bims-amsmem 2023-07-02 papers

bims-amsmem

Biomed News

on AMPK signaling mechanism in energy metabolism

Issue of 2023–07–02
nine papers selected by
Dipsikha Biswas, Københavns Universitet

CaMKK2 is not involved in contraction-stimulated AMPK activation and glucose uptake in skeletal muscle.
Empagliflozin Attenuates Vascular Calcification in Mice with Chronic Kidney Disease by Regulating the NFR2/HO-1 Anti-Inflammatory Pathway through AMPK Activation.
Metformin protects against pulmonary hypertension-induced right ventricular dysfunction in an age- and sex-specific manner independent of cardiac AMPK.
Autotaxin promotes the degradation of the mucus layer by inhibiting autophagy in mouse colitis.
Metabolic orchestration of cell death by AMPK-mediated phosphorylation of RIPK1.
Chrysin 7-O-β-D-glucopyranoside increases hepatic low-density lipoprotein receptor expression through AMP-activated protein kinase activation.
Sex and age differences in AMPK phosphorylation, mitochondrial homeostasis, and inflammation in hearts from inflammatory cardiomyopathy patients.
Peripheral and Central Impact of Methionine Source and Level on Growth Performance, Circulating Methionine Levels and Metabolism in Broiler Chickens.
Isoflurane Enhances Autophagy by Activating AMPK/ULK1, Inhibits NLRP3, and Reduces Cognitive Impairment After Cerebral Ischemia-Reperfusion Injury in Rats.

Mol Metab. 2023 Jun 26. pii: S2212-8778(23)00095-9. [Epub ahead of print] 101761

CaMKK2 is not involved in contraction-stimulated AMPK activation and glucose uptake in skeletal muscle.

Florentina Negoita, Alex B Addinsall, Kristina Hellberg, Conchita Fraguas Bringas, Paul S Hafen, Tyler J Sermersheim, Marianne Agerholm, Christopher T A Lewis, Danial Ahwazi, Naomi X Y Ling, Jeppe K Larsen, Atul S Deshmukh, Mohammad A Hossain, Jonathan S Oakhill, Julien Ochala, Jeffrey J Brault, Uma Sankar, David H Drewry, John W Scott, Carol A Witczak, Kei Sakamoto.

   OBJECTIVE: The AMP-activated protein kinase (AMPK) gets activated in response to energetic stress such as contractions and plays a vital role in regulating various metabolic processes such as insulin-independent glucose uptake in skeletal muscle. The main upstream kinase that activates AMPK through phosphorylation of α-AMPK Thr172 in skeletal muscle is LKB1, however some studies have suggested that Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) acts as an alternative kinase to activate AMPK. We aimed to establish whether CaMKK2 is involved in activation of AMPK and promotion of glucose uptake following contractions in skeletal muscle.
METHODS: A recently developed CaMKK2 inhibitor (SGC-CAMKK2-1) alongside a structurally related but inactive compound (SGC-CAMKK2-1N), as well as CaMKK2 knock-out (KO) mice were used. In vitro kinase inhibition selectivity and efficacy assays, as well as cellular inhibition efficacy analyses of CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) were performed. Phosphorylation and activity of AMPK following contractions (ex vivo) in mouse skeletal muscles treated with/without CaMKK inhibitors or isolated from wild-type (WT)/CaMKK2 KO mice were assessed. Camkk2 mRNA in mouse tissues was measured by qPCR. CaMKK2 protein expression was assessed by immunoblotting with or without prior enrichment of calmodulin-binding proteins from skeletal muscle extracts, as well as by mass spectrometry-based proteomics of mouse skeletal muscle and C2C12 myotubes.
RESULTS: STO-609 and SGC-CAMKK2-1 were equally potent and effective in inhibiting CaMKK2 in cell-free and cell-based assays, but SGC-CAMKK2-1 was much more selective. Contraction-stimulated phosphorylation and activation of AMPK were not affected with CaMKK inhibitors or in CaMKK2 null muscles. Contraction-stimulated glucose uptake was comparable between WT and CaMKK2 KO muscle. Both CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) and the inactive compound (SGC-CAMKK2-1N) significantly inhibited contraction-stimulated glucose uptake. SGC-CAMKK2-1 also inhibited glucose uptake induced by a pharmacological AMPK activator or insulin. Relatively low levels of Camkk2 mRNA were detected in mouse skeletal muscle, but neither CaMKK2 protein nor its derived peptides were detectable in mouse skeletal muscle tissue.
CONCLUSIONS: We demonstrate that pharmacological inhibition or genetic loss of CaMKK2 does not affect contraction-stimulated AMPK phosphorylation and activation, as well as glucose uptake in skeletal muscle. Previously observed inhibitory effect of STO-609 on AMPK activity and glucose uptake is likely due to off-target effects. CaMKK2 protein is either absent from adult murine skeletal muscle or below the detection limit of currently available methods.

