Lipopolysaccharide affects energy metabolism and elevates nicotinamide N-methyltransferase level in human aortic endothelial cells (HAEC)

This study aimed to investigate the putative role of nicotinamide N-methyltransferase in the metabolic response of human aortic endothelial cells. This enzyme catalyses S-adenosylmethionine-mediated methylation of nicotinamide to methylnicotinamide. This reaction is accompanied by the reduction of the intracellular nicotinamide and S-adenosylmethionine content. This may affect NAD+ synthesis and various processes of methylation, including epigenetic modifications of chromatin. Particularly high activity of nicotinamide N-methyltransferase is detected in liver, many neoplasms as well as in various cells in stressful conditions. The elevated nicotinamide N-methyltransferase content was also found in endothelial cells treated with statins. Although the exogenous methylnicotinamide has been postulated to induce a vasodilatory response, the specific metabolic role of nicotinamide N-methyltransferase in vascular endothelium is still unclear. Treatment of endothelial cells with bacterial lipopolysaccharide evokes several metabolic and functional consequences which built a multifaceted physiological response of endothelium to bacterial infection. Among the spectrum of biochemical changes substantially elevated protein level of nicotinamide N-methyltransferase was particularly intriguing. Here it has been shown that silencing of the nicotinamide N-methyltransferase gene influences several changes which are observed in cells treated with lipopolysaccharide. They include altered energy metabolism and rearrangement of the mitochondrial network. A complete explanation of the mechanisms behind the protective consequences of the nicotinamide N-methyltransferase deficiency in cells treated with lipopolysaccharide needs further investigation

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Arachidonic acid-induced apoptosis in rat hepatoma AS-30D cells is mediated by reactive oxygen species

Arachidonic acid at micromolar concentrations produced a drastic increase of the generation of reactive oxygen species (ROS) in rat hepatoma AS-30D cells cultivated in vitro along with an increase in the incidence of apoptotic cell death. Both processes were prevented by trolox, a water-soluble tocopherol derivative, and tempol, a known antioxidative agent. A synthetic hybrid of lipoic acid and trolox or preincubation with N-acetylcysteine were less effective. Preincubation of the cells with etomoxir, a known highly specific irreversible inhibitor of carnitine-palmitoyltransferase I, partly decreased the ROS formation induced by arachidonic acid but it did not affect the increase in apoptosis. Cumulatively, these results indicate that apoptosis induced in hepatoma cells by arachidonic acid is mediated by ROS. They also suggest that this effect is due to arachidonic acid as such and not to its mitochondrial oxidative metabolites

The Metabolic Changes between Monolayer (2D) and Three-Dimensional (3D) Culture Conditions in Human Mesenchymal Stem/Stromal Cells Derived from Adipose Tissue

Introduction: One of the key factors that may influence the therapeutic potential of mesenchymal stem/stromal cells (MSCs) is their metabolism. The switch between mitochondrial respiration and glycolysis can be affected by many factors, including the oxygen concentration and the spatial form of culture. This study compared the metabolic features of adipose-derived mesenchymal stem/stromal cells (ASCs) and dedifferentiated fat cells (DFATs) cultivated as monolayer or spheroid culture under 5% O2 concentration (physiological normoxia) and their impact on MSCs therapeutic abilities. Results: We observed that the cells cultured as spheroids had a slightly lower viability and a reduced proliferation rate but a higher expression of the stemness-related transcriptional factors compared to the cells cultured in monolayer. The three-dimensional culture form increased mtDNA content, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), especially in DFATs-3D population. The DFATs spheroids also demonstrated increased levels of Complex V proteins and higher rates of ATP production. Moreover, increased reactive oxygen species and lower intracellular lactic acid levels were also found in 3D culture. Conclusion: Our results may suggest that metabolic reconfiguration accompanies the transition from 2D to 3D culture and the processes of both mitochondrial respiration and glycolysis become more active. Intensified metabolism might be associated with the increased demand for energy, which is needed to maintain the expression of pluripotency genes and stemness state

The Metabolic Changes between Monolayer (2D) and Three-Dimensional (3D) Culture Conditions in Human Mesenchymal Stem/Stromal Cells Derived from Adipose Tissue

Introduction: One of the key factors that may influence the therapeutic potential of mesenchymal stem/stromal cells (MSCs) is their metabolism. The switch between mitochondrial respiration and glycolysis can be affected by many factors, including the oxygen concentration and the spatial form of culture. This study compared the metabolic features of adipose-derived mesenchymal stem/stromal cells (ASCs) and dedifferentiated fat cells (DFATs) cultivated as monolayer or spheroid culture under 5% O2 concentration (physiological normoxia) and their impact on MSCs therapeutic abilities. Results: We observed that the cells cultured as spheroids had a slightly lower viability and a reduced proliferation rate but a higher expression of the stemness-related transcriptional factors compared to the cells cultured in monolayer. The three-dimensional culture form increased mtDNA content, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), especially in DFATs-3D population. The DFATs spheroids also demonstrated increased levels of Complex V proteins and higher rates of ATP production. Moreover, increased reactive oxygen species and lower intracellular lactic acid levels were also found in 3D culture. Conclusion: Our results may suggest that metabolic reconfiguration accompanies the transition from 2D to 3D culture and the processes of both mitochondrial respiration and glycolysis become more active. Intensified metabolism might be associated with the increased demand for energy, which is needed to maintain the expression of pluripotency genes and stemness state

Mitofusin 2 Deficiency Affects Energy Metabolism and Mitochondrial Biogenesis in MEF Cells.

Mitofusin 2 (Mfn2), mitochondrial outer membrane protein which is involved in rearrangement of these organelles, was first described in pathology of hypertension and diabetes, and more recently much attention is paid to its functions in Charcot-Marie-Tooth type 2A neuropathy (CMT2A). Here, cellular energy metabolism was investigated in mouse embryonic fibroblasts (MEF) differing in the presence of the Mfn2 gene; control (MEFwt) and with Mfn2 gene depleted MEFMfn2-/-. These two cell lines were compared in terms of various parameters characterizing mitochondrial bioenergetics. Here, we have shown that relative rate of proliferation of MEFMfn2-/- cells versus control fibroblasts depend on serum supplementation of the growth media. Moreover, MEFMfn2-/- cells exhibited significantly increased respiration rate in comparison to MEFwt, regardless of serum supplementation of the medium. This effect was correlated with increased level of mitochondrial markers (TOM20 and NAO) as well as mitochondrial transcription factor A (TFAM) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) protein levels and unchanged total ATP content. Interestingly, mitochondrial DNA content in MEFMfn2-/- cells was not reduced. Fundamentally, these results are in contrast to a commonly accepted belief that mitofusin 2 deficiency inevitably results in debilitation of mitochondrial energy metabolism. However, we suggest a balance between negative metabolic consequences of mitofusin 2 deficiency and adaptive processes exemplified by increased level of PGC-1α and TFAM transcription factor which prevent an excessive depletion of mtDNA and severe impairment of cell metabolism