Retinyl esters form lipid droplets independently of triacylglycerol and seipin

Lipid droplets store neutral lipids, primarily triacylglycerol and steryl esters. Seipin plays a role in lipid droplet biogenesis and is thought to determine the site of lipid droplet biogenesis and the size of newly formed lipid droplets. Here we show a seipin-independent pathway of lipid droplet biogenesis. In silico and in vitro experiments reveal that retinyl esters have the intrinsic propensity to sequester and nucleate in lipid bilayers. Production of retinyl esters in mammalian and yeast cells that do not normally produce retinyl esters causes the formation of lipid droplets, even in a yeast strain that produces only retinyl esters and no other neutral lipids. Seipin does not determine the size or biogenesis site of lipid droplets composed of only retinyl esters or steryl esters. These findings indicate that the role of seipin in lipid droplet biogenesis depends on the type of neutral lipid stored in forming droplets.

The role of lipid droplets in microbial pathogenesis

The nonpolar lipids present in cells are mainly triacylglycerols and steryl esters. When cells are provided with an abundance of nutrients, these storage lipids accumulate. As large quantities of nonpolar lipids cannot be integrated into membranes, they are isolated from the cytosolic environment in lipid droplets. As specialized, inducible cytoplasmic organelles, lipid droplets have functions beyond the regulation of lipid metabolism, in cell signalling and activation, membrane trafficking and control of inflammatory mediator synthesis and secretion. Pathogens, including fungi, viruses, parasites, or intracellular bacteria can induce and may benefit from lipid droplets in infected cells. Here we review biogenesis of lipid droplets as well as the role of lipid droplets in the pathogenesis of selected viruses, bacteria, protists and yeasts.

Preferential lipolysis of DGAT1 over DGAT2 generated triacylglycerol in Huh7 hepatocytes

Hepatic steatosis is defined by accumulation of neutral lipids in lipid droplets (LDs) including triacylglycerol (TG) and steryl esters. Two distinct diacylglycerol acyltransferases (DGAT1 and DGAT2) catalyze synthesis of TG in hepatocytes. TG formed through either DGAT1 or DGAT2 appears to be preferentially directed to distinct intercellular fates, such as fatty acid production for oxidation or very-low density lipoprotein assembly, respectively. Because of the preferential use of TG generated by DGAT1 and DGAT2, we hypothesized that targeting/association of lipolytic machinery to LDs would differ depending on whether the TG stores were generated through DGAT1 or DGAT2 activities. Inhibition of DGAT1 or DGAT2 in human hepatoma cells (Huh7) incubated with oleic acid resulted in only a small change in TG accretion suggesting that the two DGATs can compensate for each other in fatty acid esterification. This compensation was not accompanied by changes in DGAT1 or DGAT2 mRNA expression. DGAT1 inhibition (TG synthesized by DGAT2) resulted in large LDs, whereas DGAT2 inhibition (TG synthesized by DGAT1) caused the accumulation of numerous small LDs. Oleic acid treatment increased mRNA and protein expression of the LD-associated protein PLIN2 but not PLIN5 or the lipase ATGL and its activator ABHD5/CGI-58. Inactivation of DGAT1 or DGAT2 did not alter expression (mRNA or protein) of ATGL, ABHD5/CGI-58, PLIN2 or PLIN5, but inactivation of both DGATs increased PLIN2 abundance despite a dramatic reduction in the number of LDs. ATGL localized preferentially to DGAT1- made LDs rather than to DGAT2-made LDs, and TG in these LDs was preferentially used for fatty acid (FA) oxidation. A combination of DGAT2 inhibitor and the pan lipase inhibitor E600 resulted in large LDs, suggesting that the small size of DGAT1-made LDs is due to a lipolytic process.

