Kinetics of GLUT4 Trafficking in Rat and Human Skeletal Muscle

Hkan K.R. Karlsson Alexander V. Chibalin Heikki A. Koistinen Jing Yang Francoise Koumanov Harriet Wallberg-Henriksson Juleen R. Zierath Geof frey D. Holman OBJECTIVE-In skeletal muscle, insulin stimulates glucose transport activity three- to fourfold, and a large part of this stimulation is associated with a net translocation of GLUT4 from an intracellular compartment to the cell surface. We examined the extent to which insulin or the AMP-activated protein kinase activator AICAR can lead to a stimulation of the exocytosis limb of the GLUT4 translocation pathway and thereby account for the net increase in glucose transport activity. RESEARCH DESIGN AND METHODS-Using a biotinylated photoaffinity label, we tagged endogenous GLUT4 and studied the kinetics of exocytosis of the tagged protein in rat and human skeletal muscle in response to insulin or AICAR. Isolated epitrochlearis muscles were obtained from male Wistar rats. Vastus lateralis skeletal muscle strips were prepared from open muscle biopsies obtained from six healthy men (age 39 11 years and BMI 25.8 0.8 kg/m2). RESULTS-In rat epitrochlearis muscle, insulin exposure leads to a sixfold stimulation of the GLUT4 exocytosis rate (with basal and insulin-stimulated rate constants of 0.010 and 0.067 min1 , respectively). In human vastus lateralis muscle, insulin stimulates GLUT4 translocation by a similar sixfold increase in the exocytosis rate constant (with basal and insulin-stimulated rate constants of 0.011 and 0.075 min1 , respectively). In contrast, AICAR treatment does not markedly increase exocytosis in either rat or human muscle. CONCLUSIONS-Insulin stimulation of the GLUT4 exocytosis rate constant is sufficient to account for most of the observed increase in glucose transport activity in rat and human muscle. Diabetes 58:847-854, 2009 - Iin type 2 diabetes (1 4) and are associated with mpairments in skeletal muscle glucose uptake occur defects in GLUT4 translocation rather than a change in the total amount of GLUT4 protein (2,5). These changes are also associated with defects in cell signaling (6,7). However, in healthy normal glucose-tolerant relatives of type 2 diabetic patients, impairments in skeletal muscle insulin-stimulated glucose transport activity occur without alterations in the phosphorylation of Akt or its downstream target protein AS160 (8). In rodents, marked impairments in insulin signaling to glycogen metabolism occur as a consequence of skeletal muscle GLUT4 knockout (9), suggesting that defects in GLUT4 traffic have the potential to feed back and inhibit early steps in insulin signaling. Changes in GLUT4 translocation may be one of the earliest molecular defects in type 2 diabetes (8). Thus, it is important to determine molecular mechanisms that link insulin signaling to GLUT4 translocation. Skeletal muscle represents the major site of postprandial glucose disposal (10); therefore, kinetic studies of GLUT4 trafficking in this tissue are important to determining the major sites of insulin action, particularly because GLUT4-trafficking defects have been observed in skeletal muscle from type 2 diabetic patients (11,12). In adipose tissue, GLUT4-trafficking kinetic studies have identified the exocytic limb of the translocation pathway as the major site of insulin action (1316). In contrast to adipocytes, skeletal muscle is a complex tissue, with diverse GLUT4 storage sites, including multiple perinuclear and satellite compartments that are close to the transverse tubules (1719). Moreover, skeletal muscle GLUT4 translocation and glucose transport are increased in response to a wide range of stimulatory factors including contraction, changes in calcium release, and changes in the cellular energy status and AMP levels (19). To determine the extent to which insulin action stimulates the exocytosis limb of the GLUT4 translocation pathway, and thereby account for the net increase in translocation, we have developed new methods for studying GLUT4 translocation in rat epitrochlearis and human vastus lateralis skeletal muscle. We have specifically examined whether the stimulation of exocytosis following exposure to insulin or the AMP precursor analogue AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) can fully account for associated increases in glucose transport activity. RESEARCH DESIGN AND METHODS Epitrochlearis muscle isolation. Male Wistar rats (150 200 g) were obtained from B&K Universal, Sollentuna, Sweden. Animals were housed under a 12-h light/12-h dark cycle and had free access to water and standard rodent chow and were fasted (5 h) before each experiment. The Stockholm North Animal Ethics Committee approved all studies. Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (5 mg/100 g body wt i.p.). Isolated epitrochlearis muscles were incubated at 30C. All incubation media were prepared from pregassed (95% O2/5% CO2) Krebs-Henseleit buffer (KHB) containing 5 mmol/l HEPES and 0.1% BSA (radioi (...truncated)