We have recently discovered a novel and evolutionarily conserved homeostatic response wherein alternative splicing of the pre-mRNA encoding troponin T, a protein that affects muscle force production, is tightly regulated in response to changes in body weight. The effect is based on weight rather than mass or compartmentation of mass within the body because an external load has the same effect as an increment in native body weight. In contrast, the response is affected by body composition, as load-induced changes in troponin T pre-mRNA alternative splicing are impaired in obese, but not lean, Zucker rats, leading to inappropriate expression of troponin T isoforms. Moreover, in preliminary studies, we have found a similar dysregulation in rats fed a high-fat diet enriched in saturated fatty acids. Notably, high-fat diet-induced changes in troponin T pre-mRNA alternative splicing manifest prior to detectable alterations in either body weight or composition, suggesting that alternative splicing is directly modulated in response to dietary macronutrients. In other preliminary studies we have found that the effect of a high-fat diet on pre-mRNA splicing is not unique to troponin T, but instead is observed for pre-mRNAs encoding an array of proteins. Hence, the objective of the studies proposed in the present application is to identify the mechanism(s) through which dietary fatty acids regulate alternative splicing of pre-mRNA, and delineate the functional consequences of such alterations. We hypothesize that consumption of a high-fat diet results in an altered pattern of pre-mRNA splicing in skeletal muscle, leading to expression of protein isoforms that exhibit reduced force production and/or calcium sensitivity, as well as altered metabolism. The hypothesis will be tested via the following three specific aims: (1) Establish optimal conditions for high-fat diet-induced changes in alternative splicing of the troponin T pre- mRNA in skeletal muscle, (2) Characterize high-fat diet-induced changes in the contractile properties of skeletal muscle, and, (3) Characterize and delineate the molecular mechanism(s) involved in high-fat diet- induced changes in alternative splicing of pre-mRNA in skeletal muscle across the transcriptome, and identify altered signaling and metabolic pathways. From these experiments, we will obtain an unprecedented scale and depth of understanding of how quantitative variation in alternative splicing is controlled, and how diet affects that regulation. In addition, the results will open a new window into how diet changes pathways involved in body weight homeostasis. Overall, the studies proposed here are highly original and will address a deficit in our knowledge about the plasticity of quantitative alternatie splicing in general, and mechanisms through which macronutrients affect and in some cases disrupt the way metazoans functionally and metabolically adapt to changes in their weight. We expect the proposed research to reveal biomarkers for pre-disease states caused by poor diet, and candidate molecules and pathways for pharmacological manipulation to provide new and innovative approaches for the prevention and treatment of metabolic disorders.
Metabolic adaptation to dietary consumption and composition involves changes in the pattern of gene expression that allows the organism to optimally utilize, and respond to, available nutrients. However, excess provision of certain macronutrients, e.g. saturated fatty acids, may interfere with the normal response, leading to maladaptation and disease, i.e. the metabolic syndrome. The goal of the research proposed here is to identify the mechanisms through which a high-fat diet enriched in saturated fatty acids leads to an impairment in the control of alternative pre-mRNA splicing, a ubiquitous and crucial mechanism for generating protein diversity and regulating gene expression.
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