The overall goal of this procedure is to measure basal and forskolin-stimulated lipolysis, using inguinal adipose tissue. This method can help answer key questions in field of adipose biology about lipolysis and triglyceride metabolism. The main advantage of this method is that it offers a direct, simple and cost-effective method to determine lipolysis in adipose tissues.
Demonstrating the procedure will be Padmamalini Baskaran, who is a senior research scientist in my laboratory. Begin by placing the mouse on its left side on a dissecting platform. Sterilize the skin with a 2x2 inch gauze soaked in 70%ethanol and make a lateral cut through the skin to reveal the underlying fatty layer.
Make a second one centimeter incision through the skin, below the ribs across the dorsal surface that connects with the first incision and use sterile forceps to slowly peel back the skin flap. The subcutaneous fat in the abdominal region is the inguinal fat, which is different from the subcutaneous fat around the upper arm, which demonstrates region specific lipolysis. Using a pair of scissors, carefully dissect the fat pad from the underlying muscle and fascia.
Place the fat pad in a petri dish containing fresh, room temperature PBS and isolate the second fat pad. Using sharp scissors, cut the fat tissue into five to eight pieces and incubate the pieces in 200 microliters of incubation medium, at 37 degrees celsius and 5%carbon dioxide for 60 minutes. At the end of the incubation, transfer the supernatant into a new container for 80 degrees celsius for storage and transfer the fat pieces into one milliliter of extraction at 37 degrees centigrade, at 100 RPM.
After one hour, discard the extraction solution and transfer the tissues into a sterile microfuge tube containing 500 microliters of lysis solution for an overnight incubation at 55 degrees centigrade and 100 RPM. Then determine the protein concentration of the tissue according to standard protocols. For forskolin-stimulated lipolysis, first, pre-incubate 20 milligrams of inguinal fat pad pieces isolated as just demonstrated.
In 200 microliters of fresh DMEM, supplemented with BSA, forskolin and tricsin C at 37 degree celsius and 5%carbon dioxide. The addition of tricsin C inhibits a cyclase synthetase, which proves the regeneration of triglycerides from glycerol and free fatty acids. Thereby allowing for measurement of forskolin-stimulated lipolysis.
After one hour, transfer the pieces into fresh DMEM, plus BSA, forskolin and tricsin C, for a second 60 minute incubation at 30 degree centigrade and 5%carbon dioxide. Collecting the supernatant at the end of the incubation for 80 degree celsius storage. Next, extract the fat from the tissue pieces with extraction solution is just demonstrated and transfer the tissue into a sterile microfuge tube containing 500 microliters of lysin solution for an overnight incubation at 55 degree celsius under vigorous shaking.
Then, determine the protein concentration according to standard protocols. To determine the glycerol content of the samples, first, thaw the supernatants from 80 degrees centigrade on ice and dilute the samples to a one to 10 concentration in de-ionized water. Next add glycerol standards in ascending concentrations to one centimeter path length disposable methacrylate cuvettes per concentration and bring the final volume in each cuvette up to 10 microliters with de-ionized water.
Add 10 microliters of each diluted sample into cuvettes labeled with the corresponding sample number and turn on the ultraviolet visible spectro photometer. Set the spectro photometer wavelength to 540 nanometers and add 10 microliters of each standard into a new series of standard cuvettes. Using a one milliliter pipette, add 8 milliliters of room temperature free glycerol reagent to each standard in sample cuvette and cover the cuvettes with a 1.5 centimeter square plastic paraffin film.
Mix the contents in each cuvette with three inversions and allow the standards to equilibrate for 10 minutes at room temperature. Then, place each cuvette into the spectrophotometer and record the absorbents. In this table, represented of basal and forskolin-stimulated lipolysis of inguinal fat pads are shown.
A high fat diet feeding has no effect on basal lipolysis, but demonstrates a significant suppression of forskolin-stimulated lipolysis. Capsaicin on the other hand, increases both basal and forskolin-stimulated lipolysis. Once mastered, this technique can be completed in two days if it is performed properly.
While attempting this procedure, it is important to remember to use fat free BSA and while performing the procedure, not to shake or invert the samples to prevent air bubbles. After each level have met, this method paved the way for researchers in the field of adipose biology for measuring lipolysis in adipose tissues in rodent models of obesity. After watching this video, you should have a good understanding of how to determine basal and forskolin=stimulated lipolysis in inguinal white adipose tissues.
Don't forget, working with a glycerol reagent can be unstable and how the reagent is stored and the lifetime of the reagent should be considered while working with it.
