Caloric restriction and autophagy in Caenorhabditis elegans

Autophagy is a catabolic process in which long-lived proteins and organelles are degraded for recycling in the cytoplasm. In the nematode Caenorhabditis elegans autophagy is associated with formation of the dauer larva, an alternative developmental stage that worms can enter under poor growth conditions. We have shown that C. elegans mutants that experience caloric restriction because they are feeding-defective also exhibit elevated autophagy and decreased levels of fat deposits, as well as smaller cells and, consequently, a smaller body size. Our results suggest novel relationships between caloric restriction, longevity, body size and autophagy.     

The life span-prolonging effect of Sirtuin-1 is mediated by autophagy

The life span of various model organisms can be extended by caloric restriction as well as by autophagy-inducing pharmacological agents. Life span-prolonging effects have also been observed in yeast cells, nematodes and flies upon the overexpression of the deacetylase Sirtuin-1. Intrigued by these observations and by the established link between caloric restriction and Sirtuin-1 activation, we decided to investigate the putative implication of Sirtuin-1 in the response of human cancer cells and Caenorhabditis elegans to multiple triggers of autophagy. Our data indicate that the activation of Sirtuin-1 (by the pharmacological agent resveratrol and/or genetic means) per se ignites autophagy, and that Sirtuin-1 is required for the autophagic response to nutrient deprivation, in both human and nematode cells, but not for autophagy triggered by downstream signals such as the inhibition of mTOR or p53. Since the life span-extending effects of Sirtuin-1 activators are lost in autophagy-deficient C. elegans, our results suggest that caloric restriction and resveratrol extend longevity, at least in experimental settings, by activating autophagy.     

Caloric restriction and resveratrol promote longevity through the Sirtuin-1-dependent induction of autophagy

Caloric restriction and autophagy-inducing pharmacological agents can prolong lifespan in model organisms including mice, flies, and nematodes. In this study, we show that transgenic expression of Sirtuin-1 induces autophagy in human cells in vitro and in Caenorhabditis elegans in vivo. The knockdown or knockout of Sirtuin-1 prevented the induction of autophagy by resveratrol and by nutrient deprivation in human cells as well as by dietary restriction in C. elegans. Conversely, Sirtuin-1 was not required for the induction of autophagy by rapamycin or p53 inhibition, neither in human cells nor in C. elegans. The knockdown or pharmacological inhibition of Sirtuin-1 enhanced the vulnerability of human cells to metabolic stress, unless they were stimulated to undergo autophagy by treatment with rapamycin or p53 inhibition. Along similar lines, resveratrol and dietary restriction only prolonged the lifespan of autophagy-proficient nematodes, whereas these beneficial effects on longevity were abolished by the knockdown of the essential autophagic modulator Beclin-1. We conclude that autophagy is universally required for the lifespan-prolonging effects of caloric restriction and pharmacological Sirtuin-1 activators.     

Autophagy is required for dietary restriction-mediated life span extension in C. elegans

Dietary restriction extends life span in diverse species including Caenorhabditis elegans. However, the downstream cellular targets regulated by dietary restriction are largely unknown. Autophagy, an evolutionary conserved lysosomal degradation pathway, is induced under starvation conditions and regulates life span in insulin signaling C. elegans mutants. We now report that two essential autophagy genes (bec-1 and Ce-atg7) are required for the longevity phenotype of the C. elegans dietary restriction mutant (eat-2(ad1113) animals. Thus, we propose that autophagy mediates the effect, not only of insulin signaling, but also of dietary restriction on the regulation of C. elegans life span. Since autophagy and longevity control are highly conserved from C. elegans to mammals, a similar role for autophagy in dietary restriction-mediated life span extension may also exist in mammals.     

CUP-5, the C. elegans ortholog of the mammalian lysosomal channel protein MLN1/TRPML1, is required for proteolytic degradation in autolysosomes

The process of macroautophagy (herein referred to as autophagy) involves the formation of a closed double-membrane structure, called the autophagosome, and its subsequent fusion with lysosomes to form an autolysosome. Lysosomes are regenerated from autolysosomes after degradation of the sequestrated materials. In this study, we showed that mutations in cup-5, encoding the C. elegans Mucolipin 1 homolog, cause defects in the autophagy pathway. In cup-5 mutants, a variety of autophagy substrates accumulate in enlarged vacuoles that display characteristics of late endosomes and lysosomes, indicating defective proteolytic degradation in autolysosomes. We further revealed that lysosomes in coelomocytes (scavenger cells located in the body cavity) are smaller in size and more numerous in mutants with loss of autophagy activity. Furthermore, the enlarged vacuole accumulation abnormality and embryonic lethality of cup-5 mutants are partially suppressed by reduced autophagy activity. Our results indicate that the basal constitutive level of autophagy activity regulates the size and number of lysosomes and provides insights into the molecular mechanisms underlying mucolipidosis type IV disease.     

Sexual Dimorphism in the Response of Adipose Mass and Cellularity to Graded Caloric Restriction

Objective: To investigate the effects of mild to moderate caloric restriction on parameters of body growth, fat mass, and adipose tissue cellularity in female and male Wistar rats. Research Methods and Procedures: Three‐month‐old female and male Wistar rats were subjected to a chronic, mild to moderate caloric restriction paradigm (5%, 10%, or 20% reduction in caloric intake from ad libitum values) for 6 months. This was accomplished using a unique automated feeder system tailored to the food consumption levels of individual rats. Body weight and length, weight of lean organs, regional adipose mass, and adipose cellularity were measured before and after the diet restriction. Results: Caloric restriction produced proportional decelerations in body weight increases in both genders, without significant changes in body length or lean organ mass. Marked and disproportional reductions in regional adipose tissue mass were produced at all levels of food restriction (even at 5% restriction). An unexpected finding was that in response to graded caloric restriction, female rats preserved adipose fat cell number at the expense of fat cell volume, whereas the converse was seen for male rats. Discussion: These studies demonstrate a sexual dimorphism in the response to mild to moderate degrees of chronic caloric restriction. At low levels of caloric restriction, it is possible to affect regional adipose mass and cellularity while preserving lean organ mass.     

Autophagy is Required for Dietary Restriction-Mediated Life Span Extension in C. elegans

Dietary restriction extends life span in diverse species including Canorhabditis elegans. However, the downstream cellular targets regulated by dietary restriction are largely unknown. Autophagy, an evolutionary conserved lysosomal degradation pathway, is induced under starvation conditions and regulates life span in insulin signaling C. elegans mutants. We now report that two essential autophagy genes (bec-1 and Ce-atg7) are required for the longevity phenotype of the C. elegans dietary restriction mutant (eat-2ad1113) animals. Thus, we propose that autophagy mediates the effect, not only of insulin signaling, but also of dietary restriction on the regulation of C. elegans life span. Since autophagy and longevity control are highly conserved from C. elegans to mammals, a similar role for autophagy in dietary restriction-mediated life span extension may also exist in mammals.     

Avoiding death by autophagy: Interactions of Listeria monocytogenes with the macrophage autophagy system

Autophagy restricts the growth of a variety of intracellular pathogens. However, cytosol-adapted pathogens have evolved ways to evade restriction by this innate immune mechanism. Listeria monocytogenes is a Gram-positive bacterial pathogen that utilizes a cholesterol-dependent pore-forming toxin, listeriolysin O (LLO), to escape from the phagosome. Autophagy targets L. monocytogenes in LLO-damaged phagosomes and also in the cytosol under some experimental conditions. However, this bacterium has evolved multiple mechanisms to evade restriction by autophagy, including actin-based motility in the cytosol and an as yet undefined mechanism mediated by bacterial phospholipases C’s (PLCs). A population of L. monocytogenes with inefficient LLO activity forms Spacious Listeria-containing Phagosomes (SLAPs), which are autophagosome-like compartments that do not mature, allowing slow bacterial growth within enlarged vesicles. SLAPs may represent a stalemate between bacterial LLO action and the host autophagy system, resulting in persistent infection.

Addendum to: Birmingham CL, Canadien V, Gouin E, Troy EB, Yoshimori T, Cossart P, Higgins DE, Brumell JH. Listeria monocytogenes evades killing by autophagy during colonization of host cells. Autophagy 2007; 3:442-51.andBirmingham CL, Canadien V, Kaniuk NA, Steinberg BE, Higgins DE, Brumell JH. Listeriolysin O allows Listeria monocytogenes replication in macrophage vacuoles. Nature 2008; 451:350-4.     

