INTRODUCTION

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UNCOUPLING PROTEIN (UCP) 1, the first identified UCP, is a mitochondrial inner membrane protein that uncouples cellular respiration from ATP synthesis by dissipating the proton electrochemical gradient (18). This gradient builds up across the inner mitochondrial membrane to drive the ATP synthetic pathway (18). Brown adipose tissue (BAT) contains numerous mitochondria and is characterized by the presence of UCP1 (13, 20), which is therefore a potentially important determinant of energy expenditure in rodents and neonates of larger mammals, including humans (18). Thermogenesis and energy expenditure through UCP1 has been shown to be under the control of sympathetic nerves (6, 13, 20). Unlike UCP1, UCP2, identified recently as a second UCP family member, is ubiquitously expressed in humans and rodents (7, 11). UCP3, a third member of the UCP family, is preferentially expressed in skeletal muscle in humans and rodents (3, 15, 27). The UCP family has been shown to respond to a variety of physiological factors such as thyroid hormones, free fatty acids (FFA), leptin, and induction of starvation or obesity (2, 8, 15, 16, 29).

Cold exposure is well known to increase energy expenditure and thermogenesis (6, 13, 20). The thermogenic response of BAT to cold exposure is mediated in part by adrenergic stimulation and thyroid hormone (1, 13, 20), which results in an acute increase in UCP gene transcription rate (20). The ob/ob and db/db mice develop severe obesity because of their leptin signaling deficiencies (4, 29). The lowered body temperature characteristic of these genetically obese mice also contributes to development of obesity (14, 12, 26, 24). However, little is known about thermogenic roles of UCPs in these obese animals when they are exposed to cold.

The present study aimed to examine whether UCP family mRNAs together with lipolytic activity in db/db obese mice are normally regulated in response to cold exposure.

	MATERIALS AND METHODS

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Subjects and experimental procedures. Mature male obese (db/db) and lean (C57Ksj) littermates (Seac Yoshitomi, Fukuoka, Japan) at 12-14 wk of age were housed in a room illuminated daily from 0700 to 1900 (a 12:12-h light-dark cycle) with temperature at 21 ± 1°C and humidity 55 ± 5%. The mice were allowed free access to standard pelleted rat chow (Clea Japan, Tokyo, Japan) and tap water. All of the mice were handled to equilibrate their arousal levels for 5 min daily during five successive days. Rectal temperature was measured at 1000-1030 by insertion of a plastic-coated thermocouple (Terumo, Tokyo, Japan) in the rectum. Mice were equally divided into the cold exposure and the room temperature control groups. Rectal temperature of mice was measured 12 and 24 h after cold (4°C) exposure and 12 h after rewarming to room temperature (21°C). The animals used were treated in accordance with the Oita Medical University Guidelines for the Care and Use of Laboratory Animals.

Measurement of FFA concentration. Each mouse was implanted with a Silastic catheter (no. 00; Shinnetsu, Tokyo, Japan) 1 wk before serum sample collection. The catheter was inserted through the right jugular vein with the inner end fixed immediately outside the right atrium. A sampling tube was attached to a 29-gauge multisampling needle (Terumo) to prevent air from being sucked in the system. Serum samples were collected at 12 and 24 h after cold exposure and 12 h after rewarming. Samples from the right atrium-implanted catheter were taken at 1100-1130 and immediately were separated in serum for storage at 20°C until measurement.

Preparation of adipose tissue and skeletal muscle. After decapitation at 12 and 24 h after cold exposure and 12 h after rewarming, tissues of the intrascapular BAT, epidydimal adipose tissue [white adipose tissue (WAT)], and soleus muscle were surgically removed, and the samples were immediately frozen in liquid nitrogen to store at 80°C for RNA extraction. Time lag between blood samplings was limited to a few minutes.

Preparation of rat cDNA probe. PCR primers of 5'-CATCTTCTGGGAGGTAGC-3' and 5'-AAGACAGGGCAGGAATGG-3' were designed for the coding region of the rat UCP2 gene, and primers 5'-GTTACCTTTCCACTGGACAC-3' and 5'-CCGTTTCAGCTGCTCATAGG-3' were designed for the UCP3 gene. Reverse transcription of 10 µg total RNA from C57Ksj mice was performed using Moloney murine leukemia virus RT (Life Technologies, Gaithersburg, MD). PCR was carried out with Taq DNA polymerase (Amersham International, Buckinghamshire, UK) and 20 pmol of primers. The reaction profiles were as follows: denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min for 30 cycles. The PCR fragment of 1,012 bp was subcloned in pCRTM2.1 vector (TA cloning kit; Invitrogen, San Diego, CA), and the nucleotide sequence of amplified cDNA was confirmed by sequencing. The nucleotide sequences were determined by the dideoxynucleotide chain termination method using synthetic oligonucleotide primers that were complementary to the vector sequence and the ABI373A automated DNA Sequencing System (Perkin-Elmer, Norwalk, CT). All DNA sequences were confirmed by reading both DNA strands. The UCP1, lipoprotein lipase (LPL; GenBank accession no. D28561, L03294), and 18S ribosomal RNA probe were generated in an analogous fashion.

RNA extraction and Northern blot analysis. Total cellular RNA was prepared from various rat tissues with the use of Isogen (Nippon Gene, Toyama, Japan) according to the manufacturer's protocol. Total RNA (20 µg) was electrophoresed on 1.2% formaldehyde-agarose gel, and the separated RNA was transferred to a Biodyne B membrane (Pall Canada, Ontario, Canada) in 20× saline-sodium citrate by capillary blotting and was immobilized by exposure to ultraviolet light (0.80 J). Prehybridization and hybridization were carried out according to the method described by Yang et al. (28). Membranes were washed under high-stringency conditions. After the membranes were washed, the hybridization signals were analyzed with a BIO-image analyzer BAS 2000 (Fuji Film Institution, Tokyo, Japan). The membranes were stripped by exposure to boiling 0.1% SDS and were rehybridized with a ribosomal RNA that was used to quantify the amounts of RNA species on the blots.

	RESULTS

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Changes in rectal temperature after exposure to cold and rewarming. Basal rectal temperature of db/db obese mice (36.6 ± 0.2°C) was lower than that of lean littermates (P < 0.05; Fig. 1). As shown in Fig. 1, reduction of rectal temperature progressed during cold exposure until 24 h in both the obese and lean groups [db/db, P < 0.01, lean, P < 0.05; F(4) = 267.1, P < 0.01 in the time course factor], although the magnitude of the decrement was greater in the obese group than that in the lean group [F(1,4) = 6.6, P < 0.01]. Rectal temperature at 24 h after cold exposure decreased by 4.1°C in the db/db obese group but by 1.3°C in the lean controls. Rectal temperature in both the db/db obese and the lean groups returned to their baseline temperature at 12 h after rewarming (obese, P > 0.05; lean, P > 0.05), leaving their rectal temperature different between these groups (P < 0.05; Fig. 1).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Time course changes in rectal temperature after exposure to cold and rewarming in db/db obese and lean littermates. Body temperature in the db/db obese group decreased more predominantly than the lean controls after cold exposure. Each temperature on top represents the room temperature at exposure to cold (4°C) and rewarming (21°C). Symbols and bars indicate means ± SE (n = 4 for each).

Changes in serum FFA concentration and WAT LPL mRNA after cold and rewarming. In the lean controls, the serum concentration of FFA was elevated 4 h after cold exposure (P < 0.01 vs. 0 h; Table 1). On the contrary, LPL mRNA expression was reduced 12 h after cold exposure (P < 0.05 vs. 0 h; Table 1). Serum FFA and LPL mRNA, which changed during cold exposure in the lean groups, returned to their baseline at 12 h after rewarming (Table 1). In the obese group, however, there was no such change after cold exposure either in FFA concentration or in LPL mRNA (Table 1). Both FFA concentration and LPL mRNA expression in the obese group were higher than in the lean controls [FFA: F(1) = 221.4, P < 0.01 in the group factor; LPL: F(1) = 207.4, P < 0.01 in the group factor; Table 1].

Basal UCP expression of BAT, WAT, and skeletal muscle in db/db obese and Ksj lean mice. Basal mRNAs of BAT UCP1 and UCP3 in db/db obese mice were lower by 0.7- and 0.8-fold than those in lean littermates, respectively (P < 0.05 for each; Fig. 2A). In contrast, basal UCP2 mRNA was higher in the obese than in the lean littermates by 2.0-fold (P < 0.01; Fig. 2B). Basal skeletal muscle UCP3 in the obese mice showed no statistical difference compared with that in the lean controls (Fig. 2B).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Basal mRNA expression of uncoupling protein (UCP) 1 and UCP3 in brown adipose tissue (BAT), UCP2 in white adipose tissue (WAT), and UCP3 in skeletal muscle in db/db obese and lean littermates. Northern blot analysis of UCPs was performed in total RNAs (20 µg/lane) of the samples from BAT (A) and WAT and skeletal muscle (B). Bars indicate means ± SE (n = 4 for each). rau, Relative arbitrary units. * P < 0.05 and ** P < 0.01 vs. corresponding lean littermates.

