Ucp1−/– mice (Fig. 3A). In mature adults (11-month-old mice), the obesity in Ucp1−/– mice fed the HF diet was morbid and associated with resistance to insulin and leptin (Fig. 3B–E). Histological analysis revealed increased fat accumulation not only in the WAT but also in the BAT and liver of Ucp1−/– mice fed the HF diet, compared with that in the Ucp1+/+ mice (Fig. 4). The morphological changes in BAT were characterized by lipid droplets of increased size and fat cells with an appearance similar to that in WAT, which changes were similar to those in BAT of β-less mice with a very low level of UCP1 (Bachman et al., 2002).
Effects of UCP1 deficiency and diet condition on the protein expression in BAT
In the tissue analysis, a remarkable hypertrophy of fat tissues, especially in BAT, was found in the obese Ucp1−/– mice fed the HF diet (Figs 1 and 4), suggesting a possible deterioration of BAT function. Because UCP2 and UCP3, homologues of UCP1, are also expressed in BAT and a compensated increase in UCP2 mRNA level in BAT of Ucp1−/– mice has been reported previously (Enerback et al., 1997), we examined the levels of mitochondrial proteins including UCPs in the BAT of the 11-month-old mice fed the standard chow or HF diet. As shown in Fig. 5, we found a significant increase in the UCP3 level (1.9-fold) in non-obese Ucp1−/– mice compared with the level in the control mice under the standard diet condition, suggesting an adaptive induction to compensate for the lack of UCP1 thermogenesis. The HF diet increased the UCP3 level (1.6-fold), as well as the UCP1 level (1.2-fold), in Ucp1+/+ mice. Conversely, the augmented expression of UCP3 in BAT of Ucp1−/– mice given the standard diet was down-regulated considerably by the HF diet. We could not detect a reliable signal for UCP2 protein, although we observed a significant increase in its mRNA level in the BAT of Ucp1−/– mice compared with that in Ucp1+/+ mice (Table 2), similar to previously reported results (Enerback et al., 1997). The cytochrome c oxidase subunit IV (COX-IV) level in BAT was also reduced significantly in Ucp1−/– mice compared with that in Ucp1+/+ mice. The manganous superoxide dismutase (Mn-SOD) levels were higher in the HF diet groups than in the standard diet groups of both genotypes, suggesting a rise of oxidative stress in the obese state.
|UCP2||2.73 ± 0.26***||1.49 ± 0.10||3.66 ± 0.70***,##|
|β3AR||2.89 ± 0.27***||1.39 ± 0.26||1.03 ± 0.09|
|PGC-1||3.49 ± 0.45***||1.23 ± 0.04||0.79 ± 0.09##|
|BAT||HSL||0.83 ± 0.08||0.67 ± 0.07*||0.50 ± 0.05**|
|ACC||0.98 ± 0.24||0.63 ± 0.13*||0.34 ± 0.09**|
|FAS||0.98 ± 0.23||0.55 ± 0.10*||0.29 ± 0.01***|
|aP2||2.18 ± 0.17***||1.20 ± 0.06||1.43 ± 0.11**,#|
|Leptin||1.87 ± 0.16**||0.83 ± 0.16||5.46 ± 1.23***,###|
|UCP2||0.97 ± 0.05||0.93 ± 0.05||1.32 ± 0.11*,##|
|β3AR||0.99 ± 0.11||0.79 ± 0.04||0.23 ± 0.02***,###|
|HSL||1.36 ± 0.17||0.98 ± 0.10||0.78 ± 0.05|
|WAT||ACC||1.58 ± 0.23||0.82 ± 0.20||0.19 ± 0.01*,#|
|FAS||1.81 ± 0.24*||0.86 ± 0.17*||0.38 ± 0.04**,#|
|aP2||1.49 ± 0.10**||1.13 ± 0.10||1.41 ± 0.06*|
|Leptin||1.23 ± 0.18||1.08 ± 0.10||7.18 ± 0.53***,###|
We also examined the levels of cAMP-dependent protein kinase (PKA), which consists of catalytic and regulatory subunits, in the BAT, because this signaling molecule plays an important role to mediate the stimulation of β3-AR for UCP1 thermogenesis and lipolysis in BAT. Under the standard diet condition, there was no significant difference in the level of PKA-C between genotypes, whereas the PKA-RIIb level was significantly higher (1.2-fold) in Ucp1−/– mice than in Ucp1+/+ mice. Under HF diet condition, however, the PKA-RIIb level was considerably reduced (42%) in Ucp1−/– mice, but not in Ucp1+/+ mice, compared with that of the standard diet group. The levels of PKA-C were also reduced to about 60% of those of chow diet group in both genotypes.
