The nuclear receptor DAF-12 regulates nutrient metabolism and reproductive growth in nematodes.

Appropriate nutrient response is essential for growth and reproduction. Under favorable nutrient conditions, the C. elegans nuclear receptor DAF-12 is activated by dafachronic acids, hormones that commit larvae to reproductive growth. Here, we report that in addition to its well-studied role in controlling developmental gene expression, the DAF-12 endocrine system governs expression of a gene network that stimulates the aerobic catabolism of fatty acids. Thus, activation of the DAF-12 transcriptome coordinately mobilizes energy stores to permit reproductive growth. DAF-12 regulation of this metabolic gene network is conserved in the human parasite, Strongyloides stercoralis, and inhibition of specific steps in this network blocks reproductive growth in both of the nematodes. Our study provides a molecular understanding for metabolic adaptation of nematodes to their environment, and suggests a new therapeutic strategy for treating parasitic diseases.

NHX-5, an Endosomal Na+/H+ Exchanger, Is Associated with Metformin Action.

Diabetes is one of the most impactful diseases worldwide. The most commonly prescribed anti-diabetic drug is metformin. In this study, we identified an endosomal Na(+)/H(+) exchanger (NHE) as a new potential target of metformin from an unbiased screen in Caenorhabditis elegans The same NHE homolog also exists in flies, where it too mediates the effects of metformin. Our results suggest that endosomal NHEs could be a metformin target and provide an insight into a novel mechanism of action of metformin on regulating the endocytic cycle.

Regulation of organelle function by metformin.

Metformin ameliorates hyperglycemia without the side effects of lactic acidosis or hypoglycemia. Metformin lowers the blood glucose level by decreasing hepatic glucose production in the liver and by increasing glucose uptake in the muscle. Recent studies show that metformin induces cell death in certain cancer cell lines by interfering with the metabolism of the cancer cells. Therefore, understanding the mechanisms of action for metformin will provide insights into how to better treat diabetes and other metabolic disorders and also into the development of new therapeutic drugs. One of the best understood molecular targets of metformin is the mitochondrial complex I. However, given metformin's broad effects on metabolism, it could act on multiple targets. In this review, we summarize current findings in metformin's mechanisms of action regarding its known targets in mitochondria and known effects in cancer cell lines. Then, we introduce endosomal Na+ /H+ exchangers and the V-ATPase as new potential targets of metformin's action. Finally, we will discuss the hypothesis that metformin directly acts on endosome/lysosome regulation so as to regulate metabolism and ultimately alleviate type 2 diabetes. © 2017 IUBMB Life, 69(7):459-469, 2017.

ASI regulates satiety quiescence in C. elegans.

In Caenorhabditis elegans, satiety quiescence mimics behavioral aspects of satiety and postprandial sleep in mammals. On the basis of calcium-imaging, genetics, and behavioral studies, here we report that a pair of amphid neurons, ASI, is activated by nutrition and regulates worms' behavioral states specifically promoting satiety quiescence; ASI inhibits the switch from quiescence to dwelling (a browsing state) and accelerates the switch from dwelling to quiescence. The canonical TGFß pathway, whose ligand is released from ASI, regulates satiety quiescence. The mutants of a ligand, a receptor and SMADs in the TGFß pathway all eat more and show less quiescence than wild-type. The TGFß receptor in downstream neurons RIM and RIC is sufficient for worms to exhibit satiety quiescence, suggesting neuronal connection from ASI to RIM and RIC is essential for feeding regulation through the TGFß pathway. ASI also regulates satiety quiescence partly through cGMP signaling; restoring cGMP signaling in ASI rescues the satiety quiescence defect of cGMP signaling mutants. From these results, we propose that TGFß and cGMP pathways in ASI connect nutritional status to promotion of satiety quiescence, a sleep-like behavioral state.

Lethargus is a Caenorhabditis elegans sleep-like state.

There are fundamental similarities between sleep in mammals and quiescence in the arthropod Drosophila melanogaster, suggesting that sleep-like states are evolutionarily ancient. The nematode Caenorhabditis elegans also has a quiescent behavioural state during a period called lethargus, which occurs before each of the four moults. Like sleep, lethargus maintains a constant temporal relationship with the expression of the C. elegans Period homologue LIN-42 (ref. 5). Here we show that quiescence associated with lethargus has the additional sleep-like properties of reversibility, reduced responsiveness and homeostasis. We identify the cGMP-dependent protein kinase (PKG) gene egl-4 as a regulator of sleep-like behaviour, and show that egl-4 functions in sensory neurons to promote the C. elegans sleep-like state. Conserved effects on sleep-like behaviour of homologous genes in C. elegans and Drosophila suggest a common genetic regulation of sleep-like states in arthropods and nematodes. Our results indicate that C. elegans is a suitable model system for the study of sleep regulation. The association of this C. elegans sleep-like state with developmental changes that occur with larval moults suggests that sleep may have evolved to allow for developmental changes.

