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Wine, sirtuins and nephroprotection: Not only resveratrol
Medical Hypotheses 75 (2010) 636–638
Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Wine, sirtuins and nephroprotection: Not only resveratrol C. Mannari a,⇑, A.A.E. Bertelli b, G. Stiaccini a, L. Giovannini a a b
Department of Neuroscience, Pharmacology Section, University of Pisa, Pisa, Italy Department of Human Morphology and Biomedical Sciences, ‘‘Città Studi”, University of Milan, Milan, Italy
a r t i c l e
i n f o
Article history: Received 27 May 2010 Accepted 1 August 2010
s u m m a r y Resveratrol (RSV), a red wine component, and red wine itself exert cardio- and nephroprotective effects by modulating the Nitric Oxide system (NO). It has been shown that one of the main actions resulting from NO modulation is sirtuin regulation, especially SIRT-1 regulation. Elucidating both upstream and downstream molecular mechanisms of the SIRT-1 pathway is an open field of investigation that can explain its role not only in long-term processes, such as aging, but also in short-term processes, such as protection against ischemic damage. Our hypothesis suggests the importance of investigating compounds that are routine dietary components and do not necessarily contain RSV. Their nephroprotective activity could involve not only eNOS-dependent, but also NO-dependent but eNOS-independent mechanisms, or other molecular alternative signaling systems. Ó 2010 Elsevier Ltd. All rights reserved.
Background Epidemiological studies have repeatedly shown that moderate wine consumption is associated with a reduction in the incidence of cardiovascular and cerebrovascular events, as well as of the onset of other disorders [1–3], and is associated with longer life expectancy. Anti-oxidant activity of wine polyphenols, including the stilbene polyphenol resveratrol, is usually considered responsible for this protective activity. However, endothelial activation has also been proposed as a protective factor [4]. The mechanisms responsible for the neuro- and cardiovascular protective effects both of wine in toto [5] and of some of its components, especially Resveratrol (RSV) [6], are activation of eNOS (endothelial Nitric Oxide Synthase) and the increase in production of Nitric Oxide (NO), besides the inhibition of Endothelin 1 synthesis [7,8]. NO-dependent activity seems to be responsible for the nephroprotective effect of RSV recorded in ‘‘in vivo” and ‘‘in vitro” experiments [9,10]. Although these data are already well known, they are of interest, because they cast some doubts on the concept that natural compounds, such as those found in wine, possess only anti-oxidant activity: on the contrary, it appears that several compounds can modulate baseline cellular activity by means of complex molecular
⇑ Corresponding author. Address: Dipartimento di Neuroscienze, Sezione Farmacologia, Università di Pisa, Via Roma, 55, 56126 Pisa, Italy. Tel.: +39 0502218714; fax: +39 0502218717. E-mail address: [email protected] (C. Mannari). 0306-9877/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2010.08.004
mechanisms. For instance, two important biological features of RSV are its capacity to induce ischemic preconditioning [11,12] and to mimic the effects of calorie restriction [13,14]. Both of these activities have been studied for some time, as they offer protection against several kinds of cytotoxic insults and increase survival, making RSV act, more generally, as an anti-aging agent [15,16]. Regarding anti-aging activity, it has been established that RSV is one of the main modulators of the expression and activity of SIRT-1, a protein belonging to the Nicotinamide Adenine Dinucleotide (NAD+) dependent histone deacetylase family, which regulates gene silencing. In view of its fundamental role in the regulation of the cell cycle, which prevents premature death [17], SIRT is also called the anti-aging protein. Its deacetylase activity is regulated by the [NAD+]/[NADH] ratio; thus, NAD + acts as activator and Nicotinamide and NADH as inhibitors [18,19]. Based on what is reported by Nisoli et al. [20], SIRT 1 appears to be the key that explains the protective effects of both Calorie Restriction and the eNOS system. Indeed, the authors show that Calorie Restriction is able to activate eNOS, producing mitochondrial biogenesis associated with and dependent on an increase in the expression of SIRT 1. It is reasonable to believe that the importance of SIRT-1 rests not only on the modulation of histones H1, H3 and H4 [21,22], but also on its capacity to protect the cell system from stressful and cytotoxic effects, such as the oxidative / free radical damage associated with ischemia: SIRT-1 also deacetylates nonhistone proteins, including various transcription factors, such as p53 [23], FOXO [24] and HIF-2a [25], which regulate a variety of molecular and cellular processes.
C. Mannari et al. / Medical Hypotheses 75 (2010) 636–638
The hypothesis Although RSV has been extensively studied just because of its SIRT modulating property, it is unlikely to be the only compound of the non alcoholic fraction of wine able to modulate sirtuin expression: some experimental and epidemiological studies show that also white wine (which does not contain RSV) and some white wine compounds exert cardioprotective effects [26–30]. Non-RSV dependent SIRT-1 modulation assumes that further non – NO dependent signals exist, both upstream and downstream. HIF-2a, one of the effectors of SIRT-1 activity, could be part of this pathway. Contrary to HIF-1a, which is upregulated by hypoxia, HIF-2a is trans-activated by oxygen-independent molecular mechanisms; following its activation, HIF-2a is able to modulate the transcription of genes Sod 2, Vefg A and Epo [26]. During hypoxia acetylation of HIF-2a increases, so the presence of SIRT-1 is crucial in the modulation of HIF-2a downstream signals [26]. Also in the kidney the efficacy of protection against ischemia– reperfusion (I/R) insults afforded by RSV and other polyphenols is related mainly to the eNOS/NO system [9,10]. This does not exclude the involvement of other signaling and second messenger systems. Notwithstanding the growing interest in the role of sirtuins, little is known about their NO-independent modulation: CD 38, a multifunctional protein able to generate second messengers and to hydrolyze NAD, has proved to be able to induce SIRT-1 in the liver [31]; more recently, Hwang JT et al. [33] has reported that AMP - activated protein kinase (AMPK) strongly up-regulates SIRT-1 in skeletal muscle cells and that other natural compounds, which are found in the non alcoholic fraction of both white and red wine, are able to modulate AMPK activity: for this reason the AMPK pathway could be an upstream modulator of SIRT-1. We have therefore hypothesized that nephroprotection is due to biologically active compounds that form the phytocomplex in wine matrix and do not necessarily include RSV. Indeed, although ROS exert a major role both in aging and acute /chronic renal failure, we suggest that the long-term effects of SIRT should be distinguished from its short-term effects. The former are mainly antiaging and are due to histone deacetylation and to the regulation of complex processes, such as mitochondrial biogenesis, whereas the latter are useful in protection against acute lesions, such as renal I/R damage, and are to be ascribed mainly to the modulation of rapid molecular signals. Testing the hypothesis Based on the information available to us, we decided to assess whether three red wines, all of the same year and produced with Lambrusco red grapes, but differing in type (indicated as L1, L2, L3) and characterized very low content in RSV (Table 1), were able to modulate the baseline expression of SIRT-1; this kind of wine was considered to be of interest in view of its widespread commercialization. For this kind of study we selected renal proximal tubular cells of human origin, as these cells are susceptible to oxidant stimuli and are involved in the damage due to I/R episodes, such as those that occur after renal transplantation and acute ischemic renal failure.
637
Cells were incubated for 24 h with each of the three selected wines called L1, L2, L3 diluted 1:1000 [34] separately or with Resveratrol 200nM as SIRT-1 expression inducer (positive control). The effect of the ethanol in the wine was not considered, as the final concentration in the wells was approximately 2 mM and previous studies demonstrate that ethanol, even at the concentration of 20 mM, is not able to modulate the expression or the activity of SIRT-1 [35]. Cell lysates were run along electrophoresis gel and transferred onto PVDF membrane; thereafter, to assess SIRT-1 expression, they were incubated overnight with rabbit polyclonal anti SIRT-1 antibody (Cell Signaling Technology - USA). After addition of HRPconjugated secondary antibody, the development and acquisition of images, as well as densitometric analysis, were carried out with dedicated software (Kodak 1D 3.6). Empirical data The results reported in Fig. 1 show how incubation of the tubular cells with RSV 200nM increased SIRT-1 expression as compared to the control group (1,77 ± 0540 a.u. p < 0.05 vs control, 1,00 ± 0.096). Furthermore, also the three wines, diluted 1:1000 in the medium, induced marked SIRT-1 expression after incubation for 24 h, which was similar to that induced by Resveratrol (L1 2.05 ± 0.655; L2 1,98 ± 0461; L3 2,44 ± 0.910, p < 0.05 vs control); this finding is particularly interesting, because the final concentration of RSV in the three selected wines, after dilution, was negligible. Consequences of the hypothesis and discussion The part reported above was needed to test the hypothesis that wine poor in RSV can modulate SIRT-1 expression through the presence of other biologically active compounds. As previously reported, several SIRT-1 substrates have been identified, but little is known about the regulators of SIRT1 activity. The clarification of the SIRT-1 pathway is a field of investigation that is an opportunity to take advantage of SIRT-1 biological potential not only in the long-term, e.g. for its anti-aging activity, but also in the short-term, e.g. for protection against ischemic damage. The same importance is to be given to the identification of the natural and synthetic compounds able to interact with this signal pathway. Our preliminary data stress the importance of investigating the compounds occurring in our diet, which may be able to modulate SIRT-1 expression and activity. In conclusion, we suggest that to better understand the molecular basis on their cardio-and nephroprotective activity, natural SIRT-1 modulating compounds should be studied taking in consideration not only their eNO-dependent activity, but also their
Table 1 Analysis of the content in Resveratrol of 3 red wines, all of the same vintage and produced with Lambrusco red grapes, but of three different types (indicated as L1, L2, L3).
trans-Resveratrol cis-Resveratrol
L1 0.7 mg/L 0.2 mg/L
L2 0.3 mg/L 0.2 mg/L
L3 0.5 mg/L 0.7 mg/L
Fig. 1. Assessment of SIRT-1 expression in tubular cell lysates incubated for 24 h with RSV 200nM or one of 3 different types of wine (indicated as L1, L2, L3) diluted 1:1000.
638
C. Mannari et al. / Medical Hypotheses 75 (2010) 636–638
Fig. 2. Proposed molecular activation network for SIRT-1 induced by the non alcoholic compounds occurring in wine.
eNOS-independent actions [29] together with other signaling pathways (Fig. 2) that regulate HIF 2a or similar transcription factors [31–33].
Conflicts of interest statement None declared. References [1] Gronbaek M, Deis A, Sorensen TI, Becker U, Schnohr P, Jensen G. Mortality associated with moderate intakes of wine, Beer, or spirits. BMJ 1995;310:1165–9. [2] Streppel MT, Ocké MC, Boshuizen HC, Kok FJ, Kromhout D. Long-term wine consumption is related to cardiovascular mortality and life expectancy independently of moderate alcohol intake: the Zutphen Study. J Epidemiol Community Health 2009;63:534–40. [3] German JB, Walzem RL. The health benefits of wine. Annu Rev Nutr 2000;20:561–93. [4] Bhat KPL, Kosmeder JW, Pezzuto JM. Biological effects of resveratrol. Antioxid Redox Signal 2001;3:1041–64. [5] Szmitko PE, Verma S. Antiatherogenic potential of red wine: clinician update. Am J Physiol Heart Circ Physiol 2005;288:H2023–30. [6] Wallerath T, Poleo D, Li H, Förstermann U. Red wine increases the expression of human endothelial nitric oxide synthase: a mechanism that may contribute to its beneficial cardiovascular effects. J Am Coll Cardiol 2003;41:471–8. [7] Fitzpatrick DF, Hirschfield SL, Coffey RG. Endothelium-dependent vasorelaxing activity of wine and other grape products. Am J Physiol 1993;265:H774–8. [8] Corder R, Douthwaite JA, Lees DM, et al. Endothelin-1 synthesis reduced by red wine. Nature 2001;414:863–4. [9] Bertelli AA, Migliori M, Panichi V, et al. Resveratrol, a component of wine and grapes, in the prevention of kidney disease. Ann NY Acad Sci 2002;957:230–8. [10] Giovannini L, Migliori M, Longoni BM, et al. Resveratrol, a polyphenol found in wine, reduces ischemia reperfusion injury in rat kidneys. J Cardiovasc Pharmacol 2001;37:262–70.
