AMPK Expression Research Articles

Taurine alleviated hepatic steatosis in oleic acid-treated-HepG2 cells and rats fed a high-fat diet

Taurine has been proven in many trials to alleviate the symptoms of metabolic associated fatty liver disease. Here its protective effect for hepatic steatosis and modulation of AMP-activated protein kinase and insulin signaling pathway were investigated. Steatotic HepG2 cell established with oleic acid (0.05 mmol/L), treated with taurine (5 mmol/L), dorsomorphin (10 μmol/L) for 24 h. Sprague Dawley rats were divided into regular and high-fat diet (HFD) groups, and their corresponding taurine (70 or 350 mg/kg BW/d) groups, fed for 8 weeks. In steatotic cell, taurine reduced the TG concentration and SREBP-1c, PPARγ, FAS, ACC, SCD1 protein levels, decreased phosphorylation of mTOR, IRS1 (Ser302), increased phosphorylation of AMPKα, LKB1, PI3K, Akt, ACC. While dorsomorphin eliminated taurine's TG-lowering effect. In HFD-fed rats, taurine reduced liver TG, serum TG, ALT, AST, IL-1β, IL-4, TNF-α. The effects of taurine on the main factors of fatty acid synthesis were mostly consistent with cell experiments, and the reduction of microRNAs (451, 33, 291b) was aligned with the improvement in LKB1 and AMPK expression in HFD rats. Taurine alleviated steatosis-induced inhibition of IRS1-PI3K-Akt pathway, but suppressed its positively regulated downstream factor mTOR. In parallel, taurine reduced steatosis by activating LKB1-AMPKα pathway via phosphorylation and no-phosphorylation manner, then inhibiting SREBP-1c directly or by suppressing mTOR phosphorylation.

A high-concentrate diet induces inflammatory injury via regulating Ca2+/CaMKKβ-mediated autophagy in mammary gland tissue of dairy cows.

Calmodulin-dependent protein kinase β (CaMKKβ) is closely related to Ca2+ concentration. An increase in Ca2+ concentration in the cytoplasm activates CaMKKβ, and activated CaMKKβ affects the activities of AMPK and mTOR and induces autophagy. A high-concentrate diet leads to Ca2+ disorder in mammary gland tissue. Therefore, this study mainly investigated the induction of mammary gland tissue autophagy by a high-concentrate diet and the specific mechanism of lipopolysaccharide (LPS)-induced autophagy in bovine mammary epithelial cells (BMECs). Twelve mid-lactation Holstein dairy cows were fed with a 40% concentrate diet (LC) and a 60% concentrate diet (HC) for 3 weeks. At the end of the trial, rumen fluid, lacteal vein blood, and mammary gland tissue were collected. The results showed that the HC diet significantly decreased rumen fluid pH, with a pH lower than 5.6 for more than 3 h, indicating successfully induction of subacute rumen acidosis (SARA). The mechanism of LPS-induced autophagy in BMECs was studied in vitro. First, the cells were divided into a Ctrl group and LPS group to study the effects of LPS on the concentration of Ca2+ and autophagy in BMECs. Then, cells were pretreated with an AMPK inhibitor (compound C) or CaMKKβ inhibitor (STO-609) to investigate whether the CaMKKβ-AMPK signaling pathway is involved in LPS-induced BMEC autophagy. The HC diet increased the concentration of Ca2+ in mammary gland tissue and pro-inflammatory factors in plasma. The HC diet also significantly increased the expression of CaMKKβ, AMPK, and autophagy-related proteins, resulting in mammary gland tissue injury. In vitro cell experiments showed that LPS increased intracellular Ca2+ concentration and upregulated protein expression of CaMKKβ, AMPK, and autophagy-related proteins. Compound C pretreatment decreased the expression of proteins related to autophagy and inflammation. In addition, STO-609 pretreatment not only reversed LPS-induced BMECs autophagy but also inhibited the protein expression of AMPK, thereby alleviating the inflammatory response in BMECs. These results suggest that inhibition of the Ca2+/CaMKKβ-AMPK signaling pathway reduces LPS-induced autophagy, thereby alleviating inflammatory injury of BMECs. Therefore, SARA may increase the expression of CaMKKβ by increasing Ca2+ levels and activate autophagy through the AMPK signaling pathway, thereby inducing inflammatory injury in mammary gland tissue of dairy cows.

