Characterization of the high-fat diet-induced effects on pre-mRNA alternative splicing in skeletal muscle: a focus on fatty acids and troponin T3
Open Access
- Author:
- Black, Adam James
- Graduate Program:
- Physiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- November 20, 2017
- Committee Members:
- Scot R Kimball, Dissertation Advisor/Co-Advisor
Scot R Kimball, Committee Chair/Co-Chair
Leonard Shelton Jefferson Jr., Committee Member
Charles H Lang, Committee Member
Ralph Lauren Keil, Outside Member
Lisa M Shantz, Committee Member - Keywords:
- alternative splicing
fatty acids
Tnnt3
high-fat diet
fast skeletal muscle troponin T
skeletal muscle
ceramide - Abstract:
- Fat-enriched diets produce metabolic changes in skeletal muscle, that in turn mediate changes in gene regulation. The alternative splicing of pre-mRNA is an important part of gene regulation that engenders multiple protein isoforms from a single gene. Our group has previously shown that the alternative splicing of fast skeletal muscle troponin T (Tnnt3) responds to both natural and experimentally-induced changes in body mass, and that this process is impaired in obese Zucker rats, suggesting that excessive calorie consumption may impair the normal regulation of pre-mRNA alternative splicing. Therefore, the overall goal of this research was to understand if dietary factors, specifically fatty acids, affect pre-mRNA alternative splicing. The overall hypothesis was that in addition to modulating Tnnt3 pre-mRNA alternative splicing, consumption of a high-fat diet leads to transcriptome-wide changes in the pattern of alternative splicing in skeletal muscle and that these changes are mediated in part by saturated fatty acids. Studies from an exon array analysis performed on the gastrocnemius/plantaris complex of rats fed a 10% or 60% fat diet for two or eight weeks demonstrated that consumption of a high-fat diet resulted in the alternative splicing of 668 and 726 exons, respectively. Tnnt3 alternative splicing was among the top-five regulated pre-mRNAs. Further validation of these studies demonstrated that the high-fat diet-induced changes in Tnnt3 alternative splicing also occurred in rats fed a 30% fat diet across a one- to eight-week treatment period independent of changes in body mass between groups. Moreover, this effect depended on fat-type, because Tnnt3 alternative splicing occurred in response to 45% fat diets enriched with lard, but not in response to diets enriched with 45% mono- or polyunsaturated fatty acids. Collectively, these findings suggest a role for saturated fatty acids in the high-fat diet-induced modifications in Tnnt3 alternative splicing. Furthermore, addition of the saturated fatty acid palmitate to myotubes in culture induced a more robust change in Tnnt3 alternative splicing compared to the mono- or polyunsaturated fatty acids, oleate and linoleate. Treatment with a downstream metabolite of palmitate, ceramide, had effects similar to palmitate on Tnnt3 alternative splicing. Pretreatment with myriocin, an inhibitor of de novo ceramide biosynthesis, attenuated the palmitate-induced effects on alternative splicing. Furthermore, pretreatment with okadaic acid to inhibit the ceramide-induced activation of protein phosphatase 2A prevented the palmitate- and ceramide-induced Tnnt3 alternative splicing. Overall the results support the hypothesis that fat enriched diets induce changes in the pattern of alternative splicing in skeletal muscle and suggest a role for saturated fatty acids and their metabolites in mediating the changes observed in Tnnt3 alternative splicing.