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Raising cellular NAD+ levels in human cells by nicotinic acid supplementation : biological consequences related to PARP-1 mediated reactions

Raising cellular NAD+ levels in human cells by nicotinic acid supplementation : biological consequences related to PARP-1 mediated reactions

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WEIDELE, Kathrin, 2012. Raising cellular NAD+ levels in human cells by nicotinic acid supplementation : biological consequences related to PARP-1 mediated reactions [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Weidele2012Raisi-19383, title={Raising cellular NAD+ levels in human cells by nicotinic acid supplementation : biological consequences related to PARP-1 mediated reactions}, year={2012}, author={Weidele, Kathrin}, address={Konstanz}, school={Universität Konstanz} }

Raising cellular NAD+ levels in human cells by nicotinic acid supplementation : biological consequences related to PARP-1 mediated reactions 2012 The cellular response to DNA damage includes a number of mechanisms to detect and repair various types of DNA damage in order to preserve the integrity and stability of the genome. One reaction involved is the poly(ADP-ribosyl)ation (PARylation) of proteins, a modification performed by nuclear poly(ADP-ribose)polymerase-1 or -2 (PARP-1 and PARP-2) immediately after DNA damage infliction. PARPs covalently attach ADP-ribose units in a sequential fashion to target proteins including themselves, synthesizing a negatively charged polymer by using nicotinamide adenine dinucleotide (NAD<sup>+</sup>) as substrate. According to the level of DNA damage and intracellular NAD+ status, the most active enzyme PARP-1 and its product PAR mediate the recruitment of DNA repair factors to sites of lesions, facilitate DNA repair and thus maintain genomic integrity under conditions of moderate stress. In this scenario a tolerable amount of total cellular NAD+ is used for polymer synthesis. In contrast, the critical expenditure of NAD<sup>+</sup> due to massive activation of PARP-1 under severe stress conditions can lead to cell death thus influencing deleterious or health-enhancing processes, as is apparent in inflammatory diseases or neurodegenerative disorders.<br /><br />One important parameter determining the cellular response to stresses is the level of available NAD<sup>+</sup>, which is crucial for adequate PAR synthesis and other NAD<sup>+</sup> dependent processes, as the energy metabolism or sirtuin functions. Sirtuins can act as deacetylases in terms of response to cellular damage and to metabolic imbalances thus regulating fundamental processes as well.<br /><br />In order to analyse the biological consequences of elevated NAD<sup>+</sup> levels in respect to PARP-1 mediated reactions and to address the question, if modulated NAD<sup>+</sup> and/or PAR levels contribute to physiological or pathophysiological outcomes, distinct end points were investigated.<br /><br />Human blood peripheral mononuclear cells (NAD<sup>+</sup>) were ex vivo supplemented with the NAD<sup>+</sup> precursor nicotinic aid (NA), which significantly raised intracellular nucleotide pools and led to an intensified PAR formation in response to genotoxic stimuli.<br /><br />It was observed that NA supplementation reduces cell death after genotoxic stress and shifts the residual fraction from necrosis to apoptosis, which is less harmful in regard to tissue integrity. To investigate, if this is a result of improved DNA repair, strand break formation and subsequent repair within the first 40 minutes was accessed. Interestingly, we observed that strand break rejoining is tightly regulated but was positively affected in NA supplemented cells under massive DNA damaging conditions and to a minor extent also under mild genotoxic stress. Furthermore, enhanced NAD<sup>+</sup> levels seem to be beneficial in context of genomic integrity, as supplemented cells displayed reduced frequencies of micronucleus formation. In addition to the impact on already mentioned cellular functions, it was monitored if higher NAD<sup>+</sup> levels favour also sirtuin activities. The SIRT-1 target p53 is acetylated in response to DNA damage. Preliminary results give evidence that the DNA damaged induced p53 acetylation is reduced in nicotinic acid supplemented cells, potentially due to increased deacetylation rate.<br /><br />In summary, increased NAD<sup>+</sup> levels by NA supplementation can be beneficial for a cellular system, most notably in context of massive damage, but also in situations of minor damage. So far no adverse effects were observed for the investigated parameters. Based on the current results, supplying nicotinic acid seems to be a valuable approach to augment NAD<sup>+</sup> levels especially in the case of acute damage or to correct suboptimal NAD<sup>+</sup> levels to prevent negative consequences, often linked with excessive or elevated PARP-1 activation. 2014-04-01T22:25:06Z eng Weidele, Kathrin Weidele, Kathrin deposit-license 2012-05-25T11:28:24Z

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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