Differential activation of ER stress and apoptosis in response to chronically elevated free fatty acids in pancreatic β-cells

Chronic exposure to elevated saturated free fatty acid (FFA) levels has been shown to induce endoplasmic reticulum (ER) stress that may contribute to promoting pancreatic β-cell apoptosis. Here, we compared the effects of FFAs on apoptosis and ER stress in human islets and two pancreatic β-cell lines, rat INS-1 and mouse MIN6 cells. Isolated human islets cultured in vitro underwent apoptosis, and markers of ER stress pathways were elevated by chronic palmitate exposure. Palmitate also induced apoptosis in MIN6 and INS-1 cells, although the former were more resistant to both apoptosis and ER stress. MIN6 cells were found to express significantly higher levels of ER chaperone proteins than INS-1 cells, which likely accounts for the ER stress resistance. We attempted to determine the relative contribution that ER stress plays in palmitate-induced β-cell apoptosis. Although overexpressing GRP78 in INS-1 cells partially reduced susceptibility to thapsigargin, this failed to reduce palmitate-induced ER stress or apoptosis. In INS-1 cells, palmitate induced apoptosis at concentrations that did not result in significant ER stress. Finally, MIN6 cells depleted of GRP78 were more susceptible to tunicamycin-induced apoptosis but not to palmitate-induced apoptosis compared with control cells. These results suggest that ER stress is likely not the main mechanism involved in palmitate-induced apoptosis in β-cell lines. Human islets and MIN6 cells were found to express high levels of stearoyl-CoA desaturase-1 compared with INS-1 cells, which may account for the decreased susceptibility of these cells to the cytotoxic effects of palmitate.

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Intermittent-Hypoxia-Induced Autophagy Activation Through the ER-Stress-Related PERK/eIF2α/ATF4 Pathway is a Protective Response to Pancreatic β-Cell Apoptosis

Cellular Physiology and Biochemistry ◽

10.1159/000496047 ◽

2018 ◽

Vol 51 (6) ◽

pp. 2955-2971 ◽

Cited By ~ 16

Author(s):

Shuling Song ◽

Jin Tan ◽

Yuyang Miao ◽

Zuoming Sun ◽

Qiang Zhang

Keyword(s):

Er Stress ◽

Signaling Pathway ◽

Cell Apoptosis ◽

Intermittent Hypoxia ◽

Autophagic Vacuole ◽

Pancreatic Β Cell ◽

Β Cell ◽

Protective Response

Background/Aims: Intermittent hypoxia (IH) causes apoptosis in pancreatic β-cells, but the potential mechanisms remain unclear. Endoplasmic reticulum (ER) stress, autophagy, and apoptosis are interlocked in an extensive crosstalk. Thus, this study aimed to investigate the contributions of ER stress and autophagy to IH-induced pancreatic β-cell apoptosis. Methods: We established animal and cell models of IH, and then inhibited autophagy and ER stress by pharmacology and small interfering RNA (siRNA) in INS-1 cells and rats. The levels of biomarkers for autophagy, ER stress, and apoptosis were evaluated by immunoblotting and immunofluorescence. The number of autophagic vacuoles was observed by transmission electron microscopy. Results: IH induced autophagy activation both in vivo and in vitro, as evidenced by increased autophagic vacuole formation and LC3 turnover, and decreased SQSTM1 level. The levels of ER-stress-related proteins, including GRP78, CHOP, caspase 12, phosphorylated (p)-protein kinase RNA-like ER kinase (PERK), p-eIF2α, and activating transcription factor 4 (ATF4) were increased under IH conditions. Inhibition of ER stress with tauroursodeoxycholic acid or 4-phenylbutyrate partially blocked IH-induced autophagy in INS-1 cells. Furthermore, inhibition of PERK with GSK2606414 or siRNA blocked the ERstress-related PERK/eIF2α/ATF4 signaling pathway and inhibited autophagy induced by IH, which indicates that IH-induced autophagy activation is dependent on this signaling pathway. Promoting autophagy with rapamycin alleviated IH-induced apoptosis, whereas inhibition of autophagy with chloroquine or autophagy-related gene (Atg5 and Atg7) siRNA aggravated pancreatic β-cell apoptosis caused by IH. Conclusion: IH induces autophagy activation through the ER-stress-related PERK/eIF2α/ATF4 signaling pathway, which is a protective response to pancreatic β-cell apoptosis caused by IH.

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1,25-(OH)2D3 protects pancreatic beta cells against H2O2-induced apoptosis through inhibiting the PERK-ATF4-CHOP pathway

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/gmaa138 ◽

2020 ◽

Author(s):

Xiaobo Hu ◽

Cong Hu ◽

Jun Liu ◽

Zhuan Wu ◽

Tingting Duan ◽

...

