Evidence that downregulation of hexose transport limits intracellular glucose in 3T3-L1 fibroblasts

Journal Article · Mon Oct 01 00:00:00 EDT 1990 · Diabetes; (USA)

DOI:https://doi.org/10.2337/diabetes.39.10.1228· OSTI ID:6283635

Whitesell, R R; Regen, D M; Pelletier, D; Abumrad, N A ^[1]

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1. Vanderbilt Univ. School of Medicine, Nashville, TN (USA)

Measurements of initial glucose entry rate and intracellular glucose concentration in cultured cells are difficult because of rapid transport relative to intracellular volume and a substantial extracellular space from which glucose cannot be completely removed by quick exchanges of medium. In 3T3-L1 cells, we obtained good estimates of initial entry of ({sup 14}C)methylglucose and D-({sup 14}C)glucose with (1) L-({sup 3}H)glucose as an extracellular marker together with the ({sup 14}C)glucose or ({sup 14}C)methylglucose in the substrate mixture, (2) sampling times as short as 2 s, (3) ice-cold phloretin-containing medium to stop uptake and rinse away the extracellular label, and (4) nonlinear regression of time courses. Methylglucose equilibrated in two phases--the first with a half-time of 1.7 s and the second with a half-time of 23 s; it eventually equilibrated in an intracellular space of 8 microliters/mg protein. Entry of glucose remained almost linear for 10 s, making its transport kinetics easier to study (Km = 5.7 mM, Vmax = 590 nmol.s-1.ml-1 cell water). Steady-state intracellular glucose concentration was 75-90% of extracellular glucose concentration. Cells grown in a high-glucose medium (24 mM) exhibited a 67% reduction of glucose-transport activity and a 50% reduction of steady-state ratio of intracellular glucose to extracellular glucose.

• OSTI ID: 6283635
• Journal Information: Diabetes; (USA), Journal Name: Diabetes; (USA) Vol. 39:10; ISSN 0012-1797; ISSN DIAEA
• Country of Publication: United States
• Language: English

Related Subjects

550501* -- Metabolism-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALDEHYDES
ANIMAL CELLS
ANIMALS
BIOLOGICAL MARKERS
CARBOHYDRATES
CARBON 14 COMPOUNDS
CELL CULTURES
CONNECTIVE TISSUE CELLS
FIBROBLASTS
GLUCOSE
HEXOSES
HYDROGEN COMPOUNDS
ISOTOPE APPLICATIONS
ISOTOPE DILUTION
LABELLED COMPOUNDS
MAMMALS
MEMBRANE TRANSPORT
MICE
MONOSACCHARIDES
ORGANIC COMPOUNDS
RODENTS
SACCHARIDES
SOMATIC CELLS
STEADY-STATE CONDITIONS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES