Zebrafish as a Model for Metabolic Diseases

Biomedicines. 2024 Mar 20; 12 (3): 693.

Authors: Angom RS, Nakka NMR.

Zebrafish represent an intriguing animal model for comprehending the pathophysiology of metabolic disorders in humans and pinpointing prospective treatment avenues. The metabolic features of zebrafish are comparable to those of humans, so they can be used to supplement information derived from other model organisms, such as rats. This possibility has been amply demonstrated by recent research that found that medications licensed for treating human metabolic disorders also worked well in a zebrafish model. Numerous potential human disease genes and loci linked to metabolic disease have been found thanks to genome-wide association studies (GWAS) and, more recently, whole-exome and whole-genome sequencing. How these genes work and how their malfunction impacts pathophysiology are the current problems in this regard. Zebrafish have been used to effectively examine variant function in vivo at a reasonably high throughput in various disease areas. In recent work, platelet production, which has implications for hematological disease, was discussed. These advantages of zebrafish can be used to make comparable advancements in the field of metabolism. In this section of our review, we focus on the use of zebrafish as an animal model for metabolic disease and their pros and cons in this application. We also examine the latest developments in modeling the associated disorders of metabolic syndrome—obesity, diabetes, fatty liver disease, and atherosclerosis—using zebrafish.

Table. 1 Zebrafish metabolic disease models.

Obesity

Studies on genetic gain and loss of function have shown that numerous genetic mechanisms influencing adiposity in zebrafish are like those in mammals. Nevertheless, deleting leptin signaling, one of the most researched signaling pathways that control body fat mass does not demonstrate a conserved function. When compared to healthy individuals, human patients with mutations impacting leptin signaling exhibit excessive eating tendencies, are morbidly obese and have lower fertility. Similar behaviors are observed in mouse models; after eating, insulin stimulates the release of leptin, which is strongly expressed in mammalian adipocytes. Zebrafish lacking the leptin receptor leper, on the other hand, exhibit normal growth, fat accumulation, eating patterns, and fertility. Zebrafish fat virtually never expresses the lep gene, but their liver experiences a rise in mRNA levels when they fast.

Zebrafish research has yielded genuinely innovative discoveries in the field of obesity. Recent research, for instance, has uncovered a mechanism of PLXND1, a gene linked in population genetics studies to variations in the distribution of fat between the hips and waists in humans and an elevated risk for type 2 diabetes mellitus (T2DM). While Plexin D1 deletion in mice results in embryonic death before the development of visceral fat, Plexin D1/zebrafish is accessible for investigation. Interestingly, visceral fat depots in the zebrafish mutant are hyperplastic, with a reduced capacity to store lipids. As visceral fat tissue could not grow under high-fat diet (HFD) conditions, surplus lipids had to be redistributed to subcutaneous depots. It was demonstrated that modifying the extracellular matrix surrounding the cell is the fundamental mechanism behind this process. This work offers an intriguing set of in vivo and ex vivo adipose tissue-imaging techniques that could be of great value for similar investigations in the future, in addition to revealing novel activities of plxnd1.

Though adipose tissue is absent in the early stages of embryonic and larval zebrafish development, other aspects of lipid metabolism can be studied; for instance, the zebrafish gastrointestinal system is anatomically and functionally similar to the mammalian system. One way to see lipid uptake and processing in enterocytes is to feed fluorescently tagged lipid moieties to zebrafish larvae. Using this technique, forward genetic search revealed a fat-free mutant, whose reduced processing of cholesterol and phospholipids was accompanied by unchanged intestinal morphology Later research revealed that this fat-free mutant is responsible for expressing Ang2, a conserved member of the Golgi-associated retrograde protein (GARP) complex, which controls autophagy, endosomal cholesterol trafficking, and lysosomal enzyme sorting.

Non-Alcoholic Fatty Liver Disease

Regardless of alcohol intake, nonalcoholic fatty liver disease (NAFLD) is the most common lipid storage disease of the liver and is mainly caused by high-fat diets and foods high in carbohydrates. The benefit of studying NAFLD in zebrafish is that whole-mount Oil Red O staining for neutral lipids allows for the visualization of steatosis in larvae. Zebrafish can be fed lipid-rich meals, such as an egg yolk emulsion or Artemia brine shrimp after feeding begins at 4 dpf. As an alternative, steatosis has been induced via fructose-based or ketogenic diets. Because the larval liver is large enough to be dissected easily, samples can be used for transcriptome, histological, and biochemical investigations. Further, several transgenic lines are available for observing hepatic cell types, such as hepatocytes, biliary cells, endothelial cells, and stellate cells. Adult zebrafish can be fed certain diets for an extended period, and the lipid content of their livers can be assessed using metabolomics or traditional enzymatic techniques.

