Triacylglycerol Degradation Research Articles

Overexpression of ATGL impairs lipid droplet accumulation by accelerating lipolysis in goat mammary epithelial cells

Adipose triglyceride lipase (ATGL) is the key enzyme for the degradation of triacylglycerols (TAGs). It functions in concert with other enzymes to mobilize TAG and supply fatty acids (FAs) for energy production. Dysregulated lipolysis leads to excess concentrations of circulating FAs, which may lead to destructive and lipotoxic effects to the organism. To understand the role of ATGL in mammary lipid metabolism, ATGL was overexpressed in goat mammary epithelial cells (GMECs) by using a recombinant adenovirus system. ATGL overexpression decreased lipid droplet (LD) accumulation and cellular TG content (p < 0.05) along with a decrease in the expression of the key enzyme that catalyzes the final step of TG synthesis (DGAT). Significant increases were observed in the expression of genes related to lipolysis (hormone-sensitive lipase [HSL]) and FA desaturation (SCD) by ATGL overexpression. Genes responsible for FA oxidation (PPARα), LD formation and secretion (ADRP and BTN1A1), and long-chain FA uptake (CD36) were all decreased by ATGL overexpression (p < 0.05). The primary products of TAG lipolysis, free FAs (FFAs), were notably increased in the ATGL-overexpressing cells. Taken together, our results demonstrated that ATGL activation impairs lipid formation partially through accelerating lipolysis in GMECs.

Finding new friends and revisiting old ones - how plant lipid droplets connect with other subcellular structures.

The number of described contact sites between different subcellular compartments and structures in eukaryotic cells has increased dramatically in recent years and, as such, has substantially reinforced the well-known premise that these kinds of connections are essential for overall cellular organization and the proper functioning of cellular metabolic and signaling pathways. Here, we discuss contact sites involving plant lipid droplets (LDs), including LD-endoplasmic reticulum (ER) connections that mediate the biogenesis of new LDs at the ER, LD-peroxisome connections, that facilitate the degradation of LD-stored triacylglycerols (TAGs), and the more recently discovered LD-plasma membrane connections, which involve at least three novel proteins, but have a yet unknown physiological function(s).

A negative-solvatochromic fluorescent probe for visualizing intracellular distributions of fatty acid metabolites

Metabolic distribution of fatty acid to organelles is an essential biological process for energy homeostasis as well as for the maintenance of membrane integrity, and the metabolic pathways are strictly regulated in response to environmental stimuli. Herein, we report a fluorescent fatty acid probe, which bears an azapyrene dye that changes its absorption and emission features depending on the microenvironment polarity of the organelle into which it is transported. Owing to the environmental sensitivity of this dye, the distribution of the metabolically incorporated probe in non-polar lipid droplets, medium-polarity membranes, and the polar aqueous regions, can be visualized in different colors. Based on density scatter plots of the fluorophore, we demonstrate that the degradation of triacylglycerols in lipid droplets occurs predominantly via lipolysis rather than lipophagy in nutrition-starved hepatocytes. This tool can thus be expected to significantly advance our understanding of the lipid metabolism in living organisms.

Exploring Camelina sativa lipid metabolism regulation by combining gene co‐expression and DNA affinity purification analyses

SUMMARYCamelina (Camelina sativa) is an annual oilseed plant that is gaining momentum as a biofuel cover crop. Understanding gene regulatory networks is essential to deciphering plant metabolic pathways, including lipid metabolism. Here, we take advantage of a growing collection of gene expression datasets to predict transcription factors (TFs) associated with the control of Camelina lipid metabolism. We identified approximately 350 TFs highly co‐expressed with lipid‐related genes (LRGs). These TFs are highly represented in the MYB, AP2/ERF, bZIP, and bHLH families, including a significant number of homologs of well‐known Arabidopsis lipid and seed developmental regulators. After prioritizing the top 22 TFs for further validation, we identified DNA‐binding sites and predicted target genes for 16 out of the 22 TFs tested using DNA affinity purification followed by sequencing (DAP‐seq). Enrichment analyses of targets supported the co‐expression prediction for most TF candidates, and the comparison to Arabidopsis revealed some common themes, but also aspects unique to Camelina. Within the top potential lipid regulators, we identified CsaMYB1, CsaABI3AVP1‐2, CsaHB1, CsaNAC2, CsaMYB3, and CsaNAC1 as likely involved in the control of seed fatty acid elongation and CsaABI3AVP1‐2 and CsabZIP1 as potential regulators of the synthesis and degradation of triacylglycerols (TAGs), respectively. Altogether, the integration of co‐expression data and DNA‐binding assays permitted us to generate a high‐confidence and short list of Camelina TFs involved in the control of lipid metabolism during seed development.

Elucidation of Triacylglycerol Overproduction in the C4 Bioenergy Crop Sorghum bicolor by Constraint-Based Analysis.

Upregulation of triacylglycerols (TAGs) in vegetative plant tissues such as leaves has the potential to drastically increase the energy density and biomass yield of bioenergy crops. In this context, constraint-based analysis has the promise to improve metabolic engineering strategies. Here we present a core metabolism model for the C4 biomass crop Sorghum bicolor (iTJC1414) along with a minimal model for photosynthetic CO2 assimilation, sucrose and TAG biosynthesis in C3 plants. Extending iTJC1414 to a four-cell diel model we simulate C4 photosynthesis in mature leaves with the principal photo-assimilatory product being replaced by TAG produced at different levels. Independent of specific pathways and per unit carbon assimilated, energy content and biosynthetic demands in reducing equivalents are about 1.3 to 1.4 times higher for TAG than for sucrose. For plant generic pathways, ATP- and NADPH-demands per CO2 assimilated are higher by 1.3- and 1.5-fold, respectively. If the photosynthetic supply in ATP and NADPH in iTJC1414 is adjusted to be balanced for sucrose as the sole photo-assimilatory product, overproduction of TAG is predicted to cause a substantial surplus in photosynthetic ATP. This means that if TAG synthesis was the sole photo-assimilatory process, there could be an energy imbalance that might impede the process. Adjusting iTJC1414 to a photo-assimilatory rate that approximates field conditions, we predict possible daily rates of TAG accumulation, dependent on varying ratios of carbon partitioning between exported assimilates and accumulated oil droplets (TAG, oleosin) and in dependence of activation of futile cycles of TAG synthesis and degradation. We find that, based on the capacity of leaves for photosynthetic synthesis of exported assimilates, mature leaves should be able to reach a 20% level of TAG per dry weight within one month if only 5% of the photosynthetic net assimilation can be allocated into oil droplets. From this we conclude that high TAG levels should be achievable if TAG synthesis is induced only during a final phase of the plant life cycle.

Investigation of the antioxidation capacity of gallic acid and its alkyl esters with different chain lengths for dried oyster during ambient storage

SummaryThe antioxidant capacity of gallic acid (GA) and its fatty alkyl esters such as butyl gallate, octyl gallate (OG), lauryl gallate and hexadecyl gallate in freeze‐dried oyster was investigated. During ambient storage, the significant increase of peroxide value and thiobarbituric acid value together with the decline for docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) contents indicated the occurrence of lipid autoxidation in freeze‐dried oysters. Meanwhile, the oxidative degradation of glycerol phosphocholine (GPC), glycerol phosphoethanolamine (GPE) and triacylglycerols (TAG) molecular species carrying DHA or EPA in oyster ranked as: TAG &gt; GPE &gt; GPC. GA and its alkyl esters could effectively be prevented dried oyster lipid from oxidation. Among them, OG stood out as the most effective individual. This work implied that the chain lengths of GA alkyl esters affect their antioxidant ability for dried oysters, which provides the information for selecting eligible antioxidants to enhance the oxidative stability of freeze‐dried seafood.

Multiplying the heterologous production of spinosad through tandem amplification of its biosynthetic gene cluster in Streptomyces coelicolor.

SummaryHeterologous expression of the biosynthetic gene cluster (BGC) is important for studying the microbial natural products (NPs), especially for those kept in silent or poorly expressed in their original strains. Here, we cloned the spinosad BGC through the Cas9‐Assisted Targeting of Chromosome segments and amplified it to five copies through a ZouA‐dependent DNA amplification system in Streptomyces coelicolor M1146. The resulting strain produced 1253.9 ± 78.2 μg l−1 of spinosad, which was about 224‐fold compared with that of the parent strain carrying only one copy of the spinosad BGC. Moreover, we further increased spinosad to 1958.9 ± 73.5 μg l−1 by the dynamic regulation of intracellular triacylglycerol degradation. Our study indicates that tandem amplification of the targeted gene cluster is particularly suitable to enhance the heterologous production of valuable NPs with efficiency and simplicity.

Comprehensive mining of storage oil related genes in developing seed of Abelmoschus esculentus

Okra (Abelmoschus esculentus), famous for its desired nutritional and medicinal values, could accumulate approximately 20% (w/w) seed oil dominated with unsaturated fatty acids (UFAs). However, the mechanism of oil biosynthesis and metabolic regulation in its seeds remains poorly understood. Here, storage oil profiles and transcriptome of okra seeds at different developmental stages were analyzed. Seed oil accumulated at a high rate during 21 to 28 days after flowering (DAF). Total of 136.6 million clean reads were obtained and 109,171 unigenes were assembled from the seed transcriptome. Differentially expressed genes among three developing stages of seeds were functionally annotated into the storage lipid metabolism pathway. Comparative analysis was performed for structural genes and transcription factor genes that participate in fatty acid biosynthesis, triacylglycerol assembly, oil body formation, and triacylglycerol degradation. A complex regulating network was built accordingly. Additionally, expressions of 12 key functional genes responsible for okra oil accumulation were examined by quantitative real-time PCR (qRT-PCR), demonstrating that RNA-sequencing data were reliable. This study will facilitate further investigation of molecular mechanism responsible for unsaturated fatty acid biosynthesis and oil accumulation in plant seed, providing valued reference for creating new okra varieties with better lipid composition and high oil yield.

The disassembly of lipid droplets in Chlamydomonas.

Lipid droplets (LDs) are ubiquitous and specialized organelles in eukaryotic cells. Consisting of a triacylglycerol core surrounded by a monolayer of membrane lipids, LDs are decorated with proteins and have myriad functions, from carbon/energy storage to membrane lipid remodeling and signal transduction. The biogenesis and turnover of LDs are therefore tightly coordinated with cellular metabolic needs in a fluctuating environment. Lipid droplet turnover requires remodeling of the protein coat, lipolysis, autophagy and fatty acid β-oxidation. Several key components of these processes have been identified in Chlamydomonas (Chlamydomonas reinhardtii), including the major lipid droplet protein, a CXC-domain containing regulatory protein, the phosphatidylethanolamine-binding DTH1 (DELAYED IN TAG HYDROLYSIS1), two lipases and two enzymes involved in fatty acid β-oxidation. Here, we review LD turnover and discuss its physiological significance in Chlamydomonas, a major model green microalga in research on algal oil.

Significance of two intracellular triacylglycerol lipases of Aspergillus oryzae in lipid mobilization: A perspective in industrial implication for microbial lipid production.

Microbial lipid production of oleaginous strains involves in a complex cellular metabolism controlling lipid biosynthesis, accumulation and degradation. Particular storage lipid, triacylglycerol (TAG), contributes to dynamic traits of intracellular lipids and cell growth. To explore a basis of TAG degradation in the oleaginous strain of Aspergillus oryzae, the functional role of two intracellular triacylglycerol lipases, AoTgla and AoTglb, were investigated by targeted gene disruption using CRISPR/Cas9 system. Comparative lipid profiling of different cultivation stages between the control, single and double disruptant strains (ΔAotgla, ΔAotglb and ΔAotglaΔAotglb strains) showed that the inactivation of either AoTgla or AoTglb led to the increase of total lipid contents, particularly in the TAG fraction. Moreover, the prolonged lipid-accumulating stage of all disruptant strains was obtained as indicated by a reduction in specific rate of lipid turnover, in which a holding capacity in maximal lipid and TAG levels was achieved. The involvement of AoTgls in spore production of A. oryzae was also discovered. In addition to the significance in lipid physiology of the oleaginous fungi, this study provides an impact on industrial practice by overcoming the limitation in short lipid-accumulating stage of the fungal strain, which facilitate the cell harvesting step at the maximum lipid production yield.

The microRNA miR-33 is a pleiotropic regulator of metabolic and developmental processes in Drosophila melanogaster.

miR-33 family members are well characterized regulators of cellular lipid levels in mammals. Previous studies have shown that overexpression of miR-33 in Drosophila melanogaster leads to elevated triacylglycerol (TAG) levels in certain contexts. Although loss of miR-33 in flies causes subtle defects in larval and adult ovaries, the effects of miR-33 deficiency on lipid metabolism and other phenotypes impacted by metabolic state have not yet been characterized. We found that loss of miR-33 predisposes flies to elevated TAG levels, and we identified genes involved in TAG synthesis as direct targets of miR-33, including atpcl, midway, and Akt1. miR-33 mutants survived longer upon starvation but showed greater sensitivity to an oxidative stressor. We also found evidence that miR-33 is a negative regulator of cuticle pigmentation and that miR-33 mutants show a reduction in interfollicular stalk cells during oogenesis. Our data suggest that miR-33 is a conserved regulator of lipid homeostasis, and its targets are involved in both degradation and synthesis of fatty acids and TAG. The constellation of phenotypes involving tissues that are highly sensitive to metabolic state suggests that miR-33 serves to prevent extreme fluctuations in metabolically sensitive tissues.

Mechanism Controlling High‐Temperature Degradation of Sunflower Oil Triacylglycerols in the Absence of Oxygen

AbstractThe aim of the present study is to describe the mechanism controlling heat‐induced formation of sunflower oil triacylglycerol and fatty acid methyl ester oligomers. The unique combination of high‐performance size‐exclusion chromatography with hyphenated electrospray ionization mass spectrometry (MS), atmospheric pressure chemical ionization‐MS, and high‐temperature gas chromatography‐MS techniques allows differentiating between radical coupling species and Diels–Alder cycloadducts. Targeted analysis of thermally degraded sunflower oils confirms the exact structures of various acyclic oligomers accompanied by less‐abundant products of pericyclic transformations. A series of model experiments simulate the impact of dienophile nature on the course of Diels–Alder reactions. Thus, α‐tocopherylquinone, δ‐tocopherylquinone, and methyl‐(E)‐11‐oxoundec‐9‐enoate are synthesized as naturally occurring dienophiles bearing electron‐withdrawing groups. The geometry of poor dienophiles does not affect concerted cyclization, while the structure of electron deficient dienophiles can overcome low reactivity.Practical Application: In the absence of oxygen, heat‐induced degradation of polyunsaturated triacylglycerols proceed predominantly via a radical pathway, whereas concerted reactions represent minor mechanisms. Sunflower oil triacylglycerol molecules in the system without propagation stage can be effectively protected by natural and/or synthetic antioxidants. Application of chelates is also recommended. However, antioxidant‐derived quinones, such as α‐tocopherylquinone, can enter the Diels–Alder reaction even more easily than dienophiles without electron‐withdrawing groups. Unsaturated core aldehydes possess the same reactivity. Examination of the mechanism controlling high‐temperature degradation of triacylglycerols is especially important for processing engineers in edible oil refineries and food technologists. New perspective may help them to minimize undesirable changes in polyunsaturated species.

The phosphatidylethanolamine-binding protein DTH1 mediates degradation of lipid droplets in Chlamydomonas reinhardtii

Lipid droplets (LDs) are intracellular organelles found in a wide range of organisms and play important roles in stress tolerance. During nitrogen (N) starvation, Chlamydomonas reinhardtii stores large amounts of triacylglycerols (TAGs) inside LDs. When N is resupplied, the LDs disappear and the TAGs are degraded, presumably providing carbon and energy for regrowth. The mechanism by which cells degrade LDs is poorly understood. Here, we isolated a mutant (dth1-1, Delayed in TAG Hydrolysis 1) in which TAG degradation during recovery from N starvation was compromised. Consequently, the dth1-1 mutant grew poorly compared to its parental line during N recovery. Two additional independent loss-of-function mutants (dth1-2 and dth1-3) also exhibited delayed TAG remobilization. DTH1 transcript levels increased sevenfold upon N resupply, and DTH1 protein was localized to LDs. DTH1 contains a putative lipid-binding domain (DTH1LBD) with alpha helices predicted to be structurally similar to those in apolipoproteins E and A-I. Recombinant DTH1LBD bound specifically to phosphatidylethanolamine (PE), a major phospholipid coating the LD surface. Overexpression of DTH1LBD in Chlamydomonas phenocopied the dth1 mutant's defective TAG degradation, suggesting that the function of DTH1 depends on its ability to bind PE. Together, our results demonstrate that the lipid-binding DTH1 plays an essential role in LD degradation and provide insight into the molecular mechanism of protein anchorage to LDs at the LD surface in photosynthetic cells.

Transcriptional Regulation of Lipid Catabolism during Seedling Establishment

Lipid catabolism in germinating seeds provides energy and substrates for initial seedling growth, but how this process is regulated is not well understood. Here, we show that an AT-hook motif-containing nuclear localized (AHL) protein regulates lipid mobilization and fatty acid β-oxidation during seed germination and seedling establishment. AHL4 was identified to directly interact with the lipid mediator phosphatidic acid (PA). Knockout (KO) of AHL4 enhanced, but overexpression (OE) of AHL4 attenuated, triacylglycerol (TAG) degradation and seedling growth. Normal seedling growth of the OE lines was restored by sucrose supplementation to the growth medium. AHL4-OE seedlings displayed decreased expression of genes involved in TAG hydrolysis and fatty acid oxidation, whereas the opposite was observed in AHL4-KOs. These genes contained AHL4-binding cis elements, and AHL4 was shown to bind to the promoter regions of genes encoding the TAG lipases SDP1 and DALL5 and acyl-thioesterase KAT5. These AHL4–DNA interactions were suppressed by PA species that bound to AHL4. These results indicate that AHL4 suppresses lipid catabolism by repressing the expression of specific genes involved in TAG hydrolysis and fatty acid oxidation, and that PA relieves AHL4-mediated suppression and promotes TAG degradation. Thus, AHL4 and PA together regulate lipid degradation during seed germination and seedling establishment.

The Lipolysome-A Highly Complex and Dynamic Protein Network Orchestrating Cytoplasmic Triacylglycerol Degradation.

The catabolism of intracellular triacylglycerols (TAGs) involves the activity of cytoplasmic and lysosomal enzymes. Cytoplasmic TAG hydrolysis, commonly termed lipolysis, is catalyzed by the sequential action of three major hydrolases, namely adipose triglyceride lipase, hormone-sensitive lipase, and monoacylglycerol lipase. All three enzymes interact with numerous protein binding partners that modulate their activity, cellular localization, or stability. Deficiencies of these auxiliary proteins can lead to derangements in neutral lipid metabolism and energy homeostasis. In this review, we summarize the composition and the dynamics of the complex lipolytic machinery we like to call “lipolysome”.

Algal Autophagy Is Necessary for the Regulation of Carbon Metabolism Under Nutrient Deficiency.

Autophagy is a mechanism to recycle intracellular constituents such as amino acids and other carbon- and nitrogen (N)-containing compounds. Although autophagy-related (ATG) genes required for autophagy are encoded by many algal genomes, their functional importance in microalgae in nutrient-deficiency has not been appraised using ATG-defective mutants. Recently, by characterization of an insertional mutant of the ATG8 encoding a ubiquitin-like protein indispensable for autophagosome formation in a green alga Chlamydomonas reinhardtii, we have provided evidence that supports the following notions. ATG8 protein is required for the degradation of lipid droplets and triacylglycerol (TAG) triggered by resupply of N to cell culture in N-deficient conditions. ATG8 protein is also necessary for starch accumulation under phosphorus-deficient conditions. Algal autophagy is not necessary for inheritance of chloroplast and mitochondrial genomes. In this review, we discuss the physiological roles of algal autophagy associated with nutrient deficiency revealed by the genetic and biochemical analyses using disruption mutants and reagents that inhibit the fatty acid biosynthesis and vacuolar H+-ATPase.

Determining Fingerprint Age with Mass Spectrometry Imaging via Ozonolysis of Triacylglycerols.

Despite the common use of fingerprints as a trusted means of identification, no method currently exists to reliably establish the time since deposition of latent fingerprints. A reproducible method of establishing latent fingerprint age would allow forensic personnel to determine if a latent fingerprint was relevant to a crime. This work investigates the ambient aging of triacylglycerols (TGs) and other lipids in latent fingerprint residue utilizing matrix-assisted laser desorption/ionization mass spectrometry imaging. Unsaturated TGs were found to undergo ambient ozonolysis resulting in a decrease over time. At the same time, two series of compounds related to the degradation of unsaturated TGs due to ambient ozonolysis emerged with time and were detectable within a single day of aging. Tracking the degradation of unsaturated TGs over time proved to be relatively reproducible in multiple individuals and is suggested as a means of establishing latent fingerprint age.

Autophagy, Hyperlipidemia, and Atherosclerosis.

Autophagy is an evolutionarily conserved process in eukaryotes that processes the turnover of intracellular substances. Atherosclerosis is a disease caused by multiple factors, it mainly occurs on the walls of large and medium blood vessels and atherosclerotic plaques form in the intima of the blood vessels. Hyperlipidemia is considered to be a very dangerous factor leading to cardiovascular and cerebrovascular diseases, especially atherosclerosis. This chapter mainly introduces the key role of autophagy in hyperlipidemia and atherosclerosis, that is, impaired lipophagy affects the degradation of triacylglycerol, cholesterol, etc., leading to hyperlipidemia in atherosclerosis. In patients, excessive levels of autophagy accelerate the rupture of atherosclerotic plaque. This chapter also describes the advances in the treatment of atherosclerosis and hyperlipidemia by targeted autophagy.

Lipid Droplet–Peroxisome Connections in Plants

Lipid droplets (LDs) are the principal subcellular sites for the storage of triacylglycerols (TAGs), and in plants, TAG degradation requires metabolism in peroxisomes. This metabolic cooperation in...

Harnessing the intracellular triacylglycerols for titer improvement of polyketides in Streptomyces.

Pharmaceutically important polyketides such as avermectin are mainly produced as secondary metabolites during the stationary phase of growth of Streptomyces species in fermenters. The source of intracellular metabolites that are funneled into polyketide biosynthesis has proven elusive. We applied multi-omics to reveal that intracellular triacylglycerols (TAGs), which accumulates in primary metabolism, are degraded during stationary phase. This process could channel carbon flux from both intracellular TAGs and extracellular substrates into polyketide biosynthesis. We devised a strategy named 'dynamic degradation of TAG' (ddTAG) to mobilize the TAG pool and increase polyketide biosynthesis. Using ddTAG we increased the titers of actinorhodin, jadomycin B, oxytetracycline and avermectin B1a in Streptomyces coelicolor, Streptomyces venezuelae, Streptomyces rimosus and Streptomyces avermitilis. Application of ddTAG increased the titer of avermectin B1a by 50% to 9.31 g l-1 in a 180-m3 industrial-scale fermentation, which is the highest titer ever reported. Our strategy could improve polyketide titers for pharmaceutical production.