Analyses of C. elegans fat metabolic pathways.
In Caenorhabdatis elegans as in other animals, fat regulation reflects the outcome of behavioral, physiological, and metabolic processes. The amenability of C. elegans to experimentation has led to utilization of this organism for elucidating the complex homeostatic mechanisms that underlie energy balance in intact organisms. The optical advantages of C. elegans further offer the possibility of studying cell biological mechanisms of fat uptake, transport, storage, and utilization, perhaps in real time. Here, we discuss the rationale as well as advantages and potential pitfalls of methods used thus far to study metabolism and fat regulation, specifically triglyceride metabolism, in C. elegans. We provide detailed methods for visualization of fat depots in fixed animals using histochemical stains and in live animals by vital dyes. Protocols are provided and discussed for chloroform-based extraction of total lipids from C. elegans homogenates used to assess total triglyceride or phospholipid content by methods such as thin-layer chromatography or used to obtain fatty acid profiles by methods such as gas chromatography/mass spectrometry. Additionally, protocols are provided for the determination of rates of intestinal fatty acid uptake and fatty acid breakdown by β-oxidation. Finally, we discuss methods for determining rates of de novo fat synthesis and Raman scattering approaches that have recently been employed to investigate C. elegans lipids without reliance on invasive techniques. As the C. elegans fat field is relatively new, we anticipate that the indicated methods will likely be improved upon and expanded as additional researchers enter this field.
Laboratório de Neurociência, INCT de Medicina Molecular, Faculdade de Medicina da UFMG, Belo Horizonte, Minas Gerais, Bolsista CNPq, Brazil.
Alexandre Guimarães de Almeida
AG
Barros
Jason
J
Liu
George A
GA
Lemieux
Brendan C
BC
Mullaney
Kaveh
K
Ashrafi
D000818:Animals; D017173:Caenorhabditis elegans / Q000502:physiology*; D002855:Chromatography, Thin Layer; D004734:Energy Metabolism / Q000502:physiology*; D005227:Fatty Acids / Q000378:metabolism*; D005456:Fluorescent Dyes; D008401:Gas Chromatography-Mass Spectrometry; D006706:Homeostasis; D007413:Intestinal Mucosa / Q000378:metabolism; D050356:Lipid Metabolism / Q000502:physiology*; D010078:Oxazines; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*; D014020:Tissue Extracts / Q000737:chemistry; D016707:Tissue Fixation; D014280:Triglycerides / Q000378:metabolism*
D005227:Fatty Acids; D005456:Fluorescent Dyes; D010078:Oxazines; D014020:Tissue Extracts; D014280:Triglycerides; C044808:nile red