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(The FASEB Journal. 1998;12:715-724.)
© 1998 FASEB

RESEARCH COMMUNICATION

Role of UCP homologues in skeletal muscles and brown adipose tissue: mediators of thermogenesis or regulators of lipids as fuel substrate?

S. Sameca, J. Seydouxa, and A. G. Dullooa,1

a Department of Physiology, Faculty of Medicine, University of Geneva, 1211 Geneva 4, Switzerland


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mRNA expressions of UCP2 and UCP3, two newly described genes with high sequence homology to the uncoupling protein UCP1 in brown adipose tissue (BAT), were examined in two skeletal muscles (gastrocnemius and soleus) as well as in interscapular BAT (IBAT) of the rat in response to food deprivation and controlled refeeding. In IBAT (a tissue highly dependent on lipids for thermogenesis), the pattern of mRNA expression of UCP2 and UCP3 closely follows that of UCP1: it was markedly down-regulated during food deprivation (when this tissue's thermogenesis and lipid fuel requirements are decreased) and restored to control levels by day 5 of refeeding. By contrast, in the gastrocnemius muscle (a mixed fiber type muscle with a high capacity to shift between glucose and lipids as fuel substrate), mRNA expression of both UCP2 and UCP3 mRNA was found to be markedly up-regulated during food deprivation (when this tissue's thermogenesis is also decreased but its lipid fuel utilization is increased). The expressions were subsequently found to be markedly down-regulated upon transition to refeeding, with mRNA levels remaining below control levels on days 3, 5, and 10 of refeeding (period of enhanced efficiency of body fat deposition). In the soleus muscle (an oxidative type muscle with higher dependency on lipids than the gastrocnemius, and hence with a lower capacity to shift between lipids and glucose as fuel substrate), UCP homologues were also found to be up-regulated during food deprivation, but changes in their mRNA expression contrast with those in the gastrocnemius muscle both in their much lower magnitude of response to food deprivation and in their more rapid restoration to control levels during refeeding. Up-regulation of UCP2 and UCP3 gene expressions in skeletal muscle during food deprivation was found to persist at thermoneutrality (i.e., under conditions of reduced thermoregulatory thermogenesis). Together, these tissue-dependent differential mRNA expressions of the UCP homologues in IBAT, gastrocnemius, and soleus muscles during food deprivation and refeeding are much more consistent with a role for UCP2 and UCP3 in the regulation of lipids as fuel substrate rather than as mediators of regulatory thermogenesis.—Samec, S., Seydoux, J., Dulloo, A. G. Role of UCP homologues in skeletal muscles and brown adipose tissue: mediators of thermogenesis or regulators of lipids as fuel substrate? FASEB J. 12, 715–724 (1998)


Key Words: obesity • cachexia • muscle • uncoupling protein • FFA


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IT IS WELL DOCUMENTED from studies of experimental starvation that in response to severe energy deficit, energy expenditure falls by a magnitude greater than can be accounted for by the loss of metabolically active tissues (1–4). This disproportionate reduction in metabolic rate is generally conceptualized as resulting from the operation of autoregulatory control systems, generally referred to as regulatory thermogenesis, whose suppression underlies an increase in metabolic efficiency and hence provides a buffer against the energy deficit (5). Studies of refeeding show that metabolic efficiency is enhanced for a considerable period of time (6–10) and that it underlies, at least in part, the disproportionately high rate of fat deposition commonly observed during weight recovery (11), whether after famine, experimental starvation, anorexia, pathophysiological conditions such as cancer and AIDS, or during the relapse of obesity after therapeutic dieting.

In an attempt to assess quantitatively the reduction in energy expenditure directed specifically at enhancing fat replenishment, we previously described and validated an experimental rat model of weight recovery in which, after substantial depletion of the fat stores by food restriction, the energy expenditure and energy-partitioning between protein and fat could be assessed by pair-feeding the refed rats with weight-matched controls (12, 13). It was demonstrated that during the first 2–3 wk of such controlled refeeding, the energetic efficiency during refeeding was increased by nearly twofold relative to the controls and that the energy thus conserved was directed at fat deposition, not to protein. Furthermore, this increased metabolic efficiency specific for accelerating fat replenishment was found to persist unabated during cold exposure or at thermoneutrality, i.e., under conditions in which sympathetic neural control of thermogenesis in the rat is markedly activated or suppressed, respectively (14). Together, these studies have led to the proposal that a component in the suppression of thermogenesis during the period of food restriction is dictated by the state of depletion of the fat stores (11) and persists during the early phase of refeeding, with the energy thus conserved being directed specifically to replenishment of the fat stores. Furthermore, this component of energy conservation can be dissociated from sympathetic neural modulation of thermogenesis (14) and, by extension, from the modulation of uncoupling protein (UCP1)2 activity in brown adipose tisssue (BAT), an important site of sympathetically mediated thermogenesis (15–20).

On the other hand, since skeletal muscle, another important site of energy conservation during starvation (21), is unresponsive to modulation by the sympathetic nervous system (SNS) in response to diet and cold (22), the possibility arises that this tissue, unlike BAT, may be an important site for mechanisms underlying thermogenic mechanisms that are SNS independent and whose modulation could underlie the elevated energetic efficiency for accelerated fat replenishment. The recent cloning of UCP3, a gene with 1) high sequence homology to UCP1 (23, 24), 2) in vitro uncoupling activity when overexpressed in yeast (25), and 3) high mRNA expression specific to skeletal muscles as well as to BAT (23–25) therefore provides a candidate gene that may underlie the postulated SNS-independent control of thermogenesis in this tissue.

To explore this possibility, we have compared, in our rat model of weight recovery, the pattern of changes in mRNA of UCP3 in skeletal muscles with those of UCP1 and UCP3 in interscapular BAT (IBAT) during food restriction and at various time points during the course of controlled refeeding. The mRNA expression of UCP3 was assessed both in the gastrocnemius (a mixed fiber muscle that possesses high activities for both glycolytic and oxidative enzymes) and the soleus (a predominantly slow-twitch fiber muscle that has a low potential for glycolytic activity and high activities for oxidative enzymes), two muscles with different capacities for shifting between glucose and lipids as fuel substrate (26–28). We have also assessed in these tissues the changes in mRNA expression of UCP2, another UCP homologue, which, unlike UCP1 and UCP3, has been shown to be ubiquitously expressed in rat and human tissues (29, 30).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and diets
Male Sprague-Dawley rats (6 wk old) (Zurich, Tierzucht, Switzerland) were adapted to room and cage environments for 1 wk. They were caged singly in a temperature-controlled room (22°C) with a 12 h light-dark cycle, maintained on a commercial pelleted laboratory chow diet (Provimi-Lacta, Cossonay, Switzerland) consisting (by energy) of 24% protein, 66% carbohydrates, and 10% fat, and had free access to tap water.

Study design
The experiment was conducted by using a design similar to that previously described in establishing the rat model of weight recovery for studying adjustments in energy expenditure and energy partitioning (12–14). Groups of rats (n=5–8) were food-restricted for 2 wk at approximately 50% of the spontaneous food intake of ad libitum-fed rats (mean intake of 30 g daily) by providing them with a fixed ration of 30 g every 2 days; the food-restricted rats were fasted on alternate days. At the end of this food restriction period (corresponding to day 0 of refeeding), two groups of rats were killed and the animals in the other groups were refed the chow diet at a level approximately equal in metabolizable energy (ME) content to the spontaneous food intake of rats matched for weight at the onset of refeeding. The refed animals were pair-fed to the weight-matched controls fed ad libitum. Two groups of these control animals (n=6) were also killed at this time (day 0); other groups (n=5–8) of refed and controls animals were killed at points corresponding to days 3, 5, 7, 10, and 14 of refeeding. All animals were killed by decapitation. Changes in body energy content, body composition (fat and protein), and energetic efficiency were determined during week 1 (wk 1) and wk 2 from groups killed on day 0, 7, or 14. Assays for UCP1 and the UCP homologues in IBAT and skeletal muscles as well as for serum free fatty acids (FFA) (NEFA C kit, Wako Chemicals GmbH, Neuss, Germany) were conducted in groups killed on days 0, 3, 5, and 10. The gastrocnemius and soleus muscles as well as IBAT were rapidly dissected out and cleaned of tissue debris, immediately frozen in liquid nitrogen, and then stored at -80dgC until later processing for assay of mRNA expression of the UCPs. The remaining carcasses were also analyzed for body fat content.