Large-scale forward genetics screens once more yielded the first zebrafish models of NAFLD, indicating that significant insights into disease mechanisms can be attained by methodically searching for mutations that cause NAFLD and examining their involvement in this illness's course. An ENU mutagenesis screen revealed 19 novel hepatomegaly-characterized mutant lines, most of which showed neutral solid lipid staining. Numerous mutants have distinct histological pathological characteristics, varying from mild cases of micro- or macro-vesicular steatosis to severe indications of nonalcoholic steatosis, which include hepatocyte necrosis and ballooning. Cloning the genes impacted by these mutations will probably provide crucial information on how chronic liver disease develops.

Atherosclerosis

Zebrafish possess features that aid in the study of atherosclerosis, such as the ability to detect lipid deposits inside blood vessels in vivo. Zebrafish experience atherogenic events more frequently than humans because cholesteryl esters in fish are redirected from protective HDL cholesterol particles to "bad" LDL Single-chain monoclonal antibodies coupled with GFP can be used to visualize the oxidation of LDL by malondialdehyde, thereby exploiting zebrafish's advantages for in vivo imaging.

The human liver X receptor (encoded by LXRA and LXRB) has functionally conserved activities in zebrafish. In contrast to mice, zebrafish have a single lxr ortholog (encoded by lxra/nr1h3), making it possible to determine its physiological roles through comparatively simple gain- and loss-of-function tests. Zebrafish with the lxra// knockout allele remains alive. However, they exhibit high blood and liver cholesterol levels following prolonged adherence to a high-fat diet (HFD), like the cholesterol intolerance shown in Lxra// and Lxrb// double knockout mice. The authors also showed through overexpression experiments that increased Lxr function, particularly in enterocytes, could have the opposite impact, i.e., a significant slowdown in the accumulation of cholesterol in the blood and liver in response to HFD. From a mechanical perspective, it was discovered that Lxr triggers an acyl-CoA synthetase (encoded by acsl3a) that breaks down ingested lipids and directs them toward lipid droplets for storage rather than rapid release into the bloodstream. This finding has significant clinical implications because multiple studies suggest that postprandial elevations in cholesterol are the primary cause of atherosclerosis. Statins, which prevent the liver's de novo manufacture of cholesterol, do not, however, mainly successfully regulate the postprandial dynamics of cholesterol.

Diabetes

The Zebrafish has been explored as a suitable model for diabetic research. A new diet-induced obesity (DIO) zebrafish has been shown to display higher blood glucose levels after fasting than normally fed ones. This hyperglycemic zebrafish is a helpful model for T2DM, measuring glucose tolerance, insulin production, and glycemia responsiveness to human anti-diabetic medications. In addition, liver–pancreas RNA-seq research shows that T2DM zebrafish share human pathogenic pathways. When responding to damage and metabolic stresses, a zebrafish's beta cell mass is remarkably malleable. Zebrafish research has primarily focused on the dissection of the genetic cues that govern the formation, maturation, and flexibility of functioning beta cells. This work is beginning to yield significant insights into therapies for replacing and regenerating beta cells. In this context, several different strategies are being investigated, ranging from the in vitro differentiation of mature beta cells from induced pluripotent stem cells to the in vivo restoration of sufficient numbers of functional beta cells through progenitor pools, non-beta cells, or remaining beta cells. Because each progenitor and cell type within the islet can be seen and tracked over time in vivo using fluorescent reporter proteins until the formation of a mature pancreas throughout the embryonic, larval, juvenile, and adult stages, zebrafish are anatomically well suited for this purpose. Zebrafish have a single primary islet located in the pancreas's head in addition to numerous minor islets spread throughout this organ. Once again, forward genetic screens provided the first research indicating that zebrafish may be used to analyze the genetic network controlling endocrine pancreas development. A line with a mutation in the homeobox gene vhnf1, which prevents normal expression of pdx1, a transcription factor gene required for the induction of the insulin gene, was found in an insertional mutagenesis screen. Lesions in these genes are known to induce a subtype of monogenic diabetes called mature-onset diabetes of the young (MODY) type V for VHNF1 and type IV for PDX1.

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Disease Models

Reference

1. Angom RS, Nakka NMR. (2024). "Zebrafish as a Model for Cardiovascular and Metabolic Disease: The Future of Precision Medicine." Biomedicines. 12 (3): 693.

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