Lipopolysaccharide inhibits hypothalamic Agouti-related protein gene
expression via activating mechanistic target of rapamycin signaling
in chicks
X.J. Wang, D. Li, H.C. Jiao, J.P. Zhao, H. Lin *
Department of Animal Science, Shandong Agricultural University, Shandong Key Lab for Animal Biotechnology and Disease Control, Taian, Shandong 271018, China
Lipopolysaccharide (LPS) induces profound anorexia in birds. However, the neuronal regulatory network underlying LPS-provoked anorexia is unclear. To determine whether any cross talk occurs among hypothalamic
mechanistic target of rapamycin (mTOR) and LPS in the regulation of appetite, we performed an intracerebroventricular injection of rapamycin (an mTOR inhibitor) on LPS-treated chicks. The results indicate that
peripheral administrations of LPS decreased the agouti-related protein (AgRP) mRNA level, but increased the
phosphorylated mTOR and nuclear factor-кB (NF-кB) protein level. Blocking mTOR significantly attenuated LPSinduced anorexia, AgRP suppression, and p-NF-кB increase. Thus, the results suggest that LPS causes anorexia via
the mTOR-AgRP signaling pathway, and mTOR signaling is also associated with the regulation of LPS in p-NF-кB.
1. Introduction
Lipopolysaccharide (LPS) is one of the major components of the cell
wall of gram-negative bacteria. It is released in the infected host and
stimulates an immune response. LPS is commonly used to experimentally induce the acute phase response and mimic inflammation (Inui,
2002). In mammals, both peripheral and central administrations of LPS
or proinflammatory cytokines cause a reduction in food intake (Inui,
2001; Inui, 2002; Plata-Salaman, 2001). The reduction in food intake
accompanies infection likely results from interactions between LPS/cytokines and brain regions that control food intake (Druce and Bloom,
2003). Hypothalamus has a major role in the regulation of feeding
behavior (Liu et al., 2016a; Hillebrand et al., 2002) and thus represents a
likely target where cytokines influence the central nervous system
(CNS). In vertebrates, hypothalamic neurons produce important orexigenic factors (food intake stimulators) such as neuropeptide Y (NPY) and
agouti-related protein (AgRP) as well as anorexigenic factors (food
intake inhibitors) such as pro-opiomelanocortin (POMC)-derived peptides (Liu and Zhu, 2012).
Transcription factor nuclear factor-кB (NF-кB) is a key regulator of
gene encoding cytokines, cytokine receptors, and cell adhesion molecules that stimulate inflammatory responses (Hayden and Ghosh, 2008).
Peripheral LPS activates NF-кB, which results in the production of proinflammatory cytokines, such as interleukin 1 (IL-1) and tumor necrosis factor α (TNF-α) (Finck et al., 1998; Johnson, 1997). LPS modulates
the transcriptional activity of NF-кB through the phosphorylation of the
NF-кB p65 subunit (Kim et al., 2011). However, the mechanism by hypothalamic NF-кB integrates the LPS signals to regulate food intake remains unclear.
Mechanistic target of rapamycin (mTOR), an evolutionarily
conserved serine-threonine kinase, promotes anabolic cellular processes
and is in response to growth factors, nutrients (energy status, amino
acids and glucose), and stress (Biondi et al., 2004; Wullschleger et al.,
2006). mTOR signaling pathway plays a vital role in regulating cell
growth and proliferation and has been studied extensively in a variety of
metabolic and cancer models. However, mTOR signaling pathway was
implicated in the regulation of food intake in mammals. In rat hypothalamus, mTOR expresses in orexigenic NPY neurons (Inhoff et al.,
2010). Central leucine administration activates hypothalamic mTOR
signaling and decreases food intake in rats, and the inhibition of mTOR
signaling with rapamycin blunts leucine’s anorectic effect (Cota et al.,
2006). However, the information about mTOR regulating of voluntary
food intake in avian species is scarce.
In the present study, we examined the changes in appetite-related
peptides, NF-кB and mTOR in hypothalamus of broiler chicks after peripheral LPS injection and determined the relationship between mTOR
* Corresponding author at: Department of Animal Science, Shandong Agricultural University, Tai’an, Shandong 271018, China.
