Search

Background: Oxidative stress and inflammatory cytokines in the hypothalamus paraventricular nucleus (PVN) have been implicated in sympathetic nerve activity and the development of hypertension, but the specific mechanisms underlying their production in the PVN remains to be elucidated. Previous studies have demonstrated that activation of nuclear transcription related factor-2 (Nrf2) in the PVN reduced the production of reactive oxygen species (ROS) and inflammatory mediators. Moreover, AMP-activated protein kinase (AMPK), has been observed to decrease ROS and inflammatory cytokine production when activated in the periphery. 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) is an AMPK agonist. However, little research has been conducted on the role of AMPK in the PVN during hypertension. Therefore, we hypothesized that AICAR in the PVN is involved in regulating AMPK/Nrf2 pathway, affecting ROS and inflammatory cytokine expression, influencing sympathetic nerve activity., Methods: Adult male Sprague-Dawley rats were utilized to induce two-kidney, one-clip (2K1C) hypertension via constriction of the right renal artery. Bilateral PVN was microinjected with either artificial cerebrospinal fluid or AICAR once a day for 4 weeks., Results: Compared to the SHAM group, the PVN of 2K1C hypertensive rats decreased p-AMPK and p-Nrf2 expression, increased Fra-Like, NAD(P)H oxidase (NOX)2, NOX4, tumor necrosis factor-α and interleukin (IL)-1β expression, elevated ROS levels, decreased superoxide dismutase 1 and IL-10 expression, and elevated plasma norepinephrine levels. Bilateral PVN microinjection of AICAR significantly ameliorated these changes., Conclusion: These findings suggest that repeated injection of AICAR in the PVN suppresses ROS and inflammatory cytokine production through the AMPK/Nrf2 pathway, reducing sympathetic nerve activity and improving hypertension., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: The metabolic master-switch AMP-activated protein kinase (AMPK) mediates insulin-independent glucose uptake in muscle and regulates the metabolic activity of brown and beige adipose tissue (BAT). The regulatory AMPKγ3 isoform is uniquely expressed in skeletal muscle and potentially in BAT. Herein, we investigated the role that AMPKγ3 plays in mediating skeletal muscle glucose uptake and whole-body glucose clearance in response to small-molecule activators that act on AMPK via distinct mechanisms. We also assessed whether γ3 plays a role in adipose thermogenesis and browning., Methods: Global AMPKγ3 knockout (KO) mice were generated. A systematic whole-body, tissue, and molecular phenotyping linked to glucose homeostasis was performed in γ3 KO and wild-type (WT) mice. Glucose uptake in glycolytic and oxidative skeletal muscle ex vivo as well as blood glucose clearance in response to small molecule AMPK activators that target the nucleotide-binding domain of the γ subunit (AICAR) and allosteric drug and metabolite (ADaM) site located at the interface of the α and β subunit (991, MK-8722) were assessed. Oxygen consumption, thermography, and molecular phenotyping with a β3-adrenergic receptor agonist (CL-316,243) treatment were performed to assess BAT thermogenesis, characteristics, and function., Results: Genetic ablation of γ3 did not affect body weight, body composition, physical activity, and parameters associated with glucose homeostasis under chow or high-fat diet. γ3 deficiency had no effect on fiber-type composition, mitochondrial content and components, or insulin-stimulated glucose uptake in skeletal muscle. Glycolytic muscles in γ3 KO mice showed a partial loss of AMPKα2 activity, which was associated with reduced levels of AMPKα2 and β2 subunit isoforms. Notably, γ3 deficiency resulted in a selective loss of AICAR-, but not MK-8722-induced blood glucose-lowering in vivo and glucose uptake specifically in glycolytic muscle ex vivo. We detected γ3 in BAT and found that it preferentially interacts with α2 and β2. We observed no differences in oxygen consumption, thermogenesis, morphology of BAT and inguinal white adipose tissue (iWAT), or markers of BAT activity between WT and γ3 KO mice., Conclusions: These results demonstrate that γ3 plays a key role in mediating AICAR- but not ADaM site binding drug-stimulated blood glucose clearance and glucose uptake specifically in glycolytic skeletal muscle. We also showed that γ3 is dispensable for β3-adrenergic receptor agonist-induced thermogenesis and browning of iWAT., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Objective: To investigate the efficacy and safety of the AMPK activator AICAR alone or in combination with decitabine on myelodysplastic syndromes (MDS)., Results: p-AMPK (Thr172) expression was lower in MDS samples than in healthy donors. AMPK agonist AICAR inhibited the proliferation of MDS cell lines (SKM1 and MDS-L) (P < 0.05). The results from flow cytometry suggested that AICAR induced G0/G1 phase arrest and apoptosis through inducing DNA damage, as confirmed by immunofluorescence analysis in MDS cell lines. AICAR alone or in combination with decitabine was applied to the two MDS cell lines, and the combination index values at all concentrations were significantly < 1. This strong synergistic effect was also corroborated in the primary MDS patient samples and in an MDS cell line xenograft mouse model. Furthermore, immunohistochemical staining showed that there was more DNA damage accumulation in the combination group than that in any other groups., Conclusion: This is the first report on how the AICAR suppresses MDS cell proliferation and synergizes with decitabine via DNA damage induction. AICAR in combination with decitabine may be a promising therapeutic strategy in MDS.

