Intramyocellular fatty-acid metabolism plays a critical role in mediating responses to dietary restriction in Drosophila melanogaster.
ABSTRACT Changes in fat content have been associated with dietary restriction (DR), but whether they play a causal role in mediating various responses to DR remains unknown. We demonstrate that upon DR, Drosophila melanogaster shift their metabolism toward increasing fatty-acid synthesis and breakdown, which is required for various responses to DR. Inhibition of fatty-acid synthesis or oxidation genes specifically in the muscle tissue inhibited life-span extension upon DR. Furthermore, DR enhances spontaneous activity of flies, which was found to be dependent on the enhanced fatty-acid metabolism. This increase in activity was found to be at least partially required for the life-span extension upon DR. Overexpression of adipokinetic hormone (dAKH), the functional ortholog of glucagon, enhances fat metabolism, spontaneous activity, and life span. Together, these results suggest that enhanced fat metabolism in the muscle and physical activity play a key role in the protective effects of DR.
- SourceAvailable from: Michael Rera[Show abstract] [Hide abstract]
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ABSTRACT: p53 Inducible gene 6 (PIG6) encodes mitochondrial proline dehydrogenase (PRODH) and is up-regulated several fold upon p53 activation. Proline dehydrogenase is proposed to generate radicals that contribute to cancer cell apoptosis. However, there are at least 10 mitochondrial sites that can produce superoxide and/or H2O2, and it is unclear whether proline dehydrogenase generates these species directly, or instead drives production by other sites. Amongst six cancer cell lines, ZR75-30 human breast cancer cells had the highest basal proline dehydrogenase levels, and mitochondria isolated from ZR75-30 cells consumed oxygen and produced H2O2 with proline as sole substrate. Insects use proline oxidation to fuel flight, and mitochondria isolated from Drosophila melanogaster were even more active with proline as sole substrate than ZR75-30 mitochondria. Using mitochondria from these two models we identified the sites involved in formation of superoxide/H2O2 during proline oxidation. In mitochondria from Drosophila the main sites were respiratory complexes I and II. In mitochondria from ZR75-30 breast cancer cells the main sites were complex I and the oxoglutarate dehydrogenase complex. Even with combinations of substrates and respiratory chain inhibitors designed to minimize the contributions of other sites and maximize any superoxide/H2O2 production from proline dehydrogenase itself, there was no significant direct contribution of proline dehydrogenase to the observed H2O2 production. Thus proline oxidation by proline dehydrogenase drives superoxide/H2O2 production, but it does so mainly or exclusively by providing anaplerotic carbon for other mitochondrial dehydrogenases and not by producing superoxide/H2O2 directly.Redox Biology. 01/2014;
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ABSTRACT: Phote-HrTH slows down or inhibits the crop lobe muscles (P5).•Phote-HrTH stimulates the muscles of the pump 4 (P4).•Crop emptying and gut filling are positively correlated with the P4 contraction rate.•Phote-HrTH promotes emptying of the crop and transfer of sugar solution to the gut.Journal of Insect Physiology 10/2014; · 2.50 Impact Factor
Intramyocellular Fatty-Acid Metabolism
Plays a Critical Role in Mediating Responses
to Dietary Restriction in Drosophila melanogaster
Subhash D. Katewa,1,* Fabio Demontis,2Marysia Kolipinski,1Alan Hubbard,3Matthew S. Gill,4Norbert Perrimon,2,5
Simon Melov,1and Pankaj Kapahi1,*
1Buck Institute for Research on Aging, 8001 Redwood Boulevard, Novato, CA 94945, USA
2Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA
3School of Public Health, University of California, Division of Biostatistics, 101 Haviland Hall, MC 7358, University of California,
Berkeley, CA 94720, USA
4Department of Metabolism and Aging, The Scripps Research Institute-Scripps Florida, 130 Scripps Way, Jupiter, FL 33458, USA
5Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA
*Correspondence: email@example.com (S.D.K.), firstname.lastname@example.org (P.K.)
Changes in fat content have been associated with
dietary restriction (DR), but whether they play a
causal role in mediating various responses to DR
remains unknown. We demonstrate that upon DR,
Drosophila melanogaster shift their metabolism
toward increasing fatty-acid synthesis and break-
down, which is required for various responses to
DR. Inhibition of fatty-acid synthesis or oxidation
genes specifically in the muscle tissue inhibited
life-span extension upon DR. Furthermore, DR
enhances spontaneous activity of flies, which was
found to be dependent on the enhanced fatty-acid
metabolism. This increase in activity was found to
be at least partially required for the life-span ex-
tension upon DR. Overexpression of adipokinetic
hormone (dAKH), the functional ortholog of glu-
cagon, enhances fat metabolism, spontaneous acti-
vity, and life span. Together, these results suggest
that enhanced fat metabolism in the muscle and
physical activity play a key role in the protective
effects of DR.
Dietary restriction (DR) is defined as the reduction of a particular
or total nutrient intake without causing malnutrition. DR slows
age-related diseases and extends life span in many species,
including both vertebrates and invertebrates (Masoro, 2003). In
mice and flies, restriction of protein or specific amino acids is
sufficient for life-span extension (De Marte and Enesco, 1986;
Min and Tatar, 2006). In Drosophila melanogaster, restriction of
yeast, the major source of protein in the fly diet, robustly extends
life span and is commonly used as a method for DR (Kapahi
et al., 2004; Mair et al., 2005). Pathways including the target
of rapamycin (TOR) and insulin-like signaling (ILS) have been
implicated in life-span extension by DR, but the biochemical
processes that mediate this life-span extension are not fully
understood (Kapahi et al., 2010).
DR, imposed using yeast restriction in D. melanogaster,
enhances resistance to starvation stress and lipid content
(Bradley and Simmons, 1997; Chippindale et al., 1993). In flies,
nutrient composition has been shown to be critical for fat
metabolism, with lower concentrations of yeast and higher
concentrations of sugar promoting increased steady-state
triglyceride levels (Skorupa et al., 2008). Elevated triglyceride
content is also observed in TOR and ILS pathway mutants that
extend life span in D. melanogaster (Bo ¨hni et al., 1999; Zhang
restriction (Harrison et al., 1984), the ability to maintain adipose
tissue levels correlates with life-span extension upon caloric
restriction in mice (Liao et al., 2011). Similarly, ob/ob mice, which
normally live shorter than controls on an ad libitum (AL) diet,
achieve a life span similar to control levels under caloric restric-
tion, yet their percentage of body fat is much greater than that of
controls (Harrison et al., 1984). Furthermore, mice that are fed on
a diet with reduced methionine content are long lived and show
that different dietary and genetic manipulations that extend life
span do not show a simple correlation with adiposity. Often,
steady-state levels of triglycerides are reported, but one cannot
deduce whether the change in triglycerides is due to alterations
in synthesis, storage, or breakdown of triglycerides. In the
current study, we characterize the dynamics of fat metabolism
upon DR and examine their causal link with extended longevity
using D. melanogaster.
DR Enhances Fat Synthesis and Utilization
in D. melanogaster
We confirmed that flies on DR (0.5% yeast extract) show an
increase in steady-state levels of triglyceride content compared
to flies on an AL diet (5% yeast extract) (Figure 1A). Triglycerides
are the major storage form of energy and are mostly stored as fat
droplets in the fat bodies of flies. We observed that the flies upon
DR show larger fat droplets in the fat body (Figures 1B and S1A).
Cell Metabolism 16, 97–103, July 3, 2012 ª2012 Elsevier Inc. 97
AL and DR flies, but overall a greater size is maintained upon DR
with age (Figure S1A). To examine changes in fat synthesis and
breakdown, we developed an assay to measure the dynamic
changes in triglyceride content upon DR. The assay is based
on the incorporation of radioactively labeled glucose into various
lipid fractions. Flies were raised on AL or DR diets for 10 days,
after which their diets were spiked with14C-labeled glucose for
24 hr. We observed a 2.8-fold increase in the rate of triglyceride
synthesis butnochange inphospholipid synthesisupon DR(Fig-
ure 1C). We ruled out the possibility that upon DR flies may be
eating more label by measuring the food intake of the flies (Fig-
ure S1B). Next, we measured triglyceride breakdown in vivo by
transferring the label-fed flies back to the respective diets
without label for 60 hr. We observed that upon DR there is an
increase in triglyceride breakdown, as there is a 63% reduction
in labeled triglyceride levels upon DR. The flies on an AL diet,
however, showed no significant decrease in labeled triglyceride
pool (Figure 1D). Next, we confirmed the increase in triglyceride
breakdown by measuring in vitro lipolytic capacity and free fatty
acids of corresponding whole fly lysates. Lipolysis was en-
hanced on DR by 30% (Figure 1E), whereas a 20% increase in
free fatty acids levels was observed in vivo upon DR (Figure 1F).
Together, these results suggest that flies show both an increase
in lipogenesis and lipolysis with DR.
