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Keywords:

  • activator protein-1;
  • cachexia;
  • c-Jun kinase;
  • inflammation;
  • obesity;
  • tumor necrosis factor

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Tumor necrosis factor-α (TNF-α) is known to participate in the wastage syndrome that accompanies cancer and severe infectious diseases. More recently, a role for TNF-α in the pathogenesis of type 2 diabetes mellitus and obesity has been shown. Much of the regulatory action exerted by TNF-α upon the control of energy stores depends on its action on the hypothalamus. In this study, we show that TNF-α activates canonical pro-inflammatory signal transduction pathways in the hypothalamus of rats. These signaling events lead to the transcriptional activation of an early responsive gene and to the induction of expression of cytokines and a cytokine responsive protein such as interleukin-1β, interleukin-6, interleukin-10 and suppressor of cytokine signalling-3, respectively. In addition, TNF-α induces the expression of neurotransmitters involved in the control of feeding and thermogenesis. Thus, TNF-α may act directly in the hypothalamus inducing a pro-inflammatory response and the modulation of expression of neurotransmitters involved in energy homeostasis.

Abbreviations used
AP-1

activator protein-1

CRF/CRH

corticotropin releasing factor/hormone

DIO

diet-induced obesity

i.c.v.

intracerebroventricular

IL-1β

interleukin-1β

IL-6

interleukin-6

IL-10

interleukin-10

JNK

c-Jun kinase

LPS

lipopolysaccharide

MCH

melanin concentrating hormone

NPY

neuropeptide Y

POMC

proopiomelanocortin

SOCS-3

suppressor of cytokine signaling-3

TNF-α

tumor necrosis factor-α

TNF-R1

TNF-α receptor type 1

TNF-R2

TNF-α receptor type 2

Much interest has centered on the anorexigenic properties of tumor necrosis factor-α (TNF-α), due to its participation in the wastage syndromes that accompany cancer and infectious diseases (Matthys and Billiau 1997; Plata-Salaman 2000; Wong and Pinkney 2004). According to Tracey and coworkers, the hypothalamic production and action of TNF-α are required in order to achieve the full clinical expression of cachexia (Tracey et al. 1990). Both infection- and lipopolysaccharide (LPS)-induced anorexia can be reverted by treatment with anti-TNF-α antibodies, with soluble TNF-α receptors or with chemical inhibition of TNF-α synthesis (Haslett 1998; Porter et al. 2000). In addition, tumor-induced anorexia and weight loss can be partially rescued by treatment with a soluble TNF-α receptor that antagonizes TNF-α activity (Torelli et al. 1999).

In recent years, a number of studies have shown that TNF-α and other pro-inflammatory proteins are produced by the adipose tissue during the development of obesity (Katsuki et al. 1998; Schmidt et al. 1999). The increased levels of some of these pro-inflammatory proteins in the blood and in insulin-sensitive tissues play an important role in the induction of insulin resistance, which is a hallmark of type 2 diabetes mellitus and occurs in most obese humans and animals (Hotamisligil et al. 1993, 1994; Hotamisligil 2003). Interestingly, insulin, acting in concert with leptin, provides the most robust adipostatic and anorexigenic signals to the hypothalamus (Folli et al. 1994; Schwartz et al. 2000; Carvalheira et al. 2001; Flier 2004), and a phenomenon of hypothalamic resistance to leptin and insulin has been demonstrated in different animal models of obesity (Carvalheira et al. 2003; Torsoni et al. 2003; Howard et al. 2004). In a recent study, we showed that, in diet-induced obesity (DIO), the expression of TNF-α and other pro-inflammatory proteins is remarkably increased in the hypothalamus of rats (De Souza et al. 2005). The inhibition of TNF-α signaling in these animals reduces caloric intake, promotes body weight loss and reverts hypothalamic resistance to insulin (De Souza et al. 2005).

