秀丽隐杆线虫总甘油三酯的定量研究

Anjali  Sandhu; Varsha  Singh

doi:10.21769/BioProtoc.3819

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Peer-reviewed

Total Triglyceride Quantification in Caenorhabditis elegans

秀丽隐杆线虫总甘油三酯的定量研究

Anjali Sandhu

Varsha Singh email

发布: 2020年11月20日第10卷第22期 DOI: 10.21769/BioProtoc.3819 浏览次数: 4295

评审: Demosthenis ChronisMadhuja SamaddarAnonymous reviewer(s)

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Cover of Aging Cell, featuring study using the protocol.

Jun 2020

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Abstract

Several studies suggest an important role of lipid metabolism in regulating longevity of Caenorhabditis elegans. Therefore, assays to quantify lipids have enormous value in understanding aging and pathologies associated with it. Approximately 70% of lipid metabolism genes in the nematode have orthologs in humans. Amenability of C. elegans to genetic manipulations has allowed investigations into the role of specific genetic factors in lipid metabolism. Here, we describe a protocol to quantify total triglycerides in C. elegans, which can be extended to studies of the effects of altered environmental and genetic factors on stored fats. This protocol quantifies the picomoles of the triglycerides, in whole worm lysate. Due to the sensitivity of the assay, it could help in identifying subtle changes in the total stored fat which are not discernible with microscopy techniques.

Keywords: Caenorhabditis elegans (线虫)

Background

Excess calories are often stored in the form of triacylglycerol or glycogen (Cohen, 2018). Abnormal levels of triglycerides (TAGs) have been implicated in heart diseases, pancreatitis, and atherosclerosis in humans (Cameron et al., 1974; Lee et al., 2003; Walther and Farese, 2012; Onal et al., 2017). TAGs are packed into lipid droplets in the cytosol. Lipid droplets are present in eukaryotic cells and vary in size from 20 nm to 100 µm (Stobart et al., 1986; Suzuki et al., 2011). Lipid droplets consist of a hydrophobic core surrounded by a phospholipid monolayer on the outer side. These organelles undergo active biogenesis, maturation, and turnover. At the time of need, such as during caloric restriction or starvation, TAGs are hydrolyzed to generate energy (Walther and Farese, 2012). Like other eukaryotes, C. elegans also stores excess energy in the form of TAGs packed into lipid droplets (Ashrafi, 2007; Mak, 2012). The droplets are present predominantly in the intestine and hypodermis and can be easily visualized by staining with Sudan black, Oil-Red-O, BODIPY (Ex/Em=493/503), or Nile Red (Kimura et al., 1997; Ashrafi et al., 2003; O’Rourke et al., 2009; Yen et al., 2010; Dixit et al., 2020). However, these techniques do not provide an estimate of the absolute amounts of triglycerides. Here, we describe a biochemical method for the quantification of absolute levels of triglycerides in C. elegans, with sensitivity in picomolar concentration. In this assay, lipases hydrolyze TAGs to release free fatty acids and glycerol. Glycerol is further oxidized to generate a product that reacts with a probe to generate fluorescence at Ex/Em of 535/587. Outline of the protocol is shown in Figure 1. Biochemical quantification is useful for the detection of the glycerol moiety of the TAG, but it does not provide information on the fatty acid composition of stored fats in terms of saturated and desaturated fatty acids. This method can be utilized to study changes in total triglyceride content due to genetic pertubations, change in diet or pharmacological perturbations.

Figure 1. Triglyceride quantification procedure overview

Materials and Reagents

1 L and 2 L Conical flasks (ThermoFisher Scientific, FisherBrand)
60 mm Petri dish with triple vent (Tarsons, catalog number: 460061 )
96-well flat bottom black fluorescent reading plates (SPL Life sciences, catalog number: 30496 )
50 ml graduated centrifuge tube (Tarsons, catalog number: 546021 )
15 ml graduated centrifuge tube (Tarsons, catalog number: 566041 )
0.45 µm filters (GE Healthcare, Whatman^TM, catalog number: 9913-2504 )
0.010-inch diameter Platinum wire
Aluminum foil (PRS associates, MYFOIL)
1.5 ml microfuge tube (Trasons)
Caenorhabditis elegans
Escherichia coli OP50 (Glycerol stock)
NaCl (ThermoFisher Scientific, Qualigens, catalog number: 7647 )
MgSO₄(ThermoFisher Scientific, SQ, catalog number: 10034 )
CaCl₂(Merk Life Science, EMPLURA, catalog number: QE5Q651429 )
K₂HPO₄(Avantor^TM, RANKEM, catalog number: P2607 )
KH₂PO₄ (ThermoFisher Scientific, Qualigens, catalog number: 7778 )
Na₂HPO₄ (ThermoFisher Scientific, Qualigens, catalog number: 7558 )
NaOH (SDFCL, catalog number: 20252 K05 )
Sodium hypochlorite solution (NaOCl) (Sigma-Aldrich, catalog number: 239305 )
Absolute ethanol (Merck Life Science, EMSURE, catalog number: 64-17-5 )
Molecular grade water (SIGMA, catalog number: W4502 )
Agar powder (HiMedia, catalog number: RM301 )
LB broth (BD Biosciences, Difco^TM LB Broth Miller (Luria-Bertani), catalog number: 244620 )
LB agar (BD Biosciences, Bacto^TM Agar, catalog number: 214010 )
Peptone (BD Biosciences, Bacto^TM Peptone, catalog number: 211677 )
Cholesterol (SIGMA, catalog number: C8667 )
Streptomycin (Alfa Aesar, catalog number: J61299 )
NP-40 solution (SIGMA, catalog number: 74385 )
BioVision triglyceride quantification colorimetric/fluorometric kit (BioVision, catalog number: K622 )
Reagents provided in the BioVision kit
Triglyceride Assay Buffer (TAB)
Triglyceride Probe
Lipase
Triglyceride Enzyme Mix
Triglyceride Standard (1 mM)
Nematode growth media (see Recipes)
1 M KPO₄(see Recipes)
1 M CaCl₂(see Recipes)
1 M MgSO₄(see Recipes)
5 mg/ml Cholesterol (see Recipes)
50 mg/ml Streptomycin (see Recipes)
5 N NaOH (see Recipes)
LB media (see Recipes)

Equipment

Water bath
Autoclave (JSR, model: JSAX-80 )
Laminar flow (JSR, model: JSCB-1200SB )
BOD incubator (JSR, model: JSBI-840C )
Rocker (SHALOM, GEL ROCKER, model: SLM-GR-100 )
Centrifuge (Hettich, Universal 320 R, 1406 )
Centrifuge (Eppendorf, Centrifuge 5424 R )
Sonicator (BRANSON, DIGITAL SONIFIER 450 )
Spectrophotometer (TECAN, model: Infinite M200 pro )

Software

i-control^TM (TECAN, https://lifesciences.tecan.com/plate_readers/infinite_200_pro?p=Software)
GraphPad Prism5 (PRISM, https://www.graphpad.com/scientific-software/prism/)

Procedure

English

中文翻译

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Sandhu, A. and Singh, V. (2020). Total Triglyceride Quantification in Caenorhabditis elegans. Bio-protocol 10(22): e3819. DOI: 10.21769/BioProtoc.3819.