Decrease in the Ratio proBDNF/BDNF in the Urine of Aging Female Patients with OAB.

Imbalance in the levels of neurotrophins, growth factors crucial in the development, function, and survival of neurons is commonly observed in many pathological states. Concentrations of brain-derived neurotrophic factor (BDNF) and its precursor (proBDNF) were measured in the urine of a cohort of aging female patients with overactive bladder disease (OAB). When reported to creatinine, levels were similar between OAB patients and healthy controls. However, the ratio proBDNF/BDNF was significantly decreased in the OAB group. Receiver operating characteristic (ROC) curve analysis of the ratio proBDNF/BDNF displayed a good diagnostic value for OAB (AUC = 0.729). Clinical questionnaires of symptom severity (OABSS and IIQ-7) were negatively correlated with this ratio. On the other hand, microRNAs (miRNA) involved in proBDNF gene translation were expressed at comparable levels between groups. However, urinary enzymatic activity of matrix metalloproteinase-9 (MMP-9), the enzyme that cleaves proBDNF into BDNF, was increased in OAB compared to controls. Levels of miR-491-5p, the main miRNA that downregulates MMP-9 synthesis, were greatly decreased in urine from OAB patients. These results suggest that the ratio proBDNF/BDNF could be useful in the phenotyping of OAB in an aging population, and the difference could originate from enhanced MMP-9 enzymatic activity rather than translational control.

Urinary Levels of miR-491-5p and miR-592 as Potential Diagnostic Biomarkers in Female Aging Patients with OAB: A Pilot Study.

Women with overactive bladder syndrome (OAB) have a lower urinary ratio of nerve growth factor (NGF) to its precursor (proNGF) compared to healthy controls. MicroRNAs related to NGF and proNGF metabolism and to their receptors may be present in urine and may possess diagnostic value. Urine and blood samples from 20 control and 20 OAB women (50-80 years) were obtained, together with validated questionnaires and other clinical parameters. The relative expression of urinary microRNAs was measured with RT-qPCR. MiR-491-5p, which negatively controls the translation of the matrix metalloproteinase-9 (MMP-9), the main enzyme degrading NGF, was significantly decreased in OAB. Similarly, miR-592, which represses p75NTR receptor synthesis, was down-regulated in OAB. Age, renal function and insulin resistance did not affect these results. ROC curves confirmed the high sensitivity of miR-491-5p and miR-592 for diagnosis. On the other hand, miRNAs involved in the expression of proNGF, of survival receptor TrkA and of markers of nerve integrity were similar between groups. The detection of miR-491-5p and miR-592 in urine could be a useful and non-invasive tool for the diagnosis of OAB syndrome in aging women.

Modulation of diabetic kidney disease markers by an antagonist of p75NTR in streptozotocin-treated mice.

Two therapeutic agents targeting p75NTR pathways have been recently developed to alleviate retinopathy and bladder dysfunction in diabetes mellitus (DM), namely the small molecule p75NTR antagonist THX-B and a monoclonal antibody (mAb) that neutralizes the receptor ligand proNGF. We herein explore these two components in the context of diabetic kidney disease (DKD). Streptozotocin-injected mice were treated for 4 weeks with THX-B or anti-proNGF mAb. Kidneys were taken for quantification of microRNAs and mRNAs by RT-qPCR and for detection of proteins by immunohistochemistry, immunoblotting and ELISA. Blood was sampled to measure plasma levels of urea, creatinine, and albumin. DM led to increases in plasma concentrations of urea and creatinine and decreases in plasma albumin. Receptor p75NTR was expressed in kidneys and its expression was decreased by DM. All these changes were reversed by THX-B treatment while the effect of mAb was less pronounced. MicroRNAs tightly linked to DKD (miR-21-5p, miR-214-3p and miR-342-3p) were highly expressed in diabetic kidneys compared to healthy ones. Also, miR-146a, a marker of kidney inflammation, and mRNA levels of Fn-1 and Nphs, two markers of fibrosis and inflammation, were elevated in DM. Treatments with THX-B or mAb partially or completely reduced the expression of the aforementioned microRNAs and mRNAs. P75NTR antagonism and proNGF mAb might constitute new therapeutic tools to treat or slow down the progression of kidney disease in DM, along with other diabetic related complications. The translational potential of these strategies is currently being investigated.

