Abstract
Insulin resistance is a pathophysiological hallmark of type 2 diabetes and nonalcoholic fatty liver disease. Under the condition of fat accumulation in the liver, suppression of hepatic glucose production by insulin is diminished. In order to gain deeper understanding of dysregulation of glucose production in metabolic diseases, in the present study, we performed an unbiased phenotypic screening in primary human hepatocytes to discover novel mechanisms that regulate gluconeogenesis in the presence of insulin. To optimize phenotypic screening process, we used a chemical genetic screening approach by building a small-molecule library with prior knowledge of activity-based protein profiling. The “positive hits” result from the screen will be small molecules with known protein targets. This makes downstream deconvolution process (i.e., determining the relevant biological targets) less time-consuming. To unbiasedly decipher the molecular targets, we developed a novel statistical method and discovered a set of genes, including DDR3 and CACNA1E that suppressed gluconeogenesis in human hepatocytes. Further investigation, including transcriptional profiling and gene network analysis, was performed to understand the molecular functions of DRD3 and CACNA1E in human hepatocytes.
Highlights
Type 2 diabetes mellitus (T2DM) is a multifactorial disease, represented by heterogeneous patient populations with various degrees of obesity, insulin resistance, and beta cell dysfunction.[1]Among these factors, insulin resistance is an independent risk factor of progressive deterioration from glucose intolerance to diabetes.[2]
Establish a chemical genetic screening approach in human primary hepatocytes In order to identify genes that are involved in gluconeogenesis in the presence of insulin, we first optimized glucose production assay for primary human hepatocytes
We found that 6-h starvation and 24-h substrates treatment was the optimal time window for the detection of glucose production from human primary hepatocytes
Summary
Type 2 diabetes mellitus (T2DM) is a multifactorial disease, represented by heterogeneous patient populations with various degrees of obesity, insulin resistance, and beta cell dysfunction.[1]. Phenotypic screen has been demonstrated to be a powerful approach to discover novel drug targets and molecules. With the advance of human genome sequencing technology and accumulated knowledge of small-molecule libraries, chemical genetics has emerged as a powerful approach to discover potential drug targets.[7] Traditional genetics approach uses gene knockout or RNAi technology to manipulate gene function and screens for correlations between gene function and a particular phenotype. We used chemical genetics approach to seek for protein–phenotype correlations that could relate insulin’s action on human hepatocytes with a set of biological targets (regulators of gluconeogenesis). DRD3 and CACNA1E are potential therapeutic targets for the treatment of T2DM
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