Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit

Podsumowanie

This protocol establishes a novel method of lipid droplet isolation and purification from mouse livers, using a well-established endoplasmic reticulum isolation kit.

Streszczenie

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral lipids encased by a monolayer of phospholipids and proteins. Hepatic LD lipids, such as ceramides, and proteins are implicated in several diseases that cause hepatic steatosis. Although previous methods have been established for LD isolation, they require a time-consuming preparation of reagents and are not designed for the isolation of multiple subcellular compartments. We sought to establish a new protocol to enable the isolation of LDs, endoplasmic reticulum (ER), and lysosomes from a single mouse liver.

Further, all reagents used in the protocol presented here are commercially available and require minimal reagent preparation without sacrificing LD purity. Here we present data comparing this new protocol to a standard sucrose gradient protocol, demonstrating comparable purity, morphology, and yield. Additionally, we can isolate ER and lysosomes using the same sample, providing detailed insight into the formation and intracellular flux of lipids and their associated proteins.

Wprowadzenie

LDs are bioactive organelles found in the cytosol of most eukaryotic cells and some prokaryotic cells^1,2,3. The LD core is composed of neutral lipids such as triglyceride (TG) and cholesterol esters. They also contain ceramides, bioactive lipids involved in cellular signaling pathways^4,5. LDs are surrounded by a phospholipid monolayer and coated with proteins, including the perilipin proteins perilipin 2 (PLIN2) and 3 (PLIN3)^1,5,6. Also present are lipogenic enzymes, lipases, and membrane trafficking proteins that have been linked to several hepatic steatotic diseases, including nonalcoholic fatty liver disease, alcoholic liver disease, and hepatitis C^{3,5,6,7,8,9,10}.

LDs are thought to form from the outer membrane of the ER and contain newly synthesized TG sourced from ER-derived free fatty acids¹¹. However, it remains unknown whether the pooled lipids bud, remain connected, or are excised from the ER^6,11, making the isolation of LDs free from ER technically challenging. Free fatty acids can be liberated from LDs by the action of surface lipases to provide for energy production or membrane synthesis. Additionally, LDs can be degraded via lipophagy as a regulatory mechanism and to produce free fatty acids¹².

Besides the ER and lysosomes, there are other organelles—such as mitochondria, endosomes, peroxisomes, and the plasma membrane—that are found within close association of LDs¹¹. This tight polygamy makes it difficult to perform a pure LD extraction. However, neutral lipids’ innate low density can be taken advantage of via centrifugation⁵.

Traditionally, LDs have been isolated using a sucrose density gradient^1,5,13. However, these methods have not been designed to isolate other organelles. Additionally, they require the time-consuming preparation of reagents. This protocol adapts a commercially available ER isolation kit (see the Table of Materials) to allow for LD isolation. We use the extraction buffer provided by the kit, adding an LD isolation step. Furthermore, the protocol can also be used for ER and lysosomal isolation using the same samples, allowing for a more comprehensive image of the life cycle of LDs. To validate this new method, we assay LD yield, morphology, size, and purity. We compare the results presented here to those obtained with a widely used protocol utilizing a sucrose gradient for LD isolation.

We used a 10- to 12-week-old, 20 g female C57BL/6 mouse fasted for 16 h (food removal at 5.00 P.M. for a 9.00 A.M. LD isolation time) with free access to water. The typical yield for TG is 0.6 mg/g liver, and for LD protein, it is 0.25 mg/g liver. This provides enough material for ~10 immunoblots by SDS PAGE. The protocol below details the reagents used and a protocol suitable for a single 1 g liver. The sucrose gradient isolation protocol is adapted from Sato¹⁴ and Wu¹⁵.

Protokół

All experiments were conducted on a laboratory bench with personal protective equipment appropriate for biosafety level 1, including a lab coat, gloves, and safety goggles. Experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pennsylvania. All efforts were made to minimize animal discomfort, and the animals were treated with humane care.

1. LD isolation using an ER isolation kit

1. Preparation
   NOTE: The listed amounts are suitable for a single 1 g mouse liver.
   1. Prepare 10 mL of 1x isotonic extraction buffer (IE buffer, from the ER isolation kit) by diluting 2 mL of 5x IE buffer with 8 mL of ultrapure water. Cool the buffer by placing it on ice.
   2. Prepare 100 mL of 1x phosphate-buffered saline (PBS) by diluting 10 mL of 10x PBS in 90 mL of ultrapure water. Cool it by placing it on ice.
   3. Prepare 10 mL of 5% polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (PGPE) by diluting 500 µL of PGPE in 9.5 mL of ultrapure water. Use a 1 mL syringe to measure out PGPE as it is viscous. Cool it by placing it on ice.
   4. Cool all tubes by placing them on ice: 1.7 mL microcentrifuge tubes, 15 mL centrifuge tubes, 50 mL high-speed centrifuge tubes, and 13 mL ultracentrifuge tubes.
   5. Cool all required centrifuges to 4 °C: a microcentrifuge, a high-capacity centrifuge for 15 mL tubes, a high-speed centrifuge for 50 mL tubes, and an ultracentrifuge.
   6. Sterilize forceps and dissection scissors by autoclaving.
2. Tissue preparation
   1. Euthanize the mouse by placing it in a chamber filled with 100% CO­₂ for 10 min. Confirm euthanasia by performing cervical dislocation.
   2. Using sterile forceps and dissection scissors, cut the skin and muscle layers of the abdomen on the posterior/anterior axis to expose the liver.
   3. Cut the ligaments connecting the liver to the diaphragm and intestine. Use forceps to pull the intestine away from the liver, toward the posterior, to expose the ligaments.
   4. Cut between the liver and the dorsal ribcage, moving from anterior to posterior until the liver is liberated.
   5. Transfer the liver to a cold 5 cm Petri dish with 10 mL of cold 1x PBS.
   6. Wash the liver 2x on a rocking platform in 10 mL of cold 1x PBS for 5 min at 4 °C in the 5 cm Petri dish. Pour off 1x PBS after the final wash.
   7. Use a size-10 sterile surgical blade (see the Table of Materials) to dice the liver into 3–5 mm pieces in the 5 cm Petri dish (Figure 1A).
   8. Wash the diced liver 2x with 10 mL of cold 1x PBS for 5 min at 4 °C in the 5 cm Petri dish.
      NOTE: For lysosome isolation, transfer 0.5 g of liver to a clean 45 mL homogenizer and follow the directions supplied by the lysosome extraction kit (see the Table of Materials).
   9. Decant 1x PBS and transfer the diced liver pieces to the cold 45 mL Dounce homogenizer. Ensure all pieces go to the bottom of the homogenizer.
   10. Add 7 mL of cold 1x IE buffer (from the ER isolation kit) and homogenize on ice by moving the pestle up and down 20x. Ensure that the pestle goes to the bottom of the homogenizer during each stroke.
   11. Transfer the homogenate (Figure 1B) to a cold 15 mL centrifuge tube.
   12. Rinse the homogenizer with 1 mL of cold 1x IE buffer (from the ER isolation kit) and add this to the rest of the homogenate.
3. LD isolation
   1. Remove nuclei by centrifuging the homogenate in a high-capacity centrifuge at 1,000 x g for 10 min at 4 °C (Figure 1C).
   2. Transfer the postnuclear supernatant (PNS) to a new, cold 50 mL centrifuge tube. To prevent LD loss, ensure that any lipid gathered at the top of the 15 mL tube is resuspended before transferring the PNS.
   3. Take a 100 µL aliquot of the PNS for purity analysis and place it in a cold 1.7 mL microcentrifuge tube. Set it aside on ice.
   4. To remove mitochondria, centrifuge the PNS sample, not the aliquot, in a refrigerated high-speed centrifuge at 12,000 x g for 15 min at 4 °C.
      1. Optional: For crude LD (CLD) isolates (with cytosol and cell membrane contamination), collect the top lipid layer using a glass pipette and transfer it to a 1.7 mL microcentrifuge tube. Continue to section 1.4.
   5. Transfer the postmitochondrial supernatant (PMS) to an ultracentrifuge tube. To prevent LD loss, ensure that any lipid gathered at the top is resuspended before transferring the PMS.
   6. Take a 100 µL aliquot of the PMS for purity analysis and place it in a cold 1.7 mL microcentrifuge tube. Set it aside on ice.
   7. Fill an ultracentrifuge tube to the brim with cold 1x IE buffer to prevent the tube collapsing during ultracentrifugation.
   8. Balance the samples and centrifuge them in an ultracentrifuge at 100,000 x g for 60 min at 4 °C (Figure 1D).
   9. To remove the LD layer, tilt the tube at a 45° angle and use a glass pipette to aspirate. Transfer the pellet to a cold 1.7 mL microcentrifuge tube.
      NOTE: The pellet contains isolated ER. Use this fraction downstream for ER isolation.
   10. Collect 100 µL of the post-ER supernatant (PER) under the lipid layer for purity analysis.
4. LD washing
   1. Add 1x PBS to the LD fraction collected in a 1.7 microcentrifuge tube, to a total volume of 1.3 mL.
   2. Vortex the sample.
   3. Centrifuge in a microcentrifuge at top speed for 5 min at 4 °C to pellet any cell debris. Warm the tube gently with hands to help resuspend the LD layer. Transfer the supernatant, including the lipid layer, to a new 1.7 mL microcentrifuge tube.
   4. Repeat steps 1.4.1–1.4.3 2x–3x, until the pellet is no longer visible.
   5. Add 1x PBS to the pellet-free LD supernatant to a final volume of 1.5 mL. Wash the lipid fraction by centrifugation in a microcentrifuge at top speed for 5 min at 4 °C.
   6. Remove 1 mL of 1x PBS (underneath the lipid layer) with a glass pipette without disturbing the lipid layer.
   7. Repeat steps 1.4.5 and 1.4.6 4x–5x, until the 1x PBS is visually transparent with no turbidity.
   8. Remove all 1x PBS below the lipid layer, using a glass pipette (Figure 1E).
   9. Use the result, a pure LD fraction, for downstream analysis.
5. LD protein quantitation by bicinchoninic acid assay
   NOTE: Do not use a bicinchoninic acid assay (BCA) if LD morphology is to be assessed.
   1. Resuspend the LD in 500 μL of 5% PGPE.
   2. Boil the samples for 5 min at 95 °C, vortex them, and incubate the samples at room temperature for 5 min to cool.
   3. Repeat step 1.5.2 an additional 2x.
   4. Sonicate the samples for 5 min with a 30 s on/off cycle, at medium intensity.
   5. Normalize the samples by measuring the protein concentration in all purity analysis samples and in the LD fraction by BCA assay.

