Integrated Analysis of Fatty Acid Metabolism and Transcriptome Involved in Olive Fruit Development to Improve Oil Composition

Olea europaea L. is an important oil crop with excellent nutritional properties. In this study, a full-length transcriptome combined with fatty acid composition was used to investigate the molecular mechanism of fatty acid (FA) metabolism of olive fruits at various stages of development (S1–S5). A total of 34 fatty acids (FAs) were measured using gas chromatography-mass spectrometry (GC-MS). All transcripts of FA metabolism during olive fruit development were studied, including glycolysis, fatty acid synthesis, triacylglycerol synthesis, and FA degradation. A total of 100 transcripts of 11 gene families, 68 transcripts of 12 gene families, 55 transcripts of 7 gene families, and 28 transcripts of 7 gene families were identified as encoding for enzymes involved in FA metabolism. Furthermore, one of the critical reactions in TAG metabolism is the activation of fatty acyl chains to fatty acyl CoA, which is catalyzed by long-chain acyl CoA synthetases (LACS). Phylogenetic analysis showed that 13 putative LACS-encoding genes clustered into five groups, of which two putative transcripts encoding LACS6/7 may participate in FA degradation. The aim of this study was to evaluate the fatty acid from synthesis to degradation pathways during olive fruit development to provide a better understanding of the molecular mechanism of FA metabolism during olive fruit maturation and provide information to improve the synthesis of oil components that are beneficial to human health.

Analysis of DGAT2 mutations reveals potential links between cancer and lipid droplet deregulation

Diacylglycerol O-acyltransferase 2 is a transmembrane protein encoded by the DGAT2 gene that functions in lipid metabolism, triacylglycerol synthesis, and lipid droplet regulation. Since cancer cells exhibit altered lipid metabolism, it has been proposed that mutations in DGAT2 may contribute to this state. Using data from the Catalogue of Somatic Mutations in Cancer (COSMIC), we analyzed all reported DGAT2 mutations in human cancers. Bioinformatics analyses were performed to highlight the connections between age, pathogenicity, and cancer tissue type. Mutations are generally associated with samples from older individuals, except for those in glioblastomas which occur earlier. We also found that several DGAT2 mutations fall within the catalytic site of the enzyme and may affect enzyme function. Thus, these mutations may contribute to altered cancer metabolism. We identified D222V as a mutation hotspot neighboring a previously discovered Y223H mutation that causes Axonal Charcot-Marie-Tooth disease. Remarkably, Y223H has not been detected in cancers indicating it is inhibitory to cancer progression. Further analysis showed that most mutations do not affect DGAT2 gene expression suggesting this change is not a major contributor to cancer development. Intriguingly, although most cancers are characterized by low DGAT2 gene expression, some show high expression levels, indicating that, at least in certain cases, over-expression is not inhibitory to cellular proliferation. This work uncovers unknown roles of DGAT2 in cancers and suggests that its function may be more complex than previously appreciated.

Heat stress leads to rapid lipid remodelling and transcriptional adaptations in Nicotiana tabacum pollen tubes

After reaching the stigma, pollen grains germinate and form a pollen tube that transports the sperm cells to the ovule. Due to selection pressure between pollen tubes, they likely evolved mechanisms to quickly adapt to temperature changes to sustain an elongation at the highest possible rate. We investigated these adaptions in Nicotiana tabacum pollen tubes grown in vitro under 22 °C and 37 °C by a multi-omic approach including lipidomic, metabolomic and transcriptomic analysis. Both glycerophospholipids and galactoglycerolipids increased in saturated acyl chains under heat stress while triacylglycerols changed less in respect to desaturation but showed higher levels. Free sterol composition was altered, and sterol ester levels decreased. The levels of sterylglycosides and several sphingolipid classes and species were augmented. Most amino acids increased during heat stress, including the non-codogenic amino acids γ-amino butyrate and pipecolate. Furthermore, the sugars sedoheptulose and sucrose showed higher levels. Also the transcriptome underwent pronounced changes with 1,570 of 24,013 genes being differentially up- and 813 being downregulated. Transcripts coding for heat shock proteins and many transcriptional regulators were most strongly upregulated, but also transcripts that have so far not been linked to heat stress. Transcripts involved in triacylglycerol synthesis were increased, while the modulation of acyl chain desaturation seemed not to be transcriptionally controlled indicating other means of regulation.

