Measurement of Basal and Forskolin-stimulated Lipolysis in Inguinal Adipose Fat Pads

All protocols follow the animal care guidelines of the University of Wyoming.

1. Animal Housing and Feeding

NOTE: Adult male wild type mice (C57BL/6) (age 12 to 24 weeks) were bred in the research animal facility as per the Institutional Animal Care and Use Committee (IACUC) approved protocols.

1. Starting from week 6 of age, house mice in groups of four in separate cages and randomly assign them into feeding groups of NCD or HFD (± 0.01% CAP) until week 38 of age.
   NOTE: CAP is an agonist of TRPV1 channel protein expressed in adipose tissues^11,17. Mix CAP with HFD in a blender inside the hood and transfer the blended mixture to a tray containing small 1 in² partitions. Keep the tray inside a -20 °C freezer. Remove the tray containing the HFD + CAP diet from the freezer after 24 h and store in a container at -20 °C freezer until use.
2. House mice in a climate-controlled environment (22.8 ± 2.0 °C, 45 – 50% humidity) with a 12/12-light/dark cycle with access to designated diet and water ad libitum.
3. At the end of 38 weeks, dissect inguinal adipose tissues and use for lipolysis experiments (sections 2-7).

2. Preparing Mice for the Experiments

1. Anesthetize mice by injecting ketamine and xylazine mixture (10 mg/kg and 80 mg/kg body weight, respectively). Inject 0.01 mL of the mixture/10 g body weight of mouse.
2. Confirm deep anesthesia by a firm toe pinch. If there is a pedal reflex, test the mouse again after at least 30 s.
3. Use vet eye ointment to prevent dryness of eyes during anesthesia. Do not leave mice unattended during any of the procedures.
4. Euthanize mice by injecting a high dose of ketamine and xylazine mixture injection (10 mg/kg and 80 mg/kg body weight, respectively) mixture (0.01 ml/10 g body weight) followed by cervical dislocation.
   NOTE: This method of euthanasia is approved by the IACUC of the University of Wyoming.

3. Isolation of Inguinal Adipose Fat Pads

1. Place the mouse (fed with NCD or HFD (± CAP) for 32 weeks) lying on its left for the procedure.
   NOTE: By placing the mouse on the left side, the left forelimb and left hindlimb will be on the dissection platform, while the right forelimb and right hindlimb will be facing away from the platform.
2. Sterilize the skin surface with a 2 inch² gauze pad soaked in about 2.5 mL of 70% ethanol. Make a 2 – 3 mm lateral cut through the skin using a scalpel to reveal the underlying fatty layer.
3. Make a 1 cm cut (depending on the size of the mouse) through the skin using a scalpel just below the rib cage across the dorsal surface joining the two lateral incisions.
4. Peel the skin flap by dragging it carefully using sterile forceps and leave the subcutaneous pad intact by not cutting the fat pads. This is the fat pad lying under the skin.
5. Dissect the fat pad carefully from the underlying muscle and fascia using a pair of scissors. Pull the fat pad as it is cut from the underlying muscle.
   NOTE: The weight and size of the fat pads depends on the type of mice (NCD or HFD (± CAP)-fed).
6. Use tweezers to transfer it to a Petri dish containing phosphate buffered saline (PBS) until the lipolysis experiments, at room temperature (~15 min).
7. Isolate the fat pads from the right side of the mouse as described in steps 3.2-3.6.

4. Basal Glycerol Release from Inguinal Fat Pads

1. Use sharp scissors to cut about 20 mg of fat tissue into 5 to 8 pieces.
2. Incubate the cut pieces of inguinal fat pads in 200 µL of incubation medium (Dulbecco's Modified Eagel's Medium (DMEM) containing 2% fatty acid free bovine serum albumin (BSA)) at 37 °C, 5% CO₂ and 95% humidity for 60 min.
   1. Collect the incubation medium and freeze at -80 °C for lipolysis assay.
      NOTE: The cut pieces are used for determination of protein concentration after lipid extraction.
   2. Transfer the cut fat pieces with the help of tweezers into 1 mL of extraction solution (chloroform:methanol (2:1, v/v) and 1% glacial acetic acid) and incubate for 60 min at 37 °C under vigorous shaking at 100 rpm. Discard the fat extraction solution.
      NOTE: This step will extract fat from the cut pieces. Discard the fat extraction solution as it will interfere with the protein determination.
   3. Transfer the tissue (from step 4.2.2) using tweezers into a sterile microfuge tube containing 500 µL of lysis solution (0.3 N NaOH containing 0.1% sodium dodecyl sulfate; SDS) and incubate overnight (12 h) at 55 °C under vigorous shaking at 100 rpm.
3. Determine the protein concentration of tissue (step 4.2.3) using the bicinchoninic acid (BCA) reagent and BSA as standard¹⁸.
4. Thaw the frozen medium (step 4.2.1) on ice and determine the glycerol content of the medium using a free glycerol reagent as described in protocol sections 6 and 7⁶.
   1. Extrapolate the concentration of glycerol in samples from the standard curve plotted using glycerol standards¹⁹ and express lipolysis as nanomoles glycerol per mg protein per h²⁰.

