Long-term activation of adenosine monophosphate-activated protein kinase attenuates pressure-overload-induced cardiac hypertrophy

Recent in vitro studies suggest that adenosine monophosphate (AMP)-activated protein kinase (AMPK) exerts inhibitory effects on cardiac hypertrophy. However, it is unclear whether long-term activation of AMPK will affect cardiac hypertrophy in vivo. In these reports, we investigate the in vivo effects of long-term AMPK activation on cardiac hypertrophy and the related molecular mechanisms. To examine the effects of AMPK activation in the development of pressure overload-induced cardiac hypertrophy, we administered 5-aminoimidazole 1 carboxamide ribonucleoside (AICAR, 0.5 mg/g body wt), a specific activator of AMPK, to rats with transaortic constriction (TAC) for 7 weeks. We found that long-term AMPK activation attenuated cardiac hypertrophy, and improved cardiac function in rats subjected to TAC. Furthermore, long-term AMPK activation attenuated protein synthesis, diminished calcineurin-nuclear factor of activated T cells (NFAT) and nuclear factor kappaB (NF-kappaB) signaling in pressure overload-induced hypertrophic hearts. Our in vitro experiments further proved that activation of AMPK by infection of AdAMPK blocked cardiac hypertrophy and NFAT, NF-kappaB, and MAPK signal pathways. The present study demonstrates for the first time that pharmacological activation of AMPK inhibits cardiac hypertrophy in through blocking signaling transduction pathways that are involved in cardiac growth. It presents a potential therapy strategy to inhibit pathological cardiac hypertrophy by increasing the activity of AMPK.     

Metabolic syndrome: adenosine monophosphate-activated protein kinase and malonyl coenzyme A

The metabolic syndrome can be defined as a state of metabolic dysregulation characterized by insulin resistance, central obesity, and a predisposition to type 2 diabetes, dyslipidemia, premature atherosclerosis, and other diseases. An increasing body of evidence has linked the metabolic syndrome to abnormalities in lipid metabolism that ultimately lead to cellular dysfunction. We review here the hypothesis that, in many instances, the cause of these lipid abnormalities could be a dysregulation of the adenosine monophosphate-activated protein kinase (AMPK)/malonyl coenzyme A (CoA) fuel-sensing and signaling mechanism. Such dysregulation could be reflected by isolated increases in malonyl CoA or by concurrent changes in malonyl CoA and AMPK, both of which would alter intracellular fatty acid partitioning. The possibility is also raised that pharmacological agents and other factors that activate AMPK and/or decrease malonyl CoA could be therapeutic targets.     

New cytotoxic dihydrochalcone and steroidal saponins from the aerial parts of Sansevieria cylindrica Bojer ex Hook

A new dihydrochalcone, 2‘,4‘-dihydroxy-3‘-methoxy-3,4-methylenedioxy-8-hydroxymethylene dihydrochalcone 1 and two new steroidal saponins, (25S)-ruscogenin-1-O-α-l-rhamnopyranosyl-(1 → 2)-β-d-glucopyranoside 2, (25S)-ruscogenin-3-O-α-l-rhamnopyranosyl-(1 → 4)-β-d-glucopyranoside 3, together with three known steroidal saponins (25S)-ruscogenin-3-O-β-d-glucopyranoside 4, (25S)-ruscogenin-1-O-α-l-rhamnopyranosyl-(1 → 2)-[β-d-xylopyranosyl-(1 → 3)]-α-l-arabinopyranoside 5 and (25R)-26-O-β-d-glucopyranosyl-furost-5-ene-1β,3β,22α,26-tetrol-1-O-α-L-rhamnopyranosyl-(1 → 2)-[β-d-xylopyranosyl-(1 → 3)]-α-l-arabinopyranoside 6 were isolated from the aerial parts of Sansevieria cylindrica. The structures of the new compounds were established by UV, IR, EI-MS, HR-ESI–MS as well as 1D (¹H,¹³C and DEPT-135) and 2D (HSQC, HMBC and TOCSY) NMR spectral analysis. The isolated compounds 1-6 were assayed for in vitro cytotoxicities against the three human tumor cell lines HT116, MCF7 and HepG2. Compound 1 showed a moderate cytotoxicity against MCF7. Compounds 2, 3 and 6 exhibited moderate cytotoxicities against the three used cell lines and compound 5 showed marked cytotoxicities against all used cell lines.     

