Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Podsumowanie

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

Streszczenie

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Wprowadzenie

Fatty acids are essential structural components of membranes as well as efficient energy storage molecules. Additionally, fatty acids can be cleaved from cellular membranes by lipases and be enzymatically modified to produce signaling effectors¹. Naturally occurring polyunsaturated fatty acids (PUFAs) contain two or more cis double bonds. The omega-3 fatty acids and the omega-6 fatty acids are distinguished from each other based on the positions of double bonds with respect to the methyl end of the fatty acid. Healthy diets require both omega-6 and omega-3 fatty acids. However, Western diets are particularly rich in omega-6 fatty acids and poor in omega-3 fatty acids. A high omega-6 to omega-3 fatty acid ratio is associated with increased risk of cardiovascular and inflammatory diseases, however, the precise beneficial and detrimental functions of specific fatty acids are not well understood². The roundworm Caenorhabditis elegans is useful in studying fatty acid function because it synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids, including an omega-3 desaturase, an activity that is absent in most animals^3,4 . Mutants lacking fatty acid desaturase enzymes fail to produce specific PUFAs, leading to a range of developmental and neurological defects^4-6.

To study the physiological effects of dietary fatty acids, we have developed a biochemical assay compatible with genetic analysis using both mutant and RNAi knock-down techniques in C. elegans. Supplementation with specific PUFAs is achieved by adding a fatty acid sodium salt solution to the agar medium prior to pouring. This results in PUFA uptake by the E. coli food source, where it accumulates in the bacterial membranes. C. elegans ingest the PUFA-containing bacteria, and this dietary supplementation is sufficient to rescue the defects of PUFA-deficient mutants. Supplementation of most fatty acids has no detrimental effects on wild type animals, however, specific omega-6 fatty acids, especially dihomo-gamma linolenic acid (DGLA, 20:3n-6) cause a permanent destruction of C. elegans germ cells^7,8 .

Gas chromatography is used to monitor the uptake of the supplemented fatty acid in the bacterial food source (either OP50 or HT115) as well as in the nematodes. The addition of the detergent Tergitol (NP-40) in the media allows for even distribution of fatty acids through the entire plate and more efficient uptake of the fatty acids by the E. coli and the nematodes. We have found that unsaturated fatty acids are readily taken up by bacteria and C. elegans, but the uptake of saturated fatty acids is much less efficient. This article will describe step-by-step how to supplement the agar media with fatty acids, as well as how to monitor fatty acid uptake in the nematode using gas chromatography.

Protokół

Polyunsaturated fatty acids are sensitive to heat, light and oxygen. Therefore, care must be taken when preparing fatty acid supplementation plates such that fatty acids are not exposed to excess heat and light. NGM media containing 0.1% Tergitol (NP-40) is autoclaved and partially cooled, after which fatty acid sodium salts are added with constant stirring. The plates are allowed to dry in the dark. Uptake of fatty acids by C. elegans cultured on these plates can then be monitored by gas chromatography.

