MicroRNA-193a-5p Regulates the Synthesis of Polyunsaturated Fatty Acids by Targeting Fatty Acid Desaturase 1 (FADS1) in Bovine Mammary Epithelial Cells.

Cardiovascular diseases (CVDs) are seriously threatening to human life and health. Polyunsaturated fatty acids (PUFAs) are known for their role in preventing CVDs. It is beneficial to population health to promote the content of PUFAs in bovine milk. In recent years, limited research based on molecular mechanisms has focused on this field. The biological roles of numerous microRNAs (miRNAs) remain unknown. In this study, a promising and negatively correlated pair of the miRNA (miRNA-193a-5p) and a fatty acid desaturase 1 (FADS1) gene are identified and screened to explore whether they are potential factors of PUFAs' synthesis in bovine milk. The targeted relationship between miRNA-193a-5p and FADS1 in bovine mammary epithelial cells (BMECs) is demonstrated by dual luciferase reporter assays. qRT-PCR and western blot assays indicate that both the expression of mRNA and the protein FADS1 show a negative correlation with miRNA-193a-5p expression in BMECs. Also, miR-193a-5p expression is positively correlated with the expression of genes associated with milk fatty acid metabolism, including ELOVL fatty acid elongase 6 (ELOVL6) and diacylglycerol O-acyltransferase 2 (DGAT2). The expression of fatty acid desaturase 2 (FADS2) is negatively correlated with miR-193a-5p expression in BMECs. The contents of triglycerides (TAG), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) have a significant positive correlation with the expression of FADS1 and a significant negative correlation with the expression of miR-193a-5p in BMECs. For the first time, this study confirms that miRNA-193a-5p regulates PUFAs metabolism in BMECs by targeting FADS1, indicating that miRNA-193a-5p and FADS1 are underlying factors that improve PUFAs content in bovine milk.

mTORC2 Regulates Lipogenic Gene Expression through PPARγ to Control Lipid Synthesis in Bovine Mammary Epithelial Cells.

The mechanistic target of rapamycin complex 2 (mTORC2) primarily functions as an effector of insulin/PI3K signaling to regulate cell proliferation and is associated with cell metabolism. However, the function of mTORC2 in lipid metabolism is not well understood. In the present study, mTORC2 was inactivated by the ATP-competitive mTOR inhibitor AZD8055 or shRNA targeting RICTOR in primary bovine mammary epithelial cells (pBMECs). MTT assay was performed to examine the effect of AZD8055 on cell proliferation. ELISA assay and GC-MS analysis were used to determine the content of lipid. The mRNA and protein expression levels were investigated by RT/real-time PCR and western blot analysis, respectively. We found that cell proliferation, mTORC2 activation, and lipid secretion were inhibited by AZD8055. RICTOR was knocked down and mTORC2 activation was specifically attenuated by the shRNA. Compared to control cells, the expression of the transcription factor gene PPARG and the lipogenic genes LPIN1, DGAT1, ACACA, and FASN was downregulated in RICTOR silencing cells. As a result, the content of intracellular triacylglycerol (TAG), palmitic acid (PA), docosahexaenoic acid (DHA), and other 16 types of fatty acid was decreased in the treated cells; the accumulation of TAG, PA, and DHA in cell culture medium was also reduced. Overall, mTORC2 plays a critical role in regulating lipogenic gene expression, lipid synthesis, and secretion in pBMECs, and this process probably is through PPARγ. This finding provides a model by which lipogenesis is regulated in pBMECs.

Multiple mechanisms contribute to increased neutral lipid accumulation in yeast producing recombinant variants of plant diacylglycerol acyltransferase 1.

The apparent bottleneck in the accumulation of oil during seed development in some oleaginous plant species is the formation of triacylglycerol (TAG) by the acyl-CoA-dependent acylation of sn-1,2-diacylglycerol catalyzed by diacylglycerol acyltransferase (DGAT, EC 2.3.1.20). Improving DGAT activity using protein engineering could lead to improvements in seed oil yield (e.g. in canola-type Brassica napus). Directed evolution of B. napus DGAT1 (BnaDGAT1) previously revealed that one of the regions where amino acid residue substitutions lead to higher performance in BnaDGAT1 is in the ninth predicted transmembrane domain (PTMD9). In this study, several BnaDGAT1 variants with amino acid residue substitutions in PTMD9 were characterized. Among these enzyme variants, the extent of yeast TAG production was affected by different mechanisms, including increased enzyme activity, increased polypeptide accumulation, and possibly reduced substrate inhibition. The kinetic properties of the BnaDGAT1 variants were affected by the amino acid residue substitutions, and a new kinetic model based on substrate inhibition and sigmoidicity was generated. Based on sequence alignment and further biochemical analysis, the amino acid residue substitutions that conferred increased TAG accumulation were shown to be present in the DGAT1-PTMD9 region of other higher plant species. When amino acid residue substitutions that increased BnaDGAT1 enzyme activity were introduced into recombinant Camelina sativa DGAT1, they also improved enzyme performance. Thus, the knowledge generated from directed evolution of DGAT1 in one plant species can be transferred to other plant species and has potentially broad applications in genetic engineering of oleaginous crops and microorganisms.

Analysis of Differentially Expressed Genes in Gastrocnemius Muscle between DGAT1 Transgenic Mice and Wild-Type Mice.

Adipose tissue was the major energy deposition site of the mammals and provided the energy for the body and released the external pressure to the internal organs. In animal production, fat deposition in muscle can affect the meat quality, especially the intramuscular fat (IMF) content. Diacylglycerol acyltransferase-1 (DGAT1) was the key enzyme to control the synthesis of the triacylglycerol in adipose tissue. In order to better understand the regulation mechanism of the DGAT1 in the intramuscular fat deposition, the global gene expression profiling was performed in gastrocnemius muscle between DGAT1 transgenic mice and wild-type mice by microarray. 281 differentially expressed transcripts were identified with at least 1.5-fold change and the p value < 0.05. 169 transcripts were upregulated and 112 transcripts were downregulated. Ten genes (SREBF1, DUSP1, PLAGL1, FKBP5, ZBTB16, PPP1R3C, CDC14A, GLUL, PDK4, and UCP3) were selected to validate the reliability of the chip's results by the real-time PCR. The finding of RT-PCR was consistent with the gene chip. Seventeen signal pathways were analyzed using KEGG pathway database and the pathways concentrated mainly on the G-protein coupled receptor protein signaling pathway, signal transduction, oxidation-reduction reaction, olfactory receptor activity, protein binding, and zinc ion binding. This study implied a function role of DGAT1 in the synthesis of TAG, insulin resistance, and IMF deposition.

Overexpression of diacylglycerol acyltransferase in Yarrowia lipolytica affects lipid body size, number and distribution.

In the oleaginous yeast Yarrowia lipolytica, the diacylglycerol acyltransferases (DGATs) are major factors for triacylglycerol (TAG) synthesis. The Q4 strain, in which the four acyltransferases have been deleted, is unable to accumulate lipids and to form lipid bodies (LBs). However, the expression of a single acyltransferase in this strain restores TAG accumulation and LB formation. Using this system, it becomes possible to characterize the activity and specificity of an individual DGAT. Here, we examined the effects of DGAT overexpression on lipid accumulation and LB formation in Y. lipolytica Specifically, we evaluated the consequences of introducing one or two copies of the Y. lipolytica DGAT genes YlDGA1 and YlDGA2 Overall, multi-copy DGAT overexpression increased the lipid content of yeast cells. However, the size and distribution of LBs depended on the specific DGAT overexpressed. YlDGA2 overexpression caused the formation of large LBs, while YlDGA1 overexpression generated smaller but more numerous LBs. This phenotype was accentuated through the addition of a second copy of the overexpressed gene and might be linked to the distinct subcellular localization of each DGAT, i.e. YlDga1 being localized in LBs, while YlDga2 being localized in a structure strongly resembling the endoplasmic reticulum.

