Spermidine is needed for fasting to trigger cell cleanup and extend lifespan

BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was used for all experiments, except for experiments with rapamycin in which we used BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0). The null mutants BY4741 ∆spe2::kanMX (Y01743↗), ∆spe3::kanMX (Y05488↗), ∆spe4::kanMX (Y06945↗) and the temperature-sensitive mutant hyp2-1:kanMX (TSA736) were obtained from Euroscarf. We generated the null mutants BY4741 ∆spe1::hphNT1, ∆lia1::natNT2 and ∆spe1::hphNT1∆lia1::natNT2 according to previously described methods⁷⁰. In brief, we amplified a gene-specific hphNT1 or natNT2 knockout cassette by PCR using the template plasmids pFA6a-hphNT1 and pFA6a-natNT2 (ref. ⁷⁰) and primers:

SPE1_pYM_S1: 5′-GTTCTACAACTTTTTCATAGTAATCAAAACCTTTGAATTTCAAACTTACTATGCGTACGCTGCAGGTCGAC-3′

SPE1_pYM_S2: 5′-CTTTTCCCACCCCCTCCGTCTCTCTTGCGAAAGTCGTGGTTAAATATATCCTTCAATCGATGAATTCGAGCTCG-3′

Forward control primer SPE1_Ctrl: 5′-TCATCAAGAGCCCCATCC-3′

Reverse control primer Control_S1: 5′-GTCGACCTGCAGCGTACG-3′

LIA1_pYM_S1: 5′-GTTAGGATAAACTGTAGTCCTTCTAACATACCACGCAAGAAAGAAAAAAAAAAACCGTACGCTGCAGGTCGAC-3′

LIA1_pYM_S2: 5′-CAAGATTATACAATGATTATTGTTACTATCATTATTGATGATGCTGATTCTTATCGATGAATTCGAGCTCG-3′

Forward control primer LIA1_Ctrl: 5′-GTTCCCAGGCGAAGAAAGAAC-3′

Reverse control primer natNT2_Ctrl: 5′-CGTGTCGTCAAGAGTGGTAC-3′

To determine the protein levels of Dys1 and Lia1, we generated genome-tagged C-terminal 6×HA fusions using the plasmid pYM16 (ref. ⁷⁰) and the following primers:

For Dys1-6×HA:

DYS1_pYM_S3: 5′-CTGCTACCTTTGCCAGTGGTAAACCAATCAAAAAAGTTAAGAATCGTACGCTGCAGGTCGAC-3′

DYS1_pYM_S2: 5′-GAAATAGTACAGATTCATTTTTTTTTTTTTCATCTCAAAATTCTCTCATCAATCGATGAATTCGAGCTCG-3′

Forward control primer DYS1_Ctrl: 5′-GCGTGACCAAGGTATGAATCGTATT-3′

Reverse control primer Control_hphNT1: 5′-CATATCCACGCCCTCCTAC -3′

For Lia1-6×HA:

LIA1_pYM_S3: 5′-GATATGTATGATTACGAAAACAGCAACGAACTAGAATATGCTCCAACTGCTAATCGTACGCTGCAGGTCGAC-3′

LIA1_pYM_S2: 5′-CAAGATTATACAATGATTATTGTTACTATCATTATTGATGATGCTGATTCTTCTAATCGATGAATTCGAGCTCG-3′

Forward control primer LIA1_Ctrl: 5′-CTAGGTGACAAGGATTCGTTGGATG-3′

Reverse control primer Control_hphNT1: 5′- CATATCCACGCCCTCCTAC-3′

For the Pho8∆N60 assay to measure nonselective autophagy, we generated PHO8 pho8∆N60-URA3 strains in BY4741 WT and ∆spe1 background (in this case, the ∆spe1 strain was obtained from Euroscarf; spe1::kanMX; Y05034↗) using the integrative pTN9-URA plasmid⁷¹. Pho8∆N60-specific alkaline phosphatase activity was determined as described previously⁷². Endogenous GFP–Atg8 fusions were generated as previously described⁷². In brief, we used the vector template pYM-pATG8 (ref. ⁷²) to construct NatNT2::P_ATG8-yeGFP-ATG8 strains with primers:

Atg8_pYM_S1: 5′-CTAATAATTGTAAAGTTGAGAAAATCATAATAAAATAATTACTAGAGACATGCGTACGCTGCAGGTCGAC-3′

Atg8_pYM_S4: 5′-GACTCCGCCTTCCTTTTTTCAAATGGATATTCAGACTTAAATGTAGACTTCATCGATGAATTCTCTGTCG-3′

Forward control primer yeGFP_F: 5′-GGTGAAGAATTATTCACTGGTGTTG-3′

Reverse control primer ATG8_R: 5′-GAACAATAGATGGCTAATGAGTCC-3′

All experiments were performed in baffled 100-ml flasks filled with 10–15 ml growth medium, incubated at 28 °C and constant shaking (145 rpm), if not stated otherwise. For general experiments, we used standard dextrose medium (CTL) containing 0.17% yeast nitrogen base (BD Diagnostics), 0.5% (NH₄)₂SO₄, 80 mg l⁻¹ histidine, 200 mg l⁻¹ leucine 320 mg l⁻¹ uracil, 30 mg l⁻¹ adenine and 30 mg l⁻¹ all other amino acids, with 2% glucose, which we sterilized as separate 10× stocks by autoclaving. For experiments with BY4742 we additionally added 90 mg l⁻¹ lysine after mixing the stocks.

All cultures were inoculated from cellular material from a YPD (1% yeast extract (BD), 2% bacto peptone (BD), 2% glucose, 2% agar) plate, which was incubated at 28 °C for 2–3 days, placed at 4 °C for at least 1 day and a maximum of 2 weeks. We used pre-cultures in CTL medium to inoculate main cultures to an OD₆₀₀ of ~0.01–0.05. In general, main cultures were grown to mid-logarithmic phase (OD₆₀₀ = 1), centrifuged at 3,000–4,000 rpm (1731–3077 rcf) for 3–5 min, washed once with sterile pre-warmed ddH₂O, and re-suspended in fresh medium. For nitrogen deprivation (−N) we re-suspended the cells in fresh CTL or −N medium, containing 0.17% yeast nitrogen base and 2% glucose. For glucose restriction, we used CTL medium with either 0.5% or 0.05% glucose. For water starvation, we used sterile ddH₂O.

Rapamycin (LC Laboratories, R-5000) stock was prepared as 1.1 mM in dimethylsulfoxide (DMSO) (stored at −20 °C) and used at a final concentration of 40 nM. Analogous to the −N experiments, we initiated rapamycin treatment by adding DMSO as a control or rapamycin to the cultures during the mid-logarithmic phase (at OD₆₀₀ = 1). The pharmacological DHS inhibitor N1-guanyl-1,7-diamine-heptane (GC7) was purchased from Merck (259545) and prepared as a sterile filtered 50 mM stock in ddH₂O, stored at −20 °C. GC7 treatment was initiated during inoculation and repeated when the medium was changed for nitrogen deprivation.

Generally, polyamines were supplemented from sterile stocks of polyamines starting with the main culture, except for synergy experiments as shown in Fig. 3k,l, Supplementary Fig. 4c and Extended Data Figs. 4m,n and 9e,f, where spermidine was added in the starvation medium only. For the other nitrogen starvation experiments, polyamine supplementation was renewed in the fresh medium. Cells were treated with either 100 µM putrescine (Sigma, D13208↗; from 3 M aqueous stock, pH 7.4), spermidine (Sigma, 85558; from 1 M aqueous stock, pH 7.4) or spermine (Sigma, 85590; from 0.9 M aqueous stock, pH 7.4) or 5 mM spermidine as indicated in the figure legends. In brief, the polyamine stocks were prepared on ice, with ddH₂O and the pH was set with HCl, as previously described²³. Stock solutions were stored at −20 °C for up to one month.

To monitor yeast autophagy, we employed established protocols using fluorescence microscopy, western blots and biochemical assays⁷³. We used endogenously tagged eGFP–Atg8 fusion strains, as previously reported⁷².