Determination of body composition
After the animals were killed, the skull, thorax, and abdominal cavity were incised and the gut was cleaned of undigested food. The whole carcasses were then dried to a constant weight in an oven maintained at 60°C and were subsequently homogenized. Triplicate samples of the homogenized carcass were analyzed for energy content by bomb calorimetry (31) and for fat content by the Soxhlet extraction method (32), which uses light petroleum ether. Body protein was determined from a general formula relating energy derived from fat, total energy value of the carcass, and energy derived from protein (12); the calorific values for body fat and protein were taken as 38.6 and 22.7 kJ/g, respectively.

Determination of energy balance and energetic efficiency
Energy balance measurements were conducted, as previously described (12–14), by the comparative carcass technique over two sequential periods of 1 wk each, during which food intake and food spillage were continuously monitored. For each period, energy expenditure was determined as the difference between energy gain and ME intake; the energetic efficiency was calculated as the percentage of energy gained per ME intake.

Extraction of total RNA and Northern blotting
Total RNA was isolated by the method of Chomczynski and Sacchi (33). Fifteen micrograms of each RNA sample were loaded onto a 1.2% formaldehyde gel, as described by Lehrach et al. (34), and electrophoresed overnight. After vacuum blotting of the gel onto a nylon membrane (Electran Nylon Blotting Membrane, BDH Laboratory Supplies, Poole, U.K.) at 60 mBarrs for 3 h, the RNAs were UV cross-linked onto the membrane. The coloration of the membrane with a solution containing 0.04% Bromophenol blue/0.5 M sodium acetate, pH 5.2, showed an equal loading.

Northern blot analysis
The UCP3 and UCP2 probes were obtained as described previously (24, 35). Hybridizations were performed in QuickHyb solution (Stratagene, La Jolla, Calif.) with a probe random labeled with [{alpha}-3232P]dCTP and at a specific activity of 109 dpm/µg DNA. The membranes were then washed in 2 x SSC (20xSSC is 3M sodium chloride, 0.3 M sodium citrate, pH 7.0), 0.1% SDS twice for 5 min at 50°C, and finally once in 0.1 x SSC, 0.1% SDS at 50°C. The blots were exposed to ECL films (Amersham, Bucks, U.K.). Equal loading and transfer were checked by using an oligonucleotide specific for 18S RNA subunit labeled with [{alpha}-32P]ATP. The signals on the autoradiograms were quantified by scanning photodensitometry using ImageQuant Software Version 3.3 (Molecular Dynamics, Sunnyvale, Calif.). To allow quantitative between-group comparison over time for a given UCP in a given tissue, the samples for refed and control animals at the four time points were loaded on the same gel. However, neither between-tissue comparisons nor between-UCP comparisons are possible because of loading limitations on a given gel and differences in the specific activity of labeled probes for the various UCPs.

