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The FEBS Journal Sep 2012The two diacylglycerol acyltransferases, DGAT1 and DGAT2, are known to have non-redundant functions, in spite of catalysing the same reaction and being present in the...
The two diacylglycerol acyltransferases, DGAT1 and DGAT2, are known to have non-redundant functions, in spite of catalysing the same reaction and being present in the same cell types. The basis for this distinctiveness, which is reflected in the very different phenotypes of Dgat1(-/-) and Dgat2(-/-) mice, has not been resolved. Using selective inhibitors of human DGAT1 and DGAT2 on HepG2 cells and gene silencing, we show that, although DGAT2 activity accounts for a modest fraction (< 20%) of overall cellular DGAT activity, inhibition of DGAT2 activity specifically inhibits (and is rate-limiting for) the incorporation of de novo synthesized fatty acids and of glycerol into cellular and secreted triglyceride to a much greater extent than it affects the incorporation of exogenously added oleate. By contrast, inhibition of DGAT1 affects equally the incorporation of glycerol and exogenous (preformed) oleate into cellular and secreted triacylglycerol (TAG). These data indicate that DGAT2 acts upstream of DGAT1, largely determines the rate of de novo synthesis of triglyceride, and uses nascent diacylglycerol and de novo synthesized fatty acids as substrates. By contrast, the data suggest that DGAT1 functions in the re-esterification of partial glycerides generated by intracellular lipolysis, using preformed (exogenous) fatty acids. Therefore, we describe distinct but synergistic roles of the two DGATs in an integrated pathway of TAG synthesis and secretion, with DGAT2 acting upstream of DGAT1.
Topics: Cell Line, Tumor; Diacylglycerol O-Acyltransferase; Diglycerides; Fatty Acids; Gene Silencing; Humans; Kinetics; Triglycerides
PubMed: 22748069
DOI: 10.1111/j.1742-4658.2012.08684.x -
BMC Biology Aug 2020When stressed, eukaryotic cells produce triacylglycerol (TAG) to store nutrients and mobilize autophagy to combat internal damage. We and others previously reported that...
BACKGROUND
When stressed, eukaryotic cells produce triacylglycerol (TAG) to store nutrients and mobilize autophagy to combat internal damage. We and others previously reported that in yeast, elimination of TAG synthesizing enzymes inhibits autophagy under nitrogen starvation, yet the underlying mechanism has remained elusive.
RESULTS
Here, we show that disruption of TAG synthesis led to diacylglycerol (DAG) accumulation and its relocation from the vacuolar membrane to the endoplasmic reticulum (ER). We further show that, beyond autophagy, ER-accumulated DAG caused severe defects in the endomembrane system, including disturbing the balance of ER-Golgi protein trafficking, manifesting in bulging of ER and loss of the Golgi apparatus. Genetic or chemical manipulations that increase consumption or decrease supply of DAG reversed these defects. In contrast, increased amounts of precursors of glycerolipid synthesis, including phosphatidic acid and free fatty acids, did not replicate the effects of excess DAG. We also provide evidence that the observed endomembrane defects do not rely on Golgi-produced DAG, Pkc1 signaling, or the unfolded protein response.
CONCLUSIONS
This work identifies DAG as the critical lipid molecule responsible for autophagy inhibition under condition of defective TAG synthesis and demonstrates the disruption of ER and Golgi function by excess DAG as the potential cause of the autophagy defect.
Topics: Autophagy; Cell Membrane; Diglycerides; Endoplasmic Reticulum; Golgi Apparatus; Homeostasis; Protein Transport; Saccharomyces cerevisiae
PubMed: 32859196
DOI: 10.1186/s12915-020-00837-w -
EMBO Reports Jul 2011Protein kinase D (PKD) mediates the actions of stimuli that promote diacylglycerol (DAG) biogenesis. By phosphorylating effectors that regulate transcription, fission... (Review)
Review
Protein kinase D (PKD) mediates the actions of stimuli that promote diacylglycerol (DAG) biogenesis. By phosphorylating effectors that regulate transcription, fission and polarized transport of Golgi vesicles, as well as cell migration and survival after oxidative stress, PKDs substantially expand the range of physiological processes controlled by DAG. Dysregulated PKDs have been linked to pathologies including heart hypertrophy and cancer invasiveness. Our understanding of PKD regulation by trans- and autophosphorylation, as well as the subcellular dynamics of PKD substrate phosphorylation, have increased markedly. Selective PKD inhibitors provide new, powerful tools for elucidating the physiological roles of PKDs and potentially treating cardiac disease and cancer.
Topics: Animals; Biological Transport; Cell Physiological Phenomena; Diglycerides; Extracellular Space; Humans; Protein Kinase C
PubMed: 21738220
DOI: 10.1038/embor.2011.139 -
PeerJ 2023Olive ( L.) oil accumulate more diacylglycerols (DAG) than mostly vegetable oils. Unsaturated fatty acids-enriched DAG consumption enhanced wellness in subjects....
BACKGROUND
Olive ( L.) oil accumulate more diacylglycerols (DAG) than mostly vegetable oils. Unsaturated fatty acids-enriched DAG consumption enhanced wellness in subjects. However, the mechanism of DAG accumulation is not yet fully understood.
METHODS
In this study, gene network of DAG accumulation and fatty acid composition in the two olive mesocarps ("Chenggu 32" (CG) and "Koroneiki" (QJ)) were investigated by integrating lipidome and transcriptome techniques.
RESULTS
A total of 1,408 lipid molecules were identified by lipidomic analysis in olive mesocarp, of which DAG (DAG36:3, DAG36:4 and DAG36:5) showed higher content, and triacylglycerols (TAG54:3, TAG54:4) exhibited opposite trend in CG. Specifically, DAG was rich in polyunsaturated fatty acids (especially C18:2) at the sn-2 position, which was inconsistent with TAG at the same positions (Primarily C18:1). Transcriptomic analysis revealed that phospholipase C (NPC, EC 3.1.4.3) were up-regulated relative to QJ, whereas diacylglycerol kinase (ATP) (DGK, EC 2.7.1.107), diacylglycerol acyltransferase (DGAT, EC 2.3.1.20), and phospholipid: diacylglycerol acyltransferase (PDAT, EC 2.3.1.158) were down-regulated.
CONCLUSION
We speculated that the non-acyl coenzyme A pathway played a significant role in DAG biosynthesis. Additionally, fatty acyl-ACP thioesterase B (FATB, EC 3.1.2.14), stearoyl [acyl-carrier-protein] 9-desaturase (SAD, EC 1.14.19.2) and omega-6 fatty acid desaturase (FAD2, EC 1.14.19.6) were highly expressed in CG and may be involved in regulating fatty acid composition. Meanwhile, phospholipase A1 (LCAT, EC 3.1.1.32) involved in the acyl editing reaction facilitated PUFA linkage at the sn-2 position of DAG. Our findings provide novel insights to increase the DAG content, improve the fatty acid composition of olive oil, and identify candidate genes for the production of DAG-rich oils.
Topics: Humans; Olea; Lipidomics; Diacylglycerol O-Acyltransferase; Diglycerides; Transcriptome; Fatty Acids; Fatty Acids, Unsaturated
PubMed: 37583911
DOI: 10.7717/peerj.15724 -
Advances in Biological Regulation Jan 2024Cancer still represents the second leading cause of death right after cardiovascular diseases. According to the World Health Organization (WHO), cancer provoked around... (Review)
Review
Cancer still represents the second leading cause of death right after cardiovascular diseases. According to the World Health Organization (WHO), cancer provoked around 10 million deaths in 2020, with lung and colon tumors accounting for the deadliest forms of cancer. As tumor cells become resistant to traditional therapeutic approaches, immunotherapy has emerged as a novel strategy for tumor control. T lymphocytes are key players in immune responses against tumors. Immunosurveillance allows identification, targeting and later killing of cancerous cells. Nevertheless, tumors evolve through different strategies to evade the immune response and spread in a process called metastasis. The ineffectiveness of traditional strategies to control tumor growth and expansion has led to novel approaches considering modulation of T cell activation and effector functions. Program death receptor 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) showed promising results in the early 90s and nowadays are still being exploited together with other drugs for several cancer types. Other negative regulators of T cell activation are diacylglycerol kinases (DGKs) a family of enzymes that catalyze the conversion of diacylglycerol (DAG) into phosphatidic acid (PA). In T cells, DGKα and DGKζ limit the PLCγ/Ras/ERK axis thus attenuating DAG mediated signaling and T cell effector functions. Upregulation of either of both isoforms results in impaired Ras activation and anergy induction, whereas germline knockdown mice showed enhanced antitumor properties and more effective immune responses against pathogens. Here we review the mechanisms used by DGKs to ameliorate T cell activation and how inhibition could be used to reinvigorate T cell functions in cancer context. A better knowledge of the molecular mechanisms involved upon T cell activation will help to improve current therapies with DAG promoting agents.
