bims-amsmem 2022-04-24 papers

bims-amsmem

Biomed News

on AMPK signaling mechanism in energy metabolism

Issue of 2022‒04‒24
twenty-two papers selected by
Dipsikha Biswas, Københavns Universitet

Rutaecarpine Promotes Adipose Thermogenesis and Protects against HFD-Induced Obesity via AMPK/PGC-1α Pathway.
Canagliflozin Inhibits Glioblastoma Growth and Proliferation by Activating AMPK.
Too hard to die: Exercise training mediates specific and immediate SARS-CoV-2 protection.
Mechanism of Action of Cyanidin 3-O-Glucoside in Gluconeogenesis and Oxidative Stress-Induced Cancer Cell Senescence.
Metformin Inhibits Na+/H+ Exchanger NHE3 Resulting in Intestinal Water Loss.
Calorie Restriction-Regulated Molecular Pathways and Its Impact on Various Age Groups: An Overview.
Atrial AMP-activated protein kinase is critical for prevention of dysregulation of electrical excitability and atrial fibrillation.
Fucoxanthin Attenuates Free Fatty Acid-Induced Nonalcoholic Fatty Liver Disease by Regulating Lipid Metabolism/Oxidative Stress/Inflammation via the AMPK/Nrf2/TLR4 Signaling Pathway.
Metformin-induced AMPK activation suppresses larval growth and molting probably by disrupting 20E synthesis and glycometabolism in fall webworm, Hyphantria cunea Drury.
FKBP5/FKBP51 on weight watch: central FKBP5 links regulatory WIPI protein networks to autophagy and metabolic control.
Pharmacological Effects of Panduratin A on Renal Cyst Development in In Vitro and In Vivo Models of Polycystic Kidney Disease.
High and stable ATP levels prevent aberrant intracellular protein aggregation in yeast.
Acute Administration of Metformin Protects Against Neuronal Apoptosis Induced by Cerebral Ischemia-Reperfusion Injury via Regulation of the AMPK/CREB/BDNF Pathway.
At the crossroads of fertility and metabolism: the importance of AMPK-dependent signaling in female infertility associated with hyperandrogenism.
Docosahexaenoic Acid Suppresses Oxidative Stress-Induced Autophagy and Cell Death via the AMPK-Dependent Signaling Pathway in Immortalized Fischer Rat Schwann Cells 1.
Atractylenolide III Improves Mitochondrial Function and Protects Against Ulcerative Colitis by Activating AMPK/SIRT1/PGC-1α.
Hawk tea prevents high-fat diet-induced obesity in mice by activating the AMPK/ACC/SREBP1c signaling pathways and regulating the gut microbiota.
Luteolin Synergistically Enhances Antitumor Activity of Oxaliplatin in Colorectal Carcinoma via AMPK Inhibition.
Spinacetin alleviates doxorubicin-induced cardiotoxicity by initiating protective autophagy through SIRT3/AMPK/mTOR pathways.
L-Methionine supplementation attenuates high-fat fructose diet-induced non-alcoholic steatohepatitis by modulating lipid metabolism, fibrosis, and inflammation in rats.
Traumatic-noise-induced hair cell death and hearing loss is mediated by activation of CaMKKβ.
Metformin reverses tamoxifen resistance through the lncRNA GAS5-medicated mTOR pathway in breast cancer.

Pharmaceuticals (Basel). 2022 Apr 13. pii: 469. [Epub ahead of print]15(4):

Rutaecarpine Promotes Adipose Thermogenesis and Protects against HFD-Induced Obesity via AMPK/PGC-1α Pathway.

Dandan Chen, Yanan Duan, Shuxiang Yu, Xinwen Zhang, Ni Li, Jingya Li.

  Pharmacological activation of adaptive thermogenesis to increase energy expenditure is considered to be a novel strategy for obesity. Peroxisome-proliferator-activated receptor γ co-activator-1α (PGC-1α), which serves as an inducible co-activator in energy expenditure, is highly expressed in brown adipose tissues (BAT). In this study, we found a PGC-1α transcriptional activator, natural compound rutaecarpine (Rut), which promoted brown adipocytes mitochondrial biogenesis and thermogenesis in vitro. Chronic Rut treatment reduced the body weight gain and mitigated insulin sensitivity through brown and beige adipocyte thermogenesis. Mechanistic study showed that Rut activated the energy metabolic pathway AMP-activated protein kinase (AMPK)/PGC-1α axis, and deficiency of AMPK abolished the beneficial metabolic phenotype of the Rut treatment in vitro and in vivo. In summary, a PGC-1α transcriptional activator Rut was found to activate brown and beige adipose thermogenesis to resist diet-induced obesity through AMPK pathway. Our findings serve as a further understanding of the natural compound in adipose tissue and provides a possible strategy to combat obesity and related metabolic disorders.

Keywords:  AMP-activated protein kinase; adipocytes; browning; obesity; peroxisome-proliferator-activated receptor γ co-activator-1α; rutaecarpine; thermogenesis

DOI:  https://doi.org/10.3390/ph15040469
Cell Mol Neurobiol. 2022 Apr 18.

Canagliflozin Inhibits Glioblastoma Growth and Proliferation by Activating AMPK.

Kenji Shoda, Shohei Tsuji, Shinsuke Nakamura, Yusuke Egashira, Yukiko Enomoto, Noriyuki Nakayama, Masamitsu Shimazawa, Toru Iwama, Hideaki Hara.

  Sodium-glucose transporter 2 (SGLT2) inhibitors are antidiabetic drugs affecting SGLT2. Recent studies have shown various cancers expressing SGLT2, and SGLT2 inhibitors attenuating tumor proliferation. We evaluated the antitumor activities of canagliflozin, a SGLT2 inhibitor, on glioblastoma (GBM). Three GBM cell lines, U251MG (human), U87MG (human), and GL261 (murine), were used. We assessed the expression of SGLT2 of GBM through immunoblotting, specimen-use, cell viability assays, and glucose uptake assay with canagliflozin. Then, we assessed phosphorylation of AMP-activated protein kinase (AMPK), p70 S6 kinase, and S6 ribosomal protein by immunoblotting. Concentrations of 5, 10, 20, and 40 μM canagliflozin were used in these tests. We also evaluated cell viability and immunoblotting using U251MG with siRNA knockdown of SGLT2. Furthermore, we divided the mice into vehicle group and canagliflozin group. The canagliflozin group was administrated with 100 mg/kg of canagliflozin orally for 10 days starting from the third days post-GBM transplant. The brains were removed and the tumor volume was evaluated using sections. SGLT2 was expressed in GBM cell and GBM allograft mouse. Canagliflozin administration at 40 μM significantly inhibited cell proliferation and glucose uptake into the cell. Additionally, canagliflozin at 40 μM significantly increased the phosphorylation of AMPK and suppressed that of p70 S6 kinase and S6 ribosomal protein. Similar results of cell viability assays and immunoblotting were obtained using siRNA SGLT2. Furthermore, although less effective than in vitro, the canagliflozin group significantly suppressed tumor growth in GBM-transplanted mice. This suggests that canagliflozin can be used as a potential treatment for GBM.

Keywords:  Glioma; Glycolysis; Inhibition of SGLT2; Warburg effect

DOI:  https://doi.org/10.1007/s10571-022-01221-8
World J Virol. 2022 Mar 25. 11(2): 98-103

Too hard to die: Exercise training mediates specific and immediate SARS-CoV-2 protection.

Konstantinos I Papadopoulos, Warachaya Sutheesophon, Tar-Choon Aw.

  Several mechanisms may explain how exercise training mechanistically confers protection against coronavirus disease 2019 (COVID-19). Here we propose two new perspectives through which cardiorespiratory fitness may protect against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Physical exercise-activated adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling induces endothelial nitric oxide (NO) synthase (eNOS), increases NO bio-availability, and inhibits palmitoylation, leading to specific and immediate SARS-CoV-2 protection. AMPK signaling also induces angiotensin 1-7 release and enhances eNOS activation thus further mediating cardio- and reno-protection. Irisin, a myokine released from skeletal muscles during aerobic exercise, also participates in the AMPK/Akt-eNOS/NO pathway, protects mitochondrial functions in endothelial cells, and antagonizes renin angiotensin system proinflammatory action leading to reductions in genes associated with severe COVID-19 outcomes. Collectively, all the above findings point to the fact that increased AMPK and irisin activity through exercise training greatly benefits molecular processes that mediate specific, immediate, and delayed SARS-CoV-2 protection. Maintaining regular physical activity levels is a safe and affordable lifestyle strategy against the current and future pandemics and may also mitigate against obesity and cardiometabolic disease syndemics. Move more because a moving target is harder to kill.

Keywords:  Adenosine monophosphate-activated protein kinase; Endothelial nitric oxide synthase; Irisin; Nitric oxide; Physical exercise; Severe acute respiratory syndrome coronavirus-2

DOI:  https://doi.org/10.5501/wjv.v11.i2.98
Antioxidants (Basel). 2022 Apr 09. pii: 749. [Epub ahead of print]11(4):

Mechanism of Action of Cyanidin 3-O-Glucoside in Gluconeogenesis and Oxidative Stress-Induced Cancer Cell Senescence.

Yaoyao Jia, Chunyan Wu, Adriana Rivera-Piza, Yeon-Ji Kim, Ji Hae Lee, Sung-Joon Lee.

  Cyanidin-3-O-glucoside (C3G) is a natural anthocyanin abundant in fruits and vegetables that interacts and possibly modulates energy metabolism and oxidative stress. This study investigated the effect of C3G on gluconeogenesis and cancer cell senescence. C3G activates adenosine monophosphate-activated protein kinase (AMPK), a cellular energy sensor involved in metabolism and the aging process. C3G suppressed hepatic gluconeogenesis by reducing the expression of gluconeogenic genes through the phosphorylation inactivation of CRTC2 and HDAC5 coactivators via AMPK. C3G did not directly interact with AMPK but, instead, activated AMPK through the adiponectin receptor signaling pathway, as demonstrated through adiponectin receptor gene knockdown experiments. In addition, C3G increased cellular AMP levels in cultured hepatocytes, and the oral administration of C3G in mice elevated their plasma adiponectin concentrations. These effects collectively contribute to the activation of AMPK. In addition, C3G showed potent antioxidant activity and induced cellular senescence, and apoptosis in oxidative-stress induced senescence in hepatocarcinoma cells. C3G increased senescence-associated β-galactosidase expression, while increasing the expression levels of P16, P21 and P53, key markers of cellular senescence. These findings demonstrate that anthocyanin C3G achieves hypoglycemic effects via AMPK activation and the subsequent suppression of gluconeogenesis and exhibits anti-cancer activity through the induction of apoptosis and cellular senescence.

Keywords:  Cyanidin 3-O-glucoside; HepG2 cells; adiponectin signaling; anti-carcinogenic activity; antioxidants; hepatic autophagy; natural products; oxidative stress; phytochemicals

DOI:  https://doi.org/10.3390/antiox11040749
Front Physiol. 2022 ;13 867244

Metformin Inhibits Na+/H+ Exchanger NHE3 Resulting in Intestinal Water Loss.

Yiran Han, C Chris Yun.

  Glycemic control is the key to the management of type 2 diabetes. Metformin is an effective, widely used drug for controlling plasma glucose levels in diabetes, but it is often the culprit of gastrointestinal adverse effects such as abdominal pain, nausea, indigestion, vomiting, and diarrhea. Diarrhea is a complex disease and altered intestinal transport of electrolytes and fluid is a common cause of diarrhea. Na+/H+ exchanger 3 (NHE3, SLC9A3) is the major Na+ absorptive mechanism in the intestine and our previous study has demonstrated that decreased NHE3 contributes to diarrhea associated with type 1 diabetes. The goal of this study is to investigate whether metformin regulates NHE3 and inhibition of NHE3 contributes to metformin-induced diarrhea. We first determined whether metformin alters intestinal water loss, the hallmark of diarrhea, in type 2 diabetic db/db mice. We found that metformin decreased intestinal water absorption mediated by NHE3. Metformin increased fecal water content although mice did not develop watery diarrhea. To determine the mechanism of metformin-mediated regulation of NHE3, we used intestinal epithelial cells. Metformin inhibited NHE3 activity and the effect of metformin on NHE3 was mimicked by a 5'-AMP-activated protein kinase (AMPK) activator and blocked by pharmacological inhibition of AMPK. Metformin increased phosphorylation and ubiquitination of NHE3, resulting in retrieval of NHE3 from the plasma membrane. Previous studies have demonstrated the role of neural precursor cell expressed, developmentally down-regulated 4-2 (Nedd4-2) in regulation of human NHE3. Silencing of Nedd4-2 mitigated NHE3 inhibition and ubiquitination by metformin. Our findings suggest that metformin-induced diarrhea in type 2 diabetes is in part caused by reduced Na+ and water absorption that is associated with NHE3 inhibition, probably by AMPK.

