Introduction

Macroautophagy (which we refer to as autophagy) is a cellular self-cannibalistic pathway in which parts of the cytosol or cytoplasmic organelles are enwrapped in double-membraned vesicles, autophagosomes, which then fuse with lysosomes (Klionsky, 2007). Autophagy plays a major role in the maintenance of cellular homeostasis, allows for the mobilization of energy reserves when external resources are limited, and is essential for the removal of damaged organelles and potentially toxic protein aggregates (Levine and Kroemer, 2008).  At the organismal level, autophagy can mediate cytoprotection (for instance neuroprotection and cardioprotection in the context of ischemic preconditioning; Moreau et al., 2010) and delay the pathogenic manifestations of aging (Levine and Kroemer, 2009). Given the potential health and longevity-promoting effects of autophagy, pharmacological agents that stimulate autophagy at a low level of toxicity are urgently needed. Rapamycin and the so-called rapalogs are the most effective clinically used inducers of autophagy yet have severe immunosuppressive effects (Hartford and Ratain, 2007). Thus, alternative, nontoxic autophagy inducers (such as rilmenidine or carbamazepine) are being characterized for their pharmacological profile in suitable preclinical models (Hidvegi et al., 2010; Rose et al., 2010). Nontoxic compounds, such as resveratrol and spermidine, are also being evaluated for their potential to induce autophagy in vivo (Eisenberg et al., 2009; Morselli et al., 2010). Resveratrol is a natural polyphenol found in grapes, red wine, berries, knotweed, peanuts, and other plants.

In these conditions, neither spermidine nor resveratrol impaired oxidative phosphorylation (Fig. S1 B), ruling out that resveratrol might induce autophagy via mitochondriotoxicity (Dörrie et al., 2001). Knockdown of SIRT1 with a specific siRNA suppressed the proautophagic activity of resveratrol (Fig. 1, A and B) yet failed to affect spermidineinduced autophagy (Fig. 1 C). Similarly, the SIRT1 inhibitor EX527 (Peck et al., 2010) abolished autophagy induction by resveratrol but not by spermidine (Fig. 1, D–F). These results indicate that resveratrol and spermidine trigger autophagy through distinct mechanisms. 

Phylogenetic conservation of sirtuin-independent autophagy induction by spermidine

 We next investigated whether the orthologues of sirt1 in Saccharomyces cerevisiae and C. elegans (sir2 and sir-2.1, respectively) are required for the proautophagic activity of spermidine. In yeast, spermidine caused the redistribution of a GFP-Atg8p chimera from a diffuse to a vacuolar localization (Fig. 2 A), the autophagy-dependent proteolytic liberation of GFP from GFP-Atg8p (Fig. 2 B; Suzuki et al., 2004), as well as an autophagy-related increase in vacuolar AP (Fig. 2 C; Noda et al., 1995). These effects were similar in wild-type (WT) and sir2 yeast strains (Fig. 2, A–C). Moreover, spermidine significantly improved the survival of aging WT yeast cultures, a beneficial effect that was attenuated, yet remained significant, in aging sir2 yeast cultures (Fig. 2 D). Accordingly, spermidine reduced the aging-associated overproduction of reactive oxygen species (measured by assessing the conversion of nonfluorescent dihydroethidine into fluorescent ethidium) both in WT and sir2 cells (Fig. 2 E). 

In C. elegans embryos, spermidine induced the autophagy-related expression and cytoplasmic aggregation of DsRed::LGG-1 (Fig. 3, A and B; Eisenberg et al., 2009). This effect was significant in both WT and sir-2.1 mutant nematodes, although the sir-2.1 mutation attenuated autophagy induction by spermidine (Fig. 3, C and D). Consistently, spermidine prolonged the lifespan of WT and sir-2.1–deficient worms by 18 and 13%, respectively. Collectively, these results indicate that spermidine can stimulate autophagy and extend the lifespan of yeast cells and nematodes that lack SIRT1 orthologues. 

Resveratrol and spermidine induce autophagy through convergent pathways

To investigate the signal transduction pathway stimulated by resveratrol and spermidine, the phosphorylation status of multiple cellular proteins was analyzed in human colon cancer HCT 116 cells by means of an antibody array. Surprisingly, spermidine and resveratrol, alone or in combination, elicited similar changes in the phosphorylation status of multiple kinases and their substrates (Fig. 4, A–C). For example, both spermidine and resveratrol mediated the dephosphorylation of the protein tyrosine kinase 2  (also known as PYK2) and the cyclindependent kinase inhibitor 1B (better known as p27Kip1). However, neither of the two agents had major effects on the phosphorylation levels of the regulatory subunit of AMPdependent kinase and its substrate acetyl–coenzyme A (CoA) carboxylase, which was in line with the hypothesis that the energy metabolism of the cells was normal.

 Moreover, spermidine and resveratrol did not affect the phosphorylation of mechanistic target of rapamycin (mTOR) nor that of its substrate ribosomal protein S6 kinase (also known as p70S6K; Fig. 4, A–C), which suggests that resveratrol and spermidine induce auto­phagy through AMP-dependent kinase/mTOR-independent convergent pathways. Accordingly, the administration of an optimal dose of resveratrol and spermidine (100 µM for both agents) did not result in higher levels of autophagy than that of either agent alone (Fig. 4 D). This kind of epistatic analysis confirms the suspected convergence of the proautophagic pathways elicited by both agents.

Convergent action of resveratrol and spermidine on the acetylproteome

Next, we comparatively explored the effects of resveratrol and spermidine on the acetylation patterns of cytosolic, mitochondrial, and nuclear proteins. To that purpose, we performed stable isotope labeling with amino acids in cell culture (SILAC) and then purified the proteins/peptides containing acetylated lysine residues and identified them by quantitative mass spectrometry (MS). Resveratrol or spermidine induced changes in the acetylation of 560 lysine-containing motifs corresponding to 375 different proteins (Table S1). Surprisingly, 170 proteins whose acetylation status was modified in response to resveratrol or spermidine treatment are part of the recently elucidated human autophagy protein network (Behrends et al., 2010).

 Cytoplasts were still able to accumulate GFP-LC3 puncta in response to spermidine or resveratrol treatment (Fig. 8, A and B), indicating that nuclei (and by extension transcription) are not required for short-term autophagy stimulation by these two agents. Next, we enforced overexpression of transgenic WT SIRT1 (which although preponderantly localizes to the nucleus, has been reported to efficiently shuttle to the cytoplasm; Tanno et al., 2007) or that of a mutant SIRT1 protein with a mutation in the nuclear localization signal (which is, therefore, virtually restricted to the cytoplasm; Fig. 8 C). Both constructs induced RFP-LC3 punctuation and LC3 lipidation with similar potency and similar kinetics (Fig. 8, C–E), suggesting that autophagy can be efficiently regulated by cytoplasmic (de)acetylation reactions.

Synergistic induction of autophagy by low doses of resveratrol and spermidine 

Resveratrol (but not spermidine) induces autophagy through the activation of the deacetylase SIRT1 (Morselli et al., 2010), whereas spermidine is thought to act as an inhibitor of acetylases (Eisenberg et al., 2009). We reasoned that low doses of resveratrol and spermidine might synergistically induce autophagy by affecting the equilibrium state of (de)acetylation. To assess this possibility, we treated HCT 116 cells with different concentrations of resveratrol or spermidine, alone or in combination, and analyzed the effects of the different pharmacological combinations in terms of autophagy induction. 