Changes in fatty-acid metabolism in UCP1-deficient mice with age and diet
From the protein analysis, the ability of energy expenditure appeared to have declined in the BAT of obese Ucp1−/– mice fed the HF diet (Fig. 5). To further understand the molecular mechanism underlying the development of obesity in Ucp1−/– mice, we examined the expression of genes involved in energy expenditure and fat metabolism in the adipose tissues of young and mature adult mice (Table 2). In the RNA analysis, we found notable differences in gene expression between genotypes and profound effects of age and HF diet. In both BAT and WAT of 3-month-old mice, the mRNA levels of genes related to fatty acid metabolism were constitutively higher in Ucp1−/– mice than in Ucp1+/+ mice. Particularly, marked induction of peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1) and β3-AR was found in the BAT of Ucp1−/– mice. The adipocyte fatty acid binding protein (aP2) level was also increased significantly in both BAT and WAT of Ucp1−/– mice. However, the expression of these genes in Ucp1−/– mice was down-regulated to levels similar to those in Ucp1+/+ mice in the mature adult (11 months old/chow). Hormone-sensitive lipase (HSL), acetyl-CoA carboxylase (ACC), or fatty acid synthase (FAS) are key molecules for generation of fatty acid, the major energy substrate, in adipose tissues. The gene expression of HSL, ACC, and FAS was decreased significantly in the BAT of Ucp1−/– mice with age. In the obese mice fed the HF diet, the expression of these genes was further attenuated in the BAT and WAT of Ucp1−/– mice compared with that in Ucp1+/+ mice. Significantly reduced expression of PGC-1 and β3-AR genes was also detected in BAT and WAT, respectively, although the mRNA levels of UCP2, aP2 and leptin were increased significantly in the BAT and WAT of the obese Ucp1−/– mice given the HF diet (Table 2). In addition, norepinephrine-stimulated lipolysis was markedly reduced in both the BAT (81%) and WAT (52%) of obese Ucp1−/– mice fed the HF diet compared with that in these tissues of Ucp1+/+ mice (Fig. 6). A significantly lower level of β3-AR mRNA was also confirmed in the BAT and WAT of the 19-month-old Ucp1−/– mice compared with those in Ucp1+/+ mice (data not shown).
In human society the prevalence of obesity increases with age until 60–70 years. Similarly, the UCP1-deficient mice developed diet-induced obesity with age under the normal housing temperature of 23 °C, although the obesity phenotype was not apparent at the young-adult stage. This phenotype in the young mice is similar to the previous study of Liu et al. (2003), who reported that two-month-old Ucp1−/– mice were resistant to obesity, suggesting an induction of alternative or additional mechanisms compensating the loss of UCP1 thermogenesis in the young animals. The resistance to obesity at a young age could be related to cold-sensitive phenotype in Ucp1−/– mice, because notable gene induction of β3-AR and PGC-1 (Puigserver et al., 1998), the latter a key regulator of cold-induced adaptive thermogenesis, was seen in the BAT of 3-month-old Ucp1−/– mice compared with that in Ucp1+/+ mice. The induction of β3-AR and PGC-1 genes for thermoregulation might contribute to the resistance to diet-induced obesity in the young mutant mice; however, the increased mRNA levels were returned to the levels in 11-month-old Ucp1+/+ mice. Likewise, we observed that the age-associated development of diet-induced obesity in Ucp1−/– mice was more remarkable in females than in males, while Liu et al. (2003) reported that male Ucp1−/– mice were resistant to diet-induced obesity. Since females are basically more sensitive to diet-induced obesity than males (Rodriguez & Palou, 2004), the effect of UCP1 deficiency on body weight regulation might be more apparent in females.
