Direct small molecule ADaM-site AMPK activators reveal an AMPKγ3-independent mechanism for blood glucose lowering.

OBJECTIVE: Skeletal muscle is an attractive target for blood glucose-lowering pharmacological interventions. Oral dosing of small molecule direct pan-activators of AMPK that bind to the allosteric drug and metabolite (ADaM) site, lowers blood glucose through effects in skeletal muscle. The molecular mechanisms responsible for this effect are not described in detail. This study aimed to illuminate the mechanisms by which ADaM-site activators of AMPK increase glucose uptake in skeletal muscle. Further, we investigated the consequence of co-stimulating muscles with two types of AMPK activators i.e., ADaM-site binding small molecules and the prodrug AICAR. METHODS: The effect of the ADaM-site binding small molecules (PF739 and 991), AICAR or co-stimulation with PF739 or 991 and AICAR on muscle glucose uptake was investigated ex vivo in m. extensor digitorum longus (EDL) excised from muscle-specific AMPKα1α2 as well as whole-body AMPKγ3-deficient mouse models. In vitro complex-specific AMPK activity was measured by immunoprecipitation and molecular signaling was assessed by western blotting in muscle lysate. To investigate the transferability of these studies, we treated diet-induced obese mice in vivo with PF739 and measured complex-specific AMPK activation in skeletal muscle. RESULTS: Incubation of skeletal muscle with PF739 or 991 increased skeletal muscle glucose uptake in a dose-dependent manner. Co-incubating PF739 or 991 with a maximal dose of AICAR increased glucose uptake to a greater extent than any of the treatments alone. Neither PF739 nor 991 increased AMPKα2ß2γ3 activity to the same extent as AICAR, while co-incubation led to potentiated effects on AMPKα2ß2γ3 activation. In muscle from AMPKγ3 KO mice, AICAR-stimulated glucose uptake was ablated. In contrast, the effect of PF739 or 991 on glucose uptake was not different between WT and AMPKγ3 KO muscles. In vivo PF739 treatment lowered blood glucose levels and increased muscle AMPKγ1-complex activity 2-fold, while AMPKα2ß2γ3 activity was not affected. CONCLUSIONS: ADaM-site binding AMPK activators increase glucose uptake independently of AMPKγ3. Co-incubation with PF739 or 991 and AICAR potentiates the effects on muscle glucose uptake and AMPK activation. In vivo, PF739 lowers blood glucose and selectively activates muscle AMPKγ1-complexes. Collectively, this suggests that pharmacological activation of AMPKγ1-containing complexes in skeletal muscle can increase glucose uptake and can lead to blood glucose lowering.

High glucose suppresses autophagy through the AMPK pathway while it induces autophagy via oxidative stress in chondrocytes.

Diabetes (DB) is a risk factor for osteoarthritis progression. High glucose (HG) is one of the key pathological features of DB and has been demonstrated to induce apoptosis and senescence in chondrocytes. Autophagy is an endogenous mechanism that can protect cells against apoptosis and senescence. The effects of HG on autophagy in cells including chondrocytes have been studied; however, the results have been inconsistent. The current study aimed to elucidate the underlying mechanisms, which could be associated with the contrasting outcomes. The present study revealed that HG can induce apoptosis and senescence in chondrocytes, in addition to regulating autophagy dynamically. The present study demonstrated that HG can cause oxidative stress in chondrocytes and suppress the AMPK pathway in a dose-dependent manner. Elimination of oxidative stress by Acetylcysteine, also called N-acetyl cysteine (NAC), downregulated autophagy and alleviated HG-stimulated apoptosis and senescence, while activation of the AMPK signaling pathway by AICAR not only upregulated autophagy but also alleviated HG-stimulated apoptosis and senescence. A combined treatment of NAC and AICAR was superior to treatment with either NAC or AICAR. The study has demonstrated that HG can suppress autophagy through the AMPK pathway and induce autophagy via oxidative stress in chondrocytes.

The Role of AMPK Activation for Cardioprotection in Doxorubicin-Induced Cardiotoxicity.