Expressional profiling of prostate cancer risk SNPs at 11q13.5 identifies DGAT2 as a new target gene.

A total of nine non-coding variants on 11q13.5 predispose men to prostate cancer (PrCa). rs200331695 within the EMSY intron is associated with aggressive PrCa and two high linkage disequilibrium (LD) groups of single-nucleotide polymorphisms (SNPs) in the intergenic region are associated with PrCa death. Here, the cis-effect of the SNPs on gene expression using expression quantitative trait loci analysis was investigated. The regulatory potential was screened in prostate tumors (n = 41) and in whole blood (n = 99). The results were validated in a second tumor set (n = 41), in lymphoblastoid cell lines (LCLs) (n = 38), and using the GTEx Portal. The effects of haplotypes were analyzed in the whole blood. The high LD SNPs (rs143975731, rs12277366, rs2155225, and rs2155222) were associated with DGAT2 expression in both tumors sets (screening P = 0.035-0.043; validation P = 0.005-0.018). The PrCa death-associated alleles decreased the expression by two-fold. rs200331695 decreased DGAT2 expression in LCLs (P = 0.006). The findings of SNPs regulating CAPN5 (P = 0.026-0.046) and AP001189.4 (P = 0.03-0.039) in the whole blood were not observed in LCLs, but the association with AP001189.4 expression was validated via the GTEx Portal (P = 8.7 × 10(-5) to 4.3 × 10(-4) ), which suggests that the high LD intergenic SNPs exert a tissue-dependent effect on the expression of two genes. No haplotypes including the risk SNPs at 11q13.5 were associated with gene expression and PrCa. The findings indicate the functionality of the PrCa death-predisposing SNPs rs143975731, rs12277366, rs2155225, and rs2155222 as DGAT2 regulators in prostate tumors. © 2016 Wiley Periodicals, Inc.

Hepatic Peroxisome Proliferator-Activated Receptor Gamma Signaling Contributes to Alcohol-Induced Hepatic Steatosis and Inflammation in Mice.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARγ) signaling has been shown to regulate lipogenesis and lipid accumulation. Previous studies have shown that hepatic PPARγ is up-regulated in steatotic liver of both animal and human. However, the effects of hepatic PPARγ signaling on alcoholic liver disease (ALD) remain elusive. METHODS: To determine the role of hepatic PPARγ signaling on ALD, wild-type (WT) and hepatocyte-specific PPARγ knockdown (PPARγ∆Hep) mice were fed a modified Lieber-DeCarli alcohol or isocaloric maltose dextrin control liquid diet for 8 weeks to induce ALD. Blood parameters, hepatic steatosis, and inflammation were measured after 8-week alcohol feeding. RESULTS: Alcohol feeding to WT mice resulted in liver damage (alanine aminotransferase [ALT], 94.68 ± 17.05 U/L; aspartate aminotransferase [AST], 55.87 ± 11.29 U/L), which was significantly alleviated by hepatic PPARγ knockdown (ALT, 57.36 ± 14.98 U/L; AST, 38.06 ± 3.35 U/L). Alcohol feeding led to marked lipid accumulation and up-regulation of lipogenic genes including fatty acid transport protein 1 (FATP1), acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), lipin1 (LIPIN1), diacylglycerol acyltransferase 1 (DGAT1), and diacylglycerol acyltransferase 2 (DGAT2) in the livers of WT mice. Knockdown of hepatic PPARγ significantly alleviated alcohol-induced lipid accumulation and abolished the up-regulation of FASN, DGAT1, and DGAT2. Silencing of PPARγ in FL83B cells significantly decreased ethanol (EtOH)-, linoleic acid-, and EtOH plus linoleic acid-induced lipid accumulation. Knockdown of hepatic PPARγ also significantly reduced alcohol-induced inflammatory chemokine (monocyte chemotactic protein 1 [MCP1], keratinocyte-derived chemokine [KC], interferon gamma-induced protein 10 [IP-10]) and inflammatory infiltration (lymphocyte antigen 6 complex, locus G [Ly6G], and F4/80). CONCLUSIONS: The results suggest that hepatic PPARγ signaling contributes to alcohol-induced liver injury by promoting hepatic steatosis and inflammation.

