# COMPARATIVE STUDIES OF ENERGY HOMEOSTASIS IN VERTEBRATES

EDITED BY : Maximilian Michel PUBLISHED IN : Frontiers in Endocrinology and Frontiers in Neuroscience

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# COMPARATIVE STUDIES OF ENERGY HOMEOSTASIS IN VERTEBRATES

Topic Editor: Maximilian Michel, University of Cologne, Germany

Cover image: Tom Mc Nemar/Shutterstock.com

A brief glimpse into new insight driving the comparative biology of energy homeostasis in vertebrates with a focus on non-mammalian vertebrates. What are the key conserved mechanisms and what aspects of feeding behavior and energy allocation are different between species?

Citation: Michel, M., ed. (2018). Comparative Studies of Energy Homeostasis in Vertebrates. Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-560-7

# Table of Contents

*05 Editorial: Comparative Studies of Energy Homeostasis in Vertebrates* Maximilian Michel

### SECTION 1

### LIPID AND GLUCOSE HANDLING

Rebecca Wafer, Panna Tandon and James E. N. Minchin

*37 Modeling Pancreatic Endocrine Cell Adaptation and Diabetes in the Zebrafish*

Lisette A. Maddison and Wenbiao Chen

### SECTION 2

### CENTRAL REGULATION AND ENERGY EXPENDITURE

*43 Nutrient Sensing Systems in Fish: Impact on Food Intake Regulation and Energy Homeostasis*

Marta Conde-Sieira and José L. Soengas

Caroline E. Geisler, Kyle P. Kentch and Benjamin J. Renquist

*108 Comparative Physiology of Energy Metabolism: Fishing for Endocrine Signals in the Early Vertebrate Pool* Iris van de Pol, Gert Flik and Marnix Gorissen

### SECTION 3

### A FOCUS ON LEPTIN BIOLOGY

*126 Assessing the Functional Role of Leptin in Energy Homeostasis and the Stress Response in Vertebrates*

Courtney A. Deck, Jamie L. Honeycutt, Eugene Cheung, Hannah M. Reynolds and Russell J. Borski

### SECTION 4

WIDER PERSPECTIVES

*170 Energy Homeostasis in Monotremes* Stewart C. Nicol

# Editorial: Comparative Studies of Energy Homeostasis in Vertebrates

#### *Maximilian Michel\**

*Institute of Zoology, University of Cologne, Cologne, Germany*

Keywords: energy homeostasis, melanocortins, leptin, food intake regulation, energy expenditure

### **Editorial on the Research Topic**

#### **Comparative Studies of Energy Homeostasis in Vertebrates**

Energy homeostasis of an organism is the sum of processes integrating energy intake with resource allocation. Its central control mechanisms are essential to an animal's life history and govern daily activity, such as searching for food, reproduction, etc. The first major inroads into our understanding of the genetic basis of energy homeostasis were made by the fortuitous finding of five monogenic obesity strains in mouse breeding experiments (1) *obese*, (2) *diabetes*, (3) *agouti*, (4) *fat*, and (5) *tubby* (1). The genes underlying these strains were eventually cloned, laying the groundwork for the analysis of the neural circuitry which underlies energy homeostasis in mice [for recent reviews, please see Ref. (2–4)]. This research quickly led to the discovery of the core genes involved in energy homeostasis in obese human patients such as the leptin system (5, 6) which is mutated in the *obese* and *diabetes* mouse strains or the melanocortin 4 receptor system (7, 8) which is impacted in the *agouti* mouse strain. By and by, the key genes involved in mammalian energy homeostasis were cloned and found to be conserved not only across mammals but also across all vertebrates. However, while the genes are conserved, the anatomical and functional data on these genes varies between species from comparable to variable. In contrast, research into the biochemical basis of energy acquisition and utilization showed clearly that large swaths of the underlying building blocks are highly conserved (9). It follows that the basic building blocks which play a role in energy homeostasis are largely conserved, for example, lipid handling by adipose tissue or glucose handling by the liver. In contrast, the central regulation of these processes differs between species. An argument can be made that these differences in regulation are likely dependent on the ecological niche of an individual species [see for example, Ronnestad et al. in this issue]. Understanding what exactly constitutes a conserved building block in energy homeostasis and how these are centrally regulated to allow an animal to exist in a given ecological niche will allow us to (1) gain insight into the evolution of biological diversity; (2) modulate growth behavior in farm animals (domestication as an ecological niche); and (3) ameliorate abnormal homeostasis patterns in humans (from metabolic syndrome on one extreme to anorexia and cancer cachexia on the other). This research topic comprises reviews and a research article focused on recent insight. Of note—this topic is complemented by a concurrent research topic on the "Neuroendocrine Control of Feeding Behavior" (10), which has a strong emphasis on mammalian systems.

The first set of contributions covers lipid and glucose handling in zebrafish. The first review by Steven Farber's group (Quinlivan and Farber) introduces the mechanisms governing embryonic and larval lipid dynamics in zebrafish around the time of the transition from egg yolk feeding to prey feeding. The authors highlight that the gut is colonized by microbiota following the animals first feeding. They show that during this time, gut microbiota has an immense impact on host absorptive processes. Finally, the authors point out the benefits of the zebrafish model system to carefully study the dynamics of lipid absorption and processing (Quinlivan and Farber). Amnon Schlegel (Schlegel) focuses the discussion on lipoprotein biology. He argues for the use of zebrafish as a model system,

#### *Edited by:*

*Cunming Duan, University of Michigan, United States*

#### *Reviewed by:*

*Chunyang Zhang, Harvard Medical School, United States*

#### *\*Correspondence:*

*Maximilian Michel<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Experimental Endocrinology, a section of the journal Frontiers in Endocrinology*

*Received: 16 March 2018 Accepted: 17 May 2018 Published: 05 June 2018*

#### *Citation:*

*Michel M (2018) Editorial: Comparative Studies of Energy Homeostasis in Vertebrates. Front. Endocrinol. 9:291. doi: 10.3389/fendo.2018.00291*

since cholesteryl ester transfer proteins, which predispose people as well as zebrafish to atherogenic lipoprotein profiles, are not conserved in rodents. This allows for the study of plaque formation in order to identify dyslipidemia and atherosclerosis modifiers—both chemical and genetic (Schlegel). Staying on the topic of adipose biology, James Minchin's group (Wafer et al.) reviews adipogenesis, in particular its master regulator peroxisome proliferator-activated receptor gamma (PPARG) in fish compared to mammals. Not only is this gene highly conserved, but surprisingly, the authors find that only a single ortholog is present across fish species which suggests a critical role in adipogenesis. Astonishingly, while target genes of PPARG seem to be conserved, ligands are more diverse. This suggests that specifically control of adipose tissue evolved but not the role it plays within the organism (Wafer et al.). Veering away from the control of lipid levels, the next contribution focuses on the control of systemic glucose levels by the pancreatic endocrine cells (Maddison and Chen). The authors showcase the utility of the zebrafish as a model system for beta cell proliferation, differentiation, and regeneration (Maddison and Chen). Taken together, these four contributions argue not only for the utility of the zebrafish as a model system but also that the basic building blocks underlying energy homeostasis are fundamentally conserved across vertebrates.

Conde-Sieira and Soengas expand this in their review on peripheral nutrient sensing systems. Importantly, the authors show that the basic glucosensory and fatty acid sensory mechanisms are conserved from different fish species to mammals. However, they see species-specific differences by how central regulation of energy homeostasis is achieved (Conde-Sieira and Soengas). The topic of central mechanisms is further developed in a paper in the concurrent research topic written by Delgado et al., where the authors specifically look at the hypothalamic integration of signals from glucose and fatty acid sensing systems as well as integration of circadian information and the stress axis to achieve food intake regulation (Delgado et al.). Boswell and Dunn reviewed the central melanocortin system in birds and present data for the conservation of the key aspects of central melanocortin action. Interestingly, the authors point to a little understood connection between the melanocortin system, growth, and food intake in birds (Boswell and Dunn), This effect, also seen in platyfish (11), mice and humans and may be mediated *via* the somatostatin axis (12). Hélène Volkoff in her contribution summarizes what we know to date on the impact of different endocrine systems in fish. While most mammalian peptides and hormones either have an effect on fish metabolic state (when injected) and/or are regulated by metabolic state, the phenotype strongly varies between species. This again suggests that the building blocks supporting metabolic state are intact and conserved but the control mechanisms vary between species. The author points out that this is possibly not all too surprising given the widespread adaptations necessary for more than 30,000 species of fish to find ecological niches (Volkoff). This aspect is further developed by Ronnestad et al., where the authors bring up examples of central control differences in fish with vastly different life history and ecological niches. Specifically, the authors review metabolic control in long-term fasting species (the arctic charr, mouthbrooders, and fasting in aquaculture), throughout life transitions (first feeding to juvenile), in voracious feeders, and in cave-living fish (Ronnestad et al.). Ben Renquist's group introduces metabolic rate in fish, an essential underlying principle in energy homeostasis. The authors concentrate on variations in proton leak, calcium cycling, and membrane potential and relate this to differences in habitat and ecological niche of the investigated species. They point out that small changes in genes involved in ion gradients and their regulation can correlate with critical biological differences and advantages to the species—allowing the organism to adapt to particularly extreme environmental conditions by changes in metabolic rate (Geisler et al.). The subsequent contribution by Marnix Gorissen (van de Pol et al.) further develops the concept of life history, focusing on what is required for an organism to live and survive in an aquatic niche. With a keen eye on the developments in the mammalian literature, the authors look at insulin and leptin biology in fish, from appetite regulation *via* sexual maturation, salt water adaptation, and glucose/adipose homeostasis to stress (van de Pol et al.).

The next set of reviews focus on mechanisms specifically controlled by leptin signaling. Russell Borski's group (Deck et al.) starts with an overview of leptin structure and points out that leptin has an anorexigenic effect throughout vertebrates. However, this overall effect is mediated by recruiting different building blocks in different species—mobilization of lipid or glucose stores. The authors then look at the mobilization of energy after osmotic stress, hypoxia, disease, and fasting in fish (Deck et al.). Richard Londraville's contribution (Londraville et al.) introduces the recent discovery of leptin in birds, where evidence suggests that leptin may not have an endocrine but rather may have an autocrine/paracrine function. The authors embark on an up to-date analysis of leptin receptor and endospanin (an overlapping transcript with modulatory function) evolution and structure, probing databanks based on recent structural insight. This insight can be used to inform future research into leptin function (Londraville et al.). The last contribution in this leptin-specific set of reviews is an original research article from the Denver lab which provides evidence on a scarcely studied aspect of leptin biology: leptin's role during development (Bender et al.). In *xenopus*, the authors show that leptin levels (secreted by adipose tissue) rise just prior to metamorphosis and stay elevated throughout metamorphosis. They find evidence that leptin mediates cell proliferation and conduct a microarray study which provides a basis for further investigation of leptin's role and regulation of neurogenesis (Bender et al.). These four articles provide a good overview on what is known about nonmammalian leptin biology and highlight the complex nature of this hormone more than 50 years after the identification of the first leptin phenotypes (13).

The last contribution forms a bridge between nonmammalian energy homeostasis research with the mammalian realm. Here, Stewart Nicol discusses energy homeostasis in monotremes (Nicol). Similar to evidence from birds and fish, leptin does not appear to play a role in adipostasis in monotremes. The author further looks at special life history adaptations, such as seasonality, hibernation, and torpor (Nicol). This is of particular note in relation to the contribution of Ronnestad et al. who discuss the role of seasonality in the arctic charr. An organism's life history needs to be taken into account when translating insight gained from one organism (for example, a fish or a mouse) to another (such as humans). It is imperative to keep Krogh's principle of comparative biology in mind (for each problem there will be an animal where it can be most conveniently studied) and the harvest this has brought to our understanding of biology. While historical examples range from action potentials in squid *via* learning in mollusks or the shark rectal gland; relevant examples in the context here could be the study of: (1) metabolic control in voracious feeders and seasonal animals; (2) the thermogenic role of muscle in birds lacking brown adipose; (3) the role of the melanocortin system in body growth in platyfish size morphs and birds; (4) metabolic control of migrating animals (eels, salmon,

### REFERENCES

birds). These contributions clearly show the variety of mechanisms which a single regulatory hormone can control. However, these reviews also point out that (a) a hormone can have one general role (such as the anorexigenic effect of leptin) throughout all vertebrates, while recruiting different building blocks to achieve this effect and (b) that different building blocks (such as control of adipose through PPARG) can be centrally controlled by different ligands depending on the species and likely its ecological niche.

### AUTHOR CONTRIBUTIONS

The author listed has made a substantial, direct, and intellectual contribution to the work and approved it for publication.