[11] Tan L, Yuand JT, Guan HS. Resveratrol exerts pharmacological preconditioning by activating PGC-1alpha. Med Hypotheses 2008;71:664–7. [12] Chander V, Chopra K. Role of nitric oxide in resveratrol-induced renal protective effects of ischemic preconditioning. J Vasc Surg 2005;42:1198–205. [13] Barger JL, Kayo T, Vann JM, et al. A low dose of dietary resveratrol partially mimics caloric restriction and retards aging parameters in mice. PLoS One 2008;3:e2264. [14] Yamamoto H, Schoonjans K, Auwerx J. Sirtuin functions in health and disease. Mol Endocrinol 2007;21:1745–55. [15] Yellon DM, Downey JM. Preconditioning the myocardium: from cellular physiology to clinical cardiology. Physiol Rev 2003;83:1113–51. [16] Fontana L. The scientific basis of caloric restriction leading to longer life. Curr Opin Gastroenterol 2009;25:144–50. [17] Vaquero A. The conserved role of sirtuins in chromatin regulation. Int J Dev Biol 2009;53:303–22. [18] Lin SJ, Ford E, Haigis M, Liszt G, Guarente L. Calorie restriction extends yeast life span by lowering the level of NADH. Genes Dev 2004;18:12–6. [19] Revollo JR, Grimm AA, Imai S, The NAD. The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells. J Biol Chem 2004;279:50754–63. [20] Nisoli E, Tonello C, Cardile A, et al. Calorie restriction promotes mitochondrial biogenesis by inducing the expression of eNOS. Science 2005;310:314–7. [21] Liou GG, Tanny JC, Kruger RG, Walz T, Moazed D. Assembly of the SIR complex, its regulation by O-acetyl-ADP-ribose, a product of NAD-dependent histone deacetylation. Cell 2005;121:515–27. [22] Vaquero A, Scher M, Lee D, Erdjument-Bromage H, Tempst P, Reinberg D, et al. SirT1 interacts with histone H1 and promotes formation of facultative heterochromatin. Mol Cell 2004;16:93–105. [23] Brooks CL, Gu W. Ubiquitination, phosphorylation and acetylation: the molecular basis for p53 regulation. Curr Opin Cell Biol 2003;15:164–71. [24] Brunet A, Sweeney LB, Sturgill JF, et al. Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science 2004;303:2011–5. [25] Dioum EM, Chen R, Alexander MS, et al. Regulation of hypoxia-inducible factor 2alpha signaling by the stress-responsive deacetylase sirtuin 1. Science 2009;324:1289–93. [26] Cui J, Tosaki A, Cordis GA, et al. Cardioprotective abilities of white wine. Ann NY Acad Sci 2002;957:308–16. [27] Perrot L, Dukic S, Charpentier M, et al. Antihypertensive effect of a low molecular weight fraction (1 kDa) of champagne wine in spontaneously hypertensive rats. In Oenologie 2003:688–91. [28] Dudley JI, Lekli I, Mukherjee S, et al. Does white wine qualify for French paradox? Comparison of the cardioprotective effects of red and white wines and their constituents: resveratrol, tyrosol, and hydroxytyrosol. J Agric Food Chem 2008;56:9362–73. [29] Vauzour D, Houseman EJ, George TW, et al. Moderate Champagne consumption promotes an acute improvement in acute endothelial-independent vascular function in healthy human volunteers. Br J Nutr 2009;30:1–11. [30] Mukherjee S, Lekli I, Gurusamy N, Bertelli AA, Das DK. Expression of the longevity proteins by both red and white wines and their cardioprotective components, resveratrol, tyrosol, and hydroxytyrosol. Free Radic Biol Med. 2009;46:573–8. [31] Aksoy P, Escande C, White TA, et al. Regulation of SIRT 1 mediated NAD dependent deacetylation: a novel role for the multifunctional enzyme CD38. Biochem Biophys Res Commun 2006;349:353–9. [32] Cantó C, Gerhart-Hines Z, Feige JN, et al. AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity. Nature 2009;458:1056–60. [33] Hwang JT, Kwon DY, Yoon SH. AMP-activated protein kinase: a potential target for the diseases prevention by natural occurring polyphenols. N Biotechnol 2009;26:17–22. [34] Frankel EN, Kanner J, German JB, Parks E, Kinsella JE. Inhibition of oxidation of human low-density lipoprotein by phenolic substances in red wine. Lancet 1993;341:454–7. [35] You M, Liang X, Ajmo JM, Ness GC. Involvement of mammalian sirtuin 1 in the action of ethanol in the liver. Am J Physiol Gastrointest Liver Physiol 2008;294:G892–8.
Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Wine, sirtuins and nephroprotection: Not only resveratrol C. Mannari a,⇑, A.A.E. Bertelli b, G. Stiaccini a, L. Giovannini a a b
Department of Neuroscience, Pharmacology Section, University of Pisa, Pisa, Italy Department of Human Morphology and Biomedical Sciences, ‘‘Città Studi”, University of Milan, Milan, Italy
a r t i c l e
i n f o
Article history: Received 27 May 2010 Accepted 1 August 2010
s u m m a r y Resveratrol (RSV), a red wine component, and red wine itself exert cardio- and nephroprotective effects by modulating the Nitric Oxide system (NO). It has been shown that one of the main actions resulting from NO modulation is sirtuin regulation, especially SIRT-1 regulation. Elucidating both upstream and downstream molecular mechanisms of the SIRT-1 pathway is an open field of investigation that can explain its role not only in long-term processes, such as aging, but also in short-term processes, such as protection against ischemic damage. Our hypothesis suggests the importance of investigating compounds that are routine dietary components and do not necessarily contain RSV. Their nephroprotective activity could involve not only eNOS-dependent, but also NO-dependent but eNOS-independent mechanisms, or other molecular alternative signaling systems. Ó 2010 Elsevier Ltd. All rights reserved.
Background Epidemiological studies have repeatedly shown that moderate wine consumption is associated with a reduction in the incidence of cardiovascular and cerebrovascular events, as well as of the onset of other disorders [1–3], and is associated with longer life expectancy. Anti-oxidant activity of wine polyphenols, including the stilbene polyphenol resveratrol, is usually considered responsible for this protective activity. However, endothelial activation has also been proposed as a protective factor [4]. The mechanisms responsible for the neuro- and cardiovascular protective effects both of wine in toto [5] and of some of its components, especially Resveratrol (RSV) [6], are activation of eNOS (endothelial Nitric Oxide Synthase) and the increase in production of Nitric Oxide (NO), besides the inhibition of Endothelin 1 synthesis [7,8]. NO-dependent activity seems to be responsible for the nephroprotective effect of RSV recorded in ‘‘in vivo” and ‘‘in vitro” experiments [9,10]. Although these data are already well known, they are of interest, because they cast some doubts on the concept that natural compounds, such as those found in wine, possess only anti-oxidant activity: on the contrary, it appears that several compounds can modulate baseline cellular activity by means of complex molecular
⇑ Corresponding author. Address: Dipartimento di Neuroscienze, Sezione Farmacologia, Università di Pisa, Via Roma, 55, 56126 Pisa, Italy. Tel.: +39 0502218714; fax: +39 0502218717. E-mail address: [email protected] (C. Mannari). 0306-9877/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2010.08.004
mechanisms. For instance, two important biological features of RSV are its capacity to induce ischemic preconditioning [11,12] and to mimic the effects of calorie restriction [13,14]. Both of these activities have been studied for some time, as they offer protection against several kinds of cytotoxic insults and increase survival, making RSV act, more generally, as an anti-aging agent [15,16]. Regarding anti-aging activity, it has been established that RSV is one of the main modulators of the expression and activity of SIRT-1, a protein belonging to the Nicotinamide Adenine Dinucleotide (NAD+) dependent histone deacetylase family, which regulates gene silencing. In view of its fundamental role in the regulation of the cell cycle, which prevents premature death [17], SIRT is also called the anti-aging protein. Its deacetylase activity is regulated by the [NAD+]/[NADH] ratio; thus, NAD + acts as activator and Nicotinamide and NADH as inhibitors [18,19]. Based on what is reported by Nisoli et al. [20], SIRT 1 appears to be the key that explains the protective effects of both Calorie Restriction and the eNOS system. Indeed, the authors show that Calorie Restriction is able to activate eNOS, producing mitochondrial biogenesis associated with and dependent on an increase in the expression of SIRT 1. It is reasonable to believe that the importance of SIRT-1 rests not only on the modulation of histones H1, H3 and H4 [21,22], but also on its capacity to protect the cell system from stressful and cytotoxic effects, such as the oxidative / free radical damage associated with ischemia: SIRT-1 also deacetylates nonhistone proteins, including various transcription factors, such as p53 [23], FOXO [24] and HIF-2a [25], which regulate a variety of molecular and cellular processes.
