A conserved SREBP-1/phosphatidylcholine feedback circuit regulates lipogenesis in metazoans.

Sterol regulatory element-binding proteins (SREBPs) activate genes involved in the synthesis and trafficking of cholesterol and other lipids and are critical for maintaining lipid homeostasis. Aberrant SREBP activity, however, can contribute to obesity, fatty liver disease, and insulin resistance, hallmarks of metabolic syndrome. Our studies identify a conserved regulatory circuit in which SREBP-1 controls genes in the one-carbon cycle, which produces the methyl donor S-adenosylmethionine (SAMe). Methylation is critical for the synthesis of phosphatidylcholine (PC), a major membrane component, and we find that blocking SAMe or PC synthesis in C. elegans, mouse liver, and human cells causes elevated SREBP-1-dependent transcription and lipid droplet accumulation. Distinct from negative regulation of SREBP-2 by cholesterol, our data suggest a feedback mechanism whereby maturation of nuclear, transcriptionally active SREBP-1 is controlled by levels of PC. Thus, nutritional or genetic conditions limiting SAMe or PC production may activate SREBP-1, contributing to human metabolic disorders.

Homeostatic control of stearoyl desaturase expression via patched-like receptor PTR-23 ensures the survival of C. elegans during heat stress.

Organismal responses to temperature fluctuations include an evolutionarily conserved cytosolic chaperone machinery as well as adaptive alterations in lipid constituents of cellular membranes. Using C. elegans as a model system, we asked whether adaptable lipid homeostasis is required for survival during physiologically relevant heat stress. By systematic analyses of lipid composition in worms during and before heat stress, we found that unsaturated fatty acids are reduced in heat-stressed animals. This is accompanied by the transcriptional downregulation of fatty acid desaturase enzymes encoded by fat-1, fat-3, fat-4, fat-5, fat-6, and fat-7 genes. Conversely, overexpression of the Δ9 desaturase FAT-7, responsible for the synthesis of PUFA precursor oleic acid, and supplementation of oleic acid causes accelerated death of worms during heat stress. Interestingly, heat stress causes permeability defects in the worm's cuticle. We show that fat-7 expression is reduced in the permeability defective collagen (PDC) mutant, dpy-10, known to have enhanced heat stress resistance (HSR). Further, we show that the HSR of dpy-10 animals is dependent on the upregulation of PTR-23, a patched-like receptor in the epidermis, and that PTR-23 downregulates the expression of fat-7. Consequently, abrogation of ptr-23 in wild type animals affects its survival during heat stress. This study provides evidence for the negative regulation of fatty acid desaturase expression in the soma of C. elegans via the non-canonical role of a patched receptor signaling component. Taken together, this constitutes a skin-gut axis for the regulation of lipid desaturation to promote the survival of worms during heat stress.

The fetal lineage is susceptible to Zika virus infection within days of fertilization.

Adults contracting Zika virus (ZIKV) typically exhibit mild symptoms, yet ZIKV infection of pregnant individuals can cause miscarriage or birth defects in their offspring. Many studies have focused on maternal-to-fetal ZIKV transmission via blood and placenta. Notably, however, ZIKV is also transmitted sexually, raising the possibility that ZIKV could infect the embryo shortly after fertilization, long before the placenta is established. Here, we evaluate the consequences of ZIKV infection in mouse embryos during the first few days of embryogenesis. We show that divergent strains of ZIKV can infect the fetal lineage and can cause developmental arrest, raising concern for the developmental consequences of sexual ZIKV transmission. This article has an associated 'The people behind the papers' interview.

Ether lipid deficiency disrupts lipid homeostasis leading to ferroptosis sensitivity.