Inhibition of Acetyl CoA Carboxylase (dACC) Inhibits
To examine whether the increase in lipogenesis plays a role in
modulating physiological changes in response to DR, we exam-
ined the effect of inhibiting dACC, the fly ortholog of a key
enzyme in fat metabolism. Inhibition of dACC was achieved by
using the RU486-inducible and ubiquitously expressing Actin
5C-GS Gal4 strain and was verified using real-time PCR (Fig-
ure S2A). As expected, dACC RNAi led to a marked inhibition
of fatty-acid incorporation into triglycerides (Figure S2B).
dACC RNAi flies on DR also showed reduced steady-state
triglyceride content (Figure S2C) and impaired triglyceride remo-
bilization in the fat body (Figure S2D). Combined, these results
suggest that dACC plays a key role in mediating the changes
in fat metabolism observed upon DR in D. melanogaster.
We examined whether changes in fat metabolism are required
for DR-dependent increase in stress resistance and life span.
In dACC RNAi flies, starvation resistance was reduced by
24% when compared to control flies under DR conditions (p <
0.0001), but no significant change was observed under AL con-
ditions (Figure 2A and Table S1). Similarly, DR-dependent
increase in cold-stress resistance was repressed upon dACC
inhibition (Figure 2B). As both starvation resistance and cold-
stress resistance phenotypes could be directly dependent on
the reduction of triglyceride levels, we measured the response
of dACC RNAi flies to oxidative stress. Upon ACC inhibition, flies
showed increased sensitivity to oxidative stress generated by
paraquat (Figure S2E), but the survival under hyperoxia was
not altered (Figure S2F), suggesting that though fat metabolism
may be required for various stress responses, it does not appear
to be important under hyperoxia. Next, we tested the hypothesis
that the switch toward increased fat metabolism upon DR is
critical for its life-span extension effects. Using the RU486-
inducible GAL4-UAS system to inhibit dACC, a significant inhibi-
tion of DR-dependent increase in life span was observed across
a range of yeast concentrations in both female (Figures 2C and
S2G) and male flies (Figures 2C and S2H). While control female
flies showed a 113% increase in life span upon DR compared
to AL conditions, upon dACC inhibition there was a significant
reduction in the life-span extension with DR (52%). Similarly,
control male flies showed a 22% increase in life span under
DR, while RNAi of dACC resulted in a 5% reduction in life span
with DR (Figures 2C). Similar effects on female life span upon
DR were observed using a different RNAi construct for dACC
(Figure S2I). We also verified that RU486 by itself does not affect
life span (Figure S2J) or fat metabolism in flies (Figure S2K).
Together, these experiments implicate an important role for
FFA (nmoles/mg fly wt)
Lipolytic activity (3H
CPM/ min/ mg protein)
(CPM/mg fly wt)
14C incorporation in TG
(CPM/mg fly wt)
TG ( g/mg fly wt)
Figure 1. Dietary Restriction Enhances Triglyceride Metabolism in
Control (w1118) Female D. melanogaster
(A) Comparison of triglyceride content in whole flies under different nutrient
conditions, DR diet (black bars), and AL diet (gray bars).
(B) Fat bodies of female flies were dissected and stained for the content and
number of lipid droplets (red are triglycerides [Nile red] and blue is F-actin
[Phalloidin]). Scale bar is 20 mm.
(C) Rate of denovolipid synthesismeasured by incorporationof14C glucose in
triglyceride and phospholipid fraction under DR and AL conditions.
spiked with14C labeled glucose for 24 hr (0 hr fraction), and then transferred to
nonlabeled food for 60 hr (60 hr fraction) and incorporation of14C glucose in
triglyceride fraction was measured.
(E) Lipolytic activity was measured from whole fly homogenates obtained from
AL- and DR-fed female flies to assess breakdown of3H-labeled triglycerides
(F) Comparison of free fatty-acid levels in whole fly homogenates under
different nutrient conditions. Error bars indicate SEM of four to five indepen-
dent preparations. (* indicates p < 0.05). See also Figure S1.
Dietary Restriction Enhances Fat Metabolism
98 Cell Metabolism 16, 97–103, July 3, 2012 ª2012 Elsevier Inc.
dACC in mediating DR-dependent changes to fat metabolism,
stress resistance, and life span.
To better understand how changes in fat metabolism modu-
late life span under DR, we conducted a genome-wide transcrip-
tional analysis. Both control and dACC RNAi flies were fed AL
or DR diets for 10 days before assessing transcriptional changes
in six independent biological replicate samples. To identify gene
functions that mediate life-span extension upon DR in a dACC-
dependent manner, expression changes that correlated with
life span for the four groups were identified. Figure 2D shows
a heat map with cluster analysis of the genes that were changed
upon DR (Table S2) but were reversed upon dACC inhibition. GO
enrichment analysis on this cluster of genes found a significant
enrichment of genes whose products are involved in muscle
structure and function (Table S3). Some of the GO categories
that changed upon DR, but were not significantly affected by
dACC RNAi, were related to proteolysis, detection of visible
light, visual perception, monovalent inorganic cation transport,
and synaptic vesicle exocytosis (Table S4). These global
gene-expression studies suggest that inhibition of dACC may
attenuate the life-span extension upon DR by reversing gene-
expression changes upon DR, especially of genes related to
Fatty-Acid Metabolism in the Muscle Tissue Modulates
DR-Dependent Life-Span Extension
To examine the role of various tissues in mediating the effects
of dACC on life span upon DR, we inhibited dACC in the fat
body, neurons, and muscle tissues. Interestingly, fat body
and neuronal inhibition of dACC using RU486-inducible fat
body (S1106-GS-Gal4) or neuronal (Elav-GS-Gal4) enhancer
traps did not reduce the life-span extension upon DR
030 60 90 120
DR ACC RNAi
AL ACC RNAi
DR ACC RNAi
AL ACC RNAi
025 50 75 100
DR ACC RNAi
AL ACC RNAi
AL DR AL DR
14C incorporation in TG
(CPM/mg muscle wt)
020 40 60 80
020 40 60 80
Figure 2. Fat Metabolism in Muscle Tissue
Responses in D. melanogaster
(A) Kaplan-Meier survival analysis for survival
under starvation in control flies (+/+; Act5c-GS-
Gal4/+; UAS-CG11198 /+, without RU486, blue)
and dACC RNAi flies (with RU486, red) under DR
(solid line) and AL (dashed line) conditions.
(B) Survival analysis after cold coma recovery
for control and dACC RNAi flies under DR or AL
(C) Median life span was calculated from Kaplan-
Meier survival analysis of female and male flies
upon dACC RNAi in whole body under different
yeast (YE) concentrations. The individual survival
curves are shown in Figures S2G (female) and S2H
(D) dACC RNAi reverses the effects of DR on
transcription of several genes that were specifi-
cally upregulated under DR. The heat map was
generated by selecting the group of genes whose
expression was significantly altered between
control and dACC RNAi flies between the DR
groups but not between AL groups.
(E) Effect of tissue-specific dACC RNAi on
DR-dependent life-span extension. Kaplan-Meier
survival analysis of female flies upon dACC RNAi
in the fat body ([+/+; +/+; S1106-Gal4/ UAS-
CG11198]; subpanel a), neurons ([+/+; +/+; Elav-
GS-Gal4 / UAS-CG11198]; subpanel b) and
muscle ([+/+; Mhc-GS-Gal4/+; UAS-CG11198/+];
subpanel c) under DR (solid line) and AL (dashed
line) conditions; control flies (without RU486, blue)
and dACC RNAi flies (with RU486, red).
(F) Triglyceride synthesis and breakdown rates
were measured in muscle from control and
muscle-specific dACC RNAi flies on DR and AL
diets. Flies were fed DR and AL diets spiked with
14C-labeled glucose for 24 hr (0 hr fraction)
and then transferred to nonlabeled food for
60 hr (60 hr fraction). Thoraces were isolated and
incorporation of14C glucose in triglyceride fraction
was measured. Error bars indicate SEM of four
to five independent preparations. (* indicates
p < 0.05).
RNAiofCG4389 ([+/+;Mhc-GS-Gal4/+; UAS-CG4389/+];subpanela)andCG7834([+/+;Mhc-GS-Gal4/+; UAS-CG7834/+];subpanel b)underDR(solidline) and
AL (dashed line) conditions; control flies (without RU486, blue) and RNAi flies (with RU486, red). See also Figure S2 and Tables S1–S4.
Dietary Restriction Enhances Fat Metabolism
Cell Metabolism 16, 97–103, July 3, 2012 ª2012 Elsevier Inc. 99
(Figures 2Ea–2Eb). However, muscle-specific inhibition of dACC
using a RU486-inducible muscle enhancer trap (Mhc-GS-Gal4)
reduced the DR-dependent increase in life span (Figure 2Ec).
