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Objectives: There is renewed interest in targeting the glucose-dependent insulinotropic polypeptide receptor (GIPR) for treatment of obesity and type 2 diabetes. G-protein coupled receptor desensitisation is suggested to reduce the long-term efficacy of glucagon-like-peptide 1 receptor (GLP-1R) agonists and may similarly affect the efficacy of GIPR agonists. We explored the extent of pancreatic GIPR functional desensitisation with sustained agonist exposure., Methods: A long-acting GIPR agonist, GIP108, was used to probe the effect of sustained agonist exposure on cAMP responses in dispersed pancreatic islets using live cell imaging, with rechallenge cAMP responses after prior agonist treatment used to quantify functional desensitisation. Receptor internalisation and β-arrestin-2 activation were investigated in vitro using imaging-based assays. Pancreatic mouse GIPR desensitisation was assessed in vivo via intraperitoneal glucose tolerance testing., Results: GIP108 treatment led to weight loss and improved glucose homeostasis in mice. Prolonged exposure to GIPR agonists produced homologous functional GIPR desensitisation in isolated islets. GIP108 pre-treatment in vivo also reduced the subsequent anti-hyperglycaemic response to GIP re-challenge. GIPR showed minimal agonist-induced internalisation or β-arrestin-2 activation., Conclusions: Although GIP108 chronic treatment improved glucose tolerance, it also resulted in partial desensitisation of the pancreatic islet GIPR. This suggests that ligands with reduced desensitisation tendency might lead to improved in vivo efficacy. Understanding whether pancreatic GIPR desensitisation affects the long-term benefits of GIPR agonists in humans is vital to design effective metabolic pharmacotherapies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Tricia MM Tan reports a relationship with Zihipp Ltd that includes: consulting or advisory. Stephen R Bloom reports a relationship with Zihipp Ltd that includes: employment. James S Minnion reports a relationship with Zihipp Ltd that includes: employment. Ben Jones reports a relationship with Metsera Inc that includes: consulting or advisory. Ben Jones reports a relationship with Eli Lilly and Company that includes: funding grants. Ben Jones reports a relationship with Sun Pharmaceutical Industries Ltd that includes: funding grants. Stephen R Bloom has patent #PCT/GB2024/050504 licensed to IP2IPO INNOVATIONS LIMITED. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Aims: Interleukin 11 (IL11) was initially thought important for platelet production, which led to recombinant IL11 being developed as a drug to treat thrombocytopenia. IL11 was later found to be redundant for haematopoiesis, and its use in patients is associated with unexplained and severe cardiac side effects. Here, we aim to identify, for the first time, direct cardiomyocyte toxicities associated with IL11, which was previously believed cardioprotective., Methods and Results: We injected recombinant mouse lL11 (rmIL11) into mice and studied its molecular effects in the heart using immunoblotting, qRT-PCR, bulk RNA-seq, single nuclei RNA-seq (snRNA-seq), and assay for transposase-accessible chromatin with sequencing (ATAC-seq). The physiological impact of IL11 was assessed by echocardiography in vivo and using cardiomyocyte contractility assays in vitro. To determine the activity of IL11 specifically in cardiomyocytes, we made two cardiomyocyte-specific Il11ra1 knockout (CMKO) mouse models using either AAV9-mediated and Tnnt2-restricted (vCMKO) or Myh6 (m6CMKO) Cre expression and an Il11ra1 floxed mouse strain. In pharmacologic studies, we studied the effects of JAK/STAT inhibition on rmIL11-induced cardiac toxicities. Injection of rmIL11 caused acute and dose-dependent impairment of left ventricular ejection fraction (saline: 62.4% ± 1.9; rmIL11: 32.6% ± 2.9, P < 0.001, n = 5). Following rmIL11 injection, myocardial STAT3 and JNK phosphorylation were increased and bulk RNA-seq revealed up-regulation of pro-inflammatory pathways (TNFα, NFκB, and JAK/STAT) and perturbed calcium handling. snRNA-seq showed rmIL11-induced expression of stress factors (Ankrd1, Ankrd23, Xirp2), activator protein-1 (AP-1) transcription factor genes, and Nppb in the cardiomyocyte compartment. Following rmIL11 injection, ATAC-seq identified the Ankrd1 and Nppb genes and loci enriched for stress-responsive, AP-1 transcription factor binding sites. Cardiomyocyte-specific effects were examined in vCMKO and m6CMKO mice, which were both protected from rmIL11-induced left ventricular impairment and molecular pathobiologies. In mechanistic studies, inhibition of JAK/STAT signalling with either ruxolitinib or tofacitinib prevented rmIL11-induced cardiac dysfunction., Conclusions: Injection of IL11 directly activates IL11RA/JAK/STAT3 in cardiomyocytes to cause acute heart failure. Our data overturn the earlier assumption that IL11 is cardioprotective and explain the serious cardiac side effects associated with IL11 therapy., Competing Interests: Conflict of interest: S.A.C. is a co-inventor on a number of patent applications relating to the role of IL11 in human diseases that include the published patents: WO2017103108, WO2017103108 A2, WO 2018/109174 A2, and WO 2018/109170 A2. S.A.C. is also a co-founder and shareholder of Enleofen Bio PTE LTD and VVB PTE LTD., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

We present a novel, highly customizable glutathione-responsive nanogel (NG) platform for efficient mRNA delivery with precise mRNA payload release control. Optimization of various cationic monomers, including newly synthesized cationic polyarginine, polyhistidine, and acrylated guanidine monomers, allowed fine-tuning of NG properties for mRNA binding. By incorporating a poly(ethylene) glycol-based disulphide crosslinker, we achieved glutathione-triggered mRNA release, enabling targeted intracellular delivery. Our NGs demonstrated superior encapsulation (up to 89.3 %) and loading (10.7 %) efficiencies, with controlled mRNA release kinetics at intracellular glutathione concentrations. NGs outperformed commercial transfection reagents across multiple cell lines, including traditionally difficult-to-transfect lines. We demonstrate the platform's versatility by successfully delivering GFP mRNA, Mango II RNA aptamers, and functionally relevant β2-AMPK mRNA. Furthermore, we used TIRF microscopy to measure exact RNA copy number within the NGs. Notably, mechanistic cellular uptake studies revealed that disulphide-containing NGs exhibit enhanced cellular uptake and endosomal escape, potentially due to interactions with cell surface thiols. This work represents a highly tuneable, efficient, and biocompatible platform for mRNA delivery with relevance for gene therapy and vaccine development., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2025 The Authors. Published by Elsevier Ltd.)

