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Division of Molecular Biology, School of Life and Health Sciences, University of Delaware, Newark, Delaware 19716-2590
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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This study determined the effects of
feeding status on basal and lipopolysaccharide (LPS)-stimulated
cytokine and neuropeptide gene expression in the hypothalamus. With the
use of RNase protection assays, we measured mRNA levels of
interleukin-1
(IL-1), IL-1 receptor antagonist (IL-1RA), IL-1
receptor type I (IL-1RI), IL-1R accessory proteins (AcP I and II),
tumor necrosis factor-
(TNF-), transforming growth factor-1
(TGF-1), glycoprotein 130 (Gp 130), leptin receptor (OB-R),
neuropeptide Y (NPY), preprodynorphin, and proopiomelanocortin (POMC).
Analyses were done in ad libitum-fed, fasted, and fasted and refed rats
treated with the intracerebroventricular administration of
physiological saline or LPS. The data show that food deprivation
increases the basal mRNA expression of IL-1, IL-1RA, TNF-,
IL-1RI, and IL-1R AcP I, whereas mRNA levels of POMC showed a decrease.
Five hours of refeeding returned cytokine levels to those observed in
the ad libitum-fed group. LPS administration induced a robust
upregulation of IL-1, TNF-, and IL-1RI during all three feeding
conditions. Acute food deprivation did not modulate LPS-induced changes
in hypothalamic cytokine mRNA profiles. These findings show that
1) cytokine modulation occurs as an
adaptive response to the stress of acute fasting and
2) acute fasting does not affect
LPS-induced cytokine mRNA modulation in the hypothalamus. The data have
implications to gram-negative infections associated with acute anorexia.
food deprivation; fasting; growth factor; interleukin; nervous system; opioids; stress; tumor necrosis factor
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FEEDING STATUS CAN AFFECT immune system activity, including cytokine production and action (4, 9). Previous studies reported that intracerebral administration of Escherichia coli bacterial lipopolysaccharide (LPS) significantly modulated cytokine mRNA expression in specific brain regions under ad libitum feeding conditions (10). LPS induced similar modulation of cytokine components in brain regions under refeeding after acute fasting (6). However, the effects of acute fasting on LPS action in the brain are unknown. Acute fasting could act as a stressful stimulus with influences on cytokine gene expression in the brain. In addition, there is no information on a direct comparison of different feeding conditions and LPS-induced cytokine response.
Here, we investigated the effects of acute food deprivation on
LPS-stimulated cytokine and neuropeptide mRNA production in the
hypothalamus, an important brain site for LPS action including LPS-induced anorexia. We also compared these profiles with those observed in ad libitum feeding and fasting-refeeding conditions. We
assayed for mRNA levels of interleukin-1 (IL-1) and tumor necrosis factor- (TNF-), two pivotal proinflammatory cytokines that induce their own expression and that of other cytokines (5, 18).
We also measured mRNA levels of the inhibitory or anti-inflammatory cytokines IL-1 receptor antagonist (IL-1RA, an endogenous competitive inhibitor of IL-1 action) and transforming growth factor-1
(TGF-1) (18, 26). Moreover, to understand the modifications that
take place within a cytokine system, we analyzed mRNA changes for
various other components of the IL-1 system. That is, we examined
the IL-1 receptor type I (IL-1RI, an IL-1 signaling receptor) (29) and
IL-1 receptor accessory proteins (IL-1R AcP I and II, membrane bound
and soluble forms, respectively) (8, 30). For other cytokine systems,
we also determined mRNA levels of leptin receptor (OB-R) and
glycoprotein 130 (Gp 130), a common signal transducer among receptors
for members of the IL-6 subfamily (23) that is homologous to the OB-R
(1). Both Gp 130 and OB-R may be affected by feeding status.