Loss of AMPKα2 promotes melanoma tumor growth and brain metastasis

AMP-activated protein kinase (AMPK) is a critical cellular energy sensor at the interface of metabolism and cancer. However, the role of AMPK in carcinogenesis remains unclear. Here, through analysis of the TCGA melanoma dataset, we found that PRKAA2 gene that encodes the α2 subunit of AMPK is mutated in ∼9% of cutaneous melanomas, and these mutations tend to co-occur with NF1 mutations. Knockout of AMPKα2 promoted anchorage-independent growth of NF1-mutant melanoma cells, whereas ectopic expression of AMPKα2 inhibited their growth in soft agar assays. Moreover, loss of AMPKα2 accelerated tumor growth of NF1-mutant melanoma and enhanced their brain metastasis in immune-deficient mice. Our findings support that AMPKα2 serves as a tumor suppressor in NF1-mutant melanoma and suggest that AMPK could be a therapeutic target for treating melanoma brain metastasis.

Dietary α-ketoglutarate alleviates glycinin and β-conglycinin induced damage in the intestine of mirror carp (Cyprinus carpio).

This study investigated the glycinin and β-conglycinin induced intestinal damage and α-ketoglutarate alleviating the damage of glycinin and β-conglycinin in intestine. Carp were randomly divided into six dietary groups: containing fish meal (FM) as the protein source, soybean meal (SM), glycinin (FMG), β-conglycinin (FMc), glycinin+1.0% α-ketoglutarate (AKG) (FMGA), β-conglycinin+1.0% AKG (FMcA). The intestines were collected on 7th, and the hepatopancreas and intestines were collected on 56th. Fish treated with SM and FMc displayed reduced weight gain, specific growth rate, and protein efficiency. On 56th day, Fish fed on SM, FMG and FMc presented lower superoxide dismutase (SOD) activities. FMGA and FMcA had higher SOD activity than those fed on the FMG and FMc, respectively. In intestine, fish fed on the SM diets collected on 7th presented upregulated the expression of transforming growth factor beta (TGFβ1), AMP-activated protein kinase beta (AMPKβ), AMPKγ, and acetyl-CoA carboxylase (ACC). Fish fed FMG presented upregulated expression of tumor necrosis factor alpha (TNF-α), caspase9, and AMPKγ, while downregulated the expression of claudin7 and AMPKα. FMc group presented upregulated expression of TGFβ1, caspase3, caspase8, and ACC. Fish fed FMGA showed upregulated expression of TGFβ1, claudin3c, claudin7, while downregulating the expression of TNF-α and AMPKγ when compared to fish fed FMG diet. FMcA upregulated the expression of TGFβ1, claudin3c than fed on the FMc. In intestine, the villus height and mucosal thickness of the proximal intestine (PI) and the distal intestine (DI) were decreased and crypt depth of the PI and mid intestine (MI) were increased in SM, FMG and FMc. In addition, fish fed on SM, FMG and FMc presented lower citrate synthase (CS), isocitrate dehydrogenase (ICD), α-ketoglutarate dehydrogenase complex (α-KGDHC) Na+/K+-ATPase activity in DI. FMGA had higher CS, ICD, α-KGDHC, and Na+/K+-ATPase activity in PI and MI than those fed on the FMG. FMcA had higher Na+/K+-ATPase activity in MI. In conclusion, dietary soybean meal destroys the intestine's health, the adverse effects are related to the presence of β-conglycinin and glycinin, especially glycinin. AKG may regulate intestinal energy via tricarboxylic acid cycle, thereby alleviating the damage intestinal morphology caused by the dietary soybean antigen proteins.

Anti-fatigue properties of the ethanol extract of Moringa oleifera leaves in mice.

