AMP-activated Protein Kinase Agonist AICAR Research Articles

Kimchi attenuates endoplasmic reticulum stress-induced hepatic steatosis in HepG2 cells and C57BL/6N mice

Kimchi is a traditional fermented food that contains abundant nutrients and functional ingredients with various health benefits. We previously reported that kimchi active components suppress hepatic steatosis caused by endoplasmic reticulum (ER) stress in vitro and in vivo. Therefore, we assessed the effect of kimchi on the inhibition of hepatic steatosis caused by ER stress in HepG2 cells and C57BL/6N mice to verify the hypothesis that kimchi may potentially inhibit nonalcoholic fatty liver disease. We investigated the effect of kimchi on cell viability and triglyceride concentrations in cells and on lipid profile, lipid accumulation, and expression of related genes in cells and mice with hepatic steatosis. A mechanistic study was also performed using the liver X receptor α agonist T0901317 and the AMP-activated protein kinase agonist AICAR. Kimchi was noncytotoxic and effectively reduced triglyceride concentrations and suppressed hepatic steatosis-related gene expression in cells and mice. Additionally, kimchi recovered weight loss, lowered the serum and liver tissue lipid profiles, suppressed lipid accumulation, and reduced the effects of T0901317 and AICAR on lipogenic gene expression in tunicamycin-treated mice. Our results highlight that kimchi could prevent hepatic steatosis caused by ER stress in cells and mice.

Perfluorooctane sulfonate promotes hepatic lipid accumulation and steatosis in high-fat diet mice through AMP-activated protein kinase/acetyl-CoA carboxylase (AMPK/ACC)pathway.

Perfluorooctane sulfonate (PFOS) is a hepatotoxic environmental organic pollutant that can cause aberrant lipid accumulation in the liver. However, the molecular mechanism underlying PFOS-induced hepatic steatosis remains unclear. Our research showed that subchronic PFOS exposure inhibited AMP-activated protein kinase (AMPK) phosphorylation, leading to increased acetyl-CoA carboxylase (ACC) activity, attenuated fatty acid β-oxidation, and consequent liver lipid accumulation. We found that 1 mg/kg/day PFOS exposure significantly aggravated steatosis in high-fat diet (HFD)-fed mice, along with reduced AMPK activity. Oil Red O results showed that PFOS exposure caused fat accumulation in HepG2 cells. As predicted, PFOS treatment reduced the level of phosphorylated AMPK in a concentration-dependent manner, leading to subsequent increase in ACC activity and lipid droplet accumulation in HepG2 cells. Treatment with 200-μM AMPK agonist AICAR alleviated PFOS-induced ACC activation and lipid accumulation. In summary, our data highlight a crucial role of AMPK/ACC pathway in PFOS-mediated liver lipid metabolic disorders.

Exercise-induced FKBP8-mediated Mitophagy Via AMPK Activation In Soleus Muscle Of Rats

