Magdolna Szántó

Poly(ADP-ribose) polymerase-2 depletion reduces doxorubicin-induced damage through SIRT1 induction

AIMS Doxorubicin (DOX) is widely used in cytostatic treatments, although it may cause cardiovascular dysfunction as a side effect. DOX treatment leads to enhanced free radical production that in turn causes DNA strand breakage culminating in poly(ADP-ribose) polymerase (PARP) activation and mitochondrial and cellular dysfunction. DNA nicks can activate numerous enzymes, such as PARP-2. Depletion of PARP-2 has been shown to result in a protective phenotype against free radical-mediated diseases, suggesting similar properties in the case of DOX-induced vascular damage. METHODS AND RESULTS PARP-2(+/+) and PARP-2(-/-) mice and aortic smooth muscle (MOVAS) cells were treated with DOX (25 mg/kg or 3 μM, respectively). Aortas were harvested 2-day post-treatment while MOVAS cells were treated with DOX for 7 hours. Aortas from PARP-2(-/-) mice displayed partial protection against DOX toxicity, and the protection depended on the conservation of smooth muscle but not on the conservation of endothelial function. DOX treatment evoked free radical production, DNA breakage and PARP activation. Importantly, depletion of PARP-2 did not quench any of these phenomena, suggesting an alternative mechanism. Depletion of PARP-2 prevented DOX-induced mitochondrial dysfunction through SIRT1 activation. Genetic deletion of PARP-2 resulted in the induction of the SIRT1 promoter and consequently increased SIRT1 expression both in aortas and in MOVAS cells. SIRT1 activation enhanced mitochondrial biogenesis, which provided protection against DOX-induced mitochondrial damage. CONCLUSION Our data identify PARP-2 as a mediator of DOX toxicity by regulating vascular SIRT1 activity and mitochondrial biogenesis. Moreover, to the best of our knowledge, this is the first report of SIRT1 as a protective factor in the vasculature upon oxidative stress.

Glycogen phosphorylase inhibitor N-(3,5-dimethyl-Benzoyl)-N'-(β-D-glucopyranosyl)urea improves glucose tolerance under normoglycemic and diabetic conditions and rearranges hepatic metabolism.

Glycogen phosphorylase (GP) catalyzes the breakdown of glycogen and largely contributes to hepatic glucose production making GP inhibition an attractive target to modulate glucose levels in diabetes. Hereby we present the metabolic effects of a novel, potent, glucose-based GP inhibitor (KB228) tested in vitro and in vivo under normoglycemic and diabetic conditions. KB228 administration enhanced glucose sensitivity in chow-fed and obese, diabetic mice that was a result of higher hepatic glucose uptake. Besides improved glucose sensitivity, we have observed further unexpected metabolic rearrangements. KB228 administration increased oxygen consumption that was probably due to the overexpression of uncoupling protein-2 (UCP2) that was observed in animal and cellular models. Furthermore, KB228 treatment induced mammalian target of rapamycin complex 2 (mTORC2) in mice. Our data demonstrate that glucose based GP inhibitors are capable of reducing glucose levels in mice under normo and hyperglycemic conditions. Moreover, these GP inhibitors induce accommodation in addition to GP inhibition - such as enhanced mitochondrial oxidation and mTORC2 signaling – to cope with the glucose influx and increased glycogen deposition in the cells, however the molecular mechanism of accommodation is unexplored.

Deletion of PARP-2 induces hepatic cholesterol accumulation and decrease in HDL levels

Poly(ADP-ribose) polymerase-2 (PARP-2) is acknowledged as a DNA repair enzyme. However, recent investigations have attributed unique roles to PARP-2 in metabolic regulation in the liver. We assessed changes in hepatic lipid homeostasis upon the deletion of PARP-2 and found that cholesterol levels were higher in PARP-2(-/-) mice as compared to wild-type littermates. To uncover the molecular background, we analyzed changes in steady-state mRNA levels upon the knockdown of PARP-2 in HepG2 cells and in murine liver that revealed higher expression of sterol-regulatory element binding protein (SREBP)-1 dependent genes. We demonstrated that PARP-2 is a suppressor of the SREBP1 promoter, and the suppression of the SREBP1 gene depends on the enzymatic activation of PARP-2. Consequently, the knockdown of PARP-2 enhances SREBP1 expression that in turn induces the genes driven by SREBP1 culminating in higher hepatic cholesterol content. We did not detect hypercholesterolemia, higher fecal cholesterol content or increase in serum LDL, although serum HDL levels decreased in the PARP-2(-/-) mice. In cells and mice where PARP-2 was deleted we observed decreased ABCA1 mRNA and protein expression that is probably linked to lower HDL levels. In our current study we show that PARP-2 impacts on hepatic and systemic cholesterol homeostasis. Furthermore, the depletion of PARP-2 leads to lower HDL levels which represent a risk factor to cardiovascular diseases.

