Triacylglycerol Storage Research Articles

A multigene approach secures hydroxy fatty acid production in Arabidopsis.

A central goal of green chemistry is to produce industrially useful fatty acids in oilseed crops. Although genes encoding suitable fatty acid-modifying enzymes are available from more than a dozen wild species, progress has been limited because expression of these enzymes in transgenic plants produces only low yields of the desired products. For example, fatty acid hydroxylase 12 (FAH12) from castor (Ricinus communis) produces only 17% hydroxy fatty acids (HFAs) when expressed in Arabidopsis (Arabidopsis thaliana), compared with 90% HFAs in castor seeds. The transgenic plants also have reduced oil content and seed vigor. Here, we review experiments that have provided for steady increased HFA accumulation and oil content. This research has led to exciting new discoveries of enzymes and regulatory processes in the pathways of both seed oil synthesis and lipid metabolism in other parts of the plant. Recent investigations have revealed that HFA-accumulating seeds are unable to rapidly mobilize HFA-containing triacylglycerol (TAG) storage lipid after germination to provide carbon and energy for seedling development, resulting in reduced seedling establishment. These findings present a new opportunity to investigate a different, key area of lipid metabolism-the pathways of TAG lipolysis and β-oxidation in germinating seedlings.

Both full length-cholesteryl ester transfer protein and exon 9-deleted cholesteryl ester transfer protein promote triacylglycerol storage in cultured hepatocytes.

We previously reported that overexpression of full-length cholesteryl ester transfer protein (FL-CETP), but not its exon 9-deleted variant (∆E9-CETP), in an adipose cell line reduces their triacylglycerol (TAG) content. This provided mechanistic insight into several in vivo studies where FL-CETP levels are inversely correlated with adiposity. However, increased FL-CETP is also associated with elevated hepatic lipids, suggesting that the effect of CETP on cellular lipid metabolism may be tissue-specific. Here, we directly investigated the role of FL-CETP and ∆E9-CETP in hepatic lipid metabolism. FL- or ∆E9-CETP was overexpressed in HepG2-C3A by adenovirus transduction. Overexpression of either FL or ∆E9-CETP in hepatocytes increased cellular TAG mass by 25% but reduced TAG secretion. This cellular TAG was contained in larger and more numerous lipid droplets. Analysis of TAG synthetic and catabolic pathways showed that this elevated TAG content was due to increased incorporation of fatty acid into TAG (24%), and higher de novo synthesis of fatty acid (50%) and TAG from acetate (40%). siRNA knockdown of CETP had the opposite effect on TAG synthesis and lipogenesis, and decreased cellular TAG. This novel increase in cellular TAG by FL-CETP overexpression was reproduced in Caco-2 intestinal epithelial cells. We conclude that, unlike that seen in adipocyte cells, overexpression of either CETP isoform in lipoprotein-secreting cells promotes the accumulation of TAG. These data suggest that the in vivo correlation between CETP levels and hepatic steatosis can be explained, in part, by a direct effect of CETP on hepatocyte cellular metabolism.

Convergent somatic mutations in metabolism genes in chronic liver disease.

The progression of chronic liver disease to hepatocellular carcinoma is caused by the acquisition of somatic mutations that affect 20-30 cancer genes1-8. Burdens of somatic mutations are higher and clonal expansions larger in chronic liver disease9-13 than in normal liver13-16, which enables positive selection to shape the genomic landscape9-13. Here we analysed somatic mutations from 1,590 genomes across 34 liver samples, including healthy controls, alcohol-related liver disease and non-alcoholic fatty liver disease. Seven of the 29 patients with liver disease had mutations in FOXO1, the major transcription factor in insulin signalling. These mutations affected a single hotspot within the gene, impairing the insulin-mediated nuclear export of FOXO1. Notably, six of the seven patients with FOXO1S22W hotspot mutations showed convergent evolution, with variants acquired independently by up to nine distinct hepatocyte clones per patient. CIDEB, which regulates lipid droplet metabolism in hepatocytes17-19, and GPAM, which produces storage triacylglycerol from free fatty acids20,21, also had a significant excess of mutations. We again observed frequent convergent evolution: up to fourteen independent clones per patient with CIDEB mutations and up to seven clones per patient with GPAM mutations. Mutations in metabolism genes were distributed across multiple anatomical segments of the liver, increased clone size and were seen in both alcohol-related liver disease and non-alcoholic fatty liver disease, but rarely in hepatocellular carcinoma. Master regulators of metabolic pathways are a frequent target of convergent somatic mutation in alcohol-related and non-alcoholic fatty liver disease.

