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Functional architecture of pancreatic islets identifies a population of first responder cells that drive the first-phase calcium response [1]

['Vira Kravets', 'Department Of Bioengineering', 'University Of Colorado', 'Anschutz Medical Campus', 'Aurora', 'Colorado', 'United States Of America', 'Barbara Davis Center For Childhood Diabetes', 'Jaeann M. Dwulet', 'Wolfgang E. Schleicher']

Date: 2022-09

Insulin-secreting β-cells are functionally heterogeneous. Whether there exist cells driving the first-phase calcium response in individual islets, has not been examined. We examine “first responder” cells, defined by the earliest [Ca 2+ ] response during first-phase [Ca 2+ ] elevation, distinct from previously identified “hub” and “leader” cells. We used islets isolated from Mip-Cre ER ; Rosa-Stop-Lox-Stop-GCamP6s mice (β-GCamP6s) that show β-cell-specific GCamP6s expression following tamoxifen-induced CreER-mediated recombination. First responder cells showed characteristics of high membrane excitability and lower electrical coupling to their neighbors. The first-phase response time of β-cells in the islet was spatially organized, dependent on the cell’s distance to the first responder cell, and consistent over time up to approximately 24 h. When first responder cells were laser ablated, the first-phase [Ca 2+ ] was slowed down, diminished, and discoordinated compared to random cell ablation. Cells that were next earliest to respond often took over the role of the first responder upon ablation. In summary, we discover and characterize a distinct first responder β-cell state, critical for the islet first-phase response to glucose.

Funding: This work was supported by Juvenile Diabetes Research Foundation Grant 5-CDA-2014-198-A-N (to RKPB); National Institute of Health (NIH) grants R01 DK102950 (to RKPB), R01 DK106412 (to RKPB); by Juvenile Diabetes Research Foundation grant 3-PDF-2019-741-A-N (to VK); by Human Islets Research Network subaward (HIRN, RRID:SCR_014393; UC24 DK1041162) (to VK), by Burroughs Wellcome Fund - Career At Scientific Interfaces (Project Number 25B1756) (to VK); by National Institute of Health grant F31 DK126360 (to JMD). by a Diabetes UK R.D. Lawrence (12/0004431) Fellowship (to DJH), a Welcome Trust Institutional Support Award (to DJH), and Medical Research Council (MR/N00275X/1 and MR/S025618/1) (to DJH), and Diabetes UK (17/0005681) Project Grant (to DJH). This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (Starting Grant 715884 to DJH). The funders had no role in the study design, data collection, and analysis, decisions to publish, or preparation of the manuscript.

Both in rodents and in humans, first and second phases of insulin secretion have been distinguished [ 25 , 26 ]. Glucose-stimulated [Ca 2+ ] influx into the cell is necessary for both first and second phases of insulin secretion [ 27 ]. β-cell [Ca 2+ ] is also bi-phasic [ 28 ] and is correlated with insulin secretion dynamics [ 29 ]. The first-phase of [Ca 2+ ] response following low-to-high glucose is transitional, where cells with intrinsic differences in metabolic activity or other properties may be responding differently. The second-phase [Ca 2+ ] response at high glucose is steady-state where the majority of cells are equally likely to fire [ 14 ], but with [Ca 2+ ] oscillations showing differing amplitudes, temporal delay (phase lag), and oscillation frequency [ 30 , 31 ]. While different β-cell subpopulations have been examined during this second-phase [Ca 2+ ] response, the role of functional β-cell states during the first-phase [Ca 2+ ] has not been examined.

