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Endocrinology Vol. 147, No. 8 3906-3914
Copyright © 2006 by The Endocrine Society

Ciliary Neurotrophic Factor Suppresses Hypothalamic AMP-Kinase Signaling in Leptin-Resistant Obese Mice

Gregory R. Steinberg, Matthew J. Watt, Barbara C. Fam, Joseph Proietto, Sofianos Andrikopoulos, Andrew M. Allen, Mark A. Febbraio and Bruce E. Kemp

St. Vincent’s Institute (G.R.S., B.E.K.) and Department of Medicine (G.R.S., B.E.K.), St. Vincent’s Hospital, Fitzroy 3065, Australia; Department of Medicine (B.C.F., J.P., S.A.), Heidelberg Repatriation Hospital, 3081 Heidelberg, Germany; Department of Physiology (A.M.A.), University of Melbourne, and Commonwealth Scientific and Industrial Research Organization Molecular and Health Technologies (B.E.K.), Parkville 3052, Australia; and Cellular and Molecular Metabolism Laboratory (M.J.W., M.A.F.), School of Medical Sciences, Royal Melbourne Institute of Technology, Bundoora, Victoria 3083, Australia

Address all correspondence and requests for reprints to: Gregory R. Steinberg, St. Vincent’s Institute, 9 Princes Street, Fitzroy, Victoria 3065, Australia. E-mail: gsteinberg{at}svi.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We examined the actions of a second-generation ciliary neurotrophic factor analog (CNTFAx15) on AMP-activated protein kinase (AMPK), a known regulator of food intake. Unlike leptin CNTFAx15 has been shown to reduce food intake in obese rodents and humans. Intraperitoneal injection of CNTFAx15 acutely (45 min) reduced hypothalamic AMPK{alpha}2 activity, AMPK{alpha}2Thr172 phosphorylation, and acetyl-coenzyme A carboxylase phosphorylation, effects not observed 2 or 6 h after injection. Intracerebroventricular CNTFAx15 reduced food intake, increased arcuate nucleus (ARC) signal transducer and activator of transcription 3 phosphorylation, and reduced AMPK signaling but not in the paraventricular nucleus (PVN), posterior hypothalamus, or cortex. To compare the effects of leptin and CNTFAx15 in a diet-induced model of obesity, mice were fed a control carbohydrate or high-fat diet (HFD) for 12 wk. Leptin treatment ip reduced food intake in control mice but not in mice fed a HFD. In contrast, ip CNTF markedly reduced food intake in both control and HFD animals. Both leptin and CNTF reduced AMPK activity and acetyl-coenzyme A carboxylase phosphorylation in the ARC and PVN of control-fed mice. A HFD blunted leptin but not CNTF effects on AMPK signaling in the ARC and PVN. In summary, these data demonstrate that CNTFAx15 bypasses diet-induced leptin resistance to reduce hypothalamic AMPK activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OBESITY, THE PRIMARY risk factor for developing the metabolic syndrome characterized by dyslipidemia, cardiovascular disease, and type 2 diabetes, is increasing in Western cultures. In recent years significant interest has focused on the role of AMP-activated protein kinase (AMPK) as a potential therapeutic target for the treatment of metabolic disorders. AMPK is a ubiquitously expressed serine threonine kinase that is activated in response to an increase in the cellular AMP to ATP ratio (1, 2). Once activated, AMPK triggers ATP-generating pathways such as fatty acid oxidation through inhibition/phosphorylation of acetyl-coenzyme A carboxylase (ACC), while suppressing ATP-consuming pathways such as protein synthesis, to quickly restore cellular energy balance (2). AMPK activity is dependent on phosphorylation at Thr172 within the catalytic {alpha}-subunit activation loop by the upstream kinases, LKB1 (3, 4) and calmodulin-dependent kinase kinase (5, 6, 7). Whereas most research has focused on mechanisms mediating AMPK activity in peripheral tissues such as skeletal muscle and liver, more recently a critical role of AMPK as a regulator of food intake has been revealed. The inhibition of AMPK activity mediates the hypothalamic suppression of appetite by leptin (8, 9), {alpha}-lipoic acid (10), and the fatty synthase inhibitor, C75 (11, 12), whereas activation of AMPK by glucose deprivation (9) and ghrelin (8) stimulates appetite.

Ciliary neurotrophic factor (CNTF) is a 22-kDa cytokine that was found to induce severe anorexia and weight loss during clinical trials for the treatment of amyotrophic lateral sclerosis (13). Whereas CNTF-induced weight loss was initially attributed to a cachectic response, subsequent studies in diet-induced (14) and genetic (ob/ob, MC4R–/–) obesity (15, 16) demonstrated that low doses of CNTF and the CNTF homolog, axokine (CNTFAx15) induced weight loss without causing the typical deleterious effects of other related cytokines, such as IL-1. Indeed, the safety and efficacy of CNTFAX15 as a weight loss agent have been confirmed in a recent clinical trial, although the development of neutralizing antibodies may restrict its general application (17). Obese rodents (14, 18) and humans (17) chronically administered CNTF exhibit reduced rebound weight gain after the cessation of treatment due to a sustained reduction in caloric intake, which results in the prolonged maintenance of weight loss. This effect has recently been attributed to hypothalamic neurogenesis, which appears to be capable of resetting the energy-balance set point (18).

The IL-6 family of cytokines consisting of IL-6, IL-11, leukemia inhibitory factor, oncostatin M, cardiotrophin-1, and CNTF are highly redundant in their ability to transduce biological activities, but specificity of responses is ensured by tissue-specific expression of their ligand-specific {alpha}-subunits (19). Both systemic leptin (20) and CNTF (15) reduce food intake and body mass in leptin-deficient ob/ob mice. CNTF receptor expression is localized within the hypothalamic neurons of the arcuate nucleus (ARC), an area that overlaps substantially with neurons activated by leptin (18). The effects of both leptin and CNTF are attributed to the similar expression patterns and signaling homology of both the leptin and CNTF receptor within hypothalamic regions involved in food intake and similar activation of the signal transducer and activator of transcription (STAT) 3 signaling (14, 21). The activation of STAT3 within the ARC is associated with the suppression of the synthesis of orexigenic peptides such as neuropeptide Y (NPY) and agouti-related peptide (AgRP) that in turn leads to suppression of food intake through a diverse and multifaceted signaling cascade (for review see Ref. 22).

In addition to regulation by STAT3, orexigenic peptides are also regulated by AMPK. In elegant experiments using an in vivo adenovirus approach in which dominant-negative (DN) and constitutively active (CA) mutations of AMPK were injected into the ventral medial hypothalamus. Minokoshi et al. (9) showed that a CA-AMPK increased body weight and food intake, whereas a DN-AMPK had the opposite effect. Increased food intake in animals treated with a CA-AMPK was related to increased expression of arcuate NPY and AgRP when in the fasted state, whereas the reciprocal relationship was observed in the fed state with the DN-AMPK. Lee et al. (23) extended these findings in a neuroblastoma cell line by showing that the modulation of cellular ATP and therefore AMPK by glucose, 2-deoxyglucose, pyruvate, or ATP synthesis inhibitors also altered the expression of AgRP. Similarly, the fatty acid synthase inhibitor C75 was also found to suppress ARC AMPK and NPY expression, an effect that was reversed in the presence of the AMPK activator 5-amino-imidazole-4-carboxamide-1-ß-D-ribofuranoside (12). In agreement with these observations, elevated hypothalamic AMPK{alpha}2 activity in diabetic rats was associated with elevated NPY and suppressed proopiomelanocortin (POMC) mRNA, effects that were reversed in the presence of the AMPK inhibitor, compound C (24).

