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.
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.
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.
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
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
3. Caudal Fin Regeneration Studies
4. Generation of Metabolic Memory (MM) Zebrafish
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 deficit can be quantified and as shown in Figure 2C DM and MM zebrafish exhibit a deficit of approximately 40% at 72 hr when compared to control fish. Although the data included in Figure 2C ends at 90 days this same impairment has been observed as far out as 150 days.
DAY | PROCEDURE |
1 | DM = STZ Injection (350 mg/dl), Control = saline injection |
3 | DM = STZ Injection (350 mg/dl), Control = saline injection |
5 | DM = STZ Injection (350 mg/dl), Control = saline injection |
12 | DM = STZ Injection (350 mg/dl), Control = saline injection |
19 | DM = STZ Injection (350 mg/dl), Control = saline injection |
21 | Either perform assay of interest for DM fish or proceed to make MM groups by removal of STZ pressure. |
51 | Amputate Fins 30 days following the last STZ injection of controls, DM and STZ groups in order to generate MM tissue. |
81 | Re-amputate fins of all groups in the tissue that was grown between day 51 and 81 to perform regeneration study. Alternatively treat fish/tissue with the assay of interest. |
Table 1. Protocol Summary.
Figure 1. Cartoon depicting amputation sites for metabolic memory experiments. The blue color represents tissue that was exposed to the previous hyperglycemic state. The green color indicates the tissue that was grown from 30-60 days post hyperglycemia. The black dotted line indicates the first amputation site performed at day 30 and the red indicates a potential amputation site that would occur at 60 days.
Figure 2. Caudal Fin Regeneration is Reduced in Diabetic (DM) and Metabolic Memory (MM) Zebrafish. A. A representative caudal fin image from a control injected fish showing a normal amount of regenerative growth 72 hr post amputation. The white dotted line represents the amputation plane and the pink solid line demarks the regenerative outgrowth. The amount of regeneration is determined by tracing the area contained within the pink and white lines divided by the length of the white line to normalize fin size differences. B. A representative caudal fin image from either DM or MM illustrating a reduced amount of regenerative growth 72 hr post amputation. The lines and area measurements are the same as for panel A. C. Graphic presentation of the relative regeneration rate of DM and MM zebrafish as compared to controls. The relative percentage (to controls set at 100%) of DM and MM zebrafish regenerative outgrowth at 72 hr is shown. The time in days depicted is relative to when STZ administration was halted for the metabolic memory group. These data where generate by several researchers and incorporate over 1,000 fish per group. Figure 2A and Figure 2B were adapted with permission from Olsen et al 43.
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 metabolic memory (restored glucose control) zebrafish. We further described the methodology that can be employed to separate epigenetic contributions from the potentially complicating components of the previous diabetic state. We wish to emphasize that fish can be examined at any point with any assay of interest to the particular researcher and therefore the downstream applications for future discovery are endless.
There are several steps in the protocol that warrant some further discussion and emphasis. In our experience the 0.3% STZ in solution deteriorates and loses its effectiveness after approximately 20 minutes. Therefore we suggest that a timer be used and a fresh solution be made at 20 minute intervals. During our initial attempts to generate diabetic zebrafish we were only using one injection during the first week and were successful with approximately 40% of the fish. As such, there is not a strict requirement for three injections, however, when three are performed the rate of success exceeds 95%. Secondly, when injecting STZ into the zebrafish it is important that the needle is inserted such that the bevel of the needle is fully inside the fish to allow for proper dispensing of the solution, however, caution must be taken that it does penetrate too far to prevent internal damage. Once STZ or control solution is administered the fish are incubated at a reduced temperature (22 °C – 24 °C). We cannot over emphasize the importance of the reduced temperature as without it the zebrafish will regenerate their beta cells (STZ injected) and hyperglycemia cannot be efficiently induced. Lastly, in our hands zebrafish blood clots very quickly which prevents the necessary capillary action required for efficient glucometer use and therefore we do not advocate their use. We have found that the QuantiChrome assay described is not only the most reliable but the easiest to perform and to teach laboratory personnel. Collectively the techniques described in the protocol are not difficult and if the proper precautions are taken with the steps described above the generation of diabetic zebrafish is all but assured.
There are very few limitations within the procedure described in this manuscript, however all drug induced models of disease always have the criticism of off target effects leveled at them. We refer the reader to our initial manuscript detailing this model as we provided 5 independent lines of evidence (including direct STZ injection into fins) documenting that there are no off target effects of STZ 43. Another potential limitation does not come from the procedure itself but from the fact that the reagents for zebrafish research are not yet at the level of other model organisms such as mice. Fortunately, as the zebrafish is being increasingly used as a model organism for human disease this deficiency is being quickly remedied.
In summary, as detailed in the introduction, the zebrafish model of diabetes mellitus described here has several advantages over other model organisms. Importantly, it allows for the examination of the purely epigenetic components that support the metabolic memory phenomenon. As it is projected that 400 Million people will be afflicted with this disorder we feel that the contribution from studies utilizing this model could have significant impact on human health.
The authors have nothing to disclose.
This work was supported by a research grant from the Iacocca Family Foundation, Rosalind Franklin University start-up funds, and National Institutes of Health Grant DK092721 (to R.V.I.). The authors wish to thank Nikki Intine for aid in manuscript preparation.
Name of the reagent | Company | Catalogue number | Comments (optional) |
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. |