While the control flies showed a 47.2% life-span extension
upon DR, flies with muscle-specific inhibition of dACC failed to
show any increase upon DR (Figure 2Ec). To gain further insight,
we examined changes in fat metabolism in dissected muscle
tissue. Inhibition of dACC in the muscle diminished the increase
in triglyceride synthesis and breakdown observed under DR
conditions (Figure 2F). A significant reduction in life-span exten-
muscle driver (Mhc-Gal4) (Figure S2L). To verify the role of
fatty-acid breakdown in muscle tissue on DR-dependent life-
span extension, we examined two genes that are involved in
the breakdown of fatty acids. CG4389 and CG7834 encode for
mitochondrial long-chain-3-hydroxyacyl-CoA dehydrogenase
and electron transport flavoprotein b subunit proteins respec-
tively. Muscle specific induction of RNAi of CG4389 and
CG7834 showed 25%–35% reduction in mRNA (Figure S2M).
Similar to inhibition of dACC, muscle specific knock down of
CG4389 (Figure 2Ga) or CG7834 (Figure 2Gb) significantly
reduced the DR-dependent life-span extension. The percentage
increase in lifespan upon DR was only 20% in flies with CG4389
RNAi compared to 123% in controls (Figure 2Ga). Similarly,
CG7834 RNAi flies showed only a 14% increase compared to
55% in the respective control flies (Figure 2Gb). Combined,
these experiments demonstrate that DR-dependent increase
in muscle fatty-acid metabolism is critical for its life-span exten-
DR Enhances Muscle Activity, which Is Required
for the Maximal Life-Span Extension upon DR
in a variety of species including flies, rodents, and primates
(Bross et al., 2005; McCarter et al., 1997; Weed et al., 1997).
Increased movement is likely to be part of an evolutionary adap-
tation that facilitates foraging behavior under conditions of nutri-
tional scarcity. We examined if changes in fat metabolism are
required for the increase in spontaneous activity upon DR.
Upon DR, control flies showed significantly higher levels of
spontaneous activity compared to AL flies over a 24 hr period
(Figure S3A). Control flies on DR also showed significantly higher
flies at all ages (Figure 3A). We also examined whether dACC
would influence the age-related decline in muscle function.
Muscle strength and function were assessed using an assay
that measures the ability of flies to land on the side of a cylinder
when dropped from a height (Palladino et al., 2002). dACC
inhibition reduced the protective effects of DR on age-related
decline in muscle activity (Figures 3B and S3B). These results
suggest that enhanced fat metabolism is critical to preserve
age-related decline in muscle function upon DR.
Next, we examined whether increased spontaneous activity
under DR condition plays a causal role in life-span extension.
We examined this by using flies with reduced movement due
to wing defects. Flies with ablated wings caused by overex-
pressing reaper (UAS-Rpr) with a wing specific Gal4 enhancer
trap (1096-Gal4) showed only 14% extension in life span com-
pared to control flies which showed a 61% extension upon
DR (Figure 3C). We also used partial clipping of the wings, as
an alternate method, to reduce physical activity (Figure S3C).
Similar to the ablated-winged flies, the clipped-wing flies
showed a modest 33% increase while the control flies showed
a 97% life-span extension upon DR (Figure 3D). This reduction
in life-span extension upon DR by curtailing movement was
specific to control flies as life span was not further reduced
with dACC inhibition in clipped-wing flies (Figure 3E). Cox
regression analysis performed on two independent repeats of
this experiment suggests that the impact of clipped wings on
the effect of DR on longevity is significantly less in dACC RNAi
020 40 60 80
025 50 75 100
Figure 3. Enhanced Movement and Muscle Func-
tion Is Critical for DR-Dependent Life-Span Exten-
sion in Female D. melanogaster
(A) Age-dependent measurementof totalactivity incontrol
flies (+/+; Act5c-GS-Gal4/+; UAS-CG11198 /+, without
RU486) and dACC RNAi flies (with RU486). Daily activity
was measured in the Drosophila activity monitors
(statistical analysis by two-way ANOVA; DR control/DR
RNAi[F1,3=27.9,p=0.0129];ALcontrol/DR control [F1,3=
92.9, p = 0.0025; AL control/AL RNAi [F1,3= 77.8, p =
(B) The effects of dACC RNAi on the ability of female flies
to maintain flight under different nutrient conditions. Flying
ability was measured in young (day 10) and old flies
(day 40) (Error bars indicate SEM of 4-5 independent
observations, * indicates p < 0.001, when compared to
young, for complete analysis see Figure S3B).