Despite the pronounced effects of leptin on reducing food intake in a model of leptin deficiency (ob/ob mice), these effects are abrogated in diet-induced mice fed a high-fat diet (25). In contrast, the effect of CNTF on food intake persists in diet-induced obesity and in db/db mice, which lack a functional leptin receptor (15). In this study, we tested the hypothesis that like leptin, CNTFAx15 might also suppress hypothalamic AMPK signaling. Moreover, in light of the observations of the persistent effects of CNTFAx15 but not leptin on food intake in diet-induced obesity, we examined the effects of both cytokines on AMPK activity after 12 wk of high-fat feeding.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal experimental procedure
All experimental protocols used in this study were approved by St. Vincent’s Hospital Animal Ethics Committee. All mice used in experimental procedure were male C57BL6/J mice obtained at 4 wk of age (Walter Eliza Hall Institute of Medical Research, Kew, Australia) and were maintained on a 12-h light, 12-h dark cycle with lights on at 0700 h. For high-fat diet (HFD) experiments, mice were provided with water and one of two diets for 12 wk; a control (CON) standard rodent chow diet/low-fat diet (n = 36) containing 4% kcal fat and less than 1.0% kcal fructose (3.3 kcal/g, no. 8664; Harlan Teklad, Madison, WI) or a HFD (n = 36) containing 36% kcal fat and 15% kcal fructose (5.42 kcal/g, no. F4837; BioServ, Frenchtown, NJ). To assess the effectiveness of the HFD to induce insulin resistance, glucose and insulin tolerance tests were performed after 12 wk of feeding. Food was withdrawn from mice 6 h before testing. Mice were anesthetized (50 mg/kg sodium pentobarbital), and a resting blood sample was obtained by tail bleeding. After an ip glucose (1 g/kg in 0.9% NaCl) or insulin (0.85 U/kg) injection, blood samples were obtained at 15, 30, 45, 60, 90, and 120 min and immediately analyzed for glucose (MediSense Optium, Abbot Diagnostics, Doncaster, Australia). Mice were placed back on their respective diets for 1 wk before experimental procedures were undertaken.

In preliminary experiments to test our ability to isolate specific sections of the hypothalamus, experiments in six untreated C57BL6/J mice were conducted. The following four hypothalamic dissections were made: 1) an area containing predominantly the paraventricular nucleus (PVN), 2) an area containing predominantly the anterior hypothalamus, 3) an area containing predominantly the ARC and 4) an area containing predominantly posterior hypothalamus (PH). These hypothalamic sections were dissected on dry ice as previously described for rats with minor modifications (26). Briefly, mice were decapitated and coronal slices were made with the first between the optic chiasm and approximately 0.5 mm caudal hemisected into a dorsal region that contained the PVN and a ventral region containing the anterior hypothalamus. The remainder of the hypothalamus was dissected into a ventral region containing the ARC and a dorsal region containing the PH. A sample of the cortex was also collected. All samples were freeze clamped and stored at –80 C until analysis by real-time quantitative PCR analysis for POMC and AgRP mRNA, which are markers for the ARC. POMC and AgRP mRNA were highly enriched in the ARC samples but were not detected in samples from the PVN, PH, or cortex (Table 1Go). These data suggest that the hypothalamic regions isolated were correct and representative of their respective nuclei, although it is also likely that our dissections contained other regions of the brain not described such as ventromedial nuclei in the case of the ARC and the lateral hypothalamus and perifornical area in the case of the PVN.


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  		TABLE 1. POMC and AgRP mRNA expression in ARC, PVN, PH, and cortex of untreated C57BL6/J mice assessed by quantitative real-time PCR

 
To test whether CNTF at doses known to reduce food intake, independent of cachexia or increased corticosterone (14), altered AMPK activity PBS (vehicle) or 300 µg/kg CNTFAx15 [a second-generation CNTF analog that corresponds to recombinant human CNTF (1–185 ({Delta}186–200), Cys17Ala, Arg63Gln)]; Regeneron Pharmaceuticals, Tarrytown, NY) was injected ip into conscious mice, and hypothalami were excised on dry ice after decapitation after 45 min, 2h, and 6 h, freeze clamped, and stored at –80 C until further analysis.

The ip administration of CNTFAx15 at a dose of 300 µg/kg·d reduces food intake and body weight to a similar degree to leptin (1.0 mg kg·d) in ob/ob mice (15). This leptin concentration has been demonstrated to reduce hypothalamic AMPK activity (8). Individually housed mice were injected ip at the start of the light cycle on two occasions separated over a 24-h period with PBS (vehicle), 300 µg CNTFAx15/kg body mass, or 1 mg leptin/ kg body mass (R&D Systems, Minneapolis, MN). Food intake and body mass were monitored daily over the 48-h period after treatments. Mice were then allowed to recover for 6 d on their respective diets. On the day before terminal experiments, mice were fasted overnight and the following morning injected ip with PBS, leptin, or CNTFAx15 as described above. Forty-five minutes after injection, mice were decapitated and the brain rapidly removed as described above.

Intracerebroventricular experiments
Mice were anesthetized with xylazine (10 mg/kg) and ketamine (75 mg/kg) via ip injection and secured in a stereotaxic frame. An incision was made in the skin and the skull was then leveled between lambda and bregma. Using a dental drill, a small hole was made in the skull to enable a 30-gauge guide cannula (Plastics One, Roanoke, VA) to be lowered toward the lateral ventricle (0.3 mm posterior to bregma, 1.0 mm lateral to the midline, and 3.0 mm below the surface of the skull). The cannula was then secured using dental acrylic and fixed in place with a skull screw. The cannula was blocked by a capped insert until the time of injection to maintain cannula patency and sterility and individually housed to avoid removal of cannula. Correct placement of the cannula into the lateral ventricle was confirmed the following day by injection of angiotensin II (50 ng). Animals not displaying a prompt and sustained drinking response were excluded from further study. This was approximately 8% of animals.

One week after these initial procedures PBS (vehicle) or CNTFAx15 (90 ng) were microinjected, at the start of the light cycle, into the lateral ventricle in a volume of 1 µl over 30 sec as previously described (27), and food intake was monitored over a 24-h period. Mice were allowed to recover for 6 d. On the day before terminal experiments, mice were fasted overnight and the following morning injected intracerebroventricularly (icv) with PBS or CNTFAx15. Forty-five minutes after injection, animals were killed via decapitation for harvesting of the brain as described above.

Analytical methods
Real-time quantitative PCR.
RNA was isolated from hypothalamic sections using the RNeasy minikit (QIAGEN, Doncaster, Australia) for total RNA isolation. An on-column DNase treatment was performed using an RNase-free DNase set (QIAGEN). Reverse transcription of mRNA (1.5 µg) was performed using the thermoscript RT-PCR system (Invitrogen, Mount Waverly, Australia) with random hexamer priming as recommended by the manufacturer. Quantitative real-time PCR was performed on the Rotorgene 3000 (Corbett Research, Sydney, Australia) using Assay-on-Demand gene expression kits for mouse POMC and AgRp (Applied Biosystems, Foster City, CA). cDNA was amplified using 20 µl TaqMan PCR containing 3 mM MgCl2, 200 mM deoxynucleotide triphosphates, 100 nm primers, 50 nM TaqMan probe, 1x gold reaction buffer, and 0.5 U Amplitaq gold (Applied Biosystems). Assays were performed in triplicate and normalized using 18s ribosomal RNA (Applied Biosystems) as an internal control. The relative quantities of each transcript were then calculated using the comparative critical threshold method.

AMPK activity assay.
Hypothalamic sections were homogenized in ice-cold buffer [20 mM HEPES (pH 7.5), 2 mm EDTA, 50 mM NaF, 5 mm Na4P2O7, 1% Nonidet P40 + 1% protease inhibitor cocktail (Complete, Roche, Stockholm, Sweden)] centrifuged at 14,000 x g for 25 min, and the supernatant was incubated with 10 µl AMPK{alpha}1 and AMPK{alpha}2 antibody-bound protein A agarose beads for 16 h. Immunocomplexes were washed and suspended in 50 mM Tris (pH 7.5) buffer for the AMPK assay in the presence of 200 µM AMP (28). Activities were calculated as picomoles of phosphate incorporated into the SAMS synthetic peptide per minute per milligram protein subjected to immunoprecipitation as previously described (28). Five microliters of the remaining beads were solubolized in sample buffer exposed to SDS-PAGE and transferred to polyvinyl difluoride membranes, blocked, and immunoblotted with antibodies detecting AMPK Thr172 phosphorylation and AMPK{alpha}1 and -{alpha}2 as previously described (29). After incubation with horseradish peroxidase-conjugated secondary antibody (1:2000; Amersham Biosciences, Castle Hill, New South Wales, Australia), the immunoreactive proteins were detected with enhanced chemiluminescence (PerkinElmer, Rowville, Victoria, Australia) and quantified by densitometry. For the determination of ACC samples, lysates were immunoprecipitated with streptavidin and immunoblotted as described (29).

