Early-life starvation alters lipid metabolism in adults to cause developmental pathology in Caenorhabditis elegans.

Early-life malnutrition increases adult disease risk in humans, but the causal changes in gene regulation, signaling, and metabolism are unclear. In the roundworm Caenorhabditis elegans, early-life starvation causes well-fed larvae to develop germline tumors and other gonad abnormalities as adults. Furthermore, reduced insulin/IGF signaling during larval development suppresses these starvation-induced abnormalities. How early-life starvation and insulin/IGF signaling affect adult pathology is unknown. We show that early-life starvation has pervasive effects on adult gene expression which are largely reversed by reduced insulin/IGF signaling following recovery from starvation. Early-life starvation increases adult fatty-acid synthetase fasn-1 expression in daf-2 insulin/IGF signaling receptor-dependent fashion, and fasn-1/FASN promotes starvation-induced abnormalities. Lipidomic analysis reveals increased levels of phosphatidylcholine in adults subjected to early-life starvation, and supplementation with unsaturated phosphatidylcholine during development suppresses starvation-induced abnormalities. Genetic analysis of fatty-acid desaturases reveals positive and negative effects of desaturation on development of starvation-induced abnormalities. In particular, the ω3 fatty-acid desaturase fat-1 and the Δ5 fatty-acid desaturase fat-4 inhibit and promote development of abnormalities, respectively. fat-4 is epistatic to fat-1, suggesting that arachidonic acid-containing lipids promote development of starvation-induced abnormalities, and supplementation with ARA enhanced development of abnormalities. This work shows that early-life starvation and insulin/IGF signaling converge on regulation of adult lipid metabolism, affecting stem-cell proliferation and tumor formation.

Alternative somatic and germline gene-regulatory strategies during starvation-induced developmental arrest.

Nutrient availability governs growth and quiescence, and many animals arrest development when starved. Using C. elegans L1 arrest as a model, we show that gene expression changes deep into starvation. Surprisingly, relative expression of germline-enriched genes increases for days. We conditionally degrade the large subunit of RNA polymerase II using the auxin-inducible degron system and analyze absolute expression levels. We find that somatic transcription is required for survival, but the germline maintains transcriptional quiescence. Thousands of genes are continuously transcribed in the soma, though their absolute abundance declines, such that relative expression of germline transcripts increases given extreme transcript stability. Aberrantly activating transcription in starved germ cells compromises reproduction, demonstrating important physiological function of transcriptional quiescence. This work reveals alternative somatic and germline gene-regulatory strategies during starvation, with the soma maintaining a robust transcriptional response to support survival and the germline maintaining transcriptional quiescence to support future reproductive success.

Using population selection and sequencing to characterize natural variation of starvation resistance in Caenorhabditis elegans.

Starvation resistance is important to disease and fitness, but the genetic basis of its natural variation is unknown. Uncovering the genetic basis of complex, quantitative traits such as starvation resistance is technically challenging. We developed a synthetic-population (re)sequencing approach using molecular inversion probes (MIP-seq) to measure relative fitness during and after larval starvation in Caenorhabditis elegans. We applied this competitive assay to 100 genetically diverse, sequenced, wild strains, revealing natural variation in starvation resistance. We confirmed that the most starvation-resistant strains survive and recover from starvation better than the most starvation-sensitive strains using standard assays. We performed genome-wide association (GWA) with the MIP-seq trait data and identified three quantitative trait loci (QTL) for starvation resistance, and we created near isogenic lines (NILs) to validate the effect of these QTL on the trait. These QTL contain numerous candidate genes including several members of the Insulin/EGF Receptor-L Domain (irld) family. We used genome editing to show that four different irld genes have modest effects on starvation resistance. Natural variants of irld-39 and irld-52 affect starvation resistance, and increased resistance of the irld-39; irld-52 double mutant depends on daf-16/FoxO. DAF-16/FoxO is a widely conserved transcriptional effector of insulin/IGF signaling (IIS), and these results suggest that IRLD proteins modify IIS, although they may act through other mechanisms as well. This work demonstrates efficacy of using MIP-seq to dissect a complex trait and it suggests that irld genes are natural modifiers of starvation resistance in C. elegans.

