Inhibits AMP-activated Protein Kinase Activity Research Articles

Analyzing the impact of acupuncture in conjunction with a Quantum lipid-lowering device on hyperlipidemic rats: insights into adenosine monophosphate-activated protein kinase signaling, blood lipids, and gut microbiota.

We explored the impact of acupuncture (ACUP) in conjunction with a quantum lipid-lowering device (Quantum) on the blood lipids and gut microbiota in hyperlipidemic rats, focusing on the adenosine monophosphate- (AMP)-activated protein kinase (AMPK) signaling pathway. Fifty Sprague-Dawley rats were randomly allocated into five groups: Normal, Model, Acup + Quantum, Acup, and Quantum. Hyperlipidemic models were established in all groups except Normal. The Model group did not receive any intervention after modeling. The Acup + Quantum group received both treatments, the Acup group received only acupuncture, and the Quantum group received only the quantum lipid-lowering device. We used ELISA to measure serum lipid and liver enzyme levels, hematoxylin and eosin (HE) staining for liver pathology, Western blot for protein expression, and 16S rRNA sequencing to analyze intestinal microbiota diversity in rats. Elisa results showed that compared with the model group, Acup + Quantum group could reduce the levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein-cholesterol (LDL-C), aspartate transaminase (AST) and aspartate transaminase (ALT) in rats with hyperlipidemia (P<0.01), and increase the level of high-density lipoprotein-cholesterol (HDL-C) (P<0.01). HE staining results showed that compared with the model group, the hepatocytes of rats in the Acup + Quantum group looked round and full, the liver plates were arranged regularly and neatly, and there was no obvious abnormality in the liver sinusoids. Western blot results showed that compared with the model group, the Acup + Quantum group inhibited AMPK activation, increased P-AMPK/AMPK protein expression (P<0.05), and decreased phospho-acetyl-CoA carboxylases (P-ACC/ACC), Sterol regulatory element-binding transcription factor-1C (SREBP-1C), and FAS protein expression (P<0.05; P<0.01; P<0.01), which resulted in lipid-lowering effect. The results of intestinal flora showed that Acup + Quantum group improved the intestinal microbial microenvironment of hyperlipidemic rats by regulating the structure of intestinal microflora, increasing the abundance of Firmicutes flora, and decreasing the abundance of harmful bacteria, such as Bacteroidetes and Proteobacteria. Acupuncture combined with quantum lipid-lowering device can improve the blood lipid and liver function levels and regulate the intestinal microbial microenvironment of hyperlipidemic rats. This therapeutic outcome is likely achieved through the activation of the AMPK pathway and the beneficial modulation of the intestinal microbiota of rats.

Phosphorylation of AMPKα at Ser485/491 Is Dependent on Muscle Contraction and Not Muscle-Specific IGF-I Overexpression.

Glucose is an important fuel for highly active skeletal muscles. Increased adenosine monophosphate (AMP)/adenosine triphosphate (ATP) ratios during repetitive contractions trigger AMP-activated protein kinase (AMPK), indicated by phosphorylation of AMPKαThr172, which promotes glucose uptake to support heightened energy needs, but it also suppresses anabolic processes. Inhibition of AMPK can occur by protein kinase B (AKT)-mediated phosphorylation of AMPKαSer485/491, releasing its brake on growth. The influence of insulin-like growth factor I (IGF-I) on glucose uptake and its interplay with AMPK activation is not well understood. Thus, the goal of this study was to determine if increased muscle IGF-I altered AMPKα phosphorylation and activity during muscle contraction. Adult male mice harboring the rat Igf1a cDNA regulated by the fast myosin light chain promoter (mIgf1+/+) and wildtype littermates (WT) were used in the study. mIgf1+/+ mice had enhanced glucose tolerance and insulin-stimulated glucose uptake, but similar exercise capacity. Fatiguing stimulations of extensor digitorum longus (EDL) muscles resulted in upregulated AMPKα phosphorylation at both Thr172 and Ser485/491 in WT and mIgf1+/+ muscles. No differences in the phosphorylation response of the downstream AMPK target TBC1D1 were observed, but phosphorylation of raptor was significantly higher only in WT muscles. Further, total raptor content was elevated in mIgf1+/+ muscles. The results show that high muscle IGF-I can enhance glucose uptake under resting conditions; however, in contracting muscle, it is not sufficient to inhibit AMPK activity.