Keywords:  AMPK; NHE3; Nedd4-2; intestinal epithelial cell; type 2 diabetes; ubiquitin

DOI:  https://doi.org/10.3389/fphys.2022.867244
DNA Cell Biol. 2022 Apr 22.

Calorie Restriction-Regulated Molecular Pathways and Its Impact on Various Age Groups: An Overview.

Anuradha Rachakatla, Rajender Rao Kalashikam.

  Calorie restriction (CR) if planned properly with regular exercise at different ages can result in healthy weight loss. CR can also have different beneficial effects on improving lifespan and decreasing the age-associated diseases by regulating physiological, biochemical, and molecular markers. The different pathways regulated by CR include:(1) AMP-activated protein kinase (AMPK), which involves PGC-1α, SIRT1, and SIRT3. AMPK also effects myocyte enhancer factor 2 (MEF2), peroxisome proliferator-activated receptor delta, and peroxisome proliferator-activated receptor alpha, which are involved in mitochondrial biogenesis and lipid oxidation; (2) Forkhead box transcription factor's signaling is related to the DNA repair, lipid metabolism, protection of protein structure, autophagy, and resistance to oxidative stress; (3) Mammalian target of rapamycin (mTOR) signaling, which involves key factors, such as S6 protein kinase-1 (S6K1), mTOR complex-1 (mTORC1), and 4E-binding protein (4E-BP). Under CR conditions, AMPK activation and mTOR inhibition helps in the activation of Ulk1 complex along with the acetyltransferase Mec-17, which is necessary for autophagy; (4) Insulin-like growth factor-1 (IGF-1) pathway downregulation protects against cancer and slows the aging process; (5) Nuclear factor kappa B pathway downregulation decreases the inflammation; and (6) c-Jun N-terminal kinase and p38 kinase regulation as a response to the stress. The acute and chronic CR both shows antidepression and anxiolytic action by effecting ghrelin/GHS-R1a signaling. CR also regulates GSK3β kinase and protects against age-related brain atrophy. CR at young age may show many deleterious effects by effecting different mechanisms. Parental CR before or during conception will also affect the health and development of the offspring by causing many epigenetic modifications that show transgenerational transmission. Maternal CR is associated with intrauterine growth retardation effecting the offspring in their adulthood by developing different metabolic syndromes. The epigenetic changes with response to paternal food supply also linked to offspring health. CR at middle and old age provides a significant preventive impact against the development of age-associated diseases.

Keywords:  AMPK; ROS; calorie restriction; mTOR

DOI:  https://doi.org/10.1089/dna.2021.0922
JCI Insight. 2022 Apr 22. pii: e141213. [Epub ahead of print]7(8):

Atrial AMP-activated protein kinase is critical for prevention of dysregulation of electrical excitability and atrial fibrillation.

Kevin N Su, Yina Ma, Marine Cacheux, Zeki Ilkan, Nour Raad, Grace K Muller, Xiaohong Wu, Nicole Guerrera, Stephanie L Thorn, Albert J Sinusas, Marc Foretz, Benoit Viollet, Joseph G Akar, Fadi G Akar, Lawrence H Young.

  Metabolic stress is an important cause of pathological atrial remodeling and atrial fibrillation. AMPK is a ubiquitous master metabolic regulator, yet its biological function in the atria is poorly understood in both health and disease. We investigated the impact of atrium-selective cardiac AMPK deletion on electrophysiological and structural remodeling in mice. Loss of atrial AMPK expression caused atrial changes in electrophysiological properties and atrial ectopic activity prior to the onset of spontaneous atrial fibrillation. Concomitant transcriptional downregulation of connexins and atrial ion channel subunits manifested with delayed left atrial activation and repolarization. The early molecular and electrophysiological abnormalities preceded left atrial structural remodeling and interstitial fibrosis. AMPK inactivation induced downregulation of transcription factors (Mef2c and Pitx2c) linked to connexin and ion channel transcriptional reprogramming. Thus, AMPK plays an essential homeostatic role in atria, protecting against adverse remodeling potentially by regulating key transcription factors that control the expression of atrial ion channels and gap junction proteins.

Keywords:  Arrhythmias; Cardiology; Protein kinases

DOI:  https://doi.org/10.1172/jci.insight.141213
Mar Drugs. 2022 Mar 25. pii: 225. [Epub ahead of print]20(4):

Fucoxanthin Attenuates Free Fatty Acid-Induced Nonalcoholic Fatty Liver Disease by Regulating Lipid Metabolism/Oxidative Stress/Inflammation via the AMPK/Nrf2/TLR4 Signaling Pathway.

Jiena Ye, Jiawen Zheng, Xiaoxiao Tian, Baogui Xu, Falei Yuan, Bin Wang, Zuisu Yang, Fangfang Huang.

  Fucoxanthin, a xanthophyll carotenoid abundant in brown algae, is reported to have several biological functions, such as antioxidant, anti-inflammatory, and anti-tumor activities, in mice. We investigated the effects and mechanisms of fucoxanthin in the mixture oleate/palmitate = 2/1(FFA)-induced nonalcoholic fatty liver disease (NAFLD) cell model in this study. The results showed that the content of superoxide dismutase in the FFA group was 9.8 ± 1.0 U/mgprot, while that in the fucoxanthin high-dose (H-Fx) group (2 μg/mL) increased to 22.9 ± 0.6 U/mgprot. The content of interleukin-1β in the FFA group was 89.3 ± 3.6 ng/mL, while that in the H-Fx group was reduced to 53.8 ± 2.8 ng/mL. The above results indicate that fucoxanthin could alleviate the FFA-induced oxidative stress and inflammatory levels in the liver cells. Oil red-O staining revealed visible protrusions and a significant decrease in the number of lipid droplets in the cytoplasm of cells in the fucoxanthin group. These findings on the mechanisms of action suggest that fucoxanthin can repair FFA-induced NAFLD via the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway and nuclear factor erythroid-2-related factor 2-mediated (Nrf2) signaling pathway, as well as by downregulating the expression of the Toll-like receptor 4-mediated (TLR4) signaling pathway. Fucoxanthin exhibited alleviating effects in the FFA-induced NAFLD model and could be explored as a potential anti-NAFLD substance.

