Rosario Casale

Poor glycaemic control in type 2 diabetes patients reduces endothelial progenitor cell number by influencing SIRT1 signalling via platelet-activating factor receptor activation

Aims/hypothesisDownregulation of levels of endothelial progenitor cells (EPCs) during in-vitro short-term exposure to high glucose concentrations relates to reduced activity of silent information regulator 1 (SIRT1) and increased synthesis of platelet-activating factor (PAF). We investigated the possible relationship between PAF and SIRT1 pathways in EPCs during altered glucose homeostasis.MethodsSIRT1 and PAF receptor (PAF-R) levels were determined by western blot, RT-PCR and confocal laser-scanning microscopy. In-vivo experiments were performed on 48 type 2 diabetic patients (25 with poor glycaemic control and 23 with good glycaemic control) and 20 control individuals. In-vitro experiments with the PAF-R antagonist CV3988 were performed on EPCs isolated from leucocyte-rich buffy coat of healthy human donors.ResultsDecreased SIRT1 protein levels were observed in EPCs from type 2 diabetic patients compared with control individuals (p < 0.01). Notably, the SIRT1 level was consistently lower in patients with poor glycaemic control than in those with good glycaemic control (p < 0.01). Diabetic patients also showed an upregulation of PAF-Rs; this response occurred to a greater extent in individuals with poor glycaemic control than in those with good glycaemic control. In-vitro experiments confirmed that EPCs respond to PAF stimulation with decreased SIRT1 protein and SIRT1 mRNA levels. Moreover, reduction of SIRT1 levels and activity were abolished by CV3988.Conclusions/interpretationThese findings unveil a link between PAF and SIRT1 pathways in EPCs that contributes to the deleterious effect of hyperglycaemia on the functional properties of EPCs, crucial in diabetes and peripheral vascular complications.

Sirtuins in vascular diseases: Emerging roles and therapeutic potential.

Silent information regulator-2 (Sir-2) proteins, or sirtuins, are a highly conserved protein family of histone deacetylases that promote longevity by mediating many of the beneficial effects of calorie restriction which extends life span and reduces the incidence of cancer, cardiovascular disease (CVD), and diabetes. Here, we review the role of sirtuins (SIRT1-7) in vascular homeostasis and diseases by providing an update on the latest knowledge about their roles in endothelial damage and vascular repair mechanisms. Among all sirtuins, in the light of the numerous functions reported on SIRT1 in the vascular system, herein we discuss its roles not only in the control of endothelial cells (EC) functionality but also in other cell types beyond EC, including endothelial progenitor cells (EPC), smooth muscle cells (SMC), and immune cells. Furthermore, we also provide an update on the growing field of compounds under clinical evaluation for the modulation of SIRT1 which, at the state of the art, represents the most promising target for the development of novel drugs against CVD, especially when concomitant with type 2 diabetes.

An uncommon redox behavior sheds light on the cellular antioxidant properties of ergothioneine.

Ergothioneine (ESH), an aromatic thiol occurring in the human diet and which accumulates in particular cells, is believed to act as an antioxidant. However, its redox mechanism remains unclear and it does not seem to provide any advantage compared to other antioxidants, such as alkylthiols, which are better reducing agents and generally present in cells at higher levels. Here, we investigated by ESI-MS the products of ESH oxidation produced by neutrophils during oxidative burst and, to further elucidate ESH redox behavior, we also analyzed the oxidation products of the reaction of ESH with hypochlorite in cell-free solutions. Indeed, neutrophils are the main source of hypochlorite in humans. Furthermore, we also tested other biologically relevant oxidants, such as peroxynitrite and hydrogen peroxide. Our results indicate that treatment of human neutrophils with phorbol 12-myristate 13-acetate in the presence of ESH leads to a remarkable production of the sulfonated form (ESO3H), a compound never described before, and hercynine (EH), the desulfurated form of ESH. Similar results were obtained when ESH was subjected to cell-free oxidation in the presence of hypochlorite, as well as hydrogen peroxide or peroxynitrite. Furthermore, when the disulfide of ESH was reacted with those oxidants, we found that it was also oxidized, with production of EH and ESO3H, whose amount was dependent on the oxidant strength. These data reveal a unique ESH redox behavior, entirely different from that of alkylthiols, and suggest a mechanism, so far overlooked, through which ESH performs its antioxidant action in cells.

Ergothioneine oxidation in the protection against high-glucose induced endothelial senescence: Involvement of SIRT1 and SIRT6

Ergothioneine (Egt), the betaine of 2-mercapto-L-histidine, is a dietary antioxidant protecting against many diseases, including cardiovascular disease (CVD), through a redox mechanism different from alkylthiols. Here, experiments were designed to evaluate the mechanisms underlying the beneficial effect of Egt against hyperglycaemia-induced senescence in endothelial cells. To this end, cells were incubated with increasing concentrations of Egt (0.01-1.00mM) for 12h followed by incubation for 48h with high-glucose (25mM). Cell evaluation indicated that viability was not affected by mM concentrations of Egt and that the high-glucose cytotoxicity was prevented with the highest efficacy at 0.5mM Egt. The cytoprotective effect of Egt was paralleled by reduced ROS production, cell senescence, and, interestingly, the formation of hercynine (EH), a betaine we recently found to be produced during the Egt oxidation pathway. Notably, the Egt beneficial effect was exerted through the upregulation of sirtuin 1 (SIRT1) and sirtuin 6 (SIRT6) expression and the downregulation of p66Shc and NF-κB. SIRT1 activity inhibition and SIRT6 gene silencing by small interfering RNA abolished the protective effect of Egt against the high-glucose-induced endothelial senescence. These data provide the first evidence of the Egt ability to interfere with endothelial senescence linked to hyperglycaemia through the regulation of SIRT1 and SIRT6 signaling, thus further strengthening the already assessed role of these two histone deacetylases in type 2 diabetes.

Citrus genus plants contain N-methylated tryptamine derivatives and their 5-hydroxylated forms.

The occurrence and distribution in Citrus genus plants of N-methylated derivatives of tryptamine and their 5-hydroxylated forms are reported. Tryptamine, N-methyltryptamine, N,N-dimethyltryptamine, N,N,N-trimethyltryptamine, 5-hydroxytryptamine (serotonin), 5-hydroxy-N-methyltryptamine, 5-hydroxy-N,N-dimethyltryptamine (bufotenine), and 5-hydroxy-N,N,N-trimethyltryptamine (bufotenidine) were quantitated by LC-ESI-MS/MS. Leaves of all citrus plants examined contained N,N,N-trimethyltryptamine, a compound that we first discovered in the bergamot plant. Interestingly, we also found out that all plants examined contained 5-hydroxy-N,N-dimethyltryptamine and 5-hydroxy-N,N,N-trimethyltryptamine, compounds never described so far in the Citrus genus. As N,N,N-trimethyltryptamine and 5-hydroxy-N,N,N-trimethyltryptamine possess nicotine-like activity by exerting their action on acetylcholine receptors, it is conceivable that both represent the arrival point of a biosynthetic pathway aimed to provide Citrus plants with chemical defense against aggressors. This hypothesis is supported by our finding that leaves and seeds, which are more frequently attacked by biotic agents, are the parts of the plant where the highest levels of those compounds were found.

Homostachydrine (pipecolic acid betaine) as authentication marker of roasted blends of Coffea arabica and Coffea canephora (Robusta) beans.

The occurrence of pipecolic acid betaine (homostachydrine) and its biosynthetic precursor N-methylpipecolic acid was detected for the first time in green coffee beans of Robusta and Arabica species. The analyses were conducted by HPLC-ESI tandem mass spectrometry and the metabolites identified by product ion spectra and comparison with authentic standards. N-methylpipecolic acid was found at similar levels in green coffee beans of Robusta and Arabica, whereas a noticeable difference of homostachydrine content was observed between the two green coffee bean species. Interestingly, homostachydrine content was found to be unaffected by coffee bean roasting treatment because of a noticeable heat stability, a feature that makes this compound a candidate marker to determine the content of Robusta and Arabica species in roasted coffee blends. To this end, a number of certified pure Arabica and Robusta green beans were analyzed for their homostachydrine content. Results showed that homostachydrine content was 1.5±0.5mg/kg in Arabica beans and 31.0±10.0mg/kg in Robusta beans. Finally, to further support the suitability of homostachydrine as quality marker of roasted blends of Arabica and Robusta coffee beans, commercial samples of roasted ground coffee blends were analyzed and the correspondence between the derived percentages of Arabica and Robusta beans with those declared on packages by manufacturers was verified.

