Cell selectivity in succinate receptor SUCNR1/GPR91 signaling in skeletal muscle.

Succinate is released by skeletal muscle during exercise and activates SUCNR1/GPR91. Signaling of SUCNR1 is involved in metabolite-sensing paracrine communication in skeletal muscle during exercise. However, the specific cell types responding to succinate and the directionality of communication are unclear. We aim to characterize the expression of SUCNR1 in human skeletal muscle. De novo analysis of transcriptomic datasets demonstrated that SUCNR1 mRNA is expressed in immune, adipose, and liver tissues, but scarce in skeletal muscle. In human tissues, SUCNR1 mRNA was associated with macrophage markers. Single-cell RNA sequencing and fluorescent RNAscope demonstrated that in human skeletal muscle, SUCNR1 mRNA is not expressed in muscle fibers but coincided with macrophage populations. Human M2-polarized macrophages exhibit high levels of SUCNR1 mRNA and stimulation with selective agonists of SUCNR1 triggered Gq- and Gi-coupled signaling. Primary human skeletal muscle cells were unresponsive to SUCNR1 agonists. In conclusion, SUCNR1 is not expressed in muscle cells and its role in the adaptive response of skeletal muscle to exercise is most likely mediated via paracrine mechanisms involving M2-like macrophages within the muscle.NEW & NOTEWORTHY Macrophages but not skeletal muscle cells respond to extracellular succinate via SUCNR1/GPR91.

Understanding Ischemic Retinopathies: The Role of Succinate and Its Receptor in Retinal Pigment Epithelium.

Retinopathy is the general name for all condition of the eyes in which blood vessels that supply oxygen to the retina are damaged. These include diabetic retinopathy, retinopathy of prematurity, hypertensive retinopathy, and arteriosclerotic retinopathy. Although the initial trigger that leads to insufficient perfusion of the retina may be different, once a critical level of ischemia is achieved, all types of retinopathies seem to follow a common sequence-oxidative stress, followed by hypoxia-induced formation of morphologically abnormal vessels. This preretinal vascular growth is the most severe aspect of the retinopathy, as the outcome is often retinal detachment and eventually blindness. Regardless of which therapy strategy is followed, a deeper understanding of both normal retinal growth and the underlying molecular mechanisms of retinopathies is needed in order to come up with more effective therapies. This chapter focuses on the citric acid cycle intermediate succinate and its G protein-coupled receptor SUCNR1 in ischemic retinopathies, which were identified as potent mediators of vessel growth in the settings of both normal retinal development and proliferative ischemic retinopathies.

Cancer-derived extracellular succinate: a driver of cancer metastasis.

Succinate is a tricarboxylic acid (TCA) cycle intermediate normally confined to the mitochondrial matrix. It is a substrate of succinate dehydrogenase (SDH). Mutation of SDH subunits (SDHD and SDHB) in hereditary tumors such as paraganglioma or reduction of SDHB expression in cancer results in matrix succinate accumulation which is transported to cytoplasma and secreted into the extracellular milieu. Excessive cytosolic succinate is known to stabilize hypoxia inducible factor-1α (HIF-1α) by inhibiting prolyl hydroxylase. Recent reports indicate that cancer-secreted succinate enhances cancer cell migration and promotes cancer metastasis by activating succinate receptor-1 (SUCNR-1)-mediated signaling and transcription pathways. Cancer-derived extracellular succinate enhances cancer cell and macrophage migration through SUCNR-1 â†’ PI-3 K â†’ HIF-1α pathway. Extracellular succinate induces tumor angiogenesis through SUCNR-1-mediated ERK1/2 and STAT3 activation resulting in upregulation of vascular endothelial growth factor (VEGF) expression. Succinate increases SUCNR-1 expression in cancer cells which is considered as a target for developing new anti-metastasis drugs. Furthermore, serum succinate which is elevated in cancer patients may be a theranostic biomarker for selecting patients for SUCNR-1 antagonist therapy.

The Role of Mitochondrial Enzymes, Succinate-Coupled Signaling Pathways and Mitochondrial Ultrastructure in the Formation of Urgent Adaptation to Acute Hypoxia in the Myocardium.

The effect of a single one-hour exposure to three modes of hypobaric hypoxia (HBH) differed in the content of O2 in inhaled air (FiO2-14%, 10%, 8%) in the development of mitochondrial-dependent adaptive processes in the myocardium was studied in vivo. The following parameters have been examined: (a) an urgent reaction of catalytic subunits of mitochondrial enzymes (NDUFV2, SDHA, Cyt b, COX2, ATP5A) in the myocardium as an indicator of the state of the respiratory chain electron transport function; (b) an urgent activation of signaling pathways dependent on GPR91, HIF-1α and VEGF, allowing us to assess their role in the formation of urgent mechanisms of adaptation to hypoxia in the myocardium; (c) changes in the ultrastructure of three subpopulations of myocardial mitochondria under these conditions. The studies were conducted on two rat phenotypes: rats with low resistance (LR) and high resistance (HR) to hypoxia. The adaptive and compensatory role of the mitochondrial complex II (MC II) in maintaining the electron transport and energy function of the myocardium in a wide range of reduced O2 concentrations in the initial period of hypoxic exposure has been established. The features of urgent reciprocal regulatory interaction of NAD- and FAD-dependent oxidation pathways in myocardial mitochondria under these conditions have been revealed. The data indicating the participation of GPR91, HIF-1a and VEGF in this process have been obtained. The ultrastructure of the mitochondrial subpopulations in the myocardium of LR and HR rats differed in normoxic conditions and reacted differently to hypoxia of varying severity. The parameters studied together are highly informative indicators of the quality of cardiac activity and metabolic biomarkers of urgent adaptation in various hypoxic conditions.

Receptor GPR91 contributes to voiding function and detrusor relaxation mediated by succinate.

AIM: Succinate activates the receptor GPR91 identified in the bladder. The present study aims to unravel the mechanisms of bladder relaxation by succinate and how the receptor is involved in structural and functional changes of the bladder. METHODS: Physiological recordings of bladder function were carried out by cystometry and organ bath from C57BL/6 mice, homozygous GPR91-/- mice, and Sprague-Dawley (SD) rats. GPR91 expression was confirmed by polymerase chain reaction and tissue morphology was examined by light (Masson trichrome) and fluorescence microscopy. Nitric oxide (NO) and ATP secretion were measured. RESULTS: Bladders of GPR91 KO mice had a greater mass to body weight ratio with a thicker bladder wall compared to C57BL/6 mice. They also displayed increased basal and maximal bladder pressures, and decreased intercontraction intervals, bladder capacity, micturition volume, and compliance. During cystometry, bladders of SD rats and C57BL/6 mice instilled with succinate (10 mM) showed signs of relaxation while bladders of GPR91 KO mice were unresponsive. Similarly, in organ bath, succinate relaxed bladder strips preincubated with carbachol, except GPR91 KO ones. Relaxation was stronger in the presence of urothelium and independent of NO synthesis. Bladder strips from all mice groups showed similar responses to KCl, carbachol, and electrical stimulation. In vitro, succinate increased NO secretion in urothelial cell culture of both C57BL6 and GPR91 KO mice while ATP secretion was potently decreased by succinate in C57BL6 culture only. CONCLUSION: Succinate through GPR91 is essential to bladder structure and contraction. GPR91 relaxes the detrusor partially by decreasing urothelial ATP secretion.

GPR91 antagonist and TGF-ß inhibitor suppressed collagen production of high glucose and succinate induced HSC activation.