Rotenone Modulates Caenorhabditis elegans Immunometabolism and Pathogen Susceptibility.

Mitochondria are central players in host immunometabolism as they function not only as metabolic hubs but also as signaling platforms regulating innate immunity. Environmental exposures to mitochondrial toxicants occur widely and are increasingly frequent. Exposures to these mitotoxicants may pose a serious threat to organismal health and the onset of diseases by disrupting immunometabolic pathways. In this study, we investigated whether the Complex I inhibitor rotenone could alter C. elegans immunometabolism and disease susceptibility. C. elegans embryos were exposed to rotenone (0.5 µM) or DMSO (0.125%) until they reached the L4 larval stage. Inhibition of mitochondrial respiration by rotenone and disruption of mitochondrial metabolism were evidenced by rotenone-induced detrimental effects on mitochondrial efficiency and nematode growth and development. Next, through transcriptomic analysis, we investigated if this specific but mild mitochondrial stress that we detected would lead to the modulation of immunometabolic pathways. We found 179 differentially expressed genes (DEG), which were mostly involved in detoxification, energy metabolism, and pathogen defense. Interestingly, among the down-regulated DEG, most of the known genes were involved in immune defense, and most of these were identified as commonly upregulated during P. aeruginosa infection. Furthermore, rotenone increased susceptibility to the pathogen Pseudomonas aeruginosa (PA14). However, it increased resistance to Salmonella enterica (SL1344). To shed light on potential mechanisms related to these divergent effects on pathogen resistance, we assessed the activation of the mitochondrial unfolded protein response (UPRmt), a well-known immunometabolic pathway in C. elegans which links mitochondria and immunity and provides resistance to pathogen infection. The UPRmt pathway was activated in rotenone-treated nematodes further exposed for 24 h to the pathogenic bacteria P. aeruginosa and S. enterica or the common bacterial food source Escherichia coli (OP50). However, P. aeruginosa alone suppressed UPRmt activation and rotenone treatment rescued its activation only to the level of DMSO-exposed nematodes fed with E. coli. Module-weighted annotation bioinformatics analysis was also consistent with UPRmt activation in rotenone-exposed nematodes consistent with the UPR being involved in the increased resistance to S. enterica. Together, our results demonstrate that the mitotoxicant rotenone can disrupt C. elegans immunometabolism in ways likely protective against some pathogen species but sensitizing against others.

Chromosome-Level Reference Genomes for Two Strains of Caenorhabditis briggsae: An Improved Platform for Comparative Genomics.

The publication of the Caenorhabditis briggsae reference genome in 2003 enabled the first comparative genomics studies between C. elegans and C. briggsae, shedding light on the evolution of genome content and structure in the Caenorhabditis genus. However, despite being widely used, the currently available C. briggsae reference genome is substantially less complete and structurally accurate than the C. elegans reference genome. Here, we used high-coverage Oxford Nanopore long-read and chromosome-conformation capture data to generate chromosome-level reference genomes for two C. briggsae strains: QX1410, a new reference strain closely related to the laboratory AF16 strain, and VX34, a highly divergent strain isolated in China. We also sequenced 99 recombinant inbred lines generated from reciprocal crosses between QX1410 and VX34 to create a recombination map and identify chromosomal domains. Additionally, we used both short- and long-read RNA sequencing data to generate high-quality gene annotations. By comparing these new reference genomes to the current reference, we reveal that hyper-divergent haplotypes cover large portions of the C. briggsae genome, similar to recent reports in C. elegans and C. tropicalis. We also show that the genomes of selfing Caenorhabditis species have undergone more rearrangement than their outcrossing relatives, which has biased previous estimates of rearrangement rate in Caenorhabditis. These new genomes provide a substantially improved platform for comparative genomics in Caenorhabditis and narrow the gap between the quality of genomic resources available for C. elegans and C. briggsae.

