Fiber-type Specific Differences in Glucose Uptake and Abundance of Key Metabolic Proteins in Single Fibers from Rat Skeletal Muscle.

Abstract

Skeletal muscle is the major tissue for insulin-mediated glucose disposal. Each skeletal muscle is composed of hundreds to thousands of individual fibers. Because of the marked cellular diversity among fibers, it is advantageous to understand muscle function at the single fiber level. This thesis aimed to provide insights on skeletal muscle fiber type heterogeneity, concentrating on single fibers from the rat epitrochlearis muscle. Research from this thesis developed and validated techniques for simultaneous assessment in a single fiber of: glucose uptake (GU), fiber type (by myosin heavy chain isoform expression), fiber volume, and abundance of multiple proteins (by immunoblotting). GU capacity differed by both fiber type and stimulus: 1) IIA fibers consistently had the greatest insulin-stimulated GU capacity (IIA>IIB, IIX, IIB/X) across a wide range of the lifespan (2-, 5-, 9-, and 25-months-old) and 2) IIB fibers had the greatest AICAR-stimulated GU capacity. The primary insulin-responsive glucose transporter (GLUT4) was most abundant in IIA fibers, supporting the idea that insulinstimulated GU capacity is, in part, a function of the number of GLUT4 transporters present. To better understand insulin resistance, GU was measured by single fibers from obese Zucker (OZ) versus lean Zucker (LZ) rats, and there was significant insulin resistance (44-58%) for each fiber type. Lower levels of IκB-β in OZ fibers supported the idea for greater inflammation and NFκB activation contributing to the insulin resistance. In vivo electroporation of an ectopic gene was successful, allowing xiii visualization of fluorescent protein (DsRed) expression in single fibers without altering GU. Strong evidence was provided to support the combination of single fiber analysis with genetic manipulation to study the role that a gene-of-interest has on fiber typespecific GU capacity. Overall, results from this dissertation provided the first data showing conclusive evidence for fiber type-specific GU differences within a skeletal muscle. The extensive characterization of single fibers in this thesis provides unique insights for understanding muscle biology. The novel methods and knowledge from this work, combined with continued investigation of single fibers, offer the opportunity to advance our understanding of pathophysiological conditions, providing options for improved therapeutic targets to treat metabolic disorders.