quantification-lipid-abundance-evaluation-lipid-distribution

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in <em>Caenorhabditis elegans</em> by Nile Red and Oil Red O Staining

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

University of Southern California

A recent study reported that longevity in Caenorhabditits elegans can be inherited over several generations. This is probably achieved through the following epigenetic mechanism: inherited demethylated histones at some central loci, such as miRNA, transcription factors or signaling regulators affect the expression of certain genes leading to the longevity phenotype.

Longevity and the long arm of epigenetics: acquired parental marks influence lifespan across several generations.

SKN-1/Nrf plays multiple essential roles in development and cellular homeostasis. We demonstrate that SKN-1 executes a specific and appropriate transcriptional response to changes in available nutrients, leading to metabolic adaptation. We isolated gain-of-function (gf) alleles of skn-1, affecting a domain of SKN-1 that binds the transcription factor MXL-3 and the mitochondrial outer membrane protein PGAM-5. These skn-1(gf) mutants perceive a state of starvation even in the presence of plentiful food. The aberrant monitoring of cellular nutritional status leads to an altered survival response in which skn-1(gf) mutants transcriptionally activate genes associated with metabolism, adaptation to starvation, aging, and survival. The triggered starvation response is conserved in mice with constitutively activated Nrf and may contribute to the tumorgenicity associated with activating Nrf mutations in mammalian somatic cells. Our findings delineate an evolutionarily conserved metabolic axis of SKN-1/Nrf, further establishing the complexity of this pathway.

Mitochondrial SKN-1/Nrf mediates a conserved starvation response.

Intricate and interconnected pathways modulate longevity, but screens to identify the components of these pathways have not been saturating. Because biological processes are often executed by protein complexes and fine-tuned by regulatory factors, the first-order protein-protein interactors of known longevity genes are likely to participate in the regulation of longevity. Data-rich maps of protein interactions have been established for many cardinal organisms such as yeast, worms, and humans. We propose that these interaction maps could be mined for the identification of new putative regulators of longevity. For this purpose, we have constructed longevity networks in both humans and worms. We reasoned that the essential first-order interactors of known longevity-associated genes in these networks are more likely to have longevity phenotypes than randomly chosen genes. We have used C. elegans to determine whether post-developmental inactivation of these essential genes modulates lifespan. Our results suggest that the worm and human longevity networks are functionally relevant and possess a high predictive power for identifying new longevity regulators.

Prediction of C. elegans longevity genes by human and worm longevity networks.

Diet has a substantial impact on cellular metabolism and physiology. Animals must sense different food sources and utilize distinct strategies to adapt to diverse diets. Here we show that Caenorhabditis elegans lifespan is regulated by their adaptive capacity to different diets, which is controlled by alh-6, a conserved proline metabolism gene. alh-6 mutants age prematurely when fed an Escherichia coli OP50 but not HT115 diet. Remarkably, this diet-dependent aging phenotype is determined by exposure to food during development. Mechanistically, the alh-6 mutation triggers diet-induced mitochondrial defects and increased generation of ROS, likely due to accumulation of its substrate 1-pyrroline-5-carboxylate. We also identify that neuromedin U receptor signaling is essential for diet-induced mitochondrial changes and premature aging. Moreover, dietary restriction requires alh-6 to induce longevity. Collectively, our data reveal a homeostatic mechanism that animals employ to cope with potential dietary insults and uncover an example of lifespan regulation by dietary adaptation.

Adaptive capacity to bacterial diet modulates aging in C. elegans.

Mechanisms that coordinate different metabolic pathways, such as glucose and lipid, have been recognized. However, a potential interaction between amino acid and lipid metabolism remains largely elusive. Here we show that during starvation of Caenorhabditis elegans, proline catabolism is coupled with lipid metabolism by SKN-1. Mutation of alh-6, a conserved proline catabolic enzyme, accelerates fat mobilization, enhances the expression of genes involved in fatty acid oxidation and reduces survival in response to fasting. This metabolic coordination is mediated by the activation of the transcription factor SKN-1/Nrf2, possibly due to the accumulation of the alh-6 substrate P5C, and also requires the transcriptional co-regulator MDT-15. Constitutive activation of SKN-1 induces a similar transcriptional response, which protects animals from fat accumulation when fed a high carbohydrate diet. In human cells, an orthologous alh-6 enzyme, ALDH4A1, is also linked to the activity of Nrf2, the human orthologue of SKN-1, and regulates the expression of lipid metabolic genes. Our findings identify a link between proline catabolism and lipid metabolism, and uncover a physiological role for SKN-1 in metabolism.

SKN-1 and Nrf2 couples proline catabolism with lipid metabolism during nutrient deprivation.

