AICAR

CD137L promotes immune surveillance in melanoma via HLTF regulation.

Immune checkpoint blockers (ICBs) have demonstrated substantial efficacy across various malignancies, yet the benefits of ICBs are limited to a subset of patients. Therefore, it is essential to identify novel therapeutic targets. By integrating multi-omics data from cohorts of patients with melanoma treated with ICBs, a positive correlation is observed between tumor CD137L expression and the efficacy of PD-1 blockade. Functionally, CD137L induction in cancer cells significantly enhances anti-tumor immunity by promoting CD8+ T cell survival, both in vivo and in vitro. Mechanistically, helicase-like transcription factor (HLTF) is identified as a pivotal transcriptional regulator of CD137L, controlling its expression through phosphorylation of serine at position 398. Therapeutically, the AMPK agonist AICAR (acadesine) as an inducer of CD137L, exhibiting synergistic effects with PD-1 or CTLA-4 blockade. In summary, our findings elucidate a mechanism controlling CD137L expression and highlight a promising combination therapy to enhance the efficacy of ICBs in melanoma. One Sentence Summary: Inducing co-stimulatory immune checkpoint CD137L expression in melanoma cells enhances T cell-mediated anti-tumor immunity.

Administration of AICAR, an AMPK Activator, Prevents and Reverses Diabetic Polyneuropathy (DPN) by Regulating Mitophagy.

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes in both Type 1 (T1D) and Type 2 (T2D). While there are no specific medications to prevent or treat DPN, certain strategies can help halt its progression. In T1D, maintaining tight glycemic control through insulin therapy can effectively prevent or delay the onset of DPN. However, in T2D, overall glucose control may only have a moderate impact on DPN, although exercise is clearly beneficial. Unfortunately, optimal exercise may not be feasible for many patients with DPN because of neuropathic foot pain and poor balance. Exercise has several favorable effects on health parameters, including body weight, glycemic control, lipid profile, and blood pressure. We investigated the impact of an exercise mimetic, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), on DPN. AICAR treatment prevented or reversed experimental DPN in mouse models of both T2D and T1D. AICAR in high-fat diet (HFD-fed) mice increased the phosphorylation of AMPK in DRG neuronal extracts, and the ratio of phosphorylated AMPK to total AMPK increased by 3-fold (HFD vs. HFD+AICAR; p < 0.001). Phospho AMP increased the levels of dynamin-related protein 1 (DRP1, a mitochondrial fission marker), increased phosphorylated autophagy activating kinase 1 (ULK1) at Serine-555, and increased microtubule-associated protein light chain 3-II (LC3-II, a marker for autophagosome assembly) by 2-fold. Mitochondria isolated from DRG neurons of HFD-fed had a decrease in ADP-stimulated state 3 respiration (120 &#xb1; 20 nmol O2/min in HFD vs. 220 &#xb1; 20 nmol O2/min in control diet (CD); p < 0.001. Mitochondria isolated from HFD+AICAR-treated mice had increased state 3 respiration (240 &#xb1; 30 nmol O2/min in HFD+AICAR). However, AICAR's protection in DPN in T2D mice was also mediated by its effects on insulin sensitivity, glucose metabolism, and lipid metabolism. Drugs that enhance AMPK phosphorylation may be beneficial in the treatment of DPN.

AICAr, a Widely Used AMPK Activator with Important AMPK-Independent Effects: A Systematic Review.

5-Aminoimidazole-4-carboxamide ribonucleoside (AICAr) has been one of the most commonly used pharmacological modulators of AMPK activity. The majority of early studies on the role of AMPK, both in the physiological regulation of metabolism and in cancer pathogenesis, were based solely on the use of AICAr as an AMPK-activator. Even with more complex models of AMPK downregulation and knockout being introduced, AICAr remained a regular starting point for many studies focusing on AMPK biology. However, there is an increasing number of studies showing that numerous AICAr effects, previously attributed to AMPK activation, are in fact AMPK-independent. This review aims to give an overview of the present knowledge on AMPK-dependent and AMPK-independent effects of AICAr on metabolism, hypoxia, exercise, nucleotide synthesis, and cancer, calling for caution in the interpretation of AICAr-based studies in the context of understanding AMPK signaling pathway.

Diverse Inhibitors of De Novo Purine Synthesis Promote AICAR-Induced AMPK Activation and Glucose Uptake in L6 Myotubes.

Methotrexate, an immunosuppressant and anticancer drug, promotes glucose uptake and lipid oxidation in skeletal muscle via activation of AMP-activated protein kinase (AMPK). Methotrexate promotes AMPK activation by inhibiting 5-aminoimidazole-4-carboxamide ribonucleotide (ZMP) formyltransferase/inosine monophosphate (IMP) cyclohydrolase (ATIC), which converts ZMP, an endogenous purine precursor and an active form of the pharmacological AMPK activator AICAR, to IMP during de novo purine synthesis. In addition to methotrexate, inhibition of purine synthesis underpins the therapeutic effects of a number of commonly used immunosuppressive, anticancer, and antimicrobial drugs, raising the question of whether activation of AMPK in skeletal muscle could be a recurrent feature of these drugs. Using L6 myotubes, we found that AICAR-induced AMPK activation and glucose uptake were enhanced by inhibitors of the conversion of IMP to GMP (mycophenolate mofetil) or of IMP to AMP (alanosine) as well as by indirect inhibitors of human (trimetrexate) and bacterial ATIC (sulfamethoxazole). 6-Mercaptopurine, which inhibits the conversion of IMP to GMP and AMP, activated AMPK, increased glucose uptake, and suppressed insulin signaling, but did not enhance the effect of AICAR. As determined by measuring oxygen consumption rate, none of these agents suppressed mitochondrial function. Overall, our results indicate that IMP metabolism is a gateway for the modulation of AMPK and its metabolic effects in skeletal muscle cells.

Activation of AMPK and its Impact on Exercise Capacity.

Activation of the adenosine monophosphate (AMP)-activated kinase (AMPK) contributes to beneficial effects such as improvement of the hyperglycemic state in diabetes as well as reduction of obesity and inflammatory processes. Furthermore, stimulation of AMPK activity has been associated with increased exercise capacity. A study published in 2008, directly before the Olympic Games in Beijing, showed that the AMPK activator AICAR (5-amino-1-&#x3b2;-D-ribofuranosyl-imidazole-4-carboxamide) increased the running capacity of mice without any training and thus, prompted the World Anti-Doping Agency (WADA) to include certain AMPK activators in the list of forbidden drugs. This raises the question as to whether all AMPK activators should be considered for registration or whether the increase in exercise performance is only associated with specific AMPK-activating substances. In this review, we intend to shed light on currently published AMPK-activating drugs, their working mechanisms, and their impact on body fitness.

AMPK activator AICAR promotes 5-FU-induced apoptosis in gastric cancer cells.

The aim of the present study was to determine the effect of AICAR, an AMPK activator, on apoptosis in gastric carcinoma cells (SGC-7901) with or without 5-fluorouracil (5-FU). SGC-7901 cells were treated with AICAR (0.2-5&#xa0;mM, for 24-48&#xa0;h) with or without 5-FU. Cell viability was determined using MTT assay, while apoptosis were measured through the evaluation of active caspase-3 activity and DNA fragmentation. Real-time PCR was employed to determine the expression of tumor suppressor and multi-drug resistant (mdr1) gene. Cleaved caspase-3 and phosphorylated AMPK (p-AMPK) were measured by Western blot. AICAR significant reduced cellular viability but increased apoptosis in a time- and dose-dependent manner, which is associated with an increase in p-AMPK levels. Importantly, AICAR enhanced the sensitivity to 5-FU-induced reduction of cellular viability and increased apoptosis in SGC-7901 cells. Furthermore, AICAR increased tumor suppressor genes [F-box and WD repeat domain containing 7 (FBXW7), semaphorin III/F (SEMA3F), and p21(Cip1) (p21)] but reduced mdr1 expression. Finally, p-AMPK levels were reduced in 5-FU-resistant gastric cancer cells compared to human immortalized gastric epithelial cell line and 5-FU-sensitive gastric cancer cells. AICAR not only induces apoptosis alone but also enhances pro-apoptotic effect of 5-FU in SGC-7901 cells, which lays an experimental foundation to develop AICAR as a chemotherapeutic sensitizer against gastric cancer.

