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Endocrinology, doi:10.1210/en.2008-0229
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Endocrinology Vol. 149, No. 9 4544-4553
Copyright © 2008 by The Endocrine Society

Glucocorticoids Increase Neuropeptide Y and Agouti-Related Peptide Gene Expression via Adenosine Monophosphate-Activated Protein Kinase Signaling in the Arcuate Nucleus of Rats

Hiroshi Shimizu, Hiroshi Arima, Minemori Watanabe, Motomitsu Goto, Ryoichi Banno, Ikuko Sato, Nobuaki Ozaki, Hiroshi Nagasaki and Yutaka Oiso

Department of Endocrinology and Diabetes (H.S., H.A., M.W., M.G., R.B., I.S., N.O., Y.O.), Nagoya University Graduate School of Medicine, and Department of Metabolic Medicine (H.N.), Nagoya University School of Medicine, Showa-ku, Nagoya 466-8550, Japan

Address all correspondence and requests for reprints to: Dr. H. Arima, Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: arima105{at}med.nagoya-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent studies suggest that the AMP-activated protein kinase (AMPK) signaling in the hypothalamus is the master regulator of energy balance. We reported in previous studies that glucocorticoids play a permissive role in the regulation of orexigenic neuropeptide Y (Npy) gene expression in the arcuate nucleus. In this study, we examined whether any cross talk occurs between glucocorticoids and AMPK signaling in the hypothalamus to regulate Npy as well as agouti-related peptide (Agrp) gene expression in the arcuate nucleus. In the hypothalamic organotypic cultures, the addition to the medium of the AMPK activator, 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside, increased phosphorylated AMPK (p-AMPK) as well as phosphorylated acetyl-coenzyme A carboxylase (p-ACC) in the explants, accompanied by significant increases in Npy and Agrp gene expression in the arcuate nucleus. The incubation with dexamethasone (DEX) also activated AMPK signaling in the explants, accompanied by significant increases in Npy and Agrp gene expression in the arcuate nucleus. The addition of the AMPK inhibitor compound C to the medium, which blocked increases of p-AMPK and p-ACC by DEX, significantly attenuated Npy and Agrp gene expression stimulated by DEX. Furthermore, p-AMPK and p-ACC levels in the arcuate nucleus were significantly decreased in adrenalectomized rats compared with sham-operated rats, and a replacement of glucocorticoids reversed the AMPK signaling in adrenalectomized rats. Thus, our data demonstrated that glucocorticoids up-regulate the Npy and Agrp gene expression in the arcuate nucleus through AMPK signaling, suggesting that the activation of the hypothalamic APMK signaling by glucocorticoids might be essential to the energy homeostasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PERIPHERAL SIGNALS reflecting energy balance are supposed to be integrated at the arcuate nucleus in the hypothalamus in which orexigenic neuropeptide Y (NPY) and agouti-related peptide (AGRP) as well as anorexigenic proopiomelanocortin are expressed (1). Among the orexigenic peripheral signals are glucocorticoids which are synthesized in and released from the adrenal glands (2). An excess of glucocorticoids causes obesity, whereas their depletion leads to marked anorexia in humans (2). In rodents, genetic or diet-induced obesity is reportedly prevented by adrenalectomy and restored by glucocorticoid replacement (3, 4, 5, 6, 7, 8). Previously, we demonstrated that glucocorticoids stimulated Npy gene expression in the arcuate nucleus in hypothalamic organotypic cultures, and that ghrelin and insulin, representative orexigenic and anorexigenic hormones, respectively, affected Npy gene expression only in the presence of glucocorticoids (9, 10). These data suggest that glucocorticoids not only stimulate food intake but also play a permissive role in the regulation of energy balance, and that NPY neurons in the arcuate nucleus are the possible site of action for glucocorticoids.

Recent studies suggest that AMP-activated protein kinase (AMPK) signaling pathways in the hypothalamus are involved in the regulation of energy balance (11, 12, 13, 14). AMPK is activated by phosphorylation at Thr172 within the {alpha}-subunit in metabolic stresses such as nutrient starvation (15). It has been shown that hypothalamic AMPK signaling is activated under fasting conditions (16) and is affected by various peripheral signals related to energy balance including insulin, leptin and ghrelin (17, 18). As constitutive-active AMPK and dominant-negative AMPK expressed in the hypothalamus increased and decreased food intake as well as body weight, respectively (11), hypothalamic AMPK has been supposed to function as the master regulator of the energy balance that integrates nutritional and hormonal signals. Previous studies have also demonstrated that NPY neurons express AMPK, the phosphorylation of which is increased by fasting (16), and that the activation of AMPK signaling increased Npy and Agrp gene expression in cell lines as well as in hypothalamic cultures (19, 20), suggesting the possibility that the stimulatory effects of the hypothalamic AMPK on energy balance are mediated, at least in part, via the NPY neurons.