Doxorubicin is a commonly used chemotherapeutic agent for the treatment of a range of cancers, but despite its success in improving cancer survival rates, doxorubicin is cardiotoxic and can lead to congestive heart failure. Therapeutic options for this patient group are limited to standard heart failure medications with the only drug specific for doxorubicin cardiotoxicity to reach FDA approval being dexrazoxane, an iron-chelating agent targeting oxidative stress. However, dexrazoxane has failed to live up to its expectations from preclinical studies while also bringing up concerns about its safety. Despite decades of research, the molecular mechanisms of doxorubicin cardiotoxicity are still poorly understood and oxidative stress is no longer considered to be the sole evil. Mitochondrial impairment, increased apoptosis, dysregulated autophagy and increased fibrosis have also been shown to be crucial players in doxorubicin cardiotoxicity. These cellular processes are all linked by one highly conserved intracellular kinase: adenosine monophosphate-activated protein kinase (AMPK). AMPK regulates mitochondrial biogenesis via PGC1α signalling, increases oxidative mitochondrial metabolism, decreases apoptosis through inhibition of mTOR signalling, increases autophagy through ULK1 and decreases fibrosis through inhibition of TGFß signalling. AMPK therefore sits at the control point of many mechanisms shown to be involved in doxorubicin cardiotoxicity and cardiac AMPK signalling itself has been shown to be impaired by doxorubicin. In this review, we introduce different agents known to activate AMPK (metformin, statins, resveratrol, thiazolidinediones, AICAR, specific AMPK activators) as well as exercise and dietary restriction, and we discuss the existing evidence for their potential role in cardioprotection from doxorubicin cardiotoxicity.

AICAR and nicotinamide treatment synergistically augment the proliferation and attenuate senescence-associated changes in mesenchymal stromal cells.

BACKGROUND: Mesenchymal stromal cell (MSC) stemness capacity diminishes over prolonged in vitro culture, which negatively affects their application in regenerative medicine. To slow down the senescence of MSCs, here, we have evaluated the in vitro effects of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, and nicotinamide (NAM), an activator of sirtuin1 (SIRT1). METHODS: Human adipose-derived MSCs were cultured to passage (P) 5. Subsequently, the cells were grown in either normal medium alone (control group), the medium supplemented with AICAR (1 mM) and NAM (5 mM), or in the presence of both for 5 weeks to P10. Cell proliferation, differentiation capacity, level of apoptosis and autophagy, morphological changes, total cellular reactive oxygen species (ROS), and activity of mTORC1 and AMPK were compared among different treatment groups. RESULTS: MSCs treated with AICAR, NAM, or both displayed an increase in proliferation and osteogenic differentiation, which was augmented in the group receiving both. Treatment with AICAR or NAM led to decreased expression of ß-galactosidase, reduced accumulation of dysfunctional lysosomes, and characteristic morphologic features of young MSCs. Furthermore, while NAM administration could significantly reduce the total cellular ROS in aged MSCs, AICAR treatment did not. Moreover, AICAR-treated cells possess a high proliferation capacity; however, they also show the highest level of cellular apoptosis. The observed effects of AICAR and NAM were in light of the attenuated mTORC1 activity and increased AMPK activity and autophagy. CONCLUSIONS: Selective inhibition of mTORC1 by AICAR and NAM boosts autophagy, retains MSCs' self-renewal and multi-lineage differentiation capacity, and postpones senescence-associated changes after prolonged in vitro culture. Additionally, co-administration of AICAR and NAM shows an additive or probably a synergistic effect on cellular senescence.

Neuroprotective and vision-protective effect of preserving ATP levels by AMPK activator.

Progression of blinding diseases, such as age-related macular degeneration, is accelerated by light exposure. However, no particular intervention is applied to the photostress. Here, we report neuroprotective effects of the adenosine monophosphate (AMP)-activated protein kinase (AMPK) activator, 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), on light-induced visual function impairment, photoreceptor disorders and death in mice. Increase in retinal ATP levels in response to photostress was transient, because oxygen consumption rate (OCR) and cytochrome c oxidase (CcO) activity were reduced under photostress. However, AICAR treatment preserved OCR, CcO activity, and high levels of retinal ATP after light exposure. AMPK knockdown in the photoreceptor-derived cell line revealed that AMPK targeted CcO activity. Further, our data indicated that photostress reduced mitochondrial respiratory function and ATP levels, while AICAR treatment promoted neuronal survival and retained visual function, stabilizing ATP levels through preserved CcO activity. The current study has provided proof of concept for providing cells with sufficient energy to promote cell survival in the presence of cellular stress. This is in contrast to the previous reports which primarily investigated therapeutic approaches to suppress stress signals. Hence, stabilization of the ATP supply may serve as a novel therapeutic approach to support tissue survival under stress and prevent neurodegeneration.