Impairment of AMPK-&#x3b1;2 augments detrusor contractions in bladder ischemia.

Ischemia disrupts cellular energy homeostasis. Adenosine monophosphate-activated protein kinase alpha-2 (AMPK-&#x3b1;2) is a subunit of AMPK that senses cellular energy deprivation and signals metabolic stress. Our goal was to examine the expression levels and functional role of AMPK-&#x3b1;2 in bladder ischemia. Iliac artery atherosclerosis and bladder ischemia were engendered in apolipoprotein E knockout rats by partial arterial endothelial denudation using a balloon catheter. After eight weeks, total and phosphorylated AMPK-&#x3b1;2 expression was analyzed by western blotting. Structural integrity of AMPK-&#x3b1;2 protein was assessed by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). Functional role of AMPK-&#x3b1;2 was examined by treating animals with the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D ribofuranoside (AICAR). Tissue contractility was measured in the organ bath and bladder nerve density was examined by immunostaining. Total AMPK-&#x3b1;2 expression increased in bladder ischemia, while phosphorylated AMPK-&#x3b1;2 was significantly downregulated. LC-MS/MS suggested post-translational modification of AMPK-&#x3b1;2 functional domains including phosphorylation sites, suggesting accumulation of catalytically inactive AMPK-&#x3b1;2 in bladder ischemia. Treatment of rats with AICAR diminished the force of overactive detrusor contractions and increased bladder capacity but did not have a significant effect on the frequency of bladder contractions. AICAR diminished contractile reactivity of ischemic tissues in the organ bath and prevented loss of nerve fibers in bladder ischemia. Ischemia induces post-translational modification of AMPK-&#x3b1;2 protein. Impairment of AMPK-&#x3b1;2 may contribute to overactive detrusor contractions and loss of nerve fibers in bladder ischemia. AMPK activators may have therapeutic potential against detrusor overactivity and neurodegeneration in bladder conditions involving ischemia.

AMPK activator, AICAR, inhibits palmitate-induced apoptosis in osteoblast.

Osteoblast apoptosis reduces bone mineral density. Apoptosis can be induced in a variety of cells by palmitate, which is one of the most common saturated fatty acids in dietary fat. The AMPK activator, AICAR, has been shown to inhibit palmitate-induced apoptosis. However, the role of palmitate in osteoblast apoptosis is currently unknown. This study examined whether palmitate could induce apoptosis in osteoblasts, and if so, whether AICAR could alleviate palmitate-induced apoptosis. Palmitate reduced cell survival and induced apoptosis in a dose- and time-dependent manner in human fetal osteoblasts (hFOB) 1.19. While the long-chain acyl-CoA synthetase inhibitor, triacsin C, inhibited palmitate-induced apoptosis, anti-oxidants and ceramide synthesis inhibitors did not attenuate the apoptosis. AICAR prevented palmitate-induced apoptosis and the inhibition of AICAR-mediated increase in fatty acid oxidation by etomoxir did not affect the prevention of apoptosis by AICAR. Constitutively-active AMPK also inhibited palmitate-induced apoptosis. Treatment with an AMPK inhibitor (compound C) and a dominant-negative AMPK adenovirus suppressed the inhibitory effect of AICAR on apoptosis. Palmitate impaired the activation of ERK by fetal bovine serum, which was blocked by AICAR. Moreover, AICAR increased ERK activation, and ERK inhibitors, PD98059 and U0126, as well as a dominant-negative MEK1, abolished the inhibitory effect of AICAR on palmitate-induced apoptosis. AICAR also inhibited palmitate-induced apoptosis in osteoblastic differentiated cells from human bone marrow, which was accompanied by recovered ERK activity. These results suggest that palmitate induces apoptosis in osteoblasts through the impaired activation of ERK, and the activation of AMPK inhibits palmitate-induced apoptosis by activating ERK.

AMPK mediates energetic stress-induced liver GDF15.

Growth differentiating factor-15 (GDF15) is an emerging target for the treatment of obesity and metabolic disease partly due to its ability to suppress food intake. GDF15 expression and secretion are thought to be regulated by a cellular integrated stress response, which involves endoplasmic reticulum (ER) stress. AMPK is another cellular stress sensor, but the relationship between AMPK, ER stress, and GDF15 has not been assessed in vivo. Wildtype (WT), AMPK &#x3b2;1 deficient (AMPK&#x3b2;1-/- ), and CHOP-/- mice were treated with three distinct AMPK activators; AICAR, which is converted to ZMP mimicking the effects of AMP on the AMPK&#x3b3; isoform, R419, which indirectly activates AMPK through inhibition of mitochondrial respiration, or A769662, a direct AMPK activator which binds the AMPK&#x3b2;1 isoform ADaM site causing allosteric activation. Following treatments, liver Gdf15, markers of ER-stress, AMPK activity, adenine nucleotides, circulating GDF15, and food intake were assessed. AICAR and R419 caused ER and energetic stress, increased GDF15 expression and secretion, and suppressed food intake. Direct activation of AMPK &#x3b2;1 containing complexes by A769662 increased hepatic Gdf15 expression, circulating GDF15, and suppressed food intake, independent of ER stress. The effects of AICAR, R419, and A769662 on GDF15 were attenuated in AMPK&#x3b2;1-/- mice. AICAR and A769662 increased GDF15 to a similar extent in WT and CHOP-/- mice. Herein, we provide evidence that AMPK plays a role in mediating the induction of GDF15 under conditions of energetic stress in mouse liver in vivo.

Effects of acadesine on myocardial infarction, stroke, and death following surgery. A meta-analysis of the 5 international randomized trials. The Multicenter Study of Perioperative Ischemia (McSPI) Research Group.

To determine the effects of a purine nucleoside, acadesine, on the incidence of fatal and nonfatal cardiovascular and cerebrovascular complications following coronary artery bypass graft (CABG) surgery. Individual patient data from 5 randomized, placebo-controlled, double-blind clinical trials, including 81 international medical centers of the United States, Canada, and Europe. All patients from all clinical trials were included: a total of 4043 patients undergoing CABG surgery, evaluable for efficacy, and randomized to receive either placebo (n= 2031) or acadesine (0.1 mg x kg(-1) x min(-1); n=2012) by intravenous infusion for 7 continuous hours and via the cardioplegia solution. Individual patient data were collected prospectively using standardized forms and methods and double-data entry. A general parametric approach and analysis-by-patient meta-analysis were used, including both fixed effects and random effects models. Inclusion and exclusion criteria, general methodology, and outcome assessment techniques were similar for all trials. Acadesine decreased the incidence of the primary outcome, perioperative myocardial infarction (MI) by 27% (odds ratio [OR], 0.69; 95% confidence interval [CI], 0.51-0.95; P=.02), decreased the incidence of cardiac death through postoperative day 4 by 50% (OR, 0.52; 95% Cl, 0.27-0.98; P=.04), and decreased the incidence of combined outcome (MI, stroke, or cardiac death) by 26% (OR, 0.73; 95% Cl, 0.57-0.93; P=.01). The random effects models for these outcomes also yielded significant results. The incidence of cerebrovascular accident was not significantly reduced by acadesine (OR, 0.69; 95% Cl, 0.44-1.08; P=.10). A secondary analysis of cardiac death following MI through postoperative day 4 demonstrated that acadesine decreased by 89% the number of deaths from 13.3% (13 deaths/98 MIs) in the placebo group to 1.4% (1 death/71 MIs) in acadesine-treated patients (P=.003). Acadesine also reduced the use of ventricular-assistance devices for severe postoperative heart failure by approximately one third (P=.05). Finally, regarding safety, the incidence of adverse events was similar in the acadesine vs placebo groups, with the exception of a transient increase in serum uric acid in the acadesine group. The results of this meta-analysis indicate that in patients undergoing CABG surgery, treatment with acadesine before and during surgery can reduce early cardiac death, MI, and combined adverse cardiovascular outcomes.

Acadesine suppresses TNF-&#x3b1; induced complement component 3 (C3), in retinal pigment epithelial (RPE) cells.

