CIRCADIAN CONTROL OF LIPID METABOLISM IN PROLIFERATIVE HEPG2 CELLS

MONJES, NATALIA M.; GUIDO, MARIO E.

Modalidad virtual

Congreso; SAIB-SAMIGE; 2020

Biological clocks present in organs, tissues and even in individual cells control physiological processes in a time-dependent manner, driving transcriptional and metabolic rhythms. The disruption of circadian rhythmicity in modern life (continuous artificial light, shiftwork, jetlag, etc.) may promote higher cancer risk and metabolic disorders, but little is known about clock function in tumor cells. We have previously demonstrated that glycerophospholipid (GPL) synthesis is highly regulated by the biological clock under physiological conditions in retinal cells (Guido et al 2001, Garbarino-Pico et al 2004, 2005), in the mice liver (Gorne et al 2014), and in fibroblast (NIH3T3 cells) cultures (Marquez et al 2004, Acosta et al 2013). Here we evaluated the circadian variations in redox state and lipid metabolism in hepatocarcinoma HepG2 (control) and Bmal1 knockdown HepG2 cell cultures under proliferating conditions to assess the molecular clock work and its link with the lipid metabolism. Bmal1 knockdown (KD) was performed by CRISPR-Cas9 technology. We assessed: i) mRNA expression and protein content of Clock Genes and clock controlled genes and ii) the metabolic state and lipid content in both cellular conditions. For this, we assessed mRNA expression and protein content of enzymes involved in the GPL biosynthesis, reactive oxygen species (ROS), endogenous level of GPLs and lipid droplets (LDs) in number, size and % area variations over time. Cells grown in 10% FBS-DMEM were synchronized with 100 nM Dexamethasone (DEX) for 60 min, maintained in 5% FBS-DMEM and collected at different times. mRNA levels were quantified by qPCR. Cell cytometry was assessed with MTT. Lipid content was determined by TLC. ROS were determined with 2,7-dichlorodihydrofluorescein diacetate (2 μM). LDs were stained with Nile Red (1,5 µg/ml) and visualized by confocal microscopy. Results showed that synchronized HepG2 cells displayed significant circadian rhythms in the expression of clock components (Bmal1, PER1, Rev-Erb) and choline kinase (ChoK)-like proteins, in the content of endogenous GPLs (PC and PE) and in levels of LDs (number and size and fluorescence intensity). Remarkably, when the circadian clock was perturbed by Bmal1 KD, LDs levels and lipid enzymes were severely affected, and rhythms damped out. We found an active time-dependent control of gene expression and metabolism in proliferating HepG2 cells strongly suggesting that an intrinsic metabolic clock continues to function in proliferating liver tumor cells and its perturbation severely affected lipid metabolism.