Keyword(s):

Er Stress ◽

Cell Apoptosis ◽

Pancreatic Beta Cells ◽

Critical Role ◽

Active Form ◽

Cell Counting ◽

Pancreatic Β Cell ◽

Β Cell ◽

Min6 Cells ◽

Cell Destruction

Abstract Endoplasmic reticulum (ER) stress plays a critical role in pancreatic β cell destruction which leads to the pathogenesis of type 1 diabetes mellitus (T1DM). Vitamin D (VD) has been reported to reduce the risk of T1DM; however, it remains unknown whether VD affects ER stress in pancreatic β cells. In this study, we investigated the role of the active form of VD, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), in ER stress-induced β cell apoptosis and explored its potential mechanism in mouse insulinoma cell line mouse insulinoma 6 (MIN6). The results of cell counting kit-8 (CCK8) and flow cytometric analyses showed that 1,25-(OH)2D3 caused a significant increase in the viability of MIN6 cells injured by H2O2. The protein kinase like ER kinase (PERK) signal pathway, one of the most conserved branches of ER stress, was found to be involved in this process. H2O2 activated the phosphorylation of PERK, upregulated the activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) expression, and subsequently initiated cell apoptosis, which were significantly reversed by 1,25-(OH)2D3 pretreatment. In addition, GSK2606414, a specific inhibitor of PERK, suppressed PERK phosphorylation and reduced the expressions of ATF4 and CHOP, leading to a significant decrease in β cell apoptosis induced by H2O2. Taken together, the present findings firstly demonstrated that 1,25-(OH)2D3 could prevent MIN6 cells against ER stress-associated apoptosis by inhibiting the PERK-ATF4-CHOP pathway. Therefore, our results suggested that 1,25-(OH)2D3 might serve as a potential therapeutic target for preventing pancreatic β cell destruction in T1DM.

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Dexamethasone induces pancreatic β-cell apoptosis through upregulation of TRAIL death receptor

Journal of Molecular Endocrinology ◽

10.1530/jme-20-0238 ◽

2021 ◽

Author(s):

Kanchana Suksri ◽

Namoiy Semprasert ◽

Mutita Junking ◽

Suchanoot Kutpruek ◽

Thawornchai Limjindaporn ◽

...

Keyword(s):

Cell Apoptosis ◽

Molecular Mechanisms ◽

Cultured Cells ◽

Death Receptor ◽

Caspase 8 ◽

Pancreatic Β Cell ◽

Β Cell ◽

Apoptotic Protein ◽

Induced Apoptosis ◽

Trail Death Receptor

Long-term medication with dexamethasone (a synthetic glucocorticoid (GC) drug) results in hyperglycemia, or steroid-induced diabetes. Although recent studies revealed dexamethasone directly induces pancreatic β-cell apoptosis, its molecular mechanisms remain unclear. In our initial analysis of mRNA transcripts, we discovered the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway may be involved in dexamethasone-induced pancreatic β-cell apoptosis. In the present study, a mechanism of dexamethasone-induced pancreatic β-cell apoptosis through the TRAIL pathway was investigated in cultured cells and isolated mouse islets. INS-1 cells were cultured with and without dexamethasone in the presence or absence of a glucocorticoid receptor (GR) inhibitor, RU486. We found that dexamethasone induced pancreatic β-cell apoptosis in association with the upregulation of TRAIL mRNA and protein expression. Moreover, dexamethasone upregulated the TRAIL death receptor (DR5) protein but suppressed the decoy receptor (DcR1) protein. Similar findings were observed in mouse isolated islets: dexamethasone increased TRAIL and DR5 compared to that of control mice. Furthermore, dexamethasone stimulated pro-apoptotic signaling including superoxide production, caspase-8, -9, and -3 activities, NF-B, and Bax, but repressed the anti-apoptotic protein, Bcl-2. All these effects were inhibited by the GR-inhibitor, RU486. Furthermore, knock down DR5 decreased dexamethasone-induced caspase 3 activity. Caspase-8 and caspase-9 inhibitors protected pancreatic β-cells from dexamethasone-induced apoptosis. Taken together, dexamethasone induced pancreatic β-cell apoptosis by binding to the GR and inducing DR5 and TRAIL pathway.

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Enhancer of Zeste Homolog 2 (EZH2) Mediates Glucolipotoxicity-Induced Apoptosis in β-Cells

International Journal of Molecular Sciences ◽

10.3390/ijms21218016 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8016

Author(s):

Tina Dahlby ◽

Christian Simon ◽

Marie Balslev Backe ◽

Mattias Salling Dahllöf ◽

Edward Holson ◽

...

