Near-infrared light increases ATP, extends lifespan and improves mobility in aged Drosophila melanogaster

Abstract

Ageing is an irreversible cellular decline partly driven by failing mitochondrial integrity. Mitochondria accumulate DNA mutations and reduce ATP production necessary for cellular metabolism. This is associated with inflammation. Near-infrared exposure increases retinal ATP in old mice via cytochrome c oxidase absorption and reduces inflammation. Here, we expose fruitflies daily to 670 nm radiation, revealing elevated ATP and reduced inflammation with age. Critically, there was a significant increase in average lifespan: 100–175% more flies survived into old age following 670 nm exposure and these had significantly improved mobility. This may be a simple route to extending lifespan and improving function in old age.

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Research
Cite this article: Begum R, Calaza K, Kam JH,
Salt TE, Hogg C, Jeffery G. 2015 Near-infrared
light increases ATP, extends lifespan and
improves mobility in aged Drosophila
melanogaster.Biol. Lett. 11: 20150073.
http://dx.doi.org/10.1098/rsbl.2015.0073
Received: 30 January 2015
Accepted: 23 February 2015
Subject Areas:
health and disease and epidemiology,
behaviour
Keywords:
lifespan, ATP, fly, inflammation
Author for correspondence:
Glen Jeffery
e-mail: g.jeffery@ucl.ac.uk
Physiology
Near-infrared light increases ATP, extends
lifespan and improves mobility in aged
Drosophila melanogaster
Rana Begum1, Karin Calaza2, Jaimie Hoh Kam1, Thomas E. Salt1, Chris Hogg3
and Glen Jeffery1
1
Institute of Ophthalmology, University College London, London EC1V 9EL, UK
2
Program of Neuroscience, Institute de Biologia, Universidade Federal Fluminense, Rio de Janeiro 24210130, Brazil
3
Moorfields Eye Hospital, London EC1V 2PD, UK
Ageing is an irreversible cellular decline partly driven by failing mitochondrial
integrity. Mitochondria accumulate DNA mutations and reduce ATP
production necessary for cellular metabolism. This is associated with
inflammation. Near-infrared exposure increases retinal ATP in old mice via
cytochrome coxidase absorption and reduces inflammation. Here, we expose
fruitflies daily to 670 nm radiation, revealing elevated ATP and reduced inflam-
mation with age. Critically, there was a significant increase in average lifespan:
100– 175% more flies survived into old age following 670 nm exposure and
these had significantly improved mobility. This may be a simple route to
extending lifespan and improving function in old age.
1. Introduction
Mitochondria provide cellular energy via adenosine triphosphate (ATP). But,
their DNA (mtDNA) suffers from progressive mutations resulting in reduced
ATP production, which is thought to run concomitantly with an increase in
pro-inflammatory reactive oxygen species (ROS) [1,2]. Hence, hallmarks of
ageing are reduced cellular energy and progressive systemic inflammation. Meta-
bolic demand also plays a role as tissues and organisms with high metabolic rates
generally suffer from rapid ageing [3,4]. The retina has the greatest metabolic
demand in the body [5], but ATP decline in the central nervous system can be
significantly improved by near-infrared/infrared light (NIR/IR, [6]). Specific
wavelengths in this range are absorbed by cytochrome coxidase in mitochondrial
respiration, improving its efficiency [7– 10]. These wavelengths improve mito-
chondrial membrane potentials, significantly reduce inflammation and reduce
macrophage numbers with brief exposures of around 60–90 s repeated over
approximately a week [11,12]. NIR/IR also reduces experimental pathology
when insult impacts on mitochondrial function, as in experimental Parkinson’s
disease, where NIR significantly reduces cell death in the substantia nigra [13].
However, NIR/IR studies have largely used light for short periods and their
impact on lifespan has not been assessed [7,11,12]. If NIR improves mitochondrial
function we predict it may extend life. The fly has been used here because of
its relatively short life [14]. Hence, we ask if long-term exposure to 670 nm in
Drosophila melanogaster can increase lifespan and improve function in old age.
