Lipid Droplet Formation In Cells Research Articles

ARID3A plays a key regulatory role in palmitic acid-stimulated milk fat synthesis in mouse mammary epithelial cells.

Palmitic acid (PA) can stimulate milk fat synthesis in mammary gland, but the specific mechanism is still unclear. In our research, we aim to explore the role and corresponding mechanism of AT-rich interaction domain 3A (ARID3A) in milk fat synthesis stimulated by PA. We found that ARID3A protein level in mouse mammary gland tissues during lactation was much higher than that during puberty and involution. ARID3A knockdown and gene activation showed that ARID3A stimulated the synthesis of triglycerides and cholesterol in HC11 cells, secretion of free fatty acids from cells and lipid droplet formation in cells. ARID3A also promoted the expression and maturation of SREBP1 in HC11 cells. PA stimulated ARID3A protein expression and SREBP1 expression and maturation in a dose-dependent manner, and the PI3K specific inhibitor LY294002 blocked the stimulation of PA on ARID3A expression. ARID3A knockdown blocked the stimulation of PA on SREBP1 protein expression and maturation. We further showed that ARID3A was localized in the nucleus and PA stimulated this localization, and ARID3A knockdown blocked the stimulation of PA on the mRNA expression of SREBP1. To sum up, our data reveal that ARID3A is a key mediator for PA to promote SREBP1 mRNA expression and stimulate milk fat synthesis in mammary epithelial cells.

Daidzein stimulates fatty acid-induced fat deposition in C2C12 myoblast cells via the G protein-coupled receptor 30 pathway

Fat deposition in skeletal muscle is an important aspect of improving meat quality. Isoflavones can promote animal anabolism, but whether and how they regulate muscle fat deposition remain largely unclear. In this study, we explored the role and corresponding molecular mechanism of one of the major isoflavones, daidzein, in fat deposition in C2C12 myoblast cells. In the absence of fatty acids (FAs), daidzein did not promote triglyceride synthesis and lipid droplet formation in cells but increased sterol regulatory element-binding protein 1c (SREBP-1c) expression and maturation. In the presence of FAs, daidzein enhanced FAs-induced fat deposition and the SREBP-1c signaling. Daidzein promoted FAs-induced nuclear factor κB1 (NFκB1) phosphorylation and activated the SREBP-1c signaling in a PI3K-dependent manner. G protein-coupled receptor 30 (GPR30) knockdown but not estrogen receptor α (ERα) knockdown blocked the stimulation of daidzein on the PI3K-NFκB1-SREBP-1c signaling pathway, while both knockdown did not affect the stimulation of FAs on this signaling. qRT-PCR and ChIP-qPCR further detected that daidzein stimulated NFκB1-targeted SREBP-1c transcription. Daidzein did not affect ERα expression in cells, but it stimulated GPR30 expression and cytoplasmic localization. These results reveal that daidzein promotes FAs-induced fat deposition through the GPR30 signaling in C2C12 myoblast cells.

TNF-α induces acyl-CoA synthetase 3 to promote lipid droplet formation in human endothelial cells

Chronic inflammation contributes to cardiovascular disease. Increased levels of the inflammatory cytokine, TNF-α, are often present in conditions associated with cardiovascular disease risk, and TNF-α induces a number of pro-atherogenic effects in macrovascular endothelial cells, including expression of adhesion molecules and chemokines, and lipoprotein uptake and transcytosis to the subendothelial tissue. However, little is known about the roles of acyl-CoA synthetases (ACSLs), enzymes that esterify free fatty acids into their acyl-CoA derivatives, or about the effects of TNF-α on ACSLs in endothelial cells. Therefore, we investigated the effects of TNF-α on ACSLs and downstream lipids in cultured human coronary artery endothelial cells and human umbilical vein endothelial cells. We demonstrated that TNF-α induces ACSL1, ACSL3, and ACSL5, but not ACSL4, in both cell types. TNF-α also increased oleoyl-CoA levels, consistent with the increased ACSL3 expression. RNA-sequencing demonstrated that knockdown of ACSL3 had no marked effects on the TNF-α transcriptome. Instead, ACSL3 was required for TNF-α-induced lipid droplet formation in cells exposed to oleic acid. These results demonstrate that increased acyl-CoA synthesis as a result of ACSL3 induction is part of the TNF-α response in human macrovascular endothelial cells.