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Michel. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Lipid Uptake, Metabolism, and Transport in the Larval Zebrafish

*Vanessa H. Quinlivan1,2 and Steven A. Farber1,2\**

*1Carnegie Institution for Science (CIS), Baltimore, MD, United States, 2 The Johns Hopkins University, Baltimore, MD, United States*

The developing zebrafish is a well-established model system for studies of energy metabolism, and is amenable to genetic, physiological, and biochemical approaches. For the first 5 days of life, nutrients are absorbed from its endogenous maternally deposited yolk. At 5 days post-fertilization, the yolk is exhausted and the larva has a functional digestive system including intestine, liver, gallbladder, pancreas, and intestinal microbiota. The transparency of the larval zebrafish, and the genetic and physiological similarity of its digestive system to that of mammals make it a promising system in which to address questions of energy homeostasis relevant to human health. For example, apolipoprotein expression and function is similar in zebrafish and mammals, and transgenic animals may be used to examine both the transport of lipid from yolk to body in the embryo, and the trafficking of dietary lipids in the larva. Additionally, despite the identification of many fatty acid and lipid transport proteins expressed by vertebrates, the cell biological processes that mediate the transport of dietary lipids from the intestinal lumen to the interior of enterocytes remain to be elucidated. Genetic tractability and amenability to live imaging and a range of biochemical methods make the larval zebrafish an ideal model in which to address open questions in the field of lipid transport, energy homeostasis, and nutrient metabolism.

Keywords: lipid metabolism, zebrafish, lipoproteins, comparative physiology, enterocytes

## INTRODUCTION

The developing digestive system of the embryonic and larval zebrafish is a well-established model system for the study of vertebrate gastrointestinal physiology and metabolism. Metabolic and regulatory pathways for gastrointestinal system development, intestinal and liver cell differentiation, digestion, and nutrient uptake and transport are highly conserved between zebrafish and humans (1–7). The functional regionalization of the intestine also appears to be conserved among vertebrates including zebrafish with respect to transcription factor expression in epithelial cells over the length of the intestine (8). Additionally, the transparency of the developing larva makes it ideal for live imaging experiments: The larval zebrafish has a functional and visible liver, pancreas, gallbladder, intestine, and intestinal microbiota by 5 days post-fertilization (dpf) when it begins to feed. The zebrafish is also suitable for large-scale and high-throughput experiments due to its small size and high fertility (a single pair can produce hundreds of embryos in a day). Finally, as the importance of the gut microbiome to studies of nutritional physiology is becoming increasingly clear; the larval zebrafish microbiota are well-characterized, and germ-free and gnotobiotic models are available (9).

#### *Edited by:*

*Maximilian Michel, University of Michigan, United States*

#### *Reviewed by:*

*Mingyu Li, Vanderbilt University, United States Satoshi Ogawa, Monash University Malaysia, Malaysia James Minchin, University of Edinburgh, United Kingdom*

#### *\*Correspondence:*

*Steven A. Farber<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Experimental Endocrinology, a section of the journal Frontiers in Endocrinology*

*Received: 14 July 2017 Accepted: 01 November 2017 Published: 20 November 2017*

#### *Citation:*

*Quinlivan VH and Farber SA (2017) Lipid Uptake, Metabolism, and Transport in the Larval Zebrafish. Front. Endocrinol. 8:319. doi: 10.3389/fendo.2017.00319*

The zebrafish zygote contains a large yolk cell which is absorbed over the first 5 days of life and supplies the developing embryo with nutrients. The yolk consists of a lipid and protein rich core with a cellular syncytium at its periphery, called the yolk syncytial layer (YSL). The YSL exports amino acids, hydrolyzes complex lipids to release fatty acids, and synthesizes lipoproteins, which export lipid to the developing embryo until it is able to feed independently (10). The intestine of the larval zebrafish is open at both ends and ready to absorb exogenous food at 5 dpf, though the non-enterocyte secretory cell populations do not differentiate until later larval stages (11). Once the intestinal tract is open, the gut microbiota are acquired from the media. At this time, colonization occurs essentially immediately and is maintained throughout life with the main source of variation in bacterial community composition being changes in diet (12).

Both the embryonic and larval zebrafish are valuable models of lipid uptake and trafficking, respectively, from the yolk cell and the diet. This review encompasses the roles of lipid remodeling, lipoproteins, intestinal lipid transport proteins, and the gut microbiota in lipid processing during zebrafish development.

### YOLK LIPID UPTAKE IN THE EMBRYONIC AND LARVAL ZEBRAFISH

### Lipoproteins Transport Yolk Lipids to the Body of the Developing Zebrafish Embryo

The majority of the mass of a zebrafish zygote consists of the yolk, a lipid-rich structure that is gradually depleted by transport of its contents to the embryo as it develops into a free-feeding larva. Yolk lipids are packaged into lipoproteins in the YSL before being exported to the body of the developing zebrafish. Lipoproteins are lipid-transporting structures consisting of a neutral lipid interior bounded by a phospholipid (PL) and cholesterol monolayer, carrying one or more apolipoproteins. Apolipoproteins mediate interactions among lipoproteins, cellular receptors, and lipid-processing enzymes. The zebrafish genome contains analogs of every major human apolipoprotein, but there are some differences in patterns of expression and function. Due to the teleost genome duplication, zebrafish have multiple paralogs of each apolipoprotein gene. Human lipid metabolism genes with corresponding zebrafish paralogs discussed in this review are summarized in **Table 1**. There are 11 apolipoprotein genes in the *apoB*, *apoA-IV*, *apoE*, and *apoA-I* families, and all are expressed in the YSL (13) (**Figure 1**). Whole-mount *in situ* hybridization reveals that expression of some apolipoprotein genes is localized to subregions of the YSL, suggesting a previously uncharacterized compartmentalization of this structure. For example, mRNA encoding *apoA-IV* appears to be specific to the yolk extension at earlier stages (though different paralogs in this family are concentrated here at different points in development), while members of the other apolipoprotein families are expressed more evenly throughout the YSL (13). The significance of these potential YSL subdomains has yet to be described, but it is possible that there is a relationship to the regionalization of the developing intestine.

TABLE 1 | Zebrafish express multiple paralogs of human lipid metabolism genes.

Although the expression of apolipoprotein genes in the developing embryo and larva has been thoroughly characterized, the lipoprotein profile at these stages is less well defined. Most work on fish lipoproteins has focused on adults, likely due to the

difficulty of obtaining adequate blood samples from larvae (19). Secretion of very low-density lipoprotein (VLDL) particles from the yolk has been demonstrated by electron microscopy (20, 21). The YSL also expresses *apoA-I* and *apoA-II*, which are found in HDL (high-density lipoprotein) particles and chylomicrons but not LDL (low-density lipoprotein) or VLDL (13, 14). ApoB, which is a component of chylomicrons, LDL, and VLDL, has a vital role in the export of yolk lipids. Microsomal triglyceride (TG) transfer protein (MTP) packages TGs into ApoB-containing lipoproteins. ApoB is degraded if it is not associated with lipid so, in the absence of MTP function, ApoB is not functional (22). In *mtp<sup>−</sup>/<sup>−</sup>* mutant zebrafish larvae, lipids are trapped in the yolk (characterized by retention of yolk volume, an increase in yolk opacity, and a reduction in neutral lipid in the body) and larvae do not survive beyond 5 days (23). Additionally, unlike their wild-type siblings, *mtp<sup>−</sup>/<sup>−</sup>* embryos retain fluorescent fatty acid injected into the yolk and do not export it or its fluorescent products to the circulation (18). The ability of *mtp−/−* larvae to grow and survive to 5 dpf suggests that some lipid must be transported out of the yolk in order for membranes to be synthesized, possibly through the synthesis of HDL-like particles that contain ApoA-I and do not require MTP for their assembly.

### Lipid Composition of the Embryo Changes over the Course of Yolk Absorption

According to a recently published developmental study of lipid composition performed by liquid chromatography-mass spectrometry (LC-MS), at the time of fertilization, embryo lipids are approximately 40% cholesterol, 35% PL, and 9% TG, with less abundant species, including mono- and di-glycerides, cholesterol esters (CE), ceramides, and lysophospholipids, making up the remainder (24). Over the first 5 days of life, a linear decrease in the molar amount of most lipid species is observed in the yolk with a corresponding increase in the embryonic/larval body. Some exceptions have been observed: TG in the body remains consistently low as it is depleted from the yolk, suggesting that yolk TGs are primarily broken down and either oxidized for energy or resynthesized into other lipid products. Interestingly, CE, the other "energy storage" lipid class, is exchanged evenly from the yolk to the body during this period of development with the total amount remaining the same (24). Cholesterol synthesis in animal cells is tightly controlled in response to the cholesterol content of membranes *via* regulation of HMG-CoA reductase expression, and esterification is a major mechanism by which excess cholesterol is neutralized (25). One possible reason that CE is not depleted during the lecithotrophic (yolk-feeding) period of development is that breaking down CE for fatty acid oxidation would result in an overabundance of cholesterol. Favoring glycerolipids as an early energy source, therefore, would be important for cholesterol homeostasis, while CE from the yolk could be repackaged into intracellular lipid droplets for later oxidation or storage in adipocytes. Free cholesterol in the yolk and the body decrease and increase, respectively, at the same apparent rate between 24 h and 5 days of development, but the cholesterol content of the body at 5 dpf is less than the initial amount in the yolk (24). It is likely that this portion of the cholesterol is directed to synthesis of steroid hormones and bile, though these compounds were not measured in this study.

Phospholipid dynamics in the developing embryo also appear to be more complex than simple yolk to body trafficking: while other PL classes seem to move gradually from the yolk to the body, phosphatidylcholine (PC) levels in the yolk increase over the first 24 h, then decrease over the next 4 days while remaining relatively constant in the body (24). Though the specific lipid composition of zebrafish embryonic lipoproteins has not been investigated, one possible explanation is that the initial increase in PC goes to building the outer monolayer on lipoproteins exported from the yolk. It is possible that when this lipoproteinassociated PC reaches the body, it is in excess and is either oxidized or remodeled.

Although Fraher and colleagues' published analysis of their LC-MS data set was limited to discussion of developmental changes in lipid classes, quantitation of all individual lipid species was published as a supplement to the manuscript. These data provide an opportunity to examine the changes in individual lipid species that occur during the first 5 days of zebrafish development. For example, the major PL classes are defined by head group (e.g., PCs, phosphatidylethanolamines, phosphatidylserines, etc.), but each of these classes comprises thousands of different molecules with different types of fatty acid "tails." Modern mass spectrometry technologies optimized for lipidomics can differentiate between individual lipid species at this level of resolution because they can precisely determine the mass to charge ratio (*m/z*) of each analyte in a mixture and because they employ a second step in which the molecules are fragmented and the subsequent *m/z* values of these fragments are also determined. Complex lipids such as PLs are identified using *m/z* values calculated from molecular formulas and expected fragmentation patterns, and are annotated in Fraher's supplemental data and other lipidomics data sets as "Head Group (FA 1/FA 2)." The most abundant PL in animal cell membranes, for example, is PC with the saturated 16-carbon fatty acid palmitate and the monounsaturated 18-carbon fatty acid oleate and is annotated as PC(16:0/18:1). When the specific fatty acid composition of a complex lipid cannot be determined, only the total fatty acid carbon chain length and number of unsaturated carbon–carbon bonds is given [e.g., PC(34:1)].

When trends in the amounts of individual lipids in Fraher's data set are examined, results suggest that changes in the PL profile are consistent with an increase in membrane PL in the larval body that is expected to occur with increasing growth. However, the trends in total amounts of PL present in the yolk and body are skewed by changes in individual PL species. Specifically, PC(18:2/20:4) is a major PL in the body at the start of development and shows a large decrease by 5 dpf. However, the expected major PC components of cell membranes including PC(34:1) and other PCs with total chain lengths in the low 30s increase over the course of larval development as expected. It is possible that longer-chain PLs predominate in lipoproteins but are a minor species in cell membranes, a model supported by a large increase in the amount of PC(18:2/20:4) in the yolk over the course of development (this species is the only PL in the yolk whose total molar amount increases over 1–5 days, though other PL species increase in the yolk in terms of percentage of total lipid). PLs containing the fatty acid arachidonic acid (20:4) are the precursor of eicosanoids, a class of signaling molecules with roles in regulating inflammation, vascular physiology, and stem cell activity (26, 27).

This finding suggests eicosanoids as an important area of interest in the ongoing characterization of yolk utilization in the zebrafish. Although the physiological implications of changes in individual lipids were not within the scope of this published work, the rich MS data set that was produced highlights the importance of examining behavior of individual lipids in studies of metabolism and transport.

