Activates AMP-activated Protein Kinase Activation Research Articles

Aqueous Oldenlandia diffusa extracts inhibits colorectal cancer cells via activating AMP-activated protein kinase signalings.

Here we evaluated the anti-cancer activity of aqueous Oldenlandia diffusa (OD) extracts (ODE) in colorectal cancer (CRC) cells. We showed that ODE exerted potent anti-proliferative, cytotoxic and pro-apoptotic activities against a panel of established CRC lines (HCT-116, DLD-1, HT-29 and Lovo) and primary (patient-derived) human CRC cells. ODE activated AMP-activated protein kinase (AMPK) signaling, which led to subsequent mTORC1 inhibition and Bcl-2/HIF-1α downregulation in CRC cells. In ODE-treated CRC cells, AMPKα1 formed a complex with p53. This might be important for p53 activation and subsequent cancer cell apoptosis. Inhibition of AMPK signaling, though dominant negative (dn) mutation or shRNA/siRNA knockdown of AMPKα1 attenuated ODE-exerted CRC cytotoxicity. In vivo, i.p. administration of ODE inhibited HCT-116 xenograft tumor growth in SCID mice. In addition, AMPK activation, mTORC1 inhibition and p53 activation were observed in ODE-treated HCT-116 xenograft tumors. These results suggest that ODE inhibits CRC cells in vitro and in vivo, possibly via activation of AMPK-dependent signalings.

Metformin Protects Cells from Mutant Huntingtin Toxicity Through Activation of AMPK and Modulation of Mitochondrial Dynamics.

Huntington's disease (HD) is a devastating neurodegenerative disease caused by the pathological elongation of the CAG repeats in the huntingtin gene. Caloric restriction (CR) has been the most reproducible environmental intervention to improve health and prolong life span. We have demonstrated that CR delayed onset and slowed disease progression in a mouse model of HD. Metformin, an antidiabetic drug, mimics CR by acting on cell metabolism at multiple levels. Long-term administration of metformin improved health and life span in mice. In this study, we showed that metformin rescued cells from mutant huntingtin (HTT)-induced toxicity, as indicated by reduced lactate dehydrogenase (LDH) release from cells and preserved ATP levels in cells expressing mutant HTT. Further mechanistic study indicated that metformin activated AMP-activated protein kinase (AMPK) and that inhibition of AMPK activation reduced its protective effects on mutant HTT toxicity, suggesting that AMPK mediates the protection of metformin in HD cells. Furthermore, metformin treatment prevented mitochondrial membrane depolarization and excess fission and modulated the disturbed mitochondrial dynamics in HD cells. We confirmed that metformin crossed the blood-brain barrier after oral administration and activated AMPK in the mouse brain. Our results urge further evaluation of the clinical potential for use of metformin in HD treatment.

Carnosic Acid Prevents Beta-Amyloid-Induced Injury in Human Neuroblastoma SH-SY5Y Cells via the Induction of Autophagy.

Beta-amyloid (Aβ), the hallmark protein in Alzheimer's disease (AD), induces neurotoxicity that involves oxidative stress and mitochondrial dysfunction, leading to cell death. Carnosic acid (CA), a polyphenolic diterpene isolated from the herb rosemary (Rosemarinus officinalis), was investigated in our study to assess its neuroprotective effect and underlying mechanism against Aβ-induced injury in human neuroblastoma SH-SY5Y cells. We found that CA pretreatment alleviated the Aβ25-35-induced loss of cell viability, inhibited both Aβ1-42 accumulation and tau hyperphosphorylation, reduced reactive oxygen species generation, and maintained the mitochondrial membrane potential. Moreover, CA increased the microtubule-associated protein light chain 3 (LC3)-II/I ratio and decreased SQSTM1(p62), indicating that CA could induce autophagy. Autophagy inhibitor 3-methyladenine (3-MA) attenuated the neuroprotective effect of CA, suggesting that autophagy was involved in the neuroprotection of CA. It was also observed that CA activated AMP-activated protein kinase (AMPK) but inhibited mammalian target of rapamycin (mTOR). Furthermore, blocking AMPK with si-AMPKα successfully inhibited the upregulation of LC3-II/I, prevented the downregulation of phosphorylation of mTOR and SQSTM1(p62), indicating that CA induced autophagy in SH-SY5Y cells via the activation of AMPK. These results suggested that CA might be a potential agent for preventing AD.

