The effect of Nrf2 pathway activation on human pancreatic islet cells.

BACKGROUND: Pancreatic islets are known to contain low level of antioxidants that renders them vulnerable to oxidative stress. Nrf2 is the master regulator of numerous genes, encoding antioxidant, detoxifying, and cytoprotective molecules. Activation of Nrf2 pathway induces up-regulation of numerous genes encoding antioxidant and phase II detoxifying enzymes and related proteins. However, little is known regarding the role of this pathway in human islet cells. The aim was to investigate the effect of Nrf2 activator (dh404, CDDO-9,11-dihydro-trifluoroethyl amide) on human islet cells. METHODS: Human islets were obtained from cadaveric donors. After dh404 treatment, Nrf2 translocation, mRNA expression, and protein abundance of its key target gene products were examined. The proportion of dh404-treated or non-treated viable islet beta cells was analyzed using flowcytemetry. The cytoprotective effects against oxidative stress and production of inflammatory mediators, and in vivo islet function after transplantation were determined. RESULTS: Nrf2 nuclear translocation was confirmed by con-focal microscope within 2 hours after treatment, which was associated with a dose-dependent increase in mRNA expression of anti-oxidants, including NQO1, HO-1, and GCLC. Enhanced HO-1 expression in dh404 treated islets was confirmed by Western Blot assay. Islet function after transplantation (2000 IEQ/mouse) to diabetic nude mice was not affected with or without dh404 treatment. After induction of oxidative stress with hydrogen peroxide (200 µM) the proportion of dh404-treated viable islet cells was significantly higher in the dh404-treated than untreated islets (74% vs.57%; P<0.05). Dh404 significantly decreased production of cytokines/chemokines including IL-1ß, IL-6, IFN-γ and MCP-1. CONCLUSION: Treatment of human pancreatic islets with the potent synthetic Nrf2 activator, dh404, significantly increased expression of the key anti-oxidants enzymes, decreased inflammatory mediators in islets and conferred protection against oxidative stress in beta cells.

Pharmacological activation of Nrf2 pathway improves pancreatic islet isolation and transplantation.

Oxidative stress is a major cause of islet damage and loss during the islet isolation process. The Nrf2 pathway plays a critical role in protecting the cells against oxidative stress. The aim of this study was to investigate the effect of an Nrf2 activator (dh404) on islet isolation and transplantation in a rodent model. Islet isolation was conducted using Nrf2-deficient and wild-type mice and vehicle-treated and Nrf2 activator (dh404)-treated rats. Islet yield, viability, and Nrf2 pathway activity were determined. An in vivo islet potency test was done. Islet yield and viability in Nrf2-deficient mice was significantly lower compared to wild-type (p < 0.05) mice. Furthermore, administration of dh404 to normal Sprague-Dawley rats enhanced nuclear translocation of Nrf2 and elevated HO-1 expression in the pancreas. Islet yield and viability in dh404-treated rats was significantly higher compared to the vehicle-treated group (p < 0.05). The diabetes cure rate in nude mice with chemically induced diabetes was significantly greater in those transplanted with islets from the dh404-treated group (6/9) than vehicle-treated rats (2/9, p < 0.05). The Nrf2 pathway plays a significant role in protecting islets against stress caused by the isolation process. Pharmacological activation of the Nrf2 pathway significantly increased HO-1 expression, improved islet yield, viability, and function after transplantation.

Distribution and size of mucous glands in the ferret tracheobronchial tree.

A transgenic ferret model of cystic fibrosis has recently been generated. It is probable that malfunction of airway mucous glands contributes significantly to the airway pathology of this disease. The usefulness of the ferret model may therefore depend in part on how closely the airway glands of ferrets resemble those of humans. Here, we show that in the ferret trachea glands are commonest in its most ventral aspect and disappear about half way up the lateral walls; they are virtually absent from the dorsal membranous portion. Further, the aggregate volume of glands per unit mucosal surface declines progressively by about 60% between the larynx and the carina. The average frequency of glands openings for the ferret trachea as a whole is only about one-fifth that in humans (where gland openings are found at approximately the same frequency throughout the trachea). Glands in the ferret trachea are on average about one-third the size of those in the human. Therefore, the aggregate volume of tracheal glands (per unit mucosal surface area) in the ferret is only about 6% that in humans. As in other mammalian species, airway glands in the ferret disappear at an airway internal diameter of ∼1 mm, corresponding approximately in this species to airway generation 6.

Chloride secretion by cultures of pig tracheal gland cells.

Malfunction of airway submucosal glands contributes to the pathology of cystic fibrosis (CF), and cell cultures of CF human airway glands show defects in Cl(-) and water transport. Recently, a transgenic pig model of CF (the CF pig) has been developed. Accordingly, we have developed cell cultures of pig airway gland epithelium for use in investigating alterations in gland function in CF. Our cultures form tight junctions (as evidenced by high transepithelial electrical resistance) and show high levels of active anion secretion (measured as amiloride-insensitive short-circuit current). In agreement with recent results on human airway glands, neurohumoral agents that elevate intracellular Ca(2+) potently stimulated anion secretion, while elevation of cAMP was comparatively ineffective. Our cultures express lactoferrin and lysozyme (serous gland cell markers) and MUC5B (the main mucin of airway glands). They are, therefore, potentially useful in determining if CF-related alterations in anion transport result in altered secretion of serous cell antimicrobial agents or mucus.