Optimized expression and purification of adipose triglyceride lipase improved hydrolytic and transacylation activities in vitro

Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis, the mobilization of stored triacylglycerol. This work provides an important basis for generating reproducible and detailed data on the hydrolytic and transacylation activities of ATGL. We generated full-length and C-terminally truncated ATGL variants fused with various affinity tags and analyzed their expression in different hosts, namely E.coli, the insect cell line Sf9, and the mammalian cell line human embryonic kidney 293T. Based on this screen, we expressed a fusion protein of ATGL covering residues M1-D288 flanked with N-terminal and C-terminal purification tags. Using these fusions, we identified key steps in expression and purification protocols, including production in the E. coli strain ArcticExpress (DE3) and removal of copurified chaperones. The resulting purified ATGL variant demonstrated improved lipolytic activity compared with previously published data, and it could be stimulated by the coactivator protein comparative gene identification-58 and inhibited by the protein G0/G1 switch protein 2. Shock freezing and storage did not affect the basal activity but reduced coactivation of ATGL by comparative gene identification 58. In vitro, the truncated ATGL variant demonstrated acyl-CoA–independent transacylation activity when diacylglycerol was offered as substrate, resulting in the formation of fatty acid as well as triacylglycerol and monoacylglycerol. However, the ATGL variant showed neither hydrolytic activity nor transacylation activity upon offering of monoacylglycerol as substrate. To understand the role of ATGL in different physiological contexts, it is critical for future studies to identify all its different functions and to determine under what conditions these activities occur.

WNT6/ACC2-induced storage of triacylglycerols in macrophages is exploited by Mycobacterium tuberculosis.

In view of emerging drug-resistant tuberculosis (TB), host-directed adjunct therapies are urgently needed to improve treatment outcomes with currently available anti-TB therapies. One approach is to interfere with the formation of lipid-laden "foamy" macrophages in the host, as they provide a nutrient-rich host cell environment for Mycobacterium tuberculosis (Mtb). Here, we provide evidence that Wnt family member 6 (WNT6), a ligand of the evolutionarily conserved Wingless/Integrase 1 (WNT) signaling pathway, promotes foam cell formation by regulating key lipid metabolic genes including acetyl-CoA carboxylase 2 (ACC2) during pulmonary TB. Using genetic and pharmacological approaches, we demonstrated that lack of functional WNT6 or ACC2 significantly reduced intracellular triacylglycerol (TAG) levels and Mtb survival in macrophages. Moreover, treatment of Mtb-infected mice with a combination of a pharmacological ACC2 inhibitor and the anti-TB drug isoniazid (INH) reduced lung TAG and cytokine levels, as well as lung weights, compared with treatment with INH alone. This combination also reduced Mtb bacterial numbers and the size of mononuclear cell infiltrates in livers of infected mice. In summary, our findings demonstrate that Mtb exploits WNT6/ACC2-induced storage of TAGs in macrophages to facilitate its intracellular survival, a finding that opens new perspectives for host-directed adjunctive treatment of pulmonary TB.