Because LPS is a potent anorectic agent with action in the hypothalamus, we also determined the profiles of two feeding-associated neuropeptide mRNAs, neuropeptide Y (NPY) and preprodynorphin, as well as proopiomelanocortin (POMC), a precursor of feeding regulatory opioids (15, 27). Thus the approach we used was designed to obtain comprehensive information on LPS-induced modulation of gene transcription of pivotal cytokine and neuropeptide components under various feeding conditions.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Subjects and Maintenance
Male Wistar VAF rats weighing 250-275 g at the beginning of the experiments were used. Rats were randomly assigned to groups and placed in individual cages. They were maintained ad libitum on powdered rat food (Labdiet, PMI, St. Louis, MO) and tap water as previously described (23). Room temperature was maintained at 21 ± 2°C, and artificial illumination was from 0600 to 1800 (12:12-h light-dark cycle). All rats were handled daily. After several days of adaptation to the home cages, brain cannulas were implanted.Implantation of Brain Cannulas
Under intraperitoneal ketamine (100 mg/kg) plus xylazine (5 mg/kg) anesthesia, a 23-gauge stainless steel guide cannula (18.0-mm long, 0.64-mm OD, 0.39-mm ID) was implanted into the third cerebral ventricle at stereotaxic coordinates: 2.1 mm posterior and 0.0 mm lateral with respect to bregma and 7.5-8.0 mm ventral from the brain surface as in previous studies (23). The location of the cannula tip into the third ventricle was verified by the free outflow of cerebrospinal fluid through the guide cannula. A sterile 29-gauge stainless steel obturator was used to ensure that the cannula remained patent.Feeding Protocols
After at least 20 days postsurgery, rats were randomly assigned to one of the following feeding groups: 1) fed: the rats were allowed to feed ad libitum at all times; 2) fasted: the rats were food deprived for 39.5 h before LPS treatment. Rats did not receive food following LPS administration [The 39.5-h food deprivation period was designed to include 2 complete nighttime periods and 1 daytime period, that is, 36 + 3.5 h in the daytime that corresponds to a low feeding period in the ad libitum-fed animals. We used this design in previous studies with consistent results (6)]; and 3) refed: the rats were fasted for 39.5 h, then allowed a 5-h ad libitum feeding period after the administration of LPS. The 39.5-h fasting period lasted from 1630 to 0800.Intracerebroventricular Microinfusion
Microinfusions were made in the third cerebral ventricle because of the importance of hypothalamic regions in LPS action. Intracerebroventricular microinfusions (10 µl/rat, in unrestrained and undisturbed animals) were at the rate of 1 µl/60 s by using an infusion pump (Harvard Apparatus, South Natick, MA). Each animal was infused between 0730 and 0800.Randomly assigned groups within each feeding design received either vehicle (sterile physiological saline, 10 µl/rat) or 500-ng/rat bacterial LPS (Escherichia coli serotype 0111:B4; Calbiochem-Novabiochem, La Jolla, CA) dissolved in 10-µl sterile physiological saline. The same LPS lot and stock solution were used for all experiments. The concentration of LPS was selected based on our previous studies that used the acute intracerebroventricular administration to study behavioral and molecular responses (6, 10, 24). The time selected for tissue sampling (5 h after the acute intracerebroventricular administration) was also based on the behavioral and molecular profiles exhibited by rats receiving LPS (6, 10, 26).
Dissection of Brain Regions
Five hours after LPS or saline administration, rats were decapitated, and their brains were quickly removed (<30 s from the time of decapitation). Each brain was immediately placed in oxygenated phosphate-buffered saline solution at 2-4°C. Brains were rinsed several times, and the hypothalamus was dissected. Each hypothalamus was immediately homogenized in a solution of guanidine thiocyanate and phenol (Tri Reagent, Molecular Research Center, Cincinnati, OH) using a microtissue grinder and frozen at
85°C for further processing and analyses. The complete brain
dissection, homogenization, and storage took <6 min.
RNA Isolation and RNase Protection Assays
Total cell RNA was isolated individually from the homogenized samples according to our previous studies (6, 10, 25). RNA concentration was determined by spectrophotometry at an absorbance of 260 nm. RNA integrity was assessed by agarose gel electrophoresis and ethidium bromide staining. The levels of-actin mRNA and cyclophilin mRNA
were RNase protection assayed to confirm consistency and that an equal
amount of total cell RNA was used for each assay.
Riboprobes were prepared as previously described (6, 10, 25). Probe
synthesis was conducted with 1 mM each of CTP, ATP, and UTP, 9.38 µM
of [32P]GTP (800 Ci/mmol), and 25 µM unlabeled GTP.