Age-related macular degeneration (AMD) is the most prevalent form of irreversible blindness in the developed world. Aging, inflammation and complement dysregulation affecting the retinal pigment epithelium (RPE), are considered significant contributors in its pathogenesis and several evidences have linked tumor necrosis factor alpha (TNF-&#x3b1;) and complement component 3 (C3) with AMD. Acadesine, an analog of AMP and an AMP-activated protein kinase (AMPK) activator, has been shown to have cytoprotective effects in human clinical trials as well as having anti-inflammatory and anti-vascular exudative effects in animals. The purpose of this study was to evaluate if acadesine is able to suppress TNF-&#x3b1; induced C3 in RPE cells. ARPE-19 and human primary RPE cells were cultured and allowed to grow to confluence. TNF-&#x3b1; was used for C3 induction in the presence or absence of acadesine. Small molecule inhibitors and siRNA were used to determine if acadesine exerts its effect via the extracellular or intracellular pathway and to evaluate the importance of AMPK for these effects. The expression level of C3 was determined by immunoblot analysis. Acadesine suppresses TNF-&#x3b1; induced C3 in a dose dependent manner. When we utilized the adenosine receptor inhibitor dipyridamole (DPY) along with acadesine, acadesine's effects were abolished, indicating the necessity of acadesine to enter the cell in order to exert it's action. However, pretreatment with 5-iodotubericidin (5-Iodo), an adenosine kinase (AK) inhibitor, didn't prevent acadesine from decreasing TNF-&#x3b1; induced C3 expression suggesting that acadesine does not exert its effect through AMP conversion and subsequent activation of AMPK. Consistent with this, knockdown of AMPK &#x3b1; catalytic subunit did not affect the inhibitory effect of acadesine on TNF-&#x3b1; upregulation of C3. Our results suggest that acadesine suppresses TNF-&#x3b1; induced C3, likely through an AMPK-independent pathway, and could have potential use in complement over activation diseases.

AICAR, an AMPK activator, protects against cisplatin-induced acute kidney injury through the JAK/STAT/SOCS pathway.

Cisplatin causes acute kidney injury (AKI) through proximal tubular injury. We investigated the protective effect of the adenosine monophosphate protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) against cisplatin-induced AKI. We investigated whether the AMP-kinase activator AICAR ameliorates cisplatin-induced AKI through the JAK/STAT/SOCS pathway. Male Sprague-Dawley (SD) rats were randomly divided into four groups: control, AICAR, cisplatin, and cisplatin&#xa0;+&#xa0;AICAR. As appropriate to their treatment group, the rats were injected with a single dose of cisplatin (7&#xa0;mg/kg, i.p.). AICAR was administered to the rats at 100&#xa0;mg/kg i.p. daily. Blood urea nitrogen (BUN) and serum creatinine were measured. Renal damage was analyzed in sections stained with hematoxylin and eosin (H&E). Renal tissues were also examined by immunohistochemistry and western blot for p-AMPK, Kim-1, cleaved caspase 3, and JAK/STAT/SOCS. For in&#xa0;vitro studies, NRK-52E normal rat kidney cells were treated with cisplatin and/or AICAR. By western blot, we confirmed the expression of p-AMPK and the JAK/STAT/SOCS pathway in NRK-52E cells. AICAR was protective against cisplatin-induced acute tubular injury by up-regulating p-AMPK expression in NRK-52E cells. Protein expression levels of JAK2/STAT1 were markedly ameliorated in NRK-52E cells by AICAR. The protective mechanism of AICAR may be associated with suppression of the JAK2/STAT1 pathway and up-regulation of SOCS1, an inhibitor of the JAK2/STAT1 pathway. The present study demonstrates the protective effects of AICAR against cisplatin-induced AKI and shows a new renoprotective mechanism through the JAK2/STAT1/SOCS1 pathway and apoptosis inhibition. This study suggests that activation of the AMPK activator AICAR might ameliorate cisplatin-induced AKI.

AMPK activation enhances osteoblast differentiation on a titanium disc via autophagy.

The acquisition of osseointegration during implant therapy is slower and poorer in patients with diabetes compared with healthy persons. The serum concentration of adiponectin in patients with type II diabetes is lower than that of healthy persons via the suppression of AMP-activated protein kinase (AMPK). Therefore, we hypothesized that the AMPK activation enhances bone formation around implants, resulting in the improved acquisition of osseointegration. The purpose of this study was to evaluate the impact of AMPK activation on osteoblast differentiation and its mechanism of downstream signaling on titanium disc (Ti). Confluent mouse pre-osteoblasts (MC3T3-E1) cells (1&#x2009;&#xd7;&#x2009;105 cells/well) were cultured with BMP-2 for osteoblast differentiation, in the presence or absence AICAR, an AMPK activator. We examined the effects of AMPK activation on osteoblast differentiation and the underlying mechanism on a Ti using a CCK8 assay, a luciferase assay, quantitative RT-PCR, and western blotting. Although the proliferation rate of osteoblasts was not different between a Ti and a tissue culture polystyrene dish, the addition of AICAR, AMPK activator slightly enhanced osteoblast proliferation on the Ti. AICAR enhanced the BMP-2-dependent transcriptional activity on the Ti, leading to upregulation in the expression of osteogenesis-associated molecules. AICAR simultaneously upregulated the expression of autophagy-associated molecules on the Ti, especially LC3-II. AdipoRon, an adiponectin receptor type1/type2 activator activated AMPK, and upregulated osteogenesis-associated molecules on Ti. AMPK activation enhances osteoblast differentiation on a Ti via autophagy, suggesting that it promotes the acquisition of osseointegration during implant therapy.

AMP-dependent kinase stimulates the expression of &#x3b1;Klotho.

Renal &#x3b1;Klotho along with fibroblast growth factor 23 regulates phosphate and vitamin D metabolism. Its cleavage yields soluble Klotho controlling intracellular processes. &#x3b1;Klotho has anti-inflammatory and antioxidant effects and is nephro- and cardioprotective. AMP-dependent kinase (AMPK) is a nephro- and cardioprotective energy sensor. Given that both &#x3b1;Klotho and AMPK have beneficial effects in similar organs, we studied whether AMPK regulates &#x3b1;Klotho gene expression in Madin-Darby canine kidney, normal rat kidney 52E, and human kidney 2 cells. Using quantitative real-time PCR and western blotting, we measured &#x3b1;Klotho expression upon pharmacological manipulation or siRNA-mediated knockdown of AMPK&#x3b1;. AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) enhanced &#x3b1;Klotho expression, an effect reduced in the presence of AMPK inhibitor compound C or siRNA targeting AMPK catalytic &#x3b1;-subunits (&#x3b1;1 and &#x3b1;2). Similarly, AMPK activators metformin and phenformin upregulated &#x3b1;Klotho transcripts. Taken together, our results suggest that AMPK is a powerful inducer of &#x3b1;Klotho and could thereby contribute to the development of future therapeutic interventions.

Acadesine, an adenosine-regulating agent with the potential for widespread indications.

Acadesine is an adenosine-regulating agent that increases bioavailability of adenosine and has important metabolic effects, partly through activation of the key metabolic regulatory enzyme, AMP-activated protein kinase. This review aimed to summarise and critique available data on the mechanism of action and clinical utility of acadesine, with a focus on treatment of ischaemic reperfusion injury, B-cell chronic lymphocytic leukaemia and diabetes mellitus. The literature was acquired through numerous avenues including Medline, Pubmed, institutional libraries and relevant pharmaceutical companies using keyword search criteria for all trade and common names of acadesine and its derivatives. Acadesine has proven intravenous efficacy in the amelioration of ischaemic reperfusion injury associated with coronary artery bypass graft surgery in Phase III clinical trials. Acadesine is active only in metabolically stressed tissues in the presence of ATP catabolism and therefore has fewer unwanted peripheral side effects than systemic administration of adenosine. Metabolism of the drug is through the endogenous purine pathway and acadesine has been proven to be safe and well tolerated. More recently, acadesine has entered Phase I trials for B-cell chronic lymphocytic leukaemia to compete with purine antagonists that are used at present. AMPK-activating agents with high oral bioavailability have potential application in impaired glucose tolerance, insulin resistance and types 1 and 2 diabetes, however the poor oral bioavailability of acadesine precludes such application. This review highlights that, although limited to intravenous application, acadesine is a potentially viable therapy for ischaemic reperfusion injury following coronary artery bypass surgery. Further studies are required to determine the efficacy of acadesine for other ischaemic indications, including during percutaneous transluminal coronary angioplasty for acute myocardial infarction.

AMPK activation attenuates central sensitization in a recurrent nitroglycerin-induced chronic migraine mouse model by promoting microglial M2-type polarization.

Energy metabolism disorders and neurogenic inflammation play important roles in the central sensitization to chronic migraine (CM). AMP-activated protein kinase (AMPK) is an intracellular energy sensor, and its activation regulates inflammation and reduces neuropathic pain. However, studies on the involvement of AMPK in the regulation of CM are currently lacking. Therefore, this study aimed to explore the mechanism underlying the involvement of AMPK in the central sensitization to CM. Mice with recurrent nitroglycerin (NTG)-induced CM were used to detect the expression of AMPK protein in the trigeminal nucleus caudalis (TNC). Following intraperitoneal injection of the AMPK activator 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) and inhibitor compound C, the mechanical pain threshold, activity level, and pain-like behaviors in the mice were measured. The expression of calcitonin gene-related peptide (CGRP) and cytokines, M1/M2 microglia, and NF-&#x3ba;B pathway activation were detected after the intervention. Repeated NTG injections resulted in a gradual decrease in AMPK protein expression, and the negative regulation of AMPK by increased ubiquitin-like plant homeodomain and RING finger domain 1 (UHRF1) expression may counteract AMPK activation by increasing ADP/ATP. AICAR can reduce the hyperalgesia and pain-like behaviors of CM mice, improve the activity of mice, reduce the expression of CGRP, IL-1&#x3b2;, IL-6, and TNF-&#x3b1; in the TNC region, and increase the expression of IL-4 and IL-10. Moreover, AMPK in TNC was mainly located in microglia. AICAR could reduce the expression of inducible NO synthase (iNOS) in M1 microglia and increase the expression of Arginase 1 (Arg1) in M2 microglia by inhibiting the activation of NF-&#x3ba;B pathway. AMPK was involved in the central sensitization of CM, and the activation of AMPK reduced neuroinflammation in NTG-induced CM mice. AMPK may provide new insights into interventions for energy metabolism disorders and neurogenic inflammation in migraine.

AMPK activator acadesine fails to alleviate isoniazid-caused mitochondrial instability in HepG2 cells.

Isoniazid (INH) is a first-line antituberculosis drug that is adversely associated with hepatotoxicity. Recently, impairment of mitochondrial homeostasis involved in this side effect has been noticed. Mitochondrial homeostasis is achieved by the balance between the generation of functional mitochondria by biogenesis and elimination of dysfunctional mitochondria by autophagy. AMP-activated protein kinase (AMPK) can maintain mitochondrial stability through positive control of these two processes. In this study, we showed that AMPK activator acadesine (AICAR) alleviated INH-caused impairment of mitochondrial biogenesis by activation of silent information regulator two ortholog 1 (SIRT1)-peroxisome proliferator-activated receptor &#x3b3; coactivator 1&#x3b1; (PGC1 &#x3b1;) pathway in HepG2 cells. However, mitochondrial instability and apoptosis were caused by AICAR along with an unexpected decrease in INH-induced cytoprotective autophagy. Therefore, AICAR failed to alleviate INH-caused mitochondrial instability in HepG2 cells due to its inhibitory effect on autophagy induced by INH. Copyright &#xa9; 2017 John Wiley & Sons, Ltd.

AICAR Stimulates the Pluripotency Transcriptional Complex in Embryonic Stem Cells Mediated by PI3K, GSK3&#x3b2;, and &#x3b2;-Catenin.

Pluripotent stem cells maintain the property of self-renewal and differentiate into all cell types under clear environments. Though the gene regulatory mechanism for pluripotency has been investigated in recent years, it is still not completely understood. Here, we show several signaling pathways involved in the maintenance of pluripotency. To investigate whether AMPK is involved in maintaining the pluripotency in mouse embryonic stem cells (mESCs) and elucidating the possible molecular mechanisms, implicated D3 and R1/E mESC lines were used in this study. Cells were cultured in the absence or presence of LIF and treated with 1 mM and 0.5 mM 5-aminoimidazole-4-carboxamide-1-&#x3b2;-d-ribofuranoside (AICAR), 2 mM metformin, compound C, and the PI3K inhibitor LY294002 for 24, 72, and 120 h. The levels of Nanog, Oct3/4, and REX1 and Brachyury, Notch2, and Gata4 mRNAs and Nanog or OCT3/4 protein levels were analyzed. Alkaline phosphatase and the cellular cycle were determined. The pGSK3&#x3b2;, GSK3&#x3b2;, p-&#x3b2;-catenin, and &#x3b2;-catenin protein levels were also investigated. We found that AMPK activators such as AICAR and metformin increase mRNA expression of pluripotency markers and decrease mRNA expression of differentiation markers in R1/E and D3 ES cells. AICAR increases phosphatase activity and arrests the cellular cycle in the G1 phase in these cells. We describe that AICAR effects were mediated by AMPK activation using a chemical inhibitor or by silencing this gene. AICAR effects were also mediated by PI3K, GSK3&#x3b2;, and &#x3b2;-catenin in R1/E ES cells. According to our findings, we provide a mechanism by which AICAR increases and maintains a pluripotency state through enhanced Nanog expression, involving AMPK/PI3K and p-GSK3&#x3b2; Ser21/9 pathways backing up the AICAR function as a potential target for this drug controlling pluripotency. The highlights of this study are that AICAR (5-aminoimidazole-4-carboxamied-1-b-riboside), an AMP protein kinase (AMPK) activator, blocks the ESC differentiation and AMPK is a key enzyme for pluripotency and shows valuable data to clarify the molecular pluripotency mechanism.

Acadesine: preclinical overview.

Common and distinct roles of AMPK&#x3b3; isoforms in small-molecule activator-stimulated glucose uptake in mouse skeletal muscle.

Small-molecule activators targeting the allosteric drug and metabolite (ADaM) site of AMPK enhance insulin-independent glucose uptake in skeletal muscle and lower glucose in preclinical models of hyperglycemia. The regulatory AMPK&#x3b3; subunit plays a central role in energy sensing. While the skeletal muscle-selective &#x3b3;3 isoform is essential for AMP/ZMP-induced glucose uptake, it is dispensable for ADaM site-binding activators. We hypothesized that the predominant &#x3b3;1 isoform is required for ADaM site activator-stimulated glucose uptake in skeletal muscle. Single-nucleus RNA sequencing (snRNA-seq) was performed on mouse and human skeletal muscle mapping AMPK subunit isoform distribution across resident cell types. To determine &#x3b3; isoform-specific requirements for activator-stimulated glucose uptake, skeletal muscle-specific inducible AMPK&#x3b3;1/&#x3b3;3 double knockout (im&#x3b3;1-/-/&#x3b3;3-/-) and single knockout (im&#x3b3;1-/- and im&#x3b3;3-/-) mice were generated. Ex vivo glucose uptake was measured following treatment with AICAR (AMP-mimetic) or MK-8722 (ADaM site activator), and in vivo MK-8722-induced blood glucose lowering was assessed. snRNA-seq revealed distinct AMPK isoform distribution: &#x3b3;1 was ubiquitously expressed, whereas &#x3b3;3 was enriched in glycolytic myofibers in both mouse and human skeletal muscle. Ex vivo, glucose uptake stimulated by either AICAR or MK-8722 was severely blunted in im&#x3b3;1-/-/&#x3b3;3-/- muscle, and MK-8722-induced blood glucose lowering was significantly blunted in vivo. AICAR but not MK-8722-stimulated muscle glucose uptake was abolished in im&#x3b3;3-/-, whereas both activators fully retained effects on glucose uptake and glucose lowering in im&#x3b3;1-/- mice. While &#x3b3;1 predominates in stabilizing the AMPK&#x3b1;2&#x3b2;2&#x3b3;1 complex, it is dispensable for AMPK activator-stimulated glucose uptake in skeletal muscle, whether mediated via the nucleotide-binding or ADaM site.

Acadesine during fluid resuscitation from shock and abdominal sepsis.