Given that both glucocorticoids and hypothalamic AMPK pathways play integral roles in energy balance, it is possible that cross talk might occur between the two signalings; a recent study suggested that glucocorticoids could stimulate hypothalamic AMPK signaling (21). In the present study, we examined whether or not glucocorticoids regulate Npy and Agrp gene expression in the arcuate nucleus through AMPK signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Hypothalamic organotypic cultures
Hypothalamic slice-explant cultures were performed as described previously (22, 23, 24). Sprague Dawley (SD) pups, 7–9 d old (Chubu Science Materials, Nagoya, Japan; lights on from 0900 to 2100 h), were killed by decapitation, and hypothalamic tissues were sectioned at 350 µm thickness on a Mcllwain tissue chopper (Mickle Laboratory Engineering Co., Surrey, UK). Three coronal slices containing arcuate nucleus were separated and placed in Hanks’ buffered salt solution (Invitrogen, Grand Island, NY) enriched with glucose. The standard culture medium was composed of 50% Earle’s MEM (Invitrogen), 1 mM L-glutamine (Invitrogen) and 33 mM glucose. The serum-free medium was composed of 75% Earle’s MEM, 25% HBSS, 25 U/ml penicillin/streptomycin (Invitrogen), 1 mM L-glutamine, and 5.5 mM glucose. Cultures were maintained in the standard medium for 15 d until the slices became thin enough to perform in situ hybridization, and the medium was changed to a defined serum-free medium for an additional 2 d before subjecting slices to different experimental conditions. The standard medium was changed three times a week, and the serum-free medium was changed every 24 h. All experiments were performed on d 17.

Effects of 5-aminoimidazole-4-carboxamide-1-b- D-ribofuranoside (AICAR) on AMPK signaling in hypothalamic cultures
To observe the time-course effects of AICAR (Biomol International, Ambler, PA), which is known to stimulate AMPK signaling (25), on p-AMPK and p-ACC levels, slices were incubated with 2 mM AICAR dissolved in H2O for 3, 6, 12, and 24 h; the control slices were incubated with vehicle for 24 h.

Effects of AICAR on Npy and Agrp mRNA expression in hypothalamic cultures
To examine the time-course effects of AICAR on Npy and Agrp mRNA expression, slices were incubated with 2 mM AICAR for 6, 12, and 24 h; the control slices were incubated with vehicle for 24 h. To examine the dose-response effects of AICAR on Npy and Agrp mRNA expression, slices were incubated with 0.5, 1, or 2 mM AICAR for 24 h; control slices were incubated with vehicle. To assess whether the effects of AICAR on Npy and Agrp mRNA expression were dependent on action potentials, slices were incubated with 2 mM AICAR or vehicle for 24 h in the presence or absence of the sodium channel blocker tetrodotoxin (TTX; 1 µM, Sankyo, Tokyo, Japan).

Effects of dexamethasone (DEX) on AMPK signaling in hypothalamic cultures
To examine the effects of glucocorticoids on AMPK signaling in the hypothalamic cultures, slices were incubated with 10–8 M DEX (Sigma, St. Louis, MO) dissolved in ethanol for 3–24 h, whereas the control slices were incubated with vehicle for 24 h.

Effects of DEX on AMPK signaling in the absence of gene transcription in hypothalamic cultures
To examine whether the effects of glucocorticoids on AMPK signaling are dependent on gene transcription in the hypothalamic cultures, slices were incubated with 10–8 M DEX for 3 or 24 h in the presence of 150 µM 5,6-dichloro-1-D-ribofuranosylbenzimidazole (DRB) (Sigma) added to the medium for 24 h, which blocks gene transcription in hypothalamic cultures (23).

Effects of compound C (CC) on AMPK signaling in hypothalamic cultures
To observe the time-course effects of CC (Calbiochem, San Diego, CA), which is the specific AMPK inhibitor (26), on the levels of p-AMPK and p-ACC, slices were incubated with CC dissolved in dimethyl sulfoxide (Sigma) for 3–48 h; control slices were incubated with vehicle for 48 h.

Effects of DEX and AICAR on Npy and Agrp mRNA expression
To see whether there are additive effects of DEX and AICAR on Npy and Agrp mRNA expression, slices were incubated with 10–8 M DEX, 2 mM AICAR or both for 24 h; control slices were incubated with vehicle for 24 h.

Effects of blockade of AMPK signaling on Npy and Agrp mRNA expression stimulated by DEX
To assess the effects of blockade of AMPK signaling on Npy and Agrp gene expression stimulated by DEX, slices were preincubated with CC for 24 h and then incubated with DEX or vehicle for an additional 24 h in the presence of CC.

Adrenalectomy (ADX)
Eight-week-old male SD rats (body weight 250 g; Chubu Science Materials) were housed individually and habituated by handling every day under controlled conditions (23.0 ± 0.5 C, lights on from 0900 to 2100 h). ADX or sham-operation (Sham) was performed under pentobarbital (50 mg/kg) anesthesia. Through a dorsal midline incision, small incisions were made through the muscle layer below the rib cage on each flank. Adrenal glands from both sides were isolated by blunt dissection and removed in their capsules. After operation, rats were provided with 0.9% saline in place of water.

Effects of ADX and ip injection of DEX on AMPK signaling as well as Npy and Agrp gene expression in arcuate nucleus
Seven days after the operation, rats were injected ip with DEX (5 mg/kg) and dissolved in isotonic saline or vehicle at 0900 h. After ip injection, they were deprived of food and killed at 4 h. Blood samples were collected into chilled tubes and separated by centrifugation (3500 rpm, 4 C, 15 min), and serum was stored at –30 C until the corticosterone determination. To confirm the effectiveness of ADX, serum corticosterone levels were measured with a RIA commercial kit (MP Biomedicals, Costa Mesa, CA). Rats that had plasma corticosterone concentrations of more than 25 ng/ml in the ADX group were excluded from the study. All procedures were performed in accordance with the institutional guidelines for animal care at the Nagoya University Graduate School of Medicine.