Resveratrol prevents hyperglycemia-induced endothelial dysfunction via activation of adenosine monophosphate-activated protein kinase

Endothelial dysfunction secondary to persistent hyperglycemia plays a key role in the development of type 2 diabetic vascular disease. The aim of the present study was to examine the protective effect of resveratrol against hyperglycemia-induced endothelial dysfunction. In cultured human umbilical vein endothelial cells (HUVECs), resveratrol (10-100 μM) concentration dependently enhanced phosphorylation of endothelial nitric oxide synthesis (eNOS) at Ser1177 and nitric oxide (NO) production. In addition, resveratrol can increase the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) at Thr172 and suppress high glucose-induced generation of superoxide anion. In mouse aortic rings, resveratrol (1-100 μM) elicited endothelium-dependent vasodilatations and alleviated high glucose-mediated endothelial dysfunction. All these beneficial effects of resveratrol on the endothelium were abolished by pharmacological antagonism of AMPK by compound C. These results provide new insight into the protective properties of resveratrol against endothelial dysfunction caused by high glucose, which is attributed to the AMPK mediated reduction of superoxide level.     

Irisin ameliorates hepatic glucose/lipid metabolism and enhances cell survival in insulin-resistant human HepG2 cells through adenosine monophosphate-activated protein kinase signaling

Irisin is a newly identified myokine that promotes the browning of white adipose tissue, enhances glucose uptake in skeletal muscle and modulates hepatic metabolism. However, the signaling pathways involved in the effects on hepatic glucose and lipid metabolism have not been resolved. This study aimed to examine the role of irisin in the regulation of hepatic glucose/lipid metabolism and cell survival, and whether adenosine monophosphate-activated protein kinase (AMPK), a master metabolic regulator in the liver, is involved in irisin’s actions. Human liver-derived HepG2 cells were cultured in normal glucose-normal insulin (NGNI) or high glucose-high insulin (HGHI/insulin-resistant) condition. Hepatic glucose and lipid metabolism was evaluated by glucose output and glycogen content or triglyceride accumulation assays, respectively. Our results showed that irisin stimulated phosphorylation of AMPK and acetyl-CoA-carboxylase (ACC) via liver kinase B1 (LKB1) rather than Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ) in HepG2 cells. Irisin ameliorated hepatic insulin resistance induced by HGHI condition. Irisin reduced hepatic triglyceride content and glucose output, but increased glycogen content, with those effects reversed by dorsomorphin, an AMPK inhibitor. Furthermore, irisin also stimulated extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and promoted cell survival in an AMPK-dependent manner. In conclusion, our data indicate that irisin ameliorates dysregulation of hepatic glucose/lipid metabolism and cell death in insulin-resistant states via AMPK activation. These findings reveal a novel irisin-mediated protective mechanism in hepatic metabolism which provides a scientific basis for irisin as a potential therapeutic target for the treatment of insulin resistance and type 2 diabetes mellitus.     

Hyaluronan synthesis is inhibited by adenosine monophosphate-activated protein kinase through the regulation of HAS2 activity in human aortic smooth muscle cells

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan (GAG) involved in cell motility, proliferation, tissue remodeling, development, differentiation, inflammation, tumor progression, and invasion and controls vessel thickening in cardiovascular diseases. Therefore, the control of HA synthesis could permit the fine-tuning of cell behavior, but the mechanisms that regulate HA synthesis are largely unknown. Recent studies suggest that the availability of the nucleotide-sugar precursors has a critical role. Because the formation of UDP-sugars is a highly energetically demanding process, we have analyzed whether the energy status of the cell could control GAG production. AMP-activated protein kinase (AMPK) is the main ATP/AMP sensor of mammalian cells, and we mimicked an energy stress by treating human aortic smooth muscle cells (AoSMCs) with the AMPK activators 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside and metformin. Under these conditions, HA synthesis, but not that of the other GAGs, was greatly reduced. We confirmed the inhibitory effect of AMPK using a specific inhibitor and knock-out cell lines. We found that AMPK phosphorylated Thr-110 of human HAS2, which inhibits its enzymatic activity. In contrast, the other two HAS isoenzymes (HAS1 and HAS3) were not modified by the kinase. The reduction of HA decreased the ability of AoSMCs to proliferate, migrate, and recruit immune cells, thereby reducing the pro-atherosclerotic AoSMC phenotype. Interestingly, such effects were not recovered by treatment with exogenous HA, suggesting that AMPK can block the pro-atherosclerotic signals driven by HA by interaction with its receptors.     

