Bioluminescent reporter assay for monitoring ER stress in human beta cells

miRNAs are a class of small non-coding RNAs that regulate gene expression. Type 1 diabetes is an autoimmune disease characterized by insulitis (islets inflammation) and pancreatic beta cell destruction. The pro-inflammatory cytokines interleukin 1 beta (IL1B) and interferon gamma (IFNG) are released during insulitis and trigger endoplasmic reticulum (ER) stress and expression of pro-apoptotic members of the BCL2 protein family in beta cells, thus contributing to their death. The nature of miRNAs that regulate ER stress and beta cell apoptosis remains to be elucidated. We have performed a global miRNA expression profile on cytokine-treated human islets and observed a marked downregulation of miR-211-5p. By real-time PCR and Western blot analysis, we confirmed cytokine-induced changes in the expression of miR-211-5p and the closely related miR-204-5p and downstream ER stress related genes in human beta cells. Blocking of endogenous miRNA-211-5p and miR-204-5p by the same inhibitor (it is not possible to block separately these two miRs) increased human beta cell apoptosis, as measured by Hoechst/propidium Iodide staining and by determination of cleaved caspase-3 activation. Interestingly, miRs-211-5p and 204-5p regulate the expression of several ER stress markers downstream of PERK, particularly the pro-apoptotic protein DDIT3 (also known as CHOP). Blocking CHOP expression by a specific siRNA partially prevented the increased apoptosis observed following miR-211-5p/miR-204-5p inhibition. These observations identify a novel crosstalk between miRNAs, ER stress and beta cell apoptosis in early type 1 diabetes.

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Hyperglycemia-Induced Proliferation of Adult Human Beta Cells Engrafted Into Spontaneously Diabetic Immunodeficient NOD-Rag1null IL2rγnull Ins2Akita Mice

Pancreas ◽

10.1097/mpa.0b013e31821ffabe ◽

2011 ◽

Vol 40 (7) ◽

pp. 1147-1149 ◽

Cited By ~ 13

Author(s):

Philip diIorio ◽

Agata Jurczyk ◽

Chaoxing Yang ◽

Waldemar J. Racki ◽

Michael A. Brehm ◽

...

Keyword(s):

Beta Cells ◽

Adult Human ◽

Human Beta Cells

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Human Beta Cells Produce and Release Serotonin to Inhibit Glucagon Secretion from Alpha Cells

Cell Reports ◽

10.1016/j.celrep.2016.11.072 ◽

2016 ◽

Vol 17 (12) ◽

pp. 3281-3291 ◽

Cited By ~ 58

Author(s):

Joana Almaça ◽

Judith Molina ◽

Danusa Menegaz ◽

Alexey N. Pronin ◽

Alejandro Tamayo ◽

...

Keyword(s):

Beta Cells ◽

Glucagon Secretion ◽

Alpha Cells ◽

Human Beta Cells

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Eukaryotic Translation Initiation Factor 2A (eIF2A) Protects Pancreatic Beta Cells During ER Stress-Induced ApoptosisImage 6

Canadian Journal of Diabetes ◽

10.1016/j.jcjd.2016.08.038 ◽

2016 ◽

Vol 40 (5) ◽

pp. S13

Author(s):

Evgeniy Panzhinskiy ◽

Farnaz Taghizadeh ◽

Kwan Yi Chu ◽

Yu Hsuan Yang ◽

Eric Jan ◽

...

Keyword(s):

Er Stress ◽

Beta Cells ◽

Translation Initiation ◽

Pancreatic Beta Cells ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Eukaryotic Translation Initiation Factor ◽

Eukaryotic Translation ◽

Eukaryotic Translation Initiation

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Cloning and quantitative determination of the human Ca 2+ /calmodulin-dependent protein kinase II (CaMK II) isoforms in human beta cells

Diabetologia ◽

10.1007/s001250051330 ◽

2000 ◽

Vol 43 (4) ◽

pp. 465-473 ◽

Cited By ~ 18

Author(s):

H. Rochlitz ◽

A. Voigt ◽

B. Lankat-Buttgereit ◽

B. G�ke ◽

H. Heimberg ◽

...

