AMP-activated Protein Kinase Induction Research Articles

Lemon Balm and Corn Silk Mixture Alleviates Metabolic Disorders Caused by a High-Fat Diet.

We recently reported that varying combination ratios of lemon balm (Mellissa officinalis L.) and corn silk extracts (Stigma of Zea mays L. fruit) could reduce the obesity caused by a high-fat diet (HFD). The present study investigated the dose-dependent effect of a 1:1 (w:w) mixture of lemon balm and corn silk extracts (M-LB/CS) on HFD-mediated metabolic disorders and compared the effect with metformin. Oral administration of 50–200 mg/kg of M-LB/CS for 84 days significantly inhibited HFD-induced body weight gain, adipocyte hypertrophy, and lipogenic gene induction without affecting food consumption in mice. Biochemical analyses showed that M-LB/CS blocked abnormal lipid accumulation in the blood by escalating fecal lipid excretion. In addition, M-LB/CS prevented HFD-mediated pancreatic atrophy, decreased the number of insulin- and glucagon-immunoreactive cells, and inhibited increases in glycated hemoglobin, glucose, and insulin. Moreover, M-LB/CS also reduced hepatic injury, lipid accumulation, gluconeogenesis, and lipid peroxidation in parallel with the induction of AMP-activated protein kinase and antioxidant enzymes. Furthermore, M-LB/CS protected the kidney by inhibiting tubular vacuolation and reducing serum creatinine and blood urea nitrogen levels. The prophylactic effect of 100 mg/kg M-LB/CS-administration was comparable to that of metformin. Therefore, M-LB/CS may be an alternative option for managing obesity and its related metabolic disorders.

Withaferin A exerts an anti-obesity effect by increasing energy expenditure through thermogenic gene expression in high-fat diet-fed obese mice

BackgroundThe enhancement of energy expenditure has attracted attention as a therapeutic target for the management of body weight. Withaferin A (WFA), a major constituent of Withania somnifera extract, has been reported to possess anti-obesity properties, however the underlying mechanism remains unknown. PurposeTo investigate whether WFA exerts anti-obesity effects via increased energy expenditure, and if so, to characterize the underlying pathway. MethodsC57BL/6 J mice were fed a high-fat diet (HFD) for 10 weeks, and WFA was orally administered for 7 days. The oxygen consumption rate of mice was measured at 9 weeks using an OxyletPro™ system. Hematoxylin and eosin (H&E), immunohistochemistry, immunoblotting, and real-time PCR methods were used. ResultsTreatment with WFA ameliorated HFD-induced obesity by increasing energy expenditure by improving of mitochondrial activity in brown adipose tissue (BAT) and promotion of subcutaneous white adipose tissue (scWAT) browning via increasing uncoupling protein 1 levels. WFA administration also significantly increased AMP-activated protein kinase (AMPK) phosphorylation in the BAT of obese mice. Additionally, WFA activated mitogen-activated protein kinase (MAPK) signaling, including p38/extracellular signal-regulated kinase MAPK, in both BAT and scWAT. ConclusionWFA enhances energy expenditure and ameliorates obesity via the induction of AMPK and activating p38/extracellular signal-regulated kinase MAPK, which triggers mitochondrial biogenesis and browning-related gene expression.

Liposome-Encapsulated Bacillus Calmette-Guérin Cell Wall Skeleton Enhances Antitumor Efficiency for Bladder Cancer In Vitro and In Vivo via Induction of AMP-Activated Protein Kinase.

Simple SummaryWe engineered novel nanoparticles consisting of liposome-encapsulated Bacillus Calmette–Guérin cell well skeleton (BCG-CWS) for intravesical instillation in bladder cancer. The liposome-encapsulated BCG-CWS nanoparticles had antitumoral effects in an orthotopic bladder cancer mouse model, and the BCG-CWS nanoparticles can be further developed as a non-toxic substitute for live BCG with improved dispensability, stability, and size compatibility. This is significant because we succeeded in the intravesical delivery of BCG-CWS through the intravesical route using a catheter in an orthotopic bladder cancer mouse model to specifically target tumor cells. This is the first study on the BCG-CWS-induced activation of AMPK in urothelial carcinoma cells, suggesting that AMPK-mediated reactive oxygen species (ROS) production and ER stress is a cellular signaling pathway in tumors sensitive to BCG-CWS. These results have the potential for significant ramifications in targeted therapy using a predictive marker for bladder cancer.The Mycobacterium Bacillus Calmette-Guérin cell wall skeleton (BCG-CWS), the main immune active center of BCG, is a potent candidate non-infectious immunotherapeutic drug and an alternative to live BCG for use against urothelial carcinoma. However, its application in anticancer therapy is limited, as BCG-CWS tends to aggregate in both aqueous and non-aqueous solvents. To improve the internalization of BCG-CWS into bladder cancer cells without aggregation, BCG-CWS was nanoparticulated at a 180 nm size in methylene chloride and subsequently encapsulated with conventional liposomes (CWS-Nano-CL) using an emulsified lipid (LEEL) method. In vitro cell proliferation assays showed that CWS-Nano-CL was more effective at suppressing bladder cancer cell growth compared to nonenveloped BCG-CWS. In an orthotopic implantation model of luciferase-tagged MBT2 bladder cancer cells, encapsulated BCG-CWS nanoparticles could enhance the delivery of BCG-CWS into the bladder and suppress tumor growth. Treatment with CWS-Nano-CL induced the inhibition of the mammalian target of rapamycin (mTOR) pathway and the activation of AMP-activated protein kinase (AMPK) phosphorylation, leading to apoptosis, both in vitro and in vivo. Furthermore, the antitumor activity of CWS-Nano-CL was mediated predominantly by reactive oxygen species (ROS) generation and AMPK activation, which induced endoplasmic reticulum (ER) stress, followed by c-Jun N-terminal kinase (JNK) signaling-mediated apoptosis. Therefore, our data suggest that the intravesical instillation of liposome-encapsulated BCG-CWS nanoparticles can facilitate BCG-CW cellular endocytosis and provide a promising drug-delivery system as a therapeutic strategy for BCG-mediated bladder cancer treatment.

