Supporting Material for

Fat Metabolism Links Germline Stem Cells and Longevity in C. elegans

Meng C. Wang, Eyleen J. O’Rourke, Gary Ruvkun*
*To whom correspondence should be addressed. E-mail: ruvkun@molbio.mgh.harvard.edu
Published 7 November 2008, Science 322, 957 (2008)
DOI: 10.1126/science.1162011
This PDF file includes:
Materials and Methods
Figs. S1 to S8
Tables S1 to S4
References
Supporting Online Material

Materials and methods

Strains

N2 Bristol was used as the wild-type strain. We have used the following C. elegans

strains and mutant alleles: glp-1(e2141)III, glp-4(bn2)I, fem-3(q20)IV, fem-3(e2006)IV,

glp-1(ar202)III, gld-1(q485)I, lag-2(q420)V, daf-12(rh61rh411)X, daf-2(e1370)III, glp-

1(e2141)III; mgEx[K04A8.5p::K04A8.5::gfp] and mgEx[ges-1p::K04A8.5::gfp] .

Nile Red, C1-BODIPY-C12 and Sudan Black staining

250
l of Nile Red (Molecular Probes) solution (1
g ml-1 in 1 X PBS) or C1-BODIPY-

C12 (Molecular Probes) solution (2
M in 1 X PBS) was applied to the surface of NGM

(Nematode Growth Media) plates (~12 ml agar) seeded with E. coli OP50. Plates were

used immediately after dry in a laminar flow hood (for C1-BODIPY-C12 staining) or

allowed to equilibrate overnight at room temperature (for Nile Red staining). For Nile

Red staining, synchronized animals were grown on Nile Red-containing plates since L1

larval stage and taken pictures at adulthood. For C1-BODIPY-C12 staining, animals were

transferred from NGM plates onto C1-BODIPY-C12-containing plates at L4 larval stage

and taken pictures at adulthood.

Quantification of Nile Red and C1-BODIPY-C12 staining was performed according to

Mak et al (1). Fluorescence images were captured using a Zeiss Axioplan microscope and

a CCD camera (Hamamatsu, ORCA-ER). To quantify fluorescence intensity, images of

1
the anterior intestine were captured from animals whose anterior intestine was not coved

by the gonad. The images were collected at 12-bit with the raw pixel values within the

linear range of the CCD camera that is controlled by an “automatic exposure” function

with the Openlab software (Improvision). To calculate the fluorescence intensity, the

mean pixel intensity of all fluorescence staining lipid droplets in the first 3 pairs of

intestinal cells were divided by the exposure time (Openlab, Improvision). For each

genotype, the average of 15~20 animals was presented.

For Sudan Black staining, 2-day-old adults were fixed in 1% paraformaldehyde in M9

and were then subjected to three freeze-thaw cycles. After incubated on ice for 10

minutes, fixed worms were washed with M9 and dehydrated through an ethanol series.

After staining overnight with 50% Sudan Black B solution in 70% ethanol, the animals

were rehydrated and photographed. Wild type and glp-1 mutants were in the same tube

during all the treatments. Around 20 animals of each genotype were mounted, and the

glp-1 mutants were distinguished by the sterile phenotype.

Measurement of pharyngeal pumping, defecation and food absorption rates

For pharyngeal pumping rate, 2-day-old well-fed adults after temperature shift at L4

larval stage were recorded with a video camera for 5 minutes. The number of pharyngeal

contractions in each second interval was calculated. For each genotype, the average of

15~20 animals was noted.

2
For defecation rate, 2-day-old well-fed adults after temperature shift at L4 larval stage

were monitored. For each animal, the duration of two consecutive cycles was recorded 5

times and the average of 5~10 animals is presented for each genotype.

Food absorption rate were measured as in O’Rourke et al. (in preparation). Briefly, 2-day

old animals were placed onto C1-BODIPY-C12-containing NGM plates. After 6hr

incubation in darkness, fluorescence images were captured every hour for 4 hours. C1-

BODIPY-C12 staining fluorescence was quantified as shown above. The accumulation

rate of C1-BODIPY-C12 overtime was calculated for both wild type and glp-1 mutants.

The values of the mutants were then normalized to wild type. For each genotype at each

time point, the average of 5 animals was noted.

Determination of locomotory activities

10 3-day-old well-fed adults after temperature shift at L4 larval stage were placed onto a

fresh NGM plate seeded with 50
L of OP50 bacteria. 5 minutes Digital videos were

captured using Zeiss Discovery V.12 stereoscope, Zeiss AxioCam Hsc camera and

AxioVision software. Each individual was followed by the “worm tracking” function

with the AxioVision software to quantify velocity. For each genotype, 3 plates were

prepared to take videos and 20 individuals’ activities were analyzed.

RNAi lifespan assay

3
RNAi bacteria were cultured 12 h in LB with 50
g/ml ampicillin and seeded onto RNAi

agar plates containing 5mM isopropylthiogalactoside (IPTG). The plates were allowed to

dry in a laminar flow hood and incubated at room temperature overnight to induce

dsRNA expression. Approximately 20~30 synchronized L1 larvae were placed onto

RNAi-containing agar plates, allowed to develop at 25°C for 40 hours and shifted to

20°C. The animals then were kept at 20°C and transferred to fresh RNAi plates every

two days or every seven days during or past the reproductive period respectively. For

each genotype and treatment, 4 plates were prepared and scored every day by gentle

prodding with a platinum wire to test for viability. Lifespan is defined as the first day of

adulthood (adult lifespan =1) to when they were scored as dead. Statistical analyses were

performed using SPSS software (http://www.spss.com). Each RNAi-treatment population

is compared with that of the population treated with control RNAi (bacteria carrying the

empty vector) using the log rank test.

RNAi Nile Red assay

Agar plates containing RNAi bacteria were prepared as described above. After dry in the

hood, 250
l of Nile Red solution (1
g ml-1 in 1 X PBS) was placed onto the surface of

RNAi plates (~12 ml agar). The plates were allowed to dry in the hood and incubate at

room temperature to equilibrate Nile Red and induce dsRNA expression. Approximately

30~40 synchronized L1 larvae were placed onto RNAi/Nile Red-containing plates.

Stress Assays

4
To perform heat shock assays, 2-day-old well-fed N2 adults were washed off plates and

filtered through 35
m nylon mesh to separate adults from eggs and L1 larvae. Adult were

then placed onto fresh NGM plates and heat shocked at 35 °C. For paraquat assays, 2-

day-old well-fed N2 adults were washed, filtered and submerged in 200
L of 200mM

paraquat dissolved in S-basal buffer at 20 °C.

Quantitative RT-PCR

Total RNA was isolated using TRIzol (invitrogen), and RT-PCR was performed as

previously described (2). Briefly, ~1000 1-day-old adults of each genotype and RNAi

treatment were harvested to isolate total RNA (eggs and L1 larvae were filtered away

using nylon mesh). All reactions were performed in triplicate. Data presented are from

three independent experiments and all values are normalized to rpl-32 as internal control

as well as to transcript levels in wild type.

Primer included: K04A8.5 (sense, 5’-ATGGCCGAGAAGTTCCTACATCGT-3’, and

antisense, 5’ GGTGAATTGGCGACCCAATCGAAA-3’) and rpl-32 (sense, 5’-

AGGGAATTGATAACCGTGTCCGCA-3’ and antisense, 5’-

TGTAGGACTGCATGAGGAGCATGT-3’).

References:

1. H. Y. Mak, L. S. Nelson, M. Basson, C. D. Johnson, G. Ruvkun, Nat Genet 38,

363 (Mar, 2006).
2. M. C. Wang, D. Bohmann, H. Jasper, Dev Cell 5, 811 (Nov, 2003).

5
 Figure S1
Wang et al

N2

glp-1

Fig. S1. C1-BODIPY-C12 staining of wild type and glp-1 mutants.

(A). C1-BODIPY-C12 staining of 2-d-old adult wild type (N2).

(B). C1-BODIPY-C12 staining shows a decrease in lipid accumulation in glp-1.

 Figure S2
Wang et al

N2

glp-1

Fig. S2. Sudan black staining of wild type and glp-1 mutants.

(A). Sudan black staining shows lipid accumulation in the anterior gut of 2-d-old adult wild type (N2).

(B). Sudan black staining shows a decrease in lipid accumulation in glp-1.

 Figure S3
Wang et al

A N2

B glp-1

C N2

D glp-4

Fig. S3. Lipid accumulation in the glp mutants is normal at permissive temperature.

(A). Nile Red fluorescence shows fat content of wild type (N2) at 20°C.

(B). Nile Red fluorescence shows lipid accumulation in the glp-1 mutant at 20°C.

(C). Nile Red fluorescence shows fat content of wild type (N2) at 15°C.

(D). Nile Red fluorescence shows fat content in the glp-4 mutant at 15°C.
 Figure S4
Wang et al

Fig. S4. Intestinal expression of the K04A8.5 lipase gene.