Adaptation to partial urethral obstruction in healthy aging LOU rats and the role of nerve growth factor signaling pathway in the bladder.

AIM: Aging is associated with poor ability to adapt to stress and abnormal nerve growth factor (NGF) profile. Lower urinary tract symptoms frequently disturb the quality of life of the aging population with no optimal treatment for both genders. The aim of the study was to compare the bladder response to bladder outflow obstruction in young and old LOU rats, a model of healthy aging that does not develop insulin resistance, and its relation to proNGF/NGF imbalance. METHODS: 6- and 36-month-old female LOU rats were subjected to partial bladder urethral obstruction (PUO) for 2 weeks. Morphometric parameters (body and bladder weight) and glycemia were evaluated. Cystometry was carried out to measure functional parameters followed by ex vivo assessment of muscle strip contractile characteristics. Tissue proteins were examined by immunoblotting and morphology was examined by microscopy. RESULTS: Body weight and glycaemia were not affected by surgery. PUO increases significantly bladder weight with increased thickness and fibrosis of the bladder wall as revealed by histological examination in both age groups. Cystometry showed that old PUO rats had a significant reduction in the intercontraction interval and the bladder capacity, a pattern opposite to young rats with PUO. Contractile properties of bladder strip were not affected by age or PUO. On the molecular level, the old rats had lower abundance of the mature NGF relative to proNGF, with signs of p75NTR activation suggested by the higher expression of TNF-α and JNK phosphorylation in the bladder tissue. CONCLUSION: Bladder adaptation to PUO occurs only in young LOU rats to maintain efficient bladder contractility. Old LOU rats display proNGF/NGF imbalance and the associated p75NTR activation. This can further induce tissue damage and degeneration through activation of JNK pathway and release of TNF-α which in turn interferes with the necessary bladder adaptation.

Osr1 Is Required for Mesenchymal Derivatives That Produce Collagen in the Bladder.

The extracellular matrix of the bladder consists mostly of type I and III collagen, which are required during loading. During bladder injury, there is an accumulation of collagen that impairs bladder function. Little is known about the genes that regulate production of collagens in the bladder. We demonstrate that the transcription factor Odd-skipped related 1 (Osr1) is expressed in the bladder mesenchyme and epithelium at the onset of development. As development proceeds, Osr1 is mainly expressed in mesenchymal progenitors and their derivatives. We hypothesized that Osr1 regulates mesenchymal cell differentiation and production of collagens in the bladder. To test this hypothesis, we examined newborn and adult mice heterozygous for Osr1, Osr1+/-. The bladders of newborn Osr1+/- mice had a decrease in collagen I by western blot analysis and a global decrease in collagens using Sirius red staining. There was also a decrease in the cellularity of the lamina propria, where most collagen is synthesized. This was not due to decreased proliferation or increased apoptosis in this cell population. Surprisingly, the bladders of adult Osr1+/- mice had an increase in collagen that was associated with abnormal bladder function; they also had a decrease in bladder capacity and voided more frequently. The results suggest that Osr1 is important for the differentiation of mesenchymal cells that give rise to collagen-producing cells.

Imbalance of nerve growth factor metabolism in aging women with overactive bladder syndrome.