The fungicide Iprodione is widely applied in vegetables and raises concern for human health. The A549 human lung carcinoma cell line is a suitable model for assessing the toxicological effects of drugs. The goal of this work was to evaluate the genotoxicity and oxidative stress in the A549 cell line exposed to sublethal concentrations from 3 to 100 µg/mL Iprodione considering LC50 = 243.4 µg/mL Iprodione, as determined by the MTT assay. Generalized Linear Mixed Models (GLMM) were performed to determine the association between the responses NDI, MNi m and MNi b and the explanatory variables. Iprodione and solvent were relativized to the control whereas the concentration was included as numeric variable. ANOVA was used for the comparison of treatments. The coefficients of linear association between the explanatory variables and NDI, and the coefficients of logistic association between explanatory variables and MNi m were not significant. However, these coefficients showed significant association with MNi b only for Iprodione treatment but not for Iprodione concentration, indicating lack of dose-response relationship. Genotoxicity risk assessment indicated that the increase in Iprodione concentrations increased slightly the probability of belonging to the genotoxic category. ANOVA showed significant differences in MNi b , and non-significant differences in NDI and MNi m among treatments. The oxidative stress analysis performed at 3, 12, and 25 μg/mL Iprodione showed a significant and linear increase in SOD, and a significant and linear decrease in GSH and GST. The Dunnett test was significant for GSH at 12 and SOD at 25 μg/mL.

A novel nanocomposite poly(ethylene-co-vinyl acetate) (EVA) film with controlled in vitro release of iprodione (ID) was prepared. Chitosan (CS) was used as the reinforcement which enhances the water and oxygen permeability of films. ID loaded poly(ethylene glycol)-poly(ε-caprolactone) (PEG-PCL) (IPP) micelles were used as the drug carrier which endows the films with antifungal and controlled release ability. IPP micelles with spherical shape and uniform size were obtained, and the maximum encapsulation efficacy (EE) was 91.17 ± 5.03% by well controlling the feeding amount of ID. Incorporation CS could improve the oxygen and moisture permeability of films, and the maximum oxygen permeability (OP) and water vapor transmission rate (WVTR) were 477.84 ± 13.03 cc/(m 2 ·d·0.1 MPa) and 8.60 ± 0.25 g m -2 d -1 , respectively. After loading IPP micelles, the films showed an improved antifungal ability and temperature-sensitive drug release behavior, and were found to enhance the quality of grapes by pre-harvest spraying., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

High consumption of saturated fats links to the development of hypertension. AMP-activated protein kinase (AMPK), a nutrient-sensing signal, is involved in the pathogenesis of hypertension. We examined whether early intervention with a direct AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR) during pregnancy or lactation can protect adult male offspring against hypertension programmed by high saturated fat consumption via regulation of nutrient sensing signals, nitric oxide (NO) pathway, and oxidative stress. Pregnant Sprague-Dawley rats received regular chow or high saturated fat diet (HFD) throughout pregnancy and lactation. AICAR treatment was introduced by intraperitoneal injection at 50 mg/kg twice a day for 3 weeks throughout the pregnancy period (AICAR/P) or lactation period (AICAR/L). Male offspring ( n = 7-8/group) were assigned to five groups: control, HFD, AICAR/P, HFD + AICAR/L, and HFD + AICAR/P. Male offspring were killed at 16 weeks of age. HFD caused hypertension and obesity in male adult offspring, which could be prevented by AICAR therapy used either during pregnancy or lactation. As a result, we demonstrated that HFD downregulated AMPK/SIRT1/PGC-1α pathway in offspring kidneys. In contrast, AICAR therapy in pregnancy and, to a greater extent, in lactation activated AMPK signaling pathway. The beneficial effects of AICAR therapy in pregnancy is related to restoration of NO pathway. While AICAR uses in pregnancy and lactation both diminished oxidative stress induced by HFD. Our results highlighted that pharmacological AMPK activation might be a promising strategy to prevent hypertension programmed by excessive consumption of high-fat food., Competing Interests: The authors declare no conflict of interest.

AMP-activated protein kinase (AMPK) is a member of Ser/Thr kinases that has been shown to regulate energy balance. Although recent studies have demonstrated the function of AMPK in adipose tissue using different fat-specific AMPK knockout mouse models, the results were somewhat inconsistent. For this study, we tested the hypothesis that AMPK in adipose tissue regulates whole body glucose metabolism. To determine the role of adipose tissue AMPK in vivo, we generated fat-specific AMPKα1/α2 knockout mice (AMPKFKO) using the Cre-loxP system. Body weights of AMPKFKO mice were not different between 8 and 27 weeks of age. Furthermore, tissue weights (liver, kidney, muscle, heart and white and brown adipose tissue) were similar to wild type littermates and DEXA scan analysis revealed no differences in percentages of body fat and lean mass. Knockout of AMPKα1/α2 in adipose tissue abolished basal and AICAR-stimulated phosphorylation of AMPK and Acetyl-CoA Carboxylase, a downstream of AMPK. Despite of the ablation of AICAR-stimulated AMPK phosphorylation, the blood glucose-lowering effect of AICAR injection (i.p.) was normal in AMPKFKO mice. In addition, AMPKFKO displayed normal fasting blood glucose concentration, glucose tolerance, insulin tolerance, and insulin signaling, indicating normal whole body glucose metabolism. These data demonstrate that adipose tissue AMPK plays a minimum role in whole body glucose metabolism on a chow diet., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Many motor disorders are associated with depolarization of the membrane of skeletal muscle fibers due to the impaired functioning of Na,K-ATPase. Here, we studied the role of ouabain (specific Na,K-ATPase ligand) and AMP-activated protein kinase (key regulator of muscle metabolism) in the maintenance of muscle electrogenesis; the levels of these endogenous factors are directly related to the motor activity. After 4-day intraperitoneal administration of ouabain (1 µg/kg daily), a hyperpolarization of sarcolemma was registered in isolated rat diaphragm muscles due to an increase in the electrogenic activity of Na,K-ATPase. In acute experiments, addition of nanomolar ouabain concentrations to the bathing solution resulted in the muscle membrane hyperpolarization within 15 min. The effect of ouabain reversed to membrane depolarization with the increase in the external potassium concentration. It is possible that Na,K-ATPase activation by ouabain may be regulated by such factors as specific subcellular location, interaction with molecular partners, and changes in the ionic balance. Preventive administration of the AMP-activated protein kinase activator AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside; 400 mg/kg body weight daily for 7 days) in chronic experiments resulted in the stabilization of the endplate structure and abolishment of depolarization of the rat soleus muscle membrane caused by the motor activity cessation. The obtained data can be useful for creating approaches for correction of muscle dysfunction, especially at the early stages, prior to the development of muscle atrophy.

