Following stimulation, pancreatic β-cells must orchestrate a plethora of signalling events to ensure the appropriate release of insulin and maintenance of normal glucose homeostasis. Failure at any point in this cascade leads to impaired insulin secretion, elevated blood levels of glucose and eventually type 2 diabetes mellitus. Likewise, β-cell replacement or regeneration strategies for the treatment of both type 1 and type 2 diabetes mellitus might fail if the correct cell signalling phenotype cannot be faithfully recreated. However, current understanding of β-cell function is complicated because of the highly dynamic nature of their intracellular and intercellular signalling as well as insulin release itself. β-Cells must precisely integrate multiple signals stemming from multiple cues, often with differing intensities, frequencies and cellular and subcellular localizations, before converging these signals onto insulin exocytosis. In this respect, optical approaches with high resolution in space and time are extremely useful for properly deciphering the complexity of β-cell signalling. An increased understanding of β-cell signalling might identify new mechanisms underlying insulin release, with relevance for future drug therapy and de novo stem cell engineering of functional islets.
- MeSH
- beta-buňky metabolismus MeSH
- diabetes mellitus 2. typu genetika metabolismus MeSH
- genetická predispozice k nemoci epidemiologie MeSH
- glukosa metabolismus MeSH
- hodnocení rizik MeSH
- homeostáza genetika MeSH
- incidence MeSH
- lidé MeSH
- sekrece inzulinu fyziologie MeSH
- senzitivita a specificita MeSH
- signální transdukce MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The secretion of insulin from β-cells depends on extracellular factors, in particular glucose and other small molecules, some of which act on G-protein-coupled receptors. Fatty acids (FAs) have been discussed as exogenous secretagogues of insulin for decades, especially after the FA receptor GPR40 (G-protein-coupled receptor 40) was discovered. However, the role of FAs as endogenous signaling factors has not been investigated until now. In the present work, we demonstrate that lowering endogenous FA levels in β-cell medium by stringent washing or by the application of FA-free (FAF) BSA immediately reduced glucose-induced oscillations of cytosolic Ca2+ ([Ca2+]i oscillations) in MIN6 cells and mouse primary β-cells, as well as insulin secretion. Mass spectrometry confirmed BSA-mediated removal of FAs, with palmitic, stearic, oleic, and elaidic acid being the most abundant species. [Ca2+]i oscillations in MIN6 cells recovered when BSA was replaced by buffer or as FA levels in the supernatant were restored. This was achieved by recombinant lipase-mediated FA liberation from membrane lipids, by the addition of FA-preloaded FAF-BSA, or by the photolysis of cell-impermeant caged FAs. Our combined data support the hypothesis of FAs as essential endogenous signaling factors for β-cell activity and insulin secretion.
- MeSH
- beta-buňky metabolismus MeSH
- buněčné linie MeSH
- chromatografie kapalinová MeSH
- ELISA MeSH
- hmotnostní spektrometrie MeSH
- inzulin metabolismus MeSH
- konfokální mikroskopie MeSH
- myši MeSH
- receptory spřažené s G-proteiny genetika metabolismus MeSH
- sekrece inzulinu MeSH
- sérový albumin hovězí farmakologie MeSH
- signální transdukce fyziologie MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Despite the urgent need for assays to visualize insulin secretion there is to date no reliable method available for measuring insulin release from single cells. To address this need, we developed a genetically encoded reporter termed RINS1 based on proinsulin superfolder GFP (sfGFP) and mCherry fusions for monitoring insulin secretion. RINS1 expression in MIN6 β cells resulted in proper processing yielding single-labeled insulin species. Unexpectedly, glucose or drug stimulation of insulin secretion in β cells led to the preferential release of the insulin-sfGFP construct, while the mCherry-fused C-peptide remained trapped in exocytic granules. This physical separation was used to monitor glucose-stimulated insulin secretion ratiometrically by total internal reflection fluorescence microscopy in single MIN6 and primary mouse β cells. Further, RINS1 enabled parallel monitoring of pulsatile insulin release in tolbutamide-treated β cells, demonstrating the potential of RINS1 for investigations of antidiabetic drug candidates at the single-cell level.
- MeSH
- beta-buňky cytologie účinky léků metabolismus sekrece MeSH
- biosenzitivní techniky MeSH
- buněčné linie MeSH
- fluorescenční mikroskopie MeSH
- glukosa farmakologie MeSH
- hypoglykemika farmakologie MeSH
- inzulin metabolismus sekrece MeSH
- luminescentní proteiny genetika metabolismus MeSH
- myši MeSH
- rekombinantní fúzní proteiny metabolismus sekrece MeSH
- reportérové geny MeSH
- tolbutamid farmakologie MeSH
- vápník metabolismus MeSH
- zelené fluorescenční proteiny genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1-domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains toward the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans. As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control and are broadly applicable tools to study DAG signaling.
- MeSH
- Caenorhabditis elegans enzymologie metabolismus účinky záření MeSH
- diglyceridy chemie metabolismus účinky záření MeSH
- fotochemické procesy účinky záření MeSH
- myši MeSH
- optické jevy MeSH
- proteinkinasa C chemie metabolismus účinky záření MeSH
- signální transdukce účinky záření MeSH
- ultrafialové záření * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Photoactivation of caged biomolecules has become a powerful approach to study cellular signalling events. Here we report a method for anchoring and uncaging biomolecules exclusively at the outer leaflet of the plasma membrane by employing a photocleavable, sulfonated coumarin derivative. The novel caging group allows quantifying the reaction progress and efficiency of uncaging reactions in a live-cell microscopy setup, thereby greatly improving the control of uncaging experiments. We synthesized arachidonic acid derivatives bearing the new negatively charged or a neutral, membrane-permeant coumarin caging group to locally induce signalling either at the plasma membrane or on internal membranes in β-cells and brain slices derived from C57B1/6 mice. Uncaging at the plasma membrane triggers a strong enhancement of calcium oscillations in β-cells and a pronounced potentiation of synaptic transmission while uncaging inside cells blocks calcium oscillations in β-cells and causes a more transient effect on neuronal transmission, respectively. The precise subcellular site of arachidonic acid release is therefore crucial for signalling outcome in two independent systems.
- MeSH
- beta-buňky metabolismus účinky záření MeSH
- buněčná membrána metabolismus účinky záření MeSH
- HeLa buňky MeSH
- kumariny chemie metabolismus MeSH
- kyselina arachidonová chemie metabolismus MeSH
- lidé MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- neurony metabolismus účinky záření MeSH
- světlo MeSH
- vápník metabolismus MeSH
- vápníková signalizace účinky záření MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
