AMP-activated Protein Kinase Target Research Articles

AMPK targets PDZD8 to trigger carbon source shift from glucose to glutamine.

The shift of carbon utilization from primarily glucose to other nutrients is a fundamental metabolic adaptation to cope with decreased blood glucose levels and the consequent decline in glucose oxidation. AMP-activated protein kinase (AMPK) plays crucial roles in this metabolic adaptation. However, the underlying mechanism is not fully understood. Here, we show that PDZ domain containing 8 (PDZD8), which we identify as a new substrate of AMPK activated in low glucose, is required for the low glucose-promoted glutaminolysis. AMPK phosphorylates PDZD8 at threonine 527 (T527) and promotes the interaction of PDZD8 with and activation of glutaminase 1 (GLS1), a rate-limiting enzyme of glutaminolysis. In vivo, the AMPK-PDZD8-GLS1 axis is required for the enhancement of glutaminolysis as tested in the skeletal muscle tissues, which occurs earlier than the increase in fatty acid utilization during fasting. The enhanced glutaminolysis is also observed in macrophages in low glucose or under acute lipopolysaccharide (LPS) treatment. Consistent with a requirement of heightened glutaminolysis, the PDZD8-T527A mutation dampens the secretion of pro-inflammatory cytokines in macrophages in mice treated with LPS. Together, we have revealed an AMPK-PDZD8-GLS1 axis that promotes glutaminolysis ahead of increased fatty acid utilization under glucose shortage.

AMPK Drives both Glycolytic and Oxidative Metabolism in Murine and Human T Cells During Graft-versus-host Disease

Allogeneic T cells reprogram their metabolism during acute graft-versus-host disease (GVHD) in a process involving the cellular energy sensor AMP-activated protein kinase (AMPK). Deletion of AMPK in donor T cells limits GVHD but still preserves homeostatic reconstitution and graft-versus-leukemia (GVL) effects. In the current studies, murine AMPK KO T cells decreased oxidative metabolism at early timepoints post-transplant and lacked a compensatory increase in glycolysis following inhibition of the electron transport chain. Immunoprecipitation using an antibody specific to phosphorylated targets of AMPK determined that AMPK modified interactions of several glycolytic enzymes including aldolase, enolase, pyruvate kinase M (PKM), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and enzyme assays indicated impaired aldolase and GAPDH activity in AMPK KO T cells. Importantly, these changes in glycolysis correlated with both an impaired ability of AMPK KO T cells to produce significant amounts of interferon gamma (IFNγ) upon antigenic re-stimulation and a decrease in the total number of donor CD4 T cells recovered at later time points post-transplant. Human T cells lacking AMPK gave similar results, with glycolytic compensation impaired both in vitro and following expansion in vivo. GVHD results also mirrored those of the murine model, with reduced CD4/CD8 ratios and a significant improvement in disease severity. Together these data highlight a significant role for AMPK in controlling oxidative and glycolytic metabolism in both murine and human T cells and endorse further study of AMPK inhibition as a potential clinical target for future GVHD therapies.

AMP-activated protein kinase activation suppresses leptin expression independently of adipogenesis in primary murine adipocytes.

Adipogenesis, defined as the development of mature adipocytes from stem cell precursors, is vital for the expansion, turnover and health of adipose tissue. Loss of adipogenic potential in adipose stem cells, or impairment of adipogenesis is now recognised as an underlying cause of adipose tissue dysfunction and is associated with metabolic disease. In this study, we sought to determine the role of AMP-activated protein kinase (AMPK), an evolutionarily conserved master regulator of energy homeostasis, in adipogenesis. Primary murine adipose-derived stem cells were treated with a small molecule AMPK activator (BI-9774) during key phases of adipogenesis, to determine the effect of AMPK activation on adipocyte commitment, maturation and function. To determine the contribution of the repression of lipogenesis by AMPK in these processes, we compared the effect of pharmacological inhibition of acetyl-CoA carboxylase (ACC). We show that AMPK activation inhibits adipogenesis in a time- and concentration-dependent manner. Transient AMPK activation during adipogenic commitment leads to a significant, ACC-independent, repression of adipogenic transcription factor expression. Furthermore, we identify a striking, previously unexplored inhibition of leptin gene expression in response to both short-term and chronic AMPK activation irrespective of adipogenesis. These findings reveal that in addition to its effect on adipogenesis, AMPK activation switches off leptin gene expression in primary mouse adipocytes independently of adipogenesis. Our results identify leptin expression as a novel target of AMPK through mechanisms yet to be identified.

