Anti-IgM–mediated B lymphocyte activation is associated with an increase in GLUT4 expression and cell surface localization. (A) NBLs isolated from whole blood were incubated with or without anti–human IgM F(ab′)2 for the indicated durations, and viable cell quantities were determined by MTS assay (represented by absorbance at 490 nm) and normalized to control cells. Data are mean ± SEM (n ≥ 2). (B) NBLs incubated with or without anti-IgM for 15 hours and pretreated (or not) with 100μM phloretin before evaluation of glucose consumption rates via 2-NBDG uptake assay and flow cytometric quantification of fluorescence intensity. A representative histogram is shown. (C) Quantification of data represented in panel B is shown. Median fluorescence intensity values were derived from 3 independent experiments. Data are mean ± SEM (n = 3). (D) Expression of GLUTs 1 to 12 was determined in a time-course analysis of B lymphocytes incubated with anti-IgM for 0, 3, and 15 hours by quantitative real-time RT-PCR. Relative quantification (RQ) is displayed and normalized to the MM.1S cell line for reference to expression levels in myeloma. GLUTs 2, 7, 10, and 12 were undetected. Data are mean ± SEM (n ≥ 2). (E) B lymphocytes were incubated with or without anti-IgM for 15 hours before analysis of GLUT1 and GLUT4 intracellular distribution via confocal immunofluorescence microscopy. Arrows indicate regions of cell surface GLUT4 immunoreactivity. Representative images are shown (n = 4).
