Oncotarget, Advance Publications 2015

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Rapamycin preserves gut homeostasis during Drosophila aging

Xiaolan Fan1,*, Qing Liang1,*, Ting Lian1, Qi Wu1, Uma Gaur1, Diyan Li1, Deying
Yang1, Xueping Mao1, Zhihua Jin2, Ying Li1 and Mingyao Yang1
1

Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University,
Chengdu, P.R. China
2

School of Biotechnology and Chemical Engineering, Ningbo Institute of Technology, Zhejiang University, Zhejiang, P.R.
China
*

These authors have contributed equally to this work

Correspondence to: Mingyao Yang, email: [email protected]

Keywords: Drosophila, gut homeostasis, intestinal stem cell, rapamycin, aging, Gerotarget
Received: September 15, 2015

Accepted: September 22, 2015

Published: September 29, 2015

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and source are credited.

ABSTRACT
Gut homeostasis plays an important role in maintaining the overall body health
during aging. Rapamycin, a specific inhibitor of mTOR, exerts prolongevity effects
in evolutionarily diverse species. However, its impact on the intestinal homeostasis
remains poorly understood. Here, we demonstrate that rapamycin can slow down
the proliferation rate of intestinal stem cells (ISCs) in the aging guts and induce
autophagy in the intestinal epithelium in Drosophila. Rapamycin can also significantly
affect the FOXO associated genes in intestine and up-regulate the negative regulators
of IMD/Rel pathway, consequently delaying the microbial expansion in the aging guts.
Collectively, these findings reveal that rapamycin can delay the intestinal aging by
inhibiting mTOR and thus keeping stem cell proliferation in check. These results will
further explain the mechanism of healthspan and lifespan extension by rapamycin
in Drosophila.

INTRODUTION

homeostasis is shown to play a key role in lifespan

determination in Drosophila [13-15]. Tissue homeostasis
in the midgut is maintained by multipotent intestinal stem
cells (ISCs), which are distributed along the basement
membrane [16-18]. Division of an ISC gives rise to
one daughter cell that retains stem cell fate and another
daughter cell that becomes an enteroblast (EB), both
expressing a transcription factor called Escargot (esg).
Thus, expression of esg is often used as a surrogate
marker for ISCs and EBs. After ISC division, the
daughter EB does not divide again and differentiates
into either a large, polyploid enterocyte (EC) or a
small, diploid entero-endocrine (ee) cell. In response to
stress conditions, however, ISC proliferation is strongly
increased, a regenerative response that allows restoring
large parts of the intestinal epithelium in response to
damaging agents, such as pathogens, genotoxins, or
ROS inducing compounds [19-21]. This regenerative
function of ISCs have deleterious consequences for the
organism, as excessive proliferation in response to stress
is accompanied by the accumulation of mis-differentiated

Lifespan in many organisms can be extended

by improving genetic factors, for example reducing
activity of the mechanistic target of rapamycin (mTOR)
[1] and Insulin/IGF1 signaling (IIS) [2-4], as well as
environmental conditions, such as dietary restriction [57] and decreasing oxidative stress [8,9]. The different
mechanisms are likely to act through related processes,
particularly by increasing stress-protective gene
expression in differentiated somatic cells, prolonging
their functional lifespan and delaying tissue deterioration
[10-12]. In addition to such stress protective mechanisms,
metazoans also maintain tissue homeostasis through
regenerative processes that mainly rely on the long-term
maintenance of a functional population of somatic stem
and progenitor cells. For these cells, relationship between
stress protection and lifespan is expected, as their longterm maintenance is critical to conserve regenerative
capacity [13].
In recent years, maintenance of intestinal
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cells in the intestine, which ultimately disrupts epithelial