Effects of three simultaneous demands on glucose transport, resting metabolism and morphology of laboratory mice

In nature, animals must successfully respond to many simultaneous demands from their environment in order to survive and reproduce. We examined physiological and morphological responses of mice given three demands: intestinal parasite infection with Heligmosomoides polygyrus followed by caloric restriction (70% of ad libitum food intake versus ad libitum for 10 days) and/or cold exposure (5°C vs. 23°C for 10 days). We found significant interactions between these demands as well as independent effects. Small intestine structure and function changed with demands in both independent and interactive ways. Body mass decreased during caloric restriction and this decrease was greater for cold-exposed than warm-exposed mice. In ad libitum fed mice, body mass did not change with either cold exposure or parasite infection but body composition (fat versus lean mass of whole body or organs) changed with both demands. Generally, organ masses decreased with caloric restriction (even after accounting for body mass effects) and increased with cold exposure and parasite infection whereas fat mass decreased with both caloric restriction and parasite infection. Mass adjusted resting metabolic rate (RMR) increased with cold exposure, decreased with caloric restriction but, unlike previous studies with laboratory mice, did not change with parasite infection. Our results demonstrate that the ability of mice to respond to a demand is influenced by other concurrent demands and that mice show phenotypic plasticity of morphological and physiological features ranging from the tissue level to the level of the whole organism when given three simultaneous demands.     

The tissue- and developmental stage-specific involvement of autophagy genes in aggrephagy

ABSTRACT

Genetic screens have identified two sets of genes that act at distinct steps of basal autophagy in higher eukaryotes: the pan-eukaryotic ATG genes and the metazoan-specific EPG genes. Very little is known about whether these core macroautophagy/autophagy genes are differentially employed during multicellular organism development. Here we analyzed the function of core autophagy genes in autophagic removal of SQST-1/SQSTM1 during C. elegans development. We found that loss of function of genes acting at distinct steps in the autophagy pathway causes different patterns of SQST-1 accumulation in different tissues and developmental stages. We also identified that the calpain protease clp-2 acts in a cell context-specific manner in SQST-1 degradation. clp-2 is required for degradation of SQST-1 in the hypodermis and neurons, but is dispensable in the body wall muscle and intestine. Our results indicate that autophagy genes are differentially employed in a tissue- and stage-specific manner during the development of multicellular organisms.

Abbreviations: ATG: autophagy related; CLP: calpain family; EPG: ectopic PGL granules; ER: endoplasmic reticulum; ESCRT: endosomal sorting complex required for transport; GFP: green fluorescent protein; LGG-1/LC3: LC3, GABARAP and GATE-16 family; MIT: microtubule interacting and transport; PGL: P granule abnormality protein; SQST-1: sequestosome-related; UPS: ubiquitin-proteasome system     

CUP-5, the C. elegans ortholog of the mammalian lysosomal channel protein MLN1/TRPML1, is required for proteolytic degradation in autolysosomes

The process of macroautophagy (herein referred to as autophagy) involves the formation of a closed double-membrane structure, called the autophagosome, and its subsequent fusion with lysosomes to form an autolysosome. Lysosomes are regenerated from autolysosomes after degradation of the sequestrated materials. In this study, we showed that mutations in cup-5, encoding the C. elegans Mucolipin 1 homolog, cause defects in the autophagy pathway. In cup-5 mutants, a variety of autophagy substrates accumulate in enlarged vacuoles that display characteristics of late endosomes and lysosomes, indicating defective proteolytic degradation in autolysosomes. We further revealed that lysosomes in coelomocytes (scavenger cells located in the body cavity) are smaller in size and more numerous in mutants with loss of autophagy activity. Furthermore, the enlarged vacuole accumulation abnormality and embryonic lethality of cup-5 mutants are partially suppressed by reduced autophagy activity. Our results indicate that the basal constitutive level of autophagy activity regulates the size and number of lysosomes and provides insights into the molecular mechanisms underlying mucolipidosis type IV disease.     

Disruption of Autophagy Results in Constitutive Oxidative Stress in Arabidopsis

Plant cells frequently encounter oxidative stress, leading to oxidative damage and inactivation of proteins. We have recently demonstrated that oxidative stress induces autophagy in Arabidopsis seedlings in an AtATG18a-dependent manner and that RNAi-AtATG18a transgenic lines, which are defective in autophagosome formation, are hypersensitive to reactive oxygen species. Analysis of protein oxidation indicated that oxidized proteins are degraded in the vacuole after uptake by autophagy, and this degradation is impaired in RNAi-AtATG18a lines. Our results also suggest that in the absence of a functional autophagy pathway, plants are under increased oxidative stress, even under normal growth conditions.