Activated hepatic stellate cells (HSCs) play a central role in fibrillary collagen production, the primary cause of liver fibrosis. Although it is known that primary cultured HSCs are activated by plastic culture dish stiffness, HSC activation and quiescent-state-reversion mechanisms are still unclear. In this study, we used cultured normal rat HSCs on 3.2 kPa collagen normal liver stiffness equivalent gel, to determine whether high glucose or high succinate conditions induce HSC activation, and examined whether activated HSCs reverted to a quiescent state when suppressed by GPR91 antagonists or TGF-ß1 receptor inhibitors. We measured the gene expression levels of α-SMA and type I collagen HSC activation markers using real-time PCR. Our data indicated that high glucose conditions induced HSC activation, and showed that under continuous high glucose exposure HSC activation progressed. A GPR91 antagonist, 2 d, and a TGF-ß1 receptor inhibitor, SB525334, suppressed the Col1α mRNA expression level of these activated HSCs. Similarly, under extended high succinate exposure, 2 d and SB525334 reduced Col1α mRNA expression levels of activated HSCs. From the above, we determined that even though HSCs had already been activated by high glucose or succinate conditions which persisted after activation, the GPR91 antagonist and the TGF-ß1 receptor inhibitor were able to reduce the production of type I collagen from activated HSCs.

Succinate-GPR-91 receptor signalling is responsible for nonalcoholic steatohepatitis-associated fibrosis: Effects of DHA supplementation.

BACKGROUND AND AIMS: Treatment of non-alcoholic steatohepatitis (NASH) is challenging, because suppressing fibrotic progression has not been achieved consistently by drug candidates currently in clinical trials. The aim of this study was to investigate the molecular interplays underlying NASH-associated fibrosis in a mouse NASH model and human specimens. METHODS: Mice were divided into 4 groups: Controls; NASH (high fat/Calorie diet plus high fructose and glucose in drinking water, HFCD-HF/G) for 16 weeks; HFCD-HF/G plus docosahexaenoic acid (DHA) for 16 or 8 weeks. RESULTS: Along with NASH progression, fibrotic deposition was documented in HFCD-HF/G-fed mice. Liver succinate content was significantly increased along with decreased expression of succinate dehydrogenase-A (SDH-A) in these mice; whereas, GPR-91 receptor expression was much enhanced in histology compared to control mice, and co-localized histologically with hepatic stellate cells (HSCs). Succinate content was increased in fatty acid-overloaded primary hepatocytes with significant oxidant stress and lipotoxicity. Exposure to succinate led to up-regulation of GPR-91 receptor in primary and immortalized HSCs. In contrast, suppression of GPR-91 receptor expression abolished succinate stimulatory role in GPR-91 expression and extracellular matrix production in HSCs. All these changes were minimized or abrogated by DHA supplementation in vivo or in vitro. Moreover, GPR-91 receptor expression correlates with severity of fibrosis in human NASH biopsy specimens. CONCLUSION: Succinate accumulation in steatotoic hepatocytes may result in HSC activation through GPR-91 receptor signalling in NASH progression, and the cross-talk between hepatocytes and HSC through GPR-91 signalling is most likely to be the molecular basis of fibrogenesis in NASH.

Cell-surface G-protein-coupled receptors for tumor-associated metabolites: A direct link to mitochondrial dysfunction in cancer.

Mitochondria are the sites of pyruvate oxidation, citric acid cycle, oxidative phosphorylation, ketogenesis, and fatty acid oxidation. Attenuation of mitochondrial function is one of the most significant changes that occurs in tumor cells, directly linked to oncogenesis, angiogenesis, Warburg effect, and epigenetics. In particular, three mitochondrial enzymes are inactivated in cancer: pyruvate dehydrogenase (PDH), succinate dehydrogenase (SDH), and 3-hydroxy-3-methylglutaryl CoA synthase-2 (HMGCS2). These enzymes are subject to regulation via acetylation/deacetylation. SIRT3, the predominant mitochondrial deacetylase, directly targets these enzymes for deacetylation and maintains their optimal catalytic activity. SIRT3 is a tumor suppressor, and deacetylation of these enzymes contributes to its biological function. PDH catalyzes the oxidative decarboxylation of pyruvate into acetyl CoA, SDH oxidizes succinate into fumarate, and HMGCS2 controls the synthesis of the ketone body ß-hydroxybutyrate. As the activities of these enzymes are decreased in cancer, tumor cells accumulate lactate and succinate but produce less amounts of ß-hydroxybutyrate. Apart from their role in cellular energetics, these metabolites function as signaling molecules via specific cell-surface G-protein-coupled receptors. Lactate signals via GPR81, succinate via GPR91, and ß-hydroxybutyrate via GPR109A. In addition, lactate activates hypoxia-inducible factor HIF1α and succinate promotes DNA methylation. GPR81 and GPR91 are tumor promoters, and increased production of lactate and succinate as their agonists drives tumorigenesis by enhancing signaling via these two receptors. In contrast, GPR109A is a tumor suppressor, and decreased synthesis of ß-hydroxybutyrate as its agonist suppresses signaling via this receptor, thus attenuating the tumor-suppressing function of GPR109A. In parallel with the opposing changes in lactate/succinate and ß-hydroxybutyrate levels, tumor cells upregulate GPR81 and GPR91 but downregulate GPR109A. As such, these three metabolite receptors play a critical role in cancer and represent a new class of drug targets with selective antagonists of GPR81 and GPR91 for cancer treatment and agonists of GPR109A for cancer prevention.

