Autophagy links lipid metabolism to longevity in C. elegans

The cellular recycling process of autophagy is emerging as a central player in many of the conserved longevity pathways in C. elegans, but the underlying mechanisms that link autophagy and life span remain unclear. In a recent study, we provided evidence to suggest that autophagy modulates aging through an effect on lipid homeostasis. Specifically, we identified a role for autophagy in a longevity model in which germline removal in C. elegans extends life span. Life-span extension in these animals is achieved, at least in part, through increased expression of the lipase LIPL-4. We found that autophagy and LIPL-4-dependent lipolysis are both upregulated in germline-less animals and work interdependently to prolong life span. While these genetic results lend further support to a growing link between autophagy and lipid metabolism, our findings are the first to suggest a possible molecular mechanism by which autophagy modulates organismal aging.     

Aging networks in Caenorhabditis elegans: AMP-activated protein kinase (aak-2) links multiple aging and metabolism pathways

Molecular genetics in lower organisms has allowed the elucidation of pathways that modulate the aging process. In certain instances, evolutionarily conserved genes and pathways have been shown to regulate lifespan in mammals as well. Many gene products known to affect lifespan are intimately involved in the control of energy metabolism, including the fuel sensor AMP-activated protein kinase (AMPK). We have shown previously that over-expression of an AMPK alpha subunit in Caenorhabditis elegans, designated aak-2, increases lifespan. Here we show the interaction of aak-2 with other pathways known to control aging in worms. Lifespan extension caused by daf-2/insulin-like signaling mutations was highly dependent on aak-2, as was the lifespan extension caused by over-expression of the deacetylase, sir-2.1. Similarly, there was partial requirement for aak-2 in lifespan extension by mitochondrial mutations (isp-1 and clk-1). Conversely, aak-2 was not required for lifespan extension in mutants lacking germline stem cells (glp-1) or mutants of the eating response (eat-2). These results show that aging is controlled by overlapping but distinct pathways and that AMPK/aak-2 represents a node in a network of evolutionarily conserved biochemical pathways that control aging.     

Rtg2 protein links metabolism and genome stability in yeast longevity

Mitochondrial dysfunction induces a signaling pathway, which culminates in changes in the expression of many nuclear genes. This retrograde response, as it is called, extends yeast replicative life span. It also results in a marked increase in the cellular content of extrachromosomal ribosomal DNA circles (ERCs), which can cause the demise of the cell. We have resolved the conundrum of how these two molecular mechanisms of yeast longevity operate in tandem. About 50% of the life-span extension elicited by the retrograde response involves processes other than those that counteract the deleterious effects of ERCs. Deletion of RTG2, a gene that plays a central role in relaying the retrograde response signal to the nucleus, enhances the generation of ERCs in cells with (grande) or in cells without (petite) fully functional mitochondria, and it curtails the life span of each. In contrast, overexpression of RTG2 diminishes ERC formation in both grandes and petites. The excess Rtg2p did not augment the retrograde response, indicating that it was not engaged in retrograde signaling. FOB1, which is known to be required for ERC formation, and RTG2 were found to be in converging pathways for ERC production. RTG2 did not affect silencing of ribosomal DNA in either grandes or petites, which were similar to each other in the extent of silencing at this locus. Silencing of ribosomal DNA increased with replicative age in either the presence or the absence of Rtg2p, distinguishing silencing and ERC accumulation. Our results indicate that the suppression of ERC production by Rtg2p requires that it not be in the process of transducing the retrograde signal from the mitochondrion. Thus, RTG2 lies at the nexus of cellular metabolism and genome stability, coordinating two pathways that have opposite effects on yeast longevity.     

Neuronal control of lipid metabolism by STR‐2 G protein‐coupled receptor promotes longevity in Caenorhabditis elegans

The G protein‐coupled receptor (GPCR) encoding family of genes constitutes more than 6% of genes in Caenorhabditis elegans genome. GPCRs control behavior, innate immunity, chemotaxis, and food search behavior. Here, we show that C. elegans longevity is regulated by a chemosensory GPCR STR‐2, expressed in AWC and ASI amphid sensory neurons. STR‐2 function is required at temperatures of 20°C and higher on standard Escherichia coli OP50 diet. Under these conditions, this neuronal receptor also controls health span parameters and lipid droplet (LD) homeostasis in the intestine. We show that STR‐2 regulates expression of delta‐9 desaturases, fat‐5, fat‐6 and fat‐7, and of diacylglycerol acyltransferase dgat‐2. Rescue of the STR‐2 function in either AWC and ASI, or ASI sensory neurons alone, restores expression of fat‐5, dgat‐2 and restores LD stores and longevity. Rescue of stored fat levels of GPCR mutant animals to wild‐type levels, with low concentration of glucose, rescues its lifespan phenotype. In all, we show that neuronal STR‐2 GPCR facilitates control of neutral lipid levels and longevity in C. elegans.     

