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Vol. 96, No. 1, 2012
Issue release date: July 2012
Editor's Choice -- Free Access
Neuroendocrinology 2012;96:24–31
(DOI:10.1159/000333963)

Intracerebroventricular Administration of Metformin Inhibits Ghrelin-Induced Hypothalamic AMP-Kinase Signalling and Food Intake

Stevanovic D.a · Janjetovic K.b, d · Misirkic M.b, d · Vucicevic L.b, d · Sumarac-Dumanovic M.c · Micic D.c · Starcevic V.a · Trajkovic V.b
Institutes of aMedical Physiology, bMicrobiology and Immunology, and cEndocrinology, Diabetes and Diseases of Metabolism, School of Medicine, and dInstitute for Biological Research ‘Sinisa Stankovic’, University of Belgrade, Belgrade, Serbia
email Corresponding Author

Abstract

Background/Aims: The antihyperglycaemic drug metformin reduces food consumption through mechanisms that are not fully elucidated. The present study investigated the effects of intracerebroventricular administration of metformin on food intake and hypothalamic appetite-regulating signalling pathways induced by the orexigenic peptide ghrelin. Methods: Rats were injected intracerebroventricularly with ghrelin (5 µg), metformin (50, 100 or 200 µg), 5-amino-imidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR, 25 µg) and L-leucine (1 µg) in different combinations. Food intake was monitored during the next 4 h. Hypothalamic activation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), regulatory-associated protein of mTOR (Raptor), mammalian target of rapamycin (mTOR) and p70 S6 kinase 1 (S6K) after 1 h of treatment was analysed by immunoblotting. Results: Metformin suppressed the increase in food consumption induced by intracerebroventricular ghrelin in a dose-dependent manner. Ghrelin increased phosphorylation of hypothalamic AMPK and its targets ACC and Raptor, which was associated with the reduced phosphorylation of mTOR. The mTOR substrate, S6K, was activated by intracerebroventricular ghrelin despite the inhibition of mTOR. Metformin treatment blocked ghrelin-induced activation of hypothalamic AMPK/ACC/Raptor and restored mTOR activity without affecting S6K phosphorylation. Metformin also reduced food consumption induced by the AMPK activator AICAR while the ghrelin-triggered food intake was inhibited by the mTOR activator L-leucine. Conclusion: Metformin could reduce food intake by preventing ghrelin-induced AMPK signalling and mTOR inhibition in the hypotalamus.


 Outline


 goto top of outline Key Words

  • Metformin
  • Ghrelin
  • Food intake
  • Hypothalamus
  • AMPK

 goto top of outline Abstract

Background/Aims: The antihyperglycaemic drug metformin reduces food consumption through mechanisms that are not fully elucidated. The present study investigated the effects of intracerebroventricular administration of metformin on food intake and hypothalamic appetite-regulating signalling pathways induced by the orexigenic peptide ghrelin. Methods: Rats were injected intracerebroventricularly with ghrelin (5 µg), metformin (50, 100 or 200 µg), 5-amino-imidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR, 25 µg) and L-leucine (1 µg) in different combinations. Food intake was monitored during the next 4 h. Hypothalamic activation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), regulatory-associated protein of mTOR (Raptor), mammalian target of rapamycin (mTOR) and p70 S6 kinase 1 (S6K) after 1 h of treatment was analysed by immunoblotting. Results: Metformin suppressed the increase in food consumption induced by intracerebroventricular ghrelin in a dose-dependent manner. Ghrelin increased phosphorylation of hypothalamic AMPK and its targets ACC and Raptor, which was associated with the reduced phosphorylation of mTOR. The mTOR substrate, S6K, was activated by intracerebroventricular ghrelin despite the inhibition of mTOR. Metformin treatment blocked ghrelin-induced activation of hypothalamic AMPK/ACC/Raptor and restored mTOR activity without affecting S6K phosphorylation. Metformin also reduced food consumption induced by the AMPK activator AICAR while the ghrelin-triggered food intake was inhibited by the mTOR activator L-leucine. Conclusion: Metformin could reduce food intake by preventing ghrelin-induced AMPK signalling and mTOR inhibition in the hypotalamus.

