Objective of this study was to characterize osmotically-induced insulin secretion in two tumor cell lines. We compared response of freshly isolated rat pancreatic islets and INS-1 and INS-1E tumor cell lines to high glucose, 30 % hypotonic medium and 20 % hypertonic medium. In Ca(2+)-containing medium glucose induced insulin release in all three cell types. Hypotonicity induced insulin secretion from islets and INS-1 cells but not from INS-1E cells, in which secretion was inhibited despite similar increase in cell volume in both cell types. GdCl(3) (100 micromol/l) did not affect insulin response from INS-1E cells to hypotonic challenge. Hypertonic medium inhibited glucose-induced insulin secretion from islets but not from tumor cells. Noradrenaline (1 micromol/l) inhibited glucose-induced but not swelling-induced insulin secretion from INS-1 cells. Surprisingly, perifusion with Ca(2+)-depleted medium showed distinct secretory response of INS-1E cells to hypotonicity while that of INS-1 cells was partially inhibited. Functioning glucose-induced insulin secretion is not sufficient prerequisite for hypotonicity-induced response in INS-1E cells suggesting that swelling-induced exocytosis is not essential step in the mechanism mediating glucose-induced insulin secretion. Both cell lines are resistant to inhibitory effect of hyperosmolarity on glucose-induced insulin secretion. Response of INS-1E cells to hypotonicity is inhibited by the presence of Ca(2+) in medium.

• MeSH
• Exocytosis * MeSH
• Gadolinium pharmacology   MeSH
• Glucose metabolism   MeSH
• Hypertonic Solutions MeSH
• Hypotonic Solutions MeSH
• Insulin metabolism   MeSH
• Insulinoma metabolism   MeSH
• Rats MeSH
• Islets of Langerhans drug effects   metabolism   MeSH
• Cell Line, Tumor MeSH
• Pancreatic Neoplasms metabolism   MeSH
• Norepinephrine metabolism   MeSH
• Osmotic Pressure MeSH
• Rats, Wistar MeSH
• Insulin Secretion MeSH
• Calcium deficiency   metabolism   MeSH
• Cell Size MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• gadolinium chloride MeSH Browser
• Gadolinium MeSH
• Glucose MeSH
• Hypertonic Solutions MeSH
• Hypotonic Solutions MeSH
• Insulin MeSH
• Norepinephrine MeSH
• Calcium MeSH

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.

• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Insulin-Secreting Cells metabolism   MeSH
• Cell Line MeSH
• Cyclic CMP analogs & derivatives   metabolism   MeSH
• Down-Regulation MeSH
• Glucose metabolism   MeSH
• Rats MeSH
• RNA, Small Interfering genetics   MeSH
• ATPase Inhibitory Protein MeSH
• Cyclic AMP-Dependent Protein Kinases metabolism   MeSH
• Proteins genetics   metabolism   MeSH
• Insulin Secretion * MeSH
• Signal Transduction MeSH
• Up-Regulation MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• Cyclic CMP MeSH
• dibutyryl cyclic-3',5'-cytidine monophosphate MeSH Browser
• Glucose MeSH
• RNA, Small Interfering MeSH
• Cyclic AMP-Dependent Protein Kinases MeSH
• Proteins MeSH

Pancreatic-β-cell-specifying transcription factor Nkx6.1, indispensable for embryonic development of the pancreatic epithelium and commitment to β-cell lineage, directly controls the expression of a glucose transporter (Glut2), pyruvate carboxylase (Pcx), and genes for insulin processing (endoplasmic reticulum oxidoreductase-1β, Ero1lb; zinc transporter-8, Slc30a8). The Nkx6.1 decline in aging diabetic Goto-Kakizaki rats contributes to β-cell trans-differentiation into δ-cells. Elucidating further Nkx6.1 roles, we studied Nkx6.1 ablation in rat INS-1E cells, prepared by CRISPR/Cas9 gene editing from single colonies. INS-1ENkx6.1-/- cells exhibited unchanged glucose-stimulated insulin secretion (GSIS), moderately decreased phosphorylating/non-phosphorylating respiration ratios at high glucose; unchanged but delayed ATP-elevation responses to glucose; delayed uptake of fluorescent glucose analog, but slightly improved cytosolic Ca2+-oscillations, induced by glucose; despite approximately halved Glut2, Pcx, Ero1lb, and Slc30a8 expression, and reduced nuclear receptors Nr4a1 and Nr4a3. Thus, ATP synthesis was time-compensated, despite the delayed GLUT2-mediated glucose uptake and crippled pyruvate-malate redox shuttle (owing to the PCX-deficiency) in INS-1ENkx6.1-/- cells. Nkx6.1 thus controls the expression of genes that are not essential for acute insulin secretion, the function of which can be compensated for. Considerations that Nkx6.1 deficiency is an ultimate determinant of β-cell pathology beyond cell trans-(de-)differentiation or β-cell identity are not supported by our results.

• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Insulin-Secreting Cells * metabolism   MeSH
• Glucose metabolism   MeSH
• Homeodomain Proteins * genetics   metabolism   MeSH
• Insulin * metabolism   MeSH
• Rats MeSH
• Insulin Secretion MeSH
• Transcription Factors genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• Glucose MeSH
• Homeodomain Proteins * MeSH
• Insulin * MeSH
• Nkx6-1 protein, rat MeSH Browser
• Transcription Factors MeSH

Increased ATP/ADP ratio resulting from enhanced glycolysis and oxidative phosphorylation represents a plausible mechanism controlling the glucose-stimulated insulin secretion (GSIS) in pancreatic beta-cells. Although specific bioenergetics might be involved, parallel studies of cell respiration and mitochondrial membrane potential (DeltaPsi(m)) during GSIS are lacking. Using high resolution respirometry and parallel DeltaPsi(m) monitoring by two distinct fluorescence probes we have quantified bioenergetics in rat insulinoma INS-1E cells representing a suitable model to study in vitro insulin secretion. Upon glucose addition to glucose-depleted cells we demonstrated a simultaneous increase in respiration and DeltaPsi(m) during GSIS and showed that the endogenous state 3/state 4 respiratory ratio hyperbolically increased with glucose, approaching the maximum oxidative phosphorylation rate at maximum GSIS. Attempting to assess the basis of the "toxic" effect of fatty acids on insulin secretion, GSIS was studied after linoleic acid addition, which diminished respiration increase, DeltaPsi(m) jump, and magnitude of insulin release, and reduced state 3/state 4 dependencies on glucose. Its effects were due to protonophoric function, i.e. uncoupling, since without glucose, linoleic acid accelerated both state 3 and state 4 respiration by similar extent. In turn, state 3 respiration increased marginally with linoleic acid at 10-20mM glucose. We conclude that upon glucose addition in physiological range, the INS-1E cells are able to regulate the oxidative phosphorylation rate from nearly zero to maximum and that the impairment of GSIS by linoleic acid is caused by mitochondrial uncoupling. These findings may be relevant to the pathogenesis of type 2 diabetes.

• MeSH
• Adenosine Diphosphate metabolism   MeSH
• Adenosine Triphosphate metabolism   MeSH
• Glucose pharmacology   MeSH
• Insulinoma metabolism   MeSH
• Rats MeSH
• Linoleic Acid pharmacology   MeSH
• Islets of Langerhans drug effects   metabolism   MeSH
• Membrane Potential, Mitochondrial drug effects   MeSH
• Mitochondria drug effects   metabolism   MeSH
• Tumor Cells, Cultured MeSH
• Pancreatic Neoplasms metabolism   MeSH
• Rats, Wistar MeSH
• Oxygen Consumption drug effects   MeSH
• Microscopy, Electron, Transmission MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Diphosphate MeSH
• Adenosine Triphosphate MeSH
• Glucose MeSH
• Linoleic Acid MeSH

Mitochondria in numerous cell types, especially in cultured cells, form a reticular network undergoing constant fusion and fission. The three dimensional (3D) morphology of these networks however has not been studied in detail to our knowledge. We have investigated insulinoma INS-1E and hepatocellular carcinoma HEP-G2 cells transfected with mitochondria-addressed GFP. Using 4Pi microscopy, 3D morphology changes responding to decreased oxidative phosphorylation and/or energetic status could be observed in these cells at an unprecedented 100 nm level of detail. In INS-1E cells cultivated at 11 mM glucose, the mitoreticulum appears predominantly as one interconnected mitochondrion with a nearly constant 262+/-26 nm tubule diameter. If cultured at 5 mM glucose, INS-1E cells show 311+/-36 nm tubules coexisting with numerous flat cisternae. Similar interconnected 284+/-38 nm and 417+/-110 nm tubules were found in HEP-G2 cells cultivated at 5 mM and hyperglycaemic 25 mM glucose, respectively. With rotenone inhibiting respiration to approximately 10%, disintegration into several reticula and numerous approximately 300 nm spheres or short tubules was observed. De-energization by uncoupling additionally led to formation of rings and bulky cisternae of 1.4+/-0.4 microm diameter. Rotenone and uncoupler acted synergically in INS-1E cells and increased fusion (ongoing with fission) forming bowl-like shapes. In HEP-G2 cells fission partially ceased with FCCP plus rotenone. Thus we have revealed previously undescribed details for shapes upon mitochondrial disintegration and clearly demonstrate that high resolution 3D microscopy is required for visualization of mitochondrial network. We recommend 4Pi microscopy as a new standard.

• MeSH
• Energy Metabolism * MeSH
• Carcinoma, Hepatocellular MeSH
• Insulinoma MeSH
• Microscopy, Confocal MeSH
• Rats MeSH
• Humans MeSH
• Mitochondria metabolism   pathology   ultrastructure   MeSH
• Cell Line, Tumor MeSH
• Liver Neoplasms MeSH
• Pancreatic Neoplasms MeSH
• Oxidative Phosphorylation * MeSH
• Image Processing, Computer-Assisted MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

ATPase Inhibitory factor 1 (IF1) is an endogenous regulator of mitochondrial ATP synthase, which is involved in cellular metabolism. Although great progress has been made, biological roles of IF1 and molecular mechanisms of its action are still to be elucidated. Here, we show that IF1 is present in pancreatic β-cells, bound to the ATP synthase also under normal physiological conditions. IF1 silencing in model pancreatic β-cells (INS-1E) increases insulin secretion over a range of glucose concentrations. The left-shifted dose-response curve reveals excessive insulin secretion even under low glucose, corresponding to fasting conditions. A parallel increase in cellular respiration and ATP levels is observed. To conclude, our results indicate that IF1 is a negative regulator of insulin secretion involved in pancreatic β-cell glucose sensing.

• Keywords
• ATP synthase, IF1, INS-1E cells, diabetes, insulin secretion, β-cells,
• MeSH
• Insulin-Secreting Cells cytology   metabolism   MeSH
• Glucose metabolism   MeSH
• Insulin metabolism   MeSH
• Rats MeSH
• Cell Line, Tumor MeSH
• Rats, Wistar MeSH
• ATPase Inhibitory Protein MeSH
• Proteins metabolism   MeSH
• Oxygen Consumption physiology   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Letter MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Glucose MeSH
• Insulin MeSH
• Proteins MeSH

OBJECTIVE: Enzymatic fingerprinting of key enzymes of glucose metabolism is a valuable analysis tool in cell physiological phenotyping of plant samples. Yet, a similar approach for mammalian cell line samples is missing. In this study, we applied semi-high throughput enzyme activity assays that were originally designed for plant samples and tested their feasibility in extracts of six frequently used mammalian cell lines (Caco2, HaCaT, C2C12, HEK293, HepG2 and INS-1E). RESULTS: Enzyme activities for aldolase, hexokinase, glucose-6-phosphate dehydrogenase, phosphoglucoisomerase, phosphoglucomutase, phosphofructokinase could be detected in samples of one or more mammalian cell lines. We characterized effects of sample dilution, assay temperature and repeated freeze-thaw cycles causing potential biases. After careful selection of experimental parameters, the presented semi-high throughput methods could be established as useful tool for physiological phenotyping of cultured mammalian cells.

• Keywords
• 96 well format, Aldolase, Enzyme assays, Glucose-6-phosphate dehydrogenase, Hexokinase, INS-1E, Phosphofructokinase, Phosphoglucoisomerase, Phosphoglucomutase,
• MeSH
• Fructose-Bisphosphate Aldolase metabolism   MeSH
• Cell Line MeSH
• Hep G2 Cells MeSH
• Caco-2 Cells MeSH
• Enzyme Assays methods   MeSH
• Phenotype MeSH
• Phosphofructokinases metabolism   MeSH
• Phosphoglucomutase metabolism   MeSH
• Phosphotransferases (Alcohol Group Acceptor) metabolism   MeSH
• Glucosephosphate Dehydrogenase metabolism   MeSH
• Glucose metabolism   MeSH
• Glycolysis * MeSH
• HEK293 Cells MeSH
• Hexokinase metabolism   MeSH
• Humans MeSH
• Carbohydrate Metabolism * MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Pilot Projects MeSH
• Feasibility Studies MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Fructose-Bisphosphate Aldolase MeSH
• Phosphofructokinases MeSH
• Phosphoglucomutase MeSH
• Phosphotransferases (Alcohol Group Acceptor) MeSH
• Glucosephosphate Dehydrogenase MeSH
• Glucose MeSH
• Hexokinase MeSH
• phosphoglucokinase MeSH Browser

Insulin is produced and stored inside the pancreatic β-cell secretory granules, where it is assumed to form Zn2+-stabilized oligomers. However, the actual storage forms of this hormone and the impact of zinc ions on insulin production in vivo are not known. Our initial X-ray fluorescence experiment on granules from native Langerhans islets and insulinoma-derived INS-1E cells revealed a considerable difference in the zinc content. This led our further investigation to evaluate the impact of the intra-granular Zn2+ levels on the production and storage of insulin in different model β-cells. Here, we systematically compared zinc and insulin contents in the permanent INS-1E and BRIN-BD11 β-cells and in the native rat pancreatic islets by flow cytometry, confocal microscopy, immunoblotting, specific messenger RNA (mRNA) and total insulin analysis. These studies revealed an impaired insulin production in the permanent β-cell lines with the diminished intracellular zinc content. The drop in insulin and Zn2+ levels was paralleled by a lower expression of ZnT8 zinc transporter mRNA and hampered proinsulin processing/folding in both permanent cell lines. To summarize, we showed that the disruption of zinc homeostasis in the model β-cells correlated with their impaired insulin and ZnT8 production. This indicates a need for in-depth fundamental research about the role of zinc in insulin production and storage.

• Keywords
• insulin, pancreatic islets, proinsulin, zinc ions, znt8, β-cells,
• MeSH
• Insulin-Secreting Cells metabolism   ultrastructure   MeSH
• Chemical Fractionation MeSH
• Cytoplasmic Granules metabolism   MeSH
• Gene Expression * MeSH
• Glucose metabolism   MeSH
• Insulin genetics   metabolism   MeSH
• Rats MeSH
• Islets of Langerhans metabolism   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Flow Cytometry methods   MeSH
• Zinc metabolism   MeSH
• Zinc Transporter 8 MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Glucose MeSH
• Insulin MeSH
• RNA, Messenger MeSH
• Slc30a8 protein, rat MeSH Browser
• Zinc MeSH
• Zinc Transporter 8 MeSH

Transcript levels for selected ATP synthase membrane FO-subunits-including DAPIT-in INS-1E cells were found to be sensitive to lowering glucose down from 11 mM, in which these cells are routinely cultured. Depending on conditions, the diminished mRNA levels recovered when glucose was restored to 11 mM; or were elevated during further 120 min incubations with 20-mM glucose. Asking whether DAPIT expression may be elevated by hyperglycemia in vivo, we studied mice with hyaluronic acid implants delivering glucose for up to 14 days. Such continuous two-week glucose stimulations in mice increased DAPIT mRNA by >5-fold in isolated pancreatic islets (ATP synthase F1α mRNA by 1.5-fold). In INS-1E cells, the glucose-induced ATP increment vanished with DAPIT silencing (6% of ATP rise), likewise a portion of the mtDNA-copy number increment. With 20 and 11-mM glucose the phosphorylating/non-phosphorylating respiration rate ratio diminished to ~70% and 96%, respectively, upon DAPIT silencing, whereas net GSIS rates accounted for 80% and 90% in USMG5/DAPIT-deficient cells. Consequently, the sufficient DAPIT expression and complete ATP synthase assembly is required for maximum ATP synthesis and mitochondrial biogenesis, but not for insulin secretion as such. Elevated DAPIT expression at high glucose further increases the ATP synthesis efficiency.

• Keywords
• ATP synthase oligomers mitochondrial cristae morphology, USMG5/DAPIT, glucose-induced expression, glucose-stimulated insulin secretion, membrane subunits of ATP synthase, mitochondria,
• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Insulin-Secreting Cells cytology   drug effects   metabolism   MeSH
• Cell Culture Techniques MeSH
• Cell Line MeSH
• Glucose administration & dosage   pharmacology   MeSH
• Protein Conformation MeSH
• Rats MeSH
• Hyaluronic Acid chemistry   MeSH
• Membrane Proteins chemistry   genetics   metabolism   MeSH
• DNA, Mitochondrial drug effects   genetics   MeSH
• Mitochondria drug effects   genetics   metabolism   MeSH
• Models, Molecular MeSH
• Mice MeSH
• Up-Regulation * MeSH
• DNA Copy Number Variations drug effects   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• Atp5md protein, rat MeSH Browser
• Glucose MeSH
• Hyaluronic Acid MeSH
• Membrane Proteins MeSH
• DNA, Mitochondrial MeSH

Pancreatic β-cells are vulnerable to oxidative stress due to their low content of redox buffers, such as glutathione, but possess a rich content of thioredoxin, peroxiredoxin, and other proteins capable of redox relay, transferring redox signaling. Consequently, it may be predicted that cytosolic antioxidants could interfere with the cytosolic redox signaling and should not be recommended for any potential therapy. In contrast, mitochondrial matrix-targeted antioxidants could prevent the primary oxidative stress arising from the primary superoxide sources within the mitochondrial matrix, such as at the flavin (IF) and ubiquinone (IQ) sites of superoxide formation within respiratory chain complex I and the outer ubiquinone site (IIIQ) of complex III. Therefore, using time-resolved confocal fluorescence monitoring with MitoSOX Red, we investigated various effects of mitochondria-targeted antioxidants in model pancreatic β-cells (insulinoma INS-1E cells) and pancreatic islets. Both SkQ1 (a mitochondria-targeted plastoquinone) and a suppressor of complex III site Q electron leak (S3QEL) prevented superoxide production released to the mitochondrial matrix in INS-1E cells with stimulatory glucose, where SkQ1 also exhibited an antioxidant role for UCP2-silenced cells. SkQ1 acted similarly at nonstimulatory glucose but not in UCP2-silenced cells. Thus, UCP2 can facilitate the antioxidant mechanism based on SkQ1+ fatty acid anion- pairing. The elevated superoxide formation induced by antimycin A was largely prevented by S3QEL, and that induced by rotenone was decreased by SkQ1 and S3QEL and slightly by S1QEL, acting at complex I site Q. Similar results were obtained with the MitoB probe, for the LC-MS-based assessment of the 4 hr accumulation of reactive oxygen species within the mitochondrial matrix but for isolated pancreatic islets. For 2 hr INS-1E incubations, some samples were influenced by the cell death during the experiment. Due to the frequent dependency of antioxidant effects on metabolic modes, we suggest a potential use of mitochondria-targeted antioxidants for the treatment of prediabetic states after cautious nutrition-controlled tests. Their targeted delivery might eventually attenuate the vicious spiral leading to type 2 diabetes.

• MeSH
• Antioxidants pharmacology   MeSH
• Insulin-Secreting Cells drug effects   metabolism   pathology   MeSH
• Phenanthridines MeSH
• Cells, Cultured MeSH
• Mitochondrial Membranes drug effects   metabolism   pathology   MeSH
• Mitochondria drug effects   metabolism   pathology   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Organophosphorus Compounds MeSH
• Oxidation-Reduction MeSH
• Oxidative Stress drug effects   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Uncoupling Protein 2 metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antioxidants MeSH
• Phenanthridines MeSH
• MitoSox Red MeSH Browser
• Organophosphorus Compounds MeSH
• Reactive Oxygen Species MeSH
• Uncoupling Protein 2 MeSH