Reduced synchroneity of intra-islet Ca2+ oscillations in vivo in Robo-deficient β cells.
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ABSTRACT:
SUBMITTER: Adams MT
PROVIDER: S-EPMC8289414 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Islet β-cells are responsible for secreting all circulating insulin in response to rising plasma glucose concentrations. These cells are a phenotypically diverse population that express great functional heterogeneity. In mice, certain β-cells (termed 'hubs') have been shown to be crucial for dictating the islet response to high glucose, with inhibition of these hub cells abolishing the coordinated Ca2+ oscillations necessary for driving insulin secretion. These β-cell hubs were found to be highly metabolic and susceptible to pro-inflammatory and glucolipotoxic insults. In this study, we explored the importance of hub cells in human by constructing mathematical models of Ca2+ activity in human islets. Our simulations revealed that hubs dictate the coordinated Ca2+ response in both mouse and human islets; silencing a small proportion of hubs abolished whole-islet Ca2+ activity. We also observed that if hubs are assumed to be preferentially gap junction coupled, then the simulations better adhere to the available experimental data. Our simulations of 16 size-matched mouse and human islet architectures revealed that there are species differences in the role of hubs; Ca2+ activity in human islets was more vulnerable to hub inhibition than mouse islets. These simulation results not only substantiate the existence of β-cell hubs, but also suggest that hubs may be favorably coupled in the electrical and metabolic network of the islet, and that targeted destruction of these cells would greatly impair human islet function.
| S-EPMC6113907 | biostudies-literature
Project description:Aims/hypothesisPrediabetic pancreatic beta cells can adapt their function to maintain normoglycaemia for a limited period of time, after which diabetes mellitus will manifest upon beta cell exhaustion. Understanding sex-specific beta cell compensatory mechanisms and their failure in prediabetes (impaired glucose tolerance) is crucial for early disease diagnosis and individualised treatment. Our aims were as follows: (1) to determine the key time points of the progression from beta cells' functional adaptations to their failure in vivo; and (2) to mechanistically explain in vivo sex-specific beta cell compensatory mechanisms and their failure in prediabetes.MethodsIslets from male and female transgenic Ins1CreERT2-GCaMP3 mice were transplanted into the anterior chamber of the eye of 10- to 12-week-old sex-matched C57BL/6J mice. Recipient mice were fed either a control diet (CD) or western diet (WD) for a maximum of 4 months. Metabolic variables were evaluated monthly. Beta cell cytoplasmic free calcium concentration ([Ca2+]i) dynamics were monitored in vivo longitudinally by image fluorescence of the GCaMP3 reporter islets. Global islet beta cell [Ca2+]i dynamics in line with single beta cell [Ca2+]i analysis were used for beta cell coordination studies. The glucagon receptor antagonist L-168,049 (4 mmol/l) was applied topically to the transplanted eyes to evaluate in vivo the effect of glucagon on beta cell [Ca2+]idynamics. Human islets from non-diabetic women and men were cultured for 24 h in either a control medium or high-fat/high-glucose medium in the presence or absence of the glucagon receptor antagonist L-168,049. [Ca2+]i dynamics of human islets were evaluated in vitro after 1 h exposure to Fura-10.ResultsMice fed a WD for 1 month displayed increased beta cell [Ca2+]i dynamics linked to enhanced insulin secretion as a functional compensatory mechanism in prediabetes. Recruitment of inactive beta cells in WD-fed mice explained the improved beta cell function adaptation observed in vivo; this occurred in a sex-specific manner. Mechanistically, this was attributable to an intra-islet structural rearrangement involving alpha cells. These sex-dependent cytoarchitecture reorganisations, observed in both mice and humans, induced enhanced paracrine input from adjacent alpha cells, adjusting the glucose setpoint and amplifying the insulin secretion pathway. When WD feeding was prolonged, female mice maintained the adaptive mechanism due to their intrinsically high proportion of alpha cells. In males, [Ca2+]i dynamics progressively declined subsequent to glucose stimulation while insulin secretion continue to increase, suggesting uncoordinated beta cell function as an early sign of diabetes.Conclusions/interpretationWe identified increased coordination of [Ca2+]i dynamics as a beta cell functional adaptation mechanisms in prediabetes. Importantly, we uncovered the mechanisms by which sex-dependent beta cell [Ca2+]i dynamics coordination is orchestrated by an intra-islet structure reorganisation increasing the paracrine input from alpha cells on beta cell function. Moreover, we identified reduced [Ca2+]i dynamics coordination in response to glucose as an early sign of diabetes preceding beta cell secretory dysfunction, with males being more vulnerable. Alterations in coordination capacity of [Ca2+]i dynamics may thus serve as an early marker for beta cell failure in prediabetes.
