Sphingosine 1-Phosphate Regulates Obesity and Glucose Homeostasis.

One of the major global health and welfare issues is the treatment of obesity and associated metabolic disorders, such as type 2 diabetes mellitus and nonalcoholic fatty liver disease. Obesity, caused by the excessive accumulation of triglycerides in adipose tissues, induces adipocyte dysfunction, followed by inflammation, in adipose tissues and lipotoxicity in nonadipose tissues. Several studies have shown that obesity and glucose homeostasis are influenced by sphingolipid mediators, including ceramide and sphingosine 1-phosphate (S1P). Cellular accumulation of ceramide impairs pancreatic ß-cell survival, confers insulin resistance in the liver and the skeletal muscle, and deteriorates adipose tissue inflammation via unknown molecular mechanisms. The roles of S1P are more complicated, because there are five cell-surface S1P receptors (S1PRs: S1P1-5) which have altered functions, different cellular expression patterns, and inapparent intracellular targets. Recent findings, including those by our group, support the notable concept that the pharmacological activation of S1P1 or S1P3 improves obesity and associated metabolic disorders, whereas that of S1P2 has the opposite effect. In addition, the regulation of S1P production by sphingosine kinase (SphK) is an essential factor affecting glucose homeostasis. This review summarizes the current knowledge on SphK/S1P/S1PR signaling in and against obesity, insulin resistance, and associated disorders.

Amino Acids in Health and Disease: The Good, the Bad, and the Ugly.

The Special Issue "Amino Acid Metabolism and Regulation in Health and Disease 2 [...].

Pharmacological Inhibition and Genetic Deletion of Cystathionine Gamma-Lyase in Mice Protects against Organ Injury in Sepsis: A Key Role of Adhesion Molecules on Endothelial Cells.

Hydrogen sulfide (H2S), synthesized by cystathionine gamma-lyase (Cth), contributes to the inflammatory response observed in sepsis. This study examines the effect of Cth-derived H2S in adhesion molecules on endothelial cells of vital organs in mice in a cecal ligation puncture (CLP)-induced model of sepsis, using two different and complementary approaches: Cth gene deletion and pharmacological inhibition. Our findings revealed a decreased level of H2S-synthesizing activity (via Cth) in both Cth-/- mice and PAG-treated wild-type (WT) mice following CLP-induced sepsis. Both treatment groups had reduced MPO activity and expression of chemokines (MCP-1 and MIP-2α), adhesion molecules (ICAM-1 and VCAM-1), ERK1/2 phosphorylation, and NF-κB in the liver and lung compared with in CLP-WT mice. Additionally, we found that PAG treatment in Cth-/- mice had no additional effect on the expression of ERK1/2 phosphorylation, NF-κB, or the production of chemokines and adhesion molecules in the liver and lung compared to Cth-/- mice following CLP-induced sepsis. The WT group with sepsis had an increased immunoreactivity of adhesion molecules on endothelial cells in the liver and lung than the WT sham-operated control. The Cth-/-, PAG-treated WT, and Cth-/- groups of mice showed decreased immunoreactivity of adhesion molecules on endothelial cells in the liver and lung following sepsis. Inhibition of H2S production via both approaches reduced adhesion molecule expression on endothelial cells and reduced liver and lung injury in mice with sepsis. In conclusion, this study demonstrates that H2S has an important role in the pathogenesis of sepsis and validates PAG use as a suited tool for investigating the Cth/H2S-signalling axis in sepsis.

Differential Roles of Cystathionine Gamma-Lyase and Mercaptopyruvate Sulfurtransferase in Hapten-Induced Colitis and Contact Dermatitis in Mice.

