Peripheral blood mononuclear cell (PBMC) bioenergetic profiling has emerged as a promising minimally invasive tool for assessing immunometabolic health, yet traditional venepuncture presents practical barriers for certain populations and longitudinal studies. This study examined PBMC bioenergetic profiles from upper-arm capillary blood using the Tasso+ device and compared these to traditional venepuncture. Healthy adults provided paired capillary and venous blood samples at rest and following maximal exercise. PBMCs were isolated and assessed using Seahorse XF technology for mitochondrial respiration and glycolytic function, alongside haematological and T-cell immunophenotyping via flow cytometry. Bland–Altman analysis demonstrated narrow limits of agreement and small mean bias between sampling methods for T-cell subsets and normalised bioenergetic parameters, whereas absolute bioenergetic rates showed wider limits of agreement with a consistently greater negative bias, indicating systematically lower values in capillary compared with venous samples. Formal equivalence testing confirmed method equivalence for only total CD4+ T cells and CD4+ naïve T cells at rest. For bioenergetic parameters, equivalence was demonstrated for proton leak respiration, mitochondrial health index, ATP supply rate, and glycolytic metrics. Absolute mitochondrial and glycolytic rates were not equivalent between methods. Both sampling methods preserved PBMC activation capacity following PMA/ionomycin stimulation. Our findings support capillary blood sampling using the Tasso+ device as a minimally invasive alternative to venepuncture for PBMC metabolic phenotyping and immunological assessments. However, caution should be exercised if using the methods interchangeably, given that only selected bioenergetic parameters and T-cell subsets demonstrate acceptable equivalence.

Guanylate-binding proteins (GBPs) are part of a family of large interferon gamma (IFNψ)-inducible GTPases, with ascribed roles in infection control and induction of programmed cell death. While pathogen-specific functions of GBPs have been studied in detail, their broader regulation of frontline immune defences remain unexplored. Here, we analysed the global contribution of human GBP1-5 to cellular metabolism in IFNψ-stimulated macrophages. We found a robust role of GBP2 and GBP5 in macrophage glycolysis. Only GBP5, and not GBP2 deficiency impaired surface expression and cytokine production of classically IFNψ/LPS-activated macrophages. The GTPase activity of GBP5 was required for the regulation of glycolysis and cytokine production. We found that GBP5 deficiency impaired cellular glucose uptake and lactate production specifically. Isotopic tracing with [U- 13 C 6 ]-Glucose confirmed a decrease in several glycolytic intermediates, including glucose 6-phosphate, pyruvate, and lactate, but stable levels of traced tricarboxylic acid cycle (TCA) intermediates. Elevated ribose-5-phosphate and glycerol levels suggest an altered cytosolic redox balance and enhanced breakdown of fatty acids. GBP5 localised predominantly to the cis-Golgi and in the absence of GBP5 we observed increased Golgi fragmentation, however the total Golgi size remained unchanged. Our results underscore the fundamental role of GBP5 in glycolytic fluxes and Golgi integrity in IFNψ-stimulated macrophages, highlighting its significance in immune function in general and immunometabolism specifically.

Many cardiac pathologies are characterised by increased stiffness of the myocardium, due to excess deposition of extracellular matrix (ECM) proteins and structural remodelling, impacting the behaviour of cardiomyocytes (CMs). Metabolism of CMs shifts in cardiac pathologies, with the healthy heart primarily utilising fatty acids as its source of energy production, whilst the diseased heart switches to utilise glucose. The shift in metabolic source with stiffness of the myocardium has not been investigated. To investigate the effect of ECM stiffnesses on iPSC-CM metabolism, iPSC-CMs were cultured on polydimethylsiloxane (PDMS) substrates of healthy and fibrotic stiffness (20 kPa and 130 kPa respectively) and plastic. Cellular metabolism of iPSC-CMs was assessed through isotope-labelled mass spectrometry with central carbon tracing as well as real-time cellular bioenergetics using extracellular flux analysis. Key metabolic genes were investigated at transcript and protein level, with proteomics analysis conducted to identify protein profiles on substrate stiffnesses. Mass spectrometry data revealed greater utilisation of glucose in iPSC-CMs cultured on plastic compared to softer PDMS substrates, indicating greater glycolytic activity. Extracellular flux analysis demonstrated greater lactic acid efflux from iPSC-CMs cultured on plastic substrates, reflective of increased glycolytic flux and a shift towards aerobic glycolysis as the primary source of ATP synthesis. This study revealed culture of iPSC-CMs on traditional cell culture plastics or glass coverslips displaying pathological metabolism, highlighting the use of physiological substrates for metabolic investigation.