Based on cancer, infection and, more recently, on obesity studies, we can assume that TNF-α and other pro-inflammatory cytokines play important roles in the control of body energy stores. In some cases, they participate in energy wastage, whereas in others, they take part in body weight gain. Much of this modulatory function seems to be dependent on cytokine signaling in the hypothalamus. However, little is known about the signaling pathways involved in TNF-α action in the hypothalamus and on the control exerted by this cytokine upon local protein and neurotransmitter expression. Therefore, the objective of this study was to evaluate the property of TNF-α to activate pro-inflammatory signaling and protein expression in the hypothalamus of rats.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Antibodies, chemicals and buffers

Reagents for sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting were from Bio-Rad (Richmond, CA, USA). HEPES, phenylmethylsulfonyl fluoride, aprotinin, dithiothreitol, Triton X-100, Tween 20, glycerol, bovine serum albumin (fraction V), and angiotensin II were from Sigma (St. Louis, MO, USA). Protein A-Sepharose 6MB was from Pharmacia (Uppsala, Sweden), 125I-protein A was from ICN Biomedicals (Costa Mesa, CA, USA), and nitrocellulose paper (BA85, 0.2 µm) was from Amersham (Aylesbury, UK). Sodium thiopental (Amytal) and human recombinant insulin (Humulin R) were from Lilly (Indianapolis, IN, USA). Mouse recombinant TNF-α was from Calbiochem (Darmstadt, Germany). The anti-TNF-α monoclonal antibody, Infliximab, was from Centocor (Horsham, PA, USA). Anti-phospho-c-Jun kinase (JNK) (mouse monoclonal, recognizing JNK phosphorylated at Thr 183 and Tyr 185, sc-6254), anti-phospho-p38 (mouse monoclonal, recognizing p38 phosphorylated at Tyr 182, sc-7973), anti-c-Fos (rabbit polyclonal, sc-7202), anti-c-Jun (rabbit polyclonal, sc-1694), anti-interleukin-1β (IL-1β) (rabbit polyclonal, sc-7884), anti-IL-6 (goat polyclonal, sc-1265), anti-IL-10 (goat polyclonal, sc-1783), anti-supressor of cytokine signaling-3 (SOCS-3) (rabbit polyclonal, sc-9023) and CRF/CRH (corticotropin-releasing factor/hormone) (rabbit polyclonal, sc-10718) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Experimental animals

Eight-week old male Wistar rats (Rattus norvegicus) from the University of Campinas Central Animal Breeding Center were used in the experiments. The rats were allowed access to standard rodent chow and water ad libitum. Food was withdrawn 12 h before the experiments. All experiments were conducted in accord with the principles and procedures described by the NIH Guidelines for the Care and Use of Experimental Animals and were approved by the State University of Campinas Ethical Committee. In all experiments, the rats were cannulated intracerebroventricularly (i.c.v.) and submitted to treatment with TNF-α (accompanied or not by Infliximab or insulin, according to the protocols described below).

Intracebroventricular cannulation

All rats were stereotaxically instrumented using a Stoelning stereotaxic apparatus, according to a method previously described (Torsoni et al. 2003). Cannula efficiency was tested 1 w after cannulation by the evaluation of the drinking response elicited by i.c.v. angiotensin II (Johnson and Epstein 1975).

Protocol for food ingestion determination

Rats cannulated i.c.v. were food deprived for 6 h (from 12 to 18 h) and at 18 h were treated i.c.v. with insulin (2.0 µL, 10−6mol/L), TNF-α (2.0 µL, 10−8 mol/L), TNF-α followed by insulin (after 1 h), Infliximab (2.0 µL, 10 mg/mL, i.c.v.) followed by TNF-α (after 1 h) and then by insulin (after 1 h), or saline (2.0 µL). Food ingestion was determined over the next 12 h.