Hydrogen sulfide (H2S) has been shown to act as both anti-inflammatory and pro-inflammatory mediators. Application of H2S donors generally protects against inflammation; however, experimental results using mice lacking endogenous H2S-producing enzymes, such as cystathionine γ-lyase (CTH) and mercaptopyruvate sulfurtransferase (MPST), are often contradictory. We herein examined two types of model hapten-induced inflammation models, colitis (an inflammatory bowel disease model of mucosal immunity) and contact dermatitis (a type IV allergic model of systemic immunity), in CTH-deficient (Cth-/-) and MPST-deficient (Mpst-/-) mice. Both mice exhibited no significant alteration from wild-type mice in trinitrobenzene sulfonic acid (Th1-type hapten)-induced colitis (a Crohn's disease model) and oxazolone (Th1/Th2 mix-type; Th2 dominant)-induced colitis (an ulcerative colitis model). However, Cth-/- (not Mpst-/-) mice displayed more exacerbated phenotypes in trinitrochlorobenzene (TNCB; Th1-type)-induced contact dermatitis, but not oxazolone, at the delayed phase (24 h post-administration) of inflammation. CTH mRNA expression was upregulated in the TNCB-treated ears of both wild-type and Mpst-/- mice. Although mRNA expression of pro-inflammatory cytokines (IL-1ß and IL-6) was upregulated in both early (2 h) and delayed phases of TNCB-triggered dermatitis in all genotypes, that of Th2 (IL-4) and Treg cytokines (IL-10) was upregulated only in Cth-/- mice, when that of Th1 cytokines (IFNγ and IL-2) was upregulated in wild-type and Mpst-/- mice at the delayed phase. These results suggest that (upregulated) CTH or H2S produced by it helps maintain Th1/Th2 balance to protect against contact dermatitis.

The function of SARS-CoV-2 spike protein is impaired by disulfide-bond disruption with mutation at cysteine-488 and by thiol-reactive N-acetyl-cysteine and glutathione.

Viral spike proteins play important roles in the viral entry process, facilitating attachment to cellular receptors and fusion of the viral envelope with the cell membrane. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds to the cellular receptor angiotensin converting enzyme-2 (ACE2) via its receptor-binding domain (RBD). The cysteine residue at position 488, consisting of a disulfide bridge with cysteine 480 is located in an important structural loop at ACE2-binding surface of RBD, and is highly conserved among SARS-related coronaviruses. We showed that the substitution of Cys-488 with alanine impaired pseudotyped SARS-CoV-2 infection, syncytium formation, and cell-cell fusion triggered by SARS-CoV-2 spike expression. Consistently, in vitro binding of RBD and ACE2, spike-mediated cell-cell fusion, and pseudotyped viral infection of VeroE6/TMPRSS2 cells were inhibited by the thiol-reactive compounds N-acetylcysteine (NAC) and a reduced form of glutathione (GSH). Furthermore, we demonstrated that the activity of variant spikes from the SARS-CoV-2 alpha and delta strains were also suppressed by NAC and GSH. Taken together, these data indicate that Cys-488 in spike RBD is required for SARS-CoV-2 spike functions and infectivity, and could be a target of anti-SARS-CoV-2 therapeutics.

Functional and Structural Insights into Human PPARα/δ/γ Subtype Selectivity of Bezafibrate, Fenofibric Acid, and Pemafibrate.

Among the agonists against three peroxisome proliferator-activated receptor (PPAR) subtypes, those against PPARα (fibrates) and PPARγ (glitazones) are currently used to treat dyslipidemia and type 2 diabetes, respectively, whereas PPARδ agonists are expected to be the next-generation metabolic disease drug. In addition, some dual/pan PPAR agonists are currently being investigated via clinical trials as one of the first curative drugs against nonalcoholic fatty liver disease (NAFLD). Because PPARα/δ/γ share considerable amino acid identity and three-dimensional structures, especially in ligand-binding domains (LBDs), clinically approved fibrates, such as bezafibrate, fenofibric acid, and pemafibrate, could also act on PPARδ/γ when used as anti-NAFLD drugs. Therefore, this study examined their PPARα/δ/γ selectivity using three independent assays-a dual luciferase-based GAL4 transactivation assay for COS-7 cells, time-resolved fluorescence resonance energy transfer-based coactivator recruitment assay, and circular dichroism spectroscopy-based thermostability assay. Although the efficacy and efficiency highly varied between agonists, assay types, and PPAR subtypes, the three fibrates, except fenofibric acid that did not affect PPARδ-mediated transactivation and coactivator recruitment, activated all PPAR subtypes in those assays. Furthermore, we aimed to obtain cocrystal structures of PPARδ/γ-LBD and the three fibrates via X-ray diffraction and versatile crystallization methods, which we recently used to obtain 34 structures of PPARα-LBD cocrystallized with 17 ligands, including the fibrates. We herein reveal five novel high-resolution structures of PPARδ/γ-bezafibrate, PPARγ-fenofibric acid, and PPARδ/γ-pemafibrate, thereby providing the molecular basis for their application beyond dyslipidemia treatment.