E-mail address: [email protected] (H. Lin).
Contents lists available at ScienceDirect
General and Comparative Endocrinology
journal homepage: www.elsevier.com/locate/ygcen
https://doi.org/10.1016/j.ygcen.2021.113876
Received 10 June 2021; Received in revised form 25 July 2021; Accepted 2 August 2021
General and Comparative Endocrinology 313 (2021) 113876
signaling and the LPS-induced anorexia by blocking mTOR.
2. Material and methods
2.1. Animals
Male broiler (Arbor Acres) chicks (avoiding the effect of estrogen)
were obtained from a local hatchery at 1 day of age and reared in an
environmentally controlled room. Temperature and lighting were
maintained according to commercial conditions. All chicks received a
starter diet containing 21.5% crude protein and 12.33 MJ/kg of
metabolizable energy. All birds had free access to food and water during
the rearing period. All study procedures were approved by the Shandong
Agriculture University Animal Care and Use Committee (SDAUA-2013-
019) and were conducted in accordance with the Guidelines for Experimental Animals established by the Ministry of Science and Technology
(Beijing, China).
2.2. Food intake measurements after LPS injection
In the previous study, intraperitoneal LPS injection of 0.5–1.5 mg/kg
BW significantly suppressed feeding behaviour in chicks (Yousefi et al.,
2021; Yang et al., 2019). In our study, chicks (10-day-old) were
randomly divided into four groups, with 8 chicks in each group, and all
chicks were fasted for 24 h before the experiment. The chicks were
injected intraperitoneally with LPS (Escherichia coli 055:B5, Sigma) at
dosages of 0, 0.5, 1, or 2 mg/kg BW using saline as vehicle, and were
then given immediate access to food. Food intake was recorded after 1,
2, 4, 6, 8, 12, 18 and 24 h. Time course was used to determine the
response to LPS treatment.
2.3. Effects of LPS injection on mTOR signaling and appetite-related gene
expression
We found that LPS treatment of 0.5, 1, or 2 mg/kg BW induced a
significant decrease in food intake in the present study. So we chose the
lowest dosage LPS administration group (0.5 mg/kg BW) to determine
the levels of the mTOR signaling and appetite-related gene expression.
The birds (10-day-old) fasted for 24 h received an intraperitoneal injection of 0.5 mg/kg BW of LPS or vehicle (saline). After continuous
fasting for 4 h, the chicks were sacrificed by exsanguination. The hypothalamus was dissected from the ventral surface of the brain. Two
transverse cuts were made at the apex of the optic chiasm and the rostral
margin of the mammillary bodies. Next, 2 mm bilateral cuts were made
on either side of the midline and the whole hypothalamus was removed
according to the method described in Yuan et al. (2009). After being
snap-frozen in liquid nitrogen, the hypothalamus tissue samples were
stored at − 80 ℃ until RNA and protein extraction.
2.4. Implantation of intracerebroventricular cannula
At 7 days of age, all chicks were anesthetized by intravenous injection in the wing vein with pentobarbital sodium at a dose of 25 mg/kg
BW. The chicks were mounted on a stereotaxic apparatus (Huaibei
Zhenghua, China), and a small incision was made in the flesh along the
midline. A thin-walled stainless steel guide cannula was stereotaxically
implanted into the third ventricle as described by Puelles et al. (2007).
The chicks were allowed at least 3 days of recovery before injection
(Dunbar et al., 1997).
2.5. Effects of mTOR signaling blockade on food intake and appetiterelated gene expression after intraperitoneal injection of LPS
After a 24 h fast, four groups of chicks (10-day-old) with 8 chicks in
each group were intracerebroventricularly (ICV) injected with rapamycin or the vehicle; rapamycin was dissolved in dimethyl sulfoxide
(DMSO) and was used at a final concentration of 20 µg/2 µL (Cota et al.,
2006) per chick. Following the infusion, the guide cannula remained
inserted for approximately 30 s to allow the drug to diffuse away from
the cannula tip. One hour later, each group of chicks were given an
intraperitoneal injection of either 0.5 mg/kg BW LPS or vehicle (saline).
