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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&#160;Table of Materials.
<ol>
	<li>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&#160;Table 1&#160;in a Class II biosafety cabinet. Protect FD-glucose from light throughout the experiment by turning off all the lights under the hood.</li>
	<li>Use ready-to-use cell culture reagents: Trypsin-EDTA, phosphate-buffered saline (PBS), and glucose-free and phenol red-free Dulbecco&#39;s Modified Eagle Medium (hereafter, glucose-free DMEM).</li>
</ol>
2. Ex vivo&#160;measurement of extracellular FD-glucose depletion in organs
<ol>
	<li>Pretreat mice with compounds stimulating glucose metabolism before the organ dissection, as follows.</li>
	<li>Use nine-week-old&#160;Lepob&#160;male mice (n = 11). Feed all mice with a regular chow diet before the experiment.</li>
	<li>Assign mice randomly into three groups for intraperitoneal (i.p.) injections.
	<ol>
		<li>Inject mice from the control&#160;Lepob&#160;group with 0.1 mL of sterile PBS (n = 4).</li>
		<li>Inject mice from the insulin&#160;Lepob&#160;group with 0.1 mL of sterile PBS, containing 12 IU human insulin per gram of body weight (BW) (n= 3).</li>
		<li>Inject mice from the AAC2&#160;Lepob&#160;group with 0.1 mL of sterile PBS, containing 0.1 nmol AAC2 per gram of body weight (BW) (n = 4).</li>
	</ol>
	</li>
	<li>After 15 min, subject mice to inhalation of 5% isoflurane and exsanguinate blood by cardiac puncture from the anesthetized animals.</li>
	<li>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.</li>
	<li>Prepare FD-glucose (0.29 mM) working solution in glucose-free DMEM in a Class II Biosafety cabinet without light.</li>
	<li>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.</li>
	<li>Incubate the harvested tissues/organs in a 6-well plate containing PBS for 1 min. Handle each tissue or organ in a separate well.</li>
	<li>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 &#181;L of glucose-free DMEM and incubate for 2 min.</li>
	<li>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 &#176;C (5% CO2&#160;and 95% humidity).</li>
	<li>Collect 100 &#181;L 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.</li>
	<li>Transfer 100 &#181;L 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.</li>
	<li>Normalize fluorescence to the 0 min value (100%) for each organ in each animal (Figure 1).</li>
</ol>
Table 1: Preparation of culture media and FD-glucose solutions.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution/Medium</td>
			<td>Components</td>
		</tr>
		<tr>
			<td>Ethanol:DMSO (1:1/v/v)</td>
			<td>Ethanol (200 &#181;L) and DMSO (200 &#181;L) in 1-1.5 mL tube; use cell culture grade ethanol and DMSO</td>
		</tr>
		<tr>
			<td>Medium 1</td>
			<td>DMEM (89 mL), Penicillin/streptomycin (1%) (1 mL) and Calf serum (10%) (10 mL) in sterile 50 mL tube</td>
		</tr>
		<tr>
			<td>Centrifugation Medium 2</td>
			<td>DMEM (89 mL), Penicillin/streptomycin (1%) (1 mL) and Bovine serum (10%) (10 mL) in sterile 50 mL tube</td>
		</tr>
		<tr>
			<td>Storage FD-glucose solution 5 mg/mL (14.5 mM)</td>
			<td>FD glucose (1 mg) and Ethanol:DMSO (1:1/v/v) (200 &#181;L) in 0.5 mL tube; store at -80 &#176;C, preferentially under argon or nitrogen atmosphere</td>
		</tr>
		<tr>
			<td>Working FD-glucose solution 5 &#181;g/mL (14.5 mM)</td>
			<td>Storage FD glucose solution 5 mg/mL (1 &#181;L), Glucose free DMEM (999 &#181;L); prepare immediately before experiment.</td>
		</tr>
	</tbody>
</table>

<table><tbody><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>&amp;nbsp;Mice</td></tr><tr><td>BioTek Synergy H1 modular multimode microplate reader (Fisher Scientific, US)</td><td>Fisher Scientific, US</td><td>Fisher Scientific, US</td><td>Device</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>Isoflurane, 5%</td><td>Henry Schein</td><td>NDC 11695-6776-2</td><td>Anestaetic</td></tr><tr><td>Phosphate buffered solution</td><td>Sigma-Aldrich</td><td>DA537-500 mL</td><td>Cell culture</td></tr><tr><td>Penicillin/streptomycin (P/S)</td><td>Gibco/ThermoFisher</td><td>15140-122</td><td>Cell culture</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>Fluorescent 2-deoxy-2-[(7-nitro-2,1,3- benzoxadiazol-4-yl) amino]-Dglucose)</td><td>Sigma</td><td>72987-1MG</td><td>Assay</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Gibco/ThermoFisher</td><td>11965-092</td><td>Cell culture</td></tr><tr><td>Bovine serum</td><td>Gibco/ThermoFisher</td><td>161790-060</td><td>Cell culture</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>E7023-500mL</td><td>Reagent</td></tr></tbody></table>

measurement of extracellular fluorescent glucose analog depletion: a surrogate measurement technique to assess glucose uptake in mouse organs ex vivo

