Free [NADH]/[NAD + ] regulates sirtuin expression Juan Gambini a , Mari Carmen Gomez-Cabrera a , Consuelo Borras a , Soraya L. Valles a , Raul Lopez-Grueso a , Vladimir E. Martinez-Bello a , Daniel Herranz b , Federico V. Pallardo a , Jesus A.F. Tresguerres c , Manuel Serrano b , Jose Viña a,⇑ a Department of Physiology, Faculty of Medicine, University of Valencia, Fundacion Investigacion Hospital Clinico Universitario INCLIVA, Spain b Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid E-28029, Spain c Department of Physiology, Faculty of Medicine, University Complutense, Madrid, Spain article info Article history: Received 26 January 2011 and in revised form 29 April 2011 Available online 7 May 2011 Keywords: Ethanol Exercise Lactate Pyruvate Rat Drosophila abstract Sirtuins are deacetylases involved in metabolic regulation and longevity. Our aim was to test the hypoth- esis that they are subjected to redox regulation by the [NADH]/[NAD + ] ratio. We used NIH3T3 fibroblasts in culture, Drosophila fed with or without ethanol and exercising rats. In all three models an increase in [NADH]/[NAD + ] came up with an increased expression of sirtuin mRNA and protein. PGC-1a (a substrate of sirtuins) protein level was significantly increased in fibroblasts incubated with lactate and pyruvate but this effect was lost in fibroblasts obtained from sirtuin-deficient mice. We conclude that the expression of sirtuins is subject to tight redox regulation by the [NADH]/[NAD + ] ratio, which is a major sensor for metabolite availability conserved from invertebrates to vertebrates. Ó 2011 Published by Elsevier Inc. Introduction Sirtuins are deacetylases that have been involved in a variety of processes, including longevity, cancer protection and metabolic regulation [1,2]. They use NAD + as a substrate and their reaction produces nicotinamide. They are considered as nutrient sensors [3]. Since nutrient availability affects the [NADH]/[NAD + ] ratio [4], we reasoned that sirtuin expression might be redox regulated via this ratio, which is similar in the cytosol and in the nucleus [5]. The [NADH]/[NAD + ] ratio cannot be measured directly, i.e. measur- ing NAD + and NADH because this presents two major problems, one is that a significant amount of these co-enzymes are bound to dehydrogenases and therefore their free levels are very difficult to measure as they are released from proteins when acid extracts are used. The second, and perhaps more important one, is that these direct measurements of NAD + and NADH fail to differentiate between the various cell compartments particularly mitochondria and cytosol. The [NADH]/[NAD + ] ratio in these two major cell compartments is known to be significantly different [4]. However [NADH]/[NAD + ] ratio can be calculated by measuring reduced and oxidised substrates for suitable NAD + -dependent dehydrogen- ases, in the case of cytosol, lactate and pyruvate [6]. With this framework in mind, we tested, in a variety of cells in culture as well as in invertebrate and vertebrate animal models, whether changing the [NADH]/[NAD + ] ratio could affect the expression of sirtuins. Our results are consistent with the hypothesis that in- creases in the [NADH]/[NAD + ] pair activate the expression of sirtu- ins in all the experimental models used. Materials and methods Cell culture NIH3T3 fibroblasts and mouse embryo fibroblasts (MEFs) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum in 25 or 75 cm 2 flasks. The gas phase was 5% CO 2 in air at 37 °C. Ethanol (10 or 20 mM) or lactate and pyruvate (10/1 mM) were added to the medium from 3 to 24 h depending on the experiment. Lactate and pyruvate determinations Lactate and pyruvate were measured spectrophotometrically. To determine L-lactate we used the method of Gutmann and Wahlefeld 0003-9861/$ - see front matter Ó 2011 Published by Elsevier Inc. doi:10.1016/j.abb.2011.04.020 Abbreviations used: ECL, enhanced chemiluminiscence; GAPDH, glyceraldehyde- 3P-dehydrogenase; L/P, lactate/pyruvate; MEFs, mouse embrionary fibroblasts; NAD, nicotinamide adenine dinucleotide; NIH3T3, mouse embryonic fibroblast cell line; SIRT1, sirtuin1; SDS, sodium dodecyl sulfate; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis. ⇑ Corresponding author. Address: Department of Physiology, Faculty of Medicine, Av. Blasco Ibañez 15, Valencia 46010, Spain. Fax: +34 96 386 46 42. E-mail addresses: jose.vina@uv.es, jvina@uv.es (J. Viña). Archives of Biochemistry and Biophysics 512 (2011) 24–29 Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi