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Pancreatic beta-cells synthesize and secrete insulin maintaining an organism's energy homeostasis. In humans, beta-cell dysfunction and death contribute to the pathogenesis of type 2 diabetes (T2D). Although the causes of beta-cell dysfunction are complex, obesity-induced low-grade systemic inflammation plays a role. For example, obese individuals exhibiting increased levels of proinflammatory cytokines IL-6 and IL-1beta have a higher risk of beta-cell dysfunction and T2D. Interestingly, obesity-induced inflammation changes the expression of several cellular metal regulating genes, prompting this study to examine changes in the beta-cell metallome after exposure to proinflammatory-cytokines. Primary mouse beta-cells were exposed to a combination of IL-6 and IL-1beta for 48 hours, were chemically fixed and imaged by synchrotron X-ray fluorescent microscopy. Quantitative analysis showed a surprising 2.4-fold decrease in the mean total cellular content of zinc from 158 ± 57.7 femtograms (fg) to 65.7 ± 29.7 fg; calcium decreased from 216 ± 67.4 to 154.3 ± 68.7 fg (control vs. cytokines, respectively). The mean total cellular iron content slightly increased from 30.4 ± 12.2 to 47.2 ± 36.4 fg after cytokine treatment; a sub-population of cells (38%) exhibited larger increases of iron density. Changes in the subcellular distributions of zinc and calcium were observed after cytokine exposure. Beta-cells contained numerous iron puncta that accumulated still more iron after exposure to cytokines. These findings provide evidence that exposure to low levels of cytokines is sufficient to cause changes in the total cellular content and/or subcellular distribution of several metals known to be critical for normal beta-cell function., (© The Author(s) 2021. Published by Oxford University Press.)

Islets of Langerhans, found in the pancreas, are microorgans essential for glucose homeostasis within the body. Many cells are found with an islet, such as beta cells (~70%), alpha cells (~20%), delta cells (~5%), F cells (~4%), and epsilon cells (1%), each with its own unique function. To better understand the roles of these cells and how cell communication alters their function, several techniques have been established such as islet isolation and beta cell dispersion. Here we describe how to isolate primary rodent islets, disperse pancreatic islets, measure intracellular calcium, and use immunofluorescent staining to distinguish beta cells and alpha cells.

Pancreatic islets produce pulses of insulin and other hormones that maintain normal glucose homeostasis. These micro-organs possess exquisite glucose-sensing capabilities, allowing for precise changes in pulsatile insulin secretion in response to small changes in glucose. When communication among these cells is disrupted, precision glucose sensing falters. We measured intracellular calcium patterns in 6-mM-steps between 0 and 16 mM glucose, and also more finely in 2-mM-steps from 8 to 12 mM glucose, to compare glucose sensing systematically among intact islets and dispersed islet cells derived from the same mouse pancreas in vitro. The calcium activity of intact islets was uniformly low (quiescent) below 4 mM glucose and active above 8 mM glucose, whereas dispersed beta-cells displayed a broader activation range (2-to-10 mM). Intact islets exhibited calcium oscillations with 2-to-5-min periods, yet beta-cells exhibited longer 7-10 min periods. In every case, intact islets showed changes in activity with each 6-mM-glucose step, whereas dispersed islet cells displayed a continuum of calcium responses ranging from islet-like patterns to stable oscillations unaffected by changes in glucose concentration. These differences were also observed for 2-mM-glucose steps. Despite the diversity of dispersed beta-cell responses to glucose, the sum of all activity produced a glucose dose-response curve that was surprisingly similar to the curve for intact islets, arguing against the importance of "hub cells" for function. Beta-cells thus retain many of the features of islets, but some are more islet-like than others. Determining the molecular underpinnings of these variations could be valuable for future studies of stem-cell-derived beta-cell therapies., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Extracellular ATP plays a critical role in regulating insulin secretion in pancreatic β cells. The ATP released from insulin secretory vesicles has been proposed to be a major source of extracellular ATP. Currently, the mechanism by which ATP accumulates into insulin secretory granules remains elusive. In this study, the authors identified the expression of a vesicular nucleotide transporter (VNUT) in mouse pancreas, isolated mouse islets, and MIN6 cells, a mouse β cell line. Immunohistochemistry and immunofluorescence revealed that VNUT colocalized extensively with insulin secretory granules. Functional studies showed that suppressing endogenous VNUT expression in β cells by small hairpin RNA knockdown greatly reduced basal- and glucose-induced ATP release. Importantly, knocking down VNUT expression by VNUT small hairpin RNA in MIN6 cells and isolated mouse islets dramatically suppressed basal insulin release and glucose-stimulated insulin secretion (GSIS). Moreover, acute pharmacologic blockade of VNUT with Evans blue, a VNUT antagonist, greatly attenuated GSIS in a dose-dependent manner. Exogenous ATP treatment effectively reversed the insulin secretion defect induced by both VNUT knockdown and functional inhibition, indicating that VNUT-mediated ATP release is essential for maintaining normal insulin secretion. In contrast to VNUT knockdown, overexpression of VNUT in β cells resulted in excessive ATP release and enhanced basal insulin secretion and GSIS. Elevated insulin secretion induced by VNUT overexpression was reversed by pharmacologic inhibition of P2X but not P2Y purinergic receptors. This study reveals VNUT is expressed in pancreatic β cells and plays an essential and novel role in regulating insulin secretion through vesicular ATP release and extracellular purinergic signaling.