N-Methylated Derivatives of Tyramine in Citrus Genus Plants: Identification of N,N,N-Trimethyltyramine (Candicine)

The distribution of tyramine and its methylated derivatives, N-methyltyramine and N,N-dimethyltyramine, was investigated in tissue parts (leaves and fruits) of several plants of Citrus genus by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In the course of our study we discovered the occurrence of N,N,N-trimethyltyramine in all citrus plants examined. This quaternary ammonium compound, known to act in animals as a neurotoxin, was recognized and characterized by mass spectrometric analysis. The substance, never described before in the Citrus genus, is also known as candicine or maltoxin. Results indicate that N,N,N-trimethyltyramine is consistently expressed in leaves of clementine, bitter orange, and lemon. Conversely, low levels were found in the leaves of orange, mandarin, chinotto (Citrus myrtifolia), bergamot, citron, and pomelo. In the edible part of the fruits, N,N,N-trimethyltyramine was found at trace levels.

Betaines and related ammonium compounds in chestnut (Castanea sativa Mill.)

Chestnut fruits, being poor of simple sugars and consisting mainly of fibers and starch, are among the constituents of Mediterranean diet. While numerous studies report on content of proteins and amino acids in chestnut, no one has appeared so far on betaines, an important class of nitrogen compounds ubiquitous in plants for their protective action in response to abiotic stress. In this study, we analyzed by HPLC-ESI-tandem mass spectrometry, in fruits and flours of varieties of chestnut cultivated in Italy, the composition of betaines and ammonium compounds intermediates of their biosynthesis. Besides the parent amino acids, the compounds quantified were choline, glycerophosphocholine, phosphocholine, glycine betaine, N-methylproline, proline betaine (stachydrine), β-alanine betaine, 4-guanidinobutyric acid, trigonelline, N,N,N-trimethyllysine. Interestingly, some uncommon derivatives of pipecolic acid, such as N-methylpipecolic acid, 4-hydroxypipecolic acid and 4-hydroxy-N-methylpipecolic acid were identified for the first time in chestnut samples and characterized by MS(n) tandem mass spectrometry.

Determination of Homoarginine, Arginine, NMMA, ADMA, and SDMA in Biological Samples by HPLC-ESI-Mass Spectrometry

NG,NG-dimethyl-l-arginine (ADMA) and NG-methyl-l-arginine (NMMA) are endogenous inhibitors of nitric oxide synthase (NOS). In contrast, NG,N′G-dimethyl-Larginine (SDMA) possesses only a weak inhibitory potency towards neuronal NOS and it is known to limit nitric oxide (NO) production by competing with l-arginine for cellular uptake. The inhibition of NOS is associated with endothelial dysfunction in cardiovascular diseases as well in chronic renal failure. l-Homoarginine (HArg), a structural analog of l-arginine (Arg), is an alternative but less efficient substrate for NOS. Besides, it inhibits arginase, leading to an increased availability of l-arginine for NOS to produce NO. However, its relation with cardiovascular disease remains unclear. To date, several analytical methods for the quantitative determination of Arg, HArg, NMMA, AMDA, and SDMA in biological samples have been described. Here, we present a simple, fast, and accurate HPLC-ESI-MS/MS method which allows both the simultaneous determination and quantification of these compounds without needing derivatization, and the possibility to easily modulate the chromatographic separation between HArg and NMMA (or between SDMA and ADMA). Data on biological samples revealed the feasibility of the method, the minimal sample preparation, and the fast run time which make this method very suitable and accurate for analysis in the basic and clinical settings.

Serotonin 5-O-β-Glucoside and Its N-Methylated Forms in Citrus Genus Plants

Citrus genus is characterized by a specific presence of indole metabolites deriving from the N-methylation of tryptamine and its hydroxylated form, 5-hydroxytryptamine (serotonin), which are likely involved in plant defense mechanisms. In this study, we identified for the first time the occurrence in Citrus plants of serotonin 5-O-β-glucoside and all its N-methylated derivatives, that is, N-methylserotonin 5-O-β-glucoside, N,N-dimethylserotonin (bufotenine) 5-O-β-glucoside, and N,N,N-trimethylserotonin (bufotenidine) 5-O-β-glucoside. The identification of the glucosylated compounds was based on mass spectrometric studies, hydrolysis by glucosidase, and in some cases, comparison to authentic compounds. Beside leaves, the distribution of the glucosylated forms and their aglycones in some Citrus species was evaluated in flavedo, albedo, juice, and seeds. The simultaneous presence of serotonin and its N-methylated derivatives, together with the corresponding glucosylated forms, is consistent with the occurrence of a metabolic pathway, specific for Citrus, aimed at potentiating the defensive response to biotic stress through the optimization of the production and use of the most toxic of such metabolites.