Systemic regulation of
starvation response in
Chanhee Kang1and Leon Avery
Department of Molecular Biology, University of Texas
Southwestern Medical Center, Dallas, Texas 75390, USA
When the supply of environmental nutrients is limited,
multicellular animals can make both physiological and
behavioral changes so as to cope with nutrient starvation.
Although physiological and behavioral effects of starva-
tion are well known, the mechanisms by which animals
sense starvation systemically remain elusive. Further-
more, what constituent of food is sensed and how it
modulates starvation response is still poorly understood.
In this study, we use a starvation-hypersensitive mutant
to identify molecules and mechanisms that modulate
starvation signaling. We found that specific amino acids
could suppress the starvation-induced death of gpb-2
mutants, and that MGL-1 and MGL-2, Caenorhabditis
elegans homologs of metabotropic glutamate receptors,
were involved. MGL-1 and MGL-2 acted in AIY and AIB
neurons, respectively. Treatment with leucine suppressed
starvation-induced stress resistance and life span exten-
sion in wild-type worms, and mutation of mgl-1 and mgl-
2 abolished these effects of leucine. Taken together, our
results suggest that metabotropic glutamate receptor
homologs in AIY and AIB neuron may modulate a sys-
temic starvation response, and that C. elegans senses
specific amino acids as an anti-hunger signal.
Supplemental material is available at http://www.genesdev.org.
Received July 31, 2008; revised version accepted November
During nutritional deprivation, individual cells can re-
spond to starvation by modulating intracellular signaling,
which in turn induces a starvation response and thereby
enhances their survival (Levine and Yuan 2005; Lum et al.
2005; Levineand Kroemer 2008). An importantstarvation
response of individual cells is a change of metabolism
(inhibiting anabolic pathways and activating catabolic
pathways), so as to generate metabolic substrates to
maintain basal cellular activities (Lum et al. 2005). In
multicellular organisms, however, the situation is com-
plicated by the need to induce behavioral changes in
addition to physiological changes, and more importantly
by the danger that uncoordinated starvation responses
in individual cells could be harmful to the organism. In
Caenorhabditis elegans, for example, we reported pre-
viously that excessive autophagy in pharyngeal muscle
causes its malfunction, which eventually prevents the
recovery of worms from starvation (Kang et al. 2007).
Thus, it is important that multicellular organisms ensure
their starvation response is coordinated between individ-
ual cells, and therefore it is plausible to assume that there
are mechanisms by which animals sense starvation
Since animals cannot synthesize several amino acids,
so-called ‘‘essential acids,’’ they must ingest these amino
acids from external food sources to maintain homeostasis
(Gietzen and Rogers 2006). C. elegans also requires
certain amino acids in the diet (Szewczyk et al. 2003).
This fact leads to the intriguing possibility that amino
acids act as a food or anti-hunger signal. Indeed, treat-
ment with amino acids can inhibit starvation-induced
autophagy in cultured mammalian cells (Codogno and
Meijer 2005). Furthermore, at the organismal level, intra-
cerebroventricular administration of leucine causes a de-
crease in food intake in the rat (Cota et al. 2006),
suggesting that the possibility could be true. But it is
not known whether amino acids modulate a starvation
response in multicellular organisms.
Results and Discussion
To examine the effect of amino acids on starvation
signaling in Caenorhabditis elegans, we screened several
amino acids for suppression of the death of the starvation-
hypersensitive gpb-2 mutant. We showed previously that
starvation signaling is overactivated in this background
(You et al. 2006; Kang et al. 2007; Kang and Avery 2008).
We found that treatment with a subset of amino acids
including leucine, glutamine, alanine, valine, and iso-
leucine can rescue the death of gpb-2 mutants during
starvation, whereas other amino acids did not rescue or
worsened survival (Fig. 1A,B; Supplemental Fig. 1). Given
the specificity of the rescue effect, it is unlikely that
amino acids act solely as a carbon source to maintain
nutrient homeostasis; rather, it suggests that amino acids
might act as signaling molecules that modulate a starva-
Since the amino acids observed to have a rescue effect
do not fall into specific subgroups (essential amino acids/
nonessential amino acids or polar/nonpolar), we reasoned
that there must be a mechanism(s) to sense a broad
spectrum of amino acids. It has recently been shown that
a broad spectrum of amino acids can be sensed by class 3
G-protein-coupled receptors, which include the extracel-
lular calcium sensing receptor, heterodimeric taste re-
ceptors and GPRC6A (Conigrave and Hampson 2006).
MGL-1 and MGL-2 metabotropic G-protein-coupled glu-
tamate receptors are the closest homologs of CaR in the
C. elegans genome (Dillon et al. 2006). Thus, we hypoth-
esized that MGL-1 and MGL-2 were involved in the
modulation of amino acid response and starvation re-
sponse. To test this, we made gpb-2; mgl-1, gpb-2 mgl-2
and gpb-2 mgl-2; mgl-1 mutants. Mutation of either mgl-1
or mgl-2 partially rescued death of gpb-2 mutants during
starvation, and double mutation caused an additive effect,
suggesting that mgl-1 and mgl-2 modulate a starvation
response in a parallel manner (Fig. 1C). Mutation of either
mgl-1 or mgl-2 did not abolish the response to leucine,
while mutations of both mgl-1 and mgl-2 made gpb-2
[Keywords: Starvation; amino acid response; autophagy; hormesis]
E-MAIL Chanhee.Kang@UTSouthwestern.edu; FAX (214) 648-1488.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1723409.
12GENES & DEVELOPMENT 23:12–17 ? 2009 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/09; www.genesdev.org
mutants resistant to leucine treatment, suggesting that
both mgl-1 and mgl-2 are involved. Until 5 d of starvation,
there is no decrease in the survival of wild-type or mgl-1,
mgl-2, and mgl-2; mgl-1 mutants, suggesting that the
resistance to leucine treatment observed in gpb-2 mgl-2;
mgl-1 mutants does not result from full rescue of gpb-2
mutants by mutations of mgl-1 and mgl-2 (Kang et al.
2007; data not shown). We observed similar results with
alanine and glutamine (Supplemental Fig. 2A).
One cause of death in gpb-2 mutants during starvation
is excessive autophagy in pharyngeal muscle, which
causes malfunction of the pharynx, the C. elegans feeding
organ (Kang et al. 2007). To test whether mgl-1 and mgl-2
are involved in the modulation of autophagy in pharyn-
geal muscle of gpb-2 mutants during starvation, we
generated mutant strains carrying an integrated trans-
gene that expressed a GFP-tagged version of LGG-1,
a specific marker for autophagy (Melendez et al. 2003;
Kang et al. 2007; Klionsky et al. 2008). We found that
mutation of either mgl-1 or mgl-2 decreased the excessive
levels of autophagy in pharyngeal muscle, and double
mutation showed an additive effect on decreasing autoph-
agy, suggesting that mgl-1 and mgl-2 can modulate
autophagy in a parallel manner (Fig. 2). Treatment with
leucine also decreased excessive levels of autophagy
during starvation, consistent with the hypothesis that
amino acids act as anti-hunger signals. However, leucine
had no effect on levels of autophagy when mgl-1 and mgl-
2 were both mutated, suggesting that they are necessary
for the modulation of autophagy by amino acids. We also
observed similar results with alanine and glutamine (Sup-
slightly increased levels of autophagy ingpb-2 mutants, yet
glutamate had no effect on levels of autophagy in gpb-2
mgl-2; mgl-1 mutants, consistent with survival data (Sup-
plemental Fig. 2B).
It has been reported that mgl-1 and mgl-2 are mainly
expressed in a limited number of neurons (http://www.
wormbase.org). These expression patterns, together with
the results showing that mgl-1 and mgl-2 can modulate
levels of autophagy in pharyngeal muscles (Fig. 2), lead to
the intriguing hypothesis that mgl-1 and mgl-2 can
modulate a starvation response in a cell-nonautonomous
manner. To test this, we examined whether expression
of mgl-1 or mgl-2 in specific neurons could restore the
starvation-sensitivity of gpb-2; mgl-1 and gpb-2 mgl-2
mutants. Among the neurons where mgl-1 and mgl-2 are
expressed, AIYand AIB are particularly interesting because
it has been reported recently that AIY and AIB neurons
control food- and odor-evoked behaviors (Chalasani et al.
2007). We found that AIY-specific expression of mgl-1 and
AIB-specific expression of mgl-2 could restore the starva-
tion-sensitivity of gpb-2; mgl-1 and gpb-2 mgl-2 mutants,
respectively (Fig. 3A,B). In contrast, expressing either
mgl-1 or mgl-2 in a subset of MGL-1- or MGL-2-express-
ing neurons using either the glr-4 promoter (to express in
RMD, RIB, and 17 additional neurons) or nmr-2 promoter
(to express in AVE and five additional neurons) did not
restore the starvation-sensitivity (Supplemental Fig. 3A–
C). These data support our hypothesis that mgl-1 and
mgl-2 mainly act in AIYand AIB neurons, respectively, to
modulate a starvation response.
Based on homology, mgl-1 encodes a group II glutamate
receptor, expected to inhibit adenylyl cyclase activity,
which is likely to decrease neuronal activity, whereas
sitivity of gpb-2 mutants, and mgl-1 and mgl-2 are involved in the
process. Starvation survival analyses were performed as described in
the Materials and Methods. (A) Treatment with leucine partially
rescues the starvation hypersensitivity of gpb-2 mutants. Percent of
worms surviving to adulthood on NGM plates with HB101 bacteria
after incubation in M9 buffer in the absence of food with or without
leucine for the indicated time. Error bars are standard errors
estimated assuming a Poisson distribution, and similar results were
obtained in three independent experiments. (*) P < 0.001 (x2test of
independence). (B) Summary of the rescue effect of amino acids on
the survival of gpb-2 mutants during starvation. (+) Positive effects
on survival; (0) no effects on survival; (?) negative effects on
survival. For details, see Supplemental Figure 1. (C) Mutation of
either mgl-1 or mgl-2 can rescue death of gpb-2 mutants during
starvation, and double mutation causes an enhanced rescue effect.
Percent of worms surviving to adulthood on NGM plates with
HB101 bacteria after incubation in M9 buffer in the absence of food
with or without leucine for the indicated time. Error bars are
standard errors estimated assuming a Poisson distribution, and
similar results were obtained in three independent experiments.
A subset of amino acids rescues the starvation hypersen-
muscles of gpb-2 mutants during starvation. (A) Representative
images of gpb-2 mutants with or without leucine after 3 d of star-
vation. The arrows show representative GFPTLGG-1-positive punctate
structures that label preautophagosomal and autophagosomal struc-
tures. (B) Quantification of autophagy in the pharyngeal muscles of
worms of the indicated genotype after 3 d of starvation with or without
leucine. Data come from three independent experiments (n = 80–141).
mgl-1 and mgl-2 modulate autophagy in the pharyngeal
Anti-hunger signaling in C. elegans
GENES & DEVELOPMENT13
mgl-2 encodes a group I glutamate receptor, expected to
stimulate phospholipase C and likely increase neuronal
activity (Dillon et al. 2006). To test whether mgl-1 and
mgl-2 modulate starvation response by regulating the
activity of AIY and AIB neurons, respectively, we killed
AIYand AIB neurons by AIY- and AIB-specific expression
of egl-1, the BH3-only protein that promotes cell death
(Conradt and Horvitz 1998; Chang et al. 2006). We found
that AIY-specific expression of egl-1 exacerbates the
starvation sensitivity of gpb-2 mutants and that AIB-
specific expression of egl-1 rescues gpb-2 mutants (Fig.
3C,D), suggesting that changes in the neuronal activities
of AIY and AIB are sufficient to modulate the starvation
response. We confirmed the AIY killing results using
a ttx-3 mutation (Supplemental Fig. 3D), which is known
to disrupt the function of AIY (Altun-Gultekin et al.
2001). These results, together with recent findings show-
ing that AIY is activated by food or odor presentation and
AIB is activated by food or odor removal (Chalasani et al.
2007), suggest that AIY inhibits the starvation response,
whereas AIB activates it.
