Introduction
We established a primary human skeletal muscle cell culture system using differentiated myotubes. Through the use of primary muscle cultures it should be possible to a) enhance the availability of biological material, b) increase the length of incubation time and range of experimental manipulations and c) to transfect candidate genes to engineer cells with improved insulin action on glucose metabolism.
Methods
Rectus abdominal muscle biopsies (1-3 g) were obtained during surgery from 7 healthy subjects (58 +/- 2.4 yrs). Muscle biopsies were collected in cold phosphate-buffered saline (4oC) supplemented with 1% PeSt (100 units/ml penicillin/100 mg/ml streptomycin) and processed 2-3 days post isolation to inactivate lymphocytes and fat cells. Cells were seeded and the differentiation programme was initiated when cells were >70% confluent. Cells were harvested at day 0, 3, 5, 7 and 14 days after differentiation and insulin action on glucose uptake and metabolism was assessed.
Results
At day 5 of incubation the majority of muscle cells were highly differentiated, as evident by theirmultinucleated morphology. Importantly, insulin increased glucose uptake (2-fold; p<0.05) and glycogen synthesis (2-fold; p<0.01), consistent with results from the creatine kinase assay and Giemsa/Wright staining confirming differentiation of cells into functional myotubes. Initiation of insulin responsiveness in the muscle cells coincided with an increased expression of a number of proteins known to be important for insulin action (including the insulin receptor, constitutes of the insulin signal transduction cascade and GLUT4 glucose transporter). As these cells express GLUT1, insulin-stimulated increment in glucose uptake can be masked by high rates of GLUT1-mediated glucose uptake. Cell surface GLUT4 content was assessed using a bis-mannose photolabel coupled to biotin. This allows for studies of insulin action on GLUT4 in the presence of GLUT1. At day 5 of differentiation, when several of the key proteins in insulin action reached greatest expression, preliminary studies reveal that insulin leads to a 6-fold increase in cell surface GLUT4 content. Recombinant adenovirus transferred the b-galactosidase gene into >80% of the myotubes compared to <1% transfer using more traditional methods. Thus, adenovirus transfection systems are superior to plasmid transfection systems in achieving a high degree of gene transfer efficiency.
Conclusions
Primary human skeletal muscle cultures (myotubes) are suitable systems to study insulin action on glucose uptake and metabolism. Use of the bismannose photolabel is a powerful tool to dissect insulin action on GLUT4 translocation. Day 5 of differentiation represents the most insulin sensitive stage of the muscle culture. Use of adenovirus transfection systems in cultured human muscle will allow for metabolic engineering to improve glucose metabolism in insulin resistant muscle.
References