Aims: Acute hypoglycaemia promotes pro-inflammatory cytokine production, increasing the risk for cardiovascular events in diabetes. AMP-activated protein kinase (AMPK) is regulated by and influences the production of pro-inflammatory cytokines. We sought to examine the mechanistic role of AMPK in low glucose-induced changes in the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF), which is elevated in people with diabetes., Methods: Macrophage cell line Raw264.7 cells, primary macrophage bone marrow-derived macrophages obtained from wild-type mice or AMPK γ1 gain-of-function mice, were used, as were AMPKα1/α2 knockout mouse embryonic fibroblasts (MEFs). Allosteric AMPK activators PF-06409577 and BI-9774 were used in conjunction with inhibitor SBI-0206965. We examined changes in protein phosphorylation/expression using western blotting and protein localisation using immunofluorescence. Metabolic function was assessed using extracellular flux analyses and luciferase-based ATP assay. Cytokine release was quantified by enzyme-linked immunosorbent assay (ELISA). Oxidative stress was detected using a fluorescence-based reactive oxygen species (ROS) assay, and cell viability was examined using flow cytometry., Results: Macrophages exposed to low glucose showed a transient and modest activation of AMPK and a metabolic shift towards increased oxidative phosphorylation. Moreover, low glucose increased oxidative stress and augmented the release of macrophage MIF. However, pharmacological activation of AMPK by PF-06409577 and BI-9774 attenuated low glucose-induced MIF release, with a similar trend noted with genetic activation using AMPKγ1 gain-of-function (D316A) mice, which produced a mild effect on low glucose-induced MIF release. Inhibition of NFĸB signalling diminished MIF release and AMPK activation modestly but significantly reduced low glucose-induced nuclear translocation of NFĸB., Conclusions: Taken together, these data indicate that pharmacological AMPK activation suppresses the release of MIF from macrophages caused by energy stress, suggesting that AMPK activation could be a useful strategy for mitigating hypoglycaemia-induced inflammation., (© 2024 The Author(s). Diabetic Medicine published by John Wiley & Sons Ltd on behalf of Diabetes UK.)

Efficient delivery of sensitive nucleic acid payloads, including mRNA, in Caenorhabditis elegans remains challenging, especially with traditional, labor-intensive transgenesis methods. We addressed these challenges using polymeric nanogels (NGs) as an advanced platform for mRNA delivery in C. elegans . These polymeric delivery vehicles can be engineered to suit desired applications owing to their chemical versatility, resulting from the ability to conjugate multiple functional groups onto the same backbone. Here, we validate the in vivo RNA delivery potential of redox-responsive NGs. The NGs showed up to 72.4 % RNA encapsulation and 6.61 % loading efficiencies and facilitated the controlled release of the mRNA payloads at intracellular concentrations of the reducing agent glutathione, where most of the RNA was released within 24 hours. As a proof of concept, we successfully delivered green fluorescent protein (GFP)-expressing mRNA using NGs in C. elegans for the first time. Physicochemical characterization revealed uniform NG size and charge, and fluorescence microscopy confirmed GFP expression in the gut after 24 hours of treatment. Our findings show NGs' potential as an mRNA delivery system in C. elegans ., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2024 by the authors.)

In eukaryotic cells, AMP-activated protein kinase (AMPK) plays a central role in responding to nutrient limitation by switching-off ATP-consuming (anabolic) pathways and switching-on ATP generating (catabolic) pathways. Over the last 30 years or so, a considerable body of research has been carried out that has provided us with a wealth of knowledge regarding the regulation and role of AMPK. Despite this, there is still much to learn about AMPK and the field remains highly active, with many groups around the world continuing to explore new roles for AMPK, providing insight into its biological function. This review series was inspired by recent AMPK-focused meetings in Scotland (2022) and Australia (2023) and draws on some of the research presented at those meetings., (© 2024 The Author(s).)

Objective: Glucagon has long been proposed as a component of multi-agonist obesity therapeutics due to its ability to induce energy expenditure and cause weight loss. However, chronic glucagon-receptor agonism has been associated with a reduction in circulating amino acids and loss of lean mass. Importantly, it is currently not known whether the metabolic benefits of glucagon can be maintained under contexts that allow the defence of lean mass., Methods: We investigate the metabolic effects of the long-acting glucagon receptor agonist, G108, when administered to obese mice at low-doses, and with dietary protein supplementation., Results: Dietary protein supplementation can only fully defend lean mass at a low dose of G108 that is sub-anorectic and does not reduce fat mass. However, in this context, G108 is still highly effective at improving glucose tolerance and reducing liver fat in obese mice. Mechanistically, liver RNA-Seq analysis reveals that dietary protein supplementation defends anabolic processes in low-dose G108-treated mice, and its effects on treatment-relevant glucose and lipid pathways are preserved., Conclusion: Glucagon-mediated energy expenditure and weight loss may be mechanistically coupled to hypoaminocidemia and lean mass loss. However, our data suggest that glucagon can treat MAFLD at doses which allow full defence of lean mass given sufficient dietary protein intake. Therefore, proportionate glucagon therapy may be safe and effective in targeting hepatocytes and improving in glycaemia and liver fat., Competing Interests: Declaration of competing interest TMMT is a consultant and shareholder in Zihipp/Metsera. JSM and SRB are employees and shareholders in Zihipp/Metsera, which is developing gut hormone analogues for treatment of metabolic disease., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

For healthspan and lifespan, ERK, AMPK and mTORC1 represent critical pathways and inflammation is a centrally important hallmark 1-7 . Here we examined whether IL-11, a pro-inflammatory cytokine of the IL-6 family, has a negative effect on age-associated disease and lifespan. As mice age, IL-11 is upregulated across cell types and tissues to regulate an ERK-AMPK-mTORC1 axis to modulate cellular, tissue- and organismal-level ageing pathologies. Deletion of Il11 or Il11ra1 protects against metabolic decline, multi-morbidity and frailty in old age. Administration of anti-IL-11 to 75-week-old mice for 25 weeks improves metabolism and muscle function, and reduces ageing biomarkers and frailty across sexes. In lifespan studies, genetic deletion of Il11 extended the lives of mice of both sexes, by 24.9% on average. Treatment with anti-IL-11 from 75 weeks of age until death extends the median lifespan of male mice by 22.5% and of female mice by 25%. Together, these results demonstrate a role for the pro-inflammatory factor IL-11 in mammalian healthspan and lifespan. We suggest that anti-IL-11 therapy, which is currently in early-stage clinical trials for fibrotic lung disease, may provide a translational opportunity to determine the effects of IL-11 inhibition on ageing pathologies in older people., (© 2024. The Author(s).)

Objectives: Co-agonists at the glucagon-like peptide-1 and glucagon receptors (GLP1R/GCGR) show promise as treatments for metabolic dysfunction-associated steatotic liver disease (MASLD). Although most co-agonists to date have been heavily GLP1R-biased, glucagon directly acts on the liver to reduce fat content. The aims of this study were to investigate a GCGR-biased co-agonist as treatment for hepatic steatosis in mice., Methods: Mice with diet-induced obesity (DIO) were treated with Dicretin, a GLP1/GCGR co-agonist with high potency at the GCGR, Semaglutide (GLP1R monoagonist) or food restriction over 24 days, such that their weight loss was matched. Hepatic steatosis, glucose tolerance, hepatic transcriptomics, metabolomics and lipidomics at the end of the study were compared with Vehicle-treated mice., Results: Dicretin lead to superior reduction of hepatic lipid content when compared to Semaglutide or equivalent weight loss by calorie restriction. Markers of glucose tolerance and insulin resistance improved in all treatment groups. Hepatic transcriptomic and metabolomic profiling demonstrated many changes that were unique to Dicretin-treated mice. These include some known targets of glucagon signaling and others with as yet unclear physiological significance., Conclusions: Our study supports the development of GCGR-biased GLP1/GCGR co-agonists for treatment of MASLD and related conditions., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: TM-MT, JM and SRB declare that they are shareholders in and consultants for Zihipp Ltd., an Imperial College spin-out company that develops gut hormone analogues for the treatment of obesity and associated metabolic disorders., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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., (© 2024 The Author(s).)

