Triacylglycerol Storage Research Articles

Fads2 knockout mice reveal that ALA prevention of hepatic steatosis is dependent on delta-6 desaturase activity

The production of the omega-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from alpha-linolenic acid (ALA) relies on the delta-6 desaturase (D6D) enzyme encoded by the Fads2 gene. While EPA and DHA reduce hepatic triacylglycerol (TAG) storage and regulate lipogenesis, the independent impact of ALA is less understood. To address this gap in knowledge, hepatic fatty acid metabolism was investigated in male wild-type (WT) and Fads2 knockout (KO) mice fed diets (16% kcal from fat) containing either lard (no n-3 LCPUFA), flaxseed oil (ALA-rich), or menhaden oil (EPA/DHA rich) for 21 weeks. Fat content and composition, as well as markers of lipogenesis, glyceroneogenesis, and TAG synthesis, were analyzed using histology, gas chromatography, and reverse transcription quantitative PCR (RT-qPCR). Mice fed the menhaden diet had significantly lower hepatic TAG compared to both lard- and flax-fed mice, concomitant with changes in n-3 and n-6 LCPUFA in both TAG and phospholipid (PL) fractions (all P < 0.05). Flax-fed WT mice had lower liver TAG content compared to their KO counterparts. Menhaden-fed mice had significantly lower expression of key lipogenic (Scd1, Srebp-1c, Fasn, Fads1, and Fads2), glyceroneogenic (Pck1), and TAG synthesis (Agpat3) genes compared to lard, with flax-fed mice showing some intermediate effects. Gene expression effects were independent of D6D activity, since no differences were detected between WT and KO mice fed the same diet. This study demonstrates that EPA/DHA and not ALA itself is critical for the prevention of hepatic steatosis.

Lipidomics of infant mesenchymal stem cells associate with the maternal milieu and child adiposity.

Our objective was to interrogate mesenchymal stem cell (MSC) lipid metabolism and gestational exposures beyond maternal body mass that may contribute to child obesity risk. MSCs were cultured from term infants of mothers with obesity (n = 16) or normal weight (n = 15). In MSCs undergoing myogenesis in vitro, we used lipidomics to distinguish phenotypes by unbiased cluster analysis and lipid challenge (24-hour excess fatty acid [24hFA]). We measured MSC AMP-activated protein kinase (AMPK) activity and fatty acid oxidation (FAO), and a composite index of maternal glucose, insulin, triglycerides, free fatty acids, TNF-α, and high-density lipoprotein and total cholesterol in fasting blood from mid and late gestation (~17 and ~27 weeks, respectively). We measured child adiposity at birth (n = 29), 4-6 months (n = 29), and 4-6 years (n = 13). Three MSC clusters were distinguished by triacylglycerol (TAG) stores, with greatest TAGs in Cluster 2. All clusters increased acylcarnitines and TAGs with 24hFA, although Cluster 2 was more pronounced and corresponded to AMPK activation and FAO. Maternal metabolic markers predicted MSC clusters and child adiposity at 4-6 years (both highest in Cluster 3). Our data support the notion that MSC phenotypes are predicted by comprehensive maternal metabolic milieu exposures, independent of maternal BMI, and suggest utility as an at-birth predictor for child adiposity, although validation with larger longitudinal samples is warranted.

Toxoplasma acyl-CoA synthetase TgACS3 is crucial to channel host fatty acids in lipid droplets and for parasite propagation

Apicomplexa comprise important pathogenic parasitic protists that heavily depend on lipid acquisition to survive within their human host cells. Lipid synthesis relies on the incorporation of an essential combination of fatty acids (FAs) either generated by a metabolically adaptable de novo synthesis in the parasite or by scavenging from the host cell. The incorporation of FAs into membrane lipids depends on their obligate metabolic activation by specific enzyme groups, acyl-CoA synthetases (ACSs). Each ACS has its own specificity, so it can fulfill specific metabolic functions. Whilst such functionalities have been well studied in other eukaryotic models, their roles and importance in Apicomplexa are currently very limited, especially for Toxoplasma gondii. Here, we report the identification of seven putative ACSs encoded by the genome of T. gondii (TgACS), which localize to different sub-cellular compartments of the parasite, suggesting exclusive functions. We show that the perinuclear/cytoplasmic TgACS3 regulates the replication and growth of Toxoplasma tachyzoites. Conditional disruption of TgACS3 shows that the enzyme is required for parasite propagation and survival, especially under high host nutrient content. Lipidomic analysis of parasites lacking TgACS3 reveals its role in the activation of host-derived FAs that are used for i) parasite membrane phospholipid and ii) storage triacylglycerol (TAG) syntheses, allowing proper membrane biogenesis of parasite progenies. Altogether, our results reveal the role of TgACS3 as the bulk FA activator for membrane biogenesis allowing intracellular division and survival in T. gondii tachyzoites, further pointing to the importance of ACS and FA metabolism for the parasite.

The somatic genome of Eptatretus okinoseanus reveals the adaptation to deep-sea oligotrophic environment

BackgroundHagfishes are fascinating creatures that typically inhabit the deep sea. The deep sea is characterized by its lack of sunlight, primary productivity, and diminishing biomass with increasing ocean depth. Therefore, hagfishes living in this environment must develop effective survival strategies to adapt to the limited food supply. Deep-sea hagfishes have been observed to survive without food intake for up to one year. In this study, we have assembled a high-quality somatic genome of the deep-sea hagfish (Eptatretus okinoseanus) captured below 1,000 m. We compared the genome of E. okinoseanus with the genomes of inshore hagfish, lampreys, and other related species to investigate the genetic factors underlying the deep-sea hagfish adaptations to the environment.ResultsThe E. okinoseanus somatic genome was estimated to be 1.89 Gb and assembled into 17 pseudochromosomes. Phylogenetic analysis showed that shallow-sea and deep-sea hagfishes diverged approximately 58.8 million years ago. We found Perilipin gene family was significantly expanded in deep sea E. okinoseanus, which promotes triacylglycerol storage. Furthermore, a series of genes involved in fatty acid synthesis and metabolism, blood glucose regulation, and metabolic rate regulation were also expanded, rapid evolution or positive selection, and these changes contribute to their efficiency in energy utilization. Among these genes, the positively selected gene JNK may play an important role in energy metabolism. In addition, the olfactory receptors of the deep-sea hagfish were significantly expanded to 86, and three conserved motifs present only in hagfishes olfactory receptors were identified, which may facilitate the rapid localization of carrion.ConclusionsThis study provides valuable genomic resources for insights into the survival strategies of deep-sea hagfishes in oligotrophic environments.

Dynamical modelling of lipid droplet formation suggests a key function of membrane phospholipids.

Cells store triacylglycerol (TAG) within lipid droplets (LDs). A dynamic model describing complete LD formation at the endoplasmic reticulum (ER) membrane does not yet exist. A biochemical-biophysical model of LD synthesis is proposed. It describes the time-dependent accumulation of TAG in the ER membrane as the formation of a potential LD (pLD) bounded by spherical caps of the inner and outer monolayers of the membrane. The expansion rate of the pLD depends on the TAG supply, the elastic properties of the ER membrane, and the recruitment of phospholipids (PLs) to the cap-covering monolayers. Model simulations provided the following insights: (a) Marginal differences in the surface tension of the cap monolayers are sufficient to fully drive the expansion of the pLD towards the cytosol or lumen. (b) Selective reduction of PL supply to the luminal monolayer ensures stable formation of cytosolic LDs, irrespective of variations in the elasto-mechanical properties of the ER membrane. (c) The rate of TAG supply to the cytosolic monolayer has a major effect on the size and maturation time of LDs but has no significant effect on the TAG export per individual LD. The recruitment of additional PLs to the cap monolayers of pLDs critically controls the budding direction, size, and maturation time of LDs. The ability of cells to acquire additional LD initiation sites appears to be key to coping with acutely high levels of potentially toxic free fatty acids.

