Pseudoislet formation enhances gene expression, insulin secretion and cytoprotective mechanisms of clonal human insulin-secreting 1.1B4 cells

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 (1.3-3.4-fold 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 effective insulin secretion and for protection of human beta cells against cytotoxicity.

Implanting 1.1B4 human β-cell pseudoislets improves glycaemic control in diabetic severe combined immune deficient mice

“…Furthermore, energy and fluid balance, body weight, blood glucose and glucose tolerance improved gradually in these mice. This difference is most likely due to improved insulin secretory function in 1.1B4 pseudoislets compared to single isolated cells as described previously in vitro [25,36,37,43]. This better regulated insulin release is supported by similar insulin contents of the two types of resected β-cell masses.…”

“…1.1B4 cells exhibit marked decreases in secretory function and viability following prolonged exposure to high levels of glucose[33,37]. As a result, mice with chemically-induced diabetes were given insulin therapy for 9-27 d after STZ to moderate blood glucose levels during the engraftment of implanted 1.1B4 cell suspensions and pseudoislets.…”

“…To form pseudoislets, 1.1B4 cells were seeded at a density of 1 × 10 5 cells/well into ultra-low-attachment, six-well, flat-bottomed plates (Corning Inc., NY, United States) with 5-mL/well culture medium. Cells typically formed three-dimensional pseudoislet clusters, each comprising 5000-6000 cells, within 5-7 d of seeding[37]. …”

“…These pseudoislets are morphologically similar to isolated primary islets and show increased expression of cell-cell communication genes together with remarkable potentiation of insulin secretory responses to glucose and other secretagogues in vitro [25,36]. Moreover, 1.1B4 cells showed significantly enhanced resistance to cytotoxicity when configured as pseudoislets compared to monolayers[37]. Transplantation of cells configured as pseudoislets may represent an attractive model to improve graft survival, function and resistance to hyperglycaemia.…”

Esculentin-2CHa(1–30) and its analogues: stability and mechanisms of insulinotropic action

“…Plasma membrane integrity was assessed by measuring lactate dehydrogenase (LDH) in cell incubation buffer using CytoTox 96 nonradioactive cytotoxicity assay kit (Promega) according to the manufacturer's instructions. In a second set of experiments, insulin-releasing effects of esculentin-2CHa(1-30) and selected analogues were examined over a similar concentration range using 1.1B4 human clonal beta cells (McCluskey et al 2011, Green et al 2015. In a third set of experiments, pancreatic islets isolated from NIH Swiss mice by collagenase digestion (Gotoh et al 1985) were incubated with 10 −6 and 10 −8 M of esculentin-2CHa(1-30) and selected analogues for 1 h in Krebs-Ringer bicarbonate (KRB) buffer supplemented with 3 or 20 mM glucose.…”