Tissue extraction, immunoblotting and immunoprecipitation

Rats cannulated i.c.v. were anesthetized and acutely treated with saline (2.0 µL) or TNF-α (in most experiments the dose of TNF-α was 10−8 mol/L, 2.0 µL, except in dose–response experiments when the concentration ranged from 10−12 to 10−6 mol/L). Some rats were pre-treated with Infliximab (2.0 µL, 10 mg/mL, i.c.v.) 1 h before TNF-α injection. Some rats were treated with insulin (2.0 µL, 10–6 m, i.c.v., alone or 1 h after TNF-α). After 10 min, as determined by time-course experiments, the hypothalami were obtained and immediately homogenized in solubilization buffer at 4°C [1% Triton X-100, 100 mmol/L Tris-HCl (pH 7.4), 100 mmol/L sodium pyrophosphate, 100 mmol/L sodium fluoride, 10 mmol/L EDTA, 10 mmol/L sodium orthovanadate, 2.0 mmol/L phenylmethylsulfonyl fluoride and 0.1 mg aprotinin/mL] with a Polytron PTA 20S generator (model PT 10/35; Brinkmann Instruments, Westbury, NY, USA). Insoluble material was removed by centrifugation for 20 min at 9000 g in a 70.Ti rotor (Beckman) at 4°C. The protein concentration of the supernatants was determined by the Bradford dye binding method. Aliquots of the resulting supernatants containing 2.0 mg of total protein were used for immunoprecipitation with antibodies against c-Fos at 4°C overnight, followed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transfer to nitrocellulose membranes and blotting with anti-c-Jun. In direct immunoblot experiments, 0.2 mg of protein extracts were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and blotted with anti-phospho-JNK, anti-phospho-p38, anti-c-Fos, anti-c-Jun, anti-IL-1β, anti-IL-6, anti-IL-10, anti-SOCS-3 and anti-CRF/CRH antibodies, as described (Araujo et al. 2005).

Plasmid transfection and reporter gene assay

A plasmid containing the positions −250 to +150 of the c-jun promoter fused to the firefly luciferase gene (pJC6GL3) was kindly donated by Dr Ron Prywes (Columbia University, New York, NY, USA). Rats cannulated i.c.v. received 0.1 µg of internal control SV40-renila luciferase (Invitrogen, Carlsbad, CA, USA) with or without 2.0 µg of pJC6GL3 and, after 6 days, the rats were treated i.c.v. with saline (2.0 µL) or TNF-α (10−8 M, 2.0 µL). After 6 h the hypothalamus was obtained and a protein extract was prepared and used for determination of luciferase activity according to the recommendations of the manufacturer (Dual-Luciferase Reporter Assay System, Promega). This protocol has been previously optimized (Nadruz et al. 2005).

RNA preparation for reverse transcription–polymerase chain reaction

Total hypothalamic RNA was extracted using Trizol (Life Technologies, Gaithersburg, MD, USA) reagent, according to the manufacturer's recommendations. Total RNA was rendered genomic DNA-free by digestion with RNAse-free DNAse (RQ1; Promega, Madison, Wisconsin).

Semiquantitative reverse transcription–polymerase chain reaction

Seven micrograms of total RNA were reverse-transcribed with SuperScript reverse transcriptase (200 U/µL) using oligo (dT) (50 mmol/L) in a 30 µL reaction volume (5 × RT buffer, 10 mmol/L dNTP and 40 U/µL RNAse free inhibitor). The reverse transcriptions involved a 50-min incubation at 42°C and a 15 min incubation at 70°C. The PCR products were submitted to 1.5% agarose gel electrophoresis containing ethidium bromide and visualized by excitation under UV light. Photo-documentation was performed using the Nucleovision System (NucleoTech, San Mateo, CA, USA) and band quantification was performed using the Gel Expert Software (NucleoTech). In all samples, the amplification of RPS-29 was performed and used as an internal control for quantity and quality. The semiquantitative expression (SE) of neuropeptide Y (NPY) was calculated using the formula: SE = pixel area of product/pixel area of RPS-29 × 100. The primers used, and the PCR conditions were: RPS-29 (NCBI: NM012876), sense: 5′-AGGCAAGATGGGTCACCAGC-3′, antisense: 5′-AGTCGAATCATCCATTCAGGTCG-3′ (fragment: 202 bp; Tm: 57°C; amplification: 27 cycles); MCH – melanin concentrating hormone (NCBI: M29712), sense: 5′-TACGGAGCAGCAAACA-3′, antisense: 5′-ACAGCCAGACTGAGGG-3′ (fragment: 323 bp; Tm: 55°C; amplification: 25 cycles); POMC – proopiomelanocortin (NCBI: AF510391), sense: 5′-CTCCTGCTTCAGACCTCCAT-3′, antisense: 5′-TTGGGGTACACCTTCACAGG-3′ (fragment: 398 bp;Tm: 63°C; amplification: 32 cycles); NPY – neuropeptide Y (NCBI: NM012614), sense: 5′-AGAGATCCAGCCCTGAGACA-3′, antisense: 5′-AACGACAACAAGGGAAATGG-3′ (fragment: 236 bp; Tm: 62°C; amplification: 31 cycles); TNF-R1 – TNF-α receptor type 1 (NCBI: NM013091), sense: 5′-CGGGCTTACTGGATACGAGA-3′, antisense: 5′-CACACACCTCGCAGACTGTT-3′ (fragment: 456 bp; Tm: 52°C; amplification: 22–42 cycles); TNF-R2 – TNF-α receptor type 2 (NCBI: NM012610), sense: 5′-ACTCAGACGAAGCCAACCAC-3′, antisense: 5′-CACCTCTTGAAAGCAATATAG-3′ (fragment: 364 bp; Tm: 54°C; amplification: 22–42 cycles).

