Insulin-Responsive Compartments Containing GLUT4 in 3T3-L1 and CHO Cells: Regulation by Amino Acid Concentrations

Abstract

In fat and muscle, insulin stimulates glucose uptake by rapidly mobilizing the GLUT4 glucose transporter from a specialized intracellular compartment to the plasma membrane. We describe a method to quantify the relative proportion of GLUT4 at the plasma membrane, using flow cytometry to measure a ratio of fluorescence intensities corresponding to the cell surface and total amounts of a tagged GLUT4 reporter in individual living cells. Using this assay, we demonstrate that both 3T3-L1 and CHO cells contain intracellular compartments from which GLUT4 is rapidly mobilized by insulin and that the initial magnitude and kinetics of redistribution to the plasma membrane are similar in these two cell types when they are cultured identically. Targeting of GLUT4 to a highly insulin-responsive compartment in CHO cells is modulated by culture conditions. In particular, we find that amino acids regulate distribution of GLUT4 to this kinetically defined compartment through a rapamycin-sensitive pathway. Amino acids also modulate the magnitude of insulin-stimulated translocation in 3T3-L1 adipocytes. Our results indicate a novel link between glucose and amino acid metabolism.The GLUT4 glucose transporter is expressed predominantly in adipose and muscle tissues, where it accounts for the bulk of insulin-stimulated glucose uptake (12,84,95). In the presence of insulin, GLUT4 is redistributed from an intracellular compartment to the plasma membrane, where it facilitates the diffusion of glucose into the cell (15,35,73,77,102). Another glucose transporter isoform, GLUT1, is also expressed in fat and muscle tissues and is present at high levels in many other cell types and in cultured cell lines. A large proportion of GLUT1 is present on the plasma membrane even in the absence of insulin. Thus, while both GLUT1 and GLUT4 recycle at the plasma membrane, only GLUT4 recycling is characterized by significant intracellular sequestration, resulting from a slow rate of exocytosis, in the absence of insulin. Insulin increases the rate of GLUT4 exocytosis, with little or no decrease in its rate of endocytosis, so that in adipocytes the proportion of GLUT4 at the cell surface increases from Ͻ10% in the absence of insulin to 35 to 50% in its presence (41,55,85,113,114).Characterization of the intracellular insulin-responsive GLUT4-containing compartment is complicated by the fact that GLUT4 resides in several morphologically distinct locations within the cell. Ultrastructural studies have shown that GLUT4 is present in tubulovesicular structures distinct from lysosomes, as well as in a perinuclear compartment that is in close vicinity to the trans-Golgi network (39,(96)(97)(98). Recent work demonstrates that approximately 40 to 45% of intracellular GLUT4 localizes in a transferrin receptor (TfnR)-positive endosomal compartment, while 50 to 60% is in a second, TfnR-negative compartment; it is GLUT4 in this TfnR-negative compartment that is rapidly mobilized upon insulin addition (1,32,46,55,57,65,76). In primary adipocytes, this TfnR-negative ...

Protein kinase-ζ interacts with munc18c: role in GLUT4 trafficking

“…The level of insulin stimulation of glucose uptake in the CHOmycGLUT4 cells was approximately 2.5-fold (Fig. 4a), which accorded well with the published literature on insulin-stimulated trafficking of GLUT4 to the plasma membrane of CHO cells [27]. Transient transfection of Xpress-tagged munc18c constructs was then carried out to test if they modulated glucose uptake in response to insulin.…”

“…Such cells have been used in various studies to investigate the mechanism whereby insulin stimulation promotes GLUT4 translocation to the plasma membrane [23,26,27,30,31]. The CHO cells, like 3T3-L1 adipocytes, possess GLUT4 storage vesicles which translocate to the plasma membrane in response to insulin stimulation.…”

“…Such cells are known to show insulin-stimulated glucose transport and to share many similarities with 3T3-L1 adipocytes, a canonical cell line used to investigate insulin-stimulated glucose transport [26,27]. The cells were cultured in DMEM as this has been shown to be required for insulin-responsive trafficking [27].…”

Quantitative proteomic analysis of S-nitrosated proteins in diabetic mouse liver with ICAT switch method

“…Moreover, abnormal amino acid metabolism in the liver may change levels of amino acids throughout the whole body. Some reports suggest that high levels of amino acids can have both positive (Tremblay and Marette, 2001;Nobukuni et al, 2005) and negative (Armstrong et al, 2001;Bogan et al, 2001) effects on glucose metabolism and insulin action in peripheral tissues. Amino acid levels can also affect insulin secretion in pancreatic islet β-cells (Newsholme et al, 2007).…”

A sesquiterpene quinone, dysidine, from the sponge Dysidea villosa, activates the insulin pathway through inhibition of PTPases

“…After the cell density reached 100%, F12 medium was changed to 3T3-L1 medium for 2 d [29] . Cells were stimulated with insulin for 5 min after an 8-h serum starvation, fixed with 3.7% formaldehyde for 15 min, and labeled with anti-myc monoclonal antibody and secondary antibody (Alexa Fluor 647 conjuncted anti-mouse antibody).…”