PURPOSE: Given the disputable link between nerve growth factor (NGF) and overactive bladder syndrome (OAB) and the lack of studies on its precursor (proNGF) in OAB, the aim of the study was to identify changes in the urinary levels of NGF and its proteolytic enzymes in aging women with OAB. METHODS: We examined the urinary proNGF/NGF ratio and its processing enzymes in aging women (50-80 years), comparing 20 controls and 20 subjects with OAB. RESULTS: In contrast to previous reports correlating NGF to OAB symptoms, we found that proNGF/NGF ratio in the OAB group was twice as high compared to controls (p = 0.009) with a lower NGF levels in women with OAB without statistical significance [1.36 (Q1, Q3: 0.668, 2.39) vs. 1.7 (Q1, Q3: 1.27, 3.045) pg/mg creatinine in control group, p = 0.05]. Enzymatic activity of MMP-7, the main enzyme for extracellular proNGF maturation, was significantly increased in the OAB group and correlated positively with scores of OAB symptoms questionnaires. However, this was counteracted by several-folds increase in the MMP-9 enzyme responsible for NGF proteolysis. While these findings highlight the importance of changes in the proteolytic enzymes to maintain proNGF/NGF balance in OAB, analysis of covariates showed that these changes were attributed to age, insulin resistance and renal function. CONCLUSION: NGF proteolysis imbalance can be clinically meaningful in OAB related to aging, rendering it as a potential therapeutic target. However, other age-related factors such as insulin resistance and renal function may contribute to the relationship between NGF and aging-related OAB phenotype.

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.

Antagonism of proNGF or its receptor p75NTR reverses remodelling and improves bladder function in a mouse model of diabetic voiding dysfunction.

AIMS/HYPOTHESIS: Although 80% of diabetic patients will suffer from voiding difficulties and urinary symptoms, defined as diabetic voiding dysfunction (DVD), therapeutic targets and treatment options are limited. We hypothesise that the blockade of the pro-nerve growth factor (NGF)/p75 neurotrophin receptor (p75NTR) axis by an anti-proNGF monoclonal antibody or by a small molecule p75NTR antagonist (THX-B) can restore bladder remodelling (represented by bladder weight) in an animal model of DVD. Secondary outcomes of the study include improvements in bladder compliance, contractility and morphology, as well as in voiding behaviour, proNGF/NGF balance and TNF-α expression. METHODS: In a streptozotocin-induced mouse model of diabetes, diabetic mice received either a blocking anti-proNGF monoclonal antibody or a p75NTR antagonist small molecule as weekly systemic injections for 4 weeks. Animals were tested at baseline (at 2 weeks of diabetes induction), and after 2 and 4 weeks of treatment. Outcomes measured were voiding function with voiding spot assays and cystometry. Bladders were assessed by histological, contractility and protein expression assays. RESULTS: Diabetic mice showed features of DVD as early as 2 weeks after diabetes diagnosis (baseline) presented by hypertrophy, reduced contractility and abnormal cystometric parameters. Following treatment initiation, a twofold increase (p < 0.05) in untreated diabetic mouse bladder weight and thickness compared with non-diabetic controls was observed, and this change was reversed by p75NTR antagonism (37% reduction in bladder weight compared with untreated diabetic mice [95% CI 14%, 60%]) after 4 weeks of treatment. However, blocking proNGF did not help to reverse bladder hypertrophy. While diabetic mice had significantly worse cystometric parameters and contractile responses than non-diabetic controls, proNGF antagonism normalised bladder compliance (0.007 [Q1-Q3; 0.006-0.009] vs 0.015 [Q1-Q3; 0.014-0.029] ml/cmH2O in untreated diabetic mice, representing 62% reduction [95% CI 8%, 110%], p < 0.05) and contractility to KCl, carbachol and electrical field stimulation (p < 0.05 compared with the diabetic group) after 2 weeks of treatment. These effects were not observed after 4 weeks of treatment with proNGF antagonist. p75NTR antagonism did not show important improvements in cystometric parameters after 2 weeks of treatment. Slightly improved bladder compliance (0.01 [Q1-Q3; 0.009-0.012] vs 0.013 [Q1-Q3; 0.011-0.016] ml/cmH2O for untreated diabetic mice) was seen in the p75NTR antagonist-treated group after 4 weeks of treatment with significantly stabilised contractile responses to KCl, carbachol and electric field stimulation (p < 0.05 for each) compared with diabetic mice. Bladder dysfunction observed in diabetic mice was associated with a significant increase in bladder proNGF/NGF ratio (3.1 [±1.2] vs 0.26 [±0.04] ng/pg in control group, p < 0.05 at week 2 of treatment) and TNF-α (p < 0.05). The proNGF/NGF ratio was partially reduced (about 60% reduction) with both treatments (1.03 [±0.6] ng/pg for proNGF antibody-treated group and 1.4 [±0.76] ng/pg for p75NTR blocker-treated group after 2 weeks of treatment), concomitant with a significant decrease in the bladder levels of TNF-α (p < 0.05), despite persistent hyperglycaemia. CONCLUSIONS/INTERPRETATION: Our findings indicate that blockade of proNGF and the p75NTR receptor in diabetes can impede the development and progression of DVD. The reported improvements in morphological and functional features in our DVD model validates the proNGF/p75NTR axis as a potential therapeutic target in this pathology. Graphical abstract.

