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Am J Physiol Endocrinol Metab 292: E1899-E1905, 2007. First published March 6, 2007; doi:10.1152/ajpendo.00116.2006
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Activation of AMP-activated protein kinase stimulates proopiomelanocortin gene transcription in AtT20 corticotroph cells

Yasumasa Iwasaki,1 Mitsuru Nishiyama,1 Takafumi Taguchi,1 Machiko Kambayashi,1 Masato Asai,2 Masanori Yoshida,2 Takeshi Nigawara,3 and Kozo Hashimoto1

1Department of Endocrinology, Metabolism, and Nephrology, Kochi Medical School, Kochi University, Nankoku; and Departments of 2Medicine and 3Clinical Pathophysiology, Nagoya University Graduate School of Medicine and Hospital, Nagoya, Japan

Submitted 12 March 2006 ; accepted in final form 21 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Starvation is known to activate the hypothalamo-pituitary-adrenal (HPA) axis, a representative antistress system in the living organism. In this study, we investigated in vitro whether activation of the AMP-activated protein kinase (AMPK), which is known to occur in intracellular energy depletion, influences the expression of POMC gene that encodes adrenocorticotropin. We first confirmed that each subunit of AMPK was expressed in the AtT20 corticotroph cell line. We then found that AICAR, a cell-permeable AMP analog and an activator of AMPK, potently stimulated the 5'-promoter activity of POMC gene in a dose-dependent manner. The effects were promoter specific because AICAR enhanced the AP1-mediated POMC promoter activities but did not influence other transcription factor-induced transcription. The effect of AICAR on POMC gene transcription was completely eliminated by specific AMPK inhibitor compound C or by dominant negative AMPK, whereas overexpression of constitutively active AMPK mimicked the effect of AICAR. Finally, experiments using specific kinase inhibitors suggested that the PI 3-kinase-mediated signaling pathway is at least partly involved in the effect. Our results suggest that intracellular energy depletion with the resultant activation of AMPK directly stimulates the HPA axis at the pituitary level by increasing the expression of POMC gene.

adenosine 5'-monophosphate; adrenocorticotropin; energy homeostasis; pituitary; starvation

STARVATION IS ONE OF THE POTENT CAUSES of stress in the living organism (19). Indeed, starvation activates the hypothalamo-pituitary-adrenal (HPA) axis, a representative antistress system, and stimulates adrenal glucocorticoid hormone production (6). Glucocorticoid, as the name implies, acts to maintain plasma glucose levels by stimulating gluconeogenesis in the liver. Thus, maintenance of plasma glucose, a major energy source of cellular metabolism, is the primary role of the HPA axis. Recently, Laugero and colleagues (16, 17) have shown that the activated HPA axis is relieved by sucrose supplementation in adrenalectomized rats, suggesting the importance of glucose homeostasis in the negative feedback regulation of the HPA axis.

The molecular mechanism whereby starvation activates the HPA axis, however, is not completely understood. Corticotropin-releasing hormone (CRH) is the major hypothalamic hormone regulating the HPA axis (19), but the expression of CRH is known to be decreased during starvation (11, 20, 27), and the involvement of other factor(s) such as vasopressin is assumed (7). Furthermore, the factor(s) that maintains the synthesis of adrenocorticotropin (ACTH), a key stimulator of adrenal corticosteroid production expressed in the pituitary, during starvation has not been clarified.

In this study, we investigated the effect of culture condition mimicking cellular energy depletion on the transcriptional regulation of proopiomelanocortin (POMC) gene that encodes ACTH in corticotroph cells in vitro. Lack of an energy source increases the intracellular AMP/ATP ratio, which in turn activates the AMP-activated protein kinase (AMPK) and subsequent signaling events to maintain energy homeostasis (8, 9). We used 5-amino-imidazole carboxamide riboside (AICAR), a cell-permeable AMP analog, to mimic the intracellular energy depletion (28). We found that the activation of AMPK by AICAR potently stimulates the transcriptional activity of POMC gene, thus suggesting the direct stimulatory effect of starvation stress on the HPA axis at the pituitary level.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials. AICAR was purchased from Toronto Research Chemical (North York, ON, Canada), and the reporter plasmids (pCRE-, pSRE-, pAP1-, and pNF-{kappa}B-luciferase) were from Stratagene (La Jolla, CA). Expression vectors of constitutively active and dominant negative AMPK{alpha}1 were a kind gift from Prof. David Carling (Imperial College School of Medicine, London, UK) (29). H89, PD98059, SB203580, RO32-0432, KN62, and LY294002 were obtained from Calbiochem (San Diego, CA). Wortmannin, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and thenoyltrifluoroacetone (TTFA) were from Sigma (St. Louis, MO). AMPK inhibitor compound C was obtained from Merck Research Laboratories (Rahway, NJ) (30). About 1.2 kb of the human CRH gene 5'-promoter (–907 to +171)-luciferase fusion gene was constructed by PCR and standard molecular cloning techniques.

