bims-amsmem 2023-01-15 papers

bims-amsmem

Biomed News

on AMPK signaling mechanism in energy metabolism

Issue of 2023–01–15
nineteen papers selected by
Dipsikha Biswas, Københavns Universitet

AMPK inhibits liver gluconeogenesis: fact or fiction?
Cross-talk between AMP-activated protein kinase and the sonic hedgehog pathway in the high-fat diet triggered colorectal cancer.
Loss of WNK1 Suppressed the Malignant Behaviors of Hepatocellular Carcinoma Cells by Promoting Autophagy and Activating AMPK Pathway.
Canagliflozin Attenuates Lipotoxicity in Cardiomyocytes by Inhibiting Inflammation and Ferroptosis through Activating AMPK Pathway.
Ebastine exerts antitumor activity and induces autophagy by activating AMPK/ULK1 signaling in an IPMK-dependent manner in osteosarcoma.
Effects of imeglimin on mitochondrial function, AMPK activity, and gene expression in hepatocytes.
Metformin Attenuates Slow-to-Fast Fiber Shift and Proteolysis Markers Increase in Rat Soleus after 7 Days of Rat Hindlimb Unloading.
The protective effects of Esculentoside A through AMPK in the triple transgenic mouse model of Alzheimer's disease.
Metformin and Its Immune-Mediated Effects in Various Diseases.
Ferroptosis is involved in PM2.5-induced acute nasal epithelial injury via AMPK-mediated autophagy.
Osthole: A potential AMPK agonist that inhibits NLRP3 inflammasome activation by regulating mitochondrial homeostasis for combating rheumatoid arthritis.
Lespedeza bicolor extract supplementation reduced hyperglycemia-induced skeletal muscle damage by regulation of AMPK/SIRT/PGC1α-related energy metabolism in type 2 diabetic mice.
Empagliflozin inhibits excessive autophagy through the AMPK/GSK3β signaling pathway in diabetic cardiomyopathy.
Pterostilbene alleviated NAFLD via AMPK/mTOR signaling pathways and autophagy by promoting Nrf2.
Qinling liquid ameliorates renal immune inflammatory damage via activating autophagy through AMPK/Stat3 pathway in uric acid nephropathy.
Xanthosine, a purine glycoside mediates hepatic glucose homeostasis through inhibition of gluconeogenesis and activation of glycogenesis via regulating the AMPK/ FoxO1/AKT/GSK3β signaling cascade.
Red ginseng extract improves skeletal muscle energy metabolism and mitochondrial function in chronic fatigue mice.
Quantification and Proteomic Characterization of β-hydroxybutyrylation Modification in the Hearts of AMPKα2 Knockout Mice.
Sestrin2 mediates FOXM1 expression to block the EMT process in non-small cell lung cancer through the AMPK/YAP pathway.

Biochem J. 2023 Jan 13. 480(1): 105-125

AMPK inhibits liver gluconeogenesis: fact or fiction?

Manuel Johanns, Louis Hue, Mark H Rider.

  Is there a role for AMPK in the control of hepatic gluconeogenesis and could targeting AMPK in liver be a viable strategy for treating type 2 diabetes? These are frequently asked questions this review tries to answer. After describing properties of AMPK and different small-molecule AMPK activators, we briefly review the various mechanisms for controlling hepatic glucose production, mainly via gluconeogenesis. The different experimental and genetic models that have been used to draw conclusions about the role of AMPK in the control of liver gluconeogenesis are critically discussed. The effects of several anti-diabetic drugs, particularly metformin, on hepatic gluconeogenesis are also considered. We conclude that the main effect of AMPK activation pertinent to the control of hepatic gluconeogenesis is to antagonize glucagon signalling in the short-term and, in the long-term, to improve insulin sensitivity by reducing hepatic lipid content.

Keywords:  AMPK; glucagon; gluconeogenesis; liver; metformin

DOI:  https://doi.org/10.1042/BCJ20220582
Arch Biochem Biophys. 2023 Jan 04. pii: S0003-9861(22)00386-1. [Epub ahead of print]735 109500

Cross-talk between AMP-activated protein kinase and the sonic hedgehog pathway in the high-fat diet triggered colorectal cancer.

T B Beena, Mathew A Jesil, K B Harikumar.

  The major cause of colorectal cancer (CRC) related mortality is due to its metastasis. Signaling pathways play a definite role in the development and progression of CRC. Recent studies demonstrate that the regulation of the sonic hedgehog (Shh) pathway is beneficial in the CRC treatment strategy. Also, 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a well-known regulator of metabolism and inflammation, making it a suitable treatment option for CRC. Consumption of a high-fat diet (HFD) is a significant cause of CRC genesis. Also, the lipids play an indispensable role in aberrant activation of the Shh pathway. This review explains in detail the interconnection between HFD consumption, Shh pathway activation, and the progression of CRC. According to recent studies and literature, AMPK is a potential regulator that can control the complexities of CRC and reduce lipid levels and may directly inhibit shh signalling. The review also suggests the possible risk elements of AMPK activation in CRC due to its context-dependent role. Also, the activation of AMPK in HFD-induced CRC may modulate cancer progression by regulating the Shh pathway and metabolism.

Keywords:  5ʹ-adenosine monophosphate (AMP)-Activated protein kinase; Colorectal cancer; Crosstalk; Hedgehog proteins; High fat diet; Sonic hedgehog

DOI:  https://doi.org/10.1016/j.abb.2022.109500
Dis Markers. 2022 ;2022 6831224

Loss of WNK1 Suppressed the Malignant Behaviors of Hepatocellular Carcinoma Cells by Promoting Autophagy and Activating AMPK Pathway.