AMPK: Potential Therapeutic Target for Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder. The pathogenesis of AD is very complicated. For decades, the amyloid hypothesis has influenced and guided research in the field of AD. Meanwhile, researchers gradually realized that AD is caused by multiple concomitant factors, such as autophagy, mitochondrial quality control, insulin resistance and oxidative stress. In current clinical trials, the improvement strategies of AD, such as Aß antibody immunotherapy and gamma secretase inhibitors, are limited. There is mounting evidence of neurodegenerative disorders indicated that activation of AMP-activated protein kinase (AMPK) may have broad neuroprotective effects. We reviewed the researches on AMPK for AD, the results demonstrated that activation of AMPK is controversial in Aß deposition and tau phosphorylation, but is positive to promote autophagy, maintain mitochondrial quality control, reduce insulin resistance and relieve oxidative stress. It is concluded that AMPK might be a new target for AD by aggressively treating the risk factors in the future.

Acadesine Circumvents Azacitidine Resistance in Myelodysplastic Syndrome and Acute Myeloid Leukemia.

Myelodysplastic syndrome (MDS) defines a group of heterogeneous hematologic malignancies that often progresses to acute myeloid leukemia (AML). The leading treatment for high-risk MDS patients is azacitidine (Aza, Vidaza®), but a significant proportion of patients are refractory and all patients eventually relapse after an undefined time period. Therefore, new therapies for MDS are urgently needed. We present here evidence that acadesine (Aca, Acadra®), a nucleoside analog exerts potent anti-leukemic effects in both Aza-sensitive (OCI-M2S) and resistant (OCI-M2R) MDS/AML cell lines in vitro. Aca also exerts potent anti-leukemic effect on bone marrow cells from MDS/AML patients ex-vivo. The effect of Aca on MDS/AML cell line proliferation does not rely on apoptosis induction. It is also noteworthy that Aca is efficient to kill MDS cells in a co-culture model with human medullary stromal cell lines, that mimics better the interaction occurring in the bone marrow. These initial findings led us to initiate a phase I/II clinical trial using Acadra® in 12 Aza refractory MDS/AML patients. Despite a very good response in one out 4 patients, we stopped this trial because the highest Aca dose (210 mg/kg) caused serious renal side effects in several patients. In conclusion, the side effects of high Aca doses preclude its use in patients with strong comorbidities.

5-Amino-1-ß-D-Ribofuranosyl-Imidazole-4-Carboxamide (AICAR) Reduces Peripheral Inflammation by Macrophage Phenotype Shift.

The stimulation of the AMP-activated kinase (AMPK) by 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) has been associated with antihyperalgesia and the inhibition of nociceptive signaling in the spinal cord in models of paw inflammation. The attenuated nociception comes along with a strongly reduced paw edema, indicating that peripheral antiinflammatory mechanisms contribute to antinociception. In this study, we investigated the impact of AICAR on the immune cell composition in inflamed paws, as well as the regulation of inflammatory and resolving markers in macrophages. By using fluorescence-activated cell sorting (FACS) analysis and immunofluorescence, we found a significantly increased fraction of proresolving M2 macrophages and anti-inflammatory interleukin (IL)-10 in inflamed tissue, while M1 macrophages and proinflammatory cytokines such as IL-1 were decreased by AICAR in wild type mice. In AMPKα2 knock-out mice, the M2 polarization of macrophages in the paw was missing. The results were supported by experiments in primary macrophage cultures which also showed a shift to a proresolving phenotype with decreased levels of proinflammatory mediators and increased levels of antiinflammatory mediators. However, in the cell cultures, we did not observe differences between the AMPKα2+/+ and -/- cells, thus indicating that the AICAR-induced effects are at least partially AMPK-independent. In summary, our results indicate that AICAR has potent antiinflammatory and proresolving properties in inflammation which are contributing to a reduction of inflammatory edema and antinociception.