Maf1 is a conserved repressor of RNA polymerase (Pol) III transcription; however, its physiological role in the context of a multicellular organism is not well understood. Here, we show that C. elegans MAFR-1 is functionally orthologous to human Maf1, represses the expression of both RNA Pol III and Pol II transcripts, and mediates organismal fecundity and lipid homeostasis. MAFR-1 impacts lipid transport by modulating intestinal expression of the vitellogenin family of proteins, resulting in cell-nonautonomous defects in the developing reproductive system. MAFR-1 levels inversely correlate with stored intestinal lipids, in part by influencing the expression of the lipogenesis enzymes fasn-1/FASN and pod-2/ACC1. Animals fed a high carbohydrate diet exhibit reduced mafr-1 expression and mutations in the insulin signaling pathway genes daf-18/PTEN and daf-16/FoxO abrogate the lipid storage defects associated with deregulated mafr-1 expression. Our results reveal physiological roles for mafr-1 in regulating organismal lipid homeostasis, which ensure reproductive success.

Physiological roles for mafr-1 in reproduction and lipid homeostasis.

Maf1 was first identified in yeast, and studies in metazoans have primarily focused on examining its role in the repression of transcription that is dependent on RNA polymerase III. Recent work has revealed a novel and conserved function for Maf1 in the maintenance of intracellular lipid pools in Caenorhabditis elegans, mice, and cancer cell lines. Although additional Maf1 targets are likely, they have not been identified, and these recent findings begin to define specific activities for Maf1 in multicellular organisms beyond the regulation of RNA polymerase III transcription and suggest that Maf1 plays a more diverse role in organismal physiology. We will discuss these newly defined physiological roles of Maf1 that point to its placement as an important new player in lipid metabolism with implications in human metabolic diseases such as obesity and cancer, which display prominent defects in lipid homeostasis.

Emerging Roles for Maf1 beyond the Regulation of RNA Polymerase III Activity.

Animals must continually assess nutrient availability to develop appropriate strategies for survival and reproductive success. It is no secret that nutritional state plays a large role in both aging and health.(1-7) Appropriate cellular energy usage is not only crucial for animal starvation survival, but is also important for diseases such as obesity and cancer, which characteristically have metabolic dysfunction.(8-10) C. elegans are exceptionally well poised to handle bouts of starvation as resource availability in the wild varies greatly.(11,12) We recently discovered an evolutionarily conserved pathway, regulated by the cytoprotective transcription factor SKN-1/Nrf2, which integrates diet composition and availability with utilization for survival.(13,14) These responses have potent impact on organismal physiology and remarkably are influenced by current and parental life history events, including choice of diet. In this commentary we will focus on recent insights concerning dietary intake and the impact that this can have throughout the life history of the nematode, Caenorhabditis elegans. In light of the strong impact that diet plays throughout life we urge caution when interpreting previous studies that make use of only one diet and suggest a reinvestigation utilizing a different diet is warranted.

The SKN-1 hunger games: May the odds be ever in your favor.

Animals in nature are continually challenged by periods of feast and famine as resources inevitably fluctuate, and must allocate somatic reserves for reproduction to abate evolutionary pressures. We identify an age-dependent lipid homeostasis pathway in Caenorhabditis elegans that regulates the mobilization of lipids from the soma to the germline, which supports fecundity but at the cost of survival in nutrient-poor and oxidative stress environments. This trade-off is responsive to the levels of dietary carbohydrates and organismal oleic acid and is coupled to activation of the cytoprotective transcription factor SKN-1 in both laboratory-derived and natural isolates of C. elegans. The homeostatic balance of lipid stores between the somatic and germ cells is mediated by arachidonic acid (omega-6) and eicosapentaenoic acid (omega-3) precursors of eicosanoid signaling molecules. Our results describe a mechanism for resource reallocation within intact animals that influences reproductive fitness at the cost of somatic resilience.

Omega-3 and -6 fatty acids allocate somatic and germline lipids to ensure fitness during nutrient and oxidative stress in Caenorhabditis elegans.

Diet is the most variable aspect of life history, as most individuals have a large diversity of food choices, varying in the type and amount that they ingest. In the short-term, diet can affect metabolism and energy levels. However, in the long run, the net deficiency or excess of calories from diet can influence the progression and severity of age-related diseases. An old and yet still debated question is: how do specific dietary choices impact health- and lifespan? It is clear that genetics can play a critical role - perhaps just as important as diet choices. For example, poor diet in combination with genetic susceptibility can lead to metabolic disorders, such as obesity and type 2 diabetes. Recent work in Caenorhabditis elegans has identified the existence of diet-gene pairs, where the consequence of mutating a specific gene is only realized on specific diets. Many core metabolic pathways are conserved from worm to human. Although only a handful of these diet-gene pairs has been characterized, there are potentially hundreds, if not thousands, of such interactions, which may explain the variability in the rates of aging in humans and the incidence and severity of age-related diseases.

Gene-diet interactions and aging in C. elegans.