[32P]GTP not
incorporated in 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, IL-1RI, IL-1R AcPs, IL-1RA, TNF-,
TGF-1, preprodynorphin, Gp 130, OB-R, POMC, NPY, -actin, and
cyclophilin mRNAs.
Hybridization reactions containing 6.0 µg of total cell RNA and 2.5 × 104 counts/min each of
IL-1, IL-1RI, IL-1R AcP, IL-1RA, TGF-1, and 1.5 × 104 counts/min of TNF-
antisense probe; 6.0 µg of total cell RNA and 2.5 × 104 counts/min each of
preprodynorphin, Gp 130, OB-R, POMC, and NPY antisense probes, or 3.0 µg of total cell RNA and 2.0 × 105 counts/min each of -actin
and cyclophilin antisense probes in 30 µl of hybridization buffer
[80% formamide, 0.4 M NaCl, 1 mM EDTA, 40 mM PIPES (pH
6.4)] were heated to 85°C for 5 min and then incubated at
48°C for 12-18 h. After hybridization, 280 µl of RNase
digestion buffer [50 mM sodium acetate (pH 4.5), 2 mM EDTA]
was added with 30 U of T1 RNase (Sigma, St. Louis, MO) for all assays
followed by incubation at 30°C for 60 min. After digestion, 700 µl of 7% Tri Reagent in 100% ethanol was added to the reaction followed by the addition of 70 µg of yeast transfer RNA (11). Samples
were allowed to precipitate at 20°C for at least 2 h before
pelleting the RNA by centrifugation. RNA was dissolved in loading
buffer [80% formamide, 2 mM EDTA (pH 7.4) containing 0.05%
bromophenol blue and 0.05% xylene cyanol], denatured at 85°C
for 5 min, and resolved on 5% acrylamide/8 M urea gels using a buffer
containing (in mM) 89 Tris (pH 8.0), 89 boric acid, and 2.7 EDTA. Gels
were autoradiographed, and results were quantified with an image
analyzer (Image Quant, Molecular Dynamics, Sunnyvale, CA).
Densitometric measurements for each mRNA analyzed were converted to
percent values of the total values for a particular mRNA.
In control experiments on hybridization specificity, 6.0 µg of yeast
transfer RNA were hybridized and processed as described above. No
signal corresponding to IL-1, IL-1RI, IL-1R AcPs, IL-1RA, TNF-,
TGF-1, preprodynorphin, Gp 130, OB-R, POMC, NPY, -actin, or
cyclophilin was detected.
Riboprobe Templates
Rat IL-1 expression plasmid containing the entire mature peptide
coding sequence of rat IL-1, with one extra Met codon at the
5' end cloned into plasmid pET-21d (Novagen, Madison, WI) between
EcoR I and
Nco I sites was used. Plasmid rat
riboprobe IL-1 (rRIL-1) was generated by cloning IL-1 containing
Xba
I-EcoR I fragment of the rat IL-1
expression plasmid into pGEM-2 between Xba I and
EcoR I sites. Plasmid rRIL-1 was
linearized with Hind III and transcribed with SP6 RNA polymerase to generate an
~570-nucleotide-long antisense probe that protects 492 nucleotides in
the rat IL-1 mRNA.
Plasmid rat IL-1RI 3 clone containing a 3 fragment (nucleotides 473-1826 of accession #M95578) of the rat IL-1RI cDNA cloned into the Sma I site of pbluescript SK (Stratagene) was used. Linearization of this plasmid with Hind III and transcription with T3 RNA polymerase produced an ~520-nucleotide-long antisense probe that protects 436 nucleotides in the rat IL-1RI mRNA.
Rat IL-1R AcP cDNA (nucleotides 874-1212 of accession #U48592) cloned into EcoR V site of pbluescript SK was used. Linearization of this plasmid by Hind III and transcription by T3 RNA polymerase resulted in an ~420-nucleotide-long antisense probe that 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.
Plasmid pBS-RA containing a fragment (nucleotides 3-451 of accession #M63101) of the rat IL-1RA cDNA cloned into the Sma I site of pbluescript SK was linearized with Xmn I and transcribed with T3 RNA polymerase to produce an ~250-nucleotide-long antisense probe that protects 207 nucleotides in the rat IL-1RA mRNA.
