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
Diabetes is a metabolic disease that involves the death or dysfunction of the insulin-secreting β cells in the pancreas. Consequently, most diabetes research is aimed at understanding the molecular and cellular bases of pancreatic development, islet formation, β-cell survival, and insulin secretion. Complex interactions of signaling pathways and transcription factor networks regulate the specification, growth, and differentiation of cell types in the developing pancreas. Many of the same regulators continue to modulate gene expression and cell fate of the adult pancreas. The transcription factor NEUROD1 is essential for the maturation of β cells and the expansion of the pancreatic islet cell mass. Mutations of the Neurod1 gene cause diabetes in humans and mice. However, the different aspects of the requirement of NEUROD1 for pancreas development are not fully understood. In this study, we investigated the role of NEUROD1 during the primary and secondary transitions of mouse pancreas development. We determined that the elimination of Neurod1 impairs the expression of key transcription factors for α- and β-cell differentiation, β-cell proliferation, insulin production, and islets of Langerhans formation. These findings demonstrate that the Neurod1 deletion altered the properties of α and β endocrine cells, resulting in severe neonatal diabetes, and thus, NEUROD1 is required for proper activation of the transcriptional network and differentiation of functional α and β cells.
- MeSH
- beta-buňky cytologie metabolismus MeSH
- buněčná diferenciace MeSH
- buněčný rodokmen MeSH
- diabetes mellitus genetika MeSH
- inzulin metabolismus MeSH
- Langerhansovy ostrůvky cytologie metabolismus ultrastruktura MeSH
- myši inbrední C57BL MeSH
- myši transgenní MeSH
- novorozená zvířata MeSH
- pankreas cytologie embryologie MeSH
- proliferace buněk MeSH
- transkripční faktory bHLH genetika metabolismus MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články 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
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.
- MeSH
- beta-buňky metabolismus ultrastruktura MeSH
- chemická frakcionace MeSH
- cytoplazmatická granula metabolismus MeSH
- exprese genu * MeSH
- glukosa metabolismus MeSH
- inzulin genetika metabolismus MeSH
- krysa rodu rattus MeSH
- Langerhansovy ostrůvky metabolismus MeSH
- messenger RNA genetika metabolismus MeSH
- průtoková cytometrie metody MeSH
- zinek metabolismus MeSH
- zinkový transportér 8 MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Magnetoliposomes (MLs) were synthesized and tested for longitudinal monitoring of transplanted pancreatic islets using magnetic resonance imaging (MRI) in rat models. The rat insulinoma cell line INS-1E and isolated pancreatic islets from outbred and inbred rats were used to optimize labeling conditions in vitro. Strong MRI contrast was generated by islets exposed to 50 µg Fe/ml for 24 hours without any increased cell death, loss of function or other signs of toxicity. In vivo experiments showed that pancreatic islets (50-1000 units) labeled with MLs were detectable for up to 6 weeks post-transplantation in the kidney subcapsular space. Islets were also monitored for two weeks following transplantation through the portal vein of the liver. Hereby, islets labeled with MLs and transplanted under the left kidney capsule were able to correct hyperglycemia and had stable MRI signals until nephrectomy. Interestingly, in vivo MRI of streptozotocin induced diabetic rats transplanted with allogeneic islets demonstrated loss of MRI contrast between 7-16 days, indicative of loss of islet structure. MLs used in this study were not only beneficial for monitoring the location of transplanted islets in vivo with high sensitivity but also reported on islet integrity and hereby indirectly on islet function and rejection.