Two Prenylated Chalcones, 4-Hydroxyderricin, and Xanthoangelol Prevent Postprandial Hyperglycemia by Promoting GLUT4 Translocation via the LKB1/AMPK Signaling Pathway in Skeletal Muscle Cells.

Stimulation of glucose uptake in the skeletal muscle is crucial for the prevention of postprandial hyperglycemia. Insulin and certain polyphenols enhance glucose uptake through the translocation of glucose transporter 4 (GLUT4) in the skeletal muscle. The previous study reports that prenylated chalcones, 4-hydroxyderricin (4-HD), and xanthoangelol (XAG) promote glucose uptake and GLUT4 translocation in L6 myotubes, but their underlying molecular mechanism remains unclear. This study investigates the mechanism in L6 myotubes and confirms antihyperglycemia by 4-HD and XAG. In L6 myotubes, 4-HD and XAG promote glucose uptake and GLUT4 translocation through the activation of adenosine monophosphate-activated protein kinase (AMPK) and liver kinase B1 (LKB1) signaling pathway without activating phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) and Janus kinases (JAKs)/signal transducers and activators of transcriptions (STATs) pathways. Moreover, Compound C, an AMPK-specific inhibitor, as well as siRNA targeting AMPK and LKB1 completely canceled 4-HD and XAG-increased glucose uptake. Consistently, oral administration of 4-HD and XAG to male ICR mice suppresses acute hyperglycemia in an oral glucose tolerance test. In conclusion, LKB1/AMPK pathway and subsequent GLUT4 translocation in skeletal muscle cells are involved in Ashitaba chalcone-suppressed acute hyperglycemia.

METFORMIN MITIGATES SEPSIS-ASSOCIATED PULMONARY FIBROSIS BY PROMOTING AMPK ACTIVATION AND INHIBITING HIF-1α-INDUCED AEROBIC GLYCOLYSIS.

Recent research has revealed that aerobic glycolysis has a strong correlation with sepsis-associated pulmonary fibrosis (PF). However, at present, the mechanism and pathogenesis remain unclear. We aimed to test the hypothesis that the adenosine monophosphate-activated protein kinase (AMPK) activation and suppression of hypoxia-inducible factor 1α (HIF-1α)-induced aerobic glycolysis play a central role in septic pulmonary fibrogenesis. Cellular experiments demonstrated that lipopolysaccharide increased fibroblast activation through AMPK inactivation, HIF-1α induction, alongside an augmentation of aerobic glycolysis. By contrast, the effects were reversed by AMPK activation or HIF-1α inhibition. In addition, pretreatment with metformin, which is an AMPK activator, suppresses HIF-1α expression and alleviates PF associated with sepsis, which is caused by aerobic glycolysis, in mice. Hypoxia-inducible factor 1α knockdown demonstrated similar protective effects in vivo . Our research implies that targeting AMPK activation and HIF-1α-induced aerobic glycolysis with metformin might be a practical and useful therapeutic alternative for sepsis-associated PF.

AMPK Controls Glycolytic Compensation in Gvhd-Causing T Cells

Background: The heightened metabolic requirements of alloreactive T cells following allogeneic stem cell transplantation represents a promising area for therapeutic intervention. We have shown that during the initiation of acute graft-versus-host-disease (aGVHD), murine T cells upregulate activity of AMP-activated protein kinase (AMPK), a cellular energy sensor that regulates oxidative and mitochondrial metabolism. Further, transfer of allogeneic T cells deficient in AMPK (AMPK KO) increased recipient survival and decreased disease severity while still preserving leukemia clearance. In the current studies, we demonstrate a novel role for AMPK in controlling glycolytic compensation, both in vitro and vivo, and provide a mechanism as to why cells lacking AMPK are unable to perform this vital function. Together, these data highlight the potential clinical utility in targeting metabolic decision-making to curtail T cell-mediated inflammatory responses, with significant therapeutic implications for the prevention and treatment of aGVHD. Methods: AMPK-deficient murine T cells from AMPKa1/a2 dKO C57BL/6J mice were transplanted into allogeneic B6D2F1 recipients and recovered on day 7 post-transplant. To delete AMPK in primary human T cells, T cells were isolated from healthy donors and electroporated with Cas 9 ribonucleoprotein complexes targeting the AMPKa1 locus. Glycolytic compensation was assessed by measuring extracellular acidification rates (ECAR) using the Agilent Seahorse analyzer and a mitochondrial stress kit. Intracellular cytokine expression for IFNgamma was assessed in day 7 murine T cells following re-stimulation with F1 splenocytes in the presence of Brefeldin A. Aldolase A and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) enzymatic assays were run on cell lysates with Biovision-Abcam kits according to manufacturer instructions. To measure GAPDH activity, T cells were positively selected for CD45.1 from day 7 recipient spleens, followed by cell lysis and detection of enzymatic activity. Results: AMPK KO murine T cells recovered on day 7 were unable to mediate a compensatory increase in glycolysis following inhibition of the electron transport chain (Figure 1A), (-6.499±5.156 vs 29.11±7.064, p<0.0001). Human T cells lacking AMPK gave similar results, with glycolytic compensation impaired both in vitro and following human T cell expansion in vivo in a xenogeneic model of GVHD. Immunoprecipitation of proteins from day 7 murine T cells, using an antibody specific to phosphorylated AMPK targets, recovered lower levels of multiple glycolytic enzymes including aldolase, enolase, pyruvate kinase M, and GAPDH (Figure 1B). Functionally, murine T cells lacking AMPK exhibited impaired aldolase activity following anti-CD3/CD28 stimulation and a decrease in GAPDH activity on day 7 post-transplant. Importantly, these changes in glycolysis correlated with an impaired ability of AMPK KO T cells to produce significant amounts of IFNγ upon antigenic re-stimulation with F1 splenocytes. Discussion: Our results demonstrate that in addition to influencing oxidative metabolism, AMPK also plays a role in glycolytic metabolism during GVHD in both murine and human T cells. Further, our data suggest that the more prominent lesion in human T cells post-transplant was in glycolysis , a result supported by the novel observation that a set of glycolysis-related proteins are immunoprecipitated together at much lower rates in the absence of AMPK. These findings suggest a model in which AMPK phosphorylation provides a nidus for important interactions among glycolytic enzymes and that in the absence of AMPK, these interactions are lessened, and glycolysis is consequently reduced. Important to our understanding of GVHD pathogenesis, rates of T cell glycolysis can be directly linked to IFNγ production, where reduced glycolytic activity frees GAPDH to sequester IFNγ transcripts and thereby limit IFNγ protein translation. Altogether, these results document a prominent role for AMPK in controlling T cell glycolytic activity during GVHD development and offer an additional mechanistic explanation for the decreased severity of GVHD seen following transplantation of AMPK KO T cells. Consistently similar results in human xenogeneic T cells provides a further strong rationale for development of AMPK inhibition as a clinically translatable strategy for GVHD prevention and treatment.

Prior Treatment with AICAR Causes the Selective Phosphorylation of mTOR Substrates in C2C12 Cells.

