Diacylglycerol Acyltransferase Activity Research Articles

Five new meroterpenes from the seeds of Psoralea corylifolia and their diacylglycerol acyltransferase activity

Five new meroterpenes (1−5), have been isolated and elucidated from the seeds of Psoralea corylifolia together with four known bakuchiol analogues (6−9). The structures of these compounds were elucidated by extensive spectroscopic analysis (NMR, HRESIMS and CD). Among them, compounds 1–9 were found to exhibit selective inhibitory activity on DGAT1 with IC50 values ranging from 54.4 ± 1.3 to 98.2 ± 1.1 μM.

Arabidopsis diacylglycerol acyltransferase1 mutants require fatty acid desaturation for normal seed development.

Diacylglycerol acyltransferase1 (DGAT1) is the major enzyme that synthesizes triacylglycerols (TAG) during Arabidopsis seed development. Mutant dgat1 seeds possess low oil content in addition to a high polyunsaturated fatty acid (PUFA) composition. Two genes encoding endoplasmic reticulum localized desaturase enzymes, fatty acid desaturase2 (FAD2) and fatty acid desaturase3 (FAD3), were upregulated in both dgat1-1 and dgat1-2 developing seeds. Crosses between both dgat1 mutant alleles and fad2-1 failed to generate plants homozygous for both dgat1 and fad2. Reciprocal crosses with wild-type plants demonstrated that both male and female dgat1 fad2 gametophytes were viable. Siliques from DGAT1/dgat1-1 fad2-1/fad2-1 and dgat1-1/dgat1-1 FAD2/fad2-1 possessed abnormal looking seeds that were arrested in the torpedo growth stage. Approximately 25% of the seeds exhibited this arrested phenotype, genetically consistent with them possessing the double homozygous dgat1 fad2 genotype. In contrast, double homozygous dgat1-1 fad3-2 mutant plants were viable. Seeds from these plants possessed higher levels of 18:2 while their fatty acid content was lower than dgat1 mutant controls. The results are consistent with a model where in the absence of DGAT1 activity, desaturation of fatty acids by FAD2 becomes essential to provide PUFA substrates for phospholipid:diacylglycerol acyltransferase (PDAT) to synthesize TAG. In a dgat1 fad2 mutant, seed development is aborted because TAG is unable to be synthesized by either DGAT1 or PDAT.

Identification of lipid synthesis genes in Schizochytrium sp. and their application in improving eicosapentaenoic acid synthesis in Yarrowia lipolytica

BackgroundEicosapentaenoic acid (EPA) is widely used in the functional food and nutraceutical industries due to its important benefits to human health. Oleaginous microorganisms are considered a promising alternative resource for the production of EPA lipids. However, the storage of EPA in triglyceride (TG) becomes a key factor limiting its level.ResultsThis study aimed to incorporate more EPA into TG storage through metabolic engineering. Firstly, key enzymes for TG synthesis, the diacylglycerol acyltransferase (DGAT) and glycerol-3-phosphate acyltransferase (GPAT) genes from Schizochytrium sp. HX-308 were expressed in Yarrowia lipolytica to enhance lipid and EPA accumulation. In addition, engineering the enzyme activity of DGATs through protein engineering was found to be effective in enhancing lipid synthesis by replacing the conserved motifs “HFS” in ScDGAT2A and “FFG” in ScDGAT2B with the motif “YFP”. Notably, combined with lipidomic analysis, the expression of ScDGAT2C and GPAT2 enhanced the storage of EPA in TG. Finally, the accumulation of lipid and EPA was further promoted by identifying and continuing to introduce the ScACC, ScACS, ScPDC, and ScG6PD genes from Schizochytrium sp., and the lipid and EPA titer of the final engineered strain reached 2.25 ± 0.03 g/L and 266.44 ± 5.74 mg/L, respectively, which increased by 174.39% (0.82 ± 0.02 g/L) and 282.27% (69.70 ± 0.80 mg/L) compared to the initial strain, respectively.ConclusionThis study shows that the expression of lipid synthesis genes from Schizochytrium sp. in Y. lipolytica effectively improves the synthesis of lipids and EPA, which provided a promising target for EPA-enriched microbial oil production.

