TAG Content Research Articles

Dichlorvos evokes systemic lipid dysmetabolism in Wistar rats: rescindment influence of curcumin

Organophosphorus pesticides such as dichlorvos (DDVP) are often employed to eradicate pests, especially in low and medium-income countries. However, they have several negative impacts on the visceral organs. Astonishingly, curcumin protects organs from the detrimental effects of xenobiotics via the maintenance of redox homeostasis; unfortunately, its role in dichlorvos-provoked multi-organ impairment vis-à-vis lipid homeostasis has not been examined. Therefore, this undertaking probed the remedial efficacy of curcumin on DDVP-prompted combined systemic toxicity and major lipid distribution. Randomization was engaged to dedicate rats (40) into seven groups (6 rats/group): control, DDVP only (20 mg kg-1day-1), DDVP administered with either curcumin (50 and 100 mg kg-1day-1) or reference medication atropine (0.2 mg kg-1day-1), and curcumin only (50 and 100 mg kg-1day-1). DDVP was dispensed orally for one week, followed by two weeks of curcumin treatment. Twenty-four hours after the last administration, we sacrificed the rats and collected their blood and viscera (liver, kidney, heart, lung, and brain) for bioassays. Curcumin remarkably (p<0.05) rescinded DDVP-mediated increases in plasma, LDL, and systemic cholesterol; markedly (p<0.05) attenuated DDVP-elicited decreases in HDL-cholesterol and TAG contents in all the compartments except erythrocyte and liver; and significantly (p<0.05) abated DDVP-induced plasma phospholipidaemia, multi-organ phospholipidosis, and up-regulation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA-R) activity. This finding demonstrated that curcumin reverses DDVP-triggered anomalous lipid dynamics by abating cholesterogenesis and phospholipidosis and restoring HMG-CoA-R activity.

Effect of NH4Cl supplementation on growth, photosynthesis, and triacylglycerol content in Chlamydomonas reinhardtii under mixotrophic cultivation.

Ammonium chloride (NH4Cl) is one of the nitrogen sources for microalgal cultivation. An excessive amounts of NH4Cl are toxic for microalgae. However, combining mixotrophic conditions and excessive quantities of NH4Cl positively affects microalgal biomass and lipid production. In this study, we investigated the impact of NH4Cl on the growth, biomass, and triglyceride (TAG) content of the green microalga Chlamydomonas reinhardtii especially under mixotrophic conditions. Under photoautotrophic conditions (without organic carbon supplementation), adding 25 mM NH4Cl had no significant effect on microalgal growth or TAG content. However, under mixotrophic condition (with acetate supplementation), NH4Cl interfered with microalgal growth while inducing TAG content. To explore these effects further, we conducted a two-step cultivation process and found that NH4Cl reduced microalgal growth, but induced total lipid and TAG content, especially after 4-day cultivation. The photosynthesis performances showed that NH4Cl completely inhibited oxygen evolution on day 4. However, NH4Cl slightly reduced the Fv/Fm ratio indicating that the NH4Cl supplementation directly affects microalgal photosynthesis. To investigate the TAG induction effect by NH4Cl, we compared the protein expression profiles of microalgae grown mixotrophically with and without 25 mM NH4Cl using a proteomics approach. This analysis identified 1782 proteins, with putative acetate uptake transporter GFY5 and acyl-coenzyme A oxidase being overexpressed in the NH4Cl-treated group. These findings suggested that NH4Cl supplementation may stimulate acetate utilization and fatty acid synthesis pathways in microalgae cells. Our study indicated that NH4Cl supplementation can induce microalgal biomass and lipid production, particularly when combined with mixotrophic conditions.