DAF-18/PTEN inhibits germline zygotic gene activation during primordial germ cell quiescence.

Quiescence, an actively-maintained reversible state of cell cycle arrest, is not well understood. PTEN is one of the most frequently lost tumor suppressors in human cancers and regulates quiescence of stem cells and cancer cells. The sole PTEN ortholog in Caenorhabditis elegans is daf-18. In a C. elegans loss-of-function mutant for daf-18, primordial germ cells (PGCs) divide inappropriately in L1 larvae hatched into starvation conditions, in a TOR-dependent manner. Here, we further investigated the role of daf-18 in maintaining PGC quiescence in L1 starvation. We found that maternal or zygotic daf-18 is sufficient to maintain cell cycle quiescence, that daf-18 acts in the germ line and soma, and that daf-18 affects timing of PGC divisions in fed animals. Importantly, our results also implicate daf-18 in repression of germline zygotic gene activation, though not in germline fate specification. However, TOR is less important to germline zygotic gene expression, suggesting that in the absence of food, daf-18/PTEN prevents inappropriate germline zygotic gene activation and cell division by distinct mechanisms.

Selfing is the safest sex for Caenorhabditis tropicalis.

Mating systems have profound effects on genetic diversity and compatibility. The convergent evolution of self-fertilization in three Caenorhabditis species provides a powerful lens to examine causes and consequences of mating system transitions. Among the selfers, Caenorhabditis tropicalis is the least genetically diverse and most afflicted by outbreeding depression. We generated a chromosomal-scale genome for C. tropicalis and surveyed global diversity. Population structure is very strong, and islands of extreme divergence punctuate a genomic background that is highly homogeneous around the globe. Outbreeding depression in the laboratory is caused largely by multiple Medea-like elements, genetically consistent with maternal toxin/zygotic antidote systems. Loci with Medea activity harbor novel and duplicated genes, and their activity is modified by mito-nuclear background. Segregating Medea elements dramatically reduce fitness, and simulations show that selfing limits their spread. Frequent selfing in C. tropicalis may therefore be a strategy to avoid Medea-mediated outbreeding depression.

Reactive oxygen species production by BP-1,6-quinone and its effects on the endothelial dysfunction: Involvement of the mitochondria.

Strong epidemiological evidence supports the association between increased air pollution and the risk of developing atherosclerotic cardiovascular diseases (CVDs). However, the mechanism remains unclear. As an environmental air pollutant and benzo-a-pyrene (BP) metabolite, BP-1,6-quinone (BP-1,6-Q) is present in the particulate phase of air pollution. This study was undertaken to examine the redox activity of BP-1,6-Q and mechanisms associated with it using EA.hy926 endothelial cells. BP-1,6-Q at 0.01-1 µM significantly stimulated the production of reactive oxygen species (ROS)·in intact cells and isolated mitochondria. Furthermore, BP-1,6-Q-induced ROS was altered by mitochondrial electron transport chain (METC) inhibitors of complex I (rotenone) and complex III (antimycin A), denoting the involvement of mitochondrial electron transport chain (METC) in BP-1,6-Q mediated ROS production. In METC deficient cells, interestingly, BP-1,6-Q-mediated ROS production was enhanced, suggesting that overproduction of ROS by BP-1,6-Q is not only produced from mitochondria but can also be from the cell outside of mitochondria (extramitochondrial). BP-1,6-Q also triggered endothelial-monocyte interaction and stimulated expression of vascular adhesion molecule-1 (VCAM-1). In conclusion, these results demonstrate that BP-1,6-Q can generate ROS within both mitochondria and outside of mitochondria, resulting in stimulation of adhesion of monocytes to endothelial cells, a key event in the pathogenesis of atherosclerosis.

Insulin/IGF Signaling and Vitellogenin Provisioning Mediate Intergenerational Adaptation to Nutrient Stress.