Topics: Animals; Mice; Diacylglycerol Kinase; Diglycerides; T-Lymphocytes; Neoplasms; Immunotherapy
PubMed: 37949728
DOI: 10.1016/j.jbior.2023.100999 -
Journal of Lipid Research Mar 2012The translocation of lipids across membranes (flip-flop) is an important biological process. Slow exchange on a physiological timescale allows the creation of asymmetric...
The translocation of lipids across membranes (flip-flop) is an important biological process. Slow exchange on a physiological timescale allows the creation of asymmetric distributions of lipids across cellular membranes. The location of lipids and their rate of exchange have important biological consequences, especially for lipids involved in cellular signaling. We investigated the translocation of cholesterol, ceramide, and diacylglycerol in two model bilayers using molecular dynamics simulations. We estimate half times for flip-flop for cholesterol, diacylglycerol, and ceramide of 20 μs, 30 μs, and 10 ms in a POPC bilayer, compared with approximately 30 min, 30 ms, and 30 s in a model raft bilayer (1:1:1 PSM, POPC, and cholesterol). Cholesterol has a large (54 kJ/mol) free energy of exchange between the POPC and raft bilayer, and therefore, it strongly prefers the more ordered and rigid raft bilayer over the more liquid POPC bilayer. Ceramide and diacylglycerol have relatively small free energies of exchange, suggesting nearly equal preference for both bilayers. This unexpected result may have implications for ceramide and diacylglycerol signaling and membrane localization.
Topics: Biological Transport; Ceramides; Cholesterol; Diglycerides; Lipid Bilayers; Molecular Dynamics Simulation
PubMed: 22246847
DOI: 10.1194/jlr.M022491 -
Molecules (Basel, Switzerland) Jul 2021Sulfoquinovosyldiacylglycerol (SQDG) is a glycolipid ubiquitously found in photosynthetically active organisms. It has attracted much attention in recent years due to...
Sulfoquinovosyldiacylglycerol (SQDG) is a glycolipid ubiquitously found in photosynthetically active organisms. It has attracted much attention in recent years due to its biological activities. Similarly, the increasing demand for vegan and functional foods has led to a growing interest in micronutrients such as sulfolipids and their physiological influence on human health. To study this influence, reference materials are needed for developing new analytical methods and providing enough material for model studies on the biological activity. However, the availability of these materials is limited by the difficulty to isolate and purify sulfolipids from natural sources and the unavailability of chemical standards on the market. Consequently, an alternative synthetic route for the comprehensive preparation of sulfolipids was established. Here, the synthesis of a sulfolipid with two identical saturated fatty acids is described exemplarily. The method opens possibilities for the preparation of a diverse range of interesting derivatives with different saturated and unsaturated fatty acids.
Topics: Diglycerides; Fatty Acids; Glycolipids; Lipids
PubMed: 34299550
DOI: 10.3390/molecules26144275 -
Journal of Lipid Research Mar 2024Diacylglycerol kinases (DGKs) are lipid kinases that mediate the phosphorylation of diacylglycerol (DAG) leading to the production of phosphatidic acid (PtdOH). To...
Diacylglycerol kinases (DGKs) are lipid kinases that mediate the phosphorylation of diacylglycerol (DAG) leading to the production of phosphatidic acid (PtdOH). To examine the role of phosphorylation on DGK-θ, we first identified the phosphorylated sites on endogenous DGK-θ from mouse brain and found four sites: S15, S17, which we refer to phosphomotif-1 sites, and S22 and S26 which we refer to as phosphomotif-2 sites. This study focused on the role of these phosphorylated sites on enzyme activity, membrane binding, thermal stability, and cellular half-life of DGK-θ. After generating a construct devoid of all non-catalytic phosphorylation sites (4A), we also generated other constructs to mimic phosphorylation of these residues by mutating them to glutamate (E). Our data demonstrate that an increase in membrane affinity requires the phosphorylation of all four endogenous sites as the phosphomimetic 4E but not other phosphomimietics. Furthermore, 4E also shows an increase in basal activity as well as an increase in the Syt1-induced activity compared to 4A. It is noteworthy that these phosphorylations had no effect on the thermal stability or cellular half-life of this enzyme. Interestingly, when only one phosphorylation domain (phosphomotif-1 or phosphomotif-2) contained phosphomimetics (S15E/S17E or S22E/S26E), the basal activity was also increased but membrane binding affinity was not increased. Furthermore, when only one residue in each domain mimicked an endogenous phosphorylated serine (S15E/S22E or S17E/S26E), the Syt1-induced activity as well as membrane binding affinity decreased relative to 4A. These results indicate that these endogenous phosphorylation sites contribute differentially to membrane binding and enzymatic activity.