Copyright © 2012 S. Karger AG, Basel


goto top of outline Introduction

Metformin [(1-(diaminomethylidene)-3,3-dimethylguanidine] is an antihyperglycaemic drug widely used for the management of type 2 diabetes [1]. The glucoregulatory properties of metformin are mainly attributed to reduced hepatic glucose production and augmented glucose uptake by the peripheral tissues [1]. Metformin has also been suggested to reduce weight in diabetic and non-diabetic patients, in contrast to sulphonylureas, thiazolidinediones and insulin, which all induce weight gain [2]. The positive effect of metformin on weight control has been associated with reduced food intake both in humans and experimental animals [3,4,5,6,7,8,9], but the mechanisms responsible for the metformin-mediated reduction of food consumption have not been fully clarified. Consistent with the ability of orally administered metformin to readily cross the blood-brain barrier [10], some recent data suggest that its anorexigenic effect might result from a direct action on the hypothalamic centres regulating satiety and feeding [11,12,13]. In diet-induced obese rats, metformin enhanced the hypothalamic phosphorylation of STAT3 induced by acute intracerebroventricular administration of the anorexigenic hormone leptin [11] and increased hypothalamic leptin receptor expression [12]. Additionally, metformin reduced glucose deprivation-triggered release of the potent orexigenic mediator neuropeptide Y (NPY) in primary hypothalamic neuronal cell cultures [13].

Peripheral metabolic effects of metformin at least partly depend on the stimulation of AMP-activated protein kinase (AMPK) [14,15], an intracellular energy sensor that is activated by raising AMP and acts by switching on ATP-generating catabolic pathways while switching off ATP-requiring anabolic processes [3]. However, the same dose of intraperitoneally injected metformin that readily activated hepatic AMPK, failed to increase hypothalamic AMPK phosphorylation in rats [16]. Moreover, metformin completely blocked glucose deprivation-induced AMPK phosphorylation in rat primary hypothalamic neurons in vitro [13], suggesting a different regulation of central and peripheral AMPK by this antidiabetic drug.

Ghrelin is a 28-amino-acid peptide that promotes food intake [17,18,19,20] mainly by acting on hypothalamic NPY and agouti-related protein systems [21]. It has been suggested that the orexigenic action of ghrelin is mediated by stimulation of AMPK [22,23], which controls the feeding behaviour through integration of orexigenic and anorexigenic signals [23,24]. The intracellular signals downstream of AMPK activation include phosphorylation of regulatory-associated protein of the mammalian target of rapamycin (mTOR) (Raptor) and subsequent inactivation of mTOR and its target p70 S6 kinase 1 (S6K) [25]. The activation of hypothalamic mTOR/S6K has been proposed as an important anorexigenic signal [26,27,28], thus making plausible that the orexigenic action of ghrelin might involve central AMPK-mediated mTOR/S6K inactivation. While metformin has recently been found to increase plasma ghrelin levels in patients with type 2 diabetes [29], the effects of metformin on ghrelin-induced food intake and hypothalamic AMPK/mTOR signalling have not been investigated.

Based on the above findings, we hypothesized that metformin could reduce food intake by inhibiting AMPK and consequently restoring mTOR/S6K activity in the hypothalamus. To test this assumption, we examined the influence of centrally applied metformin on ghrelin-triggered acute increase in food intake and hypothalamic AMPK/mTOR signalling.

 

goto top of outline Materials and Methods

goto top of outline Animal Preparation

Eight-week-old male Wistar rats (body weight 200 ± 20 g) were obtained from the Institute of Biomedical Research Galenika (Belgrade, Serbia). They were kept in individual cages under a 12:12 h light/dark cycle, at 22 ± 2°C, and were accustomed to daily handling for 2 weeks (body weight 252 ± 15 g). The animals were anaesthetized with intramuscular ketamine (50 mg/kg; Pfizer, New York, N.Y., USA) -xylazine (80 mg/kg; Bayer, Leverkusen, Germany) and equipped with a headset for intracerebroventricular injection, consisting of a silastic-sealed 20-gauge cannula positioned in the right lateral cerebral ventricle (1 mm posterior and 1.5 mm lateral to the bregma, and 3 mm below the cortical surface) [30]. A small stainless steel anchor screw was placed at a remote site on the skull. The cannula and screw were cemented to the skull with standard dental acrylic. Following surgery, the animals received a single subcutaneous dose of 0.28 mg/kg buprenorphin (Reckitt Benckiser Healthcare, Mannheim, Germany) and 1 week of recovery was allowed before the experiments. Only animals demonstrating progressive weight gain after surgery were used in subsequent experiments. All rats had ad libitum access to rodent chow (D.D. Veterinarski zavod Subotica, Subotica, Serbia) and water during experimental testing.

