Sirtuin biology is fiendishly complex1; that of SIRT1 in particular involves several feedback loops and multiple reciprocal interactions with other proteins. Disentangling these interconnectivities has been difficult due to its role as a hub protein for diverse signalling pathways5, and an intrinsic capacity for autoregulation23. While its role in ageing/lifespan regulation remains controversial, its contribution to healthspan is not in doubt. Hence the use of dSTACs such as resveratrol and related allosteric activators for healthspan extension is of great interest. Although crystal structures have confirmed that these molecules bind directly to the STACs-AD10,11, it is unclear how their signals are transduced by SIRT1 or how those signals are translated for healthspan extension. The data reported here strongly suggest that dSTACs may impart health benefits including delaying ageing across taxa by exploiting their structural and functional mimicry of gonadal steroids to co-opt a conserved mechanism of sirtuin signalling through steroid receptors. This mechanism does not exclude allostery but indicates that dSTACs signals may come under endocrine control in vivo. As natural selective modulators of SIRT1 signalling through the ERs, dSTACs therefore have the potential to provide all the benefits of oestradiol such as protection from cognitive impairment and metabolic diseases42,43,44,45,46,47,48,49.

However abundant caution is required in ascribing biological relevance to these data because of potentially important confounding variables. Although it is recognized that SIRT1 and dSTACs prevent MARDs2,3,4, it is presently unclear from these data whether they do so through the ERs. Furthermore because dSTACs are poorly soluble in aqueous media, it is not known what fraction was absorbed and whether they underwent modification in cell culture. In vivo, their poor bioavailability and potential to undergo postprandial first-pass metabolism further complicates the biological validity of the data. For example, isoflavone methylation or glucosidation reduces their oestrogenicity50, whereas methylation of quercetin increases its potency51. In the case of resveratrol, although the native aglycone form can persist for hours postprandially, only small amounts are rapidly absorbed while a relatively large proportion gets glucuronidated within the small intestine52. It is unclear if those modified forms would be more or less bioactive than parental resveratrol in modulating SIRT1 signalling in vivo. Another possible layer of complexity is in the fact that some dSTACs can be metabolized by intestinal microbiota. For example, daidzein and resveratrol undergo biotransformation in vivo to equol and lunularin respectively but only in individuals with the requisite gut bacteria53,54,55. Since these metabolites appear to be more oestrogenic and also regulate food intake and body weight better than their precursors, it is tempting to speculate that these dSTACs may postprandially elicit stronger SIRT1-ER signalling than they show in these in vitro data.

Resveratrol was particularly remarkable on two levels. First, its dosage-sensitivity or non-monotonicity hints at a potential for hormetic signalling, an indication that it may evoke adaptive stress responses in vivo consistent with the xenohormesis hypothesis that dSTACs may be plant stress signalling molecules56. This may also be useful cautionary information because it suggests that contrary to popular lore, high doses of resveratrol may not be necessary to activate sirtuin signalling and may in fact be harmful, i.e. less is more. Instead, low dietary doses57 may suffice to elicit the hormetic responses required to prime defence mechanisms against incipient disease58. Secondly and more importantly, resveratrol elicited oestrogenicity in low doses but in large amounts it behaved like an antioestrogen, reducing sirtuin signalling through the ERs both in the absence and presence of E2. This janus-faced feature has been previously described as mixed agonism22,59,60, and may enable it to protect against a spectrum of diseases. For example, in low doses resveratrol induces gene expression signatures that mimic calorie restriction, delays ageing-related neurodegeneration, protects against obesity and non-alcoholic fatty liver disease, improves motor and cognitive functions and increases insulin-sensitivity in patients with type 2 diabetes61,62,63,64,65; all these effects remarkably overlap those of oestradiol42,43,44,45,46,47,48,49. In small amounts (5 µM) resveratrol also increased worm lifespan66. It is therefore interesting to speculate that in low doses it may mimic oestradiol, enabling it to up-regulate SIRT1 signalling through the ERs to confer all of the above health benefits as well as protect against other diseases associated with SIRT1 including the metabolic syndrome. Interestingly, SIRT1 has been shown to form transcriptional complexes with PGC-1α on regions of mitochondrial DNA that bind both ERα and ERβ67,68. This may partly explain why mice fed a resveratrol diet showed extended lifespan, increased mitochondrial biogenesis and metabolic capacity, and were protected from the damaging effects of high-calorie diets61,69,70. Of note, Sirt1 or Sir-2.1 ablation in cell culture and in animals severely attenuated responses to resveratrol and related dSTACs36,71 while the converse was true with sirtuin overexpression (see ref. 12 and references therein). Importantly, Sirt1 deletion reduced the ability of resveratrol to protect against skin cancer in mice72. Together these studies strongly suggest that these sirtuins may be critically important for transducing and translating the health benefits of dSTACs signals perhaps through steroid hormone receptors. In very low doses, resveratrol also rapidly and ER-dependently induces nitric oxide in endothelial cells73, indicating a possible mechanism by which it might protect against cardiovascular disease. Conversely, its antagonism of the ER in high doses hints at how resveratrol may reduce the risk of oestrogen-dependent cancers74,75,76; however, these effects may not be solely due to antioestrogenicity but also to its ability to induce cytostasis at those doses60. Hence these results are intriguing because they may partly explain the French paradox, i.e. the reduced incidence of cardiovascular diseases and certain types of cancer in some populations despite their consumption of high-fat diets57,77.