Statistical analysis
Results are presented as the mean ± SEM. Data were analyzed for differences by one-way ANOVA with specific differences located with a Tukey’s post hoc test. P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Forty-five minutes after ip injection with CNTFAx15, AMPK{alpha}1 activity (Fig. 1AGo) tended to be reduced (P = 0.057), a trend that was not observed at subsequent time points (Fig. 1AGo). AMPK{alpha}1 Thr172 phosphorylation (Fig. 1BGo) was not altered by CNTFAx15. CNTF Ax15 reduced hypothalamic AMPK{alpha}2 activity (Fig. 1CGo) and phosphorylation (Fig. 1DGo). Suppressed AMPK activity after CNTFAx15 treatment was associated with reduced ACC phosphorylation (Fig. 1EGo) 45 minutes after injection but was without effect at 2 or 6 h relative vehicle (P > 0.05).


Figure 1
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  		FIG. 1. CNTFAx15 acutely reduces hypothalamic AMPK signaling. AMPK{alpha}1 and -{alpha}2 activity (A and C) and Thr172 phosphorylation (B and D, representative blot of several experiments above and quantification of densitometry below) and ACC phosphorylation (E, representative blot of several experiments above and quantification of densitometry below) were determined in mice fasted for 12 h and injected ip with vehicle (saline) or CNTFAx15 (300 µg/ kg body mass) before hypothalami were removed 45 min, 2 h, or 6 h after injection. Values shown are means ± SE (n = 7) for that time point. a, Significantly different from vehicle for that time point. Representative blots are of several experiments.

 
To determine whether the effects of CNTFAx15 on hypothalamic AMPK were mediated centrally via direct effects on AMPK, we injected CNTFAx15 icv. Intracerebroventricular CNTFAx15 reduced food intake (Fig. 2AGo). Reduced food intake was associated with elevated ARC STAT3 phosphorylation (Fig. 2BGo) and suppressed AMPK{alpha}2 activity and phosphorylation (Fig. 2Go, C and D, respectively). Although AMPK{alpha}1 phosphorylation was suppressed (Fig. 2DGo), the reduction in AMPK{alpha}1 activity did not achieve statistical significance after icv CNTFAx15 (Fig. 2CGo). Reduced food intake and AMPK activity/phosphorylation was associated with suppressed ARC ACC phosphorylation (Fig. 2EGo). Intracerebroventricular CNTFAx15 did not alter STAT3 phosphorylation (data not shown), AMPK activity or ACC phosphorylation in the PVN, PH, or cortex (Table 2Go).


Figure 2
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  		FIG. 2. Intracerebroventricular injection of CNTFAx15 reduces food intake and arcuate nucleus AMPK signaling. Saline (vehicle) or CNTFAx15 (90 ng) was microinjected into the lateral ventricle and food intake was monitored over a 24-h period (A). The following week after an overnight fast, icv saline or CNTFAx15 was injected and hypothalami sections removed 45 min after injection. ARC STAT3 phosphorylation (B), AMPK{alpha}1 and -{alpha}2 activity (C), AMPK{alpha}1 and -{alpha}2 Thr 172 phosphorylation (representative blot of several experiments above and quantification of densitometry below) (D), and ACC phosphorylation (representative blot of several experiments above and densitometry below) (E). Values shown are means ± SE (n = 9). a, Significantly different from vehicle.