goto top of outline Experiment 1

Effect of Intracerebroventricular Metformin on Ghrelin-Induced Food Intake
In this experiment, the rats were treated intracerebroventricularly with 5 µg ghrelin (Bachem, Weil am Rhein, Germany), metformin hydrochloride (50, 100 or 200 µg; 99.9% Hemofarm, Vrsac, Serbia) or ghrelin and metformin (n = 6 in each group). The orexigenic concentration of ghrelin (5 µg) was chosen based on a previous study [23]. The concentrations of metformin (50–200 µg) which did not cause overt neurotoxicity after a single intracerebroventricular injection were selected based on previous reports [16,31]. Both ghrelin and metformin were applied in 2 µl of phosphate-buffered saline (PBS; Sigma-Aldrich, St. Louis, Mo., USA) using a 5-µl Hamilton syringe. Metformin was administered 30 min before ghrelin. The animals in the ghrelin-only group received 2 µl PBS 30 min before treatment while those in the metformin-only group received 2 µl PBS 30 min after the treatment. Control animals received 2 × 2 µl PBS. Food intake was measured each hour up to 4 h after the second injection.

goto top of outline Experiment 2

Effect of Intracerebroventricular Metformin on Ghrelin-Induced AMPK/mTOR Signalling
The rats were treated as described in experiment 1 (n = 6 in each group), but metformin was used at the concentration of 100 µg. One hour after the last injection, the rats were killed by decapitation under deep isoflurane anaesthesia; hypothalamic tissues were collected and immediately frozen in liquid nitrogen for immunoblot analysis.

goto top of outline Experiment 3

Effect of Intracerebroventricular Metformin on 5-Aminoimidazole-4-Carboxamide 1-β-D-Ribofuranoside-Induced Food Intake
Rats were treated as described in experiment 1 (n = 5 in each group), except that 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR; Sigma-Aldrich) (25 µg in 2 µl dimethylsulphoxide, DMSO) was used instead of ghrelin while metformin was used at 100 µg. The orexigenic dose of AICAR was chosen based on a previous study [23]. The animals in the metformin-only group received 2 µl DMSO 30 min after metformin treatment while control animals received 2 µl PBS + 2 µl DMSO. Food intake was measured each hour up to 4 h after the second injection.

goto top of outline Experiment 4

Effect of L-Leucine on Ghrelin-Induced Food Intake
Rats were treated as described in experiment 1 (n = 5 in each group), except that L-leucine (Sigma-Aldrich) (1 µg in 2 µl PBS) was used instead of ghrelin. Food intake was measured each hour up to 4 h after the second injection. The concentration of intracerebroventricular L-leucine was selected based on its ability to suppress post-fasting food intake in rats [32].

goto top of outline Immunoblot Analysis

Western blot followed by protein detection with specific antibodies was used to assess phosphorylation (activation) of various members of the AMPK/mTOR signalling pathway [AMPK, acetyl-CoA carboxylase (ACC), Raptor, mTOR and S6K]. The hypothalamic tissue was lysed in a RIPA buffer (Sigma-Aldrich) on ice for 30 min, centrifuged at 14,000 g for 15 min at 4°C, and the supernatants were collected. Equal amounts of total protein from each sample (10 µg for actin blot and 25 µg for all other proteins) were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, Marnes-la-Coquette, France). All blots were performed on separate gels. Following incubation with primary antibodies against phospho-AMPKα (Thr172), AMPK, phospho-ACC (Ser79), phospho-Raptor (Ser792), Raptor, phospho-mTOR (Ser2448), phospho-S6K (Thr389), S6K or actin (Cell Signaling Technology, Beverly, Mass., USA) and peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, Pa., USA) as the secondary antibody, specific protein bands were visualized using enhanced-chemiluminescence reagents for Western blot analysis (Amersham Pharmacia Biotech, Piscataway, N.J., USA). The signal intensity was determined by densitometry using Image J software and the results were presented as phospho/total protein signal ratio, which was arbitrarily set to 1 in control.

