AMPK Phosphorylation Research Articles

Melatonin alleviates lead-induced fatty liver in the common carps (Cyprinus carpio) via gut-liver axis

As a widespread aquatic environmental contaminant, Lead (Pb) can provoke hepatic injury in various animals. Melatonin (MT) plays a crucial role in the regulation of inflammatory response. Accumulating evidence elucidates exogenous toxins can elicit hepatic lipid metabolic disorders by influencing the gut microbiome. Nevertheless, the effects of Pb on gut microbiota and hepatic lipid metabolism of the common carps, and whether MT can prevent and cure Pb-induced toxicity via regulating microbiome remains unknown. Here, metagenomic and transcriptomic analysis were subsequently implemented to identify the Pb exposure-triggered prominent alternation of gut-liver signal. In the present study the severe intestinal injury and fatty liver formation caused by Pb in common carp were preliminarily determined. Metagenomic analysis confirmed that the gut microbiome dominant phyla, family and genus of the common carps were Fusobacteria, Fusobacteriaceae and Cetobacterium. Meanwhile, lipopolysaccharide (LPS) biosynthesis pathway was regarded as one of the main responsible for Pb exposure. Subsequently, LPS was demonstrated as the Pb-triggered microbial-derived signal of the common carps by ELISA analysis, and involves in the hepatic metabolic disorders via deteriorating the intestinal barrier. Additionally, it confirmed that hepatocytes ferroptosis associated with Pb-evoked fatty liver of the common carps, and the aggravation of lysosomal dyshomeostasis as well as inhibition of AMPK phosphorylation were referred to lipid metabolic disorders. The results of the present study demonstrated microbial-derived signal induced by aquatic Pb contaminant cause fatty liver formation in the common carps, and the protective effects of MT on Pb toxicity were performed by receding LPS over-synthesis, restraining microbiota-sourced LPS transport, along with attenuation of hepatocytes ferroptosis.

Metformin suppresses SARS-CoV-2 in cell culture

Comorbidities such as diabetes worsen COVID-19 severity and recovery. Metformin, a first-line medication for type 2 diabetes, has antiviral properties and certain studies have also indicated its prognostic potential in COVID-19. Here, we report that metformin significantly inhibits SARS-CoV-2 growth in cell culture models. First, a steady increase in AMPK phosphorylation was detected as infection progressed, suggesting its important role during viral infection. Activation of AMPK in Calu3 and Caco2 cell lines using metformin revealed that metformin suppresses SARS-CoV-2 infectious titers up to 99%, in both naïve as well as infected cells. IC50 values from dose-variation studies in infected cells were found to be 0.4 and 1.43 mM in Calu3 and Caco2 cells, respectively. Role of AMPK in metformin's antiviral suppression was further confirmed using other pharmacological compounds, AICAR and Compound C. Collectively, our study demonstrates that metformin is effective in limiting the replication of SARS-CoV-2 in cell culture and thus possibly could offer double benefits as diabetic COVID-19 patients by lowering both blood glucose levels and viral load.

Quercetin 3-O-glucuronide-rich lotus leaf extract promotes a Brown-fat-phenotype in C3H10T1/2 mesenchymal stem cells

Lotus (Nelumbo nucifera Gaertn.) is an aquatic perennial crop planted worldwide and its leaf (also called “He-Ye”) has therapeutic effects on obesity. However, whether the underlying mechanism leads to increased energy expenditure by activation of brown adipocytes has not been clarified. Here, murine C3H10T1/2 mesenchymal stem cells (MSCs) were employed to investigate the effects of ethanol extracts from lotus leaf (LLE) on brown adipocytes formation and the underlying molecular mechanisms. The results showed LLE was rich in polyphenols (383.7 mg/g) and flavonoids (178.3 mg/g), with quercetin 3-O-glucuronide (Q3G) the most abundant (128.2 μg/mg). In LLE-treated C3H10T1/2 MSCs, the expressions of lipolytic factors (e.g., ATGL, HSL, and ABHD5) and brown regulators (e.g., Sirt1, PGC-1α, Cidea, and UCP1) were significantly upregulated compared to that in the untreated MSCs. Furthermore, LLE promoted mitochondrial biogenesis and fatty acid β-oxidation, as evidenced by increases in the expression of Tfam, Cox7A, CoxIV, Cox2, Pparα, and Adrb3. Likewise, enhanced browning and mitochondrial biogenesis were also observed in Q3G-stimulated cells. Importantly, LLE and Q3G induced phosphorylation of AMPK accompanied by a remarkable increase in the brown fat marker UCP1, while pretreatment with Compound C (an AMPK inhibitor) reversed these changes. Moreover, stimulating LLE or Q3G-treated cells with CL316243 (a beta3-AR agonist) increased p-AMPKα/AMPKα ratio and UCP1 protein expression, indicating β3-AR/AMPK signaling may involve in this process. Collectively, these observations suggested that LLE, especially the component Q3G, stimulates thermogenesis by activating brown adipocytes, which may involve the β3-AR/AMPK signaling pathway.