2. LD isolation using sucrose density gradient

1. Preparation
   1. Make 200 mL of TE buffer (10 mM Tris-HCl [pH 7.4], 1 mM EDTA [pH 8.0]). Cool it by placing it on ice.
   2. Make 100 mL of 60% (w/v) sucrose solution in TE buffer by dissolving 60 g of sucrose in TE buffer, bringing the final volume to 100 mL. Cool it by placing it on ice.
   3. Make 6 mL of 40% (w/v) sucrose solution by adding 4 mL of 60% sucrose to 2 mL of TE buffer. Cool it by placing it on ice.
   4. Make 6 mL of 25% (w/v) sucrose solution by adding 2.5 mL of 60% sucrose to 3.5 mL of TE buffer. Cool it by placing it on ice.
   5. Make 4 mL of 10% (w/v) sucrose solution by adding 0.7 mL of 60% sucrose to 3.3 mL of TE buffer. Cool it by placing it on ice.
   6. Make 10 mL of protease and phosphatase inhibitor solution by adding one tablet of both protease and phosphatase inhibitors to 10 mL of TE buffer.
   7. Prepare tissue homogenate buffer by adding 4 mL of protease and phosphatase inhibitor solution to 50 mL of 60% sucrose stock solution. Cool it by placing it on ice.
   8. Prepare overlay buffer by adding 2 mL of protease and phosphatase inhibitor solution to 50 mL of TE buffer. Cool it by placing it on ice.
   9. Prepare 100 mL of 1x PBS by diluting 10 mL of 10x PBS in 90 mL of ultrapure water. Cool it by placing it on ice.
   10. Prepare 10 mL of 5% PGPE by diluting 500 µL of PGPE in 9.5 mL of ultrapure water. Use a 1 mL syringe to measure out PGPE as it is viscous. Cool it by placing it on ice.
   11. Cool the following centrifuge tubes by placing them on ice: 1.7 mL microcentrifuge tubes, 15 mL centrifuge tubes, 50 mL high-speed centrifuge tubes, and 13 mL ultracentrifuge tubes.
   12. Cool the required centrifuges to 4 °C: a microcentrifuge, a high-capacity centrifuge (for 15 mL tubes), a high-speed centrifuge (for 50 mL tubes), and an ultracentrifuge.
2. Tissue preparation
   1. Use steps 1.2.1–1.2.9 to prepare the tissue for homogenization.
   2. Add 7 mL of cold tissue homogenate buffer and homogenize it on ice by moving the pestle up and down 20x. During the first five strokes, ensure the pestle goes to the bottom of the homogenizer.
   3. Transfer the homogenate to a cold 15 mL centrifuge tube.
   4. Rinse the homogenizer with 1 mL of cold tissue homogenate buffer and transfer it to the previously used 15 mL centrifuge tube.
   5. Bring the volume of the homogenate up to 10 mL with tissue homogenate buffer.
   6. Centrifuge the sample in a high-capacity centrifuge at 1,000 x g for 10 min at 4 °C.
   7. Transfer 8 mL of supernatant to a 50 mL centrifuge tube.
   8. Transfer 100 µL of the remaining supernatant as PNS sample for purity analysis.
3. Sucrose gradient
   1. Tilt the 50 mL centrifuge tube containing the 8 mL of PNS at a 45° angle. Carefully add 6 mL of 40% sucrose solution, ensuring the two phases stay separate.
   2. In a similar fashion, slowly add 6 mL of 25% sucrose solution; then, add 4 mL of 10% sucrose solution; finally, add 8 mL of overlay buffer.
   3. Centrifuge the sample in a high-speed centrifuge at 30,000 x g at 4 °C for 30 min.
   4. Transfer the top layer containing the LDs to a 1.7 mL microcentrifuge tube.
   5. For LD washing, follow the steps in section 1.4.