GFP–Atg8 localization was assessed by collecting 100 µl from yeast cultures by centrifugation (1,000 rcf, 1 min) and re-suspension of the cell pellet in 50 µl propidium iodide (PI) staining solution (100 ng ml⁻¹ PI in PBS, pH 7.4). Cells were pelleted again (500 rcf, 30 s), transferred to a glass microscopy slide, and covered with a cover slip. Imaging was performed with a Leica DM6B-Z fluorescence microscope using a ×100/1.40 HC Pl APO oil objective and a Leica-DFC9000GT-VSC09095↗ sCMOS camera. eGFP images were taken with a GFP filter (Ex₄₇₀/Em₅₂₅) filter and 2 s exposure. PI images were taken with a Texas Red filter (Ex₅₆₀/Em₆₃₀) and 75-ms exposure. Raw 16-bit images were exported and merged with constant contrast settings (eGFP 3,000–30,000; PI 200–50,000) in ImageJ⁷⁴. Cells were classified manually in a blinded fashion using the ImageJ CellCounter tool.

Additionally, we performed Pho8∆N60 assays⁴³, as previously reported⁷². In brief, we collected three OD₆₀₀ equivalents at the indicated time points by full-speed centrifugation in a standard tabletop centrifuge for 2 min. After a washing step with ddH₂O, we re-suspended the cell pellet in 200 µl cold assay buffer (250 mM Tris-HCl (pH 9), 10 mM MgSO₄ and 10 μM ZnSO₄) and transferred it to pre-cooled plastic reaction tubes filled with 100 µl acid-washed glass beads. The cells were homogenized in a BeadBeater (2 × 1 min with 1-min pause between the cycles) in a liquid nitrogen-cooled metal rack. After homogenization we centrifuged the samples at 10,000 rcf for 10 min at 4 °C and 100 µl supernatant was transferred into a fresh pre-cooled tube. We used a Bio-Rad protein assay (Bio-Rad, 5000006) to determine the protein concentration of the supernatant. Protein extract corresponding to 1.5 µg protein was transferred to a well of a 96-deep-well plate in duplicate and filled with assay buffer to 550 µl at room temperature (22–25 °C). The addition of 50 µl α-naphthyl phosphate solution (55 mM in assay buffer, pH 9) started the reaction. After mixing the plate by vortexing, it was incubated for 20 min at 30 °C. The reaction was stopped by adding 200 μl stopping buffer (2 M glycine/NaOH, pH 11) to each well, followed by vortexing. Then, 100 µl of each well was transferred to a 96-well plate (black bottom) in duplicate and measured in a TECAN plate reader (Ex₃₄₀ and Em₄₈₅). For the correction of background phosphatase activity, control strains (without the Pho8∆N60 mutation) were processed in parallel and subtracted.

A culture volume corresponding to three OD₆₀₀ units was collected at each time point by centrifugation at 3,000 rcf for 3 min. After a washing step with ddH₂O, the cell pellet was frozen at −20 °C until further processing. Whole-cell extracts were generated by re-suspending the cell pellets in 300 µl lysis buffer (1.85 M NaOH and 7.5% 2-mercaptoethanol) and incubation on ice for 10 min. Proteins were precipitated by adding 300 µl 55% trichloroacetic acid (TCA), incubation on ice for 10 min, centrifugation at 10,000 rcf at 4 °C for 10 min and removal of the supernatant. After an additional centrifugation step (10,000 rcf at 4 °C for 2 min) residual supernatant was removed and pellets were solubilized in final sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 8.7% glycerol, 0.004% bromophenol blue and 120 mM dithiothreitol (DTT)) and 1 M Tris was added to neutralize residual TCA until the samples turned blue. In brief, before electrophoresis, samples were boiled at 95 °C for 5 min and centrifuged at maximum speed in a tabletop centrifuge for 15 s. For protein separation, 12–15 µl of the supernatant was loaded on 4–12% or 12% (for GFP–Atg8) NuPAGE Bis-Tris gels (Thermo Fisher Scientific). Electrophoresis was performed at 100–130 V with MOPS SDS running buffer (Thermo Fisher Scientific, NP000102). Proteins were wet-transferred to methanol-activated 0.45-µm PVDF membranes (Roth, T830.1) at 220 mA for 60–90 min using transfer buffer (10 mM CAPS/NaOH, pH 11 and 10% methanol). After blotting, membranes were blocked with blocking solution (1% dry milk powder in Tris-buffered saline (TBS), pH 7.4) for 1 h and then incubated with the primary antibodies overnight at 4 °C. After three washing steps in TBS + 0.1% Triton X-100 for 5 min, membranes were incubated with secondary, horse radish peroxidase (HRP)-linked antibodies for 1 h at room temperature. After three washing steps in TBS + 0.1% Triton X-100 for 5 min, signals were detected with a ChemiDoc detection system (Bio-Rad) and Clarity Western ECL Substrate (Bio-Rad) using the ‘optimal exposure’ setting. For re-probing membranes, Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific, 46430) was used according to the manufacturer’s protocol. Band intensities were quantified using ImageLab v.5.2 (Bio-Rad) using the rectangular volume tool with local background adjustment. Primary antibodies were anti-GFP (Roche, 1814460, 1:5,000 dilution in blocking solution), anti-hypusine (Merck, ABS1064-I, 1:1,000 dilution in blocking solution), anti-GAPDH clone GA1R (Thermo Fisher Scientific, MA5-15738, 1:10,000 dilution in blocking solution) and anti-HA (Sigma, H-9658, 1:10,000 dilution in blocking solution). Secondary antibodies were HRP-linked anti-mouse IgG (Sigma, A9044, 1:10,000 in blocking solution) or HRP-linked anti-rabbit IgG (Sigma, A0545, 1:10,000 dilution in blocking solution).

Chronological aging experiments were performed as previously described⁷⁵. In brief, we used PI staining to identify dead cells using an LSR II Fortessa flow cytometer equipped with a high-throughput sampler (BD), using FACSDiva software (BD). For that purpose, 20-µl aliquots of the cultures were transferred to 96-well U-bottom plates at the indicated time points and stained with 150 µl PI staining solution (100 ng ml⁻¹ in PBS), incubated in the dark for 15 min at room temperature and centrifuged at 2,500 rcf for 5 min. The staining solution was removed by tapping the plate and the cells were re-suspended in 150 µl fresh PBS. Then, 30,000 cells were measured per sample and PI-positive (dead)/negative (live) cells were identified and quantified by flow cytometry at Ex₄₈₈/Em₆₇₀.

At selected time points, we additionally performed live qualitative fluorescence microscopy, for which we took 100-µl aliquots of the cultures, centrifuged them for 1 min at room temperature in a standard tabletop centrifuge and re-suspended the pellet in 100 µl PI staining solution. After 2 min of incubation in the dark, we transferred a droplet of this suspension onto glass microscopy slides and covered them with coverslips. Images were taken with a Leica DM6B-Z microscope, using the Texas Red filter (Ex₅₆₀/Em630, 300-ms exposure). For image processing, we used ImageJ⁷⁴.

Of note, yeast chronological lifespan is affected by the medium pH⁷⁶, which was measured with a Thermo Scientific Orion Star A221 pH meter and only minimally affected during nitrogen starvation by the SPE1 knockout (Supplementary Fig. 4a).

Yeast replicative lifespan was performed as previously described^77,78. To summarize, yeast was pulled from −80 °C freezer stocks onto YPD plates and were allowed to grow at 30 °C for 2 days. Single colonies from each sample were picked and patched for two consecutive days before being plated to RLS assay plates (YPD (1% yeast extract, 2% peptone and 2%, 0.5% or 0.05% glucose) unless otherwise stated). Virgin cells were selected as new mother cells and each generation of daughter cells were separated and counted via microdissection. Cell death is considered when mother cells are no longer capable of dividing. Mother cells that divided two or fewer times or were lost during the experiment were censored from the dataset.

In vivo TORC1 activity was assayed as previously described⁷⁹. In brief, a 10-ml cell culture was mixed with TCA at a final concentration of 6%. After centrifugation, the pellet was washed with cold acetone and dried in a SpeedVac. The pellet was re-suspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 6 M urea and 1% SDS), the amount being proportional to the OD₆₀₀ of the original cell culture. Proteins were extracted by mechanical disruption in a Precellys machine after the addition of glass beads. Subsequently, an amount of 2× sample buffer (350 mM Tris-HCl (pH 6.8), 30% glycerol, 600 mM DTT, 10% SDS and BBF) was added to the whole-cell extract and the mix was boiled at 98 °C for 5 min. The analysis was carried out by SDS–PAGE by loading 15 µl extracts on 7.5% polyacrylamide gels. The transfer on nitrocellulose membranes was performed with a Trans-Blot Turbo Transfer System (Bio-Rad) with Bjerrum Schafer-Nielsen buffer (48 mM Tris-HCl (pH 9.2), 39 mM glycine and 20% methanol). Protein detection was carried out using custom phosphospecific rabbit anti-Sch9-pThr737 (1:10,000 dilution in TBS 1% milk) and goat anti-Sch9 (1:1,000 dilution in TBS 5% milk) antibodies and a commercial rabbit anti-Adh1 antibody (Calbiochem, 126745, 1:50,000 dilution in TBS 5% milk). Band intensities from independent biological replicates were quantified with ImageJ.

