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Endocrinology, doi:10.1210/en.2007-0381
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Endocrinology Vol. 148, No. 11 5220-5229
Copyright © 2007 by The Endocrine Society

A Central Role for Neuronal Adenosine 5'-Monophosphate-Activated Protein Kinase in Cancer-Induced Anorexia

Eduardo R. Ropelle, José R. Pauli, Karina G. Zecchin, Mirian Ueno, Cláudio T. de Souza, Joseane Morari, Marcel C. Faria, Lício A. Velloso, Mario J. A. Saad and José B. C. Carvalheira

Department of Internal Medicine, Faculty of Medical Sciences, State University of Campinas-UNICAMP, 13083-970 Campinas, São Paulo, Brazil

Address all correspondence and requests for reprints to: José B. C. Carvalheira, M.D., Department of Internal Medicine, FCM, State University of Campinas-UNICAMP, 13083-970 Campinas, São Paulo, Brazil. E-mail: carvalheirajbc{at}uol.com.br.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The pathogenesis of cancer anorexia is multifactorial and associated with disturbances of the central physiological mechanisms controlling food intake. However, the neurochemical mechanisms responsible for cancer-induced anorexia are unclear. Here we show that chronic infusion of 5-amino-4imidazolecarboxamide-riboside into the third cerebral ventricle and a chronic peripheral injection of 2 deoxy-D-glucose promotes hypothalamic AMP-activated protein kinase (AMPK) activation, increases food intake, and prolongs the survival of anorexic tumor-bearing (TB) rats. In parallel, the pharmacological activation of hypothalamic AMPK in TB animals markedly reduced the hypothalamic production of inducible nitric oxide synthase, IL-1β, and TNF-{alpha} and modulated the expression of proopiomelanocortin, a hypothalamic neuropeptide that is involved in the control of energy homeostasis. Furthermore, the daily oral and intracerebroventricular treatment with biguanide antidiabetic drug metformin also induced AMPK phosphorylation in the central nervous system and increased food intake and life span in anorexic TB rats. Collectively, the findings of this study suggest that hypothalamic AMPK activation reverses cancer anorexia by inhibiting the production of proinflammatory molecules and controlling the neuropeptide expression in the hypothalamus, reflecting in a prolonged life span in TB rats. Thus, our data indicate that hypothalamic AMPK activation presents an attractive opportunity for the treatment of cancer-induced anorexia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
APPROXIMATELY 50% OF cancer patients report abnormalities in eating behavior on diagnosis (1). Anorexia and reduced energy intake also negatively affect quality of life, which is an important end point in the management of patients with cancer and the design of clinical trials (2). Accumulating evidences indicate that cancer anorexia is multifactorial in its pathogenesis, and most of the hypothalamic neuronal signaling pathways modulating energy intake are likely to be involved (3, 4, 5, 6).

Tumor growth is frequently associated with the development of anorexia. Evidence suggests that multiple proinflammatory-cytokines (IL-1β, IL-6, and TNF-{alpha}) in the central nervous system are mediators of anorexia in tumor-bearing hosts (7). Several studies have sought to explain how peripheral cytokines enter the brain and affect neuronal pathways involved in food intake regulation (8, 9). This could be done by persistent stimulation of anorexigenic neuropeptides, such as corticotropin-releasing factor, as well as by inhibition of the neuropeptide Y (NPY) orexigenic network (10, 11).

Analogous to leptin, proopiomelanocortin (POMC)-containing neurons in the arcuate hypothalamus also contain receptors to cytokines such as IL-1β and leukemia inhibitory factor, and the injections of each of these molecules into the third ventricle up-regulates POMC and decreases feeding behavior (12, 13). In addition, the high levels of IL-1β, IL-6, and TNF-{alpha} interfere with NPY release and neurotransport to a greater degree than gene expression (7, 14). Some studies reported that hypothalamic mRNA and peptide levels of orexigen NPY are decreased in different models of anorectic tumor-bearing rats (11, 15).

Multiple hypothalamic neuronal signaling pathways and the cross talk between these pathways are involved in the control of energy intake (16, 17). Recently hypothalamic AMP-activated protein kinase (AMPK) signaling has become an important focus of interest in the control of food intake. It has been proposed that changes in neuronal energy status affect AMPK phosphorylation and neuropeptide expression, leading to changes in food intake (18, 19, 20). AMPK is the central component of a protein kinase cascade that plays a key role in the regulation of energy control. AMPK is activated in response to ATP depletion, which causes an increase in the AMP to ATP ratio (21). AMPK activation occurs through the phosphorylation of a specific threonine residue (Thr-172), catalyzed by an upstream kinase that has been identified as STK11 (serine/threonine kinase 11) (22). In addition, recent studies identified calmodulin-dependent protein kinase kinase as an upstream kinase of AMPK (23, 24, 25).