C. Mannari et al. / Medical Hypotheses 75 (2010) 636–638
The hypothesis Although RSV has been extensively studied just because of its SIRT modulating property, it is unlikely to be the only compound of the non alcoholic fraction of wine able to modulate sirtuin expression: some experimental and epidemiological studies show that also white wine (which does not contain RSV) and some white wine compounds exert cardioprotective effects [26–30]. Non-RSV dependent SIRT-1 modulation assumes that further non – NO dependent signals exist, both upstream and downstream. HIF-2a, one of the effectors of SIRT-1 activity, could be part of this pathway. Contrary to HIF-1a, which is upregulated by hypoxia, HIF-2a is trans-activated by oxygen-independent molecular mechanisms; following its activation, HIF-2a is able to modulate the transcription of genes Sod 2, Vefg A and Epo [26]. During hypoxia acetylation of HIF-2a increases, so the presence of SIRT-1 is crucial in the modulation of HIF-2a downstream signals [26]. Also in the kidney the efficacy of protection against ischemia– reperfusion (I/R) insults afforded by RSV and other polyphenols is related mainly to the eNOS/NO system [9,10]. This does not exclude the involvement of other signaling and second messenger systems. Notwithstanding the growing interest in the role of sirtuins, little is known about their NO-independent modulation: CD 38, a multifunctional protein able to generate second messengers and to hydrolyze NAD, has proved to be able to induce SIRT-1 in the liver [31]; more recently, Hwang JT et al. [33] has reported that AMP - activated protein kinase (AMPK) strongly up-regulates SIRT-1 in skeletal muscle cells and that other natural compounds, which are found in the non alcoholic fraction of both white and red wine, are able to modulate AMPK activity: for this reason the AMPK pathway could be an upstream modulator of SIRT-1. We have therefore hypothesized that nephroprotection is due to biologically active compounds that form the phytocomplex in wine matrix and do not necessarily include RSV. Indeed, although ROS exert a major role both in aging and acute /chronic renal failure, we suggest that the long-term effects of SIRT should be distinguished from its short-term effects. The former are mainly antiaging and are due to histone deacetylation and to the regulation of complex processes, such as mitochondrial biogenesis, whereas the latter are useful in protection against acute lesions, such as renal I/R damage, and are to be ascribed mainly to the modulation of rapid molecular signals. Testing the hypothesis Based on the information available to us, we decided to assess whether three red wines, all of the same year and produced with Lambrusco red grapes, but differing in type (indicated as L1, L2, L3) and characterized very low content in RSV (Table 1), were able to modulate the baseline expression of SIRT-1; this kind of wine was considered to be of interest in view of its widespread commercialization. For this kind of study we selected renal proximal tubular cells of human origin, as these cells are susceptible to oxidant stimuli and are involved in the damage due to I/R episodes, such as those that occur after renal transplantation and acute ischemic renal failure.
637
Cells were incubated for 24 h with each of the three selected wines called L1, L2, L3 diluted 1:1000 [34] separately or with Resveratrol 200nM as SIRT-1 expression inducer (positive control). The effect of the ethanol in the wine was not considered, as the final concentration in the wells was approximately 2 mM and previous studies demonstrate that ethanol, even at the concentration of 20 mM, is not able to modulate the expression or the activity of SIRT-1 [35]. Cell lysates were run along electrophoresis gel and transferred onto PVDF membrane; thereafter, to assess SIRT-1 expression, they were incubated overnight with rabbit polyclonal anti SIRT-1 antibody (Cell Signaling Technology - USA). After addition of HRPconjugated secondary antibody, the development and acquisition of images, as well as densitometric analysis, were carried out with dedicated software (Kodak 1D 3.6). Empirical data The results reported in Fig. 1 show how incubation of the tubular cells with RSV 200nM increased SIRT-1 expression as compared to the control group (1,77 ± 0540 a.u. p < 0.05 vs control, 1,00 ± 0.096). Furthermore, also the three wines, diluted 1:1000 in the medium, induced marked SIRT-1 expression after incubation for 24 h, which was similar to that induced by Resveratrol (L1 2.05 ± 0.655; L2 1,98 ± 0461; L3 2,44 ± 0.910, p < 0.05 vs control); this finding is particularly interesting, because the final concentration of RSV in the three selected wines, after dilution, was negligible. Consequences of the hypothesis and discussion The part reported above was needed to test the hypothesis that wine poor in RSV can modulate SIRT-1 expression through the presence of other biologically active compounds. As previously reported, several SIRT-1 substrates have been identified, but little is known about the regulators of SIRT1 activity. The clarification of the SIRT-1 pathway is a field of investigation that is an opportunity to take advantage of SIRT-1 biological potential not only in the long-term, e.g. for its anti-aging activity, but also in the short-term, e.g. for protection against ischemic damage. The same importance is to be given to the identification of the natural and synthetic compounds able to interact with this signal pathway. Our preliminary data stress the importance of investigating the compounds occurring in our diet, which may be able to modulate SIRT-1 expression and activity. In conclusion, we suggest that to better understand the molecular basis on their cardio-and nephroprotective activity, natural SIRT-1 modulating compounds should be studied taking in consideration not only their eNO-dependent activity, but also their
Table 1 Analysis of the content in Resveratrol of 3 red wines, all of the same vintage and produced with Lambrusco red grapes, but of three different types (indicated as L1, L2, L3).
trans-Resveratrol cis-Resveratrol
L1 0.7 mg/L 0.2 mg/L
L2 0.3 mg/L 0.2 mg/L
L3 0.5 mg/L 0.7 mg/L
Fig. 1. Assessment of SIRT-1 expression in tubular cell lysates incubated for 24 h with RSV 200nM or one of 3 different types of wine (indicated as L1, L2, L3) diluted 1:1000.
638
C. Mannari et al. / Medical Hypotheses 75 (2010) 636–638
Fig. 2. Proposed molecular activation network for SIRT-1 induced by the non alcoholic compounds occurring in wine.
eNOS-independent actions [29] together with other signaling pathways (Fig. 2) that regulate HIF 2a or similar transcription factors [31–33].
Conflicts of interest statement None declared. References [1] Gronbaek M, Deis A, Sorensen TI, Becker U, Schnohr P, Jensen G. Mortality associated with moderate intakes of wine, Beer, or spirits. BMJ 1995;310:1165–9. [2] Streppel MT, Ocké MC, Boshuizen HC, Kok FJ, Kromhout D. Long-term wine consumption is related to cardiovascular mortality and life expectancy independently of moderate alcohol intake: the Zutphen Study. J Epidemiol Community Health 2009;63:534–40. [3] German JB, Walzem RL. The health benefits of wine. Annu Rev Nutr 2000;20:561–93. [4] Bhat KPL, Kosmeder JW, Pezzuto JM. Biological effects of resveratrol. Antioxid Redox Signal 2001;3:1041–64. [5] Szmitko PE, Verma S. Antiatherogenic potential of red wine: clinician update. Am J Physiol Heart Circ Physiol 2005;288:H2023–30. [6] Wallerath T, Poleo D, Li H, Förstermann U. Red wine increases the expression of human endothelial nitric oxide synthase: a mechanism that may contribute to its beneficial cardiovascular effects. J Am Coll Cardiol 2003;41:471–8. [7] Fitzpatrick DF, Hirschfield SL, Coffey RG. Endothelium-dependent vasorelaxing activity of wine and other grape products. Am J Physiol 1993;265:H774–8. [8] Corder R, Douthwaite JA, Lees DM, et al. Endothelin-1 synthesis reduced by red wine. Nature 2001;414:863–4. [9] Bertelli AA, Migliori M, Panichi V, et al. Resveratrol, a component of wine and grapes, in the prevention of kidney disease. Ann NY Acad Sci 2002;957:230–8. [10] Giovannini L, Migliori M, Longoni BM, et al. Resveratrol, a polyphenol found in wine, reduces ischemia reperfusion injury in rat kidneys. J Cardiovasc Pharmacol 2001;37:262–70.
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