MPST sulfurtransferase maintains mitochondrial protein import and cellular bioenergetics to attenuate obesity.

Given the clinical, economic, and societal impact of obesity, unraveling the mechanisms of adipose tissue expansion remains of fundamental significance. We previously showed that white adipose tissue (WAT) levels of 3-mercaptopyruvate sulfurtransferase (MPST), a mitochondrial cysteine-catabolizing enzyme that yields pyruvate and sulfide species, are downregulated in obesity. Here, we report that Mpst deletion results in fat accumulation in mice fed a high-fat diet (HFD) through transcriptional and metabolic maladaptation. Mpst-deficient mice on HFD exhibit increased body weight and inguinal WAT mass, reduced metabolic rate, and impaired glucose/insulin tolerance. At the molecular level, Mpst ablation activates HIF1α, downregulates subunits of the translocase of outer/inner membrane (TIM/TOM) complex, and impairs mitochondrial protein import. MPST deficiency suppresses the TCA cycle, oxidative phosphorylation, and fatty acid oxidation, enhancing lipid accumulation. Sulfide donor administration to obese mice reverses the HFD-induced changes. These findings reveal the significance of MPST for white adipose tissue biology and metabolic health and identify a potential new therapeutic target for obesity.

Mapping the Endothelial Cell S-Sulfhydrome Highlights the Crucial Role of Integrin Sulfhydration in Vascular Function.

BACKGROUND: In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (H2Sn), that exert their biological actions via cysteine S-sulfhydration of target proteins. This study set out to map the "S-sulfhydrome" (ie, the spectrum of proteins targeted by H2Sn) in human endothelial cells. METHODS: Liquid chromatography with tandem mass spectrometry was used to identify S-sulfhydrated cysteines in endothelial cell proteins and ß3 integrin intraprotein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress-induced cell alignment, blood pressure measurements, and flow-induced vasodilatation in endothelial cell-specific CSE knockout mice and in a small collective of patients with endothelial dysfunction. RESULTS: Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low); (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression; and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing H2Sn donor, SG1002. The endothelial cell "S-sulfhydrome" consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on ß3 integrin in detail we found that S-sulfhydration affected intraprotein disulfide bond formation and was required for the maintenance of an extended-open conformation of the ß leg. ß3 integrin S-sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of S-sulfhydration impaired interactions between ß3 integrin and Gα13 (guanine nucleotide-binding protein subunit α 13), resulting in the constitutive activation of RhoA (ras homolog family member A) and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low H2Sn generation, impaired flow-induced dilatation, and failure to detect ß3 integrin S-sulfhydration, all of which were rescued after the administration of an H2Sn supplement. CONCLUSIONS: Vascular disease is associated with marked changes in the S-sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short-term H2Sn supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.

3-Mercaptopyruvate sulfurtransferase supports endothelial cell angiogenesis and bioenergetics.

BACKGROUND AND PURPOSE: During angiogenesis, quiescent endothelial cells (ECs) are activated by various stimuli to form new blood vessels from pre-existing ones in physiological and pathological conditions. Many research groups have shown that hydrogen sulfide (H2 S), the newest member of the gasotransmitter family, acts as a proangiogenic factor. To date, very little is known about the regulatory role of 3-mercaptopyruvate sulfurtransferase (3-MST), an important H2 S-producing enzyme in ECs. The aim of our study was to explore the potential role of 3-MST in human EC bioenergetics, metabolism, and angiogenesis. EXPERIMENTAL APPROACH: To assess in vitro angiogenic responses, we used EA.hy926 human vascular ECs subjected to shRNA-mediated 3-MST attenuation and pharmacological inhibition of proliferation, migration, and tube-like network formation. To evaluate bioenergetic parameters, cell respiration, glycolysis, glucose uptake, and mitochondrial/glycolytic ATP production were measured. Finally, global metabolomic profiling was performed to determine the level of 669 metabolic compounds. KEY RESULTS: 3-MST-attenuated ECs subjected to shRNA or pharmacological inhibition of 3-MST significantly reduced EC proliferation, migration, and tube-like network formation. 3-MST silencing also suppressed VEGF-induced EC migration. From bioenergetic and metabolic standpoints, 3-MST attenuation decreased mitochondrial respiration and mitochondrial ATP production, increased glucose uptake, and perturbed the entire EC metabolome. CONCLUSION AND IMPLICATIONS: 3-MST regulates bioenergetics and morphological angiogenic functions in human ECs. The data presented in the current report support the view that 3-MST pathway may be a potential candidate for therapeutic modulation of angiogenesis. LINKED ARTICLES: This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc.