Resveratrol via activation of LKB1-AMPK signaling suppresses oxidative stress to prevent endothelial dysfunction in diabetic mice

ABSTRACTBackground: Resveratrol, a phytoalexin, is reported to activate AMP-activated protein kinase (AMPK) in vascular cells. Whether resveratrol via activation of AMPK improves endothelial dysfunction in diabetes remains unknown. Here, we reported that activation of AMPK is required for resveratrol-induced improvement of endothelial function in diabetic mice. Methods: AMPK phosphorylation and activity, productions of reactive oxygen species, endothelium-dependent/independent relaxation were determined in vitro or in vivo. Results: Exposure of cultured human umbilical vein endothelial cells (HUVECs) to resveratrol activates AMPK by increasing the thr172 phosphorylation and its activities in time/dose-dependent manner. Loss function of liver kinase B1 by siRNA or mutant abolished resveratrol-induced AMPK activation. Incubation of endothelial cells with high glucose (HG) markedly induced oxidative stress of HUVECs, which was abolished by resveratrol in AMPK-dependent manner. In isolated mice aortas, resveratrol, AICAR or apocynin improved endothelium-dependent relaxation impaired by HG. In animal studies, streptozotocin-induced hyperglycemia dramatically increased oxidative stress and caused endothelial dysfunction, which were reversed by administration of resveratrol. Conclusions: We conclude that AMPK activation is required for resveratrol to improve endothelial function in diabetic mice.

Black ginger extract increases physical fitness performance and muscular endurance by improving inflammation and energy metabolism

We previously reported that polymethoxyflavones (PMFs) in black ginger (Kaempferia parviflora) extract (KPE) increased energy production by activating AMP-activated protein kinase (AMPK) in C2C12 myoblasts. We herein evaluated the effects of KPE on physical fitness performance and muscular endurance in mice. Male mice were orally administered KPE for 4 weeks, and then forced swimming test, open-field test, inclined plane test, and wire hanging test were performed. KPE significantly increased the swimming time, motility after swimming, and grip strength. IL-6 and TNF-α mRNA expression levels were decreased in the soleus muscle, whereas peroxisome proliferator-activated receptor γ coactivator (PGC)-1α and glycogen synthase mRNA expression levels, mitochondrial number, and glycogen content were increased. These results were in agreement with those obtained for KPE and PMFs in C2C12. Therefore, the activation of AMPK by PMFs may be one of the mechanisms by which KPE improves physical fitness performance and muscular endurance.

Inhibition of mTOR by apigenin in UVB-irradiated keratinocytes: A new implication of skin cancer prevention

Ultraviolet B (UVB) radiation is the major environmental risk factor for developing skin cancer, the most common cancer worldwide, which is characterized by aberrant activation of Akt/mTOR (mammalian target of rapamycin). Importantly, the link between UV irradiation and mTOR signaling has not been fully established. Apigenin is a naturally occurring flavonoid that has been shown to inhibit UV-induced skin cancer. Previously, we have demonstrated that apigenin activates AMP-activated protein kinase (AMPK), which leads to suppression of basal mTOR activity in cultured keratinocytes. Here, we demonstrated that apigenin inhibited UVB-induced mTOR activation, cell proliferation and cell cycle progression in mouse skin and in mouse epidermal keratinocytes. Interestingly, UVB induced mTOR signaling via PI3K/Akt pathway, however, the inhibition of UVB-induced mTOR signaling by apigenin was not Akt-dependent. Instead, it was driven by AMPK activation. In addition, mTOR inhibition by apigenin in keratinocytes enhanced autophagy, which was responsible, at least in part, for the decreased proliferation in keratinocytes. In contrast, apigenin did not alter UVB-induced apoptosis. Taken together, our results indicate the important role of mTOR inhibition in UVB protection by apigenin, and provide a new target and strategy for better prevention of UV-induced skin cancer.

Hypoxia promotes drug resistance in osteosarcoma cells via activating AMP-activated protein kinase (AMPK) signaling

PurposeDrug resistance has been recognized to be a major obstacle to the chemotherapy for osteosarcoma. And the potential importance of hypoxia as a target to reverse drug resistance in osteosarcoma has been indicated, though the mechanism underlining such role is not clarified. The present study aims to investigate the role of hypoxia in the drug resistance in osteosarcoma cells via activating AMP-activated protein kinase (AMPK) signaling. Experimental designWe investigated the promotion of the resistance to doxorubicin of osteosarcoma MG-63 and U2-os cells in vitro, and then determined the role of hypoxia-inducible factor-1 (HIF-1)α and HIF-1β, the activation and regulatory role of AMPK in the osteosarcoma U2-os cells which were treated with doxorubicin under hypoxia. ResultsIt was demonstrated that hypoxia significantly reduced the sensitivity of MG-63 and U2-os cells to doxorubicin, indicating an inhibited viability reduction and a reduced apoptosis promotion. And such reduced sensitivity was not associated with HIF-1α, though it was promoted by hypoxia in U2-os cells. Interestingly, the AMPK signaling was significantly promoted by hypoxia in the doxorubicin-treated U2-os cells, with a marked upregulation of phosphorylated AMPK (Thr 172) and phosphorylated acetyl-CoA carboxylase (ACC) (Ser 79), which were sensitive to the AMPK activator, AICAR and the AMPK inhibitor, Compound C. Moreover, the promoted AMPK activity by AICAR or the downregulated AMPK activity by Compound C significantly reduced or promoted the sensitivity of U2-os cells to doxorubicin. ConclusionThe present study confirmed the AMPK signaling activation in the doxorubicin-treated osteosarcoma cells, in response to hypoxia, and the chemical upregulation or downregulation of AMPK signaling reduced or increased the chemo-sensitivity of osteosarcoma U2-os cells in vitro. Our study implies that AMPK inhibition might be a effective strategy to sensitize osteocarcoma cells to chemotherapy.

Copper Ion from Cu2O Crystal Induces AMPK-Mediated Autophagy via Superoxide in Endothelial Cells.

Copper is an essential element required for a variety of functions exerted by cuproproteins. An alteration of the copper level is associated with multiple pathological conditions including chronic ischemia, atherosclerosis and cancers. Therefore, copper homeostasis, maintained by a combination of two copper ions (Cu+ and Cu2+), is critical for health. However, less is known about which of the two copper ions is more toxic or functional in endothelial cells. Cubic-shaped Cu2O and CuO crystals were prepared to test the role of the two different ions, Cu+ and Cu2+, respectively. The Cu2O crystal was found to have an effect on cell death in endothelial cells whereas CuO had no effect. The Cu2O crystals appeared to induce p62 degradation, LC3 processing and an elevation of LC3 puncta, important processes for autophagy, but had no effect on apoptosis and necrosis. Cu2O crystals promote endothelial cell death via autophagy, elevate the level of reactive oxygen species such as superoxide and nitric oxide, and subsequently activate AMP-activated protein kinase (AMPK) through superoxide rather than nitric oxide. Consistently, the AMPK inhibitor Compound C was found to inhibit Cu2O-induced AMPK activation, p62 degradation, and LC3 processing. This study provides insight on the pathophysiologic function of Cu+ ions in the vascular system, where Cu+ induces autophagy while Cu2+ has no detected effect.

Resveratrol attenuates lipopolysaccharide-induced dysfunction of blood-brain barrier in endothelial cells via AMPK activation.