Background: Current strategies to inhibit androgen receptor (AR) are circumvented in castration-resistant prostate cancer (CRPC). Cyclin-dependent kinase 7 (CDK7) promotes AR signalling, in addition to established roles in cell cycle and global transcription, providing a rationale for its therapeutic targeting in CRPC., Methods: The antitumour activity of CT7001, an orally bioavailable CDK7 inhibitor, was investigated across CRPC models in vitro and in xenograft models in vivo. Cell-based assays and transcriptomic analyses of treated xenografts were employed to investigate the mechanisms driving CT7001 activity, alone and in combination with the antiandrogen enzalutamide., Results: CT7001 selectively engages with CDK7 in prostate cancer cells, causing inhibition of proliferation and cell cycle arrest. Activation of p53, induction of apoptosis, and suppression of transcription mediated by full-length and constitutively active AR splice variants contribute to antitumour efficacy in vitro. Oral administration of CT7001 represses growth of CRPC xenografts and significantly augments growth inhibition achieved by enzalutamide. Transcriptome analyses of treated xenografts indicate cell cycle and AR inhibition as the mode of action of CT7001 in vivo., Conclusions: This study supports CDK7 inhibition as a strategy to target deregulated cell proliferation and demonstrates CT7001 is a promising CRPC therapeutic, alone or in combination with AR-targeting compounds., (© 2023. The Author(s).)

Emerging evidence indicates that metabolic dysregulation drives prostate cancer (PCa) progression and metastasis. AMP-activated protein kinase (AMPK) is a master regulator of metabolism, although its role in PCa remains unclear. Here, we show that genetic and pharmacological activation of AMPK provides a protective effect on PCa progression in vivo. We show that AMPK activation induces PGC1α expression, leading to catabolic metabolic reprogramming of PCa cells. This catabolic state is characterized by increased mitochondrial gene expression, increased fatty acid oxidation, decreased lipogenic potential, decreased cell proliferation, and decreased cell invasiveness. Together, these changes inhibit PCa disease progression. Additionally, we identify a gene network involved in cell cycle regulation that is inhibited by AMPK activation. Strikingly, we show a correlation between this gene network and PGC1α gene expression in human PCa. Taken together, our findings support the use of AMPK activators for clinical treatment of PCa to improve patient outcome., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Glucagon analogs show promise as components of next-generation, multi-target, anti-obesity therapeutics. The biology of chronic glucagon treatment, in particular, its ability to induce energy expenditure and weight loss, remains poorly understood. Using a long-acting glucagon analog, G108, we demonstrate that glucagon-mediated body weight loss is intrinsically linked to the hypoaminoacidemia associated with its known amino acid catabolic action. Mechanistic studies reveal an energy-consuming response to low plasma amino acids in G108-treated mice, prevented by dietary amino acid supplementation and mimicked by a rationally designed low amino acid diet. Therefore, low plasma amino acids are a pre-requisite for G108-mediated energy expenditure and weight loss. However, preventing hypoaminoacidemia with additional dietary protein does not affect the ability of G108 to improve glycemia or hepatic steatosis in obese mice. These studies provide a mechanism for glucagon-mediated weight loss and confirm the hepatic glucagon receptor as an attractive molecular target for metabolic disease therapeutics., Competing Interests: Declaration of interests J.S.M., T.M.M.T., and S.R.B. are employees and/or shareholders in Zihipp, Ltd., which is developing gut hormone analogs for treatment of metabolic disease. T.A.L. has a collaboration with Novo Nordisk that is unrelated to the work presented here., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Competing Interests: The author declares that no conflict of interest exists. D.C. holds the position of Editorial Board Member for Life Metabolism, and is blinded from reviewing or making decisions for the manuscript.

Objective: To determine whether glucagon receptor (GCGR) actions are modulated by cellular cholesterol levels., Methods: We determined the effects of experimental cholesterol depletion and loading on glucagon-mediated cAMP production, ligand internalisation and glucose production in human hepatoma cells, mouse and human hepatocytes. GCGR interactions with lipid bilayers were explored using coarse-grained molecular dynamic simulations. Glucagon responsiveness was measured in mice fed a high cholesterol diet with or without simvastatin to modulate hepatocyte cholesterol content., Results: GCGR cAMP signalling was reduced by higher cholesterol levels across different cellular models. Ex vivo glucagon-induced glucose output from mouse hepatocytes was enhanced by simvastatin treatment. Mice fed a high cholesterol diet had increased hepatic cholesterol and a blunted hyperglycaemic response to glucagon, both of which were partially reversed by simvastatin. Simulations identified likely membrane-exposed cholesterol binding sites on the GCGR, including a site where cholesterol is a putative negative allosteric modulator., Conclusions: Our results indicate that cellular cholesterol content influences glucagon sensitivity and indicate a potential molecular basis for this phenomenon. This could be relevant to the pathogenesis of non-alcoholic fatty liver disease, which is associated with both hepatic cholesterol accumulation and glucagon resistance., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

IL11 initiates fibroblast activation but also causes epithelial cell dysfunction. The mechanisms underlying these processes are not known. We report that IL11-stimulated ERK/P90RSK activity causes the phosphorylation of LKB1 at S325 and S428, leading to its inactivation. This inhibits AMPK and activates mTOR across cell types. In stromal cells, IL11-stimulated ERK activity inhibits LKB1/AMPK which is associated with mTOR activation, ⍺SMA expression, and myofibroblast transformation. In hepatocytes and epithelial cells, IL11/ERK activity inhibits LKB1/AMPK leading to mTOR activation, SNAI1 expression, and cell dysfunction. Across cells, IL11-induced phenotypes were inhibited by metformin stimulated AMPK activation. In mice, genetic or pharmacologic manipulation of IL11 activity revealed a critical role of IL11/ERK signaling for LKB1/AMPK inhibition and mTOR activation in fatty liver disease. These data identify the IL11/mTOR axis as a signaling commonality in stromal, epithelial, and cancer cells and reveal a shared IL11-driven mesenchymal program across cell types., Competing Interests: A.A.W. and S.A.C. are co-inventors of the patent applications: US Patent App. 16/865,259 (Treatment and prevention of metabolic diseases), US Patent 11319368 (treatment of hepatotoxicity with IL-11 antibody), and US Patent 11339216 (treatment of kidney injury) . S.A.C is a co-inventor of the patent: WO/2017/103108 (treatment of fibrosis), WO/2018/109174 (IL11 ANTIBODIES), WO/2018/109170 (IL11RA antibodies) and a co-founder and shareholder of Enleofen Bio PTE LTD, a company that developed anti-IL11 therapeutics, which were acquired for further development by Boehringer Ingelheim. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2022 The Author(s).)

Aims/hypothesis: Although targeted in extrapancreatic tissues by several drugs used to treat type 2 diabetes, the role of AMP-activated protein kinase (AMPK) in the control of insulin secretion is still debatable. Previous studies have used pharmacological activators of limited selectivity and specificity, and none has examined in primary pancreatic beta cells the actions of the latest generation of highly potent and specific activators that act via the allosteric drug and metabolite (ADaM) site., Methods: AMPK was activated acutely in islets isolated from C57BL6/J mice, and in an EndoC-βH3 cell line, using three structurally distinct ADaM site activators (991, PF-06409577 and RA089), with varying selectivity for β1- vs β2-containing complexes. Mouse lines expressing a gain-of-function mutation in the γ1 AMPK subunit (D316a) were generated to examine the effects of chronic AMPK stimulation in the whole body, or selectively in the beta cell., Results: Acute (1.5 h) treatment of wild-type mouse islets with 991, PF-06409577 or RA089 robustly stimulated insulin secretion at high glucose concentrations (p<0.01, p<0.05 and p<0.001, respectively), despite a lowering of glucose-induced intracellular free Ca 2+ dynamics in response to 991 (AUC, p<0.05) and to RA089 at the highest dose (25 μmol/l) at 5.59 min (p<0.05). Although abolished in the absence of AMPK, the effects of 991 were observed in the absence of the upstream kinase, liver kinase B1, further implicating 'amplifying' pathways. In marked contrast, chronic activation of AMPK, either globally or selectively in the beta cell, achieved using a gain-of-function mutant, impaired insulin release in vivo (p<0.05 at 15 min following i.p. injection of 3 mmol/l glucose) and in vitro (p<0.01 following incubation of islets with 17 mmol/l glucose), and lowered glucose tolerance (p<0.001)., Conclusions/interpretation: AMPK activation exerts complex, time-dependent effects on insulin secretion. These observations should inform the design and future clinical use of AMPK modulators., (© 2022. The Author(s).)

Neuroblastoma is the most common paediatric solid tumour and prognosis remains poor for high-risk cases despite the use of multimodal treatment. Analysis of public drug sensitivity data showed neuroblastoma lines to be sensitive to indisulam, a molecular glue that selectively targets RNA splicing factor RBM39 for proteosomal degradation via DCAF15-E3-ubiquitin ligase. In neuroblastoma models, indisulam induces rapid loss of RBM39, accumulation of splicing errors and growth inhibition in a DCAF15-dependent manner. Integrative analysis of RNAseq and proteomics data highlight a distinct disruption to cell cycle and metabolism. Metabolic profiling demonstrates metabolome perturbations and mitochondrial dysfunction resulting from indisulam. Complete tumour regression without relapse was observed in both xenograft and the Th-MYCN transgenic model of neuroblastoma after indisulam treatment, with RBM39 loss, RNA splicing and metabolic changes confirmed in vivo. Our data show that dual-targeting of metabolism and RNA splicing with anticancer indisulam is a promising therapeutic approach for high-risk neuroblastoma., (© 2022. The Author(s).)

No approved therapies are available for nonalcoholic steatohepatitis (NASH). Adenosine monophosphate-activated protein kinase (AMPK) is a central regulator of cell metabolism; its activation has been suggested as a therapeutic approach to NASH. Here we aimed to fully characterize the potential for direct AMPK activation in preclinical models and to determine mechanisms that could contribute to efficacy for this disease. A novel small-molecule direct AMPK activator, PXL770, was used. Enzyme activity was measured with recombinant complexes. De novo lipogenesis (DNL) was quantitated in vivo and in mouse and human primary hepatocytes. Metabolic efficacy was assessed in ob/ob and high-fat diet-fed mice. Liver histology, biochemical measures, and immune cell profiling were assessed in diet-induced NASH mice. Direct effects on inflammation and fibrogenesis were assessed using primary mouse and human hepatic stellate cells, mouse adipose tissue explants, and human immune cells. PXL770 directly activated AMPK in vitro and reduced DNL in primary hepatocytes. In rodent models with metabolic syndrome, PXL770 improved glycemia, dyslipidemia, and insulin resistance. In mice with NASH, PXL770 reduced hepatic steatosis, ballooning, inflammation, and fibrogenesis. PXL770 exhibited direct inhibitory effects on pro-inflammatory cytokine production and activation of primary hepatic stellate cells. Conclusion: In rodent models, direct activation of AMPK is sufficient to produce improvements in all core components of NASH and to ameliorate related hyperglycemia, dyslipidemia, and systemic inflammation. Novel properties of direct AMPK activation were also unveiled: improved insulin resistance and direct suppression of inflammation and fibrogenesis. Given effects also documented in human cells (reduced DNL, suppression of inflammation and stellate cell activation), these studies support the potential for direct AMPK activation to effectively treat patients with NASH., (© 2021 Poxel SA. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Objectives: Receptor Activity-Modifying Protein 2 (RAMP2) is a chaperone protein which allosterically binds to and interacts with the glucagon receptor (GCGR). The aims of this study were to investigate the effects of RAMP2 on GCGR trafficking and signalling in the liver, where glucagon (GCG) is important for carbohydrate and lipid metabolism., Methods: Subcellular localisation of GCGR in the presence and absence of RAMP2 was investigated using confocal microscopy, trafficking and radioligand binding assays in human embryonic kidney (HEK293T) and human hepatoma (Huh7) cells. Mouse embryonic fibroblasts (MEFs) lacking the Wiskott-Aldrich Syndrome protein and scar homologue (WASH) complex and the trafficking inhibitor monensin were used to investigate the effect of halted recycling of internalised proteins on GCGR subcellular localisation and signalling in the absence of RAMP2. NanoBiT complementation and cyclic AMP assays were used to study the functional effect of RAMP2 on the recruitment and activation of GCGR signalling mediators. Response to hepatic RAMP2 upregulation in lean and obese adult mice using a bespoke adeno-associated viral vector was also studied., Results: GCGR is predominantly localised at the plasma membrane in the absence of RAMP2 and exhibits remarkably slow internalisation in response to agonist stimulation. Rapid intracellular accumulation of GCG-stimulated GCGR in cells lacking the WASH complex or in the presence of monensin indicates that activated GCGR undergoes continuous cycles of internalisation and recycling, despite apparent GCGR plasma membrane localisation up to 40 min post-stimulation. Co-expression of RAMP2 induces GCGR internalisation both basally and in response to agonist stimulation. The intracellular retention of GCGR in the presence of RAMP2 confers a bias away from β-arrestin-2 recruitment coupled with increased activation of G αs proteins at endosomes. This is associated with increased short-term efficacy for glucagon-stimulated cAMP production, although long-term signalling is dampened by increased receptor lysosomal targeting for degradation. Despite these signalling effects, only a minor disturbance of carbohydrate metabolism was observed in mice with upregulated hepatic RAMP2., Conclusions: By retaining GCGR intracellularly, RAMP2 alters the spatiotemporal pattern of GCGR signalling. Further exploration of the effects of RAMP2 on GCGR in vivo is warranted., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

AMP-activated protein kinase (AMPK) plays a key role in the cellular response to low energy stress and has emerged as an attractive therapeutic target for tackling metabolic diseases. Whilst significant progress has been made regarding the physiological role of AMPK, its function in the kidney remains only partially understood. We use a mouse model expressing a constitutively active mutant of AMPK to investigate the effect of AMPK activation on kidney function in vivo. Kidney morphology and changes in gene and protein expression were monitored and serum and urine markers were measured to assess kidney function in vivo. Global AMPK activation resulted in an early-onset polycystic kidney phenotype, featuring collecting duct cysts and compromised renal function in adult mice. Mechanistically, the cystic kidneys had increased cAMP levels and ERK activation, increased hexokinase I (Hk I) expression, glycogen accumulation and altered expression of proteins associated with autophagy. Kidney tubule-specific activation of AMPK also resulted in a polycystic phenotype, demonstrating that renal tubular AMPK activation caused the cystogenesis. Importantly, human autosomal dominant polycystic kidney disease (ADPKD) kidney sections revealed similar protein localisation patterns to that observed in the murine cystic kidneys. Our findings show that early-onset chronic AMPK activation leads to a polycystic kidney phenotype, suggesting dysregulated AMPK signalling is a contributing factor in cystogenesis., (© 2021 The Author(s).)

