Peripheral Circadian Clocks Mediate Dietary Restriction ...

Article

Peripheral Circadian Cloc
ks Mediate DietaryRestriction-Dependent Changes in Lifespan and FatMetabolism in Drosophila
Graphical Abstract

Highlights

d DR enhances the magnitude of cyclic expression of circadian

clock genes

d timeless (tim) and period are required for the optimal DR

response in Drosophila

d tim regulates fat metabolism and cycling of medium chain

triglycerides upon DR

d Overexpression of tim increases amplitude and extends

lifespan under AL conditions

Katewa et al., 2016, Cell Metabolism 23, 143–154January 12, 2016 ª2016 Elsevier Inc.http://dx.doi.org/10.1016/j.cmet.2015.10.014

Authors

Subhash D. Katewa, Kazutaka Akagi,

Neelanjan Bose, ..., Amita Sehgal,

Jadwiga M. Giebultowicz,

Pankaj Kapahi

[email protected] (S.D.K.),[email protected] (P.K.)

In Brief

Dietary restriction (DR) is known to boost

fat metabolism and lifespan. Katewa et al.

reveal that peripheral circadian clocks

play a key role in this response and that

DR increases clock gene expression.

Overexpression of timeless mimics the

effects of DR and extends fly lifespan

under ad libitum conditions.

mailto:[email protected]


http://dx.doi.org/10.1016/j.cmet.2015.10.014

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Cell Metabolism

Article

Peripheral Circadian Clocks Mediate DietaryRestriction-Dependent Changes in Lifespanand Fat Metabolism in DrosophilaSubhash D. Katewa,1,* Kazutaka Akagi,1 Neelanjan Bose,1 Kuntol Rakshit,2 Timothy Camarella,1 Xiangzhong Zheng,3

David Hall,1 Sonnet Davis,1 Christopher S. Nelson,1 Rachel B. Brem,1 Arvind Ramanathan,1 Amita Sehgal,3

Jadwiga M. Giebultowicz,2 and Pankaj Kapahi1,*1Buck Institute for Research on Aging, 8001 Redwood Blvd, Novato, CA 94945, USA2Department of Integrative Biology, Oregon State University, 3029 Cordley Hall, Corvallis, OR 97331, USA3Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, 3400 Civic Center Blvd, Philadelphia,

PA 19104, USA

*Correspondence: [email protected] (S.D.K.), [email protected] (P.K.)http://dx.doi.org/10.1016/j.cmet.2015.10.014

SUMMARY

Endogenous circadian clocks orchestrate severalmetabolic and signaling pathways that are knownto modulate lifespan, suggesting clocks as potentialtargets for manipulation of metabolism and lifespan.We report here that the core circadian clock genes,timeless (tim) and period (per), are required forthe metabolic and lifespan responses to DR inDrosophila. Consistent with the involvement of acircadian mechanism, DR enhances the amplitudeof cycling of most circadian clock genes, includingtim, in peripheral tissues. Mass-spectrometry-basedlipidomic analysis suggests a role of tim in cyclingof specific medium chain triglycerides under DR.Furthermore, overexpression of tim in peripheral tis-sues improves its oscillatory amplitude and extendslifespan under ad libitum conditions. Importantly,effects of tim on lifespan appear to be mediatedthrough enhanced fat turnover. These findings iden-tify a critical role for specific clock genes in modu-lating the effects of nutrient manipulation on fatmetabolism and aging.

INTRODUCTION

Circadian rhythms, such as daily cycles of sleep and activity and

oscillations in metabolic, physiological, and endocrine functions,

are vital to maintaining temporal homeostasis. These rhythms

are controlled by endogenous clocks located in the brain and

many peripheral tissues. Rhythmic activity of clock molecules

drives cyclic expression of many other genes, resulting in rhyth-

mic activities of metabolic and signaling pathways in mammals

as well as in the fruit fly, Drosophila melanogaster (Buhr and

Takahashi, 2013; Schibler and Sassone-Corsi, 2002; Xu et al.,

2011). Disruption of circadian rhythms is associated with cancer

and metabolic disorders that accelerate aging, including dia-

betes and obesity (Kondratov, 2007; Turek et al., 2005).

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Metabolic homeostasis is intimately linked to longevity. Die-

tary restriction (DR), leads to metabolic reprogramming, which

enhances fat turnover andmitochondrial function that is required

for its protective effects on lifespan extension in the fly (Katewa

et al., 2012). Similarly, inmice, calorie restriction has been shown

to increase fat turnover (Bruss et al., 2010). The improved cellular

homeostasis upon DR delays the onset of a number of age-

related diseases and aging inmultiple species (Bishop andGuar-

ente, 2007; Fontana et al., 2010; Kapahi et al., 2010; Mair and

Dillin, 2008). Conservation of the protective effects of DR in

Drosophila offers an opportunity to examine the fundamental

molecular mechanisms of nutritional modulation of aging and

age-related diseases (Katewa and Kapahi, 2011; Mair and Dillin,

2008; Tatar, 2007).

Given the well-established links between circadian rhythms

and metabolism (DiAngelo et al., 2011; Green et al., 2008; Xu

et al., 2011), we examined the role of circadian clocks in lifespan

extension by DR in Drosophila. Circadian behavioral rhythms

have been studied previously in the context of aging in both flies

and mice, with studies showing that rhythms of rest:activity or

sleep:wake break down with age (Koh et al., 2006; Turek et al.,

2005). However, it is not known whether interventions that lead

to life extension, such as DR, require functional circadian clocks,

and if so by which mechanisms.

Here we show that circadian regulation is critical for DR-

dependent increase in lifespan. Flies mutant for circadian

clock genes timeless or period showed an attenuated response

to DR-dependent changes in lifespan extension and fat

metabolism. In support of the idea that clock genes con-

tribute to effects of DR, we also show that DR improves the

amplitude of circadian gene cycling. Previously, we showed

that DR-dependent increase in fat metabolism is required

for lifespan extension (Katewa et al., 2012). Now here we

demonstrate that specific clock mutants can abrogate the

changes in fat turnover upon DR, while overexpression of time-

less enhances fat metabolism on ad libitum (AL) diet. Mass-

spectrometry-based lipidomic analysis of control and timeless

mutant flies shows cycling of several triglycerides (TGs)

under DR conditions. Specifically, we have identified a novel

group of medium chain TGs (MCTs) that cycle under DR in a

TIM-dependent manner. Overexpression of tim improves fat

etabolism 23, 143–154, January 12, 2016 ª2016 Elsevier Inc. 143




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Figure 1. Increase in Amplitude of Circadian Gene Expression upon DR

(A andB) Daily relativemRNA concentration profiles of core clock genes in (A) heads and (B) bodies of control CS females fed on AL andDR diets for 10 days. Data

are normalized to the trough (ZT4/16) values set at 1 for flies on AL diet. White and black horizontal bars mark periods of light and dark, respectively. Each data

point represents mean ± SEM (error bars) of three independent RNA samples. Statistical significance between AL and DR values was determined using two-way

ANOVA with Bonferonni’s post hoc test, is denoted by ***p < 0.001, **p < 0.01, and *p < 0.05, and is provided in Table S1. See also Figure S1.

turnover and extends lifespan on AL food, thereby mimicking

the effects of DR.

RESULTS

DREnhances themRNA and Protein Oscillations of CoreClock GenesThe oscillations of clock genes in peripheral tissues become

weaker with age (Luo et al., 2012; Rakshit et al., 2012). If mainte-

nance of these oscillations is important to delay aging, then DR

may act by enhancing the amplitude of circadian oscillations.

To assess how nutrients impact circadian clocks, we obtained

daily mRNA expression profiles of clock genes timeless and

period in control wild-type Canton-S (CS) flies subjected to DR

or AL feeding for 10 days. In Drosophila, restriction of yeast,

particular amino acids, or total calories increases the lifespan

(Bruce et al., 2013; Mair et al., 2003; Min and Tatar, 2006). As

reduction of yeast is sufficient to extend lifespan independent

of the calorie content, DR is implemented mostly as restriction

of dietary yeast (Chippindale et al., 1993; Kapahi et al., 2004; Ka-

tewa et al., 2012; Lee et al., 2008; Mair et al., 2005). In our study,

the DRdiet contains 0.5%yeast extract (YE) and 5%sugar, while

the AL diet contains 5% YE and 5% sugar (additional details are

provided in Supplemental Experimental Procedures).

In flies, the timeless (tim) and period (per) genes are the major

components of the cellular clock and are expressed rhythmically

in several tissues as a result of negative feedback by their protein

products (Zheng and Sehgal, 2008). The PER and TIM proteins

negatively regulate transcription by inhibiting activity of the

144 Cell Metabolism 23, 143–154, January 12, 2016 ª2016 Elsevier I

Clock (CLK) and cycle (CYC) transcriptional activators. We

observed the expected daily oscillations of tim and per mRNA

levels in animals on an AL diet with a trough at Zeitgeber Time

4 (ZT4) and peak at ZT16 in both heads (Figure 1A) and bodies

of female flies (Figure 1B; for statistical analysis, see Table S1).

