PARP2 poly(ADP-ribosyl)ates nuclear factor erythroid 2-related factor 2 (NRF2) affecting NRF2 subcellular localization.

PARP2 is a member of the PARP enzyme family. Although, PARP2 plays role in DNA repair, it has regulatory roles in mitochondrial and lipid metabolism, it has pivotal role in bringing about the adverse effects of pharmacological PARP inhibitors. Previously, we showed that the ablation of PARP2 induces oxidative stress and, consequently, mitochondrial fragmentation. In attempt to identify the source of the reactive species we assessed the possible role of a central regulator of cellular antioxidant defense, nuclear factor erythroid 2-related factor 2 (NRF2). The silencing of PARP2 did not alter either the mRNA or the protein expression of NRF2, but changed its subcellular localization, decreasing the proportion of nuclear, active fraction of NRF2. Pharmacological inhibition of PARP2 partially restored the normal localization pattern of NRF2 and in line with that, we showed that NRF2 is PARylated that is absent in the cells in which PARP2 was silenced. Apparently, the PARylation of NRF2 by PARP2 has pivotal role in regulating the subcellular (nuclear) localization of NRF2. The silencing of PARP2 rearranged the expression of genes encoding proteins with antioxidant function, among these a subset of NRF2-dependent genes.

Methods to Assess the Role of PARPs in Regulating Mitochondrial Oxidative Function.

PARP enzymes are involved in metabolic regulation and impact on a plethora of cellular metabolic pathways, among them, mitochondrial oxidative metabolism. The detrimental effects of PARP1 overactivation upon oxidative stress on mitochondrial oxidative metabolism was discovered in 1998. Since then, there was an enormous blooming in the understanding of the interplay between PARPs and mitochondria. Mitochondrial activity can be assessed by a comprehensive set of methods that we aim to introduce here.

Nicotinamide-riboside shifts the differentiation of human primary white adipocytes to beige adipocytes impacting substrate preference and uncoupling respiration through SIRT1 activation and mitochondria-derived reactive species production.

Beige adipocytes play key roles in organismal energy and metabolic balance. In this study, we assessed whether the supplementation of human white adipocytes, differentiated from human adipose tissue-derived stem cells, with nicotinamide riboside (NR), a potent NAD + precursor, can shift differentiation to beige adipocytes (beiging). NR induced mitochondrial biogenesis and the expression of beige markers (TBX1 and UCP1) in white adipocytes demonstrating that NR can declutch beiging. NR did not induce PARP activity but supported SIRT1 induction, which plays a key role in beiging. NR induced etomoxir-resistant respiration, suggesting increases in the oxidation of carbohydrates, carbohydrate breakdown products, or amino acids. Furthermore, NR boosted oligomycin-resistant respiration corresponding to uncoupled respiration. Enhanced etomoxir and oligomycin-resistant respiration were dependent on mitochondrial reactive-species production. Taken together, NR supplementation can induce beiging and uncoupled respiration, which are beneficial for combatting metabolic diseases.

Olaparib induces browning of in vitro cultures of human primary white adipocytes.

Mitochondrial biogenesis is a key feature of energy expenditure and organismal energy balance. Genetic deletion of PARP1 or PARP2 was shown to induce mitochondrial biogenesis and energy expenditure. In line with that, PARP inhibitors were shown to induce energy expenditure in skeletal muscle. We aimed to investigate whether pharmacological inhibition of PARPs induces brown or beige adipocyte differentiation. SVF fraction of human pericardial adipose tissue was isolated and human adipose-derived mesenchymal stem cells (hADMSCs) were differentiated to white and beige adipocytes. A subset of hADMSCs were differentiated to white adipocytes in the presence of Olaparib, a potent PARP inhibitor currently in clinical use, to induce browning. Olaparib induced morphological changes (smaller lipid droplets) in white adipocytes that is a feature of brown/beige adipocytes. Furthermore, Olaparib induced mitochondrial biogenesis in white adipocytes and enhanced UCP1 expression. We showed that Olaparib treatment inhibited nuclear and cytosolic PAR formation, induced NAD+/NADH ratio and consequently boosted SIRT1 and AMPK activity and the downstream transcriptional program leading to increases in OXPHOS. Olaparib treatment did not induce the expression of beige adipocyte markers in white adipocytes, suggesting the formation of brown or brown-like adipocytes. PARP1, PARP2 and tankyrases are key players in the formation of white adipose tissue. Hereby, we show that PARP inhibition induces the transdifferentiation of white adipocytes to brown-like adipocytes suggesting that PARP activity could be a determinant of the differentiation of these adipocyte lineages.