SUCNR1 regulates insulin secretion and glucose elevates the succinate response in people with prediabetes.

Pancreatic ß-cell dysfunction is a key feature of type 2 diabetes, and novel regulators of insulin secretion are desirable. Here we report that the succinate receptor (SUCNR1) is expressed in ß-cells and is up-regulated in hyperglycemic states in mice and humans. We found that succinate acts as a hormone-like metabolite and stimulates insulin secretion via a SUCNR1-Gq-PKC-dependent mechanism in human ß-cells. Mice with ß-cell-specific Sucnr1 deficiency exhibit impaired glucose tolerance and insulin secretion on a high-fat diet, indicating that SUCNR1 is essential for preserving insulin secretion in diet-induced insulin resistance. Patients with impaired glucose tolerance show an enhanced nutritional-related succinate response, which correlates with the potentiation of insulin secretion during intravenous glucose administration. These data demonstrate that the succinate/SUCNR1 axis is activated by high glucose and identify a GPCR-mediated amplifying pathway for insulin secretion relevant to the hyperinsulinemia of prediabetic states.

A specific gut microbiota signature is associated with an enhanced GLP-1 and GLP-2 secretion and improved metabolic control in patients with type 2 diabetes after metabolic Roux-en-Y gastric bypass.

Objective: To determine changes in incretins, systemic inflammation, intestinal permeability and microbiome modifications 12 months after metabolic RYGB (mRYGB) in patients with type 2 diabetes (T2D) and their relationship with metabolic improvement. Materials and methods: Prospective single-center non-randomized controlled study, including patients with class II-III obesity and T2D undergoing mRYGB. At baseline and one year after surgery we performed body composition measurements, biochemical analysis, a meal tolerance test (MTT) and lipid test (LT) with determination of the area under the curve (AUC) for insulin, C-peptide, GLP-1, GLP-2, and fasting determinations of succinate, zonulin, IL-6 and study of gut microbiota. Results: Thirteen patients aged 52.6 ± 6.5 years, BMI 39.3 ± 1.4 kg/m2, HbA1c 7.62 ± 1.5% were evaluated. After mRYGB, zonulin decreased and an increase in AUC after MTT was observed for GLP-1 (pre 9371 ± 5973 vs post 15788 ± 8021 pM, P<0.05), GLP-2 (pre 732 ± 182 vs post 1190 ± 447 ng/ml, P<0.001) and C- peptide, as well as after LT. Species belonging to Streptococaceae, Akkermansiacea, Rickenellaceae, Sutterellaceae, Enterobacteriaceae, Oscillospiraceae, Veillonellaceae, Enterobacterales_uc, and Fusobacteriaceae families increased after intervention and correlated positively with AUC of GLP-1 and GLP-2, and negatively with glucose, HbA1c, triglycerides and adiposity markers. Clostridium perfringens and Roseburia sp. 40_7 behaved similarly. In contrast, some species belonging to Lachnospiraceae, Erysipelotricaceae, and Rumnicocaceae families decreased and showed opposite correlations. Higher initial C-peptide was the only predictor for T2D remission, which was achieved in 69% of patients. Conclusions: Patients with obesity and T2D submitted to mRYGB show an enhanced incretin response, a reduced gut permeability and a metabolic improvement, associated with a specific microbiota signature.

Increased STAT3 Activation in Periodontitis Drives Inflammatory Bone Loss.

Periodontitis is one of the most prevalent human inflammatory diseases. It is characterized by periodontal tissue destruction, progressively driven by the host response. In this regard, cytokines associated with tissue destruction, such as interleukin (IL)-6 and IL-23, use a common signaling pathway mediated by STAT3. This transcription factor is also needed for IL-17A production, a key mediator in periodontitis pathogenesis. Although several studies have reported increased activation of STAT3 in experimental periodontitis, a detailed characterization of STAT3 activation in human gingival tissues and its involvement in alveolar bone loss has yet to be explored. Using a cross-sectional study design, we detected increased proportions of pSTAT3-positive cells during periodontitis compared with health, particularly in epithelial cells and T cells. Other cell types of hematopoietic and nonhematopoietic origin also display STAT3 activation in gingival tissues. We detected increased STAT3 phosphorylation and expression of STAT3-related genes during experimental periodontitis. Next, we evaluated the role of STAT3 in alveolar bone destruction using a mouse model of STAT3 loss of function (mut-Stat3 mice). Compared with controls, mut-Stat3 mice had reduced alveolar bone loss following ligature-induced periodontitis. We also evaluated pharmacologic inhibition of STAT3 in ligature-induced periodontitis. Like mut-Stat3 mice, mice treated with STAT3 small-molecule inhibitor had reduced bone loss compared with controls. Our results demonstrate that STAT3 activation is increased in epithelial and T cells during periodontitis and indicate a pathogenic role of STAT3 in inflammatory alveolar bone loss.

Protective effects of the succinate/SUCNR1 axis on damaged hepatocytes in NAFLD.

OBJECTIVE: Succinate and succinate receptor 1 (SUCNR1) are linked to fibrotic remodeling in models of non-alcoholic fatty liver disease (NAFLD), but whether they have roles beyond the activation of hepatic stellate cells remains unexplored. We investigated the succinate/SUCNR1 axis in the context of NAFLD specifically in hepatocytes. METHODS: We studied the phenotype of wild-type and Sucnr1-/- mice fed a choline-deficient high-fat diet to induce non-alcoholic steatohepatitis (NASH), and explored the function of SUCNR1 in murine primary hepatocytes and human HepG2 cells treated with palmitic acid. Lastly, plasma succinate and hepatic SUCNR1 expression were analyzed in four independent cohorts of patients in different NAFLD stages. RESULTS: Sucnr1 was upregulated in murine liver and primary hepatocytes in response to diet-induced NASH. Sucnr1 deficiency provoked both beneficial (reduced fibrosis and endoplasmic reticulum stress) and detrimental (exacerbated steatosis and inflammation and reduced glycogen content) effects in the liver, and disrupted glucose homeostasis. Studies in vitro revealed that hepatocyte injury increased Sucnr1 expression, which when activated improved lipid and glycogen homeostasis in damaged hepatocytes. In humans, SUCNR1 expression was a good determinant of NAFLD progression to advanced stages. In a population at risk of NAFLD, circulating succinate was elevated in patients with a fatty liver index (FLI) ≥60. Indeed, succinate had good predictive value for steatosis diagnosed by FLI, and improved the prediction of moderate/severe steatosis through biopsy when added to an FLI algorithm. CONCLUSIONS: We identify hepatocytes as target cells of extracellular succinate during NAFLD progression and uncover a hitherto unknown function for SUCNR1 as a regulator of hepatocyte glucose and lipid metabolism. Our clinical data highlight the potential of succinate and hepatic SUCNR1 expression as markers to diagnose fatty liver and NASH, respectively.

SUCNR1 signaling in adipocytes controls energy metabolism by modulating circadian clock and leptin expression.

Adipose tissue modulates energy homeostasis by secreting leptin, but little is known about the factors governing leptin production. We show that succinate, long perceived as a mediator of immune response and lipolysis, controls leptin expression via its receptor SUCNR1. Adipocyte-specific deletion of Sucnr1 influences metabolic health according to nutritional status. Adipocyte Sucnr1 deficiency impairs leptin response to feeding, whereas oral succinate mimics nutrient-related leptin dynamics via SUCNR1. SUCNR1 activation controls leptin expression via the circadian clock in an AMPK/JNK-C/EBPα-dependent manner. Although the anti-lipolytic role of SUCNR1 prevails in obesity, its function as a regulator of leptin signaling contributes to the metabolically favorable phenotype in adipocyte-specific Sucnr1 knockout mice under standard dietary conditions. Obesity-associated hyperleptinemia in humans is linked to SUCNR1 overexpression in adipocytes, which emerges as the major predictor of adipose tissue leptin expression. Our study establishes the succinate/SUCNR1 axis as a metabolite-sensing pathway mediating nutrient-related leptin dynamics to control whole-body homeostasis.

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

BACKGROUND: Succinate 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. RESULTS: Using 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. CONCLUSIONS: Overall, our work uncovers O. laneus as a promising next-generation probiotic to deplete succinate and improve glucose tolerance and obesity-related inflammation. Video Abstract.

Toward real applicability of electro-ozonizers: Paying attention to the gas phase using actual commercial PEM electrolyzers technology.

This work focuses on increasing the TRL of electro-ozonizer technology by evaluating the effect of electrolyte composition and operation conditions on the production of ozone, using an actual commercial cell, CONDIAPURE®, in conditions similar to what could be expected in a real application. Not only is attention paid to the changes in the concentration of ozone in the liquid phase, but also to those observed in the gas phase. The electrolyte and its recirculation flowrate, as well as operation temperatures and pressures are found to have significant influence on production rates. The most efficient way to produce ozone is operating at low temperatures and high pressures. In this work, 0.25 and 0.21 mg O3/min were obtained operating at 10 A in electrolytes consisting of aqueous solutions of perchloric and sulfuric acid, respectively, in tests carried out at 13 °C and 2 bars of gauge pressure. The negative effect of scavengers that appear electrochemically along the production of ozone is very important and seems to be partially compensated when organics are present in the solution due to the competition between the reaction of these scavengers with ozone or organics.