Succinate receptor deficiency attenuates arthritis by reducing dendritic cell traffic and expansion of Th17 cells in the lymph nodes.

Rheumatoid arthritis is a chronic inflammatory disease that leads to significant changes in metabolic activity. Succinate, an intermediate of the tricarboxylic acid cycle, has emerged as a metabolic mediator of the innate immune response. However, the involvement of succinate in the generation of the adaptive immune response and establishment of autoimmune response has not been addressed thus far. Here we demonstrated that the succinate-sensing receptor (Sucnr1/GPR91) plays a critical role in the development of immune-mediated arthritis. We found that Sucnr1 acts as a chemotactic gradient sensor that guides dendritic cells (DCs) into the lymph nodes, orchestrating the expansion of the T helper (Th)17-cell population and the development of experimental antigen-induced arthritis. Sucnr1-/- mice show reduced articular hyperalgesia, neutrophil infiltration and inflammatory cytokines in the joint, and reduced frequency of Th17 cells in draining lymph nodes. Adoptive transfer of wild-type (WT) DCs into Sucnr1-/- mice restored the development of arthritis. Moreover, DC-depleted mice transferred with Sucnr1-/- DCs developed less arthritis than mice transferred with WT DCs. In contrast, succinate given together with the immunization boosted the recruitment of DCs and the frequency of Th17 cells in draining lymph nodes, increasing arthritis severity. Therefore, the blockade of Sucnr1 may represent a novel therapeutic target of arthritis.-Saraiva, A. L., Veras, F. P., Peres, R. S., Talbot, J., de Lima, K. A., Luiz, J. P., Carballido, J. M., Cunha, T. M., Cunha, F. Q., Ryffel, B., Alves-Filho, J. C. Succinate receptor deficiency attenuates arthritis by reducing dendritic cell traffic and expansion of Th17 cells in the lymph nodes.

Metformin ameliorates activation of hepatic stellate cells and hepatic fibrosis by succinate and GPR91 inhibition.

BACKGROUND: Chronic liver disease is becoming a major cause of morbidity and mortality worldwide. During liver injury, hepatic stellate cells (HSCs) trans-differentiate into activated myofibroblasts, which produce extracellular matrix. Succinate and succinate receptor (G-protein coupled receptor91, GPR91) signaling pathway has now emerged as a regulator of metabolic signaling. A previous study showed that succinate and its specific receptor, GPR91, are involved in the activation of HSCs and the overexpression of α-smooth muscle actin (α-SMA). Metformin, a well-known anti-diabetic drug, inhibits hepatic gluconeogenesis in the liver. Many studies have shown that metformin not only prevented, but also reversed, steatosis and inflammation in a nonalcoholic steatohepatitis (NASH) animal model. However, the role of metformin in HSC activation and succinate-GPR91 signaling has not been clarified. METHODS: The immortalized human HSCs, LX-2 cells, were used for the in vitro study. For the in vivo study, male C57BL/J6 mice were randomly divided into 3 groups and were fed with a methionine-choline-deficient diet (MCD diet group) as a nonalcoholic steatohepatitis (NASH) mouse model with or without 0.1% metformin for 12 weeks, or were fed a control methionine-choline-sufficient diet (MCS diet group). RESULTS: In our study, metformin and 5-aminoimidazole-4-carboxamide 1-ß-d-ribofuranoside (AICAR), which is an analog of adenosine monophosphate, were shown to suppress α-SMA expression via enhanced phosphorylation of AMP-activated protein kinase (AMPK) and inhibition of succinate-GPR91 signaling in activated LX-2 cells induced by palmitate- or succinate. Metformin and AICAR also reduced succinate concentration in the cell lysates when LX-2 cells were treated with palmitate. Moreover, metformin and AICAR reduced interleukin-6 and, transforming growth factor-ß1 production in succinate-treated LX-2 cells. Both metformin and AICAR inhibited succinate-stimulated HSC proliferation and cell migration. Mice fed a MCD diet demonstrated increased steatohepatitis and liver fibrosis compared to that of mice fed control diet. Metformin ameliorated steatohepatitis, liver fibrosis, inflammatory cytokine production and decreased α -SMA and GPR91expression in the livers of the MCD diet-fed mice. CONCLUSION: This study shows that metformin can attenuate activation of HSCs by activating the AMPK pathway and inhibiting the succinate-GPR91 pathway. Metformin has therapeutic potential for treating steatohepatitis and liver fibrosis.

Succinate decreases bladder function in a rat model associated with metabolic syndrome.

AIMS: Succinate and its receptor, GPR91, have been implicated in different aspects of metabolic syndrome. As GPR91 is expressed in the urinary bladder, the aim of this study is to show the effect of chronically increased succinate levels on bladder function. MATERIALS AND METHODS: Healthy Sprague-Dawley (SD) rats and hypertensive Dahl rats received an intraperitoneal injection of either saline or succinate (50 mg/kg) daily for a period of 4 weeks. Conscious cystometry was performed at the end of this period. Bladders were collected and used for contractility studies and morphological assessment. Two-way ANOVA was performed to compare between the two strains and student t-tests to compare treatment groups within each strain. RESULTS: Compared to SD rats, Dahl rats showed signs of bladder dysfunction. Succinate treatment led to higher urinary succinate levels and lower bladder capacities compared to saline-treated animals. In SD rats, this was associated with higher collagen content, lower GPR91 expression and an altered bladder nerve profile in the bladder. In succinate-treated Dahl rats, detrusor contractility was reduced and associated with decreased cholinergic innervation and increased collagen content. CONCLUSIONS: It is suggested that succinate negatively affects bladder function via effects through its receptor, GPR91, and that its effects are enhanced in the presence of metabolic disturbance. These findings contribute to our understanding of the pathophysiology of bladder dysfunction, specifically in a metabolic syndrome setting.

Succinate accumulation impairs cardiac pyruvate dehydrogenase activity through GRP91-dependent and independent signaling pathways: Therapeutic effects of ginsenoside Rb1.

Altered mitochondrial oxidation increases vulnerability to cardiac ischemia/reperfusion (I/R) injury in metabolic disorders. However, the metabolic signaling responsible for the dysfunction remains partly unknown. We sought to test whether or not hypoxic succinate accumulation could inhibit pyruvate dehydrogenase (PDH) activity and subsequently aggravate I/R injury. Results showed that saturated fatty acid palmitate stimulation increased fatty acid oxidation and induced hypoxia in cardiomyocytes, leading to succinate accumulation. Intracellular succinate induced hypoxia inducible factor-1α (HIF-1α) expression and impaired PDH activity via upregulation of pyruvate dehydrogenase kinase 4 (PDK4) expression. Luciferase reporter assay showed that succinate increased PDK4 expression through gene promoter induction in a HIF-1α-dependent manner. Palmitate also induced the release of succinate into extracellular space. By activating GRP91, extracellular succinate induced the translocation of PKCδ to mitochondria and further exacerbated PDH impairment. These results demonstrated that succinate impaired PDH activity via GPR91-dependent and independent pathways. Ginsenoside Rb1 (a major compound isolated from ginseng) and trimetazidine (fatty acid ß-oxidation inhibitor) prevented hypoxic succinate accumulation in cardiomyocytes and improved PDH activity by blocking succinate-associated HIF-1α activation and GPR91 signaling. Through improving PDH activity, Rb1 and trimetazidine prevented cardiac acidification, ameliorated mitochondrial dysfunction and thereby reduced apoptosis during hypoxia/reoxygenation insult. In isolated working rat hearts perfused with palmitate and in high-fat diet-fed mice, early intervention of Rb1 and trimetazidine reduced succinate production and resultantly increased heart resistance to ischemia/reperfusion injury. Taken together, our findings demonstrated that early intervention by targeting inhibition of succinate accumulation-induced PDH impairment is an effective strategy to alleviate I/R injury.