Fang Wang, Xianxia Yan, Guangyan Shi, Lei Zhang, Xu Jing.

Background: WNK lysine deficient protein kinase 1 (WNK1) has been shown to be highly expressed in hepatocellular carcinoma (HCC) samples and related to poor prognosis of HCC patients based on bioinformatics analysis. However, the specific function of WNK1 in HCC has not been analyzed. This study is aimed at exploring the function of WNK1 in HCC progression as well as its related molecular mechanism.
Methods: After knockdown of WNK1 by small interference RNA, cell counting kit-8, colony formation, western blot, Transwell, and wound healing assays were employed to evaluate the biological behaviors of HCC cells. Immunofluorescent staining was applied to detect the effect of WNK1 on LC3 II. GSK690693 or si-AMPK was applied to block AMPK pathway. The expression of autophagy and AMPK pathway related molecules was examined by western blot assay.
Results: WNK1 was highly expressed in HCC cell lines and loss of WNK1 inhibited HCC cell proliferation, cell cycle, migration, and invasion. Additionally, we demonstrated that loss of WNK1 promoted the autophagy and activated AMPK pathway in HCC cells. While, GSK690693 treatment or si-AMPK transfection suppressed the autophagy and promoted HCC cells proliferation. However, WNK1 knockdown counteracted the effect of GSK690693 or si-AMPK in regulating HCC cell proliferation. Finally, we demonstrated that WNK1 regulated the malignant behaviors of HCC cells by modulating autophagy and AMPK pathway.
Conclusions: The above results indicated that WNK1 may be a worthwhile target to be considered for therapy of HCC.

DOI: https://doi.org/10.1155/2022/6831224
Int J Mol Sci. 2023 Jan 03. pii: 858. [Epub ahead of print]24(1):

Canagliflozin Attenuates Lipotoxicity in Cardiomyocytes by Inhibiting Inflammation and Ferroptosis through Activating AMPK Pathway.

Wanqiu Zhang, Jinghua Lu, Yangyang Wang, Pengbo Sun, Tong Gao, Naihan Xu, Yaou Zhang, Weidong Xie.

  Diabetic cardiomyopathy (DCM) is a myocardial disease independent of other cardiovascular diseases, such as coronary heart disease, hypertension, etc. Lipotoxicity is closely related to DCM. In this study, we investigated the mechanism of lipid metabolism disturbance in DCM in HL-1 cells. Through bioinformatics and Western blotting analysis, we found that canagliflozin (CAN) significantly inhibited the expression of inflammatory factors cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Ferroptosis is mediated by lipid peroxidation. We demonstrated the presence of ferroptosis in cardiomyocytes by detecting intracellular Fe2+ content and the levels of reactive oxygen species (ROS), malondialdehyde (MDA), reduced glutathione (GSH), and mitochondrial membrane potential (MMP). CAN could significantly regulate the indicators of ferroptosis. By using specific inhibitors celecoxib (coxib), S-methylisothiourea sulfate (SMT), Ferrostatin-1 (Fer-1), and Compound C, we further found that CAN regulated inflammation and ferroptosis through AMP-activated protein (AMPK), and inflammation interacted with ferroptosis. Our study indicated that CAN attenuated lipotoxicity in cardiomyocytes by regulating inflammation and ferroptosis through activating the AMPK pathway. This study provides a new direction of myocardial lipotoxicity and some new information for the treatment of DCM.

Keywords:  AMPK; canagliflozin; cardiomyocytes; ferroptosis; inflammation; lipotoxicity

DOI:  https://doi.org/10.3390/ijms24010858
Int J Biol Sci. 2023 ;19(2): 537-551

Ebastine exerts antitumor activity and induces autophagy by activating AMPK/ULK1 signaling in an IPMK-dependent manner in osteosarcoma.

Zhen Pan, Shi-Jie Li, Hua Guo, Zhao-Hui Li, Xiang Fei, Shi-Min Chang, Qing-Cheng Yang, Dong-Dong Cheng.

  Numerous studies have confirmed that in addition to interfering with the tumor inflammatory environment, anti-inflammatory agents can directly increase apoptosis and sensitivity to conventional therapies and decrease invasion and metastasis, making them useful candidates for cancer therapy. Here, we first used high-throughput screening and had screened one compound candidate, ebastine (a H1-histamine receptor antagonist), for osteosarcoma therapy. Cell viability assays, colony formation assays, wound healing assays, and Transwell assays demonstrated that ebastine elicited antitumor effects in osteosarcoma cells. In addition, ebastine treatment exerted obvious effects on cell cycle arrest, metastasis inhibition, apoptosis and autophagy induction both in vitro and in vivo. Mechanistically, we observed that ebastine treatment triggered proapoptotic autophagy by activating AMPK/ULK1 signaling in osteosarcoma cells. Treatment with the AMPK inhibitor dorsomorphin reversed ebastine-induced apoptosis and autophagy. More importantly, we found that IPMK interacted with AMPK and functioned as a positive regulator of AMPK protein in osteosarcoma cells. A rescue study showed that the induction of autophagy and activation of the AMPK/ULK1 signaling pathway by ebastine treatment were reversed by IPMK knockdown, indicating that the activity of ebastine was IPMK dependent. We provide experimental evidence demonstrating that ebastine has antitumor activity in osteosarcoma and promotes autophagy by activating the AMPK/ULK1 signaling pathway, which is IPMK dependent. Our results provide insight into the clinical application potential of ebastine, which may represent a new potential therapeutic candidate for the treatment of osteosarcoma.