Cell culture and transfection. AtT20PL, a clone of the AtT20 cell line in which {approx}0.7 kb of the rat POMC gene 5'-promoter (–708 to +64; +1 indicates the transcription start site)-luciferase fusion gene is stably incorporated, is described elsewhere (2). AtT20 or AtT20PL cells were maintained in a T75 culture flask with DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Invitrogen) and antibiotics (50 U/ml penicillin and 50 µg/ml streptomycin; Invitrogen) under a 5% CO2-95% atmosphere at 37°C. The BE(2)C human neuroblastoma cell line (14) was maintained with DMEM-F-12 (Invitrogen) with 10% FBS and antibiotics under the same condition as AtT20. For both cell lines, culture medium was changed twice/wk, and the cells were subcultured once/wk. In some experiments, cells were transfected transiently with the plasmids, using the lipofection method. The experimental procedure was otherwise the same as that for AtT20PL cells.

Experimental protocols. In all the experiments, the culture condition of the cells was basically the same as previously described (2), with minor modification. In brief, cells were plated in 24-well plates with {approx}50% confluency and cultured in DMEM with 1% FBS for 4 days. Then AICAR, in x100 solution, was added to the culture medium of each well, and the cells were harvested after the defined time interval for luciferase assay. In some experiments, cells were incubated with a variety of kinase inhibitors, in x1,000 solution, from 30 min prior to the addition of AICAR to the end of experiment.

Western blotting. The AtT20PL cells were cultured in 3.5-cm-diameter dishes and treated without or with AICAR (2 mM, for 0.5 or 1 h), and cellular protein was extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL). Then the extracts were applied for electrophoresis and incubated with antibodies to either AMPK{alpha} or phospho-AMPK{alpha} (Thr172; Cell Signaling, Beverly, MA) and with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (Santa Cruz Biotechnology, Santa Cruz, CA). Finally, the bands were detected by chemiluminescence using ECL plus Western blotting detection system (Amersham Pharmacia Biotech, Buckinghamshire, UK), following the manufacturer's instructions.

Electrophoretic mobility shift assay. Electrophoretic mobility shift assay (EMSA) was carried out using a commercially available nonradioisotopic EMSA kit (LightShift Chemiluminescent EMSA kit; Pierce). Briefly, the AtT20 PL cells were treated without or with AICAR (2 mM, for 5 h), and nuclear extract was prepared using NE-PER. The extract was then incubated for 6 h with the double-stranded, 3'-end-biotinylated oligonucleotide probe (50 fmol) containing the AP1 site of the rat POMC gene promoter (sense 5'-TAAGGAGCAGTGACTAAGAGAGGCCAC-3'-biotin, antisense 5'-GTGGCCTCTCTTAGTCACTGCTCCTTA-3' biotin; bold letters indicate the POMC AP1 site) or a probe in which the POMC AP1 is changed to the canonical AP1 (sense 5'-TAAGGAGCAGTGAGTCAGAGAGGCCAC-3' biotin, antisense 5'-GTGGCCTCTCTGACTCACTGCTCCTTA-3' biotin; bold letters indicate the canonical AP1 site). A double-stranded, nonlabeled probe containing the canonical AP1 (sense 5'-CGCTTGATGACTCAGCCGGAA-3', antisense 5'-TTCCGGCTGAGTCATCAAGCG-3') was used for a cold competition study. Finally, the mixture was subjected to 4% nondenaturing polyacrylamide gel (160 V for 4 h), the biotinylated DNA was transferred to a nylon membrane and was cross-linked, and then the biotin-labeled DNA was detected with digital imaging apparatus (LightCapture; ATTO, Tokyo, Japan).