Statistical analysis

Specific protein bands present on the blots were quantified by densitometry. Mean values ± SEM obtained from densitometric scans were compared utilizing Student's t-test for paired samples or by repeat-measures analysis of variance (one-way or two-way anova) followed by post hoc analysis of significance (Bonferroni test) when appropriate. A p < 0.05 was accepted as statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Tumor necrosis factor-α activates pro-inflammatory signal transduction and early inducible gene expression in the hypothalamus

Initially, by using RT–PCR we detected the presence of mRNA for both type 1 and type 2 TNF-α receptors in the hypothalamus. Optimal amplifications were obtained by 38 and 31 cycles for TNF-R1 and TNF-R2, respectively (n = 4). To establish the optimal dose and time for TNF-α to activate pro-inflammatory signaling in the hypothalamus, rats cannulated i.c.v. were treated with 2.0 µL of 10−8 mol/L TNF-α and after times ranging from 0 to 30 min the hypothalamus was obtained. In parallel studies, 2.0 µL TNF-α, in concentrations ranging from 10−12 to 10−6 mol/L, were injected i.c.v. and the hypothalamus was obtained after 10 min for use in immunoblot experiments. As shown in Figs 1(a) and (b), TNF-α promoted maximum phosphorylation of JNK at the dose of 10−8 mol/L and at the time of 10 min. TNF-α was also able to produce the phosphorylation of p38 at the optimal time of 10 min (Fig. 1c). To evaluate the effect of TNF-α on the activation of a pro-inflammatory transcription factor, rats cannulated i.c.v. were treated with 2.0 µL of 10−8 mol/L TNF-α and, after the times ranging from 0 to 180 min, the hypothalamus was obtained and used in immunoprecipitation assays with anti-c-Fos antibody. The immunoprecipitates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and blotted with anti-c-Jun antibody. AP-1 complex formation started as early as 5 min, reaching a peak at 15 min and returning to basal levels at 60 min (Fig. 1d). Finally, TNF-α (2.0 µL, 10−8 mol/L) injected i.c.v. was able to induce hypothalamic c-Fos (Fig. 1e) and c-Jun (Fig. 1f) expression, which was detectable at 60 min for c-Fos, and started at 15 min, increasing progressively until 60 min for c-Jun. To test the specificity of the signaling events described above, rats cannulated i.c.v. were treated with the anti-TNF-α monoclonal antibody Infliximab and after 60 min with TNF-α (2.0 µL, 10−8 mol/L). The pre-treatment with Infliximab completely inhibited TNF-α-induced activation of JNK (Fig. 2a) and p38 (Fig. 2b).

image

Figure 1.  Tumor necrosis factor-α (TNF-α)-induced signal transduction and activation of early responsive gene expression in the hypothalamus. Anesthetized rats were i. c.v. treated with 2.0 µL of 10−8 mol/L TNF-α (a, c–f) or at the concentrations depicted in (b). After the time frames, as depicted in (a, c–f) or after 10 min (b), hypothalami were obtained for protein extract preparation. For direct immunoblot analysis (a–c, e, f), samples containing 0.2 mg protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and blotted (IB) with specific antibodies to phospho-JNK (p-JNK) (a, b), phospho-p38 (p-p38) (c), c-Fos (e) and c-Jun (f). For immunoprecipitation followed by immunoblot, samples containing 2.0 mg total protein were submitted to immunoprecipitation (IP) with antibodies to c-Fos (d); immunoprecipitates were collected with Protein A-Sepharose and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and blotted with antibodies to c-Jun. Specific bands were quantified by densitometric analysis. In all experiments, n = 4; *p < 0.05 vs. time 0 (a, c–f) or vs. concentration 0 (b). Results are presented as mean ± SEM.

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image

Figure 2.  Specificity of the tumor necrosis factor-α (TNF-α)-induced signal transduction in the hypothalamus. Anesthetized rats were treated intracerebroventricularly (i.c.v.) with 2.0 µL, 10−8 mol/L TNF-α or pre-treated with Infliximab 2.0 µL, 10 mg/mL and after 1 h with 2.0 µL of 10−8 mol/L TNF-α (I + T). After 10 min, hypothalami were obtained for protein extract preparation. Samples containing 0.2 mg protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and blotted (IB) with specific antibodies to phospho-JNK (p-JNK) (a) or phospho-p38 (p-p38) (b). Specific bands were quantified by densitometric analysis. In all experiments, n = 4; *p < 0.05 vs. control (C) (treated i.c.v. with similar volume of saline). Results are presented as mean ± SEM.

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Tumor necrosis factor-α activates gene transcription through activator protein-1 in the hypothalamus

To examine whether acute TNF-α treatment induces transcriptional regulation of c-jun, rats cannulated i.c.v. were transfected with the plasmid pJC6GL3 containing the c-jun promoter fused to the firefly luciferase gene (Fig. 3a). As depicted in Fig. 3(b), the treatment with TNF-α promoted a 30-fold increase in the luciferase activity in the hypothalamus at 6 h.

image

Figure 3.  Activation of the c-jun promoter by tumor necrosis factor-α (TNF-α) in the hypothalamus. The schematic representation of the c-jun promoter fused to the firefly luciferase gene is depicted in (a). In (b), rats cannulated intracerebroventricularly were treated with 0.1 µg internal control SV40-renila luciferase without (C) or with (c-jun) 2.0 µg of the pJC6GL3 construct containing the c-jun promoter. After 6 days, rats were treated with a single dose of TNF-α (10−8 mol/L, 2.0 µL) and after 6 h, hypothalami were obtained for determination of luciferase activity. In all experiments, n = 4; *p < 0.05 vs. control (C). Results are presented as mean ± SEM.

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Tumor necrosis factor-α induces the expression of cytokines and supressor of cytokine signalling-3 in the hypothalamus

To evaluate the ability of TNF-α to modulate cytokine and cytokine responsive protein expression in the hypothalamus rats cannulated i.c.v. were treated with a single dose of TNF-α (2.0 µL, 10−8 mol/L) and after times ranging from 0 to 180 min the hypothalami were obtained and used in immunoblotting experiments. As depicted in Fig. 4, TNF-α induced the hypothalamic expression of IL-1β, starting at 15 min and increasing continuously up to 180 min (Fig. 4a). TNF-α also induced the expression of IL-6, starting at 15 min and increasing continuously up to 180 min (Fig. 4b), and of IL-10, starting at 15 min and increasing continuously up to 180 min (Fig. 4c). Finally, TNF-α was able to induce the expression of the inhibitor of cytokine signaling, SOCS-3, which was detected at 120 and 180 min (Fig. 4d).

image

Figure 4.  Tumor necrosis factor-α (TNF-α) induces the expression of cytokines and a cytokine responsive protein in the hypothalamus. Anesthetized rats were treated intracerebroventricularly with 2.0 µL of 10−8 mol/L TNF-α and after the time frames depicted in the figure, the hypothalami were obtained for protein extract preparation. Samples containing 0.2 mg protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and blotted (IB) with specific antibodies to interleukin-1β (IL-1β) (a), IL-6 (b); IL-10 (c) or supressor of cytokine signaling-3 (SOCS-3) (d). Specific bands were quantified by densitometric analysis. In all experiments, n = 4; *p < 0.05 vs. control (0). Results are presented as mean ± SEM.