Metabolic stress in skeletal muscle cells causes sustained metabolic changes, but the mechanisms of the prolonged effects are not fully known. In this study, we tested C2C12 cells with the AMP-activated protein kinase (AMPK) stimulator AICAR and measured the changes in the metabolic pathways and signaling kinases. AICAR caused an acute increase in the phosphorylation of the AMPK target ULK1, the mTORC1 substrate S6K, and the mTORC2 target Akt. Intriguingly, prior exposure to AICAR only decreased glucose-6 phosphate dehydrogenase activity when it underwent three-hour recovery after exposure to AICAR in a bicarbonate buffer containing glucose (KHB) instead of Dulbecco's Minimum Essential Medium (DMEM). The phosphorylation of the mTORC1 target S6K was increased after recovery in DMEM but not KHB, although this appeared to be specific to S6K, as the phosphorylation of the mTORC1 target site on ULK1 was not altered when the cells recovered in DMEM. The phosphorylation of mTORC2 target sites was also heterogenous under these conditions, with Akt increasing at serine 473 while other targets (SGK1 and PKCα) were unaffected. The exposure of cells to rapamycin (an mTORC1 inhibitor) and PP242 (an inhibitor of both mTOR complexes) revealed the differential phosphorylation of mTORC2 substrates. Taken together, the data suggest that prior exposure to AICAR causes the selective phosphorylation of mTOR substrates, even after prolonged recovery in a nutrient-replete medium.

Activation of mitogen-activated protein kinase signaling and development of papillary thyroid carcinoma in thyroid-stimulating hormone receptor D633H knockin mice.

Nonautoimmune hyperthyroidism (NAH) is rare and occurs due to a constitutively activating thyroid stimulating hormone receptor (TSHR) mutation. In contrast to other thyroid nodules, no further evaluation for malignancy is recommended for hot thyroid nodules. In the first model for NAH in mice nearly all homozygous mice had developed papillary thyroid cancer by 12 months of age. To further evaluate these mice, whole exome sequencing and phosphoproteome analysis were employed in a further generation of mice to identify any other mutations potentially responsible and to identify the pathways involved in thyroid carcinoma development. Only three genes (Nrg1, Rrs1, Rasal2) were mutated in all mice examined, none of which were known primary drivers of papillary thyroid cancer development. Wild-type and homozygous TSHR D633H knockin mice showed distinct phosphoproteome profiles with an enrichment of altered phosphosites found in ERK/mitogen-activated protein kinase (MAPK) signaling. Most importantly, phosphosites with known downstream effects included BRAF p.S766, which forms an inhibitory site: a decrease of phosphorylation at this site suggests an increase in MEK/ERK pathway activation. The decreased phosphorylation at BRAF p.S766 would suggest decreased AMP-activated protein kinase (AMPK) signaling, which is supported by the decreased phosphorylation of STIM1 p.S257, a downstream AMPK target. The modified phosphoproteome profile of the homozygous mice in combination with human literature suggests a potential signaling pathway from constitutive TSHR signaling and cAMP activation to the activation of ERK/MAPK signaling. This is the first time that a specific mechanism has been identified for a possible involvement of TSH signaling in thyroid carcinoma development.

Review of Patents for Anticancer Agents Targeting Adenosine Monophosphate-Activated Protein Kinase.

Cancer, caused by abnormal and excessive cellular proliferation, can invade and destroy surrounding tissues and organs through the spreading of cancer cells. A general strategy for developing anticancer agents is to identify biomarkers that, if targeted, can produce a robust cytotoxic effect with minimal side effects. Cell-cycle regulators, checkpoint regulatory genes, and apoptosis-related genes are well-known biomarkers that inhibit cancer cell proliferation. Several compounds that target such biomarkers have been patented and more are being developed as novel therapies. Recent additions to this list include anticancer drugs that target signaling pathway proteins, such as 5' adenosine monophosphate-activated protein kinase (AMPK), which plays a vital role in cancer and normal cell metabolism. Herein, we have reviewed recent patents related to AMPK-targeting anticancer drugs and discussed the mechanisms of action of these drugs. We conclude that these recently published patents include several attractive compounds and methods for targeting AMPK. Further research and clinical trials are required to elucidate the comprehensive role of AMPK in cancer cell metabolism, identify its associated signal transduction systems, and develop novel activators that may find applications in cancer therapy. Clinical Trial Registration number: NCT01904123.

