Article pubs.acs.org/jpr
Specific Lipidome Signatures in Central Nervous System from Methionine-Restricted Mice Mariona Jové,†,‡ Victòria Ayala,†,‡ Omar Ramírez-Núñez,† Alba Naudí,† Rosanna Cabré,† Corinne M. Spickett,§ Manuel Portero-Otín,† and Reinald Pamplona*,† †
Department of Experimental Medicine, Faculty of Medicine, University of Lleida-IRBLleida, Lleida, Spain School of Life and Health Sciences, Aston University, Birmingham, United Kingdom
§
S Supporting Information *
ABSTRACT: Membrane lipid composition is an important correlate of the rate of aging of animals. Dietary methionine restriction (MetR) increases lifespan in rodents. The underlying mechanisms have not been elucidated but could include changes in tissue lipidomes. In this work, we demonstrate that 80% MetR in mice induces marked changes in the brain, spinal cord, and liver lipidomes. Further, at least 50% of the lipids changed are common in the brain and spinal cord but not in the liver, suggesting a nervous system-specific lipidomic profile of MetR. The differentially expressed lipids includes (a) specific phospholipid species, which could reflect adaptive membrane responses, (b) sphingolipids, which could lead to changes in ceramide signaling pathways, and (c) the physiologically redox-relevant ubiquinone 9, indicating adaptations in phase II antioxidant response metabolism. In addition, specific oxidation products derived from cholesterol, phosphatidylcholine, and phosphatidylethanolamine were significantly decreased in the brain, spinal cord, and liver from MetR mice. These results demonstrate the importance of adaptive responses of membrane lipids leading to increased stress resistance as a major mechanistic contributor to the lowered rate of aging in MetR mice. KEYWORDS: membrane unsaturation, phospholipids oxidation, rate of aging, mitochondria, free radicals, phase-II antioxidants
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INTRODUCTION Membrane lipid composition is an important correlate of the rate of aging of animals.1−3 A low degree of unsaturation of cellular membranes is a characteristic of long-lived animal species, both vertebrates and invertebrates.2 Besides fatty acid unsaturation, lipid classes also change with aging: it is known that both human serum4 and cellular5 metabolome/lipidome changes with age. In rodents, aging affects brain phospholipids classes,6 and this relationship extends to mitochondrial lipids where it is particularly apparent in phosphatidylcholines (PC) and phosphatidylethanolamines (PE).7,8 Among the cellular constituents, lipids (cholesterol, phospholipids and others) are relevant targets of oxidative attack leading to the formation and accumulation of lipid peroxidation (LPO) products, such as oxysterols, hydroperoxides and endoperoxides.9 The fragmentation of these LPO products produces a wide range of reactive carbonyl intermediates which can, finally, react and damage cellular proteins, aminophospholipids and DNA.2,3,10 Although these molecules play a role as physiological mediators of cellular antioxidant signaling pathways,2 progressive accumulation of their modifications on target molecules results in tissue and cell dysfunction, a typical feature of aging and oxidative-stress related diseases.11 In this sense, it has been known for many © 2013 American Chemical Society
years that fatty acids differ dramatically in their susceptibility to peroxidation. Polyunsaturated fatty acids (PUFAs) are the most susceptible, and the greater the degree of unsaturation of a PUFA molecule the greater its susceptibility to peroxidation, while both saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) are almost incapable of being peroxidized by physiological oxidants.3,12 In long-lived animal species, including birds and mammals, a low degree of membrane fatty acid unsaturation is associated with a low sensitivity to LPO in vivo and in vitro and, consequently, a low steady-state level of lipoxidation-derived adducts in both tissue and mitochondrial proteins of skeletal muscle, heart, liver, and brain.2 In this scenario, tissues with postmitotic cells (like brain, heart or skeletal tissues) tend to accumulate high quantities of LPO products with aging.2,3 Indeed, lipofuscin accumulation (basically derived from LPO reactions) is a cellular hallmark of aging.13 In particular, the central nervous system (CNS) is a very sensitive target for LPO damage because of the high level of polyunsaturated lipids in neuronal cell membranes, high metabolic rate and relatively poor antioxidant defense.14,15 Accordingly, oxidative stress is very pronounced in a number of Received: January 22, 2013 Published: April 16, 2013 2679
dx.doi.org/10.1021/pr400064a | J. Proteome Res. 2013, 12, 2679−2689
Journal of Proteome Research
Article
(Supplementary Table S1, Supporting Information). In order to ensure same food and energy intake of both groups, a pairfeeding experimental method was applied. Daily visual inspection of the mice cages indicated that there were no differences in food spillage between control and MetR animals. After 6 months of dietary treatment the animals were sacrificed. The evolution of body weights during the experiment were represented in Supplementary Figure S1, Supporting Information. As described previously,16,17 no gross morphological changes were evident under macroscopic examination of major organs. The tissues (brain, spinal cord and liver) were immediately stored at −80 °C until analysis. All experimental procedures were approved by the Institutional Animal Care Committee of IRBLleida and were conformed with the Directive 2010/63/EU of the European Parliament.
age-related neurodegenerative disorders such as Azheimer’s disease, Parkinson’s disease or Amyotrophic Lateral Sclerosis, among others.15 Although numerous studies have documented the decrease in the rate of aging by calorie restriction (CR), the dietary factors that cause these beneficial changes are currently unclear. Previous studies demonstrated that methionine restriction (MetR) without changes in energy intake is also capable of increasing maximum lifespan in rodents.16,17 MetR appeared to have a similar effect on lifespan to CR but the potential mechanisms involved are apparently different.18 In common with CR, MetR can decrease mitochondrial free radical production, as well as oxidative damage to mitochondrial DNA and proteins in mitochondria from rat heart and liver,19 and also in the brain,20 suggesting that the decrease in methionine intake could be responsible, at least in part, for the decrease in aging rate observed during CR. However, no information was available on the potential effects of MetR in biologically relevant lipids. In the current study, we addressed this outstanding issue by investigating MetR-induced changes to a wide range of phospholipids and other esterified lipids because there is increasing evidence for specific biological effects of different phospholipid classes, as well as the occurrence of nonrandom oxidation between phospholipids.21 To achieve this aim, we used a QTOF-based high-throughput lipidomic approach in order to understand the specific cell adaptive response in front to methionine restriction. We have focused on nervous tissue, due to its relevance in neurodegenerative diseases, its sensitivity to age-related changes and its structural dependence on cellular lipid composition.
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Sample Processing
Five-hundred micrograms of brain, spinal cord and liver were homogenized separately in 1 mL of 0.88% KCl (w/v), with a Potter−Elvehjem device at 4 °C. Protein concentration was measured using the Lowry assay (Bio-Rad Laboratories, München, Germany) with bovine serum albumin as a standard. Total lipids from tissue samples were extracted with chloroform/methanol (2:1, v/v) in the presence of 0.01% butylated hydroxytoluene to avoid artifactual oxidation.23 Lipidomic Analyses
For LC-Q-TOF-based lipid molecular species analyses, lipid extracts were subjected to mass-spectrometry using a HPLC 1200 series coupled to ESI-Q-TOF MS/MS 6520 (Agilent Technologies, Barcelona, Spain).24 Lipid class representative internal standards were added (Supplementary Table S2).25 The samples were evaporated using a Speed Vac (Thermo Fisher Scientific, Barcelona, Spain) and resuspended in chloroform/methanol (1:3, v/v). One μL of sample was applied onto a reversed-phase column (Luna C5, 3.5 μm, 4.6 × 50 mm, Phenomenex, Los Angeles, CA) equipped with a reversed-phase precolumn (C4, 3.5 μm, 2 × 20 mm, Phenomenex) for positive ionization, and onto a reverse phase column (Gemini C18, 3.5 μm, 4.6 × 50 mm, Phenomenex) equipped with a reverse phase precolumn (C18, 3.5 μm, 2 × 20 mm, Phenomenex) for negative ionization. The flow rate was 200 μL/min with solvent A composed of 95% water, 5% methanol containing 0.1% formic acid and 5 mM ammonium formate for positive ionization or 0.1% ammonium hydroxide for negative ionization, and solvent B composed of 65% isopropanol, 30% methanol, 5% water containing corresponding counterions. The gradient consisted of solvent B from 0 to 20% in 5 min, from 20 to 100% in 60 min, return to 0% B in 20 min, and re-equilibrated at 0% solvent B for 10 min. Data were collected in positive and negative electrospray ionization mode TOF operated in full-scan mode at 100 to 3000 m/z in an extended dynamic range (2 GHz), using N2 as nebulizer gas (5 L/min, 350 °C). The capillary voltage was 3500 V with a scan rate of 1 scan/s. The MassHunter Data Analysis Software (Agilent Technologies, Barcelona, Spain) was used to collect the results and the MassHunter Qualitative Analysis Software (Agilent Technologies, Barcelona, Spain) to obtain the molecular features of the samples, representing different, comigrating ionic species of a given molecular entity using the Molecular Feature Extractor algorithm (Agilent Technologies, Barcelona, Spain).26 Finally, the MassHunter Mass Profiler Professional Software (Agilent Technologies,
EXPERIMENTAL SECTION
Chemicals
Potassium chloride, chloroform, ammonium formate and ammonium hydroxide were from Sigma-Aldrich (St. Louis, MO), methanol was from Carlo Erba (Milano, Italy), acetone was from Riedel-de-Häen (Seelze, Germany) and LC−MSgrade isopropanol and formic acid were from Baker (Phillipsburg, NJ). Chloroform is a toxic agent and therefore should be handled under a fume hood following Regulations for its use and waste disposal.22 Animals and Diet
Fourteen B6SJL/C57BL6 1 month old mice (6 male and 7 female) of 15.06 ± 0.16 g (male) and 13.11 ± 1.91g (female) body weight were obtained from Jackson Laboratories (Bar Harbour, ME). The animals were caged and maintained in a 12:12 (light/dark) cycle at 22 ± 2 °C and 50 ± 10% relative humidity. Control animals (3 male and 4 female) were fed a semipurified diet prepared by MP Biochemicals (Irvine, CA). The detailed composition of the two diets is shown in Supplementary Table S1, Supporting Information. The composition of the 80% MetR diets was similar to that of the control diet except that L-methionine was present at 0.172%, which corresponds to amounts 80% lower than the Lmethionine content of the control diet (0.86%). The percent decrease in L-methionine in the 80% MetR diets was compensated by increases in all the rest of the dietary components in proportion to their presence in the diet. Since the percent absolute decrease in L-methionine was small, with this procedure the percent presence of all the rest of the dietary components was almost the same in both experimental diets 2680
dx.doi.org/10.1021/pr400064a | J. Proteome Res. 2013, 12, 2679−2689
Journal of Proteome Research
Article
(NaF), 1 mM butylated hydroxytoluene, and a commercial protease inhibitors (Amershan Bioscience, Madrid Spain)) with a Potter−Eljeveim device at 4 °C. After sample homogenization, protein concentration was measured using the BIORAD protein assay kit (BioRad Laboratories, München, Germany). For immunodetection, after SDS−PAGE, proteins were transferred using a Mini Trans Blot Transfer Cell (BioRad, Barcelona, Spain) to PVDF membranes (Millipore, Bedford, MA). The protein levels of mitochondrial respiratory chain complexes, NQO1, SOD1 and HO1 were estimated using Western Blot analysis. Immunodetection of respiratory chain complexes was performed using specific antibodies for the 39 kDa (NDUFA9) subunit of complex I (1:2000), 70 kDa subunit (Flavoprotein) of complex II (1:2000), and 48 kDa subunit (CORE 2) of complex III (1:2000) (antibodies A21344, A11142, A11143 from Molecular Probes, Eugene, OR, respectively). NOQ1C-terminal was detected using N5288 from Sigma-Aldrich, St. Louis, MO (1:2000), SOD1 using AB1237 from Millipore, Madrid, Spain (1:2000) and HO1 using P109 from Cell Signaling Technology, Inc., Danvers, MA (1:1000). An antibody to porin (1:20000, A31855, Molecular Probes, Eugene, OR) was used as a control for total mitochondrial mass; beta-actin (1:2000, A5441, from SigmaAldrich, St. Louis, MO) or tubulin (1:1000, ab7291, Abcam, Cambridge, U.K.) were employed for total protein charge. Appropriate peroxidase-coupled secondary antibodies (sheep antimouse (1:7500, GE Amersham, Barcelona, Spain); antirabbit (1:50000, Pierce, Barcelona, Spain) and chemiluminescence HRP substrate (Millipore Spain, Barcelona, Spain) were used for detection. Signal quantification and recording was performed with a ChemiDoc instrument (BioRad, Barcelona, Spain). Control experiments showed that omission of primary or secondary antibody addition produced blots with no detectable signal.
Barcelona, Spain) was used to perform a nontargeted lipidomic analysis over the extracted features. Only common features (found in at least 75% of the samples of the same condition) were taken into account to correct for individual bias. PCA was obtained using this software. The masses representing significant differences by Student t test (fold change ≥ 2, p < 0.05 with Benjamini-Hochberg Multiple Testing Correction) were searched against the LIPID MAPS27 database (exact mass ppm