Data analysis and statistics
The data were analyzed by using two-factor analysis of variance (ANOVA) for the main effects of groups and time as well as for the group x time interaction. Between-group comparisons at specific time points were conducted using the rank sum two-sample (Mann-Whitney) test; the level of statistical significance was taken as P < 0.05. All statistical analyses were performed using the computer software STATISTIX, version 4.0 (Analytical Software, St. Paul, Minnesota, Minn.).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Energy balance and body composition
The data on body weight, body composition, and energy balance in the refed and control animals during wks 1 and 2 of the experiment are shown in Table 1. Body weight and protein at days 0, 7, and 14, as well as the gain in weight and protein between days 0–7 (wk 1) and days 7–14 (wk 2), are not significantly different for the refed and control groups. By contrast, body fat, which is reduced by approximately 50% below control levels at the end of food restriction (day 0 of refeeding), is found to be higher in the refed group than in controls on days 7 and 14; ANOVA indicates a significant group effect as well as a significant interaction between group and time (P<0.001), thereby suggesting a highly significant difference between refed and controls groups across time. Indeed, actual calculation of the gain in body fat suggests that body fat was laid down approximately 2.8-fold faster in the refed rats than in controls during both wk 1 and wk 2. As indicated by the results of ANOVA for body fat and body energy gain, the significant group effect but nonsignificant interaction term suggest that the differences between refed and controls in these parameters are not dependent on the time periods, i.e., they are significantly higher in the refed group than in the control group during both wk 1 and wk 2, despite no between-group difference in energy intake. By contrast, energy expenditure is significantly lower in the refed than in controls during both wk 1 (-17%) and wk 2 (-16%). This is reflected in the energetic efficiency, which is found to be greater in the refed than in controls by approximately twofold during both wk 1 and wk 2.


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  		Table 1. Energy balance and changes in body composition during two successive weeks in refed and control groups

UCP mRNA expression
These data on body fat at time points 0, 7, and 14, together with data for body fat measured in other groups of animals at time points 0, 3, 5, and 10, are presented in Fig. 1, which shows the more rapid gain in body fat in the refed than in controls during all time intervals (0–3, 3–5, 5–7, 7–10, 10–14), even though food intake did not differ between these two groups. The results on mRNA expression of the various UCPs, measured at days 0, 3, 5, and 10, are hence described in relation to 1) the time point at the end of food restriction (day 0 of refeeding) when body fat is 50% depleted, with serum FFA being elevated; and 2) at time points (3, 5, and 10) during this phase of increased efficiency of fat gain (validated in the energy balance study conducted during the two sequential periods described above), with serum FFA being restored to control levels by day 5 of refeeding.



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  		Figure 1. Pattern of changes of body fat, serum FFA, and mRNA expression of UCP1 and UCP3 in interscapular brown adipose tissue (IBAT) and of UCP3 in two skeletal muscles (gastrocnemius and soleus) at the end of food restriction (day 0) and at various time points during controlled refeeding (days 3, 5, and 10). Note that the refed group (RF) was pair-fed to the control group (C) on a day-to-day basis. The asterisk indicates a statistically significant difference between the two groups at a given specific time point (P<0.05). The `T' symbol at time point day 0 under Soleus-UCP3 indicates a P value of 0.08: the difference just failed to reach statistical significance by the Mann-Whitney test, but was found to be statistically different (P<0.05) according to the median test. Results of the statistical treatment of these data for UCP1 and UCP3 by ANOVA, together with that for UCP2, are shown in Table 2.

In IBAT, mRNA expression of UCP3 in the refed group follows the same pattern as for UCP1. The expressions are markedly lower than in controls at the end of food restriction (day 0) and have returned to the levels found in controls by day 5 of refeeding, a time point that is still early in the phase of accelerated fat deposition. As shown in Fig. 1, the greater rate of body fat gain in the refed group persisted beyond 10 days of refeeding.

In both skeletal muscles, UCP3 mRNA expressions are found to be elevated relative to controls at the end of food restriction (day 0), an effect that is more marked in the gastrocnemius (5-fold increase, P<0.01) than in the soleus (2.5-fold increase, P=0.08). On day 3 of refeeding, UCP3 mRNA expression in both skeletal muscles is found to be lower than in controls, and again the effect is much more pronounced in the gastrocnemius (12-fold reduction, P<0.01) than in the soleus muscle (2.6-fold reduction, P<0.05). Furthermore, whereas the mRNA level of UCP3 in the gastrocnemius muscle remains markedly below control levels on day 5 of refeeding and remains incompletely restored relative to controls by day 10, in the soleus muscle, by contrast, it is found to be completely restored to control levels by day 5 of refeeding.

