RNA seq to determine differential gene expression in pgph-2 oe C.elegans animals and the subset of HLH-30-dependent genes on normal growth and excess glucose conditions.
Metabolic stress caused by excess nutrients accelerates aging. We recently demonstrated that the newly-discovered enzyme glycerol-3-phosphate phosphatase (G3PP; gene Pgp), which operates an evolutionarily conserved glycerol shunt that hydrolyzes glucose-derived glycerol-3-phosphate to glycerol, counters metabolic stress and promotes healthy aging in C. elegans. However, the mechanism whereby G3PP activation extends healthspan and lifespan, particularly under glucotoxicity, remained unknown. We demonstrated that G3PP (PGPH-2) overexpression activates three key longevity factors, AMPK, the TFEB homolog HLH-30, and autophagy, and is an attractive target for age-related metabolic disorders linked to excess nutrients. This transcriptomics analysis was designed to determine genes that are differentially regulated in pgph-2 oe animals in comparison to wild-type animals on normal and excess glucose conditions. To determine the role of HLH-30 in the regulation of gene expression, we also used pgph-2 oe; hlh-30 and hlh-30 mutant animals and found a signature mediated by pgph-2 oe and HLH-30-dependent. Overall design: Synchronized young adult WT, pgph-2 o/e, pgph-2 o/e; hlh-30 and hlh-30 animals and grown on normal growth medium or plates supplemented with 2% glucose are harvested. Total RNA is extracted using trizol and purified with RNA purification kit and subjected to seq analysis.