A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Summary

Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.

Abstract

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.

We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

Introduction

Diabetes mellitus (DM) is a serious and growing health problem that results in reduced life expectancy due to disease specific microvascular (retinopathy, nephropathy, neuropathy, impaired wound healing) and macrovascular (heart disease and stroke) complications ¹. Once initiated, diabetic complications continue to progress uninterrupted even when glycemic control is achieved ^2,3 and this phenomenon has been termed metabolic memory or the legacy effect. The presence of this phenomenon was recognized clinically during the early 1990s as the "The Diabetes Control and Complications Trial (DCCT)" progressed and since has been supported by multiple ad....

Protocol

All procedures are performed following the guidelines described in "Principles of Laboratory Animal Care" (National Institutes of Health publication no. 85-23, revised 1985) and the approved Rosalind Franklin University Institutional Animal Care and Use Committee animal protocol 08-19.

There are 2 important abbreviations that are used in this manuscript. 1) DM: refers to fish that are in an acute (300 mg/dl) hyperglycemic state and have been for at least 3 weeks. 2) MM: refers to fish that were 21 day (see protocol) DM fish and allowed to restore glycemic control through pancreatic regeneration. This is achieved within (14) days of drug rem....

Results

Type I diabetic zebrafish not only display the known secondary complications of retinopathy and nephropathy, but also, exhibit an additional complication: impaired caudal fin regeneration. This later complication persists due to metabolic memory in fish that have restored normal glucose control following a hyperglycemic period. In Figure 2A (control) and Figure 2B (metabolic memory) representative images of regenerating fins that were captured at 72 hr post-amputation are presented. The .......

Discussion

Diabetes mellitus is a disease of metabolic dysregulation, initially diagnosed as hyperglycemia, that ultimately results in blood vessel damage leading to many complications which all persist even after euglycemia is achieved though pharmaceutical intervention. This persistence of complications is referred to as metabolic memory and several recent studies have examined the role that epigenetic mechanisms play in this phenomenon. Here we have detailed a protocol that allows for the generation of both acute diabetic and me.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Streptozocin	Sigma Aldrich	S0130
2 phenoxyethanol	Sigma Aldrich	P1126
Scalpel (size 10)	Fisher Scientific	089275A
Petri Dishes	Fisher Scientific	08-757-13
½ cc syringe, with 27 1/2 gauge needle	Fisher Scientific	305620
QuantiChrome glucose assay kit.	Bioassay Systems	DIGL-100
Sodium Chloride	Sigma Aldrich	S3014
Dissecting Microscope	Nikon	TMZ-1500	Any dissecting microscope is fine.
Camera for Imaging	Nikon	Q imaging	Any camera is suitable.
Image J software	National Institutes of Health	NIH Image
NIS Elements	Nikon		Any imaging software is suitable.