PKA activation via β3-AR in adipose tissues has a pivotal role in the regulation of cold-induced thermogenesis and lipolysis (Prusiner et al., 1968; Cummings et al., 1996; Lowell & Spiegelman, 2000). In the cold, the sympathetic nervous system is activated, which then stimulates vasoconstriction (to suppress heat loss) and heat production. PKA-RIIb is the principal PKA regulatory subunit in BAT (Cummings et al., 1996). PKA activates HSL, increasing the concentration of FFA, which in turn increases the activity of UCPs (Echtay et al., 2002). Since UCP3 has been implicated in the mechanism of adaptive thermogenesis (Clapham et al., 2000; Lowell & Spiegelman, 2000), the significant increases in the levels of UCP3 and PKA-RIIb in the BAT of adult Ucp1−/– mice fed the standard diet may indicate enhanced β-adrenergic responsiveness similar to that seen in cold exposure, contributing to thermogenesis and fatty acid metabolism. The thermogenic role of UCP3 is still controversial, but Mills et al. (2003) recently indicated in their report based on a study using UCP3-deficient mice that UCP3 was required for the rise in skeletal and core temperature associated with the administration of an amphetamine-type stimulant.
In contrast, the increased expression of UCP3 and PKA under the standard diet condition was down-regulated markedly in the BAT of Ucp1−/– mice under the HF diet condition; and the Ucp1−/– mice developed severe obesity with insulin and leptin resistance with age. Since the primary role of UCP3 has also been demonstrated to be the regulation of oxidative stress or the outward translocation of fatty acids away from the mitochondrial matrix (Samec et al., 1998; Himms-Hagen & Harper, 2001; Echtay et al., 2002; Schrauwen et al., 2003), a reduction in the UCP3 level would increase the accumulation of non-esterified fatty acids within the mitochondrial matrix. This process may increase lipid peroxidation and impair mitochondrial function. Increased lipid peroxidation and/or mitochondrial damage has been observed in the skeletal muscle of mice lacking UCP3 (Brand et al., 2002) or of diabetic subjects with a decreased level of UCP3 (Schrauwen et al., 2001). So, the augmentation of the Mn-SOD level in the mitochondria of the obese mice could be a defense mechanism against oxidative stress and mitochondrial damage. We also found that the level of COX-IV in BAT was considerably lower in Ucp1−/– mice than in Ucp1+/+ mice. We do not know why the COX-IV level was reduced in Ucp1−/– mice. However, when we calculated the ratios of UCP1 or UCP3/COX-IV (our unpublished data), an increase of UCP3-dependent uncoupling in Ucp1−/– mice was suggested under the standard diet condition. HF diet significantly increased the ratios of UCP1/COX-IV and UCP3/COX-IV in BAT of Ucp1+/+ mice; however, further increase in UCP3/COX-IV ratio was not observed in Ucp1−/– mice fed the HF diet. This may suggest a limitation of energy dispensation (i.e. balance between coupling and uncoupling) in UCP1-deficient mice.
Given the results on norepinephrine-induced lipolysis, the obesity phenotype in Ucp1−/– mice appears to be caused at least in part by the large attenuation of β-adrenergic responsiveness in the animals due to the combination of aging and HF diet, which interpretation is also supported by the data on the change in gene expression of HSL and β3-AR in the adipose tissues. The remarkable decrease in the β3-AR mRNA level in the WAT of the obese Ucp1−/– mice is consistent with that found in the study of Nadler et al. (2000), who suggested that some degree of dedifferentiation had taken place in the adipose tissue of the obese mice. Moreover, the marked reduction in ACC and FAS in the BAT and WAT of Ucp1−/– mice strongly suggests a decrease in de novo synthesis of fatty acid in the adipose tissues. Because fatty acid is not only an energy substrate but also an activator of UCPs (Echtay et al., 2002), the reduced fatty acid generation may also affect the function of UCPs in Ucp1−/– mice. Taken together, the data suggest that the attenuation of fatty acid turnover might contribute to fat accumulation in the Ucp1−/– mice given the HF diet, in which the substrate is not sufficiently burned and released as heat.
With respect to lifespan of UCP1-deficient mice, Golozoubova et al. (2001) have demonstrated that, unlike Ucp1+/+ mice, Ucp1−/– mice cannot live in a room at 4 °C, although the mutant mice survived in the cold for several months. This phenotype of Ucp1−/– mice is similar to that of humans. Although a very low level of UCP1 can be detected in humans, this amount does not allow us to survive in the cold, indicating an essential role of UCP1 thermogenesis in maintaining mammalian life in the cold. However, the influence of UCP1 deficiency on lifespan at normal housing temperature was unknown. Unexpectedly, UCP1 deficiency did not affect the lifespan of mice at 23 °C in this study. Therefore, the lack of UCP1 may not act negatively on lifespan in humans by itself, if we live in a well-controlled environment including diet condition.