Keyword(s):

Er Stress ◽

Molecular Mechanisms ◽

Selective Inhibition ◽

Human Islets ◽

Β Cells ◽

Β Cell ◽

Enhancer Of Zeste ◽

Cell Gene Expression ◽

Nfκb Pathway ◽

Induced Apoptosis

Selective inhibition of histone deacetylase 3 (HDAC3) prevents glucolipotoxicity-induced β-cell dysfunction and apoptosis by alleviation of proapoptotic endoplasmic reticulum (ER) stress-signaling, but the precise molecular mechanisms of alleviation are unexplored. By unbiased microarray analysis of the β-cell gene expression profile of insulin-producing cells exposed to glucolipotoxicity in the presence or absence of a selective HDAC3 inhibitor, we identified Enhancer of zeste homolog 2 (EZH2) as the sole target candidate. β-Cells were protected against glucolipotoxicity-induced ER stress and apoptosis by EZH2 attenuation. Small molecule inhibitors of EZH2 histone methyltransferase activity rescued human islets from glucolipotoxicity-induced apoptosis. Moreover, EZH2 knockdown cells were protected against glucolipotoxicity-induced downregulation of the protective non-canonical Nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) pathway. We conclude that EZH2 deficiency protects from glucolipotoxicity-induced ER stress, apoptosis and downregulation of the non-canonical NFκB pathway, but not from insulin secretory dysfunction. The mechanism likely involves transcriptional regulation via EZH2 functioning as a methyltransferase and/or as a methylation-dependent transcription factor.

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Dihydrochalcone Derivative Induces Breast Cancer Cell Apoptosis via Intrinsic, Extrinsic, and ER Stress Pathways but Abolishes EGFR/MAPK Pathway

BioMed Research International ◽

10.1155/2019/7298539 ◽

2019 ◽

Vol 2019 ◽

pp. 1-18 ◽

Cited By ~ 3

Author(s):

Wasitta Rachakhom ◽

Patompong Khaw-on ◽

Wilart Pompimon ◽

Ratana Banjerdpongchai

Keyword(s):

Breast Cancer ◽

Er Stress ◽

Cell Lines ◽

Cell Apoptosis ◽

Mapk Pathway ◽

Annexin V ◽

Mapk Signaling ◽

Dependent Manner ◽

Induced Apoptosis ◽

Mcf 7

Dihydrochalcone derivatives are active compounds that have been purified from the Thai medicinal plant Cyathostemma argenteum. The objectives of this study were to investigate the effects of two dihydrochalcone derivatives on human breast cancer MDA-MB-231 and MCF-7 cell proliferation and to study the relevant mechanisms involved. The two dihydrochalcone derivatives are 4′,6′-dihydroxy-2′,4-dimethoxy-5′-(2″-hydroxybenzyl)dihydrochalcone (compound 1) and calomelanone (2′,6′-dihydroxy-4,4′-dimethoxydihydrochalcone, compound 2), both of which induced cytotoxicity toward both cell lines in a dose-dependent manner by using MTT assay. Treatment with both derivatives induced apoptosis as determined by annexin V-FITC/propidium iodide employing flow cytometry. The reduction of mitochondrial transmembrane potential (staining with 3,3′-dihexyloxacarbocyanine iodide, DiOC6, employing a flow cytometer) was established in the compound 1-treated cells. Compound 1 induced caspase-3, caspase-8, and caspase-9 activities in both cell lines, as has been determined by specific colorimetric substrates and a spectrophotometric microplate reader which indicated the involvement of both the extrinsic and intrinsic pathways. Calcium ion levels in mitochondrial and cytosolic compartments increased in compound 1-treated cells as detected by Rhod-2AM and Fluo-3AM intensity, respectively, indicating the involvement of the endoplasmic reticulum (ER) stress pathway. Compound 1 induced cell cycle arrest via enhanced atm and atr expressions and by upregulating proapoptotic proteins, namely, Bim, Bad, and tBid. Moreover, compound 1 significantly inhibited the EGFR/MAPK signaling pathway. In conclusion, compound 1 induced MDA-MB-231 and MCF-7 cell apoptosis via intrinsic, extrinsic, and ER stress pathways, whereas it ameliorated the EGFR/MAPK pathway in the MCF-7 cell line. Consequently, it is believed that compound 1 could be effectively developed for cancer treatments.