Cells (equivalent to 15 OD₆₀₀, grown as described before) of BY4741 and BY4741 ∆spe1 after 6 or 24 h of nitrogen deprivation were collected by centrifugation at 4,000 rpm (3,077 rcf) for 3 min at 4 °C, washed once with ice-cold ddH₂O, frozen in liquid nitrogen and stored at −80 °C until further processing. The pellets were re-suspended in 400 µl ice-cold methanol (−20 °C) and 200 µl MilliQ H₂O and transferred to a tube containing Precellys beads (1.4-mm zirconium oxide beads, Bertin Technologies, Villeurbanne, France) for homogenization on a Precellys 24 homogenizer for two cycles of 20 s with 5,000 rpm and 10 s breaks. Cell debris was pelleted by centrifugation at 13,000 rpm (15,871 rcf) for 30 min (4 °C). Supernatants were lyophilized and dissolved in 500 µl NMR buffer (0.08 M Na₂HPO₄, 5 mM 3-(trimethylsilyl) propionic acid-2,2,3,3-d₄ sodium salt (TSP), 0.04% (w/v) NaN₃ in D₂O, pH adjusted to 7.4 with 8 M HCl and 5 M NaOH) for NMR measurements.

All NMR experiments were acquired at 310 K using a Bruker 600 MHz Avance Neo spectrometer equipped with a TXI probe head. The one-dimensional (1D) CPMG (Carr–Purcell–Meiboom–Gill) pulse sequence (‘cpmgpr1d’; 512 scans; size of free induction decay (FID) 73,728; 11,904.76 Hz spectral width; recycle delay 4 s), with water signal suppression using presaturation, was recorded for ¹H 1D NMR experiments. In brief, and as described before⁸⁰, data were processed in Bruker Topspin v.4.0.6 using one-dimensional exponential window multiplication of the FID, Fourier transformation and phase correction. The NMR data were then imported into Matlab2014b; TSP was used as the internal standard for chemical-shift referencing (set to 0 ppm); regions around the water, TSP and methanol signals were excluded; the NMR spectra were aligned; and a probabilistic quotient normalization was performed. The statistical significance of the determined differences was validated by the quality assessment statistic Q². This measure provides information about cross-validation and is a qualitative measure of consistency between the predicted and original data with a maximum value of 1.

Metabolite identification was carried out using Chenomx NMR Suite v.8.4 (Chenomx) and reference compounds. Quantification of metabolites was carried out by signal integration of normalized spectra. For each metabolite, a representative peak with no overlapping signals was identified, the start and end points of the integration were chosen to revolve around that peak, and the area of the peak was integrated by summing up the value for each point. Peaks which could not be unambiguously identified are shown with their ppm value at maximal intensity.

We used MetaboAnalyst v.5.0 for data analysis and visualization⁸¹, using mean-centred normalization. For metabolite set enrichment analysis we used the integrated KEGG database. sPLS-DA analysis was performed with ten features for each component and heatmaps were generated using Ward’s method and Euclidian distance settings.

Polyamine extraction and quantification was performed as previously described⁸². In brief, yeast cells were grown as previously mentioned and at the indicated time points, one to three OD₆₀₀ were collected by centrifugation, washed once with ddH₂O and mixed with 5% TCA containing stable-isotope labelled standards of indicated polyamines at a final concentration of 100 ng ml⁻¹ each, followed by incubation on ice for 60 min with vortexing every 15 min. After centrifugation at 25,000 rcf at 4 °C for 10 min, 150 µl supernatant were transferred to 1.5-ml LoBind reaction tubes and 37.5 µl ammonium formate (2 M), 800 µl of ultra-pure H₂O and 125 µl of saturated Na₂CO₃ buffer were added.

For derivatization, 20 µl isobutyl chloroformate was added, followed by a 15-min incubation at 35 °C. After centrifugation for 1 min at 15,000 rpm/21,130 rcf, 800 µl supernatant was then transferred to LoBind 96-deep-well plates (VWR International, 737-2544) for storage at −80 °C. Polyamine derivatives were extracted offline by solid-phase extraction (SPE) (Strata-X, Polymeric Reversed Phase, 96-well plate). SPE was conditioned with 500 µl acetonitrile, equilibrated with 500 µl distilled water containing 0.2% acetic acid. Derivatized TCA extracts were loaded onto the SPE and after two washing steps with 500 µl 0.2% acetic acid, samples were eluted with 250 µl 80% acetonitrile containing 0.2% acetic acid. Eluted SPE extracts were subjected to liquid chromatography (LC)–MS/MS (mobile phase, isocratic 80% acetonitrile containing 0.2% acetic acid; flow rate 250 µl min⁻¹; HPLC column: Kinetex 2.6 µm C18 100A 50 mm × 2.1 mm TSQ Quantum Access Max coupled to an Ultimate 3000). MS conditions were set as previously published⁸³. LC–MS/MS data were acquired and processed using Xcalibur v.4.0 Software (Thermo Fisher Scientific). The final data were normalized to OD₆₀₀ and the control conditions for each polyamine.

The main cultures were grown to log phase in medium containing ¹³C₆-labelled arginine (30 mg l⁻¹; Eurisotop, CLM-2265) instead of non-labelled arginine. The control cells received fresh medium containing ¹³C₆-labelled arginine, whereas the nitrogen-starved cells were cultivated in standard −N medium for another 6 h, before collecting by centrifugation as described above. Metabolites were measured following a previously described protocol⁸⁴, but without internal standards in the extraction buffer. In brief, 50-µl samples were vortexed for 5 min with 500 µl ice-cold extraction mixture (methanol:water, 9:1, −20 °C) and then centrifuged (10 min at 15,000 rpm/21,130 rcf, 4 °C). Several fractions were then split to be analysed by LC–MS and gas chromatography (GC)–MS⁸⁴. One fraction was analysed by the LC–MS Orbitrap q-Exactive Thermo in full profiling. Tracking of arginine and its isotopes was performed by post-acquisition treatment with the software Xcalibur Thermo (v.2.2). Polyamines analyses were performed by LC–MS/MS with a 1290 UHPLC (ultra-high performance liquid chromatography) (Agilent Technologies) coupled to a QQQ 6470 (Agilent Technologies) and were previously described⁸⁵. The MRM method was adapted to track polyamines but also their isotopes. Data were processed by MassHunter Quantifier (Agilent) software (v.10.1).

Yeast samples were processed in each sequence (experiment) in one MS run to reduce batch effects. For cell lysis 5% SDS, 50 mM ammonium bicarbonate (used in the GC7-pertubation experiment) or 8 M urea, 20 mM HEPES (used in the SPD-rescue experiment) and zirconia beads (1:1 v/v) were added to the frozen pellet and lysed in a BeadBeater in three cycles (2 min, 15 s rest, 1,500 rpm per cycle) (Spex Geno Grinder). Then, 150 µg protein of each sample were alkylated with 5 mM tris(2-carboxyethyl)phosphine (TCEP), reduced with 10 mM chloroacetamide (CAA) and digested with trypsin (1:50 wt/wt) as well as purified utilizing the S-Trap mini columns (Protifi), following the manufacturer’s instructions: S-Trap mini spin column digestion protocol. Peptides were lyophilized, re-suspended in 0.1% formic acid and diluted to 200 ng µl⁻¹, whereby 1 µl was used per MS injection.

Samples were analysed on a timsTOF ion mobility mass spectrometer (with PASEF technology, Bruker Daltonics) in-line with UltiMate 3000 RSLCnano UHPLC system (Thermo Scientific). Peptides were separated on a reversed-phase C18 Aurora column (25 cm × 75 µm) with an integrated Captive Spray Emitter (IonOpticks). Mobile phases A 0.1% (v/v) formic acid in water and B 0.1% (v/v) formic acid in ACN with a flow rate of 300 nl min⁻¹, respectively. Fraction B was linearly increased from 2% to 25% in the 90-min gradient, increased to 40% for 10 min, and a further increase to 80% for 10 min, followed by re-equilibration. The spectra were recorded in data-independent acquisition (DIA) mode as previously described⁸⁶.

