TRANSLATIONAL CONTROL OF GENE EXPRESSION IN RAT LIVER IN RELATION TO MAMMALIAN TARGET OF RAPAMYCIN SIGNALING

Open Access
- Author:
- Reiter, Ali Kathleen
- Graduate Program:
- Physiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- August 24, 2004
- Committee Members:
- Leonard Shelton Jefferson Jr., Committee Chair/Co-Chair
Scot R Kimball, Committee Member
Charles H Lang, Committee Member
Kathryn F Lanoue, Committee Member
Ian Alexander Simpson, Committee Member
Peter A Farrell, Committee Member - Keywords:
- eukaryotic translation initiation factor
translation
mTOR
gene expression
liver
AMPK
exercise - Abstract:
- Previous research demonstrated a correlation between changes in RNA content and protein synthesis in the liver following food intake or endurance exercise. A central protein implicated in the regulation of protein synthesis and ribosome biogenesis is referred to as the mammalian target of rapamycin (mTOR). However, while the protein kinase referred to as the mTOR has been implicated in the regulation of ribosome biogenesis in the liver, inhibition of mTOR does not alter the rate of protein synthesis. The overall objective of the research presented herein was to gain insight into the translational regulation of gene expression in rat liver in response to two physiological perturbations, food intake and endurance exercise. In response to food intake, phosphorylation of two substrates of mTOR, the eukaryotic initiation factor (eIF)4E binding protein-1 4E-BP1 and the ribosomal protein (rp) S6 kinase S6K1 and its substrate rpS6, was increased within 15 min and was sustained for at least 180 min. Activation of S6K1 has been linked to upregulated translation of a subset of mRNAs. To identify translationally regulated mRNAs, polysomal (i.e. actively translated) and nonpolysomal (non-translated) fractions were isolated and subjected to microarray analysis. The mRNAs encoding 78 proteins, including 42 proteins involved in protein synthesis, exhibited increased abundance in polysomes in response to feeding. In contrast, the mRNAs encoding 50 proteins displayed decreased abundance in polysomes in response to feeding. In another study, the effect of a single bout of treadmill running on signaling through mTOR in liver was examined in 12 h fasted rats. Phosphorylation of a downstream target of mTOR, 4E-BP1, was decreased after exercise to values even lower than the fasting-induced basal levels. Phosphorylation of another mTOR target, S6K1, was already maximally repressed by fasting and was not further reduced by exercise, and phosphorylation of rpS6 was unaltered by endurance exercise. Neither the global rate of protein synthesis nor the proportion of the mRNA encoding b-actin associated with polysomes was changed after exercise from the basal fasting state. However, the proportion of the mRNAs containing a 5’-TOP sequence associated with polysomes was significantly lower in liver after exercise suggesting that the translation of mRNAs encoding ribosomal proteins was specifically repressed. Activation of AMP-activated protein kinase (AMPK) occurs in response to extended food deprivation and endurance exercise and could act as a common mediator for the changes in mTOR signaling. For this reason, the role of AMPK activation in the regulation of mTOR was evaluated in the intact liver. Phosphorylation of AMPK either in vivo or in situ was associated with a repression of protein synthesis as well as decreased phosphorylation of a number of targets of mTOR signaling including S6K1, eIF4G, and 4E-BP1. Together, the results demonstrate a coordinated response in the liver to changes in nutrient and hormonal status to alter gene expression through translational control mechanisms in the liver.