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Intracellular Na elevation in the heart is a hallmark of pathologies where both acute and chronic metabolic remodelling occurs. Here, we assess whether acute (75 μM ouabain 100 nM blebbistatin) or chronic myocardial Na i load (PLM 3SA mouse) are causally linked to metabolic remodelling and whether the failing heart shares a common Na-mediated metabolic ‘fingerprint’. Control (PLM WT ), transgenic (PLM 3SA ), ouabain-treated and hypertrophied Langendorff-perfused mouse hearts are studied by 23 Na, 31 P, 13 C NMR followed by 1 H-NMR metabolomic profiling. Elevated Na i leads to common adaptive metabolic alterations preceding energetic impairment: a switch from fatty acid to carbohydrate metabolism and changes in steady-state metabolite concentrations (glycolytic, anaplerotic, Krebs cycle intermediates). Inhibition of mitochondrial Na/Ca exchanger by CGP37157 ameliorates the metabolic changes. In silico modelling indicates altered metabolic fluxes (Krebs cycle, fatty acid, carbohydrate, amino acid metabolism). Prevention of Na i overload or inhibition of Na/Ca mito may be a new approach to ameliorate metabolic dysregulation in heart failure. The failing heart is characterised by both alterations in mitochondrial metabolism and an elevation of cytosolic sodium. Here, the authors use 23 Na NMR and metabolic profiling to show these are related, and that elevation in intracellular Na reprograms cardiac substrate utilisation via effects on mitochondrial Na/Ca exchange.

Biological characterisation of membrane proteins lags behind that of soluble proteins. This reflects issues with the traditional use of detergents for extraction, as the surrounding lipids are generally lost, with adverse structural and functional consequences. In contrast, styrene maleic acid (SMA) copolymers offer a detergent-free method for biological membrane solubilisation to produce SMA-lipid particles (SMALPs) containing membrane proteins together with their surrounding lipid environment. We report the development of a reverse-phase LC-MS/MS method for bacterial phospholipids and the first comparison of the profiles of SMALP co-extracted phospholipids from three exemplar bacterial membrane proteins with different topographies: FtsA (associated membrane protein), ZipA (single transmembrane helix), and PgpB (integral membrane protein). The data showed that while SMA treatment per se did not preferentially extract specific phospholipids from the membrane, SMALP-extracted ZipA showed an enrichment in phosphatidylethanolamines and depletion in cardiolipins compared to the bulk membrane lipid. Comparison of the phospholipid profiles of the 3 SMALP-extracted proteins revealed distinct lipid compositions for each protein: ZipA and PgpB were similar, but in FtsA samples longer chain phosphatidylglycerols and phosphatidylethanolamines were more abundant. This method offers novel information on the phospholipid interactions of these membrane proteins.

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.

Background: Skeletal muscle is central to whole body metabolic homeostasis, with age and disease impairing its ability to function appropriately to maintain health. Inadequate NAD + availability is proposed to contribute to pathophysiology by impairing metabolic energy pathway use. Despite the importance of NAD + as a vital redox cofactor in energy production pathways being well-established, the wider impact of disrupted NAD + homeostasis on these pathways is unknown. Methods: We utilised skeletal muscle myotube models to induce NAD + depletion, repletion and excess and conducted metabolic tracing to provide comprehensive and detailed analysis of the consequences of altered NAD + metabolism on central carbon metabolic pathways. We used stable isotope tracers, [1,2-13C] D-glucose and [U- 13 C] glutamine, and conducted combined 2D-1H,13C-heteronuclear single quantum coherence (HSQC) NMR spectroscopy and GC-MS analysis. Results: NAD + excess driven by nicotinamide riboside (NR) supplementation within skeletal muscle cells resulted in enhanced nicotinamide clearance, but had no effect on energy homeostasis or central carbon metabolism. Nicotinamide phosphoribosyltransferase (NAMPT) inhibition induced NAD + depletion and resulted in equilibration of metabolites upstream of glyceraldehyde phosphate dehydrogenase (GAPDH). Aspartate production through glycolysis and TCA cycle activity was increased in response to low NAD + , which was rapidly reversed with repletion of the NAD + pool using NR. NAD + depletion reversibly inhibits cytosolic GAPDH activity, but retains mitochondrial oxidative metabolism, suggesting differential effects of this treatment on sub-cellular pyridine pools. When supplemented, NR efficiently reversed these metabolic consequences. However, the functional relevance of increased aspartate levels after NAD + depletion remains unclear, and requires further investigation. Conclusions: These data highlight the need to consider carbon metabolism and clearance pathways when investigating NAD + precursor usage in models of skeletal muscle physiology.

