Chemico-Biological Interactions 277 (2017) 101e109

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Magnolol protects pancreatic b-cells against methylglyoxal-induced cellular dysfunction Kwang Sik Suh a, 1, Suk Chon a, 1, Woon-Won Jung b, Eun Mi Choi a, * a b

Dept. of Endocrinology & Metabolism, School of Medicine, Kyung Hee University, Seoul, 02447, Republic of Korea Dept. of Biomedical Laboratory Science, College of Health Sciences, Cheongju University, Cheongju, Chungbuk, 28503, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 July 2017 Received in revised form 28 August 2017 Accepted 13 September 2017 Available online 15 September 2017

Chronic hyperglycemia aggravates insulin resistance, in part due to increased formation of advanced glycation end-products (AGEs). Methylglyoxal (MG), a major precursor of AGEs, accumulates abnormally in various tissues and organs and participates in oxidative damage. We investigated the insulinotropic benefits of magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, in pancreatic b-cells exposed to MG in vitro. When exposed to cytotoxic levels of MG for 48 h, RIN-m5F b-cells exhibited a significant loss of viability and impaired insulin secretion, whereas pretreatment with magnolol protected against MG-induced cell death and decreased insulin secretion. Moreover, magnolol increased the expression of genes involved in b-cell survival and function, including Ins2 and PDX1. Furthermore, magnolol increased the levels of AMPK phosphorylation, SIRT1, and PGC1a in RIN-5F bcells. In addition, magnolol increased the activity of glyoxalase I and decreased the levels of MG-modified protein adducts, which suggests that magnolol protects against MG-induced protein glycation. Taken together, the results indicate the potential application of magnolol as an intervention against MGinduced hyperglycemia. © 2017 Elsevier B.V. All rights reserved.

Keywords: Magnolol Methylglyoxal Oxidative stress Pancreatic beta cells Mitochondrial biogenesis

1. Introduction Diabetes mellitus is a chronic and progressive metabolic disorder characterized by hyperglycemia. While type 1 diabetes is caused by a loss of insulin secretion due to the destruction of pancreatic b-cells [1], progressive b-cell failure is the central component of the onset and progression of type 2 diabetes [2]. Gradual reductions in b-cell mass and dysfunction of b-cells ultimately lead to insulin deficiency in patients with type 2 diabetes [3]. Methylglyoxal (MG), a reactive sugar metabolite, is increased in hyperglycemic states such as diabetes mellitus [4]. Importantly, MG is found in significantly elevated levels in the plasma of uncontrolled diabetics at baseline, reaching as high as 6 mM while nondiabetic control plasma had a concentration near 1 mM [5]. MG tends to interact readily with amino acid side-chains in select proteins to form irreversible advanced glycation end products

* Corresponding author. Dept. of Endocrinology & Metabolism, School of Medicine, Kyung Hee University, 1, Hoegi-dong, Dongdaemun-gu, Seoul, 02447, Republic of Korea. E-mail address: [email protected] (E.M. Choi). 1 These authors are contributed equally to this work. http://dx.doi.org/10.1016/j.cbi.2017.09.014 0009-2797/© 2017 Elsevier B.V. All rights reserved.

(AGEs). MG is the most reactive AGE precursor [6]. Intracellular concentrations of AGEs can be increased via auto-oxidation of glucose, resulting in the formation of glyoxal [7], which can undergo a final fragmentation to produce MG [8]. The diet is considered the main exogenous source of AGEs contributing to the development of diabetes [9]. High-temperature cooking methods result in the highest production of AGEs, and dietary fats are the main contributors to AGE formation [10]. Thus, both endogenous and exogenous AGEs can be risk factors involved in metabolic diseases. Protein glycation is associated with disease exacerbation, especially the chronic complications associated with diabetes [11,12]. Under normal conditions, cells are protected against MGinduced cellular damage by glyoxalase defense mechanisms [13]. Oxidative stress, which may induce b-cell death and decrease b-cell mass, is involved in the pathological process of diabetes [14,15]. Pancreatic b-cells are susceptible to reactive oxygen species (ROS), and the action of ROS on these cells is a potential mechanism of glucose toxicity in diabetes [14]. Several studies have shown that increased MG production stimulates ROS production [16,17]. Overproduction of ROS during oxidative phosphorylation can upregulate mitochondrial uncoupling protein 2, leading to inward

