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polar body transfer is complicated by the

need to identify pronuclear gender, which

is relatively easy in mice though more

complicated in primates.

The ultimate objective of this body of

work is to initiate human clinical trials, in

carrier women with a prior affected child,

in efforts to prevent disease transmission

in subsequent children. Since mitochon-

drial replacement involves germline gene

therapy, however, such approaches are

currently restricted. Regulatory agencies

in the United States and United Kingdom

are evaluating safety and efficacy issues

based on animal model studies such as

Wang et al., 2014 and those described

herein (Callaway, 2014); however, it re-

mains to be seen when authorization will

be forthcoming and whether polar body

transfer will be included as an approved

approach.

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Mitobolites: The Elixir of Life

Subhash D. Katewa,1 Amit Khanna,1 and Pankaj Kapahi1,* 1Buck Institute for Research on Aging, Novato, CA 94945, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cmet.2014.06.013

One of the biggest challenges in biology is to understand how mitochondria influence aging and age-related diseases. Chin et al. (2014) reveal how a mitochondrial metabolite (mitobolite) inhibits mitochondrial ATPase and extends lifespan by mimicking dietary restriction in worms.

The free radical theory of aging proposes

that accumulating macromolecular dam-

age due to increased reactive oxygen

species over time causes aging. As

mitochondria are believed to be the

main contributors of free radicals,

reducing mitochondrial electron transport

chain function would be expected to in-

crease lifespan. Several studies in nema-

todes, flies, and mice have corroborated

this idea by demonstrating that genetic

inhibition of several mitochondrial com-

ponents, especially those of the electron

transport chain (ETC) complexes, can

extend lifespan (Aguilaniu et al., 2005).

However, several findings in the field

have diminished the enthusiasm for the

idea that simply the inhibition of mito-

chondrial function or even free radicals

will extend lifespan in mammals. Further-

more, restricting nutrients in mammals or

flies is accompanied by enhancement

of mitochondrial biogenesis and function

(Guarente, 2008; Zid et al., 2009). This

in principle opposes the ‘‘Warburg ef-

fect,’’ as the enhancement of mitochon-

drial function helps the organism switch

from glycolysis to oxidative metabolism,

which mediates the protective effects of

dietary restriction, such as slowing aging

and decreased cancer rates. How and

when an organism reduces mitochon-

drial function to extend lifespan, how-

ever, remains unclear. Recent work by

Jing Huang and colleagues suggests

that a mitochondrial metabolite (mito-

bolite) extends lifespan by inhibition of

mitochondrial ATPase through mecha-

nisms overlapping with dietary restriction

in Caenorhabditis elegans (Chin et al.,

2014).

Mitochondrial tricarboxylic acid cycle

(TCA) metabolites, such as pyruvate,

fumarate, malate, and oxaloacetate,

have been previously shown to extend

lifespan upon feeding in C. elegans (Mou-

chiroud et al., 2011; Williams et al., 2009),

though the mechanistic underpinnings

of the prolongevity effects remain to be

delineated. Chin et al. now add a-ketoglu-

tarate (KG) to the list of ‘‘youthful’’ TCA

intermediates. Using a novel, unbiased

technique, DARTS (drug affinity respon-

sive target stability), they identify the bind-

ing proteins of KG that may explain its

effects on aging. In this assay, cell lysates

were incubated in varying concentrations

of KG, followed by a complete proteoly-

sis by a mixture of proteases, with the

assumption that if KG is binding to a

protein, that protein or the associated

fragment will be protected from protease

action. Uncleaved fragments were sepa-

rated on a gel and further identified

by liquid chromatography-tandem mass

spectrometry. Chin et al. found that KG

binds to several proteins, including two

subunits of mitochondrial ATPase. Using

several biochemical assays, including

8 Cell Metabolism 20, July 1, 2014 ª2014 Elsevier Inc.

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measurement of ATPase activity, respira-

tion in isolated mitochondria, and mea-

surement of ATP levels in worms after

KG feeding, the authors confirmed that

KG indeed inhibits mitochondrial ATPase

activity. Consistently, reduction of atp2

(C. elegans mitochondrial ATPase b sub-

unit) by RNAi led to increased lifespan,

which was not further increased by KG

feeding.

One consequence of inhibition of mito-

chondrial ATPase is reduction in cellular

ATP levels. This in turn could lead to

increased AMP and activation of the

energy-sensing kinases such as AMP ki-

nase. The authors observed a reduction

in ATP levels, though the lifespan exten-

sion upon KG feeding was not dependent

on AMPK or hypoxia-inducible factor-1a.

