Dynamic glucoregulation and mammalian-like responses to metabolic and developmental disruption in zebrafish

University of Massachusetts Medical School, Diabetes Center of Excellence, 373 Plantation Street, Suite 218, Worcester, MA 01605, USA.
General and Comparative Endocrinology (Impact Factor: 2.47). 10/2010; 170(2):334-45. DOI: 10.1016/j.ygcen.2010.10.010
Source: PubMed


Zebrafish embryos are emerging as models of glucose metabolism. However, patterns of endogenous glucose levels, and the role of the islet in glucoregulation, are unknown. We measured absolute glucose levels in zebrafish and mouse embryos, and demonstrate similar, dynamic glucose fluctuations in both species. Further, we show that chemical and genetic perturbations elicit mammalian-like glycemic responses in zebrafish embryos. We show that glucose is undetectable in early zebrafish and mouse embryos, but increases in parallel with pancreatic islet formation in both species. In zebrafish, increasing glucose is associated with activation of gluconeogenic phosphoenolpyruvate carboxykinase1 (pck1) transcription. Non-hepatic Pck1 protein is expressed in mouse embryos. We show using RNA in situ hybridization, that zebrafish pck1 mRNA is similarly expressed in multiple cell types prior to hepatogenesis. Further, we demonstrate that the Pck1 inhibitor 3-mercaptopicolinic acid suppresses normal glucose accumulation in early zebrafish embryos. This shows that pre- and extra-hepatic pck1 is functional, and provides glucose locally to rapidly developing tissues. To determine if the primary islet is glucoregulatory in early fish embryos, we injected pdx1-specific morpholinos into transgenic embryos expressing GFP in beta cells. Most morphant islets were hypomorphic, not a genetic, but embryos still exhibited persistent hyperglycemia. We conclude from these data that the early zebrafish islet is functional, and regulates endogenous glucose. In summary, we identify mechanisms of glucoregulation in zebrafish embryos that are conserved with embryonic and adult mammals. These observations justify use of this model in mechanistic studies of human metabolic disease.

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Available from: Agata Jurczyk, Oct 01, 2015
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    • "Most importantly, we identified that activation of glucagon is required for β cell regeneration from both precursor pools. Furthermore, our results demonstrate that key physiological actions of glucagon in regulating blood glucose are conserved with mammals, just as for insulin (Kinkel and Prince, 2009; Jurczyk et al., 2011; Andersson et al., 2012); in particular, we found that in zebrafish glucagon activity is correlated with blood glucose levels, and that glucagon expression is upregulated in the absence of β cells. These findings further validate zebrafish as a model for studying human metabolic disease. "
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    ABSTRACT: The interconversion of cell lineages via transdifferentiation is an adaptive mode of tissue regeneration and an appealing therapeutic target. However, its clinical exploitation is contingent upon the discovery of contextual regulators of cell fate acquisition and maintenance. In murine models of diabetes, glucagon-secreting alpha cells transdifferentiate into insulin-secreting beta cells following targeted beta cell depletion, regenerating the form and function of the pancreatic islet. However, the molecular triggers of this mode of regeneration are unknown. Here, using lineage-tracing assays in a transgenic zebrafish model of beta cell ablation, we demonstrate conserved plasticity of alpha cells during islet regeneration. In addition, we show that glucagon expression is upregulated after injury. Through gene knockdown and rescue approaches, we also find that peptides derived from the glucagon gene are necessary for alpha-to-beta cell fate switching. Importantly, whereas beta cell neogenesis was stimulated by glucose, alpha-to-beta cell conversion was not, suggesting that transdifferentiation is not mediated by glucagon/GLP-1 control of hepatic glucose production. Overall, this study supports the hypothesis that alpha cells are an endogenous reservoir of potential new beta cells. It further reveals that glucagon plays an important role in maintaining endocrine cell homeostasis through feedback mechanisms that govern cell fate stability. © 2015. Published by The Company of Biologists Ltd.
    Development 04/2015; 142(8):1407-17. DOI:10.1242/dev.117911 · 6.46 Impact Factor
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    • "These two peaks coincide with the two periods when the animals exhibit an increase in glucose levels [8], [16], suggesting that high glucose levels stimulate a compensatory increase in beta-cell number. Whether glucose exerts a direct effect on beta-cell proliferation or whether the increase in proliferation is the result of transient insulin resistance during these two periods, as shown in mouse models [5], [16], remains to be determined. Importantly, these analyses also revealed that from 72 to 96 hpf, a majority of beta-cells enter a period of quiescence. "
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    ABSTRACT: Inducing beta-cell mass expansion in diabetic patients with the aim to restore glucose homeostasis is a promising therapeutic strategy. Although several in vitro studies have been carried out to identify modulators of beta-cell mass expansion, restoring endogenous beta-cell mass in vivo has yet to be achieved. To identify potential stimulators of beta-cell replication in vivo, we established transgenic zebrafish lines that monitor and allow the quantification of cell proliferation by using the fluorescent ubiquitylation-based cell cycle indicator (FUCCI) technology. Using these new reagents, we performed an unbiased chemical screen, and identified 20 small molecules that markedly increased beta-cell proliferation in vivo. Importantly, these structurally distinct molecules, which include clinically-approved drugs, modulate three specific signaling pathways: serotonin, retinoic acid and glucocorticoids, showing the high sensitivity and robustness of our screen. Notably, two drug classes, retinoic acid and glucocorticoids, also promoted beta-cell regeneration after beta-cell ablation. Thus, this study establishes a proof of principle for a high-throughput small molecule-screen for beta-cell proliferation in vivo, and identified compounds that stimulate beta-cell proliferation and regeneration.
    PLoS ONE 08/2014; 9(8):e104112. DOI:10.1371/journal.pone.0104112 · 3.23 Impact Factor
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    • "Glucose measurements were done using a fluorescencebased enzymatic detection kit (Biovision, Inc., Mountain View, CA, USA) as described previously (Jurczyk et al. 2011). RNA deep sequencing (RNA-seq) "
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    ABSTRACT: Type 2 diabetes, obesity and metabolic syndrome are pathologies where insulin resistance plays a central role and that have an impact on a large population worldwide. These pathologies are usually associated with a dysregulation of insulin secretion leading to a chronic exposure of the tissues to high insulin levels (i.e. hyperinsulinemia), which diminishes the concentration of key downstream elements causing insulin resistance. The complexity of the study of insulin resistance arises from the heterogeneity of the metabolic states where it is observed. To contribute to the understanding of the mechanisms triggering insulin resistance we have developed a zebrafish model to study insulin metabolism and its associated disorders. Zebrafish larvae appeared to be sensitive to human recombinant insulin, becoming insulin resistant when exposed to a high dose of the hormone. Moreover RNAseq-based transcriptomic profiling of these larvae revealed a strong down regulation of a number of immune relevant genes as a consequence of the exposure to hyperinsulinemia. Interestingly, as an exception, the negative immune modulator ptpn6 appeared to be up regulated in insulin resistant larvae. Knockdown of ptpn6 showed to counteract the observed down regulation of the immune system and insulin signaling pathway caused by hyperinsulinemia. These results show that ptpn6 is a mediator of the metabolic switch between insulin sensitive and insulin resistant states. Our zebrafish model for hyperinsulinemia has therefore demonstrated its suitability to discover novel regulators of insulin resistance. In addition, our data will be very useful to further study the function of immunological determinants in a non-obese model system.
    Journal of Endocrinology 06/2014; DOI:10.1530/JOE-14-0178 · 3.72 Impact Factor
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