Plasmid prTNFtrans(T7) containing the peptide coding portion of rat
TNF- cDNA (nucleotides 1-708 of accession #S40199) was used.
Linearization of this plasmid by EcoR
I and transcription with SP6 RNA polymerase produced an
~790-nucleotide-long antisense probe that protects 708 nucleotides in
the rat TNF- mRNA.
Rat TGF-1 cDNA (accession #X52498) cloned into pbluescript II KS
vector (Stratagene) was used. Linearization of this plasmid by
BamH I and transcription with T3 RNA
polymerase produced an ~320-nucleotide-long antisense probe that
protects 244 nucleotides in the rat TGF-1 mRNA.
Rat Gp 130 cDNA (accession #M92340) cloned into EcoR I site of pBS vector was used. This cDNA was linearized with Bsa I and transcribed with T3 RNA polymerase to produce an ~590-nucleotide-long antisense probe that protects 530 nucleotides in the rat Gp 130 mRNA.
Leptin receptor riboprobe (Ob-Rb) template was generated by cloning a Hinc II-BamH I fragment of rat Ob-R cDNA into respective sites of pGEM-2. Ob-Rb was linearized by Hind III, and SP6 transcription resulted in an ~450-nucleotide-long antisense probe that protects 415 nucleotides (corresponding to nucleotides 686-1101 of accession #U52966) in the rat OB-R mRNA.
Rat POMC cDNA cloned between BamH I and EcoR I sites of pbluescript KS (+) vector was used. Plasmid was linearized with Xmn I, and T3 RNA polymerase transcription resulted in an ~320-nucleotide-long antisense probe that protects 264 nucleotides in the rat POMC mRNA (predominant form).
Plasmid pBLNPY-1 containing a 511-bp insert (accession #M20373)
comprising most of the cDNA of rat prepro-NPY ligated into the
EcoR I site of vector Bluescribe M13
() was used. Linearization of the plasmid with
Bbs I and transcription with T3 RNA
polymerase produced an ~242-nucleotide-long antisense probe that
protects 180 nucleotides in the rat NPY mRNA.
Rat antisense template pTRI--actin-125-Rat (Ambion; which contains a
126-bp cDNA fragment of the rat -actin gene) was used to generate
the -actin antisense probe. Rat antisense template pTRI-Cyclophilin
(Ambion; which contains a 103-bp cDNA fragment of the rat cyclophilin
gene) was used to generate the cyclophilin antisense probe.
Preparation of Rat Preprodynorphin Template
Primers 5'-AGAAGCCTGCCAGCGACAAAG-3' (corresponding to nucleotides 717-737 of rat dynorphin gene exon 1; accession #M32781) and 5'-CAGAGATGCAATTAACCCTCACTAAAGGGAGAGTCCTCGTCCCCAGTCATCTC-3' (corresponding to nucleotides 453-473 of rat dynorphin gene exon 4, accession #M32784 and including an additional sequence comprising T3 RNA polymerase promoter) were designed to amplify a fragment of the rat preprodynorphin cDNA. Reverse transcription was performed with 1 µg of total rat brain RNA and random hexamers in a final volume of 20 µl using RNA PCR Core Kit according to the protocol of the manufacturer (Perkin Elmer, Foster City, CA). Amplification was performed by using Taq 2000 DNA polymerase in a final volume of 100 µl according to the protocol of the manufacturer; this was subjected to 35 cycles denaturation at 95°C for 60 s, annealing at 60°C for 70 s, and extension at 72°C for 90 s. The PCR product was purified by the Wizard PCR Preps DNA Purification System (Promega, Madison, WI) and used directly for rat preprodynorphin antisense probe synthesis. The identity of the PCR product was confirmed by sequencing using the Sanger method with T3 RNA polymerase promoter specific primers.Data Analyses
Results are expressed as means ± SE. Data were analyzed using ANOVA followed by post hoc tests for pairwise comparisons (Student-Newman-Keuls method) if there was a significant main effect. Kruskal-Wallis ANOVA on ranks was applied (followed by post hoc tests) when data did not pass the normality (Kolmogorov-Smirnov) and equal variance (Levene Median) tests. Data were also analyzed using t-test or Mann-Whitney test (when data did not pass normality and equal variance tests). Differences were considered to be significant only for P < 0.05.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The same brain region samples were used to analyze the IL-1 system,
TNF-, TGF-1, Gp 130, OB-R, NPY, POMC, preprodynorphin, -actin,
and cyclophilin mRNA levels. Figures 1 and
2 present examples of the RNase protection
assays used. Hypothalamic samples from fed, fasted, and fasted-refed
animals were analyzed concomitantly. All samples were analyzed
individually, from which all means ± SE were generated
(n = 7). The levels of
cyclophilin mRNA (Fig. 1) and -actin mRNA (Fig. 2) were relatively
constant across the hypothalamic samples examined. This demonstrates
equivalent processing and that equal amounts of total RNA were used.