Expression and localization of GPR91 and GPR99 in murine organs.

Energy substrates and metabolic intermediates are proven ligands of a growing number of G-protein coupled receptors. In 2004, GPR91 and GPR99 were identified as receptors for the citric acid cycle intermediates, succinate and α-ketoglutarate, respectively. GPR91 seems to act as a first responder to local stress and GPR99 participates in the regulation of the acid-base balance through an intrarenal paracrine mechanism. However, a systematic analysis of the distribution of both receptors in mouse organs is still missing. The aim of this study was to examine the expression of GPR91 and GPR99 in a large number of different murine organs both at mRNA and protein level. Whereas GPR91 mRNA was detectable in almost all organs, GPR99 mRNA was mainly expressed in neuronal tissues. Widespread expression of GPR91 was also detected at the protein level by western blotting and immunohistochemistry. In addition to neuronal cells, GPR99 protein was found in renal intercalated cells and epididymal narrow cells. Double-labeling immunohistochemistry demonstrated the colocalization of GPR99 with the B1 subunit isoform of vacuolar H(+)-ATPases which is expressed only by a very limited number of cell types. In summary, our detailed expression analysis of GPR91 and GPR99 in murine tissues will allow a more directed search for additional functions of both receptors.

Specific Features of Immediate Expression of Succinate-Dependent Receptor GPR91 in Tissues during Hypoxia.

Under normoxic conditions, succinate-dependent receptor GPR91 was found in varying amounts in all analyzed aerobic tissues except erythrocytes. The maximum density of the receptor was observed in the myocardium: by 2.3 and 1.7 times higher than in the kidneys and brain cortex, respectively. Hypoxic expression of GPR91 was tissue-specific, depended on the duration and severity of hypoxia, and did not correlate with the basal level of this receptor. It was maximum in the brain cortex, which confirms the high importance of this signal pathway for brain functioning. Single hypoxic exposure induced immediate expression of GPR91 in the brain cortex within 15-60 min, which correlated with the development of urgent tolerance to hypoxia of the body. Induction of immediate expression of GPR91 in brain cortex occurred during first 15-60 min and correlated to the forming of immediate tolerance of organism to hypoxia. Brain cortex-specific immediate expression of GPR91 during hypoxia was related to activity of the GABA-bypass that acts as the source of succinate for the receptor under these conditions.

Succinate causes α-SMA production through GPR91 activation in hepatic stellate cells.

Succinate acts as an extracellular signaling molecule as well as an intermediate in the citric acid cycle. It binds to and activates its specific G protein-coupled receptor 91 (GPR91). GPR91 is present in hepatic stellate cells (HSCs), but its role in hepatic fibrogenesis remains unclear. Cultured HSCs treated with succinate showed increased protein expression of GPR91 and α-smooth muscle actin (α-SMA), markers of fibrogenic response. Succinate also increased mRNA expression of α-SMA, transforming growth factor ß (TGF-ß), and collagen type I. Transfection of siRNA against GPR91 abrogated succinate-induced increases in α-SMA expression. Malonate, an inhibitor of succinate dehydrogenase (SDH), increased succinate levels in cultured HSCs and increased GPR91 and α-SMA expression. Feeding mice a methionine- and choline-deficient (MCD) diet is a widely used technique to create an animal model of nonalcoholic steatohepatitis (NASH). HSCs cultured in MCD media showed significantly decreased SDH activity and increased succinate concentration and GPR91 and α-SMA expression. Similarly, palmitate treatment significantly decreased SDH activity and increased GPR91 and α-SMA expression. Finally, C57BL6/J mice fed the MCD diet had elevated succinate levels in their plasma. The MCD diet also decreased SDH activity, increased succinate concentration, and increased GPR91 and α-SMA expression in isolated HSCs. Collectively, our results show that succinate plays an important role in HSC activation through GPR91 induction, and suggest that succinate and GPR91 may represent new therapeutic targets for modulating hepatic fibrosis.