The changes in UCP2 mRNA expression in various tissues during food restriction and refeeding followed the same pattern as that found for UCP3 ( Table 2). Like UCP1 and UCP3 mRNA expressions in IBAT, the mRNA level of UCP2 is also lower in this tissue at the end of food restriction (day 0) and is restored to control levels by day 5 of refeeding; for all UCPs in IBAT, ANOVA indicates a highly significant interaction between the two groups (refed and control) over time (P<0.001). In skeletal muscle, mRNA expression of UCP2, like that for UCP3, is found to be increased both in the gastrocnemius and soleus at the end of food restriction (day 0). Although UCP2 and UCP3 expression in the gastrocnemius muscle is markedly decreased (below control levels) on day 3 of refeeding and remains incompletely restored even on day 10 of refeeding, its levels in the soleus muscle are not different from control values as early as day 3 of refeeding. ANOVA indicates a highly significant interaction between the refed and control groups over time for UCP2 and UCP3 mRNA levels in the gastrocnemius muscle (P<0.002), but no significant group effect or interaction between groups over time is found for UCP2 and UCP3 in the soleus muscle. Representative Northern blots showing the levels of mRNA expression of UCP1 in IBAT, and of UCP homologues in all three tissues from refed and control animals, are presented in Fig. 2.


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  		Table 2. UCP mRNA expressions in skeletal muscles (gastrocnemius and soleus) and in interscapular brown adipose tissue (IBAT) in control and refed groupsa



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  		Figure 2. Northern blots of UCP1, UCP2, and UCP3 in IBAT and UCP2 and UCP3 in the gastrocnemius and soleus muscles at the end of food restriction (day 0) and at time points (days 3, 5, 10) during refeeding. The refed animals showed higher efficiency of fat deposition during the first 14 days of refeeding.

Additional experiment: fasting at thermoneutrality
In view of the findings described above of an unexpected increase in UCP3 and UCP2 mRNA expressions in skeletal muscles during food deprivation, we have conducted additional studies to investigate whether this up-regulation of skeletal muscle UCP homologues could be related to the increased thermoregulatory needs of the body consequential to the starvation-induced loss of body weight (hence increased ratio of surface area-to-volume) and loss of body fat (hence reduced insulation). To this end, two separate experiments assessed the effect of 48 h fasting on UCP2 and UCP3 mRNA expression in the gastrocnemius and soleus: one was conducted at laboratory temperature (22°C) and the other under conditions of thermoneutrality (29°C). In both experiments, the fed animals were allowed ad libitum access to food and water, whereas the fasted animals had access only to water containing 0.45% NaCl. The fasting period started 1 wk after adaptation to housing conditions and environmental temperature. The results show that in both gastrocnemius and soleus muscles, mRNA expression of UCP3 and UCP2 is markedly higher in fasted animals than in fed controls, and the fasting-induced up-regulation of muscle UCP homologues occurs even when the experiment is conducted under conditions of thermoneutrality ( Fig. 3).



