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 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.
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.
1. Preparation of Cell Culture Media and Solutions
2. Culture of Human Primary Muscle Cells
3. Insulin Stimulation
4. Glucose Uptake
5. Cell Lysis
6. Determination of Radiolabeled Glucose
7. Rate of Glucose Uptake
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 <0.01). The insulin stimulated glucose uptake rate is significantly lower in myotubes treated with PALM (p <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 <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.
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 µm. Please click here to view a larger version of this figure.
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 ± sem of four independent experiments using myotubes from four different donors. #P <0.05 compared with the basal value; *P <0.05 compared with BSA insulin stimulation value. Please click here to view a larger version of this figure.
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 <0.05 compared to BSA condition (n = 4). Please click here to view a larger version of this figure.
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.
The authors have nothing to disclose.
The authors acknowledge Anne Charrié at the Radiobiology service (Lyon-Sud hospital) and the Fond National Suisse (FNS) for their financial support.
Human primary muscle cell | In house preparation from human skeletal muscle biopsies | In house preparation from human skeletal muscle biopsies | If not available, use commercial source |
Human primary muscle cell | Promocell | C-12530 | Should be cultured with associated media C23060 and C23061 |
6-well plate | Corning | 356400 | BioCoat Collagen I Multiwell Plates |
Ham's F10 | Dutscher | L0145-500 | 1 g/l glucose |
Glutamine | Dutscher | X0551-100 | |
penicilin/streptomycin 100x | Thermo fisher scientific | 15140122 | |
Serum substitute UltroserG | Pall France | 15950.017 | serum substitute in text |
DMEM low glucose | Dutscher | L0064-500 | 1 g/l glucose |
Fetal Calf Serum | Eurobio | CVFSVF00-01 | |
Dulbecco's Phosphate-Buffered Saline | Dutscher | L0625-500 | Contains Mg2+ (0.5 mM) and Ca2+ (0.9 mM) |
Insulin solution human | Sigma-Aldrich | I9278 | |
2-deoxy-D-glucose | Sigma-Aldrich | D6134 | |
Albumin bovine | euromedex | 04-100-812-E | |
fatty acid-free BSA | Roche | 10,775,835,001 | |
palmitate | Sigma-Aldrich | P0500 | |
Deoxy-D-glucose, 2-[1,2-3H (N)] | PerkinElmer | NET328A001MC | Specific Activity: 5-10Ci (185-370GBq)/mmol, 1mCi (37MBq |
Cytochalasin B | Sigma-Aldrich | c2743 | |
PICO PRIAS VIAL 6ml | PerkinElmer | 6000192 | |
ultima gold MW CA | PerkinElmer | 6013159 | scintillation liquid |
bêta counter | PerkinElmer | 2900TR |