Steryl Sinapate as a New Antioxidant to Improve Rapeseed Oil Quality during Accelerated Shelf Life

In recent years, steryl esters have become an attractive for the cosmetic, pharmaceutical, and food industries. Hence, the effect of exogenous antioxidant, β-sitosteryl sinapate on oxidative stability and antioxidant activity (AA) of refined rapeseed oil was evaluated by the accelerated shelf-life test. Oxidative parameters of refined rapeseed oil—peroxide value (PV), anisidine value (p-AnV), acid value (AV), and spectrophotometric indices (K232, K268)—increased during storage. However, the addition of β-sitosteryl sinapate caused a decrease of the primary and secondary oxidation products in the supplemented oils in comparison with the control sample. Moreover, oils with steryl ester had higher AA than oil without the synthetic antioxidant. The accelerated storage negatively affected the antioxidant potential of refined and enriched oils causing the AA decrease by 25–54% and 7–15%, respectively. Studies have consistently demonstrated beneficial associations between the presence of β-sitosteryl sinapate in oil samples and the inhibition of their oxidative degradation under the accelerated conditions. Additionally, the possibility of using the synchronous fluorescence (SF) spectroscopy and excitation–emission matrix (EEM) fluorescence spectroscopy for identification and observing changes in main fluorescent components present in non-supplemented and supplemented rapeseed oils during the accelerated storage was attempted.

Impact of regulative noise exposure to biodiesel production due to enhanced lipid droplet production in Saccharomyces cerevisiae: Preliminary results from a laboratory experiment

Lipid Droplet (LD) is a ubiquitous cellular organelle that stores natural lipids as an energy and carbon source. It has emerged as a highly active organelle, engaged in lipid synthesis, protein storage, protein degradation, transportation, and metabolism. It stores natural lipids in the form of triacylglycerols (TAG) and steryl esters. TAGs consider promising biotechnological importance to produce biodiesel; thus, LD is considered a tremendous scientific concern in the modern era. The TAG accumulation is found in various feedstocks, but amongst the microorganisms becomes an evident alternative against animal and plant-derived sources due to economic reasons. Amid microorganisms, the Saccharomyces cerevisiae is a better alternative for industrial utilization but has low production of TAGs. Thus, to enhance the LD concentration, novel research was designed to induce alternate high and low sound frequency at a regular interval on a yeast model organism. The control and treated yeast samples further investigated using biochemical, biophysical, and computational tools to conclude that cells increase lipid droplet production under regulative noise exposure. The results endorsed that noise induces yeast LD yield is significantly higher than control, which could be considered a milestone in the biodiesel industry development and the biodiesel policy. This analysis also helps researchers to understand the novel function of LDs and their regulation in cell metabolism.

Synthesis of Steryl Hydroxycinnamates to Enhance Antioxidant Activity of Rapeseed Oil and Emulsions

In recent years, steryl esters have found potential applications in food, pharmaceutical and cosmetic industries. Therefore, three hydroxycinnamate steryl esters (HSEs): β-sitosteryl sinapate (β-SSA), β-sitosteryl caffeate (β-SCA), and β-sitosteryl ferulate (β-SFA) were synthesized by chemical approach and their antioxidant activity (AA) were analyzed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays. The values of inhibitory concentration (IC50) of each ester needed to inhibit 50% of the DPPH radical (IC50(DPPH) = 238.9, 78.3, 290.0 µmol/L for β-SSA, β-SCA, and β-SFA, respectively) and ABTS radical cation (IC50(ABTS) = 174.6, 106.7, 206.0 µmol/L for β-SSA, β-SCA, and β-SFA, respectively) were estimated and compared with antioxidant potential of phenolic acids. Moreover, the effect of HSEs addition in the concentrations range between 0.01% and 0.5% on the AA of refined rapeseed oil, mayonnaise and margarine was evaluated. Chemical structures of the synthesized HSEs and their concentrations strongly affect the AA of fat products. Oil and emulsions supplemented with higher concentrations of HSEs had significantly higher AA than control samples. Unfortunately, lower concentrations of HSEs (0.01% and 0.02%) did not increase the AA of fat products. However, steryl phenolates added in higher amounts can be considered as potential antioxidants delaying the oxidation processes of studied fats.

The surface of lipid droplets constitutes a barrier for endoplasmic reticulum residential integral membrane spanning proteins

Lipid droplets (LDs) are globular subcellular structures that mainly serve to store energy in form of neutral lipids, particularly triacylglycerols and steryl esters. LDs are closely associated with the membrane of the endoplasmic reticulum (ER), and are limited by a monolayer membrane of phospholipids harboring a specific set of proteins. Most of these proteins associate with LDs through either an amphipathic helix or a membrane-embedded hairpin motif. Here we address the question whether integral membrane spanning proteins could localize to the surface of LDs. To test this, we fused perilipin 3 (PLIN3), a mammalian LD-targeted protein, to ER resident proteins, such as Wbp1 (a N-glycosyl transferase complex subunit), Sec61 (a translocon subunit), and Pmt1 (a protein O-mannosyltransferase). The resulting fusion proteins localize to the periphery of LDs in both yeast and mammalian cells. This peripheral LD localization of the fusion proteins, however, is due to redistribution of the ER around LDs, as revealed by bimolecular fluorescence complementation between ER- and LD-localized partners in cells coexpressing the membrane-anchored perilipin. A LD-tethering function of PLIN3-containing membrane proteins was confirmed by fusing PLIN3 to the cytoplasmic domain of OM14, an outer mitochondrial membrane protein. Expression of OM14-PLIN3 resulted in close apposition of mitochondria and LDs. Taken together, these data indicate that the LD surface constitutes a barrier for ER-localized integral membrane spanning proteins.

Synchrotron multimodal imaging in a whole cell reveals lipid droplet core organization

A lipid droplet (LD) core of a cell consists mainly of neutral lipids, triacylglycerols and/or steryl esters (SEs). The structuration of these lipids inside the core is still under debate. Lipid segregation inside LDs has been observed but is sometimes suggested to be an artefact of LD isolation and chemical fixation. LD imaging in their native state and in unaltered cellular environments appears essential to overcome these possible technical pitfalls. Here, imaging techniques for ultrastructural study of native LDs in cellulo are provided and it is shown that LDs are organized structures. Cryo soft X-ray tomography and deep-ultraviolet (DUV) transmittance imaging are showing a partitioning of SEs at the periphery of the LD core. Furthermore, DUV transmittance and tryptophan/tyrosine auto-fluorescence imaging on living cells are combined to obtain complementary information on cell chemical contents. This multimodal approach paves the way for a new label-free organelle imaging technique in living cells.

Steryl Ester Formation and Accumulation in Steroid-Degrading Bacteria

ABSTRACT Steryl esters (SEs) are important storage compounds in many eukaryotes and are often prominent components of intracellular lipid droplets. Here, we demonstrate that selected Actino- and Proteobacteria growing on sterols are also able to synthesize SEs and to sequester them in cytoplasmic lipid droplets. We found cholesteryl ester (CE) formation in members of the actinobacterial genera Rhodococcus, Mycobacterium, and Amycolatopsis, as well as several members of the proteobacterial Cellvibrionales order. CEs maximally accumulated under nitrogen-limiting conditions, suggesting that steryl ester formation plays a crucial role for storing excess energy and carbon under adverse conditions. Rhodococcus jostii RHA1 was able to synthesize phytosteryl and cholesteryl esters, the latter reaching up to 7% of its cellular dry weight and 69% of its lipid droplets. Purified lipid droplets from RHA1 contained CEs, free cholesterol, and triacylglycerols. In addition, we found formation of CEs in Mycobacterium tuberculosis when it was grown with cholesterol plus an additional fatty acid substrate. This study provides a basis for the application of bacterial whole-cell systems in the biotechnological production of SEs for use in functional foods and cosmetics. IMPORTANCE Oleaginous bacteria exhibit great potential for the production of high-value neutral lipids, such as triacylglycerols and wax esters. This study describes the formation of steryl esters (SEs) as neutral lipid storage compounds in sterol-degrading oleaginous bacteria, providing a basis for biotechnological production of SEs using bacterial systems with potential applications in the functional food, nutraceutical, and cosmetic industries. We found cholesteryl ester (CE) formation in several sterol-degrading Actino- and Proteobacteria under nitrogen-limiting conditions, suggesting an important role of this process in storing energy and carbon under adverse conditions. In addition, Mycobacterium tuberculosis grown on cholesterol accumulated CEs in the presence of an additional fatty acid substrate.