Protective effect of nicotinamide on high glucose/palmitate-induced glucolipotoxicity to INS-1 beta cells is attributed to its inhibitory activity to sirtuins

on July 23rd, 2018 by admin | Comments Off on Protective effect of nicotinamide on high glucose/palmitate-induced glucolipotoxicity to INS-1 beta cells is attributed to its inhibitory activity to sirtuins

These present study was undertaken to elucidate the protective mechanism of NAM in HG/PA-induced glucolipotoxicity to beta cells. As NAM’s protective mechanism in HG/PA-induced beta cell death was reported to be due to its antioxidant activity, the effect of other anti-oxidants on HG/PA-induced INS-1 cell death was ini- tially investigated. NAC and reduced GSH did not protected HG/PA- induced cell death. The mitochondria-targeted anti-oxidant Mito- TEMPOL did not have any protective effect on the HG/PA-induced INS-1 cell death, either. As NAD+ supplementation was reported to contribute to NAM’s protective effect on damaged neuron cells occurred by various insults, effect of NAD+ supplementation on HG/PA-induced INS-1 cell death was investigated. Direct supple- mentation of NAD+ or indirect supplementation through Bioavailable NMN or kynurenine did not show any protective effect on HG/PA-induced INS-1 cell death. In particular, inhibition or knockdown of NAMPT as an enzyme of NAD+ salvage pathway did not augment HG/PA-induced INS-1 cell death. Furthermore, inhibition or knockdown of PARP as a critical NAD+-consuming enzyme did not protect HG/PA-induced cell INS-1 death. On the other hand, overexpression of SIRT3 and SIRT4 augmented HG/PA-induced cell death whereas knockdown of SIRT3 and SIRT4 protected the death. These data suggest that selective activation of mito- chondrial sirtuins such as Sirt3 and 4 may be involved in HG/ PA-induced beta cell death and that NAM may have a protective role in HG/PA-induced glucolipotoxicity as an inhibitor of the SIRTs. Treatment with NAM in the presence of HG/PA restored HG/PA-induced reduction of survival phospho-Akt signal, but re- duced damaged ER stress signals such as phospho-JNK and CHOP.

Fig. 6. Inhibitory effect of nicotinamide on HG/PA-induced ER stress. INS-1 cells were treated with 20 mM NAM in the presence of HG/PA for the indicated times. Levels of each signal protein were determined by western blot with different antibodies. The band intensity was determined by densitometric analysis using a one-dimensional Quantity OneÒ 1D image analysis system. Maximum intensity was converted to 1 and the relative intensity on the basis of maximum was then calculated. P-Cas 3; pro- caspase 3, C-Cas 3; cleaved caspase 3, Cal; calnexin, C-Cal; cleaved calnexin. ⁄P < 0.05; ⁄⁄P < 0.01, vs. band intensity of NAM-untreated INS-1 cells.

It has been reported that treatment with cytokines including IL- 1b, INF-c, and TNF-a stimulates production of NO through signal activation of NFjB in beta cells, with NO subsequently participat- ing in cell death through DNA damage [41,42]. Thus, NAM as an inhibitor of PARP is supposed to prevent cytokine-induced beta cell death. However, presently, NAM treatment did not bestow any protective effect on cytokine-induced INS-1 cell death. A recent re- port that cytokine-induced beta-cell damage is not mediated by the hyper-activation of PARP-1 supports the postulated non-pro- tective role of NAM in cytokine-induced INS-1 cell death [43]. On the other hand, NAM had been reported to protect both HG/PA-in- duced and STZ-induced INS-1 cell death [30,34]. However, the death mechanism of STZ-induced beta cell death seems to be quite different from that of HG/PA-induced death. Presently, oxidative stress induced by ROS and hyper-activation of PARP were critically involved in STZ-induced INS-1 cell death, since all anti-oxidants and PARP inhibitors demonstrated significant protective effect on STZ-induced INS-1 cell death whereas the anti-oxidants and inhib- itors were not protective against HG/PA-induced INS-1 cell death.

Our results are consistent with early reports that hyper-activation of PARP plays a key role in STZ-induced beta cell death [44,45]. However, the data that supplementation of NAD+ itself did not have any protective effect on STZ-induced INS-1 cell death argues Okamoto’s early hypothesis that consumption of NAD+ and subse- quent depletion of energy through PARP hyper-activation is critical in STZ-induced beta cell death [44]. It remains to be studied whether PAR polymers produced by PARP activation are involved in STZ-induced beta cell death, because a toxic role of PAR poly- mers independent of NAD+ depletion has recently been reported in PARP activation-induced cell death [26]. While sirtinol as a Sirt inhibitor did not influence STZ-induced INS-1 cell death, resvera- trol as a SIRT activator showed a protective effect on the STZ-in- duced death (Supplemental 5C). A recent report that resveratrol treatment prevented STZ-induced beta cell apoptosis in a rat mod- el supports the protective role in SIRT activation in STZ-induced cytotoxicity [46].

Production of ROS and subsequent oxidative damage by ROS has been suggested to be main cause for HG-induced glucotoxicity, FFA-induced lipotoxicity, and HG/PA-induced glucolipotoxcity [6,9,47]. Recently, mitochondrial ROS was reported to be a key mediator for beta cell glucolipotoxicity [48]. However, according to another study, ROS and oxidative stress were not involved in HG/PA-induced beta cell glucolipotoxiicty since various antioxi- dants and ROS scavenging agents, including NAC, amino guanidine, lipoic acid, and vitamin E failed to curtail HG/PA-induced apoptotic death [49]. Although it remains to be studied why there is this dis- crepancy, experimental conditions and different cell lines may be involved in this difference. Our data also demonstrate that cellular and mitochondrial ROS are not likely to be involved in HG/PA-in- duced INS-1 cell death since all antioxidants such as NAC, GSH or Mito-TEMPOL did not protect the death.

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