Expression Of Diacylglycerol Acyltransferase Research Articles

Epichloë fungal endophyte interactions in perennial ryegrass (Lolium perenne L.) modified to accumulate foliar lipids for increased energy density

BackgroundCommercial cultivars of perennial ryegrass infected with selected Epichloë fungal endophytes are highly desirable in certain pastures as the resulting mutualistic association has the capacity to confer agronomic benefits (such as invertebrate pest deterrence) largely due to fungal produced secondary metabolites (e.g., alkaloids). In this study, we investigated T2 segregating populations derived from two independent transformation events expressing diacylglycerol acyltransferase (DGAT) and cysteine oleosin (CO) genes designed to increase foliar lipid and biomass accumulation. These populations were either infected with Epichloë festucae var. lolii strain AR1 or Epichloë sp. LpTG-3 strain AR37 to examine relationships between the introduced trait and the endophytic association. Here we report on experiments designed to investigate if expression of the DGAT + CO trait in foliar tissues of perennial ryegrass could negatively impact the grass-endophyte association and vice versa. Both endophyte and plant characters were measured under controlled environment and field conditions.ResultsExpected relative increases in total fatty acids of 17–58% accrued as a result of DGAT + CO expression with no significant difference between the endophyte-infected and non-infected progeny. Hyphal growth in association with DGAT + CO expression appeared normal when compared to control plants in a growth chamber. There was no significant difference in mycelial biomass for both strains AR1 and AR37, however, Epichloë-derived alkaloid concentrations were significantly lower on some occasions in the DGAT + CO plants compared to the corresponding null-segregant progenies, although these remained within the reported range for bioactivity.ConclusionsThese results suggest that the mutualistic association formed between perennial ryegrass and selected Epichloë strains does not influence expression of the host DGAT + CO technology, but that endophyte performance may be reduced under some circumstances. Further investigation will now be required to determine the preferred genetic backgrounds for introgression of the DGAT + CO trait in combination with selected endophyte strains, as grass host genetics is a major determinant to the success of the grass-endophyte association in this species.

Intron-mediated enhancement of DIACYLGLYCEROL ACYLTRANSFERASE1 expression in energycane promotes a step change for lipid accumulation in vegetative tissues

BackgroundMetabolic engineering for hyperaccumulation of lipids in vegetative tissues is a novel strategy for enhancing energy density and biofuel production from biomass crops. Energycane is a prime feedstock for this approach due to its high biomass production and resilience under marginal conditions. DIACYLGLYCEROL ACYLTRANSFERASE (DGAT) catalyzes the last and only committed step in the biosynthesis of triacylglycerol (TAG) and can be a rate-limiting enzyme for the production of TAG.ResultsIn this study, we explored the effect of intron-mediated enhancement (IME) on the expression of DGAT1 and resulting accumulation of TAG and total fatty acid (TFA) in leaf and stem tissues of energycane. To maximize lipid accumulation these evaluations were carried out by co-expressing the lipogenic transcription factor WRINKLED1 (WRI1) and the TAG protect factor oleosin (OLE1). Including an intron in the codon-optimized TmDGAT1 elevated the accumulation of its transcript in leaves by seven times on average based on 5 transgenic lines for each construct. Plants with WRI1 (W), DGAT1 with intron (Di), and OLE1 (O) expression (WDiO) accumulated TAG up to a 3.85% of leaf dry weight (DW), a 192-fold increase compared to non-modified energycane (WT) and a 3.8-fold increase compared to the highest accumulation under the intron-less gene combination (WDO). This corresponded to TFA accumulation of up to 8.4% of leaf dry weight, a 2.8-fold or 6.1-fold increase compared to WDO or WT, respectively. Co-expression of WDiO resulted in stem accumulations of TAG up to 1.14% of DW or TFA up to 2.08% of DW that exceeded WT by 57-fold or 12-fold and WDO more than twofold, respectively. Constitutive expression of these lipogenic “push pull and protect” factors correlated with biomass reduction.ConclusionsIntron-mediated enhancement (IME) of the expression of DGAT resulted in a step change in lipid accumulation of energycane and confirmed that under our experimental conditions it is rate limiting for lipid accumulation. IME should be applied to other lipogenic factors and metabolic engineering strategies. The findings from this study may be valuable in developing a high biomass feedstock for commercial production of lipids and advanced biofuels.Graphical abstract

Mutation c.-379 C>T in DGAT1 affects intramyocellular lipid content by altering MYOD1 binding affinity.