Scientific Abstract PURPOSE: To investigate the effects of downhill running on mitochondrial structure/function and expression levels of mitophagy-related proteins in rat skeletal muscle, and whether AMP-activated protein kinase (AMPK) plays a regulatory role in this context. METHODS: Male Sprague-Dawley rats were divided into a control (C, n = 8) or an exercise (E, n = 56) group. Rats in the E group were exercised on a treadmill down a 16° incline at 16 m/min for 90 min, and were further divided into 0 h (E0), 12 h (E12), 24 h (E24), 48 h (E48) and 72 h (E72) post-exercise sub-groups. Rats in the E0 and E12 sub-groups received acute AMPK agonist AICAR treatment (500 mg/kg via I.P.) or saline (n = 8 each) 30 min before exercise. At each time point the soleus muscle was collected under full anesthesia. Mitochondrial ultrastructural changes in skeletal muscle were observed by using a transmission electron microscope. Protein expression levels of skeletal muscle p-AMPKα1/2(T172), total AMPKα, heat shock protein 60 (HSP60), cyclophilin D (CypD), mitochondrial FK506 binding protein 8 (FKBP8/FKBP38), microtubule-associated protein 1 light chain 3 (LC3) and cytosolic cytochrome C (Cyt C) were determined by using Western blot. LC3 co-localizations with mitochondria and FKBP8 were measured by the immunofluorescence technique. One-way and two-way ANOVAs were used for data analysis. RESULTS: After downhill running, skeletal muscle mitochondrial structure appeared to be abnormal and contained a large number of mitophagosomes; the expression levels of AMPK phosphorylation (E12 = 1.81 ± 0.54), FKBP8 (E12 = 3.11 ± 0.26), LC3 (E12 = 1.80 ± 0.12), CypD (E0 = 1.41 ± 0.26) and Cyt C (E0 = 2.31 ± 0.68) and the co-localization of FKBP8 and LC3 were significantly higher, whereas the expression levels of mitochondrial quantitative protein HSP60 (E12 = 0.62 ± 0.09) were significantly lower than those in the C group (p < 0.05 to p < 0.01). AICAR treatment enhanced AMPK phosphorylation in exercised muscle and the expression levels of mitochondrial FKBP8 and LC3 involved in mitophagy. CONCLUSION: A session of downhill running activated the AMPK phosphorylation and LC3 co-localizations with mitochondria and FKBP8, and induced mitophagy and mitochondrial damage within rat skeletal muscle. (Supported by Natural Science Foundation of China grant 31900842).

FLT4/VEGFR3 activates AMPK to coordinate glycometabolic reprogramming with autophagy and inflammasome activation for bacterial elimination

ABSTRACT Macrophages rapidly undergo glycolytic reprogramming in response to macroautophagy/autophagy, inflammasome activation and pyroptosis for the clearance of bacteria. Identification the key molecules involved in these three events will provide critical potential therapeutic applications. Upon S. typhimurium infection, FLT4/VEGFR3 and its ligand VEGFC were inducibly expressed in macrophages, and FLT4 signaling inhibited CASP1 (caspase 1)-dependent inflammasome activation and pyroptosis but enhanced MAP1LC3/LC3 activation for elimination of the bacteria. Consistently, FLT4 mutants lacking the extracellular ligand-binding domain increased production of the proinflammatory metabolites such as succinate and lactate, and reduced antimicrobial metabolites including citrate and NAD(P)H in macrophages and liver upon infection. Mechanistically, FLT4 recruited AMP-activated protein kinase (AMPK) and phosphorylated Y247 and Y441/442 in the PRKAA/alpha subunit for AMPK activation. The AMPK agonist AICAR could rescue glycolytic reprogramming and inflammasome activation in macrophages expressing the mutant FLT4, which has potential translational application in patients carrying Flt4 mutations to prevent recurrent infections. Collectively, we have elucidated that the FLT4-AMPK module in macrophages coordinates glycolytic reprogramming, autophagy, inflammasome activation and pyroptosis to eliminate invading bacteria.

Peripheral Administration of a Cell-Penetrating MOTS-c Analogue Enhances Memory and Attenuates Aβ1-42- or LPS-Induced Memory Impairment through Inhibiting Neuroinflammation.

MOTS-c is a 16-amino acid mitochondrial derivative peptide reported to be involved in regulating insulin and metabolic homeostasis via the AMP activated protein kinase (AMPK). AMPK agonist AICAR has been reported to improve cognition. Previous reports also pointed out that MOTS-c may be effective as a therapeutic option toward the prevention of the aging processes. Therefore, we investigated the roles of MOTS-c in the memory recognition process. The results showed that central MOTS-c not only enhanced object and location recognition memory formation and consolidation but also ameliorated the memory deficit induced by Aβ1-42 or LPS. The memory-ameliorating effects of MOTS-c could be blocked by AMPK inhibitor dorsomorphin. Moreover, MOTS-c treatment significantly increased the phosphorylation of AMPK but not ERK, JNK, and p38 in the hippocampus. The underlying mechanism of MOTS-c neuroprotection may involve inhibiting the activation of astrocytes and microglia and production of proinflammatory cytokines. In addition, we found that peripheral administration of MOTS-c does not cross the blood-brain barrier (BBB) and plays an effect. In order to improve the brain intake of MOTS-c, we screen out (PRR)5, a cell penetrating peptides, as a carrier for MOTS-c into the brain. Then in the NOR task, intranasal or intravenous MP (cell-penetrating MOTS-c analogue) showed good memory performance on memory formation, memory consolidation, and memory impairment. Near-infrared fluorescent experiments showed the real-time biodistribution in brain after intranasal or intravenous infusion of MP. These results suggested that MOTS-c might be a new potential target for treatment of cognitive decline in AD.