Metabolomic Profiling Revealed Diversion of Cytidinediphosphate-Diacylglycerol and Glycerol Pathway towards Denovo Triacylglycerol Synthesis in Rhodosporidium toruloides

Oleaginous yeast Rhodosporidium toruloides has great biotechnological potential and scientific interest, yet the molecular rationale of its cellular behavior to carbon and nitrogen ratios with concurrent lipid agglomeration remains elusive. Here, metabolomics adaptations of the R. toruloides in response to varying glucose and nitrogen concentrations have been investigated. In preliminary screening we found that 5% glucose (w/v) was optimal for further analysis in Rhodosporidium toruloides 3641. Hereafter, the effect of complementation to increase lipid agglomeration was evaluated with different nitrogen sources and their concentration. The results obtained illustrated that the biomass (13 g/L) and lipid (9.1 g/L) production were maximum on 5% (w/v) glucose and 0.12% (NH4)2SO4. Furthermore, to shed lights on lipid accumulation induced by nitrogen-limitation, we performed metabolomic analysis of the oleaginous yeast R. toruloides 3641. Significant changes were observed in metabolite concentrations by qualitative metabolomics through gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), which were mapped onto the governing metabolic pathways. Notable finding in this strain concerns glycerol and CDP-DAG metabolism wherein reduced production of glycerol and phospholipids induced a bypass leading to enhanced de-novo triacylglyceride synthesis. Collectively, our findings help in understanding the central carbon metabolism of R. toruloides which may assist in developing rationale metabolic models and engineering efforts in this organism.

The relationship between polymorphism rs12449964 of the phosphatidylethanolamine- N-methyltransferase gene and hypertriglyceridemia and obesity in patients with type 2 diabetes

Phosphatidylethanolamine N-methyltransferase (PEMT) is the enzyme of lipid metabolism that catalyzes the conversion of phosphatidylethanolamine to phosphatidylcholine in a series of three methylation reactions. Low activity of the enzyme can increase the availability of phosphatidic acid for triacylglycerol synthesis and thus favor obesity, one of the most important risk factors for type 2 diabetes (T2D). The aim of the study: To study the relationship of the rs12449964 (C>T) in the regulatory region of the PEMT (phosphatidylethanolamine-N-methyltransferase) gene with blood plasma triglycerides, as well as the risk of obesity and T2D in population of Central Russia. Materials and methods: The study included 2060 unrelated individuals of Slavic origin, including 1024 patients with T2D and 1036 healthy volunteers. Genotyping of PEMT gene polymorphism (C>T, rs12449964) was performed by laser desorption / ionization time-of-flight mass spectrometry using the MassArray Analyzer 4 platform (Agena Bioscience). SNPStats online program was used for statistical analysis of the obtained data. Results: Linear regression analysis did not reveal an association of rs12449964 of the PEMT gene with a risk of developing T2D regardless of body mass index (P>0,05). However, the T/T genotype of the studied SNP is associated with an increased risk of obesity in patients with type 2 diabetes (OR 1.66; 95% CI 1.11-2.46; P = 0.011, adjusted for sex and age, recessive model). In addition, carriage of the T/T genotype was associated with a higher level of triacylglycerols in the blood plasma of patients with T2D, both in the presence of obesity and without it (P<0.05). According to GTEx Portal, the rs12449964T allele is associated with decreased PEMT expression in various tissues. Conclusion: The study revealed for the first time the association of rs12449964 of the PEMT gene with hypertriglyceridemia and an increased risk of obesity in patients with T2D, which may be due to the low transcriptional activity of the phosphatidylethanolamine- N-methyltransferase gene in carriers of the alternative allele of the studied SNP.

