AMP-activated protein kinase can be allosterically activated by ADP but AMP remains the key activating ligand.

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. When activated by increases in ADP:ATP and/or AMP:ATP ratios (signalling energy deficit), AMPK acts to restore energy balance. Binding of AMP to one or more of three CBS repeats (CBS1, CBS3, CBS4) on the AMPK-γ subunit activates the kinase complex by three complementary mechanisms: (i) promoting α-subunit Thr172 phosphorylation by the upstream kinase LKB1; (ii) protecting against Thr172 dephosphorylation; (iii) allosteric activation. Surprisingly, binding of ADP has been reported to mimic the first two effects, but not the third. We now show that at physiologically relevant concentrations of Mg.ATP2- (above those used in the standard assay) ADP binding does cause allosteric activation. However, ADP causes only a modest activation because (unlike AMP), at concentrations just above those where activation becomes evident, ADP starts to cause competitive inhibition at the catalytic site. Our results cast doubt on the physiological relevance of the effects of ADP and suggest that AMP is the primary activator in vivo. We have also made mutations to hydrophobic residues involved in binding adenine nucleotides at each of the three γ subunit CBS repeats of the human α2ß2γ1 complex and examined their effects on regulation by AMP and ADP. Mutation of the CBS3 site has the largest effects on all three mechanisms of AMP activation, especially at lower ATP concentrations, while mutation of CBS4 reduces the sensitivity to AMP. All three sites appear to be required for allosteric activation by ADP.

Discovery of natural adenosine monophosphate­activated protein kinase activators through virtual screening and activity verification studies.

The adenosine monophosphate­activated protein kinase (AMPK) is a promising target in drug development for various metabolic diseases. In the present study, the aim was to discover natural direct AMPK activators from natural sources, thus a virtual screening for direct AMPK activators was conducted by combining ligand­based and structure­based screening. A common­feature pharmacophore model (HipHop1) was generated with two hydrogen bond acceptor lipid features and one hydrophobic region feature. A total of 1,235 natural products were screened using the HipHop1 hypothesis and CDOCKER protocol successively. According to the docking score, seven hit compounds were selected for AMPK activation assays. Ultimately, (­)­catechin (compound 522) and licochalcone A (compound 1148) exhibited the highest AMPK activation activity. These findings may contribute to the development of AMPK activators from medicinal plants.

Poricoic acid A activates AMPK to attenuate fibroblast activation and abnormal extracellular matrix remodelling in renal fibrosis.

BACKGROUND: In chronic kidney disease, although fibrosis prevention is beneficial, few interventions are available that specifically target fibrogenesis. Poricoic acid A (PAA) isolated from Poria cocos exhibits anti-fibrotic effects in the kidney, however the underlying mechanisms remain obscure. PURPOSE: We isolated PAA and investigated its effects and the underlying mechanisms in renal fibrosis. STUDY DESIGN: Unilateral ureteral obstruction (UUO) and 5/6 nephrectomy (Nx) animal models and TGF-ß1-induced renal fibroblasts (NRK-49F) were used to investigate the anti-fibrotic activity of PAA and its underlying mechanisms. METHODS: Western blots, qRT-PCR, immunofluorescence staining, co-immunoprecipitation and molecular docking methods were used. Knock-down and knock-in of adenosine monophosphate-activated protein kinase (AMPK) in the UUO model and cultured NRK-49F cells were employed to verify the mechanisms of action of PAA. RESULTS: PAA improved renal function and alleviated fibrosis by stimulating AMPK and inhibiting Smad3 specifically in Nx and UUO models. Reduced AMPK activity was associated with Smad3 induction, fibroblast activation, and the accumulation and aberrant remodelling of extracellular matrix (ECM) in human renal puncture samples and cultured NRK-49F cells. PAA stimulated AMPK activity and decreased fibrosis in a dose-dependent manner, thus showing that AMPK was essential for PAA to exert its anti-fibrotic effects. AMPK deficiency reduced the anti-fibrotic effects of PAA, while AMPK overexpression enhanced its effect. CONCLUSION: PAA activated AMPK and further inhibited Smad3 specifically to suppress fibrosis by preventing aberrant ECM accumulation and remodelling and facilitating the deactivation of fibroblasts.