The roundworm C. elegans reversibly arrests larval development during starvation [1], but extended early-life starvation reduces reproductive success [2, 3]. Maternal dietary restriction (DR) buffers progeny from starvation as young larvae, preserving reproductive success [4]. However, the developmental basis of reduced fertility following early-life starvation is unknown, and it is unclear how maternal diet modifies developmental physiology in progeny. We show here that extended starvation in first-stage (L1) larvae followed by unrestricted feeding results in a variety of developmental abnormalities in the reproductive system, including proliferative germ-cell tumors and uterine masses that express neuronal and epidermal cell fate markers. We found that maternal DR and reduced maternal insulin/insulin-like growth factor (IGF) signaling (IIS) increase oocyte provisioning of vitellogenin lipoprotein, reducing penetrance of starvation-induced abnormalities in progeny, including tumors. Furthermore, we show that maternal DR and reduced maternal IIS reduce IIS in progeny. daf-16/FoxO and skn-1/Nrf, transcriptional effectors of IIS, are required in progeny for maternal DR and increased vitellogenin provisioning to suppress starvation-induced abnormalities. daf-16/FoxO activity in somatic tissues is sufficient to suppress starvation-induced abnormalities, suggesting cell-nonautonomous regulation of reproductive system development. This work reveals that early-life starvation compromises reproductive development and that vitellogenin-mediated intergenerational insulin/IGF-to-insulin/IGF signaling mediates adaptation to nutrient availability.

Food perception without ingestion leads to metabolic changes and irreversible developmental arrest in C. elegans.

BACKGROUND: Developmental physiology is very sensitive to nutrient availability. For instance, in the nematode Caenorhabditis elegans, newly hatched L1-stage larvae require food to initiate postembryonic development. In addition, larvae arrested in the dauer diapause, a non-feeding state of developmental arrest that occurs during the L3 stage, initiate recovery when exposed to food. Despite the essential role of food in C. elegans development, the contribution of food perception versus ingestion on physiology has not been delineated. RESULTS: We used a pharmacological approach to uncouple the effects of food (bacteria) perception and ingestion in C. elegans. Perception was not sufficient to promote postembryonic development in L1-stage larvae. However, L1 larvae exposed to food without ingestion failed to develop upon return to normal culture conditions, instead displaying an irreversible arrest phenotype. Inhibition of gene expression during perception rescued subsequent development, demonstrating that the response to perception without feeding is deleterious. Perception altered DAF-16/FOXO subcellular localization, reflecting activation of insulin/IGF signaling (IIS). The insulin-like peptide daf-28 was specifically required, suggesting perception in chemosensory neurons, where it is expressed, regulates peptide synthesis and possibly secretion. However, genetic manipulation of IIS did not modify the irreversible arrest phenotype caused by food perception, revealing that wild-type function of the IIS pathway is not required to produce this phenotype and that other pathways affected by perception of food in the absence of its ingestion are likely to be involved. Gene expression and Nile red staining showed that food perception could alter lipid metabolism and storage. We found that starved larvae sense environmental polypeptides, with similar molecular and developmental effects as perception of bacteria. Environmental polypeptides also promoted recovery from dauer diapause, suggesting that perception of polypeptides plays an important role in the life history of free-living nematodes. CONCLUSIONS: We conclude that actual ingestion of food is required to initiate postembryonic development in C. elegans. We also conclude that polypeptides are perceived as a food-associated cue in this and likely other animals, initiating a signaling and gene regulatory cascade that alters metabolism in anticipation of feeding and development, but that this response is detrimental if feeding does not occur.

Transgenerational Effects of Extended Dauer Diapause on Starvation Survival and Gene Expression Plasticity in Caenorhabditis elegans.

