D-amino acids are being recognized in mammals as important molecules with function. This is a first identification of endogenous D-cysteine in mammalian pancreas. D-cysteine is synthesized by serine racemase (SR) and SR−/− mice produce 6-10 fold higher levels of insulin in the pancreas and plasma including higher glycogen and ketone bodies in the liver. The excess insulin is stored as amyloid in secretory vesicles and exosomes. In glucose stimulated insulin secretion studies in mouse and human islets, equimolar amount of D-cysteine showed higher inhibition of insulin secretion compared to D-serine, another closely related stereoisomer synthesized by SR. In mouse models of diabetes (STZ and NOD) and human pancreas, the diabetic state showed increased expression of D-cysteine compared to D-serine followed by increased expression of SR. SR−/− mice show decreased cAMP in the pancreas followed by reduced phosphorylation of CREB (S133), lower DNA methyltransferase enzymatic and promoter activities resulting in decreased methylation of the Ins1 promoter. D-cysteine is efficiently metabolized by D-amino acid oxidase and transported by ASCT2 and Asc1. Dietary supplementation with methyl donors restored the high insulin levels and low DNMT enzymatic activity in SR−/− mice. Our data show that endogenous D-cysteine in the mammalian pancreas is a regulator of insulin secretion. Highlights 1. Serine Racemase also functions as a cysteine racemase. 2. Lack of Serine Racemase results in significantly high levels of insulin in the pancreas, plasma and larger islets. 3. D-cysteine shows greater inhibition of insulin secretion compared to D-serine. 4. Endogenous D-cysteine signals via cyclic AMP that mediates downstream CREB-DNMT1 interaction. 5. CREB-DNMT1 interaction results in hypomethylation of Ins1 promoter that can be rescued by high methyl donor dietary supplementation rescuing high insulin levels. ### Competing Interest Statement The authors have declared no competing interest.

Background: D-amino acids are being recognized as important molecules in mammals with function. This is a first identification of endogenous D-cysteine in mammalian pancreas. Methods: Using a novel stereospecific bioluminescent assay, chiral chromatography, enzyme kinetics and a transgenic mouse model we identify endogenous D-cysteine. We elucidate its function in two mice models of type 1 diabetes (STZ and NOD), and in tests of Glucose Stimulated Insulin Secretion in isolated mouse and human islets and INS-1 832/13 cell line. Results and Discussion: D-cysteine is synthesized by serine racemase (SR) and SR-/- mice produce 6-10 fold higher levels of insulin in the pancreas and plasma including higher glycogen and ketone bodies in the liver. The excess insulin is stored as amyloid in secretory vesicles and exosomes. In glucose stimulated insulin secretion in mouse and human islets, equimolar amount of D-cysteine showed higher inhibition of insulin secretion compared to D-serine, another closely related stereoisomer synthesized by SR. In mouse models of diabetes (Streptozotocin (STZ) and Non Obese Diabetes (NOD) and human pancreas, the diabetic state showed increased expression of D-cysteine compared to D-serine followed by increased expression of SR. SR-/- mice show decreased cAMP in the pancreas, lower DNA methyltransferase enzymatic and promoter activities followed by reduced phosphorylation of CREB (S133), resulting in decreased methylation of the Ins1 promoter. D-cysteine is efficiently metabolized by D-amino acid oxidase and transported by ASCT2 and Asc1. Dietary supplementation with methyl donors restored the high insulin levels and low DNMT enzymatic activity in SR-/- mice. Conclusions: Our data show that endogenous D-cysteine in the mammalian pancreas is a regulator of insulin secretion.