Keywords:  AMPK/ULK1 pathway; Ebastine; IPMK; autophagy; osteosarcoma

DOI:  https://doi.org/10.7150/ijbs.69541
Sci Rep. 2023 Jan 13. 13(1): 746

Effects of imeglimin on mitochondrial function, AMPK activity, and gene expression in hepatocytes.

Kaori Hozumi, Kenji Sugawara, Takaya Ishihara, Naotada Ishihara, Wataru Ogawa.

Imeglimin is a recently launched antidiabetic drug structurally related to metformin. To provide insight into the pharmacological properties of imeglimin, we investigated its effects on hepatocytes and compared them with those of metformin. The effects of imeglimin on mitochondrial function in HepG2 cells or mouse primary hepatocytes were examined with an extracellular flux analyzer and on gene expression in HepG2 cells by comprehensive RNA-sequencing analysis. The effects of the drug on AMPK activity in HepG2 cells, mouse primary hepatocytes, and mouse liver were also examined. Treatment of HepG2 cells or mouse primary hepatocytes with imeglimin reduced the oxygen consumption rate coupled to ATP production. Imeglimin activated AMPK in these cells whereas the potency was smaller than metformin. Bolus administration of imeglimin in mice also activated AMPK in the liver. Whereas the effects of imeglimin and metformin on gene expression in HepG2 cells were similar overall, the expression of genes encoding proteins of mitochondrial respiratory complex III and complex I was upregulated by imeglimin but not by metformin. Our results suggest that imeglimin and metformin exert similar pharmacological effects on mitochondrial respiration, AMPK activity, and gene expression in cultured hepatocytes, whereas the two drugs differ in their effects on the expression of certain genes related to mitochondrial function.

DOI: https://doi.org/10.1038/s41598-023-27689-y
Int J Mol Sci. 2022 Dec 28. pii: 503. [Epub ahead of print]24(1):

Metformin Attenuates Slow-to-Fast Fiber Shift and Proteolysis Markers Increase in Rat Soleus after 7 Days of Rat Hindlimb Unloading.

Kristina A Sharlo, Irina D Lvova, Svetlana P Belova, Ksenia A Zaripova, Boris S Shenkman, Tatiana L Nemirovskaya.

  Muscle unloading leads to signaling alterations that cause muscle atrophy and weakness. The cellular energy sensor AMPK can regulate myofiber-type shift, calcium-dependent signaling and ubiquitin-proteasome system markers. We hypothesized that the prevention of p-AMPK downregulation during the first week of muscle unloading would impede atrophy development and the slow-to-fast shift of soleus muscle fibers, and the aim of the study was to test this hypothesis. Thirty-two male Wistar rats were randomly assigned to four groups: placebo control (C), control rats treated with metformin (C + M), 7 days of hindlimb suspension (HS) + placebo (7HS), and 7 days of HS + metformin administration (7HS + M). In the soleus of the 7HS rats, we detected a slow-to-fast fiber-type shift as well as a significant downregulation of MEF-2D and p300 in the nuclei. In the 7HS group, we also found decreases in p-ACC (AMPK target) protein level and in the expression of E3 ubiquitin ligases and p-CaMK II protein level vs. the C group. The 7-day metformin treatment for soleus muscle unloading (1) prevented slow-to-fast fiber-type shift; (2) counteracted changes in the p-ACC protein level; (3) hindered changes in the nuclear protein level of the slow myosin expression activators MEF-2D and p300, but did not affect NFATc1 signaling; and (4) attenuated the unloading-induced upregulation of MuRF-1, atrogin-1, ubiquitin and myostatin mRNA expression, but did not prevent soleus muscle atrophy. Thus, metformin treatment during muscle disuse could be useful to prevent the decrease in the percentage of slow-type fatigue-resistant muscle fibers.

Keywords:  AMPK; atrophy; hindlimb unloading; myosin; proteolysis

DOI:  https://doi.org/10.3390/ijms24010503
Phytomedicine. 2023 Jan;pii: S0944-7113(22)00643-2. [Epub ahead of print]109 154555

The protective effects of Esculentoside A through AMPK in the triple transgenic mouse model of Alzheimer's disease.

Zhijun He, Huajie Zhang, Xiaoqian Li, Sixin Tu, Zi Wang, Shuangxue Han, Xiubo Du, Liming Shen, Nan Li, Qiong Liu.