Is AMP-Activated Protein Kinase Associated to the Metabolic Changes in Primary Ovarian Insufficiency?

Primary ovarian insufficiency (POI) is a critical fertility defect characterized by anticipated impairment of the follicular reserve, which pathophysiological mechanisms have not yet been identified. We have investigated the possible relationship between AMP-activated protein kinase (AMPK) and the pathophysiology of POI. We studied 35 POI patients with altered levels of follicle-stimulating hormone (FSH) and estradiol and increased percentage of overweight compared with 20 healthy women. Blood mononuclear cells from POI patients showed reduced levels of phosphorylated AMPK, adenosine triphosphate, and high serum levels of leptin and tumor necrosis factor-alpha. All these alterations were also observed in granulosa cells, a more closer environment of the ovary. Significant negative correlations between AMPK and body mass index, FSH and leptin levels, and a positive correlation between AMPK and estradiol levels were observed. Oral metformin supplementation restored biochemical parameters. Interestingly, AMPK levels from patients were negatively correlated with age, suggesting an accelerated aging in POI mediated by the impairment of AMPK. These results lead to the hypothesis that an impairment of AMPK could be implicated in the metabolic pathophysiology of POI indicating at AMPK induction as a possible new therapeutic target.

Interleukin-22 drives a metabolic adaptive reprogramming to maintain mitochondrial fitness and treat liver injury.

Rationale: Interleukin 22 (IL-22) is an epithelial survival cytokine that is at present being explored as therapeutic agents for acute and chronic liver injury. However, its molecular basis of protective activities remains poorly understood.Methods: Here we demonstrate that IL-22 inhibits the deteriorating metabolic states induced by stimuli in hepatocytes. Utilizing cell biological, molecular, and biochemical approaches, we provide evidence that IL-22 promotes oxidative phosphorylation (OXPHOS) and glycolysis and regulates the metabolic reprogramming related transcriptional responses.Results: IL-22 controls metabolic regulators and enzymes activity through the induction of AMP-activated protein kinase (AMPK), AKT and mammalian target of rapamycin (mTOR), thereby ameliorating mitochondrial dysfunction. The upstream effector lncRNA H19 also participates in the controlling of these metabolic processes in hepatocytes. Importantly, amelioration of liver injury by IL-22 through activation of metabolism relevant signaling and regulation of mitochondrial function are further demonstrated in cisplatin-induced liver injury and steatohepatitis.Conclusions: Collectively, our results reveal a novel mechanism underscoring the regulation of metabolic profiles of hepatocytes by IL-22 during liver injury, which might provide useful insights from the bench to the clinic in treating and preventing liver diseases.

The hepatoprotective effect of myricetin against lipopolysaccharide and D-galactosamine-induced fulminant hepatitis