Growing experimental evidences suggests the antiinflammatory effects of AMPK because the pharmacological activation of AMPK with 5-amino-4-imidazole carboxamide ribose (AICAR) blocks lipopolysaccharide (LPS)-induced inflammatory processes by blocking the expression of proinflammatory cytokines, inducible nitric oxide synthase (iNOS), cyclooxygenase-2, and MnSOD genes in astroglial cells (26, 27). Furthermore, the activation of AMPK inhibits LPS-induced expression of proinflammatory cytokines in primary rat astrocytes, microglia, and peritoneal macrophages (26, 28). Although growing evidence demonstrates the importance of AMPK activity in regulating the inflammatory process (in vitro and in vivo) (26) and food intake and body weight in different models of animals (18, 19, 29), the role of neuronal AMPK activity in cancer-induced anorexia is not known.

The present data demonstrate that different pharmacological activators of AMPK signaling, including oral administration of the antidiabetic drug metformin, increase AMPK phosphorylation in the central nervous system, reverse cancer anorexia, and increase the life span in tumor-bearing (TB) rats through the inhibition of the production of proinflammatory molecules and by controlling neuropeptide expression in the hypothalamus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents for SDS-PAGE and immunoblotting were from Bio-Rad Laboratories (Hercules, CA). Tris, aprotinin, ATP, dithiothreitol, phenylmethylsulfonyl fluoride, Triton X-100, Tween 20, glycerol, and BSA (fraction V) were from Sigma Chemical Co. (St. Louis, MO). Nitrocellulose paper (BA85, 0.2 mm) was from Schleicher & Schuell (Keene, NH). Ketamine hydrochloride was from Cristália (Itapira SP, Brazil). Antiphospho-[Ser79] acetyl-coenzyme A carboxylase (ACC) was from Upstate Biotechnology (Charlottesville, VA). Anti-ACC, anti-iNOS, anti-{alpha}-tubulin, anti-IL-1β, and anti-TNF-{alpha} were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) Antiphospho-[Thr172] AMPK and anti-AMPK were from Cell Signaling Technology (Beverly, MA). AICAR, 2-deoxy-D-glucose (2DG), and metformin (1,1-dimethylbiguanide hydrochloride) were obtained from Sigma-Aldrich (St. Louis, MO) and dissolved in sterile saline. The doses administrated in each experimental group are given below.

Animals and surgical procedures
Adult male Wistar rats (250–300 g) were used in all of the experiments in accordance with the guidelines of the Brazilian College for Animal Experimentation; the Ethics Committee at the State University of Campinas approved experiments. Room temperature was maintained (28 ± 1 C), and rats were housed in individual cages, subjected to a standard light-dark cycle (0600–1800/1800–0600 h) and provided with standard rodent chow and water ad libitum. After an overnight fast, the rats were anesthetized with ketamine hydrochloride (100 mg/kg, ip) and positioned on a Stoelting stereotaxic apparatus. The implantation of an intracerebroventricular (i.c.v.) catheter into the third ventricle has been previously described (16). After 5 d recovery period, cannula placement was confirmed by a positive drinking response after administration of angiotensin II (40 ng per 2 µl), and animals that did not drink 5 ml of water within 15 min after treatment were not included in the experiments.

The Walker 256 tumor cell line (originally obtained from the Christ Hospital Line, National Cancer Institute Bank, Cambridge, UK) is currently maintained frozen in liquid nitrogen in our laboratory. Walker 256 tumor cells were obtained from the ascitic fluid of the peritoneal cavity, 5 d after the ip injection of 20 x 106 carcinoma cells. After cell harvesting, the percentage of viable cells was determined by using 1% Trypan blue solution in a Neubauer chamber. Tumor cells (~2 x 106 cells in 1 ml saline solution) were then sc injected in the right flank after the surgical implantation of the i.c.v. cannula.

Definition of cancer anorexia and treatments
The metabolic and feeding experiments were performed 1 wk after complete recovery from the stereotaxic surgery according to the protocol depicted in Fig. 1Go. Each animal’s individual baseline 24-h food intake was defined as the average daily food intake over a period of 3 consecutives days. Subsequent food intake data are expressed as individual percentage, baseline daily food intake. In TB animals, cancer anorexia was defined as a single value less than 70% of baseline occurring after a steady decline of at least 3 d duration.


Figure 1
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  		FIG. 1. Schematic representation of the experimental procedures. Surgical implantation of i.c.v. cannula was performed approximately 3 wk before the in vivo study. Full recovery of body weight and food intake was achieved after 7 d. When criteria for anorexia had been met, i.c.v. infusions were started, and blood chemistries, food intake, and/or survival analysis were assessed.

 
When criteria for anorexia had been met, TB animals were daily treated with ip injection of vehicle or 2DG (500 mg/kg); i.c.v. injection (3 µl bolus injection) of AICAR (0.5 or 2.0 mM), metformin (1 mM), anti-IL1β antibody, anti-TNF-{alpha} antibody, or vehicle; or oral gavage with metformin (500 mg/kg). These treatments were performed during 4 d before Western blot and cumulative food intake analysis and the daily chronic treatment were conducted for survival curve determination. In preliminary experiments we determined water intake in TB animals that received i.c.v. AICAR, ip 2DG, and oral and i.c.v. metformin. Water intake was not altered by these treatments, compared with the respective TB+saline group.

Physiological and metabolic parameters
After 6 h of fasting, rats were submitted to an insulin tolerance test (1 U/kg body weight of insulin). After 30 min of i.c.v. AICAR, 180 min of ip 2DG and 180 min of oral metformin administration, the rats were injected with insulin and the blood samples were collected at 0, 4, 8, 12, and 16 min from the tail for serum glucose determination. The rate constant for plasma glucose disappearance was calculated using the formula 0.693/(t1/2). The plasma glucose t1/2 was calculated from the slope of last square analysis of the plasma glucose concentration during the linear phase of decline (30). Plasma glucose was determined using a glucose meter (Roche Diagnostic, Rotkreuze, Switzerland), and RIA was used to measure serum insulin, according to a previous description (31). Leptin concentrations were determined using a commercially available ELISA kit (Crystal Chem Inc., Chicago, IL). Rats were killed after 4 d of the different treatments and the body, tumor weight (wt), and spleen index were obtained. The spleen index was calculated as follows (32):

Formula

Proinflammatory-cytokine determination
Cytokine levels (IL-1β and TNF-{alpha}) were determined in samples of hypothalamic protein extracts (2.0 mg/ml) as well as serum TNF-{alpha} using ELISA kits (Pierce Endogen, Rockford, IL), according to the manufacturer’s instructions.

Real-time quantitative PCR
NPY and POMC neuropeptides were measured in the hypothalami of controls and treated and untreated TB animals rats under fasting conditions (12 h). AICAR (2.0 mM) or vehicle was injected 6 h before the hypothalamic extraction. Six rats were anesthetized, decapitated, and their hypothalamus immediately dissected on ice and snap frozen in liquid nitrogen. Frozen samples were immersed in TRIzol and homogenized for 30 sec at maximum speed. Total RNA was isolated according to the manufacturer’s guidelines and quantified by a spectrophotometer. The integrity of RNA was verified on ethidium bromide-stained 1% agarose gel, and the fluorescence intensity ratio of 28S/18S rRNA was determined (Eagle Eye; Stratagene, La Jolla, CA). Only samples that met the criteria of quality (both 260/280 nm and 28S/18S > 1.8) were included in the experiments. RNA samples were DNase treated at 37 C for 30 min and the enzyme inactivated at 65 C for 10 min. All samples were column purified, and 1.6 µg of total RNA were reverse transcribed at the same time using a master mix containing oligo (dT) primer and SuperScript III in a final volume of 20 µl.

Intron-skipping primers were optimized for mRNA encoding NPY, POMC, and glyceraldehyde-3-phosphate dehydrogenase and are listed below: NPY, forward, 5'-accaggcagagatatggcaaga-3', reverse, 5'-ggacattttctgtgctttctctcatta-3'; POMC, forward, 5'-cgctcctactctatggagcactt-3', reverse, 5'-tcacctaccagctccctcttg 3'; glyceraldehyde-3-phosphate dehydrogenase, forward, 5'-aacgaccccttcattgac-3', reverse, 5'-tccacgacatactcagcac-3'. The specificities of the products were confirmed by BLAST analyses and electrophoresis on an ethidium bromide-stained 3% agarose gel. Real-time PCR analysis of gene expression was carried out in an ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA). The optimal concentration of cDNA and primers as well as the maximum efficiency of amplification were obtained through five-point, 2-fold dilution curve analysis for each gene. Each PCR contained 2.5 or 5.0 ng of reverse-transcribed RNA (depending on the gene), 200 nM of each specific primer, SYBR Green PCR master mix, and RNase-free water to a 20-µl final volume. cDNA samples from hypothalamus were processed at the same time in triplicate for each gene and the negative controls included for each primer. The PCR conditions were 10 min at 95 C, followed by 40 cycles at 95 C for 15 sec and 60 C for 60 sec, and a melting step (dissociation curve) was performed after each run to further confirm the specificity of the products and the absence of primer dimers. Real-time data were analyzed using the Sequence Detector System 1.7 (Applied Biosystems).

Immunoblotting
Four days after the respective treatments, rats were anesthetized with sodium amobarbital (15 mg/kg body weight, ip) and used as soon as anesthesia was assured by the loss of pedal and corneal reflexes. The rats were killed and the basal diencephalon, including the preoptic area and the hypothalamus, was removed at the time points indicated, minced coarsely, and homogenized immediately in solubilization buffer containing (mM) 100 Tris (pH 7.6), 1% Triton X-100, 10 Na3VO4, 100 NaF, 10 Na4P2O7, 4 EDTA, 150 NaCl, 0.1 mg aprotinin, and 35 mg phenylmethylsulfonyl fluoride per milliliter, using a polytron PTA 20S generator (model PT 10/35; Brinkmann Instruments, Westbury, NY) operated at maximum speed for 30 sec and clarified by centrifugation. Hypothalami (200 µg protein) were used for immunoblotting followed by Western blot analysis with the indicated antibodies. Blots were exposed to preflashed XAR film (Kodak, Rochester, NY) with Cronex Lightning Plus intensifying screens at –80 C for 12–48 h. Band intensities were quantitated by optical densitometry (Scion Image software; Scion Corp., Frederick, MD).

