Confocal analysis of hepatocellular long chain fatty acid
uptake.
Elsing, Christoph, Ulrike Winn-B[diaeresis]orner, and Wolfgang
Stremmel.
Department of Medicine, University of Heidelberg
APStracts 2:0110G, 1995.
Transmembrane transport and cytosolic accumulation of fatty acids were
investigated using confocal laser scanning microscopy (cLSM). A Zeiss
LSM 310 system was used to determine the uptake of the fluorescent
fatty acid derivative 12-N-methyl-7-nitrobenzo-2-oxa-1,3-diazol-amino
stearate (12-NBD-stearate) (C18) in single rat hepatocytes. Uptake
was a saturable process with a Km of 68 nM. Initial uptake velocity
was dependent on extracellular presence of albumin and [beta]
-lactoglobulin. Absence of albumin reduced uptake to 32+/- 16 %
(p<0.01) of control values. In presence of unlabeled stearate
uptake of 12-NBD-stearate was lowered to 49+/-12% (p<0.01). Ion
substitution experiments showed no sodium dependency of uptake.
Increase in membrane potential led to an pronounced accumulation of
the fatty acid derivative within the plasma membrane and in the
adjacent cytoplasmic compartment, whereas membrane depolarization had
no effect on uptake rates. In separate experiments line scans through
representative hepatocytes were analysed to generate "X-T"
plots. 12-NBD-stearate showed a fluorescence pattern with prominent
staining of the area of the plasma membrane and the adjacent
cytoplasm, dependent on the presence of extracellular albumin. For
the hepatocellular cytosolic accumulation process of 12-NBD-stearate
a diffusion constant of 22.2 +/- 6.2 x 10-9 cm2/s was calculated. In
contrast to the long chain fatty acid derivative 12-NBD-stearate,
short (C5) and medium (C11) chain fatty acids revealed no membrane
interaction with hepatocytes. Erythrocytes also lacked a membrane
interaction process for 12-NBD-stearate. In conclusion, it was
demonstrated that cLSM is capable to evaluate directly the cellular
fatty acid uptake process at a subcellular level.
Received 13 June 1994; accepted in final form 27 May 1995.
APS Manuscript Number G225-4.
Article publication pending Am. J. Physiol. (Gastrointest. Liver
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 8 June 1995.