Bi Zhang1,2,3, Heejin Jun2,3, Jun Wu2,3, Jianfeng Liu1, X Z Shawn Xu2,3. 1. College of Life Science and Technology, Key Laboratory of Molecular Biophysics of MOE, Huazhong University of Science and Technology, Wuhan, Hubei, 430074, China. 2. Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA. 3. Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Abstract
The role of food nutrients in mediating the positive effect of dietary restriction (DR) on longevity has been extensively characterized, but how non-nutrient food components regulate lifespan is not well understood. Here, we show that food-associated odors shorten the lifespan of C. elegans under DR but not those fed ad libitum, revealing a specific effect of food odors on DR-mediated longevity. Food odors act on a neural circuit comprising the sensory neurons ADF and CEP, and the interneuron RIC. This olfactory circuit signals the gut to suppress DR-mediated longevity via octopamine, the mammalian homolog of norepinephrine, by regulating the energy sensor AMPK through a Gq-PLCβ-CaMKK-dependent mechanism. In mouse primary cells, we find that norepinephrine signaling regulates AMPK through a similar mechanism. Our results identify a brain-gut axis that regulates DR-mediated longevity by relaying olfactory information about food abundance from the brain to the gut.
The role of food nutrients in mediating the positive effect of dietary restriction (DR) on longevity has been extensively characterized, but how non-nutrient food components regulate lifespan is not well understood. Here, we show that food-associated odors shorten the lifespan of C. elegans under DR but not those fed ad libitum, revealing a specific effect of food odors on DR-mediated longevity. Food odors act on a neural circuit comprising the sensory neurons ADF and CEP, and the interneuron RIC. This olfactory circuit signals the gut to suppress DR-mediated longevity via octopamine, the mammalian homolog of norepinephrine, by regulating the energy sensor AMPK through a Gq-PLCβ-CaMKK-dependent mechanism. In mouse primary cells, we find that norepinephrine signaling regulates AMPK through a similar mechanism. Our results identify a brain-gut axis that regulates DR-mediated longevity by relaying olfactory information about food abundance from the brain to the gut.
Food is a primary environmental factor that affects aging[1,2].
Dietary restriction (DR) represents one of the most effective interventions to
extend lifespan in all tested organisms, ranging from yeast to mammals[1,2]. Previous efforts have mostly focused on investigating how
nutrients in the food affect aging. This has led to the identification of several
nutrient signaling pathways, particularly AMPK and mTOR signaling, that regulate
longevity[2-4].However, food is not solely composed of nutrients, as other components are
also associated with food, such as volatile odors and various types of non-volatile,
non-nutrient chemicals. Like nutrients, these food components may also affect
aging[5]. For example, in
C. elegans, worms defective in chemosensation show altered
lifespan, suggesting a role of chemical cues in lifespan regulation[6,7]; in Drosophila, odorants from food can inhibit
DR longevity[8]. However, it is
unclear how non-nutrient chemicals in the food regulate longevity. In particular, it
is not known whether and how these non-nutrient food chemicals interact with the
brain, and if so, how the brain then engages the rest of the body to alter lifespan.
Here, we sought to address these questions in C. elegans, a genetic
model organism widely used for studying the biology of aging.
Results
Food odors suppress the lifespan of worms under dietary restriction (DR) but
not those fed ad libitum (AL)
Lifespan assays in C. elegans are usually conducted on
NGM agar plates. Though not as common, lifespan may also be performed in liquid
culture. As such, two major types of DR regimens have been developed:
solid-phase DR (sDR) assayed on agar plates, and liquid-phase DR (bDR) carried
out in liquid culture, with each supplied with a reduced amount of bacteria
food[9]. Different DR
regimens may engage different signaling mechanisms[4,9].
sDR has been widely used in the community due to its easy handing. Because of
this and the fact that it is rather difficult to expose food odors to worms
cultured in liquid, we chose to focus on sDR. In this protocol, bacteria are
killed to prevent their further growth[10]. Previous effort found that application of the
supernatant from bacteria culture to the assaying plates shortened the lifespan
of worms deprived of food (bacteria deprivation or DD)[11,12]. However, as this protocol did not separate volatile odors
from various water-soluble chemicals, it remains unclear whether food odors have
an effect on worm longevity.To directly test the effect of food odors, we developed a protocol to
deliver food odors to worms in lifespan assays. To do so, we placed a small
piece of NGM agar with a thin lawn of live OP50 bacteria on the lid of the NGM
plate (Fig.1a). As a control, the same size
of NGM agar piece was placed on the lid but without OP50 bacteria. Worms on the
plate did not make direct contact with bacteria on the lid, but were exposed to
the odors emitted from these bacteria (Fig.1a). Exposing such food-derived odors to worms under DR-mediated
longevity by ~50%, while exposing food odors to worms fed ad
libitum (AL) did not have a notable effect on lifespan (Fig.1b), revealing a specific effect of food
odors on DR longevity.
Fig.1 |
Food odors suppress the lifespan of worms under DR but not those fed AL, and
this requires neurotransmission.
(a-b) Food-derived odors shorten the lifespan of worms
under DR, but not those fed AL. (a) Schematic describing the assay.
(b) Lifespan curves.
(c) Food odors do not change the pumping rate of worms
under DR. Pumping rate was counted at 1 hr after worms were transferred to the
DR assaying plates. 2xDR: twice amount of bacterial food. n = 30
(DR), 30 (DR + odor), 16 (2xDR) and 16 (2xDR + odor) biologically independent
animals.
(d-e) Food odors do not change the amount of bacteria
ingested by worms under DR. tdTomato-expressing OP50 bacteria were used as food
source. Scale bar, 200 μm. (d) Sample images.
(e) Bar graph. n = 34 (DR), 40 (DR + odor), 40
(2xDR) and 40 (2xDR + odor) biologically independent animals.
(f-n) Food odors shorten the lifespan of WT
(f), eat-4 mutant (k),
unc-17 mutant (l), unc-25
mutant (m), and unc-31 mutant (n)
worms under DR condition. However, loss of tph-1
(g), cat-2 (h),
tbh-1 (i), or tdc-1
(j) blocks the ability of food odors to suppress DR
longevity.
Data are presented as mean ± s.e.m. p values were
calculated with one-way ANOVA with Bonfferronìs test.
See Supplementary
Table 1 for lifespan statistics.
One potential concern is that food odors might have stimulated feeding,
thereby shortening lifespan due to increased food ingestion. This, however, was
not the case, as the pharyngeal pumping rate of DR worms was not affected by
food odors compared to the control (Fig.1c
and Extended Data Fig.1a–b). In addition, the amount of ingested
bacteria was similar under the two conditions, which was determined by
quantifying the fluorescence of worm gut bacteria expressing tdTomato (Fig.1d–e). Slightly increasing the amount of bacteria food (2x) fed to DR
worms stimulated their pharyngeal pumping rate and increased the amount of
ingested bacteria in the gut (Fig.1c–e), yet had no
notable effect on lifespan (Extended Data
Fig.1c). By contrast, the inhibitory effect of food odors on the
lifespan of DR worms persisted under this condition (Extended Data Fig.1c). This set of control experiments
indicates that the two assays used have the sensitivity to detect small
increases in feeding and food intake that would have been induced by food odors.
We thus conclude that food odors inhibit longevity in a DR-dependent manner in
C. elegans.
Extended Data Fig. 1
Additional data related to food odor suppression of DR longevity without
increasing food ingestion.
(a-b) Food odors do not change the pumping rate of
worms under DR. Pumping rate was counted at 24 hr (a), and 96
hr (b), after worms were transferred to the DR assaying plates.
2xDR: twice amount of bacterial food, which stimulated the pumping rate.
n = 30 (DR), 30 (DR + odor), 16 (2xDR) and 16 (2xDR + odor)
biologically independent animals in (a). n = 30
(DR), 30 (DR + odor), 15 (2xDR) and 14 (2xDR + odor) biologically
independent animals in (b). Data are presented as
mean ± s.e.m. p values were calculated with one-way
ANOVA with Bonfferronìs test. See Fig.1c for data of the pumping rate counted at 1 hr
post-transfer. (c) Feeding worms with twice amount of bacteria
food (2x DR) does not affect DR longevity, but food odors can still suppress
the lifespan of these worms.
Food odor-induced suppression of DR longevity requires
neurotransmission
How do food odors suppress DR longevity? As odors are usually detected by
the nervous system, which involves neurotransmission, we first asked whether and
which types of neurotransmission mediate the odor effect. By screening different
neurotransmission mutants, we found that mutations abolishing the synthesis of
serotonin (tph-1), dopamine (cat-2),
octopamine (tbh-1), or tyramine (tdc-1) all
prevented food odors from inhibiting DR longevity (Fig.1f–j), indicating a
requirement of these neurotransmitters. Other neurotransmitter mutants such as
eat-4 (glutamate), unc-17 (ACh),
unc-25 (GABA) and unc-31 (neuropeptides)
did not exhibit a notable defect (Fig.1k–n). These results
raise the possibility that serotonin, dopamine and octopamine/tyramine neurons
may form a chemosensory circuit to sense and process odor signals to inhibit DR
longevity.
ADF, CEP and RIC neurons form an olfactory circuit mediating odor-induced
suppression of DR longevity
Among serotonin, dopamine and octopamine/tyramine neurons, only ADFserotonin neurons are chemosensory neurons[13]; the other major type of serotonin neurons NSM is
classified as motor neurons[14],
while dopamine neurons (CEP/ADE/PDE) are mechanosensory neurons[15-17], and octopamine/tyramine neurons (RIC
and RIM) are interneurons[18].
As ADF neurons are known to sense volatile odors[19], ADF neurons might function as the
olfactory neurons in the circuit to sense food odors. To test this, we recorded
the activity of both ADF and NSM serotonin neurons in response to food odors by
calcium imaging. ADF neurons from DR worms responded robustly to medium
containing odors from OP50 bacteria food (Fig.2a, 2d). Strikingly, ADF
neurons from AL worms responded very weakly to food odors (Fig.2a, 2d),
revealing a DR-specific effect. By contrast, food odors evoked no notable
response in NSM serotonin neurons (Extended Data
Fig.2b, 2c), suggesting that NSM
neurons are not part of the circuit. To test whether ADF neurons sense food
odors cell-autonomously, we repeated the imaging experiment in
unc-13 and unc-31 mutants, and obtained a
similar result (Fig.2b, 2d). Mutations in unc-13 and
unc-31 block neurotransmitter release from synaptic
vesicles and dense core vesicles, respectively[20,21]. Thus, ADF neurons likely responded to food odors
cell-autonomously, suggesting that these chemosensory neurons act as primary
olfactory sensory neurons to sense food odors in DR worms.
Fig.2 |
Food odors act on an olfactory circuit, which is composed of ADF, CEP and RIC
neurons, to suppress DR longevity.
(a) ADF neurons from DR worms respond robustly to medium
containing odors from OP50 bacteria food, while very weak, if any, calcium
response is evoked by food odors in ADF neurons from AL worms. G-CaMP6f was
expressed as a transgene in ADF neurons under tph-1(L)
promoter. DsRed was co-expressed to enable ratiometric imaging.
(b) Food odor-evoked calcium response in AFD neurons
remains normal in unc-13 and unc-31 mutant
worms.
(c) Blocking dopamine signaling (using
cat-2 mutation) or octopamine signaling (using
tbh-1 mutation) does not notably affect food odor-evoked
calcium response in ADF neurons.
(d) Bar graph summarizing the data in (a),
(b), and (c). n=8 (WT- AL), 10 (WT - DR), 10
(unc-13 - AL), 10 (unc-13 - DR), 10
(unc-31 - AL), 11 (unc-31 - DR), 11
(cat-2 - AL), 11 (cat-2 - DR), 10
(tbh-1 - AL) and 10 (tbh-1 - DR)
biological independent animals.
(e) CEP neurons from DR worms but not AL worms respond to
food odors. To facilitate dendrite imaging, we expressed myr-GCaMP6f
(membrane-targeted) in CEP using the dat-1 promoter. Note: the
dat-1 promoter also drives expression in ADE and PDE
dopamine neurons.
(f) Blocking serotonin signaling (using either
tph-1 mutation or ADF::TeTx transgene)
eliminates CEP calcium response evoked by food odors.
(g) Blocking octopamine signaling (using
tbh-1 mutation) has no effect on CEP calcium response
evoked by food odors.
(h) Bar graph summarizing data in (e),
(f), and (g). n=11 (WT - AL), 9 (WT - DR), 13
(tph-1 - AL), 13 (tph-1 - DR), 10
(ADF::TeTx - AL), 11 (ADF::TeTx - DR), 10
(tbh-1 - AL) and 10 (tbh-1 - DR)
biological independent animals.
(i) RIC neurons from DR worms but not AL worms respond to
food odors. tbh-1 promoter was used to drive transgene
expression in RIC.
(j-k) Blocking serotonin signaling (using
tph-1 mutation or ADF::TeTx transgene
(j)) or inhibiting dopamine signaling (using
cat-2 mutation or CEP::TeTx transgene)
(k)) abolishes RIC calcium response evoked by food odors.
(l) Bar graph summarizing the data in (i),
(j), and (k). n=11 (WT - AL), 14 (WT - DR), 12
(tph-1 - AL), 14 (tph-1 - DR), 8
(ADF::TeTx - AL), 8 (ADF::TeTx - DR), 13
(cat-2 - AL), 15 (cat-2 - DR), 8
(CEP::TeTx - AL) and 9 (CEP::TeTx - DR)
biological independent animals.
(m-o) Blocking the output from ADF (m), CEP
(n), and RIC (o) neurons using
TeTx transgene abrogates the ability of food odors to
suppress DR longevity. A fragment of the bas-1 promoter
(bas-1(prom7)), dat-1 promoter and
tbh-1 promoter was used to drive TeTx
expression in ADF, CEP and RIC neurons, respectively.
Data are presented as mean ± s.e.m. Shades along
the calcium imaging traces represent error bars ( ± s.e.m).
p values were calculated with one-way ANOVA with Bonfferronìs test.
See Supplementary
Table 1 for lifespan statistics.
Extended Data Fig. 2
Additional data related to the olfactory circuit.
a) ADF dendrite, soma and axon from DR, but not AL
worms, all respond robustly to medium containing food odors. GCaMP6f was
expressed as a transgene in ADF neurons under tph-1 long
promoter. DsRed was co-expressed to enable ratiometric imaging. Shades along
the traces represent error bars (SEM).
(b) NSM neurons (dendrite/soma/axon) from DR and AL
worms do not respond to medium containing food odors. GCaMP6f was expressed
as a transgene in the NSM neurons using tph-1(s) promoter.
DsRed was co-expressed to enable ratiometric imaging. Shades along the
traces represent error bars (SEM).
(d) CEP dendrite from DR worms, but not AL worms,
responds robustly to medium containing food odors. GCaMP6f was expressed as
a transgene in CEP neurons using dat-1 promoter. DsRed was
co-expressed to enable ratiometric imaging. Shades along the traces
represent error bars (SEM). (e) CEP soma and axon from both DR
and AL worms respond to medium containing food odors, showing no specificity
towards DR. Shades along the traces represent error bars (SEM).
(f) Bar graph summarizing data in (d) and
(e). n=6(CEP dendrite - AL), 8(CEP dendrite - DR), 13(CEP
soma - AL), 20(CEP soma - DR), 12(CEP axon - AL) and 17(CEP axon - DR)
biologically independent animals. (g-h) ADE and PDE neurons
(soma and processes) from DR and AL worms do not respond to medium
containing food odors. GCaMP6f was expressed as a transgene in ADE and PDE
neurons using dat-1 promoter. DsRed was co-expressed to
enable ratiometric imaging. Shades along the traces represent error bars
(SEM). (i) Bar graph summarizing data in (g) and
(h). n=11 (ADE soma - AL), 10 (ADE soma - DR), 13 (ADE
processes - AL), 15 (ADE processes - DR), 10 (PDE soma - AL), 11 (PDE soma -
DR), 10 (PDE processes - AL) and 11 (PDE processes - DR) biologically
independent animals. (j) RIC soma and processes from DR worms,
but not AL worms, respond robustly to medium containing food odors. GCaMP6f
was expressed as a transgene in RIC neurons under tbh-1
promoter. DsRed was co-expressed to enable ratiometric imaging. Shades along
the traces represent error bars (SEM). The soma curves are duplicates of
those presented in Fig.2i, as these experiments were performed at
the same time. (k) RIM soma and axon from both DR and AL worms
respond to medium containing food odors, showing no specificity towards DR.
GCaMP6f was expressed as a transgene in RIM neurons using
cex-1 promoter. DsRed was co-expressed to enable
ratiometric imaging. Shades along the traces represent error bars (SEM).
(l) Bar graph summarizing data in (j) and
(k). n=11 (RIC soma - AL), 14 (RIC soma - DR), 11 (RIC
processes - AL), 12 (RIC processes - DR), 12 (RIM soma - AL), 12 (RIM soma -
DR), 12 (RIM processes - AL) and 10 (RIM processes - DR) biologically
independent animals. (m-n) RNAi knockdown of
odr-3 and ocr-2 specifically in ADF
neurons eliminates food odor-evoked calcium responses in these neurons.
dsRNA against odr-3 and ocr-2 gene was
expressed as a transgene specifically in ADF neurons using the
bas-1(prom7) promoter. (m) Calcium imaging
traces. Shades along the traces represent error bars (SEM). (n)
Bar graph summarizing the data in (m). n=10 (WT - AL), 9 (WT - DR), 10
(ADF odr-3(RNAi) - AL), 10 (ADF
odr-3(RNAi) - DR), 10 (ADF ocr-2(RNAi) - AL)
and 10 (ADF ocr-2(RNAi) - DR) biologically independent
animals. (o) NSM neurons are not required for food odors to
suppress DR longevity. tph-1(s) promoter was used to drive
the expression of TeTx transgene specifically in NSM
neurons.
(p) Blocking the output of RIC neuron shortens DR
longevity. tbh-1 promoter was used to drive the expression
of TeTx transgene in RIC neurons. Data are presented as
mean ± s.e.m. p values in c,
f, i and l: two-tailed student`s
t test. P values in n: one-way ANOVA with Bonfferronìs
test.
We then asked whether ADF neurons are important for mediating the
inhibitory effort of food odors on DR longevity. We ablated the output of ADF
neurons by expressing TeTx (tetanus toxin) as a transgene specifically in these
neurons. TeTx cleaves synaptobrevin, an essential SNARE subunit, to block
exocytosis[22]. Ablating
the output of ADF neurons but not NSM neurons abolished the effect of food odors
(Fig.2m and Extended Data Fig. 2o), indicating that ADF neurons
are required for food odors to suppress DR longevity. This data, together with
our calcium imaging results, suggests that ADF chemosensory neurons act as
primary olfactory sensory neurons to sense food odors to suppress longevity in
DR worms.Using the same strategy, we interrogated the potential roles of dopamine
neurons (CEP/ADE/PDE), octopamine neurons (RIC), and tyramine neurons (RIM) in
the circuit. CEP dopamine neurons responded robustly to food odors in a
DR-dependent manner (Fig.2e, 2h), while ADE and PDE dopamine neurons did not (Extended Data Fig.2g–i). Interestingly, RIC neurons also displayed
DR-dependent calcium response to food odors, except that food odors inhibited
rather than stimulated their activity (Fig.2i–l). These results
suggest that CEP and RIC neurons are part of the circuit. One notable
observation is that while different sub-compartments of ADF and RIC neurons
(dendrites/soma/axon for ADF and soma/processes for RIC) all responded similarly
to food odors (Extended Data Fig.2a, 2c, 2j,
and 2l), in CEP neurons only the dendrites
responded to food odors in a DR-dependent manner (Extended Data Fig.2d–f).
This is not surprising, as many worm neurons show compartmentalized calcium
responses[23,24]. By contrast, RIM neurons exhibited no
specificity towards DR, as these neurons from DR and AL worms responded
similarly to food odors (Extended Data
Fig.2k–l), suggesting
that RIM neurons are not part of the circuit. These calcium imaging results
suggest that in addition to ADF neurons, CEP and RIC neurons are also part of
the circuit. Consistent with this model, blocking the output of CEP and RIC
neurons using a TeTx transgene eliminated the inhibitory effect of food odors on
DR longevity (Fig.2n–o), indicating that CEP and RIC are required for food
odors to suppress lifespan in DR worms. We thus conclude that ADF, CEP and RIC
are essential components of an olfactory circuit that senses and processes odor
signals from food to suppress longevity in DR worms.We then sought to map the position of ADF, CEP and RIC neurons in the
circuit. Given that our data showed that ADF neurons are the primary olfactory
neurons sensing food odors, CEP and RIC would be expected to act downstream of
ADF in the circuit. If so, blocking the output of the upstream ADF sensory
neurons shall abolish the sensitivity of the downstream CEP and RIC neurons to
food odors. This appears to be the case: blunting the output of ADF with a TeTx
transgene rendered both CEP and RIC neurons insensitive to food odors (Fig.2f, 2h, 2j, and 2l); so did mutations in tph-1 that
abolished serotonin release from ADF neurons (Fig.2f, 2h, 2j, and 2l). On
the other hand, eliminating the output of the downstream neurons CEP and RIC
using cat-2 mutation (blocking dopamine release from CEP) or
tbh-1 mutation (blocking octopamine release from RIC),
respectively, had no effect on the response of ADF to food odors (Fig.2c–d).
This set of experiment places ADF neurons upstream of CEP and RIC neurons.Using a similar strategy, we tested CEP and RIC neurons. While blocking
the output of CEP neurons (using a TeTx transgene and cat-2
mutation) abrogated RIC’s odor sensitivity (Fig. 2k–l), inhibiting
RIC’s output (using tbh-1 mutation) had no effect on
CEP’s sensitivity to food odors (Fig.2g–h). This places
CEP upstream of RIC neurons. As food odors stimulated ADF and CEP neurons but
inhibited RIC neurons, this suggests a circuit mechanism by which food odors
suppress DR longevity by stimulating ADF and CEP neurons to inhibit RIC neurons
(Fig.3l). Though CEP neurons are
mechanosensory neurons, as they act downstream of ADF, these CEP neurons may in
fact function as interneurons in the circuit.
Fig.3 |
The molecular basis by which the olfactory circuit senses and processes odor
signals from food.
(a-c) Mutations in odr-3 (a)
and ocr-2 (b) abolish food odor-evoked calcium
response in ADF neurons, a phenotype rescued by transgenic expression of
wild-type odr-3 and ocr-2 genes in ADF
neurons. A fragment of the bas-1 promoter
(bas-1(prom7)) was used to drive expression of
odr-3 and ocr-2 cDNA specifically in ADF
neurons. (c) Bar graph summarizing the data in (a) and
(b). n=17 (WT - AL), 20 (WT - DR), 9 (odr-3 -
AL), 8 (odr-3 - DR), 13 (odr-3 rescue - AL),
12 (odr-3 rescue - DR), 9 (ocr-2 - AL), 9
(ocr-2 - DR), 13 (ocr-2 rescue - AL) and
13 (ocr-2 rescue - DR) biological independent animals.
(d-e) Mutations in odr-3 and
ocr-2 prevent food odors from suppressing DR longevity, a
phenotype rescued transgenic expression of wild-type odr-3 and
ocr-2 cDNA in ADF neurons.
(f) Loss of ser-5 eliminates the ability
of food odors to suppress DR longevity, a phenotype rescued by transgenic
expression ser-5 cDNA in CEP neurons using the
dat-1 promoter.
(g-h) Loss of ser-5 abolishes CEP calcium
response evoked by food odors, a defect rescued by transgenic expression
ser-5 cDNA in CEP neurons. (h) Bar graph. n=11
(WT - AL), 12 (WT - DR), 14 (ser-5 - AL), 13
(ser-5 - DR), 15 (ser-5 rescue - AL) and
12 (ser-5 rescue - DR) biological independent animals.
(i) Loss of dop-6 prevents food odors from
suppressing DR longevity, a phenotype rescued by transgenic expression
dop-6 cDNA in RIC neurons.
(j-k) Loss of dop-6 abolishes RIC calcium
response evoked by food odors, a defect rescued by transgenic expression
dop-6 cDNA in RIC neurons. (k) Bar graph with
individual datapoints. n=12 (WT - AL), 14 (WT - DR), 15 (dop-6
- AL), 17 (dop-6 - DR), 14 (dop-6 rescue - AL)
and 12 (dop-6 rescue - DR) biological independent animals.
(l) A schematic model of the olfactory circuit that senses
and processes odor signals from food to suppress DR longevity.
Data are presented as mean ± s.e.m. Shades along
the calcium imaging traces represent error bars ( ± s.e.m).
p values were calculated with one-way ANOVA with Bonfferronìs test.
See Supplementary
Table 1 for lifespan statistics.
The molecular mechanisms by which the olfactory circuit senses and processes
odor signals
To gain a molecular understanding of how the olfactory circuit senses
and processes odor signals from food, we went on to identify the receptors that
act in each neuron to sense and process odor signals. Olfactory transduction in
C. elegans sensory neurons is a G protein-mediated process
that culminates in the opening of downstream transduction channels, leading to
sensory neuron activation[13].
We thus examined the G protein ODR-3 and the transduction channel subunit OCR-2
known to function in ADF sensory neurons to mediate chemosensation[13,25]. Mutations in both odr-3 and
ocr-2 not only abolished ADF neuron’s calcium
response to food odors (Fig.3a–c), but also the ability of food odors to
suppress DR longevity (Fig.3d–e). Both phenotypes were rescued by
transgenic expression of wild-type odr-3 and
ocr-2 genes in ADF neurons (Fig.3a–e), demonstrating
that ODR-3 and OCR-2 act in ADF neurons to sense food odors. Additional evidence
came from cell-specific knockdown experiments, where we expressed dsRNA of
odr-3 or ocr-2 as a transgene specifically
in ADF neurons. RNAi of odr-3 and ocr-2 in ADF
neurons abolished their sensitivity to food odors (Extended Data Fig.2m–n),
providing further evidence that the G protein ODR-3 and the transduction channel
OCR-2 act in ADF neurons to sense food odors. These experiments are also
consistent with the notion that ADF neurons are primary olfactory sensory
neurons[19].ADF neurons are serotoninergic. Given that CEP neurons act downstream of
ADF neurons, we reasoned that a serotonin receptor might act in CEP neurons to
transmit odor signals by responding to serotonin released from ADF neurons. We
thus examined all the five serotonin receptor genes encoded by the worm genome:
ser-1, ser-4, ser-5, ser-7 and
mod-1[26]. Mutations in ser-5, but not the other
four serotonin receptor genes, blocked the ability of food odors to suppress DR
longevity, a phenotype that was rescued by transgenic expression of wild-type
ser-5 gene in CEP neurons (Fig.3f and Extended Data
Fig.3a–d). Thus, SER-5
may function as the serotonin receptor in CEP neurons to transmit odor signals.
In support of this idea, no food odor-evoked calcium response in CEP neurons was
detected in ser-5 mutant worms, and this phenotype was rescued
by a ser-5 transgene expressed in CEP neurons (Fig.3g–h).
These results identify SER-5 as the serotonin receptor that acts in CEP neurons
to transmit odor signals in the circuit.
Extended Data Fig. 3
Other serotonin receptors are not required for food odors to suppress DR
longevity.
Food odors can still suppress DR longevity in ser-1
(a), ser-4 (b),
ser-7 (c) and mod-1
(d) mutant worms. (a-d) share the same control
group, as these experiments were performed at the same time
CEP neurons are dopaminergic. We thus hypothesized that a dopamine
receptor may act in the downstream RIC neurons to transmit odor signals by
responding to dopamine released from CEP neurons. By screening mutants of all
the six dopamine receptor genes encoded by the worm genome: dop-1,
dop-2, dop-3, dop-4, dop-5 and dop-6[26], we found that food odors lost
the ability to suppress DR longevity in dop-6 but not the other
five dop mutant worms (Fig.3i and Extended Data
Fig.4a–e). Similarly,
RIC neurons in dop-6 mutant worms lost the ability to respond
to food odors in calcium imaging assay (Fig.3j–k). Both
phenotypes were rescued by transgenic expression of wild-type
dop-6 gene in RIC neurons (Fig.3i–k). Thus, DOP-6
may function as the dopamine receptor in RIC neurons to transmit odor signals in
the circuit.
Extended Data Fig. 4
Other dopamine receptors are not required for food odors to suppress DR
longevity.
Food odors can still suppress DR longevity in dop-1
(a), dop-2 (b),
dop-3 (c) , dop-4
(d) and dop-5 (e) mutant worms.
(a) and (c) share the same control group, as
these experiments were performed at the same time. (b),
(d) and (e) share the same control group, as
these experiments were performed at the same time.
Together, our data suggest a model that food odors suppress DR longevity
via an olfactory circuit comprising three pairs of neurons: ADF, CEP and RIC
(Fig.3l). In this circuit, we suggest
that ADF neurons function as primary sensory neurons to sense food odors through
a G protein-mediated transduction mechanism; ADF neurons then stimulate CEP
neurons, which in turn inhibit RIC neurons; SER-5 and DOP-6 function in CEP and
RIC neurons to transmit odor signals by responding to the neurotransmitter
serotonin and dopamine released from upstream neurons, respectively (Fig.3l). As food odors inhibit RIC neurons to
suppress DR longevity, this suggests that the normal output of RIC neurons is to
promote longevity. If so, then inhibiting the output of RIC neurons should mimic
the inhibitory effect of food odors on DR longevity. Indeed, inhibiting the
output of RIC neurons with a TeTx transgene shortened DR longevity (Extended Data Fig.2p), providing further
evidence that food odors suppress DR longevity by inhibiting the output of RIC
octopamine neurons.
The olfactory circuit signals the intestine to regulate the energy sensor
AMPK
We then wondered how the olfactory circuit engages the rest of the
animal body to regulate DR longevity. As DR longevity pathways all converge on
nutrient signaling[2-4], we first asked which nutrient
signaling underlies DR longevity in our lifespan assay. AMPK and mTOR signaling
are the two primary nutrient signaling that regulate DR longevity in
worms[2,3]. Consistent with previous work[10], we found that loss of the
C. elegansAMPKα ortholog AAK-2 abolished the
ability of DR to extend lifespan (Fig.4a),
demonstrating that AAK-2 is required for DR longevity. This supports the notion
that AMPK mediates the longevity under the sDR regimen[10]. Notably, aak-2 mutant
worms were also insensitive to food odors (Fig.4a), indicating a requirement of AMPK for food odors to suppress
DR longevity. By contrast, DR longevity persisted in raga-1 and
rict-1 mutant worms (Extended Data Fig.5a, 5c),
which were deficient in mTORC1 and mTORC2 signaling, respectively [27], and these two mutants also
remained sensitive to food odors (Extended Data
Fig.5b, 5d). Thus, both DR
longevity and its sensitivity to food odors require AMPK but not mTOR signaling
in our DR regimen. Notably, transgenic expression of wild-type
aak-2 gene in the intestine rescued both the longevity and
odor sensitivity defects in aak-2 mutant worms (Fig.4b). By contrast, expression of
aak-2 as a transgene in neurons only had a slight rescue
effect on the longevity defect (Extended Data
Fig.5e). Neuronal expression of aak-2 also failed to
rescue the odor sensitivity defect in aak-2 mutant worms. Thus,
AAK-2 primarily acts in the intestine. Additional evidence came from an RNAi
experiment, in which we knocked down aak-2 expression in the
intestine by expressing dsRNA of aak-2 as a transgene
specifically in the intestine, and found that it blocked the ability of food
odors to suppress DR longevity (Fig.4c).
This experiment was conducted in sid-1 background to restrict
RNAi to the tissue of interest[28]. These results together suggest that the olfactory circuit
signals the intestine to regulate the energy sensor AMPK to suppress DR
longevity, revealing a brain-gut signaling axis.
Fig.4 |
The olfactory circuit signals the intestine to regulate the energy sensory
AAK-2/AMPK via Gq-PLCβ-CaMKK-dependent norepinephrine signaling.
(a) aak-2 mutant worms are completely
insensitive to DR and food odors.
(b) Transgenic expression of wild-type
aak-2 gene in the intestine rescues both the longevity and
odor sensitivity phenotypes in aak-2 mutant worms.
(c) RNAi of aak-2 in the intestine
abolishes the ability of food odors to suppress DR longevity. dsRNA against
aak-2 was expressed as a transgene specifically in the
intestine using the ges-1 promoter. All experiments were
carried out in sid-1 mutant background where the systemic
effect of RNAi is absent.
(d) Loss of ser-3 abolishes the ability of
food odors to suppress DR longevity, a phenotype rescued by transgenic
expression of wild-type ser-3 gene in the intestine using the
ges-1 promoter.
(e) Intestine-specific RNAi of ser-3 by a
dsRNA transgene prevented food odors from suppressing DR longevity.
(f-h) Intestine-specific RNAi of egl-30/Gq
(f), egl-8/PLCβ (g) or
ckk-1/CaMKK (h) by dsRNA transgenes abolishes
the ability of food odors to suppress DR longevity. (f),
(g) and (h) share the same sid-1
control curves, as these experiments were performed at the same time.
(i) aak-2(T181D) transgene rescues the DR
longevity defect but not the odor sensitivity defect of aak-2
mutant worms.
(j-l) Intestinal expression of AAK-2/AMPK slows down the
age-dependent decline in intestinal barrier function. The blue dye (FD&C
blue), which is impermeable to the intestine epithelium, was taken up into the
intestine by the worm. The dye was confined inside the intestinal lumen in young
worms; but as worms age, the dye leaked into the body cavity. Worms were
cultured at 25oC. (j) Young wild-type worm (day 1).
(k) Aged wild-type worm (day 6), showing that the dye leaked
into the body cavity. Arrows point to the dye outside of the intestine. Scale
Bar: 25 μm. (l) Bar graph showing that the intestinal
aak-2 transgene Pges-1::aak-2 slowed down
the age-dependent dye leakage. The percentage of worms showing body-cavity dye
leakage was quantified. Each data point was derived from 10 worms and repeated
five times. Data are presented as mean ± s.e.m. p values
were calculated with two-way ANOVA with Bonfferronìs test.
See Supplementary
Table 1 for lifespan statistics.
Extended Data Fig. 5
Additional data related to regulation of DR longevity by AMPK and
octopamine signaling.
a) DR can extend the lifespan of
raga-1 mutant worms. (b) Food odors can
suppress DR longevity in raga-1 mutant worms.
(c) DR can extend the lifespan of rict-1
mutant worms. (d) Food odors can suppress DR longevity in
rict-1 mutant worms. (e) Pan-neuronal
expression of aak-2 gene only has a slight rescue effect on
the longevity defect of aak-2 mutant worms. This
aak-2 neuronal transgene also does not rescue the odor
sensitivity defect of aak-2 mutant worms.
rgef-1 promoter was used to drive the expression of
aak-2 cDNA in neurons. (f-g) Food odors
can still suppress DR longevity in ser-6 (f)
and octr-1 (g) mutant worms. (f)
and (g) share the same control group, as these experiments were
performed at the same time. (h) Intestine-specific knock-down
of par-4/LKB1 by dsRNA transgene
(Pges-1::par-4(RNAi)) does not prevent food odors from
suppressing DR longevity, though it partially inhibits DR longevity.
(i) Intestine-specific knock-down of
mom-4/TAK1 by dsRNA transgene
(Pges-1::mom-4(RNAi)) does not prevent food odors from
suppressing DR longevity; nor does it affect DR longevity.
(j-l) Mutations in egl-30
(j), egl-8 (k), and
ckk-1 (l) abolish the ability of food
odors to suppress DR longevity, a defect that is rescued by transgenic
expression of corresponding wild-type genes in the intestine using
ges-1 promoter.
Given that AAK-2/AMPK expression in the intestine extended lifespan
(Extended Data Fig.6a), we wondered if
it could also promote the health of the intestine. A primary function of the
intestine epithelium is to forms a selective barrier that allows it to absorb
nutrients, ions and water but remain impermeable to toxic substances and
microorganisms[29,30]. Like other animals[29,31], the barrier function of C. elegans
intestine declines with age progressively, which renders the intestine of aged
worms permeable to otherwise impermeable chemicals, resulting in their leak into
the body cavity[30] (Fig. 4j–k). Intestinal expression of AAK-2 greatly slowed down the
aged-dependent decline in the intestinal barrier function (Fig.4l), indicating that AMPK can promote the health
of the intestine.
(a) Lifespan extension mediated by intestinal
expression of aak-2 requires daf-16.
daf-16 RNAi blocked the lifespan-extension effect of
the intestinal aak-2 transgene. (b-d)
Intestinal expression of aak-2 promotes
sod-3 gene expression in multiple tissues in a
daf-16-dependent manner. sod-3::gfp is
a transgene reporting the expression level of sod-3 gene.
(b) Sample images showing a low level of
sod-3::gfp expression. Left: bright field image. Right:
fluorescent image. (c) Sample images showing that the
Pges-1::aak-2 transgene increased the expression of
sod-3::gfp. Top left: bright field image. Top right:
fluorescent image. Bottom: zoomed-in images showing
sod-3::gfp expression in multiple tissues, including
pharynx (head), neurons (head), body-wall muscles, vulval muscles
(mid-body), intestine, etc. Scale Bar: 100 μm. (d) Bar
graph summarizing the data in (b) and (c). n=24
(WT), 20 (Pges-1::aak-2), 43
(daf-16(RNAi)) and 22 (daf-16(RNAi);
Pges-1::aak-2) biologically independent animals. Data are
presented as mean ± s.e.m. p values were calculated
with one-way ANOVA with Bonfferronìs test.
Gq-PLCβ-CaMKK-dependent octopamine signaling regulates AMPK in the
intestine
How does the olfactory circuit signal the intestine to regulate AMPK? As
RIC neurons are octopamine neurons acting downstream in the circuit (Fig.3l), we reasoned that RIC neurons might
signal the intestine via octopamine. Consistent with this model, food odors
failed to suppress DR longevity in tbh-1 mutant worms deficient
in octopamine production (Fig.1i). We then
attempted to identify the octopamine receptor that functions in the intestine to
transmit odor signals to regulate AMPK. Among all the three worm octopamine
receptor genes (i.e. ser-3, ser-6 and
octr-1)[26], loss of ser-3 but not the other two
receptor genes abrogated the ability of food odors to suppress DR longevity
(Fig.4d and Extended Data Fig.5f–g). Transgenic expression of wild-type
ser-3 gene in the intestine rescued the odor sensitivity
defect in ser-3 mutant worms (Fig.4d). In addition, RNAi of ser-3 specifically in
the intestine of wild-type worms recapitulated the ser-3 mutant
phenotype (Fig.4e). These data identify
SER-3 as the octopamine receptor that acts in the intestine to transmit signals
from the olfactory circuit to regulate AMPK.The question arises as to how the octopamine receptor SER-3 regulates
AMPK in the intestine. SER-3 is coupled to Gq/PLCβ-mediated calcium
signaling[32]. We thus
examined the worm Gq ortholog EGL-30 and PLCβ ortholog EGL-8. RNAi of
egl-30 and egl-8 specifically in the
intestine prevented food odors from suppressing DR longevity (Fig.4f–g),
indicating a requirement for Gq and PLCβ in the pathway. Then how does
Gq-PLCβ couple SER-3 to AMPK? AMPK activation requires phosphorylation of
its catalytic α subunit by an AMPK kinase[33]. Among the three AMPK kinases CaMKK,
LKB1 and TAK1, CaMKK is the only one that is activated by calcium
signaling[33]. As
Gq-PLCβ activation triggers calcium signaling[34], CaMKK emerges as a candidate AMPK
kinase that couples SER-3-Gq-PLCβ to AMPK. Indeed, RNAi of the worm CaMKK
gene ckk-1 specifically in the intestine prevented food odors
from suppressing DR longevity (Fig.4h),
while RNAi of the other two putative AMPK kinase genes
par-4/LKB1 and mom-4/TAK1 did not (Extended Data Fig.5h–i). This result suggests that CKK-1/CaMKK is an AMPK
kinase that acts downstream of Gq-PLCβ to activate AMPK. As a
complementary approach, we examined egl-30, egl-8, and
ckk-1 mutant worms, and found that they all lost the
sensitivity to food odors in lifespan assay, a phenotype that was rescued by
transgenic expression of wild-type egl-30, egl-8, and
ckk-1 genes in the intestine, respectively (Extended Data Fig.5j–l). These observations together suggest that the
octopamine receptor SER-3 regulates AMPK in the intestine via a
Gq-PLCβ-CaMKK-dependent mechanism.To provide additional evidence, we overexpressed the octopamine receptor
SER-3 as a transgene in the intestine, and found that it extended lifespan under
normal condition (Fig.5a), suggesting that
activation of octopamine signaling in the intestine promotes longevity. This is
also consistent with our observation that the output of RIC octopamine neurons
in the olfactory circuit was to promote longevity. Importantly, the
SER-3-dependent longevity was fully suppressed by intestine-specific RNAi of all
the downstream components, i.e. egl-30/Gq,
egl-8/PLCβ, ckk-1/CaMKK, and
aak-2/AMPK (Fig.5a–d). This provides
additional evidence that the octopamine receptor SER-3 regulates AMPK in the
intestine via a Gq-PLCβ-CaMKK-dependent mechanism.
Fig. 5 |
Octopamine signaling in the intestine promotes lifespan and stimulates AMPK
phosphorylation via a Gq-PLCβ-CaMKK-dependent mechanism.
(a-d) Transgenic expression of the octopamine receptor
SER-3 in the intestine (Pges-1::ser-3) extends lifespan under
normal condition (a), and this lifespan-extending effect is blocked
by intestine-specific RNAi of egl-30/Gq (a),
egl-8/PLCb (b), ckk-1/CaMKK
(c), and aak-2 (d) with
corresponding dsRNA transgenes. (b) and (c) share the
same sid-1 control curves, as these experiments were performed
at the same time.
(e) Transgenic expression of SER-3 in the intestine
(Pges-1::ser-3) stimulates AAK-2/AMPK phosphorylation.
Samples collected from aak-2 mutant worms was used to
demonstrate the specificity of the antibody. Actin (ACT-1) was used as a loading
control.
(f-h) Loss of egl-30/Gq (f),
egl-8/PLCb (g) or ckk-1/CaMKK
(h) inhibits SER-3-depedent stimulation of AAK-2/AMPK
phosphorylation.
See Supplementary
Table 1 for lifespan statistics.
To obtain further evidence, we assessed whether the octopamine receptor
SER-3 can promote AMPK activity, and if so, whether it acts in a
Gq-PLCβ-CaMKK-dependent manner. Transgenic expression of SER-3 in the
intestine stimulated AAK-2 phosphorylation (Fig.5e), demonstrating that intestinal SER-3 can promote the
activity of AMPK. Importantly, this SER-3-dependent AAK-2 activation was
abrogated in egl-30/Gq, egl-8/PLCβ and
ckk-1/CaMKK mutant worms (Fig.5f–h). This
experiment provides biochemical evidence that the octopamine receptor SER-3
promotes AMPK activity in a Gq-PLCβ-CaMKK-dependent manner.
Activation of norepinephrine signaling in mouse primary cells regulate AMPK
via a similar Gq-PLCβ-CaMKK-dependent mechanism
Octopamine is the invertebrate homolog of norepinephrine. We then
wondered if norepinephrine signaling could regulate AMPK in mammalian cells via
a similar Gq-PLCβ-CaMKK-dependent mechanism. In C.
elegans, the intestine also fulfills many of the complex functions
of mammalian fat and liver tissues[35]. We thus characterized mouse primary subcutaneous
adipocytes and primary hepatocytes, both of which express the Gq-coupled
α1A adrenergic receptor[36,37]. Selective
activation of α1A adrenergic receptor with cirazoline stimulated AMPK
phosphorylation in both mouse primary subcutaneous adipocytes and primary
hepatocytes (Fig.6a–b), indicating that activation of norepinephrine
signaling can promote AMPK activity in these primary cells. Importantly, this
AMPK activation was blocked by inhibitors of Gq (YM254890), PLC (U73122 but not
its inactive analog U73343), and CaMKK (STO-609) (Fig.6c–h). This set of
data suggests that activation of norepinephrine signaling in mouse primary cells
can stimulate AMPK via a Gq-PLCβ-CaMKK-dependent mechanism.
Fig.6 |
Activation of norepinephrine signaling stimulates AMPK via a
Gq-PLCβ-CaMKK-dependent mechanism in mouse primary cells.
(a) Selective activation of α1A-adrenergic receptor
activates AMPK in mouse primary subcutaneous adipocytes. Cirazoline (100
μM), a selective agonist for α1A-adrenergic receptor, can
stimulate the phosphorylation of T172 site in AMPKα. AICAR (500
μM, 15 min), an AMPK activator, was used as a positive control.
(b) Selective activation of α1A-adrenergic receptor
activates AMPK in mouse primary hepatocytes. Cirazoline (100 μM), a
selective agonist for α1A-adrenergic receptor, can stimulate the
phosphorylation of T172 site in AMPKα. AICAR (500 μM, 15 min), an
AMPK activator, was used as a positive control.
(c-e) Inhibition of Gq (c), PLCβ
(d) or CaMKK (e) abolishes cirazoline-induced (100
mM, 60 min) phosphorylation of T172 site in AMPKα in mouse primary
subcutaneous adipocytes. Cells were pre-treated with the following inhibitors
for 30 min: the Gq inhibitor YM254890 (25 μM), the PLC inhibitor U73122
(5 μM), U73343 (5 μM) that is an inactive analog of U73122, and
the CaMKK inhibitor STO-609 (5 μM).
(f-h) Inhibition of Gq (f), PLCβ
(g) or CaMKK (h) abolishes cirazoline-induced (100
μM, 15 min) phosphorylation of T172 site in AMPKα in mouse primary
hepatocytes. Cells were pre-treated with the following inhibitors for 30 min:
the Gq inhibitor YM254890 (25 μM), the PLC inhibitor U73122 (5
μM), U73343 (5 μM) that is an inactive analog of U73122, and the
CaMKK inhibitor STO-609 (5 μM).
(i) Schematic model. 5-HT: serotonin. DA: dopamine. OA:
octopamine.
Food odors and DR converge on AMPK to regulate longevity
One notable observation is that among all the genes characterized in
this study, AMPK is unique in that it is the only one that, when mutated,
blocked the effects from both DR and food odors. Namely,
aak-2/AMPK mutants were completely insensitive to both DR and
food odors (Fig.4a). By contrast, though
mutants of other genes in the olfactory circuit and the downstream octopamine
signaling failed to respond to food odors, they remained sensitive to DR at
least partially in lifespan assay (Supplementary Table 1). This unique
feature of AMPK suggests that food odors and DR may converge on AMPK to regulate
lifespan. AMPK is an energy sensor sensitive to DR[3,33]. Specifically, DR leads to an increase in AMP::ATP ratio,
resulting in activation of AMPK[3,33]. Notably, AMPK
is under dual-regulation, as its activation also requires phosphorylation by
AMPK kinases such as CaMKK[3,33], which we showed is regulated
by octopamine signaling residing downstream of the olfactory circuit. Thus, AMPK
may sit at a unique position to integrate signals from DR and food odors (Fig.6i). To test this, we mutated the
CKK-1/CaMKK phosphorylation site in AAK-2/AMPK from Thr to Asp (i.e.
AAK-2(T181D)) to render it insensitive to CKK-1/CaMKK, thereby uncoupling it
from food odors. This AAK-2(T181D) is functional, as it rescued the DR longevity
defect in aak-2 mutant worms (Fig.4i). Remarkably, worms expressing AAK-2(T181D) were insensitive
to food odors (Fig.4i), indicating that
this mutant form of AAK-2/AMPK lost the ability to integrate signals from DR and
food odors. This provides further evidence that food odors and DR converge on
AAK-2/AMPK to regulate longevity.
Discussion
In the current study, we investigated how food-associated odors regulate DR
longevity in C. elegans. The fact that food odors suppress the
lifespan of worms under DR but not those fed AL highlights the notion that in
addition to the actual food abundance (nutrient level), the perception of food
abundance (from food odors) is also important for longevity[5]. Our results suggest a model that food odors
suppress DR longevity by acting on an olfactory circuit, which signals the gut
intestine via octopamine, the invertebrate homology of norepinephrine, to regulate
the energy sensor AMPK through a Gq-PLCβ-CaMKK-dependent mechanism (Fig.6i). Interestingly, norepinephrine signaling
can also regulate AMPK through a similar mechanism in mouse primary cells. These
results identify a brain-gut axis through which food odors suppress DR-mediated
longevity, illustrating how non-nutrient food components may regulate lifespan by
altering animals’ perception of food abundance.The olfactory circuit comprises three pairs of neurons: ADF, CEP and RIC. In
this circuit, food odors stimulate ADF to inhibit RIC via CEP (Fig.6i). We also characterized the molecular mechanisms by
which the circuit senses and process odor signals by identifying some of the key
components mediating sensory transduction and sensory processing (Fig.6i). While CEP forms direct connections with RIC, no
such connections are found between ADF and CEP[14], suggesting that part of this circuit is neuroendocrine by nature. We thus do not exclude the possibility that other neurons or cells may also be part of this circuit. For example, some
other chemosensory neurons have also been suggested to modulate longevity[6,12,38]; however, whether
these neurons can directly sense food odors to regulate lifespan remains to be
determined. One interesting observation is that food odors elicited robust calcium
responses in ADF/CEP/RIC neurons from DR worms, but not those fed AL, revealing a
DR-specific effect. It is possible that DR sensitized the neurons in the circuit.
Alternatively but not mutually exclusively, AL may suppress the circuit
responsiveness. In humans, fasting potentiates olfactory sensitivity, while satiety
suppresses it[39], unveiling an
interesting similarity between worms and mammals. Future studies will determine how
DR and AL differentially regulate the sensitivity of the olfactory system to food
odors.AMPK sits at a unique position to integrate signals from both food odors and
DR due to its dual-regulation mode by AMP::ATP ratio (DR) and CaMKK (food odors).
AMPK primarily acts in the intestine to mediate this effect. Nevertheless, we do not
exclude a role for AMPK in other tissues such as neurons, given that AMPK has the
capacity to regulate lifespan cell-non-autonomously[40]. AMPK is probably not the only substrate
regulated by both DR and food odors. For example, DR also regulates the sensitivity
of the olfactory circuit to food odors. Nonetheless, our results suggest that AMPK
is a primary site where food odors and DR converge to regulate longevity. Then what
act downstream of AMPK? One downstream effector of AMPK is the transcription factor
DAF-16/FOXO[10], which is a
master regulator of longevity. AMPK regulates DAF-16/FOXO via direct
phosphorylation[10,41]. We found that
AAK-2/AMPK-dependent lifespan extension requires DAF-16 (Extended Data Fig.6a), and that AAK-2/AMPK expression in
the intestine promoted the expression of the DAF-16/FOXO target gene
sod-3 in a DAF-16-dependent manner (Extended Data Fig.6b–d). SOD-3 expression was up-regulated cell-non-autonomously in many
other tissues, indicating that the longevity signal was disseminated throughout the
body (Extended Data Fig.6c), consistent with
the notion that the intestine is a signaling hub for longevity regulation[1]. Another effector of AMPK could be
mitochondria, as AMPK regulates mitochondria function and dynamics to modulate
longevity[42]. Thus, it is
likely that multiple AMPK effectors may act downstream to regulate DR longevity.We identify octopamine as a signaling molecule that mediates brain-gut
communications. Specifically, our data suggest that food odors suppress octopamine
signaling in the intestine by inhibiting octopamine release from the olfactory
circuit. We also show that octopamine signaling regulates AMPK in the intestine via
a Gq-PLCβ-CaMKK-dependent mechanism. Octopamine is the invertebrate homolog
of norepinephrine. Remarkably, activation of norepinephrine signaling in mouse
primary cells can stimulate AMPK in a similar manner, suggesting a conserved
mechanism. Interestingly, blocking olfactory sensation in mice stimulates
norepinephrine release from sympathetic nerves and promotes norepinephrine signaling
in fat tissues, leading to improved energy metabolism and protection against
obesity[43]. Conversely,
enhancing olfaction in mice causes insulin resistance and obesity[43]. This raises the intriguing
possibility that olfaction might inhibit longevity in mice through norepinephrine
signaling, pointing to another potential similarity between worms and mice. It would
be interesting to test whether those olfaction deficient mice are long-lived.
Materials and methods
Strains
C. elegans strains were maintained at 20°C on
nematode growth medium (NGM) plates seeded with OP50 bacteria unless otherwise
specified. Transgenic lines were generated by injecting plasmid DNA directly
into hermaphrodite gonad following standard protocol. Mutant strains were
outcrossed at least six times before use. For genetic crosses, all genotypes
were confirmed using PCR and, if necessary, followed by Sanger sequencing to
verify single nucleotide mutations. The strains used are:
Chemical reagents
Collagenase D (#11088882001) and dispase II (#04942078001) were
purchased from Roche. All the cell culture media, including DMEM
(#11995–073), DMEM-low glucose (#11885–084) and DMEM/F12 GlutaMAX
(#10565–042), were obtained from Life Technologies. Erioglaucine disodium
salt (#861146), 5-Fluoro-2’-deoxyuridine (FUDR, #F0503),
3-Isobutyl-1-methylxanthine (IBMX, #I7018), dexamethasone (#D4902), insulin
(#I5500) and fetal bovine serum (FBS) (#F2442) were acquired from Sigma Aldrich.
Cirazoline hydrochloride (#0888) and AICAR (#2840) were purchased from Tocris.
STO-609 (#15325) and rosiglitazone (#71740) were purchased from Cayman Chemical.
YM2548909 (#257–00631) was purchased from FUJIFILM Wako Chemicals.
Collagenase Type IV (#LS004188) was obtained from Worthington Biochemical.
Mice
Animal care and experimental protocol were reviewed and approved by the
Institutional Animal Care and Use Committee at the University of Michigan.
Wild-type C57BL/6J mice (Stock no. 00664) were obtained from the Jackson
Laboratory and housed under 12 h light/12 h dark cycle with a standard rodent
chow diet (5L0D, PicoLab).
Molecular biology
The following promoters were used to drive gene expression in specific
neurons and tissues: tph-1(L) promoter (2.1 kb 5’UTR and
0.5 kb coding region) in serotonin neurons, including both ADF and NSM;
tph-1(s) promoter (1.7 kb) in NSM neurons;
bas-1(prom7) promoter (166 bp) in ADF neurons;
cex-1 promoter (1.1 kb) in RIM neurons[44]; dat-1 promoter (0.8
kb) in CEP, ADE and PDE neurons; tbh-1 promoter (4.4 kb) in RIC
neurons; rgef-1 promoter (3.4 kb) in all neurons;
ges-1 promoter (3.2 kb) in the intestine.
odr-3, ocr-2, ser-5,
dop-6, ser-3, egl-30,
egl-8, ckk-1, aak-2 cDNA
were cloned by RT-PCR from total RNA isolated from wild-type (N2) worms. To
generate dsRNA plasmids, ser-3, egl-30,
egl-8, ckk-1, aak-2,
par-4, and mom-4 fragments were amplified
from genomic DNA extracted from wild-type (N2) worms. The first 12 amino acids
from NCS-2 protein were fused with GCaMP6f to make myr-GCaMP6f, a
membrane-targeting form of GCaMP6f. The dat-1 promoter was used
to drive myr-G-CaMP6f and DsRed expression in CEP neurons to facilitate imaging
of the dendritic compartments of these neurons. To make tdTomato-expressing OP50
bacteria, tdTomato coding sequence was cloned into pGEX-5x-3
vector and transformed into OP50 component cells.
Lifespan assays
Lifespan experiments were performed on NGM plates seeded with OP50 at
20°C. In all experiments, the first day of adulthood was scored as day 1.
Worms that crawled off the plate, exploded or bagged were censored. DR lifespan
assays (sDR) were performed as described in previous literature[45]. All assays were conducted on
OP50. To harvest bacteria food, fresh OP50 colonies were inoculated in LB medium
overnight (14–16 hr), and bacteria pellets were collected by high-speed
centrifugation (4000 rpm, 4°C, 20 min). Bacteria pellets were
re-suspended in 10 ml fresh LB medium and pelleted again by centrifugation, and
this step was repeated one more time. Lastly, bacteria pellets were re-suspended
in fresh LB medium containing 5 mg/mL carbenicillin to a final concentration of
1011 cfu/mL. Ad libitum (AL) plates were
prepared with a bacteria concentration of 1011 cfu/mL (200
μl/per plate), and DR plates with a bacteria concentration of
109 cfu/mL (200 μl/per plate).
5-Fluoro-2’-deoxyuridine (FUDR) was added to NGM plates at a final
concentration of 2.5 μg/ml to prevent egg-laying and bagging. Unless
indicated otherwise, about 80–100 worms were transferred onto AL and DR
plates at Day 4 and then transferred every 2–4 days to fresh AL or DR NGM
plates at a density of 15 worms per plate until day 19–20 for DR and
15–16 for AL conditions. For lifespan assays under normal condition,
about 80–100 worms of each strain were included and transferred every
2–4 days until day 11–12 as described previously[46]. All statistical analyses were
performed using GraphPad Prism 6 (GraphPad Software, Inc.) and IBM SPSS
Statistics 21 (IBM, Inc.). p values were calculated using the
log-rank (Kaplan-Meier) method.
Feeding and food ingestion measurements
To quantify feeding rate, day 4 adult worms were transferred to NGM
plates seeded with DR or 2xDR amount of bacteria food and randomly divided into
two groups, with one group exposed to food odors and the other group not.
Pharyngeal pumping rate was then counted under a stereoscope at 1 hr, 24 hr, and
96 hr after worms were transferred to DR plates at 20oC.To quantify food ingestion, OP50-tdTomato bacteria were used as food.
Worms were transferred to NGM plates seeded with DR or 2×DR amount of
bacteria food and randomly divided into two groups, with one group exposed to
food-derived odors and the other group not. At 1 hr post-transfer, worms were
immobilized in 20 mM sodium-azide/M13 solution, mounted on 2% agarose pads, and
imaged on Olympus IX73 inverted microscope under a 10x objective using
ORCA-Flash4.0 LT+ Digital CMOS camera (Hammatsu Inc.) with MetaMorph software
(Molecular Devices Inc.). ImageJ software was used to quantify the images.
Calcium imaging
A microfluidic system was used to perform calcium imaging as previously
described[47].
1–2 days after transferred onto AL or DR plates, worms were loaded onto a
microfluidic device mounted on Olympus IX73 inverted microscope and incubated in
M13 solution (30 mM Tris-Cl, 100 mM NaCl, 10 mM KCl, pH7.0). Images were
acquired under a 40x objective using an ORCA-Flash4.0 LT+ Digital CMOS camera
(Hammatsu Inc.) with MetaFluor software (Molecular Devices Inc.) at 1 Hz.
GCaMP6f and DsRed were co-expressed as a transgene in specific neurons using
corresponding promoters to enable ratiometric imaging under blue (484 nm) and
yellow (565 nm) light. As food odor-evoked calcium responses are similar in
different sub-compartments in ADF and RIC neurons, we focused on recording soma
signals in these two neurons. For CEP neurons, we focused on recording dendritic
signals. This is because only dendritic responses in CEP neurons are
DR-dependent, while soma and axon responses do not depend on DR or the input
from the upstream ADF neurons, indicating that CEP soma and axon responses are
irrelevant to the DR pathway. We expressed myr-GCaMP6f in CEP to facilitate
dendritic imaging. Worms were first exposed to blue-yellow light cycles for 2
min in LB medium to establish a basal line before challenged with LB medium
containing food odors. To prepare LB medium containing food odors, OP50 bacteria
were cultured in LB medium overnight (14–16 hr), and the medium was
collected by two rounds of high-speed centrifugation (each at 4000 rpm,
4°C, 20 min) to remove all bacteria, followed by filtering through a 0.45
μm filter. Such medium was freshly prepared each time for the imaging
experiment. Background fluorescence was subtracted when calculating the ratio of
GCaMP/DsRed. The peak fold change in the ratio of GCaMP/DsRed fluorescence was
analyzed.
Mouse primary cell isolation and culture
To prepare mouse primary subcutaneous adipocytes, the stromal vascular
fraction (SVF) was first isolated from inguinal adipose tissues of C57BL/6J mice
(age: 6–8 weeks; gender: male and female) cultured and differentiated as
described previously[48].
Specifically, inguinal adipose tissues were dissected, washed in PBS, minced and
digested in PBS containing collagenase D (1.5 U/mL), dispase II (2.4 U/mL) and
10 mM CaCl2 for 20 min in a 37 °C water bath with agitation.
Digestion reaction was terminated by addition of culture medium consisting of
DMEM/F12 GlutaMAX supplemented with 10 % FBS and 1% penicillin/streptomycin.
Tissue suspension was passed through a 100 μm cell strainer and
centrifuged at 300–500 × g for 5 min to pellet SVF cells. The cell
pellet was resuspended in culture medium, filtered through a 40 μm cell
strainer and centrifuged as above. Isolated SVF cells were plated onto a
collagen-coated 10 cm cell culture dish and grown in culture medium.
Adipogenesis was induced with culture medium supplemented with 0.5 μg/mL
insulin, 5 μM dexamethasone, 1 μM rosiglitazone and 0.5 mM IBMX in
confluent cells. After 2 days of induction, cells were maintained in culture
medium containing 0.5 μg/mL insulin for 3 days. Differentiated adipocytes
were stimulated with 100 μM Cirazoline for varying time durations as
indicated in the figure or with 500 μM AICAR (15 min). To test
inhibitors, cells were pre-treated with 25 μM YM254890, 5 μM
U73122, 5 μM U73343 or 5 μM STO-609 for 30 min, followed by 100
μM Cirazoline treatment for 60 min before harvested for
immunoblotting.Primary hepatocytes were isolated from C57BL/6J mice as previously
reported [49]. Mouse was
anesthetized, and the liver was perfused via an inferior vena cava with washing
buffer (HBSS buffer containing 0.5 mM EGTA and 25 mM HEPES, pH 7.4), followed by
digestion medium (DMEM-low glucose supplemented with 200 mg/L CaCl2,
1% penicillin/streptomycin, 15 mM HEPES and 100 U/mL collagenase IV). The
digested liver was excised, diced in digestion medium and filtered through a 70
μm cell strainer. Isolated hepatocytes were washed twice and plated onto
collagen-coated culture plates with isolation medium (DMEM/F12 GlutaMAX
supplemented with 10% FBS, 1% penicillin/streptomycin, 1 μM dexamethasone
and 0.1 μM insulin) for one hour. The medium was replaced with culture
medium (DMEM-low glucose containing 10% FBS, 1% penicillin/streptomycin, 0.1
μM dexamethasone and 1 nM insulin). After 3 hours, hepatocytes were
maintained in culture medium without FBS overnight until treatment with 100
μM Cirazoline (varying time durations as indicated in the figure) or 500
μM AICAR (15 min). To test inhibitors, hepatocytes were pre-treated with
25 μM YM254890, 5 μM U73122, 5 μM U73343 or 5 μM
STO-609 for 30 min, followed by 100 μM Cirazoline treatment for 15 min
before harvested for immunoblotting.
Immunoblotting.
Mouse primary cells were lysed in ice-cold RIPA buffer (50 mM Tris-HCl,
pH 7.5, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM
phenylmethylsulfonyl fluoride) supplemented with a protease inhibitor cocktail
(Roche) and phosphatase inhibitors (10 mM NaF, 60 mM β-glycerolphosphate,
pH 7.5, 2 mM sodium orthovanadate and 10 mM sodium pyrophosphate). Proteins were
separated by SDS-PAGE and transferred to nitrocellulose membranes. The blots
were probed with the following primary antibodies obtained from Cell Signaling
Technology: phospho-AMPKαT172 (#2531), AMPKα (#2532),
GAPDH (#5174) and HSP90 (#4874).Total proteins from ~120 day-1 adult worms grown on OP50 bacteria at
20°C were extracted with 120 μl RIPA lysis buffer supplemented
with protease inhibitor cocktail (Roche) and phosphatase inhibitors
(Sigma-Aldrich) by ultrasound sonication. The samples were then heated to
100°C for 10 min, followed by high-speed centrifugation (13000 rpm,
4°C, 5 min). Proteins were separated by SDS-PAGE and transferred to
nitrocellulose membranes, which were probed with the following primary
antibodies obtained from Cell Signaling Technology:
phospho-AMPKαT172 (#2531), β-actin (#4970). The
phospho-AMPKαT172 antibody recognizes the phosphorylated
form of C. elegansAAK-2/AMPK [50]. As AMPKα (#2532) antibody does
not recognize C. elegansAAK-2/AMPK, we used β-actin as
a control for protein loading.
Intestinal barrier function assay
Worms were cultured at 25°C and were removed at different days
from NGM plates and incubated for 3 hours in M9 medium containing fresh
E.coli OP50 (OD = 0.5–0.6) mixed with 5% FD&C
blue No.1 dye (Erioglaucine disodium salt, Sigma-Aldrich). Subsequently, worms
were collected and washed with fresh M9 buffer for 3 times and transferred to
fresh NGM plates with fresh OP50 bacteria. Next, worms were immobilized on an
agarose pad with 5 mM sodium azide, and images were acquired on an Olympus
upright microscope with a digital camera (Canon). We quantified the percentage
of worms with body-cavity leakage, characterized by the presence of the dye
outside the intestine.
Statistics and reproducibility
Samples were randomized and treated under the same conditions. The
sample sizes were not pre-determined with a statistical method, but they are
similar to those reported previously[46,51,52]. The number of independent replicates
was indicated in Supplemental
Table 1 for lifespan assay. For other assays, experiments were
repeated independently at least twice with similar results. Data collection and
analysis were not performed blindly. We assumed data distribution to be normal,
but did not test it formally. No data were excluded from the analysis.
Quantification and statistical parameters were indicated in the legends of each
figure or directly marked in the figures, including the statistical method,
error bars, n numbers, and p values. We applied one-way ANOVA, two-way ANOVA,
student`s t-test and Log-Rank test to determine statistical significance.
Specifically, for those analyses involving multiple group comparisons, we
applied one-way ANOVA followed by a post hoc test (Bonferroni test). In the case
of factor analysis (Fig. 4l), we applied
two-way ANOVA followed by a post hoc test (Bonferroni test). For those only
involving two groups, we applied two-tailed student`s t-test. Life span
comparisons were calculated by Log-Rank test. P values less than 0.05 are
considered statistically significant. One-way or two-way ANOVA tests were
performed using Prism 8 (GraphPad Software). Two-tailed student`s t tests were
performed using Excel (Microsoft Office). All Lifespan analyses were performed
using SPSS Statistics software (IBM, Inc).
Reporting Summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this article.
Additional data related to food odor suppression of DR longevity without
increasing food ingestion.
(a-b) Food odors do not change the pumping rate of
worms under DR. Pumping rate was counted at 24 hr (a), and 96
hr (b), after worms were transferred to the DR assaying plates.
2xDR: twice amount of bacterial food, which stimulated the pumping rate.
n = 30 (DR), 30 (DR + odor), 16 (2xDR) and 16 (2xDR + odor)
biologically independent animals in (a). n = 30
(DR), 30 (DR + odor), 15 (2xDR) and 14 (2xDR + odor) biologically
independent animals in (b). Data are presented as
mean ± s.e.m. p values were calculated with one-way
ANOVA with Bonfferronìs test. See Fig.1c for data of the pumping rate counted at 1 hr
post-transfer. (c) Feeding worms with twice amount of bacteria
food (2x DR) does not affect DR longevity, but food odors can still suppress
the lifespan of these worms.
Additional data related to the olfactory circuit.
a) ADF dendrite, soma and axon from DR, but not AL
worms, all respond robustly to medium containing food odors. GCaMP6f was
expressed as a transgene in ADF neurons under tph-1 long
promoter. DsRed was co-expressed to enable ratiometric imaging. Shades along
the traces represent error bars (SEM).(b) NSM neurons (dendrite/soma/axon) from DR and AL
worms do not respond to medium containing food odors. GCaMP6f was expressed
as a transgene in the NSM neurons using tph-1(s) promoter.
