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  • Free Radical Biology & Medicine 49 (2010) 1522–1533

    Contents lists available at ScienceDirect

    Free Radical Biology & Medicine

    j ourna l homepage: www.e lsev ie r.com/ locate / f reeradb iomed

    Original Contribution

    Carbonyl reductase 1 protects pancreatic β-cells against oxidative stress-induced apoptosis in glucotoxicity and glucolipotoxicity

    M.A. Rashid a, Seonmin Lee a, Eunyoung Tak a, Jisun Lee a, Tae Gyu Choi a, Joo-Won Lee b, Jae Bum Kim b, Jang H. Youn a,c, Insug Kang a, Joohun Ha a, Sung Soo Kim a,⁎ a Medical Science and Engineering Research Center for Bioreaction to Reactive Oxygen Species and Biomedical Science Institute (BK-21), Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, Seoul 130-701, Korea b Department of Biological Sciences, Seoul National University, Kwanak-Gu, Seoul, Korea c Department of Physiology and Biophysics, University of Southern California Keck School of Medicine, Los Angeles, CA 90089, USA

    Abbreviations: ACC, acetyl-CoA carboxylase; ABCA1, aldehyde dehydrogenase; AR, aldose reductase; Bip/Gr protein/glucose-regulated protein-78; CBR1, carbony dichlorodihydrofluorescein diacetate; FAS, fatty acid syn glucose-stimulated insulin secretion; HNE, 4-hydroxyno 2-en-4-one; MDA, malondialdehyde; MTT, 3-(4,5-dimet tetrazolium bromide; NAC, N-acetyl-L-cysteine; ONA, 4-o enal; PDI, protein disulfide isomerase; ROS, reactive ox regulatory element binding protein (SREBP) 1c; TG, trig ⁎ Corresponding author. Fax: +82 2 959 8168.

    E-mail address: [email protected] (S.S. Kim).

    0891-5849/$ – see front matter © 2010 Elsevier Inc. A doi:10.1016/j.freeradbiomed.2010.08.015

    a b s t r a c t

    a r t i c l e i n f o

    Article history: Received 5 February 2010 Revised 5 August 2010 Accepted 12 August 2010 Available online 19 August 2010

    Keywords: Carbonyl reductase 1 Reactive oxygen species Lipogenesis Lipid peroxidation ER stress Pancreatic β-cell failure Type 2 diabetes Free radicals

    Carbonyl reductase 1 (CBR1) plays an important role in the detoxification of reactive lipid aldehydes. Oxidative stress has been implicated in the pathogenesis of pancreatic β-cell failure. However, the functional role of CBR1 in pancreatic β-cell failure has not been studied yet. Therefore, we investigated the role of CBR1 in pancreatic β-cell failure under glucotoxic and glucolipotoxic conditions. Under both conditions, knockdown of CBR1 by specific siRNA increased β-cell apoptosis, expression of lipogenic enzymes (such as ACC, FAS, and ABCA1), intracellular lipid accumulation, oxidative stress, ER stress, and nuclear SREBP1c, but decreased glucose-stimulated insulin secretion. In contrast, overexpression of CBR1 showed the opposite effects. The antioxidants N-acetyl-L-cysteine and Tiron, as well as the FAS inhibitor cerulenin, reversed the effects of CBR1 knockdown. Interestingly, the expression level and enzyme activity of CBR1 were significantly decreased in pancreatic islets of db/db mice, compared with those of wild-type mice. In conclusion, CBR1 protects pancreatic β-cells against oxidative stress and promotes their survival in glucotoxicity and glucolipotoxicity.

    cholesterol transporter; ALDH, p78, immunoglobulin-binding l reductase 1; DCF-DA, 2′,7′- thase; FFA, free fatty acid; GSIS, n-2-enal; HNO, 1-hydroxynon- hylthiazol-2-yl)-2,5-diphenyl- xononanal; ONE, 4-oxonon-2- ygen species; SREBP1c, sterol lyceride.

    ll rights reserved.

    © 2010 Elsevier Inc. All rights reserved.

    Pancreaticβ-cell failure plays a key role in the pathogenesis of type 2 diabetes. Although the exact mechanism underlying β-cell destruction is not known, it has been suggested that both hyperglycemia and hyperlipidemia contribute to β-cell destruction. Both glucotoxicity and lipotoxicity are important in thepathogenesis of type2diabetes because they lead to interference with insulin signal transduction and thus insulin resistance on the one hand and β-cell destruction on the other [1,2]. The functional consequences of glucotoxicity and glucolipotoxicity for β-cells include inhibition of glucose-stimulated insulin secretion (GSIS), impairment of insulin gene expression, and induction of cell death by apoptosis.

    Hyperglycemia is one of the major factors contributing to oxidative stress in pancreatic β-cells. Under normal glucose concentrations, pancreatic β-cells metabolize glucose easily via the glycolysis pathway and tricarboxylic acid (TCA) cycle, during which production of reactive oxygen species (ROS) from mitochondria remains manageable. How- ever, under high glucose concentrations, mitochondria produce exces- sive amounts of ROS as they utilize alternative glucose-metabolizing pathways prone to inductionof oxidative stress. Thesepathways include glyceraldehyde autoxidation to methylglyoxal and glycation, enediol and α-ketoaldehyde formation, glucosamine and hexosamine metabo- lism, and sorbitol metabolism [3,4]. In addition to increasing the production of ROS by mitochondria, glucose is known to induce an increase in ROS generated by NADPH oxidase in the cell membrane. The administration of glucose to normal subjects results in a marked increase in ROS generation even without an increase in glucose to supranormal levels. Thus, glucose is a powerful inducer of oxidative stress [5]. Both sources of ROS, the mitochondria and NADPH oxidase, are likely to induce a marked increase in the oxidative stress to theβ-cells [6,7]. As far as lipids are concerned, an increase in free fatty acid concentrations is known to occur in insulin-resistant states such as obesity and the metabolic syndrome. When free fatty acid (FFA) concentrations are increased in normal subjects to the levels found in the obese, marked increases in ROS generation and inflammatory stress

    http://dx.doi.org/10.1016/j.freeradbiomed.2010.08.015 mailto:[email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2010.08.015 http://www.sciencedirect.com/science/journal/08915849

  • 1523M.A. Rashid et al. / Free Radical Biology & Medicine 49 (2010) 1522–1533

    are induced [8]. Indeed, obesity is associated with increased lipolysis and FFA concentrations, chronic oxidative stress, inflammation, and insulin resistance [9–11]. This statemay also contribute to oxidative and inflammatory stress in theβ-cells. In addition, pancreatic β-cells are not well equipped with antioxidative defense mechanisms, and thus are easily overwhelmed by redox imbalance arising from the overpro- duction of ROS. Eventually, when hyperglycemia occurs, it provokes further excessive ROSproduction and oxidative damage of nucleic acids, proteins, and membrane lipids [12–14]. These processes damage pancreatic β-cells and may eventually lead to marked losses of β-cell function in type 2 diabetes.

    Carbonyl reductase 1 (CBR1) is a NADPH-dependent, monomeric, and cytosolic enzyme belonging to a family of short-chain dehydro- genases/reductases. The enzyme consists of 277 amino acid residues and is widely distributed in human tissues such as liver, epidermis, stomach, small intestine, kidney, neuronal cells, and smooth muscle fibers [15]. The best substrates of CBR1 are quinones, including ubiquinone-1 and tocopherolquinone (vitamin E). Ubiquinones (coenzyme Q) are constitutive parts of the respiratory chain, and tocopherolquinone protects lipids of biological membranes against lipid peroxidation, indicating that CBR1 may play an important role as an oxidation–reduction catalyst in biological processes [16]. Furthermore, CBR1 inactivates highly reactive lipid aldehydes, such as 4-oxonon-2-enal (ONE), 4-hydroxynon-2-enal (HNE), and acrolein, which are able to modify protein and DNA damage within cells [17]. A mutation in the gene encoding a homologue of CBR1 causes oxidative stress-induced neurodegeneration in Drosophila melanogaster [18], and overexpression of the human CBR1 in NIH3T3 cells protects from ROS-induced cellular damage [19], both supporting CBR1 as a major contributor to the control of oxidative stress.

    Although ROS have been implicated in the pathogenesis of pancreatic β-cell failure and HNE–albumin adducts were shown to be increased in the serum of type 2 diabetes patients [20], the role of CBR1 has never been explored in type 2 diabetes. Therefore, we hypothesized that (i) CBR1 exerts a beneficial role in protecting pancreatic β-cells from oxidative stress under glucotoxic and glucolipotoxic conditions, in vitro, and (ii) pancreatic islets from the diabetic db/db mice show low levels of CBR1 expression and enzyme activity.

    Materials and methods

    Materials

    RPMI 1640 medium and fetal bovine serum were purchased from Lonza (Walkersville, MD, USA) and G418 from Duchefa (St. Louis, MO, USA). T0901317 was purchased from Calbiochem (San Diego, CA, USA). Cerulenin,N-acetyl-L-cysteine (NAC), Tiron, 2′,7′-dichlorodihydrofluor- escein diacetate (DCF-DA), Hoechst 33342, 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT), and oil red O were acquired fromSigma (St. Louis,MO,USA). CBR1 antibodywaspurchased from Abnova (Taipei, Taiwan), acetyl-CoA carboxylase (ACC) from Upstate Biotechnology (Lake Placid, NY, USA), and cholesterol trans- porter (ABCA1) from Novus Biologicals (Littleton, CO, USA). Antibodies specific to fatty acid synthase (FAS), HNE, andmalondialdehyde (MDA) were purchased from Abcam (Cambridge, UK). Antibodies to aldose reductase (AR), aldehyde dehydrogenase (ALDH), actin, immunoglob- ulin-binding protein/glucose-regulated protein-78 (Bip/Grp78), insu- lin, SREBP1c, and lamin B were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against C/EBP-homologous protein (CHOP), phosphorylationof the eukaryotic initiation factor2α (p-eIF2α), poly(ADP ribose) pol