Formation Of Cytosolic Lipid Droplets Research Articles

Cytosolic lipid droplets as engineered organelles for production and accumulation of terpenoid biomaterials in leaves

Cytosolic lipid droplets are endoplasmic reticulum-derived organelles typically found in seeds as reservoirs for physiological energy and carbon to fuel germination. Here, we report synthetic biology approaches to co-produce high-value sesqui- or diterpenoids together with lipid droplets in plant leaves. The formation of cytosolic lipid droplets is enhanced in the transient Nicotiana benthamiana system through ectopic production of WRINKLED1, a key regulator of plastid fatty acid biosynthesis, and a microalgal lipid droplet surface protein. Engineering of the pathways providing the universal C5-building blocks for terpenoids and installation of terpenoid biosynthetic pathways through direction of the enzymes to native and non-native compartments boost the production of target terpenoids. We show that anchoring of distinct biosynthetic steps onto the surface of lipid droplets leads to efficient production of terpenoid scaffolds and functionalized terpenoids. The co-produced lipid droplets “trap” the terpenoids in the cells.

Keeping the Heart Fitm2 during Chemotherapy.

Keeping the Heart Fitm2 during Chemotherapy.

Metabolic transition of milk triacylglycerol synthesis in response to varying levels of palmitate in porcine mammary epithelial cells

BackgroundMilk in mammals is a key source of lipids for offspring, providing both critical energy and essential fatty acids. For lactating sows, palmitic acid is one of the most abundant fatty acids in milk, providing 10~12% of the suckling pig total dietary energy supply. However, the effects of exogenous palmitic acid on milk fat synthesis in sow mammary glands are not well-known. In this study, we investigated the effects of palmitic acid on lipogenic genes in porcine mammary epithelial cells (pMECs) to explore the role of exogenous palmitic acid in mediating milk triacylglycerols (TAG) synthesis.MethodsPorcine mammary epithelial cells were cultured for 24 h in the presence of different concentrations of palmitate (0, 25, 50, 100, 200, 400, and 600 μM). The effect of palmitate on cell viability was tested via MTT assay. Intracellular lipid accumulation was measured through Oil Red O staining, and TAG levels were quantified by enzymatic colorimetric methods. Expression of genes and proteins involved in milk fat biosynthesis were assayed with quantitative real-time polymerase chain reaction (qPCR) and Western blotting, respectively.ResultsIncubation with palmitate promoted cellular lipid synthesis in a dose-dependent manner, as reflected by the increased TAG content and enhanced formation of cytosolic lipid droplets. The increased lipid synthesis by palmitate was probably attributable to the upregulated mRNA expression of genes associated with milk fat biosynthesis, including long-chain fatty acid uptake (LPL, CD36), intracellular activation and transport (ACSL3, FABP3), TAG synthesis (GPAM, AGPAT6, DGAT1), lipid droplet formation (PLIN2), and regulation of transcription (PPARγ). Western blot analysis of CD36 and DGAT1 proteins confirmed the increased lipid synthesis with increasing incubation of palmitate. However, the genes involved in fatty acid de novo synthesis (ACACA, FASN), fatty acid desaturation (SCD), and regulation of transcription (SREBP1, INSIG1) were inversely affected by incubation with increasing concentrations of palmitate. Western blot analysis of ACACA protein confirmed this decrease associated with increasing levels of palmitate.ConclusionsResults from this study suggest that palmitate stimulated the cytosolic TAG accumulation in pMECs, probably by promoting lipogenic genes and proteins that are involved in lipid synthesis. However, addition of palmitate decreased the fatty acid de novo synthesis in pMECs.

Acute loss of iron–sulfur clusters results in metabolic reprogramming and generation of lipid droplets in mammalian cells

Iron-sulfur (Fe-S) clusters are ancient cofactors in cells and participate in diverse biochemical functions, including electron transfer and enzymatic catalysis. Although cell lines derived from individuals carrying mutations in the Fe-S cluster biogenesis pathway or siRNA-mediated knockdown of the Fe-S assembly components provide excellent models for investigating Fe-S cluster formation in mammalian cells, these experimental strategies focus on the consequences of prolonged impairment of Fe-S assembly. Here, we constructed and expressed dominant-negative variants of the primary Fe-S biogenesis scaffold protein iron-sulfur cluster assembly enzyme 2 (ISCU2) in human HEK293 cells. This approach enabled us to study the early metabolic reprogramming associated with loss of Fe-S-containing proteins in several major cellular compartments. Using multiple metabolomics platforms, we observed a ∼12-fold increase in intracellular citrate content in Fe-S-deficient cells, a surge that was due to loss of aconitase activity. The excess citrate was generated from glucose-derived acetyl-CoA, and global analysis of cellular lipids revealed that fatty acid biosynthesis increased markedly relative to cellular proliferation rates in Fe-S-deficient cells. We also observed intracellular lipid droplet accumulation in both acutely Fe-S-deficient cells and iron-starved cells. We conclude that deficient Fe-S biogenesis and acute iron deficiency rapidly increase cellular citrate concentrations, leading to fatty acid synthesis and cytosolic lipid droplet formation. Our findings uncover a potential cause of cellular steatosis in nonadipose tissues.

The Epigenetic Drug 5-Azacytidine Interferes with Cholesterol and Lipid Metabolism

DNA methylation and histone acetylation inhibitors are widely used to study the role of epigenetic marks in the regulation of gene expression. In addition, several of these molecules are being tested in clinical trials or already in use in the clinic. Antimetabolites, such as the DNA-hypomethylating agent 5-azacytidine (5-AzaC), have been shown to lower malignant progression to acute myeloid leukemia and to prolong survival in patients with myelodysplastic syndromes. Here we examined the effects of DNA methylation inhibitors on the expression of lipid biosynthetic and uptake genes. Our data demonstrate that, independently of DNA methylation, 5-AzaC selectively and very potently reduces expression of key genes involved in cholesterol and lipid metabolism (e.g. PCSK9, HMGCR, and FASN) in all tested cell lines and in vivo in mouse liver. Treatment with 5-AzaC disturbed subcellular cholesterol homeostasis, thereby impeding activation of sterol regulatory element-binding proteins (key regulators of lipid metabolism). Through inhibition of UMP synthase, 5-AzaC also strongly induced expression of 1-acylglycerol-3-phosphate O-acyltransferase 9 (AGPAT9) and promoted triacylglycerol synthesis and cytosolic lipid droplet formation. Remarkably, complete reversal was obtained by the co-addition of either UMP or cytidine. Therefore, this study provides the first evidence that inhibition of the de novo pyrimidine synthesis by 5-AzaC disturbs cholesterol and lipid homeostasis, probably through the glycerolipid biosynthesis pathway, which may contribute mechanistically to its beneficial cytostatic properties.

The yeast lipin orthologue Pah1p is important for biogenesis of lipid droplets

Lipins are phosphatidate phosphatases that generate diacylglycerol (DAG). In this study, we report that yeast lipin, Pah1p, controls the formation of cytosolic lipid droplets. Disruption of PAH1 resulted in a 63% decrease in droplet number, although total neutral lipid levels did not change. This was accompanied by an accumulation of neutral lipids in the endoplasmic reticulum (ER). The droplet biogenesis defect was not a result of alterations in neutral lipid ratios. No droplets were visible in the absence of both PAH1 and steryl acyltransferases when grown in glucose medium, even though the strain produces as much triacylglycerol as wild type. The requirement of PAH1 for normal droplet formation can be bypassed by a knockout of DGK1. Nem1p, the activator of Pah1p, localizes to a single punctum per cell on the ER that is usually next to a droplet, suggesting that it is a site of droplet assembly. Overall, this study provides strong evidence that DAG generated by Pah1p is important for droplet biogenesis.

