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The Journal of Clinical Investigation Feb 1990Recent evidence has suggested that pancreatic islets isolated from rats synthesize 1,2-diacyl-sn-glycerol (DAG) de novo from glucose and that this process may constitute...
Recent evidence has suggested that pancreatic islets isolated from rats synthesize 1,2-diacyl-sn-glycerol (DAG) de novo from glucose and that this process may constitute the long-sought link between the metabolism of glucose and the induction of insulin secretion. The cell-permeant diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol (200 microM) has been found here to amplify both the first and second phases of insulin secretion from perifused human islets. Measurements of the mass of endogenous DAG in human pancreatic islets by enzymatic and by mass spectrometric methods indicate that levels of 200 microM may be achieved under physiologic conditions. Conversion of [14C]glucose to [14C]DAG has been demonstrated here to occur within 60 s of exposure of rat and human islets to stimulatory concentrations of glucose. This process has been found to be a quantitatively minor contributor to the total islet DAG mass after acute stimulation with glucose, however, and glucose has been found not to induce a rise in total islet DAG content within 20 min of induction of insulin secretion. In contrast to the case with rodent islets, two pharmacologic inhibitors of DAG-induced activation of protein kinase C (staurosporine and sphingosine) have been found not to influence glucose-induced insulin secretion from isolated human islets. These findings indicate that de novo synthesis of DAG from glucose does not participate in acute signal-response coupling in islets.
Topics: Alkaloids; Animals; Culture Techniques; Diglycerides; Glucose; Glycerides; Humans; Insulin; Insulin Secretion; Islets of Langerhans; Male; Perfusion; Protein Kinase C; Rats; Rats, Inbred Strains; Sphingosine; Staurosporine
PubMed: 2405021
DOI: 10.1172/JCI114463 -
Food Chemistry Oct 2022This study developed an alpha-linolenic acid (ALA) supplement with emulsion form using ALA-rich diacylglycerol (ALA-DAG) and ALA-DAG stearin (DAG-SF) as a new source of...
This study developed an alpha-linolenic acid (ALA) supplement with emulsion form using ALA-rich diacylglycerol (ALA-DAG) and ALA-DAG stearin (DAG-SF) as a new source of ALA and emulsifier. Stable, commercial surfactant-free W/O emulsions with 90 wt% oil phase (including DAG-SF and ALA-DAG with 10:90 - 20:80 wt ratio) was fabricated. Microstructure and Raman spectra revealed that the compact crystal networks and high amounts of solid acyl chains were responsible for high emulsion stability. These emulsions exhibited good potential in improving the ALA nutritional status (with ALA release level of 60.49% - 62.98%). Furthermore, the emulsifier-to-oil ratio greatly impacted the emulsion texture (solid-like or liquid-like) and emulsions showed great oxidation stability (2.80 - 3.09 meq/kg lipid of peroxide value at 6th week). The tunable texture and high oxidation stability make this emulsion system useful for a wide range of food products. This developed emulsion system could provide valuable information for other important fatty acids supplement.
Topics: Digestion; Diglycerides; Emulsifying Agents; Emulsions; Water; alpha-Linolenic Acid
PubMed: 35609461
DOI: 10.1016/j.foodchem.2022.133201 -
Methods in Molecular Biology (Clifton,... 2013Diacylglycerol (DAG) is an important intermediate of lipid metabolism and a component of phospholipase C signal transduction. Quantification of DAG in plant membranes...
Diacylglycerol (DAG) is an important intermediate of lipid metabolism and a component of phospholipase C signal transduction. Quantification of DAG in plant membranes represents a challenging task because of its low abundance. DAG can be measured by direct infusion mass spectrometry (MS) on a quadrupole time-of-flight mass spectrometer after purification from the crude plant lipid extract via solid-phase extraction on silica columns. Different internal standards are employed to compensate for the dependence of the MS and MS/MS signals on the chain length and the presence of double bonds in the acyl moieties. Thus, using a combination of single MS and MS/MS experiments, quantitative results for the different molecular species of DAGs from Arabidopsis can be obtained.
