Spermidine, a naturally occurring polyamine, has recently emerged as exhibiting anti-aging properties. Its supplementation increases lifespan and resistance to stress, and decreases the occurrence of age-related pathology and loss of locomotor ability. Its mechanisms of action are just beginning to be understood. Objectives: An up-to-date overview of the so far identified mechanisms of action of spermidine and other polyamines on aging is presented. Methods: Studies of aging and of the molecular effects of polyamines in general and spermidine in particular are used to synthesize our knowledge on what molecular mechanisms spermidine and other polyamines trigger to positively affect aging. Results: Autophagy is the main mechanism of action of spermidine at the molecular level. However, recent research shows that spermidine can act via other mechanisms, namely inflammation reduction, lipid metabolism and regulation of cell growth, proliferation and death. It is suggested that the main pathway used by spermidine to trigger its effects is the MAPK pathway. Conclusions: Given that polyamines can interact with many molecules, it is not surprising that they affect aging via several mechanisms. Many of these mechanisms discovered so far have already been linked with aging and by acting on all of these mechanisms, polyamines may be strong regulators of aging. 

Aging is a multifaceted process, probably caused by a myriad of interacting factors and with consequences at all levels of the organism. Research into the subject has revealed that factors leading to aging are as varied as sustained exposure to cellular stress, chronic inflammation, dysregulation of lipid metabolism, autophagy and cell survival and death. These factors will impinge upon each other in complex interactions. Effective interventions against aging will need to be able to impact as many as possible of the factors causing aging and their interactions. Dietary restriction may be one such intervention with its wide-ranging effects. Polyamines, especially spermidine, have also emerged as strong potential candidates and this review will give evidence to support the involvement of polyamines in aging. Spermidine belongs to the family of polyamines. Polyamines are polycations that will interact readily with negatively charged molecules, including DNA, RNA and lipids. This ability to bind such different molecules means polyamines are involved in many important processes such as DNA stability, cell growth, proliferation and death. To regulate these processes, a set of specific enzymes tightly regulate polyamine synthesis and catabolism ( fig. 1 ) [reviewed in 1 ]. Polyamine metabolism is very dynamic and many studies have investigated the role of all three main polyamines (putrescine, spermidine and spermine) on aging, so this review will discuss all three polyamines and not only spermidine.

It has been known for some time that polyamine levels decrease with age [reviewed in 1 ]. However, it is only recently that the effect of polyamines, especially spermidine, on aging has been investigated. Indeed, Pucciarelli et al. [2] recently observed that spermidine levels in humans aged between 60 and 80 were lower than in humans below 50 but humans older than 90 have levels similar to people below 50. These results suggest that maintaining spermidine levels in aging may contribute to longevity. Polyamine levels can be increased in both model organisms and humans by supplementation in food or water [3–5] , so the effect of polyamines on aging can be investigated relatively easily. Examples of food and drinks containing high levels of polyamines are rice bran, green pepper, broccoli, soybeans, mushrooms, oranges, and green tea. We have shown that yeast cells Saccharomyces cerevisiae , nematode worms Caenorhabditis elegans , fruit flies Drosophila melanogaster as well as human peripheral blood mononuclear cells supplemented with spermidine had increased lifespans [3] . Yeast cells unable to synthesize polyamines are short-lived and spermidine addition rescued this lifespan defect [3] . Soda et al. [5, 6] also reported that a high-polyamine diet decreased mortality in mice. However in both cases, mice were sacrificed at 88 weeks of age for use in other measurements, precluding a full lifespan measurement

. The same authors also showed that a high-polyamine diet decreased the incidence of age-related kidney glomerular atrophy. Aging triggered DNA methylation changes in kidneys and the high-polyamine diet suppressed these changes [6] . Finally, they reported that these mice kept thicker coat and higher activity levels with age, although these variables were not quantified [5] . Recently, we found that spermidine reduced the age-related decline of climbing activity in D. melanogaster [Minois, unpubl. results]. It was also reported that spermidine feeding reSpermine Acetyl spermidine Putrescine Spermidine SMO Spm Syn H2N NH2 H N N H H2N N NH2 H Spd Syn SSAT PAO PAO H2N NH2 PAO Acetyl spermine Diacetyl spermine SSAT SSAT Antizyme ODC Ornithine Arginase Arginine Fig. 1. Polyamine metabolism. ODC = Ornithine decarboxylase; PAO = polyamine oxidase; SMO = spermine oxidase; Spd Syn = spermidine synthase; Spm Syn = spermine synthase; SSAT = spermidine/ spermine N 1 -acetyltransferase [for more details, see 1] . The levels of polyamines in many mammalian tissues are in the order spermidine > spermine > putrescine, with the ratios between them easily varying from 2 to 20. Polyamines and Aging Gerontology 2014;60:319–326 DOI: 10.1159/000356748 321 duced levels of age-related oxidative damage in mice [3] and age-related overproduction of reactive oxygen species in yeast [7] . Although not measured as a function of age, spermidine has positive effects on variables related to aging, namely resistance to stress. Yeast cells fed spermidine were more resistant to heat and hydrogen peroxide [3] . We also observed that spermidine-treated D. melanogaster was more resistant to paraquat and hydrogen peroxide [8] .