Quantitative RT-PCR analysis. Reagents, software, and equipment were from Applied Biosystems (Foster City, CA). TaqMan reactions were performed using SuperScript III reverse transcriptase (Invitrogen) and the ABI PRISM 7700 sequence detection system, and then analysis was performed using the sequence detection system software. Mouse POMC primers and TaqMan probe were designed on the mouse POMC gene exon 2–3 junction as follows: mouse POMC forward, 5'-GGAAGATGCCGAGATTCTGCTA-3'; reverse, 5'-GCGAGAGGTCGAGTTTGCA-3'; TaqMan probe, 5'-TCAGACCTCCATAGATGTG-3'. The quantity of the target mRNA in an unknown sample was determined from the cycle threshold value using the standard curve. A control without a template was included in each experiment. Nontemplate controls, standard dilutions, and samples were assayed in duplicate.

Measurements and statistics. Luciferase activity was measured as described (12). ACTH concentration in the culture medium was measured by immunoradiometric assay (ACTH IRMA kit; Mitsubishi Chemical, Tokyo, Japan) (2).

Results are expressed as means ± SE of triplicate or quadruplicate dishes in each group. The statistical significance of differences among the groups was analyzed by ANOVA, followed by a Fisher's protected least significant difference test at a significance level of 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
AMPK subunit mRNAs are expressed in AtT20PL cells. We first analyzed the expression of each of the subunits of AMPK in AtT20 cells by RT-PCR. We found that bands corresponding to the {alpha}2-, beta1-, beta2-, and {gamma}2-subunits of AMPK mRNAs were amplified (Fig. 1).


Figure 1
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  		Fig. 1. Expression of the AMP-activated protein kinase (AMPK) subunits analyzed by RT-PCR in AtT20PL cells. cDNA produced from an RT reaction using total RNA from AtT20PL cells was amplified by PCR with pairs of oligonucleotide primers specific for each cDNA. MW, molecular weight marker.

 
AICAR stimulates POMC but inhibits CRH gene expression. We then examined the effect of AICAR, an AMP analog and the activator of AMPK, on the 5'-promoter activity of POMC and CRH genes. The results showed that AICAR potently stimulated the transcriptional activity of POMC gene (Fig. 2A). In contrast, a significant inhibitory effect of AICAR was observed on the 5'-promoter activity of CRH gene (Fig. 2B).


Figure 2
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  		Fig. 2. Effects of 5-amino-imidazole carboximide riboside (AICAR) on the transcriptional activity of the proopiomelanocortin (POMC) and corticotropin-releasing hormone (CRH) genes. A: AtT20PL cells were treated with AICAR (2 mM) for 8 h, and the 5'-promoter activity was determined by luciferase assay. B: BE(2)C cells were transfected transiently with CRH907-luciferase plasmid and treated with AICAR (2 mM) for 6 h, and then the 5'-promoter activity of CRH gene was determined as above. C: AtT20PL cells were treated with AICAR (2 mM) for 0 and 4–24 h. D: AtT20PL cells were treated with various doses of AICAR (0, 0.1–2 mM) for 8 h. *P < 0.05 vs. control (C) or value at time 0.

 
Further analysis of the AICAR effect on POMC expression revealed that AICAR increased the 5'-promoter activity of POMC gene in a dose- and time-related manner (Fig. 2, C and D). The maximal effect was obtained at 8 h, with a 2- to 2.5-fold increase by 2 mM of AICAR. The minimal effective concentration of AICAR was 0.1 mM.

Involvement of AMPK in POMC gene transcription. Since AICAR is an activator of AMPK, we studied the role of AMPK in the transcriptional regulation of POMC gene. We found that overexpression of constitutively active AMPK{alpha} significantly stimulated the 5'-promoter activity of POMC gene in a dose-dependent manner (Fig. 3A). We also found that the positive effect of AICAR on POMC gene transcription was completely eliminated when cells were treated with specific AMPK inhibitor compound C or when dominant negative AMPK{alpha} was overexpressed (Fig. 3, B and C), indicating the involvement of AMPK in the AICAR effect. Moreover, the 5'-promoter activity of POMC gene was significantly stimulated when cells were treated with the mitochondrial respiration inhibitors CCCP or TTFA, confirming that cellular energy depletion stimulates POMC gene transcription (Fig. 3D). We also examined the effect of AICAR and energy depletion on endogenous POMC mRNA level using quantitative RT-PCR and found that treatment of the cells with AICAR (2 mM, 6 h) or low glucose (1 mM, 6 h) significantly increased the amount of POMC mRNA (Fig. 3E).