1. Preparation of Fatty Acid Supplemented Media

1. Measure out media components into an appropriately sized flask. Per 1 L, add 17 g Bacto-agar, 2.5 g tryptone, 3 g NaCl, 1 ml 5 mg/ml cholesterol dissolved in ethanol, and 10 ml 10% Tergitol dissolved in water.
2. Add 80% of final desired volume of Millipore water and autoclave the media along with empty glass bottles (equal to the number of different fatty acid concentrations to be tested), as well as appropriate graduated cylinders.
3. Cool media in a water bath set to 55 °C. While the media is cooling prepare the working stock solution of the fatty acid sodium salt by breaking open the glass vial, using safety precautions and ensuring glass particles do not mix in with the fatty acids. Weigh out enough fatty acid to make a 100 mM working stock.
4. Bring the fatty acid solution to a final concentration of 100 mM with purified water. Fully dissolving the fatty acid sodium salt typically takes about 20-30 min. Purge the working stock of fatty acid with argon or nitrogen, because contact with an inert gas prevents oxidation. Cap the vial and store the working stock in the dark until media has cooled.
5. (Optional) If RNAi media is desired, measure ampicillin and Isopropyl β-D-1-thiogalactopyranoside (IPTG) solutions here.
6. When the agar has cooled to 55 °C add per 1 L: 1 ml of 1 M MgSO₄, 1 ml of 1 M CaCl₂, and 25 ml of phosphate buffer (108.3 g KH₂PO₄ and 35.6 g K₂HPO₄ per 1 L autoclaved). Add the filter sterilized ampicillin and IPTG solutions if making RNAi plates. For all types of plates, add sterile water to bring the final media volume to 1 L.
7. Near a flame, transfer media to an autoclaved and appropriately sized graduated cylinder and then add warm sterile water to desired volume. Transfer media back to initial flask and mix by stirring.
8. Near a flame, transfer media into a number of bottles equal to the number of concentrations to be tested by measuring with an autoclaved and appropriately sized graduated cylinder. Maintain the aliquoted media as liquid using a water bath until the fatty acid working stock has fully dissolved.
9. Place one bottle on a stir plate and stir until the media is warm to the touch, but not hot. Make sure to leave yourself enough time to stir in the fatty acid stock solution and pour plates before the media begins to solidify. If stir plate has temperature control, set it to 55 °C.
10. Dilute the 100 mM fatty acid working stock into the media for the final concentration desired. Stir media until the white precipitate is in solution (approximately 1 min). Media may still be slightly cloudy afterwards.
11. (Optional) If RNAi media is desired add ampicillin to 0.1 mg/ml and IPTG to 2 mM final concentration.
12. Pour media using an automated pipette aid and a sterile 25 ml pipette, adding 8 ml of media per 60 mm plate or 25 ml of media per 100 mm plate. Repeat fatty acid addition and plate pouring steps for the remaining bottles.
13. After the agar has solidified, cover the fatty acid-supplemented plates with a box or store at room temperature in a well-vented drawer to protect from light oxidation.
14. Seed E. coli OP50 onto plates two days after plates have dried. For 60 mm plates, pipette 300 µl of an overnight OP50 culture (grown at 37 °C). If RNAi plates were poured, seed with 300 µl of RNAi bacteria after the plates have dried for four days. Plate drying time may need to be adjusted due to differences in humidity in various lab environments.
15. Incubate the plates in a dark environment at room temperature while the bacterial lawn is drying.

2. Inducing Germ Cell Destruction by Supplementation of DGLA

1. C. elegans grown on plates containing 0.3 mM DGLA (20:3n-6) become sterile due to the destruction of germ cells in both larval stage and adult nematodes.
   1. Prepare a synchronized population of L1 larvae by treating gravid hermaphrodites with alkaline hypochlorite solution(for a 10 ml solution: 0.5 ml of 5M NaOH, 2.5 ml of household bleach, and 7 ml H₂O). Gently rock the gravid hermaphrodites in this solution until the adult worms dissolve. Eggs will be preserved, and can be pelleted by low speed centrifugation.
   2. Wash egg preparation 3 times in M9 buffer (3 g KH₂PO₄, 6 g Na₂HPO₄, 5 g NaCl, 1 ml 1 M MgSO₄, H₂O to 1 L. Add MgSO₄ after autoclaving). To provide enough oxygen, resuspend the eggs in a 15 ml plastic tube to a final volume of 5 ml of M9 buffer and rock on a shaker overnight.
   3. L1 larvae should be added to the supplemented plates two days after seeding with OP50, or one day after plating the RNAi bacteria. Incubate worms at 20 °C for three days or until they reach the adult stage.
2. Adult worms can be scored for sterility using a dissecting microscope with high enough magnification to visualize embryos developing in the uterus. Successful germ cell loss will appear as a clear uterus void of eggs.
3. Worms can also be scored by fixing and staining with the nucleic acid dye diamindinophenylindole (DAPI), which facilitates the visualization of nuclei in germ cells and developing embryos. A rapid DAPI stain is achieved by picking worms into a drop of M9 buffer on a watchglass⁹.
   1. Flood watch-glass with 1 ml of a 0.2 ng/m. DAPI in 95% ethanol solution, let sit for approximately 5 min.
   2. Pick worms into a drop of VectaShield on a slide, and then cover with a coverslip.
   3. Seal coverslip and store at 4 °C in the dark overnight for optimum staining, however slides can also be viewed immediately. Score for presence or absence of germ cell nuclei using a fluorescence microscope equipped with a UV lamp and filter.

3. Confirming Fatty Acid Uptake by Gas Chromatography

Overall fatty acid composition of C. elegans can be determined by producing fatty acid methyl esters (FAMEs) which are then separated and quantified using gas chromatography⁴.

