Adipolysis Assay

The ability to modulate the number of adipocytes and/or the size of adipocytes is a key in the development and physiology of obesity as well as the origin of chronic disease. Triglycerides can be broken down into free fatty acids (FFA) and glycerol by a process of adipolysis (lipolysis). Adipolysis is a highly regulated process that allows for appropriate FFA delivery to meet energy needs.

3T3-L1 cells are well-characterized models for studying lipid metabolism including the uptake and release of fatty acids from fat cells. This model shares similar morphology, gene expression, and metabolism with adipocytes in vivo. During terminal differentiation, the fibroblast-like preadipocytes undergo a series of morphological and biochemical changes to eventually accumulate lipid droplets. Triglycerides stored in these lipid droplets can be stimulated by known factors or compounds to release FFA and glycerol. The latter can be determined by incubation with glycerol kinase, glycerol phosphate oxidase, and horseradish peroxidase in the presence of a colorimetric substrate to produce a chromophore at 540 nm. The amount of glycerol released into the medium is proportional to the level of triglyceride storage and/or degree of adipolysis. Adipolysis assay allows investigators to screen compounds involved in lipid storage and metabolism.

Materials and Reagents

1) IMBX Solution (0.5 M 3-isobutyl-1-methylxanthine (IBMX) in DMSO)
2) Insulin Solution (10 mg/mL recombinant human insulin)
3) Dexamethasone Solution (10 mM dexamethasone in ethanol)
4) Isoproterenol Solution (10 mM isoproterenol in water)
5) Glycerol Standard Solution (0.26 mg/mL glycerol standard)
6) Wash Solution (Hanks' Balanced Salt Solution)
7) Incubation Solution (Hanks' Balanced Salt Solution)
8) 3T3-L1 Preadipocyte
9) Spectrophotometer or 96-well plate reader
10) Induction Medium (adding 10 μL of IMBX Solution, Insulin Solution and Dexamethasone Solution to 10 mL of DMEM containing 10% FBS)
11) Insulin Medium (adding 10 μL of Insulin Solution to 10 mL of DMEM containing 10% FBS)
12) Free Glycerol Assay Reagent (0.75 mM ATP, 3.75 mM Magnesium salt, 0.188 mM 4-aminoantipyrine, 2.11 mM sodium-N-ethyl-N (3-sulfopropyl) m-anisidine, 1.25 units/mL microbial glycerol kinase, 2.5 units/mL microbial glycerol phosphate oxidase, 2.5 units/mL horseradish peroxidase, 0.05% sodium azide, pH 7.0)

3T3-L1 Preadipocyte Cell Differentiation

1) Seed a 96-well plate with 3 x 10⁴ cells/well.
2) Grow preadipocytes to confluence in DMEM containing 10% calf serum.
3) Two days post confluence (Day 0), change the medium to either Induction Medium (treatment group) or fresh DMEM containing 10% FBS (control group).
4) Three days after the induction (Day 3), change the medium to Insulin Medium (treatment group) or fresh DMEM containing 10% FBS (control group).
5) Five days after the induction (Day 5), change the medium to fresh Insulin Medium (treatment group) or DMEM containing 10% FBS (control group).
6) Monitor visible accumulation of lipid droplets under a microscope two days later (Day 7). If more differentiation is desired, change medium to fresh Insulin Medium (treatment group) or DMEM containing 10% FBS (control group) and every three days monitor the degree of differentiation.
7) More than 80% of the cells are usually differentiated by Day 7. Cells may be maintained for up to two weeks after this point for the Adipolysis Assay.

Glycerol Standard Curve

1) Obtain eight clean test tubes and label them #1 through #8.
2) Aliquot 100 μL of culture medium into tubes #2 - #8.
3) Transfer 200 μL of Glycerol Standard Solution into tube #1.
4) Serially dilute the standard by removing 100 μL from tube #1 and placing it into tube #2; mix thoroughly. Next remove 100 μL from tube #2 and place it into tube #3; mix thoroughly. Repeat it for tubes #4, #5, #6, and #7.
5) Do not add any standard to tube #8.

Adipolysis Assay Procedure

1) Carefully aspirate the culture medium from each well.
2) Replace with fresh medium containing test compounds at desired concentrations. The Isoproterenol Solution may be used as a positive control by diluting to yield a 10 μM solution. Negative controls with medium alone should also be included.
3) Incubate cells at 37°C for the desired time period. Isoproterenol begins to induce adipolysis within one hour and the amount of detectable free glycerol increases linearly afterwards. The optimal incubation time period must be determined for each experimental compound.
4) Collect cell culture supernatants from each well into glycerol-free containers. Samples may be assayed immediately or stored at -20°C.
5) To perform the assay, transfer 25 μL of the standards (tubes 1-8) prepared above into a new 96-well plate. We recommend that the standards be run in duplicate.
6) Transfer 25 μL of each supernatant collected in step 4) to the corresponding wells on the new plate. Depending on the experimental compounds being tested, samples may need to be diluted with culture medium prior to addition to the plate in order to fall within the range of the standard curve.
7) Add 100 μL of diluted Free Glycerol Assay Reagent per well and incubate for 15 minutes at room temperature.
8) Read absorbance at 540 nm.
Note: Each plate should contain a glycerol standard curve, wells containing medium only, and wells containing supernatants from samples treated with and without test compounds. It is recommended that standards be run in duplicate and that each treatment be performed in triplicate.

Data Analysis

1) Plotting the standard curve - Make a plot of absorbance at 540 nm as a function of glycerol concentration and determine the equation of the line.
2) Determination of Glycerol concentration by the following formula:

Troubleshooting

For research use only. Not for any other purpose.