The DIA data were quantified with DIA-NN v.1.8.0 (ref. ⁸⁷) by creating a synthetic fasta library using the reviewed Uni-Prot S.cerevisiae protein database (downloaded 24 September 2021). The MS proteomics raw data of both experiments together with the processing log files have been deposited to the ProteomeXchange Consortium using the PRIDE partner repository (https://www.ebi.ac.uk/pride/↗) with the dataset identifier PXD035909↗. In the SPD-rescue experiment, a total of 4,684 distinct protein groups were identified. Notably, 82% of these groups demonstrated consistent presence across all samples, indicating a robust and pervasive occurrence. Furthermore, close to 90% of the samples exhibited the presence of approximately 4,207 protein groups, corroborating their widespread and persistent detection within the experimental set. In the GC7-perturbation experiment, 4,019 unique protein groups were quantified. Among these, 45% were identified across every sample, with approximately 58% detected in at least 90% of the samples.

PCA was performed by singular-value decomposition of the centred and scaled protein groups (n = 4,684), whereby missing values were imputed with zero. Protein complex enrichment analyses were performed based on KEGG yeast pathways from ConsensusPathDB (downloaded 27 April 2022)⁸⁸, as well as complex portal annotations of the TORC complex⁸⁹. Aggregated z-values were calculated for each protein complex resulting from averaged absolute protein expression of each complex member. The z-scores for each protein were calculated as followed \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$z=\frac{\left(x-\mu \right)}{\sigma }$$\end{document}z=x−μσ, where x is the expression, μ is the protein expression across all samples and σ is the s.d. of the protein expression across all samples.

Data were visualized using R⁹⁰ by utilizing the packages prcomp^91,92, ggplot⁹³ and pheatmap⁹⁴.

All fly strains had a w¹¹¹⁸ genetic background. The heterozygous mutant strains were backcrossed to the laboratory-specific w¹¹¹⁸ at least six times to ensure isogenicity. The heterozygous odc1 mutant was purchased from Bloomington Drosophila Stock center (Indiana University, 56103). The heterozygous eIF5A mutant carrying a point mutation at the lysine site of hypusination was created as described previously⁵³. Fly food was prepared according to the Bloomington medium recipe with minor modifications, which we refer to as ‘normal food’ (per litre: 4.2 g agar–agar, 85.3 g sugar beet syrup, 7.5 g baker’s yeast, 8.3 g soybean flour, 66.7 g cornmeal, 1.3 g methyl p-hydroxybenzoate dissolved in 4.2 ml ethanol and 5.25 ml propionic acid). Difluoromethylornithine (DFMO) (a kind gift from P. M. Woster, Medical University of South Carolina), freshly prepared as a sterile filtered 50 mM stock in ddH₂O, was added to the food after cooling the freshly cooked food to ~40 °C. Agar-only food (0.6%) prepared with a 1:1 mixture of deionized and tap water was used as the fasting food. The flies were reared under standard laboratory conditions, as previously reported (25 °C, 70% humidity with a constant 12-h light–dark cycle)^53,95. Flies used in all experiments were F1. WT flies and mutant flies were collected within 24–48 h after eclosion and considered as 1-day-old flies. The w¹¹¹⁸ virgin females were used to cross with males from w¹¹¹⁸ and mutant strains, respectively. Female and male flies were separated after 24 h of mating on fresh food by light CO₂ anaesthesia and were flipped to fresh normal food every other day in portions of 20–25 flies per vial.

IF regimes were initiated 1 day after sorting and maintained throughout the flies’ lifespan. We used a 12-h feeding–fasting cycle (IF^12:12), providing food during daylight hours (8:00 to 20:00). Control groups were flipped in the same rhythm but had food access during the whole 24 h. We changed food vials every 2–3 days and kept vials at 4 °C when not used. Flies that escaped or died due to unnatural causes were censored. Dead flies were identified by their unresponsiveness to mechanical stimuli using a small brush.

Drosophila body weight was measured throughout a fasting cycle as wet weight of snap-frozen fly bodies. The start time of the experiments was 20:00, followed by measurement time points after 12 and 24 h of fasting, as well as after 12 h of re-feeding. The ad libitum-fed group had access to food for the whole time. Data were normalized to each ad libitum group.

Food consumption was monitored similarly to the previously described capillary feeding (CAFE) assay⁹⁶: For each replicate, five flies were anaesthetized on ice and transferred to a chamber with two 5-µl glass capillaries (VWR International, 612-2401) inserted in the top caps. Capillaries were either filled with liquid food containing 5% (w/v) sucrose (Roth, 4661.4) and 5% (w/v) yeast extract (BD, 288620) (ad libitum) or tap water (fasting). Data were collected twice per day (8:00 and 20:00) for at least 3 days after a 24-h acclimatization period. IF flies had access to capillaries containing food from 8:00 to 20:00 Passive evaporation was accounted for with three empty chambers without flies. Data are presented as microlitres of food per fly per hour.

Twenty flies (wet weight of snap-frozen fly bodies was estimated and used for normalization) were placed into 1.5-ml LoBind reaction tubes (Eppendorf, 0030108116), snap-frozen in liquid nitrogen and homogenized for 30 s on ice in 600 µl 5% TCA containing stable-isotope labelled standards of indicated polyamines at a final concentration of 100 ng ml⁻¹ each using a Turrax homogenizer (IKA T10 basic). Further polyamine extraction and quantification was performed as previously described with adapted analyte concentrations in the calibration solutions⁸² (see also ‘Yeast’ section).

For fly protein extraction, ten snap-frozen fly heads were homogenized in lysis buffer (1× PBS, pH 7, 0.5% Triton X-100, 2% SDS and 1× complete protease inhibitor mix) for 30 s with a motorized pestle (5 µl lysis buffer per fly head) followed by an incubation for 60 min at 4 °C with continuous rotation. The homogenized samples were centrifuged for 10 min at 10,000 rcf at 4 °C. The final sample buffer (final concentration of 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 8.7% glycerol, 0.004% bromophenol blue and 120 mM DTT) was added to the supernatant and samples were stored at −20 °C until further processing. In brief, before electrophoresis, samples were boiled at 95 °C for 5 min and centrifuged at 10,000 rcf for 1 min. Then, 12.5 µl supernatant (equivalent to two heads) were loaded onto 4–12% Bis-Tris gels and run at 90–120 V. Proteins were transferred to 0.45-μm PVDF membranes for 90 min at 220 mA using transfer buffer (10 mM CAPS pH 11 and 10% methanol). The membranes were then incubated in blocking solution (3% dry milk powder in TBS, pH 7.4) for 1 h and subsequently incubated with the primary antibody overnight at 4 °C. Membranes were washed three times with TBST (TBS + 0.1% Tween-20) for 5 min and subsequently incubated with the secondary antibody for 1 h at room temperature. After three washing steps in TBST for 5 min, signals were detected with a ChemiDoc detection system (Bio-Rad) and Clarity Western ECL Substrate (Bio-Rad) using the ‘optimal exposure’ setting. Band intensities were quantified using ImageLab v.5.2 (Bio-Rad) using the rectangular volume tool with local background adjustment. Primary antibody was anti-hypusine (Merck, ABS1064-I, 1:1,000 dilution in 1% dry milk powder in TBS, pH 7.4). The secondary antibody was HRP-linked anti-rabbit IgG (Sigma, A0545, 1:10,000 dilution in 1% dry milk powder in TBS pH 7.4). As a loading control, HRP anti-β actin monoclonal antibody (Abcam, ab197277, 1:2,000 dilution in TBS + 0.1% Tween +1% BSA) was used.

For assessing the locomotor function, 5–20 flies were placed in custom-made three-dimensional-printed climbing chambers with nine slots per chamber followed by a defined acclimation period of 30 min in the dark. The chambers were individually attached to a custom-built climbing platform to ensure a standardized impact force and tapped down four times with an interval of 30 s (four rounds). Flies climbing up the rack were recorded by video tracking (Canon EOS 700D). The videos were then analysed with a fly tracking program, which tracks flies based on previously described methods⁹⁷. For the calculation of climbing parameters (height, average height reached by the flies in the given climbing duration; speed, speed of the flies during the climbing duration in mm s⁻¹; and distance, the distance travelled by the flies during the climbing duration), flies were tracked over 10 s after the tapping impact. For analysis, the area under the curve (AUC) was calculated for each parameter and strain and normalized to the respective control. Every data point corresponds to the average of the flies assessed in one chamber.

