bims-amsmem 2023-06-25 papers

bims-amsmem

Biomed News

on AMPK signaling mechanism in energy metabolism

Issue of 2023–06–25
nine papers selected by
Dipsikha Biswas, Københavns Universitet

Metformin inhibits methylglyoxal-induced retinal pigment epithelial cell death and retinopathy via AMPK-dependent mechanisms: Reversing mitochondrial dysfunction and upregulating glyoxalase 1.
A paradigm shift: AMPK negatively regulates ULK1 activity.
α-amanitin induces autophagy through AMPK-mTOR-ULK1 signaling pathway in hepatocytes.
Cholesterol sulfate inhibits osteoclast differentiation and survival by regulating the AMPK-Sirt1-NF-κB pathway.
Targeted Activation of HNF4α by AMPK Inhibits Apoptosis and Ameliorates Neurological Injury Caused by Cardiac Arrest in Rats.
Metformin Disrupts Signaling and Metabolism in Fetal Hepatocytes.
GLUT4 dynamic subcellular localization is controlled by AMP kinase activation as revealed by proximal proteome mapping in human muscle cells.
CaMKK2 alleviates myocardial ischemia/reperfusion injury by inhibiting oxidative stress and inflammation via the action on the AMPK-AKT-GSK-3β/Nrf2 signaling cascade.
Activation of AMPKα2 attenuated doxorubicin-induced cardiotoxicity via inhibiting lipid peroxidation associated ferroptosis.

Redox Biol. 2023 Jun 15. pii: S2213-2317(23)00187-8. [Epub ahead of print]64 102786

Metformin inhibits methylglyoxal-induced retinal pigment epithelial cell death and retinopathy via AMPK-dependent mechanisms: Reversing mitochondrial dysfunction and upregulating glyoxalase 1.

Ponarulselvam Sekar, George Hsiao, Shu-Hao Hsu, Duen-Yi Huang, Wan-Wan Lin, Chi-Ming Chan.

  Diabetic retinopathy (DR) is a major cause of blindness in adult, and the accumulation of advanced glycation end products (AGEs) is a major pathologic event in DR. Methylglyoxal (MGO), a highly reactive dicarbonyl compound, is a precursor of AGEs. Although the therapeutic potential of metformin for retinopathy disorders has recently been elucidated, possibly through AMPK activation, it remains unknown how metformin directly affects the MGO-induced stress response in retinal pigment epithelial cells. Therefore, in this study, we compared the effects of metformin and the AMPK activator A769662 on MGO-induced DR in mice, as well as evaluated cytotoxicity, mitochondrial dynamic changes and dysfunction in ARPE-19 cells. We found MGO can induce mitochondrial ROS production and mitochondrial membrane potential loss, but reduce cytosolic ROS level in ARPE-19 cells. Although these effects of MGO can be reversed by both metformin and A769662, we demonstrated that reduction of mitochondrial ROS production rather than restoration of cytosolic ROS level contributes to cell protective effects of metformin and A769662. Moreover, MGO inhibits AMPK activity, reduces LC3II accumulation, and suppresses protein and gene expressions of MFN1, PGC-1α and TFAM, leading to mitochondrial fission, inhibition of mitochondrial biogenesis and autophagy. In contrast, these events of MGO were reversed by metformin in an AMPK-dependent manner as evidenced by the effects of compound C and AMPK silencing. In addition, we observed an AMPK-dependent upregulation of glyoxalase 1, a ubiquitous cellular enzyme that participates in the detoxification of MGO. In intravitreal drug-treated mice, we found that AMPK activators can reverse the MGO-induced cotton wool spots, macular edema and retinal damage. Functional, histological and optical coherence tomography analysis support the protective actions of both agents against MGO-elicited retinal damage. Metformin and A769662 via AMPK activation exert a strong protection against MGO-induced retinal pigment epithelial cell death and retinopathy. Therefore, metformin and AMPK activator can be therapeutic agents for DR.

Keywords:  AMPK; Diabetic retinopathy; Glyoxalase 1; Metformin; Methylglyoxal; Mitochondria; Retinal pigment epithelial cells

DOI:  https://doi.org/10.1016/j.redox.2023.102786
Autophagy. 2023 Jun 20. 1-3

A paradigm shift: AMPK negatively regulates ULK1 activity.

Ji-Man Park, Do-Hyung Kim.