Fulminant hepatitis (FH) is a severe liver disease characterized by extensive hepatic necrosis, oxidative stress, and inflammation. Myricetin (Myr), a botanical flavonoid glycoside, is recognized to exert antiapoptosis, anti-inflammatory, and antioxidant properties. In the current study, we focused on exploring the protective effects and underlying mechanisms of Myr against lipopolysaccharide (LPS) and D-galactosamine (D-GalN)-induced FH. These data indicated that Myr effectively protected from LPS/D-GalN-induced FH by lowering the mortality of mice, decreasing ALT and AST levels, and alleviating histopathological changes, oxidative stress, inflammation, and hepatic apoptosis. Moreover, Myr could efficiently mediate multiple signaling pathways, displaying not only the regulation of caspase-3/9 and P53 protein, inhibition of toll-like receptor 4 (TLR4)-nuclear factor-kappa B (NF-κB) activation, and -mitogen-activated protein kinase (MAPK), but also the increase of heme oxygenase-1 (HO-1) and nuclear factor-erythroid 2-related factor 2 (Nrf2) expression, as well as induction of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation in mice with LPS/D-GalN-induced FH. Importantly, our further results in vitro suggested that Myr remarkably attenuated H2O2-triggered hepatotoxicity and ROS generation, activated Keap1-Nrf2/HO-1 and AMPK/ACC signaling pathway. However, Myr-enhanced the expression of HO-1 and Nrf2 protein was reversed by Keap1-overexpression, Nrf2-null and AMPK inhibitor. Meanwhile, Myr-relieved hepatotoxicity excited by H2O2 was blocked by Nrf2-null and AMPK inhibitor. Taken together, Myr exhibits a protective role against LPS/D-GalN-induced FH by suppressing hepatic apoptosis, inflammation, and oxidative stress, likely involving in the regulation of apoptosis-related protein, TLR4-NF-κB/-MAPK and NLRP3 inflammasome, and AMPK-Nrf2/HO-1 signaling pathway.

Differential regulation of AMP-activated protein kinase in healthy and cancer cells explains why V-ATPase inhibition selectively kills cancer cells

The cellular energy sensor AMP-activated protein kinase (AMPK) is a metabolic hub regulating various pathways involved in tumor metabolism. Here we report that vacuolar H+-ATPase (V-ATPase) inhibition differentially affects regulation of AMPK in tumor and nontumor cells and that this differential regulation contributes to the selectivity of V-ATPase inhibitors for tumor cells. In nonmalignant cells, the V-ATPase inhibitor archazolid increased phosphorylation and lysosomal localization of AMPK. We noted that AMPK localization has a prosurvival role, as AMPK silencing decreased cellular growth rates. In contrast, in cancer cells, we found that AMPK is constitutively active and that archazolid does not affect its phosphorylation and localization. Moreover, V-ATPase-independent AMPK induction in tumor cells protected them from archazolid-induced cytotoxicity, further underlining the role of AMPK as a prosurvival mediator. These observations indicate that AMPK regulation is uncoupled from V-ATPase activity in cancer cells and that this makes them more susceptible to cell death induction by V-ATPase inhibitors. In both tumor and healthy cells, V-ATPase inhibition induced a distinct metabolic regulatory cascade downstream of AMPK, affecting ATP and NADPH levels, glucose uptake, and reactive oxygen species production. We could attribute the prosurvival effects to AMPK's ability to maintain redox homeostasis by inhibiting reactive oxygen species production and maintaining NADPH levels. In summary, the results of our work indicate that V-ATPase inhibition has differential effects on AMPK-mediated metabolic regulation in cancer and healthy cells and explain the tumor-specific cytotoxicity of V-ATPase inhibition.

IGFBP-2 stimulates calcium/calmodulin-dependent protein kinase kinase 2 activation leading to AMP-activated protein kinase induction which is required for osteoblast differentiation.

Insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins-2 (IGFBP-2) function coordinately to stimulate osteoblast differentiation. Induction of AMP-activated protein kinase (AMPK) is required for differentiation and is stimulated by these two factors. These studies were undertaken to determine how these two peptides lead to activation of AMPK. Enzymatic inhibitors and small interfering RNA were utilized to attenuate calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) activity in osteoblasts, and both manipulations resulted in failure to activate AMPK, thereby resulting in inhibition of osteoblast differentiation. IGFBP-2 and IGF-I stimulated an increase in CaMKK2, and inhibition of IGFBP-2 binding its receptor resulted in failure to induce CaMKK2 and AMPK activation. Injection of a peptide that contained the IGFBP-2 receptor-binding domain into IGFBP-2-/- mice activated CaMKK2 and injection of a CaMKK2 inhibitor into normal mice inhibited both CamKK2 and AMPK activation in osteoblasts. We conclude that induction of CaMKK2 by IGFBP-2 and IGF-I in osteoblasts is an important signaling event that occurs early in differentiation and is responsible for activation of AMPK, which is required for optimal osteoblast differentiation.