2. Material and methods
Drosophila melanogaster were used. Hatched male flies were housed on 12/12 light
cycle at 258C within a season. Half were exposed to 670 nm for 20 min per day at
&2015 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution
License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original
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40 mW cm
22
in clear plastic 50 cm
3
(28 mm wide) containers,
illuminating flies from either side, which were counted weekly.
Room illumination was 2 mW cm
22
. 670 nm energies were
approximately 100 times lower than indirect sunlight, consistent
with earlier studies [7]. Light devices were built by C. H. Elec-
tronics UK and contained 50 670 nm LEDS over 20 cm
2
. Six
independent replicates were used in lifespan experiments (n¼
620 flies). ATP, inflammation and mobility were assessed at
seven weeks, when ATP and mobility are known to decline [15].
ATP was measured by luciferin –luciferase assay (Enliten
w
ATP
Assay System, Promega). Flies were killed with liquid nitrogen,
transferred to 2.5% trichoroacetic acid (TCA), then homogenized
at 48C. Supernatant was collected and the TCA was neutralized
with 1 M Tris– acetate buffer (pH 7.75, final TCA concentration
0.0625%); 10 ml of neutralized solution was added to 100 ml of luci-
ferin– luciferase in fresh buffer. ATP was measured using an Orion
microplate luminometer (Berthold Detection Systems GmbH) and
data normalized to fly numbers.
Tissues were homogenized in 2% sodium dodecyl sulfate (SDS)
with protease inhibitor cocktail for Western blot (Roche Diagnos-
tics), and centrifuged; the supernatant was pipetted out,
separated with 10% SDS– PAGEand electrophoretically transferred
onto nylon membranes. Immunoblotting was undertaken for
complement component C3 (Cappel, MP Biomedicals), which is
highly conserved [16]. Protein was quantified by densitometric
X-ray scanning and values were normalized to a-tubulin.
Fly mobility assessment was as Bjedov et al. [14]. Flies were
placed in 100 ml clear cylinders (seven flies per trial), tapped to
the bottom and then videoed, the last two steps repeated three
times. Using the videos, the number of flies above the 50 ml
mark (9 cm from the bottom) was counted after 1 min. Individual
flies were traced, with absolute distance travelled measured.
Data were analysed with GraphPad PRISM v. 5 and statistical
analysis was undertaken using Mann– Whitney Unon-parametric
and log-rank tests.
3. Results
(a) ATP levels are elevated and systemic inflammation
reduced
Whole body ATP declines with age only after approximately
seven weeks [14], when ATP was measured here. ATP concen-
trations were significantly greater, by approximately 80%, in
670 nm exposed animals compared with unexposed (figure 1a,
Mann–Whitney test p¼0.028). At seven weeks, Western blots
were undertaken for inflammatory marker complement com-
ponent C3. This was reduced in 670 nm exposed flies
compared with controls (figure 1b). Hence, 670 nm radiation
elevates ATP and reduces inflammation.
(b) Lifespan increases
Fly numbers in experimental and control groups were similar
in the two weeks post-hatching. From week 3, fly deaths were
greater in controls than 670 nm exposed flies and they
remained so at each time point until week 11– 12, when all
flies were dead in both groups. This difference was significant
(figure 2, log-rank test p¼0.008).
The progressive mean percentage increase in 670 nm flies
alive over controls is given in figure 2b. Group differences
accelerated from week 4, when 10% extra 670 nm treated
flies were alive compared with controls, to approximately
50% extra when the control population had halved. By the
time the control population was reduced by 80%, at week
8, more than 100% extra 670 nm treated flies remained
alive. Subsequently, group differences reached almost 180%
before declining to zero in both groups at week 11– 12.
Hence, 670 nm did not extend absolute lifespan.