Structural characteristics and catalysis properties of a dual-functional enzyme Lip10 from Mucor circinelloides

Mucor circinelloides lipase Lip10 is a dual-functional enzyme with lipolytic activity and phospholipid:diacylglycerol acyltransferase (PDAT) activity in aqueous phase. However, the catalytic properties of Lip10 and its structural characteristics remain unknown. The present study systematically investigated its PDAT activity and lipolytic activity using six phospholipid substrates, 18:0 triacylglycerol and 16:0 1,2-diacylglycerol, predict the 3D structure of Lip10 using AlphaFold2, and evaluated residues’ flexibility through molecular dynamics analysis. Lip10 exhibited a preference for phosphorylethanolamine (PE) with a long-chain fatty acyl group and showed the lipolytic activity to 18:0 triacylglycerol and 16:0 1.2-diacylglycerol. 20 mM Mg2+ and Fe2+ can significantly increase the PDAT activity of Lip10 by 50–80%. Molecular docking analysis suggest that the lower binding energy for 18:0 PE with Lip10 contributes to the higher structural stability of the complex of 18:0 PE and Lip10. Mutations of Phe565Asp and Glu318Met increase PDAT activity of Lip10 by 3.6-fold and 2.7-fold, while the TAG content produced by mutants Lip10Phe103Pro, Lip10Ile487Lys and Lip10Phe570Asp decrease by 33%− 43%. The present study demonstrated structural properties of the dual-functional lipase Lip10 and provide a theoretical basis to study its roles in the lipid metabolism of M. circinelloides in the future.

Biochemical characterization of lipid metabolic genes of Aurantiochytrium limacinum

Docosahexaenoic acid (DHA) is known to have numerous health benefits and immense dietary value. There is a pressing need to have a deeper understanding of DHA metabolism. Acyl CoA: Diacylglycerol Acyltransferase (DGAT) is an important enzyme of lipid anabolism and an essential piece of the puzzle. Aurantiochytrium limacinum, a primary producer of DHA, is a good model for studying DHA metabolism. Thus, we aimed to investigate important lipid metabolic genes from A. limacinum. We cloned four putative DGATs (DGAT2a, DGAT2b, DGAT2c, and DGAT2d) from A. limacinum and performed detailed in vivo and in vitro characterization. Functional characterization showed that not all the studied genes exhibited DGAT activity. DGAT2a and DGAT2d conferred DGAT activity whereas DGAT2b showed wax synthase (WS) activity and DGAT2c showed dual function of both WS and DGAT. Based on their identified function, DGAT2b and DGAT2c were renamed as AlWS and AlWS/DGAT respectively. DGAT2a was found to exhibit a preference for DHA as a substrate. DGAT2d was found to have robust activity and emerged as a promising candidate for genetic engineering aimed at increasing oil yield. The study enriches our knowledge of lipid biosynthetic enzymes in A. limacinum, which can be utilized to design suitable application strategies.

Diacylglycerol acyltransferase (DGAT) in Crangon crangon and Pandalus montagui (Decapoda, Caridea) - Implications for lipid storage capacities and life history traits.

Lipids play essential roles in cell-structuring, cell-signaling, and as efficient metabolic energy stores. Lipid storage capacities determine life history traits of organisms and, thus, their ecological function. Among storage lipids, triacylglycerols (TAGs) are widespread in marine invertebrates. However, abilities to accumulate TAGs can vary even between closely related species, such as the caridean shrimps Crangon crangon and Pandalus montagui. The first species shows low TAG levels throughout the year in the main storage organ, the midgut gland, while the latter accumulates high TAG-levels, peaking in summer. TAGs synthesis is facilitated by the terminal step of the Kennedy-pathway, where the enzyme diacylglycerol-acyltransferase (DGAT) catalyzes the esterification of diacylglycerols with activated fatty acids. We investigated DGAT activity in the midgut gland using a fluorescent enzyme assay. Sequence information was extracted from whole transcriptome shotgun assembly data, that was publicly available on NCBI, and catalytic properties were deduced from molecular structure analysis. C. crangon showed significantly lower TAG synthesis rates than P. montagui, which explains the native TAG levels. Transcriptome data yielded several isoforms of DGAT enzymes in both species. C. crangon DGAT showed point mutations, which are capable of obstructing the catalytic capacity. The consequences are limited starvation resistance and, thus, presumably restricting C. crangon to a habitat with year-round sufficient food. In contrast, higher TAG synthesis rates presumably enable P. montagui to extend into northern subarctic habitats with limited food availability in winter. Moreover, the limited TAG synthesis and accumulation in the midgut gland may force C. crangon to direct energy into the ovaries, which results in multiple spawnings.

Garcinia mangostana Suppresses Triacylglycerol Synthesis in Hepatocytes and Enterocytes.

Regulation of diacylglycerol acyltransferase (DGAT) and pancreatic lipase (PL) activities is important in the treatment of triacylglycerol (TG)-related metabolic diseases. Garcinia mangostana, also known as mangosteen, is a traditional medicine ingredient used in the treatment of inflammation in Southeast Asia. In this study, The ethanolic extract of G. mangostana peel inhibited human recombinant DGAT1 and DGAT2, and PL enzyme activities in vitro. The inhibitory activity of DGAT1 and DGAT2 enzymes of four representative bioactive substances in mangosteen was confirmed. In addition, G. mangostana was confirmed to suppress the serum TG levels in C57 mice by inhibiting the absorption and synthesis of TG in the gastrointestinal tract. Through this study, it was revealed that G. mangostana extract could be useful for the prevention and amelioration of TG-related metabolic diseases such as obesity and fatty liver.

Dietary Medium-Chain Fatty Acids Raise and (n-3) Polyunsaturated Fatty Acids Lower Hepatic Triacylglycerol Synthesis in Rats

The hypothesis tested was that dietary medium-chain or (n-3) polyunsaturated fatty acids, when compared with (n-6) polyunsaturated fatty acids, alter plasma triacylglycerol levels by affecting hepatic triacylglycerol synthesis as reflected by the activities of acetyl-CoA carboxylase, fatty acid synthase and diacylglycerol acyltransferase in liver. In two separate experiments rats were fed purified diets containing (n-6) polyunsaturated fatty acids in the form of corn oil and either (n-3) polyunsaturated fatty acids in the form of fish oil or medium-chain triacylglycerols (MCT). Consumption of MCT significantly raised plasma triacylglycerol concentrations, whereas fish oil feeding had a lowering effect compared with the corn oil-fed group. In individual rats, the hepatic triacylglycerol concentration was directly correlated with the plasma triacylglycerol concentration (r = 0.60, P < 0.001). The MCT oil diet vs. the corn oil diet markedly raised the activities of hepatic acetyl-CoA carboxylase, fatty acid synthase and diacylglycerol acyltransferase. In the rats fed fish oil, the activities of fatty acid synthase and diacylglycerol acyltransferase were significantly reduced, whereas the activity of acetyl-CoA carboxylase was not affected relative to activities in rats fed corn oil. The activities of the three enzymes were directly correlated with plasma triacylglycerol concentrations in individual rats (r = 0.60–0.75, P < 0.001). The type of fat in the diet probably affects the rate of hepatic triacylglycerol synthesis which is an important determinant of plasma triacylglycerol concentrations.

Transmembrane Protein 68 Functions as an MGAT and DGAT Enzyme for Triacylglycerol Biosynthesis.

Triacylglycerol (TG) biosynthesis is an important metabolic process for intracellular storage of surplus energy, intestinal dietary fat absorption, attenuation of lipotoxicity, lipid transportation, lactation and signal transduction in mammals. Transmembrane protein 68 (TMEM68) is an endoplasmic reticulum (ER)-anchored acyltransferase family member of unknown function. In the current study we show that overexpression of TMEM68 promotes TG accumulation and lipid droplet (LD) formation in a conserved active sites-dependent manner. Quantitative targeted lipidomic analysis showed that diacylglycerol (DG), free fatty acid (FFA) and TG levels were increased by TMEM68 expression. In addition, TMEM68 overexpression affected the levels of several glycerophospholipids, such as phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, as well as sterol ester contents. TMEM68 exhibited monoacylglycerol acyltransferase (MGAT) and diacylglycerol acyltransferase (DGAT) activities dependent on the conserved active sites in an in vitro assay. The expression of lipogenesis genes, including DGATs, fatty acid synthesis-related genes and peroxisome proliferator-activated receptor γ was upregulated in TMEM68-overexpressing cells. These results together demonstrate for the first time that TMEM68 functions as an acyltransferase and affects lipogenic gene expression, glycerolipid metabolism and TG storage in mammalian cells.