Addendum to:

Degradation of Oxidized Proteins by Autophagy during Oxidative Stress in Arabidopsis

Y. Xiong, A.L. Contento, N.Q. Phan and D.C. Bassham

Plant Physiol 2007; 143:291-9     

Regulation of cell growth by autophagy

Cell growth–the primary determinant of cell size–has an intimate relationship with proliferation; cells divide only after they reach a critical size. Despite its developmental and medical significance, little is known about cellular pathways that mediate the growth of cells. Accumulating evidence demonstrates a role for autophagy–a mechanism of eukaryotic cells to digest their own constituents during development or starvation–in cell size control. Increasing autophagic activity by prolonged starvation, rapamycin treatment inhibiting TOR (target of rapamycin) signaling, or genetic intervention, causes cellular atrophy in worms, flies and mammalian cell cultures. In contrast, we have shown that in the nematode Caenorhabditis elegans mutational inactivation of two autophagy genes, unc-51/Atg1 and bec-1/Atg6, confers reduced cell size. We argue that physiological levels of autophagy are required for normal cell size, whereas both insufficient and excessive levels of autophagy lead to retarded cell growth. Furthermore, we discuss data suggesting that the insulin/IGF-1 (insulin-like growth factor receptor-1) and TGFβ (transforming growth factor-beta) signaling systems acting as major growth regulatory pathways converge on autophagy genes to control cell size. Thus, autophagy may act as a central regulatory mechanism of cell growth.

Addendum to: Aladzsity I, Tóth ML, Sigmond T, Szabó E., Bicsák B, Barna J, Reg?s A, Orosz L, Kovács AL, Vellai T. Autophagy genes unc-51 and bec-1 are required for normal cell size in Caenorhabditis elegans. Genetics 2007; 177:655-60, DOI: 10.1534/genetics.107.075762     

Resistance Training Improves Cardiovascular Risk Factors in Obese Women Despite a Significative Decrease in Serum Adiponectin Levels

Increased circulating adiponectin and insulin sensitivity are usually observed after body fat loss induced by a weight‐loss diet. Progressive resistance training (PRT) without a concomitant weight‐loss diet significantly decreases visceral fat, thus improving insulin sensitivity. Therefore, the purpose of this study was to ascertain the effects of combined 16‐week PRT and weight‐loss diet on circulating adiponectin and insulin sensitivity index. Thirty‐four obese (BMI: 30–40 kg/m²) women, aged 40–60 year, were randomized to three groups: a control group (C; n = 9); a diet group (WL; n = 12) with a caloric restriction of 500 kcal/d; and a diet plus resistance training group (WL+RT; n = 13) with the same caloric restriction as group WL and a 16‐week supervised whole body PRT of two sessions/week. Both WL and WL+RT groups showed similar decreases in body mass (?6.3% and ?7.7%) and visceral fat (?19.9% and ?20.5%). WL resulted in an expected increase in circulating levels of adiponectin (P = 0.07) and insulin sensitivity. However, circulating total adiponectin decreased (P < 0.05) in WL+RT group, whereas an improvement in different cardiovascular risk factors (insulin sensitivity, low‐density lipoprotein cholesterol (LDL‐C), etc.) was observed. In conclusion, in obese women a 16‐week combined PRT and weight‐loss diet is accompanied by significant improvements in different cardiovascular risk factors in spite of a significant decrease of circulating adiponectin.     

Astrocytes as Perspective Targets of Exercise- and Caloric Restriction‐Mimetics

Enhanced mental and physical activity can have positive effects on the function of aging brain, both in the experimental animals and human patients, although cellular mechanisms underlying these effects are currently unclear. There is a growing evidence that pre-clinical stage of many neurodegenerative diseases involves changes in interactions between astrocytes and neurons. Conversely, astrocytes are strategically positioned to mediate the positive influence of physical activity and diet on neuronal function. Thus, development of therapeutic agents which could improve the astroglia-neuron communications in ageing brain is of crucial importance. Recent advances in studies of cellular mechanisms of brain longevity suggest that astrocyte-neuron communications have a vital role in the beneficial effects of caloric restriction, physical exercise and their pharmacological mimetics on synaptic homeostasis and cognitive function. In particular, our recent data indicate that noradrenaline uptake inhibitor atomoxetine can enhance astrocytic Ca²⁺-signaling and astroglia-driven modulation of synaptic plasticity. Similar effects were exhibited by caloric restriction-mimetics metformin and resveratrol. The emerged data also suggest that astrocytes could be involved in the modulatory action of caloric restriction and its mimetics on neuronal autophagy. Still, the efficiency of astrocyte-targeting compounds in preventing age-related cognitive decline is yet to be fully explored, in particular in the animal models of neurodegenerative diseases and autophagy impairment.