1. Collect 500-1,000 adult worms by washing them off of the plates with water and transferring to a silanized 13 mm x 100 mm glass screw top tube.
2. Let worms settle by gravity, and then remove as much water as possible with a glass Pasteur pipette.
3. Wash worms once with water, and then again remove as much water as possible.
4. FAMEs are formed by adding 1 ml of 2.5 % H₂SO₄ in methanol, and then heating at 70 °C for 1 hr in a water bath.
5. Remove the tubes from the water bath and cool for 1 min.
6. Extract FAMEs by adding 1.5 ml water and 0.25 ml of hexane.
7. Recap tubes and shake vigorously.
8. Centrifuge tubes in a tabletop clinical centrifuge for 1 min at maximum speed to separate hexane from aqueous solvent.
9. Transfer hexane (top layer) to a GC vial insert within a GC vial, being careful not to transfer any of the aqueous phase. For GC analysis, 1-2 μl of FAMEs in hexane is injected onto a polar capillary gas chromatography column suitable for FAMEs analysis. The Agilent 7890 GC injector is set at 250 °C, with a flow rate of 1.4 ml/min, and the GC oven is programmed for an initial temperature of 130 °C, which is held for 1 min. Subsequently, the temperature is ramped 10 °C/min until 190 °C, and then ramped again at 5 °C/min until 210 °C and held for an additional 1 min.
10. To ensure that uptake of DGLA has occurred, analyze FAMEs by flame ionization detection (FID) or mass spectrometry (MS) detection, using authentic standards for the identification of the C. elegans fatty acids.

Wyniki

Supplementation of the C. elegans diet is limited by the ability of the bacterial food source to uptake and incorporate fatty acid into the bacterial membrane. To determine the ability of E. coli OP50 to assimilate various fatty acids into its membranes, OP50 was plated onto media with no supplement, 0.1 mM and 0.3 mM concentrations of stearic acid (18:0), sodium oleate (18:1n-9), and sodium DGLA (20:3n-6). Plates were dried at room temperature for 2 days in the dark, and incubated at 20 °C for 3 d...

Dyskusje

Here we describe a method of supplementation of C. elegans with dietary unsaturated fatty acids. As mentioned above, care must be taken in the preparation of PUFA supplemented plates because the reactive nature of the double bonds in PUFAs causes these fatty acids to be sensitive to oxidation through heat and light¹¹. To avoid oxidation, it is important to add the PUFA to the liquid agar medium after the media has cooled to 55 °C and stores plates in a dark environment.

Materiały

Name	Company	Catalog Number	Comments
Bacto-Agar	Difco	214010
Tryptone	Difco	211705
NaCl	J.T. Baker	3624-05
Tergitol	Sigma	NP40S-500mL
Cholesterol	Sigma	C8667-25G	(5 mg/mL in ethanol)
MgSO4	J.T. Baker	2504-01
CaCl2	J.T. Baker	1311-01
K2HPO4	J.T. Baker	3254-05
KH2PO4	J.T. Baker	3246-05
Sodium dihomogamma linolenate	NuCHEK	S-1143
Warm sterile Millipore water
Sterile water for collecting worms
Nuclease-free Water for DGLA stock solution	Ambion	AM9932
Ampicillin	Fisher Scientific	BP1760-25	100 mg/ml in water (for RNAi plates)
Isopropyl-beta-D-thiogalactopyranoside (IPTG)	Gold Biotechnology	12481C100	1 M in water (for RNAi plates)
HSO4	J.T. Baker	9681-03
Methanol	Fisher Scientific	A452-4
Hexane	Fisher Scientific	H302-4
diamindinophenylindole (DAPI)	Sigma	D9542
VectaShield	Vector Laboratories	H-1000
Glass Flask	Corning	4980-2L
Autoclaveable Glass bottles with stirbars	Fisherbrand	FB-800
Autoclaveable Glass Graduated Cylinder	Fisherbrand	08-557
Stir Plate	VWR	97042-642
Waterbath at 55+ °C	Precision Scientific Inc.	66551
Screwcap Brown Glass Vial	Sun SRI	200 494
Argon gas tank
Automated Pipette aid	Pipette-Aid	P-90297
Sterile Serological Pipettes (25 ml)	Corning	4489
Bunsen Burner	VWR	89038-534
Dissection microscope	Leica	TLB3000
Silanized glass tube	Thermo Scientific	STT-13100-S	for FAMEs derivitization
PTFE Screw caps	Kimble-Chase	1493015D
Clinical tabletop centrifuge	IEC
GC Crimp Vial	SUN SRi	200 000
GC Vial Insert	SUN SRi	200 232
GC Vial cap	SUN SRi	200 100
Gas Chromatograph	Agilent	7890A
Mass Spectrometry Detector	Agilent	5975C
Column for gas chromatography	Suppelco	SP 2380	30 m x 0.25 mm fused silica capillary column