Lipid Sequestration Research Articles

Innovating Lubrication with Polyelectrolyte Hydrogels: Sustained Performance Through Lipid Dynamics

AbstractThe lubrication process of natural joint cartilage involves a series of coordinated mechanisms, a key aspect of which is its ability to continuously extract lubricating components from synovial fluid to achieve sustained lubrication. Inspired by this natural phenomenon, this study reports a novel polyelectrolyte hydrogel, specifically integrating ε‐poly‐L‐lysine (ε‐PL), adeptly captures lipids from environment to achieve effective lubrication similar to human joints. The ε‐PL within the hydrogel facilitates the dynamic sequestration of lipids, fostering interfacial self‐assembly. This setup, enriched with highly hydrated lipid head groups, enhances boundary lubrication capabilities for extended performance. Through rigorous evaluation of friction coefficients and supramolecular interactions between the hydrogel and lipids, it identified hydrogen bonding, charge‐dipole, and hydrophobic interactions as key to this self‐assembly. The findings affirm the versatility of polyelectrolytes in synthesizing lubricating hydrogels, bridging the gap to the creation of biomimetic hydrogels that mimic natural lubrication with enhanced durability and efficiency.

A versatile delivery vehicle for cellular oxygen and fuels or metabolic sensor? A review and perspective on the functions of myoglobin.

A canonical view of the primary physiological function of myoglobin (Mb) is that it is an oxygen (O2) storage protein supporting mitochondrial oxidative phosphorylation, especially as the tissue O2 partial pressure (Po2) drops and Mb off-loads O2. Besides O2 storage/transport, recent findings support functions for Mb in lipid trafficking and sequestration, interacting with cellular glycolytic metabolites such as lactate (LAC) and pyruvate (PYR), and "ectopic" expression in some types of cancer cells and in brown adipose tissue (BAT). Data from Mb knockout (Mb-/-) mice and biochemical models suggest additional metabolic roles for Mb, especially regulation of nitric oxide (NO) pools, modulation of BAT bioenergetics, thermogenesis, and lipid storage phenotypes. From these and other findings in the literature over many decades, Mb's function is not confined to delivering O2 in support of oxidative phosphorylation but may serve as an O2 sensor that modulates intracellular Po2- and NO-responsive molecular signaling pathways. This paradigm reflects a fundamental change in how oxidative metabolism and cell regulation are viewed in Mb-expressing cells such as skeletal muscle, heart, brown adipocytes, and select cancer cells. Here, we review historic and emerging views related to the physiological roles for Mb and present working models illustrating the possible importance of interactions between Mb, gases, and small-molecule metabolites in regulation of cell signaling and bioenergetics.

Myoglobin: A versatile age-old oxygen carrying hemoprotein and its emerging role in metabolite regulation in diverse tissues

Myoglobin (Mb) is an age-old heme containing cytoplasmic protein primarily expressed in heart and skeletal muscles, which is capable of rapid release of O2 during the periods of hypoxia or anoxia. The canonical view of the primary physiological function of Mb is that it is a tissue oxygen (O2) storage protein supporting mitochondrial oxidative phosphorylation especially as the tissue O2 partial pressure ( pO2) drops and Mb increasingly offloads O2. For the past 50 years, many investigators are constantly exploring wide-range of physiological functions and metabolic regulation activities of Mb. Advanced genetic engineering tools and molecular techniques revealed important new insights and provided additional functions of Mb. However, accumulating evidence indicates that this paradigm is too narrow, especially in light of recent findings supporting plausible functions for Mb in lipid traffcking and sequestration, binding of small molecules such as lactate (LAC), pyruvate (PYR), glucose (GLU), polyamines, and glutathione (GSH), and “ectopic” expression in some types of cancer cells. Data from Mb knockout mice (Mb−/−) and biochemical models suggest additional metabolic roles for Mb, especially regulation of nitric oxide (NO) pools and modulation of thermogenic brown adipose tissue (BAT) bioenergetics and lipid storage phenotypes. From these and other findings in the literature over many decades, it may be contemplated that the primary role for Mb under most conditions for Mb besides delivering O2 in support of oxidative phosphorylation, but also serve as an O2-sensor that modulates intracellular O2- and NO-responsive molecular signaling pathways. This paradigm shift reflect a fundamental change in how oxidative metabolism and cell regulation are viewed in Mb-expressing cells such as skeletal muscle, heart, brown adipocytes, and select cancer cells. Herein, we present historic and emerging views related to the physiological roles for Mb, and working models illustrating the possible importance of interactions between Mb, gases, and small molecule metabolites in regulation of cell signaling and bioenergetics. USDA-ARS project 6026-51000-010-06S, Sturgis Foundation Grant AWD00054750 and ACRI-ABI Investigator Initiated Grant Award GR037175-4450S ABI. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Gut ribotoxic stress responses facilitate dyslipidemia via metabolic reprogramming: an environmental health prediction.

