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On the interdependence of ketone body oxidation, glycogen content, glycolysis and energy metabolism in the heart.
In heart, glucose and glycolysis are important for anaplerosis and potentially therefore for d-β-hydroxybutyrate (βHB) oxidation. As a glucose store, glycogen may also furnish anaplerosis. We determined the effects of glycogen content on βHB oxidation and glycolytic rates, and their downstream effects on energetics, in the isolated rat heart. High glycogen (HG) and low glycogen (LG) containing hearts were perfused with 11 mM [5-3 H]glucose and/or 4 mM [14 C]βHB to measure glycolytic rates or βHB oxidation, respectively, then freeze-clamped for glycogen and metabolomic analyses. Free cytosolic [NAD+ ]/[NADH] and mitochondrial [Q+ ]/[QH2 ] ratios were estimated using the lactate dehydrogenase and succinate dehydrogenase reaction, respectively. Phosphocreatine (PCr) and inorganic phosphate (Pi ) concentrations were measured using 31 P-nuclear magnetic resonance spectroscopy. Rates of βHB oxidation in LG hearts were half that in HG hearts, with βHB oxidation directly proportional to glycogen content. βHB oxidation decreased glycolysis in all hearts. Glycogenolysis in glycogen-replete hearts perfused with βHB alone was twice that of hearts perfused with βHB and glucose, which had significantly higher levels of the glycolytic intermediates fructose 1,6-bisphosphate and 3-phosphoglycerate, and higher free cytosolic [NAD+ ]/[NADH]. βHB oxidation increased the Krebs cycle intermediates citrate, 2-oxoglutarate and succinate, the total NADP/H pool, reduced mitochondrial [Q+ ]/[QH2 ], and increased the calculated free energy of ATP hydrolysis (∆GATP ). Although βHB oxidation inhibited glycolysis, glycolytic intermediates were not depleted, and cytosolic free NAD remained oxidised. βHB oxidation alone increased Krebs cycle intermediates, reduced mitochondrial Q and increased ∆GATP . We conclude that glycogen facilitates cardiac βHB oxidation by anaplerosis. KEY POINTS: Ketone bodies (d-β-hydroxybutyrate, acetoacetate) are increasingly recognised as important cardiac energetic substrates, in both healthy and diseased hearts. As 2-carbon equivalents they are cataplerotic, causing depletion of Krebs cycle intermediates; therefore their utilisation requires anaplerotic supplementation, and intra-myocardial glycogen has been suggested as a potential anaplerotic source during ketone oxidation. It is demonstrated here that cardiac glycogen does indeed provide anaplerotic substrate to facilitate β-hydroxybutyrate oxidation in isolated perfused rat heart, and this contribution was quantified using a novel pulse-chase metabolic approach. Further, using metabolomics and 31 P-MR, it was shown that glycolytic flux from myocardial glycogen increased the heart's ability to oxidise βHB, and βHB oxidation increased the mitochondrial redox potential, ultimately increasing the free energy of ATP hydrolysis.
Abnormal whole-body energy metabolism in iron-deficient humans despite preserved skeletal muscle oxidative phosphorylation.
Iron deficiency impairs skeletal muscle metabolism. The underlying mechanisms are incompletely characterised, but animal and human experiments suggest the involvement of signalling pathways co-dependent upon oxygen and iron availability, including the pathway associated with hypoxia-inducible factor (HIF). We performed a prospective, case-control, clinical physiology study to explore the effects of iron deficiency on human metabolism, using exercise as a stressor. Thirteen iron-deficient (ID) individuals and thirteen iron-replete (IR) control participants each underwent 31P-magnetic resonance spectroscopy of exercising calf muscle to investigate differences in oxidative phosphorylation, followed by whole-body cardiopulmonary exercise testing. Thereafter, individuals were given an intravenous (IV) infusion, randomised to either iron or saline, and the assessments repeated ~ 1 week later. Neither baseline iron status nor IV iron significantly influenced high-energy phosphate metabolism. During submaximal cardiopulmonary exercise, the rate of decline in blood lactate concentration was diminished in the ID group (P = 0.005). Intravenous iron corrected this abnormality. Furthermore, IV iron increased lactate threshold during maximal cardiopulmonary exercise by ~ 10%, regardless of baseline iron status. These findings demonstrate abnormal whole-body energy metabolism in iron-deficient but otherwise healthy humans. Iron deficiency promotes a more glycolytic phenotype without having a detectable effect on mitochondrial bioenergetics.
The effects of endogenously- and exogenously-induced hyperketonemia on exercise performance and adaptation.