Resveratrol, a phytoalexin, is reported to activate AMP-activated protein kinase (AMPK) in vascular cells. The blood-brain barrier (BBB), formed by specialized brain endothelial cells that are interconnected by tight junctions, strictly regulates paracellular permeability to maintain an optimal extracellular environment for brain homeostasis. The aim of this study was to elucidate the effects of resveratrol and the role of AMPK in BBB dysfunction induced by lipopolysaccharide (LPS). Exposure of human brain microvascular endothelial cells (HBMECs) to LPS (1 µg/ml) for 4 to 24 hours week dramatically increased the permeability of the BBB in parallel with lowered expression levels of occluding and claudin-5, which are essential to maintain tight junctions in HBMECs. In addition, LPS significantly increased the reactive oxygen species (ROS) productions. All effects induced by LPS in HBVMCs were reversed by adenoviral overexpression of superoxide dismutase, inhibition of NAD(P) H oxidase by apocynin or gain-function of AMPK by adenoviral overexpression of constitutively active mutant (AMPK-CA) or by resveratrol. Finally, upregulation of AMPK by either AMPK-CA or resveratrol abolished the levels of LPS-enhanced NAD(P)H oxidase subunits protein expressions. We conclude that AMPK activation by resveratrol improves the integrity of the BBB disrupted by LPS through suppressing the induction of NAD(P)H oxidase-derived ROS in HBMECs.

Anti-Tumor Activity of Yuanhuacine by Regulating AMPK/mTOR Signaling Pathway and Actin Cytoskeleton Organization in Non-Small Cell Lung Cancer Cells.

Yuanhuacine (YC), a daphnane diterpenoid from the flowers of Daphne genkwa, exhibited a potential growth inhibitory activity against human non-small cell lung cancer (NSCLC) cells. YC also suppressed the invasion and migration of lung cancer cells. However, the precise molecular mechanisms remain to be elucidated. In the present study, we report that YC significantly activated AMP-activated protein kinase (AMPK) signaling pathway and suppressed mTORC2-mediated downstream signaling pathway in H1993 human NSCLC cells. AMPK plays an important role in energy metabolism and cancer biology. Therefore, activators of AMPK signaling pathways can be applicable to the treatment of cancer. YC enhanced the expression of p-AMPKα. The co-treatment of YC and compound C (an AMPK inhibitor) or metformin (an AMPK activator) also confirmed that YC increases p-AMPKα. YC also suppressed the activation of the mammalian target of rapamycin (mTOR) expression, a downstream target of AMPK. Further study revealed that YC modulates mTORC2-associated downstream signaling pathways with a decreased expressions of p-Akt, p-protein kinase C alpha (PKCα), p-ras-related C3 botulinum toxin substrate 1 (Rac1) and filamentous actin (F-actin) that are known to activate cell growth and organize actin cytoskeleton. In addition, YC inhibited the tumor growth in H1993 cell-implanted xenograft nude mouse model. These data suggest the YC could be a potential candidate for cancer chemotherapeutic agents derived from natural products by regulating AMPK/mTORC2 signaling pathway and actin cytoskeleton organization.

AMPK activation by liquiritigenin inhibited oxidative hepatic injury and mitochondrial dysfunction induced by nutrition deprivation as mediated with induction of farnesoid X receptor.

Nutrition is indispensable for cell survival and proliferation. Thus, loss of nutrition caused by serum starvation in cells could induce formation of reactive oxygen species (ROS), resulting in cell death. Liquiritigenin (LQ) is an active flavonoid in licorice and plays a role in the liver as a hepatic protectant. This study investigated the effect of LQ, metformin [an activator of activated AMP-activated protein kinase (AMPK)] and GW4064 [a ligand of farnesoid X receptor (FXR)] on mitochondrial dysfunction and oxidative stress induced by serum deprivation as well as its molecular mechanism, as assessed by immunoblot and flow cytometer assays. Serum deprivation in HepG2, H4IIE and AML12 cells successfully induced oxidative stress and apoptosis, as indicated by depletion of glutathione, formation of ROS, and altered expression of apoptosis-related proteins such as procaspase-3, poly(ADP-ribose) polymerase, and Bcl-2. However, LQ pretreatment significantly blocked these pathological changes and mitochondrial dysfunction caused by serum deprivation. Moreover, LQ activated AMPK in HepG2 cells and mice liver, as shown by phosphorylation of AMPK and ACC, and this activation was mediated by its upstream kinase (i.e., LKB1). Experiments using a chemical inhibitor of AMPK with LKB1-deficient Hela cells revealed the role of the LKB1-AMPK pathway in cellular protection conferred by LQ. LQ also induced protein and mRNA expression of both FXR as well as small heterodimer partner, which is important since treatment with FXR ligand GW4064 protected hepatocytes against cell death and mitochondrial damage induced by serum deprivation. AMPK activators such as LQ can protect hepatocytes against oxidative hepatic injury and mitochondrial dysfunction induced by serum deprivation, and the beneficial effect might be mediated through the LKB1 pathway as well as FXR induction.