Rationale: The gut and its accessory organ, the liver, are crucial determinants of metabolic homeostasis via the regulation of circulating lipids for cardiovascular health. In response to environmental insults, cells undergo diverse adaptation or pathophysiological processes via stress-responsive eukaryotic initiation factor 2 alpha (eIF2α) kinase signaling and subsequent cellular reprogramming. We noted that patients with inflammatory gut distress display enhanced levels of ribosomal stress-responsive eIF2α kinase, which is notably associated with lipid metabolic process genes. Based on an assumption that eukaryotic ribosomes are a promising stress-responsive module for molecular reprogramming, chemical ribosome-inactivating stressors (RIS) were assessed for their involvement in enterohepatic lipid regulation. Methods: Experimental assessment was based on prediction using the clinical transcriptome and single-cell RNA-sequencing analysis of inflammatory bowel diseases and obesity. The prediction was verified using RIS exposure models of mice, gut organoids, and intestinal cells. The lipidomic profiling was performed to address RIS-induced intracellular fat alterations. Biochemical processes of the mechanisms were evaluated using RT-PCR, western blot analysis, luciferase reporter assays, and confocal microscopy of genetically ablated or chemically inhibited mice, organoids, and cells. Results: Chemical RIS including deoxynivalenol promoted enterohepatic lipid sequestration while lowering blood LDL cholesterol in normal and diet-induced obese mice. Although ribosomal stress caused extensive alterations in cellular lipids and metabolic genes, the cholesterol import-associated pathway was notably modulated. In particular, ribosomal stress enhanced gut levels of the low-density lipoprotein receptor (LDLR) via both transcriptional and post-transcriptional regulation. Subsequently, LDLR facilitated enterohepatic cholesterol accumulation, leading to dyslipidemia in response to ribosomal stress. Moreover, genetic features of stress-responsive LDLR modulators were consistently proven in the inflammation- and obesity-associated gut model. Conclusion: The elucidated ribosome-linked gut lipid regulation provides predictive insights into stress-responsive metabolic rewiring in chronic human diseases as an environmental health prediction.

Dihydromyricetin supplementation improves ethanol-induced lipid accumulation and inflammation.

Excessive alcohol consumption leads to a myriad of detrimental health effects, including alcohol-associated liver disease (ALD). Unfortunately, no available treatments exist to combat the progression of ALD beyond corticosteroid administration and/or liver transplants. Dihydromyricetin (DHM) is a bioactive polyphenol and flavonoid that has traditionally been used in Chinese herbal medicine for its robust antioxidant and anti-inflammatory properties. It is derived from many plants, including Hovenia dulcis and is found as the active ingredient in a variety of popular hangover remedies. Investigations utilizing DHM have demonstrated its ability to alleviate ethanol-induced disruptions in mitochondrial and lipid metabolism, while demonstrating hepatoprotective activity. Female c57BL/6J mice (n = 12/group) were treated using the Lieber DeCarli forced-drinking and ethanol (EtOH) containing liquid diet, for 5 weeks. Mice were randomly divided into three groups: (1) No-EtOH, (2) EtOH [5% (v/v)], and (3) EtOH [5% (v/v)] + DHM (6 mg/mL). Mice were exposed to ethanol for 2 weeks to ensure the development of ALD pathology prior to receiving dihydromyricetin supplementation. Statistical analysis included one-way ANOVA along with Bonferroni multiple comparison tests, where p ≤ 0.05 was considered statistically significant. Dihydromyricetin administration significantly improved aminotransferase levels (AST/ALT) and reduced levels of circulating lipids including LDL/VLDL, total cholesterol (free cholesterol), and triglycerides. DHM demonstrated enhanced lipid clearance by way of increased lipophagy activity, shown as the increased interaction and colocalization of p62/SQSTM-1, LC3B, and PLIN-1 proteins. DHM-fed mice had increased hepatocyte-to-hepatocyte lipid droplet (LD) heterogeneity, suggesting increased neutralization and sequestration of free lipids into LDs. DHM administration significantly reduced prominent pro-inflammatory cytokines commonly associated with ALD pathology such as TNF-α, IL-6, and IL-17. Dihydromyricetin is commercially available as a dietary supplement. The results of this proof-of-concept study demonstrate its potential utility and functionality as a cost-effective and safe candidate to combat inflammation and the progression of ALD pathology.