goto top of outline Statistical Analysis

The data obtained from each sample were averaged per experimental group and the standard deviation of the mean (SD) was calculated. A one-way analysis of variance (ANOVA), followed by a Student-Newman-Keuls test for multiple comparisons, was used to assess differences between the groups. A p value of less than 0.05 was considered statistically significant.

 

goto top of outline Results

goto top of outline Centrally Applied Metformin Inhibits Ghrelin-Induced Food Intake

We first investigated the ability of centrally applied metformin to influence the orexigenic effect of ghrelin. In the absence of ghrelin, intracerebroventricular administration of metformin (50, 100 or 200 µg) did not significantly affect food intake (fig. 1a). Expectedly, intracerebroventricular injection of ghrelin (5 µg) caused a time-dependent increase in cumulative food intake over the 4-hour post-injection period (fig. 1b). Pretreatment with metformin reduced the orexigenic effect of ghrelin at each time point in a dose-dependent manner (fig. 1b). These data demonstrate that metformin can counteract the orexigenic effect of ghrelin at the hypothalamic level.

FIG01
F01B
Fig. 1. Centrally applied metformin inhibits ghrelin-induced food intake. Rats (n = 6 per group) were injected intracerebroventricularly with PBS (control; a, b), 50–200 µg metformin (a), 5 µg ghrelin (b), or ghrelin and metformin (b). Cumulative food intake was measured during 4 h at the indicated time points. The results from the same experiment (presented separately for clarity) are mean ± SD values (a p < 0.05 and b p < 0.05 refer to control and ghrelin-treated rats, respectively).

goto top of outline Metformin Modulates Hypothalamic AMPK/mTOR Signalling in Ghrelin-Treated Rats

We next investigated the influence of metformin on hypothalamic AMPK/mTOR signalling in ghrelin-treated rats. Central administration of ghrelin (5 µg) increased phosphorylation of hypothalamic AMPK and its downstream targets ACC and Raptor, which was associated with reduced phosphorylation of mTOR, but increased activation of its direct substrate S6K (fig. 2). While treatment with metformin (100 µg) did not alter hypothalamic AMPK/mTOR signalling in the absence of ghrelin, it significantly suppressed ghrelin-induced activation of AMPK/ACC/Raptor and restored the phosphorylation of ghrelin-inactivated mTOR without affecting S6K phosphorylation (fig. 2). Therefore, centrally applied metformin can interfere with hypothalamic AMPK/mTOR signalling, but not with S6K activation in rats treated with intracerebroventricular ghrelin.

FIG02
Fig. 2. Metformin modulates hypothalamic AMPK/mTOR signalling in ghrelin-treated rats. Rats (n = 6 per group) were injected intracerebroventricularly with PBS (control), metformin (100 µg), ghrelin (5 µg), or ghrelin and metformin. After 1 h, the levels of phosphorylated (p) and total AMPK, ACC, Raptor, mTOR and S6K were assessed by immunoblotting, with actin used as a loading control. The representative blots are presented in a while the data in b are mean ± SD values (n = 6) of phospho/total protein signal ratio (a p < 0.05 and b p < 0.05 refer to control and ghrelin-treated rats, respectively).

goto top of outline Metformin Inhibits Food Intake Induced by AMPK Activator AICAR

We next assessed whether metformin could inhibit food intake induced by intracerebroventricular injection of AICAR, a pharmacological AMPK activator with orexigenic activity in rats [23]. In comparison with control animals, rats treated with AICAR (25 µg) consumed more food during the 4-hour follow-up period, with a significant increase in food intake observed after 3 and 4 h (fig. 3). AICAR-induced hyperphagia was significantly reduced at both time points by co-administration of metformin (100 µg).

FIG03
Fig. 3. Metformin inhibits AICAR-induced food intake. Rats (n = 5 per group) were injected intracerebroventricularly with PBS (control), metformin (100 µg), AICAR (25 µg), or AICAR and metformin. Cumulative food intake was measured during 4 h at the indicated time points. The results are presented as mean ± SD values (a p < 0.05 and b p < 0.05 refer to control and AICAR-treated rats, respectively).

goto top of outline mTOR Activator L-Leucine Inhibits Ghrelin-Induced Food Intake

Finally, we tested the ability of L-leucine, an mTOR activator with anorexigenic activity [32], to affect ghrelin-mediated food intake. Treatment with ghrelin (5 µg, i.c.v.) induced a sustained increase in food consumption for 4 h after injection (fig. 4). Intracerebroventricularly injected L-leucine (1 µg) did not interfere with food intake in control animals (fig. 4). On the other hand, it significantly reduced the ghrelin-triggered increase in food consumption at each of the time points (fig. 4).

FIG04
Fig. 4.L-Leucine inhibits ghrelin-induced food intake. Rats (n = 5 per group) were injected intracerebroventricularly with PBS (control), L-leucine (1 µg), ghrelin (5 µg), or ghrelin and L-leucine. Cumulative food intake was measured during 4 h at the indicated time points. The results are presented as mean ± SD values (a p < 0.05 and b p < 0.05 refer to control and ghrelin-treated rats, respectively).

 

goto top of outline Discussion

The present study, for the first time, demonstrates the ability of centrally applied metformin to suppress acute ghrelin-induced increase in food intake. The observed effect was associated with the inhibition of ghrelin-triggered activation of hypothalamic AMPK, as well as with the restoration of mTOR activity. These data suggest that the previously well-documented anorexigenic effect of metformin [3,4,5,6,7,8,9] could, at least partly, be mediated at the hypothalamic level through interference with ghrelin-induced orexigenic AMPK signalling.

Our hypothesis that the appetite-reducing effect of metformin could be due to suppression of AMPK activation is consistent with the proposed role of this intracellular energy sensor in ghrelin-mediated food intake [22,23]. In accordance with the present study, both central and systemic ghrelin administration activate hypothalamic AMPK [22,23], and experimental genetic activation or inactivation of hypothalamic AMPK leads to increased or decreased food intake, respectively [24]. It has been shown that the calcium/calmodulin-dependent protein kinase 2 and sirtuin 1/p53 pathway are required for hypothalamic AMPK activation by ghrelin [33,34,35], but the appetite-controlling signals downstream of AMPK activation have not been fully delineated. mTOR is a plausible AMPK target which has been suggested to mediate the anorexigenic effects of leptin [26,27,28], an important adipocyte-derived negative regulator of energy balance that counteracts the metabolic actions of ghrelin [36]. Our findings that ghrelin-induced hyperphagia coincided with downregulation of mTOR activity and was suppressed by the mTOR activator L-leucine are indeed consistent with the proposed role of hypothalamic mTOR as an anorexigenic signal [26,27,28]. Moreover, in our study, metformin-mediated blockade of ghrelin-triggered food intake and AMPK activation were associated with restoration of hypothalamic mTOR activity, thus indicating that the anorexigenic effect of metformin might depend on interference with AMPK-mediated mTOR downregulation in the hypothalamus. This assumption is consistent with the in vitro data demonstrating metformin-mediated AMPK inhibition in glucose-deprived rat primary hypothalamic neurons [13]. However, it seems that the inhibitory effect of metformin on AMPK might be restricted to the hypothalamus or might be species specific as in two recent studies metformin activated AMPK and inhibited mTOR in mouse hypocampal slices and cultured cortical neurons [37,38].

While, as discussed above, metformin might target hypothalamic AMPK/mTOR signalling to block the orexigenic action of ghrelin, it should be noted that the changes in the activation of the mTOR substrate S6K in our experiments did not correlate with the activation status of mTOR. Namely, ghrelin-mediated mTOR downregulation was paradoxically associated with S6K activation, which was not further altered by restoring mTOR phosphorylation with metformin. These data actually concur with the recent findings by Villanueva et al. [39], who reported activation of S6K in the arcuate nucleus of mice treated intracerebroventricularly with ghrelin or exposed to fasting, a state associated with an increase in the levels of circulating ghrelin [40,41]. Moreover, both S6K1- and S6K2-knockout mice responded to ghrelin by increasing food consumption comparably to their wild-type counterparts, indicating that S6K modulation is dispensable for ghrelin-induced hyperphagia [Stevanovic et al., submitted for publication]. While the role of hypothalamic S6K in the orexigenic effect of ghrelin remains to be fully elucidated, the dissociated mTOR and S6K activation status in our experiments clearly indicates that ghrelin could activate S6K independently of mTOR. Accordingly, mTOR-independent regulation of S6K has previously been reported [42]. In addition, our data imply that the putative involvement of mTOR inhibition as an orexigenic signal downstream of AMPK is mediated independently of S6K, or that alternatively, mTOR-independent mechanisms are responsible for the orexigenic action of AMPK. The latter assumption is supported by previous demonstration that ghrelin-mediated activation of hypothalamic AMPK increases food intake through a hypothalamic fatty acid oxidation pathway involving ACC/malonyl-CoA, carnitine palmitoyltransferase 1 and uncoupling protein 2 [43,44,45]. Indeed, we have observed an increase in ACC activation upon intracerebroventricular injection of ghrelin, and this effect was efficiently counteracted by co-administration of metformin. It is therefore possible that hypothalamic ACC and/or other AMPK downstream targets different from mTOR/S6K could be responsible for the anorexigenic action of metformin. In the present study, we did not investigate the interference of metformin with ghrelin orexigenic signals that are further downstream of hypothalamic AMPK activation, such as NPY release. In contrast to the in vitro inhibitory effect of metformin on NPY release by glucose-deprived rat hypothalamic neurons [13], the anorexigenic effect of metformin in genetically obese Zucker rats is independent of the changes in hypothalamic NPY content [46]. We are currently investigating the role of NPY modulation in metformin-mediated suppression of ghrelin-induced hyperphagia.

Finally, it is interesting to note that metformin treatment increased plasma ghrelin concentrations in patients with type-2 diabetes [29]. This is somewhat unexpected considering the orexigenic action of ghrelin [17,18,19,20] and the well-documented inhibitory effect of metformin on food intake [3,4,5,6,7,8,9]. Our data, however, resolve this discrepancy by demonstrating that metformin could actually block the action of ghrelin in the hypothalamus, thus presumably counteracting the stimulatory effect on circulating ghrelin levels.

In conclusion, our data indicate a novel mechanism of the anorexigenic action of metformin involving downregulation of ghrelin-induced activation of the AMPK signalling pathway. A different regulation of AMPK activation in the hypothalamus and peripheral tissues might contribute to the beneficial metabolic effects of metformin as peripheral AMPK activation will increase energy expenditure while hypothalamic AMPK inhibition will reduce food intake. It should be noted, however, that the metformin concentrations used in our study and in previous studies [16,31] seem rather high compared with those applied therapeutically [47]. Nevertheless, lower concentrations of chronically administered metformin might still affect hypothalamic AMPK signalling. Therefore, further studies are required to explore the mechanisms underlying central AMPK downregulation by metformin and its potential therapeutic significance in metabolic disorders.

 

goto top of outline Acknowledgements

This study was supported by the Ministry of Science and Technological Development of the Republic of Serbia (grant No. 41025 and 175067). The authors thank Dr. Dragomir Marisavljevic (Hemofarm, Vrsac, Serbia) for kindly providing metformin hydrochloride, Katarina Zivanovic and Marina Halasev-Dikovic for excellent technical support, and Ljubica Harhaji-Trajkovic for critically reading the manuscript.