### Complex Lipid Synthesis and Remodeling Occurs in the Embryonic and Larval Zebrafish Yolk

The embryonic and larval zebrafish yolk is metabolically active not just in lipid transport, but also in the synthesis and remodeling of complex lipids, as was demonstrated through the injection of radioactive and fluorescently labeled lipids into the larval yolk followed by thin layer chromatography (TLC) analysis of the products of these metabolic tracers (18). Fatty acids labeled with BODIPY-FL (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza*s*-indacene; a green fluorescent small molecule tag) or radioactive fatty acids injected into the yolk of 3 dpf larval zebrafish were both metabolized into complex lipids including PL, CE, and TG and transported throughout the developing body. Furthermore, injection of radioactive oleate showed that the yolk synthesizes complex lipids at the earliest stages of development, as radioactive TG and PL products were found in embryos injected as early as 0.75 hours post-fertilization (hpf). While the rate of incorporation of radioactive oleate into each PL class was consistent in embryos and larvae aged 0.75–3 dpf, larvae injected at 3 dpf were the only group to synthesize labeled CE, and there was a large increase in the amount of radioactive TG at later stages as well (18). When BODIPY-C12 was injected into the yolk of 24 hpf zebrafish embryos and yolk and body lipids were analyzed separately by TLC 1–6 h post injection (hpi), fluorescent complex lipids including TG, CE, and several unidentified species were produced in the yolk at early time points. Some fluorescent complex lipids were detected in the body at 6 hpi (24). (It is not known whether fluorescent PL was synthesized in this experiment as the assay only detected nonpolar lipids.) Injection of fluorescent PL into the yolk at 24 hpf resulted in fluorescent diglyceride and unidentified complex lipid species in the yolk, but no identified products in the body up to 6 hpi (24). Taken together, this and other evidence shows that the yolk is metabolically active throughout development and can both break down and synthesize complex lipids (18, 24, 28) (**Table 2**).

### DIETARY LIPID UPTAKE IN THE LARVAL ZEBRAFISH

### Digestion and Absorption of Dietary Complex Lipids

The larval zebrafish undergoes a switch from a lecithotrophic state to a free-feeding animal during its fifth day of development,

so by the time its yolk supply is depleted it must be able to digest and absorb nutrients from exogenous food sources. The ability to precisely control timing of the first meal is an advantage of this model as processing of dietary lipids by enterocytes can be observed without interference from lipids absorbed from previous meals. Additionally, because the larva retains its transparency for several weeks after it becomes free-feeding, it is possible to perform live imaging experiments with either single meals or ongoing defined diets in the same system.

Most dietary lipid consumed by animals enters the intestine not in the form of free fatty acids, but in complex lipids. Dietary TGs, PLs, and CE must be broken down by intestinal lipases in the lumen before the components of these molecules can cross the enterocyte membrane. As the fatty acids in these molecules are all linked by ester bonds, the intestinal lipases secreted by the exocrine pancreas are versatile and process a wide range of dietary lipids so that they can be absorbed (30). Following lipolysis, dietary lipid products form micelles in the intestinal lumen, which are emulsified in this aqueous environment by bile. The composition of bile varies between species and there are significant differences between teleost fish and humans, but its function is conserved (31, 32).

### Enteroendocrine Cells in the Intestine Regulate Digestion and Are Influenced by the Microbiota

As they do in mammals, enteroendocrine cells in zebrafish secrete a wide range of hormones including serotonin, which influences motility and appetite, and cholecystokinin (CCK), which stimulates gall bladder contraction and release of digestive enzymes from the pancreas (33, 34). The zebrafish genome contains two CCK paralogs; *ccka* is expressed in the digestive system of adults (no data are available for larvae at this time) and both *ccka* and *cckb* are expressed in the brain starting at 24 hpf (35, 36). In mammals, CCK promotes lipid digestion by stimulating the gall bladder to secrete bile, but does not increase lipase activity (37, 38). Similarly, larval zebrafish treated with a CCK receptor antagonist show reduced protease activity while intestinal phospholipase activity is unaffected (30). Enteroendocrine cells expressing serotonin begin to appear in the larval zebrafish intestine at 5 dpf. They may be detected by immunohistochemistry for serotonin, and are distinguished from the enteric neurons (which also express serotonin) by their shape and location in the epithelium. By 8 dpf, 10–18 enteroendocrine cells per larva may be observed in the distal intestine (posterior to the swim bladder) (11). A notable difference is that the larval zebrafish intestine does not have crypts, where enteroendocrine cells would be located in mammals.

The intestinal microbiota is required for normal enteroendocrine cell development (11). In germ-free larval zebrafish, 0–6 enteroendocrine cells were observed at 8 dpf (the total number of cells in the distal intestinal epithelium did not vary between germ-free and conventional groups). Larvae raised germ-free until 5 dpf, and then colonized with the conventional microbiota, developed normal numbers of enteroendocrine cells, suggesting that the yet-unidentified signal from the microbiota that promotes enteroendocrine cell development is not required before 5 dpf. Higher gut motility was observed in zebrafish larvae raised germ-free, suggesting a possible connection to digestive problems (including irritable bowel disease) observed in humans when the gut microbiota is disrupted (11). The lower number of serotonin-positive cells could explain this physiological effect as serotonin regulates gut motility in humans (33).

### Lipid Transport into Enterocytes

Dietary lipids are imported from the intestinal lumen across the apical enterocyte membrane by several different mechanisms depending on their class. After complex lipids (including both glycerolipids and CE) are digested to yield fatty acids, monoglycerides, and/or lysophospholipids, these products may cross membranes by a variety of transport processes conserved among zebrafish and mammals.

Cholesterol is taken up by enterocytes by a mechanism that requires the Niemann-Pick C1-Like 1 (NPC1L1) transport protein (39, 40). This membrane-associated protein is located at the brush border of enterocytes and is translocated to an intracellular compartment when cells are exposed to cholesterol; current models postulate a clathrin-dependent endocytic mechanism in which NPC1L1 is internalized along with a cholesterol cargo, which then moves through endosomes to the endoplasmic reticulum where it can be packaged into membranes or used to synthesize cholesterol ester (41, 42). NPC1L1 is encoded in the zebrafish genome, and several lines with point mutations in this gene have been created through the Sanger Institute Zebrafish Mutation Project (43). Ezetimibe, an inhibitor of NPC1L1-mediated cholesterol absorption that is used to treat hypercholesterolemia in humans, also blocks dietary cholesterol absorption in larval zebrafish (44–46). This creates an opportunity to use the zebrafish model to study physiological effects of modulating metabolic availability of a single component of a mixed-lipid diet. Regulation of NPC1L1 activity remains largely uncharacterized, although there is evidence from studies in humans given statins (inhibitors of cholesterol synthesis) that NPC1L1 expression levels increase in response to low intracellular cholesterol levels, suggesting that there may be an unidentified genetic mechanism that regulates NPC1L1 expression that could counteract the effects of statins by upregulating import of dietary cholesterol (47).

Fatty acid transfer proteins (FATPs) are a family of integral membrane proteins that facilitate transport of fatty acids into cells, including transport of dietary fatty acids into enterocytes. FATPs act in concert with acyl-coA synthetases (ACSs), which activate the newly imported fatty acids so that they are ready to form ester bonds and be incorporated into complex lipids (48, 49). There is evidence from mammalian and cell culture models that both the FATP and ACS families play roles in regulating preferential uptake of some dietary fatty acids over others, and in the partitioning of dietary fatty acids among complex lipids (48, 50). The zebrafish genome encodes 9 ACSL (ACSs specific to long-chain fatty acids, the type of fatty acid most abundant in animals including zebrafish) gene paralogs in six families. Expression of this class of genes is ubiquitous in adults, with proteins corresponding to seven of nine paralogs detectable by Western blot in most tissues including the gut (51). Expression of ACSL genes in the larva is more regionalized: in the *acsl1* family, *acsl1b* mRNA is detectable in the YSL and gut in early larval stages. The *acsl1a* paralog is not expressed in the YSL, and no expression data are available for early-gut development (17). Only *acsl1a* is expressed in the gut in adults (51). *Acsl4a* mRNA is present in both the YSL and the larval gut (52). Expression of *acsl4b* and *acsl5* is detectable in the YSL, but expression data are not available from larval stages after the gut has begun to develop (17) (**Figure 2**). Expression data are unavailable for the other *acsl* paralogs at any embryonic or larval stage, but what is known about expression of *acsl* genes in this model suggests potential division of function among paralogs similar to that suggested by regionalized apolipoprotein gene expression.

Compared with the ACSs, there is far less coverage of zebrafish FATPs in the current literature. As of now, no studies of FATP function in this model system have been published and only one genomic sequence is annotated as a FATP in the Ensembl database; FATP3/ACSVL3/SLC27A3 [with 7 paralogs, all annotated as members of solute carrier family 27 (*slc27*)]. The other six putative FATP paralogs are annotated as SLC27A1A and B {both with 65% protein sequence identity to human SLC27A1/FATP1/ ACSVL5 [a mitochondrial long-chain FATP (54)], using the NCBI protein BLAST tool}, SLC27A2A (47% protein sequence identity to human SLC27A2/FATP2/ACSVL1), SLC27A2B (55% protein sequence identity to human SLC27A2/FATP2/ACSVL1),

FIGURE 2 | Acyl-CoA synthetases are expressed in the larval zebrafish yolk syncytial layer (YSL) and intestine. (A) *In situ* hybridization reveals expression of *acsl1b*, *acsl4b*, and *acsl5* in the YSL at 24 hpf, and *acsl1b* in the developing gut at 2 dpf. Adapted from Ref. (17). (B) acsl4a is expressed in the gut and central nervous system (cns) of the 4 dpf larval zebrafish, and in the YSL at 24 hpf and earlier. Reprinted from Ref. (52), Figure S1E in Supplementary Material, under a CC-BY license. (C) *fatp4*/*acsvl4* is expressed in the gut (especially the anterior bulb) of the 5 dpf larval zebrafish. Adapted from Ref. (53).

SLC27A4 (70% protein sequence identity to human SLC27A4/ FATP4/ACSVL4), and SLC27A6 (57% protein sequence identity to human SLC27A6/FATP6/ACSVL2). The chromosomal locations of all of these putative *fatp* genes are conserved between the human and zebrafish genomes (syntenic analysis by ZFIN). Zebrafish SLC27A2A is expressed in the adult liver (55), and SLC27A4 is expressed in the anterior gut at 5 dpf (53) (**Figure 2**). No expression data are available for other adult organs, earlier larval stages, or the other putative FATPs at this time. However, as FATP4 is the primary fatty acid transporter on the apical brush border of human enterocytes, the similarity in expression between zebrafish and humans supports the larval zebrafish as a model in the investigation of FATP function in dietary fatty acid absorption (56).

The relative contributions of FATP4, other membraneassociated fatty acid-binding proteins, and passive diffusion to uptake of dietary fatty acids by enterocytes in larval zebrafish are not known. A recent review proposes a model in which the transmembrane receptor protein CD36, Caveolin 1 (Cav1), and FATP4 all act as fatty acid transporters at the enterocyte brush border, and in which passive diffusion of long-chain fatty acid salts across the enterocyte membrane plays a major role in adsorption (57). Larval zebrafish express CD36 and Cav1 in the intestine as well as FATP4 and, therefore, present an opportunity to apply live wholeanimal imaging tools toward investigations of the roles of each of these proteins in dietary fatty acid processing (58, 59). [Cav1 is located on the basolateral membrane of enterocytes in zebrafish and not at the brush border and, therefore, is unlikely to participate directly in uptake of fatty acids from the intestinal lumen (59).] In sum, despite tight conservation of FATPs and other fatty acid transporters, and their intestinal expression throughout the vertebrates, their physiological role in the intestine remains unclear.

### The Intestinal Microbiota Influences Dietary Lipid Uptake

The bacterial population of the intestine also plays an important role in dietary lipid uptake and metabolism. Fermentation by the gut microbiota allows host animals to utilize dietary plant polysaccharides that would otherwise be indigestible by converting them to metabolizable short-chain fatty acids and monosaccharides (60). Multiple studies over the last decade have shown effects of changes in composition of the gut microbiota on adiposity, serum lipids, and tissue lipids in mammals (61–66). However, determining mechanisms by which bacteria may cause global changes in vertebrate host physiology has been difficult as the composition of the gut microbiota also changes in response to changes in diet (67, 68). The larval zebrafish model was recently used to investigate aspects of the relationship between gut bacteria and lipids involving processes other than short-chain fatty acid synthesis: when larvae raised germ-free were given a high-fat meal labeled with fluorescent fatty acids, less fluorescence accumulated in the intestinal epithelium when compared with conventionally raised larvae, showing that at least some members of the microbiota are necessary to promote uptake of dietary lipids. Monoassociated larvae (larvae raised germ-free and then inoculated with a single bacterial species) colonized with the Firmicutes strain *Exiguobacterium* sp. were used to demonstrate that this bacterial strain alone was sufficient to promote intestinal fatty acid uptake to a point where fluorescence could be observed in extra-intestinal tissues. Furthermore, experiments using conditioned media from this strain and two others also revealed significant increases in enterocyte lipid droplet number over untreated germ-free larvae, suggesting that a factor secreted by these species is involved in promoting dietary lipid uptake (69). The exact mechanism for this host–microbe relationship is currently uncharacterized, as is the evolutionary advantage of promoting host lipid uptake for these microbial species.