Apolipoprotein B in the Rough Endoplasmic Reticulum: Translation, Translocation and the Initiation of Lipoprotein Assembly

Apolipoprotein (apo) B and the microsomal triglyceride transfer protein are essential for the hepatic assembly and secretion of triglyceride-rich VLDL. To understand how apoB initiates the process of lipoprotein formation, interest has focused on the biogenesis of its amino terminal globular domain (α1 domain). When only this domain is expressed in hepatoma cells, no lipoprotein particle will form. However, proper folding of the α1 domain is essential for the internal lipophilic regions of apoB to engage in cotranslational lipid recruitment. The essential function of this domain may be related to its capacity to promote a specific physical interaction with the microsomal triglyceride transfer protein, necessary for apoB's proper folding and lipidation. Alternatively, this domain may promote an autonomous lipid recruitment step that nucleates microsomal triglyceride transfer protein-dependent lipid sequestration by apoB. Forms of apoB that fail to initiate particle assembly or forms associated with aberrant underlipidated particles are targeted for intracellular turnover. Two sites of apoB degradation have been identified. In hepatocarcinoma-derived cells, misassembled apoB may undergo progressive reverse translocation from the endoplasmic reticulum lumen to the cytosol, a process that is mechanistically coupled to polyubiquitination and proteasome-mediated degradation on the cytosolic side of the membrane. Alternatively, studies in primary hepatocytes reveal that apoB may undergo sorting to a post-endoplasmic reticulum compartment for presecretory degradation. In either case, the balance between assembly and presecretory degradation of apoB may represent a control point for the production of hepatic VLDL.

Host-Erythrocytic Sphingosine-1-Phosphate Regulates Plasmodium Histone Deacetylase Activity and Exhibits Epigenetic Control over Cell Death and Differentiation.

The evolution of resistance to practically all antimalarial drugs poses a challenge to the current malaria elimination and eradication efforts. Given that the epigenome of Plasmodium falciparum governs several crucial parasite functions, pharmaceutical interventions with transmission-blocking potential that target epigenetic molecular markers and regulatory mechanisms are likely to encounter drug resistance. In the malaria parasite, histone deacetylases (HDACs) are essential epigenetic modulators that regulate cellular transcriptional rearrangements, notably the molecular mechanisms underlying parasite proliferation and differentiation. We establish "lipid sequestration" as a mechanism by which sphingolipids, specifically Sphingosine-1-Phosphate (S1P) (a metabolic product of Sphingosine Kinase 1 [SphK-1]), regulate epigenetic reprogramming in the parasite by interacting with, and modulating, the histone-deacetylation activity of PfHDAC-1, thereby regulating Plasmodium pathogenesis. Furthermore, we demonstrate that altering host S1P levels with PF-543, a potent and selective Sphk-1 inhibitor, dysregulates PfHDAC-1 activity, resulting in a significant increase in the global histone acetylation signals and, consequently, transcriptional modulation of genes associated with gametocytogenesis, virulence, and proliferation. Our findings point to a hitherto unrecognized functional role for host S1P-mediated sphingolipid signaling in modulating PfHDAC-1's enzymatic activity and, as a result, the parasite's dynamic genome-wide transcriptional patterns. The epigenetic regulation of parasite proliferation and sexual differentiation offers a novel approach for developing host-targeted therapeutics to combat malaria resistance to conventional regimens. IMPORTANCE Sphingolipid is an 18-carbon amino-alcohol-containing lipid with a sphingosine backbone, which when phosphorylated by sphingosine kinase 1 (SphK-1), generates sphingosine-1-phosphate (S1P), an essential lipid signaling molecule. Dysregulation of S1P function has been observed in a variety of pathologies, including severe malaria. The malaria parasite Plasmodium acquires a host S1P pool for its growth and survival. Here, we describe the molecular attuning of histone deacetylase-1 (PfHDAC-1), a crucial epigenetic modulator that contributes to the establishment of epigenetic chromatin states and parasite survival, in response to S1P binding. Our findings highlight the host lipid-mediated epigenetic regulation of malaria parasite key genes.