Phospholipid oxidation generates a wide variety of products with potentially novel biological activities that may be associated with disease pathogenesis. To understand their role in disease requires precise information about their abundance in biological samples. Liquid chromatography-mass spectrometry (LCMS) is a sensitive technique that can provide detailed information about the oxidative lipidome, but challenges still remain. The work in this thesis developed improved methods for detection of OxPLs by improvement of chromatographic separation through the comparison and optimisation of several HPLC columns such as C8, C18 and C30 reverse phase, polystyrene-divinylbenzene based monolithic, and mixed-mode hydrophilic interaction (HILIC) columns & solvent systems, with use of semi-targeted mass spectrometry approaches. The results suggests that the monolithic column was the most robust method for separating short chain oxPLs from long chain oxidised and native PLs. In addition, several approaches for method validation were explored such as testing of reproducibility and repeatability of the methods, together with the reanalysis of samples on a high resolution QToF mass spectrometer with automated quantitative data analysis using the Progenesis QI software to validate the identification. The combination of the developed methods allowed the identification of several oxPLs in biological samples. These were: i) ascites fluid of lean and obese rat model of acute pancreatitis; ii) isolated components of red blood cells (RBCs) infected with the malarial parasite Plasmodium falciparum; and iii) plasma samples of healthy and diabetic patients. In addition, an evaluation of post-acquisition data handling to minimise inherent biological variation was performed. Quantitative differences in oxPLs were observed in isolated malarial components as well as other studied disease models. Overall, several protocols were developed that provide improved performance for the identification of OxPL in biological samples that can be used as a reference method by research laboratories interested in oxidative lipidomics work.

Background: Skeletal muscle is central to whole body metabolic homeostasis, with age and disease impairing its ability to function appropriately to maintain health. Inadequate NAD + availability is proposed to contribute to pathophysiology by impairing metabolic energy pathway use. Despite the importance of NAD + as a vital redox cofactor in energy production pathways being well-established, the wider impact of disrupted NAD + homeostasis on these pathways is unknown. Methods: We utilised skeletal muscle myotube models to induce NAD + depletion, repletion and excess and conducted metabolic tracing to provide comprehensive and detailed analysis of the consequences of altered NAD + metabolism on central carbon metabolic pathways. We used stable isotope tracers, [1,2-13C] D-glucose and [U- 13 C] glutamine, and conducted combined 2D-1H,13C-heteronuclear single quantum coherence (HSQC) NMR spectroscopy and GC-MS analysis. Results: NAD + excess driven by nicotinamide riboside (NR) supplementation within skeletal muscle cells results in enhanced nicotinamide clearance, but had no effect on energy homeostasis or central carbon metabolism. Nicotinamide phosphoribosyltransferase (NAMPT) inhibition induced NAD + depletion and resulted in equilibration of metabolites upstream of glyceraldehyde phosphate dehydrogenase (GAPDH). Aspartate production through glycolysis and TCA cycle activity is increased in response to low NAD + , which is rapidly reversed with repletion of the NAD + pool using NR. NAD + depletion reversibly inhibits cytosolic GAPDH activity, but retains mitochondrial oxidative metabolism, suggesting differential effects of this treatment on sub-cellular pyridine pools. When supplemented, NR efficiently reverses these metabolic consequences. However, the functional relevance of increased aspartate levels after NAD + depletion remains unclear, and requires further investigation. Conclusions: These data highlight the need to consider carbon metabolism and clearance pathways when investigating NAD + precursor usage in models of skeletal muscle physiology.

Phospholipid oxidation (OxPL) generates a wide variety of products with potentially novel biological activities that may be associated with disease pathogenesis. To understand their role in disease requires precise information about their abundance in biological samples. Liquid chromatography-mass spectrometry (LCMS) is a sensitive technique that can provide detailed information about the oxidative lipidome, but challenges remain. Furthermore, variation in charge of the polar head groups and the extreme diversity of oxidised species make analysis of several classes of OxPLs within one analytical run challenging. The work in this study aims to develop improved methods for detection of OxPLs by improvement of chromatographic separation through the serial coupling of polystyrene-divinylbenzene based monolithic, and mixed-mode hydrophilic interaction (HILIC) with use of semi-targeted mass spectrometry approaches. The results suggests that by serially coupling two columns, HILIC and monolith, provided the better coverage of OxPL species in a single analytical run. We tested in-vitro generated oxidized species for phosphatidylcholine (PC) and phosphatidylethanolamine (PE) class and the combination of orthogonal chromatographic separation allowed separation of oxdised species from both the classes, which otherwise coeluted.