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Resveratrol and curcumin enhance pancreatic β-cell function by inhibiting phosphodiesterase activity

Journal of Endocrinology ◽

10.1530/joe-14-0335 ◽

2014 ◽

Vol 223 (2) ◽

pp. 107-117 ◽

Cited By ~ 53

Author(s):

Michael Rouse ◽

Antoine Younès ◽

Josephine M Egan

Keyword(s):

Insulin Secretion ◽

Cell Lines ◽

Cell Function ◽

Human Islets ◽

Dependent Manner ◽

Pancreatic Β Cell ◽

Β Cells ◽

Β Cell ◽

Pde Inhibitors ◽

Β Cell Function

Resveratrol (RES) and curcumin (CUR) are polyphenols that are found in fruits and turmeric, and possess medicinal properties that are beneficial in various diseases, such as heart disease, cancer, and type 2 diabetes mellitus (T2DM). Results from recent studies have indicated that their therapeutic properties can be attributed to their anti-inflammatory effects. Owing to reports stating that they protect against β-cell dysfunction, we studied their mechanism(s) of action in β-cells. In T2DM, cAMP plays a critical role in glucose- and incretin-stimulated insulin secretion as well as overall pancreatic β-cell health. A potential therapeutic target in the management of T2DM lies in regulating the activity of phosphodiesterases (PDEs), which degrade cAMP. Both RES and CUR have been reported to act as PDE inhibitors in various cell types, but it remains unknown if they do so in pancreatic β-cells. In our current study, we found that both RES (0.1–10 μmol/l) and CUR (1–100 pmol/l)-regulated insulin secretion under glucose-stimulated conditions. Additionally, treating β-cell lines and human islets with these polyphenols led to increased intracellular cAMP levels in a manner similar to 3-isobutyl-1-methylxanthine, a classic PDE inhibitor. When we investigated the effects of RES and CUR on PDEs, we found that treatment significantly downregulated the mRNA expression of most of the 11 PDE isozymes, including PDE3B, PDE8A, and PDE10A, which have been linked previously to regulation of insulin secretion in islets. Furthermore, RES and CUR inhibited PDE activity in a dose-dependent manner in β-cell lines and human islets. Collectively, we demonstrate a novel role for natural-occurring polyphenols as PDE inhibitors that enhance pancreatic β-cell function.

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A novel secretagogin/ATF4 pathway is involved in oxidized LDL-induced endoplasmic reticulum stress and islet β-cell apoptosis

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/gmaa142 ◽

2020 ◽

Author(s):

Li Wu ◽

Yuncheng Lv ◽

Ying Lv ◽

Sunmin Xiang ◽

Zhibo Zhao ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Transcription Factor ◽

Er Stress ◽

Cell Apoptosis ◽

Initiation Factor ◽

Β Cells ◽

Β Cell ◽

Immunoprecipitation Assay ◽

Min6 Cells ◽

Stress Markers

Abstract Excessive accumulation of cholesterol in β cells initiates endoplasmic reticulum (ER) stress and associated apoptosis. We have reported that excessive uptake of cholesterol by MIN6 cells decreases the expression of secretagogin (SCGN) and then attenuates insulin secretion. Here, we aimed to determine whether cholesterol-induced SCGN decrease is involved in the modulation of ER stress and apoptosis in pancreatic β cells. In this study, MIN6 cells were treated with oxidized low-density lipoprotein (ox-LDL) for 24 h, and then intracellular lipid droplets and cell apoptosis were quantified, and SCGN and ER stress markers were identified by western blot analysis. Furthermore, small interfer RNA (siRNA)-mediated SCGN knockdown and recombinant plasmid-mediated SCGN restoration experiments were performed to confirm the role of SCGN in ER stress and associated cell apoptosis. Finally, the interaction of SCGN with ATF4 was computationally predicted and then validated by a co-immunoprecipitation assay. We found that ox-LDL treatment increased the levels of ER stress markers, such as phosphorylated protein kinase-like endoplasmic reticulum kinase, phosphorylated eukaryotic initiation factor 2 alpha, activating transcription factor 4 (ATF4), and transcription factor CCAAT-enhancer-binding protein homologous protein, and promoted MIN6 cell apoptosis; in addition, the expression of SCGN was downregulated. siRNA-mediated SCGN knockdown exacerbated β-cell ER stress by increasing ATF4 expression. Pretreatment of MIN6 cells with the recombinant SCGN partly antagonized ox-LDL-induced ER stress and apoptosis. Furthermore, a co-immunoprecipitation assay revealed an interaction between SCGN and ATF4 in MIN6 cells. Taken together, these results demonstrated that pancreatic β-cell apoptosis induced by ox-LDL treatment can be attributed, in part, to an SCGN/ATF4-dependent ER stress response.