Abstract A12: The metabolic stress mediator LKB1 is required for ovarian cancer metastasis

Abstract Most epithelial ovarian cancer (EOC) patients with metastatic disease initially respond to cytotoxic chemotherapy, yet almost all will relapse with resistant disease. Thus, improving patient outcomes will require novel approaches to limit metastasis and overcome chemo-resistance. Liver Kinase B1 (LKB1), encoded by the STK11 gene, is a key intracellular regulator of metabolic stress and is considered a putative tumor suppressor in some cancers. However, we have demonstrated that LKB1 is intact and required for EOC cell viability and growth in an in vitro spheroid model of ovarian cancer metastasis. We propose that LKB1 signaling enables malignant EOC cells to maintain viability and survive in metabolically challenging environments like that encountered during intraperitoneal metastasis. To further investigate the therapeutic potential of targeting LKB1 activity in metastatic EOC, we generated STK11-knockout cell lines—normal FT190 cells, and EOC cell lines OVCAR8, HeyA8, and iOvCa147—using CRISPR technology. STK11KO resulted in decreased malignant properties of EOC cells in vitro, including clonogenicity and anchorage-independent growth; however, loss of LKB1 in FT190 cells had no effect on cell proliferation, clonogenicity, or anchorage-independent growth, indicating LKB1 does not likely act as a tumor suppressor in EOC. Loss of LKB1 sensitized EOC cells to the growth-inhibiting effects of specific metabolic stresses. OVCAR8-STK11KO cells were more sensitive to nutrient deprivation in adherent culture, and to carboplatin and paclitaxel treatment in spheroid culture, as compared with OVCAR8 cells. Among the three EOC cell lines, STK11KO yielded variable sensitivity to inhibition of mitochondrial ATP production via oligomycin treatment. Interestingly, STK11KO did not affect induction of AMP-activated protein kinase (AMPK) phosphorylation in EOC spheroids, indicating that metabolic stress signaling to support EOC cell survival in spheroids during metastasis may occur via alternative pathways. In support of our previous knockdown results, EOC spheroids completely lacking LKB1 had markedly impaired growth in suspension culture compared to parental cell controls. In contrast, FT190 spheroids exhibited rapid cell attrition in spheroid culture regardless of LKB1 status. These results indicate that LKB1 may be specifically required in EOC cells to evade anoikis during metastatic spread. Finally, to test directly whether loss of LKB1 activity affects the metastatic potential of EOC cells, we performed intraperitoneal injections of OVCAR8-STK11KO and HeyA8-STK11KO cells with their respective parental cell controls. Loss of LKB1 in both aggressive EOC cell lines exhibited a dramatic reduction on tumor burden. STK11KO significantly decreased the establishment of large, solid tumor masses, reduced adhesion of OVCAR8 tumor nodules, and even changed the metastatic trajectory of HeyA8 cells with evidence of tumor cell growth only as a thin layer on peritoneal walls. Histologic analysis revealed evidence of extensive necrosis in STK11KO tumors, which was likely the major contributor to reduced tumor burden. These results strongly indicate that loss of LKB1 activity abrogates the metabolic stress response necessary during EOC metastasis both in spheroids and establishment of intraperitoneal tumors. Overall, LKB1 or its AMPK-independent signaling mediators represent unique yet very potent therapeutic vulnerabilities in metastatic EOC. Citation Format: Adrian Buensuceso, Yudith R. Valdes, Rene Figueredo, Gabriel E. DiMattia, Trevor G. Shepherd. The metabolic stress mediator LKB1 is required for ovarian cancer metastasis. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A12.

Digitoxin Suppresses Human Cytomegalovirus Replication via Na+, K+/ATPase α1 Subunit-Dependent AMP-Activated Protein Kinase and Autophagy Activation.

Host-directed therapeutics for human cytomegalovirus (HCMV) requires elucidation of cellular mechanisms that inhibit HCMV. We report a novel pathway used by cardiac glycosides to inhibit HCMV replication: induction of AMP-activated protein kinase (AMPK) activity and autophagy flux through the Na+,K+/ATPase α1 subunit. Our data illustrate an intricate balance between the autophagy regulators AMPK, mammalian target of rapamycin (mTOR), and ULK1 during infection and treatment with the cardiac glycoside digitoxin. Both infection and digitoxin induced AMPK phosphorylation, but ULK1 was differentially phosphorylated at unique sites leading to opposing effects on autophagy. Suppression of autophagy during infection occurred via ULK1 phosphorylation at Ser757 by enhanced mTOR activity. Digitoxin continuously phosphorylated AMPK, leading to ULK1 phosphorylation at Ser317, and suppressed mTOR, resulting in increased autophagy flux and HCMV inhibition. In ATG5-deficient human fibroblasts, digitoxin did not inhibit HCMV, supporting autophagy induction as a mechanism for virus inhibition. Drug combination studies with digitoxin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) further confirmed the role of autophagy activation in HCMV inhibition. Individually, each compound phosphorylated AMPK, but their combination reduced autophagy rather than inducing it and was antagonistic against HCMV, resulting in virus replication. The initial ULK1 activation by digitoxin was counteracted by AICAR, which prevented the downstream interaction of Beclin1 and phosphatidylinositol 3-kinase class III (PI3K-CIII), further supporting digitoxin-mediated HCMV inhibition through autophagy. Finally, the α1 subunit was required for autophagy induction, since in α1-deficient cells neither AMPK nor autophagy was activated and HCMV was not inhibited by digitoxin. In summary, induction of a novel pathway (α1-AMPK-ULK1) induces autophagy as a host-directed strategy for HCMV inhibition.IMPORTANCE Infection with human cytomegalovirus (HCMV) creates therapeutic challenges in congenitally infected children and transplant recipients. Side effects and selection of resistant mutants with the limited drugs available prompted evaluation of host-directed therapeutics. We report a novel mechanism of HCMV inhibition by the cardiac glycoside digitoxin. At low concentrations that inhibit HCMV, digitoxin induced signaling through the α1 subunit of the Na+,K+/ATPase pump and the cellular kinase AMPK, resulting in binding and phosphorylation of ULK1 (Ser317) and autophagy activation. HCMV suppressed autophagy through ULK1 phosphorylation (Ser757) by activating the mTOR kinase. The pump-autophagy pathway was required for HCMV inhibition, since in α1- or ATG5-deficient cells the virus was not inhibited. Furthermore, the AMPK activator AICAR antagonized digitoxin activity against HCMV, a phenomenon resulting from opposing effects downstream in the autophagy pathway, at the Beclin1 stage. In summary, autophagy may provide a strategy for harnessing HCMV replication.