(C) Effect of genetic ablation of wings on DR-dependent
increase in life span. Kaplan-Meier survival analysis of
wings-ablated (1096-Gal4/+; UAS-rpr/+; +/+) and control
(1096-Gal4/+; +/+; +/+) female flies.
(D) Effect of mechanically clipping wings of control flies on
DR-dependent increase in life span. Kaplan-Meier survival
analysis of control female flies with nonclipped wings (blue) and clipped wings (black) under a DR (solid line) or AL (dashed line) diet.
(E) Effect of mechanically clipping wings on DR-dependent life-span extension in dACC RNAi flies. Kaplan-Meier survival analysis of dACC RNAi female flies
with nonclipped wings (red) and clipped wings (black) under a DR or AL diet. See also Figure S3 and Table S1.
Dietary Restriction Enhances Fat Metabolism
100 Cell Metabolism 16, 97–103, July 3, 2012 ª2012 Elsevier Inc.
flies compared to control flies (Supplemental Experimental
Procedures).Thecontrol andclipped-wing fliesshowedasimilar
The muscle-specific RNAi of dACC, CG7834, or CG4389 re-
sulted in a significant reduction in movement of flies upon DR
(Figure S3E). Together these observations further support the
hypothesis that enhanced muscle activity due to enhanced fat
metabolism plays a critical role in ensuring extended life span
dAKH Overexpression Increases Fat Metabolism,
Spontaneous Activity, and Life Span in a Nutrient-
Glucagon, a catabolic hormone, is critical for maintaining
glucose and triglyceride homeostasis in both mammals and
insects. In flies and other insects, AKH is a circulating peptide
that is considered a functional ortholog of glucagon based on
its role in glucose and lipid metabolism (Kim and Rulifson,
2004; Lee and Park, 2004). We examined whether increasing
AKH levels would improve fat metabolism and extend life span.
Flies overexpressing dAKH in a ubiquitous manner showed
significant increase in triglyceride synthesis and breakdown
under AL conditions (Figure 4A). dAKH overexpression was
sufficient to increase spontaneous movement under both AL
(148%) and DR (154%) conditions (Figure 4B) and life span on
AL (33%) conditions (Figure 4C). However, despite an increase
in movement under DR conditions, life span was not increased,
overlapping mechanisms to increase life span (Figure 4C).
Together, these experiments suggest that AKH modulates
changes in fat metabolism, movement, and life span in response
to changes in nutrient status.
The relationship between survival, fat metabolism, and life span
remains poorly understood. In D. melanogaster, elevated lipid
content has been observed in both long-lived mutants of TOR
and ILS pathways and the short-lived mutants lacking Brummer
and AKHR (AKH receptor) lipases (Gro ¨nke et al., 2007). These
apparently contradictory observations may be reconciled if one
takes into account the rate of fat synthesis and breakdown
rather than the steady-state levels of the measured triglycerides.
Our data demonstrates that even though flies under DR show
higher steady-state triglyceride levels, the rates of synthesis
and breakdown are also enhanced in these flies. In contrast flies
on AL show little to no change in TG breakdown even after 60 hr
of unlabeled food intake. This was surprising, as this would
suggest a continuous synthesis and accumulation of low levels
of TG in flies on an AL diet. As our assay measures TGs synthe-
sized from labeled glucose and not the TG obtained from the AL
diet itself, one possibility is that the basal level of TG synthesized
de novo from sugars, incorporated and distributed in different
tissues, might be getting recycled rather than utilized. Our data
from D. melanogaster is consistent with previous data from
mice where increased fat synthesis and oxidation is observed
during caloric restriction (Bruss et al., 2010). A positive correla-
tion between life-span extension upon caloric restriction and
the ability to maintain fat levels was also observed in a large
number of inbred mice strains (Liao et al., 2011). Similarly, by
taking advantage of radiolabeled nucleotides from nuclear
testing, fat turnover in humans has also been directly measured,
showing that the low levels of lipid turnover are associated with
multiple metabolic diseases (Arner et al., 2011).