A High-Methionine Diet for One-Week Induces a High Accumulation of Methionine in the Cerebrospinal Fluid and Confers Bipolar Disorder-like Behavior in Mice.

Methionine (Met) is considered the most toxic amino acid in mammals. Here, we investigated biochemical and behavioral impacts of ad libitum one-week feeding of high-Met diets on mice. Adult male mice were fed the standard rodent diet that contained 0.44% Met (1×) or a diet containing 16 graded Met doses (1.2×-13×). High-Met diets for one-week induced a dose-dependent decrease in body weight and an increase in serum Met levels with a 2.55 mM peak (versus basal 53 µM) on the 12×Met diet. Total homocysteine (Hcy) levels were also upregulated while concentrations of other amino acids were almost maintained in serum. Similarly, levels of Met and Hcy (but not the other amino acids) were highly elevated in the cerebrospinal fluids of mice on the 10×Met diet; the Met levels were much higher than Hcy and the others. In a series of behavioral tests, mice on the 10×Met diet displayed increased anxiety and decreased traveled distances in an open-field test, increased activity to escape from water soaking and tail hanging, and normal learning/memory activity in a Y-maze test, which were reflections of negative/positive symptoms and normal cognitive function, respectively. These results indicate that high-Met ad libitum feeding even for a week can induce bipolar disorder-like disease models in mice.

Crystal Structures of the Human Peroxisome Proliferator-Activated Receptor (PPAR)α Ligand-Binding Domain in Complexes with a Series of Phenylpropanoic Acid Derivatives Generated by a Ligand-Exchange Soaking Method.

Peroxisome proliferator-activated receptor (PPAR)α, a member of the nuclear receptor family, is a transcription factor that regulates the expression of genes related to lipid metabolism in a ligand-dependent manner, and has attracted attention as a target for hypolipidemic drugs. We have been developing phenylpropaonic acid derivatives as PPARα-targeted drug candidates for the treatment of metabolic diseases. Recently, we have developed the "ligand-exchange soaking method," which crystallizes the recombinant PPARα ligand-binding domain (LBD) as a complex with intrinsic fatty acids derived from an expression host Escherichia (E.) coli and thereafter replaces them with other higher-affinity ligands by soaking. Here we applied this method for preparation of cocrystals of PPARα LBD with its ligands that have not been obtained with the conventional cocrystallization method. We revealed the high-resolution structures of the cocrystals of PPARα LBD and the three synthetic phenylpropaonic acid derivatives: TIPP-703, APHM19, and YN4pai, the latter two of which are the first observations. The overall structures of cocrystals obtained from the two methods are identical and illustrate the close interaction between these ligands and the surrounding amino acid residues of PPARα LBD. This ligand-exchange soaking method could be applicable to high throughput preparations of co-crystals with another subtype PPARδ LBD for high resolution X-ray crystallography, because it also crystallizes in complex with intrinsic fatty acid(s) while not in the apo-form.