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electron leak and reduced ATP synthesis, which can impair insulin secretion [18,19]. Furthermore, ROS cause auto-oxidation of sugars and their derivatives, unsaturated fatty acids, and proteins in plasma membranes, resulting in increased production of malondialdehyde [20]. Chronic hyperglycemia increases oxidative stress, leading to glucose toxicity with concomitant impairment of glucose-induced insulin secretion and insulin gene expression and reduced b-cell mass via apoptosis [21,22]. This creates a cycle of continual b-cell destruction that eventually leads to overt diabetes. Most oral hypoglycemic treatment agents have side effects. However, plants and their derivatives have been used effectively to treat diabetes, because they are considered to be less toxic and to have fewer side effects than synthetic treatments [23,24]. Magnolol, extracted from Magnolia officinalis, is a natural biphenolic neolignan. The properties of magnolol have been investigated extensively and include anti-oxidant [25], anti-inflammatory [26], and anti-tumorigenic activities [27]. Magnolol was effective against the hepatic oxidative damage, hyperglycemia and hyperlipemia of diabetic rats [28], and magnolol stimulated glucose uptake in insulin-sensitive and insulin-resistant murine and human adipocytes using the insulin signaling pathway [29]. In addition, the use of magnolol in non-obese type 2 diabetic rats resulted in good blood glucose control and could prevent or retard development of diabetic complications such as diabetic nephropathy [30]. We previously reported that magnolol has potential beneficial effects in osteoblasts, as magnolol prevented antimycin A-induced cellular damage in the osteoblastic MC3T3-E1 cell line [31,32]. Given the mounting evidence for a major role of pancreatic b-cells in the pathogenesis of type 2 diabetes, we investigated the effects of magnolol on MG-induced oxidative cell damage in pancreatic bcells and its underlying mechanisms of action.

was added to dissolve the formazan products, and the plates were shaken for 5 min. The absorbance was measured using the Zenyth 3100 multimode detector (Anthos Labtec Instruments, Wals/Salzburg, Austria) at 570 nm. Cells incubated with the culture medium alone were set at 100% viability and included as a control in all experiments to allow estimation of percent viability. 2.4. Apoptosis assay Apoptosis was detected by assaying cytoplasmic histoneassociated DNA fragments (mononucleosome and oligonucleosomes) formed during apoptosis using a Cell Death Detection ELISAPlus kit (Roche Molecular Biochemicals, Germany), according to the instructions of the manufacturer. 2.5. Measurement of insulin secretion Cells were seeded in a 24-well plate at a density of 2  104 cells/ well in culture medium. After 48 h, cells were pre-incubated with various concentrations of magnolol or 400 mM aminoguanidine (AG) before treatment with 300 mM MG for 48 h. Cell culture supernatants were collected for measurement of secreted insulin levels using an ultrasensitive rat insulin ELISA kit (Mercodia Inc., Uppsala, Sweden) according to the manufacturer's instructions. Insulin in the sample reacts with anti-insulin antibodies bound to microtitration wells and peroxidase-conjugated anti-insulin antibodies in the solution. Protein concentrations were determined using the Bio-Rad Protein assay reagent (Bio-Rad, Hercules, CA, USA). 2.6. RNA isolation and quantitative reverse-transcription PCR (qRTPCR)

2. Materials and methods 2.1. Reagents Magnolol (purity: 98%) isolated from Magnolia officinalis was purchased from ChromaDex Inc. (Irvine, CA, USA). The magnolol was dissolved in dimethylsulfoxide (DMSO) and diluted with culture media to a final DMSO concentration
0.05% (v/v). a-Modified minimal essential medium and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). Other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2.2. Cell culture RIN-m5F cells derived from rat pancreatic b-cells were purchased from the American Type Culture Collection (Manassas, VA, USA). RIN-m5F cells were maintained in RPMI1640 medium (2 g/L glucose) supplemented with 10% FBS and 1% penicillin/streptomycin solution under saturated humidity atmospheric conditions containing 5% CO2 at 37  C. The medium was renewed every 3 days. Forty-eight hours after seeding, confluent monolayers of the cells were pre-incubated for 1 h with medium containing 0.1% FBS and experimental reagents before treatment with MG for 48 h. 2.3. Cell viability The RIN-m5F cells were seeded in a 24-well plate at a density of 2  104 cells/well. After 48 h, the cells were treated with different concentrations of magnolol in the absence or presence of 300 mM methylglyoxal (MG) for 48 h. Cell viability was assessed by the MTT method. MTT in phosphate buffered salt solution, pH 7.4 (20 mL; 5 mg/mL) was added to each well, and the plates were incubated for an additional 2 h. After removing the solution from the well, DMSO

