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

Summary

This protocol describes the method of determining basal and forskolin-stimulated lipolysis in inguinal fat pads obtained from normal chow diet (NCD) or high fat diet (HFD) ± capsaicin fed wild type mice. As an index for lipolysis, glycerol release was measured from inguinal adipose fat pads.

Abstract

Lipolysis is a process by which the lipid stored as triglycerides in adipose tissues are hydrolyzed into glycerol and fatty acids. This article describes the method for the measurement of basal and forskolin (FSK)-stimulated lipolysis in the inguinal fat pads isolated from wild type mice fed either normal chow diet (NCD), high fat diet (HFD) or a high fat diet containing 0.01% of capsaicin (CAP; transient receptor potential vanilloid subfamily 1 (TRPV1) agonist) for 32 weeks. The method described here for performing ex vivo lipolysis is adopted from Schweiger et al.¹ We present a detailed protocol for measuring glycerol levels by UV-Visible (UV/VIS) spectrophotometry. The method described here can be used to successfully isolate inguinal fat pads for lipolysis measurements to obtain consistent results. The protocol described for inguinal fat pads can readily be extended to measure lipolysis in other tissues.

Introduction

Adipose tissues store energy as fat² and fatty acid oxidation is required for thermogenesis^3,4. Fatty acids ingested through diets are packaged along with apoproteins into chylomicrons and delivered to different tissues in the body via blood circulation. Although most cells in the body store a reserve of energy, adipose tissue stores excess energy as fat^5,6. Lipolysis in adipose tissue is regulated by complex processes and the molecular details of lipolysis still remain vague⁷.

Lipolysis is a process by which triglycerides (TGL) stored in adipose tissue are hydrolyzed to produce glycerol and fatty acids (FA) by the enzyme adipose triglyceride lipase (ATGL)⁸. Alterations in basal and stimulated lipolysis is a characteristic feature of obesity. The basal lipolysis is regulated by ATGL activation⁹, which converts TGL to diacylglycerol (DAG), that is subsequently hydrolyzed to monoacyl glycerol (MAG). Activation of hormone sensitive lipase (HSL) via adenylyl cyclase activates cyclic adenosine monophosphate (cAMP) dependent protein kinase A (PKA) stimulation and causes lipolysis. Measurement of lipolysis, basal and stimulated, is, therefore, important to analyze the activity of proteins involved in this process. Also, unraveling the molecular regulation of lipolysis may be beneficial to develop novel therapeutic strategies against obesity¹⁰. Since, molecules that stimulate lipolysis and fatty acid oxidation are potential candidates for decreasing fats stored in depots, it is important to employ a robust assay for reproducibility.

Previously published data suggest that activation of TRPV1 protein expressed in white adipose tissue by CAP enhanced basal and FSK (adenylyl cyclase activator)-stimulated lipolysis in inguinal fat pads¹¹. Previous research also suggests that long-term activation of TRPV1 by CAP activates PKA¹². Since the activation of PKA stimulates lipolysis^13,14, measuring both basal and PKA-dependent stimulated lipolysis in inguinal fat pads isolated from NCD or HFD (± CAP)-fed mice after 32 weeks of feeding the respective diets will validate the role of TRPV1 activation in lipolysis.

This article describes an efficient method of determining basal and stimulated lipolysis. Although other methods that employ radioactive isotopes of glycerol and tedious high performance liquid chromatography or gas chromatography/mass spectrometry for measurements^15,16 are available, this method offers a more direct, simple and cost effective technique to determine lipolysis in adipose tissues.

Protocol

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.

Results

To evaluate the effect of CAP on basal and stimulated lipolysis, this research measured lipolysis in inguinal adipose fat pads isolated from NCD or HFD (± CAP)-fed wild type mice. The representative results for the basal and FSK-stimulated lipolysis for the inguinal fat pads are given in Table I. Basal and FSK-stimulated glycerol release in the presence of Triacsin C, which inhibits acyl coA synthetase and prevents the regeneration of TGL. As shown in

Discussion

The breakdown process of TGL into glycerol and fatty acids is catalyzed by ATGL⁹ during basal lipolysis and orchestrated by an array of proteins including the activation of adenylyl cyclase/PKA-dependent pathway during stimulated lipolysis^21,22,23. Enhancement of lipolysis increases the plasma levels of fatty acids for transportation and energy use²⁴. Fatty acids are taken up by mi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Capsaicin	Sigma, USA	M2028	TRPV1 agonist
Forskolin	Sigma, USA	F6886	Adenylyl cyclase activator
DMEM	GE healthcare and life sciences, UT, USA	SH30081.01
High fat diet	Research diets, New Brunswick, USA	D12492	Abbreviated as HFD
Tris	Amresco, USA	O497
Sodium chloride	Thermofisher Scientific	BP358-212
Sodium deoxycholate	Sigma, USA	D6750
Dithiothreitol	Sigma, USA	D9163
Sodium orthovanadate	Sigma, USA	S6508
Protease inhibitor cocktail	Sigma, USA	P8340
Free Glycerol reagent	Sigma USA	F6428
DMSO	Sigma, USA	D8779
Triacsin C	Sigma, USA	T4540	Acyl CoA transferase inhibitor
Bovine serum albumin	Sigma, USA	A7030
Chloroform	Sigma Aldrich	31998-8
Methanol	Thermofisher Scientific, USA	A412-1
Sodium hydroxide	Amresco, USA	O583
Sodium dodecyl sulfate	Sigma, USA	L3371
Bicinchoninic acid reagent	Sigma, USA	BCA1-1KT
UV-VIS Spectrophotometer	Pharmacia Biotech, NJ, USA	Ultrospec 2000
Normal chow diet	Labdiet.com	500I	abreviated as NCD
C57BL/6 mice	Jackson Laboratory, CT, USA	Stock number000664	wild type mice
Parafilm	Heathrow Scientific, USA	HS 234526A
Glycerol standard	Sigma, USA	G7793