Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids.
Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de novo from acetyl-CoA and are primarily present in eukaryotes in the form of triglycerides (TGs) and sterol-esters (SEs). The enzymes responsible for the synthesis of NLs are highly conserved from Saccharomyces cerevisiae (yeast) to humans, making yeast a useful model organism to dissect the function and regulation of NL metabolism enzymes. While much is known about how acetyl-CoA is converted into a diverse set of NL species, mechanisms for regulating NL metabolism enzymes, and how mis-regulation can contribute to cellular pathologies, are still being discovered. Numerous methods for the isolation and characterization of NL species have been developed and used over decades of research; however, a quantitative and simple protocol for the comprehensive characterization of major NL species has not been discussed. Here, a simple and adaptable method to quantify the de novo synthesis of major NL species in yeast is presented. We apply 14C-acetic acid metabolic labeling coupled with thin layer chromatography to separate and quantify a diverse range of physiologically important NLs. Additionally, this method can be easily applied to study in vivo reaction rates of NL enzymes or degradation of NL species over time.
Acetyl-CoA is the fundamental building block of diverse biomolecules including neutral lipids (NLs), which serve as a versatile biomolecular currency for building membranes, generating ATP, and regulating cell signaling1,2. The availability of NLs to be shunted into any of these respective pathways is, in part, regulated by their storage. Lipid droplets (LDs), cytoplasmic organelles composed of hydrophobic cores of triglycerides (TGs) and sterol-esters (SEs), are the main storage compartments of most cellular NLs. As such, LDs sequester and regulate NLs, which can be degraded and subsequently utilized for biochemical and metabolic processes3,4. It is known that the mis-regulation of NL and LD-associated proteins is correlated with the onset of pathologies including lipodystrophy and metabolic syndromes5,6. Because of this, current LD research is intensely focused on how NL synthesis is regulated spatially, temporally, and across distinct tissues of multi-cellular organisms. Due to the ubiquitous cellular roles for NLs, many enzymes responsible for the synthesis and regulation of NLs are conserved throughout eukaryotes7. Indeed, even some prokaryotes store NLs in LDs8. Therefore, genetically tractable model organisms such as Saccharomyces cerevisiae (budding yeast) have been useful for the study of NL synthesis and regulation.
The separation and quantification of NLs from cell extracts can be accomplished in a myriad of ways, including gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS)9,10,11. Perhaps the simplest method for separating NLs is via thin layer chromatography (TLC), which allows for subsequent densitometric quantification from a standard curve12,13. Although TLC provides only a course-grained separation of NLs, it remains a powerful technique because it is inexpensive, and it allows for the rapid separation of NLs from several samples simultaneously. Two of the most considerable challenges facing the study of NLs via TLC are: 1) the broad range of cellular abundances of NL species and their intermediates, and 2) the range of hydrophilicity/hydrophobicity of lipid intermediates within NL synthesis pathways. Consequently, the quantification of NL species via TLC is typically restricted to the most abundant species; however, introduction of a 14C-acetic acid radiolabel can significantly enhance the detection of low abundance intermediates within NL pathways. Acetic acid is rapidly converted into acetyl-CoA by the acetyl-CoA synthetase ACS214, which makes 14C-acetic acid a suitable radiolabeling substrate in yeast15. Additionally, separation of both hydrophobic NLs and hydrophilic intermediates of NLs can be achieved by TLC through the use of multiple solvent systems16. Here, a method is presented for the separation of NLs using 14C-acetic acid metabolic labeling in yeast. Lipids labeled during the pulse period are subsequently isolated by a well-established total lipid isolation protocol17, followed by the separation of NL species by TLC. Developing of TLC plates by both autoradiography to visualize labeled lipids, and a chemical spray to visualize total lipids, permits for multiple methods of quantification. Individual lipid bands can also be easily extracted from the TLC plate using a razor blade, and scintillation counting can be used to quantify amount of radiolabeled material within the band.
1. Growth and labeling of yeast cells with 14C-acetic acid
2. Isolation of total lipids from yeast
NOTE: The following protocol for lipid isolation is based on a well-established and frequently used method that efficiently extracts most neutral lipid species17,18.
CAUTION: When using organic solvent, always wear appropriate PPE and work inside of a fume hood when possible. During lipid extraction, avoid using plastics that are incompatible with organic solvents. Polypropylene tubes are suitable for the following protocol.