 

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  		TABLE 2. AMPK{alpha}2 and AMPK{alpha}1 activities and ACC phosphorylation in PVN, PH, and cortex 45 min after icv injection of vehicle (saline) or CNTFAx15 (90 ng) (n = 6–9)

 
To determine the efficacy of CNTFAx15 to suppress food intake and AMPK signaling in a rodent model of obesity, male C57BL6/J mice were fed a HFD for 12 wk. Animals fed a HFD had greater body mass (P < 0.001) (Fig. S1A, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://endo.endojournals.org). Area under the curve after insulin (P = 0.02) (Fig. S1B) and glucose tolerance tests (P = 0.054) (Fig. S1C) was greater after a HFD demonstrating the presence of insulin resistance. A HFD led to significantly greater plasma leptin suggestive of the presence of leptin resistance (Fig. S1D).

To assess the effects of leptin and CNTFAx15 on food intake and body mass, mice were injected ip with saline, leptin, or CNTF Ax15 for 2 d at 0900 h. Both CNTFAx15 and leptin treatment reduced food intake to a similar degree in animals fed a CON diet (Fig. 3AGo). Animals fed a HFD had a greater daily caloric intake than CON-fed animals (Fig. 3AGo). Importantly despite the pronounced effects of leptin in animals fed a CON diet, leptin was ineffective in reducing food intake in HFD animals, whereas CNTFAx15 reduced food intake to a similar degree as that observed in animals fed the CON diet (Fig. 3AGo). In line with reductions in food intake, both CNTFAx15 and leptin reduced body mass over the 2-d treatment period in animals fed a CON diet, whereas only CNTFAx15 reduced body mass in animals fed a HFD (Fig. 3BGo). It should be noted that reductions in body mass were greater than expected from calorie restriction alone and were likely attributed to increased energy expenditure as previously reported (30).


Figure 3
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  		FIG. 3. CNTFAx15 and leptin reduce food intake and body mass in CON-fed animals, but only CNTFAx15 effects are preserved after a HFD. CON or HFD animals were injected ip with vehicle (saline), leptin (1 mg/kg body mass), or CNTFAx15 (300 µg/kg body mass) twice separated by 24 h. A, Mean daily food intake over 48 h. B, Change in body mass over 48 h relative before treatment. Values shown are means ± SE (n = 7–9). a, Significantly different from vehicle; b, significantly different from CON leptin; c, significantly different from HFD leptin.

 
To examine effects on AMPK signaling, saline, leptin, or CNTFAx15 was delivered at the same doses used in the experiments examining food intake described above. Intraperitoneal injection of leptin reduced ARC and PVN AMPK{alpha}2 activity (Fig. 4Go, A and C) and ACC phosphorylation (Fig. 4Go, B and D) in CON but had no effect in HFD. In CON CNTFAx15 reduced AMPK{alpha}2 activity in ARC and tended (P = 0.11) to do so in the PVN (Fig. 4Go, A and C) and suppressed ACC phosphorylation in both the ARC and PVN (Fig. 4Go, B and D), effects that were preserved after a HFD. Neither leptin nor CNTFAx15 altered AMPK{alpha}2 activity or ACC phosphorylation in the PH or cortex (Table 3Go). AMPK{alpha}1 activity was unaltered by leptin or CNTFAx15 in the ARC, PVN, PH, and cortex (data not shown).


Figure 4
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  		FIG. 4. In CON-fed animals CNTFAx15 and leptin reduce ARC and PVN AMPK{alpha}2 activity and ACC phosphorylation, but only CNTFAx15 effects are preserved after a HFD. CON or HFD animals were injected ip with vehicle (saline), leptin (1 mg/kg body mass), or CNTFAx15 (300 µg/ kg body mass) and hypothalami sections removed 45 min after injection. A and B, ARC. A, AMPK{alpha}2 activity. B, ACCSer218 phosphorylation (representative blot of several experiments above and quantification of densitometry below). C and D, PVN. C, AMPK{alpha}2 activity. D, ACCSer218 phosphorylation (representative blot above and quantification of densitometry below). Values shown are means ± SE (n = 7–9). a, Significantly different from vehicle; b, significantly different from HFD leptin; c, significantly different from CON vehicle.