Objective: Adenosine monophosphate-activated protein kinase (AMPK) is a cellular energy sensor whose phosphorylation increases energy production. We sought to evaluate the placenta-specific effect of AMPK activation on the handling of nutrients required for fetal development., Methods: Explants were isolated from term placenta of 29 women (pregravid body mass index: 29.1 ± 9.9 kg/m 2 ) and incubated for 24 hours with 0 to 100 µmol/L resveratrol or 0 to 1 mmol/L of 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR). Following treatment, uptake and metabolism of radiolabeled fatty acids and glucose were measured. Phosphorylation of AMPK was measured by Western blotting. Adenosine diphosphate (ATP) production was assessed using the mitochondrial ToxGlo assay kit. P < .05 was considered statistically significant., Results: Resveratrol and AICAR increased AMPK phosphorylation in human placental explants. Exposure to resveratrol decreased the uptake of polyunsaturated fatty acids, arachidonic acid, and docosahexaenoic acid at 100 µmol/L ( P < .0001). Fatty acid oxidation was decreased by 100 µmol/L ( P < .05) resveratrol, while esterification was unchanged. Resveratrol decreased glucose uptake at the 50 and 100 µmol/L doses ( P < .05). Glycolysis was not significantly affected. AICAR had similar effects, decreasing fatty acid uptake and glycolysis ( P < .05). Production of ATP declined at doses found to decrease nutrient metabolism ( P < .05)., Conclusions: Activation of AMPK in the human placenta leads to global downregulation of metabolism, with mitotoxicity induced at the doses of resveratrol and AICAR used to activate AMPK. Although activation of this pathway has positive metabolic effects on other tissues, in the placenta there is potential for harm, as inadequate placental delivery of critical nutrients may compromise fetal development.

Premature baboons exhibit peripheral insulin resistance and impaired insulin signaling. 5' AMP-activated protein kinase (AMPK) activation improves insulin sensitivity by enhancing glucose uptake (via increased glucose transporter type 4 [GLUT4] translocation and activation of the extracellular signal-regulated kinase [ERK]/ atypical protein kinase C [aPKC] pathway), and increasing fatty acid oxidation (via inhibition of acetyl-CoA carboxylase 1 [ACC]), while downregulating gluconeogenesis (via induction of small heterodimer partner [SHP] and subsequent downregulation of the gluconeogenic enzymes: phosphoenolpyruvate carboxykinase [PEPCK], glucose 6-phosphatase [G6PASE], fructose- 1,6-bisphosphatase 1 [FBP1], and forkhead box protein 1 [FOXO1]). The purpose of this study was to investigate whether pharmacologic activation of AMPK with AICAR (5-aminoimidazole-4-carboximide riboside) administration improves peripheral insulin sensitivity in preterm baboons. 11 baboons were delivered prematurely at 125±2 days (67%) gestation. 5 animals were randomized to receive 5 days of continuous AICAR infusion at a dose of 0.5 mg·g-1·day-1. 6 animals were in the placebo group. Euglycemic hyperinsulinemic clamps were performed at 5±2 and 14±2 days of life. Key molecules potentially altered by AICAR (AMPK, GLUT4, ACC, PEPCK, G6PASE, FBP1, and FOXO1), and the insulin signaling molecules: insulin receptor (INSR), insulin receptor substrate 1 (IRS-1), protein kinase B (AKT), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) were measured using RT-PCR and western blotting. AICAR infusion did not improve whole body insulin-stimulated glucose disposal in preterm baboons (12.8±2.4 vs 12.4±2.0 mg/(kg·min), p = 0.8, placebo vs AICAR). One animal developed complications during treatment. In skeletal muscle, AICAR infusion did not increase phosphorylation of ACC, AKT, or AMPK whereas it increased mRNA expression of ACACA (ACC), AKT, and PPARGC1A (PGC1α). In the liver, INSR, IRS1, G6PC3, AKT, PCK1, FOXO1, and FBP1 were unchanged, whereas PPARGC1A mRNA expression increased after AICAR infusion. This study provides evidence that AICAR does not improve insulin sensitivity in premature euglycemic baboons, and may have adverse effects., Competing Interests: The authors have declared that no competing interests exist.

Myotonic dystrophy type 1 (DM1) is a debilitating multisystemic disorder caused by a triplet repeat expansion in the 3' untranslated region of dystrophia myotonica protein kinase mRNAs. Mutant mRNAs accumulate in the nucleus of affected cells and misregulate RNA-binding proteins, thereby promoting characteristic missplicing events. However, little is known about the signaling pathways that may be affected in DM1. Here, we investigated the status of activated protein kinase (AMPK) signaling in DM1 skeletal muscle and found that the AMPK pathway is markedly repressed in a DM1 mouse model (human skeletal actin-long repeat, HSALR) and patient-derived DM1 myoblasts. Chronic pharmacological activation of AMPK signaling in DM1 mice with 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) has multiple beneficial effects on the DM1 phenotype. Indeed, a 6-week AICAR treatment of DM1 mice promoted expression of a slower, more oxidative phenotype, improved muscle histology and corrected several events associated with RNA toxicity. Importantly, AICAR also had a dose-dependent positive effect on the spliceopathy in patient-derived DM1 myoblasts. In separate experiments, we also show that chronic treatment of DM1 mice with resveratrol as well as voluntary wheel running also rescued missplicing events in muscle. Collectively, our findings demonstrate the therapeutic potential of chronic AMPK stimulation both physiologically and pharmacologically for DM1 patients.