Phospholipid oxidation by adventitious damage generates a wide variety of products with potentially novel biological activities that can modulate inflammatory processes associated with various diseases such as atherosclerosis, acute Pancreatitis and Type 2 diabetes. To understand the biological importance of oxidised phospholipids (OxPL) and their potential role as disease biomarkers requires precise information about the abundance of these compounds in cells and tissues. There are many chemiluminescence and spectroscopic assays available for detecting oxidised phospholipids, but they all have some limitations. Mass spectrometry coupled with liquid chromatography is a powerful and sensitive approach but its application to complex biological samples remains challenging. The aim of this work was to develop improved methods for detection of OxPLs, specifically by using targeted mass spectrometry approaches (precursor ion [PIS] and neutral loss [NL] scanning), high resolution mass spectrometry and alternative chromatographic approaches. Initial experiments were carried out using oxidation products generated in vitro from a commercially available phosphatidylcholine (PC) and phosphatidylethanolamine (PE) mixture in order to optimise the chromatography separation parameters and mass spectrometry parameters. The chromatographic separation of oxidised phosphatidylcholines (OxPCs) and oxidised phosphatidylethanolamines (OXPEs) was evaluated using C8, C18 and C30 reverse phase, polystyrene divinylbenzene based monolithic and mixed mode hydrophilic interaction (HILIC) columns, interfaced with mass spectrometry. Our results suggest that the divinylbenzene based reverse phase monolithic column gave best separation of short chain OxPCs and OxPEs from long chain oxidised and native PCs and PEs. Targeted mass spectrometric approaches for the selective identification of short chain OxPCs using PIS for m/z 184 Da and NL for m/z 34 Da for identification of hydroperoxides were tested on OxPC mixture, it enabled identification of low abundant oxidation products such as: hydroxy alkenals and alkenoates and saturated aldehydes collectively termed as short chain oxidation products such as PONPC, POVPC and HOOA-PC. The combination of these chromatographic and MS methods allowed identification of several oxidised molecular species in plasma of diabetic patients. Quantitative differences in oxidised products were observed in diabetic samples and the trend showed high abundance of oxidised phosphatidylcholine species in diabetic samples, compared to healthy plasma samples. However, the difference in abundance was statistically not significant when the samples were analysed using Progenesis QI software, performing global normalisation and ANOVA analysis because of inherent biological variability observed for OxPC species in samples.

The alpha ketoglutarate-dependent dioxygenase, prolyl-4-hydroxylase 3 (PHD3), is a hypoxia-inducible factor target that uses molecular oxygen to hydroxylate proline. While PHD3 has been reported to influence cancer cell metabolism and liver insulin sensitivity, relatively little is known about effects of this highly conserved enzyme in insulin-secreting β-cells. Here, we show that deletion of PHD3 specifically in β-cells (βPHD3KO) is associated with impaired glucose homeostasis in mice fed high fat diet. In the early stages of dietary fat excess, βPHD3KO islets energetically rewire, leading to defects in the management of pyruvate fate and a shift away from glycolysis. However, βPHD3KO islets are able to maintain oxidative phosphorylation and insulin secretion by increasing utilization of fatty acids to supply the tricarboxylic acid cycle. This nutrient-sensing switch cannot be sustained and βPHD3KO islets begin to show signs of failure in response to prolonged metabolic stress, including 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 by suppressing the use of fatty acids as a primary fuel source under obesogenic and insulin resistant states. Competing Interest Statement The authors have declared no competing interest.

Nonalcoholic fatty liver disease (NAFLD) affects ~88% of obese individuals and is characterised by hepatic lipid accumulation. Mitochondrial metabolic dysfunction is a feature of NAFLD. We used a human pluripotent stem cell-based system to determine how mitochondrial dysfunction is linked to hepatic lipid accumulation. We induced lipid accumulation in hepatocyte-like cells (HLCs) using lactate, pyruvate and octanoate (LPO). Transcriptomic analysis revealed perturbation of mitochondrial respiratory pathways in LPO exposed cells. Using 13C isotopic tracing, we identified truncation of the TCA cycle in steatotic HLCs. We show that increased purine nucleotide cycle (PNC) activity fuels fumarate accumulation and drives lipid accumulation in steatotic cells. These findings provide new insights into the pathogenesis of hepatic steatosis and may lead to an improved understanding of the metabolic and transcriptional rewiring associated with NAFLD. Competing Interest Statement Professor David Hay is a founder, shareholder and director in Stemnovate Limited.