Intramuscular fat (IMF) is one of the most important indexes of pork taste quality. Diacylglycerol acyltransferase 1 (DGAT1), belonging to the acyl-coenzyme A: DGAT enzymes family, is a rate-limiting enzyme responsible for the final step of triglyceride (TG) synthesis. It is involved in TG storage in skeletal muscle; however, the underlying mechanism is not well understood. This study aimed to uncover functional mutations that can influence DGAT1 expression and consequently affect IMF deposition in pork. Two experimental groups containing individuals with high and low IMF content (6.23 ± 0.20 vs. 1.25 ± 0.05, p < 0.01) were formed from 260 Duroc × Large White × Yorkshire (D × L × Y) cross-bred pigs. A novel SNP c.-379 C>T was uncovered in the DGAT1 gene using comparative sequencing with pool DNA of high- and low-IMF groups. The IMF content of CT genotype individuals (3.19 ± 0.11%) was higher than that of CC genotype individuals (2.86 ± 0.11%) when analyzing 260 D × L × Y pigs (p < 0.05). The DGAT1 expression levels revealed a significant positive correlation with IMF content (r=0.33, p < 0.01). Luciferase assay revealed that the DGAT1 promoter with the c.-379 T allele has a higher transcription activity than that bearing the C allele in C2C12 myoblast cells, but not in 3T3-L1 pre-adipocytes. Online prediction followed by chromatin immunoprecipitation-polymerase chain reaction assay confirmed that myogenic determination factor 1 (MYOD1) binds to the DGAT1 promoter with the c.-379 C allele but not the T allele. In vitro experiments demonstrated that MYOD1 represses DGAT1 transcription and lipogenesis. As a muscle-specific transcription factor, MYOD1 can inhibit the transcription of DGAT1 with the c.-379 C allele in muscle cells. However, in the absence of MYOD1 binding to the mutated DGAT1 promoter with the c.-379 T allele, DGAT1 expresses at a higher level in the muscle cells of the c.-379 T genotype, leading to more intramyocellular lipid accumulation than in the muscle cells of the c.-379 C genotype. The SNP c.-379 C>T in the promoter region of the DGAT1 gene provides a promising molecular marker for improving pork IMF content without affecting other fat depots.

Perinatal exposure to di-(2-ethylhexyl) phthalate induces hepatic lipid accumulation mediated by diacylglycerol acyltransferase 1.

Di-(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer in consumer products and medical devices. It is also suspected to exacerbate the development of fatty liver. However, the mechanisms underlying excessive lipid synthesis and its deposition in the liver are yet to be identified. This study was aimed to evaluate the molecular mechanisms of hepatic lipid accumulation in adult male offspring after perinatal exposure to DEHP. Corn oil and DEHP (0.75 mg/kg/day) were administered once per day to dam from gestation day 6 to postnatal day (PND) 21 by oral gavage. After the weaning period, DEHP treated male pups were categorized into early life stage- and lifelong period group. Male rats both control and early life stage group administered corn oil, and lifelong period group administered DEHP from PND 22 to 70. Histological examination and triglyceride (TG) levels in the liver were analyzed. Expressions of transcription factors associated with lipid accumulation in the liver were analyzed. Both early life stage- and lifelong period group, hepatic TG levels, and mRNA and protein expression of diacylglycerol acyltransferase 1 (DGAT1) were significantly higher than control (TG: all p < 0.05, mRNA & protein: p < 0.05 and p < 0.001, respectively). The average body weight from PND 35 to 63, and mRNA and protein expression of sterol regulatory element binding protein 1c in lifelong period group were significantly lower than control (all p < 0.05); however, alanine transaminase were significantly higher than control (p < 0.01). Perinatal exposure to DEHP may induce the hepatic lipid accumulation through up-regulation of DGAT1 expression.

An altered lipid metabolism characterizes Charcot-Marie-Tooth type 2B peripheral neuropathy

Charcot-Marie Tooth type 2B (CMT2B) is a rare inherited peripheral neuropathy caused by five missense mutations in the RAB7A gene, which encodes a small GTPase of the RAB family. Currently, no cure is available for this disease. In this study, we approached the disease by comparing the lipid metabolism of CMT2B-derived fibroblasts to that of healthy controls. We found that CMT2B cells showed increased monounsaturated fatty acid level and increased expression of key enzymes of monounsaturated and polyunsaturated fatty acid synthesis. Moreover, in CMT2B cells a higher expression of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), key enzymes of de novo fatty acid synthesis, with a concomitantly increased [1-14C]acetate incorporation into fatty acids, was observed. The expression of diacylglycerol acyltransferase 2, a rate-limiting enzyme in triacylglycerol synthesis, as well as triacylglycerol levels were increased in CMT2B compared to control cells. In addition, as RAB7A controls lipid droplet breakdown and lipid droplet dynamics have been linked to diseases, we analyzed these organelles and showed that in CMT2B cells there is a strong accumulation of lipid droplets compared to control cells, thus reinforcing our data on abnormal lipid metabolism in CMT2B. Furthermore, we demonstrated that ACC and FAS expression levels changed upon RAB7 silencing or overexpression in HeLa cells, thus suggesting that metabolic modifications observed in CMT2B-derived fibroblasts can be, at least in part, related to RAB7 mutations.

