Embryonic exposure to Mono(2-ethylhexyl) phthalate (MEHP) disrupts pancreatic organogenesis in zebrafish (Danio rerio).

Mono(2-ethylhexyl) phthalate (MEHP) is the bioactive metabolite of di(2-ethylhexyl) phthalate, a plasticizing agent and persistent environmental contaminant associated with obesity, developmental abnormalities, and oxidative stress. Nrf2 (Nfe2l2) is a transcription factor that regulates cytoprotective genes as part of the adaptive antioxidant response. We previously identified the pancreas as a sensitive target of oxidative stress during embryonic development. The goals of this study were to 1) characterize the effects of MEHP exposure on pancreatic development, and 2) determine whether oxidative stress contributes to MEHP embryotoxicity. Zebrafish (Danio rerio) embryos from AB wildtype and Tg(ins:GFP;nrf2afh318/fh318) were exposed to 0 or 200 µg/L MEHP at 3 h post fertilization (hpf) through 168 hpf to assess pancreatic organogenesis. MEHP exposure significantly decreased ß-cell area at all timepoints (48, 72, 96, 168 hpf), but Nrf2a did not significantly protect against islet hypomorphism. Tg(gcga:GFP) embryos exposed to MEHP showed a decrease in α-cell area in the islet across the same timepoints. Tg(ptf1a:GFP) embryos were assessed at 80 and 168 hpf for exocrine pancreas length. MEHP exposure decreased growth of the exocrine pancreas. Expression of pancreas genes insa, sst2 and ptf1a was significantly reduced by MEHP exposure compared to controls. Glutathione (GSH) concentrations and redox potentials were quantified at 72 hpf by HPLC, but no significant changes were observed. However, expression of the GSH-related genes gstp1 and gsr were significantly altered by MEHP exposure. These data indicate that the developing pancreas is a sensitive target tissue of embryonic exposure to MEHP.

Expression and function of ATP-dependent potassium channels in zebrafish islet ß-cells.

ATP-sensitive potassium channels (KATP channels) are critical nutrient sensors in many mammalian tissues. In the pancreas, KATP channels are essential for coupling glucose metabolism to insulin secretion. While orthologous genes for many components of metabolism-secretion coupling in mammals are present in lower vertebrates, their expression, functionality and ultimate impact on body glucose homeostasis are unclear. In this paper, we demonstrate that zebrafish islet ß-cells express functional KATP channels of similar subunit composition, structure and metabolic sensitivity to their mammalian counterparts. We further show that pharmacological activation of native zebrafish KATP using diazoxide, a specific KATP channel opener, is sufficient to disturb glucose tolerance in adult zebrafish. That ß-cell KATP channel expression and function are conserved between zebrafish and mammals illustrates the evolutionary conservation of islet metabolic sensing from fish to humans, and lends relevance to the use of zebrafish to model islet glucose sensing and diseases of membrane excitability such as neonatal diabetes.

Embryonic exposures to perfluorooctanesulfonic acid (PFOS) disrupt pancreatic organogenesis in the zebrafish, Danio rerio.

Perfluorooctanesulfonic acid (PFOS) is a ubiquitous environmental contaminant, previously utilized as a non-stick application for consumer products and firefighting foam. It can cross the placenta, and has been repeatedly associated with increased risk for diabetes in epidemiological studies. Here, we sought to establish the hazard posed by embryonic PFOS exposures on the developing pancreas in a model vertebrate embryo, and develop criteria for an adverse outcome pathway (AOP) framework to study the developmental origins of metabolic dysfunction. Zebrafish (Danio rerio) embryos were exposed to 16, 32, or 64 µM PFOS beginning at the mid-blastula transition. We assessed embryo health, size, and islet morphology in Tg(insulin-GFP) embryos at 48, 96 and 168 hpf, and pancreas length in Tg(ptf1a-GFP) embryos at 96 and 168 hpf. QPCR was used to measure gene expression of endocrine and exocrine hormones, digestive peptides, and transcription factors to determine whether these could be used as a predictive measure in an AOP. Embryos exposed to PFOS showed anomalous islet morphology and decreased islet size and pancreas length in a U-shaped dose-response curve, which resemble congenital defects associated with increased risk for diabetes in humans. Expression of genes encoding islet hormones and exocrine digestive peptides followed a similar pattern, as did total larval growth. Our results demonstrate that embryonic PFOS exposures can disrupt pancreatic organogenesis in ways that mimic human congenital defects known to predispose individuals to diabetes; however, future study of the association between these defects and metabolic dysfunction are needed to establish an improved AOP framework.