MAF1 is a conserved negative regulator of RNA polymerase (pol) III and intracellular lipid homeostasis across species. Here, we show that the MAF1 C-box region negatively regulates its activity. Mutations in Caenorhabditis elegans mafr-1 that truncate the C-box retain the ability to inhibit the transcription of RNA pol III targets, reduce lipid biogenesis, and lower reproductive output. In human cells, C-box deletion of MAF1 leads to increased MAF1 nuclear localization and enhanced repression of ACC1 and FASN, but with impaired repression of RNA pol III targets. Surprisingly, C-box mutations render MAF1 insensitive to rapamycin, further defining a regulatory role for this region. Two MAF1 species, MAF1and MAF1, are regulated by the C-box YSY motif, which, when mutated, alters species stoichiometry and proteasome-dependent turnover of nuclear MAF1. Our results reveal a role for the C-box region as a critical determinant of MAF1 stability, activity, and response to cellular stress.

The C-Box Region of MAF1 Regulates Transcriptional Activity and Protein Stability.

Cellular adaptation to stress is essential to ensure organismal survival. NRF2/NFE2L2 is a key determinant of xenobiotic stress responses, and loss of negative regulation by the KEAP1-CUL3 proteasome system is implicated in several chemo- and radiation-resistant cancers. Advantageously using C. elegans alongside human cell culture models, we establish a new WDR23-DDB1-CUL4 regulatory axis for NRF2 activity that operates independently of the canonical KEAP1-CUL3 system. WDR23 binds the DIDLID sequence within the Neh2 domain of NRF2 to regulate its stability; this regulation is not dependent on the KEAP1-binding DLG or ETGE motifs. The C-terminal domain of WDR23 is highly conserved and involved in regulation of NRF2 by the DDB1-CUL4 complex. The addition of WDR23 increases cellular sensitivity to cytotoxic chemotherapeutic drugs and suppresses NRF2 in KEAP1-negative cancer cell lines. Together, our results identify WDR23 as an alternative regulator of NRF2 proteostasis and uncover a cellular pathway that regulates NRF2 activity and capacity for cytoprotection independently of KEAP1.

WDR23 regulates NRF2 independently of KEAP1.

Organisms must execute metabolic defenses to survive nutrient deprivation. We performed a genome-wide RNAi screen in Caenorhabditis elegans to identify fat regulatory genes indispensable for starvation resistance. Here, we show that opposing proteostasis pathways are principal determinants of starvation survival. Reduced function of cytoplasmic aminoacyl tRNA synthetases (ARS genes) increases fat mass and extends starvation survival, whereas reduced proteasomal function reduces fat and starvation survival. These opposing pathways converge on AMP-activated protein kinase (AMPK) as the critical effector of starvation defenses. Extended starvation survival in ARS deficiency is dependent upon increased proteasome-mediated activation of AMPK. When the proteasome is inhibited, neither starvation nor ARS deficiency can fully activate AMPK, leading to greatly diminished starvation survival. Thus, activity of the proteasome and AMPK are mechanistically linked and highly correlated with starvation resistance. Conversely, aberrant activation of the proteostasis-AMPK axis during nutritional excess may have implications for obesity and cardiometabolic diseases.

Article	Year
Longevity and the long arm of epigenetics: acquired parental marks influence lifespan across several generations. BioEssays : news and reviews in molecular, cellular and developmental biology\| PubMed ID: 22674543	2012
Mitochondrial SKN-1/Nrf mediates a conserved starvation response. Cell metabolism\| PubMed ID: 23040073	2012
Prediction of C. elegans longevity genes by human and worm longevity networks. PloS one\| PubMed ID: 23144747	2012
Adaptive capacity to bacterial diet modulates aging in C. elegans. Cell metabolism\| PubMed ID: 24440036	2014
SKN-1 and Nrf2 couples proline catabolism with lipid metabolism during nutrient deprivation. Nature communications\| PubMed ID: 25284427	2014
Physiological roles for mafr-1 in reproduction and lipid homeostasis. Cell reports\| PubMed ID: 25497095	2014
Emerging Roles for Maf1 beyond the Regulation of RNA Polymerase III Activity. Journal of molecular biology\| PubMed ID: 26173035	2015
The SKN-1 hunger games: May the odds be ever in your favor. Worm\| PubMed ID: 26430571	2015
Omega-3 and -6 fatty acids allocate somatic and germline lipids to ensure fitness during nutrient and oxidative stress in Caenorhabditis elegans. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 26621724	2015
Gene-diet interactions and aging in C. elegans. Experimental gerontology\| PubMed ID: 26924670	2016
The C-Box Region of MAF1 Regulates Transcriptional Activity and Protein Stability. Journal of molecular biology\| PubMed ID: 27986570	2017
WDR23 regulates NRF2 independently of KEAP1. PLoS genetics\| PubMed ID: 28453520	2017
Genome-wide RNAi Screen for Fat Regulatory Genes in C. elegans Identifies a Proteostasis-AMPK Axis Critical for Starvation Survival. Cell reports\| PubMed ID: 28723566	2017

Sean P. Curran

Biography

JoVE Journal Publications

Other Publications