AMP-activated Protein Kinase Activation Research Articles

Limonin, a novel AMPK activator, protects against LPS-induced acute lung injury

AMP-activated protein kinase (AMPK) activation plays crucial roles in the treatment of many oxidative stress- and inflammation-induced diseases, including acute lung injury (ALI). Limonin is a naturally occurring tetracyclic triterpenoid extracted from the plants of Rutaceae and Meliaceae. Limonin also serves as an AMPK activator with anti-inflammatory and anti-oxidation effects. However, the potential beneficial effects of limonin on ALI and the possible mechanisms have never been disclosed till now. Here, the effects of limonin on lipopolysaccharide (LPS)-induced ALI in C57 BL/6 mice, plus bone marrow-derived macrophages (BMDM) stimulated with LPS to induce in vitro ALI model were investigated. Limonin significantly improved pulmonary function and alleviated lung pathological injury in LPS-induced mice. Meanwhile, limonin also markedly decreased inflammation and oxidative stress in lung tissues from LPS-treated mice. In vitro experiments also unveiled that limonin could decrease inflammation and oxidative stress in LPS-induced BMDM in a concentration-dependent manner. Mechanically, limonin could promote the activation of AMPKα and upregulate the expression of nuclear factor erythroid 2-related factor 2 (NRF2) in lung tissues and BMDM. Pharmacological inhibition of AMPKα by Compound C or AMPKα knockout could abolish the pulmonary protection from limonin during ALI. In conclusion, limonin mediates the activation of AMPKα/NRF2 pathway, providing an attractive therapeutic target for ALI in the future.

HSP90/CDC37 inactivation promotes degradation of LKB1 protein to suppress AMPK signaling in bronchial epithelial cells exposed to sulfur mustard analog, 2-chloroethyl ethyl sulfide

To investigate the role of the liver kinase (LK) B1 protein, an activator of AMP-activated protein kinase (AMPK), in AMPK signaling suppression when exposed to vesicant, a kind of chemical warfare agent. Cultured human bronchial epithelial cells were inflicted with sulfur mustard (SM) analog, 2-chloroethyl ethyl sulfide (CEES) of 0.2–1.0 mM concentration, and cell proliferation, apoptosis, autophagy, and cellular ATP level were analyzed up to 24 h after the exposure. Focusing on LKB1, heat shock protein (HSP) 90, and cell division cycle (CDC) 37 proteins, the protein expression, phosphorylation, and interaction were examined with western blot, immunofluorescence staining, and/or immunoprecipitation. AMPK signaling was found to be inhibited 24 h after being exposed to either sub-cytotoxic (0.5 mM) or cytotoxic (1.0 mM) concentration of CEES based on MTS assay. Consistently, the degradation of the LKB1 protein and its less interaction with the HSP90/CDC37 complex was confirmed. It was found that 1.0, not 0.5 mM CEES also decreased the CDC37 protein, proteasome activity, and cellular ATP content that modulates HSP90 protein conformation. Inhibiting proteasome activity could alternatively activate autophagy. Finally, either 0.5 or 1.0 mM CEES activated HSP70 and autophagy, and the application of an HSP70 inhibitor blocked autophagy and autophagic degradation of the LKB1 protein. In conclusion, we reported here that AMPK signaling inactivation by CEES was a result of LKB1 protein loss via less protein complex formation and enhanced degradation.

Krill oil inhibited adipogenic differentiation by inducing the nuclear Nrf2 expression and the AMPK activity.

The current study investigated the antiadipogenic mechanism of krill oil from the 3T3-L1 adipocytes. The krill oil adhered to the criteria as a food standard by showing 50.8% of the total phospholipid, 5.27% myristic acid, and 1.63% linoleic acid. The lipid accumulation that was measured in the 3T3-L1 cells using oil red O staining was reduced up to 54% by the krill oil. The krill oil treatment reduced the adipogenic transcription factors by downregulating the sterol regulatory element binding protein 1 (SREBP1) and acetyl-CoA carboxylase (ACC), phospho-ACC, and AMP-activated protein kinase (AMPK) phosphorylation. The current study confirmed that the krill oil inhibited adipogenesis by downregulating SREBP1 and ACC via the upregulation of the AMPK and nuclear factors E2-related factor 2 (Nrf2) signaling pathway in the 3T3-L1 adipocytes. These findings suggest that krill oil is a good source of phospholipid and phosphatidylcholine, which could be a potential natural antiobesity ingredient by inhibiting adipogenesis.

