Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.
Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).
The mouse is currently the predominant model for obesity research. The short generation interval and genetic tools available in the mouse have been unmatched to date. However, the zebrafish also has a short generation interval (3 – 4 months) and surpasses even the mouse in ease of genetic manipulation 1,2. The zebrafish maintains nearly 90% of mammalian genes, while far exceeding the mouse in number of offspring and potential for use in genetic and drug screens 3.
To tap the potential of the zebrafish model for studies in obesity, assays must be developed to investigate factors that influence body weight regulation, including energy expenditure. As metabolites are processed through β-oxidation and the tricarboxylic acid cycle oxygen is consumed and NADH2 is produced. Thus, NADH2 is a direct indicator of the flux of metabolites through metabolic pathways (metabolite oxidation). In poikilotherms, H+ leak through the inner mitochondrial membrane, which uncouples NADH2 oxidation from ATP synthesis, is 4 – 5 fold lower than in homeotherms 4. Accordingly, in zebrafish NADH2 is very tightly linked to ATP production through oxidative phosphorylation. Herein, we describe an assay that measures NADH2 production in larval zebrafish as a proxy for energy expenditure5.
Oxygen consumption is the gold standard for measuring energy expenditure. Yet, to best take advantage of the high throughput potential of the zebrafish, assays of energy expenditure must be amenable to high-throughput. Oxygen consumption systems that depend on a closed chamber circulating system are limited in throughput by the number of chambers available6. Open air O2 consumption/CO2 production assays have also been applied in the zebrafish5,7. These open air 96-well plate based systems are amenable to high throughput. Unfortunately, gas exchange with the environment limits sensitivity of these assays. We recently published the application of an assay that monitors NADH2 using the redox indicator alamarBlue5. This assay overcomes the limitations in throughput and sensitivity common to analyses of oxygen consumption in the zebrafish.
The zebrafish is becoming an increasingly important model for studies of whole body energy homeostasis. In part, because zebrafish are amenable to use in forward genetic screens and drug screens. Moreover, targeted genetic manipulation, including knockdown and knock-in, can be quickly applied. We have previously shown that this assay can be combined with bath drug application and genetic knockdown or knockout to identify compounds and genes that alter metabolic rate5. Moreover, this assay is designed to exploit the high throughput advantages inherent to the zebrafish.
Note: This protocol follows the guidelines of the University of Arizona Office for the Responsible Conduct of Research and has been approved by the Institutional Animal Care and Use Committee
1. Zebrafish Breeding and Embryo Maintenance
2. Prepare Assay Solution
Ingredient | % of Total | Volume (µl/10 ml) |
60x E3 Embryo Medium | 1.667 | 166.7 |
Double deionized (ddi) H20 | 96.233 | 9623.3 |
Alamar Blue | 1 | 100 |
400 mM NaOH | 1 | 100 |
DMSO | 0.1 | 10 |
Table 1. Assay Medium
3. Perform Assay
4. Assess the Relative Change in Fluorescence Associated with Treatment
Assay solution does not increase fluorescence in the absence of embryonic fish (Figure 1). However, the assay is highly sensitive to small changes in metabolic rate within the well. A single fish within a well creates a signal significantly different from blank wells within 1 hr (P<0.0001). Figure 2 provides a visual representation of signals generated by embryonic fish after 24 hr of incubation. For the purpose of this picture clear sided wells were used. Yet, black sided wells are preferred for assay performance to prevent leaching of the fluorescent signal from nearby wells.
Because the NADH2 induced reduction of alamarBlue is non-reversible, the signal accumulates with time. This allows for small changes to be amplified. To show that differences accumulate and thus increase with time, we monitored the relative change in fluorescence induced by 1 to 5 fish at 1, 2, and 4 hr (Figure 3). At each number of fish/well the relative change in fluorescence significantly increased with time (P<0.001). More importantly, because signal accumulates with time, the magnitude of differences in fluorescence change that result from manipulating the number of fish/well is more robust at 4 hr than at 1 hr of incubation. Thus, by increasing the duration of exposure we can enhance the ability to detect differences associated with a given treatment.
