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ABSTRACT: Whether the well-known metabolic switch AMP-activated protein kinase (AMPK) is involved in the insulin-sensitizing effect of calorie restriction (CR) is unclear. In this study, we investigated the role of AMPK in the insulin-sensitizing effect of CR in skeletal muscle. Wild-type (WT) and AMPK-α2(-/-) mice received ad libitum (AL) or CR (8 weeks at 60% of AL) feeding. CR increased the protein level of AMPK-α2 and phosphorylation of AMPK-α2. In WT and AMPK-α2(-/-) mice, CR induced comparable changes of body weight, fat pad weight, serum triglycerides, serum nonesterified fatty acids, and serum leptin levels. However, decreasing levels of fasting/fed insulin and fed glucose were observed in WT mice but not in AMPK-α2(-/-) mice. Moreover, CR-induced improvements of whole-body insulin sensitivity (evidenced by glucose tolerance test/insulin tolerance test assays) and glucose uptake in skeletal muscle tissues were abolished in AMPK-α2(-/-) mice. Furthermore, CR-induced activation of Akt-TBC1D1/TBC1D4 signaling, inhibition of mammalian target of rapamycin-S6K1-insulin receptor substrate-1 pathway, and induction of nicotinamide phosphoribosyltransferase-NAD(+)-sirtuin-1 cascade were remarkably impaired in AMPK-α2(-/-) mice. CR serum increased stability of AMPK-α2 protein via inhibiting the X chromosome-linked ubiquitin-specific protease 9-mediated ubiquitylation of AMPK-α2. Our results suggest that AMPK may be modulated by CR in a ubiquitylation-dependent manner and acts as a chief dictator for the insulin-sensitizing effects of CR in skeletal muscle.
Diabetes 03/2012; 61(5):1051-61. · 8.29 Impact Factor
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ABSTRACT: To analyze the (i) expression of AMPK in a variety of epithelial cancer cells, (ii) regulation of AMPK subunit expression by ionizing radiation (IR) and (iii) impact of AMPK on signaling pathways regulating cell cycle and survival.
Human lung, prostate, and breast normal and cancer cells were treated with 0 or 8 Gy IR and mRNA and protein levels of AMPK were evaluated by RT-PCR and immunoblotting 24 or 48 h later. Untreated and radiated wild type (WT) and AMPKα(-/-) mouse embryonic fibroblasts (MEFs) were analyzed by immunoblotting using total- and phosphorylation-specific antibodies. Histone H2Ax was examined by fluorescence microscopy. The cell cycle and survival of WT and AMPKα(-/-) MEFs was also evaluated following 8 Gy by IR.
AMPK subunits were found widely expressed in normal and cancer epithelial cells. IR increased subunit protein levels and stimulated gene transcription in cancer cells. AMPKα(-/-)-MEFs showed enhanced basal total levels of ATM and phosphorylation of its substrates histone H2Ax, but inhibited response of these markers and of checkpoint kinase Chk2 phosphorylation to IR. AMPKα(-/-)-MEFs showed increased basal levels of p53 and cyclin-dependent kinase inhibitors p21(cip1), but lack of response of both genes to IR. These cells had increased basal levels and activation of the Akt-mTOR-p70(S6K)/4-EBP1 signalling pathway. IR increased Akt, p70(S6K) and 4-EBP1 phosphorylation in WT-MEFs, but this was reduced in AMPKα(-/-)-MEFs. AMPKα(-/-)-MEFs failed to arrest at the G2-M checkpoint after IR and showed a trend for radio-resistance in proliferation assays.
AMPK is widely expressed in human normal and cancer epithelial cells and its gene transcription, protein levels, and enzymatic activity is stimulated by IR. Work with AMPKα knockout cells suggests that AMPK (i) may mediate a suppressive regulation on basal expression and activity of ATM and its downstream effector pathways Chk2/p53-p21(cip1) and Akt-mTOR, (ii) facilitates the normal response of these pathways to IR and, (iii) mediates the IR-induced G2-M checkpoint.
