Apigenin Alleviates Endoplasmic Reticulum Stress-Mediated Apoptosis in INS-1 β-Cells

Abstract

The improvement of type 2 diabetes mellitus induced by naturally occurring polyphenols, known as flavonoids, has received considerable attention. However, there is a dearth of information regarding the effect of the trihydroxyflavone apigenin on pancreatic β-cell function. In the present study, the anti-diabetic effect of apigenin on pancreatic β-cell insulin secretion, apoptosis, and the mechanism underlying its anti-diabetic effects, were investigated in the INS-ID β-cell line. The results showed that apigenin concentration-dependently facilitated 11.1-mM glucose-induced insulin secretion, which peaked at 30 µM. Apigenin also concentration-dependently inhibited the expression of endoplasmic reticulum (ER) stress signaling proteins, CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) and cleaved caspase-3, which was elevated by thapsigargin in INS-1D cells, with peak suppression at 30 µM. This was strongly correlated with the results of flow cytometric analysis of annexin V/propidium iodide (PI) staining and DNA fragmentation analysis. Moreover, the increased expression of thioredoxin-interacting protein (TXNIP) induced by thapsigargin was remarkably reduced by apigenin in a concentration-dependent manner. These results suggest that apigenin is an attractive candidate with remarkable and potent anti-diabetic effects on β-cells, which are mediated by facilitating glucose-stimulated insulin secretion and preventing ER stress-mediated β-cell apoptosis, the latter of which may be possibly mediated by reduced expression of CHOP and TXNIP, thereby promoting β-cell survival and function.

INTRODUCTION

Globally, the development of strategies for the prevention and treatment of diabetes remains a challenge. Diabetes is a chronic and complex disease characterized by hyperglycemia and other metabolic disorders, which results from pancreatic β-cell mass loss and insulin secretion deficiency. The involvement of β-cell apoptosis in such β-cell dysfunction has been reported.¹⁾ Hence, strategies preventing or suppressing β-cell apoptosis, which is expected to improve β-cell function and survival, would be valuable therapeutic approaches for the prevention and treatment of type 2 diabetes.

Numerous studies have shown that flavonoids with phenolic structures have beneficial roles in the prevention and treatment of diabetes.²⁾ Apigenin, 4′,5,7-trihydroxyflavone (Fig. 1), is one of the most common plant flavonoids commonly present in fruits, vegetables, nuts, olives, and other plant-derived beverages.³⁾ Apigenin has been shown to have numerous pharmacological activities, such as anti-oxidant,⁴⁾ anti-inflammatory,⁵⁾ immunomodulatory,⁶⁾ antibacterial,⁷⁾ and antiviral effects.⁸⁾ Although apigenin has been reported to exert antihyperglycemic effects in pancreatic β-cell lines,^9,10) the mechanisms for the effects of apigenin on β-cell functions have not been fully elucidated.

Fig. 1. Chemical Structure of Apigenin

In the present study, we aimed to elucidate the mechanism for the anti-diabetic effects of apigenin by determining whether apigenin facilitates insulin secretion and inhibits endoplasmic reticulum (ER) stress-mediated apoptosis in pancreatic β-cells. We particularly examined the hypothesis that the reduction of thioredoxin-interacting protein (TXNIP), an inhibitor of the anti-oxidant protein thioredoxin, may be involved in apigenin-mediated β-cell protective effects.

MATERIALS AND METHODS

Reagents

Apigenin (Fig. 1) with 95% purity was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Apigenin was dissolved in dimethyl sulfoxide (DMSO) to make a stock solution (100 mM). Once diluted in appropriate concentrations with DMSO just before experiments, the apigenin solution was further diluted in the RPMI1640 medium. The final concentration of DMSO was less than 0.3%, which was confirmed not to affect the function of INS-1 cells in our preliminary experiments. Control experiments were performed without the addition of the solvent.

