David Carling

AMP‐activated protein kinase (AMPK) is a key evolutionarily conserved sensor of energy homeostasis and plays a central role in metabolic health and disease. AMPK has also been implicated in ageing; however, most in vivo drug studies rely on the use of indirect activators, such as metformin, which have complex modes‐of‐action, therefore making conclusions on the specific role of AMPK more challenging. Here, we demonstrate that direct activation of AMPK with the compound 991 extends lifespan in Drosophila melanogaster , Caenorhabditis elegans and Schizosaccharomyces pombe . In mice, 991 treatment induces a pro‐longevity proteomic profile, highlighting the potential for translation to mammals. Overall, our study provides important proof‐of‐principle for AMPK as a pharmacological target with longevity benefits.

Background and Aims: Dilated cardiomyopathy (DCM) is a genetically heterogeneous cause of heart failure and sudden cardiac death. Many patients remain without a molecular diagnosis. MAP3K7 encodes TAK1, a serine/threonine kinase important for cardiac homeostasis . MAP3K7 variants are an established cause of syndromic disease, including cardiospondylocarpofacial syndrome (CSCF), in which DCM has been occasionally reported. Here, we demonstrate that MAP3K7 variants can cause apparently isolated DCM, expanding the phenotypic spectrum of MAP3K7 -related disorders. Methods: We integrated four lines of genetic evidence: de novo variation, common variant association, familial segregation, and rare variant enrichment, alongside functional characterisation. Results: In 117 paediatric cardiomyopathy trios from the 100,000 Genomes Project, two probands harboured rare de novo MAP3K7 missense variants, significantly more than expected (Bonferroni-adjusted p=0.036). Independent GWAS implicated MAP3K7 as a susceptibility locus for adult DCM. Across global DCM cohorts, we identified families harbouring rare MAP3K7 variants, including one with segregation in nine affected relatives (p.(Tyr125Cys), estimated LOD = 2.7). Rare damaging non-truncating variants were enriched in DCM versus INTERVAL (OR 22, P=0.007) and gnomAD controls (OR 14, P=6.2×10⁻⁴), while truncating variants were associated with DCM in Genomics England and increased left ventricular volumes in UK Biobank. DCM-associated variants reduced TAK1 kinase activity, supporting a loss-of-function mechanism consistent with CSCF-associated alleles. Conclusion: Multiple independent lines of evidence establish an association between MAP3K7 loss of function variants and DCM. Several affected individuals lacked overt syndromic features, demonstrating that MAP3K7 -related disease may present as apparently isolated DCM across the lifespan and supporting inclusion of MAP3K7 in DCM diagnostic pipelines. GRAPHICAL Key Question: Do MAP3K7 variants cause isolated dilated cardiomyopathy?: MAP3K7 variants are known to cause rare syndromic disease, including cardiospondylocarpofacial syndrome (CSCF). Some patients with CSCF develop dilated cardiomyopathy (DCM), however we do not know whether MAP3K7 variants cause primary or isolated DCM without overt syndromic disease. Key Finding: We present multiple orthogonal data sources supporting an association between MAP3K7 loss-of-function variants and DCM. The collective evidence for this gene-disease association encompasses paediatric trio de novo discovery, GWAS, familial segregation, rare variant case-control analysis and variant categorisation in vitro . Strong segregation evidence was obtained for p.(Tyr125Cys), with nine clinically affected genotype-positive individuals in one pedigree (estimated LOD score 2.7). Further, p.(Cys266del) was observed in three affected individuals in one family and a de novo paediatric case. Take-Home Message: Loss-of-function variants in MAP3K7 are associated with DCM and several affected individuals lack overt syndromic features. MAP3K7 -related disease may present as apparently isolated DCM across the lifespan. These findings support the incorporation of MAP3K7 testing into DCM diagnostic pipelines.

AMP-activated protein kinase (AMPK) plays an important role in maintaining energy homeostasis in mammals. AMPK is a heterotrimer of an α catalytic subunit and two regulatory subunits, β and γ. In mammals, each subunit has different isoforms (α1/α2, β1/ β2, and γ1/γ2/γ3) encoded by separate genes, leading to the potential expression of 12 AMPK complexes. Here, we show that AMPK containing the long forms of γ2 (γ2a, encoding a protein of 569 amino acids, and γ2c, 525 amino acids) binds to 14-3-3. In contrast to AMPK containing the short form of γ2 (γ2b, 328 amino acids), bacterial expression of AMPK containing the long forms of γ2 requires co-expression with 14-3-3 and prior phosphorylation of Thr172 within the α subunit. AMPKγ2-14-3-3 complexes have reduced activity compared with AMPKγ1 or AMPKγ2b but retain allosteric activation by AMP and the AMPK activator, 991. We found that two predicted 14-3-3 binding sites within γ2a (T97 and S122) were phosphorylated in the bacterially expressed AMPK complex. Furthermore, we show that a peptide spanning these two phosphorylated sites binds to 14-3-3 in vitro and determined the crystal structure of this 14-3-3-peptide co-complex. These results indicate that 14-3-3 binds to the N-terminal region of γ2a/c, reducing the activity of AMPK relative to AMPKγ1 and AMPKγ2b. Our findings reveal a new mode of regulation of AMPK containing the long forms of γ2. While the biological significance of 14-3-3 binding to AMPKγ2a/c complexes remains to be determined, our studies provide the starting point to begin to address this issue.

High-throughput proteomics requires efficient and highly reproducible sample processing, yet workflows─particularly for PTM profiling─remain complex and costly to fully automate. Here, we present a practical intermediate solution using manually operated 96-channel devices: the Gilson Platemaster P220 pipettor and VP Scientific 96-well magnetic pin device. Using this setup, we achieved robust and reproducible phosphoproteomics in a 96-well format, completing protein aggregation capture (PAC/SP3) digestion, desalting, phosphopeptide enrichment, and a second desalting step within 2 days while minimizing operator workload and variability. Several innovations enable this workflow. First, we describe a cost-efficient method to generate 96-well solid-phase extraction plates by directly packing the Oasis HLB sorbent into tapered filter plates. We extensively characterize these plates in terms of loading capacity, lipid removal efficiency, and suitability for high-pH fractionation. Second, we demonstrate that efficient PAC digestion does not require continuous bead suspension; instead, digestion can be achieved by briefly aspirating beads in protease solution, eliminating the need for orbital shaking and simplifying automation. The presented workflow familiarizes users with 96-channel devices and hence serves as a good step toward full automation.

It has been shown that in most cells, low glucose leads to activation of AMP-activated protein kinase (AMPK) via the lysosomal glucose-sensing pathway, where glycolytic aldolase acts as the glucose sensor. Here, we show that ALDOC (aldolase C), the predominant isozyme of aldolase in mouse and rat oligodendrocyte precursor cells (OPCs), is acetylated at lysine 14, making the lysosomal glucose-sensing AMPK pathway unable to operate. We find that the blockage of AMPK activation is required for the proper proliferation and differentiation of OPCs into mature oligodendrocytes for myelination during development and for remyelination in areas of demyelination where the local glucose levels are low. Therefore, the acetylation of aldolase acts as a checkpoint for AMPK activation in response to low glucose to ensure the proliferation and differentiation of OPCs for myelination, and remyelination of demyelinated neurons.

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.

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.

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.