Phenotypic plasticity is facilitated by epigenetic regulation, and remnants of such regulation may persist after plasticity-inducing cues are gone. However, the relationship between plasticity and transgenerational epigenetic memory is not understood. Dauer diapause in Caenorhabditis elegans provides an opportunity to determine how a plastic response to the early-life environment affects traits later in life and in subsequent generations. We report that, after extended diapause, postdauer worms initially exhibit reduced reproductive success and greater interindividual variation. In contrast, F3 progeny of postdauers display increased starvation resistance and lifespan, revealing potentially adaptive transgenerational effects. Transgenerational effects are dependent on the duration of diapause, indicating an effect of extended starvation. In agreement, RNA-seq demonstrates a transgenerational effect on nutrient-responsive genes. Further, postdauer F3 progeny exhibit reduced gene expression plasticity, suggesting a trade-off between plasticity and epigenetic memory. This work reveals complex effects of nutrient stress over different time scales in an animal that evolved to thrive in feast and famine.

daf-16/FoxO promotes gluconeogenesis and trehalose synthesis during starvation to support survival.

daf-16/FoxO is required to survive starvation in Caenorhabditis elegans, but how daf-16IFoxO promotes starvation resistance is unclear. We show that daf-16/FoxO restructures carbohydrate metabolism by driving carbon flux through the glyoxylate shunt and gluconeogenesis and into synthesis of trehalose, a disaccharide of glucose. Trehalose is a well-known stress protectant, capable of preserving membrane organization and protein structure during abiotic stress. Metabolomic, genetic, and pharmacological analyses confirm increased trehalose synthesis and further show that trehalose not only supports survival as a stress protectant but also serves as a glycolytic input. Furthermore, we provide evidence that metabolic cycling between trehalose and glucose is necessary for this dual function of trehalose. This work demonstrates that daf-16/FoxO promotes starvation resistance by shifting carbon metabolism to drive trehalose synthesis, which in turn supports survival by providing an energy source and acting as a stress protectant.

Luteolin protects against vascular inflammation in mice and TNF-alpha-induced monocyte adhesion to endothelial cells via suppressing IΚBα/NF-κB signaling pathway.

Vascular inflammation plays a significant role in the pathogenesis of atherosclerosis. Luteolin, a naturally occurring flavonoid present in many medicinal plants and some commonly consumed fruits and vegetables, has received wide attention for its potential to improve vascular function in vitro. However, its effect in vivo and the molecular mechanism of luteolin at physiological concentrations remain unclear. Here, we report that luteolin as low as 0.5 µM significantly inhibited tumor necrosis factor (TNF)-α-induced adhesion of monocytes to human EA.hy 926 endothelial cells, a key event in triggering vascular inflammation. Luteolin potently suppressed TNF-α-induced expression of the chemokine monocyte chemotactic protein-1 (MCP-1) and adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), key mediators involved in enhancing endothelial cell-monocyte interaction. Furthermore, luteolin inhibited TNF-α-induced nuclear factor (NF)-κB transcriptional activity, IκBα degradation, expression of IκB kinase ß and subsequent NF-κB p65 nuclear translocation in endothelial cells, suggesting that luteolin can inhibit inflammation by suppressing NF-κB signaling. In an animal study, C57BL/6 mice were fed a diet containing 0% or 0.6% luteolin for 3 weeks, and luteolin supplementation greatly suppressed TNF-α-induced increase in circulating levels of MCP-1/JE, CXCL1/KC and sICAM-1 in C57BL/6 mice. Consistently, dietary intake of luteolin significantly reduced TNF-α-stimulated adhesion of monocytes to aortic endothelial cells ex vivo. Histology shows that luteolin treatment prevented the eruption of endothelial lining in the intima layer of the aorta and preserved elastin fibers' delicate organization as shown by Verhoeff-Van Gieson staining. Immunohistochemistry studies further show that luteolin treatment also reduced VCAM-1 and monocyte-derived F4/80-positive macrophages in the aorta of TNF-α-treated mice. In conclusion, luteolin protects against TNF-α-induced vascular inflammation in both in vitro and in vivo models. This anti-inflammatory effect of luteolin may be mediated via inhibition of the NF-κB-mediated pathway.