Brain Research 795 Ž1998.

77–86

Research report

Lipopolysaccharide Ž LPS. - and muramyl dipeptide Ž MDP. -induced anorexia

during refeeding following acute fasting: characterization of brain cytokine
and neuropeptide systems mRNAs
)
´
Dave Gayle, Sergey E. Ilyin, Mark C. Flynn, Carlos R. Plata-Salaman
DiÕision of Molecular Biology, School of Life and Health Sciences, UniÕersity of Delaware, Newark, DE 19716-2590, USA
Accepted 10 March 1998

Abstract

We investigated the effectiveness of lipopolysaccharide ŽLPS. and muramyl dipeptide ŽMDP. administered into the brain to induce
anorexia in acutely fasted Wistar rats allowed to refeed. We also assayed for changes in mRNA levels of IL-1 system components,
TNF-a , TGF-b 1, glycoprotein 130 Žgp 130., leptin receptor ŽOB-R., pro-opiomelanocortin ŽPOMC., neuropeptide Y ŽNPY., glucocorti-
coid receptor ŽGR., and CRF receptor ŽCRF-R. in selected brain regions. The data show that LPS and MDP induced anorexia
differentially during refeeding. LPS-induced anorexia was of a stronger magnitude and duration than that of MDP. RNase protection
assays showed that LPS and MDP significantly increased the expression of IL-1 b , IL-1 receptor type I, and TNF-a mRNAs in the
cerebellum, hippocampus, and hypothalamus; LPS was more potent in all cases. MDP treatment, on the other hand, induced a stronger
increase in hypothalamic levels of IL-1 receptor antagonist ŽIL-1Ra. and TGF-b 1 mRNAs relative to LPS. In addition, competitive
RT–PCR analysis showed that LPS induced an eleven-fold increase in IL-1 a mRNA in the hypothalamus relative to vehicle. These
findings suggest that LPS and MDP mediate anorexia through different cytokine mechanisms. A stronger up-regulation of anti-inflamma-
tory cytokines ŽIL-1Ra and TGF-b 1. mRNA expression by MDP may be involved in the weaker MDP-induced anorexia relative to LPS.
No significant changes were observed in the peptide components examined except for an up-regulation in cerebellar gp 130 mRNA and
down-regulation of hypothalamic GR mRNA expression in response to LPS or MDP. This study shows that LPS and MDP induce
anorexia in fasted rats allowed to refeed, and suggests an important role for endogenous cytokine–cytokine interactions. q 1998 Elsevier
Science B.V. All rights reserved.

Keywords: Interleukin; Tumor necrosis factor; Leptin; Neuropeptide Y; Opioid; Glucocorticoid; Corticotropin; Endotoxin; Nervous system; Cerebellum;
Hippocampus; Hypothalamus; Feeding; Food intake; RNase protection; Competitive RT–PCR

1. Introduction refeed ad libitum. We also assayed mRNA levels of vari-

ous components of peptide systems that have been shown
Bacterial products such as lipopolysaccharide ŽLPS,
to modulate feeding behavior w18,19,33x. It is unknown
from Gram negative bacteria. and muramyl dipeptide
ŽMDP, from Gram positive bacteria. have been shown to whether LPS or MDP induce anorexia in fasted rats al-
lowed to refeed by modulating cytokines andror feeding-
reduce food intake w8,16x and to increase cytokine produc-
regulatory peptides. Hence, this approach allows the study
tion w9,17,30x. On the other hand, acute fasting induces
of cytokine–cytokine and cytokine–peptide interactions
hyperphagia when animals are allowed to refeed w6x. To
that may underlie anorexia induced by LPS or MDP.
characterize LPS- and MDP-induced anorexia during a
Specifically, animals were fasted for 39.5 h, then chal-
condition of enhanced feeding drive Ži.e., following food
lenged with the intracerebroventricular Ži.c.v.. administra-
deprivation., we investigated the feeding response and
tion of LPS or MDP, followed by 5 h of ad libitum
mRNA response profiles of various cytokines to LPS and
feeding. We then determined brain Žcerebellar, hippocam-
MDP administration into the brain of fasted rats allowed to
pal, hypothalamic. mRNA levels of interleukin-1 ŽIL-1.
system components: the ligands IL-1 a and IL-1 b , two
)
Corresponding author. Fax: q 1-302-831-2647; E-mail: pro-inflammatory cytokines w7,20x; IL-1 receptor type I
crps@udel.edu ŽIL-1RI., the signaling receptor w28x; IL-1 receptor acces-

0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.

PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 2 8 0 - 7
78 D. Gayle et al.r Brain Research 795 (1998) 77–86
sory proteins ŽIL-1R AcP I and II, membrane bound and
soluble forms, respectively., with the membrane bound
form conferring responsiveness w11,21,36x; and IL-1 recep-
tor antagonist ŽIL-1Ra., an endogenous competitive in-
hibitor of IL-1 action w1x. We also determined mRNA
levels for tumor necrosis factor-a ŽTNF-a ., a pro-in-
flammatory cytokine w20x, and transforming growth factor-
b 1 ŽTGF-b 1., a potential anti-inflammatory cytokine
w4,20,31x. Messenger RNA levels of the following compo-
nents were also assayed: pro-opiomelanocortin ŽPOMC.,
the precursor of b-endorphin and MSH; neuropeptide Y
ŽNPY., a potent feeding inducer peptide w5,18,27x which
can interact antagonistically with IL-1 b w29x; glycoprotein
130 Žgp 130., a common signal transducer among recep-
tors for members of the IL-6 subfamily w23x that is homol-
ogous to the leptin receptor w2,32x; leptin receptor ŽOB-R.;
glucocorticoid receptor ŽGR.; and corticotropin releasing
factor receptor ŽCRF-R..
Our previous work found that levels of IL-1 a mRNA
were below the sensitivity of RNase protection assay.
Hence, we used the more sensitive competitive RT–PCR
analysis to determine IL-1 a mRNA profile in response to
LPS treatment. RNase protection assays were used for all
other components.
The results show that LPS and MDP inhibit feeding in
fasted rats allowed to refeed, and differentially modulate
the mRNA expression of various cytokines and peptide
system components.
2. Materials and methods
2.1. Subjects and maintenance
Male Wistar ŽVAF. rats weighing between 250 and 275
g at the beginning of the experiments were used. Rats were
randomly assigned into groups and placed in individual
cages. They were maintained ad libitum on powdered rat
food ŽLabdiet, PMI Feeds, St. Louis, MO. and tap water as
previously described w26x. Room temperature was main-
tained at 21 " 28C, and artificial illumination was from
0600 to 1800 h Ž12:12 h light:dark cycle.. All rats were
handled daily. After several days of adaptation to the home
cages, brain cannulas were implanted.
2.2. Food intake
The measurement of powdered food intake was the
same as in previous studies w26x. Food intake was mea-
sured to within 0.1 g. Before and after the cannula implan-
tation, the rats were fed ad libitum daily, except between
1630 and 1800 h when food was removed to be measured
and replaced, rats were handled and their body weight was
measured. To verify normal feeding throughout mainte-
nance of the animals, food intake was measured daily
during the nighttime Ž1800 to 0600 h. and total daily Ž1800
to 1630 h. periods. At the beginning of the experimental
period, food was removed at 1630 h and returned 39.5 h
later Žat 0800 h.. Food intake was measured at 0900 h Ž1-h
intake., 1100 h Ž3-h intake. and at 1300 h Ž5-h intake..
Rats were sacrificed following the 5-h measurement.
2.3. Implantation of brain cannulas
Under intraperitoneal Ži.p.. ketamine Ž100 mgrkg. plus
xylazine Ž5 mgrkg. anesthesia, a 23-gauge stainless steel
guide cannula Ž18.0 mm long, 0.64 mm o.d., 0.39 mm i.d..
was implanted into the third ventricle at stereotaxic coordi-
nates: 2.1 mm posterior and 0.0 mm lateral with respect to
bregma, and 7.5–8.0 ventral from the brain surface as in
previous studies w26x. The location of the cannula tip into
the third ventricle was verified by the free outflow of CSF
through the guide cannula. A sterile 29-gauge stainless
steel obturator was used to ensure that the cannula re-
mained patent.
2.4. IntracerebroÕentricular microinfusion
Microinfusions were made into the third ventricle be-
cause of the importance of hypothalamic regions in the
control of feeding. In this study, test solutions were admin-
istered following a recovery period of at least 20 days after
surgery. I.c.v. microinfusions Ž10 m lrrat, into unre-
strained, undisturbed animals. were at the rate of 1 m l per
60 s by using an infusion pump ŽHarvard Apparatus, South
Natick, MA.. Each animal was infused between 0730 and