3. Separation and quantification of radioisotope-labeled NLs by thin layer chromatography
4. Visualization and quantification of TLC separated lipids
In this protocol, we have demonstrated that the labeling, detection, and quantification of NL species can be accomplished by 14C-acetic acid metabolic labeling. Major NL species can be separated in a solvent system of 50:40:10:1 (v/v/v/v%) Hexane:Petroleum ether:Diethyl ether:Acetic acid (Figure 1A,B). Phosphor imaging allows for visualization of labeled free fatty acid (FFA), triacylglycerol (TG), diacylglycerol (DG), cholesterol (Chol), and squalene (SQ) (Figure 1A). Although SEs can be separated from other NL species in this solvent, none are detected in the autoradiogram following a 20-minute pulse. This may be attributed to slow SE synthesis during the stationary phase of growth in yeast. It is also demonstrated that purified lipid species can be separated in this method and subsequently visualized by spraying of the TLC plate with p-anisaldehyde reagent (Figure 1B). While NL species are well separated in this solvent, polar species like phosphatidylcholine (PC) stay at the origin (Figure 1B). By applying a chase period in radiolabel-free media following the pulse, relative flux through NL pathways can be measured (Figure 1C). After a 10-min chase, the major pool of SQ has disappeared, and total Chol is elevated. Similarly, the appearance of DG in the chase period correlates with a decrease in the FFA signal.
Figure 1: 14C-Acetic acid radiolabeling allows for detection of multiple NL species. (A) Autoradiogram of lipids separated by TLC isolated from yeast radiolabeled with 14C-acetic acid in stationary phase. Clearly detectable species include free fatty acid (FFA), triglyceride (TG), diacylglycerol (DG), cholesterol (Chol), and squalene (SQ). Unlabeled bands are unidentified NL species. (B) Purified lipid species separated by TLC and visualized by p-anisaldehyde staining. Visualized species include all lipids mentioned in (A) in addition to sterol-esters (SE) and phosphatidylcholine (PC). (C) Autoradiogram of lipids separated by TLC isolated from yeast pulsed with 14-acetic acid in stationary phase followed by a 10 min chase period in radiolabel-free media. Disappearance of SQ is met with increase in Chol. Rise in DG in the chase period is accompanied by decrease in FFA species. Please click here to view a larger version of this figure.
Supplementary File: Recipes for buffers, media, and solutions. Please click here to download this file.
Here, a versatile radiolabeling protocol to quantitatively monitor the synthesis of NL species in yeast is presented. This protocol is very modular, which allows for the procedure to be finished within 3-6 days. Additionally, a wealth of literature exists on the use of TLC to separate lipid species and metabolites, which should permit the user to detect several lipid species of interest with a simple change of TLC solvent systems16,19.This protocol is conducive to the separation, detection, and quantification of radiolabeled lipids. It can also be coupled with a chase period in un-labeled media to detect the turnover time of labeled NLs. Collectively, this procedure gives a useful structure to begin exploring the radiolabeling of NL species.
Other methods, such as HPLC, GC-MS, and UPLC-MS provide higher resolution of lipid separation and quantification; however, it is typically not optimal to run radiolabeled samples through MS, although this can be overcome by using stable-isotopes. Nevertheless, this radiolabel method provides high detection sensitivity and versatility for many lipid species. Another advantage of this protocol compared to MS is its affordability. TLC separation of lipids is relatively simple, requires no extravagant equipment, and relies on common laboratory materials. Regarding limitations: certain low-abundance species, like lyso-lipids, may not be detectable even following incorporation of a 14C label. Additionally, most TLC approaches are not suitable for 'lipidomic' characterizations, due to the course-grained separation of lipid species within a given solvent.
Yeast offer a convenient, genetically tractable model system for the study of lipids via radiolabel biochemical approaches. However, it should be noted that in specific genetic backgrounds, or during a particular metabolic growth conditions, the cellular uptake of radiolabeled-acetic acid or other radiolabels may be reduced. Labeling of cells with 14C-acetic acid in the absence of glucose robustly increases the uptake of the radiolabel. Long incubations in the absence of glucose will proportionally increase radiolabel uptake; however, this may also influence the pathways in question. Therefore, labeling efficiency for a particular growth condition should be established prior to following the 14C-acetic acid radiolabeling protocol in full. In particular, pay attention to the length of the radiolabeling period. The labeling time should be kept as short as possible to detect the lipid species-of-interest. Altogether, this procedure allows for the study of important lipid synthesis reactions and should permit the investigation of NL regulation in intact cells.
The authors have nothing to disclose.