Trypanosoma cruzi modulates lipid metabolism and highjacks phospholipids from the midgut of Rhodnius prolixus

Chagas disease is potentially life-threatening and caused by the protozoan parasite Trypanosoma cruzi. The parasite cannot synthesize some lipids and depends on the uptake of these lipids from its vertebrate and invertebrate hosts. To achieve this, T. cruzi may need to modify the physiology of the insect host for its own benefit. In this study, we investigated the interaction of T. cruzi (Y strain) with its insect vector Rhodnius prolixus and how it manipulates the vector lipid metabolism. We observed a physiological change in lipid flux in of infected insects. In the fat body of infected insects, triacylglycerol levels decreased by 80.6% and lipid storage droplet-1(LSD-1) mRNA levels were lower, when compared to controls. Lipid sequestration by infected midguts led to increased levels of 5′ AMP-activated protein kinase (AMPK) phosphorylation and activation in the fat body, inhibiting the synthesis of fatty acids and stimulating their oxidation. This led to reduced lipid levels in the fat body of infected insets, despite the fact that T. cruzi does not colonize this tissue. There was a 3-fold increase, in lipid uptake and synthesis in the midgut of infected insects. Finally, our results suggest that the parasite modifies the lipid flux and metabolism of its vector R. prolixus through the increase in lipid delivery from the fat body to midgut that are then scavenge by T cruzi.

Metabolic Engineering Strategies for Improved Lipid Production and Cellular Physiological Responses in Yeast Saccharomyces cerevisiae

Microbial lipids have been a hot topic in the field of metabolic engineering and synthetic biology due to their increased market and important applications in biofuels, oleochemicals, cosmetics, etc. This review first compares the popular hosts for lipid production and explains the four modules for lipid synthesis in yeast, including the fatty acid biosynthesis module, lipid accumulation module, lipid sequestration module, and fatty acid modification module. This is followed by a summary of metabolic engineering strategies that could be used for enhancing each module for lipid production. In addition, the efforts being invested in improving the production of value-added fatty acids in engineered yeast, such as cyclopropane fatty acid, ricinoleic acid, gamma linoleic acid, EPA, and DHA, are included. A discussion is further made on the potential relationships between lipid pathway engineering and consequential changes in cellular physiological properties, such as cell membrane integrity, intracellular reactive oxygen species level, and mitochondrial membrane potential. Finally, with the rapid development of synthetic biology tools, such as CRISPR genome editing tools and machine learning models, this review proposes some future trends that could be employed to engineer yeast with enhanced intracellular lipid production while not compromising much of its cellular health.

Neuronal ROS-induced glial lipid droplet formation is altered by loss of Alzheimer’s disease–associated genes

A growing list of Alzheimer's disease (AD) genetic risk factors is being identified, but the contribution of each variant to disease mechanism remains largely unknown. We have previously shown that elevated levels of reactive oxygen species (ROS) induces lipid synthesis in neurons leading to the sequestration of peroxidated lipids in glial lipid droplets (LD), delaying neurotoxicity. This neuron-to-glia lipid transport is APOD/E-dependent. To identify proteins that modulate these neuroprotective effects, we tested the role of AD risk genes in ROS-induced LD formation and demonstrate that several genes impact neuroprotective LD formation, including homologs of human ABCA1, ABCA7, VLDLR, VPS26, VPS35, AP2A, PICALM, and CD2AP Our data also show that ROS enhances Aβ42 phenotypes in flies and mice. Finally, a peptide agonist of ABCA1 restores glial LD formation in a humanized APOE4 fly model, highlighting a potentially therapeutic avenue to prevent ROS-induced neurotoxicity. This study places many AD genetic risk factors in a ROS-induced neuron-to-glia lipid transfer pathway with a critical role in protecting against neurotoxicity.