Triacylglycerol Biosynthesis: Another Cellular Lipid Pathway Essential to HCV Replication

Evaluation of: Herker E, Harris C, Hernandez C et al.: Efficient hepatitis C virus particle formation requires diacyglycerol acyltransferase-1. Nat. Med. 16, 1295–1298 (2010). Two diacylglycerol acyltransferase (DGAT) enzymes catalyze the final step of triglyceride synthesis, with structural, topological and physiological differences. They also contribute to the formation of cytosolic lipid droplets, which have been shown to play an essential role in HCV replication. Selective inhibition or silencing of DGAT1, but not of DGAT2, dramatically impaired the production and secretion of infectious virus, while leaving HCV RNA replication unaffected. An interaction between DGAT1 and core protein was demonstrated. The interaction between the two proteins appears to be independent of the catalytic activity of the enzyme, while its inhibition decreases the localization of core and HCV RNA onto the surface of lipid droplets. This study not only provides another piece of evidence of the pivotal role of lipids and lipid droplets in the viral life cycle, but also adds triglyceride synthesis to the cellular lipid pathways that are essential to the production of infectious viral particles.

Cholesterol rich lipid raft microdomains are gateway for acute phase protein, SERPINA1

Cholesterol is the most abundant lipid component of the plasma membrane, and thus the equilibrium between free cholesterol and raft cholesterol act as a determinant of raft function and cell signalling. The mechanisms that regulate the lipid raft cholesterol levels are largely unknown. Here we demonstrate that SERPINA1 (α1-antitrypsin), an acute phase protein and the classical neutrophil elastase inhibitor, is localized within lipid rafts in primary human monocytes in vitro. SERPINA1 association with monocytes is inhibited by cholesterol depleting/efflux-stimulating agents (nystatin, filipin, MβCD (methyl-beta-cyclodextrin) and oxidized low-density lipoprotein (oxLDL) and conversely, enhanced by free cholesterol. Furthermore, SERPINA1/monocyte association per se depletes lipid raft cholesterol as characterized by the activation of extracellular signal-regulated kinase 2, formation of cytosolic lipid droplets, and a complete inhibition of oxLDL uptake by monocytes. Our findings for the first time highlight that the entry and cell association of SERPINA1 is dependent on lipid raft cholesterol and that SERPINA1 depletes lipid raft cholesterol.

Hypoxia Converts Human Macrophages Into Triglyceride-Loaded Foam Cells

Atherosclerotic lesions have regions that are hypoxic. Because the lesion contains macrophages that are loaded with lipid, we investigated whether hypoxia can influence the accumulation of lipids in these cells. Exposure of human macrophages to hypoxia for 24 hours resulted in an increased formation of cytosolic lipid droplets and an increased accumulation of triglycerides. Exposure of the macrophages to oxidized low-density lipoprotein (oxLDL) increased the accumulation of cytosolic lipid droplets because of an increase in cellular cholesterol esters. The accumulation of lipid droplets in oxLDL-treated cells was further increased after hypoxia, caused by an increased level of triglycerides. Expression analyses combined with immunoblot or RT-PCR demonstrated that hypoxia increased the expression of several genes that could promote the accumulation of lipid droplets. Hypoxia increased the mRNA and protein levels of adipocyte differentiation-related protein (ADRP). It is well known that an increased expression of ADRP increases the formation of lipid droplets. Hypoxia decreased the expression of enzymes involved in beta-oxidation (acyl-coenzyme A synthetase and acyl-coenzyme A dehydrogenase) and increased the expression of stearoyl-coenzyme A desaturase, an important enzyme in the fatty acid biosynthesis. Moreover, exposure to hypoxia decreased the rate of beta-oxidation, whereas the accumulation of triglycerides increased. The results demonstrate that exposure of human macrophages to hypoxia causes an accumulation of triglyceride-containing cytosolic lipid droplets. This indicates that the hypoxia present in atherosclerotic lesions can contribute to the formation of the lipid-loaded macrophages that characterize the lesion and to the accumulation of triglycerides in such lesions.

