A High-Throughput Fluorescence-Based Assay System for Appetite-Regulating Gene and Drug Screening

Shimada, Yasuhito; Hirano, Minoru; Nishimura, Yuhei; Tanaka, Toshio

doi:10.1371/journal.pone.0052549

Cited by 67 publications

(61 citation statements)

References 67 publications

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“…A strategy to track food intake and its digestion is to stain the food source with fluorescent-labeled macrospheres, phospholipids, or membrane dyes in these transparent vertebrates. In past efforts, researchers have implemented such strategies and generated key insights into digestive physiology (3,9,14,15,34). However, all of these were based on euthanized or anesthetized larvae, which makes dynamic measurements of intestinal food content difficult.…”

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confidence: 99%

A high-throughput assay for quantifying appetite and digestive dynamics

Jordi

Guggiana-Nilo

Soucy

et al. 2015

American Journal of Physiology-Regulatory, Integrative and Comp

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Jordi J, Guggiana-Nilo D, Soucy E, Song EY, Wee CL, Engert F. A high-throughput assay for quantifying appetite and digestive dynamics. Am J Physiol Regul Integr Comp Physiol 309: R345-R357, 2015. First published June 24, 2015 doi:10.1152/ajpregu.00225.2015.-Food intake and digestion are vital functions, and their dysregulation is fundamental for many human diseases. Current methods do not support their dynamic quantification on large scales in unrestrained vertebrates. Here, we combine an infrared macroscope with fluorescently labeled food to quantify feeding behavior and intestinal nutrient metabolism with high temporal resolution, sensitivity, and throughput in naturally behaving zebrafish larvae. Using this method and rate-based modeling, we demonstrate that zebrafish larvae match nutrient intake to their bodily demand and that larvae adjust their digestion rate, according to the ingested meal size. Such adaptive feedback mechanisms make this model system amenable to identify potential chemical modulators. As proof of concept, we demonstrate that nicotine, L-lysine, ghrelin, and insulin have analogous impact on food intake as in mammals. Consequently, the method presented here will promote large-scale translational research of food intake and digestive function in a naturally behaving vertebrate.appetite; hunger; satiation; satiety; DiR' dye FOOD INTAKE AND DIGESTION are key physiological processes that provide nutrients to drive all bodily functions. Nutrient intake is matched to nutritional needs by the brain-a process termed nutrient homeostasis-using an intertwined organism-wide array of extrinsic and intrinsic cues coding food availability and demand (2, 42). Dysfunction of feeding and digestive behavior is at the root of global health problems, such as obesity, malnutrition, and Type 2 diabetes, among many others, and, therefore, a deeper understanding of feeding and digestive behavior is of high importance (25).Drugs or genetic manipulations that alter food intake or digestion are highly desirable remedies for food-related disorders. To screen for genes or small molecules with clinically desirable impact, the technology of choice needs to support the analyses of hundreds to thousands of individual animals. Until now, large-scale studies of feeding behavior have solely been feasible in small invertebrates such as Drosophila, but were constrained to 20 -50 conditions in vertebrates, thereby making large-scale screens elusive (12,17,35). Technically, even more demanding than measuring food intake is the quantification of nutrient digestion, as there is no direct optical access to the gastrointestinal tract in most species. More specialized technologies, such as bioluminescence, MRI, or computed tomography, have been valuable to generate insights into in vivo dynamics of digestive function (10,18,19,31). However, all of these methods require immobilization of the experimental subject to reduce motion artifacts, thereby making concurrent behavioral observations impossible. Consequently, quantifying the dy...

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mentioning

confidence: 99%

A high-throughput assay for quantifying appetite and digestive dynamics

Jordi

Guggiana-Nilo

Soucy

et al. 2015

American Journal of Physiology-Regulatory, Integrative and Comp

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“…High throughput assays are essential for fully appreciating the value of the zebrafish model to studies of energy homeostasis. Innovative tools designed to assess phagic drive will allow for screens focused on energy intake, while high throughput lipid storage assays will lead to studies on whole body lipid homeostasis [15][16][17] . The assay we describe here provides a high throughput tool to assess the role of genes, compounds or their interactions on whole body energy expenditure.…”

Section: Discussionmentioning

confidence: 99%

High Throughput <em>Danio Rerio</em> Energy Expenditure Assay

Williams

Renquist

2016

JoVE

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Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96-or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH 2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH 2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers). Video LinkThe video component of this article can be found at

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“…Knockdown of npy decreases feeding (zebrafish) [165] Agouti-related protein (AgRP) ↑ Food consumption and ↓ Locomotion…”

Section: Genesmentioning

confidence: 99%

“…Knockdown of agrp decreases metabolism and locomotion; overexpression increases food intake (zebrafish) [87,165] Orexin (ORX) ↑ Food consumption Intracerebroventricular (ICV) administration of orexin-A increases food (zebrafish) [113] Cannabinoid receptor 1 (CNR1) ↑ Food consumption and ↓ Locomotion…”

Section: Genesmentioning

confidence: 99%