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Protein Kinase B/Akt Prevents Fatty Acid-induced Apoptosis in Pancreatic β-Cells (INS-1)

Journal of Biological Chemistry ◽

10.1074/jbc.m208756200 ◽

2002 ◽

Vol 277 (51) ◽

pp. 49676-49684 ◽

Cited By ~ 182

Author(s):

Christian E. Wrede ◽

Lorna M. Dickson ◽

Melissa K. Lingohr ◽

Isabelle Briaud ◽

Christopher J. Rhodes

Keyword(s):

Growth Factor ◽

Protein Kinase ◽

Cell Apoptosis ◽

Glycogen Synthase ◽

Protein Kinase B ◽

Glycogen Synthase Kinase ◽

Pancreatic Β Cell ◽

Β Cells ◽

Β Cell ◽

Induced Apoptosis

Free fatty acids (FFA) have been reported to reduce pancreatic β-cell mitogenesis and to increase apoptosis. Here we show that the FFA, oleic acid, increased apoptosis 16-fold in the pancreatic β-cell line, INS-1, over a 18-h period as assessed by Hoechst 33342/propidium iodide staining and caspase-3 and -9 activation, with negligible necrosis. A parallel analysis of the phosphorylation activation of protein kinase B (PKB) showed this was reduced in the presence of FFA that correlated with the incidence of apoptosis. At stimulatory 15 mmglucose and/or in the added presence of insulin-like growth factor 1, FFA-induced β-cell apoptosis was lessened compared with that at a basal 5 mmglucose. However, most strikingly, adenoviral mediated expression of a constitutively active PKB, but not a “kinase-dead” PKB variant, essentially prevented FFA-induced β-cell apoptosis under all glucose/insulin-like growth factor 1 conditions. Further analysis of pro-apoptotic downstream targets of PKB, implicated a role for PKB-mediated phosphorylation inhibition of glycogen synthase kinase-3α/β and the forkhead transcription factor, FoxO1, in protection of FFA-induced β-cell apoptosis. In addition, down-regulation of the pro-apoptotic tumor suppresser protein, p53, via PKB-mediated phosphorylation of MDM2 might also play a role in partially protecting β-cells from FFA-induced apoptosis. Adenoviral mediated expression of wild type p53 potentiated FFA-induced β-cell apoptosis, whereas expression of a dominant negative p53 partly inhibited β-cell apoptosis by ∼50%. Hence, these data demonstrate that PKB activation plays an important role in promoting pancreatic β-cell survival in part via inhibition of the pro-apoptotic proteins glycogen synthase kinase-3α/β, FoxO1, and p53. This, in turn, provides novel insight into the mechanisms involved in FFA-induced β-cell apoptosis.

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The Hippo kinase LATS2 impairs pancreatic β-cell survival in diabetes through the mTORC1-autophagy axis

Nature Communications ◽

10.1038/s41467-021-25145-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ting Yuan ◽

Karthika Annamalai ◽

Shruti Naik ◽

Blaz Lupse ◽

Shirin Geravandi ◽

...

Keyword(s):

Cell Survival ◽

Cell Apoptosis ◽

Molecular Mechanisms ◽

Cell Mass ◽

Human Islets ◽

Hippo Signaling ◽

Pancreatic Β Cell ◽

Β Cells ◽

Β Cell ◽

Large Tumor

AbstractDiabetes results from a decline in functional pancreatic β-cells, but the molecular mechanisms underlying the pathological β-cell failure are poorly understood. Here we report that large-tumor suppressor 2 (LATS2), a core component of the Hippo signaling pathway, is activated under diabetic conditions and induces β-cell apoptosis and impaired function. LATS2 deficiency in β-cells and primary isolated human islets as well as β-cell specific LATS2 ablation in mice improves β-cell viability, insulin secretion and β-cell mass and ameliorates diabetes development. LATS2 activates mechanistic target of rapamycin complex 1 (mTORC1), a physiological suppressor of autophagy, in β-cells and genetic and pharmacological inhibition of mTORC1 counteracts the pro-apoptotic action of activated LATS2. We further show a direct interplay between Hippo and autophagy, in which LATS2 is an autophagy substrate. On the other hand, LATS2 regulates β-cell apoptosis triggered by impaired autophagy suggesting an existence of a stress-sensitive multicomponent cellular loop coordinating β-cell compensation and survival. Our data reveal an important role for LATS2 in pancreatic β-cell turnover and suggest LATS2 as a potential therapeutic target to improve pancreatic β-cell survival and function in diabetes.

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