integrity with a dysplastic phenotype [22]. In the aging
gut, such dysplasia is widely observed under normal
culture conditions, suggesting that an age-related overproliferation of ISCs contribute to the loss of intestinal
function and to the increased mortality of aged flies [23,
24]. ISC self-renewal and differentiation is controlled by
the Notch and mTOR signaling pathways. The long-term
stem cell maintenance is further ensured by mechanisms
that prevent activation of mTOR signaling [25-27].
Rapamycin is the most specific TOR inhibitor
known and it acts through association with the
intracellular protein FKBP12, which binds to the FKBP12rapamycin-binding (FRB) domain of TOR, inhibiting
TORC1 activity. Although rapamycin does not bind to the
catalytic domain of TOR, it reduces phosphorylation of
two downstream TORC1 targets, S6K and 4E-BP [1]. The
effect of rapamycin on lifespan extension has been studied
in many species but its effect on gut homeostasis is not
fully elucidated.
In order to examine whether rapamycin can preserve
gut homeostasis during aging, we used Drosophila
intestine as an accessible model system. Our results
revealed a significant correlation between rapamycin
intake and slowing down of the intestinal aging. Most
importantly, we showed that rapamycin limits the
proliferation rates of intestinal stem cells by moderately
inhibiting mTOR leading to delay in the microbial
expansion during gut aging. Our findings demonstrate that
maintenance of the guts homeostasis during aging could
be one of the important effect of rapamycin that extends
lifespan in Drosophila.

found that the proliferation of ISCs in the guts of young

flies (3 days old), is maintained at low level in both control
and rapamycin groups (Figure 1B-1C). Whereas in aging
flies guts (20 Days old) there was a significant decrease in
the number of GFP and Delta positive cells in rapamycin
treated group when compared to the control group (Figure
1D-1F). The result showed that addition of rapamycin in
the food can slow down the proliferation rate of ISCs in
the aging guts and therefore can contribute towards the
lifespan extension in Drosophila.

Rapamycin slows down the intestinal barrier

dysfunction and activates autophagy in the aging
guts
Flies with intestinal barrier dysfunction display
increased expression of antimicrobial peptides (AMPs),
impaired IIS and reduced metabolic stores compared with
age-matched flies without intestinal barrier defects [14].
We tested the barrier dysfunction display to highlight
the intestinal aging at the tissue level. Loss of intestinal
integrity can be assayed in living flies by monitoring the
presence of non-absorbed dye (FD&C blue no. 1) outside
of the digestive tract post feeding [30]. As expected,
we observed that in young flies (10 days old) the dye is
restricted to the proboscis and digestive tract after feeding
with FD&C blue no. 1 (Figure 2A), however, in aged flies
(35 days old) we observed that a fraction of flies displayed
a strikingly different phenotype. In these flies, the blue
dye was clearly visible throughout the body after feeding;
subsequently, these flies were referred to as Smurf flies
(Figure 2B). In control flies, the percentage of Smurf
flies in the population increases dramatically with age,
from 0% at day 10 to 23% at day 50. Interestingly, flies
upon rapamycin treatment retards the age-related onset
of the Smurf phenotype (Figure 2C). Therefore,
rapamycin improved intestinal integrity in aged flies,
which is consistent with the delay in disruption of apical
basal polarity in intestines from aging flies [14].
It is previously reported that the intestinal stem cell
population in the midgut of catalase mutant flies was
increased compared to controls [31]. Catalase is a very
important enzyme in protecting the cell from oxidative
damage by reactive oxygen species (ROS). We also
tested the catalase expression level in the aging guts
and the result showed that the catalase is significantly
over-expressed in rapamycin treated group (Figure 3A).
This suggests that rapamycin can improve the intestinal
capacity for antioxidants. We also assayed markers of
autophagy in the intestine upon rapamycin treatment.
Interestingly, we observed increased mRNA levels of
Atg1, Atg5, and Atg8b in intestinal tissue (Figure 3B-3D),
which indicates that rapamycin can induce autophagy and
that may play a role in lifespan extension|.

RESULTS
Rapamycin slows ISCs proliferation rate and
extends lifespan
The addition of rapamycin in dose 200M in the
food extended Drosophila lifespan in our study (Figure
1A), which is similar to the previous report [1]. Whether
rapamycin exerts an effect on lifespan extension by
maintaining the intestinal homeostasis remains to be
explored. To test this hypothesis, we first assessed the
relationship between the proliferation rate of ISCs and
lifespan in the aging guts in presence of rapamycin.
We used a heat-inducible system in which esg-Gal4 is
combined with a temperature-sensitive Gal80 (TARGET
system), and the flies were maintained at 25 oC until the
day before dissection and shifted to 29 oC for 24h, to
allow expression of the GFP in ISCs and EBs. Only GFP
positive cells can show the ISC proliferation rates as esg
is specifically expressed in ISCs and EBs whereas Delta
specific expression is shown only by ISCs in intestine. We
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Figure 1: Rapamycin slows ISCs proliferation rates. A., Rapamycin treatment extends the life span of wDah females. Compared to

flies on control food (0M of rapamycin), flies on food having 200M of rapamycin have increased median life spans (T test p < 0.0001).
B and C, esg-positive (esg+) cells (green) in control B., and rapamycin treated C., young (3 Days old) fly guts. D.-D, esg+cells (green) and
ISCs (by Delta) in control aging (20Days old) fly guts. E.-E, esg+cells (green) and ISCs (by Dl) in rapamycin treated aging (20Days old)
fly guts.F., Quantification of midgut GFP cells in control and rapamycin treated 20Days old guts. MeanSD is shown. n = 8-10 guts. **p
< 0.01.

Figure 2: Rapamycin slows down the intestinal barrier dysfunction. A., A 10 days old fly after consuming a non-absorbed food

dye (FD&C blue dye #1). The dye is restricted to the proboscis and digestive tract. B., A 40 days old aged Smurf fly after consuming the
same food dye. The blue dye is seen throughout the body due to loss of intestinal integrity. C., Analysis of intestinal integrity as a function
of age. In control flies, the percentage of Smurf flies in the population increases with age. Rapamycin pretreatment improves intestinal
integrity in aged flies. (***p < 0.001, n >60 females for each genotype), Data are represented as mean SEM.
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Effect of rapamycin treatment on FOXO

associated genes in the intestine

activate AMP genes through both Rel/NFkB-dependent

and independent mechanisms [30-33]. For further
uncovering the mechanism of rapamycin induced slow
down of the intestinal aging, we detected the RNA
expression level of diptericin (dpt, one of AMPs genes) in
the aging guts. The result showed that, the dpt expression
level in the aging intestinal tissue decreased four folds in
rapamycin treatment group when compared to the control
(Figure 4C). We also tested the dual oxidase (Duox) gene
which is associated with ROS level and is a likely cause
of age-related loss of epithelial homeostatis [23]. In aging
fly intestine the duox expression level is significantly
decreased when the rapamycin is added (Figure 4D),
indicating that rapamycin may reduce the intestinal ROS
accumulation in the aging flies, which means lower level
of ROS accumulations are helpful in maintaining the
epithelial homeostasis.

Rapamycin induces the phosphorylation of FOXO1

without any significant change in the RNA expression
level. Rapamycin can elevate the expression levels
of Insulin receptor (Inr) [32], which is present at the
beginning of Insulin pathway and can repress FOXO
phosphorylation by AKT1. When we checked the Foxo
RNA expression level in aged guts by qRT-PCR, we
didnt find any significant change (Figure 4A), whereas
rapamycin treatment resulted in significantly elevated Inr
expression (Figure 4B).
Intestinal barrier dysfunction display is always
accompanied by increased AMPs expression level during
the aging process. IIS-regulated Foxo can transcriptionally

Figure 3: Rapamycin activating the autophagy in the aging guts. A., Intestinal expression of antioxidant gene catalase in 30

days old female fly guts. B.-D., Expression levels of autophagy genes in 30 days old female fly guts. (*p < 0.05, ** p < 0.01,***p < 0.001,
n = 3 of RNA extracted from 7 intestines/replicate).
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Rapamycin treatment delayed the microbiota

intestinal dysplasia by up-regulating the negative
regulators of IMD/Rel pathway

to the control at 7 days of age, the number of CFUs in

the gut of flies increased exponentially for all analyzed
bacterial phylotypes at 40 days of age. While there was
not any obvious gut size change observed after longterm rapamycin treatment, addition of rapamycin in the
food can significantly reduce the number of CFUs for all
analyzed bacterial phylotypes at 40 days of age (Figure
5A), indicating that rapamycin can delay the microbial
expansion in the aging guts.
At the same time management of the commensal
flora and innate immune responses to pathogens are
achieved primarily in Drosophila ECs by activation of
the immune deficiency (IMD/Relish) pathway, which
activates the NFkB-like transcription factor Relish (Rel).
We found that the Rel activity in the rapamycin treated gut
is lower than the control fly gut (Figure 5B). In order to

Duox is transcriptionally induced in ECs and

activated in response to a microbial challenge [34].
We found that the duox expression level is reduced in
the rapamycin treated aging guts (Figure 4D). We first
confirmed that age-related dysplasia was associated with
increased microbial loads in control flies. We quantified
overall microbial load by measuring colony-forming
units (CFUs) in dissected guts of young and aging flies,
using selective plates to identify Enterobacteriaceae,
Lactobacillae, Acetobacteriaceae and other bacteria
growing on nutrient-rich medium. When compared

Figure 4: Rapamycin treatment impacts FOXO associated genes in the intestine. A., Expression levels of gene Foxo in

intestines of 30 days old flies at control and rapamycin induction. B., Expression levels of insulin receptor (InR) in intestines of 30 days old
flies at control and rapamycin induction. C., Expression levels of AMP gene Dpt in intestines of 30 days old flies at control and rapamycin
induction. D., Duox mRNA expression were quantified by RT-PCR in intestines of 30 days old flies at control and rapamycin induction.
(***p < 0.001, *p < 0.05).
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assess whether rapamycin delay the microbial expansion

in the aging guts, we examined the gene expression of
the negative regulators of IMD/Rel pathway, Caudal,
USP36, PGRPs-SC, which are important in maintaining
homeostasis. The result showed that they all are
significantly up-regulated (Figure 5C-5E), suggesting that
rapamycin can up-regulate the negative regulators of IMD/
Rel pathway therefore keeping the immune homeostasis
and delaying the microbial expansion in the aging guts.

Stem cell aging is a cause of organismal aging

especially in high-turnover tissues such as animal
intestine and dysregulation of such processes is likely
to promote cancer or degenerative diseases. According
to the previous reports, limited rate of ISC proliferation
in the aging intestine can extend the lifespan [13-15,
30]. We hypothesize that rapamycin can slow the ISCs
proliferation rates and ultimately promote longevity.
Evidently ISCs become dysregulated in aging flies,
over proliferate and produce a large number of cells
that undergo incomplete differentiation [20]. This
triggers intestinal dysplasia, which is characterized by
loss of apico-basal organization of the epithelium and
accumulation of mis-differentiated cells at the basement
membrane [14]. In our results the number of ISCs and
EB cells are significantly less in rapamycin treated aging
guts in comparison to age-matched control flies. During
aging the TORC1 hyperactivation leads to ISCs lost,
but treatment with rapamycin in adult stage may help to
maintain the ISCs [26].
A microbial challenge in the gut will induce the
duox expression in ECs [34]. In our study we found that

DISCUSSION
By using Drosophila as a model, we have shown
that rapamycin can slow down the proliferation rate
of intestinal stem cells (ISCs) in the aging guts; induce
autophagy in the intestinal epithelium; affect the FOXO
associated genes in intestine; and up-regulate the negative
regulators of IMD/Rel pathway. The results uncovered that
rapamycin can delay the intestinal aging by keeping the
proliferation of the stem cells in control. Possibly it could
be one of mechanisms underlying extension of lifespan in
Drosophila.

Figure 5: Rapamycin treatment delayed the commensal intestinal dysplasia by up-regulating negative regulators of
IMD/Rel pathway. A., CFUs in intestinal extracts of control and rapamycin treated flies. Midgut homogenates from flies at 7 days or

40 days of age were plated on nutrient-rich medium (NR) or on selective plates allowing growth of Lactobacilli (LMRS), Acetobacteria
(ACE), or Enterobacteria (ENT). B., qRT-PCR for rel expression C.-E., qRT-PCR for pgrp-sc1, pgrp-sc2, caudal and usp36 expression (n
= 3; *p < 0.05,**p < 0.01,***p < 0.001).
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MATERIALS AND METHODS

duox expression level is reduced in the rapamycin treated

aged guts which indicates that rapamycin is delaying
the microbial expansion during gut aging process. The
age-related decline of proliferative homeostasis seems
to be fundamentally a consequence of dysregulation of
the interaction between the intestinal epithelium and the
commensal bacterial population, resulting in commensal
dysbiosis [15]. Drosophila lifespan is affected by
intestinal dysplasia, and the dysplasia can be prevented by
maintaining flies axenically. An age-related impairment in
the ability to manage the intestinal microflora thus appears
to be the underlying cause of intestinal dysplasia, limiting
lifespan [15]
In the aging intestine of Drosophila, chronic
activation of the transcription factor Foxo reduces
expression of the IMD/Rel pathway negative regulators
(PGRP-SC et al) [15]. Rapamycin inhibits the TOR
pathway thereby the negative feedback loop is not
effective, which activates the PI3K/Akt signaling
pathway and up-regulates the IRS-1. The Akt1 can induce
the phosphorylation of FOXO1 and prevent its entry
to the nucleus, that can reduce the chronic activation
of FOXO [35, 36]. In the aging guts this repression
causes dysregulation of Rel/NFkB activity, resulting
in commensal dysbiosis. We observed the InR, caudal,
usp36 and pgrp-sc1, are significantly up-regulated in the
rapamycin treated aged guts. It seems that rapamycin may
improve innate immune homeostasis to stabilize the gut
microbiota that help to maintain the gut homeostasis and
improve health and lifespan.
In the present study we found that the AMP gene
dpt expression level is significantly decreased and the
intestinal dysfunction is delayed in the aged flies gut.
Intestinal barrier dysfunction plays a critical role in the
etiology and/or mortality associated with many age-related
diseases [37-41]. Flies with intestinal barrier dysfunction
displayed increased expression of AMPs, impaired IIS and
reduced metabolic stores compared to age-matched flies
without intestinal barrier defects [14].
Here we have shown that rapamycin treatment
preserves gut homeostasis during the Drosophila aging,
which contributes to the healthspan and lifespan extension.
Due to the high degree of evolutionary conservation in the
mTOR pathway, we expect that the mechanism underlying
rapamycin treatment for maintaining gut homeostasis
could apply in other species as well. Future studies to
investigate the effects of rapamycin in disease model
systems may reveal potential common therapies for a wide
range of age-related ailments.

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Fly stocks and husbandry

The wild-type stock Dahomey was collected in 1970
in Dahomey (now Benin) and has since been maintained
in large population cages with overlapping generations on
a 12L:12D cycle at 25oC. esgGal4, UAS-GFP, tub Gal80ts
(gift from N. Perrimon). All stocks were maintained and
experiments were conducted at 25oC on a 12 hr:12 hr
light:dark cycle at constant humidity using standard sugar/
yeast/agar (SYA) media [28]. For all experiments, flies
were reared at standard larval density and eclosed adults
were collected over a 12 hr period. Flies were mated for
48 hr before sorting into single sexes.