Structural Basis for Anti-non-alcoholic Fatty Liver Disease and Diabetic Dyslipidemia Drug Saroglitazar as a PPAR α/γ Dual Agonist.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptor-type transcription factors that consist of three subtypes (α, γ, and ß/δ) with distinct functions and PPAR dual/pan agonists are expected to be the next generation of drugs for metabolic diseases. Saroglitazar is the first clinically approved PPARα/γ dual agonist for treatment of diabetic dyslipidemia and is currently in clinical trials to treat non-alcoholic fatty liver disease (NAFLD); however, the structural information of its interaction with PPARα/γ remains unknown. We recently revealed the high-resolution co-crystal structure of saroglitazar and the PPARα-ligand binding domain (LBD) through X-ray crystallography, and in this study, we report the structure of saroglitazar and the PPARγ-LBD. Saroglitazar was located at the center of "Y"-shaped PPARγ-ligand-binding pocket (LBP), just as it was in the respective region of PPARα-LBP. Its carboxylic acid was attached to four amino acids (Ser289/His323/His449/Thr473), which contributes to the stabilization of Activating Function-2 helix 12, and its phenylpyrrole moiety was rotated 121.8 degrees in PPARγ-LBD from that in PPARα-LBD to interact with Phe264. PPARδ-LBD has the consensus four amino acids (Thr253/His287/His413/Tyr437) towards the carboxylic acids of its ligands, but it seems to lack sufficient space to accept saroglitazar because of the steric hindrance between the Trp228 or Arg248 residue of PPARδ-LBD and its methylthiophenyl moiety. Accordingly, in a coactivator recruitment assay, saroglitazar activated PPARα-LBD and PPARγ-LBD but not PPARδ-LBD, whereas glycine substitution of either Trp228, Arg248, or both of PPARδ-LBD conferred saroglitazar concentration-dependent activation. Our findings may be valuable in the molecular design of PPARα/γ dual or PPARα/γ/δ pan agonists.

Homocysteine Hypothesis on the Impaired Peripheral but Not Central Nervous System Oxytocin Responses in Cystathionine γ-Lyase-Deficient Dam Mice.

An elevated plasma homocysteine level is an independent risk factor for cardiovascular diseases, neurological disorders, and pregnancy complications. We recently demonstrated partial lactation failure in cystathionine γ-lyase-deficient (Cth-/-) dam mice and their defective oxytocin responses in peripheral tissues: uterine (ex vivo) and mammary gland (in vivo). We reasoned that elevated levels of circulatory homocysteine in Cth-/- dam mice counteract with oxytocin-dependent milk ejection from the mammary gland. Based on our observation that those mice displayed normal maternal behaviors against their pups and adult Cth-/- male mice exhibited normal social behaviors against adult wild-type female mice, both of which are regulated by oxytocin in the central nervous system (CNS), we conducted the present study to investigate the amino acid profiles, including total homocysteine, in both blood and cerebrospinal fluid (CSF) of wild-type and Cth-/- female mice before pregnancy and at day 1 of lactation (L1). Serum levels of total homocysteine in wild-type and Cth-/- L1 dam mice were 9.44 and 188 µmol/L, respectively, whereas their CSF levels were below 0.21 (limit of quantification) and 3.62 µmol/L, respectively. Their CSF/serum level ratio was the lowest (1/51.9) among all 20 proteinogenic amino acids, sulfur-containing amino acids, and citrulline/ornithine in Cth-/- mice. Therefore, we hypothesize that the blood-brain barrier protects the CNS from high levels of circulatory homocysteine in Cth-/- dam mice, thereby conferring normal oxytocin-dependent maternal behaviors.

Hydrogen Sulfide Treatment Improves Post-Infarct Remodeling and Long-Term Cardiac Function in CSE Knockout and Wild-Type Mice.

Hydrogen sulfide (H2S) is recognized as an endogenous gaseous signaling molecule generated by cystathionine γ-lyase (CSE) in cardiovascular tissues. H2S up-regulation has been shown to reduce ischemic injury, and H2S donors are cardioprotective in rodent models when administered concurrent with myocardial ischemia. We evaluated the potential utility of H2S therapy in ameliorating cardiac remodeling with administration delayed until 2 h post-infarction in mice with or without cystathionine γ-lyase gene deletion (CSE-/-). The slow-release H2S donor, GYY4137, was administered from 2 h after surgery and daily for 28 days following myocardial infarction (MI) induced by coronary artery ligation, comparing responses in CSE-/- with wild-type (WT) mice (n = 5-10/group/genotype). Measures of cardiac function and expression of key genes associated with cardiac hypertrophy, fibrosis, and apoptosis were documented in atria, ventricle, and kidney tissues. Post-MI GYY4137 administration reduced infarct area and restored cardiac function, accompanied by reduction of the elevated ventricular expression of genes mediating cardiac remodeling to near-normal levels. Few differences between WT and CSE-/- mice were observed, except CSE-/- mice had higher blood pressures, and higher atrial Mir21a expression across all treatment groups. These findings suggest endogenous CSE gene deletion does not substantially exacerbate the long-term response to MI. Moreover, the H2S donor GYY4137 administered after onset of MI preserves cardiac function and protects against adverse cardiac remodeling in both WT and CSE-deficient mice.