AMPK agonist AICAR ameliorates portal hypertension and liver cirrhosis via NO pathway in the BDL rat model.

Recent studies have indicated that the Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway is closely involved in liver fibrosis and other fibrotic diseases. However, whether targeting the AMPK pathway can rescue liver fibrosis and its complications, such as portal hypertension, is unknown. This study aimed to explore the therapeutic value of AICAR (5-aminoimidazole-4-carboxyamide ribonucleoside), an agonist of the AMPK pathway, on liver fibrosis and portal hypertension in bile duct ligation (BDL) rats. In vitro experiments showed that the gene expression levels of TGF-b, a-SMA, and collagen 1 in primary rat hepatic stellate cells (HSCs) were significantly decreased after AICAR treatment. The p-eNOS expression and nitric oxide (NO) production were increased by AICAR administration in sinusoidal endothelial cells (SECs). For in vivo animal studies, AICAR acutely decreased portal pressure in the BDL and CCL4 fibrotic rats, but not in the partial portal vein ligation (PVL) rats, without changes in systemic hemodynamics. It was also observed by using intravital fluorescence microscopy that AICAR led to sinusoidal vasodilation in situ experiment. We propose that the relevant mechanisms may be related to the activation of the AMPK/NO pathway in SECs and that this activation promoted NO production in the liver, thereby promoting hepatic sinusoid microcirculation and decreased intrahepatic resistance. The results were verified using the NO inhibitor L-NAME. Chronic AICAR treatment also showed profound beneficial effects on the BDL model rats. The hemodynamic condition was greatly improved, but the positive effect could be partially blocked by L-NAME. Moreover, AICAR also decreased hepatic fibrogenesis in the BDL rats. KEY MESSAGES: Acute and chronic use of AICAR could alleviate portal pressure without changing systemic hemodynamics. AICAR induced sinusoidal vasodilation by improving NO bioavailability and ameliorating endothelial dysfunction in vivo and in vitro. AICAR could alleviate liver cirrhosis in the BDL model rats.

AMPK activation attenuates inflammatory pain through inhibiting NF-κB activation and IL-1ß expression.

BACKGROUND: Chronic pain is a major clinical problem with limited treatment options. Previous studies have demonstrated that activation of adenosine monophosphate-activated protein kinase (AMPK) can attenuate neuropathic pain. Inflammation/immune response at the site of complete Freund's adjuvant (CFA) injection is known to be a critical trigger of the pathological changes that produce inflammatory pain. However, whether activation of AMPK produces an analgesic effect through inhibiting the proinflammatory cytokines, including interleukin-1ß (IL-1ß), in inflammatory pain remains unknown. METHODS: Inflammatory pain was induced in mice injected with CFA. The effects of AICAR (5-aminoimidazole-4-carboxyamide ribonucleoside, an AMPK activator), Compound C (an AMPK inhibitor), and IL-1ra (an IL-1 receptor antagonist) were tested at day 4 after CFA injection. Inflammatory pain was assessed with von Frey filaments and hot plate. Immunoblotting, hematoxylin and eosin (H&E) staining, and immunofluorescence were used to assess inflammation-induced biochemical changes. RESULTS: The AMPK activator AICAR produced an analgesic effect and inhibited the level of proinflammatory cytokine IL-1ß in the inflamed skin in mice. Moreover, activation of AMPK suppressed CFA-induced NF-κB p65 translocation from the cytosol to the nucleus in activated macrophages (CD68+ and CX3CR1+) of inflamed skin tissues. Subcutaneous injection of IL-1ra attenuated CFA-induced inflammatory pain. The AMPK inhibitor Compound C and AMPKα shRNA reversed the analgesic effect of AICAR and the effects of AICAR on IL-1ß and NF-κB activation in inflamed skin tissues. CONCLUSIONS: Our study provides new information that AMPK activation produces the analgesic effect by inhibiting NF-κB activation and reducing the expression of IL-1ß in inflammatory pain.

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 + AICAR. As appropriate to their treatment group, the rats were injected with a single dose of cisplatin (7 mg/kg, i.p.). AICAR was administered to the rats at 100 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 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.

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.