Inhibition of hepatitis C virus replication through adenosine monophosphate-activated protein kinase-dependent and -independent pathways

Persistent infection with hepatitis C virus (HCV) is closely correlated with type 2 diabetes. In this study, replication of HCV at different glucose concentrations was investigated by using J6/JFH1-derived cell-adapted HCV in Huh-7.5 cells and the mechanism of regulation of HCV replication by AMP-activated protein kinase (AMPK) as an energy sensor of the cell analyzed. Reducing the glucose concentration in the cell culture medium from 4.5 to 1.0 g/L resulted in suppression of HCV replication, along with activation of AMPK. Whereas treatment of cells with AMPK activator 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) suppressed HCV replication, compound C, a specific AMPK inhibitor, prevented AICAR's effect, suggesting that AICAR suppresses the replication of HCV by activating AMPK in Huh-7.5 cells. In contrast, compound C induced further suppression of HCV replication when the cells were cultured in low glucose concentrations or with metformin. These results suggest that low glucose concentrations and metformin have anti-HCV effects independently of AMPK activation.     

Galloyl, caffeoyl and hexahydroxydiphenoyl esters of dihydrochalcone glucosides from Balanophora tobiracola

Seven galloyl, caffeoyl and (S)-hexahydroxydiphenoyl (HHDP) esters of dihydrochalcone glucosides were isolated from Balanophora tobiracola; based on spectroscopic and chemical evidence, their structures were determined to be 6'-O-galloyl-, 3',4'-di-O-galloyl-, 4',6'-di-O-galloyl-, 4',6'-O-(S)-HHDP-, 3'-O-galloyl-4',6'-O-(S)-HHDP-, 3'-O-caffeoyl-4',6'-O-(S)-HHDP-3-hydroxyphloretin 4'-O-beta-D-glucosides and 3'-O-galloyl-4',6'-O-(S)-HHDP-phloretin 4'-O-beta-D-glucoside, respectively. By contrast, these compounds were not found in the taxonomically related B. japonica. The 3'-galloyl-4',6'-HHDP esters of the dihydrochalcone glucosides showed strong inhibitory activities against alpha-glucosidase. Four known compounds were also isolated namely, (+/-)-eriodictyol 7-O-beta-D-glucoside, 1-O-caffeoyl-3-O-galloyl-beta-D-glucose, phloretin 4'-O-beta-D-glucoside, and 3-hydroxyphloretin 4'-O-beta-D-glucoside.     

Development of protein kinase activators: AMPK as a target in metabolic disorders and cancer

AMP-activated protein kinase (AMPK) is a cellular energy sensor activated by metabolic stresses that either inhibit ATP synthesis or accelerate ATP consumption. Activation of AMPK in response to an increase in the cellular AMP:ATP ratio results in inhibition of ATP-consuming processes such as gluconeogenesis and fatty acid synthesis, while stimulating ATP-generating processes, including fatty acid oxidation. These alterations in lipid and glucose metabolism would be expected to ameliorate the pathogenesis of obesity, type 2 diabetes and other metabolic disorders. Recently, AMPK has also been identified as a potential target for cancer prevention and/or treatment. Cell growth and proliferation are energetically demanding, and AMPK may act as an “energy checkpoint” that permits growth and proliferation only when energy reserves are sufficient. Thus, activators of AMPK could have potential as novel therapeutics both for metabolic disorders and for cancer, which together constitute two of the most prevalent groups of diseases worldwide.     

Biphasic regulation of macrophage attachment by activators of cyclic adenosine monophosphate-dependent kinase and protein kinase C

A method is described that enabled us to study the adhesiveness of J-774 murine macrophages. Cell attachment was stimulated by activators of kinase C (i.e., phorbol esters) as well as kinase A (cyclic adenosine monophosphate; cAMP). This novel effect of cAMP was observed when its levels were increased via receptor triggering (prostaglandin E1, beta-adrenergic agonists), activation of Ns (cholera toxin), or inhibition of phosphodiesterase (Ro 20-1724) or when the kinase was directly activated by Br8-cAMP. The simultaneous treatment with kinase A and kinase C activators at the time of attachment resulted in a partially additive response. On the other hand, preincubation of the cells in suspension with one of the activators rendered them refractory to subsequent stimulation at the onset of the adhesion assay, whatever agent was used. Such a refractoriness was also observed in cells preincubated with oleoyl-acetyl-glycerol (OAG). On the other hand, when added at the time of attachment, this near-physiological activator of kinase C evoked a biphasic response: the early stimulation of cell attachment was followed by an accelerated rate of "detachment." In conclusion, kinase C and kinase A play a role in the sequence of events leading to cell adhesion. The cross desensitization observed is distal and takes place at or beyond the kinase step.     