(c) Aged mobility increases
Mobility of 670 nm treated and control flies was measured at
seven weeks. Significantly more 670 nm treated flies climbed
above the 50 ml level (9 cm) and significantly more travelled
a greater distance than controls (Mann–Whitney test p¼
0.028, p¼0.014, respectively). Twice as many 670 nm flies
climbed above 50 ml (9 cm) compared with controls and
these travelled twice the distance in 1 min compared with
controls (figure 2c,d). Hence, 670 nm exposure significantly
improves both lifespan and mobility.
4. Discussion
Drosophila melanogaster has been widely used in lifespan
studies as they are short lived and their genomic sequence
is relatively well understood [14,17], hence their adoption
experimentally here to extend lifespan. Our results reveal
[ATP] (nM)/fly
ATP level(a)
C3 expression (Western blot)(b)
control
absolute intensity
670
control 670
control
C3 ~ 110 kDa
a-tubulin 55 kDa
670
*
0
0
500
1000
200
400
600
Figure 1. Exposure to 670 nm radiation increases ATP in aged flies and reduces
inflammation. (a) Seven week old flies exposed to 670 nm had a significant increase
in whole body ATP compared with controls, p¼0.028. n¼25 flies per group.
(b) Whole body inflammation (C3) was measured in seven week flies using Western
blot. This was reduced in 670 nm exposed flies by approximately 15%. Here, flies
were pooled within groups as C3 protein levels were low in individuals. Hence there
are no error bars. n¼15 flies per group. (Online version in colour.)
rsbl.royalsocietypublishing.org Biol. Lett. 11: 20150073
2

that when flies are exposed to 670 nm radiation they have
reduced inflammation, improved ATP, improved mobility
and extended average lifespans. These data are consistent
with the majority of studies undertaken using 670 nm on
mammals, showing reduced inflammation in experimental
models and in ageing, and improved ATP levels [6,7]. How-
ever, it would be difficult to undertake lifespan experiments
in mice as the light would not penetrate the entire body as it
does in flies and hence its influence would not be systemic.
There are many factors and pathways in ageing, and nine
candidate hallmarks have been suggested, which may be
separate, but also are likely to have interactions [1]. Mito-
chondrial function is one. Previously, mitochondrial
function and ageing were viewed within a framework of pro-
gressive mtDNA mutations/deletions resulting in reduced
ATP and increased ROS. The balance of these factors was
seen as a driver in the mitochondrial theory of ageing [18].
However, evidence has undermined the role of ROS in
ageing [19,20]. Hence, some mutant mice have reduced life-
span as a result of mtDNA mutations/deletions not
associated with increased ROS [21,22]. Further, increased
ROS can prolong lifespan in yeast and Caenorhabditis elegans
[22,23], and in mammals it does not accelerate ageing [20].
These data are reviewed by Lopez-Otin et al. [1], who argue
that low ROS may activate compensatory mechanisms and
not directly contribute to ageing. Such data may undermine
the ROS element in Harman’s mitochondrial theory [18]. If
correct, it places greater potential emphasis on ATP in ageing.
NIR has been successful in treating induced pathology [7]
and ageing, particularly in the retina, where progressive age-
related inflammation is marked owing to high metabolic rate
[11,12]. These wavelengths penetrate deeply and 670 nm
trans-illuminated our flies at 40 mW cm
22
. In relation to this,
it may be significant that, while old domestic incandescentlight-
ing contained significant NIR elements, none is present in
modern strip lighting or energy-saving domestic lighting [12].
The absence of these wavelengths from artificial lighting may
have long-term consequences. As longer wavelengths penetrate
deeply, this may be of significance not only for the ageing eye,
but also potentially for other tissues.
Ethics statement. Fly research is free of legal ethical constraint.
Data accessibility. All data are presented in the manuscript.
Acknowledgement. We thank Iris Salecker, Giovanna Vinti and Tobi
Weinrrich for technical assistance.
Author contributions. G.J. designed experiments and wrote the manu-
script. All authors undertook the experiments and approved the
final version of the manuscript. R.B. analysed the data.
Funding statement. Supported by the Rosetrees Trust UK. K.C. was a
research fellow from CAPES Brazil ( proc. 18134/12-2).
Competing interests. We have no competing interests.
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