A yeast‐based tool for screening mammalian diacylglycerol acyltransferase inhibitors

Dysregulation of lipid metabolism is associated with obesity and metabolic diseases but there is also increasing evidence of a relationship between lipid body excess and cancer. Lipid bodysynthesis requires diacylglycerol acyltransferases (DGATs) which catalyze the last step of triacylglycerol synthesis from diacylglycerol and acyl-coenzyme A. The DGATs and in particular DGAT2, are therefore considered potential therapeutic targets for the control of these pathologies. Here, the murine and the human DGAT2 were overexpressed in the oleaginous yeast Yarrowia lipolytica deleted for all DGAT activities, to evaluate the functionality of the enzymes in this heterologous host and DGAT activity inhibitors. This work provides evidence that mammalian DGATs expressed in Y. lipolytica are a useful tool for screening chemical libraries to identify potential inhibitors or activators of these enzymes of therapeutic interest.

Downregulation of Elovl5 promotes breast cancer metastasis through a lipid-droplet accumulation-mediated induction of TGF-β receptors

Metastatic breast cancer cannot be cured, and alteration of fatty acid metabolism contributes to tumor progression and metastasis. Here, we were interested in the elongation of very long-chain fatty acids protein 5 (Elovl5) in breast cancer. We observed that breast cancer tumors had a lower expression of Elovl5 than normal breast tissues. Furthermore, low expression of Elovl5 is associated with a worse prognosis in ER+ breast cancer patients. In accordance with this finding, decrease of Elovl5 expression was more pronounced in ER+ breast tumors from patients with metastases in lymph nodes. Although downregulation of Elovl5 expression limited breast cancer cell proliferation and cancer progression, suppression of Elovl5 promoted EMT, cell invasion and lung metastases in murine breast cancer models. The loss of Elovl5 expression induced upregulation of TGF-β receptors mediated by a lipid-droplet accumulation-dependent Smad2 acetylation. As expected, inhibition of TGF-β receptors restored proliferation and dampened invasion in low Elovl5 expressing cancer cells. Interestingly, the abolition of lipid-droplet formation by inhibition of diacylglycerol acyltransferase activity reversed induction of TGF-β receptors, cell invasion, and lung metastasis triggered by Elovl5 knockdown. Altogether, we showed that Elovl5 is involved in metastasis through lipid droplets-regulated TGF-β receptor expression and is a predictive biomarker of metastatic ER+ breast cancer.

Soil drought decreases oil synthesis and increases protein synthesis in cottonseed kernel during the flowering and boll formation of cotton

In cotton production, drought is particularly prominent in the flowering and boll formation stage, which significantly affects the cottonseed yield and quality. As the two main storage products in cottonseed kernel, the formation of oil and protein will be affected inescapably under soil drought. Still, the effects of soil drought on the synthesis of cottonseed oil and protein are poorly investigated. To this end, experiments were conducted using two cultivars with contrasting drought tolerance (Dexiamian 1 and Yuzaomian 9110) under three water levels [soil relative water content: control (75 ± 5)%, mild drought (60 ± 5)% and severe drought (45 ± 5)%] from 2017 to 2019. We examined the responses of metabolic activities in cottonseed kernels to drought. Otherwise, 13 CO 2 and 15 N-urea isotope labeling were applied to verify the trend of oil and protein accumulation under soil drought. Results showed that drought decreased cottonseed yield per plant due to the decreases of single-seed weight and cottonseed number. And drought decreased oil content (% dry weight −1 ) and increased protein content (% dry weight −1 ) in cottonseed kernel. According to the findings, drought decreased 13 C content and limited the flow of carbon into oil synthesis pathway; moreover, drought decreased the expression of GhPEPC1 and GhDGAT and activities of phosphoenolpyruvate carboxylase (PEPCase) and diacylglycerol acyltransferase (DGAT), which restrained oil accumulation. Increased protein content under drought was attributed to the increased translocation of plant nitrogen to cottonseed kernel, higher expression of GhGS and GhGOGAT , glutamine synthetase (GS) and glutamate synthase (GOGAT) activities. The Yuzaomian 9110 was more sensitive to drought stress with a higher loss of cottonseed yield and yield components than Dexiamian 1. This study provides insight into the physiological mechanisms of cottonseed oil and protein synthesis under drought and provides a theoretical basis for the utilization of cottonseed oil and protein in the future. Annotation: PEP, phosphoenolpyruvic acid; OAA, oxaloacetic acid; TCA, tricarboxylic acid cycle; Ace-CoA, acetyl coenzyme A; Mal-CoA, Malonyl mono-CoA; G3P, glyceraldehyde 3-phosphate; DAG, diacylglycerol; TAG, triacylglycerol; NADPH, nicotinamide adenine dinucleotide phosphate; PEPCase, Phosphoenolpyruvate carboxylase; ACCase, acetyl-CoA carboxylase; DGAT, diacylglycerol acyltransferase; GS, glutamine synthetase; GOGAT, glutamate synthase; AST, aspartate aminotransferase; ALT, alanine aminotransferase. • Cottonseed yield was significantly decreased under soil drought. • Drought stress significantly restrained 13 C into oil in cottonseed kernels, and enhanced 15 N into kernels. • The oil content was restrained under drought due to lower carbon substrates and enzyme activities. • The increase of nitrogen accumulation and protein synthesis enhanced kernel protein content under drought.

ATGL is a biosynthetic enzyme for fatty acid esters of hydroxy fatty acids

Branched fatty acid (FA) esters of hydroxy FAs (HFAs; FAHFAs) are recently discovered lipids that are conserved from yeast to mammals1,2. A subfamily, palmitic acid esters of hydroxy stearic acids (PAHSAs), are anti-inflammatory and anti-diabetic1,3. Humans and mice with insulin resistance have lower PAHSA levels in subcutaneous adipose tissue and serum1. PAHSA administration improves glucose tolerance and insulin sensitivity and reduces inflammation in obesity, diabetes and immune-mediated diseases1,4–7. The enzyme(s) responsible for FAHFA biosynthesis in vivo remains unknown. Here we identified adipose triglyceride lipase (ATGL, also known as patatin-like phospholipase domain containing 2 (PNPLA2)) as a candidate biosynthetic enzyme for FAHFAs using chemical biology and proteomics. We discovered that recombinant ATGL uses a transacylation reaction that esterifies an HFA with a FA from triglyceride (TG) or diglyceride to produce FAHFAs. Overexpression of wild-type, but not catalytically dead, ATGL increases FAHFA biosynthesis. Chemical inhibition of ATGL or genetic deletion of Atgl inhibits FAHFA biosynthesis and reduces the levels of FAHFA and FAHFA-TG. Levels of endogenous and nascent FAHFAs and FAHFA-TGs are 80–90 per cent lower in adipose tissue of mice in which Atgl is knocked out specifically in the adipose tissue. Increasing TG levels by upregulating diacylglycerol acyltransferase (DGAT) activity promotes FAHFA biosynthesis, and decreasing DGAT activity inhibits it, reinforcing TGs as FAHFA precursors. ATGL biosynthetic transacylase activity is present in human adipose tissue underscoring its potential clinical relevance. In summary, we discovered the first, to our knowledge, biosynthetic enzyme that catalyses the formation of the FAHFA ester bond in mammals. Whereas ATGL lipase activity is well known, our data establish a paradigm shift demonstrating that ATGL transacylase activity is biologically important.

Expression of Chlamydomonas reinhardtii chloroplast diacylglycerol acyltransferase 3 is induced by light in concert with triacylglycerol accumulation.

Considerable progress has been made towards the understanding of triacylglycerol (TAG) accumulation in algae. One key aspect is finding conditions that trigger TAG production without reducing cell division. Previously, we identified a soluble diacylglycerol acyltransferase (DGAT), related to plant DGAT3, with heterologous DGAT activity. In this work, we demonstrate that Chlamydomonas reinhardtii DGAT3 localizes to the chloroplast and that its expression is induced by light, in correspondence with TAG accumulation. Dgat3 mRNAs and TAGs increase in both wild-type and starch-deficient cells grown with acetate upon transferring them from dark or low light to higher light levels, albeit affected by the particularities of each strain. The response of dgat3 mRNAs and TAGs to light depends on the pre-existing levels of TAGs, suggesting the existence of a negative regulatory loop in the synthesis pathway, although an effect of TAG turnover cannot be ruled out. Altogether, these results hint towards a possible role of DGAT3 in light-dependent TAG accumulation in C. reinhardtii.

Increasing lipid production of Desmodesmus sp. through atmospheric and room temperature plasma orientated with malonic acid: Performance and biochemical mechanism

Desmodesmus sp. strain b15 with a high lipid yield was obtained through atmospheric and room temperature plasma (ARTP) mutagenesis orientated by malonic acid (MA) resistance screening. Key enzyme activity and gene expression were measured to elucidate the biochemical mechanisms regulating the high lipid synthesis of the mutant algal strains. MA further significantly enhanced the screening efficiency of the targeted lipid-rich algal strains mutated by ARTP, where the number of potential target algal strains was 3.5 times higher than the mutant strains by single ARTP. The triglyceride and total lipid content of strain b15 reached 330.21 and 373.27 mg L−1, which were 48.98% and 114.99% higher than the mutant a17 strain from single ARTP and the control strain, respectively. The maximum quantum yield (Fv/Fm) and relative electron transport rate (ETR) for the photosynthetic activity of b15 were lower than the control group. Additionally, the activities of acetyl-CoA carboxylase (ACCase) and diacylglycerol acyltransferase (DGAT) related to triglyceride synthesis were higher in b15, but malate dehydrogenase (MDH) and succinate dehydrogenase (SDH) were greatly inhibited in the tricarboxylic acid cycle (TCA) by MA. The expressions of key regulatory genes of accD and dgat7566 for lipid synthesis in mutant b15 were also significantly higher than those in a17 and the control group (P < 0.05), and the decreased expression of SDH2 in the TCA also verified the superior lipid synthesis performance of strain b15.

Identification of key genes and enzymes contributing to nutrition conversion of Torreya grandis nuts during post-ripening process

The seeds of Torreya grandis are necessary to go through a ripening process, which eventually leads to nutrition conversion and the production of edible nuts. However, the molecular basis of nutrition conversion remains unclear. Here, transcriptome sequencing was performed on seeds treated with different temperature and humidity. A total of 881 unigenes related to nutrition conversion were identified. The correlations between nutrient content and gene expression suggested that sucrose phosphate synthase (SPS), dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex (DLST), glycerol-3-phosphate acyltransferase (GPAT) and Pyruvate kinase (PK) may play key roles in nutrition conversion. Transient over-expression of TgDLST, TgPK and TgGPAT in tobacco leaves promoted nutritional conversion. Moreover, enzyme activity analysis indicated that diacylglycerol acyltransferase (DGAT) and pyruvate dehydrogenase (PDH) activities may also accelerate the nutritional conversion. This study uncovers the molecular basis of nutrition conversion in T. grandis seeds, which critical for shortening the time of nutrition conversion.

Effects of elevated air temperature coupling with soil drought on carbohydrate metabolism and oil synthesis during cottonseed development.

Cotton, as the fifth-largest oilseed crop, often faces the coupling stress of heat and drought. Still, the effects of combined stress on cottonseed oil synthesis and its closely related carbon metabolism are poorly investigated. To this end, experiments were conducted with two cultivars (Sumian 15 and PHY370WR) under two temperature regimes: ambient temperature (AT) and elevated temperature (ET, which was 2.5°C-2.7°C higher than AT) and three water regimes: optimum soil moisture (soil relative water content [SRWC] at 75% ± 5%), and drought (SD) including SRWC 60% ± 5% and SRWC 45% ± 5%, during 2016-2018. Results showed that ET plus SD decreased cottonseed kernel yield, seed index, kernel weight, and kernel percentage more than either single stress. The content of hexoses, the carbon skeleton source for oil synthesis, was decreased by ET while increased by SD. The combined stress increased the hexose content by increasing the activities of sucrose synthase (SuSy, EC 2.4.1.13) and invertase (Inv, EC 3.2.1.26) and upregulating GhSuSy expression; however, hexose content under combined stress was lower than that under SD alone. Increased oil content under SD was attributed to the high phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31), acetyl-CoA carboxylase (ACCase, EC 6.4.1.2), and diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) activities, whereas the opposite effects were seen under ET. Under combined stress, although ACCase activity decreased, PEPCase and DGAT activities, and GhPEPC-1 and GhDGAT-1 expression upregulated, enhancing carbon flow into oil metabolism and triacylglycerol synthesis, ultimately generating higher oil content.

Evolutionary and biochemical characterization of a Chromochloris zofingiensis MBOAT with wax synthase and diacylglycerol acyltransferase activity.