In fibroblasts, TGFβ1 stimulates IL11 upregulation that leads to an autocrine loop of IL11-dependent pro-fibrotic protein translation. The signaling pathways downstream of IL11, which acts via IL6ST, are contentious with both STAT3 and ERK implicated. Here we dissect IL11 signaling in fibroblasts and study IL11-dependent protein synthesis pathways in the context of approved anti-fibrotic drug mechanisms of action. We show that IL11-induced ERK activation drives fibrogenesis and while STAT3 phosphorylation (pSTAT3) is also seen, this appears unrelated to fibroblast activation. Ironically, recombinant human IL11, which has been used extensively in mouse experiments to infer STAT3 activity downstream of IL11, increases pSTAT3 in Il11ra1 null mouse fibroblasts. Unexpectedly, inhibition of STAT3 was found to induce severe proteotoxic ER stress, generalized fibroblast dysfunction and cell death. In contrast, inhibition of ERK prevented fibroblast activation in the absence of ER stress. IL11 stimulated an axis of ERK/mTOR/P70RSK protein translation and its selectivity for Collagen 1 synthesis was ascribed to an EPRS-regulated, ribosome stalling mechanism. Surprisingly, the anti-fibrotic drug nintedanib caused dose-dependent ER stress and lesser pSTAT3 expression. Pirfenidone had no effect on ER stress whereas anti-IL11 specifically inhibited the ERK/mTOR axis while reducing ER stress. These studies define the translation-specific signaling pathways downstream of IL11, intersect immune and metabolic signaling and reveal unappreciated effects of nintedanib., Competing Interests: SC and SS are co-inventors of the patent applications: WO/2017/103108 (TREATMENT OF FIBROSIS), WO/2018/109174 (IL11 ANTIBODIES), WO/2018/109170 (IL11RA ANTIBODIES). SC and SS are co-founders and shareholders of Enleofen Bio PTE LTD. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Widjaja, Viswanathan, Jinrui, Singh, Tan, Wei Ting, Lamb, Shekeran, George, Schafer, Carling, Adami and Cook.)

Objective: Skeletal muscle is an attractive target for blood glucose-lowering pharmacological interventions. Oral dosing of small molecule direct pan-activators of AMPK that bind to the allosteric drug and metabolite (ADaM) site, lowers blood glucose through effects in skeletal muscle. The molecular mechanisms responsible for this effect are not described in detail. This study aimed to illuminate the mechanisms by which ADaM-site activators of AMPK increase glucose uptake in skeletal muscle. Further, we investigated the consequence of co-stimulating muscles with two types of AMPK activators i.e., ADaM-site binding small molecules and the prodrug AICAR., Methods: The effect of the ADaM-site binding small molecules (PF739 and 991), AICAR or co-stimulation with PF739 or 991 and AICAR on muscle glucose uptake was investigated ex vivo in m. extensor digitorum longus (EDL) excised from muscle-specific AMPKα1α2 as well as whole-body AMPKγ3-deficient mouse models. In vitro complex-specific AMPK activity was measured by immunoprecipitation and molecular signaling was assessed by western blotting in muscle lysate. To investigate the transferability of these studies, we treated diet-induced obese mice in vivo with PF739 and measured complex-specific AMPK activation in skeletal muscle., Results: Incubation of skeletal muscle with PF739 or 991 increased skeletal muscle glucose uptake in a dose-dependent manner. Co-incubating PF739 or 991 with a maximal dose of AICAR increased glucose uptake to a greater extent than any of the treatments alone. Neither PF739 nor 991 increased AMPKα2β2γ3 activity to the same extent as AICAR, while co-incubation led to potentiated effects on AMPKα2β2γ3 activation. In muscle from AMPKγ3 KO mice, AICAR-stimulated glucose uptake was ablated. In contrast, the effect of PF739 or 991 on glucose uptake was not different between WT and AMPKγ3 KO muscles. In vivo PF739 treatment lowered blood glucose levels and increased muscle AMPKγ1-complex activity 2-fold, while AMPKα2β2γ3 activity was not affected., Conclusions: ADaM-site binding AMPK activators increase glucose uptake independently of AMPKγ3. Co-incubation with PF739 or 991 and AICAR potentiates the effects on muscle glucose uptake and AMPK activation. In vivo, PF739 lowers blood glucose and selectively activates muscle AMPKγ1-complexes. Collectively, this suggests that pharmacological activation of AMPKγ1-containing complexes in skeletal muscle can increase glucose uptake and can lead to blood glucose lowering., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Cell competition is emerging as a quality-control mechanism that eliminates unfit cells in a wide range of settings from development to the adult. However, the nature of the cells normally eliminated by cell competition and what triggers their elimination remains poorly understood. In mice, 35% of epiblast cells are eliminated before gastrulation. Here we show that cells with mitochondrial defects are eliminated by cell competition during early mouse development. Using single-cell transcriptional profiling of eliminated mouse epiblast cells, we identify hallmarks of cell competition and mitochondrial defects. We demonstrate that mitochondrial defects are common to a range of different loser cell types and that manipulating mitochondrial function triggers cell competition. Moreover, we show that in the mouse embryo, cell competition eliminates cells with sequence changes in mt-Rnr1 and mt-Rnr2, and that even non-pathological changes in mitochondrial DNA sequences can induce cell competition. Our results suggest that cell competition is a purifying selection that optimizes mitochondrial performance before gastrulation., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Inflammatory bowel diseases are the most common chronic intestinal inflammatory conditions, and their incidence has shown a dramatic increase in recent decades. Limited efficacy and questionable safety profiles with existing therapies suggest the need for better targeting of therapeutic strategies. Adenosine monophosphate-activated protein kinase (AMPK) is a key regulator of cellular metabolism and has been implicated in intestinal inflammation. Macrophages execute an important role in the generation of intestinal inflammation. Impaired AMPK in macrophages has been shown to be associated with higher production of proinflammatory cytokines; however, the role of macrophage AMPK in intestinal inflammation and the mechanism by which it regulates inflammation remain to be determined. In this study, we investigated the role of AMPK with a specific focus on macrophages in the pathogenesis of intestinal inflammation., Methods: A dextran sodium sulfate-induced colitis model was used to assess the disease activity index, histological scores, macroscopic scores, and myeloperoxidase level. Proinflammatory cytokines such as tumor necrosis factor-α, interleukin-6, and interleukin-1β were measured by enzyme-linked immunosorbent assay. Transient transfection of AMPKβ1 and LC3-II siRNA in RAW 264.7 cells was performed to elucidate the regulation of autophagy by AMPK. The expression of p-AMPK, AMPK, and autophagy markers (eg, LC3-II, p62, Beclin-1, and Atg-12) was analyzed by Western blot., Results: Genetic deletion of AMPKβ1 in macrophages upregulated the production of proinflammatory cytokines, aggravated the severity of dextran sodium sulfate-induced colitis in mice, which was associated with an increased nuclear translocation of nuclear factor-κB, and impaired autophagy both in vitro and in vivo. Notably, the commonly used anti-inflammatory 5-aminosalicylic acid (ie, mesalazine) and sodium salicylate ameliorated dextran sodium sulfate-induced colitis through the activation of macrophage AMPK targeting the β1 subunit., Conclusions: Together, these data suggest that the development of therapeutic agents targeting AMPKβ1 may be effective in the treatment of intestinal inflammatory conditions including inflammatory bowel disease., (© 2020 Crohn’s & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aims: Atherosclerotic vascular disease has an inflammatory pathogenesis. Heme from intraplaque haemorrhage may drive a protective and pro-resolving macrophage M2-like phenotype, Mhem, via AMPK and activating transcription factor 1 (ATF1). The antidiabetic drug metformin may also activate AMPK-dependent signalling. Hypothesis: Metformin systematically induces atheroprotective genes in macrophages via AMPK and ATF1, thereby suppresses atherogenesis., Methods and Results: Normoglycaemic Ldlr-/- hyperlipidaemic mice were treated with oral metformin, which profoundly suppressed atherosclerotic lesion development (P < 5 × 10-11). Bone marrow transplantation from AMPK-deficient mice demonstrated that metformin-related atheroprotection required haematopoietic AMPK [analysis of variance (ANOVA), P < 0.03]. Metformin at a clinically relevant concentration (10 μM) evoked AMPK-dependent and ATF1-dependent increases in Hmox1, Nr1h2 (Lxrb), Abca1, Apoe, Igf1, and Pdgf, increases in several M2-markers and decreases in Nos2, in murine bone marrow macrophages. Similar effects were seen in human blood-derived macrophages, in which metformin-induced protective genes and M2-like genes, suppressible by si-ATF1-mediated knockdown. Microarray analysis comparing metformin with heme in human macrophages indicated that the transcriptomic effects of metformin were related to those of heme, but not identical. Metformin-induced lesional macrophage expression of p-AMPK, p-ATF1, and downstream M2-like protective effects., Conclusion: Metformin activates a conserved AMPK-ATF1-M2-like pathway in mouse and human macrophages, and results in highly suppressed atherogenesis in hyperlipidaemic mice via haematopoietic AMPK., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Failure of neural tube closure during embryonic development can result in anencephaly, one of the most common birth defects in humans. A family with recurrent anencephalic fetuses was investigated to understand its etiology and pathogenesis. Exome sequencing revealed a recessive germline 21-bp in-frame deletion in NUAK2 segregating with the disease. In vitro kinase assays demonstrated that the 7-amino acid truncation in NUAK2, a serine/threonine kinase, completely abrogated its catalytic activity. Patient-derived disease models including neural progenitor cells and cerebral organoids showed that loss of NUAK2 activity led to decreased Hippo signaling via cytoplasmic YAP retention. In neural tube-like structures, endogenous NUAK2 colocalized apically with the actomyosin network, which was disrupted in patient cells, causing impaired nucleokinesis and apical constriction. Our results establish NUAK2 as an indispensable kinase for brain development in humans and suggest that a NUAK2-Hippo signaling axis regulates cytoskeletal processes that govern cell shape during neural tube closure., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Bonnard et al.)