Flies at 6 days old were either fed ad libitum or fasted for 12 or 24 h. Then the fly brains were dissected for brain immunostaining. Adult brains were dissected in HL3 solution on ice and immediately fixed in cold 4% paraformaldehyde (w/v) for 30 min at room temperature. After fixation, samples were washed three times for 10 min each with 0.7% PBT (PBS containing 0.7% Triton X-100 v/v) and then blocked with 10% normal goat serum in PBT (v/v) for 2 h at room temperature. After blocking, samples were incubated in 0.7% PBT containing 5% normal goat serum and primary antibodies for 48 h at 4 °C. After primary antibody incubation, brains were washed in 0.7% PBT six times for 30 min each at room temperature and then incubated in 0.7% PBT with 5% normal goat serum containing the secondary antibodies overnight at 4 °C. Brains were washed six times for 30 min each with PBT at room temperature and mounted in Vectashield (Vector Labs). The following antibodies and dilutions were used in whole-mount adult brain staining: rabbit anti-hypusine antibody (Merck, ABS1064; 1:1,000 dilution), guinea pig anti-eIF5A antibody (customized; 1:200 dilution), goat anti-guinea pig Alexa 555 (Invitrogen; 1:200 dilution), goat anti-rabbit Cy5 (Invitrogen; 1:200 dilution).

Image stacks of specimens were imaged on a Leica TCS SP8 confocal microscope (Leica Microsystems) using a ×40, 1.3 NA oil objective for whole-brain imaging with a voxel size of 0.3788 × 0.3788 × 0.9997 micron³. Images were quantified using ImageJ software. In brief, the average intensity z-projection was performed with ~25 stacks of each brain from the beginning of the antennal lobe to the end of the antennal lobe and the mean grey value of the central brain was measured.

We followed standard procedures for C.elegans maintenance and other genetic manipulations⁹⁸. The nematode-rearing temperature was kept at 20 °C unless noted otherwise. The following strains, available at the Caenorhabditis Genetics Center, were used: N2: WT Bristol isolate, HZ589: bpIs151 (sqst-1p::sqst-1::GFP + unc-76(+)), MAH215: sqIs11 (lgg-1p::mCherry::GFP::lgg-1 + rol-6).

Lifespan assays were performed at 20 °C. Synchronous animal populations were generated by hypochlorite treatment of gravid adults to obtain tightly synchronized embryos that were allowed to develop into adulthood under appropriate, defined conditions.

For RNAi lifespan experiments, worms were placed on Nematode Growth Medium plates containing 2 mM IPTG and seeded with HT115 (DE3) bacteria transformed with either the pL4440 vector or the test RNAi construct. The progeny was grown at 20 °C unless noted otherwise, through the L4-young adult larval stage and then transferred to fresh plates in groups of around 30 worms per plate for a total of at least 155 individuals per experiment. Animals were transferred to fresh plates every 2 days thereafter and examined every day for touch-provoked movement and pharyngeal pumping until death. Worms that died owing to internally hatched eggs, an extruded gonad or desiccation due to crawling on the edge of the plates were censored and incorporated as such into the dataset. Each survival assay was repeated at least four times.

For IF (IF^48:48), synchronized young adult worms raised on Nematode Growth Medium plates with live HT115 were transferred to plates containing floxuridine (120 μM), seeded with HT115 RNAi-transformed bacteria, which were UV-killed before animal transfer. On day 2 of adulthood, worms were divided into ad libitum and IF. Worms in the ad libitum groups were fed ultraviolet-killed HT115 throughout their lifespan. Worms in IF were placed on plates with UV-killed HT115 or without food (seeded bacteria) alternatively every other day. All worms were transferred to freshly seeded plates every 2 days⁹⁹.

Lifespan assays with spermidine and rapamycin supplementation were performed as previously described^23,100. In brief, SPD (Sigma, S0266) was used at a final concentration of 0.2 mM. Sterilized water solution of SPD was applied to the top of the RNAi bacterial lawn after being killed by UV irradiation for 15 min (0.5 J) using a UV cross-linker (bio-Link BLX-E365;Vilber Lourmat). Rapamycin (LC Laboratories) was dissolved in DMSO and added to the agar plate to a final concentration of 50 μM. Control plates contained an appropriate concentration of DMSO (<0.1%). SPD and rapamycin experiments were performed simultaneously, thus sharing the same control. Drug administration was performed on late-L4 worms unless otherwise noted. RNAi, SPD or rapamycin treatments were continued throughout life. In autophagy experiments, identical concentrations of SPD (0.2 mM) and rapamycin (50 μΜ) were administered and autophagic events were assessed at the first round of IF.

RNAi was performed by feeding methods¹⁰¹ and was performed lifelong, starting from egg hatching. To generate the RNAi constructs, gene-specific fragments of interest were amplified by PCR directly from the C.elegans genomic DNA using appropriate primer sets. The PCR-generated fragments were initially inserted into the TOPO-pCRII vector and then sub-cloned in the pL4440 plasmid vector. The final constructs were transformed into HT115 (DE3) Escherichiacoli bacteria.

For argn-1(RNAi), a 1,409-bp fragment was amplified by using the primer set:

For_argn-1: 5′-ATGAAAAAGTCTACACAACTCGCCAGA-3′ and

Rev_argn-1: 5′-TCACATTGCTCTTGTAATTTTCTGAGATTG-3′.

For dhps-1(RNAi), a 2,420-bp fragment was amplified by using the primer set:

For_dhps-1: 5′-ATGAGCACCAACGAAGCAGCAG-3′ and

Rev_dhps-1: 5′-ATGCTTGGCCGCCCAATACA-3′.

For odc-1(RNAi), a 1,327-bp fragment was amplified by using the primer set:

odc-1_For: 5′-ATGATTTCTCAATTCGAAATTATTGGTGAC-3′ and

odc-1_Rev: 5′-ATCACATACATCGGCACAGGCTTC-3′.

For smd-1(RNAi), we amplified a 1,724-bp fragment by using the primer set:

For_smd-1: 5′-ATGTCTGCCACGTCTGCCAC-3′ and

Rev_smd-1: 5′-TCCTCGTCGCTCGATGATGA-3′.

For spds-1(RNAi), a 1,714-bp fragment was amplified by using the primer set:

For_spds-1: 5′-ATGAACAAGCTGCACAAGGGATG-3′ and

rev_spds-1: 5′-CTTGCGATGACAAAATTCCATCCTC-3′.

Quantification of mRNA levels was performed as previously described¹⁰². Total mRNA was isolated from synchronized adults, lysed in 250 μl TRIzol by freeze-cracking (Invitrogen). For complementary DNA synthesis, mRNA was reverse transcribed using an iScriptTM cDNA Synthesis kit (Bio-Rad). Quantitative PCR was performed in triplicate using a Bio-Rad CFX96 Real-Time PCR system (Bio-Rad). The housekeeping gene act-3 was used as an internal normalization control. In each experiment, three technical replicates were used per sample.

For act-3 mRNA detection, the following set of primers was used:

act-3 RT Fw: 5′-ATCCGTAAGGACTTGTACGCCAAC-3′ and

act-3 RT Rev: 5′-CGATGATCTTGATCTTCATGGTTC-3′.

For smd-1 mRNA detection, the following set of primers was used:

smd-1RT_For: 5′-CTCAAACAGGATAGATGGTACCTTTACACAT-3′ and

smd-1RT_Rev: 5′-TTGTCAATACCGGCTCGCATG-3′.

For spds-1 mRNA detection, the following set of primers was used:

spds-1RT_For: 5′-GATGTGCTGGTTTTTGAGAGCACA-3′ and

spds-1RT_Rev: 5′-ATCTCCTCCACCAATGATAAGTACACGT-3′.

For dhps-1 mRNA detection, the following set of primers was used:

dhps-1RT_For: 5′-GGTGACCAGTGGGCTTCGTGAGGTAC-3′ and

dhps-1RT_Rev: 5′-TTATCATTCGGAATGAGCACATTGC-3′.

For dohh-1 mRNA detection, the following set of primers was used:

dohh-1_RTFor: 5′-ATTGCTGATCCTTCAGTCAAAGATGTCCTC-3′ and

dohh-1_RTRev: 5′-GTTGAAGCTGATGGTGTTGGATCAA-3′.