Cells were seeded in a 100 mm dish at a density of 106 cells/well in culture medium. After 48 h, cells were pre-incubated with magnolol or AG before treatment with 300 mM MG for 48 h. Total RNA from each well was extracted from RIN-m5F cells using the RNeasy Mini kit (Qiagen NV, Venlo, The Netherlands). Samples are first lysed and then homogenized. Ethanol is added to the lysate to provide ideal binding conditions. The lysate is then loaded onto the RNeasy silica membrane. RNA binds, and all contaminants are efficiently washed away. Pure, concentrated RNA is eluted in 100 ml water. cDNA was synthesized using the PrimeScript First Strand DNA Synthesis kit (TaKaRa Bio, Inc., Otsu, Shiga, Japan) according to the manufacturer's instructions. qRT-PCR was performed using the SYBR Premix Ex Taq kit (TaKaRa) and the ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA) to determine gene expression levels. Each PCR reaction was performed in a 20 mL solution containing 0.8 mL (10 mM) of the forward and reverse primers each, 10 mL Premix Ex Taq DNA polymerase, 0.4 mL ROX reference dye, 6 mL dH2O, and 2 mL reverse-transcription reaction products. The qRT-PCR primers used in the experiment are shown in Table 1. All experiments were performed in quadruplicate. Relative expression was determined using the 2eDDCt method with the housekeeping gene G6PD as the internal control, and foldchange expression was calculated relative to the corresponding control group. 2.7. Measurement of IL-1b Cells were seeded in a 24-well plate at a density of 2  104 cells/ well in culture medium. After 48 h, cells were pre-incubated with various concentrations of magnolol or AG before treatment with 300 mM MG for 48 h. Cellular IL-1b contents were measured using an enzyme immunoassay system (R&D Systems Inc., Minneapolis,

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Table 1 Primer sequences used in this study. Gene

Forward primer

Reverse primer

Insulin 2 PDX1 GPX1 G6PD

50 -CGA AGT GGA GGA CCC ACA-30 50 -ACC CGT ACA GCC TAC ACT CG-30 50 -AGA AGG CTC ACC CGC TCT-30 50 -TGC AGC AGC TGT CCT CTA TG-30

50 -TGC TGG TGC AGC ACT GAT-30 50 -GCC GGG AGA TGT ATT TGT TAA A-30 50 -GGA TCG TCA CTG GGT GCT-30 50 -ACT TCA GCT TTG CGC TCA TT-30

MN, USA) according to the manufacturer's instructions. A polyclonal antibody specific for rat IL-1b has been pre-coated onto a microplate. Standards, Control, and samples are pipetted into the wells and any rat IL-1b present is bound by the immobilized antibody. After washing away any unbound substances, an enzymelinked polyclonal antibody specific for rat IL-1b is added to the wells. Following a wash to remove any unbound antibodyenzyme reagent, a substrate solution is added to the wells. The enzyme reaction yields a blue product that turns yellow when the Stop Solution is added. The intensity of the color measured is in proportion to the amount of rat IL-1b bound in the initial step. The sample values are then read off the standard curve. 2.8. Measurement of intracellular ROS Cells were seeded in a 24-well plate at a density of 2  104 cells/ well in culture medium. After 48 h, cells were pre-incubated with various concentrations of magnolol or AG before treatment with 300 mM MG for 48 h. Formation of intracellular ROS was measured using 20 ,70 -dichlorodihydrofluorescin diacetate (H2DCFDA) [33]. Viable cells can deacetylate H2DCFDA into the non-fluorescent derivative 20 ,70 -dichlorofluorescin (DCF), which reacts with oxygen species and provides an index of intracellular oxidant production. To load the cells with the fluorescent dye, they were incubated with H2DCFDA in Hank's solution at a final concentration of 10 mM for 45 min at 37  C in the dark. After washing the cells with Dulbecco's Phosphate Buffered Saline (DPBS), the fluorescence intensity was measured (excitation 485 nm, emission 515 nm) using the Zenyth 3100 multimode detector.

Diego, CA, USA). This enzyme immunoassay was developed for rapid detection and quantitation of MG-H1 (methyl-glyoxal-hydroimidazolone) protein adducts. The MG-protein adducts present in the sample or standard were probed using a specific anti-MG monoclonal antibody, followed by a horseradish peroxidaseconjugated secondary antibody. The quantity of MG adducts in the protein samples was determined by comparing their absorbances against an MG-BSA standard curve.

2.11. Glyoxalase I activity Cells were seeded in a 6-well plate at a density of 2  105 cells/ well in culture medium. After 48 h, cells were pre-incubated with various concentrations of magnolol or AG before treatment with 300 mM MG for 48 h. Cells were rinsed in ice-cold DPBS and homogenized in DPBS using a glass Dounce homogenizer (Taylor Scientific) on ice. The resulting suspension was subjected to two freeze-thaw cycles to further disrupt the cell membranes. Cell homogenates were centrifuged at 13,000  g for 15 min at 4  C, and the resulting supernatant was used to measure glyoxalase I activity and protein content. Glyoxalase I activity was measured using a modification of a previously published method [34]. To measure glyoxalase I activity, 50 mL sample were loaded onto a UV microplate (Microtiter; ThermoFisher Scientific), and 200 mL reaction mix were added. The reaction mix consisted of 60 mM sodium phosphate buffer, pH 6.6, 4 mM GSH, and 4 mM MG and was preincubated for 10 min at 37  C. S-Lactoylglutathione synthesis was determined by measuring the absorbance at 240 nm for 5 min at 25  C.