Development of a potent and orally active activator of adenosine monophosphate-activated protein kinase (AMPK), ASP4132, as a clinical candidate for the treatment of human cancer

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) plays a key role in maintaining cellular metabolism. AMP or adenosine diphosphate (ADP) levels rise during metabolic stress, such as during nutrient starvation, hypoxia and muscle contraction, and bind to AMPK to induce activity. Recently, activation of AMPK has been considered an attractive therapeutic strategy in the field of human oncology. Structural optimization of lead compound 2, a new type of AMPK activator with potent AMPK activation activity and attractive selective growth inhibition against human cancer cells, improved aqueous solubility, metabolic stability and animal pharmacokinetics (PK) and culminated in the identification of (5-{1-[(6-methoxypyridin-3-yl)methyl]piperidin-4-yl}-1H-benzimidazol-2-yl)(4-{[4-(trifluoromethyl)phenyl]methyl}piperazin-1-yl)methanone ditosylate, ASP4132 (28). Studies on ASP4132 had advanced to clinical trials for the treatment of cancer.     

Activation of adenosine monophosphate activated protein kinase inhibits growth of multiple myeloma cells

The role of adenosine monophosphate activated protein kinase (AMPK) in regulating multiple myeloma (MM) cell growth is not yet clear. In this study, we show that the AMPK activators 5-aminoimidazole-4-carboxamide riboside (AICAr) and D942 inhibit cell growth in MM cell lines. AICAr also induced an S-phase cell cycle arrest in all four tested cell lines and led to phosphorylation and thus activation of AMPK. Furthermore, the inhibition of a nucleoside transporter by nitrobenzyl-thio-9-beta-d-ribofuranosylpurine (NBTI), inhibition of the adenosine kinase by iodotubericidine and inhibition of AMPK by AMPKI Compound C reversed AICAr effects, indicating that the cellular effects of AICAr were mediated by AMPK. Activation of AMPK inhibited basal extracellular signal-regulated kinase (ERK), mammalian target of rapamycin (mTOR) and P70S6 kinase (P70S6K) as well as AKT phosphorylation, and blocked IL-6, IGF-1, and HS-5 stromal cell conditioned medium-induced increase of cell growth. Troglitazone, which has previously been shown to activate AMPK, similarly inhibited MM cell growth, activated AMPK, and decreased ERK and P70S6K phosphorylation. Our results suggest that activation of AMPK inhibits MM cell growth despite stimulation with IL-6, IGF-1, or HS-5 stromal cell conditioned medium and represents a potential new target in the therapy of MM.     

Rapid dephosphorylation of protein kinase C substrates by protein kinase A activators results from inhibition of diacylglycerol release

The biochemical events encompassing the dephosphorylation of protein kinase C substrates by protein kinase A activators have been investigated in a neurotumor cell line, NCB-20. Treatment of [32P]orthophosphate-labeled cells with protein kinase A activators (e.g. forskolin, dibutyryl cAMP, prostaglandin E1) resulted in an inhibition of protein kinase C activity due to a failure of the protein kinase C complex to translocate into the membrane. Phospholipase C activity, as measured by the synchronous release of diacylglycerol and inositol phosphates (inositol 1,4,5-trisphosphate, inositol 1,4-bisphosphate, and inositol 1-phosphate) in response to bradykinin, was inhibited up to 50% following exposure to protein kinase A activators. At the same time, phospholipase C-specific inositol phospholipid substrates (phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate) were found to accumulate in NCB-20 cells following treatment with protein kinase A activators. This suggests that phospholipase C may be altered through protein kinase A-mediated protein phosphorylation. Second messenger generation (inositol phosphates, diacylglycerol, and Ca2+) is therefore inhibited through cyclic AMP-mediated shutdown of the inositol lipid cycle at the level of phospholipase C.     