The effects of UCP2 on autophagy through the AMPK signaling pathway in septic cardiomyopathy and the underlying mechanism.

BackgroundMitochondrial dysfunction plays an important role in the development of septic cardiomyopathy. This study aimed to reveal the protective role of uncoupling protein 2 (UCP2) in mitochondria through AMP-activated protein kinase (AMPK) on autophagy during septic cardiomyopathy.MethodsUCP2 knockout mice via a cecal ligation and puncture (CLP) model and the H9C2 cardiomyocyte cell line in response to lipopolysaccharide (LPS) in vitro were used to study the effect. The myocardial morphological alterations, indicators of mitochondrial injury and levels of autophagy-associated proteins (pAMPK, pmTOR, pULK1, pTSC2, Beclin-1, and LC3-I/II) were assessed. In addition, the mechanism of the interaction between UCP2 and AMPK was further studied through gain- and loss-of-function studies.ResultsCompared with the wild-type mice, the UCP2 knockout mice exhibited more severe cardiomyocyte injury after CLP, and the AMPK agonist AICAR protected against such injury. Consistent with this result, silencing UCP2 augmented the LPS-induced pathological damage and mitochondrial injury in the H9C2 cells, limited the upregulation of autophagy proteins and reduced AMPK phosphorylation. AICAR protected the cells from morphological changes and mitochondrial membrane potential loss and promoted autophagy. The silencing and overexpression of UCP2 led to correlated changes in the AMPK upstream kinases pLKB1 and CAMKK2.ConclusionsUCP2 exerts cardioprotective effects on mitochondrial dysfunction during sepsis via the action of AMPK on autophagy.

Activation of AMP-activated protein kinase signaling pathway ameliorates steatosis in laying hen hepatocytes

The fatty liver hemorrhage syndrome in laying hens is a disease of lipid metabolism disorders. Importantly, energy sensor AMP-activated protein kinase (AMPK) plays an essential role in homeostasis regulation of liver lipid. The current research aims to investigate the relationship between AMPK signaling pathway and lipid metabolism in laying hen hepatocytes and explore the underlying mechanisms. The steatotic hepatocytes model of laying hen was established and treated with AMPK agonist AICAR and inhibitor compound C. The results showed that the levels of triglyceride, total cholesterol, and low-density lipoprotein cholesterol significantly declined while high-density lipoprotein cholesterol level increased in the AICAR-treated steatosis group compared with the steatosis group. Furthermore, the mRNA levels of liver kinase B1 and AMP-activated protein kinase α1 declined significantly in the steatosis group compared with those in the normal group. However, AMPK activation significantly upregulated the mRNA levels of peroxisome proliferator-activated receptor α and carnitine palmitoyl transferase-1 while downregulated the mRNA levels of acetyl CoA carboxylase, fatty acid synthase, 3-hydroxy-3-methyl glutaryl coenzyme A reductase, Sn-glycerol-3-phosphate acyltransferase, and hepatocyte nuclear factor 4α. These results suggest that activated AMPK signaling pathway increases fatty acid oxidation and reduces lipid synthesis in laying hen hepatocytes, thereby ameliorating liver steatosis.

Asprosin promotes β-cell apoptosis by inhibiting the autophagy of β-cell via AMPK-mTOR pathway.

