Measuring peroxidasin activity in live cells using bromide addition for signal amplification.

Peroxidasin (PXDN) is involved in the crosslinking of collagen IV, a major constituent of basement membranes. Disruption of basement membrane integrity as observed in genetic alterations of collagen IV or PXDN can result in developmental defects and diverse pathologies. Hence, the study of PXDN activity in (patho)physiological contexts is highly relevant. So far, measurements of PXDN activity have been reported from purified proteins, cell lysates and de-cellularized extracellular matrix. Here, for the first time we report the measurement of PXDN activity in live cells using the Amplex Red assay with a signal amplifying modification. We observe that bromide addition enhances the obtained signal, most likely due to formation of HOBr. Abrogation of signal amplification by the HOBr scavenger carnosine supports this hypothesis. Both, pharmacological inhibition as well as complementary genetic approaches confirm that the obtained signal is indeed related to PXDN activity. We validate the modified assay by investigating the effect of Brefeldin A, to inhibit the secretory pathway and thus the access of PXDN to the extracellular Amplex Red dye. Our method opens up new possibilities to investigate the activity of PXDN in (patho)physiological contexts.

Characterization of the Proprotein Convertase-Mediated Processing of Peroxidasin and Peroxidasin-like Protein.

Peroxidasin (PXDN) and peroxidasin-like protein (PXDNL) are members of the peroxidase-cyclooxygenase superfamily. PXDN functions in basement membrane synthesis by forming collagen IV crosslinks, while the function of PXDNL remains practically unknown. In this work, we characterized the post-translational proteolytic processing of PXDN and PXDNL. Using a novel knock-in mouse model, we demonstrate that the proteolytic cleavage of PXDN occurs in vivo. With the help of furin-specific siRNA we also demonstrate that the proprotein-convertase, furin participates in the proteolytic processing of PXDN. Furthermore, we demonstrate that only the proteolytically processed PXDN integrates into the extracellular matrix, highlighting the importance of the proteolysis step in PXDN's collagen IV-crosslinking activity. We also provide multiple lines of evidence for the importance of peroxidase activity in the proteolytic processing of PXDN. Finally, we show that PXDNL does not undergo proteolytic processing, despite containing sequence elements efficiently recognized by proprotein convertases. Collectively, our observations suggest a previously unknown protein quality control during PXDN synthesis and the importance of the peroxidase activity of PXDN in this process.

Peroxidasin-mediated crosslinking of collagen IV is independent of NADPH oxidases.

Collagen IV is a major component of the basement membrane in epithelial tissues. The NC1 domains of collagen IV protomers are covalently linked together through sulfilimine bonds, the formation of which is catalyzed by peroxidasin. Although hydrogen peroxide is essential for this reaction, the exact source of the oxidant remains elusive. Members of the NOX/DUOX NADPH oxidase family are specifically devoted to the production of superoxide and hydrogen peroxide. Our aim in this study was to find out if NADPH oxidases contribute in vivo to the formation of collagen IV sulfilimine crosslinks. We used multiple genetically modified in vivo model systems to provide a detailed assessment of this question. Our data indicate that in various peroxidasin-expressing tissues sulfilimine crosslinks between the NC1 domains of collagen IV can be readily detected in the absence of functioning NADPH oxidases. We also analyzed how subatmospheric oxygen levels influence the collagen IV network in collagen-producing cultured cells with rapid matrix turnover. We showed that collagen IV crosslinks remain intact even under strongly hypoxic conditions. Our hypothesis is that during collagen IV network formation PXDN cooperates with a NOX/DUOX-independent H2O2 source that is functional also at very low ambient oxygen levels.

Interaction between p22phox and Nox4 in the endoplasmic reticulum suggests a unique mechanism of NADPH oxidase complex formation.

