Biology subjects

Thakker, A.

Publications and source records attributed to Thakker, A..

4 recordsLinked to original sources

bioRxiv2020

Macrovesicular steatosis in nonalcoholic fatty liver disease is a consequence of purine nucleotide cycle driven fumarate accumulation

SummaryNonalcoholic 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 StatementProfessor David Hay is a founder, shareholder and director in Stemnovate Limited.View Full Text

biochemistry↗

bioRxiv2020

The prolyl hydroxylase PHD3 maintains β-cell glucose metabolism during fatty acid excess

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 {beta}-cells. Here, we show that deletion of PHD3 specifically in {beta}-cells ({beta}PHD3KO) is associated with impaired glucose homeostasis in mice fed high fat diet. In the early stages of dietary fat excess, {beta}PHD3KO islets energetically rewire, leading to defects in the management of pyruvate fate and a shift away from glycolysis. However, {beta}PHD3KO 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 {beta}PHD3KO 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 {beta}-cell glucose metabolism machinery by suppressing the use of fatty acids as a primary fuel source, under obesogenic and insulin resistant states. SIGNIFICANCE STATEMENTProlyl-4-hydroxylase 3 (PHD3) is involved in the oxygen-dependent regulation of cell phenotype. A number of recent studies have shown that PHD3 might operate at the interface between oxygen availability and metabolism. To understand how PHD3 influences insulin secretion, which depends on intact glucose metabolism, we generated mice lacking PHD3 specifically in pancreatic {beta}-cells. These mice, termed {beta}PHD3KO, are apparently normal until fed high fat diet at which point their {beta}-cells switch to fatty acids as a fuel source. This switch cannot be tolerated and {beta}-cells in {beta}PHD3KO mice eventually fail. Thus, PHD3 maintains glucose-stimulated insulin secretion in {beta}-cells during states of fatty acid excess, such as diabetes and obesity.

physiology↗

bioRxiv2019

Serial coupling of chromatographic columns having orthogonal selectivity to improve the coverage of oxidised lipidome for mass spectrometry analysis

1Phospholipid 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.\n\nThe 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.

biochemistry↗

bioRxiv2019

A Targeted Mass Spectrometry Approach to detect and quantify Oxidised Phospholipids in plasma samples of Diabetic patients

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.\n\nThe 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.\n\nTargeted 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: {gamma}-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.

biochemistry↗