AMPK and TOR: The Yin and Yang of Cellular Nutrient Sensing and Growth Control.

The AMPK (AMP-activated protein kinase) and TOR (target-of-rapamycin) pathways are interlinked, opposing signaling pathways involved in sensing availability of nutrients and energy and regulation of cell growth. AMPK (Yin, or the "dark side") is switched on by lack of energy or nutrients and inhibits cell growth, while TOR (Yang, or the "bright side") is switched on by nutrient availability and promotes cell growth. Genes encoding the AMPK and TOR complexes are found in almost all eukaryotes, suggesting that these pathways arose very early during eukaryotic evolution. During the development of multicellularity, an additional tier of cell-extrinsic growth control arose that is mediated by growth factors, but these often act by modulating nutrient uptake so that AMPK and TOR remain the underlying regulators of cellular growth control. In this review, we discuss the evolution, structure, and regulation of the AMPK and TOR pathways and the complex mechanisms by which they interact.

Mechanism of Activation of AMPK by Cordycepin.

Cordycepin (3'-deoxyadenosine) is a major bioactive agent in Cordyceps militaris, a fungus used in traditional Chinese medicine. It has been proposed to have many beneficial metabolic effects by activating AMP-activated protein kinase (AMPK), but the mechanism of activation remained uncertain. We report that cordycepin enters cells via adenosine transporters and is converted by cellular metabolism into mono-, di-, and triphosphates, which at high cordycepin concentrations can almost replace cellular adenine nucleotides. AMPK activation by cordycepin in intact cells correlates with the content of cordycepin monophosphate and not other cordycepin or adenine nucleotides. Genetic knockout of AMPK sensitizes cells to the cytotoxic effects of cordycepin. In cell-free assays, cordycepin monophosphate mimics all three effects of AMP on AMPK, while activation in cells is blocked by a γ-subunit mutation that prevents activation by AMP. Thus, cordycepin is a pro-drug that activates AMPK by being converted by cellular metabolism into the AMP analog cordycepin monophosphate.

Discovery of 3,5-Dimethylpyridin-4(1H)-one Derivatives as Activators of AMP-Activated Protein Kinase (AMPK).

Novel 3,5-dimethylpyridin-4(1H)-one scaffold compounds were synthesized and evaluated as AMP-activated protein kinase (AMPK) activators. Unlike direct AMPK activators, this series of compounds showed selective cell growth inhibitory activity against human breast cancer cell lines. By optimizing the lead compound (4a) from our library, 2-[({1'-[(4-fluorophenyl)methyl]-2-methyl-1',2',3',6'-tetrahydro[3,4'-bipyridin]-6-yl}oxy)methyl]-3,5-dimethylpyridin-4(1H)-one (25) was found to have potent AMPK activating activity. Compound 25 also showed good aqueous solubility while maintaining the unique selectivity in cell growth inhibitory activity.

AMPK in microvascular complications of diabetes and the beneficial effects of AMPK activators from plants.

BACKGROUND: Diabetes mellitus is a multifactorial disorder with the risk of micro- and macro-vascular complications. High glucose-induced derangements in metabolic pathways are primarily associated with the initiation and progression of secondary complications namely, diabetic nephropathy, neuropathy, and retinopathy. Adenosine monophosphate-activated protein kinase (AMPK) has emerged as an attractive therapeutic target to treat various metabolic disorders including diabetes mellitus. It is a master metabolic regulator that helps in maintaining cellular energy homeostasis by promoting ATP-generating catabolic pathways and inhibiting ATP-consuming anabolic pathways. Numerous pharmacological and plant-derived bioactive compounds that increase AMP-activated protein kinase activation has shown beneficial effects by mitigating secondary complications namely retinopathy, nephropathy, and neuropathy. PURPOSE: The purpose of this review is to highlight current knowledge on the role of AMPK and its activators from plant origin in diabetic microvascular complications. METHODS: Search engines such as Google Scholar, PubMed, Science Direct and Web of Science are used to extract papers using relevant key words. Papers mainly focusing on the role of AMPK and AMPK activators from plant origin in diabetic nephropathy, retinopathy, and neuropathy was chosen to be highlighted. RESULTS: According to results, decrease in AMPK activation during diabetes play a causative role in the pathogenesis of diabetic microvascular complications. Some of the plant-derived bioactive compounds were beneficial in restoring AMPK activity and ameliorating diabetic microvascular complications. CONCLUSION: AMPK activators from plant origin are beneficial in mitigating diabetic microvascular complications. These pieces of evidence will be helpful in the development of AMPK-centric therapies to mitigate diabetic microvascular complications.