### Lipid Processing in Enterocytes for Storage and Export

Fatty acids taken up by enterocytes are repackaged into complex lipids at the endoplasmic reticulum and are subsequently stored in enterocyte lipid droplets or directed to lipoprotein synthesis for export. Lipid droplets are composed primarily of TGs and CE in the interior, and bounded by a PL monolayer with associated proteins such as perilipins (70). Though the mechanisms by which lipid droplets grow and shrink are well characterized, the regulation of lipid droplet size and number in various tissues is not as well understood, and most current research efforts focus on adipose and hepatic lipid droplets (71). As the intestine is not a site of long-term lipid storage in vertebrates including larval zebrafish, enterocyte lipid droplets are highly dynamic, temporary structures that respond with high sensitivity to the nutritive state of the animal. This property combined with the relative ease of live imaging in the larval zebrafish intestine compared with other animal models makes for an ideal system for the study of lipid droplet dynamics and regulation. When 5 dpf larvae are fed a high-fat/high-cholesterol meal of chicken egg yolk, both the average lipid droplet number per enterocyte and total area of the cell covered by lipid droplets increase significantly by 1 h postfeeding. Lipid droplet number peaks at 1 h and then gradually decreases, while total lipid droplet area is maintained up to 3 h following the meal, suggesting that smaller lipid droplets fuse as they mature (72). The gut microbiota also influence enterocyte lipid droplet number and size. Intestinal lipid droplets are both larger and more numerous in conventionally raised larvae after feeding than in germ-free larvae. Furthermore, conditioned media from a Firmicutes bacterial strain found to promote dietary fatty acid uptake and export to the liver was sufficient to increase enterocyte lipid droplet number but not the average lipid droplet size (69). These results have begun to reveal the diverse mechanisms by which different members of the gut microbiota influence lipid droplet dynamics and dietary lipid metabolism.

Lipoproteins are essential for the export of the products of dietary lipid from enterocytes into the circulation. Expression and function of apolipoproteins in the zebrafish is similar to that observed in mammals; at least one paralog from each of the ApoA-I, ApoB, ApoE, and ApoA-IV families is expressed in the larval zebrafish intestine (13). There is evidence that division of apolipoprotein function among organs is regulated by different mechanisms that achieve the same end in zebrafish and mammals: while different variants of ApoB are produced in the mammalian intestine and liver *via* RNA editing, larval zebrafish produce mRNA for the ApoB paralog b.1 in the intestine and liver and ApoBb.2 in the liver only. Similar compartmentalization of paralog expression between the liver and intestine is observed in the other apolipoprotein families as well (13) (**Figure 3**). Intestinal lipid accumulation in animals treated with an MTP inhibitor shows that as in the larval

FIGURE 3 | Zebrafish apolipoprotein genes are expressed in the larval digestive system. *In situ* hybridization reveals expression of 10 of the 11 zebrafish apolipoprotein genes in the *apoB*, *apoA-IV*, *apoE*, and *apoA-I* families in the liver and/or intestine of the 6 dpf larva. Dissected intestines probed for *apoA-Ia* are shown, and the gut of a larva probed for *apoA-1b* is magnified below the image of the whole larva. L, liver, I, intestine. Adapted and reprinted from Ref. (13), Figures 2–5, under a CC-BY license.

zebrafish yolk, availability of functional ApoB in necessary for normal rates of lipid export from the intestine, and that enterocyte lipid droplets are the destination of excess dietary fatty acids when export is slowed (73–75). The MTP inhibitor lomitapide is effective in larval zebrafish (72). It has also been observed that in mammals as the dietary fat content increases, chylomicron number reaches a plateau but average chylomicron size continues to increase, suggesting that apolipoprotein expression is the limiting factor in the rate of lipid export from the intestine (76).

### Total Lipid Biochemistry of the Larval Zebrafish Reveals Global Effects of Diet on Lipid Composition, and Facilitates Metabolic Labeling Studies

The larval zebrafish intestine is not only an excellent model for the study of lipid droplet and lipoprotein packaging, but also a site of differential channeling of dietary fatty acids depending on their chemical properties. The amenability of this model to biochemistry due to the ease of obtaining large numbers

Fluorescent fatty acids (BODIPY-FL-C12 depicted) are trafficked and metabolized along with native yolk or dietary lipids when delivered to the developing zebrafish by yolk injection or feeding. LD, cytoplasmic lipid droplet, LP, lipoprotein, VLDL, very low-density lipoprotein. Embryo and larva illustrations adapted from Ref. (18).

of embryos and larvae and performing lipid extractions from them, combined with the transparency of the larva, provides an opportunity unique among vertebrates to perform live imaging and metabolic labeling experiments in parallel using the same fluorescent lipid reagents (28, 77–79). Additionally, the whole-body lipid composition of the larval zebrafish is highly sensitive to changes in diet: the TG content of the 6 dpf larva increases 10-fold 24 h after a single high-fat meal (compared with a standard low-fat diet, and allowing time for the intestinal lumen to clear) (29). (In these experiments, the high-fat meal was chicken egg yolk; ~50% lipid dry weight, and the low-fat meal was SERA Micron larval growth food; 7% lipid. The lipid content of "standard chow" for zebrafish larvae is typically 5–15%.) Working at developmental stages before adipose tissue appears (~14 dpf) avoids signal to noise problems that may occur when the neutral lipid stored in adipose is included in the whole-body lipid profile. Also, at these early developmental stages examination of dietary lipid processing in the intestine can be isolated from potential regulatory influences from adipose tissue.

Though it was beyond the scope of our recent metabolic labeling study (29), the biochemical techniques described therein could be applied to later-stage larvae in order to examine potential crosstalk between adipose tissue and the enterocytes that could influence dietary lipid partitioning. We have also developed methods for using fluorescent fatty acids as metabolic labels in the context of standard and lipid-enriched diets in larval zebrafish (**Table 2**). In addition to exploring the metabolic labeling potential of fluorescent lipids whose product profiles were not previously described, we have also applied HPLC with charged aerosol (total lipid detection) and fluorescence detection to obtain a greater depth of information than previous studies using fluorescent TLC (28). Initial findings indicate that the partitioning of saturated

### REFERENCES

fluorescent fatty acids among complex lipid classes varies with carbon chain length, the total fat and cholesterol content of the diet, and the type of fluorescent tag (29). Metabolic labeling with fluorescent fatty acids in the context of lipid metabolism by the larval zebrafish is summarized in **Figure 4**.

Potential mechanisms regulating the rate of lipid export from the intestine beyond lipoprotein levels, the regulation and physiological effects of the size of enterocyte lipid droplets, and the channeling of newly absorbed dietary fatty acids into the different classes of complex lipids are currently largely uncharacterized. The optically clear and genetically tractable larval zebrafish model presents an ideal system in which to investigate these questions relating to energy homeostasis with a combined live imaging and biochemical approach.

### AUTHOR CONTRIBUTIONS

VQ wrote the manuscript. SF edited the manuscript and provided guidance on the topic and scope.

### FUNDING

This work was supported in part by the National Institute on Alcohol Abuse and Alcoholism (NIAAA) F31AA023142 to VQ, National Institute of Diabetes and Digestive and Kidney (NIDDK) R01DK093399 to SF, and National Institute of General Medicine (GM) R01GM63904 to the Zebrafish Functional Genomics Consortium (Stephen Ekker and SF). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health (NIH). Additional support for this work was provided by the G. Harold and Leila Y. Mathers Charitable Foundation to the laboratory of SF.

morphology and lipid-dependent transcriptional responses. *J Biol Chem* (2016) 291:23804–16. doi:10.1074/jbc.M116.749358

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer JM declared a past collaboration with one of the authors SF to the handling editor.

*Copyright © 2017 Quinlivan and Farber. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Zebrafish Models for Dyslipidemia and Atherosclerosis Research

#### *Amnon Schlegel1,2,3,4\**

*1 University of Utah Molecular Medicine Program, School of Medicine, University of Utah, Salt Lake City, UT, USA, 2Department of Internal Medicine, Division of Endocrinology, Metabolism and Diabetes, University of Utah, Salt Lake City, UT, USA, 3Department of Biochemistry, School of Medicine, University of Utah, Salt Lake City, UT, USA, 4Department of Nutrition and Integrative Physiology, College of Health, University of Utah, Salt Lake City, UT, USA*

Atherosclerotic cardiovascular disease is the leading cause of death. Elevated circulating concentrations of lipids are a central pathogenetic driver of atherosclerosis. While numerous effective therapies for this condition have been developed, there is substantial unmet need for this pandemic illness. Here, I will review nutritional, physiological, genetic, and pathological discoveries in the emerging zebrafish model for studying dyslipidemia and atherosclerosis. The technical and physiological advantages and the pharmacological potential of this organism for discovery and validation of dyslipidemia and atherosclerosis targets are stressed through summary of recent findings. An emerging literature shows that zebrafish, through retention of a *cetp* ortholog gene and high sensitivity to ingestion of excess cholesterol, rapidly develops hypercholesterolemia, with a pattern of distribution of lipid species in lipoprotein particles similar to humans. Furthermore, recent studies leveraging the optical transparency of zebrafish larvae to monitor the fate of these ingested lipids have provided exciting insights to the development of dyslipidemia and atherosclerosis. Future directions for investigation are considered, with particular attention to the potential for *in vivo* cell biological study of atherosclerotic plaques.

#### *Edited by:*

*Maximilian Michel, Vanderbilt University, USA*

#### *Reviewed by:*

*Peggy Biga, University of Alabama at Birmingham, USA Anna-Pavlina Haramis, Leiden University, Netherlands*

### *\*Correspondence:*

*Amnon Schlegel<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Experimental Endocrinology, a section of the journal Frontiers in Endocrinology*

*Received: 03 November 2016 Accepted: 02 December 2016 Published: 16 December 2016*

#### *Citation:*

*Schlegel A (2016) Zebrafish Models for Dyslipidemia and Atherosclerosis Research. Front. Endocrinol. 7:159. doi: 10.3389/fendo.2016.00159*

Keywords: atherosclerosis, dyslipidemia, zebrafish, genetics, physiology and metabolism

### INTRODUCTION

Atherosclerosis is the leading cause of death (1). This chronic, progressive build-up of cholesterol, cellular debris, and calcium can narrow the lumens of critical arteries supplying the heart, brain, limbs, and organs. Plaques are mechanically weak structures and are prone to rupture. Once ruptured, a rapid thrombosis cascade is activated at the site of the plaque, occluding the artery and causing ischemic death to the supplied organ. Persons who have sustained an ischemic event in any artery are at substantially increased risk of repeated plaque rupture and thrombosis. While the last several decades have witnessed a decrease in the incidence of myocardial infarction and ischemic cerebrovascular accident, cardiovascular death is predicted to remain the leading killer for decades to come. A confluence of cardiovascular risk factors including tobacco exposure, hypertension, obesity in children and adults, type 2 diabetes mellitus, and non-alcoholic fatty liver disease is to blame for this trajectory (2–6). Moreover, there is widespread underutilization of effective antiplatelet, antihypertensive, and lipid lowering therapies (3, 7).

In the face of this clinical reality, I will argue in this minireview that zebrafish is an excellent system to discover and characterize new diagnostic and therapeutic targets for atherosclerosis. Those properties that make the study of lipid physiology and atherosclerosis in zebrafish potentially transformative will be reviewed, with an emphasis on original work as throughout the remainder of this article, stressing studies published since others and I last reviewed this topic (8, 9).

### ZEBRAFISH MODEL OVERVIEW

The general strengths of zebrafish for biomedical research are well known, owing to its facile husbandry and low cost of housing and maintenance. This organism can be used to generate large numbers of externally fertilized embryos. These animals develop rapidly and are a mainstay of embryological, forward genetic, and pharmacological research (10–12). In the last decade, a full array of modern genome editing tools has been deployed in zebrafish, including very promising knock-in technologies (13–15). These advances have been married to progress in working in zebrafish late larvae, juveniles, and adults, where numerous aspects of physiology pertinent to atherosclerosis emerge.

### GENERAL FEATURES OF LIPOPROTEIN METABOLISM IN ZEBRAFISH

### Lipoprotein Biology in Zebrafish

Elevated serum cholesterol and non-fasting triacylglycerol (TG) are central drivers of atherosclerosis (16–18). Understanding how lipids are absorbed from the diet, metabolized in tissues, and modified in atherosclerosis are central areas of investigation in developing newer and more effective therapies to treat atherosclerosis. A highly conserved system for transporting water-insoluble lipids is present in all animals (19). In particular, the apolipoprotein B (APOB)-coated particles produced by the intestine (chylomicrons) and liver [very low-density lipoprotein (VLDL) particles] are the carriers of the bulk of absorbed and resynthesized neutral lipids, cholesteryl esters (CE), and TG. These "β-lipoprotein" particles also carry fat-soluble vitamins A, D, and E from their sites of absorption or synthesis to their sites of use or storage (**Figure 1**).

