Succinate Receptor Research Articles

Succinate aggravates intestinal injury in mice with necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is the most prevalent gastrointestinal disorder that predominantly threatens preterm newborns. Succinate is an emerging metabolic signaling molecule that was recently studied in relation to the regulation of intestinal immunity and homeostasis. We aimed to investigate the relationship between NEC and gut luminal succinate and preliminarily explored the effect of succinate on NEC pathogenesis. Fecal samples from human neonates and mouse pups were analyzed by HPLC - MS/MS and 16S rRNA gene sequencing. C57BL/6 mice were randomly divided into four groups: control, NEC, Lsuc, and Hsuc. The mortality, weight gain, and intestinal pathological changes in four mouse groups were observed. Inflammatory cytokines and markers of macrophages were identified by quantitative real-time PCR. Succinate receptor 1 (SUCNR1) localization was visualized by immunohistochemistry. The protein levels of SUCNR1 and hypoxia-inducible factor 1a (HIF-1a) were quantified by western blotting. The levels of succinate in feces from NEC patients were higher than those in feces from non-NEC patients (P <0.05). In the murine models, succinate levels in intestinal content samples were also higher in the NEC group than in the control group (P <0.05). The change in succinate level was closely related to intestinal flora composition. In samples from human neonates, relative to the control group, the NEC group showed a higher abundance of Enterobacteriaceae and a lower abundance of Lactobacillaceae and Lactobacillus (P <0.05). In the murine models, relative to the control group, increased abundance was observed for Clostridiaceae, Enterococcaceae, Clostridium_sensu_stricto_1, and Enterococcus, whereas decreased abundance was observed for Lactobacillaceae and Lactobacillus (P <0.05). Increased succinate levels prevented mice from gaining weight, damaged their intestines, and increased their mortality; upregulated the gene expression of interleukin-1β (IL-1β), IL-6, IL-18 and tumor necrosis factor (TNF); and downregulated the gene expression of IL-10 and transforming growth factor (TGF)-β. Exogenous succinic acid increased inducible nitric oxide synthase (iNOS) gene expression but decreased Arginase-1 (Arg1) gene expression; and increased the protein expression of SUCNR1 and HIF-1a. Succinate plays an important role in the development of necrotizing enterocolitis severity, and the activation of the HIF-1a signaling pathway may lead to disease progression.

Gut microbiota-derived succinate aggravates acute lung injury after intestinal ischaemia/reperfusion in mice.

Acute lung injury (ALI) is a major cause of morbidity and mortality after intestinal ischaemia/reperfusion (I/R). The gut microbiota and its metabolic byproducts act as important modulators of the gut-lung axis. This study aimed to define the role of succinate, a key microbiota metabolite, in intestinal I/R-induced ALI progression. Gut and lung microbiota of mice subjected to intestinal I/R were analysed using 16S rRNA gene sequencing. Succinate level alterations were measured in germ-free mice or conventional mice treated with antibiotics. Succinate-induced alveolar macrophage polarisation and its effects on alveolar epithelial apoptosis were evaluated in succinate receptor 1 (Sucnr1)-deficient mice and in murine alveolar macrophages transfected with Sucnr1-short interfering RNA. Succinate levels were measured in patients undergoing cardiopulmonary bypass, including intestinal I/R. Succinate accumulated in lungs after intestinal I/R, and this was associated with an imbalance of succinate-producing and succinate-consuming bacteria in the gut, but not the lungs. Succinate accumulation was absent in germ-free mice and was reversed by gut microbiota depletion with antibiotics, indicating that the gut microbiota is a source of lung succinate. Moreover, succinate promoted alveolar macrophage polarisation, alveolar epithelial apoptosis and lung injury during intestinal I/R. Conversely, knockdown of Sucnr1 or blockage of SUCNR1 in vitro and in vivo reversed the effects of succinate by modulating the phosphoinositide 3-kinase-AKT/hypoxia-inducible factor-1α pathway. Plasma succinate levels significantly correlated with intestinal I/R-related lung injury after cardiopulmonary bypass. Gut microbiota-derived succinate exacerbates intestinal I/R-induced ALI through SUCNR1-dependent alveolar macrophage polarisation, identifying succinate as a novel target for gut-derived ALI in critically ill patients.

Targeting the succinate receptor effectively inhibits periodontitis.

SUMMARYPeriodontal disease (PD) is one of the most common inflammatory diseases in humans and is initiated by an oral microbial dysbiosis that stimulates inflammation and bone loss. Here, we report an abnormal elevation of succinate in the subgingival plaque of subjects with severe PD. Succinate activates succinate receptor-1 (SUCNR1) and stimulates inflammation. We detected SUCNR1 expression in the human and mouse periodontium and hypothesize that succinate activates SUCNR1 to accelerate periodontitis through the inflammatory response. Administration of exogenous succinate enhanced periodontal disease, whereas SUCNR1 knockout mice were protected from inflammation, oral dysbiosis, and subsequent periodontal bone loss in two different models of periodontitis. Therapeutic studies demonstrated that a SUCNR1 antagonist inhibited inflammatory events and osteoclastogenesis in vitro and reduced periodontal bone loss in vivo. Our study reveals succinate’s effect on periodontitis pathogenesis and provides a topical treatment for this disease.

Orally administered Odoribacter laneus improves glucose control and inflammatory profile in obese mice by depleting circulating succinate

BackgroundSuccinate is produced by both human cells and by gut bacteria and couples metabolism to inflammation as an extracellular signaling transducer. Circulating succinate is elevated in patients with obesity and type 2 diabetes and is linked to numerous complications, yet no studies have specifically addressed the contribution of gut microbiota to systemic succinate or explored the consequences of reducing intestinal succinate levels in this setting.ResultsUsing germ-free and microbiota-depleted mouse models, we show that the gut microbiota is a significant source of circulating succinate, which is elevated in obesity. We also show in vivo that therapeutic treatments with selected bacteria diminish the levels of circulating succinate in obese mice. Specifically, we demonstrate that Odoribacter laneus is a promising probiotic based on its ability to deplete succinate and improve glucose tolerance and the inflammatory profile in two independent models of obesity (db/db mice and diet-induced obese mice). Mechanistically, this is partly mediated by the succinate receptor 1. Supporting these preclinical findings, we demonstrate an inverse correlation between plasma and fecal levels of succinate in a cohort of patients with severe obesity. We also show that plasma succinate, which is associated with several components of metabolic syndrome including waist circumference, triglycerides, and uric acid, among others, is a primary determinant of insulin sensitivity evaluated by the euglycemic-hyperinsulinemic clamp.ConclusionsOverall, our work uncovers O. laneus as a promising next-generation probiotic to deplete succinate and improve glucose tolerance and obesity-related inflammation.3xBZTCGcPCuPE54e-xtBbBVideo

The Succinate Receptor SUCNR1 Resides at the Endoplasmic Reticulum and Relocates to the Plasma Membrane in Hypoxic Conditions.

The GPCR SUCNR1/GPR91 exerts proangiogenesis upon stimulation with the Krebs cycle metabolite succinate. GPCR signaling depends on the surrounding environment and intracellular localization through location bias. Here, we show by microscopy and by cell fractionation that in neurons, SUCNR1 resides at the endoplasmic reticulum (ER), while being fully functional, as shown by calcium release and the induction of the expression of the proangiogenic gene for VEGFA. ER localization was found to depend upon N-glycosylation, particularly at position N8; the nonglycosylated mutant receptor localizes at the plasma membrane shuttled by RAB11. This SUCNR1 glycosylation is physiologically regulated, so that during hypoxic conditions, SUCNR1 is deglycosylated and relocates to the plasma membrane. Downstream signal transduction of SUCNR1 was found to activate the prostaglandin synthesis pathway through direct interaction with COX-2 at the ER; pharmacologic antagonism of the PGE2 EP4 receptor (localized at the nucleus) was found to prevent VEGFA expression. Concordantly, restoring the expression of SUCNR1 in the retina of SUCNR1-null mice renormalized vascularization; this effect is markedly diminished after transfection of the plasma membrane-localized SUCNR1 N8A mutant, emphasizing that ER localization of the succinate receptor is necessary for proper vascularization. These findings uncover an unprecedented physiologic process where GPCR resides at the ER for signaling function.