A sulfonamide chalcone AMPK activator ameliorates hyperglycemia and diabetic nephropathy in db/db mice

Diabetic nephropathy (DN) is a serious complication of diabetes mellitus (DM), which currently lacks effective treatments. AMP-activated protein kinase (AMPK) stimulation by chalcones, a class of polyphenols abundantly found in plants, is proposed as a promising therapeutic approach for DM. This study aimed to identify novel chalcone derivatives with improved AMPK-stimulating activity in human podocytes and evaluate their mechanisms of action as well as in vivo efficacy in a mouse model of DN. Among 133 chalcone derivatives tested, the sulfonamide chalcone derivative IP-004 was identified as the most potent AMPK activator in human podocytes. Western blot analyses, intracellular calcium measurements and molecular docking simulation indicated that IP-004 activated AMPK by mechanisms involving direct binding at allosteric site of calcium-dependent protein kinase kinase β (CaMKKβ) without affecting intracellular calcium levels. Interestingly, eight weeks of intraperitoneal administration of IP-004 (20 mg/kg/day) significantly decreased fasting blood glucose level, activated AMPK in the livers, muscles and glomeruli, and ameliorated albuminuria in db/db type II diabetic mice. Collectively, this study identifies a novel chalcone derivative capable of activating AMPK in vitro and in vivo and exhibiting efficacy against hyperglycemia and DN in mice. Further development of AMPK activators based on chalcone derivatives may provide an effective treatment of DN.

Mannose antagonizes GSDME-mediated pyroptosis through AMPK activated by metabolite GlcNAc-6P

Pyroptosis is a type of regulated cell death executed by gasdermin family members. However, how gasdermin-mediated pyroptosis is negatively regulated remains unclear. Here, we demonstrate that mannose, a hexose, inhibits GSDME-mediated pyroptosis by activating AMP-activated protein kinase (AMPK). Mechanistically, mannose metabolism in the hexosamine biosynthetic pathway increases levels of the metabolite N-acetylglucosamine-6-phosphate (GlcNAc-6P), which binds AMPK to facilitate AMPK phosphorylation by LKB1. Activated AMPK then phosphorylates GSDME at Thr6, which leads to blockade of caspase-3-induced GSDME cleavage, thereby repressing pyroptosis. The regulatory role of AMPK-mediated GSDME phosphorylation was further confirmed in AMPK knockout and GSDMET6E or GSDMET6A knock-in mice. In mouse primary cancer models, mannose administration suppressed pyroptosis in small intestine and kidney to alleviate cisplatin- or oxaliplatin-induced tissue toxicity without impairing antitumor effects. The protective effect of mannose was also verified in a small group of patients with gastrointestinal cancer who received normal chemotherapy. Our study reveals a novel mechanism whereby mannose antagonizes GSDME-mediated pyroptosis through GlcNAc-6P-mediated activation of AMPK, and suggests the utility of mannose supplementation in alleviating chemotherapy-induced side effects in clinic applications.

Computational modeling of AMPK and mTOR crosstalk in glutamatergic synapse calcium signaling

Neuronal energy consumption is vital for information processing and memory formation in synapses. The brain consists of just 2% of the human body’s mass, but consumes almost 20% of the body’s energy budget. Most of this energy is attributed to active transport in ion signaling, with calcium being the canonical second messenger of synaptic transmission. Here, we develop a computational model of synaptic signaling resulting in the activation of two protein kinases critical in metabolic regulation and cell fate, AMP-Activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) and investigate the effect of glutamate stimulus frequency on their dynamics. Our model predicts that frequencies of glutamate stimulus over 10 Hz perturb AMPK and mTOR oscillations at higher magnitudes by up to 36% and change the area under curve (AUC) by 5%. This dynamic difference in AMPK and mTOR activation trajectories potentially differentiates high frequency stimulus bursts from basal neuronal signaling leading to a downstream change in synaptic plasticity. Further, we also investigate the crosstalk between insulin receptor and calcium signaling on AMPK and mTOR activation and predict that the pathways demonstrate multistability dependent on strength of insulin signaling and metabolic consumption rate. Our predictions have implications for improving our understanding of neuronal metabolism, synaptic pruning, and synaptic plasticity.

