A Zebrafish Model of Diabetes Mellitus and Metabolic Memory Reveals Epigenetic Alterations

The zebrafish (Danio Rerio) is an established model organism for the study of developmental processes, human disease and tissue regeneration. In the first part of this study we report that limb regeneration is severely impaired in our newly developed adult zebrafish model of type I diabetes. Intraperitoneal streptozocin (STZ) injection of adult, wild type zebrafish results in a sustained hyperglycemic state as determined by elevated fasting blood glucose values and increased glycation of serum protein. Serum insulin levels are also decreased and pancreas immunohistochemisty revealed a lesser amount of insulin signal in hyperglycemic fish. Additionally, the diabetic complications of retinal thinning and glomerular basement membrane thickening (early signs of retinopathy and nephropathy) resulting from the hyperglycemic state were evident in streptozocin injected fish at three weeks. Most significantly, limb regeneration, following caudal fin amputation, is severely impaired in diabetic zebrafish. Nonspecific toxic effects outside the pancreas were not found to contribute to impaired limb regeneration. This experimental system using adult zebrafish facilitates a broad spectrum of genetic and molecular approaches to study regeneration in the diabetic background.;The objective of the second part of the study is to determine if diabetic metabolic memory (MM) is heritable to daughter cells in vivo and if the epigenetic process of DNA methylation contributes to MM. Adult zebrafish were induced into a diabetic-hyperglycemic state using STZ and allowed to return to a normal physiological state of glucose homeostasis by cessation of STZ administration (due to pancreatic islet regeneration). Limb regeneration (caudal fin regeneration) was used to monitor tissue repair/regeneration in the diabetic state and the post-diabetic state, termed the MM state. Parameters of the diabetic state and MM state were analyzed by (1) ELISA, (2) immunohistochemistry, (3) RNA and DNA extraction and quantitative real-time PCR, and (4) MeDIP sequencing for determination of genome wide DNA methylation. Caudal fin regeneration and skin wound healing were found to be impaired when adult zebrafish are in a hyperglycemic state. When STZ was withdrawn, pancreatic islet regeneration occurred and serum insulin and glucose returned to normal levels within two weeks. Despite return to a euglycemic state, fin regeneration and skin wound healing remained impaired to the same extent. The impairment was heritable and transmittable to daughter cell tissue that was never exposed to the hyperglycemic state. AGEs and ROSs did not persist in MM regenerating tissue while genome-wide DNA hypomethylation was detected in both diabetic and MM fish. As analyzed in an adult zebrafish model of Type I diabetes, evidence indicates that diabetic metabolic memory correlates with heritable transmission of DNA hypo-methylation.