Tumor cells dictate anti-tumor immune responses by altering pyruvate utilization and succinate signaling in CD8+ T cells

The tumor microenvironment (TME) is a unique metabolic niche that can inhibit Tcell metabolism and cytotoxicity. To dissect the metabolic interplay between tumors and Tcells, we establish an invitro system that recapitulates the metabolic niche of the TME and allows us to define cell-specific metabolism. We identify tumor-derived lactate as an inhibitor of CD8+ Tcell cytotoxicity, revealing an unexpected metabolic shunt in the TCA cycle. Metabolically fit cytotoxic Tcells shunt succinate out of the TCA cycle to promote autocrine signaling via the succinate receptor (SUCNR1). Cytotoxic Tcells are reliant on pyruvate carboxylase (PC) to replenish TCA cycle intermediates. By contrast, lactate reduces PC-mediated anaplerosis. The inhibition of pyruvate dehydrogenase (PDH) is sufficient to restore PC activity, succinate secretion, and the activation of SUCNR1. These studies identify PDH as a potential drug target to allow CD8+ Tcells to retain cytotoxicity and overcome a lactate-enriched TME.

S251: DISRUPTION OF SUCCINATE RECEPTOR SIGNALING PROMOTES MYELOPROLIFERATION AND ACUTE MYELOID LEUKAEMIA

Background: Unlike healthy haematopoietic stem cells (HSC), leukaemia stem cells (LSC) have high oxidative phosphorylation and hence targeting the tricarboxylic acid (TCA) cycle seems a promising therapeutic strategy in acute myeloid leukemia (AML) treatment. Accumulation of the TCA cycle oncometabolite succinate is associated with tumourigenesis, but its potential involvement in HSC function and leukaemia is unkown. Succinate stabilizes the oxygen-sensing transcription factor hypoxia-inducible factor (HIF)-1-α and activates interleukin (IL)-1β production in macrophages. Succinate may also be exported and has cell extrinsic effects through its receptor, succinate receptor-1 (SUCNR1), which may be pro- or anti-inflammatory. Aims: To investigate the in vivo role and molecular mechanisms of succinate and its receptor in normal and malignant haematopoiesis. Methods: To study the effect of succinate receptor on haematopoiesis, we characterized the immunophenotype of Sucnr1-KO mice. To dissect the role of Sucnr1 on healthy haematopoiesis via cell-autonomous effects, we transplanted fl/Sucnr1/fl control or Mx1-Cre fl/Sucnr1/fl bone marrow (BM) cells into wild-type (WT) C57BL/6J mice, which were later injected with pIpC. To study the role of Sucnr1 through non-cell autonomous effects, we transplanted WT BM into Nes-CreERT2fl/Sucnr1/fl recipients or fl/Sucnr1/fl controls, which were induced with tamoxifen. We characterized the effects of Sucnr1 deletion at the molecular level by bulk and single-cell RNA-sequencing (scRNA-seq), and aimed to uncover the molecular mechanisms mediating the effects of Sucnr1 ablation using in vivo tageting with drugs. To study the effect of succinate on leukaemic progression, we used two different mouse models, i.e. Mx1-Cre NrasG12D and MLL-AF9 knock-in (KI). In human AML, we quantified SUCNR1 expression in peripheral blood mononuclear cells (PBMC) from AML patients versus healthy volunteers, and treated xenograft recipients of CD34+ AML cells with succinate or vehicle. Results:In vivo deletion of Sucnr1 induced myeloid bias and expansion of the haematopoietic stem and progenitor cell (HSPC) compartment. In vivo deletion of Sucnr1 from Nestin+ stromal cells promoted expansion of long-term HSC (LT-HSC) versus intact recipients. Conversely, in vivo deletion of Sucnr1 from the haematopoietic system induced expansion of multipotent progenitors (MPP) versus intact haematopoietic cells. RNA-seq in HSPC from Sucnr1-KO versus WT mice showed changes in genes associated with myeloid output, and pinpointed related pathways potentially involved. scRNA-seq confirmed expansion of LT-HSC in Sucnr1-KO versus WT mice. In vivo treatments targeted signalling pathways at least partially responsible for the haematopoietic abnormalities in Sucnr1-KO. We found higher content of succinate in the BM extracellular fluid of leukaemic Mx1-Cre NrasG12D. Succinate injection to xenograft recipients of CD34+ AML cells increased leukaemic output versus vehicle, promoted myeloid bias in Mx1-Cre NrasG12D mice and reduced survival in mice transplanted with BM of MLL-AF9 leukaemic donors. PBMC from AML patients expressed lower level of SUCNR1 versus healthy donors and BM nucleated cells from Mx1-Cre NrasG12D also expressed less Sucnr1 than their healthy counterparts. Summary/Conclusion: We show a novel role for succinate and its receptor in the haematopoietic system and pinpoint this pathway as a novel means of communication within the BM HSC microenvironment, whose disruption may be involved in myeloid malignancy. These findings may help design new therapeutic strategies for AML patients.

The Gut Microbiota Metabolite Succinate Promotes Adipose Tissue Browning in Crohn's Disease.

Crohn's disease [CD] is associated with complex microbe-host interactions, involving changes in microbial communities, and gut barrier defects, leading to the translocation of microorganisms to surrounding adipose tissue [AT]. We evaluated the presence of beige AT depots in CD and questioned whether succinate and/or bacterial translocation promotes white-to-beige transition in adipocytes. Visceral [VAT] and subcutaneous [SAT] AT biopsies, serum and plasma were obtained from patients with active [n = 21] or inactive [n = 12] CD, and from healthy controls [n = 15]. Adipose-derived stem cells [ASCs] and AT macrophages [ATMs] were isolated from VAT biopsies. Plasma succinate levels were significantly higher in patients with active CD than in controls and were intermediate in those with inactive disease. Plasma succinate correlated with the inflammatory marker high-sensitivity C-reactive protein. Expression of the succinate receptor SUCNR1 was higher in VAT, ASCs and ATMs from the active CD group than from the inactive or control groups. Succinate treatment of ASCs elevated the expression of several beige AT markers from controls and from patients with inactive disease, including uncoupling protein-1 [UCP1]. Notably, beige AT markers were prominent in ASCs from patients with active CD. Secretome profiling revealed that ASCs from patients with active disease secrete beige AT-related proteins, and co-culture assays showed that bacteria also trigger the white-to-beige switch of ASCs from patients with CD. Finally, AT depots from patients with CD exhibited a conversion from white to beige AT together with high UCP1 expression, which was corroborated by in situ thermal imaging analysis. Succinate and bacteria trigger white-to-beige AT transition in CD. Understanding the role of beige AT in CD might aid in the development of therapeutic or diagnostic interventions.

Succinic acid exacerbates experimental autoimmune uveitis by stimulating neutrophil extracellular traps formation via SUCNR1 receptor

AimsTo investigate the effect of succinic acid on the development of experimental autoimmune uveitis (EAU) and the underlying mechanism.MethodsSuccinic acid was administrated intraperitoneally to evaluate its effects on immune response...

SUCNR1 Mediates the Priming Step of the Inflammasome in Intestinal Epithelial Cells: Relevance in Ulcerative Colitis.

Intestinal epithelial cells (IECs) constitute a defensive physical barrier in mucosal tissues and their disruption is involved in the etiopathogenesis of several inflammatory pathologies, such as Ulcerative Colitis (UC). Recently, the succinate receptor SUCNR1 was associated with the activation of inflammatory pathways in several cell types, but little is known about its role in IECs. We aimed to analyze the role of SUCNR1 in the inflammasome priming and its relevance in UC. Inflammatory and inflammasome markers and SUCNR1 were analyzed in HT29 cells treated with succinate and/or an inflammatory cocktail and transfected with SUCNR1 siRNA in a murine DSS model, and in intestinal resections from 15 UC and non-IBD patients. Results showed that this receptor mediated the inflammasome, priming both in vitro in HT29 cells and in vivo in a murine chronic DSS-colitis model. Moreover, SUNCR1 was also found to be involved in the activation of the inflammatory pathways NFкB and ERK pathways, even in basal conditions, since the transient knock-down of this receptor significantly reduced the constitutive levels of pERK-1/2 and pNFкB and impaired LPS-induced inflammation. Finally, UC patients showed a significant increase in the expression of SUCNR1 and several inflammasome components which correlated positively and significantly. Therefore, our results demonstrated a role for SUCNR1 in basal and stimulated inflammatory pathways in intestinal epithelial cells and suggested a pivotal role for this receptor in inflammasome activation in UC.

Succinate/IL-1β Signaling Axis Promotes the Inflammatory Progression of Endothelial and Exacerbates Atherosclerosis.

Inflammation is an important driver of atherosclerosis. Succinate is a new extracellular inflammatory alarm released by activated macrophages. Succinate is sensed by succinate receptor 1 (Sucnr1) and then transferred to effector cells. It is worth exploring whether succinate is capable of facilitating the inflammatory response in atherosclerosis. In this study, we firstly found that arterial serum of Coronary Heart Disease (CHD) patients contained significantly higher succinate and interleukin (IL)-1β than Health control (HC) subjects, and succinate was positively correlated with IL-1β. As demonstrated by the in vitro study, succinate/hypoxia-inducible factor 1α (Hif)-1α/IL-1β signal axis existed and significantly facilitated the inflammatory program in human umbilical vein endothelial cells (HUVECs). Under the coculture, activated macrophages released succinate, which would be transferred to HUVECs via Sucnr1 and then activate Hif-1α to produce a greater amount of IL-1β. Likewise, the aortic sinus’s inflammatory phenotype was found to be more significant within Apoe-/- mice that were injected with succinate. Furthermore, Sucnr1 inhibitor (NF-56-EJ40) could significantly interrupt succinate/IL-1β signal in HUVECs and macrophages. As revealed by this study, glycolytic metabolism following the release of succinate could be found in atherosclerotic pathology, and succinate would drive succinate/IL-1β signal dependent on Sucnr1 and then exacerbate inflammatory responses. Sucnr1 might be a novel target for cutting off the transduction of succinate signal to prevent the inflammation of atherosclerosis.

A183 SUCCINATE IN THE RELATIONSHIP BETWEEN INFLAMMATORY BOWEL DISEASE AND OBESITY IN SUCCINATE RECEPTOR DEFICIENT MICE

BackgroundThe incidence of Inflammatory Bowel Disease (IBD) among the obese pediatric populations is increasing. Succinate has been identified as a possible metabolite linking the two diseases. Succinate receptor 1 (SUCNR1) gene knockout (KO) mice are less susceptible to dextran sulfate sodium (DSS)-induced colitis.AimsTo determine whether succinate plays a role in mice becoming obese and colitis in obesity, and whether an obese-inducing diet would change the gut microbiota. We hypothesized that SUCNR1-KO mice would not become obese and would experience less colonic inflammation despite the diet.MethodsC57BL/6 (WT) were bred with SUCNR1-KO mice (generously provided by Amgen), and the heterozygous (HZ) F1 offspring bred to obtain F2. Two of 3 F2 litters included SUCNR1-KO mice, which were caged by sex but not genotype. These F2 mice began a high-fat/high-sugar (obese, Dytes) diet at 5 wks of age for 5 wks. Weights were recorded and stool collected. All mice then had 3% DSS replace their water, for 5 days. Mice were observed for diarrhea and occult blood. After the DSS, facility water was returned for 1 day prior to postmortem analyses. Mice were scanned using dual-energy X-ray absorptiometry (DEXA) for measures of fat, lean and fat mass, and bone density. Their colons were resected and fixed for histopathology. Stool was banked frozen until processed for sequencing using 16S Ribosomal primers.ResultsThree F2 litters were comprised of ratios of HZ:WT:SUCNR1-KO of 14:7:2. SUCNR1-KO mice (n=2 female) had a greater increase in weight compared to other genotypes during the obesity-induction phase. Weight loss during the DSS phase was similar across all genotypes. All mice had blood in their stool. DEXA measures did not differ between genotypes. All genotypes of mice had inflamed colons.ConclusionsSUCNR1-KO mice are not resistant to obesity, nor from colitis when consuming the obese diet, outcomes that do not support our hypothesis. It remains to be determined whether the diet alters the microbiome resulting in SUCNR1-KO mice being suscueptible to colitis. Microbial sequencing is underway.Funding AgenciesIWK Health Project Grant

Activation of succinate receptor 1 boosts human mast cell reactivity and allergic bronchoconstriction.