Nuclear and cytoplasmic huntingtin inclusions exhibit distinct biochemical composition, interactome and ultrastructural properties

Despite the strong evidence linking the aggregation of the Huntingtin protein (Htt) to the pathogenesis of Huntington’s disease (HD), the mechanisms underlying Htt aggregation and neurodegeneration remain poorly understood. Herein, we investigated the ultrastructural properties and protein composition of Htt cytoplasmic and nuclear inclusions in mammalian cells and primary neurons overexpressing mutant exon1 of the Htt protein. Our findings provide unique insight into the ultrastructural properties of cytoplasmic and nuclear Htt inclusions and their mechanisms of formation. We show that Htt inclusion formation and maturation are complex processes that, although initially driven by polyQ-dependent Htt aggregation, also involve the polyQ and PRD domain-dependent sequestration of lipids and cytoplasmic and cytoskeletal proteins related to HD dysregulated pathways; the recruitment and accumulation of remodeled or dysfunctional membranous organelles, and the impairment of the protein quality control and degradation machinery. We also show that nuclear and cytoplasmic Htt inclusions exhibit distinct biochemical compositions and ultrastructural properties, suggesting different mechanisms of aggregation and toxicity.

Angiopoietin-like protein 8 differentially regulates ANGPTL3 and ANGPTL4 during postprandial partitioning of fatty acids

Angiopoietin-like protein (ANGPTL)8 has been implicated in metabolic syndrome and reported to regulate adipose FA uptake through unknown mechanisms. Here, we studied how complex formation of ANGPTL8 with ANGPTL3 or ANGPTL4 varies with feeding to regulate LPL. In human serum, ANGPTL3/8 and ANGPTL4/8 complexes both increased postprandially, correlated negatively with HDL, and correlated positively with all other metabolic syndrome markers. ANGPTL3/8 also correlated positively with LDL-C and blocked LPL-facilitated hepatocyte VLDL-C uptake. LPL-inhibitory activity of ANGPTL3/8 was >100-fold more potent than that of ANGPTL3, and LPL-inhibitory activity of ANGPTL4/8 was >100-fold less potent than that of ANGPTL4. Quantitative analyses of inhibitory activities and competition experiments among the complexes suggested a model in which localized ANGPTL4/8 blocks the LPL-inhibitory activity of both circulating ANGPTL3/8 and localized ANGPTL4, allowing lipid sequestration into fat rather than muscle during the fed state. Supporting this model, insulin increased ANGPTL3/8 secretion from hepatocytes and ANGPTL4/8 secretion from adipocytes. These results suggest that low ANGPTL8 levels during fasting enable ANGPTL4-mediated LPL inhibition in fat tissue to minimize adipose FA uptake. During feeding, increased ANGPTL8 increases ANGPTL3 inhibition of LPL in muscle via circulating ANGPTL3/8, while decreasing ANGPTL4 inhibition of LPL in adipose tissue through localized ANGPTL4/8, thereby increasing FA uptake into adipose tissue. Excessive caloric intake may shift this system toward the latter conditions, possibly predisposing to metabolic syndrome.

Synergistic effects between autotrophy and heterotrophy in optimization of mixotrophic cultivation of Chlorella sorokiniana in bubble-column photobioreactors

Mixotrophic cultivation of microalgae enables inorganic carbon capture through photosynthesis along with simultaneous organic carbon uptake. Extremophilic microalga Chlorella sorokiniana, capable of tolerating high CO2 levels, was cultivated mixotrophically in 1 L bubble-column photobioreactors. This study shows how the biomass yield increases as the algal CO2 uptake process shifts from being driven by Carbon Concentrating Mechanism (CCM) to being driven by direct diffusion, as the CO2 concentration increases from very low (0%) and low (atmospheric: 0.04%) to high (1%, 2%, 3%, 5%). The biomass yield and productivity increase from 1.05 g/L and 0.33 g/L/day, respectively, at atmospheric CO2 to 1.85 g/L and 0.48 g/L/day, respectively, at 3% CO2, with a corresponding doubling of the lipid yield. Using acetic acid as the organic carbon source allows it to directly enter the metabolic pathways at the acetyl-CoA junction and enhance fatty acid synthesis, while enhanced reactor illumination (below photo-inhibition) synergizes the phototrophic and photo-heterotrophic pathways. These three reactor scale parameters, namely, organic (acetic acid) and inorganic (CO2) carbon loading and illumination, are co-optimized to activate the synergies between the metabolic pathways for autotrophy and heterotrophy in mixotrophic algal growth, which, in turn, significantly enhances the biomass and the macromolecular yields at the cellular scale. This multiscale synergy leads to a four-fold increase in the biomass yield (from 0.7 to 2.79 g/L), a five-fold increase in biomass productivity (from 0.124 to 0.593 g/L/day), and a six-fold increase in the total lipid yield (from 0.12 to 0.75 g/L), while eliminating night-time biomass losses. Such an approach of co-optimising reactor-scale parameters to synergize the autotrophic and the heterotrophic metabolic pathways can be implemented in mass cultivation of microalgae, resulting in faster carbon sequestration and higher yields of biomass, lipids and other value-added co-products.

Lipid Transfer Proteins (AaLTP3 and AaLTP4) Are Involved in Sesquiterpene Lactone Secretion from Glandular Trichomes in Artemisia annua.

In Artemisia annua plants, glandular trichomes (GTs) are responsible for the biosynthesis and secretion of sesquiterpene lactones including artemisinin/arteannuin B. Nonspecific lipid transfer proteins (LTPs) in plants bind and carry lipid molecules across the cell membrane and are also known as secretary proteins. Interestingly, the transcripts of LTP genes are exceptionally abundant in the GTs of A. annua. In the present study, we isolated two trichome-specific LTP genes (AaLTP3 and AaLTP4) from a Korean ecotype of A. annua. AaLTP3 was expressed abundantly in shoots, whereas AaLTP4 was expressed in flowers. The GUS signal driven by the AaLTP3 or AaLTP4 promoter in transgenic A. annua plants revealed that the AaLTP3 promoter was active on hair-like non-GTs and that the AaLTP4 promoter was active on GTs. Analysis of enhanced cyan fluorescent protein (ECFP) fluorescence fused with the AaLTP3 or AaLTP4 protein in transgenic tobacco revealed that ECFP florescence was very bright on secreted lipids of long GTs. Moreover, the florescence was also bright on the head cells of short trichomes and their secreted granules. Immunoblotting analysis of GT exudates in petioles of A. annua revealed a strong positive signal against the AaLTP4 antibody. Overexpression of AaLTP3 or AaLTP4 in transgenic A. annua plants resulted in enhanced production of sesquiterpene lactones (arteannuin B, artemisinin, dihydroartemisinic acid and artemisinic acid) compared with those of wild type. The present study shows that LTP genes (AaLTP3 or AaLTP4) play important roles in the sequestration and secretion of lipids in GTs of A. annua, which is useful for the enhanced production of sesquiterpene lactones by genetic engineering.

Flow-cytometry-based physiological characterisation and transcriptome analyses reveal a mechanism for reduced cell viability in yeast engineered for increased lipid content

BackgroundYeast has been the focus of development of cell biofactories for the production of lipids and interest in the field has been driven by the need for sustainably sourced lipids for use in a broad range of industrial applications. Previously, we reported a metabolic engineering strategy for enhanced lipid production in yeast which delivered high per-cell lipid but with low cell growth and compromised physiology. To investigate the relationship between lipid engineering and cellular physiological responses and to identify further metabolic engineering targets, we analysed transcriptomes and measured cell physiology parameters in engineered strains.ResultsIn the engineering strategy, the central carbon pathway was reprogrammed to provide more precursors for lipid production and lipid accumulation and sequestration steps were enhanced through the expression of heterologous genes. Genes coding for enzymes within the pentose phosphate, beta-oxidation pathways, ATP and NADPH biosynthesis had lower transcript levels in engineered cells. Meanwhile, flow-cytometry analysis of fluorescent-dye stained cells showed the highest reactive oxygen species (ROS) levels and mitochondrial membrane potential (Δψm) in cells with the highest lipid content, supporting the known relationship between mitochondrial activity and ROS generation. High intracellular ROS and low membrane integrity were not ameliorated by application of antioxidants.ConclusionsThe limited intracellular energy supplies and the unbalanced redox environment could be regarded as targets for further lipid engineering, similarly for native lipid accumulation genes that were upregulated. Thus, lipid pathway engineering has an important effect on the central carbon pathway, directing these towards lipid production and sacrificing the precursors, energy and cofactor supply to satisfy homeostatic metabolic requirements.