Study of Valproic Acid-Enhanced Hepatocyte Steatosis.

Valproic acid (VPA) is one of the most widely used antiepilepsy drugs. However, several side effects, including weight gain and fatty liver, have been reported in patients following VPA treatment. In this study, we explored the molecular mechanisms of VPA-induced hepatic steatosis using FL83B cell line-based in vitro model. Using fluorescent lipid staining technique, we found that VPA enhanced oleic acid- (OLA-) induced lipid accumulation in a dose-dependent manner in hepatocytes; this may be due to upregulated lipid uptake, triacylglycerol (TAG) synthesis, and lipid droplet formation. Real-time PCR results showed that, following VPA treatment, the expression levels of genes encoding cluster of differentiation 36 (Cd36), low-density lipoprotein receptor-related protein 1 (Lrp1), diacylglycerol acyltransferase 2 (Dgat2), and perilipin 2 (Plin2) were increased, that of carnitine palmitoyltransferase I a (Cpt1a) was not affected, and those of acetyl-Co A carboxylase α (Acca) and fatty acid synthase (Fasn) were decreased. Furthermore, using immunofluorescence staining and flow cytometry analyses, we found that VPA also induced peroxisome proliferator-activated receptor γ (PPARγ) nuclear translocation and increased levels of cell-surface CD36. Based on these results, we propose that VPA may enhance OLA-induced hepatocyte steatosis through the upregulation of PPARγ- and CD36-dependent lipid uptake, TAG synthesis, and lipid droplet formation.

Metabolic engineering of Pichia pastoris to produce ricinoleic acid, a hydroxy fatty acid of industrial importance.

Ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) has many specialized uses in bioproduct industries, while castor bean is currently the only commercial source for the fatty acid. This report describes metabolic engineering of a microbial system (Pichia pastoris) to produce ricinoleic acid using a "push" (synthesis) and "pull" (assembly) strategy. CpFAH, a fatty acid hydroxylase from Claviceps purpurea, was used for synthesis of ricinoleic acid, and CpDGAT1, a diacylglycerol acyl transferase for the triacylglycerol synthesis from the same species, was used for assembly of the fatty acid. Coexpression of CpFAH and CpDGAT1 produced higher lipid contents and ricinoleic acid levels than expression of CpFAH alone. Coexpression in a mutant haploid strain defective in the Δ12 desaturase activity resulted in a higher level of ricinoleic acid than that in the diploid strain. Intriguingly, the ricinoleic acid produced was mainly distributed in the neutral lipid fractions, particularly the free fatty acid form, but with little in the polar lipids. This work demonstrates the effectiveness of the metabolic engineering strategy and excellent capacity of the microbial system for production of ricinoleic acid as an alternative to plant sources for industrial uses.

Anti-obesity effect of total phenylpropanoid glycosides from Ligustrum robustum Blume in fatty diet-fed mice via up-regulating leptin.