In summary, we showed that UCP1-deficiency increased the susceptibility to diet-induced obesity with age, although the loss of UCP1 function does not simply cause obesity nor shorten the lifespan in mice under the conditions of low-fat diet and normal room temperature. The increased expression of molecules involved in the mechanisms of thermogenesis and fatty acid utilization in Ucp1−/– mice, which may be a mechanism compensating for UCP1 deficiency, was attenuated with successive intake of high-fat diet and aging. This etiology of obesity in Ucp1−/– mice seems to be similar to that seen in adult humans who cannot undergo UCP1 thermogenesis sufficiently, because of a mutation of the β3-AR gene and/or an age-related decline in β-adrenergic responsiveness (Lean, 1992; Clement et al., 1995; and 1996; Kogre et al., 1998; Schwartz et al., 1990). In this context, the UCP1-deficient mice would serve as a unique model for research to circumvent the late-onset obesity and diabetes in humans, although further details of the compensatory mechanism remain to be revealed.
Animals. Ucptm1 knockout mice on a congenic C57BL/6 J background were established by backcrossing and typing with microsatellite markers as described (Hofmann et al., 2001). The mice used in this study were N12–N15 generations. The mice were bred and reared at 23 ± 1 °C under artificial lighting for 12 h per day on a standard chow diet (Diet No.CE-2, 344 kCal/100 g, 11.6% kCal from fat, CLEA JAPAN, INC.) and tap water provided ad libitum in the animal facility of the National Institute for Longevity Sciences. Although the size of Ucp1–/– mice was smaller than that of Ucp1+/+ mice at weaning, the mutant mice grew normally and there was no difference in size or in various biochemical parameters between the genotypes at 3 months of age (Table 1). These mice were housed in groups of 2–4 and were used for the longitudinal study according to the institutional guidelines for animal care.
Diet study. In the longitudinal analysis of phenotypes, 3-month-old mice in the first cohort were fed a standard chow diet or a high-fat diet (HF: Diet No. B15040, 423 kCal/100 g, 41.9% kCal from fat, CLEA JAPAN, INC) which consists of CE-2 (80%) and N-Neopowder-T (20%). N-Neopowder-T contains beef tallow (80%), milk sugar (9%), dextrin (4.7%), casein (4%), glyceric acid ester (2%), and NaH2PO4 (0.3%). The components of fatty acids in beef tallow was 2.8% myristic acid (14 : 0), 22.0% palmitic acid (16 : 0), 7.1% palmitoleic acid (16 : 1), 15.2% stearic acid (18 : 0), 50.5% oleic acid (18 : 1), 1.9% linolic acid (18 : 2), and 0.6% linolenic acid (18 : 3). The numbers of mice were 10 males and 10 females in the Ucp1+/+ standard diet group, 10 males and 12 females in the Ucp1−/– standard diet group, 9 males and 7 females in the Ucp1+/+ HF diet group, and 8 males and 11 females in the Ucp1−/– HF diet group. The changes in body weight and energy intake were examined every month. The energy intake was calculated from the measure of food intake in each cage for 1 week at the beginning of every month. The mice fed the standard diet were maintained until death to evaluate the lifespan. The female mice in the second cohort (n = 23 for each genotype) were sampled to determine the effects of diet and aging on physiological function and pathogenesis of obesity in the mice. The left pads of inguinal, gonadal, and retroperitoneal white adipose tissues (I-, G-, R-WAT, respectively) and gastrocnemius skeletal muscles were used for the analysis of tissue mass.
Biochemical analysis. Intraperitoneal glucose tolerance test (IPGTT) using 1.5 mg of glucose/g body weight was performed after 17 h of starvation. Blood samples were collected from a tail vein before and 30, 60, and 120 min after the glucose injection and used immediately to determine the glucose level by use of a glucometer (NovoAssist Plus, Novo Nordisk Tokyo, Japan). The serum insulin, leptin, free fatty acid (FFA), and total cholesterol levels were measured by using commercial assay kits (Ultrasensitive insulin ELISA, Mercodia, Uppsala, Sweden; Enzyme Immunoassay kit for leptin, Cayman, Ann Arbor, MI, USA; NEFA C-test and T-Cho E-test, Wako Pure Chemical, Osaka, Japan). The protein concentration in tissue samples was measured by conducting BCA protein assays (PIERCE, Rockford, IL, USA).