Statistical analysis
The survival curves were estimated using Kaplan-Meier’s estimates, and curves were compared using the log-rank test and the level of significance was set at P < 0.001. Where appropriate, results are expressed as the means ± SEM accompanied by the indicated number of rats used in experiments. Comparisons among groups were made using parametric one-way ANOVA; where F ratios were significant, further comparisons were made using the Bonferroni test. The level of significance was set at P < 0.05. The results of blots are presented as direct comparisons of bands in autoradiographs and quantified by densitometry.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Physiological and metabolic parameters
Table 1Go shows comparative data for controls and TB groups with respective treatments. As previously shown (33, 34), 4 d after the criteria of anorexia had been met, all the TB groups showed weight loss, reduction in the 6 h-fasting serum insulin and leptin levels, and insulin resistance when compared with age-matched controls. When the tumor weights were excluded, treatments with i.c.v. AICAR, ip 2DG and oral metformin were observed to increase the body weight when compared with saline treatment in TB animals. All the TB groups presented an increased serum TNF-{alpha} and spleen index, and 4 d after the beginning of the different treatments, there was a significant reduction in serum TNF-{alpha} and splenomegaly, compared with the TB+saline group. In addition, AICAR, 2DG, and metformin increased the 6-h fasting serum insulin and leptin and improved insulin sensitivity, compared with saline treatment in TB rats. No significant variations were found in 6-h fasting serum glucose levels between the groups, and the tumor weights were similar between TB groups.


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  		TABLE 1. Characteristics of TB rats and their age-matched controls

 
Central infusion of AICAR, peripheral injection of 2DG, or oral administration of metformin increase hypothalamic AMPK phosphorylation in TB rats
We first examined the hypothalamic AMPK phosphorylation in TB and control rats under feeding and 12-h fasting states. Under feeding conditions, AMPK phosphorylation was similar in the hypothalamus of TB and control rats; however, in the 12-h fasting state, the hypothalamic AMPK phosphorylation in the TB animals was reduced by 2.1-fold when compared with the control group (Fig. 2AGo, upper panel). The protein expression of AMPK and {alpha}-tubulin were similar in the hypothalamus of controls and TB rats in both conditions (Fig. 2AGo, middle and lower panels).


Figure 2
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  		FIG. 2. The basal levels of hypothalamic AMPK phosphorylation and the time course and dose response after central infusion of AICAR, peripheral administration of 2DG, or oral metformin in TB rats. Representative Western blots demonstrating the effect of 12 h fasting on hypothalamic AMPK phosphorylation in control and TB animals (A), the hypothalamic AMPK phosphorylation in a time- and dose-dependent manner after central infusions of AICAR (B), and after ip injections of 2DG in TB rats (C). Representative Western blots demonstrate the effect of oral metformin on hypothalamic AMPK phosphorylation in a time-dependent manner (D). Representative Western blots demonstrate the effect of oral metformin on hypothalamic AMPK and ACC phosphorylation (E and F). Tissue extracts were immunoblotted (IB) with antiphospho AMPK antibody and antiphospho ACC (upper panels), anti-AMPK-{alpha}, anti-ACC, and anti-{alpha}-tubulin antibodies (lower panels). The results of scanning densitometry were expressed as arbitrary units. Bars, Means ± SEM of six to eight rats. #, P < 0.05 vs. control fasted; *, P < 0.05, vs. respective vehicles.

 
The i.c.v. microinfusion of AICAR and ip injection of 2DG in TB rats after a 12-h fast caused an increase in hypothalamic AMPK phosphorylation in a dose- and time-manner dependent (Fig. 2Go, B and C). The i.c.v. infusion of AICAR (0.5 and 2.0 mM) increased AMPK phosphorylation in the hypothalamus of TB rats by 2.2- and 4.9-fold at 60 min, respectively, when compared with vehicle (Fig. 2BGo, upper panel). The 2DG (250 and 500 mg/kg) ip injection increased the hypothalamic AMPK phosphorylation by 1.2- and 3.6-fold, respectively, in the hypothalamus of TB rats at 180 min, when compared with vehicle (Fig. 2CGo, upper panel). The AMPK and {alpha}-tubulin protein levels did not differ between the groups after AICAR or 2DG administration (Fig. 2Go, B and C, middle and lower panels).

To determine whether the antidiabetic drug, metformin, induces hypothalamic AMPK phosphorylation in TB rats, we administered metformin using oral gavage during cancer-induced anorexia in rats after a 12-h fast. The oral administration of metformin (500 mg/kg) in TB rats caused an increase in hypothalamic AMPK phosphorylation in a time-manner dependent, with maximal response at 180 min after oral gavage (Fig. 2DGo, upper panel). The total AMPK and {alpha}-tubulin protein levels were not different between the groups after metformin administration (Fig. 2DGo, middle and lower panels).