Cell Culture

The rat pancreatic β cell-line INS-ID, obtained from Dr. C. Wollheim (University Medical Center, Geneva, Switzerland), was cultured in 11.1-mM glucose containing RPMI-1640 medium supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 100 µg/mL streptomycin, 100 U/mL penicillin G, 50 mM 2-mercaptoethanol, 10% fetal bovine serum (Gibco, Carlsbad, CA, U.S.A.), and 10 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) in 95% air-5% CO₂ at 37 °C.¹¹⁾ Medium exchange was performed every three days. Subcultures were performed once a week by trypsin-ethylenediaminetetraacetic acid (EDTA) treatment. The cells used in the present study were within passage 28 to 38.

Insulin Secretion Measurement

INS-ID cells were seeded in 24 well plate at a density of 2.5 × 10⁵ cells per well and cultured for 48 h, allowing for attachment. The cells were preincubated for 60 min at 37 °C in 2.8-mM glucose-containing HEPES-buffered Krebs (HK) solution (in mM: NaCl, 129; NaHCO₃, 5.0; KCl, 4.7; KH₂PO₄, 1.2; CaCl₂, 1.0; MgSO₄, 1.2; and HEPES, 10; adjusted to pH 7.4 with NaOH), and then stimulated with 11.1-mM glucose-containing HK solution in the absence or presence of various concentrations of apigenin. The supernatants of the incubation media were aspirated and stored at −20 °C until radioimmunoassay (RIA). The amount of insulin secretion was measured using a rat insulin RIA kit (EMD Millipore, Billerica, MA, U.S.A.).

Immunoblotting

INS-1D cells were seeded in 60 mm petri dishes at a density of 3 × 10⁶ cells/dish and cultured for 48 h. The cells were treated with thapsigargin (300 nM) for 14 h in the absence or presence of various concentrations of apigenin. The cells were then sonicated in ice-cold homogenization buffer containing 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 250 mM sucrose, 50 µg/mL phenylmethylsulfonylfluoride, 2 mM EDTA, snd 20 mM Tris–HCl (pH 7.4). The protein samples were then boiled in a buffer containing 2% sodium dodecyl sulfate (SDS), 10% glycerol, 5% 2-mercaptoethanol, and 0.0005% bromophenol blue, and 62.5 mM Tris–HCl (pH 6.8). The samples were separated in a polyacrylamide gel (10 or 12%) and transferred to a polyvinylidene difluoride membrane. After blocking with 5% skimmed milk or 3% bovine serum albumin (BSA) in a buffer consisting of 10 mM Tris–HCl, 137 mM NaCl, and 0.1% Tween 20 (TBS-T), the membrane was incubated overnight at 4 °C with a primary antibody against cleaved caspase-3 (#9661, cysteine-aspartic protease-3, Cell Signaling Technology, Danvers, MA, U.S.A.), CCAAT/enhancer binding protein homologous protein (CHOP) (#5554, Cell Signaling Technology), TXNIP (#K0205-3, Medical & Biological Laboratories Co., Ltd., Nagoya, Japan), or β-actin (#A5441, Sigma, St. Louis, MO, U.S.A.). After washing with TBS-T several times, the membrane was incubated with horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin G (IgG) antibody (#7074, Cell Signaling Technology) or anti-mouse goat IgG (#A4416, Sigma). Bands were visualized with ECL plus Western blotting reagent (GE Healthcare, Little Chalfont, U.K.) or Immunostar LD (Wako Pure Chemical Corporation) using a chemiluminescent imager (C-DiGit Blot Scanner, Li-COR, Lincoln, NE, U.S.A.; or Amersham Imager 680 QC, Cytiva, Marlborough, MA, U.S.A.). The molecular weight of the protein bands was determined by Precision Plus Protein Standards (Bio-Rad; Hercules, CA, U.S.A.). The densitometric analysis of the bands was performed using the NIH software ImageJ.