  In glucose-starved cells, macroautophagy (hereafter referred to as autophagy) is considered to serve as an energy-generating process contributing to cell survival. AMPK (adenosine monophosphate-activated protein kinase) is the primary cellular energy sensor that is activated during glucose starvation. According to the current paradigm in the field, AMPK promotes autophagy in response to energy deprivation by binding and phosphorylating ULK1 (UNC-51 like kinase 1), the protein kinase responsible for autophagy initiation. However, conflicting findings have been reported casting doubts about the current established model. In our recent study, we have thoroughly reevaluated the role of AMPK in autophagy. Contrary to the current paradigm, our study revealed that AMPK functions as a negative regulator of ULK1 activity. The study has elucidated the underlying mechanism and demonstrated the significance of the negative role in controlling autophagy and maintaining cellular resilience during energy depletion.Abbreviations: AMPK: adenosine monophosphate-activated protein kinase; ULK1: UNC-51 like kinase 1; MTORC1: mechanistic target of rapamycin complex 1; ATG14: autophagy-related protein 14; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; ATP: adenosine triphosphate; VPS34: vacuolar protein sorting 34; BECN1: Beclin 1; AMPKα: AMPK catalytic subunit α; LKB1: liver kinase B1; PIK3R4: phosphatidylinositol 3-kinase regulatory subunit 4.

Keywords:  AMPK; LKB1; MTORC1; ULK1; energy stress; glucose starvation

DOI:  https://doi.org/10.1080/15548627.2023.2223465
Toxicol Lett. 2023 Jun 15. pii: S0378-4274(23)00204-7. [Epub ahead of print]

α-amanitin induces autophagy through AMPK-mTOR-ULK1 signaling pathway in hepatocytes.

Yue Xu, Shangwen Wang, Chi-Kwan Leung, Hao Chen, Chan Wang, Huijie Zhang, Shuwei Zhang, Yi Tan, Haowei Wang, Lin Miao, Yi Li, Yizhen Huang, Xiaoxing Zhang, Genmeng Yang, Ruilin Zhang, Xiaofeng Zeng.

  Amanitin poisoning is one of the most life-threatening mushroom poisonings. α-Amanitin plays a key role in Amanita phalloides intoxication. α-Amanitin shows toxic effects on the liver. However, the mechanism by which α-amanitin induces liver injury has not been elucidated. Autophagy plays a crucial role in maintaining cellular homeostasis and is closely related to the occurrence of a variety of diseases. Studies have shown that autophagy may play an important role in the process of α-amanitin-induced liver injury. However, the mechanism of α-amanitin-induced autophagy remains unclear. Thus, this study aimed to explore the mechanisms of α-amanitin in inducing hepatotoxicity in Sprague Dawley (SD) rats and the normal human liver cell line L02 cells. The SD rats and L02 cells exposed to α-amanitin were observed to determine whether α-amanitin could induce the autophagy of rat liver and L02 cells. The regulatory relationship between autophagy and the AMPK-mTOR- ULK pathway by exposing the autophagy agonist (rapamycin (RAPA)), autophagy inhibitor (3-methylademine (3-MA)), and AMPK inhibitor (compound C) was also explored. Autophagy-related proteins and AMPK-mTOR-ULK pathway-related proteins were detected using Western blot. The results of the study indicated that exposure to different concentrations of α-amanitin led to morphological changes in liver cells and significantly elevated levels of ALT and AST in the serum of SD rats. Additionally, the expression levels of LC3-II, Beclin-1, ATG5, ATG7, AMPK, p-AMPK, mTOR, p-mTOR, and ULK1 were significantly increased in the rat liver. And we found that L02 cells exposed to 0.5μM α-amanitin for 6h significantly induced autophagy and activated the AMPK-mTOR-ULK1 pathway. Pretreated with RAPA, 3-MA, and compound C for 1h, the expression levels of autophagy-related proteins and AMPK-mTOR-ULK pathway-related proteins significantly changed. Our results indicates that autophagy and the AMPK-mTOR-ULK pathway are involved in the process of α-amanitin-induced liver injury. This study may foster the identification of actionable therapeutic targets for A. phalloides intoxication.

Keywords:  AMPK-mTOR-ULK1 signaling pathway; L02 cells; autophagy; liver injury; α-amanitin

DOI:  https://doi.org/10.1016/j.toxlet.2023.06.004
J Cell Physiol. 2023 Jun 19.

