Koen Houthoofd

Life extension via dietary restriction is independent of the Ins/IGF-1 signalling pathway in Caenorhabditis elegans

Dietary restriction (DR) increases life span in a wide variety of animals. In Caenorhabditis elegans both reduced bacterial concentration (BDR) and culture on non-bacterial, semi-defined, axenic food sources (ADR) increased longevity. An Ins/IGF-1-like (IIF) signalling pathway has been shown to specify life span in C. elegans and it has been suggested that this IIF signalling pathway mediates life extension via DR. We show that both ADR and BDR act independently with mutations in the IIF pathway to increase longevity, stress resistance, and specific activities of superoxide dismutase and catalase. Moreover, these effects are not dependent on daf-16, which is known to suppress other mutations that act through the IIF pathway. We conclude that DR extends life span by mechanisms distinct from those specified by the IIF pathway.

Axenic growth up-regulates mass-specific metabolic rate, stress resistance, and extends life span in Caenorhabditis elegans

Culture in axenic medium causes two-fold increases in the length of development and adult life span in Caenorhabditis elegans. We asked whether axenic medium imposes dietary restriction (ADR), and causes changes in metabolic activity and stress resistance. Eat mutants, which have a reduced food intake, were studied in parallel with wild-type worms to assess potential synergistic actions of axenic culture and food restriction. We found that axenic culture enhances metabolic activity as assessed by mass-specific oxygen consumption rate and heat production. Axenic culture also caused higher activities of the antioxidant enzymes superoxide dismutase and catalase, and led to increased resistance to high temperature, which was further exacerbated by mutation in eat-2. These results show that axenic medium up-regulates a variety of somatic maintenance functions including oxidative and thermal stress resistance and that food restriction due to axenic growth and to mutation in eat-2 are very similar but not identical.

No reduction of metabolic rate in food restricted Caenorhabditis elegans.

Dietary restriction (DR) is the most consistent means of extending life span throughout the animal kingdom. Multiple mechanisms by which DR may act have been proposed but none are clearly predominant. We asked whether metabolic rate and stress resistance is altered in Caenorhabditis elegans in response to DR. DR was imposed in two complementary ways: by growing wild-type worms in liquid medium supplemented with reduced concentrations of bacteria and by using eat-2 mutants, which have a feeding defect. Metabolic rate was not reduced when we fed wild-type worms reduced food and was up-regulated in the eat-2 mutants in liquid culture, as assessed by oxygen consumption rate and heat production. The specific activity levels of the antioxidant enzymes superoxide dismutase (SOD) and catalase showed small increases when we reduced food in wild-type worms, but restricted worms acquired no elevated protection against paraquat and hydrogen peroxide. eat-2 mutants showed elevated specific activities of SOD and catalase relative to wild type in liquid culture. These results indicate that the effects imparted by DR and the eat-2 mutation are not identical, and they contradict, at least in C. elegans, the widespread belief that CR acts by lowering the rate of metabolism.

Assaying metabolic activity in ageing Caenorhabditis elegans

Accurate measures of physiological and metabolic condition could provide more insight into how longevity genes and signalling pathways affect global metabolic activity and life span. The present study is essentially a methodological treatise in which we describe and evaluate a number of methods to assess changes of metabolic activity in ageing Caenorhabditis elegans. Oxygen consumption and CO(2) production rate assays, and measurement of the heat output by microcalorimetry are performed using live worms. For other assays, frozen (-75 degrees C) samples can be used. A lucigenin-mediated light production assay provides information on the metabolic capacity (scope for metabolic activity) of the worms just before freezing. Assaying ATP and ADP levels provides a measure of the instantly available energy. The XTT assay measures the activity of enzymes that can reduce XTT. Blue fluorescence emitted at 420-470 nm is a potentially useful biomarker of the rate of ageing. A protein quantification protocol for normalising all data for quantitative comparisons is presented. We illustrate how these methods can validate or disprove models of gene action inferred from molecular identification.

Assessing metabolic activity in aging Caenorhabditis elegans: concepts and controversies.

