Objectives: Skeletal muscle cells are responsible for 80-90% of the insulin-induced glucose uptake in the body. Insulin signaling in skeletal muscle results in the targeted trafficking of glucose transporter type 4 (GLUT4) onto the cell membrane, enabling glucose uptake. Insulin resistant cells show defects in insulin-induced GLUT4 exocytosis. The eight-protein exocyst complex has an essential role in the insulin-induced exocytosis of GLUT4 vesicles in cultured adipocytes but it is not known if the exocyst-mediated molecular mechanism is conserved in other, insulin-responsive tissues, such as the skeletal muscle. We hypothesized that the exocyst complex is essential for the insulin-induced exocytosis of GLUT4-containing vesicles in skeletal muscle as well and that the exocyst is a master regulator of glucose homeostasis in insulin-responsive tissues. Methods/Results: We have generated a tamoxifen-inducible skeletal muscle-specific knockout mouse strain of exocyst central subunit Exoc5 (Exoc5-SMKO) to assess the exocyst’s role in glucose homeostasis in vivo. Exoc5 knockout does not affect grip strength, motor coordination or locomotor activity levels in these animals. Both male and female Exoc5-SMKO mice present with elevated fasting glucose levels, as compared to control littermates. Glucose tolerance testing revealed an impaired glucose clearance in Exoc5-SMKO mice, while insulin tolerance, fasting insulin levels, and A1C levels were similar between knockouts and controls. Conclusion: Our findings suggest that Exoc5 and the exocyst are necessary for insulin-stimulated glucose uptake in skeletal muscle. Ongoing work will further investigate the molecular mechanism of exocyst-mediated GLUT4 trafficking in skeletal muscle. Disclosure B. Fujimoto: None. L.T. Carter: None. A.M. Wong: None. M.W. Pitts: None. R.K. Villiger: None. M. Young: None. B. Fogelgren: None. N. Polgar: None. Funding National Institute of General Medical Sciences (5P20GM113134)
Objectives: Skeletal muscle cells are responsible for 80-90% of the insulin-induced glucose uptake in the body. Insulin activation of muscle cells triggers a signaling cascade that results in the exocytosis of membrane-bound glucose transporter type 4 (GLUT4) to the plasma membrane. The eight-protein complex called the exocyst is recognized as having an essential role in the insulin-induced exocytosis of GLUT4 vesicles in cultured adipocytes. We hypothesize that Sec10, a central component of the exocyst complex, is essential for the insulin-induced exocytosis of GLUT4 vesicles in skeletal myoblasts and that the exocyst is a master regulator of glucose homeostasis in metabolic tissues. Methods/Results: To analyze exocyst-mediated intracellular trafficking in skeletal muscle in vitro, we used L6 GLUT4-myc rat skeletal myoblasts, and CRISPR/Cas9 to create Sec10 knockout (Sec10-KO) clones from these cells. Immunofluorescent staining shows co-localization of exocyst Sec10 and GLUT4 upon insulin signal in L6 myoblasts. Cellular fractionation reveals that GLUT4 delivery to the plasma membrane in response to insulin is impaired in Sec10-KO cells. Also, glucose uptake rates are significantly decreased in Sec-10-KO L6 myoblasts compared to wild type cells upon insulin stimulus. We have also generated a tamoxifen-activated skeletal muscle-specific Sec10-knockout mouse strain to assess the exocyst’s role in glucose homeostasis in vivo. Sec10 knockout mice demonstrate impaired glucose tolerance compared to littermate controls. Conclusion: Based on our findings, Sec10 and the exocyst are necessary for insulin stimulated glucose uptake in skeletal muscle. Ongoing work will further investigate the molecular mechanism of exocyst-mediated GLUT4 trafficking in skeletal muscle. Disclosure B. Fujimoto: None. A. Lee: None. A.M. Wong: None. L.T. Carter: None. B. Fogelgren: None. N. Polgar: None.
There has been increasing interest in methods to generate synthetic lipid membranes as key constituents of artificial cells or to develop new tools for remodeling membranes in living cells. However, the biosynthesis of phospholipids involves elaborate enzymatic pathways that are challenging to reconstitute in vitro. An alternative approach is to use chemical reactions to non-enzymatically generate natural or non-canonical phospholipids de novo. Previous reports have shown that synthetic lipid membranes can be formed in situ using various ligation chemistries, but these methods lack biocompatibility and/or suffer from slow kinetics at physiological pH. Thus, it would be valuable to develop chemoselective strategies for synthesizing phospholipids from water-soluble precursors that are compatible with synthetic or living cells Here, we demonstrate that amide-forming ligations between lipid precursors bearing hydroxylamines and α-ketoacids (KAs) or potassium acyltrifluoroborates (KATs) can be used to prepare non-canonical phospholipids in physiologically relevant conditions. The generated amide-linked phospholipids spontaneously self-assemble into cell-like micron-sized vesicles similar to natural phospholipid membranes. We show that lipid synthesis using KAT ligation proceeds extremely rapidly, and the high selectivity and biocompatibility of the approach facilitates the in situ synthesis of phospholipids and associated membranes in living cells.
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