0800 h.
Randomly assigned groups received either vehicle
Žsterile physiological saline solution, 10 m lrrat., or 500
ngrrat bacterial lipopolysaccharide Ž Escherichia coli
serotype 0111:B4; Calbiochem–Novabiochem, La Jolla,
CA., or 500 ngrrat N-acetylmuramyl-L-alanyl-D-iso-
glutamine ŽCalbiochem–Novabiochem. dissolved in 10 m l
sterile physiological saline. The same LPS or MDP lot and
stock solutions were used for all experiments. The concen-
tration of LPS was selected based on our previous studies
that used the acute i.c.v. administration w24x. The time
selected for tissue sampling Ž5-h after the acute i.c.v.
administration. was based on the behavioral profile exhib-
ited by rats receiving LPS and MDP.
2.5. Dissection of brain regions
Following the 5-h refeeding period, rats were decapi-
tated and their brains were quickly removed Ž- 30 s from
the time of decapitation.. Each brain was immediately
placed in oxygenated phosphate-buffered saline solution at
2–48C. Brains were rinsed several times and selected
regions Žcerebellum, hippocampus, and hypothalamus.
were dissected. Each brain region sample was immediately
homogenized in guanidine thiocyanaterphenol solution
ŽTri Reagent, Molecular Research Center, Cincinnati, OH.
 D. Gayle et al.r Brain Research 795 (1998) 77–86
using a microtissue grinder, and frozen at y858C. The
brain samples were number coded for further processing
and analyses. The complete brain dissection, homogeniza-
tion and storage took less than 6 min.
2.6. RNA isolation and RNase protection assay of IL-1b ,
IL-1RI, IL-1R AcPs, IL-1Ra, TNF-a , TGF-b 1, gp 130,
OB-R, POMC, NPY, GR, CRF-R, GAPDH and cyclophilin
mRNAs
Total cell RNA was isolated individually from the
homogenized samples according to our previous studies
w14,15x. RNA concentration was determined by spec-
trophotometry at an absorbance of 260 nm. RNA integrity
was assessed by agarose gel electrophoresis and ethidium
bromide staining. The level of rat glyceraldehyde 3-phos-
phate dehydrogenase ŽGAPDH. mRNA and cyclophilin
mRNA were RNase protection assayed to confirm consis-
tency and that an equal amount of total cell RNA was used
for each assay.
Riboprobes were prepared as previously described
w10,15,25x. Probe synthesis was conducted with 1 mM each
of CTP, ATP, and UTP; and 9.38 m M of w32 PxGTP Ž800
Cirmmol. and 25 m M of unlabeled GTP. w32 PxGTP not
incorporated into the probe was removed by two ethanol
precipitations in the presence of 2.5 M ammonium acetate.
RNase protection assays were used to detect the IL-1 b ,
IL-1RI, IL-1R AcPs, IL-1Ra, TNF-a , TGF-b 1, gp 130,
OB-R, POMC, NPY, GR, CRF-R, GAPDH and cy-
clophilin mRNAs.
Hybridization reactions containing 6.0 m g of total cell
RNA and 2.5 = 10 4 c.p.m. each of IL-1 b , IL-1RI, IL-1R
AcP, IL-1Ra, TGF-b 1, and 1.5 = 10 4 c.p.m. of TNF-a
antisense probe; 6.0 m g of total cell RNA and 2.5 = 10 4
c.p.m. each of gp 130, OB-R, POMC, NPY, GR, and
CRF-R antisense probes, or 3.0 m g of total cell RNA and
2.0 = 10 5 c.p.m. each of GAPDH and cyclophilin anti-
sense probes in 30 m l of hybridization buffer Ž80% for-
mamide, 0.4 M NaCl, 1 mM EDTA, 40 mM PIPES ŽpH
6.4.. were heated to 858C for 5 min and then incubated at
488C for 12 to 18 h. After hybridization, 280 m l of RNase
digestion buffer Ž50 mM sodium acetate ŽpH 4.5., 2 mM
EDTA. was added with 30 U of T1 RNase ŽSigma, St.
Louis, MO. for all assays followed by incubation at 308C
for 60 min. RNase digestion was terminated by the addi-
tion of 10 m l of 20% SDS and 50 m g of proteinase K and
incubation for 30 min at 378C. RNA was extracted with
phenolrchloroform and precipitated with 70 m g of yeast
transfer RNA by the addition of ethanol. RNA was dis-
solved in loading buffer Ž80% formamide, 2 mM EDTA
ŽpH 7.4. containing 0.05% bromophenol blue and 0.05%
xylene cyanol., denatured at 858C for 5 min, and resolved
on 5% acrylamider8 M urea gels using TBE-buffer Ž89
mM Tris pH 8.0, 89 mM boric acid and 2.7 mM EDTA..
Gels were autoradiographed and results were quantified
with an image analyzer ŽImage Quant, Molecular Dynam-
79
ics, Sunnyvale, CA.. Densitometric values for each mRNA
analyzed were converted to percentage values of the total
values for a particular mRNA.
In control experiments on hybridization specificity, 6.0
m g of yeast transfer RNA was hybridized and processed as
described above. No signal corresponding to IL-1 b , IL-
1RI, IL-1R AcPs, IL-1Ra, TNF-a , TGF-b 1, gp 130, OB-R,
POMC, CRF-R, GR, NPY or POMC was detected.
2.7. Preparation of rat interleukin-1a competitor RNA
Prim ers 5 X -CTTTACATTCTTGTCCTTAGG-3 X
Žnucleotides 1641–1661. and 5X-TCCCGTCTTTAGATG-
GTTAGC-3X Ž1796–1816. were designed to amplify a 176
bp fragment of the rat interleukin-1 a cDNA Žaccession a
D00403.. Reverse transcription was performed with 1 m g
of rat brain total RNA and random hexamers in a final
volume of 20 m l using RNA PCR Core Kit according to
the manufacturer’s protocol ŽPerkin Elmer, Foster City,
CA.. For amplification, the PCR reaction mixture was
added directly to the reverse transcription tube to a final
concentration of 1 = PCR buffer ŽPerkin Elmer., 4 mM
MgCl 2 , 200 m M dNTPs, 0.15 m M each of the primers,
and 0.025 Urm l AmpliTaq DNA polymerase ŽPerkin
Elmer. in a final volume of 100 m l; this was subjected to
35 cycles of denaturation at 958C for 45 s, and annealing-
extension at 608C for 45 s.
The generated IL-1 a cDNA fragment was used in a
second round of PCR amplification in which the upstream
primer was substituted by a competitor primer; all other
conditions were the same. The competitor primer 5X-CTT-
TACATTCTTGTCCTTAGGTGTTCACTTCGTTCATT-3X
was generated by combining the IL-1 a upstream primer
with another 17-bp sequence taken from the 5X terminus of
the target cDNA. This deleted 30 bp between the 17-bp
sequence and the upstream primer. The competitor DNA
was purified by the Wizard PCR Preps DNA Purification
System ŽPromega, Madison, WI. and ligated into Srf I site
of pCR-Script SKŽq. vector according to the manufac-
turer’s protocol ŽpCR-Script Amp SKŽq. Cloning Kit,
Stratagene Cloning Systems, La Jolla, CA.. The resulting
plasmid DNA was linearized by SacI and transcribed with
T7 RNA polymerase to generate an IL-1 a RNA competi-
tor.
2.8. ReÕerse transcription and competitiÕe PCR for IL-1a
Reverse transcription was performed according to the
manufacturer’s protocol ŽPerkin Elmer. with 0.2 m g of
total cell RNA and a concentration of RNA competitor that
was varied three- to nine-fold between samples. Amplifica-
tion was performed as described above in the presence of
w32 PxdCTP. Following amplification, aliquots were ana-
lyzed on 5% acrylamide gel in TBE buffer, autoradio-
graphed and quantified with an image analyzer ŽImage
Quant.. The template-to-competitor ratio vs. the relative
80 D. Gayle et al.r Brain Research 795 (1998) 77–86
amount of competitor was compared on double logarithmic
plots, and a best fit line through the data was used to
determine the relative amount of competitor RNA that
yielded a 1:1 template-to-competitor ratio w22x. The iden-
tity of the target was confirmed by sequencing.
2.9. Riboprobe templates
Rat IL-1 b expression plasmid containing the entire
mature peptide coding sequence of rat IL-1 b , with one
extra Met codon at the 5X-end cloned into plasmid pET-21d
ŽNovagen, Madison, WI. between EcoRI and NcoI sites
was used. Plasmid rat riboprobe IL-1 b ŽrRIL-1 b . was
generated by cloning IL-1 b containing XbaI–EcoRI frag-
ment of the rat IL-1 b expression plasmid into pGEM-2
between XbaI and EcoRI sites. Plasmid rRIL-1 b was
linearized with HindIII, and transcribed with SP6 RNA
polymerase to generate an approximately 570
nucleotides–long antisense probe which protects 492 nu-
cleotides in the rat IL-1 b mRNA.
Plasmid rat IL-1RI 3X clone containing a 3X fragment
Žnucleotides 473–1826 of accession a M95578. of the rat
IL-1RI cDNA cloned into the SmaI site of pBluescript SK
ŽStratagene. was used. Linearization of this plasmid with
HindIII and transcription with T3 RNA polymerase pro-
duced an approximately 520 nucleotides–long antisense
probe which protects 436 nucleotides in the rat IL-1RI
mRNA.
Rat IL-1R AcP cDNA Žnucleotides 874–1212 of acces-
sion a U48592. cloned into EcoRV site of pBluescript SK
was used. Linearization of this plasmid by HindIII and
transcription by T3 RNA polymerase resulted in an ap-
proximately 420 nucleotides–long antisense probe which
protects 339 nucleotides in the membrane bound form of
rat IL-1R AcP; additionally, this probe protects a shorter
fragment corresponding to the soluble form of IL-1R AcP
w15x.
Plasmid pBS-RA containing a fragment Žnucleotides
3–451 of accession a M63101. of the rat IL-1Ra cDNA
cloned into the SmaI site of pBluescript SK was linearized
with XmnI, and transcribed with T3 RNA polymerase to
produce an approximately 250 nucleotides–long antisense
probe which protects 207 nucleotides in the rat IL-1Ra
mRNA.
Plasmid prTNFtransŽT7. containing the peptide coding
portion of rat TNF-a cDNA Žnucleotides 1–708 of acces-
sion a S40199. was used. Linearization of this plasmid by
EcoRI and transcription with SP6 RNA polymerase pro-
duced an approximately 790 nucleotides–long antisense
probe which protects 708 nucleotides in the rat TNF-a
mRNA.
Rat TGF-b 1 cDNA Žaccession a X52498. cloned into
pBluescript II KS vector ŽStratagene. was used. Lineariza-
tion of this plasmid by BamHI and transcription with T3
RNA polymerase produced an approximately 320 nu-
cleotides–long antisense probe which protects 244 nu-
cleotides Žbases 1342–1585. in the rat TGF-b 1 mRNA.
Rat gp 130 cDNA Žaccession a M92340. cloned into
EcoRI site of pBS vector was used. This cDNA was
linearized with BsaI and transcribed with T3 RNA poly-
merase to produce an approximately 590 nucleotides–long
antisense probe which protects 530 nucleotides Žbases
1972–2501. in the rat gp 130 mRNA.
Leptin receptor riboprobe template was generated by
cloning a HincII–BamHI fragment of rat leptin receptor
ŽOb–Rb isoform. cDNA into respective sites of pGEM-2.
Leptin receptor riboprobe was linearized by HindIII and
SP6 transcription resulted in an approximately 450 nu-
cleotides–long antisense probe which protects 415 nu-
cleotides Žcorresponding to nucleotides 686–1101 of ac-
cession a U52966. in the rat OB-R mRNA.
Rat POMC cDNA cloned between BamHI and EcoRI
sites of pBluescript KSŽq. vector was used. Plasmid was
linearized with XmnI and T3 RNA polymerase transcrip-
tion resulted in an approximately 320 nucleotides–long
antisense probe which protects 264 nucleotides Žbases
774–808 in exon 1 plus 49–199 in exon 2 plus 89–166 in
exon 3. in the rat POMC mRNA Žpredominant form..
Plasmid pBLNPY-1 containing a 511-bp insert Žacces-
sion a M20373. comprising most of the cDNA of rat
prepro-neuropeptide Y ligated into the EcoRI site of vec-
tor Bluescribe M13Žy. was used. Linearization of the
plasmid with BbsI and transcription with T3 RNA poly-
merase produced an approximately 242 nucleotides–long
antisense probe which protects 180 nucleotides Žbases
332–511. in the rat NPY mRNA.
Rat GR cDNA Žnucleotides 482–1640 of accession a
M14053. cloned between PstI and XhoI sites of pBlue-
script II KS was linearized with BsaI and transcribed with
T7 RNA polymerase to produce an approximately 384
nucleotides–long antisense probe which protects 324 nu-
cleotides in the rat GR mRNA.
Rat CRF-R cDNA Žaccession a L24096. cloned into
Pst I site of pBluescript II SK was used. Linearization of
the cDNA with BsaI and transcription with T7 RNA
polymerase resulted in an approximately 368 nucleotides–
long antisense probe which protects 302 nucleotides Žbases
1099–1400. in the rat CRF-R mRNA.
Rat antisense template pTRI-GAPDH ŽAmbion; which
contains a 316-bp fragment of the rat glyceraldehyde
3-phosphate dehydrogenase gene. was used to generate the
GAPDH antisense probe. Antisense template pTRI-
Cyclophilin-Rat ŽAmbion; which contains a 103-bp cDNA
fragment of the rat cyclophilin gene. was used to generate
the cyclophilin antisense probe.
2.10. Data analyses
Results are expressed as means " S.E. Data were ana-
lyzed using analysis of variance ŽANOVA. followed by
 D. Gayle et al.r Brain Research 795 (1998) 77–86 81

post-hoc tests for pairwise comparisons ŽStudent–New- 3. Results

man–Keuls method. if there was a significant main effect.
Kruskal–Wallis test was applied Žfollowed by post-hoc
tests. when data did not pass the normality ŽKolmogorov–
Smirnov. and equal variance ŽLevene Median. tests. Data
were also analyzed using t-test or Mann–Whitney test
Žwhen data did not pass normality and equal variance
tests.. To test for quantitative relationships, Pearson prod-
uct-moment correlation coefficients Ž r ’s. were calculated.
Differences were considered to be significant only for
p - 0.05.
3.1. Food intake
The treatments differentially affected cumulative food
intake over the 5-h refeeding period Ž F Ž2,24. s 7, p -
0.004.. Cumulative food intake data collected at 1-h and
3-h post-infusion show that both LPS Ž n s 9. and MDP
Ž n s 9. significantly Ž p - 0.05. decreased food intake rel-
ative to the vehicle Ž n s 9.. Values at 1-h were: 6.2 " 0.3
g for LPS, 7.4 " 1.0 g for MDP, and 9.0 " 0.6 g for

Fig. 1. Top panel, RNase-protection assay of TNF-a ŽTNF-a ., interleukin-1 b ŽIL-1 b ., IL-1 receptor type I ŽIL-1RI., IL-1 receptor accessory proteins
ŽIL-1R AcP. I and II, transforming growth factor-b 1 ŽTGF-b 1., IL-1 receptor antagonist ŽIL-1Ra., and glyceraldehyde 3-phosphate dehydrogenase
ŽGAPDH. mRNA levels in the cerebellum ŽCER., hippocampus ŽHIP., and hypothalamus ŽHYP.; bottom panel, RNase-protection assay of glycoprotein
130 Žgp 130., leptin receptor ŽOB-R., pro-opiomelanocortin ŽPOMC., neuropeptide Y ŽNPY., glucocorticoid receptor ŽGR., corticotropin releasing factor
receptor ŽCRF-R., and cyclophilin. Fasted male Wistar rats were treated with the acute intracerebroventricular Ži.c.v.. microinfusion of vehicle ŽV., MDP
ŽM, 500 ngrrat. or LPS ŽL, 500 ngrrat.. Brain tissue samples were collected at the end of the 5-h refeeding period.
82 D. Gayle et al.r Brain Research 795 (1998) 77–86
Fig. 2. IL-1 b mRNA levels in response to the i.c.v. administration of
vehicle Žcontrol., MDP Ž500 ngrrat. and LPS Ž500 ngrrat.. Values
Žmean"S.E.; ns9 for each group. were standardized to arbitrary units.
Abbreviations are as for Fig. 1. ), p- 0.05 from vehicle; ‡, p- 0.05
between MDP and LPS Žpost-hoc tests following ANOVA..
vehicle; and at 3-h: 6.3 " 0.3 g for LPS; 8.1 " 0.8 g for
MDP; and 9.9 " 0.6 g for vehicle. Pairwise comparisons
also showed significant differences at 5-h between LPS
Ž7.0 " 0.3 g. and MDP Ž9.6 " 0.9 g., and between LPS
and vehicle Ž10.2 " 0.5 g.; the difference between MDP
and vehicle was not significant. It is feasible that MDP no
longer induced anorexia at 5-h post-infusion due to the
production of anti-inflammatory cytokines which might
have attenuated the anorectic response Žsee below.. There
were no significant differences in cumulative water intake
during the refeeding period at 0–1, 0–3, or 0–5 h post-in-
fusion. This suggests that LPS and MDP selectively af-
fected food intake.
3.2. RNase protection assay
The same rats were used to characterize the behavioral
profile and to analyze the IL-1 system, TNF-a , TGF-b 1,
gp 130, OB-R, POMC, NPY, GR, CRF-R, GAPDH, and
cyclophilin mRNA levels in selected brain regions. Fig. 1
is an example of the RNase protection assays used. As
Fig. 3. IL-1RI mRNA levels in response to the i.c.v. administration of
vehicle Žcontrol., MDP, and LPS. Other explanations are as for Fig. 2.
Fig. 4. IL-1R AcP I mRNA levels in response to the i.c.v. administration
of vehicle Žcontrol., MDP, and LPS. Other explanations are as for Fig. 2.
shown, samples from vehicle-, MDP- and LPS-treated
animals from all three brain regions were analyzed con-
comitantly. Consistency was verified by analyzing all sam-
ples individually, from which all means " S.E. were gener-
ated. It should be noted that each individual sample was
obtained from a different rat, that the same rat provided all
three brain regions, and that the same samples were ana-
lyzed with all antisense probes as described in Section 2.
Levels of rat GAPDH mRNA ŽFig. 1, top panel. and
cyclophilin mRNA ŽFig. 1, bottom panel. were relatively
constant between treatments within a brain region. This
suggests consistency of processing and that equal amounts
of total RNA were used. In addition, the specificity of the
probes used has been demonstrated previously w15,25x.
3.3. IL-1 system components mRNA leÕels
The profile of IL-1 b mRNA in response to treatments
is summarized in Fig. 2. ANOVA revealed that treatments
differed significantly in the induction of cerebellar Ž H2
. Ž
degrees of freedom Ž2 d f . s 21.6, p - 0.0001 , hippocampal H 2
. Ž
d f s 21.6, p - 0.0001 , and hypothalamic H 2 d f s 21.0,
p - 0.0001. IL-1 b mRNA levels. Similarly, treatments
Fig. 5. IL-1R AcP II mRNA levels in response to the i.c.v. administration
of vehicle Žcontrol., MDP, and LPS. Other explanations are as for Fig. 2.
 D. Gayle et al.r Brain Research 795 (1998) 77–86 83

Fig. 6. IL-1Ra mRNA levels in response to the i.c.v. administration of

vehicle Žcontrol., MDP, and LPS. Other explanations are as for Fig. 2. Fig. 8. TNF-a mRNA levels in response to the i.c.v. administration of
vehicle Žcontrol., MDP, and LPS. Other explanations are as for Fig. 2.