MiRNA-7b-5p attenuates the progression of osteoporosis by inhibiting adipose differentiation of hMSCs via regulating IRS2.

To elucidate whether microRNA-7b-5p (miRNA-7b-5p) could inhibit adipose differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) through regulating IRS2, thereby alleviating the progression of osteoporosis. Expression levels of miRNA-7b-5p and IRS2 in hMSCs at different stages of adipogenic differentiation and osteogenic differentiation were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot. After transfection of miRNA-7b-5p mimic or pcDNA-IRS2 in hMSCs, lipid droplet formation in cells was observed by oil red O staining. Expressions of C/EBPα and PPARγ were detected by qRT-PCR and Western blot. The potential target gene of miRNA-7b-5p was predicted by bioinformatics and verified by dual-luciferase reporter gene assay. Finally, expressions of IRS2 in hMSCs transfected with miRNA-7b-5p-NC, miRNA-7b-5p mimic or co-transfected with miRNA-7b-5p mimic and pcDNA-IRS2 were examined. Expressions of miRNA-7b-5p and IRS2 gradually decreased with the prolongation of adipogenic differentiation, but increased during osteogenic differentiation of hMSCs. Transfection of miRNA-7b-5p mimic reduced oil red O staining after adipogenic differentiation and downregulated mRNA and protein levels of C/EBPα and PPARγ. Transfection of pcDNA-IRS2 increased oil red O staining after osteogenic differentiation and upregulated mRNA and protein levels of C/EBPα and PPARγ. Dual-luciferase reporter gene results showed that miRNA-7b-5p could bind to IRS2. Overexpression of IRS2 reversed the downregulated mRNA and protein levels of adipogenic-related genes C/EBPα and PPARγ due to the overexpression of miRNA-7b-5p. MiRNA-7b-5p inhibits the adipogenic differentiation of hMSCs through IRS2, thus alleviating the development of osteoporosis.

Polybasic RKKR motif in the linker region of lipid droplet (LD)–associated protein CIDEC inhibits LD fusion activity by interacting with acidic phospholipids

Lipid droplets (LDs) are intracellular organelles and a central site for lipid synthesis, storage, and mobilization. The size of LDs reflects the dynamic regulation of lipid metabolism in cells. Previously, we found that cell death-inducing DFFA-like effector C (CIDEC) mediates LD fusion and growth by lipid transfer through LD-LD contact sites in adipocytes and hepatocytes. The CIDE-N domains of CIDEC molecules form homodimers, whereas the CIDE-C domain plays an important role in LD targeting and enrichment. Here, using targeted protein deletions and GFP expression coupled with fluorescence microscopy, we identified a polybasic RKKR motif in the linker region that connects the CIDE-N and CIDE-C domains of CIDEC and functions as a regulatory motif for LD fusion. We found that deletion of the linker region or mutation of the RKKR motif increases the formation of supersized LDs compared with LD formation in cells with WT CIDEC. This enhanced LD fusion activity required the interaction between CIDE-N domains. Mechanistically, we found that the RKKR motif interacts with acidic phospholipids via electrostatic attraction. Loss of this motif disrupted the protein-lipid interaction, resulting in enhanced lipid droplet fusion activity and thus formation of larger LDs. In summary, we have uncovered a CIDEC domain that regulates LD fusion activity, a finding that provides insights into the inhibitory regulation of LD fusion through CIDEC-lipid interactions.

Knocking down clock control gene CRY1 decreases adipogenesis via canonical Wnt/β-catenin signaling pathway

Cryptochrome gene 1(CRY1) is a member of circadian clock genes, which play an important role in adipocyte biology. CRY1 was reported to be related with the lipid metabolism, but the molecule mechanism of CRY1 in regulating the adipogenesis remains unclear. Here we report that CRY1 is a key regulator in adipogenic differentiation. We found that the expression levels of CRY1 in 3T3-L1 cells and C3H10T1/2 cells gradually increased during the process of adipogenic differentiation. Knockdown of endogenous CRY1 significantly inhibited the expression of adipogenic markers and lipid droplet formation in cells under adipogenic induction. In addition, knockdown of endogenous CRY1 promoted the expression and nuclear accumulation of β-catenin, the critical signal molecular in the canonical canonical Wnt signaling pathway, suggesting the regulation effect of CRY1 in adipogenesis was mediated by canonical Wnt/β-catenin signaling. Taken together, our study suggests that CRY1 regulates adipogenic differentiation through modulating the canonical Wnt/β-catenin signaling pathway.