Gap junction coupling is non-uniform throughout the islet [ 17 ]. β-cells are also heterogeneous in glucose metabolism and excitability [ 18 ]. As a result, at low glucose some β-cells are suppressed more than others by hyperpolarizing currents transmitted from neighboring cells [ 19 ]. Conversely at elevated glucose, some β-cells are recruited and/or coordinated more than others by depolarizing currents transmitted from neighboring cells. As such, the response of each β-cell within the islet to glucose is different, reflecting both its intrinsic heterogeneity and its context within the islet. Several studies have sought to identify and characterize functional β-cell states within the islet based on the [Ca 2+ ] response under glucose stimulation, together with the use of optogenetic-based constructs and laser ablation. For example, β-cells that show significantly increased connectivity, termed “hubs” or “hub cells” [ 20 , 21 ], disproportionately suppressed islet [Ca 2+ ] following targeted hyperpolarization via optogenetic stimulation. Conversely, a population of β-cells disproportionately activated islet [Ca 2+ ] following targeted depolarization via optogenetic stimulation [ 22 ]. Furthermore, cells that show [Ca 2+ ] oscillations that precede the rest of the islet, termed “leader” cells or “wave-origin” [ 22 – 24 ], have also been suggested to drive the oscillatory dynamics of [Ca 2+ ].

Diabetes mellitus is a disease characterized by high blood glucose, caused by insufficient secretion of insulin relative to insulin requirements. β-cells within pancreatic islets of Langerhans secrete insulin and are compromised in diabetes. Early work showed that in mechanically dispersed islets, single β-cells are heterogeneous in the level of insulin release [ 1 ]. More recent studies have discovered markers that separate β-cells into distinct populations with differing functional properties. This includes markers that subdivides proliferative-competent β-cells from mature β-cells ([ 2 ]; subdivides β-cells with different levels of insulin gene expression, granularity, and secretion [ 3 ]; or subdivides β-cells that have differing responsiveness to insulin secretagogues [ 4 ]). Furthermore, single-cell high-throughput approaches such as single-cell RNA sequencing (scRNAseq) or mass cytometry separate distinct β-cell populations [ 5 , 6 ]. However, the role of the heterogeneity in the function of the islet is poorly understood.

Results

Clusters of first responders are spatially consistent We reasoned that if cells respond randomly to glucose stimulation, then first responder cells identified during the initial glucose elevation will not be the same first responder cells during a repeated glucose elevation. Conversely, if there exists a functional hierarchy of cells within the islet, then there should be consistency in the location of the first responder cells. We first stimulated islets to elevated (11 mM) glucose, lowered back to basal levels, and restimulated over the course of 1 to 2 h (Fig 2A). The location of the first responder cluster in the islet remained consistent between the initial and repeated glucose elevation (Fig 2B and 2C). Some initial first responder cells were first responders in the repeated glucose stimulation (35% of cases), whereas some surrendered their role either to the nearest neighbor cell (35%), or to the 2nd neighbor (18%), or to a more distant cell (12%). PPT PowerPoint slide

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TIFF original image Download: Fig 2. Consistency of first responder cells. (A) Representative time course of [Ca2+] dynamics within an islet under repeated glucose stimulation. Vertical scale bar represents 30% of the fluorescence intensity change. (B) Representative image of the islet with the location of first responders (red) and last responders (green) during initial and repeated glucose elevation. (C) Spatial consistency: spatial location of new first responder cells (during repeated glucose elevation) relative to the old first responder cells (during original elevation) (n = 10 islets from 4 mice). (D) Time of response of cells identified as first responders during the initial glucose elevation and upon repeated glucose elevation (n = 10 islets from 4 mice). (E) As in (D) for last responders. (F) Representative time course of [Ca2+] dynamics within an islet under 11 mM glucose stimulation (dashed curve) and under 10 μM glibenclamide (solid curve) stimulation for first responder cell (red) and islet average (gray). (G) Representative image of the islet with the location of first responders (red) and last responders (green) during glucose and glibenclamide stimulation. Scale bar indicates 20 μm. (H) Time of response of cells identified as first responders during glucose stimulation and upon glibenclamide stimulation (n = 13 islets from 7 mice). (I) False-color map indicating [Ca2+] response time to glucose elevation during the first phase, recorded in the same region of the islet over 48 h at 6 h intervals. Scale bar indicates 20 μm. White arrows point to the 1st responder cell identified at that time. (J) AUC for [Ca2+] elevation over 0–5 min for the islet average, for each time window indicated. Data are normalized to the [Ca2+] AUC at 0 h. (K) Time evolution of the response time of the cells identified as first and (L) as in (H) for last responders (n = 7 islets from 3 mice). Statistical analysis in D, E utilized 1-sample t test (with the null hypothesis of initial or repeated difference from the islet being 0). Statistical analysis in H utilized 1-sample t test (with the null hypothesis of glucose or glibenclamide difference from the islet being 0). K, L utilized LMEM (S1 Statistical analysis LMEM). † in K indicates p = 0.06. See S2 Data file for values used in each graph, and Statistical analysis–Source data for LMEM and 1-sample t test details. AUC, area under the curve; LMEM, linear mixed-effects model. https://doi.org/10.1371/journal.pbio.3001761.g002 We examined location of the first and last responder cells, as well as leader cells in 3D (S3 Fig). Within 3 cell layers, each separated by 10 μm, the locations of each β-cell subpopulation were conserved, suggesting functional organization in 3D, as we observed in 2D.