Topics: Arabidopsis; Chemical Fractionation; Diglycerides; Mass Spectrometry; Molecular Weight; Plant Leaves; Reference Standards; Solid Phase Extraction; Statistics as Topic
PubMed: 23681522
DOI: 10.1007/978-1-62703-401-2_5 -
Yeast (Chichester, England) Jan 2020The 3-acetyl-1,2-diacylglycerols (acTAGs) are the molecules that are structurally similar to triacylglycerols (TAGs). They are naturally produced by plants of the family...
Overexpression of diacylglycerol acetyltransferase from Euonymus europaeus in Yarrowia lipolytica leads to the production of single-cell oil enriched with 3-acetyl-1,2-diacylglycerols.
The 3-acetyl-1,2-diacylglycerols (acTAGs) are the molecules that are structurally similar to triacylglycerols (TAGs). They are naturally produced by plants of the family Celastraceae and animals such as Cervus nippon and Eurosta solidaginis. The presence of acetate in the sn-3 position of the glycerol backbone confers advantages to these compounds, for example, lower viscosity and calorific value compared to classical TAGs. In this work, the gene EeDAcT, which encodes diacylglycerol acetyltransferase in a species of bush (Euonymus europaeus), was overexpressed in strains Po1d (capable of accumulating storage lipids) and JMY1877 (incapable of accumulating storage lipids) of Yarrowia lipolytica, to test the activity of the gene EeDAcT and the production of acTAGs in oleaginous and nonoleaginous genetic backgrounds. It was observed that both the strains containing the gene EeDAcT (YL33 and YL35 for Po1d and JMY1877 strains, respectively) produced acTAGs. The strain YL33 accumulated up to 20% intracellular lipids, 20% of which was acTAGs, and 40% was TAGs. On the other hand, the strain YL35, which showed interrupted TAGs accumulation, produced up to 10% acTAGs as the only storage lipid. Unfortunately, the quantity of acTAGs produced in YL35 was insignificant, as the overall lipid accumulated in the strain was not more than 4% of the biomass. The fatty acid profile of acTAGs produced by the YL33 strain was remarkably similar to TAGs, and both of these structures were rich in oleic (45%) and palmitic (25%) acids.
Topics: Biomass; Diacylglycerol O-Acyltransferase; Diglycerides; Euonymus; Lipid Metabolism; Microorganisms, Genetically-Modified; Oleic Acid; Palmitic Acid; Plant Proteins; Triglycerides; Yarrowia
PubMed: 31509617
DOI: 10.1002/yea.3442 -
The Journal of Biological Chemistry Dec 1991The mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated phosphatidylcholine biosynthesis in HeLa cells was investigated. TPA caused a 3-fold increase in...
The mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated phosphatidylcholine biosynthesis in HeLa cells was investigated. TPA caused a 3-fold increase in particulate CTP:phosphocholine cytidylyltransferase activity in HeLa cells which correlated with decreased cytidylyltransferase activity in the cytosol. The increase in membrane-associated cytidylyltransferase was confirmed by immunoblotting. Immunoprecipitation studies suggested that TPA had no effect on the phosphorylation state of cytidylyltransferase. Enhanced binding of cytidylyltransferase to diacylglycerol-enriched membranes has previously been shown. Diacylglycerol levels in TPA-treated HeLa cells increased approximately 2-fold (2.29 to 4.02 nmol/mg of protein) after 1 h of TPA treatment. A time course experiment showed a temporal relationship in which production of diacylglycerol appeared to signal translocation of cytidylyltransferase to membranes followed by a stimulation of phosphatidylcholine biosynthesis. Diacylglycerol was further evaluated as a translocator of cytidylyltransferase by depleting HeLa cells of protein kinase C and incubating with dioctanoylglcerol. This treatment increased both membrane-associated cytidylyltransferase activity and the rate of phosphatidylcholine biosynthesis approximately 2-fold. A time course experiment with dioctanoylglycerol showed a strong positive correlation (r2 = 0.89) between the amount of particulate cytidylyltransferase activity and the rate of phosphatidylcholine biosynthesis. Therefore, TPA stimulates phosphatidylcholine biosynthesis by causing a translocation of cytidylyltransferase from the cytosol to membranes, which appears to be mediated by increased diacylglycerol.