ETHNOPHARMACOLOGICAL RELEVANCE: In Chinese folk medicine, the leaves of Ligustrum robustum Blume (LR) were commonly used in the treatment of obesity and hyperlipidemia. This study aimed to evaluate the anti-obesity effect and mechanisms of total phenylpropanoid glycosides from Ligustrum robustum Blume (LRTPG) in fatty diet-fed C57BL/6J mice. MATERIALS AND METHODS: C57BL/6J mice were divided randomly into 6 groups, i.e., control, model, positive (Orlistat 0.12g/kg), and LRTPG at three dosages (0.3, 0.6 or 1.2g/kg), respectively. Control mice were fed with standard diet; the others were fed with fatty diet. After 4 weeks׳ modeling, therapy mice were intragastrically administrated with positive drug or LRTPG for 5 weeks, respectively. Pharmacodynamic effects including body weight, fat weight, Lee׳s index, serum lipid levels, morphological changes and adipocyte area ratio were evaluated. The mechanisms were explored as the factors related to lipids metabolism in gene expressions by real-time PCR, and assured as the protein level of differential gene by Western blotting. RESULTS: The anti-obesity effects of LRTPG in all treated mice were shown as decreased body weight, fat mass, Lee׳s index, total cholesterol (TC) level, and adipocyte area. The mechanisms were demonstrated as elevated mRNA and protein levels of adipose leptin, and consequently decreasing mRNA of adipose acyl coenzyme A: diacylglycerol acyltransferase (DGAT) with increasing mRNA of hepatic cholesterol 7α-hydroxylase (CYP7A1), which led to inhibition of triglyceride (TG) synthesis and promotion of cholesterol catabolism. CONCLUSIONS: The anti-obesity effect of LRTPG in fatty diet-fed mice was related to the up-regulation of leptin, which may provide scientific evidence supporting the traditional usage of LR on obesity in China.

Intestinal triacylglycerol synthesis in fat absorption and systemic energy metabolism.

The intestine plays a prominent role in the biosynthesis of triacylglycerol (triglyceride; TAG). Digested dietary TAG is repackaged in the intestine to form the hydrophobic core of chylomicrons, which deliver metabolic fuels, essential fatty acids, and other lipid-soluble nutrients to the peripheral tissues. By controlling the flux of dietary fat into the circulation, intestinal TAG synthesis can greatly impact systemic metabolism. Genes encoding many of the enzymes involved in TAG synthesis have been identified. Among TAG synthesis enzymes, acyl-CoA:monoacylglycerol acyltransferase 2 and acyl-CoA:diacylglycerol acyltransferase (DGAT)1 are highly expressed in the intestine. Their physiological functions have been examined in the context of whole organisms using genetically engineered mice and, in the case of DGAT1, specific inhibitors. An emerging theme from recent findings is that limiting the rate of TAG synthesis in the intestine can modulate gut hormone secretion, lipid metabolism, and systemic energy balance. The underlying mechanisms and their implications for humans are yet to be explored. Pharmacological inhibition of TAG hydrolysis in the intestinal lumen has been employed to combat obesity and associated disorders with modest efficacy and unwanted side effects. The therapeutic potential of inhibiting specific enzymes involved in intestinal TAG synthesis warrants further investigation.

The functional characterization of long noncoding RNA SPRY4-IT1 in human melanoma cells.

Expression of the long noncoding RNA (lncRNA) SPRY4-IT1 is low in normal human melanocytes but high in melanoma cells. siRNA knockdown of SPRY4-IT1 blocks melanoma cell invasion and proliferation, and increases apoptosis. To investigate its function further, we affinity purified SPRY4-IT1 from melanoma cells and used mass spectrometry to identify the protein lipin 2, an enzyme that converts phosphatidate to diacylglycerol (DAG), as a major binding partner. SPRY4-IT1 knockdown increases the accumulation of lipin2 protein and upregulate the expression of diacylglycerol O-acyltransferase 2 (DGAT2) an enzyme involved in the conversion of DAG to triacylglycerol (TAG). When SPRY4-IT1 knockdown and control melanoma cells were subjected to shotgun lipidomics, an MS-based assay that permits the quantification of changes in the cellular lipid profile, we found that SPRY4-IT1 knockdown induced significant changes in a number of lipid species, including increased acyl carnitine, fatty acyl chains, and triacylglycerol (TAG). Together, these results suggest the possibility that SPRY4-IT1 knockdown may induce apoptosis via lipin 2-mediated alterations in lipid metabolism leading to cellular lipotoxicity.