Wyniki

We have performed the LD isolation with the ER kit on approximately 30 mice and compared the results with those following the sucrose isolation protocol on approximately 40 mice. The reported findings are typical for both protocols. Mice were fasted overnight with free access to water, to increase the LD yield. LD isolation using a sucrose gradient was run in parallel with the ER kit LD isolation protocol. Samples of PNS, PMS, PER, CLDs, and LDs were collected throughout the ER kit LD iso...

Dyskusje

Hepatic LD biology is increasingly being recognized as a critical regulator of hepatocellular physiology in both health and disease. As bona fide organelles, LDs are dynamic, interact with other cellular structures, and contain within them bioactive components involved in both lipid and glucose homeostasis. The sucrose gradient is routinely used for LD isolation and enables investigators to study LD structure, but it requires them to make numerous buffers. We have demonstrated that the use of a commercially available ER ...

Materiały

Name	Company	Catalog Number	Comments
BioExpress Vortex Mixer	GeneMate	S-3200-1	Vortex Mixer
Centrifuge 54242 R	Eppendorf	54242 R	Refrigerated Counter Microcentrifuge Rotor: FA-45-24-11
Centrifuge Sorvall RC6 +	Thermo Scientific	RC6+	Refrigerated High Speed Centrifuge Rotor: F21S-8x50y
Centrifuge Sorvall Legend RT+	Thermo Scientific	Legend RT+	Refrigerated High Capacity Centrifuge Rotor: 75006445 Bucket: 75006441
cOmplete Protease Inhibitor Cocktail	Sigma Aldrich	04 693 116 001	Protease Inhibitor Tablets
Diagenode Bioruptur UCD-200	Diagenode	UCD-200	Sonication System
Dounce Tissue Grinder (45 mL)	Wheaton	357546	Use Loose pestle
Dulbecco's Phosphate-Buffered Saline (1x)	Corning	21-031-CM
Endoplasmic Reticulum Isolation Kit	Sigma Aldrich	ER0100	For ER kit Extraction: Contains: Isotonic Extraction Buffer 5X 100 mL Hypotonic Extraction Buffer 10 mL Calcium chloride, 2.5 M solution 5 mL OptiPrep Density Gradient Medium 100 mL Blunt Nosed Needle 1 ea.
External light source for flourescent excitation	Leica	Leica EL6000
Hydrochloric acid	Sigma Aldrich	H1758	Hydrochloric Acid
ImageJ			Image Analysis Software
Inverted Modulating Contrast Microscope	Leica	Leica DM IRB
Lysosome Enrichment Kit for Tissues and Cultured Cells	Thermo Fisher Scientific	89839	For Lysosome Isolation
Microscope Camera	Qimaging	QICAM fast 1394
Optima XL-100K Ultracentrifuge	Beckman-Coulter	XL-100K	Ultracentrifuge Rotor: SW41Ti
Pasteur Pipettes	Fisher Scientific	22-042817
Petri Dish 5 cm	Fisher Scientific	FB0875713A
Pierce BCA Protein Assay Kit	Thermo Fisher Scientific	23225
PhosSTOP	Sigma Aldrich	04 906 845 001	Phosphatase Inhibior Tablets
Sterile Surgical Blade #10	Pioneer	S2646
Sucrose	Fisher Scientific	S5-500	Crystalline/Certified ACS
Triglyceride Liquicolor Kit	Stanbio	2200-225	Colorimetric Triglyceride kit
Tris Base	Fisher Scientific	BP152-1	For TE buffer
Triton X-100	Fisher BioReagents	BP151-100	5%, Polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether, Protein Lysis Reagent
UltraPure EDTA (0.5M, pH 8.0)	Thermo Fisher Scientific	15575-038	For TE buffer