Heilbrigðisskýrslur - 01.12.1990, Side 142

By blocking cAMP hydrolysis with the phosphodiesterase inhibitor isobutyl
methyl xanthine (IBMX) (ÍO'3 M), the cellular cAMP response to glucagon was
greatly enhanced (Fig. 2), and suppression of VLDL secretion was increased
(triacvlglvcerol: glucagon + IBMX. 101 ± 11 ug mg-1 24 h'1: cholesterohglucagQIl
+ IBMX. 2.3 ± 0.7 pg mg1 24 h'1; P <0.05 compared with glucagon alone),
emphasizing the role of cAMP as an intracellular messenger involved in
regulating VLDL secretion. Despite suppressed VLDL secretion, little or no net
cellular accumulation of lipids was observed in the present studies. Presumably,
this is due to suppressed fatty acid synthesis and enhanced B-oxidation of fatty
acids caused by cAMP [15].
There was little or no change in the rate of ketogenesis over the 3-day culture
period when glucagon was absent (Fig. 3). However, a rather steep fall in
ketogenesis was observed in the presence of glucagon (Fig. 3). Addition of
carnitine (final conc. 0.5 mM) [17] on the third day of culture enhanced
ketogenesis and suppressed both cellular triacylglycerol concentration and
VLDL output. This suggests that carnitine may become a limiting factor for
hepatocellular lipid metabolism during longer periods of culture.
It should be noted, however, that on the first day of culture, addition of carnitine
had no effect on the secretion of VLDL triacylglycerol (data not shown). Although
on day 3 the cells retained their ability to increase cAMP conc. in response to
glucagon (Fig. 2), their ketogenic response to this hormone was reduced
compared to that on day 1 of culture (Fig. 3). This decreased response was also
observed in the presence of carnitine (Fig. 4), and suggests that cAMP was less
able to stimulate fatty acid oxidation after 3 days in culture. Nevertheless, VLDL
secretion remained responsive to the inhibitory effect of forskolin [18] under these
conditions (Fig. 4), which suggests an uncoupling of the cAMP mediated
regulation of hepatic lipid oxidation and lipid secretion [19].
Primary culture of rat hepatocytes is an experimental system or tool, which
offers unique opportunities for studying long-term effects of hormones and other
extracellular mediators on hepatocytes' metabolism in a chemically defined
medium without interference of other humoral or neural factors or the influence
of other cell types from the liver. This advantage may, however, be gained at the
cost of some of the physiological characteristics of the cells, one of which may be
the loss of carnitine from the cells during long-term culture. Co-culture (culture
of hepatocytes together with other cell types from the liver, such as hepatic
epithelial or endothelial cells) may prolong cell life and help to maintain the
physiological characteristics of the cells [7]. However, since the cell population is
not homogeneous, results from such co-culture experiments may be difficult to
interpret in metabolic terms. During the past several years secondary cultures of
hepatocytes, such as human hepatoma cells (HepG2 cells), have been extensively
studied. HepG2 cells can be maintained in culture for years, but they secrete little
VLDL and triacylglycerol, and they are reported to respond poorly to glucagon
[20].
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