Since mgl-1 and mgl-2 are similar to metabotropic
glutamate receptors, it is possible that glutamate neuro-
transmission is involved in the modulation of mgl-1 and
mgl-2. To test this possibility, we looked at the effect of
mgl-1 and mgl-2 on starvation survival in an eat-4 mutant
background. eat-4 encodes a vesicular glutamate trans-
porter, and glutamate neurotransmission is impaired in
eat-4 mutants (Lee et al. 1999), including transmission
from the known presynaptic partners of AIY and AIB
(Chalasani et al. 2007). If amino acids modulate the
effects of mgl-1, mgl-2, and leucine. However, this is not
the case. mgl-1, mgl-2, and leucine have pronounced
effects on starvation survival in the absence of eat-4. This
suggests that glutamatergic neurotransmission is not
necessary for the activation of MGL-1 and MGL-2. We
activity of mgl-1 and mgl-2 (Supplemental Fig. 4). An
alternative possibility, however, is that other vesicular
glutamate transporters function redundantly with eat-4,
Our data in gpb-2 mutants indicate that amino acids
act as an anti-hunger signal and that mgl-1 and mgl-2 are
involved in the systemic starvation response. To evaluate
whether treatment with amino acids can also suppress
and mgl-2 are involved in that process, we took advantage
of the concept of starvation-induced hormesis. Hormesis
indicates beneficial effects of low doses of treatments
known to be harmful at higher doses (Gems and Partridge
2008). It has been reported that short-term starvation (1–2
d) increases oxidative-stress resistance and extends life
span in C. elegans (Cypser et al. 2006; Weinkove et al.
2006). We confirmed that starvation induces heat-shock
resistance, oxidative stress resistance, and life span ex-
tension in wild-type worms, and found that treatment
with leucine partially suppressed these effects of starva-
tion, suggesting that amino acids act as an anti-hunger
signal in wild-type worms (Fig. 4A–C). More interestingly,
we saw no effect of leucine on the starvation-induced
heat-shock resistance, oxidative stress resistance, and life
span extension in mgl-2; mgl-1 mutants, suggesting that
respectively. (A–D) Percent of worms surviving to adulthood on
NGM plates with HB101 bacteria after incubation in M9 buffer in
the absence of food for the indicated time. Error bars are standard
errors estimated assuming a Poisson distribution, and similar results
were obtained in three independent experiments. (*) P < 0.05; (**) P
< 0.01; (***) P < 0.001 (x2test of independence). (A,B) AIY-specific
expression of mgl-1 and AIB-specific expression of mgl-2 restore the
starvation-sensitivity of gpb-2; mgl-1, and gpb-2 mgl-2, respectively.
Data for gpb-2 single mutants in A and B are from Figure 1A. Similar
results were obtained in two independent transgenic lines. (C,D)
AIY-specific expression of egl-1 exacerbates death of gpb-2 mutants
during starvation, whereas AIB-specific expression of egl-1 rescues it.
mgl-1 and mgl-2 mainly act in AIY and AIB neurons,
partially suppresses it in wild-type worms, and the effect of leucine
is abolished in mgl-2; mgl-1 mutants. (A,B) Indexes of starvation-
induced heat-shock resistance and oxidative stress resistance were
calculated as described in the Materials and Methods. Starvation
induces heat-shock resistance and oxidative stress resistance, treat-
ment with leucine partially suppresses it in wild-type worms, and
the effect of leucine is not observed in mgl-2; mgl-1 mutants. Data
were combined from at least four independent experiments (n =
389;2458 for each heat-shock experiment and n = 213;1181 for
each oxidative stress resistance). (*) P < 0.05; (**) P < 0.01 (Student’s
t-test, paired); (#) P < 0.05 (Student’s t-test, unpaired). (C,D) Life span
curves represent combined data from two independent experiments
(n = 76;101). Starvation extends life span, and treatment with
leucine partially suppresses the life span extension in wild-type
worms (starved N2, 16.74 6 0.04 vs. starved N2 with leucine, 14.61
6 0.05, P < 0.005, Mantel-Cox Log-rank test). Effect of leucine is not
observed in mgl-2; mgl-1 mutants (starved mgl-2; mgl-1, 16.23 6
0.03 vs. starved mgl-2; mgl-1 with leucine, 16.73 6 0.04).
Starvation induces hormesis, treatment with leucine
Kang and Avery
14GENES & DEVELOPMENT
mgl-1 and mgl-2 are likely involved in wild-type amino
acid responses, as they are in the gpb-2 starvation re-
sponse (Fig. 4A,B,D). AIY-specific expression of mgl-1 and
AIB-specific expression of mgl-2 restored the effect of
leucine on the starvation-induced heat-shock and oxida-
tive stress resistance in mgl-2; mgl-1 mutants, further
supporting our hypothesis that mgl-1 and mgl-2 acts in
AIY and AIB neurons (Supplemental Fig. 5).
It is reasonable to suppose that the stress response
should be coordinated in multicellular organisms, because
uncoordinated stress responses in individual cells within
a multicellular organism could impair the function of
tissues, and eventually be harmful for the survival of the
organism. However, little is known about whether the
stress response previously thought to be cell-autonomous
can be systemically regulated. A recent study elegantly
showed that, in C. elegans, the heat-shock response is sys-
temically regulated through the activity of AFD (Prahlad
etal. 2008),whichwasknown previously tosense ambient
temperature (Mori 1999). By analogy, our study suggests
that the starvation response can be systemically regulated
through mgl-1 and mgl-2, presumably regulating the ac-
tivity of AIY and AIB interneurons (Fig. 5), which were
known previously to modulate behaviors in response to
Since both AIY and AIB are amphid interneurons, one
might expect that that they do not directly sense environ-
mental amino acids. It is possible that AIYand AIB receive
synapses from certain chemosensory neurons (for example,
ASE, AWC, or ASI, based on known neural connectivity)
acids, so as to modulate a systemic starvation response
that AIYand AIB directly respond to internal amino acids,
previously absorbed from the environment. The latter
possibility is plausible based on the fact that, in rodents,
a sensory role for either taste or odor is not involved in the
sensing of essential amino acids, and it is suggested that
a chemosensor for essential amino acids is in the brain
(Gietzen and Rogers 2006). More importantly, our eat-4
in response to amino acids, suggesting the possibility that
in the case of extracellular calcium sensing receptor. It
would be interesting to test if AIY and AIB could directly
sense amino acids by calcium imaging.
Surprisingly, treatment with leucine decreased survival
of gpb-2 mgl-2 mutants during starvation. One possible
explanation is that leucine has an additional negative
effect on starvation survival, which is downstream from
mgl-1, and is masked by the positive effect of leucine
through mgl-2. Indeed, in our preliminary experiments, we
observed that treatment with leucine decreased mgl-2
mutants survival at later times (9 d of starvation), com-
pared with wild-type or mgl-1 and mgl-2; mgl-1 mutants
(data not shown), supporting the possibility.
In summary our data suggest that mgl-1 and mgl-2 are
involved in the two related systemic processes: amino
acid response and starvation response. Our current model
(Fig. 5) suggests that the amino acid signal, which may be
a component of the food signal, activates AIY neurons
and inhibits AIB neurons by modulating the activities of
MGL-1 and MGL-2, respectively. AIY then inhibits the
starvation response, whereas AIB activates it. Given that
there is no direct connection between AIY/AIB neurons
and pharyngeal muscle, it is reasonable to think that spe-
cific hormones, perhaps neuropeptides, act downstream
from AIY and AIB neurons to modulate the systemic
starvation response in several tissues. Further experi-
ments are needed to elucidate whether the systemic
signal is indeed one or more neuropeptides and, if so,
which peptides regulate the systemic starvation response
downstream from AIY and AIB. Our model system, the
starvation-hypersensitive gpb-2 mutants, might be help-
ful to find such peptide signals.
While this paper was under review, Greer et al. (2008)
published a study describing the role of metabotropic
glutamate receptorsin thefat accumulation of C.elegans.