Moringa oleifera (M. oleifera) leaves are rich in nutrients and bioactive ingredients. This study was aimed at evaluating the anti-fatigue effect of the ethanol extract of M. oleifera leaves (MLEE) on mice and its primary mechanism of action using a weight-loaded forced swimming test. In the present study, MLEE was prepared by ultrasound-assisted extraction, and its anti-fatigue effect and antioxidant capacity were evaluated in mice. Mice were administrated MLEE (320 mg kg-1 body weight) for 15 days. MLEE supplementation significantly increased levels of glucose and non-esterified fatty acids (NEFA), while decreasing levels of lactate and blood urea nitrogen in serum (P < 0.05); the levels of glycogen in the liver and muscle were also increased, as was the activity of glycogen synthase and the level of NEFA in muscle (P < 0.05). According to a Western blot analysis, MLEE increased the expression of AMPKα1, JNK, AKT and STAT3 in the muscle of mice. Our findings indicate that MLEE has an anti-fatigue effect via the AMPK-linked route, which enables it to control energy metabolism and enhance antioxidant enzyme activity. © 2023 Society of Chemical Industry.

A free fatty acid receptor agonist inducing autophagy in HT-29 cells by downregulating the AKT/mTOR signaling pathway.

GW9508, a free fatty acid receptor agonist acts in a G-coupled protein receptor 40 (GPR40)-dependent pathway. Here, we investigated the induction of stress oxidative and autophagy by GW9508 in the human colorectal cancer cell line (HT-29) and the crosstalk between autophagy and apoptotic in HT-29 cells. HT-29 was treated with GW9508 at a concentrations range of 50-500 μM in fibrin gel. Cell viability was investigated using an MTT assay. Induction of autophagy and apoptosis was assessed through Western blotting for associated proteins, acridine orange staining, MDC staining, qRT-PCR, and electron microscopy. Also, we estimated the molecular interactions between GW9805 and some markers through molecular docking. GW9508 inhibited HT-29 cell proliferation, induced apoptosis, and resulted in autophagy. The induced autophagy in cells was confirmed by the observation of autophagosomes, the presence of autophagy markers, including beclin-1, LC3, AMPK, and lack expression of mTOR and AKT. Moreover, GW9508 treatment significantly increased the expression of catalase and superoxide dismutase in cells. Our results indicated that GW9508 could induce autophagy by inhibiting the Akt/mTOR in HT-29. Hence, GW9508 is suggested as a novel anticancer reagent.

Pretreatment of kaempferol-3-O-rutinoside protects H9c2 cells against LPS-induced inflammation through the AMPK/SIRT1 pathway

Kaempferol-3-O-rutinoside (KR) is a flavonoid glycoside derived from traditional Chinese medicine, plants, and tea, and has good myocardial protection. This study focuses on the mechanism of myocardial protection with KR and whether myocardial protection can be achieved by activating the adenosine monophosphate-activated protein kinase–silent mating-type information regulation 2 homolog 1 (sirtuin) (AMPK/SIRT1) signal pathway. Molecular docking technique was used to predict the binding affinity of KR to AMPK. The inflammatory injury model of H9c2 cells was established by lipopolysaccharide (LPS) induction. H9c2 cell proliferation was detected by cell counting kit -8 assay. The apoptosis rate was measured by flow cytometry. Levels of interlukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) were determined by enzyme-linked-immunosorbent serologic assay. AMPK and SIRT1 expression levels were quantified through reverse transcription-polymerase chain reaction and Western blotting assay. Results indicated that KR has a certain binding affinity with AMPK. KR could increase the growth of H9c2 inhibited by LPS, reduce apoptosis rate, and reverse the elevated levels of IL-1β, IL-6, and TNF-α. Furthermore, KR could suppress the expression levels of AMPK and SIRT1, and AMPK-specific inhibitor (Compound C) could significantly counteract the activation of KR, indicating that its anti-inflammatory effect is achieved by regulating the AMPK/SIRT1 signaling pathway.

VCE-005.1, an hypoxia mimetic betulinic acid derivative, induces angiogenesis and shows efficacy in a murine model of traumatic brain injury

One of the main global causes of mortality and morbidity is traumatic brain injury (TBI). Neuroinflammation and brain-blood barrier (BBB) disruption play a pivotal role in the pathogenesis of acute and chronic TBI onset. The activation of the hypoxia pathway is a promising approach for CNS neurodegenerative diseases, including TBI. Herein, we have studied the efficacy of VCE-005.1, a betulinic acid hydroxamate, against acute neuroinflammation in vitro and on a TBI mouse model. The effect of VCE-005.1 on the HIF pathway in endothelial vascular cells was assessed by western blot, gene expression, in vitro angiogenesis, confocal analysis and MTT assays. In vivo angiogenesis was evaluated through a Matrigel plug model and a mouse model of TBI induced by a controlled cortical impact (CCI) was used to assess VCE-005.1 efficacy. VCE-005.1 stabilized HIF-1α through a mechanism that involved AMPK and stimulated the expression of HIF-dependent genes. VCE-005.1 protected vascular endothelial cells under prooxidant and pro-inflammatory conditions by enhancing TJ protein expression and induced angiogenesis both in vitro and in vivo. Furthermore, in CCI model, VCE-005.1 greatly improved locomotor coordination, increased neovascularization and preserved BBB integrity that paralleled with a large reduction of peripheral immune cells infiltration, recovering AMPK expression and reducing apoptosis in neuronal cells. Taken together, our results demonstrate that VCE-005.1 is a multitarget compound that shows anti-inflammatory and neuroprotective effects mainly by preventing BBB disruption and has the potential to be further developed pharmacologically in TBI and maybe other neurological conditions that concur with neuroinflammation and BBB disruption.

Grain-sized moxibustion at Zusanli (ST36) promotes hepatic autophagy in rats with hyperlipidemia by regulating the ULK1 and TFEB expression through the AMPK/mTOR signaling pathway

ObjectiveGrain-sized moxibustion is an effective treatment for hyperlipidemia, but how it regulates dyslipidemia and liver lipid deposits still needs to be fully understood. This study explored the molecular biological mechanism of grain-sized moxibustion to regulate hepatic autophagy in hyperlipidemic rats by affecting ULK1 and TFEB through the AMPK/mTOR signaling pathway. MethodsThirty male Sprague-Dawley (SD) rats were fed a high-fat diet for eight weeks to induce hyperlipidemia. Hyperlipidemic rats were divided into the HFD group, HFD + Statin group, HFD + CC + Moxi group, and grain-sized moxibustion intervention group (HFD + Moxi group). The control (Blank) group consisted of normal rats without any intervention. Grain-sized moxibustion and drug interventions were initiated eight weeks after high-fat diet induction and continued for ten weeks. Serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL), as well as hepatic triglyceride (TG), were measured after treatment. Hepatic steatosis and the expression of LC3I, LC3II, p62, p-AMPK, AMPK, p-mTOR, mTOR, ULK1, p-ULK1, and TFEB in the liver were analyzed. ResultsCompared with the HFD group, grain-sized moxibustion improved hyperlipidemia and hepatocyte steatosis, increased the LC3, p-AMPK, p-ULK1, and nuclear TFEB expression in the liver, but decreased the p62 and p-mTOR expression. ConclusionGrain-sized moxibustion at ST36 acupoints could regulate the blood lipid level of SD rats with hyperlipidemia, increase the expression level of ULK1 and TFEB by activating the AMPK/mTOR signaling pathway in liver tissues, and initiate the transcription of autophagy genes such as LC3.

Protective Effect of Ferulic Acid on Lipopolysaccharide-Induced BV2 Microglia Inflammation via AMPK/mTOR Signaling Pathway

In neurodegenerative diseases, microglial activation and neuroinflammation are essential for the control and progression of neurodegenerative diseases. Mitigating microglium-induced inflammation is one strategy for hindering the progression of neurodegenerative diseases. Ferulic acid (FA) is an effective anti-inflammatory agent, but its potential role and regulation mechanism in neuroinflammatory reactions have not been fully studied. In this study, the neuroinflammation model was established by lipopolysaccharide (LPS), and the inhibitory effect of FA on neuroinflammation of BV2 microglia was studied. The results showed that FA significantly reduced the production and expression of reactive oxygen species (ROS), tumor necrosis factor-α (TNF-α), leukocyte-6 (IL-6) and interleukin-1β (IL-1β). We further studied the mechanism of FA's regulation of LPS-induced BV2 neuroinflammation and found that FA can significantly reduce the expression of mTOR in BV2 microglia induced by LPS, and significantly increase the expression of AMPK, indicating that FA may have an anti-inflammatory effect by activating the AMPK/mTOR signaling pathway to regulate the release of inflammatory mediators (such as NLRP3, caspase-1 p20 and IL-1β). We further added an autophagy inhibitor (3-MA) and an AMPK inhibitor (compound C, CC) for reverse verification. The results showed that FA's inhibitory effects on TNF-α, IL-6 and IL-1β and its regulatory effect on AMPK/mTOR were destroyed by 3-MA and CC, which further indicated that FA's inhibitory effect on neuroinflammation is related to its activation of the AMPK/mTOR autophagy signaling pathway. In a word, our experimental results show that FA can inhibit LPS-induced neuroinflammation of BV2 microglia by activating the AMPK/mTOR signaling pathway, and FA may be a potential drug for treating neuroinflammatory diseases.

Chronic voluntary wheel running exercise ameliorates metabolic dysfunction via PGC-1α expression independently of FNDC5/irisin pathway in high fat diet-induced obese mice

Exercise is an effective intervention to ameliorate metabolic diseases including obesity and insulin resistance, but the mechanisms involved in the metabolic amelioration have not yet been fully elucidated. This study aimed to determine whether AMPK–SIRT1–PGC-1α–FNDC5/Irisin-UCP1 expression is activated and whether metabolic dysfunction is ameliorated by chronic voluntary wheel running (VWR) in high-fat diet (HFD) induced obese mice. C57BL6J mice were randomly assigned into three groups at the age of 7 weeks for 10 weeks: normal chow diet (CON) group, HFD group, and HFD + VWR group. Chronic VWR ameliorates metabolic parameters and leads to increases in the expression of PGC-1α in the gastrocnemius muscle in HFD-induced obese mice. In contrast, the expression of AMPKα, SIRT1, and FNDC5, or circulating irisin levels did not lead to alteration. Improvement of metabolic health was partly mediated via PGC-1α expression by chronic VWR, but not FNDC5/Irisin pathway in HFD-induced obese mice.

Potent Effect of Phlorotannins Derived from Sargassum linifolium as Antioxidant and Antidiabetic in a Streptozotocin-Induced Diabetic Rats Model

Phlorotannins are phenolic compounds existing in large amounts in Phaeophyta, with this amount differing according to the season and collection area. There are many pharmacological properties of phlorotannins, such as being antioxidant, antidiabetic, and anti-cancer. In this work, phlorotannins from the Phaeophyta Sargassum linifolium were extracted, characterized, and identified, for use as an antioxidant and an anti-diabetic in a streptozotocin-induced diabetes rat model. Phlorotanins were characterized using ultraviolet (UV) and Fourier transform infrared (FT-IR) analysis, dimethoxy benzaldehyde assay (DMBA), and Folin–Ciocalteu assays. Groups of rats were tested as follows: normal control (negative control) (G1), normal rats treated with 60 mg/kg body weight of phlorotannins (G2), positive control diabetic rats injected with one dose of streptozotocin (G3), and a diabetic group treated with phlorotannins at 60 mg kg−1 body weight (G4). The biochemical parameters were determined after 4 weeks of treatment. The results demonstrated that the extracted compound was a phlorotannin, which had antioxidant properties. An in vivo study confirmed that the glucose and insulin levels in G4 were relatively similar to those in the normal control G1. The glucosidase, alpha-amylase, glutathione, and catalase levels were 0.11 ± 0.097, 420.5 ± 13, 11.27 ± 3.3, and 8.01 ± 1.31 µmol/min/g in G1, and 0.04 ± 0.016, 184.75 ± 55.24, 12.78 ± 2.1, and 11.28 ± 1.74 µmol/min/g) in G4, respectively. There were no side effects in the kidney function of both G2 and G4, and the levels of cholesterol and triglyceride were also normal. The results demonstrated that phlorotannins have antioxidant properties in vivo and that the diabetic rats had an activated AMPK expression. According to the histological analysis, phlorotannins improved the islet size and reversed necrotic and fibrotic alterations in the pancreas. The results of the present study suggest the use of phlorotannins derived from Sargassum linifolium as an antioxidant and anti-diabetic for an in vivo study. They could be used in developing medicinal preparations for treating diabetes and its related symptoms.

