Browse
Submit Data
Databases
API
Help

Dataset Information

31 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Glucose stimulation induces dynamic change of mitochondrial morphology to promote insulin secretion in the insulinoma cell line INS-1E.

ABSTRACT: Fission and fusion of mitochondrial tubules are the major processes regulating mitochondrial morphology. However, the physiological significance of mitochondrial shape change is poorly understood. Glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells requires mitochondrial ATP production which evokes Ca(2+) influx through plasma membrane depolarization, triggering insulin vesicle exocytosis. Therefore, GSIS reflects mitochondrial function and can be used for evaluating functional changes associated with morphological alterations of mitochondria. Using the insulin-secreting cell line INS-1E, we found that glucose stimulation induced rapid mitochondrial shortening and recovery. Inhibition of mitochondrial fission through expression of the dominant-negative mutant DLP1-K38A eliminated this dynamic mitochondrial shape change and, importantly, blocked GSIS. We found that abolishing mitochondrial morphology change in glucose stimulation increased the mitochondrial inner membrane proton leak, and thus significantly diminished the mitochondrial ATP producing capacity in response to glucose stimulation. These results demonstrate that dynamic change of mitochondrial morphology is a previously unrecognized component for metabolism-secretion coupling of pancreatic β-cells by participating in efficient ATP production in response to elevated glucose levels.

SUBMITTER: Jhun BS

PROVIDER: S-EPMC3614983 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Sfrp5 increases glucose-stimulated insulin secretion in the rat pancreatic beta cell line INS-1E.

Project description:Previous studies reported that secreted frizzled-related protein-5 (Sfrp5) decreases beta cell proliferation and increases fasting insulin levels, but studies on direct effects of Sfrp5 on insulin secretion and its underlying mechanisms are missing. This study examined effects of Sfrp5 on (i) beta cell viability and proliferation, (ii) basal and glucose-stimulated insulin secretion and (iii) canonical and non-canonical Wnt signalling pathways. We incubated rat INS-1E cells with 0.1, 1 or 5 μg/ml recombinant Sfrp5 for 24h. We measured basal and glucose-stimulated insulin secretion at glucose concentrations of 2.5 and 20 mmol/l. Phosphorylated and total protein content as well as mRNA levels of markers of cell proliferation, canonical and non-canonical Wnt signalling pathways were examined using Western blotting and real-time PCR. Differences between treatments were analysed by repeated measurement one-way ANOVA or Friedman's test followed by correction for multiple testing using the Benjamini-Hochberg procedure. At 5 μg/ml, Sfrp5 reduced mRNA levels of cyclin-B1 by 25% (p<0.05). At 1 and 5 μg/ml, Sfrp5 increased glucose-stimulated insulin secretion by 24% and by 34% (both p<0.05), respectively, but had no impact on basal insulin secretion. Sfrp5 reduced the phosphorylation of the splicing forms p46 and p54 of JNK by 39% (p<0.01) and 49% (p<0.05), respectively. In conclusion, Sfrp5 reduced markers of cell proliferation, but increased in parallel dose-dependently glucose-stimulated insulin secretion in INS-1E cells. This effect is likely mediated by reduced JNK activity, an important component of the non-canonical Wnt signalling pathway.

| S-EPMC6438539 | biostudies-literature

Glucose and GLP-1 stimulate cAMP production via distinct adenylyl cyclases in INS-1E insulinoma cells.

Project description:In beta cells, both glucose and hormones, such as GLP-1, stimulate production of the second messenger cAMP, but glucose and GLP-1 elicit distinct cellular responses. We now show in INS-1E insulinoma cells that glucose and GLP-1 produce cAMP with distinct kinetics via different adenylyl cyclases. GLP-1 induces a rapid cAMP signal mediated by G protein-responsive transmembrane adenylyl cyclases (tmAC). In contrast, glucose elicits a delayed cAMP rise mediated by bicarbonate, calcium, and ATP-sensitive soluble adenylyl cyclase (sAC). This glucose-induced, sAC-dependent cAMP rise is dependent upon calcium influx and is responsible for the glucose-induced activation of the mitogen-activated protein kinase (ERK1/2) pathway. These results demonstrate that sAC-generated and tmAC-generated cAMP define distinct signaling cascades.

| S-EPMC2518727 | biostudies-literature

Metabolic response of Insulinoma 1E cells to glucose stimulation studied by fluorescence lifetime imaging.

Project description:A cascade of highly regulated biochemical processes connects glucose stimulation to insulin secretion in specialized cells of mammalian pancreas, the ?-cells. Given the importance of this process for systemic glucose homeostasis, noninvasive and fast strategies capable to monitor the response to glucose in living cells are highly desirable. Here, we use the phasor-based approach to Fluorescence Lifetime IMaging (FLIM) microscopy to quantify the ratio between protein-bound and free Nicotinamide adenine dinucleotide (phosphate) species in their reduced form (NAD(P)H), and the Insulinoma cell line INS-1E as a ?-like cellular model. Phasor-FLIM analysis shows that the bound/free ratio of NAD(P)H species increases upon pulsed glucose stimulation. Such response is impaired by 48-hours preincubation of cells under hyperglycemic conditions. Phasor-FLIM concomitantly monitors the appearance of long-lifetime species (LLS) as characteristic products of hyperglycemia-induced oxidative stress.

| S-EPMC7354695 | biostudies-literature

Overexpression of native IF1 downregulates glucose-stimulated insulin secretion by pancreatic INS-1E cells.

Project description:We have previously reported that transient knock-down of ATPase inhibitory factor 1 (IF1) by siRNA upregulates ATP levels and subsequently augments insulin secretion in model pancreatic β-cells INS-1E. Here we investigated how long-term IF1-overexpression impacts pancreatic β-cell bioenergetics and insulin secretion. We generated INS-1E cell line stably overexpressing native IF1. We revealed that IF1 overexpression leads to a substantial decrease in ATP levels and reduced glucose-stimulated insulin secretion. A decrease in total cellular ATP content was also reflected in decreased free ATP cytosolic and mitochondrial levels, as monitored with ATeam biosensor. Consistently, cellular respiration of IF1-overexpressing cells was decreased. 3D structured illumination microscopy (SIM) revealed a higher amount of insulin granules with higher volume in IF1-overexpressing cells. Similar effects occurred when cells were incubated at low glucose concentrations. Noteworthy, activation of PKA by dibutyryl cAMP entirely abolished the inhibitory effect of IF1 overexpression on ATP production and insulin secretion. Mitochondrial network morphology and cristae ultrastructure in INS-1E overexpressing IF1 remained mostly unchanged. Finally, we show that INS-1E cells decrease their IF1 protein levels relative to ATP synthase α-subunit in response to increased glucose. In conclusion, IF1 actively downregulates INS-1E cellular metabolism and reduces their ability to secrete insulin.

| S-EPMC6994519 | biostudies-literature

High-dose clevudine impairs mitochondrial function and glucose-stimulated insulin secretion in INS-1E cells.

Project description:BACKGROUND: Clevudine is a nucleoside analog reverse transcriptase inhibitor that exhibits potent antiviral activity against hepatitis B virus (HBV) without serious side effects. However, mitochondrial myopathy has been observed in patients with chronic HBV infection taking clevudine. Moreover, the development of diabetes was recently reported in patients receiving long-term treatment with clevudine. In this study, we investigated the effects of clevudine on mitochondrial function and insulin release in a rat clonal ?-cell line, INS-1E. METHODS: The mitochondrial DNA (mtDNA) copy number and the mRNA levels were measured by using quantitative PCR. MTT analysis, ATP/lactate measurements, and insulin assay were performed. RESULTS: Both INS-1E cells and HepG2 cells, which originated from human hepatoma, showed dose-dependent decreases in mtDNA copy number and cytochrome c oxidase-1 (Cox-1) mRNA level following culture with clevudine (10 ?M-1 mM) for 4 weeks. INS-1E cells treated with clevudine had reduced total mitochondrial activities, lower cytosolic ATP contents, enhanced lactate production, and more lipid accumulation. Insulin release in response to glucose application was markedly decreased in clevudine-treated INS-1E cells, which might be a consequence of mitochondrial dysfunction. CONCLUSIONS: Our data suggest that high-dose treatment with clevudine induces mitochondrial defects associated with mtDNA depletion and impairs glucose-stimulated insulin secretion in insulin-releasing cells. These findings partly explain the development of diabetes in patients receiving clevudine who might have a high susceptibility to mitochondrial toxicity.

| S-EPMC3288815 | biostudies-literature

RyR2/IRBIT regulates insulin gene transcript, insulin content, and secretion in the insulinoma cell line INS-1.

Project description:The role of ER Ca2+ release via ryanodine receptors (RyR) in pancreatic β-cell function is not well defined. Deletion of RyR2 from the rat insulinoma INS-1 (RyR2KO) enhanced IP3 receptor activity stimulated by 7.5 mM glucose, coincident with reduced levels of the protein IP3 Receptor Binding protein released with Inositol 1,4,5 Trisphosphate (IRBIT). Insulin content, basal (2.5 mM glucose) and 7.5 mM glucose-stimulated insulin secretion were reduced in RyR2KO and IRBITKO cells compared to controls. INS2 mRNA levels were reduced in both RyR2KO and IRBITKO cells, but INS1 mRNA levels were specifically decreased in RyR2KO cells. Nuclear localization of S-adenosylhomocysteinase (AHCY) was increased in RyR2KO and IRBITKO cells. DNA methylation of the INS1 and INS2 gene promotor regions was very low, and not different among RyR2KO, IRBITKO, and controls, but exon 2 of the INS1 and INS2 genes was more extensively methylated in RyR2KO and IRBITKO cells. Exploratory proteomic analysis revealed that deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. These results suggest that RyR2 regulates IRBIT levels and activity in INS-1 cells, and together maintain insulin content and secretion, and regulate the proteome, perhaps via DNA methylation.

| S-EPMC9095623 | biostudies-literature

Pro-inflammatory cytokines attenuate glucose-stimulated insulin secretion from INS-1E insulinoma cells by restricting mitochondrial pyruvate oxidation capacity - Novel mechanistic insight from real-time analysis of oxidative phosphorylation.

Project description:Pro-inflammatory cytokines cause pancreatic beta cell failure during the development of type 2 diabetes. This beta cell failure associates with mitochondrial dysfunction, but the precise effects of cytokines on mitochondrial respiration remain unclear. To test the hypothesis that pro-inflammatory cytokines impair glucose-stimulated insulin secretion (GSIS) by inhibiting oxidative ATP synthesis, we probed insulin release and real-time mitochondrial respiration in rat INS-1E insulinoma cells that were exposed to a combination of 2 ng/mL interleukin-1-beta and 50 ng/mL interferon-gamma. We show that 24-h exposure to these cytokines dampens both glucose- and pyruvate-stimulated insulin secretion (P < 0.0001 and P < 0.05, respectively), but does not affect KCl-induced insulin release. Mirroring secretory defects, glucose- and pyruvate-stimulated mitochondrial respiration are lowered after cytokine exposure (P < 0.01). Further analysis confirms that cytokine-induced mitochondrial respiratory defects occur irrespective of whether fuel oxidation is coupled to, or uncoupled from, ATP synthesis. These observations demonstrate that pro-inflammatory cytokines attenuate GSIS by restricting mitochondrial pyruvate oxidation capacity. Interleukin-1-beta and interferon-gamma also increase mitochondrial superoxide levels (P < 0.05), which may reinforce the inhibition of pyruvate oxidation, and cause a modest (20%) but significant (P < 0.01) loss of INS-1E cells. Cytokine-induced INS-1E cell failure is insensitive to palmitoleate and linoleate, which is at odds with the cytoprotection offered by unsaturated fatty acids against harm caused by nutrient excess. Our data disclose a mitochondrial mechanism for cytokine-impaired GSIS in INS-1E cells, and suggest that inflammatory and nutrient-related beta cell failure emerge, at least partly, through distinct paths.

| S-EPMC6023166 | biostudies-literature

Menaquinone-4 Amplified Glucose-Stimulated Insulin Secretion in Isolated Mouse Pancreatic Islets and INS-1 Rat Insulinoma Cells.