Isoliquiritigenin Activates Nuclear Factor Erythroid-2 Related Factor 2 to Suppress the NOD-Like Receptor Protein 3 Inflammasome and Inhibits the NF-κB Pathway in Macrophages and in Acute Lung Injury

Among the cellular response mechanisms, the nuclear factor erythroid-2 related factor 2 (Nrf2) pathway is considered a survival pathway that alleviates oxidative injury, while both the NOD-like receptor protein 3 (NLRP3) and NF-κB pathways are pro-inflammatory pathways that cause damage to cells. These pathways are implicated in the development and resolution of acute lung injury (ALI). Isoliquiritigenin (ISL), a flavonoid from the liquorice compound, is suggested to be a regulator of the above pathways, but the mechanisms of how the NLRP3/NF-κB pathway interacts with Nrf2 and its protective effects in ALI remain unknown. In the present study, ISL inhibited reactive oxygen species (ROS) generation and cytotoxicity induced by t-BHP and pro-inflammatory enzymes production induced by LPS in RAW 264.7 cells. Such cytoprotective effects coincided with the induction of AMP-activated protein kinase (AMPK)/Nrf2/antioxidant response element (ARE) signaling and the suppression of the NLRP3 and NF-κB pathways. Consistent with these findings, ISL treatment significantly alleviated lung injury in LPS-induced ALI mice, which was reflected by reductions in histopathological changes, pulmonary edema, and protein leakage. At the same time, the increased levels of inflammatory cell exudation and pro-inflammatory mediators, the enhanced production of ROS, myeloperoxidase, and malondialdehyde, and the depleted expression of GSH and superoxide dismutase induced by LPS were ameliorated by ISL. Furthermore, ISL notably activated AMPK/Nrf2/ARE signaling and inhibited LPS-induced NLRP3 and NF-κB activation in the lung. Moreover, although inhibition of the LPS-induced histopathological changes and ROS production were attenuated in Nrf2-deficient mice, the repression of the NLRP3 and NF-κB pathways by ISL was Nrf2-dependent and Nrf2-independent, respectively. In conclusion, our results are the first to highlight the beneficial role and relevant mechanisms of ISL in LPS-induced ALI and provide novel insight into its application.

Asiatic Acid Exhibits Anti-inflammatory and Antioxidant Activities against Lipopolysaccharide and d-Galactosamine-Induced Fulminant Hepatic Failure.

Inflammation and oxidative stress are essential for the pathogenesis of fulminant hepatic failure (FHF). Asiatic acid (AA), which is a pentacyclic triterpene that widely occurs in various vegetables and fruits, has been reported to possess antioxidant and anti-inflammatory properties. In this study, we investigated the protective effects of AA against lipopolysaccharide (LPS) and d-galactosamine (GalN)-induced FHF and the underlying molecular mechanisms. Our findings suggested that AA treatment effectively protected against LPS/d-GalN-induced FHF by lessening the lethality; decreasing the alanine transaminase and aspartate aminotransferase levels, interleukin (IL)-1β, IL-6, and tumor necrosis factor-α production, malondialdehyde formation, myeloperoxidase level and reactive oxygen species generation (i.e., H2O2, NO, and ), and increasing the glutathione and superoxide dismutase contents. Moreover, AA treatment significantly inhibited mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathway activation via the partial induction of programmed cell death 4 (PDCD4) protein expressions, which are involved in inflammatory responses. Furthermore, AA treatment dramatically induced the expression of the glutamate-cysteine ligase modifier subunit, the glutamate-cysteine ligase catalytic subunit, heme oxygenase-1, and NAD (P) H: quinoneoxidoreductase 1 (NQO1), which are largely dependent on activation of the nuclear factor-erythroid 2-related factor 2 (Nrf2) through the induction of AMP-activated protein kinase (AMPK) and glycogen synthase kinase-3β (GSK3β) phosphorylation. Accordingly, AA exhibited protective roles against LPS/d-GalN-induced FHF by inhibiting oxidative stress and inflammation. The underlying mechanism may be associated with the inhibition of MAPK and NF-κB activation via the partial induction of PDCD4 and upregulation of Nrf2 in an AMPK/GSK3β pathway activation-dependent manner.

