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 compounds that decrease the efficiency of energy conversion, such as mitochondrial uncouplers, can be caloric restriction mimetics. Treatment of mice with low doses of the protonophore 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.

Experimental procedures Animal care Experiments were approved by the Comitê de Ética em Cuidados e Uso Animal, and follow NIH guidelines. Female Swiss Webster outbred albino mice, purchased originally from Taconic Farms, were bred and lodged at the Biotério de Produção e Experimentação da Faculdade de Ciências Farmacêuticas e Instituto de Química, with HEPA‐filtered air and under controlled temperature (22 °C), humidity, light (12‐h light/dark cycles) and pressure. The colony is specific pathogen free submitted to sanitary controls thrice a year. After weaning, animals were allowed free access to standard irradiated laboratory rodent diet (Nuvital CR1, Colombo, Brazil) containing 21.6% protein and 4.0% lipid. Starting at 18 weeks, the DNP group continuously received drinking water containing 1 mg L−1 DNP, prepared biweekly, in light‐protected bottles. DNP did not alter water ingestion at any time point measured (data shown represent animals at 75 weeks). Based on water ingestion and body weight, DNP doses ranged between 30 and 105 µg kg−1 day−1. A group of 60 animals (30 treated with DNP and 30 controls) was kept in the animal facilities until the end of their natural lifespan. These animals were submitted to weekly weighing, temperature measurements, and food and water ingestion analysis. Other DNP‐treated and control animals were sacrificed at 22 or 32 weeks (1 month and 5 months treatment, respectively) to collect organs and serum. Body temperatures Rectal temperatures were measured using a digital thermometer (BD Basic, Becton Dickinson, São Paulo, Brazil). Animals were adapted to rapid comfortable immobilization and measurements. Temperatures were recorded between 14:00 and 15:00 hours. No differences in temperatures were noted at any time point measured. Data shown are averages of 5 days of measurements per animal, at 75 weeks of age. Water and food ingestion Mice were kept in groups of two in metabolic cages (Beira‐Mar, São Paulo, Brazil), with free access to food and water. After 24‐h adaptation, food ingestion and water intake were measured over a further 24 h. No differences between the treated and untreated groups were noted at any time point measured. Data shown were collected at 75 weeks of age. Fecal and urinary quantities were also measured, and standard urinalysis was conducted, presenting no significant differences (results not shown). Body weight and efficiency of energy conversion Animals were individually weighed weekly throughout their natural lifespan. The data shown represent averages of measurements made every 10 weeks, for sake of clarity. Weight gain/ingestion was calculated over weeks 18 through 38. Oxygen consumption Brain, liver and heart tissues were collected and segmented into fine (~1 mm) pieces in phosphate‐buffered saline (PBS) (137 mm NaCl, 10 mm phosphate, 2.7 mm KCl, pH 7.4). The suspension was added to the temperature‐controlled (37 °C) chamber of a Clark‐type electrode (Hansatech Instruments, Norfolk, UK). Oxygen consumption was recorded over 20 min, assuming solubility at 37 °C was 210 µmol mL−1. Samples were homogenized for protein content determination. H 2 O 2 release Tissues were processed as described for oxygen consumption measurements, and incubated for 20 min in 37 °C PBS. Fifty micromolar Amplex Red plus 1 U mL−1 horseradish peroxidase were added to the supernatant to determine H 2 O 2 concentrations. Baseline fluorescence in the same media was subtracted from all measurements. Amplex Red reacts with peroxidase‐H 2 O 2 complexes with 1 : 1 stoichiometry to produce fluorescent resorufin (Zhou et al., 1997). Fluorescence was measured at Ex = 563 and Em = 587 nm, using 5 nm slits on an Hitachi 4500 fluorescence spectrophotometer. Data are expressed as percentages of control fluorescence since different tissues promoted changes in baseline fluorescence, rendering calibrations imprecise. Protein carbonyl detection Protein carbonylation was quantified by derivatization with 2,4‐dinitrophenylhydrazine followed by immunological detection (Shacter et al., 1994). Organs were homogenized in buffer containing 320 mm sucrose, 5 mm MgCl 2 , 10 mm Tris‐HCl, 0.1 mm desferroxamine and protease inhibitors (1 mm sodium orthovanadate, 1 mm phenylmethanesulphonylfluoride, 2 µg mL−1 aprotinin, 2 µg mL−1 pepstanin, 2 µg mL−1 leupeptin). The homogenates were stored at – 80 °C. Thawed samples (4 mg protein mL−1) were incubated with 5 mm 2,4‐dinitrophenylhydrazine and 6% sodium dodecyl sulphate (SDS) for 30 min, followed by neutralization with 25%β‐mercaptoethanol. Eighteen micrograms of protein were then applied to an SDS‐PAGE (5% stacking, 8% running gel). Proteins were transferred to a nitrocellulose membrane and Western blot was developed using primary anti‐dinitrophenylhydrazine rabbit antibody (Calbiochem, Darmstadt, Germany) at 1 : 5000 dilution and secondary peroxidase‐linked anti‐rabbit IgG from Pierce KLP (Rockford, IL, USA). Images were analyzed using ImageQuant software, integrating intensities of all visible bands in a fixed selected area, relative to the background. Decreases in carbonylation in DNP samples were widespread, and did not significantly differ between specific protein bands. DNA extraction and enzymatic hydrolysis DNA was isolated from tissues kept at –80 °C using the method described by Wang et al. (1994), with some modifications. The tissue (300 mg) was suspended and washed in 2 mL of a lysis solution (1% w/v Triton X‐100, 320 mm sucrose, 5 mm MgCl 2 , 10 mm Tris‐HCl, pH 7.5). After centrifugation at 1500 g for 10 min, the nucleus pellets were suspended in 3 mL of 10 mm Tris‐HCl buffer, pH 8.0, containing 5 mm EDTA and 0.15 mm deferoxamine mesylate salt. RNAse A (30 µL of a 10 g L−1 solution in 10 mm sodium‐acetate pH 5.2, heated for 15 min at 100 °C) and RNAse T1 (4 µL of a 20 U µL−1 solution in 10 mm Tris‐HCl buffer, pH 7.4, containing 1 mm EDTA and 2.5 mm deferoxamine mesylate salt) were added with 200 µL of 10% (w/v) SDS. The reaction mixture was incubated at 37 °C for 1 h. Thirty microliters of proteinase K (20 g L−1) were added, followed by additional incubation at 37 °C for 1 h. For brain samples, 1 : 1 chloroform was added, followed by homogenization and phase separation to remove excess lipid. The aqueous phase followed the same subsequent steps as all tissues. After centrifugation at 5000 g for 15 min, the liquid phase was collected and 4 mL of isopropanol were added. The content was homogenized by inversion until a whitish precipitate appeared. The precipitate was collected by centrifugation at 5000 g for 15 min and washed with 1 mL of 60% (v/v) isopropanol followed by 1 mL of 70% (v/v) ethanol. After additional centrifugation at 5000 g for 15 min, the DNA pellet was solubilized in 0.1 mm deferoxamine mesylate. The DNA concentration was measured spectrophotometrically at 260 nm and its purity was assessed by ensuring A 260 /A 280 ≥ 1.7. DNA hydrolysis was performed as described by Fiala et al. (1989) with some adaptations. For the hydrolysis of 100 µg of DNA, 2.0 µL of 1 m sodium acetate buffer pH 5.0 and 1 U of nuclease P 1 were added to the sample, which was incubated at 37 °C for 30 min. Four microliters of 1 m Tris‐HCl buffer (pH 7.4) and 4 µL of 500 mm potassium acetate buffer (pH 7.0) containing 100 mm of Tris‐acetate and 100 mm magnesium acetate were added, followed by the addition of 3 U of alkaline phosphatase. The final volume of the sample was adjusted to 100 µL with water. The reaction mixture was incubated at 37 °C for 1 h. High performance liquid chromatography–electrochemical detection (HPLC/EC) of 8‐oxodGuo Analysis was performed as described by Fiala et al. (1989) with some adaptations. Samples (100 µg) of digested DNA were injected into the HPLC/EC system, consisting of a Shimadzu LC‐10 AD pump (Shimadzu, Tokyo, Japan) connected to a Luna C18 analytical column (250 mm × 4.6 mm i.d., 5 µm; Phenomenex, Torrance, CA, USA), kept at 18 °C by a Shimadzu CTO‐10AS VP column oven. The isocratic eluent was 25 mm potassium phosphate buffer (pH 5.5) and 8% methanol at a flow rate of 1 mL min−1. Coulometric detection was provided by a Coulochem II detector (ESA, Chelmsford, MA, USA) and spectrophotometric detection by Shimadzu SPD‐10 A. The potential of the electrode was set at 280 mV. Elution of unmodified nucleosides was simultaneously monitored by a Shimadzu SPD‐10AV/VP UV detector set at 254 nm. Shimadzu Class‐LC10 1.6 software was used to calculate the peak areas. The molar ratio of 8‐oxodGuo to dGuo in each DNA sample was determined based on coulometric detection at 280 mV for 8‐oxodGuo and absorbance at 254 nm for dGuo in each injection. Glycemia, triglyceridemia and insulinemia Blood was collected from fasted 32‐week animals and centrifuged at 780 g for 15 min. Serum was analyzed for glucose and triglyceride levels by the Laboratório de Análises Clínicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, using enzymatic colorimetric assays (Labtest Diagnóstica, Lagoa Santa, Brazil). Insulin levels were determined in frozen serum samples using an ELISA kit from Linco Research (St. Charles, MO, USA). Data analysis Data were analyzed using GraphPad Prism and Origin Software. Figures 1–3 represent averages ± SEM of measurements from 7–30 different animals. DNP and control groups were compared using unpaired t‐tests. Two‐tailed p‐values under 0.05 were considered significant. Survival curves (Fig. 4) were compared by log rank tests.

Acknowledgments The authors thank Edson A. Gomes for excellent technical assistance, Marilene Demasi for help with protein carbonyl measurements, Silvania M. P. Neves, Renata S. Fontes, Flavia M. P. Ong and Maria de Fátima Rodrigues for outstanding animal husbandry and care, and Professor Ohara Augusto for critical reading of the manuscript. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Instituto do Milênio Redoxoma and the John Simon Guggenheim Memorial Foundation.