DNA Laddering

DNA fragmentation in INS-ID cells was quantified by agarose gel electrophoresis according to our previous method with slight modifications.¹²⁾ After washed with phosphate-buffered saline (PBS), the harvested cells (approximately 1.3 × 10⁶) were lysed with lysis buffer consisting of 0.5% sodium-N-lauroyl sarcosinate, 10 mM EDTA-2Na, and 50 mM Tris–HCl (pH 8.0). The lysates were then incubated in the lysis buffer supplemented with 0.33 mg/mL ribonuclease (RNase) A at 50 °C for 30 min, and further incubated in the lysis buffer supplemented with 0.33 mg/mL proteinase K at 50 °C for 1 h. After quantification, DNA was electrophoresed in a 2.0% agarose gel. The gels were stained with GEL Red (Wako Pure Chemical Corporation) and visualized under UV light.

Flow Cytometry

Apoptosis was analyzed by an Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining kit (#15342, Nacalai Tesque, Kyoto, Japan) using flow cytometry. INS-1D cells were grown in a 6 well plate at a density of 8.0 × 10⁵ cells per well for 48 h. The cells were incubated for 18 h in 11.1-mM glucose containing RPMI-1640 medium in the absence or presence of various concentrations of apigenin. The cells were harvested and pelleted by centrifugation at 100 × g for 5 min at room temperature. The cells were washed with PBS and resuspended in a binding buffer consisting of 150 mM NaCl, 1.8 mM CaCl₂, and 10 mM HEPES. One hundred µL of the solution was gently mixed with 5 µL annexin V-FITC and 5 µL propidium iodide and incubated in the dark for 15 min. Four hundred µL of the binding buffer was then added. The assay was carried out using CytoFLEX flow cytometry (Beckman Coulter, Brea, CA, U.S.A.).

Statistics

Results are expressed as mean ± standard error of the mean (S.E.M.). Comparisons were made using an unpaired t-test or Tukey’s or Dunnett’s multiple comparison test and p < 0.05 was considered statistically significant.

RESULTS

Effect of Apigenin on Insulin Secretion

Apigenin concentration-dependently facilitated 11.1-mM glucose-induced insulin secretion from INS-1D cells, peaking at 30 µM. However, at 100 µM, the insulinotropic effects of apigenin disappeared (Fig. 2).

Fig. 2. Effect of Apigenin on Insulin Secretion from INS-1 β-cells

Effect of apigenin on glucose-stimulated insulin secretion (GSIS) in INS-ID cells incubated for 1 h in HEPES-buffered Krebs solution containing 2.8- or 11.1-mM glucose in the presence or absence of apigenin at the indicated concentrations. Each data represents the mean ± S.E.M. of twelve independent experiments. ** p < 0.01.

Effect of Apigenin on ER Stress-Induced Apoptosis of β-Cells

Phase contrast microscopy images of cells revealed that INS-1D cells treated with thapsigargin for 14 h had numerous floating and apoptotic cells, whereas INS-1D cells treated with apigenin as well as thapsigargin had fewer floating and apoptotic cells (Fig. 3A).

Fig. 3. Effect of Apigenin on Thapsigargin-Induced β-Cell Apoptosis

A: Morphological changes in INS-ID cells exposed to thapsigargin (300 nM) for 14 h in the presence or absence of indicated treatments with apigenin. B: Gel electrophoresis of DNA extracts from INS-ID cells exposed to thapsigargin (300 nM) for 14 h in the presence of the indicated apigenin concentrations. Nobiletin was used as a positive control. C: Flow cytometry analysis of the anti-apoptotic effect of apigenin using Annexin V/PI staining. Upper: Representative flow cytometry charts of annexin V-FITC/PI staining of cells untreated and treated with thapsigargin (300 nM) for 14 h in the presence of the indicated concentrations of apigenin. Lower: Quantitative analysis of the percentage of early apoptotic cells under each condition. Each data represents the mean ± S.E.M. of three independent experiments. *p < 0.05, **p < 0.01.

DNA was extracted from INS-ID cells treated with thapsigargin (300 nM) for 14 h, which was used to induce ER stress, in the absence or presence of various concentrations of apigenin. We confirmed that in the cells treated with thapsigargen, DNA laddering indicating apoptosis was observed. Apigenin markedly and concentration-dependently decreased the thapsigargin-induced DNA laddering (Fig. 3B).