Genetic Ablation of PARP-1 Protects Against Oxazolone-Induced Contact Hypersensitivity by Modulating Oxidative Stress

Contact hypersensitivity (CHS) reaction is a form of delayed-type of hypersensitivity caused by contact allergens such as oxazolone (OXA). In previous studies it has been shown that poly(ADP-ribose) polymerase (PARP) inhibition reduces the extent of inflammation in CHS. We aimed to shed light on the molecular events causing the protective effect of PARP inhibitors. PARP-1 and -2 knockout mice were sensitized by abdominal delivery of OXA, and a week later CHS was induced by applying OXA on the ears of the mice. PARP-1(-/-) mice were protected against OXA-induced CHS in contrast to PARP-2(-/-) mice. In PARP-1(-/-) mice, neutrophil infiltration was reduced in line with the suppressed expression of proinflammatory cytokines, cell adhesion factors, and matrix metalloproteinase-9, which is likely because of impaired activation of NF-κB p65 and activating transcription factor-2, the two redox-sensitive transcription factors. Moreover, reduced nitrosative and oxidative stress was observed under inflammatory conditions in the PARP-1(-/-) mice when compared with PARP-1(+/+). In conclusion, PARP-1 activation is necessary for proinflammatory gene expression through which PARP-1 enhances neutrophil infiltration and hence oxidative/nitrosative stress, forming a vicious circle, and further aggravating the inflammatory process. Our data identify PARP-1 as a possible target in CHS.

Combined Treatment of MCF-7 Cells with AICAR and Methotrexate, Arrests Cell Cycle and Reverses Warburg Metabolism through AMP-Activated Protein Kinase (AMPK) and FOXO1

Cancer cells are characterized by metabolic alterations, namely, depressed mitochondrial oxidation, enhanced glycolysis and pentose phosphate shunt flux to support rapid cell growth, which is called the Warburg effect. In our study we assessed the metabolic consequences of a joint treatment of MCF-7 breast cancer cells with AICAR, an inducer of AMP-activated kinase (AMPK) jointly with methotrexate (MTX), a folate-analog antimetabolite that blunts de novo nucleotide synthesis. MCF7 cells, a model of breast cancer cells, were resistant to the individual application of AICAR or MTX, however combined treatment of AICAR and MTX reduced cell proliferation. Prolonged joint application of AICAR and MTX induced AMPK and consequently enhanced mitochondrial oxidation and reduced the rate of glycolysis. These metabolic changes suggest an anti-Warburg rearrangement of metabolism that led to the block of the G1/S and the G2/M transition slowing down cell cycle. The slowdown of cell proliferation was abolished when mitotropic transcription factors, PGC-1α, PGC-1β or FOXO1 were silenced. In human breast cancers higher expression of AMPKα and FOXO1 extended survival. AICAR and MTX exerts similar additive antiproliferative effect on other breast cancer cell lines, such as SKBR and 4T1 cells, too. Our data not only underline the importance of Warburg metabolism in breast cancer cells but nominate the AICAR+MTX combination as a potential cytostatic regime blunting Warburg metabolism. Furthermore, we suggest the targeting of AMPK and FOXO1 to combat breast cancer.

AMP-Activated Kinase (AMPK) Activation by AICAR in Human White Adipocytes Derived from Pericardial White Adipose Tissue Stem Cells Induces a Partial Beige-Like Phenotype.