Probes for Npy and Agrp gene expression
The plasmids containing the cDNA for rat preproNPY were kindly provided by Dr. S. L. Sabol (Laboratory of Biochemical Genetics, National Institutes of Health, Bethesda, MD). The plasmids containing the cDNA for rat AGRP were kindly provided by Dr. K. L. Grove (Division of Neuroscience, Oregon Regional Primate Research Center, Oregon Health & Science University, Beaverton, OR). Plasmids containing a 733-bp fragment localized entirely within intron 1 of the rat Npy gene were used to generate probes for Npy heteronuclear RNA (hnRNA), a sensitive indicator of gene transcription (27). The specificity of the probe was demonstrated in a previous study (9). Highly specific RNA probes were synthesized as described previously (23).

In situ hybridization
Hypothalamic slices from the organotypic cultures were fixed with 4% formaldehyde in PBS for 30 min, washed twice in PBS, mounted on poly-L-lysine-coated slides, dried, and kept at –80 C until the procedure. Brains from the in vivo experiments were removed immediately after decapitation, frozen on dry ice, and stored at –80 C until sectioning for in situ hybridization. Coronal sections (12 µm) at 2.8 mm caudal from the bregma, according to the brain atlas of Paxinos and Watson (28), were cut on a cryostat, mounted onto poly-L-lysine-coated slides (three brain sections per slide), and then stored at –80 C until in situ hybridization. Prehybridization, hybridization, and posthybridization procedures were performed as described previously (23). The ODs of the autoradiograph were quantified using a computer image analysis system (Imaging Research, St. Catharines, Ontario, Canada) and a public domain National Institutes of Health Image program. Changes in gene expression were quantified by measurements of the integrated OD (OD x area) of the film images. In hypothalamic cultures, the total sum of OD signals in the bilateral arcuate nuclei in three explants from each rat was used for the analysis. In each culture, control explants were involved, and their expression levels were expressed as 100.

Western blotting
Hypothalamic slices from the organotypic cultures were washed twice with ice-cold PBS, which was removed by centrifugation, then kept at –80 C. Brains from the in vivo experiments were removed immediately after decapitation, frozen on dry ice, and stored at –80 C. Coronal sections (2 mm) at 2.8–4.8 mm caudal from the bregma, according to the brain atlas of Paxinos and Watson (28), were cut on a cryostat, and the arcuate nuclei were punched out with an 18-gauge stainless steel needle. Samples of hypothalamic explants as well as punched-out arcuate nuclei were lysed in 50 µl of a buffer containing 50 mM NaF, 150 mM NaCl, 20 mM HEPES (pH 7.5), 0.5 mM Na2VO3, 1 mM EDTA, 1% Triton X-100 (Sigma) and 1% protease inhibitor mix (Rosch, Stockholm, Sweden). After centrifuging the samples, protein concentrations in the supernatant were determined by the bicinchoninic acid kit (Sigma). Thirty micrograms protein per sample was run on a 10% sodium dodecyl sulfate-polyacrylamide gradient gel and transferred onto nitrocellulose membranes. Blots were blocked for 1 h in TBST solution [10 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% Tween] containing 5% skimmed milk. Membranes were incubated with either AMPK{alpha}, phospho-AMPK (Thr172), ACC, or phospho-ACC (Ser79) antibody (Cell Signaling, Beverly, MA). Each antibody was used at a 1:1000 dilution. The signals were developed using an enhanced chemiluminescence Western blot analysis detection kit (Amersham, Little Chalfont, UK). The membranes were stripped, and hybridized again with β-actin antibody (1:3000; Abcam, Cambridge, UK) to allow normalization. Densitometry analysis of the bands was performed by Scion Image (Scion, Frederick, MD).

Statistical analyses
Statistical analyses were performed with ANOVA followed by Fisher’s protected least significant difference test. Results are expressed as means ± SE, and differences were considered statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of AICAR on AMPK signaling in hypothalamic cultures
Incubation with 2 mM AICAR for 3 h significantly increased the levels of p-AMPK (control: 100 ± 8; AICAR: 151 ± 27 arbitrary units, P < 0.05) as well as p-ACC (control: 100 ± 15; AICAR: 144 ± 11 arbitrary units, P < 0.05), and the stimulatory effects were continued until 24 h in the hypothalamic organotypic cultures. On the other hand, AICAR did not significantly affect the t-AMPK or t-ACC levels until 24 h (data not shown).

Effects of AICAR on Npy and Agrp mRNA expression in hypothalamic cultures
Levels of Npy mRNA expression in the arcuate nucleus were significantly increased with the incubation of 2 mM AICAR for 24 h in the hypothalamic organotypic cultures (Fig. 1Go, A and B). Levels of Agrp mRNA expression in the arcuate nucleus were also increased significantly in the hypothalamic organotypic cultures with the incubation of 1 or 2 mM AICAR for 24 h (Fig. 2Go, A and B). The incubation with 2 mM AICAR for 24 h significantly increased Npy and Agrp mRNA expression even in the presence of 1 µM TTX (Figs. 1CGo and 2CGo), which has been shown to abolish action potentials in hypothalamic organotypic cultures (29), indicating that the action of AICAR on Npy and Agrp mRNA expression was independent of action potentials. Representative photographs showing the effects of AICAR on Npy and Agrp mRNA expression are presented in Figs. 1DGo and 2DGo.


