Methotrexate (MTX) is the mainstay treatment for juvenile idiopathic arthritis (JIA), however approximately 30% of children will fail to respond to the drug. Identification of genetic predictors of response to MTX would be invaluable in developing optimal treatment strategies for JIA. Using a candidate gene approach, single nucleotide polymorphisms (SNPs) within genes in the metabolic pathway of MTX, were investigated for association with response to treatment in JIA cases.

5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR), an analog of AMP is widely used as an activator of AMP-kinase (AMPK), a protein that regulates the responses of the cell to energy change. Recently, we showed that AICAR-induced AMPK activation inhibits the growth of retinoblastoma cells in vitro by decreasing cyclins and by inducing apoptosis and S-phase arrest. In this study, we investigated the effects of AMPK activator AICAR on the growth of retinoblastoma in vivo. Intraperitoneal injection of AICAR resulted in 48% growth inhibition of Y79 retinoblastoma cell tumors in mice. Tumors isolated from mice treated with AICAR had decreased expression of Ki67 and increased apoptotic cells (TUNEL positive) compared with the control. In addition, AICAR treatment suppressed significantly tumor vessel density and macrophage infiltration. We also showed that AICAR administration resulted in AMPK activation and mTOR pathway inhibition. Paradoxically observed down-regulation of p21, which indicates that p21 may have a novel function of an oncogene in retinoblastoma tumor. Our results indicate that AICAR treatment inhibited the growth of retinoblastoma tumor in vivo via AMPK/mTORC1 pathway and by apoptogenic, anti-proliferative, anti-angiogenesis mechanism. AICAR is a promising novel non-chemotherapeutic drug that may be effective as an adjuvant in treating Retinoblastoma.

All-trans retinoic acid (ATRA)-based treatment of acute promyelocytic leukemia (APL) is the most successful pharmacological treatment of acute myeloid leukemia (AML). Recent development of inhibitors of mutated isocitrate dehydrogenase and dihydroorotate dehydrogenase (DHODH) has revived interest in differentiation therapy of non-APL AML. Our previous studies demonstrated that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) induced differentiation of monocytic cell lines by activating the ATR/Chk1 via pyrimidine depletion. In the present study, the effects of AICAr on the viability and differentiation of primary AML blasts isolated from bone marrow of patients with non-APL AML were tested and compared with the effects of DHODH inhibitor brequinar and ATRA.

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.

Pharmacological modulators of AMP-dependent kinase (AMPK) have been suggested in treatment of cancer. The biguanide metformin and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) have been reported to inhibit proliferation of solid tumors and hematological malignancies, but their role in differentiation is less explored. Our previous study demonstrated that AICAR alone induced AMPK-independent expression of differentiation markers in monocytic U937 leukemia cells, and no such effects were observed in response to metformin. The aim of this study was to determine the mechanism of AICAR-mediated effects and to test for the possible role of autophagy in differentiation of leukemia cells. The results showed that AICAR-mediated effects on the expression of differentiation markers were not mimicked by A769662, a more specific direct AMPK activator. Long-term incubation of U937 cells with AICAR and other differentiation agents, all-trans-retinoic acid (ATRA) and phorbol 12-myristate 13-acetate, increased the expression of the autophagy marker LC3B-II, and these effects were not observed in response to metformin. Western blot and immunofluorescence analyses of U937 cells treated with bafilomycin A1 or transfected with mRFP-GFP-LC3 proved that the increase in the expression of LC3B-II was due to an increase in autophagy flux, and not to a decrease in lysosomal degradation. 3-Methyladenine inhibited the expression of differentiation markers in response to all inducers, but had stimulatory effects on autophagy flux at dose that effectively inhibited the production of phosphatidylinositol 3-phosphate. The small inhibitory RNA-mediated down-modulation of Beclin 1 and hVPS34 had no effects on AICAR and ATRA-mediated increase in the expression of differentiation markers. These results show that AICAR and other differentiation agents induce autophagy flux in U937 cells and that the effects of AICAR and ATRA on the expression of differentiation markers do not depend on the normal levels of key proteins of the classical or canonical autophagy pathway.