The authors would like to thank the members of the Henne lab for help and conceptual advice in the completion of this study. W.M.H. is supported by funds from the Welch Foundation (I-1873), the NIH NIGMS (GM119768), the Ara Paresghian Medical Research Fund, and the UT Southwestern Endowed Scholars Program. S.R has been supported by a T32 program grant (5T32GM008297).
[1-C14] Acetic acid sodium salt specific activity: 45-60mCi | PerkinElmer | NEC084H001MC | |
18:1 1,2 dioleoyl-sn-glycerol | Avanti | 800811O | |
200 proof absolute ethanol | Sigma | 459836 | |
Acid washed glass beads 425-600um | Sigma | G8772 | |
Amber bulbs for Pastuer pipettes | Fisher | 03-448-24 | |
Ammonium Sulfate >99% | Sigma | A4418 | |
Beckman LS6500 scintillation counter | PerkinElmer | A481000 | |
Chloroform (HPLC grade) | Fisher | C607SK | |
Cholesterol >99% | Sigma | C8667 | |
Cholesteryl-linoleate >98% | Sigma | C0289 | |
Concentrated sulfuric acid | Sigma | 339741 | |
Corning 50mL conical tubes, polypropylene with centristar cap | Sigma | CLS430829 | |
Dextrose, anhydrous grade | Sigma | D9434 | |
Diethyl ether anhydrous grade | Sigma | 296082 | |
Drying oven | Fisher | 11-475-155 | |
EcoLume scintillation liquid | VWR | IC88247001 | |
Eppendorf 5424R centrifuge | Fisher | 05-401-205 | |
GE Storage phosphor screen | Sigma | GE28-9564-75 | |
GE Typhoon FLA9500 imager | |||
Glacial acetic acid, ACS grade | Sigma | 695092 | |
Glass 6mL scintillation vials | Sigma | M1901 | |
Glass centrifuge tube caps | Fisher | 14-595-36A | |
Glass centrifuge tubes | Fisher | 14-595-35A | |
Glass Pasteur pipette | Fisher | 13-678-20C | |
Hexane, anhydrous grade | Sigma | 296090 | |
L-Adenine >99% | Sigma | A8626 | |
L-Alanine >98% | Sigma | A7627 | |
L-Arginine >99% | Sigma | A1270000 | |
L-Asparagine >98% | Sigma | A0884 | |
L-Aspartate >98% | Sigma | A9256 | |
L-Cysteine >97% | Sigma | W326305 | |
L-Glutamic acid monosodium salt monohydrate >98% | Sigma | 49621 | |
L-Glutamine >99% | Sigma | G3126 | |
L-Glycine >99% | Sigma | G8898 | |
L-Histidine >99% | Sigma | H8000 | |
L-Isoleucine >98% | Sigma | I2752 | |
L-Leucine >98% | Sigma | L8000 | |
L-Lysine >98% | Sigma | L5501 | |
L-Methionine, HPLC grade | Sigma | M9625 | |
L-Phenylalanine, reagent grade | Sigma | P2126 | |
L-Proline >99% | Sigma | P0380 | |
L-Serine >99% | Sigma | S4500 | |
L-Theronine, reagent grade | Sigma | T8625 | |
L-Tryptophan >98% | Sigma | T0254 | |
L-Tyrosine >98% | Sigma | T3754 | |
L-Uracil >99% | Sigma | U0750 | |
L-Valine >98% | Sigma | V0500 | |
Methanol, ACS grade | Fisher | A412 | |
Oleic acid >99% | Sigma | O1008 | |
p-anisaldehyde | Sigma | A88107 | |
Petroleum ether, ACS grade | Sigma | 184519 | |
Phosphatidylcholine, dipalmitoyl >99% | Sigma | P1652 | |
Pipettes | Eppendorf | 2231000713 | |
Potassium chloride, ACS grade | Sigma | P3911 | |
Sodium Hydroxide pellets, certified ACS | Fisher | S318-100 | |
Squalene >98% | Sigma | S3626 | |
Succinic Acid crystalline/certified | Fisher | 110-15-6 | |
TLC saturation pad | Sigma | Z265225 | |
TLC silica gel 60G glass channeled plate | Fisher | NC9825743 | No fluorescent indicators |
Transparency plastic film | Apollo | 829903 | |
Tricine | Sigma | T0377 | |
Triolein >99% | Sigma | T7140 | |
Vortex mixer | Fisher | 02-215-414 | |
Whatman exposure cassette | Sigma | WHA29175523 | |
Yeast nitrogen base without ammonium sulfate and amino acids | Sigma | Y1251 |