Chemicals

All chemicals were obtained from Sigma-Aldrich (Munich, Germany) unless stated otherwise.

Statistical analyses

Data are expressed as means±s.d. unless otherwise indicated. Statistical analyses for all C. elegans data except life span and stress resistance assays were performed by Student’s t-test (unpaired, two-tailed) after testing for equal distribution of the data and equal variances within the data set. For comparing significant distributions between different groups in the life span assays and stress resistance assays, statistical calculations were performed using JMP software version 9.0 (SAS Institute Inc., Cary, NC, USA) applying the log-rank test.

Statistical analyses for all murine data except life span analyses were performed by two-way ANOVA including the ‘treatment by sex’ interaction to test for potential sex-specific interactions. In parallel, sex-specific analyses were performed using Student’s t-test (unpaired, two-tailed). Mortality rates during the murine survival study were assessed by using log-rank and Cox regression tests to compare survival using SPSS Version 20 (IBM, Armonk, NY, USA). Cox regression was performed including the ‘treatment by sex’ interaction to test for potential sex-specific interactions. Additional calculations were performed using Excel 2007 (Microsoft, Albuquerque, NM, USA). Except for promoter analyses (see below), a P-value <0.05 was considered as statistically significant; a P-value <0.1 was considered as ‘significant by trend’.

Nematode strains and maintenance

C. elegans strains used for this publication were provided by the Caenorhabditis Genetics Center (University of Minnesota, USA). Nematodes were grown and maintained on NGM agar plates at 20 °C using OP50 bacteria as food source52. Treatment of C. elegans was carried out on NGM agar plates containing 100 μM GlcN, if not stated otherwise. All agar plates were prepared from the same batch of NGM agar, whereas treatment plates were supplemented with the respective compound and control plates with water. After plates were poured and dried, they were sealed and stored at 4 °C. Freshly prepared E. coli OP50 were spotted on plates on the previous evening and allowed to dry and settle overnight. Incubations with compounds started 64 h after synchronization of the population, by washing the synchronized, young adult worms and then transferring them to the respective treatment plates using S-Buffer16.

Nematodal life span assays

All life span assays were performed at 20 °C. Sixty-four hours after egg preparation around synchronized 150 nematodes were manually transferred to fresh incubation plates containing the respective compounds. For the first 10–14 days, worms were transferred every day and afterwards every second day. Nematodes that show no reaction to gentle stimulation were scored as dead. Those animals that crawled off the plates or displayed non-natural death particularly because of internal hatching were censored.

Quantification of E. coli growth

Fifty millilitres of liquid Luria Broth (LB) or NGM media were supplemented respectively with either 50 μl H 2 O or 50 μl of a 100 mM GlcN solution. Four Erlenmeyer flasks for each medium and condition were used. Flask were inoculated with 100 μl of an E. coli OP50 suspension freshly prepared from an overnight culture. Flasks were put in an orbital shaker (GFL, Burgwedel, Germany) set at 37 °C and 200 r.p.m. Bacterial concentration was determined by measuring optical density at 600 nm in a micro-titer plate reader (FLUOstar Omega, BMG Labtech, Offenburg, Germany).

RNAi-mediated gene knockdown experiments

For RNAi gene knock-down experiments, we applied E. coli HT115 to the worms. Clones for RNAi against aat-1 and F21D5.1 were derived from Ahringer library (Source BioScience, Nottingham, UK) and C. elegans ORF Collection (Thermo Scientific, Waltham, MA, USA) (Supplementary Table 1) respectively, and were sequenced prior use. The bacteria were spotted on NGM plates containing additionally 1 mM IPTG, 100 μg ml−1 Ampicillin and, if required, 12.5 μg ml−1 tetracycline (all from Applichem, Darmstadt, Germany).

[14C]Glucose oxidation rates

Uniformly labelled [14C]D-glucose was purchased from GE Healthcare. Specific activity of the batch used was 300 mCi mmol−1. Animals were maintained as described above on plates containing 5′-fluorouridine and the respective treatment for 24 h. Equal numbers of animals were placed on each plate when treatment was initiated. After collection and three subsequent washes in S-buffer, worm pellets were resuspended in the incubation buffer. Two millilitres of the suspension was transferred to 4 cm Petri dishes. The latter were placed in 10 cm Petri dishes together with a second 4 cm Petri dish containing 600 μl of 0.1 M KOH solution to trap CO 2 53. Consequently, each 10 cm dish contained two 4 cm dishes, one carrying nematodes and the other containing KOH. As a substrate, labelled glucose was added to a final concentration of 17.1 μM U-[14C]D-glucose (5 μCi ml−1) in the nematode suspension. Non-radioactive glucose was added to a final concentration of 0.5 mM to the control samples or the samples pre-treated with GlcN, respectively. The 10 cm Petri dishes were covered, sealed with Parafilm in an air-tight manner and incubated at 20 °C for 60 min. Subsequently, an aliquot of 500 μl of KOH was immersed in 10 ml of scintillation fluid and placed in a liquid scintillation counter (Beckman LS 6000, Global Medical Instrumentation, Inc.) to quantify the amount of trapped 14CO 2 . An aliquot was used for protein determination for normalization.

