Name: extracellular glucose depletion as an indirect measure of glucose uptake in cells and tissues ex vivo
Uploaded: 2022-04-06T14:20:00
Description: Watch this Scientific Journal Video about Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo at JoVE.com

The project was supported by Ralph and Marian Falk Medical Research Catalyst Award and Kathleen Kelly Award. Other supports included the National Center for Research Resources UL1RR025755 and NCI P30CA16058 (OSUCCC), the NIH Roadmap for Medical Research. The content is solely the responsibility of the authors and does not represent the official views of the National Center for Research Resources or the NIH.

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.
<ol>
	<li>Prepare Medium 1, centrifugation Medium 2, and storage and working...

<ol>
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A meta-analysis.</a> Diabetes &amp; Metabolic Syndrome: Clinical Research &amp; Review. 14 (4), 535-545 (2020).</li><li>Lee, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+prognostic+impact+of+glucose+uptake+in+visceral+adipose+tissue+according+to+sex+in+patients+with+colorectal+cancer.">Different prognostic impact of glucose uptake in visceral adipose tissue according to sex in patients with colorectal cancer.</a> Scientific Reports. 11 (1), 21556 (2021).</li><li>Miner, M. W. 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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 &#956;g FD-glucose/mL appear to provide the optimal range. Extracellular FD-glucose does not require normalization to cell number or protein concentrations ...

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, cancer4,5, 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 replication7,8,9. 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 vitro16,17,18 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&#160;amino acid compound 2 (AAC2) described elsewhere18.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3T3-L1 mouse fibroblasts</td>
			<td>ATCC</td>
			<td>CL-173</td>
			<td>Cell line</td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Falcon</td>
			<td>353227</td>
			<td>Plastic ware</td>
		</tr>
		<tr>
			<td>B6.V-Lepob/J male mice</td>
			<td>Jackson Laboratory</td>
			<td>stock number 000632</td>
			<td>Mice</td>
		</tr>
		<tr>
			<td>BioTek Synergy H1 modular multimode microplate reader (Fisher Scientific, US)</td>
			<td>Fisher Scientific, US</td>
			<td>&#160;B-SHT</td>
			<td>Device</td>
		</tr>
		<tr>
			<td>Bovine serum</td>
			<td>Gibco/ThermoFisher</td>
			<td>161790-060</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Calf serum</td>
			<td>Gibco/ThermoFisher</td>
			<td>26010-066</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Cell incubator</td>
			<td>Forma</td>
			<td>Series II Water Jacket</td>
			<td>Device</td>
		</tr>
		<tr>
			<td>Diet (mouse/rat diet, irradiated)</td>
			<td>Envigo</td>
			<td>Teklad LM-485</td>
			<td>Diet</td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide (DMSO)</td>
			<td>Sigma LifeScience</td>
			<td>D2650-100mL</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco/ThermoFisher</td>
			<td>&#160;11965-092</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma Aldrich</td>
			<td>E7023-500mL</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Fluorescent 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose)</td>
			<td>Sigma</td>
			<td>72987-1MG</td>
			<td>Assay</td>
		</tr>
		<tr>
			<td>Glucose-free and phenol red-free DMEM</td>
			<td>Gibco/ThermoFisher</td>
			<td>A14430-01</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Human insulin 10 mg/mL</td>
			<td>MilliporeSigma, Cat N 91077C</td>
			<td>Cat N 91077C</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Isoflurane, 5%</td>
			<td>Henry Schein</td>
			<td>NDC 11695-6776-2</td>
			<td>Anestaetic</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin (P/S)</td>
			<td>Gibco/ThermoFisher</td>
			<td>15140-122</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Phosphate buffered solution</td>
			<td>Sigma-Aldrich</td>
			<td>DA537-500 mL</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Pierce bicinchoninic acid (BCA) protein assay</td>
			<td>ThermoFisher</td>
			<td>Cat N23225</td>
			<td>Assay</td>
		</tr>
		<tr>
			<td>Radioimmunoprecipitation assay lysis buffer</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-24948</td>
			<td>Assay</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%)</td>
			<td>Gibco/ThermoFisher</td>
			<td>&#160;25300-054</td>
			<td>Cell culture</td>
		</tr>
	</tbody>
</table>

extracellular glucose depletion as an indirect measure of glucose uptake in cells and tissues ex vivo

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.

Intracellular intake and extracellular glucose depletion were measured in 3T3-L1 preadipocytes, in response to different concentrations of FD-glucose (Figure&#160;2) with and without insulin stimulation. Figure&#160;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 <strong class="xfig"...

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

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.

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

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic ...