Recent study in our laboratory has demonstrated that BEFV-induced autophagy via activation of the PI3K/Akt/NF-κB and Src/JNK pathways and suppression of the PI3K-AKt-mTORC1 pathway is beneficial for virus replication. In the current study, we found that both aspirin and 5-aminoimidazole-4-carboxamide-1-β-riboside (AICAR) siginificantly attenuated virus replication by inhibiting BEFV-induced autophagy via suppressing the BEFV-activated PI3K/Akt/NF-κB and Src/JNK pathways as well as inducing reversion of the BEFV-suppressed PI3K-Akt-mTORC1 pathway. AICAR reversed the BEFV-activated PI3K/Akt/NF-κB and Src/JNK pathways at the early to late stages of infection and induced reversion of the BEFV-suppressed PI3K-AKt-mTORC1 pathway at the late stage of infection. Our findings reveal that inhibition of BEFV-induced autophagy by AICAR is independent of AMPK. Furthermore, we found that AICAR transcriptionally downregulates the ATG related genes ULK1, Beclin 1, and LC3 and enhances Atg7 degradation by the proteasome pathway. Aspirin suppresses virus replication by inhibiting BEFV-induced autophagy. It directly suppressed the NF-κB pathway and reversed the BEFV-activated Src/JNK pathway at the early stage of infection and reversed the BEFV-suppressed PI3K/Akt/mTOR pathway at the late stage of infection. The current study provides mechanistic insights into the effects of aspirin and AICAR on BEFV replication through suppression of BEFV-induced autophagy.

Skeletal muscle is a major contributor to whole-body glucose homeostasis and is an important endocrine organ. To date, few studies have undertaken the large-scale identification of skeletal muscle-derived secreted proteins (myokines), particularly in response to stimuli that activate pathways governing energy metabolism in health and disease. Whereas the AMP-activated protein kinase (AMPK) and insulin-signaling pathways have received notable attention for their ability to independently regulate skeletal muscle substrate metabolism, little work has examined their ability to re-pattern the secretome. The present study coupled the use of high-resolution MS-based proteomics and bioinformatics analysis of conditioned media derived from 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR-an AMPK activator)- and insulin-treated differentiated C2C12 myotubes. We quantified 858 secreted proteins, including cytokines and growth factors such as fibroblast growth factor-21 (Fgf21). We identified 377 and 118 proteins that were significantly altered by insulin and AICAR treatment, respectively. Notably, the family of insulin growth factor binding-proteins (Igfbp) was differentially regulated by each treatment. Insulin- but not AICAR-induced conditioned media increased the mitochondrial respiratory capacity of myotubes, potentially via secreted factors. These findings may serve as an important resource to elucidate secondary metabolic effects of insulin and AICAR stimulation in skeletal muscle.

Activation of AMPK has been associated with pro-atrophic signaling in muscle. However, AMPK also has anti-inflammatory effects, suggesting that in cachexia, a syndrome of inflammatory-driven muscle wasting, AMPK activation could be beneficial. Here we show that the AMPK agonist AICAR suppresses IFNγ/TNFα-induced atrophy, while the mitochondrial inhibitor metformin does not. IFNγ/TNFα impair mitochondrial oxidative respiration in myotubes and promote a metabolic shift to aerobic glycolysis, similarly to metformin. In contrast, AICAR partially restored metabolic function. The effects of AICAR were prevented by the AMPK inhibitor Compound C and were reproduced with A-769662, a specific AMPK activator. AICAR and A-769662 co-treatment was found to be synergistic, suggesting that the anti-cachectic effects of these drugs are mediated through AMPK activation. AICAR spared muscle mass in mouse models of cancer and LPS induced atrophy. Together, our findings suggest a dual function for AMPK during inflammation-driven atrophy, wherein it can play a protective role when activated exogenously early in disease progression, but may contribute to anabolic suppression and atrophy when activated later through mitochondrial dysfunction and subsequent metabolic stress.

Purine nucleotide synthesis in Plasmodium falciparum takes place solely by the purine salvage pathway in which preformed purine base(s) are salvaged from the host and acted upon by a battery of enzymes to generate AMP and GMP. Inhibitors of this pathway have a potent effect on the in vitro growth of P. falciparum and are hence, implicated as promising leads for the development of new generation anti-malarials. Here, we describe the mechanism of inhibition of the intraerythrocytic growth of P. falciparum by the purine nucleoside precursor, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). Our results show that AICAR toxicity is mediated through the erythrocyte in which AICAR is phosphorylated to its nucleotide, ZMP. Further, purine metabolite labeling of the parasitized erythrocytes by [(3)H]-hypoxanthine, in the presence of AICAR, showed a significant decrease in radioactive counts in adenylate fractions but not in guanylate fractions. The most dramatic effect on parasite growth was observed when erythrocytes pretreated with AICAR were used in culture. Pretreatment of erythrocytes with AICAR led to significant intracellular accumulation of ZMP and these erythrocytes were incapable of supporting parasite growth. These results implicate that in addition to the purine salvage pathway in P. falciparum, AICAR alters the metabolic status of the erythrocytes, which inhibits parasite growth. As AICAR and ZMP are metabolites in the human serum and erythrocytes, our studies reported here throw light on their possible role in disease susceptibility, and also suggests the possibility of AICAR being a potential prophylactic or chemotherapeutic anti-malarial compound.

The effect of deer antler extract on muscle differentiation and muscle atrophy were evaluated to minimize muscle loss following aging. Various deer antler extracts (HWE, hot water extract of deer antler; FE, HWE of fermented deer antler; ET, enzyme-assisted extract of deer antler; UE, extract prepared by ultrasonication of deer antler) were evaluated for their effect on muscle differentiation and inhibition of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)-induced muscle atrophy in C2C12 cells. Morphological changes according to the effect of antler extracts on muscle differentiation were confirmed by Jenner-Giemsa staining. In addition, the expression levels of genes related to muscle differentiation and atrophy were confirmed through qRT-PCR. In the presence of antler extracts, the length and thickness of myotubes and myogenin differentiation 1 (MyoD1) and myogenic factor 5 (Myf5) gene expression were increased compared to those in the control group (CON). Gene expression of AMP-activated protein kinase (AMPK), MyoD1, and myogenin, along with the muscle atrophy factors muscle RING finger-1 (MuRF-1) and forkhead box O3a (FoxO3a) upon addition of deer antler extracts to muscle-atrophied C2C12 cells was determined by qRT-PCR after treatment with AICAR. The expression of MuRF-1 and FoxO3a decreased in the groups treated with antler extracts compared to that in the group treated with AICAR alone. In addition, gene expression of MyoD1 and myogenin in the muscle atrophy cell model was significantly increased compared that into the CON. Therefore, our findings indicate that antler extract can increase the expression of MyoD1, Myf5 and myogenin, inhibit muscle atrophy, and promote muscle differentiation.

It is debatable whether AMP-activated protein kinase (AMPK) activation is involved in anti-apoptotic or pro-apoptotic signaling. AICAR treatment increases AMPK-α1 phosphorylation, decreases intracellular reactive oxygen species (ROS) levels, and significantly increases Annexin V-positive cells, DNA laddering, and caspase activity in human myeloid cell. AMPK activation is therefore implicated in apoptosis. However, AMPK-α1-knockdown THP-1 cells are more sensitive to apoptosis than control THP-1 cells are, suggesting that the apoptosis is AMPK-independent. Low doses of AICAR induce cell proliferation, whereas high doses of AICAR suppress cell proliferation. Moreover, these effects are significantly correlated with the downregulation of intracellular ROS, strongly suggesting that AICAR-induced apoptosis is critically associated with the inhibition of NADPH oxidase by AICAR. Collectively, our results demonstrate that in AICAR-induced apoptosis, intracellular ROS levels are far more relevant than AMPK activation.

In Salmonella enterica, aminoimidazole carboxamide ribotide (AICAR) is a purine biosynthetic intermediate and a substrate of the AICAR transformylase/IMP cyclohydrolase (PurH) enzyme. When purH is eliminated in an otherwise wild-type strain, AICAR accumulates and indirectly inhibits synthesis of the essential coenzyme thiamine pyrophosphate (TPP). In this study, untargeted metabolomics approaches were used to i) corroborate previously defined metabolite changes, ii) define the global consequences of AICAR accumulation and iii) investigate the metabolic effects of mutations that restore thiamine prototrophy to a purH mutant. The data showed that AICAR accumulation led to an increase in the global regulator cyclic AMP (cAMP) and that disrupting central carbon metabolism could decrease AICAR and/or cAMP to restore thiamine synthesis. A mutant (icc) blocked in cAMP degradation that accumulated cAMP but had wild-type levels of AICAR was used to identify changes in the purH metabolome that were a direct result of elevated cAMP. Data herein describe the use of metabolomics to identify the metabolic state of mutant strains and probe the underlying mechanisms used by AICAR to inhibit thiamine synthesis. The results obtained provide a cautionary tale of using metabolite concentrations as the only data to define the physiological state of a bacterial cell.