Keywords:  NAFLD; Toll-like receptor 4-mediated (TLR4); adenosine monophosphate-activated protein kinase (AMPK); fucoxanthin; nuclear factor erythroid-2-related factor 2-mediated (Nrf2)

DOI:  https://doi.org/10.3390/md20040225
Pestic Biochem Physiol. 2022 May;pii: S0048-3575(22)00050-5. [Epub ahead of print]183 105083

Metformin-induced AMPK activation suppresses larval growth and molting probably by disrupting 20E synthesis and glycometabolism in fall webworm, Hyphantria cunea Drury.

Haifeng Zou, Hang Zou, Xingpeng Li, Qian Qiu, Nannan Geng, Bihan Zhang, Gaige Yan, Zhidong Zhang, Shengyu Zhang, Bin Yao, Guocai Zhang, Chuanshan Zou.

  Metformin, considered to be a potent AMPK activator, is widely used for clinical therapy of cancer and diabetes due to its distinct function in regulating cell energy balance and body metabolism. However, the effect of metformin-induced AMPK activation on the growth and development of insects remains largely unknown. In the present study, we focused on the role of metformin in regulating the growth and development of Hyphantria cunea, a notorious defoliator in the forestry. Firstly, we obtained the complete coding sequences of HcAMPKα2, HcAMPKβ1, HcAMPKγ2 from H. cunea, which encoded a protein of 512, 281, and 680 amino acids respectively. Furthermore, the phylogenetic analysis revealed that these three subunits were highly homologous with the AMPK subunits from other lepidopteran species. According to the bioassay, we found metformin remarkably restrained the growth and development of H. cunea larvae, and caused molting delayed and body weight reduced. In addition, expressions of HcAMPKα2, HcAMPKβ1, and HcAMPKγ2 were upregulated 3.30-, 5.93- and 5.92-folds at 24 h after treatment, confirming that metformin activated AMPK signaling at the transcriptional level in H. cunea larvae. Conversely, the expressions of two vital Halloween genes (HcCYP306A1 and HcCYP314A1) in the 20E synthesis pathway were remarkably suppressed by metformin. Thus, we presumed that metformin delayed larval molting probably by impeding 20E synthesis in the H. cunea larvae. Finally, we found that metformin accelerated glycogen breakdown, elevated in vivo trehalose level, promoted chitin synthesis, and upregulated transcriptions of the genes in chitin synthesis pathway. Taken together, the findings provide a new insight into the molecular mechanisms by which AMPK regulates carbohydrate metabolism and chitin synthesis in insects.

Keywords:  20E signal; AMP-activated protein kinase (AMPK); Chitin synthesis; Glycometabolism; Growth and molting; Metformin

DOI:  https://doi.org/10.1016/j.pestbp.2022.105083
Autophagy. 2022 Apr 19. 1-3

FKBP5/FKBP51 on weight watch: central FKBP5 links regulatory WIPI protein networks to autophagy and metabolic control.

Thomas Bajaj, Alexander S Häusl, Mathias V Schmidt, Nils C Gassen.

  Stress and changes in energy stores are perceived by hormone- and nutrient-sensing nuclei of the hypothalamus, which orchestrate an adaptive physiological body response to maintain homeostasis. Macroautophagy/autophagy is a fundamental lysosomal degradation system contributing to preservation of proteome balance and metabolic homeostasis. Its dysregulation is linked to diverse human pathologies, including neuropsychiatric and metabolic disorders. Autophagy is coordinated by cellular nutrient sensors, including AMPK and MTORC1 that interact with WIPI proteins. Studies suggest that WDR45/WIPI4 interacts with the stress-sensitive co-chaperone FKBP5/FKBP51, which has emerged as a key autophagy scaffold. However, the impact of FKBP5 on autophagy signaling in response to metabolic challenges, such as a high-fat diet, is elusive. Therefore, we manipulated FKBP5 in the mediobasal hypothalamus (MBH) and studied autophagy signaling and protein interactions in their physiological context. We identified FKBP5 as a scaffold of the STK11/LKB1-AMPK complex with WDR45/WIPI4 and TSC2 with WDR45B/WIPI3 in response to metabolic challenges, positioning FKBP5 in major nutrient-sensing and autophagy-regulating networks. Intriguingly, we could demonstrate that FKBP5 deletion in the MBH strongly induces obesity, whereas its overexpression protects against high-fat diet-induced obesity. Our findings suggest a crucial regulatory and adaptive function of FKBP5-regulated autophagy within the MBH in response to metabolic challenges.Abbreviations: AKT: thymoma viral proto-oncogene; AMPK: AMP-activated protein kinase; BECN1: beclin 1, autophagy related; eWAT: epididymal white adipose tissue; FKBP5/FKBP51: FK506 binding protein 5; KO, knockout; MBH, mediobasal hypothalamus; MTORC1, mechanistic target of rapamycin kinase complex 1; p: phosphorylated; PHLPP: PH domain and leucine rich repeat protein phosphatase; RPS6KB/p70S6K: ribosomal protein S6 kinase; SKP2: S-phase kinase-associated protein 2; SM: soleus muscle; SQSTM1/p62, sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TSC: TSC complex; ULK1: unc-51 like kinase 1; WIPI: WD repeat domain, phosphoinositide interacting; WT: wild type.

Keywords:  AMPK; FKBP5/FKBP51; WIPI; autophagy; metabolic stress

DOI:  https://doi.org/10.1080/15548627.2022.2063006
Int J Mol Sci. 2022 Apr 14. pii: 4328. [Epub ahead of print]23(8):

Pharmacological Effects of Panduratin A on Renal Cyst Development in In Vitro and In Vivo Models of Polycystic Kidney Disease.

Kanlayanee Tonum, Nipitpon Srimai, Napason Chabang, Somsak Fongsupa, Patoomratana Tuchinda, Jacob A Torres, Thomas Weimbs, Sunhapas Soodvilai.

  Renal cyst expansion in polycystic kidney disease (PKD) involves abnormalities in both cyst-lining-cell proliferation and fluid accumulation. Suppression of these processes may retard the progression of PKD. Evidence suggests that the activation of 5' AMP-activated protein kinase (AMPK) inhibits cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride secretion, leading to reduced progression of PKD. Here we investigated the pharmacological effects of panduratin A, a bioactive compound known as an AMPK activator, on CFTR-mediated chloride secretion and renal cyst development using in vitro and animal models of PKD. We demonstrated that AMPK was activated in immortalized normal renal cells and autosomal dominant polycystic kidney disease (ADPKD) cells following treatment with panduratin A. Treatment with panduratin A reduced the number of renal cyst colonies corresponding with a decrease in cell proliferation and phosphorylated p70/S6K, a downstream target of mTOR signaling. Additionally, panduratin A slowed cyst expansion via inhibition of the protein expression and transport function of CFTR. In heterozygous Han:Sprague-Dawley (Cy/+) rats, an animal model of PKD, intraperitoneal administration of panduratin A (25 mg/kg BW) for 5 weeks significantly decreased the kidney weight per body weight ratios and the cystic index. Panduratin A also reduced collagen deposition in renal tissue. Intraperitoneal administration of panduratin A caused abdominal bleeding and reduced body weight. However, 25 mg/kg BW of panduratin A via oral administration in the PCK rats, another non-orthologous PKD model, showed a significant decrease in the cystic index without severe adverse effects, indicating that the route of administration is critical in preventing adverse effects while still slowing disease progression. These findings reveal that panduratin A might hold therapeutic properties for the treatment of PKD.