In amniotes, two different protein products are encoded by a single *APOB* locus. In enterocytes of reptiles, birds, and mammals, the *APOB* pre-mRNA undergoes cytosine deamination (catalyzed by APOBEC) to generate a transcript encoding a truncated protein (APOB48) that is found exclusively on chylomicrons (21). The full-length *APOB* transcript can be translated in both the liver and intestine (encoding APOB100). APOB48-coated chylomicron remnants are susceptible to rapid postprandial clearance by the liver, whereas APOB100-coated chylomicron remnants and VLDL remnants [intermediate density lipoprotein (IDL) particles] can mature into the long-lived and atherogenic low-density lipoprotein (LDL) particles (**Figure 1**). Thus, it is important to appreciate that Apob (operationally equivalent to "Apob100")-coated zebrafish chylomicrons are, most likely, not cleared rapidly. Furthermore, zebrafish chylomicrons carry the potential to mature into LDL stoichiometrically (22). This lack of Apob48 might contribute to the rapid dyslipidemia and atherogenesis seen in dietary and genetic studies of zebrafish that will be discussed in subsequent sections. Finally, there are two zebrafish *apob* paralogs (two *apob* genes on different chromosomes). The contribution (expression and incorporation into chylomicrons and VLDL) of these Apob paralogs to circulating β-lipoproteins and atherogenesis is not known; however, their larval expression patterns are different, and their encoded proteins are structurally dissimilar, raising the possibility that they have unique functions (23).

Immediately beyond these critical issues of Apob biology, zebrafish utilizes highly conserved β-lipoprotein assembly proteins and transport mechanisms. Gene expression survey and knockdown approaches confirmed that the central Apob-coated lipoprotein particle-producing enzyme microsomal triglyceride transfer protein (encoded by *mtp* and having orthologs in all species ranging from insects to mammals) is present and functional in zebrafish yolk cell layer, liver, and intestine (24–26). More recently, studies on the intracellular trafficking of nascent chylomicrons have confirmed that the zebrafish model is well suited to investigating the molecular and cellular machinery of dietary energy harvest: the enterocyte undergoes stereotypical changes in ultrastructure when absorbing fats, and its secretory apparatus uses proteins conserved in evolution to pack and traffic nascent chylomicrons (27, 28). Finally, the major determinant of clearance of LDL particles from the circulation, the LDL receptor (Ldlr), has conserved function in zebrafish (29). In short, zebrafish has a complement of conserved lipid trafficking genes that renders study of lipid transport in this model organism relevant to human physiology. The next section will consider one additional, critical circulating protein that makes zebrafish lipoprotein biology particularly useful for modeling human lipoprotein biology.

## Cholesteryl Ester Transfer Protein (CETP)

Following release into the circulation, lipoproteins are modified in zebrafish blood by enzymatic machinery that is also highly conserved with humans. Specifically, zebrafish carries an ortholog of the human *CETP* gene (30). CETP encodes a circulating protein that transfers CE from HDL particles to LDL particles in exchange for TG (**Figure 2**). Once loaded with TG and subject to additional modification, HDL is rendered more prone to rapid clearance, decreasing its "ability" to engage in atheroprotective processes such as reverse cholesterol transport (i.e., retrieving cholesterol from tissue macrophages to delivery to the liver and intestine for elimination). Likewise, increased CE loading of and depletion of TG from LDL contribute to atherogenesis by producing readily modifiable (oxidizable) small dense particles that can enter the subintimal space and drive atherogenesis (31–33). The net effect of Cetp function is to leave the organism with a higher concentration of atherogenic LDL particles and a lower concentration of atheroprotective HDL particles in circulation (the so-called "β-dominant" lipoprotein profile). In zebrafish, the fasting lipoprotein profile is β-dominant (34). This similarity to human lipoprotein composition reflects retention of a *cetp* ortholog in the zebrafish genome. As discussed below, this conservation of a critical human lipoprotein-modifying enzyme

(TG), cholesteryl esters (CE), and phospholipids (not shown) and packaged into chylomicrons, whose signature coat protein in humans is Apob48 (one molecule per particle). This particle enters the vasculature and acquires an Apoc2 molecule from an HDL particle (not shown). Apoc2 is a required binding partner for lipoprotein lipase (LPL), an enzyme tethered to the apical surface of capillary bed endothelial cells in muscle and adipose tissues (20). LPL liberates free fatty acids for use by these tissues. The partially lipid-depleted chylomicron remnant is rapidly cleared by the liver through the action of Apoe-binding LRP receptors and Apob-binding low-density lipoprotein receptors (LDLR). The liver synthesizes very low-density lipoprotein (VLDL) particles from *de novo* lipogenesis-derived fatty acids and re-esterified fatty acids that reach the liver after adipocyte hydrolysis (and has relatively less CE in it). Human VLDL's signature coat protein is Apob100. Following LPL-catalyzed lipid hydrolysis, VLDL remnants, intermediate density lipoprotein (IDL) particles, are either rapidly cleared by the liver or mature into LDL. LDL particles have a long circulating half-life, and they can deposit under vascular endothelial cells, undergo oxidation, and trigger an inflammatory atherosclerotic reaction with subsequent plaque rupture and thrombosis causing ischemia to the supplied tissue.

renders zebrafish susceptible to a dyslipidemia with short dietary interventions. This conservation of a critical aspect of lipoprotein biology also opens the door to pharmacological intervention studies in zebrafish: many other, non-rodent laboratory models also fall short of recapitulating human lipoprotein composition and atherogenesis (35).

A Cetp ortholog is absent in commonly used rodent models of dyslipidemia, rendering the study of atherosclerosis inherently difficult in these species. Specifically, rodents are resistant to atherosclerosis because, among other things, they lack this enzyme's action. Rodents have high circulating HDL concentrations and low LDL concentrations (the so-called "α-dominant" lipoprotein pattern) as a consequence of losing the *Cetp* gene (35). The most commonly used genetic strategy to trigger dyslipidemia in mice is to study dietary and genetic interventions in the context of deleting the *Apoe* or *Ldlr* genes (36–40). Although they are widely used, these models do not capture the full biology of the corresponding human Mendelian diseases, familial dysbetalipoproteinemia (in the case of *Apoe*), and familial hypercholesterolemia (in the case of *Ldlr*): the HDL-cholesterol in both *Apoe*<sup>−</sup>/<sup>−</sup> or *Ldlr*<sup>−</sup>/<sup>−</sup> mice is still higher than in humans with *APOE2*/*<sup>2</sup>* or *LDLR*<sup>−</sup>/<sup>−</sup> genotypes, and in both models, it is mainly the VLDL (and not the IDL and LDL, respectively) that increases. Furthermore, studies with only *Apoe*<sup>−</sup>/<sup>−</sup> or *Ldlr*<sup>−</sup>/<sup>−</sup> mouse models are often limited in generalizability: there is incomplete agreement in the findings with these two models (41). Beyond the limitations of standard mouse genetic models in driving atherogenic dyslipidemia, the atheromatous plaques that do form in mice lack features of "complex" human lesions. Indeed, to generate atheromatous plaques that appear more like human plaques, extreme physical stress is required in *Apoe*<sup>−</sup>/<sup>−</sup> animals deliberately maintained on a mixed genetic background (42). Even with this severe stress, plaque rupture (as in myocardial infarction) does not occur in rodents. Another supraphysiological approach to studying plaque rupture in mice that has met with some criticism because of its artificial nature involves causing a prolonged pharmacological hypertensive crisis in *Apoe*−/− animals; this paradigm causes plaque rupture in the brachiocephalic artery, an uncommon site of rupture in humans (43).

## ZEBRAFISH DYSLIPIDEMIA MODELS

### High-Cholesterol Diet (HCD) Paradigm

The nutritional requirements of zebrafish are now known (44, 45). This knowledge has allowed several groups to establish conditions to induce metabolic stress by altering standard diets. For instance, zebrafish is susceptible to high-fat diet (HFD)-induced obesity, hyperglycemia, and dyslipidemia (46). The major breakthrough in applying zebrafish to the study of dyslipidemia was the development of HCD (34, 47, 48). Not only do larvae and adults readily ingest such diets but also these animals demonstrate a series of responses that firmly established the utility of this organism in studying dyslipidemia and atherogenesis. First, HCD-challenge caused β-dominant hypercholesterolemia in adults; second, vascular intimal lipid accumulation can be seen in larvae after short exposure to HCD, and these accumulated lipids attracted circulating monocytes; third, the extravasated LDL undergoes oxidation (to generate high-affinity ligands for innate immune receptors that are central for driving the inflammation of plaques); and fourth, the oxidized LDL particles can be tracked with live imaging (34, 47, 48). This last observation was made *via* transgenic overexpression of the human monoclonal antibody IK17, which binds to malondialdehyde-modified LDL. The sustained overexpression of IK17 prevented HCD-induced sub-intimal lipid accumulation (47). This is the first proof-of-principle demonstration that atherogenesis can be prevented in zebrafish through, presumably, accelerating immune complex-mediated clearance of modified LDL particles from the circulation (before they deposit in the walls of arteries). This constellation of findings sets the stage for future live imaging of atheromas *in vivo*, as discussed below.

### CETP Pharmacology

Natural compound extracts of cinnamon, clove, grape skin, laurel, loquat, and turmeric contain inhibitors of zebrafish Cetp (30, 49, 50). These extracts protect zebrafish from highcholesterol diet-induced dyslipidemia. Conversely, artificial sweeteners increase HDL particle-carried Cetp activity and drive hyperlipidemia (51, 52). Whether such zebrafish studies will translate into better inhibitors of human, CETP is difficult to predict; moreover, artificial sweeteners appear to exert multiple pathological effects, including triggering glucose intolerance by altering the gut microbiome (53). This pharmaceutical research space has been marked by several abandoned small molecules; one ongoing cardiovascular outcomes trial of a CETP inhibitor (http://ClinicalTrials.gov identifier NCT01252953) and a possible study of another inhibitor might provide opportunities for not only using this approach in humans but also to identify additional questions that might be answered with a zebrafish model (54, 55).

### Zebrafish APOC2 Deficiency

A further advance in developing zebrafish dyslipidemia models comes from the targeted deletion of the *apoc2* gene. Humans lacking APOC2 have familial chylomicronemia, a condition marked by high serum TG concentrations and propensity to recurrent bouts of pancreatitis (56). Zebrafish *apoc2<sup>−</sup>*/*<sup>−</sup>* mutants were generated with genome editing tools (22). These mutants demonstrated the hallmark finding of human APOC2 deficiency: decreased plasma lipase activity and severe hypertriglyceridemia (**Figure 1**). Their lipoprotein pattern is predominantly large, β-lipoproteins, as quantified with sizeexclusion chromatography and scanning electron microscopy techniques. Imaging of the vasculature in *apoc2<sup>−</sup>*/*<sup>−</sup>* mutants reveals accumulation of lipids and lipid-laden macrophages, both hallmarks of atherosclerotic plaques. This powerful dyslipidemia model might prove particularly useful in studying the steps of LDL extravasation, oxidation, and engulfment by vascular wall macrophages.

### Zebrafish Liver X Receptor (LXR) Deletion

Lxrs are central inducers of cholesterol catabolism (57). These nuclear receptor transcription factors regulate metabolism through engaging oxysterol ligands and altering expression of functionally integrated genes involved in reverse cholesterol transport, lipoprotein modification, intestinal cholesterol absorption and excretion, liver fatty acid and TG regulation, bile transport, and immune and inflammatory signaling (57). There are two Lxr paralogs in mammals. Lxrα, which arose in fish, is mainly expressed in tissues involved in tissue macrophages, liver, and intestine, whereas Lxrβ, which arose in amphibians, is more widely expressed (58). Lxrα upregulates hepatic lipogenic enzymes and increases blood TG levels (59, 60). This seemingly self-defeating function—driving elimination of cholesterol while triggering fatty acid synthesis—has been a major impediment to developing Lxr-based therapeutics. Indeed, the LXRβ-selective agonist BMS-852927 not only promotes reverse cholesterol transport in humans but also induces hepatic *de novo* lipogenesis and attendant hypertriglyceridemia; BMS-852927 also causes a rapid decrease in circulating neutrophil counts in humans, but not in cynomolgus monkeys, underscoring the challenge of drug development (61).

Zebrafish bearing a targeted deletion mutation of the Lxrα gene *nr1h3* develop severe hypercholesterolemia and hepatic steatosis when fed HCD and HFD (62). Conversely, overexpression of *nr1h3* in enterocytes confers protection from dyslipidemia and hepatic steatosis when animals are fed a HFD; this metabolically beneficial effect of *nr1h3* overexpression is due to the induction of a transcriptional program resulting in temporary enterocyte storage of lipids, delaying an *en masse* delivery of atherogenic lipoprotein particles in the circulation. As noted above, zebrafish chylomicrons likely mature into LDL particles because of their full-length Apob-coat protein. As such, the *nr1h3* gene deletion and intestine-limited overexpression models might be useful for studying atherogenesis in that the increase in LDL-cholesterol seen in *nr1h3<sup>−</sup>*/*<sup>−</sup>* mutants is substantial. Furthermore, these studies may lead to the rational development of intestine-limited LXR agonists to blunt the development of dyslipidemia and atherosclerosis.

