The Lipid-Metabolism-Associated Anti-Obesity Properties of Rapeseed Diacylglycerol Oil.

To investigate the effects of rapeseed diacylglycerol oil (RDG) intake on lipid accumulation and metabolism in C57BL/6J mice, obese mice were fed a high-fat diet in which 45% of the total energy content came from RDG (RDGM group) or rapeseed triacylglycerol oil (RTGM group). This diet intervention was conducted for 12 weeks following the establishment of the obese mouse model. By the end of the experiment, the serum glucose levels of the mice in the RTGM and RDGM groups were 13.0 ± 1.3 mmol/L and 9.7 ± 1.5 mmol/L, respectively. Meanwhile, the serum triglyceride level in the RDGM group was 26.3% lower than that in the RTGM group. The weight-loss effect in the RDGM group was accompanied by a significant decrease in the white adipose tissue (WAT) index. The RDG intervention did not significantly change the antioxidant and anti-inflammatory properties of the rapeseed oil in vivo. The RDG diet improved the liver lipid metabolism abnormalities induced by a high-fat diet, leading to decreased liver damage index values (AST and ALT). Additionally, compared to that in the RTGM group, the expression of the adipogenic genes PPAR-γ and DGAT decreased in both the liver and intestine by 21.7% and 16.7% and by 38.7% and 47.2%, respectively, in the RDGM group. Further, most lipolytic genes in BAT showed no significant change after the RDG intervention. This implies that RDG regulates lipid metabolism by altering the expression of adipogenic genes in the liver, intestine, and adipose tissue, thereby reducing the accumulation of WAT. Furthermore, the RDG diet enhanced gut flora diversity, increasing the relative levels of unclassified Muribaculaceae and decreasing the levels of Dubosiella and Faecalibaculum in the mouse gut, potentially accelerating lipid metabolism. Thus, a three-month RDG diet intervention in obese mice exhibited benefits in regulating the somatotype, serum obesity-related indices, gut flora structure, and lipid metabolism in the adipose tissue, liver, and intestine.

Functional Characterization of the Effects of CsDGAT1 and CsDGAT2 on Fatty Acid Composition in Camelina sativa.

Triacylglycerols (TAGs) are the storage oils of plant seeds, and these lipids provide energy for seed germination and valuable oils for human consumption. Three diacylglycerol acyltransferases (DGAT1, DGAT2, and DGAT3) and phospholipid:diacylglycerol acyltransferases participate in the biosynthesis of TAGs. DGAT1 and DGAT2 participate in the biosynthesis of TAGs through the endoplasmic reticulum (ER) pathway. In this study, we functionally characterized CsDGAT1 and CsDGAT2 from camelina (Camelina sativa). Green fluorescent protein-fused CsDGAT1 and CsDGAT2 localized to the ER when transiently expressed in Nicotiana benthamiana leaves. To generate Csdgat1 and Csdgat2 mutants using the CRISPR/Cas9 system, camelina was transformed with a binary vector carrying Cas9 and the respective guide RNAs targeting CsDGAT1s and CsDGAT2s via the Agrobacterium-mediated floral dip method. The EDD1 lines had missense and nonsense mutations in the CsDGAT1 homoeologs, suggesting that they retained some CsDGAT1 function, and their seeds showed decreased eicosaenoic acid (C20:1) contents and increased C18:3 contents compared to the wild type (WT). The EDD2 lines had a complete knockout of all CsDGAT2 homoeologs and a slightly decreased C18:3 content compared to the WT. In conclusion, CsDGAT1 and CsDGAT2 have a small influence on the seed oil content and have an acyl preference for C20:1 and C18:3, respectively. This finding can be applied to develop oilseed plants containing high omega-3 fatty acids or high oleic acid.

The synthesis of triacylglycerol by diacylglycerol acyltransferases (CsDGAT1A and CsDGAT2D) is essential for tolerance of cucumber's resistance to low-temperature stress.

KEY MESSAGE: CsDGAT1A and CsDGAT2D play a positive regulatory role in cucumber's response to low-temperature stress and positively regulate the synthesis of triacylglycerol (TAG). Triacylglycerol (TAG), a highly abundant and significant organic compound in plants, plays crucial roles in plant growth, development, and stress responses. The final acetylation step of TAG synthesis is catalyzed by diacylglycerol acyltransferases (DGATs). However, the involvement of DGATs in cucumber's low-temperature stress response remains unexplored. This study focused on two DGAT genes, CsDGAT1A and CsDGAT2D, investigating their function in enhancing cucumber's low-temperature stress tolerance. Our results revealed that both proteins were the members of the diacylglycerol acyltransferase family and were predominantly localized in the endoplasmic reticulum. Functional analysis demonstrated that transient silencing of CsDGAT1A and CsDGAT2D significantly compromised cucumber's low-temperature stress tolerance, whereas transient overexpression enhanced it. Furthermore, the TAG content quantification indicated that CsDGAT1A and CsDGAT2D promoted TAG accumulation. In conclusion, this study elucidates the lipid metabolism mechanism in cucumber's low-temperature stress response and offers valuable insights for the cultivation of cold-tolerant cucumber plants.

Identification of key genes for triacylglycerol biosynthesis and storage in herbaceous peony (Paeonia lactifolra Pall.) seeds based on full-length transcriptome.

BACKGROUND: The herbaceous peony (Paeonia lactiflora Pall.) is extensively cultivated in China due to its root being used as a traditional Chinese medicine known as 'Radix Paeoniae Alba'. In recent years, it has been discovered that its seeds incorporate abundant unsaturated fatty acids, thereby presenting a potential new oilseed plant. Surprisingly, little is known about the full-length transcriptome sequencing of Paeonia lactiflora, limiting research into its gene function and molecular mechanisms. RESULTS: A total of 484,931 Reads of Inserts (ROI) sequences and 1,455,771 full-Length non-chimeric reads (FLNC) sequences were obtained for CDS prediction, TF analysis, SSR analysis and lncRNA identification. In addition, gene function annotation and gene structure analysis were performed. A total of 4905 transcripts were related to lipid metabolism biosynthesis pathway, belonging to 28 enzymes. We use these data to identify 10 oleosin (OLE) and 5 diacylglycerol acyltransferase (DGAT) gene members after de-redundancy. The analysis of physicochemical properties and secondary structure showed them similarity in gene family respectively. The phylogenetic analysis showed that the distribution of OLE and DGAT family members was roughly the same as that of Arabidopsis. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses revealed expression changes in different seed development stages, and showed a trend of increasing and then decreasing. CONCLUSION: In summary, these results provide new insights into the molecular mechanism of triacylglycerol (TAG) biosynthesis and storage during the seedling stage in Paeonia lactiflora. It provides theoretical references for selecting and breeding oil varieties and understanding the functions of oil storage as well as lipid synthesis related genes in Paeonia lactiflora.

Palmitic Acid Exerts Anti-Tumorigenic Activities by Modulating Cellular Stress and Lipid Droplet Formation in Endometrial Cancer.

Epidemiological and clinical evidence have extensively documented the role of obesity in the development of endometrial cancer. However, the effect of fatty acids on cell growth in endometrial cancer has not been widely studied. Here, we reported that palmitic acid significantly inhibited cell proliferation of endometrial cancer cells and primary cultures of endometrial cancer and reduced tumor growth in a transgenic mouse model of endometrial cancer, in parallel with increased cellular stress and apoptosis and decreased cellular adhesion and invasion. Inhibition of cellular stress by N-acetyl-L-cysteine effectively reversed the effects of palmitic acid on cell proliferation, apoptosis, and invasive capacity in endometrial cancer cells. Palmitic acid increased the intracellular formation of lipid droplets in a time- and dose-dependent manner. Depletion of lipid droplets by blocking DGAT1 and DGAT2 effectively increased the ability of palmitic acid to inhibit cell proliferation and induce cleaved caspase 3 activity. Collectively, this study provides new insight into the effect of palmitic acid on cell proliferation and invasion and the formation of lipid droplets that may have potential clinical relevance in the treatment of obesity-driven endometrial cancer.

HuR promotes triglyceride synthesis and intestinal fat absorption.

