Targeted metabolomics combined with network pharmacology to reveal the protective role of luteolin in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease that significantly endangers human health, where metabolism may drive pathogenesis: a shift from mitochondrial oxidation to glycolysis occurs in diseased pulmonary vessels and the right ventricle. An increase in pulmonary vascular resistance in patients with heart failure with a preserved ejection fraction portends a poor prognosis. Luteolin exists in numerous foods and is marketed as a dietary supplement assisting in many disease treatments. However, little is known about the protective effect of luteolin on metabolism disorders in diseased pulmonary vessels. In this study, we found that luteolin apparently reversed the pulmonary vascular remodeling of PAH rats by inhibiting the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs). Moreover, network pharmacology and metabolomics results revealed that the arachidonic acid pathway, amino acid pathway and TCA cycle were dysregulated in PAH. A total of 14 differential metabolites were significantly changed during the PAH, including DHA, PGE2, PGD2, LTB4, 12-HETE, 15-HETE, PGF2α, and 8-iso-PGF2α metabolites in the arachidonic acid pathway, and L-asparagine, oxaloacetate, N-acetyl-L-ornithine, butane diacid, ornithine, glutamic acid metabolites in amino acid and TCA pathways. However, treatment with luteolin recovered the LTB4, PGE2, PGD2, 12-HETE, 15-HETE, PGF2α and 8-iso-PGF2α levels close to normal. Meanwhile, we showed that luteolin also downregulated the gene and protein levels of COX 1, 5-LOX, 12-LOX, and 15-LOX in the arachidonic acid pathway. Collectively, this work highlighted the metabolic mechanism of luteolin-protected PAH and showed that luteolin would hold great potential in PAH prevention.

Metabolism of malate in bovine adrenocortical mitochondria studied by 13C-NMR spectroscopy.

13C-NMR spectroscopy was used to study the metabolism of [13C]malate in bovine coupled adrenocortical mitochondria. The most apparent difference between the mitochondria from steroidogenic tissues and mitochondria from other tissues is the presence, in addition to the normal respiratory chain, of a second electron-transport system responsible for steroid hydroxylation. [13C]malate was synthesized from [13C]succinate by isolated adrenocortical mitochondria. The basic functional suspension consisted of oxygenated mitochondria to which were added ADP, inorganic phosphate (Pi) and [13C]malate, both in the absence or presence of the steroid substrate, deoxycorticosterone. These mitochondria synthesized [13C]citrate and [13C]pyruvate from [13C]malate. The 13C labeling of these two metabolites demonstrated an important role of the malic enzyme and the kinetics depended on the presence of the steroid substrate; the citric acid cycle was stopped during the hydroxylation pathway. The addition of cyanide, a strong inhibitor of the respiratory chain, confirmed an increased malic enzyme activity when hydroxylation occurred, since pyruvate was trapped by formation of a cyanohydrin. The relative enzymic activities of malic enzyme and isocitrate dehydrogenase were compared, both in the absence or presence of the steroid substrate, by supplementing the basic suspension with unlabeled exogenous metabolites, such as pyruvate or oxaloacetate.

Novel oxaloacetate effect on mitochondrial Ca2+ movement.

Mitochondrial Ca2+ movement was investigated in the presence of oxaloacetate, which is widely known as a 'Ca(2+)-releasing' agent [1978, Proc. Natl. Acad. Sci. USA 75, 1690-1694]. It is demonstrated that rat liver mitochondrial are capable of net Ca2+ accumulation from the oxaloacetate supplemented assay mixture. Both the membrane energization and the cation uniport at the expense of oxaloacetate are shown to be specifically blocked by either arsenite or ammonium chloride. With respiratory inhibitors present, ADP is shown to be a prerequisite for a high Ca2+ capacity, which can be alternatively enlarged with a concomitant loss of the arsenite effect by an addition of an NADP(+)-specific reductant (isocitrate). Arsenite-sensitive production of NADPH is observed, thus suggesting coupling between pyridine nucleotide transhydrogenation and the cation uniport in mitochondria. The role of such a coupling mechanism in the uniporter-mediated Ca2+ fluxes in mitochondria is discussed.

Limited proteolysis of inactive tetrameric chloroplast NADP-malate dehydrogenase produces active dimers.

Carboxy-terminal amino acids of NADP-dependent malate dehydrogenase (EC 1.1.1.82) from pea chloroplasts were removed by treatment with carboxypeptidase Y. This results in the activation of the inactive oxidized enzyme, while activation by light in vivo is thought to occur via reduction of an intrasubunit disulfide bridge. After proteolytic activation the oxidized enzyme had a specific activity of 100 U/mg protein, which is 50% of the maximal activity of the control enzyme in the reduced state. When the truncated enzyme was reduced with dithiothreitol (DTT), the specific activity was further increased to 1200 U/mg. While the native enzyme is composed of four identical subunits of 38,900 Da, the truncated malate dehydrogenase forms dimers composed of two subunits of 38,000 Da. No further change of molecular mass or activity was noticed subsequent to prolonged incubation of native NADP-malate dehydrogenase with carboxypeptidase Y for several days. When the enzyme is denatured by 2 M guanidine-HCl, the proteolytic activation proceeds more rapidly, but only transiently. The truncated enzyme is less accessible to activation by reduced thioredoxin, but the stimulation of activity by DTT alone is more rapid than that of the native enzyme. These results indicate that only a small carboxy-terminal peptide of native NADP-malate dehydrogenase from pea chloroplasts is accessible to proteolytic degradation and that this peptide is involved in the regulation of activity, tetramer formation, and thioredoxin binding. While the pH optimum for catalytic activity of the intact reduced enzyme is at pH 8.0-8.5, it is shifted to more acidic values upon proteolysis of NADP-malate dehydrogenase. At pH values below 8 the reduced truncated enzyme exhibits substrate inhibition by oxaloacetate.

Dissociation of NAD(P)+-stimulated mitochondrial Ca2+ efflux from swelling and membrane damage.

NAD(P)+-stimulated Ca2+ efflux from mitochondria in a high-sucrose medium is irreversible and is accompanied by large-amplitude mitochondrial swelling and membrane damage. If sucrose is partially replaced by polyethylene glycol (Mr approximately equal to 1000) as osmolar supporting medium, Ca2+ efflux is still stimulated by NAD(P)+ but mitochondrial swelling is eliminated. Other experiments in a high-sucrose medium showed that the lag phase between NAD(P)H oxidation and the beginning of net Ca2+ efflux decreases with increasing temperature. At 37 degrees C Ca2+ efflux precedes mitochondrial swelling, even in a high-sucrose medium, showing that the mitochondrial damage, as reflected by large-amplitude swelling, is not obligatory for Ca2+ efflux induced by the oxidized state of mitochondrial NAD(P)+. If a high-sucrose medium is supplemented with 20 mM potassium acetate, longer periods of Ca2+ release can be observed before the appearance of swelling. Under these experimental conditions the release of Ca2+ can be completely reversed if the rereduction of NAD(P)+ is brought about by the addition of the reductants beta-hydroxybutyrate and isocitrate.