By comparing lipid droplet storage between control and Ano6 mutant mice, we want to test if ANO6 is involved in lipid metabolism, and then elucidate the pathway underlying the lipid droplet regulation process, to identify the physiological and pathological involvement of ANO6 in inflammation and neurodegeneration.

Anoctamins (ANO), in the transmembrane proteins 16 (TMEM16) family, are highly conserved intracellular calcium (Ca²⁺)–activated proteins. ANO6 mediates Ca²⁺-dependent phospholipid scramblase activity. It is an intriguing open question whether ANO6 is involved in lipid metabolism. Given that abnormal lipid metabolism is implicated in aging and degeneration, understanding the mechanism of ANO6 in lipid droplet regulation will guide us on finding therapy.

Primary astrocytes were cultured from pups of postnatal day 2. Ano6 KO HeLa cells were generated with CRISPR-Cas9 technology. Immunostaining of periventricular hypothalamus brain tissue and small intestine tissue from control and Ano6 mutant mice. 

Lipid droplets were visualized on a confocal microscope (Leica SP5) after cells were stained with Lipidtox. Free fatty uptake was measured in a plate reader after treating cells with QBT^TM.

We found significant Lipid droplet storage decrease in primary astrocytes, brain and small intestine tissue from Ano6 KO mice. To determine the resource of fatty acid that forming lipid droplet, we block different pathways that contribute to this process. We found that free fatty acid uptake from extracellular media, but not de novo fatty acid synthesis or membrane hydrolysis, accounts for the decrease in lipid droplet formation in Ano6 mutant cells. Interestingly, we also noticed a suppression in the chemokines level in Ano6 mutant astrocytes.

We have found an obvious decrease in the lipid droplet storage in Ano6 mutant mice, which is mediated by decreased fatty acid uptake. Our study revealing a suppressed inflammatory level in Ano6 mutant mice may facilitate future therapeutic developments targeting inflammation related disease.