The authors acknowledge Anne Charri&#233; at the Radiobiology service (Lyon-Sud hospital) and the Fond National Suisse (FNS) for their financial support.

1. Preparation of Cell Culture Media and Solutions
<ol>
	<li>Preparation of culture media
	<ol>
		<li>Prepare proliferation medium (PM) by supplementing Ham&#39;s F-10 medium with glutamine (2 mM), penicillin/streptomycin (5 &#181;g/mL final), 2% Fetal Calf Serum (FCS) and 2% serum substitute.</li>
		<li>Prepare differentiation medium (DM) by supplementing Dulbecco&#39;s Modified Eagle Medium (DMEM) with glutamine (2 mM), penicillin/streptomycin (5 &#181;g/mL final), and 2% FCS.</li>
	</ol>
	</li>
	<li>Preparation of glucose uptake solutions 
	Caution: Handling of radioactive material is only allowed in a restricted and controlled area by authorized personnel. Material and waste must be handled according to appropriate procedures, guidelines and local legislation.
	<ol>
		<li>To prepare X-Dulbecco&#39;s phosphate-buffered saline (X-DPBS), make a solution of DPBS containing 0.2% (w/v) (final concentration) bovine serum albumin (BSA). Filter the solution through a 0.2 &#181;m filter. Store at 4 &#176;C.</li>
		<li>To prepare cold 2-deoxy-D-glucose (2dG) solution, weigh 16.4 mg of 2dG and solubilize in 10 mL distilled water to obtain a 10 mM solution. Store at 4 &#176;C.</li>
		<li>Add 600 &#181;L of cold 2dG and 6 &#181;L of radiolabeled [3H] 2dG to 5400 &#181;L of X-DPBS to obtain the radiolabeled 2dG (2dG*) solution. 
		NOTE: The final concentration is 1 mM 2dG and labeling is 1 &#181;Ci/mL.
		<ol>
			<li>Set aside a 20 &#181;L aliquot (TC20) of radiolabeled 2dG* solution.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of incubation mixtures
	<ol>
		<li>For the cytochalasin B mixture, add 2 &#181;L of 20 mM cytochalasin B to 2 mL of radiolabeled 2dG* solution. 
		NOTE: Stock solution of cytochalasin B is at 10 mg/mL in dimethyl sulfoxide (DMSO).</li>
		<li>For the DMSO mixture, add 4 &#181;L of DMSO to the remaining 4 mL of radiolabeled 2dG* solution.</li>
	</ol>
	</li>
</ol>
2. Culture of Human Primary Muscle Cells
<ol>
	<li>Seeding of 6-well plates with human muscle satellite cells 
	NOTE: Use in-house (see reference 24 for details) or commercially available human muscle satellite cells from a frozen vial (containing 250,000 cells). The following procedure is given for 250,000 cells in order to obtain one 6-well plate necessary for the measurement of glucose uptake in a single condition.
	<ol>
		<li>Rapidly thaw frozen vials of in-house24 or commercial preparations of human muscle satellite cells in pre-warmed water (37 &#176;C) until only a small ice block remains in the vial.</li>
		<li>Pour directly into a 50 mL plastic tube containing 10 mL of pre-warmed (37 &#176;C) PM.</li>
		<li>Centrifuge for 5 min at 500 x g and discard the supernatant.</li>
		<li>Gently resuspend the cell pellet with 18 mL of pre-warmed PM (to obtain 42,000 cells per 3 mL of medium). Distribute 3 mL in each well of a 6-well plate (9.6 cm2). 
		NOTE: The six individual wells of a plate are required to perform a duplicate measurement of glucose uptake for the following conditions: passive transport inhibition (wells 1 and 2), basal rate (wells 3 and 4) and insulin stimulated rate (wells 5 and 6). Repeat as many six-well plates as distinct treatments are needed.</li>
		<li>Incubate in standard culture conditions (37 &#176;C, 5% CO2) until cells reach 90% confluence. 
		NOTE: This step takes between 48 - 72 h depending on the cell batch. Do not change medium during this step.</li>
	</ol>
	</li>
	<li>Differentiation of muscle cells
	<ol>
		<li>Remove PM (after 48-72 h) and replace with pre-warmed DM (3 mL per well). Incubate at 37 &#176;C, 5% CO2. 
		NOTE: Differentiation takes five days to reach a stable state where cells are aligned and polynucleated. Typically, the primary myotubes are cultured in a 1 g/L glucose medium. Therefore, to avoid glucose depletion during culture, fill the plate with 3 mL medium to ensure that enough glucose substrate is available for the cells at all time.</li>
		<li>Replace DM medium every two days. 
		NOTE: From this point, myotubes are stable for up to 7 days without any significant change and glucose uptake measurement can be performed at any time.</li>
	</ol>
	</li>
