The present study demonstrates that subchronic (7 day) combined DA+NE reuptake inhibition causes weight loss by increasing locomotor activity and interscapular temperature and not by inhibiting food intake. Combined catecholamine reuptake inhibition with either BUP or GBR+NIS increased both activity and temperature, but BUP did not cause significant weight loss owing to a compensatory increase in food intake. Because the effect of combined selective DA+NE reuptake inhibitors on activity and temperature is different from the individual effects of these drugs, elevated energy expenditure is likely the result of an interaction between DA and NE systems. This study also provides new evidence that the weight loss observed with BUP treatment in obese humans is possibly the result of elevated energy expenditure by increased activity and thermogenesis caused by a combined dopaminergic and noradrenergic mechanism.

Body weight is determined by energy intake and energy expenditure. Energy expenditure can be further divided into basal metabolism, mechanical work (locomotor activity), and adaptive thermogenesis (Spiegelman and Flier, 2001). Thus, elevated locomotor activity and interscapular temperature probably caused increased energy expenditure. Pharmacological and genetic studies support this assertion. Elevated activity and temperature have already been demonstrated to account for increased energy expenditure caused by treatment with the weight loss drug sibutramine (Liu et al, 2002; Golozoubova et al, 2006). In mice on a high-fat diet, elevated activity and temperature prevent weight gain. However, mice lacking the melanocortin-4 receptor do not increase activity and temperature in response to a high-fat diet and exhibit accelerated weight gain (Butler et al, 2001). This data set does not prove that the magnitude of the increase in interscapular temperature and locomotor activity regulates energy expenditure enough to produce changes in body weight. However, they provide a persuasive argument that this is the case. Changes in temperature and locomotor activity are in the appropriate qualitative direction, and in the case of BUP were sufficient to produce no net change in body weight, in spite of increased food intake. We cannot discount the formal possibility that increased interscapular temperature could be due to increased local blood flow, but the large increases in locomotor activity observed in this study would certainly cause an increase in energy use.

Remote biotelemetry allows for simultaneous and continuous measurement of locomotor activity and temperature in the animals' home cage, minimizing confounding variables and allowing for temporal data acquisition (Harkin et al, 2002). By using biotelemetry to measure mechanical work and adaptive thermogenesis in mice during both acute and chronic drug treatment, we were able to record the two types of energy expenditure that vary with energy state (Spiegelman and Flier, 2001).

Because E-Mitters were implanted underneath IBAT, the main organ for adaptive thermogenesis in small mammals, temperature fluctuations probably reflect activity of IBAT (Lowell and Spiegelman, 2000; Avram et al, 2005). The idea that BAT may be a viable target for obesity pharmacotherapy has already been proposed. Increasing BAT activity elevates metabolic rate and prevents diet-induced obesity in rodents, whereas genetic mutation and pharmacological inhibition of BAT can cause obesity (Spiegelman and Flier, 2001; Crowley et al, 2002). Because adaptive thermogenesis is believed to contribute to the decreased energy expenditure characteristic of obesity and caloric deficit (dieting), drugs that increase adaptive thermogenesis (by activating BAT) have been suggested as a possible means to achieve safe and sustained energy expenditure (Major et al, 2007).

Acute and chronically administered BUP has repeatedly been demonstrated to dose dependently increase locomotor activity in rodents with similar potency, regardless of species, age, or strain (Soroko et al, 1977; Nielsen et al, 1986; Zarrindast and Hosseini-Nia, 1988; Vassout et al, 1993; Redolat et al, 2005; Mitchell et al, 2006; Billes and Cowley, 2007). The current finding that BUP, DA, and DA+NE reuptake inhibition all transiently increased locomotor activity is consistent with the idea that inhibition of the DA transporter is sufficient to cause a significant short-term increase in locomotion and even reverse the decrease in activity caused by inhibition of NE reuptake. The transient increase in locomotor activity caused by subchronic BUP infusion is identical to the effect caused by subchronic infusion of various selective DA reuptake inhibitors (Izenwasser et al, 1999). A dopaminergic mechanism for increased locomotor activity by BUP is consistent with increased striatal DA concentrations in rats following acute BUP administration (Vassout et al, 1993), the failure of BUP to increase locomotor activity in rats with selective ablation of DA neurons (Cooper et al, 1980), and a recent report by Mitchell et al (2006) demonstrating that acute BUP increases locomotor activity in NE transporter knockout mice. DA reuptake inhibition also accounts for increased locomotion by other mixed monoamine reuptake inhibitors including psychostimulants and the weight loss drug sibutramine (Missale et al, 1998; Izenwasser et al, 1999; Golozoubova et al, 2006; Mitchell et al, 2006). Thus, the finding that BUP increases locomotor activity is widely supported.

