Studies from human cells, rats, and zebrafish have documented that hyperglycemia (HG) induces the demethylation of specific cytosines throughout the genome. We previously documented that a subset of these changes become permanent and may provide, in part, a mechanism for the persistence of complications referred to as the metabolic memory phenomenon. In this report, we present studies aimed at elucidating the molecular machinery that is responsible for the HG-induced DNA demethylation observed. To this end, RNA expression and enzymatic activity assays indicate that the ten-eleven translocation (Tet) family of enzymes are activated by HG. Furthermore, through the detection of intermediates generated via conversion of 5-methyl-cytosine back to the unmethylated form, the data were consistent with the use of the Tet-dependent iterative oxidation pathway. In addition, evidence is provided that the activity of the poly(ADP-ribose) polymerase (Parp) enzyme is required for activation of Tet activity because the use of a Parp inhibitor prevented demethylation of specific loci and the accumulation of Tet-induced intermediates. Remarkably, this inhibition was accompanied by a complete restoration of the tissue regeneration deficit that is also induced by HG. The ultimate goal of this work is to provide potential new avenues for therapeutic discovery.

We previously reported a zebrafish model of type I diabetes mellitus (DM) that can be used to study the hyperglycemic (HG) and metabolic memory (MM) states within the same fish. Clinically, MM is defined as the persistence of diabetic complications even after glycemic control is pharmacologically achieved. In our zebrafish model, MM occurs following β-cell regeneration, which returns fish to euglycemia. During HG, fish acquire tissue deficits reflective of the complications seen in patients with DM and these deficits persist after fish return to euglycemia (MM). The unifying mechanism for the induction of diabetic complications involves a cascade of events that is initiated by the HG stimulation of poly-ADP ribose polymerase enzyme (Parp) activity. Additionally, recent evidence shows that the HG induction of Parp activity stimulates changes in epigenetic mechanisms that correlate with the MM state and the persistence of complications. Here we report that wound-induced angiogenesis is impaired in DM and remains impaired when fish return to a euglycemic state. Additionally, inhibition of Parp activity prevented the HG-induced wound angiogenesis deficiency observed. This approach can identify molecular targets that will provide potential new avenues for therapeutic discovery as angiogenesis imbalances are associated with all HG-damaged tissues.

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al. 17 that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required. Video LinkThe video component of this article can be found at

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