Apoptotic Cells Activate AMP-activated Protein Kinase (AMPK) and Inhibit Epithelial Cell Growth without Change in Intracellular Energy Stores

Apoptosis plays an indispensable role in the maintenance and development of tissues. We have shown that receptor-mediated recognition of apoptotic target cells by viable kidney proximal tubular epithelial cells (PTECs) inhibits the proliferation and survival of PTECs. Here, we examined the effect of apoptotic targets on PTEC cell growth (cell size during G1 phase of the cell cycle). Using a cell culture model, we show that apoptotic cells potently activate AMP-activated protein kinase (AMPK), a highly sensitive sensor of intracellular energy stores. AMPK activation leads to decreased activity of its downstream target, ribosomal protein p70 S6 kinase (p70S6K), and concomitant inhibition of cell growth. Importantly, these events occur without detectable change in intracellular levels of AMP, ADP, or ATP. Inhibition of AMPK, either pharmacologically by compound C or molecularly by shRNA, diminishes the effects of apoptotic targets and largely restores p70S6K activity and cell size to normal levels. Apoptotic targets also inhibit Akt, a second signaling pathway regulating cell growth. Expression of a constitutively active Akt construct partially relieved cell growth inhibition but was less effective than inhibition of AMPK. Inhibition of cell growth by apoptotic targets is dependent on physical interaction between apoptotic targets and PTECs but independent of phagocytosis. We conclude that receptor-mediated recognition of apoptotic targets mimics the effects of intracellular energy depletion, activating AMPK and inhibiting cell growth. By acting as sentinels of environmental change, apoptotic death may enable nearby viable cells, especially nonmigratory epithelial cells, to monitor and adapt to local stresses.

Salidroside improves endothelial function and alleviates atherosclerosis by activating a mitochondria-related AMPK/PI3K/Akt/eNOS pathway

Salidroside (SAL) is a phenylpropanoid glycoside isolated from the medicinal plant Rhodiola rosea. A recent study has reported that SAL can efficiently decrease atherosclerotic plaque formation in low-density lipoprotein receptor-deficient mice. This study was to investigate the molecular mechanism of antiatherogenic effects of SAL. Given the importance of endothelial nitric oxide synthase (eNOS) in atherosclerosis, we sought to elucidate whether SAL could stimulate eNOS activation and also to explore its upstream signaling pathway. Six-week old apoE−/− male mice were fed a high-fat diet for 8weeks and then were administered with SAL for another 8weeks. SAL significantly improved endothelial function associated with increasing eNOS activation, thus reduced the atherosclerotic lesion area. SAL increased eNOS-Ser1177 phosphorylation and decreased eNOS-Thr495 phosphorylation, indicative of eNOS activation in endothelium. The aortic sinus lesions in SAL treated mice displayed reduced inflammation. SAL significantly activated AMP-activated protein kinase (AMPK). Both AMPK inhibitor and AMPK small interfering RNA (siRNA) abolished SAL-induced Akt-Ser473 and eNOS-Ser1177 phosphorylation. In contrast, LY294002, the PI3k/Akt pathway inhibitor, abolished SAL-induced phosphorylation and expression of eNOS. High performance liquid chromatography (HPLC) analysis revealed that SAL decreased cellular ATP content and increased the cellular AMP/ATP ratio, which was associated with the activation of AMPK. SAL was found to decrease the mitochondrial membrane potential (ΔΨm), which is a likely consequence of reduced ATP production. The action of SAL to reduce atherosclerotic lesion formation may at least be attributed to its effect on improving endothelial function by promoting nitric oxide (NO) production, which was associated with mitochondrial depolarization and subsequent activation of the AMPK/PI3K/Akt/eNOS pathway. Taken together, our data described the effects of SAL on mitochondria, which played critical roles in improving endothelial function in atherosclerosis.