Topics: Cell Membrane Permeability; Choline-Phosphate Cytidylyltransferase; Digitonin; Diglycerides; HeLa Cells; Humans; Kinetics; Microsomes; Nucleotidyltransferases; Phosphatidylcholines; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate
PubMed: 1660890
DOI: No ID Found -
Biochimica Et Biophysica Acta Jun 1989The conversion of [3H]- or [14C]diacylglycerol to labelled phosphatidylethanolamine by dialysed, particulate fraction of Escherichia coli was studied. The reaction...
The conversion of [3H]- or [14C]diacylglycerol to labelled phosphatidylethanolamine by dialysed, particulate fraction of Escherichia coli was studied. The reaction occurred in the presence of hydroxylamine, under which conditions, the synthesis of [14C]phosphatidylethanolamine from CDP-diacylglycerol and [14C]serine did not occur. The conversion was enhanced by addition of dilauroyl- or dioleoylphosphatidylethanolamine. A conversion of [3H]- or [14C]phosphatidylethanolamine to labelled diacylglycerol could also be readily demonstrated provided unlabelled diacylglycerol was added. Double-labelled [acyl-3H, 32P]phosphatidylethanolamine was converted to labelled diacylglycerol without formation of labelled water-soluble products. The formation of double-labelled phosphatidylethanolamine from [3H]diacyl[14C]glycerol or of double-labelled diacylglycerol from [acyl-3H,glycerol-14C]phosphatidylethanolamine occurred without significant change in isotope ratio. When [acyl-3H]phosphatidylethanolamine was incubated with increasing concentrations of [acyl-14C]diacylglycerol, correspondingly increasing concentrations of [14C]phosphatidylethanolamine were formed which matched the concentrations of [3H]diacylglycerol produced concurrently. It was concluded that E. coli extracts can catalyze an exchange between the diacylglycerol moiety of phosphatidylethanolamine and free diacylglycerol with complete sparing of the phosphoethanolamine moiety.
Topics: Diglycerides; Escherichia coli; Glycerides; Phosphatidylethanolamines
PubMed: 2659083
DOI: 10.1016/0005-2760(89)90260-9 -
Methods in Enzymology 2023Diacylglycerols (DAGs) are anabolic precursors to membrane lipid and storage triacylglycerol biosynthesis, metabolic intermediates of lipid catabolism, and potent...
Diacylglycerols (DAGs) are anabolic precursors to membrane lipid and storage triacylglycerol biosynthesis, metabolic intermediates of lipid catabolism, and potent cellular signaling molecules. The different DAG molecular species that accumulate over development or in different tissues reflect the changing aspects of cellular lipid metabolism. Consequently, an accurate determination of DAG molecular species in biological samples is essential to understand various metabolic processes and their diagnostic relevance. However, quantification of DAG molecular species in various biological samples represents a challenging task because of their low abundance, hydrophobicity, and instability. This chapter describes the most common chromatographic (TLC and HPLC) and mass spectrometry (MS) methods used to analyze DAG molecular species. In addition, we directly compared the three methods using DAG obtained by phospholipase C hydrolysis of phosphatidylcholine purified from a Nicotiana benthamiana leaf extract. We conclude that each method identified similar major molecular species, however, the exact levels of those varied mainly due to sensitivity of the technique, differences in sample preparation, and processing. This chapter provides three different methods to analyze DAG molecular species, and the discussion of the benefits and challenges of each technique will aid in choosing the right method for your analysis.
Topics: Diglycerides; Chromatography, High Pressure Liquid; Spectrometry, Mass, Electrospray Ionization; Phosphatidylcholines
PubMed: 37087188
DOI: 10.1016/bs.mie.2022.09.011 -
The FEBS Journal Feb 2024Diacylglycerol kinases (DGKs) control local and temporal amounts of diacylglycerol (DAG) and phosphatidic acid (PA) by converting DAG to PA through phosphorylation in...