Cholesterol sulfate inhibits osteoclast differentiation and survival by regulating the AMPK-Sirt1-NF-κB pathway.

Jin Ha Park, Jiae Lee, Gong-Rak Lee, Minjeong Kwon, Hye In Lee, Narae Kim, Hee Jin Kim, Mi-Ock Lee, Woojin Jeong.

  Cholesterol sulfate (CS) is an activator of retinoic acid-related orphan receptor α (RORα). CS treatment or RORα overexpression attenuates osteoclastogenesis in a collagen-induced arthritis mouse model. However, the mechanism by which CS and RORα regulate osteoclast differentiation remains largely unknown. Thus, we aimed to investigate the role of CS and RORα in osteoclastogenesis and their underlying mechanism. CS inhibited osteoclast differentiation, but RORα deficiency did not affect osteoclast differentiation and CS-mediated inhibition of osteoclastogenesis. CS enhanced adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and sirtuin1 (Sirt1) activity, leading to nuclear factor-κB (NF-κB) inhibition by decreasing acetylation at Lys310 of p65. The NF-κB inhibition was restored by AMPK inhibitor, but the effects of CS on AMPK and NF-κB were not altered by RORα deficiency. CS also induced osteoclast apoptosis, which may be due to sustained AMPK activation and consequent NF-κB inhibition, and the effects of CS were significantly reversed by interleukin-1β treatment. Collectively, these results indicate that CS inhibits osteoclast differentiation and survival by suppressing NF-κB via the AMPK-Sirt1 axis in a RORα-independent manner. Furthermore, CS protects against bone destruction in lipopolysaccharide- and ovariectomy-mediated bone loss mouse models, suggesting that CS is a useful therapeutic candidate for treating inflammation-induced bone diseases and postmenopausal osteoporosis.

Keywords:  AMPK; NF-κB; Sirt1; cholesterol sulfate; osteoclast

DOI:  https://doi.org/10.1002/jcp.31064
Neurochem Res. 2023 Jun 20.

Targeted Activation of HNF4α by AMPK Inhibits Apoptosis and Ameliorates Neurological Injury Caused by Cardiac Arrest in Rats.

Haohong Zhan, Qiang Zhang, Chenyu Zhang, Jingge Cheng, Yilin Yang, Cong Liu, Shuhao Li, Chuyue Wang, Junqin Yang, Hanmei Ge, Dawang Zhou, Bo Li, Hongyan Wei, Chunlin Hu.

  Previous studies have shown that AMPK plays an important role in cerebral ischemia-reperfusion injury by participating in apoptosis, but the exact mechanism and target of action remains unclear. This study aimed to investigate the protective mechanism of AMPK activation on brain injury secondary to cardiac arrest. HE, Nills and TUNEL assays were used to evaluate neuronal damage and apoptosis. The relationships between AMPK, HNF4α and apoptotic genes were verified by ChIP-seq, dual-luciferase and WB assays. The results showed that AMPK improved the 7-day memory function of rats, and reduced neuronal cell injury and apoptosis in the hippocampal CA1 region after ROSC, while the use of HNF4α inhibitor weakened the protective effect of AMPK. Further research found that AMPK positively regulated the expression of HNF4α, and AMPK could promote the expression of Bcl-2 and inhibit the expression of Bax and Cleaved-Caspase 3. In vitro experiments showed that AMPK ameliorated neuronal injury by inhibiting apoptosis through the activation of HNF4α. Combined with ChIP-seq, JASPAR analysis and Dual-luciferase assay, the binding site of HNF4α to the upstream promoter of Bcl-2 was found. Taken together, AMPK attenuates brain injury after CA by activating HNF4α to target Bcl-2 to inhibit apoptosis.

Keywords:  AICAR; AMPK; Apoptosis; Cardiopulmonary resuscitation; HNF4α; Neuroprotection

DOI:  https://doi.org/10.1007/s11064-023-03957-1
Diabetes. 2023 Jun 22. pii: db230089. [Epub ahead of print]

Metformin Disrupts Signaling and Metabolism in Fetal Hepatocytes.

Karli S Swenson, Dong Wang, Amanda K Jones, Michael J Nash, Rebecca O'Rourke, Diana L Takahashi, Paul Kievit, Jon D Hennebold, Kjersti M Aagaard, Jacob E Friedman, Kenneth L Jones, Paul J Rozance, Laura D Brown, Stephanie R Wesolowski.