Ozone induced multigenerational glucose and lipid metabolism disorders in Drosophila

Ozone (O3), a persistent pollutant, poses a significant health threat. However, research on its multigenerational toxicity remains limited. Leveraging the Drosophila model with its short lifespan and advanced genetic tools, we explored the effects of O3 exposure across three generations of fruit flies. The findings revealed that O3 disrupted motility, body weight, stress resistance, and oxidative stress in three generations of flies, with varying effects observed among them. Transcriptome analysis highlighted the disruption of glucose metabolism-related pathways, encompassing gluconeogenesis/glycolysis, galactose metabolism, and carbon metabolism. Hub genes were identified, and RT-qPCR results indicated that O3 decreased their transcription levels. Comparative analysis of their human orthologs was conducted using Comparative Toxicogenomics Database (CTD) and DisGeNET databases. These genes are linked to various metabolic diseases, including diabetes, hypoglycemia, and obesity. The trehalose content was reduced in F0 generation flies but increased in F1-F2 generations after O3 exposure. While the trehalase and glucose levels were decreased across F0-F2 generations. TAG synthesis-related genes were significantly upregulated in F0 generation flies but downregulated in F1-F2 generations. The expression patterns of lipolysis-related genes varied among the three generations of flies. Food intake was increased in F0 generation flies but decreased in F1-F2 generations. Moreover, TAG content was significantly elevated in F0 generation flies by O3 exposure, while it was reduced in F2 generation flies. These differential effects of O3 across three generations of flies suggest a metabolic reprogramming aimed at mitigating the damage caused by O3 to flies. The study affirms the viability of employing the Drosophila model to investigate the mechanisms underlying O3-induced glucose and lipid metabolism disorders while emphasizing the importance of studying the long-term health effects of O3 exposure. Moreover, this research highlights the Drosophila model as a viable tool for investigating the multigenerational effects of pollutants, particularly atmospheric pollutants.

Cultivation of Chlorella vulgaris in wastewater: biodiesel potential and wastewater remediation.

The present investigation has evaluated the use of effluents from a secondary municipal wastewater treatment plant for biomass production and potential of the biomass for biodiesel production. Cultivations of Chlorella vulgaris using wastewater, wastewater with supplementation, and WC medium were carried out. Effect of wastewater collected in different months on biomass productivity (BP) and lipid composition was studied. Methods based on NMR and GC-MS techniques were applied for determining the composition of the lipids and their fatty acid profile including poly unsaturated fatty acids (PUFAs). Lipids extracted are comprised of both neutral (tri acyl glycerides, TAG; free fatty acids, FFA) and polar (glyco glycero/phospho) lipids. The TAG content of the extracted lipids was determined in the range of 22.5-41.3% w/w. The NMR and GC-MS compositional results of microalgal lipids of biomasses cultivated in wastewater without nutrient supplementation, collected in different months, showed potential for biodiesel production. The fatty acid profiles of neutral and polar lipids, which are mainly comprised of saturated and unsaturated long alkyl chain (C16-C22) fatty acids, are potential sources for the biodiesel and food industry. The concentration of nitrates (45-78mg L-1) in wastewater without supplementation, collected in different months, was found to be optimum to enable cultivation of biomasses with reasonably good BP of 21.5-28.1mg L-1day-1. Similar results have been obtained in the present work as well as reported in the literature in the case of WC medium (nitrate, 69mg L-1) with BP of 25.5-28.2mg L-1day-1, thus highlighted the significance of the presented work.

Fasting and postprandial plasma metabolite responses to a 12-wk dietary intervention in tissue-specific insulin resistance: a secondary analysis of the PERSonalized glucose Optimization through Nutritional intervention (PERSON) randomized trial