NSBP-1 mediates the effects of cholesterol on insulin/IGF-1 signaling in Caenorhabditis elegans.

Nematode sterol-binding protein 1 (NSBP-1) is a homolog of nucleosome assembly protein 1 in mammals that is expressed widely in Caenorhabditis elegans. NSBP-1 mutants are biologically lethal, demonstrating the significance of the gene in growth and development. We investigated how cholesterol influences the insulin signaling pathway through this novel sterol-binding protein in C. elegans. Here we report that NSBP-1 influences many biological processes mediated by insulin signaling, such as longevity, dauer formation, fat storage, and resistance to oxidative stress. We found that NSBP-1 is phosphorylated by AKT-1 downstream of insulin signaling. In the absence of insulin signaling, NSBP-1 is translocated to the nucleus and binds to DAF-16, a FOXO transcription factor, in a cholesterol-dependent manner. Moreover, NSBP-1 and DAF-16 regulate a common set of genes that can directly modulate fat storage, longevity, and resistance to stress. Together, our results present a new steroid-binding molecule that can connect sterol signaling to insulin signaling through direct interaction with FOXO.

Food preference-based screening method for identification of effectors of substance use disorders using Caenorhabditis elegans.

Substance use disorder (SUD) affects over 48 million Americans aged 12 and over. Thus, identifying novel chemicals contributing to SUD will be critical for developing efficient prevention and mitigation strategies. Considering the complexity of the actions and effects of these substances on human behavior, a high-throughput platform using a living organism is ideal. We developed a quick and easy screening assay using Caenorhabditis elegans. C. elegans prefers high-quality food (Escherichia coli HB101) over low-quality food (Bacillus megaterium), with a food preference index of approximately 0.2, defined as the difference in the number of worms at E. coli HB101 and B. megaterium over the total worm number. The food preference index was significantly increased by loperamide, a µ-opioid receptor (MOPR) agonist, and decreased by naloxone, a MOPR antagonist. These changes depended on npr-17, a C. elegans homolog of opioid receptors. In addition, the food preference index was significantly increased by arachidonyl-2'-chloroethylamide, a cannabinoid 1 receptor (CB1R) agonist, and decreased by rimonabant, a CB1R inverse agonist. These changes depended on npr-19, a homolog of CB1R. These results suggest that the conserved opioid and endocannabinoid systems modulate the food preference behaviors of C. elegans. Finally, the humanoid C. elegans strains where npr-17 was replaced with human MOPR and where npr-19 was replaced with human CB1R phenocopied the changes in food preference by the drug treatment. Together, the current results show that this method can be used to rapidly screen the potential effectors of MOPR and CB1R to yield results highly translatable to humans.

Neuronal E93 is required for adaptation to adult metabolism and behavior.

OBJECTIVE: Metamorphosis is a transition from growth to reproduction, through which an animal adopts adult behavior and metabolism. Yet the neural mechanisms underlying the switch are unclear. Here we report that neuronal E93, a transcription factor essential for metamorphosis, regulates the adult metabolism, physiology, and behavior in Drosophila melanogaster. METHODS: To find new neuronal regulators of metabolism, we performed a targeted RNAi-based screen of 70 Drosophila orthologs of the mammalian genes enriched in ventromedial hypothalamus (VMH). Once E93 was identified from the screen, we characterized changes in physiology and behavior when neuronal expression of E93 is knocked down. To identify the neurons where E93 acts, we performed an additional screen targeting subsets of neurons or endocrine cells. RESULTS: E93 is required to control appetite, metabolism, exercise endurance, and circadian rhythms. The diverse phenotypes caused by pan-neuronal knockdown of E93, including obesity, exercise intolerance and circadian disruption, can all be phenocopied by knockdown of E93 specifically in either GABA or MIP neurons, suggesting these neurons are key sites of E93 action. Knockdown of the Ecdysone Receptor specifically in MIP neurons partially phenocopies the MIP neuron-specific knockdown of E93, suggesting the steroid signal coordinates adult metabolism via E93 and a neuropeptidergic signal. Finally, E93 expression in GABA and MIP neurons also serves as a key switch for the adaptation to adult behavior, as animals with reduced expression of E93 in the two subsets of neurons exhibit reduced reproductive activity. CONCLUSIONS: Our study reveals that E93 is a new monogenic factor essential for metabolic, physiological, and behavioral adaptation from larval behavior to adult behavior.