5. FSK-stimulated lipolysis

1. Preincubate about 20 mg of inguinal fat pad (5 to 8 cut pieces) obtained from NCD or HFD (± CAP)-fed wild type mouse in 200 µL DMEM containing 2% fatty acid free BSA, 10 µM FSK and 5 µM Triacsin C at 37 °C in 5% CO₂ and 95% humidity for 60 min.
   1. Transfer the tissue pieces using tweezers to an identical medium and incubate for a further 60 min at 37 °C, 5% CO₂ and 95% humidity. Collect the incubation medium and store at -80 °C until the lipolysis assay.
      NOTE: Stimulation of lipolysis causes a rapid release of fatty acids and glycerol within 15 min, which is linear during the first hour, and plateaus thereafter. Since the rate of stimulated lipolysis is higher and stable between the first and second hour of FSK stimulation, this protocol measured lipolysis in the stimulated state following the second incubation period as described previously¹. So, both the experimental steps (5.1 and 5.1.1) are necessary.
2. To extract fat from inguinal fat pads, transfer the cut pieces of inguinal fat pads (obtained from NCD or HFD (± CAP)-fed mice) from step 5.1 with the help of tweezers into 1 mL of extraction solution (chloroform:methanol (2:1. v/v) and 1% glacial acetic acid) and incubate for 60 min at 37 °C under vigorous shaking at 100 rpm. Discard the extracted fat.
3. Transfer the tissue (from step 5.2) using tweezers into a sterile microfuge tube containing 500 µL of lysis solution (0.3 N NaOH containing 0.1% SDS) and incubate overnight (12 h) at 55 °C under vigorous shaking at 100 rpm.
4. Determine the protein concentration and glycerol content of tissue as described in steps 4.3 and 4.4, respectively.

6. Preparation of Free Glycerol Reagent

1. Reconstitute the glycerol reagent¹⁹ in 40 mL of deionized water in an amber colored glass vial, place a stopper on the vial and mix well by inverting 10 times. Do not mix by shaking.
2. Store the vial at 4 °C inside a refrigerator protected from light by covering the vial completely with aluminum foil.
3. Proceed to the preparation of glycerol standard (section 7).

7. Preparation of Glycerol Standard and Determination of Glycerol Content

1. Make a stock of 1 mM by diluting 35 µL of the supplied stock with 65 µL of deionized water.
   NOTE: The manufacturer supplied glycerol standard stock is 2.8 mM glycerol.
2. Take five 1-cm path length disposable methacrylate cuvettes. Label the cuvettes using a marker as 0, 1.25, 2.5, 5 and 10 nmol standard.
3. Add 0, 1.25, 2.5, 5 and 10 µL of 1 mM glycerol standard to the respective labeled cuvettes. Make up the volume of each cuvette to 10 µL with deionized water.
4. Make 1 to 10 dilution of all samples (from step 4.4 or 5.1.1) by adding 10 µL of the sample to 90 µL of deionized water into fresh 500-µL prelabeled centrifuge tubes. Add 10 µL of the diluted samples into the cuvettes labeled with the respective sample identification number.
5. Switch on the UV-VIS spectrophotometer and set the wavelength to 540 nm.
6. Warm the free glycerol reagent to room temperature by keeping the vial (step 6.2) at room temperature for 15 min.
7. Set up a series of labelled cuvettes as blank "0" nmol standard (step 7.3), standards (step 7.3) and samples (step 7.4).
8. Add 10 µL of each of the standard and sample into the respective labeled cuvette. Use "0" nmol standard as blank.
9. Add 0.8 mL of free glycerol reagent into each cuvette, containing the glycerol standards using a 1 mL pipette.
10. Cover the cuvette with a 1.5 cm square plastic paraffin film and mix the contents by inverting the cuvette for 3 times and set aside at room temperature for 10 min.
11. Place the cuvette in the UV-VIS spectrophotometer and record the absorbance (540 nm wavelength).
12. Plot the standard curve using the concentrations of the standards (nmol) in X-axis and the recorded absorbance in the Y-axis.
13. Calculate the concentration of glycerol in the samples (nmol) by extrapolation using the standard curve plotted in step 7.12.
14. Multiply the concentration of samples (nmol/cuvette) with the dilution factor 10 (refer to step 7.4) and 20 (total volume)
15. Divide the concentration of glycerol in each sample (step 7.13) by the mg of protein calculated by the BCA method.
    NOTE: Since the inguinal fat pad samples are incubated for 60 min, represent the results as nmol of glycerol/mg protein/h.