Keyword(s):

Protein Kinase ◽

Quantitative Determination ◽

Beta Cells ◽

Dependent Protein Kinase ◽

Protein Kinase Ii ◽

Human Beta Cells ◽

Dependent Protein

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Disruption of Beta-Cell Mitochondrial Networks by the Orphan Nuclear Receptor Nor1/Nr4a3

Cells ◽

10.3390/cells9010168 ◽

2020 ◽

Vol 9 (1) ◽

pp. 168 ◽

Cited By ~ 2

Author(s):

Anne-Françoise Close ◽

Nidheesh Dadheech ◽

Hélène Lemieux ◽

Qian Wang ◽

Jean Buteau

Keyword(s):

Beta Cell ◽

Beta Cells ◽

Nuclear Receptor ◽

Pancreatic Beta Cells ◽

Orphan Nuclear Receptor ◽

Atp Production ◽

Human Beta Cells ◽

Glucose Stimulated Insulin Secretion ◽

Mitochondrial Networks

Nor1, the third member of the Nr4a subfamily of nuclear receptor, is garnering increased interest in view of its role in the regulation of glucose homeostasis. Our previous study highlighted a proapoptotic role of Nor1 in pancreatic beta cells and showed that Nor1 expression was increased in islets isolated from type 2 diabetic individuals, suggesting that Nor1 could mediate the deterioration of islet function in type 2 diabetes. However, the mechanism remains incompletely understood. We herein investigated the subcellular localization of Nor1 in INS832/13 cells and dispersed human beta cells. We also examined the consequences of Nor1 overexpression on mitochondrial function and morphology. Our results show that, surprisingly, Nor1 is mostly cytoplasmic in beta cells and undergoes mitochondrial translocation upon activation by proinflammatory cytokines. Mitochondrial localization of Nor1 reduced glucose oxidation, lowered ATP production rates, and inhibited glucose-stimulated insulin secretion. Western blot and microscopy images revealed that Nor1 could provoke mitochondrial fragmentation via mitophagy. Our study unveils a new mode of action for Nor1, which affects beta-cell viability and function by disrupting mitochondrial networks.

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Stearoyl CoA desaturase is a gatekeeper that protects human beta cells against lipotoxicity and maintains their identity

Diabetologia ◽

10.1007/s00125-019-05046-x ◽

2019 ◽

Vol 63 (2) ◽

pp. 395-409 ◽

Cited By ~ 4

Author(s):

Masaya Oshima ◽

Séverine Pechberty ◽

Lara Bellini ◽

Sven O. Göpel ◽

Mélanie Campana ◽

...

Keyword(s):

Type 2 Diabetes ◽

Endoplasmic Reticulum ◽

Endoplasmic Reticulum Stress ◽

Beta Cell ◽

Beta Cells ◽

Cell Identity ◽

Human Beta Cells ◽

Human Beta Cell ◽

Stearoyl Coa Desaturase

Abstract Aims/hypothesis During the onset of type 2 diabetes, excessive dietary intake of saturated NEFA and fructose lead to impaired insulin production and secretion by insulin-producing pancreatic beta cells. The majority of data on the deleterious effects of lipids on functional beta cell mass were obtained either in vivo in rodent models or in vitro using rodent islets and beta cell lines. Translating data from rodent to human beta cells remains challenging. Here, we used the human beta cell line EndoC-βH1 and analysed its sensitivity to a lipotoxic and glucolipotoxic (high palmitate with or without high glucose) insult, as a way to model human beta cells in a type 2 diabetes environment. Methods EndoC-βH1 cells were exposed to palmitate after knockdown of genes related to saturated NEFA metabolism. We analysed whether and how palmitate induces apoptosis, stress and inflammation and modulates beta cell identity. Results EndoC-βH1 cells were insensitive to the deleterious effects of saturated NEFA (palmitate and stearate) unless stearoyl CoA desaturase (SCD) was silenced. SCD was abundantly expressed in EndoC-βH1 cells, as well as in human islets and human induced pluripotent stem cell-derived beta cells. SCD silencing induced markers of inflammation and endoplasmic reticulum stress and also IAPP mRNA. Treatment with the SCD products oleate or palmitoleate reversed inflammation and endoplasmic reticulum stress. Upon SCD knockdown, palmitate induced expression of dedifferentiation markers such as SOX9, MYC and HES1. Interestingly, SCD knockdown by itself disrupted beta cell identity with a decrease in mature beta cell markers INS, MAFA and SLC30A8 and decreased insulin content and glucose-stimulated insulin secretion. Conclusions/interpretation The present study delineates an important role for SCD in the protection against lipotoxicity and in the maintenance of human beta cell identity. Data availability Microarray data and all experimental details that support the findings of this study have been deposited in in the GEO database with the GSE130208 accession code.

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