Towards Simazine Monitoring in Agro-Zootechnical Productions: A Yeast Cell Bioprobe for Real Samples Screening.

Simazine is an herbicide that is able to contaminate surface waters, ground waters, and milk/dairy products, thus posing concerns in both environmental health and food safety. A yeast-based bioprobe was utilized to detect simazine in spiked real samples of livestock drinking water and raw cow’s milk. Yeast aerobic respiration was taken as short-term toxicological endpoint. We carried out comparative measures of yeast oxygen consumption between simazine-spiked samples and blank samples. Percentage interference (%ρ) on yeast aerobic respiration was calculated through the comparison of aerobic respiration of simazine-exposed and non-exposed yeast cells. The method was optimized for raw cow’s milk samples by using boric acid as fungistatic agent in order to avoid cellular proliferation. Overall, the results have shown that simazine can be detected up to concentrations five times below the EU legal concentration limits for drinking water (0.02 ppb) and cow’s milk (2 ppb) (%ρ values of 18.53% and 20.43% respectively; %RSD ≤ 15%). Dose-effect relationships of simazine were assessed. The findings of the bioassays match reasonably well with known mechanisms of toxicity and intracellular detoxification in yeast. A correlation between fat content in milk samples and analytical performance of the bioprobe was established. Results suggest the involvement of a matrix effect, presumably due to lipid sequestration of simazine. The yeast-based bioprobe has proved to be sensitive and suitable for the detection of simazine in real samples in concentrations of interest.

Parsing the Potential Neuroendocrine Actions of FGF21 in Primates.

Fibroblast growth factor (FGF) 21, a unique, largely liver-derived endocrine member of the FGF superfamily, is often thought of as a fasting factor owing to its induction in rodents during starvation. However, FGF21 is not increased by fasting for periods of <7 days in humans; instead, it rises sharply after acute alcohol and sugar intake and also after several days of overfeeding, suggesting another role in states of positive energy balance. Recent studies suggest that in the postingestive state, FGF21 may regulate energy intake and discourage consumption of alcohol and sugars, most likely through effector circuits in the central nervous system. FGF21 also increases fat oxidation in the liver, improves markers of insulin sensitivity, and stimulates adiponectin production. Thus, in primates, FGF21 may defend against hepatic nutrient overload by promoting adaptations that reduce ectopic lipid storage, including inhibiting sugar and alcohol appetite and promoting lipid sequestration in adipose tissue.

T cell autoreactivity directed toward CD1c itself rather than toward carried self lipids.

The hallmark function of αβ T cell antigen receptors (TCRs) involves the highly specific co-recognition of a major histocompatibility complex molecule and its carried peptide. However, the molecular basis of the interactions of TCRs with the lipid antigen-presenting molecule CD1c is unknown. We identified frequent staining of human T cells with CD1c tetramers across numerous subjects. Whereas TCRs typically show high specificity for antigen, both tetramer binding and autoreactivity occurred with CD1c in complex with numerous, chemically diverse self lipids. Such extreme polyspecificity was attributable to binding of the TCR over the closed surface of CD1c, with the TCR covering the portal where lipids normally protrude. The TCR essentially failed to contact lipids because they were fully seated within CD1c. These data demonstrate the sequestration of lipids within CD1c as a mechanism of autoreactivity and point to small lipid size as a determinant of autoreactive T cell responses.

Potential CO2 fixation and optimal Dunaliella tertiolecta cultivation: Influence of fertilizer, wavelength of light-emitting diodes, salinity and carbon supply strategy

NPK-fertilizer based optimum culture medium was formulated for Dunaliella tertiolecta cultivation using 100% CO2 and different wavelength light emitting diodes (LEDs) involving green, white, red, pink and blue. The influence of decision variables like fertilizer, salinity, carbon supply strategy and LEDs with different wavelength was investigated to maximize algae-biomass, lipid and CO2 sequestration. NaCl was used to vary salinity of the culture medium, whereas carbon supply strategy was adopted for algae-biomass growth by varying NaOH dozing. Five levels and four variables response surface method (RSM) was used to fix the optimum values of the decision variables and the efficacy of RSM-oriented results were compared with the binary coded genetic algorithm (GA). Algae-biomass growth, lipid production and CO2 sequestration were enhanced significantly using optimally formulated fertilizer medium over standard artificial seawater based f/2 medium. Carbon supply strategy was found to be the most important variable for optimal algal cultivation, whereas (NPK-NaOH, NaCl-NaOH), (NaCl-LED, NPK-LED, NPK-NaOH) and (NaOH-LED, NPK-LED) were the most influencing interactions to maximize biomass growth, lipid productivity and CO2 sequestration, respectively. Following cultivation parameters, i.e., fertilizer (gL−1), NaCl (gL-1), LED, and NaOH (gL-1) were obtained to maximize algae-biomass (1.9696gL-1), lipid (0.7068gL-1) and CO2 sequestration (0.2434gL-1 d-1): (0.43, 32.99, red-72%/pink-28%, 1.09), (0.47, 59.24, red-86%/pink-14%, 1.29) and (0.46, 41.99, red-81%/pink-19%, 1.26), respectively, using simple GA. Three multi-objective optimization.

Abstract 363: Lysosomal Lipid Accumulation and Dysregulation of Downstream Cholesterol Homeostasis in Human Arterial Smooth Muscle Cells

Background and Hypothesis: Previously we reported that ≥ 50% of the foam cells in human coronary artery atherosclerosis are smooth muscle cell (SMC) derived, and that the rate-limiting cholesterol exporter, ATP-binding cassette transporter protein A1 (ABCA1), has reduced expression in SMCs compared to leukocytes in the intima. Upregulation of ABCA1 expression is dependent on normal flux of lipoprotein-derived cholesterol out of lysosomes and the subsequent generation of 27-hydroxycholesterol (27-OHC). Excess lipoprotein-derived cholesterol is also converted to cholesteryl esters (CE) through the actions of acyl-coA cholesterol acyltransferase (ACAT). Processes by which macrophages store lipoprotein-derived cholesterol in cytosolic and lysosomal compartments are well described, whereas less is known about SMCs. Hypothesis: We hypothesize that preferential SMC foam cell formation and the observed derangement in ABCA1 expression are associated with increased lysosomal sequestration of atherogenic lipids. Methods and Results: Human aortic SMCs and human monocyte-derived macrophages (HMMs) were loaded with aggregated LDL (agLDL) for 24 hrs, followed by a 24 hr equilibration without lipids to allow processing of lipoproteins. Data are presented as mean±SEM. In response to agLDL, CE content (nmol/mg protein) measured by LC/MS/MS increased from 2.2±1.3 to 277.0±18.2 in HMMs, and 3.5±5.3 to 46.8±14.5 in SMCs (n=9). Confocal microscopy indicated elevated lysosomal lipid accumulation in SMCs compared to HMMs. Levels of 27-OHC (ng/mg protein) as measured by LC/MS/MS increased from 80.9±9.5 to 281.4±22.7 (3.5-fold) in HMMs, and 0.7±0.2 to 1.5±0.3 (1.4-fold) in SMCs (n=9). ACAT activity, measured by the incorporation of 14 C-labelled oleate (pmol/mg protein), increased from 21.0±1.7 to 678.1±110.9 (32.2-fold) in HMMs, and 55.6±3.3 to 133.7±15.2 (2.4-fold) in SMCs (n=16). By Western blot, fold change in ABCA1 with exposure to agLDL was 2.3±0.3 in HMMs and 1.3±0.2 in SMCs (n=7-10). Conclusions: Accumulation of atherogenic lipids in the lysosomes of SMCs provides a potential mechanism for the reduced ABCA1 expression and preferential formation of foam cells by arterial SMCs.