Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

Podsumowanie

Extracellular depletion of fluorescently labeled glucose correlates with glucose uptake and could be used for high-throughput screening of glucose uptake in excised organs and cell cultures.

Streszczenie

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic factors affecting glucose uptake. The measurement of intracellular fluorescence is a widely used method to test the uptake of fluorescently-labeled glucose (FD-glucose) in cells in vitro, or for imaging glucose-consuming tissues in vivo. This assay assesses glucose uptake at a chosen time point. The intracellular analysis assumes that the metabolism of FD-glucose is slower than that of endogenous glucose, which participates in catabolic and anabolic reactions and signaling. However, dynamic glucose metabolism also alters uptake mechanisms, which would require kinetic measurements of glucose uptake in response to different factors. This article describes a method for measuring extracellular FD-glucose depletion and validates its correlation with intracellular FD-glucose uptake in cells and tissues ex vivo. Extracellular glucose depletion may be potentially applicable for high-throughput kinetic and dose-dependent studies, as well as identifying compounds with glycemic activity and their tissue-specific effects.

Wprowadzenie

The demand for measuring glucose uptake rises together with the critical need to address an epidemic increase in a multitude of diseases dependent on glucose metabolism. Underlying mechanisms of degenerative metabolic diseases, neurological and cognitive disorders¹, inflammatory² and infectious diseases³, cancer^4,5, as well as aging⁶, depend on glucose metabolism for energy and its storage, anabolic processes, protein, and gene modification, signaling, regulation of genes, and nucleic acids synthesis and replication^7,8,9. Diabetes mellitus (DM) is directly related to malfunction of glucose uptake regulation. DM is a spectrum of chronic diseases such as type-1, -2, and -3 diabetes mellitus, gestational diabetes, maturity-onset diabetes of the young, and other types of this disease induced by environmental and/or genetic factors. In 2016, the first WHO Global report on diabetes demonstrated that the number of adults living with the most widespread DM has almost quadrupled since 1980 to 422 million adults¹⁰, and this number of DM patients has been rising exponentially for the last few decades. In 2019 alone, an estimate of 1.5 million deaths was directly caused by DM¹⁰. This dramatic upsurge is due to the rise in type-2 DM and the conditions driving it, including overweight and obesity¹⁰. The COVID-19 pandemic revealed a two-fold increase in mortality in patients with DM compared to the general population, suggesting the profound yet poorly understood role of glucose metabolism in the immune defense³. Prevention, early diagnosis, and treatment of DM, obesity, and other diseases require optimization of measurements of glucose uptake by different tissues, and identification of environmental¹¹, nutritional¹², endocrine¹³, genetic¹⁴, and epigenetic¹⁵ factors affecting glucose uptake.

In research, intracellular and/or tissue uptake of glucose is commonly measured by fluorescently-labeled glucose (FD-glucose) in vitro^16,17,18 and in vivo¹⁹. FD-glucose became a preferred method compared to more precise methods using radioactively-labeled glucose²⁰, analytical mass spectroscopy analysis²¹, metabolomics²², nuclear magnetic resonance methods²³, and positron emission tomography/computed tomography (PET/CT)^5,24. Unlike FD-glucose uptake, analytical methods requiring more biological material may involve a multi-step sample preparation, expensive instruments, and complex data analysis. Effective and inexpensive measurements of FD-glucose uptake in cell cultures have been utilized in proof-of-concept experiments and may require validation by other methods.

The basis of FD-glucose application for glucose uptake studies is the reduced metabolism of FD-glucose compared to endogenous glucose²⁵. Nonetheless, both endogenous glucose and FD-glucose are dynamically distributed among all cellular compartments for use in anabolic, catabolic, and signaling processes. The compartmentalization and time-dependent processing²⁵ of FD-glucose interfere with the fluorescence measurements, and represent the major limiting factors for the use of this assay in high-throughput screening experiments, kinetic analysis, 3D cell culture, co-cultures, and tissue explant experiments. Here, we provide data demonstrating a high correlation between the extracellular depletion of FD-glucose and its intracellular uptake, suggesting the extracellular depletion of FD-glucose as a surrogate measurement for intracellular glucose uptake. The measurement of extracellular depletion of glucose was applied to validate tissue-specific differences in glucose uptake in mice treated with insulin and an experimental drug¹⁸ to provide a proof-of-principle of this method.