Inhibition of target of rapamycin (TOR),

which mediates the effects of dietary

restriction in multiple species (Kapahi

et al., 2010), did not further extend life-

span upon KG feeding. However, the

longevity effects were dependent on

pha-4, known to be critical for life-

span extension by dietary restriction in

C. elegans (Panowski et al., 2007). The au-

thors also demonstrated that KG inhibits

TOR in both human and mouse cell lines,

though the mechanisms of this interaction

remain to be elucidated. Consistent

with the effects of inhibition of TOR, auto-

phagy was enhanced in worms fed with

KG, treated with oligomycin (ATPase

inhibitor), or carrying atp-2 mutations.

Together, these results suggest that KG-

dependent ATPase inhibition modulates

autophagy in a TOR-dependent manner

to extend lifespan.

When defining the impact of modulating

mitochondrial function, there tends to be

an overemphasis on measuring ROS and

ATP levels, which fails to take into consid-

eration the dynamic role of ‘‘mitobolites’’

(Figure 1). Mitochondria originated by

endosymbiosis, and over time they lost

most of their DNA, which normally plays

an important role in responding to envi-

ronmental perturbations. However, mito-

bolites may act as the key sensors of

mitochondrial perturbations and mediate

nuclear-mitochondrial interactions. The

study by Chin et al. adds to the emerging

evidence on the growing role of such

mitobolites in modulating metabolism,

growth, disease processes, and longevity

(Chandel, 2014). Many unanswered ques-

tions remain about mitobolites. How is the

mitochondrial metabolite flux regulated?

How are levels of various metabolites in-

tegrated to code for certain responses?

What are the different targets, and their

tissue specificity, that are regulated by

the secreted mitobolites? Also, what is

the role of non-TCA mitobolites in aging

such as those generated by fatty acid

oxidation, urea cycle, cardiolipin syn-

thesis, heme, quinone, and steroid

biosynthesis? Recent studies have indi-

cated that the exometabolome (metabo-

lite excreted in the environment by the

worm) of long-lived mitochondrial mu-

tants is significantly different from short-

lived mitochondrial mutants or other

animals (such as insulin receptor mutant,

daf-2), supporting the possibility that

mitobolites can further extend lifespan in

certain long-lived mutants (Butler et al.,

2010, 2013). Thus, one may have to inte-

grate the levels of various mitobolites

and the cellular state to decipher how

each perturbation will influence the down-

stream events to modulate aging.

The elucidation of the mechanism by

which KG inhibits mitochondrial ATPase

and mimics the effects of dietary re-

striction underscores the important role

mitobolites play as potential signaling

molecules. Wider appreciation of the role

of mitobolites will not only help resolve

some of the paradoxes in the field but

also provide excellent therapeutic targets

to counteract aging and age-related dis-

eases in mammals.

ACKNOWLEDGMENTS

This work was funded by grants from the American Federation for Aging Research and the NIH (R01 AG038012 and RO1 AG038688 to P.K.).

REFERENCES

Aguilaniu, H., Durieux, J., and Dillin, A. (2005). Genes Dev. 19, 2399–2406.

Butler, J.A., Ventura, N., Johnson, T.E., and Rea, S.L. (2010). FASEB J. 24, 4977–4988.

Butler, J.A., Mishur, R.J., Bhaskaran, S., and Rea, S.L. (2013). Aging Cell 12, 130–138.

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Chin, R.M., Fu, X., Pai, M.Y., Vergnes, L., Hwang, H., Deng, G., Diep, S., Lomenick, B., Meli, V.S., Monsalve, G.C., et al. (2014). Nature. Published online May 14, 2014. http://dx.doi.org/10.1038/ nature13264.

Guarente, L. (2008). Cell 132, 171–176.

Kapahi, P., Chen, D., Rogers, A.N., Katewa, S.D., Li, P.W., Thomas, E.L., and Kockel, L. (2010). Cell Metab. 11, 453–465.

Mouchiroud, L., Molin, L., Kasturi, P., Triba, M.N., Dumas, M.E., Wilson, M.C., Halestrap, A.P., Rous- sel, D., Masse, I., Dallière, N., et al. (2011). Aging Cell 10, 39–54.

Panowski, S.H., Wolff, S., Aguilaniu, H., Durieux, J., and Dillin, A. (2007). Nature 447, 550–555.

Williams, D.S., Cash, A., Hamadani, L., and Die- mer, T. (2009). Aging Cell 8, 765–768.

Zid, B.M., Rogers, A.N., Katewa, S.D., Vargas, M.A., Kolipinski, M.C., Lu, T.A., Benzer, S., and Kapahi, P. (2009). Cell 139, 149–160.

Figure 1. The Role of Mitochondria-Derived Metabolites, ‘‘Mitobolites,’’ in Modulating Metabolism, Growth, and Longevity Interventions that modulate mitochondrial functions regulate the flux of mitobolites. Mitobolites (red dots) potentially regulate mitochondrial proteins, including mitochondrial ATPase (black structures), and once released from mitochondria, these mitobolites may also regulate cellular signaling pathways. This in turn modulates various physiological processes like metabolism, growth, and longevity.

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