Asprosin is a newly discovered adipokine. A recent study showed that asprosin promoted the β-cell apoptosis of MIN6 cells. This study aims to determine whether asprosin induces the apoptosis in β-cell via regulating autophagy. Mouse insulinoma MIN6 cells were divided into the following four groups: control, vehicle, high glucose, and asprosin groups. MIN6 cells in the asprosin group were transfected with recombinant asprosin-T2A-GFP vector. MIN6 cells in the vehicle group were transfected with vector only. Then the apoptosis of MIN6 cells was detected using flow cytometry. Real-time polymerase chain reaction and western blot were used to determine the expression of caspase 3, LC3-I, LC3-II, beclin 1, P62, AMP activated protein kinase (AMPK), and mammalian target of rapamycin (mTOR). AMPK pathway was enhanced using AMPK agonist AICAR. High glucose and asprosin induced the apoptosis of MIN6 cells and elevated expression of caspase 3. In addition, high glucose and asprosin resulted in reduced expression of LC3-II/LC3-I, beclin 1, and increased expression of P62. p-AMPK expression was decreased and p-mTOR expression was increased after high glucose and asprosin treatment. AMPK agonist AICAR was used to activate the AMPK pathway. Treatment with AICAR significantly partly restored decreased autophagy and increased apoptosis of β-cell, which was induced by asprosin. Asprosin promotes the apoptosis of β-cell by inhibiting the autophagy of β-cell via AMPK-mTOR pathway.

DIHYDROMYRICETIN ATTENUATES HIGH GLUCOSE-INDUCED CASPASE 3 EXPRESSION, ROS PRODUCTION AND INCREASES AMPK PHOSPHONYLATION IN PC12 CELLS

Dihydromyricetin (DMY) has a protective effect on neural function under central nervous system dysfunction conditions. There is growing interest concerning the beneficial effects of DMY on treating diabetic neuropathy (DN). This study was carried to detect protective effects of DMY on high glucose (HG)-induced cell damage and related mechanisms. The effect of DMY on cell survival was detected by MTT assay. Caspase-3 and phosphorylated AMP-activated protein kinase (AMPK) was evaluated by Western blotting. The effects of DMY and AMPK agonist AICAR on ROS production was determined. Our results showed that DMY treatment protect against HG-induced cell damage. DMY treatment significantly reduced the expression of caspase-3 and phosphorylated AMPK. ROS production was inhibited by DMY or AMPK agonist AICAR treatment. These studies demonstrate that DMY may inhibit ROS production, caspase-3 expression through AMPK pathway.&#x0D; Keywords: dihydromyricetin, caspase, oxidative stress

Sirtuin4 suppresses the anti‐neuroinflammatory activity of infiltrating regulatory T cells in the traumatically injured spinal cord

The neuroinflammation following traumatic spinal cord injury (SCI) is a critical process that impacts both the injury and the recovery of spinal cord parenchyma. Infiltrating regulatory T (Treg) cells are potent anti-inflammatory cells that restrain post-SCI neuroinflammation. To understand the molecular mechanisms underlying the activity of infiltrating Treg cells, we used a mouse spinal cord compression injury model to analyze the role of Sirtuins (SIRTs) in the modulation of infiltrating Treg cell functions. We found that the expressions of SIRT4 and SIRT6 were up-regulated in infiltrating Treg cells. Using lentivirus-mediated gene expression or RNA interference, we revealed that SIRT4 substantially inhibited the expression of Foxp3, interleukin-10, and transforming growth factor-β in Treg cells, whereas SIRT6 had little effect on Treg cells. Consistently, SIRT4 overexpression weakened the suppressive effect of Treg cells on lipopolysaccharide-stimulated spinal cord CD11b+ myeloid cells. Knock-down of SIRT4 enhanced the anti-inflammatory activity of infiltrating Treg cells in the parenchyma of injured spinal cords. Additionally, SIRT4 overexpression blocked in vitro Treg cell generation from conventional T cells. Furthermore, SIRT4 down-regulated 5' AMP-activated protein kinase (AMPK) signaling in Treg cells, whereas the AMPK agonist AICAR restored the expression of Foxp3 and interleukin-10 in SIRT4-overexpressing Treg cells. In conclusion, our research unveils a new mechanism by which the post-SCI neuroinflammation is regulated.