Resveratrol via activation of AMPK lowers blood pressure in DOCA-salt hypertensive mice

Objective: Resveratrol, a phytoalexin, is reported to activate AMP-activated protein kinase (AMPK) in vascular cells. Activation of AMPK induces vasorelaxation to lower blood pressure (BP). Whether resveratrol via activation of AMPK decreases, BP remains unknown. Methods: Male wild-type (WT) mice and mice deficient in AMPKα2 (AMPKα2−/−) were fed with resveratrol (400 mg/kg). After 7 d, mice were implanted with deoxycorticosterone acetate (DOCA)-salt (150 mg/kg) for 35 d. BP was detected by the radiotelemetry method. Vessel contraction was determined by organ chamber. Active RhoA, Rho-associated kinase (ROCK) activity, phosphorylations of myosin light chain (MLC), and myosin phosphatase targeting subunit 1 (MYPT1) were assayed by western blot. Results: Implantation of DOCA-salt dramatically increased systemic BPs (systolic BP and diastolic BP) in WT and AMPKα2−/− mice. However, treatment of resveratrol significantly decreased systemic BP in WT mice but not in AMPKα2−/− mice. In the organ chamber study, resveratrol inhibited agonist-induced vessel relaxation in WT mice aortas. Loss of AMPKα2 or AMPK inhibition by compound C reversed resveratrol-suppressed vasoconstriction in isolated mice aortas. In cultured vascular smooth muscle cells (VSMCs), activation of AMPK by resveratrol inhibited phenylephrine-enhanced MLC phosphorylation in a dose-dependent manner. Conclusions: Resveratrol via activation of AMPK lowers BP in DOCA-hypertensive mice through an AMPK/RhoA/ROCK2/MLCMLC pathway.

Regulation of Pancreatic β Cell Mass by Cross-Interaction between CCAAT Enhancer Binding Protein β Induced by Endoplasmic Reticulum Stress and AMP-Activated Protein Kinase Activity.

During the development of type 2 diabetes, endoplasmic reticulum (ER) stress leads to not only insulin resistance but also to pancreatic beta cell failure. Conversely, cell function under various stressed conditions can be restored by reducing ER stress by activating AMP-activated protein kinase (AMPK). However, the details of this mechanism are still obscure. Therefore, the current study aims to elucidate the role of AMPK activity during ER stress-associated pancreatic beta cell failure. MIN6 cells were loaded with 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) and metformin to assess the relationship between AMPK activity and CCAAT enhancer binding protein β (C/EBPβ) expression levels. The effect of C/EBPβ phosphorylation on expression levels was also investigated. Vildagliptin and metformin were administered to pancreatic beta cell-specific C/EBPβ transgenic mice to investigate the relationship between C/EBPβ expression levels and AMPK activity in the pancreatic islets. When pancreatic beta cells are exposed to ER stress, the accumulation of the transcription factor C/EBPβ lowers the AMP/ATP ratio, thereby decreasing AMPK activity. In an opposite manner, incubation of MIN6 cells with AICAR or metformin activated AMPK, which suppressed C/EBPβ expression. In addition, administration of the dipeptidyl peptidase-4 inhibitor vildagliptin and metformin to pancreatic beta cell-specific C/EBPβ transgenic mice decreased C/EBPβ expression levels and enhanced pancreatic beta cell mass in proportion to the recovery of AMPK activity. Enhanced C/EBPβ expression and decreased AMPK activity act synergistically to induce ER stress-associated pancreatic beta cell failure.