FABP5 Deficiency Impaired Macrophage Inflammation by Regulating AMPK/NF-κB Signaling Pathway.

Fatty acid binding protein 5 (FABP5) is mainly involved in the uptake, transport, and metabolism of fatty acid in the cytoplasm, and its role in immune cells has been recognized in recent years. However, the role of FABP5 in macrophage inflammation and its underlying mechanisms were not fully addressed. In our study, the acute liver injury and sepsis mouse models were induced by i.p. injection of LPS and cecal contents, respectively. Oleic acid (0.6 g/kg) was injected four times by intragastric administration every week, and this lasted for 1 wk before the LPS or cecal content challenge. We found that myeloid-specific deletion of FABP5 mitigated LPS-induced acute liver injury with reduced mortality of mice, histological liver damage, alanine aminotransferase, and proinflammatory factor levels. Metabolic analysis showed that FABP5 deletion increased the intracellular unsaturated fatty acids, especially oleic acid, in LPS-induced macrophages. The addition of oleic acid also decreased LPS-stimulated macrophage inflammation in vitro and reduced acute liver injury in LPS-induced or cecal content-induced sepsis mice. RNA-sequencing and molecular mechanism studies showed that FABP5 deletion or oleic acid supplementation increased the AMP/ATP ratio and AMP-activated protein kinase (AMPK) activation and inhibited the NF-κB pathway during the inflammatory response to LPS stimulation of macrophages. Inhibiting AMPK activation or expression by chemical or genetic approaches significantly rescued the decreased NF-κB signaling pathway and inflammatory response in LPS-treated FABP5-knockout macrophages. Our present study indicated that inhibiting FABP5 or supplementation of oleic acid might be used for the treatment of sepsis-caused acute liver injury.

AMP-Activated Protein Kinase Contributes to Apoptosis Induced by the Bcl-2 Inhibitor Venetoclax in Acute Myeloid Leukemia.

Simple SummaryThe Bcl2 inhibitor venetoclax is a breakthrough therapy in acute myeloid leukemia (AML). We show that venetoclax induces caspase-dependent degradation of AMPK, a central regulator of cellular energy metabolism, with implications in the anti-Leukemic activity of venetoclax in AML.The treatment of acute myeloid leukemia (AML) remains a challenge especially among the elderly. The Bcl-2 inhibitor venetoclax recently showed significant survival benefits in AML patients when combined to low-dose cytarabine or azacitidine. Bcl-2 inhibition initiate mitochondrial apoptosis, but also respiration and cellular ATP production in AML. AMP-Activated Protein Kinase (AMPK) is a central energy sensor activated by increased AMP:ATP ratio to restore the cellular energy balance. Unexpectedly, we observed that venetoclax inhibited AMPK activity through caspase-dependent degradation of AMPK subunits in AML cells. On the other hand, genetic models of AMPK invalidation and re-expression suggested that AMPK participated to the early stages of apoptotic response through a negative regulation of multi-domain anti-apoptotic effectors such as Mcl-1 or Bcl-xL. Together our results suggested a new link between AMPK and Bcl-2-dependent mitochondrial apoptosis that participated to the anti-leukemic activity of venetoclax in AML.

Nuclear UHRF1 is a gate-keeper of cellular AMPK activity and function.

The AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis. Although much has been learned on how low energy status and glucose starvation activate AMPK, how AMPK activity is properly controlled in vivo is still poorly understood. Here we report that UHRF1, an epigenetic regulator highly expressed in proliferating and cancer cells, interacts with AMPK and serves to suppress AMPK activity under both basal and stressed conditions. As a nuclear protein, UHRF1 promotes AMPK nuclear retention and strongly suppresses nuclear AMPK activity toward substrates H2B and EZH2. Importantly, we demonstrate that UHRF1 also robustly inhibits AMPK activity in the cytoplasm compartment, most likely as a consequence of AMPK nucleocytoplasmic shuttling. Mechanistically, we found that UHRF1 has no obvious effect on AMPK activation by upstream kinases LKB1 and CAMKK2 but inhibits AMPK activity by acting as a bridging factor targeting phosphatase PP2A to dephosphorylate AMPK. Hepatic overexpression of UHRF1 showed profound effects on glucose and lipid metabolism in wild-type mice but not in those with the liver-specific knockout of AMPKα1/α2, whereas knockdown of UHRF1 in adipose tissue led to AMPK activation and reduced sizes of adipocytes and lipogenic activity, highlighting the physiological significance of this regulation in glucose and lipid metabolism. Thus, our study identifies UHRF1 as a novel AMPK gate-keeper with critical roles in cellular metabolism.