The dynamin-related protein DRP-1 and the insulin signaling pathway cooperate to modulate Caenorhabditis elegans longevity

Here, we report that inactivation of the Caenorhabditis elegans dynamin-related protein DRP-1, a key component responsible for mitochondrial fission and conserved from yeast to humans, dramatically enhanced the effect of reduced insulin signaling (IIS) to extend lifespan. This represents the first report of a beneficial impact of manipulating mitochondrial dynamics on animal lifespan and suggests that mitochondrial morphology and IIS cooperate to modulate aging.     

The FAR protein family of the nematode Caenorhabditis elegans. Differential lipid binding properties,structural characteristics,and developmental regulation

Parasitic nematodes of humans and plants secrete a structurally novel type of fatty acid- and retinol-binding protein, FAR, into the tissues they occupy. These proteins may interfere with intercellular lipid signaling to manipulate the defense reactions of the host or acquire essential lipids for the parasites. The genome of the nematode Caenorhabditis elegans encodes eight FAR-like proteins (Ce-FAR-1 to -8). These fall into three discrete groups as indicated by phylogenetic sequence comparisons and intron positions, the proteins from parasitic nematodes falling into group A. Recombinant Ce-FAR-1 to -7 were produced in Escherichia coli and tested for lipid binding in fluorescence-based assays. Ce-FAR-1 to -6 bound DAUDA (11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid), cis-parinaric acid, and retinol with dissociation constants in the micromolar range, whereas Ce-FAR-7 bound the latter two lipids relatively poorly. Each protein produced a characteristic shift in peak fluorescence emission of DAUDA, and one (Ce-FAR-5) produced a shift greater than has been observed previously for any lipid-binding protein. Selected Ce-FAR proteins were analyzed by circular dichroism (CD) and differential scanning calorimetry, were found to be helix-rich, and exhibited high thermal stability (transition midpoint, 82.7 degrees C). CD and secondary structure predictions, however, both indicated that Ce-FAR-7 possesses substantially less helix than the other FAR proteins. The genes encoding the Ce-FAR proteins were found to be transcribed differentially through the life cycle of C. elegans, such that Ce-far-4 was transcribed at highest levels in the fourth larval stage, and Ce-far-3 and -7 predominated in males.     

Proteomic study and marker protein identification of Caenorhabditis elegans lipid droplets

Lipid droplets (LDs) are a neutral lipid storage organelle that is conserved across almost all species. Many metabolic syndromes are directly linked to the over-storage of neutral lipids in LDs. The study of LDs in Caenorhabditis elegans (C. elegans) has been difficult because of the lack of specific LD marker proteins. Here we report the purification and proteomic analysis of C. elegans lipid droplets for the first time. We identified 306 proteins, 63% of these proteins were previously known to be LD-proteins, suggesting a similarity between mammalian and C. elegans LDs. Using morphological and biochemical analyses, we show that short-chain dehydrogenase, DHS-3 is almost exclusively localized on C. elegans LDs, indicating that it can be used as a LD marker protein in C. elegans. These results will facilitate further mechanistic studies of LDs in this powerful genetic system, C. elegans.     

HPL-2/HP1 Prevents Inappropriate Vulval Induction in Caenorhabditis elegans by Acting in Both HYP7 and Vulval Precursor Cells

A current model for Caenorhabditis elegans vulval cell fate specification is that SynMuv genes act redundantly in the hyp7 hypodermal syncytium to repress the LIN-3/EGF inducer and prevent ectopic vulval induction of vulva precursor cells (VPCs). Here we show that the SynMuv gene hpl-2/HP1 has an additional function in VPCs, where it may act through target genes including LIN-39/Hox.     

Genetic, behavioral and environmental determinants of male longevity in Caenorhabditis elegans

Males of the nematode Caenorhabditis elegans are shorter lived than hermaphrodites when maintained in single-sex groups. We observed that groups of young males form clumps and that solitary males live longer, indicating that male-male interactions reduce life span. By contrast, grouped or isolated hermaphrodites exhibited the same longevity. In one wild isolate of C. elegans, AB2, there was evidence of copulation between males. Nine uncoordinated (unc) mutations were used to block clumping behavior. These mutations had little effect on hermaphrodite life span in most cases, yet many increased male longevity even beyond that of solitary wild-type males. In one case, the neuronal function mutant unc-64(e246), hermaphrodite life span was also increased by up to 60%. The longevity of unc-4(e120), unc-13(e51), and unc-32(e189) males exceeded that of hermaphrodites by 70-120%. This difference appears to reflect a difference in sex-specific life span potential revealed in the absence of male behavior that is detrimental to survival. The greater longevity of males appears not to be affected by daf-2, but is influenced by daf-16. In the absence of male-male interactions, median (but not maximum) male life span was variable. This variability was reduced when dead bacteria were used as food. Maintenance on dead bacteria extended both male and hermaphrodite longevity.     