It is widely believed that normal by‐products of oxidative metabolism and the subsequent molecular damage inflicted by them couple the aging process to metabolic rate. Accordingly, high metabolic rates would be expected to accelerate aging, and life‐extending interventions are often assumed to act by attenuating metabolic rate. Notorious examples in Caenorhabditis elegans are food restriction, mutation in the Clock genes and several genes of the insulin‐like signalling pathway. Here we discuss how metabolic rate can be accurately measured and normalized, and how to deal with differences in body size. These issues are illustrated using experimental data of the long‐lived mutant strains clk‐1(e2519) and daf‐2(e1370). Appropriate analysis shows that metabolic rates in wild‐type and in the clk‐1 mutant are very similar. In contrast, the metabolic rate profiles point to a metabolic shift toward enhanced efficiency of oxidative phosphorylation in the daf‐2 worms.

Metabolism, physiology and stress defense in three aging Ins/IGF‐1 mutants of the nematode Caenorhabditis elegans

The insulin/insulin‐like growth factor‐1 (Ins/IGF‐1) pathway regulates the aging rate of the nematode Caenorhabditis elegans. We describe other features of the three Ins/IGF‐1 mutants daf‐2, age‐1 and aap‐1. We show that the investigated Ins/IGF‐1 mutants all have a reduced body volume, reduced reproductive capacity, increased ATP concentrations and an elevated stress resistance. We also observed that heat production is lower in these mutants, although the respiration rate was similar or higher compared with wild‐type individuals, suggesting a metabolic shift in these mutants.

Alternative Oxidase Dependent Respiration Leads to an Increased Mitochondrial Content in Two Long-Lived Mutants of the Ageing Model Podospora anserina

The retrograde response constitutes an important signalling pathway from mitochondria to the nucleus which induces several genes to allow compensation of mitochondrial impairments. In the filamentous ascomycete Podospora anserina, an example for such a response is the induction of a nuclear-encoded and iron-dependent alternative oxidase (AOX) occurring when cytochrome-c oxidase (COX) dependent respiration is affected. Several long-lived mutants are known which predominantly or exclusively respire via AOX. Here we show that two AOX-utilising mutants, grisea and PaCox17::ble, are able to compensate partially for lowered OXPHOS efficiency resulting from AOX-dependent respiration by increasing mitochondrial content. At the physiological level this is demonstrated by an elevated oxygen consumption and increased heat production. However, in the two mutants, ATP levels do not reach WT levels. Interestingly, mutant PaCox17::ble is characterized by a highly increased release of the reactive oxygen species (ROS) hydrogen peroxide. Both grisea and PaCox17::ble contain elevated levels of mitochondrial proteins involved in quality control, i. e. LON protease and the molecular chaperone HSP60. Taken together, our work demonstrates that AOX-dependent respiration in two mutants of the ageing model P. anserina is linked to a novel mechanism involved in the retrograde response pathway, mitochondrial biogenesis, which might also play an important role for cellular maintenance in other organisms.

Rebuttal to Van Voorhies: ‘The influence of metabolic rate on longevity in the nematode Caenorhabditis elegans’