Figure 3
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  		Fig. 3. Effects of AMPK overexpression/inhibition and energy depletion. A: AtT20 cells were transfected transiently with constitutively active AMPK{alpha}1 expression vector and POMC-luciferase plasmid and incubated for 36 h. The ratio of plasmid amount used is as follows: low (L), AMPK:POMC = 0.2:1.0; high (H), AMPK:POMC = 1.0:1.0. *P < 0.05 vs. C; +P < 0.05 vs. L group. B: AtT20PL cells were treated with vehicle (C) or AMPK inhibitor compound C (10 mM) and then with vehicle (C) or AICAR (2 mM) for 8 h. *P < 0.05 vs. C. C: AtT20 cells were transfected transiently with dominant negative AMPK{alpha}1 expression vector (DN-AMPK) and POMC-luciferase plasmid and incubated for 36 h. Then the cells were treated with vehicle or AICAR (2 mM) for 8 h. *P < 0.05 vs. control (C). D: AtT20PL cells were treated with vehicle (C) or with either carbonyl cyanide m-chlorophenylhydrazone (CCCP; 5 mM) or thenoyltrifluoroacetone (TTFA; 10 mM) and incubated for 7 h. *P < 0.05 vs. C. E: AtT20PL cells were treated with either AICAR (2 mM) or low-glucose medium (1 mM; LG) for 6 h, and endogenous POMC mRNA levels were determined by quantitative RT-PCR analysis. *P < 0.05 vs. C.

 
AICAR induces AMPK phosphorylation. To confirm that AICAR activates AMPK, we examined the effect of AICAR on AMPK phosphorylation by Western blotting. When AtT20PL cells were treated with AICAR, a time-dependent increase in the amount of phospho-AMPK{alpha} was observed, whereas the amount of total AMPK{alpha} was not changed (Fig. 4). The result indicates that AICAR indeed activates AMPK in corticotroph cells.


Figure 4
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  		Fig. 4. Effect of AICAR on AMPK phosphorylation. AtT20PL cells were incubated with vehicle or with AICAR (2 mM) for 0.5 or 1 h. Then the cellular protein was extracted and applied for Western blot analysis of either total or phosphorylated AMPK{alpha} (see MATERIALS AND METHODS). M, molecular size marker.

 
Effect of AICAR on ACTH secretion. In contrast to the effect on POMC gene transcription, AICAR (2 mM, 8 h) did not influence ACTH secretion into culture medium in AtT20PL cells (Fig. 5).


Figure 5
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  		Fig. 5. Effect of AICAR on ACTH secretion. AtT20PL cells were incubated with AICAR (2 mM) for 8 h, and the amount of ACTH released into culture medium during the incubation was determined by specific immunoradiometric assay. NS, not significant.

 
Effect of signal transduction inhibitors on the AICAR-mediated POMC gene expression. To elucidate the intracellular signaling pathway(s) responsible for the AICAR-induced POMC gene transcription, we tested the effects of a variety of signal transduction inhibitors, i.e., H89 (30 µM), PD98059 (20 µM), SB203580 (10 µM), RO32-0432 (5 µM), KN62 (5 µM), and wortmannin (1 µM)/LY294002 (10 µM) to inhibit protein kinase A, p42/44 MAP kinase, p38 MAP kinase, protein kinase C, calmodulin kinase, and phosphatidylinositol 3-kinase (PI3K), respectively. We found that wortmannin completely, and LY294002 substantially, although not completely, eliminated the stimulatory effect of AICAR (Fig. 6). Significant but much smaller inhibitory effects were also observed with H89, PD98059, and SB203580.


