Flow cytometry protocol for GLUT4-myc detection on cell surfaces.

Insulin and muscle contraction trigger GLUT4 translocation to the plasma membrane, which increases glucose uptake by muscle cells. Insulin resistance and Type 2 diabetes are the result of impaired GLUT4 translocation. Quantifying GLUT4 translocation is essential for comprehending the intricacies of both physiological and pathophysiological processes involved in glucose metabolism. The most commonly used methods for measuring GLUT4 translocation are the ELISA-type assay and the immunofluorescence assay. While some reports suggest that flow cytometry could be useful in quantifying GLUT4 translocation, this technique is not frequently used. Much of our current understanding of the regulation of GLUT4 has been based on experiments using the rat myoblast cell line (L6 cell) which expresses GLUT4 with a myc epitope on the exofacial loop. In the present study, we use the L6-GLUT4myc cell line to develop a flow cytometry-based approach to detect GLUT4 translocation. Flow cytometry offers the advantages of both immunofluorescence and ELISA-based assays. It allows easy identification of separate cell populations in the sample, similar to immunofluorescence, while providing results based on a population-level analysis of multiple individual cells, like an ELISA-based assay. Our results demonstrate a 0.6-fold increase with insulin stimulation compared with basal conditions. Finally, flow cytometry consistently yielded results across different experiments and exhibited sensitivity under the tested conditions.

The light chain of tetanus toxin bound to arginine-rich cell-penetrating peptide inhibits cortical reaction in mouse oocytes.

Introduction: Cortical reaction is a secretory process that occurs after a spermatozoon fuses with the oocyte, avoiding the fusion of additional sperm. During this exocytic event, the cortical granule membrane fuses with the oocyte plasma membrane. We have identified several molecular components involved in this process and confirmed that SNARE proteins regulate membrane fusion during cortical reaction in mouse oocytes. In those studies, we microinjected different nonpermeable reagents to demonstrate the participation of a specific protein in the cortical reaction. However, the microinjection technique has several limitations. In this work, we aimed to assess the potential of cell-penetrating peptides (CPP) as biotechnological tools for delivering molecules into oocytes, and to evaluate the functionality of the permeable tetanus toxin (bound to CPP sequence) during cortical reaction. Methods: Arginine-rich cell-penetrating peptides have demonstrated the optimal internalization of small molecules in mammalian cells. Two arginine-rich CPP were used in the present study. One, labeled with 5-carboxyfluorescein, to characterize the factors that can modulate its internalization, and the other, the permeable light chain of tetanus toxin, that cleaves the SNAREs VAMP1 and VAMP3 expressed in mouse oocytes. Results: Results showed that fluorescent CPP was internalized into the oocyte cytoplasm and that internalization was dependent on the concentration, time, temperature, and maturation stage of the oocyte. Using our functional assay to study cortical reaction, the light chain of tetanus toxin bound to arginine-rich cell-penetrating peptide inhibited cortical granules exocytosis. Discussion: Results obtained from the use of permeable peptides demonstrate that this CPP is a promising biotechnological tool to study functional macromolecules in mouse oocytes.

Complexin-2 redistributes to the membrane of muscle cells in response to insulin and contributes to GLUT4 translocation.

Insulin stimulates glucose uptake in muscle cells by rapidly redistributing vesicles containing GLUT4 glucose transporters from intracellular compartments to the plasma membrane (PM). GLUT4 vesicle fusion requires the formation of SNARE complexes between vesicular VAMP and PM syntaxin4 and SNAP23. SNARE accessory proteins usually regulate vesicle fusion processes. Complexins aide in neuro-secretory vesicle-membrane fusion by stabilizing trans-SNARE complexes but their participation in GLUT4 vesicle fusion is unknown. We report that complexin-2 is expressed and homogeneously distributed in L6 rat skeletal muscle cells. Upon insulin stimulation, a cohort of complexin-2 redistributes to the PM. Complexin-2 knockdown markedly inhibited GLUT4 translocation without affecting proximal insulin signalling of Akt/PKB phosphorylation and actin fiber remodelling. Similarly, complexin-2 overexpression decreased maximal GLUT4 translocation suggesting that the concentration of complexin-2 is finely tuned to vesicle fusion. These findings reveal an insulin-dependent regulation of GLUT4 insertion into the PM involving complexin-2.