Beta-3 Adrenoceptor Signaling Pathways in Urothelial and Smooth Muscle Cells in the Presence of Succinate.

Succinate, an intermediate metabolite of the Krebs cycle, can alter the metabolomics response to certain drugs and controls an array of molecular responses in the urothelium through activation of its receptor, G-protein coupled receptor 91 (GPR91). Mirabegron, a ß3-adrenergic receptor (ß3-AR) agonist used to treat overactive bladder syndrome (OAB), increases intracellular cAMP in the detrusor smooth muscle cells (SMC), leading to relaxation. We have previously shown that succinate inhibits forskolin-stimulated cAMP production in urothelium. To determine whether succinate interferes with mirabegron-mediated bladder relaxation, we examined their individual and synergistic effect in urothelial-cell and SMC signaling. We first confirmed ß3-AR involvement in the mirabegron response by quantifying receptor abundance by immunoblotting in cultured urothelial cells and SMC and cellular localization by immunohistochemistry in rat bladder tissue. Mirabegron increased cAMP levels in SMC but not in urothelial cells, an increase that was inhibited by succinate, suggesting that it impairs cAMP-mediated bladder relaxation by mirabegron. Succinate and mirabegron increased inducible nitric oxide synthesis and nitric oxide secretion only in urothelial cells, suggesting that its release can indirectly induces SMC relaxation. Succinate exposure decreased the expression of ß3-AR protein in whole bladder in vivo and in SMC in vitro, indicating that this metabolite may lead to impaired pharmacodynamics of the bladder. Together, our results demonstrate that increased levels of succinate in settings of metabolic stress (e.g., the metabolic syndrome) may lead to impaired mirabegron and ß3-AR interaction, inhibition of cAMP production, and ultimately requiring mirabegron dose adjustment for its treatment of OAB related to these conditions.

Succinate, increased in metabolic syndrome, activates GPR91 receptor signaling in urothelial cells.

Metabolic syndrome is associated with overactive bladder syndrome (OAB) and increased circulating levels of succinate, an intermediate of the Krebs cycle. The urothelium is an essential regulator of bladder muscle contraction. This study aimed to determine if GPR91, the succinate receptor, is expressed and functional in the bladder. Urothelial and smooth muscle cells (SMCs) were cultured and characterized. PCR revealed that urothelial cells express GPR91, twice as much as SMCs. Incubation of cells with succinate stimulated phosphorylation of ERK and JNK in urothelial cells. Succinate also potently inhibited forskolin-stimulated cyclic AMP production in urothelial cells, an effect prevented by a protein Gi inhibitor. ERK phosphorylation stimulated by succinate was abolished by inhibitors of protein Gq, phospholipase C, MAPK pathway and PKC. Incubation of urothelial cells with succinate potently increased iNOS synthesis and secretion of nitric oxide (NO), and decreased secretion of prostaglandin E2 (PGE2). Finally, succinate triggered entry of calcium in urothelial cells. GPR91 knockdown by shRNA abolished most of these signaling effects. We conclude that in the bladder, urothelial cells are a primary target of succinate through its receptor GPR91. Its activation leads to signaling via phospholipase C, MAPK, PKC pathway and protein Gq and Gi. Succinate binding to GPR91 triggers a rise in intracellular calcium, an increase in secretion of NO and a decrease in the release of PGE2. Succinate might be essential in the understanding of OAB that occurs in metabolic syndrome.

Regulation of leptin receptor expression in human polarized Caco-2/15 cells.

BACKGROUND: Leptin receptors (LEPR) are expressed in intestinal epithelial cells from the duodenum to the colon. Since their role is fundamental for the proper control of nutrient absorption, mucus secretion and mucosa renewal, the regulation of LEPR expression is for the first time investigated as a function of various potential effectors. METHODOLOGY/PRINCIPAL FINDINGS: Fully differentiated Caco-2/15 cells were incubated for 24 hours with nutrients [carbohydrates, fatty acids (FA), amino acids and sterols], hormones (leptin, insulin, hydrocortisone and epithelial growth factor), inflammatory agents (Interferon-γ, LPS, TNF-α), and PPAR agonists (rosiglitazone and WY14643). Levels of LEPR mRNA and protein expressions were measured by RT-PCR and Western blots, respectively. RESULTS: Long (219.1) and short (219.3) isoforms of the LEPR were detected in Caco-2/15 cells, while absence of the isoform 219.2 was noted. Their gene expression was modulated by carbohydrates, FA, PPAR agonists, biliary salts, insulin and leptin itself. On the other hand, LEPR protein expression was modulated by FA, cholesterol, biliary salts, PPAR agonists and insulin. Interestingly, the same effectors may have opposite effects on the short and the long LEPR isoforms, as well as on mRNA and protein levels. Finally, Caco-2/15 cells were found to be sensitive to the effector location, i.e. apical or basolateral compartment. CONCLUSIONS/SIGNIFICANCE: Our results suggest that (i) the expression of LEPR in Caco-2/15 cell line is not constitutive; (ii) the agents present in the apical or basolateral medium have different effects on LEPR mRNA and/or protein levels; and (iii) short and long isoforms of LEPR follow different patterns of regulation.

Cross-talk between adipose and gastric leptins for the control of food intake and energy metabolism.

The understanding of the regulation of food intake has become increasingly complex. More than 20 hormones, both orexigenic and anorexigenic, have been identified. After crossing the blood-brain barrier, they reach their main site of action located in several hypothalamic areas and interact to balance satiety and hunger. One of the most significant advances in this matter has been the discovery of leptin. This hormone plays fundamental roles in the control of appetite and in regulating energy expenditure. In accordance with the lipostatic theory stated by Kennedy in 1953, leptin was originally discovered in white adipose tissue. Its expression by other tissues was later established. Among them, the gastric mucosa has been shown to secrete large amounts of leptin. Both the adipose and the gastric tissues share similar characteristics in the synthesis and storage of leptin in granules, in the formation of a complex with the soluble receptor and a secretion modulated by hormones and energy substrates. However while adipose tissue secretes leptin in a slow constitutive endocrine way, the gastric mucosa releases leptin in a rapid regulated exocrine fashion into the gastric juice. Exocrine-secreted leptin survives the extreme hydrolytic conditions of the gastric juice and reach the duodenal lumen in an intact active form. Scrutiny into transport mechanisms revealed that a significant amount of the exocrine leptin crosses the intestinal wall by active transcytosis. Leptin receptors, expressed on the luminal and basal membrane of intestinal epithelial cells, are involved in the control of nutrient absorption by enterocytes, mucus secretion by goblet cells and motility, among other processes, and this control is indeed different depending upon luminal or basal stimulus. Gastric leptin after transcytosis reaches the central nervous system, to control food intake. Studies using the Caco-2, the human intestinal cell line, in vitro allowed analysis of the mechanisms of leptin actions on the intestinal mucosa, identification of the mechanisms of leptin transcytosis and understanding the modulation of leptin receptors by nutrients and hormones. Exocrine-secreted gastric leptin thus participates in a physiological axis independent in terms of time and regulation from that of adipose tissue to rapidly control food intake and nutrient absorption. Adipocytes and gastric epithelial cells are two cell types the metabolism of which is closely linked to food intake and energy storage. The coordinated secretion of adipose and gastric leptins ensures proper management of food processing and energy storage.