differentially induced IL-1RI mRNA ŽFig. 3. across brain

regions: cerebellum Ž H2 d f s 12.4, p - 0.002.; hippocam-
pus Ž H2 d f s 15.5, p - 0.0004.; and hypothalamus Ž H2
.
d f s 18.5, p - 0.0001 . In all cases, the responses to MDP
and LPS were significantly different from each other and
from the vehicle infusion. The highest induction of IL-1 b
and IL-1RI mRNAs was in response to LPS in all three
brain regions.
Lipopolysaccharide treatment significantly up-regulated
IL-1R AcP I mRNA levels in all three brain regions ŽFig.
4., while MDP treatment induced a significant response
only in the hippocampus. Fig. 5 shows that both treatments
increased the hypothalamic levels of IL-1R AcP II mRNA,
while only LPS increased the hippocampal levels.
Lipopolysaccharide and MDP treatments also signifi-
cantly increased hypothalamic levels of IL-1Ra mRNA
ŽFig. 6.. In addition, competitive RT–PCR revealed that
LPS treatment induced an eleven-fold ŽLPS: 161.1 " 18.6;
vehicle: 14.7 " 4.9, mean " S.E. in arbitrary units. in-
crease in IL-1 a mRNA levels in the hypothalamus relative
to the control ŽFig. 7..
3.4. TNF-a and TGF-b 1 mRNA leÕels
As shown in Fig. 8, treatments differentially induced
TNF-a mRNA levels in the cerebellum Ž H2 d f s 13.0,
p - 0.002., hippocampus Ž F Ž2,24. s 20, p - 0.0001., and
hypothalamus Ž H2 d f s 19.8, p - 0.0001.. LPS induction
of TNF-a mRNA was significantly higher relative to MDP
in all cases. On the other hand, only MDP treatment
significantly increased TGF-b 1 mRNA expression in the
hypothalamus ŽFig. 9..
3.5. Gp 130, OB-R, POMC, NPY, GR, and CRF-R mRNA
leÕels
The data is summarized in Table 1. MDP and LPS
significantly down-regulated hypothalamic GR mRNA and
up-regulated cerebellar gp 130 mRNA expression. LPS
treatment also significantly decreased hippocampal GR
mRNA and increased cerebellar OB-R mRNA levels. On

Fig. 7. Competitive RT–PCR analysis of hypothalamic IL-1 a mRNA levels in response to the i.c.v. administration of vehicle Žcontrol. and LPS Ž500
ngrrat.. A series of concentrations of internal standards were added to each tube containing 0.2 m g of total RNA. The competitor concentration was
decreased three-fold in each subsequent lane Ž1 through 6. and nine-fold from lanes 6 to 7. The same concentrations of competitor were used for vehicle
and LPS samples. Žy.RT, control Žreaction without reverse transcriptase..
84 D. Gayle et al.r Brain Research 795 (1998) 77–86