Increased Urinary 3-Mercaptolactate Excretion and Enhanced Passive Systemic Anaphylaxis in Mice Lacking Mercaptopyruvate Sulfurtransferase, a Model of Mercaptolactate-Cysteine Disulfiduria.

Mercaptopyruvate sulfurtransferase (Mpst) and its homolog thiosulfate sulfurtransferase (Tst = rhodanese) detoxify cyanide to thiocyanate. Mpst is attracting attention as one of the four endogenous hydrogen sulfide (H2S)/reactive sulfur species (RSS)-producing enzymes, along with cystathionine ß-synthase (Cbs), cystathionine γ-lyase (Cth), and cysteinyl-tRNA synthetase 2 (Cars2). MPST deficiency was found in 1960s among rare hereditary mercaptolactate-cysteine disulfiduria patients. Mpst-knockout (KO) mice with enhanced liver Tst expression were recently generated as its model; however, the physiological roles/significances of Mpst remain largely unknown. Here we generated three independent germ lines of Mpst-KO mice by CRISPR/Cas9 technology, all of which maintained normal hepatic Tst expression/activity. Mpst/Cth-double knockout (DKO) mice were generated via crossbreeding with our previously generated Cth-KO mice. Mpst-KO mice were born at the expected frequency and developed normally like Cth-KO mice, but displayed increased urinary 3-mercaptolactate excretion and enhanced passive systemic anaphylactic responses when compared to wild-type or Cth-KO mice. Mpst/Cth-DKO mice were also born at the expected frequency and developed normally, but excreted slightly more 3-mercaptolactate in urine compared to Mpst-KO or Cth-KO mice. Our Mpst-KO, Cth-KO, and Mpst/Cth-DKO mice, unlike semi-lethal Cbs-KO mice and lethal Cars2-KO mice, are useful tools for analyzing the unknown physiological roles of endogenous H2S/RSS production.

Hydrogen sulfide inhibits NLRP3 inflammasome activation and reduces cytokine production both in vitro and in a mouse model of inflammation.

A variety of stimuli, including monosodium urate (MSU) crystals, activate the NLRP3 inflammasome, and this activation involves several molecular mechanisms including xanthine oxidase (XO) up-regulation and mitochondrial dysfunction. Upon oligomerization of apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1 becomes active and cleaves the proinflammatory cytokine IL-1ß into its active secreted form. Hydrogen sulfide (H2S), a gasotransmitter mainly produced by cystathionine γ-lyase (CSE) in macrophages, could modulate inflammation. Here, we sought to investigate the effects of exogenous and endogenous H2S on NLRP3 inflammasome activation in vitro and in vivo Primed bone marrow-derived macrophages (BMDM) isolated from wildtype (wt) or CSE-deficient mice and human macrophages (THP1 cells and primary macrophages), were stimulated with MSU crystals in the presence or absence of a H2S donor, sodium thiosulfate (STS) or GYY4137 (GYY). In murine and human macrophages in vitro, both STS and GYY inhibited MSU crystal-induced IL-1ß secretion in a dose-dependent manner. Moreover, the H2S donors inhibited MSU crystal-induced XO/caspase-1 activities, mitochondrial reactive oxygen species (ROS) generation, and ASC oligomerization. Accordingly, IL-1ß secretion and XO/caspase-1 activities were higher in CSE-deficient BMDMs than in wt BMDMs. For in vivo studies, we experimentally induced peritonitis by intraperitoneal injection of MSU crystals into mice. GYY pretreatment ameliorated inflammation, evidenced by decreased IL-6/monocyte chemoattractant protein-1 (MCP-1) released into peritoneal lavages. Taken together, our results suggest that both exogenous (via H2S donors) and endogenous (via CSE) H2S production may represent approaches for managing, for example, acute gout or other inflammation conditions.

Abnormal Amino Acid Profiles of Blood and Cerebrospinal Fluid from Cystathionine ß-Synthase-Deficient Mice, an Animal Model of Homocystinuria.