Figure 1
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  		FIG. 1. Effects of AICAR on Npy mRNA expression in hypothalamic cultures. A, Time-course effects of 2 mM AICAR on Npy mRNA expression. B, Dose-response effects of AICAR on Npy mRNA expression. C, Effects of AICAR on Npy mRNA expression in the presence of TTX (1 µM). D, Representative autoradiographs showing the effects of AICAR on Npy mRNA expression. Levels at time 0 (A), without AICAR (B) or without AICAR and TTX (C) are expressed as 100. Results are expressed as means ± SE (n = 10). *, P < 0.05 vs. values at time 0 (A), without AICAR (B).

 

Figure 2
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  		FIG. 2. Effects of AICAR on Agrp mRNA expression in hypothalamic cultures. A, Time-course effects of 2 mM AICAR on Agrp mRNA expression. B, Dose-response effects of AICAR on Agrp mRNA expression. C, Effects of AICAR on Agrp mRNA expression in the presence of TTX (1 µM). D, Representative autoradiographs showing the effects of AICAR on Agrp mRNA expression. Levels at time 0 (A), without AICAR (B) or without AICAR and TTX (C) are expressed as 100. Results are expressed as means ± SE (n = 10). *, P < 0.05 vs. values at time 0 (A), without AICAR (B).

 
Effects of DEX on AMPK signaling in hypothalamic cultures
The levels of p-AMPK as well as p-ACC were significantly increased with the incubation of 10–8 M DEX for 3–24 h in hypothalamic organotypic cultures (Fig. 3Go, A and B). The incubation with 10–8 M DEX significantly increased t-AMPK protein expression at 12 and 24 h (Fig. 3CGo), and t-ACC protein expression at 24 h (Fig. 3DGo).


Figure 3
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  		FIG. 3. Effects of DEX on AMPK signaling in hypothalamic cultures. Levels of p-AMPK (A), p-ACC (B), t-AMPK (C), and t-ACC (D) were normalized with β-actin levels. Levels at time 0 are expressed as 100. Results are expressed as means ± SE (n = 5). *, P < 0.05 vs. values at time 0.

 
Effects of DEX on AMPK signaling in the absence of gene transcription in hypothalamic cultures
The levels of p-AMPK as well as p-ACC were significantly increased with the incubation of 10–8 M DEX for 3 h in the presence of DRB, which blocks gene transcription (23) in hypothalamic organotypic cultures (Fig. 4Go, A and B). On the other hand, the levels of p-AMPK or p-ACC were not changed significantly with the incubation of 10–8 M DEX for 24 h in the presence of DRB in hypothalamic organotypic cultures (Fig. 4Go, C and D). The stimulatory effects of DEX on t-AMPK and t-ACC expression at 24 h were abolished in the presence of DRB (data not shown).


Figure 4
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  		FIG. 4. Effects of DEX on AMPK signaling in the absence of gene transcription in hypothalamic cultures. Slices were incubated with 10–8 M DEX for 3 (A and B) or 24 h (C and D) in the absence or presence of DRB which blocks gene transcription. Levels of p-AMPK (A and C) and p-ACC (B and D) were normalized with β-actin levels. Levels in control (without DEX and DRB) are expressed as 100. Results are expressed as means ± SE (n = 5). n.s., Not significant.

 
Effects of CC on AMPK signaling in hypothalamic cultures
The levels of p-AMPK as well as p-ACC were significantly reduced with the incubation of the specific AMPK inhibitor CC (20 µM) for 6–48 h in hypothalamic organotypic cultures (Fig. 5Go, A and B). The stimulatory effects of DEX on the p-AMPK and p-ACC levels were completely abolished in the presence of CC (Fig. 5Go, C and D).


Figure 5
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  		FIG. 5. Effects of compound C (CC) on AMPK signaling in hypothalamic cultures. A, Time-course effects of CC (20 µM) on p-AMPK levels. B, Time-course effects of CC on p-ACC levels. C, Effects of DEX on p-AMPK levels in the absence or presence of CC. D, Effects of DEX on p-ACC levels in the absence or presence of CC. Levels at time 0 (A and B) or those without CC and DEX (C and D) are expressed as 100. Levels of p-AMPK (A and C) and p-ACC (B and D) were normalized with β-actin levels. Results are expressed as means ± SE (n = 5). *, P < 0.05 vs. values at time 0. n.s., Not significant.

 
Effects of AICAR and DEX on Npy and Agrp mRNA expression in hypothalamic cultures
The levels of Npy and Agrp mRNA stimulated by DEX were significantly elevated than those stimulated by AICAR, and there were no additive effects of DEX and AICAR on Npy and Agrp mRNA expression (Fig. 6Go, A and B).


Figure 6
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  		FIG. 6. AMPK-dependent and -independent action of DEX on Npy and Agrp mRNA expression in hypothalamic cultures. A, Effects of AICAR and DEX on Npy mRNA expression. B, Effects of AICAR and DEX on Agrp mRNA expression. C, Effects of DEX on Npy mRNA expression in the absence or presence of CC. D, Effects of DEX on Agrp mRNA expression in the absence or presence of CC. Expression levels without AICAR and DEX (A and B), or without DEX and CC (C and D) are expressed as 100. Results are expressed as means ± SE (n = 10). *, P < 0.05 vs. values without AICAR and DEX (A and B), without DEX and CC (C, D). n.s., Not significant.

 
Effects of blockade of AMPK signaling on Npy and Agrp mRNA expression stimulated by DEX in hypothalamic cultures
Although the incubation with CC did not affect significantly the basal expression levels of Npy or Agrp mRNA, the stimulatory effects of DEX on Npy and Agrp mRNA expression were partially but significantly attenuated in the presence of CC (Fig. 6Go, C and D).

