Assessing isoproterenol-stimulated whole-body lipolysis in vivo

Background

Lipolysis is the hydrolysis of triglycerides into glycerol and free fatty acids. Lipolysis is induced through activation of beta adrenergic receptors. Isoproterenol/Isoprenaline is a non-selective beta adrenergic agonist structurally similar to adrenaline. By administering isoproterenol in vivo, it is possible to artificially stimulate whole-body lipolysis and assess changes in the concentrations of the products of lipolysis (i.e., glycerol and free fatty acids) in the blood.

Reagents Needed

1. (−)-Isoproterenol hydrochloride (Sigma cat# I6504)
2. PBS
3. Isofluorane

Experimental protocol

Note: Mice do not need to be in a fasted state prior to this test.

1. Briefly anesthetize mice with isofluorane and collect blood via retro orbital bleed. Allow blood to clot over ice.
2. Inject 10 mg/kg isoproterenol (prepared fresh in sterile PBS) via intraperitoneal injection. This will be 10mg in 10mL then inject 10uL/g mouse.
3. Wait 15 minutes.
4. Briefly anesthetize mice with isoflurane and collect blood via retro orbital bleed. Allow blood to clot over ice. ***Note: Take care when anesthetizing mice the second time. They will go under VERY quickly, so watch the mouse closely***
5. Centrifuge blood samples and collect serum.
6. Assay serum for NEFA's and Glycerol/Triglycerides using Wako and Sigma assay kits, respectively.

NEFA determination from serum (use Wako Diagnostics NEFA-HR(2) Microtiter Kit)

• Note: The manufacturers protocol says to use 225 uL of reaction buffer A and 75 uL of reaction buffer B with 4 uL of sample, read at 560:670 nm. This uses more reagent than is necessary for the volume of serum/concentrations of NEFA that we would typically analyze.

1. To wells of a clear 96 well plate, add 4uL of water (as blank), appropriate standard (the kit comes with stock of 1 mEq/L. Dilute the stock in ddH20 to give 0.25 and 0.5 mEq/L standards. For the high standard, add 8 uL of the 1 mEq/L stock. Due to the extra volume, the concentration of this last standard is actually 1.97 mEq/L).
2. To remaining wells, add 4 uL of serum samples. ***Note: You may need to add more/less serum, depending on the NEFA concentration of your samples. The assay is linear between 0.01-4.00 mEq/L***
3. To each well, add 100 uL of reaction buffer A, mix gently and incubate at 37 deg C for ~5 minutes.
4. Allow plate to return to room temperature before reading at 560:660 nm. These are the initial readings.
   1. Note: If the plate reader being used does not allow for measuring two wavelengths simultaneously, read the plate at 560 nm. The rationale behind measuring two wavelengths simultaneously is to account for possible sample contamination by products of haemolysis, which can interfere with the assay.
5. To each well, add 50 uL of reaction buffer B and incubate at 37 deg C for ~5 minutes. The wells will turn purple. If they do not, check whether the reagents are within the best before date.
6. Allow plate to return to room temperature before re-reading at 560:660 nm. These are the final readings.
7. Calculations
   1. For both initial and final readings, subtract the absorbance values obtained at the 670 nm wavelength from those obtained at the 560 nm wavelength.
   2. Subtract the initial absorbance reading from the final reading, then calculate sample concentrations from the standard curve.

Glycerol and Triglyceride determination from serum (use Sigma Diagnostics Triglyceride Assay Kit)

1. To wells of a clear 96 well plate, add 3 uL of water (as blank), the appropriate volume of standard (the kit comes with a stock standard of 2.5 mg/mL triolein; to replicate wells, add 1, 2, 3 and 4 uL of this stock. The assay is only linear up to concentrations of 10 mg/mL, which is 4 uL of 2.5 ug/mL standard).
2. To each well, add 80 uL of glycerol reagent.
3. To each well, add 3-8 uL of serum samples and appropriate volume of standards (3 if you expect a lot of lipolysis, and greater volume if you expect less).
4. Incubate plate at 37 deg C for 5 minutes (or room temperature for 30 min).
5. launch Gen5 Software on the computer
6. Choose Glycerol Assay protocol
7. Under the procedures menu, choose wavelength 540 nm and set the shaking orbital for 2 seconds prior to reading the plate
8. Under the layout menu specify what wells you would like to be read
9. Read the plate at 540 nm. This is the initial reading. (if the readings do not seem correct, check for air bubbles and reread the plate)
10. Save the file
11. To each well, add 20 uL of triglyceride reagent. Incubate plate at 37 deg C for ~5 minutes. The triglyceride reagent contains a lipase that breaks down the triglycerides into glycerol and fatty acids. The assay measures the glycerol in the sample, so the first reading tells us how much glycerol is present.
12. Re-read the plate at 540 nm. This is the final reading. The second reading tells us how much glycerol is present after the triglycerides present in the serum have been broken down.
13. Save the file
14. export both files onto excel
15. Calculations
    1. Subtract the initial reading from the final reading. This tells us what proportion of the glycerol in the sample came from triglycerides.
    2. For each of the initial, final and calculated absorbances, calculate the concentration of the samples from the standard curve.