Inhibition of AMP-activated protein kinase in respiratory syncytial virus infection activates lipid metabolism.

Respiratory syncytial virus (RSV) is most commonly associated with upper respiratory tract infections during childhood. The lipid composition of cells and lipogenic enzymes play an important role in RSV infection. There are controversial data about whether lipid biosynthesis regulators such as AMP-activated protein kinase (AMPK) are deregulated by RSV. Hence, we examined whether the activation state of AMPK is altered in RSV-infected HEp-2 cells. Our data show that RSV infection inhibits AMPK activity, favoring the activation of downstream lipogenic effectors and cellular lipid anabolism in HEp-2 cells.

Microbial Imidazole Propionate Affects Responses to Metformin through p38γ-Dependent Inhibitory AMPK Phosphorylation

SummaryMetformin is the first-line therapy for type 2 diabetes, but there are large inter-individual variations in responses to this drug. Its mechanism of action is not fully understood, but activation of AMP-activated protein kinase (AMPK) and changes in the gut microbiota appear to be important. The inhibitory role of microbial metabolites on metformin action has not previously been investigated. Here, we show that concentrations of the microbial metabolite imidazole propionate are higher in subjects with type 2 diabetes taking metformin who have high blood glucose. We also show that metformin-induced glucose lowering is not observed in mice pretreated with imidazole propionate. Furthermore, we demonstrate that imidazole propionate inhibits AMPK activity by inducing inhibitory AMPK phosphorylation, which is dependent on imidazole propionate-induced basal Akt activation. Finally, we identify imidazole propionate-activated p38γ as a novel kinase for Akt and demonstrate that p38γ kinase activity mediates the inhibitory action of imidazole propionate on metformin.

Ubiquitin-dependent proteasomal degradation of AMPK gamma subunit by Cereblon inhibits AMPK activity

Cereblon (CRBN), a substrate receptor for Cullin-ring E3 ubiquitin ligase (CRL), is a major target protein of immunomodulatory drugs. An earlier study demonstrated that CRBN directly interacts with the catalytic α subunit of AMP-activated protein kinase (AMPK), a master regulator of energy homeostasis, down-regulating the enzymatic activity of AMPK. However, it is not clear how CRBN modulates AMPK activity. To investigate the mechanism of CRBN-dependent AMPK inhibition, we measured protein levels of each AMPK subunit in brains, livers, lungs, hearts, spleens, skeletal muscles, testes, kidneys, and embryonic fibroblasts from wild-type and Crbn−/− mice. Protein levels and stability of the regulatory AMPKγ subunit were increased in Crbn−/− mice. Increased stability of AMPKγ in Crbn−/− MEFs was dramatically reduced by exogenous expression of Crbn. In wild-type MEFs, the proteasomal inhibitor MG132 blocked degradation of AMPKγ. We also found that CRL4CRBN directly ubiquitinated AMPKγ. Taken together, these findings suggest that CRL4CRBN regulates AMPK through ubiquitin-dependent proteasomal degradation of AMPKγ.

AMPK activation increases postoperative cognitive impairment in intermittent hypoxia rats via direct activating PAK2

The pathogenesis of postoperative cognitive impairment of obstructive sleep apnea-hypopnea syndrome (OSAHS) individuals remains unclear. AMP activated protein kinase (AMPK) is a ubiquitous sensor/effector of cell stresses. Thus we detected the role and underlying mechanisms of AMPK in postoperative cognitive impairment of OSAHS individuals in intermittent hypoxia rats. Cognitive function was evaluated by novel object recognition test and Barnes maze during the first 4 days after laparotomy. We found that laparotomy induced postoperative cognitive impairment and AMPK activation in intermittent hypoxia rats, but not in adult rats. Inhibiting AMPK activation via Compound C during laparotomy improved postoperative cognitive impairment and alleviated surgery-induced upregulation of p-PAK2, AMPK-PAK2 complex, and neuroinflammation (marked by microglial activation and IL-1β level) in intermittent hypoxia rats. These data suggested that AMPK played an important role in postoperative cognitive impairment of OSAHS individuals via directly activating PAK2.

Electroacupuncture Improves Synaptic Function in SAMP8 Mice Probably via Inhibition of the AMPK/eEF2K/eEF2 Signaling Pathway.

Synaptic loss and dysfunction is associated with cognitive impairment in Alzheimer's disease (AD). Recent evidence indicates that the AMP-activated protein kinase (AMPK)/eukaryotic elongation factor-2 kinase (eEF2K)/eukaryotic elongation factor-2 (eEF2) pathway was implicated in synaptic plasticity in AD. Therapeutic strategies for AD treatment are currently limited. Here, we investigate the effects of electroacupuncture (EA) on synaptic function and the AMPK/eEF2K/eEF2 signaling pathway in male senescence-accelerated mouse-prone 8 (SAMP8) mice. Male 7-month-old SAMP8 and SAMR1 mice (senescence-accelerated mouse resistant 1) were randomly divided into 3 groups: SAMR1 control group (Rc), SAMP8 control group (Pc), and SAMP8 electroacupuncture group (Pe). The Pe group was treated with EA for 30 days. Transmission electron microscopy (TEM) was used to observe the structure of synapse. The protein and mRNA expression of synaptophysin (SYN) and postsynaptic density 95 (PSD95) was examined by immunohistochemistry, western blot, and real-time RT-PCR. The activity of AMPK and eEF2K was studied by western blot. Our results showed that EA ameliorated synaptic loss, increased the expression of SYN and PSD95, and inhibited AMPK activation and eEF2K activity. Collectively, these findings suggested that the mechanisms of EA improving synaptic function in AD may be associated with the inhibition of the AMPK/eEF2K/eEF2 signaling pathway.

Repression of TERRA Expression by Subtelomeric DNA Methylation Is Dependent on NRF1 Binding.

Chromosome ends are transcribed into long noncoding telomeric repeat-containing RNA (TERRA) from subtelomeric promoters. A class of TERRA promoters are associated with CpG islands embedded in repetitive DNA tracts. Cytosines in these subtelomeric CpG islands are frequently methylated in telomerase-positive cancer cells, and demethylation induced by depletion of DNA methyltransferases is associated with increased TERRA levels. However, the direct evidence and the underlying mechanism regulating TERRA expression through subtelomeric CpG islands methylation are still to establish. To analyze TERRA regulation by subtelomeric DNA methylation in human cell line (HeLa), we used an epigenetic engineering tool based on CRISPR-dCas9 (clustered regularly interspaced short palindromic repeats – dead CRISPR associated protein 9) associated with TET1 (ten-eleven 1 hydroxylase) to specifically demethylate subtelomeric CpG islands. This targeted demethylation caused an up-regulation of TERRA, and the enhanced TERRA production depended on the methyl-sensitive transcription factor NRF1 (nuclear respiratory factor 1). Since AMPK (AMP-activated protein kinase) is a well-known activator of NRF1, we treated cells with an AMPK inhibitor (compound C). Surprisingly, compound C treatment increased TERRA levels but did not inhibit AMPK activity in these experimental conditions. Altogether, our results provide new insight in the fine-tuning of TERRA at specific subtelomeric promoters and could allow identifying new regulators of TERRA.

166-OR: AMPK-Dependent Regulation of Skeletal Muscle Mitochondrial Function by FNIP1

Skeletal muscle mitochondria are essential for maintaining metabolic homeostasis in response to a myriad of physiologic or pathophysiological stresses. The folliculin (FLCN)-interacting protein 1 (FNIP1) forms a complex with AMP-activated protein kinase (AMPK), and we previously demonstrated that FNIP1 inhibits AMPK activity in myocytes. Using gain- and loss-of-function approaches in mice, we discovered that FNIP1 is necessary, but not sufficient, to suppress AMPK-dependent mitochondrial function in skeletal muscle. Muscles in FNIP1 knockout (KO) mice show enhanced red coloration, oxidative mitochondrial activity, and type I fiber transformation. Surprisingly, no change in mitochondrial oxidative capacity and fiber type switching was observed in transgenic mice overexpressing FNIP1 in muscle (FNIP1Tg), while restoration of the expression of FNIP1 in the KO muscle (FNIP1Tg/KO) reversed the mitochondrial oxidative metabolism program. RNA-seq profiling revealed that 3328 genes showed over 1.5-fold altered in FNIP1 KO muscle, and the majority number (2856) was regulated in a FNIP1-dependent manner. Gene ontology based analysis revealed that the primary FNIP1-altered genes were mitochondrial metabolic genes. Mitochondrial respiration analysis confirmed that both pyruvate- and succinate-driven state 3 respiration rates were significantly higher in FNIP1 KO muscle, whereas restoration of the expression of FNIP1 in KO muscle reduced the mitochondrial function. Importantly, AMPK, but not mTOR, was markedly induced in FNIP1 KO muscle but reduced in FNIP1Tg/KO muscle. Muscle-specific knockout of the two AMPKα subunits (AMPKα1α2 dmKO) reduced mitochondrial function in FNIP1 KO muscle. Similar results were obtained in skeletal myocytes in culture, using AMPK loss-of-function approaches in FNIP1 KO myocyte. Thus, FNIP1 represents a previously unrecognized negative regulator of muscle mitochondrial function and AMPK is vital for FNIP1 actions in skeletal muscle. Disclosure L. Xiao: None. J. Liu: None. Z. Sun: None. Y. Yin: None. Y. Mao: None.

Dichloroacetate Ameliorates Cardiac Dysfunction Caused by Ischemic Insults Through AMPK Signal Pathway—Not Only Shifts Metabolism

Dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase (PDK), regulates substrate metabolism in the heart. AMP-activated protein kinase (AMPK) is an age-related energy sensor that protects the heart from ischemic injury. This study aims to investigate whether DCA can protect the heart from ischemic injury through the AMPK signaling pathway. Young (3-4 months) and aged (20-24 months) male C57BL/6J mice were subjected to ligation of the left anterior descending coronary artery (LAD) for an in vivo ischemic model. The systolic function of the hearts was significantly decreased in both young and aged mice after 45 min of ischemia and 24 h of reperfusion. DCA treatment significantly improved cardiac function in both young and aged mice. The myocardial infarction analysis demonstrated that DCA treatment significantly reduced the infarction size caused by ischemia/reperfusion (I/R) in both young and aged mice. The isolated-cardiomyocyte experiments showed that DCA treatment ameliorated contractile dysfunction and improved the intracellular calcium signal of cardiomyocytes under hypoxia/reoxygenation (H/R) conditions. These cardioprotective functions of DCA can be attenuated by inhibiting AMPK activation. Furthermore, the metabolic measurements with an ex vivo working heart system demonstrated that the effects of DCA treatment on modulating the metabolic shift response to ischemia and reperfusion stress can be attenuated by inhibiting AMPK activity. The immunoblotting results showed that DCA treatment triggered cardiac AMPK signaling pathway by increasing the phosphorylation of AMPK's upstream kinase liver kinase B1 (LKB1) under both sham operations and I/R conditions. Thus, except from modulating metabolism in hearts, the cardioprotective function of DCA during I/R was mediated by the LKB1-AMPK pathway.

Activation of AMP-activated protein kinase (AMPK) aggravated postoperative cognitive dysfunction and pathogenesis in aged rats

The upstream signal molecule modulating neuro-inflammation and synaptic changes during the pathogenesis of postoperative cognitive dysfunction (POCD) is still elusive. Here, we examined the effects and mechanisms of energy sensor AMP-activated protein kinase (AMPK) in the pathogenesis of POCD. Our data showed that surgery significantly increased the expression of p-AMPK in aged rats (p < 0.05), but not in adult rats (p > 0.05). Moreover, inhibiting AMPK activation via compound C during operation significantly improved surgery-induced impairment of the learning and memory of aged rats in water maze (p < 0.05). Further mechanism studies showed that corresponding to the impairment of learning and memory after surgery, surgery significantly increased the activation of microglia, decreased the expressions of NR2B and p-NR2B, and increased the expressions of Tau and p-Tau, which also were obviously restored by inhibiting AMPK during operation. In contrast, Inhibiting AMPK activation during operation didn’t change ATP level in the hippocampus of aged rats after surgery. These data suggest that surgery induced activation of AMPK in hippocampus in an age-dependent manner. AMPK plays important roles in POCD of aged rats via multiple mechanisms, and is a possible molecular target for the prevention and treatment of POCD.