Receptor-Mediated Transcytosis of Leptin through Human Intestinal Cells In Vitro.

Gastric Leptin is absorbed by duodenal enterocytes and released on the basolateral side towards the bloodstream. We investigated in vitro some of the mechanisms of this transport. Caco-2/15 cells internalize leptin from the apical medium and release it through transcytosis in the basal medium in a time- temperature-dependent and saturable fashion. Leptin receptors are revealed on the apical brush-border membrane of the Caco-2 cells. RNA-mediated silencing of the receptor led to decreases in the uptake and basolateral release. Leptin in the basal medium was found bound to the soluble form of its receptor. An inhibitor of clathrin-dependent endocytosis (chlorpromazine) decreased leptin uptake. Confocal immunocytochemistry and the use of brefeldin A and okadaic acid revealed the passage of leptin through the Golgi apparatus. We propose that leptin transcytosis by intestinal cells depends on its receptor, on clathrin-coated vesicles and transits through the Golgi apparatus.

Adiponectin stimulates phosphorylation of AMP-activated protein kinase alpha in renal glomeruli.

Adiponectin receptor ADIPOR1 activates the intracellular second messenger AMP-activated protein kinase (AMPK) that participates in the control of the oxidative stress and apoptosis. This study reveals the presence of a functional ADIPOR1 receptor in all the cells of the renal glomeruli. Isolated glomeruli were incubated in vitro with adiponectin and proteins analysed by western blot. Electron microscopy using immunogold labeling was carried out on kidney sections. ADIPOR1 and catalytic AMPK sub-units alpha1 and alpha2 were revealed in normal rat glomeruli and incubation of freshly isolated rat glomeruli with either adiponectin or AICAR led to the activation by phosphorylation of catalytic AMPK. Electron microscopy localized with high resolution these proteins at the plasma membrane of the three glomerular cells, namely the endothelial, the mesangial and the podocyte cells, as well as on Bowman's capsule epithelial cells. It is concluded that glomerular cells express a functional adiponectin receptor ADIPOR1 which, through activation of AMPK, may play important roles in the control of oxidative stress and cell survival within the glomerulus.

Control of glycogen synthase through ADIPOR1-AMPK pathway in renal distal tubules of normal and diabetic rats.

Diabetic nephropathies are characterized by glycogen accumulation in distal tubular cells, which eventually leads to their apoptosis. The present study aims to determine whether adiponectin and AMPK are involved in the regulation of glycogen synthase (GS) in these structures. Western blots of isolated distal tubules revealed the presence of adiponectin receptor ADIPOR1, catalytic AMPK subunits alpha(1) and alpha(2), their phosphorylated active forms, and the glycogen-binding AMPK subunit beta(2). ADIPOR2 was not detected. Expression levels of ADIPOR1, AMPKalpha(1), AMPKalpha(2), and AMPKbeta(2) were increased in streptozotocin-treated diabetic rats, whereas phosphorylated active AMPK levels were strongly decreased. Immunohistochemistry revealed the presence of ADIPOR1 on the luminal portion of distal tubules and thick ascending limb cells. Catalytic subunits alpha(1) and alpha(2), their phosphorylated active forms, and the glycogen-binding subunit beta(2) were also found in the same cells, confirming immunoblot results. In vitro, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR; 2 mM) and globular adiponectin (10 mug/ml) activated catalytic AMPK in distal tubules isolated from kidneys of normal rats but much more weakly in those from diabetic rats. GS inhibition paralleled AMPK activation in both groups of animals: active GS levels were low in control animals and elevated in diabetic ones. Finally, glucose-6-phosphate, an allosteric activator of GS, was also increased in diabetic rats. These results demonstrate that in distal tubular cells, adiponectin through luminal ADIPOR1 activates AMPK, leading to the inhibition of GS. During hyperglycemia, this regulation is altered, which may explain, at least in part, the accumulation of large glycogen deposits.

