Resveratrol and Sirtuins

Low calorie diet leads in many organisms to retardation of the aging processes and to longer life. The molecular mechanisms, at least in higher organisms, are still a matter of debate. However, there are good points to be made for a hypothesis that the principles are not so fundamentally different in all organisms.

Limiting organisms with a low calorie supply activates a specific set gene of genes that leads to longer life for the individual cell, and one of the effects is that DNA repair processes operate more efficiently than in cells living in gluttony. The exciting is that this effect apparently can be mimicked by certain low molecular plant natural products, e.g. some flavonoids (e.g. quercetin) and most efficiently: resveratrol (life extension by up to 70% in yeast!). The beneficial effects of calorie restriction have been shown in all types of organisms, including rhesus monkey, i.e. mammals not too far away from Homo sapiens. Resveratrol indeed had some comparable effects in mice.  Naturally, it is not that easy to prove such beneficial effects of resveratrol, like life span extension, with humans, but the hopes are high.

Resveratrol has only been found in plants.  However, it is not present in most of our important crops. Actually, the presence of resveratrol in many wines (especially red wines!) may well provide the explanation for the "French paradox", i.e. that the French have less heart problems than for example North Americans, despite living on a high-fat diet and with high cholesterol levels.

The explanations given for yeast may also help explaining reports that resveratrol protects against heart disease, raises the "good" HDL cholesterol, inhibits blood clots and stops viral replication.

(Resveratrol and other derivatives from grapes (cis- and trans-forms, with the trans-form more abundant than the cis-form) and the other major stilbene components, pterostilbene (dimethylated resveratrol), piceid (resveratrol glucoside), epsilon-viniferin (a resveratrol dimer), and alpha-viniferin (a resveratrol trimer) were first discovered as phytoalexins in leaves and skin. particular resveratrol, its glucoside piceid, and pterostilbene were found in all tissues investigated. Others were first identified in cell suspension cultures, e.g. piceatannol and its glucoside (astringin) ,delta-viniferin and pallidol).

Enzymes involved in resveratrol synthesis:

cDNAs for resveratrol synthase (RS), the key enzyme in resveratrol biosynthesis.

The functional analysis with recombinant enzymes showed that 4-coumaroyl-CoA is the preferred substrate, as would be expected from the abundance of resveratrol. piceatannol and astringin are probably synthesized from caffeoyl-CoA, the starter molecule containing already two vicinal hydroxyl groups. in humans resveratrol can be hydroxylated to piceatannol by cytochrome P450 CYP1B1.

RS is expressed in all plant tissues, and is induced by a variety of stress conditions, particularly localization in the berries, expression during ripening, wilting, and UV-treatment and the induction by UV which could lead to a substantial increase in resveratrol yield.

The synthesis of pterostilbene requires an O-methyltransferase (OMT), and a cDNA for such a protein has been described. Interestingly, the transgenic co-expression of RS and this OMT in tobacco led to pterostilbene, indicating that the OMT carries out both methylations.

The formation of piceid and astringin requires a glucosyltransferase, and a cDNA for such protein has been described. the analysis of a recombinant protein showed that it is poly-functional: it glucosylates resveratrol, flavonoids, and coumarins at higher pH, and hydroxybenzoic acids and hydroxycinnamic acids at a lower pH.

Pinosylvin is a stilbene closely related to resveratrol: just replace the 4-coumaroyl-CoA in the biosynthesis by cinnamoyl-CoA , and carry out the standard stilbene synthase reaction. Cinnamoyl-CoA is simply 4-coumaroyl-CoA without the hydroxyl group (-OH) at the aromatic ring system. This stilbene also has interesting medical potential, but not so much work focused on it.

Some references:

1) Austin,M.B.; Bowman,M.E.; Ferrer,J.-L.; Schröder,J.; Noel,J.P.: An aldol switch discovered in stilbene synthases mediates cyclization specificity of type III polyketide synthases. Chemistry & Biology 11, 1179-1194 (2004)

2) Schröder, G., Brown, J.W.S. and Schröder, J.: Molecular analysis of resveratrol synthase: cDNA, genomic clones and relationship with chalcone synthase. European Journal of Biochemistry 172, 161-169 (1988).

  

3) Fliegmann, J., Schröder, G., Schanz, S., Britsch, L. and Schröder, J.: Molecular analysis of chalcone and dihydropinosylvin synthase from Scots pine (Pinus sylvestris), and differential regulation of these and related enzyme activities in stressed plants. Plant Molecular Biology 18, 489-503 (1992).

  

4) Schanz, S., Schröder, G. and Schröder, J.: Stilbene synthase from Scots pine (Pinus sylvestris). FEBS Letters 313, 71-74 (1992).

Sirtuins:

Sirtuins were first described in yeast, under various names, and even the early results showed already  that the proteins had important functions in metabolism, cell cycle and mating in these 'simple' organisms (The protein discovered first was named Sir2, the abbreviation of 'silent information regulator'). The human sirtuins have attracted considerable interest because of the immense importance of sirtuins for health and longevity that emerged in the last few years. I will attempt to give a very simple overview of these proteins and their enzymatic activities in humans.

