No abstract

Caloric restriction can decrease the incidence of metabolic diseases such as obesity and type 2 diabetes mellitus (T2DM). The mechanisms underlying the benefits of caloric restriction involved in insulin secretion and glucose homeostasis are not fully understood. Intercellular communication within the islets of Langerhans, mediated by Connexin36 (Cx36) gap junctions, regulates insulin secretion dynamics and glucose homeostasis. The goal of this study was to determine if caloric restriction can protect against decreases in Cx36 gap junction coupling and altered islet function induced in models of obesity and prediabetes. C57BL6 mice were fed with a high fat diet (HFD), showing indications of prediabetes after 2 months, including weight gain, insulin resistance, and elevated fasting glucose and insulin levels. Subsequently, mice were submitted to one month of 40% caloric restriction (2g/day of HFD). Mice under 40% caloric restriction showed reversal in weight gain and recovered insulin sensitivity, fasting glucose and insulin levels. In islets of mice fed the HFD, caloric restriction protected against obesity-induced decreases in gap junction coupling and preserved glucose-stimulated calcium signaling, including Ca2+ oscillation coordination and oscillation amplitude. Caloric restriction also promoted a slight increase in glucose metabolism, as measured by increased NAD(P)H autofluorescence, as well as recovering glucose-stimulated insulin secretion. We conclude that declines in Cx36 gap junction coupling that occur in obesity can be completely recovered by caloric restriction and obesity reversal, improving Ca2+ dynamics and insulin secretion regulation. This suggests a critical role for caloric restriction in the context of obesity to prevent islet dysfunction.

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 [Ca2+] response during first-phase [Ca2+] elevation, distinct from previously identified “hub” and “leader” cells. We used islets isolated from Mip-CreER; 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 [Ca2+] 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.

Insulin-secreting β-cells are functionally heterogeneous. Subpopulations of β-cells can control islet function and the regulation of hormone release, such as driving the second (oscillatory) phase of free-calcium ([Ca2+]) following glucose elevation. Whether there exists a subpopulation that drives the first-phase response, critical for effective insulin secretion and disrupted early in diabetes, has not been examined. Here, we examine a ‘first responder’ cell population, defined by the earliest [Ca2+] response during first-phase [Ca2+] elevation. We record [Ca2+] dynamics in intact mouse islets, via β-cell specific expression of the [Ca2+] indicator GCamP6s. We identify multiple β-cell subpopulations based on signatures of their [Ca2+] dynamics and investigate the role of ‘first responder’ cells in islet function by means of 2-photon laser ablation. We further characterize the functional properties of ‘first responder’ cells by NAD(P)H autofluorescence, fluorescent recovery after photobleaching, glibenclamide stimulation, and network analysis. We also investigate which functional characteristics of these cells are critical by a computational model of islet electrophysiology. Based on the dynamics of [Ca2+] responses, first responder cells are distinct from previously identified functional subpopulations. The first-phase response time of β-cells is spatially organized, dependent on the cell’s distance to the first responder cells, and consistent over time up to ~24 h. First responder cells showed characteristics of high membrane excitability and slightly lower than average coupling to their neighbors. When first responder cells were ablated, the first-phase [Ca2+] diminished, compared to ablating a random cell. We also observed a hierarchy of the first-phase response time, where cells that were next earliest to respond often take over the role of the first responder cell upon ablation. In summary, we discover and characterize a distinct first responder β-cell subpopulation, based on [Ca2+] response timing, which is critical for the islet first-phase response to glucose.

Superparamagnetic iron oxide (Fe3O4) and highly anisotropic barium hexaferrite (BaFe12O19) nanoparticles were coated with an anti-inflammatory drug and magnetically transported through mucus produced by primary human airway epithelial cells. Using wet planetary ball milling, dl-2-amino-3-phosphonopropionic acid-coated BaFe12O19 nano-particles (BaNPs) of 1–100 nm in diameter were prepared in water. BaNPs and conventional 20–30-nm Fe3O4 nanoparticles (FeNPs) were then encased in a polymer (PLGA) loaded with dexamethasone (Dex) and tagged for imaging. PLGA-Dex-coated BaNPs and FeNPs were characterized using dynamic light scattering (DLS), transmission electron microscopy (TEM), and superconducting quantum interference device (SQUID) magnetometry. Both PLGA-Dex-coated BaNPs and FeNPs were transferred to the surface of a ~100-μm thick mucus layer of air-liquid interface cultured primary normal human tracheobronchial epithelial (NHTE) cells. Within 30 min, the nanoparticles were pulled successfully through the mucus layer by a permanent neodymium magnet. The penetration time of the nanomedicine was monitored using confocal microscopy and tailored by varying the thickness of the PLGA-Dex coating around the particles.