Ejaculatory training lengthens the ejaculation latency and facilitates the functioning of the spinal generator for ejaculation of rats with rapid ejaculation.

A spinal pattern generator controls the ejaculatory response. Central pattern generators (CPGs) may be entrained to improve the motor patterns under their control. In the present study we tested the hypothesis that training of the spinal generator for ejaculation (SGE) by daily copulation until ejaculation, could promote substantive changes in its functioning permitting a better SGE control of the genital motor pattern of ejaculation (GMPE) and, as a consequence, a normalization of the ejaculation latency of rats with rapid ejaculation. To that aim, we evaluated in sexually experienced male rats with rapid ejaculation (1) the effects of daily copulation to ejaculation, following different entrainment schedules, on their ejaculation latencies, (2) the impact of these different ejaculatory entrainment schedules upon the parameters of the GMPE and (3) the possible emergence of persistent changes in the functioning of the SGE associated to the daily ejaculation entrainment schedules. The data obtained show that intense ejaculatory training of rats with rapid ejaculation lengthens the ejaculation latency during copulation and augments the ejaculatory capacity of the SGE in this population when spinalized. Thus, present data reveal that like other CPGs, the SGE can be trained and put forward that training of the SGE by daily copulation to ejaculation might be a promising alternative that should be taken into consideration for the treatment of premature ejaculation.

Rhythmic motor patterns accompanying ejaculation in spinal cord-transected male rats.

A spinal pattern generator controls the ejaculatory response. Activation of this spinal generator elicits rhythmic motor patterns of the striated musculature that surrounds the genital tract that contributes to the expulsion of seminal secretions. In the present study, we elicited ejaculation in spinal cord-transected male rats by mechanically stimulating the urethra and registered rhythmic motor patterns in the cremasteric, iliopsoas and pubococcygeus muscles. The rhythmic motor activity recorded in these muscles was compared with that elicited in the bulbospongiosus muscles; the results revealed similarities in the motor parameters among all the muscles. Data of this study, showing the occurrence of rhythmic motor behaviour in the cremasteric, iliopsoas and pubococcygeus muscles during ejaculation, suggest that these muscles might be under the control of the spinal generator for ejaculation.

Bilateral synchronous granulomatous orchitis in a patient with erectile disfunction: clinical and pathologic study of the case.

A 50-year-old male patient presented with erectile failure and loss of libido. In the physical examination, there were stone-hard indurations in his bilateral testes. The ultrasonographic study demonstrated multiple hypoechoic areas in the testes and normal epididymis. Since the lesion was presumed as malignancy, bilateral inguinal exploration was performed and intraoperative frozen biopsies were studied and diagnosed as inflammatory process. Nevertheless, we decided to perform left orchiectomy to a deeper histopathologic analysis which revealed granulomatous orchitis, mastocytosis, and severe depletion of Leydig cells at the testicular interstitium. Differential diagnosis between testicular tumor and granulomatous orchitis is very difficult in any examination except by histological findings. Bilateral cases of this pathology are relatively rare, but it is necessary to distinguish them from the testicular tumor before surgical intervention to avoid an unnecessary orchiectomy.

Predictors of improved seminal parameters and fertility after varicocele repair in young adults.

The aim of our study was to determine hormonal or biochemical markers in patients with clinically palpable left varicocele but without a history of infertility, with especial emphasis on nitric oxide, related with improved seminal parameters after varicocelectomy. Semen samples were obtained from 202 patients with left varicocele grade II or III. Nitric oxide levels in seminal plasma were determined by the Griess technique. Testicular volume was determined ultrasonographically in both testes and hormonal profile was measured. The post-operative sperm concentration increased significantly in patients with normal sperm count or moderate oligozoospermia, but we did not find an increment in sperm count in patients with mild and severe oligozoospermia after surgery. The mean percentage of normal motility significantly increased after surgery, but we did not observe a significant increment in morphologically normal sperm count and testicular volume after varicocele repair. Moreover, we did not find any correlation between nitric oxide concentrations and severity of oligozoospermia, asthenozoospermia or abnormal sperm morphology in this population. It is concluded that in the general male population, varicocele repair is not associated with an improved semen profile in all cases. We did not observe a significant correlation between nitric oxide concentrations and semen profile.