Exercise training impacts skeletal muscle remodelling induced by metabolic syndrome in ZSF1 rats through metabolism regulation

Metabolic syndrome (MetS), characterized by a set of conditions that include obesity, hypertension, and dyslipidemia, is associated with increased cardiovascular risk. Exercise training (EX) has been reported to improve MetS management, although the underlying metabolic adaptations that drive its benefits remain poorly understood. This work aims to characterize the molecular changes induced by EX in skeletal muscle in MetS, focusing on gastrocnemius metabolic remodelling. 1H NMR metabolomics and molecular assays were employed to assess the metabolic profile of skeletal muscle tissue from lean male ZSF1 rats (CTL), obese sedentary male ZSF1 rats (MetS-SED), and obese male ZF1 rats submitted to 4 weeks of treadmill EX (5 days/week, 60 min/day, 15 m/min) (MetS-EX). EX did not counteract the significant increase of body weight and circulating lipid profile, but had an anti-inflammatory effect and improved exercise capacity. The decreased gastrocnemius mass observed in MetS was paralleled with glycogen degradation into small glucose oligosaccharides, with the release of glucose-1-phosphate, and an increase in glucose-6-phosphate and glucose levels. Moreover, sedentary MetS animals' muscle exhibited lower AMPK expression levels and higher amino acids' metabolism such as glutamine and glutamate, compared to lean animals. In contrast, the EX group showed changes suggesting an increase in fatty acid oxidation and oxidative phosphorylation. Additionally, EX mitigated MetS-induced fiber atrophy and fibrosis in the gastrocnemius muscle. EX had a positive effect on gastrocnemius metabolism by enhancing oxidative metabolism and, consequently, reducing susceptibility to fatigue. These findings reinforce the importance of prescribing EX programs to patients with MetS.

AMPK/mTOR/p70S6K axis prevents apoptosis of Porphyromonas gingivalis-infected gingival epithelial cells via BadSer136 phosphorylation.

Epithelial disruption is the initiation of most infectious disease. Regulation of epithelium apoptosis may play a key role in balance the survival competition between resident bacteria and host cells. The role of the mTOR/p70S6K pathway in preventing apoptosis of human gingival epithelial cells (hGECs) infected with Porphyromonas gingivalis (Pg) was investigated in order to further understand the survival strategy of the epithelial cells in during Pg infecting. hGECs was challenged with Pg for 4, 12, and 24h. Additionally, hGECs was pretreated with LY294002 (PI3K signaling inhibitor) or Compound C (AMPK inhibitor) for 12h and exposed them to Pg for 24h. Subsequently, apoptosis was detected using flow cytometry, and expression and activity of Bcl-2, Bad, Bax, PI3K, AKT, AMPK, mTOR, and p70S6K proteins were analyzed using western blotting. Pg-infecting did not increase apoptosis of hGECs; but the expression ratio of Bad to Bcl-2 was increased after infecting. In contrast, BadSer136 phosphorylation was promoted, accompanied by a significant reduction of mTOR/p70S6K and PI3K/AKT signaling, along with the upregulation of AMPKThr172 signaling. Morrover, the PI3K inhibitor LY294002 promoted Pg-mediated reduction of mTOR/p70S6K expression, and the increase of AMPK signaling and BadSer136 phosphorylation rate, eventually decreasing apoptosis. While Compound C inhibited Pg-mediated activation of AMPK and downregulation of mTOR/p70S6K signaling, significantly reduced the BadSer136 phosphorylation rate, thereby increasing apoptosis. Thus, hGECs prevent apoptosis via an inherent cellular-homeostasis, pro-survival mechanism during Pg infection, the AMPK/mTOR/p70S6K pathway helps prevent apoptosis in hGECs infected with Pg by regulating BadSer136 phosphorylation.

WSSV exploits AMPK to activate mTORC2 signaling for proliferation by enhancing aerobic glycolysis

AMPK plays significant roles in the modulation of metabolic reprogramming and viral infection. However, the detailed mechanism by which AMPK affects viral infection is unclear. The present study aims to determine how AMPK influences white spot syndrome virus (WSSV) infection in shrimp (Marsupenaeus japonicus). Here, we find that AMPK expression and phosphorylation are significantly upregulated in WSSV-infected shrimp. WSSV replication decreases remarkably after knockdown of Ampkα and the shrimp survival rate of AMPK-inhibitor injection shrimp increases significantly, suggesting that AMPK is beneficial for WSSV proliferation. Mechanistically, WSSV infection increases intracellular Ca2+ level, and activates CaMKK, which result in AMPK phosphorylation and partial nuclear translocation. AMPK directly activates mTORC2-AKT signaling pathway to phosphorylate key enzymes of glycolysis in the cytosol and promotes expression of Hif1α to mediate transcription of key glycolytic enzyme genes, both of which lead to increased glycolysis to provide energy for WSSV proliferation. Our findings reveal a novel mechanism by which WSSV exploits the host CaMKK-AMPK-mTORC2 pathway for its proliferation, and suggest that AMPK might be a target for WSSV control in shrimp aquaculture.

Targeted Inhibition of Hsp90 in Combination with Metformin Modulates Programmed Cell Death Pathways in A549 Lung Cancer Cells.

The pathophysiology of lung cancer is dependent on the dysregulation in the apoptotic and autophagic pathways. The intricate link between apoptosis and autophagy through shared signaling pathways complicates our understanding of how lung cancer pathophysiology is regulated. As drug resistance is the primary reason behind treatment failure, it is crucial to understand how cancer cells may respond to different therapies and integrate crosstalk between apoptosis and autophagy in response to them, leading to cell death or survival. Thus, in this study, we have tried to evaluate the crosstalk between autophagy and apoptosis in A549 lung cancer cell line that could be modulated by employing a combination therapy of metformin (6mM), an anti-diabetic drug, with gedunin (12µM), an Hsp90 inhibitor, to provide insights into the development of new cancer therapeutics. Our results demonstrated that metformin and gedunin were cytotoxic to A549 lung cancer cells. Combination of metformin and gedunin generated ROS and promoted MMP loss and DNA damage. The combination further increased the expression of AMPKα1 and promoted the nuclear localization of AMPKα1/α2. The expression of Hsp90 was downregulated, further decreasing the expression of its clients, EGFR, PIK3CA, AKT1, and AKT3. Inhibition of the EGFR/PI3K/AKT pathway upregulated TP53 and inhibited autophagy. The combination was promoting nuclear localization of p53; however, some cytoplasmic signals were also detected. Further increase in the expression of caspase 9 and caspase 3 was observed. Thus, we concluded that the combination of metformin and gedunin upregulates apoptosis by inhibiting the EGFR/PI3K/AKT pathway and autophagy in A549 lung cancer cells.

Curcumin (CUMINUP60®) mitigates exercise fatigue through regulating PI3K/Akt/AMPK/mTOR pathway in mice

Curcumin is a chemical constituent extracted from Curcuma longa L. Several clinical and preclinical studies have demonstrated that it can mitigate exercise fatigue, but the exact mechanism is still unknown. Therefore, we applied a mouse model of exercise fatigue to investigate the possible molecular mechanisms of curcumin's anti-fatigue effect. Depending on body mass, Kunming mice were randomly divided into control, caffeine (positive drug), and curcumin groups, and were given 28 days intragastric administration. Both the caffeine group and curcumin group showed significant improvement in exercise fatigue compared to the control group, as evidenced by the increase in time to exhaustion, as well as the higher quadriceps coefficient, muscle glycogen (MG) content, and increase in the expression of Akt, AMPK, PI3K, and mTOR proteins. While the curcumin group also significantly improved the exercise fatigue of the mice, demonstrating a lower AMP/ATP ratio and lactic acid (LA) content, and increased glycogen synthase (GS), and myonectin content compared to the caffeine group. Therefore, in the present study, we found that curcumin can exert a similar anti-fatigue effect to caffeine and may act by regulating energy metabolism through modulating the expression of the proteins in the PI3K/Akt/AMPK/mTOR pathway.

NR3C2 inhibits the proliferation of colorectal cancer via regulating glucose metabolism and phosphorylating AMPK

We aim to investigate the roles and mechanisms of NR3C2 in colorectal cancer (CRC). The expression of NR3C2 in CRC tumours and paired paracancerous tissues of 71 CRC patients and five CRC cell lines was detected by western blotting, immunohistochemistry and real-time reverse-transcription PCR. Moreover, NR3C2 was overexpressed or knocked down in CRC cells by lentiviral vector transfection. The proliferation of cells was measured by MTT, colony formation assay and flow cytometry. Glucose metabolism was assessed by detecting lactate production, glucose consumption and ATP production. Western blotting and real-time reverse-transcription PCR were used to detect the expression of AMPK, LDHA and HK2. The expression of NR3C2 was significantly decreased in CRC tumours compared to paracancerous tissues, which was correlated with distant metastasis, poor prognosis and advanced stages of CRC patients. Overexpressing NR3C2 suppressed the proliferation and promoted the G2/M cell cycle arrest of CRC cells. Furthermore, NR3C2 inhibited glucose metabolism by decreasing the expression of HK2 and LDHA. The phosphorylation of AMPK was also downregulated in CRC cells overexpressing NR3C2. This study demonstrated that NR3C2 inhibited the proliferation of CRC by inhibiting glucose metabolism and phosphorylation of AMPK which may serve as a therapeutic target for CRC.

AMP-activated protein kinase-farnesoid X receptorpathway contributes to oleanolic acid-induced liver injury.

Natural pentacyclic triterpenoid oleanolic acid (OA) is used as an over-the-counter drug for acute and chronic hepatitis. However, clinical use of OA-containing herbal medicines has been reported to cause cholestasis, and the specific mechanism is unknown. The purpose of this study was to explore how OA causes cholestatic liver injury via the AMP-activated protein kinase (AMPK)-farnesoid X receptor (FXR) pathway. In animal experiments, it was found that OA treatment activated AMPK and decreased FXR and bile acid efflux transport proteins expression. When intervened with the specific inhibitor Compound C (CC), it was observed that AMPK activation was inhibited, the reduction of FXR and bile acid efflux transport protein expression was effectively alleviated, serum biochemical indicators were significantly reduced, and liver pathological damage brought about by OA was effectively ameliorated. In addition, OA was found to downregulate the expression of FXR and bile acid efflux transport proteins by activating the ERK1/2-LKB1-AMPK pathway in cellular experiments. The ERK1/2 inhibitor U0126 was used to pretreat primary hepatocytes, and this drastically reduced the phosphorylation levels of LKB1 and AMPK. The inhibition effects of OA on FXR and bile acid efflux transport proteins were also effectively alleviated after pretreatment with CC. In addition, OA-induced downregulation of FXR gene and protein expression levels was significantly prevented after silencing AMPKα1 expression in AML12 cells. Our study demonstrated that OA inhibited FXR and bile acid efflux transporters through the activation of AMPK, thus leading to cholestatic liver injury.

Response to acute hyperglycemia and high fructose in cultured tenocytes.

High monosaccharide levels are intimately associated with diabetes and impact tendon cells through inflammation and impairment in metabolic homeostasis. Experiments were designed to understand the responses elicited by cultured tenocytes under monosaccharide stress induced by hyperglycemia and hyperfructosemia. We simulated hyperglycemia and hyperfructosemia in vitro by treating tenocytes with media containing sublethal concentrations of glucose and fructose, respectively. Exposure of tenocytes to high glucose and high fructose altered the levels of IL-1β, IL-2, IL-6, IL10 and IL-17A. AMPK expression was increased in high-glucose and decreased in high-fructose groups. High fructose increased the level of IRS-1 compared with the control. Increased mitochondrial superoxide levels and compromised mitochondrial membrane integrity were exhibited by both the groups. The findings from the network analysis revealed many altered genes that are related to pathways for enzyme-linked receptor protein signaling, positive regulation of metabolic processes, transmembrane receptor tyrosine kinase pathway, insulin receptor signaling and regulation of cytokine production. Overall, the data suggest that the tenocytes under high monosaccharide levels exhibit survival responses by altering the expression status of cytokines and metabolic mediators that are involved in the underlying pathogenesis of tendinopathy.