K04A8.5::GFP expression is detected in the intestine of the glp-1 mutants at non-permissive temperature.
 Figure S5
Wang et al

glp-1 N2

A C
vector

B
B D
daf-16

E G
vector

F H
kri-1

Fig. S5. Regulation of fat metabolism by germline stem cell proliferation dependently of
Kri-1/Daf-16 signaling.

(A-D). Reducing daf-16 activity by RNAi restores lipid accumulation in glp-1, but does not affect
fat storage in wild type.

(E-H). kri-1 RNAi suppresses the decreased fat storage in glp-1, but does not affect wild type.
 Figure S6
Wang et al

A
heat shock heat shock
3000 hsp-16.1 K04A8.5
Fold Change (Q-PCR)

1
2000

0.5
1000

0 0
0hr 1.5hr 3hr 0hr 1.5hr 3hr

B
paraquat paraquat
3
ctl-2 K04A8.5
Fold Change (Q-PCR)

1
2

1 0.5

0 0
ctrl 1hr 1.5hr ctrl 1hr 1.5hr

Fig. S6. Unchanged levels of the lipase gene upon heat shock and paraquat treatment.

(A). After heat shock, the Daf-16 target hsp-16.1 is greatly up-regulated. However the
K04A8.5 gene is not induced by heat shock.

(B). Paraquat treatment induces ctl-2, a target gene of Daf-16, but not K04A8.5.
 Figure S7
Wang et al

N2 glp-1

A C
vector

B D
daf-12

E F
N2 K04A8.5
glp-1
Fold Change (Nile Red)

1.0

Fold Change (Q-PCR)

1.0

0.5
0.5

0 0
vector daf-12 vector daf-12

Fig. S7. Regulation of fat metabolism by germline stem cell proliferation indepen-
dently of Daf-12 signaling.

(A-D). Reducing daf-12 activity by RNAi slightly decreases lipid accumulation in wild type
and glp-1 mutants.

(B). Quantification shows that daf-12 RNAi causes an 18.2% decrease in wild type
(p<0.0001) and a 22.8% reduction in glp-1 (p<0.0001).

(C). The up-regulation of K04A8.5 is not suppressed by subjecting glp-1 to daf-12 RNAi.
 Figure S8
Wang et al

N2 glp-1

A C
Vector

B D
daf-2

Fig. S8. Synergistic effects of daf-2 and glp-1 in regulation of fat storage.

(A, B). Reducing daf-2 activities specifically at adulthood decreases fat storage in wild type.

(C, D). Adult specific daf-2 RNAi further reduces fat storage in the glp-1 mutants.
 Table S1
Wang et al