Mental retardation is the most common feature among inborn errors of amino acid metabolism. Patients with homocystinuria/homocysteinemia caused by cystathionine ß-synthase (CBS) deficiency suffer from thromboembolism and mental retardation from early ages; therefore, detection by newborn screening is performed. Furthermore, elevated levels of serum homocysteine during pregnancy are associated with the occurrence of neural tube defects (NTDs) in newborns. However, the causes of such central nervous system (CNS) defects are unknown. We found previously impaired learning abilities in Cbs-deficient (Cbs-/-) mice (but not NTD births). Here, we investigated the amino acid profiles of serum and cerebrospinal fluid (CSF) from Cbs-/- mice. Mice deficient in cystathionine γ-lyase (Cth), a downstream enzyme of CBS in transsulfuration, as well as wild-type mice, were analyzed as controls. Cbs-/- and Cth-/- mice were smaller than wild-type mice, and CSF yields in Cbs-/- mice were lower than the others. CSF amino acid levels were generally lower than those in serum, and compared with the dramatic amino acid level alterations in Cbs-/- mouse serum, alterations in CSF were less apparent. However, marked upregulation (versus wild-type) of aspartic acid/asparagine (Asp/Asn), glutamine (Gln), serine (Ser), threonine (Thr), phenylalanine (Phe), tyrosine (Tyr), methionine (Met), total homocysteine, and citrulline, and downregulation of lysine (Lys) were found in Cbs-/- mouse CSF. Because similar regulation of total homocysteine/citrulline/Lys was observed in the CSF of Cth-/- mice, which are free of CNS dysfunction, the reduced CSF volumes and the level changes of other amino acids could be relevant to Cbs-/--specific CNS defects.

Reactive sulfur species regulate tRNA methylthiolation and contribute to insulin secretion.

The 2-methylthio (ms2) modification at A37 of tRNAs is critical for accurate decoding, and contributes to metabolic homeostasis in mammals. However, the regulatory mechanism of ms2 modification remains largely unknown. Here, we report that cysteine hydropersulfide (CysSSH), a newly identified reactive sulfur species, is involved in ms2 modification in cells. The suppression of intracellular CysSSH production rapidly reduced ms2 modification, which was rescued by the application of an exogenous CysSSH donor. Using a unique and stable isotope-labeled CysSSH donor, we show that CysSSH was capable of specifically transferring its reactive sulfur atom to the cysteine residues of ms2-modifying enzymes as well as ms2 modification. Furthermore, the suppression of CysSSH production impaired insulin secretion and caused glucose intolerance in both a pancreatic ß-cell line and mouse model. These results demonstrate that intracellular CysSSH is a novel sulfur source for ms2 modification, and that it contributes to insulin secretion.

Preeclampsia-Like Features and Partial Lactation Failure in Mice Lacking Cystathionine γ-Lyase-An Animal Model of Cystathioninuria.

Elevated plasma homocysteine levels are considered as a risk factor for cardiovascular diseases as well as preeclampsia-a pregnancy disorder characterized by hypertension and proteinuria. We previously generated mice lacking cystathionine γ-lyase (Cth) as cystathioninuria models and found them to be with cystathioninemia/homocysteinemia. We investigated whether Cth-deficient (Cth-/-) pregnant mice display any features of preeclampsia. Cth-/- females developed normally but showed mild hypertension (~10 mmHg systolic blood pressure elevation) in late pregnancy and mild proteinuria throughout development/pregnancy. Cth-/- dams had normal numbers of pups and exhibited normal maternal behavior except slightly lower breastfeeding activity. However, half of them could not raise their pups owing to defective lactation; they could produce/store the first milk in their mammary glands but not often provide milk to their pups after the first ejection. The serum oxytocin levels and oxytocin receptor expression in the mammary glands were comparable between wild-type and Cth-/- dams, but the contraction responses of mammary gland myoepithelial cells to oxytocin were significantly lower in Cth-/- dams. The contraction responses to oxytocin were lower in uteruses isolated from Cth-/- mice. Our results suggest that elevated homocysteine or other unknown factors in preeclampsia-like Cth-/- dams interfere with oxytocin that regulates milk ejection reflex.

Cystathionine-Gamma-Lyase-Derived Hydrogen Sulfide-Regulated Substance P Modulates Liver Sieve Fenestrations in Caecal Ligation and Puncture-Induced Sepsis.