Diacylglycerol kinases (DGKs) control local and temporal amounts of diacylglycerol (DAG) and phosphatidic acid (PA) by converting DAG to PA through phosphorylation in cells. Certain DGK enzymes possess C-terminal sequences that encode potential PDZ-binding motifs (PBMs), which could be involved in their recruitment into supramolecular signaling complexes. In this study, we used two different interactomic approaches, quantitative native holdup (nHU) and qualitative affinity purification (AP), both coupled to mass spectrometry (MS) to investigate the PDZ partners associated with the potential PBMs of DGKs. Complementing these results with site-specific affinity interactomic data measured on isolated PDZ domain fragments and PBM motifs, as well as evolutionary conservation analysis of the PBMs of DGKs, we explored functional differences within different DGK groups. All our results indicate that putative PBM sequences of type II enzymes, namely DGKδ, DGKη, and DGKκ, are likely to be nonfunctional. In contrast, type IV enzymes, namely DGKζ and DGKι, possess highly promiscuous PBMs that interact with a set of PDZ proteins with very similar affinity interactomes. The combination of various interactomic assays and evolutionary analyses provides a useful strategy for identifying functional domains and motifs within diverse enzyme families.
Topics: Diacylglycerol Kinase; Diglycerides; Signal Transduction; Phosphorylation
PubMed: 37942667
DOI: 10.1111/febs.16994 -
Journal of Lipid Research Jun 2017In mammals, ether lipids exert a wide spectrum of signaling and structural functions, such as stimulation of immune responses, anti-tumor activities, and enhancement of...
In mammals, ether lipids exert a wide spectrum of signaling and structural functions, such as stimulation of immune responses, anti-tumor activities, and enhancement of sperm functions. Abnormal accumulation of monoalkyl-diacylglycerol (MADAG) was found in Wolman's disease, a human genetic disorder defined by a deficiency in lysosomal acid lipase. In the current study, we found that among the nine recombinant human lipid acyltransferases examined, acyl-CoA:diacylglycerol acyltransferase (DGAT)1, DGAT2, acyl-CoA:monoacylglycerol acyltransferase (MGAT)2, MGAT3, acyl-CoA:wax-alcohol acyltransferase 2/MFAT, and DGAT candidate 3 were able to use 1-monoalkylglycerol (1-MAkG) as an acyl acceptor for the synthesis of monoalkyl-monoacylglycerol (MAMAG). These enzymes demonstrated different enzymatic turnover rates and relative efficiencies for the first and second acylation steps leading to the synthesis of MAMAG and MADAG, respectively. They also exhibited different degrees of substrate preference when presented with 1-monooleoylglycerol versus 1-MAkG. In CHO-K1 cells, treatment with DGAT1 selective inhibitor, XP-620, completely blocked the synthesis of MADAG, indicating that DGAT1 is the predominant enzyme responsible for the intracellular synthesis of MADAG in this model system. The levels of MADAG in the adrenal gland of DGAT1 KO mice were reduced as compared with those of the WT mice, suggesting that DGAT1 is a major enzyme for the synthesis of MADAG in this tissue. Our findings indicate that several of these lipid acyltransferases may be able to synthesize neutral ether lipids in mammals.
Topics: Acylation; Acyltransferases; Animals; CHO Cells; Chlorocebus aethiops; Cricetulus; Diglycerides; Ethers; Humans
PubMed: 28420705
DOI: 10.1194/jlr.M073445 -
FEBS Letters Dec 2011We show that diacylglycerol kinase-ε (DGKε) has less preference for the acyl chain at the sn-1 position of diacylglycerol (DAG) than the one at the sn-2 position....
We show that diacylglycerol kinase-ε (DGKε) has less preference for the acyl chain at the sn-1 position of diacylglycerol (DAG) than the one at the sn-2 position. Although DGKε discriminates between 1-stearoyl-2-arachidonoyl-DAG and 1-palmitoyl-2-arachidonoyl-DAG, it has similar substrate preference for 1-stearoyl-2-arachidonoyl-DAG and 1,2-diarachidonoyl-DAG. We suggest that in addition to binding to the enzyme, the acyl chain at the sn-1 position may contribute to the depth of insertion of the DAG into the membrane. Thus, the DAG intermediate of the PI-cycle, 1-stearoyl-2-arachidonoyl-DAG, is not the only DAG that is a good substrate for DGKε, the DGK isoform involved in PI-cycling.
Topics: Diacylglycerol Kinase; Diglycerides; Enzyme Inhibitors; Humans; Kinetics; Phosphatidylinositols; Substrate Specificity
PubMed: 22108654
DOI: 10.1016/j.febslet.2011.11.016