AICAR

Abstract

This study aims to elucidate the synergistic protective mechanism of the AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) in ischemia-reperfusion injury -associated acute kidney injury (IRI-AKI). By establishing a hypoxia/reoxygenation (H/R) injury model using human proximal tubule cells (HK-2) and IRI-AKI rat model, and employing molecular techniques including qRT-PCR, western blotting, serum biochemical assays, renal tissue hematoxylin and eosin staining, immunofluorescence, and transmission electron microscopy (TEM), we demonstrated that AICAR activates AMPK, leading to the significant downregulation of TXNIP and NLRP3, blocks Caspase-1-dependent release of IL-1β and IL-18, and ultimately suppresses pyroptosis, thereby alleviating renal inflammatory injury. Furthermore, AICAR restored mitochondrial membrane potential and ATP levels in H/R-treated HK-2 cells, reduced reactive oxygen species production in renal tissues of IRI-AKI rats, and elevated levels of antioxidant enzymes. Concurrently, utilizing targeted metabolomics technology, we discovered that AICAR effectively restores the levels of multiple metabolites associated with glycolysis, the TCA cycle, the urea cycle, and tryptophan metabolism and alleviates lipid deposition in IRI-AKI. This confirms that AICAR alleviates IRI-AKI by activating AMPK to restore impaired cellular energy metabolism, improve mitochondrial function, and ameliorate oxidative stress. Notably, this study is the first to reveal that AICAR, via AMPK activation, synchronously regulates dual protective pathways: "pyroptosis inhibition" and "energy metabolism remodeling." This synergistic protective mechanism may represent the core advantage distinguishing AICAR from other potential therapeutic strategies, highlighting its substantial translational potential as a multi-mechanism synergistic therapeutic agent. Our findings provide an innovative dual-regulatory ("pyroptosis-energy metabolism") therapeutic strategy for the clinical prevention and treatment of IRI-AKI.

Abstract

AMP-activated protein kinase (AMPK) is activated by reduced cellular energy charge and mimics the action of insulin in muscle by stimulating increased trafficking of GLUT4 to the plasma membrane. In contrast, we have previously reported that short-term activation of AMPK in adipocytes has no effect on glucose uptake. Whether prolonged AMPK activation influences adipocyte glucose uptake remains poorly characterised. To investigate the effect of sustained AMPK activation on glucose uptake in adipocytes, glucose uptake and insulin signalling were assessed in 3T3-L1 adipocytes stimulated with AICAR and 991, which activate AMPK by different mechanisms, for 24 h. Furthermore, glucose uptake and GLUT4 levels were assessed in adipocytes or adipose tissue from mice lacking AMPKα1 as a model of prolonged AMPK downregulation. AICAR, but not 991, markedly inhibited insulin-stimulated glucose uptake in 3T3-L1 adipocytes. This effect of AICAR was associated with impaired trafficking of GLUT4 to the plasma membrane but did not alter cellular GLUT4 levels or insulin signalling via AKT. The effect of AICAR did, however, require phosphorylation to the nucleotide ZMP and was associated with altered insulin-stimulated MEK1/2-ERK1/2 phosphorylation. Sustained AMPK downregulation had no effect on adipocyte glucose uptake or GLUT4 levels. Taken together, these data demonstrate that sustained changes in AMPK activity do not alter adipocyte glucose uptake. Furthermore, AICAR reduces insulin-stimulated GLUT4 translocation and glucose uptake in adipocytes by a mechanism that is independent of AMPK but requires phosphorylation of AICAR to ZMP.

Abstract

<p>Introduction: Diabetic cardiomyopathy (DCM) is a common complication of diabetes characterized by chronic low-grade inflammation. Exercise has been recognized as an effective intervention for DCM; however, its underlying mechanisms remain incompletely understood.

Abstract

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.

Abstract

AMP-activated protein kinase (AMPK) is a fundamental energy sensor fine-tuning cellular activity based on ATP availability. On the other hand, BK-channel current is tightly regulated by leptin, which in turn regulates neuronal excitability by modulating ion channels such as the BK-channel. However, this mechanism remains unclear to date. In this work we aimed to determine whether AMPK mediates the leptin regulation on BK-channel. We hypothesized that leptin regulation of BK-channel through AMPK underlies the modulating changes in neuronal excitability of CA1 hippocampal neurons. By using patch-clamping methods on CA1 pyramidal neurons in brain slices and biochemical reagents, we found that AMPK activation with AICAR inhibits BK-channel current, while AMPK inhibition with Compound C enhances BK-channel activity. Remarkably, AMPK activation reverses BK-channel current enhanced by leptin supporting an AMPK-dependent metabolic regulation of BK. Accordingly, current-clamp experiments revealed that AMPK manipulations significantly affect leptin responses on CA1 neuronal firing. These results support AMPK as a key mediator of the interplay between leptin and neuronal excitability, readily integrating metabolic signals with the computing state of firing outputs in CA1 hippocampal neurons.