IL-10 gene therapy prevents TNBS-induced colitis.

The transfer of genes encoding immunomodulatory proteins to the intestinal mucosa is a promising new approach to the treatment of Crohn's disease (CD). This study investigates the therapeutic efficacy of an adenoviral vector encoding IL-10 (AdvmuIL-10) in experimental colitis. BALB/c mice were treated with a single intravenous injection of AdvmuIL-10, empty cassette virus (Adv0) or PBS prior to the induction of trinitrobenzene sulphonic acid (TNBS) colitis. AdvmuIL-10 treatment prevented the severe loss of body weight associated with TNBS administration. In addition, AdvmuIL-10 therapy led to a significant reduction in both stool markers of inflammation (IL-1beta and TNFR-II) and acute phase response (serum amyloid protein). Finally, the histological scores of mice with TNBS colitis treated with AdvmuIL-10 were significantly lower than Adv0- or PBS-treated controls. The therapeutic efficacy of AdvmuIL-10 was associated with a decrease in the IFN-gamma and IL-6 levels detected in colonic homogenates from mice with TNBS colitis, whereas no effect was observed on cytokine release from stimulated systemic lymphocytes. Thus, AdvmuIL-10 is an effective therapy in the TNBS model of colitis. Gene therapy strategies using adenoviral vectors encoding IL-10 may prove to be a potent therapy for chronic inflammatory conditions such as CD.

Modulation of histamine H(2) receptor signalling by G-protein-coupled receptor kinase 2 and 3.

To evaluate the role of G-protein-coupled receptor kinases (GRK) in the desensitization of the histamine H(2) receptor, the H(2) receptor was transiently cotransfected with GRK2, 3, 5 or 6 in COS-7 cells and the cyclic AMP levels in response to histamine were studied. Coexpression of the H(2) receptor with GRK2 and 3 significantly decreased both the basal cyclic AMP levels and the cyclic AMP response to 100 microM histamine. Moreover, preincubation with 100 microM histamine desensitized the H(2) receptor response to 53+/-8%. Coexpression of GRK2 and 3 increased the H(2) receptor desensitization to 27+/-4% and 24+/-4% respectively. No effect on either cyclic AMP response or desensitization was found when GRK5, GRK6 or dominant negative mutants of GRK2 or 3 (GRK2K(220)R and GRK3K(220)R) were coexpressed. To study the role of the C-terminal tail in the GRK-mediated desensitization of the H(2) receptor, three truncations of C-tail were constructed: H(2)T295, H(2)T307 and H(2)T341. H(2)T307 and 341 H(2)T341 expressed and responded normally to 100 microM histamine. The interaction of the H(2) receptor with GRK2 and 3 was also not altered upon truncation of the C-terminal tail. These findings strongly suggest a role of GRK2 and 3 in the desensitization of the H(2) receptor. Furthermore, the finding that C-terminal truncations of the H(2) receptor did not abolish the effect of GRK2 and 3 suggests that the C-terminus is not involved in the GRK mediated desensitization of the histamine H(2) receptor.

Dopamine enhances somatostatin receptor-mediated inhibition of adenylate cyclase in rat striatum and hippocampus.

Although there is evidence that suggests that dopamine (DA) has stimulatory effects on somatostatinergic transmission, it is unknown to date if DA increases the activity of the somatostatin (SS) receptor-effector system in the rat brain. In this study, we evaluated the effects of the administration of DA and the DA D1-like (D1, D5) receptor antagonist SCH 23390 and the D2-like (D2, D3, D4) receptor antagonist spiperone on the SS receptor-adenylate cyclase (AC) system in the Sprague-Dawley rat striatum and hippocampus. An intracerebroventricular injection of DA (0.5 microgram/rat) increased the number of SS receptors and decreased their apparent affinity in the striatum and hippocampus 15 hr after its administration. The simultaneous administration of the DA receptor antagonists SCH 23390 (0.25 mg/kg, ip) and spiperone (0.1 mg/kg, ip) before DA injection partially prevented the DA-induced increase in SS binding. The administration of SCH 23390 plus spiperone alone produced a significant decrease in the number of SS receptors in both brain areas studied at 15 hr after injection, an effect that disappeared at 24 hr. The increased number of SS receptors in the DA-treated rats was associated with an increased capacity of SS to inhibit basal and forskolin (FK)-stimulated (AC) activity in the striatum and hippocampus at 15 hr after injection. This effect had disappeared at 24 hr. By contrast, basal and FK-stimulated enzyme activities were unaltered after DA injection. No significant changes in the levels of the alpha i (alpha i1 + alpha i2) subunits were found in DA-treated rats as compared with control rats. In addition, the immunodetection of the alpha i1 or alpha i2 subunits showed no significant changes in their levels in DA-treated rats when compared with controls. DA injection also induced an increase in SS-like immunoreactive content in the rat striatum but not hippocampus at 15 hr after administration and returned to control values at 24 hr. These results provide direct evidence of a functional linkage between the dopaminergic and somatostatinergic systems at the molecular level.

