A Comprehensive Overview of the Effects of Diet on the Hepatic Transcriptome and Translatome in Rat Liver

Restricted (Penn State Only)
Author:
Welles, Jaclyn
Graduate Program:
Biomedical Sciences
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
January 26, 2021
Committee Members:
  • Scot R Kimball, Dissertation Advisor/Co-Advisor
  • Scot R Kimball, Committee Chair/Co-Chair
  • Leonard Shelton Jefferson, Jr., Committee Member
  • Lisa M Shantz, Committee Member
  • David James Degraff, Outside Member
  • David L Waning, Committee Member
  • Ralph Lauren Keil, Program Head/Chair
Keywords:
  • mtorc1
  • high-fat diet
  • western-diet
  • fatty liver
  • nafld
  • nash
  • mrna transcription
  • mrna translation
  • regulatory RNA motif
  • protein synthesis
Abstract:
Consumption of excess calories is associated with alterations in lipid and carbohydrate metabolism in the liver, and often leads to development of metabolic diseases in the liver such as, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), and diabetes. Even though hepatic lipid accumulation begins with as little as 1-wk of high-fat diet i.e. HFD consumption, previous studies searching for the mechanism(s) involved have focused almost universally on time points ≥1 month. Moreover, no studies have assessed possible changes in mRNA translation as a mechanism involved in hepatic fatty acid accumulation, even though changes in mRNA translation have been shown to explain ~40% of alterations in protein expression. Thus, for the studies presented in this thesis, we hypothesized that the translation of specific mRNAs essential in the regulation of metabolic processes in the liver, may be detrimentally dysregulated, contributing to the onset and pathogenesis of metabolic diseases. In chapter 3 we hypothesized that short-term (i.e. 2-wk) consumption of a Western-diet (WD), high in saturated fat, sucrose, and cholesterol, significantly alters the translation of specific mRNA transcripts e.g. lipoprotein lipase (LPL), hormone sensitive lipase E (LIPE), and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), in the liver, contributing to the onset of metabolic disorders such as NASH and NAFLD. Notably, the 5’ and 3’ untranslated region (UTR) of all 3 mRNAs contain regulatory motifs e.g., upstream open reading frames (uORF), internal ribosomal entry sites (IRES), terminal oligopyrimidine (TOP) motif, which may be modulating their translational control as observed in studies performed in chapter 3. Moreover, the expression of several mRNAs i.e. LPL, LIPE, glucokinase (GCK), and fibroblast growth factor 21 (FGF21), was significantly lower in the livers of both fasted and refed rats fed a WD, when compared to rats fed a CD. Overall, the results presented in chapter 3 suggest that alterations in liver metabolism occur rapidly after placing rats on a WD, and that such changes manifest as a consequence of alterations in gene expression occurring at both the transcriptional and translational levels. Surprisingly, feeding-induced phosphorylation of 4E-BP1 and S6, was blunted in WD-fed rats when compared to CD, suggesting that hepatic mTORC1 activity may be, at least in part, responsible for some of the translational changes observed in this study. Thus, the changes observed between fasted and refed CD and WD-fed rats, suggest that the results demonstrating modulations in the expression, and/or translation of several mRNAs, critical in function in the regulation of metabolic processes (i.e. glucose and lipid metabolism), may be occurring through a mechanism dependent oft hepatic mTORC1-activity. Interestingly, in studies performed in chapter 3, the expression of FGF21 mRNA was significantly upregulated in the livers of fasted rats when compared to refed rats, a finding previously demonstrated in studies found in the literature. Studies recently published by Cyphert et. al. demonstrated how the fasting secretagogue glucagon increased the secretion of FGF21 into the media in three hepatoma cell lines, without affecting total FGF21 mRNA abundance suggesting either translational or post-translational control of FGF21 mRNA translation in the liver. Notably, several studies have found that in cases of obesity, glucagon concentrations are significantly dysregulated, leading to glucagon-dependent changes, such as changes in liver weight, hepatic triglycerides, gluconeogenesis, and glycogenolysis. Moreover, in studies where mice lacking the expression of the glucagon receptor (GCGR), were challenged using diet-induced obesity (DIO) by HFD, or streptozotocin treatments to model Type 2 Diabetes, mice lacking GCGR expression, were protected against hyperglycemia, beta cell loss, and hepatic steatosis, directly associating glucagon activity with improvements in metabolic control (93). Together, these findings delineate glucagon as a potential therapeutic against diet-induced obesogenic pathologies. Thus, we hypothesized that FGF21 mRNA was being translationally upregulated by glucagon in the liver. Notably, results from studies performed in chapter 4 of this thesis, demonstrated how the translation and secretion of FGF21 was significantly upregulated by the peptide hormone, glucagon, in rat H4IIE hepatoma cells. Furthermore, glucagon also inhibited mTORC1 activity concurrently with upregulation of FGF21 mRNA secretion, suggesting a mechanism which may be dependent on mTORC1 activity in the liver. Finally, we also observed significant increases in the translation of FGF21 mRNA in the livers of fasted rats when compared to refed, changes independent of changes to total FGF21 mRNA abundance. All in all, results from these studies highlight mRNA translation, as a method of gene regulation, which may be influencing the onset and pathogenesis of metabolic diseases driven by the liver. Studies are currently underway exploring possible regulatory motifs in RNA e.g. uORFs; 5’-TOP; IRES; RNA binding, motifs that may be coordinating translational control of mRNAs identified in chapters 3 and 4.