Abstract 5170: The effect of vitamin K on aggressiveness, lipid metabolism and gene expression in triple-negative breast cancer cells

Abstract Vitamin K is an essential cofactor in the γ-carboxylation of glutamate (Glu) to γ-carboxyglutamate (Gla), a post-translational modification mediated by the gamma-glutamyl carboxylase enzyme (GGCX) and vitamin K oxidoreductases (VKORC1 or VCORC1L1). All three of these genes are expressed in most breast cancer cell lines and The Cancer Genome Atlas (TCGA) data indicate that upregulation of these genes is associated with reduced survival in breast cancer patients. Since vitamin K is not typically present in cell culture media, γ-carboxylation is limited in vitro despite the presence of GGCX and VKORs. To determine the functional significance of the vitamin K pathway in breast cancer cells, we assessed the effect of vitamin K treatment on cell survival, migration and mammosphere formation. SUM159 and Hs578T cells were maintained in vitamin K1 (5 ug/mL) for at least 3 passages for all assays. Cells passaged with vitamin K produced γ-carboxylated proteins and exhibited increased cell survival after 72h, increased mammosphere formation after 8 days and increased cell migration after 48h compared to cells passaged in ethanol vehicle. Inhibition of VKORC1 with 2 uM warfarin decreased the production of γ-carboxylated proteins and reduced the effects of vitamin K on cell survival and migration but not on mammosphere formation. Microarray profiling and pathway analysis indicated upregulation of several known oncogenic pathways, including PI3K-AKT, VEGF-VEGFR, RANK-RANKL and JAK/STAT in vitamin K-treated SUM159 and Hs578T cells. Thus, γ-carboxylation of proteins driven by vitamin K might indirectly activate these pathways to enhance cell survival, cell migration and mammosphere formation. Additional pathways upregulated in vitamin K-treated cells included adipogenesis, fatty acid biosynthesis and lipid metabolism. We therefore examined lipid droplet formation in cells exposed to vitamin K using Bodipy assays. We detected an increase in lipid droplet formation after 72 hours of vitamin K treatment, which was inhibited by warfarin. Since aggressive breast cancers are often associated with accumulation of cytoplasmic lipid droplets, further studies on the mechanism by which vitamin K increases lipid accumulation are warranted. In conclusion, our data indicate that vitamin K enhances γ-carboxylation, breast cancer aggressiveness and lipid metabolism, suggesting that blocking the vitamin K pathway might be of therapeutic value for patients whose tumors express GGCX and VKORs. Citation Format: Leila Kokabee, Sarah Beaudin, JoEllen Welsh. The effect of vitamin K on aggressiveness, lipid metabolism and gene expression in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5170.

Lipid Droplet Formation in Cells of the Filamentous Green Alga Klebsormidium nitens as Revealed by BODIOY-DiOC6 and BODIPY-Nile Red Double-Staining Microscopy

Lipid Droplet Formation in Cells of the Filamentous Green Alga Klebsormidium nitens as Revealed by BODIOY-DiOC6 and BODIPY-Nile Red Double-Staining Microscopy

Expression of oleosin and perilipins in yeast promote formation of lipid droplets from the endoplasmatic reticulum