The Liver Circadian Clock Modulates Biochemical and Physiological Responses to Metformin.

Metformin is widely used in the treatment of type 2 diabetes to lower blood glucose. Although metformin is a relatively safe and effective drug, its clinical efficacy is variable and under certain circumstances it may contribute to life-threatening lactic acidosis. Thus, additional understanding of metformin pharmacokinetics and pharmacodynamics could provide important information regarding therapeutic use of this widely prescribed drug. Here we report a significant effect of time of day on acute blood glucose reduction in response to metformin administration and on blood lactate levels in healthy mice. Furthermore, we demonstrate that while metformin transport into hepatocytes is unaltered by time of day, the kinetics of metformin-induced activation of AMP-activated protein kinase (AMPK) in the liver are remarkably altered with circadian time. Liver-specific ablation of Bmal1 expression alters metformin induction of AMPK and blood glucose response but does not completely abolish time of day differences. Together, these data demonstrate that circadian rhythms affect the biological responses to metformin in a complex manner.

Calcium channels in chicken sperm regulate motility and the acrosome reaction.

Intracellular cytoplasmic calcium ([Ca(2+) ]i ) has an important regulatory role in gamete functions. However, the biochemical components involved in Ca(2+) transport are still unknown in birds, an animal class that has lost functional sperm-specific CatSper channels. Here, we provide evidence for the presence and expression of various Ca(2+) channels in chicken sperm, including high voltage-activated channels (L and R types), the store-operated Ca(2+) channel (SOC) component Orai1, the transient receptor potential channel (TRPC1) and inositol-1,4,5-trisphosphate receptors (IP3 R1). L- and R-type channels were mainly localized in the acrosome and the midpiece, and T-type channels were not detected in chicken sperm. Orai1 was found in all compartments, but with a weak, diffuse signal in the flagellum. TRCP1 was mainly localized in the acrosome and the midpiece, but a weak diffuse signal was also observed in the nucleus and the flagellum. IP3 R1 was mainly detected in the nucleus. The L-type channel inhibitor nifedipine, the R-type channel inhibitor SNX-482 and the SOC inhibitors MRS-1845, 2-APB and YM-58483 decreased [Ca(2+) ]i sperm motility and acrosome reaction capability, with the SOC inhibitors inhibiting these functions most efficiently. Furthermore, we showed that Ca(2+) -mediated induction of AMP-activated protein kinase (AMPK) phosphorylation was blocked by SOC inhibition. Our identification of important regulators of Ca(2+) signaling in avian sperm suggests that SOCs play a predominant role in gamete function, whereas T-type channels may not be involved. In addition, Ca(2+) entry via SOCs appears to be the most likely pathway for AMPK activation and energy-requiring sperm functions such as motility and the acrosome reaction.

IGF-I and IGFBP-2 Stimulate AMPK Activation and Autophagy, Which Are Required for Osteoblast Differentiation.

IGF-I/insulin-like growth factor binding protein 2 (IGFBP-2) coordinately stimulate osteoblast differentiation but the mechanisms by which they function have not been determined. AMP-activated protein kinase (AMPK) is induced during differentiation and AMPK knockout mice have reduced bone mass. IGF-I modulates AMPK in other cell types; therefore, these studies determined whether IGF-I/IGFBP-2 stimulate AMPK activation and the mechanism by which AMPK modulates differentiation. Calvarial osteoblasts and MC-3T3 cells expressed activated AMPK early in differentiation and AMPK inhibitors attenuated differentiation. However, expression of constitutively activated AMPK inhibited differentiation. To resolve this discrepancy we analyzed the time course of AMPK induction. AMPK activation was required early in differentiation (day 3-6) but down-regulation of AMPK after day 9 was also necessary. IGF-I/IGFBP-2 induced AMPK through their respective receptors and blocking-receptor activation blocked AMPK induction. To determine the mechanism by which AMPK functioned we analyzed components of the autophagosome. Activated AMPK stimulated ULK-1 S555 phosphorylation as well as beclin-1 and microtubule-associated protein 1A/1B light-chain phosphatidylethanolamine conjugate (LC3II) induction. Inhibition of AMPK attenuated these changes and direct inhibition of autophagy inhibited differentiation. Conversely, expression of activated AMPK was associated with persistence of these changes beyond day 9 and inhibited differentiation. Blocking AMPK activation after day 9 down-regulated these autophagosome components and rescued differentiation. This allowed induction of mechanistic target of rapamycin and AKT, which suppressed autophagy. The results show that early induction of AMPK in response to IGF-I/IGFBP-2 followed by suppression is required for osteoblast differentiation. AMPK functions through stimulation of autophagy. The findings suggest that these early catabolic changes are important for determining the energy source for osteoblast respiration and down-regulation of these components may be required for induction of glycolysis, which is required during the final anabolic stages of differentiation.