Nitrogen starvation leads to TOR kinase-mediated downregulation of fatty acid synthesis in the algae Chlorella sorokiniana and Chlamydomonas reinhardtii

BackgroundWhen subject to stress conditions such as nutrient limitation microalgae accumulate triacylglycerol (TAG). Fatty acid, a substrate for TAG synthesis is derived from de novo synthesis or by membrane remodeling. The model industrial alga Chlorellasorokiniana accumulates TAG and other storage compounds under nitrogen (N)-limited growth. Molecular mechanisms underlying these processes are still to be elucidated.ResultPreviously we used transcriptomics to explore the regulation of TAG synthesis in C. sorokiniana. Surprisingly, our analysis showed that the expression of several key genes encoding enzymes involved in plastidic fatty acid synthesis are significantly repressed. Metabolic labeling with radiolabeled acetate showed that de novo fatty acid synthesis is indeed downregulated under N-limitation. Likewise, inhibition of the Target of Rapamycin kinase (TOR), a key regulator of metabolism and growth, decreased fatty acid synthesis. We compared the changes in proteins and phosphoprotein abundance using a proteomics and phosphoproteomics approach in C. sorokiniana cells under N-limitation or TOR inhibition and found extensive overlap between the N-limited and TOR-inhibited conditions. We also identified changes in the phosphorylation status of TOR complex proteins, TOR-kinase, and RAPTOR, under N-limitation. This indicates that TOR signaling is altered in a nitrogen-dependent manner. We find that TOR-mediated metabolic remodeling of fatty acid synthesis under N-limitation is conserved in the chlorophyte algae Chlorella sorokiniana and Chlamydomonas reinhardtii.ConclusionOur results indicate that under N-limitation there is significant metabolic remodeling, including fatty acid synthesis, mediated by TOR signaling. This process is conserved across chlorophyte algae. Using proteomic and phosphoproteomic analysis, we show that N-limitation affects TOR signaling and this in-turn affects the metabolic status of the cells. This study presents a link between N-limitation, TOR signaling and fatty acid synthesis in green-lineage.

Juvenile hormone-induced microRNA miR-iab-8 regulates lipid homeostasis and metamorphosis in Drosophila melanogaster.

Metamorphosis plays an important role in the evolutionary success of insects. Accumulating evidence indicated that microRNAs (miRNAs) are involved in the regulation of processes associated with insect metamorphosis. However, the miRNAs coordinated with juvenile hormone (JH)-regulated metamorphosis remain poorly reported. In the present study, using high-throughput miRNA sequencing combined with Drosophila genetic approaches, we demonstrated that miR-iab-8, which primarily targets homeotic genes to modulate haltere-wing transformation and sterilitywas up-regulated by JH and involved in JH-mediated metamorphosis. Overexpression of miR-iab-8 in the fat body resulted in delayed development and failure of larval-pupal transition. Furthermore, metabolomic analysis results revealed that overexpression of miR-iab-8 caused severe energy metabolism defects especially the lipid metabolism, resulting in significantly reduced triacylglycerol (TG) content and glycerophospholipids but enhanced accumulation of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). In line with this, Nile red staining demonstrated that during the third larval development, the TG content in the miR-iab-8 overexpression larvae was continuously decreased, which is opposite to the control. Additionally, the transcription levels of genes committed to TG synthesis and breakdown were found to be significantly increased and the expression of genes responsible for glycerophospholipids metabolism were also altered. Overall, we proposed that JH induced miR-iab-8 expression to perturb the lipid metabolism homeostasis especially the TG storage in the fat body, which in turn affected larval growth and metamorphosis.

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

BackgroundThe 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.ResultsA 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.ConclusionIn 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.

A CTL − Lys immune function maintains insect metamorphosis by preventing gut bacterial dysbiosis and limiting opportunistic infections

BackgroundGut bacteria are beneficial to the host, many of which must be passed on to host offspring. During metamorphosis, the midgut of holometabolous insects undergoes histolysis and remodeling, and thus risks losing gut bacteria. Strategies employed by holometabolous insects to minimize this risk are obscure. How gut bacteria affect host insects after entering the hemocoel and causing opportunistic infections remains largely elusive.ResultsWe used holometabolous Helicoverpa armigera as a model and found low Lactobacillus load, high level of a C-type lectin (CTL) gene CD209 antigen-like protein 2 (CD209) and its downstream lysozyme 1 (Lys1) in the midgut of the wandering stage. CD209 or Lys1 depletion increased the load of midgut Lactobacillus, which further translocate to the hemocoel. In particular, CD209 or Lys1 depletion, injection of Lactobacillus plantarum, or translocation of midgut L. plantarum into the hemocoel suppressed 20-hydroxyecdysone (20E) signaling and delayed pupariation. Injection of L. plantarum decreased triacylglycerol and cholesterol storage, which may result in insufficient energy and 20E available for pupariation. Further, Lysine-type peptidoglycan, the major component of gram-positive bacterial cell wall, contributed to delayed pupariation and decreased levels of triacylglycerols, cholesterols, and 20E, in both H. armigera and Drosophila melanogaster.ConclusionsA mechanism by which (Lactobacillus-induced) opportunistic infections delay insect metamorphosis was found, namely by disturbing the homeostasis of lipid metabolism and reducing 20E production. Moreover, the immune function of CTL − Lys was characterized for insect metamorphosis by maintaining gut homeostasis and limiting the opportunistic infections.

