Discordant transcriptional regulation of gluconeogenic and lipogenic gene expression

Abstract Dysregulated glucose and lipid metabolism are hallmarks of insulin resistance and type 2 diabetes. In the liver, this is manifested as the inability of insulin to suppress hepatic glucose output, while de novo lipogenesis remains elevated even in the fasted state but is still capable of displaying an insulin-stimulated increase. The inability of insulin to suppress hepatic glucose output while activating de novo lipogenesis has been referred to as selective insulin resistance. The acute regulation of both gluconeogenesis and de novo lipogenesis primarily results from rapid changes in allosteric regulation, however, in chronic states such as insulin resistance and type 2 diabetes there are marked changes in gluconeogenic and lipogenic gene expression. We have obtained preliminary data indicating that the prototypical lipogenic genes (i.e. FASN, SCD1) are primarily regulated by transcription initiation whereas gluconeogenic genes (i.e.: PCK1, G6Pc) are primarily regulated at the level of transcriptional elongation. During diet induced hepatic insulin resistance, the elongation factor Spt5 is functionally dysregulated thereby increasing gluconeogenic gene expression in the fed state. In addition, using single cell RNAseq we have identified the differential engagement of different hepatocyte subsets during the fasting/feeding cycle that are also altered during the development of hepatocyte insulin resistance. Based upon these data, we propose two specific aims to examine several novel molecular and cellular mechanisms that are important regulatory components of normal physiologic hepatocyte lipogenic and gluconeogenic gene expression that are subsequently dysregulated during diet-induced insulin resistance. Specifically, we use 1) ChIP-Seq to determine the changes in various initiation and elongation factors in their DNA occupancy binding, 2) ATAC-Seq to determine chromatin organization, and 3) ChRO-Seq to directly measure the rates of transcription across gene bodies. These analyses of total hepatocyte function will be complemented with single cell molecular analyses that include 1) single cell RNA-Seq to directly determine differential function of hepatocyte subsets, 2) single cell ATAC-Seq for individual hepatocyte chromatin organization changes, and 3) single molecule FISH to assess individual cellular transcriptional activity and identification of their anatomical zonation. These approaches will allow us to determine the molecular mechanisms responsible for the activation of transcriptional initiation and elongation between lipogenic and gluconeogenic gene expression, and will expand our understanding of the individual hepatocyte cellular responses that account for these changes in under normal fasting/feeding states and in diet-induced liver insulin resistance.