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  		Figure 3. Effect of 48 h fasting on mRNA expression of UCP3 and UCP2 in gastrocnemius and soleus muscles from experiments conducted either at standard laboratory temperature (A) or at thermoneutrality (B). Under both experimental conditions of environmental temperature (22 or 29°C), fasting resulted in marked increases in the mRNA expression of UCP3 and UCP2 in both skeletal muscles. In each experiment (22 or 29 °C), n = 5 and a between-group comparison (fasted vs. fed) was conducted using the Mann-Whitney test, with the level of statistical significance indicated as follows: *P < 0.05; **P < 0.01.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Animal model of weight recovery
Several earlier studies have demonstrated that refeeding after partial or total food deprivation is accompanied by an enhanced efficiency of energy utilization and higher rates of fat deposition (8–10), a phenomenon recognized to be a carryover effect of the suppression of thermogenesis that occurred in the preceeding period of food deprivation. The assessment of its quantitative importance in the excessive fat recovery has, however, been obscured by confounding factors that complicate the interpretation of differences in energy expenditure between refed and control animals, in particular 1) by compensatory hyperphagia that often characterizes the early phase of food reavailability in refed animals, and 2) by differences in body size and lean body mass between refed animals and their larger age-matched controls. By pair-feeding the refed animals to ad lib-fed controls matched for similar body weight and lean tissue mass at the onset of refeeding, our experimental approach bypasses the problems associated with a comparison between animals of different body sizes and hence provides a means of assessing the contribution of regulatory adjustments in energy expenditure to the phenomenon of accelerated fat deposition during refeeding. Although such comparisons between refed and weight-matched control groups introduce an age difference because these controls are inevitably younger than those that are refed, previous validation studies of this model of weight recovery in our laboratory have shown that, within the limits of our experimental design, the age difference between the refed and weight-matched controls has little or no effect on the difference in their energy expenditures (12, 13). Consequently, the lower energy expenditure in the refed animals relative to the weight-matched controls during the first 2 wk of refeeding reflects essentially a sustained suppression of thermogenesis. An important component in the adaptive reduction in thermogenesis that is known to occur during food deprivation persists during refeeding. As shown in the present study, its magnitude during wk 2 of refeeding is as important as during wk 1, since during both weeks the energy expenditure in the refed group was lower than in the control group by ~16%. These findings, coupled with the data in Fig. 1showing that gain in body fat was higher in refed than in controls over all time intervals, as well as the lack of between-group differences in body protein and protein gain ( Table 1), suggest that virtually all the energy conserved in the refed animals was directed specifically at enhancing fat deposition throughout the 14-day refeeding period.

Dissociation of high efficiency of fat recovery from SNS-BAT-UCP1 axis
The results of this study pertaining to the pattern of mRNA expression of UCP1 during food restriction and refeeding provide evidence complementary to our previous suggestion that this component of suppressed thermogenesis for rapid fat deposition can be dissociated from the sympathetic control of thermogenesis (14), and by extension, to BAT, a tissue whose thermogenic activity is primarily under sympathetic control of its uncoupling protein, UCP1 (15–20). Figure 1shows the marked down-regulation of UCP1 mRNA levels in IBAT in response to food deprivation is in accordance with its well-established role in energy conservation during food deprivation (16–20). However, the present findings that its expression is restored to control levels by day 5, a time point still early in the phase of accelerated fat deposition (which lasts for ~14 days or more), is in line with our contention that tissues such as BAT, whose thermogenic activity is under SNS control, are unlikely be important sites for the component of reduced thermogenesis that underlies the elevated efficiency of fat recovery. Indeed, under conditions whereby the dietary modulations of SNS activity and BAT thermogenic capacity have been shown to be overridden by thermoregulatory needs—i.e., in the cold (36) and at thermoneutrality (37)—the efficiency of fat deposition in the refed animals remains higher than in controls (14).

Dissociation of regulatory thermogenesis from skeletal muscle UCPs
Since SNS activity in skeletal muscle is unresponsive to modulation by diet and cold exposure (22) and because this tissue is an important site of energy conservation during starvation (21), the possibility arises that the skeletal muscle is an important site for this component of suppressed thermogenesis, which is SNS independent. This led us to examine whether skeletal muscle UCP3 and UCP2, the two recently cloned genes with high sequence homology to BAT UCP1, could be implicated in energy conservation during food restriction and refeeding. Our results on the pattern of UCP mRNA expressions in both gastrocnemius and soleus muscles are at variance, however, with a role for skeletal muscle UCP homologues in dietary regulation of thermogenesis in general and in regulatory thermogenesis underlying energy conservation in particular. First, expression of these genes is found to be up-regulated during food deprivation, a directional change in mRNA expression that is opposite to that expected for any putative uncoupling protein with regulatory functions vis-à-vis energy conservation. Second, the possibility that the up-regulation of skeletal muscle UCPs during food deprivation may reflect their functional role as `inhibitory modulators' of thermogenesis in this tissue is not tenable, since one would expect such `inhibitory modulation' to be either sustained or to return gradually toward control levels during refeeding, when energy conservation persists in our animal model, rather than to be down-regulated below control levels. Third, it has been suggested that the mRNA up-regulation of skeletal muscle UCPs during food deprivation may still be reflecting an uncoupling effect, but whose functional importance is to meet the increased thermoregulatory needs of the body consequential to starvation-induced body wasting, loss of insulation, and overall energy conservation (25). However, this explanation is difficult to reconcile with 1) the demonstration, by blood flow studies coupled with regional arteriovenous oxygen differences, that skeletal muscle is a quantitatively important site of energy conservation during starvation (21), and 2) the findings that mRNA expressions of these UCP homologues are down-regulated (i.e., below control levels) during refeeding, a phase when the energy conservation phenomenon is still very much evident and has been shown to persist independent of the thermoregulatory needs of the rat (14). Furthermore, the data presented here ( Fig. 3) that fasting-induced up-regulation of UCP2 and UCP3 mRNA in skeletal muscles occurs even when the animals are maintained at thermoneutrality (i.e., under conditions of reduced thermoregulatory needs), coupled with previous reports that cold exposure has no effect on these UCP homologues in muscle (24, 35), also argue against a functional role for these UCPs in thermoregulatory thermogenesis.