AMPK activation improves recovery from pneumonia-induced lung injury via reduction of er-stress and apoptosis in alveolar epithelial cells

BackgroundBacterial pneumonia and related lung injury are among the most frequent causes of mortality in intensive care units, but also inflict serious and prolonged respiratory complications among survivors. Given that endoplasmic reticulum (ER) stress is a hallmark of sepsis-related alveolar epithelial cell (AEC) dysfunction, we tested if AMP-activated protein kinase (AMPK) affects recovery from ER stress and apoptosis of AECs during post-bacterial infection.MethodsIn a murine model of lung injury by P. aeruginosa non-lethal infection, therapeutic interventions included AMPK activator metformin or GSK-3β inhibitor Tideglusib for 96 h. Recovery from AEC injury was evidenced by accumulation of soluble T-1α (AEC Type 1 marker) in BAL fluids along with fluorescence analysis of ER-stress (CHOP) and apoptosis (TUNEL) in lung sections. AMPK phosphorylation status and mediators of ER stress were determined via Immunoblot analysis from lung homogenates. Macrophage-dependent clearance of apoptotic cells was determined using flow cytometry assay.ResultsP. aeruginosa-induced lung injury resulted in accumulation of neutrophils and cellular debris in the alveolar space along with persistent (96 h) ER-stress and apoptosis of AECs. While lung infection triggered AMPK inactivation (de-phosphorylation of Thr172-AMPK), metformin and Tideglusib promptly restored the AMPK activation status. In post infected mice, AMPK activation reduced indices of lung injury, ER stress and related apoptosis of AECs, as early as 24 h post administration of AMPK activators. In addition, we demonstrate that the extent of apoptotic cell accumulation is also dependent on AMPK-mediated clearance of apoptotic cells by macrophages.ConclusionsOur study provides important insights into AMPK function in the preservation of AEC viability after bacterial infection, in particular due reduction of ER-stress and apoptosis, thereby promoting effective recovery from lung injury after pneumonia.

An Update on Molecular Modes Implicated in Diastolic Impairment in Early Diabetic Cardiomyopathy; Probable Modes of Therapy &Avoidance-A Narrative Review

In case of diabetic patients cardiomyopathy is a significant etiology of heart failure (HF); howeverno clarification exists regarding its pathophysiology. Myocardial hypertrophy as well as diastolic impairment are the logo for Diabetic cardiomyopathy (DbCM), whereas systolic function gets impacted in the latter phase of this disease. Previously we had reviewed Diabetic cardiomyopathy (DbCM with emphasis on epigenetic alterations ,miR changes apart from adipocyte impairment in HF induction, role of SGLT2 hampering agents in beneficial Cardiovascular Outcome Trials (CVOT’s)and renal benefits, role of sirtuins in macrophage Polarization for Diabesity therapy .Here our aim was to posit the pathophysiological mode implicated in myocardial hypertrophy and escalated myocardial stiffness resulting in diastolic impairment .This myocardial stiffness takes place from cellular and extracellular matrix(ECM ) stiffness &cell- matrix crosstalk. Escalated inherent cardiomyocytes stiffness plausibly is the maximum significant cause responsible for myocardial stiffness. It leads to dysfunctional cardiomyocytes cytoskeleton stiffness. Various other modes are implicatedbyT2DM; particularly having significant influence on myocardial stiffening i.e dysfunctional nitric oxide (NO), coronary microvascular impairment, escalated inflammation, as well as Oxidative stress(OS), myocardial Sodium –glucose specific cotransporter 2(SGLT2) modulated actions. Greater insight aids in planning better therapy.Anti diabeticagents, SGLT2 hampering agents have revealed newer ways of benefit ,like sirtuins action , upregulation of nutrient deprivation signaling in addition to downregulation of nutrient surplus signaling, escalated expression &activity of AMP-activated protein kinase(AMPK), Sirtuins (SIRT1), SIRT3 andSIRT6, & Peroxisome Proliferator Activated Receptor γCoactivator -1α(PGC-1α) ,reduction in activation of mammalian target of rapamycin inhibitors (mTOR). Recently Ghosh et al. (2023),advocated targeting sarcoplasmic / cytosolic endoplasmic reticulum Ca2+ -ATPase(SERCA2) ,use of imeglimin which escalates insulin action& along with reverses Pancreatic βcells impairment ,use of anti oxidants, angiotensin receptor neprilysin hampering agents(ARNi),NO stimulating agents. More work is being done regarding combination of empagliflozin with linagliptin &others like semaglutide with .Gradually we will get answers by getting more insight.

TIMELESS promotes the proliferation and migration of lung adenocarcinoma cells by activating EGFR through AMPK and SPHK1 regulation

BackgroundLung adenocarcinoma (LUAD) has high morbidity and is prone to recurrence. TIMELESS (TIM), which regulates circadian rhythms in Drosophila, is highly expressed in various tumors. Its role in LUAD has gained attention, but the detailed function and mechanism have not been clarified completely at present. MethodsTumor samples from patients with LUAD patient data from public databases were used to confirm the relationship of TIM expression with lung cancer. LUAD cell lines were used and siRNA of TIM was adopted to knock down TIM expression in LUAD cells, and further cell proliferation, migration and colony formation were analyzed. By using Western blot and qPCR, we detected the influence of TIM on epidermal growth factor receptor (EGFR), sphingosine kinase 1 (SPHK1) and AMP-activated protein kinase (AMPK). With proteomics analysis, we comprehensively inspected the different changed proteins influenced by TIM and did global bioinformatic analysis. ResultsWe found that TIM expression was elevated in LUAD and that this high expression was positively correlated with more advanced tumor pathological stages and shorter overall and disease-free survival. TIM knockdown inhibited EGFR activation and also AKT/mTOR phosphorylation. We also clarified that TIM regulated the activation of SPHK1 in LUAD cells. And with SPHK1 siRNA to knock down the expression level of SPHK1, we found that EGFR activation were inhibited greatly too. Quantitative proteomics techniques combined with bioinformatics analysis clarified the global molecular mechanisms regulated by TIM in LUAD. The results of proteomics suggested that mitochondrial translation elongation and termination were altered, which were closely related to the process of mitochondrial oxidative phosphorylation. We further confirmed that TIM knockdown reduced ATP content and promoted AMPK activation in LUAD cells. ConclusionsOur study revealed that siTIM could inhibit EGFR activation through activating AMPK and inhibiting SPHK1 expression, as well as influencing mitochondrial function and altering the ATP level; TIM's high expression in LUAD is an important factor and a potential key target in LUAD.

Numerous Trigger-like Interactions of Kinases/Protein Phosphatases in Human Skeletal Muscles Can Underlie Transient Processes in Activation of Signaling Pathways during Exercise.

Optimizing physical training regimens to increase muscle aerobic capacity requires an understanding of the internal processes that occur during exercise that initiate subsequent adaptation. During exercise, muscle cells undergo a series of metabolic events that trigger downstream signaling pathways and induce the expression of many genes in working muscle fibers. There are a number of studies that show the dependence of changes in the activity of AMP-activated protein kinase (AMPK), one of the mediators of cellular signaling pathways, on the duration and intensity of single exercises. The activity of various AMPK isoforms can change in different directions, increasing for some isoforms and decreasing for others, depending on the intensity and duration of the load. This review summarizes research data on changes in the activity of AMPK, Ca2+/calmodulin-dependent protein kinase II (CaMKII), and other components of the signaling pathways in skeletal muscles during exercise. Based on these data, we hypothesize that the observed changes in AMPK activity may be largely related to metabolic and signaling transients rather than exercise intensity per se. Probably, the main events associated with these transients occur at the beginning of the exercise in a time window of about 1-10 min. We hypothesize that these transients may be partly due to putative trigger-like kinase/protein phosphatase interactions regulated by feedback loops. In addition, numerous dynamically changing factors, such as [Ca2+], metabolite concentration, and reactive oxygen and nitrogen species (RONS), can shift the switching thresholds and change the states of these triggers, thereby affecting the activity of kinases (in particular, AMPK and CaMKII) and phosphatases. The review considers the putative molecular mechanisms underlying trigger-like interactions. The proposed hypothesis allows for a reinterpretation of the experimental data available in the literature as well as the generation of ideas to optimize future training regimens.

Chrysin 7-O-β-d-glucopyranoside increases hepatic low-density lipoprotein receptor expression through AMP-activated protein kinase activation.

Elevated plasma low-density lipoprotein (LDL) cholesterol level is a risk factor for developing atherosclerosis. Increased LDL receptor (LDLR) expression is expected to reduce the risk of atherosclerotic disease since hepatic LDLR is essential for clearing plasma LDL cholesterol. Here, we screened human LDLR promoter effectors and observed that extracts from peduncles of sweet cherry (Prunus avium) 'Sato-Nishiki' induce LDLR gene promoter activity. We used several analytical and chemical methods to show that chrysin 7-O-β-d-glucopyranoside (chrysin-7G) is one of the compounds that stimulate LDLR gene promoter activity in cherry peduncle extracts. Furthermore, synthetic chrysin-7G increased the expression and activity of LDLR. The chrysin-7G-mediated increase in LDLR expression and activity was completely abolished by treatment with an AMP-activated protein kinase (AMPK) inhibitor, compound C. These results indicate that chrysin-7G increases LDLR expression through AMPK activation and may be a useful compound that can be recycled from waste parts of agricultural products.

Hydroxypropyl-beta-Cyclodextrin embedded resveratrol regulates gut microbiota to prevent NAFLD via activating AMPK signaling pathway

Non-alcoholic fatty liver disease (NAFLD) has become a globally rising issue that can cause liver-related morbidity and mortality. Recent studies have revealed that a high-fat diet (HFD) highly contributes to the prevalence and progression of NAFLD via impacting gut microbiota and lipid metabolism signal pathways. Resveratrol (RSV), a natural bioactive compound, has exhibited potential for preventing and alleviating NAFLD. However, due to the poor bioavailability of RSV, its strengths and underlying mechanisms for NAFLD therapeutic potential are poorly understood. To address this, we utilized Hydroxypropyl-beta-Cyclodextrin (HBC) to encapsulate RSV to enhance its water solubility and conducted prevention and intervention experiments in HFD-fed mice. The results showed that the HBC has significantly enhanced the water solubility of RSV by 250-fold and the HBC-RSV better prevented and alleviated the liver steatosis, obesity and abnormal lipid metabolism induced by HFD than RSV alone. Meanwhile, combining HFD and HBC-RSV or RSV prevented HFD mice progressing to NAFLD. Besides, further investigation indicated that RSV could resist liver injury and obesity by modulating gut microbiota, raising the levels of short-chain fatty acids (SCFAs) and activating AMP-activated protein kinase (AMPK) signaling pathway. The activated pathway down-regulated sterol receptor element binding protein 1c (SREBP1c) and acetyl-coenzyme A carboxylase (ACC) to decrease lipid synthesis and up-regulated peroxisome proliferators-activated receptorα (PPARα) to promote the fatty acid oxidation, thus preventing NAFLD. Our findings suggested that water solubility-enhanced RSV beneficially modulated gut microbiota, altered gut microbiota-derived SCFAs, and activated lipid metabolism regulatory pathways, providing potential for NAFLD prevention and alleviation.

AMPK is mitochondrial medicine for neuromuscular disorders.

Duchenne muscular dystrophy (DMD), myotonic dystrophy type 1 (DM1), and spinal muscular atrophy (SMA) are the most prevalent neuromuscular disorders (NMDs) in children and adults. Central to a healthy neuromuscular system are the processes that govern mitochondrial turnover and dynamics, which are regulated by AMP-activated protein kinase (AMPK). Here, we survey mitochondrial stresses that are common between, as well as unique to, DMD, DM1, and SMA, and which may serve as potential therapeutic targets to mitigate neuromuscular disease. We also highlight recent advances that leverage a mutation-agnostic strategy featuring physiological or pharmacological AMPK activation to enhance mitochondrial health in these conditions, as well as identify outstanding questions and opportunities for future pursuit.

Metformin protects against pulmonary hypertension-induced right ventricular dysfunction in an age- and sex-specific manner independent of cardiac AMPK.

Right ventricular (RV) function is the strongest predictor of survival in age-related heart failure as well as other clinical contexts in which aging populations suffer significant morbidity and mortality. However, despite the significance of maintaining RV function with age and disease, mechanisms of RV failure remain poorly understood and no RV-directed therapies exist. The antidiabetic drug and AMP-activated protein kinase (AMPK) activator metformin protects against left ventricular dysfunction, suggesting cardioprotective properties may translate to the RV. Here, we aimed to understand the impact of advanced age on pulmonary hypertension (PH)-induced right ventricular dysfunction. We further aimed to test whether metformin is cardioprotective in the RV and whether the protection afforded by metformin requires cardiac AMPK. We used a murine model of PH by exposing adult (4-6 mo) and aged (18 mo) male and female mice to hypobaric hypoxia (HH) for 4 wk. Cardiopulmonary remodeling was exacerbated in aged mice compared with adult mice as evidenced by elevated RV weight and impaired RV systolic function. Metformin attenuated HH-induced RV dysfunction but only in adult male mice. Metformin still protected the adult male RV even in the absence of cardiac AMPK. Together, we suggest that aging exacerbates PH-induced RV remodeling and that metformin may represent a therapeutic option for this disease in a sex- and age-dependent manner, but in an AMPK-independent manner. Ongoing efforts are aimed at elucidating the molecular basis for RV remodeling as well as delineating the mechanisms of cardioprotection provided by metformin in the absence of cardiac AMPK.NEW & NOTEWORTHY Right ventricular (RV) function predicts survival in age-related disease, yet mechanisms of RV failure are unclear. We show that aged mice undergo exacerbated RV remodeling compared with young. We tested the AMPK activator metformin to improve RV function and show that metformin attenuates RV remodeling only in adult male mice via a mechanism that does not require cardiac AMPK. Metformin is therapeutic for RV dysfunction in an age- and sex-specific manner independent of cardiac AMPK.