Confirmatory investigation of the effect of apigenin on β-cell apoptosis was further conducted by flow cytometry with annexin V/PI staining. The treatment with thapsigargin (300 nM) for 14 h caused a significant increase in the number of early apoptotic cells (annexin V-positive and PI-negative cells in the upper charts of Fig. 3C). Apigenin at concentrations between 10 and 100 µM significantly suppressed the apoptosis in a concentration-dependent manner (Fig. 3C).

Mechanism for the Anti-apoptotic Effect of Apigenin

Thapsigargin (300 nM; for 14 h) elicited a marked increase in the expression of CHOP (Fig. 4A), protein kinase RNA-like ER kinase (PERK), a downstream factor of the ER stress sensor protein, and cleaved caspase-3, a marker of cellular apoptosis, in INS-1D cells (Fig. 4B). Apigenin significantly and concentration-dependently suppressed thapsigargin-induced increases in the expression of CHOP (Fig. 4A) and cleaved caspase-3 (Fig. 4B).

Fig. 4. Effects of Apigenin on Apoptosis-Related Proteins

Effects of apigenin on the expression of apoptotic markers CHOP (A) and cleaved caspase-3 (B) in INS-ID cells exposed to thapsigargin (300 nM) for 14 h. Data represent the mean ± S.E.M. of six independent experiments. * p < 0.05, *** p < 0.001, **** p < 0.0001.

To explore the mechanism underlying the reduction of β-cell apoptosis induced by apigenin, we investigated the possible involvement of TXNIP, which has been shown to be a potential mediator of β-cell glucose toxicity and oxidative stress,¹³⁾ in the anti-apoptotic effect of apigenin. Immunoblot analysis showed that the treatment of INS-1D cells with thapsigargin (300 nM) for 14 h increased the expression of TXNIP (Fig. 5). Apigenin remarkably reduced the increased expression of TXNIP in a concentration-dependent manner (Fig. 5), suggesting that the decreased expression of TXNIP may be involved in the anti-apoptotic effect of apigenin.

Fig. 5. Effects of Apigenin on TXNIP Protein Expression

Effects of apigenin on the expression of TXNIP in INS-ID cells exposed to thapsigargin (300 nM) for 14 h. Data represent mean ± S.E.M. of four independent experiments. * p < 0.05.

DISCUSSION

Numerous studies have indicated the effective and beneficial role of flavonoids, including apigenin, in glucose homeostasis and diabetes.²⁾ Observational evidence has revealed that the consumption of flavonoid-rich vegetables and fruits reduces the incidence of type 2 diabetes mellitus.^14,15) In our previous study, the citrus peel flavonoid nobiletin was shown to suppress β-cell apoptosis and ameliorate glucose tolerance.^16,17) The present study showed that the dietary flavonoid apigenin inhibits ER stress-mediated apoptosis in INS-1D pancreatic β-cells. The present study further suggests that apigenin may inhibit β-cell apoptosis by reducing the expression of TXNIP, a protein involved in cellular redox state.

Apigenin, a naturally occurring dietary flavone, has gained growing interest owing to its pharmacological activity, minimal side effects, and affordability.⁵⁾ Numerous previous studies have reported that apigenin promotes cancer cell apoptosis through its autophagy-promoting effect.^18–20) In contrast, the cytoprotective effect of apigenin against oxidative stress, inflammation, apoptosis, and oxidative and ER stresses has been demonstrated in various cell types.^3,21) Although the cytoprotective effect of apigenin has also been reported in pancreatic β-cell lines RINm5F and HIT15,^10,22) its effects and mechanisms on ER stress-mediated apoptosis have been unknown.

