In this study, we developed a zebrafish model of DIO. DIO zebrafish exhibited increased BMI, hypertriglyceridemia and hepatosteatosis. Comparative transcriptome analysis using visceral AT revealed that the DIO zebrafish and obese mammals share common pathophysiological pathways. These findings suggest that the DIO zebrafish model can be used to identify putative pharmacological targets and to test novel drugs for the treatment of human obesity.
Zebrafish can be used as a useful animal model of DIO
The regulation of feeding behavior occurs via close interactions between peripheral regions and the brain [1–4, 35–38]. Peripheral endocrine and metabolic factors convey information regarding nutritional status to the brain. The peripheral signals include satiety signals, such as PYY , GLP-1  and ghrelin , which originate from the gastrointestinal tract, as well as adiposity signals, such as adiponectin  and insulin , the blood levels of which are proportional to body nutrient stores . The brain then processes this peripheral information and induces neuropeptide signaling (for example, via NPY , α-MSH , AgRP  and orexin ), mainly from the hypothalamus, to stimulate or inhibit feeding. These peripheral and central factors controlling food intake are well conserved in zebrafish [20, 22, 39–45].
Probably because of the low satiating effect of fat consumption , high dietary fat intake is associated with an increased risk of obesity [1, 25]. Because of the relatively high fat content in Artemia, as compared with flake foods, we fed the zebrafish a diet consisting solely of Artemia to induce obesity. We have demonstrated that zebrafish overfed with Artemia showed significant increases in BMI and plasma TG levels and hepatosteatosis, consistent with obesity observed in humans and rodent models of DIO [1, 6, 7]. Of note, there seemed to be no decrease in physical activity in the zebrafish overfed Artemia (data not shown), indicating that the development of obesity is most likely to be a result of the increased intake of a high-fat diet.
There are several advantages to the DIO zebrafish model. First, zebrafish (AB line) respond well to the Artemia diet because almost all of the zebrafish overfed Artemia developed obesity. The C57BL/6J (B6) line has been widely used for DIO in mice because these mice are very susceptible to obesity when fed a high-fat diet [6–9]. However, there are variations in adiposity among individual B6 mice [46, 47]. Similarly, when outbred Sprague-Dawley rats are fed a high-fat diet, about half become obese, while the other half are resistant to DIO [6–10]. The relatively homogenous responses of zebrafish to overfeeding with Artemia suggest that these fish represent an excellent alternative model species for experimental research on DIO. Furthermore, the dietary protocol to induce obesity in zebrafish is simple and can be applied to other zebrafish lines. For example, we are currently applying the protocol to the Casper line, which have transparent abdomens, even in the adult stage . Using DIO Casper zebrafish, visceral AT can be visualized in a living animal under a fluorescent microscope by staining the AT with a fluorescent dye, such as Nile Red . This feature makes it possible to monitor the short- and long-term effects of a therapeutic intervention on the amount of visceral AT in live DIO zebrafish. Finally, zebrafish are small and easy to maintain in large stocks because of their high fecundity, thus making zebrafish amenable for medium-to-high throughput screening for early drug discovery .
One limitation of the zebrafish DIO model is the apparent absence of brown AT . The development of obesity in mammals not only depends on the balance between food intake and calorie utilization, but also on the balance between white AT and brown AT . Therefore, zebrafish DIO may not be suitable to identify signaling pathways related to brown AT.
Comparative transcriptomics of visceral AT revealed common pathophysiological pathways in zebrafish and mammalian obesity
Genome-wide expression assays using DNA microarrays allow rapid screening and quantification of differences in large groups of functionally related genes and are thus well-suited to studies of pathways dysregulated in obesity . We compared the visceral AT expression profiles of zebrafish, rat, mouse and human obesity. The comparative transcriptome analysis revealed that several genes involved in blood coagulation, platelet activation, fatty acid metabolism, cholesterol efflux, and triglyceride metabolism were dysregulated in both zebrafish and mammalian obesity. IL-6, IL-1β and APOH were identified as regulatory factors involved in blood coagulation and platelet activation in zebrafish and mammalian obesity. Similarly, SREBP1, PPARα/γ, NR3H1 and LEP were identified as common regulatory factors for fatty acid metabolism, cholesterol efflux and triglyceride metabolism in zebrafish and mammalian obesity.
Visceral AT dysfunction can play a causal role in the prothrombotic state observed in obesity by affecting hemostasis, coagulation and fibrinolysis [53–55]. It has also been shown that IL-6 and IL-1β, secreted from visceral AT, induce the biosynthesis of fibrinogen from visceral AT and liver. Fibrinogen is a substrate of F2 (thrombin) in the final step of the coagulation cascade, and its presence is essential for platelet aggregation . Of interest, plasma fibrinogen levels were found to be significantly higher in obese subjects than in age- and sex-matched non-obese individuals . Significant correlations have been reported between fibrinogen and BMI and the waist-to-hip ratio . It has also been reported that substantial weight loss reduces fibrinogen levels more effectively than modest weight reduction . Consistent with these reports, OF and CR significantly induced and reduced, respectively, the expression of fibrinogen in zebrafish (Figures 3A and 5A, Additional files 1 and 2: Supplemental Table S1 and S2). APOH binds to activated protein C (APC), an anticoagulant enzyme that is activated by activation of the protein C zymogen by the thrombin-thrombomodulin complex on the surface of endothelial cells, platelets and monocytes . However, the effect of APOH binding to APC is inconclusive and the functional role of APOH in obesity remains to be elucidated.
Dyslipidemia is commonly seen in obesity [57, 58] and is characterized by an increased flux of free fatty acids, elevated TG levels, low high-density lipoprotein cholesterol levels and increased low-density lipoprotein levels . Fatty acid metabolism, cholesterol efflux and triglyceride metabolism are closely related to these pathways. This study revealed that SREBP1, PPARα/γ, NR3H1 and LEP are key regulatory factors in these pathways and are expressed in zebrafish and mammalian obesity. PPARs mediate adaptive metabolic responses to increased systemic lipid availability and are activated by endogenous or dietary lipids . PPARα promotes lipid clearance by increasing tissue fat oxidization [59, 60] while PPARγ promotes lipid storage in white AT, as well as preadipocyte differentiation to mature adipocytes . SREBP1 is an important transcription factor that regulates the transcription of many lipid genes and participates in adipocyte differentiation by stimulating PPARγ [61, 62]. NR3H1, also known as liver X receptor A (LXRA), has been shown to regulate lipid and carbohydrate homeostasis . LEP, an adiposity hormone produced by white AT, reflects total fat mass . Although we did not detect a statistically significant difference between OF8W and OF1W in terms of the mRNA expression level of lep measured by qPCR (data not shown), the expression of apoa1, a transcriptional target gene of leptin , was significantly induced and reduced by OF and CR, respectively (Figures 4A and 5B) [Additional files 1, 2 and 7: Supplemental Tables S1 and S2, and Figure S1], suggesting that leptin protein levels were likely to be increased in zebrafish DIO. It is noteworthy that the functional importance of SREBP1, PPARα/γ, NR3H1 and LEP in obesity has been shown in many genetic studies [2, 11, 63, 66].