The papers by Van Voorhies in Free Radical Biology & Medicine ( 33 , 587–596, 2002) and in this Journal claim that the major longevity-extending mutations in C. elegans essentially act by reducing metabolic rate as predicted by the rate-of-living theory, and do not alter any metabolically independent mechanism specific to aging. In contrast, we found no evidence of a reduction in metabolic rate in these mutants using different experimental approaches. Now, Van Voorhies challenges the accuracy of our experimental results. Some of the criticisms raised by Van Voorhies point to misunderstanding. For example, he is puzzled by the fact that the energy value of the oxygen consumed fails to match the measured heat flux. The simple answer is that this conversion is inappropriate since different instruments involving different environmental conditions are used, as explained in our contribution. Van Voorhies also assumes that the ATP content in the worms is bound to remain constant because of the well known principle that ATP is made as needed, and he infers that our method must be inappropriate ‘because significant amounts of ATP can hydrolyse before heat inactivation of ATPases occurs’. The fact is that we submerge frozen ( − 75 ° C) microvials, containing no more than 100 μ L worm suspension, in boiling water. It takes little time for the sample to reach boiling temperature. The ATP concentrations thus measured still decrease exponentially with age. Do we really lose ATP as an exponential function of age? Van Voorhies suggests that ATPase activity can be responsible for this effect. Because ATP shows a 7–10-fold (not 30-fold as mentioned in the Van Voorhies paper) decrease and oxygen consumption decreases a mere 3-fold, this implies that ATPase activity should increase 3-fold with age. This would be a major discovery. However, in our opinion it is very unlikely that ATPases, an impressive group of enzymes involved in a myriad of cellular functions, would increase their activity 3 times (on average) over life span. The alternative explanation is that, in C. elegans , ATP effectively decreases with age in vivo , although the mechanism responsible for this change remains unknown. We hypothesize that the efficiency of ATP production may decline, which would also explain the faster decline of ATP relative to oxygen consumption and heat output. Van Voorhies criticises the lucigenin-mediated light production assay. As explained in our contribution, this assay measures the light that is produced when freeze-thawed worms are suspended in assay medium containing lucigenin, NADH, NADPH and KCN (blocking cytochrome oxidase and Cu/ZnSOD). The nicotineamide coenzymes drive reactions that produce superoxide to a maximal speed limited by the activity of the biochemical reactions involved. Superoxide thus produced reacts with lucigenin, resulting in luminescence. Superoxide is produced at several sites, but we have estimated that respiration contributes well over 70% of all superoxide produced (Braeckman et al. , Mech. Aging Dev ., 123 , 105–119, 2002). It should be stressed that this assay measures a capacity, a maximum metabolic (mostly mitochondrial) output under the artificial assay conditions. The decrease of luminescence with age reflects a progressive reduction of metabolic (mostly mitochondrial) capacity, not metabolic rate. In the case of the mitochondrial contribution, the light production assay reflects the activity of complex I and downstream elements to transmit electrons under uncoupled conditions (resulting from membrane damage during freezing). Since cytochrome oxidase is blocked, the upstream electron transport system will be in a reduced form, resulting in enhanced superoxide production which remains dependent, however, on the flux of electrons from complex I to cytochrome oxidase. This flux declines with age probably because of increasing failure of components of the electron transport chain. We first saw a similar age-specific decline of lucigenin-mediated light production in microsome fractions prepared from fer-15 and fer-15 ; age-1 mutant worms. Age-dependent decline was attenuated in the Age mutant (Vanfleteren, Biochem. J ., 292 , 605–608, 1993). Van Voorhies also sees conceptual problems in the fact that light production levels decrease far more quickly than measures of oxygen consumption. Our explanation is that the light production assay measures a capacity, i.e. an activity in vitro under forced artificial conditions. The performance measured under these conditions can very well decline before any effect becomes visible in vivo , under natural conditions. Correspondence Jacques R. Vanfleteren, Department of Biology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium. Tel.: 32 9264 52 12; fax: 32 9264 87 93; e-mail: Jacques.Vanfleteren@rug.ac.be

Metabolism and life span determination in C. elegans

Publisher Summary This chapter discusses the metabolism and life span determination in Caenorhabditis elegans . Several mechanisms can extend longevity of C. elegans , including caloric restriction and single gene mutations notably in the Ins/IGF pathway and in any one of the clk genes. All of them affect metabolism, and it is important to establish the changes consistently associated with aging. Caloric restriction causes increases rather than decreases in metabolic rate, measured as respiration and heat production rates. Reduced Ins/IGF signaling does not affect respiration rates, however lowers heat dissipation and possibly CO 2 production. Mutation in any one of the clk genes deregulates, and on average slows down, a number of temporal processes, however does only mildly, at most, affect respiration and heat production rates. Longevity is generally associated with increased resistance to stress, particularly oxidative stress, which is also reflected by co-ordinated increases in catalase and superoxide dismutase (SOD) activities. Consistent up-regulation of these enzyme activities is not seen in clk mutants. Life extension by dietary restriction is independent of the Ins/IGF signaling pathway in C. elegans , like in the mouse.

Chapter 5. Energy metabolism, anti-oxidant defense and aging in Caenorhabditis elegans

Conditional Senescence in Prokaryotes.- Aging and Mitochondrial Dysfunction in the Filamentous Fungus Podospora anserina.- Caulobacter: Model for Prokaryotic Replicative Senescence and Trade-off Theories of Aging.- Mitochondria, Metabolism and Aging: the Retrograde Response and Calorie Restriction.- Mitochondrial ROS production and Apoptosis-like Processes in Yeast.- Energy Metabolism, Anti-oxidant Defense and Aging in Caenorhabditis elegans.- Do Green Plants Age and, if so, How? Mammalian Aging, Oxygen Radicals and Restricted Feeding.- Aging and the Programmed Death Phenomena.- The Human Werner Syndrome as a Model for Aging.- Towards a Role of Subcytotoxic Stress in Tissue Ageing.-