Flies subjected to DR had higher magnitude tim and per expres-

sion, in particular at the normal peak time of ZT 12–20, resulting

in increased amplitude of cycling, compared to flies reared on an

AL diet. This effect was particularly strong in body clocks (Fig-

ure 1B). We confirmed this further by measuring the cycling of

tim and per in isolated fat bodies (Figure S1A). Male flies from

either CS or w1118 also showed a similar increase in expression

of tim and per mRNA upon DR (Figure S1B). However, we have

used female flies for all other experiments as they typically

show a stronger response to variation of yeast in the diet (Katewa

et al., 2012; Vargas et al., 2010). We also observed that a mini-

mum of 6 days of DR treatment is required to see a robust

response in clock gene amplitude (data not shown). mRNA

expression profiles of Par Domain Protein 1ε (Pdp1ε) and vrille

(vri) were also significantly more robust in females on DR, and

the same was true for the amplitude of Clk oscillations (Figures

1A and 1B). DR did not have a significant effect on the daily os-

cillations of cryptochrome (cry), which encodes the Drosophila

circadian photoreceptor and functions as a clock component

in some peripheral tissues (Figures 1A and 1B) (Krishnan et al.,

2001). Because the amplitude of clock gene oscillations declines

with age, we also measured circadian gene expression in

33-day-old flies. While the overall amplitude was reduced

with aging in both AL and DR conditions, DR flies sustained

nc.

Figure 2. Increase in Magnitude of Clock Protein Expression

upon DR

(A) TIM and PER expression in adult fat body at ZT 4 and 18 in control females

fed AL and DR diets for 10 days. (Left) TIM staining. (Center) PER staining.

(Right) Merged images (TIM, PER, and DAPI). Scale bars indicate 15 mm.

(B) Quantification of both TIM and PER expressions in the nucleus. n = 90 from

six fat bodies in each conditions. Error bars indicate SEM (*p < 0.05 by t test).

significantly higher oscillations of all clock genes (except cry) in

both heads and abdomens (Figure S1C). To determine if the in-

crease in mRNA expression of clock genes is translated into

more clock proteins, we measured the protein levels in adult

fat bodies at ZT4 and ZT18. Control (w1118) female flies were

fed AL or DR diet for 10 days, after which the adult fat bodies

were removed, fixed, and stained for TIM and PER proteins. As

expected, levels of TIM and PER were significantly higher at

ZT18 on both food types when compared to ZT4 (Figures 2A

and 2B). Two-way ANOVA indicated significant effect of diet

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(p = 0.0432) and time (p = 0.0010) for TIM and significant effect

of time (p = 0.0019) for PER (Figure 2B). Combined the results

(Figures 1 and 2) suggest that DR not only increases the magni-

tude of mRNA expression of clock genes but also increases the

clock protein levels in peripheral tissues.

Circadian Regulation Is Required for DR-DependentIncrease in LifespanNext, we asked whether circadian clocks are required for DR-

dependent responses by measuring the survival of female flies

maintained under constant light conditions (LL), which disrupt

the molecular clock and eliminate overt rhythms (Emery and

Clayton, 2001). Wild-type CS female flies were maintained in in-

cubators with 12 hr light:12 hr dark (LD) cycles or 24 hr LL. Flies

maintained in LD showed significantly greater extension in life-

span upon DR than did flies in LL (Figure 3A, diet*genotype inter-

action p value = 1.343 10�7; for statistical analysis of all survival

assays see, Table S2, and for replications, see Table S3). To

verify that the reduction in DR-dependent lifespan extension

was due to the loss of circadian regulation, we tested the effects

of DR in flies lacking the timeless gene (tim01). tim01mutants (out-

crossed eight times into the control CS background) maintained

on DR showed a small increase in lifespan (10%) in contrast to a

large extension in control flies (51%) (Figure 3B, interaction

p value = 5.36 3 10�6; Tables S2 and S3). tim01 flies in another

control (w1118) background also showed impaired lifespan

extension upon DR compared to controls (Figure 3C, interaction

p value = 6.683 10�6). For finer resolution of the changes in life-

span upon yeast variation, survival assays were also done at five

different concentrations of YE in the diet (Figure 3D; Tables S2

and S3). Loss of TIM significantly decreased the increase in life-

span produced by lowering YE in the diet (Figure 3D). It had less

effect at yeast concentrations that produced little to no change in

lifespan. To determine whether reduced lifespan extension re-

flects a circadian function of tim, we also measured the effect

of DR on lifespan in flies lacking the PERIOD (PER) protein, which

is the partner of TIM in the molecular clock. Indeed, the per01

mutant flies showed a similar reduction in DR-dependent life-

span extension (Figure 3E, interaction p value = 1.853 10�6; Ta-

bles S2 and S3). Surprisingly, despite showing a similar response

in terms of mRNA expression (Figure S1B), male flies belonging

to either tim01 or per01 groups were not significantly different

from control animals on DR (Figure S2). Combined, the results

indicate that DR improves temporal homeostasis in both sexes

and that functional circadian clocks contribute to maximal DR-

dependent lifespan extension in females.

Circadian Clocks Are Required for DR-DependentChanges in Fat MetabolismEnhanced TG turnover is necessary for DR-dependent lifespan

extension in flies (Katewa et al., 2012). The improved fat meta-

bolism in dietary-restricted flies also increases their survival in

response to acute starvation (Katewa et al., 2012). To determine

a role of circadian clocks in regulating fat metabolism, we first

measured the starvation response of circadian mutant flies. Prior

exposure to DR substantially increased the survival of control

flies upon starvation but had much less of an effect on tim01 (Fig-

ure 4A, interaction p value = 6.323 10�5) and per01mutants (Fig-

ure 4B, interaction p value = 1.34 3 10�7). Thus, per and tim


Figure 3. Reduction of Lifespan Extension upon DR in Flies with Mutations in Circadian Clock Genes

(A–C) DR-mediated lifespan extension was reduced in (A) female flies maintained in 24 hr LL, (B) tim01 mutant (in CS background) under LD, and (C) tim01 mutant

(in w1118 background) under LD.

(D) tim01 flies showed reduced response to varying YE concentration in the diet.

(E) Lifespan extension upon DR was reduced in per01 mutants under LD. Statistical analysis of the survival curves number of flies, and data from additional TG

repeats are provided in Tables S2 and S3. See also Figure S2.

mutants are impaired in the resistance to starvation that normally

occurs following DR.

We next askedwhether the effect on starvation reflects altered

synthesis or breakdown of TG. For this, we measured fat turn-

over in tim01 mutant and control flies using a radiolabeled

glucose tracer method (Katewa et al., 2012). Consistent with

extended survival upon starvation, wild-type flies showed a

4-fold increase in the rate of TG synthesis after DR conditions,

whereas the tim01mutants showed only a 1.6-fold increase, indi-

cating a reduced rate of TG synthesis in tim01 flies (Figure 4C).

Furthermore, the control flies showed a 51%decrease of labeled

TG after 60 hr, whereas tim01 flies showed a 30% reduction sug-

gesting decreased fat turnover in tim01 flies under DR (Figure 4C).

These results support the idea that clock genes contribute to the

enhanced synthesis and breakdown of TGs upon DR.

To identify the TG that might cycle under DR and investigate

their possible regulation by circadian clocks, we performed a

detailed analysis of lipids on DR. A quantitative mass spectrom-

etry (MS) approach was used to identify features (see Supple-

mental Experimental Procedures for details) in fly lipid extracts

at different time points in control and tim01 flies maintained on

DR food conditions. A preliminary analysis identified about 253

features (Figure 4D) from fly lipid extracts, and approximately

50 showed some cycling properties (had one clear peak and

one trough in a 24 hr time period) (Figure S3) in a tim-dependent

fashion. Based on our previous results (Katewa et al., 2012) and

the effect of tim01 on TG synthesis (Figure 4C), we focused on

TGs and broadly classified them in three groups: Group 1 was

non-cycling TGs, group 2 was cycling TGs with a peak in the

night (at ZT16–20), and group 3 showed cycling with a peak at

daytime (ZT4–8) (Figure 4E). Consistent with the involvement of

the clock (Figure 4C), tim01 flies showed reduced levels of all


TGs. Next, we used high-resolution MS to identify specific TGs

among the cycling features belonging to similar phased groups

(Table S4) and observed that group 2 contained MCTs (contain-

ing medium-chain fatty acids [MCFAs] with 8 to14 carbons). The

most prominent tim-dependent cycling TG (feature 88, matched

to TG(36:0) by high-resolution MS analysis) was followed up with

subsequent high-resolution MS/MS analysis to elucidate it’s

constituent fatty acids (Figures 4F and 4G). Under our high per-

formance liquid chromatography (HPLC) conditions, TGs most

predominantly formed the corresponding [M+NH4]+ adduct

ions (Figure 4D; Supplemental Experimental Procedures).

When fragmented with a collision gas, TG [M+NH4]+ adducts un-

dergo a characteristic neutral loss of [RCO2H+NH3] (where

RCO2H is a constituent fatty acid; e.g., R = C11H23 for the satu-

ratedC12-fatty acid, lauric acid) for each fatty acid component of

the TG (King et al., 2015). Our MS/MS analyses revealed only a

single [RCO2H+NH3] neutral loss fragment ion from feature 88,

corresponding to [C11H23CO2H + NH3] (Figures 4F and 4G). To

corroborate this, only a single fatty acyl chain fragment ion

([C11H23C = O]+) was further observed (Figures 4F and 4G).

These data suggest that feature 88 is actually a symmetric TG,

trilauryl glycerol (TLG), i.e., TG(12:0/12:0/12:0) (Figure 4G). Addi-

tionally, we found that the cycling property of TLG in flies is

strictly tim-dependent upon DR food conditions (Figures 4E

and S4).

Overexpression of timeless in Peripheral TissuesIncreases Lifespan and Fat Metabolism on a RichNutrient DietAging causes a reduction in the amplitude of clock gene expres-

sion in peripheral tissues of both flies and mammals, but it is not

known whether increasing the amplitude might slow aging (Luo

nc.

et al., 2012; Rakshit et al., 2012; Yamazaki et al., 2002). To deter-

mine whether increased expression or oscillations of tim could

affect aging, we induced overexpression of tim using an Actin-

5C-Gene switch-GAL4 driver (a drug inducible promoter that is

expressed in whole flies) and measured survival. Expression of

this driver is induced by adding RU486 to the medium, and so

survival can be compared in induced and un-induced genetically

identical strains. We observed a significant increase in survival in

tim-overexpressing flies on AL but not on DR diet. Flies on AL

diet showed a 38% (16 day) increase in lifespan (Figure 5A, inter-

action p value = 6.443 10�15), while flies on DR showed no sig-

nificant difference in survival when tim was overexpressed in all

tissues (Figure 5A; Tables S2 and S3).We also tried overexpress-

ing per (two different transgenes) in all tissues but did not see a

comparable effect to tim overexpression (Figure S5A). This sug-

gests either a tim-specific effect or that it is limiting in some tis-

sues. To identify the tissue responsible for lifespan extending

effects of tim, we overexpressed tim in different tissues of

the flies (by using tissue-specific promoter-driven expression)

including neurons, fat bodies, gut, andMalpighian tubules. Over-

expression in neurons had no effect on lifespan (Figures 5B,

interaction p value = 0.991, and S5B), whereas overexpression

in all other tissues caused a small but significant increase in life-

span under AL but not under DR conditions (Figures S5C–S5E).

Overexpression of tim in the fat body extended lifespan by 26%

(Figure 5C, interaction p value = 4.763 10�5), overexpression in

gut extended lifespan by 14% (Figure 5D, interaction p value =

0.00152), and overexpression in tubules increased lifespan by

17% (Figure 5E, interaction p value = 0.00374). As the lifespan

extension from individual tissues was lower than what we

observed with whole-body overexpression of tim (Figure 5A),

we infer that multiple peripheral clocks contribute to the lifespan

extension by tim overexpression.

One caveat of the overexpression of any circadian gene is that

the overexpressed mRNA may not follow the regular circadian

expression pattern of the gene itself, so we also determined

how the overexpression of tim affects the circadian nature of

tim expression. Overexpression of tim increased the abundance

of tim mRNA significantly at ZT16-20 in flies fed AL (Figure 6A),

resulting in a 5-fold increase in the amplitude of the tim

mRNA oscillation, similar to flies on DR, which showed a 3-fold

increase in amplitude as peak and trough levels were elevated

(Figure S6A). In addition, Clock was increased significantly

(p > 0.05) near the peak time at ZT4. However, this increase in

rhythmicity was not observed for per, suggesting a timeless-spe-

cific effect of overexpression (Figure S6B). This could possibly

explain why period overexpression did not increase lifespan on

AL food, as per overexpression may not have maintained cyclic

expression.

Next, we examined the effect of tim overexpression on fat

metabolism. As DR increases fat metabolism (Katewa et al.,

2012) and we observed that tim and permutants showed dimin-

ished fat metabolism, we hypothesize that increasing clock os-

cillations results in more robust rhythmicity of fat metabolism.

Overexpression of tim led to significantly higher de novo TG syn-

thesis from 14C -labeled glucose and a higher rate of breakdown

in flies on AL diet (Figure 6B), while it had little effect on fliesmain-

tained under DR (Figure S6C). Next, we examined whether tis-

sue-specific overexpression of tim modulates fat metabolism

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in the same or other tissues? tim overexpression in fat bodies

increased fat synthesis and breakdown in fat bodies and to a

smaller extent in the thorax but no effect was observed in heads

(Figure 6C). The effect in thorax suggests a possibility of either an

increased mobilization from fat bodies or a tissue autonomous

effect of fat body clocks. Finally, to determine if fat metabolism

is critical for the observed increase in lifespan under tim overex-

pression conditions, we measured survival in transgenic flies

overexpressing tim while inhibiting acetyl CoA carboxylase

(ACC). We have previously shown that upon inhibition of ACC

there is a significant reduction of de novo synthesis of TG, lead-

ing to reduced fat turnover and a significant reduction in lifespan

extension upon DR (Katewa et al., 2012). We observed that un-

der AL conditions where tim overexpression increases lifespan,

co-expression with ACC RNAi inhibits the observed extension

in lifespan (Figures 6D, 6E, S6D, and S6E). We also evaluated

if the levels of TLG are changing under this conditions. While

ACCRNAi reduced the levels of TLG, the levels were significantly

upregulated upon tim overexpression and co-expression with

ACC RNAi resulted in a smaller but significant reduction (Fig-

ure 6F). Combined, the results indicate that increasing the rhyth-

mic expression of timmRNA is sufficient to increase fat turnover

that mediates lifespan extension under rich nutrient conditions.

DISCUSSION

Clocks Are Required for DR-Dependent LifespanExtensionWithin the last decade, there have been important mechanistic

insights into the protective effects of DR. Several genetic and

metabolic pathways including the insulin/IGF-1 and target of ra-

pamycin (TOR), AMPK, and sirtuins have been shown to play a

role in mediating DR responses (Bordone and Guarente, 2005;

Kapahi et al., 2004; Mair and Dillin, 2008; Mair et al., 2011; Pan-

owski et al., 2007). Our results show for the first time that

circadian clock genes also play a role in modulating lifespan

extension in response to DR. Flies that were maintained in con-

stant light conditions, which disrupt the functioning of themolec-

ular clock, showed a reduced response to DR in terms of lifespan

extension. Significantly reduced response to DR was also found

in flies mutant for either the tim or the per gene, further suggest-

ing the importance of clocks for effects of DR on lifespan. As

both per and timmutant animals show some level of rhythmicity

under normal 12 hr LD (light-dark) cycle (Sehgal et al., 1994;

Wheeler et al., 1993), the strong reduction in lifespan under con-

stant light conditions and in the two circadian mutants argues

that lifespan extension upon DR is not simply a consequence

of altered circadian behaviors but more likely linked to clock

controlled genes regulated by tim and per. It is important to

note here that animals lacking clocks as well as those on con-

stant light still display some lifespan extension, suggesting

possible contributions of clock-independent pathways in the

response to DR. This is not surprising as several pathways are

likely to work in concert to mediate the maximal lifespan exten-

sion by DR in flies (Kapahi et al., 2010; Mair and Dillin, 2008).

Though some circadian mutants are also slightly shorter lived

on AL diet, we consistently observed that the reduction on

DR food is higher in the circadian mutant animals. Additionally,

it is reasonable to expect that certain manipulations such as


(legend on next page)

148 Cell Metabolism 23, 143–154, January 12, 2016 ª2016 Elsevier Inc.

Figure 5. Overexpression of tim Increases Survival in a Diet-Dependent Manner

Kaplan Meier survival analysis of female flies upon tim overexpression under DR (solid line) and AL (dashed line) conditions, control flies (without RU486, blue),

and overexpression flies (with RU486, red).

(A) Overexpression of tim in whole-body increases lifespan on an AL diet.

(B) Overexpression of tim specifically in neurons has no effect on lifespan.

(C–E) Overexpression of tim specifically in (C) fat body or (D) gut or (E) tubules increases lifespan on AL diet. Statistical analysis of the survival curves, complete

genotype, number of flies, and data from additional independent repeats are provided in Tables S2 and S3. See also Figure S5.

circadian disruption that limits DR-dependent lifespan may

shorten lifespan under all diets as these cellular processes

may be critical for ensuring a normal lifespan. Indeed, lifespan-

shortening effects are also observed for mutants of daf-16/

dfoxO, which is a key effector of the lifespan extension produced

by inhibiting the insulin signaling pathway in both C. elegans and

Drosophila.

DR Increases and Maintains the Magnitude ofExpression of Clock Genes and ProteinsOur results demonstrate that DR increases the magnitude of

circadian gene expression, which can attenuate the age-related

Figure 4. Mutations in Circadian Clock Genes Reduce TG Homeostasi

(A and B) Starvation resistance was reduced in tim01 (A) and per01mutants (B). Sta

TG repeats are provided in Tables S2 and S3.

(C) tim01 mutant flies show reduced TG turnover upon DR. Flies were fed AL and

measured (shown as 0 hr) by measuring the amount of 14C in the TG fraction of

experiment where the 24 hr fed flies were transferred to unlabelled food for 60 hr

mean ± SEM (error bars) of five independent preparations. Statistical significance

denoted by a, b, or c, when compared between time points and by (*) between g

(D–G) MS-based lipidomics to identify TGs that cycle upon DR in a timeless-dep

(D) Extracted ion chromatograms (XICs) for (top) a mixture of synthetic d5-di and T

and optimize conditions for HPLC-MS and -MS/MS analyses and (middle) �253

visualization, each chromatogram is plotted to show a 3-min window around the p

labeled with the corresponding feature number (e.g., feature 88/ F88) to avoid an

(E) Levels of specific TGs (error bars, SEM) at different time points in control and t

on their cycling nature—group 1: non-cycling; group 2: cycling, peak at night; an

(F) High resolution (HR) HPLC-MS/MS spectra for the most prominent cycling fe

(G) Schematic annotations for the molecular ion ([M+NH4]+) and the observed frag

See also Figures S3 and S4.

Cell M

loss of circadian oscillations that has been observed in several

species (Asai et al., 2001; Rakshit et al., 2012; Yamazaki et al.,

2002; Zhdanova et al., 2008). Aging is known to attenuate

various circadian behaviors such as daily rhythms in hormone

levels, body temperature, sleep/wake cycles, etc., so con-

versely, improving oscillations might be expected to slow

down the age-related decline in function (Koh et al., 2006; Toui-

tou and Haus, 2000; Weinert and Waterhouse, 2007; Zhdanova

et al., 2011). Longevity in golden hamsters was reduced with

noninvasive disruption of circadian rhythmicity but was

increased in older animals when given suprachiasmatic implants

that restored high-amplitude rhythms in the animals (Hurd and

s upon DR

tistical analysis of the survival curves, number of flies, and data from additional

DR diet containing 14C-labeled glucose for 24 hr, and the synthesis of TG was

the isolated lipids from the flies. Breakdown was measured by a pulse-chase

and the remaining 14C label in TG fraction was measured (60 Hrs). Values are

was determined using Student’s t test and is denoted between time points is

roups. c or *** indicates p < 0.001, b or **p < 0.01, and a or *p < 0.05.

endent fashion.

Fs (predominantly forming the corresponding [M+NH4]+ ions) used to establish

features identified from HPLC-MS analysis of DR fly lipid extracts. For ease of

eak of interest. Further, for fly lipid features (bottom), only exemplary peaks are

y overlap.

im01 flies maintained on DR food conditions, segregated in three groups based

d group 3: cycling, peak during day.

ature (F88).

ment ions (Figure 4F), based on analysis of HRMS data of feature 88/TG(36:0).


Figure 6. Overexpression of tim Enhances Fat Metabolism, which Mediates Lifespan Extension

(A) Overexpression of tim in whole-body increases relative abundance of tim mRNA and circadian amplitude of tim expression. The data are normalized to the

trough (ZT4) levels seen in control flies on AL diet. Values are mean ± SEM (error bars) of three independent preparations.

(B) Overexpression of tim in whole-body increases TG turnover upon AL. Values are mean ± SEM of four independent preparations. Statistical significance was

determined using Student’s t test and is denoted by ***p < 0.001, **p < 0.01, and *p < 0.05.

(C) Fat-body-specific overexpression of tim increases TG synthesis specifically in the abdomen and thorax of the flies on AL diet. Values are mean ± SEM (error

bars) of four to five independent preparations. Statistical significance was determined using Student’s t test, and *denotes p < 0.05.

(D) Co-expression of tim overexpression and ACC RNAi in whole-body abrogates the AL-dependent increase in survival. KaplanMeier survival analysis of female

flies with tim overexpression under AL (with RU486, red) conditions, control flies (without RU486, blue), ACC RNAi (with RU486, green), and tim overexpression

with ACC RNAi (with RU486, black).

(E) The mean lifespan observed in survival curves shown in (6D). Statistical analysis of the survival curves, complete genotype and number of flies are provided in

Table S2, and for an independent repeat, see Figure S6.

(F) Levels of TLG are increased in flies overexpressing tim and are reduced upon ACC RNAi in whole body on AL diet. Values are mean ± SEM of four (error bars)

independent preparations. Statistical significance was determined using Student’s t test and is denoted by *p < 0.05, **p < 0.01, and ***p < 0.001.

Ralph, 1998). In Drosophila, overexpression of cryptochrome

(cry) in all clock cells was shown to maintain strong rest/activity

rhythms in older animals (Rakshit and Giebultowicz, 2013). How-

ever, overexpression of CRY in central clock neurons alone was

not sufficient to restore rest/activity rhythms, suggesting a

possible role of peripheral clocks in delaying behavioral and

physiological aging. Our results are in line with these observa-

tions as we show that DR improves the oscillations and levels

of the clock gene expression and proteins in peripheral tissues.

We also show that overexpression of tim in peripheral tissues

increased fat turnover and increased lifespan, even under AL

food conditions. Although tim overexpression in most of the pe-

ripheral tissues tested increased lifespan, we observed that the

lifespans of the control flies (without RU486) in this experiment

were shorter than those of other controls due to variability across

GAL4 strains. Thus, at the present time, we cannot distinguish

between compensation by TIM for lifespan-shortening variations

versus an ability to extend lifespan beyond normal. Neverthe-

less, it is clear that TIM overexpression is beneficial for lifespan

in, at least, some contexts.


The molecular mechanism of how DR enhances clock robust-

nesswill require futurestudiesexamining reciprocal linksbetween

nutrient sensingpathways and theclocks in various tissues. There

are three possible explanation of how a reduction in dietary pro-

tein or yeast (in case of flies) leads to regulation of circadian

clocks. The first is that yeast restriction could influence clocks

through the inhibition of TOR/insulin signaling. It was previously

shown that Drosophila foxomodulates stress sensitivity of circa-

dian clocks (Zheng et al., 2007). Furthermore, they also showed a

role of TOR/TSC and AKT in central clocks, likely through modu-

lation of GSK3b, which phosphorylates central clock proteins

(Zheng and Sehgal, 2010). Second, protein restriction modulates

TOR/ 4EBP signaling (Kapahi et al., 2010; Katewa and Kapahi,

2010; Zid et al., 2009), which in turn could influence the translation

of clock proteins. Finally, protein restriction influences the activity

of nuclear de-acetylating proteins (such as HDACs, SIRT1, or

SIRT6), which in turn could influence the transcriptional levels of

clock-dependent genes. Recently, Sassone-Corsi lab showed a

role of SIRT1 and SIRT6 in regulating circadian gene expression

in peripheral clocks (Masri et al., 2014).

nc.

Clocks Regulate Fat Metabolism under DRCircadian clocks are critical regulators of metabolism (Green

et al., 2008; Kohsaka et al., 2007; Panda et al., 2002; Ramsey

et al., 2007; Xu et al., 2011) and maintain temporal organization

that optimizes various cellular functions (Kovac et al., 2009; Lo-

boda et al., 2009; Ramsey et al., 2007). Genome-wide circadian

expression profiling studies have uncovered potential connec-

tions between circadian clocks and many aspects of meta-

bolism, including energy, carbohydrate, amino acid, lipid, and

protein metabolism, as well as detoxification (Beaver et al.,

2012; Panda and Hogenesch, 2004; Wijnen and Young, 2006).

In flies, recent studies have highlighted the regulation of lipid

and carbohydrate metabolism by circadian clocks (DiAngelo

et al., 2011; Seay and Thummel, 2011; Xu et al., 2011). Although

the levels of total TGs do not show circadian rhythmicity, flies

mutant for circadian genes as such tim (tim01) and cry (cry01)

show reduction in total levels of TGs (Seay and Thummel,

2011). Recently, we showed that upon DR, Drosophila mela-

nogaster shift their metabolism toward increasing both fatty

acid synthesis and breakdown, and these changes are required

for various responses to DR. Furthermore, flies that tend to in-

crease fat turnover on AL food show increased lifespan (Katewa

et al., 2012). However, it is not clear how flies undertake both

increased breakdown and synthesis of TGs. Our results here

support the idea that circadian clock genes play a critical role

in this improved fat turnover. Both tim and per mutant flies

showed reduced starvation resistance under DR conditions,

and this was associated with reduced fat synthesis and reduced

breakdown. Overexpression of tim on the other hand resulted in

increased fat turnover under rich nutrient conditions in both

whole flies or dissected tissues (Figures 6B and 6C). As the life-

span of tim01 female flies does not differ significantly from control

flies on AL food, we reasoned that themost effective way of iden-

tifying the group of lipids related with lifespan effects in tim01

would be lipidomic analysis under DR food conditions. We found

that one group of cycling lipids consists primarily of MCTs (Fig-

ure 4E). One of the MCTs, TLG, showed an almost 2-fold in-

crease between ZT4 and ZT16 under DR conditions, and its

levels were dependent on tim (Figures 4E, S4, and 6F). Reducing

de novo TG synthesis by inhibition of ACC led to a reduction of

the protective effects of tim overexpression, further supporting

the critical role of fat turnover in lifespan extension under both

DR (Katewa et al., 2012) as well as tim overexpression (Figures

6E, 6F, and S6D). The question of whether it is timeless alone

or also other clock components that regulate DR response is still

not clear. Although, the mRNA levels of other core clock genes

are not influenced by timeless overexpression, our results

showing that both tim and per mutants show reduction in DR-

dependent starvation resistance, and lifespan extension, indi-

cates a role of circadian clocks in DR responses. However, while

overexpression of timeless increases fat turnover and lifespan

under AL conditions, period overexpression has no effect on life-

span. It is possible that per is not limiting for these effects, but tim

could be the major driver here, with per mutants showing an

effect only because of their effect on nuclear expression of TIM

(Zheng and Sehgal, 2008).

MCTs are TGs containing MCFAs (C8 to C12 length). MCTs

were originally used for dietary treatment of malabsorption

syndromes in humans because of their rapid absorption and

Cell M

breakdown compared to long-chain TGs (LCT) (Scheig, 1968).

In animals, replacing dietary LCT byMCT causes a rise in energy

expenditure, increases thermogenesis, depresses food intake,

and causes lowering of body fat (St-Onge and Jones, 2002). In

humans also, feedingMCT increases energy expenditure relative

to LCT feeding (Mumme and Stonehouse, 2015). Based on these

effects, MCT-based interventions are being considered for die-

tary treatment of obesity and are proposed for weight-loss treat-

ments (St-Onge and Jones, 2002). Despite being used in both

animals and humans over several decades, ours is the first report

of de novo synthesis of MCTs in response to DR in any animal.

However, it’s still not clear why DR animals would enhance levels

of MCTs. One hypothesis is that MCTs are faster to synthesize

and easier to break down by cells for energy purposes. Another

possibility is that asMCT feeding results in increased b-oxidation

of not only MCTs but also of LCTs (Ronis et al., 2013), this would

lead to increase fatty acid turnover andwill reduce the time avail-

able for fatty acids to undergo oxidation (lipid peroxidation, etc.).

Finally, as their levels are regulated by clocks, changing concen-

trations of MCTs could act as signaling molecules and modulate

activities of kinases or nuclear transcription factors. Support for

this hypothesis comes from a recent study showing that MCT

feeding ameliorates insulin resistance and inflammation in

high-fat-diet-induced obese mice (Geng et al., 2015). In conclu-

sion, our results strongly support a key role of clock regulated

metabolic adaptation under DR and pave the way for future

longevity studies involving manipulations of clocks via environ-

mental or pharmacological interventions.

EXPERIMENTAL PROCEDURES

Fly Stocks, Husbandry, and Survival Assays: Fly Stocks

The following fly strains were used: Act5C-GS-GAL4 (w; P{Act5C(-FRT)

GAL4.Switch.PR}255B) (Ford et al., 2007), S1106 GAL4- (w1118; P{Switch1}

106) (Roman et al., 2001), Elav-GS-GAL4 (yw;P{elav-Switch.O}GSG301) (Os-

terwalder et al., 2001), tim01 (Myers et al., 1995), per01 (Konopka and Benzer,

1971), UAS-tim, UAS-per24 (Yang and Sehgal, 2001), UAS-per10 (Stoleru

et al., 2007), C42-GAL4 (w[*]; P{w[+mW.hs] = GawB}c42), and 5966-GS-

GAL4 (Guo et al., 2014) and ACC- RNAi (w[1118];P{GD3482}v8105) (Katewa

et al., 2012). All survival assays were carried out on AL or DR media as

described previously (Katewa et al., 2012; Zid et al., 2009). Adult female flies

were transferred within 2 to 3 days of eclosion to media differing only in the

amount of YE in the diet and were maintained at 25�C temperature, 60% hu-

midity, and 12 hr light and 12 hr dark conditions for their entire lifespan. About

25–30 mated females were maintained per vial, and the flies were transferred

every 2 to 3 days onto fresh media vials and deaths recorded. Additional de-

tails about various fly media recipes that were used in the study are provided

in the Supplemental Experimental Procedures.

Circadian Fly Sample Collections

Fly vials were removed every 4 hr (starting at ZT0 [8.00 AM], ZT4, ZT8, ZT12,

ZT16, ZT20, and ZT24 [next day 8.00 AM]) and were either frozen immediately

(for mRNA measurements) or were kept on ice for fat body dissection. Flies

selected for circadian analysis were transferred into individual racks during

the light period so that removal of one vial during the night doesn’t disturb other

flies that would be frozen later in the night.

Fat Turnover and Starvation Assay

After 10 days feeding on AL and DR media, 240 flies were transferred to AL

or DR media with 2 mCi of 14C-labeled glucose added on top of the media.

After 24 hr of feeding, half of the flies were snap-frozen in liquid nitrogen

(and are referred to as 0 hr sample). The other half were then transferred

to fresh non-radioactive AL or DR media and were kept on this media for


the next 60 hr and then immediately frozen (these samples were referred as

60 hr sample). The frozen samples (20 flies/replicate) were homogenized

in chloroform-methanol (2:1), and total lipid was extracted by the Folch

method (Folch et al., 1957). Total lipid was resuspended in 500 ml of chloro-

form and was further fractionated into TG fraction by using DSC-NH2

cartridges and different solvents as described previously (Katewa et al.,

2012). The fractions were dried under nitrogen, re-suspended in the scintilla-

tion fluid, and counted in a scintillation counter. 0 hr samples indicate the

rate of incorporation of glucose in fatty acids, and 60 hr sample indicate

the breakdown of the labeled fatty acids. For starvation assays, female flies

(day 10 on AL/DR media) were transferred to vials containing 1% agar. The

flies were transferred to fresh vials every 24 hr, and deaths were recorded

every 6–12 hr.

Lipid Extraction and HPLC—MS Sample Preparation

Fly lipid extraction was performed based on a previously reported protocol

(Hammad et al., 2011) with some modifications. Briefly, five flies for each

time point for a genotype (control and tim01) were weighed, flash-frozen

over liquid nitrogen, and subsequently homogenized ultrasonically using a

Fisher Scientific’s 550 Sonic Dismembrator with 100 ml of 0.9% NaCl soution.

Three 20-s pulses at amplitude setting 4 of the instrument (on ice) were suf-

ficient to completely homogenize fly bodies. The homogenates were then

transferred to clean glass vials, and the original tubes were washed twice

with 50 ml of the 0.9% NaCl solution to ensure complete transfer. To this,

1,000 ml of 2:1 dichloromethane (DCM):methanol (containing 5 mM of each

of the following internal standards: d5-1,3-DG(15:0/15:0) and d5-TG(17:0/

17:1/17:0)) was added and each sample vortexed for five times over a period

of �30 min (each 30 s long). Subsequently, the samples were centrifuged at

4,000 rpm for 10 min—at this point, two clear layers separated, and the fly

debris was collected at the interphase of the layers. 500 ml of the lower

organic layer was quantitatively collected using a clean glass syringe and

5 ml injected directly for qualitative high-resolution HPLC-MS analysis, or

diluted 1:5 with 1:1 DCM:methanol and then 5 ml injected for quantitative

unit resolution HPLC-MS analysis without any further processing. Between

each subsequent extract collection, the glass syringe was thoroughly

washed with the 1:1 DCM:methanol solution. Additional details of HPLC-

MS instrumentation and methods are provided in Supplemental Experimental

Procedures.

HPLC-MS and -MS/MS Analyses

A preliminary analysis revealed that the control/ZT 16 sample to contain high

(or most prominently detectable; i.e., with the highest signal-to-noise ratio) in-

tensities for most peaks and hence was thoroughly examined to tabulate a list

of mass spectrometric features (�350), defined as a specificm/z at a particular

retention time. The primary focus of our study was to identify cycling TGs;

since a standard mix of synthetic d5-TGs (Figure 4D) showed predominantly

[M+NH4]+ under our HPLC-MS conditions, care was taken to specifically tabu-

late the [M+NH4]+ adduct ions and eliminate features that indicated low (but

still detectable) levels of the corresponding [M+H]+ or [M+Na]+ adducts.

Further, features originating from solvent contaminants (and/or other exoge-

nous sources) were carefully selected and removed prior to quantitative anal-

ysis. Subsequently, peak areas for �253 features (Figure 4D) were computed

from the unit resolution HPLC-MS data for fly lipid extracts from the QTRAP

MS instrument across each of four replicates for eight time points (ZT 0, 4,

8, 12, 16, 20, and 24 hr) of control and tim01 flies (total of 56 samples). Peak

areaswere subsequently normalized first by the flyweight for the sample (repli-

cate), calculated prior to lipid extraction, and then by the corresponding peak

area of the DG-internal standard [M+NH4]+ adduct ion for each replicate run

to account for variability during the biphasic lipid extraction process and/or

sample to sample variability in MS response across �60 hr of continuous

acquisition.

The cycling features were characterized further using the corresponding

HRMS data acquired using the QTOF MS instrument (see Table S4) to anno-

tate features to specific TGs and the most prominent cycling feature 88,

TG(36:0) was subjected to high-resolution MS/MS analysis (Figures 4F and

4G). The high-resolution molecular ion and fragment ions were matched

against LIPID MAPS’ (Sud et al., 2007) database using their online MS tools

(Fahy et al., 2007) for corresponding structural elucidation (Figure 4G).