Theaflavin inhibits African swine fever virus replication by disrupting lipid metabolism through activation of the AMPK signaling pathway in virto

African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), which is one of the most harmful swine diseases in the pig industry because of its nearly 100% mortality rate in domestic pigs and results in incalculable economic loss. Ever since ASF was initially reported, scientists have worked to develop anti-ASF vaccines; however, currently no clinically effective vaccine for ASF is available. Therefore, the development of novel measures to prevent ASFV infection and transmission is essential. In this study, we aimed to investigate the anti-ASF activity of theaflavin (TF), a natural compound mainly isolated from black tea. We found that TF potently inhibited ASFV replication at non-cytotoxic concentrations ex vivo in primary porcine alveolar macrophages (PAMs). Mechanistically, we found that TF inhibited ASFV replication by acting on cells rather than interacting directly with ASFV to inhibit viral replication. Further, we found that TF upregulated the AMPK (5′-AMP-activated protein kinase) signaling pathway in ASFV-infected and uninfected cells, and treatment with the AMPK agonist MK8722 upregulated the AMPK signaling pathway and inhibited ASFV proliferation in a dose-dependent manner. Notably, the effects of TF on AMPK activation and ASFV inhibition were partially reversed by the AMPK inhibitor dorsomorphin. In addition, we found that TF down-regulated the expression of genes related to lipid synthesis and decreased the intracellular accumulation of total cholesterol and total triglycerides in ASFV-infected cells, suggesting that TF may inhibit ASFV replication by disrupting lipid metabolism. In summary, our results demonstrated that TF is an ASFV infection inhibitor and revealed the mechanism by which ASFV replication is inhibited, providing a novel mechanism and potential lead compound for the development of anti-ASFV drugs.

CaMKK2 is not involved in contraction-stimulated AMPK activation and glucose uptake in skeletal muscle

ObjectiveThe AMP-activated protein kinase (AMPK) gets activated in response to energetic stress such as contractions and plays a vital role in regulating various metabolic processes such as insulin-independent glucose uptake in skeletal muscle. The main upstream kinase that activates AMPK through phosphorylation of α-AMPK Thr172 in skeletal muscle is LKB1, however some studies have suggested that Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) acts as an alternative kinase to activate AMPK. We aimed to establish whether CaMKK2 is involved in activation of AMPK and promotion of glucose uptake following contractions in skeletal muscle. MethodsA recently developed CaMKK2 inhibitor (SGC-CAMKK2-1) alongside a structurally related but inactive compound (SGC-CAMKK2-1N), as well as CaMKK2 knock-out (KO) mice were used. In vitro kinase inhibition selectivity and efficacy assays, as well as cellular inhibition efficacy analyses of CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) were performed. Phosphorylation and activity of AMPK following contractions (ex vivo) in mouse skeletal muscles treated with/without CaMKK inhibitors or isolated from wild-type (WT)/CaMKK2 KO mice were assessed. Camkk2 mRNA in mouse tissues was measured by qPCR. CaMKK2 protein expression was assessed by immunoblotting with or without prior enrichment of calmodulin-binding proteins from skeletal muscle extracts, as well as by mass spectrometry-based proteomics of mouse skeletal muscle and C2C12 myotubes. ResultsSTO-609 and SGC-CAMKK2-1 were equally potent and effective in inhibiting CaMKK2 in cell-free and cell-based assays, but SGC-CAMKK2-1 was much more selective. Contraction-stimulated phosphorylation and activation of AMPK were not affected with CaMKK inhibitors or in CaMKK2 null muscles. Contraction-stimulated glucose uptake was comparable between WT and CaMKK2 KO muscle. Both CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) and the inactive compound (SGC-CAMKK2-1N) significantly inhibited contraction-stimulated glucose uptake. SGC-CAMKK2-1 also inhibited glucose uptake induced by a pharmacological AMPK activator or insulin. Relatively low levels of Camkk2 mRNA were detected in mouse skeletal muscle, but neither CaMKK2 protein nor its derived peptides were detectable in mouse skeletal muscle tissue. ConclusionsWe demonstrate that pharmacological inhibition or genetic loss of CaMKK2 does not affect contraction-stimulated AMPK phosphorylation and activation, as well as glucose uptake in skeletal muscle. Previously observed inhibitory effect of STO-609 on AMPK activity and glucose uptake is likely due to off-target effects. CaMKK2 protein is either absent from adult murine skeletal muscle or below the detection limit of currently available methods.

Insights into the therapeutic outcomes of trimetazidine/doxorubicin combination in Ehrlich solid-phase carcinoma mouse tumor model

One of the key features of cancer is metabolic reprogramming that can be exploited to sensitize cancer cells to chemotherapy. Trimetazidine (TMZ) is a metabolic anti-ischemic drug that blocks the activity of long-chain 3-ketoacyl CoA thiolase leading to the inhibition of fatty acid oxidation. AimsThe objective of the current investigation was to evaluate the idea that TMZ could synergize the antitumor activity of doxorubicin (DOX). Main methodsThe hypothesis was examined in vitro using the human breast cancer cell lines MCF-7 and MDA-MB231. In addition, the in vivo experiments were conducted using the Ehrlich solid phase carcinoma model. Key findingsIn vitro cytotoxicity experiments demonstrated that TMZ improved the potency of DOX in MCF-7 cell lines in a synergistic manner. In vivo testing confirmed that DOX/TMZ combination exhibits synergistic effect at both DOX/TMZ 1:10 and 1:5 ratios, where DOX was administered at one tenth and one fifth of its original dose, respectively. The co-treatment (1:5 ratio) significantly reduced tumor Nicotinamide adenine dinucleotide (NAD)+/NADH ratio (6.1-fold) and Adenosine triphosphate (ATP) levels (61 %) with concurrent activation of AMP-activated protein kinase (AMPK) (2.2-fold) and peroxisome proliferator-activated receptor-gamma coactivator (PGC)1-α (5.5-fold) protein expression versus control. The same treatment decreased the nuclear levels of NF-κB (p65) (57.5 %) and induced tumor apoptosis as evidenced by elevated Bax/Bcl-2 ratio (6.8-fold) along with active caspase-3 levels (6.6-fold) against control. SignificanceThe current investigation constitutes a proof-of-concept study that provided preclinical evidence for the anticancer activity of DOX/TMZ combination and warrants further investigation for repurposing TMZ in DOX protocols.

1507-P: Carnosic Acid–Induced Browning in 3T3-L1 Adipocytes Is Dependent on AMPK Activation

Background: Increased adiposity has long been associated with insulin resistance and adipose tissue plays a crucial role in regulating metabolic homeostasis, as its dysfunction in obesity leads to insulin resistance and type 2 diabetes (T2D). The primary function of white adipose tissue (WAT) is to store energy as lipids while brown adipose tissue (BAT) regulates thermogenesis by dissipating energy in a form of heat. The process of browning involves transdifferention of WAT into brown-like or beige adipocytes, which exhibit the same functional properties as BAT. Browning of WAT is an attractive approach against obesity and insulin resistance. In addition, evidence indicate that activation of the energy sensor AMP-activated protein kinase (AMPK) could counteract insulin resistance. The aim of this study is to examine if carnosic acid (CA), induces browning via activation of AMPK in 3T3-L1 white adipocytes. Material and methods: Cell morphology, lipid accumulation, mitochondrial density, and browning markers such as uncoupling protein-1 (UCP-1) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) levels were assessed using Cytation5 microscope, Oil-O-Red (ORO), MitoTracker Red, immunoblotting and immunocytochemistry. Results: Microscopic investigation revealed that CA reduced the size and number of lipid droplets in the cells, giving a rise to a brown-like phenotype. The reduction of lipid accumulation was also confirmed by ORO stain. Next, CA increased mitochondrial density and protein expression of UCP-1 and PGC-1-α in 3T3-L1 adipocytes. Most importantly, pre-treatment with compound C, prevented the CA-induced reduction in lipid accumulation, and mitochondrial density and blocked the CA-induced increase in UCP-1 and PGC-1 expression. Conclusion: This study demonstrates that CA has a pronounced potential in stimulating browning of adipocytes via AMPK-dependent mechanism. Future in vivo studies are required to fully examine the effects of CA. Disclosure F.Vlavcheski: None. N.Tsakiridis: None. A.Giacca: None. E.Tsiani: None. Funding Natural Science and Engineering Research Council of Canada (RGPIN-2018-06666)

1554-P: Carnosic Acid Protects against Short-Term Lipid-Infusion–Induced Glucose Intolerance in Mice

Background: Obesity and type 2 diabetes (T2D) are associated with elevated plasma free fatty acids (FFA). In vivo exposure to FFAs, such as palmitate, by lipid infusion results in insulin resistance as observed in obesity and T2D. In recent years, compounds found in herbs and plants have attracted attention for prevention and treatment of insulin resistance and T2D. In recent studies by our group, carnosic acid (CA), found in rosemary, increased glucose uptake and attenuated the FFA (palmitate)-induced insulin resistance in muscle cells and adipocytes. In addition, CA caused a robust activation of AMP-activated protein kinase (AMPK). Our present study aimed to examine the effects of CA in short-term FFA-induced glucose intolerance in mice. Material and Methods: C57Bl/6J mice were cannulated in their jugular veins and infused for 48h with ethylpalmitate (EtP, nontoxic method of elevating circulating palmitate because of hydrolysis to palmitate and ethanol by plasma esterases), mimicking elevated FFA levels seen in obesity. In addition, a group of mice were infused with EtP plus 50 mg/kg CA for 48h. Ethanol vehicle with or without CA were two additional control groups. At the end of the infusions, the mice were subjected to a 2 hr hyperglycemic clamp (20 mM glucose) and the glucose infusion rate was measured to assess glucose tolerance. Results: Preliminary data revealed that mice infused with EtP and CA had a higher glucose infusion rate (GIR; mg/kg*min) (66.5±10, mean±SEM) than mice infused with EtP alone (22.7±10). The EtP and CA was comparable to the GIR of ethanol vehicle infused mice (73±9). Future Directions: Further analysis of collected plasma and tissue will be used to assess insulin resistance and beta cell function in vivo and to elucidate the mechanisms of CA alleviation of FFA-induced insulin resistance. Conclusions: Our data indicate that CA has the potential to counteract FFA-induced glucose intolerance in mice. Disclosure N.Tsakiridis: None. F.Vlavcheski: None. E.Tsiani: None. A.Giacca: None.

259-LB: Loss of AMPK Induces Elongation of Preexisting Hepatic Fatty Acids, without Affecting DNL, during Fasting–Refeeding

AMP-activated protein kinase (AMPK) is a cellular energy sensor that conserves energy by inhibiting anabolic processes, such as de novo lipogenesis (DNL). Paradoxically, fructose metabolism activates AMPK by reducing the energy state (elevated AMP/ATP ratio), yet also promotes DNL. However, the comprehensive effects of fructose metabolism and AMPK activity on hepatic DNL has not been thoroughly investigated. Here, we examine how loss of AMPK function impacts DNL in response to fasting followed by refeeding a fructose or chow diet. After a 16 hour fast, 12 liver-specific AMPKα1α2 knock-out mice (AMPK-KO) and 12 AMPK α1α2 lox/lox mice (control) were fed either high fructose or chow diets (6 per a group) and administered D2O drinking water for 7 hours prior to blood collection and tissue harvest. After Folch extraction, fatty acids in liver triglyceride (TG-FA) were analyzed by proton and deuterium NMR spectroscopy to determine 2H incorporation and estimate DNL, elongation and re-esterification fluxes. Body water enrichment (~3.5%) was similar between groups. After correcting for food intake and body weight, preliminary data indicates that the AMP/ATP ratio was greater in AMPK-KO liver compared to controls, but neither chow nor fructose diets affected the energy state. Overall, neither hepatic steatosis nor DNL differed between groups. Furthermore, loss of AMPK function did not affect the amount of various FA species (i.e. saturated, unsaturated, DHA and ω3, linoleic, and non-essential). Instead, AMPK-KO liver had increased TG-FAs that were elongated compared to controls. This effect also tended to be greater in fructose fed mice (AMPK-KO and controls) compared to chow fed mice, even though fructose fed mice consumed fewer calories. In conclusion, loss of AMPK function resulted in increased elongation of hepatic TG-FAs, but not DNL, or the amount of FAs in the liver in response to fasting and refeeding with either fructose or chow diets. Disclosure J. A. Fletcher: None. S. Deja: None. S. C. Burgess: None. X. Fu: None. M. R. Inigo: None. J. Browning: None. B. Kucejova: None. Funding National Institutes of Health (K01AA030327)