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Gene Expression Patterns in Synchronized Islet Populations

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Gene Expression Patterns in Synchronized Islet Populations bioRxiv preprint doi: https://doi.org/10.1101/377317; this version posted July 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Gene expression patterns in synchronized islet populations Nikita Mukhitov1 and Michael G. Roper1* 1Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL. * Corresponding author E-mail: [email protected] Abstract In vivo levels of insulin are oscillatory with a period of ~5-10 minutes, implying that the numerous islets of Langerhans within the pancreas are synchronized. While the synchronizing factors are still under investigation, one result of this behavior is expected to be coordinated 2+ 2+ intracellular [Ca ] ([Ca ]i) oscillations throughout the islet population. The role that 2+ coordinated [Ca ]i oscillations have on controlling gene expression within pancreatic islets was examined by comparing gene expression levels in islets that were synchronized using a 2+ low amplitude glucose wave and an unsynchronized population. The [Ca ]i oscillations in the synchronized population were homogeneous and had a significantly lower drift in their oscillation period as compared to unsynchronized islets. This reduced drift in the synchronized population was verified by comparing the drift of in vivo and in vitro profiles from published reports. Microarray profiling indicated a number of Ca2+-dependent genes were differentially regulated between the two islet populations. Gene set enrichment analysis revealed that the synchronized population had reduced expression of gene sets related to protein translation, protein turnover, energy expenditure, and insulin synthesis, while those that were related to maintenance of cell morphology were increased. It is speculated that these gene expression patterns in the synchronized islets results in a more efficient utilization of intra-cellular resources and response to environmental changes. 1 bioRxiv preprint doi: https://doi.org/10.1101/377317; this version posted July 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Introduction Pancreatic islets are key modulators of glucose homeostasis through the release of insulin and other hormones.1 Secretion of these hormones is dynamic occurring in a multi- oscillatory secretion rhythm that encompasses timescales from minutes to hours. While circadian2 and ultradian3 insulin oscillations are well understood, the rapid pulses observed in vivo with periods ranging from 5- to 10-min,4-7 are less characterized. However, it has been shown that diminished insulin oscillations are one of the first observable features of type 2 diabetes,8 further emphasizing the significance of the dynamic profile of hormone release. The pulsatile nature of insulin secretion originates from the smallest structural unit within the islets, the pancreatic beta cell. Within beta cells, increases in the ratio of ATP/ADP due to increased flux through glycolysis and oxidative phosphorylation closes ATP-sensitive potassium channels with a concomitant depolarization in the cell membrane potential.9,10 Depolarization opens L-type Ca2+ channels resulting in an influx of extracellular Ca2+ and a 2+ 2+ 2+ rise in intracellular [Ca ] ([Ca ]i). This increase in [Ca ]i initiates insulin secretion through Ca2+-dependent processes.11 It is widely believed that ~5-min oscillations in glucose metabolism results in the oscillatory dynamics of the downstream processes mentioned 9,10 2+ 12,13 above, producing oscillations in membrane potential, [Ca ]i [9-12], and insulin release . The individual beta cells within an islet are synchronized by electrical coupling and diffusion of glycolytic intermediates via gap-junctions9,10. An elusive question that remains is how do the ~1,000,000 islets within an individual synchronize to produce a pulsatile output of hormones from the pancreas? One long standing hypothesis is that islet synchronization occurs through classic insulin-glucose feedback loops that result in oscillatory glucose and insulin levels, both of which have been observed in vivo.14-17 This feedback loop has been mimicked in a microfluidic system by delivering glucose levels to a population of islets and iteratively adjusting the glucose levels by use of a mathematical model that mimicked insulin-dependent glucose uptake.16,17 With the appropriate parameters of the model, the islets synchronized resulting in 2+ population level oscillations of [Ca ]i, insulin secretion, and glucose levels. The average period to which the system converged to was ~5 min, similar to the period of insulin secretion observed in vivo.16,18 To simplify this feedback model, these parameters can be mimicked by delivering a 5-min glucose wave to an islet population.8, 18-20 These “open-loop” experiments 2+ result in [Ca ]i and insulin oscillations that are phase-locked, or entrained, to the glucose wave. In this study, we set out to determine if there are advantages to islet synchronization. 