Statistical Analysis

Circadian expression data were statistically analyzed with GraphPad Prism

(v.5.0) and GraphPad Instat (v.3.0). qRT-PCR data for circadian gene expres-

sion were evaluated by two-way ANOVAwith Bonferroni’s post hoc test. Other

data were analyzed by Student’s t test. Each survival assay was repeated at

least twice, and the shown figures represent a typical curve. Survival curves

were created using the product-limit method of Kaplan and Meier. The log-

rank (Mantel-Cox) test was used to evaluate differences between survivals

and determine p values. We used the Prism software package (GraphPad

Software) to carry out statistical analysis and to determine lifespan values.

Additionally, we have used Cox proportional hazards analysis implemented

in the R package ‘‘survival’’ to analyze the significance of the interaction

between two variables in several of the survival outcomes. We report the prob-

ability that B1,2 = 0, from fitting the formula phenotype = B1 , variable1+B2 ,

variable2+B1,2 , (variable1 , variable2). The respective p values are included

in the text.

SUPPLEMENTAL INFORMATION

Supplemental Information includes six figures, four tables, and Supplemental

Experimental Procedures and can be found with this article online at http://dx.

doi.org/10.1016/j.cmet.2015.10.014.

ACKNOWLEDGMENTS

We thank M. Kolipinski, J. Beck, and M. Vargas for lifespan data collection

and members of the Kapahi Lab for discussions and suggestions. We would

also like to thank Prof. Daniel Promislow for help with survival analysis. This

work was funded by grants from the American Federation of Aging Research

(S.D.K.), Larry L. Hillblom Foundation (S.D.K.), NIH (R01AG038688,

AG038012, and AG045835 [P.K.]; R01 AG045830 [J.M.G.]), and from the Elli-

son Medical Foundation (A.S.). A.S. is an HHMI investigator.

Received: January 23, 2015

Revised: June 22, 2015

Accepted: October 25, 2015

Published: November 25, 2015

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Cell Metabolism, Volume 23

Supplemental Information

Peripheral Circadian Clocks Mediate Dietary

Restriction-Dependent Changes in Lifespan

and Fat Metabolism in Drosophila Subhash D. Katewa, Kazutaka Akagi, Neelanjan Bose, Kuntol Rakshit, Timothy Camarella, Xiangzhong Zheng, David Hall, Sonnet Davies, Christopher Nelson, Rachel B. Brem, Arvind Ramanathan, Amita Sehgal, Jadwiga M. Giebultowicz, and Pankaj Kapahi

Supplemental Figures and legends

Figure S1, related to Figure 1. Increase in amplitude of circadian gene expression upon DR. (A) Increased amplitude of circadian gene expression after dietary restriction in fat bodies isolated from female flies. (B) Increased amplitude of circadian gene expression after dietary restriction in male flies. Daily mRNA profiles of core clock genes tim and per in control (i and ii) Canton S and (iii and iv) w1118 flies fed on AL (5% YE, 5% sugar) and DR (0.5% YE, 5% sugar) diets for 10 days. Data are normalized to the ZT4 values set at 1 for flies on each gene. (C) DR improves the amplitude of circadian gene expression in old flies. Daily mRNA profiles of clock genes in (i) heads and (ii) bodies of Canton S females fed on AL or DR diets for 30 days, starting on day 3. The data are normalized to the trough (ZT4/16) values set at 1 for flies on AL diet. White and black horizontal bars mark periods of light and dark respectively. Each data point represents mean ± SEM of three independent RNA samples. Statistical significance between AL and DR values was determined using two-way ANOVA with Bonferonni’s post hoc test, and is denoted by ***p< 0.001, **p<0.01, and *p<0.05.

Figure S2, related to Figure 3. Male tim01 and per01 flies respond to DR in a similar way as controls. (A) tim01 mutant males and controls showed similar response to DR. Kaplan Meier survival analysis for in control flies (blue) and tim01flies (red) under DR (solid line) and AL (dashed line) conditions. Following median lifespan were observed for control DR (50 days (n=137) and control AL (44 days (n=125)); tim01 DR (52 days (n=144) and tim01AL (42 days (n=149)). (B) DR dependent increase in lifespan was similar in male per01 and control flies. Kaplan Meier survival analysis for control flies (blue) and per01 flies (red) under DR (solid line) and AL (dashed line) conditions. Following median lifespan were observed for control DR (57 days (n=117) and control AL (41 days (n=123)); per01 DR (48 days (n=132) and per01 AL (36 days (n=111)).

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Figure S3, related to Figure 4. Daily quantitative profiles of 250 lipid like features in control and tim01 flies on DR. Lipids were extracted from adult female flies fed DR diet for 10 days. The lipids were separated and analyzed by LC-MS/MS and were normalized for both internal standards and fly wts. Error bars indicate S.E.M of 4 independent biological repeats.

0

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1.2 F189F231F174F186F239

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Ctrl tim01 Ctrl tim01

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0.15 F137F155F141F191F169

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0.15 F161F54F26F279F298

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0.12 F130F103F229F242F158

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0.09 F287F163F201F154F135

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0.09 F203F270F205F278F150

0

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0.09 F108F204F47F21F123

0

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0.09 F244F118F268F296F219

Figure S3, related to Figure 4 (Continued). Daily quantitative profiles of 250 lipid like features in control and tim01 flies on DR. Lipids were extracted from adult female flies fed DR diet for 10 days. The lipids were separated and analyzed by LC-MS/MS and were normalized for both internal standards and fly wts. Error bars indicate S.E.M of 4 independent biological repeats.

Ctrl tim01

Zeitgeber Time

Ctrl tim01 Ctrl tim01 Ctrl tim01

0

0.02

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0.06 F171F52F182F116F238

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0.045 F258F300F285F333F299

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0.045 F210F166F140F263F44

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0.045 F198F243F218F107F294

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0.045 F286F146F179F252F256

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0.03 F284F70F248F275F30

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0.03 F222F159F156F227F188

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0.03 F40F29F338F265F170

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0.03 F197F22F322F225F20

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0.03 F336F43F232F262F200

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Figure S4, related to Figure 4. Daily quantitative profiles of trilauryl glycerol (TLG, feature 88) in control and tim01 flies on DR. Lipids were extracted from control and tim01 female flies fed DR diet for 10 days. The lipids were separated and analyzed by LC-MS/MS and were normalized for both internal standards and fly wts. Error bars indicate S.E.M of 4 independent biological repeats. Statistical significance between the two groups was determined using two-way ANOVA with Bonferonni’s post hoc test, and is denoted by ***p< 0.001, **p<0.01, and *p<0.05.

Figure S5, related to Figure 5. Overexpression of per and tim had no effect on lifespan under DR conditions. (A) Overexpression of UAS-per24 had no effect on lifespan under DR conditions but reduced lifespan under AL conditions. Kaplan Meier survival analysis for control flies (without RU486, blue) and per24overexpression flies (with RU486, red) under DR (solid line) and AL (dashed line) conditions. Following median lifespan were observed for control DR (80 days (n=119) and AL (68 days (n=139)); per24overexpression, DR (77 days (n=109) and AL (59 days (n=122)). (B) Overexpression of UAS-per10 had no effect on lifespan under DR or AL conditions. Kaplan Meier survival analysis for control flies (without RU486, blue) and tim overexpression flies (with RU486, red) under DR (solid line) and AL (dashed line) conditions. Following median lifespan were observed for control DR (78 days (n=127) and AL (50 days (n=136)); per10 overexpression, DR (83 days (n=145) and AL (48 days (n=130). (C-F) Overexpression of tim has no effect on lifespan on a DR diet. Overexpression of tim had no effect on lifespan under DR conditions when over-expressed specifically in (C) neurons, (D) fat body (E) gut (F) and Malpighian tubules. Statistical analysis of the survival curves, genotype and the number of flies used in each group are provided in Table S2. For additional independent repeats of the data see Table S3.

B C

D E F

A

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Figure S6, related to Figure 6. Overexpression of tim increases expression, fat metabolism and lifespan in a diet dependent manner. (A) Overexpression of tim increased the magnitude of expression of tim mRNA on a DR diet. Circadian expression of tim mRNA levels under induced and un-induced conditions. The data are normalized to the trough (ZT4) levels seen on an AL (-RU486) diet. The error bars indicate S.E.M of 3 independent preparations. (B) Overexpression of tim has no effect on the expression of other clock genes. Daily mRNA concentration profiles of core clock genes in flies overexpressing tim in the whole body. The data are normalized to the trough (ZT4) levels seen in control flies on AL diet. Values are mean ± SEM of 3 independent preparations. (C) Overexpression of tim has no effect on fat metabolism on a DR diet. Triglyceride turnover was similar under DR conditions in both tim overexpression induced (+RU486) and un-induced (- RU486) flies. The error bars indicate S.E.M of 4 independent preparations. Statistical significance was determined using Student’s t test and is denoted by ***p< 0.001, **p<0.01, and *p<0.05. (D-E) Co-expression of tim overexpression and ACC RNAi in whole body abrogates the AL dependent increase in survival. (D) Kaplan Meyers survival analysis for flies overexpressing timeless in presence or absence of ACC RNAi in whole fly (with Act5c-GS-GAL4 driver). RU486 addition was used to induce overexpression. (E) represents the median lifespan observed in survival curves shown in (D). Statistical analysis of the survival curves, genotype and the number of flies used in each group are provided in Table S2.