The chicks were then returned to their cages, and were granted immediate access to food. The cumulative food intake was recorded at 1, 2, 4,
6, 8, 12, 18 and 24 h from food re-offered. The experiment was repeated
twice. During the second experiment, the chicks were continuously
fasted for 4 h after LPS administration, and the hypothalami were
collected for RNA and protein extraction.
2.6. RNA isolation and analysis
Total RNA extraction and qRT-PCR were performed as described
previously (Liu et al., 2014). Sequences of primers are shown in Table 1.
The PCR data were analyzed with the 2− ΔΔCT method. 18 s rRNA and
GAPDH were used as the housekeeping gene. The final results of the
relative mRNA quantification were verified with GAPDH levels (△CT).
Amplification product was consistent with the target gene sequence in
NCBI after gene sequence alignment.
2.7. Protein preparation and western blotting
Protein concentrations were determined using a bicinchoninic acid
assay kit (Beyotime, Jiangsu, China). After boiling at 100 ◦C, the protein
extracts (30 μg) were electrophoresed in 7.5 ~ 10% sodium dodecyl
sulfate polyacrylamide gels following the procedure described by
Laemmli (1970). The membranes were blocked and immunoblotted
with the following primary antibodies: p-mTORSer2448, mTOR, ribosomal p70S6 kinase (p70S6K), p-p70S6KThr389, p-Nf-κB p65Ser536 antibody and NF-κB p65 antibody. Protein detection was performed using
goat anti-rabbit IgG (H + L) HRP-conjugated secondary antibody (BioRad, Richmond, CA, USA) or HRP-labeled goat anti-mouse IgG (H + L)
secondary antibody by enhanced chemiluminescence using western
blotting detection reagents (Beyotime). Monoclonal mouse anti-β-actin
antibody was used as a loading control. Western blots were developed
and quantified using BioSpectrum 810 with VisionWorksLS 7.1 software neral and Comparative Endocrinology 313 (2021) 113876
2.8. Statistical analysis
The data are presented as the mean ± SEM. All data were subjected
to t-text analysis. P < 0.05 was considered statistically significant.
3. Results
3.1. Effects of LPS on food intake, appetite-related signaling and gene
expression
Consistent with previous studies, we found that LPS treatment of
0.5–2 mg/kg BW induced a significant decrease in food intake (P < 0.05;
Fig. 1A). So we chose the lowest dosages LPS administration group (0.5
mg/kg BW) to determine the levels of the pro-inflammatory cytokines
and appetite gene expression. The results showed LPS administration did
not significantly affect the gene expression of NPY, POMC and TNF-α
compared to the control group (P > 0.05, Fig. 1B). The gene expression
of AgRP was significantly down-regulated following LPS injection relative to the control (P < 0.05, Fig. 1B). In contrast, the mRNA levels of IL-
1β was significantly increased after LPS administration compared to the
control group (P < 0.05, Fig. 1B). We further detected the appetiterelated signaling pathways, and the results showed that the phosphorylation of the NF-кB p65, mTOR and p70S6K proteins elevated significantly following LPS administration (P < 0.05, Fig. 1C).
3.2. Effects of mTOR signaling blockade on food intake, appetite-related
signaling and gene expression following LPS treatment
Rapamycin was used to investigate the effect of mTOR inhibition on
the LPS-induced anorexia. Pre-treatment with rapamycin significantly
attenuated the lower food intake caused by LPS compared with the LPS
treatment alone (P < 0.05; Fig. 2A). Pre-treatment with rapamycin
significantly attenuated the LPS-stimulated expression of mTOR,
p70S6K and NF-кB and the LPS-inhibited expression of AgRP compared
with the LPS treatment alone (P < 0.05; Fig. 2B, 2C). Compared with the
LPS treatment, pre-treatment with rapamycin did not alter the stimulated effect of IL-1β gene expression (P > 0.05; Fig. 2B). Compared with
the control alone, ICV treatment with rapamycin significantly decreased
the p-mTOR protein level (P < 0.05; Fig. 2C), but significantly stimulated the food intake of chicks at 18 and 24 h (P < 0.05; Fig. 2A).
