Elevated GLUT4 Levels in Human Skeletal Muscle Microtissues is Accompanied by Functional Insulin Dependence

Insulin resistance in skeletal muscle is a hallmark of type 2 diabetes mellitus (T2D). While two-dimensional myotube cultures offer a controlled environment for studying T2D-related metabolic dysfunction, insulin-dependent glucose transporter type 4 (GLUT4) levels are limited and insulin-independent glucose transporter type 1 (GLUT1) expression dominates; reducing physiological relevance. Three-dimensional skeletal muscle microtissue cultures offer a promising alternative, and unlike 2D myotubes, are amenable to repeated contractile stimulation. However, microtissue GLUT1 and GLUT4 glucose transporter profiles remain under-characterized, particularly under physiological glucose and insulin conditions, which is evaluated herein. We report that GLUT1 levels trended [~]3.0-fold lower in microtissues compared with myotubes in 2D culture, although not statistically significant (p = 0.072), while GLUT4 levels were [~]12-fold higher (p < 0.0001), leading to a [~]60-fold increase in the GLUT4:GLUT1 ratio (p = 0.023). Notably, the microtissue GLUT4:GLUT1 profile approached, but did not match that of native human muscle. Microtissues required supraphysiological insulin conditions for the development of maximal contractility, while physiological glucose levels were sufficient. Insulin withdrawal restored insulin responsiveness but impaired microtissue contractile strength (p < 0.0001) and fatigue resistance (p = 0.015). Our findings indicate that the glucose transporter profile of microtissues offers improved physiological relevance. However, their reliance on insulin to maintain contractile function limits their suitability for modeling T2D. The implementation of a robust, insulin-free differentiation protocol would facilitate the development of a microtissue-based T2D model which can be applied to study contraction-mediated increases in insulin sensitivity as a therapeutic approach.