Method for the Detection of Insulin‐independent Glucose Uptake in Skeletal Muscle
Authors: Norman Natalie J. , Landero-Figueroa Julio A. , Heiny Judith J. 
Published: April 2020

The quantification of glucose uptake into tissues, alongside co‐solutes flux, is essential to advance basic knowledge of glucose transport and metabolism. However, comprehensive understanding of glucose metabolism has been elusive. A major player in this crucial process is muscle tissue ‐ its large mass, high catabolic rate and storage capabilities make a major contribution to glucose homeostasis. The traditional insulin‐dependent model has proven insufficient to explain a well‐documented clinical observation: Type II diabetic, insulin resistant patients can lower their blood sugar levels with frequent exercise. One challenge in studying a complex mechanism like glucose uptake in muscles is the lack of experimental tools with multi‐channel capabilities, able to quantify most of, if not all the possible related solute fluxes within current models, in‐vivo or ex‐vivo. Our goal was to develop a method to measure glucose, K+, Na+, Ca2+, Fe, Cu, Zn2+ and Mn flux in biological samples using Inductively Coupled Plasma – Mass Spectroscopy (ICP‐MS). ICP‐MS is well established as a sensitive and versatile tool for direct detection and quantification of specific elements.In this study, we developed a new ratiometric method, based on stable‐isotope dilution, to quantify 13C‐based molecules using the molar ratio of 13C/12C. This, added to the multi‐metal detection capabilities of ICP‐MS was combined for a multi‐channel ion flux measurement in mouse EDL muscle during contraction‐stimulated glucose uptake. The preliminary results demonstrated a correlation of K+ uptake with glucose uptake. We showed involvement of the Na+K+ATPase (NKA) with this process using ouabain. An isolated EDL muscle was either stimulated at 90 Hz for 5 min or left unstimulated in 11mM 13C glucose uptake solution at 32°C. The muscle was then washed at 0°C for 20 minutes to wash away excess glucose on the surface of the muscle and to inhibit transporter activity. The muscle was then digested in 1:1 HCl and H2SO4 to break it down to inorganic forms. Finally, we used ICP‐MS to quantify glucose and co‐solute transport. The transport rate for glucose (nM/g min) for stimulated tissues was 601 as compared to 9 for unstimulated tissues. The stimulated muscles also took up K+, as measured through Rb+ tracer, indicating that NKA was active during contraction‐stimulated glucose uptake. To determine which isoform of Na,K,ATPase (NKA) the glucose was coupled to, we used 0.75uM ouabain. This concentration is enough to maintain muscle contractions as well as inhibit NKA α2 but not α1. Ouabain reduced the average glucose uptake rate by 81 %. These preliminary results show that NKA α2 may be coupled to a possible novel glucose transporter in skeletal muscle tissue or to GLUT‐4 in an undescribed way. The development of a method to measure glucose concentrations in biological samples together with coupled fluxes of other ions will advance knowledge of glucose handling in health and disease. In addition, our method to reproducibly quantify C‐based organic molecules by ICP‐MS will advance applications of ICP‐MS to many other fields of biomedical research.Support or Funding InformationNIH RO1 AR 063710