The present study showed that apigenin at concentrations up to 30 µM facilitated glucose-induced insulin secretion from pancreatic β-cells. Our results are consistent with those previously reported on the stimulation of GSIS by extra virgin oil flavonoids containing apigenin in INS-IE cells.²³⁾ Although the exact mechanism responsible for the facilitation of insulin secretion in INS-I cells is still unknown and remains to be investigated, mounting evidence from a previous report suggests the involvement of the activation of Akt and cAMP response element-binding protein (CREB).²²⁾ The insulinotropic effect of apigenin was abolished at the higher concentration of 100 µM. Flavonoids are known to have cytotoxic properties²⁴⁾ and our previous study also showed that tea catechins, a type of flavonoid, have an inhibitory effect on insulin secretion.²⁵⁾ Apigenin at high concentrations may suppress insulin secretion by activating signaling pathways other than those involved in its insulinotropic effect.

Thapsigargin, an inhibitor of ER Ca²⁺-ATPase, induces ER stress-mediated apoptosis. The present study showed that the treatment of INS-1D cells with thapsigargin induced the pro-apoptotic stress transcription factor CHOP and robustly led to the generation of the active form of caspase-3, which was accompanied by a dramatic exacerbation of DNA laddering; hence, the treatment could be regarded as successfully mimicking the ER stress scenario and consequently the cellular injuries associated with diabetic pathogenesis. Apigenin treatment seemed to attenuate thapsigargin-induced pancreatic β-cell damage by its protective effect on DNA fragmentation and suppression of CHOP and cleaved caspases-3 induction. The results of flow cytometry were in agreement with the DNA electrophoresis and cell morphology results. In several diseases, including diabetes, the ER stress pathway is identified as an apoptosis signaling pathway.²¹⁾ The transcription factors, CHOP and cleaved caspase-3, are good markers of ER stress, as they are expressed under conditions of ER dysfunction in pancreatic β-cells.²⁶⁾ Apigenin exhibits anti-apoptotic activity by protecting cells against ER stress, accompanied by decreased levels of apoptotic markers, CHOP, and cleaved caspase-3.

TXNIP has been recognized as a key regulator of diabetic β-cell apoptosis and dysfunction, potentially mediating β-cell glucotoxicity and oxidative stress.^26–28) Its expression is tightly regulated by metabolic stimuli and stress including ER-stress.^28,29) The present study showed that the expression of TXNIP, which was increased by the thapsigargin treatment, was downregulated in INS-1D cells in response to apigenin. This was well correlated with the apigenin-induced reduction of apoptotic markers, CHOP and cleaved caspase-3. The upregulation of TXNIP during ER stress has been shown to be mediated by inositol-requiring enzyme 1 (IRE1) and PERK in β-cells.³⁰⁾ This reduction of TXNIP expression may therefore be essential for the anti-apoptotic effect of apigenin, as shown by the results that apigenin blunted the thapsigargin-induced increase in apoptotic factors. Thapsigargin is known to induce apoptosis through not only through ER stress-mediated pathway but also through mitochondrial dysfunction.³¹⁾ It has also been reported that TXNIP deficiency in β-cells inhibits apoptosis mediated by mitochondrial dysfunction.²⁸⁾ Therefore, the apigenin-induced reduction of TXNIP expression not only reduces ER stress response but also may ameliorate mitochondrial dysfunction, thereby inhibiting apoptosis. However, since the current in vitro studies did not examine the effect of apigenin under the condition that TXNIP signaling was blocked, further investigation is needed.