Interplay between obesity and aging on myocardial geometry and function: Role of leptin-STAT3-stress signaling

BackgroundUncorrected obesity facilitates premature aging and cardiovascular anomalies. This study examined the interaction between obesity and aging on cardiac remodeling and contractile function. Methods: Cardiac echocardiographic geometry, function, morphology, intracellular Ca2+ handling, oxidative stress (DHE fluorescence), STAT3 and stress signaling were evaluated in young (3-mo) and old (12- and 18-mo) lean and leptin deficient ob/ob obese mice. Cardiomyocytes from young and old lean and ob/ob mice were treated with leptin (1 nM) for 4 h in vitro prior to assessment of mechanical and biochemical properties. High fat diet (45% calorie from fat) and the leptin receptor mutant db/db obese mice at young and old age were evaluated for comparison. Results: Our results displayed reduced survival in ob/ob mice. Obesity but less likely older age dampened echocardiographic, geometric, cardiomyocyte function and intracellular Ca2+ properties, elevated O2− and p47phox NADPH oxidase levels with a more pronounced geometric change at older age. Immunoblot analysis revealed elevated p47phox NADPH oxidase and dampened phosphorylation of STAT3, with a more pronounced response in old ob/ob mice, the effects were restored by leptin. Obesity and aging inhibited phosphorylation of Akt, eNOS, AMPK, and p38 while promoting phosphorylation of JNK and IκB. Leptin reconciled cardiomyocyte dysfunction, O2− yield, p47phox upregulation, STAT3 dephosphorylation and stress signaling in ob/ob mice although its action on stress signaling cascades were lost at old age. High fat diet-induced and db/db obesity displayed aging-associated cardiomyocyte anomalies reminiscent of ob/ob model albeit lost leptin response. ConclusionsOur data suggest disparate age-associated obesity response in cardiac remodeling and contractile dysfunction due to phosphorylation of Akt, eNOS and stress signaling-related oxidative stress.

Taxifolin-3-O-glucoside from Osbeckia nepalensis Hook. mediates antihyperglycemic activity in CC1 hepatocytes and in diabetic Wistar rats via regulating AMPK/G6Pase/PEPCK signaling axis

Ethnopharmacological relevanceOsbeckia nepalensis Hook. f. is an ICMR documented plant well known for its antidiabetic uses among the folk people of Northeast Region of India. In-depth study with scientific substantiation of the plant may uphold the therapeutic potential against the treatment of type 2 diabetes mellitus (T2DM). Aim of the studyThe present study evaluates the traditionally claimed prophylactic potential of O. nepalensis and its extracts along with the isolated compound taxifolin-3-O-glucoside (TG) against the downregulation of T2DM related hepatic gluconeogenesis through in vitro, in vivo and in silico conditions as a means of ameliorating hyperglycemia. Materials and methodsAntidiabetic potential of O. nepalensis was carried out in both CC1 hepatocytes (in vitro) and STZ-induced diabetic male Wistar rats (in vivo). Enriched bioactive fraction and bioactive molecules were isolated through bioactivity-guided fractionation, yielding two major molecules, taxifolin-3-O-glucoside and quercitin-3-O-rhamnoside. The bioactivity of taxifolin-3-O-glucoside was validated through immunoblotting techniques aided by in silico molecular docking and simulations. ResultsMethanolic extract of O. nepalensis and taxifolin-3-O-glucoside (TG) isolated thereof enhanced the uptake of glucose in CC1 hepatocytes and downregulates the gluconeogenic enzymes (G6Pase and PEPCK) and its related transcription factors (FOXO1, HNF4α and PGC1α) through the stimulation of AMPK phosphorylation in in vitro condition. Moreover, in in vivo experiments, the in vitro most active fraction BuSFr1 (consisting of the two active major compounds taxifolin-3-O-glucoside and quercitin-3-O-rhamnoside) exhibited a substantial decrease in elevated blood glucose level and increase the glucose tolerance as well as plasma insulin level. In silico molecular docking and simulations for TG with the protein G6Pase inferred the docking sites and stability and showed taxifolin-3-O-glucoside as more potent and non-toxic as compared to quercitin-3-O-rhamnoside. ConclusionThe traditionally claimed antidiabetic effect of O. nepalensis has been proved to be effective in lowering the blood glucose level through in vitro, in vivo and in silico analysis which will pave a way for the development of antidiabetic phytopharmaceutical drugs which can be validated through further clinical studies.

Exercise-induced specialized proresolving mediators stimulate AMPK phosphorylation to promote mitochondrial respiration in macrophages

ObjectivePhysical activity has been shown to reduce the risk of CVD mortality in large-cohort longitudinal studies; however, the mechanisms underpinning the beneficial effects of exercise remain incompletely understood. Emerging data suggest that the risk reducing effect of exercise extends beyond changes in traditional CVD risk factors alone and involves alterations in immunity and reductions in inflammatory mediator production. Our study aimed to determine whether exercise-enhanced production of proresolving lipid mediators contribute to alterations in macrophage intermediary metabolism, which may contribute to the anti-inflammatory effects of exercise. MethodsChanges in lipid mediators and macrophage metabolism were assessed in C57Bl/6 mice following 4 weeks of voluntary exercise training. To investigate whether exercise-stimulated upregulation of specialized proresolving lipid mediators (SPMs) was sufficient to enhance mitochondrial respiration, both macrophages from control mice and human donors were incubated in vitro with SPMs and mitochondrial respiratory parameters were measured using extracellular flux analysis. Compound-C, an ATP-competitive inhibitor of AMPK kinase activity, was used to investigate the role of AMPK activity in SPM-induced mitochondrial metabolism. To assess the in vivo contribution of 5-lipoxygenase in AMPK activation and exercise-induced mitochondrial metabolism in macrophages, Alox5−/− mice were also subjected to exercise training. ResultsFour weeks of exercise training enhanced proresolving lipid mediator production, while also stimulating the catabolism of inflammatory lipid mediators (e.g., leukotrienes and prostaglandins). This shift in lipid mediator balance following exercise was associated with increased macrophage mitochondrial metabolism. We also find that treating human and murine macrophages in vitro with proresolving lipid mediators enhances mitochondrial respiratory parameters. The proresolving lipid mediators RvD1, RvE1, and MaR1, but not RvD2, stimulated mitochondrial respiration through an AMPK-dependent signaling mechanism. Additionally, in a subset of macrophages, exercise-induced mitochondrial activity in vivo was dependent upon 5-lipoxygenase activity. ConclusionCollectively, these results suggest that exercise stimulates proresolving lipid mediator biosynthesis and mitochondrial metabolism in macrophages via AMPK, which might contribute to the anti-inflammatory and CVD risk reducing effect of exercise.

Topiramate Reprofiling for the Attenuation of Cadmium-Induced Testicular Impairment in Rats: Role of NLRP3 Inflammasome and AMPK/mTOR-Linked Autophagy.

Topiramate, a promising drug classically used for the management of neurological disorders including epilepsy and migraine, has demonstrated marked anti-inflammatory and anti-apoptotic actions in murine models of cardiac post-infarction inflammation, wound healing, and gastric/intestinal injury. However, its potential impact on cadmium-induced testicular injury remains to be elucidated. Herein, the present study aimed to explore the effect of topiramate against cadmium-invoked testicular impairment with emphasis on the molecular mechanisms linked to inflammation, apoptosis, and autophagy. Herein, administration of topiramate (50 mg/kg/day, by gavage) continued for 60 days and the testes were examined by histology, immunohistochemistry, and biochemical assays. The present data demonstrated that serum testosterone, sperm count/abnormalities, relative testicular weight, and histopathological aberrations were improved by topiramate administration to cadmium-intoxicated rats. The rescue of testicular dysfunction was driven by multi-pronged mechanisms including suppression of NLRP3/caspase-1/IL-1β cascade, which was evidenced by dampened caspase-1 activity, lowered IL-1β/IL-18 production, and decreased nuclear levels of activated NF-κBp65. Moreover, curbing testicular apoptosis was seen by lowered Bax expression, decreased caspase-3 activity, and upregulation of Bcl-2. In tandem, testicular autophagy was activated as seen by diminished p62 SQSTM1 accumulation alongside Beclin-1 upregulation. Autophagy activation was associated with AMPK/mTOR pathway stimulation demonstrated by decreased mTOR (Ser2448) phosphorylation and increased AMPK (Ser487) phosphorylation. In conclusion, combating inflammation/apoptosis and enhancing autophagic events by topiramate were engaged in ameliorating cadmium-induced testicular impairment.

