Modulation of white adipose tissue proteome by aging and calorie restriction Adamo Valle, Jordi Sastre-Serra, Pilar Roca and Jordi Oliver Grupo Multidisciplinar de Oncologı´a Traslacional, Institut Universitari d’Investigacio ´ en Cie ` ncies de la Salut, Universitat de les Illes Balears; Ciber Fisiopatologı ´a Obesidad y Nutricio ´n (CB06 ⁄ 03) Instituto Salud Carlos III, Cra Valldemossa km 7.5, E-07122, Palma de Mallorca, Illes Balears, Spain Summary Aging is associated with an accrual of body fat, progres- sive development of insulin resistance and other obesity comorbidities that contribute to decrease life span. Calo- ric restriction (CR), which primarily affects energy stores in adipose tissue, is known to extend life span and retard the aging process in animal models. In this study, a prote- omic approach combining 2-DE and MS was used to iden- tify proteins modulated by aging and CR in rat white adipose tissue proteome. Proteomic analysis revealed 133 differentially expressed spots, 57 of which were unambig- uously identified by MS. Although CR opposed part of the age-associated protein expression patterns, many effects of CR were on proteins unaltered by age, suggesting that the effects of CR on adipose tissue are only weakly related to those of aging. Particularly, CR and aging altered glu- cose, intermediate and lipid metabolism, with CR enhanc- ing the expression of enzymes involved in oxalacetate and NADPH production, lipid biosynthesis and lipolysis. Consistently, insulin-b and b3-adrenergic receptors were also increased by CR, which denotes improved sensitivity to lipogenic ⁄ lipolytic stimuli. Other beneficial outcomes of CR were an improvement in oxidative stress, prevent- ing the age-associated decrease in several antioxidant enzymes. Proteins involved in cytoskeleton, iron storage, energy metabolism and several proteins with novel or unknown functions in adipose tissue were also modu- lated by age and ⁄ or CR. Such orchestrated changes in expression of multiple proteins provide insights into the mechanism underlying CR effects, ultimately allowing the discovery of new markers of aging and targets for the development of CR-mimetics. Key words: adipose tissue; aging; caloric restriction; life- span; proteomics. Introduction Caloric restriction (CR) without malnutrition is the most robust, reproducible and simplest manipulation able to extend life span and delay the onset of age-associated disorders in a wide range of animal models. In rodents, 30–40% reduction in caloric intake has been shown to extend maximum life span 30–50% and reduce numerous pathological phenotypes, including type II diabetes (Astrup, 2001), neurodegenerative disorders (Patel et al., 2005), oxidative stress (Yu, 2006) and inflammation (Kalani et al., 2006). Although attenuation of oxidative stress and reduction of glycemia and insulinemia have been proposed to be involved in the actions of CR, the exact underlying mechanism and the main target tissue involved in the benefits of this intervention are still debatable (Sohal & Weindruch, 1996). Aging is known to be associated with obesity phenotype, alterations in body fat distribution and the development of insu- lin and leptin resistance (Enzi et al., 1986; Fraze et al., 1987; Wang et al., 2001; Ma et al., 2002). Several reports have sug- gested that reduction in white adipose tissue (WAT) may be a primary factor in the longevity induced by CR. Thus, mice engi- neered to have reduced WAT have been reported to live longer, even though these animals do not eat less (Bluher et al., 2003). Another supporting point is that surgical removal of visceral fat has also been shown to prevent obesity-induced and age-associ- ated insulin resistance in rats (Barzilai et al., 1999; Gabriely et al., 2002). Nevertheless, it is worth noting that CR benefits are not simply mediated through a reduction in overall adiposity, as evidenced by CR-induced life span extension in genetically obese mice (Harrison et al., 1984). In addition to its role as fat store and energy buffer, WAT acts as an active endocrine organ which secretes adipokines contributing to regulate several physiological processes such as energy homeostasis, reproduction or inflammation. During adaptation to CR, fat is mobilized from WAT but adipocytes, with minimal fat stores, are sustained in the tissue (Higami et al., 2006; Bouwman et al., 2009). These metabolically reprogrammed cells may have endocrine and functional prop- erties that differ from white adipocytes of ad libitum-fed animals and, therefore, may help to explain the metabolic and endocrine shift underlying the health advantages of CR diet. Several studies based on gene profiling have focused on WAT reprogramming after weight loss or CR diet (Viguerie et al., 2005; Higami et al., 2006; Linford et al., 2007; Swindell, 2009). Although DNA array is a powerful tool, the Correspondence Dr. Adamo Valle, Department Biologia Fonamental I Cie `ncies de la Salut, Universitat de les Illes Balears. Cra. Valldemossa km 7.5 E-07122- Palma de Mallorca, Spain. Tel.: +34 971 259 644; fax: +34 971 173 184; e-mail: adamo.valle@uib.es Accepted for publication 23 July 2010 882 ª 2010 The Authors Aging Cell ª 2010 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland Aging Cell (2010) 9, pp882–894 Doi: 10.1111/j.1474-9726.2010.00613.x Aging Cell