Temporal consistency of first responder cells in not rigid We next sought to determine whether an early response time was a consistent feature of first responder cells. As above, we first stimulated islets to elevated (11 mM) glucose, lowered back to basal levels, and restimulated over the course of 1 to 2 h (Fig 2A). The period of the observed slow oscillations was in the same range as previously reported [32], (T = 246 ± 89 s). No significant difference was observed in the frequency of the oscillations during the repeated stimulation (0.011 ± 0.008 Hz versus 0.007 ± 0.001 Hz). Response times for all cells during the initial and repeated glucose stimulation are shown in S4 Fig for individual islets. When considering all cells within the islet, there was no significant correlation between the response time of a cell during the initial stimulation compared to the response time upon the repeated stimulation. However, first responder cells in the majority of islets still remained consistent. Last responder cells were not consistent (S4 Fig). We found that temporal consistency of first responder cells (Fig 2D) was substantial, but not fully rigid. Upon restimulation, the initial first responders retained a response that was significantly earlier than the median islet response time: on average in the fastest 25% of the whole T resp distribution. In contrast, the last-responding cells lacked consistency upon repeated glucose elevation, with a response time close to the islet average (Fig 2E). We performed sequential glucose and glibenclamide stimulation, in the same manner as repeated glucose stimulation experiments. Glibenclamide is a K ATP channel blocker, resulting in the cell membrane depolarization, mimicking that which happens under glucose-stimulated K ATP channel closure. Following each stimulation, we identified first responder cells (Fig 2F and 2G). Those cells that responded first to glucose, also showed a significantly lower than average response time to glibenclamide (Fig 2G and 2H) (p = 0.046). In contrast, those cells that responded last to glucose did not show a response time different to the islet average under glibenclamide. When all cells in the islet plane were considered, we did not observe a significant correlation between the response time during glucose stimulation as compared to the response time upon glibenclamide stimulation. This suggested that while differing K ATP conductance (or resting depolarizing current) may be one factor in defining the earlier response time for a first responder cell, other factors are also likely involved.

First responder cells represent a cell state rather than a stable subpopulation To test whether first responder cells are maintained over a longer time, we measured the response time upon elevated glucose within the same cell layer in the islet for 48 h at 6 h intervals. At each time point, we defined the first responder and last responder cells (Fig 2I). There was no significant difference in the total [Ca2+] influx in the islet at each time point, suggesting islet remained functional during the culture (Fig 2J). The first responder cells remained consistent during the first 12 h, where they showed significantly earlier [Ca2+] response time than the islet average. However, at 18 to 24 h, the first responder cells became less distinguishable from the islet average, and at >24 h, their response time was indistinguishable from the islet average (Fig 2K). A similar temporal pattern was observed for the last responder cells (Fig 2L). Thus, not all β-cells responded randomly to glucose stimulation. Rather, a first responder β-cell state consistently led this response, but this was maintained only over an approximately 24 h time period.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001761

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