Insulin, cGMP, and TGF-beta signals regulate food intake and quiescence in C. elegans: a model for satiety.

Despite the prevalence of obesity and its related diseases, the signaling pathways for appetite control and satiety are not clearly understood. Here we report C. elegans quiescence behavior, a cessation of food intake and movement that is possibly a result of satiety. C. elegans quiescence shares several characteristics of satiety in mammals. It is induced by high-quality food, it requires nutritional signals from the intestine, and it depends on prior feeding history: fasting enhances quiescence after refeeding. During refeeding after fasting, quiescence is evoked, causing gradual inhibition of food intake and movement, mimicking the behavioral sequence of satiety in mammals. Based on these similarities, we propose that quiescence results from satiety. This hypothesized satiety-induced quiescence is regulated by peptide signals such as insulin and TGF-beta. The EGL-4 cGMP-dependent protein kinase functions downstream of insulin and TGF-beta in sensory neurons including ASI to control quiescence in response to food intake.

Oocyte Quiescence: From Formation to Awakening.

Decades of work using various model organisms have resulted in an exciting and emerging field of oocyte maturation. High levels of insulin and active mammalian target of rapamycin signals, indicative of a good nutritional environment, and hormones such as gonadotrophin, indicative of the growth of the organism, work together to control oocyte maturation to ensure that reproduction happens at the right timing under the right conditions. In the wild, animals often face serious challenges to maintain oocyte quiescence under long-term unfavorable conditions in the absence of mates or food. Failure to maintain oocyte quiescence will result in activation of oocytes at the wrong time and thus lead to exhaustion of the oocyte pool and sterility of the organism. In this review, we discuss the shared mechanisms in oocyte quiescence and awakening and a conserved role of noradrenergic signals in maintenance of the quiescent oocyte pool under unfavorable conditions in simple model organisms.

Starvation activates MAP kinase through the muscarinic acetylcholine pathway in Caenorhabditis elegans pharynx.

Starvation activates MAPK in the pharyngeal muscles of C. elegans through a muscarinic acetylcholine receptor, Gqalpha, and nPKC as shown by the following results: (1) Starvation causes phosphorylation of MAPK in pharyngeal muscle. (2) In a sensitized genetic background in which Gqalpha signaling cannot be downregulated, activation of the pathway by a muscarinic agonist causes lethal changes in pharyngeal muscle function. Starvation has identical effects. (3) A muscarinic antagonist blocks the effects of starvation on sensitized muscle. (4) Mutations and drugs that block any step of signaling from the muscarinic receptor to MAPK also block the effects of starvation on sensitized muscle. (5) Overexpression of MAPK in wild-type pharyngeal muscle mimics the effects of muscarinic agonist and of starvation on sensitized muscle. We suggest that, during starvation, the muscarinic pathway to MAPK is activated to change the pharyngeal muscle physiology to enhance ingestion of food when food becomes available.

Regulation of Satiety Quiescence by Neuropeptide Signaling in Caenorhabditis elegans.

Sleep and metabolism are interconnected homeostatic states; the sleep cycle can be entrained by the feeding cycle, and perturbation of the sleep often results in dysregulation in metabolism. However, the neuro-molecular mechanism by which metabolism regulates sleep is not fully understood. We investigated how metabolism and feeding regulate sleep using satiety quiescence behavior as a readout in Caenorhabditis elegans, which shares certain key aspects of postprandial sleep in mammals. From an RNA interference-based screen of two neuropeptide families, RFamide-related peptides (FLPs) and insulin-like peptides (INSs), we identified flp-11, known to regulate other types of sleep-like behaviors in C. elegans, as a gene that plays the most significant role in satiety quiescence. A mutation in flp-11 significantly reduces quiescence, whereas over-expression of the gene enhances it. A genetic analysis shows that FLP-11 acts upstream of the cGMP signaling but downstream of the TGFß pathway, suggesting that TGFß released from a pair of head sensory neurons (ASI) activates FLP-11 in an interneuron (RIS). Then, cGMP signaling acting in downstream of RIS neurons induces satiety quiescence. Among the 28 INSs genes screened, ins-1, known to play a significant role in starvation-associated behavior working in AIA is inhibitory to satiety quiescence. Our study suggests that specific combinations of neuropeptides are released, and their signals are integrated in order for an animal to gauge its metabolic state and to control satiety quiescence, a feeding-induced sleep-like state in C. elegans.