Cystathionine-γ-lyase (CSE) isa hydrogen sulfide (H2S)-synthesizing enzyme that promotesinflammation by upregulating H2S in sepsis. Liver sinusoidal endothelial cells (LSECs) are fenestrated endothelial cells (liver sieve) that undergo alteration during sepsis and H2S plays a role in this process. Substance P (SP) is encoded by the preprotachykinin A (PPTA) gene, and promotes inflammation in sepsis; however, its regulation by H2S is poorly understood. Furthermore, the interaction between H2S and SP in modulating LSEC fenestrations following sepsis remains unclear. This study aimed to investigate whether CSE/H2S regulates SP and the neurokinin-1 receptor (NK-1R) andmodulates fenestrations in LSECs following caecalligation and puncture (CLP)-induced sepsis. Here we report thatthe absence of either CSE or H2S protects against liver sieve defenestration and gaps formation in LSECsin sepsis by decreased SP-NK-1R signaling. Following sepsis, there is an increased expression of liver CSE and H2S synthesis, and plasma H2S levels, which were aligned with higher SP levels in the liver, lungs and plasma and NK-1R in the liver and lungs. The genetic deletion of CSE led to decreased sepsis-induced SP and NK-1R in the liver, lungs and plasma SP suggesting H2S synthesized through CSE regulates the SP-NK-1R pathway in sepsis. Further, mice deficient in the SP-encoding gene (PPTA) preservedsepsis-induced LSEC defenestrationand gaps formation, as seen by maintenance of patent fenestrations and fewer gaps. In conclusion, CSE/H2S regulates SP-NK-1R and modulates LSEC fenestrations in sepsis.

The Capture of Cadmium by Reactive Polysulfides Attenuates Cadmium-Induced Adaptive Responses and Hepatotoxicity.

Cadmium (Cd) is an environmental electrophile that modifies protein nucleophiles, thereby modulating cellular signaling and toxicity. While reactive persulfides/polysulfides exhibit relatively high nucleophilic properties, their roles in the altered gene expression and toxicity caused by Cd remain unclear. Exposing primary mouse hepatocytes to Cd caused heat shock protein 70 (HSP70) and metallothionein (MT)-I/II to be upregulated and cytotoxicity to occur. These effects were blocked in the presence of polysulfide sodium tetrasulfide (Na2S4). Electrospray ionization mass spectrometry analysis indicated that cadmium sulfide (CdS) and cadmium thiosulfate (CdS2O3) were produced when Cd reacted with Na2S4. Authentic CdS did not cause cellular signaling responses to be activated or hepatotoxic effects, while CdS2O3 had effects similar to those of Cd. HSP70 and MT-I/II upregulation and hepatotoxicity caused by exposure to Cd were significantly enhanced by the deletion of cystathionine γ-lyase (CSE), which catalyzes the formation of reactive persulfides/polysulfides. Deleting CSE also exacerbated Cd-mediated liver injury, whereas little hepatic damage was found when CdS or Na2S4 along with Cd was administered. Overall, the results suggest that the persulfide/polysulfide-mediated formation of sulfur adducts of Cd such as CdS rather than CdS2O3 is, at least in part, involved in decreasing the level of Cd-mediated activation of cellular signaling and toxicity.

Immobilization of the Gas Signaling Molecule H2 S by Radioisotopes: Detection, Quantification, and In Vivo Imaging.

Hydrogen sulfide (H2 S) has multifunctional roles as a gas signaling molecule in living systems. However, the efficient detection and imaging of H2 S in live animals is very challenging. Herein, we report the first radioisotope-based immobilization technique for the detection, quantification, and in vivo imaging of endogenous H2 S. Macrocyclic (64) Cu complexes that instantly reacted with gaseous H2 S to form insoluble (64) CuS in a highly sensitive and selective manner were prepared. The H2 S concentration in biological samples was measured by a thin-layer radiochromatography method. When (64) Cu-cyclen was injected into mice, an elevated H2 S concentration in the inflamed paw was clearly visualized and quantified by Cerenkov luminescence and positron emission tomography (PET) imaging. PET imaging was also able to pinpoint increased H2 S levels in a millimeter-sized infarcted lesion of the rat heart.