	<li>Muscle cells treatment (optional) 
	NOTE: Primary myotubes can be treated for several days to induce modification (drug test, inhibitors/activators of signaling pathway, etc.) before insulin stimulation and glucose uptake measurements. Muscle cells can be submitted to any treatment that may have an impact on insulin sensitivity, and glucose uptake measurement will quantify this impact. For example, incubation of muscle cells with the saturated fatty acid palmitate promotes insulin resistance, and cells display reduced insulin stimulated glucose uptake.
	<ol>
		<li>Prepare 12 mL of DM supplemented with 10% BSA (fatty acid free) and 0.5 mL palmitate (PALM). Prepare 12 mL of DM supplemented with 10% BSA (fatty acid free) only.</li>
		<li>Prepare two 6-well plates with human primary myotubes, and culture them as described in sections 2.1 and 2.2 (with 5 days of differentiation).</li>
		<li>On day 5, wash each well with 2 mL of PBS. To one plate, add 2 mL of DM containing PALM. To the other plate add 2 mL of BSA only containing DM.</li>
		<li>Incubate for 48 h at 37 &#176;C, 5% CO2.</li>
	</ol>
	</li>
</ol>
3. Insulin Stimulation
<ol>
	<li>Wash differentiated muscle cells twice with 2 mL PBS.</li>
	<li>Remove PBS carefully and incubate with 3 mL of DM without FCS for 3 h (37 &#176;C, 5% CO2) for serum depletion.</li>
	<li>Replace media in all wells with 3 mL of DM without FCS. Add 100 nM insulin to wells 5 and 6.</li>
	<li>Incubate human myotubes culture for 1 h (37 &#176;C, 5% CO2).</li>
</ol>
4. Glucose Uptake
<ol>
	<li>After 1 h of insulin stimulation, wash wells twice with X-DPBS (1 mL per wash).</li>
	<li>Add 1 mL of cytochalasin B mixture to wells 1 and 2, and 1 mL of DMSO mixture to wells 3 - 6. Incubate for 15 min (37 &#176;C, 5% CO2). At the end of the incubation, immediately place the plate on ice.</li>
</ol>
5. Cell Lysis
<ol>
	<li>Wash the cells twice with 1 mL of ice cold PBS.</li>
	<li>Lyse the cells in each well with 600 &#181;L of 50 mM NaOH. Incubate on ice for 5 min and mix gently with slow orbital rotation. 
	NOTE: If the lysate is too viscous, dilute with up to 1.5 mL NaOH.</li>
	<li>Using a pipette, resuspend and collect the cell lysate.</li>
</ol>
6. Determination of Radiolabeled Glucose
<ol>
	<li>Put 400 &#181;L of each cell lysate in a liquid scintillation counting vial. Prepare a negative control vial with 400 &#181;L of 50 mM NaOH, and a positive control vial with 20 &#181;L of TC20 (from step 1.2.3.1).</li>
	<li>Add 4 mL of liquid scintillation solution to each vial. Close the cap and mix each vial thoroughly (1-2 s).</li>
	<li>Insert each vial in a liquid scintillation counter and measure the radioactivity according to the manufacturer&#39;s instruction. Record counts per min (CPM) for each scintillation vial for 10 min. 
	NOTE: CPM = &#34;disintegrations per minute&#34; x &#34;counting efficiency&#34;.</li>
</ol>
7. Rate of Glucose Uptake
<ol>
	<li>Use the remaining lysate (200 &#181;L; from step 5.2) to measure the protein concentration. Determine the protein concentration of each cell lysate using Bradford25 or an equivalent method. Calculate total protein quantity (Q) in mg for each well.</li>
	<li>To obtain TC1 (the value for 1 &#181;L of radiolabeled 2dG*), divide the CPM value of TC20 by 20.</li>
	<li>For each vial, calculate the rate of glucose uptake as follows: 
	<img alt="Equation" src="/files/ftp_upload/55743/55743eq1.jpg" /> 
	NOTE: R is measured in pmol/mg/min. Mean of R for wells 1 - 2 gives passive transport rate, Rp. Mean of R for wells 3-4 gives basal total transport rate, Rbt. Mean of R for wells 5-6 gives insulin stimulated total transport rate, Rit.
	<ol>
		<li>Calculate the basal active transport rate (Rba) as follows: 
		<img alt="Equation" src="/files/ftp_upload/55743/55743eq2.jpg" /></li>
		<li>Calculate the insulin stimulated active transport rate (Ria) as follows: 
		<img alt="Equation" src="/files/ftp_upload/55743/55743eq3.jpg" /> 
		NOTE: In insulin responsive cells like myotubes, glucose uptake rates are usually represented by three values: Rba, Ria, and the fold insulin stimulation as Ria/Rba.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Glucose uptake is a key biological measurement for testing activators or inhibitors on cell culture and how they impact glucose use, and the ability of the cell to respond to insulin. The method described here has been shown to be quick and reliable and has been widely used in many studies using primary myotubes from healthy subjects and/or metabolically affected patients6,7,10,12,21,26,27,36,37. With only one 6-well plate, rates can be obtained in duplicates for total basal transport, basal active transport, and insulin-stimulated active transport. These three values fully describe the insulin sensitivity of the in vitro cultured muscle cells.
Primary muscle cells are routinely cultured on collagen-coated plates. When using cell lines like C2C12 or L6, non-coated plates can be used. To maximize insulin response, it is important to deplete insulin from the muscle cells before stimulation in order to have the lowest possible basal state. The 3 h incubation time indicated in this protocol can be modified as per cell stability upon insulin and/or serum starvation. The 3 h incubation with insulin is followed by a 15-min incubation with radiolabeled glucose. The purpose of the one-hour incubation with insulin is to allow translocation of any available GLUT4 containing vesicles to the membrane in order to achieve maximal glucose uptake rate. Previous tests showed that shorter incubation times (15 and 30 min) did not achieve maximal rates. Persistence of insulin stimulation during the 15 min with radiolabeled 2dG is not mandatory as it did not affect the measurement (personal data). Moreover, the same radiolabeled 2dG mixture can be added to all tested wells. For cell lysis, the volume of NaOH can be modified according to the cell characteristics. When working with cells that contain high levels of fatty acids i.e. for very viscous lysates, the volume of NaOH can be increased up to 1.5 mL.
The method uses radiolabeled material that enhances the sensitivity of the measurement, but requires that the protocol be performed in controlled areas. Materials and wastes need to be handled according to the local safety guidelines. For non-equipped laboratories, other colorimetric and fluorescence quantification38 methods are available. In non-radioactive assays, fluorescence intensity can be measured after incubation of the cells with a fluorescent D-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG). With this analog, some groups report that insulin lost its physiological effects in L6 cells39, whereas other groups showed that insulin still increases glucose uptake (in human primary muscle cells)38. Due to low GLUT4 expression, quantification of insulin stimulated glucose uptake in cultured muscle cells is reduced compared to in vivo conditions6,16. It is thus important to further characterize insulin responsiveness of the cultured muscle cells through other approaches. Measurements of phosphorylation status of key proteins of the insulin-signaling pathway (like Akt/PKB and/or GSK3) using immuno-detection can be used14,21,31. At the enzymatic level, glycogen synthesis, glucose and/or lipid oxidation can also be assessed in conditions with and without insulin14,26,31. Any other cell type (whether it responds to insulin or not) could theoretically be analyzed as all cells can actively or passively uptake glucose. The insulin-stimulated wells can thus be replaced by any other appropriate treatment.
The use of human primary myotube implies that muscle cells have reached a differentiation state consistent with enough expression of GLUT4 protein. If the preparation contains non-muscle cells or essentially myoblasts, glucose uptake values in insulin stimulation condition will not be different from the basal state (Ria versus Rba), mainly due to the predominance of GLUT1 transporter.
Insulin can be added to the primary muscle cell culture medium to promote and enhance muscle cell proliferation and differentiation. Nevertheless, insulin has also been demonstrated to induce insulin resistance in chronic stimulation40,41,42. In our experiments, human primary muscle cells properly differentiate only upon medium change with reduced serum. We thus prefer not to add external insulin during culture to preserve the cells from chronic stimulation before assessment of insulin responsiveness. Finally, the method requires several washing steps. Therefore, care must be taken to not detach cells from the plate as it can happen with primary cells.

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. This insulin responsive tissue is the primary site of the glucose uptake that is triggered by insulin stimulation1.
In type 2 diabetes, insulin resistance is observed in several tissues, including skeletal muscle, and leads to above normal blood glucose concentration. Thus, it is of major relevance to determine the level of insulin sensitivity of this tissue and its cells, whether the aim is to characterize a defect in a subject, or to evaluate the efficiency of a treatment intending to improve it. In human or animal subjects, the gold standard technique to assess insulin sensitivity is the hyperinsulinemic-euglycemic clamp. Introduced by DeFronzo in 19792 and modified since3,4 then, the method allows to quantify whole body and tissues insulin responsiveness measured as the rate of glucose to be perfused under insulin stimulation to maintain normal blood glucose concentration.
Insulin sensitivity exploration can also be performed at the cell level using in vitro muscle models, and measurement of glucose uptake rates remains an efficient and reliable tool to quantify the biological response of the cell to insulin stimulation5,6,7. Indeed, glucose uptake measurement quantifies the cell biological response to insulin stimulation, from the binding of insulin to its receptor to translocation of GLUT4 enriched vesicles, and including intracellular signaling and phosphorylation cascades8.
This is of major interest with human samples, as differentiated myotubes maintain many features of the donor phenotype, including metabolic properties and disorders observed in the patient9,10,11,12. The myotubes displays structural, metabolic and phenotypic similarities to the skeletal muscle13,14, including the expression of glucose transporters15 and the cellular insulin signaling machinery16. Thus, measurement of the glucose uptake in primary myotubes is of relevance to characterizing the muscle phenotype of a donor, or investigating the effect of an intervention (drug, nutrition, or physical activity) on the insulin sensitivity in the muscle cell.
The measurement of glucose uptake on cultured myotubes also is a reliable tool when performing experiments that modify insulin sensitivity17,18. The in vitro model is suitable for the test of any compounds that could improve insulin responsiveness, or could prevent or reverse acquired or induced insulin resistance19,20,21,22,23.
Here we describe a detailed protocol to culture and differentiate human myotubes and to measure cell glucose uptake rates. The method is applicable to any source of human muscle precursor cells, whether they come from in-lab preparations, collaboration, or commercially available suppliers. Immortalized muscle cell lines, like C2C12 and L6, respectively from mouse and rat origin, can also be used for glucose uptake measurement with this protocol7.
We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H]2dG. The use of a labeled glucose analog allows accurate determination of glucose entry with reduced starting material, a common condition when working with primary cells. The modified glucose molecule is unable to enter metabolic pathways, and thus, accumulates within the cell, allowing reliable quantification via total cell radioactivity. Experimental conditions include the use of a glucose transport inhibitor (cytochalasin B), and measurements are performed with and without insulin. This combination allows the determination of glucose active entry rates, as well as the calculation of fold change for the insulin response index. The method is presented with one dose of insulin during a single incubation time, but the protocol can easily be modified for dose response or time course experiments12.

