Type 2 diabetes (T2D) affects approximately 8.3% of the United States population. Family history is one of the major risk factors for T2D noncoding genetic variation in the transcription factor 7-like 2 (TCF7L2) gene is significantly associated with T2D in multiple ethnic populations, yet the biological mechanism(s) that lead to increased risk are still unclear. Evidence for involvement of the central nervous system (CNS) in regulating fasting and postprandial glucose levels continues to grow, and TCF7L2 localization in the hypothalamus suggests a potential role in glucose regulation. However, the genes and networks regulated by TCF7L2 in the hypothalamus are not known. This project will elucidate those genes and networks in human neurons using chromatin immunoprecipitation sequencing (ChIP-Seq) and gene expression profiling of induced pluripotent stem cells (iPSC)-derived neurons from adult male subjects of the San Antonio Family Diabetes Study (SAFDS). Two iPSC from each subject will be differentiated into NPY-expressing glutamatergic neurons. siRNA will be used to knockdown TCF7L2 expression. Using these human neuronal cultures, ChIP- Seq will be performed pre- and post-knockdown to identify potential target genes of TCF7L2. From the same neuronal cultures, gene expression profiles will be assayed using RNAseq in order to correlate the bound sites with changes in expression of the target genes. Bioinformatic analysis such as gene set enrichment analysis will be used to identify pathways TCF7L2 may influence. We will also investigate genes expression profiles during various environmental challenges such as high glucose, leptin and adiponectin to determine if regulation of biological networks changes in response. Regulation of glucose homeostasis by CNS and TCF7L2 will be further elucidated with an in vivo mouse model containing a conditional knockdown of TCF7L2 specific to the hypothalamus. Tamoxifen-induced knockdown will be implemented in adult mice. We will detect alterations in glucose homeostasis through several physiological measures. We shall also isolate the hypothalamus to measure changes in RNA and protein expression in the same metabolic pathways as described previously. Information regarding diabetes-related regulatory function of TCF7L2 in the hypothalamus may help to elucidate CNS pathways potentially disrupted in diabetes pathogenesis and therefore targets for intervention. This study is the first to address TCF7L2 function of glucose homeostasis in human neurons. This study also applies iPSC technology providing an ample supply of otherwise difficult to obtain neurons. The training outlined here will provide a solid foundation in research techniques, data interpretation, planning, scientific communication and professionalism to prepare me for a career as an independent research scientist.
Genetic variation in the gene transcription factor 7-like 2 (TCF7L2) has been significantly associated with type 2 diabetes risk in human populations across the globe. The central nervous system is a key regulator of glucose homeostasis and is therefore a potential target organ for treatment of type 2 diabetes. Our studies will establish TCF7L2 genetic targets in CNS neurons and examine a neuron-specific role for TCF7L2 in glucose homeostasis.