Relationship between skeletal muscle UCP and altered lipid fuel utilization
By contrast, the tissue-dependent differential expression of UCP homologues in response to dietary manipulation in our study reveals a much more coherent relationship between the pattern of transcription of these genes and the tissue requirements for lipids as fuel substrates. First, mRNA up-regulation of UCP2 and UCP3 in both gastrocnemius and soleus muscles at the end of food restriction correlates with the increased circulating levels of FFA and hence with the expected increased uptake and utilization of lipids as fuel substrates by skeletal muscles during starvation. Second, down-regulation of UCPs in these skeletal muscles (in particular, the gastrocnemius muscle) during refeeding correlates with the need to reduce the utilization of lipids as a fuel substrate to levels well below those of controls, since the refed rats have to `spare' lipids for storage, given the drive (underlain by mechanisms that suppress thermogenesis) to accelerate fat deposition during weight recovery. Indeed, the still incomplete recovery of both UCP2 and UCP3 mRNA expression in the gastrocnemius muscle even on day 10 of refeeding correlates well with the phenomenon of the accelerated rate of fat deposition lasting for more than 10 days in our animal model. The close association between enhanced fat recovery and the expression of UCP homologues observed in gastrocnemius muscle contrasts with the weaker association found between enhanced fat recovery and the expression of UCP1 and UCP homologues in BAT, and hence could underlie a more important role for skeletal muscles than for BAT as a site for the modulation of fat utilization during weight recovery after starvation. Third, the differential responses of the two skeletal muscles studied (gastrocnemius and the soleus) to food deprivation vis-à-vis mRNA expression of UCP2 and UCP3, as well as differences in their pattern of expression during the course of refeeding, are also consistent with the heterogeneity in skeletal muscle types in their glycolytic and oxidative enzymes activities and hence in their capacity to shift between lipids and glucose as fuel substrates (26–28). In the gastrocnemius muscle (mixed fiber type), with its great capacity to switch between lipids and glucose for fuel substrates, the magnitude of increase in its UCP mRNA expressions during food restriction (period of increased lipid uptake and utilization) was severalfold greater than in the soleus muscle, which is an oxidative (red fiber) type muscle that has a low glycolytic capacity, high dependency on lipids as fuel substrates even in the fed state, and thus has less capacity to increase lipid utilization further during food deprivation. The same explanation is also valid for the differential responses of UCP expression in these two muscle types during refeeding. The much more sustained down-regulation of UCP2 and UCP3 mRNA expression in the gastrocnemius vs. the soleus muscle can be attributed to the much larger glycolytic capacity of the gastrocnemius, and hence to its greater ability to sustain a shift toward glucose utilization in order to `spare' lipids for storage during a phase of increased efficiency of fat deposition. Indeed, the greater dependency of the soleus muscle (than the gastrocnemius muscle) on lipids as fuel substrates is reflected in the close association of its UCP mRNA expressions with circulating FFA both during food restriction and refeeding ( Fig. 1). These observations also raise the possibility that FFA might be a modulator of gene transcription of these UCP homologues in highly oxidative skeletal muscles, and investigations using both pharmacological and nutritional approaches are under way in our laboratory to test this hypothesis.