 Spermidine treatment decreased nitric oxide and prostaglandin E 2 production in a dose-dependent manner, as well as the mRNA expression of pro-inflammatory cytokines, among them interleukin-6 and tumor necrosis factor-α. The authors’ work suggested that this anti-inflammatory response was triggered by the suppression of the translocation in the nucleus of NF-κB p65 subunit, leading to a decreased phosphorylation of Akt and MAPK. Recently, Paul and Kang [11] studied the effect of polyamines on ear edema in mice. They induced an inflammatory response in the ear by applying 12-O-tetradecanoylphorbol-13-acetate (TPA). Prior to TPA application, mice were topically treated with saline, hydrocortisone (positive anti-inflammatory control) or the main three polyamines (putrescine, spermidine or spermine). The authors showed that application of polyamines reduced ear thickness in a dose-dependent manner to levels similar to the positive control. Spermidine and spermine induced the highest water content decrease. Polyamines also reduced neutrophil infiltration in the ear tissues. The authors then induced inflammation in mice macrophages with LPS. Polyamines decreased nitric oxide production triggered by the LPS treatment, spermine having the strongest effect. They also inhibited the expression of interleukin-1β and tumor necrosis factor-α. It has to be kept in mind that these studies focus on acute inflammation, in contrast with the chronic, silent inflammation observed in aging. Work on the effects of polyamines on chronic inflammation is therefore needed. However, the present research suggests that spermidine in particular and polyamines in general could promote their anti-aging properties by reducing inflammation.

Although these last studies showed an important involvement of spermidine in adipogenesis, other studies pointed to more complex interactions between polyamines and adipogenesis. For instance, in 3T3-L1 pre adipocytes, inhibiting spermidine synthase or spermine synthase respectively decreased and increased TAG accumulation. These inhibitions had opposite effects in mature adipocytes. Polyamine catabolism also regulates adipogenesis in these cells [14] . We have recently discovered that spermidine altered lipid metabolism in whole D. melanogaster [Minois, unpubl. results]. Spermidine supplementation increased TAG levels and altered lipid profile. We observed that spermidine supplementation changed the relative abundance of fatty acids (for instance a decline in C14:1, C14:0, C20:3 and C20:2 and an increase in C18:2 in females) and phospholipids (for instance an increase with spermidine in ethanolamine phosphate ceramide EPCd38:1, phosphatidylcholine PC32:2 and phosphatidylethanolamine PE38 in males). Spermidine also triggered changes in the ratio of saturated over unsaturated fatty acids and some ceramide phosphoethanolamine species, the main sphingolipid in invertebrates. Most of these changes are autophagy-dependent. The changes in lipids profile will modulate membrane fluidity and proneness to oxidative damage as well as signalling. How this can affect aging will need to be further studied.

Furthermore, spermidine decreases the phosphorylation status of PKB/Akt in HCT116 cells [21] and of Akt in LPS-activated BV2 microglial cells [10] . It will be interesting to study whether the effect of spermidine on lifespan overlaps with the IIS pathway. A likely candidate for regulating the anti-aging effects of spermidine is the MAPK pathway, another pathway involved in autophagy modulation [23] . In addition to regulating autophagy, it is known that the MAPK pathway interacts with polyamines [24] . CK2, a ubiquitous kinase, phosphorylates KSR (kinase suppressor of ras that serves as scaffold and can act as raf activator) and also directly raf. Polyamines change the phosphorylation of raf by CK2, either acting as inhibitor (spermine) or activator (spermine plus spermidine or putrescine) of raf. CK2 could act as a sensor for polyamine levels and translate the information to the MAPK Furthermore, some MAPKs show altered phosphorylation status upon spermidine treatment in HTC116 cells [21] and MAPKs phosphorylation is reduced by spermidine in LPS-activated BV2 microglial cells [10] . In tomato green fruits exposed to spermidine (1 m M for 30 min) at high temperature, spermidine upregulates MAPK family genes [25] suggesting more involvement of polyamines with the MAPK pathway. Finally, the expression of transcription factors targets of MAPKs have been shown to be altered upon polyamines depletion and then re-addition of spermidine in NIH3T3 mouse fibroblasts [16] .