Engineering of Saccharomyces cerevisiae for the accumulation of high amounts of triacylglycerol

BackgroundFatty acid-based substances play an important role in many products, from food supplements to pharmaceutical products and biofuels. The production of fatty acids, mainly in their esterified form as triacylglycerol (TAG), has been intensively studied in oleaginous yeasts, whereas much less effort has been invested into non-oleaginous species. In the present work, we engineered the model yeast Saccharomyces cerevisiae, which is commonly regarded as non-oleaginous, for the storage of high amounts of TAG, comparable to the contents achieved in oleaginous yeasts.ResultsWe investigated the effects of several mutations with regard to increased TAG accumulation and identified six of them as important for this phenotype: a point mutation in the acetyl-CoA carboxylase Acc1p, overexpression of the diacylglycerol acyltransferase Dga1p, deletions of genes coding for enzymes involved in the competing pathways glycogen and steryl ester synthesis and TAG hydrolysis, and a deletion of CKB1, the gene coding for one of the regulatory subunits of casein kinase 2. With the combination of these mutations in a S. cerevisiae strain with a relatively high neutral lipid level already in the non-engineered state, we achieved a TAG content of 65% in the dry biomass. High TAG levels were not only obtained under conditions that favor lipid accumulation, but also in defined standard carbon-limited media.ConclusionsBaker's yeast, which is usually regarded as inefficient in the storage of TAG, can be converted into a highly oleaginous strain that could be useful in processes aiming at the synthesis of fatty acid-based products. This work emphasizes the importance of strain selection in combination with metabolic engineering to obtain high product levels.

The Roles of Cytoplasmic Lipid Droplets in Modulating Intestinal Uptake of Dietary Fat.

Dietary fat absorption is required for health but also contributes to hyperlipidemia and metabolic disease when dysregulated. One step in the process of dietary fat absorption is the formation of cytoplasmic lipid droplets (CLDs) in small intestinal enterocytes; these CLDs serve as dynamic triacylglycerol storage organelles that influence the rate at which dietary fat is absorbed. Recent studies have uncovered novel factors regulating enterocyte CLD metabolism that in turn influence the absorption of dietary fat. These include peroxisome proliferator-activated receptor α activation, compartmentalization of different lipid pools, the gut microbiome, liver X receptor and farnesoid X receptor activation, obesity, and physiological factors stimulating CLD mobilization. Understanding how enterocyte CLD metabolism is regulated is key in modulating the absorption of dietary fat in the prevention of hyperlipidemia and its associated metabolic disorders.

Toxoplasma LIPIN is essential in channeling host lipid fluxes through membrane biogenesis and lipid storage

Apicomplexa are obligate intracellular parasites responsible for major human diseases. Their intracellular survival relies on intense lipid synthesis, which fuels membrane biogenesis. Parasite lipids are generated as an essential combination of fatty acids scavenged from the host and de novo synthesized within the parasite apicoplast. The molecular and metabolic mechanisms allowing regulation and channeling of these fatty acid fluxes for intracellular parasite survival are currently unknown. Here, we identify an essential phosphatidic acid phosphatase in Toxoplasma gondii, TgLIPIN, as the central metabolic nexus responsible for controlled lipid synthesis sustaining parasite development. Lipidomics reveal that TgLIPIN controls the synthesis of diacylglycerol and levels of phosphatidic acid that regulates the fine balance of lipids between storage and membrane biogenesis. Using fluxomic approaches, we uncover the first parasite host-scavenged lipidome and show that TgLIPIN prevents parasite death by ‘lipotoxicity’ through effective channeling of host-scavenged fatty acids to storage triacylglycerols and membrane phospholipids.

Into the Deep: New Data on the Lipid and Fatty Acid Profile of Redfish Sebastes mentella Inhabiting Different Depths in the Irminger Sea.

New data on lipid and fatty acid profiles are presented, and the dynamics of the studied components in muscles in the males and females of the beaked redfish, Sebastes mentella, in the depth gradient of the Irminger Sea (North Atlantic) is discussed. The contents of the total lipids (TLs), total phospholipids (PLs), monoacylglycerols (MAGs), diacylglycerols (DAGs), triacylglycerols (TAGs), cholesterol (Chol), Chol esters, non-esterified fatty acids (NEFAs), and wax esters were determined by HPTLC; the phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylcholine (PC), and lysophosphatidylcholine (LPC) were determined by HPLC; and fatty acids of total lipids were determined using GC. The Chol esters prevailed in muscles over the storage TAGs, and the wax ester content was high, which is a characteristic trait of vertically migrating species. Specific dynamics in certain PL in redfish were found to be depended on depth, suggesting that PLs are involved in the re-arrangement of the membrane physicochemical state and the maintenance of motor activity under high hydrostatic pressure. The high contents of DHA and EPA were observed in beaked redfish muscles is the species’ characteristic trait. The MUFAs in muscles include dietary markers of zooplankton (copepods)—20:1(n-9) and 22:1(n-11), whose content was found to be lower in fish sampled from greater depths.