Maintenance of quiescent oocytes by noradrenergic signals.

All females adopt an evolutionary conserved reproduction strategy; under unfavorable conditions such as scarcity of food or mates, oocytes remain quiescent. However, the signals to maintain oocyte quiescence are largely unknown. Here, we report that in four different species - Caenorhabditis elegans, Caenorhabditis remanei, Drosophila melanogaster, and Danio rerio - octopamine and norepinephrine play an essential role in maintaining oocyte quiescence. In the absence of mates, the oocytes of Caenorhabditis mutants lacking octopamine signaling fail to remain quiescent, but continue to divide and become polyploid. Upon starvation, the egg chambers of D. melanogaster mutants lacking octopamine signaling fail to remain at the previtellogenic stage, but grow to full-grown egg chambers. Upon starvation, D. rerio lacking norepinephrine fails to maintain a quiescent primordial follicle and activates an excessive number of primordial follicles. Our study reveals an evolutionarily conserved function of the noradrenergic signal in maintaining quiescent oocytes.

Behavioral States.

Caenorhabditis elegans' behavioral states, like those of other animals, are shaped by its immediate environment, its past experiences, and by internal factors. We here review the literature on C. elegans behavioral states and their regulation. We discuss dwelling and roaming, local and global search, mate finding, sleep, and the interaction between internal metabolic states and behavior.

The EGL-4 PKG acts with KIN-29 salt-inducible kinase and protein kinase A to regulate chemoreceptor gene expression and sensory behaviors in Caenorhabditis elegans.

The regulation of chemoreceptor (CR) gene expression by environmental signals and internal cues may contribute to the modulation of multiple physiological processes and behavior in Caenorhabditis elegans. We previously showed that KIN-29, a homolog of salt-inducible kinase, acts in sensory neurons to regulate the expression of a subset of CR genes, as well as sensory behaviors. Here we show that the cGMP-dependent protein kinase EGL-4 acts partly in parallel with KIN-29 to regulate CR gene expression. Sensory inputs inhibit both EGL-4 and KIN-29 functions, and KIN-29 function is inhibited in turn by cAMP-dependent protein kinase (PKA) activation. EGL-4 and KIN-29 regulate CR gene expression by antagonizing the gene repression functions of the class II HDAC HDA-4 and the MEF-2 transcription factor, and KIN-29, EGL-4, and PKA target distinct residues in HDA-4 to regulate its function and subcellular localization. While KIN-29 acts primarily via MEF-2/HDA-4 to regulate additional sensory signal-regulated physiological processes and behaviors, EGL-4 acts via both MEF-2-dependent and -independent pathways. Our results suggest that integration of complex sensory inputs via multiple signaling pathways allows animals to precisely regulate sensory gene expression, thereby appropriately modulating physiology and behavior.

Satiety behavior is regulated by ASI/ASH reciprocal antagonism.

Appropriate decision-making is essential for ensuring survival; one such decision is whether to eat. Overall metabolic state and the safety of food are the two factors we examined using C. elegans to ask whether the metabolic state regulates neuronal activities and corresponding feeding behavior. We monitored the activity of sensory neurons that are activated by nutritious (or appetitive) stimuli (ASI) and aversive stimuli (ASH) in starved vs. well-fed worms during stimuli presentation. Starvation reduces ASH activity to aversive stimuli while increasing ASI activity to nutritious stimuli, showing the responsiveness of each neuron is modulated by overall metabolic state. When we monitored satiety quiescence behavior that reflects the overall metabolic state, ablation of ASI and ASH produce the opposite behavior, showing the two neurons interact to control the decision to eat or not. This circuit provides a simple approach to how neurons handle sensory conflict and reach a decision that is translated to behavior.

BLIMP-1/BLMP-1 and Metastasis-Associated Protein Regulate Stress Resistant Development in Caenorhabditis elegans.

Environmental stress triggers multilevel adaptations in animal development that depend in part on epigenetic mechanisms. In response to harsh environmental conditions and pheromone signals, Caenorhabditis elegans larvae become the highly stress-resistant and long-lived dauer. Despite extensive studies of dauer formation pathways that integrate specific environmental cues and appear to depend on transcriptional reprogramming, the role of epigenetic regulation in dauer development has remained unclear. Here we report that BLMP-1, the BLIMP-1 ortholog, regulates dauer formation via epigenetic pathways; in the absence of TGF-ß signaling (in daf-7 mutants), lack of blmp-1 caused lethality. Using this phenotype, we screened 283 epigenetic factors, and identified lin-40, a homolog of metastasis-associate protein 1 (MTA1) as an interactor of BLMP-1 The interaction between LIN-40 and BLMP-1 is conserved because mammalian homologs for both MTA1 and BLIMP-1 could also interact. From microarray studies, we identified several downstream target genes of blmp-1: npr-3, nhr-23, ptr-4, and sams-1 Among them S-adenosyl methionine synthase (SAMS-1), is the key enzyme for production of SAM used in histone methylation. Indeed, blmp-1 is necessary for controlling histone methylation level in daf-7 mutants, suggesting BLMP-1 regulates the expression of SAMS-1, which in turn may regulate histone methylation and dauer formation. Our results reveal a new interaction between BLMP-1/BLIMP-1 and LIN-40/MTA1, as well as potential epigenetic downstream pathways, whereby these proteins cooperate to regulate stress-specific developmental adaptations.

Fat Metabolism Regulates Satiety Behavior in C. elegans.

Animals change feeding behavior depending on their metabolic status; starved animals are eager to eat and satiated animals stop eating. C. elegans exhibits satiety quiescence under certain conditions that mimics many aspects of post-prandial sleep in mammals. Here we show that this feeding behavior depends on fat metabolism mediated by the SREBP-SCD pathway, an acetyl-CoA carboxylase (ACC) and certain nuclear hormone receptors (NRs). Mutations of the genes in the SREBP-SCD pathway reduce satiety quiescence. An RNA interference (RNAi) screen of the genes that regulate glucose and fatty acid metabolism identified an ACC necessary for satiety quiescence in C. elegans. ACC catalyzes the first step in de novo fatty acid biosynthesis known to be downstream of the SREBP pathway in mammals. We identified 28 NRs by microarray whose expression changes during refeeding after being starved. When individually knocked down by RNAi, 11 NRs among 28 affect both fat storage and satiety behavior. Our results show that the major fat metabolism pathway regulates feeding behavior and NRs could be the mediators to link the feeding behavior to the metabolic changes.

An opioid-like system regulating feeding behavior in C. elegans.

Neuropeptides are essential for the regulation of appetite. Here we show that neuropeptides could regulate feeding in mutants that lack neurotransmission from the motor neurons that stimulate feeding muscles. We identified nlp-24 by an RNAi screen of 115 neuropeptide genes, testing whether they affected growth. NLP-24 peptides have a conserved YGGXX sequence, similar to mammalian opioid neuropeptides. In addition, morphine and naloxone respectively stimulated and inhibited feeding in starved worms, but not in worms lacking NPR-17, which encodes a protein with sequence similarity to opioid receptors. Opioid agonists activated heterologously expressed NPR-17, as did at least one NLP-24 peptide. Worms lacking the ASI neurons, which express npr-17, did not response to naloxone. Thus, we suggest that Caenorhabditis elegans has an endogenous opioid system that acts through NPR-17, and that opioids regulate feeding via ASI neurons. Together, these results suggest C. elegans may be the first genetically tractable invertebrate opioid model.

A HIF-independent mediator of transcriptional responses to oxygen deprivation in Caenorhabditis elegans.

The adaptive response to hypoxia is accompanied by widespread transcriptional changes that allow for prolonged survival in low oxygen. Many of these changes are directly regulated by the conserved hypoxia-inducible factor-1 (HIF-1) complex; however, even in its absence, many oxygen-sensitive transcripts in Caenorhabditis elegans are appropriately regulated in hypoxia. To identify mediators of these non-HIF-dependent responses, we established a hif-1 mutant reporter line that expresses GFP in hypoxia or when worms are treated with the hypoxia mimetic cobalt chloride (CoCl2). The reporter is selective and HIF independent, in that it remains insensitive to a number of cellular stresses, but is unaffected by mutation of the prolyl hydroxylase egl-9, suggesting that the regulators of this response pathway are different from those controlling the HIF pathway. We used the HIF-independent reporter to screen a transcription factor RNA interference (RNAi) library and identified genes that are required for hypoxia-sensitive and CoCl2-induced GFP expression. We identified the zinc finger protein BLMP-1 as a mediator of the HIF-independent response. We show that mutation of blmp-1 renders animals sensitive to hypoxic exposure and that blmp-1 is required for appropriate hypoxic-induced expression of HIF-independent transcripts. Further, we demonstrate that BLMP-1 is necessary for an increase of hypoxia-dependent histone acetylation within the promoter of a non-HIF-dependent hypoxia response gene.