Peripheral Gonadotropin-Inhibitory Hormone (GnIH) Acting as a Novel Modulator Involved in Hyperphagia-Induced Obesity and Associated Disorders of Metabolism in an In Vivo Female Piglet Model.

Apart from the well-established role of the gonadotropin-inhibitory hormone (GnIH) in the regulation of the reproductive functions, much less is known about the peripheral role of the GnIH and its receptor in the metabolic processes. On account of pig being an excellent model for studies of food intake and obesity in humans, we investigated the peripheral effects of the GnIH on food intake and energy homeostasis and revealed the underlying mechanism(s) in female piglets in vivo. Compared to the vehicle-treated group, intraperitoneally injected GnIH significantly increased the food intake and altered the meal microstructure both in the fasting and ad libitum female piglet. GnIH-triggered hyperphagia induced female piglet obesity and altered islet hormone secretion in the pancreas, accompanied with dyslipidemia and hyperglycemia. Interestingly, GnIH decreased the glucose transport capacity and glycogen synthesis, whereas it increased the gluconeogenesis in the liver, while it also induced an insulin resistance in white adipose tissue (WAT) via inhibiting the activity of AKT-GSK3-β signaling. In terms of the lipid metabolism, GnIH reduced the oxidation of fatty acids, whereas the elevated fat synthesis ability in the liver and WAT was developed though the inhibited AMPK phosphorylation. Our findings demonstrate that peripheral GnIH could trigger hyperphagia-induced obesity and an associated glycolipid metabolism disorder in female piglets, suggesting that GnIH may act as a potential therapeutic agent for metabolic syndrome, obesity and diabetes.

Inhibition of USP1 activates ER stress through Ubi-protein aggregation to induce autophagy and apoptosis in HCC

The deubiquitinating enzyme USP1 (ubiquitin-specific protease 1) plays a role in the progression of various tumors, emerging as a potential therapeutic target. This study aimed to determine the role of USP1 as a therapeutic target in hepatocellular carcinoma (HCC). We detected USP1 expression in the tumor and adjacent tissues of patients with HCC using immunohistochemical staining. We evaluated the effect of the USP1 inhibitor ML-323 on HCC cell proliferation and cell cycle using a CCK-8 cell-counting kit and plate cloning assays, and propidium iodide, respectively. Apoptosis was detected by annexin V-FITC/Propidium Iodide (PI) staining and caspase 3 (casp3) activity. Transmission electron microscopy and LC3B immunofluorescence were used to detect autophagy. Western blotting was used to detect the accumulation of ubiquitinated proteins, the expression of endoplasmic reticulum (ER) stress-related proteins, and the AMPK-ULK1/ATG13 signaling pathway. We demonstrated that ML-323 inhibits the growth of HCC cells and induces G1 phase cell cycle arrest by regulating cyclin expression. ML-323 treatment resulted in the accumulation of ubiquitinated proteins, induced ER stress, and triggered Noxa-dependent apoptosis, which was regulated by the Activating Transcription Factor 4(ATF4). Moreover, active ER stress induces protective autophagy by increasing AMPK phosphorylation; therefore, we inhibited ER stress using 4-Phenylbutyric acid (4-PBA), which resulted in ER stress reduction, apoptosis, and autophagy in ML-323-treated HCC cells. In addition, blocking autophagy using the AMPK inhibitor compound C (CC), chloroquine (CQ), or bafilomycin A1 (BafA1) enhanced the cytotoxic effect of ML-323. Our findings revealed that targeting USP1 may be a potential strategy for the treatment of HCC.

Abstract 11789: Exosomes Released by Human Cardiac Fibroblasts Promote the Maturation of Human Induced-Pluripotent Stem Cells Derived Cardiomyocyte via AMPK Signal Pathway

Background: Human induced-pluripotent stem cell derived cardiomyocytes (hiPS-CMs) is phenotypically less matured than adult cardiomyocytes. The aim of this study was to explore the potential mechanism by which human cardiac fibroblast-derived exosomes (hc-FB-EXOs) enhance the maturation of hiPS-CMs. Method: Exosomes were extracted from human cardiac fibroblasts (hc-FBs) which were cultured with exosome-free FBS medium. The cultured hiPS-CMs were randomly devided into two groups: co-culture with (EXO) and without (NC) hc-FB-EXOs. The morphology of hiPS-CMs was assessed via immunostaining. Mitochondrial functions assay were measured by Seahorse XF. Potential mechanism was assessed via mRNA-Seq, real-time PCR and western blot. Results: Morphologically, the cells treated in EXO group was significantly larger than the in NC group in terms of cell area and sarcomere length (Panel A). In cellular metabolism, the mitochondrial oxygen consumption rate of EXO group was significantly higher than that of NC group, including basal respiration, proton leak, maximal respiration and spare respiratory capacity (Panel B). The expression of cardiac Troponin I (cTnI), Myosin Heavy Chain 7 (MYH7), Cyclin Dependent Kinase Inhibitor 2C (CDKN2C or p18), phospho-WEE1 and CAV3 (caveolin3) were significant higher, while the expression of of Myosin Heavy Chain 6 (MYH6) was significant lower in EXO group than in NC group (Panel C). Furthermore, qPCR showed that TNNI1, TNNT2, TNNI3/TNNI1, total titin, N2B, MYH7, LPL, ND1, N2BA, and MYH6 were significantly upregulated in EXO group compared with the NC group. Furthermore, western blot validation of RNA-Seq data confirmed that significantly upregulated of phospho-AMPK and downregulated of ACC1 and SCD1 were detected in EXO group compared with the NC group (Panel D). Conclusion: hc-FB-EXOs could significantly facilitate maturation of hiPS-CMs via the phosphorylation of AMPK.

Abstract 12910: Exercise-Induced Specialized Proresolving Mediators Stimulate AMPK Phosphorylation to Promote Mitochondrial Respiration in Macrophages

Emerging epidemiological data suggests that exercise imparts its CVD risk-reducing effects in part through improvements in systemic inflammation. The mechanisms underpinning the salutary effects of physical activity, however, remain incompletely understood. We recently reported that exercise enhances the production of specialized proresolving mediators (SPMs), which act as agonist of inflammation resolution through actions on macrophages (Mϕ). In this study, we find that wheel running accelerated the metabolism of proinflammatory lipid mediators LTB 4 and PGD 2 into their respective metabolites 20-OH-LTB 4 (Sed 27.14±5.88 vs Exe 70.40±10.82 pg/mL; P<0.05; n =3-4) and PGJ 2 (Sed 92.63±16.08 vs Exe 227.90±30.23 pg/mL; P<0.05; n =3-4), resulting in an increase in the ratio of proresolving-to-proinflammatory lipid mediator levels (RvE1+RvD1:leukotrienes; Sed 0.19±0.02 vs Exe 0.32±0.90; P<0.05; n =3-4). These findings were associated with enhanced mitochondrial metabolism in Mϕ, as evidenced by increased basal (Sed 0.12±0.06 vs Exe 1.53±0.07; P<0.05; n =3-4), maximum (Sed 9.20±0.30 vs Exe 12.02±0.82; P<0.05; n =3-4), and ATP-linked oxygen consumption rate (Sed 1.25±0.06 vs Exe 1.47±0.09 pmol/min/μg protein; P<0.05; n =3-4). We also find that SPMs RvD1, RvD2, RvE1 or MaR1 stimulate basal (Ctrl 6.11±0.1 vs RvD1 7.76±0.6 vs RvD2 9.48±0.9 vs RvE1 7.20±0.5 vs MaR1 8.73±0.9 pmol/min/μg protein; P<0.05; n =3) and ATP-linked respiration (Ctrl 2.83±0.2 vs RvD1 4.50±0.3 vs RvD2 4.39±0.2 vs RvE1 4.71±0.3 vs MaR1 4.20±0.2 pmol/min/μg protein; P<0.05; n =3). We find that an increase in basal (Ctrl 1.80±0.13 vs RvD1 2.43±0.19 pmol/min/μg protein; P<0.05; n =3-5) and ATP-linked respiration (Ctrl 1.44±0.10 vs RvD1 1.70±0.15 pmol/min/μg protein; P<0.05; n =3-5) with RvD1 treatment occurs concomitantly with AMPK phosphorylation (Ctrl 1.00±0.07 vs RvD1 1.40±0.02 pAMPK/AMPK fold change; P<0.05; n =3) and inhibiting the kinase activity of AMPK with Compound C abrogates SPM-induced mitochondrial respiration. Taken together, these data suggest that exercise-enhanced SPM production stimulates mitochondrial metabolism in Mϕ via AMPK, which may contribute to exercise-induced amelioration of chronic inflammation.

Eugenol improves high-fat diet/streptomycin-induced type 2 diabetes mellitus (T2DM) mice muscle dysfunction by alleviating inflammation and increasing muscle glucose uptake.

Eugenol has been used in dietary interventions for metabolic diseases such as diabetes and obesity. However, the protective effect of eugenol on muscle function in diabetes is unclear. In this study, a high-fat diet (HFD) with a streptozocin (STZ) injection induced type II diabetes mellitus in a mouse model. Oral eugenol lowered blood glucose and insulin resistance of HFD/STZ-treated mice. Eugenol reduced HFD/STZ-induced muscle inflammation and prevented muscle weakness and atrophy. Eugenol administration significantly increased GLUT4 translocation and AMPK phosphorylation in skeletal muscle, thereby enhancing glucose uptake. By silencing the transient receptor potential vanilloid channel 1 (TRPV1) gene in C2C12 myotube cells, eugenol was found to increase intracellular Ca2+ levels through TRPV1, which then activated calmodulin-dependent protein kinase-2 (CaMKK2) and affected AMPK protein phosphorylation. In conclusion, eugenol is a potential nutraceutical for preventing high-glucose-induced muscle impairments, which could be explained by its mediating effects on glucose absorption and inflammatory responses in the muscle.

Anesthetic sevoflurane simultaneously regulates autophagic flux and pyroptotic cell death-associated cellular inflammation in the hypoxic/re-oxygenated cardiomyocytes: Identification of sevoflurane as putative drug for the treatment of myocardial ischemia-reperfusion injury

Preand post-conditioning of sevoflurane attenuate cardiomyocyte death and protects against myocardial ischemia/reperfusion (I/R) injury, and this process is considered to be associated with cell autophagy and pyroptosis, but the detailed molecular mechanisms regarding to this issue have not been fully studied. In this study, we verified that sevoflurane exerted its protective effects in myocardial I/R injury by synergistically regulating the AMPK/ULK1 pathway-mediated autophagy and NLRP3-mediated pyroptotic cell death, and the interplays between cell autophagy and pyroptosis were also preliminarily investigated. Specifically, sevoflurane conditioning suppressed NLRP3 and cleaved caspase-1 expressions to inactivate cell pyroptosis, upregulated LC3B-II/I ratio, facilitated autophagosome formation and accelerated p62 degradation to trigger autophagy, and promoted the expressions of CDK2, CDK6 and Cyclin D1 to recover cell cycle in I/R mouse myocardial tissues in vivo and hypoxic/re-oxygenated (H/R) cardiomyocytes in vitro. Further experiments validated that sevoflurane promoted the phosphorylation of both AMPK (p-AMPK) and ULK1 (p-ULK1) to activate the AMPK/ULK1 pathway, and the promoting effects of sevoflurane on cell autophagy in H/R cardiomyocytes were abrogated by co-treating cells with AMPK inhibitor (compound C) and ULK1 inhibitor (SBI-0206965). Moreover, it was verified that compound C, SBI-0206965 and autophagy blocker chloroquine reversed H/R-induced cell death and pyroptosis in cardiomyocytes. Taken together, we concluded that sevoflurane activated the AMPK/ULK1 pathway to trigger autophagic flux and suppress NLRP3-mediated pyroptotic cell death in I/R or H/R-treated cardiomyocytes, which further ameliorated myocardial I/R injury.

Role of AMPK-SREBP Signaling in Regulating Fatty Acid Binding-4 (FABP4) Expression following Ethanol Metabolism.

Fatty acid binding protein-4 (FABP4) is not normally expressed in the liver but is induced in alcohol-dependent liver disease (ALD)). This study sought to identify mechanisms whereby ethanol (EtOH) metabolism alters triglyceride accumulation and FABP4 production. Human hepatoma cells which were stably transfected to express alcohol dehydrogenase (ADH) or cytochrome P4502E1 (CYP2E1) were exposed to EtOH in the absence/presence of inhibitors of ADH (4-methylpyrazole) or CYP2E1 (chlormethiazole). Cells were analyzed for free fatty acid (FFA) content and FABP4 mRNA, then culture medium assayed for FABP4 levels. Cell lysates were analyzed for AMP-activated protein kinase-α (AMPKα), Acetyl-CoA carboxylase (ACC), sterol regulatory element binding protein-1c (SREBP-1c), and Lipin-1β activity and localization in the absence/presence of EtOH and pharmacological inhibitors. CYP2E1-EtOH metabolism led to increased FABP4 mRNA/protein expression and FFA accumulation. Analysis of signaling pathway activity revealed decreased AMPKα activation and increased nuclear-SREBP-1c localization following CYP2E1-EtOH metabolism. The role of AMPKα-SREBP-1c in regulating CYP2E1-EtOH-dependent FFA accumulation and increased FABP4 was confirmed using pharmacological inhibitors and over-expression of AMPKα. Inhibition of ACC or Lipin-1β failed to prevent FFA accumulation or changes in FABP4 mRNA expression or protein secretion. These data suggest that CYP2E1-EtOH metabolism inhibits AMPKα phosphorylation to stimulate FFA accumulation and FABP4 protein secretion via an SREBP-1c dependent mechanism.

OR2AT4, an Ectopic Olfactory Receptor, Suppresses Oxidative Stress-Induced Senescence in Human Keratinocytes.

Olfactory receptors (ORs) are the largest protein superfamily in mammals. Certain ORs are ectopically expressed in extranasal tissues and regulate cell type-specific signal transduction pathways. OR2AT4 is ectopically expressed in skin cells and promotes wound healing and hair growth. As the capacities of wound healing and hair growth decline with aging, we investigated the role of OR2AT4 in the aging and senescence of human keratinocytes. OR2AT4 was functionally expressed in human keratinocytes (HaCaT) and exhibited co-expression with G-protein-coupled receptor signaling components, Golfα and adenylate cyclase 3. The OR2AT4 ligand sandalore modulates the intracellular calcium, inositol phosphate, and cyclic adenosine monophosphate (cAMP) levels. The increased calcium level induced by sandalore was attenuated in cells with OR2AT4 knockdown. OR2AT4 activation by sandalore inhibited the senescent cell phenotypes and restored cell proliferation and Ki-67 expression. Sandalore also inhibited the expression of senescence-associated β-galactosidase and increased p21 expression in senescent HaCaT cells in response to hydrogen peroxide. Additionally, sandalore activated the CaMKKβ/AMPK/mTORC1/autophagy signaling axis and promoted autophagy. OR2AT4 knockdown attenuated the increased in the intracellular calcium level, cell proliferation, and AMPK phosphorylation induced by sandalore. These findings demonstrate that the effects of sandalore are mediated by OR2AT4 activation. Our findings suggest that OR2AT4 may be a novel therapeutic target for anti-aging and anti-senescence in human keratinocytes.

Plasma exosomal miR-122 regulates the efficacy of metformin via AMPK in type 2 diabetes and hepatocellular carcinoma

Metformin is a drug that has been applied in clinical use for many years for the treatment of type 2 diabetes mellitus (T2DM). It achieves its function through multiple targets and modulation of multiple signaling pathways. To date, the mechanism of the action of metformin is still not fully understood. Along with glycemic control, metformin has shown good inhibitory effects on the development of many tumors. Here, we elucidated that plasma exosomal microRNA-122-5p (miR-122) is closely related to the mechanism of metformin. MiR-122 regulates glycogen-glucose metabolism in hepatocytes or hepatocellular carcinoma cells (HCC) by inhibiting the phosphorylation of AMPK. Since miR-122 and metformin regulate glucose metabolism homeostasis through similar mechanisms, miR-122 can antagonize the effects of metformin. MiR-122 expression increases the sensitivity of hepatocytes or HCC to metformin. Conversely, decreased expression of miR-122 results in hepatocyte insensitivity to metformin. Therefore, significantly elevated levels of miR-122 in plasma exosomes of hepatocellular carcinoma patients could enhance their sensitivity to metformin. The results of the present study revealed a key regulatory role of plasma exosomal miR-122 on the molecular mechanism of metformin. The regulation of key molecules of related signaling pathways by miR-122 may lead to similar glycemic lowering and tumor suppression therapeutic effects as metformin. This provides new ideas for the development of new therapeutic strategies for hepatocellular carcinoma based on the mechanism of miR-122 and metformin.

New mechanism of metformin action mediated by lysosomal presenilin enhancer 2.

Formation of the PEN2-ATP6AP1 complex induced by the binding of metformin to PEN2 results in the inhibition of v-ATPase activity and in the recruitment of AXIN/LKB1 to lysosomes, which in turn results in the phosphorylation and activation of AMPK.

Metformin-induced AMPK activation promotes cisplatin resistance through PINK1/Parkin dependent mitophagy in gastric cancer.

Gastric cancer (GC) is one of the most common tumors worldwide, and cisplatin is a standard chemotherapeutic reagent for GC treatment. However, chemoresistance is an inherent challenge which limits its application and effectiveness in clinic. This study aims to investigate the mechanism of metformin-induced cisplatin resistance in GC. Intriguingly, the upregulation of mitophagy markers, mitochondrial fission, autophagy and mitophagosome were observed in SGC-7901/DDP cells compared to those in the SGC-7901 cells. Treatment with metformin significantly increased mitochondrial fission and mitophagy in both AGS and SGC-7901 cells, resulting in decreased ATP production, which unexpectedly protected GC cells against the cytotoxicity of cisplatin. In contrast, application of Chloroquine and 3-methyladenine, two inhibitors of autophagy, significantly alleviated the protective effect of metformin on SGC-7901 and AGS cells against cytotoxicity of cisplatin. Moreover, metformin also stimulated the phosphorylation of AMPK (Thr172) and increased the expression of mitophagy markers including Parkin and PINK1 in the AMPK signaling-dependent manner. Consistently, the cell viability and cell apoptosis assay showed that metformin-induced cisplatin resistance was prevented by knockdown of AMPKα1. Taken together, all data in this study indicate that metformin induced AMPK activation and PINK1/Parkin dependent mitophagy, which may contribute to the progression of cisplatin resistance in GC.

Modulation of hypothalamic AMPK phosphorylation by olanzapine controls energy balance and body weight

BackgroundSecond-generation antipsychotics (SGAs) are a mainstay therapy for schizophrenia. SGA-treated patients present higher risk for weight gain, dyslipidemia and hyperglycemia. Herein, we evaluated the effects of olanzapine (OLA), widely prescribed SGA, in mice focusing on changes in body weight and energy balance. We further explored OLA effects in protein tyrosine phosphatase-1B deficient (PTP1B-KO) mice, a preclinical model of leptin hypersensitivity protected against obesity. MethodsWild-type (WT) and PTP1B-KO mice were fed an OLA-supplemented diet (5 mg/kg/day, 7 months) or treated with OLA via intraperitoneal (i.p.) injection or by oral gavage (10 mg/kg/day, 8 weeks). Readouts of the crosstalk between hypothalamus and brown or subcutaneous white adipose tissue (BAT and iWAT, respectively) were assessed. The effects of intrahypothalamic administration of OLA with adenoviruses expressing constitutive active AMPKα1 in mice were also analyzed. ResultsBoth WT and PTP1B-KO mice receiving OLA-supplemented diet presented hyperphagia, but weight gain was enhanced only in WT mice. Unexpectedly, all mice receiving OLA via i.p. lost weight without changes in food intake, but with increased energy expenditure (EE). In these mice, reduced hypothalamic AMPK phosphorylation concurred with elevations in UCP-1 and temperature in BAT. These effects were also found by intrahypothalamic OLA injection and were abolished by constitutive activation of AMPK in the hypothalamus. Additionally, OLA i.p. treatment was associated with enhanced Tyrosine Hydroxylase (TH)-positive innervation and less sympathetic neuron-associated macrophages in iWAT. Both central and i.p. OLA injections increased UCP-1 and TH in iWAT, an effect also prevented by hypothalamic AMPK activation. By contrast, in mice fed an OLA-supplemented diet, BAT thermogenesis was only enhanced in those lacking PTP1B. Our results shed light for the first time that a threshold of OLA levels reaching the hypothalamus is required to activate the hypothalamus BAT/iWAT axis and, therefore, avoid weight gain. ConclusionOur results have unraveled an unexpected metabolic rewiring controlled by hypothalamic AMPK that avoids weight gain in male mice treated i.p. with OLA by activating BAT thermogenesis and iWAT browning and a potential benefit of PTP1B inhibition against OLA-induced weight gain upon oral treatment.

Larotrectinib induces autophagic cell death through AMPK/mTOR signalling in colon cancer.

Larotrectinib (Lar) is a highly selective and potent small-molecule inhibitor used in patients with tropomyosin receptor kinase (TRK) fusion-positive cancers, including colon cancer. However, the underlying molecular mechanisms specifically in patients with colon cancer have not yet been explored. Our data showed that Lar significantly suppressed proliferation and migration of colon cancer cells. In addition, Lar suppressed the epithelial-mesenchymal transition (EMT) process, as evidenced by elevation in E-cadherin (E-cad), and downregulation of vimentin and matrix metalloproteinase (MMP) 2/9 expression. Furthermore, Lar was found to activate autophagic flux, in which Lar increased the ratio between LC3II/LC3I and decreased the expression of p62 in colon cancer cells. More importantly, Lar also increased AMPK phosphorylation and suppressed mTOR phosphorylation in colon cancer cells. However, when we silenced AMPK in colon cancer cells, Lar-induced accumulation of autolysomes as well as Lar-induced suppression of the EMT process were significantly diminished. An in vivo assay also confirmed that tumour volume and weight decreased in Lar-treated mice than in control mice. Taken together, this study suggests that Lar significantly suppresses colon cancer proliferation and migration by activating AMPK/mTOR-mediated autophagic cell death.