They found that daf-7 inactivation (which mimics the
status under unfavorable environmental conditions such
as reduced food availability) causes fat accumulation via
metabotropic glutamate receptors, supporting our model
that metabotropic glutamate receptors are involved in the
Materials and methods
Strains were maintained as described (Brenner 1974) at 19°C. All
worms were maintained and grown on Escherichia coli HB101
bacteria. The following strains were generated using standard
X, gpb-2(ad541) mgl-2(tm355) I, gpb-2(ad541) mgl-2(tm355) I;
mgl-1(tm1811) X, gpb-2(ad541) I; adIs2122[GFPTlgg-1 rol-6(d)],
gpb-2(ad541) I; mgl-1(tm1811) X; adIs2122, gpb-2(ad541) mgl-
X; adIs2122, gpb-2(ad541) I; mgl-1(tm1811) X; adEx2241[pttx-3T
mgl-1 rol-6(d)], gpb-2(ad541) mgl-2(tm355) I; adEx2242[podr-
2bTmgl-2 rol-6(d)], gpb-2(ad541) I; adEx2243[pttx-3Tegl-1 rol-
6(d)], gpb-2(ad541) I; adEx2244[podr-2bTegl-1 rol-6(d)], gpb-2
(ad541) I; eat-4(ky5) III, gpb-2(ad541) I; eat-4(ky5) III; mgl-
1(tm1811) X, gpb-2(ad541) mgl-2(tm355) I; eat-4(ky5) III, mgl-
2(tm355) I; mgl-1(tm1811) X, mgl-2(tm355) I; mgl-1(tm1811)
X;[pttx-3Tmgl-1 podr-2bTmgl-2 rol-6(d)], gpb-2(ad541) I; ttx-
3(ks5) X, gpb-2(ad541) I; mgl-1(tm1811); [pglr-4Tmgl-1 rol-6(d)],
gpb-2(ad541) I mgl-2(tm355); [pglr-4Tmgl-2 rol-6(d)], gpb-2
(ad541) I mgl-2(tm355); [pnmr-2Tmgl-2 rol-6(d)].
cDNA corresponding to the entire coding sequence of egl-1 was
amplified and cloned under cell-specific promoters as indicated.
in C. elegans. Amino acids or food signal modulates the activity of
AIY and AIB neurons, probably through MGL-1 and MGL-2, re-
spectively, which in turn regulate the systemic starvation response.
Model of the systemic regulation of starvation response
Anti-hunger signaling in C. elegans
GENES & DEVELOPMENT15
Primer sequences were as follows: egl-1: 59-GCTCTAGAAT
GTCCAACGTTTTTGACGTTCAATCT-39 and 59-CGCTCG
egl-1 DNA in pBluescript from Scott Cameron and Malia Potts
(University of Texas Southwestern Medical Center). Expression
in AIY and AIB was achieved using the ttx-3 and odr-2b
promoters, respectively, from Cornelia Bargmann (The Rock-
cDNA corresponding to the entire coding sequence of mgl-2
was obtained using SpeI and XhoI restriction enzymes from mgl-
2 cDNA in pBluescript from Isao Katsura (National Institute of
Genetics) and cloned under the odr-2b promoter.
