DHA and 19,20-EDP induce lysosomal-proteolytic-dependent cytotoxicity through de novo ceramide production in H9c2 cells with a glycolytic profile

Masahiko Hirafuji,Ayman O. S. El-Kadi,Tomoko Endo,John M. Seubert,Ahmed A. El-Sherbeni,Victor Samokhvalov,Takuji Machida,Kevin M. W. Khey,Ahmed M. Darwesh

doi:10.1038/s41420-018-0090-1

Masahiko Hirafuji, Ayman O. S. El-Kadi + Show 7 more

Open Access

https://doi.org/10.1038/s41420-018-0090-1

Copy DOI

Abstract

Docosahexaenoic acid (DHA) and their CYP-derived metabolites, epoxydocosapentaenoic acids (EDPs), are important fatty acids obtained from dietary sources. While it is known that they have significant biological effects, which can differ between cell type and disease state, our understanding of how they work remains limited. Previously, we demonstrated that DHA and 19,20-EDP triggered pronounced cytotoxicity in H9c2 cells correlating with increased ceramide production. In this study, we examine whether DHA- and 19,20-EDP-induced cell death depends on the type of metabolism (glycolysis or OXPHOS). We cultivated H9c2 cells in distinct conditions that result in either glycolytic or oxidative metabolism. Our major findings suggest that DHA and its epoxy metabolite, 19,20-EDP, trigger cytotoxic effects toward H9c2 cells with a glycolytic metabolic profile. Cell death occurred through a mechanism involving activation of a lysosomal-proteolytic degradation pathway. Importantly, accumulation of ceramide played a critical role in the susceptibility of glycolytic H9c2 cells to cytotoxicity. Furthermore, our data suggest that an alteration in the cellular metabolic profile is a major factor determining the type and magnitude of cellular toxic response. Together, the novelty of this study demonstrates that DHA and 19,20-EDP induce cell death in H9c2 cells with a glycolytic metabolicwct 2 profile through a lysosomal-proteolytic mechanism.

Highlights

Introduction Long chain n3 polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA, C22:6n-3) and eicosapentaenoic acids (C20:5n-3) are important fatty acids obtained from dietary sources
H9c2 cells grown in 25 mM glucose media had an respiration control ratio (RCR) of 1.44 ± 0.18, while cells cultivated in 5.5 mM glucose media had RCR up to 6.9 ± 0.87, demonstrating that there was a shift from glycolysis to oxidative phosphorylation (OXPHOS) in H9c2 cells grown in 5.5 mM glucose media
It is well documented that cytochrome P450 (CYP) epoxygenases catalyze the enzymatic transformation of PUFA into various potent epoxylipid including omega-3 epoxides of Docosahexaenoic acid (DHA) referred as epoxydocosapentaenoic acids (EDPs)

Summary

Introduction

3 polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA, C22:6n-3) and eicosapentaenoic acids (C20:5n-3) are important fatty acids obtained from dietary sources These essential fatty acids are required components of phospholipid membranes and serve as precursors to numerous lipid mediators with various biological properties. Ceramide is a central lipid component of sphingolipid structure that is biosynthesized by three pathways, which include de novo synthesis from palmitoyl-CoA and serine, hydrolysis of sphingomyelin or a salvage pathway[17] It is an important lipid mediator regulating various cellular responses like cell death, and recent evidence suggests a role in various metabolic pathways influencing mitochondrial function[18]. We investigated the effects of DHA and 19,20-EDP in undifferentiated H9c2 cells cultured under conditions triggering glycolytic or oxidative phosphorylation-mediated metabolism

Methods

Results

Conclusion