Pflugers Arch - Eur J Physiol DOI 10.1007/s00424-014-1681-1
SIGNALING AND CELL PHYSIOLOGY
Pseudoislet formation enhances gene expression, insulin secretion and cytoprotective mechanisms of clonal human insulin-secreting 1.1B4 cells Alastair D. Green & Srividya Vasu & Neville H. McClenaghan & Peter R. Flatt
Received: 20 October 2014 / Revised: 8 December 2014 / Accepted: 18 December 2014 # Springer-Verlag Berlin Heidelberg 2015
Abstract We have studied the effects of cell communication on human beta cell function and resistance to cytotoxicity using the novel human insulin-secreting cell line 1.1B4 configured as monolayers and pseudoislets. Incubation with the incretin gut hormones GLP-1 and GIP caused dose-dependent stimulation of insulin secretion from 1.1B4 cell monolayers and pseudoislets. The secretory responses were 1.5–2.7-fold greater than monolayers. Cell viability (MTT), DNA damage (comet assay) and apoptosis (acridine orange/ethidium bromide staining) were investigated following 2-h exposure of 1.1B4 monolayers and pseudoislets to ninhydrin, H2O2, streptozotocin, glucose, palmitate or cocktails of proinflammatory cytokines. All agents tested decreased viability and increased DNA damage and apoptosis in both 1.1B4 monolayers and pseudoislets. However, pseudoislets exhibited significantly greater resistance to cytotoxicity (1.5–2.7-fold increases in LD50) and lower levels of DNA damage (1.3–3.4fold differences in percentage tail DNA and olive tail moment) and apoptosis (1.3–1.5-fold difference) compared to monolayers. Measurement of gene expression by reverse-transcription, real-time PCR showed that genes involved with insulin secretion (INS, PDX1, PCSK1, PCSK2, GLP1R and GIPR), cell-cell communication (GJD2, GJA1 and CDH1) and antioxidant defence (SOD1, SOD2, GPX1 and CAT) were significantly upregulated in pseudoislets compared to monolayers, whilst the expression of proapoptotic genes (NOS2, MAPK8, MAPK10 and NFKB1) showed no significant differences. In summary, these data indicate cell-communication associated with three-dimensional islet architecture is important both for Alastair D. Green and Srividya Vasu contributed equally to this work. A. D. Green : S. Vasu (*) : N. H. McClenaghan : P. R. Flatt SAAD Centre for Pharmacy and Diabetes, University of Ulster, Cromore Road, Coleraine BT52 1SA, Northern Ireland, UK e-mail:
[email protected]
effective insulin secretion and for protection of human beta cells against cytotoxicity. Keywords 1.1B4 cells . Beta cell . Cell communication . Insulin secretion . Cytotoxicity
Introduction Pancreatic beta cells rely on cell-to-cell communication to efficiently regulate glucose-stimulated insulin secretion. Beta cell coupling and formation of intercellular gap junctions allow for propagation and synchronisation of chemical signalling between neighbouring cells [4, 5, 11, 28]. Uncoupling of pancreatic beta cells has been shown to considerably diminish their secretory function [3, 10, 25]. Similarly, reaggregation of dispersed beta cells from various species into threedimensional islet-like spheroids termed pseudoislets has been demonstrated to enhance secretory function [3, 9, 10, 14, 15, 25]. Due to the limited availability of primary human islets and difficulties in generating stable human beta cell lines, there has been limited study of the effects of cell-to-cell communication on the function and survival of human beta cells. The novel human beta cell line 1.1B4 created by electrofusion of freshly isolated human beta cells and immortal PANC-1 ductal epithelial cells has recently shown promise as a model for the study of human beta cell physiology and function [9, 21, 30–32]. Furthermore, configuration of 1.1B4 cells as pseudoislets markedly potentiated insulin secretion in response to glucose and other modulators of insulin release and significantly increased the expression of proteins involved
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in stimulus-secretion coupling and communication, including connexins 36 and 43, and E-cadherin [9, 21]. Recent studies suggest that cell-to-cell communication may also play a role in beta cell survival and resistance to cytotoxicity. Downregulation or upregulation of the gap-junction protein connexin 36 in transgenic mice was accompanied by a respective increase or decrease in beta cell apoptosis in response to cytotoxic drugs and cytokines [17]. The cytotoxic effects and adaptive responses of 1.1B4 cell monolayers have been demonstrated following exposure to various chemical assaults commonly associated with beta cell destruction and dysfunction including hyperglycaemia, hyperlipidaemia and toxic concentrations of proinflammatory cytokines. The responses reported were similar to those for primary rodent and human islets, indicating that this novel 1.1B4 cell line is a suitable model for studies of beta cell function and responses to cytotoxicity [30–32]. Here, we studied the effects of cell communication on human beta cell function and resistance to cytotoxicity using the clonal 1.1B4 beta cell line. For this, we assessed the differences in gene expression, cell viability, insulin release, DNA integrity and apoptosis between 1.1B4 cell monolayers and pseudoislets.
Materials and methods Culture of 1.1B4 cells The generation and characterisation of the human 1.1B4 beta cell line has been described previously [21]. The cells were maintained at 37 °C with 5 % CO2 in RPMI-1640 media (Gibco® Invitrogen, Paisley, UK) containing 11.1 mol l−1 glucose and 2.0 mol l−1 L-glutamine supplemented with 10 % (v/ v) foetal calf serum (Gibco® Invitrogen, Paisley, UK) and antibiotics (100 U ml−1 penicillin and 0.1 g l−1 streptomycin) (Gibco® Invitrogen, Paisley, UK). Cells were given fresh media every 2–3 days as necessary and were routinely used from passage 25–35. The cell line is available to purchase from Sigma-Aldrich (Dorset, UK). Formation and dissociation of 1.1B4 cell pseudoislets To form pseudoislets, 1.1B4 cells were seeded at a density of 1×105 cells/well into ultra-low-attachment, six-well, flatbottomed plates (Corning Inc., NY, USA) with 5-ml/well culture medium [9, 21]. Cells typically formed three-dimensional pseudoislet clusters within 5–7 days of seeding. Where necessary for experimental comparison with monolayer cells, pseudoislets were pelleted by centrifugation at 10,000g for 5 min and dissociated by resuspension in enzyme-free cell dissociation buffer (Sigma-Aldrich, Dorset, UK) for 10 min followed by gentle agitation by pipetting to produce a single cell suspension. Cell suspensions derived from both
monolayers and pseudoislets were then exposed to cytotoxic agents in order to compare responses. This protocol was chosen to enable direct comparison of the two cell populations avoiding differences due to diffusion into pseudoislets whilst preserving functional characteristics such as gene expression. Measurement of insulin release Insulin release from 1.1B4 cell monolayers and pseudoislets was determined as described previously [9, 21]. Briefly, cell monolayers were seeded at a density of 1.5×105 cells/well into flatbottomed 24-well tissue culture plates (Iwaki Glass, Funabashi, Japan) and allowed to attach overnight. Pseudoislets were picked under a microscope with a pipette immediately prior to experimentation and placed into 1.5-ml Eppendorf tubes (five pseudoislets/tube). For acute tests, cells/pseudoislets were first preincubated for 40 min at 37 °C in Krebs-Ringer bicarbonate buffer (KRBB) (115 mmol l−1 NaCl, 4.7 mmol l−1 KCl, 1.28 mmol l−1 CaCl2, 1.2 mmol l−1 MgSO4, 10 mmol l−1 NaHCO3, 20 mmol l−1 HEPES) containing 1.1 mmol l−1 glucose supplemented with 0.1 %w/v bovine serum albumin (BSA) (Gibco® Invitrogen, Paisley, UK), before being incubated for a further 60 min in KRBB supplemented with (v/v) 0.1 % BSA and a range of concentrations of glucose and modulators of insulin secretion as described in the figures. Following acute tests, supernatants were stored at −20 °C until insulin analysis by radioimmunoassay [7]. Cells were lysed by overnight incubation at 4 °C with acid-ethanol (1.5 % (v/v) HCl, 70 % (v/v) ethanol) to determine insulin content. Cytotoxin treatments To investigate the relative cytoprotective capabilities of 1.1B4 cell monolayers and pseudoislets, cells were incubated for 2 h at 37 °C with cytotoxins at the concentrations indicated in the tables, figures and their legends. Cytotoxins included ninhydrin, H2O2, streptozotocin (STZ), glucose, palmitate (all procured from Sigma-aldrich, Dorset, UK) and cocktails of the proinflammatory cytokines containing interleukin 1 beta (IL1B), interferon gamma (IFNg) and tumour necrosis factor alpha (TNFa). Working solutions were prepared by diluting concentrated stocks appropriately in tissue culture media immediately before use, except for glucose which was added directly to culture media with no intermediate stock. Assessment of cell viability Viability of 1.1B4 cell suspensions derived from cell culture as monolayers and pseudoislets was determined following incubation with a range of cytotoxic agents as indicated in the figures. Viability was measured by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [26].
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Measurement of cellular apoptosis Apoptosis of 1.1B4 was assessed by staining with acridine orange and ethidium bromide. Cells configured as monolayers or pseudoislets were exposed to cytotoxins, harvested and then resuspended in phosphate buffered saline (PBS) at a density of 5×105 cells ml−1. The cell suspension was then stained with solutions of acridine orange and ethidium bromide (20 μg ml−1 each) for 5 min at room temperature. Cells were mounted with antifade mounting medium and viewed under 488- and 594-nm filters using fluorescent microscope (Olympus System microscope, BX51) and photographed using the DP70 camera adapter system. The apoptotic state of cells was categorised as follows: bright green nuclei—healthy cells; dense green nuclei (evidence of chromatin condensation)—early apoptosis; bright yellow nuclei and yellow cytoplasm—late apoptosis; and orange/red nuclei, orange cytoplasm—late apoptosis/ necrosis. Membrane blebs and apoptotic bodies were also taken into account for analysis. Approximately 100 cells per replicate (n=4) were analysed using ImageJ software.
(ACTB) were used. A negative control containing RNasefree water in place of cDNA was included for each set of primers used. Data were acquired using a MiniOpticon twocolour real-time PCR detection system (BioRad, Hertfordshire, UK), and the results were analysed using the ΔΔCt method, with mRNA expression normalised to that of ACTB.
Statistical analysis Results are expressed as mean±SEM for a specific number of observations (n). Data sets were compared using Student’s unpaired t test with two-tailed p values and 95 % confidence intervals and one-way analysis of variance (ANOVA) with Bonferroni post hoc test where applicable (GraphPad Prism Software, USA). Two groups of data were considered to be significantly different if p