Towards multi-target antidiabetic agents: Discovery of biphenyl-benzimidazole conjugates as AMPK activators.

Type 2 diabetes mellitus is a complex metabolic disorder requiring polypharmacology approaches for effective treatment. Combinatorial library of fifteen new tricyclic benzimidazole derivatives have been designed and synthesized to combine fragments commonly found in allosteric AMPK activators and AT1 receptor antagonists. It was found that 2'-cyanobiphenyl serves as the pharmacophore of AMPK-activating activity, which also increases with the expansion of the external hydrogenated cycle. Also, pronounced antiplatelet activity is characteristic of the studied compounds. One of derivatives was identified as a potent inhibitor of the formation of advanced protein glycation end-products with reactive dicarbonyl scavenging activity. Two submicromolar AMPK activators 2b and 3b prevents inflammatory activation of murine macrophages. Along with good water solubility and synthetic availability, these results render biphenyl derivatives of fused benzimidazoles as a valuable starting point for the development of AMPK activators with multi-target antidiabetic activity.

Activation of AMPK by Medicinal Plants and Natural Products: Its Role in Type 2 Diabetes Mellitus.

Type-2 Diabetes (T2D) is a metabolic disease characterized by permanent hyperglycemia, whose development can be prevented or delayed by using therapeutic agents and implementing lifestyle changes. Some therapeutic alternatives include regulation of glycemia through modulation of different mediators and enzymes, such as AMP-activated protein kinase (AMPK), a highly relevant cellular energy sensor for metabolic homeostasis regulation, with particular relevance in the modulation of liver and muscle insulin sensitivity. This makes it a potential therapeutic target for antidiabetic drugs. In fact, some of them are standard drugs used for treatment of T2D, such as biguanides and thiazolidindiones. In this review, we compile the principal natural products that are activators of AMPK and their effect on glucose metabolism, which could make them candidates as future antidiabetic agents. Phenolics such as flavonoids and resveratrol, alkaloids such as berberine, and some saponins are potential natural activators of AMPK with a potential future as antidiabetic drugs.

Recent advances in the role of AMP-activated protein kinase in metabolic reprogramming of metastatic cancer cells: targeting cellular bioenergetics and biosynthetic pathways for anti-tumor treatment.

Growing data indicate that tumor progression and metastasis is dependent on the reprograming of cellular metabolism. Rapidly growing cancer cells undergo metabolic stress in a harsh microenvironment. AMP-activated protein kinase (AMPK) is an energy sensing factor that regulates bioenergetics and biosynthetic pathways within the cell, but its role under metastasis is in dispute. The best studied phenotype of cancer cells is aerobic glycolysis (the Warburg effect), an increased catabolism of glucose to lactate. However, glycolysis and mitochondrial oxidative phosphorylation may operate simultaneously in cancer cells. Many tumors may switch between these pathways accordingly to the current requirements. The alterations in metabolism of cancer cells combined with the overexpression of oncogenes (c-Myc) and transcription factors (Hypoxia-inducible factor 1a) confer a great advantage to malignant cells to avoid reactive oxygen species induced apoptosis. The determination of the role of AMPK network in metabolic reprogramming of metastatic cancer cells may help to identify the selective molecular targets for efficient anti-cancer therapies. In this review, we discuss the implications of AMPK activation in metabolic reprogramming of cancer cells and we present several potential therapeutic strategies targeting cancer cell metabolism. AMPK activator, biguanide metformin, either alone or in combination with other drugs, may selectively modulate signaling pathways, expresses the chemopreventive potential and can be used in current anti-cancer approaches. However, the ambiguous data suggest that the activation of AMPK may induce multiple effects and thus potential therapeutic anti-cancer approach should be carefully considered in relation to metabolic network of cancer cell signaling and other determinants such tumor stage and origin as well.