BackgroundWe previously showed that dietary intervention effects on cardiometabolic health were driven by tissue-specific insulin resistance (IR) phenotype: individuals with predominant muscle IR (MIR) benefited more from a low-fat, high-protein, and high-fiber (LFHP) diet, whereas individuals with predominant liver insulin resistance (LIR) benefited more from a high-monounsaturated fatty acid (HMUFA) diet. ObjectivesTo further characterize the effects of LFHP and HMUFA diets and their interaction with tissue-specific IR, we investigated dietary intervention effects on fasting and postprandial plasma metabolite profile. MethodsAdults with MIR or LIR (40–75 y, BMI 25–40 kg/m2) were randomly assigned to a 12-wk HMUFA or LFHP diet (n = 242). After the exclusion of statin use, 214 participants were included in this prespecified secondary analysis. Plasma samples were collected before (T = 0) and after (T = 30, 60, 120, and 240 min) a high-fat mixed meal for quantification of 247 metabolite measures using nuclear magnetic resonance spectroscopy. ResultsA larger reduction in fasting VLDL-triacylglycerol (TAG) and VLDL particle size was observed in individuals with MIR following the LFHP diet and those with LIR following the HMUFA diet, although no longer statistically significant after false discovery rate (FDR) adjustment. No IR phenotype-by-diet interactions were found for postprandial plasma metabolites assessed as total area under the curve (tAUC). Irrespective of IR phenotype, the LFHP diet induced greater reductions in postprandial plasma tAUC of the larger VLDL particles and small HDL particles, and TAG content in most VLDL subclasses and the smaller LDL and HDL subclasses (for example, VLDL-TAG tAUC standardized mean change [95% CI] LFHP = −0.29 [−0.43, −0.16] compared with HMUFA = −0.04 [−0.16, 0.09]; FDR-adjusted P for diet × time = 0.041). ConclusionsDiet effects on plasma metabolite profiles were more pronounced than phenotype-by-diet interactions. An LFHP diet may be more effective than an HMUFA diet for reducing cardiometabolic risk in individuals with tissue-specific IR, irrespective of IR phenotype. Am J Clin Nutr 20xx;x:xx.This trial was registered at the clinicaltrials.gov registration (https://clinicaltrials.gov/study/NCT03708419?term=NCT03708419&rank=1) as NCT03708419 and CCMO registration (https://www.toetsingonline.nl/to/ccmo_search.nsf/fABRpop?readform&unids=3969AABCD9BA27FEC12587F1001BCC65) as NL63768.068.17.

Effects of beef fat enriched with trans vaccenic acid and cis9, trans11-CLA on glucose homoeostasis and hepatic lipid accumulation in high-fat diet-induced obese mice.

Trans vaccenic acid (TVA, trans11-18:1) and cis9, trans11-CLA (also known as rumenic acid; RA) have received widespread attention as potentially beneficial trans-FA due to their putative health benefits, including anti-diabetic properties. The objective of this study was to determine the effects of beef fat naturally enriched with TVA and RA on parameters related to glucose homoeostasis and associated metabolic markers in diet-induced obese (DIO) mice. Thirty-six male C57BL/6J mice (8 weeks old) were fed for 19 weeks with either a control low-fat diet (CLF), a control high-fat diet (CHF), or a TVA+RA-enriched high-fat diet (EHF). Compared with CLF, feeding either CHF or EHF resulted in adverse metabolic outcomes associated with high-fat diets, including adiposity, impaired glucose control and hepatic steatosis. However, the EHF diet induced a significantly higher liver weight TAG content and elevated plasma alanine transaminase levels compared with the CHF diet. Collectively, the findings from this study suggest that EHF does not improve glucose tolerance and worsens liver steatosis in DIO mice. However, the adverse effects of EHF on the liver could be in part related to the presence of other trans-FA in the enriched beef fat.

Atractylodes macrocephala Koidz. and Cuscuta chinensis Lam. extract relieves insulin resistance via PI3K/Akt signalling in diabetic Drosophila

Background and aimType-2 diabetes mellitus (T2DM) is mainly characterized by insulin resistance (IR) induced by hyperglycaemia and insufficient insulin secretion. We employed a diabetic fly model to examine the effect and molecular mechanism of Atractylodes macrocephala Koidz. and Cuscuta chinensis Lam. (AMK–CCL) extract as traditional Chinese medicine in treating IR and T2DM. Experimental procedureThe contents of the active ingredients (rhamnose, xylose, mannose, and hyperoside) in AMK–CCL extract were determined by high-performance liquid chromatography. Wild-type (Cg-GAL4/+) or diabetic (Cg > InRK1409A) Drosophila flies were divided into the control group or metformin group and AMK–CCL (0.0125, 0.025, 0.05, 0.1 g/ml) groups. Food intake, haemolymph glucose and trehalose, protein, weight, triglycerides (TAG), and glycogen were measured to assess glycolipid metabolism. Phosphatidylinositol-3-kinase (PI3K)/Akt signalling was detected using fluorescent reporters [tGPH, Drosophila forkhead box O (dFoxO)–green fluorescent protein (GFP), Glut1–GFP, 2-NBDG] in vivo. Glut1/3 mRNA levels and Akt phosphorylation levels were detected by quantitative polymerase chain reaction and western blotting, respectively, in vitro. ResultsAMK–CCL extract contained 0.038 % rhamnose, 0.017 % xylose, 0.69 % mannose, and 0.039 % hyperoside. AMK–CCL at 0.0125 g/mL significantly suppressed the increase in circulating glucose, and the decrease in body weight, TAG, and glycogen contents of diabetic flies. AMK–CCL improved PI3K activity, Akt phosphorylation, Glut1/3 expression, and glucose uptake in diabetic flies, and also rescued diabetes-induced dFoxO nuclear localisation. ConclusionsThese findings indicate that AMK–CCL extract ameliorates IR-induced diabetes via the PI3K/Akt signalling pathway, providing an experimental basis for clinical treatment.