To determine properties of acadesine, the prototype adenosine regulating agent, in an experimental model in which abdominal sepsis is superimposed onto hemorrhagic shock. Randomized, blinded animal study. University-based animal research facility. Twenty-eight anesthetized mongrel pigs (35.5 +/- 1.1 kg). The cecum was ligated and punctured to produce abdominal sepsis. To produce hemorrhagic shock, 45% to 47% of the estimated blood volume was withdrawn. After 1 hr, shed blood plus supplemental crystalloid (twice the shed blood volume) plus either acadesine (5 mg/kg bolus + 1 mg/kg x 60 min, n = 10) or its vehicle (n = 10) was administered. All animals were awakened and observed for 48 hrs. At 48 hrs, cardiac function, bacterial cultures from the septic focus, and inflammatory changes in the abdomen were quantified. After resuscitation with acadesine vs. vehicle, we observed the following: a) arterial blood pressure and cardiac filling pressures were similar but cardiac index, systemic oxygen delivery, and systemic oxygen consumption were increased; b) plasma lactate was higher, systemic vascular resistance was lower, but ileal mucosal blood flow was not measurably altered; c) lipopolysaccharide-evoked tumor necrosis factor production in whole blood ex vivo was reduced; d) in those animals that survived 48 hrs (10/10 vs. 8/10), sepsis-induced cardiac depression, amount of free intraperitoneal fluid, extra abscess inflammatory reaction, abscess wall formation, abscess bacterial counts, and peritoneal bacterial counts, were all similar, but blood bacterial counts were higher. Fluid resuscitation with acadesine produced no adverse hemodynamic consequences and probably improved washout of metabolites from the reperfused microcirculation in sites other than the small intestine or heart. Taken together, these observations suggest that adenosine regulating agents might have therapeutic potential during fluid resuscitation from trauma. However, at least in these extreme conditions, the acute salutary effects of acadesine were probably overwhelmed by polymicrobial sepsis. Further studies must determine whether supplemental adjuvants to boost host defense during recovery from trauma will optimize adenosine-based resuscitation solutions.

Mechanisms of aerobic exercise on doxorubicin-induced cardiomyocyte apoptosis: based on AMPK/PI3K/AKT signalling pathway.

Doxorubicin (Dox), a common anthracycline used in cancer therapy, is constrained by its cardiotoxic side effects. Preclinical research indicates that aerobic exercise might reduce Dox-induced cardiac injury. However, the precise mechanisms remain unclear. The aim of this study is to investigate whether aerobic exercise alleviates Dox-induced cardiomyocyte apoptosis by modulating the AMPK/PI3K/AKT signalling pathway. An in vivo model of Dox-induced cardiotoxicity was established by using C57BL/6&#xa0;J mice, followed by aerobic exercise intervention. Cardiac function, morphology, apoptosis, and proliferation, as well as the activity AMPK/PI3K/AKT pathway were assessed. In addition, an in vitro model of Dox-induced cardiomyocyte injury was established by using H9c2 cells. Cells were treated with 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR, AMPK activator) and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002, PI3K inhibitor), and cardiomyocyte apoptosis, proliferation, as well as the activity of PI3K/AKT pathway were evaluated. In vivo demonstrated that Dox administration resulted in cardiac dysfunction, morphological disarray, myocardial fibrosis, pathological cardiomyocyte hypertrophy, and cardiomyocyte apoptosis, and significantly suppressed the activity of the AMPK and PI3K/AKT signalling pathways, while aerobic exercise reversed these effects. In vitro, the AICAR reduced Dox-induced cardiomyocyte apoptosis and promoted cardiomyocyte proliferation by activating the PI3K/AKT signalling pathway. Our findings indicate aerobic exercise may protect against myocardial damage by activating the AMPK/PI3K/AKT signalling pathway, offering insights into the molecular mechanisms of cardioprotection and highlighting the potential benefits of exercise.

AMPK-driven Macrophage Responses Are Autophagy Dependent in Experimental Bronchopulmonary Dysplasia.

The pathogenesis of bronchopulmonary dysplasia (BPD) remains incompletely understood. Recent studies suggest insufficient AMP-activated protein kinase (AMPK) activation as a potential cause of impaired autophagy in rodent and nonhuman primate models of BPD. Impaired autophagy is associated with enhanced inflammatory signaling in alveolar macrophages (AMs) and increased severity of murine BPD induced by neonatal hyperoxia exposure. The goal of this study was to determine the role of autophagy and AMPK activation in macrophage responses in murine BPD. C57BL/6J mice were exposed to neonatal hyperoxia starting on postnatal day (P)1 and treated with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) between P3 and P6. Mice were euthanized on P7, and markers of AMPK activation and autophagy were assessed by immunoblotting. Alveolarization was assessed using radial alveolar counts, mean linear intercept measurements, and quantification of alveolar septal myofibroblasts. Relative mRNA expression of M1-like and M2-like genes was assessed in AMs isolated from BAL fluid from wild-type, LysMCre--Becn1fl/fl, and LysMCre+-Becn1fl/fl mice after neonatal hyperoxia exposure. AICAR treatment resulted in AMPK activation and induction of autophagic activity in whole-lung and BAL cell lysates and attenuated hyperoxia-induced alveolar simplification in neonatal lungs. AICAR-treated control but not Beclin1-deficient AMs demonstrated significantly decreased expression of M1-like markers and significantly increased expression of M2-like markers. In conclusion, pharmacologic activation of AMPK by AICAR resulted in induction of autophagy and played a protective role, at least in part, through attenuation of proinflammatory signaling in AMs via autophagy-dependent mechanisms in a murine model of BPD.

AICAR Protects against High Palmitate/High Insulin-Induced Intramyocellular Lipid Accumulation and Insulin Resistance in HL-1 Cardiac Cells by Inducing PPAR-Target Gene Expression.

Here we studied the impact of 5-aminoimidazole-4-carboxamide riboside (AICAR), a well-known AMPK activator, on cardiac metabolic adaptation. AMPK activation by AICAR was confirmed by increased phospho-Thr(172)-AMPK and phospho-Ser(79)-ACC protein levels in HL-1 cardiomyocytes. Then, cells were exposed to AICAR stimulation for 24 h in the presence or absence of the AMPK inhibitor Compound C, and the mRNA levels of the three PPARs were analyzed by real-time RT-PCR. Treatment with AICAR induced gene expression of all three PPARs, but only the Ppara and Pparg regulation were dependent on AMPK. Next, we exposed HL-1 cells to high palmitate/high insulin (HP/HI) conditions either in presence or in absence of AICAR, and we evaluated the expression of selected PPAR-targets genes. HP/HI induced insulin resistance and lipid storage was accompanied by increased Cd36, Acot1, and Ucp3 mRNA levels. AICAR treatment induced the expression of Acadvl and Glut4, which correlated to prevention of the HP/HI-induced intramyocellular lipid build-up, and attenuation of the HP/HI-induced impairment of glucose uptake. These data support the hypothesis that AICAR contributes to cardiac metabolic adaptation via regulation of transcriptional mechanisms.

The AMPK Activator Aicar Ameliorates Age-Dependent Myocardial Injury in Murine Hemorrhagic Shock.

The development of myocardial dysfunction in patients with hemorrhagic shock is significantly impacted by the patient age. AMP-activated protein kinase (AMPK) is a pivotal orchestrator of energy homeostasis, which coordinates metabolic recovery after cellular stress. We investigated whether AMPK-regulated pathways are age-dependent in hemorrhage-induced myocardial injury and whether AMPK activation by 5-amino-4-imidazolecarboxamide riboside (AICAR) affords cardioprotective effects. Anesthetized C57/BL6 young (3-5 months old) and mature (9-12 months old) male mice were subjected to hemorrhagic shock by blood withdrawing followed by resuscitation with shed blood and Lactated Ringer's solution. Mice were sacrificed at 3&#x200a;h after resuscitation, and plasma and hearts were harvested for biochemical assays. Vehicle-treated mature mice exhibited higher myocardial injury and higher levels of plasma biomarkers of cardiovascular injury (endocan and follistatin) when compared with young mice. Cardiac cell mitochondrial structure was also markedly impaired in vehicle-treated mature mice when compared with young mice. At molecular analysis, an increase of the phosphorylated catalytic subunit pAMPK&#x3b1; was associated with nuclear translocation of the peroxisome proliferator-activated receptor &#x3b3; coactivator-&#x3b1; in young, but not mature mice. No changes in autophagy were observed as evaluated by the conversion of the light-chain (LC)3B-I protein to LC3B-II form. Treatment with AICAR ameliorated myocardial damage in both age groups. However, AICAR therapeutic effects were less effective in mature mice than young mice and involved distinct mechanisms of action. Thus, our data demonstrate that during hemorrhagic shock AMPK-dependent metabolic mechanisms are important for mitigating myocardial injury. However, these mechanisms are less competent with age.