Cows are not mice: the role of cyclic AMP, phosphodiesterases, and adenosine monophosphate-activated protein kinase in the maintenance of meiotic arrest in bovine oocytes

Meiotic maturation in mammalian oocytes is initiated during fetal development, and is then arrested at the dictyate stage - possibly for several years. Oocyte meiosis resumes in preovulatory follicles in response to the lutenizing hormone (LH) surge or spontaneously when competent oocytes are removed from follicles and cultured. The mechanisms involved in meiotic arrest and resumption in bovine oocytes are not fully understood, and several studies point to important differences between oocytes from rodent and livestock species. This paper reviews earlier and contemporary studies on the effects of cAMP-elevating agents and phosphodiesterase (PDE) enzyme inhibitors on the maintenance of meiotic arrest in bovine oocytes in vitro. Contrary to results obtained with mouse oocytes, bovine oocyte meiosis is inhibited by activators of the energy sensor adenosine monophosphate-activated protein kinase (AMPK, mammalian gene PRKA), which is activated by AMP, the degradation product of cAMP. It is not clear whether or not the effects were due to AMPK activation, and they may depend on culture conditions. Evidence suggests that other signaling pathways (for example, the cGMP/nitric oxide pathway) are involved in bovine oocyte meiotic arrest, but further studies are needed to understand the interactions between the signaling pathways that lead to maturation promoting factor (MPF) being inactive or active. An improved understanding of the mechanisms involved in the control of bovine oocyte meiosis will facilitate better control of the process in vitro, resulting in increased developmental competence and increased efficiency of in vitro embryo production procedures.     

Isolation and structural elucidation of novel cholestane glycosides and spirostane saponins from Polygonatum odoratum

Much attention has been paid to cholestane-type steroidal glycosides because of their importance from the perspectives of both chemical diversity and significant biological activities. A phytochemical investigation of the rhizomes of Polygonatum odoratum (Liliaceae) resulted in the isolation of three novel cholestane-type steroidal glycosides (1–3) with unique Δ^14,16-unsaturated D-ring structures as well as two novel spirostane-type steroidal saponins (4 and 5) and three known steroidal glycosides (6–8). Their structures were determined by various spectroscopic methods and chemical reactions. Steroidal saponin 7 showed significant antifungal activity against Candida albicans JCM1542 (MIC 3.1 μg/mL) and Aspergillus fumigatus JCM1738 (MIC 6.3 μg/mL).     

秦岭地区玉竹根茎的脂溶性成分及其抑菌活性研究

从秦岭产玉竹（Polygonatum odoratum）根茎的石油醚萃取物中分离得到8个化合物,其中有5个化合物为首次从黄精属植物中得到,其结构分别为：（1）（24R/S）-9,19-环阿尔廷-25-烯-3β,24-二醇;（2）α-棕榈酸甘油酯;（3）棕榈酸甲酯;（4）二十八碳酸;（5）（Z）-6-十九碳烯酸。首次对化合物1的抗菌活性进行了测定,发现化合物1在浓度为10μg/mL时,对黄瓜炭疽病原菌的抑制率达到100%;在浓度为100μg/mL时,对灵杆菌的抑菌能力与红霉素相当。     

Tumor necrosis factor receptors and cytocidal activity are down-regulated by activators of protein kinase C

Human HeLa cells and murine L(S) cells are highly sensitive to the cytocidal activity of tumor necrosis factor (TNF) when simultaneously treated with the inhibitor of protein synthesis cycloheximide. This cytocidal activity of TNF was inhibited up to 90% in both cell lines after a 15-60-min pretreatment with 3-10 ng/ml of phorbol 12-myristate 13-acetate (PMA). This inhibition was long lasting for HeLa cells but transient for L(S) cells. The protection afforded by PMA was most effective when the cells were pretreated with this phorbol ester, but it decreased when PMA was added together with TNF or after TNF addition. This finding suggested that PMA interfered with one of the early steps in the mechanism of action of TNF. A pretreatment with the calcium ionophore A23187 also reduced the cytocidal activity of TNF in both HeLa and L(S) cells to about the same extent. Treatment of these cells with either PMA or A23187 significantly decreased the binding of 125I-TNF to cell surface receptors. This decrease paralleled the time course and dose-response of the inhibition of cytocidal activity. In addition, treatment of HeLa cells with 1-oleyl-2-acetyl-glycerol (OAG) also induced a rapid loss of TNF binding capacity. Since OAG, PMA, and A23187 are all activators of protein kinase C (Ca2+/phospholipid-dependent enzyme), these results suggest that this kinase is involved in modulation of TNF sensitivity. Furthermore, depletion or inhibition of protein kinase C antagonized PMA-induced effects on TNF cytotoxicity and binding to receptors. Internalization of bound TNF was not significantly affected by PMA treatment, and Scatchard analysis of binding data indicated that PMA decreased TNF receptor binding affinity rather than the number of TNF-binding sites. These findings suggest that protein kinase C may have a physiological role in mediating TNF sensitivity.