Activation of AMP-activated protein kinase (AMPK) in endothelial cells by vascular endothelial growth factor (VEGF) via the Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) represents a pro-angiogenic pathway, whose regulation and function is incompletely understood. This study investigates whether the VEGF/AMPK pathway is regulated by cAMP-mediated signalling. We show that cAMP elevation in endothelial cells by forskolin, an activator of the adenylate cyclase, and/or 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases, triggers protein kinase A (PKA)-mediated phosphorylation of CaMKK2 (serine residues S495, S511) and AMPK (S487). Phosphorylation of CaMKK2 by PKA led to an inhibition of its activity as measured in CaMKK2 immunoprecipitates of forskolin/IBMX-treated cells. This inhibition was linked to phosphorylation of S495, since it was not seen in cells expressing a non-phosphorylatable CaMKK2 S495C mutant. Phosphorylation of S511 alone in these cells was not able to inhibit CaMKK2 activity. Moreover, phosphorylation of AMPK at S487 was not sufficient to inhibit VEGF-induced AMPK activation in cells, in which PKA-mediated CaMKK2 inhibition was prevented by expression of the CaMKK2 S495C mutant. cAMP elevation in endothelial cells reduced basal and VEGF-induced acetyl-CoA carboxylase (ACC) phosphorylation at S79 even if AMPK was not inhibited. Together, this study reveals a novel regulatory mechanism of VEGF-induced AMPK activation by cAMP/PKA, which may explain, in part, inhibitory effects of PKA on angiogenic sprouting and play a role in balancing pro- and anti-angiogenic mechanisms in order to ensure functional angiogenesis., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Rationale: The efficient resolution of tissue hemorrhage is an important homeostatic function. In human macrophages in vitro, heme activates an AMPK (AMP-activated protein kinase)/ATF1 (activating transcription factor-1) pathway that directs Mhem macrophages through coregulation of HO-1 (heme oxygenase-1; HMOX1 ) and lipid homeostasis genes., Objective: We asked whether this pathway had an in vivo role in mice., Methods and Results: Perifemoral hematomas were used as a model of hematoma resolution. In mouse bone marrow-derived macrophages, heme induced HO-1, lipid regulatory genes including LXR (lipid X receptor), the growth factor IGF1 (insulin-like growth factor-1), and the splenic red pulp macrophage gene Spic . This response was lost in bone marrow-derived macrophages from mice deficient in AMPK ( Prkab1 -/ - ) or ATF1 ( Atf1 -/- ). In vivo, femoral hematomas resolved completely between days 8 and 9 in littermate control mice (n=12), but were still present at day 9 in mice deficient in either AMPK ( Prkab1 -/- ) or ATF1 ( Atf1 -/- ; n=6 each). Residual hematomas were accompanied by increased macrophage infiltration, inflammatory activation and oxidative stress. We also found that fluorescent lipids and a fluorescent iron-analog were trafficked to lipid-laden and iron-laden macrophages respectively. Moreover erythrocyte iron and lipid abnormally colocalized in the same macrophages in Atf1 -/- mice. Therefore, iron-lipid separation was Atf1 -dependent., Conclusions: Taken together, these data demonstrate that both AMPK and ATF1 are required for normal hematoma resolution. Graphic Abstract: An online graphic abstract is available for this article.

Metabolic inflexibility, defined as the inability to respond or adapt to metabolic demand, is now recognised as a driving factor behind many pathologies associated with obesity and the metabolic syndrome. Adipose tissue plays a pivotal role in the ability of an organism to sense, adapt to and counteract environmental changes. It provides a buffer in times of nutrient excess, a fuel reserve during starvation and the ability to resist cold-stress through non-shivering thermogenesis. Recent advances in single-cell RNA sequencing combined with lineage tracing, transcriptomic and proteomic analyses have identified novel adipocyte progenitors that give rise to specialised adipocytes with diverse functions, some of which have the potential to be exploited therapeutically. This review will highlight the common and distinct functions of well-known adipocyte populations with respect to their lineage and plasticity, as well as introducing the most recent members of the adipocyte family and their roles in whole organism energy homeostasis. Finally, this article will outline some of the more preliminary findings from large data sets generated by single-cell transcriptomics of mouse and human adipose tissue and their implications for the field, both for discovery and for therapy., (© 2020 The Author(s).)