PCR construction of mgl-1 with ttx-3 promoter was achieved
as follows: The ttx-3 promoter region was amplified from a Pttx-
3Tmod-1 cDNATGFP plasmid (from Cornelia Bargmann, The
Rockefeller University) using primers 59-GTCTCATTTAAATTTT
CAGAGCTTAAAAATGG-39 and 59-TGTTTGCCTCATATTGA
CACCGAAGACAATTATT-39 (PCR#1). cDNA corresponding to
the entire coding sequence of mgl-1 was amplified from mgl-1
cDNA in pMW118 (from Isao Katsura, National Institute of
Genetics) using primers 59-CTTCGGTGTCAATATGAGGCAAA
CATTTCGGAA-39 and 59-TCATAAGAAAGTATCGTGAGCA
G-39 (PCR#2). PCR#1 and #2 were fused using primers 59-
GTCTCATTTAAATTTTCAGAGCTTAAAAATGG-39 and 59-
Expression in RMD/RIB and AVE was achieved using the glr-4
(RMD, RIB, AVA, SMD, SAA, SIB, RIM, AVH, FLP, RMG, DVA,
AUA, PVD, URY, URA, SAB, RIF, DB, and PVU) and nmr-2 (AVE,
Starvation survival analyses
Starvation survival analyses were performed as described (You
et al. 2006) with a few modifications. After collection of L1
worms from synchronization by egg preparation, we incubated
them in 3 mL of sterilized M9 buffer with or without leucine for
the time indicated in the figures at 19°C. At each time point, an
aliquot from each sample tube was placed on a plate seeded with
E. coli HB101. The number of worms surviving to L4 or adult-
hood was determined after 3 d further growth at 19°C. The
number from day 1 of starvation was used as control and as the
denominator to calculate the percentage of worms recovering
after starvation. Since starvation-survival was influenced by
assay conditions, all relevant experimental data were examined
and compared within the same experiment. For initial screening,
we used 10.9 mM amino acids. Since we found in preliminary
exploratory experiments that high concentrations of leucine less
efficiently rescued survival of gpb-2 mutants, we used 3.6 mM
leucine in all subsequent experiments except the hormesis
experiments (Fig. 4), in which we used 7.2 mM leucine. Leucine
has a rescueeffect on gpb-2 mutants in the rangeof 3.6–10.9mM.
C. elegans autophagy analysis
Autophagy analysis was performed as described (Kang et al.
2007). For light microscopic analysis of autophagy, starved L1
animals carrying anintegrated
a GFPTLGG-1 fusion were collected at 3 d of starvation. GFP-
positive punctate regions were visualized in the pharyngeal
muscles of L1 animals using a Zeiss Axioplan 2 compound
microscope (Zeiss Corporation).
Starvation-induced heat-shock resistance analyses
After collection of L1 worms after synchronization by egg
preparation, we incubated them in 3 mL of M9 buffer with or
without leucine for 0 d (nonstarved group) or 2 d (starved group).
We equally divided each group into two subgroups and gave
a heat shock (90 min, 35°) to one of them (heat-shock subgroup).
We calculated the index of starvation-induced heat-shock
resistance as follows: index of starvation-induced heat-shock
resistance = (number of surviving starved worms with heat
shock/number of surviving starved worms without heat shock) –
(number of surviving unstarved worms with heat shock/number
of surviving unstarved worms without heat shock).
Starvation-induced oxidative stress resistance analyses
After collection of L1 worms after synchronization by egg
preparation, we incubated them in 3 mL of M9 buffer with or
without leucine for 0 d (nonstarved group) or 2 d (starved group).
We equally divided each group into two subgroups and gave an
oxidative stress (H2O210 mM, 40 min) to one of them (H2O2
group). We calculated the index of starvation-induced oxidative
stress resistance as follows: index of starvation-induced oxida-
tive stress resistance = (number of nonarrested starved worms
with H2O2/number of nonarrested starved worms without
H2O2) – (number of nonarrested unstarved worms with H2O2/
number of nonarrested unstarved worms without H2O2).
Starvation-induced life span extension analyses
Life span analyses were performed as described (Tullet et al.
2008) with a few modifications. After collection of L1 worms
after synchronization by egg preparation, animals were allowed
to develop at 19°C. When animals reached adulthood, they were
collected and incubated in sterilized M9 buffer with or without
leucine for 0 h (nonstarved group) or 40 h (starved group). They
were transferred to a plate seeded with E. coli HB101, kept at
19°C, and score every 2 d as dead or alive. Animals that crawled
off the plate, exploded, or died as bags of worms were excluded
from analysis at the time of death. Life spans were measured
from adulthood. Survival curve P values were calculated by the
Mantel-Cox Log Rank test using Prism statistical software
We thank S. Cameron, B. Levine, M. Cobb, and C. Bargmann for
helpful discussions; S. Cameron, C. Bargmann, K. Ashrafi, I.
Katsura, and T. Ishihara for providing plasmids; the C. elegans
Genetics Center and S. Mitani (National Bioresource Project) for
providing strains; and Y. Choi for helping statistic analyses. C.K.
thanks M.S. Kim and D. Kang for unfailing support and encour-
agement. This work was supported by research grant HL46154
from the U.S. Public Health Service.
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