miR-25 modulates triacylglycerol and lipid accumulation in goat mammary epithelial cells by repressing PGC-1beta

Background: The goat(Caprahircus) is one of the most important livestock animals. Goat milk fat is an important component in the nutritional quality of goat milk. Growing evidence points to the critical roles of microRNAs(miRNAs) in lipid metabolism.Results: Using a highly sensitive method of S-poly(T) plus for miRNAs detection, we analyze the expression patterns of 715 miRNAs in goat mammary gland tissues at different stages of lactation. We observed that miR-25 expression had an inverse relationship with milk production. Overexpression of miR-25 significantly repressed triacylglycerol synthesis and lipid droplet accumulation. To explore the regulatory mechanism of miR-25 in milk lipid metabolism,we analyzed its putative target genes with bioinformatics analysis followed by 3′-UTR assays. Peroxisome proliferative activated receptor gamma coactivator 1 beta(PGC-1 beta), a key regulator of lipogenics was identified as a direct target of miR-25 with three specific sites within its 3′-UTR. In addition, miR-25 mimics in goat mammary epithelial cells reduced the expressions of genes involved in lipid metabolism.Conclusions: Taken together, our results show miR-25 is potentially involved in lipid metabolism and we reveal the function of the miR-25/PGC-1 beta regulatory axis during lactation.

The Light Regime Effect on Triacylglycerol Accumulation of Isochrysis zhangjiangensis

Stress state of microalgal cells is caused under unfavorable conditions such as disordered light regime and depleted nitrogen. The stress state can impair photosynthetic efficiency, inhibit cell growth and result in the accumulation of triacylglycerol(TAG) from protective mechanisms. Continuous light or nitrogen starvation was applied on microalgae and performed effectively on inducing TAG production. To evaluate the light regime effect on inducing TAG production, the effect of different light regimes on nitrogen-starved Isochrysis zhangjiangensis was investigated in this work. The continuous light and nitrogen starvation elevated TAG content of biomass by 73% and 193%, respectively. Furthermore, the TAG accumulation of I. zhangjiangensis cell under nitrogen starvation decreased under aggravated stress from continuous illumination. Our results demonstrated that culturing the cells with 14 L: 10 D light regime under nitrogen starvation is the optimal mode to achieve maximal accumulation of TAG. A recovery in light regime was necessary for I. zhangjiangensis cultivation.

Phospholipase D antagonist 1-butanol inhibited the mobilization of triacylglycerol during seed germination in Arabidopsis

Storage oil breakdown plays an important role in the life cycle of many plants by providing the carbon skeletons that support seedling growth immediately following germination. 1-Butanol, a specific inhibitor of phospholipase D(PLD)-dependent production of the signalling molecule phosphatidic acid(PA), inhibited Arabidopsis seed germination. N-Acylethanolamines(NAEs), which have been shown to inhibits PLDa1 activity, have no effect on seed germination. However, mobilization profile of triacylglycerols(TAG) that induced by each compound has not been reported. To gain deeper insights into the mode of mobilization of TAG during NAE 12:0 or 1-butanol treatment, we conducted a detailed comparative analysis of the effect of NAE 12:0, DMSO, 1-butanol and tert-butanol on Arabidopsis seed germination and fatty acid composition, tert-butanol and DMSO served as the corresponding controls treatment respectively. Our data show that 1-butanol, but not the inactive tert-butanol isomer, inhibited Arabidopsis seed germination, which is accompanied by a with retardation of the mobilization of triacylglycerols(TAG). In contrast, NAE 12:0 did not affect mobilization of TAG, nor did it significantly delay seed germination as monitored by radicle and cotyledon emergence. 1-Butanol induced RNA degradation in seeds and seedlings. We speculate that the large-scale degradation of RNA under the induction of 1-butanol may lead to abnormal gene expression in genes necessary for seed germination, including the genes needed for the mobilization of oil bodies, and thus cause a delay of seed germination. To the best of our knowledge, we report for the first time that 1-butanol delays the mobilization of TAG.

A Study on Triacylglycerol Composition and the Structure of High-Oleic Rapeseed Oil

The composition of fatty acids in triacylglycerides （TAGs） and their position on the glycerol backbone de- termine the nutritional value of vegetable oil. In this study, gas chromatography and high-performance liquid chromatography-tandem mass spectrometry （HPLC-MS/MS） were used to analyze the compo- sition and distribution of fatty acids in TAGs of different rapeseed oils. Our results show the content of oleic acid in higb-oleic-acid rapeseed oil to be about 80%. In terms of the number of acyl carbon atoms （CN）, TAGs with CN52-54 were most abundant, with a maximum concentration at CN54 （80%）. The main type of TAG was oleic-oleic-oleic （OOO）, accounting for 71.75%, while oleic-oleic-linoleic （OOL） accounted for ？.56%, oleic-oleic-linolenic （OOLn） accounted for 4.81%, and stearic-oleic-oleic （SO0） accounted for 4.74%. Oleic acid in high-oleic-acid rapeseed oil was distributed in the following order of preference： sn-2 〉 sn-1/3. In high-erucic-acid rapeseed oil, however, oleic acid was enriched at the sn-1/3. These data show that the content of oleic acid can be as high as about 80% in high-oleic-acid material. This finding suggests that high-oleic-acid rapeseed oil has high nutritional value.

Genetic Manipulation of Non-Classic Oilseed Plants for Enhancement of Their Potential as a Biofactory for Triacylglycerol Production

Global demand for vegetable oil is anticipated to double by 2030. The current vegetable oil production platforms, including oil palm and temperate oilseeds, are unlikely to produce such an expansion. Therefore, the exploration of novel vegetable oil sources has become increasingly important in order to make up this future vegetable oil shortfall. Triacylglycerol （TAG）, as the dominant form of vegetable oil, has recently attracted immense interest in terms of being produced in plant vegetative tissues via genetic engineering technologies. Multidiscipline-based ＂-omics＂ studies are increasingly enhancing our understanding of plant lipid biochemistry and metabolism. As a result, the identification of biochemical pathways and the annotation of key genes contributing to fatty acid biosynthesis and to lipid assembly and turnover have been effectively updated. In recent years, there has been a rapid development in the genetic enhancement of TAG accumulation in high-biomass plant vegetative tissues and oilseeds through the genetic manipulation of the key genes and regulators involved in TAG biosynthesis. In this review, current genetic engineering strategies ranging from single-gene manipulation to multigene stacking aimed at increasing plant biomass TAG accumulation are summarized. New directions and suggestions for plant oil production that may help to further alleviate the potential shortage of edible oil and biodiesel are discussed.

Cyclic adenosine monophosphate signal pathway is involved in regulation of triacylglycerol biosynthesis following nitrogen deprivation in Chlamydomonas reinhardtii

The unicellular green alga,Chlamydomonas reinhardtii is a model organism for studying various biological processes,such as photosynthesis,flagellar motility,and lipid metabolism.To find some novel genes regulating the lipid metabolism under various stress conditions,the paromomycin resistance gene aphVIII was transferred into the genome of C.reinhardtii to establish a mutant library.Two genes mutated in two of the TAG-reduced mutants(Cre06.g278111 in M2 mutant,Cre06.g278110 in M6 mutants)were neighboring in the genome,and their expression levels were down-regulated in their corresponding mutants in parallel with their reduced TAG levels following N deprivation.The proteins encoded by these two genes(KCN11 by Cre06.g278111,ACYC3 by Cre06.g278110)contained a conversed cyclic mononucleotide phosphate(cNMP)binding protein and an adenylate domain,respectively.Since cNMP binding protein and adenylate domain have been known as important components of cyclic adenosine monophosphate(cAMP)signaling pathway,suggesting that these two genes might af fect cellular TAG biosynthesis through cAMP signal pathway.

Comparison of Oxidative Stability of Monogalactosyl Diacylglycerol, Digalactosyl Diacylglycerol, and Triacylglycerol Containing Polyunsaturated Fatty Acids

Oxidative stability of three different lipid classes, namely, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) from spinach and edible brown seaweed (Akamoku) and triacylglycerol (TAG) of linseed oil was compared. Analysis of oxygen consumption and polyunsaturated fatty acid (PUFA) composition demonstrated that spinach DGDG had the highest oxidative stability, followed by Akamoku DGDG, Akamoku MGDG, spinach MGDG, and linseed TAG. These results disagree with the order of oxidative stability expected from the average number of bis-allylic positions of each lipid. Additionally, DGDG constituents of both spinach and Akamoku showed higher oxidative stability than their MGDG constituents. The unusual oxidative stability of MGDG and DGDG could be conferred by the protection of bis-allylic positions of the PUFA against oxidative attack by the galactosyl moiety of the GL.

Composition of Triacylglycerols in Fats of Cow and Goat Milk Produced in Four Zones of Mexico

The study of the triacylglycerols (TAG) by gas chromatography (GC) using capillary columns is an efficient technique for the determination of some characteristics of quality of fats and oils. The objective of the present study was to determine by GC the content of TAG present in fat of cow and goat milk produced in four zones of Mexico. According to criteria established in Mexican Standardization, 25 samples were obtained of 1 L of ultra-pasteurized cow milk (UHT) and 27 and 48 of raw cow and goat milk, respectively. The fat was extracted from all of the milk samples by detergent solution, and was stored at –20℃ until its analysis. The chromatographic conditions made it possible to identify and quantify TAG of 28 to 54 numbers of carbons, which were analyzed under descriptive and inferential statistical techniques. For the cow milk fat, the statistical analyses indicated significant difference (p < 0.05) in the TAG C34, C50 and C52, and for the goat milk fat in the TAG of C36 to C52. No equality of means was found among the TAG of the cow and goat milk fat. This study offers an advance in the characterization of the TAG present in the cow and goat milk fat produced in Mexico.

Rapid semi-quantification of triacylglycerols,phosphatidylcholines,and free fatty acids in the rice bran of one grain

We developed a microplate assay method for determining the contents of triacylglycerols（TAGs）, phosphatidylcholines（PCs）, and free fatty acids（FFAs） in the rice bran of one grain using enzymatic reactions. In this method, enzymes from commercially available kits were used. Optimum reaction conditions were established. It was found that Nonidet P-40 was the optimal among the three surfactants used（Triton X-100, Tween 40, and Nonidet P-40） when lipid was dissolved in a reaction solution. Using this method, it was possible to quantify TAGs, PCs, and FFAs in concentration ranges of 7–150, 5–70, and 8–200 mg L-（–1）, respectively. Furthermore, when the TAG contents in the rice bran were measured, the values closely corresponded to those obtained by extracting from large amounts of rice bran. However, sufficient data on the PC and FFA contents in rice bran are not available for valid comparisons. Although this method can accurately quantify the TAG contents in the rice bran of one grain, the accuracy of the PC and FFA contents has not been verified. Hence, future study is necessary.

Fast determination and quantification of triacylglycerols in plant oil by direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS)

Triacylglycerols(TGs)are the main constituents(95%-98%)of plant oil with high nutritional value.They are usually determined by high performance liquid chromatography coupled with mass spectrometry(HPLC-MS)which takes a relatively long time during chromatographic separation.Therefore,in this study,a method about fast determination and quantification of TGs in plant oil based on MALDI-TOF-MS(matrix-assisted laser desorption ionization time-of-flight mass spectrometry)was established.OCNT(oxidized carbon nanotube)and CNT(carbon nanotube)were firstly used as matrix of MALDI-TOF-MS for TGs identification.Satisfyingly,OCNT showed good ionization efficiency,especially when used with CHCA(α-Cyano-4-hydroxy cinnamic acid).Sandwich method was optimized to solve the problem that how matrix and sample were deposited to the plate well and to improve ionization efficiency.Nine calibration curves were established for TGs quantification analysis in plant oil.To verify the accuracy of this method,relative content(%)of fatty acids(FAs)calculated from TGs in peanut oil,rapeseed oil,and soybean oil,respectively,by MALDI-TOF-MS were compared with that calculated from FAMEs(fatty acid methyl esters)by gas chromatography.In general,the results were relatively consistent,proving that this method was convenient,fast and accurate for the determination of main TG molecular species in plant oil.

Research progress on unusual acetyl triacylglycerol

Acetyl triacylglycerol (acetyl-TAG) containing acetyl at sn-3 position is rare in nature. It is optically active asymmetric TAG with low viscosity, having improved cold temperature property and low calorific content compared to regular TAGs. It gains increasingly attentions due to its potential applications. Metabolic engineering of plant or microorganism for production of acetyl-TAG is currently attractive. Here we summarize the researches on acetyl-TAG, with emphasis on gene discovery, protein analysis and its potential applications. Comprehensive understanding of current development in acetyl-TAG biosynthesis pathway may contribute to the increase of production and more applications.

Impact of plant polyphenols on triacylglycerols and phospholipids in rat liver fed with high fat diet

The effect of plant (rapeseed and grape seed) polyphenols on lipid metabolism of rat fed with high fat diet was studied. 30 rats were divided into 5 groups including group Z (basal diet), C (high fat diet without plant polyphenol), S (high fat diet with rapeseed polyphenols), R (high fat diet with grape seed polyphenols) and F (combined with rapeseed and grape seed polyphenols). Levels of lipids including TAGs and PLs in rats were investigated by shotgun-electrospray ionization tandem mass spectrometry (shotgun-ESI-MS). Results showed that high fat diet would give rise to the total TAGs and decreased total phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) (p < 0.05) in group C, S, R and F, compared with group Z. Results also showed that supplementary with rapeseed polyphenols or grape seed polyphenols reduced total TAGs and increased total PC, PE and PS (p < 0.05) in group S, R and F, compared with group C. Grape seed polyphenols and rapeseed polyphenols had certain regulatory effect on metabolism diseases such as fatty liver.

高三酰甘油血症-腰围表型与2型糖尿病发病风险:一项前瞻性队列研究

背景高三酰甘油血症-腰围表型是2型糖尿病(T2DM)的风险因素,不同性别、不同地区、不同年龄段及超重/肥胖人群中T2DM发病风险可能不同。目的探讨不同性别、不同地区、不同年龄段及超重/肥胖人群高三酰甘油血症-腰围表型与T2DM发病风险的关系,为糖尿病防控提供科学依据。方法本研究为前瞻性队列研究,2010年采取分层整群随机抽样方法,以贵州省12个县(市、区)18岁及以上9280名常住居民为队列人群进行基线调查。于2016—2020年进行随访,共随访到8163人,剔除基线时糖尿病患者、相关指标信息缺失者、失访和死亡的对象,最终纳入6743名调查对象。根据基线腰围(WC)和血清三酰甘油(TG)水平将调查对象分为4组,正常腰围和正常血清三酰甘油(NWNT)组:男性腰围<90 cm或女性腰围<85 cm、血清三酰甘油<1.7 mmol/L;正常腰围和高血清三酰甘油(NWHT)组:男性腰围<90 cm或女性腰围<85 cm、血清三酰甘油≥1.7 mmol/L;腰围增大和正常血清三酰甘油(EWNT)组:男性腰围≥90 cm或女性腰围≥85 cm、血清三酰甘油<1.7 mmol/L;腰围增大和高血清三酰甘油(EWHT)组:男性腰围≥90 cm或女性腰围≥85 cm、血清三酰甘油≥1.7 mmol/L。采用Cox比例风险回归模型分析高三酰甘油血症-腰围表型分组与新发T2DM之间的关系。结果中位随访时间6.58年,6743名调查对象中新发T2DM 706例,发病率为10.47%。在调整了相关的混杂因素后,Cox比例风险回归分析显示,EWHT组T2DM发病风险是NWNT组的1.486倍(HR=1.486,95%CI=1.185~1.865,P=0.001)。亚组多因素Cox比例风险回归分析显示,在男性、农村、年龄<45岁、超重/肥胖人群中,EWHT组T2DM发病风险分别是NWNT组的1.792倍(HR=1.792,95%CI=1.297~2.476,P<0.001)、1.483倍(HR=1.483,95%CI=1.115~1.971,P=0.007)、1.540倍(HR=1.540,95%CI=1.083~2.190,P=0.016)、1.614倍(HR=1.614,95%CI=1.123~2.321,P=0.010)。结论对大样本人群进行中位随访6.58年后,高三酰甘油血症-腰围表型是贵州省T2DM的发病风险,在男性、农村、年龄<45岁、超重/肥胖人群中更为显著。因此,对这类人群应该加强健康教育,通过合理饮食、适当运动从而控制体质量,降低血清三酰甘油水平是预防T2DM发病的必要手段和有效的方法。

Involvement of phosphatidate phosphatase in the biosynthesis of triacylglycerols in Chlamydomonas reinhardtii

Lipid biosynthesis is essential for eukaryotic cells, but the mechanisms of the process in microalgae remain poorly understood. Phosphatidic acid phosphohydrolase or 3-sn-phosphatidate phosphohydrolase(PAP) catalyzes the dephosphorylation of phosphatidic acid to form diacylglycerols and inorganic orthophosphates. This reaction is integral in the synthesis of triacylglycerols. In this study, the mRNA level of the PAP isoform CrPAP2 in a species of Chlamydomonas was found to increase in nitrogen-free conditions. Silencing of the CrPAP2 gene using RNA interference resulted in the decline of lipid content by 2.4%–17.4%. By contrast, over-expression of the CrPAP2 gene resulted in an increase in lipid content by 7.5%–21.8%. These observations indicate that regulation of the CrPAP2 gene can control the lipid content of the algal cells. In vitro CrPAP2 enzyme activity assay indicated that the cloned CrPAP2 gene exhibited biological activities.

Effect of different exogenous fatty acids on the cytosolic triacylglycerol content in bovine mammary cells

The objective of this study was to determine how cytosolic triacylglycerols(TAG) are stored in mammary cells and whether this depends on the individual chemical configuration of fatty acids(FA). This objective was accomplished by addition of different FA to a FA-free medium used to culture mammary alveolar cells-large T antigen cells(MAC-T). Treatments consisted of adding FA(palmitate, stearate, oleate, linoleate,rumenic acid [CLA], elaidate and vaccinate) solutions to the medium at 100, 200, 300 and 400 mmol/L concentrations for a 24-h incubation period. At the end of each incubation period, cytosolic TAG, DNA and protein contents were measured. Palmitate, vaccenate, linoleate and CLA increased(P < 0.05) cytosolic TAG(μg/mg protein). Palmitate and CLA increased(P < 0.05) cytosolic TAG adjusted for DNA content. Overall, effects on cytosolic TAG accumulation depended on individual FA structure(chain length, degree of saturation, and number and orientation of FA double bonds). In addition, the long-chain FA used in this study did not have a detrimental effect on MAC-T cells as indicated by cytosolic protein and DNA contents reflecting their biological role in lipid accumulation.

The role of glutathione-mediated triacylglycerol synthesis in the response to ultra-high cadmium stress in Auxenochlorella protothecoides

Under ultra-high cadmium(Cd)stress,large amounts of glutathione are produced in Auxenochlorella protothecoides UTEX 2341,and the lipid content increases significantly.Glutathione is the best reductant that can effectively remove Cd,but the relationship between lipid accumulation and the cellular response to Cd stress has not been ascertained.Integrating analyses of the transcriptomes and lipidomes,the mechanism of lipid accumulation to Cd tolerance were studied from the perspectives of metabolism,transcriptional regulation and protein glutathionylation.Under Cd stress,basic metabolic pathways,such as purine metabolism,translation and pre-m RNA splicing process,were inhibited,while the lipid accumulation pathway was significantly activated.Further analysis revealed that the transcription factors(TFs)and genes related to lipid accumulation were also activated.Analysis of the TF interaction sites showed that ABI5,MYBrel and NF-YB could further regulate the expression of diacylglycerol acyltransferase through glutathionylation/deglutathionylation,which led to increase of the triacylglycerol(TAG)content.Lipidomes analysis showed that TAG could help maintain lipid homeostasis by adjusting its saturation/unsaturation levels.This study for the first time indicated that glutathione could activate TAG synthesis in microalga A.protothecoides,leading to TAG accumulation and glutathione accumulation under Cd stress.Therefore,the accumulation of TAG and glutathione can confer resistance to high Cd stress.This study provided insights into a new operation mode of TAG accumulation under heavy metal stress.

