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.

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.

Heat shock proteins 27, 60, 70, 90alpha, and 20S proteasome in on-pump versus off-pump coronary artery bypass graft patients.

BACKGROUND: The secretion of heat shock protein (HSP) 27, HSP60, HSP70, HSP90alpha, 20S proteasome, and their correlations to proinflammatory cytokine interleukin-6 is unknown in patients undergoing on-pump versus off-pump coronary artery bypass graft (CABG) operation. METHODS: Forty patients were included in this explorative study (on- versus off-pump CABG, each n = 20). Serum samples were obtained before and 30 minutes, 60 minutes, and 24 hours after CABG operation. Enzyme-linked immunosorbent assay technique was utilized to determine soluble HSP27, 60, 70, and 90alpha, 20S proteasome, and levels of interleukin-6. RESULTS: Serum levels of HSP are increased in patients undergoing on-pump CABG operation as compared with off-pump CABG technique. These differences were highly significant for HSP27, 70, and 90alpha at 60 minutes after initiation of cardiopulmonary bypass (all, p < 0.001). Concentrations of soluble 20S proteasome were increased 24 hours after operation in on- and off-pump CABG patients (p < 0.001) and correlated significantly with the serum content of HSP 27, 70, and 90alpha at 60 minutes after initiation of cardiopulmonary bypass (p < 0.001). No correlation was found when comparing interleukin-6 levels with intravascular leakage of HSP and 20S proteasome after CABG operation. CONCLUSIONS: We conclude from our data that the innate immune system is activated owing to spillage of known immune modulatory and apoptosis-associated proteins after CABG operation.

Apoptosis-specific activation markers in on- versus off-pump coronary artery bypass graft (CABG) patients.

BACKGROUND: The relation of epithelial/endothelial apoptosis and secretion of death-inducing receptors (DIR) in comparison to vascular adhesion molecules is not known in patients undergoing the On- versus Off-pump coronary artery bypass graft (CABG) procedure. METHODS: 30 patients were prospectively included in the study (On- vs. Off-pump CABG, each n = 15). Serum samples were obtained prior to, and 30 minutes, 60 minutes and 24 hours after CABG operation. ELISA was utilized to detect caspase-cleaved cytokeratin-18 (CK18) by means of M30 antibody, soluble VCAM-1, soluble ICAM-1, and soluble DIR TNFR-1 and CD95. RESULTS: Soluble caspase-cleaved CK18 was increased and leveled to initial values at 24 hrs. sICAM-1 showed a significant decrease at 30 minutes and 60 minutes in comparison to preoperative values. sTNFR-1/sCD95 showed a rise that was not significant to preoperative values. CONCLUSION: These results indicate for the first time that epithelial/endothelial apoptosis is occurring in patients undergoing bypass operation, irrespective of the CABG procedure selected.

Soluble ST2 protein in cardiac surgery: a possible negative feedback loop to prevent uncontrolled inflammatory reactions.

BACKGROUND: Recent reports have demonstrated that cardiopulmonary bypass (CBP) utilization leads to a TH2 cytokine bias in patients undergoing coronary artery bypass grafting (CABG) operation. The relation of soluble ST2 and secretion of IL-10, markers of TH2 T-cell activation, and IL-13 in relation to immunoglobulin isotope production is not known in patients undergoing On- versus Off-pump (CABG) procedure. METHODS: 30 patients were prospectively included in the study (On- vs Off-pump CABG, each n = 15). Serum samples were obtained prior to, and 30 min, 60 min and 24hrs after operation. ELISA was utilized to detect sST2 and IL-10, IL-13 and immunoglobulin isotype production. RESULTS: In both cohorts we could demonstrate a significant rise of ST2 24 hours after the CABG procedure. In the On-pump group ST2 levels (pg/ml) before the operation, at 30 and 60 minutes and after 24 hours were 115.3 +/- 25, 71.2 +/- 15, 114.1 +/- 26 and 4231.9 +/- 520, respectively. In the Off-pump group they were 200.3 +/- 109, 91.2 +/- 20, 137 +/- 29 and 4144.9 +/- 488 (both, p < 0.0001, p < 0.0001, respectively). IL-10 (pg/ml) levels rose from preoperative values of 6.2 +/- 1.6 in the On-pump group and 7.91 +/- 1.8 in the Off-pump group to 33.14 +/- 8.7 and 13.72 +/- 3 after 60 minutes (p 0.0189, p 0.0397, respectively). IL-13 levels and immunoglobulin production did not change significantly within the study period irrespective of the operation procedure used. CONCLUSION: In conclusion, our results demonstrate that sST2 and IL-10, markers of TH2 cytokine producing cells, are increased in CABG operation, irrespective of the procedure selected, and settles a longstanding controversy concerning the shift from Th1 to Th2 cells.