Metformin is used by women during pregnancy to manage diabetes and crosses the placenta, yet its effects on the fetus are unclear. We show that the liver is a site of metformin action in fetal sheep and macaques, given relatively abundant OCT1 transporter expression and hepatic uptake following metformin infusion into fetal sheep. To determine the effects of metformin action, we performed studies in primary hepatocytes from fetal sheep, fetal macaques, and juvenile macaques. Metformin increases AMP-activated protein kinase (AMPK) signaling, decreases mammalian target of rapamycin (mTOR) signaling, and decreases glucose production in fetal and juvenile hepatocytes. Metformin also decreases oxygen consumption in fetal hepatocytes. Unique to fetal hepatocytes, metformin activates stress pathways (e.g., increased PGC1A gene expression, NRF-2 protein abundance, and phosphorylation of eIF2α and CREB proteins) alongside perturbations in hepatokine expression (e.g., increased growth/differentiation factor 15 (GDF15) and fibroblast growth factor 21 (FGF21) expression and decreased insulinlike growth factor 2 (IGF2) expression). Similarly, in liver tissue from sheep fetuses infused with metformin in vivo, AMPK phosphorylation, NRF-2 protein, and PGC1A expression are increased. These results demonstrate disruption of signaling and metabolism, induction of stress, and alterations in hepatokine expression in association with metformin exposure in fetal hepatocytes.

DOI: https://doi.org/10.2337/db23-0089
bioRxiv. 2023 Jun 07. pii: 2023.06.06.543897. [Epub ahead of print]

GLUT4 dynamic subcellular localization is controlled by AMP kinase activation as revealed by proximal proteome mapping in human muscle cells.

Anuttoma Ray, Jennifer Wen, Lucie Yammine, Jeff Culver, Jeonifer Garren, Liang Xue, Katherine Hales, Qing Xiang, Morris J Birnbaum, Bei B Zhang, Mara Monetti, T E McGraw.

Regulation of glucose transport into muscle and adipocytes, central for control of whole-body metabolism, is determined by the amount of GLUT4 glucose transporter in the plasma membrane ( PM ). Physiologic signals (activated insulin receptor or AMP kinase [ AMPK ]), acutely increase PM GLUT4 to enhance glucose uptake. Here we show in kinetic studies that intracellular GLUT4 is in equilibrium with the PM in unstimulated cultured human skeletal muscle cells, and that AMPK promotes GLUT4 redistribution to the PM by regulating both exocytosis and endocytosis. AMPK-stimulation of exocytosis requires Rab10 and Rab GTPase activating protein TBC1D4, requirements shared with insulin control of GLUT4 in adipocytes. Using APEX2 proximity mapping, we identify, at high-density and high-resolution, the GLUT4 proximal proteome, revealing GLUT4 traverses both PM proximal and distal compartments in unstimulated muscle cells. These data support intracellular retention of GLUT4 in unstimulated muscle cells by a dynamic mechanism dependent on the rates of internalization and recycling. AMPK promoted GLUT4 translocation to the PM involves redistribution of GLUT4 among the same compartments traversed in unstimulated cells, with a significant redistribution of GLUT4 from the PM distal Trans Golgi Network Golgi compartments. The comprehensive proximal protein mapping provides an integrated, whole cell accounting of GLUT4's localization at a resolution of ∼20 nm, a structural framework for understanding the molecular mechanisms regulating GLUT4 trafficking downstream of different signaling inputs in physiologically relevant cell type and as such, sheds new light on novel key pathways and molecular components as potential therapeutic approaches to modulate muscle glucose uptake.

DOI: https://doi.org/10.1101/2023.06.06.543897
Inflamm Res. 2023 Jun 20.

CaMKK2 alleviates myocardial ischemia/reperfusion injury by inhibiting oxidative stress and inflammation via the action on the AMPK-AKT-GSK-3β/Nrf2 signaling cascade.

Chengliang Li, Jiajia Hao, Huichang Qiu, Hong Xin.