ApoL6 associates with lipid droplets and disrupts Perilipin1-HSL interaction to inhibit lipolysis.

Adipose tissue stores triacylglycerol (TAG) in lipid droplets (LD) and release fatty acids upon lipolysis during energy shortage. We identify ApoL6 as a LD-associated protein mainly found in adipose tissue, specifically in adipocytes. ApoL6 expression is low during fasting but induced upon feeding. ApoL6 knockdown results in smaller LD with lower TAG content in adipocytes, while ApoL6 overexpression causes larger LD with higher TAG content. We show that the ApoL6 affects adipocytes through inhibition of lipolysis. While ApoL6, Perilipin 1 (Plin1), and HSL can form a complex on LD, C-terminal ApoL6 directly interacts with N-terminal Plin1 to prevent Plin1 binding to HSL, to inhibit lipolysis. Thus, ApoL6 ablation decreases white adipose tissue mass, protecting mice from diet-induced obesity, while ApoL6 overexpression in adipose brings obesity and insulin resistance, making ApoL6 a potential future target against obesity and diabetes.

Intrabacterial lipid inclusion-associated proteins: a core machinery conserved from saprophyte Actinobacteria to the human pathogen Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), the aetiologic agent of tuberculosis (TB), stores triacylglycerol (TAG) in the form of intrabacterial lipid inclusions (ILI) to survive and chronically persist within its host. These highly energetic molecules represent a major source of carbon to support bacterial persistence and reactivation, thus playing a leading role in TB pathogenesis. However, despite its physiological and clinical relevance, ILI metabolism in Mtb remains poorly understood. Recent discoveries have suggested that several ILI-associated proteins might be widely conserved across TAG-producing prokaryotes, but still very little is known regarding the nature and the biological functions of these proteins. Herein, we performed an in silico analysis of three independent ILI-associated proteomes previously reported to computationally define a potential core ILI-associated proteome, referred to as ILIome. Our investigation revealed the presence of 70 orthologous proteins that were strictly conserved, thereby defining a minimal ILIome core. We further narrowed our analysis to proteins involved in lipid metabolism and discuss here their putative biological functions, along with their molecular interactions and dynamics at the surface of these bacterial organelles. We also highlight the experimental limitations of the original proteomic investigations and of the present bioinformatic analysis, while describing new technological approaches and presenting biological perspectives in the field. The in silico investigation presented here aims at providing useful datasets that could constitute a scientific resource of broad interest for the mycobacterial community, with the ultimate goal of enlightening ILI metabolism in prokaryotes with a special emphasis on Mtb pathogenesis.

Glycerol-3-Phosphate Acyltransferase GPAT9 Enhanced Seed Oil Accumulation and Eukaryotic Galactolipid Synthesis in Brassica napus.

Glycerol-3-phosphate acyltransferase GPAT9 catalyzes the first acylation of glycerol-3-phosphate (G3P), a committed step of glycerolipid synthesis in Arabidopsis. The role of GPAT9 in Brassica napus remains to be elucidated. Here, we identified four orthologs of GPAT9 and found that BnaGPAT9 encoded by BnaC01T0014600WE is a predominant isoform and promotes seed oil accumulation and eukaryotic galactolipid synthesis in Brassica napus. BnaGPAT9 is highly expressed in developing seeds and is localized in the endoplasmic reticulum (ER). Ectopic expression of BnaGPAT9 in E. coli and siliques of Brassica napus enhanced phosphatidic acid (PA) production. Overexpression of BnaGPAT9 enhanced seed oil accumulation resulting from increased 18:2-fatty acid. Lipid profiling in developing seeds showed that overexpression of BnaGPAT9 led to decreased phosphatidylcholine (PC) and a corresponding increase in phosphatidylethanolamine (PE), implying that BnaGPAT9 promotes PC flux to storage triacylglycerol (TAG). Furthermore, overexpression of BnaGPAT9 also enhanced eukaryotic galactolipids including monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), with increased 36:6-MGDG and 36:6-DGDG, and decreased 34:6-MGDG in developing seeds. Collectively, these results suggest that ER-localized BnaGPAT9 promotes PA production, thereby enhancing seed oil accumulation and eukaryotic galactolipid biosynthesis in Brassica napus.