Soluble epoxide hydrolase promotes astrocyte survival in retinopathy of prematurity.

Polyunsaturated fatty acids such as docosahexaenoic acid (DHA) positively affect the outcome of retinopathy of prematurity (ROP). Given that DHA metabolism by cytochrome P450 and soluble epoxide hydrolase (sEH) enzymes affects retinal angiogenesis and vascular stability, we investigated the role of sEH in a mouse model of ROP. In WT mice, hyperoxia elicited tyrosine nitration and inhibition of sEH and decreased generation of the DHA-derived diol 19,20-dihydroxydocosapentaenoic acid (19,20-DHDP). Correspondingly, in a murine model of ROP, sEH-/- mice developed a larger central avascular zone and peripheral pathological vascular tuft formation than did their WT littermates. Astrocytes were the cells most affected by sEH deletion, and hyperoxia increased astrocyte apoptosis. In rescue experiments, 19,20-DHDP prevented astrocyte loss by targeting the mitochondrial membrane to prevent the hyperoxia-induced dissociation of presenilin-1 and presenilin-1-associated protein to attenuate poly ADP-ribose polymerase activation and mitochondrial DNA damage. Therapeutic intravitreal administration of 19,20-DHDP not only suppressed astrocyte loss, but also reduced pathological vascular tuft formation in sEH-/- mice. Our data indicate that sEH activity is required for mitochondrial integrity and retinal astrocyte survival in ROP. Moreover, 19,20-DHDP may be more effective than DHA as a nutritional supplement for preventing retinopathy in preterm infants.

Hydrogen Sulfide Preserves Endothelial Nitric Oxide Synthase Function by Inhibiting Proline-Rich Kinase 2: Implications for Cardiomyocyte Survival and Cardioprotection.

Hydrogen sulfide (H2S) exhibits beneficial effects in the cardiovascular system, many of which depend on nitric oxide (NO). Proline-rich tyrosine kinase 2 (PYK2), a redox-sensitive tyrosine kinase, directly phosphorylates and inhibits endothelial NO synthase (eNOS). We investigated the ability of H2S to relieve PYK2-mediated eNOS inhibition and evaluated the importance of the H2S/PYK2/eNOS axis on cardiomyocyte injury in vitro and in vivo. Exposure of H9c2 cardiomyocytes to H2O2 or pharmacologic inhibition of H2S production increased PYK2 (Y402) and eNOS (Y656) phosphorylation. These effects were blocked by treatment with Na2S or by overexpression of cystathionine γ-lyase (CSE). In addition, PYK2 overexpression reduced eNOS activity in a H2S-reversible manner. The viability of cardiomyocytes exposed to Η2Ο2 was reduced and declined further after the inhibition of H2S production. PYK2 downregulation, l-cysteine supplementation, or CSE overexpression alleviated the effects of H2O2 on H9c2 cardiomyocyte survival. Moreover, H2S promoted PYK2 sulfhydration and inhibited its activity. In vivo, H2S administration reduced reactive oxygen species levels, as well as PYK2 (Y402) and eNOS (Y656) phosphorylation. Pharmacologic blockade of PYK2 or inhibition of PYK2 activation by Na2S reduced myocardial infarct size in mice. Coadministration of a PYK2 inhibitor and Na2S did not result in additive effects on infarct size. We conclude that H2S relieves the inhibitory effect of PYK2 on eNOS, allowing the latter to produce greater amounts of NO, thereby affording cardioprotection. Our results unravel the existence of a novel H2S-NO interaction and identify PYK2 as a crucial target for the protective effects of H2S under conditions of oxidative stress.