Elevating blood ketones may enhance exercise capacity and modulate adaptations to exercise training; however, these effects may depend on whether hyperketonemia is induced endogenously through dietary carbohydrate restriction, or exogenously through ketone supplementation. To determine this, we compared the effects of endogenously- and exogenously-induced hyperketonemia on exercise capacity and adaptation. Trained endurance athletes undertook 6 days of laboratory based cycling ("race") whilst following either: a carbohydrate-rich control diet (n = 7; CHO); a carbohydrate-rich diet + ketone drink four-times daily (n = 7; Ex Ket); or a ketogenic diet (n = 7; End Ket). Exercise capacity was measured daily, and adaptations in exercise metabolism, exercise physiology and postprandial insulin sensitivity (via an oral glucose tolerance test) were measured before and after dietary interventions. Urinary β-hydroxybutyrate increased by ⁓150-fold and ⁓650-fold versus CHO with Ex Ket and End Ket, respectively. Exercise capacity was increased versus pre-intervention by ~5% on race day 1 with CHO (p 0.05) with End Ket. There was an ⁓3-fold increase in fat oxidation from pre- to post-intervention (p
Unlocking Intracellular Protein Delivery by Harnessing Polymersomes Synthesized at Microliter Volumes using Photo-PISA.
Efficient delivery of therapeutic proteins and vaccine antigens to intracellular targets is challenging due to generally poor cell membrane permeation and endolysosomal entrapment causing degradation. Herein, these challenges are addressed by developing an oxygen-tolerant photoinitiated polymerization-induced self-assembly (Photo-PISA) process, allowing for the microliter-scale (10 µL) synthesis of protein-loaded polymersomes directly in 1536-well plates. High-resolution techniques capable of analysis at a single particle level are employed to analyze protein encapsulation and release mechanisms. Using confocal microscopy and super-resolution stochastic optical reconstruction microscopy (STORM) imaging, their ability to deliver proteins into the cytosol following endosomal escape is subsequently visualized. Lastly, the adaptability of these polymersomes is exploited to encapsulate and deliver a prototype vaccine antigen, demonstrating its ability to activate antigen-presenting cells and support antigen cross-presentation for applications in subunit vaccines and cancer immunotherapy. This combination of ultralow volume synthesis and efficient intracellular delivery holds significant promise for unlocking the high throughput screening of a broad range of otherwise cost-prohibitive or early-stage therapeutic protein and vaccine antigen candidates that can be difficult to obtain in large quantities. The versatility of this platform for rapid screening of intracellular protein delivery can result in significant advancements across the fields of nanomedicine and biomedical engineering.
Transfer learning Bayesian optimization for competitor DNA molecule design for use in diagnostic assays.
With the rise in engineered biomolecular devices, there is an increased need for tailor-made biological sequences. Often, many similar biological sequences need to be made for a specific application meaning numerous, sometimes prohibitively expensive, lab experiments are necessary for their optimization. This paper presents a transfer learning design of experiments workflow to make this development feasible. By combining a transfer learning surrogate model with Bayesian optimization, we show how the total number of experiments can be reduced by sharing information between optimization tasks. We demonstrate the reduction in the number of experiments using data from the development of DNA competitors for use in an amplification-based diagnostic assay. We use cross-validation to compare the predictive accuracy of different transfer learning models, and then compare the performance of the models for both single objective and penalized optimization tasks.
Cardiomyocyte-derived C-type natriuretic peptide diminishes myocardial ischaemic injury by promoting revascularisation and limiting fibrotic burden.
BACKGROUND: C-type natriuretic peptide (CNP) is a significant player in the maintenance of cardiac and vascular homeostasis regulating local blood flow, platelet and leukocyte activation, heart structure and function, angiogenesis and metabolic balance. Since such processes are perturbed in myocardial infarction (MI), we explored the role of cardiomyocyte-derived CNP, and pharmacological administration of the peptide, in offsetting the pathological consequences of MI. METHODS: Wild type (WT) and cardiomyocyte-restricted CNP null (cmCNP-/-) mice were subjected to left anterior descending coronary artery (LADCA) ligation and acute effects on infarct size and longer-term outcomes of cardiac repair explored. Heart structure and function were assessed by combined echocardiographic and molecular analyses. Pharmacological administration of CNP (0.2 mg/kg/day; s.c.) was utilized to assess therapeutic potential. RESULTS: Compared to WT littermates, cmCNP-/- mice had a modestly increased infarct size following LADCA ligation but without significant deterioration of cardiac structural and functional indices. However, cmCNP-/- animals exhibited overtly worse heart morphology and contractility 6 weeks following MI, with particularly deleterious reductions in left ventricular ejection fraction, dilatation, fibrosis and revascularization. This phenotype was largely recapitulated in animals with global deletion of natriuretic peptide receptor (NPR)-C (NPR-C-/-). Pharmacological administration of CNP rescued the deleterious pathology in WT and cmCNP-/-, but not NPR-C-/-, animals. CONCLUSIONS AND IMPLICATIONS: Cardiomyocytes synthesize and release CNP as an intrinsic protective mechanism in response to MI that reduces cardiac structural and functional deficits; these salutary actions are primarily NPR-C-dependent. Pharmacological targeting of CNP may represent a new therapeutic option for MI.