Tissue- and paralogue-specific functions of acyl-CoA-binding proteins in lipid metabolism in Caenorhabditis elegans

ACBP (acyl-CoA-binding protein) is a small primarily cytosolic protein that binds acyl-CoA esters with high specificity and affinity. ACBP has been identified in all eukaryotic species, indicating that it performs a basal cellular function. However, differential tissue expression and the existence of several ACBP paralogues in many eukaryotic species indicate that these proteins serve distinct functions. The nematode Caenorhabditis elegans expresses seven ACBPs: four basal forms and three ACBP domain proteins. We find that each of these paralogues is capable of complementing the growth of ACBP-deficient yeast cells, and that they exhibit distinct temporal and tissue expression patterns in C. elegans. We have obtained loss-of-function mutants for six of these forms. All single mutants display relatively subtle phenotypes; however, we find that functional loss of ACBP-1 leads to reduced triacylglycerol (triglyceride) levels and aberrant lipid droplet morphology and number in the intestine. We also show that worms lacking ACBP-2 show a severe decrease in the β-oxidation of unsaturated fatty acids. A quadruple mutant, lacking all basal ACBPs, is slightly developmentally delayed, displays abnormal intestinal lipid storage, and increased β-oxidation. Collectively, the present results suggest that each of the ACBP paralogues serves a distinct function in C. elegans.     

Cloning and developmental expression analysis of ltd-1, the Caenorhabditis elegans homologue of the mouse kyphoscoliosis (ky) gene

We have characterized the developmental expression pattern of the Caenorhabditis elegans homologue of the mouse ky gene. The Ky protein has a putative key function in muscle development and has homologues in invertebrates, fungi and a cyanobacterium. The C. elegans Ky homologue gene has been named ltd-1 for LIM and transglutaminase domains gene. The LTD-1::GFP construct is expressed in developing hypodermal cells from the twofold stage embryo through adulthood. These data define the ltd-1 gene as a novel marker for C. elegans epithelial cell development.     

Mitochondrial uncoupling protein 2 (UCP2) in glucose and lipid metabolism

Nutrient availability is critical for the physiological functions of all tissues. By contrast, an excess of nutrients such as carbohydrate and fats impair health and shorten life due by stimulating chronic diseases, including diabetes, cancer and neurodegeneration. The control of circulating glucose and lipid levels involve mitochondria in both central and peripheral mechanisms of metabolism regulation. Mitochondrial uncoupling protein 2 (UCP2) has been implicated in physiological and pathological processes related to glucose and lipid metabolism, and in this review we discuss the latest data on the relationships between UCP2 and glucose and lipid sensing from the perspective of specific hypothalamic neuronal circuits and peripheral tissue functions. The goal is to provide a framework for discussion of future therapeutic strategies for metabolism-related chronic diseases.     

Contribution of sams-1 and pmt-1 to lipid homoeostasis in adult Caenorhabditis elegans

Accumulation of lipids inside the cell is primarily caused by disorders of lipid metabolism. S-adenosylmethionine synthetase (SAMS) produces SAM, an important methyl donor in various phospholipid methyltransferase reactions catalysed by phosphoethanolamine N-methyltransferase (PMT-1). A gel-based, quantitative proteomic analysis of the RNA interference (RNAi)-mediated inactivation of the pod-2 gene, which encodes acetyl-CoA carboxylase, showed a substantial down-regulation of SAMS-1. Consequently, RNAi of either sams-1 or pmt-1 caused a significant increase in lipid droplet size in the intestine of Caenorhabditis elegans. Lipid droplets exhibited increased triacylglycerol (TG) and decreased phosphatidylcholine (PC) levels, suggesting a reciprocal relationship between TG and PC regulation. These lipid-associated phenotypes were rescued by choline feeding. Among the five fat metabolism-related genes examined, two genes were highly induced by inactivation of sams-1 or pmt-1: pod-2 and stearoyl-CoA desaturase (fat-7). Thus, both SAMS-1 and PMT-1 were shown to contribute to the homoeostasis of TG and PC levels in C. elegans, which would provide an important survival strategy under harsh environmental conditions.