AMP-activated protein kinase negatively regulates heat treatment-induced lactate secretion in cultured boar sertoli cells

Lactate secreted by Sertoli cells plays an important role in spermatogenesis. Heat stress changes AMP-activated protein kinase (AMPK) activity in many tissues and cells, and enhances lactate secretion in Sertoli cells. However, it is unclear whether heat stress affects lactate secretion by regulating the phosphorylation level of AMPK in boar immature Sertoli cells. In this study, immature boar Sertoli cells were treated at 43 °C for 30 min in an incubator. From 0 to 48 h post-heat stress, lactate secretion was enhanced and reached the maximum level (175% of the control) at 12 h. However, with increased recovery time, the phosphorylation level of AMPK decreased gradually, and reached the minimum level (58% of the control) at 12 h. Compared with heat treatment alone, pretreatment with the AMPK agonist AICAR (2 mmol/L, 2 h) reduced lactate secretion by 42.6%. Additionally, AICAR significantly decreased the lactate dehydrogenase (LDH) activity, and the mRNA and protein expression levels of GLUT3, LDHA, and MCT1. In addition, AMPK overexpression reduced lactate secretion by 22.5%, significantly decreased the LDH activity, and mRNA and protein expression levels of GLUT3, LDHA, and MCT1. These results showed that AMPK reduces heat-induced lactate secretion by decreasing the expression levels of GLUT3, LDHA and MCT1. The results also suggested that AMPK is a negative regulator of heat treatment-induced lactate secretion in cultured boar Sertoli cells.

Pharmacological eEF 2K activation promotes cell death and inhibits cancer progression

Activation of the elongation factor 2 kinase (eEF2K) leads to the phosphorylation and inhibition of the elongation factor eEF2, reducing mRNA translation rates. Emerging evidence indicates that the regulation of factors involved in protein synthesis may be critical for controlling diverse biological processes including cancer progression. Here we show that inhibitors of the HIV aspartyl protease (HIV-PIs), nelfinavir in particular, trigger a robust activation of eEF2K leading to the phosphorylation of eEF2. Beyond its anti-viral effects, nelfinavir has antitumoral activity and promotes cell death. We show that nelfinavir-resistant cells specifically evade eEF2 inhibition. Decreased cell viability induced by nelfinavir is impaired in cells lacking eEF2K. Moreover, nelfinavir-mediated anti-tumoral activity is severely compromised in eEF2K-deficient engrafted tumors in vivo Our findings imply that exacerbated activation of eEF2K is detrimental for tumor survival and describe a mechanism explaining the anti-tumoral properties of HIV-PIs.

AMPK Suppresses Connexin43 Expression in the Bladder and Ameliorates Voiding Dysfunction in Cyclophosphamide-induced Mouse Cystitis.

Bladder voiding dysfunction is closely related to local oxidation, inflammation, and enhanced channel activities. Given that the AMP-activated protein kinase (AMPK) has anti-oxidative, anti-inflammatory and channel-inhibiting properties, we examined whether and how AMPK affected bladder activity. AMPK activation in rat bladder smooth muscle cells (BSMCs) using three different AMPK agonists resulted in a decrease in connexin43 (Cx43) expression and function, which was associated with reduced CREB phosphorylation, Cx43 promoter activity and mRNA expression, but not Cx43 degradation. Downregulation of CREB with siRNA increased Cx43 expression. A functional analysis revealed that AMPK weakened BSMC contraction and bladder capacity. AMPK also counteracted the IL-1β- and TNFα-induced increase in Cx43 in BSMCs. In vivo administration of the AMPK agonist AICAR attenuated cyclophosphamide-initiated bladder oxidation, inflammation, Cx43 expression and voiding dysfunction. Further analysis comparing the responses of the wild-type (Cx43+/+) and heterozygous (Cx43+/−) Cx43 mice to cyclophosphamide revealed that the Cx43+/− mice retained a relatively normal micturition pattern compared to the Cx43+/+ mice. Taken together, our results indicate that AMPK inhibits Cx43 in BSMCs and improves bladder activity under pathological conditions. We propose that strategies that target AMPK can be developed as novel therapeutic approaches for treating bladder dysfunction.