2.9. Measurement of SIRT1, PGC-1a, and AMPK Cells were seeded in a 24-well plate at a density of 2  104 cells/ well in culture medium. After 48 h, cells were pre-incubated with various concentrations of magnolol or AG before treatment with 300 mM MG for 48 h. Cells were rinsed in ice-cold DPBS and homogenized in DPBS on ice using a glass Dounce homogenizer (Taylor Scientific, St. Louis, MO, USA). The resulting suspension was subjected to two freeze-thaw cycles to further disrupt the cell membranes. Cell homogenates were centrifuged at 13,000  g for 15 min at 4  C, and the supernatant was used for ELISA and measurement of protein content. Sirtuin-1 (SIRT1) was measured using the Sirtuin 1 ELISA Kit (Cloud-Clone Corp., Houston, TX, USA). Mouse Peroxisome PGC-1a and phosphorylated adenosine monophosphate activated protein kinase (AMPK) were measured using ELISA kit (MyBioSource, Inc., San Diego, CA, USA). 2.10. Quantification of MG-modified protein adducts Cells were seeded in a 24-well plate at a density of 2  104 cells/ well in culture medium. After 48 h, cells were pre-incubated with various concentrations of magnolol or AG before treatment with 300 mM MG for 48 h. The quantification of MG-modified proteins (MG-protein adducts) was determined using an OxiSelect™ methylglyoxal competitive ELISA kit from Cell BioLabs, Inc. (San

2.12. Statistics Results are expressed as means ± SEM. Statistical significance was determined by one-way analysis of variance followed by Dunnett's test (p < 0.05).

3. Results 3.1. Effects of magnolol on the viability of RIN-m5F cells To determine whether magnolol had a protective effect against MG-induced cytotoxicity, cells were preincubated with magnolol for 1 h and then cultured with 300 mM MG for 48 h. Magnolol at concentrations of
1 mM had no effect on the viability of RIN-m5F cells in the absence of MG (Table 2). However, pretreatment with magnolol (0.01e1 mM) significantly inhibited MG-induced cytotoxicity. Morphology of RIN-m5F cells cultured in MG and/or magnolol-treated media appeared as Fig. 1. We further examined the effect of magnolol on MG-induced apoptosis. As shown in Fig. 2, magnolol reduced MG-induced apoptosis at concentrations of 0.1e1 mM, which indicates that magnolol exerts its cytoprotective effects in RIN-m5F cells through the reduction of apoptosis.

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Table 2 Effects of magnolol on the viability of RIN-m5F cells. Magnolol (mM)

Absorbance Without MG

0 0.01 0.1 1

0.533 0.563 0.522 0.520

± ± ± ±

With MG a

0.022 0.060a 0.008a 0.030a

0.291 0.455 0.448 0.446

± ± ± ±

0.009*b 0.017a 0.007a 0.012a

RIN-m5F cells were treated with magnolol in the absence or presence of 300 mM methylglyoxal (MG) for 48 h, and then cell viability was assessed by MTT assay. * p < 0.05, compared with untreated cells. Groups with different letters in the same column are significantly different from each other (n ¼ 6) according to analysis of variance.

3.2. Magnolol relieved MG-abrogated insulin secretion and increased the expression of insulin 2 (Ins2) and pancreatic and duodenal homeobox protein 1 (Pdx1) To evaluate the protective effect of magnolol on b-cell function, insulin secretion was assessed. Treatment with 300 mM MG resulted in an impressive decrease in insulin secretion by RIN-m5F cells (Fig. 3A), indicating a stress-induced loss of function. Interestingly, the decrease in insulin secretion by RIN-m5F cells subjected to MG was completely restored when these cells were pretreated with magnolol (0.01e1 mM). These results indicate that magnolol preserves not only RIN-m5F cell viability but also insulin secretion, the most important b-cell function. Alterations in insulin signal transduction could lead to b-cell dysfunction, contributing to the pathogenesis of type 2 diabetes. To investigate whether magnolol modulates the expression profile of genes important for b-cell function, we compared the expression levels of regulators of insulin secretion in pancreatic b-cells. As shown in Fig. 3, MG significantly downregulated the transcript levels of Ins2 relative to those in control cells. However, there was no difference in PDX-1 transcription levels between control and MG treated cells. Pretreatment with 0.01 mM magnolol significantly mitigated the MG-induced

decreases in expression of Pdx1 and Ins2. Aminoguanidine (AG) (400 mM), a carbonyl scavenger, also restored insulin secretion inhibited by MG, but had no effect on Ins2 and PDX1 levels (Fig. 3B and C). 3.3. Magnolol decreased production of IL-1b in MG-treated RINm5F cells MG promotes the formation of proinflammatory cytokines in various cell types. Thus, we investigated whether magnolol modulates the production of IL-1b in MG-treated cells (Fig. 4). When 300 mM MG was added to the cells, the production of IL-1b increased significantly. However, MG-induced IL-1b production was significantly inhibited by pretreatment with magnolol at concentrations of 0.01e1 mM. AG also decreased MG-induced IL-1b production. 3.4. Magnolol decreased ROS production and increased glutathione peroxidase (GPX) gene expression in MG-treated cells Intracellular ROS were measured using the oxidation-sensitive probe H2DCFDA. As shown in Fig. 5A, treatment with 300 mM MG significantly enhanced ROS levels compared with those in control cells. Under conditions of MG toxicity, magnolol (0.01e1 mM) or AG significantly suppressed ROS production, suggesting that the cytoprotective effect of magnolol is partly attributed to its regulation of ROS. The effect of magnolol on GPX expression in RIN-m5F b-cells was also examined. We found that GPX expression was increased significantly in MG-treated cells relative to control cells (Fig. 5B). Pretreatment of RIN-m5F b-cells with magnolol (0.01 mM) further increased GPX gene expression relative to that in cells treated with MG alone. AG restored the MG-induced changes. These findings suggest that magnolol enhances the antioxidant status of MG-treated RIN-m5F b-cells by upregulating the expression of GPX.

Fig. 1. Representative images of cells with MG and/or magnolol. RIN-m5F cells were pre-incubated with magnolol (1 mM) before treatment with 300 mM MG for 48 h. Cells were observed by inverted microscopy. (A) Control; (B) 1 mM Magnolol; (C) 300 mM MG; (D) 300 mM MG þ 1 mM Magnolol.

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Fig. 2. Effects of magnolol on apoptosis of RIN-m5F cells in the presence of MG. RIN-m5F cells were pre-incubated with magnolol (Mag) or 400 mM aminoguanidine (AG) before treatment with 300 mM MG for 48 h #p < 0.05, compared with untreated cells; *p < 0.05, compared with cells treated with MG alone.

3.5. Effects of magnolol on mitochondrial metabolic factors in MGtreated RIN-m5F cells AMPK acts as a key regulator of mitochondrial biogenesis. As shown in Fig. 6A, magnolol (0.01e1 mM) significantly stimulated the activation of AMPK under MG-treated conditions, indicating that magnolol is a potent inducer of AMPK. SIRT1 likely regulates multiple pathways involved in mitochondrial biogenesis. As shown in Fig. 6B, SIRT1 levels were increased by magnolol (0.01e1 mM) or AG treatment. PGC-1a is also considered a key regulator of mitochondrial biogenesis in multiple tissues. Since SIRT1 may also directly deacetylate PGC-1a and increase its activity, we measured PGC-1a levels in pancreatic b-cells. PGC-1a was significantly increased by magnolol (0.01e1 mM) or AG treatment (Fig. 6C). These data indicate that magnolol enhances pancreatic b-cell function by activating the AMPK/SIRT1/PGC-1a pathway. 3.6. Inhibitory effect of magnolol on MG-induced glycation in RINm5F cells MG reacts with free amino groups and thiols to form AGE protein adducts, thereby altering protein function. We investigated whether magnolol treatment reduces the formation of protein adducts in RIN-m5F b-cells. As shown in Fig. 7A, protein adducts accumulated in cells treated with 300 mM MG. However, pretreatment with magnolol (0.01e1 mM) or AG decreased protein adduct formation induced by MG. This indicates that magnolol can block MG-derived protein glycation in RIN-m5F b-cells, which might be part of its mechanism of inhibiting RIN-m5F b-cell death. Because MG is detoxified by the glyoxalase system, we also examined the effect of magnolol on glyoxalase I activity in RIN-m5F b-cells. As shown in Fig. 7B, we found a significant decrease in glyoxalase I activity in RIN-m5F b-cells treated with MG (300 mM). However, glyoxalase I activity was recovered by magnolol (0.01 and 0.1 mM) or AG treatment. These data suggest that magnolol decreases MGinduced glycation in part by increasing glyoxalase I activity. 4. Discussions MG is a highly reactive dicarbonyl metabolite formed mainly during glucose metabolism [35], and it is a major precursor of AGEs

Fig. 3. Effects of magnolol on insulin secretion and related gene expression in methylglyoxal (MG)-treated cells. RIN-m5F cells were pre-incubated with magnolol (Mag) or 400 mM aminoguanidine (AG) before treatment with 300 mM MG for 48 h. (A) The level of insulin content in the control was 34.93 ± 1.15 ng/mg protein. The expression levels of Ins2 (B) and PDX1 (C) mRNAs were determined by real-time PCR and were normalized to those of G6PD as an internal standard. Values are shown as mean ± SEM and analyzed by one-way ANOVA with Dunnett's test. #p < 0.05, compared with untreated cells; *p < 0.05, compared with cells treated with MG alone.

