Glucose Uptake and Insulin Response in Tissue-engineered Human Skeletal Muscle

Abstract

Background: Tissue-engineered muscles ("myobundles") offer a promising platform for developing a human in vitro model of healthy and diseased muscle for drug development and testing. Compared to traditional monolayer cultures, myobundles better model the three-dimensional structure of native skeletal muscle and are amenable to diverse functional measures to monitor the muscle health and drug response. Characterizing the metabolic function of human myobundles is of particular interest to enable their utilization in mechanistic studies of human metabolic diseases, identification of related drug targets, and systematic studies of drug safety and efficacy.

Methods: To this end, we studied glucose uptake and insulin responsiveness in human tissue-engineered skeletal muscle myobundles in the basal state and in response to drug treatments.

Results: In the human skeletal muscle myobundle system, insulin stimulates a 50% increase in 2-deoxyglucose (2-DG) uptake with a compiled EC₅₀ of 0.27 ± 0.03 nM. Treatment of myobundles with 400 µM metformin increased basal 2-DG uptake 1.7-fold and caused a significant drop in twitch and tetanus contractile force along with decreased fatigue resistance. Treatment with the histone deacetylase inhibitor 4-phenylbutyrate (4-PBA) increased the magnitude of insulin response from a 1.2-fold increase in glucose uptake in the untreated state to a 1.4-fold increase after 4-PBA treatment. 4-PBA treated myobundles also exhibited increased fatigue resistance and increased twitch half-relaxation time.

Conclusion: Although tissue-engineered human myobundles exhibit a modest increase in glucose uptake in response to insulin, they recapitulate key features of in vivo insulin sensitivity and exhibit relevant drug-mediated perturbations in contractile function and glucose metabolism.

Keywords: Insulin; Muscle; Myobundles; Skeletal; Tissue engineering.

Fig. 1

Characterization of insulin sensitivity and dose response in myobundles. A Basal and insulin-mediated 2-DG uptake were measured in myobundles at 2 weeks post-differentiation. The 2-DG uptake rates were normalized to the basal uptake rate for each donor. Using a curve fitting tool, the values for the R_max and EC₅₀ parameters were determined. B Individual donor experimental values for the maximum fold-change in 2-DG uptake with insulin stimulation and the insulin concentration at which that maximum fold-change occurred, as well as the individual donor R_max and EC₅₀ values. Donor S111 exhibited glucose uptake rates in response to insulin stimulation that were lower than the basal 2-DG uptake, which precluded the determination of the EC₅₀ and R_max values for that donor. The numbers denoted with * represent values obtained when limiting analysis to only physiologically-relevant insulin concentrations of ≤ 10 nM, while the un-starred numbers represent values obtained when considering the full range of insulin doses. For each donor, 2-DG uptake was obtained for n = 3−7 myobundles per insulin concentration. Data presented as mean ± S.E.M

Fig. 2

Compilation of insulin responsiveness in 2-D and 3-D. Data are compiled from experiments shown in this study. The magnitude of insulin response is calculated as a ratio of 2-DG uptake in response to 10 nM insulin over the 2-DG uptake in response to 0 nM insulin (where a magnitude of 1 indicates equivalent uptake rates at 0 and 10 nM insulin). Each data point is obtained by averaging the responses of 3-4 wells (for 2-D) or myobundles (for 3-D), The inlayed graph depicts the average magnitude of insulin response of myotubes in 2-D culture and myobundles in 3-D culture from all donors, shown as mean ± S.E.M

Fig. 3

Effect of metformin treatment on myotube and myobundle function. A Myotubes were differentiated in a 6-well plate and treated with 400 µM metformin for 18 h immediately prior to 2-DG uptake at the 2 week differentiation time point. N = 3 donors, n = 3 wells per condition per donor. B Myobundles were treated with 400 µM metformin for 18 h immediately prior to 2-DG uptake at the 2 week differentiation time point. N = 3 donors, n = 3−4 myobundles per condition per donor. C, D Myobundles were treated with 400 µM metformin for 18 h immediately prior to measuring twitch and tetanus contractile force and percent fatigue at the 2 week differentiation time point. N = 3 donors, n = 3-4 myobundles per condition per donor. E) Analysis of time to maximum force and half-relaxation time of twitch contractions. N = 3 donors, n = 3−4 myobundles per condition per donor. * p < 0.05, **p < 0.01, *** p < 0.001 when compared with the same insulin condition or contractile function measurement in the no treatment group. Data in all panels presented as mean ± S.E.M

Fig. 4

Effect of metformin treatment on myobundle gene expression. Myobundles were treated with 400 µM metformin for 18 h immediately prior to flash freezing. Gene expression of A, B glucose transporters GLUT1, GLUT4, and GLUT3 and metabolic regulator PGC-1α and C, D myosin heavy chain isoforms was determined. For all panels, N = 3 donors, n = 3 myobundles per treatment condition per donor. **p < 0.01, ***p < 0.001 when compared with the no treatment group. Data in all panels presented as mean ± S.E.M

Fig. 5

Effect of the general HDAC inhibitor 4-PBA on myotube and myobundle function. A Myotubes were differentiated in a 6-well plate and treated with 5 mM 4-PBA for 18 h immediately prior to 2-DG uptake at the 2 week differentiation time point. N = 3 donors, n = 3 wells per condition per donor. B Myobundles were treated with 5 mM 4-PBA for 18 h immediately prior to 2-DG uptake at the 2 week differentiation time point. N = 3 donors, n = 3−4 myobundles per condition per donor. C–E Myobundles were treated with 5 mM 4-PBA for 18 h immediately prior to measuring twitch and tetanus contractile force, percent fatigue, and twitch kinetics at the 2 week differentiation time point. N = 3 donors, n = 3−4 myobundles per condition per donor. For all panels, N = 3 donors, n = 3 myobundles per condition per donor. *p < 0.05, **p < 0.01, ***p < 0.001 when compared with the same insulin condition or contractile function measurement in the no treatment group.# p < 0.05 when compared with the no insulin condition within the treatment group. Data in all panels presented as mean ± S.E.M

Fig. 6

Effect of 4-PBA treatment on myobundle gene and protein expression. Myobundles were treated with 5 mM 4-PBA for 18 h immediately prior to flash freezing. A Gene expression of glucose transporters GLUT1, GLUT4, and GLUT3 and metabolic regulator PGC-1α. N = 3 donors, n = 3 myobundles per treatment condition per donor. B Quantification of Western blots for glucose transporters GLUT1 and GLUT4. N = 3 donors, n = 6−9 myobundles per treatment condition per donor. C Representative Western blots for one donor. Each lane contains pooled protein from 3 myobundles. D Gene expression of myosin heavy chain isoforms. N = 3 donors, n = 3 myobundles per treatment condition per donor. *p < 0.05, **p < 0.01, ***p < 0.001 when compared with the no treatment group. Data in all panels presented as mean ± S.E.M