Alzheimer's disease (AD) pathology shows characteristic 'plaques' rich in amyloid beta (Abeta) peptide deposits. Inflammatory process-related proteins such as pro-inflammatory cytokines have been detected in AD brain suggesting that an inflammatory immune reaction also plays a role in the pathogenesis of AD. Glial cells in culture respond to LPS and Abeta stimuli by upregulating the expression of cytokines TNF-alpha, IL-1beta, and IL-6, and also the expression of proinflammatory genes iNOS and COX-2. We have earlier reported that LPS/Abeta stimulation-induced ceramide and ROS generation leads to iNOS expression and nitric oxide production in glial cells. The present study was undertaken to investigate the neuroprotective function of AICAR (a potent activator of AMP-activated protein kinase) in blocking the pro-oxidant/proinflammatory responses induced in primary glial cultures treated with LPS and Abeta peptide.

Metabolic stress, as well as several antidiabetic agents, increases hepatic nucleotide monophosphate (NMP) levels, activates AMP-activated protein kinase (AMPK), and suppresses glucose production. We tested the necessity of hepatic AMPK for the in vivo effects of an acute elevation in NMP on metabolism. 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR; 8 mg·kg(-1)·min(-1))-euglycemic clamps were performed to elicit an increase in NMP in wild type (α1α2(lox/lox)) and liver-specific AMPK knock-out mice (α1α2(lox/lox) + Albcre) in the presence of fixed glucose. Glucose kinetics were equivalent in 5-h fasted α1α2(lox/lox) and α1α2(lox/lox) + Albcre mice. AMPK was not required for AICAR-mediated suppression of glucose production and increased glucose disappearance. These results demonstrate that AMPK is unnecessary for normal 5-h fasting glucose kinetics and AICAR-mediated inhibition of glucose production. Moreover, plasma fatty acids and triglycerides also decreased independently of hepatic AMPK during AICAR administration. Although the glucoregulatory effects of AICAR were shown to be independent of AMPK, these studies provide in vivo support for the AMPK energy sensor paradigm. AICAR reduced hepatic energy charge by ∼20% in α1α2(lox/lox), which was exacerbated by ∼2-fold in α1α2(lox/lox) + Albcre. This corresponded to a ∼6-fold rise in AMP/ATP in α1α2(lox/lox) + Albcre. Consistent with the effects on adenine nucleotides, maximal mitochondrial respiration was ∼30% lower in α1α2(lox/lox) + Albcre than α1α2(lox/lox) livers. Mitochondrial oxidative phosphorylation efficiency was reduced by 25%. In summary, these results demonstrate that the NMP capacity to inhibit glucose production in vivo is independent of liver AMPK. In contrast, AMPK promotes mitochondrial function and protects against a more precipitous fall in ATP during AICAR administration.

Insulin-induced microvascular recruitment is important for optimal muscle glucose uptake. 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR, an activator of AMP-activated protein kinase), can also induce microvascular recruitment, at doses that do not acutely activate glucose transport in rat muscle. Whether low doses of AICAR can augment physiologic insulin action is unknown. In the present study we used the euglycemic hyperinsulinemic clamp to assess whether insulin action is augmented by low dose AICAR.

What is the central question of this study? Does fukutin deficiency in skeletal muscle cause mitochondrial dysfunction, and if so, can AMP-activated protein kinase (AMPK) stimulation via 5-aminoimidazole-4-carboxamide ribonucleotide attenuate this through regulation of mitochondrial biogenesis and autophagy? What is the main finding and its importance? Mitochondrial dysfunction is associated with fukutin deficiency and AMPK stimulation may benefit muscle contractility to a greater extent than mitochondrial function.

Tumor cells generally require large amounts of nucleotides, and thus activate de novo purine synthesis (dnPS). In the dnPS reactions, 10-formyltetrahydorofolate (10-fTHF) supplied by one-carbon metabolism is utilized as a formyl group donor. We focused on aldehyde dehydrogenase 1 family member L1 (ALDH1L1), which metabolizes 10-fTHF to tetrahydrofolate and whose expression is often attenuated in hepatocellular carcinoma (HCC). We generated ALDH1L1-expressing HuH-7 cells to perform metabolome analysis and found that intracellular levels of serine were reduced and glycine was increased. In addition, 5-aminoimidazole-4-carboxamide ribonucleotide (ZMP), a dnPS intermediate, accumulated due to the consumption of 10-fTHF by ALDH1L1, which inhibited ZMP formylation. Importantly, ALDH1L1-expressing cells showed reduced ZMP sensitivity and higher mitochondrial activity. The suppression of mitochondrial serine catabolism by ALDH1L1 expression was speculated to be closely related to this phenotype. Gene set enrichment analysis utilizing The Cancer Genome Atlas data revealed that genes related to oxidative phosphorylation were enriched in HCC patients with high ALDH1L1 expression. Moreover, drug sensitivity data analysis demonstrated that HCC cell lines with low expression of ALDH1L1 were sensitive to ZMP and cordycepin, a structural analog of ZMP and AMP. Our study revealed that ZMP and AMP analogs might be effective in the pharmacotherapy of HCC patients with low expression of ALDH1L1.

Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy affecting children. Despite significant progress and success in the treatment of ALL, a significant number of children continue to relapse and for them, outcome remains poor. Therefore, the search for novel therapeutic approaches is warranted. The aim of this study was to investigate the AMP activated protein kinase (AMPK) as a potential target in childhood acute lymphoblastic leukemia (ALL) subtypes characterized by non-random translocation signature profiles. We evaluated the effects of the AMPK activator AICAR on cell growth, cell cycle regulators and apoptosis of various childhood ALL cells.

BACKGROUND The development and progression of hepatocellular carcinoma (HCC) are associated with obesity and hepatosteatosis. AMP-activated protein kinase (AMPK) regulates metabolic homeostasis. This study aimed to investigate the effects of treatment with the adenosine monophosphate (AMP) analog, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) on hepatosteatosis in a mouse model fed a high-fat diet (HFD), and on hepatocellular carcinoma (HCC) induced by diethylnitrosamine (DEN) in the HFD mouse model. MATERIAL AND METHODS Male C57BL/6 male mice from two weeks of age were fed a high-fat diet, resulting in hepatosteatosis. HFD mice (15-20 per group) were treated with AICAR and without AICAR. HFD mice were treated with DEN, with and without AICAR. Mouse liver tissues were examined histologically using lipid histochemical stains, immunohistochemistry, and immunofluorescence. Levels of cytokines, alanine transaminase (ALT), triacylglyceride (TAG), and apoptosis were determined. Western blot was used to detect AMPK, pAMPK, STAT3, and pSTAT3. Real-time polymerase chain reaction (RT-PCR) detected expression of the ACL, FAS, CD36, ATGL, CPT1, and IL6 genes. RESULTS In the HFD mouse model, AICAR treatment inhibited hepatic lipid synthesis and IL-6 expression. In the DEN-treated mice, AICAR treatment reduced tumorigenesis, IL-6 signaling, and STAT3 activation. Short-term AICAR treatment had no significant effect in advanced HCC. CONCLUSIONS In an HFD mouse model, treatment with AICAR reduced the development of hepatosteatosis, and following treatment with the liver carcinogen, DEN, AICAR reduced the development of HCC. These preliminary findings support further studies on the role of AICAR in fatty liver disease and HCC.

This study tested whether chronic systemic administration of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) could attenuate hyperphagia, reduce lean and fat mass losses, and improve whole-body energy homeostasis in insulin-deficient rats. Male Wistar rats were first rendered diabetic through streptozotocin (STZ) administration and then intraperitoneally injected with AICAR for 7 consecutive days. Food and water intake, ambulatory activity, and energy expenditure were assessed at the end of the AICAR-treatment period. Blood was collected for circulating leptin measurement and the hypothalami were extracted for the determination of suppressor of cytokine signaling 3 (SOCS3) content, as well as the content and phosphorylation of AMP-kinase (AMPK), acetyl-CoA carboxylase (ACC), and the signal transducer and activator of transcription 3 (STAT3). Rats were thoroughly dissected for adiposity and lean body mass (LBM) determinations. In non-diabetic rats, despite reducing adiposity, AICAR increased (∼1.7-fold) circulating leptin and reduced hypothalamic SOCS3 content and food intake by 67% and 25%, respectively. The anorexic effect of AICAR was lost in diabetic rats, even though hypothalamic AMPK and ACC phosphorylation markedly decreased in these animals. Importantly, hypothalamic SOCS3 and STAT3 levels remained elevated and reduced, respectively, after treatment of insulin-deficient rats with AICAR. Diabetic rats were lethargic and displayed marked losses of fat and LBM. AICAR treatment increased ambulatory activity and whole-body energy expenditure while also attenuating diabetes-induced fat and LBM losses. In conclusion, AICAR did not reverse hyperphagia, but it promoted anti-catabolic effects on skeletal muscle and fat, enhanced spontaneous physical activity, and improved the ability of rats to cope with the diabetes-induced dysfunctional alterations in glucose metabolism and whole-body energy homeostasis.