A model of hepatic steatosis with declined viability and function in a liver-organ-on-a-chip

Nonalcoholic fatty liver disease (NAFLD) begins with benign steatosis caused by ectopic storage of triacylglycerols in the liver. Persistent steatosis, in combination with other genetic and environmental factors, leads to nonalcoholic steatohepatitis (NASH) characterized by functional impairment, inflammation, and fibrosis. However, it remains unclear how persistent steatosis directly contributes to the progression of NAFLD, which may represent a therapeutic target. The organ-on-a-chip (OOC) has emerged as a new culture platform to recapitulate human pathological conditions under which drug candidates can be screened. Here, we developed a simple OOC steatosis model using the Mimetas OrganoPlate with a human liver cell line, HepG2. Treating the HepG2 OOCs with fatty acid overload induced steatosis within 24 h. Moreover, persistent steatosis for 6 days impaired OOC viability and hepatic function, as measured by a WST-8 assay and albumin production, respectively. Lastly, the HepG2 OOCs were exposed to drugs being tested in clinical trials for NAFLD/NASH during the 6-day period. Pioglitazone improved the OOC viability while elafibranor reduced the steatosis in association with reduced viability and albumin production. In conclusion, we show that the HepG2 steatosis OOC model is a useful tool on which the efficacy and toxicity of various therapeutic candidates can be tested.

Native promoter-mediated transcriptional regulation of crucial oleosin protein OLE1 from Prunus sibirica for seed development and high oil accumulation

Oleosin (OLE) is vital to stabilize lipid droplet for seed triacylglycerol (TAG) storage. This work aimed to determine key OLE and to unravel mechanism that governed seed oil accumulation of Prunus sibirica for developing biodiesel. An integrated assay of global identification of LD-related protein and the cross-accessions/developing stages comparisons associated with oil accumulative amount and OLE transcript level was performed on seeds of 12 plus trees of P. sibirica to identify OLE1 (15.5 kDa) as key oleosin protein crucial for high seed oil accumulation. The OLE1 gene and its promoter were cloned from P. sibirica seeds, and overexpression of PsOLE1 in Arabidopsis was conducted under the controls of native promoter and constitutive CaMV35S promoter, respectively. PsOLE1 promoter had seed-specific cis-elements and showed seed specificity, by which PsOLE1 was specifically expressed in seeds. Ectopic overexpression of PsOLE1, especially driven by its promoter, could facilitate seed development and oil accumulation with an increase in unsaturated FAs, and upregulate transcript of TAG assembly enzymes, but suppress transcript of LD/TAG-hydrolyzed lipases and transporters, revealing a role of native promoter-mediated transcription of PsOLE1 in seed development and oil accumulation. PsOLE1 and its promoter have considerable potential for engineering oil accumulation in oilseed plants.

Heterologous expression of MiMLDP, a major lipid droplet protein of the microalga Myrmecia incisa, in Saccharomyces cerevisiae promotes more triacylglycerol storage principally by enlarging lipid droplet size

In some algae, lipid droplets (LDs) are surrounded by phospholipid monolayers containing specialized proteins such as major lipid droplet protein (MLDP), which serves as a principal structural component and makes significant contributions to the enrichment of LDs. In the present study, a new MLDP gene from green alga Myrmecia incisa was reported for the first time, and was comprehensively investigated with respect to its identification, localization and function. The gene product MiMLDP was 35.46 % identical to the reported ortholog in the synonym Lobosphaera incisa. A polyclonal antibody was prepared and the subcellular localization was confirmed to be on the LDs. By flow cytometry and TLC-FID analyses, MiMLDP was demonstrated to improve the accumulation of triacylglycerol. By fusing the coding sequence with eYFP and transforming into yeast strain BY4741, MiMLDP was found to be able to increase the size of LDs and decrease the content of steryl esters, which consequently enhanced the triacylglycerol storage.