Effects of ADX and ip injection DEX on AMPK signaling in the arcuate nucleus
Levels of p-AMPK as well as p-ACC in the arcuate nucleus were significantly decreased in ADX rats compared with Sham rats (Fig. 7Go, A and B). The ip injection of DEX significantly increased levels of p-AMPK and p-ACC in ADX rats (Fig. 7Go, A and B). Levels of t-AMPK or t-ACC were not affected significantly by ADX or the administration of DEX (Fig. 7Go, C and D).


Figure 7
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  		FIG. 7. Effects of glucocorticoids on p-AMPK and p-ACC levels in the arcuate nucleus in vivo. Rats in the sham-operated (Sham) group were injected with isotonic saline, and those in adrenalectomized (ADX) groups were injected with either isotonic saline or dexamethasone (DEX; 5 mg/kg) 4 h before the rats were killed. The arcuate nuclei were punched out, and the levels of p-AMPK (A), p-ACC (B), t-AMPK (C), and t-ACC (D) were examined. Levels were normalized with those of β-actin. Sham group levels are expressed as 100. Results are expressed as means ± SE (n = 5).

 
Effects of ADX and ip injection DEX on Npy and Agrp gene expression in the arcuate nucleus
Although levels of Npy or Agrp mRNA expression in the arcuate nucleus were not significantly different between Sham and ADX rats (Fig. 8Go, A–D), those of Npy hnRNA (a measure of transcription; see Materials and Methods) were significantly decreased in ADX rats compared with Sham rats (Fig. 8Go, E and F). The administration of DEX significantly increased the levels of Npy and Agrp mRNA as well as Npy hnRNA expression in ADX rats (Fig. 8Go).


Figure 8
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  		FIG. 8. Effects of glucocorticoids on Npy and Agrp gene expression in the arcuate nucleus in vivo. Rats in the sham-operated (Sham) group were injected with isotonic saline, and those in adrenalectomized (ADX) groups were injected with either isotonic saline or dexamethasone (DEX; 5 mg/kg) 4 h before the rats were killed. Expression levels of Npy mRNA (A and B), Agrp mRNA (C and D), and Npy hnRNA (E and F) in the arcuate nucleus are shown. Sham group levels are expressed as 100. Results are expressed as means ± SE (n = 8).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we examined whether or not glucocorticoids stimulate Npy and Agrp gene expression in the arcuate nucleus by activating AMPK signaling in the hypothalamus. Our data on the organotypic cultures demonstrated that: 1) glucocorticoids stimulate AMPK signaling in the hypothalamus; 2) the activation of AMPK signaling increases Npy and Agrp mRNA expression in the arcuate nucleus; and 3) the inhibition of AMPK signaling attenuates Npy and Agrp expression stimulated by glucocorticoids. Our in vivo experiments showed that AMPK signaling in the hypothalamus is decreased by ADX and stimulated by peripheral injection of glucocorticoids, accompanied by increases in Npy and Agrp expression in the arcuate nucleus.

It has been demonstrated that the activation or blockade of AMPK signaling at levels of the hypothalamus affected Npy and Agrp expression in the arcuate nucleus in vivo (11). Furthermore, it was shown that the activation of AMPK signaling increases Npy and Agrp gene expression in cell lines and in hypothalamic primary cultures (19, 20). On the other hand, a recent study demonstrated that Npy or Agrp gene expression levels did not differ between wild-type mice and those lacking AMPK{alpha}2 in the AGRP neurons (30), suggesting that AMPK signaling is not pivotal in the regulation of Npy and Agrp gene expression in the arcuate nucleus. However, this does not necessarily exclude the role of AMPK signaling in the regulation of Npy or Agrp gene expression because it is possible that some compensational mechanisms might occur in these mice. In the present study, we examined the regulation of Npy and Agrp gene expression in the arcuate nucleus in the organotypic cultures which maintain the intrinsic properties (9, 10, 22, 23, 24). Our data demonstrated that activating AMPK signaling increased both Npy and Agrp gene expression in the arcuate nucleus, and that the action was independent of action potentials.

Several hormonal and nutrient signals including ghrelin, leptin, and insulin are shown to affect AMPK signaling in the periphery as well (17, 18, 31, 32, 33), and it is supposed that the coordinated regulation of the AMPK signaling in the hypothalamus and peripheral tissues plays a critical role in energy homeostasis (12, 13, 14). Glucocorticoids also reportedly affected AMPK signaling in the periphery (34, 35), and a recent study demonstrated glucocorticoids stimulated hypothalamic AMPK signaling as well (21). Although glucocorticoids exert their effects after binding to the cytoplasmic glucocorticoid receptor that belongs to the nuclear receptor superfamily (2), it is also demonstrated that some effects of glucocorticoids on cellular responses are independent of gene transcription (36, 37, 38). We demonstrated in the present study that, although the phosphorylation of AMPK and ACC was accompanied by increases in protein levels in the late phase, glucocorticoids induced the phosphorylation without affecting protein levels in the early phase. Experiments using DRB, which blocks gene transcription (23), suggest that the stimulatory effects of glucocorticoids on AMPK signaling are mediated via nongenomic action in the early phase (3 h) and via genomic action in the late phase (24 h). Thus, the mode of action of glucocorticoids on hypothalamic AMPK signaling is complex, and elucidation of the detailed mechanisms is an important task in future.