Blackcurrant Extract Ameliorates Hyperglycemia in Type 2 Diabetic Mice in Association with Increased Basal Secretion of Glucagon-Like Peptide-1 and Activation of AMP-Activated Protein Kinase.

Blackcurrants are berries that contain high levels of anthocyanins, particularly delphinidin 3-rutinoside (D3R). Several studies have reported that the consumption of blackcurrant extract (BCE) lowers blood glucose levels and ameliorates glucose tolerance, but the mechanism underlying this effect remains unclear. Glucagon-like peptide-1 (GLP-1) and AMP-activated protein kinase (AMPK) are considered one of the most significant molecular targets for the prevention and treatment of type 2 diabetes. In this study, we showed that dietary BCE significantly reduced blood glucose concentration and improved glucose tolerance in type 2 diabetic mice (KK-Ay). The basal GLP-1 concentration in plasma was significantly increased in the BCE group accompanied by upregulation of prohormone convertase 1/3 (PC1/3), the enzyme that processes intestinal proglucagon. Moreover, the level of phospho-AMPKα protein in skeletal muscle was significantly increased in the BCE group, and this was increase accompanied by significant upregulation of glucose transporter 4 (Glut4) proteins in the plasma membrane of BCE group. In conclusion, dietary BCE significantly reduced blood glucose concentration and improved glucose tolerance in association with increased basal GLP-1 concentration in plasma, upregulation of PC1/3 expression, and translocation of Glut4 to the plasma membrane of skeletal muscle in type 2 diabetic mice; furthermore, these effects were accompanied by activation of AMPK. Our findings demonstrated that D3R-rich BCE may help prevent diabetes and allow the dosages of diabetes drugs to be reduced.

Anti-obesity effects of Spirulina maxima in high fat diet induced obese rats via the activation of AMPK pathway and SIRT1.

To evaluate the anti-obesity effect of Spirulina maxima (SM), obese rats fed with a high-fat diet for 6 weeks were orally treated with SM or green tea extract (GTE, positive control) for an additional 4 weeks. The results demonstrated that the administration of SM not only prevented weight gain and reduced the index of white adipose tissues, but also attenuated changes of metabolic parameters in serum, such as adiponectin, leptin, TNF-α, glucose, insulin and lipid profile. Furthermore, an increase of adipocyte size was also inhibited by treatment with SM. In mesenteric white adipose tissue and skeletal muscle, the administration of SM activated AMP-activated protein kinase (AMPK) pathway and sirtuin 1 (SIRT1), leading to the suppression of the development of pathophysiological mechanism associated with obesity, including promoted lipid synthesis and blocked lipid oxidation. Taken together, SM improves obese phenotype induced by the consumption of a high-fat diet, suggesting that SM might be a potential agent to prevent or treat obesity.

Phytol stimulates the browning of white adipocytes through the activation of AMP-activated protein kinase (AMPK) α in mice fed high-fat diet.

Stimulating the browning of white adipocytes contributes to the restriction of obesity and related metabolic disorders. This study aimed to investigate the browning effects of phytol on mice inguinal subcutaneous white adipose tissue (iWAT) and explore the underlying mechanisms. Our results demonstrated that phytol administration decreased body weight gain and iWAT index, and stimulated the browning of mice iWAT, with the increased expression of brown adipocyte marker genes (UCP1, PRDM16, PGC1α, PDH, and Cyto C). In addition, phytol treatment activated the AMPKα signaling pathway in mice iWAT. In good agreement with the in vivo findings, the in vitro results showed that 100 µM phytol stimulated brown adipogenic differentiation and formation of brown-like adipocytes in the differentiated 3T3-L1 by increasing the mitochondria content and oxygen consumption, and promoting mRNA and/or protein expression of brown adipocyte markers (UCP1, PRDM16, PGC1α, PDH, Cyto C, Cidea and Elovl3) and beige adipocyte markers (CD137 and TMEM26). Meanwhile, phytol activated the AMPKα signaling pathway in the differentiated 3T3-L1. However, the inhibition of AMPKα with Compound C totally abolished phytol-stimulated brown adipogenic differentiation and formation of brown-like adipocytes. In conclusion, these results showed that phytol stimulated the browning of mice iWAT, which was coincident with the increased formation of brown-like adipocytes in the differentiated 3T3-L1, and appeared to be primarily mediated by the AMPKα signaling pathway. These data provided new insight into the role of phytol in regulating the browning of WAT and suggested the potential application of phytol as a nutritional intervention for the restriction of obesity and related metabolic disorders.