Tunable Hybrid Hydrogels of Alginate and Cell-Derived dECM to Study the Impact of Matrix Alterations on Epithelial-to-Mesenchymal Transition.
Epithelial-to-mesenchymal transition (EMT) is crucial for tumor progression, being linked to alterations in the extracellular matrix (ECM). Understanding the ECM's role in EMT can uncover new therapeutic targets, yet replicating these interactions in vitro remains challenging. It is shown that hybrid hydrogels of alginate (ALG) and cell-derived decellularized ECM (dECM), with independently tunable composition and stiffness, are useful 3D-models to explore the impact of the breast tumor matrix on EMT. Soft RGD-ALG hydrogels (200 Pa), used as neutral bulk material, supported mammary epithelial cells morphogenesis without spontaneous EMT, allowing to define the gene, protein, and biochemical profiles of cells at different TGFβ1-induced EMT states. To mimic the breast tumor composition, dECM from TGFβ1-activated fibroblasts (adECM) are generated, which shows upregulation of tumor-associated proteins compared to ndECM from normal fibroblasts. Using hybrid adECM-ALG hydrogels, it is shown that the presence of adECM induces partial EMT in normal epithelial cells, and amplifes TGF-β1 effects compared to ALG and ndECM-ALG. Increasing the hydrogel stiffness to tumor-like levels (2.5 kPa) have a synergistic effect, promoting a more evident EMT. These findings shed light on the complex interplay between matrix composition and stiffness in EMT, underscoring the utility of dECM-ALG hydrogels as a valuable in vitro platform for cancer research.
Comparative Analysis of Volatile Components in Chi-Nan and Ordinary Agarwood Aromatherapies: Implications for Sleep Improvement.
Agarwood, a precious traditional medicinal herb and fragrant material, is known for its sedative and sleep-improving properties. This study explores the mechanisms underlying the aromatherapy effects of Chi-Nan agarwood and ordinary agarwood in improving sleep. Using a combination of gas chromatography-mass spectrometry (GC-MS), network pharmacology, and molecular docking techniques, we identified and c ompared the chemical compositions and potential molecular targets of both types of agarwood. The GC-MS analysis detected 87 volatile components across six types of agarwood aromatherapy, with 51 shared between Chi-Nan and ordinary agarwood, while each type also had 18 unique components. Ordinary agarwood was found to be richer in sesquiterpenes and small aromatic molecules, whereas Chi-Nan agarwood contained higher levels of chromones. These differences in chemical composition are likely responsible for the distinct sleep-improving effects observed between the two types of agarwood. Through network pharmacology, 100, 65, and 47 non-repetitive target genes related to sleep improvement were identified for components shared by both types of agarwood (CSBTs), components unique to common agarwood (CUCMs), and components unique to Chi-Nan agarwood (CUCNs), respectively. The constructed protein-protein interaction (PPI) networks revealed that key targets such as MAOA, MAOB, SLC6A4, and ESR1 are involved in the sleep-improving mechanisms of agarwood aromatherapy. Molecular docking further confirmed the strong binding affinities of major active components, such as 5-Isopropylidene-6-methyldeca-369-trien-2-one and 2-(2-Phenylethyl)chromone, with these core targets. The results suggest that agarwood aromatherapy enhances sleep quality through both hormonal and neurotransmitter pathways, with ordinary agarwood more deeply mediating hormonal regulation, while Chi-Nan agarwood predominantly influences neurotransmitter pathways, particularly those involving serotonin and GABA. This study provides valuable insights into the distinct therapeutic potentials of Chi-Nan and ordinary agarwood, highlighting their roles in sleep improvement and offering a foundation for future research in the clinical application of agarwood-based aromatherapy.
Lipid Droplets Big and Small: Basic Mechanisms That Make Them All.
Lipid droplets (LDs) are dynamic storage organelles with central roles in lipid and energy metabolism. They consist of a core of neutral lipids, such as triacylglycerol, which is surrounded by a monolayer of phospholipids and specialized surface proteins. The surface composition determines many of the LD properties, such as size, subcellular distribution, and interaction with partner organelles. Considering the diverse energetic and metabolic demands of various cell types, it is not surprising that LDs are highly heterogeneous within and between cell types. Despite their diversity, all LDs share a common biogenesis mechanism. However, adipocytes have evolved specific adaptations of these basic mechanisms, enabling the regulation of lipid and energy metabolism at both the cellular and organismal levels. Here, we discuss recent advances in the understanding of both the general mechanisms of LD biogenesis and the adipocyte-specific adaptations controlling these fascinating organelles.