Stability to oxidation and interfacial behavior at the air/water interface of minimally-processed versus processed walnut oil-bodies

Oil bodies (OB), the form of triacylglycerol storage in seeds, are interesting natural assemblies for nutritional applications. In walnuts, OB contain an important amount of polyunsaturated fatty acids that could be interesting food ingredients but may be prone to oxidation. The oxidative and interfacial behavior of walnut OB, either minimally-processed or after processing, were compared with processed complex walnut juice. The good oxidative stability of minimally-processed OB over 10 days (PV ≤ 8.4 meq O2/kg, TBARS = 1.4 mmol eq MDA/kg) and of processed walnut complex matrixes over 20 days (PV ≤ 4.8 meq O2/kg, TBARS = 1.4 mmol eq MDA/kg) was evidenced. In comparison, processing of OB promoted their oxidation. The interfacial studies led to the proposition of a new model of adsorption for minimally-processed OB that will be useful to design functional emulsion or foam in which OB act as emulsifiers.

High oil accumulation in tuber of yellow nutsedge compared to purple nutsedge is associated with more abundant expression of genes involved in fatty acid synthesis and triacylglycerol storage

BackgroundYellow nutsedge is a unique plant species that can accumulate up to 35% oil of tuber dry weight, perhaps the highest level observed in the tuber tissues of plant kingdom. To gain insight into the molecular mechanism that leads to high oil accumulation in yellow nutsedge, gene expression profiles of oil production pathways involved carbon metabolism, fatty acid synthesis, triacylglycerol synthesis, and triacylglycerol storage during tuber development were compared with purple nutsedge, the closest relative of yellow nutsedge that is poor in oil accumulation.ResultsCompared with purple nutsedge, high oil accumulation in yellow nutsedge was associated with significant up-regulation of specific key enzymes of plastidial RubisCO bypass as well as malate and pyruvate metabolism, almost all fatty acid synthesis enzymes, and seed-like oil-body proteins. However, overall transcripts for carbon metabolism toward carbon precursor for fatty acid synthesis were comparable and for triacylglycerol synthesis were similar in both species. Two seed-like master transcription factors ABI3 and WRI1 were found to display similar transcript patterns but were expressed at 6.5- and 14.3-fold higher levels in yellow nutsedge than in purple nutsedge, respectively. A weighted gene co-expression network analysis revealed that ABI3 was in strong transcriptional coordination with WRI1 and other key oil-related genes.ConclusionsThese results implied that pyruvate availability and fatty acid synthesis in plastid, along with triacylglycerol storage in oil bodies, rather than triacylglycerol synthesis in endoplasmic reticulum, are the major factors responsible for high oil production in tuber of yellow nutsedge, and ABI3 most likely plays a critical role in regulating oil accumulation. This study is of significance with regard to understanding the molecular mechanism controlling carbon partitioning toward oil production in oil-rich tuber and provides a valuable reference for enhancing oil accumulation in non-seed tissues of crops through genetic breeding or metabolic engineering.

Whole Community Metatranscriptomes and Lipidomes Reveal Diverse Responses Among Antarctic Phytoplankton to Changing Ice Conditions

The transition from winter to spring represents a major shift in the basal energy source for the Antarctic marine ecosystem from lipids and other sources of stored energy to sunlight. Because sea ice imposes a strong control on the transmission of sunlight into the water column during the polar spring, we hypothesized that the timing of the sea ice retreat influences the timing of the transition from stored energy to photosynthesis. To test the influence of sea ice on water column microbial energy utilization we took advantage of unique sea ice conditions in Arthur Harbor, an embayment near Palmer Station on the western Antarctic Peninsula, during the 2015 spring–summer seasonal transition. Over a 5-week period we sampled water from below land-fast sea ice, in the marginal ice zone at nearby Palmer Station B, and conducted an ice removal experiment with incubations of water collected below the land-fast ice. Whole-community metatranscriptomes were paired with lipidomics to better understand how lipid production and utilization was influenced by light conditions. We identified several different phytoplankton taxa that responded similarly to light by the number of genes up-regulated, and in the transcriptional complexity of this response. We applied a principal components analysis to these data to reduce their dimensionality, revealing that each of these taxa exhibited a strikingly different pattern of gene up-regulation. By correlating the changes in lipid concentration to the first principal component of log fold-change for each taxa we could make predictions about which taxa were associated with different changes in the community lipidome. We found that genes coding for the catabolism of triacylglycerol storage lipids were expressed early on in phytoplankton associated with a Fragilariopsis kerguelensis reference transcriptome. Phytoplankton associated with a Corethron pennatum reference transcriptome occupied an adjacent niche, responding favorably to higher light conditions than F. kerguelensis. Other diatom and dinoflagellate taxa had distinct transcriptional profiles and correlations to lipids, suggesting diverse ecological strategies during the polar winter–spring transition.

Hormone-sensitive lipase couples intergenerational sterol metabolism to reproductive success.

Triacylglycerol (TG) and steryl ester (SE) lipid storage is a universal strategy to maintain organismal energy and membrane homeostasis. Cycles of building and mobilizing storage fat are fundamental in (re)distributing lipid substrates between tissues or to progress ontogenetic transitions. In this study, we show that Hormone-sensitive lipase (Hsl) specifically controls SE mobilization to initiate intergenerational sterol transfer in Drosophila melanogaster. Tissue-autonomous Hsl functions in the maternal fat body and germline coordinately prevent adult SE overstorage and maximize sterol allocation to embryos. While Hsl-deficiency is largely dispensable for normal development on sterol-rich diets, animals depend on adipocyte Hsl for optimal fecundity when dietary sterol becomes limiting. Notably, accumulation of SE but not of TG is a characteristic of Hsl-deficient cells across phyla including murine white adipocytes. In summary, we identified Hsl as an ancestral regulator of SE degradation, which improves intergenerational sterol transfer and reproductive success in flies.

Using 14C-acetate Pulse-chase Labeling to Study Fatty Acid and Glycerolipid Metabolism in Plant Leaves.

Lipids metabolism is comprised of networks of reactions occurred in different subcellular compartments. Isotopic labeling is a good way to track the transformations and movements of metabolites without perturbing overall cellular metabolism. Fatty acids, the building blocks of membrane lipids and storage triacylglycerols, are synthesized in plastids. The immediate precursor for fatty acid synthesis is acetyl-CoA. Exogenous acetate is rapidly incorporated into fatty acids in leaves and isolated plastids because it can diffuse freely through cellular membranes, enter the plastid where it is rapidly metabolized to acetyl-CoA. Therefore, isotope-labeled acetate is often used as a tracer for the investigation of fatty acid synthesis and complex lipid metabolism in plants and other organisms. The basic principle of isotope labeling and its recent technological advances have been reviewed ( Allen et al., 2015 ). The present protocol describes the use of 14C-labeled acetate to determine rates of fatty acid synthesis and degradation and to track the metabolism of glycerolipids in leaves. This method, which is often referred to as acetate pulse-chase labeling, has been widely used to probe various aspects of lipid metabolism ( Allen et al., 2015 ), including the role of autophagy in membrane lipid turnover ( Fan et al., 2019 ) and the interplay between lipid and starch metabolism pathways ( Yu et al., 2018 ).

Plin2 deletion increases cholesteryl ester lipid droplet content and disturbs cholesterol balance in adrenal cortex

AbstractCholesteryl esters (CEs) are the water-insoluble transport and storage form of cholesterol. Steroidogenic cells primarily store CEs in cytoplasmic lipid droplet (LD) organelles, as contrasted to the majority of mammalian cell types that predominantly store triacylglycerol (TAG) in LDs. The LD-binding Plin2 binds to both CE- and TAG-rich LDs, and although Plin2 is known to regulate degradation of TAG-rich LDs, its role for regulation of CE-rich LDs is unclear. To investigate the role of Plin2 in the regulation of CE-rich LDs, we performed histological and molecular characterization of adrenal glands from Plin2+/+ and Plin2−/− mice. Adrenal glands of Plin2−/− mice had significantly enlarged organ size, increased size and numbers of CE-rich LDs in cortical cells, elevated cellular unesterified cholesterol levels, and increased expression of macrophage markers and genes facilitating reverse cholesterol transport. Despite altered LD storage, mobilization of adrenal LDs and secretion of corticosterone induced by adrenocorticotropic hormone stimulation or starvation were similar in Plin2+/+ and Plin2−/− mice. Plin2−/− adrenals accumulated ceroid-like structures rich in multilamellar bodies in the adrenal cortex-medulla boundary, which increased with age, particularly in females. Finally, Plin2−/− mice displayed unexpectedly high levels of phosphatidylglycerols, which directly paralleled the accumulation of these ceroid-like structures. Our findings demonstrate an important role of Plin2 for regulation of CE-rich LDs and cellular cholesterol balance in the adrenal cortex.

Isolation of Lipid Droplets for Protein and Lipid Analysis.

Cytosolic lipid droplets (LDs) are organelles which emulsify a variety of hydrophobic molecules in the aqueous cytoplasm of essentially all plant cells. Most familiar are the LDs from oilseeds or oleaginous fruits that primarily store triacylglycerols and serve a storage function. However, similar hydrophobic particles are found in cells of plant tissues that package terpenoids, sterol esters, wax esters, or other types of nonpolar lipids. The various hydrophobic lipids inside LDs are coated with a phospholipid monolayer, mostly derived from membrane phospholipids during their ontogeny. Various proteins have been identified to be associated with LDs, and these may be cell-type, tissue-type, or even species specific. While major LD proteins like oleosins have been known for decades, more recently a growing list of LD proteins has been identified, primarily by proteomics analyses of isolated LDs and confirmation of their localization by confocal microscopy. LDs, unlike other organelles, have a density less than that of water, and consequently can be isolated and enriched in cellular fractions by flotation centrifugation for composition studies. However, due to its deep coverage, modern proteomics approaches are also prone to identify contaminants, making control experiments necessary. Here, procedures for the isolation of LDs, and analysis of LD components are provided as well as methods to validate the LD localization of proteins.

Coordinated control of adiposity and growth by anti-anabolic kinase ERK7.

Energy storage and growth are coordinated in response to nutrient status of animals. How nutrient‐regulated signaling pathways control these processes in vivo remains insufficiently understood. Here, we establish an atypical MAP kinase, ERK7, as an inhibitor of adiposity and growth in Drosophila. ERK7 mutant larvae display elevated triacylglycerol (TAG) stores and accelerated growth rate, while overexpressed ERK7 is sufficient to inhibit lipid storage and growth. ERK7 expression is elevated upon fasting and ERK7 mutant larvae display impaired survival during nutrient deprivation. ERK7 acts in the fat body, the insect counterpart of liver and adipose tissue, where it controls the subcellular localization of chromatin‐binding protein PWP1, a growth‐promoting downstream effector of mTOR. PWP1 maintains the expression of sugarbabe, encoding a lipogenic Gli‐similar family transcription factor. Both PWP1 and Sugarbabe are necessary for the increased growth and adiposity phenotypes of ERK7 loss‐of‐function animals. In conclusion, ERK7 is an anti‐anabolic kinase that inhibits lipid storage and growth while promoting survival on fasting conditions.

The Regulation of Fat Metabolism During Aerobic Exercise.

