Label-free quantitative analysis of lipid metabolism in living Caenorhabditis elegant

Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA.
The Journal of Lipid Research (Impact Factor: 4.42). 09/2009; 51(3):672-7. DOI: 10.1194/jlr.D000638
Source: PubMed


The ubiquity of lipids in biological structures and functions suggests that lipid metabolisms are highly regulated. However, current invasive techniques for lipid studies prevent characterization of the dynamic interactions between various lipid metabolism pathways. Here, we describe a noninvasive approach to study lipid metabolisms using a multifunctional coherent anti-Stokes Raman scattering (CARS) microscope. Using living Caenorhabditis elegans as a model organism, we report label-free visualization of coexisting neutral and autofluorescent lipid species. We find that the relative expression level of neutral and autofluorescent lipid species can be used to assay the genotype-phenotype relationship of mutant C. elegans with deletions in the genes encoding lipid synthesis transcription factors, LDL receptors, transforming growth factor beta receptors, lipid desaturation enzymes, and antioxidant enzymes. Furthermore, by coupling CARS with fingerprint confocal Raman analysis, we analyze the unsaturation level of lipids in wild-type and mutant C. elegans. Our study shows that complex genotype-phenotype relationships between lipid storage, peroxidation, and desaturation can be rapidly and quantitatively analyzed in a single living C. elegans.

Download full-text


Available from: Chang-Deng Hu
  • Source
    • "Scattering (SRS) microscopy (Hellerer et al., 2007; Le et al., 2010; Wang et al., 2011). Both techniques are compatible with live, anesthetized C. elegans samples and work by excitation of particular Raman vibrational frequencies of endogenous biomolecules although they depend on distinct optical configurations and measure different features of the Raman emission. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Abstract C. elegans provides a genetically tractable system for deciphering the homeostatic mechanisms that underlie fat regulation in intact organisms. Here, we provide an overview of the recent advances in the C. elegans fat field with particular attention to studies of C. elegans lipid droplets, the complex links between lipases, autophagy, and lifespan, and analyses of key transcriptional regulatory mechanisms that coordinate lipid homeostasis. These studies demonstrate the ancient origins of mammalian and C. elegans fat regulatory pathways and highlight how C. elegans is being used to identify and analyze novel lipid pathways that are then shown to function similarly in mammals. Despite its many advantages, study of fat regulation in C. elegans is currently faced with a number of conceptual and methodological challenges. We critically evaluate some of the assumptions in the field and highlight issues that we believe should be taken into consideration when interpreting lipid content data in C. elegans.
    Full-text · Article · Sep 2014 · Critical Reviews in Biochemistry and Molecular Biology
  • Source
    • "Second, it is estimated that more than half of C. elegans genes are homologous to genes implicated in human diseases [7, 8]. Third, this model organism is maintained under simple experimental conditions in the laboratory and has an optically transparent body that is amenable to the use of fluorescent probes to visualize oxidative stress within the nematodes in vivo [9, 10]. C. elegans contains many cell types that represent the major tissues of complex metazoans, such as muscle, intestinal, nervous, and epithelial tissue [11]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Caenorhabditis elegans is a powerful model organism that is invaluable for experimental research because it can be used to recapitulate most human diseases at either the metabolic or genomic level in vivo. This organism contains many key components related to metabolic and oxidative stress networks that could conceivably allow us to increase and integrate information to understand the causes and mechanisms of complex diseases. Oxidative stress is an etiological factor that influences numerous human diseases, including diabetes. C. elegans displays remarkably similar molecular bases and cellular pathways to those of mammals. Defects in the insulin/insulin-like growth factor-1 signaling pathway or increased ROS levels induce the conserved phase II detoxification response via the SKN-1 pathway to fight against oxidative stress. However, it is noteworthy that, aside from the detrimental effects of ROS, they have been proposed as second messengers that trigger the mitohormetic response to attenuate the adverse effects of oxidative stress. Herein, we briefly describe the importance of C. elegans as an experimental model system for studying metabolic disorders related to oxidative stress and the molecular mechanisms that underlie their pathophysiology.
    Full-text · Article · May 2014 · Oxidative medicine and cellular longevity
  • Source
    • "Despite their utility, optical imaging techniques can suffer from limited imaging depth penetration due to scattering of light within the turbid environment of most biological tissues [18], [22]. As light passes through tissue such as nerves, photons are scattered due to particles, layers, and other inhomogeneities that alter the local index of refraction, resulting in a perturbed focal spot geometry, reduced power at the focal plane, and signal loss due to emission signal attenuation. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Peripheral nerve injury (PNI), a common injury in both the civilian and military arenas, is usually associated with high healthcare costs and with patients enduring slow recovery times, diminished quality of life, and potential long-term disability. Patients with PNI typically undergo complex interventions but the factors that govern optimal response are not fully characterized. A fundamental understanding of the cellular and tissue-level events in the immediate postoperative period is essential for improving treatment and optimizing repair. Here, we demonstrate a comprehensive imaging approach to evaluate peripheral nerve axonal regeneration in a rodent PNI model using a tissue clearing method to improve depth penetration while preserving neural architecture. Sciatic nerve transaction and end-to-end repair were performed in both wild type and thy-1 GFP rats. The nerves were harvested at time points after repair before undergoing whole mount immunofluorescence staining and tissue clearing. By increasing the optic depth penetration, tissue clearing allowed the visualization and evaluation of Wallerian degeneration and nerve regrowth throughout entire sciatic nerves with subcellular resolution. The tissue clearing protocol did not affect immunofluorescence labeling and no observable decrease in the fluorescence signal was observed. Large-area, high-resolution tissue volumes could be quantified to provide structural and connectivity information not available from current gold-standard approaches for evaluating axonal regeneration following PNI. The results are suggestive of observed behavioral recovery in vivo after neurorrhaphy, providing a method of evaluating axonal regeneration following repair that can serve as an adjunct to current standard outcomes measurements. This study demonstrates that tissue clearing following whole mount immunofluorescence staining enables the complete visualization and quantitative evaluation of axons throughout nerves in a PNI model. The methods developed in this study could advance PNI research allowing both researchers and clinicians to further understand the individual events of axonal degeneration and regeneration on a multifaceted level.
    Full-text · Article · Apr 2014 · PLoS ONE
Show more