Approaches to Early Parkinson's Disease Subtyping.
Parkinson's disease (PD) unfolds with pathological processes and neurodegeneration well before the emergence of noticeable motor symptoms, providing a window for early identification. The extended prodromal phase allows the use of risk stratification measures and prodromal markers to pinpoint individuals likely to develop PD. Importantly, a growing body of evidence emphasizes the heterogeneity within prodromal and clinically diagnosed PD. The disease likely comprises distinct subtypes exhibiting diverse clinical manifestations, pathophysiological mechanisms, and patterns of α-synuclein progression in the central and peripheral nervous systems. There is a pressing need to refine the definition and early identification of these prodromal subtypes. This requires a comprehensive strategy that integrates genetic, pathological, imaging, and multi-omics markers, alongside careful observation of subtle motor and non-motor symptoms. Such multi-dimensional classification of early PD subtypes will improve our understanding of underlying disease pathophysiology, improve predictions of clinical endpoints, progression trajectory and medication response, contribute to drug discovery and personalized medicine by identifying subtype-specific disease mechanisms, and facilitate drug trials by reducing confounding effects of heterogeneity. Here we explore different subtyping methodologies in prodromal and clinical PD, focusing on clinical, imaging, genetic and molecular subtyping approaches. We also emphasize the need for refined, theoretical a priori disease models. These will be prerequisite to understanding the biological underpinnings of biological subtypes, which have been defined by large scale data-driven approaches and multi-omics fingerprints.
Glial cells undergo rapid changes following acute chemogenetic manipulation of cortical layer 5 projection neurons.
Bidirectional communication between neurons and glial cells is crucial to establishing and maintaining normal brain function. Some of these interactions are activity-dependent, yet it remains largely unexplored how acute changes in neuronal activity affect glial-to-neuron and neuron-to-glial dynamics. Here, we use excitatory and inhibitory designer receptors exclusively activated by designer drugs (DREADD) to study the effects of acute chemogenetic manipulations of a subpopulation of layer 5 cortical projection and dentate gyrus neurons in adult (Rbp4Cre) mouse brains. We show that acute chemogenetic neuronal activation reduces synaptic density, and increases microglia and astrocyte reactivity, but does not affect parvalbumin (PV+) neurons, only perineuronal nets (PNN). Conversely, acute silencing increases synaptic density and decreases glial reactivity. We show fast glial response upon clozapine-N-oxide (CNO) administration in cortical and subcortical regions. Together, our work provides evidence of fast, activity-dependent, bidirectional interactions between neurons and glial cells.
In Vivo Two-Photon Microscopy Reveals Sensory-Evoked Serotonin (5-HT) Release in Adult Mammalian Neocortex.
The recent development of genetically encoded fluorescent neurotransmitter biosensors has opened the door to recording serotonin (5-hydroxytryptamine, 5-HT) signaling dynamics with high temporal and spatial resolution in vivo. While this represents a significant step forward for serotonin research, the utility of available 5-HT biosensors remains to be fully established under diverse in vivo conditions. Here, we used two-photon microscopy in awake mice to examine the effectiveness of specific 5-HT biosensors for monitoring 5-HT dynamics in somatosensory cortex. Initial experiments found that whisker stimulation evoked a striking change in 5-HT biosensor signal. However, similar changes were observed in controls expressing green fluorescent protein, suggesting a potential hemodynamic artifact. Subsequent use of a second control fluorophore with emission peaks separated from the 5-HT biosensor revealed a reproducible, stimulus-locked increase in 5-HT signal. Our data highlight the promise of 5-HT biosensors for in vivo application, provided measurements are carried out with appropriate optical controls.
Midbrain dopamine neurons signal phasic and ramping reward prediction error during goal-directed navigation.
Goal-directed navigation requires learning to accurately estimate location and select optimal actions in each location. Midbrain dopamine neurons are involved in reward value learning and have been linked to reward location learning. They are therefore ideally placed to provide teaching signals for goal-directed navigation. By imaging dopamine neural activity as mice learned to actively navigate a closed-loop virtual reality corridor to obtain reward, we observe phasic and pre-reward ramping dopamine activity, which are modulated by learning stage and task engagement. A Q-learning model incorporating position inference recapitulates our results, displaying prediction errors resembling phasic and ramping dopamine neural activity. The model predicts that ramping is followed by improved task performance, which we confirm in our experimental data, indicating that the dopamine ramp may have a teaching effect. Our results suggest that midbrain dopamine neurons encode phasic and ramping reward prediction error signals to improve goal-directed navigation.