Since the lipid profile is altered by physical activity, the study of lipid metabolism is a remarkable element in understanding if and how physical activity affects the health of both professional athletes and sedentary subjects. Although not fully defined, it has become clear that resistance exercise uses fat as an energy source. The fatty acid oxidation rate is the result of the following processes: (a) triglycerides lipolysis, most abundant in fat adipocytes and intramuscular triacylglycerol (IMTG) stores, (b) fatty acid transport from blood plasma to muscle sarcoplasm, (c) availability and hydrolysis rate of intramuscular triglycerides, and (d) transport of fatty acids through the mitochondrial membrane. In this review, we report some studies concerning the relationship between exercise and the aforementioned processes also in light of hormonal controls and molecular regulations within fat and skeletal muscle cells.

Spatially Resolved Activity-based Proteomic Profiles of the Murine Small Intestinal Lipases

Despite the crucial function of the small intestine in nutrient uptake our understanding of the molecular events underlying the digestive function is still rudimentary. Recent studies demonstrated that enterocytes do not direct the entire dietary triacylglycerol toward immediate chylomicron synthesis. Especially after high-fat challenges, parts of the resynthesized triacylglycerol are packaged into cytosolic lipid droplets for transient storage in the endothelial layer of the small intestine. The reason for this temporary storage of triacylglycerol is not completely understood. To utilize lipids from cytosolic lipid droplets for chylomicron synthesis in the endoplasmic reticulum, stored triacylglycerol has to be hydrolyzed either by cytosolic lipolysis or lipophagy. Interestingly, triacylglycerol storage and chylomicron secretion rates are unevenly distributed along the small intestine, with the proximal jejunum exhibiting the highest intermittent storage capacity. We hypothesize that correlating hydrolytic enzyme activities with the reported distribution of triacylglycerol storage and chylomicron secretion in different sections of the small intestine is a promising strategy to determine key enzymes in triacylglycerol remobilization. We employed a serine hydrolase specific activity-based labeling approach in combination with quantitative proteomics to identify and rank hydrolases based on their relative activity in 11 sections of the small intestine. Moreover, we identified several clusters of enzymes showing similar activity distribution along the small intestine. Merging our activity-based results with substrate specificity and subcellular localization known from previous studies, carboxylesterase 2e and arylacetamide deacetylase emerge as promising candidates for triacylglycerol mobilization from cytosolic lipid droplets in enterocytes.

Insulin receptor deficiency reduces lipid synthesis and reproductive function in the insect Rhodnius prolixus

Rhodnius prolixus, a vector of Chagas disease, is a hematophagous insect that feeds exclusively on blood. Each blood meal is digested within the first fourteen days after feeding, providing substrates for lipid synthesis for storage and egg production. These events are precisely regulated and emerging evidence points to a key function of insulin-like peptides (ILPs) in this control. Here we investigated the role of insulin receptor in the regulation of nutrient metabolism in fed adult females. The expression of insulin receptor (RhoprIR) gene was determined in adult organs, and it was highest in ovaries and previtellogenic follicles. We generated insects with RNAi-mediated knockdown of RhoprIR to address the physiological role of this receptor. RhoprIR deficiency improved longevity and reduced triacylglycerol storage in the fat body, whereas blood digestion remained unchanged for seven days after blood meal. The lower lipid content was attributable to decreased de novo lipogenesis as well as reduced incorporation of hemolymph-derived fatty acids into newly synthesized lipids within this organ. Consistent with that, fat bodies from RhoprIR-deficient insects exhibited decreased gene expression levels of lipophorin receptor (RhoprLpR), glycerol-3-phosphate acyltransferase 1 and 4 (RhoprGpat1 and RhoprGpat4), and carnitine palmitoyltransferase 1 (RhoprCpt1). Although hemolymph lipid profile was not affected by RhoprIR disruption, the concentration of circulating vitellogenin was increased. In line with these changes, RhoprIR-deficient females exhibited smaller ovaries and a marked reduction in oviposition. Taken together, these findings support a key role of insulin receptor in nutrient homeostasis, lipid synthesis and egg production following a blood meal.