4. Discussion
In the present study, we investigated which hypothalamic peptides
were involved in the anorexia of chicks after an intraperitoneal injection
of LPS and whether the mTOR pathway was involved. Our data
demonstrated that LPS induce anorexia of chicks by decreasing hypothalamic AgRP level, and the inhibition of mTOR signaling attenuated
anorexia and the AgRP expression caused by LPS. The results suggest
that LPS inhibit food intake via the mTOR pathway in chicks. Besides,
the regulating process of LPS in hypothalmic NF-кB is also associated
with mTOR signaling. The proposed model of mTOR signaling on
appetite regulation of chicks in case of LPS is shown in Fig. 3.
4.1. LPS inhibits food intake via suppressing AgRP gene expression
In line with a previous report in birds (Webel et al., 1998), the present data demonstrate that peripheral LPS injection decreased food
intake. This result was also in agreement with previous report that
showed ICV injection of LPS (Faggioni et al., 1995). Compared with the
vehicle-infused control, chicks receiving ICV AgRP administration
exhibited marked hyperphagia (Small et al., 2003). In present study, the
decreased hypothalamic AgRP mRNA level after peripheral LPS treatment in chicks was similar to previous reports in mice (Liu et al., 2016b),
which suggest that AgRP is an important target in LPS-induced anorexia.
Intraperitoneal LPS administration significantly increased POMC
gene expression in the rat hypothalamus (Sergeyev et al., 2001; Yue
et al., 2015). The POMC mRNA level in present work was not
Fig. 1. The cumulative (A) food intake (g) was measured for the chicks with intraperitoneal LPS injection of 0, 0.5, 1, or 2 mg/kg BW. The effect of intraperitoneal
LPS injection (0.5 mg/kg BW) on the hypothalamic mRNA levels of (B) POMC, AgRP, NPY, TNF-α, and IL-1β and the protein expressions of (C) p-mTOR, p-p70S6K
and p-Nf-κB in chicks compared to the controls (saline). The values are expressed as the mean ± SEM (for food intake, n = 8; for the protein and mRNA levels, n = 6). a, b
The means differ significantly (P < 0.05).
X.J. Wang et al.
General and Comparative Endocrinology 313 (2021) 113876
significantly changed by LPS, suggesting the different regulation of the
POMC signals in response to LPS between birds and mammals. In
agreement with the previous study in rat hypothalamus (Sergeyev et al.,
2001; Yue et al., 2015), NPY mRNA level did not change significantly
after LPS treatment. All the data suggest POMC and NPY were not
essential in LPS-induced anorexia in chicks.
4.2. mTOR is involved in LPS-induced anorexia via suppressing AgRP
gene expression
Hypothalamic mTOR acts as the master regulator of energy balance,
autophagy, integrating nutritional and hormonal signals. In the rat,
mTOR signaling is controlled by energy status in specific regions of the
hypothalamus and colocalizes with NPY and POMC neurons in the
arcuate nucleus (ARC). The activation of hypothalamic mTOR signaling
decreases food intake (Cota et al., 2006). However, the function of
mTOR in poultry with regard to appetite regulation has not been thoroughly investigated. In the present study, the inhibiting hypothalamic
mTOR signaling via ICV injection of rapamycin increased the food
intake. The results suggest the appetite control of mTOR in birds was
similar with in mammals. Autophagy also could regulate food intake.
Low glucose availability induced autophagy, leading to changes in NPY
and POMC expression in hypothalamic neuronal cells (Oh et al., 2016).
Rapamycin reduced mTOR activity leading to autophagy induction (Oh
et al., 2016). In our study, LPS maybe regulate hypothalamic autophagy
via mTOR, which further affect appetite.
In the regulation of appetite, cell growth and proliferation of mammals, p70S6K is downstream of mTOR (Wullschleger et al., 2006; Cota
et al., 2006). However, we found that the inhibiting hypothalamic
mTOR significantly increased the p-p70S6K protein level, which were
inconsistent with the previous observations that indicating the activating mTOR-induced increases in p-p70S6K protein levels in rat hypothalamic (Cota et al., 2006). Therefore, the relationship between
mTOR and this specific downstream target in avian species remains
unclear and warrants further investigation (Liu et al., 2015).