Respiration assays

Respiration was quantified using a DW1/AD Clark-type electrode (Hansatech, King’s Lynn, England; Great Britain). After the individual incubation period, worms were harvested, washed and transferred into the DW1 chamber. Oxygen consumption was monitored for at least 10 min. Afterwards, worms were carefully removed from the chamber and collected for a subsequent protein determination. Therefore, worms were sonicated three times and centrifuged for 10 min at 12,000 g. Supernatant was used for protein determination using BCA, as described below.

Cell culture

HepG2 hepatoma cells were obtained from LGC Standards (Wesel, Germany) and were grown in RPMI media containing 10% heat-inactivated fetal bovine serum at 37 °C in 5% CO 2 /20% O 2 . Cells were serum starved for 5 h and incubated with or without 5 mM GlcN for the indicated time before collection.

ATP determination

Worms and cells were harvested and immediately shock frozen in liquid nitrogen. The frozen pellet was grinded in a nitrogen chilled mortar to yield powder. Guanidinium-hydrochloric acid (Guanidium HCl) (4 M) was prepared, heated to 100 °C and then mixed with the frozen powder to destroy ATPase activity and to further lyse samples. The mixture was boiled for 15 min at 100 °C with a subsequent centrifugation step (30 min at 13,200g and 4 °C). The supernatant was diluted with ddH 2 O 1:200 and analysed using a commercially available kit (CellTiter Glo; Promega, Fitchburg, WI, USA) according to the manufacturer’s instructions. For normalization of the luminescence signal, protein was determined as described below.

Immunoblotting

Frozen worm pellets were grinded in a nitrogen-chilled mortar and suspended in phosphate buffer containing protease and phosphatase inhibitors (Complete protease inhibitor cocktail (Roche, Penzberg, Germany) with the addition of 2 mM sodium fluoride, 2 mM sodium ortho-vanadate, 1 mM PMSF and 2 mM EDTA). Cells were harvested with identical buffer. Extracts were sonicated three times and centrifuged for 7 min at 12,000 g. Supernatants were used for protein quantification and an aliquot was boiled in Laemmli buffer and applied to SDS-PAGE. Antibodies against the phospho-AMPKα (Thr172; Cell Signaling, no. 2531), basal AMPK (Cell Signaling, no. 2532), phospho-p38 (Thr180/182; Cell Signaling, no. 9211), basal p38 (Cell Signaling, no. 9212) and alpha-tubulin (clone DM1A; Sigma-Aldrich, T9026) were used. Primary antibodies were diluted to 1:1,000 and secondary antibody to 1:10,000 before use on the membranes. Full-length images of immunoblots are shown in Supplementary Fig. 5.

Protein quantification

Protein content in nematodes and cells was determined by the Bradford method54 or the BCA method55. Assays were performed in 96-well plates using commercial available kits (Bio-Rad Laboratories AG, Cressier, Switzerland, and Thermo Scientific). Absorbance was measured in a microplate reader (FLUOstar Optima, BMG Labtech, Offenburg, Germany).

Nematodal quantification of mitochondrial ROS formation

Before ROS measurement MitoTracker Red CM-H 2 X ROS (Invitrogen, Carlsbad, CA, USA) incubation plates were prepared as follows: for each treatment, 500 μl heat-inactivated OP50 (65 °C and 30 min) were mixed with 100 μl MitoTracker Red CM-H 2 X stock solution (100 μM) and spotted on a large NGM agar plate that was allowed to dry for ~20 min32. Nematodes were incubated without GlcN, then washed off the plates with S-buffer and then allowed to settle by gravitation to remove offspring. Worms were washed two additional times with S-buffer and centrifuged (300 g, 30 s). The worm pellet was transferred to freshly prepared MitoTracker Red CM-H 2 X solution and incubated for 2 h at 20 °C. To remove excess dye from the gut, worms were transferred to NGM agar plates with the corresponding compound or, as a positive control, to plates containing 1 μM rotenone for 1 h at 20 °C. Aliquots of 100 μl worm suspension were distributed into 96-well Fluotrac plates (Greiner Bio-One, Frickenhausen, Germany). Fluorescence intensity was measured in a microplate reader (FLUOstar Optima) using well-scanning mode (excitation wavelength (ex): 570 nm, emission wavelength (em): 610 nm). To normalize fluorescence signal, the remaining worm suspension was used for protein determination.