The presence of endogenous d-stereoisomers of amino acids in mammals dispels a long-standing dogma about their existence. d-Serine and d-aspartate function as novel neurotransmitters in mammals. However, the stereoisomer with the fastest, spontaneous in vitro racemization rate, d-cysteine, has not been reported. We utilized a novel, stereospecific, bioluminescent assay to identify endogenous d-cysteine in substantial amounts in the eye, brain, and pancreas of mice. d-Cysteine is enriched in mice embryonic brains at day E9.5 (4.5 mM) and decreases progressively with development (μM levels). d-Cysteine is also present in significantly higher amounts in the human brain white matter compared with gray matter. In the luciferase assay, d-cysteine conjugates with cyano hydroxy benzothiazole in the presence of a base and reducing agent to form d-luciferin. d-Luciferin, subsequently, in the presence of firefly luciferase and ATP, emits bioluminescence proportional to the concentration of d-cysteine. The assay is stereospecific and allows the quantitative estimation of endogenous d-cysteine in tissues in addition to its specificity for d-cysteine. Future efforts aimed at bioluminescent in vivo imaging of d-cysteine may allow a more noninvasive means of its detection, thereby elucidating its function.

Endogenous D-stereoisomers of amino acids are poorly understood in mammals. Here we report the identification and function of endogenous D-cysteine in the pancreas. D-cysteine is present in substantial amounts in the eyes and pancreas of mice. Serine Racemase (SR) is the biosynthetic enzyme for D-cysteine, as it can racemize L-cysteine to D-cysteine. To investigate endogenous D-cysteine, we used SR deficient mice lacking racemizing ability and show 3.5 fold reduction in pancreatic D-cysteine. SR-/- mice produce 6-10 fold higher levels of insulin and are hypoglycemic. The excess insulin is stored in secretory vesicles and plasma exosomes as amyloid. Lack of endogenous D-cysteine results in decreased levels of cAMP. This results in reduced phosphorylation of CREB (S133) leading to lower expression of DNA methyltransferase (DNMT) 1, 3A and 3B, resulting in reduced DNMT and DNMT1 promoter activities in the pancreas and eventual decrease in DNA methylation globally and specifically of the Ins1 promoter. D-cysteine is efficiently metabolized by D-amino acid oxidase and transported by ASCT2 and Asc1. Methyl donor dietary supplementation rescues the high insulin levels and low DNMT activity in SR-/- mice. Our data show that endogenous D-cysteine in the pancreas is a physiologic down regulator of insulin promoter methylation.

Significance d -amino acids are increasingly recognized as important signaling molecules in the mammalian central nervous system. Cysteine is the amino acid with the fastest in vitro spontaneous racemization rate, but its d -stereoisomer has not been examined. Here, we establish the presence of endogenous d -cysteine in the mammalian brain. Using sensitive and specific assays, we delineate its actions as a negative regulator of growth factor signaling during cortical development and identify a putative binding partner mediating these effects. By describing the newest member of the d -amino acid family, we open an avenue of research into the functions of these multifaceted signaling molecules.

The inositol pyrophosphates, molecular messengers containing an energetic pyrophosphate bond, impact a wide range of biologic processes. They are generated primarily by a family of three inositol hexakisphosphate kinases (IP6Ks), the principal product of which is diphosphoinositol pentakisphosphate (IP7). We report that IP6K2, via IP7 synthesis, is a major mediator of cancer cell migration and tumor metastasis in cell culture and in intact mice. IP6K2 acts by enhancing cell-matrix adhesion and decreasing cell-cell adhesion. This action is mediated by IP7-elicited nuclear sequestration and inactivation of the tumor suppressor liver kinase B1 (LKB1). Accordingly, inhibitors of IP6K2 offer promise in cancer therapy.

Systemic responses to hypoxia vary among human subjects and in different strains of rats. We examined whether these inherent variations in hypoxic response to carotid body (CB) O2 sensing and involve CO‐H2S signaling. Experiments were performed on age and gender matched Sprague‐Dawley (SD), Brown‐Norway (BN) and Spontaneous Hypertensive (SH) rats. Compared to SD, BN rats exhibited impaired carotid body response to hypoxia, and developed pulmonary edema as a consequence of poor ventilatory adaptation to hypobaric hypoxia. SH rat carotid bodies displayed inherent hypersensitivity to hypoxia and develop hypertension. BN CBs have higher CO and lower H2S levels than SD, while SH carotid bodies have reduced CO and greater H2S generation. Reducing CO levels in BN carotid body increased H2S generation, restoring CB hypoxic sensing and preventing high‐altitude pulmonary edema. Increasing CO levels in SH carotid bodies reduced H2S generation, preventing hypersensitivity to hypoxia and controlling hypertension. Supported by NIH‐HL‐90554.

