- Klíčová slova
- hyperglukagonémie,
- MeSH
- beta-buňky metabolismus sekrece účinky léků MeSH
- buňky vylučující glukagon metabolismus sekrece účinky léků MeSH
- diabetes mellitus 2. typu * enzymologie farmakoterapie metabolismus MeSH
- financování organizované MeSH
- glukagon * metabolismus sekrece škodlivé účinky MeSH
- glukagonu podobný peptid 1 aplikace a dávkování škodlivé účinky terapeutické užití MeSH
- glukosa metabolismus škodlivé účinky terapeutické užití MeSH
- hyperglykemie * komplikace patofyziologie prevence a kontrola MeSH
- inhibitory dipeptidylpeptidasy 4 aplikace a dávkování škodlivé účinky terapeutické užití MeSH
- inzuliny aplikace a dávkování škodlivé účinky terapeutické užití MeSH
- Langerhansovy ostrůvky fyziologie patofyziologie sekrece MeSH
- lidé MeSH
- metformin aplikace a dávkování škodlivé účinky terapeutické užití MeSH
- Check Tag
- lidé 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.
- Klíčová slova
- léčba DM založená na inkretinech,
- MeSH
- aminokyseliny, peptidy a proteiny fyziologie metabolismus sekrece MeSH
- beta-buňky * enzymologie metabolismus sekrece MeSH
- dipeptidylpeptidasa 4 farmakologie metabolismus sekrece MeSH
- enteroendokrinní buňky cytologie enzymologie metabolismus MeSH
- glukagonu podobný peptid 1 * fyziologie metabolismus sekrece MeSH
- inkretiny fyziologie izolace a purifikace metabolismus MeSH
- inzulin * fyziologie izolace a purifikace sekrece MeSH
- L buňky (buněčná linie) fyziologie metabolismus účinky léků MeSH
- Langerhansovy ostrůvky cytologie fyziologie sekrece MeSH
- lidé MeSH
- lipidy fyziologie sekrece MeSH
- mastné kyseliny fyziologie sekrece MeSH
- nervový systém enzymologie metabolismus sekrece MeSH
- potravní vláknina metabolismus využití MeSH
- sacharidy fyziologie sekrece MeSH
- statistika jako téma MeSH
- tenké střevo enzymologie fyziologie sekrece MeSH
- žlučové kyseliny a soli fyziologie metabolismus sekrece MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
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 Ca2+-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. GdCl3 (100 µmol/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 µmol/l) inhibited glucose-induced but not swelling-induced insulin secretion from INS-1 cells. Surprisingly, perifusion with Ca2+-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 Ca2+ in medium.
- Klíčová slova
- Cell volume, Insulin secretion, Pancreatic islets, Tumor cell line, Ca2+ and exocytosis,
- MeSH
- exocytóza MeSH
- financování organizované MeSH
- gadolinium farmakologie MeSH
- glukosa metabolismus MeSH
- hypertonické roztoky MeSH
- hypotonické roztoky MeSH
- inzulin sekrece MeSH
- inzulinom metabolismus sekrece MeSH
- krysa rodu rattus MeSH
- Langerhansovy ostrůvky metabolismus sekrece účinky léků MeSH
- nádorové buněčné linie MeSH
- nádory slinivky břišní metabolismus sekrece MeSH
- noradrenalin metabolismus MeSH
- osmotický tlak MeSH
- potkani Wistar MeSH
- vápník metabolismus nedostatek MeSH
- velikost buňky MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata 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
- adenosindifosfát metabolismus MeSH
- adenosintrifosfát metabolismus MeSH
- financování organizované MeSH
- glukosa farmakologie MeSH
- inzulinom sekrece MeSH
- krysa rodu rattus MeSH
- kyselina linolová farmakologie MeSH
- Langerhansovy ostrůvky sekrece účinky léků MeSH
- membránový potenciál mitochondrií účinky léků MeSH
- mitochondrie metabolismus účinky léků MeSH
- nádorové buňky kultivované MeSH
- nádory slinivky břišní sekrece MeSH
- potkani Wistar MeSH
- spotřeba kyslíku účinky léků MeSH
- transmisní elektronová mikroskopie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
Pro diabetes mellitus 2. typu je typická hyperglykemie, která je důsledkem kombinace dvou poruch – účinku inzulinu a jeho sekrece. V poslední době se ukazuje, že při hodnocení účinku léčby nejsou rozhodující jen hodnoty glykosylovaného hemoglobinu a glykemie nalačno, ale také glykemie postprandiální, jež mohou mít dokonce větší hodnotu pro předpověď úmrtí než hodnoty nalačno.
- MeSH
- diabetes mellitus 2. typu farmakoterapie MeSH
- fenylalanin analogy a deriváty terapeutické užití MeSH
- hodnocení léčiv MeSH
- hyperglykemie farmakoterapie MeSH
- hypoglykemika aplikace a dávkování farmakokinetika farmakologie MeSH
- Langerhansovy ostrůvky sekrece účinky léků MeSH
- lidé MeSH
- nateglinid MeSH
- postprandiální období MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- randomizované kontrolované studie MeSH