Role of UCP homologues in adipose tissues
Finally, the proposed role of UCP2 and UCP3 in regulating lipids as fuel substrate in the skeletal muscles can be extended to adipose tissues. In BAT, a tissue that is highly dependent on lipids to fuel its thermogenesis, and in which increased glucose uptake (e.g., during cold exposure) is generally directed at lipogenesis to meet the high FFA demands for fuel rather than for substrate oxidation (38, 39), regulation of its lipids for fuel is intimately linked with the regulation of this tissue's thermogenic state. This is well illustrated in the present study, where the pattern of changes in IBAT mRNA expression of UCP1 (the established functional uncoupler) is similar to that for UCP2 and UCP3 in this tissue: all are markedly down-regulated during food deprivation and simultaneously restored during refeeding. These findings, together with other reports that expression is up-regulated in this tissue in response to cold (35), would also be consistent with a role for UCP2 and UCP3 homologues in regulating lipids as fuel substrates for UCP-1-mediated thermogenesis in BAT. In white adipose tissue, on the other hand, reports that mRNA expression of UCP2 and/or of UCP3 are up-regulated under conditions of increased lipid utilization—specifically, 1) in rats resisting obesity when fed a high-fat diet (29), 2) treated with adrenoceptor agonists (25), or 3) infused with a recombinant adenovirus containing leptin (40)—are also in line with a role for these UCP homologues as regulators of lipids as a fuel substrate.

In summary, this study reveals tissue dependency in transcriptional changes in these UCP2 and UCP3 homologues (not only between IBAT and the skeletal muscles, but also between muscle types) that are consistent both with the differential requirements of these tissues for lipids during food deprivation and in their capacity to shift from lipids to glucose during refeeding. These findings are therefore much more consistent with a functional role for UCP2 and UCP3 as regulators of lipids as fuel substrate rather than as mediators of regulatory thermogenesis. Additional studies would need to dissect out the exact control points at which these UCPs homologues may be acting in this postulated regulation of fuel lipids and, among some of the contenders, whether this can be pinpointed to regulation at the level of competition between lipids and carbohydrates utilization (Randle glucose–FFA cycle), to the regulation of lipolysis within the tissue's triglyceride pool, to FFA carriers across the mitochondrial membrane, or to the regulation of lipid oxidation and peroxidation. The development of assays for the protein products and their exact localization within the cell would no doubt be the first steps in elucidating the exact functional role of these UCP homologues. A better understanding of the mechanisms underlying the regulation of fuel lipids will have wide implications in the field of metabolism and in clinical medicine, particularly in elucidating the metabolic basis of obesity and its relapse, in the etiology of non-insulin-dependent diabetes, and in pathophysiological cachexic conditions (e.g., AIDS, cancer, sepsis) in which nutritional rehabilitation is characterized by the tendency to recover fat rather than lean tisssue, leading to difficulties in restoring body functions.


   ACKNOWLEDGMENTS
 
We are grateful to Claudette Duret and Martine Vollenweider for excellent technical assistance and to Florence Strub and Raynald Schumacher for the illustrations. We thank O. Boss and J. P. Giacobino for kindly providing us the probes for UCP1 as well as UCP2 and UCP3 homologues. This work was supported by grant (No. 31–47211.96) of the Swiss National Science Foundation.


   FOOTNOTES
 
1 Correspondence: Department of Physiology, Faculty of Medicine (CMU), 1 rue Michel-Servet, 1211 Geneva 4, Switzerland.

2 Abbreviations: BAT, brown adipose tissue; SNS, sympathetic nervous system; FFA, free fatty acids; UCP, uncoupling protein; IBAT, intercapsular BAT; wk 1, week 1; ME, metabolizable energy; ANOVA, analysis of variance; UCP, uncoupling protein.

Received for publication December 2, 1997. Accepted for publication January 22, 1998.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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