Activation of AMPK/MnSOD signaling mediates anti-apoptotic effect of hepatitis B virus in hepatoma cells.

To investigate the anti-apoptotic capability of the hepatitis B virus (HBV) in the HepG2 hepatoma cell line and the underlying mechanisms. Cell viability and apoptosis were measured by MTT assay and flow cytometry, respectively. Targeted knockdown of manganese superoxide dismutase (MnSOD), AMP-activated protein kinase (AMPK) and hepatitis B virus X protein (HBx) genes as well as AMPK agonist AICAR and antagonist compound C were employed to determine the correlations of expression of these genes. HBV markedly protected the hepatoma cells from growth suppression and cell death in the condition of serum deprivation. A decrease of superoxide anion production accompanied with an increase of MnSOD expression and activity was found in HepG2.215 cells. Moreover, AMPK activation contributed to the up-regulation of MnSOD. HBx protein was identified to induce the expression of AMPK and MnSOD. Our results suggest that HBV suppresses mitochondrial superoxide level and exerts an anti-apoptotic effect by activating AMPK/MnSOD signaling pathway, which may provide a novel pharmacological strategy to prevent HCC.

Ampelopsin protects endothelial cells from hyperglycemia-induced oxidative damage by inducing autophagy via the AMPK signaling pathway.

Diabetic angiopathy is a major diabetes-specific complication that often begins with endothelial dysfunction induced by hyperglycemia; however, the pathological mechanisms of this progression remain unclear. Ampelopsin is a natural flavonol that has strong antioxidant activity, but little information is available regarding its antidiabetic effect. This study focused on the effect of ampelopsin on hyperglycemia-induced oxidative damage and the underlying mechanism of this effect in human umbilical vein endothelial cells (HUVECs). We found that hyperglycemia impaired autophagy in HUVECs through the inhibition of AMP-activated protein kinase (AMPK), which directly led to endothelial cell damage. Ampelopsin significantly attenuated the detrimental effect of hyperglycemia-induced cell dysfunction in a concentration-dependent manner in HUVECs. Ampelopsin significantly upregulated LC3-II, Beclin1, and Atg5 protein levels but downregulated p62 protein levels in HUVECs. Transmission electron microscopy and confocal microscopy indicated that ampelopsin notably induced autophagosomes and LC3-II dots, respectively. Additionally, the autophagy-specific inhibitor 3-MA, as well as Atg5 and Beclin1 siRNA pretreatment, markedly attenuated ampelopsin-induced autophagy, which subsequently abolished the protective effect of ampelopsin against hyperglycemia in HUVECs. Moreover, ampelopsin also increased AMPK activity and inhibited mTOR (mammalian target of rapamycin) complex activation. Ampelopsin-induced autophagy was attenuated by the AMPK antagonist compound C but strengthened by the AMPK agonist AICAR (5-minoimidazole-4-carboxamide ribonucleotide). Furthermore, AMPK siRNA transfection eliminated ampelopsin's alleviation of cell injury induced by hyperglycemia. The protective effect of ampelopsin against hyperglycemia-induced cell damage, which functions by targeting autophagy via AMPK activation, makes it a promising pharmacological treatment for type-2 diabetes.

AMP-activated protein kinase inhibits transforming growth factor-β-mediated vascular smooth muscle cell growth: implications for a Smad-3-dependent mechanism.