LEW.1WR1 Rats have increased insulin levels and liver lipid lipolytic gene expression during their type 1 diabetes susceptibility window.

LEW.1WR1(1WR1) rats have increased type 1 diabetes (T1D) onset with the administration of polyinosinic‐polycytidylic acid (PIC). The susceptibility window of inducible T1D in the 1WR1 rat occurs between 21–35 days of life. The susceptibility of this model is related to FAT10 overexpression, a ubiquitin‐like protein that plays an unclear role in disease induction and the regulation of lipid metabolism. We hypothesized that these rodents have altered liver lipid metabolism due to increased FAT 10 gene expression in their livers and increased circulating insulin levels which can lead to decreased liver insulin sensitivity and increased beta‐cell sensitivity to inflammation. The objective of this study was to compare the insulitis status and liver gene expression in both control and treated 1WR1 rats during disease induction with a T1D‐resistant strain of rat, the LEW/SSNHsd.Using immunohistochemistry we observed higher T cell infiltration in the 1WR1 treated rats compared to the control strain, the LEW/SsNHsd(SsNHsd) PIC‐treated rat. By correlating plasma glucose levels measured by glucometer with this finding we confirmed that the 1WR1 rats were not undergoing significant insulitis at the time point we selected. We also observed that the rats have significantly fewer total islets than the SsNHsd rats but remained normoglycemic. Interestingly the 1WR1 treated rat had significantly increased insulin levels compared to the SsNHsd rats, as previous studies showed a 3 fold increase in the 1WR1 rat compared to Wistar Furth rats in a similar study(unpublished data). Both groups of the 1WR1 rats also had increased liver FAT10 gene expression compared to the SsNHsd. We measured liver lipid lipolytic gene expression to assess the metabolic status of the livers at this time point. We observed 1WR1 rats have increased expression of genes related to lipid catabolism like SREBP‐1 (Sterol Regulatory Element‐binding Protein‐1), ATGL (Adipose triglyceride lipase), DGAT1(diacylglycerol acyltransferase‐1), DGAT2(diacylglycerol acyltransferase‐2) and lower expression of glucose metabolism‐related genes like GLUT2(Glucose transporter 2), suggesting that the 1WR1 rats may have had a liver sensitivity response to the hyperinsulinemia. In conclusion, 1WR1 rats have reduced insulin sensitivity in the context of increased inflammation due to PIC injection which led to beta‐cell sensitivity and eventual T‐cell mediated destruction.Support or Funding InformationDepartment of Chemistry, UAHDepartment of Biology, UAHCollege of Science, UAHOffice of Provost, UAH

Genome-wide analysis and functional characterization of Acyl-CoA:diacylglycerol acyltransferase from soybean identify GmDGAT1A and 1B roles in oil synthesis in Arabidopsis seeds

Acyl-CoA:diacylglycerol acyltransferase (DGAT) is a key enzyme in the Kennedy pathway of triacylglycerol (TAG) synthesis. It catalyzes the acyl-CoA-dependent acylation of sn-1, 2-diacylglycerol to form TAG. DGATs in soybean (Glycine max) have been reported, but their functions are largely unclear. Here we cloned three members of DGAT1 and four members of DGAT2 family from soybean, named GmDGAT1A to GmDGAT1C, and GmDGAT2A to GmDGAT2D, respectively. GmDGAT1A and GmDGAT1C were expressed at a high level in immature seeds, GmDGAT2B in mature seeds, and GmDGAT2C in older leaves. The seven genes were transformed into the H1246 quadruple mutant yeast strain, in which GmDGAT1A, GmDGAT1B, GmDGAT1C, GmDGAT2A, and GmDGAT2B had the ability to produce TAG. Six genes were transformed into Arabidopsis respectively, and constitutive expression of GmDGAT1A and GmDGAT1B resulted in an increase in oil content at the cost of reduced protein content in seeds. Overexpression of GmDGAT1A produced heavier weight of individual seed, but did not affect the weight of total seeds from a plant. Our results reveal the functions of soybean DGATs in seed oil synthesis using transgenic Arabidopsis. The implications for the biotechnological modification of the oil contents in soybeans by altering DGAT expression are discussed.

The MYB96 Transcription Factor Regulates Triacylglycerol Accumulation by Activating DGAT1 and PDAT1 Expression in Arabidopsis Seeds.

Maturing seeds stimulate fatty acid (FA) biosynthesis and triacylglycerol (TAG) accumulation to ensure carbon and energy reserves. Transcriptional reprogramming is a key regulatory scheme in seed oil accumulation. In particular, TAG assembly is mainly controlled by the transcriptional regulation of two key enzymes, acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) and phospholipid:diacylglycerol acyltransferase 1 (PDAT1), in Arabidopsis seeds. However, the transcriptional regulators of these enzymes are as yet unknown. Here, we report that the R2R3-type MYB96 transcription factor regulates seed oil accumulation by activating the genes encoding DGAT1 and PDAT1, the rate-limiting enzymes of the last step of TAG assembly. Total FA levels are significantly elevated in MYB96-overexpressing transgenic seeds, but reduced in MYB96-deficient mutant seeds. Notably, MYB96 regulation of TAG accumulation is independent of WRINKLED 1 (WRI1)-mediated FA biosynthesis. Taken together, our findings indicate that FA biosynthesis and TAG accumulation are under independent transcriptional control, and MYB96 is mainly responsible for TAG assembly in seeds.

Intestinal Failure and Aberrant Lipid Metabolism in Patients With DGAT1 Deficiency

Background & AimsCongenital diarrheal disorders are rare inherited intestinal disorders characterized by intractable, sometimes life-threatening, diarrhea and nutrient malabsorption; some have been associated with mutations in diacylglycerol-acyltransferase 1 (DGAT1), which catalyzes formation of triacylglycerol from diacylglycerol and acyl-CoA. We investigated the mechanisms by which DGAT1 deficiency contributes to intestinal failure using patient-derived organoids.MethodsWe collected blood samples from 10 patients, from 6 unrelated pedigrees, who presented with early-onset severe diarrhea and/or vomiting, hypoalbuminemia, and/or (fatal) protein-losing enteropathy with intestinal failure; we performed next-generation sequencing analysis of DNA from 8 patients. Organoids were generated from duodenal biopsies from 3 patients and 3 healthy individuals (controls). Caco-2 cells and patient-derived dermal fibroblasts were transfected or transduced with vectors that express full-length or mutant forms of DGAT1 or full-length DGAT2. We performed CRISPR/Cas9-guided disruption of DGAT1 in control intestinal organoids. Cells and organoids were analyzed by immunoblot, immunofluorescence, flow cytometry, chromatography, quantitative real-time polymerase chain reaction, and for the activity of caspases 3 and 7.ResultsIn the 10 patients, we identified 5 bi-allelic loss-of-function mutations in DGAT1. In patient-derived fibroblasts and organoids, the mutations reduced expression of DGAT1 protein and altered triacylglycerol metabolism, resulting in decreased lipid droplet formation after oleic acid addition. Expression of full-length DGAT2 in patient-derived fibroblasts restored formation of lipid droplets. Organoids derived from patients with DGAT1 mutations were more susceptible to lipid-induced cell death than control organoids.ConclusionsWe identified a large cohort of patients with congenital diarrheal disorders with mutations in DGAT1 that reduced expression of its product; dermal fibroblasts and intestinal organoids derived from these patients had altered lipid metabolism and were susceptible to lipid-induced cell death. Expression of full-length wildtype DGAT1 or DGAT2 restored normal lipid metabolism in these cells. These findings indicate the importance of DGAT1 in fat metabolism and lipotoxicity in the intestinal epithelium. A fat-free diet might serve as the first line of therapy for patients with reduced DGAT1 expression. It is important to identify genetic variants associated with congenital diarrheal disorders for proper diagnosis and selection of treatment strategies.

Prepartum fatty acid supplementation in sheep. II. Supplementation of eicosapentaenoic acid and docosahexaenoic acid during late gestation alters the fatty acid profile of plasma, colostrum, milk and adipose tissue, and increases lipogenic gene expression of adipose tissue.

The objectives of this study were as follows: 1) to establish whether feeding a source of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to ewes during late gestation changes the fatty acid profile of colostrum, milk, ewe adipose tissue, and plasma and subsequently lamb plasma and red blood cells (RBC), and 2) to investigate the effects of EPA and DHA on mRNA expression in ewe adipose tissue. Eighty-four gestating ewes (28 pens, three per pen) were blocked by lambing day and assigned to a diet with an addition of fat at 0.39% of the DM during the last 50 d of gestation using Ca salts of a palm fatty acid distillate (PFAD) high in palmitic and oleic acids or EPA + DHA. Blood samples were taken from ewes on days 20, 1 (parturition), and 30 and from lambs on days 1 and 30 for plasma fatty acid analysis. Fatty analysis of lamb RBC was performed on day 1. Colostrum samples were taken at lambing and milk samples on day 30 for fatty acid analysis. Subcutaneous adipose tissue biopsies were taken from one ewe per pen on day 20 for fatty acid analysis and gene expression analysis of 27 genes. Treatment × day interactions (P < 0.10) were observed for several isomers of C18:1, with concentrations that were greater in plasma of EPA + DHA ewes on day 20, but were not different on day 1 or 30. Plasma concentrations of EPA tended to be greater (P = 0.07), whereas DHA was greater (P < 0.001) in EPA + DHA ewes compared with PFAD ewes. There was no difference in EPA or DHA in adipose tissue with EPA + DHA vs. PFAD supplementation (P > 0.10). Concentrations of fatty acids with 6 to 10 carbons were significantly increased (P < 0.05) in colostrum and milk of EPA + DHA ewes. There was a treatment × day interaction with EPA + DHA ewes yielding greater EPA (P = 0.03) and DHA (P = 0.04) concentrations than PFAD in colostrum, but not in milk. Treatment × day interactions (P < 0.05) were observed for several C18:1 isomers with concentrations that were greater in EPA + DHA ewe colostrum, but were not different between treatments in milk. In lamb plasma and RBC, EPA and DHA were not different between treatments (P > 0.10). The expression of fatty acid synthase and leptin was significantly increased (P < 0.05), whereas the expression of diacylglycerol acyltransferase 2 tended to be increased (P = 0.08) by supplementation of EPA + DHA vs. PFAD. These results suggest that supplementation with EPA and DHA to ewes during late gestation alters the fatty acid profile of plasma, colostrum, and milk and may increase lipogenesis.

Two Acyltransferases Contribute Differently to Linolenic Acid Levels in Seed Oil.

Acyltransferases are key contributors to triacylglycerol (TAG) synthesis and, thus, are of great importance for seed oil quality. The effects of increased or decreased expression of ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) or PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE (PDAT) on seed lipid composition were assessed in several Camelina sativa lines. Furthermore, in vitro assays of acyltransferases in microsomal fractions prepared from developing seeds of some of these lines were performed. Decreased expression of DGAT1 led to an increased percentage of 18:3n-3 without any change in total lipid content of the seed. The tri-18:3 TAG increase occurred predominantly in the cotyledon, as determined with matrix-assisted laser desorption/ionization-mass spectrometry, whereas species with two 18:3n-3 acyl groups were elevated in both cotyledon and embryonal axis. PDAT overexpression led to a relative increase of 18:2n-6 at the expense of 18:3n-3, also without affecting the total lipid content. Differential distributions of TAG species also were observed in different parts of the seed. The microsomal assays revealed that C.sativa seeds have very high activity of diacylglycerol-phosphatidylcholine interconversion. The combination of analytical and biochemical data suggests that the higher 18:2n-6 content in the seed oil of the PDAT overexpressors is due to the channeling of fatty acids from phosphatidylcholine into TAG before being desaturated to 18:3n-3, caused by the high activity of PDAT in general and by PDAT specificity for 18:2n-6. The higher levels of 18:3n-3 in DGAT1-silencing lines are likely due to the compensatory activity of a TAG-synthesizing enzyme with specificity for this acyl group and more desaturation of acyl groups occurring on phosphatidylcholine.

Spatio-temporal expression and stress responses of DGAT1, DGAT2 and PDAT responsible for TAG biosynthesis in Camelina sativa

Camelina (Camelina sativa L.) is embraced as a dedicated Brassicaceae oilseed for its valued seed oil wildly used for food, feed and biofuel. The high resistance to environmental stresses of camelina enabled its cultivation expanding to increase largely across the world recently. To gain a better understanding of the molecular mechanism underlying camelina oil biosynthesis and great environmental adaptability, expression analysis was conducted for phospholipid diacylglycerol acyltransferase (PDAT), diacylglycerol acyltransferase 1(DGAT1) and diacylglycerol acyltransferase 2 (DGAT2), three genes responsible for the final acylation step during triacylglycerol (TAG) synthesis, in camelina seed and other tissues. Genome-wide identification revealed that each of those gene families had three members in camelina. The three families and their members showed various expression patterns in camelina seed development and other tissues. The positive correlation between the expression peaks of the CsDGATs and oil rapid accumulation in developing seeds indicates that both CsDGAT1-A and CsDGAT2-C contribute importantly in seed oil accumulation with CsDGAT1-A as the major player. CsPDAT3 and CsDGAT1-C highly expressed in leaf and flower, indicating that both enzymes, particular CsPDAT3 is the major contributor for oil accumulation in leaf and flower. CsDGAT2-A was active in root and stem. Under cold stress, with oil elevating, expressions of CsDGAT2-B and CsPDAT1 were induced to increase in the seedlings by 11-folds and 6-folds respectively compared to the control of 3-week-old plants, suggesting that CsDGAT2-B and CsPDAT1 mainly function for oil accumulation in plants beneficial for camelina against cold stress. Furthermore, salt stress induced CsDGAT1-B and CsPDAT2 to express up by 8-15 folds in the seedlings, demonstrating that CsDGAT1-B and CsPDAT2 play important roles in camelina salt resistance. The present data provide a new insight into the regulatory mechanism of oil accumulation, particularly functions of CsDGAT1, CsDGAT2 and CsPDAT family members in plant stress responses.

Genetic enhancement of oil content in potato tuber (Solanum tuberosum L.) through an integrated metabolic engineering strategy.

SummaryPotato tuber is a high yielding food crop known for its high levels of starch accumulation but only negligible levels of triacylglycerol (TAG). In this study, we evaluated the potential for lipid production in potato tubers by simultaneously introducing three transgenes, including WRINKLED 1 (WRI1), DIACYLGLYCEROL ACYLTRANSFERASE 1 (DGAT1) and OLEOSIN under the transcriptional control of tuber‐specific (patatin) and constitutive (CaMV‐35S) promoters. This coordinated metabolic engineering approach resulted in over a 100‐fold increase in TAG accumulation to levels up to 3.3% of tuber dry weight (DW). Phospholipids and galactolipids were also found to be significantly increased in the potato tuber. The increase of lipids in these transgenic tubers was accompanied by a significant reduction in starch content and an increase in soluble sugars. Microscopic examination revealed that starch granules in the transgenic tubers had more irregular shapes and surface indentations when compared with the relatively smooth surfaces of wild‐type starch granules. Ultrastructural examination of lipid droplets showed their close proximity to endoplasmic reticulum and mitochondria, which may indicate a dynamic interaction with these organelles during the processes of lipid biosynthesis and turnover. Increases in lipid levels were also observed in the transgenic potato leaves, likely due to the constitutive expression of DGAT1 and incomplete tuber specificity of the patatin promoter. This study represents an important proof‐of‐concept demonstration of oil increase in tubers and provides a model system to further study carbon reallocation during development of nonphotosynthetic underground storage organs.

696 MEK and BRAF inhibitors augment the production and accumulation of sebum in hamster sebocytes

The activation of RAS-RAF-MEK-ERK signal pathway has been associated with tumor progression. Selective MEK and BRAF protein kinase inhibitors, trametinib and vemurafenib, respectively, exhibit anti-tumorigenic activity in patients with metastatic melanoma. Many patients treated with trametinib or vemurafenib develop common adverse events such as acne-like rash. Although acne is characterized as a functional disorder in sebaceous glands and pilosebaceous units, e.g., excess sebum production, secretion, and accumulation, there have been no reports to date that trametinib and vemurafenib may modulate sebum production in sebaceous glands. In the present study, therefore, we examined whether or not trametinib and vemurafenib directly influenced the sebum production in hamster sebocytes (HamSEB) in vitro. Intracellular lipid-droplet formation was augmented by trametinib and vemurafenib in HamSEB. In addition, both trametinib and vemurafenib were found to increase the production of triacylglycerols (TG), a major component of sebum, in HamSEB. Furthermore, the gene expression of diacylglycerol acyltransferase-1 (DGAT-1) and perilipin-1, which participate in sebum production and accumulation, respectively, was found to be augmented by both reagents. Thus, trametinib and vemurafenib are likely to directly facilitate sebum production and accumulation along with the increased expression of DGAT-1 and perilipin in HamSEB. These findings may increase the clinical understanding of the side effects of trametinib and vemurafenib.

Impact of temperature on the expression of Kennedy Pathway genes in developing soybean seeds

McNaughton, A. J. M., Shelp, B. J. and Rajcan, I. 2015. Impact of temperature on the expression of Kennedy Pathway genes in developing soybean seeds. Can. J. Plant Sci. 95: 87–101. The main oil storage molecule, triacylglycerol (TAG), can be created by the Kennedy Pathway. The objective of this study was to determine the impact of growing temperature on the expression of the Kennedy Pathway genes, glycerol-3-phosphate acyltransferase (G3PAT), lysophosphatidic acid acyltransferase (LPAAT), and diacylglycerol acyltransferase 1 (DGAT1), in developing seed of four soybean genotypes with altered fatty acid composition using quantitative polymerase chain reaction. The three growing temperatures were: high, 30°C day/25°C night; normal, 25°C day/20°C night; and low, 20°C day/15°C night. The expression of G3PAT steadily declined following 15 d after flowering (DAF), suggesting that it is likely to be more highly expressed earlier in development than was measured in the study. As a result, the expression of G3PAT did not correspond to fatty acid accumulation. LPAAT expression coincided with accumulation of oleic acid (18:1∆9) and linolenic acid (18:3∆9,12,15) in a temperature-dependent manner. The expression of DGAT1 corresponded to accumulation of linoleic acid (18:2∆9,12), which varied among the soybean genotypes, indicating a genotypic effect on the expression of DGAT1. This study suggests that the expression of the acyltransferase enzymes of the Kennedy Pathway influences the fatty acid composition in seed of four altered fatty acid soybean genotypes.

Abstract 17736: Cardiomyocyte Specific Loss of Diacylglycerol Acyl Transferase 1 (Dgat1) Reproduces the Abnormalities in Lipids Found in Severe Heart Failure

Diacylglycerol acyl transferase 1 (DGAT1) catalyzes the final step in triglyceride (TG) synthesis, the conversion of diacylglycerol (DAG) to TG. Dgat1-/- mice exhibit a number of beneficial metabolic effects including reduced obesity and improved insulin sensitivity and no known cardiac dysfunction. In contrast, failing human hearts have severely reduced DGAT1 expression associated with accumulation of DAGs and ceramides. To test whether DGAT1 loss alone affects heart function we created cardiomyocyte specific DGAT1 knockout (hDgat1-/-) mice. hDgat1-/- mice hearts had 95% increased DAG and 85% increased ceramides compared to floxed controls. 50% of these mice died by 9 months of age. The heart failure marker brain natriuretic peptide (Bnp) increased 5-fold in hDgat1-/- hearts and fractional shortening (FS) was reduced. This was associated with a 30% increase in PPARalpha and a 40% increase in Cd36. We crossed hDgat1-/- mice with previously described enterocyte-specific Dgat1 knockout mice (hiDgat1-/-). This corrected the early mortality, improved FS 40%, and reduced cardiac ceramide and DAG content. Treatment of hDgat1-/- mice with GLP-1 receptor agonist exenatide for 1 week reduced Bnp mRNA by 50%, improved FS, and reduced heart DAG and ceramide content by 30-40%. Increased fatty acid uptake into hDgat1-/- hearts was normalized by exenatide. Reduced activity of protein kinase Cα (PKCα), which is known to be increased by DAG and ceramides, paralleled the reductions in these lipids. Our mouse studies show that loss of DGAT1 reproduces the lipid abnormalities seen in severe human heart failure.

Cardiomyocyte-specific Loss of Diacylglycerol Acyltransferase 1 (DGAT1) Reproduces the Abnormalities in Lipids Found in Severe Heart Failure

Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final step in triglyceride synthesis, the conversion of diacylglycerol (DAG) to triglyceride. Dgat1(-/-) mice exhibit a number of beneficial metabolic effects including reduced obesity and improved insulin sensitivity and no known cardiac dysfunction. In contrast, failing human hearts have severely reduced DGAT1 expression associated with accumulation of DAGs and ceramides. To test whether DGAT1 loss alone affects heart function, we created cardiomyocyte-specific DGAT1 knock-out (hDgat1(-/-)) mice. hDgat1(-/-) mouse hearts had 95% increased DAG and 85% increased ceramides compared with floxed controls. 50% of these mice died by 9 months of age. The heart failure marker brain natriuretic peptide increased 5-fold in hDgat1(-/-) hearts, and fractional shortening (FS) was reduced. This was associated with increased expression of peroxisome proliferator-activated receptor α and cluster of differentiation 36. We crossed hDgat1(-/-) mice with previously described enterocyte-specific Dgat1 knock-out mice (hiDgat1(-/-)). This corrected the early mortality, improved FS, and reduced cardiac ceramide and DAG content. Treatment of hDgat1(-/-) mice with the glucagon-like peptide 1 receptor agonist exenatide also improved FS and reduced heart DAG and ceramide content. Increased fatty acid uptake into hDgat1(-/-) hearts was normalized by exenatide. Reduced activation of protein kinase Cα (PKCα), which is increased by DAG and ceramides, paralleled the reductions in these lipids. Our mouse studies show that loss of DGAT1 reproduces the lipid abnormalities seen in severe human heart failure.

Palmitic Acid and DGAT1 Deficiency Enhance Osteoclastogenesis, while Oleic Acid-Induced Triglyceride Formation Prevents It

Both obesity and diabetes mellitus are associated with alterations in lipid metabolism as well as a change in bone homeostasis and osteoclastogenesis. We hypothesized that increased fatty acid levels affect bone health by altering precursor cell differentiation and osteoclast activation. Here we show that palmitic acid (PA, 16:0) enhances receptor activator of NF-κB ligand (RANKL)-stimulated osteoclastogenesis and is sufficient to induce osteoclast differentiation even in the absence of RANKL. TNFα expression is crucial for PA-induced osteoclastogenesis, as shown by increased TNFα mRNA levels in PA-treated cells and abrogation of PA-stimulated osteoclastogenesis by TNFα neutralizing antibodies. In contrast, oleic acid (OA, 18:1) does not enhance osteoclast differentiation, leads to increased intracellular triglyceride accumulation, and inhibits PA-induced osteoclastogenesis. Adenovirus-mediated expression of diacylglycerol acyl transferase 1 (DGAT1), a gene involved in triglyceride synthesis, also inhibits PA-induced osteoclastogenesis, suggesting a protective role of DGAT1 for bone health. Accordingly, Dgat1 knockout mice have larger bone marrow-derived osteoclasts and decreased bone mass indices. In line with these findings, mice on a high-fat PA-enriched diet have a greater reduction in bone mass and structure than mice on a high-fat OA-enriched diet. Thus, we propose that TNFα mediates saturated fatty acid-induced osteoclastogenesis that can be prevented by DGAT activation or supplementation with OA.

ABA-insensitive (ABI) 4 and ABI5 synergistically regulate DGAT1 expression in Arabidopsis seedlings under stress

Triacylglycerol (TAG) accumulation is essential for seed maturation in plants. Diacylglycerol acyltransferase 1 (DGAT1) is the rate-limiting enzyme in TAG biosynthesis. In this study, we show that TAG accumulation in Arabidopsis seedlings is correlated with environmental stress, and both ABI4 and ABI5 play important roles in regulating DGAT1 expression. Tobacco transient assays revealed the synergistic effect of ABI4 with ABI5 in regulating DGAT1 expression. Taken together, our findings indicate ABI5 is an important accessory factor with ABI4 in the activation of DGAT1 in Arabidopsis seedlings under stress.

Adapalene suppresses sebum accumulation via the inhibition of triacylglycerol biosynthesis and perilipin expression in differentiated hamster sebocytes in vitro

BackgroundAcne is a chronic inflammatory disease in sebaceous glands and pilosebaceous units where excess sebum production and follicular hyperkeratinization are observed. Adapalene, which exerts comedolytic and anti-inflammatory effects, is used for the topical treatment of mild to moderate acne. ObjectiveWe examined the effect of adapalene on sebum production and accumulation in sebaceous gland cells (sebocytes). MethodsThe regulation of sebum production was examined by oil red O and nile red staining and the measurement of triacylglycerols (TGs) in differentiated hamster sebocytes. The gene expression and production of diacylglycerol acyltransferase-1 (DGAT-1) and perilipin 1 (PLIN1) were analyzed using real-time PCR and Western blotting, respectively. ResultsAdapalene suppressed sebum accumulation as lipid droplets in spontaneously and insulin-differentiated hamster sebocytes. The TG production, and the gene expression and production of DGAT-1, a rate-limiting enzyme of TG biosynthesis, were dose-dependently inhibited by adapalene in insulin-, 5α-dihydrotestosterone- or a peroxisome proliferator activating receptor γ agonist, troglitazone-differentiated hamster sebocytes. In addition, the inhibition of TG production by adapalene interfered with antagonists against nuclear retinoic acid and retinoid X receptors (CD2665 and UVI3006, respectively) in the differentiated sebocytes. Furthermore, the production of PLIN1, a lipid storage droplet protein, was transcriptionally inhibited by adapalene in the differentiated sebocytes. ConclusionsThese results suggest that adapalene exerts an inhibitory action for sebum accumulation due to the suppression of TG and PLIN1 production in differentiated hamster sebocytes. Furthermore, these findings may contribute to a novel understanding of the molecular mechanisms of adapalene for acne treatment and prevention.