Differential effects of low-fat and high-fat diets on fed-state hepatic triacylglycerol secretion, hepatic fatty acid profiles, and DGAT-1 protein expression in obese-prone Sprague-Dawley rats.

The purpose of this study was to compare the effects of short-term low-fat (LF) and high-fat (HF) diets on fed-state hepatic triacylglycerol (TAG) secretion, the content of proteins involved in TAG assembly and secretion, fatty acid oxidation (FAO), and the fatty acid profile of stored TAG. Using selectively bred obese-prone Sprague-Dawley rats, we directly measured fed-state hepatic TAG secretion, using Tyloxapol (a lipoprotein lipase inhibitor) and a standardized oral mixed meal (45% carbohydrate, 40% fat, 15% protein) bolus in animals fed a HF or LF diet for 2 weeks, after which the rats were maintained on their respective diet for 1 week (washout) prior to the liver being excised to measure protein content, FAO, and TAG fatty acid profiles. Hepatic DGAT-1 protein expression was ∼27% lower in HF- than in LF-fed animals (p < 0.05); the protein expression of all other molecules was similar in the 2 diets. The fed-state hepatic TAG secretion rate was ∼39% lower (p < 0.05) in HF- (4.62 ± 0.18 mmol·h(-1)) than in LF- (7.60 ± 0.57 mmol·h(-1)) fed animals. Hepatic TAG content was ∼2-fold higher (p < 0.05) in HF- (1.07 ± 0.15 nmol·g(-1) tissue) than in LF- (0.50 ± 0.16 nmol·g(-1) tissue) fed animals. In addition, the fatty acid profile of liver TAG in HF-fed animals closely resembled the diet, whereas in LF-fed animals, the fatty acid profile consisted of mostly de novo synthesized fatty acids. FAO was not altered by diet. LF and HF diets differentially alter fed-state hepatic TAG secretion, hepatic fatty acid profiles, and DGAT-1 protein expression.

Loss of ß-carotene 15,15'-oxygenase in developing mouse tissues alters esterification of retinol, cholesterol and diacylglycerols.

We provide novel insights into the function(s) of ß-carotene-15,15'-oxygenase (CMOI) during embryogenesis. By performing in vivo and in vitro experiments, we showed that CMOI influences not only lecithin:retinol acyltransferase but also acyl CoA:retinol acyltransferase reaction in the developing tissues at mid-gestation. In addition, LC/MS lipidomics analysis of the CMOI-/- embryos showed reduced levels of four phosphatidylcholine and three phosphatidylethanolamine acyl chain species, and of eight triacylglycerol species with four or more unsaturations and fifty-two or more carbons in the acyl chains. Cholesteryl esters of arachidonate, palmitate, linoleate, and DHA were also reduced to less than 30% of control. Analysis of the fatty acyl CoA species ruled out a loss in fatty acyl CoA synthetase capability. Comparison of acyl species suggested significantly decreased 18:2, 18:3, 20:1, 20:4, or 22:6 acyl chains within the above lipids in CMOI-null embryos. Furthermore, LCAT, ACAT1 and DGAT2 mRNA levels were also downregulated in CMOI-/- embryos. These data strongly support the notion that, in addition to cleaving ß-carotene to generate retinoids, CMOI serves an additional function(s) in retinoid and lipid metabolism and point to its role in the formation of specific lipids, possibly for use in nervous system tissue.

Overexpression of NgAUREO1, the gene coding for aurechrome 1 from Nannochloropsis gaditana, into Saccharomyces cerevisiae leads to a 1.6-fold increase in lipid accumulation.