4. Discussion

This study shows that i.c.v. administered LPS and MDP

induce anorexia of different magnitudes and durations in
fasted rats allowed to refeed ad libitum. Treatments also
differentially affected cytokine and peptide system compo-
nents mRNA levels in the brain regions examined. Specifi-
cally, LPS and MDP administration significantly increased
IL-1 b , IL-1RI, and TNF-a mRNA levels in the cerebel-
lum, hippocampus, and hypothalamus. MDP also signifi-
cantly increased hippocampal IL-1R AcP I and hypothala-
mic IL-1R AcP II, IL-1Ra, and TGF-b 1 mRNA levels.
LPS treatment increased levels of IL-1R AcP I mRNA in
Fig. 9. TGF-b 1 mRNA levels in response to the administration of vehicle
Žcontrol., MDP, and LPS. Other explanations are as for Fig. 2. all three brain regions, IL-1R AcP II mRNA in the hip-
pocampus and hypothalamus, and IL-1Ra mRNA in the
hypothalamus. In addition, LPS induced an eleven-fold
increase in IL-1 a mRNA.
the other hand, treatments did not significantly affect the Our findings suggest that LPS and MDP administration
POMC, NPY, and CRF-R mRNA profiles in any brain can significantly inhibit fasting-induced hyperphagia. LPS
region examined. induced a stronger anorexia which lasted throughout the
5-h refeeding period. MDP-induced anorexia, however,
was no longer evident 3 h post-infusion. This and other
Table 1 findings w16x suggest that LPS and MDP mediate anorexia
Gp 130, OB-R, POMC, NPY, GR, and CRF-R mRNAs in brain regions through different mechanisms.
in response to treatments
In support of this hypothesis, molecular analyses show
Cerebellum Hippocampus Hypothalamus that LPS induced higher mRNA expressions of the pro-in-
Gp 130 mRNA flammatory cytokines IL-1 b Žand its signaling receptor
Vehicle 3.9"0.4 3.5"0.3 5.7"0.6 IL-1RI. and TNF-a relative to MDP. In contrast, MDP
MDP 7.0"1.2) 4.6"0.4 7.2"0.6
induced higher hypothalamic levels of the anti-inflamma-
LPS 7.1"0.5) 4.1"0.4 6.8"0.5
tory cytokines IL-1Ra and TGF-b 1 mRNAs. The differen-
OB-R mRNA tial cytokine induction by LPS and MDP, especially in the
Vehicle 11.3"1.4 1.0"0.2 1.8"0.2 hypothalamus, could explain differences in their anorexi-
MDP 13.6"2.4 1.2"0.2 2.1"0.2
genic effectiveness. It is possible that both treatments
LPS 15.7"1.1) 1.3"0.1 2.0"0.3
initially induce anorexia due to up-regulation of the pro-in-
POMC mRNA flammatory cytokines, but subsequent or simultaneous in-
Vehicle y y 14.5"1.4 duction of anti-inflammatory cytokines, particularly by
MDP y y 14.4"1.2
MDP, attenuates the magnitude and duration of the
LPS y y 14.0"1.2
anorexia. Determination of cytokine protein content will
NPY mRNA test this hypothesis.
Vehicle 2.8"0.4 7.0"0.5 5.4"0.3 The observed LPS-induced up-regulation of IL-1 a
MDP 3.7"1.2 7.0"0.7 7.0"0.6
mRNA in the hypothalamus suggests that this predomi-
LPS 2.9"0.4 7.6"0.6 6.5"0.6
nantly intracellular form of IL-1 w7x may also contribute to
GR mRNA LPS-induced anorexia. This study further confirms our
Vehicle 7.4"0.8 4.2"0.3 5.0"0.2 previous findings w13x that cytokine–cytokine interactions
MDP 10.6"1.3 4.2"0.3 4.2"0.2)
are critical in determining the magnitude of the anorexia
LPS 7.6"0.5 3.1"0.2) 3.6"0.2)
induced by LPS or MDP in ad libitum rats.
CRF-R mRNA Our study also shows that LPS and MDP treatments
Vehicle 12.1"1.0 1.5"0.8 2.5"0.8 decreased GR mRNA levels in the hypothalamus suggest-
MDP 14.9"2.4 1.8"0.6 1.5"0.6
ing that glucocorticoids and the CRF system may be
LPS 11.6"0.9 2.2"0.8 2.0"0.4
involved in the anorectic response to LPS or MDP. Evi-
Values are means"S.E. Žstandardized to arbitrary units. from eight dence suggests that IL-1 b , which is up-regulated by LPS
individual samples examined for gp 130, OB-R, POMC, and NPY and MDP, induces anorexia that is mediated, at least in
mRNAs; and six individual samples for GR and CRF-R mRNA assays. part, by CRF w34x. LPS has also been shown to increase
Vehicle, 0.15 M NaCl; MDP, muramyl dipeptide Ž500 ngrrat.; LPS,
lipopolysaccharide Ž500 ngrrat.. ), significantly Ž p- 0.05. different
plasma concentrations of cortisol while inducing anorexia
from vehicle; y, below sensitivity of detection. All mRNA signals were w35x. We also observed high basal levels of OB-R mRNA
examined in the same brain region samples. in cerebellum which were modulated by LPS treatment.
 D. Gayle et al.r Brain Research 795 (1998) 77–86
High basal levels of cerebellar OB-R mRNA have been
reported previously w12x, but its functional significance is
yet to be determined.
Previous studies reported that food deprivation in-
creased mRNA levels of various neuropeptides in the
hypothalamus w3,27x. It is possible that we did not observe
mRNA changes in other peptide system components be-
cause of normalization during the refeeding period. Studies
are currently underway to determine cytokine and peptide
systems mRNA levels in fasted non-refed rats.
In conclusion, this study demonstrates that LPS and
MDP administration into the brain of acutely fasted rats,
differentially induce anorexia during refeeding. LPS and
MDP also differentially modulate cytokine components
mRNA expression. This suggests that interactions among
cytokines Že.g., pro-inflammatory—IL-1 a , IL-1 b , TNF-a ;
and anti-inflammatory—IL-1Ra, TGF-b 1. may be critical
in determining the magnitude of the anorexia induced by
LPS or MDP.
Acknowledgements
We thank Dr. Ronald P. Hart ŽDepartment of Biological
Sciences, Rutgers University. for providing the rat IL-1 b ,
IL-1Ra, IL-1RI and IL-1R AcP cDNAs, Dr. Karl Decker
ŽBiochemisches Institut der Albert Ludwigs Universitat ¨.
for providing the rat TNF-a cDNA, Dr. David Danielpour
ŽNational Cancer Institute. for providing the rat TGF-b 1
cDNA, Dr. Gerald M. Fuller ŽDepartment of Cell Biology
and Anatomy, University of Alabama at Birmingham. for
providing the rat glycoprotein 130 cDNA, Dr. Charles I.
Rosenblum ŽMerck Research Laboratories, Rahway, NJ.
for providing the rat leptin receptor ŽOb–Rb isoform.
cDNA, Dr. Andrea Gore ŽCenter for Neurobiology, The
Mount Sinai Medical Center. for providing the rat pro-
opiomelanocortin cDNA, Dr. Steven L. Sabol ŽLaboratory
of Biochemical Genetics, National Heart, Lung, and Blood
Institute, NIH. for providing the rat NPY cDNA, and Dr.
Jeffrey Rosen ŽDepartment of Psychology, University of
Delaware. for providing the rat GR and CRF-R cDNAs.
Research supported by funds from the University of
Delaware and the National Institutes of Health to C.R.P.-S.
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