DsRed was co-expressed to enable ratiometric imaging. Shades along the
traces represent error bars (SEM).(c) Bar graph summarizing data in (a) and
(b). n=11 (ADF dendrite - AL), 10 (ADF dendrite - DR), 8
(ADF soma - AL), 9 (ADF soma - DR), 11 (ADF axon - AL), 10 (ADF axon - DR),
11 (NSM soma - AL), 14 (NSM soma - DR), 13 (NSM processes - AL), and 10 (NSM
processes - DR) biologically independent animals.(d) CEP dendrite from DR worms, but not AL worms,
responds robustly to medium containing food odors. GCaMP6f was expressed as
a transgene in CEP neurons using dat-1 promoter. DsRed was
co-expressed to enable ratiometric imaging. Shades along the traces
represent error bars (SEM). (e) CEP soma and axon from both DR
and AL worms respond to medium containing food odors, showing no specificity
towards DR. Shades along the traces represent error bars (SEM).
(f) Bar graph summarizing data in (d) and
(e). n=6(CEP dendrite - AL), 8(CEP dendrite - DR), 13(CEP
soma - AL), 20(CEP soma - DR), 12(CEP axon - AL) and 17(CEP axon - DR)
biologically independent animals. (g-h) ADE and PDE neurons
(soma and processes) from DR and AL worms do not respond to medium
containing food odors. GCaMP6f was expressed as a transgene in ADE and PDE
neurons using dat-1 promoter. DsRed was co-expressed to
enable ratiometric imaging. Shades along the traces represent error bars
(SEM). (i) Bar graph summarizing data in (g) and
(h). n=11 (ADE soma - AL), 10 (ADE soma - DR), 13 (ADE
processes - AL), 15 (ADE processes - DR), 10 (PDE soma - AL), 11 (PDE soma -
DR), 10 (PDE processes - AL) and 11 (PDE processes - DR) biologically
independent animals. (j) RIC soma and processes from DR worms,
but not AL worms, respond robustly to medium containing food odors. GCaMP6f
was expressed as a transgene in RIC neurons under tbh-1
promoter. DsRed was co-expressed to enable ratiometric imaging. Shades along
the traces represent error bars (SEM). The soma curves are duplicates of
those presented in Fig.2i, as these experiments were performed at
the same time. (k) RIM soma and axon from both DR and AL worms
respond to medium containing food odors, showing no specificity towards DR.
GCaMP6f was expressed as a transgene in RIM neurons using
cex-1 promoter. DsRed was co-expressed to enable
ratiometric imaging. Shades along the traces represent error bars (SEM).
(l) Bar graph summarizing data in (j) and
(k). n=11 (RIC soma - AL), 14 (RIC soma - DR), 11 (RIC
processes - AL), 12 (RIC processes - DR), 12 (RIM soma - AL), 12 (RIM soma -
DR), 12 (RIM processes - AL) and 10 (RIM processes - DR) biologically
independent animals. (m-n) RNAi knockdown of
odr-3 and ocr-2 specifically in ADF
neurons eliminates food odor-evoked calcium responses in these neurons.
dsRNA against odr-3 and ocr-2 gene was
expressed as a transgene specifically in ADF neurons using the
bas-1(prom7) promoter. (m) Calcium imaging
traces. Shades along the traces represent error bars (SEM). (n)
Bar graph summarizing the data in (m). n=10 (WT - AL), 9 (WT - DR), 10
(ADFodr-3(RNAi) - AL), 10 (ADFodr-3(RNAi) - DR), 10 (ADFocr-2(RNAi) - AL)
and 10 (ADFocr-2(RNAi) - DR) biologically independent
animals. (o) NSM neurons are not required for food odors to
suppress DR longevity. tph-1(s) promoter was used to drive
the expression of TeTx transgene specifically in NSM
neurons.(p) Blocking the output of RIC neuron shortens DR
longevity. tbh-1 promoter was used to drive the expression
of TeTx transgene in RIC neurons. Data are presented as
mean ± s.e.m. p values in c,
f, i and l: two-tailed student`s
t test. P values in n: one-way ANOVA with Bonfferronìs
test.
Other serotonin receptors are not required for food odors to suppress DR
longevity.
Food odors can still suppress DR longevity in ser-1
(a), ser-4 (b),
ser-7 (c) and mod-1
(d) mutant worms. (a-d) share the same control
group, as these experiments were performed at the same time
Other dopamine receptors are not required for food odors to suppress DR
longevity.
Food odors can still suppress DR longevity in dop-1
(a), dop-2 (b),
dop-3 (c) , dop-4
(d) and dop-5 (e) mutant worms.
(a) and (c) share the same control group, as
these experiments were performed at the same time. (b),
(d) and (e) share the same control group, as
these experiments were performed at the same time.
Additional data related to regulation of DR longevity by AMPK and
octopamine signaling.
a) DR can extend the lifespan of
raga-1 mutant worms. (b) Food odors can
suppress DR longevity in raga-1 mutant worms.
(c) DR can extend the lifespan of rict-1
mutant worms. (d) Food odors can suppress DR longevity in
rict-1 mutant worms. (e) Pan-neuronal
expression of aak-2 gene only has a slight rescue effect on
the longevity defect of aak-2 mutant worms. This
aak-2 neuronal transgene also does not rescue the odor
sensitivity defect of aak-2 mutant worms.
rgef-1 promoter was used to drive the expression of
aak-2 cDNA in neurons. (f-g) Food odors
can still suppress DR longevity in ser-6 (f)
and octr-1 (g) mutant worms. (f)
and (g) share the same control group, as these experiments were
performed at the same time. (h) Intestine-specific knock-down
of par-4/LKB1 by dsRNA transgene
(Pges-1::par-4(RNAi)) does not prevent food odors from
suppressing DR longevity, though it partially inhibits DR longevity.
(i) Intestine-specific knock-down of
mom-4/TAK1 by dsRNA transgene
(Pges-1::mom-4(RNAi)) does not prevent food odors from
suppressing DR longevity; nor does it affect DR longevity.(j-l) Mutations in egl-30
(j), egl-8 (k), and
ckk-1 (l) abolish the ability of food
odors to suppress DR longevity, a defect that is rescued by transgenic
expression of corresponding wild-type genes in the intestine using
ges-1 promoter.
(a) Lifespan extension mediated by intestinal
expression of aak-2 requires daf-16.
daf-16 RNAi blocked the lifespan-extension effect of
the intestinal aak-2 transgene. (b-d)
Intestinal expression of aak-2 promotes
sod-3 gene expression in multiple tissues in a
daf-16-dependent manner. sod-3::gfp is
a transgene reporting the expression level of sod-3 gene.
(b) Sample images showing a low level of
sod-3::gfp expression. Left: bright field image. Right:
fluorescent image. (c) Sample images showing that the
Pges-1::aak-2 transgene increased the expression of
sod-3::gfp. Top left: bright field image. Top right:
fluorescent image. Bottom: zoomed-in images showing
sod-3::gfp expression in multiple tissues, including
pharynx (head), neurons (head), body-wall muscles, vulval muscles
(mid-body), intestine, etc. Scale Bar: 100 μm. (d) Bar
graph summarizing the data in (b) and (c). n=24
(WT), 20 (Pges-1::aak-2), 43
(daf-16(RNAi)) and 22 (daf-16(RNAi);
Pges-1::aak-2) biologically independent animals. Data are
presented as mean ± s.e.m. p values were calculated
with one-way ANOVA with Bonfferronìs test.
TQ3030
N2 (Wild-type)
TQ4931
tph-1(mg280) II
TQ4935
cat-2(e1112) II
TQ4927
tbh-1(n3247) X
TQ4929
tdc-1(n3419) II
TQ4933
eat-4(ky5) III
TQ6452
unc-17(e245) IV
TQ4168
unc-25(e156) III
TQ1280
unc-31(e169) IV
TQ9677
xuEx255a[Ptph-1(L)::GCaMP6f+Ptph-1(L)::mcherry2]
TQ9984
xuEx3388[Pdat-1::myr-GCaMP6f+Pdat-1::mcherry2]
TQ10132
xuEx3312[Ptbh-1::GCaMP6f+Ptbh-1::mcherry2]
TQ10049
xuEx255a[Ptph-1(L)::GCaMP6f+Ptph-1(L)::mcherrry2];
unc-13(e51) I
TQ10046
xuEx255a[Ptph-1(L)::GCaMP6f+Ptph-1(L)::mcherry2];
unc-31(e169) IV
TQ10047
xuEx255a[Ptph-1(L)::GCaMP6f+Ptph-1(L)::mcherry2];
cat-2(e1112) II
TQ10042
xuEx255a[Ptph-1(L)::GCaMP6f+Ptph-1(L)::mcherry2];
tbh-1(n3247) X
TQ10003
xuEx3388[Pdat-1::myr-GCaMP6f+Pdat-1::mcherry2];
tph-1(mg280) II
Authors: Eric L Greer; Dara Dowlatshahi; Max R Banko; Judit Villen; Kimmi Hoang; Daniel Blanchard; Steven P Gygi; Anne Brunet Journal: Curr Biol Date: 2007-09-27 Impact factor: 10.834
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Authors: Christi M Gendron; Tuhin S Chakraborty; Brian Y Chung; Zachary M Harvanek; Kristina J Holme; Jacob C Johnson; Yang Lyu; Allyson S Munneke; Scott D Pletcher Journal: Annu Rev Physiol Date: 2019-10-21 Impact factor: 22.163
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Fig.1 |
Extended Data Fig. 1
Fig.2 |
Extended Data Fig. 2
Fig.3 |
Extended Data Fig. 3
Extended Data Fig. 4
Fig.4 |
Extended Data Fig. 5
Extended Data Fig. 6
Fig. 5 |
Fig.6 |