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Fig. 4. Effect of magnolol on the levels of IL-1b in methylglyoxal (MG)-treated cells. RIN-m5F cells were pre-incubated with magnolol (Mag) or 400 mM aminoguanidine (AG) before treatment with 300 mM MG for 48 h. The level of IL-1b in the control was 47.61 ± 22.32 pg/mg protein. #p < 0.05, compared with untreated cells; *p < 0.05, compared with cells treated with MG alone.

involved in the pathogenesis of diabetes and inflammation. Studies suggest that AGEs and MG can generate large amounts of proinflammatory cytokines through AGE receptor activation, which is related to the modulation of inflammatory molecules through oxidative stress [35]. Pancreatic b-cell failure is a critical metabolic disorder involved in the development of type 2 diabetes. Decreased viability and dysfunction of b-cells accelerates the diabetic pathogenesis associated with increased mortality [36]. Pancreatic islet cells are more susceptible to oxidative stress than are cells of other tissues, as they contain low levels of antioxidant enzymes [37]. Damage to pancreatic islets leads to decreased insulin secretion, resulting in a hyperglycemic condition and continuous generation of additional oxidative stress, creating a vicious cycle that eventually leads to cell failure. In the present study, we investigated the effects of magnolol on RIN-5F cells under MG-treated conditions and its underlying mechanism of action. We showed that magnolol efficiently preserves the functionality of insulin-secreting RIN-5F cells against MG-induced oxidative stress by reducing ROS overproduction, recovering altered antioxidant defenses, and restoring insulin-secreting machinery. The search for natural compounds as preventive and therapeutic agents to delay the onset of diabetic complications has received wide attention. A recent report showed that grape seed procyanidins improve b-cell functions in vitro [38], and Cai and Lin [39] previously showed that tea flavonol epigallocatechin gallate activates insulin signaling. Here, we report for the first time that magnolol prevents MG-induced damage of b-cell insulin secretion. According to Kittl et al. [40], in INS-1 cells quercetin acutely stimulates insulin release, presumably by transient KATP channel inhibition and simultaneous transient stimulation of voltage sensitive Ca2þ channels. In rat insulinoma-m5F cells under glucose-induced toxicity, treatment of flavan-3-ols led to improvement in the insulin secretory function of b-cells through increased expression of insulin receptor substrate 2 (IRS2), AKT, forkhead box protein O1 (FOXO1), and pancreatic duodenal homeobox-1 (PDX-1) [39]. Therefore, magnolol may play a protective role against diseases, such as type 2 diabetes. Among several transcription factors that bind to the insulin promoter, PDX1 has been shown to be important for the maintenance of insulin biosynthesis as well as b-cell mass [41]. Transcription of the human insulin gene is activated by PDX1 binding to

Fig. 5. Effects of magnolol on ROS levels and GPX expression in methylglyoxal (MG)-treated cells. RIN-m5F cells were pre-incubated with magnolol (Mag) or 400 mM aminoguanidine (AG) before treatment with 300 mM MG for 48 h. The data show changes in levels of ROS (A), measured using the DCF fluorescence method. The expression level of GPX (B) mRNA was determined by real-time PCR and was normalized to that of G6PD as an internal standard. Values are shown as mean ± SEM and analyzed by one-way ANOVA with Dunnett's test. #p < 0.05, compared with untreated cells; *p < 0.05, compared with cells treated with MG alone.

several sites within its promoter [42]. Mice homozygous for a targeted disruption of Pdx1 fail to develop a pancreas, and specific inactivation of Pdx1 in mouse b-cells decreased b-cell mass and insulin expression [41]. PDX1 deficiency contributes to impaired proliferation and enhanced apoptosis via transcriptional mechanisms in models of type 2 diabetes [43]. In response to acute elevations of glucose and survival factors, such as insulin, PDX1 is phosphorylated and translocated to the nucleus [44]. By contrast, stimuli associated with diabetes, such as oxidative stress [45] and free fatty acids [46], cause nuclear exclusion of PDX1 [46]. Several lines of evidence support the suggestion that insulin modulates PDX1 DNA-binding activity and insulin promoter activity [47]. PDX1 also modulates mRNA expression of glucokinase, which catalyzes the first step of glycolysis, and thereby regulates glucose responsiveness for insulin release [48]. Here we demonstrated in RIN-5F cells that magnolol treatment enhances the expression of the Pdx1 and insulin genes. Thus, magnolol exerts a beneficial effect on the expression of genes involved in preserving critical b-cell functions.

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Fig. 7. Effects of magnolol on methylglyoxal (MG)-induced protein adduct formation and glyoxalase 1 activity in MG-treated cells. RIN-m5F cells were preincubated with magnolol (Mag) or 400 mM aminoguanidine (AG) before treatment with 300 mM MG for 48 h. The protein adduct levels and glyoxalase 1 activity in the control were 2.603 ± 0.348 ng/mg protein and 0.013 ± 0.001 DOD/min/mg protein, respectively. Values are shown as mean ± SEM and analyzed by one-way ANOVA with Dunnett's test. #p < 0.05, compared with untreated cells; *p < 0.05, compared with cells treated with MG alone.

