The roles of Elovl3 and Elovl4 in lipid homeostasis in Harderian glands of mice

Abstract

Purpose : The mouse Harderian gland (HG) is an exocrine organ which produces lipids (HGL) whose physiological roles involve (together with meibum which is produced by Meibomian glands, MG) protection and lubrication of the cornea, among other functions. Our aim was to establish the roles of two major lipid biosynthesis-related genes, Elovl3 and Elovl4, in maintaining lipid homeostasis in HG and MG, by conducting lipid analyses of HGL and meibum of wild type (WT) and ELOVL3/ELOVL4-compromised mice.

Methods : The HG specimens were evaluated using histological and immunohistochemical approaches. The HGL and meibum specimens of WT mice and their age- and gender-matched Elovl3^-/-, Elovl3^+/-, Elovl4^+/-, Elovl3^-/-/Elovl4^+/- littermates were analyzed using liquid chromatography-mass spectrometry (MS). The structures of lipids were established using high resolution Time-of-Flight MS, fragmentation of lipids using multi-stage ion trap MS, and authentic lipid standards as control.

Results : Both singly and doubly mutated mice produced higly abnormal HG phenotypes. The major lipid components of HGL were found to belong to the class of glycerol ether-diesters (GEDEs) of the C₅₇H₁₀₈O₅- C₆₅H₁₂₅O₅ families. Much smaller pools of cholesteryl esters, wax esters and triacyl glycerols were also observed. The ten most prominent GEDEs were those with molecular formulas of C₅₇H₁₀₉O₅, C₅₉H₁₁₂O₅, C₆₁H₁₁₄O₅, C₆₁H₁₁₆O₅, C₆₂H₁₁₄O₅, C₆₃H₁₁₈O₅, C₆₃H₁₂₀O₅, C₆₄H₁₁₈O₅, C₆₅H₁₂₃O₅, and C₆₅H₁₂₅O₅, all of which existed as multiple isobaric homologs. Inactivation of Elovl3 caused a decline in GEDEs longer than C₆₃ and fatty acid (FA) longer than C₂₀. Considering that at least one of the FA residues of the latter GEDEs is about C₂₀ or longer, the effect of the Elovl3 inactivating mutation replicated its inhibitory effect on elongation of FA in MG. Partial inactivation of Elovl4, in conjunction with loss of Elovl3 function, decreased the biosynthesis of C₆₃ and longer homologs even further.

Conclusions : The inactivating mutations in Elovl3 and Elovl4 genes caused suppression of FA elongation and changed the profile of lipids that contain very long chain FA in HGL and meibum. Specifically, HGL with FA longer than C₂₀ were shown to be products of the ELOVL3/ELOVL4 pathway, which replicated the FA elongation pathway in MG. Harderian glands may be a useful model for studying effects of inactivation of various genes on lipid metabolism in the eye and adnexa.

This is a 2020 ARVO Annual Meeting abstract.