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Project description:The ability of a single Ca2+ ion to play an important role in cell biology is highlighted by the need for cells to form Ca2+ signals in the dimensions of space, time, and amplitude. Thus, spatial and temporal changes in intracellular Ca2+ concentration are important for determining cell fate. Optogenetic technology has been developed to provide more precise and targeted stimulation of cells. Here, U2OS cells overexpressing Ca2+ translocating channelrhodopsin (CatCh) were used to mediate Ca2+ influx through blue light illumination with various parameters, such as intensity, frequency, duty cycle, and duration. We identified that several Ca2+ -dependent transcription factors and certain kinases can be activated by specific Ca2+ waves. Using a wound-healing assay, we found that low-frequency Ca2+ oscillations increased cell migration through the activation of NF-κB. This study explores the regulation of cell migration by Ca2+ signals. Thus, we can choose optical parameters to modulate Ca2+ waves and achieve activation of specific signaling pathways. This novel methodology can be applied to clarify related cell-signaling mechanisms in the future.
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Project description:Bariatric surgery improves both insulin sensitivity and secretion and can induce diabetes remission. However, the mechanisms and time courses of these changes, particularly the impact on β cell function, are difficult to monitor directly. In this study, we investigated the effect of Vertical Sleeve Gastrectomy (VSG) on β cell function in vivo by imaging Ca2+ dynamics in islets engrafted into the anterior eye chamber. Mirroring its clinical utility, VSG in mice results in significantly improved glucose tolerance, and enhanced insulin secretion. We reveal that these benefits are underpinned by augmented β cell function and coordinated activity across the islet. These effects involve changes in circulating GLP-1 levels which may act both directly and indirectly on the β cell, in the latter case through changes in body weight. Thus, bariatric surgery leads to time-dependent increases in β cell function and intra-islet connectivity which are likely to contribute to diabetes remission.
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Project description:Assessing the response of pancreatic islet cells to glucose stimulation is important for understanding β-cell function. Zebrafish are a promising model for studies of metabolism in general, including stimulus-secretion coupling in the pancreas. We used transgenic zebrafish embryos expressing a genetically-encoded Ca2+ sensor in pancreatic β-cells to monitor a key step in glucose induced insulin secretion; the elevations of intracellular [Ca2+]i. In vivo and ex vivo analyses of [Ca2+]i demonstrate that β-cell responsiveness to glucose is well established in late embryogenesis and that embryonic β-cells also respond to free fatty acid and amino acid challenges. In vivo imaging of whole embryos further shows that indirect glucose administration, for example by yolk injection, results in a slow and asynchronous induction of β-cell [Ca2+]i responses, while intravenous glucose injections cause immediate and islet-wide synchronized [Ca2+]i fluctuations. Finally, we demonstrate that embryos with disrupted mutation of the CaV1.2 channel gene cacna1c are hyperglycemic and that this phenotype is associated with glucose-independent [Ca2+]i fluctuation in β-cells. The data reveal a novel central role of cacna1c in β-cell specific stimulus-secretion coupling in zebrafish and demonstrate that the novel approach we propose - to monitor the [Ca2+]i dynamics in embryonic β-cells in vivo - will help to expand the understanding of β-cell physiological functions in healthy and diseased states.
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Project description:In most species, fertilization induces Ca2+ transients in the egg. In mammals, the Ca2+ rises are triggered by phospholipase Cζ (PLCζ) released from the sperm; IP3 generated by PLCζ induces Ca2+ release from the intracellular Ca2+ store through IP3 receptor, termed IP3-induced Ca2+ release. Here, we developed new fluorescent IP3 sensors (IRIS-2s) with the wider dynamic range and higher sensitivity (Kd = 0.047-1.7 μM) than that we developed previously. IRIS-2s employed green fluorescent protein and Halo-protein conjugated with the tetramethylrhodamine ligand as fluorescence resonance energy transfer (FRET) donor and acceptor, respectively. For simultaneous imaging of Ca2+ and IP3, using IRIS-2s as the IP3 sensor, we developed a new single fluorophore Ca2+ sensor protein, DYC3.60. With IRIS-2s and DYC3.60, we found that, right after fertilization, IP3 concentration ([IP3]) starts to increase before the onset of the first Ca2+ wave. [IP3] stayed at the elevated level with small peaks followed after Ca2+ spikes through Ca2+ oscillations. We detected delays in the peak of [IP3] compared to the peak of each Ca2+ spike, suggesting that Ca2+-induced regenerative IP3 production through PLC produces small [IP3] rises to maintain [IP3] over the basal level, which results in long lasting Ca2+ oscillations in fertilized eggs.
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Project description:The vast osteocytic network is believed to orchestrate bone metabolic activity in response to mechanical stimuli through production of sclerostin, RANKL, and osteoprotegerin (OPG). However, the mechanisms of osteocyte mechanotransduction remain poorly understood. We've previously shown that osteocyte mechanosensitivity is encoded through unique intracellular calcium (Ca2+) dynamics. Here, by simultaneously monitoring Ca2+ and actin dynamics in single cells exposed to fluid shear flow, we detected actin network contractions immediately upon onset of flow-induced Ca2+ transients, which were facilitated by smooth muscle myosin and further confirmed in native osteocytes ex vivo. Actomyosin contractions have been linked to the secretion of extracellular vesicles (EVs), and our studies demonstrate that mechanical stimulation upregulates EV production in osteocytes through immunostaining for the secretory vesicle marker Lysosomal-associated membrane protein 1 (LAMP1) and quantifying EV release in conditioned medium, both of which are blunted when Ca2+ signaling was inhibited by neomycin. Axial tibia compression was used to induce anabolic bone formation responses in mice, revealing upregulated LAMP1 and expected downregulation of sclerostin in vivo. This load-related increase in LAMP1 expression was inhibited in neomycin-injected mice compared to vehicle. Micro-computed tomography revealed significant load-related increases in both trabecular bone volume fraction and cortical thickness after two weeks of loading, which were blunted by neomycin treatment. In summary, we found mechanical stimulation of osteocytes activates Ca2+-dependent contractions and enhances the production and release of EVs containing bone regulatory proteins. Further, blocking Ca2+ signaling significantly attenuates adaptation to mechanical loading in vivo, suggesting a critical role for Ca2+-mediated signaling in bone adaptation.
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Project description:Transcriptionally mature and immature β-cells co-exist within the adult islet. How such diversity contributes to insulin release remains poorly understood. Here we show that subtle differences in β-cell maturity, defined using PDX1 and MAFA expression, contribute to islet operation. Functional mapping of rodent and human islets containing proportionally more PDX1HIGH and MAFAHIGH β-cells reveals defects in metabolism, ionic fluxes and insulin secretion. At the transcriptomic level, the presence of increased numbers of PDX1HIGH and MAFAHIGH β-cells leads to dysregulation of gene pathways involved in metabolic processes. Using a chemogenetic disruption strategy, differences in PDX1 and MAFA expression are shown to depend on islet Ca2+ signaling patterns. During metabolic stress, islet function can be restored by redressing the balance between PDX1 and MAFA levels across the β-cell population. Thus, preserving heterogeneity in PDX1 and MAFA expression, and more widely in β-cell maturity, might be important for the maintenance of islet function.
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Project description:Mutations of large conductance Ca2+- and voltage-activated K+ channels (BK) are associated with cognitive impairment. Here we report that CA1 pyramidal neuron-specific conditional BK knock-out (cKO) mice display normal locomotor and anxiety behavior. They do, however, exhibit impaired memory acquisition and retrieval in the Morris Water Maze (MWM) when compared to littermate controls (CTRL). In line with cognitive impairment in vivo, electrical and chemical long-term potentiation (LTP) in cKO brain slices were impaired in vitro. We further used a genetically encoded fluorescent K+ biosensor and a Ca2+-sensitive probe to observe cultured hippocampal neurons during chemical LTP (cLTP) induction. cLTP massively reduced intracellular K+ concentration ([K+]i) while elevating L-Type Ca2+ channel- and NMDA receptor-dependent Ca2+ oscillation frequencies. Both, [K+]i decrease and Ca2+ oscillation frequency increase were absent after pharmacological BK inhibition or in cells lacking BK. Our data suggest that L-Type- and NMDAR-dependent BK-mediated K+ outflow significantly contributes to hippocampal LTP, as well as learning and memory.
| S-EPMC10663188 | biostudies-literature
Project description:Pancreatic β-cells release insulin upon a rise in blood glucose. The precise mechanisms of stimulus-secretion coupling, and its failure in Diabetes Mellitus Type 2, remain to be elucidated. The consensus model, as well as a class of currently prescribed anti-diabetic drugs, are based around the observation that glucose-evoked ATP production in β-cells leads to closure of cell membrane ATP-gated potassium (KATP) channels, plasma membrane depolarisation, Ca2+ influx, and finally the exocytosis of insulin granules. However, it has been demonstrated by the inactivation of this pathway using genetic and pharmacological means that closure of the KATP channel alone may not be sufficient to explain all β-cell responses to glucose elevation. We have previously proposed that NAADP-evoked Ca2+ release is an important step in stimulus-secretion coupling in pancreatic β-cells. Here we show using total internal reflection fluorescence (TIRF) microscopy that glucose as well as the Ca2+ mobilising messenger nicotinic acid adenine dinucleotide phosphate (NAADP), known to operate in β-cells, lead to highly localised elementary intracellular Ca2+ signals. These were found to be obscured by measurements of global Ca2+ signals and the action of powerful SERCA-based sequestration mechanisms at the endoplasmic reticulum (ER). Building on our previous work demonstrating that NAADP-evoked Ca2+ release is an important step in stimulus-secretion coupling in pancreatic β-cells, we provide here the first demonstration of elementary Ca2+ signals in response to NAADP, whose occurrence was previously suspected. Optical quantal analysis of these events reveals a unitary event amplitude equivalent to that of known elementary Ca2+ signalling events, inositol trisphosphate (IP3) receptor mediated blips, and ryanodine receptor mediated quarks. We propose that a mechanism based on these highly localised intracellular Ca2+ signalling events mediated by NAADP may initially operate in β-cells when they respond to elevations in blood glucose.
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