Complex interplay between three gases—oxygen, carbon monoxide, and hydrogen sulfide—is necessary to control breathing.

Previous studies showed that O2 sensing by the carotid body (CB) requires carbon monoxide (CO) generation by heme oxygenase (HO)‐2 and hydrogen sulfide (H2S) synthesis by cystathionine‐γ‐lyase (CSE). However, the mechanism(s) underlying O2‐dependent gaseous messenger generation are not known. Here, we report that CO but not H2S generation is sensitive to changes in O2 levels. The O2‐dependent CO generation requires two cysteine residues (C265 and C282) located in the heme‐regulatory motif of HO‐2. Carbon monoxide, in turn inhibits H2S generation from CSE via protein kinase G (PKG)‐dependent phosphorylation of serine 377. Hypoxia, by reducing CO generation, decreases the inhibition of CSE resulting in elevated H2S, which mediates the increased CB neural activity. These results demonstrate that PKG‐dependent regulation of H2S governs O2 sensing by the carotid body (Supported by NIH‐HLBI PO1 HL‐090554).

Inositol polyphosphates containing an energetic pyrophosphate bond are formed primarily by a family of three inositol hexakisphosphate (IP6) kinases (IP6K1-3). The Cullin-RING ubiquitin ligases (CRLs) regulate diverse biological processes through substrate ubiquitylation. CRL4, comprising the scaffold Cullin 4A/B, the E2-interacting Roc1/2, and the adaptor protein damage-specific DNA-binding protein 1, is activated by DNA damage. Basal CRL4 activity is inhibited by binding to the COP9 signalosome (CSN). UV radiation and other stressors dissociate the complex, leading to E3 ligase activation, but signaling events that trigger signalosome dissociation from CRL4 have been unclear. In the present study, we show that, under basal conditions, IP6K1 forms a ternary complex with CSN and CRL4 in which IP6K1 and CRL4 are inactive. UV dissociates IP6K1 to generate IP7, which then dissociates CSN-CRL4 to activate CRL4. Thus, IP6K1 is a novel CRL4 subunit that transduces UV signals to mediate disassembly of the CRL4-CSN complex, thereby regulating nucleotide excision repair and cell death.

Oxygen (O-2) sensing by the carotid body and its chemosensory reflex is critical for homeostatic regulation of breathing and blood pressure. Humans and animals exhibit substantial interindividual variation in this chemosensory reflex response, with profound effects on cardiorespiratory functions. However, the underlying mechanisms are not known. Here, we report that inherent variations in carotid body O-2 sensing by carbon monoxide (CO)-sensitive hydrogen sulfide (H2S) signaling contribute to reflex variation in three genetically distinct rat strains. Compared with SpragueDawley (SD) rats, Brown-Norway (BN) rats exhibit impaired carotid body O-2 sensing and develop pulmonary edema as a consequence of poor ventilatory adaptation to hypobaric hypoxia. Spontaneous Hypertensive (SH) rat carotid bodies display inherent hypersensitivity to hypoxia and develop hypertension. BN rat carotid bodies have naturally higher CO and lower H2S levels than SD rat, whereas SH carotid bodies have reduced CO and greater H2S generation. Higher CO levels in BN rats were associated with higher substrate affinity of the enzyme heme oxygenase 2, whereas SH rats present lower substrate affinity and, thus, reduced CO generation. Reducing CO levels in BN rat carotid bodies increased H2S generation, restoring O-2 sensing and preventing hypoxia-induced pulmonary edema. Increasing CO levels in SH carotid bodies reduced H2S generation, preventing hypersensitivity to hypoxia and controlling hypertension in SH rats.