SIRT1 (Enzyme function: DAC , Localization: Nucleus (Euchromatin), cytoplasm)

SIRT2 (Enzyme function: DAC and ART , Localization: cytoplasm)

SIRT3 (Enzyme function: DAC and ART , Localization: Mitochondria)

SIRT4 (Enzyme function: ART, Localization: Mitochondria)

SIRT5 (Enzyme function: DAC, Localization: Mitochondria)

SIRT6 (Enzyme function: DAC and ART, Localization: Nucleus(Heterochromatin))

SIRT7 (Enzyme function: Not known, Localization: Nucleus (Nucleolus))

Enzyme function: DAC=NAD+ Dependent Deacetylase

                             ART= Mono-ADP-Ribosyl Transferase

Sirtuins Activators:

A publication by Howitz et al. (2003) certainly was a key event in the development of the resveratrol story: they studied the capacity of small natural molecules to stimulate sirtuin activities. The screen with recombinant human SIRT1 led to the identification of several molecules, and resveratrol turned to be the most active one. Others, but with lower activity, were another stilbene (piceatannol), two chalcones (butein, isoliquiritigenin), and two flavonols (fisetin, quercetin)

These assays measured the deacetylation of acetylated peptides conjugated to a nonphysiological fluorophore. There are good reasons question whether the effects of these natural products were really caused by direct affects on the SIRT1 protein. The in vivo effects with yeast cells looked much better: butein, fisetin, and resveratrol increased average life span by 31%, 55%, and 70%, respectively

The next important advance was the demonstration that resveratrol improved health and survival of mammals (mice) on a high-calorie diet.

Few concerns with the application of resveratrol to humans: Bioavailability, Tissue specificity and down regulation of p53.

More pronounced research would lead to Sirtuin specific activator (resveratrol) devoid of any concerns with pharmaceutical industries like bioavailability, metabolism problems and multiple targets.

Some References:

1)Alcain, F. J., Villalba, J. M., 2009a. Sirtuin activators. Expert Opinion on Therapeutic Patents 19, 403-414.

2) Kopp, P., 1998. Resveratrol, a phytoestrogen found in red wine. A possible explanation for the conundrum of the 'French paradox'? Eur. J. Endocrinol. 138, 619-620.

3) Constant, J., 1997. Alcohol, ischemic heart disease, and the French paradox. Coronary Artery Disease 8, 645-649.

4)Soleas, G. J., Diamandis, E. P., Goldberg, D. M., 1997. Resveratrol: a molecule whose time has come? And gone? Clinical Biochemistry 30, 91-113.

5) Soleas, G.J., Diamandis, E.P. and Goldberg, D.M., 1997. Wine as a biological fluid: history, production, and role in disease prevention. J. Clin. Lab. Analysis 11, 287-313.

6) Rimm, E.B., Klatsky, A., Grobbee, D. and Stampfer, M.J., 1996. Review of moderate alcohol consumption and reduced risk of coronary heart disease - is the effect due to beer, wine, or spirits. British Medical Journal 312, 731-736.

Sirtuin Inhibitors:

SIRT1: Apart from all the beneficial effects, SIRT1 activation has at least one property with a Janus head, i.e. it can be good or bad. That is the SIRT1 catalyzed deacetylation of the tumour suppressor protein p53 (Vaziri et al., 2001) that leads to inactivation of p53.

That is good for normal cells because it increases their survival and prolongs life span, but it is not so desirable for tumorous cells because it might stimulate the survival and replication of tumour cells.

Re-activating p53, i.e. inhibiting SIRT1 instead of stimulating it, could trigger strong apoptosis in tumour cells, and thus could eliminate tumours.

SIRT2: This protein is predominantly in the cytoplasm, with highest expression in the brain; it co-localizes with microtubules, deacetylates tubulin and plays a role in the cell cycle. It is noteworthy that p53 and histones H3 and H4 are additional substrates of SIRT2, suggesting a broader role (Heltweg et al., 2006), and thus SIRT2 activation could potentially as undesirable as SIRT1 activation. On the other hand, SIRT2 also deacetylates the transcription regulator FOXO3a (Wang et al., 2007), activating it, and in consequence promotes cell death under severe stress.

SIRT2 it is a potential tumour suppressor, and it is actually severely reduced in a large number of brain tumour cell lines.

Some of the sirtuins inhibitors are Splitomycin and Derivatives, Sirtinol, AGK2, Tenovin, Gambinol and Salermide.

Some references:

Alcain, F. J., Villalba, J. M., 2009b. Sirtuin inhibitors. Expert Opinion on Therapeutic Patents 19, 283-294.

All these beneficial effects have been described, but it also should be noted that the conclusions are from studies with model systems.

But honestly, how could it be otherwise? And who wants to wait for 125 years to prove longevity achieved by daily doses of resveratrol?

Would it not be nice to counteract all the bad effects by taking a simple pill? Without actually having to change eating habits? Is there really an easy way to a long healthy life?

Biotivia LLC is the leading manufacturer of resveratrol rich tested and approved supplements. Please visit the website for more information www.biotivia.com.