Next, we explored the effects of oral gavage of metformin on hypothalamic AMPK and ACC phosphorylation in the TB animals. After 12-h fasting conditions, AMPK and ACC phosphorylation were reduced by 3.1- and 4.6-fold in the hypothalamus of TB rats when compared with the control group, respectively. Metformin administration increased hypothalamic AMPK and ACC phosphorylation by 2.0- and 3.9-fold, when compared with saline-treated TB rats (Fig. 2EGo and F, upper panels). The AMPK, ACC, and {alpha}-tubulin protein levels were not different between the groups after metformin administration (Fig. 2Go, E and F, middle and lower panels).

AMPK activation attenuates cancer anorexia and increases survival in tumor bearing rats
To determine whether the hypothalamic AMPK activation increases food intake and survival in TB animals, we administered i.c.v. AICAR or ip 2DG during the period of anorexia in TB rats. Figure 3Go, A and D, shows that the tumor development markedly reduced food intake during 24 h in TB rats after saline treatment when compared with control animals. The central or peripheral administration of AMPK activators in TB rats caused an increase in food intake that was apparent 4 h after treatment and lasted for 12 and 24 h (Fig. 3Go, A and D). When criteria for anorexia had been met, we administered a daily injection of i.c.v. AICAR or ip 2DG during 4 d in TB rats to evaluate the cumulative food intake. In TB animals the cumulative food intake was reduced by about 75% when compared with respective control groups; however, the chronic treatment with AICAR (i.c.v.) or 2DG (ip) increased the cumulative food intake by about 25 and 35%, respectively, when compared with TB rats treated with i.c.v. or ip saline (Fig. 3Go, B and E). Because AMPK is the only enzyme known to be activated by both AICAR and 2DG, these data suggest that tumor-induced anorexia is dependent on the activation of AMPK.


Figure 3
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  		FIG. 3. The effects of i.c.v. infusion of AICAR, ip 2DG, and oral or i.c.v. metformin on food intake and survival in TB rats. Determination of 24 h of food intake after AICAR microinfusion (A), after 2DG injection (D), after oral metformin (G), and after i.c.v. metformin (J). Determination of 4 d of cumulative food intake after AICAR (B), 2DG treatment (E), oral metformin (H), or i.c.v. metformin (K). Results represent means ± SEM of six to eight rats. #, P < 0.05 vs. saline; *, P < 0.05 vs. TB rats + saline. Representative survival curves demonstrating the effect of chronic administration of AICAR (C), 2DG (F), oral metformin (I), or i.c.v. metformin (L) on life span of TB animals. The survival curves were estimated using Kaplan-Meier’s estimates, and curves were compared using the log-rank test, P < 0.001 (n = 10).

 
In addition, we investigated whether the hypothalamic AMPK activation increases survival in TB animals after chronic AICAR or 2DG treatment. As shown in the Kaplan-Maier graphs, a daily central infusion of AICAR or a peripheral injection of 2DG statistically prolonged the survival in TB animals, whereas the median survival of TB rats after chronic administration of saline via i.c.v. or ip was approximately 5.5 d, and chronic administration of AICAR or 2DG increased the median survival to 9.5 and 8.0 d, respectively (Fig. 3Go, C and F).

Figure 3GGo shows that the tumor development markedly reduced food intake during 24 h in TB rats after saline treatment when compared with control animals. Furthermore, oral administration of metformin in TB rats caused an increase in food intake that was apparent 4 h after treatment and lasted for 12 and 24 h (Fig. 3GGo). When criteria for anorexia had been met, the cumulative food intake was evaluated after a daily oral gavage treatment with saline or metformin during 4 d in TB animals. The cumulative food intake was reduced by about 75% when compared with control animals; however, the daily oral treatment with metformin increased the cumulative food intake by about 28% when compared with TB rats treated with oral saline (Fig. 3HGo).

We also investigated whether the hypothalamic AMPK activation increases the survival in TB animals after chronic oral treatment with metformin. Figure 3IGo shows a daily oral administration of metformin significantly increases survival, whereas the median survival of oral saline-treated TB rats was 5.5 d and chronic administration of oral metformin increased the median survival to 8.5 d.

To explore the specific action of metformin in the hypothalamus, we injected i.c.v. metformin (1 mM) in TB rats to evaluate food intake and survival. Figure 3JGo shows that the tumor development markedly reduced food intake during 24 h in TB rats after saline treatment when compared with control animals. The i.c.v. administration of metformin in TB rats, caused an increase in food intake that was apparent 4 h after treatment and lasted for 12 and 24 h (Fig. 3JGo). When criteria for anorexia had been met, the cumulative food intake was evaluated after a daily i.c.v. treatment with saline or metformin during 4 d in TB animals. The cumulative food intake was reduced by about 75% when compared with control animals; however, the daily treatment with metformin increased the cumulative food intake by about 32%, when compared with TB rats treated with i.c.v. saline (Fig. 3KGo).

We finally investigated whether the hypothalamic AMPK activation increases the survival in TB animals after chronic i.c.v. treatment with metformin. Figure 3LGo shows that daily i.c.v. administration of metformin significantly increases survival, whereas the median survival of oral saline-treated TB rats was 5.5 d and chronic administration of i.c.v. metformin increased the median survival to 9 d.