Ferroptosis is an iron-dependent form of regulated cell death associated with uncontrolled membrane lipid peroxidation and destruction. Previously, we showed that dietary dihomo-gamma-linolenic acid (DGLA; 20: 3(n-6)) triggers ferroptosis in the germ cells of the model organism, Caenorhabditis elegans. We also demonstrated that ether lipid-deficient mutant strains are sensitive to DGLA-induced ferroptosis, suggesting a protective role for ether lipids. The vinyl ether bond unique to plasmalogen lipids has been hypothesized to function as an antioxidant, but this has not been tested in animal models. In this study, we used C. elegans mutants to test the hypothesis that the vinyl ether bond in plasmalogens acts as an antioxidant to protect against germ cell ferroptosis as well as to protect from whole-body tert-butyl hydroperoxide (TBHP)-induced oxidative stress. We found no role for plasmalogens in either process. Instead, we demonstrate that ether lipid-deficiency disrupts lipid homeostasis in C. elegans, leading to altered ratios of saturated and monounsaturated fatty acid (MUFA) content in cellular membranes. We demonstrate that ferroptosis sensitivity in both wild type and ether-lipid deficient mutants can be rescued in several ways that change the relative abundance of saturated fats, MUFAs and specific polyunsaturated fatty acids (PUFAs). Specifically, we reduced ferroptosis sensitivity by (1) using mutant strains unable to synthesize DGLA, (2) using a strain carrying a gain-of-function mutation in the transcriptional mediator MDT-15, or (3) by dietary supplementation of MUFAs. Furthermore, our studies reveal important differences in how dietary lipids influence germ cell ferroptosis versus whole-body peroxide-induced oxidative stress. These studies highlight a potentially beneficial role for endogenous and dietary MUFAs in the prevention of ferroptosis.

A compendium of kinetic modulatory profiles identifies ferroptosis regulators.

Cell death can be executed by regulated apoptotic and nonapoptotic pathways, including the iron-dependent process of ferroptosis. Small molecules are essential tools for studying the regulation of cell death. Using time-lapse imaging and a library of 1,833 bioactive compounds, we assembled a large compendium of kinetic cell death modulatory profiles for inducers of apoptosis and ferroptosis. From this dataset we identify dozens of ferroptosis suppressors, including numerous compounds that appear to act via cryptic off-target antioxidant or iron chelating activities. We show that the FDA-approved drug bazedoxifene acts as a potent radical trapping antioxidant inhibitor of ferroptosis both in vitro and in vivo. ATP-competitive mechanistic target of rapamycin (mTOR) inhibitors, by contrast, are on-target ferroptosis inhibitors. Further investigation revealed both mTOR-dependent and mTOR-independent mechanisms that link amino acid metabolism to ferroptosis sensitivity. These results highlight kinetic modulatory profiling as a useful tool to investigate cell death regulation.

TEAD4, YAP1 and WWTR1 prevent the premature onset of pluripotency prior to the 16-cell stage.

In mice, pluripotent cells are thought to derive from cells buried inside the embryo around the 16-cell stage. Sox2 is the only pluripotency gene known to be expressed specifically within inside cells at this stage. To understand how pluripotency is established, we therefore investigated the mechanisms regulating the initial activation of Sox2 expression. Surprisingly, Sox2 expression initiated normally in the absence of both Nanog and Oct4 (Pou5f1), highlighting differences between embryo and stem cell models of pluripotency. However, we observed precocious ectopic expression of Sox2 prior to the 16-cell stage in the absence of Yap1, Wwtr1 and Tead4 Interestingly, the repression of premature Sox2 expression was sensitive to LATS kinase activity, even though LATS proteins normally do not limit activity of TEAD4, YAP1 and WWTR1 during these early stages. Finally, we present evidence for direct transcriptional repression of Sox2 by YAP1, WWTR1 and TEAD4. Taken together, our observations reveal that, while embryos are initially competent to express Sox2 as early as the four-cell stage, transcriptional repression prevents the premature expression of Sox2, thereby restricting the pluripotency program to the stage when inside cells are first created.

Emerging roles for specific fatty acids in developmental processes.

Animals synthesize a vast range of fatty acids serving diverse cellular functions. The roles of specific fatty acids in early development are just beginning to be characterized. In the March 15, 2012, issue of Genes & Development, Kniazeva and colleagues (pp. 554-566) describe how the particular combination of a branched chain fatty acid and an acyl-CoA synthetase is required for critical cellular processes during early embryogenesis in Caenorhabditis elegans.

The biotin-ligating protein BPL-1 is critical for lipid biosynthesis and polarization of the Caenorhabditis elegans embryo.