Table S1. Metabolic genes and potential candidates

Sequence Name Brief Desciption Sequence Name Brief Desciption
B0272.3 3-hydroxyacyl-CoA dehydrogenase H04M03.1 Phosphoenolpyruvate carboxykinase
B0303.3 Acetyl-CoA acetyltransferase H14A12.2 Fumarase
C02B10.1 Isovaleryl-CoA dehydrogenase H25P06.1 Hexokinase
C03G5.1 Succinate dehydrogenase K02B2.1 phospho-fructo-kinase
C03H5.4 phospholipase K04A8.5 Triglyceride lipase
C04C3.3 Pyruvate dehydrogenase K05F1.3 acyl-CoA dehydrogenase
C05E4.9 Malate Synthase K06A5.6 acyl-CoA dehydrogenase
C05G5.4 Succinyl-CoA synthetase K07A3.1 Fructose-1,6-bisphosphatase
C07E3.9 phospholipase K08E3.5 UDP-glucose pyrophosphorylase
C17C3.12 acyl-CoA dehydrogenase K10B3.7 Glyceraldehyde 3-phosphate dehydrogenase
C23H3.7 Neutral trehalase K10B3.8 Glyceraldehyde 3-phosphate dehydrogenase
C28C12.9 Very-long-chain acyl-CoA dehydrogenase K11D12.4 Carnitine O-acyltransferase CPT
C29F3.1 Hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase K11H3.1 Glycerol-3-phosphate dehydrogenase
C30F12.7 Isocitrate dehydrogenase LLC1.3 Dihydrolipoamide dehydrogenase
C30H6.7 Dihydrolipoamide acetyltransferase R06F6.9 Enoyl-CoA hydratase/isomerase
C31H5.6 Peroxisomal long chain acyl-CoA thioesterase I R07C3.4 Acyl-CoA synthetase
C34B2.7 Succinate dehydrogenase R07H5.2 Carnitine O-acyltransferase CPT
C34F6.8 NADP-dependent isocitrate dehydrogenase R09B5.6 3-hydroxyacyl-CoA dehydrogenase
C37E2.1 Isocitrate dehydrogenase R09E10.3 Long-chain acyl-CoA synthetases
C40H1.2 Predicted lipase R09E10.4 Acyl-CoA synthetase
C44B7.8 Peroxisomal long-chain acyl-CoA transporter R11A5.4 Phosphoenolpyruvate carboxykinase
C44B7.9 Peroxisomal long-chain acyl-CoA transporter R11F4.1 Glycerol Kinase
C46C11.1 hormone sensitive lipase T02D1.5 Peroxisomal long-chain acyl-CoA transporter
C46F4.2 Acyl-CoA synthetase T02G5.4 Acetyl-CoA acetyltransferase
C46F4.2 Acyl-CoA synthetase T02G5.7 Acetyl-CoA acetyltransferase
C48B4.1 Acyl-CoA oxidase T02G5.8 Acetyl-CoA acetyltransferase
C50F4.2 phospho-fructo-kinase T03F1.3 3-phosphoglycerate kinase
C50F7.4 Succinyl-CoA synthetase T05A12.2 Neutral trehalase
C55B7.4 acyl-CoA dehydrogenase T05C12.3 Enoyl-ACP reductase
D1005.2 UDP-glucose pyrophosphorylase T05D4.1 Fructose-biphosphate aldolase
D2023.2 Pyruvate carboxylase T05G5.6 Enoyl-CoA hydratase
E04F6.5 acyl-CoA dehydrogenase T05H10.6 Pyruvate dehydrogenase
F01F1.12 Fructose-biphosphate aldolase T07C4.7 Succinate dehydrogenase
F01G10.2 Hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase T08B1.6 Long chain fatty acid acyl-CoA ligase
F01G10.3 Hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase T08B2.7 3-hydroxyacyl-CoA dehydrogenase
F01G4.2 3-hydroxyacyl-CoA dehydrogenase T08G2.3 Medium-chain acyl-CoA dehydrogenase
F08A8.1 Acyl-CoA oxidase T10E9.9 Short-chain acyl-CoA dehydrogenase
F08A8.2 Acyl-CoA oxidase T13F2.1 Delta 6-fatty acid desaturase/delta-8 sphingolipid desaturase
F08A8.3 Acyl-CoA oxidase T20G5.2 Citrate synthase
F08A8.4 Acyl-CoA oxidase T22B7.7 Acyl-CoA thioesterase
F09E10.3 Mitochondrial beta-ketoacyl-ACP reductase T21B10.2 Enolase
F09F3.9 Carnitine O-acyltransferase CPT T22B11.5 2-oxoglutarate dehydrogenase
F10D2.9 Fatty acid desaturase T22F3.3 Glycogen phosphorylase
F14B4.2 Hexokinase T22G5.2 Fatty acid-binding protein FABP
F15A2.2 Neutral trehalase T22G5.6 Fatty acid-binding protein FABP
F19H8.1 Trehalose-6-phosphate synthase T25G12.5 acyl-CoA dehydrogenase
F20H11.3 Malate dehydrogenase T25G3.4 Glycerol-3-phosphate dehydrogenase
F23B12.5 Dihydrolipoamide acetyltransferase VF13D12L.3 malate dehydrogenase
F23H11.3 Succinyl-CoA synthetase VZK822L.1 Fatty acid desaturase
F25C8.1 Acyl-CoA oxidase W01A11.5 Carnitine O-acyltransferase CPT
F25H5.3 Pyruvate Kinase W01C9.4 Enoyl-ACP reductase
F28A10.6 acyl-CoA dehydrogenase W02A2.1 Oleate desaturase/linoleate desaturase
F28D1.9 Long-chain fatty acid transport protein W02D3.5 Fatty acid-binding protein FABP
F28F8.2 Long chain fatty acid acyl-CoA ligase W02D3.7 Fatty acid-binding protein FABP
F32H2.5 acetyl-CoA carboxylase W02F12.5 2-oxoglutarate dehydrogenase
F32H2.6 fatty acid synthase W05E10.4 Neutral trehalase
F33D4.4 Fatty acid desaturase W05G11.6 Phosphoenolpyruvate carboxykinase
F33H1.2 Glyceraldehyde 3-phosphate dehydrogenase W06D12.3 Fatty acid desaturase
F36A2.3 malate dehydrogenase W08D2.4 Delta 6-fatty acid desaturase/delta-8 sphingolipid desaturase
F37C12.7 Acyl-CoA synthetase W09B6.1 fatty acid synthase
F38H4.8 Enoyl-CoA isomerase Y105E8A.4 Enoyl-CoA hydratase
F40F4.2 Fatty acid-binding protein FABP Y110A7A.6 phospho-fructo-kinase
F40F4.3 Fatty acid-binding protein FABP Y17G7B.7 Triosephosphate isomerase
F40F4.4 Fatty acid-binding protein FABP Y40B10A.1 Fatty acid-binding protein FABP
F41C3.3 Acyl-CoA synthetase Y46G5A.31 Glycogen synthase
F42A8.2 Succinate dehydrogenase Y48B6A.12 NADP+-dependent malic enzyme
F43G9.1 Isocitrate dehydrogenase Y48G9A.10 Carnitine O-acyltransferase CPT
F43H9.1 Enoyl-CoA hydratase Y50E8A.6 Glycerol-3-phosphate dehydrogenase
F46E10.1 Acyl-CoA synthetase Y54E5A.1 Fatty acid desaturase
F46E10.10 Malate dehydrogenase Y57A10C.6 Peroxisomal 3-ketoacyl-CoA-thiolase
F47B10.1 Succinyl-CoA synthetase Y65B4BL.5 Acyl-CoA synthetase
F47B8.10 GLUCOSE-6-PHOSPHATE TRANSPORTER Y67H2A.8 Oleate desaturase/linoleate desaturase
F47G4.3 Glycerol-3-phosphate dehydrogenase Y71H10A.1 phospho-fructo-kinase
F47G6.2 Acyl-CoA synthetase Y76A2B.3 Acyl-CoA synthetase
F48E8.3 Succinate dehydrogenase Y77E11A.1 Hexokinase
F53A2.7 Acetyl-CoA acetyltransferase Y87G2A.8 Glucose-6-phosphate isomerase
F53C11.3 Enoyl-ACP reductase ZK455.1 Aconitase/homoaconitase
F54C8.1 3-hydroxyacyl-CoA dehydrogenase ZK54.2 Trehalose-6-phosphate synthase
F54H12.1 Aconitase/homoaconitase ZK593.1 Pyruvate Kinase
F57B10.3 Phosphoglycerate mutase ZK742.5 Fatty acid-binding protein FABP
F57B10.7 Neutral trehalase ZK836.2 2-oxoglutarate dehydrogenase
F59F4.1 Acyl-CoA oxidase

