Abstract
Purpose :
Omega-3 fatty acids (ω3 FAs) have been reported to improve signs and symptoms of dry eye disease, and increase production of neutral lipid in an immortalized human Meibomian gland epithelial cell line (hMGEC). Since the molecular mechanisms regulating these effects are unknown, we have hypothesized that ω3 FAs activate the lipid sensitive nuclear receptor, PPARγ, leading to meibocyte differentiation, lipid synthesis, and downstream cellular disintegration of hMGEC.
Methods :
HMGEC (a gift from D. Sullivan) were exposed to eicosapentaenoic acid (EPA) or in combination with docosahexaenoic acid (DHA) at 10-100 μM for 2 to 6 days. Cultures were also exposed to specific PPARγ antagonist, T0070907, to block PPARγ signaling. Expression of PPARγ response genes, ADFP and ELOVL4, were evaluated by quantitative PCR. During differentiation, cellular disintegration was monitored using the Autophagy Tandem Sensor (RFP-GFP-LC3B) kit (Molecular Probes) and LysoTracker probes (Molecular Probes) to evaluate and quantify the formation of autophagosomes, autophagolysosomes, and lysosomes, respectively. Accumulated lipid droplets were measured using LipidTox staining, and the intracellular compartmental localization of lipid was identified using live Lipi-Blue (Dojindo Molecular Tech) staining in combination with ER-Tracker (Molecular Probes), LysoTracker, and the Autophagy Tandem Sensor. Activation of autophagy was also investigated using western blotting for LC3BII and phospho-AMPK.
Results :
EPA alone or EPA/DHA induced accumulation of lipid droplets in a time and dose dependent manner. EPA upregulated expression of ADFP and ELOVL4 mRNAs and T0070907 significantly abrogated this response when treated prior to FAs. EPA also increased the formation of autophagolysosomes, the expression of LC3BII and phosphorylation of AMPK. Lipid droplets were localized to the ER compartment and were not associated with either the autophagosomal, autophagolysosomal or lysosomal cellular compartment.
Conclusions :
ω3 FAs induces accumulation of lipid droplets within the ER through PPARγ receptor pathway involving the up regulation of PPARγ response genes. PPARγ induced meibocyte differentiation also leads to activation of autophagy, which we hypothesize is involved in meibocyte disintegration and lipid secretion in the meibomian gland.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.