Preferential lipolysis of DGAT1 over DGAT2 generated triacylglycerol in Huh7 hepatocytes

Two distinct diacylglycerol acyltransferases (DGAT1 and DGAT2) catalyze the final committed step of triacylglycerol (TG) synthesis in hepatocytes. After its synthesis in the endoplasmic reticulum (ER) TG is either stored in cytosolic lipid droplets (LDs) or is assembled into very low-density lipoproteins in the ER lumen. TG stored in cytosolic LDs is hydrolyzed by adipose triglyceride lipase (ATGL) and the released fatty acids are converted to energy by oxidation in mitochondria. We hypothesized that targeting/association of ATGL to LDs would differ depending on whether the TG stores were generated through DGAT1 or DGAT2 activities. Individual inhibition of DGAT1 or DGAT2 in Huh7 hepatocytes incubated with oleic acid did not yield differences in TG accretion while combined inhibition of both DGATs completely prevented TG synthesis suggesting that either DGAT can efficiently esterify exogenously supplied fatty acid. DGAT2-made TG was stored in larger LDs, whereas TG formed by DGAT1 accumulated in smaller LDs. Inactivation of DGAT1 or DGAT2 did not alter expression (mRNA or protein) of ATGL, the ATGL activator ABHD5/CGI-58, or LD coat proteins PLIN2 or PLIN5, but inactivation of both DGATs increased PLIN2 abundance despite a dramatic reduction in the number of LDs. ATGL was found to preferentially target to LDs generated by DGAT1 and fatty acids released from TG in these LDs were also preferentially used for fatty acid oxidation. Combined inhibition of DGAT2 and ATGL resulted in larger LDs, suggesting that the smaller size of DGAT1-generated LDs is the result of increased lipolysis of TG in these LDs.

Lipid droplets proteome reveals dynamic changes of lipid droplets protein during embryonic development of Carya cathayensis nuts

Lipid droplets (LD) is an important intracellular organelle for triacylglycerols (TAGs) storage. A variety of proteins on the surface of LD coordinately control the contents, size, stability and biogenesis of LD. However, the LD proteins in Chinese hickory (Carya cathayensis) nuts, which is rich in oil and composed of unsaturated fatty acids, have not been identified and their roles in LD formation still remain largely unknown. In present study, LD fractions from three developmental stages of Chinese hickory seed were enriched and the LD fraction accumulated proteins were then isolated and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein compositions throughout the various developmental phases were calculated using label-free intensity-based absolute quantification (iBAQ) algorithm. The dynamic proportion of high abundance lipid droplets proteins such as oleosins 2 (OLE2), caleosins 1 (CLO1) and steroleosin 5 (HSD5) increased parallelly with the embryo development. For low abundance lipid droplets proteins, seed LD protein 2 (SLDP2), sterol methyltransferase 1 (SMT1) and LD-associated protein 1 (LDAP1) were the predominant proteins. Moreover, 14 low abundance OB proteins such as oil body-associated protein 2 A (OBAP2A) were selected for future investigation that may associate with embryo development. Overall, 62 differentially expressed proteins (DEPs) were determined by label free quantification (LFQ) algorithms and may involve in LD biogenesis. Furthermore, the subcellular localization validation indicated that selected LD proteins were targeted to the lipid droplets, confirming the promising of proteome data. Taken together, this comparative study may shed light on further study to understand the lipid droplets function in the seed, which contains high oil content.

Delta-6 desaturase (Fads2) deficiency alters triacylglycerol/fatty acid cycling in murine white adipose tissue

The Δ-6 desaturase (D6D) enzyme is not only critical for the synthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from α-linolenic acid (ALA), but recent evidence suggests that it also plays a role in adipocyte lipid metabolism and body weight; however, the mechanisms remain largely unexplored. The goal of this study was to investigate if a D6D deficiency would inhibit triacylglycerol storage and alter lipolytic and lipogenic pathways in mouse white adipose tissue (WAT) depots due to a disruption in EPA and DHA production. Male C57BL/6J D6D knockout (KO) and wild-type (WT) mice were fed either a 7% w/w lard or flax (ALA rich) diet for 21 weeks. Energy expenditure, physical activity, and substrate utilization were measured with metabolic caging. Inguinal and epididymal WAT depots were analyzed for changes in tissue weight, fatty acid composition, adipocyte size, and markers of lipogenesis, lipolysis, and insulin signaling. KO mice had lower body weight, higher serum nonesterified fatty acids, smaller WAT depots, and reduced adipocyte size compared to WT mice without altered food intake, energy expenditure, or physical activity, regardless of the diet. Markers of lipogenesis and lipolysis were more highly expressed in KO mice compared to WT mice in both depots, regardless of the diet. These changes were concomitant with lower basal insulin signaling in WAT. Collectively, a D6D deficiency alters triacylglycerol/fatty acid cycling in WAT by promoting lipolysis and reducing fatty acid re-esterification, which may be partially attributed to a reduction in WAT insulin signaling.

Functional genome annotation and transcriptome analysis of Pseudozyma hubeiensis BOT-O, an oleaginous yeast that utilizes glucose and xylose at equal rates

Pseudozyma hubeiensis is a basidiomycete yeast that has the highly desirable traits for lignocellulose valorisation of being equally efficient at utilization of glucose and xylose, and capable of their co-utilization. The species has previously mainly been studied for its capacity to produce secreted biosurfactants in the form of mannosylerythritol lipids, but it is also an oleaginous species capable of accumulating high levels of triacylglycerol storage lipids during nutrient starvation. In this study, we aimed to further characterize the oleaginous nature of P. hubeiensis by evaluating metabolism and gene expression responses during storage lipid formation conditions with glucose or xylose as a carbon source.The genome of the recently isolated P. hubeiensis BOT-O strain was sequenced using MinION long-read sequencing and resulted in the most contiguous P. hubeiensis assembly to date with 18.95 Mb in 31 contigs. Using transcriptome data as experimental support, we generated the first mRNA-supported P. hubeiensis genome annotation and identified 6540 genes. 80% of the predicted genes were assigned functional annotations based on protein homology to other yeasts. Based on the annotation, key metabolic pathways in BOT-O were reconstructed, including pathways for storage lipids, mannosylerythritol lipids and xylose assimilation. BOT-O was confirmed to consume glucose and xylose at equal rates, but during mixed glucose-xylose cultivation glucose was found to be taken up faster. Differential expression analysis revealed that only a total of 122 genes were significantly differentially expressed at a cut-off of |log2 fold change| ≥ 2 when comparing cultivation on xylose with glucose, during exponential growth and during nitrogen-starvation. Of these 122 genes, a core-set of 24 genes was identified that were differentially expressed at all time points. Nitrogen-starvation resulted in a larger transcriptional effect, with a total of 1179 genes with significant expression changes at the designated fold change cut-off compared with exponential growth on either glucose or xylose.

Gene cloning, expression pattern, and response to dietary total lipids and phospholipids of hormone-sensitive lipase (HSL) in the Oriental river prawnMacrobrachium nipponenseDe Haan, 1849 (Decapoda: Caridea: Palaemonidae)

AbstractHormone-sensitive lipase (HSL) is an important regulator of cellular lipid homeostasis and catalyzes the hydrolysis of stored triacylglycerol. We identified and cloned for the first time the full-length cDNA sequence of HSL of the prawn Macrobrachium nipponense De Haan, 1849 [De Haan, 1833–1850] from a hepatopancreas cDNA library. The complete HSL sequence is 3,575 bp and encoded a 785 amino acid peptide with the catalytic core (GXSXG) containing a serine residue. The phylogenetic tree revealed that the gene of HSL of M. nipponense is closely related with that of Penaeus vanmameiBoone, 1931. The tissue distribution showed that the mRNA expression level of HSL in the hepatopancreas was significantly higher than that in other tissues (P &amp;lt; 0.05). Furthermore, the HSL expression in hepatopancreas was upregulated with the increase of dietary lipids but partially inhibited when the ratio of phospholipids was increased in the lipid mixture. These results demonstrate that HSL is involved in the lipid metabolism of M. nipponense and highlights the importance of phospholipids in lipid metabolism.