Virtual Substrates Screening Model of Triacylglycerol Lipase from Bacillus thermocatenulanatus

A reliable 3-D structure of Triacylglycerol lipase from Bacillus thermocatenulanatus was constructed by homology modeling. Under molecular dynamics simulation, it was refined and checked. The model was further used as a receptor to search binding sites and carry out flexible docking with a range of substrates, whose enzyme activities were already measured. By inputting a series of docking results, virtual substrates screening models were established and assessed. Monadic nonlinear solution demanded less data but was weak in fitting enzyme activity data with little difference; its mean absolute percentage error （MAPE） of regression was 0.67 and mean square error （MSE） was 1.73 U/mg. Both quadratic stepwise regression and BP neural network were good in regression and prediction; however, more data were required. In the cross-validation of 12 groups, overall MAPE of regression and prediction for the former were 0.18 and 0.49, while the latter was 0.55 and 0.36. MSE values for these two methods were 0.95 and 1.20 U/mg, respectively. Therefore, monadic nonlinear regression model can be used as a preliminary screening one; quadratic stepwise regression and BP neural network approach can be applied to precise screening.

Triacylglycerol lipases of the yeast

All eukaryotes including the yeast contain a lipid storage compartment which is named lipid particle,lipid droplet or oil body.Lipids accumulating in this subcellular fraction serve as a depot of energy and building blocks for membrane lipid synthesis.In the yeast,the major storage lipids are triacylglycerols(TGs)and steryl esters(SEs).An important step in the life cycle of these non-polar lipids is their mobilization from their site of storage and channeling of their degradation components to the appropriate metabolic pathways.A key step in this mobilization process is hydrolysis of TG and SE which is accomplished by lipases and hydrolases.In this review,we describe our recent knowledge of TG lipases from the yeast based on biochemical,molecular biological and cell biological information.We report about recent findings addressing the versatile role of TG lipases in lipid metabolism,and discuss non-polar lipid homeostasis and its newly discovered links to various cell biological processes in the yeast.

Synthesis of symmetrical medium-and long-chain triacylglycerols rich in arachidonic acid at sn-2 position for infant formula

Medium-and long-chain triacylglycerols(MLCT)rich in arachidonic acid(ARA)at sn-2 position were synthesized by a two-step enzymatic method.Firstly,sn-2 monoacylglycerols(MAG)were synthesized at a temperature of 25℃ by enzymatic alcoholysis.The MAG with 69.42%ARA at sn-2 position were obtained by solvent extraction and low temperature solvent crystallization.Secondly,the MLCT rich in ARA at sn-2 position and capric acid(CA)at sn-1,3 positions were produced by enzymatic esterification.Under the optimal conditions(MAG:CA=1:3(mol/mol),0.05 MPa vacuum,8% Lipozyme RM IM,5 h,25℃),the content of triacylglycerol was up to 93.60%.The triacylglycerol in the form of C10:0-C20:4-C10:0(including isomers)was about 40.43%.The ARA contents in the total and sn-2 fatty acid composition of the final product were 32.35%and 51.12%,respectively.MLCT rich in ARA at sn-2 position were successfully produced and the product has the potential application for functional food and infant formula.

畜禽动物油甘油二酯的酶法制备及应用研究进展

1,3-甘油二酯(diacylglycerol,DAG)是公认的安全食品,在人体中的代谢途径不同于甘油三酯,具有提高人体新陈代谢水平以及预防肥胖和心血管疾病等功效。然而,天然DAG在食用油中的质量分数不及10%,因此,近年来高纯度DAG的制备受到了广泛关注。与化学法相比,酶法因绿色、安全、选择性高等优点成为了油脂工业制备DAG的首选。畜禽动物油具有特殊的风味和一定的营养价值,但其高饱和脂肪、高胆固醇和高熔点等理化特征致使其在食品工业的利用受到了极大限制。鉴于此,合成畜禽动物油DAG成为了实现畜禽动物油高值化利用的一种有效途径。本文综述了酶法制备畜禽动物油DAG的研究现状及其潜在的应用价值,以期为畜禽动物油DAG的酶法加工及高值化利用提供一定的思路。