Acetyl-coenzyme A acetyltransferase 1 promotes brown adipogenesis by activating the AMPK-PGC1α signaling pathway

Brown adipose tissue (BAT) is thermogenic, expressing high levels of uncoupling protein-1 to convert nutrient energy to heat energy, bypassing ATP synthesis. BAT is a promising therapeutic target for treatment of obesity and type 2 diabetes since it converts fatty acids into heat but mechanisms controlling brown adipogenesis remain unclear. Knockdown of acetyl-Coenzyme A acetyltransferase 1 (ACAT1) in C3H10T1/2 cells suppressed brown adipocyte maturation during the current study and ACAT1 overexpression promoted brown adipocyte maturation. The downstream target of AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor gamma coactivator-1-α (PGC1α), was involved in the action of ACAT1 on brown adipocyte maturation. ACAT1 overexpression enhanced AMPK phosphorylation and promoted PGC1α expression. It is suggested that ACAT1 promotes brown adipocyte maturation by activating the AMPK-PGC1α signaling pathway.

Phosphorylation of AMPKα at Ser485/491 Is Dependent on Muscle Contraction and Not Muscle-Specific IGF-I Overexpression.

Glucose is an important fuel for highly active skeletal muscles. Increased adenosine monophosphate (AMP)/adenosine triphosphate (ATP) ratios during repetitive contractions trigger AMP-activated protein kinase (AMPK), indicated by phosphorylation of AMPKαThr172, which promotes glucose uptake to support heightened energy needs, but it also suppresses anabolic processes. Inhibition of AMPK can occur by protein kinase B (AKT)-mediated phosphorylation of AMPKαSer485/491, releasing its brake on growth. The influence of insulin-like growth factor I (IGF-I) on glucose uptake and its interplay with AMPK activation is not well understood. Thus, the goal of this study was to determine if increased muscle IGF-I altered AMPKα phosphorylation and activity during muscle contraction. Adult male mice harboring the rat Igf1a cDNA regulated by the fast myosin light chain promoter (mIgf1+/+) and wildtype littermates (WT) were used in the study. mIgf1+/+ mice had enhanced glucose tolerance and insulin-stimulated glucose uptake, but similar exercise capacity. Fatiguing stimulations of extensor digitorum longus (EDL) muscles resulted in upregulated AMPKα phosphorylation at both Thr172 and Ser485/491 in WT and mIgf1+/+ muscles. No differences in the phosphorylation response of the downstream AMPK target TBC1D1 were observed, but phosphorylation of raptor was significantly higher only in WT muscles. Further, total raptor content was elevated in mIgf1+/+ muscles. The results show that high muscle IGF-I can enhance glucose uptake under resting conditions; however, in contracting muscle, it is not sufficient to inhibit AMPK activity.

AMPK promotes lysosomal and mitochondrial biogenesis via folliculin:FNIP1.

The AMP-activated protein kinase (AMPK) is known to maintain the integrity of cellular mitochondrial networks by (i) promoting fission, (ii) inhibiting fusion, (iii) promoting recycling of damaged components via mitophagy, (iv) enhancing lysosomal biogenesis to support mitophagy, and (v) promoting biogenesis of new mitochondrial components. While the AMPK targets underlying the first three of these effects are known, a recent paper suggests that direct phosphorylation of the folliculin-interacting protein 1 (FNIP1) by AMPK may be involved in the remaining two.