Source: Kumar, S. B. et al., <a href="https://app.jove.com/v/63681">Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo.</a>&#160;J. Vis. Exp.&#160;(2022)
In this video, we demonstrate an ex vivo technique to measure extracellular glucose analog depletion in harvested mouse organs. It involves incubating the mouse tissue in a solution containing fluorescently-labeled deoxyglucose, which is taken up by the cells. The glucose analog uptake can be indirectly determined by measuring the fluorescence of the incubated medium.

<img alt="Figure 1" src="/files/ftp_upload/21213/21213_Figure1.jpg" /> 
Figure 1: Kinetics of extracellular FD-glucose depletion in different organs&#160;ex vivo.&#160;(A-C)&#160;Lepob&#160;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...

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

Source: Kumar, S. B. et al., Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo.&#160;J. Vis. ...

In tissues, extracellular glucose enters cells via facilitated diffusion through specific glucose transporters.
Glucose gets broken down to pyruvate in the cell cytoplasm via glycolysis, a metabolic pathway, generating energy in the form of adenosine triphosphate, or ATP, for cellular metabolic processes.
To measure glucose uptake ex vivo, begin with freshly harvested small-sized mouse liver tissue. Add suitable buffer to maintain physiological conditions.
Next, transfer the tissue to a multi-well plate containing glucose-free media. Follow it by incubation in glucose-free media supplemented with deoxyglucose, a fluorescently-labeled glucose analog. The small size of the tissue allows the entry of deoxyglucose.
Deoxyglucose enters the cells through glucose transporters, in the absence of glucose. Within the cells, deoxyglucose undergoes phosphorylation but is not metabolized further, leading to its accumulation.
Collect the media containing unutilized fluorescently-labeled deoxyglucose at desired time points. Using a microplate reader, measure the fluorescence of the extracellular deoxyglucose, which can be correlated with the intracellular tissue uptake of deoxyglucose.

measurement-extracellular-fluorescent-glucose-analog-depletion

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149 Views. Source: Kumar, S. B. et al., Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo. J. Vis. Exp. (2022) In this video, we demonstrate an ex vivo technique to measure extracellular glucose analog depletion in harvested mouse organs. It involves incubating the mouse tissue in a solution containing fluorescently-labeled deoxyglucose, which is taken up by the cells. The glucose analog uptake can be indirectly determined by measuring the fluo...

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

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

JoVE Journal

Immunology and Infection

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Biology

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been reported that skeletal muscle may increase the extraction of glucose from the blood by up to 50-fold during exercise compared to resting conditions. The increase in muscle glucose uptake during exercise and insulin stimulation is dependent on the translocation of glucose transporter 4 (GLUT4) from intracellular compartments to the muscle cell surface membrane, as well as phosphorylation of glucose to glucose-6-phosphate by hexokinase II. Isolation and incubation of mouse muscles such as m. soleus and m. extensor digitorum longus (EDL) is an appropriate ex vivo model to study the effects of insulin and electrically-induced contraction (a model for exercise) on glucose uptake in mature skeletal muscle. Thus, the ex vivo model permits evaluation of muscle insulin sensitivity and makes it possible to match muscle force production during contraction ensuring uniform recruitment of muscle fibers during measurements of muscle glucose uptake. Moreover, the described model is suitable for pharmacological compound testing that may have an impact on muscle insulin sensitivity or may be of help when trying to delineate the regulatory complexity of skeletal muscle glucose uptake.
Here we describe and provide a detailed protocol on how to measure insulin- and contraction-stimulated glucose uptake in isolated and incubated soleus and EDL muscle preparations from mice using radiolabeled [3H]2-deoxy-D-glucose and [14C]mannitol as an extracellular marker. This allows accurate assessment of glucose uptake in mature skeletal muscle in the absence of confounding factors that may interfere in the intact animal model. In addition, we provide information on metabolic viability of incubated mouse skeletal muscle suggesting that the method&#160;applied possesses some caveats under certain conditions when studying muscle energy metabolism.

Intact regulation of muscle glucose uptake is important for maintaining whole body glucose homeostasis. This protocol presents assessment of insulin- and contraction-stimulated glucose uptake in isolated and incubated mature skeletal muscle when delineating the impact of various physiological interventions on whole body glucose metabolism.

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been ...

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

Transcript

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

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice