Name: glucose uptake measurement and response to insulin stimulation in in vitro cultured human primary myotubes
Uploaded: 2017-06-25T14:00:00
Description: Watch this Scientific Journal Video about Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes at JoVE.com

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...

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,<s...

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 border="0" cellpadding="0" cellspacing="0" width="868">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Human primary muscle cell</td>
			<td style="width:171px;">In house preparation from human skeletal muscle biopsies</td>
			<td style="width:180px;">In house preparation from human skeletal muscle biopsies</td>
			<td style="width:257px;">If not available, use commercial source</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Human primary muscle cell</td>
			<td style="width:171px;">Promocell</td>
			<td>C-12530</td>
			<td style="width:257px;">Should be cultured with associated media C23060 and C23061</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">6-well plate</td>
			<td style="width:171px;">Corning</td>
			<td style="width:180px;">356400</td>
			<td style="width:257px;">BioCoat Collagen I Multiwell Plates</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Ham&#39;s F10</td>
			<td style="width:171px;">Dutscher</td>
			<td style="width:180px;">L0145-500</td>
			<td style="width:257px;">1 g/l glucose</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Glutamine</td>
			<td style="width:171px;">Dutscher</td>
			<td style="width:180px;">X0551-100</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">penicilin/streptomycin 100x</td>
			<td style="width:171px;">Thermo fisher scientific</td>
			<td style="width:180px;">15140122</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Serum substitute UltroserG</td>
			<td style="width:171px;">Pall France</td>
			<td style="width:180px;">15950.017</td>
			<td style="width:257px;">serum substitute in text</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">DMEM low glucose</td>
			<td style="width:171px;">Dutscher</td>
			<td style="width:180px;">L0064-500</td>
			<td style="width:257px;">1 g/l glucose</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Fetal Calf Serum</td>
			<td style="width:171px;">Eurobio</td>
			<td style="width:180px;">CVFSVF00-01</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td style="width:171px;">Dutscher</td>
			<td style="width:180px;">L0625-500</td>
			<td style="width:257px;">Contains Mg2+ (0.5 mM) and Ca2+ (0.9 mM)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Insulin solution human</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:180px;">I9278</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">2-deoxy-D-glucose&#160;</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:180px;">D6134</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Albumin bovine</td>
			<td style="width:171px;">euromedex</td>
			<td style="width:180px;">04-100-812-E</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">fatty acid-free BSA</td>
			<td style="width:171px;">Roche</td>
			<td style="width:180px;">10,775,835,001</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">palmitate</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:180px;">P0500</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Deoxy-D-glucose, 2-[1,2-3H (N)]</td>
			<td style="width:171px;">PerkinElmer</td>
			<td style="width:180px;">NET328A001MC</td>
			<td style="width:257px;">Specific Activity: 5-10Ci (185-370GBq)/mmol, 1mCi (37MBq</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Cytochalasin B</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:180px;">c2743</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PICO PRIAS VIAL 6ml</td>
			<td style="width:171px;">PerkinElmer</td>
			<td style="width:180px;">6000192</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">ultima gold MW CA&#160;</td>
			<td style="width:171px;">PerkinElmer</td>
			<td>6013159</td>
			<td style="width:257px;">scintillation liquid</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">b&#234;ta counter&#160;</td>
			<td style="width:171px;">PerkinElmer</td>
			<td style="width:180px;">2900TR</td>
			<td style="width:257px;"></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...

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

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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