Triacylglyceride (TAG) synthesis in the small intestine determines the absorption of dietary fat, but the underlying mechanisms remain to be further studied. Here, we report that the RNA-binding protein HuR (ELAVL1) promotes TAG synthesis in the small intestine. HuR associates with the 3' UTR of Dgat2 mRNA and intron 1 of Mgat2 pre-mRNA. Association of HuR with Dgat2 3' UTR stabilizes Dgat2 mRNA, while association of HuR with intron 1 of Mgat2 pre-mRNA promotes the processing of Mgat2 pre-mRNA. Intestinal epithelium-specific HuR knockout reduces the expression of DGAT2 and MGAT2, thereby reducing the dietary fat absorption through TAG synthesis and mitigating high-fat-diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) and obesity. Our findings highlight a critical role of HuR in promoting dietary fat absorption.

Rab1b facilitates lipid droplet growth by ER-to-lipid droplet targeting of DGAT2.

Lipid droplets (LDs) comprise a triglyceride core surrounded by a lipid monolayer enriched with proteins, many of which function in LD homeostasis. How proteins are targeted to the growing LD is still unclear. Rab1b, a GTPase regulating secretory transport, was recently associated with targeting proteins to LDs in a Drosophila RNAi screen. LD formation was prevented in human hepatoma cells overexpressing dominant-negative Rab1b. We thus hypothesized that Rab1b recruits lipid-synthesizing enzymes, facilitating LD growth. Here, FRET between diacylglycerol acyltransferase 2 (DGAT2) and Rab1b and activity mutants of the latter demonstrated that Rab1b promotes DGAT2 ER to the LD surface redistribution. Last, alterations in LD metabolism and DGAT2 redistribution, consistent with Rab1b activity, were caused by mutations in the Rab1b-GTPase activating protein TBC1D20 in Warburg Micro syndrome (WARBM) model mice fibroblasts. These data contribute to our understanding of the mechanism of Rab1b in LD homeostasis and WARBM, a devastating autosomal-recessive disorder caused by mutations in TBC1D20.

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

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

Identification of triacylglycerol remodeling mechanism to synthesize unusual fatty acid containing oils.

Typical plant membranes and storage lipids are comprised of five common fatty acids yet over 450 unusual fatty acids accumulate in seed oils of various plant species. Plant oils are important human and animal nutrients, while some unusual fatty acids such as hydroxylated fatty acids (HFA) are used in the chemical industry (lubricants, paints, polymers, cosmetics, etc.). Most unusual fatty acids are extracted from non-agronomic crops leading to high production costs. Attempts to engineer HFA into crops are unsuccessful due to bottlenecks in the overlapping pathways of oil and membrane lipid synthesis where HFA are not compatible. Physaria fendleri naturally overcomes these bottlenecks through a triacylglycerol (TAG) remodeling mechanism where HFA are incorporated into TAG after initial synthesis. TAG remodeling involves a unique TAG lipase and two diacylglycerol acyltransferases (DGAT) that are selective for different stereochemical and acyl-containing species of diacylglycerol within a synthesis, partial degradation, and resynthesis cycle. The TAG lipase interacts with DGAT1, localizes to the endoplasmic reticulum (with the DGATs) and to puncta around the lipid droplet, likely forming a TAG remodeling metabolon near the lipid droplet-ER junction. Each characterized DGAT and TAG lipase can increase HFA accumulation in engineered seed oils.

Biochemical characterization of acyl-CoA:diacylglycerol acyltransferase2 from the diatom Phaeodactylum tricornutum and its potential effect on LC-PUFAs biosynthesis in planta.