Modulation of AMPK Significantly Alters Uveal Melanoma Tumor Cell Viability

Introduction: Uveal melanoma (UM) responds poorly to targeted therapies or immune checkpoint inhibitors. Adenosine monophosphate-activated protein kinase (AMPK) is a pivotal serine/threonine protein kinase that coordinates vital processes such as cell growth. Targeting AMPK pathway, which represents a critical mechanism mediating the survival of UM cells, may prove to be a novel treatment strategy for UM. We aimed to demonstrate the effects of AMPK modulation on UM cells. Methods: In silico analyses were performed to compare UM and normal melanocyte cells via Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA). The effects of AMPK modulation on cell viability and proliferation in UM cell lines with different molecular profiles (i.e., 92-1, MP46, OMM2.5, and Mel270) were investigated via XTT cell viability and proliferation assays after treating the cells with varying concentrations of A-769662 (AMPK activator) or dorsomorphin (AMPK inhibitor). Results: KEGG/GSEA studies demonstrated that genes implicated in the AMPK signaling pathway were differentially regulated in UM. Gene sets comprising genes involved in AMPK signaling and genes involved in energy-dependent regulation of mammalian target of rapamycin by liver kinase B1-AMPK were downregulated in UM. We observed gradual decreases in the numbers of viable UM cells as the concentration of A-769662 treatment increased. All UM cells demonstrated statistically significant decreases in cell viability when treated with 200 µm A-769662. Moreover, the effects of AMPK inhibition on UM cells were potent, since low doses of dorsomorphin treatment resulted in significant decreases in viabilities of UM cells. The half maximal inhibitory concentration (IC50) values confirmed the potency of dorsomorphin treatment against UM in vitro. Conclusion: AMPK may act like a friend or a foe in cancer depending on the context. As such, the current study contributes to the literature in determining the effects of therapeutic strategies targeting AMPK in several UM cells. We propose a new perspective in the treatment of UM. Targeting AMPK pathway may open up new avenues in developing novel therapeutic approaches to improve overall survival in UM.

The role of 5′-adenosine monophosphate-activated protein kinase (AMPK) in skeletal muscle atrophy

As a key coordinator of metabolism, AMP-activated protein kinase (AMPK) is vitally involved in skeletal muscle maintenance. AMPK exerts its cellular effects through its function as a serine/threonine protein kinase by regulating many downstream targets and plays important roles in the development and growth of skeletal muscle. AMPK is activated by phosphorylation and exerts its function as a kinase in many processes, including synthesis and degradation of proteins, mitochondrial biogenesis, glucose uptake, and fatty acid and cholesterol metabolism. Skeletal muscle atrophy is a result of various diseases or disorders and is characterized by a decrease in muscle mass. The pathogenesis and therapeutic strategies of skeletal muscle atrophy are still under investigation. In this review, we discuss the role of AMPK in skeletal muscle metabolism and atrophy. We also discuss targeting AMPK for skeletal muscle treatment, including exercise, AMPK activators including 5-amino-4-imidazolecarboxamide ribonucleoside and metformin, and low-level lasers. These studies show the important roles of AMPK in regulating muscle metabolism and function; thus, the treatment of skeletal muscle atrophy needs to take into account the roles of AMPK.

Nutrient-sensing mTORC1 and AMPK pathways in chronic kidney diseases.

Nutrients such as glucose, amino acids and lipids are fundamental sources for the maintenance of essential cellular processes and homeostasis in all organisms. The nutrient-sensing kinases mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) are expressed in many cell types and have key roles in the control of cell growth, proliferation, differentiation, metabolism and survival, ultimately contributing to the physiological development and functions of various organs, including the kidney. Dysregulation of these kinases leads to many human health problems, including cancer, neurodegenerative diseases, metabolic disorders and kidney diseases. In the kidney, physiological levels of mTOR and AMPK activity are required to support kidney cell growth and differentiation and to maintain kidney cell integrity and normal nephron function, including transport of electrolytes, water and glucose. mTOR forms two functional multi-protein kinase complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Hyperactivation of mTORC1 leads to podocyte and tubular cell dysfunction and vulnerability to injury, thereby contributing to the development of chronic kidney diseases, including diabetic kidney disease, obesity-related kidney disease and polycystic kidney disease. Emerging evidence suggests that targeting mTOR and/or AMPK could be an effective therapeutic approach to controlling or preventing these diseases.

AMP-activated protein kinase - a journey from 1 to 100 downstream targets.

