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
- adenosintrifosfát metabolismus MeSH
- beta-buňky * metabolismus MeSH
- glukosa metabolismus MeSH
- homeodoménové proteiny * genetika metabolismus MeSH
- inzulin * metabolismus MeSH
- krysa rodu rattus MeSH
- sekrece inzulinu MeSH
- transkripční faktory genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Statins are primary drugs in the treatment of hyperlipidemias. This group of drugs is known for its beneficial pleiotropic effects (e.g., reduction of inflammatory state). However, a growing body of evidence suggests its diabetogenic properties. The culpable mechanism is not completely understood and might be related to the damage to pancreatic beta cells. Therefore, we conceived an in vitro study to explore the impact of atorvastatin on pancreatic islet beta cells line (1.1.E7). We evaluated the influence on viability, insulin, low-density lipoprotein (LDL) receptor, and proprotein convertase subtilisin/kexin type 9 (PCSK9) expression. A significant drop in mRNA for proinsulin and insulin expression was noted. Concurrently, a rise in LDL receptor at the protein level in cells exposed to atorvastatin was noted. Further experiments have shown that exenatide - belonging to glucagon-like peptide 1 (GLP-1) analogs that are used in a treatment of diabetes and known for its weight reducing properties - can alleviate the observed alterations. In this case, the mechanism of action of exenatide was dependent on a protein kinase A pathway. In conclusion, our results support the hypothesis that statin may have diabetogenic properties, which according to our study is related to reduced insulin expression. The concomitant use of GLP-1 receptor agonist seemed to successfully revert insulin expression.
- MeSH
- atorvastatin farmakologie metabolismus MeSH
- beta-buňky * MeSH
- exenatid farmakologie metabolismus MeSH
- inzulin metabolismus MeSH
- proproteinkonvertasa subtilisin/kexin typu 9 metabolismus farmakologie MeSH
- proteinkinasy závislé na cyklickém AMP metabolismus farmakologie MeSH
- receptory LDL metabolismus MeSH
- sekrece inzulinu MeSH
- statiny * farmakologie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Significance: Mitochondria determine glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells by elevating ATP synthesis. As the metabolic and redox hub, mitochondria provide numerous links to the plasma membrane channels, insulin granule vesicles (IGVs), cell redox, NADH, NADPH, and Ca2+ homeostasis, all affecting insulin secretion. Recent Advances: Mitochondrial redox signaling was implicated in several modes of insulin secretion (branched-chain ketoacid [BCKA]-, fatty acid [FA]-stimulated). Mitochondrial Ca2+ influx was found to enhance GSIS, reflecting cytosolic Ca2+ oscillations induced by action potential spikes (intermittent opening of voltage-dependent Ca2+ and K+ channels) or the superimposed Ca2+ release from the endoplasmic reticulum (ER). The ATPase inhibitory factor 1 (IF1) was reported to tune the glucose sensitivity range for GSIS. Mitochondrial protein kinase A was implicated in preventing the IF1-mediated inhibition of the ATP synthase. Critical Issues: It is unknown how the redox signal spreads up to the plasma membrane and what its targets are, what the differences in metabolic, redox, NADH/NADPH, and Ca2+ signaling, and homeostasis are between the first and second GSIS phase, and whether mitochondria can replace ER in the amplification of IGV exocytosis. Future Directions: Metabolomics studies performed to distinguish between the mitochondrial matrix and cytosolic metabolites will elucidate further details. Identifying the targets of cell signaling into mitochondria and of mitochondrial retrograde metabolic and redox signals to the cell will uncover further molecular mechanisms for insulin secretion stimulated by glucose, BCKAs, and FAs, and the amplification of secretion by glucagon-like peptide (GLP-1) and metabotropic receptors. They will identify the distinction between the hub β-cells and their followers in intact and diabetic states. Antioxid. Redox Signal. 36, 920-952.
- MeSH
- adenosintrifosfát metabolismus MeSH
- beta-buňky * metabolismus MeSH
- glukosa metabolismus MeSH
- inzulin metabolismus MeSH
- Langerhansovy ostrůvky * metabolismus MeSH
- mitochondrie metabolismus MeSH
- NAD metabolismus MeSH
- NADP metabolismus MeSH
- sekrece inzulinu MeSH
- sekretagoga metabolismus MeSH
- vápník metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- MeSH
- beta-buňky fyziologie patologie účinky léků MeSH
- cévy patologie účinky léků MeSH
- diabetes mellitus 2. typu * epidemiologie etiologie komplikace mortalita patofyziologie MeSH
- diabetické angiopatie etiologie MeSH
- endotel patofyziologie účinky léků MeSH
- inzulinová rezistence MeSH
- kouření patofyziologie škodlivé účinky MeSH
- lidé MeSH
- nikotin * škodlivé účinky MeSH
- oxidační stres fyziologie účinky léků MeSH
- rizikové faktory MeSH
- sekrece inzulinu MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
We adapted a radioligand receptor binding assay for measuring insulin levels in unknown samples. The assay enables rapid and accurate determination of insulin concentrations in experimental samples, such as from insulin-secreting cells. The principle of the method is based on the binding competition of insulin in a measured sample with a radiolabeled insulin for insulin receptor (IR) in IM-9 cells. Both key components, radiolabeled insulin and IM-9 cells, are commercially available. The IR binding assay was used to determine unknown amounts of insulin secreted by MIN6 β cell line after stimulation with glucose, arginine, ornithine, dopamine, and serotonin. The experimental data obtained by the IR binding assay were compared to the results determined by RIA kits and both methods showed a very good agreement of results. We observed the stimulation of glucose-induced insulin secretion from MIN6 cells by arginine, weaker stimulation by ornithine, but inhibitory effects of dopamine. Serotonin effects were either stimulatory or inhibitory, depending on the concentration of serotonin used. The results will require further investigation. The study also clearly revealed advantages of the IR binding assay that allows the measuring of a higher throughput of measured samples, with a broader range of concentrations than in the case of RIA kits. The IR binding assay can provide an alternative to standard RIA and ELISA assays for the determination of insulin levels in experimental samples and can be especially useful in scientific laboratories studying insulin production and secretion by β cells and searching for new modulators of insulin secretion.
- MeSH
- arginin metabolismus MeSH
- beta-buňky metabolismus MeSH
- buněčné linie MeSH
- dopamin metabolismus MeSH
- glukosa metabolismus MeSH
- inzulin analýza metabolismus MeSH
- krysa rodu rattus MeSH
- Langerhansovy ostrůvky metabolismus MeSH
- lidé MeSH
- myši MeSH
- ornithin metabolismus MeSH
- potkani Wistar MeSH
- radioimunoanalýza metody MeSH
- radioligandová zkouška metody MeSH
- sekrece inzulinu * MeSH
- serotonin metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Diabetes mellitus je stav chronické hyperglykemie, který může být způsoben mnoha zevními a genetickými faktory. Nejčastější v populaci je diabetes mellitus 2. typu, v jehož patogenezi se uplatňuje inzulinová rezistence a porucha inzulinové sekrece. Diabetici patří mezi vysoce rizikové pacienty a přítomnost diabetu je hodnocena přímo jako ekvivalent kardiovaskulárního onemocnění. Součástí léčby pacientů s diabetem 2. typu jsou režimová opatření, redukce hmotnosti, současně v době diagnostiky zahajujeme léčbu metforminem, pokud nejsou kontraindikace k jeho použití. Při nedostatečném efektu intenzifikujeme léčbu přidáním dalšího perorálního antidiabetika nebo inzulinu.
Diabetes mellitus means presence of chronic hyperglycaemia caused by many external and internal conditions. The most frequently present type is Type 2 diabetes. In its pathogenesis play important role insulin resistance and disturbance of insulin secretion. Diabetic patients are extremely risky in development of macrovascular complications and diabetes is considered to be an equivalent of present cardiovascular disorder. In treatment of diabetes we start with lifestyle changes, weight reduction and metformin at the same time. In not optimal metabolic compensation, we add to metformin another drug or insulin.
- MeSH
- diabetes mellitus 2. typu * diagnóza farmakoterapie MeSH
- glifloziny terapeutické užití MeSH
- hypoglykemika terapeutické užití MeSH
- inkretiny terapeutické užití MeSH
- inzulin terapeutické užití MeSH
- inzulinová rezistence MeSH
- lidé MeSH
- metformin terapeutické užití MeSH
- pioglitazon terapeutické užití MeSH
- sekrece inzulinu MeSH
- sekretagoga terapeutické užití MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Aims: Glucose-stimulated insulin secretion (GSIS) in pancreatic β cells was expected to enhance mitochondrial superoxide formation. Hence, we elucidated relevant redox equilibria. Results: Unexpectedly, INS-1E cells at transitions from 3 (11 mM; pancreatic islets from 5 mM) to 25 mM glucose decreased matrix superoxide release rates (MitoSOX Red monitoring validated by MitoB) and H2O2 (mitoHyPer, subtracting mitoSypHer emission). Novel double-channel fluorescence lifetime imaging, approximating free mitochondrial matrix NADHF, indicated its ∼20% decrease. Matrix NAD+F increased on GSIS, indicated by the FAD-emission lifetime decrease, reflecting higher quenching of FAD by NAD+F. The participation of pyruvate/malate and pyruvate/citrate redox shuttles, elevating cytosolic NADPHF (iNAP1 fluorescence monitoring) at the expense of matrix NADHF, was indicated, using citrate (2-oxoglutarate) carrier inhibitors and cytosolic malic enzyme silencing: All changes vanished on these manipulations. 13C-incorporation from 13C-L-glutamine into 13C-citrate reflected the pyruvate/isocitrate shuttle. Matrix NADPHF (iNAP3 monitored) decreased. With decreasing glucose, the suppressor of Complex III site Q electron leak (S3QEL) suppressor caused a higher Complex I IF site contribution, but a lower superoxide fraction ascribed to the Complex III site IIIQo. Thus, the diminished matrix NADHF/NAD+F decreased Complex I flavin site IF superoxide formation on GSIS. Innovation: Mutually validated methods showed decreasing superoxide release into the mitochondrial matrix in pancreatic β cells on GSIS, due to the decreasing matrix NADHF/NAD+F (NADPHF/NADP+F) at increasing cytosolic NADPHF levels. The developed innovative methods enable real-time NADH/NAD+ and NADPH/NADP+ monitoring in any distinct cell compartment. Conclusion: The export of reducing equivalents from mitochondria adjusts lower mitochondrial superoxide production on GSIS, but it does not prevent oxidative stress in pancreatic β cells.
- MeSH
- adenosintrifosfát metabolismus MeSH
- beta-buňky metabolismus MeSH
- buněčné dýchání MeSH
- chromatografie kapalinová MeSH
- energetický metabolismus MeSH
- flavinadenindinukleotid metabolismus MeSH
- glukosa metabolismus MeSH
- hmotnostní spektrometrie MeSH
- krysa rodu rattus MeSH
- kyselina citronová metabolismus MeSH
- membránový potenciál mitochondrií MeSH
- metabolické sítě a dráhy MeSH
- metabolomika metody MeSH
- mitochondrie metabolismus MeSH
- NAD metabolismus MeSH
- peroxid vodíku metabolismus MeSH
- sekrece inzulinu * MeSH
- signální transdukce MeSH
- superoxidy metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ideální bazální inzulin by měl mít co nejmenší nebo žádný vrchol účinku, téměř konstantní vliv na snížení glykemie a zároveň co nejmenší variabilitu. Bylo prokázáno, že zvýšená glykemická variabilita je spjata s rizikem rozvoje závažné hypoglykemie. Studie EDITION JUNIOR prokázala u pacientů v dětském a adolescentním věku numerické snížení incidence závažné hypoglykemie i incidence hyperglykemie s ketózou ve skupině léčené inzulinem glargin 300 j/ml (Gla-300) vs. inzulinem glargin 100 j/ml (Gla-100). Systémy kontinuálního sledování glykemie (CGM) nám v současnosti umožňují účinně posoudit variabilitu glykemie a procento času v jejím cílovém rozmezí. Máme již k dispozici data CGM u pacientů s diabetem 2. typu, u nichž byl průměrný čas strávený v hypoglykemii signifikantně kratší při léčbě Gla-300 vs. inzulinem degludek U 100 (Deg) (p = 0,002).20 Na podzim tohoto roku má být ukončena studie InRange, využívající CGM ke srovnání léčby Deg a Gla-300 u pacientů s diabetem 1. typu a v roce 2021/2022 můžeme očekávat publikaci výsledků.
An ideal basal insulin would have little or no peak and instead produce a near-continuous level of blood glucose- -lowering action while minimizing variability. Elevated glycaemic variability was identified as independent determinant of severe hypoglycemia risk. The EDITION JUNIOR study showed a numerical reduction in the incidence of severe hypoglycaemia and the incidence of hyperglycaemia with ketosis in the Gla-300 vs. Gla-100 group in pediatric and adolescent patients. CGM systems currently allow us to effectively assess glycemic variability and percentage of time within the target glycemic range. We already have CGM data for type 2 diabetes patients, where the mean time spent in hypoglycaemia with Gla-300 vs. Deg was significantly shorter (p = 0,002). In the autumn should be finished InRange trial using CGM for comparison of Deg and Gla-300 treatment in type 1 diabetic patients and publication of results we can expect in 2021/2022.
NADPH facilitates glucose-stimulated insulin secretion (GSIS) in pancreatic islets (PIs) of β-cells through an as yet unknown mechanism. We found NADPH oxidase isoform 4 (NOX4) to be the main producer of cytosolic H2O2, which is essential for GSIS; an increase in ATP alone was insufficient for GSIS. The fast GSIS phase was absent from PIs from NOX4-null, β-cell-specific knockout mice (NOX4βKO) (though not from NOX2 knockout mice) and from NOX4-silenced or catalase-overexpressing INS-1E cells. Lentiviral NOX4 overexpression or H2O2 rescued GSIS in PIs from NOX4βKO mice. NOX4 silencing suppressed Ca2+ oscillations, and the patch-clamped KATP channel opened more frequently when glucose was high. Mitochondrial H2O2, decreasing upon GSIS, provided alternative redox signaling when 2-oxo-isocaproate or fatty acid oxidation formed superoxides through electron-transfer flavoprotein:Q-oxidoreductase. Unlike GSIS, such insulin secretion was blocked with mitochondrial antioxidant SkQ1. Both NOX4 knockout and NOX4βKO mice exhibited impaired glucose tolerance and peripheral insulin resistance. Thus, the redox signaling previously suggested to cause β-cells to self-check hypothetically induces insulin resistance when it is absent. In conclusion, increases in ATP and H2O2 constitute an essential signal that switches on insulin exocytosis for glucose and branched-chain oxoacids as secretagogues (it does so partially for fatty acids). Redox signaling could be impaired by cytosolic antioxidants; hence, those targeting mitochondria should be preferred for clinical applications to treat (pre)diabetes at any stage.
- MeSH
- draslíkové kanály fyziologie MeSH
- glukosa farmakologie MeSH
- inzulinová rezistence MeSH
- kultivované buňky MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- NADPH-oxidasa 4 fyziologie MeSH
- peroxid vodíku metabolismus MeSH
- sekrece inzulinu * MeSH
- signální transdukce fyziologie MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVE: Reparixin is an inhibitor of CXCR1/2 chemokine receptor shown to be an effective anti-inflammatory adjuvant in a pilot clinical trial in allotransplant recipients. RESEARCH DESIGN AND METHODS: A phase 3, multicenter, randomized, double-blind, parallel-assignment study (NCT01817959) was conducted in recipients of islet allotransplants randomized (2:1) to reparixin or placebo in addition to immunosuppression. Primary outcome was the area under the curve (AUC) for C-peptide during the mixed-meal tolerance test at day 75 ± 5 after the first and day 365 ± 14 after the last transplant. Secondary end points included insulin independence and standard measures of glycemic control. RESULTS: The intention-to-treat analysis did not show a significant difference in C-peptide AUC at both day 75 (27 on reparixin vs. 18 on placebo, P = 0.99) and day 365 (24 on reparixin vs. 15 on placebo, P = 0.71). There was no statistically significant difference between treatment groups at any time point for any secondary variable. Analysis of patient subsets showed a trend for a higher percentage of subjects retaining insulin independence for 1 year after a single islet infusion in patients receiving reparixin as compared with patients receiving placebo (26.7% vs. 0%, P = 0.09) when antithymocyte globulin was used as induction immunosuppression. CONCLUSIONS: In this first double-blind randomized trial, islet transplantation data obtained with reparixin do not support a role of CXCR1/2 inhibition in preventing islet inflammation-mediated damage.
- MeSH
- časové faktory MeSH
- diabetes mellitus 1. typu farmakoterapie terapie MeSH
- dospělí MeSH
- dvojitá slepá metoda MeSH
- imunosupresiva aplikace a dávkování MeSH
- kombinovaná terapie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- placeba MeSH
- pooperační období MeSH
- receptory interleukinu-8A antagonisté a inhibitory MeSH
- receptory interleukinu-8B antagonisté a inhibitory MeSH
- rozvrh dávkování léků MeSH
- sekrece inzulinu účinky léků MeSH
- senioři MeSH
- sulfonamidy aplikace a dávkování škodlivé účinky farmakologie MeSH
- transplantace Langerhansových ostrůvků * MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- klinické zkoušky, fáze III MeSH
- multicentrická studie MeSH
- práce podpořená grantem MeSH
- randomizované kontrolované studie MeSH