Pre-treatment with metformin activates Nrf2 antioxidant pathways and inhibits inflammatory responses through induction of AMPK after transient global cerebral ischemia.

Global cerebral ischemia arises in patients who have a variety of clinical conditions including cardiac arrest, shock and asphyxia. In spite of advances in understanding of the brain ischemia and stroke etiology, therapeutic approaches to improve ischemic injury still remain limited. It has been established that metformin can attenuate cell death in cerebral ischemia. One of the main functions of metformin is proposed to be conducted via AMP-activated protein kinase (AMPK)-dependent pathway in the experimental cerebral ischemia model. It is also established that metformin can suppress inflammation and activate Nuclear factor erythroid 2-related factor (Nrf2) pathways in neurons. In the current study, the role of metformin in regulating inflammatory and antioxidant pathways in the global cerebral ischemia was investigated. Our results indicated that pretreatment of rats by metformin attenuated cellular levels of nuclear factor-κB, Tumor Necrosis Factor alpha and Cyclooxygenase-2 which are considered as three important proteins involved in the inflammation pathway. Pretreatment by metformin increased the level of Nrf2 and heme oxygenase-1 in the hippocampus of ischemic rats compared with untreated ischemic group. Moreover, pretreatment by metformin enhanced the level of glutathione and catalase activities compared with them in ischemic group. Such protective changes detected by metformin pretreatment were reversed by injecting compound c, an AMPK inhibitor. These findings suggested that metformin might protect cells through modulating inflammatory and antioxidant pathways via induction of AMPK. However, more experimental and clinical trial studies regarding neuroprotective potential of metformin and the involved mechanisms, especially in the context of cerebral ischemic injuries, are necessary.

Metformin lowers Ser-129 phosphorylated α-synuclein levels via mTOR-dependent protein phosphatase 2A activation

Phospho-Ser129 α-synuclein is the modified form of α-synuclein that occurs most frequently within Parkinson's disease pathological inclusions. Here we demonstrate that the antidiabetic drug metformin significantly reduces levels of phospho-Ser129 α-synuclein and the ratio of phospho-Ser129 α-synuclein to total α-synuclein. This effect was documented in vitro in SH-SY5Y and HeLa cells as well as in primary cultures of hippocampal neurons. In vitro work also elucidated the mechanisms underlying metformin's action. Following metformin exposure, decreased phospho-Ser129 α-synuclein was not strictly dependent on induction of AMP-activated protein kinase, a primary target of the drug. On the other hand, metformin-induced phospho-Ser129 α-synuclein reduction was consistently associated with inhibition of mammalian target of rapamycin (mTOR) and activation of protein phosphatase 2A (PP2A). Evidence supporting a key role of mTOR/PP2A signaling included the finding that, similar to metformin, the canonical mTOR inhibitor rapamycin was capable of lowering the ratio of phospho-Ser129 α-synuclein to total α-synuclein. Furthermore, no decrease in phosphorylated α-synuclein occurred with either metformin or rapamycin when phosphatase activity was inhibited, supporting a direct relationship between mTOR inhibition, PP2A activation and protein dephosphorylation. A final set of experiments confirmed the effectiveness of metformin in vivo in wild-type C57BL/6 mice. Addition of the drug to food or drinking water lowered levels of phospho-Ser129 α-synuclein in the brain of treated animals. These data reveal a new mechanism leading to α-synuclein dephosphorylation that could be targeted for therapeutic intervention by drugs like metformin and rapamycin.

Fueling the engine: induction of AMP-activated protein kinase in trout skeletal muscle by swimming

AMP-activated protein kinase (AMPK) is well known to be induced by exercise and to mediate important metabolic changes in the skeletal muscle of mammals. Despite the physiological importance of exercise as a modulator of energy use by locomotory muscle, the regulation of this enzyme by swimming has not been investigated in fish. We found that sustained swimming (40 days at 0.75 body lengths s(-1)) increased AMPK activity in red and white trout skeletal muscle (3.9- and 2.2-fold, respectively) as well as the expression of AMPK target genes involved in energy use: lipoprotein lipase and citrate synthase in red and white muscle and CPT1β1b and PGC-1α in red muscle. Furthermore, electrical pulse stimulation of cultured trout myotubes increased AMPK activity and glucose uptake (1.9- and 1.2-fold, respectively) in an AMPK-dependent manner. These results suggest that AMPK may play an important mediatory role in the metabolic adaptation to swimming in fish skeletal muscle.