Regulation and role of endogenously produced hydrogen sulfide in angiogenesis.

Recent studies have implicated endogenously produced H2S in the angiogenic process. On one hand, pharmacological inhibition and silencing of the enzymes involved in H2S synthesis attenuate the angiogenic properties of endothelial cells, including proliferation, migration and tube-like structure network formation. On the other hand, enhanced production of H2S by substrate supplementation or over-expression of H2S-producing enzymes leads to enhanced angiogenic responses in cultured endothelial cells. Importantly, H2S up-regulates expression of the key angiogenic factor vascular endothelial growth factor (VEGF) and contributes to the angiogenic signaling in response to VEGF. The signaling pathways mediating H2S-induced angiogenesis include mitogen-activated protein kinases, phosphoinositide-3 kinase, nitric oxide/cGMP-regulated cascades and ATP-sensitive potassium channels. Endogenously produced H2S has also been shown to facilitate neovascularization in prototypical model systems in vivo, and to contribute to wound healing, post-ischemic angiogenesis in the heart and other tissues, as well as in tumor angiogenesis. Targeting of H2S synthesizing enzymes might offer novel therapeutic opportunities for angiogenesis-related diseases.

The natural olive constituent oleuropein induces nutritional cardioprotection in normal and cholesterol-fed rabbits: comparison with preconditioning.

Ischemic preconditioning, which is mediated by cell signaling molecules, protects the heart from ischemia-reperfusion injury by limiting the infarct size. Oleuropein, the main polyphenolic constituent of olives, reduced the infarct size in normal and cholesterol-fed rabbits when it was administered at a nutritional dose. The aim of the present study was to compare the effects of oleuropein and preconditioning in terms of the cell signaling and metabolism pathways underlying myocardial protection. Rabbits were randomly divided into six groups: the control group received 5 % dextrose for six weeks, the preconditioning group was subjected to two cycles of preconditioning with 5 min ischemia/10 min reperfusion, the O6 group was treated with oleuropein for six weeks, the Chol group was fed a cholesterol-enriched diet and 5 % dextrose for six weeks, and the CholO6 and CholO3 groups were treated with cholesterol and oleuropein for six and three weeks, respectively; oleuropein was dissolved in 5 % dextrose solution and was administered orally at a dose of 20 mg × kg(-1) × day(-1). All animals were subsequently subjected to 30 min myocardial ischemia followed by 10 min of reperfusion. At that time, myocardial biopsies were taken from the ischemic areas for the assessment of oxidative and nitrosative stress biomarkers (malondialdehyde and nitrotyrosine), and determination of phosphorylation of signaling molecules involved in the mechanism of preconditioning (PI3K, Akt, eNOS, AMPK, STAT3). The tissue extracts NMR metabolic profile was recorded and further analyzed by multivariate statistics. Oxidative biomarkers were significantly reduced in the O6, CholO6, and CholO3 groups compared to the control, preconditioning, and Chol groups. Considering the underlying signaling cascade, the phosphorylation of PI3K, Akt, eNOS, AMPK, and STAT-3 was significantly higher in the preconditioning and all oleuropein-treated groups compared to the control and Chol groups. The NMR-based metabonomic study, performed through the analysis of spectroscopic data, depicted differences in the metabolome of the various groups with significant alterations in purine metabolism. In conclusion, the addition of oleuropein to a normal or hypercholesterolemic diet results in a preconditioning-like intracellular effect, eliminating the deleterious consequences of ischemia and hypercholesterolemia, followed by a decrease of oxidative stress biomarkers. This effect is exerted through inducing preconditioning-involved signaling transduction. Nutritional preconditioning may support the low cardiovascular morbidity and mortality associated with the consumption of olive products.