The current protocol describes intracellular and extracellular (Figure 1) measurements of FD-glucose uptake in 3T3-L1 cells. Protocol sections 1-7 explain the culture and growth of cells for 48 h; cell starvation, stimulation, and baseline extracellular measurements; and post-stimulation measurements of extracellular FD-glucose and intracellular measurements of FD-glucose and protein. Protocol section 8 describes the ex vivo measurement of extracellular uptake of FD-glucose in tissues dissected from ob/ob mice in the presence and absence of insulin and amino acid compound 2 (AAC2) described elsewhere¹⁸.

Protokół

Animal studies were approved by the Institutional Animal Care and Use Committee of The Ohio State University (OSU, protocol 2007A0262-R4).

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).
3. Use 20 mL of radioimmunoprecipitation assay (RIPA) lysis buffer.
   ​NOTE: In the following sections, extracellular depletion and intracellular uptake of different FD-glucose doses are compared in 3T3-L1 fibroblasts with and without insulin stimulation (Figure 2).

2. 3T3-L1 cell culture and maintenance

1. Culture 3T3-L1 mouse fibroblasts by plating 1 x 10⁶ cells diluted in 20 mL of Medium 1 in two 96-well plates. Plate 100 μL of cell suspension per well, ensuring to maintain homogeneity of this suspension by mixing. Grow cells for 48 h in a 37 °C incubator (5% CO₂ and 95% humidity) without changing media until about 70%-80% confluency.
2. Maintain cells confluent and fasted by culturing them in the same medium for 48 h. Vary the incubation time from 24-72 h depending on the desired fasting catabolic state.

3. Starvation of 3T3-L1 cells

1. Decant media from cells in the 96-well plates. Absorb the remaining fluid using sterile paper towels.
2. Rinse the 96-well plate with 100 μL of PBS per well and absorb the remaining fluid with sterile paper towels.
3. Add 100 μL of glucose-free DMEM per well and incubate for 40 min. Adjust the time of incubation depending on cell type, stimuli, and desired level of fasting.
   ​NOTE: The time of incubation may require optimization depending on the season.

4. Preparation of FD-glucose solutions with different concentrations

NOTE: The experiment in Figure 2 examines extracellular and intercellular media with and without stimulation with insulin.

1. Use eight replicates (one row in a 96-well plate) for each stimulation condition. Therefore, prepare 1 mL for each stimulation condition (specified in Table 2 and below).
2. Use eight stimulation conditions without insulin: FD-glucose of 2.5 μg/mL, 1 μg/mL, 0.5 μg/mL, 0.2 μg/mL, 0.1 μg/mL, 0.05 μg/mL, and 0 μg /mL (control without FD-glucose) in one 96-well plate.
3. Use eight stimulation conditions with insulin (10 μg/mL, final concentration): FD-glucose of 2.5 μg/mL, 1 μg/mL, 0.5 μg/mL, 0.2 μg/mL, 0.1 μg/mL, 0.05 μg/mL, and 0 μg/mL (control without FD-glucose) in another 96-well plate.
4. To prepare stimulation conditions, dilute 1 μL of 5 mg/mL FD-glucose working solution (Table 1) in 999 μL of glucose-free DMEM to obtain 1 mL of working stock solution (1:1000 dilution or 5 μg/mL).
   1. Using this working stock solution, prepare 1:2000, 1:5000, 1:10,000, 1:25,000, 1:50,000, and 1:100,000 dilutions of FD-glucose to obtain 2.5 μg/mL, 1 μg/mL, 0.5 μg/mL, 0.2 μg/mL, 0.1 μg/mL, and 0.05 μg/mL (Table 2) in 2 mL tubes immediately before experiments in a biosafety cabinet without lights.
5. Repeat step 4.4 and add 1 μL of insulin (10 mg/mL, final concentration) to each of the conditions specified above.
   ​NOTE: The final concentration of insulin in these samples is 10 μg/mL = 1.7 nmol/mL. To achieve homogenization, add insulin to the tubes before adding other solutions.

5. Treating starved 3T3-L1 cells

1. After 40 min of incubation, decant the glucose-free DMEM from each well in 96-well plates and absorb the remaining fluid with sterile paper towels.
2. Add 100 μL (per well) each from the various treatment conditions described above to wells along one column, and 100 μL (per well) from the same treatment condition to the wells along the row (eight replicates) in both 96-well plates. Label the plates that utilized the conditions with and without insulin. Add glucose-free DMEM alone to the control wells (n = 8).
3. Incubate the plate for 40 min in a cell culture incubator (37 °C, 5% CO₂, and 95% humidity) in the dark.