In line with the study in rats (Yue et al., 2015), we observed that LPS
treatment could activate mTOR signaling through increasing the phosphorylation level of mTOR protein in hypothalamus stimulated, which
implies that the mTOR pathway may be associated with the suppressed
food consumption by LPS. Therefore, we proposed that inhibition of
hypothalamic mTOR could block the anorexigenic effects of LPS. In ICV
rapamycin experiment, we found that the suppressed food intake and
AgRP gene expression by LPS were fully restored after mTOR pathway
blockade, demonstrating that the mTOR signaling pathway plays an
important role in LPS-induced anorexia and low AgRP gene expression.
4.3. NF-кB pathway is involved in LPS and mTOR-induced anorexia
Hypothalamic NF-кB plays an important role in feeding regulation.
Inhibiting NF-кB attenuated the reductions in body weight and food
intake induced by tumors (Kawamura et al., 1999). NF-кB directly
Fig. 2. The effects of central rapamycin treatment (20 µg per chick) on (A) the cumulative food intake (g), the hypothalamic mRNA levels of (B) AgRP and IL-1β and
the protein expression of (C) p-mTOR, p-p70S6K and p-Nf-κB in chicks exposed to LPS (0.5 mg/kg BW) or saline. The values are expressed as the mean ± SEM (for
food intake, n = 8; for the protein and mRNA levels, n = 6). a, b, c The means differ significantly (P < 0.05).
Fig. 3. Proposed model of the effect of LPS on hypothalamic AgRP gene
expression in chicks (↑increase;↓decrease; → stimulatory; ┤inhibitory, …|
might inhibitory).
X.J. Wang et al.
General and Comparative Endocrinology 313 (2021) 113876
bound the POMC or AgRP gene to promote or inhibit transcription in
response to LPS and leptin treatment (Jang et al., 2010). In our study, we
found that intraperitoneal administration of LPS activated hypothalamic
NF-кB by increasing the phosphorylation of the p65 subunit, indicating
that hypothalamic NF-кB plays a critical role in LPS-induced anorexia. In
rat hypothalamus, NF-кB signaling is involved in the appetite regulation
of hypothalamic mTOR signaling and intratracheal LPS administration
(Yue et al., 2015). In the present experiment, the increased phosphorylation protein level of NF-кB after LPS treatment was attenuated by
mTOR inhibition via ICV injection of rapamycin. What’s more, single
inhibition of mTOR decreased the phosphorylation level of NF-кB. All
the data demonstrated that LPS could increase the phosphorylation level
of NF-кB via mTOR signaling.
NF-кB also regulates the release of pro-inflammatory cytokines (e.g.,
IL-1) (Wong and Pinkney, 2004). These cytokines may be associated
with the induction of anorexia (Laye et al., 2000). Hypothalamic inflammatory signals, rather than peripheral, have been identified as the
major causes of LPS-induced anorexia (Wisse et al., 2007). In line with
previous studies, the mRNA levels of cytokines such as IL-1β were
significantly increased in hypothalamus after LPS administration. In
hypothalamus or peripheral tissue, mTOR signaling is involved in the
regulation of the cytokine-induced inflammatory response (Lee et al.,
2007; Yue et al., 2015). But we observed that the inhibition of mTOR did
not attenuate significantly LPS-stimulated IL-1β gene expression, and
signal inhibition of mTOR also did not alter the IL-1β mRNA level, which
were discrepant with the results in rats (Yue et al., 2015). These results
suggest that hypothalamic mTOR did not regulate the release of IL-1β
caused by LPS in birds, and IL-1β was not essential for LPS-induced
anorexia.
5. Conclusion
Our present results demonstrated that LPS induce anorexia in chicks
though hypothalamic mTOR-AgRP signaling pathway. Besides, mTOR
signaling is also associated with the regulation of LPS in p-NF-кB.
Author contributions
W.X.J. designed research and wrote the paper; L.D. executed
experiment and collected data; J.H.C. provided essential reagents; Z.J.P.
executed PCR test; H.L. had offered the funds support and primary responsibility for final content.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by grants from the National Key Research
and Development Program (2016YFD0500510, 2018YFE0128200),
Natural Science Foundation of China (31672441), Key Technology
Research and Development Program of Shandong Province
(2019JZZY020602), Modern Agro-industry Technology Research System (CARS-40-K09), Taishan Scholars Program (201511023) and Funds
of Shandong ‘Double Tops’ Program (2019).
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