Circadian disruption alters hepatic calcium hemostasis, endocannabinoidome and mitochondria through N -docosahexaenoyl ethanolamide-GPR110 signaling

Circadian rhythm disruption is associated with metabolic and inflammatory disorders; however, the mechanisms linking circadian dysfunction to endocannabinoidome (eCBome) signaling and mitochondrial metabolism remain unclear. In our previous in vivo study, constant light exposure altered hepatic eCBome profiles, reduced N -acylethanolamines (NAEs), increased monoacylglycerols (MAGs), and elevated inflammatory cytokines. Here, we investigated the underlying mechanisms using CRISPR/Cas9-generated BMAL1 knockout (KO) HepG2 cells as an in vitro model of circadian alteration. The BMAL1 KO model showed broad lipid remodeling characterized by increased fatty acids, prostaglandins, and MAGs together with reduced NAEs and enhanced lipid accumulation. These changes were accompanied by increased inflammatory signaling and cytokine production. Among the assessed genes, GPR110 was significantly altered in mice exposed to constant light ( in vivo study) and BMAL1 KO model and emerged as a potential mediator linking circadian signaling to mitochondrial function. BMAL1 KO cells also exhibited significantly increased calcium (Ca² ⁺ ) levels in mitochondria and the endoplasmic reticulum (ER), along with attenuation of mitochondrial and glycolytic ATP production. BMAL1KO did not abolish the rhythmicity of NAEs level over 24 hours from medium deprivation and read ministration except for N -docosahexaenoyl-ethanolamide (DHEA). Further, experiments showed that DHEA acts through GPR110 and suppress inflammatory lipid-associated pathways, enhances ATP production, and increases mitochondrial and ER Ca² ⁺ accumulation and inflammatory signaling. Together, these mitochondrial Ca² ⁺ signaling, and inflammation in hepatocytes, highlighting DHEA-GPR110 signaling as a potential regulator of hepatic metabolic homeostasis.