5-Aminoimidazole-4-carboxamide-1-&#x3b2;-D-ribofuranoside-attenuates LPS/D-Gal-induced acute hepatitis in mice.

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-&#x3b2;-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-&#x3b1;, 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.

Acute activation of endothelial AMPK surprisingly inhibits endothelium-dependent hyperpolarization-like relaxations in rat mesenteric arteries.

Endothelium-dependent hyperpolarizations (EDHs) contribute to the regulation of peripheral resistance. They are initiated through opening of endothelial calcium-activated potassium channels (KCa ); the potassium ions released then diffuse to the underlying smooth muscle cells, causing hyperpolarization and thus relaxation. The present study aimed to examine whether or not AMPK modulates EDH-like relaxations in rat mesenteric arteries. Arterial rings were isolated for isometric tension recording. AMPK activity and protein level were measured by ELISA and western blotting respectively. The AMPK activator, AICAR, reduced ACh-induced EDH-like relaxations and increased AMPK activity in preparations with endothelium; these responses were prevented by compound C, an AMPK inhibitor. AICAR inhibited relaxations induced by SKA-31 (opener of endothelial KCa ) but did not affect potassium-induced, hyperpolarization-attributable relaxations or increase AMPK activity in preparations without endothelium. A769662, another AMPK activator, not only caused a similar inhibition of relaxations to ACh and SKA-31 in preparations with endothelium but also inhibited hyperpolarization-attributable relaxations and augmented AMPK activity in rings without endothelium. Protein levels of total AMPK&#x3b1;, AMPK&#x3b1;1, or AMPK&#x3b2;1/2 were comparable between preparations with and without endothelium. Activation of endothelial AMPK, by either AICAR or A769662, acutely inhibits EDH-like relaxations of rat mesenteric arteries. Furthermore, A769662 inhibits signalling downstream of smooth muscle hyperpolarization. In view of the major blunting effect of AMPK activation on EDH-like relaxations, caution should be applied when administering therapeutic agents that activate AMPK in patients with endothelial dysfunction characterized by reduced production and/or bioavailability of NO.

AMPK activator AICAR ameliorates ischaemia reperfusion injury in the rat kidney.

BACKGROUND AND PURPOSE Renal ischaemia/reperfusion (RI/R) injury is a major cause of acute kidney injury (AKI) and an important determinant of long-term kidney dysfunction. AMP-kinase and histone deacetylase sirtuin 1 (SIRT1) regulate cellular metabolism and are activated during hypoxia. We investigated whether AMP-kinase activator AICAR (5-amino-4-imidazolecarboxamide riboside-1-&#x3b2;-D-ribofuranoside) ameliorates RI/R injury and whether SIRT1 is involved in the pathogenesis. EXPERIMENTAL APPROACH Eight-week-old Sprague Dawley rats were divided into five groups: (i) sham-operated group; (ii) I/R group (40 min bilateral ischaemia followed by 24 h of reperfusion; (iii) I/R group + AICAR 50 mg&#xb7;kg(-1) i.v. given 60 min before operation; (iv). I/R group + AICAR 160 mg&#xb7;kg(-1) i.v; (v) I/R group + AICAR 500 mg&#xb7;kg(-1) i.v. Serum creatinine and urea levels were measured. Acute tubular necrosis (ATN), monocyte/macrophage infiltration and nitrotyrosine expression were scored. Kidney AMP-activated protein kinase (AMPK) and SIRT1 expressions were measured. KEY RESULTS Highest dose of AICAR decreased serum creatinine and urea levels, attenuated I/R injury-induced nitrosative stress and monocyte/macrophage infiltration, and ameliorated the development of ATN. Kidney I/R injury was associated with decreased AMPK phosphorylation and a fivefold increase in kidney SIRT1 expression. AICAR increased pAMPK/AMPK ratio and prevented the I/R-induced increase in renal SIRT1 expression. CONCLUSIONS AND IMPLICATIONS AICAR protects against the development of ATN after kidney I/R injury. Activators of kidney AMP kinase may thus represent a novel therapeutic approach to patients susceptible to AKI and to those undergoing kidney transplantation. The present study also suggests a role for SIRT1 in the pathogenesis of RI/R injury.

CCAL1 enhances osteoarthritis through the NF-&#x3ba;B/AMPK signaling pathway.

Osteoarthritis (OA) is a chronic joint disease characterized by articular cartilage degeneration and secondary osteogenesis. It has been previously demonstrated that the CCAL1 locus is the gene encoding tumor necrosis factor receptor superfamily member 11B (TNFRSF11B). The purpose of this study was to demonstrate the role of CCAL1 in OA progression and to elucidate its molecular mechanisms. We report that CCAL1 is highly expressed in the cartilage of OA patients and its expression level is positively correlated with the severity of OA. We found that CCAL1 causes a switch to the fibrosis-prone phenotype of Human Chondrocyte-Osteoarthritis (HC-OA) cells. In addition, CCAL1 enhances cell viability and promotes the proliferation of HC-OA cells. Finally, the detection of proteins associated with the NF-&#x3ba;B/AMPK signaling pathway by western blot suggested that CCAL1 exerts its role on HC-OA cells by activating the NF-&#x3ba;B signaling pathway and inhibiting the AMPK signaling pathway, which was verified through the addition of NF-&#x3ba;B inhibitor caffeic acid phenethyl ester (CAPE) and AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR). In summary, we report that CCAL1 may promote OA through the NF-&#x3ba;B and AMPK signaling pathways.

Sustained protection by acadesine against ischemia- and reperfusion-induced injury. Studies in the transplanted rat heart.

We have shown that acadesine (AICAr: 5-amino-4-imidazole carboxamide riboside) improves the early recovery of function of the ischemic and reperfused rat heart. In the present studies we used the transplanted rat heart, with reperfusion for up to 24 hours, to assess whether the beneficial effect of acadesine is a transient or a sustained phenomenon (i.e., to determine whether the drug improves the extent of recovery or only the rate). Hearts (n = 8 per group) were excised and immediately arrested with an infusion (2 minutes at 20 degrees C) of the St. Thomas' Hospital cardioplegic solution with or without the addition of acadesine (20 mumol/l). They were then subjected to 4 hours of global ischemia (20 degrees C), and the cardioplegic solution (with or without acadesine) was infused for 2 minutes every 30 minutes. The hearts then were transplanted (1 hour additional ischemic time) into the abdomens of recipient rats, which had been given acadesine (100 mg/kg i.v.) or saline. They were reperfused in situ for 30 minutes or 24 hours and then excised and perfused aerobically for 20 minutes. Contractile function was assessed, and the hearts were taken for metabolite analysis. Two sets of four groups (n = 8 per group) were studied (one set with 30 minutes and the other with 24 hours of reperfusion): group A, acadesine-free control; group B, acadesine during cardioplegia alone; group C, acadesine during reperfusion alone; and group D, acadesine during both cardioplegia and reperfusion. With 30 minutes of reperfusion, a significant improvement in functional recovery was seen in the two groups (groups B and D) in which acadesine had been added to the cardioplegic solution. Left ventricular developed pressure (LVDP) at 12 mm Hg of left ventricular end-diastolic pressure (LVEDP) was 104 +/- 3 mm Hg in both groups versus 88 +/- 3 mm Hg in the acadesine-free controls (p less than 0.05). No protection was observed after 30 minutes of reperfusion when acadesine had been added during reperfusion alone (89 +/- 4 mm Hg). In contrast, after 24 hours of reperfusion there was a significant improvement in postischemic LVDP in all acadesine-treated groups (group B, 104 +/- 6 mm Hg; group C, 106 +/- 7 mm Hg; and group D, 117 +/- 3 mm Hg versus only 73 +/- 6 mm Hg in the acadesine-free controls; p less than 0.05 in each case). Metabolite analysis indicated that at the end of ischemia ATP was less depleted and levels of tissue adenosine were higher in the acadesine group. During early (30 minutes) reperfusion, acadesine produced higher mean ATP contents, although this achieved a level of statistical significance only when the drug was administered during both cardioplegia and reperfusion. After 24 hours of reperfusion, the adenine nucleotide pools were similar in all groups. Acadesine can afford sustained functional protection against injury during extended periods of ischemia and reperfusion. We present evidence that the beneficial effect of acadesine may be mediated by two different components, with one operative during ischemia and early reperfusion and the other acting later in the reperfusion period.