     

Why Sick Cells Produce Tumors: The Protective Role of Autophagy

Cells exploit autophagy for survival to metabolic stress in vitro as well as in tumors where it localizes to regions of metabolic stress suggesting its role as a survival pathway. Consistent with this survival function, deficiency in autophagy impairs cell survival, but also promotes tumor growth, creating a paradox that the loss of a survival pathway leads to tumorigenesis. There is evidence that autophagy is a homeostatic process functioning to limit the accumulation of poly-ubiquitinated proteins and mutant protein aggregates associated with neuronal degeneration. Interestingly, we found that deficiency in autophagy caused by monoallelic loss of beclin1 or deletion of atg5 leads to accelerated DNA damage and chromosomal instability demonstrating a mutator phenotype. These cells also exhibit enhanced chromosomal gains or losses suggesting that autophagy functions as a tumor suppressor by limiting chromosomal instability. Thus the impairment of survival to metabolic stress due to deficiency in autophagy may be compensated by an enhanced mutation rate thereby promoting tumorigenesis. The protective role of autophagy may be exploited in developing novel autophagy modulators as rational chemotherapeutic as well as chemopreventive agents.

Addendum to:

Autophagy Supresses Tumor Progression by Limiting Chromosomal Instability

R. Mathew, S. Kongara, B. Beaudoin, C.M. Karp, K. Bray, K. Degenhardt, G. Chen, S. Jin and E. White

Genes Dev 2007; 21:1367-81     

Metformin alleviates hepatosteatosis by restoring SIRT1-mediated autophagy induction via an AMP-activated protein kinase-independent pathway

Metformin activates both PRKA and SIRT1. Furthermore, autophagy is induced by either the PRKA-MTOR-ULK1 or SIRT1-FOXO signaling pathways. We aimed to elucidate the mechanism by which metformin alleviates hepatosteatosis by examining the molecular interplay between SIRT1, PRKA, and autophagy. ob/ob mice were divided into 3 groups: one with ad libitum feeding of a standard chow diet, one with 300 mg/kg intraperitoneal metformin injections, and one with 3 g/d caloric restriction (CR) for a period of 4 wk. Primary hepatocytes or HepG2 cells were treated with oleic acid (OA) plus high glucose in the absence or presence of metformin. Both CR and metformin significantly improved body weight and glucose homeostasis, along with hepatic steatosis, in ob/ob mice. Furthermore, CR and metformin both upregulated SIRT1 expression and also stimulated autophagy induction and flux in vivo. Metformin also prevented OA with high glucose-induced suppression of both SIRT1 expression and SIRT1-dependent activation of autophagy machinery, thereby alleviating intracellular lipid accumulation in vitro. Interestingly, metformin treatment upregulated SIRT1 expression and activated PRKA even after siRNA-mediated knockdown of PRKAA1/2 and SIRT1, respectively. Taken together, these results suggest that metformin alleviates hepatic steatosis through PRKA-independent, SIRT1-mediated effects on the autophagy machinery.     

Systemic regulation of autophagy in Caenorhabditis elegans

When no supply of environmental nutrients is available, cells induce autophagy, thereby generating a source of emergency metabolic substrates and energy to maintain the basal cellular activity needed for survival. This autophagy response to starvation has been well characterized in various multicellular organisms, including worms, flies, and mice. Although prosurvival effects of autophagy in response to starvation are well known in animals, the mechanisms by which animals regulate and coordinate autophagy systemically remain elusive. Using C. elegans as a model system, we found that specific amino acids could regulate starvation-induced autophagy, and that MGL-1 and MGL-2, Caenorhabditis elegans homologs of metabotropic glutamate receptors, were involved. MGL-1 and MGL-2 specifically acted in AIY and AIB neurons, respectively, to modulate the autophagy response in other tissues such as pharyngeal muscle. Our recent study suggests that the autophagy response to starvation, previously thought to be cell-autonomous, can be systemically regulated, and that there is a specific sensor for monitoring systemic amino acids levels in Caenorhabditis elegans.