Simon Wyn Jones

GLP-1 and GIP/GLP-1 receptor agonists produce substantial weight loss in clinical trials but significant loss of lean body mass is reported. Whether this reflects a direct pharmacological effect on skeletal muscle or an indirect consequence of caloric restriction and reduced mechanical loading is unknown. Primary myoblasts were isolated from skeletal muscle of older adults with obesity undergoing orthopaedic surgery. GIPR and GLP-1R expression was characterised by RT-qPCR and flow cytometry. Differentiated myotubes were treated with semaglutide or GIP peptide and assessed for atrophy-related gene expression (qPCR), secretome perturbation (Olink Reveal), mitochondrial and glycolytic bioenergetics (Seahorse XF Real-Time ATP Rate Assay, glucose uptake, lactate secretion) and myotube morphology and myogenesis (immunofluorescence). GIPR mRNA was consistently detected across all donors; GLP-1R mRNA was undetectable by PCR, though LUXendin645 flow cytometry in a subset of three donors identified low-level surface GLP-1R protein in 51–66% of myoblasts. Neither semaglutide nor GIP altered atrophy-related gene expression or the secretome, with no proteins reaching significance. Semaglutide reduced glycolytic and total ATP production rates, accompanied by reduced lactate secretion, suggesting modest suppression of glycolytic flux; mitochondrial parameters were unaffected. Neither treatment impaired myotube thickness or differentiation; GIP increased myotube thickness after 8 days. Direct GLP-1 and GIP receptor activation did not produce major atrophic, inflammatory, morphological, myogenic, or mitochondrial effects on primary human skeletal muscle myotubes. These findings are consistent with an indirect mechanism underlying the lean mass loss observed with incretin-based therapies, a question of particular relevance as these agents are increasingly used in older adults at risk of sarcopenia.

Chondrosarcoma is a hyper-vascularised, chemoresistant cartilage malignancy driven by VEGF-centred angiogenesis, and local adipose depots are increasingly recognised as paracrine drivers of tumour angiogenesis via adipokines and extracellular vesicles (EVs). The infrapatellar fat pad (IFP), an inflammatory adipose depot within the articular joint in direct cartilage contact, is a key local source of adipose-derived EVs, and thus a candidate driver of angiogenesis in chondrosarcoma. The aim of this study was to determine whether the IFP is a productive source of EVs, and whether IFP-derived EVs induce angiogenesis in articular chondrocytes. The IFP released significantly more EVs than subcutaneous fat (n = 8 per depot; p = 0.027). Treating primary human articular chondrocytes with IFP EVs for 24 h upregulated VEGFA (+1.6-fold, p = 0.036) and downregulated BMP4 (−2.4-fold, p = 0.011), engaging the VEGF/eNOS/ERK axis that drives chondrosarcoma angiogenesis. Re-analysis of a previously published phospho-kinase dataset from the same donor EVs, corroborated by a pooled donor-group analysis (n = 3), supported activation of eNOS, ERK1/2, PLC-γ1 and HSP27. These findings identify the IFP as a dominant source of EVs within the articular joint, which can induce a pro-angiogenic, VEGF-axis switch in articular cartilage cells, supporting a signalling model relevant to chondrosarcoma angiogenesis.

GLP-1 and GIP/GLP-1 receptor agonists produce substantial weight loss in clinical trials but significant loss of lean body mass is reported. Whether this reflects a direct pharmacological effect on skeletal muscle or an indirect consequence of caloric restriction and reduced mechanical loading is unknown. Primary myoblasts were isolated from skeletal muscle of older adults with obesity undergoing orthopaedic surgery. GIPR and GLP-1R expression was characterised by RT- qPCR and flow cytometry. Differentiated myotubes were treated with semaglutide or GIP peptide and assessed for atrophy-related gene expression (qPCR), secretome perturbation (Olink Reveal), mitochondrial and glycolytic bioenergetics (Seahorse XF Real-Time ATP Rate Assay, glucose uptake, lactate secretion) and myotube morphology and myogenesis (immunofluorescence). GIPR mRNA was consistently detected across all donors; GLP-1R mRNA was undetectable by PCR, though LUXendin645 flow cytometry identified low-level surface GLP-1R protein in 51–66% of myoblasts. Neither semaglutide nor GIP altered atrophy-related gene expression or the secretome, with no proteins reaching significance. Semaglutide reduced glycolytic and total ATP production rates, accompanied by reduced lactate secretion, suggesting modest suppression of glycolytic flux; mitochondrial parameters were unaffected. Neither treatment impaired myotube thickness or differentiation; GIP increased myotube thickness after 8 days. Direct GLP-1 and GIP receptor activation does not substantively perturb atrophic signalling, myogenesis, or the secretome of primary human skeletal muscle myotubes. These findings suggest that lean mass loss with incretin-based therapies is unlikely to be driven by direct pharmacological action on skeletal muscle - particularly relevant as these agents are increasingly used in older adults at risk of sarcopenia.

Adolescent idiopathic scoliosis (AIS) is associated with dysregulated bone remodelling, yet the molecular underpinnings remain unclear. To investigate site-specific osteoblast phenotypes at the spinal curve apex, we performed bulk RNA sequencing on primary matched osteoblasts isolated from the convex, concave, and non-curve regions of AIS patients. Principal component analysis revealed distinct transcriptional clustering by spinal site, independent of patient-specific factors. Differential expression analysis identified region-specific molecular profiles in convex and concave osteoblasts compared to non-curve controls, with the mTOR pathway being highlighted as one of the most dysregulated. Rapamycin, an mTOR inhibitor, reduced Alkaline phosphatase (ALP) activity, Osteoprotegerin (OPG) secretion, and mineralization, while modulating osteogenic gene expression, including sustained upregulation of RUNX2 and COL1A1 . In AIS patient-derived osteoblasts, rapamycin elicited pronounced inhibition of mTOR signalling and osteogenic activity in convex cells compared to control. Convex osteoblasts also showed elevated mTOR expression but reduced downstream translation-related signalling, suggesting dysregulated or uncoupled mTOR activity. Notably, mTOR expression level correlated with curve severity, reinforcing the link between mTOR dysregulation and AIS pathology. These findings identify mTOR signalling as a key regulatory pathway in AIS osteoblast dysfunction and highlight rapamycin as a potential, though complex, therapeutic candidate.

Obesity represents a major global healthcare crisis, with childhood obesity rising at an alarming rate. Children with obesity are highly likely to carry it into adulthood, bringing numerous associated health risks. Even more troubling is the emerging understanding of “obesity memory”, which contributes to the frequent issue of weight regain. Here, we show that obesity imprints CD4 T cells through DNA methylation, leading to a long-time lag, spanning years, before adaptive immune homeostasis is restored after weight loss. Differential DNA methylation analysis highlights autophagy and immune senescence as potential key mechanisms underpinning this memory of obesity in CD4 T cells. In addition, particularly palmitate could be a key saturated fatty acid that can contribute to epigenetic alterations in CD4 T cells, potentially perpetuating this altered state. We identify molecular candidates (i.e., Stk26 and Cdkn1c) underpinning key cell functions (autophagy and immune senescence) that could be targeted to promote a return to immune homeostasis alongside weight loss. These findings raise the possibility that targeting such pathways could support the restoration of immune homeostasis alongside weight loss therapies.

Background: Ageing is a complex, multi-dimensional process, underpinned by interacting biological hallmarks that collectively contribute to functional decline and increased susceptibility to disease. While considerable progress has been made in delineating individual ageing pathways, translation into human studies has been hindered by methodological heterogeneity and a lack of standardised, multi-system approaches. Here, we describe a validated, high-resolution toolkit for the simultaneous quantification of multiple ageing hallmarks in clinically accessible human samples, encompassing cellular senescence, immune ageing, inflammation, mitochondrial function, mTOR signalling, autophagy, genomic instability, and stem cell exhaustion. Methods: Blood (25ml) was obtained from young and aged donors (26–81y). Deep immunophenotyping was performed using a novel 30-colour spectral flow cytometry panel. T-cell mTOR activation and autophagic flux were assessed by flow cytometry. Metabolic flux was measured by Seahorse. From whole blood (4 ml), muscle, and adipose tissue (AT) (obtained during elective hip arthroplasty) RNA, DNA, AT stem cells, and myoblasts were isolated. DNA copy number and senescent cell burden were assessed by q-PCR and SA-β-gal staining, respectively. Findings: Utilising this toolkit, we identified pronounced age-related immune remodelling, increased senescent T-cell burden, diminished mitochondrial capacity and altered mTOR–autophagy signalling between healthy young and aged donors. Furthermore, metabolism was significantly affected by anti-coagulant and freezing sample before analysis. Interpretation: This integrated platform provides a foundation for reproducible, cross-study analyses and facilitates translational investigation of interventions targeting health-span extension. Funding: Wellcome Leap Dynamic Resilience program (co-funded by Temasek Trust).

Background: Sarcopenic obesity, where excess body fat coexists with reduced muscle mass and function, is becoming increasingly common in ageing populations and contributes to poor physical and metabolic health. Although adipose tissue–secreted factors are implicated in muscle decline, the specific mechanisms remain unclear. Extracellular vesicles (EVs), which carry regulatory cargo such as microRNAs (miRNAs) between cells, may play a key role in this adipose–muscle communication. Methods: EVs were isolated from adipose‐conditioned media (ACM) collected from lean and non‐lean human donors using ultracentrifugation. Donors were grouped by BMI (lean: 20.7–24.4; non‐lean: 25.3–39.3) and age (younger: 31–56 years; older: 60–84 years). EVs were characterised using nanoparticle tracking analysis (NTA), ExoView, nanoscale flow cytometry (CytoFLEX Nano) and transmission electron microscopy (TEM). Primary human myoblasts were differentiated into myotubes and treated for 24 h with lean or non‐lean EVs (1.3 × 10 ⁹ particles/mL) or left untreated. Myotube thickness was measured by immunofluorescence microscopy. Transcriptomic changes were assessed by bulk RNA sequencing. EV miRNA cargo was profiled by small RNA‐seq and validated by qPCR. The role of miR‐150‐5p was tested using antagomir inhibition. Results: Non‐lean EVs significantly reduced myotube thickness in older adult‐derived myotubes compared to both untreated controls (8.7 ± 1.66 μm vs. 12.4 ± 1.72 μm, p < 0.01) and lean EV‐treated myotubes (8.7 ± 1.66 μm vs. 13.2 ± 3.84 μm, p < 0.05), indicating a donor BMI‐specific effect. This atrophy was restricted to myotubes derived from older donors. The same experimental approach was applied to younger adult‐derived myotubes; no reduction in myotube thickness was observed. MAFbx expression was significantly increased in response to non‐lean EVs ( p < 0.05). RNA‐seq revealed 471 differentially expressed genes (DEGs) in EV‐treated versus untreated cells and 293 DEGs between lean and non‐lean EV conditions, with enrichment in inflammatory (TNF and IL1B), oxidative stress, mitochondrial and chromatin pathways. Small RNA‐seq identified seven differentially expressed miRNAs (annotated using miRBase release 22.1), including miR‐150‐5p and miR‐193b‐5p, both significantly upregulated in non‐lean EVs and validated by qPCR. Inhibiting miR‐150‐5p partially rescued myotube thickness (10.5 ± 1.37 μm vs. 8.7 ± 1.66 μm, p < 0.05) and reduced MAFbx expression. Conclusions: EVs from non‐lean adipose tissue drive muscle atrophy and transcriptional changes in an age‐dependent manner. These effects are partially mediated by miR‐150‐5p, highlighting a mechanistic role for EV cargo in adipose‐muscle signalling. Targeting EV‐derived miRNAs may offer a novel strategy to combat muscle loss in obesity and ageing.

Background: Patients with end‐stage liver disease (ESLD) often present with sarcopenia, defined as loss of skeletal muscle mass and quality, which is associated with reduced quality of life and increased mortality. However, the molecular mechanisms driving sarcopenia in ESLD are not fully understood and there are currently no therapeutic interventions. This study aimed to identify potential circulating factors contributing to sarcopenia progression in ESLD by assessing their role in driving transcriptomic alterations in skeletal muscle. Methods: Quadriceps muscle tissue, plasma and serum were obtained from ESLD patients ( n = 24) and age/sex‐matched healthy controls (HC; n = 18) (Clinical Trial ID: NCT04734496, Ethical Approval 18/WM/0167). Total RNA from snap‐frozen vastus lateralis muscle biopsies underwent RNA sequencing (Illumina). Serum concentrations of 60 cytokines were profiled by Luminex and ELISA, with comparisons made both between ESLD and HC, and across ESLD aetiologies (alcohol‐related, NAFLD, viral hepatitis, other). In vitro, primary human myotubes (from non‐ESLD aged donors, NRES #16/SS/0172) were treated with 10% ESLD or HC plasma (24 h, n = 6 per group) followed by RNA sequencing (BGI Genomics). Differentially expressed genes ( p < 0.05, fold‐change > 1.5) were identified via Qlucore and DESeq2, and pathway analysis performed using Ingenuity (Qiagen). The impact of physiological concentrations of candidate cytokines (IL‐1α, GDF‐15 and HGF) on myotube thickness, differentiation and mitochondrial function was assessed by immunofluorescence microscopy, RT‐qPCR and metabolic flux assays. Results: In ESLD muscle, 387 and 225 genes were significantly up‐ and downregulated compared to HC, respectively, with cellular senescence identified as a top dysregulated function. Upstream regulator analysis predicted activation of hepatocyte growth factor (HGF) and interleukin‐1 signalling. Subgroup analysis revealed distinct transcriptomic profiles based on disease aetiology. Serum profiling identified 15 cytokines significantly elevated ( p < 0.05) and five reduced ( p < 0.05) in ESLD, including increased HGF and reduced interleukin‐1 receptor antagonist. Stratified analysis also revealed aetiology specific cytokine profiles, with only GDF‐15 significantly ( p < 0.0001) elevated in all groupsTwenty‐four‐hour ESLD plasma treatment induced 423 differentially expressed genes in human myotubes, which were again associated with significant activation of senescence pathways, with IL‐1 identified as a key upstream driver. In vitro, IL‐1α, GDF‐15, and HGF significantly reduced myotube thickness, nuclear fusion index and perturbed metabolism (increased glycolysis, impaired oxidative phosphorylation). Conclusions: Collectively, these findings suggest that sarcopenia in ESLD is driven by aetiology‐specific mechanisms, highlighting the potential for targeted therapies to improve muscle mass and function.

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Background: Sarcopenic obesity, where excess body fat coexists with reduced muscle mass and function, is becoming increasingly common in ageing populations and contributes to poor physical and metabolic health. Although adipose tissue-secreted factors are implicated in muscle decline, the specific mechanisms remain unclear. Extracellular vesicles (EVs), which carry regulatory cargo such as microRNAs (miRNAs) between cells, may play a key role in this adipose-muscle communication. Methods: EVs were isolated from adipose-conditioned media (ACM) collected from lean and non-lean human donors using ultracentrifugation. Donors were grouped by BMI (lean: 20.7-24.4; non-lean: 25.3-39.3) and age (younger: 31-56 years; older: 60-84 years). EVs were characterised using nanoparticle tracking analysis (NTA), ExoView, nanoscale flow cytometry (CytoFLEX Nano), and transmission electron microscopy (TEM). Primary human myoblasts were differentiated into myotubes and treated for 24 hours with lean or non-lean EVs (1.3x10^9 particles/ml) or left untreated. Myotube thickness was measured by immunofluorescence microscopy. Transcriptomic changes were assessed by bulk RNA sequencing. EV miRNA cargo was profiled by small RNA-seq and validated by qPCR. The role of miR-150-5p was tested using antagomir inhibition. Results: Non-lean EVs significantly reduced myotube thickness compared to both untreated controls (8.7 +/- 1.66 um vs. 12.4 +/- 1.72 um, p < 0.01) and lean EV-treated myotubes (8.7 +/- 1.66 um vs. 13.2 +/- 3.84 um, p < 0.05), indicating a donor BMI-specific effect. This atrophy was restricted to myotubes derived from older donors. MAFbx expression was significantly increased in response to non-lean EVs (p < 0.05). RNA-seq revealed 471 differentially expressed genes (DEGs) in EV-treated vs. untreated cells and 293 DEGs between lean and non-lean EV conditions, with enrichment in inflammatory (TNF, IL1B), oxidative stress, mitochondrial, and chromatin pathways. Small RNA-seq identified 7 differentially expressed miRNAs, including miR-150-5p and miR-193b-5p, both significantly upregulated in non-lean EVs and validated by qPCR. Inhibiting miR-150-5p partially rescued myotube thickness (10.5 +/- 1.37 um vs. 8.7 +/- 1.66 um, p < 0.05) and reduced MAFbx expression. Conclusions: EVs from non-lean adipose tissue drive muscle atrophy and transcriptional changes in an age-dependent manner. These effects are partially mediated by miR-150-5p, highlighting a mechanistic role for EV cargo in adipose-muscle signalling. Targeting EV-derived miRNAs may offer a novel strategy to combat muscle loss in obesity and ageing.