Ginger extract adjuvant to doxorubicin in mammary carcinoma: study of some molecular mechanisms.

PURPOSE: The present study aimed to investigate the molecular mechanisms underlying the anticancer properties of ginger extract (GE) in mice bearing solid Ehrlich carcinoma (SEC) and to evaluate the use of GE in combination with doxorubicin (DOX) as a complementary therapy against SEC. METHODS: SEC was induced in 60 female mice. Mice were divided into four equal groups: SEC, GE, DOX and GE + DOX. GE (100 mg/kg orally day after day) and DOX (4 mg/kg i.p. for 4 cycles every 5 days) were given to mice starting on day 12 of inoculation. On the 28th day, blood samples were collected, mice were scarified, tumor volume was measured, and tumor tissues were excised. RESULTS: The anti-cancer effect of GE was mediated by activation of adenosine monophosphate protein kinase (AMPK) and down-regulation of cyclin D1 gene expression. GE also showed pro-apoptotic properties as evidenced by elevation of the P53 and suppression of nuclear factor-kappa B (NF-κB) content in tumor tissue. Co-administration of GE alongside DOX markedly increased survival rate, decreased tumor volume, and increased the level of phosphorylated AMPK (PAMPK) and improved related pathways compared to DOX group. In addition, the histopathological results demonstrated enhanced apoptosis and absence of multinucleated cells in tumor tissue of GE + DOX group. CONCLUSION: AMPK pathway and cyclin D1 gene expression could be a molecular therapeutic target for the anticancer effect of GE in mice bearing SEC. Combining GE and DOX revealed a greater efficacy as anticancer therapeutic regimen.

AMPK modulatory activity of olive-tree leaves phenolic compounds: Bioassay-guided isolation on adipocyte model and in silico approach.

SCOPE: Olive-tree polyphenols have demonstrated potential for the management of obesity-related pathologies. We aimed to explore the capacity of Olive-tree leaves extract to modulate triglyceride accumulation and AMP-activated protein kinase activity (AMPK) on a hypertrophic adipocyte model. METHODS: Intracellular triglycerides and AMPK activity were measured on the hypertrophic 3T3-L1 adipocyte model by AdipoRed and immunofluorescence microscopy, respectively. Reverse phase high performance liquid chromatography coupled to time-of-flight mass detection with electrospray ionization (RP-HPLC-ESI-TOF/MS) was used for the fractionation of the extract and the identification of the compounds. In-silico molecular docking of the AMPK alpha-2, beta and gamma subunits with the identified compounds was performed. RESULTS: Olive-tree leaves extract decreased the intracellular lipid accumulation through AMPK-dependent mechanisms in hypertrophic adipocytes. Secoiridoids, cinnamic acids, phenylethanoids and phenylpropanoids, flavonoids and lignans were the candidates predicted to account for this effect. Molecular docking revealed that some compounds may be AMPK-gamma modulators. The modulatory effects of compounds over the alpha and beta AMPK subunits appear to be less probable. CONCLUSIONS: Olive-tree leaves polyphenols modulate AMPK activity, which may become a therapeutic aid in the management of obesity-associated disturbances. The natural occurrence of these compounds may have important nutritional implications for the design of functional ingredients.

Adenosine Monophosphate (AMP)-Activated Protein Kinase: A New Target for Nutraceutical Compounds.

