DIRECT EVIDENCE FOR A ROLE OF THE SPOT-14 PROTEIN IN THE REGULATION OF LIPID-SYNTHESIS

''Spot 14'' is a nuclear protein that is rapidly induced by thyroid hormone (T-3) and dietary carbohydrate in liver, We used an antisense oligonucleotide to inhibit induction of spot 14 protein by T-3 and glucose in primary cultures of rat hepatocytes to test the hypothesis that the protein could function in the regulation of lipid synthesis. Spot 14 protein was undetectable in hepatocytes maintained in 5.5 mM glucose without T-3, and was induced within 4 h after addition of 27.5 mM glucose and 50 nM T-3 to the culture medium, reaching a maximal level within 24 h. Accumulation of spot 14 protein was markedly inhibited in hepatocytes transfected with a spot 14 antisense oligonucleotide, but not in those treated with a control oligonucleotide. Transfection of the antisense, but not control, oligonucleotide also abrogated the increase in lipogenesis induced by T-3 and glucose. Reduced triglyceride formation accounted for the diminished net lipid synthesis, In contrast to lipogenesis, glucose uptake was not significantly affected by the transfections. Antisense transfection inhibited the induction of both ATP citrate lyase and fatty acid synthase immunoreactivities, as well as malic enzyme activity, indicating that the observed reduction in lipogenesis could be explained by diminished cellular content of lipogenic enzymes. Reduced malic enzyme activity in antisense-transfected hepatocytes was accompanied by lowered relative abundance of malic enzyme mRNA, suggesting that the antisense effects on lipogenic enzymes were mediated at the pretranslational level. The oligonucleotides did not significantly affect lipogenesis in a rat hepatoma cell line that does not express detectable spot 14 mRNA or protein. These data directly implicate the spot 14 protein in the transduction of hormonal and dietary signals for increased lipid metabolism in hepatocytes.