Biotin is an essential cofactor for multiple metabolic reactions catalyzed by carboxylases. Biotin is covalently linked to apoproteins by holocarboxylase synthetase (HCS). Accordingly, some mutations in HCS cause holocarboxylase deficiency, a rare metabolic disorder that can be life-threatening if left untreated. However, the long-term effects of HCS deficiency are poorly understood. Here, we report our investigations of bpl-1, which encodes the Caenorhabditis elegans ortholog of HCS. We found that mutations in the biotin-binding region of bpl-1 are maternal-effect lethal and cause defects in embryonic polarity establishment, meiosis, and the integrity of the eggshell permeability barrier. We confirmed that BPL-1 biotinylates four carboxylase enzymes, and we demonstrate that BPL-1 is required for efficient de novo fatty acid biosynthesis. We also show that the lack of larval growth defects as well as nearly normal fatty acid composition in young adult worms is due to sufficient fatty acid precursors provided by dietary bacteria. However, BPL-1 disruption strongly decreased levels of polyunsaturated fatty acids in embryos produced by bpl-1 mutant hermaphrodites, revealing a critical role for BPL-1 in lipid biosynthesis during embryogenesis and demonstrating that dietary fatty acids and lipid precursors are not adequate to support early embryogenesis in the absence of BPL-1. Our findings highlight that studying BPL-1 function in C. elegans could help dissect the roles of this important metabolic enzyme under different environmental and dietary conditions.

Conserved role of SIRT1 orthologs in fasting-dependent inhibition of the lipid/cholesterol regulator SREBP.

The sterol regulatory element-binding protein (SREBP) transcription factor family is a critical regulator of lipid and sterol homeostasis in eukaryotes. In mammals, SREBPs are highly active in the fed state to promote the expression of lipogenic and cholesterogenic genes and facilitate fat storage. During fasting, SREBP-dependent lipid/cholesterol synthesis is rapidly diminished in the mouse liver; however, the mechanism has remained incompletely understood. Moreover, the evolutionary conservation of fasting regulation of SREBP-dependent programs of gene expression and control of lipid homeostasis has been unclear. We demonstrate here a conserved role for orthologs of the NAD(+)-dependent deacetylase SIRT1 in metazoans in down-regulation of SREBP orthologs during fasting, resulting in inhibition of lipid synthesis and fat storage. Our data reveal that SIRT1 can directly deacetylate SREBP, and modulation of SIRT1 activity results in changes in SREBP ubiquitination, protein stability, and target gene expression. In addition, chemical activators of SIRT1 inhibit SREBP target gene expression in vitro and in vivo, correlating with decreased hepatic lipid and cholesterol levels and attenuated liver steatosis in diet-induced and genetically obese mice. We conclude that SIRT1 orthologs play a critical role in controlling SREBP-dependent gene regulation governing lipid/cholesterol homeostasis in metazoans in response to fasting cues. These findings may have important biomedical implications for the treatment of metabolic disorders associated with aberrant lipid/cholesterol homeostasis, including metabolic syndrome and atherosclerosis.

Activation of the endoplasmic reticulum unfolded protein response by lipid disequilibrium without disturbed proteostasis in vivo.

The Mediator is a conserved transcriptional coregulator complex required for eukaryotic gene expression. In Caenorhabditis elegans, the Mediator subunit mdt-15 is essential for the expression of genes involved in fatty acid metabolism and ingestion-associated stress responses. mdt-15 loss of function causes defects in reproduction and mobility and shortens lifespan. In the present study, we find that worms with mutated or depleted mdt-15 (mdt-15 worms) exhibit decreased membrane phospholipid desaturation, especially in phosphatidylcholine. Accordingly, mdt-15 worms exhibit disturbed endoplasmic reticulum (ER) homeostasis, as indicated by a constitutively activated ER unfolded protein response (UPR(ER)). Activation of this stress response is only partially the consequence of reduced membrane lipid desaturation, implicating other mdt-15-regulated processes in maintaining ER homeostasis. Interestingly, mdt-15 inactivation or depletion of the lipid metabolism enzymes stearoyl-CoA-desaturases (SCD) and S-adenosyl methionine synthetase (sams-1) activates the UPR(ER) without promoting misfolded protein aggregates. Moreover, these worms exhibit wild-type sensitivity to chemically induced protein misfolding, and they do not display synthetic lethality with mutations in UPR(ER) genes, which cause protein misfolding. Therefore, the constitutively activated UPR(ER) in mdt-15, SCD, and sams-1 worms is not the consequence of proteotoxic stress but likely is the direct result of changes in ER membrane fluidity and composition. Together, our data suggest that the UPR(ER) is induced directly upon membrane disequilibrium and thus monitors altered ER homeostasis.