Abstract

The systemic administration of cisplatin has been shown to substantially reduce skeletal muscle mass. This is a serious concern, as muscle loss is correlated with increased mortality in patients with cancer. Cisplatin also contributes to cognitive decline, but the exact mechanism thereof remains unclear. In this study, we focused on fibronectin type III domain-containing 5 (Fndc5), a gene that produces irisin, a myokine that is important for brain health. Male C57BL/6J mice (8-9 weeks old) were injected with cisplatin or saline for 4 consecutive days. Twenty-four h after final injection of cisplatin, quadriceps muscles were isolated. C2C12 myotubes were treated with cisplatin with/without AICAR. In male C57BL/6J mice treated with cisplatin, a reduced expression of the key regulator PGC-1α was observed, along with reduced levels of Fndc5/irisin mRNA and protein in the mice quadriceps muscles. Similar findings were seen in cisplatin-treated C2C12 myotube cells, where the activation of PGC-1α with AICAR partially offset these effects. These results suggest that cisplatin inhibits the synthesis of Fndc5/irisin and may contribute to the metabolic changes and cognitive decline observed in patients with cancer who receive this treatment.

Abstract

AMPK is a master regulator of metabolism and is highly conserved and ubiquitously expressed. Activation of AMPK stimulates the catabolic pathway (glucose utilization and β-oxidation) and inhibits the anabolic pathway (gluconeogenesis, protein synthesis, and lipogenesis), leading to improvement of cellular energy status. However, the mechanisms of maintaining low cellular AMPK activity are not fully understood. We and other investigators showed that activated PKA in the glucagon-cAMP signaling pathway and insulin-activated AKT both can directly phosphorylate AMPKα1/2 at S496/491 to inhibit AMPK activity. In the current study, we found that activation of AMPK by an activator, AICAR, led to elevated and prolonged phosphorylation of AMPKα1/2 at S496/S491, reflecting a feedback inhibition of AMPK activity. In an in vitro assay, functional AMPKα1β1γ1 or AMPKα2β1γ1 can phosphorylate AMPKα1 at S496 or AMPKα2 at S491, respectively. We designed and successfully screened a new AMPKα2-targeting peptide to activate AMPK through competitively blocking the negative phosphorylation, resulting in suppression of gluconeogenic gene expression and promotion of mitochondrial fission in hepatocytes.

Abstract

AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin complex 1 (mTORC1) are crucial kinase signalling hubs that regulate the balance between catabolism and anabolism in skeletal muscle. The scaffold protein AXIN1 has been proposed to regulate the switch between these pathways and be required for GLUT4 translocation in skeletal muscle and adipocyte cell lines. Muscle-specific AXIN1 knockout (KO) mice exhibit no discernable phenotype, possibly due to compensation by AXIN2 upon AXIN1 loss. Thus we generated and characterized muscle-specific inducible AXIN1 and AXIN2 double knockout (dKO) mice. Surprisingly AXIN1/2 dKO mice displayed normal AMPK and mTORC1 signalling and glucose uptake in response to 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), insulin and in situ muscle contraction. These findings suggest that AXIN proteins are not essential for the regulation of AMPK and mTORC1 signalling or glucose uptake in skeletal muscle. This study challenges the previously indicated critical roles of AXIN1 in exercise-stimulated AMPK activation and GLUT4-mediated glucose uptake in skeletal muscle. KEY POINTS: Phenotyping of tamoxifen-inducible muscle-specific AXIN1/2 double knockout (dKO) mice. We find no evidence for AXIN-dependent AMPK or mTORC1 regulation in skeletal muscle by insulin, AMPK activation or contraction. Glucose uptake regulation by insulin and AMPK activation is normal in AXIN1/2 dKO mice.