Regulating Lipid Storage

The accumulation of intracellular lipid stores is an essential process, but excess storage is also associated with metabolic diseases such as insulin resistance. Furthermore, lipid-loaded macrophages, called foam cells, are associated with atherosclerosis. Andersson et al . show that insulin signals through phospholipase D1 (PLD1) and extracellular signal-regulated kinase 2 (ERK2) to stimulate lipid droplet formation in NIH 3T3 cells. Even in the absence of stimuli, overexpression of PLD1 but not PLD2, or ERK2 but not ERK1, promoted the formation of lipid droplets. RNAi technology demonstrated that reduction in PLD1 or ERK2 inhibited lipid droplet formation, and short-interfering RNA (siRNA) against ERK2 prevented the increase in lipid droplets caused by overexpression of PLD1, suggesting that ERK2 is downstream of PLD1. However, the abundance of PLD1 did not alter the abundance of phosphorylated (and presumably active) ERK2. Thus, the exact relationship between PLD1 and ERK2 remains to be determined. The insulin-mediated increase in lipid storage was blocked by siRNA against PLD1 or by the presence of an ERK2 inhibitor (Ste-Mek1 13 ). Analysis of the phosphorylated proteins in cell homogenates exposed to active ERK2 showed that the microtubule motor dynein was phosphorylated. Microtubules have been implicated in fusion of lipid particles that bud from the microsomal membranes, which is the basis for the formation of large lipid storage droplets. Phosphorylated dynein was isolated from lipid droplets, and in vitro the association of dynein with lipid droplets was increased when the cell lysates were exposed to active ERK2. Inhibition of dynein by microinjection of an antibody against dynein intermediate chain prevented lipid droplet formation by inhibiting fusion. Thus, these results provide evidence for an ERK2-specific function in lipid storage. L. Andersson, P. Boström, J. Ericson, M. Rutberg, B. Magnusson, D. Marchesan, M. Ruiz, L. Asp, P. Huang, M. A. Frohman, J. Borén, S.-O. Olofsson, PLD1 and ERK2 regulated cytosolic lipid droplet formation. J. Cell Sci. 119 , 2246-2257 (2006). [Abstract] [Full Text]

PLD1 and ERK2 regulate cytosolic lipid droplet formation

We have previously uncovered roles for phospholipase D (PLD) and an unknown cytosolic protein in the formation of cytosolic lipid droplets using a cell-free system. In this report, PLD1 has been identified as the relevant isoform, and extracellular signal-regulated kinase 2 (ERK2) as the cytosolic protein. Increased expression of PLD1 increased lipid droplet formation whereas knockdown of PLD1 using siRNA was inhibitory. A role for ERK2 in basal lipid droplet formation was revealed by overexpression or microinjection, and ablation by siRNA knockdown or pharmacological inhibition. Similar manipulations of other Map kinases such as ERK1, JNK1 or JNK2 and p38alpha or p38beta were without effect. Insulin stimulated the formation of lipid droplets and this stimulation was inhibited by knockdown of PLD1 (by siRNA) and by inhibition or knockdown (by siRNA) of ERK2. Inhibition of ERK2 eliminated the effect of PLD1 on lipid droplet formation without affecting PLD1 activity, suggesting that PLD1 functions upstream of ERK2. ERK2 increased the phosphorylation of dynein which increased the amount of the protein on ADRP-containing lipid droplets. Microinjection of antibodies to dynein strongly inhibited the formation of lipid droplets, demonstrating that dynein has a central role in this formation. Thus dynein is a possible target for ERK2.