Transcytosis of gastric leptin through the rat duodenal mucosa.

Leptin is secreted into the gastric juice by epithelial Chief cells and reaches the duodenum in a biologically intact active form. We assessed the possibility that this gastric leptin crosses the intestinal mucosa by transcytosis through enterocytes to reach blood circulation. Endogenous gastric leptin secretion was triggered by cholinergic stimulation. In another set of experiments, recombinant leptin was inserted in vivo into the duodenal lumen. Plasma levels of leptin were assessed by enzyme immunoassay and Western blot, and duodenal tissue was processed for immunocytochemistry. We first observed that leptin was found inside duodenal enterocytes from fed rats but not inside those from fasted ones. Stimulation of gastric secretion by a cholinergic agent led to rapid increases in plasma leptin levels (202 +/- 39%) except when the pylorus was clamped. Insertion of recombinant leptin into the duodenal lumen raised plasma leptin concentrations (558 +/- 34%) quite rapidly, whereas carrier solution without leptin had no effect. The use of FITC-tagged leptin reinforced these results. Light and electron microscopy revealed the cellular compartments involved in its transcytosis, namely, the enterocyte microvilli, the endocytotic vesicles, the Golgi complex, and the basolateral interdigitations. Leptin was also present in the lamina propria, in capillary endothelial cell plasmalemmal vesicles, and in capillary lumina. These results demonstrate that gastric exocrine leptin is internalized by duodenal enterocytes and delivered to the lamina propria and blood circulation. It may thus be able to play important paracrine and endocrine functions for the control of gastric emptying and nutrient absorption.

Leptin biosynthetic pathway in white adipocytes.

The aim of this study was to determine through morphological and biochemical means the biosynthetic and secretory pathway followed by leptin in adipocytes. Immunocytochemistry revealed the presence of leptin in the rough endoplasmic reticulum, the Golgi apparatus, and in numerous small vesicles along the plasma membrane of white adipo cytes. In vitro, isolated adipocytes under nonstimulated conditions (basal) continuously secreted leptin while their intra cellular content remained unchanged. When adipocytes were stimulated with insulin, leptin cellular content and secretion increased in parallel and were significantly different from basal secretion only after 45 min. L-leucine and L-glutamate also strongly stimulated leptin synthesis and secretion. These stimulating effects were abolished by cycloheximide and brefeldin A. The transcriptional inhibitor actinomycin D did not have any effects in either basal or stimulated conditions. Leptin mRNA levels were not affected by any stimulating or inhibiting agents. Finally, norepinephrine, isoproterenol, CL316243, and palmitate inhibited the effects of insulin, L-leucine, and L-glutamate on leptin synthesis. We thus conclude that (i) adipocytes continuously synthesize and secrete leptin along a rough endoplasmic reticulum-Golgi secretory vesicles pathway, (ii) an increase in leptin secretion requires increased de novo synthesis, and (iii) short-term leptin secretion does not involve changes in mRNA levels.

Secretion of soluble leptin receptors by exocrine and endocrine cells of the gastric mucosa.