Hypothalamic AMPK activation decreases the expression of proinflammatory molecules in TB rats
We next investigated the antiinflammatory properties of AICAR in the hypothalamus of TB rats. To test the hypothesis that AMPK activation reduces the production of proinflammatory cytokines in TB rats, we injected the AMPK activator and evaluated the hypothalamic expression of iNOS, IL-1β, and TNF-{alpha}. The protein levels of iNOS increased by 3.8-fold in the hypothalamus of TB rats when compared with control animals, and the microinfusion of AICAR reduced iNOS expression by 1.5-fold in the hypothalamus of TB rats when compared with saline-treated TB rats (Fig. 4AGo, upper panel). The {alpha}-tubulin protein levels were not different between the groups after AICAR administration (Fig. 4AGo, lower panel). In addition, the protein levels of IL-1β and TNF-{alpha} were increased by 7.9- and 2.0-fold, respectively, in the hypothalamus of TB rats when compared with the control group, and AMPK activation reduced the hypothalamic expression of IL-1β by 1.9-fold and TNF-{alpha} by 1.5-fold when compared with TB rats treated with i.c.v. saline (Fig. 4Go, B and C, respectively).


Figure 4
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  		FIG. 4. Intracerebroventricular infusion of AICAR attenuated the production of proinflammatory molecules in the hypothalamus of TB rats. Representative Western blots demonstrate the effects of AICAR on iNOS (A) production in the hypothalamus of TB rats. Bars, means ± SEM of six rats. Tissue extracts were immunoblotted (IB) with anti-iNOS antibody (upper panel) and with anti-{alpha}-tubulin antibody (lower panel). ELISA demonstrated the antiinflammatory proprieties of AICAR on IL-1β (B) and TNF-{alpha} (C) expression in the hypothalamus of TB rats. Determination of cumulative food intake after chronic treatment with i.c.v. anti-IL-1β or anti-TNF{alpha} antibodies (D) (n = 5 per group). #, P < 0.05 vs. Saline (Sal); *, P < 0.05 vs. TB rats + saline.

 
To explore the hypothesis that the reduction in inflammatory molecules in the hypothalamus attenuates anorexia in the of TB rats, we injected a specific anti-IL-1β or anti-TNF{alpha} antibodies via i.c.v. in TB animals. In TB rats, the cumulative food intake was reduced by about 75% when compared with the respective control group; however, the chronic treatment with anti-IL-1β antibody (i.c.v.) increased the cumulative food intake by about 72%, when compared with TB rats treated with i.c.v. saline (Fig. 4DGo). Chronic i.c.v. treatment with anti-TNF{alpha} antibody produced a slight increase in cumulative food intake, but we did not observe a significant difference when compared with saline i.c.v. treatment in TB animals (Fig. 4DGo).

Central infusion of AICAR reduced POMC mRNA levels in the hypothalamus of TB rats
To explore the mechanism(s) by which AMPK activation regulates food intake in TB rats, we examined the expression of hypothalamic neuropeptides involved in the control of energy homeostasis in TB animals. After 12 h of fasting, the NPY mRNA levels were reduced by 1.7-fold in the hypothalamus of TB rats when compared with the control group, and the i.c.v. microinfusion of AICAR was not able to increase hypothalamic NPY mRNA levels in TB rats when compared with saline-treated TB rats (Fig. 5AGo). On the other hand, POMC mRNA levels were increased by 2.2-fold in the hypothalamus of TB rats when compared with the control group, and the i.c.v. microinfusion of AICAR decreased hypothalamic POMC mRNA levels by 1.4-fold in TB rats when compared with saline-treated TB rats (Fig. 5BGo).


Figure 5
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  		FIG. 5. The effect of central infusion of AICAR on NPY and POMC mRNA levels in the hypothalamus of TB rats after a 12-h fast. The real-time PCR demonstrated the NPY (A) and POMC (B) mRNA levels after AICAR microinfusion. Results for real-time PCR are presented as transcript amount per hypothalamus. Bars, means ± SEM of six rats. #, P < 0.05 vs. saline; *, P < 0.05 vs. TB rats + saline.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrate that central AMPK activation can attenuate cancer-induced anorexia, whereas the activation of neuronal AMPK inhibits the hypothalamic expression of proinflammatory molecules, suppresses POMC mRNA levels in the hypothalamus of anorectic TB rats. Interestingly, different pharmacological activators of AMPK signaling, including i.c.v. microinfusion of AICAR, ip injection of 2DG, and oral or i.c.v. administration of metformin, activate AMPK in the central nervous system, increase food consumption, and prolong life span in the model of cancer-induced anorexia.