- MeSH
- experimentální diabetes mellitus chemicky indukované metabolismus patologie MeSH
- hyperglykemie metabolismus patologie MeSH
- inzulin metabolismus MeSH
- játra metabolismus patologie MeSH
- kontrastní látky metabolismus MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- Langerhansovy ostrůvky metabolismus patologie MeSH
- longitudinální studie MeSH
- magnetická rezonanční tomografie metody MeSH
- magnetické nanočástice aplikace a dávkování MeSH
- potkani inbrední LEW MeSH
- potkani Wistar MeSH
- streptozocin farmakologie MeSH
- transplantace Langerhansových ostrůvků metody MeSH
- vena portae metabolismus patologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Subcutaneously implanted polymeric scaffolds represent an alternative transplantation site for pancreatic islets (PIs) with the option of vascularisation enhancement by mesenchymal stem cells (MSC). Nevertheless, a proper timing of the transplantation steps is crucial. In this study, scaffolds supplemented with plastic rods were implanted into diabetic rats and two timing schemes for subsequent transplantation of bioluminescent PIs (4 or 7 days after rod removal) were examined by multimodal imaging. The cavities were left to heal spontaneously or with 10 million injected MSCs. Morphological and vascularisation changes were examined by MRI, while the localisation and viability of transplanted islets were monitored by bioluminescence imaging. The results show that PIs transplanted 4 days after rod removal showed the higher optical signal and vascularisation compared to transplantation after 7 days. MSCs slightly improved vascularisation of the graft but hindered therapeutic efficiency of PIs. Long-term glycaemia normalisation (4 months) was attained in 80% of animals. In summary, multimodal imaging confirmed the long-term survival and function of transplanted PIs in the devices. The best outcome was reached with PIs transplanted on day 4 after rod removal and therefore the suggested protocol holds a potential for further applications.
- MeSH
- alografty MeSH
- experimentální diabetes mellitus * diagnostické zobrazování chirurgie MeSH
- krysa rodu rattus MeSH
- Langerhansovy ostrůvky * krevní zásobení diagnostické zobrazování metabolismus patologie MeSH
- luminiscenční měření * MeSH
- mezenchymální kmenové buňky metabolismus patologie MeSH
- potkani transgenní MeSH
- přežívání štěpu * MeSH
- tkáňové podpůrné struktury * MeSH
- transplantace Langerhansových ostrůvků metody MeSH
- transplantace mezenchymálních kmenových buněk MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: The mechanisms and relevance of impaired glucose homeostasis in advanced heart failure (HF) are poorly understood. The study goals were to examine glucose regulation, pancreatic endocrine function, and metabolic factors related to prognosis in patients with nondiabetic advanced HF. METHODS AND RESULTS: In total, 140 advanced HF patients without known diabetes mellitus and 21 sex-, age-, and body mass index-matched controls underwent body composition assessment, oral glucose tolerance testing, and measurement of glucose-regulating hormones to model pancreatic β-cell secretory response. Compared with controls, HF patients had similar fasting glucose and insulin levels but higher levels after oral glucose tolerance testing. Insulin secretion was not impaired, but with increasing HF severity, there was a reduction in glucose, insulin, and insulin/glucagon ratio-a signature of starvation. The insulin/C-peptide ratio was decreased in HF, indicating enhanced insulin clearance, and this was correlated with lower cardiac output, hepatic insufficiency, right ventricular dysfunction, and body wasting. After a median of 449 days, 41% of patients experienced an adverse event (death, urgent transplant, or assist device). Increased glucagon and, paradoxically, low fasting plasma glucose displayed the strongest relations to outcome (P=0.01). Patients in the lowest quartile of fasting plasma glucose (3.8-5.1 mmol·L-1, 68-101 mg·dL-1) had 3-times higher event risk than in the top quartile (6.0-7.9 mmol·L-1, 108-142 mg·dL-1; relative risk: 3.05 [95% confidence interval, 1.46-6.77]; P=0.002). CONCLUSIONS: Low fasting plasma glucose and increased glucagon are robust metabolic predictors of adverse events in advanced HF. Pancreatic insulin secretion is preserved in advanced HF, but levels decrease with increasing HF severity due to enhanced insulin clearance that is coupled with right heart failure and cardiac cachexia.