Abstract

We previously demonstrated that neurotensin, a 13-amino-acid gut hormone peptide, enhances small intestinal epithelial cell fatty acid uptake through inhibition of AMPK. Here, utilizing Drosophila and mouse models in vivo, as well as mouse and human small intestinal epithelial organoids or monolayers ex vivo, we determine the targets of neurotensin and AMPK associated with obesity and aging. High-fat diet and aging decreased AMPK and insulin signaling, which was prevented by neurotensin deficiency. High-fat diet feeding increased FABP1 protein expression in wild-type mice; this effect was attenuated in neurotensin-deficient mice. AICAR and metformin increased AMPK phosphorylation in young but not in aged small intestinal epithelial cells. By contrast, AICAR and metformin inhibited FABP1 mRNA and protein expression. Moreover, cytosolic colocalization of AMPKα1 and FABP1 was noted in IEC-6 cells. AMPK phosphorylation and FABP1 expression was decreased in aged wild-type small intestinal epithelial cells; however, this effect was reversed in neurotensin-deficient cells. Results from human duodenal organoids confirm the effects of neurotensin, palmitic acid and metformin on AMPK phosphorylation and FABP1. Finally, overexpressing neurotensin in enteroendocrine cells reduced the lifespan of Drosophila; neurotensin deficiency extended the lifespan of mice fed a high-fat diet. Our findings indicate that neurotensin inhibits AMPK and increases FABP1 in small intestinal epithelial cells under conditions of obesity. Neurotensin deficiency preserves AMPK and FABP1 levels, thus attenuating some of the negative effects of obesity and aging.

Abstract

Methotrexate (MTX) is the conventional synthetic disease-modifying anti-rheumatic drug (csDMARD) recommended as the first-choice anti-rheumatic drug for rheumatoid arthritis (RA). However, responses to MTX may be influenced by genetic variants. We aim to evaluate the association of the rs2372536, rs4673990, and rs4673993 genetic variants of the ATIC gene with therapeutic failure of MTX in patients with RA. A case-control study was performed. Disease activity was measured using the disease activity score based on erythrocyte sedimentation rate (DAS28-ESR). RA patients were classified into two groups: (a) responders (DAS28-ESR ≤ 3.2), which is the group of patients who did respond to methotrexate, and (b) non-responders (DAS28-ESR > 3.2), which is the group of patients who did not respond to methotrexate. Serum levels of the 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) enzyme and Interleukin-6 (IL-6) were quantified using an enzyme-linked immunosorbent assay (ELISA). Genotyping of ATIC genetic variants was performed with quantitative polymerase chain reaction (qPCR) using TaqMan probes. A total of 260 patients with RA were included. In total, 142 (54.6%) were non-responders to MTX. IL-6 levels were increased in the non-responder group (p = 0.002), while no statistical differences were observed in the AICAR levels. The variables associated with non-response were higher HAQ-Di, weekly MTX dose, glucocorticoid use, erythrocyte sedimentation rate, and carriers of the polymorphic homozygous variant of rs4673993 (OR = 4.5, 95% CI: 1.04-19.34; p = 0.04). The use of sulfazaline offered protective effects. Our findings indicate that the polymorphism rs4673993 gene variant of the AICAR protein may significantly influence MTX resistance. Therefore, these results support the importance of the pathway generating extracellular adenosine and its effects on promoting the immune regulation for the mechanism of MTX therapy of RA.

Abstract

In early life, RNA probably played the central role and, in the corresponding RNA world, the main produced amino acids and small peptides had to react continuously with RNA, ribonucleos(t)ides and nucleobases, especially with purines. A RNA-peptide world and key metabolic pathways have emerged from the corresponding chemical modifications such as the translation process performed by the ribosome. Some interesting reactions of the purine bicycle and of the corresponding ribonucleos(t)ides are performed under plausible prebiotic conditions and described RNA chemical lesions are reviewed with the prospect to highlight their connection with some major steps of the purine and histidine biosynthetic pathways that are, in an intriguingly way, related through two key metabolites, adenosine 5'-triphosphate and the imidazole ribonucleotide 5-aminoimidazole-4-carboxamide ribonucleotide. Ring-opening reactions of purines stand out as efficient accesses to imidazole ribonucleotides and to formamidopyrimidine (Fapy) ribonucleotides suggesting that biosynthetic pathway' first steps have emerged from RNA and ribonucleos(t)ide damages. Also, are summarized the works on the formation and catalytic properties, under plausible prebiotic conditions, of N6-derivatives of the purine base adenine as potential surrogates of histidine in catalysis accordingly to their structural relationship.