G-protein-coupled receptor 91 and succinate are key contributors in neonatal postcerebral hypoxia-ischemia recovery.

OBJECTIVE: Prompt post-hypoxia-ischemia (HI) revascularization has been suggested to improve outcome in adults and newborn subjects. Other than hypoxia-inducible factor, sensors of metabolic demand remain largely unknown. During HI, anaerobic respiration is arrested resulting in accumulation of carbohydrate metabolic intermediates. As such succinate readily increases, exerting its biological effects via a specific receptor, G-protein-coupled receptor (GPR) 91. We postulate that succinate/GPR91 enhances post-HI vascularization and reduces infarct size in a model of newborn HI brain injury. APPROACH AND RESULTS: The Rice-Vannucci model of neonatal HI was used. Succinate was measured by mass spectrometry, and microvascular density was evaluated by quantification of lectin-stained cryosection. Gene expression was evaluated by real-time polymerase chain reaction. Succinate levels rapidly increased in the penumbral region of brain infarcts. GPR91 was foremost localized not only in neurons but also in astrocytes. Microvascular density increased at 96 hours after injury in wild-type animals; it was diminished in GPR91-null mice leading to an increased infarct size. Stimulation with succinate led to an increase in growth factors implicated in angiogenesis only in wild-type mice. To explain the mode of action of succinate/GPR91, we investigated the role of prostaglandin E2-prostaglandin E receptor 4, previously proposed in neural angiogenesis. Succinate-induced vascular endothelial growth factor expression was abrogated by a cyclooxygenase inhibitor and a selective prostaglandin E receptor 4 antagonist. This antagonist also abolished succinate-induced neovascularization. CONCLUSIONS: We uncover a dominant metabolic sensor responsible for post-HI neurovascular adaptation, notably succinate/GPR91, acting via prostaglandin E2-prostaglandin E receptor 4 to govern expression of major angiogenic factors. We propose that pharmacological intervention targeting GPR91 could improve post-HI brain recovery.

Identification of lead compounds for (99m)Tc and (18)F GPR91 radiotracers.

To develop the first radiotracer targeting GPR91, a cell membrane-bound receptor that modulates the cellular response to hyperglycemia and hypoxia, we designed and prepared a small series of compounds based on a published series of 1,8-naphthyridines with high affinity to GPR91. Our approach provides a mechanism to incorporate radioactive atoms ((99m)Tc and (18)F) into the GPR91 pharmacophore as the final synthetic step. Pharmacological assays confirmed lead compounds for (99m)Tc and (18)F GPR91 radiotracers within the series.

miR-369-3p ameliorates diabetes-associated atherosclerosis by regulating macrophage succinate-GPR91 signaling.