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Tumor necrosis factor-α inhibits food intake and modulates orexigenic/anorexigenic neurotransmitter expression in the hypothalamus

The effects of acute treatment with TNF-α were evaluated upon the expression of two orexigenic (NPY and MCH) and two anorexigenic (POMC and CRH) neuropeptides. TNF-α induced the expression of NPY (Fig. 5a), POMC (Fig. 5b) and MCH (Fig. 5c) mRNAs, starting at 15 min and lasting for at least 120 min. TNF-α also induced CRF/CRH protein expression (Fig. 5d), beginning at 120 min. Of note, is the finding that the TNF-α-induced increase in the orexigenic neurotransmitter levels reached a maximum of 1.3-fold, whereas the increase in anorexigenic neurotransmitter levels reached 1.8-fold for CRF/CRH and 8.0-fold for POMC. Finally, the acute effect of TNF-α on spontaneous food intake was determined and compared with the effect of insulin. As depicted in Fig. 5(e), insulin promoted a 45% reduction in 12-h food intake, whereas TNF-α induced a 25% reduction in feeding during the same time frame.

image

Figure 5.  Tumor necrosis factor-α (TNF-α) regulates hypothalamic neurotransmitter expression and food intake. Anesthetized rats were treated intracerebroventricularly (i. c.v.) with 2.0 µL of 10−8 mol/L TNF-α and, after the time frames depicted in the figure the hypothalami were obtained for RNA (a–c) or protein extract (d) preparation. In (a–c), RT–PCR reactions were conducted according to the settings presented in Materials and methods section. The amounts of neuropeptide Y (NPY) (a), proopiomelanocortin (POMC) (B) or melanin concentrating hormone (MCH) (c) were calculated as a fraction of the internal control RPS-29. In (d), samples containing 0.2 mg protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and blotted (IB) with a specific antibody to corticotropin releasing factor/hormone (CRF/CRH). Specific bands were quantified by densitometric analysis. In (e), rats cannulated i.c.v. were food deprived for 6 h (from 12 to 18 h) and at 18 h were treated i.c.v. with insulin (2.0 µL, 10−6mol/L), TNF-α (2.0 µL, 10−8 mol/L) or saline (S) (2.0 µL). Food ingestion was determined over the next 12 h. In all experiments, n = 4; *p < 0.05 vs. control (0) (a–d) and saline (S) (e); §p < 0.05 vs. insulin (e). Results are presented as mean ± SEM.

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Acute treatment with tumor necrosis factor-α does not modulate the anorexigenic effect of insulin in the hypothalamus

To evaluate the effect of TNF-α upon insulin action in the hypothalamus, rats cannulated i.c.v. were treated with TNF-α and after 1 h with insulin. Spontaneous food intake was determined over the next 12 h. As depicted in Fig. 6(a), a single acute dose of TNF-α before insulin did not modify the capacity of insulin to inhibit approximately 50% food intake. In addition, although a single acute dose of insulin was capable of inducing the phosphorylation/activation of JNK (Fig. 6b) and p38 (Fig. 6c), the combined treatment of TNF-α plus insulin did not modify the level of phosphorylation/activation of these two intermediaries, imposed by a single dose of TNF-α.

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Figure 6.  Effect of tumor necrosis factor-α (TNF-α) on insulin action in the hypothalamus. In (a), rats cannulated intracerebroventricularly (i. c.v.) were food deprived for 6 h (from 12 to 18 h). Twelve hours food intake was determined in rats treated with insulin (Ins) (2.0 µL, 10−6 mol/L), TNF-α (2.0 µL, 10−8 mol/L), saline (S) (2.0 µL), TNF-α followed, after 1 h, by insulin (T + Ins), or Infliximab (I) (2.0 µL, 10 mg/mL) followed, after 1 h, by TNF-α and, after 1 h, by insulin (I + T + Ins). In (b) and (c), anesthetized rats were i.c.v. treated with 2.0 µL, 10−8 mol/L TNF-α, 2.0 µL, 10−6mol/L insulin (Ins), or pre-treated with TNF-α and after 1 h with insulin (T + Ins). After 10 min, hypothalami were obtained for protein extract preparation. Samples containing 0.2 mg protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and blotted (IB) with specific antibodies to phospho-JNK (p-JNK) (b) or phospho-p38 (p-p38) (c). Specific bands were quantified by densitometric analysis. In all experiments, n = 4; *p < 0.05 vs. control (C) (b and c) and (S) (a); §p < 0.05 vs. insulin (b and c). Results are presented as mean ± SEM.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

TNF-α signal transduction is dependent on ligand binding to at least two distinct receptors, TNF-R1 and TNF-R2 (Locksley et al. 2001). TNF-R2 is expressed mostly in cells of the immune system and is thought to play an important role in the ontogenesis and differentiation of lymphoid tissues (Locksley et al. 2001), whereas TNF-R1 is widely distributed and mediates TNF signaling towards activation of pro-inflammatory responses, control of gene expression, and regulation of apoptosis, amongst a number of other functions (Wajant et al. 2003).

In cells of the immune system and in other tissues, the intracellular outcomes of TNF-α signaling have been thoroughly studied and the capacity of this cytokine to activate signal transduction through NF-kappaB, JNK, p38 and intermediaries of apoptosis is well known. However, in the central nervous system and more specifically in the hypothalamus, little is known about the transduction of the TNF-α signal (Owens et al. 2005).

Here, we have evaluated TNF-α signal transduction and activation of gene expression in the hypothalamus of rats. Although the mRNA for both types of receptor were detected in the hypothalamus, we believe that most, if not all of the effects of TNF-α determined in this study were dependent on TNF-R1, as it has been previously shown that TNF-R2 protein is almost absent in the brain of rodents not exposed to any pro-inflammatory or growth-inducing stimulus (Bette et al. 2003). In the first part of the study, we showed that an acute i.c.v. injection of TNF-α is capable of inducing the activation of JNK and p38. These events were accompanied by the induction of AP-1 and the expression of c-Jun and c-Fos. We also showed that these effects are specifically dependent on TNF-α, as the pre-treatment of rats with the anti-TNF-α monoclonal antibody, Infliximab, completely prevented the activation of signal transduction. Finally, by evaluating the outcomes of i.c.v. TNF-α treatment upon the activation of the transfected construct containing the c-jun promoter fused to the firefly luciferase gene, we provide strong evidence for the integrity of the TNF-α signaling system, from membrane to nucleus, in cells of the hypothalamus.

No previous study has looked into JNK and p38 engagement by TNF-α in the hypothalamus. In other regions of the central nervous system and in brain cell cultures, TNF-α signaling has been shown to induce the activation of JNK and p38 in conditions such as acute stimulation with TNF-α, experimental trauma and viral infections (Zhang et al. 1996; de Bock et al. 1998; Ladiwala et al. 1999). In most cases, TNF-α or TNF-α-inducing stimuli promote an early activation of JNK and p38 signaling in a pattern very similar to the one observed here in the hypothalamus. Thus, we can assume that the hypothalamus of rats is fully equipped with the molecular apparatus required for TNF-α signal transduction through JNK and p38.

We next evaluated the effects of TNF-α upon the expression of two pro-inflammatory cytokines (IL-1β and IL-6) and one anti-inflammatory (IL-10) cytokine and of a cytokine responsive protein, SOCS-3, in the hypothalamus. As a rule, all three cytokines were rapidly induced in response to TNF-α, starting as early as 15 min and increasing continuously to up to 3 h. In cell culture systems (Sawada et al. 1992; Norris et al. 1994; Sheng et al. 1995) and in different regions of the brain (Bristulf and Bartfai 1995; Pitossi et al. 1997; Zhai et al. 1997; Didier et al. 2003), TNF-α has been shown to induce the expression of cytokines. In general, the response to TNF-α stimulus is rapid, suggesting that this is a primary event. This seems to be the case here. We believe that, in our experiments, the induction of IL-1β, IL-6 and IL-10 were all primarily dependent on TNF-α and required no intermediaries. With regard to SOCS-3, it was initially thought that its expression would be controlled exclusively by cytokine signaling through type I cytokine receptors (Auernhammer and Melmed 2001). More recently, it was realized that SOCS-3 could be induced also by cytokine and hormone signaling through other classes of receptors, including TNF-α (Bjorbaek et al. 1998; Emanuelli et al. 2001; Rui et al. 2002; Calegari et al. 2003; Torsoni et al. 2004). The capacity of TNF-α to induce SOCS-3 was shown in adipose tissue (Emanuelli et al. 2001), in liver (Campbell et al. 2001; Sass et al. 2005) and in cells of the immune system (Bode et al. 2003). However, no previous study has demonstrated the capacity of TNF-α to induce SOCS-3 (or other SOCS family members) in neural tissues. Due to the moderately long time frame required for TNF-α to induce SOCS-3 in our study, we cannot be sure if this is a primary event or if it depends on the expression of intermediaries. It is of particular interest that IL-6 has been shown to induce SOCS-3 in the brain (Lebel et al. 2000); however, in this case, and also in most other situations that lead to the induction of SOCS-3, there is a requirement for a moderately long time for the protein to be detected. Thus, at this point we can assume that TNF-α is capable of inducing SOCS-3 expression in the hypothalamus but whether this is a primary or a secondary event remains to be determined.

In the last part of the study, we evaluated the capacity of TNF-α to modulate neurotransmitter expression in the hypothalamus. Using RT–PCR or immunoblot, we observed that both orexigenic and anorexigenic peptides were significantly and rapidly induced by TNF-α treatment. Due to the involvement of TNF-α in the cachexia syndrome that accompanies cancer and some infectious diseases, much interest has been drawn upon the ability of TNF-α to modulate the expression of hypothalamic peptides involved in energy homeostasis; however, in most studies the effects of TNF-α were evaluated indirectly. There are quite a number of studies showing that TNF-α and other pro-inflammatory cytokines, such as IL-1β and IL-6, can modulate the hypothalamic amounts of serotonin, tryptophan and norepinephrine (for a comprehensive review see Dunn 2000). In addition, some studies have shown that, in most systems tested, TNF-α is capable of inducing increases in the expressions of CRF/CRH (Bernardini et al. 1990; Watanobe and Takebe 1992) and POMC/MSH (Wisse et al. 2003). With respect to NPY, most studies could not detect any significant changes in its expression in response to TNF-α (King et al. 2000); however, the effect of TNF-α injected directly in the hypothalamus has not been tested. Finally, we could find no study that has evaluated the direct effect of TNF-α on MCH expression. It is interesting to note that, in the present study, although we detected significant increases in the expression of both orexigenic and anorexigenic neurotransmitters, the effect upon the anorexigenic peptites was of a remarkably higher magnitude. Thus, we believe that the balance of neuropeptide expression favoring anorexigenic inputs may have an impact on the outcome of TNF-α action in the hypothalamus, which is clearly anorexigenic in the doses used in this study. Nevertheless, it must be emphasized that in such a complex signaling environment, the local regulation of neurotransmitter expression may be responsible only for part of the effects of cytokines on feeding and thermogenesis. Interestingly, we observed no effect of an acute dose of TNF-α upon the anorexigenic properties of insulin in the hypothalamus. Considering that in peripheral tissues TNF-α can inhibit insulin action by inducing serine phosphorylation of important intermediaries of the insulin signaling pathway (Hotamisligil 2003), it will be interesting to test if under chronic stimulation this cytokine will promote any modulation of the insulin action and signal transduction.

In conclusion, this study shows that TNF-α can activate a cascade of signal transduction in the hypothalamus of rats. These signaling events induce the expression of cytokines and other proteins related to inflammatory signaling that, on a long-term basis, may have implications in the local response to a pro-inflammatory stimulus. In addition, TNF-α can, per se, modulate the expression of neurotransmitters involved in the control of energy homeostasis, favoring energy wastage.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Mr L. Janeri for technical assistance. The costs for these studies were defrayed by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). We thank Dr Nicola Conran for English language editing.

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  6. Acknowledgements
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