<table><tbody><tr><td>Human primary muscle cell</td><td>In house preparation from human skeletal muscle biopsies</td><td>In house preparation from human skeletal muscle biopsies</td><td>If not available, use commercial source</td></tr><tr><td>Human primary muscle cell</td><td>Promocell</td><td>C-12530</td><td>Should be cultured with associated media C23060 and C23061</td></tr><tr><td>6-well plate</td><td>Corning</td><td>356400</td><td>BioCoat Collagen I Multiwell Plates</td></tr><tr><td>Ham&#039;s F10</td><td>Dutscher</td><td>L0145-500</td><td>1 g/L glucose</td></tr><tr><td>Glutamine</td><td>Dutscher</td><td>X0551-100</td><td></td></tr><tr><td>penicilin/streptomycin 100x</td><td>Thermo fisher scientific</td><td>15140122</td><td></td></tr><tr><td>Serum substitute UltroserG</td><td>Pall France</td><td>15950.017</td><td>serum substitute in text</td></tr><tr><td>DMEM low glucose</td><td>Dutscher</td><td>L0064-500</td><td>1 g/L glucose</td></tr><tr><td>Fetal Calf Serum</td><td>Eurobio</td><td>CVFSVF00-01</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline</td><td>Dutscher</td><td>L0625-500</td><td>Contains Mg&lt;sup&gt;2+&lt;/sup&gt; (0.5 mM) and Ca&lt;sup&gt;2+&lt;/sup&gt; (0.9 mM)</td></tr><tr><td>Insulin solution human</td><td>Sigma-Aldrich</td><td>I9278</td><td></td></tr><tr><td>2-deoxy-D-glucose&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D6134</td><td></td></tr><tr><td>Albumin bovine</td><td>euromedex</td><td>04-100-812-E</td><td></td></tr><tr><td>fatty acid-free BSA</td><td>Roche</td><td>10,775,835,001</td><td></td></tr><tr><td>palmitate</td><td>Sigma-Aldrich</td><td>P0500</td><td></td></tr><tr><td>Deoxy-D-glucose, 2-[1,2-3H (N)]</td><td>PerkinElmer</td><td>NET328A001MC</td><td>Specific Activity: 5 - 10 Ci (185-370GBq)/mmol, 1 mCi (37MBq</td></tr><tr><td>Cytochalasin B</td><td>Sigma-Aldrich</td><td>c2743</td><td></td></tr><tr><td>PICO PRIAS VIAL 6 mL</td><td>PerkinElmer</td><td>6000192</td><td></td></tr><tr><td>ultima gold MW CA&amp;nbsp;</td><td>PerkinElmer</td><td>6013159</td><td>scintillation liquid</td></tr><tr><td>b&amp;ecirc;ta counter&amp;nbsp;</td><td>PerkinElmer</td><td>2900TR</td><td></td></tr></tbody></table>

