Aging Cell (2008) 7, pp552–560 Doi: 10.1111/j.1474-9726.2008.00407.x 552 © 2008 The Authors Journal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2008 Blackwell Publishing Ltd Mild mitochondrial uncoupling in mice affects energy metabolism, redox balance and longevity Camille C. Caldeira da Silva,* Fernanda M. Cerqueira,* Lívea F. Barbosa, Marisa H. G. Medeiros and Alicia J. Kowaltowski Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil *Authors contributed equally. Summary Caloric restriction is the most effective non-genetic intervention to enhance lifespan known to date. A major research interest has been the development of therapeutic strategies capable of promoting the beneficial results of this dietary regimen. In this sense, we propose that com- pounds that decrease the efficiency of energy conversion, such as mitochondrial uncouplers, can be caloric restriction mimetics. Treatment of mice with low doses of the pro- tonophore 2,4-dinitrophenol promotes enhanced tissue respiratory rates, improved serological glucose, triglyceride and insulin levels, decrease of reactive oxygen species levels and tissue DNA and protein oxidation, as well as reduced body weight. Importantly, 2,4-dinitrophenol-treated animals also presented enhanced longevity. Our results demonstrate that mild mitochondrial uncoupling is a highly effective in vivo antioxidant strategy, and describe the first therapeutic intervention capable of effectively reproducing the physiological, metabolic and lifespan effects of caloric restriction in healthy mammals. Key words: caloric restriction; 2,4-dinitrophenol; energy conversion; free radicals; life span. Introduction Caloric restriction, or the limitation of dietary calories without lack of essential nutrients, extends lifespan in a variety of species, including yeast, worms, flies, mice, rats and, probably, nonhuman primates (Sohal & Weindruch, 1996; Partridge & Gems, 2002; Roth et al., 2004). In humans, caloric restriction leads to improvements in blood glucose and plasma lipid levels similar to those seen in other animals (Walford et al., 2002). One of the central effects of caloric restriction in many models is to promote changes in mitochondrial respiratory rates (Lin et al., 2002; Merry, 2004; Barros et al., 2004; Bonawitz et al., 2007; Guarente, 2008). In Saccharomyces cerevisiae, caloric restriction augments replicative and chronological lifespan by increasing mitochondrial respiration (Lin et al., 2002; Barros et al., 2004; Fabrizio et al., 2005; Tahara et al., 2007), enhancing the activity of the Sir2p histone deacetylase (Lin et al., 2000) and preventing the build-up of mitochondrially generated reactive oxygen species (ROS; Barros et al., 2004; Tahara et al., 2007). Indeed, a variety of interventions that enhance or inhibit mitochondrial respiration in yeast augment or decrease lifespan, respectively (Lin et al., 2002; Barros et al., 2004; Bonawitz et al., 2007). Dietary restriction also leads to increased respiration and longevity in Caenorhabditis elegans (Bishop & Guarente, 2007). In Drosophila melanogaster, NF1 gene mutants have shortened lifespans associated with decreased respiratory rates and elevated ROS formation, while flies overexpressing NF1 present increased lifespan and respiration, along with lower ROS production (Tong et al., 2007). Increasing respiration in flies by expression of uncoupling protein decreases ROS production and enhances lifespan (Fridell et al., 2005). In addition, treating larvae with the chemical uncoupler 2,4-dinitrophenol (DNP) enhances average lifespan (Padalko, 2005). In mammals, many studies (but not all, see Lambert & Merry, 2005; Ferguson et al., 2007) demonstrate that caloric restriction stimulates respiratory rates (see Guarente, 2008, for a review). Increases in respiration involve enhanced biogenesis and increases in mitochondrial density in tissues (Lambert et al., 2004; Nisoli et al., 2005) as well as decreases in coupling between oxygen consumption and oxidative phosphorylation (Lambert & Merry, 2004; Merry, 2004; Xiao et al., 2004). In addition, Speakman et al. (2004) elegantly demonstrated that mice which spontaneously exhibit enhanced lifespans present higher oxygen consumption rates, strongly suggesting a direct association between mitochondrial respiration and the aging process. Respiratory rates are well known to affect mitochondrial ROS production (Korshunov et al., 1997; Skulachev, 1998; Balaban et al., 2005) and caloric restriction is widely associated with a decrease in oxidative damage (see Sohal & Weindruch, 1996; Merry, 2004, for reviews). In addition, antioxidants targeted to mitochondria increase lifespan in mice (Schriner et al., 2005; Skulachev, 2007), suggesting that mitochondrially generated ROS are a cause of lifespan limitation. On the other hand, decreases in ROS release measured in mitochondria from calorically restricted animals (Sohal & Weindruch, 1996; Merry, 2004) are not consistently found when using intact cells (Lambert & Merry, 2005), and accumulation of oxidative damage in mitochondria is not necessarily associated to functional defects and enhanced aging (Stuart et al., 2005). Correspondence Alicia J. Kowaltowski, Av. Prof. Lineu Prestes, 748, Cidade Universitária, São Paulo, SP 05508-900, Brazil. Tel.: +55 11 30913810; fax: +55 11 38155579; e-mail: alicia@iq.usp.br Accepted for publication 10 May 2008