Contractile function and insulin sensitivity are inversely regulated by insulin in primary myoblast human skeletal...

Insulin resistance in skeletal muscle is a hallmark of Type 2 diabetes mellitus (T2D). Three-dimensional skeletal muscle microtissues (hMMTs) offer advantages over two-dimensional myotube cultures, including enhanced physiological relevance and amenability to repeated contractile stimulation. However, standard hMMT differentiation protocols rely on supraphysiological insulin concentrations. This creates a fundamental conundrum, since the very conditions required to generate functional hMMTs are expected to render them insulin-unresponsive, limiting their utility for modeling T2D. Whether hMMTs can be differentiated under more physiological conditions while maintaining myotube formation, contractile strength, and glucose transporter type 4 (GLUT4) expression and function, and whether insulin responsiveness can be restored post-differentiation, remains unknown.

We systematically evaluated the effects of nutrient and insulin conditions on myotube morphometry, contractile function, and glucose transporter profiles (GLUT4, GLUT1, insulin receptor β) in human primary myoblast-derived 2D myotubes and hMMTs. We tested whether reducing insulin or glucose to physiological levels during differentiation impacted hMMT contractility. We then evaluated an insulin re-sensitization protocol involving insulin withdrawal to restore insulin responsiveness and assessed its impact on contractile function, glucose uptake, and AKT signaling.

Insulin supplementation during differentiation was essential for myonuclear accretion in 2D cultures and for contractile hMMT formation, with supraphysiological insulin (≥ 0.86 µM) levels required for maximal hMMT strength. Physiological glucose levels were sufficient to support myotube formation in 2D and 3D settings and even enhanced hMMT contractile force as compared to standard supraphysiological glucose formulations. hMMTs exhibited ~ 12-fold higher GLUT4 levels and a ~ 40-fold increase in GLUT4:GLUT1 ratio compared to 2D myotubes (p < 0.0001 and p = 0.044, respectively), approaching but not matching native adult human muscle. hMMTs differentiated in supraphysiological insulin appeared to display blunted insulin responsiveness. A 4-day insulin withdrawal period was associated with a partial restoration of insulin sensitivity, increasing insulin-stimulated glucose uptake ~ 1.5-fold and activating AKT signaling (N = 2). However, re-sensitization significantly compromised hMMT contractile strength (p < 0.0001) and fatigue resistance (p = 0.015).

We demonstrate that hMMTs derived from a 19-year-old female primary myoblast cell line require supraphysiological insulin during differentiation to achieve optimal contractile function and physiologically relevant GLUT4:GLUT1 ratios, but this renders them insulin-resistant. Although insulin withdrawal may partially restore insulin responsiveness, it impairs contractile integrity. These findings highlight a critical trade-off that may limit hMMT-based T2D modeling. Development of insulin-free differentiation protocols that preserve both contractile function and insulin sensitivity is important to unlock the potential of hMMTs for studying contraction-mediated therapeutic interventions in T2D.