High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Summary

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

Abstract

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.

Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

Introduction

We are in a time where obesity, and its associated economic burdens, is a worldwide problem¹. Two out of every three Americans are overweight or obese with related heart pathologies, the primary cause of death within the adult population². New efficient methods are needed to adequately investigate the genetic and molecular components implicated in the regulation of metabolic syndrome using model organisms. For this reason, we choose the fruit fly Drosophila model because it shares the most basic biological processes with mammals, including mice and humans^3,4

Protocol

1. HFD Feeding Protocol

Table 1. Fly food recipe.
This table summarizes the different ingredients used to prepare our control food. Once made, 10 mL of the food is poured into vials, cooled and stored at 4 °C for long term storage.

1. HFD preparation
   1. To make 1 kg of high fat food, weigh 700 g of pre-made normal fly food (see Table 1) and....

Results

In D. melanogaster, as is the case with other species, there is sexual dimorphism between males and females²². It is well known that females are larger, with more fat in their abdomens, than males²². To test the effectiveness of our protocol, we performed TAG assays to determine the differences in TAG content between males and females of standard laboratory wildtype (w¹¹¹⁸) flies. The data show that females have .......

Discussion

Obesity induction in mice can take months^19,20. In flies, this HFD feeding protocol allows for induction of excess fat accumulation in a matter of days or less, causing increases in fat accumulation only after 18 h (see Figure 2). HFD feeding with the described protocol increases glucose content ¹² and decreases Bmm lipase and PGC-1 expression²⁴. This is in contrast to fasting .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Talboys Ball dropper/bead Dispenser	Talboys	#: 930150
Talboys High Throughput Homogenizer	Talboys	#: 930145
Grinding Balls, Stainless Steel	OPS Diagnostics, LLC	# GBSS 156-5000-01	5000 balls
Masterblock 96 Well deep Microplates	Greiner Bio-One	# T-3058-1	case of 80 plates
Greiner  96 well microplate flat bottom	Sigma Aldrich	# M4436	40 plates
Greiner CapMat for sealing multiwell plates	Sigma Aldrich	# C3606	50 sealing plates
Reagent Reservoirs	Thomas Scientific	# 1192T71	12/PK
Thermo Scientific Finnpipette 4661040	Thermo Scientific	# 4661040	1-10 ul multipipette
Thermo Scientific Finnpipette 4661070	Thermo Scientific	# 4661070	30-300ul multipipette
Thermo Scientific Finnpipette 4661020	Thermo Scientific	#4661020	10-100ul multipipette
Multichannel tips	Denville Scientific Inc	# P3131-S	for 10 uL pipette
Multichannel tips	Denville Scientific Inc	# P3133-S	for 200 uL pipette
Multichannel tips	Denville Scientific Inc	#P1125	for 100 uL pipette
Forceps	Roboz Surgical	# 5 Dumonts	Super fine forceps
Mettler Toledo Excellence XS Analytical Balance Mfr# XS64	Cole-Parmer scientific experts	# EW-11333-00
Metler Toledo Excellence XS Toploading Balance	Cole-Parmer scientific experts	# EW-11333-49
96-Well microplate Centrifuge	Hettich Zentrifugen	# Rotina 420R
Microplate Reader	Molecular devices	# SpectraMax 190
Lab-Line Bench Top Orbit Environ Shaker Incubator	Biostad	# 3527
Infinity Triglycerides reagent	Thermo Scientific	# TR22421
Triglyceride Standard	Stanbio	#2103 - 030
Quick Start Bradford Protein Assay	Bio-RAD	# 500-0205	1x dye Reagent
Coconut oil	Nutiva	# 692752200014	15 0z jar
PBS 10X	Thermo Scientific	# AM9625	500 ml
Triton X-100	Sigma Aldrich	# 9002-93-1
Gas-permeable Foil	Macherey-Nagel	# 740675	50 pieces
filter Paper	VWR	# 28317-241	Pack of 100
Drosophila vials	Genesee Scientific	Cat #: 32-116SB
Quick Start Bovine Serum Albumin Standard	Bio-Rad	# 5000206
FlyNap Anesthetic	Carolina	# 173025	100 mL
Kimwipes Low-Lint	Uline	# S-8115	1-Ply, 4.4 x 8.4"