Our data in hypothalamic cultures showed that the stimulation of AMPK signaling by glucocorticoids was accompanied by increases in Npy and Agrp expression in the arcuate nucleus, and that the stimulatory effects of glucocorticoids on their gene expression were significantly attenuated by the blockade of AMPK signaling. These data strongly suggest that glucocorticoids stimulate Npy and Agrp gene expression via AMPK signaling. The findings that there were no additive effects of DEX and AICAR on the levels of Npy and Agrp gene expression suggest that DEX stimulated AMPK signaling fully in the hypothalamic cultures. Our previous studies demonstrated that ghrelin and insulin affected Npy gene expression in the arcuate nucleus only in the presence of glucocorticoids (9, 10). Because it is also reported that AMPK plays a permissive role in the regulation of Npy gene expression (11), it is possible that the activation of AMPK signaling by glucocorticoids in the NPY neurons is essential to the integration of various signals related to energy balance. On the other hand, the levels of Npy and Agrp gene expression stimulated by DEX were significantly more elevated than those stimulated by AICAR. Furthermore, the blockade of AMPK signaling only partially affected Npy and Agrp expression stimulated by DEX. These data suggest that glucocorticoids stimulate Npy and Agrp gene expression by mechanisms other than AMPK signaling as well. Further studies of such mechanisms are warranted that might involve cAMP-CREB and JAK-Stat signaling, both of which are shown to affect Npy gene expression in the hypothalamus (16, 39, 40, 41) and possibly be affected by glucocorticoids (42, 43).

To determine whether or not glucocorticoids could affect AMPK signaling in the hypothalamus in vivo as well, we compared AMPK signaling between Sham and ADX rats. Although body weight and food intake decreased in ADX compared with Sham rats as reported previously (3, 4, 5, 6, 7, 8), the levels of p-AMPK as well as p-ACC in the arcuate nucleus were reduced in ADX rats. This finding is noteworthy because decreases in body weight reportedly increased hypothalamic AMPK signaling (16). Thus, our data suggest that glucocorticoids are required to maintain the basal levels of AMPK signaling in the hypothalamus, and they provide support for the hypothesis that AMPK signaling is downstream of glucocorticoids. To further explore the relationship between glucocorticoids and hypothalamic AMPK signaling, we injected ADX rats with DEX. The impact of peripheral administration of glucocorticoids on energy balance could be time and dose dependent because it would induce the release of anorexigenic hormones such as insulin and leptin (3, 44, 45, 46). By examining the acute effects of relatively high doses of DEX, we clearly demonstrated that increases in glucocorticoids in the periphery activate hypothalamic AMPK signaling, at least during the time course we used.

Consistent with the findings in the organotypic cultures, the activation of the hypothalamic AMPK signaling by replacement of glucocoticoids in ADX rats lead to increases in Npy and Agrp expression in the arcuate nucleus in the present study. On the other hand, ADX per se did not affect significantly the levels of Npy and Agrp mRNA expression in the arcuate nucleus, as reported in previous studies (9). We have found, however, that the levels of Npy hnRNA, a sensitive indicator for gene transcription (27), were significantly decreased by ADX. Together with the findings that the AMPK signaling was decreased by ADX, it is suggested that Npy gene transcription in the arcuate nucleus is tonically stimulated by glucocorticoids via AMPK signaling in vivo. Although further studies are warranted, the discrepancy between mRNA and hnRNA levels might be explained by the fact that Npy mRNA stability was increased to compensate for the decrease in gene transcription in ADX rats.

In conclusion, we demonstrated that glucocorticoids up-regulate the Npy and Agrp gene expression in the arcuate nucleus through AMPK signaling, at least in part. The activation of AMPK signaling by glucocorticoids may well be essential for the integration of various signals related to energy balance.


   Footnotes
 
Disclosure Statement: All authors have nothing to declare.

First Published Online June 5, 2008

Abbreviations: ADX, Adrenalectomy; AGRP, agouti-related peptide; AICAR, 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside; AMPK, AMP-activated protein kinase; CC, compound C; DRB, 5,6-dichloro-1-D-ribofuranosylbenzimidazole; NPY, neuropeptide Y; p-ACC, phosphorylated acetyl-coenzyme A carboxylase; SD, Sprague Dawley; TTX, tetrodotoxin.

Received February 15, 2008.