 

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  		TABLE 3. AMPK{alpha}2 activity (pmol/min/mg protein) and ACC phosphorylation (arbitrary units) in PH and cortex 45 min after ip treatment with vehicle (saline), leptin (1 mg/kg body mass), or CNTFAx15 (300 µg/kg body mass)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we investigated the acute effects of CNTFAx15 on hypothalamic AMPK signaling in both control and leptin-resistant, diet-induced obese rodents. We show that both ip and icv CNTFAx15 acutely reduces hypothalamic AMPK signaling and food intake. Importantly, we demonstrate that the effects of CNTFAx15 persist in diet-induced obesity, whereas the effects of leptin on AMPK signaling are blunted. The capacity of CNTFAx15 to bypass leptin resistance to reduce hypothalamic AMPK signaling highlights its potential role in the therapeutic treatment of obesity. We recently found that CNTF stimulates AMPK signaling and fatty acid oxidation in skeletal muscle, and this is also insensitive to diet-induced obesity (31).

We have shown that like leptin, CNTFAx15 injected both ip and icv selectively reduces hypothalamic AMPK{alpha}2 activity acutely, effects that are not maintained 2 or 6 h after ip injection. This transient reduction of AMPK{alpha}2 activity is consistent with the short half-life (~45 min) of CNTFAx15 in circulation (30), and our icv experiments support a direct role for CNTFAx15 in acutely reducing hypothalamic AMPK activity, an effect associated with chronic reductions in food intake. It should be noted that the icv injection of CNTFAx15 was not specific to hypothalamic neurons and may have had effects on other regions of the brain accessible by the ventricular system. Indeed, supporting this possibility, Bjorbaek et al. (32) demonstrated the rapid stimulation of the suppressor of cytokine signaling 3 (SOCS3) in the cerebellum after CNTF administration. Therefore, in the present study, we cannot discount the possibility that extrahypothalamic regions may have contributed to reductions in food intake by CNTFAx15.

Acute reductions in hypothalamic AMPK{alpha}2 activity after CNTFAx15 were associated with reduced phosphorylation of ACC, a downstream target of AMPK. Reduced phosphorylation of ACC is associated with elevated levels of malonyl-CoA, thereby reducing carnitine palmitoyltransferase (CPT)-1 activity (33). Chemical and genetic inhibition of hypothalamic CPT-1 have been shown to reduce food intake (34); thus, our results showing an inhibition of AMPK by CNTFAx15 are consistent with the role of CPT-1 as a regulator of appetite and suggests a mechanism by which reduced AMPK activity may alter food intake. In addition to direct modulation of CPT-1 activity, AMPK has been demonstrated to have direct effects on the expression of orexigenic NPY and AgRP neurons (9). Previous studies (14, 35) reported reduced NPY after CNTFAx15 treatment, and our data support the idea that AMPK is a transcriptional regulator of NPY. Taken together, our data suggest that the acute modulation of AMPK activity by CNTFAx15 may chronically alter food intake by both inhibition of CPT-1 and transcriptional regulation of AgRP and NPY. To assess the quantitative importance of hypothalamic AMPK in the regulation of food intake by CNTFAx15, future studies in hypothalamic-specific AMPK null mice are warranted.

Leptin signaling in the hypothalamus is essential for the regulation of body mass and neuroendocrine homeostasis (36). These effects are attributed, in part, to the binding of leptin to the long form of the leptin receptor (LRb) within hypothalamic nuclei, which regulates food intake and energy expenditure. Specifically, tyrosine phosphorylation of 1138 of LRb by leptin is central to these regulatory effects (37). Despite the pronounced effects of leptin in models of leptin deficiency (20) or lipodystrophy (38), rodents fed a HFD are resistant to the effects of leptin (25). Human obesity is similarly characterized by elevated levels of leptin (39), and recombinant leptin infusion has minimal effects on body mass and food intake under these conditions (40). In the present study, we demonstrate that diet-induced obesity blunts the acute effects of leptin on AMPK signaling, suggesting that the inability of leptin to reduce AMPK signaling may be a contributing factor to the elevated caloric intake observed in high-fat-fed animals, but hypothalamic-specific deletion of AMPK will be necessary to directly test this hypothesis.

A potential mediator of leptin resistance is SOCS3. Elegant studies by Bjorbaek et al. (41, 42, 43) illustrated that SOCS3 inhibits leptin signaling through Src homology 2 binding to Tyr 985 of the leptin receptor, resulting in suppressed hypothalamic STAT3 activation. More recently elevated SOCS3 has been observed within the ARC after high-fat feeding, an effect associated with suppressed leptin-dependent STAT3 phosphorylation (44). In support of a critical role of SOCS3 in the regulation of hypothalamic leptin resistance, studies in SOCS3 hypothalamic-specific null mice (45) or SOCS3 mice with haplo-insufficiency (46) demonstrated protection from diet-induced leptin and insulin resistance. Whereas the effects of SOCS3 on food intake is considered to be due to the inhibition of STAT3, SOCS3 also inhibits leptin activation of ERK signaling (47). These data indicate that SOCS3 inhibits alternative leptin signaling pathways and suggest that elevated SOCS3 after diet-induced obesity may also inhibit leptin’s effects on AMPK signaling.

Previous studies (14, 18, 21, 48) have shown that CNTF increases phosphorylation of STAT3 in hypothalamic neurons that overlap with those activated by leptin. Surprisingly, despite the similarities in signaling between leptin and CNTF in chow-fed animals, HFDs suppressed leptin but not CNTFAx15 effects on AMPK signaling. Whereas SOCS3 has been demonstrated to bind to the SHP-2-binding site of gp130 (49), we demonstrate that CNTFAx15 suppression of hypothalamic AMPK is not blunted by high-fat feeding and that CNTFAx15 is capable of reducing food intake in leptin-resistant animals. Whereas the LRb shares significant sequence homology with both gp130 and the leukemia inhibitory factor receptor (50), signaling is independent of these IL-6 class of receptors (51). The mechanism(s) mediating the sustained sensitivity to CNTF but not leptin are presently not understood.

In summary, the present study has demonstrated that acute reductions in hypothalamic AMPK signaling by CNTFAx15 represent a potentially important mechanism mediating CNTFAx15 effects on food intake. Importantly, the effects of CNTFAx15 are maintained in diet-induced obesity, whereas the suppressive effects of leptin on AMPK signaling are blunted, highlighting a potential mechanism contributing to leptin resistance and highlighting the potential therapeutic potential of CNTFAx15 as a treatment for human obesity. Further investigations examining the role of hypothalamic AMPK in mediating the chronic effects of CNTFAx15 for the treatment of obesity are warranted.


   Acknowledgments
 
We thank Mark Sleeman (Regeneron Pharmaceuticals, Tarrytown, NY) for kindly supplying the CNTFAx15 used in this study.


   Footnotes
 
This work was supported by the National Health and Medical Research Council of Australia (NHMRC; to M.A.F., B.E.K., and G.R.S.); Australian Research Council and National Heart Foundation (to B.E.K.); Royal Melbourne Institute of Technology Faculty of Life Sciences (to M.J.W.). M.J.W. is a Peter Doherty Postdoctoral Fellow, and M.A.F. is a Senior Research Fellow supported by the NHMRC. B.E.K. is an Australian Research Council Federation Fellow. G.R.S. is supported by a Target Obesity Fellowship from the Canadian Diabetes Association, Heart and Stroke Foundation, and Canadian Institutes of Health Research.

The authors declare no potential conflicts of interest.

First Published Online May 4, 2006

Abbreviations: ACC, Acetyl-coenzyme A carboxylase; AgRP, agouti-related peptide; ARC, arcuate nucleus; AMPK, AMP-activated protein kinase; CA, constitutively active; CNTF, ciliary neurotrophic factor; CNTFAx15, CNTF homolog, axokine; CON, control; CPT, carnitine palmitoyltransferase; DN, dominant negative; HFD, high-fat diet; icv, intracerebroventricularly; LRb, long form of the leptin receptor; NPY, neuropeptide Y; PH, posterior hypothalamus; POMC, proopiomelanocortin; PVN, paraventricular nucleus; SOCS3, suppressor of cytokine signaling 3; STAT, signal transducer and activator of transcription.

Received December 15, 2005.

Accepted for publication April 24, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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