Radiotherapy and Oncology 03/2012; 102(3):459-65. · 5.58 Impact Factor
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ABSTRACT: Pharmacoepidemiologic studies provide evidence that use of metformin, a drug commonly prescribed for type II diabetes, is associated with a substantial reduction in cancer risk. Experimental models show that metformin inhibits the growth of certain neoplasms by cell autonomous mechanisms such as activation of AMP kinase with secondary inhibition of protein synthesis or by an indirect mechanism involving reduction in gluconeogenesis leading to a decline in insulin levels and reduced proliferation of insulin-responsive cancers. Here, we show that metformin attenuates paraquat-induced elevations in reactive oxygen species (ROS), and related DNA damage and mutations, but has no effect on similar changes induced by H(2)0(2), indicating a reduction in endogenous ROS production. Importantly, metformin also inhibited Ras-induced ROS production and DNA damage. Our results reveal previously unrecognized inhibitory effects of metformin on ROS production and somatic cell mutation, providing a novel mechanism for the reduction in cancer risk reported to be associated with exposure to this drug.
Cancer Prevention Research 02/2012; 5(4):536-43. · 4.91 Impact Factor
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ABSTRACT: Although stress can suppress growth and proliferation, cells can induce adaptive responses that allow them to maintain these functions under stress. While numerous studies have focused on the inhibitory effects of stress on cell growth, less is known on how growth-promoting pathways influence stress responses. We have approached this question by analyzing the effect of mammalian target of rapamycin (mTOR), a central growth controller, on the osmotic stress response. Our results showed that mammalian cells exposed to moderate hypertonicity maintained active mTOR, which was required to sustain their cell size and proliferative capacity. Moreover, mTOR regulated the induction of diverse osmostress response genes, including targets of the tonicity-responsive transcription factor NFAT5 as well as NFAT5-independent genes. Genes sensitive to mTOR-included regulators of stress responses, growth and proliferation. Among them, we identified REDD1 and REDD2, which had been previously characterized as mTOR inhibitors in other stress contexts. We observed that mTOR facilitated transcription-permissive conditions for several osmoresponsive genes by enhancing histone H4 acetylation and the recruitment of RNA polymerase II. Altogether, these results reveal a previously unappreciated role of mTOR in regulating transcriptional mechanisms that control gene expression during cellular stress responses.
Nucleic Acids Research 01/2012; 40(10):4368-84. · 8.03 Impact Factor
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Laurent Bultot,
Bruno Guigas,
Alexander Von Wilamowitz-Moellendorff,
Liliane Maisin,
Didier Vertommen,
Nusrat Hussain,
Monique Beullens,
Joan J Guinovart,
Marc Foretz, Benoît Viollet,
Kei Sakamoto,
Louis Hue,
Mark H Rider
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ABSTRACT: Recombinant muscle GYS1 (glycogen synthase 1) and recombinant liver GYS2 were phosphorylated by recombinant AMPK (AMP-activated protein kinase) in a time-dependent manner and to a similar stoichiometry. The phosphorylation site in GYS2 was identified as Ser7, which lies in a favourable consensus for phosphorylation by AMPK. Phosphorylation of GYS1 or GYS2 by AMPK led to enzyme inactivation by decreasing the affinity for both UDP-Glc (UDP-glucose) [assayed in the absence of Glc-6-P (glucose-6-phosphate)] and Glc-6-P (assayed at low UDP-Glc concentrations). Incubation of freshly isolated rat hepatocytes with the pharmacological AMPK activators AICA riboside (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) or A769662 led to persistent GYS inactivation and Ser7 phosphorylation, whereas inactivation by glucagon treatment was transient. In hepatocytes from mice harbouring a liver-specific deletion of the AMPK catalytic α1/α2 subunits, GYS2 inactivation by AICA riboside and A769662 was blunted, whereas inactivation by glucagon was unaffected. The results suggest that GYS inactivation by AMPK activators in hepatocytes is due to GYS2 Ser7 phosphorylation.
