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 additional clinical trials ^{4,5,6,7,8,9,10,11,12,13,14}. Animal models of DM have been critical for discoveries related to the patho-physiology of diabetic complications and metabolic memory. In fact, the persistence of diabetic complications was first documented in a canine model of diabetic retinopathy which has since been supported by several lines of experimental evidence using a variety of in vitro culture systems and animal models ^{15,16,17,18,19,20,21}. These studies clearly show that an initial hyperglycemic period results in permanent abnormalities (including aberrant gene expression) of target organs/cells and mechanistically suggests the involvement of the epigenome.

Epigenomes consist of all the chromatin modifications for a given cell type and are responsible for a cell's unique gene expression profile. The chromosome modifications are dynamic during development, support cell differentiation, are responsive to external stimuli, are mitotically stably inherited ^22,23 and can be altered in disease ^24,25,26. These epigenetic mechanisms include: post translational histone modifications, non-canonical histone variant inclusion in octomers, chromatin access changes through DNA methylation, and gene expression control through non-coding micro RNAs ^27,28,29,30. Altogether, epigenetic processes allow cells/organisms to quickly respond to changing environmental stimuli ^31,32,33 , they also confer the ability for the cell to "memorize" these encounters once the stimulus is removed ^23,22. Therefore, as altered gene expression profiles resulting from epigenetic processes are stable in the absence of the signal(s) that initiated them and are heritable through cell division, they have gained great interest as underlying molecular mechanisms of human pathologies including metabolic memory. The results that are emerging in the context of DM and epigenetics parallel advancements in other diseases in that a plethora of epigenetic changes induced by hyperglycemia cause remarkable persistent changes in transcriptional networks of cells (reviewed in ^{34,35,36,37,38}).

The zebrafish has long been a premier model organism to study vertebrate development however the last 15 years has seen an exponential growth in utilizing this organism for study of human disease. ³⁹. Zebrafish models of human disease have been established spanning a wide range of human pathologies including genetic disorders and acquired disease ^40,41,42. The many advantages of the zebrafish over other vertebrate model organisms include high fecundity, short generation time, transparency through early adulthood, reduced housing costs and an array of tools for gene manipulation. Moreover, due to the extensive conservation of genetic pathways and cellular physiology among the vertebrates and the capacity to perform high throughput drug screenings, the zebrafish has been successfully used for pharmaceutical discovery.

We have developed an adult zebrafish model of type I diabetes mellitus using the diabetogenic drug, streptozocin. We have characterized this model to show that diabetic zebrafish not only display the known human secondary complications but in addition, exhibit impaired limb regeneration (caudal fin regeneration) as a consequence of the hyperglycemic environment. In addition, we have reported that hyperglycemic zebrafish revert back to normal glycemia within 2 weeks of drug removal due to regeneration of endogenous pancreatic beta cells resulting in a physiologically normal glycemic state. However, in contrast, limb regeneration in these fish remains impaired to the same extent as in the acute diabetic state indicating this complication persists and is susceptible to metabolic memory. The main impetus for generating this model was to provide a system to study the mitotically stable epigenetic components that support the metabolic memory phenomenon in the absence of the background noise of the previous hyperglycemic environment. At the conclusion of the protocol provided here the zebrafish and or selective tissues can be processed by any assay suitable to the researchers needs. We have successfully used this procedure to identify the genome-wide persistent changes in DNA methylation induced by hyperglycemia that are maintained in the metabolic memory state ²¹.

We feel that this zebrafish model of type I diabetes mellitus has several innovative advantages over other model systems for examining metabolic memory. 1) All of our studies can be conducted in vivo and as the previous hyperglycemic fish return to euglycemia through regeneration of endogenous insulin production, they do not require exogenous insulin injections. Therefore, this avoids the complicating spikes and valleys in glycemic control that may occur in animals requiring exogenous insulin. 2) As described above, the background stimulation from the previous diabetic state (i.e. the continued presence of advanced glycation end-products and reactive oxygen species markers) are eliminated and therefore one can examine the purely epigenetic factors of metabolic memory. 3) The experiments can be performed rapidly as it takes approximately 80 days from diabetes induction until metabolic memory examination. 4) Caudal fin regeneration is experimentally very approachable and allows for easy genetic and experimental manipulation for which there are a vast array of tools. 5) Caudal fin regeneration provides a very simple and quantifiable method to assess metabolic memory and therefore will allow for future drug discovery.

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 removal. The fish are considered MM fish from this point onward. It is also important to note that fish that are referred to as control are treated identically as DM or MM fish in terms of the number of injections (saline only), the incubation times at the various temperatures and the number and timing of caudal fin amputations.