Leptin is a hormone secreted by the gastric mucosa into the lumen of the stomach. It is present in its intact form in the intestine where it regulates nutrient absorption and intestinal mucosa integrity. We have identified the binding protein that protects leptin from the harsh conditions of the gastric juice. Immunoprecipitations and Western blot analyses demonstrated that leptin is present in the gastric mucosa and the gastric juice, bound to a protein corresponding to the extracellular domain of the leptin receptor. In the absence of this soluble receptor, leptin is rapidly degraded. Immunocytochemistry on rat gastric mucosa identified the cells and intracellular compartments involved in secretion of this complex. Leptin receptor extracellular domain and leptin are present along the rough endoplasmic reticulum-Golgi-granules secretory pathways and form a complex in the secretory granules of Chief and specific endocrine cells. The long-form membrane leptin receptor OB-Rb, the protease activator furin, and proprotein convertase 7 were found in Chief cell granules but not in those of endocrine cells. The shedding of the receptor occurs in the immature granules. It is concluded that in the immature secretory granules of Chief cells, furin activates proprotein convertase 7 that, in turn, cleaves the extracellular portion of membrane-bound leptin receptors. Leptin bound to its soluble receptor forms a complex that is resistant to the gastric juice. Endocrine cells, on the other hand, generate a soluble leptin receptor by mechanisms different from those of the exocrine cells.

Endocrine and exocrine secretion of leptin by the gastric mucosa.

Leptin is a hormone that plays important roles in nutritional status and in obesity. By means of immunocytochemistry, two populations of leptin-secreting cells were found in the lower half of the gastric mucosa. One consists of numerous large cells located around the gastric pits, the Chief epithelial cells, whereas the second refers to much smaller cells, strongly stained, few in number, and scattered between the gastric pits, the endocrine cells. By double immunostaining, leptin and pepsinogen were colocalized in the Chief cells, whereas the endocrine cells were positive only for leptin. Immunoelectron microscopy showed that leptin is present along the rough endoplasmic reticulum-Golgi-granules secretory pathways of the Chief and endocrine cells. On the other hand, leptin-receptor (long and short forms) immunolabelings, although absent in the gastric epithelial cell plasma membranes, were present in enterocytes at the level of their apical and basolateral membranes. Duodenal, jejunal, and ileal enterocytes displayed similar labelings for the leptin receptor. Thus, exocrine and endocrine secretions of leptin together with the presence of leptin receptors on enterocyte plasma membranes constitute a gastroenteric axis that coordinates the role played by leptin in the digestive tract.

Regulation of leptin secretion from white adipocytes by insulin, glycolytic substrates, and amino acids.

The aim of the present study was to determine the respective roles of energy substrates and insulin on leptin secretion from white adipocytes. Cells secreted leptin in the absence of glucose or other substrates, and addition of glucose (5 mM) increased this secretion. Insulin doubled leptin secretion in the presence of glucose (5 mM), but not in its absence. High concentrations of glucose (up to 25 mM) did not significantly enhance leptin secretion over that elicited by 5 mM glucose. Similar results were obtained when glucose was replaced by pyruvate or fructose (both 5 mM). L-Glycine or L-alanine mimicked the effect of glucose on basal leptin secretion but completely prevented stimulation by insulin. On the other hand, insulin stimulated leptin secretion when glucose was replaced by L-aspartate, L-valine, L-methionine, or L-phenylalanine, but not by L-leucine (all 5 mM). Interestingly, these five amino acids potently increased basal and insulin-stimulated leptin secretion in the presence of glucose. Unexpectedly, L-glutamate acutely stimulated leptin secretion in the absence of glucose or insulin. Finally, nonmetabolizable analogs of glucose or amino acids were without effects on leptin secretion. These results suggest that 1) energy substrates are necessary to maintain basal leptin secretion constant, 2) high availability of glycolysis substrates is not sufficient to enhance leptin secretion but is necessary for its stimulation by insulin, 3) amino acid precursors of tricarboxylic acid cycle intermediates potently stimulate basal leptin secretion per se, with insulin having an additive effect, and 4) substrates need to be metabolized to increase leptin secretion.