GLUT1 is associated with sphingolipid-organized, cholesterol-independent domains in L929 mouse fibroblast cells

• Published in: Biochimie Vol. 162; pp. 88 - 96
• Main Authors: Rylaarsdam, Lauren E., Johnecheck, Grace N., Looyenga, Brendan D., Louters, Larry L.
• Format: Journal Article
• Language: English
• Published: France Elsevier B.V 01-07-2019
• Subjects: Animals; beta-Cyclodextrins - pharmacology; Biological Transport; Cell Line; Detergent resistant microdomains; Fatty Acids, Monounsaturated - pharmacology; Fibroblasts - metabolism; Glucose - metabolism; Glucose Transporter Type 1 - antagonists & inhibitors; Glucose Transporter Type 1 - metabolism; Glucose uptake; GLUT1; Lipid rafts; Membrane Lipids - analysis; Membrane Microdomains - metabolism; Mice; Octoxynol - pharmacology; Sphingolipid-rich microdomains; Sphingomyelin Phosphodiesterase - pharmacology; GLUT1; Glucose uptake; Detergent resistant microdomains; Lipid rafts; Sphingolipid-rich microdomains
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2019.04.010

Glucose is a preferred metabolite in most mammalian cells, and proper regulation of uptake is critical for organism homeostasis. The glucose transporter 1 (GLUT1) is responsible for glucose uptake in a wide variety of cells and appears to be regulated in a tissue specific manner. Therefore, a better understanding of GLUT1 regulation within its various cellular environments is essential for developing therapeutic strategies to treat disorders associated with glucose homeostasis. Previous findings suggest that plasma membrane subdomains called lipid rafts may play a role in regulation of GLUT1 uptake activity. While studying this phenomenon in L929 mouse fibroblast cells, we observed that GLUT1 associates with a low density lipid microdomain distinct from traditionally-defined lipid rafts. These structures are not altered by cholesterol removal with methyl-β-cyclodextrin and lack resistance to cold Triton X-100 extraction. Our data indicate that the GLUT1-containing membrane microdomains in L929 cells, as well as GLUT1's basal activity, are instead sphingolipid-dependent, being sensitive to both myriocin and sphingomyelinase treatment. These microdomains appear to be organized primarily by their lipid composition, as disruption of the actin cytoskeleton or microtubules does not alter the association of GLUT1 with them. Furthermore, the association of GLUT1 with these microdomains appears not to require palmitoylation or glycosylation, as pharmacologic inhibition of these processes had no impact on GLUT1 density in membrane fractions. Importantly, we find no evidence that GLUT1 is actively translocated into or out of low density membrane fractions in response to acute activation in L929 cell.
•GLUT1 is located in cholesterol-independent low density microdomains in L929 cells.•Activation of GLUT1 is independent of translocation into or out of low-density domains.•Myriocin and sphingomyelinase treatment reduces both density and activity of GLUT1.•GLUT1 is found in sphingolipid rich microdomains in L929 fibroblast cells.•GLUT1 in low-density domains is independent of glycosylation, palmitoylation, actin cytoskeleton and microtubules.