AIMS: Diabetes leads to dysregulated macrophage immunometabolism, contributing to accelerated atherosclerosis progression. Identifying critical factors to restore metabolic alterations and promote resolution of inflammation remains an unmet goal. MicroRNAs (miRs) orchestrate multiple signaling events in macrophages, yet their therapeutic potential in diabetes-associated atherosclerosis remains unclear. METHODS AND RESULTS: MiRNA profiling revealed significantly lower miR-369-3p expression in aortic intimal lesions from Ldlr-/- mice on a high-fat sucrose containing (HFSC) diet for 12 weeks. miR-369-3p was also reduced in peripheral blood mononuclear cells (PBMCs) from diabetic patients with coronary artery disease (CAD). Cell-type expression profiling showed miR-369-3p enrichment in aortic macrophages. In vitro, oxLDL treatment reduced miR-369-3p expression in mouse bone marrow-derived macrophages (BMDMs). Metabolic profiling in BMDMs revealed that miR-369-3p overexpression blocked the oxLDL-mediated increase in the cellular metabolite succinate and reduced mitochondrial respiration (OXPHOS) and inflammation (lL-1ß, TNF-a, IL-6). Mechanistically, miR-369-3p targeted the succinate receptor (GPR91) and alleviated the oxLDL-induced activation of inflammasome signaling pathways. Therapeutic administration of miR-369-3p mimics in HFSC-fed Ldlr-/- mice reduced GPR91 expression in lesional macrophages and diabetes-accelerated atherosclerosis, evident by a decrease in plaque size and pro-inflammatory Ly6Chi monocytes. RNA-seq analyses showed more pro-resolving pathways in plaque macrophages from miR-369-3p treated mice, consistent with an increase in macrophage efferocytosis in lesions. Finally, a GPR91 antagonist attenuated oxLDL-induced inflammation in primary monocytes from human subjects with diabetes. CONCLUSION: These findings establish a therapeutic role for miR-369-3p in halting diabetes-associated atherosclerosis by regulating GPR91 and macrophage succinate metabolism.

Succinate promotes pulmonary fibrosis through GPR91 and predicts death in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is believed to be associated with a notable disruption of cellular energy metabolism. By detecting the changes of energy metabolites in the serum of patients with pulmonary fibrosis, we aimed to investigate the diagnostic and prognostic value of energy metabolites in IPF, and further elucidated the mechanism of their involvement in pulmonary fibrosis. Through metabolomics research, it was discovered that the TCA cycle intermediates changed dramatically in IPF patients. In another validation cohort of 55 patients with IPF compared to 19 healthy controls, it was found that succinate, an intermediate product of TCA cycle, has diagnostic and prognostic value in IPF. The cut-off levels of serum succinate were 98.36 µM for distinguishing IPF from healthy controls (sensitivity, 83.64%; specificity, 63.16%; likelihood ratio, 2.27, respectively). Moreover, a high serum succinate level was independently associated with higher rates of disease progression (OR 13.087, 95%CI (2.819-60.761)) and mortality (HR 3.418, 95% CI (1.308-8.927)). In addition, accumulation of succinate and increased expression of the succinate receptor GPR91 were found in both IPF patients and BLM mouse models of pulmonary fibrosis. Reducing succinate accumulation in BLM mice alleviated pulmonary fibrosis and 21d mortality, while exogenous administration of succinate can aggravate pulmonary fibrosis in BLM mice. Furthermore, GPR91 deficiency protected against lung fibrosis caused by BLM. In vitro, succinate promoted the activation of lung fibroblasts by activating ERK pathway through GPR91. In summary, succinate is a promising biomarker for diagnosis and prognosis of IPF. The accumulation of succinate may promote fibroblast activation through GPR91 and pulmonary fibrosis.

Methionine secreted by tumor-associated pericytes supports cancer stem cells in clear cell renal carcinoma.

Here, we identify a subset of vascular pericytes, defined by expression of platelet-derived growth factor receptor beta (PDGFR-ß) and G-protein-coupled receptor 91 (GPR91), that promote tumorigenesis and tyrosine kinase inhibitors (TKIs) resistance by functioning as the primary methionine source for cancer stem cells (CSCs) in clear cell renal cell carcinoma (ccRCC). Tumor-cell-derived succinate binds to GPR91 on pericyte to activate autophagy for methionine production. CSCs use methionine to create stabilizing N6-methyladenosine in ATPase-family-AAA-domain-containing 2 (ATAD2) mRNA, and the resulting ATAD2 protein complexes with SRY-box transcription factor 9 to assemble super enhancers and thereby dictate its target genes that feature prominently in CSCs. Targeting PDGFR-ß+GPR91+ pericytes with specific GRP91 antagonists reduce intratumoral methionine level, eliminate CSCs, and enhance TKIs sensitivity. These results unraveled the mechanisms by which PDGFR-ß+GPR91+ pericytes provide supportive niche for CSCs and could be used to develop targets for treating ccRCC.