Dysfunctional vascular growth is a major contributor to cardiovascular disease, the leading cause of morbidity and mortality worldwide. Growth factor-induced activation of vascular smooth muscle cells (VSMCs) results in a phenotypic switch from a quiescent, contractile state to a proliferative state foundational to vessel pathology. Transforming growth factor-β (TGF-β) is a multifunctional signaling protein capable of growth stimulation via Smad signaling. Although Smad signaling is well characterized in many tissues, its role in VSM growth disorders remains controversial. Recent data from our lab and others implicate the metabolic regulator AMP-activated protein kinase (AMPK) in VSM growth inhibition. We hypothesized that AMPK inhibits VSMC proliferation by reducing TGF-β-mediated growth in a Smad-dependent fashion. Treatment of rat VSMCs with the AMPK agonist AICAR significantly decreased TGF-β-mediated activation of synthetic Smad2 and Smad3 and increased inhibitory Smad7. Flow cytometry and automated cell counting revealed that AICAR reversed TGF-β-mediated cell cycle progression at 24 h and elevated cell numbers at 48 h. TGF-β/Smad signaling increased the G0/G1 inducers cyclin D1/cyclin-dependent kinase (CDK) 4 and cyclin E/CDK2; however, AICAR reversed these events while increasing cytostatic p21. The specific role of Smad3 in AMPK-mediated reversal of TGF-β-induced growth was then explored using adenovirus-mediated Smad3 overexpression (Ad-Smad3). Ad-Smad3 cells increased cell cycle progression and cell numbers compared with Ad-GFP control cells, and these were restored to basal levels with concomitant AICAR treatment. These findings support a novel AMPK target in TGF-β/Smad3 for VSMC growth control and support continued investigation of AMPK as a possible therapeutic target for reducing vascular growth disorders.

Activation of AMPK restricts coxsackievirus B3 replication by inhibiting lipid accumulation

Coxsackievirus B3 (CVB3) is the major pathogen of human viral myocarditis. CVB3 has been found to manipulate and modify the cellular lipid metabolism for viral replication. The cellular AMP-activated protein kinase (AMPK) is a key regulator of multiple metabolic pathways, including lipid metabolism. Here we explore the potential roles AMPK plays in CVB3 infection. We found that AMPK is activated by the viral replication during CVB3 infection in Hela cells and primary myocardial cells. RNA interference mediated inhibition of AMPK could increase the CVB3 replication in cells, indicating that AMPK contributed to restricting the viral replication. Next, we showed that CVB3 replication could be inhibited by several different pharmacological AMPK activators including metformin, A769662 and AICAR. And the constitutively active AMPK mutant (CA-AMPK) could also inhibit the CVB3 replication. Furthermore, we found that CVB3 infection increased the cellular lipid levels and showed that the AMPK agonist AICAR both restricted CVB3 replication and reduced lipid accumulation through inhibiting the lipid synthesis associated gene expression. We further found that CVB3 infection would also induce AMPK activated in vivo. The AMPK agonist metformin, which has been widely used in diabetes therapy, could decrease the viral replication and further protect the mice from myocardial histological and functional changes in CVB3 induced myocarditis, and improve the survival rate of infected mice. Lastly, it was demonstrated that the AICAR-mediated restriction of viral replication could be rescued partially by exogenous palmitate, the first product of fatty acid biosynthesis, demonstrating that AMPK activation restricted CVB3 infection through its inhibition of lipid synthesis. Taken together, these data in the present study suggest a model in which AMPK is activated by CVB3 infection and restricts viral replication by inhibiting the cellular lipid accumulation, and inform a potential novel therapeutic strategy for CVB3-associated diseases.

Salinomycin Activates AMP-Activated Protein Kinase-Dependent Autophagy in Cultured Osteoblastoma Cells: A Negative Regulator against Cell Apoptosis

BackgroundThe malignant osteoblastoma has poor prognosis, thus the search for novel and more efficient chemo-agents against this disease is urgent. Salinomycin induces broad anti-cancer effects both in vivo and in vitro, however, its role in osteoblastoma is still not clear. Key FindingsSalinomycin induced both apoptosis and autophagy in cultured U2OS and MG-63 osteoblastoma cells. Inhibition of autophagy by 3-methyladenine (3-MA), or by RNA interference (RNAi) of light chain 3B (LC3B), enhanced salinomycin-induced cytotoxicity and apoptosis. Salinomycin induced a profound AMP-activated protein kinase (AMPK) activation, which was required for autophagy induction. AMPK inhibition by compound C, or by AMPKα RNAi prevented salinomycin-induced autophagy activation, while facilitating cancer cell death and apoptosis. On the other hand, the AMPK agonist AICAR promoted autophagy activation in U2OS cells. Salinomycin-induced AMPK activation was dependent on reactive oxygen species (ROS) production in osteoblastoma cells. Antioxidant n-acetyl cysteine (NAC) significantly inhibited salinomycin-induced AMPK activation and autophagy induction. ConclusionsSalinomycin activates AMPK-dependent autophagy in osteoblastoma cells, which serves as a negative regulator against cell apoptosis. AMPK-autophagy inhibition might be a novel strategy to sensitize salinomycin’s effect in cancer cells.

Transcription factor Krüppel-like factor 2 plays a vital role in endothelial colony forming cells differentiation.

Endothelial colony forming cells (ECFCs) participate in post-natal vasculogenesis. We previously reported that vascular endothelial growth factor (VEGF) promotes human ECFC differentiation through AMP-activated protein kinase (AMPK) activation. However, the mechanisms underlying transcriptional regulation of ECFC differentiation still remain largely elusive. Here, we investigated the role of transcription factor Krüppel-like factor 2 (KLF2) in the regulation of ECFC function. Human ECFCs were isolated from cord blood and cultured. Treatment with VEGF significantly increased endothelial markers in ECFCs and their capacity for migration and tube formation. The mRNA and protein levels of KLF2 were also significantly up-regulated. This up-regulation was abrogated by AMPK inhibition or by knockdown of KLF2 with siRNA. Furthermore, adenovirus-mediated overexpression of KLF2 promoted ECFC differentiation by enhancing expression of endothelial cell markers, reducing expression of progenitor cell markers, and increasing the capacity for tube formation in vitro, indicating the important role of KLF2 in ECFC-mediated angiogenesis. Histone deacetylase 5 (HDAC5) was phosphorylated by AMPK activity induced by VEGF and the AMPK agonist AICAR (5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide). In vivo angiogenesis assay revealed that overexpression of KLF2 in bone-marrow-derived pro-angiogenic progenitor cells promoted vessel formation when the cells were implanted in C57BL/6 mice. Up-regulation of KLF2 by AMPK activation constitutes a novel mechanism of ECFC differentiation, and may have therapeutic value in the treatment of ischaemic heart disease.

Metformin preconditioning protects Daphnia pulex from lethal hypoxic insult involving AMPK, HIF and mTOR signaling

Hypoxic tolerance is subjected to modification during environmental stress. Understanding the mechanisms underlying the plasticity of hypoxic tolerance will be helpful for clinical applications such as stroke prevention. In a freshwater invertebrate, Daphnia pulex, we found that preconditioning with the antidiabetic drug metformin protects the animals from hypoxic insult. Metformin is known to activate the cellular energy sensor, AMP-activated protein kinase (AMPK). Application of the AMPK antagonist, compound C, effectively abolished the protective action by metformin. Meanwhile, the AMPK agonist AICAR failed to mimic the protective effect of metformin. At transcript level, metformin treatment increased the expression of hypoxia-inducible factor (HIF) α and β genes, as well as the HIF target genes in an AMPK-dependent manner, while AICAR treatment alone failed to increase the expression of HIF genes. Metformin treatment also increased the expression of AMPK α and γ genes, and this effect was blocked by compound C. These observations suggest that HIF activation and HIF target gene expression are possibly involved in metformin-mediated protection, while AMPK activation is necessary, but not sufficient for metformin-induced protection. Since increased hypoxic tolerance involves regenerative responses and thus protein synthesis, we measured the gene expression of the components of mTOR signaling pathway. Metformin increased the gene expression of raptor, a component of mTORC1 and known to control protein synthesis, and such increase was also eliminated by compound C. Taken together, metformin preconditioning activates multiple signaling pathways involved in gene expression and protein synthesis.