Cellular Application of Genetically Encoded Sensors and Impeders of AMPK.

Unraveling the spatiotemporal dynamics of 5'-AMP-activated protein kinase (AMPK) signaling is necessary to bridge the gap between nutrient signaling and downstream function. Three genetically encoded Förster Resonance Energy Transfer (FRET)-based AMPK biosensors are available yielding insight into how AMPK-derived signal propagates throughout a cell in response to particular inputs. These findings, together with accumulating evidence obtained from biochemical techniques, promise to give a holistic understanding of the AMPK signaling. In this protocol, we describe the procedures and materials required for imaging intracellular AMPK activity in an organelle-specific manner, with a focus on ABKAR, a FRET-based biosensor. In addition, we introduce a novel AMPK inhibitor peptide that allows us to inhibit AMPK activity at specific subcellular compartments.

MicroRNA-25 targets PKCζ and protects osteoblastic cells from dexamethasone via activating AMPK signaling

AMP-activated protein kinase (AMPK) activation could protect osteoblasts from dexamethasone (Dex). This study aims to provoke AMPK activation via microRNA downregulation of its negative regulator protein kinase C ζ (PKCζ). Results show that microRNA-25-5p (miR-25-5p) targets PKCζ's 3’ untranslated regions (UTRs). Forced-expression of miR-25 downregulated PKCζ and activated AMPK in human osteoblastic cells (OB-6 and hFOB1.19 lines), which thereafter protected cells from Dex. Reversely, expression of antagomiR-25, the miR-25 inhibitor, upregulated PKCζ and inhibited AMPK activation, exacerbating Dex damages. Notably, PKCζ shRNA knockdown similarly activated AMPK and protected osteoblastic cells from Dex. AMPK activation was required for miR-25-induced osteoblastic cell protection. AMPKα shRNA or dominant negative mutation almost completely blocked miR-25-induced cytoprotection against Dex. Further studies showed that miR-25 expression increased NADPH activity and suppressed Dex-induced oxidative stress in osteoblastic cells. Such effects by miR-25 were abolished with AMPKα knockdown or mutation. Significantly, miR-25-5p level was increased in patients’ necrotic femoral head tissues, which was correlated with PKCζ downregulation and AMPK hyper-activation. These results suggest that miR-25-5p targets PKCζ and protects osteoblastic cells from Dex possibly via activating AMPK signaling.

AMPK-dependent and independent effects of AICAR and compound C on T-cell responses.

As a master metabolic sensor, AMP-activated protein kinase (AMPK) is involved in different fundamental cellular processes. Regulation of AMPK activity either by agonists (e.g., AICAR) or by antagonists (e.g., Compound C) has been widely employed to study the physiological functions of AMPK. However, mounting evidence indicates AMPK-independent effects for these chemicals and how they regulate immune cell functions remains largely unknown. Herein, using T cells from AMPK conditional knockout mice and their wild type littermates, we demonstrate that AICAR and Compound C can, indeed, activate or inhibit AMPK activity in T cells, respectively. Specifically, AICAR inhibits, but Compound C promotes, Ca2+-induced T cell death in an AMPK-dependent manner. In contrast, our data also demonstrate that AICAR and Compound C inhibit T cell activation and cytokine production in an AMPK-independent manner. Moreover, we find that the AMPK-independent activity of AICAR and Compound Cis mediated via the mTOR signaling pathway in activated T cells. Our results not only reveal the critical role of AMPK in regulating T cell survival and function, but also demonstrate AMPK-dependent and independent rolesof AICAR/Compound C in regulating T cell responses, thus suggesting a context-dependent effect of these “AMPK regulators”.

AMPK-dependent and independent effects of AICAR and Compound C on T-cell responses

Abstract As a master metabolic sensor, AMP-activated protein kinase (AMPK) is involved in different fundamental cellular processes. Regulation of AMPK activity either by agonists (e.g., AICAR) or by antagonists (e.g., Compound C) has been widely employed to study the physiological functions of AMPK. However, mounting evidence indicates AMPK-independent effects for these chemicals. Herein, using T cells from AMPK conditional knockout mice and their wild type littermates, we demonstrate that AICAR and Compound C can, indeed, activate or inhibit AMPK activity in T cells, respectively. Specifically, AICAR inhibits, but Compound C promotes, Ca2+-induced T cell death in an AMPK-dependent manner. In contrast, our data also demonstrate that AICAR and Compound C inhibit T cell activation and cytokine production in an AMPK-independent manner. Moreover, we find that the AMPK-independent activity of AICAR and Compound C is mediated via the mTOR signaling pathway in activated T cells. Our results not only reveal the critical role of AMPK in regulating T cell survival and function, but also demonstrate AMPK-dependent and independent roles of AICAR/Compound C in regulating T cell responses, thus suggesting a context-dependent effect of these “AMPK regulators”.

ATP-citrate lyase regulates cellular senescence via an AMPK- and p53-dependent pathway.

ATP citrate lyase (ACLY) is a key enzyme that is involved in de novo lipogenesis by catalyzing conversion of cytosolic citrate into acetyl CoA and oxaloacetate. Up-regulation of ACLY in various types of tumors enhances fatty acid synthesis and supplies excess acetyl CoA for histone acetylation. However, there is evidence that its enzymatic activity alone is insufficient to explain ACLY silencing-mediated growth arrest in tumor cells. In this study, we found that ACLY knockdown in primary human cells triggers cellular senescence and activation of tumor suppressor p53. Provision of acetyl CoA to ACLY knockdown cells did not alleviate ACLY silencing-induced p53 activation, suggesting an independent role for ACLY activity. Instead, ACLY physically interacted with the catalytic subunit of AMP-activated protein kinase (AMPK) and inhibited AMPK activity. The activation of AMPK under ACLY knockdown conditions may lead to p53 activation, ultimately leading to cellular senescence. In cancer cells, ACLY silencing-induced p53 activation facilitated DNA damage-induced cell death. Taken together, our results suggest a novel function of ACLY in cellular senescence and tumorigenesis.

Protein phosphatase 2C-alpha knockdown reduces angiotensin II-mediated skeletal muscle wasting via restoration of mitochondrial recycling and function.

BackgroundCirculating angiotensin II (AngII) is elevated in congestive heart failure (CHF), and leads to skeletal muscle wasting, which is strongly associated with poor patient outcomes. We previously found that AngII upregulates protein phosphatase 2C-alpha (PP2Cα) and dephosphorylates AMP-activated protein kinase (AMPK), a critical regulator of cellular metabolism, in skeletal muscle.MethodsTo determine the role of PP2Cα in AngII-induced wasting, gastrocnemius (Gas) muscles of FVB mice were injected with scrambled or PP2Cα siRNA and mice were infused with saline or AngII for 4 days.ResultsKnockdown of PP2Cα reduced AngII wasting, blocked AngII upregulation of PP2Cα, increased p-T172-AMPK, and inhibited AngII-mediated reductions in peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (TFAM), in complex IV activity, and in ATP levels. AngII impaired the rate of autophagy as determined by a 2.4-fold increase in p62/SQSTM1 (p62) accumulation. This induction was reduced by PP2Cα knockdown, which also increased beclin-1 expression and microtubule-associated protein 1 light chain 3 (LC3)-II conversion in AngII-infused Gas. AngII reduced activating S555 phosphorylation of UNC-51-like kinase 1 (ULK1), a critical regulator of autophagosome formation, and increased inhibitory S757 ULK1 phosphorylation and these effects were prevented by PP2Cα siRNA.ConclusionsAngII inhibited AMPK activity and reduced PGC-1α and TFAM expression (thereby inhibiting mitochondrial biogenesis) and impaired ULK1 activation and autophagy (thereby also inhibiting clearance of damaged mitochondria), resulting in mitochondrial dysfunction, decreased ATP, and wasting. Knockdown of PP2Cα normalized AMPK activity, PGC-1α, NRF1, and TFAM levels and blocked AngII inhibition of ULK1, leading to improved mitochondrial biogenesis/recycling/function, energy production, and inhibition of AngII-induced wasting. These results demonstrate novel effects of AngII on cellular metabolism that are likely critical in mediating the muscle wasting that is a hallmark of CHF.