1. Generation of Zebrafish with Diabetes mellitus, DM Fish

1. Prepare both recovery and anesthetic water tanks. Recovery water is normal fish water. For anesthetic water add sufficient 2-phenoxyethanol so that a 1:1,000 dilution in normal fish water is achieved.
2. Prepare a 0.3% solution (in a fume hood) of streptozocin (STZ) by adding 6 mg of STZ to 2 ml of 0.09% sodium chloride and immediately place the solution on ice . This will provide enough injection solution for the injection of approximately 20 fish in 20 min. If you exceed the 20 minute mark, stop, and make a fresh STZ solution before proceeding. In a separate tube aliquot enough saline solution for control fish. Once the STZ is solubilized all subsequent steps do not require fume hood use.
3. Fill a ½ cc syringe equipped with a 27 1/2 gauge needle with the STZ or control solutions ensuring that no air bubbles are trapped.
4. Anesthetize each fish individually by placing the fish in anesthetic water, and wait until their swimming motion ceases (1-2 min).
5. Once anesthetized briefly place the fish on a paper towel to absorb any excess water, place the fish in weigh boat and measure the mass of the fish.
6. Place the fish on a firm surface (Petri dish lid) for injection.
7. Inject the STZ or control solution into the peritoneal cavity of the fish by inserting the needle past the bevel into the posterior aspect of the ventral peritoneum.
8. 0.35 mg/g (350 mg/kg) of STZ should be delivered to each fish and the volume of the 0.3% solution required can be calculated in the following manner.
   1. Multiply mass of fish (g) by 0.35 to yield the amount of STZ in mg required.
   2. divide the product generated above by 3 to yield the volume of the 0.3% solution required for injection in μl.
      Sample: For a 0.5 g fish : a) 0.5 x 0.35 = 0.175 b) .0175/3= 0.058 ml = 58 μl.
      The same volume without the STZ would be injected for a control fish. A sheet for volumes to inject per fish mass should be generated and used for quick reference.
9. Following injection place the fish in the recovery water tank and monitor them for normal swimming activity. Once this has been achieved the fish are transferred to a normal living tank that is maintained at the reduced temperature range of 22 °C - 24 °C. This reduced temperature is critical for efficient induction of hyperglycemia (diabetes mellitus, DM).
10. Although hyperglycemia is detected within 24 hr of the first injection, in order to induce a prolonged state of very high hyperglycemia the zebrafish require a frequent injection induction phase followed by weekly maintenance injections as shown below.

Week 1: 3 injections (Day 1, 3, 5), Week 2: 1 injection (Day 12), Week 3: 1 injection (Day 19),
Week 4: (Day 21) Perform assay of interest.

At this point the zebrafish are considered to have been in a prolonged state of hyperglycemia and exhibit the diabetic complications of retinopathy, nephropathy and also impaired fin regeneration. These are referred to as DM fish. Additionally if desired the fish can be maintained in the hyperglycemic state with weekly maintenance injections. Approximately 5% death during this process should be expected.

2. Blood Collection and Fasting Blood Glucose Level (FBGL) Determination

1. Each group of DM and control fish must include enough fish to accurately determine the average FBGL of the group as this assay requires these fish to be sacrificed.
2. Prepare a labeled PCR tube for each blood sample containing 5 μl of sterile normal saline.
3. For blood collection, anesthetize fish as above, remove all water, place the fish on a microscope slide and using a scalpel, remove the head of the fish at the base of the operculum.
4. Collect the blood (up to 2 μl) that is released from the fish onto the slide and quickly add it to the 5 μl of sterile normal saline pipetting up and down to make sure that the blood does not clog. Immediately place the sample on ice.
5. Determine the blood sample volume by measuring the total volume of the liquid (saline + blood) and then subtracting out the 5 μl of saline.
6. Transfer 5 μl of the diluted blood from each PCR tube into a 1.5 ml microfuge tube and determine the blood glucose concentration using the QuantiChrome Glucose Assay Kit. This is performed by following the manufacturer's protocol without exception. Expected results: normal/control fish 60 mg/dl and DM fish 310 mg/dl.

3. Caudal Fin Regeneration Studies

1. Anesthetize fish as described in 1.0-1.4.
2. Place fish on a Petri dish lid and amputate the caudal fin in a straight line using a sterile size 10 scalpel proximal to the first lepidotrichia branching point while viewing the fin through a dissecting microscope.
3. Allow the fish to recover as in 1.10 but place the fish at 33 °C for the regenerative growth phase of the assay. This is an established temperature for accelerated fin regeneration analysis.
4. The fins can be imaged at any time post amputation however we routinely examine the regenerative growth at 24, 28 and 72 hr following trans-section.
5. Anesthetize fish as before (see 1.0-1.4), place the fish under a dissecting microscope equipped with a camera (we use a Nikon SMZ-1500 equipped with a Q-imaging Camera) and collect all fin images at 1X magnification with NIS elements software. The fin should be spread for imaging so that it is fully extended without stretching or damaging the tissue and should be free of any water droplets. For consistency always place the dorsal side to the right.
6. Print images and measure the regenerative growth area using Image J software and the use of a drawing pad. Trace around the whole area of new growth and determine the area. Each measurement should be made five times and averaged to ensure accuracy of tracing. It is important that the images used do not have shadows or water droplets as these contribute to errors in measuring the outgrowth area.
7. Measure the length of the amputation site along the dorsal-ventral axis and divide the area determined in 3.6 by this measurement. This allows fish of different sizes to be compared directly.

4. Generation of Metabolic Memory (MM) Zebrafish

1. Initiate a group of DM fish and their appropriate controls as described in section 1.
2. At 21 days determine the FBGL for a subset of the group and divide the DM fish into 2 subgroups. Continue weekly STZ injections for one of the groups as DM controls for the duration of the experiment. Cease STZ injection for the second group and incubate the fish at normal temperature. Within 14 days these zebrafish will restore normal blood insulin and glucose control through pancreas regeneration. These fish are now called MM (metabolic memory fish).
3. At day 30 post drug removal amputate the caudal fins of control, DM and MM fish via the method described in 3.2.
4. Return fish to normal water conditions for fin regeneration for 30 days. This allows the growth of what we term, metabolic memory tissue.
5. At day 60 perform a second amputation (as described in 3.2) within the tissue that was regenerated in the period from 30-60 days and perform the caudal fin regeneration assay (3.1-3.7) Figure 1.
6. Isolate tissue and perform assay of interest. See Table 1 for a protocol summary.

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.