Glucose uptake is the key to understanding health and disease. As glucose plays a pivotal role in metabolic and structural demands in the human body, as well as playing a vital role in regulation. The extracellular measurement of glucose allows for the development of high throughput assays for kinetics of glucose uptake in cells, tissues, and organs of different genetic background in response to nutrients or drugs.This assay would be a valuable tool for the discovery of therapeutics and nutrition for diabetes, cancer, degenerative diseases of the central nervous system, obesity, and all the diseases of glucose and metabolism. The assay needs an adjustment of time for starvation and stimulation that should be based on metabolic specifics for different tissues and cell cultures. Culture 3T3-L1 mouse fibroblasts by plating 10 to the 6 cells, diluted in 20 milliliters of medium 1 in 296 well plates.Plate 100 microliters of cell suspension per well, ensuring to maintain homogeneity of this suspension by mixing. Grow cells for 48 hours in a 37 degree Celsius incubator without changing media until about 70 to 80%confluency. Maintain cells confluent and fasted by culturing them in the same medium for 48 hours.Vary the incubation time from 24 to 72 hours, depending on the desired fasting catabolic state. Decant media from cells in the 96 well plates. Absorb the remaining fluid using sterile paper towels.Rinse the 96 well plate with 100 microliters of PBS per well, and absorb the remaining fluid with sterile paper towels. Add 100 microliters of glucose free DMEM per well, and incubate for 40 minutes. Adjust the incubation time depending on cell type stimuli, and desired level of fasting.Use eight replicates for each simulation condition. Therefore, prepare one milliliter for each simulation condition. Use seven simulation conditions without insulin.FD glucose of 2.5 micrograms per milliliter, 1 microgram per milliliter, 0.5 microgram per milliliter, 0.2 microgram per milliliter, 0.1 microgram per milliliter, 0.05 microgram per milliliter, and 0 milligram per milliliter in 196 well plate. Use seven stimulation conditions with insulin. FD glucose of 2.5 micrograms per milliliter, 1 microgram per milliliter, 0.5 microgram per milliliter, 0.2 microgram per milliliter, 0.1 microgram per milliliter, 0.05 microgram per milliliter, and 0 microgram per milliliter in another 96 well plate.To prepare stimulation conditions, dilute one microliter of five milligrams per milliliter FD glucose working solution, and 999 microliters of glucose free DMEM to obtain one milliliter of working stock solution. Using this working stock solution, prepare 1 to 2000, 1 to 5, 000, 1 to 10, 000, 1 to 25, 000, 1 to 50, 000, and 1 to 100, 000 delusions of FD glucose to obtain 2.5 micrograms per milliliter, 1 microgram per milliliter, 0.5 microgram per milliliter, 0.2 microgram per milliliter, 0.1 microgram per milliliter, 0.05 microgram per milliliter in two milliliter tubes immediately before experiments in a biosafety cabinet without lights. Add one microliter of insulin to each of these conditions.After 40 minutes of incubation, decant the glucose free DMEM from each well in 96 well plates, and absorb the remaining fluid with sterile paper towels. Add 100 microliters each from the various treatment conditions described before, to wells along one column. And 100 microliters from the same treatment condition to the wells along the row in both 96 wall plates.Label the plates that utilized the conditions, with and without insulin. Add glucose free DMEM alone to the control wells. Incubate the plate for 40 minutes in the cell culture incubator in the dark.After the cells have been stimulated for 40 minutes. Transfer the stimulation media from both the plates into new 96 well plates, maintaining the same experimental layout. Wash the cells with PBS.Remove PBS and decant any remaining solution on sterile paper towels. Add a protease inhibitor to the radio immunoprecipitation assay buffer to protect proteins. Place plates containing radio immunoprecipitation assay in a shaker for 30 minutes.Using a microplate reader, measure fluorescence at excitation and emission wavelengths at 485 and 535 nanometers respectively. First in the medium containing extracellular FD glucose, and then the plate with radioimmunoprecipitation assay lized cells at the end of 30 minutes incubation. Perform BCA protein assay according to manufacturer's instructions.Quantify the protein concentrations in each well containing radio immunoprecipitation assay lized cells. Use 10 microliters of the radio immunoprecipitation assay homogenate, and measure protein concentrations, and triplicate to increase the accuracy of measurements in 96 well plates. Quantify protein based on protein standards analyzed together with samples on each 96 well plate.Incubate the harvested tissues or organs in a six well plate containing PBS for one minute. Handle each tissue or organ in a separate well. After one minute, place each tissue on a sterile paper towel to absorb PBS.Transfer tissues or organs in a separate six wall plate containing 4, 000 microliters of glucose free DMEM, and incubate for two minutes. After two minutes, remove and transfer the tissues into wells of a six well plate containing 0.29 Millimolar FD glucose working solution. Incubate the six well plates containing tissues in FD glucose working solution at 37 degrees Celsius.Collect 100 microliters of FD glucose working solution from each well after 0, 10, 20, 30, 40, 60, 90, and 120 minutes of incubation, and transfer collected 100 microliters of FD glucose working solution into 96 well plates to analyze the kinetics of extracellular FD glucose depletion. Shake before and after collection. Measure the fluorescence at excitation in emission wavelengths at 485 and 535 nanometer respectively using a microplate reader.Dose dependency in the intracellular uptake of FD glucose was significantly increased in the presence of insulin. The insulin stimulation led to significantly decreased extracellular FD glucose levels compared to the samples without insulin stimulation. Extracellular depletion of FD glucose can measure a change in glucose uptake with comparable accuracy as intracellular FD glucose uptake.The extracellular FD glucose was not depleted in non stimulated control visceral fat during 120 minutes of incubation. In contrast, pretreatment of mice with insulin or AAC2 before the dissection, led to significant depletion of extracellular FD glucose within a time interval of 30 minutes, and 60 minutes respectively. Extracellular FD glucose was not depleted in insulin stimulated liver explants, compared with non stimulated liver explants.Extracellular FD glucose was significantly decreased in the time dependent manner, and liver explants pretreated with AAC2.22%depletion of glucose was observed in the extracellular medium containing the non-treated brain after 60 minutes of incubation. The medium incubated with brains from insulin treated mice have shown a 5%linear decrease in FD glucose. AAC2 stimulated brains led to a profound rapid decrease to 67.4%of the extracellular FD glucose during the first 20 minutes.Substance involving washing are especially important for removing traces of glucose, blood, or serum within medium and, or organs, as it can interfere with the fluorescence reading of these mediums. After high throughput identification of compounds of metabolites in genes with glycemic properties, as well as the optimal conditions for their action in vivo experiments, could be validated with labeled FD glucose and PET scanning to confirm glucose accumulation in specific organs. This technique for discovery of glycemic action is both beneficial in deletion of numerous metabolites, therapeutics or their combinations, and highlights their actions in different organs.

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