BackgroundSUCNR1 is a sensor of extracellular succinate, a Krebs cycle intermediate generated in excess during oxidative stress and has been linked to metabolic regulation and inflammation. While mast cells express SUCNR1, its role in mast cell reactivity and allergic conditions such as asthma remains to be elucidated.MethodsCord blood‐derived mast cells and human mast cell line LAD‐2 challenged by SUCNR1 ligands were analyzed for the activation and mediator release. Effects on mast cell‐dependent bronchoconstriction were assessed in guinea pig trachea and isolated human small bronchi challenged with antigen and anti‐IgE, respectively.ResultsSUCNR1 is abundantly expressed on human mast cells. Challenge with succinate, or the synthetic non‐metabolite agonist cis‐epoxysuccinate, renders mast cells hypersensitive to IgE‐dependent activation, resulting in augmented degranulation and histamine release, de novo biosynthesis of eicosanoids and cytokine secretion. The succinate‐potentiated mast cell reactivity was attenuated by SUCNR1 knockdown and selective SUCNR1 antagonists and could be tuned by pharmacologically targeting protein kinase C and extracellular signal‐regulated kinase. Both succinate and cis‐epoxysuccinate dose‐dependently potentiated antigen‐induced contraction in a mast cell‐dependent guinea pig airway model, associated with increased generation of cysteinyl‐leukotrienes and histamine in trachea. Similarly, cis‐epoxysuccinate aggravated IgE‐receptor‐induced contraction of human bronchi, which was blocked by SUCNR1 antagonism.ConclusionSUCNR1 amplifies IgE‐receptor‐induced mast cell activation and allergic bronchoconstriction, suggesting a role for this pathway in aggravation of allergic asthma, thus linking metabolic perturbations to mast cell‐dependent inflammation.

Succinate receptor 1 inhibits mitochondrial respiration in cancer cells addicted to glutamine.

Cancer cells display metabolic alterations to meet the bioenergetic demands for their high proliferation rates. Succinate is a central metabolite of the tricarboxylic acid (TCA) cycle, but was also shown to act as an oncometabolite and to specifically activate the succinate receptor 1 (SUCNR1), which is expressed in several types of cancer. However, functional studies focusing on the connection between SUCNR1 and cancer cell metabolism are still lacking. In the present study, we analyzed the role of SUCNR1 for cancer cell metabolism and survival applying different signal transduction, metabolic and imaging analyses. We chose a gastric, a lung and a pancreatic cancer cell line for which our data revealed functional expression of SUCNR1. Further, presence of glutamine (Gln) caused high respiratory rates and elevated expression of SUCNR1. Knockdown of SUCNR1 resulted in a significant increase of mitochondrial respiration and superoxide production accompanied by an increase in TCA cycle throughput and a reduction of cancer cell survival in the analyzed cancer cell lines. Combination of SUCNR1 knockdown and treatment with the chemotherapeutics cisplatin and gemcitabine further increased cancer cell death. In summary, our data implicates that SUCNR1 is crucial for Gln-addicted cancer cells by limiting TCA cycle throughput, mitochondrial respiration and the production of reactive oxygen species, highlighting its potential as a pharmacological target for cancer treatment.

Mechanism of ligand binding pathways and activation of the succinate receptor

Molecular dynamic (MD) simulations of high-resolution X-ray structures of GPR91 in the presence of free succinate molecules demonstrate that the acidic metabolite initially binds between TM6 and TM7 in the extracellular vestibule (ECV) before moving down through an intermediate stage to its final binding site between transmembrane helices. The low energy binding in the initial and the final binding stage with a higher energy barrier in the intermediate stage were investigated by well-tempered metadynamic.