In summary, apigenin exerts anti-diabetic effects in pancreatic β-cells, which are mediated by increasing glucose-stimulated insulin secretion and preventing ER stress-mediated β-cell apoptosis, thereby promoting β-cell function and survival. The inhibitory effect of apigenin on TXNIP expression is likely to be involved in its anti-apoptotic effects of apigenin.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA, Butler PC. β-Cell deficit and increased β-cell apoptosis in humans with type 2 diabetes. Diabetes, 52, 102–110 (2003).
• 2) Ghorbani A, Rashidi R, Shafiee-Nick R. Flavonoids for preserving pancreatic beta cell survival and function: a mechanistic review. Biomed. Pharmacother., 111, 947–957 (2019).
• 3) Salehi B, Venditti A, Sharifi-rad M, Kręgiel D, Sharifi-rad J, Durazzo A, Lucarini M, Santini A, Souto EB, Novellino E, Antolak H, Azzini E. The therapeutic potential of apigenin. Int. J. Mol. Sci., 20, 1305 (2019).
• 4) Sánchez-Marzo N, Pérez-Sánchez A, Ruiz-Torres V, Martínez-Tébar A, Castillo J, Herranz-López M, Barrajón-Catalán E. Antioxidant and photoprotective activity of apigenin and its potassium salt derivative in human keratinocytes and absorption in caco-2 cell monolayers. Int. J. Mol. Sci., 20, 2148 (2019).
• 5) Ginwala R, Bhavsar R, Chigbu DGI, Jain P, Khan ZK. Potential role of flavonoids in treating chronic inflammatory diseases with a special focus on the anti-inflammatory activity of apigenin. Antioxidants (Basel), 8, 35 (2019).
• 6) Hosseinzade A, Sadeghi O, Naghdipour Biregani A, Soukhtehzari S, Brandt GS, Esmaillzadeh A. Immunomodulatory effects of flavonoids: Possible induction of T CD4+ regulatory cells through suppression of mTOR pathway signaling activity. Front. Immunol., 10, 51 (2019).
• 7) Nayaka HB, Londonkar RL, Umesh MK, Tukappa A. Antibacterial attributes of apigenin, isolated from Portulaca oleracea L. Int. J. Bacteriol., 2014, 175851 (2014).
• 8) Wang L, Song J, Liu A, Xiao B, Li S, Wen Z, Lu Y, Du G. Research progress of the antiviral bioactivities of natural flavonoids. Nat. Prod. Bioprospect., 10, 271–283 (2020).
• 9) Panda S, Kar A. Apigenin (4′,5,7-trihydroxyflavone) regulates hyperglycaemia, thyroid dysfunction and lipid peroxidation in alloxan-induced diabetic mice. J. Pharm. Pharmacol., 59, 1543–1548 (2010).
• 10) Wang N, Yi WJ, Tan L, Zhang JH, Xu J, Chen Y, Qin M, Yu S, Guan J, Zhang R. Apigenin attenuates streptozotocin-induced pancreatic b cell damage by its protective effects on cellular antioxidant defense. In Vitro Cell. Dev. Biol. Anim., 53, 554–563 (2017).
• 11) Asfari M, Janjic D, Meda P, Li G, Halban PA, Wollheim CB. Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines. Endocrinology, 130, 167–178 (1992).
• 12) Noguchi A, Takada M, Nakayama K, Ishikawa T. cGMP-independent anti-apoptotic effect of nitric oxide on thapsigargin-induced apoptosis in the pancreatic β-cell line INS-1. Life Sci., 83, 865–870 (2008).
• 13) Chen J, Saxena G, Mungrue IN, Lusis AJ, Shalev A. Thioredoxin-Interacting protein: a critical link between glucose toxicity and β-Cell apoptosis. Diabetes, 57, 938–944 (2008).
• 14) Wedick NM, Pan A, Cassidy A, Rimm EB, Sampson L, Rosner B, Willett W, Hu FB, Sun Q, van Dam RM. Dietary flavonoid intakes and risk of type 2 diabetes in U.S. men and women. Am. J. Clin. Nutr., 95, 925–933 (2012).
