"Spot 14" is a nuclear protein that is rapidly induced by thyroid hormone (T3) and dietary carbohydrate in liver. We used an antisense oligonucleotide to inhibit induction of spot 14 protein by T3 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 T3, and was induced within 4 h after addition of 27.5 mM glucose and 50 nM T3 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 T3 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.
We generated a Spot 14 null mouse to assess the role of Spot 14 in de novo lipid synthesis and report the Spot 14 null mouse exhibits a phenotype in the lactating mammary gland. Spot 14 null pups nursed by Spot 14 null dams gain significantly less weight than wild-type pups nursed by wild-type dams. In contrast, Spot 14 null pups nursed by heterozygous dams show similar weight gain to wild-type littermates. We found the triglyceride content in Spot 14 null milk is significantly reduced. We demonstrate this reduction is the direct result of decreased de novo lipid synthesis in lactating mammary glands, corroborated by a marked reduction of medium-chain fatty acids in the triglyceride pool. Importantly, the reduced lipogenic rate is not associated with significant changes in the activities or mRNA of key lipogenic enzymes. Finally, we report the expression of a Spot 14-related gene in liver and adipose tissue, which is absent in the lactating mammary gland. We suggest that expression of both the Spot 14 and Spot 14-related proteins is required for maximum efficiency of de novo lipid synthesis in vivo and that these proteins impart a novel mechanism regulating de novo lipogenesis.
Thyroid hormone (TH) is necessary for normal axonal myelination. Myelin basic protein (MBP) is a structural protein essential for myelin function. In this study, we demonstrate that perinatal hypothyroidism regulates MBP mRNA levels via indirect mechanisms. We observed decreased MBP mRNA accumulation in the hypothyroid rat brain at postnatal (PN) d 10 and 50. Acute TH replacement did not rescue hypothyroid MBP mRNA levels at PN5, 10, or 50. TH is necessary for normal intrahemispheric commissure development including the anterior commissure (AC) and the corpus callosum (CC). We determined that perinatal hypothyroidism decreases AC area and cellularity in the developing rat brain by PN10 and 50. In the developing CC, hypothyroidism initially increases area and cellularity by PN5, but then ultimately decreases area and cellularity by PN50. MBP-expressing oligodendrocytes are a recognized target of TH and are responsible for myelination within intrahemispheric commissures. We found that hypothyroidism reduces the number of mature oligodendrocytes within both the AC and CC. This reduction is noted at PN5, 10, and 50 in the AC and by PN10 and 50 in the CC. Together, these data suggest that TH regulates MBP mRNA levels through indirect mechanisms. These data demonstrate the complex mechanisms whereby TH regulates myelination in the developing brain.
Spot 14 (S14) is a protein whose mRNA is rapidly up-regulated by lipogenic stimuli including thyroid hormone and a high-carbohydrate diet. Previous investigation into the role of S14 suggested that it is involved in de novo lipogenesis. Knockout of the gene in mice has given further support to this hypothesis. The lack of S14 in different tissues resulted in varying phenotypic effects. In the lactating mammary gland, levels of lipogenesis, specifically the production of medium chain fatty acids, were decreased, whereas hepatic lipogenesis was not decreased. In fact, hepatic lipogenesis was increased, and the increase may be due to compensation by a paralog of S14 called S14-R. S14-R is expressed in the liver but not the mammary gland. Importantly, S14 knockout mice did not have reduced levels of lipogenic enzymes, implying that it does not affect the transcriptional rate of those enzymes. Instead, S14 may act in the cytoplasm to affect lipogenesis.
We have compared the affinities for T3 and the T3 analog binding characteristics of the in vitro translational products of seven c-erbA cDNAs (chicken c-erbA alpha; human placental c-erbA beta; rat c-erbA beta-1; rat c-erbA alpha-1; rat c-erbA alpha-2; human testis c-erbA alpha-2; and human kidney c-erbA alpha-2). Four of these (chicken c-erbA alpha, human placental c-erbA beta, rat c-erbA beta-1, rat c-erbA alpha-1) bound T3 with high affinity as previously described. When compared under identical conditions of synthesis and [125I]T3 binding, there was no significant difference between the affinity of the chicken c-erb A alpha-1 and the human c-erbA beta but in a more limited series the affinity of rat c-erbA beta-1 for T3 was 4.6-fold higher than that of the rat c-erbA alpha-1. In vitro translational products of the beta-probes showed a characteristic 2.2-fold higher triiodothyroacetic acid/T3 ratio than did the products of the alpha-probes, regardless of the species of origin of the probe. As previously established, the rat c-erbA alpha-2 product did not bind T3. However, in contrast to two published reports, the human testis and kidney alpha-2 probe products also failed to bind T3. These findings indicate that highly conserved C-terminal 37-40 residues are important for high affinity T3 binding by proteins encoded by the c-erb A family of genes.
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