Bioaccumulation of resveratrol metabolites in myocardial tissue is dose-time dependent and related to cardiac hemodynamics in diabetic rats

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Abstract

Background and aims

Trans-resveratrol (RSV) is a natural compound occurring in different foods and plants, which in vivo is rapidly conjugated with glucuronic acid and sulfate. Despite its demonstrated cardioprotective activity, the bioaccumulation of RSV or its metabolites in cardiac tissue is still unknown.

Methods and results

Diabetic rats were randomized to 1, 3 or 6 weeks of RSV treatment at two different doses (1 or 5 mg/kg/day). A dose and time-dependent accumulation was observed, with no detectable levels of RSV metabolites found in heart tissues after 1 week and significant concentrations of RSV-3-sulfate and RSV-3-glucuronide after 6 weeks of treatment (0.05 nmol/g of tissue and 0.01 nmol/g of tissue, respectively). Tissue accumulation of RSV metabolites was accompanied by an improvement of cardiac function in long-term diabetes, when myocardial morpho-functional damage is more evident, with an almost complete recovery of all hemodynamic parameters, at the highest RSV dose.

Conclusion

Even if a higher concentration of RSV in tissues cannot be ruled out after constant oral administration, an accumulation coherent with what is usually evaluated in cell based mechanistic studies is largely unattainable and the RSV unconjugated form would not be present in this paradigm. The current investigation provides data on myocardial tissue concentrations of RSV metabolites, after short/medium term RSV treatment. This knowledge constitutes a basic requirement for future studies aimed at reliably defining the molecular pathways underlying RSV-mediated cardioprotective effects and opens up new perspectives for research focused on testing phenolic compounds as adjuvants in degenerative heart diseases.

Introduction

Resveratrol (RSV: trans-3,5,4′-trihydroxystilbene) is a natural compound occurring in different foods and plants, such as red wine, grapes, peanuts, pistachios and berries [1].

Over recent decades, a number of studies have linked RSV consumption to beneficial and protective effects on human health. In particular, RSV has been described as an antioxidant, modulator of lipoprotein metabolism, inhibitor of platelet aggregation, vasorelaxant and cancer chemopreventive [2], [3]. It is also considered a potentially effective therapeutic adjuvant to prevent the occurrence of cardiovascular complications in both type 1 and type 2 diabetes [4], [5], [6], [7]. In addition to antioxidant, antiapoptotic/anti-inflammatory effects, RSV may possess several other cardio-protective roles due to a wide range of direct and indirect target molecules mediating its biological actions, including the modulation of enzymes, cell signaling pathways and gene expression [5], [7], [8], [9], [10], [11], [12].

Despite a wide body of experimental evidence indicating that RSV can have positive effects on the diabetic heart, leading to functional and structural recovery, proof of efficacy of RSV in humans has been more elusive and to date there is a paucity of convincing human studies, pertaining to cardiovascular disease prevention or treatment [5], [13]. The current evidence base does not justify the therapeutic administration of RSV to humans, beyond that which can be achieved via dietary sources [13]. Major challenges remain, concerning the safety and efficacy of chronic RSV administration as well as optimal doses, due to the well-known hormetic actions of the compound, which demonstrates cardio-protective properties at lower doses and detrimental effects at higher doses [14]. When considering the potential future use of RSV in the clinical setting, to prevent and/or treat cardiovascular complications of diabetes such as cardiomyopathy, a number of issues remain to be addressed. An expanding body of preclinical evidence has been largely focused on characterizing the pharmacokinetics and metabolism of RSV. However, the effects of time and doses on cardiac tissue distribution as well as excretion of RSV and its main structurally-related metabolites remain limited.

Although several studies have already demonstrated the ability of RSV to protect against several diseases [15], some questions arise due to its low bioavailability [2], due to rapid metabolism, following oral absorption [16]. In animals and humans, at enterocyte and hepatocyte levels, RSV is rapidly metabolized [16] and conjugated with glucuronic acid and sulfate due to the action of UDP-glucuronosyl-transferases and sulfotransferases [17] and these are the major circulating forms of this molecule. However, the biological activity of these metabolites formed in humans and experimental animals needs to be elucidated fully. There is a lack of studies investigating organ, fluid and tissue distribution of RSV and its metabolites with the exception of some work in normal rodents [18], [19], [20], [21], rabbits [22] and pigs [23], [24].

Particularly, little is known about the heart tissue distribution of RSV and its active metabolites after chronic treatment with different doses of the compound [20]. This preliminary knowledge is required to perform “in vitro” mechanistic studies aimed at reliably defining the molecular pathways underlying the “in vivo” measured effects of the compound, and the possible combined effects of chronic exposure to RSV with other well characterized bioactive compounds. Indeed, the majority of “in vitro” available data have been obtained by employing the un-conjugated form of RSV, at concentrations which largely exceed those that can be reached in vivo, at both plasma and tissue levels [25].

In the present study we specifically addressed these issues, in a rat model of type-1 diabetes induced by Streptozotocin injection [26], with a view to defining, for the first time, the distribution of RSV and its active metabolites in myocardial tissue after short and medium-term treatment with the compound, at different low doses. Dose-time dependent effects of RSV administration on glycemic control and functional properties of the diabetic heart have also been assessed.

Section snippets

Methods

The investigation was approved by the Veterinary Animal Care and Use Committee of the University of Parma-Italy and conforms to the National Ethical Guidelines of the Italian Ministry of Health (Permit number: 41/2009-B) and the Guide for the Care and Use of Laboratory Animals (National Institute of Health, Bethesda, MD,USA, revised 1996). All surgery was performed under ketamine chloride anesthesia, and all efforts were made to minimize suffering.

Tissue and urine distribution study of RSV and active metabolites

An example of UHPLC traces and mass spectra of RSV sulfate detected in heart samples and a comparison with the pure standard is reported (Fig. 1).

Concerning RSV sulfate recovered in rat hearts, no dose of RSV led to bioaccumulation in myocardial tissue after the first week of treatment. Starting from 3 weeks of RSV administration, low concentrations of RSV metabolites were recovered in the heart of animals treated with 5 mg/kg/day (Fig. 2A). After 6 weeks of treatment the RSV sulfate myocardial

Discussion

Despite the vast literature on RSV cardioprotective action, little is known about the heart tissue distribution of RSV and its active metabolites after chronic treatment with different doses of the compound. To our knowledge, this is the first study where RSV metabolite accumulation in diabetic rat heart after a short-term (1 week) and medium-term exposure (3–6 weeks) to different doses of RSV has been investigated in relation to specific functional beneficial effects.

RSV is a stilbene with a

Acknowledgments

This study has been partly supported by “Progetto AGER” (grant no. 2011-0283) and by the National Institute for Cardiovascular Research (INRC).

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    These authors equally contributed to this work.

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