Time course changes of UCPs in response to cold and rewarming. As shown in Fig. 3, time course upregulation of BAT, WAT, and skeletal muscle UCPs was more predominant in the lean group than in the obese group [BAT UCP1: F(1,3) = 26.3, P < 0.01; BAT UCP3: F(1,3) = 18.6, P < 0.01; WAT UCP2: F(1,3) = 13.1, P < 0.01; skeletal muscle UCP3: F(1,3) = 6.5, P < 0.01]. UCP levels in the lean group returned to baseline levels 12 h after replacement to 21°C room temperature (Fig. 3). Time course changes in UCP expression were not significant in the db/db obese group.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Time course changes in mRNA expression of UCP1 and UCP3 in BAT, UCP2 in WAT, and UCP3 in skeletal muscle after cold exposure. UCP expression in the lean controls was elevated in response to 4°C and returned to the baseline levels after replacement to 21°C room temperature. Northern blot analysis of UCP expression was performed in total RNA (20 µg/lane) of the samples from BAT (A and B), WAT (C), and skeletal muscle (D). Symbols and bars indicate means ± SE (n = 4 for each).

	DISCUSSION

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In agreement with previous reports (12, 14, 24, 26), the present study confirms that db/db obese mice were impaired in maintaining normal rectal temperature during cold exposure because the obese mice had a lower basal temperature that decreased more than that in lean littermates after cold exposure. In addition, the results showed that the thermogenic capacity of db/db obese mice was diminished throughout the time course of the acclimation to cold.

Expression of UCP1 and UCP3 mRNAs in BAT, UCP2 mRNA in WAT, and UCP3 mRNA in skeletal muscle was downregulated in db/db obese mice in response to cold exposure. UCP1 mRNA expression in BAT is well known to be upregulated mainly by activation of sympathetic nerves that abundantly innervate BAT (13, 20). Sympathetic regulation plays a major role in upregulation of BAT UCP1 in response to cold exposure (20). UCP1 in brown fat is thus well suited for thermoregulation in rodents. Basal UCP1 mRNA expression in BAT and basal UCP2 (unpublished data) and UCP3 mRNA expression in BAT were impaired in the present db/db obese mice. Indeed, a previous report that ob/ob and db/db mice did not respond to cold exposure supports our results (12, 14, 23-25). These responses of the UCP family to changes in ambient and inner circumstance may affect thermogenic activity, although the relation between BAT UCP2 and UCP3 expression and thermogenesis has not been clarified to date. The findings can be taken to indicate that some part of the signaling process from afferent perception of cold exposure to the efferent effects on target organs seems to be disrupted in db/db mice. At the present time, however, the mechanism of the impairment in these obese animals remains obscure. It seems reasonable to suggest that leptin is a probable candidate to explain the defect. To support the assumption, 1) leptin has been shown to increase sympathetic innervation (20), 2) leptin upregulates BAT, WAT, and skeletal muscle UCPs (5, 22), and 3) db/db obese mice are leptin receptor-mutated animals and are deficient in leptin action (4).

Recent years have seen an increase in our understanding of FFA as a modulator of UCP regulation (10, 21). Evidence to date reveals that FFA increases mitochondrial UCP2 expression in WAT and UCP3 in skeletal muscle, providing a mechanism to dispose of excess fatty acids (21). LPL is an adipocyte-born enzyme that cleaves fatty acids from circulating lipoproteins (19). Cold exposure reportedly increased plasma levels of FFA and decreased LPL mRNA in WAT immediately after cold exposure (9, 25). In the present results from lean mice, serum FFA concentration was increased and LPL mRNA in WAT was decreased by cold exposure. The findings are very much in line with the foregoing discussion. Unlike the lean data, those from db/db obese mice showed higher levels of both serum FFA concentration and LPL mRNA expression. Precisely why elevation of FFA levels can not drive upregulation of UCP2 and UCP3 remains unclear. We previously reported that chronic elevation of serum FFA concentration failed to upregulate UCP family expression in genetically obese mice, although acute elevation of FFA was essential for upregulation of UCP2 and UCP3 expression in mice (10). In this view, it would be very much consistent with the present results. Impaired responses of FFA and LPL in db/db mice may cause poor responsiveness of the UCP family to cold exposure, which contributes to abnormalities of energy metabolism in these obese mice during cold. However, the functional connection between UCP2 and -3 expression and thermogenesis has not been clarified to date. Further studies are required to specifically address the relation functionally.

In conclusion, responses of the UCP family to cold exposure regulated by sympathetic innervation and/or lipolytic fatty acids was demonstrated to be impaired in db/db obese mice, a model of genetic obesity. The impaired lipolytic activity may contribute to the inactivation of the UCP family in db/db obese mice.

Perspectives