Assessment of Toxicological Perturbations and Variants of Pancreatic Islet Development in the Zebrafish Model.

The pancreatic islets, largely comprised of insulin-producing beta cells, play a critical role in endocrine signaling and glucose homeostasis. Because they have low levels of antioxidant defenses and a high perfusion rate, the endocrine islets may be a highly susceptible target tissue of chemical exposures. However, this endpoint, as well as the integrity of the surrounding exocrine pancreas, is often overlooked in studies of developmental toxicology. Disruption of development by toxicants can alter cell fate and migration, resulting in structural alterations that are difficult to detect in mammalian embryo systems, but that are easily observed in the zebrafish embryo model (Danio rerio). Using endogenously expressed fluorescent protein markers for developing zebrafish beta cells and exocrine pancreas tissue, we documented differences in islet area and incidence rates of islet morphological variants in zebrafish embryos between 48 and 96 h post fertilization (hpf), raised under control conditions commonly used in embryotoxicity assays. We identified critical windows for chemical exposures during which increased incidences of endocrine pancreas abnormalities were observed following exposure to cyclopamine (2-12 hpf), Mono-2-ethylhexyl phthalate (MEHP) (3-48 hpf), and Perfluorooctanesulfonic acid (PFOS) (3-48 hpf). Both islet area and length of the exocrine pancreas were sensitive to oxidative stress from exposure to the oxidant tert-butyl hydroperoxide during a highly proliferative critical window (72 hpf). Finally, pancreatic dysmorphogenesis following developmental exposures is discussed with respect to human disease.

Detection of autofluorescent Mycobacterium chelonae in living zebrafish.

Mycobacterium chelonae is widespread in aquatic environments and can cause mycobacteriosis with low virulence in zebrafish. The risk of infection in zebrafish is exacerbated in closed-recirculating aquatic systems where rapidly growing mycobacteria can live on biofilms, as well as in zebrafish tissues. We have discovered a method of identifying and visualizing M. chelonae infections in living zebrafish using endogenous autofluorescence. Infected larvae are easily identified and can be excluded from experimental results. Because infection may reduce fertility in zebrafish, the visualization of active infection in contaminated eggs of transparent casper females simplifies screening. Transparent fish are also particularly useful as sentinels that can be examined periodically for the presence of autofluorescence, which can then be tested directly for M. chelonae.

Imaging beta cell regeneration and interactions with islet vasculature in transparent adult zebrafish.

Blood vessel networks provide nutrients and gaseous exchange that are essential for functions. Pancreatic islet capillaries deliver oxygen to endocrine cells while transporting hormones to organs and peripheral locations throughout the body. We have developed a zebrafish diabetes model in which adult islets can be followed in vivo during beta cell regeneration while calibrating changes in beta cell mass and fasting blood glucose levels. After genetic ablation, beta cells are initially dysfunctional or dying, and blood glucose levels increase fourfold. During a 2-week period, hyperglycemia eventually normalizes as beta cell mass regenerates. We show that mCherry-fluorescent, insulin-positive beta cells re-emerge in close contact with the vascular endothelium. Alterations in the dense vascular network of zebrafish islets were visualized by the expression of green fluorescent protein (GFP) in endothelial cells derived from the Fli transcription factor promoter. The rapid destruction and regeneration of beta cell mass was evaluated in the same animal over time, providing a functional model for investigating the interactions of islet cell types with vascular cells as well as the consequences of hyperglycemia on other tissues. Regenerating adult zebrafish can be utilized as vertebrate, metabolically active models for generating new insights into treatments for type 2 diabetes.

