scholarly journals DNA damage induces senescent phenotypes in mouse NIT1 beta cells and human islets

2021 ◽  
Author(s):  
Gabriel Brawerman ◽  
Peter J. Thompson
Keyword(s):  
Dna Damage ◽  
Beta Cell ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Transcriptional Response ◽  
Strand Break ◽  
Human Islets ◽  
Cell Senescence ◽  
Dna Double Strand Break ◽  
Beta Cell Line

Pancreatic beta cells are essential endocrine cells in the islets of Langerhans that respond to increases in blood glucose by secreting insulin and their dysfunction and death drives the development of diabetes. Beta cell senescence involving a DNA damage response (DDR) was recently shown to contribute to the pathogenesis of Type 1 Diabetes (T1D), however, a basic mechanistic understanding of how senescence develops in beta cells is lacking. Here, we investigated senescent phenotypes arising in the mouse beta cell line NIT1 derived from the T1D susceptible nonobese diabetic (NOD) mouse strain and the transcriptome response in human islets after induction of DNA damage. Sub-lethal DNA damage in NIT1 cells led to several classical hallmarks of senescence including the DDR, growth arrest, enlarged flattened morphology and a senescence-associated secretory phenotype (SASP) resembling what occurs during T1D. RNA-seq analysis on human islets with DNA double-strand break damage revealed a coordinated p53-p21 transcriptional response and upregulation of genes involved in prosurvival signaling and SASP. Taken together, these findings suggest that some of the phenotypes of mouse and human beta cell senescence during T1D can be induced by DNA damage in NIT1 cells and human islets in culture.

scholarly journals High dose of histone deacetylase inhibitors affects insulin secretory mechanism of pancreatic beta cell line

2018 ◽  
Vol 52 (1) ◽  
pp. 21-26 ◽  
Author(s):  
Eiji Yamato
Keyword(s):  
Insulin Secretion ◽  
Beta Cell ◽  
Cell Line ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Pancreatic Beta Cell ◽  
High Dose ◽  
Min6 Cells ◽  
Beta Cell Line ◽  
High Doses

Abstract Objective. Histone deacytylase inhibitors (HDACis) inhibit the deacetylation of the lysine residue of proteins, including histones, and regulate the transcription of a variety of genes. Recently, HDACis have been used clinically as anti-cancer drugs and possible anti-diabetic drugs. Even though HDACis have been proven to protect the cytokine-induced damage of pancreatic beta cells, evidence also shows that high doses of HDACis are cytotoxic. In the present study, we, therefore, investigated the eff ect of HDACis on insulin secretion in a pancreatic beta cell line. Methods. Pancreatic beta cells MIN6 were treated with selected HDACis (trichostatin A, TSA; valproic acid, VPA; and sodium butyrate, NaB) in medium supplemented with 25 mM glucose and 13% heat-inactivated fetal bovine serum (FBS) for indicated time intervals. Protein expression of Pdx1 and Mafa in MIN6 cells was demonstrated by immunohistochemistry and immunocytochemistry, expression of Pdx1 and Mafa genes was measured by quantitative RT-PCR method. Insulin release from MIN6 cells and insulin cell content were estimated by ELISA kit. Superoxide production in MIN6 cells was measured using a Total ROS/Superoxide Detection System. Results. TSA, VPA, and NaB inhibited the expression of Pdx1 and Mafa genes and their products. TSA treatment led to beta cell malfunction, characterized by enhanced insulin secretion at 3 and 9 mM glucose, but impaired insulin secretion at 15 and 25 mM glucose. Th us, TSA induced dysregulation of the insulin secretion mechanism. TSA also enhanced reactive oxygen species production in pancreatic beta cells. Conclusions. Our results showed that HDACis caused failure to suppress insulin secretion at low glucose concentrations and enhance insulin secretion at high glucose concentrations. In other words, when these HDACis are used clinically, high doses of HDACis may cause hypoglycemia in the fasting state and hyperglycemia in the fed state. When using HDACis, physicians should, therefore, be aware of the capacity of these drugs to modulate the insulin secretory capacity of pancreatic beta cells.