It may be important to note that the biphasic responses evoked by resveratrol and its non-monotonicity are typical of steroid hormones in general but of oestradiol in particular (see ref. 32 and references therein); this may prove consequential in vivo. At the molecular level, it could be speculated that in low doses, resveratrol may recruit ER coactivators such as SIRT1 and PGC-1α23. This may be supported by co-crystal structures showing that it induces a conformational change in ERα LBD that facilitates coactivator recruitment39. Alternatively, low-dose resveratrol may induce an open chromatin landscape permissive to SIRT1-ER coactivator complexes while in high doses it may either induce chromatin compaction that occludes these complexes or actively promote corepressor recruitment. It could also be that high-dose resveratrol induces a conformational change that inhibits ER binding/coactivation by SIRT1 (see Figs. 3 and 4). These assumptions may be consistent with reports that agonists and antagonists induce distinct ER conformations that respectively recruit coactivators and corepressors to the LBD78,79. Were similar conformers to be induced by low and high doses of resveratrol, they would explain its bifunctionality and the biphasic ER responses to it.

The ability of dSTACs to modulate ER subtype-selectivity and coactivation by SIRT1 may be teleologically important given that these receptors play vital physiological roles in health and disease42,43,44,45,46,47,48,49. Both ERs are expressed tissue-specifically suggesting that coactivation by SIRT1 may also be tissue-specific; for example, whereas ERα is strongly expressed in the liver compared with ERβ, the converse is true in the brain19,49. Oestrogen signalling through ERα regulates energy balance, food intake, bodyweight, glucose and lipid metabolism, and confers protection against osteoporosis, metabolic and cardiovascular diseases while ERβ is thought to protect against breast, colon and prostate cancers as well as neurodegeneration42,43,44,45,46,47,48,49,80,81,82,83,84,85. Notably the isoflavones increased ERβ coactivation by SIRT1 independently of oestradiol, suggesting how these dSTACs may protect against such conditions. Transcriptomic analyses have also shown that dSTACs induce distinct as well as overlapping gene networks regulated by ERα and ERβ86. Hence the ability of dSTACs to modulate SIRT1 signalling through both receptors independently of oestradiol could be important in non-reproductive tissues such as the liver, vascular endothelium, and adipose tissue but especially in skeletal muscle, brain and bone which markedly deteriorate with ageing. Dietary STACs may thus be most acutely beneficial in these tissues because their intrinsic oestrogenicity may subserve diverse oestradiol functions including neuroprotection46,49,87,88,89. These data may therefore provide clues to how dSTACs may reduce the incidence of MARDs.

What might underlie the ability of sirtuins to transduce diverse dSTACs signals through steroid receptors from evolutionarily distinct organisms? Phylogenetic evidence suggests that all extant steroid receptors evolved from a common ancestral ER (see ref. 90 and references therein) followed by ligand diversification from a primordial oestrogen or “paraestrol”91, i.e. steroid hormones including oestradiol and dafachronic acid evolved from a basic paraestrol template. Since this ancestral oestrogen predates phytoestrogens90, it is interesting to speculate that dSTACs mimic oestradiol because they may have been derived from the same polyphenolic backbone (see Fig. 1a,b), thus enabling them to co-opt sirtuin signalling through steroid receptors. Interestingly, ecdysteroid receptor activation by 20-hydroxyecdysone can also be modulated by dSTACs92, suggesting a common mechanism of signal transduction. Crucially, dSTACs are unable to activate the androgen, glucocorticoid, progesterone, and mineralocorticoid receptors. This selectivity appears to be due to the fact that ligands of these steroid receptors have a ketone group at their C-3 positions whereas ER agonists have a hydroxyl moiety. Both the number and positions of these hydroxyl groups determine ER-binding specificity20,90,93,94 (see Fig. 1a,b) which may partly explain why dSTACs preferentially activate the ER. In contrast and coincident with the loss of the ER in nematodes90, dSTACs are imperfect mimics of dafachronic acids which are structurally more similar to phytosterols than to phytoestrogens; this may partly explain why they could only residually activate DAF-12 in the absence of hormone. Hence dSTACs appear to be more specific to the ER than to other steroid receptors, indicating a primordial ligand-receptor pairing that persists in extant taxa. This selectivity may provide a basis to use them as templates to design functionalized and improved ER subtype-selective modulators95,96; e.g. ipriflavone is a synthetic derivative of daidzein used to prevent postmenopausal osteoporosis97,98. Resveratrol has also been modified or oligomerized into higher order molecular variants that bind differentially to ERα and regulate gene sets similar to those induced by oestradiol99,100. Interestingly the potencies of these oligomers directly correlate with the number of resveratrol repeats, leading to a spectrum of signalling outputs including tumour suppression, anti-inflammatory and lipid-lowering mechanisms101,102,103. Some synthetic forms have also been shown to be more bioactive than resveratrol in extending worm lifespan104 but whether that is mediated by Sir-2.1 is not known. It may therefore be possible to synthesize novel stilbenoids that more potently activate SIRT1-ER signalling than parental resveratrol.

In conclusion, the results shown here suggest that in addition to allosteric activation8, dSTACs such as resveratrol also ligand-independently enhance sirtuin coactivation of steroid receptors apparently by mimicking sex steroid hormones. Such exploitative mimicry and co-optation of sirtuin signalling could be especially important for hormone-independent ER coactivation in non-gonadal tissues most prone to ageing-related functional decline e.g. the skeletal muscle, brain and bone or in conditions where endogenous oestrogen synthesis may be inadequate e.g. the menopause. Hence this report may provide a new mechanistic insight into how dSTACs extend healthspan, and a basis to reimagine their potential as oestrogen substitutes to ameliorate or delay the onset of MARDs and to improve healthy ageing.