For odc-1 mRNA detection, the following set of primers was used:

odc-1RT_For: 5′-CATCTGCCTACAGGAATGCTCTTCA-3′

odc-1RT_Rev: 5′-TCACGAATTGTCTCGGCAATCTTC-3′.

For argn-1 mRNA detection, the following set of primers was used:

argn-1RT_For: 5′-AGTGCAATGTCAGGAGTTACACAGACAT-3′ and

argn-1RT_Rev: 5′-CATCAACCCAAATAAGCCCAATATCTC-3′.

For amx-3 mRNA detection, the following set of primers was used:

amx-3RT_For: 5′-TCCAGTCCGGTAGGTGAGCTT-3′ and

amx-3RT_Rev: 5′-CAGTTCAATCTAATGTTTCCCGCAGGTAT-3′.

For hpo-15 mRNA detection, the following set of primers was used:

hpo-15RT_For: 5′-ACATGATGAGAAGACTGCAAGACGCTA-3′ and

hpo-15RT_Rev: 5′-TCCACTCTTTTAGAACGTGAACACTCAGAGT-3′.

For d2023.4 mRNA detection, the following set of primers was used:

d2023.4RT_For: 5′-TGCATATTCAACATGGGTTGGGCAGTAT-3′ and

d2023.4RT_Rev: 5′-TCAACAGTGTCGTACAGAGCAATCGCAT-3′.

For iff-1 mRNA detection, the following set of primers was used:

iff-1RT_For: 5′-ATCGAAGACGGGTTCTGCTCGCT-3′ and

iff-1RT_Rev: 5′-CAGGCAGCAACGACTTGAACCAAG-3′.

Five-day-old N2 (WT) animals (after the first round of fasting) were placed at 37 °C for 6 h. After recovery overnight at 20 °C, survival was assessed. Animals that did not exhibit either touch-provoked movement or pharyngeal pumping were considered dead. Five independent experiments were performed.

To measure autophagic flux, the C.elegans strain MAH215 (sqIs11 (lgg-1p::mCherry::GFP::lgg-1 + rol-6)) was used (LGG-1 is the C.elegans orthologue of LC3/Atg8). Adult animals after the first round of fasting were measured. We calculated the number of autolysosomes (ALs) (mCherry-positive puncta). The number of mCherry-only puncta was calculated (the total number of mCherry-positive puncta − the number of GFP-positive + mCherry-positive puncta) as previously shown¹⁰³. Z-stack images were acquired using the LEICA TCS SP8 laser scanning confocal microscope unit. Measurements were performed in the terminal bulb and at the area where the pharyngeal lumen was best observed. Additionally, we calculated the ratio of mCherry:GFP signal by measuring mean fluorescence intensity in the head region of animals, as previously reported¹⁰⁴. We also calculated SQST-1::GFP (the C.elegans orthologue of SQSTM1) positive particles using the HZ589: bpIs151 (sqst-1p::sqst-1::GFP + unc-76(+)) strain. Particles were measured in the head region of the animals. Images were acquired using the EVOS Cell Imaging Systems.

Five-day-old N2 (WT) animals (after the first round of fasting) were anaesthetized and observed with ×4 magnification. Images were acquired using the EVOS Cell Imaging Systems. Animal periphery was precisely circled manually using the ‘polygon selections’ tool in ImageJ and the contained area was automatically calculated.

For sample preparation, synchronous animal populations were collected after 48 h of fasting and washed in M9 buffer. After washing, two volumes of homogenization buffer (20 mM Tris, pH 7.4, 20 mM NaCl and 1 mM MgCl₂) containing complete mini proteinase inhibitor cocktail (Roche) in a final concentration 1× was added. One volume of beads (0.5 mm zirconium oxide beads) was added and samples were placed in a Bullet Blender Homogenizer (Model BT24M, Next Advance) for 3 min at speed 10. We used Bio-Rad protein assay (Bio-Rad, 5000006) to determine the protein concentration of the supernatant. The final sample buffer (final concentration of 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 8.7% glycerol, 0.004% bromophenol blue and 120 mM DTT) was added to the supernatant. In brief, before electrophoresis, samples were boiled at 95 °C for 5 min and centrifuged at 10,000 rcf for 1 min. Protein extract corresponding to 5 µg protein was loaded onto 4–12% Bis-Tris gels and electrophoresis was performed at 90–120 V. Proteins were wet-transferred to methanol-activated 0.45-µm PVDF membranes (Roth, T830.1) at 220 mA for 60–90 min using transfer buffer (10 mM CAPS/NaOH, pH 11 and 10% methanol). After blotting, membranes were blocked with blocking solution (1% dry milk powder in TBS, pH 7.4) for 1 h and then incubated with the primary antibodies overnight at 4 °C. After three washing steps in TBST for 5 min, membranes were incubated with secondary, HRP-linked antibodies for 1 h at room temperature. After three washing steps in TBST for 5 min, signals were detected with a ChemiDoc detection system (Bio-Rad) and Clarity Western ECL Substrate (Bio-Rad) using the ‘optimal exposure’ setting. Band intensities were quantified using ImageLab v.5.2 (Bio-Rad) using the rectangular volume tool with local background adjustment. Primary antibodies were anti-hypusine (Merck, ABS1064-I, 1:1,000 dilution in blocking solution) and anti-GAPDH clone GA1R (Thermo Fisher Scientific, MA5-15738, 1:10,000 dilution in blocking solution). Secondary antibodies were HRP-linked anti-mouse IgG (Sigma, A9044, 1:10,000 dilution in blocking solution) or HRP-linked anti-rabbit IgG (Sigma, A0545, 1:10,000 dilution in blocking solution).

Male mice overexpressing human IGF1R specifically in cardiomyocytes (IGF1R^tg mice)⁴⁰ and male mice expressing a dominant negative phosphoinositide 3-kinase (PI3K) p110α mutant with impaired catalytic activity restricted to cardiomyocytes (dnPI3K mice)⁴¹ were used. In brief, a truncated p110 mutant with p85 binding domains, but lacking the kinase domain (p110Δkinase), was generated, and the p110Δkinase gene, together with FLAG epitope tag, was cloned into the αMyHC promoter construct to produce dnPI3K transgenic mice⁴¹. Both transgenic mouse models and their WT littermates were generated on the FVB/N genetic background and were killed at 3–9 months of age for heart tissue analysis, which included immunoblotting and polyamine measurements.

Various healthspan measurements were performed on IF^CR mice, with adapted protocols as detailed previously¹⁰⁵. The measurements were performed in the morning until noon and the operators were blinded to the group allocation. In brief, grip strength (two paws and four paws, on different days) was assessed with a grip strength meter (TSE), according to the manufacturer’s manual. The maximum grip strength of three repeats was counted and normalized to body weight of the animal on the test day. A visual frailty index was performed as previously described¹⁰⁶, which assesses several phenotypical alterations occurring with age in mice. Body composition of physically restrained animals was analysed with an NMR MinspecAnalyzer (Bruker). A four-limb grid-hanging test was performed at a height of 35 cm above a cushioned cage and the time to fall was recorded manually. Body surface temperature was recorded in the lower abdomen with a handheld infra-red thermometer and the maximum of three recordings was counted.

Cardiac function and dimensions were evaluated in lightly anaesthetized mice (4–5% for induction; 1–1.5% isoflurane for maintenance) by transthoracic echocardiography using a high-resolution micro-imaging system Vevo3100 (Fujifilm VisualSonics). Animal temperature was kept at 37 °C using a temperature-controlled heating platform, on which mice were placed in a supine position with their limbs in direct contact with electrode pads for heart rate assessment. Pre-warmed ultrasound transmission gel was spread on a shaved chest to obtain cardiac tracings in the parasternal long axis using 55 MHz linear-array probe. As previously described⁴², M-mode tracings were used to evaluate cardiac walls thickness and internal left ventricular (LV) dimensions at the level of the papillary muscles during systole and diastole. The ratio between peak early filling velocity of transmitral flow (E) and the corresponding mitral valve annulus velocity (e′) was assessed using pulsed wave and tissue Doppler imaging, respectively. All measures were averaged from at least three cardiac cycles under stable conditions.

BALB/cJRj mice were purchased from Janvier Labs. Mice were kept in a temperature- and humidity-controlled facility with free access to water and a 12-h light–dark cycle. All experiments were performed using age- and sex-matched littermate controls.