Decreased insulin content and secretion in RIN 1046-38 cells overexpressing alpha 2-adrenergic receptors.

Several Gi-protein-coupled receptors normally expressed in islet beta-cells inhibit insulin secretion on binding of their respective agonists. To study the effect of supraphysiologic expression of such a receptor in insulin-secreting beta-cells, we stably transfected cDNA encoding the mouse alpha 2a-adrenergic receptor into RIN 1046-38 cells. Four different cell lines were selected, each overexpressing the alpha 2a-adrenergic receptor to varying degrees. Cell lines showing the highest level of receptor expression showed significantly reduced insulin content, and reduced basal and stimulated insulin secretion. Pertussis toxin (PTX) treatment of cells was able to reverse partially the reduced insulin secretory response. Our results suggest that overexpression of a Gi-protein-coupled receptor in beta-cells causes tonic inhibition of both insulin synthesis and secretion. Abnormalities in expression or function of such receptors could be a contributory factor in the impaired insulin secretion present in type II diabetes.

Modulation of forskolin-mediated adenylyl cyclase activation by constitutively active G(S)-coupled receptors.

In transfected CHO cells constitutively active histamine H2 receptors not only increase the basal cAMP level, but also enhance forskolin-induced cAMP production. The increased forskolin response was inhibited by inverse H2 agonists with potencies similar to those determined at basal levels. The modulation of the forskolin response was also observed after H2 receptor expression in HEK-293 and Sf9 cells or TSH receptor expression in COS-7 cells. The enhancement of forskolin-induced cAMP production seems to be a general characteristic of constitutively active G(S)-coupled receptors and can be very useful to study inverse agonism at wild-type receptors.

Functional characterization of five V2 vasopressin receptor gene mutations.

COS7 cells were transiently transfected with plasmids encoding mutant forms of the V2 vasopressin receptors corresponding to mutations [Y280C, L292P, R337stop, V277A, and G12E (the latter found in the same kindred with L292P)] recently identified in subjects with X-linked nephrogenic diabetes insipidus (NDI). cAMP response to dDAVP and AVP, saturation binding experiments with [3H]-AVP, immunofluorescence, and indirect ELISA studies were performed to characterize the functional consequences of these mutations. The Y280C, L292P, and R337stop mutant V2 receptors show substantially decreased cell surface expression and are functionally inactive. The V277A mutant receptor, though well expressed at the cell surface as seen by immunofluorescence and ELISA and having a dissociation constant with AVP similar to the wild type receptor, was functionally less active as seen by a substantially decreased receptor number (Bmax) and reduced cAMP stimulation by dDAVP. The G12E mutant was functionally the same as the wild type V2 receptor in both cAMP stimulation and binding. These results provide insight into residues critical for V2 receptor expression and function and also provide direct evidence that Y280C, L292P, R337stop and V277A mutations are the cause of X-linked NDI in affected subjects.

Insulin-like growth factor-I stimulates neurogenesis in chick retina by regulating expression of the alpha 6 integrin subunit.

Insulin-like growth factor I (IGF-I) strongly stimulates the generation of differentiated neurons in cultures of neuroepithelial cells of the embryonic chick neural retina in the presence of a laminin-1 tissue culture substrate. Treatment of cultured neuroepithelial cells with IGF-I rapidly up-regulated the mRNA coding for the alpha 6 integrin subunit whereas specific reduction of alpha 6 subunit levels by treatment with an alpha 6 integrin antisense oligonucleotide resulted in reduced neuronal differentiation in vitro. Although IGF-I immunoreactivity is seen throughout the neural retina, expression of IGF-I mRNA is confined to the pigment epithelium during the period of neurogenesis in vivo. Neutralization of the endogenous IGF-I with a blocking antibody down-regulated levels of alpha 6 integrin mRNA and reduced the production of differentiated retinal neurons in vivo. These data indicate a role for IGF-I in the generation of retinal neurons mediated by the interaction of laminin with its alpha 6 integrin subunit-containing receptor.