   BACKGROUND: Neurofibrillary tangles comprising hyperphosphorylated tau are vital factors associated with the pathogenesis of Alzheimer's disease (AD). The elimination or reduction of hyperphosphorylated and abnormally aggregated tau is a valuable measure in AD therapy. Esculentoside A (EsA), isolated from Phytolacca esculenta, exhibits pharmacotherapeutic efficacy in mice with amyloid beta-induced AD. However, whether EsA affects tau pathology and its specific mechanism of action in AD mice remains unclear.
PURPOSE: To investigate the roles and mechanisms of EsA in cognitive decline and tau pathology in a triple transgenic AD (3 × Tg-AD) mouse model.
METHODS: EsA (5 and 10 mg/kg) was administered via intraperitoneal injection to 8-month-old AD mice for eight consecutive weeks. Y-maze and novel object recognition tasks were used to evaluate the cognitive abilities of mice. Potential signaling pathways and targets in EsA-treated AD mice were assessed using quantitative proteomic analysis. The NFT levels and hippocampal synapse numbers were investigated using Gallyas-Braak silver staining and transmission electron microscopy, respectively. Western blotting and immunofluorescence assays were used to measure the expression of tau-associated proteins.
RESULTS: EsA administration attenuated memory and recognition deficits and synaptic damage in AD mice. Isobaric tags for relative and absolute quantitation proteomic analysis of the mouse hippocampus revealed that EsA modulated the expression of some critical proteins, including brain-specific angiogenesis inhibitor 3, galectin-1, and Ras-related protein 24, whose biological roles are relevant to synaptic function and autophagy. Further research revealed that EsA upregulated AKT/GSK3β activity, in turn, inhibited tau hyperphosphorylation and promoted autophagy to clear abnormally phosphorylated tau. In hippocampus-derived primary neurons, inhibiting AMP-activated protein kinase (AMPK) activity through dorsomorphin could eliminate the effect of EsA, as revealed by increased tau hyperphosphorylation, downregulated activity AKT/GSK3β, and blocked autophagy.
CONCLUSIONS: To our knowledge, this study is the first to demonstrate that EsA attenuates cognitive decline by targeting the pathways of both tau hyperphosphorylation and autophagic clearance in an AMPK-dependent manner and it shows a high reference value in AD pharmacotherapy research.

Keywords:  AMP-activated protein kinase (AMPK); Alzheimer's disease (AD); Esculentoside A (EsA); Isobaric tags for relative and absolute quantitation (iTRAQ); Microtubule-associated protein tau (Tau)

DOI:  https://doi.org/10.1016/j.phymed.2022.154555
Int J Mol Sci. 2023 Jan 01. pii: 755. [Epub ahead of print]24(1):

Metformin and Its Immune-Mediated Effects in Various Diseases.

Ichiro Nojima, Jun Wada.

  Metformin has been a long-standing prescribed drug for treatment of type 2 diabetes (T2D) and its beneficial effects on virus infection, autoimmune diseases, aging and cancers are also recognized. Metformin modulates the differentiation and activation of various immune-mediated cells such as CD4+ and CD+8 T cells. The activation of adenosine 5'-monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin complex 1 (mTORC1) pathway may be involved in this process. Recent studies using Extracellular Flux Analyzer demonstrated that metformin alters the activities of glycolysis, oxidative phosphorylation (OXPHOS), lipid oxidation, and glutaminolysis, which tightly link to the modulation of cytokine production in CD4+ and CD+8 T cells in various disease states, such as virus infection, autoimmune diseases, aging and cancers.

Keywords:  AMPK; CD8 T cells; OXPHOS; aging; autoimmune disease; cancer; mTORC

DOI:  https://doi.org/10.3390/ijms24010755
Int Immunopharmacol. 2023 Jan 04. pii: S1567-5769(22)01143-2. [Epub ahead of print]115 109658

Ferroptosis is involved in PM2.5-induced acute nasal epithelial injury via AMPK-mediated autophagy.

Wenjing Gu, Tianhua Hou, Hongwei Zhou, Laiyu Zhu, Wei Zhu, Yusheng Wang.

  PM2.5 is one of the main harmful environmental pollutants and can damage nasal epithelial carriers to worsen allergic rhinitis. Ferroptosis is a novel form of regulated cell death with iron-dependent lipid peroxidation. However, whether ferroptosis is involved in PM2.5-induced nasal epithelial injury has not been elucidated. To verify the vital role of ferroptosis in PM2.5-induced nasal epithelial injury and further explore the potential mechanism, we detected intracellular iron content, ROS release and lipid peroxidation and ferroptosis-related proteins in vitro as well as the pathological changes in the nasal epithelium and the levels of proinflammatory factors in nasal lavage fluid in vivo. Our results showed that PM2.5 exposure led to oxidative stress, labile iron accumulation and lipid peroxidation in HNEPCs. In addition, the expression levels of xCT, GPx4, FTH1 and FTL in HNEPCs were greatly inhibited by PM2.5. Treatment with the ferroptosis inhibitors deferoxamine (DFO) and ferrostatin-1 (Fer-1) significantly reversed the toxicity of PM2.5 to human nasal epithelial cells (HNEPCs). Mechanistically, AMPK-mediated autophagy was initiated during PM2.5 exposure, which drove ferroptosis of HNEPCs. Autophagy inhibitor remarkably improved cell death, oxidative stress, labile iron accumulation, lipid peroxidation, and the downregulated expression of xCT, GPx4, FTH1 and FTL in HNEPCs induced by PM2.5. Furthermore, an AMPK inhibitor (Compound C, CC) and siRNA-AMPKα suppressed autophagy activation and ferroptosis stimulated by PM2.5. In vivo, Fer-1 reduced nasal epithelial injury and mucus secretion in PM2.5-exposed mice. In addition, CC significantly improved nasal epithelial damage and proinflammatory factor production in mice caused by PM2.5 intranasal treatment. In addition, CC greatly inhibited autophagy activation but reversed the downregulation of GPX4 and FTH1 induced by PM2.5 in the nasal epithelium of mice. Together, these data suggest that AMPK-mediated autophagy plays an important role in PM2.5-induced ferroptosis and that AMPK might be a potential treatment target for PM2.5-induced nasal epithelial injury.

