@article{246412ce78c046c9ac3ae31700917c41,
title = "Cytoprotective effects of N-acetylcysteine on streptozotocin-induced oxidative stress and apoptosis in RIN-5F pancreatic β-cells",
abstract = "Background/Aims: Numerous studies have reported overproduction of reactive oxygen species (ROS) and alterations in mitochondrial energy metabolism in the development of diabetes and its complications. The potential protective effects of N-acetylcysteine (NAC) in diabetes have been reported in many therapeutic studies. NAC has been shown to reduce oxidative stress and enhance redox potential in tissues protecting them against oxidative stress associated complications in diabetes. In the current study, we aimed to investigate the molecular mechanisms of the protective action of NAC on STZ-induced toxicity in insulin secreting Rin-5F pancreatic β-cells. Methods: Rin-5F cells were grown to 80% confluence and then treated with 10mM STZ for 24h in the presence or absence of 10mM NAC. After sub-cellular fractionation, oxidative stress, GSH-dependent metabolism and mitochondrial respiratory functions were studied using spectrophotometric, flow cytometric and Western blotting techniques. Results: Our results showed that STZ-induced oxidative stress and apoptosis caused inhibition in insulin secretion while NAC treatment restored the redox homeostasis, enhanced insulin secretion in control cells and prevented apoptosis in STZ-treated cells. Moreover, NAC attenuated the inhibition of mitochondrial functions induced by STZ through partial recovery of the mitochondrial enzymes and restoration of membrane potential. STZ-induced DNA damage and expression of apoptotic proteins were significantly inhibited in NAC-treated cells. Conclusion: Our results suggest that the cytoprotective action of NAC is mediated via suppression of oxidative stress and apoptosis and restoration of GSH homeostasis and mitochondrial bioenergetics. This study may, thus, help in better understanding the cellular defense mechanisms of pancreatic β-cells against STZ-induced cytotoxicity.",
keywords = "Apoptosis, N-acetylcysteine, Oxidative stress, Pancreatic Rin-5F cells, Streptozotocin",
author = "Al-Nahdi, {Arwa M.T.} and Annie John and Haider Raza",
note = "Funding Information: GSH is a master regulator of redox homeostasis, therefore, there is a growing interest in studying the therapeutic effects of N-acetylcysteine (NAC), a pro-drug and a GSH precursor, in the prevention of diseases characterized by increased oxidative stress, such as diabetes [44]. In the present study, our aim was to elucidate the cytoprotective effects of NAC on STZ-induced toxicity in Rin-眀F cells. STZ is a known pancreatic beta-cell speci 퀀ic cytotoxin and is therefore being widely used for its diabetogenic properties [45, 46]. We have previously demonstrated that diabetes (both in vivo and in vitro models) induces oxidative stress and mitochondrial respiratory dysfunction [10, 28, 47]. In the present study we intended to investigate the cytoprotective mechanisms of NAC on oxidative stress, mitochondrial bioenergetics, GSH metabolism and apoptosis. The selection of the STZ dose and time point treatment were based on our pervious study on Rin-眀F cells [ 球礂က and HepG 琀 [ 甃砂က where lower dose (10mM) for short time period (24h) had minimal effect on the cell viability. A signi ?icant recovery of altered GSH metabolism by NAC treatment was observed in STZ-treated cells suggesting the potential mechanism of NAC action in protecting cells from oxidative stress. NAC has also been shown to exert its antioxidant and anticancer effects by modulating the glutathione metabolism and the biotransformation of carcinogenic substances in vivo [48]. Studies have shown that glutamate dehydrogenase plays a key role in regulating redox homeostasis through the activation of GSH-Px [ 瘃笂ခ In our study we found that STZ treatment signi 퀀icantly decreased the activity of GDH and increased ROS production, which might have activated the ROS scavenger enzyme, GSH-Px, to regulate the redox homeostasis. NAC treatment signi 퀀icantly decreased the ROS levels, subsequently reducing the activity of GSH-Px. Endogenous ROS production plays an important role in the regulation of glycolytic activity and expression of glycolytic enzymes, including HK, which could be reversed by NAC pre-treatment [ 眃爂ခ Our study also indicated a similar decrease in HK activity after NAC pretreatment, which could be related to its antioxidant effects. The key parameter for evaluating mitochondrial function is mitochondrial membrane potential, which drives the synthesis of ATP [ 眃猂ခ A signi 퀀icant loss of ??P was observed in Rin-眀F cells after treatment with STZ accompanied by a decrease in the activities of the respiratory complexes, Complex I and Complex IV as well as that of the mitochondrial matrix enzyme, aconitase. NAC treatment had signi 퀀icantly recovered the ??P and the activity of ROS-sensitive aconitase enzyme. However, NAC treatment inhibited the activity of Complex I but not Complex IV. This could be because NAC acts as a redox sensor by inhibiting the Complex I activity and limiting the oxygen and NADH utilization, thus reducing oxidative stress [ 眃琂ခ Complex I may also be inhibited by excess GSH through glutathionylation [ 眃甂က since NAC treatment results in an increase in GSH levels. ?oreover, the suppression of Complex I activity could also be a result of S-nitrosation of Complex I by NO, which is an effective mechanism to prevent ROS formation, thus protecting the cells from oxidative stress [ 眃瘁 眃?]. We have previously shown that NAC treatment partially protected the oxidative modi 퀀ication of mitochondrial complexes and helped in preserving the mitochondrial function in HepG 琀 cells [ 眃砂ခ Studies have also shown membrane permeabilization to be related to apoptosis [ 眃礂ခ Our present study has also demonstrated activation of caspase-甁 caspase-笀 and increased ratio of Bax/Bcl-琀 expression, which con 퀀irmed the increased oxidative stress and thus the increased apoptosis after STZ treatment, which recovered signi 퀀icantly after NAC pre-treatment. Another important 퀀inding of our study was the marked reduction in insulin secretion, which, however was not normalized by NAC pre-treatment. This could be due to NAC scavenging the ROS, physiologically required for insulin secretion. However, a moderate increase in insulin secretion was observed in cells treated with NAC alone. This could be due to the improvement of pancreatic physiological and mitochondrial functions, which control the glucose-stimulated insulin secretion. This is supported by previous studies suggesting the improvement of insulin secretion by NAC treatment associated with improvement of GLUT Publisher Copyright: {\textcopyright} 2018 The Author(s). Published by S. Karger AG, Basel.",
year = "2018",
month = nov,
day = "1",
doi = "10.1159/000495200",
language = "English",
volume = "51",
pages = "201--216",
journal = "Cellular Physiology and Biochemistry",
issn = "1015-8987",
publisher = "S. Karger AG",
number = "1",
}