   OBJECTIVE: Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) can regulate numerous biological processes and is implicated in diverse pathological processes. Yet its role in myocardial ischemia/reperfusion (MI/R) injury remains unknown. This project explored the possible functions and mechanisms of CaMKK2 in MI/R injury.
METHODS: A rat model of MI/R in vivo was established using the left anterior descending coronary artery ligation method. Rat cardiomyocytes were exposed to hypoxia/reoxygenation (H/R) in vitro to establish a cell model. Overexpression of CaMKK2 was achieved by infecting recombinant adeno-associated virus or adenovirus expressing CaMKK2. Real-time quantitative PCR, immunoblotting, TTC staining, TUNEL assay, ELISA, oxidative stress detection assays, flow cytometry, and CCK-8 assay were carried out.
RESULTS: A decline in CaMKK2 levels was induced by MI/R in vivo or H/R in vitro. Up-modulation of CaMKK2 in rats ameliorated the cardiac injury evoked by MI/R injury accompanied by suppression of cardiac apoptosis, oxidative stress, and proinflammatory response. Rat cardiomyocytes with CaMKK2 overexpression were also protected from H/R damage by inhibiting apoptosis, oxidative stress, and proinflammatory response. CaMKK2 overexpression led to increased phosphorylation of AMPK, AKT, and GSK-3β, and enhanced activation of Nrf2 under MI/R or H/R conditions. Inhibition of AMPK abolished CaMKK2-mediated Nrf2 activation and relevant cardioprotective effect. Restraint of Nrf2 also diminished CaMKK2-mediated relevant cardioprotective effect.
CONCLUSIONS: Up-regulation of CaMKK2 provides a therapeutic benefit in the rat model of MI/R injury by boosting the Nrf2 pathway through regulation of AMPK/AKT/GSK-3β, which suggests CaMKK2 as a new molecular target for the treatment of MI/R injury.

Keywords:  CaMKK2; Inflammation; Myocardial ischemia/reperfusion; Nrf2; Oxidative stress

DOI:  https://doi.org/10.1007/s00011-023-01756-6
Free Radic Biol Med. 2023 Jun 17. pii: S0891-5849(23)00486-0. [Epub ahead of print]205 275-290

Activation of AMPKα2 attenuated doxorubicin-induced cardiotoxicity via inhibiting lipid peroxidation associated ferroptosis.

Hai-Han Liao, Wen Ding, Nan Zhang, Zi-Ying Zhou, Zheng Ling, Wen-Jing Li, Si Chen, Qi-Zhu Tang.

  Ferroptosis has been suggested to involve in doxorubicin (DOX)-induced cardiotoxicity. However, the underlying mechanisms and regulatory targets of cardiomyocyte ferroptosis remains to be understood. This study demonstrated that the up-regulation of ferroptosis associated proteins genes were accompanied with the down-regulation of AMPKα2 phosphorylation in DOX treated mouse heart or neonatal rat cardiomyocytes (NRCMs). AMPKα2 knockout (AMPKα2-/-) significantly exacerbated mouse cardiac dysfunction, increased mortality, promoting ferroptosis associated mitochondrial injuries, enhanced ferroptosis associated proteins and genes expression, and lead to accumulation of lactate dehydrogenase (LDH) and malondialdehyde (MDA) in mouse serum and hearts respectively. Ferrostatin-1 administration markedly improved cardiac function, decreased mortality, inhibited mitochondrial injuries and ferroptosis associated proteins and genes expression, and depressed accumulation of LDH and MDA in DOX treated AMPKα2-/- mouse. Moreover, Adeno-associated virus serotype 9 AMPKα2 (AAV9-AMPKα2) or AICAR treatment mediated AMPKα2 activation could significantly improve cardiac function and depress ferroptosis in mouse. AMPKα2 activation or silence could also inhibit or promote ferroptosis associated injuries in DOX treated NRCMs respecitively. Mechanistically, AMPKα2/ACC mediated lipid metabolism has been suggested to involve in regulating DOX-treatment induced ferroptosis other than mTORC1 or autophagy dependent pathway. The metabolomics analysis exhibited that AMPKα2-/- significantly enhanced accumulation of polyunsaturated fatty acids (PFAs), oxidized lipid, and phosphatidylethanolamine (PE). Finally, this study also demonstrated that metformin (MET) treatment could inhibit ferroptosis and improve cardiac function via activating AMPKα2 phosphorylation. The metabolomics analysis exhibited that MET treatment significantly depressed PFAs accumulation in DOX treated mouse hearts. Collectively, this study suggested that AMPKα2 activation might protect against anthracycline chemotherapeutic drugs mediated cardiotoxicity via inhibiting ferroptosis.

Keywords:  AMPKα2; Doxorubicin; Ferroptosis; Heart; Lipid metabolism

DOI:  https://doi.org/10.1016/j.freeradbiomed.2023.06.004