Intracerebroventricular administration of metformin inhibits ghrelin-induced Hypothalamic AMP-kinase signalling and food intake.

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-ß-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.

Protective effect of autophagy in laser-induced glioma cell death in vitro.

BACKGROUND AND OBJECTIVE: Laser phototherapy could be potentially used for cancer treatment, but the mechanisms of laser-induced cell death are not completely understood. Autophagy is the process in which the damaged cellular proteins and organelles are engulfed by and destroyed in acidified multiple-membrane vesicles. The aim of the present study was to investigate the role of autophagy in laser-induced tumor cell death in vitro. STUDY DESIGN/MATERIALS AND METHODS: The monolayers of U251 human glioma tumor cells were exposed to 532 nm laser light from a single mode frequency-doubled Nd-YVO4 laser. A flattened Gaussian radial profile of laser beam (0.5-4 W) was used to uniformly illuminate entire colony of cells for various amounts of time (15-120 seconds) in the absence of cell culture medium. The cells were grown for 24 hours and the cell viability was determined by crystal violet or MTT assay. The presence of autophagy was assessed after 16 hours by fluorescence microscopy/flow cytometric analysis of acridine orange-stained autophagolysosomes and Western blot analysis of the autophagosome-associated LC3-II protein. The concentration of the principal pro-autophagic protein beclin-1 was determined after 6 hours by cell-based ELISA. RESULTS: The intracytoplasmic accumulation of autophagic vesicles, increase in LC3-II and up-regulation of beclin-1 expression were clearly observed under irradiation conditions that caused approximately 50% cytotoxicity. Post-irradiation addition of three different autophagy inhibitors (bafilomycin A1, chloroquine, or wortmannin) further increased the laser-induced cytotoxicity, without affecting non-irradiated cells. CONCLUSIONS: These data indicate that beclin-1-dependent induction of autophagy can protect glioma cells from laser-mediated cytotoxicity.

Synergistic antiglioma action of hyperthermia and nitric oxide.

To explore combined antiglioma effect of nitric oxide (NO) and hyperthermia, the rat C6 and human U251 glioma cells were exposed to NO-releasing agents sodium nitroprusside(SNP), S-nitrosoglutathione or PAPA-NONOate, followed by hyperthermia (1 h, 43 degrees C). While each treatment alone showed only moderate efficiency, a synergistic cytotoxicity of NO donors and hyperthermia was clearly demonstrated by crystal violet and MTT cytotoxicity assays. The flow cytometric analysis with the appropriate reporter fluorochromes confirmed that hyperthermia and SNP cooperated in inducing oxidative stress, mitochondrial depolarization, caspase activation and DNA fragmentation, leading to both necrotic and caspase-dependent apoptotic cell death. The acridine orange staining of intracellular acidic compartments revealed that SNP completely blocked hyperthermia-induced autophagy, while the inhibition of autophagy by 3-methyl adenine mimicked SNP-triggered oxidative stress, caspase activation and cell death in hyperthermia-exposed cells. Therefore, the synergistic cytotoxicity of SNP and hyperthermia could result from NO-mediated suppression of protective autophagic response in glioma cells.