- MeSH
- biologické markery krev MeSH
- časové faktory MeSH
- dysfunkce pravé srdeční komory krev diagnóza patofyziologie MeSH
- funkce pravé komory srdeční MeSH
- glukagon krev MeSH
- glukózový toleranční test MeSH
- homeostáza MeSH
- inzulin krev MeSH
- kachexie krev diagnóza patofyziologie MeSH
- Kaplanův-Meierův odhad MeSH
- krevní glukóza metabolismus MeSH
- Langerhansovy ostrůvky metabolismus patofyziologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- prognóza MeSH
- rizikové faktory MeSH
- senioři MeSH
- srdeční selhání krev diagnóza patofyziologie MeSH
- studie případů a kontrol MeSH
- stupeň závažnosti nemoci MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- alanin fyziologie chemie metabolismus MeSH
- diabetes mellitus 2. typu * enzymologie etiologie metabolismus MeSH
- disacharidy fyziologie chemie metabolismus MeSH
- glukoneogeneze fyziologie imunologie MeSH
- glukosa * fyziologie chemie metabolismus MeSH
- glutamin fyziologie chemie metabolismus MeSH
- glykogen fyziologie chemie metabolismus MeSH
- hepatocyty cytologie fyziologie metabolismus MeSH
- hyperglykemie etiologie krev metabolismus MeSH
- hypoglykemie etiologie krev metabolismus MeSH
- inzulin fyziologie chemie metabolismus MeSH
- inzulinová rezistence fyziologie genetika imunologie MeSH
- Langerhansovy ostrůvky cytologie fyziologie metabolismus MeSH
- lidé MeSH
- lipoproteiny VLDL fyziologie chemie metabolismus MeSH
- mastné kyseliny fyziologie chemie metabolismus MeSH
- proteiny usnadňující transport glukosy fyziologie chemie metabolismus MeSH
- statistika jako téma MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Variability of pancreatic donors may significantly impact the success of islet isolation. The aim of this study was to evaluate donor factors associated with isolation failure and to investigate whether immunohistology could contribute to organ selection. Donor characteristics were evaluated for both successful (n = 61) and failed (n = 98) islet isolations. Samples of donor pancreatic tissue (n = 78) were taken for immunohistochemical examination. Islet isolations with 250000 islet equivalents were considered successful. We confirmed that BMI of less than 25 kg/m(2) (P < 0.001), cold ischemia time more than 8 hours (P < 0.01), hospitalization longer than 96 hours (P < 0.05), higher catecholamine doses (P < 0.05), and edematous pancreases (P < 0.01) all unfavorably affected isolation outcome. Subsequent immunohistochemical examination of donor pancreases confirmed significant differences in insulin-positive areas (P < 0.001). ROC analyses then established that the insulin-positive area in the pancreas could be used to predict the likely success of islet isolation (P < 0.001). At the optimal cutoff point (>1.02%), sensitivity and specificity were 89% and 76%, respectively. To conclude, while the insulin-positive area, determined preislet isolation, as a single variable, is sufficient to predict isolation outcome and helps to improve the success of this procedure, its combination with the established donor scoring system might further improve organ selection.
- MeSH
- dárci tkání statistika a číselné údaje MeSH
- délka pobytu statistika a číselné údaje MeSH
- diabetes mellitus 1. typu chirurgie MeSH
- edém epidemiologie MeSH
- hospitalizace MeSH
- imunohistochemie MeSH
- index tělesné hmotnosti MeSH
- inzulin metabolismus MeSH
- Langerhansovy ostrůvky metabolismus chirurgie MeSH
- lidé MeSH
- pankreas MeSH
- retrospektivní studie MeSH
- studená ischemie statistika a číselné údaje MeSH
- transplantace Langerhansových ostrůvků metody MeSH
- vazokonstriktory terapeutické užití MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3) in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information from 48 hrs onwards.