Structural characteristics and catalysis properties of a dual-functional enzyme Lip10 from Mucor circinelloides

Mucor circinelloides lipase Lip10 is a dual-functional enzyme with lipolytic activity and phospholipid:diacylglycerol acyltransferase (PDAT) activity in aqueous phase. However, the catalytic properties of Lip10 and its structural characteristics remain unknown. The present study systematically investigated its PDAT activity and lipolytic activity using six phospholipid substrates, 18:0 triacylglycerol and 16:0 1,2-diacylglycerol, predict the 3D structure of Lip10 using AlphaFold2, and evaluated residues’ flexibility through molecular dynamics analysis. Lip10 exhibited a preference for phosphorylethanolamine (PE) with a long-chain fatty acyl group and showed the lipolytic activity to 18:0 triacylglycerol and 16:0 1.2-diacylglycerol. 20 mM Mg2+ and Fe2+ can significantly increase the PDAT activity of Lip10 by 50–80%. Molecular docking analysis suggest that the lower binding energy for 18:0 PE with Lip10 contributes to the higher structural stability of the complex of 18:0 PE and Lip10. Mutations of Phe565Asp and Glu318Met increase PDAT activity of Lip10 by 3.6-fold and 2.7-fold, while the TAG content produced by mutants Lip10Phe103Pro, Lip10Ile487Lys and Lip10Phe570Asp decrease by 33%− 43%. The present study demonstrated structural properties of the dual-functional lipase Lip10 and provide a theoretical basis to study its roles in the lipid metabolism of M. circinelloides in the future.

Comparative 4D Label-Free Quantitative Proteomic Analysis of Bombus terrestris Provides Insights into Proteins and Processes Associated with Diapause.

Diapause, an adaptative strategy for survival under harsh conditions, is a dynamic multi-stage process. Bombus terrestris, an important agricultural pollinator, is declining in the wild, but artificial breeding is possible by imitating natural conditions. Mated queen bees enter reproductive diapause in winter and recover in spring, but the regulatory mechanisms remain unclear. Herein, we conducted a comparative 4D label-free proteomic analysis of queen bees during artificial breeding at seven timepoints, including pre-diapause, diapause, and post-diapause stages. Through bioinformatics analysis of proteomic and detection of substance content changes, our results found that, during pre-diapause stages, queen bees had active mitochondria with high levels of oxidative phosphorylation, high body weight, and glycogen and TAG content, all of which support energy consumption during subsequent diapause. During diapause stages, body weight and water content were decreased but glycerol increased, contributing to cold resistance. Dopamine content, immune defense, and protein phosphorylation were elevated, while fat metabolism, protein export, cell communication, signal transduction, and hydrolase activity decreased. Following diapause termination, JH titer, water, fatty acid, and pyruvate levels increased, catabolism, synaptic transmission, and insulin signaling were stimulated, ribosome and cell cycle proteins were upregulated, and cell proliferation was accelerated. Meanwhile, TAG and glycogen content decreased, and ovaries gradually developed. These findings illuminate changes occurring in queen bees at different diapause stages during commercial production.

How Crowd Worker Factors Influence Subjective Annotations: A Study of Tagging Misogynistic Hate Speech in Tweets

Crowdsourced annotation is vital to both collecting labelled data to train and test automated content moderation systems and to support human-in-the-loop review of system decisions. However, annotation tasks such as judging hate speech are subjective and thus highly sensitive to biases stemming from annotator beliefs, characteristics and demographics. We conduct two crowdsourcing studies on Mechanical Turk to examine annotator bias in labelling sexist and misogynistic hate speech. Results from 109 annotators show that annotator political inclination, moral integrity, personality traits, and sexist attitudes significantly impact annotation accuracy and the tendency to tag content as hate speech. In addition, semi-structured interviews with nine crowd workers provide further insights regarding the influence of subjectivity on annotations. In exploring how workers interpret a task — shaped by complex negotiations between platform structures, task instructions, subjective motivations, and external contextual factors — we see annotations not only impacted by worker factors but also simultaneously shaped by the structures under which they labour.

Effects of choline deficiency and supplementation on lipid droplet accumulation in bovine primary liver cells in vitro

Rumen-protected choline (RPC) supplementation in the periparturient period has in some instances prevented and alleviated fatty liver disease in dairy cows. Mechanistically, however, it is unclear how choline prevents the accumulation of lipid droplets (LD) in liver cells. In this study, primary liver cells isolated from liver tissue obtained via puncture biopsy from 3 nonpregnant mid-lactation multiparous Holstein cows (∼160 d postpartum) were used. Analyses of LD via oil red O staining, protein abundance via Western blotting, and phospholipid content and composition measured by thin-layer chromatography and HPLC/mass spectrometry were performed in liver cells cultured in choline-deficient medium containing 150 μmol/L linoleic acid for 24 h. In a subsequent experiment, lipophagy was assessed in liver cells cultured with 30, 60, or 90 µmol/L choline-chloride. All data were analyzed statistically using SPSS 20.0 via t-tests or one-way ANOVA. Compared with liver cells cultured in Dulbecco's Modified Eagle Medium alone, choline deficiency increased the average diameter of LD (1.59 vs. 2.10 µm), decreased the proportion of small LD (<2 µm) from 75.3% to 56.6%, and increased the proportion of large LD (>4 µm) from 5.6% to 15.0%. In addition, the speed of LD fusion was enhanced by the absence of choline. Among phospholipid species, the phosphatidylcholine (PC) content of liver cells decreased by 34.5%. Seventeen species of PC (PC [18:2_22:6], PC [15:0_16:1], PC [14:0_20:4], and so on) and 6 species of lysophosphatidylcholine (LPC; LPC [15:0/0:0]), PC (22:2/0:0), LPC (20:2/0:0), and so on] were decreased, while PC (14:1_16:1) and LPC (0:0/20:1) were increased. Choline deficiency increased the triglyceride (TAG) content (0.57 vs. 0.39 μmol/mg) in liver cells and increased the protein abundance of sterol regulatory element binding protein 1, sterol regulatory element binding protein cleavage activation protein, and fatty acid synthase by 23.5%, 17%, and 36.1%, respectively. Upon re-supplementation with choline, the phenotype of LD (TAG content, size, proportion, and phospholipid profile) was reversed, and the ratio of autophagy marker LC3II/LC3I protein was significantly upregulated in a dose-dependent manner. Overall, at least in vitro in mid-lactation cows, these data demonstrated that PC synthesis is necessary for normal LD formation, and both rely on choline availability. According to the limitation of the source of liver cells used, further work should be conducted to ascertain that these effects are applicable to liver cells from postpartum cows, the physiological stage where the use of RPC has been implemented for the prevention and treatment of fatty liver.

Multivessel Coronary Artery Disease Complicated by Diabetes Mellitus Has a Relatively Small Effect on Endothelial and Lipoprotein Lipases Expression in the Human Atrial Myocardium and Coronary Perivascular Adipose Tissue.

Endothelial (EL) and lipoprotein (LPL) lipases are enzymes involved in lipoproteins metabolism and formation of atherosclerosis, a pathological feature of coronary artery disease (CAD). This paper examines the role of the lipases in the right atrial appendage (RAA) and coronary perivascular adipose tissue (PVAT) of patients with CAD alone or with accompanying diabetes. Additionally, correlation analysis for plasma concentration of the lipases, apolipoproteins (ApoA-ApoJ) and blood lipids (Chol, HDL-C, LDL-C, TAG) was performed. We observed that CAD had little effect on the lipases gene/protein levels in the RAA, while their transcript content was elevated in the PVAT of diabetic CAD patients. Interestingly, the RAA was characterized by higher expression of EL/LPL (EL: +1-fold for mRNA, +5-fold for protein; LPL: +2.8-fold for mRNA, +12-fold for protein) compared to PVAT. Furthermore, ApoA1 plasma concentration was decreased, whereas ApoC1 and ApoH were increased in the patients with CAD and/or diabetes. The concentrations of ApoC3 and ApoD were strongly positively correlated with TAG content in the blood, and the same was true for ApoB with respect to LDL-C and total cholesterol. Although plasma concentrations of EL/LPL were elevated in the patients with diabetes, CAD alone had little effect on blood, myocardial and perivascular fat expression of the lipases.

Analysis of Lipid Molecule Profiling and Conversion Pathway in Mandarin Fish (Siniperca chuatsi) during Fermentation via Untargeted Lipidomics

Lipids are the key aroma contributors and nutrients in fermented fish products. A total of 376 lipid molecules were identified in mandarin fish during fermentation by untargeted lipidomics, including glycerolipids, glycerophospholipids, lysoglycerophospholipid, sphingolipids, fatty acids (FAs), and sterol lipids. Both lipid composition and content changed dynamically during fermentation. Triglyceride (TAG, 30.05%) and phosphatidylcholine (PC, 14.87%) were the two major lipids, with especially 39.36% saturated FAs in PCs and 35.34% polyunsaturated FAs in TAGs. The content of TAGs and PCs reached a peak point at 0 and 6 days, respectively. Fermented mandarin fish expressed a high nutritional value, and the ratio of total linoleic acid/total linolenic acid was about 5:1. Glycerophospholipid metabolism was a potential metabolic pathway, and the oxidation of derived FAs contributed to flavor. These data progress in understanding lipid dynamic variation during fermentation and provide thoughts on controlling the flavor quality and safety of fermented fish products.

Diet supplementation of organic zinc positively affects growth, antioxidant capacity, immune response and lipid metabolism in juvenile largemouth bass, Micropterus salmoides.

Znis an important trace element involved in various biochemical processes in aquatic species. An 8-week rearing trial was thus conducted to investigate the effects of Zn on juvenile largemouth bass (Micropterus salmoides) by feeding seven diets, respectively, supplemented with no Zn (Con), 60 and 120 mg/kg inorganic Zn (Sul60 and Sul120), and 30, 60, 90 and 120 mg/kg organic Zn (Bio30, Bio60, Bio90 and Bio120). Sul120 and Bio120 groups showed significantly higher weight gain and specific growth rate than Con group, with Bio60 group obtaining the lowest viscerosomatic index and hepatosomatic index. 60 or 90 mg/kg organic Zn significantly facilitated whole body Zn retention. Up-regulation of hepatic superoxide dismutase, glutathione peroxidase and catalase activities and decline of malondialdehyde contents indicated augmented antioxidant capacities by organic Zn. Zn treatment also lowered plasma aminotransferase levels while promoting acid phosphatase activity and hepatic transcription levels of alp1, acp1 and lyz-c than deprivation of Zn. The alterations in whole body and liver crude lipid and plasma TAG contents illustrated the regulatory effect of Zn on lipid metabolism, which could be possibly attributed to the changes in hepatic expressions of acc1, pparγ, atgl and cpt1. These findings demonstrated the capabilities of Zn in potentiating growth and morphological performance, antioxidant capacity, immunity as well as regulating lipid metabolism in M. salmoides. Organic Zn could perform comparable effects at same or lower supplementation levels than inorganic Zn, suggesting its higher efficiency. 60 mg/kg supplementation of organic Zn could effectively cover the requirements of M. salmoides.

Auxin supplementation under nitrogen limitation enhanced oleic acid and MUFA content in Eustigmatos calaminaris biomass with potential for biodiesel production

Due to their lipid accumulation potential, microalgae are widely studied in terms of their use in the production of biodiesel. The present study was focused on determination of changes in the biomass production, biochemical composition, accumulation and distribution of fatty acids in neutral lipids, glycolipids, phospholipids and biodiesel properties of soil microalga Eustigmatos calaminaris in response to various levels of nitrogen stress and indole-3-acetic acid supplementation. The highest growth rate, the highest lipid content and daily lipid productivity were noted at the nitrogen limitation up to 25% with IAA supplementation. The increase in NL was associated with nutrient stress. An increase in the level of GL and PL were recorded upon the reduction of the nitrogen content (25% N) and the addition of IAA. The gas chromatography/mass spectrometry analysis demonstrated that C16:0, C16:1, and C18:1 were the main fatty acids in E. calaminaris lipids. As shown by the lipidomic analysis, the IAA supplementation in the nitrogen limitation variants enhanced the content of TAGs in C18:1 and monounsaturated fatty acids. The current findings indicated a potential strategy to improve the fatty acid profile in neutral lipids and high potential of E. calaminaris for biodiesel applications.