Catecholamine modulation of calcium currents in clonal pancreatic beta-cells

1989 ◽  
Vol 257 (6) ◽  
pp. C1171-C1176 ◽  
Author(s):  
H. H. Keahey ◽  
A. E. Boyd ◽  
D. L. Kunze
Keyword(s):  
Beta Cell ◽  
G Protein ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Calcium Influx ◽  
Pancreatic Beta Cell ◽  
Cytosolic Calcium ◽  
Calcium Currents ◽  
Secretory Process ◽  
Beta Cell Line

The mechanisms by which norepinephrine and epinephrine activate alpha 2-adrenergic receptors and inhibit insulin release from the pancreatic beta-cell (19, 21, 23) are not yet clear but may involve modulation at several sites. Because intracellular calcium has been implicated in the secretory process, it has been suggested that catecholamines may inhibit secretion by blocking calcium influx, thus reducing the free cytosolic calcium concentration (23). The present study examines the effects of epinephrine, norepinephrine, and clonidine on calcium current in an SV40-transformed hamster beta-cell line (HIT cells). Under voltage-clamp conditions, calcium currents were reversibly inhibited by norepinephrine, epinephrine, and clonidine in the low nanomolar range. The effects were blocked by 1) the alpha 2-antagonist yohimbine, 2) preincubation of the cells with pertussis toxin (PTX), and 3) guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), the nonhydrolyzable GDP analogue that competitively inhibits the interaction of GTP with G proteins. In contrast, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) caused irreversible blockade by catecholamines. These effects could not be overcome by adenosine 3',5'-cyclic monophosphate (cAMP), suggesting that the adenylate cyclase pathway is not involved in the G protein coupling with the channels. These studies show that catecholamines inhibit calcium currents in beta-cells through an alpha 2-adrenoreceptor PTX-sensitive G protein pathway and could inhibit insulin secretion by this mechanism.

scholarly journals PCSK9 affects expression of key surface proteins in human pancreatic beta cells through intra- and extracellular regulatory circuits

2021 ◽  
Author(s):  
kevin Saitoski ◽  
Maria Ryaboshapkina ◽  
Ghaith Hamza ◽  
Andrew F Jarnuczak ◽  
claire berthault ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Beta Cell ◽  
Pancreatic Islets ◽  
Cell Line ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Pancreatic Beta Cell ◽  
Loss Of Function ◽  
Beta Cell Line ◽  
Gain And Loss

Aims/hypothesis: Proprotein convertase subtilisin/kexin 9 (PCSK9) is involved in the degradation of LDLR. However, PCSK9 can target other proteins in a cell-type specific manner. While PCSK9 has been detected in pancreatic islets, its expression in insulin-producing pancreatic beta cells is debated. Herein, we studied PCSK9 expression, regulation and function in the human pancreatic beta cell line EndoC-βH1. Methods: We assessed PCSK9 expression in mouse and human pancreatic islets, and in the pancreatic beta cell line EndoC-βH1. We also studied PCSK9 regulation by cholesterol, lipoproteins, Mevastatin, and by SREBPs transcription factors. To evaluate PCSK9 function in pancreatic beta cells, we performed PCSK9 gain-and loss-of-function experiments in EndoC-βH1 using siPCSK9 or recombinant PCSK9 treatments, respectively. Results: We demonstrate that PCSK9 is expressed and secreted by pancreatic beta cells. In EndoC-βH1 cells, PCSK9 expression is regulated by cholesterol and by SREBPs transcription factors. Importantly, PCSK9 knockdown results in multiple transcriptome, proteome and secretome deregulations and impaired insulin secretion. By gain- and loss-of- function experiments, we observed that PCSK9 regulates the expression levels of LDLR and VLDLR through an extracellular mechanism while CD36, PD-L1 and HLA-ABC are regulated through an intracellular mechanism. Conclusions/interpretation: Collectively, these results highlight PCSK9 as an important regulator of CD36, PD-L1 and HLA-ABC cell surface expression in pancreatic beta cells. Data availability: RNA-seq data have been deposited to GEO database with accession number GSE182016. Mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the following identifiers: PXD027921, PXD027911 and PXD027913.

Pancreatic beta-cell somatostatin receptors

1990 ◽  
Vol 259 (2) ◽  
pp. E216-E224 ◽  
Author(s):  
K. Thermos ◽  
M. D. Meglasson ◽  
J. Nelson ◽  
K. M. Lounsbury ◽  
T. Reisine
Keyword(s):  
Beta Cell ◽  
Physical Properties ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Pancreatic Beta Cell ◽  
Hormone Secretion ◽  
Isolated Islets ◽  
Binding Studies ◽  
Beta Cell Line ◽  
Hit Cells