Keywords:  ADPKD; AMP-activated protein kinase (AMPK); cell proliferation; cystic fibrosis transmembrane conductance regulator (CFTR); cystogenesis; renal fluid secretion

DOI:  https://doi.org/10.3390/ijms23084328
Elife. 2022 Apr 19. pii: e67659. [Epub ahead of print]11

High and stable ATP levels prevent aberrant intracellular protein aggregation in yeast.

Masak Takaine, Hiromi Imamura, Satoshi Yoshida.

  Adenosine triphosphate (ATP) at millimolar levels has recently been implicated in the solubilization of cellular proteins. However, the significance of this high ATP level under physiological conditions and the mechanisms that maintain ATP remain unclear. We herein demonstrated that AMP-activated protein kinase (AMPK) and adenylate kinase (ADK) cooperated to maintain cellular ATP levels regardless of glucose levels. Single-cell imaging of ATP-reduced yeast mutants revealed that ATP levels in these mutants underwent stochastic and transient depletion, which promoted the cytotoxic aggregation of endogenous proteins and pathogenic proteins, such as huntingtin and α-synuclein. Moreover, pharmacological elevations in ATP levels in an ATP-reduced mutant prevented the accumulation of α-synuclein aggregates and its cytotoxicity. The present study demonstrates that cellular ATP homeostasis ensures proteostasis and revealed that suppressing the high volatility of cellular ATP levels prevented cytotoxic protein aggregation, implying that AMPK and ADK are important factors that prevent proteinopathies, such as neurodegenerative diseases.

Keywords:  AMPK; ATP; S. cerevisiae; adenylate kinase; cell biology; homeostasis; proteostasis; yeast

DOI:  https://doi.org/10.7554/eLife.67659
Front Pharmacol. 2022 ;13 832611

Acute Administration of Metformin Protects Against Neuronal Apoptosis Induced by Cerebral Ischemia-Reperfusion Injury via Regulation of the AMPK/CREB/BDNF Pathway.

Ke Liu, Lulu Li, Zhijun Liu, Gang Li, Yanqing Wu, Xingjun Jiang, Mengdie Wang, Yanmin Chang, Tingting Jiang, Jianheng Luo, Jiahui Zhu, Hongge Li, Yong Wang.

  Metformin is a first-line anti-diabetic agent with a powerful hypoglycemic effect. Several studies have reported that metformin can improve the prognosis of stroke patients and that this effect is independent of its hypoglycemic effect; however, the specific mechanism remains unclear. In this research, we explored the effect and specific mechanism of metformin in cerebral ischemia-reperfusion (I/R) injury by constructing a transient middle cerebral artery occlusion model in vivo and a glucose and oxygen deprivation/reoxygenation (OGD/R) model in vitro. The results of the in vivo experiments showed that acute treatment with low-dose metformin (10 mg/kg) ameliorated cerebral edema, reduced the cerebral infarction volume, improved the neurological deficit score, and ameliorated neuronal apoptosis in the ischemic penumbra. Moreover, metformin up-regulated the brain-derived neurotrophic factor (BDNF) expression and increased phosphorylation levels of AMP-activated protein kinase (AMPK) and cAMP-response element binding protein (CREB) in the ischemia penumbra. Nevertheless, the above-mentioned effects of metformin were reversed by Compound C. The results of the in vitro experiments showed that low metformin concentrations (20 μM) could reduce apoptosis of human umbilical vein endothelial cells (HUVECs) under OGD/R conditions and promote cell proliferation. Moreover, metformin could further promote BDNF expression and release in HUVECs under OGD/R conditions via the AMPK/CREB pathway. The Transwell chamber assay showed that HUVECs treated with metformin could reduce apoptosis of SH-SY5Y cells under OGD/R conditions and this effect could be partially reversed by transfection of BDNF siRNA in HUVECs. In summary, our results suggest that metformin upregulates the level of BDNF in the cerebral ischemic penumbra via the AMPK/CREB pathway, thereby playing a protective effect in cerebral I/R injury.

Keywords:  AMP-activated protein kinase; brain-derived neurotrophic factor; cerebral ischemia-reperfusion injury; metformin; neuronal apoptosis

DOI:  https://doi.org/10.3389/fphar.2022.832611
Hum Reprod. 2022 Apr 23. pii: deac067. [Epub ahead of print]

At the crossroads of fertility and metabolism: the importance of AMPK-dependent signaling in female infertility associated with hyperandrogenism.

Pascal Froment, Ingrid Plotton, Cecilia Giulivi, Stephane Fabre, Rita Khoueiry, Nizar I Mourad, Sandrine Horman, Christelle Ramé, Charlène Rouillon, Jeremy Grandhaye, Yves Bigot, Claire Chevaleyre, Remy Le Guevel, Patricia Mallegol, Ramaroson Andriantsitohaina, Fabrice Guerif, Jérôme Tamburini, Benoit Viollet, Marc Foretz, Joelle Dupont.