Lifespan analysis
Flies that eclosed over a 36 hour period were
collected and allowed to mate for approximately 60 hours.
Females were randomly allocated to the experimental food
treatments and housed in plastic vials containing food at
a density of 10 flies per vial, with 10 vials per condition
(n = 100). Flies were transferred to a fresh food source
3 times per week, during which any deaths and censors
were recorded. Rapamycin (Sigma) was dissolved in
ethanol and added to SYA food at concentrations 200m.
For control food ethanol alone was added.

Immunostaining and fluorescence microscopy

In all experiments, only the female posterior midgut
was analyzed. The immunostaining of intestines was
performed as previously described [29]. The primary
antibodies used were mouse mAb anti-Dl (C594.9B, 1:50).
All images were captured by a Zeiss LSM780 inverted
confocal microscope and processed in Adobe Photoshop
and Illustrator.

Analysis of gene expression

Total RNA from 7 guts, were extracted using
Trizol and cDNA was synthesized using Superscript II
(Invitrogen). Real time PCR was performed in triplicates
using SYBR Green, on Biorad IQ5 instrument and the
following primer pairs were used.
(Actin5C-5CTCGCCACTTGCGTTTACAGT,
Actin5C-3TCCATATCGTCCCAGTTGGTC.
catalaes-5ATGCGGCTTCCAATCAGTTGAT,
catalaes-3CGAAGTGCGACATCTCATCCA.
Atg1-5GCCAGCTCCATCGAAAATAACC,
Atg13GCGGCGCAGCAGGCACAG.
Atg5-5GCCCCTGCGACTTCACTATCC, Atg5-3
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CCATTAAATCGGCCAAACTCTTCT.
Atg8b-5AATGTGATCCCACCGACATC, Atg8b3TTGAGCGAGTAGTGCCAATG.
Dpt-5
GGCTTATCCGATGCCCGACG, Dpt3
TCTGTAGGTGTAGGTGCTTCCC.
Duox-5
GTCGCACGCCAACCACAAGAGACT,
Duox3CACGCGCAGCAGGATGTAAGGTTT.
Foxo-5 TGTCGCTGCACAACCGCTTTATGA,
Foxo-3TTGCCGGAAATCGGGCGATAATTG.
InR-5
CATCGGAAGGGAGGCGTAA, InR3CGTTTGCCTAATCGTCGAACA.
Rel5
ACAGCTACAGGAACTGCATCAGGAA, Rel3TCATCCTCCTCGAAGAACCTCACT.
Pgrp-sc2-5 AACTACCTGAGCTACGCCGTGAT,
Pgrp-sc2-3
AGCAGAGGTGAGGGTGTTGGTATT.
Pgrp-sc1-5 CTATGTCGTCTCCAAGGCGGAGT,
Pgrp-sc1-3
CGATCAGGAAGTTGTAGCCGATGT.
cad-5
CGCCATCGAAGCCGCCATAC, cad3
ACCGCCGCCAAGGAGTGGCT.
Usp36-5 GCCAAGAGCGGCGAGGACAC,
Usp36-3CAATTGGCCAGGGCGGGTATG.).
Fold changes in expression were calculated using
the 2-Ct method and normalized to actin (house keeping
gene) levels. Results are average+/-standard deviation of
at least 3 independent biological samples run in triplicates.

All media were autoclaved at 121 degree for 15 min.

ACKNOWLEDGMENTS
We thank all lab members for invaluable comments
on the manuscript. We thank Prof. N. Perrimon for
providing us the fly line.

GRANT SUPPORT
This work was supported by The National Natural
Science Foundation of China (31471998) and the Research
Program of Sichuan Bureau of Education (15ZB0015
,14TD0002) and the Thousand Talents Program in
Sichuan.

CONFLICTS OF INTEREST
The authors declare no conflict of interests.

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