Acute combined DA+NE reuptake inhibition, either with BUP or coadministration of selective DA+NE reuptake inhibitors, caused a brief decrease in interscapular temperature. The only other study examining the effects of BUP on temperature in mice reported that acute BUP causes rapid and pronounced hypothermia (Zarrindast and Abolfathi-Araghi, 1992). More recent studies in rats have demonstrated the opposite effect, instead showing that peripheral BUP causes a small increase in colonic and core temperature and also O 2 consumption (Liu et al, 2002, 2004; Hasegawa et al, 2005). Many variables may account for the conflicting acute effects of BUP on temperature. Factors such as species (mice vs rats), dose, route of administration, time of day, duration of temperature measurement, location of temperature measurement (core, peripheral, or IBAT), or ambient temperature could have influenced BUP's effects on temperature. Ambient temperature has been demonstrated to influence the effect of cocaine on body temperature (Lomax and Daniel, 1990). Similar to cocaine, BUP has little effect on temperature in humans in a thermoneutral environment, but may increase temperature in a warm environment due to a higher set point for hyperthermia and reduced compensatory heat loss (Griffith et al, 1983; Watson et al, 2005). Finally, because the current acute studies were performed in the light phase (when animals are less active and have lower body temperature) in fasted animals that were subsequently fed, these results may reflect an acute attenuation of the thermic effect of food by BUP, rather than a decrease in steady-state temperature (Lowell and Spiegelman, 2000). In fact, the mild but significant increase in temperature between 1 and 2 h post injection supports the larger collection of evidence that BUP, and perhaps its pharmacologically active metabolites, increase temperature (Ascher et al, 1995; Liu et al, 2002, 2004; Hasegawa et al, 2005).

NE released from sympathetic nerve terminals activates β 3 -adrenoceptors on brown adipocytes in BAT and leads to lipolysis, increased activity of uncoupling protein-1 (UCP-1), and thermogenesis (Avram et al, 2005; Fan et al, 2005). By blocking NE reuptake in sympathetic nerve terminals, an NE reuptake inhibitor could increase β 3 -adrenoceptor activation, thereby increasing oxidative phosphorylation and the production of heat (Iversen, 1971). In rats, antagonism of the β 3 -adrenoceptor attenuates the increased O 2 consumption caused by BUP (Liu et al, 2004) and the weight loss drug sibutramine (Connoley et al, 1999), suggesting that BAT activation accounts for a significant proportion of the increased energy expenditure with these drugs. The current finding that selective NE reuptake inhibition decreased interscapular temperature emphasizes that additional dopaminergic (with BUP treatment) or serotonergic (with sibutramine treatment) input is important to maintain increased sympathetic tone and cause thermogenesis.

Because thermogenesis through BAT activation is a metabolic regulatory process that is controlled by the hypothalamus via descending sympathetic fibers (Lowell and Spiegelman, 2000), catecholamine reuptake inhibitors may act both centrally and peripherally to influence interscapular temperature (Wellman, 2005). BUP has been shown to increase DA and NE in hypothalamic nuclei that regulate body temperature such as the preoptic area and anterior hypothalamus (Hasegawa et al, 2005). Catecholamine reuptake inhibitors may also affect activity of cells in the hypothalamic melanocortin system, which regulates caloric intake and metabolic rate in response to energy availability (Fan et al, 2005; Ramos et al, 2005). For example, activation of the dopamine D2 receptor increases expression of anorexic pro-opiomelanocortin mRNA and decreases expression of orexigenic neuropeptide Y mRNA within the arcuate nucleus of the hypothalamus (Pelletier and Simard, 1991; Tong and Pelletier, 1992). By increasing extracellular DA, DA reuptake inhibitors could indirectly increase D2 receptor activation and affect activity of neurons in the melanocortin system.

