Measurement of Extracellular Fluorescent Glucose Analog Depletion: A Surrogate Measurement Technique to Assess Glucose Uptake in Mouse Organs Ex Vivo

Protokół

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

NOTE: All procedures must be done in a class II biosafety cabinet with the blower on and the lights off.

1. Preparation of materials

NOTE: All materials are listed in the Table of Materials.

1. Prepare Medium 1, centrifugation Medium 2, and storage and working solutions of fluorescent 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG or FD-glucose) according to Table 1 in a Class II biosafety cabinet. Protect FD-glucose from light throughout the experiment by turning off all the lights under the hood.
2. Use ready-to-use cell culture reagents: Trypsin-EDTA, phosphate-buffered saline (PBS), and glucose-free and phenol red-free Dulbecco's Modified Eagle Medium (hereafter, glucose-free DMEM).

2. Ex vivo measurement of extracellular FD-glucose depletion in organs

1. Pretreat mice with compounds stimulating glucose metabolism before the organ dissection, as follows.
2. Use nine-week-old Lep^ob male mice (n = 11). Feed all mice with a regular chow diet before the experiment.
3. Assign mice randomly into three groups for intraperitoneal (i.p.) injections.
   1. Inject mice from the control Lep^ob group with 0.1 mL of sterile PBS (n = 4).
   2. Inject mice from the insulin Lep^ob group with 0.1 mL of sterile PBS, containing 12 IU human insulin per gram of body weight (BW) (n= 3).
   3. Inject mice from the AAC2 Lep^ob group with 0.1 mL of sterile PBS, containing 0.1 nmol AAC2 per gram of body weight (BW) (n = 4).
4. After 15 min, subject mice to inhalation of 5% isoflurane and exsanguinate blood by cardiac puncture from the anesthetized animals.
5. Dissect visceral epidydimal white adipose tissue (200 mg), liver (200 mg), and whole brain from each animal. Use a Class II biosafety hood for tissue handling.
6. Prepare FD-glucose (0.29 mM) working solution in glucose-free DMEM in a Class II Biosafety cabinet without light.
7. Measure the fluorescence of FD-glucose working solution (0.29 mM) at excitation and emission wavelengths of 485 and 535 nm, respectively, using a microplate reader.
8. Incubate the harvested tissues/organs in a 6-well plate containing PBS for 1 min. Handle each tissue or organ in a separate well.
9. After 1 min, place each tissue on a sterile paper towel to absorb PBS. Transfer tissues/organs into a separate 6-well plate containing 4,000 µL of glucose-free DMEM and incubate for 2 min.
10. After 2 min, remove and transfer the tissues into wells of a 6-well plate containing 0.29 mM FD-glucose working solution (1 mL/well). Incubate the 6-well plates containing tissues in FD-glucose working solution at 37 °C (5% CO₂ and 95% humidity).
11. Collect 100 µL of FD-glucose working solution from each well after 0, 10, 20, 30, 40, 60, 90, and 120 min of incubation, to analyze the kinetics of extracellular FD glucose depletion. Shake before and after collection.
12. Transfer 100 µL of FD-glucose working solution into 96-well plates to measure the fluorescence at excitation and emission wavelengths of 485 and 535 nm, respectively, using a microplate reader.
13. Normalize fluorescence to the 0 min value (100%) for each organ in each animal (Figure 1).

Table 1: Preparation of culture media and FD-glucose solutions.

Solution/Medium	Components
Ethanol:DMSO (1:1/v/v)	Ethanol (200 µL) and DMSO (200 µL) in 1-1.5 mL tube; use cell culture grade ethanol and DMSO
Medium 1	DMEM (89 mL), Penicillin/streptomycin (1%) (1 mL) and Calf serum (10%) (10 mL) in sterile 50 mL tube
Centrifugation Medium 2	DMEM (89 mL), Penicillin/streptomycin (1%) (1 mL) and Bovine serum (10%) (10 mL) in sterile 50 mL tube
Storage FD-glucose solution 5 mg/mL (14.5 mM)	FD glucose (1 mg) and Ethanol:DMSO (1:1/v/v) (200 µL) in 0.5 mL tube; store at -80 °C, preferentially under argon or nitrogen atmosphere
Working FD-glucose solution 5 µg/mL (14.5 mM)	Storage FD glucose solution 5 mg/mL (1 µL), Glucose free DMEM (999 µL); prepare immediately before experiment.

Wyniki

Figure 1: Kinetics of extracellular FD-glucose depletion in different organs ex vivo. (A-C) Lep^ob mice were injected with vehicle (PBS, n = 4), insulin (12 IU/kg BW, n = 3), and AAC2 (1 nmol/g BW, n = 3). After 15 min, tissues were dissected and isolated. Explants of (A) visceral fat, (B) liver, an...

Materiały

Name	Company	Catalog Number	Comments
96-well plates	Falcon	353227	Plastic ware
B6.V-Lepob/J male mice	Jackson Laboratory	stock number 000632	Mice
BioTek Synergy H1 modular multimode microplate reader (Fisher Scientific, US)	Fisher Scientific, US	Fisher Scientific, US	Device
Cell incubator	Forma	Series II Water Jacket	Device
Diet (mouse/rat diet, irradiated)	Envigo	Teklad LM-485	Diet
Isoflurane, 5%	Henry Schein	NDC 11695-6776-2	Anestaetic
Phosphate buffered solution	Sigma-Aldrich	DA537-500 mL	Cell culture
Penicillin/streptomycin (P/S)	Gibco/ThermoFisher	15140-122	Cell culture
Glucose-free and phenol red-free DMEM	Gibco/ThermoFisher	A14430-01	Cell culture
Fluorescent 2-deoxy-2-[(7-nitro-2,1,3- benzoxadiazol-4-yl) amino]-Dglucose)	Sigma	72987-1MG	Assay
Dulbecco's Modified Eagle Medium	Gibco/ThermoFisher	11965-092	Cell culture
Bovine serum	Gibco/ThermoFisher	161790-060	Cell culture
Ethanol	Sigma-Aldrich	E7023-500mL	Reagent