6. Extracellular and intercellular measurements for stimulated 3T3-L1 cells

1. After the cells have been stimulated for 40 min, transfer the stimulation media from both the plates into new 96-well plates maintaining the same experimental layout.
2. Decant any remaining solution from the original stimulated 96-well plates with cells on sterile paper towels.
3. Optionally, wash cells with PBS. Remove PBS and decant any remaining solution on sterile paper towels.
4. Add 100 μL of the RIPA lysis buffer to each well. Optionally, add protease inhibitor to the RIPA buffer to protect proteins. Place plates containing RIPA in a shaker for 30 min.
5. Using a microplate reader (Table of Materials), measure fluorescence at excitation (Ex) and emission (Em) wavelengths of 485 and 535 nm, respectively, first in the medium containing extracellular FD-glucose, and then, the plate with RIPA-lysed cells at the end of 30 min incubation (see step 6.4).

7. Protein-based normalization of intracellular glucose uptake

NOTE: Intracellular FD-glucose uptake depends on the cell number. Protein levels in cellular lysates are proportional to the cell numbers that allow normalizing intracellular FD-glucose levels to the number of cells in each well.

1. Perform BCA protein assay according to manufacturer's instructions (see Table of Materials). Quantify the protein concentrations in each well containing RIPA-lysed cells.
2. Use 10 μL of the RIPA homogenate and measure protein concentrations in triplicate to increase the accuracy of measurements in 96-well plates.
3. Quantify protein based on protein standards (0, 10, 20, 30, 40, 50, and 60 μg/mL protein) analyzed together with samples on each 96-well plate.
4. Measure the absorbance at 562 nm and quantify protein concentrations in each sample based on the linear regression formula obtained for the protein standard.
5. Normalize the levels of intracellular FD-glucose by using fluorescent value/protein concentration in each well. Extracellular FD-glucose depletion does not require normalization.
6. Optionally, use the average baseline values in control samples for additional normalization (100%).

8. 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 animals²⁶.
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 3).

Wyniki

Intracellular intake and extracellular glucose depletion were measured in 3T3-L1 preadipocytes, in response to different concentrations of FD-glucose (Figure 2) with and without insulin stimulation. Figure 2A demonstrates a dose-dependent increase in the intracellular uptake of FD-glucose, which was significantly increased in the presence of insulin. The concomitant decrease in extracellular FD-glucose in the same cells is shown in

Dyskusje

The direct comparison of extracellular FD-glucose depletion with normalized intracellular glucose uptake in cells culture showed a high correlation, suggesting that extracellular glucose depletion could be a surrogate measurement for glucose uptake assessment. The measurement of extracellular FD-glucose can use a broad range of FD glucose concentrations, also 0.5-2.5 μg FD-glucose/mL appear to provide the optimal range. Extracellular FD-glucose does not require normalization to cell number or protein concentrations ...

Materiały

Name	Company	Catalog Number	Comments
3T3-L1 mouse fibroblasts	ATCC	CL-173	Cell line
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	B-SHT	Device
Bovine serum	Gibco/ThermoFisher	161790-060	Cell culture
Calf serum	Gibco/ThermoFisher	26010-066	Cell culture
Cell incubator	Forma	Series II Water Jacket	Device
Diet (mouse/rat diet, irradiated)	Envigo	Teklad LM-485	Diet
Dimethylsulfoxide (DMSO)	Sigma LifeScience	D2650-100mL	Reagent
Dulbecco's Modified Eagle Medium	Gibco/ThermoFisher	11965-092	Cell culture
Ethanol	Sigma Aldrich	E7023-500mL	Reagent
Fluorescent 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose)	Sigma	72987-1MG	Assay
Glucose-free and phenol red-free DMEM	Gibco/ThermoFisher	A14430-01	Cell culture
Human insulin 10 mg/mL	MilliporeSigma, Cat N 91077C	Cat N 91077C	Reagent
Isoflurane, 5%	Henry Schein	NDC 11695-6776-2	Anestaetic
Penicillin/streptomycin (P/S)	Gibco/ThermoFisher	15140-122	Cell culture
Phosphate buffered solution	Sigma-Aldrich	DA537-500 mL	Cell culture
Pierce bicinchoninic acid (BCA) protein assay	ThermoFisher	Cat N23225	Assay
Radioimmunoprecipitation assay lysis buffer	Santa Cruz Biotechnology	sc-24948	Assay
Trypsin-EDTA (0.05%)	Gibco/ThermoFisher	25300-054	Cell culture