Part of the efficacy of drugs like BUP is that they affect multiple systems, sometimes resulting in favorable drug interactions that could not be predicted based on the individual effects of selective DA and NE reuptake inhibitors (Kaplan, 2005). An example of a drug interaction that was not predicted is the effect of subchronic DA+NE reuptake coadministration on IBAT temperature. During the light phase, subchronic DA+NE reuptake inhibition caused an average 0.4°C increase in IBAT temperature, even though temperature was essentially unaffected by DA reuptake inhibition and decreased by NE reuptake inhibition. Although adult humans do not have defined peripheral BAT deposits, recent evidence shows that brown adipocytes may be dispersed within white adipose tissue deposits. Additional studies linking human obesity to β 3 -adrenoceptor and UCP-1 genetic polymorphisms suggest a role for BAT in energy expenditure in humans (Avram et al, 2005).

This study emphasizes that although acute studies may infer long-term drug effects, they do not necessarily predict a drug's chronic effects. Even though the chronic studies presented here were limited (by method of administration and drug solubility) to 1 week of treatment, they were sufficient to illustrate differences between the acute and subchronic effects of catecholamine reuptake inhibitors. Further studies assessing the effects of chronic (2–3 week) drug treatment could potentially offer a more comprehensive analysis of the chronic effects of catecholamine reuptake inhibitors on energy balance, but may be confounded by different methods of drug administration. Even daily peripheral BUP injection may not produce the same neuronal adaptations as chronic BUP infusion via minipump, which produces more stable drug and metabolite levels (Ferris and Beaman, 1983; Klimek et al, 1985; Ascher et al, 1995). Increasing evidence suggests that the cellular and behavioral effects of chronically administered catecholamine reuptake inhibitors and psychostimulants also depend on whether chronically administered drugs are delivered via daily bolus injection or continuous infusion (Ansah et al, 1996; Davidson et al, 2005). Because previous studies on the cellular effects of chronic BUP were conducted in rodents that received BUP orally or via daily injection, further studies examining how chronic infusion of BUP or selective DA+NE reuptake inhibitors via osmotic minipump affects neuronal signaling are necessary to begin to elucidate the most probable mechanism through which catecholamine reuptake inhibition affects energy balance.

It has been established in vitro and in vivo that BUP inhibits both the DA and NE transporters (Nomikos et al, 1989; Ferris and Cooper, 1993; Wellman, 2005). We previously demonstrated that coadministration of selective DA+NE reuptake inhibitors produces similar effects on food intake and body weight as BUP (Billes and Cowley, 2007). We extend our previous finding by demonstrating that coadministration of DA+NE reuptake inhibitors mimics the effects of BUP on locomotor activity and interscapular temperature. This study also corroborates the modest effect of BUP on body weight in humans. More importantly, we illustrate the individual effects of selective DA or selective NE reuptake inhibition on activity and interscapular temperature and demonstrate that coadministration of selective DA+NE reuptake inhibitors increased activity and temperature and caused weight loss in lean mice. It remains to be determined whether catecholamine reuptake inhibition would have a greater effect on energy balance in an obese rodent model, as has been suggested by acute studies with BUP, NIS, and GBR (Billes and Cowley, 2007) and also sibutramine (Strack et al, 2002). The results of this study shed light on the mechanism through which catecholamine reuptake inhibitors cause weight loss and provide additional insight into the role of catecholamines in regulation of energy balance. The relevance for humans is not that combined catecholamine reuptake inhibitors may actually increase energy expenditure, but rather that they may prevent diet-induced decreases in adaptive thermogenesis, thereby facilitating weight loss.