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