Fig. 6. Effects of magnolol on the levels of AMPK, SIRT1, and PGC-1a in methylglyoxal (MG)-treated cells. RIN-m5F cells were pre-incubated with magnolol (Mag) or 400 mM aminoguanidine (AG) before treatment with 300 mM MG for 48 h. The levels of AMPK, SIRT1, and PGC-1a in the control were 15.27 ± 0.886 U/mg protein, 18.1 ± 3.3 ng/mg protein, and 96.3 ± 14.98 ng/mg protein, respectively. Values are shown as mean ± SEM and analyzed by one-way ANOVA with Dunnett's test. # p < 0.05, compared with untreated cells; *p < 0.05, compared with cells treated with MG alone.

Pro-inflammatory cytokines have been implicated in the pathogenesis of both type 1 and type 2 diabetes mellitus. Cytokines such as IL-1b may be produced and secreted by b-cells under highglucose conditions and have been reported to increase the concentration of ROS [49]. IL-1b interferes with b-cell function by inhibiting insulin transcription. Treatment with IL-1b decreases mRNA levels of proinsulin-converting enzymes in pancreatic islets, indicating that IL-1b can decrease proinsulin conversion [50]. Through mitogen-activated protein kinase kinase kinase 1, IL-1b inhibits insulin gene transcription and does so, at least in part, by decreasing MafA transcriptional activity at the rat insulin promoter element 3b control element [51]. In addition, IL-1b-induced pancreatic b-cell dysfunction is due mainly to the activation of nuclear factor-kB (NF-kB) [52]. We demonstrated a profound inhibitory effect of magnolol on MG-induced IL-1b production in bcells. The protective effects of magnolol against MG toxicity in RIN5F pancreatic b-cells may be mediated by anti-inflammatory

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actions. This investigation shows that magnolol can protect rat pancreatic islets in vitro against the noxious effects induced by MG. AMPK is a master sensor of cellular energy balance that plays a key role in the regulation of energy homeostasis. Dysregulation of AMPK has been implicated in a number of metabolic diseases, including type 2 diabetes and obesity. Activation of AMPK enhances mitochondrial biogenesis and b-oxidation by regulating a cotranscriptional regulatory factor, PGC-1a [53]. PGC-1a, a master regulator of mitochondrial biogenesis, directly increases the synthesis of nuclear respiratory factors and mitochondrial transcription factor A, all of which increase mitochondrial DNA, an indicator of mitochondrial function [54]. A nicotinamide adenine dinucleotide-dependent deacetylase, SIRT1, is also an important functional regulator of PGC-1a that induces the transcription of mitochondrial fatty acid oxidation genes [55]. Mitochondrial ROS production is dependent on mitochondrial density, as more mitochondria work at lower levels of respiratory activity to generate the same degree of ATP [56]. Our data revealed that magnolol can increase the activities of AMPK, SIRT1, and PGC-1a. Especially, the enhancement in the AMPK activity was higher with lower doses of magnolol. Therefore, the beneficial effects of magnolol on RIN-5F cells could be mediated by enhanced mitochondrial biogenesis via activation of the AMPK/SIRT1/PGC-1a pathway. ROS-induced oxidative damage in pancreatic b-cells plays an important role in the pathology of diabetes. ROS level reduction and antioxidant treatment reportedly improve b-cell structure and function in vitro [57]. To evaluate the role of free radicals in the protective activity of magnolol, we used H2DCF-DA assays to analyze its effects on MG-induced ROS generation. Treatment with MG alone significantly increased intracellular ROS generation. Subsequent treatment with magnolol decreased ROS generation. This decrease in MG-induced ROS levels may account for the observed cytoprotective effect of magnolol. Our results indicate that magnolol treatment enables RIN-5F cells to maintain cell function and escape cell death in the presence of increasing levels of ROS induced by MG. The cytoprotective effects are mediated mainly by upregulation of intracellular antioxidant enzyme activity. GPX is an intracellular antioxidant enzyme that is an essential part of the cellular defense against oxidative insults, and it consequently plays a major role in cellular adaptation to overcome oxidative stress [58]. Pancreatic b-cells have been reported to have a lower capacity to degrade hydrogen peroxide and to express lower levels of endogenous antioxidant enzymes, particularly GPX [59]. In the present study, significant increase in GPX gene expression was observed in response to MG treatment, compared with the control. Our results showed that the MG-treated cells fought against ROS production by raising the expression of GPX. The magnololpretreated cells showed further increase in GPX expression compared with cells without magnolol. The increase in GPX expression indicates that GPX enzyme may be stable or important for stress tolerance. However, the fact that exogenous magnolol increased GPX expression and suppressed the production of ROS level indicates that magnolol is able to reduce MG-induced oxidative damage by increasing GPX expression. Therefore, magnololpretreated MG-stressed cells significantly enhanced the expression of GPX, and thus contributed to the reduction of H2O2, O2 and lipid hydroperoxide levels under MG stress, and subsequently affirmed higher tolerance to MG stress. This suggests that magnolol effectively decreased MG-induced intracellular ROS generation and reduced pancreatic b-cell death by increasing GPX expression. Protein glycation is viewed as a post-translational modification that accumulates mostly on extracellular proteins. Protein adduct concentrations are increased by increased protein modifications and/or decreased protein turnover [60]. Protein damage by MG is suppressed by glyoxalase 1 of the cytoplasmic glyoxalase system.

Glyoxalase 1 catalyzes the conversion of MG to a non-toxic hemithioacetal metabolite, using GSH as a cofactor. It was reported that glyoxalase 1 overexpression in Caenorhabditis elegans decreased hyperglycemia-induced accumulation of AGEs and oxidative stress and enhanced lifespan [61]. Overexpression of glycoxalase 1 reduces hyperglycemia-induced levels of carbonyl stress, AGEs, and oxidative stress in diabetic rats, indicating that glycoxalase 1 plays an important role in the prevention of glycation reactions under hyperglycemic conditions [62]. In this study, magnolol decreased the production of MG-protein adducts in cultured RIN-5F cells. In addition, glyoxalase 1 activity was increased in these magnololtreated cells, indicating the importance of glyoxalase 1 in scavenging MG-protein adducts in RIN-5F cells treated with MG. However, glyoxalase 1 activity seemed not to be related to the amount of MG-protein adducts. In conclusion, our present study suggests that MG negatively affects b-cell function, and that magnolol may ameliorate MGinduced pancreatic b-cell damage. The mechanism of magnolol action likely involves potentiation of AMPK/SIRT-1/PGC-1a signaling, increased glyoxalase 1 activity, elevated gene expression of Pdx1, Ins2, and GPX, and protection against detrimental oxidative and inflammatory damage. Magnolol may preserve and/or improve b-cell function in diabetics suffering from elevated circulating levels of toxic aldehydes due to chronic hyperglycemia. Conflict of interest Authors have no conflict of interest. Acknowledgements This research was supported by a grant from the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (2016R1D 1A1B03930082). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2017.09.014. References [1] J.S. Lee, Y.R. Kim, J.M. Park, S.J. Ha, Y.E. Kim, N.I. Baek, et al., Mulberry fruit extract protects pancreatic b-cells against hydrogen peroxide-induced apoptosis via antioxidative activity, Molecules 19 (2014) 8904e8915. [2] M. Prentki, C.J. Nolan, Islet b cell failure in type 2 diabetes, J. Clin. Invest 116 (2006) 1802e1812. [3] M. Stumvoll, B.J. Goldstein, T.W. van Haeften, Type 2 diabetes: principles of pathogenesis and therapy, Lancet 365 (2005) 1333e1346. [4] J. Lu, E. Randell, Y. Han, K. Adeli, J. Krahn, Q.H. Meng, Increased plasma methylglyoxal level, inflammation, and vascular endothelial dysfunction in diabetic nephropathy, Clin. Biochem. 44 (2011) 307e311. [5] P.J. Thornalley, N.I. Hooper, P.E. Jennings, C.M. Florkowski, A.F. Jones, J. Lunec, A.H. Barnett, The human red blood cell glyoxalase system in diabetes mellitus, Diabetes Res. Clin. Pract. 7 (2) (1989) 115e120. [6] M.P. Kalapos, Methylglyoxal in living organisms: chemistry, biochemistry, toxicology and biological implications, Toxicol. Lett. 110 (1999) 145e175. [7] K.J. Wells-Knecht, D.V. Zyzak, J.E. Litchfield, S.R. Thorpe, J.W. Baynes, Mechanism of autoxidative glycosylation: identification of glyoxal and arabinose as intermediates in the autoxidative modification of proteins by glucose, Biochemistry 34 (1995) 3702e3709. [8] P.J. Thornalley, The glyoxalase system: new developments towards functional characterization of a metabolic pathway fundamental to biological life, Biochem. J. 269 (1990) 1e11. [9] H. Vlassara, Advanced glycation in health and disease: role of the modern environment, Ann. N. Y. Acad. Sci. 1043 (2005) 452e460. [10] T. Goldberg, W. Cai, M. Peppa, V. Dardaine, B.S. Baliga, J. Uribarri, H. Vlassara, Advanced glycoxidation end products in commonly consumed foods, J. Am. Diet. Assoc. 104 (2004) 1287e1291. [11] D.S. Fosmark, P.A. Torjesen, B.K. Kilhovd, T.J. Berg, L. Sandvik, K.F. Hanssen, et

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