Keywords:  AMPK; Autophagy; Ferroptosis; Nasal epithelium; PM2.5

DOI:  https://doi.org/10.1016/j.intimp.2022.109658
Phytomedicine. 2023 Jan 01. pii: S0944-7113(22)00727-9. [Epub ahead of print]110 154640

Osthole: A potential AMPK agonist that inhibits NLRP3 inflammasome activation by regulating mitochondrial homeostasis for combating rheumatoid arthritis.

Xiaoli Jiang, Zhuojian Lu, Qian Zhang, Jialin Yu, Dong Han, Jinhong Liu, Ping Li, Fei Li.

   BACKGROUND: Osthole (OST), a characteristic coumarin compound in Angelicae pubescentis radix (APR), has shown potent efficacy in the treatment of rheumatoid arthritis (RA), but its specific targets and potential mechanism are limited.
PURPOSE: This study aimed to explore the potential targets and molecular mechanisms of OST against RA using computer-assisted techniques in combination with RA fibroblast-like synoviocytes (FLS) inflammation model and CIA rat model.
METHODS: Network pharmacology and molecular docking were applied to initially predict the potential targets of OST for the treatment of RA. Thereafter, TNFα was used to stimulate FLS to build an in vitro model of inflammation, combined with RNA-seq technology and molecular biology such as qPCR to investigate the anti-inflammatory effects and related mechanisms of OST. Finally, the anti-RA effect of OST was demonstrated by establishing a CIA rat model.
RESULTS: The network model results showed that the anti-RA effect of OST was mainly related to its anti-inflammatory effect, and AMPK was identified as a potential target for the potency of OST. In the TNFα-induced FLS cells, OST inhibited the secretion of FLS inflammatory factors, which was attributed to the ability of OST to activate AMPK to inhibit the activation of the NLRP3 inflammasome. Further, it was observed that the activation of AMPK by OST facilitated mitochondrial biogenesis, and corrected abnormal mitochondrial dynamics in FLS, which was favoured to the restoration of mitochondrial homeostasis, and further promoted the occurrence of apoptosis and the decrease of ROS in FLS. Consistent with in vivo studies, administration of OST significantly improved joint deformity and toe erythema, reduced arthritis index scores and inhibited synovial inflammation in CIA rats.
CONCLUSION: Our study proposed for the first time that AMPK, served as a potential target of OST, positively participated in the anti-RA therapeutic effect of OST. By regulating mitochondrial homeostasis and function, OST can effectively inhibit the activation of inflammasome and the secretion of inflammatory factors in vitro and in vivo, and finally achieve beneficial effects in the treatment of RA, which provides support and greater possibility to make further efforts on pharmacological research and clinical application of OST.

Keywords:  AMPK; FLS; Mitochondria; NLRP3 inflammasome; Rheumatoid arthritis

DOI:  https://doi.org/10.1016/j.phymed.2022.154640
Nutr Res. 2022 Dec 22. pii: S0271-5317(22)00150-6. [Epub ahead of print]110 1-13

Lespedeza bicolor extract supplementation reduced hyperglycemia-induced skeletal muscle damage by regulation of AMPK/SIRT/PGC1α-related energy metabolism in type 2 diabetic mice.

Heaji Lee, Sun Yeou Kim, Yunsook Lim.

  Lespedeza bicolor (LB) is known to have antidiabetic activities; however, the underlying molecular mechanisms of LB in hyperglycemia-induced skeletal muscle damage is unclear. Inflammation and oxidative stress caused by type 2 diabetes mellitus (T2DM) not only contributes to insulin resistance, but also promotes muscle atrophy via decreased muscle protein synthesis and increased protein degradation, leading to frailty and sarcopenia. In this study, we hypothesized that LB extract (LBE) supplementatin has an ameliorative effect on hyperglycemia-induced skeletal muscle damage by activation of 5' adenosine monophosphate-activated protein kinase (AMPK)/sirtuin (SIRT)/proliferator-activated receptor γ coactivator 1α (PGC1α)-associated energy metabolism in mice with T2DM. Diabetes was induced by a high-fat diet with a 2-time streptozotoxin injection (30 mg/kg body weight) in male C57BL/6J mice. After diabetes was induced (fasting blood glucose level ≥140 mg/dL), the mice were administered with LBE at a low dose (100 mg/kg/d) or high dose (250 mg/kg/d) by gavage for 12 weeks. LBE supplementation ameliorated glucose tolerance and hemoglobin A1c (%) in mice with T2DM. Moreover, LBE supplementation upregulated protein levels of insulin receptor subunit-1 and Akt accompanied by increased translocation of glucose transporter 4 in mice with T2DM. Furthermore, LBE increased mitochondrial biogenesis by activating SIRT1, SIRT3, SIRT4, and peroxisome PGC1α in diabetic skeletal muscle. Meanwhile, LBE supplementation reduced oxidative stress and inflammation in mice with T2DM. Taken together, the current study suggested that LBE could be a potential therapeutic to prevent skeletal muscle damage by regulation AMPK/SIRT/PGC1α-related energy metabolism in T2DM.

Keywords:  Energy metabolism; Insulin signaling; Lespedeza bicolor; Skeletal muscle; Type 2 diabetes mellitus

DOI:  https://doi.org/10.1016/j.nutres.2022.12.007
Cardiovasc Res. 2023 Jan 11. pii: cvad009. [Epub ahead of print]

Empagliflozin inhibits excessive autophagy through the AMPK/GSK3β signaling pathway in diabetic cardiomyopathy.