  STUDY QUESTION: What biological processes are linked to the signaling of the energy sensor 5'-AMP-activated protein kinase (AMPK) in mouse and human granulosa cells (GCs)?SUMMARY ANSWER: The lack of α1AMPK in GCs impacted cell cycle, adhesion, lipid metabolism and induced a hyperandrogenic response.
WHAT IS KNOWN ALREADY: AMPK is expressed in the ovarian follicle, and its activation by pharmacological medications, such as metformin, inhibits the production of steroids. Polycystic ovary syndrome (PCOS) is responsible for infertility in approximately 5-20% of women of childbearing age and possible treatments include reducing body weight, improving lifestyle and the administration of a combination of drugs to improve insulin resistance, such as metformin.
STUDY DESIGN, SIZE, DURATION: AMPK signaling was evaluated by analyzing differential gene expression in immortalized human granulosa cells (KGNs) with and without silencing α1AMPK using CRISPR/Cas9. In vivo studies included the use of a α1AMPK knock-out mouse model to evaluate the role of α1AMPK in folliculogenesis and fertility. Expression of α1AMPK was evaluated in primary human granulosa-luteal cells retrieved from women undergoing IVF with and without a lean PCOS phenotype (i.e. BMI: 18-25 kg/m2).
PARTICIPANTS/MATERIALS, SETTING, METHODS: α1AMPK was disrupted in KGN cells and a transgenic mouse model. Cell viability, proliferation and metabolism were evaluated. Androgen production was evaluated by analyzing protein levels of relevant enzymes in the steroid pathway by western blots, and steroid levels obtained from in vitro and in vivo models by mass spectrometry. Differential gene expression in human GC was obtained by RNA sequencing. Analysis of in vivo murine folliculogenesis was performed by histology and immunochemistry, including evaluation of the anti-Müllerian hormone (AMH) marker. The α1AMPK gene expression was evaluated by quantitative RT-PCR in primary GCs obtained from women with the lean PCOS phenotype (n = 8) and without PCOS (n = 9).
MAIN RESULTS AND THE ROLE OF CHANCE: Silencing of α1AMPK in KGN increased cell proliferation (P < 0.05 versus control, n = 4), promoted the use of fatty acids over glucose, and induced a hyperandrogenic response resulting from upregulation of two of the enzymes involved in steroid production, namely 3β-hydroxysteroid dehydrogenase (3βHSD) and P450 side-chain cleavage enzyme (P450scc) (P < 0.05, n = 3). Female mice deficient in α1AMPK had a 30% decrease in their ovulation rate (P < 0.05, n = 7) and litter size, a hyperandrogenic response (P < 0.05, n = 7) with higher levels of 3βHSD and p450scc levels in the ovaries, and an increase in the population of antral follicles (P < 0.01, n = 10) compared to controls. Primary GCs from lean women with PCOS had lower α1AMPK mRNA expression levels than the control group (P < 0.05, n = 8-9).
LARGE SCALE DATA: The FastQ files and metadata were submitted to the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB46048.
LIMITATIONS, REASONS FOR CAUTION: The human KGN is a not fully differentiated, transformed cell line. As such, to confirm the role of AMPK in GC and the PCOS phenotype, this model was compared to two others: an α1AMPK transgenic mouse model and primary differentiated granulosa-lutein cells from non-obese women undergoing IVF (with and without PCOS). A clear limitation is the small number of patients with PCOS utilized in this study and that the collection of human GCs was performed after hormonal stimulation.
WIDER IMPLICATIONS OF THE FINDINGS: Our results reveal that AMPK is directly involved in steroid production in human GCs. In addition, AMPK signaling was associated with other processes frequently reported as dysfunctional in PCOS models, such as cell adhesion, lipid metabolism and inflammation. Silencing of α1AMPK in KGN promoted folliculogenesis, with increases in AMH. Evaluating the expression of the α1AMPK subunit could be considered as a marker of interest in infertility cases related to hormonal imbalances and metabolic disorders, including PCOS.
STUDY FUNDING/COMPETING INTEREST(S): This study was financially supported by the Institut National de la Recherche Agronomique (INRA) and the national programme « FERTiNERGY » funded by the French National Research Agency (ANR). The authors report no intellectual or financial conflicts of interest related to this work. R.K. is identified as personnel of the International Agency for Research on Cancer/World Health Organization. R.K. alone is responsible for the views expressed in this article and she does not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer/World Health Organization.
TRIAL REGISTRATION NUMBER: N/A.

Keywords:  AMP-activated protein kinases; AMPK; androgens; anti-Müllerian hormone; fertility; granulosa cells; ovary; polycystic ovary syndrome; testosterone

DOI:  https://doi.org/10.1093/humrep/deac067
Int J Mol Sci. 2022 Apr 15. pii: 4405. [Epub ahead of print]23(8):

Docosahexaenoic Acid Suppresses Oxidative Stress-Induced Autophagy and Cell Death via the AMPK-Dependent Signaling Pathway in Immortalized Fischer Rat Schwann Cells 1.

Yasuaki Tatsumi, Ayako Kato, Naoko Niimi, Hideji Yako, Tatsuhito Himeno, Masaki Kondo, Shin Tsunekawa, Yoshiro Kato, Hideki Kamiya, Jiro Nakamura, Koji Higai, Kazunori Sango, Koichi Kato.

  Autophagy is the process by which intracellular components are degraded by lysosomes. It is also activated by oxidative stress; hence, autophagy is thought to be closely related to oxidative stress, one of the major causes of diabetic neuropathy. We previously reported that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) induced antioxidant enzymes and protected Schwann cells from oxidative stress. However, the relationship between autophagy and oxidative stress-induced cell death in diabetic neuropathy has not been elucidated. Treatment with tert-butyl hydroperoxide (tBHP) decreased the cell survival rate, as measured by an MTT assay in immortalized Fischer rat Schwann cells 1 (IFRS1). A DHA pretreatment significantly prevented tBHP-induced cytotoxicity. tBHP increased autophagy, which was revealed by the ratio of the initiation markers, AMP-activated protein kinase, and UNC51-like kinase phosphorylation. Conversely, the DHA pretreatment suppressed excessive tBHP-induced autophagy signaling. Autophagosomes induced by tBHP in IFRS1 cells were decreased to control levels by the DHA pretreatment whereas autolysosomes were only partially decreased. These results suggest that DHA attenuated excessive autophagy induced by oxidative stress in Schwann cells and may be useful to prevent or reduce cell death in vitro. However, its potentiality to treat diabetic neuropathy must be validated in in vivo studies.

Keywords:  AMPK; DHA; Schwann cells; autophagy; oxidative stress

DOI:  https://doi.org/10.3390/ijms23084405
Mediators Inflamm. 2022 ;2022 9129984

Atractylenolide III Improves Mitochondrial Function and Protects Against Ulcerative Colitis by Activating AMPK/SIRT1/PGC-1α.

Jieru Han, Wenhao Li, Guangyu Shi, Yunlei Huang, Xutao Sun, Na Sun, Deyou Jiang.

Ulcerative colitis (UC) is a complex inflammatory bowel disease (IBD) associated with mitochondrial function. Atractylenolide III (AT III) is a natural product with anti-inflammatory effects. The aim of this work is to investigate the protective effect of AT III on UC and its underlying mechanisms. Herein, dextran sulfate sodium- (DSS-) induced mice and lipopolysaccharide- (LPS-) stimulated intestinal epithelial cells (IEC-6) were employed to mimic UC pathologies in vivo and in vitro. The results showed that in DSS-induced mice, AT III significantly reversed the body weight loss, colon length reduction, disease activity index (DAI) increase, and histological damage. The production of proinflammatory factors and reduction of antioxidants in colitis were suppressed by AT III. In addition, we demonstrated that AT III attenuated the intestinal epithelial barrier destruction and mitochondrial dysfunction induced by DSS, which was similar to the in vitro results in LPS-treated IEC-6 cells. The protein levels of p-AMPK, SIRT1, and PGC-1α along with acetylated PGC-1α were also upregulated by AT III in vivo and in vitro. In conclusion, these findings support that AT III may protect against mitochondrial dysfunction by the activation of the AMPK/SIRT1/PGC-1α signaling pathway during UC development.

DOI: https://doi.org/10.1155/2022/9129984
Food Funct. 2022 Apr 19.

Hawk tea prevents high-fat diet-induced obesity in mice by activating the AMPK/ACC/SREBP1c signaling pathways and regulating the gut microbiota.

Wei Tao, Weiguo Cao, Bao Yu, Huan Chen, Ruixue Gong, Quji Luorong, Juan Luo, Ling Yao, Dan Zhang.

Scope: Hawk tea, a non-Camellia tea, is an ancient tea drink from southwest China and has been proven to exhibit significant hypoglycaemic and lipid-lowering effects. The aim of this study was to evaluate whether Hawk tea extract (HTE) can improve obesity induced by a high-fat diet (HFD) in a mouse model and to determine whether its anti-obesity effects are related to improvements in lipid metabolism and the gut microbiota. Methods and results: We tested the ability of HTE to prevent obesity and regulate gut microbiota in C57BL/6J mice fed with a HFD. We found that HTE significantly reduced body weight, fat deposition, serum triglyceride (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels, and significantly increased serum levels of high-density lipoprotein cholesterol (HDL-C) induced by HFD. HTE also increased the levels of AMPK and ACC phosphorylation, up-regulated the expression of CPT-1, and downregulated the expression of SREBP1c and FAS. In addition, the administration of HTE significantly altered the composition of the gut microbiota, reduced the ratio of Firmicutes to Bacteroidetes, increased the relative abundance of Akkermansia muciniphila, Bacteroides-vulgatus, and Faecalibaculum_rodentium, and decreased the relative abundance of Desulfovibrionaceae and Lachnospiraceae. Conclusions: Collectively, our data demonstrate that HTE can prevent HFD-induced obesity by regulating the AMPK/ACC/SREBP1c signaling pathways and the gut microbiota.

DOI: https://doi.org/10.1039/d1fo04260b
Antioxidants (Basel). 2022 Mar 24. pii: 626. [Epub ahead of print]11(4):

Luteolin Synergistically Enhances Antitumor Activity of Oxaliplatin in Colorectal Carcinoma via AMPK Inhibition.

Chan Ho Jang, Nayoung Moon, Jinkyung Lee, Min Jeong Kwon, Jisun Oh, Jong-Sang Kim.

  Luteolin is a naturally-occurring polyphenolic compound that is known to have antioxidative and antitumor activities in vitro. This study aimed to examine the in vivo anticancer efficacy of luteolin in conjunction with oxaliplatin treatment using a colorectal carcinoma xenograft mouse model. HCT116 human colorectal carcinoma cells were subcutaneously implanted into BALB/c nude mice, followed by the intraperitoneal administration of luteolin at a dose of 50 mg/kg body weight (BW)/day with or without oxaliplatin at a dose of 10 mg/kg BW/day three times per week for a total of 3 weeks. The combined luteolin and oxaliplatin treatment resulted in the synergistic suppression of the growth of HCT116 xenograft tumors when compared to treatment with luteolin or oxaliplatin alone. In addition, the combined treatment significantly increased the expression of cleaved PARP and p53 in the xenograft tumors compared with the vehicle control, but only marginally affected the level of heme oxygenase-1 (HO-1). These results indicated that luteolin treatment retarded oxaliplatin-induced tumor growth by facilitating apoptotic cell death and inhibiting HO-1-mediated cytoprotection. Therefore, these findings suggest the synergistic potential of dietary luteolin in conjunction with conventional chemotherapy for the treatment of colorectal cancer.

Keywords:  AMPK inhibition; colorectal cancer; luteolin; oxaliplatin; xenograft tumor

DOI:  https://doi.org/10.3390/antiox11040626
Phytomedicine. 2022 Apr 12. pii: S0944-7113(22)00176-3. [Epub ahead of print]101 154098

Spinacetin alleviates doxorubicin-induced cardiotoxicity by initiating protective autophagy through SIRT3/AMPK/mTOR pathways.

Dawei Liu, Lei Zhao.

  BACKGROUND: Doxorubicin-induced myocardiopathy is a massive obstacle in administering chemotherapeutic drugs in cancer patients.PURPOSES: In the present study, we aim to investigate the effects of spinacetin, a flavonoid glycoside, on doxorubicin-induced cardiotoxicity.
STUDY DESIGN: The doxorubicin-induced cardiotoxicity mice model was established to evaluate the cardioprotective effects of SP. The H9C2 cell line was used to study SP's potential mechanisms of action. Dexrazoxane (180 mg/kg) was used as the positive control.
METHODS: The CCK-8 cell proliferation assay, hematoxylin and eosin (HE) staining, detection of serum biomarkers, flow cytometry for apoptosis, dansylcadaverine (MDC) staining, and Western blot for crucial molecules were conducted in the present study.
RESULTS: SP significantly increased the survival rate of primary cardiomyocytes and decreased the serum LDH, CK-MB, TrT, and myocardial MDA level. The apoptosis of cardiomyocytes significantly decreased by SP, with upregulation of autophagy. In the H9C2 cell line, SP protects the cells from doxorubicin-induced cytotoxicity, decreases apoptosis, and increases autophagy. The subsequent mechanism study showed that the activation of AMPK/mTOR signaling was involved in the protective effects of SP on doxorubicin-induced cardiotoxicity through upregulating the expression level of SIRT3.
CONCLUSION: We concluded that SP could protect against doxorubicin-induced cardiotoxicity both in vitro and in vivo by initiating protective autophagy through SIRT3/AMPK/mTOR pathways, which has not been reported previously. SP could be treated as a potential candidate for cardioprotective usage during chemotherapy. The further clinical study is still urgently needed to investigate the safety and effectiveness of SP in patients.

Keywords:  Apoptosis; Autophagy; Doxorubicin-induced cardiotoxicity; SIRT3; Spinacetin

DOI:  https://doi.org/10.1016/j.phymed.2022.154098
Food Funct. 2022 Apr 19.

L-Methionine supplementation attenuates high-fat fructose diet-induced non-alcoholic steatohepatitis by modulating lipid metabolism, fibrosis, and inflammation in rats.

Umashanker Navik, Vaibhav G Sheth, Nisha Sharma, Kulbhushan Tikoo.

Recently, the protective effects of a methionine-rich diet on hepatic oxidative stress and fibrosis have been suggested but not adequately studied. We, therefore, hypothesized that L-methionine supplementation would ameliorate the progression of hepatic injury in a diet-induced non-alcoholic steatohepatitis (NASH) model and aimed to investigate the underlying mechanism. NASH was developed in male Sprague Dawley rats by feeding them with a high-fat-fructose diet (HFFrD) for 10 weeks. The results demonstrated that L-methionine supplementation to NASH rats for 16 weeks improved the glycemic, lipid, and liver function profiles in NASH rats. Histological analysis of liver tissue revealed a remarkable improvement in the three classical lesions of NASH: steatosis, inflammation, and ballooning. Besides, L-methionine supplementation ameliorated the HFFrD-induced enhanced lipogenesis and lipid peroxidation. An anti-inflammatory effect of L-methionine was also observed through the inhibition of the release of proinflammatory cytokines. Furthermore, the hepatic SIRT1/AMPK signaling pathway was associated with the beneficial effects of L-methionine. This study demonstrates that L-methionine supplementation in HFFrD-fed rats improves their liver pathology via regulation of lipogenesis, inflammation, and the SIRT1/AMPK pathway, thus encouraging its clinical evaluation for the treatment of NASH.

DOI: https://doi.org/10.1039/d1fo03403k
Cell Mol Life Sci. 2022 Apr 19. 79(5): 249

Traumatic-noise-induced hair cell death and hearing loss is mediated by activation of CaMKKβ.

Fan Wu, Kayla Hill, Qiaojun Fang, Zuhong He, Hongwei Zheng, Xianren Wang, Hao Xiong, Su-Hua Sha.

  BACKGROUND: The Ca2+/calmodulin-dependent protein kinase kinases (CaMKKs) are serine/threonine-directed protein kinases that are activated following increases in intracellular calcium, playing a critical role in neuronal signaling. Inner-ear-trauma-induced calcium overload in sensory hair cells has been well documented in the pathogenesis of traumatic noise-induced hair cell death and hearing loss, but there are no established pharmaceutical therapies available due to a lack of specific therapeutic targets. In this study, we investigated the activation of CaMKKβ in the inner ear after traumatic noise exposure and assessed the prevention of noise-induced hearing loss (NIHL) with RNA silencing.RESULTS: Treatment with short hairpin RNA of CaMKKβ (shCaMKKβ) via adeno-associated virus transduction significantly knocked down CaMKKβ expression in the inner ear. Knockdown of CaMKKβ significantly attenuated noise-induced hair cell loss and hearing loss (NIHL). Additionally, pretreatment with naked CaMKKβ small interfering RNA (siCaMKKβ) attenuated noise-induced losses of inner hair cell synapses and OHCs and NIHL. Furthermore, traumatic noise exposure activates CaMKKβ in OHCs as demonstrated by immunolabeling for p-CaMKI. CaMKKβ mRNA assessed by fluorescence in-situ hybridization and immunolabeling for CaMKKβ in OHCs also increased after the exposure. Finally, pretreatment with siCaMKKβ diminished noise-induced activation of AMPKα in OHCs.
CONCLUSIONS: These findings demonstrate that traumatic-noise-induced OHC loss and hearing loss occur primarily via activation of CaMKKβ. Targeting CaMKKβ is a key strategy for prevention of noise-induced hearing loss. Furthermore, our data suggest that noise-induced activation of AMPKα in OHCs occurs via the CaMKKβ pathway.

Keywords:  Activation of CaMKKβ after traumatic noise exposure; Adeno-associated virus-mediated gene silencing; Fluorescence in-situ hybridization in adult mouse cochleae; Prevention of noise-induced hearing loss by RNA silencing in-vivo

DOI:  https://doi.org/10.1007/s00018-022-04268-4
Ann Transl Med. 2022 Mar;10(6): 366

Metformin reverses tamoxifen resistance through the lncRNA GAS5-medicated mTOR pathway in breast cancer.

Yiyan Jiang, Ting Qian, Suxia Li, Yuanyuan Xie, Min Tao.

  Background: Metformin (Met) has antitumor effects on various cancers, but it is still unclear whether it exerts a reversible effect on endocrine resistance in breast cancer (BC) patients. In the present survey, metformin's effects on tamoxifen-resistant MCF cells (The cell line was established at the Michigan Cancer Foundation, hence the name MCF cell) were evaluated and the molecular mechanism was explored.Methods: We constructed a tamoxifen-resistant BC cell line MCF-7R, then applied Cell Counting Kit-8 (CCK-8), flow cytometry, and EdU assessments to determine the growth and apoptosis of MCF-7R cells. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to evaluate the expression level of lncRNA GAS5 and the mTOR-associated proteins. Overexpression and downregulation of lncRNA GAS5 were carried out to explore the molecular mechanism of Met.
Results: The combined effect of Met and OHT (4-hydroxytamoxifen, main active metabolite of tamoxifen) was to remarkably reduce the growth and increase the apoptosis of MCF-7R cells compared with OHT alone. The levels of the mTOR, p-mTOR and p-P70S6K proteins in MCF-7R cells decreased after Met treatment, but the p-AMPK2 and PTEN proteins significantly increased. The overexpression of lncRNA GAS5 in MCF-7R cells inhibited cell growth with decreased mTOR, p-mTOR, PCNA, and Bcl-2 proteins. Metformin had no significant effect on MCF-7R cells with lncRNA GAS5 knockdown but contrarily activated p-mTOR.
Conclusions: Metformin can inhibit overactivation of the mTOR signaling pathway through upregulating lncRNA GAS5 expression, thereby inhibiting the growth and inducing the apoptosis of BC cells, providing a new clinical treatment for BC.

Keywords:  Breast Cancer (BC); GAS5 long non-coding RNA; drug resistance; mTOR protein; metformin

DOI:  https://doi.org/10.21037/atm-22-795