Muscle RING-finger protein 1 (MuRF1, gene name: TRIM63) is well known as a critical molecular regulator in skeletal muscle atrophy. Despite the identification of several substrates and interaction partners for MuRF1, the precise molecular mechanisms by which MuRF1 causes skeletal muscle atrophy remain unclear. To gain further insight into the underlying mechanism of skeletal muscle atrophy, we applied targeted biochemical approaches and identified tripartite motif-containing protein 72 (TRIM72) as a novel MuRF1-interacting protein. Subsequent analysis using MuRF1 knockout and rescue experiments showed that TRIM72 protein abundance is dependent on the presence of MuRF1 protein. Furthermore, TRIM72 protein level was increased by dexamethasone treatment in C2C12 myotubes, alongside increased MuRF1 protein level. Dexamethasone decreases IRS1/Akt signaling and glucose uptake specifically in wild type myotubes, but not in MuRF1 KO myotubes. Further analysis showed that overexpression of TRIM72 impairs IRS1/Akt signaling without the presence of MuRF1, indicating that MuRF1 induces a negative impact on insulin signaling through a plausible cooperation with TRIM72. Our findings provide novel non-degradative molecular roles of MuRF1 that link together skeletal muscle atrophy and impaired insulin sensitivity.

Background/Introduction:The utility of measuring real-time cellular bioenergetics of peripheral blood mononuclear cells (PBMCs) as biomarkers in disease monitoring, such as the bioenergetic health index, is of emerging interest. However, various experimental factors can impact the accuracy and reproducibility of these measurements.Methods: PBMC bioenergetics were probed in real-time using extracellular flux analysis to identify optimal seeding density and injection protocol. Using a modified protocol, we assessed the extent to which blood processing time and isolation method (SepMate™ vs. EasySep™ Direct) influence PBMC bioenergetics under basal and stimulated conditions. Advanced metabolic control analysis including mitochondrial and glycolytic ATP supply flux, respiratory control ratio, bioenergetic health index, and mitochondrial toxicity index were used to identify and quantify PBMC bioenergetics. Results:Measures of metabolic profiling such as mitochondrial respiration, glycolytic activity, ATP supply flux, and respiratory control ratio were significantly diminished in PBMCs due to blood processing delay (48-72 hours) and were influenced by isolation method. Extended blood processing time significantly lowered T cell activation capacity in PBMCs, evidenced by decreased responses of mitochondrial and glycolytic ATP supply to CD3/CD28 activation. Discussion/Conclusion:This study demonstrates that key experimental variables including blood processing time and isolation method critically affect the reliability and biological relevance of PBMC metabolic assessments, highlighting the importance of protocol standardisation for accurate bioenergetic biomarker measurements.

Alström syndrome (AS) is an inherited rare ciliopathy characterised by multi-organ dysfunction and premature cardiovascular disease. This may manifest as an infantile-onset dilated cardiomyopathy with significant associated mortality. An adult-onset restrictive cardiomyopathy may also feature later in life. Loss of function pathogenic variants in ALMS1 have been identified in AS patients, leading to a lack of ALMS1 protein. The biological role of ALMS1 is unknown, particularly in a cardiovascular context. To understand the role of ALMS1 in infantile cardiomyopathy, the reduction of ALMS1 protein seen in AS patients was modelled using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), in which ALMS1 was knocked out. MuscleMotion analysis and calcium optical mapping experiments suggest that ALMS1 knockout (KO) cells have increased contractility, with altered calcium extrusion and impaired calcium handling dynamics compared to wildtype (WT) counterparts. Seahorse metabolic assays showed ALMS1 knockout iPSC-CMs had increased glycolytic and mitochondrial respiration rates, with ALMS1 knockout cells portraying increased energetic demand and respiratory capacity than WT counterparts. Using senescence associated β-galactosidase (SA-β gal) staining assay, we identified increased senescence of ALMS1 knockout iPSC-CMs. Overall, this study provides insights into the molecular mechanisms in AS, particularly the role of ALMS1 in infantile cardiomyopathy in AS, using iPSC-CMs as a ‘disease in a dish’ model to provide insights into multiple aspects of this complex disease.

Aims Reduced left atrial PITX2 is associated with atrial cardiomyopathy and atrial fibrillation (AF). PITX2 is restricted to left atrial cardiomyocytes (aCMs) in the adult heart. The links between PITX2 deficiency, atrial cardiomyopathy, and AF are not fully understood.Methods and results To identify mechanisms linking PITX2 deficiency to AF, we generated and characterized PITX2-deficient human aCMs derived from human induced pluripotent stem cells (hiPSC) and their controls. PITX2-deficient hiPSC-derived atrial cardiomyocytes showed shorter and disorganized sarcomeres and increased mononucleation. Electron microscopy found an increased number of smaller mitochondria compared with isogenic controls. Mitochondrial protein expression was altered in PITX2-deficient hiPSC-derived atrial cardiomyocytes. Single-nuclear RNA-sequencing found differences in cellular respiration pathways and differentially expressed mitochondrial and ion channel genes in PITX2-deficient hiPSC-derived atrial cardiomyocytes. PITX2 repression in hiPSC-derived atrial cardiomyocytes replicated dysregulation of cellular respiration. Mitochondrial respiration was shifted to increased glycolysis in PITX2-deficient hiPSC-derived atrial cardiomyocytes. PITX2-deficient human hiPSC-derived atrial cardiomyocytes showed higher spontaneous beating rates. Action potential duration was more variable with an overall prolongation of early repolarization, consistent with metabolic defects. Gene expression analyses confirmed changes in mitochondrial genes in left atria from 42 patients with AF compared with 43 patients with sinus rhythm. Dysregulation of left atrial mitochondrial (COX7C) and metabolic (FOXO1) genes was associated with PITX2 expression in human left atria.Conclusion PITX2 deficiency causes atrial mitochondrial dysfunction and a metabolic shift to glycolysis in human aCMs. PITX2-dependent metabolic changes can contribute to the structural and functional defects found in PITX2-deficient atria. Graphical Abstract Deficiency in PITX2, a gene with left atrial and skeletal muscle expression in adults leads to mitochondrial dysfunction. PITX2 deficiency is likely to underlie the genomic basis for atrial fibrillation (AF). Reduced PITX2 in atrial cardiomyocytes (aCMs) conveys electrical changes and structural alterations. The cellular mechanisms linking PITX2 deficiency to AF are not fully understood. PITX2 deficiency increases cellular and functional heterogeneity in human iPSC-derived aCMs. These experiments show that PITX2 alters mitochondrial function and metabolism by altering gene and protein expression in aCMs, creating a metabolic shift away from respiration towards glycolysis. Left atrial tissue from patients with AF shows similar changes in gene expression patterns of mitochondrial genes and their association with PITX2. Figure was generated using BioRender.com.

Cancer cells take up amino acids from the extracellular space to drive cell proliferation and viability. Similar mechanisms are applied by immune cells, resulting in the competition between conventional T cells, or indeed chimeric antigen receptor (CAR) T cells and tumor cells, for the limited availability of amino acids within the environment. We demonstrate that T cells can be re-engineered to express SLC7A5 or SLC7A11 transmembrane amino acid transporters alongside CARs. Transporter modifications increase CAR T-cell proliferation under low tryptophan or cystine conditions with no loss of CAR cytotoxicity or increased exhaustion. Transcriptomic and phenotypic analysis reveals that downstream, SLC7A5/SLC7A11-modified CAR T cells upregulate intracellular arginase expression and activity. In turn, we engineer and phenotype a further generation of CART cells that express functional arginase 1/arginase 2 enzymes and have enhanced CAR T-cell proliferation and antitumor activity. Thus, CAR T cells can be adapted to the amino acid metabolic microenvironment of cancer, a hitherto recognized but unaddressed barrier for successful CAR T-cell therapy.

Neutrophil responses are critical during inflammatory and infective events, and neutrophil dysregulation has been associated with poor patient outcomes. Immunometabolism is a rapidly growing field that has provided insights into cellular functions in health and disease. Neutrophils are highly glycolytic when activated, with inhibition of glycolysis associated with functional deficits. There is currently very limited data available assessing metabolism in neutrophils. Extracellular flux (XF) analysis assesses real time oxygen consumption and the rate of proton efflux in cells. This technology allows for the automated addition of inhibitors and stimulants to visualise the effect on metabolism. We describe optimised protocols for an XFe96 XF Analyser to (i) probe glycolysis in neutrophils under basal and stimulated conditions, (ii) probe phorbol 12-myristate 13-acetate induced oxidative burst, and (iii) highlight challenges of using XF technology to examine mitochondrial function in neutrophils. We provide an overview of how to analyze XF data and identify pitfalls of probing neutrophil metabolism with XF analysis. In summary we describe robust methods for assessing glycolysis and oxidative burst in human neutrophils and discuss the challenges around using this technique to assess mitochondrial respiration. XF technology is a powerful platform with a user-friendly interface and data analysis templates, however we suggest caution when assessing neutrophil mitochondrial respiration.

Introduction. Vascular endothelial dysfunction is characterised by lowered nitric oxide (NO) bioavailability, which may be explained by increased production of reactive oxygen species (ROS), mitochondrial dysfunction, and altered cell signalling. (-)-Epicatechin (EPI) has proven effective in the context of vascular endothelial dysfunction, but the underlying mechanisms associated with EPI’s effects remain unclear. Objective(s). Our aim was to investigate whether EPI impacts reactive oxygen and nitrogen species (RONS) production and mitochondrial function of human vascular endothelial cells (HUVECs). We hypothesised that EPI would attenuate ROS production, increase NO bioavailability, and enhance indices of mitochondrial function. Methods. HUVECs were treated with EPI (0-20 μM) for up to 48 h. Mitochondrial and cellular ROS were measured in the absence and presence of antimycin A (AA), an inhibitor of the mitochondrial electron transport protein complex III, favouring ROS production. Genes associated with mitochondrial remodelling and the antioxidant response were quantified by RT-qPCR. Mitochondrial bioenergetics were assessed by respirometry and signalling responses determined by western blotting. Results. Mitochondrial superoxide production without AA was increased 32% and decreased 53% after 5 and 10 μM EPI treatment vs. CTRL ( P < 0.001 ). With AA, only 10 μM EPI increased mitochondrial superoxide production vs. CTRL (25%, P < 0.001 ). NO bioavailability was increased by 45% with 10 μM EPI vs. CTRL ( P = 0.010 ). However, EPI did not impact mitochondrial respiration. NRF2 mRNA expression was increased 1.5- and 1.6-fold with 5 and 10 μM EPI over 48 h vs. CTRL ( P = 0.015 and P = 0.001 , respectively). Finally, EPI transiently enhanced ERK1/2 phosphorylation (2.9 and 3.2-fold over 15 min and 1 h vs. 0 h, respectively; P = 0.035 and P = 0.011 ). Conclusion(s). EPI dose-dependently alters RONS production of HUVECs but does not impact mitochondrial respiration. The induction of NRF2 mRNA expression with EPI might relate to enhanced ERK1/2 signalling, rather than RONS production. In humans, EPI may improve vascular endothelial dysfunction via alteration of RONS and activation of cell signalling.

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing cells as two-dimensional, single-cell monolayers (2D culture), spheroid cell culture promotes, regulates, and supports physiological cellular architecture and characteristics that exist in vivo, including the expression of extracellular matrix proteins, cell signaling, gene expression, protein production, differentiation, and proliferation. The importance of 3D culture has been recognized in many research fields, including oncology, diabetes, stem cell biology, and tissue engineering. Over the last decade, improved methods have been developed to produce spheroids and assess their metabolic function and fate. Extracellular flux (XF) analyzers have been used to explore mitochondrial function in 3D microtissues such as spheroids using either an XF24 islet capture plate or an XFe96 spheroid microplate. However, distinct protocols and the optimization of probing mitochondrial energy metabolism in spheroids using XF technology have not been described in detail. This paper provides detailed protocols for probing mitochondrial energy metabolism in single 3D spheroids using spheroid microplates with the XFe96 XF analyzer. Using different cancer cell lines, XF technology is demonstrated to be capable of distinguishing between cellular respiration in 3D spheroids of not only different sizes but also different volumes, cell numbers, DNA content and type. The optimal mitochondrial effector compound concentrations of oligomycin, BAM15, rotenone, and antimycin A are used to probe specific parameters of mitochondrial energy metabolism in 3D spheroids. This paper also discusses methods to normalize data obtained from spheroids and addresses many considerations that should be considered when exploring spheroid metabolism using XF technology. This protocol will help drive research in advanced in vitro spheroid models.

Acute myeloid leukaemia (AML) creates an immunosuppressive environment to conventional T cells through Arginase 2 (ARG2)-induced arginine depletion. We identify that AML blasts release the acute phase protein serum amyloid A (SAA), which acts in an autocrine manner to upregulate ARG2 expression and activity, and promote AML blast viability. Following in vitro cross-talk invariant natural killer T (iNKT) cells become activated, upregulate mitochondrial capacity, and release IFN-γ. iNKT retain their ability to proliferate and be activated despite the low arginine AML environment, due to the upregulation of Large Neutral Amino Acid Transporter-1 (LAT-1) and Argininosuccinate Synthetase 1 (ASS)-dependent amino acid pathways, resulting in AML cell death. T cell proliferation is restored in vitro and in vivo. The capacity of iNKT cells to restore antigen-specific T cell immunity was similarly demonstrated against myeloid-derived suppressor cells (MDSCs) in wild-type and Jα18−/− syngeneic lymphoma-bearing models in vivo. Thus, stimulation of iNKT cell activity has the potential as an immunotherapy against AML or as an adjunct to boost antigen-specific T cell immunotherapies in haematological or solid cancers.

Introduction Chronological ageing is associated with mitochondrial dysfunction and increased reactive oxygen species (ROS) production in skeletal muscle. However, the effects of replicative ageing on skeletal muscle cellular metabolism are not well known. Using an established myoblast model of cellular (replicative) ageing, we investigated the impact of ageing on energy metabolism in murine C2C12 myotubes. Methods Control (P7-11) and replicatively ‘aged’ (P48-51) C2C12 myoblasts were differentiated over 72-120 h. Mitochondrial bioenergetics were investigated by respirometry and mitochondrial superoxide and cellular ROS were measured in the absence and presence of antimycin A (AA). Genes related to mitochondrial remodelling and the antioxidant response were quantified by RT-qPCR. Intracellular metabolites were quantified using an untargeted 1H-NMR metabolomics pipeline. Results Mitochondrial coupling efficiency (Control: 79.5 vs. Aged: 70.3%, P=0.006) and relative oxidative ATP synthesis (Control: 48.6 vs. Aged: 31.7%, P=0.022) were higher in control vs. aged myotubes, but rates of mitochondrial superoxide production were lower (Control: 2.4×10−5 ± 0.4 × 10−5 vs. Aged: 9.7×10−5 ± 1.6×10−5 RFU/sec/cell; P=0.035). Replicatively aged myotubes had greater mRNA expression of mfn2 and Tfam compared to control. Yet, Nrf2 and PGC-1α expression were 2.8-fold and 3.0-fold higher in control versus aged myotubes over 24 h and 48 h (P<0.05), respectively. Branched chain amino acids L-leucine, L-isoleucine and L-valine, and L-carnitine were less abundant in aged versus control myotubes. Conclusion(s) Replicative ageing is associated with bioenergetic uncoupling, increased ROS production and impaired amino acid metabolism. Our findings suggest that cellular mitochondrial dysfunction and altered energy metabolism may exacerbate the age-related decline in skeletal muscle mass and function.

The α-ketoglutarate-dependent dioxygenase, prolyl-4-hydroxylase 3 (PHD3), is an HIF target that uses molecular oxygen to hydroxylate peptidyl prolyl residues. Although PHD3 has been reported to influence cancer cell metabolism and liver insulin sensitivity, relatively little is known about the effects of this highly conserved enzyme in insulin-secreting β cells in vivo. Here, we show that the deletion of PHD3 specifically in β cells (βPHD3KO) was associated with impaired glucose homeostasis in mice fed a high-fat diet. In the early stages of dietary fat excess, βPHD3KO islets energetically rewired, leading to defects in the management of pyruvate fate and a shift from glycolysis to increased fatty acid oxidation (FAO). However, under more prolonged metabolic stress, this switch to preferential FAO in βPHD3KO islets was associated with impaired glucose-stimulated ATP/ADP rises, Ca2+ fluxes, and insulin secretion. Thus, PHD3 might be a pivotal component of the β cell glucose metabolism machinery in mice by suppressing the use of fatty acids as a primary fuel source during the early phases of metabolic stress.

8 1 Institute of Metabolism and Systems Research (IMSR), University of Birmingham, 9 Birmingham, UK. 10 2 Centre for Endocrinology, Diabetes and Metabolism, Birmingham Health Partners, 11 Birmingham, UK. 12 3 Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham, 13 Birmingham, UK. 14 4 Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, 15 UK. 16 5 Oxford NIHR Biomedical Research Centre, Churchill Hospital, Oxford, UK. 17 6 Department of Physiology, Anatomy and Genetics, University of Oxford, Parks Road, Oxford, 18 UK. 19 7 Mitochondrial Profiling Centre, School of Sport, Exercise and Rehabilitation Sciences, 20 University of Birmingham, Birmingham, UK. 21 8 School of Pharmacy, University of Waterloo, Kitchener, Ontario, Canada. 22 9 Division of Systems Medicine, School of Medicine, University of Dundee, Dundee, UK. 23 24 These authors contributed equally 25

AIMS/HYPOTHESIS:This study aimed to examine if beta-aminoisobutyric acid (BAIBA) is (i) secreted by skeletal muscle in humans during exercise, (ii) associated with insulin secretory function in vivo, and (iii) directly linked with acute glucose-mediated insulin release by pancreatic beta cells in vitro. METHODS:Following 2-weeks of single-leg immobilization, plasma BAIBA concentrations were measured in the brachial artery and the femoral veins of each leg in healthy male subjects, at rest and during two-legged dynamic knee-extensor exercise. During a 2-h hyperglycamic clamp, insulin secretory function and levels of plasma BAIBA were assessed in non-diabetic individuals, non-diabetic individuals following 24-h hyperglycemia and patients with type 2 diabetes. Direct effects of BAIBA on acute glucose-mediated insulin release were probed in INS-1832/3 cells under normal and 'diabetes-like' conditions. Finally, the effect of BAIBA on mitochondrial function was assessed in INS-1832/3 cells using extracellular flux analysis. RESULTS:(i) BAIBA is released from skeletal muscle at rest and during exercise under healthy conditions but is suppressed during exercise following leg immobilization, (ii) plasma BAIBA concentrations inversely associate with insulin secretory function in humans, (iii) BAIBA lowers mitochondrial energy metabolism in INS-1 832/3 cells in parallel with decreased insulin secretionConclusion/interpretation: BAIBA is a myokine released by skeletal muscle during exercise and indepedantly alters the triggering pathway of insulin secretion in cultured INS-1832/3 cells.

Mitophagy is a key process regulating mitochondrial quality control. Several mechanisms have been proposed to regulate mitophagy, but these have mostly been studied using stably expressed non-native proteins in immortalized cell lines. In skeletal muscle, mitophagy and its molecular mechanisms require more thorough investigation. To measure mitophagy directly, we generated a stable skeletal muscle C2C12 cell line, expressing a mitophagy reporter construct (mCherry-green fluorescence protein-mtFIS1(101-152)). Here, we report that both carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment and adenosine monophosphate activated protein kinase (AMPK) activation by 991 promote mitochondrial fission via phosphorylation of MFF and induce mitophagy by similar to 20%. Upon CCCP treatment, but not 991, ubiquitin phosphorylation, a read-out of PTEN-induced kinase 1 (PINK1) activity, and Parkin E3 ligase activity toward CDGSH iron sulfur domain 1 (CISD1) were increased. Although the PINK1-Parkin signaling pathway is active in response to CCCP treatment, we observed no change in markers of mitochondrial protein content. Interestingly, our data shows that TANK-binding kinase 1 (TBK1) phosphorylation is increased after both CCCP and 991 treatments, suggesting TBK1 activation to be independent of both PINK1 and Parkin. Finally, we confirmed in non-muscle cell lines that TBK1 phosphorylation occurs in the absence of PINK1 and is regulated by AMPK-dependent signaling. Thus, AMPK activation promotes mitophagy by enhancing mitochondrial fission (via MFF phosphorylation) and autophagosomal engulfment (via TBK1 activation) in a PINK1-Parkin independent manner.

New Findings What is the topic of this review? This review discusses the evidence of the benefits of exercise training for beta-cell health through improvements in function, proliferation and survival which may have implications in the treatment of diabetes.

Skeletal muscle is an endocrine organ that secretes a variety of compounds including proteins (myokines), metabolites, microRNAs (miRNAs), and exosomes, many of which are regulated by exercise and play important roles in endocrine signaling. Interorgan communication via muscle-secreted factors therefore provides a novel area for investigation and implicates the importance of skeletal muscle in the pathophysiology of metabolic diseases such as type 2 diabetes (T2D). Given that underlying molecular mechanisms of T2D are subject of ongoing research, in light of new evidence it is probable that interorgan cross-talk between skeletal muscle and pancreatic β-cells plays an important part. To date, the number of studies published in this field provide the basis of this review. Specifically, we discuss current experimental evidence in support for a role of skeletal muscle to β-cell cross-talk, paying particular attention to muscle-secreted factors including myokines, metabolites, miRNAs, and factors contained within exosomes that influence the function and/or the survival of β-cells in health and disease. In reviewing this evidence, we provide an update on the list of known muscle-secreted factors that have potential to influence the function and/or survival of β-cells under normal and diabetic conditions. We also report limitations of current cross-talk methods and discuss future directions in this growing field.