### THE FUTURE: ATHEROMA CELL BIOLOGY, GENETIC AND PHARMACOLOGICAL SCREENS, AND CANDIDATE GENE ANALYSES

### Cell Biology of Atherosclerosis *In Vivo*

There is no *a priori* guarantee that zebrafish atheromas will model the full natural history of the human disease more closely in terms of mechanisms of development, inflammatory response, and final architecture than available models. However, if the advances in studying lipoprotein biology are any guides, the natural history of atheroma progression—from simple lipid accumulation below the vasculature to organization into "complex," cell-rich, and debris-rich plaques—should be feasible in zebrafish. In particular, live fluorescent markers of various cell types that accumulate within plaques such as vascular smooth muscles, macrophages, and other immune cells are available. These live imaging reporters could be used to monitor atherogenesis in real time. Notably, such live imaging tools have been deployed with remarkable success in studying architecturally complex mycobacterial infection and host response in zebrafish (63–65). This success in modeling a human host response to mycobacteria in zebrafish—where other laboratory species have fallen short of producing human-like granulomas—is cause for hope that zebrafish atherosclerosis will reveal conserved inflammatory and immune mechanisms. In particular, the observation that zebrafish macrophages form granulomas in response to mycobacterial infection raises the hope that these cells may very well form lipid-laden "foam cells" in atherosclerotic plaques and, thus, drive an evolutionarily conserved inflammatory response that defines "complex" lesions (i.e., recruiting additional cells to the plaque and driving inflammation). Whether zebrafish will be useful in studying all aspects of more advanced atherosclerotic plaques biology is not clear. For instance, zebrafish have much lower blood pressure than terrestrial animals. Whether this organism will be useful for generating plaque rupture models is difficult to predict. Systematic histological examination of zebrafish arteries from dyslipidemia models will be required to determine what aspects of plaque biology can be studied in this model.

### Genetic and Chemical Screens for Dyslipidemia and Atherosclerosis Modifiers

Beyond hemodynamic concerns regarding the natural history of zebrafish atherosclerosis, it remains uncertain whether genetic or pharmacological screens could be designed to look for modulators of dyslipidemia and atherogenesis in zebrafish. These complex phenotypes develop after a period of feeding in late larvae, raising the time and effort required to perform a large-scale screening project. Developing convenient reporters for the development of dyslipidemia and atherogenesis and their validation in already established zebrafish models would help guide screen design. Such an approach has proven feasible and informative in studying fasting glucose regulation in larvae (66, 67).

### Candidate Genes

A large repertoire of genetic loci has been associated on a population genetics level with lipid parameters (68). For most, the molecular and cellular bases of the associations are unknown. While it is now experimentally tractable to rapidly overexpress and delete genes in zebrafish, phenotypic characterization for atherosclerosis is still limited in comparison to examination of alterations in glucose metabolism (for which larvae phenotypes develop more rapidly and do not require feeding). For instance, my group recently explored the mechanistic association of a single nucleotide polymorphism (SNP) in the *FOXN3* locus with fasting blood glucose (69). Through a blend of primary human hepatocyte, immortalized HepG2 hepatoma cell, and transgenic zebrafish approaches, we found that this SNP increases the expression of the FOXN3 protein and that this transcriptional repressor blunts a glucose utilization transcriptional program in the liver. Overexpression of both human *FOXN3* and zebrafish *foxn3* in liver increased fasting blood glucose in adults. Whether this gene acts in other tissues to modulate blood glucose is not known. Neither is the mechanism through which the risk allele increases FOXN3 expression. An approach similar to the one used to study FOXN3 could be used to examine the effect of overexpressing candidate genes (in liver or elsewhere) on zebrafish lipoprotein metabolism, again, with the caveat that the experimental window will need to be larger. Those "hits" showing changes in circulating lipids could be explored further using a large collection of null alleles (70) or through genome editing approaches (including conditional alleles).

## CONCLUSION

Through a combination of genetic, developmental, and physiological advantages, the zebrafish has emerged as a major mechanistic discovery platform for studying dyslipidemia and atherosclerosis. Here, I highlighted individual areas of success using this organism, from dietary interventions, exploration of various aspects of lipoprotein production and processing, and genetic models for dyslipidemia and early atherosclerosis. Future work in the zebrafish system should include more thorough exploration of the composition and cellular architecture of atheromatous plaques, screenings effort to identify novel genes and small molecules that modulate atherogenesis, and candidate gene approaches to elucidating the functions of loci implicated in dyslipidemia and atherosclerosis through population genetic analyses. Collectively, this work in zebrafish may lead to the development of new and more effective therapies for dyslipidemia and atherosclerosis.

## AUTHOR CONTRIBUTIONS

The author is the sole contributor to the conceptual development and writing of this minireview.

## FUNDING

Work in my laboratory is supported by US National Institutes of Health grant R01-DK096710 and American Heart Association Grant in Aid 15GRNT24670009.

### REFERENCES

created by homologous recombination in ES cells. *Cell* (1992) 71(2):343–53. doi:10.1016/0092-8674(92)90362-G

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Schlegel. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# The Role of Peroxisome Proliferator-Activated Receptor Gamma (*PPARG*) in Adipogenesis: Applying Knowledge from the Fish Aquaculture Industry to Biomedical Research

### *Rebecca Wafer, Panna Tandon and James E. N. Minchin\**

*BHF Centre for Cardiovascular Science, The Queen's Medical Research Institute, University of Edinburgh, Edinburgh, UK*

#### *Edited by:*

*Encarnación Capilla, University of Barcelona, Spain*

#### *Reviewed by:*

*Marta Bou, Nofima, Norway Mercedes Blazquez, Consejo Superior de Investigaciones Científicas (CSIC), Spain*

> *\*Correspondence: James E. N. Minchin<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Experimental Endocrinology, a section of the journal Frontiers in Endocrinology*

*Received: 09 March 2017 Accepted: 01 May 2017 Published: 22 May 2017*

#### *Citation:*

*Wafer R, Tandon P and Minchin JEN (2017) The Role of Peroxisome Proliferator-Activated Receptor Gamma (PPARG) in Adipogenesis: Applying Knowledge from the Fish Aquaculture Industry to Biomedical Research. Front. Endocrinol. 8:102. doi: 10.3389/fendo.2017.00102*

The tropical freshwater zebrafish has recently emerged as a valuable model organism for the study of adipose tissue biology and obesity-related disease. The strengths of the zebrafish model system are its wealth of genetic mutants, transgenic tools, and amenability to high-resolution imaging of cell dynamics within live animals. However, zebrafish adipose research is at a nascent stage and many gaps exist in our understanding of zebrafish adipose physiology and metabolism. By contrast, adipose research within other, closely related, teleost species has a rich and extensive history, owing to the economic importance of these fish as a food source. Here, we compare and contrast knowledge on peroxisome proliferator-activated receptor gamma (PPARG)-mediated adipogenesis derived from both biomedical and aquaculture literatures. We first concentrate on the biomedical literature to (i) briefly review PPARG-mediated adipogenesis in mammals, before (ii) reviewing Pparg-mediated adipogenesis in zebrafish. Finally, we (iii) mine the aquaculture literature to compare and contrast Pparg-mediated adipogenesis in aquaculturally relevant teleosts. Our goal is to highlight evolutionary similarities and differences in adipose biology that will inform our understanding of the role of adipose tissue in obesity and related disease.

#### Keywords: adipose, zebrafish, adipogenesis, pparg, aquaculture

Adipogenesis—the process of progenitor cell differentiation to generate mature, lipid-laden adipocytes (fat cells) is central to physiological homeostasis. Dysregulation of adipogenesis and a reduced capacity to sequester lipid within cytoplasmic lipid droplets (LDs) of adipocytes leads to lipodystrophy, ectopic lipid deposition, systemic metabolic dysfunction, and increased risk for developing diabetes and cardiovascular disease (1–3). Members of the peroxisome proliferator-activated receptor (PPAR) family of nuclear receptors have paramount roles in lipid metabolism; and, in particular, PPAR gamma (PPARG) is critical for adipogenesis. Much is known on PPARG-mediated adipogenesis in mammalian model systems; however, extensive research has also been conducted on adipogenesis in fish species relevant to the aquaculture industry. The aim of this mini-review is to integrate findings on Pparg-mediated adipogenesis from the aquaculture industry into the larger biomedical-centered literature. This review is focused on adipogenesis in white adipose tissue (WAT); however, adipogenesis in brown adipose has also recently been reviewed (4).

### PPARG: A MASTER REGULATOR OF MAMMALIAN ADIPOGENESIS

Peroxisome proliferator-activated receptor gamma is both necessary and sufficient for WAT adipogenesis in mammals, and is considered a "master regulator" of adipogenesis. In mouse, Pparg plays an important role in placental vascularization, monocyte differentiation, and cardiac development (5, 6); however, Pparg is also required for adipogenesis both *in vitro* (7) and *in vivo* (5, 7, 8). Naturally occurring mutations within the *PPARG* coding sequence can lead to PPARG loss-of-function (LOF), severe lipodystrophy, insulin resistance, and diabetes in humans (2, 3, 9). Further, adipocyte-specific deletion of *Pparg* in mouse results in the complete absence of WAT (8). Strikingly, expression of *Pparg*, together with provision of an activating ligand, is sufficient to initiate an adipogenic program and maintain an adipocyte phenotype in previously non-adipogenic cells (10, 11). Therefore, PPARG has a central role in mammalian adipogenesis, typified by PPARG LOF in humans, which is associated with severe lipodystrophy, and metabolic dysfunction and disease.

In mammals, *PPARG* exists as two isoforms, G1 (γ1) and G2 (γ2), derived from a single gene, and transcribed by distinct promoters (12, 13). *PPARG2* contains additional 30 amino acids at the N-terminal of *PPARG1* and is specific to WAT—whereas, *PPARG1* can be expressed at low levels in non-WAT tissues (12, 13). Both γ1 and γ2 isoforms can instruct a similar adipogenic gene expression program; however, *PPARG2* exhibits a quantitatively greater adipogenic ability (14). Structurally, *PPARG* contains six protein domains (domains A–F) (**Figure 1A**): the N-terminal A/B-domain contains the ligand-independent transactivation function 1 (AF-1); the C-domain is a highly conserved DNA-binding domain (DBD), consisting of two type II zinc fingers; the D-domain is a flexible hinge region; the E-domain contains the AF-2 ligand-binding domain (LBD); and at the C-terminus, a small F-domain has been shown to interact with cofactors (15).

The function of each PPARG domain has been extensively studied. The N-terminal AF-1 domain regulates the transcriptional activity of *PPARG* by (i) influencing Pparg ubiquitination and receptor turnover (16), (ii) controlling localization of Pparg to distinct cellular compartments (17, 18), (iii) facilitating communication with the LBD and enhancing ligand-dependent transcription (19), and (iv) recruitment of coactivators (20, 21) and corepressors (22). Importantly, many AF-1 focused regulatory mechanisms rely on posttranscriptional modifications of PPARG and can be both ligand-dependent or ligand-independent (23). Accordingly, inhibiting phosphorylation of serine 112 (S112) of Pparg2 in mouse results in improved insulin sensitivity when fed a high-fat diet (24).  Together, these studies show that multiple diverse mechanisms converge on the AF-1 domain to regulate the transcriptional activity, and insulin sensitizing potential, of PPARG.

The transcriptional activity of PPARG is highly dependent on its DBD. Mutations within the DBD of human *PPARG* inhibit the transcriptional potential of PPARG and patients carrying such mutations exhibit severe insulin resistance and an increased risk for diabetes (3, 9, 26). The core DBD is highly conserved between different nuclear receptors; both within the PPAR family, and between distinct nuclear receptor families (27). Indeed, some nuclear receptors bind identical DNA motifs (28) and, in support, Pparg retains the ability to conduct an adipogenic program even when fused to alternative DBDs (29). These data suggest that the specificity of PPARG-mediated gene activation is not entirely contained within the DBD. Pparg generally binds DNA as obligate heterodimers with members of the retinoid X receptor (RXR) family of nuclear receptors (30), although some evidence suggests Pparg can also function as a homodimer (31). Strikingly, mutations within RXR DBDs have severe consequences for the transcriptional activity of PPARG:RXR heterodimers, suggesting the DNA-binding activity of RXR is also central to PPARG function (32). PPARG/RXR heterodimers bind to cis-acting peroxisome proliferator response elements (PPREs) containing direct repeats of 5′-AGGTCA-3′ separated by *n* nucleotides (DR*n*) (**Figure 1B**) (33–35). Along with an AAACT flanking sequence situated immediately 5′ to the core DR*n* motif, which helps guide selective PPAR binding (36–38). ChIP-Seq analyses for Pparg binding have identified DR1 as the canonical motif for PPARG binding (33, 39, 40), and binding is dependent on the sequence, and affinity, of specific DR1 motifs (40). Wider chromatin organization and accessibility also appear key for PPARG-mediated adipogenesis, as extensive chromatin remodeling occurs early in adipogenesis, prior to Pparg binding, and creates "hotspots" primed for future Pparg binding (41).

### THE LBD OF PPARG, KNOWN LIGANDS, AND MODULATION OF TRANSCRIPTIONAL ACTIVITY

Ligand binding regulates the transcriptional activity of PPARG and, as such, the LBD is central to the ability of PPARG to direct adipogenesis and regulate insulin sensitivity. Numerous lipid metabolites have been identified as PPARG ligands; including, polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid and linoleic acid, eicosanoids, and 15-deoxy-Δ12,14 prostanglandin J2 [PGJ(2)] (10, 42–47). Many of these ligands bind PPARG with low affinity and are unlikely to be present at concentrations required to activate PPARG *in vivo* (48). However, derivatives of linoleic acid have been shown to potently bind Pparg, and may represent an endogenous ligand for PPARG (49, 50). Intriguingly, a cAMP-induced, transient Pparg ligand is produced by 3T3-L1 adipocytes during the early stages of adipogenesis (51) and drives Pparg-mediated progenitor differentiation. Furthermore, this transient Pparg ligand is suggestive of a positive feedback loop, which is autonomous to adipocytes and acts in a paracrine manner. Synthetic ligands

functional verification (9). Yellow–brown histogram indicates the degree of conservation in PPARG between human, mouse, coelacanth, spotted gar, zebrafish, fugu, and Nile tilapia. Height and color indicate the degree of conservation.

also bind and regulate PPARG activity. Most prominently, thiazolidinediones (TZDs) are potent PPARG agonists that lower hyperglycemia, decrease plasma triacylglycerides and free fatty acids, and increase insulin sensitivity (51). As such, TZDs have incredible potential to improve insulin sensitivity and glucose homeostasis in diabetic patients. However, many TZDs have been withdrawn from clinical use, or are under extensive review, owing to toxic side effects (52, 53). In particular, TZDs induce adipogenesis in patients and can lead to increased weight gain (51, 54, 55).

### ZEBRAFISH AS A MODEL TO STUDY Pparg-MEDIATED ADIPOGENESIS

As a complement to mammalian model systems, the zebrafish has recently emerged as a tractable model for studying adipogenesis *in vivo*. Zebrafish possess adipose tissue that is morphologically similar to mammalian WAT (56–58), and which is deposited in anatomically homologous regions to mammalian WAT (56, 58, 59). Further, zebrafish adipose responds to nutritional manipulation, suggesting a conserved role for WAT as an energy store or supply during periods of caloric excess or restriction (56, 60, 61). Zebrafish possess a single *pparg* ortholog on chromosome 11 (47), which exhibits 67% overall similarity to human *PPARG* (47). The LBD and DBD of zebrafish *pparg* show especially high conservation to human *PPARG* (80.5 and 94.3% of amino acids are identical in LDB and DBD, respectively) (**Figure 1C**) (47, 62). The N-terminal AF-1 domain shows much less conservation between zebrafish and human (**Figure 1C**) (47, 62); however, this is unsurprising as the AF-1 domain is well known to exhibit low similarity even between more closely related species (23). Interestingly, zebrafish *pparg* contain multiple regions with amino acid insertions not present in mammalian *PPARG*, suggesting the potential for neo-functionalization of fish Pparg (**Figure 1C**) (47). Importantly, zebrafish *pparg* mRNA is detected in adipocytes (56, 58, 60). Moreover, many compounds known to stimulate mammalian Pparg also modulate zebrafish *pparg* mRNA; including, organotin compounds such as tributyltin (63, 64), halogenated analogs of bisphenol A (65), and PGJ(2) (66). Construction of a zebrafish transgenic line expressing the human PPARG LBD fused to a Gal4 DBD exhibits increased transcriptional activity after treatment with TZDs including rosiglitazone, pioglitazone, or troglitazone (67), thus suggesting that ligand-dependent coactivators of Pparg are conserved and functional in zebrafish. Intriguingly, recent work showed that treatment of zebrafish with the TZD rosiglitazone increased adiposity, suggesting that the role of Pparg in stimulating zebrafish adipogenesis may also be conserved to mammals (68).

## ADIPOGENESIS AND THE AQUACULTURE INDUSTRY

The use of zebrafish as a biomedical model system to study adipogenesis is at a nascent stage, and many gaps exist in our understanding. However, the aquaculture industry has conducted extensive investigation into adipogenesis in closely related fish species, owing to the fact that adipogenesis affects meat quality, animal health, and harvest yields (69). Aquaculture is defined as the farming of aquatic organisms; including fish, crustaceans, mollusks, and plants. The aquaculture industry contributes ~50% of the world's aquatic food source (70); thus representing a significant proportion of all food consumed worldwide (71). For this review, we focus on teleost species most closely related to zebrafish. For a comprehensive review of teleost phylogeny, we refer you to the following articles (72, 73). The teleost lineage is divided into three branches; clupeocephalans (including the majority of teleosts); and the relatively minor elopomorpha (including eels and tarpons), and osteoglossomorpha (fish possessing toothed or bony tongues) (72, 73). For this review, we only consider clupeocephalans, which belong to two main lineages; ostariophysi and euteleostei. In 2010, freshwater fish production was dominated by ostariophysi such as silver carp (*Hypophthalmichthys molitrix*), grass carp (*Ctenopharyngodon idella*), common carp (*Cyprinus carpio*), and the euteleostei, Nile tilapia (*Oreochromis niloticus*). The euteleostei Atlantic salmon (*Salmo salar*) was the most farmed saltwater fish (70). Extensive regional differences exist in the species of fish farmed; for example, Asian countries primarily farm ostariophysi carp species, accounting for 89% of world aquaculture (70). By contrast, Mediterranean countries farm euteleostei species including gilthead sea bream (*Sparus aurata)* (74), European sea bass (*Dicentrarchus labrax*), and flathead gray mullet (*Mugil cephalus*) (70). Northern European countries, primarily farm euteleostei salmonid species such as Atlantic salmon and rainbow trout (*Oncorhynchus mykiss)* (70). A characteristic of teleosts is a teleost-specific third whole genome duplication (Ts3R), which is estimated to have occurred ~225–333 million years ago (72, 75). Recent genome sequencing projects have revealed that, in addition to Ts3R, certain teleost lineages have undergone further extensive genome duplication; including, salmonids (76, 77) and common carp (78). Genome duplications are hypothesized to underlie the dramatic radiation of teleosts and often lead to multiple gene copies, under reduced selective constraint, and thus receptive to neo-, non-, and subfunctionalization of the ancestral gene role (73).

### EXTENSIVE SYNTENIC CONSERVATION AT TELEOST *pparg* LOCI

As *PPARG* exerts such a central role in mammalian adipogenesis, we first wished to assess whether duplicated teleost *pparg* paralogs have been retained. Only a single *pparg* ortholog was identified in 9 (of 10) teleost fish species with genome data on Ensembl (79). These data are striking, as the other members of the PPAR family (*ppara* and *ppard*) have been extensively duplicated, with paralogs retained, in teleosts (Ensembl Gene Tree: ENSGT00870000136388) (47). Teleost species with a single *pparg* ortholog include, ostariophysi such as zebrafish (cyprinidae); and euteleostei such as, Atlantic cod (gadiformes), pufferfish (tetradontiformes, both fugu and tetraodon), stickleback (gasterodae), and Nile tilapia (cichlidae) (Ensembl Gene Tree: ENSGT00870000136388) (47). The single teleost species with a retained *pparg* paralog is the ostariophysi blind cavefish (*Astyanax mexicanus*) of the chariciformes order (**Figure 2**). The striking loss of duplicated *pparg* genes in the majority of teleosts suggest stringent selective pressures for retaining Pparg copy number and function. To construct a predicted ancestral *pparg* locus, we examined synteny at the *Pparg* locus in tetrapods (mouse and human), a basal sarcopterygian (coelacanth), an actinopterygian holosteian basal to teleosts (spotted gar), and a chondrichthyan, cartilaginous fish (elephant shark) (**Figure 2**). Following the Ts3R, two *pparg* loci can be identified which each share extensive synteny to the predicted ancestral locus (**Figure 2**). Remarkably, in both ostariophysi and euteleostei, *pparg* appeared to be retained at a specific single locus (locus 1) (**Figure 2**), with the

(not shown). The *C. milii* locus contained *Cenp* and *Rab43* genes (indicated with an asterisk). Ts3R indicates the teleost-specific genome duplication. Note the inversion of *TIMP4* and *SYN2* upstream of *PPARG* in the mammalian lineage. Duplicated *raf1* paralogs (*raf1a* and *raf1b*) are only retained in zebrafish and cavefish.

exception being Atlantic salmon, which retained *pparg* at locus 2 (**Figure 2**). Strikingly, in all euteleostei species examined, the region downstream of *pparg* contained multiple new genes not found in other species (*iffo2b*, *akr7a3*, *mrto4*, *megf6b*), suggesting an euteleostei-specific recombination event that completely changed the sequence downstream of *pparg* (**Figure 2**).

## SEQUENCE HOMOLOGY OF TELEOST *pparg* GENES

Extensive synteny at teleost and mammalian *PPARG* suggest the locus is under considerable selective constraint; therefore, we next assessed whether the PPARG amino acid sequence was equally conserved. We aligned PPARG sequences from representative tetrapods, a basal sarcopterygian, a holosteian, an ostariophysan, and euteleostians (**Figure 1C**). As expected, not all elements of the mammalian PPARG sequence were conserved from mammals to fish (**Figure 1C**). Strikingly, the DBD, hinge region, and LDB exhibited high levels of conservation from mammals to fish (**Figure 1C**). However, we found a large insertion into the hinge region specific to the euteleosts, fugu, and Nile tilapia (**Figure 1C**). We further found euteleost-specific and zebrafishspecific insertions into the AF-1 domain (**Figure 1C**). Aside from these three regions inserted into teleost Pparg, conservation was extensive (**Figure 1C**). Previous studies have identified that the LDB of fish Pparg (red sea bream and Nile tilapia) often contains additional amino acids compared to human PPARG (80, 81). However, the DBD of PPARG is well conserved between fish and mammals (81). These distinct patterns of conservation have been suggested to reflect the fact that PPARG target genes are well conserved, while there may be greater diversity in ligands, which activate PPARG (81) and may explain why some human PPARG agonists are unable to stimulate *pparg* expression across teleost species (81, 82).

### CONSERVATION OF Pparg AMINO ACIDS AFFECTED BY DISEASE-ASSOCIATED GENETIC VARIATION IN HUMANS

The considerable sequence conservation between mammalian and teleost *PPARG* suggest that residues affected by naturally occurring, disease-associated, mutations in human *PPARG* may also be conserved in teleosts. To address this, we (i) collected all known human *PPARG* single nucleotide polymorphisms (SNPs) from dbSNP (347 SNPs), (ii) filtered these SNPs to identify 73 SNPs predicted to have a highly deleterious effect on PPARG function, (iii) identified amino acids altered by the deleterious SNPs, which were conserved to teleosts (39 SNPs/amino acids), (iv) filtered the conserved deleterious SNPs to ones that had been experimentally verified to have an effect on adipogenesis and PPARG function in humans (4 SNPs) (9). The resulting collection of SNPs (**Figure 1C**) represent ideal initial targets for modeling Pparg function during teleost adipogenesis and highlight the highly conserved nature of PPARG from mammals to fish.

### EXPRESSION DYNAMICS OF *pparg* IN FARMED FISH SPECIES

Although little is known regarding the expression of *pparg* in zebrafish, extensive experiments have been undertaken in farmed fish species to determine the dynamics of *pparg* during adipogenesis. In grass carp (ostariophysi), gilthead sea bream, large yellow croaker, and Atlantic salmon (all euteleostei), Pparg/*pparg* appeared coincident with early stages of adipocyte differentiation and increased gradually throughout adipogenesis (69, 83–87). These dynamics mirror those observed in mammalian 3T3-L1 cells, where *Pparg* mRNA is present at low levels in adipocyte progenitors, and increases upon stimulation of adipogenesis (10, 88). Similar to 3T3-L1 cells, *cebpb* mRNA was also induced prior to *pparg* during differentiation of adipocyte progenitors in the euteleostei, cobia (*Rachycentron canadum*), and in Atlantic Salmon (89). By contrast, in red sea bream (*Pagrus major*) (another euteleostei), *pparg* mRNA appeared to remain stable during a 10-day preadipocyte culture; however, isolated cells were maintained for 4 days prior to induction of adipogenesis; therefore, it remains possible that fluctuations in *pparg* mRNA expression occurred prior to analysis (80). However, by this method, accumulation of LDs appeared late and was not robust (80). Furthermore, in gilthead sea bream (euteleostei), *pparg* mRNA decreased in preadipocytes upon the addition of an adipogenic cocktail (83). The experimental reasons for differences in teleost pparg expression dynamics is unclear; however, in most fish species, the induction and maintenance of *pparg* mRNA during adipogenesis appears largely conserved to mammals. Furthermore, multiple *pparg* isoforms have been found in Nile tilapia and Atlantic salmon (both long and short isoforms) (81, 85, 90, 91), suggesting that teleost *pparg* is alternatively spliced similar to mammalian *PPARG*.