Abstract

Leaky gut, an increased intestinal permeability, has been described in many diseases. We have recently demonstrated that neuropeptides such as orexin in the brain improved leaky gut, suggesting that the brain is involved in maintaining intestinal barrier function. It has been suggested that AMPK in the hypothalamus play a role in food intake. Because the hypothalamus is involved in the regulation of not only feeding behavior but also gut function, the present study was performed to clarify a hypothesis that AMPK in the brain regulate gut barrier function. Colonic permeability was determined by quantifying the absorbed Evans blue within the colonic tissue in rats. Intracisternal AICAR, an AMPK activator, could reduce LPS-induced colonic hyperpermeability while peripherally administered AICAR failed to change it. The improvement of colonic hyperpermeability by intracisternal AICAR was blocked by intracisternal but not subcutaneous compound C, AMPK inhibitor, atropine or vagotomy. The improvement of colonic hyperpermeability by intracisternal AICAR was blocked by intracisternal orexin receptor antagonist but not oxytocin or GLP-1 receptor antagonist. Intracisternal compound C prevented brain oxytocin or GLP-1 but not orexin-induced improvement of colonic hyperpermeability. These results suggest that activation of brain AMPK is capable of reducing colonic hyperpermeability through brain orexin signaling and the vagus nerve. In addition, endogenous AMPK in the brain may mediate the oxytocin or GLP-induced improvement of colonic hyperpermeability. We would suggest that improvement of leaky gut by activation of brain AMPK may play a role in leaky gut-related diseases.

Abstract

The endothelial small-conductance calcium-activated potassium channels (KCa2.3) are indispensable for endothelium-dependent hyperpolarization (EDH) response, mainly in resistance arteries. We recently demonstrated in diet-induced obese mice that adenosine monophosphate-activated protein kinase (AMPK) upregulates endothelial KCa2.3 expression and improves endothelial function. However, the molecular mechanism of regulation of KCa2.3 by AMPK remains less explored. Using techniques of bioinformatics, molecular biology and wire myograph system, we examined KCa2.3 phosphorylation by AMPK in human umbilical vein endothelial cells (HUVECs), human embryonic kidney 293 (HEK-293T) cells and second-order mesenteric resistance arteries from angiotensin II-induced hypertensive mice. In HUVECs, treatment with activators of AMPK (AICAR, metformin, and MK-8722) significantly increased phosphorylation of KCa2.3 Thr106 (human), which was antagonized by AMPK inhibitor compound C. In HEK-293T cells, KCa2.3 current was enhanced by AMPK activation or phosphomimetic mutant KCa2.3 (T106D), which was abolished after de-phosphomimetic mutant (T106A) or deletion of KCa2.3 of Thr106 site (T106Del). In mice with angiotensin II infusion, 2-week treatment with AICAR or overexpressing phosphomimetic mutant KCa2.3 Thr107D (mouse) restored KCa2.3-mediated EDH-dependent relaxation in mesenteric resistance arteries together with reversal of early phase hypertension. Our study demonstrates for the first time that AMPK activation mediates KCa2.3 phosphorylation in endothelial cells with enhanced channel activity. This effect ameliorates endothelial dysfunction of mesenteric resistance arteries and alleviates angiotensin II-induced early phase hypertension in mice.

Abstract

Nicotinamide adenine dinucleotide kinase (NADK) is essential to the generation of nicotinamide adenine dinucleotide phosphate (NADP(H)), an important metabolic coupling factor involved in glucose-stimulated insulin secretion. In the present study, we showed that the expression of Nadk and Nadk2 transcripts and NADP(H) content were lower in islets of 80-week-old (aged) mice than those of 8-week-old (young) mice. This was associated with diminished oral glucose tolerance of old mice and the glucose-stimulated insulin secretion (GSIS) response of islets. Knockdown (KD) of Nadk or Nadk2 gene expression in NIT-1 cells impaired glucose-stimulated insulin secretion. Metabolomic analysis revealed that Nadk KD specifically affected purine metabolism in glucose-stimulated cells. The levels of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) were higher in KD cells than in the non-targeting control (NTC) cells. Phosphorylation of AMP-activated protein kinase (AMPK) was elevated in glucose-treated KD cells compared to that of NTC cells. Increased AICAR level and AMPKα phosphorylation were observed in the glucose-stimulated islets of the aged mice. Genetic and pharmacological inhibition of AMPK promoted glucose-stimulated insulin release by KD cells and the aged mouse islets. It is likely that NADK is modulatory to AMPK activation in pancreatic β-cells and to their GSIS response. Enhanced AICAR formation in KD cells was accompanied by significantly increased conversion from inosine monophosphate (IMP) in a tetrahydrofolate (THF)-dependent manner. Folate supplementation augmented the GSIS response of KD cells and aged mouse islets. Taken together, these findings suggest that the aging-associated decline in NADK expression may underlie the reduced insulin secretory capacity of pancreatic β-cells.

Abstract

This study aimed to investigate the regulatory role of the AMP-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR) signaling pathway in mediating transforming growth factor-beta 1 (TGF-β1)-induced cellular proliferation and transdifferentiation processes in human pterygium fibroblasts (HPFs).