Acute Succinate Administration Increases Oxidative Phosphorylation and Skeletal Muscle Explosive Strength via SUCNR1.

Endurance training and explosive strength training, with different contraction protein and energy metabolism adaptation in skeletal muscle, are both beneficial for physical function and quality of life. Our previous study found that chronic succinate feeding enhanced the endurance exercise of mice by inducing skeletal muscle fiber-type transformation. The purpose of this study is to investigate the effect of acute succinate administration on skeletal muscle explosive strength and its potential mechanism. Succinate was injected to mature mice to explore the acute effect of succinate on skeletal muscle explosive strength. And C2C12 cells were used to verify the short-term effect of succinate on oxidative phosphorylation. Then the cells interfered with succinate receptor 1 (SUCNR1) siRNA, and the SUCNR1-GKO mouse model was used for verifying the role of SUCNR1 in succinate-induced muscle metabolism and expression and explosive strength. The results showed that acute injection of succinate remarkably improved the explosive strength in mice and also decreased the ratio of nicotinamide adenine dinucleotide (NADH) to NAD+ and increased the mitochondrial complex enzyme activity and creatine kinase (CK) activity in skeletal muscle tissue. Similarly, treatment of C2C12 cells with succinate revealed that succinate significantly enhanced oxidative phosphorylation with increased adenosine triphosphate (ATP) content, CK, and the activities of mitochondrial complex I and complex II, but with decreased lactate content, reactive oxygen species (ROS) content, and NADH/NAD+ ratio. Moreover, the succinate's effects on oxidative phosphorylation were blocked in SUCNR1-KD cells and SUCNR1-KO mice. In addition, succinate-induced explosive strength was also abolished by SUCNR1 knockout. All the results indicate that acute succinate administration increases oxidative phosphorylation and skeletal muscle explosive strength in a SUCNR1-dependent manner.

Targeting Succinate Receptor Reduces Microglial Activation and Neuroinflammation

Targeting Succinate Receptor Reduces Microglial Activation and Neuroinflammation

Diethyl Succinate Modulates Microglial Polarization and Activation by Reducing Mitochondrial Fission and Cellular ROS.

Succinate is a metabolite in the tricarboxylic acid cycle (TCA) which plays a central role in mitochondrial activity. Excess succinate is known to be transported out of the cytosol, where it activates a succinate receptor (SUCNR1) to enhance inflammation through macrophages in various contexts. In addition, the intracellular role of succinate beyond an intermediate metabolite and prior to its extracellular release is also important to the polarization of macrophages. However, the role of succinate in microglial cells has not been characterized. Lipopolysaccharide (LPS) stimulates the elevation of intracellular succinate levels. To reveal the function of intracellular succinate associated with LPS-stimulated inflammatory response in microglial cells, we assessed the levels of ROS, cytokine production and mitochondrial fission in the primary microglia pretreated with cell-permeable diethyl succinate mimicking increased intracellular succinate. Our results suggest that elevated intracellular succinate exerts a protective role in the primary microglia by preventing their conversion into the pro-inflammatory M1 phenotype induced by LPS. This protective effect is SUCNR1-independent and mediated by reduced mitochondrial fission and cellular ROS production.

Paeoniflorin alleviates CFA-induced inflammatory pain by inhibiting TRPV1 and succinate/SUCNR1-HIF-1α/NLPR3 pathway