Theaflavin-3,3′-digallate, a black tea polyphenol, stimulates lipolysis associated with the induction of mitochondrial uncoupling proteins and AMPK–FoxO3A–MnSOD pathway in 3T3-L1 adipocytes

Phytochemicals have gained an immense interest in obesity management. Previously, we have shown that theaflavin-3,3′-digallate (TF3), a black tea polyphenol, prevents adipocyte-triggered metaflammation. Here, we demonstrate that TF3 attenuates triacylglycerol accumulation in adipocytes, concomitant with promoting gene expression profile that favors lipolysis and β-oxidation, and inducing energy dissipation-related genes, mitochondrial uncoupling protein-1 (UCP-1) and UCP-2. The gene expression is in line with the upregulation of peroxisome proliferator-activated receptor α (PPARα), a primary transactivator for the expression of lipolytic genes. TF3 activates AMP-activated protein kinase (AMPK), which is required for TF3 effects of PPARα upregulation, and the reversal of the inactivation of Forkhead-box-O 3A (FoxO3A) and insulin-induced suppression of manganese superoxide dismutase (MnSOD). The role of MnSOD in adipogenesis is verified in MnSOD-overexpressing adipocytes. Thus, our results demonstrate TF3 as a potent AMPK activator with anti-adiposity activity in adipocytes, suggesting its potential application in functional foods and nutraceuticals for obesity management.

Salidroside ameliorates insulin resistance through activation of a mitochondria-associated AMPK/PI3K/Akt/GSK3β pathway.

Recent reports have suggested that salidroside could protect cardiomyocytes from oxidative injury and stimulate glucose uptake in skeletal muscle cells by activating AMP-activated protein kinase (AMPK). The aim of this study was to evaluate the therapeutic effects of salidroside on diabetic mice and to explore the underlying mechanisms. The therapeutic effects of salidroside on type 2 diabetes were investigated. Increasing doses of salidroside (25, 50 and 100 mg·kg(-1) ·day(-1)) were administered p.o. to db/db mice for 8 weeks. Biochemical analysis and histopathological examinations were conducted to evaluate the therapeutic effects of salidroside. Primary cultured mouse hepatocytes were used to further explore the underlying mechanisms in vitro. Salidroside dramatically reduced blood glucose and serum insulin levels and alleviated insulin resistance. Hypolipidaemic effects and amelioration of liver steatosis were observed after salidroside administration. In vitro, salidroside dose-dependently induced an increase in the phosphorylations of AMPK and PI3K/Akt, as well as glycogen synthase kinase 3β (GSK3β) in hepatocytes. Furthermore, salidroside-stimulated AMPK activation was found to suppress the expression of PEPCK and glucose-6-phosphatase. Salidroside-induced AMPK activation also resulted in phosphorylation of acetyl CoA carboxylase, which can reduce lipid accumulation in peripheral tissues. In isolated mitochondria, salidroside inhibited respiratory chain complex I and disturbed oxidation/phosphorylation coupling and moderately depolarized the mitochondrial membrane potential, resulting in a transient increase in the AMP/ATP ratio. Salidroside exerts an antidiabetic effect by improving the cellular metabolic flux through the activation of a mitochondria-related AMPK/PI3K/Akt/GSK3β pathway.

LKB1/Mo25/STRAD Uniquely Impacts Sarcomeric Contractile Function and Posttranslational Modification