Potential Candidates
 Table S2
Wang et al

Table S2. Effect of K04A8.5 RNAi treatment on N2 and glp-1(e2141ts) lifespan

Experiment #1
Genotype RNAi Treatment Mean ± SEM (days) Animal Number % of Control p Value
N2 (wild type) vector 18.50 ± 0.44 135
K04A8.5 19.64 ± 0.47 139 106 0.0246
glp-1(e2141ts) vector 27.21 ± 0.83 95
K04A8.5 20.66 ± 0.63 106 76 <0.0001

Experiment #2
Genotype RNAi Treatment Mean ± SEM (days) Animal Number % of Control p Value
N2 (wild type) vector 19.81 ± 0.55 107
K04A8.5 19.01 ± 0.64 91 96 0.6056
glp-1(e2141ts) vector 26.19 ± 0.59 118
K04A8.5 21.93 ± 0.51 116 84 <0.0001

“Genotype” column indicates that the experiments were done in wild type (N2) or glp-1 (e2141ts) mutants.
“RNAi Treatment” column indicates that the animals were exposed to bacteria containing empty vector
plasmid (vector) or dsRNA of K04A8.5.
Experiment #1 shows in Figure 3.
 Table S3
Wang et al

Table S3. Intestine-specific over-expession of K04A8.5 promotes longevity

Genotype Mean ± SEM (days) Events/Trials % of Control p Value

Ctrl (w/o GFP) 18.67 ± 0.48 81/91
Ex[Pges-1::K04A8.5::gfp] (w/GFP) 23.23 ± 0.85 64/78 124% <0.0001

“Genotype” column indicates that the experiments were done in transgenic animals (w/GFP)
and their siblings (w/o GFP).
 Table S4
Wang et al

Table S4. Effect of K04A8.5 RNAi treatment on N2 and daf-2(e1370) lifespan

Genotype RNAi Treatment Mean ± SEM (days) Events/Trials % of Control p Value

N2 (wild type) vector 18.48 ± 0.62 91/92
K04A8.5 18.57 ± 0.61 81/83 100 0.8549
daf-2(e1370) vector 37.39 ± 2.52 60/62
K04A8.5 28.61 ± 2.04 57/62 77 <0.005

“Genotype” column indicates that the experiments were done in wild type (N2) or daf-2 (e1370) mutants.
“RNAi Treatment” column indicates that the animals were exposed to bacteria containing empty vector
plasmid (vector) or dsRNA of K04A8.5.