Figure 6
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  		Fig. 6. Effects of a variety of protein kinase inhibitors on AICAR-induced transcriptional activity of POMC gene. AtT20PL cells were treated with vehicle (open bars) or AICAR (2 mM, filled bars) for 8 h. From 1 h prior to the addition of AICAR, cells were incubated without (C) or with H89 (30 µM) PD98059 (PD; 20 µM), SB203580 (SB; 10 µM), RO32-0432 (RO; 5 µM), KN62 (KN; 5 µM), wortmannin (Wort; 1 µM), or LY294002 (LY; 10 µM), and this was continued until the end of the experiment. Each value is shown as a percentage of the value of the corresponding vehicle (no AICAR) group. *P < 0.05 vs. value of vehicle group; +P < 0.05 vs. value of AICAR alone (without inhibitors).

 
Effect of AICAR on AP1-dependent gene transcription. Finally, to further clarify the signaling pathways, we examined the effect of AICAR by using reporter genes responsive to representative signaling proteins/transcription factors and by EMSA. Among the plasmids tested, the transcriptional activity of a reporter gene containing AP1 (Fos/Jun)-binding sites was significantly enhanced by AICAR, whereas those of other reporter genes were not influenced (Fig. 7A). EMSA analysis also showed that, when cells were treated with AICAR, DNA protein complex was markedly enhanced using a probe containing a consensus AP1 motif (Fig. 7B, lane 7). A similar, though less intense, band was recognized with a probe containing a noncanonical POMC AP1 motif (Fig. 7B, lane 4). Because AP1 is known to be involved in the transcriptional regulation of POMC gene (3, 4), it seems likely that the effect of AICAR is mediated by the stress-sensitive transcription factor.


Figure 7
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  		Fig. 7. Effects of AICAR on AP1-dependent gene transcription. A: AtT20 cells were transfected transiently with pCRE-, pSRE-, pAP1-, or pNF-{kappa}B-luciferase reporter plasmids. The cells were then treated with vehicle (open bars) or AICAR (2 mM, filled bars) for 8 h. Each value is shown as a percentage of the value of corresponding vehicle (no AICAR) group. *P < 0.05 vs. value of no AICAR group. B: AtT20PL cells were treated with vehicle or AICAR (2 mM, 5 h). Then, nuclear protein was extracted and applied for electrophoretic mobility shift assay analysis using the biotin-labeled probes containing the AP1 motif in the POMC gene promoter (lanes 2–4: lane 3, vehicle; lane 4, AICAR treated) or consensus AP1 sequence (lanes 5–7: lane 6, vehicle; lane 7, AICAR treated). An excess amount (x200) of unlabeled consensus AP1 probe was used as a competitor (lanes 2 and 5). Lanes without nuclear extract (probe only) are shown as a negative control (lanes 1 and 8).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results in this study show that activation of AMPK by AICAR stimulates POMC but not CRH gene transcription. The effect is promoter specific, and PI3K and AP1 signaling molecules are, at least partly, thought to be responsible for mediating the effect. Since starvation is a major metabolic stress causing the activation of the HPA axis, intracellular energy depletion may directly stimulate the POMC gene expression at the pituitary level.

The HPA axis, the major antistress system in higher organisms, is known to be involved in the regulation of energy metabolism (19, 23). Glucocorticoid hormones, the end products of the axis, play a pivotal role in maintaining the level of plasma glucose by facilitating the gluconeogenesis in the liver, as the name glucocorticoid implies. Indeed, adrenal insufficiency is frequently accompanied by severe hypoglycemia, and starvation is known to activate the HPA axis. Recent animal experiments (16, 17) also suggest that the glucose metabolism itself is profoundly involved in the negative feedback regulation of the HPA axis in that adrenalectomy-induced enhancement of the HPA axis is attenuated by sucrose ingestion in rats. Our present data strongly suggest that energy depletion by itself activates the HPA axis at the pituitary level by stimulating the transcription of POMC gene, which encodes ACTH, a key hormone of the antistress system. Furthermore, AMPK, an energy sensor of the cells, may mediate the effect, because stimulation of AMPK by AICAR, or overexpression of AMPK itself, potently enhanced POMC gene transcription.

AMPK is a serine/threonine protein kinase that consists of three subcomponent molecules (8, 9). The enzyme is activated by intracellular energy depletion, where the AMP/ATP ratio is elevated and exerts a variety of metabolic effects, such as glucose uptake and beta-oxidation of intracellular lipid storage. Thus, AMPK works as a "fuel gauge" to maintain the cellular energy homeostasis. We confirmed the expression of all three subunits ({alpha}, beta, and {gamma}) of the enzyme necessary for the formation of a heterotrimeric complex in AtT20 cells (10), which suggested that the enzyme can take effect in these cells and may mediate the effect of AICAR on POMC transcription.