Here we demonstrate that the metabolic shift toward
enhanced fatty-acid metabolism under DR is critical for some
of its protective effects. In flies, ACC is coded by a single
gene, CG11198, whereas in mammals, two different genes
(ACC1 and ACC2) code for separate isoforms of ACC. Mice
that are null for ACC1 do not survive, whereas ACC2-null mice
are viable. Although it is not known how ACC2-null animals will
behave upon caloric restriction, upon regular diet, these animals
show an increased triglyceride breakdown, leaner phenotype,
increased insulin sensitivity—and no effect on life span (Choi
0 25 5075 100125
Total fly activity /day
Activity /fly/10 min
1 4 8 12 16 20 24
14C incorporated in TG
(CPM/mg fly wt)
Figure 4. dAKH Overexpression in Flies Enhances Fat Metabolism,
Spontaneous Activity, and Life Span in a Nutrient-Dependent
Manner in D. melanogaster
(A) Triglyceride synthesis and breakdown rates were measured in control (+/+;
Act5c-GS-GAL4/UAS-AKH; + /+; without RU486) and AKH overexpression
(with RU486) flies on DR and AL diets. Error bars indicate SEM of four to five
independent preparations (* indicates p < 0.05).
(B) Effect of dAKH overexpression on spontaneous activity. The graph shows
averaged activity (four vials per group with 25 flies in each vial) per 10 min for
control and AKH overexpression flies. The x axis represents time (in hr) after
the flies were moved to the activity monitors. The activity measurement was
started at 4:00 p.m. (subpanel a). The data in the graph is also plotted as bar
graphs representing the total activity/fly/day (subpanel b). Error bar indicates
SEM, with n = 4 for each group (* indicates p < 0.05).
(C) The effects of dAKH overexpression on nutrient-dependent changes in
life span in female flies. Kaplan-Meier survival analysis for control and AKH
overexpression flies on DR and AL diets. See also Table S1.
Dietary Restriction Enhances Fat Metabolism
Cell Metabolism 16, 97–103, July 3, 2012 ª2012 Elsevier Inc. 101
et al., 2007). Interestingly, we also observed some benefits of
dACC RNAi that were dependent on diet and tissue-specific
knockdown. dACC RNAi flies on AL show increased sponta-
neous activity (Figure 4A), and fat body-specific dACC RNAi
showed a small but significant increase in lifespan upon AL
conditions (Figure 2Ea). In some of our experiments, we noted
that though the benefits of DR were reduced, the fly strains
were short lived on both AL and DR diets. One possible explana-
tion is that our genetic and physical manipulations are very
strong, which made the flies sick under standard laboratory
conditions. This could not be ruled out as some cellular
processes, such as fat metabolism and movement, may be crit-
ical for ensuring a normal life span. For this reason, the
percentage increase in life span upon nutrient-limiting diets
was deemed critical for their importance in DR. Furthermore,
to support our conclusions, we examined the effects of dACC
inhibition using multiple tissue-specific drivers and identified
muscle as the critical tissue for its effects. Similar results using
b-oxidation genes (Figure 2G) and AKH (Figure 4) support the
role of fat metabolism in life-span extension upon DR.
Mitochondrial b-oxidation plays a critical role in enhancing
fat utilization for energy purposes. Physiological conditions like
exercise and fasting, which require increased fatty-acid oxida-
tion, show upregulation of mitochondrial biogenesis and mito-
chondrial b-oxidation (Hood, 2001) by modulating multiple
pathways. We have previously observed that flies upon DR
have enhanced mitochondrial function, which is mediated by
enhanced mRNA translation of nuclear-encoded mitochondrial
mRNAs in a d4E-BP dependent manner (Zid et al., 2009). Given
the role of mitochondria in fatty-acid oxidation, it is possible
that enhancing mitochondrial function allows for a switch to
increased fat utilization and physical activity upon DR.
Our data suggest that increased spontaneous activity might
be a critical component of DR-dependent life-span extension.
in life-span extension. This result does not agree with a recent
report showing that singly housed male flies that were restricted
in small activity tubes showed significant increase in life span in
a sugar restriction-based DR paradigm (Linford et al., 2012).
