Lipid Droplet Biogenesis and Function in the Endothelium

Abstract

Rationale: Fatty acids (FA) are transported across the capillary endothelium to parenchymal tissues. However, it is not known how endothelial cells (EC) from large vessels process a postprandial surge of FA.

Objective: This study was designed to characterize lipid droplet (LD) formation in EC by manipulating pathways leading to the formation and degradation of LD. In addition, several functions of LD-derived FA were assessed.

Methods and results: LD were present in EC lining the aorta after the peak in plasma triglycerides initiated by a gavage of olive oil in mice, in vivo. Similarly, in isolated aorta, oleic acid treatment generates LD in EC ex vivo. Cultured EC readily form LD largely via the enzyme DGAT (diacylglycerol O-acyltransferase 1) and degrade LD via ATGL (adipocyte triglyceride lipase) after FA loading. Functionally, LD-derived FA are dynamically regulated and function to protect EC from lipotoxic stress and provide FA for metabolic needs.

Conclusions: Our results delineate endothelial LD dynamics for the first time in vivo and in vitro. Moreover, LD formation protects EC from lipotoxic stress, regulates EC glycolysis, and provides a source of FA for adjacent cells in the vessel wall or tissues.

Keywords: endothelial function; endothelium; fatty acids; lipid droplet; lipids; metabolism; triglycerides.

Figure 1. LD are dynamically regulated in EC in vivo

WT (C57BL/6) mice were fasted for 16 hrs and gavaged with olive oil for plasma TG and endothelial LD analysis. (A) Plasma TG levels peak at 90 mins whereas (B) LD in EC layer were detected via en face immunostaining at 180 mins post-gavage, as quantified in (B) by numbers of LD. n=2–4 mice, 7 images per mouse. Scale bar, 50μm. Data in (A) and (C) are expressed as mean ± SEM.

Figure 2. EC utilize TG synthesis enzymes for LD formation and canonical lipases for lipolysis

(A) OA supplementation (1mM) increases LD formation in MLEC over time, as quantified in (B). (C) OA-induced LD formation in MLEC is dose-dependent and maximal at 1mM OA incubation overnight as quantified in (D). (E) mRNA expressions of TG synthetic enzymes (GPAT4, AGPAT2, Lipin2, DGAT1 and DGAT2 expressions) are induced in MLEC responding to OA treatment over time, whereas sterol ester synthetic enzymes (ACAT1 and ACAT2) are not. (F) Western blotting analyses show that ATGL and HSL levels increase in response to OA treatment whereas MGL expression remains unchanged in MLEC. (n=3) (G) MLEC express PLN family proteins (PLN2 and PLN3) shown by agarose gel of qPCR products. (n=3) (H) LD fractionation from EAhy 926 cells untreated (“−” lanes) or treated with OA “+” lanes) show that PLN2, PLN3, ATGL, CGI-58, and Cav-1 are enriched in LD fraction whereas other proteins examined, FLOT-1, Grp94 and Hsp90, are not. (n=3) Scale bars, 25μm. Data in (B), (D), and (E) are expressed as mean ± SEM (n=3, 5 images per experiment in (B) and (D)) and analyzed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.005.

Figure 3. DGAT1 and ATGL are key enzymes regulating LD dynamics in EC

(A) DGAT1 inhibition by A922500 (5μM) has a greater effect on OA-induced LD formation in MLEC than DGAT2 inhibition by PF-06424439 (10μM). Co-incubation with both inhibitors completely abolishes LD formation, as quantified in (B). (C) Basal lipolysis is significantly delayed over time by Atglistatin (10 μM) in MLEC pre-treated with OA (1mM) overnight, as quantified in (D) by TG content. (E) Thoracic aortae incubated with OA (1mM) overnight show LD formation in EC layer via en face immunostaining, where DGAT-1 inhibitor, A922500 (5μM) abolishes LD formation and ATGL inhibition by Atglistatin (10μM) enhances LD accumulation, as quantified in (F) by BODIPY intensity. (n=3) Scale bars in (A) and (C), 10μm. Scale bar in (E), 50μm. Data in (B), (D) and (F) are expressed as mean ± SEM (n=3, 5 images per experiment) and analyzed by unpaired student-t test. *p<0.05

Figure 4. LD formation protects EC from lipotoxcity and lipolysis-derived FA function as an energy resource in EC or for release to parenchymal cells

(A) MLEC were treated with OA (C18:1), PA (C16:0), SA (C18:0) all at 1mM, or mixtures of OA and PA/SA (0.5mM of each) overnight. LD formation was detected in EC treated with OA or OA with PA/SA. (n=3) (B) OA incubation induces ER stress in MLEC when both DGAT1 (5μM, A922500) and DGAT2 (10μM, PF-06424439) are inhibited, as accessed by BiP and CHOP immunoblotting. PA and SA lead to increases in ER stress where the presence of OA abrogates this induction. This reduction is reversed when both DGAT1 and DGAT2 are inhibited. (n=2) (C) OA incubation has no effect on EC general mitochondrial function measured by OCR with injections of compounds indicated (−OA and +OA). Inhibition of FAO (ETOX; 40μM) in LD-rich EC shows decreased baseline OCR and mitochondria capacity (FCCP; 1μM) (+OA +ETOX) determined by FCCP (1μM) treatment, whereas ETOX has no effect on normal EC (−OA +ETOX). (D) Glycolytic parameters were determined by measuring ECAR. LD-rich EC exhibited impaired glycolytic activity via addition of glucose (10mM) (+OA). Inhibition of fatty acid oxidation (ETOX; 40μM) in LD-rich EC shows recovery of glycolytic flux (+OA +ETOX). (E) FA released from LD-rich HDMEC in a transwell insert accumulates at both sides of the transwell over time (CTRL). Inhibition of lipolysis (Atglistatin; 10μM or CAY10499; 10μM) reduces this accumulation whereas activation of lipolysis (Forskolin; 10μM and IBMX; 0.1mM) increases FA release. (F) In the co-culture system with LD-rich EC and C2C12 myotubes in the bottom well, FA release was only detected in the apical chamber, whereas (G) C2C12 myotubes accumulated LD. (n=3, 5 images of each experiment). Scale Bar in (A), 10μm. Scale Bar in (G), 50μm. Data in (C), (D), (E) and (F) are expressed as mean ± SEM (n=3) and analyzed by two-way ANOVA. *p<0.05 to −OA group. #p<0.05 to +OA group. #p<0.05 to control. N.S., non significant.