2+ We hypothesized that a synchronized islet population would have a more stable [Ca ]i 2 bioRxiv preprint doi: https://doi.org/10.1101/377317; this version posted July 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. oscillation period than a non-synchronized population, due to the control offered by a feedback 2+ system. These stable [Ca ]i oscillation periods may induce or repress specific genes in the islets. It has been shown in multiple cells types that the frequency, amplitude, and phase of 2+ 21-26 [Ca ]i oscillations offers specificity and robustness to signal transduction pathways. Some 2+ of the functions encoded by [Ca ]i signatures include cellular proliferation and differentiation, morphogenesis, contraction, and secretion.23 Moreover, expression levels of several genes 2+ 24,26 were found to be dependent on the oscillation frequency of [Ca ]i . In one study performed 2+ with T-cells, oscillations in [Ca ]i activated NFAT and NF-kB more efficiently than a constant 2+ 27,28 [Ca ]i level. In another study with B-lymphocytes, the activation of NFAT was highest when 2+ 29 only select values of [Ca ]i oscillation frequencies were present. Finally, it has been shown that intra-portal delivery of insulin pulses induced specific gene expression in the liver as compared to the same dose of insulin delivered in a constant manner.30 While these effects of dynamic signals have been shown in other cell types, they have been largely ignored in pancreatic islets. To test the hypothesis that islet synchronization may produce advantages over non- synchronized populations, the experimental mimic of the classic insulin-glucose feedback loop was performed whereby a population of islets was synchronized by exposure to a glucose wave, lysed, and their gene expression levels compared to a group of islets that were not synchronized. Between the two populations, differential regulation of Ca2+-dependent genes and gene sets were observed, which we hypothesize may have a positive influence on cell function in the synchronized state. Materials and Methods Chemical reagents and supplies Cosmic calf serum, dimethyl sulfoxide (DMSO), fluorescein, 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES), MgCl2, NaCl, pluronic F-127, RPMI 1640, and an antibiotic/antimycotic solution were obtained from Sigma-Aldrich (St. Louis, MO). Dextrose was obtained from Fisher Scientific (Pittsburgh, PA). CaCl2, KCl, and NaOH were obtained from EMD Chemicals (Gibbstown, NJ). Fura-2 acetoxymethyl ester (Fura-2 AM) was obtained from Invitrogen (Carlsbad, CA). Poly(dimethyl siloxane) (PDMS) elastomer kit was obtained from Dow Corning (Midland, MI). For all islet stimulation experiments, a balanced salt solution (BSS) was used. BSS was composed of 125 mM NaCl, 2.4 mM CaCl2, 1.2 mM MgCl2, 5.9 mM KCl, 25 mM HEPES, 3 bioRxiv preprint doi: https://doi.org/10.1101/377317; this version posted July 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. and adjusted to pH 7.4. The BSS was further supplemented with either 3 or 13 mM glucose. Fura 2-AM stock was prepared by reconstitution in 10 µL of pluronic F127 and 10 µL of DMSO. The prepared stock was stored protected from light at room temperature. Isolation and handling of islets of Langerhans All experiments were performed under guidelines approved by the Florida State University Animal Care and Use Committee (ACUC) protocol #1519. Islets were isolated from 20-40 g male CD-1 mice (Charles River Laboratories, Wilmington, MA) as previously described.19,20 All efforts were made to minimize suffering. For each set of experiments, islets were isolated from at least three mice and equally pooled from each animal (e.g., 40 islets were taken from three mice to a total of 120). From this pooled collection, islets were randomly 2+ selected for stimulation and imaging of [Ca ]i. All experiments were conducted within two days of isolation. Experimental Setup Microfluidic devices were made from PDMS and fabricated using conventional soft lithography.31 The design used in this study was described in previous work.17 Imaging of 2+ 17 [Ca ]i was performed as previously described. Briefly, the microfluidic device was positioned on the stage of a Nikon Ti-S microscope equipped with a 10X, 0.5 NA objective (Nikon Instruments, Melville, NY). Excitation was achieved with a Xenon arc lamp equipped with an integrated shutter and filter wheel (Sutter Instruments, Novato, CA).
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