Dataset Information

Reduced synchroneity of intra-islet Ca²⁺ oscillations in vivo in Robo-deficient β cells.

ABSTRACT: The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion among β cells, yet testing this in vivo in the intact pancreas is challenging. Robo βKO mice, in which the genes Robo1 and Robo2 are deleted selectively in β cells, provide a unique model of altered islet spatial architecture without loss of β cell differentiation or islet damage from diabetes. Combining Robo βKO mice with intravital microscopy, we show here that Robo βKO islets have reduced synchronized intra-islet Ca²⁺ oscillations among β cells in vivo. We provide evidence that this loss is not due to a β cell-intrinsic function of Robo, mis-expression or mis-localization of Cx36 gap junctions, or changes in islet vascularization or innervation, suggesting that the islet architecture itself is required for synchronized Ca²⁺ oscillations. These results have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

SUBMITTER: Adams MT

PROVIDER: S-EPMC8289414 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Reduced synchroneity of intra-islet Ca2+ oscillations in vivo in Robo-deficient β cells.

Adams Melissa T MT Dwulet JaeAnn M JM Briggs Jennifer K JK Reissaus Christopher A CA Jin Erli E Szulczewski Joseph M JM Lyman Melissa R MR Sdao Sophia M SM Kravets Vira V Nimkulrat Sutichot D SD Ponik Suzanne M SM Merrins Matthew J MJ Mirmira Raghavendra G RG Linnemann Amelia K AK Benninger Richard Kp RK Blum Barak B

eLife 20210707

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion among β cells, yet testing this in vivo in the intact pancreas is challenging. Robo βKO mice, in which the genes Robo1 and Robo2 are deleted selectively in β cells, provide a unique model of altered islet spatial architecture without loss of β cell differentiation or islet damage from diabetes. Combining Robo βKO mice with intravital microscopy, we show here that Robo β ...[more]

PMID: 34231467

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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.

Project description:ObjectiveDiabetes occurs because of insufficient insulin secretion due to β-cell dysfunction within the islet of Langerhans. Elevated glucose levels trigger β-cell membrane depolarization, action potential generation, and slow sustained free-Ca2+ ([Ca2+]) oscillations, which trigger insulin release. Nuclear factor of activated T-cell (NFAT) is a transcription factor, which is regulated by the increases in [Ca2+] and calceineurin (CaN) activation. NFAT regulation links cell activity with gene transcription in many systems and regulates proliferation and insulin granule biogenesis within the β-cell. However, the link between the regulation of β-cell electrical activity and oscillatory [Ca2+] dynamics with NFAT activation and downstream transcription is poorly understood. Here, we tested whether dynamic changes to β-cell electrical activity and [Ca2+] regulate NFAT activation and downstream transcription.MethodsIn cell lines, mouse islets, and human islets, including those from donors with type 2 diabetes, we applied both agonists/antagonists of ion channels together with optogenetics to modulate β-cell electrical activity. We measured the dynamics of [Ca2+] and NFAT activation as well as performed whole transcriptome and functional analyses.ResultsBoth glucose-induced membrane depolarization and optogenetic stimulation triggered NFAT activation as well as increased the transcription of NFAT targets and intermediate early genes (IEGs). Importantly, slow, sustained [Ca2+] oscillation conditions led to NFAT activation and downstream transcription. In contrast, in human islets from donors with type2 diabetes, NFAT activation by glucose was diminished, but rescued upon pharmacological stimulation of electrical activity. NFAT activation regulated GJD2 expression and increased Cx36 gap junction permeability upon elevated oscillatory [Ca2+] dynamics. However, it is unclear if NFAT directly binds the GJD2 gene to regulate expression.ConclusionsThis study provides an insight into the specific patterns of electrical activity that regulate NFAT activation, gene transcription, and islet function. In addition, it provides information on how these factors are disrupted in diabetes.

Dataset Information

Reduced synchroneity of intra-islet Ca²⁺ oscillations in vivo in Robo-deficient β cells.

Publications

Reduced synchroneity of intra-islet Ca<sup>2+</sup> oscillations in vivo in <i>Robo</i>-deficient β cells.

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