This protocol details the determination of the rate of adipocyte lipolysis in cultured adipocytes or ex vivo adipose tissue, allowing for the comparison of lipolytic rates between murine models or treatments. This protocol uses serial sampling, which allows one to internally validate that lipolysis is being measured in the linear phase and reduces measurement error. With optimization, this protocol may be used to measure lipolysis in brown adipose tissue, adipose tissue from other organisms, or adipogenic cell lines.
Ex vivo tissues must be chopped into small chunks of consistent size and shape to allow for sequestration of the released free fatty acids by BSA in the media. To begin, prepare the 5%BSA medium by dissolving five grams of Bovine Serum Albumin, or BSA, in 100 milliliters of Dulbecco's Modified Eagle's Medium, or DMEM, without phenol red. Gently stir the solution to dissolve the BSA.
Then prepare working concentrations of control and stimulation media. Before using, warm the media to 37 degrees Celsius. Label a 96-well plate for media collection.
Inside a sterile fume hood, perform isolation and differentiation of 100%confluent primary preadipocytes. Every two to three days, change the culture media containing insulin maintaining a volume of one milliliter per well. For this study, use only cultures having differentiation rates above 90%and similar across groups.
Then culture the cells in insulin free media for 24 hours before measuring lipolysis. Wash the cells with DPBS once to remove residual serum from the culture media. Prepare a 48-well plate with one well for each replicate.
Place 400 microliters of room temperature DMEM into each well to be used. Use two-to-four control and two-to-four stimulated wells per tissue per mouse. Prepare a six-well plate with one well for each tissue to be collected from each mouse and put four milliliters of room temperature DMEM in each well to be used.
Place the collected gonadal adipose tissue into the six-well plate. Remove tissue from the well. Place it on a silicone mat and cut it into five to seven milligram chunks with scissors.
Blot the tissues on a clean towel to remove any media. Then weigh 25 to 30 milligrams, equivalent to five or six tissue chunks, for each assay well and place them in the previously prepared 48-well assay plate. Weigh the weight boat after removing the tissues and record the weight of any residue left behind.
Wipe the weight boat clean between samples and retear if necessary. Use a new weight boat for each tissue. Once all the tissue samples have been weighed, place the 48-well assay plate in a 37 degree Celsius incubator under 10%carbon dioxide for 15 minutes.
Perform media collection in a sterile fume hood. At time zero, after removing the media, add 400 microliters of either control or stimulation media per well and place the assay plate into an incubator at 37 degrees Celsius and 10%carbon dioxide. For ex vivo tissue culture, carefully remove media using a pipette and avoid applying suction.
At times one, two, three, and four hours, collect 200 microliters of media, replace it with 200 microliters of the appropriate control or stimulation media, and return the assay plate to the incubator. Store the collection plate at four degrees Celsius. Thaw and mix the samples.
Warm the reagents to room temperature and dissolve one bottle of color reagent A using one bottle of solvent A and one bottle of color reagent B using one bottle of solvent B.After creating a free fatty acid or FFA standard curve, pipette standards and samples into a 96-well assay plate. The recommended sample volume is 10 microliters per well. Add 150 microliters of reagent A to each well and mix.
Incubate the assay plate at 37 degrees Celsius for five minutes. Read the absorbance of the plate at 550 nanometers and 660 nanometers and refer to this as reading A.Add 75 microliters of reagent B to each well and mix. Incubate the assay plate at 37 degrees Celsius for five minutes.
Then after removing the plate from the incubator, read the absorbance of the plate again at 550 and 660 nanometers. Refer to this reading as reading B.Reconstitute the free glycerol reagent with 36 liters of ultrapure water and acclimatize it to room temperature. Thaw and mix the samples.
Create a glycerol standard curve by making a 7.2 fold serial dilution of the glycerol standard solution and a water blank. Pipette 25 microliters of each standard and samples into the 96-well assay plate. Add 175 microliters of free glycerol reagent to each well and mix.
Incubate the assay plate at 37 degrees Celsius for five minutes. After removing the plate from the incubator, read the absorbance of the plate at 540 nanometers. Follow the steps described in the text to calculate the lipolytic rates in the respective R square values.
Then using the FFA and glycerol production rates for each sample as an individual data point, perform statistical analysis and plot the values if different lipolytic rates are being compared. If comparing ex vivo lipolytic rates across genotypes, use two or three samples per animal as technical replicates and use the average for one data point per animal so that the sample size is equal to the number of animals. Then perform statistical analysis with the data.
FFA and glycerol production in the in vitro differentiated preadipocytes was linear during the four-hour assay. The stimulated lipolytic rates were significantly higher than basal rates. In stimulated cells, the FFA-to-glycerol molar ratio was about three, indicating triglyceride lipolysis without significant reuptake or retention.
In the unstimulated cells, the ratio was about one, suggesting a non-lipolytic source of glycerol. In ex vivo cultures, larger tissue chunks having a lower surface area to volume ratio exhibited higher FFA retention, resulting in lower FFA-to-glycerol molar ratios. In the 25 milligram tissue chunks, FFA retention negatively impacted the rates of lipolysis.
Very tiny tissue chunks also exhibited reduced lipolytic rates. Reduction of BSA in the media significantly reduced both FFA and glycerol release. The FFA accumulation in the 5%BSA media after 24 hours stimulation was five millimolar while that of the 0.5%BSA media was only 0.6 millimolar.
The FFA levels in the collected media reached the danger zone of 2.3 millimolar in three hours. The lack of increase in media FFA and glycerol at four hours suggested feedback inhibition. Excluding the four-hour time point caused a small, but significant increase in the release rates.
The rate of FFA release was linear even between the usual time points. When removing media from the ex vivo lipolysis samples, it is important to ensure the tissue chunks are not accidentally removed by the pipette. It may be necessary to measure protein or lipid content in these samples to properly normalize the results.