An increased expression of cytokines during tumor growth prevents the hypothalamus from responding appropriately to peripheral signals by persistently activating anorexigenic systems and inhibiting prophagic pathways (7). Convincing evidence suggests that cytokines may across the blood-brain barrier and have a vital role, triggering the complex neurochemical cascade, leading to the onset of cancer anorexia (7, 8). In addition, cytokine production in brain regions distant from the brain tumor site may also be involved in this clinical manifestation (35). In patients with cancer, it is probable that cytokines and anorexia are connected because the biological effects of cytokines are largely mediated by paracrine and autocrine influences (7). In accordance with our data demonstrating that the reduction in IL-1β cause the increased appetite, in the Fischer rat/MCA sarcoma model, brain IL-1β concentrations inversely correlate with food intake (36) and intrahypothalamic microinjections of an IL-1 receptor antagonist increases energy intake (37). TNF-{alpha} is one of the mediators of the hypothalamic anorexigenic signals that participate in the induction of the cachexia syndromes present in advanced stage cancer and in severe infectious (38, 39). Although we did not observe a significant increase in cumulative food intake with anti-TNF-{alpha} treatment, our data demonstrated that the reversal effect of AMPK on cancer-induced -anorexia is associated with decreased concentrations of iNOS, IL-1β, and TNF-{alpha} in the hypothalamus of TB rats. The antiinflammatory response produced by AMPK activation in the central nervous system is in accordance with several studies, suggesting that AICAR inhibits production of proinflammatory mediators (TNF-{alpha}, interferon-{gamma}, and nitric oxide) in brain glial cells, primary astrocytes, microglia, and peritoneal macrophages (26, 28). In addition, the pharmacological activation of AMPK also inhibited the production of proinflammatory molecules in serum and their expression in the central nervous system of rats injected with a sublethal dose of LPS by ip injection (26).

The mechanisms involved in the proinflammatory cytokine-dependent modulation of food intake and energy expenditure may also involve the control of hypothalamic neurotransmitter expression (40, 41); in situ stimulation of the expression of other cytokines, particularly IL-1β (42, 43); and activation of anorexigenic leptin-like signal transduction in the hypothalamus (44). IL-1β has been clearly associated with the induction of anorexia (39) by blocking NPY-induced feeding. The levels of this molecule are reduced in anorectic TB rats (11), and a correlation between food intake and brain IL-1 has been found in anorectic rats with cancer. The mechanism involved in the attenuation of NPY activity by cytokines may be related to an inhibition of cell-firing rates or NPY synthesis or an attenuation of its postsynaptic effects (45). On the other hand, it has been demonstrated that POMC-containing cells contain cytokine receptors and that these neurons are activated during acute and chronic inflammation (13). This discovery demonstrates that proinflammatory cytokines such as TNF-{alpha}, IL-1β, and IL-6 can bind to cytokine receptors on POMC-containing cells, causing these cells to increase their signaling to second-order neurons that affect outputs related to anorexia, loss of lean body mass, and increased energy expenditure. In our model of cancer-induced anorexia, we observed high levels of iNOS, IL-1β, and TNF-{alpha} in the hypothalamus of Walker TB rats and after 12 h of fasting; the mRNA levels of NPY were lower and POMC much higher than in control animals. Notably, we also observed defective 172 threonine AMPK phosphorylation in the hypothalamus of anorexic TB rats under 12-h fasting conditions when compared with normal rats, suggesting that the neuronal AMPK could be involved in cancer-mediated anorexia.

AMPK is expressed in the hypothalamic neurons involved in the regulation of food intake (46), whereas the central pharmacological activation of AMPK by AICAR leads to increased food intake and decreased energy expenditure in normal mice (19). Furthermore, acute ip administration of 2DG increased food intake and both the expression of agouti gene-related peptide and NPY in the arcuate nucleus (47). These findings were further explored in different studies, demonstrating that increasing hypothalamic AMPK activity increased the expression of orexigen neuropeptides in the arcuate nucleus of the hypothalamus, leading to increased food intake (29); moreover, preventing activity of the melanocortin system, using either genetic deletions of the melanocortin-4 receptor or small-molecule antagonists of this receptor, is effective in improving appetite and lean body mass in small animal models of cachexia in chronic disease (48, 49, 50). We demonstrate that the microinfusion of AICAR activates AMPK in neuronal cells in a time- and dose-dependent manner and that the higher dose of AICAR (2 mM) (although it does not change NPY mRNA levels) partially restores POMC mRNA levels in the hypothalamus of anorectic rats after a 12-h fast. Moreover, both central microinfusion of AICAR and ip injection of 2DG increased food intake and prolonged survival in TB animals.

Several groups have used the adenosine analog AICAR to activate AMPK. AICAR is taken up into the cell by adenosine transporters (51) and converted by adenosine kinase into the monophosphorylated nucleotide, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranotide, which mimics all of the effects of AMP on the AMPK system (52). Moreover, recent evidence demonstrates that calmodulin-dependent protein kinase kinase/AMPK axis is phosphorylated by 2DG (53). These data taken together with our findings that tumor’s inhibitory effect on food intake is blocked by either of two AMPK activators reduce the possibility of nonspecific effects of these drugs and support an important pathophysiological role for this intracellular signaling pathway.