BACKGROUND: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), belonging to ω-3 long-chain polyunsaturated fatty acids (ω3-LC-PUFAs), are essential components of human diet. They are mainly supplemented by marine fish consumption, although their native producers are oleaginous microalgae. Currently, increasing demand for fish oils is insufficient to meet the entire global needs, which puts pressure on searching for the alternative solutions. One possibility may be metabolic engineering of plants with an introduced enzymatic pathway producing ω3-LC-PUFAs. RESULT: In this study we focused on the acyl-CoA:diacylglycerol acyltransferase2b (PtDGAT2b) from the diatom Phaeodactylum tricornutum, an enzyme responsible for triacylglycerol (TAG) biosynthesis via acyl-CoA-dependent pathway. Gene encoding PtDGAT2b, incorporated into TAG-deficient yeast strain H1246, was used to confirm its activity and conduct biochemical characterization. PtDGAT2b exhibited a broad acyl-CoA preference with both di-16:0-DAG and di-18:1-DAG, whereas di-18:1-DAG was favored. The highest preference for acyl donors was observed for 16:1-, 10:0- and 12:0-CoA. PtDGAT2b also very efficiently utilized CoA-conjugated ω-3 LC-PUFAs (stearidonic acid, eicosatetraenoic acid and EPA). Additionally, verification of the potential role of PtDGAT2b in planta, through its transient expression in tobacco leaves, indicated increased TAG production with its relative amount increasing to 8%. Its co-expression with the gene combinations aimed at EPA biosynthesis led to, beside elevated TAG accumulation, efficient accumulation of EPA which constituted even 25.1% of synthesized non-native fatty acids (9.2% of all fatty acids in TAG pool). CONCLUSIONS: This set of experiments provides a comprehensive biochemical characterization of DGAT enzyme from marine microalgae. Additionally, this study elucidates that PtDGAT2b can be used successfully in metabolic engineering of plants designed to obtain a boosted TAG level, enriched not only in ω-3 LC-PUFAs but also in medium-chain and ω-7 fatty acids.

Mixed exposure to haloacetaldehyde disinfection by-products exacerbates lipid aggregation in the liver of mice.

Haloacetaldehyde disinfection by-products (HAL-DBPs) are among the top three unregulated DBPs found in drinking water. The cytotoxicity and genotoxicity of HALs are much higher than that of the regulated trihalomethanes and haloacetic acids. Previous studies have mainly focused on the toxic effects of single HAL, with few examining the toxic effects of mixed exposures to HALs. The study aimed to observe the effects of mixed exposures of 1∼1000X the realistic level of HALs on the hepatotoxicity and lipid metabolism of C57BL/6J mice, based on the component and concentration of HALs detected in the finished water of Shanghai. Exposure to realistic levels of HALs led to a significant increase in phosphorated acetyl CoA carboxylase 1 (p-ACC1) in the hepatic de novo lipogenesis (DNL) pathway. Additionally, exposure to 100X realistic levels of HALs resulted in significant alterations to key enzymes of DNL pathway, including ACC1, fatty acid synthase (FAS), and diacylglycerol acyltransferase 2 (DGAT2), as well as key proteins of lipid disposal such as carnitine palmitoyltransferase 1 (CPT-1) and peroxisome proliferator activated receptor α (PPARα). Exposure to 1000X realistic levels of HALs significantly increased hepatic and serum triglyceride levels, as well as total cholesterol, low-density lipoprotein, alanine aminotransferase, aspartate transaminase, alkaline phosphatase, and lactate dehydrogenase levels, significantly decreased high-density lipoprotein. Meanwhile, histopathological analysis demonstrated that HALs exacerbated tissue vacuolization and inflammatory cell infiltration in mice livers, which showed the typical phenotypes of non-alcoholic fatty liver disease (NAFLD). These results suggested that the HALs mixture is a critical risk factor for NAFLD and is significantly highly toxic to C57BL/6J mice.

Differences in diacylglycerol acyltransferases expression patterns and regulation cause distinct hepatic triglyceride deposition in fish.

Triglyceride (TAG) deposition in the liver is associated with metabolic disorders. In lower vertebrate, the propensity to accumulate hepatic TAG varies widely among fish species. Diacylglycerol acyltransferases (DGAT1 and DGAT2) are major enzymes for TAG synthesis. Here we show that large yellow croaker (Larimichthys crocea) has significantly higher hepatic TAG level than that in rainbow trout (Oncorhynchus mykiss) fed with same diet. Hepatic expression of DGATs genes in croaker is markedly higher compared with trout under physiological condition. Meanwhile, DGAT1 and DGAT2 in both croaker and trout are required for TAG synthesis and lipid droplet formation in vitro. Furthermore, oleic acid treatment increases DGAT1 expression in croaker hepatocytes rather than in trout and has no significant difference in DGAT2 expression in two fish species. Finally, effects of various transcription factors on croaker and trout DGAT1 promoter are studied. We find that DGAT1 is a target gene of the transcription factor CREBH in croaker rather than in trout. Overall, hepatic expression and transcriptional regulation of DGATs display significant species differences between croaker and trout with distinct hepatic triglyceride deposition, which bring new perspectives on the use of fish models for studying hepatic TAG deposition.

A novel approach to pH-Responsive targeted cancer Therapy: Inhibition of FaDu cancer cell proliferation with a pH low insertion Peptide-Conjugated DGAT1 inhibitor.

Targeting enzymes involved in lipid metabolism is increasingly recognized as a promising anticancer strategy. Efficient inhibition of diacylglycerol O-transferase 1 (DGAT1) can block fatty acid (FA) storage. This, in turn, triggers an increase in free polyunsaturated FA concentration, leading to peroxidation and ferroptosis. In this study, we report the development of a pH-sensitive peptide (pHLIP)-drug conjugate designed to selectively deliver DGAT1 inhibitors to cancer cells nested within the acidic microenvironment of tumors. We utilized two previously established pHLIP sequences for coupling with drugs. The study of DGAT1 conjugates in large unilamellar vesicles (LUVs) of different compositions did not reveal enhanced pH-dependent insertion compared to POPC LUVs. However, using in vitro 3D tumor spheroids, significant antiproliferative effects were observed upon exposure to pHLIP-T863 (DGAT1 inhibitor) conjugates, surpassing the inhibitory activity of T863 alone. In conclusion, our study provides the first evidence that pHLIP-based conjugates with DGAT1 inhibitors have the potential to specifically target the acidic compartment of tumors. Moreover, it sheds light on the limitations of LUV models in capturing the pH-dependency of such conjugates.

Neuroinvasive virus facilitates viral replication by employing lipid droplets to reduce arachidonic acid-induced ferroptosis.

Lipids have been previously implicated in the lifecycle of neuroinvasive viruses. However, the role of lipids in programmed cell death and the relationship between programmed cell death and lipid droplets (LDs) in neuroinvasive virus infection remains unclear. Here, we found that the infection of neuroinvasive virus, such as rabies virus and encephalomyocarditis virus could enhance the LD formation in N2a cells, and decreasing LDs production by targeting diacylglycerol acyltransferase could suppress viral replication. The lipidomics analysis revealed that arachidonic acid (AA) was significantly increased after reducing LD formation by restricting diacylglycerol acyltransferase, and AA was further demonstrated to induce ferroptosis to inhibit neuroinvasive virus replication. Moreover, lipid peroxidation and viral replication inhibition could be significantly alleviated by a ferroptosis inhibitor, ferrostatin-1, indicating that AA affected neuroinvasive virus replication mainly through inducing ferroptosis. Furthermore, AA was demonstrated to activate the acyl-CoA synthetase long-chain family member 4-lysophosphatidylcholine acyltransferase 3-cytochrome P450 oxidoreductase axis to induce ferroptosis. Our findings highlight novel cross-talks among viral infection, LDs, and ferroptosis for the first time, providing a potential target for antiviral drug development.

The role of DGAT1 and DGAT2 in regulating tumor cell growth and their potential clinical implications.

Lipid metabolism is widely reprogrammed in tumor cells. Lipid droplet is a common organelle existing in most mammal cells, and its complex and dynamic functions in maintaining redox and metabolic balance, regulating endoplasmic reticulum stress, modulating chemoresistance, and providing essential biomolecules and ATP have been well established in tumor cells. The balance between lipid droplet formation and catabolism is critical to maintaining energy metabolism in tumor cells, while the process of energy metabolism affects various functions essential for tumor growth. The imbalance of synthesis and catabolism of fatty acids in tumor cells leads to the alteration of lipid droplet content in tumor cells. Diacylglycerol acyltransferase 1 and diacylglycerol acyltransferase 2, the enzymes that catalyze the final step of triglyceride synthesis, participate in the formation of lipid droplets in tumor cells and in the regulation of cell proliferation, migration and invasion, chemoresistance, and prognosis in tumor. Several diacylglycerol acyltransferase 1 and diacylglycerol acyltransferase 2 inhibitors have been developed over the past decade and have shown anti-tumor effects in preclinical tumor models and improvement of metabolism in clinical trials. In this review, we highlight key features of fatty acid metabolism and different paradigms of diacylglycerol acyltransferase 1 and diacylglycerol acyltransferase 2 activities on cell proliferation, migration, chemoresistance, and prognosis in tumor, with the hope that these scientific findings will have potential clinical implications.

Investigation of pharmacokinetic drug interaction between clesacostat and DGAT2 inhibitor ervogastat in healthy adult participants.

Co-administration of clesacostat (acetyl-CoA carboxylase inhibitor, PF-05221304) and ervogastat (diacylglycerol O-acyltransferase inhibitor, PF-06865571) in laboratory models improved non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) end points and mitigated clesacostat-induced elevations in circulating triglycerides. Clesacostat is cleared via organic anion-transporting polypeptide-mediated hepatic uptake and cytochrome P450 family 3A (CYP3A); in vitro clesacostat is identified as a potential CYP3A time-dependent inactivator. In vitro ervogastat is identified as a substrate and potential inducer of CYP3A. Prior to longer-term efficacy trials in participants with NAFLD, safety and pharmacokinetics (PK) were evaluated in a phase I, non-randomized, open-label, fixed-sequence trial in healthy participants. In Cohort 1, participants (n = 7) received clesacostat 15 mg twice daily (b.i.d.) alone (Days 1-7) and co-administered with ervogastat 300 mg b.i.d. (Days 8-14). Mean systemic clesacostat exposures, when co-administered with ervogastat, decreased by 12% and 19%, based on maximum plasma drug concentration and area under the plasma drug concentration-time curve during the dosing interval, respectively. In Cohort 2, participants (n = 9) received ervogastat 300 mg b.i.d. alone (Days 1-7) and co-administered with clesacostat 15 mg b.i.d. (Days 8-14). There were no meaningful differences in systemic ervogastat exposures when administered alone or with clesacostat. Clesacostat 15 mg b.i.d. and ervogastat 300 mg b.i.d. co-administration was overall safe and well tolerated in healthy participants. Cumulative safety and no clinically meaningful PK drug interactions observed in this study supported co-administration of these two novel agents in additional studies exploring efficacy and safety in the management of NAFLD.

All members of the Arabidopsis DGAT and PDAT acyltransferase families operate during high and low temperatures.

The accumulation of triacylglycerol (TAG) in vegetative tissues is necessary to adapt to changing temperatures. It has been hypothesized that TAG accumulation is required as a storage location for maladaptive membrane lipids. The TAG acyltransferase family has five members (DIACYLGLYCEROL ACYLTRANSFERSE1/2/3 and PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE1/2), and their individual roles during temperature challenges have either been described conflictingly or not at all. Therefore, we used Arabidopsis (Arabidopsis thaliana) loss of function mutants in each acyltransferase to investigate the effects of temperature challenge on TAG accumulation, plasma membrane integrity, and temperature tolerance. All mutants were tested under one high- and two low-temperature regimens, during which we quantified lipids, assessed temperature sensitivity, and measured plasma membrane electrolyte leakage. Our findings revealed reduced effectiveness in TAG production during at least one temperature regimen for all acyltransferase mutants compared to the wild type, resolved conflicting roles of pdat1 and dgat1 by demonstrating their distinct temperature-specific actions, and uncovered that plasma membrane integrity and TAG accumulation do not always coincide, suggesting a multifaceted role of TAG beyond its conventional lipid reservoir function during temperature stress.

DGAT2 inhibition blocks SREBP-1 cleavage and improves hepatic steatosis by increasing phosphatidylethanolamine in the ER.

Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step of triglyceride (TG) synthesis. DGAT2 deletion in mice lowers liver TGs, and DGAT2 inhibitors are under investigation for the treatment of fatty liver disease. Here, we show that DGAT2 inhibition also suppressed SREBP-1 cleavage, reduced fatty acid synthesis, and lowered TG accumulation and secretion from liver. DGAT2 inhibition increased phosphatidylethanolamine (PE) levels in the endoplasmic reticulum (ER) and inhibited SREBP-1 cleavage, while DGAT2 overexpression lowered ER PE concentrations and increased SREBP-1 cleavage in vivo. ER enrichment with PE blocked SREBP-1 cleavage independent of Insigs, which are ER proteins that normally retain SREBPs in the ER. Thus, inhibition of DGAT2 shunted diacylglycerol into phospholipid synthesis, increasing the PE content of the ER, resulting in reduced SREBP-1 cleavage and less hepatic steatosis. This study reveals a new mechanism that regulates SREBP-1 activation and lipogenesis that is independent of sterols and SREBP-2 in liver.

Role of diacylglycerol O-acyltransferase 1 (DGAT1) in lipolysis and autophagy of adipose tissue from ketotic dairy cows.

High-yielding dairy cows in early lactation often encounter difficulties in meeting the energy requirements essential for maintaining milk production. This is primarily attributed to insufficient dry matter intake, which consequently leads to sustained lipolysis of adipose tissue. Fatty acids released by lipolysis can disrupt metabolic homeostasis. Autophagy, an adaptive response to intracellular environmental changes, is considered a crucial mechanism for regulating lipid metabolism and maintaining a proper cellular energy status. Despite its close relationship with aberrant lipid metabolism and cytolipotoxicity in animal models of metabolic disorders, the precise function of diacylglycerol o-acyltransferase 1 (DGAT1) in bovine adipose tissue during periods of negative energy balance is not fully understood, particularly regarding its involvement in lipolysis and autophagy. The objective of the present study was to assess the effect of DGAT1 on both lipolysis and autophagy in bovine adipose tissue and isolated adipocytes. Adipose tissue and blood samples were collected from cows diagnosed as clinically ketotic (n = 15) or healthy (n = 15) following a veterinary evaluation based on clinical symptoms and serum concentrations of BHB, which were 3.19 mM (interquartile range = 0.20) and 0.50 mM (interquartile range = 0.06), respectively. Protein abundance of DGAT1 and phosphorylation levels of unc-51-like kinase 1 (ULK1), were greater in adipose tissue from cows with ketosis, whereas phosphorylation levels of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) were lower. Furthermore, when adipocytes isolated from the harvested adipose tissue of 15 healthy cows were transfected with DGAT1 overexpression adenovirus or DGAT1 small interfering RNA followed by exposure to epinephrine (EPI), it led to greater ratios and protein abundance of phosphorylated hormone-sensitive triglyceride lipase (LIPE) to total LIPE and adipose triglyceride lipase (ATGL), while inhibiting the protein phosphorylation levels of ULK1, PI3K, AKT, and mTOR. Overexpression of DGAT1 in EPI-treated adipocytes reduced lipolysis and autophagy, whereas silencing DGAT1 further exacerbated EPI-induced lipolysis and autophagy. Taken together, these findings indicate that upregulation of DGAT1 may function as an adaptive response to suppress adipocytes lipolysis, highlighting the significance of maintaining metabolic homeostasis in dairy cows during periods of negative energy balance.

The expression of genes encoding novel Sesame oleosin variants facilitates enhanced triacylglycerol accumulation in Arabidopsis leaves and seeds.

Triacylglycerols (TAG), accumulate within lipid droplets (LD), predominantly surrounded by OLEOSINs (OLE), that protect TAG from hydrolysis. We tested the hypothesis that identifying and removing degradation signals from OLE would promote its abundance, preventing TAG degradation and enhancing TAG accumulation. We tested whether mutating potential ubiquitin-conjugation sites in a previously reported improved Sesamum indicum OLE (SiO) variant, o3-3 Cys-OLE (SiCO herein), would stabilize it and increase its lipogenic potential. SiCOv1 was created by replacing all five lysines in SiCO with arginines. Separately, six cysteine residues within SiCO were deleted to create SiCOv2. SiCOv1 and SiCOv2 mutations were combined to create SiCOv3. Transient expression of SiCOv3 in Nicotiana benthamiana increased TAG by two-fold relative to SiCO. Constitutive expression of SiCOv3 or SiCOv5, containing the five predominant TAG-increasing mutations from SiCOv3, in Arabidopsis along with mouse DGAT2 (mD) increased TAG accumulation by 54% in leaves and 13% in seeds compared with control lines coexpressing SiCO and mD. Lipid synthesis rates increased, consistent with an increase in lipid sink strength that sequesters newly synthesized TAG, thereby relieving the constitutive BADC-dependent inhibition of ACCase reported for WT Arabidopsis. These OLE variants represent novel factors for potentially increasing TAG accumulation in a variety of oil crops.