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.

On day 3, myoblasts reach confluence (Figure 1A). The myoblasts at this stage are typically mononucleated. Medium was changed and on day 8, differentiation was completed (Figure 1B) (protocol section 2). After 5 days of differentiation, myotubes are aligned and typically polynucleated. Human primary myotubes were subjected to a palmitate or a BSA-only treatment before glucose uptake rate measurement. Cells were incubated for 48 h with palmitate 0.5 mM in BSA (PALM) or BSA alone (BSA). Insulin stimulation was performed (protocol section 3) and glucose uptake was measured (protocol section 4). Figure 2 shows glucose uptake rate in a control condition (BSA) and a treatment condition (PALM). Nonspecific uptake is quantified in myotubes incubated with cytochalasin B. Basal and insulin stimulated glucose uptake rates can be compared in BSA and PALM conditions. Insulin significantly increases glucose transport in the BSA condition (p &#60;0.01). The insulin stimulated glucose uptake rate is significantly lower in myotubes treated with PALM (p &#60;0.05). Furthermore, the human myotube cell response to insulin can also be expressed as fold change. Figure 3 demonstrates that the response to insulin is decreased in myotubes treated with PALM when compared to control (p &#60;0.05).
Despite limited expression of GLUT4 protein in cultured myotubes, primary muscle cells are able to respond to insulin with an increase in glucose uptake (Figure 2). The mean fold change observed is ~1.3, similar to what was described by other groups with human muscle cells6,7,10,26,27. Induction of insulin resistance in muscle cells with saturated fatty acid is commonly described in mouse, rat and human muscle cells, and has been demonstrated to reduce glucose uptake of treated muscle cells28,29,30,31,32,33,34. We performed palmitate (0.5 mM) incubation for 48 h and observed a decrease in the rate of basal and insulin stimulated glucose uptake and a decrease in the insulin fold change, reflecting the insulin resistant state. The incubation time used in this manuscript has been chosen to fully demonstrate the inhibitory effect on glucose uptake. Shorter incubation times can also produce insulin resistance and reduced glucose uptake31,32,33,34. Lower and thus more physiological concentrations can be used in vitro, as well as dose response and/or time course experiments, and comparison with the effect of unsaturated fatty acids like oleate29,35.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55743/55743fig1.jpg" /> 
Figure 1: In Vitro Primary Human Myotubes. Images of human myotubes at 2 stages of culture obtained using an optical microscope are shown. (A) Day 3 of myoblast culture at confluence. (B) At day 8 (5 days of differentiation), myotubes are aligned. Scale bar = 200 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55743/55743fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55743/55743fig2.jpg" /> 
Figure 2: Effect of Insulin Stimulation on Glucose Uptake Rate in Human Myotubes Under BSA and PALM Conditions. Myotubes were treated with BSA alone or 0.5 mM PALM for 48 h. After medium change and 3 h serum depletion, specific glucose uptake rate (in pmol/mg/min) was measured in the absence (-) or in the presence (+) of insulin (1 h, 100 nM). Data are mean &#177; sem of four independent experiments using myotubes from four different donors. #P &#60;0.05 compared with the basal value; *P &#60;0.05 compared with BSA insulin stimulation value. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55743/55743fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55743/55743fig3.jpg" /> 
Figure 3: Fold Insulin Response in BSA and PALM Culture Conditions. Ratio of insulin stimulated on basal glucose transport rate in BSA and PALM conditions. This ratio gives the fold glucose uptake rate upon insulin stimulation (100 nM). *P &#60;0.05 compared to BSA condition (n = 4). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55743/55743fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes at JoVE.com

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

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.

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 ...

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19.8K Views.  University of Lyon.  Bij deze werkwijze worden humane primaire spiercellen in vitro gekweekt om gedifferentieerde myotubes te verkrijgen en de opname van glucose wordt gemeten. Wij bieden een gedetailleerd protocol om de tarieven in basale en insuline gestimuleerde staten te kwantificeren met radioactief gemerkte [3H] 2-deoxy-D-glucose. 

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

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.

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.

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

Biochemistry

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved F&#246;rster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved F&#246;rster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D2-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.

Here, we present homogeneous time resolved FRET (HTRF) as an efficient method for rapid detection of insulin secreted from cells.