Amplex Red-based quantification of supernatant hydrogen peroxide

Worms were removed from plates with 50 mM sodium-phosphate buffer, pH 7.4, washed twice and transferred into an upright Plexiglas cylinder (1.5 ml volume) with continuous stirring at low speed (100 r.p.m.) at 20 °C. First, determination of fluorescence was done without horse radish peroxidase only in the presence of 1 μM Amplex Red (Invitrogen) to detect possible unspecific increase in fluorescence (which was not observed). Next, 0.01 U ml−1 horse radish peroxidase was added and changes of fluorescence were recorded with a fluorescence detector (LF402 ProLine, IOM, Berlin, Germany) for at least 15 min at excitation and emission wavelengths of 571 and 585 nm, respectively42. Immediately afterwards, worms were removed and collected for protein determination to normalize fluorescence values.

Superoxide dismutase and catalase activity assays

To determine antioxidant enzyme activities (superoxide dismutase (SOD), catalase (CAT)) nematodes were harvested and washed with ice-cold buffer. The frozen pellet was grinded in a nitrogen chilled mortar together with 200 μl 50 mM phosphate buffer+1 mM EDTA) and sonicated threefold. Lysate was cleared by centrifugation for 15 min at 12,000 g and 4 °C. Supernatant was used for the subsequent measurement of catalase or superoxide dismutase activity as well as for a protein quantification to normalize enzyme activities. Determination of catalase activity was performed like previously described with minor changes56. Briefly, the diluted (25 mM potassium phosphate buffer plus 1 mM EDTA plus 0.1% BSA, pH 7.5) sample supernatant was mixed with assay buffer (100 mM potassium phosphate buffer, pH 7) and methanol (VWR, Darmstadt, Germany). Hydrogen peroxide (Applichem, 30%) was added and incubated for 20 min under continuous shaking at 20 °C. Reaction was terminated by addition of potassium hydroxide (Applichem, 10 M) and Purpald (Sigma-Aldrich, St. Louis, MO, USA; 46 mM in 0.5 M HCl) and incubated for 10 min by continuous shaking at 20 °C. Potassium periodate (Sigma-Aldrich; 192 mM in 0.5 M potassium hydroxide) was added to oxidize the Purpald–formaldehyde complex and incubated for further 5 min before the absorbance was measured at 540 nm.

SOD activity was quantified using a method described earlier57. Sample supernatant was incubated with WST-1 working solution (Tris–HCl, pH 8, diethylene-triamine-penta-acetic acid (Sigma-Aldrich, 100 μM), hypoxanthine (Applichem, 100 μM), WST-1 (180 μM) and xanthine oxidase (Sigma-Aldrich, 240 mU ml−1) for 20 min at 37 °C. Then, absorbance was measured at 450 nm.

PQ stress assay

N2 nematodes at an adult age of 6 days were transferred manually to fresh NGM plates containing 10 mM PQ (Acros Organics, Geel, Belgium) covered with heat-inactivated OP50 (30 min at 65 °C in a water bath) and attended by daily determination of the survival rate until all nematodes were deceased42. As described for life span analysis, worms were counted as censored in case of internal hatching, crawling off and bursting.

Murine breeding and housing conditions

C57BL/6NRj mice were bred in our own facilities based on founders from Janvier Sas (Le Genest Saint Isle, France); it should be noted that these mice lack the nicotinamide nucleotide transhydrogenase mutation found in C57BL/6 J-derived strains58. Animals were studied starting at an age of 100 weeks. Mice in our colony are tested every 6 months for serologic evidence of viral infection, and all such tests have been negative throughout the period of this study.

The ageing study was initiated with 71 male and 75 female mice in total. These were subjected to GlcN (34 male and 38 female) and control groups (remaining), respectively. Mice were inspected daily for health issues and deaths were recorded for each animal. Moribund animals were killed and recorded.

Mice were housed in a controlled environment (21±1 °C, 12 h/12 h light/dark cycle) and had free access to water and to pellet rodent chow containing 14 mg kg−1 alpha-tocopherol and 10 mg kg−1 ascorbic acid, as determined by HPLC. At an age of 100 weeks GlcN was supplied at a concentration of 10 g kg−1 in the diet (Ssniff Spezialdiäten GmbH, Soest, Germany). All experiments were approved by the Ethics Committee of the State Ministry of Environment, Health and Consumer Protection (Federal States of Brandenburg and Thuringia, Germany).

Murine body mass and body composition analyses

Body mass was quantified using a graded scale. Body composition was measured by use of quantitative nuclear magnetic resonance technique (Echo MRI-100 Body Composition Analyzer, Echo Medical Systems, Houston, USA) as described59.

Blood sampling

Blood samples, collected in tubes containing 21 U lithium heparin and centrifuged for 10 min at 4 °C and 8,000 r.p.m. (6,800 g), were obtained both in the fed state as well as after mice were fasted 16 h overnight by using lancets for submandibular bleeding (Goldenrod Animal Lancet, Medipoint, Mineola, NY, USA).

GlcN plasma concentrations

Plasma was derivatized60 with AccQ-Fluor Reagent Kit (Waters, Milford, MA, USA) according to the manufacturer’s instructions. HPLC was performed on Nexera sytem equipped with degasser DGU-20/A5, auto-sampler SIL-30AC, column oven CTO-20AC and fluorescence detector RF-20AXS (all obtained from Shimadzu, Kyoto, Japan). Chromatographic separation was performed on Reprospher 100 C18-DE 1.8 μm, 50 × 2 mm (Dr Maisch GmbH, Ammerbuch-Entringen, Germany) at 45 °C. Elution was performed using acetonitrile and sodium acetate buffer, pH 5.25, at flow rate of 0.8 ml min−1 using a gradient. Detection was carried out using excitation wavelength of 250 nm and emission wavelength of 395 nm.

Glucose tolerance tests

Glucose tolerance tests were performed by intraperitoneal glucose injection (D-glucose, Merck, Darmstadt, Germany) after mice were fasted 16 h overnight. Plasma was collected before and 10, 30, 60 and 120 min after administering of glucose and immediately frozen at −80 °C for measurement of glucose and insulin61.

Insulin tolerance tests

Mice were subjected to an insulin tolerance test by intraperitoneal injection of 1.5 IU kg−1 of human recombinant insulin (Insuman Rapid, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany). Glucose was determined from a tail vein blood before and 15, 30, 45, 60, 75, and 90 min after insulin injection by a glucometer (Contour, Bayer AG, Leverkusen, Germany).

Respiratory quotient and total energy expenditure

Total energy expenditure and respiratory quotients were determined by indirect calorimetry at 22 °C for 24 h with an open-circuitry calorimetry system (TSE PhenoMaster System, Inc., Midland, MI, USA). Rates of oxygen consumption (VO 2 ) and carbon dioxide production (VCO 2 ) were recorded for a 24-h period after mice were allowed to acclimate to the system for a period of 2 days. The air-tight respiratory cages were measured with a flow rate of about 0.38 l min−1. VO 2 and VCO 2 were recorded for 1.5 min in 16-min intervals for each animal, so that three or four data points were obtained every other hour. TEE (kcal h−1) was calculated with the equation TEE=16.17/VO 2 +5.03/VCO 2 −5.98/N, where N is excreted nitrogen and was assumed to be (0.1 g per day). Total energy expenditure was normalized to 24 h and metabolic body mass.

Determination of plasma parameters

Determination of glucose, triglycerides, alanine-aminotransferase, free fatty acids and total cholesterol in plasma was performed using an automated analyser (Cobas Mira S, Hoffmann-La Roche, Basel, Switzerland) with the appropriate commercially available reagent kits (Glucose HK CP, triglycerides, ALT CP, cholesterol CP, ABX, Montpellier, France; and NEFA HR, Wako, Neuss, Germany).

Quantification of D -GlcN-6-phosphate

Quantification of GlcN-6-phosphate and its metabolites was performed on an Agilent 6550 QTOF instrument by flow injection analysis time-of-flight mass spectrometry62. All samples were injected in duplicates. Ions were annotated based on their accurate mass and the Kyoto Encyclopedia of Genes and Genomes (KEGG) hsa reference list allowing a tolerance of 0.001 Da. Unknown ions and those annotated as adducts were discarded. This resulted in a total of 472 putatively annotated ions with unique m/z.