A casual decision made one evening in 1976, in a bar near the Biochemistry Department at the University of Dundee, led me to start my personal research journey by following up a paper that suggested that acetyl-CoA carboxylase (ACC) (believed to be a key regulatory enzyme of fatty acid synthesis) was inactivated by phosphorylation by what appeared to be a novel, cyclic AMP-independent protein kinase. This led me to define and name the AMP-activated protein kinase (AMPK) signalling pathway, on which I am still working 46 years later. ACC was the first known downstream target for AMPK, but at least 100 others have now been identified. This article contains some personal reminiscences of that research journey, focussing on: (i) the early days when we were defining the kinase and developing the key tools required to study it; (ii) the late 1990s and early 2000s, an exciting time when we and others were identifying the upstream kinases; (iii) recent times when we have been studying the complex role of AMPK in cancer. The article is published in conjunction with the Sir Philip Randle Lecture of the Biochemical Society, which I gave in September 2022 at the European Workshop on AMPK and AMPK-related kinases in Clydebank, Scotland. During the early years of my research career, Sir Philip acted as a role model, due to his pioneering work on insulin signalling and the regulation of pyruvate dehydrogenase.

FGFR2 Mutation p.Cys342Arg Enhances Mitochondrial Metabolism-Mediated Osteogenesis via FGF/FGFR-AMPK-Erk1/2 Axis in Crouzon Syndrome.

Background: Crouzon syndrome ([OMIM] #123500) caused by FGFR2 mutation is an autosomal dominant syndrome with craniosynostosis, the underlying mechanism of which remains obscure. Methods: First, whole exome sequencing was used to screen the possible pathogenic variant in two sporadic patients with Crouzon syndrome. The investigation of primary and secondary structures as well as the conservation analysis of FGFR2 mutation (p.Cys342Arg) was performed. Then, wild-type and mutant overexpression plasmids were constructed and transfected into pre-osteoblastic murine cell line MC3T3-E1 cells. Osteogenesis and mitochondrial metabolism were analyzed by CCK8, ALP staining and ALP activity, alizarin red staining, qRT-PCR, Western blot, seahorse assays and mitochondrial staining. The siRNA targeting FGFR2 and domain negative FGFR2 were designed for verification. Results: First, FGFR2 mutation (p.Cys342Arg) was detected in two sporadic Chinese Crouzon syndrome patients. FGFR2 p.Cys342Arg promoted the osteogenic differentiation of MC3T3-E1 cells through the upregulation of AMP-activated protein kinase (AMPK)-Erk1/2 signal pathway. Furthermore, FGFR2 p.Cys342Arg enhanced oxidative phosphorylation and converted mitochondrial fusion to the fission of MC3T3-E1, promoting osteogenic differentiation and craniosynostosis in Crouzon syndrome. Additionally, AMPK or Erk1/2 inhibitors delayed the cranial suture closure. Conclusion: FGFR2 mutation p.Cys342Arg promotes osteogenesis by enhancing mitochondrial metabolism-mediated via FGF/FGFR-AMPK-Erk1/2 axis, which indicates the potential of therapy targeting AMPK or Erk1/2 for syndromic craniosynostosis treatment.

AMP-activated protein kinase promotes breast cancer stemness and drug resistance.

ABSTRACTBreast cancer stem cells (BCSCs) are a major cause of therapy resistance and tumour progression. Currently, their regulation is not entirely understood. Previous work from our laboratory demonstrated a context-specific pro-tumorigenic role for AMP-activated protein kinase (AMPK) under anchorage-deprivation and mammosphere formation, which are hallmarks of BCSCs. Therefore, we investigated the role of AMPK in the maintenance of BCSC state/function. AMPK depletion reduces serial sphere formation in vitro and tumour initiation in vivo. Intriguingly, tumour-derived cell analysis using stem cell markers and functional assays revealed that AMPK is required for the maintenance of BCSC populations in vivo. AMPK promotes the expression of stemness genes such as NANOG, SOX2 and BMI1 through the transcriptional upregulation of TWIST via promoter acetylation. Further, AMPK-driven stemness plays a critical role in doxorubicin resistance. Significantly, AMPK activity increased after chemotherapy in patient-derived tumour samples alongside an increase in stemness markers. Importantly, AMPK depletion sensitises mouse tumours to doxorubicin treatment. Our work indicates that targeting of AMPK in conjunction with regular chemotherapy is likely to reduce the stem cell pool and improve chemosensitivity in breast cancers.