The specificity of the probes used has also been demonstrated
previously (6, 10, 26).
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Effects of Feeding Status on LPS-Stimulated Cytokine Ligand mRNA Expression
IL-1. IL-1 mRNA levels were barely detectable
in the hypothalamus from fed and fasted-refed vehicle groups (Fig.
3). However, control animals subjected to
acute food deprivation exhibited an ~10-fold difference of IL-1 mRNA
relative to both the fed and fasted-refed groups.
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LPS significantly upregulated hypothalamic IL-1 mRNA in all three
feeding conditions relative to the corresponding vehicle controls: fed,
P < 0.001; fasted,
P = 0.001; and fasted-refed, P < 0.001. There were no significant
differences in the IL-1 mRNA response to LPS treatment among the
three groups. However, when defining the net effect of LPS treatment as
the difference between vehicle and LPS-stimulated levels, the largest
effect was in the fed > fasted-refed > fasted group. That is,
although the feeding status did not affect the LPS-induced IL-1 mRNA
expression response, fasting per se decreased the net induction of
IL-1 mRNA.
IL-1RA. IL-1RA mRNA were similarly low
in the hypothalamus from fed and fasted-refed animals treated with
vehicle; fasted rats had increased levels (~3-fold increase relative
to the fed and fasted-refed groups; Fig.
4).
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IL-1RA mRNA levels were upregulated by LPS treatment in the fed (P < 0.001) and fasted-refed (P = 0.002) groups relative to the vehicle treatment (Fig. 4). There was no significant change in the fasted group. This suggests the possibility of a ceiling effect in LPS-induced modulation (i.e., the IL-1RA mRNA response to LPS treatment in the fed group was indistinguishable from that observed in the fasted group) and that fasting alone has a significant effect triggering IL-1RA mRNA expression. Interestingly, the LPS-stimulated IL-1RA mRNA levels were significantly (P = 0.008) lower in the fasted-refed group relative to the fed group. The data show that fasting attenuates the net IL-1RA mRNA response to LPS treatment: the fasted and fasted-refed groups had similar net increases of 5.7 and 6.3 arbitrary units, respectively, compared with a net increase of 20.3 arbitrary units in the fed group.
TNF-. Fasting was
associated with a fourfold (P < 0.001) induction of TNF- mRNA expression in the vehicle-treated
group (Fig. 5). There were no differences
between the fed and fasted-refed vehicle groups.
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LPS treatment upregulated TNF- mRNA levels in the fed
(P = 0.001), fasted
(P = 0.027), and fasted-refed
(P < 0.001) groups. The data show
similar net LPS-stimulated increases in the fed (24.6 arbitrary units)
and fasted-refed (19.6 arbitrary units) groups. A smaller net increase
of 12.2 arbitrary units was observed in the fasted group. All feeding
groups showed similar TNF- mRNA response to LPS treatment.
TGF-1.
No differences in TGF-1 mRNA profile were observed in any of the
three feeding conditions after vehicle administration (Fig.
6).
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LPS-induced TGF-1 mRNA levels in the fasted-refed group were lower
than those observed in the fed (P = 0.016) and fasted (P = 0.002) groups.
However, this may simply reflect the trend in TGF-1 mRNA levels
present in the fasted-refed vehicle group (Fig. 6).