Most cells store neutral lipids in a dedicated compartment, the lipid droplet (LD). These LDs are structurally and functionally conserved across species. In higher eukaryotes, LDs are covered by abundant scaffolding proteins, such as the oleosins in plants and perilipins (PLINs) in animal cells. Saccharomyces cerevisiae, however, has no homologues of these scaffolding proteins. To analyze a possible function of these proteins in the biogenesis of LDs, oleosin and perilipin family members (PLIN1, ADRP/PLIN2 and TIP47/PLIN3) were expressed in yeast cells and their targeting to LDs, membrane association and function in neutral lipid homeostasis and LD biogenesis were analyzed. When expressed in wild-type cells, these proteins were properly targeted to LDs. However, when expressed in cells lacking LDs, oleosin was localized to the ER bilayer and was rapidly degraded. PLINs, on the other hand, did not localize to the ER membrane in the absence of LDs and lost their membrane association. Photobleaching experiments revealed that PLIN2 and PLIN3 rapidly exchanged their LD association, but PLINs did not move as quickly as integral membrane proteins, such as oleosin, over the LD surface. Interestingly, expression of these scaffolding LD proteins in mutant cells containing elevated levels of neutral lipids within the ER bilayer resulted in the formation of LDs. These results suggest that these LD scaffolding proteins promote the sequestration of neutral lipids from the ER bilayer and thereby induce LD formation. Consistent with this proposition, addition of a cell-permeable diacylglycerol (DAG) was sufficient to promote LD formation in cells expressing the LD scaffolding proteins but lacking the capacity to synthesize storage lipids.

Group IVA Phospholipase A2 Is Necessary for the Biogenesis of Lipid Droplets

Lipid droplets (LD) are organelles present in all cell types, consisting of a hydrophobic core of triacylglycerols and cholesteryl esters, surrounded by a monolayer of phospholipids and cholesterol. This work shows that LD biogenesis induced by serum, by long-chain fatty acids, or the combination of both in CHO-K1 cells was prevented by phospholipase A(2) inhibitors with a pharmacological profile consistent with the implication of group IVA cytosolic phospholipase A(2) (cPLA(2)alpha). Knocking down cPLA(2)alpha expression with short interfering RNA was similar to pharmacological inhibition in terms of enzyme activity and LD biogenesis. A Chinese hamster ovary cell clone stably expressing an enhanced green fluorescent protein-cPLA(2)alpha fusion protein (EGFP-cPLA(2)) displayed higher LD occurrence under basal conditions and upon LD induction. Induction of LD took place with concurrent phosphorylation of cPLA(2)alpha at Ser(505). Transfection of a S505A mutant cPLA(2)alpha showed that phosphorylation at Ser(505) is key for enzyme activity and LD formation. cPLA(2)alpha contribution to LD biogenesis was not because of the generation of arachidonic acid, nor was it related to neutral lipid synthesis. cPLA(2)alpha inhibition in cells induced to form LD resulted in the appearance of tubulo-vesicular profiles of the smooth endoplasmic reticulum, compatible with a role of cPLA(2)alpha in the formation of nascent LD from the endoplasmic reticulum.

Effects of metallic ion toxicity on human gingival fibroblasts morphology.

Alloys used as implant materials release metal ions to surrounding tissues. Cytotoxic substances attack at the molecular level, and these effects are reflected in the structure of the cells and organelles. The objective of this study was to evaluate the cellular morphology and ultrastructural changes of cultured human gingival fibroblasts to salt solutions of ions (beryllium (Be+2), chromium (Cr+6 and Cr+3), nickel (Ni+2), molybdenum (Mo+6)) which may be released from nickel-chromium dental alloys. The concentrations chosen were based on previously conducted cell culture studies. Fibroblasts were exposed to the different ion concentrations for 24 or 72 h. Cellular morphology and ultrastructural features were examined using scanning electron microscopy and transmission electron microscopy. Ultrastructural alterations observed included irregular shaped nuclei for cells exposed to hexavalent chromium and nickel, pseudopodia for cells exposed to beryllium and molybdenum, and lipid droplet formation in cells exposed to nickel.

Flow cytometric analysis of lipid droplet formation in cells of the human monocytic cell line, U937

Active uptake of a free fatty acid, oleate, and its incorporation into triacylglycerol and phospholipid in the human monocytic cell line, U937, was identified using 14C-labelled oleate. Excess triacylglycerol accumulates in the form of lipid droplets in the cytoplasm. The extent of lipid droplet formation in each cell can be assessed in the absence of any staining by 90 degrees light scatter intensity, one of the basic parameters of flow cytometry.