The myocardium undergoes extensive metabolic and energetic remodeling during the progression of cardiac disease. Central to remodeling are changes in the adenine nucleotide pool. Fluctuations in these pools can activate AMP-activated protein kinase (AMPK), the central regulator of cellular energetics. Binding of AMP to AMPK not only allosterically activates AMPK but also promotes phosphorylation of AMPK by an upstream kinase complex, LKB1/Mo25/STRAD (liver kinase B 1, mouse protein 25, STE-related adaptor protein). AMPK phosphorylation by the LKB1 complex results in a substantial increase in AMPK activity. Molecular targeting by the LKB1 complex depends on subcellular localization and transcriptional expression. Yet, little is known about the ability of the LKB1 complex to modulate targeting of AMPK after activation. Accordingly, we hypothesized that differing stoichiometric ratios of LKB1 activator complex to AMPK would uniquely impact myofilament function. Demembranated rat cardiac trabeculae were incubated with varying ratios of the LKB1 complex to AMPK or the LKB1 complex alone. After incubation, we measured the Ca2+ sensitivity of tension, rate constant for tension redevelopment, maximum tension generation, length-dependent activation, cooperativity, and sarcomeric protein phosphorylation status. We found that the Ca2+ sensitivity of tension and cross-bridge dynamics were dependent on the LKB1 complex/AMPK ratio. We also found that the LKB1 complex desensitizes and suppresses myofilament function independently of AMPK. A phospho-proteomic analysis of myofilament proteins revealed site-specific changes in cardiac Troponin I (cTnI) phosphorylation, as well as a unique distribution of cTnI phosphospecies that were dependent on the LKB1 complex/ AMPK ratio. Fibers treated with the LKB1 complex alone did not alter cTnI phosphorylation or phosphospecies distribution. However, LKB1 complex treatment independent of AMPK increased phosphorylation of myosin-binding protein C. Therefore, we conclude that the LKB1/AMPK signaling axis is able to alter muscle function through multiple mechanisms.

AMPK-activated protein kinase activation by Impatiens balsamina L. is related to apoptosis in HSC-2 human oral cancer cells.

Objective:In the present study, we investigated the efficacy of a methanol extract from Impatiens balsamina L. (MEIB) against HSC-2 human oral cancer cells.Materials and Methods:The anti-cancer efficacies of MEIB were performed by methanethiosulfonate assay, phospho-kinase array, Western blot, 4’-6-diamidino-2-phenylindole staining, trypan blue exclusion assay and 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide assay.Results:MEIB decreased the cell viability of HSC-2 cells. According to phospho-kinase arrays, MEIB markedly activated AMP-activated protein kinase (AMPK) signaling, but inactivated mammalian target of rapamycin signaling. MEIB induced apoptosis as evidenced by activation of caspase-3, poly (ADP-ribose) polymerase cleavage and nuclear condensation. In addition, AMPK activation by two known activators (5-aminoimidazole-4-carboxamide-1-β-ribofuranoside and metformin) decreased cell viability and induced apoptosis. Moreover, MEIB increased the expression levels of mitochondria-related proteins (t-Bid, Bak and Bad), which contributed to the disruption of mitochondrial membrane potential, cytochrome C release and activation of caspase-9. Metformin also increased t-Bid expression and the subsequent release of cytochrome C into the cytosol.Conclusion:These results suggest that MEIB may be of therapeutic value for treating oral cancer and that its mechanism of action occurs through AMPK and t-Bid.

AMP-activated protein kinase regulates autophagic protection against cisplatin-induced tissue injury in the kidney.

Although the nephrotoxicity of cisplatin has been well documented as a major side effect of chemotherapy, the exact mechanism by which prosurvival and apoptotic pathways interplay to determine renal pathology remains elusive. Recent studies suggested that autophagy might serve as an adaptive mechanism to promote cell survival during acute kidney injury (AKI). We have used AKI as a disease model to investigate the mechanism regulating the cytoprotective role of autophagy in cisplatin-induced tissue damage. Pharmacological inhibitors such as chloroquine were used to manipulate autophagy during AKI, and DNA damage was evaluated by using the cellular marker γH2AX. Cisplatin induced extensive DNA damage during AKI. Autophagy activation served as a survival strategy to suppress cisplatin-induced DNA damage in the pathology of AKI both in vitro and in vivo. Interestingly, in the kidney, cisplatin treatment can activate AMP-activated protein kinase (AMPK), a signaling molecule that is also critical for p53-mediated inactivation of mammalian target of rapamycin (mTOR) pathways. As a result, inhibition or knockdown of AMPK can lead to repressed autophagy in cisplatin-induced AKI, resulting in more DNA damage. Activation of AMPK regulates autophagy during cisplatin-induced AKI. Given the fact that p53 can regulate autophagy by inactivating mTOR via AMPK, our results suggest that the p53 pathway may also play a critical role in the pathogenesis of cisplatin-induced renal damage. This study may further our understanding of the physiological roles of autophagy in the pathogenesis of renal injuries, and thus may have pathological implications in the clinical setting.