In our experimental condition, AICAR did not increase ACTH secretion. Because we unfortunately have no data regarding the effect of AICAR on intracellular POMC protein content due to the unavailability of a specific assay for the precursor protein, the reason for the discrepancy between POMC expression and ACTH secretion is not clear. AMPK is known to be an evolutionarily conserved enzyme existing in yeast and even in plants and plays a fundamental role in cellular energy metabolism (25). The enzyme is also expressed ubiquitously in a variety of tissues in higher organisms. Thus, we assume that AMPK activation influences the transcription of a variety of genes, including the POMC gene, but is not specifically linked to a short-term hormone secretion in endocrine cells. Alternatively, since the effect of AMPK activation on basal ACTH secretion appears to be relatively weak, the minimal increase might be masked by a high constitutive secretory activity of ACTH in AtT20 cells (22).

It is of particular interest that AICAR did not stimulate but rather suppressed the transcription of CRH gene, a key regulator of the HPA axis in the brain. Makino et al. (20) previously showed that, in the rat, starvation suppresses hypothalamic CRH gene expression, whereas the expression of POMC and the activation of the HPA axis are maintained in the face of high plasma cortisol levels. Since CRH is an anorectic peptide, the suppression of CRH in vivo under starvation is reasonable and coincides well with our in vitro data. In this case, however, there should be additional factor(s) other than CRH to maintain the tone of the HPA axis during starvation. Concerning the ACTH synthesis, the sustained transcription of the POMC gene may be explained by the direct effect of cellular energy depletion through AMPK as shown in this study. In addition, ACTH secretion should also be stimulated by other hypothalamic factors, and the most likely candidate is vasopressin, the expression of which is not suppressed during starvation (11).

The molecular link between the activation of AMPK and that of POMC transcription has not been completely clarified in this study. Unlike insulin, AMPK-stimulated glucose uptake is known not to be mediated by the PI3K signaling pathway (26). In this study, however, the positive effect of AICAR on POMC gene was completely abolished by wortmannin and significantly, although not completely, diminished by LY294002, whereas the impact of the other inhibitors was minimal. This suggests that PI3K is, at least in part, involved in the effect of AMPK on POMC gene transcription. Furthermore, AICAR specifically enhanced DNA binding of AP1 and stimulated AP1 (Fos/Jun)-mediated transcription among the stress-sensitive reporter genes. These data suggest that the effect of AICAR is not nonspecific and is instead comprised of promoter-specific events. Since AP1 is known to play a significant role in the regulation of POMC gene (3, 4), our data altogether indicate that malnutritional stress activates ACTH synthesis through an increase in the AMP/ATP ratio with a resultant AMPK activation, followed by PI3K, AP1, and, finally, the transcriptional activation of POMC gene. Recent studies (15, 18) suggest that AMPK is involved in the transcriptional control of a variety of genes as well as in the regulation of energy metabolism. More specifically, AMPK has recently been shown to activate MAP kinases such as p42/44, p38, and Jun NH2-terminal kinase (1, 21), which might explain the AMPK-mediated AP1 activation. PI3K-dependent activation of AP1 is also reported (5, 24).

In conclusion, our results in this study raise the possibility that metabolic derangements such as energy depletion directly activate the HPA axis. This can probably explain the fact that the POMC expression as well as the responsiveness to hypoglycemia is maintained in CRH knockout mice (13) and that the activity of the HPA axis is maintained despite suppressed CRH expression in energy-starved states. In this case, the decreased CRH under starvation is compensated at the pituitary level, and our in vitro data may support the molecular mechanism of this compensation observed in vivo.


   ACKNOWLEDGMENTS
 
We thank Prof. David Carling (Imperial College School of Medicine, London, UK) for AMPK expression vectors and Astellas Pharmaceutical for compound C.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Iwasaki, Dept. of Endocrinology, Metabolism, and Nephrology, Kochi Medical School, Kochi University, Kohasu, Oko-cho, Nankoku 783-8505, Japan (e-mail: iwasaki{at}kochi-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 RESULTS
 DISCUSSION
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