Targeting Sirt1, AMPK, Nrf2, CK2, and Soluble Guanylate Cyclase with Nutraceuticals: A Practical Strategy for Preserving Bone Mass.

There is a vast pre-clinical literature suggesting that certain nutraceuticals have the potential to aid the preservation of bone mass in the context of estrogen withdrawal, glucocorticoid treatment, chronic inflammation, or aging. In an effort to bring some logical clarity to these findings, the signaling pathways regulating osteoblast, osteocyte, and osteoclast induction, activity, and survival are briefly reviewed in the present study. The focus is placed on the following factors: the mechanisms that induce and activate the RUNX2 transcription factor, a key driver of osteoblast differentiation and function; the promotion of autophagy and prevention of apoptosis in osteoblasts/osteoclasts; and the induction and activation of NFATc1, which promotes the expression of many proteins required for osteoclast-mediated osteolysis. This analysis suggests that the activation of sirtuin 1 (Sirt1), AMP-activated protein kinase (AMPK), the Nrf2 transcription factor, and soluble guanylate cyclase (sGC) can be expected to aid the maintenance of bone mass, whereas the inhibition of the serine kinase CK2 should also be protective in this regard. Fortuitously, nutraceuticals are available to address each of these targets. Sirt1 activation can be promoted with ferulic acid, N1-methylnicotinamide, melatonin, nicotinamide riboside, glucosamine, and thymoquinone. Berberine, such as the drug metformin, is a clinically useful activator of AMPK. Many agents, including lipoic acid, melatonin, thymoquinone, astaxanthin, and crucifera-derived sulforaphane, can promote Nrf2 activity. Pharmacological doses of biotin can directly stimulate sGC. Additionally, certain flavonols, notably quercetin, can inhibit CK2 in high nanomolar concentrations that may be clinically relevant. Many, though not all, of these agents have shown favorable effects on bone density and structure in rodent models of bone loss. Complex nutraceutical regimens providing a selection of these nutraceuticals in clinically meaningful doses may have an important potential for preserving bone health. Concurrent supplementation with taurine, N-acetylcysteine, vitamins D and K2, and minerals, including magnesium, zinc, and manganese, plus a diet naturally high in potassium, may also be helpful in this regard.

Hepatocyte-specific eNOS deletion impairs exercise-induced adaptations in hepatic mitochondrial function and autophagy.

Endothelial nitric oxide synthase (eNOS) is a potential mediator of exercise-induced hepatic mitochondrial adaptations. Here, male and female hepatocyte-specific eNOS knockout (eNOShep-/- ) and intact hepatic eNOS (eNOSfl/fl ) mice performed voluntary wheel-running exercise (EX) or remained in sedentary cage conditions for 10 weeks. EX resolved the exacerbated hepatic steatosis in eNOShep-/- male mice. Elevated hydrogen peroxide emission (~50% higher in eNOShep-/- vs. eNOSfl/fl mice) was completely ablated with EX. Interestingly, EX increased [1-14 C] palmitate oxidation in eNOSfl/fl male mice, but this was blunted in the eNOShep-/- male mice. eNOShep-/- mice had lower markers of the energy sensors AMP-activated protein kinase (AMPK)/phospho- (p)AMPK and mammalian target of rapamycin (mTOR) and p-mTOR, as well as the autophagy initiators serine/threonine-protein kinase ULK1 and pULK1, compared with eNOSfl/fl mice. Females showed elevated electron transport chain protein content and markers of mitochondrial biogenesis (transcription factor A, mitochondrial, peroxisome proliferator-activated receptor-gamma coactivator 1α). Collectively, this study demonstrates for the first time, to the authors' knowledge, the requirement of eNOS in hepatocytes in the EX-induced increases in hepatic fatty acid oxidation in male mice. Deletion of eNOS in hepatocytes also appears to impair the energy-sensing ability of the cell and inhibit the activation of the autophagy initiating factor ULK1. These data uncover the important and novel role of hepatocyte eNOS in EX-induced hepatic mitochondrial adaptations.