An Exercise‐Driven, Insulin‐Independent Glucose Uptake Pathway in Contracting Skeletal Muscle

Glucose uptake by skeletal muscles, which import and store up to 80% of circulating glucose, plays a major role in glucose homeostasis. Prior research has focused largely on the insulin regulated GLUT4 transporter, which mediates glucose uptake in quiescent (non‐contracting) muscles using a canonical insulin‐dependent translocation mechanism. An ongoing paradox in the field is that actively contracting muscles take up glucose at 50‐100x greater rates than quiescent muscles, despite declining insulin levels. For this reason, exercise is a potent treatment for disorders of insulin insufficiency and insulin resistance. However, despite more than 30 years of study, the mechanism by which contracting muscles take up glucose without a requirement for insulin remains incompletely understood. It is proposed that GLUT4‐translocation occurs using signaling pathways that bypass the insulin receptor. However, no GLUT4 models have accounted for all the glucose taken up by contracting muscles or the great discrepancy between measured GLUT4 translocation (2‐fold increase in membrane density) and glucose uptake rate (50‐100‐fold increase), suggesting the existence of additional mechanisms. The goals of this study were to:i) develop a technology based on Inductively Coupled Plasma‐Mass Spectroscopy (ICP‐MS) that allows simultaneous detection of multiple transported species, including 13 C‐labeled hexoses; and ii) test the hypothesis that contraction‐stimulated glucose uptake may be linked to Na,K‐ATPase (NKA) transport, which is also dramatically stimulated during muscle contraction. We measured uptake of Rb (a congener for K + ), and 13 C‐[6C]‐D‐glucose or 13 C‐[6C]‐2‐deoxy glucose in isolated mouse EDL muscles stimulated to contract ex vivo. Muscles were obtained from WT mice. Glucose uptake was measured for 5 min at 32 C in a physiological solution containing 200 mM tracer RbCl and 11mM 13 C glucose. Following uptake, the Rb and 13 C content of the muscle were measured by ICP‐MS. We achieved a Limit of Detection of 54uM for 13 C glucose in mouse muscle. This detection power for 13 C by ICP‐MS is entirely new and was achieved by combining technical approaches: i) introduction of 13 C‐based hexose substrates; ii) modifications to sample preparation that reduce background carbon; iii) ratiometric monitoring of the 13 C/ 12 C signal; and iv) 13 C‐optimized instrument conditions. Contracting skeletal muscle took up 13 C‐[6C]‐labeled‐D‐glucose at a rate of 10.8 mM/g‐min, and Rb at a rate of 1.2 mM/g‐min. The ability to detect glucose and other carbon‐based organic compounds by ICP‐MS is expected to advance studies of coupled transport and other biological processes that require multi‐species detection.