Adenosine monophosphate-activated protein kinase (AMPK) is an important energy sensor which is activated by increases in adenosine monophosphate (AMP)/adenosine triphosphate (ATP) ratio and/or adenosine diphosphate (ADP)/ATP ratio, and increases different metabolic pathways such as fatty acid oxidation, glucose transport and mitochondrial biogenesis. In this sense, AMPK maintains cellular energy homeostasis by induction of catabolism and inhibition of ATP-consuming biosynthetic pathways to preserve ATP levels. Several studies indicate a reduction of AMPK sensitivity to cellular stress during aging and this could impair the downstream signaling and the maintenance of the cellular energy balance and the stress resistance. However, several diseases have been related with an AMPK dysfunction. Alterations in AMPK signaling decrease mitochondrial biogenesis, increase cellular stress and induce inflammation, which are typical events of the aging process and have been associated to several pathological processes. In this sense, in the last few years AMPK has been identified as a very interesting target and different nutraceutical compounds are being studied for an interesting potential effect on AMPK induction. In this review, we will evaluate the interaction of the different nutraceutical compounds to induce the AMPK phosphorylation and the applications in diseases such as cancer, type II diabetes, neurodegenerative diseases or cardiovascular diseases.

AMP-activated protein kinase (AMPK) activator A-769662 increases intracellular calcium and ATP release from astrocytes in an AMPK-independent manner.

AIM: To test the hypothesis that, given the role of AMP-activated protein kinase (AMPK) in regulating intracellular ATP levels, AMPK may alter ATP release from astrocytes, the main sources of extracellular ATP (eATP) within the brain. MATERIALS AND METHODS: Measurements of ATP release were made from human U373 astrocytoma cells, primary mouse hypothalamic (HTAS) and cortical astrocytes (CRTAS) and wild-type and AMPK α1/α2 null mouse embryonic fibroblasts (MEFs). Cells were treated with drugs known to modulate AMPK activity: A-769662, AICAR and metformin, for up to 3 hours. Intracellular calcium was measured using Fluo4 and Fura-2 calcium-sensitive fluorescent dyes. RESULTS: In U373 cells, A-769662 (100 µM) increased AMPK phosphorylation, whereas AICAR and metformin (1 mM) induced a modest increase or had no effect, respectively. Only A-769662 increased eATP levels, and this was partially blocked by AMPK inhibitor Compound C. A-769662-induced increases in eATP were preserved in AMPK α1/α2 null MEF cells. A-769662 increased intracellular calcium in U373, HTAS and CRTAS cells and chelation of intracellular calcium using BAPTA-AM reduced A-769662-induced eATP levels. A-769662 also increased ATP release from a number of other central and peripheral endocrine cell types. CONCLUSIONS: AMPK is required to maintain basal eATP levels but is not required for A-769662-induced increases in eATP. A-769662 (>50 µM) enhanced intracellular calcium levels leading to ATP release in an AMPK and purinergic receptor independent pathway.

Polyphenolics from mango (Mangifera indica L.) suppress breast cancer ductal carcinoma in situ proliferation through activation of AMPK pathway and suppression of mTOR in athymic nude mice.

The objective of this study was to assess the underlying mechanisms of mango polyphenol decreased cell proliferation and tumor volume in ductal carcinoma in situ breast cancer. We hypothesized that mango polyphenols suppress signaling along the AKT/mTOR axis while up-regulating AMPK. To test this hypothesis, mango polyphenols (0.8 mg gallic acid equivalents per day) and pyrogallol (0.2 mg/day) were administered for 4 weeks to mice xenografted with MCF10DCIS.com cells subcutaneously (n=10 per group). Tumor volumes were significantly decreased, both mango and pyrogallol groups displayed greater than 50% decreased volume compared to control. There was a significant reduction of phosphorylated protein levels of IR, IRS1, IGF-1R, and mTOR by mango; while pyrogallol significantly reduced the phosphorylation levels of IR, IRS1, IGF-1R, p70S6K, and ERK. The protein levels of Sestrin2, which is involved in AMPK-signaling, were significantly elevated in both groups. Also, mango significantly elevated AMPK phosphorylation and pyrogallol significantly elevated LKB1 protein levels. In an in vitro model, mango and pyrogallol increased reactive oxygen species (ROS) generation and arrested cells in S phase. In silico modeling indicates that pyrogallol has the potential to bind directly to the allosteric binding site of AMPK, inducing activation. When AMPK expression was down-regulated using siRNA in vitro, pyrogallol reversed the reduced expression of AMPK. This indicates that pyrogallol not only activates AMPK, but also increases constitutive protein expression. These results suggest that mango polyphenols and their major microbial metabolite, pyrogallol, inhibit proliferation of breast cancer cells through ROS-dependent up-regulation of AMPK and down-regulation of the AKT/mTOR pathway.

