We are interested in brown and beige subtypes of adipocyte with disparate energy in the form of heat. While white adipocytes generally store energy as repeat, our goal is to develop a new treatment for obesity and diabetes based on promotion of energy expenditure rather than suppression of energy intake. We have identified a transcription factor in FY as a critical regulator of lung adipocyte differentiation by a canon-wide open chromatic analysis.
In FY and master transcriptional regulator of adipogenesis prepare gamma, co-localize as brown-fat specific enhancers. In the process of isolating stromal vascular fractions from mass adipose tissue, murine method and collagenase digestion of adipose tissue can lead to experimental variability and contamination. Our method uses a tissue dissociator for collagenase digestion, which facilitate XPF isolation, reduce experimental variability and contamination, and improves reproducibility.
Experiment using individual differentiated adipocyte are indispensable for research in our field. Our protocol would offer easy and reproducible SVF isolation for subsequent adipocyte differentiation, and would accelerate research in our community. Currently we are investigating the role of NFIA on formidable metabolism, such as body weight and glucose, using gain and loss of function mass models.
We will also explore other pharmacological activation of NFIA for each downstream pathway in near future. Begin by placing a seven to eight week old euthanized male mouse on clean bench surface. Cut the abdominal skin of the mouse with a pair of scissors towards the lower abdomen.
Excise the adipose tissue from the inner thighs. Place the excised tissue into tube C on ice, containing 2.5 milliliters of an enzyme mixture of enzymes DRA, and 2.35 milliliters of DMEM/F-12 without FBS or antibiotics. Using scissors, cut the adipose tissue into approximately two square millimeter-sized pieces.
Tightly close the cap of tube C and invert the tube. Attach the tube to the sleeve of a tissue dissociator and digest the samples at 37 degrees Celsius for 40 minutes. Next, prewarm DMEM/F-12 media containing FBS and antibiotics to 37 degrees Celsius.
After incubation, remove tube C from the tissue dissociator, and stop digestion by adding five milliliters of DMEM/F-12 with FBS and antibiotics. Gently pipette four times. Centrifuge the suspension at 700 g for 10 minutes at 20 degrees Celsius.
Without disturbing the cell palate, carefully aspirate the supernatant. Re-suspend the palate in 10 milliliters of DMEM/F-12 containing FBS and antibiotics, and gently pipette five times. Filter the cell suspension through a 70 micron diameter cell strainer placed on a 50 milliliter tube.
Centrifuge the filtrate at 250 g for five minutes. Re-suspend the palate in 10 milliliters of PBS. Before plating, centrifuge the cell suspension at 500 g for five minutes.
Discard the supernatant. Re-suspend the pellet in 10 milliliters of DMEM/F-12 containing FBS and antibiotics. Mix by pipetting the suspension gently 10 times.
Plate 10 milliliters of the suspension on a 10 centimeter collagen-coated dish. Place the dish in a cell culture incubator. Aspirate the medium and wash the cells three times with three milliliters of PBS per wash.
Add 10 milliliters of DMEM/F-12 containing FBS and antibiotics, and incubate. Oil Red O staining showed lipid laden adipocytes seven days after inducing adipocyte differentiation. The degree of full differentiation was confirmed by mRNA expression analysis of adipogenesis regulators.
Rosiglitazone induced a dose-dependent effect on the expression levels of brown fat-specific genes, such as Ucp1 one and Ppargc1a. However, for Fabp4, the effect saturated at 0.1 micromole Rosiglitazone concentration. Differentiated adipocytes can be used for various functional and mechanistic analyses, such as oxygen consumption rate analysis, and chromatin immunoprecipitation.