BackgroundTreatment of chronic inflammatory pain remains a major goal in the clinic. It is thus of prime importance to characterize inherent pathophysiological pathways to design new therapeutic strategies and analgesics for pain management. Paeoniflorin (PF), a monoterpenoid glycoside from Paeonia lactiflora Pallas plants, possesses promising anti-nociceptive property. However, therapeutic effect and underlying mechanism of action of PF on inflammatory pain have not yet been fully elucidated. In this study, we aim to investigate the analgesic effect further and clarify its mechanism of action of PF on complete freund’s adjuvant (CFA)-evoked inflammatory pain. MethodsTwenty-four male mice were divided into 3 groups: sham, CFA, and CFA + PF groups (n = 8/group). Mice were treated with normal saline or PF (30 mg/kg) for 11 days. Footpad swelling (n = 8/group), mechanical (n = 8/group) and thermal hypersensitivity (n = 8/group) were measured to evaluate the analgesic effect of PF on CFA-injected mice. At the end of the animal experiment, blood and L4-L6 dorsal root ganglion neurons were collected to assess the therapeutic effect of PF on CFA-induced inflammatory pain. Next, hematoxylin and eosin, quantitative realtime PCR, ELISA, capsaicin and dimethyl succinate induced pain test (n = 8/group), motor coordination test (n = 8/group), tail flicking test (n = 8/group), pyruvate and succinate dehydrogenase assay (n = 6/group), immunohistochemical staining, were performed to clarify the action mechanism of PF on CFA-evoked inflammatory pain. Besides, the effect of PF on TRPV1 was evaluated by whole-cell patch clamp recording on primary neurons (n = 7). Finally, molecular docking further performed to evaluate the binding ability of PF to TRPV1. ResultsPF significantly relieved inflammatory pain (P < 0.001) and paw edema (P < 0.001) on a complete Freund adjuvant (CFA)-induced peripheral inflammatory pain model. Furthermore, PF inhibited neutrophil infiltration (P < 0.01), IL-1β increase (P < 0.01), and pain-related peptide substance P release (P < 0.001). Intriguingly, CFA-induced succinate aggregation was notably reversed by PF via modulating pyruvate and SDH activity (P < 0.01). In addition, PF dampened the high expression of subsequent succinate receptor SUCNR1 (P < 0.01), HIF-1α (P < 0.05), as well as the activation of NLPR3 inflammasome (P < 0.05) and TRPV1 (P < 0.05). More importantly, both capsaicin and dimethyl succinate supplementation obviously counteracted the pain-relieving effect of PF and TRPV1 (P < 0.01 or P < 0.001). ConclusionOur findings suggest that PF can significantly relieve CFA-induced paw swelling, as well as mechanical and thermal hyperalgesia. PF alleviated inflammatory pain partly through inhibiting the activation of TRPV1 and succinate/SUCNR1-HIF-1α/NLPR3 pathway. Furthermore, we found that PF exerted its analgesic effect without affecting motor coordination and pain-related cold ion-channels. In summary, this study may provide valuable evidence for the potential application of PF as therapeutic strategy for inflammatory pain treatment.

Tuft Cells Increase Following Ovine Intestinal Parasite Infections and Define Evolutionarily Conserved and Divergent Responses.

Helminth parasite infections of humans and livestock are a global health and economic problem. Resistance of helminths to current drug treatment is an increasing problem and alternative control approaches, including vaccines, are needed. Effective vaccine design requires knowledge of host immune mechanisms and how these are stimulated. Mouse models of helminth infection indicate that tuft cells, an unusual type of epithelial cell, may ‘sense’ infection in the small intestine and trigger a type 2 immune response. Currently nothing is known of tuft cells in immunity in other host species and in other compartments of the gastrointestinal (GI) tract. Here we address this gap and use immunohistochemistry and single cell RNA-sequencing to detail the presence and gene expression profile of tuft cells in sheep following nematode infections. We identify and characterize tuft cells in the ovine abomasum (true stomach of ruminants) and show that they increase significantly in number following infection with the globally important nematodes Teladorsagia circumcincta and Haemonchus contortus. Ovine abomasal tuft cells show enriched expression of tuft cell markers POU2F3, GFI1B, TRPM5 and genes involved in signaling and inflammatory pathways. However succinate receptor SUCNR1 and free fatty acid receptor FFAR3, proposed as ‘sensing’ receptors in murine tuft cells, are not expressed, and instead ovine tuft cells are enriched for taste receptor TAS2R16 and mechanosensory receptor ADGRG6. We also identify tuft cell sub-clusters at potentially different stages of maturation, suggesting a dynamic process not apparent from mouse models of infection. Our findings reveal a tuft cell response to economically important parasite infections and show that while tuft cell effector functions have been retained during mammalian evolution, receptor specificity has diverged. Our data advance knowledge of host-parasite interactions in the GI mucosa and identify receptors that may potentiate type 2 immunity for optimized control of parasitic nematodes.