Keywords:  AMP-activated protein kinase; Ca(2+)/calmodulin dependent protein kinase kinase 2; SGC-CAMKK2-1; STO-609; glucose uptake

DOI:  https://doi.org/10.1016/j.molmet.2023.101761
Int J Mol Sci. 2023 Jun 12. pii: 10016. [Epub ahead of print]24(12):

Empagliflozin Attenuates Vascular Calcification in Mice with Chronic Kidney Disease by Regulating the NFR2/HO-1 Anti-Inflammatory Pathway through AMPK Activation.

Chia-Wen Lu, Chung-Jen Lee, Yi-Jen Hsieh, Bang-Gee Hsu.

  Vascular calcification (VC) is associated with increased cardiovascular risks in patients with chronic kidney disease (CKD). Sodium-glucose cotransporter 2 inhibitors, such as empagliflozin, can improve cardiovascular and renal outcomes. We assessed the expression of Runt-related transcription factor 2 (Runx2), interleukin (IL)-1β, IL-6, AMP-activated protein kinase (AMPK), nuclear factor erythroid-2-related factor (Nrf2), and heme oxygenase 1 (HO-1) in inorganic phosphate-induced VC in mouse vascular smooth muscle cells (VSMCs) to investigate the mechanisms underlying empagliflozin's therapeutic effects. We evaluated biochemical parameters, mean artery pressure (MAP), pulse wave velocity (PWV), transcutaneous glomerular filtration rate (GFR), and histology in an in vivo mouse model with VC induced by an oral high-phosphorus diet following a 5/6 nephrectomy in ApoE-/- mice. Compared to the control group, empagliflozin-treated mice showed significant reductions in blood glucose, MAP, PWV, and calcification, as well as increased calcium and GFR levels. Empagliflozin inhibited osteogenic trans-differentiation by decreasing inflammatory cytokine expression and increasing AMPK, Nrf2, and HO-1 levels. Empagliflozin mitigates high phosphate-induced calcification in mouse VSMCs through the Nrf2/HO-1 anti-inflammatory pathway by activating AMPK. Animal experiments suggested that empagliflozin reduces VC in CKD ApoE-/- mice on a high-phosphate diet.

Keywords:  AMP-activated protein kinase; chronic kidney disease; empagliflozin; heme oxygenase 1; nuclear factor erythroid-2-related factor; vascular calcification

DOI:  https://doi.org/10.3390/ijms241210016
Am J Physiol Heart Circ Physiol. 2023 Jun 30.

Metformin protects against pulmonary hypertension-induced right ventricular dysfunction in an age- and sex-specific manner independent of cardiac AMPK.

Benjamin D McNair, Sydney M Polson, Samantha K Shorthill, Aykhan Yusifov, Lori A Walker, Mary C M Weiser-Evans, Elizabeth J Kovacs, Danielle R Bruns.

  Right ventricular (RV) function is the strongest predictor of survival in age-related heart failure as well as other clinical contexts in which aging populations suffer significant morbidity and mortality. However, despite the significance of maintaining RV function with age and disease, mechanisms of RV failure remain poorly understood and no RV-directed therapies exist. The anti-diabetic drug and AMP-activated protein kinase (AMPK) activator metformin protects against left ventricular dysfunction, suggesting cardioprotective properties may translate to the RV. Here, we aimed to understand the impact of advanced age on pulmonary hypertension induced right ventricular dysfunction. We further aimed to test whether metformin is cardioprotective in the RV and if the protection afforded by metformin requires cardiac AMPK. We used a murine model of PH by exposing adult (4-6 months) and aged (18 months) male and female mice to hypobaric hypoxia (HH) for 4 weeks. Cardiopulmonary remodeling was exacerbated in aged mice compared to adult as evidenced by elevated RV weight and impaired RV systolic function. Metformin attenuated HH induced RV dysfunction but only in adult male mice. Metformin still protected the adult male RV even in the absence of cardiac AMPK. Together, we suggest that aging exacerbates PH-induced RV remodeling and that metformin may represent a therapeutic option for this disease in a sex and age-dependent, but in an AMPK independent manner. Ongoing efforts are aimed at elucidating the molecular basis for RV remodeling as well as delineating the mechanisms of cardioprotection provided by metformin in the absence of cardiac AMPK.

Keywords:  AMPK; aging; metformin; right ventricle; sex difference

DOI:  https://doi.org/10.1152/ajpheart.00124.2023
Mol Immunol. 2023 Jun 23. pii: S0161-5890(23)00123-2. [Epub ahead of print]160 44-54

Autotaxin promotes the degradation of the mucus layer by inhibiting autophagy in mouse colitis.

Xiaoyan Chen, Hui Zhang, Xiaojiang Zhou, Yunwu Wang, Wenjie Shi.

  Autotaxin (ATX or ENPP2) is an autocrine enzyme associated with the metabolism of various phospholipids. ATX has recently been identified as a regulatory factor in immune-related and inflammation-associated diseases, such as inflammatory bowel disease, but the exact mechanism is unclear. Here, we treated mice with recombinant ATX protein or an ATX inhibitor to investigate the effect of ATX on colitis in mice and the underlying mechanism. In a mouse model of colitis, ATX expression was increased, autophagy was impaired, and the mucus barrier was disrupted. Recombinant ATX protein promoted intestinal inflammation, inhibited autophagy, and disrupted the mucus barrier, while an ATX inhibitor had the opposite effect. Next, we treated mice that received ATX with an autophagy activator and an adenosine 5'-monophosphate-activated protein kinase (AMPK) agonist. We observed that autophagy activator and AMPK agonist could repair the mucus barrier and alleviate intestinal inflammation in ATX-treated mice. In vitro, we obtained consistent results. Thus, we concluded that ATX could inhibit autophagy through the AMPK pathway, which consequently disordered the mucus barrier and aggravated intestinal inflammation.

Keywords:  AMPK; Autophagy; Autotaxin; Mucus barrier; Ulcerative colitis

DOI:  https://doi.org/10.1016/j.molimm.2023.06.002
Science. 2023 Jun 30. 380(6652): 1372-1380

Metabolic orchestration of cell death by AMPK-mediated phosphorylation of RIPK1.

Tao Zhang, Daichao Xu, Elijah Trefts, Mingming Lv, Hiroyuki Inuzuka, Guobin Song, Min Liu, Jianlin Lu, Jianping Liu, Chen Chu, Min Wang, Huibing Wang, Huyan Meng, Hui Liu, Yuan Zhuang, Xingxing Xie, Fabin Dang, Dongxian Guan, Yuqin Men, Shuwen Jiang, Cong Jiang, Xiaoming Dai, Jing Liu, Zhen Wang, Peiqiang Yan, Jingchao Wang, Zhenbo Tu, Mrigya Babuta, Emily Erickson, Alissandra L Hillis, Christian C Dibble, John M Asara, Gyongy Szabo, Piotr Sicinski, Ji Miao, Yu-Ru Lee, Lifeng Pan, Reuben J Shaw, Junying Yuan, Wenyi Wei.

Adenosine monophosphate-activated protein kinase (AMPK) activity is stimulated to promote metabolic adaptation upon energy stress. However, sustained metabolic stress may cause cell death. The mechanisms by which AMPK dictates cell death are not fully understood. We report that metabolic stress promoted receptor-interacting protein kinase 1 (RIPK1) activation mediated by TRAIL receptors, whereas AMPK inhibited RIPK1 by phosphorylation at Ser415 to suppress energy stress-induced cell death. Inhibiting pS415-RIPK1 by Ampk deficiency or RIPK1 S415A mutation promoted RIPK1 activation. Furthermore, genetic inactivation of RIPK1 protected against ischemic injury in myeloid Ampkα1-deficient mice. Our studies reveal that AMPK phosphorylation of RIPK1 represents a crucial metabolic checkpoint, which dictates cell fate response to metabolic stress, and highlight a previously unappreciated role for the AMPK-RIPK1 axis in integrating metabolism, cell death, and inflammation.

DOI: https://doi.org/10.1126/science.abn1725
FEBS Open Bio. 2023 Jun 26.