A

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ACCRNAi UAS-tim UAS-tim/ACCRNAi

Supplemental Tables

Table S1, related to Figure 1. Statistical analysis of gene expression (qPCR) data by two-way ANOVA with Bonferroni’s post-hoc test. Effect of ZT Effect of diet

Heads (Day 13)

per F6,28 = 59.75 p < 0.0001 F1,28 = 37.68 p < 0.0001

tim F6,28 = 111.49 p < 0.0001 F1,28 = 31.79 p < 0.0001

Pdp1 ε F6,28 = 96.07 p < 0.0001 F1,28 = 33.76 p < 0.0001

vri F6,28 = 550.81 p < 0.0001 F1,28 = 122.19 p < 0.0001

Clk F6,28 = 118.44 p < 0.0001 F1,28 = 130.55 p < 0.0001

cry F6,28 = 12.29 p < 0.0001 F1,28 = 0.91 p = 0.3488

Heads (Day 33)

per F6,28 = 110.38 p < 0.0001 F1,28 = 58.27 p < 0.0001

tim F6,28 = 99.74 p < 0.0001 F1,28 = 48.74 p < 0.0001

Pdp1 ε F6,28 = 89.16 p < 0.0001 F1,28 = 37.18 p < 0.0001

vri F6,28 = 53.76 p < 0.0001 F1,28 = 6.55 p < 0.05

Clk F6,28 = 21.61 p < 0.0001 F1,28 = 38.27 p < 0.0001

cry F6,28 = 8.02 p < 0.0001 F1,28 = 0.45 p = 0.5057

Bodies (Day 13)

per F6,28 = 48.82 p < 0.0001 F1,28 = 320.29 p < 0.0001

tim F6,28 = 130.93 p < 0.0001 F1,28 = 349.92 p < 0.0001

Pdp1 ε F6,28 = 191.49 p < 0.0001 F1,28 = 351.30 p < 0.0001

vri F6,28 = 34.17 p < 0.0001 F1,28 = 121.14 p < 0.0001

Clk F6,28 = 8.42 p < 0.0001 F1,28 = 114.97 p < 0.0001

cry F6,28 = 2.84 p < 0.05 F1,28 = 0.33 p = 0.9152

Bodies (Day 33)

per F6,28 = 48.97 p < 0.0001 F1,28 = 50.50 p < 0.0001

tim F6,28 = 170.72 p < 0.0001 F1,28 = 57.77 p < 0.0001

Pdp1ε F6,28 = 182.38 p < 0.0001 F1,28 = 31.72 p < 0.0001

vri F6,28 = 62.34 p < 0.0001 F1,28 = 92.78 p < 0.0001

Clk F6,28 = 16.47 p < 0.0001 F1,28 = 62.16 p < 0.0001

cry F6,28 = 13.03 p < 0.0001 F1,28 = 0.01 p = 0.9781

Subscripted values indicate the degrees of freedom in numerator (DFn) and denominator (DFd), respectively.

Table S2, related to Figures 3, 4, 5 and 6. Detailed statistical analyses of survival curves provided in the main figures 3, 4, 5 and 6 (provided as a separate excel spreadsheet)

Table S3, related to Figures 3, 4 and 5. Summary of the independent repeats of the lifespan analyses of the survival curves. Median Lifespan (in days) Group (Cross genotype )

Date Repeat # LL DR (n)

LL AL (n)

Control DR (n)

Control AL (n)

Control (Canton-S) (Figure 3A)

7/12/2011 1 58(98) 46(91) 72(110) 51(103) 2/2/2012 2 60(151) 40(140) 74(149) 43(144)

Median Lifespan (in days) Group (Cross genotype )

Date Repeat # Mutant DR (n)

Mutant AL (n)

Control DR (n)

Control AL (n)

tim01 (Canton-S background) lifespan (Figure 3B)

6/21/2011 1 44(141) 40(139) 68(140) 45(136) 2/3/2011 2 36 (159) 27(152) 55(115) 39(112) 6/2/2011 3 47.5(150) 41(140) 67(142) 47(150)



Mutant AL (n)

Control DR (n)

Control AL (n)

tim01 (W1118 back ground) (Figure 3C)

4/15/2013 1 47(91) 31(99) 75(119) 39(131) 4/13/2013 2 39(64) 21(73) 65(92) 25(80) 6/12/2013 3 54(116) 28(121) 75(133) 26(139)



Mutant AL (n)

Control DR (n)

Control AL (n)

per01 lifespan (Figure 3E)

5/28/2012 1 46(111) 37(104) 70(128) 49(125) 5/10/2012 2 60(145) 39(111) 64(133) 39(101) 8/16/2012 3 53(186) 41(160) 64(171) 43(179)

Median Lifespan (in Hrs) Group (Cross genotype )


Mutant AL (n)

Control DR (n)

Control AL (n)

tim01 starvation (Figure 4A)

7/04/2011 1 77(125) 52(114) 124(119) 72(97) 10/4/2011 2 69 (128) 45(119) 80(135) 45(119)

Median Lifespan (in Hrs) Group (Cross genotype )


Mutant AL (n)

Control DR (n)

Control AL (n)

per01 starvation (Figure 4B)

5/28/2012 1 53(132) 45(89) 77(103) 45(104) 11/18/2012 2 52(114) 41(117) 98(100) 44(131) 8/15/2012 3 53(132) 44(132) 68(131) 44(126)


Date Repeat #

Ctrl DR (n)

(+ Ru486) DR (n)

Ctrl AL (n)

(+ Ru486) AL (n)

Act5c-GS-GAL4 x UAS tim2-

5(Figure 5A)

10/31/2012 1 91(129) 84(124) 42(127) 58(140) 5/17/2012 2 71(130) 71(112) 48(128) 60(125) 2/17/2012 3 80(120) 83(100) 57(136) 62(143) 9/30/2012 4 49(131) 48(122) 38(143) 45(137)


Date Repeat #

Ctrl DR (n)

(+ Ru486) DR (n)

Ctrl AL (n)

(+ Ru486) AL (n)

Elav-GS-GAL4 x UAS tim2-

5(Figure 5B)

11/27/2012 1 76(124) 76(115) 50(133) 50(139) 1/31/2012 2 46 (125) 42(123) 33(132) 26 (135)


Date Repeat #

Ctrl DR (n)

(+ Ru486) DR (n)

Ctrl AL (n)

(+ Ru486) AL (n)

S106-GS-GAL4 x UAS tim2-5 (Figure 5C)

12/13/2012 1 53(114) 53(128) 27(140) 34(133) 6/12/2011 2 58 (131) 60 (102) 46

(135) 56 (121)

8/8/2011 3 73 (104) 70 (99) 50 (88) 56 (83)

Table S4, related to Experimental Procedures (section HPLC-MS and –MS/MS analyses).

HRMS analysis for TGs classified in groups 1-3.

Features (Group)

Retention Time (min)

Observed HRMS

LIPID MAPS

Annotated TG

Calculated HRMS

Molecular ion formula [M+NH4]+

delta (ppm)

feature231 (1) 29.8 794.7244 TG(46:1) 794.7232 C49H96NO6+ 1.5

feature174 (1) 28.6 766.6927 TG(44:1) 766.6919 C47H92NO6+ 1.0

feature266 (1) 30.7 822.756 TG(48:1) 822.7545 C51H100NO6+ 1.8

feature276 (1) 31 848.7712 TG(50:2) 848.7702 C53H102NO6+ 1.2

feature102 (2) 25.2 696.6171 TG(39:1) 696.6137 C42H82NO6+ 4.9

feature88 (2) 24.1 656.5824 TG(36:0) 656.5824 C39H78NO6+ 0

feature97 (2) 24.5 682.5989 TG(38:1) 682.598 C41H80NO6+ 1.3

feature128 (3) 27 712.6458 TG(40:0) 712.645 C43H86NO6+ 1.1

feature143 (3) 27.5 738.6611 TG(42:1) 738.6606 C45H88NO6+ 0.7

feature167 (3) 28.4 740.6769 TG(42:0) 740.6763 C45H90NO6+ 0.8

feature230 (3) 29.8 792.7095 TG(46:2) 792.7076 C49H94NO6+ 2.4

Supplemental Experimental Procedures (Related to Experimental Procedures) Fly husbandry, media and survival assays:

The description of various fly media recipes that were used in the study are provided below: Standard media: All fly stocks were maintained on standard lab fly media. The standard lab media is based on the Caltech media recipe, which includes 8.6% (w/v) Cornmeal, 1.6% (w/v) Yeast, 5% (w/v) Sucrose, 0.46% (w/v) Agar, 1% (v/v) Acid mix (6, 13, 34). To prepare the media, Cornmeal (86 gm), Sucrose (50 gm), active-dry-yeast (16 gm, "Saf-instant") and Agar (4.6 gm) were mixed in a liter of water and brought to boil with constant stirring. The media was allowed to cool down to 60°C, before 10 ml of acid mix was added and mixed in the media. Acid mix was prepared by mixing equal volumes of 10% propionic acid (v/v) and 83.6% orthophosphoric acid. The media was then poured in vials (~10ml/ vial) or bottles (25 ml/bottle) and allowed to cool down before storing at 4°C for later usage. These vials or bottles were then seeded with some live yeast just before the flies are transferred and used for maintenance of lab stocks or for collecting virgins and setting up the crosses. Media for survival analyses: All survival and other assays were performed on media with varying yeast extract (YE) concentration, which is described below. AL media: The AL media contained 8.6% (w/v) Cornmeal, 5.0% (w/v) Baker's yeast extract (#212750 Bacto™ Yeast Extract, B.D. Diagnostic Systems, Sparks, MD), 5% (w/v) Sucrose, 0.46% (w/v) Agar, 1% (v/v) Acid mix. To prepare the media, Cornmeal (86 gm), Sucrose (50 gm), Yeast extract (50 gm) and Agar (4.6 gm) were mixed in a liter of water and brought to boil with constant stirring. The media was allowed to cool down to 60°C, before 10 ml of acid mix was added and mixed in the media. The media was then poured in vials (~5ml/vial) and allowed to cool down before storing at 4°C for later usage. DR media: The DR media contained 8.6% (w/v) Cornmeal, 0.5% (w/v) Baker's yeast extract (#212750 Bacto™ Yeast Extract, B.D. Diagnostic Systems, Sparks, MD), 5% (w/v) Sucrose, 0.46% (w/v) Agar, 1% (v/v) Acid mix. To prepare the media, Cornmeal (86 gm), Sucrose (50 gm), Yeast extract (5 gm) and Agar (4.6 gm) were mixed in a liter of water and brought to boil with constant stirring. The media was allowed to cool down to 60°C, before 10 ml of acid mix was added and mixed in the media. The media was then poured in vials (~5ml/vial) and allowed to cool down before storing at 4°C for later usage. 0%, 1% and 2% YE media: These media types were used to assay the response of flies to varying concentration of yeast restriction. The media recipe is same as AL or DR media with differences only in the amount of yeast extract used. AL or DR media with RU486: For induction of tim overexpression, we used AL or DR media with additional RU486 mixed in the media. Ru486 was added in the cooling media at the same time as acid mix. RU486 was dissolved in 95% ethanol and was used at a final concentration of 100µM (the media is then refereed as 'with RU486'). The control AL or DR media contained the same volume of 95 % ethanol and is referred to as media 'without RU486'.

Genetic crosses: 10-12 young adult females (belonging to either the Gal4 stocks or the mutant (tim01 or per01) or control flies (CS or w1118) with 3-5 males were transferred to a new stock bottle and were allowed to lay eggs for few days, after which the parents were removed. 7-8 days later. The newly eclosed virgin females were collected and used to setup the genetic crosses. To set up the cross, 10-12 young virgin females were kept with 3-5 young male flies in new stock bottles. The males were either from the mutant or control or the UAS-tim over-expression flies. For example, male flies carrying UAS-tim (30) are crossed to virgin females from the RU486 inducible Act5C-GS-GAL4 driver stocks. Flies were kept in the stock bottles for four days, after which the parents were removed and the larvae were allowed to develop in standard lab conditions (25°C temperature, 60% humidity and 12 hr day and 12 hr night). The newly eclosed flies from the crosses were allowed to mate for 2-3 days before they were sorted into females and males under light CO2 anesthesia. The sorted flies were then transferred to the appropriate media for survival analyses.

Survival assays: All survival assays were carried out on AL or DR media as described previously

(Katewa et al., 2012; Zid et al., 2009). Adult female flies were transferred within 2-3 days of eclosion to media differing only in the amount of yeast extract (YE) in the diet and were maintained at 25°C temperature, 60% humidity and 12 hr light and 12 hr dark conditions for their entire lifespan. About 25-30 mated females were maintained per vial and the flies were transferred every 2-3 days onto fresh media vials and deaths recorded.

Survival assays with and without RU486: For over-expression of tim, we collected virgins from the different GAL4 stocks (Act5c-GS-GAL4, S106-GS-GAL4, Elav-GS-GAL4 , 5966-GS-GAL4 or C42-GAL4) and crossed them with males from UAS-tim overexpression stocks in bottles with stock media. The parents were removed after four days and the resulting progeny was sorted 2-3 days after eclosion. Newly sorted flies were then transferred to media (AL or DR) with or without the presence of 100µM RU486 and maintained at standard lab conditions (25°C temperature, 60% humidity and 12 hr light and 12 hr dark conditions) for various measurements. For survival assays, 25-30 mated females were maintained per vial and the flies were transferred every 2-3 days onto fresh media vials (with or without RU486) and deaths recorded.

Survival assays on continuous light (LL): Adult young mated Canton S female flies (2-3 days post-eclosion) were sorted and transferred to AL or DR media. The survival assays were then carried out at 25°C temperature, 60% humidity and 24 hrs continuous light (LL). The control group received similar lifespan conditions but with 12 hr light and 12 hr dark conditions (LD).

High performance liquid chromatography (HPLC) - mass spectrometry (MS)

instrumentation and methods: For unit resolution (quantitative) HPLC-MS analysis, HPLC was performed using a Shimadzu UFLC prominence system fitted with following modules: CBM-20A (Communication bus module), DGU-A3 (degasser), two LC-20AD (liquid chromatograph, binary pump), SIL-20AC HT (auto sampler) and connected to an Agilent Zorbax SB-C18 column (2.1 x 150 mm, 3.5 µm, maintained at 50 °C). Samples were kept at

+4°C. MS was performed using a 4000 QTRAP® LC-MS/MS mass spectrometer from AB SCIEX fitted with a TurboVTM ion source. MS was operated in positive ion scanning mode for a mass range of m/z 200-1200 with the following source conditions: curtain gas (CUR) 20, nebulizer gas (GS1) 60, auxiliary gas (GS2) 50, ionspray voltage (IS) +4500 V, and source temperature (TEM) 350 oC. AB SCiex’s Analyst® v1.6.1 was used for all forms of data acquisition and development of HPLC method. PeakView® v2.1 was used for the preliminary analysis of HPLC-MS data and feature generation. Skyline-daily® 3.1.1.7490 was used for in-depth analysis of the HPLC-MS data, specifically for calculating the peak areas for the identified features from fly lipid extracts.

For high-resolution (accurate-mass) HPLC-MS analysis, HPLC was performed using an Agilent 1260 Infinity LC system fitted with following modules: u-degasser (G1322A), binary pump (G1312B), thermostated column compartment (G1330B), HiPALS auto sampler (G1367E) and connected to an Agilent Zorbax SB-C18 column (2.1 x 150 mm, 3.5 µm, maintained at 50 °C). Samples were kept at +4°C. High-resolution MS1 and MS/MS analysis were performed using an Agilent 6520 QTOF mass spectrometer fitted with a Dual-Spray Electrospray Source (ESI). The instrument was operated at a mass resolution of ~20,000 for TOF MS1 scan and ~5,000 for product ion (MS/MS) scan using 2GHx extended dynamic range mode. The ionization parameters were set as follows: gas temperature (TEM) 350°C; drying gas, 9L/min; Vcap, 2500V; nebulizer, 35psig; fragmentor, 125V; and skimmer, 65V. MS1 acquisition was operated in the positive ion scanning mode for a mass range of 500-1200 m/z. Targeted MS/MS acquisition was composed of single MS scan (cycle time 1 spectra/sec) followed by ion specific MS/MS scans (1 spectra/sec) in the positive ion scanning mode for a mass range of 500-1200 m/z and 150-1000 m/z respectively. Collision energies were determined by linear interpolation, calculated according to the following equation: CE= (slope*precursor mass (m/z))/100 + Offset. For fly lipid extract analysis, a slope of 4V and an offset of 10V were used. The parameters for MS/MS on a target ion list were set as follows: quadrupole mass band pass for precursor isolation, medium (4 Da); precursor charge state (z), 1; and delta retention time (retention time range for accepting that a precursor is found), 2 min. HPLC-MS data was acquired using Agilent MassHunter Workstation (B.05.00). Agilent MassHunter Qualitative Analysis B.05.00 was used for the in-depth analysis of the HPLC-MS data and feature generation for the identified features from fly lipid extracts. For HPLC separation, a solvent gradient of 0.1% formic acid in 5:4:1 isopropanol: methanol: water containing 5 mM ammonium formate (aqueous) – 0.1% formic acid in 99:1 isopropanol:water containing 5 mM ammonium formate (organic) was used with 0.2 mL/min flow rate, starting with an organic content of 0% for 3 min, which was increased to 30% over 2 min and then to 95 % over the next 25 min and held at 95% for 12 min. The column was subsequently reconstituted to its initial condition (organic content of 0%) over the next 1 min and re-equilibrated for 9 min prior to the next injection. A blend of standard d5-DGs and d5-TGs from Avanti® polar lipids were used to develop this method (Figure 4D).

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