Effects of Feeding Status on LPS-Stimulated Cytokine Receptor Component mRNA Expression
Food deprivation was associated with changes in mRNA expression of various cytokine receptor components. Fasted rats exhibited increased basal mRNA levels of IL-1RI (P < 0.001; Fig. 7), IL-1R AcP I (29%, from means of 13.6-17.5 arbitrary units, P = 0.007; Table 1), and Gp 130 (21%, from means of 13.2-16 arbitrary units, P = 0.01; Table 1). However, there were no significant differences for IL-1RI, IL-1R AcP I, or Gp 130 mRNAs between fed and fasted-refed vehicle groups (Fig. 7 and Table 1). There were also no differences in IL-1R AcP II and OB-R mRNA expression between fed and fasted or fasted-refed vehicle groups (Table 1).
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LPS treatment significantly induced IL-1RI mRNA expression in the fed (P < 0.001), fasted (P = 0.012), and fasted-refed (P <0.001) groups (Fig. 7). The net increases from vehicle were highest in the fed (14.1 arbitrary units) > fasted-refed (10.9 arbitrary units) > fasted (7.1 arbitrary units). However, the data revealed no significant differences in LPS-stimulated IL-1RI mRNA response among the feeding groups.
The results for other cytokine receptor components are presented in Table 1. Gp 130 mRNA expression was upregulated by LPS treatment in the fed (P < 0.001), fasted (P = 0.003), and fasted-refed (P = 0.01) groups. The highest net increase from vehicle was observed in the fed group (5.8 arbitrary units). Fasting attenuated this increase (to 2.8). Again, no differences in LPS-stimulated Gp 130 mRNA responses were observed.
LPS also stimulated IL-1R AcP I mRNA levels in the fed (P = 0.008) and fasted-refed (P = 0.004) groups relative to the vehicle treatment; there was no significant change in fasted animals. The LPS-associated difference of IL-1R AcP II mRNA expression in fasted-refed animals was dependent on the significantly lower levels in the corresponding vehicle group. LPS treatment did not modulate mRNA levels of OB-R under any of the three feeding conditions used.
Effects of Feeding Status on LPS-Modulated Neuropeptide mRNA Profiles
Table 1 presents the neuropeptide mRNA profiles obtained in the three feeding conditions. Fasting decreased POMC mRNA expression by 25% (from means of 19.7-14.8 arbitrary units, P = 0.003), a change that was unaffected by refeeding (P < 0.003). Preprodynorphin mRNA levels were unaffected in fasted animals, whereas the fasted-refed group exhibited a 42% increase (from 14.3 to 20.3 arbitrary units, P = 0.003). In this study, 39.5 h fasting was associated with only a slight increase in basal NPY mRNA levels that was normalized by refeeding.There were no changes in POMC mRNA expression in LPS-treated animals relative to the corresponding controls. LPS treatment upregulated preprodynorphin (P < 0.001) mRNA levels in fed animals only. In addition, the preprodynorphin mRNA levels in the fed group were 85% higher relative to fasted group (from means of 11.8-21.8 arbitrary units). LPS-treated animals also exhibited a small increase (P < 0.02) in NPY mRNA levels in the fed group (Table 1).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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This study shows that acute fasting modulates mRNA levels of various cytokine and neuropeptide components in the hypothalamus. However, overall LPS-induced changes were unaffected by fasting.
Fasting-Associated mRNA Profiles
Fasted animals exhibited increases in the hypothalamic expression of IL-1, IL-1RA, TNF-, IL-1RI, IL-1R AcP I, and Gp 130 mRNAs
relative to the fed and fasted-refed groups. In all cases, refeeding
attenuated or completely blocked the increase. The cytokine mRNA
changes observed in response to fasting are specific. TGF-1 mRNA
levels, which consistently follow the trend of proinflammatory cytokines (6, 10, 26), were the same in the fasted and fed groups.
Moreover, POMC mRNA levels were similar in the fasted and fasted-refed
groups, whereas preprodynorphin mRNA levels were comparably lower in
the fasted and fed groups.
Previous studies showed that various forms of stress induce cytokine expression (3, 21, 22, 28). The present data suggest that acute fasting could act as a stressful stimulus that modulates cytokine gene expression.
Fasting decreases hypothalamic POMC mRNA levels (13, 19). Our data confirm these reports. On the other hand, we did not detect changes in preprodynorphin or NPY mRNA levels after fasting as previously described (2, 12, 13, 14, 16, 17). However, the current experimental approach does not detect subregional changes within the hypothalamus (2, 12, 13, 14). Interestingly, we observed a 42% increase in preprodynorphin mRNA in animals that were refed after fasting (Table 1). This change may be related to the proposal of endogenous opioids mediating rewarding aspects of food intake (13).
LPS-Stimulated mRNA Profiles
LPS significantly stimulated the mRNA expression of hypothalamic IL-1, IL-1RI, TNF-, and Gp 130 in all three feeding groups and of
IL-1RA and IL-1R AcP I in the fed and fasted-refed groups. The net
LPS-induced increases (difference between vehicle and LPS-stimulated
levels) were usually highest in fed > fasted-refed > fasted.
However, food deprivation did not affect the response to LPS treatment
at a dose that is sufficient to evoke a robust immune response in the
brain. These findings suggest that acute food deprivation does not
compromise the ability of brain cytokine systems to respond to LPS.
This is consistent with various studies that showed that LPS-induced
modulation of other systems was unaffected by food deprivation (7, 20).
Of course, a more prolonged fasting, which results in the depletion of
energy stores and potentially in malnutrition, may negatively impact on
the response to LPS (20). The possibility of a ceiling effect in
LPS-induced modulation of cytokine mRNA components depending on the
dose/concentration of LPS should also be considered.
Bacterial LPS increased preprodynorphin (52%) and NPY (19%) mRNAs in the fed group. Fasting attenuated these effects. These findings demonstrate that acute fasting, in fact, can selectively modulate LPS action on other hypothalamic chemical noncytokine systems.
Perspectives
Acute food deprivation induces hypothalamic expression of various, but not all, cytokine mRNAs. This cytokine mRNA response may be due to the fasting-induced stress. In turn, cytokines may mediate aspects of the fasting-associated stress response. This is consistent with the evidence of cytokine modulation in response to various stressors (21, 22, 28). Acute fasting, on the other hand, does not affect LPS-induced cytokine modulation in the hypothalamus, an important brain site for LPS action. This suggests that homeostatic adaptive mechanisms activated by acute fasting maintain LPS action on brain cytokine modulation. However, acute fasting does affect LPS-induced modulation of hypothalamic preprodynorphin and NPY mRNAs. These results show specificity of fasting as well as of LPS action. The results also have implications for gram-negative brain infections in which significant or complete anorexia frequently occurs. That is, even in extreme cases of an acute reduction in energy intake, LPS modulation of cytokine-associated transcriptional changes in the hypothalamus is fully maintained.| |
ACKNOWLEDGEMENTS |
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We thank Dr. Ronald P. Hart (Dept. of Biological Sciences, Rutgers
University) for providing the rat IL-1, IL-1RA, IL-1RI, and IL-1R
AcP cDNAs; Dr. Karl Decker (Biochemisches Institut der Albert Ludwigs
Universität) for providing the rat TNF- cDNA; Dr. David
Danielpour (National Cancer Institute) for providing the rat TGF-1
cDNA; Dr. Gerald M. Fuller (Dept. of Cell Biology and Anatomy,
University of Alabama at Birmingham) for providing the rat Gp 130 cDNA;
Dr. Charles I. Rosenblum (Merck, Rahway, NJ) for providing the rat
leptin receptor (Ob-Rb isoform) cDNA; Dr. Andrea Gore (Center for
Neurobiology, The Mount Sinai Medical Center) for providing the rat
proopiomelanocortin cDNA; and Dr. Steven L. Sabol (Laboratory of
Biochemical Genetics, National Heart, Lung, and Blood Institute) for
providing the rat NPY cDNA.
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FOOTNOTES |
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Research was supported by funds from the University of Delaware and the National Institutes of Health (to C. R. Plata-Salamán).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. R. Plata-Salamán, Division of Molecular Biology, School of Life and Health Sciences, University of Delaware, Newark, DE 19716-2590 (E-mail: crps{at}udel.edu).
Received 18 February 1999; accepted in final form 16 June 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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