Biochemical Journal 01/2012; 443(1):193-203. · 4.90 Impact Factor
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ABSTRACT: This study was to investigate the effect of nicotine on insulin sensitivity and explore the underlying mechanisms. Treatment of Sprague-Dawley rats with nicotine (3 mg/kg/day) for 6 weeks reduced 43% body weight gain and 65% blood insulin level, but had no effect on blood glucose level. Both insulin tolerance test and glucose tolerance test demonstrated that nicotine treatment enhanced insulin sensitivity. Pretreatment of rats with hexamethonium (20 mg/kg/day) to antagonize peripheral nicotinic receptors except for α7 nicotinic acetylcholine receptor (α7-nAChR) had no effect on the insulin sensitizing effect of nicotine. However, the insulin sensitizing effect but not the bodyweight reducing effect of nicotine was abrogated in α7-nAChR knockout mice. Further, chronic treatment with PNU-282987 (0.53 mg/kg/day), a selective α7-nAChR agonist, significantly enhanced insulin sensitivity without apparently modifying bodyweight not only in normal mice but also in AMP-activated kinase-α2 knockout mice, an animal model of insulin resistance with no sign of inflammation. Moreover, PNU-282987 treatment enhanced phosphorylation of signal transducer and activator of transcription 3 (STAT3) in skeletal muscle, adipose tissue and liver in normal mice. PNU-282987 treatment also increased glucose uptake by 25% in C2C12 myotubes and this effect was total abrogated by STAT3 inhibitor, S3I-201. All together, these findings demonstrated that nicotine enhanced insulin sensitivity in animals with or without insulin resistance, at least in part via stimulating α7-nAChR-STAT3 pathway independent of inflammation. Our results contribute not only to the understanding of the pharmacological effects of nicotine, but also to the identifying of new therapeutic targets against insulin resistance.
PLoS ONE 01/2012; 7(12):e51217. · 4.09 Impact Factor
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ABSTRACT: Acute ozone exposure causes lung oxidative stress and inflammation leading to lung injury. At least one mechanism underlying the lung toxicity of ozone involves excessive production of reactive oxygen and nitrogen intermediates such as peroxynitrite. In addition and beyond its major prooxidant properties, peroxynitrite may nitrate tyrosine residues altering phosphorylation of many protein kinases involved in cell signalling. It was recently proposed that peroxynitrite activates 5'-AMP-activated kinase (AMPK), which regulates metabolic pathways and the response to cell stress. AMPK activation as a consequence of ozone exposure has not been previously evaluated. First, we tested whether acute ozone exposure in mice would impair alveolar fluid clearance, increase lung tissue peroxynitrite production and activate AMPK. Second, we tested whether loss of AMP-activated protein kinase alpha1 subunit in mouse would prevent enhanced oxidative stress and lung injury induced by ozone exposure.
Control and AMPKα1 deficient mice were exposed to ozone at a concentration of 2.0 ppm for 3 h in glass cages. Evaluation was performed 24 h after ozone exposure. Alveolar fluid clearance (AFC) was evaluated using fluorescein isothiocyanate tagged albumin. Differential cell counts, total protein levels, cytokine concentrations, myeloperoxidase activity and markers of oxidative stress, i.e. malondialdehyde and peroxynitrite, were determined in bronchoalveolar lavage (BAL) and lung homogenates (LH). Levels of AMPK-Thr172 phosphorylation and basolateral membrane Na(+)-K(+)-ATPase abundance were determined by Western blot.
In control mice, ozone exposure induced lung inflammation as evidence by increased leukocyte count, protein concentration in BAL and myeloperoxidase activity, pro-inflammatory cytokine levels in LH. Increases in peroxynitrite levels (3 vs 4.4 nM, p = 0.02) and malondialdehyde concentrations (110 vs 230 μmole/g wet tissue) were detected in LH obtained from ozone-exposed control mice. Ozone exposure consistently increased phosphorylated AMPK-Thr172 to total AMPK ratio by 80% in control mice. Ozone exposure causes increases in AFC and basolateral membrane Na(+)-K(+)-ATPase abundance in control mice which did not occur in AMPKα1 deficient mice.
Our results collectively suggest that AMPK activation participates in ozone-induced increases in AFC, inflammation and oxidative stress. Further studies are needed to understand how the AMPK pathway may provide a novel approach for the prevention of ozone-induced lung injury.
Respiratory research 05/2011; 12:64. · 3.36 Impact Factor
