Poster Session 2 - E2
1,2Victoria Tokarz, 1Javier Jaldin-Fincati, 1Zhi Liu, 1,2Amira Klip
1 Cell Biology, Hospital for Sick Children; 2 Department of Physiology, University of Toronto, Toronto, ON, Canada
Introduction: Skeletal muscle insulin resistance is a consequence of obesity and a requisite precursor of type 2 diabetes (T2D). Glucose enters the muscle cell through the GLUT4 glucose transporter that undergoes redistribution to the cell surface in response to insulin (GLUT4 translocation). The activation of this transport machinery is the rate-limiting step in muscle glucose metabolism and its failure is implicated in the development of insulin resistance. GLUT4 delivery to the plasma membrane (PM) is coordinated by two parallel signalling pathways activated downstream of the insulin receptor (IR): one pathway involves Akt and insulin-responsive Rabs, and the other engages Rac1 to facilitate actin remodeling. Downstream of Akt, two insulin-activated Rabs, Rab8A and Rab13, have been shown to modulate GLUT4 traffic, together mobilizing GLUT4 containing vesicles to the PM. The most recent and major findings in the field implicate Rabs at the nexus of GLUT4 translocation and muscle glucose homeostasis. In parallel, IR signalling activates of Rac1, a Rho GTPase that facilitates actin remodeling. This dynamic cortical actin remodeling is required for GLUT4 translocation, and it has been shown that dysregulated skeletal muscle Rac1 activity is a consequence of high-fat diet-induced insulin resistance. Together, these coordinated signalling events regulate GLUT4 delivery to the plasma membrane in response to insulin.
Rationale: During insulin resistance, defects in GLUT4 translocation impair skeletal muscle glucose disposal and causes hyperglycemia. Reductions in insulin-stimulated Akt phosphorylation have been documented during insulin resistance, but defective Akt phosphorylation is insufficient to explain defective GLUT4 translocation, as near complete ablation of Akt activity is required to inhibit the process. Hence, it is unknown which specific steps in GLUT4 translocation are vulnerable during insulin resistance and are responsible for dysfunctional GLUT4 translocation.
Hypothesis: We hypothesize that specific downstream elements of the GLUT4 signal transduction cascade are uniquely vulnerable to dysfunction during insulin resistance with important consequences on whole-body glucose metabolism. We hypothesize that independent defects in both insulin-stimulated Rab activation and Rac1 activity jointly, but independently, impair GLUT4 translocation during insulin resistance.
Methods: Cultures of L6 skeletal muscle myoblasts overexpressing myc-tagged GLUT4 and the human insulin receptor were treated with palmitate (a saturated fatty acid known to cause insulin resistance) for 18 hours. Cultures were serum starved for 3 hours before stimulation with insulin and processed for the following experiments: quantitative detection of surface GLUT4, immunofluorescence or immunoblotting.
Results: Insulin (0.1nM-100nM) stimulated dose-dependent increases in surface GLUT4 in L6 skeletal muscle myoblasts. Palmitate treatment (18h) at doses between 0.250mM and 0.500mM significantly impaired insulin-stimulated GLUT4 translocation without any effects on Akt phosphorylation at Ser473 or Thr308. Additionally, immunofluorescence revealed that palmitate treatment abrogated the formation of insulin-stimulated actin ruffles (a consequence of Rac1 activation and required for GLUT4 translocation).
Conclusions/Significance: Together, these data suggest that the observed defects in insulin-stimulated GLUT4 translocation during palmitate-induced insulin resistance may be due to unique and independent defects in both arms of the GLUT4 translocation signalling pathway. Identification and characterization of these defects is imperative to our understanding of the pathogenesis of T2D. Currently, no diabetic therapy exists that targets skeletal muscle GLUT4 translocation, even though the skeletal muscle is responsible for up to 80% of whole body glucose disposal. The development of such a treatment would revolutionize diabetic care.