However, it is conceivable that restricting space may not limit
muscle activity of flies similarly to loss of wings. Furthermore,
as the sugar-restricted male flies showed an increase neither
in triglyceride content nor in total activity in response to diet, it
is possible that the mechanism of life-span extension by the
two DR paradigms is different. It is also conceivable that in our
experiments, upon wing ablation or clipping, there could be
damaging effects on muscle tissue that limits life span. Future
work exploring the relationship between physical activity and
life span upon dietary restriction will help resolve these con-
Enhanced fat metabolism in the muscle has been associated
of fatty acids in muscle for energy purposes are seen in mice
that overexpress PEPCK (phosphoenolpyruvate carboxykinase)
in the muscle tissue (Hanson and Hakimi, 2008). These mice
are long lived, store up to five times more triglyceride in their
skeletal muscle, and show increased levels of physical activity
compared to control mice (Hanson and Hakimi, 2008). However,
increased fat deposition in skeletal muscles is also associated
with insulin resistance, as is commonly seen in obesity and
that enhances insulin sensitivity also increases muscle-fat
content which is termed as the ‘‘athlete’s paradox’’ (Goodpaster
et al., 2001). One potential explanation is that athletes break-
down their muscle triglycerides much more frequently than
type 2 diabetics. Our data demonstrate that diminishing fatty-
acid oxidation in the muscle limits life-span extension, while
enhancing fat mobilization by overexpressing AKH can extend
life span. Our data also suggest that DR may induce changes
in muscle similar to those observed under endurance exercise
and that molecules like AKH that enhance fat breakdown could
serve as potential DR mimetics. The protective effects of DR
may be linked to increased spontaneous activity through
enhanced fat metabolism, which helps maintain muscle function
Fly Husbandry and Life-Span Analysis
Flies were developed on standard lab food (Caltech food recipe) and adults
were transferred within 2–3 days of eclosion to a yeast-extract (YE) diet (vari-
able concentrations of YE) as described previously (Zid et al., 2009). Details of
fly stocks, genotype, and crosses are available in the Supplemental Experi-
mental Procedures; statistical analyses of the life span, and additional repeats
data are provided in Table S1.
After 10days of feeding on an AL or DRdiet, 240flies weretransferred to an AL
or DR diet with 2 mCi of14C-labeled glucose. After 24 hr, half the flies were
snap frozen in liquid nitrogen (0 hr sample). The other half was transferred to
a fresh nonradioactive AL or DR diet, was kept on this food for the next
60 hr (60 hr sample), and then immediately frozen. The frozen samples
(20 flies/replicate) were homogenized in chloroform, and total lipid was frac-
tionated into triglyceride and phospholipid fractions by using DSC-NH2
cartridges and different solvents (Bodennec et al., 2000; Kaluzny et al.,
1985).The fractions were dried undernitrogen, resuspended inthe scintillation
fluid, and counted. Zero hour samples indicate the rate of incorporation of
glucose in fatty acids and sixty hour samples indicate the breakdown of the
labeled fatty acids. Lipolysis measurements from whole flies were carried
out by following a previously described protocol (Gro ¨nke et al., 2007). Triglyc-
eride and free fatty acid were measured using commercially available kits
(Stanbio Labs; Boerne, TX). Fat-body staining is described in detail in the
Gene-Array Expression Analysis
Total RNA was extracted from approximately 35 flies per group. Six indepen-
dent biological replicates were collected and expression array analysis was
carried out. Details of RNA extraction, amplification, labeling, hybridization,
and analyses are provided in the Supplemental Information.The Gene Expres-
sion Omnibus (GEO) accession number for the microarray data reported in
this paper is GSE37537.
Spontaneous Activity Measurements, Muscle-Strength assays, and
For measurement of spontaneous activity we used Drosophila population
activity monitors (Trikinetics Inc.; Waltham, MA). Muscle strength, starvation,
and cold coma stress assays were performed using standard protocols and
are available in detail in the Supplemental Experimental Procedures.
Supplemental Information includes Supplemental Experimental Procedures,
Supplemental References, three figures, and four tables and can be found
with this article online at http://dx.doi.org/10.1016/j.cmet.2012.06.005.
Dietary Restriction Enhances Fat Metabolism
102 Cell Metabolism 16, 97–103, July 3, 2012 ª2012 Elsevier Inc.
We thank Roger Chang, Akio Nada, Emily Chang (life-span data collection),
Krysta Felkey (microarrays) and Vishal Patel (staining and quantification). We
also thank John Tower, Haig Keshishian, and Kai Zinn for providing various
Gal4 drivers used in the study. We thank Matthew Laye, Gordon Lithgow,
Judith Campisi, Martin Brand, and members of the Kapahi Lab for helpful
discussions and suggestions. This work was funded by grants from the
AFAR and the NIH (R01 AG031337-01A1; RO1 AG038688; RL1 AAG032113
and P01 AG025901-S1) (P.K.), RL9 AG032114 (S.D.K.), R01 AG036992
(M.S.G), a Nathan Shock Award (P30AG025708) (S.M.) and R01 AR057352
(N.P.). F.D. is an EMF/AFAR postdoctoral fellow. N.P. is an HHMI investigator.
Received: August 25, 2011
Revised: February 24, 2012
Accepted: June 18, 2012
Published online: July 3, 2012
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