After 3 weeks of intermittent fasting/treatment, 6 h after re-feeding, 100 µl serum from arthritic transgenic K/BxN mice (obtained from the animal facility of the University of Erlangen) were administered to mice via intraperitoneal injection. The severity of K/BxN serum transfer arthritis¹⁰⁷ was then clinically assessed by visual evaluation of swelling and redness in each paw, including the tarsus and carpus joints as previously described¹⁰⁸. In short, the following scoring system was used: 0, no erythema or swelling; 1, erythema and swelling in up to two joints; 2, erythema and swelling in more than two joints or slight swelling of the ankle; 3, marked swelling of the ankle; and 4, substantial swelling of the whole paw, including toes and fingers. Scores of all paws were added together and AUCs of arthritis courses were calculated. IF and DFMO treatments were continued until the end of the experiment 24 days after serum transfer.

Tissues were dissected and snap-frozen in liquid nitrogen after killing animals by a final blood draw (from retro-orbital plexus) under deep isoflurane narcosis followed by cervical dislocation. Serum was obtained via 30 min incubation at room temperature, followed by centrifugation at 1,000–2,000 rcf for 10 min at 4 °C. Appropriate amounts of frozen tissue (10–50 mg) were homogenized in varying volumes of 5% TCA to yield a final SPD concentration of 50–1,000 ng ml⁻¹ (corresponds to the validated linear range of detection) in the final extract. Polyamine extraction and measurement were performed as previously described, with adapted analyte concentrations in the calibration solutions⁸² (see also ‘Yeast’ section).

For protein extraction, tissue was homogenized in lysis buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 2× Complete protease inhibitor cocktail (Roche) and 1× PhosSTOP (Roche); 400 µl lysis buffer per 30 mg of tissue) for 30 s with a Turrax (IKA T10 basic) followed by an incubation for 30 min at 4 °C with continuous rotation. The homogenized samples were centrifuged for 10 min at 20,000 rcf at 4 °C. We used a Bio-Rad protein assay (Bio-Rad 5000006) to determine the protein concentration of the supernatant. Final sample buffer (final concentration: 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 8.7% glycerol, 0.004% bromophenol blue and 120 mM DTT) was added to the supernatant. In brief before electrophoresis, samples were boiled at 95 °C for 5 min and centrifuged at 10,000 rcf for 1 min. Protein extract corresponding to 20 µg protein was loaded onto 4–12% Bis-Tris gels and electrophoresis was performed at 90–120 V. Proteins were transferred to methanol-activated 0.45-μm PVDF membranes for 90 min at 220 mA using transfer buffer (10 mM CAPS pH 11 and 10% methanol). The membranes were then incubated in blocking solution (3% dry milk powder in TBS, pH 7.4) for 1 h and subsequently incubated with the primary antibody overnight at 4 °C. Membranes were washed three times with TBST for 5 min and subsequently incubated with the secondary antibody for 1 h at room temperature. After three washing steps in TBST for 5 min, signals were detected with a ChemiDoc detection system (Bio-Rad) and Clarity Western ECL Substrate (Bio-Rad) using the ‘optimal exposure’ setting. Band intensities were quantified using ImageLab v.5.2 (Bio-Rad) using the rectangular volume tool with local background adjustment. For re-probing membranes, Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific, 46430) was used according to the manufacturer’s protocol. Primary antibodies were anti-hypusine (Merck, ABS1064-I, 1:1,000 dilution), anti-eIF5a (BD Biosciences, 611977, 1:10,000 dilution) and anti-GAPDH clone GA1R (Thermo Fisher Scientific, MA5-15738, 1:10,000 dilution) in 1% dry milk powder in TBS, pH 7.4. Secondary antibodies were HRP-linked anti-mouse IgG (Sigma, A9044, 1:10,000 dilution in 1% dry milk powder in TBS, pH 7.4) and HRP-linked anti-rabbit IgG (Sigma, A0545, 1:10,000 dilution in 1% dry milk powder in TBS, pH 7.4).

All animal experiments were performed in accordance with national and European ethical regulation (Directive 2010/63/EU) and approved by the responsible government agencies (Bundesministerium für Wissenschaft, Forschung und Wirtschaft, BMWFW; BMWFW-66.007/0029-WF/V/3b/2017, GZ 2021-0.524.242, GZ 2022-0.137.213, BMWFW-66.010/0160-WF/V/3b/2014, BMWFW-66.010/0198-WF/V/3b/2017 and BMBWF-66.010/0042-V/3b/2018). Animal studies using protocol 3 (IF^24:24) and the arthritis model were approved by the local ethical committee (District Government of Lower Franconia, 55.2-2532-2-1041-15). All experiments were conducted according to the guidelines of the Federation of European Laboratory Animal Science Associations. CR mice (protocol 4) were used in accordance with protocols approved by the Institutional Animal Care and Use Committee of the NIA.

All culture media and supplements for human cell culture were purchased from Gibco-Life Technologies. Plasticware was purchased from Greiner Bio-One. Cells used in this study were cultured in DMEM containing 10% FBS and non-essential amino acids (Gibco, 111400) at 37 °C under 5% CO₂. Hanks’ balanced salt solution (HBSS; Gibco,14025092) was used for starvation experiments. DFMO, spermidine, rapamycin, torin-1, aminoguanidine, bafilomycin and chloroquine were obtained from Sigma-Aldrich. The pharmacological DHS inhibitor N1-guanyl-1,7-diamine-heptane (GC7) was purchased from Merck (259545). DFMO and GC7 were prepared as sterile filtered 50 mM stocks in ddH₂O and stored in aliquots at −20 °C.

We used human glioblastoma H4 and human osteosarcoma U2OS cells (from ATCC), either in WT form or stably expressing GFP–LC3. Generally, cells were seeded in six-well plates at approximately 1 × 10⁵ cells per well. DFMO treatment was initiated after overnight attachment by exchanging the medium with freshly prepared DFMO-containing medium.

The plates were placed on ice and quickly washed with cold PBS (4 °C). Then, 500 µl of cold methanol:water (9:1 v/v, with internal standards) with 2% of sulfosalicylic acid were added to each well and the plate was stored overnight at −80 °C. Two wells at ~5 × 10⁵ cells per well were scrapped and pooled for one measurement in microcentrifuge tubes. For normalization purposes, we used reference wells for cell counting.

The suspension was vortexed for 30 s and centrifuged at 15,000 rpm (21,130 rcf) for 10 min at 4 °C. After the transfer of the whole supernatant to another set of microtubes, samples were evaporated. The dried samples were spiked with 200 µl of MilliQ water before injection in UHPLC–MS. Polyamine analysis was performed by LC–MS/MS with a 1290 UHPLC (Agilent Technologies) coupled to a QQQ 6470 (Agilent Technologies)⁸⁴.

DFMO treatment (48–72 h) was continuously performed, whereas GC7 was used acutely during starvation. Trypsinized GFP–LC3-expressing cells were seeded in 384-well black microplates (~4,000 cells per well). Starvation with HBSS or treatment with rapamycin (10 µM; 2 mM DMSO stock stored in aliquots at −20 °C) or Torin-1 (300 nM; 60 µM DMSO stock stored in aliquots at −20 °C) was initiated after overnight attachment. Before starvation experiments, the cells were gently washed with warm PBS (37 °C). DMSO at a final concentration of 0.5% was used as the control treatment for rapamycin and torin-1. Fresh DMEM containing FBS and non-essential amino acids was used as the control treatment for starvation experiments. Chloroquine (50 µM in sterile filtered ddH₂O) was used as an autophagic flux inhibitor and added to the wells after half of the treatment time, except for Extended Data Fig. 5r,s, where bafilomycin A1 (BafA1, 100 nM) was used.

Experiments involving spermidine supplementation (Sigma, 85558; from aqueous stocks, pH 7.4) were co-treated with aminoguanidine (from aqueous stocks, 1 mM, Sigma, 396494).

After treatment, cells were fixed with 3.7% paraformaldehyde (w/v in PBS containing 2 µg ml⁻¹ Hoechst) for 20 min at room temperature. Microscopy images were acquired with an ImageXpress Micro Confocal microscope (Molecular Devices) equipped with a ×20 PlanApo objective (Nikon). Four view fields and six z-stacks were imaged per well. We used the Custom Module Editor functionality of the MetaXpress software (Molecular Devices) to analyse the images. First, using the nuclear Hoechst staining and cytoplasmic GFP signal, cells were segmented into nuclear and cytoplasmic regions. Then, cytoplasmic GFP–LC3 puncta were detected using automated thresholding, and their number and the cellular surface covered by GFP–LC3 puncta were analysed.