κ-opioid receptor activation protects against myocardial ischemia-reperfusion injury via AMPK/Akt/eNOS signaling activation.

This study aims to investigate the effect of κ-opioid receptor activation on myocardial ischemia and reperfusion(I/R) injury and elucidate the underlying mechanisms. Myocardial I/R rat model and simulated I/R cardiomyocytes model were established. In vivo study showed that U50,488 H improved cardiac function, reduced myocardial infarct size and serum cTnT significantly. The effect of U50,488 H was abolished by nor-BNI(a κ-opioid receptor antagonist), Compound C(an AMPK inhibitor), Akt inhibitor and L-NAME(an eNOS inhibitor). AICAR, an AMPK activator, mimicked the effect of U50,488 H. U50,488 H up-regulated p-AMPK, p-Akt, and p-eNOS, which were abolished by nor-BNI. AICAR increased p-Akt and p-eNOS, which was abolished by Compound C. In vitro study showed that U50,488 H increased p-AMPK, p-Akt, and p-eNOS via κ-OR activation. The effect of U50,488 H on p-AMPK was abolished by compound C, but not Akt inhibitor and L-NAME. The effect of U50,488 H on p-Akt was abolished by compound C and Akt inhibitor, but not L-NAME. AICAR increased p-Akt and p-eNOS, which was abolished by Akt inhibitor, but not L-NAME. U50,488 H and AICAR also increased the viability of cardiomyocytes subjected to simulated I/R, the effects of U50,488 H and AICAR were blocked by nor-BNI, Compound C, Akt inhibitor, and L-NAME, respectively. In conclusion, κ-OR activation confers cardioprotection via AMPK/Akt/eNOS signaling.

The AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), but not metformin, prevents inflammation-associated cachectic muscle wasting.

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.

PIK3CA mutation sensitizes breast cancer cells to synergistic therapy of PI3K inhibition and AMPK activation.

Breast cancer has been emerging as a most common threat among women, thus many efforts were made to find drugs for fighting breast cancer. So far, PI3K (Phosphatidylinositol-4,5-bisphosphate 3-kinase) inhibitors have been believed to be effective drugs until frequent resistance emerged. Recently, PI3K H1047R mutation has been reported to sensitize breast cancer cells to PI3K inhibition by aspirin. Considering aspirin activates AMPK (AMP-activated protein kinase) simultaneously, it is possible that AMPK activators and PI3K inhibitors can synergistically inhibit breast cancers. Here we clearly observed synergistic suppression of cell growth in all three breast cancer cell lines (MCF-7, MDA-MB-361 and HCC38) when co-treating cells with PI3K inhibitor GDC-0941 and AMPK activator AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide). What is more, it is rather remarkable that the synergistic effect was much more dramatic in PIK3CA (PI3K catalytic subunit alpha) mutated (E545K) cells (MCF-7 and MDA-MB-361) than in PIK3CA wild-type cells (HCC38), which implied there is a relationship between PI3K genetic status and the efficacy of combination therapy. By using PIK3CA wild-type isogenic MCF-7 cell line, which exhibited attenuated cell proliferation compared with the parental MCF-7 cell line, we found endogenous reverse mutation of PIK3CA E545K alleles to wild-type sequence in MCF-7 cells dramatically impaired the synergy of PI3Ki&Ka (combinatorial PI3K inhibition and AMPK activation). Furthermore, PI3Ki&Ka significantly attenuated tumorigenesis of parental MCF-7 cells but not PIK3CA wild-type isogenic MCF-7 cells in tumor xenograft models. Taken together, our results suggest a promising precision therapy of PI3Ki&Ka in PIK3CA mutant breast cancers.

Effect of AICAR and 5-Fluorouracil on X-ray Repair, Cross-Complementing Group 1 Expression, and Consequent Cytotoxicity Regulation in Human HCT-116 Colorectal Cancer Cells.

Colorectal cancer (CRC) is one of the leading causes of cancer mortality and 5-Fluorouracil (5-FU) is the most common chemotherapy agent of CRC. A high level of X-ray repair cross complementing group 1 (XRCC1) in cancer cells has been associated with the drug resistance occurrence. Moreover, the activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK) has been indicated to regulate the cancer cell survival. Thus, this study was aimed to examine whether XRCC1 plays a role in the 5-FU/AMPK agonist (AICAR)-induced cytotoxic effect on CRC and the underlying mechanisms. Human HCT-116 colorectal cells were used in this study. It was shown that 5-FU increases the XRCC1 expression in HCT-116 cells and then affects the cell survival through CXCR4/Akt signaling. Moreover, 5-FU combined with AICAR further result in more survival inhibition in HCT-116 cells, accompanied with reduced CXCR4/Akt signaling activity and XRCC1 expression. These results elucidate the role and mechanism of XRCC1 in the drug resistance of HCT-116 cells to 5-FU. We also demonstrate the synergistic inhibitory effect of AMPK on 5-FU-inhibited HCT-116 cell survival under the 5-FU and AICAR co-treatment. Thus, our findings may provide a new notion for the future drug regimen incorporating 5-FU and AMPK agonists for the CRC treatment.

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 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 pAMPKα was associated with nuclear translocation of the peroxisome proliferator-activated receptor γ coactivator-α 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.

Energy Remodeling, Mitochondrial Disorder and Heart Failure.

Heart failure (HF) is a major global problem in public health with no curative treatment currently available. Energy remodeling is one of the features in HF, preceding cardiac structure remodeling. As an important energy organelle, mitochondrion plays critical roles in the progress of HF. This review focuses on the potential mechanisms linking mitochondrial functions and energy remodeling in HF including the energy starvation theory and energy substrate metabolism. It also highlights the potentials of novel drugs targeting HF energy metabolism.

Preconditioning mice with activators of AMPK ameliorates ischemic acute kidney injury in vivo.

This study had two objectives: 1) to determine whether preconditioning cultured proximal tubular cells (PTCs) with pharmacological activators of AMP-activated protein kinase (AMPK) protects these cells from apoptosis induced by metabolic stress in vitro and 2) to assess the effects of preconditioning mice with these agents on the severity of ischemic acute renal kidney injury (AKI) in vivo. We demonstrate that preconditioning PTCs with 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) or A-769662 reduces apoptosis of PTCs induced by subsequent stress. We also show that the reduction in cell death during metabolic stress associated with pretreatment by AMPK activators is associated with an increase in the cytosolic level of ATP, which is mediated by an increase in the rate of glycolysis. In addition, we provide evidence that the effect of AMPK activators on glycolysis is mediated, at least in part, by an increased uptake of glucose, and by the induction of hexokinase II (HK II) expression. Our data also show that the increased in HK II expression associated with preconditioning with AMPK activators is mediated by the activation (phosphorylation) of the cAMP-response element binding protein (CREB). We also provide entirely novel evidence that that A-79662 is substantially more effective than AICAR in mediating these alterations in PTCs in vitro. Finally, we demonstrate that preconditioning mice with AICAR or A-769662 substantially reduces the severity of renal dysfunction and tubular injury in a model of ischemic AKI in vivo and that the efficacy of AICAR and A-768662 in ameliorating ischemic AKI in vivo is comparable.

AMPK in cardiac fibrosis and repair: Actions beyond metabolic regulation.

Fibrosis is a general term encompassing a plethora of pathologies that span all systems and is marked by increased deposition of collagen. Injury of variable etiology gives rise to complex cascades involving several cell-types and molecular signals, leading to the excessive accumulation of extracellular matrix that promotes fibrosis and eventually leads to organ failure. Cardiac fibrosis is a dynamic process associated notably with ischemia, hypertrophy, volume- and pressure-overload, aging and diabetes mellitus. It has profoundly deleterious consequences on the normal architecture and functioning of the myocardium and is associated with considerable mortality and morbidity. The AMP-activated protein kinase (AMPK) is a ubiquitously expressed cellular energy sensor and an essential component of the adaptive response to cardiomyocyte stress that occurs during ischemia. Nevertheless, its actions extend well beyond its energy-regulating role and it appears to possess an essential role in regulating fibrosis of the myocardium. In this review paper, we will summarize the main elements and crucial players of cardiac fibrosis. In addition, we will provide an overview of the diverse roles of AMPK in the heart and discuss in detail its implication in cardiac fibrosis. Lastly, we will highlight the recently published literature concerning AMPK-targeting current therapy and novel strategies aiming to attenuate fibrosis.