Accepted for publication May 23, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Schwartz MW, Woods SC, Porte Jr D, Seeley RJ, Baskin DG 2000 Central nervous system control of food intake. Nature 404:661–671[Medline]
  2. Stewart PM 2002 The adrenal cortex. In: Larsen PR, Kronenberg HM, Melmed S, Polonsky KS, eds. Williams textbook of endocrinology. 10th ed. Philadelphia: Saunders; 491–551
  3. Castonguay TW, Dallman MF, Stern JS 1986 Some metabolic and behavioral effects of adrenalectomy on obese Zucker rats. Am J Physiol 251:R923–R933
  4. Freedman MR, Castonguay TW, Stern JS 1985 Effect of adrenalectomy and corticosterone replacement on meal patterns of Zucker rats. Am J Physiol 249:R584–R594
  5. Freedman MR, Horwitz BA, Stern JS 1986 Effect of adrenalectomy and glucocorticoid replacement on development of obesity. Am J Physiol 250: R595–R607
  6. Saito M, Bray GA 1984 Adrenalectomy and food restriction in the genetically obese (ob/ob) mouse. Am J Physiol 246:R20–R25
  7. King BM, Smith RL 1985 Hypothalamic obesity after hypophysectomy or adrenalectomy: dependence on corticosterone. Am J Physiol 249:R522–R526
  8. Trocki O, Baer DJ, Castonguay TW 1995 Comparison of effects of adrenalectomy and RU-486 in rats given a choice of maintenance diet and fat supplement. Am J Physiol 269:R708–R719
  9. Goto M, Arima H, Watanabe M, Hayashi M, Banno R, Sato I, Nagasaki H, Oiso Y 2006 Ghrelin increases neuropeptide Y and agouti-related peptide gene expression in the arcuate nucleus in rat hypothalamic organotypic cultures. Endocrinology 147:5102–5109[Abstract/Free Full Text]
  10. Sato I, Arima H, Ozaki N, Watanabe M, Goto M, Hayashi M, Banno R, Nagasaki H, Oiso Y 2005 Insulin inhibits neuropeptide Y gene expression in the arcuate nucleus through GABAergic systems. J Neurosci 25:8657–8664[Abstract/Free Full Text]
  11. Minokoshi Y, Alquier T, Furukawa N, Kim YB, Lee A, Xue B, Mu J, Foufelle F, Ferre P, Birnbaum MJ, Stuck BJ, Kahn BB 2004 AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus. Nature 428:569–574[CrossRef][Medline]
  12. Kahn BB, Alquier T, Carling D, Hardie DG 2005 AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 1:15–25[CrossRef][Medline]
  13. Xue B, Kahn BB 2006 AMPK integrates nutrient and hormonal signals to regulate food intake and energy balance through effects in the hypothalamus and peripheral tissues. J Physiol 574:73–83[Abstract/Free Full Text]
  14. Kim MS, Lee KU 2005 Role of hypothalamic 5'-AMP-activated protein kinase in the regulation of food intake and energy homeostasis. J Mol Med 83:514–520[CrossRef][Medline]
  15. da Silva Xavier G, Leclerc I, Salt IP, Doiron B, Hardie DG, Kahn A, Rutter GA 2000 Role of AMP-activated protein kinase in the regulation by glucose of islet β cell gene expression. Proc Natl Acad Sci USA 97:4023–4028[Abstract/Free Full Text]
  16. Kim EK, Miller I, Aja S, Landree LE, Pinn M, McFadden J, Kuhajda FP, Moran TH, Ronnett GV 2004 C75, a fatty acid synthase inhibitor, reduces food intake via hypothalamic AMP-activated protein kinase. J Biol Chem 279:19970–19976[Abstract/Free Full Text]
  17. Andersson U, Filipsson K, Abbott CR, Woods A, Smith K, Bloom SR, Carling D, Small CJ 2004 AMP-activated protein kinase plays a role in the control of food intake. J Biol Chem 279:12005–12008[Abstract/Free Full Text]
  18. Namkoong C, Kim MS, Jang PG, Han SM, Park HS, Koh EH, Lee WJ, Kim JY, Park IS, Park JY, Lee KU 2005 Enhanced hypothalamic AMP-activated protein kinase activity contributes to hyperphagia in diabetic rats. Diabetes 54:63–68[Abstract/Free Full Text]
  19. Lee K, Li B, Xi X, Suh Y, Martin RJ 2005 Role of neuronal energy status in the regulation of adenosine 5'-monophosphate-activated protein kinase, orexigenic neuropeptides expression, and feeding behavior. Endocrinology 146:3–10[Abstract/Free Full Text]
  20. Chau-Van C, Gamba M, Salvi R, Gaillard RC, Pralong FP 2007 Metformin inhibits adenosine 5'-monophosphate-activated kinase activation and prevents increases in neuropeptide Y expression in cultured hypothalamic neurons. Endocrinology 148:507–511[Abstract/Free Full Text]
  21. Christ-Crain M, Kola B, Lolli F, Fekete C, Seboek D, Wittmann G, Feltrin D, Igreja SC, Ajodha S, Harvey-White J, Kunos G, Muller B, Pralong F, Aubert G, Arnaldi G, Giacchetti G, Boscaro M, Grossman AB, Korbonits M 2008 AMP-activated protein kinase mediates glucocorticoid-induced metabolic changes: a novel mechanism in Cushing’s syndrome. FASEB J 22:1672–1683[Abstract/Free Full Text]
  22. Arima H, House SB, Gainer H, Aguilera G 2002 Neuronal activity is required for the circadian rhythm of vasopressin gene transcription in the suprachiasmatic nucleus in vitro. Endocrinology 143:4165–4171[Abstract/Free Full Text]
  23. Kuwahara S, Arima H, Banno R, Sato I, Kondo N, Oiso Y 2003 Regulation of vasopressin gene expression by cAMP and glucocorticoids in parvocellular neurons of the paraventricular nucleus in rat hypothalamic organotypic cultures. J Neurosci 23:10231–10237[Abstract/Free Full Text]