Insulin degrading enzyme (IDE) is believed to act as a junction point of Type 2 diabetes (T2D) and Alzheimer's disease (AD); however, the underlying mechanism was not completely clear yet. Transgenic APPSwe/PS1 mice were used as the AD model and were treated with streptozocin/streptozotocin (STZ) to develop a mixed mice model presenting both AD and T2D. Morris Water Maze (MWM) and recognition task were performed to trace the cognitive function. The detection of fasting plasma glucose (FPG) and plasma insulin concentration, and oral glucose tolerance test (OGTT) were used to trace the metabolism evolution. Aβ40 and Aβ42 were quantified by colorimetric ELISA kits. The mRNA or protein expression levels were determined by quantitative real-time RT-PCR and Western blotting analysis respectively. T2D contributes to the AD progress by accelerating and worsening spatial learning and recognition impairments. Metabolic parameters and glucose tolerance were significantly changed in the presence of the AD and T2D. The expression levels of IDE, PPARγ, and AMPK were down-regulated in mice with AD and T2D. PPARγ activator rosiglitazone (RSZ) or AMPK activator AICAR increased the expression level of IDE and decreased Aβ levels in mice with AD and T2D. RSZ or AICAR treatment also alleviated the spatial learning and recognition impairments in AD and T2D mice. Our results found that, in the mice with T2D and AD, the activators of PPARγ/AMPK signaling pathway significantly increased the expression level of IDE, and decreased the accumulation of Aβ40 and Aβ42, as well as alleviated the spatial learning and recognition impairments., (© 2018 The Author(s).)

Tetrandrine, a bisbenzylisoquinoline alkaloid isolated from the roots of Stephania tetrandra is a traditional Chinese medicine and exerts anticancer capacity in various types of cancers. Previous studies have shown that tetrandrine induces apoptosis in bladder cancer cells via activation of the caspase cascade. However, the underlying mechanism has not yet been reported. Autophagy is a cellular process involved in the degradation of broken proteins and aging organelles to maintain homeostasis. Recent studies indicate that autophagy is implicated in cancer therapy. Thus, we focused on the correlation between autophagy and apoptosis upon tetrandrine treatment in human bladder cancer cells. Firstly, our results observed a marked increase in autophagic double-membrane vacuoles and fluorescent puncta of red fluorescence protein-green fluorescence protein-LC3 (GRP-RFP-LC3) upon tetrandrine treatment, as evidenced by transmission electron microscopy and confocal fluorescence microscopy. Secondly, the expression of LC3-II was increased in tetrandrine-treated T24 and 5637 cells in a time- and concentration-dependent manner. Subsequently, downregulation of p62 and LC3 turnover assay further confirmed that tetrandrine induced autophagic flux in bladder cancer T24 and 5637 cells. Thirdly, the protein levels of phosphorylated-AMP-activated protein kinase (AMPK) and phosphorylated-acetyl-coenzyme A carboxylase (ACC) were upregulated in the tetrandrine-treated cells, while the mammalian target of rapamycin (mTOR)-related proteins were downregulated. Moreover, AICAR, a common AMPK activator, further increased the expression the LC3-II, while AMPK inhibitor compound C partially reversed the LC3-II protein levels in bladder cancer T24 cells. Finally, AICAR significantly reinforced the growth inhibition and apoptosis induction of tetrandrine in T24 and 5637 cells, while compound C had an opposite effect, suggesting that AMPK-mediated autophagy enhanced the cytotoxic and pro-apoptosis effect of tetrandrine in human bladder cancer cells. Taken together, the present study showed that tetrandrine induced autophagy in human bladder cancer cells by regulating the AMPK/mTOR signaling pathway, which contributed to the apoptosis induction by tetrandrine, indicating that tetrandrine may be a potential anticancer candidate for the treatment of bladder cancer, and autophagy may be a possible mechanism for cancer therapy.

SUR2A is an 'atypical' ABC protein that forms sarcolemmal ATP-sensitive K + (K ATP ) channels by binding to inward rectifier Kir6.2. Manipulation with SUR2A levels has been suggested to be a promising therapeutic strategy against ischaemic heart diseases and other diseases where increased heart resistance to stress is beneficial. Some years ago, it has been reported that high-altitude residents have lower mortality rates for ischaemic heart disease. The purpose of this study was to determine whether SUR2A is regulated by mild-to-severe hypoxic conditions (15% oxygen; oxygen tension equivalent to 3000 m above sea level) and elucidate the underlying mechanism. Mice were exposed to either to 21% (control) or 15% concentration of oxygen for 24 hrs. Twenty-four hours long exposure to 15% oxygen decreased partial pressure of O2 (PO 2 ), but did not affect blood CO 2 (PCO 2 ), haematocrit nor levels of ATP, lactate and NAD+/NADH in the heart. Cardiac SUR2A levels were significantly increased while Kir6.2 levels were not affected. Hypoxia did not induce phosphorylation of extracellular signal-regulated kinases (ERK1/2) or protein kinase B (Akt), but triggered phosphorylation of AMP activated protein kinase (AMPK). AICAR, an activator of AMPK, increased the level of SUR2A in H9c2 cells. We conclude that oxygen increases SUR2A level by activating AMPK. This is the first account of AMPK-mediated regulation of SUR2A., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) modulates cellular energy metabolism, and promotes mitochondrial proliferation and apoptosis. Previous studies have shown that AICAR has anticancer effects in various cancers, however the roles of AMPK and/or the effects of AICAR on osteosarcoma have not been reported. In the present study, we evaluated the effects of AICAR on tumor growth and mitochondrial apoptosis in human osteosarcoma both in vitro and in vivo. For in vitro experiments, two human osteosarcoma cell lines, MG63 and KHOS, were treated with AICAR, and the effects of AICAR on cell growth and mitochondrial apoptosis were assessed by WST assays, TUNEL staining, and immunoblot analyses. In vivo, human osteosarcoma-bearing mice were treated with AICAR, and the mitochondrial proliferation and apoptotic activity in treated tumors were assessed. In vitro experiments revealed that AICAR activated AMPK, inhibited cell growth, and induced mitochondrial apoptosis in both osteosarcoma cell lines. In vivo, AICAR significantly reduced osteosarcoma growth without apparent body weight loss and AICAR increased both mitochondrial proliferation and apoptotic activity in treated tumor tissues. AICAR showed anticancer effects in osteosarcoma cells through an AMPK-dependent peroxisome proliferator‑activated receptor-γ coactivator-1α (PGC-1α)/mitochondrial transcription factor A (TFAM)/mitochondrial pathway. The findings in this study strongly suggest that AICAR could be considered as a potent therapeutic agent for the treatment of human osteosarcoma.

Purpose: Resistance to chemotherapy is one of the major problems facing current cancer research. Enhancing tumor cell response to anticancer agents increases chemotherapeutic effectiveness. We have recently addressed this issue and reported on producing multifunctional nanoparticles (Fe 3 O 4 @SiO 2 (FITC)-FA/AICAR/DOX) aiming to overcome chemoresistance with synergetic effect of AICAR and DOX. In the present study, we demonstrated that these nanoparticles not only show enhanced cellular uptake and cytotoxic effect but can also show enhanced pro-apoptotic and anti-proliferative effects in five different tumor-derived cell lines (A549, HCT-116, HeLa, Jurkat and MIA PaCa-2)., Methods: The nanoparticles were examined by using flow cytometric analyses of apoptosis and cell cycle. In addition, we performed caspase-3 activity assay, which supported our flow cytometric data. Furthermore, we demonstrated the applicability of this approach in a variety of cancer types confirming the potential widespread utility of this approach., Results: With the concept of co-delivery of AICAR and DOX in the nanoparticle formulation, the use of AICAR against survivin (BIRC5) sensitized cancer cells to DOX chemotherapy which resulted in effective cancer cell elimination. These result showed that combination therapy involving both a molecularly targeted therapy and chemotherapeutic agent has the ability to retain and enhance therapeutic efficacy., Conclusion: Fe 3 O 4 @SiO 2 (FITC)-FA/AICAR/DOX nanoparticles is superior to monotherapy via the synergetic effect of AICAR and DOX and also the nanoparticle formulation could overcome issues of toxicity with targeted therapy while maintaining the potent anticancer effects of AICAR and DOX. Graphical Abstract Apoptosis analysis of A549 cells by flow cytometry-based PE-annexin-V / 7-ADD double staining treated with low-dose (10 μg/ml) concentration of (1) Fe3O4@SiO2(FITC)-FA (2) Fe3O4@SiO2(FITC)-FA/AICAR, (3) Fe3O4@SiO2(FITC)-FA/DOX or (4) Fe3O4@SiO2(FITC)-FA/AICAR/DOX nanoparticles. Viable cells labelled with PE-annexin-V(-)/7-ADD(-), early apoptotic cells labelled with PE-annexin-V(+)/7-ADD(-) and apoptotic cells labelled with PE-annexin-V(+)/ 7-ADD(+) in flow cytometric graphics.

A series of poly(ADP-ribose)polymerase (PARP)-1 inhibitors containing a novel scaffold, the 1H-thieno[3,4-d]imidazole-4-carboxamide moiety, was designed and synthesized. These efforts provided some compounds with relatively good PARP-1 inhibitory activity, and among them, 16l was the most potent one. Cellular evaluations indicated that the anti-proliferative activities of 16g, 16i, 16j and 16l against BRCA-deficient cell lines were similar to that of olaparib, while the cytotoxicities of 16j and 16l toward human normal cells were lower. In addition, ADMET prediction results indicated that these compounds might possess more favorable toxicity and pharmacokinetic properties. This study provides a basis for our further investigation.

AICAR (5-Aminoimidazole-4-carboxamide riboside or acadesine) is an AMP-activated protein kinase (AMPK) agonist, which induces cytotoxic effect to several cancer cells. Its potential activity in prostate cancer cells and the underlying signaling mechanisms have not been extensively studied. Here, we showed that AICAR primarily induced programmed necrosis, but not apoptosis, in prostate cancer cells (LNCaP, PC-3 and PC-82 lines). AICAR's cytotoxicity to prostate cancer cells was largely attenuated by the necrosis inhibitor necrostatin-1. Mitochondrial protein cyclophilin-D (CYPD) is required for AICAR-induced programmed necrosis. CYPD inhibitors (cyclosporin A and sanglifehrin A) as well as CYPD shRNAs dramatically attenuated AICAR-induced prostate cancer cell necrosis and cytotoxicity. Notably, AICAR-induced cell necrosis appeared independent of AMPK, yet requiring reactive oxygen species (ROS) production. ROS scavengers (N-acetylcysteine and MnTBAP), but not AMPKα shRNAs, largely inhibited prostate cancer cell necrosis and cytotoxicity by AICAR. In summary, the results of the present study demonstrate mechanistic evidences that AMPK-independent programmed necrosis contributes to AICAR's cytotoxicity in prostate cancer cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Diabetes is the seventh leading cause of death in the USA, and disruption of circadian rhythms is gaining recognition as a contributing factor to disease prevalence. This disease is characterized by hyperglycemia and glucose intolerance and symptoms caused by failure to produce and/or respond to insulin. The skeletal muscle is a key insulin-sensitive metabolic tissue, taking up ~80 % of postprandial glucose. To address the role of the skeletal muscle molecular clock to insulin sensitivity and glucose tolerance, we generated an inducible skeletal muscle-specific Bmal1 (-/-) mouse (iMSBmal1 (-/-))., Results: Progressive changes in body composition (decreases in percent fat) were seen in the iMSBmal1 (-/-) mice from 3 to 12 weeks post-treatment as well as glucose intolerance and non-fasting hyperglycemia. Ex vivo analysis of glucose uptake revealed that the extensor digitorum longus (EDL) muscles did not respond to either insulin or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) stimulation. RT-PCR and Western blot analyses demonstrated a significant decrease in mRNA expression and protein content of the muscle glucose transporter (Glut4). We also found that both mRNA expression and activity of two key rate-limiting enzymes of glycolysis, hexokinase 2 (Hk2) and phosphofructokinase 1 (Pfk1), were significantly reduced in the iMSBmal1 (-/-) muscle. Lastly, results from metabolomics analyses provided evidence of decreased glycolytic flux and uncovered decreases in some tricarboxylic acid (TCA) intermediates with increases in amino acid levels in the iMSBmal1 (-/-) muscle. These findings suggest that the muscle is relying predominantly on fat as a fuel with increased protein breakdown to support the TCA cycle., Conclusions: These data support a fundamental role for Bmal1, the endogenous circadian clock, in glucose metabolism in the skeletal muscle. Our findings have implicated altered molecular clock dictating significant changes in altered substrate metabolism in the absence of feeding or activity changes. The changes in body composition in our model also highlight the important role that changes in skeletal muscle carbohydrate, and fat metabolism can play in systemic metabolism.

Studies have showed that diabetes is one of the high risk factors of endometrial cancer; however, no reports describe the anti- or pro-cancer effect of a new kind of anti-diabetes drug, glucagon-like peptide-1 receptor agonist exenatide (exendin-4), on endometrial cancer. To investigate whether exenatide promotes or inhibits the growth of endometrial cancer, we used the subcutaneous human endometrial cancer cell Ishikawa xenografts in nude mouse model, and divided them into control group and exenatide-treated group. The tumor growth rate in exenatide group was slower than that in control group, and the apoptosis rate of exenatide group was higher than that in control group. In vitro, exendin-4 also attenuated Ishikawa cell viability and clone formation rate, but promoted cell apoptosis. There was an increase of phosphorylated-AMPK protein, a decrease of phosphorylated-mTOR protein both in vivo and in vitro after exenatide or exendin-4 treatment. Moreover, when treated with exendin-4 plus AICAR, an AMPK activator, cell apoptosis increased with higher ratio of phosphorylayed-AMPK/AMPK, lower ratio of phosphorylated-mTOR/mTOR and higher expression of cleaved caspase-3 than those in exendin-4 alone group, and the results were the opposite when treated with exendin-4 plus compound C, an AMPK inhibitor. Our results suggest that exenatide could attenuate the growth of endometrial cancer Ishikawa xenografts in nude mice, and AMPK may be the target of the mechanism.

Cancer cells are characterized by metabolic alterations, namely, depressed mitochondrial oxidation, enhanced glycolysis and pentose phosphate shunt flux to support rapid cell growth, which is called the Warburg effect. In our study we assessed the metabolic consequences of a joint treatment of MCF-7 breast cancer cells with AICAR, an inducer of AMP-activated kinase (AMPK) jointly with methotrexate (MTX), a folate-analog antimetabolite that blunts de novo nucleotide synthesis. MCF7 cells, a model of breast cancer cells, were resistant to the individual application of AICAR or MTX, however combined treatment of AICAR and MTX reduced cell proliferation. Prolonged joint application of AICAR and MTX induced AMPK and consequently enhanced mitochondrial oxidation and reduced the rate of glycolysis. These metabolic changes suggest an anti-Warburg rearrangement of metabolism that led to the block of the G1/S and the G2/M transition slowing down cell cycle. The slowdown of cell proliferation was abolished when mitotropic transcription factors, PGC-1α, PGC-1β or FOXO1 were silenced. In human breast cancers higher expression of AMPKα and FOXO1 extended survival. AICAR and MTX exerts similar additive antiproliferative effect on other breast cancer cell lines, such as SKBR and 4T1 cells, too. Our data not only underline the importance of Warburg metabolism in breast cancer cells but nominate the AICAR+MTX combination as a potential cytostatic regime blunting Warburg metabolism. Furthermore, we suggest the targeting of AMPK and FOXO1 to combat breast cancer.

Paclitaxel (Taxol), a potent drug of natural origin isolated from the bark of the Pacific yew, is widely used for treating ovarian, lung and breast cancers. Currently, there is little information regarding the specific mechanism underlying the anticancer activity of paclitaxel. In the present study, we found that 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR), a well-known activator of adenosine monophosphate (AMP)-activated protein kinase (AMPK), downregulated the protein and mRNA levels of elongation factor 1 α (EF1α) in breast cancer MCF7 cells. Paclitaxel increased the phosphorylation of AMPK and also downregulated the expression of EF1α in MCF7 cells. In addition, paclitaxel increased the expression, as well as the phosphorylation of forkhead box O3a (FOXO3a). Phosphorylation of FOXO3a was suppressed in the presence of compound C, a specific AMPK inhibitor, suggesting the involvement of AMPK in paclitaxel-induced FOXO3a phosphorylation. The induction and phosphorylation of FOXO3a by paclitaxel were not observed in EF1α and AMPK knockdown cells. Co-treatment with AICAR resulted in increased susceptibility of cancer cells to paclitaxel-induced suppression of their viability and further enhanced paclitaxel-induced FOXO3a phosphorylation. These results suggest that the antitumor effects of paclitaxel in breast cancer are mediated by activation of the AMPK/EF1α/FOXO3a signaling pathway.

The AMP-activated protein kinase (AMPK)-mediated energy-sensing signals play important roles in reprogramming the expression of inflammatory genes. In the present study, the potential effects of the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) were investigated in a mouse model with LPS/D-Gal-induced acute hepatitis. Our experimental data indicated that treatment with AICAR suppressed the elevation of plasma aminotransferases and alleviated the histopathological abnormalities in mice exposed to LPS/D-Gal. Treatment with AICAR also inhibited the LPS/D-Gal-induced up-regulation of TNF-α, NO and myeloperoxidase. In addition, the LPS/D-Gal-induced expression of pro-apoptotic factor Bax, cleavage of caspase-3, elevation of hepatic caspase-3, caspase-8, caspase-9 activities and induction of terminal deoxynucleotidyl transferase-mediated nucleotide nick-end labeling-positive cells were all suppressed by AICAR. These results suggested that the AMPK activator AICAR could attenuate LPS/D-Gal-induced acute hepatitis, which implies that AMPK might become a novel target for the treatment of inflammation-based liver disorders., (© The Author(s) 2015.)

Background and Purpose: This study aims to investigate whether and how pharmacological activation of AMP-activated protein kinase (AMPK) improves endothelial function by suppressing mitochondrial ROS-associated endoplasmic reticulum stress (ER stress) in the endothelium. Experimental approach Palmitate stimulation induced mitochondrial fission and ER stress-associated endothelial dysfunction. The effects of AMPK activators salicylate and AICA riboside (AICAR) on mitochondrial ROS production, Drp1 phosphorylation, mitochondrial fission, ER stress, thioredoxin-interacting protein (TXNIP)/NLRP3 inflammasome activation, inflammation, cell apoptosis and endothelium-dependent vasodilation were observed. Key results "Silencing" of TXNIP by RNA interference inhibited NLRP3 inflammasome activation in response to ER stress, indicating that TXNIP was a key link between ER stress and NLRP3 inflammasome activation. AMPK activators salicylate and AICAR prevented ROS-induced mitochondrial fission by enhancing dynamin-related protein 1 (Drp1) phosphorylation (Ser 637) and thereby attenuated IRE-1α and PERK phosphorylation, but their actions were blocked by knockdown of AMPK. Salicylate and AICAR reduced TXNIP induction and inhibited NLRP3 inflammasome activation by reducing NLRP3 and caspase-1 expression, leading to a reduction in IL-1β secretion. As a result, salicylate and AICAR inhibited inflammation and reduced cell apoptosis. Meanwhile, salicylate and AICAR enhanced eNOS phosphorylation and restored the loss of endothelium-dependent vasodilation in the rat aorta. Immunohistochemistry staining showed that AMPK activation inhibited ER stress and NLRP3 inflammasome activation in the vascular endothelium., Conclusion and Implications: Pharmacological activation of AMPK regulated mitochondrial morphology and ameliorated endothelial dysfunction by suppression of mitochondrial ROS-associated ER stress and subsequent TXNIP/NLRP3 inflammasome activation. These findings suggested that regulation of Drp1 phosphorylation by AMPK activation contributed to suppression of ER stress and thus presented a potential therapeutic strategy for AMPK activation in the regulation of endothelium homeostasis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Oocyte nuclear maturation depends on sufficient energy supply through oxidative phosphorylation and β-oxidation. AMP-activated protein kinase (AMPK) is an energy sensor controlling the oocyte energy metabolism. The main aim of this study was to examine the effect of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a potent activator of AMPK, on the ATP content and mitochondrial DNA copy number (Mt-number) of bovine oocytes and on their developmental ability. Oocytes were collected from slaughterhouse-derived bovine ovaries. When these oocytes were cultured in a maturation medium containing 0-, 50-, 250-, and 500-μM AICAR, higher AICAR concentrations reduced the rate of meiotic maturation and the ATP content in oocytes, whereas lower AICAR increased the ATP content in oocytes without affecting the maturation rate. Supplementation of the maturation medium with a low concentration of AICAR (50 and 250 μM) increased phospho-AMPK expression level, as determined by immunostaining. In addition, AICAR treatment increased the ATP content in oocytes, which remained elevated for as long as 2 days after fertilization. On culturing the oocytes with AICAR (250 μM), the fertilization outcome, rate of blastulation, and total cell number of the blastocysts significantly improved. When the proteosomal mitochondrial degradation was inhibited by supplementing the maturation medium with MG132, the Mt-number, as determined by real-time polymerase chain reaction, significantly increased. However, the treatment of oocytes with AICAR did not affect the Mt-number in the presence or absence of MG132. From these data, we conclude that low concentrations of AICAR improved the embryonic developmental ability, presumably via the upregulation of the ATP content in oocytes, but the increase in the ATP content was not due to the upregulation of mitochondrial biogeneration., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Acadesine is a nucleoside analogue with known activity against B-cell malignancies. Herein, we showed that in mantle cell lymphoma (MCL) cells acadesine induced caspase-dependent apoptosis through turning on the mitochondrial apoptotic machinery. At the molecular level, the compound triggered the activation of the AMPK pathway, consequently modulating known downstream targets, such as mTOR and the cell motility-related vasodilator-stimulated phosphoprotein (VASP). VASP phosphorylation by acadesine was concomitant with a blockade of CXCL12-induced migration. The inhibition of the mTOR cascade by acadesine, committed MCL cells to enter in apoptosis by a translational downregulation of the antiapoptotic Mcl-1 protein. In contrast, Bcl-2 protein levels were unaffected by acadesine and MCL samples expressing high levels of Bcl-2 tended to have a reduced response to the drug. Targeting Bcl-2 with the selective BH3-mimetic agent ABT-199 sensitized Bcl-2high MCL cells to acadesine. This effect was validated in vivo, where the combination of both agents displayed a more marked inhibition of tumor outgrowth than each drug alone. These findings support the notions that antiapoptotic proteins of the Bcl-2 family regulate MCL cell sensitivity to acadesine and that the combination of this agent with Bcl-2 inhibitors might be an interesting therapeutic option to treat MCL patients.

Sepsis and septic shock are enormous public health problems with astronomical financial repercussions on health systems worldwide. The central nervous system (CNS) is closely intertwined in the septic process but the underlying mechanism is still obscure. AMP-activated protein kinase (AMPK) is a ubiquitous energy sensor enzyme and plays a key role in regulation of energy homeostasis and cell survival. In this study, we hypothesized that activation of AMPK in the brain would attenuate inflammatory responses in sepsis, particularly in the lungs. Adult C57BL/6 male mice were treated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR, 20 ng), an AMPK activator, or vehicle (normal saline) by intracerebroventricular (ICV) injection, followed by cecal ligation and puncture (CLP) at 30 min post-ICV. The septic mice treated with AICAR exhibited elevated phosphorylation of AMPKα in the brain along with reduced serum levels of aspartate aminotransferase, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), compared with the vehicle. Similarly, the expressions of TNF-α, IL-1β, keratinocyte-derived chemokine and macrophage inflammatory protein-2 as well as myeloperoxidase activity in the lungs of AICAR-treated mice were significantly reduced. Moreover, histological findings in the lungs showed improvement of morphologic features and reduction of apoptosis with AICAR treatment. We further found that the beneficial effects of AICAR on septic mice were diminished in AMPKα2 deficient mice, showing that AMPK mediates these effects. In conclusion, our findings reveal a new functional role of activating AMPK in the CNS to attenuate inflammatory responses and acute lung injury in sepsis.

Hindbrain adenosine 5'-monophosphate-activated protein kinase (AMPK) activation alters hypothalamic neuronal genomic activity in an estradiol (E)-dependent manner. This study examines the premise that E regulates metabolic effector neuron reactivity to hindbrain AMPK. Paraventricular (PVH), arcuate (ARH), and ventromedial (VMH) nuclei were micropunched from brains of E- or oil (O)-implanted ovariectomized female rats that had been injected, into the fourth ventricle, with the AMPK activator 5-aminoimidazole-4-carboxamide-riboside (AICAR; A) or saline (S) and analyzed by quantitative polymerase chain reaction and Western blotting for neurotransmitter mRNA and protein expression. PVH corticotrophin-releasing hormone gene and protein profiles were decreased in O/A and E/A animals. ARH pro-opiomelanocortin (POMC) mRNA and protein were both elevated in O/A but were diminished or unchanged, respectively, in E/A animals; ARH neuropeptide Y (NPY) transcription was inhibited in O/A and E/A animals, but neuropeptide content was augmented in E/A only. VMH SF-1 mRNA and protein were reduced in O and E animals. AICAR did not alter AMPK protein in any structure but elevated PVH (↑E), did not alter ARH, and decreased VMH (↓O,↓E) pAMPK. Results demonstrate hypothalamic metabolic neurotransmitter and AMPK reactivity to hindbrain AMPK activation, including E-dependent adjustments in POMC and NPY transcription and protein expression. Dissimilar POMC (↑O vs. ↔E) and NPY (↓O vs. ↑E) neuropeptide responses to caudal fourth ventricle AICAR indicate E regulation of hindbrain AMPK signaling and/or target receptivity, implying that ARH-controlled metabolic responses may differ in the presence vs. absence of E. Evidence for variable changes in hypothalamic AMPK activity resulting from hindbrain sensor manipulation suggests that individual (or region-based groups of) AMPK-expressing neuron populations are uniquely impacted by hindbrain AMPK., (© 2014 Wiley Periodicals, Inc.)

Fos immunocytochemistry is a valuable anatomical mapping tool for distinguishing cells within complex tissues that undergo genomic activation, but it is seldom paired with corroborative molecular analytical techniques. Due to preparatory requirements that include protein cross-linking for specimen sectioning, histological tissue sections are regarded as unsuitable for those methods. Our studies show that pharmacological activation of the hindbrain energy sensor AMPK by AICAR elicits estradiol (E)-dependent patterns of Fos immunolabeling of hypothalamic metabolic loci. Here, Western blotting was applied to hypothalamic tissue removed from histological sections of E- versus oil (O)-implanted ovariectomized (OVX) female rat brain to measure levels of metabolic transmitters associated with Fos-positive structures. In both E and O rats, AICAR treatment elicited alterations in pro-opiomelanocortin, neuropeptide Y, SF-1, and orexin-A neuropeptide expression that coincided with patterns of Fos labeling of structures containing neurons that synthesize these neurotransmitters, e.g. arcuate and ventromedial nuclei and lateral hypothalamic area. O, but not E animals also exhibited parallel augmentation of tissue corticotropin-releasing hormone neuropeptide levels and paraventricular nucleus Fos staining. Data demonstrate the utility of immunoblot analysis as a follow-through technique to capitalize on Fos mapping of transactivation sites in the brain. Findings that induction of Fos immunoreactivity coincides with adjustments in hypothalamic metabolic neuropeptide expression affirms that this functional indicator reflects changes in neurotransmission in pathways governing metabolic outflow., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

mTOR - the mammalian/mechanistic target of rapamycin - has been implicated as a key signaling node for promoting survival of cancer cells. However, clinical trials that have targeted mTOR with rapamycin or rapamycin analogs have had minimal impact. In spite of the high specificity of rapamycin for mTOR, the doses needed to suppress key mTOR substrates have proved toxic. We report here that rapamycin when combined with AICAR - a compound that activates AMP-activated protein kinase makes rapamycin cytotoxic rather than cytostatic at doses that are tolerated clinically. AICAR by itself is able to suppress mTOR complex 1 (mTORC1), but also stimulates a feedback activation of mTORC2, which activates the survival kinase Akt. However, AICAR also suppresses production of phosphatidic acid (PA), which interacts with mTOR in a manner that is competitive with rapamycin. The reduced level of PA sensitizes mTORC2 to rapamycin at tolerable nano-molar doses leading reduced Akt phosphorylation and apoptosis. This study reveals how the use of AICAR enhances the efficacy of rapamycin such that rapamycin at low nano-molar doses can suppress mTORC2 and induce apoptosis in human cancer cells at doses that are clinically tolerable.