• 15) Jacques PF, Cassidy A, Rogers G, Peterson JJ, Meigs JB, Dwyer JT. Higher dietary flavonol intake is associated with lower incidence of type 2 diabetes. J. Nutr., 143, 1474–1480 (2013).
• 16) Takii M, Kaneko YK, Akiyama K, Aoyagi Y, Tara Y, Asakawa T, Inai M, Kan T, Nemoto K, Ishikawa T. Insulinotropic and anti-apoptotic effects of nobiletin in INS-1D β-cells. J. Funct. Foods, 30, 8–15 (2017).
• 17) Kaneko YK, Tara Y, Ihim SA, Yamamoto M, Kaji M, Ishikawa T. Nobiletin ameliorates glucose tolerance by protecting against β-cell loss in type-2 diabetic db/db mice. Phytomed. Plus, 2, 100367 (2022).
• 18) Kim TW, Lee HG. Apigenin induces autophagy and cell death by targeting EZH2 under hypoxia conditions in gastric cancer cells. Int. J. Mol. Sci., 22, 13455 (2021).
• 19) Sung B, Chung HY, Kim ND. Role of apigenin in cancer prevention via the induction of apoptosis and autophagy. J. Cancer Prev, 21, 216–226 (2016).
• 20) Yan X, Qi M, Li P, Zhan Y, Shao H. Apigenin in cancer therapy: anti-cancer effects and mechanisms of action. Cell Biosci., 7, 50 (2017).
• 21) Martucciello S, Masullo M, Cerulli A, Piacente S. Natural products targeting er stress, and the functional link to mitochondria. Int. J. Mol. Sci., 21, 1905 (2020).
• 22) Suh KS, Oh S, Woo J-T, Kim S-W, Kim J-W, Kim YS, Chon S. Apigenin attenuates 2-deoxy-D-ribose-induced oxidative cell damage in HIT-T15 pancreatic β-cells. Biol. Pharm. Bull., 35, 121–126 (2012).
• 23) Marrano N, Spagnuolo R, Biondi G, Cignarelli A, Perrini S, Vincenti L, Laviola L, Giorgino F, Natalicchio A. Effects of extra virgin olive oil polyphenols on β-cell function and survival. Plants (Basel), 10, 286 (2021).
• 24) Galati G, O’Brien PJ. Potential toxicity of flavonoids and other dietary phenolics: significance for their chemopreventive and anticancer properties. Free Radic. Biol. Med., 37, 287–303 (2004).
• 25) Kaneko YK, Takii M, Kojima Y, Yokosawa H, Ishikawa T. Structure-dependent inhibitory effects of green tea catechins on insulin secretion from pancreatic β-cells. Biol. Pharm. Bull., 38, 476–481 (2015).
• 26) Leibowitz G, Bachar E, Shaked M, Sinai A, Ketzinel-Gilad M, Cerasi E, Kaiser N. Glucose regulation of β-cell stress in type 2 diabetes. Diabetes Obes. Metab., 12 (Suppl. 2), 66–75 (2010).
• 27) Hong K, Xu G, Grayson TB, Shalev A. Cytokines regulate β-cell thioredoxin-interacting protein (TXNIP) via distinct mechanisms and pathways. J. Biol. Chem., 291, 8428–8439 (2016).
• 28) Shalev A. Minireview: thioredoxin-interacting protein: regulation and function in the pancreatic β-Cell. Mol. Endocrinol., 28, 1211–1220 (2014).
• 29) Lerner AG, Upton J-P, Praveen PVK, Ghosh R, Nakagawa Y, Igbaria A, Shen S, Nguyen V, Backes BJ, Heiman M, Heintz N, Greengard P, Hui S, Tang Q, Trusina A, Oakes SA, Papa FR. IRE1α induces thioredoxin-interacting protein to activate the NLRP3 inflammasome and promote programmed cell death under irremediable ER stress. Cell Metab., 16, 250–264 (2012).
• 30) Oslowski CM, Hara T, O’Sullivan-Murphy B, Kanekura K, Lu S, Hara M, Ishigaki S, Zhu LJ, Hayashi E, Hui ST, Greiner D, Kaufman RJ, Bortell R, Urano F. Thioredoxin-interacting protein mediates er stress-induced β cell death through initiation of the inflammasome. Cell Metab., 16, 265–273 (2012).
• 31) Iurlaro R, Muñoz-Pinedo C. Cell death induced by endoplasmic reticulum stress. FEBS J., 283, 2640–2652 (2016).