Project description:Vitamin K2 is indispensable for blood coagulation and bone metabolism. Menaquinone-4 (MK-4) is the predominant homolog of vitamin K2, which is present in large amounts in the pancreas, although its function is unclear. Meanwhile, β-cell dysfunction following insulin secretion has been found to decrease in patients with type 2 diabetes mellitus. To elucidate the physiological function of MK-4 in pancreatic β-cells, we studied the effects of MK-4 treatment on isolated mouse pancreatic islets and rat INS-1 cells. Glucose-stimulated insulin secretion significantly increased in isolated islets and INS-1 cells treated with MK-4. It was further clarified that MK-4 enhanced cAMP levels, accompanied by the regulation of the exchange protein directly activated by the cAMP 2 (Epac2)-dependent pathway but not the protein kinase A (PKA)-dependent pathway. A novel function of MK-4 on glucose-stimulated insulin secretion was found, suggesting that MK-4 might act as a potent amplifier of the incretin effect. This study therefore presents a novel potential therapeutic approach for impaired insulinotropic effects.

| S-EPMC6515216 | biostudies-literature

Mitochondrial uncoupling protein-2 is not involved in palmitate-induced impairment of glucose-stimulated insulin secretion in INS-1E insulinoma cells and is not needed for the amplification of insulin release.

Project description:We have recently shown that overnight exposure of INS-1E insulinoma cells to palmitate in the presence of high glucose causes defects in both mitochondrial energy metabolism and glucose-stimulated insulin secretion (GSIS). Here we report experiments designed to test the involvement of mitochondrial uncoupling protein-2 (UCP2) in these glucolipotoxic effects. Measuring real-time oxygen consumption in siRNA-transfected INS-1E cells, we show that deleterious effects of palmitate on the glucose sensitivity of mitochondrial respiration and on the coupling efficiency of oxidative phosphorylation are independent of UCP2. Consistently, palmitate impairs GSIS to the same extent in cells with and without UCP2. Furthermore, we knocked down UCP2 in spheroid INS-1E cell clusters (pseudoislets) to test whether or not UCP2 regulates insulin secretion during prolonged glucose exposure. We demonstrate that there are no differences in temporal GSIS kinetics between perifused pseudoislets with and without UCP2. We conclude that UCP2 is not involved in palmitate-induced impairment of GSIS in INS-1E insulinoma cells and is not needed for the amplification of insulin release. These conclusions inform ongoing debate on the disputed biochemical and physiological functions of the beta cell UCP2.

| S-EPMC4547158 | biostudies-literature

Proglucagon-Derived Peptides Expression and Secretion in Rat Insulinoma INS-1 Cells.

Project description:Rat insulinoma INS-1 cells are widely used to study insulin secretory mechanisms. Studies have shown that a population of INS-1 cells are bi-hormonal, co-expressing insulin, and proglucagon proteins. They coined this population as immature cells since they co-secrete proglucagon-derived peptides from the same secretory vesicles similar to that of insulin. Since proglucagon encodes multiple peptides including glucagon, glucagon-like-peptide-1 (GLP-1), GLP-2, oxyntomodulin, and glicentin, their specific expression and secretion are technically challenging. In this study, we aimed to focus on glucagon expression which shares the same amino acid sequence with glicentin and proglucagon. Validation of the anti-glucagon antibody (Abcam) by Western blotting techniques revealed that the antibody detects proglucagon (≈ 20 kDa), glicentin (≈ 9 kDa), and glucagon (≈ 3 kDa) in INS-1 cells and primary islets, all of which were absent in the kidney cell line (HEK293). Using the validated anti-glucagon antibody, we showed by immunofluorescence imaging that a population of INS-1 cells co-express insulin and proglucagon-derived proteins. Furthermore, we found that chronic treatment of INS-1 cells with high-glucose decreases insulin and glucagon content, and also reduces the percentage of bi-hormonal cells. In line with insulin secretion, we found glucagon and glicentin secretion to be induced in a glucose-dependent manner. We conclude that INS-1 cells are a useful model to study glucose-stimulated insulin secretion, but not that of glucagon or glicentin. Our study suggests Western blotting technique as an important tool for researchers to study proglucagon-derived peptides expression and regulation in primary islets in response to various metabolic stimuli.

| S-EPMC7683504 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data