Rosalinda Madonna, Stefania Moscato, Maria Concetta Cufaro, Damiana Pieragostino, Letizia Mattii, Piero Del Boccio, Sandra Ghelardoni, Riccardo Zucchi, Raffaele De Caterina.

   BACKGROUND AND AIMS: Sodium-glucose cotransporter 2 (SGLT2) inhibitors have beneficial effects on heart failure and cardiovascular mortality in diabetic and nondiabetic patients, with unclear mechanisms. Autophagy is a cardioprotective mechanism under acute stress conditions, but excessive autophagy accelerates myocardial cell death leading to autosis. We evaluated the protective role of empagliflozin (EMPA) against cardiac injury in murine diabetic cardiomyopathy.
METHODS AND RESULTS: Male mice, rendered diabetics by one single intraperitoneal injection of streptozotocin and treated with EMPA (30 mg/kg/day) had fewer apoptotic cells (4.9 ± 2.1 vs 1 ± 0.5 TUNEL-positive cells %, p < 0.05), less senescence (10.1 ± 2 vs 7.9 ± 1.2 β-gal positivity/tissue area, p < 0.05), fibrosis (0.2 ± 0.05 vs 0.15 ± 0.06, p < 0.05 fibrotic area/tissue area), autophagy (7.9 ± 0.05 vs 2.3 ± 0.6 fluorescence intensity/total area, p < 0.01), and connexin (Cx)-43 lateralization compared with diabetic mice. Proteomic analysis showed a downregulation of the 5' adenosine monophosphate-activated protein kinase (AMPK) pathway and upstream activation of sirtuins in the heart of diabetic mice treated with EMPA compared with diabetic mice. Because sirtuin activation leads to modulation of cardiomyogenic transcription factors, we analyzed the DNA binding activity to serum response elements (SRE) of serum response factor (SRF) by electromobility shift assay. Compared with diabetic mice (0.5 ± 0.01 densitometric units, DU), nondiabetic mice treated with EMPA (2.2 ± 0.01 DU, p < 0.01) and diabetic mice treated with EMPA (2.0 ± 0.1 DU, p < 0.01) significantly increased SRF binding activity to SRE, paralleled by increased cardiac actin expression (4.1 ± 0.1 vs 2.2 ± 0.01 target protein/β-actin ratio, p < 0.01). EMPA significantly reversed cardiac dysfunction on echocardiography in diabetic mice and inhibited excessive autophagy in high-glucose-treated cardiomyocytes by inhibiting the autophagy inducer GSK3β, leading to reactivation of cardiomyogenic transcription factors.
CONCLUSIONS: Taken together, our results describe a novel paradigm in which EMPA inhibits hyperactivation of autophagy through the AMPK/GSK3β signaling pathway in the context of diabetes.

Keywords:  autophagy; connexins; diabetic cardiomyopathy; empagliflozin; glycogen synthase kinase 3 beta; serum response factor; sodium-glucose cotransporter type 2 (SGLT2) inhibitors

DOI:  https://doi.org/10.1093/cvr/cvad009
Phytomedicine. 2023 Jan;pii: S0944-7113(22)00649-3. [Epub ahead of print]109 154561

Pterostilbene alleviated NAFLD via AMPK/mTOR signaling pathways and autophagy by promoting Nrf2.

Bingyu Shen, Yeling Wang, Jiaqi Cheng, Yi Peng, Qiaoling Zhang, Zheng Li, Lilei Zhao, Xuming Deng, Haihua Feng.

   BACKGROUND: NAFLD is a liver disease that is caused by liver damage or extreme lipid deposition but not alcohol. Nrf2 could mediate resistance to oxidative stress injury. Autophagy can degrade metabolic waste and accumulated toxic endogenous substances. Pterostilbene (PTE) is an active compound extracted from blueberry, and grape, that exhibits many biological effects, such as antiinflammation and antitumor.
PURPOSE: This study provides a mechanism of PTE affecting on oxidative stress and autophagy in NAFLD mice. Tyloxapol, oil acid (OA) and palmitic acid (PA) were used to induce lipid accumulation in mice and HepG2 cells.
METHODS: Western blotting, CRISPR/Cas 9 and other molecular biological approaches were applied to explore the mechanisms of PTE effected on NAFLD.
RESULTS: PTE pretreatment effectively reduced the lipid accumulation in OA and PA induced HepG2 cells and tyloxapol induced mice, and significantly promoted the expression of nNrf2, PPAR-α and HO-1, and AMPK activity, but inhibited the expression of mTORC 1 and SREBP-1c. PTE activated phosphatidylinositide 3-kinase (PI3K) and proteins in the autophagy-related gene (ATG) family, and promoted the transformation of LC3Ⅰ to LC3Ⅱ which indicated the activation of autophagy, however, these effects were abolished after Nrf2 knockout.
CONCLUSION: PTE effectively alleviated oxidative stress damage induced by excessive lipid accumulation in hepatocytes, thus promoting the metabolism and decomposition of fatty acids to improve NAFLD.

Keywords:  Autophagy; NAFLD; Nrf2; Oxidative stress; Pterostilbene

DOI:  https://doi.org/10.1016/j.phymed.2022.154561
Cytokine. 2023 Jan 09. pii: S1043-4666(22)00329-5. [Epub ahead of print]163 156120

Qinling liquid ameliorates renal immune inflammatory damage via activating autophagy through AMPK/Stat3 pathway in uric acid nephropathy.