Beige adipocytes are special cells situated in the white adipose tissue. Beige adipocytes, lacking thermogenic cues, morphologically look quite similar to regular white adipocytes, but with a markedly different response to adrenalin. White adipocytes respond to adrenergic stimuli by enhancing lipolysis, while in beige adipocytes adrenalin induces mitochondrial biogenesis too. A key step in the differentiation and function of beige adipocytes is the deacetylation of peroxisome proliferator-activated receptor (PPARγ) by SIRT1 and the consequent mitochondrial biogenesis. AMP-activated protein kinase (AMPK) is an upstream activator of SIRT1, therefore we set out to investigate the role of AMPK in beige adipocyte differentiation using human adipose-derived mesenchymal stem cells (hADMSCs) from pericardial adipose tissue. hADMSCs were differentiated to white and beige adipocytes and the differentiation medium of the white adipocytes was supplemented with 100 μM [(2R,3S,4R,5R)-5-(4-Carbamoyl-5-aminoimidazol-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl dihydrogen phosphate (AICAR), a known activator of AMPK. The activation of AMPK with AICAR led to the appearance of beige-like morphological properties in differentiated white adipocytes. Namely, smaller lipid droplets appeared in AICAR-treated white adipocytes in a similar fashion as in beige cells. Moreover, in AICAR-treated white adipocytes the mitochondrial network was more fused than in white adipocytes; a fused mitochondrial system was characteristic to beige adipocytes. Despite the morphological similarities between AICAR-treated white adipocytes and beige cells, functionally AICAR-treated white adipocytes were similar to white adipocytes. We were unable to detect increases in basal or cAMP-induced oxygen consumption rate (a marker of mitochondrial biogenesis) when comparing control and AICAR-treated white adipocytes. Similarly, markers of beige adipocytes such as TBX1, UCP1, CIDEA, PRDM16 and TMEM26 remained the same when comparing control and AICAR-treated white adipocytes. Our data point out that in human pericardial hADMSCs the role of AMPK activation in controlling beige differentiation is restricted to morphological features, but not to actual metabolic changes.

Poly(ADP) ribose polymerase-1 ablation alters eicosanoid and docosanoid signaling and metabolism in a murine model of contact hypersensitivity.

Poly(ADP‑ribose) polymerase (PARP)‑1 is a pro‑inflammatory protein. The inhibition of PARP‑1 reduces the activity of numerous pro‑inflammatory transcription factors, which results in the reduced production of pro‑inflammatory cytokines, chemokines, matrix metalloproteinases and inducible nitric oxide synthase, culminating in reduced inflammation of the skin and other organs. The aim of the present study was to investigate the effects of the deletion of PARP‑1 expression on polyunsaturated fatty acids (PUFA), and PUFA metabolite composition, in mice under control conditions or undergoing an oxazolone (OXA)‑induced contact hypersensitivity reaction (CHS). CHS was elicited using OXA in both the PARP‑1+/+ and PARP‑1/ mice, and the concentration of PUFAs and PUFA metabolites in the diseased skin were assessed using lipidomics experiments. The levels of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were shown to be increased in the PARP‑1/ mice, as compared with the control, unsensitized PARP‑1+/+ mice. In addition, higher expression levels of fatty acid binding protein 7 (FABP7) were detected in the PARP‑1/ mice. FABP7 is considered to be a specific carrier of DHA and EPA. Furthermore, the levels of the metabolites of DHA and EPA (considered mainly as anti‑inflammatory or pro‑resolving factors) were higher, as compared with the metabolites of arachidonic acid (considered mainly pro‑inflammatory), both in the unsensitized control and OXA‑sensitized PARP‑1/ mice. The results of the present study suggest that the genetic deletion of PARP‑1 may affect the PUFA‑homeostasis of the skin, resulting in an anti‑inflammatory milieu, including increased DHA and EPA levels, and DHA and EPA metabolite levels. This may be an important component of the anti‑inflammatory action of PARP‑1 inhibition.

Poly(ADP-ribose) polymerase-2 is a lipid-modulated modulator of muscular lipid homeostasis

There is a growing body of evidence that poly(ADP-ribose) polymerase-2 (PARP2), although originally described as a DNA repair protein, has a widespread role as a metabolic regulator. We show that the ablation of PARP2 induced characteristic changes in the lipidome. The silencing of PARP2 induced the expression of sterol regulatory element-binding protein-1 and -2 and initiated de novo cholesterol biosynthesis in skeletal muscle. Increased muscular cholesterol was shunted to muscular biosynthesis of dihydrotestosterone, an anabolic steroid. Thus, skeletal muscle fibers in PARP2-/- mice were stronger compared to those of their wild-type littermates. In addition, we detected changes in the dynamics of the cell membrane, suggesting that lipidome changes also affect the biophysical characteristics of the cell membrane. In in silico and wet chemistry studies, we identified lipid species that can decrease the expression of PARP2 and potentially phenocopy the genetic abruption of PARP2, including artificial steroids. In view of these observations, we propose a new role for PARP2 as a lipid-modulated regulator of lipid metabolism.