The characteristics of somatostatin (SRIF) receptors in rat pancreatic beta-cells were investigated using rat islets and the beta-cell line HIT-T15 (HIT). The biochemical properties of the SRIF receptors were examined with 125I-labeled des-Ala-1,Gly-2-desamino-Cys-3-[Tyr-11]- dicarba3,14-somatostatin (CGP 23996). 125I-CGP 23996 bound to SRIF receptors in HIT cells with high affinity and in a saturable manner. The binding of 125I-CGP 23996 to SRIF receptors was blocked by SRIF analogues with a rank order of potency of somatostatin 28 (SRIF-28) greater than D-Trp-8-somatostatin greater than somatostatin 14 (SRIF-14). To investigate the physical properties of the HIT cell SRIF receptor, the receptor was covalently labeled with 125I-CGP 23996 using photo-cross-linking techniques. 125I-CGP 23996 specifically labeled a protein of 55 kDa in HIT cell membranes. The size of the SRIF receptor in HIT cells is similar to the size of the SRIF receptor labeled with 125I-CGP 23996 in membranes of freshly isolated islets, suggesting that the physical properties of SRIF receptors in HIT cells and rat islet cells are similar. The binding studies suggest that beta-cells predominantly express a SRIF-28-preferring receptor. In freshly isolated islets, glucose- and arginine-stimulated insulin release was effectively blocked by SRIF-28 but not by SRIF-14. SRIF-14 did inhibit arginine-stimulated glucagon secretion from freshly isolated islets. The dissociation of the inhibitory effects of SRIF-28 and SRIF-14 on insulin and glucagon release from freshly isolated islets suggests that the two peptides act through different receptors in islets to regulate hormone secretion.

scholarly journals Long Noncoding RNA Meg3 Regulates Mafa Expression in Mouse Beta Cells by Inactivating Rad21, Smc3 or Sin3α

2018 ◽  
Vol 45 (5) ◽  
pp. 2031-2043 ◽  
Author(s):  
Ning Wang ◽  
Yanan Zhu ◽  
Min Xie ◽  
Lintao Wang ◽  
Feiyan Jin ◽  
...  
Keyword(s):  
Beta Cell ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Noncoding Rna ◽  
Islet Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Insulin Biosynthesis ◽  
Expression Levels ◽  
Beta Cell Line

Background/Aims: The main pathogenic mechanism of diabetes is a decrease in the number of islet beta cells or a decline in their function. Recent studies have shown that pancreatic long noncoding RNAs (lncRNAs) have a high degree of tissue specificity and may be involved in the maintenance of islet cells function and the development of diabetes. The aim of this study was to investigate the molecular regulatory mechanism of mouse maternal expressed gene 3 (Meg3) in insulin biosynthesis in pancreatic islets. Methods: Chromatin immunoprecipitation–quantitative polymerase chain reaction (qPCR) and RNA immunoprecipitation–qPCR were used to investigate the molecular mechanism of lncRNA Meg3 in insulin biosynthesis by regulating v-Maf musculoaponeurotic fibrosarcoma oncogene family, protein A (MafA), a mature beta cell marker in the MIN6 beta cell line. Further, the expression levels of Meg3, Ezh2, MafA, Rad21, Smc3, and Sin3α were analyzed in vivo and in vitro by RT-PCR and western blotting. Results: Intranuclear lncRNA Meg3 can bind EZH2, a methyltransferase belonging to the Polycomb repressive complex-2, in pancreatic islet cells. In addition, knockdown of Ezh2 can also inhibit the expression of MafA and Ins2, while expression levels of Rad21, Smc3, and Sin3α are upregulated, by interfering with Ezh2 or Meg3 in pancreatic beta cells. Knockdown of Meg3 resulted in the loss of EZH2 binding and H3K27 trimethylation occupancy of Rad21, Smc3, and Sin3α promoter regions. The inhibition of Rad21, Smc3, or Sin3α, which directly act on the MafA promoter, leads to upregulated expression of MafA in both MIN6 cells and mouse islets. Moreover, the synthesis and secretion of insulin were increased by inhibition of these transcription factors. Conclusions: Pancreatic lncRNA Meg3 can epigenetically regulate the expression of Rad21, Smc3, and Sin3α via EZH2-driven H3K27 methylation. By inhibiting the expression of Rad21, Smc3, or Sin3α, Meg3 promotes the expression of MafA and affects the production of insulin.

scholarly journals Human Islets Contain a Beta Cell Type That Expresses Proinsulin But Not the Enzyme That Converts the Precursor to Insulin

2020 ◽  
Vol 68 (10) ◽  
pp. 691-702
Author(s):  
Gladys Teitelman
Keyword(s):  
Beta Cell ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Cell Types ◽  
Human Islets ◽  
Cell Type ◽  
Human Beta Cells ◽  
Human Beta Cell ◽  
Cell Phenotypes