U2OS cells (with or without stably expressing GFP–LC3) were seeded in 384-well imaging microplates at an approximate density of 1,000 cells per well (for autophagy measurements) or in six-well plates at an approximate density of 1 × 10⁵ cells per well cells per well (for polyamine measurements). Following 1 day of attachment, cells were transfected with siRNAs against ODC1 or a cherry-picked siRNA library (Dharmacon) targeting selected genes associated with polyamine metabolism at a final concentration of 30 nM using the DharmaFECT transfection reagent (Dharmacon) according to the manufacturer’s instructions. Each gene was represented as four individual siRNAs and non-targeting siRNAs served as control (Supplementary Table 4). After 24 h, the medium was renewed and cells were left to adapt for an additional 24 h. Subsequently, cells were carefully washed with warm PBS and subjected to starvation with HBSS for 6 h.

Image acquisition was carried out as previously described. The obtained images were segmented and analysed with the freely available software R (https://www.r-project.org↗), integrated with the following packages: EBImage from the Bioconductor repository (https://www.bioconductor.org↗), MetaxpR (https://github.com/kroemerlab/MetaxpR↗), RBioFormats (https://github.com/aoles/RBioFormats↗), as well as MorphR (https://github.com/kroemerlab/MorphR↗). The primary region of interest was designated by a mask around the nucleus to enable cell enumeration, while a secondary cytoplasmic region of interest was employed for the segmentation of GFP–LC3 puncta. Following the exclusion of cellular debris and dead cells, the number of GFP–LC3 dots per cell was quantified and represented as fold change relative to the control.

For RNA extraction, the RNeasy Plus Mini kit (cat. no. 74134, QIAGEN) was utilized. Cell pellets were stored at −80 °C before purifying RNA. Buffer RLT Plus was used to lyse the cell pellets. The lysate was centrifuged and subjected to further purification following the manufacturer’s instructions. All steps of the procedure were carried out at room temperature and all centrifugation steps were performed at 20–25 °C in a standard microcentrifuge. Approximately 1 μg total RNA was reverse transcribed using a Maxima First Strand cDNA Synthesis kit (cat. no. K1642, Thermo Fisher Scientific). Quantitative reverse transcription PCR (qRT–PCR) was conducted using PowerUp SYBR Green Master Mix (cat. no. A25776, Thermo Fisher Scientific) with a StepOnePlus Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific). The 2^−ΔΔCT method was applied for the analysis of real-time PCR data with the appropriate primers. All primers used for gene expression analysis are listed in Supplementary Table 5.

For immunoblotting, 2–3 wells from six-well plates were washed with PBS, then collected on ice with RIPA cell lysis buffer. Cells were scraped off and incubated for 30 min at 4 °C before centrifugation for 20 min at 12,000 rpm (13,523 rcf). The protein concentration in the supernatant was determined with a Bradford assay and the samples were stored at −20 °C until further processing. In brief before electrophoresis, samples were mixed with final sample buffer (final concentration of 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 8.7% glycerol, 0.004% bromophenol blue and 120 mM DTT) and boiled at 95 °C for 5 min. Then, 10 µg total protein was loaded onto 4–12% Bis-Tris gels and run at 90–120 V. Proteins were transferred to 0.45-μm PVDF membranes for 90 min at 220 mA using transfer buffer (10 mM CAPS pH 11 and 10% methanol). The membranes were then incubated in blocking solution (3% dry milk powder in TBS, pH 7.4) for 1 h and subsequently incubated with the primary antibody overnight at 4 °C. Membranes were washed three times with TBST for 5 min and subsequently incubated with the secondary antibody for 1 h at room temperature. After three washing steps in TBST for 5 min, signals were detected with a ChemiDoc detection system (Bio-Rad) and Clarity Western ECL Substrate (Bio-Rad) using the ‘optimal exposure’ setting. For re-probing membranes, Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific, 46430) was used according to the manufacturer’s protocol. Band intensities were quantified using ImageLab v.5.2 (Bio-Rad) using the rectangular volume tool with local background adjustment. Primary antibodies were anti-hypusine (Merck, ABS1064-I, 1:1,000 dilution), anti-GAPDH clone GA1R (Thermo Fisher Scientific, MA5-15738, 1:10,000 dilution) and anti-eIF5A (611977, BD Biosciences, 1:10,000 dilution), diluted in 1% dry milk powder in TBST. Secondary antibodies were HRP-linked anti-mouse IgG (Sigma, A9044, 1:10,000 dilution in blocking solution) or HRP-linked anti-rabbit IgG (Sigma, A0545, 1:10,000 dilution in blocking solution).

The study protocol was approved by the medical council of Baden-Württemberg under the application number F-2018-118 and registered in the German Clinical Trials Register (ID DRKS00016657). The 109 participants were recruited among the in-house patients of the Buchinger Wilhelmi clinic Überlingen as previously described^109–111. All participants underwent a 10 ± 3-day fasting period under medical supervision. A daily caloric intake of around 250 kcal was provided by 250 ml organic juice, 250 ml vegetable soup and 20 g honey. Serum samples were collected before the start of the fast and at the end of the 10 ± 3-day fasting period. Tubes were stored at −70 °C until further analysis.

The 1-year observational study was performed in the Buchinger Wilhelmi clinic Überlingen and described in detail previously^112–115. The medical council of Baden-Württemberg and the Ethics Committee of the Charité-University Medical Center Berlin approved the study protocol under application no. EA4/054/15 that was registered in the German Clinical Trials Register (ID DRKS00010111). Blood samples before and at the end of fasting were available for a subgroup of 63 participants who fasted more than 4 days according to the Buchinger Wilhelmi fasting programme. Serum samples were stored at −70 °C.

Cohort 3 was previously described^116,117. For the purpose of this study, we took a subset of study participants with available plasma samples (23 of an initial 30 performing alternate day fasting) after 12 and 36 h of fasting, stored them at −70 °C and performed targeted MS of spermidine as described below, which was previously not quantified.

Clinical data were collected during an exploratory clinical trial. Healthy participants were included by written informed consent when presenting no manifest chronic diseases nor pregnancy or lactation. Recruitment and intervention were performed on an outpatient basis at the Immanuel Hospital Berlin, where the outpatient department of the Institute of Social Medicine, Epidemiology and Health Economics of the Charité – Universitätsmedizin Berlin is seated. The study protocol was approved by the institutional review board of Charité Universitätsmedizin Berlin (ID EA1/263/20) and is registered with ClinicalTrials.gov under NCT04739852↗.

Recruitment for healthy volunteers took place in August 2020. All measurements and the intervention were conducted in September 2020. Participants underwent a 5-day prolonged fasting period with a dietary energy intake of 200–350 kcal per day with vegetable broths and juices. These were followed by three days of gradual re-introduction of solid food intake, the meals containing mainly complex carbohydrates. PBMCs and blood were collected at baseline, after 1, 3 and 5 days of the fasting intervention, as well as on days 3 and 7 after re-introduction of food.

The blood of healthy volunteers was collected in EDTA-sampling tubes (Sarstedt, 02.1066.001) via venipuncture, carefully loaded on Biocoll solution (BioChrom, L6113) in Lecosep tubes (Z642843, Greiner) and centrifuged at 684 rcf for 30 min (brakeless running down). PBMCs were enriched by selecting the interphase of the Biocoll gradient. PBMCs of the interphase were washed twice with ice-cold PBS. Pellets were stored at −80 °C until they were further processed. This is the first publication of data from this cohort and the primary end points will be published elsewhere.

Protein extracts from PBMCs were obtained by lysing cells (approx. 1–2 × 10⁶ cells) in 80 µl T-PER extraction buffer (Thermo Scientific, 78510), supplemented with protease (Sigma, P2714) and phosphatase (Roche, 04906837001) inhibitor cocktail. Protein extracts (1–2 µg) were separated by capillary electrophoresis on Jess (ProteinSimple) using 12–230 kDa cartridges (ProteinSimple, SM-W004) and the following primary antibodies for detection: anti-hypusine (1:50 dilution, ABS1064-I, Merck), anti-eIF5a (1:50 dilution, 611977, BD Biosciences) and anti-ODC1 (1:50 dilution, PA5-21362, Thermo Scientific). The signals were normalized to total protein using the protein normalization module (DM-PN02, ProteinSimple).