Paeonol protects against endoplasmic reticulum stress-induced endothelial dysfunction via AMPK/PPARδ signaling pathway.

Endoplasmic reticulum (ER) stress in endothelial cells often leads to endothelial dysfunction which underlies the pathogenesis of cardiovascular diseases. Paeonol, a major phenolic component extracted from Moutan Cortex, possesses various medicinal benefits which have been used extensively in traditional Chinese medicine. The present study investigated the protective mechanism of paeonol against tunicamycin-induced ER stress in isolated mouse aortas and human umbilical vein endothelial cells (HUVECs). Vascular reactivity in aorta was measured using a wire myograph. The effects of paeonol on protein expression of ER stress markers, reactive oxygen species (ROS) production, nitric oxide (NO) bioavailability and peroxisome proliferator-activated receptor δ (PPARδ) activity in the vascular wall were assessed by Western blot, dihydroethidium fluorescence (DHE) or lucigenin enhanced-chemiluminescence, 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM DA) and dual luciferase reporter assay, respectively. Ex vivo treatment with paeonol (0.1µM) for 16h reversed the impaired endothelium-dependent relaxations in C57BJ/6J and PPARδ wild type (WT) mouse aortas following incubation with tunicamycin (0.5µg/mL). Elevated ER stress markers, oxidative stress and reduction of NO bioavailability induced by tunicamycin in HUVECs, C57BJ/6J and PPARδ WT mouse aortas were reversed by paeonol treatment. These beneficial effects of paeonol were diminished in PPARδ knockout (KO) mouse aortas. Paeonol increased the expression of 5' adenosine monophosphate-activated protein kinase (AMPK) and PPARδ expression and activity while restoring the decreased phosphorylation of eNOS. The present study delineates that paeonol protects against tunicamycin-induced vascular endothelial dysfunction by inhibition of ER stress and oxidative stress, thus elevating NO bioavailability via the AMPK/PPARδ signaling pathway.

Cucurbitacin E ameliorates hepatic fibrosis in vivo and in vitro through activation of AMPK and blocking mTOR-dependent signaling pathway.

The study evaluated the potential protective effect and underlying mechanism of Cucurbitacin E (CuE) in both thioacetamide-induced hepatic fibrosis and activated HSCs. CuE inhibited the proliferation of activated HSC/T-6 cells in a concentration- and time-dependent manner; triggered the activation of caspase-3, cleaved PARP, altered ratio of bcl-2-to-bax, and affected cytochrome C protein in a time- and concentration-dependent manner. CuE arrested activated HSCs at the G2/M phase. Furthermore, CuE reduced levels of p-Erk/MAPK and also inhibited the protein and mRNA expressions of α-SMA, TIMP-1 and collagen I in activated HSC-T6 cells. CuE inhibited PI3K and Akt phosphorylation, and reduced the levels of p-mTOR and p-P70S6K and increased the expression of p-AMPK, which is similar with AICAR and metformin. C57BL/6 mice were intraperitoneally injected with thioacetamide (TAA) for five continuous weeks (100 or 200mg/kg, three times per week) along with daily administration of CuE (5 or 10mg/kg/d) and curcumin (Cur, 20mg/kg). CuE treatments significantly reduced serum ALT/AST levels, α-SMA, TIMP-1, and collagen I protein expressions. HE, Masson trichrome, Sirius red and immunohistochemical staining also suggested that CuE could ameliorate hepatic fibrosis. Our findings suggest that CuE induces apoptosis of activated HSC and ameliorates TAA-induced hepatic fibrosis through activation of AMPK and blocking mTOR-dependent signaling pathway.