Aureochrome-1 (AUREO1) is a transcription factor that is induced by blue light and controls branching of Vaucheria frigida. We have cloned the gene, NgAUREO1, coding for AUREO1 from Nannochloropsis gaditana, and report that the lipid content in recombinant Saccharomyces cerevisiae was 1.6-fold more than in wild-type S. cerevisiae (6.3 % lipid increased to 10 %). Over-expression of AUREO1 in S. cerevisiae up-regulated the expression of acetyl-CoA carboxylase and acyl-CoA:diacylglycerol acyl-transferase but down-regulated the expression of long-chain-acyl CoA synthetase. This enhanced the accumulation of lipid. This study highlights a novel function of AUREO1 and allows a better understanding of the regulation mechanism of fatty acid metabolism.

Cell-type-specific regulation of genes involved in testicular lipid metabolism: fatty acid-binding proteins, diacylglycerol acyltransferases, and perilipin 2.

Male germ cell differentiation entails the synthesis and remodeling of membrane polar lipids and the formation of triacylglycerols (TAGs). This requires fatty acid-binding proteins (FABPs) for intracellular fatty acid traffic, a diacylglycerol acyltransferase (DGAT) to catalyze the final step of TAG biosynthesis, and a TAG storage mode. We examined the expression of genes encoding five members of the FABP family and two DGAT proteins, as well as the lipid droplet protein perilipin 2 (PLIN2), during mouse testis development and in specific cells from seminiferous epithelium. Fabp5 expression was distinctive of Sertoli cells and consequently was higher in prepubertal than in adult testis. The expression of Fabp3 increased in testis during postnatal development, associated with the functional differentiation of interstitial cells, but was low in germ cells. Fabp9, together with Fabp12, was prominently expressed in the latter. Their transcripts increased from spermatocytes to spermatids and, interestingly, were highest in spermatid-derived residual bodies (RB). Both Sertoli and germ cells, which produce neutral lipids and store them in lipid droplets, expressed Plin2. Yet, while Dgat1 was detected in Sertoli cells, Dgat2 accumulated in germ cells with a similar pattern of expression as Fabp9. These results correlated with polyunsaturated fatty acid-rich TAG levels also increasing with mouse germ cell differentiation highest in RB, connecting DGAT2 with the biosynthesis of such TAGs. The age- and germ cell type-associated increases in Fabp9, Dgat2, and Plin2 levels are thus functionally related in the last stages of germ cell differentiation.

Intestinal acyl-CoA:diacylglycerol acyltransferase 2 overexpression enhances postprandial triglyceridemic response and exacerbates high fat diet-induced hepatic triacylglycerol storage.

Intestinal acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) is important in the cellular and physiological responses to dietary fat. To determine the effect of increased intestinal DGAT2 on cellular and physiological responses to acute and chronic dietary fat challenges, we generated mice with intestine-specific overexpression of DGAT2 and compared them with intestine-specific overexpression of DGAT1 and wild-type (WT) mice. We found that when intestinal DGAT2 is present in excess, triacylglycerol (TG) secretion from enterocytes is enhanced compared to WT mice; however, TG storage within enterocytes is similar compared to WT mice. We found that when intestinal DGAT2 is present in excess, mRNA levels of genes involved in fatty acid oxidation were reduced. This result suggests that reduced fatty acid oxidation may contribute to increased TG secretion by overexpression of DGAT2 in intestine. Furthermore, this enhanced supply of TG for secretion in Dgat2(Int) mice may be a significant contributing factor to the elevated fasting plasma TG and exacerbated hepatic TG storage in response to a chronic HFD. These results highlight that altering fatty acid and TG metabolism within enterocytes has the capacity to alter systemic delivery of dietary fat and may serve as an effective target for preventing and treating metabolic diseases such as hepatic steatosis.

Overexpression of the active diacylglycerol acyltransferase variant transforms Saccharomyces cerevisiae into an oleaginous yeast.

Lipid production by Saccharomyces cerevisiae was improved by overexpression of the yeast diacylglycerol acyltransferase Dga1p lacking the N-terminal 29 amino acids (Dga1∆Np), which was previously found to be an active form in the ∆snf2 mutant. Overexpression of Dga1∆Np in the ∆snf2 mutant, however, did not increase lipid content as expected, which prompted us to search for a more suitable strain in which to study the role of Dga1∆Np in lipid accumulation. We found that the overexpression of Dga1∆Np in the ∆dga1 mutant effectively increased the lipid content up to about 45 % in the medium containing 10 % glucose. The high lipid content of the transformant was dependent on glucose concentration, nitrogen limitation, and active leucine biosynthesis. To better understand the effect of dga1 disruption on the ability of Dga1∆Np to stimulate lipid accumulation, the ∆dga1-1 mutant, in which the 3'-terminal 36 bp of the dga1 open reading frame (ORF) remained, and the ∆dga1-2 mutant, in which the 3'-terminal 36 bp were also deleted, were prepared with URA3 disruption cassettes. Surprisingly, the overexpression of Dga1∆Np in the ∆dga1-1 mutant had a lower lipid content than the original ∆dga1 mutant, whereas overexpression in the ∆dga1-2 mutant led to a high lipid content of about 45 %. These results indicated that deletion of the 3' terminal region of the dga1 ORF, rather than abrogation of genomic Dga1p expression, was crucial for the effect of Dga1∆Np on lipid accumulation. To investigate whether dga1 disruption affected gene expression adjacent to DGA1, we found that the overexpression of Esa1p together with Dga1∆Np in the ∆dga1 mutant reverted the lipid content to the level of the wild-type strain overexpressing Dga1∆Np. In addition, RT-qPCR analysis revealed that ESA1 mRNA expression in the ∆dga1 mutant was decreased compared to the wild-type strain at the early stages of culture, suggesting that lowered Esa1p expression is involved in the effect of dga1 disruption on Dga1∆Np-dependent lipid accumulation. These results provide a new strategy to engineer S. cerevisiae for optimal lipid production.

Identification and characterization of PtDGAT2B, an acyltransferase of the DGAT2 acyl-coenzyme A: diacylglycerol acyltransferase family in the diatom Phaeodactylum tricornutum.

Diacylglycerol acyltransferase (DGAT) plays a pivotal role in triacylglycerol (TAG) formation in some oleaginous organisms. We describe here the identification of a type 2 DGAT (PtDGAT2B) in the diatom Phaeodactylum tricornutum that contains four putative type 2 acyl-CoA:DGATs, sharing little sequence similarity with each other. TAG synthesis and lipid body formation could be completely restored in a Saccharomyces cerevisiae TAG-deficient quadruple mutant by expressing PtDGAT2B. Up-regulation of PtDGAT2B precedes the accumulation of TAG. Functional analysis of enzyme activity in vivo demonstrated that expression of PtDGAT2B can increase the proportion of unsaturated C(16) and C(18) fatty acids in yeast TAG.

Synergistic effect of WRI1 and DGAT1 coexpression on triacylglycerol biosynthesis in plants.

Metabolic engineering approaches to increase plant oil levels can generally be divided into categories which increase fatty acid biosynthesis ('Push'), are involved in TAG assembly ('Pull') or increase TAG storage/decrease breakdown ('Accumulation'). In this study, we describe the surprising synergy when Push (WRI1) and Pull (DGAT1) approaches are combined. Co-expression of these genes in the Nicotiana benthamiana transient leaf expression system resulted in TAG levels exceeding those expected from an additive effect and biochemical tracer studies confirmed increased flux of carbon through fatty acid and TAG synthesis pathways. Leaf fatty acid profile also synergistically shifts from polyunsaturated to monounsaturated fatty acids.