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  34. Kohno D, Gao HZ, Muroya S, Kikuyama S, Yada T: Ghrelin directly interacts with neuropeptide-Y-containing neurons in the rat arcuate nucleus: Ca2+ signaling via protein kinase A and N-type channel-dependent mechanisms and cross-talk with leptin and orexin. Diabetes 2003;52:948–956.
  35. Velásquez DA, Martínez G, Romero A, Vázquez MJ, Boit KD, Dopeso-Reyes IG, López M, Vidal A, Nogueiras R, Diéguez C: The central Sirtuin 1/p53 pathway is essential for the orexigenic action of ghrelin. Diabetes 2011;60:1177–1185.
  36. Pinto S, Roseberry AG, Liu H, Diano S, Shanabrough M, Cai X, Friedman JM, Horvath TL: Rapid rewiring of arcuate nucleus feeding circuits by leptin. Science 2004;304:110–115.
  37. Potter WB, O’Riordan KJ, Barnett D, Osting SM, Wagoner M, Burger C, Roopra A: Metabolic regulation of neuronal plasticity by the energy sensor AMPK. PLoS One 2010;5:e8996.
  38. Williams T, Courchet J, Viollet B, Brenman JE, Polleux F: AMP-activated protein kinase (AMPK) activity is not required for neuronal development but regulates axogenesis during metabolic stress. Proc Natl Acad Sci USA 2011;108:5849–5854.
  39. Villanueva EC, Münzberg H, Cota D, Leshan RL, Kopp K, Ishida-Takahashi R, Jones JC, Fingar DC, Seeley RJ, Myers MG Jr: Complex regulation of mammalian target of rapamycin complex 1 in the basomedial hypothalamus by leptin and nutritional status. Endocrinology 2009;150:4541–4551.
  40. Moesgaard SG, Ahrén B, Carr RD, Gram DX, Brand CL, Sundler F: Effects of high-fat feeding and fasting on ghrelin expression in the mouse stomach. Regul Pept 2004;120:261–267.
  41. Toshinai K, Date Y, Murakami N, Shimada M, Mondal MS, Shimbara T, Guan JL, Wang QP, Funahashi H, Sakurai T, Shioda S, Matsukura S, Kangawa K, Nakazato M: Ghrelin-induced food intake is mediated via the orexin pathway. Endocrinology 2003;144:1506–1512.
  42. Jaeschke A, Hartkamp J, Saitoh M, Roworth W, Nobukuni T, Hodges A, Sampson J, Thomas G, Lamb R: Tuberous sclerosis complex tumor suppressor-mediated S6 kinase inhibition by phosphatidylinositide-3-OH kinase is mTOR independent. J Cell Biol 2002;159:217–224.
  43. Andrews ZB, Liu ZW, Walllingford N, Erion DM, Borok E, Friedman JM, Tschöp MH, Shanabrough M, Cline G, Shulman GI, Coppola A, Gao XB, Horvath TL, Diano S: UCP2 mediates ghrelin’s action on NPY/AgRP neurons by lowering free radicals. Nature 2008;454:846–851.
  44. López M, Lage R, Saha AK, Pérez-Tilve D, Vázquez MJ, Varela L, Sangiao-Alvarellos S, Tovar S, Raghay K, Rodríguez-Cuenca S, Deoliveira RM, Castañeda T, Datta R, Dong JZ, Culler M, Sleeman MW, Alvarez CV, Gallego R, Lelliott CJ, Carling D, Tschöp MH, Diéguez C, Vidal-Puig A: Hypothalamic fatty acid metabolism mediates the orexigenic action of ghrelin. Cell Metab 2008;7:389–399.
  45. Lage R, Vázquez MJ, Varela L, Saha AK, Vidal-Puig A, Nogueiras R, Diéguez C, López M: Ghrelin effects on neuropeptides in the rat hypothalamus depend on fatty acid metabolism actions on BSX but not on gender. FASEB J 2010;24:2670–2679.
  46. Rouru J, Pesonen U, Koulu M, Huupponen R, Santti E, Virtanen K, Rouvari T, Jhanwar-Uniyal M: Anorectic effect of metformin in obese Zucker rats: lack of evidence for the involvement of neuropeptide Y. Eur J Pharmacol 1995;273:99–106.
  47. Graham GG, Punt J, Arora M, Day RO, Doogue MP, Duong JK, Furlong TJ, Greenfield JR, Greenup LC, Kirkpatrick CM, Ray JE, Timmins P, Williams KM: Clinical pharmacokinetics of metformin. Clin Pharmacokinet 2011;50:81–98.