Wax synthase (WS) catalyzes the last step in wax ester biosynthesis in green plants. Two unrelated sub-families of WS, including the bifunctional acyltransferase and plant-like WS have been reported, but the latter is largely uncharacterized in microalgae. Here, we functionally characterized a putative plant-like WS (CzWS1) from the emerging model green microalga Chromochloris zofingiensis. Our results showed that plant-like WS evolved under different selection constraints in plants and microalgae, with positive selection likely contributing to functional divergence. Unlike jojoba with high amounts of wax ester in seeds and a highly active WS enzyme, C. zofingiensis has no detectable wax ester but a high abundance of WS transcripts. Co-expression analysis showed that C. zofingiensis WS has different expression correlation with lipid biosynthetic genes from jojoba, and may have a divergent function. In vitro characterization indicated that CzWS1 had diacylglycerol acyltransferase activity along with WS activity, and overexpression of CzWS1 in yeast and Chlamydomonas reinhardtii affected triacylglycerol accumulation. Moreover, biochemical and bioinformatic analyses revealed the relevance of the C-terminal region of CzWS1 in enzyme function. Taken together, our results indicated a functional divergence of plant-like WS in plants and microalgae, and the importance of its C-terminal region in specialization of enzyme function.

Accumulation and deposition of triacylglycerols in the starchy endosperm of wheat grain

A combination of lipidomics, transcriptomics and bioimaging has been used to study triacylglycerol synthesis and deposition in the developing starchy endosperm of wheat. The content of TAG increased between 14 and 34 days after anthesis, from 50 to 115 mg/100 g dry wt and from about 35 to 175 mg/100 g dry wt in two experiments. The major fatty acids were C16 (palmitic C16:0 and palmitoleic C16:1) and C18 (stearic C18:0, oleic C18:1, linoleic C18:2 and linolenic C18:3), with unsaturated fatty acids accounting for about 75–80% of the total throughout development. Linoleic acid (C18:2) was the major component at all stages and the proportion increased during development. Transcript profiling indicated that predominant route to TAG synthesis and oil accumulation is via the Kennedy pathway and diacylglycerol acyltransferase (DGAT) activity. Confocal microscopy of stained tissue sections showed that TAG accumulated in droplets which are associated with protein and concentrated in the starchy endosperm cells below the sub-aleurone cells. Transcripts encoding 16kd oleosins were also expressed, indicating that the oil droplets are in part stabilised by oleosin proteins.

Hypoxia-inducible lipid droplet-associated induces DGAT1 and promotes lipid storage in hepatocytes

ObjectiveStorage of triglycerides in lipid droplets is governed by a set of lipid droplet-associated proteins. One of these lipid droplet-associated proteins, hypoxia-inducible lipid droplet-associated (HILPDA), was found to impair lipid droplet breakdown in macrophages and cancer cells by inhibiting adipose triglyceride lipase. Here, we aimed to better characterize the role and mechanism of action of HILPDA in hepatocytes. MethodsWe performed studies in HILPDA-deficient and HILPDA-overexpressing liver cells, liver slices, and mice. The functional role and physical interactions of HILPDA were investigated using a variety of biochemical and microscopic techniques, including real-time fluorescence live-cell imaging and Förster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM). ResultsLevels of HILPDA were markedly induced by fatty acids in several hepatoma cell lines. Hepatocyte-specific deficiency of HILPDA in mice modestly but significantly reduced hepatic triglycerides in mice with non-alcoholic steatohepatitis. Similarly, deficiency of HILPDA in mouse liver slices and primary hepatocytes reduced lipid storage and accumulation of fluorescently-labeled fatty acids in lipid droplets, respectively, which was independent of adipose triglyceride lipase. Fluorescence microscopy showed that HILPDA partly colocalizes with lipid droplets and with the endoplasmic reticulum, is especially abundant in perinuclear areas, and mainly associates with newly added fatty acids. Real-time fluorescence live-cell imaging further revealed that HILPDA preferentially localizes to lipid droplets that are being remodeled. Overexpression of HILPDA in liver cells increased the activity of diacylglycerol acyltransferases (DGAT) and DGAT1 protein levels, concurrent with increased lipid storage. Confocal microscopy coupled to FRET-FLIM analysis demonstrated that HILPDA physically interacts with DGAT1 in living liver cells. The stimulatory effect of HILPDA on lipid storage via DGAT1 was corroborated in adipocytes. ConclusionsOur data indicate that HILPDA physically interacts with DGAT1 and increases DGAT activity. Our findings suggest a novel regulatory mechanism by which fatty acids promote triglyceride synthesis and storage.