Since the discovery of AMP-activated protein kinase (AMPK) as a central regulator of energy homeostasis, many exciting insights into its structure, regulation and physiological roles have been revealed. While exercise, caloric restriction, metformin and many natural products increase AMPK activity and exert a multitude of health benefits, developing direct activators of AMPK to elicit beneficial effects has been challenging. However, in recent years, direct AMPK activators have been identified and tested in preclinical models, and a small number have entered clinical trials. Despite these advances, which disease(s) represent the best indications for therapeutic AMPK activation and the long-term safety of such approaches remain to be established.

: New targets are required for treating prostate cancer, particularly castrate-resistant disease. Previous studies reported that calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) expression is increased in human prostate cancer. Here, we show that Camkk2 deletion or pharmacologic inhibition protects against prostate cancer development in a preclinical mouse model that lacks expression of prostate-specific Pten. In contrast, deletion of AMP-activated protein kinase (Ampk) β1 resulted in earlier onset of adenocarcinoma development. These findings suggest for the first time that Camkk2 and Ampk have opposing effects in prostate cancer progression. Loss of CAMKK2 in vivo or in human prostate cancer cells reduced the expression of two key lipogenic enzymes, acetyl-CoA carboxylase and fatty acid synthase. This reduction was mediated via a posttranscriptional mechanism, potentially involving a decrease in protein translation. Moreover, either deletion of CAMKK2 or activation of AMPK reduced cell growth in human prostate cancer cells by inhibiting de novo lipogenesis. Activation of AMPK in a panel of human prostate cancer cells inhibited cell proliferation, migration, and invasion as well as androgen-receptor signaling. These findings demonstrate that CAMKK2 and AMPK have opposing effects on lipogenesis, providing a potential mechanism for their contrasting effects on prostate cancer progression in vivo . They also suggest that inhibition of CAMKK2 combined with activation of AMPK would offer an efficacious therapeutic strategy in treatment of prostate cancer. SIGNIFICANCE: These findings show that CAMKK2 and its downstream target AMPK have opposing effects on prostate cancer development and raise the possibility of a new combined therapeutic approach that inhibits CAMKK2 and activates AMPK., (©2018 American Association for Cancer Research.)

Mitochondrial reactive oxygen species (ROS) production is a tightly regulated redox signal that transmits information from the organelle to the cell. Other mitochondrial signals, such as ATP, are sensed by enzymes, including the key metabolic sensor and regulator, AMP-activated protein kinase (AMPK). AMPK responds to the cellular ATP/AMP and ATP/ADP ratios by matching mitochondrial ATP production to demand. Previous reports proposed that AMPK activity also responds to ROS, by ROS acting on redox-sensitive cysteine residues (Cys-299/Cys-304) on the AMPK α subunit. This suggests an appealing model in which mitochondria fine-tune AMPK activity by both adenine nucleotide-dependent mechanisms and by redox signals. Here we assessed whether physiological levels of ROS directly alter AMPK activity. To this end we added exogenous hydrogen peroxide (H 2 O 2 ) to cells and utilized the mitochondria-targeted redox cycler MitoParaquat to generate ROS within mitochondria without disrupting oxidative phosphorylation. Mitochondrial and cytosolic thiol oxidation was assessed by measuring peroxiredoxin dimerization and by redox-sensitive fluorescent proteins. Replacing the putative redox-active cysteine residues on AMPK α1 with alanines did not alter the response of AMPK to H 2 O 2 In parallel with measurements of AMPK activity, we measured the cell ATP/ADP ratio. This allowed us to separate the effects on AMPK activity due to ROS production from those caused by changes in this ratio. We conclude that AMPK activity in response to redox changes is not due to direct action on AMPK itself, but is a secondary consequence of redox effects on other processes, such as mitochondrial ATP production., (© 2018 Hinchy et al.)

AMP-activated protein kinase (AMPK) is a key regulator of cellular and systemic energy homeostasis which achieves this through the phosphorylation of a myriad of downstream targets. One target is TBC1D1 a Rab-GTPase-activating protein that regulates glucose uptake in muscle cells by integrating insulin signalling with that promoted by muscle contraction. Ser 237 in TBC1D1 is a target for phosphorylation by AMPK, an event which may be important in regulating glucose uptake. Here, we show AMPK heterotrimers containing the α1, but not the α2, isoform of the catalytic subunit form an unusual and stable association with TBC1D1, but not its paralogue AS160. The interaction between the two proteins is direct, involves a dual interaction mechanism employing both phosphotyrosine-binding (PTB) domains of TBC1D1 and is increased by two different pharmacological activators of AMPK (AICAR and A769962). The interaction enhances the efficiency by which AMPK phosphorylates TBC1D1 on its key regulatory site, Ser 237 Furthermore, the interaction is reduced by a naturally occurring R125W mutation in the PTB1 domain of TBC1D1, previously found to be associated with severe familial obesity in females, with a concomitant reduction in Ser 237 phosphorylation. Our observations provide evidence for a functional difference between AMPK α-subunits and extend the repertoire of protein kinases that interact with substrates via stabilisation mechanisms that modify the efficacy of substrate phosphorylation., (© 2018 The Author(s).)

AMPK is a conserved serine/threonine kinase whose activity maintains cellular energy homeostasis. Eukaryotic AMPK exists as αβγ complexes, whose regulatory γ subunit confers energy sensor function by binding adenine nucleotides. Humans bearing activating mutations in the γ2 subunit exhibit a phenotype including unexplained slowing of heart rate (bradycardia). Here, we show that γ2 AMPK activation downregulates fundamental sinoatrial cell pacemaker mechanisms to lower heart rate, including sarcolemmal hyperpolarization-activated current (I f ) and ryanodine receptor-derived diastolic local subsarcolemmal Ca 2+ release. In contrast, loss of γ2 AMPK induces a reciprocal phenotype of increased heart rate, and prevents the adaptive intrinsic bradycardia of endurance training. Our results reveal that in mammals, for which heart rate is a key determinant of cardiac energy demand, AMPK functions in an organ-specific manner to maintain cardiac energy homeostasis and determines cardiac physiological adaptation to exercise by modulating intrinsic sinoatrial cell behavior.

AMP-activated protein kinase (AMPK) plays a major role in regulating metabolism and has attracted significant attention as a therapeutic target for treating metabolic disorders. AMPK activity is stimulated more than 100-fold by phosphorylation of threonine 172 (Thr 172 ). Binding of AMP to the γ subunit allosterically activates the kinase. Additionally, many small molecules, e.g. 991, have been identified that bind between the kinase domain and the carbohydrate-binding module of the β subunit, stabilising their interaction and leading to activation. It was reported recently that non-phosphorylated Thr 172 AMPK is activated by AMP and A769662. We present here the crystal structure of non-phosphorylated Thr 172 AMPK in complex with AMP and 991. This structure reveals that the activation loop, as well as the complex overall, is similar to the Thr 172 phosphorylated complex. We find that in the presence of AMP and 991 non-phosphorylated Thr 172 , AMPK is much less active than the Thr 172 phosphorylated enzyme. In human cells, the basal level of Thr 172 phosphorylation is very low (∼1%), but is increased 10-fold by treatment with 2-deoxyglucose. In cells lacking the major Thr 172 kinases, LKB1 and CaMKKβ, Thr 172 phosphorylation is almost completely abolished, and AMPK activity is virtually undetectable. Our data show that AMP and 991 binding to non-phosphorylated Thr 172 AMPK can induce an ordered, active-like, conformation of the activation loop explaining how AMPK activity can be measured in vitro without Thr 172 phosphorylation. However, in a cellular context, phosphorylation of Thr 172 is critical for significant activation of AMPK., (© 2017 The Author(s).)