Co-Expression of Lipid Transporters Simultaneously Enhances Oil and Starch Accumulation in the Green Microalga Chlamydomonas reinhardtii under Nitrogen Starvation.

Lipid transporters synergistically contribute to oil accumulation under normal conditions in microalgae; however, their effects on lipid metabolism under stress conditions are unknown. Here, we examined the effect of the co-expression of lipid transporters, fatty acid transporters, (FAX1 and FAX2) and ABC transporter (ABCA2) on lipid metabolism and physiological changes in the green microalga Chlamydomonas under nitrogen (N) starvation. The results showed that the TAG content in FAX1-FAX2-ABCA2 over-expressor (OE) was 2.4-fold greater than in the parental line. Notably, in FAX1-FAX2-ABCA2-OE, the major membrane lipids and the starch and cellular biomass content also significantly increased compared with the control lines. Moreover, the expression levels of genes directly involved in TAG, fatty acid, and starch biosynthesis were upregulated. FAX1-FAX2-ABCA2-OE showed altered photosynthesis activity and increased ROS levels during nitrogen (N) deprivation. Our results indicated that FAX1-FAX2-ABCA2 overexpression not only enhanced cellular lipids but also improved starch and biomass contents under N starvation through modulation of lipid and starch metabolism and changes in photosynthesis activity. The strategy developed here could also be applied to other microalgae to produce FA-derived energy-rich and value-added compounds.

CIDEA Regulates De Novo Fatty Acid Synthesis in Bovine Mammary Epithelial Cells by Targeting the AMPK/PPARγ Axis and Regulating SREBP1.

Cell-death-inducing DNA fragmentation factor-α-like effector A (CIDEA) is a lipid-droplet-associated protein that helps to promote lipid metabolism in adipocytes of mice and humans. However, studies on the regulatory mechanism of CIDEA on lipid metabolism in the mammary glands of dairy cows are rare. Therefore, the role of CIDEA in bovine mammary epithelial cells (bMECs) was investigated in this study. The CIDEA expression levels in the mammary glands of high-fat-milk-producing cows were significantly higher compared to those in low-fat-milk-producing cows. Results of in vitro studies in bMECs showed that the inhibition of CIDEA inhibited the expression of fatty acid synthesis-related genes and triglyceride (TAG) synthesis-related genes. Conversely, the overexpression of CIDEA leads to an increase in the content of TAG and fatty acid. The results of mechanistic studies indicated that the overexpression of CIDEA inhibits AMP-activated protein kinase (AMPK) activity, which enhances the expression of peroxisome proliferator-activated receptor-γ (PPARγ) and consequently increases the TAG content. Furthermore, the overexpression of CIDEA promoted the nuclear translocation of sterol regulatory element-binding protein 1 (SREBP1). Therefore, a theoretical framework is provided by this study for the regulation of lipid metabolism in dairy cows by means of nutrition and the hormone targeting of CIDEA.

Purple acid phosphatase2 stimulates a futile cycle of lipid synthesis and degradation, and mitigates the negative growth effects of triacylglycerol accumulation in vegetative tissues.

Storage lipids (mostly triacylglycerols, TAGs) serve as an important energy and carbon reserve in plants, and hyperaccumulation of TAG in vegetative tissues can have negative effects on plant growth. Purple acid phosphatase2 (PAP2) was previously shown to affect carbon metabolism and boost plant growth. However, the effects of PAP2 on lipid metabolism remain unknown. Here, we demonstrated that PAP2 can stimulate a futile cycle of fatty acid (FA) synthesis and degradation, and mitigate negative growth effects associated with high accumulation of TAG in vegetative tissues. Constitutive expression of PAP2 in Arabidopsis thaliana enhanced both lipid synthesis and degradation in leaves and led to a substantial increase in seed oil yield. Suppressing lipid degradation in a PAP2-overexpressing line by disrupting sugar-dependent1 (SDP1), a predominant TAG lipase, significantly elevated vegetative TAG content and improved plant growth. Diverting FAs from membrane lipids to TAGs in PAP2-overexpressing plants by constitutively expressing phospholipid:diacylglycerol acyltransferase1 (PDAT1) greatly increased TAG content in vegetative tissues without compromising biomass yield. These results highlight the potential of combining PAP2 with TAG-promoting factors to enhance carbon assimilation, FA synthesis and allocation to TAGs for optimized plant growth and storage lipid accumulation in vegetative tissues.

Abstract 6181: Cell line panel with HIBIT tagged endogenous proteins to accelerate PROTAC drug discovery

Abstract Recent years have seen great advancement of targeted protein degradation technology, in particular, proteolysis targeting chimeras (PROTACs) are now widely used in developing therapeutics for treating cancer. A PROTAC is a heterobifunctional small molecule with three distinct moieties: a ligand to bind a targeted protein of interest (POI), a second ligand to recruit E3 ubiquitin ligase to form a ternary complex, and a linker for bridging the two ligands. Through the E3 ubiquitin ligase pathway, properly designed PROTACs can effectively degrade POIs with high specificity, which can be pathogenic proteins, thus regulate related pathways and inhibit tumor growth. Currently, there is no high-throughput biochemical assay to measure the POI degradation in vitro, hindering the application of the PROTAC technology. Here we report the development of a high-throughput cell based assay to quantitatively measure the endogenous drug target degradation induced by PROTACs. This assay encompasses over 800 cancer cell lines, many of which are CRISPR-engineered ones on common drug targets. A unique feature of our assay is the incorporation of HiBiT short sequence tags on either N-terminal or C-terminal of endogenous protein in E3 ligase matched cell line through site-specific gene homologous recombination technology. By highly specific and sensitive detection of HiBiT tag content in cell lysates with biochemical assay, we can determine target protein degradation by PROTAC treatment. We demonstrate the utility of the assay on a wide array of drug targets including RAS, LDHA, BTK and HPK1, and show that it can accelerate PROTAC drug discovery. Citation Format: Yunpeng Zhai, Ming Tan, Defu Liu, Guoqian Wang, Jinying Ning, Feng Hao. Cell line panel with HIBIT tagged endogenous proteins to accelerate PROTAC drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6181.

Dietary l-carnitine regulates liver lipid metabolism via simultaneously activating fatty acid β-oxidation and suppressing endoplasmic reticulum stress in large yellow croaker fed with high-fat diets.

Dietary l-carnitine (LC) is a nutritional factor that reduces liver lipid content. However, whether dietary LC can improve lipid metabolism via simultaneous activation of mitochondrial fatty acid (FA) β-oxidation and suppression of endoplasmic reticulum (ER) stress is still unknown. Large yellow croaker were fed with a high-fat diet (HFD) supplemented with dietary LC at 0, 1·2 or 2·4 ‰ for 10 weeks. The results indicated that a HFD supplemented with LC reduced the liver total lipid and TAG content and improved serum lipid profiles. LC supplementation administered to this fish increased the liver antioxidant capacity by decreasing serum and liver malondialdehyde levels and enhancing the liver antioxidant capacity, which then relieved the liver damage. Dietary LC increased the ATP dynamic process and mitochondrial number, decreased mitochondrial DNA damage and enhanced the protein expression of mitochondrial β-oxidation, biogenesis and mitophagy. Furthermore, dietary LC supplementation increased the expression of genes and proteins related to peroxisomal β-oxidation and biogenesis. Interestingly, feeding fish with LC-enriched diets decreased the protein levels indicative of ER stress, such as glucose-regulated protein 78, p-eukaryotic translational initiation factor 2a and activating transcription factor 6. Dietary LC supplementation downregulated mRNA expression relative to FA synthesis, reduced liver lipid and relieved liver damage through regulating β-oxidation and biogenesis of mitochondria and peroxisomes, as well as the ER stress pathway in fish fed with HFD. The present study provides the first evidence that dietary LC can improve lipid metabolism via simultaneously promoting FA β-oxidation capability and suppressing the ER stress pathway in fish.