Nuclear factor κB (NF-κB) is an antiapoptotic transcription factor. We show that the antiapoptotic actions of NF-κB are mediated by hydrogen sulfide (H(2)S) synthesized by cystathionine gamma-lyase (CSE). TNF-α treatment triples H(2)S generation by stimulating binding of SP1 to the CSE promoter. H(2)S generated by CSE stimulates DNA binding and gene activation of NF-κB, processes that are abolished in CSE-deleted mice. As CSE deletion leads to decreased glutathione levels, resultant oxidative stress may contribute to alterations in CSE mutant mice. H(2)S acts by sulfhydrating the p65 subunit of NF-κB at cysteine-38, which promotes its binding to the coactivator ribosomal protein S3 (RPS3). Sulfhydration of p65 predominates early after TNF-α treatment, then declines and is succeeded by a reciprocal enhancement of p65 nitrosylation. In CSE mutant mice, antiapoptotic influences of NF-κB are markedly diminished. Thus, sulfhydration of NF-κB appears to be a physiologic determinant of its antiapoptotic transcriptional activity.

Enhancement of cerebral blood flow by hypoxia is critical for brain function, but signaling systems underlying its regulation have been unclear. We report a pathway mediating hypoxia-induced cerebral vasodilation in studies monitoring vascular disposition in cerebellar slices and in intact mouse brains using two-photon intravital laser scanning microscopy. In this cascade, hypoxia elicits cerebral vasodilation via the coordinate actions of H2S formed by cystathionine β-synthase (CBS) and CO generated by heme oxygenase (HO)-2. Hypoxia diminishes CO generation by HO-2, an oxygen sensor. The constitutive CO physiologically inhibits CBS, and hypoxia leads to increased levels of H2S that mediate the vasodilation of precapillary arterioles. Mice with targeted deletion of HO-2 or CBS display impaired vascular responses to hypoxia. Thus, in intact adult brain cerebral cortex of HO-2–null mice, imaging mass spectrometry reveals an impaired ability to maintain ATP levels on hypoxia.

H(2)S generated by the enzyme cystathionine-γ-lyase (CSE) has been implicated in O(2) sensing by the carotid body. The objectives of the present study were to determine whether glomus cells, the primary site of hypoxic sensing in the carotid body, generate H(2)S in an O(2)-sensitive manner and whether endogenous H(2)S is required for O(2) sensing by glomus cells. Experiments were performed on glomus cells harvested from anesthetized adult rats as well as age and sex-matched CSE(+/+) and CSE(-/-) mice. Physiological levels of hypoxia (Po(2) ∼30 mmHg) increased H(2)S levels in glomus cells, and dl-propargylglycine (PAG), a CSE inhibitor, prevented this response in a dose-dependent manner. Catecholamine (CA) secretion from glomus cells was monitored by carbon-fiber amperometry. Hypoxia increased CA secretion from rat and mouse glomus cells, and this response was markedly attenuated by PAG and in cells from CSE(-/-) mice. CA secretion evoked by 40 mM KCl, however, was unaffected by PAG or CSE deletion. Exogenous application of a H(2)S donor (50 μM NaHS) increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in glomus cells, with a time course and magnitude that are similar to that produced by hypoxia. [Ca(2+)](i) responses to NaHS and hypoxia were markedly attenuated in the presence of Ca(2+)-free medium or cadmium chloride, a pan voltage-gated Ca(2+) channel blocker, or nifedipine, an L-type Ca(2+) channel inhibitor, suggesting that both hypoxia and H(2)S share common Ca(2+)-activating mechanisms. These results demonstrate that H(2)S generated by CSE is a physiologic mediator of the glomus cell's response to hypoxia.

