Dataset Information

Effect of diacylglycerol acyltransferase 2 overexpression in 3T3-L1 is associated to an increase in mono-unsaturated fatty acid accumulation.

ABSTRACT: BACKGROUND:Fatty acid (FA) composition is the most important parameter affecting the flavor and nutritional value of the meat. The final and the only committed step in the biosynthesis of triglycerides is catalyzed by diacylglycerol acyltransferase 2 (DGAT2). The role of DGAT2 in lipid accumulation has been demonstrated in adipocytes, However, little is known about the effect of DGAT2 on the FA composition of these cells. METHODS:To investigate the role of DGAT2 in regulating lipid accumulation, FA composition and the expression of adipogenic genes, we cloned the open reading frame of the porcine DGAT2 gene and established 3T3-L1 cells that overexpressed DGAT2. Cells were then cultured in differentiation medium (DM) without FA, with a mixture of FAs (FA-DM), or containing a (13)C stable isotope-labeled FA mixture (IFA-DM). The FA composition of adipocytes was analyzed by gas chromatography-mass spectrometry and gas chromatography-isotope ratio mass spectrometry. Quantitative PCR and western blotting were employed to detect expression of adipogenic genes in 3T3-L1 adipocytes cultured with FA-DM for 12 d. RESULTS:The triacylglyceride (TAG) content was significantly higher in 3T3-L1 adipocytes overexpressing DGAT2 than in control cells. When cultured in DM or FA-DM for 12 d, cells overexpressing DGAT2 showed a higher proportion of unsaturated FAs (C16:1 and C18:1). However, when cells overexpressing DGAT2 were cultured with FA-DM for 30 min, the FA composition was almost identical to that of controls. Further, the proportion of stable isotope-labeled FAs were similar in 3T3-L1 adipocytes overexpressing DGAT2 and control cells cultured in IFA-DM for 12 d. These results collectively indicate that the higher proportion of mono-unsaturated FAs, C16:1 and C18:1, may originate from de novo FA synthesis but not from the uptake of specific FAs from the medium. This hypothesis is further supported by evidence that both mRNA and protein expression of genes involved in FA synthesis (ACACA, FASN, SCD1, and A-FABP) were significantly higher in cells overexpressing DGAT2 than in control cells. CONCLUSIONS:In conclusion, our study revealed that TAG accumulation, the proportion of MUFAs, and the expression of adipogenic genes were higher in 3T3-L1 cells overexpressing DGAT2 than in control cells.

SUBMITTER: Zhang Z

PROVIDER: S-EPMC4075931 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Effect of diacylglycerol acyltransferase 2 overexpression in 3T3-L1 is associated to an increase in mono-unsaturated fatty acid accumulation.

Zhang Zhiqi Z Shu Gang G Zhu Xiaotong X Guo Junming J Cai Han H Wang Songbo S Wang Lina L Gao Ping P Xi Qianyun Q Zhang Yongliang Y Yuan Li L Jiang Qingyan Q

Journal of animal science and biotechnology 20140528 1

<h4>Background</h4>Fatty acid (FA) composition is the most important parameter affecting the flavor and nutritional value of the meat. The final and the only committed step in the biosynthesis of triglycerides is catalyzed by diacylglycerol acyltransferase 2 (DGAT2). The role of DGAT2 in lipid accumulation has been demonstrated in adipocytes, However, little is known about the effect of DGAT2 on the FA composition of these cells.<h4>Methods</h4>To investigate the role of DGAT2 in regulating lipi ...[more]

PMID: 24982757

Similar Datasets

Project description:We previously found that SNF2, a gene encoding a transcription factor forming part of the SWI/SNF (switching/sucrose non-fermenting) chromatin-remodelling complex, is involved in lipid accumulation, because the Deltasnf2 disruptant of Saccharomyces cerevisiae has a higher lipid content. The present study was conducted to identify other factors that might further increase lipid accumulation in the Deltasnf2 disruptant. First, expression of LEU2 (a gene encoding beta-isopropylmalate dehydrogenase), which was used to select transformed strains by complementation of the leucine axotroph, unexpectedly increased both growth and lipid accumulation, especially in the Deltasnf2 disruptant. The effect of LEU2 expression on growth and lipid accumulation could be reproduced by adding large amounts of leucine to the culture medium, indicating that the effect was not due to Leu2p (beta-isopropylmalate dehydrogenase) itself, but rather to leucine biosynthesis. To increase lipid accumulation further, genes encoding the triacylglycerol biosynthetic enzymes diacylglycerol acyltransferase (DGA1) and phospholipid:diacylglycerol acyltransferase (LRO1) were overexpressed in the Deltasnf2 disruptant. Overexpression of DGA1 significantly increased lipid accumulation, especially in the Deltasnf2 disruptant, whereas LRO1 overexpression decreased lipid accumulation in the Deltasnf2 disruptant. Furthermore, the effect of overexpression of acyl-CoA synthase genes (FAA1, FAA2, FAA3 and FAA4), which each supply a substrate for Dga1p (diacylglycerol acyltransferase), was investigated. Overexpression of FAA3, together with that of DGA1, did not further increase lipid accumulation in the Deltasnf2 disruptant, but did enhance lipid accumulation in the presence of exogenous fatty acids. Lastly, the total lipid content in the Deltasnf2 disruptant transformed with DGA1 and FAA3 overexpression vectors reached approx. 30%, of which triacylglycerol was the most abundant lipid. Diacylglycerol acyltransferase activity was significantly increased in the Deltasnf2 disruptant strain overexpressing DGA1 as compared with the wild-type strain overexpressing DGA1; this higher activity may account for the prominent increase in lipid accumulation in the Deltasnf2 disruptant with DGA1 overexpression. The strains obtained have a lipid content that is high enough to act as a model of oleaginous yeast and they may be useful for the metabolic engineering of lipid production in yeast.

Project description:Diacylglycerol acyltransferase (DGAT) is the rate-limiting enzyme in triacylglycerol biosynthesis in eukaryotic organisms. Triacylglycerols are important energy-storage oils in plants such as peanuts, soybeans and rape. In this study, Arachis hypogaea type 2 DGAT (AhDGAT2) genes were cloned from the peanut cultivar 'Luhua 14' using a homologous gene sequence method and rapid amplification of cDNA ends. To understand the role of AhDGAT2 in triacylglycerol biosynthesis, two AhDGAT2 nucleotide sequences that differed by three amino acids were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli Rosetta (DE3). Following IPTG induction, the isozymes (AhDGAT2a and AhDGAT2b) were expressed as 64.5 kDa GST fusion proteins. Both AhDGAT2a and AhDGAT2b occurred in the host cell cytoplasm and inclusion bodies, with larger amounts in the inclusion bodies. Overexpression of AhDGATs depressed the host cell growth rates relative to non-transformed cells, but cells harboring empty-vector, AhDGAT2a-GST, or AhDGAT2b-GST exhibited no obvious growth rate differences. Interestingly, induction of AhDGAT2a-GST and AhDGAT2b-GST proteins increased the sizes of the host cells by 2.4-2.5 times that of the controls (post-IPTG induction). The total fatty acid (FA) levels of the AhDGAT2a-GST and AhDGAT2a-GST transformants, as well as levels of C12:0, C14:0, C16:0, C16:1, C18:1n9c and C18:3n3 FAs, increased markedly, whereas C15:0 and C21:0 levels were lower than in non-transformed cells or those containing empty-vectors. In addition, the levels of some FAs differed between the two transformant strains, indicating that the two isozymes might have different functions in peanuts. This is the first time that a full-length recombinant peanut DGAT2 has been produced in a bacterial expression system and the first analysis of its effects on the content and composition of fatty acids in E. coli. Our results indicate that AhDGAT2 is a strong candidate gene for efficient FA production in E. coli.

Project description:Microalgae are promising sources of lipid triacylglycerol (TAG) for biodiesel production. However, to date, microalgal biodiesel is technically feasible, but not yet economically viable. Increasing TAG content and productivity are important to achieve economic viability of microalgal biodiesel. To increase TAG content, oleaginous microalga Neochloris oleoabundans was genetically engineered with an endogenous key enzyme diacylglycerol acyltransferase 2 (NeoDGAT2) responsible for TAG biosynthesis.The integration of NeoDGAT2 expression cassettes in N. oleoabundans transformant was confirmed by PCR. The neutral lipid accumulation in the transformant detected by Nile red staining was accelerated and 1.9-fold higher than in wild type; the lipid bodies in the transformant visualized under fluorescence microscope were also larger. The NeoDGAT2 transcript was two-fold higher in the transformant than wild type. Remarkably higher lipid accumulation was found in the transformant than wild type: total lipid content increased 1.6-to 2.3-fold up to 74.5 ± 4.0% dry cell weight (DCW) and total lipid productivity increased 1.6- to 3.2-fold up to 14.6 ± 2.0 mg/L/day; while TAG content increased 1.8- to 3.2-fold up to 46.1 ± 1.6% DCW and TAG productivity increased 1.6- to 4.3-fold up to 8.9 ± 1.3 mg/L/day. A significantly altered fatty acid composition was detected in the transformant compared to wild type; the levels of saturated fatty acid C16:0 increased double to 49%, whereas C18:0 was reduced triple to 6%. Long-term stability was observed in the transformant continuously maintained in solid medium over 100 generations in a period of about 4 years.Our results demonstrate the increased TAG content and productivity in N. oleoabundans by NeoDGAT2 overexpression that may offer the first step towards making microalgae an economically feasible source for biodiesel production. The strategy for genetically improved microalga presented in this study can be applied to other microalgal species possessing desired characteristics for industrial biofuel production.

Dataset Information

Effect of diacylglycerol acyltransferase 2 overexpression in 3T3-L1 is associated to an increase in mono-unsaturated fatty acid accumulation.

Publications

Effect of diacylglycerol acyltransferase 2 overexpression in 3T3-L1 is associated to an increase in mono-unsaturated fatty acid accumulation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets