Quantification of glucose uptake in the mammalian skeletal muscle

Submission note: A thesis submitted in total fulfillment of the requirements for the Degree of Doctor of Philosophy to the School of Life Sciences, College of Science, Health and Engineering, La Trobe University, Bundoora.

Until now, the ability to measure accurately the kinetic properties of glucose uptake in muscle had meant that the limitations to glucose transport and thus, dysregulation of glucose transport, could only be speculated upon. This arose in part from the inability to discriminate between barriers (such as the surface membrane and hexokinase). It also arose because of the inability to directly measure the intracellular glucose concentration. The first aim of this thesis was to describe a new and novel approach to measure the transport kinetics of glucose in single muscle fibres of known type. In Chapters 2 and 3 I describe a novel method for measuring the kinetics of glucose transport specifically via the GLUTs. I consequently identify for the first time the kinetic properties of GLUTs in situ by fibre type and determine the contribution of GLUT-1 and GLUT-4 to basal glucose transport and that the total glucose pool is not fully utilized even at high extracellular glucose concentration. Consequently, I also show that the basal rate of transport of glucose across the surface membrane is not the rate limiting step. I also measure for the first time the intracellular glucose concentration and describe a possible role of elevated intracellular glucose in the development of insulin resistance. The second major aim of this thesis was to then examine the properties of Insulin mediated glucose transport using this newly developed technique. In Chapter 4, I describe the kinetic properties of insulin mediated glucose transport. I show that kinetic properties of insulin mediated glucose transport via GLUTs are identical for both FT and ST fibres. Importantly, I was able to measure the rate of hexokinase activity and show that insulin has no effect on hexokinase. Consequently, I show that the kinetics of insulin xxviii mediated glucose uptake is predominately mediated by GLUT-4 translocation to the surface membrane. The third major aim of this thesis was to examine the effects of specific reactive oxygen and nitrosylating species (ROS/NO) on the kinetics of glucose transport. I describe for the first time an effect of ROS/NO specifically on GLUT-1 and hexokinase and show that both molecular species contribute to increased glucose uptake via an AMPK independent pathway. These results have implications in understanding the role of ROS/NO in the development of insulin resistance.