Profiling of the fetal and adult rat liver transcriptome and translatome reveals discordant regulation by the mechanistic target of rapamycin (mTOR)

Abstract

of the fetal and adult rat liver transcriptome and translatome reveals discordant regulation by the mechanistic target of rapamycin (mTOR). Am J Physiol Regul Integr Comp Physiol 309: R22-R35, 2015. First published April 29, 2015 doi:10.1152/ajpregu.00114.2015.-The mechanistic target of rapamycin (mTOR) integrates growth factor signaling, nutrient abundance, cell growth, and proliferation. On the basis of our interest in somatic growth in the late gestation fetus, we characterized the role of mTOR in the regulation of hepatic gene expression and translation initiation in fetal and adult rats. Our strategy was to manipulate mTOR signaling in vivo and then characterize the transcriptome and translating mRNA in liver tissue. In adult rats, we used the nonproliferative growth model of refeeding after a period of fasting and the proliferative model of liver regeneration following partial hepatectomy. We also studied livers from preterm fetal rats (embryonic day 19) in which fetal hepatocytes are asynchronously proliferating. All three models employed rapamycin to inhibit mTOR signaling. Analysis of the transcriptome in fasted-refed animals showed rapamycin-mediated induction of genes associated with oxidative phosphorylation. Genes associated with RNA processing were downregulated. In liver regeneration, rapamycin induced genes associated with lysosomal metabolism, steroid metabolism, and the acute phase response. In fetal animals, rapamycin inhibited expression of genes in several functional categories that were unrelated to effects in the adult animals. Translation control showed marked fetal-adult differences. In both adult models, rapamycin inhibited the translation of genes with complex 5= untranslated regions, including those encoding ribosomal proteins. Fetal translation was resistant to the effects of rapamycin. We conclude that the mTOR pathway in liver serves distinct physiological roles in the adult and fetus, with the latter representing a condition of rapamycin resistance.

Regulation of fetal liver growth in a model of diet restriction in the pregnant rat

“…The nutrient-sensing system involving the mechanistic mTOR, mTOR complex 1 (mTORC1), in particular, was an obvious candidate (46). We compared the present results to those from our recently published study (10), in which we investigated the effects of rapamycin-induced mTORC1 inhibition on liver gene expression in three models of liver growth, late fetal development, fasting followed by refeeding, and partial hepatectomy. This comparison revealed no overlap between gene sets enriched in IUGR fetuses and those from the prior study that were administered rapamycin in situ.…”

“…Western immunoblotting was carried out using methods standard for our laboratory (10). Primary antibodies for immunoblotting were as follows: Antibodies directed toward phospho-S6 (235/236), total S6, phospho-eIF2␣ (Ser 51) were obtained from Cell Signaling Technology (Danvers, MA); antibodies directed toward 4E-BP1 and total eIF2␣ were from Santa Cruz Biotechnology (Dallas, TX).…”

“…Total RNA and polysomal RNA were prepared in triplicate using the same methods employed by Boylan et al (10). Total RNA and RNA derived from translating polysomes were analyzed in parallel using Affymetrix Genechip Rat Gene ST 1.0 Arrays (Affymetrix, Santa Clara, CA).…”

“…Significance was only assigned to specific IPA categories when the P value for an individual category was lower than the P values for all categories obtained using five control data sets. As previously described (34), these control data sets, each of which was the same size as the corresponding experimental data set, were randomly selected from the list of genes that showed a fold change Ͻ1.1.Gene Set Enrichment Analysis (GSEA) was performed as previously described (10). For GSEA (52), we focused on three of the available collections (abbreviated C1, C2, C3, etc.)…”

“…However, our prior observations had indicated that both of these pathways, along with mitogenic signaling through the Ras-Raf-MEKErk pathway, are uncoupled in late-gestation fetal liver in the rat (2,9). Most recently, we studied the effects of the mTOR inhibitor, rapamycin, on three models of liver growth in the rat, partial hepatectomy in the adult, fasting followed by refeeding in the adult, and normal fetal growth late in gestation (10). We confirmed that signaling through mTOR complex 1 (mTORC1) in the adult regulated a spectrum of genes at the expression level and the translation of a defined subset of mRNAs that have complex 5= untranslated structures.…”