AMP-activated protein kinase (AMPK) plays a key role in integrating metabolic pathways in response to energy demand. AMPK activation results in a wide range of downstream responses, many of which are associated with improved metabolic outcome, making AMPK an attractive target for the treatment of metabolic diseases. AMPK is a heterotrimeric complex consisting of a catalytic subunit (α) and two regulatory subunits (β and γ). The γ-subunit harbours the nucleotide-binding sites and plays an important role in AMPK regulation in response to cellular energy levels. In mammals, there are three isoforms of the γ-subunit and these respond differently to regulation by nucleotides, but there is limited information regarding their role in activation by small molecules. Here, we determined the effect of different γ-isoforms on AMPK by a direct activator, 991. In cells, 991 led to a greater activation of γ2-containing AMPK complexes compared with either γ1 or γ3. This effect was dependent on the long N-terminal region of the γ2-isoform. We were able to rule out an effect of Ser 108 phosphorylation, since mutation of Ser 108 to alanine in the β2-isoform had no effect on activation of AMPK by 991 in either γ1- or γ2-complexes. The rate of dephosphorylation of Thr 172 was slower for γ2- compared with γ1-complexes, both in the absence and presence of 991. Our studies show that activation of AMPK by 991 depends on the nature of the γ-isoform. This finding may have implications for the design of isoform-selective AMPK activators., (© 2017 The Author(s).)

In eukaryotic cells AMP-activated protein kinase (AMPK) plays a major role in regulating cellular energy balance. AMPK responds to changes in intracellular adenine nucleotide levels, being activated by an increase in AMP/ADP relative to ATP. Activation of AMPK increases the rate of catabolic (ATP-generating) pathways and decreases the rate of anabolic (ATP-utilising) pathways. In addition to its role in maintaining intracellular energy balance, AMPK regulates whole body energy metabolism. Given its key role in controlling energy homeostasis, AMPK has attracted widespread interest as a potential therapeutic target for metabolic diseases, including type 2 diabetes and, more recently, cancer. Here I review the regulation of AMPK and its potential as a target for therapeutic intervention in human disease., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

AMP-activated protein kinase (AMPK) plays a key role in integrating metabolic pathways in response to energy demand. We identified a mutation in the γ1 subunit (γ1 D316A ) that leads to activation of AMPK. We generated mice with this mutation to study the effect of chronic liver-specific activation of AMPK in vivo. Primary hepatocytes isolated from these mice have reduced gluconeogenesis and fatty acid synthesis, but there is no effect on fatty acid oxidation compared to cells from wild-type mice. Liver-specific activation of AMPK decreases lipogenesis in vivo and completely protects against hepatic steatosis when mice are fed a high-fructose diet. Our findings demonstrate that liver-specific activation of AMPK is sufficient to protect against hepatic triglyceride accumulation, a hallmark of non-alcoholic fatty liver disease (NAFLD). These results emphasize the clinical relevance of activating AMPK in the liver to combat NAFLD and potentially other associated complications (e.g., cirrhosis and hepatocellular carcinoma)., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format., Competing Interests: CD has filed a patent on OPM.

We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation.

Folliculin (FLCN) is a tumor-suppressor protein mutated in the Birt-Hogg-Dubé (BHD) syndrome, which associates with two paralogous proteins, folliculin-interacting protein (FNIP)1 and FNIP2, forming a complex that interacts with the AMP-activated protein kinase (AMPK). Although it is clear that this complex influences AMPK and other metabolic regulators, reports of its effects have been inconsistent. To address this issue, we created a recessive loss-of-function variant of Fnip1 Homozygous FNIP1 deficiency resulted in profound B-cell deficiency, partially restored by overexpression of the antiapoptotic protein BCL2, whereas heterozygous deficiency caused a loss of marginal zone B cells. FNIP1-deficient mice developed cardiomyopathy characterized by left ventricular hypertrophy and glycogen accumulation, with close parallels to mice and humans bearing gain-of-function mutations in the γ2 subunit of AMPK. Concordantly, γ2-specific AMPK activity was elevated in neonatal FNIP1-deficient myocardium, whereas AMPK-dependent unc-51-like autophagy activating kinase 1 (ULK1) phosphorylation and autophagy were increased in FNIP1-deficient B-cell progenitors. These data support a role for FNIP1 as a negative regulator of AMPK.

The glycerophospholipid phosphatidylcholine is the most abundant phospholipid species of eukaryotic membranes and essential for structural integrity and signaling function of cell membranes required for cancer cell growth. Inhibition of choline kinase alpha (CHKA), the first committed step to phosphatidylcholine synthesis, by the selective small-molecule ICL-CCIC-0019, potently suppressed growth of a panel of 60 cancer cell lines with median GI50 of 1.12 μM and inhibited tumor xenograft growth in mice. ICL-CCIC-0019 decreased phosphocholine levels and the fraction of labeled choline in lipids, and induced G1 arrest, endoplasmic reticulum stress and apoptosis. Changes in phosphocholine cellular levels following treatment could be detected non-invasively in tumor xenografts by [18F]-fluoromethyl-[1,2-2H4]-choline positron emission tomography. Herein, we reveal a previously unappreciated effect of choline metabolism on mitochondria function. Comparative metabolomics demonstrated that phosphatidylcholine pathway inhibition leads to a metabolically stressed phenotype analogous to mitochondria toxin treatment but without reactive oxygen species activation. Drug treatment decreased mitochondria function with associated reduction of citrate synthase expression and AMPK activation. Glucose and acetate uptake were increased in an attempt to overcome the metabolic stress. This study indicates that choline pathway pharmacological inhibition critically affects the metabolic function of the cell beyond reduced synthesis of phospholipids., Competing Interests: None.

Despite significant advances in our understanding of the biology determining systemic energy homeostasis, the treatment of obesity remains a medical challenge. Activation of AMP-activated protein kinase (AMPK) has been proposed as an attractive strategy for the treatment of obesity and its complications. AMPK is a conserved, ubiquitously expressed, heterotrimeric serine/threonine kinase whose short-term activation has multiple beneficial metabolic effects. Whether these translate into long-term benefits for obesity and its complications is unknown. Here, we observe that mice with chronic AMPK activation, resulting from mutation of the AMPK γ2 subunit, exhibit ghrelin signaling-dependent hyperphagia, obesity, and impaired pancreatic islet insulin secretion. Humans bearing the homologous mutation manifest a congruent phenotype. Our studies highlight that long-term AMPK activation throughout all tissues can have adverse metabolic consequences, with implications for pharmacological strategies seeking to chronically activate AMPK systemically to treat metabolic disease., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The recent exciting advances in our understanding of the regulation of the energy sensor AMP-activated protein kinase (AMPK), together with renewed appreciation of its importance in maintaining cellular function, brought together leading scientists at a recent FASEB-sponsored meeting in September 2014. Here, we report some of the highlights of this conference., (Copyright © 2015 Elsevier Inc. All rights reserved.)