Articles by Chun Austin Changou in JoVE
Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy Chun Austin Changou1,2,3, Reni Ajoy4,5, Szu-Yi Chou4,5,6 1The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, 2Integrated Laboratory, Center of Translational Medicine, Taipei Medical University, 3Core Facility, Taipei Medical University, 4The Ph.D. Program for Neural Regenerative Medicine, College of Medical Science and Technology, Taipei Medical University, 5Graduate Institute of Neural Regenerative Medicine, College of Medical Science and Technology, Taipei Medical University, 6TMU research center for Neurotrauma and Neuroregeneration, College of Medical Science and Technology, Taipei Medical University This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin–GLUT4 signaling pathway with Deconvolution Microscopy.
Other articles by Chun Austin Changou on PubMed
CCL5/RANTES Contributes to Hypothalamic Insulin Signaling for Systemic Insulin Responsiveness Through CCR5 Scientific Reports. | Pubmed ID: 27898058 Many neurodegenerative diseases are accompanied by metabolic disorders. CCL5/RANTES, and its receptor CCR5 are known to contribute to neuronal function as well as to metabolic disorders such as type 2 diabetes mellitus, obesity, atherosclerosis and metabolic changes after HIV infection. Herein, we found that the lack of CCR5 or CCL5 in mice impaired regulation of energy metabolism in hypothalamus. Immunostaining and co-immunoprecipitation revealed the specific expression of CCR5, associated with insulin receptors, in the hypothalamic arcuate nucleus (ARC). Both ex vivo stimulation and in vitro tissue culture studies demonstrated that the activation of insulin, and PI3K-Akt pathways were impaired in CCR5 and CCL5 deficient hypothalamus. The inhibitory phosphorylation of insulin response substrate-1 at Ser302 (IRS-1(S302)) but not IRS-2, by insulin was markedly increased in CCR5 and CCL5 deficient animals. Elevating CCR5/CCL5 activity induced GLUT4 membrane translocation and reduced phospho-IRS-1(S302) through AMPKα-S6 Kinase. Blocking CCR5 using the antagonist, (Met)CCL5, abolished the de-phosphorylation of IRS-1(S302) and insulin signal activation. In addition, intracerebroventricular delivery of (Met)CCL5 interrupted hypothalamic insulin signaling and elicited peripheral insulin responsiveness and glucose intolerance. Taken together, our data suggest that CCR5 regulates insulin signaling in hypothalamus which contributes to systemic insulin sensitivity and glucose metabolism.
The CD9, CD81, and CD151 EC2 Domains Bind to the Classical RGD-binding Site of Integrin αvβ3 The Biochemical Journal. | Pubmed ID: 27993971 Tetraspanins play important roles in normal (e.g. cell adhesion, motility, activation, and proliferation) and pathological conditions (e.g. metastasis and viral infection). Tetraspanins interact with integrins and regulate integrin functions, but the specifics of tetraspanin-integrin interactions are unclear. Using co-immunoprecipitation with integrins as a sole method to detect interaction between integrins and full-length tetraspanins, it has been proposed that the variable region (helices D and E) of the extracellular-2 (EC2) domain of tetraspanins laterally associates with a non-ligand-binding site of integrins. We describe that, using adhesion assays, the EC2 domain of CD81, CD9, and CD151 bound to integrin αvβ3, and this binding was suppressed by cRGDfV, a specific inhibitor of αvβ3, and antibody 7E3, which is mapped to the ligand-binding site of β3. We also present evidence that the specificity loop of β3 directly bound to the EC2 domains. This suggests that the EC2 domains specifically bind to the classical ligand-binding site of αvβ3. αvβ3 was a more effective receptor for the EC2 domains than the previously known tetraspanin receptors α3β1, α4β1, and α6β1. Docking simulation predicted that the helices A and B of CD81 EC2 bind to the RGD-binding site of αvβ3. Substituting Lys residues at positions 116 and 144/148 of CD81 EC2 in the predicted integrin-binding interface reduced the binding of CD81 EC2 to αvβ3, consistent with the docking model. These findings suggest that, in contrast with previous models, the ligand-binding site of integrin αvβ3, a new tetraspanin receptor, binds to the constant region (helices A and B) of the EC2 domain.