Jie Wang, Xiangwei Bu, Xinping Qiu, Xiuyuan Zhang, Jianhua Gui, Honghong Zhang, Yun Wang, Chen Wang, Fengxian Meng.

   BACKGROUND: Excessive deposition of uric acid (UA) is one of the risk factors for kidney damage. Qinling liquid (QL) has a certain therapeutic effect on uric acid nephropathy (UAN), but its regulation mechanism is still unclear.
METHODS: UAN rat models and UA induced rat renal tubular epithelial cells (NRK-52E) were constructed to evaluate the functional roles of QL. We firstly evaluated the kidney function and the degree of kidney damage in rats after QL treatment. Then, effects of QL on autophagy and NLRP3 inflammasome activation were assessed. Moreover, the regulation of QL in AMPK and Stat3 phosphorylation levels and the relationship among autophagy, AMPK/Stat3 pathway and NLRP3 inflammasomes were determined.
RESULTS: QL could alleviate the inflammatory damage in UAN rats and promote the activation of autophagy. In addition, QL suppressed UA-induced activation of NLRP3 inflammasomes in rat renal tubular epithelial cells, which was partially reversed by autophagy inhibitor. Further, AMPK/Stat3 axis-mediated autophagy participated in the regulation of UA-induced NLRP3 inflammasome activation in NRK-52E cells. Finally, we confirmed that inhibiting AMPK/Stat3 pathway partly deteriorated the ameliorating effect of QL on renal immune inflammatory injury in UAN rats.
CONCLUSION: Through in vivo and in vitro experiments, we found that QL promotes autophagy by activating the AMPK/Stat3 pathway, thereby improving renal immune inflammatory injury in UAN.

Keywords:  AMPK/Stat3 pathway; Autophagy; Qinling liquid; Uric acid nephropathy

DOI:  https://doi.org/10.1016/j.cyto.2022.156120
Chem Biol Interact. 2023 Jan 07. pii: S0009-2797(23)00014-5. [Epub ahead of print] 110347

Xanthosine, a purine glycoside mediates hepatic glucose homeostasis through inhibition of gluconeogenesis and activation of glycogenesis via regulating the AMPK/ FoxO1/AKT/GSK3β signaling cascade.

Semim Akhtar Ahmed, Pranamika Sarma, Sagar Ramrao Barge, Deepsikha Swargiary, Gurumayum Shalini Devi, Jagat Chandra Borah.

  Type 2 Diabetes Mellitus (T2DM) is characterized by hepatic insulin resistance, which results in increased glucose production and reduced glycogen storage in the liver. There is no previous study in the literature that has explored the role of Xanthosine in hepatic insulin resistance. Moreover, mechanistic explanation for the beneficial effects of Xanthosine in lowering glucose production in diabetes is yet to be determined. This study for the first time investigated the beneficial effects of Tribulus terrestris (TT) and its active constituent, Xanthosine on gluconeogenesis and glycogenesis in Free Fatty Acid (FFA)-induced CC1 hepatocytes and streptozotocin (STZ)-induced Wistar rats. Xanthosine enhanced glucose uptake and decreased glucose production through phosphorylation of AMP-activated protein kinase (AMPK) and forkhead box transcription factor O1 (FoxO1), and downregulation of two rate limiting enzymes of gluconeogenesis, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) expression in FFA-induced CC1 cells. Xanthosine also prevented FFA-induced decreases in the phosphorylation of AKT/Protein kinase B, glycogen synthase kinase-3β (GSK3β), and increased glycogen synthase (GS) phosphorylation to increase the glycogen content in the hepatocytes. Moreover, in STZ-induced diabetic rats, oral administration of TT n-butanol fraction (TTBF) enriched with compound Xanthosine (10, 50 & 100 mg/kg body weight) improved insulin sensitivity, reduced fasting blood glucose levels, improved glucose homeostasis by reducing gluconeogenesis via AMPK/FoxO1-mediated PEPCK and G6Pase down-regulation and increasing glycogenesis via AKT/GSK3β-mediated GS activation. Overall, Xanthosine may be developed further for treating insulin resistance and hyperglycemia in T2DM.

Keywords:  AKT; AMPK; Gluconeogenesis; Glycogenesis; Tribulus terrestris; Type 2 diabetes; Xanthosine

DOI:  https://doi.org/10.1016/j.cbi.2023.110347
Front Pharmacol. 2022 ;13 1077249

Red ginseng extract improves skeletal muscle energy metabolism and mitochondrial function in chronic fatigue mice.

Haijing Zhang, Chunhui Zhao, Jinli Hou, Ping Su, Yifei Yang, Bing Xia, Xiaoang Zhao, Rong He, Lifang Wang, Chunyu Cao, Ting Liu, Jixiang Tian.