Figure 1. Comparison of Noise to Signal. A detectable change in fluorescence is generated by 1 zebrafish in a 96 well plate at 1 hr. Wells that do not contain fish (blanks) generate little signal. Error bars represent SEM (n = 5 – 8). Please click here to view a larger version of this figure.
Figure 2. Visual Representation of Assay Plate at 24 hr. Representative picture of results obtained using this assay. Pink wells are indicative of fish with a high metabolic rate, purple wells a moderate metabolic rate, and blue wells a low metabolic rate. Please click here to view a larger version of this figure.
Figure 3. Signal Differences Amplify With Time. The difference in fluorescent signal generated by varying the number of fish/well increases with time. Because signal accumulates with time, the magnitude of fluorescence change diverges with time betweensamples that differ in rate of signal generation. Error bars represent SEM (n = 7).
Contamination with bacteria or fungi will greatly limit application of this assay. The methylene blue in the E3 embryo medium limits the possibility of fungal contamination. Throughout embryo rearing it is critical that care is taken to remove dead embryos twice each day. Additionally, it is essential to thoroughly rinse the embryos with sterile E3 embryo medium on the day of assay initiation. These 2 steps limit the potential for bacterial contamination of the embryos. The inclusion of blank wells described in step 3.2 allows for detection of any bacterial contamination. Bacterial contamination that yields a measurable signal is rare. However, if the blank wells do show a robust signal, the assay should be repeated with care taken to maintain embryo cleanliness. If the user expects that contamination is arising prior to embryo collection, the embryos may be bleached as previously described (p. 22) 10. Alternatively, if the experimenter suspects that contamination is occurring after collection the user may use E2 with penicillin and streptomycin to rear the embryos to 4 dpf (p. 23)10.
Although this assay is valuable for application in embryos, we have not tested for validity in adult zebrafish. We suspect that functionality of this assay would be limited in adult fish by poor tissue penetration of AlamarBlue. Moreover, in feeding fish the gut microbiota may produce a profound signal, not indicative of the fish metabolism. Thus, this assay is restricted to use in yolk feeding embryonic fish. The relative inactivity of embryonic fish provides a unique advantage, as this limits the variability that would be associated with activity induced energy expenditure 11. Thus this assay is a unique tool to screen the metabolic rate response to genetic or pharmacological manipulation in a whole animal system.
The zebrafish is rapidly becoming a leading model for the metabolic perturbations that accompany obesity. In fact, there are published models of both transgenic and diet-induced zebrafish obesity 12,13. Similar to mouse models, diet induced obesity in the zebrafish increases serum triglycerides and hepatic lipid accumulation12. Furthermore, overfed zebrafish have elevated fasting glucose, suggesting glucose intolerance 14. Using the assay described here we have shown that 24 hr of pretreatment with Metformin, an insulin sensitizer, increases the metabolic rate response to insulin treatment 5. Thus, the zebrafish may prove to be an ideal whole animal model to discover drugs that enhance insulin sensitivity (embryo) and to establish their effectiveness in alleviating obesity related insulin resistance (adult).
The development of genetic and diet induced obese zebrafish models that mimic the phenotypes observed in obese mammals creates the impetus for initiating zebrafish studies focused on metabolic disease. High throughput assays are essential for fully appreciating the value of the zebrafish model to studies of energy homeostasis. Innovative tools designed to assess phagic drive will allow for screens focused on energy intake, while high throughput lipid storage assays will lead to studies on whole body lipid homeostasis 15-17. The assay we describe here provides a high throughput tool to assess the role of genes, compounds or their interactions on whole body energy expenditure.
The authors have nothing to disclose.
The authors would like to thank Drs. Roger Cone and Chao Zhang for their contributions to validating the application of this assay as previously published. This work was supported by NIH 1F32DK082167-01 (BJR).
AlamarBlue | Fisher Scientific | 10-230-103 | Manufactured by Thermo Scientific |
Fine Tip DisposableTransfer Pipette | Fisher Scientific | 13-711-26 | Manufactured by Thermo Scientific |
Fisherbrand Graduated Disposable Transfer Pipette | Fisher Scientific | 13-771-9AM | Manufactured by Thermo Scientific |
Black sided clear bottom 96-well plates | Fisher Scientific | 50-320-785 | Manufactured by E&K Scientific Products Inc. |