Deletion of GαZ protein protects against diet-induced glucose intolerance via expansion of ß-cell mass.

Insufficient plasma insulin levels caused by deficits in both pancreatic ß-cell function and mass contribute to the pathogenesis of type 2 diabetes. This loss of insulin-producing capacity is termed ß-cell decompensation. Our work is focused on defining the role(s) of guanine nucleotide-binding protein (G protein) signaling pathways in regulating ß-cell decompensation. We have previously demonstrated that the α-subunit of the heterotrimeric G(z) protein, Gα(z), impairs insulin secretion by suppressing production of cAMP. Pancreatic islets from Gα(z)-null mice also exhibit constitutively increased cAMP production and augmented glucose-stimulated insulin secretion, suggesting that Gα(z) is a tonic inhibitor of adenylate cyclase, the enzyme responsible for the conversion of ATP to cAMP. In the present study, we show that mice genetically deficient for Gα(z) are protected from developing glucose intolerance when fed a high fat (45 kcal%) diet. In these mice, a robust increase in ß-cell proliferation is correlated with significantly increased ß-cell mass. Further, an endogenous Gα(z) signaling pathway, through circulating prostaglandin E activating the EP3 isoform of the E prostanoid receptor, appears to be up-regulated in insulin-resistant, glucose-intolerant mice. These results, along with those of our previous work, link signaling through Gα(z) to both major aspects of ß-cell decompensation: insufficient ß-cell function and mass.

Regeneration of the pancreas in adult zebrafish.

OBJECTIVE: Regenerating organs in diverse biological systems have provided clues to processes that can be harnessed to repair damaged tissue. Adult mammalian beta-cells have a limited capacity to regenerate, resulting in diabetes and lifelong reliance on insulin. Zebrafish have been used as a model for the regeneration of many organs. We demonstrate the regeneration of adult zebrafish pancreatic beta-cells. This nonmammalian model can be used to define pathways for islet-cell regeneration in humans. RESEARCH DESIGN AND METHODS: Adult transgenic zebrafish were injected with a single high dose of streptozotocin or metronidazole and anesthetized at 3, 7, or 14 days or pancreatectomized. Blood glucose measurements were determined and gut sections were analyzed using specific endocrine, exocrine, and duct cell markers as well as markers for dividing cells. RESULTS: Zebrafish recovered rapidly without the need for insulin injections, and normoglycemia was attained within 2 weeks. Although few proliferating cells were present in vehicles, ablation caused islet destruction and a striking increase of proliferating cells, some of which were Pdx1 positive. Dividing cells were primarily associated with affected islets and ducts but, with the exception of surgical partial pancreatectomy, were not extensively beta-cells. CONCLUSIONS: The ability of the zebrafish to regenerate a functional pancreas using chemical, genetic, and surgical approaches enabled us to identify patterns of cell proliferation in islets and ducts. Further study of the origin and contribution of proliferating cells in reestablishing islet function could provide strategies for treating human diseases.

Sonic hedgehog is required early in pancreatic islet development.

Pancreatic organogenesis relies on a complex interplay of cell-autonomous and extracellular signals. We demonstrate that the morphogen sonic hedgehog (Shh) is required for pancreatic development in zebrafish. Genetic mutants of Shh and its signaling pathway establish this dependence as specific to endocrine, but not exocrine, pancreas. Using cyclopamine to inhibit hedgehog signaling, we show that transient Shh signaling is necessary during gastrulation for subsequent differentiation of endoderm into islet tissue. A second hedgehog-dependent activity occurring later in development was also identified and may be analogous to the known action of Shh in gut endoderm to direct localization of pancreatic development. The early action of Shh may be part of a more general process allowing neuroendocrine cells to originate in nonneuroectodermally derived tissues.