  24. Arima H, House SB, Gainer H, Aguilera G 2001 Direct stimulation of arginine vasopressin gene transcription by cAMP in parvocellular neurons of the paraventricular nucleus in organotypic cultures. Endocrinology 142:5027–5030[Abstract/Free Full Text]
  25. Corton JM, Gillespie JG, Hawley SA, Hardie DG 1995 5-Aminoimidazole-4-carboxamide ribonucleoside. A specific method for activating AMP-activated protein kinase in intact cells? Eur J Biochem 229:558–565[Medline]
  26. Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, Wu M, Ventre J, Doebber T, Fujii N, Musi N, Hirshman MF, Goodyear LJ, Moller DE 2001 Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 108:1167–1174[CrossRef][Medline]
  27. Herman JP, Schafer MK, Thompson RC, Watson SJ 1992 Rapid regulation of corticotropin-releasing hormone gene transcription in vivo. Mol Endocrinol 6:1061–1069[Abstract/Free Full Text]
  28. Paxinos G, Watson C 1998 The rat brain in stereotaxic coordinates. New York: Academic
  29. Wray S, Kusano K, Gainer H 1991 Maintenance of LHRH and oxytocin neurons in slice explants cultured in serum-free media: effects of tetrodotoxin on gene expression. Neuroendocrinology 54:327–339[CrossRef][Medline]
  30. Claret M, Smith MA, Batterham RL, Selman C, Choudhury AI, Fryer LG, Clements M, Al-Qassab H, Heffron H, Xu AW, Speakman JR, Barsh GS, Viollet B, Vaulont S, Ashford ML, Carling D, Withers DJ 2007 AMPK is essential for energy homeostasis regulation and glucose sensing by POMC and AgRP neurons. J Clin Invest 117:2325–2336[CrossRef][Medline]
  31. Minokoshi Y, Kim YB, Peroni OD, Fryer LG, Muller C, Carling D, Kahn BB 2002 Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 415:339–343[CrossRef][Medline]
  32. Gamble J, Lopaschuk GD 1997 Insulin inhibition of 5' adenosine monophosphate-activated protein kinase in the heart results in activation of acetyl coenzyme A carboxylase and inhibition of fatty acid oxidation. Metabolism 46:1270–1274[CrossRef][Medline]
  33. Towler MC, Hardie DG 2007 AMP-activated protein kinase in metabolic control and insulin signaling. Circ Res 100:328–341[Abstract/Free Full Text]
  34. Qi D, An D, Kewalramani G, Qi Y, Pulinilkunnil T, Abrahani A, Al-Atar U, Ghosh S, Wambolt RB, Allard MF, Innis SM, Rodrigues B 2006 Altered cardiac fatty acid composition and utilization following dexamethasone-induced insulin resistance. Am J Physiol Endocrinol Metab 291:E420–E427
  35. Viana AY, Sakoda H, Anai M, Fujishiro M, Ono H, Kushiyama A, Fukushima Y, Sato Y, Oshida Y, Uchijima Y, Kurihara H, Asano T 2006 Role of hepatic AMPK activation in glucose metabolism and dexamethasone-induced regulation of AMPK expression. Diabetes Res Clin Pract 73:135–142[CrossRef][Medline]
  36. Losel R, Wehling M 2003 Nongenomic actions of steroid hormones. Nat Rev Mol Cell Biol 4:46–56[CrossRef][Medline]
  37. Norman AW, Mizwicki MT, Norman DP 2004 Steroid-hormone rapid actions, membrane receptors and a conformational ensemble model. Nat Rev Drug Discov 3:27–41[CrossRef][Medline]
  38. Lowenberg M, Verhaar AP, van den Brink GR, Hommes DW 2007 Glucocorticoid signaling: a nongenomic mechanism for T-cell immunosuppression. Trends Mol Med 13:158–163[CrossRef][Medline]
  39. Higuchi H, Yang HY, Sabol SL 1988 Rat neuropeptide Y precursor gene expression. mRNA structure, tissue distribution, and regulation by glucocorticoids, cyclic AMP, and phorbol ester. J Biol Chem 263:6288–6295[Abstract/Free Full Text]
  40. Akabayashi A, Zaia CT, Gabriel SM, Silva I, Cheung WK, Leibowitz SF 1994 Intracerebroventricular injection of dibutyryl cyclic adenosine 3',5'-monophosphate increases hypothalamic levels of neuropeptide Y. Brain Res 660:323–328[CrossRef][Medline]
  41. Morton GJ, Cummings DE, Baskin DG, Barsh GS, Schwartz MW 2006 Central nervous system control of food intake and body weight. Nature 443:289–295[CrossRef][Medline]
  42. Malcher-Lopes R, Di S, Marcheselli VS, Weng FJ, Stuart CT, Bazan NG, Tasker JG 2006 Opposing crosstalk between leptin and glucocorticoids rapidly modulates synaptic excitation via endocannabinoid release. J Neurosci 26:6643–6650[Abstract/Free Full Text]
  43. Ishida-Takahashi R, Uotani S, Abe T, Degawa-Yamauchi M, Fukushima T, Fujita N, Sakamaki H, Yamasaki H, Yamaguchi Y, Eguchi K 2004 Rapid inhibition of leptin signaling by glucocorticoids in vitro and in vivo. J Biol Chem 279:19658–19664[Abstract/Free Full Text]
  44. Strack AM, Sebastian RJ, Schwartz MW, Dallman MF 1995 Glucocorticoids and insulin: reciprocal signals for energy balance. Am J Physiol 268:R142–R149
  45. Dallman MF, la Fleur SE, Pecoraro NC, Gomez F, Houshyar H, Akana SF 2004 Minireview: glucocorticoids—food intake, abdominal obesity, and wealthy nations in 2004. Endocrinology 145:2633–2638[Abstract/Free Full Text]
  46. Zakrzewska KE, Cusin I, Stricker-Krongrad A, Boss O, Ricquier D, Jeanrenaud B, Rohner-Jeanrenaud F 1999 Induction of obesity and hyperleptinemia by central glucocorticoid infusion in the rat. Diabetes 48:365–370[Abstract]



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