Topics: Animals; Mice; Phosphorylation; Diglycerides; Diacylglycerol Kinase
PubMed: 38272356
DOI: 10.1016/j.jlr.2024.100506 -
Journal of Oleo Science Jun 2017Monoacylglycerol (MAG) and diacylglycerol (DAG) are minor components of edible fats and oils, and they relate to the quality of these foods. The AOCS official method Cd...
Monoacylglycerol (MAG) and diacylglycerol (DAG) are minor components of edible fats and oils, and they relate to the quality of these foods. The AOCS official method Cd 11b-91 has been used to determine MAG and DAG contents in fats and oils. There are, however, difficulties in the determination of MAG and DAG using this analytical procedure. Therefore, we improved this method by modifying the trimethylsilyl derivatization procedure and replacing the internal standard (IS) material. In our modified method, TMS-HT (mixture of hexamethyldisilazane and trimethylchlorosilane) was used for derivatization of MAG and DAG, which was followed by liquid-liquid extraction with water and n-hexane solution containing the IS, tricaprin. Using the modified method, we demonstrated superior repeatability in comparison with that of the AOCS method by reducing procedural difficulties. The relative standard deviation of distearin peak areas was 1.8% or 2.9% in the modified method, while it was 5.6% in the AOCS method. In addition, capillary columns, such as DB-1ht and DB-5ht could be used in this method.
Topics: Chromatography, Gas; Dietary Fats; Dietary Fats, Unsaturated; Diglycerides; Food Quality; Hexanes; Liquid-Liquid Extraction; Monoglycerides; Organosilicon Compounds; Reproducibility of Results; Solutions; Triglycerides; Trimethylsilyl Compounds; Water
PubMed: 28515375
DOI: 10.5650/jos.ess16143 -
Analytical and Bioanalytical Chemistry Apr 2020The intestinal microbiome plays an important role in human health and disease and fecal materials reflect the microbial activity. Thus, analysis of fecal metabolites...
The intestinal microbiome plays an important role in human health and disease and fecal materials reflect the microbial activity. Thus, analysis of fecal metabolites provides insight in metabolic interactions between gut microbiota and host organism. In this work, we applied flow injection analysis coupled to Fourier transform mass spectrometry (FIA-FTMS) to identify and quantify lipid species in human fecal samples. Fecal homogenates were subjected to lipid extraction and analyzed by FIA-FTMS. The analysis of different subjects revealed a vast heterogeneity of lipid species abundance. The majority of samples displayed prominent signals of triacylglycerol (TG) and diacylglycerol (DG) species that could be verified by MS2 spectra. Therefore, we focused on the quantification of TG and DG. Method validation included limit of quantification, linearity, evaluation of matrix effects, recovery, and reproducibility. The validation experiments demonstrated the suitability of the method, with exception for approximately 10% of samples, where we observed coefficients of variation higher than 15%. Impaired reproducibility was related to sample inhomogeneity and could not be improved by additional sample preparation steps. Additionally, these experiments demonstrated that compared with aqueous samples, samples containing isopropanol showed higher amounts of DG, presumably due to lysis of bacteria and increased TG lipolysis. These effects were sample-specific and substantiate the high heterogeneity of fecal materials as well as the need for further evaluation of pre-analytic conditions. In summary, FIA-FTMS offers a fast and accurate tool to quantify DG and TG species and is suitable to provide insight into the fecal lipidome and its role in health and disease.
Topics: Diglycerides; Feces; Flow Injection Analysis; Humans; Mass Spectrometry; Triglycerides
PubMed: 32198533
DOI: 10.1007/s00216-020-02416-y