### THE FUNCTIONAL ROLE OF Pparg-MEDIATED ADIPOGENESIS IN FARMED FISH SPECIES

In addition to expression dynamics, extensive experiments on Pparg-mediated adipogenesis have been conducted in aquaculturally relevant fish species. Much of the evidence for Pparg-mediated adipogenesis in fish species derive from primary adipocyte progenitor, or "preadipocyte," cell culture systems. Primary preadipocyte cultures have been established in multiple species, including; Atlantic salmon (84), red sea bream (92), rainbow trout (93), grass carp (94), large yellow croaker (69), gilthead sea bream (74), and cobia (89). In all of these systems, primary stromal-vascular cells were isolated from visceral adipose tissue (VAT) [the VAT source most likely equates to the pancreatic VAT and abdominal VAT deposits described in zebrafish (59)]. The preadipocyte culture methods closely follow established methods for the growth and differentiation of mammalian 3T3-L1 cells (95) and enable the incubation of preadipocytes, and differentiated adipocytes, with a range of pharmacological and biological agents to study potential roles during Pparg-mediated adipogenesis.

To our knowledge, no functional genetic data on the role of Pparg in farmed fish species is currently published. However, extensive data exist on pharmacological manipulation of Pparg and adipogenesis. Troglitazone, an insulin sensitizing TZD, potently stimulates preadipocyte differentiation in porcine and human preadipocytes (96, 97); and co-incubation with insulin induced preadipocyte differentiation in rainbow trout (98). A second TZD tested in teleosts, ciglitazone, induced *pparg* expression in preadipocytes of red sea bream (80), suggesting that TZDs induce both *pparg* and adipogenesis in teleosts. The role of several pro- and anti-adipogenic factors have also been studied in fish. Insulin has potent stimulatory effects on *Pparg* mRNA levels, and the proliferation and differentiation of mammalian preadipocytes, acting through IRS1 and the MAPK pathway (99, 100). In large yellow croaker (Percomorpha), insulin increased *pparg* mRNA, along with stimulating preadipocyte proliferation and differentiation (69). In accordance with mammalian data, Insulin inhibited lipolysis in differentiated adipocytes of rainbow trout (69, 101). Similarly, insulin also stimulated the differentiation of adipocyte progenitors and lipid accumulation in red sea bream (92). These findings suggest that insulin has a conserved role in stimulating *pparg* expression and promoting adipogenesis. Insulin-mediated induction of *pparg* and adipogenesis is also potentially conserved to other fish species, as the insulin-IRS1-MAPK signaling axis is also functional in rainbow trout adipocytes (102). However, unlike in rainbow trout, insulin had no effect on adipocyte lipolysis in gilthead sea bream (103). Tumor necrosis factor alpha (TNFA) is secreted from mammalian adipocytes and inhibits adipogenesis (104). Treating large yellow croaker preadipocytes with human TNFA reduced *pparg* mRNA levels, suppressed proliferation and differentiation, and stimulated lipolysis in differentiated adipocytes (69). An anti-adipogenic role for TNFA was also found in rainbow trout adipocytes at both RNA and protein levels (93, 105). PUFAs inhibit the proliferation and differentiation of mammalian preadipocytes (106, 107). DHA, an omega-3 fatty acid, was used in the treatment of large yellow croaker preadipocytes and led to decreased *pparg* mRNA levels and reductions in cell proliferation (69). It has further been shown that DHA stimulates lipolysis in 3T3-L1 preadipocytes (108); however, DHA did not exert a positive effect on lipolysis within large yellow croaker adipocytes and was actually observed to have an anti-lipolytic effect (69). Interestingly, DHA reduced lipid accumulation in Atlantic salmon adipocytes, although a mechanisms by which this occurred was not identified (85). Conversely, an analog of the saturated fatty acid palmitate, 2-bromopalmitate, increased *pparg* mRNA (red sea bream) (80). *pparg* cooperates with *rxra* to transcribe *fabp4* suggesting that fish Pparg also functions as an

### REFERENCES

obligate heterodimer with Rxr proteins to guide adipogenic gene expression (Nile tilapia) (81). In Atlantic salmon, *pparg* mRNA was induced after addition of liver X receptor (lxr) agonists (109), suggesting Pparg:Lxr coordinate gene expression in teleosts as they do in mammals (110).

### CONCLUSION AND FUTURE DIRECTIONS

Peroxisome proliferator-activated receptor gamma is a master regulator of adipogenesis in mammals, and mutations deleterious to PPARG function lead to increased susceptibility to diabetes and cardiovascular disease. In this review, we assessed the literature on Pparg-mediated adipogenesis in teleost fish species, including the biomedical model system, zebrafish, and multiple aquaculturally relevant farmed fish species. We found a high degree of synteny and conservation at/in *pparg* in teleost fish, along with evidence of conserved expression, regulation, and function derived from primary preadipocyte culture studies. Altogether, information on the role of Pparg gleaned from aquaculturally relevant species is likely to be highly informative for future zebrafish and mammalian biomedical studies on adipogenesis.

### AUTHOR CONTRIBUTIONS

Background literature research, writing, and review were conducted by RW, PT, and JM. Sequence and locus analysis was conducted by JM.

### FUNDING

This mini review was funded by a British Heart Foundation and University of Edinburgh Fellowship to JM, and a Diabetes UK Early Career Small Grant to JM (16/0005494).

gamma activity through the direct binding to the activation function-1 domain. *J Biol Chem* (2010) 285:3126–32. doi:10.1074/jbc.M109.055095

targets during adipogenesis. *PLoS One* (2009) 4:e4907. doi:10.1371/journal. pone.0004907

(*Sparus aurata*) adipocytes. *Am J Physiol Regul Integr Comp Physiol* (2005) 289:R259–65. doi:10.1152/ajpregu.00574.2004

**Conflict of Interest Statement:** These authors declare no commercial or financial activities that may act as potential conflicts of interest.

*Copyright © 2017 Wafer, Tandon and Minchin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Modeling Pancreatic Endocrine Cell Adaptation and Diabetes in the Zebrafish

#### *Lisette A. Maddison and Wenbiao Chen\**

*Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, USA*

Glucose homeostasis is an important element of energy balance and is conserved in organisms from fruit fly to mammals. Central to the control of circulating glucose levels in vertebrates are the endocrine cells of the pancreas, particularly the insulin-producing β-cells and the glucagon producing α-cells. A feature of α- and β-cells is their plasticity, an ability to adapt, in function and number as a response to physiological and pathophysiological conditions of increased hormone demand. The molecular mechanisms underlying these adaptive responses that maintain glucose homeostasis are incompletely defined. The zebrafish is an attractive model due to the low cost, high fecundity, and amenability to genetic and compound screens, and mechanisms governing the development of the pancreatic endocrine cells are conserved between zebrafish and mammals. Post development, both β- and α-cells of zebrafish display plasticity as in mammals. Here, we summarize the studies of pancreatic endocrine cell adaptation in zebrafish. We further explore the utility of the zebrafish as a model for diabetes, a relevant topic considering the increase in diabetes in the human population.

#### *Edited by:*

*Yong Zhu, East Carolina University, USA*

#### *Reviewed by:*

*Amnon Schlegel, University of Utah, USA Mary Kinkel, Appalachian State University, USA*

*\*Correspondence: Wenbiao Chen<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Experimental Endocrinology, a section of the journal Frontiers in Endocrinology*

*Received: 29 November 2016 Accepted: 11 January 2017 Published: 26 January 2017*

#### *Citation:*

*Maddison LA and Chen W (2017) Modeling Pancreatic Endocrine Cell Adaptation and Diabetes in the Zebrafish. Front. Endocrinol. 8:9. doi: 10.3389/fendo.2017.00009*

Keywords: zebrafish, glucose homeostasis, **β**-cell, **α**-cell, plasticity, regeneration

### INTRODUCTION

Glucose homeostasis is a central physiological mechanism important to maintain proper energy balance and cellular function. While the bulk of the information concerning glucose homeostasis comes from studies in mammals, homeostatic mechanisms are active in many organisms including teleost fish [reviewed in Ref. (1)], which underscores the importance of maintaining tight control of circulating glucose. Maintenance of glucose homeostasis is a coordinated effort between multiple organ systems including the brain, skeletal muscle, liver, and the pancreatic endocrine cells. By appropriately secreting glucagon and insulin into the circulation to regulate the production and uptake of blood glucose, respectively, the pancreatic α- and β-cells play a central role in glucose homeostasis. Interestingly, in conditions where demand for insulin or glucagon exceeds the current secretory capacity, glucose homeostasis is still maintained through increased function and number of α- or β-cells. The regulatory mechanisms of this adaptation or compensation, particularly the compensatory increase of cell number, are not fully defined.

The zebrafish has been firmly established as an attractive animal model to explore questions in developmental biology. This has been aided by the low cost, high fecundity traits of the model (2) as well as the optical transparency (3–6), ease of genome manipulation (7–10), and the amenability toward small molecule screens (11–13). These attractive traits of zebrafish have also inspired investigators to study other biological questions including glucose homeostasis.

## GLUCOSE HOMEOSTASIS IN ZEBRAFISH

The systems regulating glucose homeostasis in zebrafish are similar to those of mammals in composition, ontogeny, and function. As in mammals, glucose homeostasis in zebrafish involves brain, skeletal muscle, liver, and the pancreatic endocrine cells. For example, the glucose transporter Glut2 has been found to be critical for proper brain development (14) supporting the importance of appropriate glucose control in the brain. In skeletal muscle, glucose transporters are present (15) and glucose uptake has been found to be insulin sensitive (16), suggesting that like in mammals, skeletal muscle is a major site of glucose disposal. The liver has a critical function in glucose homeostasis as it both uses and produces glucose. Gluconeogenesis is dynamically regulated in the zebrafish liver (17), and glucose-regulated *pck* promoter activity has been leveraged to screen for compounds that impact glucose production (18). Furthermore, risk alleles for altered fasting blood glucose in humans have been found to increase gluconeogenesis in the liver (19). This again supports the conservation of regulatory pathways of glucose homeostasis. Coordinating many aspects of glucose homeostasis are the pancreatic endocrine cells. Of primary focus has been the insulinproducing β-cells and the glucagon-producing α-cells. In the zebrafish, these cells are present as early as 1 day post fertilization and their development is regulated by pathways similar to those for mammals (20–22). The conservation of glucose homeostasis system between zebrafish and mammals supports that zebrafish is a relevant model to study mechanisms of glucose homeostasis, including aspects of pancreatic endocrine cell biology.

### PROMOTING **β**-CELL PROLIFERATION AND DIFFERENTIATION IN LARVAL STAGES WITH SMALL MOLECULES: LINEAGES AND PATHWAYS

Replenishing the β-cell mass has been an active area of investigation for many years as approaches to treat both type 1 and type 2 diabetes. To increase β-cells in adults, often the approach is to manipulate pathways active during development. But there is also a need to understand the mechanisms that promote an increase of β-cells post development, either through neogenesis or through increased proliferation as different mechanisms may be active for adaption to changes in physiology. In embryonic and early larval stages, the pancreatic endocrine cells are primarily coalesced in a single large islet referred to as the principal islet (23). At later larval stages, additional secondary islets are present (24). These secondary islets arise from centroacinar cells in the pancreatic duct (24–28). These cells are Notch sensitive (24, 25, 28) and express markers of endocrine precursors including Nkx6.1 (26) and Nkx2.2 (25). Inducing formation of secondary islets as a way to uncover pathways important in stages beyond early development was the basis of a compound screen (29, 30) and a component of another large-scale screen (30). These screens took advantage of the optical transparency of the zebrafish and transgenic lines that mark the pancreatic endocrine cells. The first screen revealed an important role for retinoic acid signaling in the differentiation of endocrine progenitors (29). Follow-up studies have shown this pathway is functionally conserved in humans (31) and that retinoic acid signaling regulated Sox9b (32), an important transcription factor in endocrine cell differentiation (33). The high-throughput screen was based on increasing endocrine cells in both the principal and secondary islets and yielded several candidate pathways controlling endocrine cell differentiation including NFκB signaling and serotonin signaling (30). Both screens captured changes in both proliferation of endocrine cells and differentiation of precursors. Another compound screen aimed solely to increase β-cell proliferation (34) and relied on expression of markers indicative of the different phases of the cell cycle (35). This screen also identified retinoic acid and serotonin signaling, as well as glucocorticoids, as regulators of proliferation (34). These compound-screening approaches identified both molecules with functions in development, such as Sox9, and also pathways such as serotonin and NFκB which likely also function in post-developmental stages. Ultimately, these compound screening approaches using zebrafish may provide molecules that can be targeted to increase β-cell mass as a treatment for adults with diabetes.

### PANCREATIC ENDOCRINE CELL PLASTICITY IN RESPONSE TO INSUFFICIENT HORMONE ACTION