Targeted analysis of BCAA catabolism

To assess activation of catabolic pathways of BCAAs, we quantified levels of methyl-butanoyl-CoA, methyl-crotonyl-CoA and succinate on a Thermo Quantum Ultra instrument by targeted ion pairing-liquid chromatography tandem mass spectrometry using multiple reaction monitoring36,37.

Quantification of mtDNA

Total DNA was isolated by standard proteinase K and phenol–chloroform methods. mtDNA copy number level was analysed by quantitative real-time PCR using Viia 7 (Applied Biosystems). The amount of 0.4 ng of total DNA was used as a template for the amplification of mtDNA in mouse or C. elegans. Level of primers against mouse mtDNA (5′-AAGACACCTTGCCTAGCCACAC-3′ and 5′-TGGCTGGCACGAAATTTACC-3′) were normalized against nuclear 18SrRNA gene (5′-AACTTTCGATGGTAGTCGCCG-3′ and 5′-CCTTGGATGTGGTAGCCGTTT-3′). The same approach was used for analysis of C. elegans samples by using primer for mtDNA (5′-CTTTTATTACTCTATATGAGCGTC-3′ and 5′-AACAAAAGAAATTCCTGGTACAAG-3′) normalized against nuclear 18SrRNA homologue (5′-GCGAAAGCATTTGCCAAGAA-3′ and 5′-ATCGCGAGATGGCATCGTT-3′).

Extraction of RNA

Total RNA was isolated using QIAzol (Qiagen, Hilden, Germany) based on the phenol–chloroform extraction method. Afterwards, the RNA was quantified photometrically with a NanoDrop 1000 (PeqLab, Erlangen, Germany) and stored at −80 °C until use.

Next-generation sequencing (RNAseq)

Total RNA was inspected for degradation using Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). For library preparation an amount of 2 μg of total RNA per sample was processed using Illumina’s TruSeq RNA Sample Prep Kit (Illumina; San Diego; CA, USA) following the manufacturer’s instruction. Each library includes its own index sequence to allow multiplexing. The libraries were sequenced using v3 sequencing chemistry and a HiSeq2000 (Illumina) in a single read approach with 50 cycles resulting in reads with a length of 50 nucleotides. Libraries were sequenced in a multiplex manner pooling four libraries per lane. Sequencing ends up with ~30–40 Mio reads per sample. Sequence data were extracted in FastQ format and used for mapping approach.

Quantification of aat-1 mRNA

Total RNA was isolated and reverse-transcribed to first-strand cDNA using high-capacity cDNA Reverse Transcription Kit (Applied Biosystems), according to manufacturer’s protocols. Quantitative real-time PCR was performed using optical 384-well plates, SYBR Select Master Mix and Viia 7 (Applied Biosystems). All samples were measured in triplicates, and non-template controls were used to confirm specificity. Expression of aat-1 (F27C8.1) was quantified using specific primers 5′-ACCGGACTTGGTCTCCTTTT-3′ and 5′-TTTGGGTTCTGCAACTCCTC-3′, and normalized against a housekeeping gene (R07G3.1) using primers 5′-CTGCTGGACAGGAAGATTACG-3′ and 5′-CTCGGACATTCTCGAATGAAG-3′.

Bioinformatics of RNA expression data

All reads were mapped against the respective genomic sequences (ce10 for C. elegans; mm10 for Mus musculus) using TopHat 1.4.1. Only uniquely mappable reads were regarded. For counting the reads per gene (raw counts) the Python package HTSeq ( http://www.huber.embl.de/users/anders/HTSeq/doc/overview.html) was used in mode ‘union’ together with gene annotation for all RefSeq genes downloaded from the UCSC website.

Raw counts for the genes were analysed using the R Statistical Computing Environment63 and edgeR64. The latter provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg's approach65 for controlling the false discovery rate. If false discovery rate values were <0.05, genes were assigned as differentially expressed. For the comparison of orthologous differentially expressed genes in C. elegans and M. musculus, the R package orthology was applied66.

Promoter analyses

The search for SKN-1 transcription factor binding sites for each gene was done within the proximal promoter region 2 kb upstream of the predicted start codon. Therefore, a FASTA file containing the promoter regions for all genes was created using WormMart. Next, the remaining sequence file was scanned for one or more matches to the position-specific scoring matrices of SKN-1 using the matrix scan function of the pattern-matching programme regulatory sequence analysis tools67. The position-specific scoring matrices contains the nucleotide frequency at each position within the binding sites and were obtained from the Transfac database68. The threshold P-value, which indicates the risk of false positive predictions, was set to 0.0001.