 As expected, both spermidine and resveratrol used at high doses (100 µM) induced GFP-LC3 punctuation and LC3 lipidation (Fig. 9 A) in cultured cells. Interestingly, although none of the two agents at low doses (10 µM) was able to significantly up-regulate autophagic flux, the combination of spermidine and resveratrol at low doses (10 µM) was as efficient in enhancing GFP-LC3 puncta formation, LC3 lipidation, and an increase in autophagic flux as were high doses of spermidine or resveratrol (Fig. 9, A and B). To try to extend these results to a physiological setting, we intraperitoneally injected optimal doses of resveratrol (25 mg/kg) or spermidine (50 mg/kg) into mice expressing a GFP-LC3 transgene to induce autophagy in an array of organs. One tenth of this optimal dose (2.5 mg/kg resveratrol or 5 mg/kg spermidine) had no major proautophagic effect in vivo when either compound was injected alone. However, the combination of low doses of both agents was highly efficient in triggering autophagy in vivo (Fig. 9, C and D). Similar results were obtained when these agents were injected into WT mice, as shown by means of LC3 lipidation and p62 degradation (Fig. 9 E). In conclusion, low doses of spermidine and resveratrol can induce autophagy in a synergistic fashion.

Discussion

Resveratrol can induce autophagy only in the presence of SIRT1 (Morselli et al., 2010), whereas SIRT1 (or its orthologues in yeast and nematodes) is dispensable for spermidine-stimulated autophagy.  

When analyzed as individual compounds, neither polyphenols nor polyamines consumed with the normal diet may reach concentrations high enough to mediate pharmacological effects. Nonetheless, it is tempting to speculate that combinations of these agents— and perhaps that of other proautophagic dietary components— may affect the autophagic rheostat, as based on their distinct yet convergent mode of action.

Materials and methods 

Chemical, cell line, and culture conditions

Unless otherwise specified, chemicals were purchased from Sigma-Aldrich, culture media and supplements for cell culture were obtained from Invitrogen, and plasticware was purchased from Corning.  Human colon carcinoma HCT 116 cells (gift from B. Volgelstein, Howard Hughes Medical Institute, Johns Hopkins University, Baltimore, MD; Bunz et al., 1999) were cultured in McCoy’s 5A medium containing 10% fetal bovine serum, 100 mg/liter sodium pyruvate, 10-mM Hepes buffer, 100 U/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate (5% CO2 at 37°C). Cells were seeded in 6- and 12-well plates or in 10- and 15-cm dishes and grown for 24 h before treatment with 10-µM EX527 (Tocris Bioscience), 10- or 100-µM resveratrol, 10- or 100-µM spermidine, and 1-µM rapamycin or 1-nM bafilomycin A1 (Tocris Bioscience) for the time indicated in each experimental figure legend.

Plasmids, transfection, and RNA interference in human cell cultures 

Cells were cultured in 12-well plates and transfected at 50% confluence with siRNAs targeting human SIRT1 (Ford et al., 2005), ATG5, or ATG7 (Thermo Fisher Scientific) or with an unrelated control siRNA by means of a transfection reagent (Oligofectamine; Invitrogen) following the manufacturer’s instructions. After 24 h, cells were transfected with a plasmid coding for a GFP-LC3 fusion (Kabeya et al., 2000). Transient plasmid transfections were performed with the Attractene reagent (QIAGEN) as suggested by the manufacturer, and unless otherwise indicated, cells were analyzed 24 h after transfection. Cells were transfected with a plasmid coding for RFP fused to LC3 (RFP-LC3; obtained from Invitrogen) in the presence of an empty vector (pcDNA3) or of different constructs for the overexpression of GFP-tagged WT SIRT1 or a SIRT1 variant mutated in the nuclear localization signal, which mostly localizes in the nucleus (Tanno et al., 2007). For fluorescence microscopy determinations, cells cultured on coverslips were fixed in paraformaldehyde (4% wt/vol) for 15 min at RT, washed three times in PBS, and mounted with mounting medium (Vectashield; Vector Laboratories).

Fluorescence microscopy

Confocal fluorescent images were captured using a confocal fluorescence microscope (TCS SP2; Leica). For experiments with HCT 116 cells, an Apochromat 63× 1.3 NA immersion objective was used, whereas for the analysis of GFP-LC3 mice tissue sections, an Apochromat 40× 1.15 NA immersion objective was used. All the acquisitions were made at RT with fixed cells/tissue slides. Images were acquired with a camera (DFC 350 FX 1.8.0; Leica) using LAS AF software (Leica) and processed with Photoshop (CS2; Adobe) software.