Immunoblotting was performed by using the mitochondrial fraction recovered from BAT and specific antibodies against UCP1 (STRATAGENE, La Jolla, CA, USA), UCP3 (CHEMICON, Temecula, CA, USA), COX-IV (Molecular Probes, Eugene, OR, USA), and Mn-SOD (StressGen, Victoria, BC, Canada) as described (Kontani et al., 2002). Equal amounts of mitochondrial protein (10 µg) were separated on 12.5% gels (Daiichi Pure Chemicals; Tokyo, Japan) and transferred onto immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were incubated with the specific antibodies for each protein. After the secondary antibody reaction for 1 h at room temperature, the specific signals were detected by using an ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Similarly, immunodetection of PKA was carried out by using 30 µg of BAT lysate and specific antibodies against the catalytic subunit (PKA-C, BD Transduction Laboratories, San Diego, CA, USA) and regulatory subunit IIβ (PKA-RIIb, CHEMICON, Temecula, CA, USA). The resulting images were quantified with NIH Image (version 1.63).
Lipolytic activity of adipose tissues was measured as described (Suzuki et al., 1984). Pieces of adipose tissue were incubated with mild shaking at 37 °C for 60 min in Krebs-Ringer bicarbonate buffer containing 2% bovine albumin (essentially fatty acid free; Sigma-Aldrich, St Louis, MO, USA) in the absence (basal lipolysis) or presence of 100 µm norepinephrine (BAT: ∼25 mg tissue/mL, WAT: 50–75 mg tissue/mL). After the reaction had been stopped by cooling the flasks in ice water for 10 min, the FFA levels in the reaction buffer were determined. The difference between norepinephrine-stimulated lipolysis and basal lipolysis was considered to be norepinephrine-induced lipolysis.
RNA analysis. Total RNA (15 µg for BAT and 10 µg for WAT), prepared from the tissues with TRIzol (Invitrogen, Carisbad, CA, USA), was analyzed by Northern blotting as described previously (Yamashita et al., 1999). Blots were hybridized successively with probes (labeled with [32P] dCTP) for the mRNAs of UCP2, β3AR, PGC-1, HSL, ACC, FAS, aP2, leptin, and 18S rRNA. As well as the probes for UCP2 and leptin mRNAs and 18S rRNA (Enerback et al., 1997; Yamashita et al., 1999), probes for β3AR, PGC-1, HSL, ACC, FAS, and aP2 mRNAs were produced by the reverse transcription PCR technique. The sequences used were the following: β3AR, positions 377–789 of the rat sequence (GenBank accession No. M74716); PGC-1, positions 737–1491 of the mouse sequence (GenBank accession No. NM008904); HSL, positions 2280–2747 of the rat sequence (GenBank accession No. X51415); ACC, positions 1638–2366 of the rat sequence (GenBank accession No. J03808); FAS, positions 5706–6970 of the mouse sequence (GenBank accession No. AF127033); and aP2, positions 3–499 of the mouse sequence (GenBank accession No. K02109). The PCR products were sequenced after subcloning into the pCRII or pCR2.1 vector (Invitogen, Carisbad, CA, USA). Hybridization signals were quantified with Fuji Bioimage.
Histological analysis. Tissues were fixed immediately in 10% formaldehyde neutral buffer solution and embedded into paraffin. Tissue sections of 3 µm were stained with hematoxylin and eosin.
Statistical analysis. Data were expressed as the mean ± SE. Significant differences among groups were assessed by analysis of variance (anova), repeated measure anova, or analysis of covariance (ancova) with Fisher's PLSD test.
The authors thank L. P. Kozak for the UCP1-deficient mice and helpful discussion. We also thank for Y. Minokoshi for critical reading of the manuscript. This work was supported by grants (11C-04, 15C-8) from the program Research Grant for Longevity Sciences and by a grant from the program Health Sciences Research Grants, Comprehensive Research on Aging and Health, of the Ministry of Health, Labor, and Welfare to H. Yamashita.
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