Abstract
Abstract Hepatocytes prepared from the livers of 18- or 19-day-old chick embryos and maintained in culture medium containing serum for 3 days exhibited marked induction of the lipogenic pathway. Incorporation of [U-14C]glucose into fatty acids and the total activities of malic enzyme, fatty acid synthetase, and ATP-citrate lyase increased by 1000-, 47-, 21-, and 4.5-fold, respectively. When l-triiodothyronine was added to the culture medium, the activities increased a total of 2500-, 140-, 40-, and 8-fold, respectively. The increases in the rate of fatty acid synthesis and total activity of malic enzyme were inhibited by bovine serum albumin, human thyroxin-binding prealbumin, and rabbit anti-thyroxin serum. Inhibition was reversed by adding a large amount of l-triiodothyronine. Thus, thyroid hormone was a major serum factor causing induction of the lipogenic pathway. Oxidation of [1-14C]acetate to CO2 and the total activity of NADP-isocitrate dehydrogenase were not affected by incubating the cells with or without l-triiodothyronine. The activity of lactate dehydrogenase decreased during the culture period. This induction of the lipogenesis pathway quantitatively mimics changes in the pathway which occur when newly hatched chicks are fed a high carbohydrate mash diet. Glucagon, cAMP, and free stearate also inhibited the induction of lipogenesis and its associated enzymes. These compounds also inhibit fatty acid synthesis and elevate fatty acyl-CoA levels in freshly prepared hepatocytes. Hence, glucagon and free fatty acids exert both long and short term regulation over fatty acid synthesis, possibly via changes in the intracellular concentration of fatty acyl-CoA.
Highlights
Morphology-Hepatocytes were completely dissociated into single cells before they were added to the culture medium (12)
Stimulation of Lipogenic Pathway in Hepatocytes in CultureWhen hepatocytes isolated from prenatal liver were incubated in Nutrient Mixture F-10 containing undialyzed sera, there was a rapid and very large increase in the capacity of the cells to synthesize fatty acids from glucose or acetate (Table I)
Addition of L-triiodothyronine to the culture medium caused a further increase in fatty acid synthesis from glucose but not from acetate (Table I)
Summary
Preparation and Maintenance of Isolated Cells-Unincubated embryonated eggs from white Leghorn chickens were incubated in an electric forced draft incubator at 37.5 + 0.5” and 6Oy, relative humidity. Neonatal chicks and prenatal embryos were killed by decapitation. Cells were isolated as previously described (12). The isolated cells were suspended in either Nutrient Mixture F-10 (13) or Waymouth Medium MD. 705/l (14) (1.5 to 2.5 mg of total cell protein per ml). One milliliter of cells (about 1 X 10’ cells) was incubated with 8 ml of F-10 or Waymouth medium plus 1 ml of the desired experimental addition. The F-10 and Waymouth media were supplemented with heat-inactivated horse and fetal calf sera (15 and 2.5yc, respectively), glutamine (1.0 mM for F-10; 2.4 mM for Waymouth), penicillin (60 mg per liter), and streptomycin (100 mg per liter)
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