In addition to the increased food intake, other possibilities beneficial effects on the survival of animals may occur after these treatments. First, AMPK activation is related to inhibition of the mammalian target of rapamycin cascade, which decreases cell proliferation (54). In this regard, we may not have seen any differences in tumor size after the different treatments, possibly due to the short duration of the treatments (4 d); moreover, AICAR and metformin i.c.v. infusion probably does not directly modulate the AMPK/mammalian target of rapamycin pathway in the tumor and tumor growth. Second, our results show that targeting the brain improves the metabolic milieu, as shown by the i.c.v. infusion of AICAR, suggesting that the brain may control some of the insulin resistance characteristics. Finally, it is also possible that AMPK activators are also acting as immune-suppressing agents and decreasing the overall inflammatory response to the tumor. Although we found a close correlation between the intensity of hypothalamic inflammation, AMPK activity, and life span, survival did not correspond to serum TNF-{alpha} and the intensity of the inflammatory response in the spleen. Direct hypothalamic activation with AICAR reproduces the effect of oral treatment with metformin and ip treatment with 2DG on food intake and survival, despite a distinct serum TNF-{alpha} and spleen index between the i.c.v. and systemic treatment. Thus, we cannot exclude the possibility that AMPK activation in the hypothalamus decreases some whole-body inflammatory response to the tumor and that this contributes to the increased survival; these results are intriguing and deserve further investigation.

Efforts have been made to implicate leptin as a modulator of food intake in tumor-induced anorexia; however, lower leptin levels were found in patients with gastrointestinal cancers, regardless of their degree of weight loss and in colon cancer patients who had no weight loss at all (55, 56, 57). In contrast, an association between leptin levels and weight loss was noted in a cohort of lung (58) and pancreatic cancer patients (59). Similarly, we found an association between leptin levels and cancer-induced weight loss. It was recently shown that both ip and i.c.v. administrations of the melanocortin agonist, melanotan II, increase IL1β mRNA expression in the mediobasal hypothalamus (60). Thus, IL-1β signaling may be downstream of melanocortin signaling. Moreover, physiological changes in leptin appear to alter signaling in only a small subset of POMC neurons in the rostral portion of the arcuate nucleus (61). Taken together these data suggest that tumor-induced anorexia is independent of the levels of leptin.

AMPK provides a candidate target, capable of mediating the beneficial metabolic effects of metformin (62). Metformin lowers blood glucose and blood lipid contents, and these effects are thought to be at least partially responsible for its therapeutic benefits (63, 64). Metformin decreases the leptin concentration in morbidly obese subjects (65, 66) and in normal-weight healthy men (67). Metformin has been suggested to act through the stimulation of AMPK in peripheral tissues; however, few studies have demonstrated whether metformin modulates AMPK activity in the neuronal cell. In Zucker rats, a single sc dose of metformin (300 mg/kg) reduced food intake only in obese animals, whereas the same dose of metformin given orally did not affect food intake in either lean or obese animals (68). In an acute study, metformin treatment increased the anorexic effect of leptin, and this was accompanied by increased levels of phosphorylated signal transducer and activator of transcription 3 in the hypothalamus of high-fat-fed obese rats (69). Metformin also inhibits AMPK activation and prevents increases in NPY expression in cultured hypothalamic neurons (70). On the other hand, our results indicated that a high dose of oral metformin (500 mg/kg) increases the phosphorylation of the AMPK/ACC pathway in a time-dependent manner in the hypothalamus of rodents in cancer-induced anorexia after 12-h fast. A daily oral or i.c.v. administration of metformin blockaded the anorectic response, leading to increased food intake and life span in TB rats. These apparent contradictory effects of metformin in the hypothalamus may be related to the different physiological and metabolic profiles observed in these studies. In addition, we used a prolonged fasting period (12 h) to evaluate the hypothalamic AMPK phosphorylation. Furthermore, we performed the same experiments with an oral administration of metformin (500 mg/kg) and i.c.v. metformin (1 mM) in normal rats, and we did not observe changes in either food intake or body weight (data not shown), suggesting that the physiological and metabolic parameters are essential to metformin-induced changes in food intake and body weight in rodents.

In summary, the findings of this study suggest that neuronal AMPK activation reverses cancer anorexia by inhibiting the production of proinflammatory molecules and controlling the expression of POMC, reflecting in the prolonged life span of TB rats. Thus, our data indicate that hypothalamic AMPK activation presents an attractive opportunity for the treatment of cancer-induced anorexia, whereas the restoration of appetite in cancer patients is likely to improve quality of life and might also improve overall patient survival.


   Acknowledgments
 
The authors thank Mr. Luiz Janeri, Jósimo Pinheiro, Leandro Macow, and Márcio A. da Cruz for technical assistance.


   Footnotes
 
This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo and Conselho Nacional de Desnevolvimento Cientifico e Tecnológico.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 23, 2007

Abbreviations: ACC, Acetyl-coenzyme A carboxylase; AICAR, 5-amino-4-imidazole carboxamide ribose; AMPK, AMP-activated protein kinase; 2DG, 2-deoxy-D-glucose; i.c.v., intracerebroventricular; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; NPY, neuropeptide Y; POMC, proopiomelanocortin; TB, tumor bearing.

Received March 22, 2007.

Accepted for publication August 14, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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