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

Medicine

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical manifestations include weight loss, dyslipidemia, and hypermetabolism; however, it remains unclear how these relate to motor neuron degeneration. Using a Drosophila model of TDP-43 proteinopathy that recapitulates several features of ALS including cytoplasmic inclusions, locomotor dysfunction, and reduced lifespan, we recently identified broad ranging metabolic deficits. Among these, glycolysis was found to be upregulated and genetic interaction experiments provided evidence for a compensatory neuroprotective mechanism. Indeed, despite upregulation of phosphofructokinase, the rate limiting enzyme in glycolysis, an increase in glycolysis using dietary and genetic manipulations was shown to mitigate locomotor dysfunction and increased lifespan in fly models of TDP-43 proteinopathy. To further investigate the effect on TDP-43 proteinopathy on glycolytic flux in motor neurons, a previously reported genetically encoded, FRET-based sensor, FLII12Pglu-700&#181;&#948;6, was used. This sensor is comprised of a bacterial glucose-sensing domain and cyan and yellow fluorescent proteins as the FRET pair. Upon glucose binding, the sensor undergoes a conformational change allowing FRET to occur. Using FLII12Pglu-700&#181;&#948;6, glucose uptake was found to be significantly increased in motor neurons expressing TDP-43G298S, an ALS causing variant. Here, we show how to measure glucose uptake, ex vivo, in larval ventral nerve cord preparations expressing the glucose sensor FLII12Pglu-700&#181;&#948;6 in the context of TDP-43 proteinopathy. This approach can be used to measure glucose uptake and assess glycolytic flux in different cell types or in the context of various mutations causing ALS and related neurodegenerative disorders.

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Glucose uptake is increased in Drosophila motor neurons affected by TAR DNA binding protein (TDP-43) proteinopathy, as indicated by a FRET-based, genetically encoded glucose sensor.

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical ...

Neuroscience

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

The goal of this protocol is to measure the effect of glucose-mediated changes to mitochondrial respiration in the presence of natural compounds on intact 832/13 beta cells using high-resolution respirometry.

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...

Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate

Performing antibody-based assays for secreted insulin post-sample collection usually requires a few hours to a day of assay time and can be expensive, depending on the specific assay. Secreted luciferase assays expedite results and lower the assay cost per sample substantially. Here we present a relatively underused approach to gauge insulin secretory activity from pancreatic &#946; cells by using Gaussia luciferase genetically inserted within the C-peptide. During proteolytic processing of proinsulin, the C-peptide is excised releasing the luciferase within the insulin secretory vesicle where it is co-secreted with insulin. Results can be obtained within minutes after sample collection because of the speed of luciferase assays. A limitation of the assay is that it is a relative measurement of insulin secretion and not an absolute quantitation. However, this protocol is economical, scalable, and can be performed using most standard luminescence plate readers. Analog and digital multichannel pipettes facilitate multiple steps of the assay. Many different experimental variations can be tested simultaneously. Once a focused set of conditions are decided upon, insulin concentrations should be measured directly using antibody-based assays with standard curves to confirm the luciferase assay results.

This protocol describes how to perform rapid low-cost luciferase assays at medium-throughput using an insulin-linked Gaussia luciferase as a proxy for insulin secretion from beta cells. The assay can be performed with most luminescence plate readers and multichannel pipettes.

Performing antibody-based assays for secreted insulin post-sample collection usually requires a few hours to a day of assay time and can be expensive, ...

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 ...

In Vitro Glucose Depletion Assay: A Fluorescence-Based Method to Quantify Cellular Glucose Uptake via Extracellular Glucose Depletion Measurement

Source: Kumar, S. B. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63681">Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues&#160;Ex Vivo.</a>&#160;J. Vis. Exp.&#160;(2022)
This video demonstrates an in vitro assay to indirectly measure intracellular glucose uptake. The assay uses a fluorescently-labeled glucose analog to establish a correlation between extracellular glucose depletion and intracellular glucose uptake.

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

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Cell-based assays

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.remotexs.ntu.edu.sg/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.

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

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