Deficiency of intestinal Bmal1 prevents obesity induced by high-fat feeding

The role of intestine clock in energy homeostasis remains elusive. Here we show that mice with Bmal1 specifically deleted in the intestine (Bmal1iKO mice) have a normal phenotype on a chow diet. However, on a high-fat diet (HFD), Bmal1iKO mice are protected against development of obesity and related abnormalities such as hyperlipidemia and fatty livers. These metabolic phenotypes are attributed to impaired lipid resynthesis in the intestine and reduced fat secretion. Consistently, wild-type mice fed a HFD during nighttime (with a lower BMAL1 expression) show alleviated obesity compared to mice fed ad libitum. Mechanistic studies uncover that BMAL1 transactivates the Dgat2 gene (encoding the triacylglycerol synthesis enzyme DGAT2) via direct binding to an E-box in the promoter, thereby promoting dietary fat absorption. Supporting these findings, intestinal deficiency of Rev-erbα, a known BMAL1 repressor, enhances dietary fat absorption and exacerbates HFD-induced obesity and comorbidities. Moreover, small-molecule targeting of REV-ERBα/BMAL1 by SR9009 ameliorates HFD-induced obesity in mice. Altogether, intestine clock functions as an accelerator in dietary fat absorption and targeting intestinal BMAL1 may be a promising approach for management of metabolic diseases induced by excess fat intake.

Comprehensive Genetic Analysis of DGAT2 Mutations and Gene Expression Patterns in Human Cancers

DGAT2 is a transmembrane protein encoded by the DGAT2 gene that functions in lipid metabolism, triacylglycerol synthesis, and lipid droplet regulation. Cancer cells exhibit altered lipid metabolism and mutations in DGAT2 may contribute to this state. Using data from the Catalogue of Somatic Mutations in Cancer (COSMIC), we analyzed all cancer genetic DGAT2 alterations, including mutations, copy number variations and gene expression. We find that several DGAT2 mutations fall within the catalytic site of the enzyme. Using the Variant Effect Scoring Tool (VEST), we identify multiple mutations with a high likelihood of contributing to cellular transformation. We also found that D222V is a mutation hotspot neighboring a previously discovered Y223H mutation that causes Axonal Charcot-Marie-Tooth disease. Remarkably, Y223H has not been detected in cancers, suggesting that it is inhibitory to cancer progression. We also identify several single nucleotide polymorphisms (SNP) with high VEST scores, indicating that certain alleles in human populations have a pathogenic predisposition. Most mutations do not correlate with a change in gene expression, nor is gene expression dependent on high allele copy number. However, we did identify eight alleles with high expression levels, suggesting that at least in certain cases, the excess DGAT2 gene product is not inhibitory to cellular proliferation. This work uncovers unknown functions of DGAT2 in cancers and suggests that its role may be more complex than previously appreciated.

A sequence variant in the diacylglycerol O-acyltransferase 2 gene influences palmitoleic acid content in pig muscle

The bulk of body fat in mammals is in the form of triacylglycerol. Diacylglycerol O-acyltransferase 2 (DGAT2) catalyses the terminal step in triacylglycerol synthesis. The proximity of DGAT2 with stearoyl-CoA desaturase (SCD) in the endoplasmic reticulum may facilitate provision of de novo SCD-mediated fatty acids as substrate for DGAT2. Here, we first searched for sequence variants in the DGAT2 gene to then validate their effect on fat content and fatty acid composition in muscle, subcutaneous fat and liver of 1129 Duroc pigs. A single nucleotide polymorphism in exon 9 (ss7315407085 G > A) was selected as a tag variant for the 33 sequence variants identified in the DGAT2 region. The DGAT2-G allele increased DGAT2 expression in muscle and had a positive impact on muscular C14 and C16 fatty acids at the expense of C18 fatty acids. Although there was no evidence for an interaction of DGAT2 with functional SCD genotypes, pigs carrying the DGAT2-G allele had proportionally more palmitoleic acid relative to palmitic acid. Our findings indicate that DGAT2 preferentially uptakes shorter rather than longer-chain fatty acids as substrate, especially if they are monounsaturated, and confirm that fatty acid metabolism in pigs is subjected to subtle tissue-specific genetic regulatory mechanisms.