Comparative Performance of Pediatric Weight Estimation Techniques: A Human Factor Errors Analysis.

OBJECTIVE: We compared performance characteristics of 7 weight estimation methods examining predictive performance and human factors errors. METHODS: This was a prospective study of 80 emergency care providers (raters) and 80 children aged 2 months to 16 years. Raters estimated weights in 5 children with the following 7 strategies: visual estimation, Advanced Pediatric Life Support, Luscombe and Owens, Broselow tape, devised weight estimation method, 2D Mercy TAPE (2DT), and 3D Mercy TAPE (3DT). Quantitative errors were determined by checking rater values against values returned with optimal method use. RESULTS: Four hundred rater-child pairings generated 2800 weight estimates. For all methods, rater-estimated weights were less accurate than weights derived by optimal application. Skill-based, perception, and judgment/decision error were observed. For visual estimation, weights were underestimated in most children. For Advanced Pediatric Life Support/Luscombe and Owens, order of operations markedly impacted errors with 23% of calculations requiring addition first performed incorrectly versus 9% of calculations requiring multiplication first. For Broselow tape, only 63% of cases were eligible for estimation with this device, yet raters assigned a weight in 96% of cases. For Devised Weight Estimation Method, 96% of overweight and 48% of obese children were classified as slim or average. For 2DT/3DT, the 2DT was prone to more errors most commonly use of the wrong side of the device (24%). The impact of rater characteristics on error was most pronounced for methods requiring calculation. CONCLUSIONS: Skill-based, perception, or judgment errors were observed in more than 1 of 20 cases. No singular strategy was used with 100% accuracy.

A Caenorhabditis elegans model for ether lipid biosynthesis and function.

Ether lipids are widespread in nature, and they are structurally and functionally important components of membranes. The roundworm, Caenorhabditis elegans, synthesizes numerous lipid species containing alkyl and alkenyl ether bonds. We isolated C. elegans strains carrying loss-of-function mutations in three genes encoding the proteins required for the initial three steps in the ether lipid biosynthetic pathway, FARD-1/FAR1, ACL-7/GNPAT, and ADS-1/AGPS. Analysis of the mutant strains show that they lack ether lipids, but possess the ability to alter their lipid composition in response to lack of ether lipids. We found that increases in de novo fatty acid synthesis and reduction of stearoyl- and palmitoyl-CoA desaturase activity, processes that are at least partially regulated transcriptionally, mediate the altered lipid composition in ether lipid-deficient mutants. Phenotypic analysis demonstrated the importance of ether lipids for optimal fertility, lifespan, survival at cold temperatures, and resistance to oxidative stress.Caenorhabditis.

Lipidomic and proteomic analysis of Caenorhabditis elegans lipid droplets and identification of ACS-4 as a lipid droplet-associated protein.

Lipid droplets are cytoplasmic organelles that store neutral lipids for membrane synthesis and energy reserves. In this study, we characterized the lipid and protein composition of purified Caenorhabditis elegans lipid droplets. These lipid droplets are composed mainly of triacylglycerols, surrounded by a phospholipid monolayer composed primarily of phosphatidylcholine and phosphatidylethanolamine. The fatty acid composition of the triacylglycerols is rich in fatty acid species obtained from the dietary Escherichia coli, including cyclopropane fatty acids and cis-vaccenic acid. Unlike other organisms, C. elegans lipid droplets contain very little cholesterol or cholesterol esters. Comparison of the lipid droplet proteomes of wild type and high-fat daf-2 mutant strains shows a very similar proteome in both strains, except that the most abundant protein in the C. elegans lipid droplet proteome, MDT-28, is relatively less abundant in lipid droplets isolated from daf-2 mutants. Functional analysis of lipid droplet proteins identified in our proteomic studies indicated an enrichment of proteins required for growth and fat homeostasis in C. elegans. Finally, we confirmed the localization of one of the newly identified lipid droplet proteins, ACS-4. We found that ACS-4 localizes to the surface of lipid droplets in the C. elegans intestine and skin. This study bolsters C. elegans as a model to study the dynamics and functions of lipid droplets in a multicellular organism.

Stress response pathways protect germ cells from omega-6 polyunsaturated fatty acid-mediated toxicity in Caenorhabditis elegans.

Polyunsaturated fatty acids serve both structural and functional roles as membrane components and precursors for a number of different factors involved in inflammation and signaling. These fatty acids are required in the human diet, although excess dietary intake of omega-6 fatty polyunsaturated fatty acids may have a negative influence on human health. In the model nematode, Caenorhabditis elegans, dietary exposure to dihomo-gamma-linolenic acid (DGLA), an omega-6 fatty acid, causes the destruction of germ cells and leads to sterility. In this study we used genetic and microscopic approaches to further characterize this phenomenon. We found that strains carrying mutations in genes involved in lipid homeostasis enhanced sterility phenotypes, while mutations reducing the activity of the conserved insulin/IGF signaling pathway suppressed sterility phenotypes. Exposure to a mild heat stress prior to omega-6 fatty acid treatment led to an adaptive or hormetic response, resulting in less sterility. Mutations in skn-1 and knockdown of genes encoding phase II detoxification enzymes led to increased sterility in the presence of dietary DGLA. Thus, detoxification systems and genetic changes that increase overall stress responses protect the germ cells from destruction. Microscopic analyses revealed that dietary DGLA leads to deterioration of germ cell membranes in the proliferative and transition zones of the developing germ line. Together, these data demonstrate that specific omega-6 polyunsaturated fatty acids, or molecules derived from them, are transported to the germ line where they disrupt the rapidly expanding germ cell membranes, leading to germ cell death.

Regulation of lipid droplet size and phospholipid composition by stearoyl-CoA desaturase.

Fatty acid desaturation regulates membrane function and fat storage in animals. To determine the contribution of stearoyl-CoA desaturase (SCD) activity on fat storage and development in the nematode Caenorhabditis elegans, we analyzed the lipid composition and lipid droplet size in the fat-6;fat-7 desaturase mutants independently and in combination with mutants disrupted in conserved lipid metabolic pathways. C. elegans with impaired SCD activity displayed both reduced fat stores and decreased lipid droplet size. Mutants in the daf-2 (insulin-like growth factor receptor), rsks-1 (homolog of p70S6 kinase, an effector of the target of rapamycin signaling pathway), and daf-7 (transforming growth factor ß) displayed high fat stores, the opposite of the low fat observed in the fat-6;fat-7 desaturase mutants. The metabolic mutants in combination with fat-6;fat-7 displayed low fat stores, with the exception of the daf-2;fat-6;fat-7 triple mutants, which had increased de novo fatty acid synthesis and wild-type levels of fat stores. Notably, SCD activity is required for the formation of large-sized lipid droplets in all mutant backgrounds, as well as for normal ratios of phosphatidylcholine (PC) to phosphatidylethanolamine (PE). These studies reveal previously uncharacterized roles for SCD in the regulation of lipid droplet size and membrane phospholipid composition.

Polyunsaturated fatty acid derived signaling in reproduction and development: insights from Caenorhabditis elegans and Drosophila melanogaster.

Polyunsaturated fatty acids (PUFAs) exhibit a diverse range of critical functions in biological systems. PUFAs modulate the biophysical properties of membranes and, along with their derivatives, the eicosanoids and endocannabinoids, form a wide array potent lipid signaling molecules. Much of our early understanding of PUFAs and PUFA-derived signaling stems from work in mammals; however, technological advances have made comprehensive lipid analysis possible in small genetic models such as Caenorhabditis elegans and Drosophila melanogaster. These models have a number of advantages, such as simple anatomy and genome-wide genetic screening techniques, which can broaden our understanding of fatty-acid-derived signaling in biological systems. Here we review what is known about PUFAs, eicosanoids, and endocannabinoids in the development and reproduction of C. elegans and D. melanogaster. Fatty acid signaling appears to be fundamental for multicellular organisms, and simple invertebrates often employ functionally similar pathways. In particular, studies in C. elegans and Drosophila are providing insight into the roles of PUFAs and PUFA-derived signaling in early developmental processes, such as meiosis, fertilization, and early embryonic cleavage.

Oocyte signals derived from polyunsaturated fatty acids control sperm recruitment in vivo.

A fundamental question in animal development is how motile cells find their correct target destinations. During mating in the nematode Caenorhabditis elegans, males inject sperm through the hermaphrodite vulva into the uterus. Amoeboid sperm crawl around fertilized eggs to the spermatheca--a convoluted tube where fertilization occurs. Here, we show that polyunsaturated fatty acids (PUFAs), the precursors of eicosanoid signalling molecules, function in oocytes to control directional sperm motility within the uterus. PUFAs are transported from the intestine, the site of fat metabolism, to the oocytes yolk, which is a lipoprotein complex. Loss of the RME-2 low-density lipoprotein (LDL) receptor, which mediates yolk endocytosis and fatty acid transport into oocytes, causes severe defects in sperm targeting. We used an RNAi screen to identify lipid regulators required for directional sperm motility. Our results support the hypothesis that PUFAs function in oocytes as precursors of signals that control sperm recruitment to the spermatheca. A common property of PUFAs in mammals and C. elegans is that these fats control local recruitment of motile cells to their target tissues.

ETS-4 is a transcriptional regulator of life span in Caenorhabditis elegans.

Aging is a complex phenotype responsive to a plethora of environmental inputs; yet only a limited number of transcriptional regulators are known to influence life span. How the downstream expression programs mediated by these factors (or others) are coordinated into common or distinct set of aging effectors is an addressable question in model organisms, such as C. elegans. Here, we establish the transcription factor ETS-4, an ortholog of vertebrate SPDEF, as a longevity determinant. Adult worms with ets-4 mutations had a significant extension of mean life span. Restoring ETS-4 activity in the intestine, but not neurons, of ets-4 mutant worms rescued life span to wild-type levels. Using RNAi, we demonstrated that ets-4 is required post-developmentally to regulate adult life span; thus uncoupling the role of ETS-4 in aging from potential functions in worm intestinal development. Seventy ETS-4-regulated genes, identified by gene expression profiling of two distinct ets-4 alleles and analyzed by bioinformatics, were enriched for known longevity effectors that function in lipid transport, lipid metabolism, and innate immunity. Putative target genes were enriched for ones that change expression during normal aging, the majority of which are controlled by the GATA factors. Also, some ETS-4-regulated genes function downstream of the FOXO factor, DAF-16 and the insulin/IGF-1 signaling pathway. However, epistasis and phenotypic analyses indicate that ets-4 functioned in parallel to the insulin/IGF-1 receptor, daf-2 and akt-1/2 kinases. Furthermore, ets-4 required daf-16 to modulate aging, suggesting overlap in function at the level of common targets that affect life span. In conclusion, ETS-4 is a new transcriptional regulator of aging, which shares transcriptional targets with GATA and FOXO factors, suggesting that overlapping pathways direct common sets of lifespan-related genes.

DNA damage induced MutS homologue hMSH4 acetylation.

Acetylation of non-histone proteins is increasingly recognized as an important post-translational modification for controlling the actions of various cellular processes including DNA repair and damage response. Here, we report that the human MutS homologue hMSH4 undergoes acetylation following DNA damage induced by ionizing radiation (IR). To determine which acetyltransferases are responsible for hMSH4 acetylation in response to DNA damage, potential interactions of hMSH4 with hTip60, hGCN5, and hMof were analyzed. The results of these experiments indicate that only hMof interacts with hMSH4 in a DNA damage-dependent manner. Intriguingly, the interplay between hMSH4 and hMof manipulates the outcomes of nonhomologous end joining (NHEJ)-mediated DNA double strand break (DSB) repair and thereby controls cell survival in response to IR. This study also shows that hMSH4 interacts with HDAC3, by which HDAC3 negatively regulates the levels of hMSH4 acetylation. Interestingly, elevated levels of HDAC3 correlate with increased NHEJ-mediated DSB repair, suggesting that hMSH4 acetylation per se may not directly affect the role of hMSH4 in DSB repair.