Chrysin 7-O-β-D-glucopyranoside increases hepatic low-density lipoprotein receptor expression through AMP-activated protein kinase activation.

Naoki Kaname, Takahiro Fujimaki, Shotaro Horikoshi, Kaito Fujimura, Manami Kodaka, Shinnosuke Wakamori, Ryo Katsuta, Ken Ishigami, Tsukasa Suzuki, Yuji Yamamoto, Jun Inoue.

Elevated plasma low-density lipoprotein (LDL) cholesterol level is a risk factor for developing atherosclerosis. Increased LDL receptor (LDLR) expression is expected to reduce the risk of atherosclerotic disease, since hepatic LDLR is essential for clearing plasma LDL cholesterol. Here, we screened human LDLR promoter effectors and observed that extracts from peduncles of sweet cherry (Prunus avium) "Sato-Nishiki" induce LDLR gene promoter activity. We used several analytical and chemical methods to show that chrysin 7-O-β-D-glucopyranoside (chrysin-7G) is one of the compounds that stimulate LDLR gene promoter activity in cherry peduncle extracts. Furthermore, synthetic chrysin-7G increased the expression and activity of LDLR. The chrysin-7G-mediated increase in LDLR expression and activity was completely abolished by treatment with an AMP-activated protein kinase (AMPK) inhibitor, compound C. These results indicate that chrysin-7G increases LDLR expression through AMPK activation and may be a useful compound that can be recycled from waste parts of agricultural products.

DOI: https://doi.org/10.1002/2211-5463.13665
Aging Cell. 2023 Jun 26. e13894

Sex and age differences in AMPK phosphorylation, mitochondrial homeostasis, and inflammation in hearts from inflammatory cardiomyopathy patients.

Maria Luisa Barcena, Greta Tonini, Natalie Haritonow, Pavelas Breiter, Hendrik Milting, Istvan Baczko, Ursula Müller-Werdan, Yury Ladilov, Vera Regitz-Zagrosek.

  Linked to exacerbated inflammation, myocarditis is a cardiovascular disease, which may lead to dilated cardiomyopathy. Although sex and age differences in the development of chronic myocarditis have been postulated, underlying cellular mechanisms remain poorly understood. In the current study, we aimed to investigate sex and age differences in mitochondrial homeostasis, inflammation, and cellular senescence. Cardiac tissue samples from younger and older patients with inflammatory dilated cardiomyopathy (DCMI) were used. The expression of Sirt1, phosphorylated AMPK, PGC-1α, Sirt3, acetylated SOD2, catalase, and several mitochondrial genes was analyzed to assess mitochondrial homeostasis. The expression of NF-κB, TLR4, and interleukins was used to examine the inflammatory state in the heart. Finally, several senescence markers and telomere length were investigated. Cardiac AMPK expression and phosphorylation were significantly elevated in male DCMI patients, whereas Sirt1 expression remained unchanged in all groups investigated. AMPK upregulation was accompanied by a preserved expression of all mitochondrial proteins/genes investigated in older male DCMI patients, whereas the expression of TOM40, TIM23, and the mitochondrial oxidative phosphorylation genes was significantly reduced in older female patients. Mitochondrial homeostasis in older male patients was further supported by the reduced acetylation of mitochondrial proteins as indicated by acetylated SOD2. The inflammatory markers NF-κB and TLR4 were downregulated in older male DCMI patients, whereas the expression of IL-18 was increased in older female patients. This was accompanied by progressed senescence in older DCMI hearts. In conclusion, older women experience more dramatic immunometabolic disorders on the cellular level than older men.

Keywords:  AMPK; acetylation; inflammatory dilated cardiomyopathy; mitochondrial biogenesis; senescence

DOI:  https://doi.org/10.1111/acel.13894
Animals (Basel). 2023 Jun 12. pii: 1961. [Epub ahead of print]13(12):

Peripheral and Central Impact of Methionine Source and Level on Growth Performance, Circulating Methionine Levels and Metabolism in Broiler Chickens.

Craig W Maynard, Elizabeth Gilbert, Frances Yan, Mark A Cline, Sami Dridi.

  The present study was designed to evaluate the effects of DL-methionine (DL-Met) 2-hydroxy-4-(methylthio) butanoic acid (HMTBa), or S-(5'-Adenosyl)-L-methionine chloride (SAM), using feeding trial and central administration, on live performance, plasma metabolites, and the expression of feeding-related hypothalamic neuropeptides in broilers raised to a market age (35 d). Final average body weight (BW) and feed conversion ratio (FCR) from the feeding trial exceeded the performance measurements published by the primary breeder. At d35, the MTBHa group had better BW and lower feed intake, which resulted in a better FCR than the DL-Met group at 87 TSAA to lysine. At the molecular levels, the expression of hypothalamic neuropeptide (NPY) and monocarboxylate transporter (MCT) 2 did not differ between all treated groups; however, the mRNA abundances of hypothalamic MCT1 and orexin (ORX) were significantly upregulated in DL-Met- treated groups compared to the control. The ICV administration of SAM significantly reduced feed intake at all tested periods (from 30 to 180 min post injection) compared to the aCSF-treated group (control). The central administration of HMTBa increased feed intake, which reached a significant level only 60 min post administration, compared to the control group. ICV administration of DL-Met slightly increased feed intake compared to the control group, but the difference was not statistically discernable. Quantitative real-time PCR analysis showed that the hypothalamic expression of NPY, cocaine- and amphetamine-regulated transcript, MCT1, and MCT2 was significantly upregulated in the ICV-HMTBa group compared to the aCSF birds. The hypothalamic expression of the mechanistic target of rapamycin (mTOR), AMP-activated protein kinase (AMPKα1), D-amino acid oxidase, and hydroxyacid oxidase was significantly upregulated in DL-Met compared to the control group. The mRNA abundances of ORX were significantly increased in the hypothalamus of both DL-Met and HMTBa groups compared to the aCSF birds; however, mTOR gene expression was significantly downregulated in the SAM compared to the control group. Taken together, these data show, for the first time, that DL-Met and HMTBa have a common downstream (ORX) pathway, but also a differential central pathway, typically NPY-MCT for HMTBa and mTOR-AMPK for methionine.

Keywords:  DL-methionine; HMTBa; ICV injection; diet supplementation; gene expression; hypothalamus

DOI:  https://doi.org/10.3390/ani13121961
J Mol Neurosci. 2023 Jun 30.

Isoflurane Enhances Autophagy by Activating AMPK/ULK1, Inhibits NLRP3, and Reduces Cognitive Impairment After Cerebral Ischemia-Reperfusion Injury in Rats.

Jingwen Zhai, Nian Li, Xu Zhang, Yan Li, Ketao Ma, Ruixue Wang, Xinlei Qin, Jiangwen Yin, Sheng Wang.

  Cerebral ischemic stroke (CIS) has become the second leading cause of death worldwide, which is largely related to cerebral ischemia reperfusion injury (CIRI). Surgical intervention is a reliable treatment for CIS, which predictably causes cerebral reperfusion. Therefore, the choice of anesthetic drugs has important clinical significance. Isoflurane (ISO), one of the most used anesthetics, attenuates cognitive impairment and has brain protective effects. However, the role of isoflurane in regulating autophagy and its regulatory mechanism on inflammation in CIRI are still unclear. The middle cerebral artery occlusion (MCAO) method was used to establish a rat model of CIRI. After 24 h of reperfusion, all rats were evaluated by mNSS scoring and dark avoidance experiment. Western blotting and immunofluorescence were used to examine the expression of key proteins. Compared with the sham group, the MCAO group showed increased neurobehavioral scores and decreased cognitive memory function (P < 0.05). As for the ISO-treated MCAO rats, the neurobehavioral score was significantly decreased, the expression of AMPK, ULK1, Beclin1, and LC3B was significantly increased, and the cognitive and memory functions were also significantly improved (P < 0.05). After inhibition of autophagy pathway or key protein AMPK in autophagy, neurobehavioral scores and protein expression of NLRP3, IL-1β, and IL-18 were significantly increased (P < 0.05). Isoflurane post-treatment may enhance autophagy by activating the AMPK/ULK1 signaling pathway and further inhibit the release of inflammatory factors from NLRP3 inflammasomes, thereby ameliorating neurological function and cognitive impairment and exerting a protective effect on the brain in CIRI rats.

Keywords:  AMPK; Autophagy; Cerebral ischemia–reperfusion injury; Isoflurane; NLRP3; ULK1

DOI:  https://doi.org/10.1007/s12031-023-02135-w