Epigallocatechin gallate increases the formation of cytosolic lipid droplets and decreases the secretion of apoB-100 VLDL

Epigallocatechin gallate (EGCG) increases the formation of cytosolic lipid droplets by a mechanism that is independent of the rate of triglyceride biosynthesis and involves an enhanced fusion between lipid droplets, a process that is crucial for their growth in size. EGCG treatment reduced the secretion of both triglycerides and apolipoprotein B-100 (apoB-100) VLDLs but not of transferrin, albumin, or total proteins, indicating that EGCG diverts triglycerides from VLDL assembly to storage in the cytosol. This is further supported by the observed increase in both intracellular degradation of apoB-100 and ubiquitination of the protein (indicative of increased proteasomal degradation) in EGCG-treated cells. EGCG did not interfere with the microsomal triglyceride transfer protein, and the effect of EGCG on the secretion of VLDLs was found to be independent of the LDL receptor. Thus, our results indicate that EGCG promotes the accumulation of triglycerides in cytosolic lipid droplets, thereby diverting lipids from the assembly of VLDL to storage in the cytosol. Our results also indicate that the accumulation of lipids in the cytosol is not always associated with increased secretion of VLDL.

RNA Interference–Mediated Choline Kinase Suppression in Breast Cancer Cells Induces Differentiation and Reduces Proliferation

Choline kinase is overexpressed in breast cancer cells and activated by oncogenes and mitogenic signals, making it a potential target for cancer therapy. Here, we have examined, for the first time, the effects of RNA interference (RNAi)-mediated down-regulation of choline kinase in nonmalignant and malignant human breast epithelial cell lines using magnetic resonance spectroscopy (MRS) as well as molecular analyses of proliferation and differentiation markers. RNAi knockdown of choline kinase reduced proliferation, as detected by proliferating cell nuclear antigen and Ki-67 expression, and promoted differentiation, as detected by cytosolic lipid droplet formation and expression of galectin-3. The functional importance of RNAi-mediated choline kinase down-regulation on choline phospholipid metabolism was confirmed by the significant reduction of phosphocholine detected by MRS. These results strongly support the targeting of choline kinase in breast cancer cells with RNAi and show the potential ability of noninvasive MRS to detect and evaluate future treatments incorporating such strategies.

Complete inhibition of mouse macrophage-derived foam cell formation by triacsin C.

Primary mouse peritoneal macrophages effectively take up and metabolize phosphatidylcholine/cholesterol liposomes containing a small amount of phosphatidylserine, which results in the massive accumulation in the cytoplasm of oil red O positive lipid droplets consisting of cholesteryl ester (CE) and triacylglycerol (TG) [Nishikawa et al. (1990) J. Biol. Chem. 265, 5226-5231]. A number of inhibitors of CE synthesis have been reported, but their effects on the lipid droplet formation have not been fully examined. Furthermore, the contribution of TG synthesis to lipid droplet formation has been poorly investigated. We have investigated the relationship between CE and TG syntheses and cytosolic lipid droplet formation in macrophages cultured in the presence of inhibitors with different modes of action. When macrophages were cultured with liposomes and [14C]oleic acid in the presence of triacsin C, a potent inhibitor of long chain acyl-CoA synthetase, both [14C]CE and [14C]TG syntheses were inhibited to similar extents with IC50 values of 0.19 and 0.10 microM, respectively. On the other hand, pregnenolone, a well-known inhibitor of cellular cholesterol transport, and CL-283,546, a potent inhibitor of acyl-CoA:cholesterol acyltransferase, inhibited [14C]CE synthesis specifically with IC50 values of 5.0 and 0.038 microM, respectively. Microscopic observation revealed that the inhibitors of cholesterol metabolism produce only a partial decrease in cytosolic lipid droplets even at the highest doses which cause almost complete inhibition of [14C]CE synthesis. However, the triacsin C-dose dependent inhibition of lipid droplet formation was almost complete at 0.59 microM, a concentration that inhibits [14C]CE and [14C]TG syntheses by about 90%. These results show that inhibiton of acyl-CoA synthetase by triacsin C causes a decrease in the cellular levels of acyl-CoA, the common substrate for CE and TG syntheses, leading to an inhibiton of neutral lipid synthesis and eventually to the complete disappearance of cytosolic lipid droplets from macrophages. These findings suggest that TG synthesis, as well as CE synthesis, is responsible for macrophage-derived foam cell formation, and is therefore a potential target for new antiatherosclerotic agents.