Alice P. Sowton

The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic, but pericellular oxygen levels, which are affected by oxygen diffusivity and consumption, are rarely reported. Here, we provide evidence that several cell types in culture actually experience local hypoxia, with important implications for cell metabolism and function. We focused initially on adipocytes, as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions, cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria, lowered HIF1α activity, and resulted in widespread transcriptional rewiring. Functionally, adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli, recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages, hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types, and that considering pericellular oxygen improves the quality, reproducibility and translatability of culture models.

The metabolic syndrome (MetS) is a cluster of cardiovascular risk factors characterised by central obesity, atherogenic dyslipidaemia, and changes in the circulating lipidome; the underlying mechanisms that lead to this lipid remodelling have only been partially elucidated. This study used an integrated “omics” approach (untargeted whole serum lipidomics, targeted proteomics, and lipoprotein lipidomics) to study lipoprotein remodelling and HDL composition in subjects with central obesity diagnosed with MetS (vs. controls). Compared with healthy subjects, MetS patients showed higher free fatty acids, diglycerides, phosphatidylcholines, and triglycerides, particularly those enriched in products of de novo lipogenesis. On the other hand, the “lysophosphatidylcholines to phosphatidylcholines” and “cholesteryl ester to free cholesterol” ratios were reduced, pointing to a lower activity of lecithin cholesterol acyltransferase (LCAT) in MetS; LCAT activity (directly measured and predicted by lipidomic ratios) was positively correlated with high-density lipoprotein cholesterol (HDL-C) and negatively correlated with body mass index (BMI) and insulin resistance. Moreover, many phosphatidylcholines and sphingomyelins were significantly lower in the HDL of MetS patients and strongly correlated with BMI and clinical metabolic parameters. These results suggest that MetS is associated with an impairment of phospholipid metabolism in HDL, partially led by LCAT, and associated with obesity and underlying insulin resistance. This study proposes a candidate strategy to use integrated “omics” approaches to gain mechanistic insights into lipoprotein remodelling, thus deepening the knowledge regarding the molecular basis of the association between MetS and atherosclerosis.

The increasing global prevalence of diabetes has been accompanied by a rise in diabetes-related conditions. This includes diabetic cardiomyopathy (DbCM), a progressive form of heart disease that occurs with both insulin-dependent (type-1) and insulin-independent (type-2) diabetes and arises in the absence of hypertension or coronary artery disease. Over time, DbCM can develop into overt heart failure. Like other forms of cardiomyopathy, DbCM is accompanied by alterations in metabolism which could lead to further progression of the pathology, with metabolic derangement postulated to precede functional changes in the diabetic heart. Moreover in the case of type-2 diabetes, underlying insulin resistance is likely to prevent the canonical substrate switch of the failing heart away from fatty acid oxidation toward increased use of glycolysis. Analytical chemistry techniques, collectively known as metabolomics, are useful tools for investigating the condition. In this article, we provide a comprehensive review of those studies that have employed metabolomic techniques, namely chromatography, mass spectrometry and nuclear magnetic resonance spectroscopy, to profile metabolic remodeling in the diabetic heart of human patients and animal models. These studies collectively demonstrate that glycolysis and glucose oxidation are suppressed in the diabetic myocardium and highlight a complex picture regarding lipid metabolism. The diabetic heart typically shows an increased reliance on fatty acid oxidation, yet triacylglycerols and other lipids accumulate in the diabetic myocardium indicating probable lipotoxicity. The application of lipidomic techniques to the diabetic heart has identified specific lipid species that become enriched and which may in turn act as plasma-borne biomarkers for the condition. Metabolomics is proving to be a powerful approach, allowing a much richer analysis of the metabolic alterations that occur in the diabetic heart. Careful physiological interpretation of metabolomic results will now be key in order to establish which aspects of the metabolic derangement are causal to the progression of DbCM and might form the basis for novel therapeutic intervention.

Nonalcoholic fatty liver disease (NAFLD) is associated with dietary folate deficiency and mutations in genes required for one‑carbon metabolism. However, the mechanism through which this occurs is unclear. To improve our understanding of this link, we investigated liver morphology, metabolism and fuel storage in adult mice with a hypomorphic mutation in the gene methionine synthase reductase (Mtrrgt). MTRR enzyme is a key regulator of the methionine and folate cycles. The Mtrrgt mutation in mice was previously shown to disrupt one‑carbon metabolism and cause a wide-spectrum of developmental phenotypes and late adult-onset macrocytic anaemia. Here, we showed that livers of Mtrrgt/gt female mice were enlarged compared to control C57Bl/6J livers. Histological analysis of these livers revealed eosinophilic hepatocytes with decreased glycogen content, which was associated with down-regulation of genes involved in glycogen synthesis (e.g., Ugp2 and Gsk3a genes). While female Mtrrgt/gt livers showed evidence of reduced β-oxidation of fatty acids, there were no other associated changes in the lipidome in female or male Mtrrgt/gt livers compared with controls. Defects in glycogen storage and lipid metabolism often associate with disruption of mitochondrial electron transfer system activity. However, defects in mitochondrial function were not detected in Mtrrgt/gt livers as determined by high-resolution respirometry analysis. Overall, we demonstrated that adult Mtrrgt/gt female mice showed abnormal liver morphology that differed from the NAFLD phenotype and that was accompanied by subtle changes in their hepatic metabolism and fuel storage.

Platelet activation plays a key role in normal haemostasis and pathological thrombosis. Platelet activation is rapid; within minutes of stimulation, platelets generate bioactive phospholipids, secrete their granule contents, activate integrins and aggregate together to form a haemostatic plug. These events are dependent on ATP synthesis. Mitochondrial function in platelets from healthy volunteers and patients with a range of diseases indicate an important role for oxygen consumption in oxidative phosphorylation in normal and pathological function. Platelets also consume oxygen during oxidation reactions, such as cyclooxygenase-dependent thromboxane A2 synthesis. In this study, we used high-resolution respirometry to investigate rapid changes in oxygen consumption during platelet activation. We demonstrated a rapid, transient increase in oxygen consumption rate within minutes of platelet stimulation by the physiological activator, thrombin. This was partly inhibited by aspirin and by oligomycin. This shows that high resolution respirometry can provide information regarding rapid and dynamic changes in oxygen consumption during platelet activation.