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

Summary

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.

Abstract

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.

Introduction

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 an....

Protocol

1. Preparation of Cell Culture Media and Solutions

1. Preparation of culture media
   1. Prepare proliferation medium (PM) by supplementing Ham's F-10 medium with glutamine (2 mM), penicillin/streptomycin (5 µg/mL final), 2% Fetal Calf Serum (FCS) and 2% serum substitute.
   2. Prepare differentiation medium (DM) by supplementing Dulbecco's Modified Eagle Medium (DMEM) with glutamine (2 mM), penicillin/streptomycin (5 µg/mL final), and 2% FCS.
2. Preparation of glucose uptake solutions
   Caution: Handling of radioactive material is only allowed in a restricted and controlled area by authoriz....

Results

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.......

Discussion

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 patients^6,7,10,12,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 - 10 Ci (185-370GBq)/mmol, 1 mCi (37MBq
Cytochalasin B	Sigma-Aldrich	c2743
PICO PRIAS VIAL 6 mL	PerkinElmer	6000192
ultima gold MW CA	PerkinElmer	6013159	scintillation liquid
bêta counter	PerkinElmer	2900TR