Replicatively senescent human fibroblasts reveal a distinct intracellular metabolic profile with alterations in NAD+ and nicotinamide metabolism

Cellular senescence occurs by proliferative exhaustion (PEsen) or following multiple cellular stresses but had not previously been subject to detailed metabolomic analysis. Therefore, we compared PEsen fibroblasts with proliferating and transiently growth arrested controls using a combination of different mass spectroscopy techniques. PEsen cells showed many specific alterations in both the NAD+ de novo and salvage pathways including striking accumulations of nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) in the amidated salvage pathway despite no increase in nicotinamide phosphoribosyl transferase or in the NR transport protein, CD73. Extracellular nicotinate was depleted and metabolites of the deamidated salvage pathway were reduced but intracellular NAD+ and nicotinamide were nevertheless maintained. However, sirtuin 1 was downregulated and so the accumulation of NMN and NR was best explained by reduced flux through the amidated arm of the NAD+ salvage pathway due to reduced sirtuin activity. PEsen cells also showed evidence of increased redox homeostasis and upregulated pathways used to generate energy and cellular membranes; these included nucleotide catabolism, membrane lipid breakdown and increased creatine metabolism. Thus PEsen cells upregulate several different pathways to sustain their survival which may serve as pharmacological targets for the elimination of senescent cells in age-related disease.


Sample Preparation for Global Metabolomics
Samples were stored at -80°C until processed. Sample preparation was carried out as described previously 1 at Metabolon, Inc. Briefly, recovery standards were added prior to the first step in the extraction process for quality control purposes. To remove protein, dissociate small molecules bound to protein or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, Three types of controls were analyzed in concert with the experimental samples: samples generated from a pool of human plasma extensively characterized by Metabolon, Inc. or generated from a small portion of each experimental sample of interest served as technical replicate throughout the data set; extracted water samples served as process blanks; and a cocktail of standards spiked into every analyzed sample allowed instrument performance monitoring. Instrument variability was determined by calculating the median relative standard deviation (RSD) for the standards that were added to each sample prior to injection into the mass spectrometers (median RSD typically = 4-6%; n ≥ 30 standards). Overall process variability was determined by calculating the median RSD for all endogenous metabolites (i.e., noninstrument standards) present in 100% of the pooled human plasma or client matrix samples (median RSD = 8-12%; n = several hundred metabolites). Experimental samples and controls were randomized across the platform run.

Mass Spectrometry Analysis
Non-targeted MS analysis was performed at Metabolon, Inc. Extracts were subjected to either GC-MS 2 or UPLC-MS/MS 1 . The chromatography was standardized and, once the method was validated no further changes were made. As part of Metabolon's general practice, all columns were purchased from a single manufacturer's lot at the outset of experiments. All solvents were similarly purchased in bulk from a single manufacturer's lot in sufficient quantity to complete all related experiments. For each sample, vacuum-dried samples were dissolved in injection solvent containing eight or more injection standards at fixed concentrations, depending on the platform. The internal standards were used both to assure injection and chromatographic consistency. Instruments were tuned and calibrated for mass resolution and mass accuracy daily.
The UPLC-MS/MS platform utilized a Waters Acquity UPLC with Waters UPLC BEH C18-2.1×100 mm, 1.7 μm columns and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. The sample extract was dried then reconstituted in acidic or basic LCcompatible solvents, each of which contained 8 or more injection standards at fixed concentrations to ensure injection and chromatographic consistency. One aliquot was analyzed using acidic, positive ionoptimized conditions and the other using basic, negative ion-optimized conditions in two independent injections using separate dedicated columns (Waters UPLC BEH C18-2.1x100 mm, 1.7 µm). Extracts reconstituted in acidic conditions were gradient eluted using water and methanol containing 0.1% formic acid, while the basic extracts, which also used water/methanol, contained 6.5mM ammonium bicarbonate. A third aliquot was analyzed via negative ionization following elution from a HILIC column (Waters UPLC BEH Amide 2.1x150 mm, 1.7 µm) using a gradient consisting of water and acetonitrile with 10mM Ammonium Formate. The MS analysis alternated between MS and data-dependent MS 2 scans using dynamic exclusion, and the scan range was from 80-1000 m/z. The samples destined for analysis by GC-MS were dried under vacuum desiccation for a minimum of 18 h prior to being derivatized under dried nitrogen using bistrimethyl-silyltrifluoroacetamide. Derivatized samples were separated on a 5% diphenyl / 95% dimethyl polysiloxane fused silica column (20 m x 0.18 mm ID; 0.18 um film thickness) with helium as carrier gas and a temperature ramp from 60° to 340°C in a 17.5 min period. All samples were analyzed on a Thermo-Finnigan Trace DSQ fast-scanning singlequadrupole MS using electron impact ionization (EI) and operated at unit mass resolving power. The scan range was from 50-750 m/z.

Compound Identification, Quantification, and Data Curation
Metabolites were identified by automated comparison of the ion features in the experimental samples to a reference library of chemical standard entries that included retention time, molecular weight (m/z), preferred adducts, and in-source fragments as well as associated MS spectra and curated by visual inspection for quality control using software developed at Metabolon 3 . Identification of known chemical entities is based on comparison to metabolomic library entries of purified standards. Commercially available purified standard compounds have been acquired and registered into LIMS for distribution to both the UPLC-MS/MS and GC-MS platforms for determination of their detectable characteristics. Peaks were quantified using area-under-the-curve. Raw area counts for each metabolite in each sample were normalized to correct for variation resulting from instrument inter-day tuning differences by the median value for each run-day, therefore, setting the medians to 1.0 for each run. This preserved variation between samples but allowed metabolites of widely different raw peak areas to be compared on a similar graphical scale. Missing values were imputed with the observed minimum after normalization.
Immunofluorescence images were quantified using the Image J program as described previously 4 .

Western blotting
Western blotting was carried out as described previously 6 using the following antibody: Anti SIRT1 Mouse monoclonal Abcam Cat # Ab110304. HeLa cell extracts were used as positive controls. False discovery rates as determined by the Welch's t-test and indicated by q values of less than 0.1 are considered most significant and are highlighted in red when accumulated and green for depleted. False discovery rates as determined by the Welch's t-test and indicated by q values of less than 0.1 are considered most significant and are highlighted in red when accumulated and green for depleted. ND = not determined. False discovery rates as determined by the Welch's t-test and indicated by q values of less than 0.1 are considered most significant and are highlighted in red when accumulated and green for depleted.