Gaseous messengers, nitric oxide and carbon monoxide are implicated in O2 sensing by the carotid body. In the present study, we examined the role of hydrogen sulfide (H2S) another gaseous messenger in the carotid body sensory response to hypoxia. Glomus cells from rat and mice carotid bodies expressed cystathionine γ‐lyase (CSE), an H2S generating enzyme. Hypoxia increased H2S generation in a stimulus‐dependent manner. Mice with genetic deletion of CSE displayed severely impaired carotid body and ventilatory responses to hypoxia as well as loss of hypoxia‐evoked H2S generation from the carotid body. Pharmacologic inhibition of CSE elicited a similar phenotype in mice and rats. Inhibition of hemeoxygenase‐2 in normoxia increased H2S generation, whereas a carbon monoxide donor inhibited hypoxia‐evoked H2S generation in the carotid body. These results demonstrate that a) hypoxia increases H2S generation via CSE, which mediates carotid body sensory response to hypoxia; and b) hypoxia‐evoked H2S generation require interaction of hemeoxygenase‐2 with CSE. Supported by HL‐90554, HL‐76537, HL‐86493 and HL‐089616.

Neonatal adrenal medullary chromaffin cells (AMC) are extremely sensitive to hypoxia, and low O2 stimulates catecholamine secretion. Given that H2S generated by cystathionine γ‐lyase (CSE) is important for mediating carotid body responses to hypoxia, we examined whether hypoxia‐evoked catecholamine secretion from neonatal AMC requires similar signaling mechanisms. Experiments were performed on rats and mice aged P10. Catecholamine secretion was monitored by amperometry using a carbon fiber electrode. AMC from rats and mice expressed CSE‐like immunoreactivity. Genetic deletion of CSE markedly impaired hypoxia‐evoked catecholamine secretion from mice AMC; whereas high potassium‐evoked catecholamine secretion was unaffected. Similar impairment of low O2‐evoked but not high K+induced catecholamine secretion was seen in rat AMC treated with the CSE inhibitor, DL‐propargyl glycine (DL‐PAG; 30–50 μM). These results demonstrate that like in the carotid body, H2S derived from CSE mediates hypoxic sensing by neonatal AMC. Supported by HL‐90554, HL‐76537, HL‐86493 and HL‐089616.

Gaseous messengers, nitric oxide and carbon monoxide, have been implicated in O2 sensing by the carotid body, a sensory organ that monitors arterial blood O2 levels and stimulates breathing in response to hypoxia. We now show that hydrogen sulfide (H2S) is a physiologic gasotransmitter of the carotid body, enhancing its sensory response to hypoxia. Glomus cells, the site of O2 sensing in the carotid body, express cystathionine γ-lyase (CSE), an H2S-generating enzyme, with hypoxia increasing H2S generation in a stimulus-dependent manner. Mice with genetic deletion of CSE display severely impaired carotid body response and ventilatory stimulation to hypoxia, as well as a loss of hypoxia-evoked H2S generation. Pharmacologic inhibition of CSE elicits a similar phenotype in mice and rats. Hypoxia-evoked H2S generation in the carotid body seems to require interaction of CSE with hemeoxygenase-2, which generates carbon monoxide. CSE is also expressed in neonatal adrenal medullary chromaffin cells of rats and mice whose hypoxia-evoked catecholamine secretion is greatly attenuated by CSE inhibitors and in CSE knockout mice.

Olson and Perry (1) commented on our recent article “H2S mediates O2 sensing in the carotid body” (2), noting that they published a study relevant to hypoxia and H2S (3). To understand the relationship of the two studies, we will summarize work from our laboratories bearing on H2S as a gasotransmitter in blood vessels (4) and the carotid body (2). Because of its chemical reactivity, H2S is known to affect multiple biological systems, including blood vessels. Whether endogenously produced H2S is a physiologic messenger molecule had not been definitively examined. Availability of mice with targeted deletion of cystathionine γ-lyase (CSE) enabled the laboratories of Rui Wang and Solomon H. Snyder to show that CSE deletion depletes peripheral tissue H2S, markedly reduces endothelial-dependent relaxing factor activity, and elicits hypertension (4). This work established that H2S is physiologically generated peripherally by CSE and is a major regulator of blood vessel function.