In pancreatic beta cells, proinsulin (ProIN) undergoes folding in endoplasmic reticulum/Golgi system and is translocated to secretory vesicles for processing into insulin and C-peptide by the proprotein convertases (PC)1/3 and PC2, and carboxypeptidase E. Human beta cells show significant variation in the level of expression of PC1/3, the critical proconvertase involved in proinsulin processing. To ascertain whether this heterogeneity is correlated with the level of expression of the prohormone and mature hormone, the expression of proinsulin, insulin, and PC1/3 in human beta cells was examined. This analysis identified a human beta cell type that expressed proinsulin but lacked PC1/3 (ProIN+PC1/3−). This beta cell type is absent in rodent islets and is abundant in human islets of adults but scarce in islets from postnatal donors. Human islets also contained a beta cell type that expressed both proinsulin and variable levels of PC1/3 (ProIN+PC1/3+) and a less abundant cell type that lacked proinsulin but expressed the convertase (ProIN−PC1/3+). These cell phenotypes were altered by type 2 diabetes. These data suggest that these three cell types represent different stages of a dynamic process with proinsulin folding in ProIN+PC1/3− cells, proinsulin conversion into insulin in ProIN+PC1/3+cells, and replenishment of the proinsulin content in ProIN−PC1/3+ cells:

TRAIL induces proliferation in rodent pancreatic beta cells via AKT activation

2021 ◽  
Author(s):  
Sevim Kahraman ◽  
Ozlem Yilmaz ◽  
Hasan Ali Altunbas ◽  
Ercument Dirice ◽  
Ahter Dilsad Sanlioglu
Keyword(s):  
Beta Cell ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Pancreatic Beta Cell ◽  
Cell Mass ◽  
Beta Cell Mass ◽  
Min6 Cells ◽  
Akt Activation ◽  
Proliferative Effect ◽  
Beta Cell Line

Strategies to increase functional pancreatic beta cell mass is of great interest in diabetes-related research. TNF-related apoptosis-inducing ligand (TRAIL) is well-known to promote proliferation and survival in various cell types, including vascular smooth muscle and endothelial cells. Correlation between the protective nature of TRAIL on these cells and its proliferative effect is noteworthy. TRAIL’s seemingly protective/therapeutic effect in diabetes prompted us to question whether it may act as an inducer of proliferation in pancreatic beta cells. We used rat primary islet cells and MIN6 mouse beta cell line to investigate TRAIL-induced proliferation. Cell viability and/or death was analysed by MTT, WST-1, and annexin-V/PI assays, while proliferation rates and pathways were assessed via immunocytochemical and Western blot analyses. Receptor neutralization antibodies identified the mediator receptors. Recombinant soluble TRAIL (sTRAIL) treatment led to 1.6-fold increased proliferation in insulin-positive cells in dispersed rat islets compared to the untreated group, while adenovirus-mediated overexpression of TRAIL increased the number of proliferating beta cells up to more than 6-fold. sTRAIL or adenoviral vector-mediated TRAIL overexpression induced proliferation in MIN6 cells also. TRAIL’s proliferative effect was mediated via AKT activation, which was suppressed upon specific inhibition. Neutralization of each TRAIL receptor reversed the proliferative effect to some degree, with the highest level of inhibition in death receptor 5 (DR5) blockage in MIN6 cells, and in decoy receptor 1 (DcR1) blockage in primary rat beta cells. Thus, TRAIL induces proliferation in rodent pancreatic beta cells through activation of the AKT pathway.

scholarly journals Oxidative Metabolism Genes Are Not Responsive to Oxidative Stress in Rodent Beta Cell Lines

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Faer Morrison ◽  
Karen Johnstone ◽  
Anna Murray ◽  
Jonathan Locke ◽  
Lorna W. Harries
Keyword(s):  
Oxidative Stress ◽  
Skeletal Muscle ◽  
Beta Cell ◽  
Beta Cells ◽  
Oxidative Metabolism ◽  
Pancreatic Beta Cells ◽  
Ros Production ◽  
Altered Expression ◽  
Beta Cell Line

Altered expression of oxidative metabolism genes has been described in the skeletal muscle of individuals with type 2 diabetes. Pancreatic beta cells contain low levels of antioxidant enzymes and are particularly susceptible to oxidative stress. In this study, we explored the effect of hyperglycemia-induced oxidative stress on a panel of oxidative metabolism genes in a rodent beta cell line. We exposed INS-1 rodent beta cells to low (5.6 mmol/L), ambient (11 mmol/L), and high (28 mmol/L) glucose conditions for 48 hours. Increases in oxidative stress were measured using the fluorescent probe dihydrorhodamine 123. We then measured the expression levels of a panel of 90 oxidative metabolism genes by real-time PCR. Elevated reactive oxygen species (ROS) production was evident in INS-1 cells after 48 hours (P<0.05). TLDA analysis revealed a significant (P<0.05) upregulation of 16 of the 90 genes under hyperglycemic conditions, although these expression differences did not reflect differences in ROS. We conclude that although altered glycemia may influence the expression of some oxidative metabolism genes, this effect is probably not mediated by increased ROS production. The alterations to the expression of oxidative metabolism genes previously observed in human diabetic skeletal muscle do not appear to be mirrored in rodent pancreatic beta cells.

scholarly journals 1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol Attenuates Streptozotocin-Induced Pancreatic Beta Cell Damage by Promoting Glucose Transporter 2 Endocytosis

2019 ◽  
Vol 39 (21) ◽  
Author(s):  
Jimin Kim ◽  
Joo Heon Kim ◽  
Ki-Young Sohn ◽  
Sun Young Yoon ◽  
Jae Wha Kim
Keyword(s):  
Beta Cell ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Glucose Transporter ◽  
Cell Damage ◽  
Serum Insulin ◽  
Pancreatic Beta Cell ◽  
Ros Generation ◽  
Glucose Transporter 2 ◽  
Beta Cell Line

ABSTRACT Streptozotocin (STZ) is widely used to induce diabetic rodent models. It is specifically toxic to pancreatic beta cells and causes severe destruction and dysfunction. We investigated the effect of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) on an STZ-induced diabetic mouse model. PLAG attenuated the glucose increase and maintained serum insulin at levels similar to those seen with control mice. In pancreatic beta cell line INS-1, STZ-induced cell apoptosis and intracellular reactive oxygen species (ROS) generation were significantly reduced to nearly normal levels after PLAG treatment. Glucose transporter 2 (GLUT2) localization analyses and glucose uptake assays showed that PLAG accelerated GLUT2 internalization, which ameliorated excessive entry of glucose, as well as STZ. STZ-induced cytotoxic effects were significantly reduced in PLAG-treated groups. The biological activity of PLAG was further confirmed in GLUT2-silenced cells, and the specificity of PLAG was verified using its derivative 1-palmitoyl-2-linoleoyl-3-hydroxyl-rac-glycerol (PLH). Our results suggest that PLAG may be a useful agent for protecting beta cells in the setting of excessive glucose influx.

scholarly journals Stable overexpression of the glucose-6-phosphatase catalytic subunit attenuates glucose sensitivity of insulin secretion from a mouse pancreatic beta-cell line

2000 ◽  
Vol 164 (3) ◽  
pp. 307-314 ◽  
Author(s):  
K Iizuka ◽  
H Nakajima ◽  
A Ono ◽  
K Okita ◽  
J Miyazaki ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Beta Cell ◽  
Cell Line ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Glucose Sensor ◽  
Pancreatic Beta Cell ◽  
Catalytic Subunit ◽  
Beta Cell Line ◽  
Glucose Stimulated Insulin Secretion

Glucose-6-phosphatase (G-6-Pase) hydrolyzes glucose-6-phosphate to glucose, reciprocal with the so-called glucose sensor, glucokinase, in pancreatic beta cells. To study the role of G-6-Pase in glucose-stimulated insulin secretion from beta cells, we have introduced rat G-6-Pase catalytic subunit cDNA and have established permanent clones with 3-, 7- and 24-fold G-6-Pase activity of the mouse beta-cell line, MIN6. In these clones, glucose usage and ATP production in the presence of 5.5 or 25 mM glucose were reduced, and glucose-stimulated insulin secretion was decreased in proportion to the increased G-6-Pase activity. In addition, insulin secretory capacity in response to d-fructose and pyruvate was unchanged; however, 25 mM glucose-stimulated insulin secretion and intracellular calcium response were completely inhibited. In the clone with 24-fold G-6-Pase activity, changes in intracellular NAD(P)H autofluorescence in response to 25 mM glucose were reduced, but the changes with 20 mM fructose and 20 mM pyruvate were not altered. Stable overexpression of G-6-Pase in beta cells resulted in attenuation of the overall glucose-stimulated metabolic responses corresponding to the degree of overexpression. This particular experimental manipulation shows that the possibility exists of modulating glucose-stimulated insulin release by thoroughly altering glucose cycling at the glucokinase/G-6-Pase step.

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