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

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Loss of insulin-producing beta cells was thought to be a patho- genic cause of type 2 diabetes as well as type 1 diabetes [1]. While destruction of beta cells by treatment with streptozotocin (STZ)1 or cytokines such as interleukin (IL)-1b, tumor necrosis factor-alpha (TNF-a), and interferon-gamma (IFN-c) has been studied to eluci- date the cellular mechanism of beta cell loss in type 1 diabetes, death by exposure of beta cells on high concentration of glucose and satu- rated fatty acid has been studied to elucidate the mechanism of beta loss in type 2 diabetes. Following its uptake into the beta cells, STZ can be split into methylnitrosourea. Owing to its alkylating proper- ties of methylnitrosourea, DNAs in beta cells are broken and poly (ADP-ribose) polymerase (PARP) as a DNA repairing enzyme is then activated. The activation of PARP gives rise to depletion of cellular nicotinamide adenine dinucleotide (NAD+) and ATP [2] and the depletion of cellular energy is then thought to be a main contributor to STZ-induced beta cell death. In addition, c-Jun NH2-terminal ki- nase (JNK) activated through reactive oxygen species (ROS) has been reported to be involved in STZ-induced beta cell death [3]. Treatment of beta cells with cytotoxic cytokines activates nuclear factor-kappa B (NF-jB) signals, which lead to the production of nitric oxide (NO) through induction of inducible nitric oxide syntase (iNOS) and ulti- mately leads to the induction of endoplasmic reticulum (ER) stress [4]. The execution of cytokine-induced b-cell death was thought to occur through activation of the stress-activated protein kinases C- Jun N-terminal kinase (JNK) and also by combined induction of death-promoting genes but reduction of survival genes [5]. On the other hand, long-term treatment with high glucose/palmitate (HG/ PA) was demonstrated to induce beta cell death, and so was termed glucolipotoxcity. In fact, glucolipotoxicity is thought to be an aug- mented form of fatty acid-induced lipotoxicity [6]. Ceramide-in- duced nitric oxide (NO) has been suggested as a mediator for glucolipotoxcity [7,8]. Chronic oxidative stress was reported to be in- volved in glucose-induced glucotoxicity and palmitate-induced lipo- toxicity [9]. Recently, endoplasmic reticulum (ER) stress induced by calcium depletion in the ER rumen and subsequent activation of death signals such as JNK and C/EBP homologous protein (CHOP), have been reported to be a critical mediator for palmitate-induced lipotoxicity [10,11].

Nicotinamide dinucleotide (NAD+) is synthesized using a de- novo pathway from dietary tryptophan through kynurenine and quinolinic acid intermediates or using a NAD+ salvage pathway from nicotinamide (NAM), an amide form of niacin (vitamin B3) [12]. While NAM is converted to nicotinamide mononucleotide (NMN) through nicotinamide phosphoribosyl transferase (NAMPT), NMN is subsequently synthesized to NAD+ through nic- otinamide mononucleotide adenyltransferase (NMNAT) [13]. NAD+ is usually consumed by NAD+-dependent deacetylases such as various sirtuins (SIRT1, 2, 3, 5 and 7) or NAD+-dependent ADP- ribosylases including SIRT4, 6 and PARP. The NAD+-consuming en- zymes produce deacetylated proteins or transfer ADP-ribose to tar- get proteins with production of NAM as a byproduct [14]. On the other hand, NAD+-consuming enzymes can be selectively inhibited by NAM itself since the enzymes contain a NAM-product site [15]. NAD+ not only serves as an adenosine donor but also as an oxi- dized form of NADH, which is a high energy source produced through glycolysis and tricarboxylic acid (TCA) cycle. Thus, deple- tion of NAD+ causes reduction of cellular energy through inhibition of ATP-producing metabolism such as glycolysis, TCA cycle, and oxidative phosphorylation [16,17]. On the other hand, supplemen- tation of NAM can be crucial in maintaining cellular energy state as it is a precursor of NAD+ or inhibitor of NAD+-consuming enzymes. Nutritional status is reported to control the level of NAD+ through modulation of NAD+-synthetic and -consuming enzymes [12,18]. Many reports have documented that NAM protects cells against various toxic insults such as anoxia, glucose deprivation, and expo- sure of free radicals, excitotoxic glutamate or genotoxic agents [19–23]. It is generally accepted that NAM offer cytoptotection through the maintenance of cellular NAD+ [24,25]. In particular, NAM is able to provide protective activity through inhibition of PARP, since the massive activation of PARP induces cytotoxic poly (ADP-ribose) polymer (PAR) accumulation [26]. Recently, SIRT inhibition was suggested to contribute to NAM-induced protection against injured cells because it can also preserve cellular NAD+ [25].

NAM has been used to protect beta cells damaged by various in- sults. In particular, NAM was able to protect beta cells which have been exposed to STZ [27]. Since a sharp drop of NAD+ was observed in STZ-treated beta cells, NAD+ depletion by PARP activation was suggested to play a critical role in STZ-induced beta cell death [28]. Reports that 3-aminobenzamide (3-AB) as a PARP inhibitor had protective effect on STZ-induced cell death and that PARP-defi- cient mice are resistant to SZT-induced death strongly support the hypothesis that the protective role of NAM in STZ-induced beta cell death is due to its inhibitory activity on PARP [29,30]. An earlier study demonstrated that the combination of three cytokines (IL- 1b, TNF-a, and IFN-c) induced NO production and subsequent DNA strand breakage and that NAM could prevent NO-dependent beta cell apoptosis by decreasing expression of nitric oxide syn- thase [31]. However, the protective effect of NAM on cytokine-in- duced beta cell death was suspected because cell death induced by cytokine mix was not affected by the presence of NAM [32,33]. On the other hand, Piro et al. demonstrated that NAM was able to protect beta cell apoptosis by high glucose and ole- ate/palmitate and suggested that protective effect of NAM on beta cell glucolipotoxicity was due to its role of antioxidant [34]. In par- ticular, NAM was also shown to inhibit the oxidative damage in- duced by reactive oxygen species such as NO or H2O2 in beta cells and therefore, NAM was regarded as a free radical scavenger [27,35].

This work was initiated to determine whether treatment with NAM protected high glucose/palmitate (HG/PA)-induced INS-1

beta cell death and to elucidate the mechanism of NAM’s protec- tive effect, if a protective effect existed. While cell viability was determined by the 3-[4,5-dimetylthiazol-2-yl]-2,5-diphenyltetra- zoilium bromide (MTT) viability assay, cell death was determined by DNA fragmentation assay and caspase 3 activation. To deter- mine the protective role of NAM as an anti-oxidant, the effect of different anti-oxidants such as N-acetyl cysteine (NAC), reduced glutathione (GSH) and Mito-TEMPOL on HG/PA-induced death was investigated. The role of NAD+ depletion in HG/PA-induced death was investigated by treatment of INS-1 cells with NAD+ or NAD+ precursors such as NMN and kynurenine. The role of NAD+ salvage pathway in HG/PA-induced INS-1 cell death was also investigated through pharmacological inhibition or knock- down of NAD+-synthetic enzymes such as NAMPT and NMNAT. As NAM is an inhibitor of NAD+-consuming enzymes such as PARP and sirtuins, the effect of the NAD+-consuming enzymes on HG/ PA-induced INS-1 cell death was investigated by pharmacological inhibition or knockdown of PARP and sirtuins. To determine how NAM protects HG/PA-induced death, the levels of HG/PA- induced signal mediators, such as phospho-JNK, phospho-Akt, phospho-eIF2a, and C/EBP homologous protein (CHOP), were also determined.

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