AMPK activation by AICAR reduces diet induced fatty liver in C57BL/6 mice.

Dysregulation of 5'-adenosine monophosphate-activated protein kinase (AMPK) occurs in metabolic disorders including non-alcoholic fatty liver disease (NAFLD) which makes it a molecular target for treatment. An AMPK activator, 5-aminoimidazole-4-carboxamide-1-&#x3b2;-D-ribofuranoside (AICAR) alleviates NAFLD in experimental rats, however the specific mechanism remains to be explored. We aimed to study the effect of AICAR on lipid levels, oxidant-antioxidant balance, AMPK and mTOR activation and FOXO3 gene expression in liver of mice model. Fatty liver was induced in two groups of C57BL/6 mice (groups 2 and 3) by providing a high fat high fructose diet (HFFD) for 10 weeks while groups 1 and 4 animals were fed normal pellet. For the last two weeks, groups 3 and 4 were administered AICAR (150&#xa0;mg/kg bw/day, i.p.) while groups 1 and 2 were administered saline. AICAR decreased fatty liver, decreased glucose and insulin in circulation, prevented the accumulation of triglycerides and collagen and ameliorated oxidative stress in HFFD fed mice. At the molecular level, AICAR upregulated FOXO3 and p-AMPK expression and reduced p-mTOR expression. AMPK activation may involve FOXO3 in protection against NAFLD. The role of AMPK, mTOR and FOXO3 crosstalk in NAFLD needs to be characterised in future.

Metformin Attenuates Inflammation and Fibrosis in Thyroid-Associated Ophthalmopathy.

The pathogenesis of thyroid-associated ophthalmopathy (TAO) is still unclear, and therapeutic drugs have great limitations. As metformin has multiple therapeutic effects in many autoimmune diseases, we explored the effects of metformin on TAO in an in vitro fibroblast model. We used orbital connective tissues and fibroblasts that were obtained from TAO patients and normal controls. The activity of adenosine monophosphate-activated protein kinase (AMPK) and the levels of inflammatory or fibrotic factors were examined by immunofluorescence (IF) and immunohistochemistry (IHC). Quantitative real-time polymerase chain reaction (qPCR), cytokine quantification by enzyme-linked immunosorbent sssay (ELISA), IF, and western blotting (WB) were used to measure the expression of factors related to inflammation, fibrosis, and autophagy. To determine the anti-inflammatory and antifibrotic mechanisms of metformin, we pretreated cells with metformin, 5-aminoimidazole-4-carboxamide 1-&#x3b2;-D-ribofuranoside (AICAR, an AMPK activator) or compound C (CC, an AMPK inhibitor) for 24 h and used WB to verify the changes in protein levels in the AMPK/mammalian target of rapamycin (mTOR) pathway. We determined that the low activity of AMPK in the periorbital tissue of TAO patients may be closely related to the occurrence and development of inflammation and fibrosis, and metformin exerts multiple effects by activating AMPK in TAO. Furthermore, we suggest that AMPK may be a potential target of TAO therapy.

The AMPK-activator AICAR in thyroid cancer: effects on CXCL8 secretion and on CXCL8-induced neoplastic cell migration.

The AMPK-activator AICAR recently raised great interest for its anti-cancer properties. With specific regard to thyroid cancer, AICAR reduces cancer cell growth, invasion and metastasis. CXCL8, a chemokine with several recognized tumorigenic effects, is abundantly secreted in thyroid cancer microenvironment. The aim of this study was to investigate if AICAR could inhibit the basal and the TNF&#x3b1;-induced CXCL8 secretion in normal human thyroid cells (NHT) and in thyroid cancer cell lines TPC-1 and BCPAP (RET/PTC and BRAFV600e mutated, respectively). The effect of AICAR on basal and CXCL8-induced cell migration was assessed. Cells were incubated with AICAR (0.05, 0.5, 1, 2&#xa0;mM) alone or in combination with TNF-&#x3b1; (10&#xa0;ng/ml) for 24&#xa0;h. CXCL8 concentrations were measured in cell supernatants. Transwell migration assays were performed in NHT, TPC-1 and BCPAP, basally and after treatment with AICAR (2&#xa0;mM) and rh-CXCL8 (50&#xa0;ng/ml) alone or in combination. AICAR dose dependently inhibited the basal secretion of CXCL8 in TPC-1 (F&#x2009;=&#x2009;4.26; p&#x2009;<&#x2009;0.007) and BCPAP (F&#x2009;=&#x2009;6.75; p&#x2009;<&#x2009;0.0001) but not in NHT. TNF&#x3b1;-induced CXCL8 secretion was dose dependently reduced by AICAR in NHT (F&#x2009;=&#x2009;9.99; p&#x2009;<&#x2009;0.0001), TPC-1 (F&#x2009;=&#x2009;9.25; p&#x2009;<&#x2009;0.0001) and BCPAP (F&#x2009;=&#x2009;6.82; p&#x2009;<&#x2009;0.0001). AICAR significantly reduced the basal migration of TPC-1 and BCPAP but not of NHT. CXCL8-induced cell migration was inhibited in NHT, TPC-1 and BCPAP. This is the first demonstration of the inhibition of CXCL8 secretion exerted by AICAR in TPC-1 and BCPAP indicating that the anti-cancer properties of AICAR are, at least in part, mediated by its ability to reduce the pro-tumorigenic effects of CXCL8.

AMPK activator AICAR in combination with anti-mouse IL10 mAb restores the functionality of intra-tumoral Tfh cells in the 4T1 mouse model.

4T1 cell-mediated TNBC breast cell carcinoma is a highly malignant mice tumor model which resembles an advanced stage of breast cancer in humans. Tumor progression occurs depending on the intra-tumoral balance of pro- and anti- tumorigenic immune cells. Enhancement of T-cell-mediated anti-tumor immunity will be advantageous for inhibiting tumor progression and improving the efficacy of cancer therapy. This study is focused on alleviating suppressed anti-tumor immune response by improving CD4+ T follicular helper cell (Tfh) response in 4T1 mice. We employed anti-IL10 mAb along with metabolic drugs 2-deoxy-D-glucose (2DG) which inhibits the glycolytic pathway and Cpt1a inhibitor Etomoxir which inhibits FAO. AMPK activator AICAR with or without anti-IL10 mAb was also used to ameliorate metabolic stress and exhaustion faced by immune cells. Our results demonstrate that synergistic treatment with 2DG/Etomoxir&#xa0;+&#xa0;anti-IL10 mAb induced Tfh cell, memory B, and GC B cell response more potently compared to treatment with 2DG or Etomoxir treatment alone as observed in several LNs and tumor tissue of 4T1 mouse. However, AICAR&#xa0;+&#xa0;anti-IL10 mAb increased the frequency of intratumoral Tfh cells, simultaneously downregulated Tfr cells; and improved humoral response by stimulating upregulation of memory B, GC B, and plasmablasts in tumor-draining, axillary, and mesenteric LNs of 4T1 mouse.

Adenylate kinase 4 (AK4) deficiency prevents vascular smooth muscle cell phenotypic switching by regulating mitochondrial dysfunction through AMPK&#x3b1; inactivation.

Mitochondrial dynamics are key mechanism regulating the conversion of vascular smooth muscle cells (VSMCs) from a contractile to a synthetic phenotype, which is involved in neointima formation and restenosis. However, the underlying mechanisms leading to mitochondrial dysfunction are not fully understood. Western blot was used to detect the expression of relevant molecules at the protein level. CCK-8, EdU assays, and transwell were used to test cell proliferation and migration capacity. Flow cytometry was used to assess cell cycle and ROS. AK4 was upregulated in 10&#xa0;% fetal bovine serum (FBS)- and 20&#xa0;ng/mL platelet-derived growth factor-BB (PDGF-BB)-induced human aortic VSMCs (HASMCs). Knockdown of AK4 suppressed the proliferation and synthetic phenotype of HASMCs, while AK4 overexpression accelerated it. Mechanistically, AK4 interacted with protein kinase AMP-activated catalytic subunit alpha (AMPK&#x3b1;) and promoted the phosphorylation of AMPK&#x3b1; at Thr172, which reduced mitochondrial oxidative damage and improved mitochondrial function. Furthermore, activation of AMPK&#x3b1; by metformin or AICAR (acadesine) reversed the inhibitory effects of AK4 deficiency on HASMC phenotypic switching. Moreover, overexpression of wild-type AMPK&#x3b1; counteracted the effects of AK4 knockdown, whereas mutational inactivation of AMPK&#x3b1; (AMPK&#x3b1;T172A) was not effective in reversing the effect on HASMCs. Our findings suggest that AK4 is a novel regulator of AMPK&#x3b1; activity and positively regulates VSMC dedifferentiation, proliferation, and migration. Targeted inhibition of AK4 may be a potential approach for the treatment of neointima formation and restenosis.

AICAR, an AMPK activator, has protective effects on alcohol-induced fatty liver in rats.

Previous work with metformin has shown that this antidiabetic agent improves nonalcoholic fatty liver in ob/ob mice. AMP-activated protein kinase (AMPK) is one of the major cellular regulators of lipid and glucose metabolism, and reportedly mediates the beneficial metabolic effects of metformin. In this study, we examined the effects of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), an AMPK activator, on an experimental model of ethanol-induced hepatic steatosis. Rats were randomly divided into three groups: (A) rats fed ethanol-containing liquid diet for six weeks; (B) rats pair-fed ethanol-containing liquid diet for six weeks, during the last three weeks of which they were subcutaneously injected with 0.5 mg AICAR/g body weight per day; (C) rats pair-fed isocaloric liquid diet without ethanol for six weeks. At the end of the six-week period, the animals were sacrificed. Serum and liver specimens were analyzed using biochemical and histologic methods, as well as real-time PCR. Chronic ethanol feeding resulted in fatty liver both histologically and biochemically, whereas AICAR administration attenuated the degree of change in the liver. AICAR also decreased the hepatic sterol regulatory factor binding protein-1c (SREBP-1c) and reduced fatty acid synthase (FAS) expression; these changes led to reduced triglyceride synthesis in rat livers. Furthermore, detection of 4-hydroxy-2-nonenal (4-HNE)-protein adducts showed that the AICAR treatment also decreased the products of lipid peroxidation. In this preclinical rat model, AICAR, an AMPK activator, appears to protect the liver from fatty changes associated with chronic alcohol use. As such, AICAR may have a role in the treatment and prevention of alcohol-induced fatty liver.

The Role of AMPK/mTOR Modulators in the Therapy of Acute Myeloid Leukemia.

Differentiation therapy of acute promyelocytic leukemia with all-trans retinoic acid represents the most successful pharmacological therapy of acute myeloid leukemia (AML). Numerous studies demonstrate that drugs that inhibit mechanistic target of rapamycin (mTOR) and activate AMP-kinase (AMPK) have beneficial effects in promoting differentiation and blocking proliferation of AML. Most of these drugs are already in use for other purposes; rapalogs as immunosuppressants, biguanides as oral antidiabetics, and 5-amino-4-imidazolecarboxamide ribonucleoside (AICAr, acadesine) as an exercise mimetic. Although most of these pharmacological modulators have been widely used for decades, their mechanism of action is only partially understood. In this review, we summarize the role of AMPK and mTOR in hematological malignancies and discuss the possible role of pharmacological modulators in proliferation and differentiation of leukemia cells.

AMP-independent activator of AMPK for treatment of mitochondrial disorders.

Mitochondrial diseases are a clinically heterogenous group of disorders caused by respiratory chain dysfunction and associated with progressive, multi-systemic phenotype. There is no effective treatment or cure, and no FDA-approved drug for treating mitochondrial disease. To identify and characterize potential therapeutic compounds, we developed an in vitro screening assay and identified a group of direct AMP-activated protein kinase (AMPK) activators originally developed for the treatment of diabetes and metabolic syndrome. Unlike previously investigated AMPK agonists such as AICAR, these compounds allosterically activate AMPK in an AMP-independent manner, thereby increasing specificity and decreasing pleiotropic effects. The direct AMPK activator PT1 significantly improved mitochondrial function in assays of cellular respiration, energy status, and cellular redox. PT1 also protected against retinal degeneration in a mouse model of photoreceptor degeneration associated with mitochondrial dysfunction and oxidative stress, further supporting the therapeutic potential of AMP-independent AMPK agonists in the treatment of mitochondrial disease.

AMPK attenuates bupivacaine-induced neurotoxicity.

Bupivacaine has been widely used as a long-acting local anesthetic. However, evidence strongly suggests that bupivacaine causes apoptosis. AMP-activated protein kinase (AMPK) regulates metabolic homeostasis and mediates cellular protection from stress. We hypothesized that AMPK may be cytoprotective in bupivacaine-treated Schwann cells. To explore this, we applied bupivacaine to the RT4-D6P2T Schwann cell line. The expression of phosphorylated AMPK was compared after bupivacaine treatment. Bupivacaine induced cell death in a time- and dose- [50% lethal dose (LD(50)) = 316 microM] dependent manner, and increased expression of phosphorylated AMPK after bupivacaine treatment. Bupivacaine-induced cytotoxicity was attenuated by AICAR (an AMPK activator), whereas compound C (an AMPK inhibitor) enhanced it. The cytoprotective effect of AICAR was reversed in the presence of iodotubercidin, an AICAR inhibitor. Our results suggest that the AMPK pathway may protect Schwann cells from bupivacaine-induced cytotoxicity.

NMDARs activation regulates endothelial ferroptosis via the PP2A-AMPK-HMGB1 axis.

N-methyl-D-aspartate receptors (NMDARs) are ligand-gated, voltage-dependent channels of the ionotropic glutamate receptor family. The present study explored whether NMDAR activation induced ferroptosis in vascular endothelial cells and its complicated mechanisms in vivo and in vitro. Various detection approaches were used to determine the ferroptosis-related cellular iron content, lipid reactive oxygen species (LOS), siRNA molecules, RNA-sequence, MDA, GSH, and western blotting. The AMPK activator Acadesine (AICAR), HMGB1 inhibitor glycyrrhizin (GLY), PP2A inhibitor LB-100, and NMDAR inhibitor MK801 were used to investigate the involved in vivo and in vitro pathways. The activation of NMDAR with L-glutamic acid (GLU) or NMDA significantly promoted cellular ferroptosis, iron content, MDA, and the PTGS2 expression, while decreasing GPX4 expression and GSH concentration in human umbilical vein endothelial cells (HUVECs), which was reversed by ferroptosis inhibitors Ferrostatin-1(Fer-1), Liproxstatin-1 (Lip-1), or Deferoxamine (DFO). RNA-seq revealed that ferroptosis and SLC7A11 participate in NMDA or GLU-mediated NMDAR activation. The PP2A-AMPK-HMGB1 pathway was majorly associated with NMDAR activation-induced ferroptosis, validated using the PP2A inhibitor LB-100, AMPK activator AICAR, or HMGB1 siRNA. The role of NMDAR in ferroptosis was validated in HUVECs induced with the ferroptosis activator errasin or RSL3 and counteracted by the NMDAR inhibitor MK-801. The in vivo results showed that NMDA- or GLU-induced ferroptosis and LOS production was reversed by MK-801, LB-100, AICAR, MK-801, and GLY, confirming that the PP2A-AMPK-HMGB1 pathway is involved in NMDAR activation-induced vascular endothelium ferroptosis. In conclusion, the present study demonstrated a novel role of NMDAR in endothelial cell injury by regulating ferroptosis via the PP2A-AMPK-HMGB1 pathway.