 goto top of outline Author Contacts

Vladimir Trajkovic
Institute of Microbiology and Immunology, School of Medicine
University of Belgrade, Dr. Subotica 1
RS–11000 Belgrade (Serbia)
Tel. +381 11 3643 233, E-Mail vtrajkovic@med.bg.ac.rs


 goto top of outline Article Information

Received: April 19, 2011
Accepted after revision: September 26, 2011
Published online: February 14, 2012
Number of Print Pages : 8
Number of Figures : 4, Number of Tables : 0, Number of References : 47


 goto top of outline Publication Details

Neuroendocrinology (International Journal for Basic and Clinical Studies on Neuroendocrine Relationships)

Vol. 96, No. 1, Year 2012 (Cover Date: July 2012)

Journal Editor: Millar R.P. (Edinburgh)
ISSN: 0028-3835 (Print), eISSN: 1423-0194 (Online)

For additional information: http://www.karger.com/NEN


Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Abstract

Background/Aims: The antihyperglycaemic drug metformin reduces food consumption through mechanisms that are not fully elucidated. The present study investigated the effects of intracerebroventricular administration of metformin on food intake and hypothalamic appetite-regulating signalling pathways induced by the orexigenic peptide ghrelin. Methods: Rats were injected intracerebroventricularly with ghrelin (5 µg), metformin (50, 100 or 200 µg), 5-amino-imidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR, 25 µg) and L-leucine (1 µg) in different combinations. Food intake was monitored during the next 4 h. Hypothalamic activation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), regulatory-associated protein of mTOR (Raptor), mammalian target of rapamycin (mTOR) and p70 S6 kinase 1 (S6K) after 1 h of treatment was analysed by immunoblotting. Results: Metformin suppressed the increase in food consumption induced by intracerebroventricular ghrelin in a dose-dependent manner. Ghrelin increased phosphorylation of hypothalamic AMPK and its targets ACC and Raptor, which was associated with the reduced phosphorylation of mTOR. The mTOR substrate, S6K, was activated by intracerebroventricular ghrelin despite the inhibition of mTOR. Metformin treatment blocked ghrelin-induced activation of hypothalamic AMPK/ACC/Raptor and restored mTOR activity without affecting S6K phosphorylation. Metformin also reduced food consumption induced by the AMPK activator AICAR while the ghrelin-triggered food intake was inhibited by the mTOR activator L-leucine. Conclusion: Metformin could reduce food intake by preventing ghrelin-induced AMPK signalling and mTOR inhibition in the hypotalamus.



 goto top of outline Author Contacts

Vladimir Trajkovic
Institute of Microbiology and Immunology, School of Medicine
University of Belgrade, Dr. Subotica 1
RS–11000 Belgrade (Serbia)
Tel. +381 11 3643 233, E-Mail vtrajkovic@med.bg.ac.rs


 goto top of outline Article Information

Received: April 19, 2011
Accepted after revision: September 26, 2011
Published online: February 14, 2012
Number of Print Pages : 8
Number of Figures : 4, Number of Tables : 0, Number of References : 47


 goto top of outline Publication Details

Neuroendocrinology (International Journal for Basic and Clinical Studies on Neuroendocrine Relationships)

Vol. 96, No. 1, Year 2012 (Cover Date: July 2012)

Journal Editor: Millar R.P. (Edinburgh)
ISSN: 0028-3835 (Print), eISSN: 1423-0194 (Online)

For additional information: http://www.karger.com/NEN


Copyright / Drug Dosage

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

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  46. Rouru J, Pesonen U, Koulu M, Huupponen R, Santti E, Virtanen K, Rouvari T, Jhanwar-Uniyal M: Anorectic effect of metformin in obese Zucker rats: lack of evidence for the involvement of neuropeptide Y. Eur J Pharmacol 1995;273:99–106.
  47. Graham GG, Punt J, Arora M, Day RO, Doogue MP, Duong JK, Furlong TJ, Greenfield JR, Greenup LC, Kirkpatrick CM, Ray JE, Timmins P, Williams KM: Clinical pharmacokinetics of metformin. Clin Pharmacokinet 2011;50:81–98.