Chemical genetic-based phenotypic screen reveals novel regulators of gluconeogenesis in human primary hepatocytes

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.

Glycerol induces G6pc in primary mouse hepatocytes and is the preferred substrate for gluconeogenesis both in vitro and in vivo

“…It is conventionally accepted that pyruvate and lactate are the main substrates for GNG, and these substrates are extensively used to test hepatic glucose production both in vitro and in vivo and are employed generally at supraphysiological concentrations (29–35). There are no studies, however, showing glucose production using physiological fasting concentrations of substrates or studies using a combination of substrates.…”

Comprehensive Molecular Analyses of a Six-Gene Signature for Predicting Late Recurrence of Hepatocellular Carcinoma

“…As the figure displayed, TTN , MUC16 , CSMD3 , AXIN1 , and CACNA1E mutations characterized the high-risk group. Notably, three of these five genes, TTN , MUC16 , and AXIN1 were reported in a previously published series as the driver genes in hepatocarcinogenesis ( 30 – 32 ), while CSMD3 acted as a tumor suppressor gene and decreased expression contributing to hepatocarcinogenesis ( 33 ). Of particular interest, CACNA1E , the major subunit of the voltage-dependent CaV2.3 Ca2+ channel, may be involved in regulating metabolism-related functions of the liver, including bile secretion, glucose and lipid metabolism, and mitochondria functions, while the specific function of hepatocytes needs more extensive study ( 34 ).…”

Mitochondria‐associated membranes contribution to exercise‐mediated alleviation of hepatic insulin resistance: Contrasting high‐intensity interval training with moderate‐intensity continuous training in a high‐fat diet mouse model

“…This was determined by evaluating hepatic steatosis severity and the phosphorylation levels of key proteins in the hepatic insulin signaling pathway (PI3K/AKT/GSK3β). Hepatic IR triggers uncontrolled hepatic gluconeogenesis, 27 leading to elevated blood glucose levels and consequently contributing to systemic IR. 28 Consistently, we also observed systemic IR phenotypes in HFD mice, including increased blood glucose, elevated blood lipids, impaired glucose tolerance, and diminished insulin sensitivity.…”