  Background: Skeletal muscles are organs with high energy requirements, especially during vigorous exercise. Adequate mitochondrial function is essential to meet the high energy needs of skeletal muscle cells. Recent studies have reported that red ginseng can significantly improve chronic fatigue; however, the specific mechanism of action is still not clear. Methods: A chronic fatigue syndrome mouse model was developed using C57BL/6J mice through long-term compound stimulation of stress factors. Following this, the animals were orally administered 200, 400, or 600 mg/kg red ginseng extracts for 28 days. Skeletal muscle lactate acid, serum lactate dehydrogenase, urea concentrations, ATP level, mitochondrial membrane potential, activities of Na+-K+-ATPase and cytochrome c oxidase were determined using assay kits or an automatic biochemical analyser detection system. Skeletal muscle mitochondria morphology was observed using electron microscopy and the expression of p-AMPK, PGC-1α, ACO2 and complex I in skeletal muscle protein was determined by western blotting. Results: Oral administration of 400 or 600 mg/kg red ginseng extract in mice with chronic fatigue reduced lactic acid, lactate dehydrogenase and urea, rescued the density and morphology of skeletal muscle mitochondria, increased the activities of Na+-K+-ATPase and cytochrome c oxidase, and activated the AMPK/PGC-1α cascade pathway, resulting in improved skeletal muscle mitochondrial function by restoring ATP level, mitochondrial membrane potential, complex I and mitochondrial biogenesis. Conclusion: The anti-fatigue effects of red ginseng are partly related to its potent mitochondrial improving activity, including decreasing mitochondrial swelling and mitochondrial membrane permeability, increasing mitochondrial biogenesis, thus ameliorating mitochondrial dysfunction.

Keywords:  AMPK; chronic fatigue; energy metabolism; mitocchondrial dysfunction; red ginseng

DOI:  https://doi.org/10.3389/fphar.2022.1077249
Mol Cell Proteomics. 2023 Jan 05. pii: S1535-9476(23)00003-8. [Epub ahead of print] 100494

Quantification and Proteomic Characterization of β-hydroxybutyrylation Modification in the Hearts of AMPKα2 Knockout Mice.

Wen-Jing Ding, Xue-Hui Li, Cong-Min Tang, Xue-Chun Yang, Yan Sun, Yi-Ping Song, Ming-Ying Ling, Rong Yan, Hai-Qing Gao, Wen-Hua Zhang, Na Yu, Jun-Chao Feng, Zhen Zhang, Yan-Qiu Xing.

  AMP-activated protein kinase alpha 2 (AMPKα2) regulates energy metabolism, protein synthesis, and glucolipid metabolism myocardial cells. Ketone bodies (KB) produced by fatty acid β-oxidation, especially β-hydroxybutyrate (β-OHB), are fatty energy-supplying substances for the heart, brain, and other organs during fasting and long-term exercise. They also regulate metabolic signaling for multiple cellular functions. Lysine β-hydroxybutyrylation (Kbhb) is a β-OHB mediated protein post-translational modification (PTMs). Histone Kbhb has been identified in yeast, mouse, and human cells. However, whether AMPK regulates protein Kbhb is yet unclear. Hence, the present study explored the changes in proteomics and Kbhb modification omics in the hearts of AMPKα2 knockout mice using a comprehensive quantitative proteomic analysis. Based on mass spectrometry (LC-MS/MS) analysis, the number of 1181 Kbhb modified sites in 455 proteins were quantified between AMPKα2 knockout (AK) mice and wild-type (WT) mice; 244 Kbhb sites in 142 proteins decreased or increased after AMPKα2 knockout (fold change >1.5 or <1/1.5, P＜0.05). The regulation of Kbhb sites in 26 key enzymes of fatty acid degradation and tricarboxylic acid cycle (TCA cycle) was noted in AMPKα2 knockout mouse cardiomyocytes. These findings, for the first time, identified proteomic features and Kbhb modification of cardiomyocytes after AMPKα2 knockout, suggesting that AMPKα2 regulates energy metabolism by modifying protein Kbhb.

Keywords:  AMPKα2; TCA cycle; fatty acid degradation; heart; proteomics; β-hydroxybutyrylation

DOI:  https://doi.org/10.1016/j.mcpro.2023.100494
Neoplasma. 2023 Jan 09. pii: 220711N705. [Epub ahead of print]

Sestrin2 mediates FOXM1 expression to block the EMT process in non-small cell lung cancer through the AMPK/YAP pathway.

Yuan-Li Wang, Qi Wang, Hua Li, Cai-Chun Wang, Yan-Lin Ma, Zhong Han.

Non-small cell lung cancer (NSCLC) is characterized by high incidence and mortality, severely threatening human health. The infinite growth and metastasis of NSCLC cells result in a poor prognosis. Therefore, our study was to investigate the mechanism of Sestrin2 on the epithelial-mesenchymal transition (EMT) process of NSCLC cells. Human embryonic lung fibroblasts, NSCLC cell lines, and nude mice were experimental subjects in this study. qRT-PCR and western blot were performed to evaluate the mRNA and protein expression of genes. CCK-8 and EdU assay were conducted to detect cell proliferation. The scratch test and Transwell assay were applied to examine cell migration and invasion. The bioinformatics analysis and Co-IP assay were employed to predict and consolidate the interaction between YAP and TEAD. We found the expression of Sestrin2 was declined but the expression of YAP was elevated in NSCLC cells. Sestrin2 sufficiency or YAP silencing could effectively impair cell growth and metastasis. Mechanistically, YAP interacted with TEAD to enhance FOXM1 expression. Additionally, the elevation of FOXM1 abolished the inhibitory influences of Sestrin2 sufficiency on NSCLC cell growth, invasion, and EMT process. Eventually, Sestrin2 elevation attenuated tumor growth in mice via modulation of the AMPK/YAP/FOXM1 axis, which was reversed by FOXM1 overexpression. Our consequences suggested Sestrin2 could inhibit the activation of YAP via prompting AMPK phosphorylation and then suppress FOXM1 expression through the interplay between YAP and TEAD to impair the capacities of NSCLC cell proliferation, migration, invasion, and EMT. This study provided a novel mechanism of Sestrin2 in NSCLC.

DOI: https://doi.org/10.4149/neo_2022_220711N705