Prolonged AMP-activated protein kinase induction impairs vascular functions

AMP-activated protein kinase (AMPK) is a regulator of cellular metabolism and is involved in the pathogenesis of several diseases, including type 2 diabetes and cardiovascular diseases. Data showing the effects of AMPK on vasculature are controversial. Therefore, the aim of this study was to determine the impact of prolonged AMPK activation on vascular functions. For this purpose we have examined the role of AMPK in endothelium-dependent and -independent relaxation and vascular contractions. For this, we incubated thoracic aortic rings, from rats, with AMPK activator 5-aminoimidazole-4-carboxamide-1-4-ribofuranoside (AICAR, 500 μmol/L or 2 mmol/L) in the presence or absence of AMPK inhibitor compound C (10 μmol/L). Next, cumulative dose-response curves to acetylcholine (ACh) (10(-9)-10(-4) mol/L), nitroglycerine (NG) (10(-9)-3 × 10(-5) mol/L), and noradrenaline (NA) (10(-9)-10(-4) mol/L) were obtained. Endothelial nitric oxide synthase (eNOS) protein expression was determined. Our results show that endothelium-dependent relaxation was inhibited after AICAR treatment, and that this effect was reversed by AMPK inhibition. Moreover, AICAR enhanced the contractile response to NA and caused a decrease in eNOS protein expression. In conclusion, prolonged AMPK induction causes endothelial impairment, possibly via increased degradation and (or) reduced expression of eNOS.

Old Drug Acquires New Target: Metformin and Sirt1

Type 2 diabetes mellitus [T2DM] is a progressive metabolic disease which requires multiple and frequent treatment plan adjustments during the course of the disease. Conventional therapeutic strategy usually starts with lifestyle interventions supported by prescription of one glucose-lowering drug. When insulin resistance progresses and the monotherapy option becomes insufficient to control plasma glucose, patients are usually switched to a double drug regimen. In the worst case scenario further progression of insulin resistance and decline in endogenous insulin production require administration of exogenous insulin which becomes an unavoidable therapeutic imperative for many T2DM patients [1]. Metformin is the oldest and most widely prescribed first choice anti-diabetic drug representing the group of guanidinebased hypoglycemic agents. Most newly diagnosed T2DM patients are placed on metformin monotherapy which is later supplemented or often substituted by sulphonylureas, thiazolidinediones and dipeptidyl peptidase-4 inhibitors [2]. The antidiabetic properties of guanidine-related compounds were acknowledged for many centuries by the widespread use of G. officinalis (also known as French lilac, Goat’s rue or Italian Fitch) in diabetes care. However, in scientific terms, the hypoglycemic effect of guanidines, in particular the effect of isoamylene guanidine, was first demonstrated by C.K.Watanabe in 1918 [3]. The toxicity of guanidines imposed a significant limit on their pharmacological exploration, until a less toxic biguanidine analog – N,N-dimethylimidodicarbonimidic diamide (metformin) was synthesized in 1922. The discovery of insulin, whose hypoglycemic activity remains unsurpassed as well as the identification of other glucose-lowering compounds, weakened interest towards clinical use of guanidine-related compounds, including metformin, for decades. Therefore, it was not until 1979 that metformin was introduced in Europe and approved for T2DM use by the FDA in 1994. The moderate hypoglycemic activity of metformin resulting in low risk of hypoglycemia is complemented by the wide spectrum of pharmacological activity of the drug. Besides controlling blood glucose, metformin is known to have some “pleiotropic effects” such as reduction of plasma free fatty acids, LDL and VLDL, promotion of body weight loss, decrease in cardiovascular mortality and morbidity, prevention of hepatosteatosis and some anti-neoplastic effect [ 4]. Despite significant clinical research on metformin use in T2DM patients, the molecular mechanisms underlying the anti-diabetic properties of the drug remain surprisingly obscure. It is believed though that hypoglycemic action of metformin reflects its direct effect on the hepatic gluconeogenic pathway and on glucose utilization in peripheral tissues. While most of the hepatic effects of metformin develop due to induction of AMP-activated protein kinase (AMPK) and/or changes in CREBP-binding protein phosphorylation, the effect of metformin on glucose uptake is rather related to enhanced expression of glucose transporters [5,6]. Recent reports have unveiled a variety of new molecular mechanisms to explain the anti-diabetic activity of metformin. Among exciting scientific findings there are newly emerging reports suggesting that metformin can mediate its anti-diabetic activity through the family of proteins called sirtuins. This piece of information comes from the report of Caton PW et al . [7] who described increased levels of SIRT1 protein and its activity in the livers of metformin-treated db/db mice and HepG2 cells, a hepatocyte