The p22phox protein is an essential component of the phagocytic- and inner ear NADPH oxidases but its relationship to other Nox proteins is less clear. We have studied the role of p22phox in the TGF-ß1-stimulated H2O2 production of primary human and murine fibroblasts. TGF-ß1 induced H2O2 release of the examined cells, and the response was dependent on the expression of both Nox4 and p22phox. Interestingly, the p22phox protein was present in the absence of any detectable Nox/Duox expression, and the p22phox level was unaffected by TGF-ß1. On the other hand, Nox4 expression was dependent on the presence of p22phox, establishing an asymmetrical relationship between the two proteins. Nox4 and p22phox proteins localized to the endoplasmic reticulum and their distribution was unaffected by TGF-ß1. We used a chemically induced protein dimerization method to study the orientation of p22phox and Nox4 in the endoplasmic reticulum membrane. This technique is based on the rapamycin-mediated heterodimerization of the mammalian FRB domain with the FK506 binding protein. The results of these experiments suggest that the enzyme complex produces H2O2 into the lumen of the endoplasmic reticulum, indicating that Nox4 contributes to the development of the oxidative milieu within this organelle.

Sleeve gastrectomy and Roux-en-Y gastric bypass alter the gut-brain communication.

This study investigated the anatomical integrity of vagal innervation of the gastrointestinal tract following vertical sleeve gastrectomy (VSG) and Roux-en-Y gastric bypass (RYGB) operations. The retrograde tracer fast blue (FB) was injected into the stomach to label vagal neurons originating from nodose ganglion (NG) and dorsal motor nucleus of the vagus (DMV). Microglia activation was determined by quantifying changes in the fluorescent staining of hindbrain sections against an ionizing calcium adapter binding molecule 1 (Iba1). Reorganization of vagal afferents in the hindbrain was studied by fluorescent staining against isolectin 4 (IB4). The density of Iba1- and IB4-immunoreactivity was analyzed using Nikon Elements software. There was no difference in the number of FB-labeled neurons located in NG and DMV between VSG and VSG-sham rats. RYGB, but not RYGB-sham rats, showed a dramatic reduction in number of FB-labeled neurons located in the NG and DMV. VSG increased, while the RYGB operation decreased, the density of vagal afferents in the nucleus tractus solitarius (NTS). The RYGB operation, but not the VSG procedure, significantly activated microglia in the NTS and DMV. Results of this study show that the RYGB, but not the VSG procedure, triggers microglia activation in vagal structures and remodels gut-brain communication.

Structure-function analysis of peroxidasin provides insight into the mechanism of collagen IV crosslinking.

Basement membranes provide structural support and convey regulatory signals to cells in diverse tissues. Assembly of collagen IV into a sheet-like network is a fundamental mechanism during the formation of basement membranes. Peroxidasin (PXDN) was recently described to catalyze crosslinking of collagen IV through the formation of sulfilimine bonds. Despite the significance of this pathway in tissue genesis, our understanding of PXDN function is far from complete. In this work we demonstrate that collagen IV crosslinking is a physiological function of mammalian PXDN. Moreover, we carried out structure-function analysis of PXDN to gain a better insight into its role in collagen IV synthesis. We identify conserved cysteines in PXDN that mediate the oligomerization of the protein into a trimeric complex. We also demonstrate that oligomerization is not an absolute requirement for enzymatic activity, but optimal collagen IV coupling is only catalyzed by the PXDN trimers. Localization experiments of different PXDN mutants in two different cell models revealed that PXDN oligomers, but not monomers, adhere on the cell surface in "hot spots," which represent previously unknown locations of collagen IV crosslinking.

Peroxidasin-like protein: a novel peroxidase homologue in the human heart.

AIMS: Peroxidases serve diverse biological functions including well-characterized activities in host defence and hormone biosynthesis. More recently, peroxidasin (PXDN) was found to be involved in collagen IV cross-linking in the extracellular matrix (ECM). The aim of this study was to characterize the expression and function of peroxidasin-like protein (PXDNL), a previously unknown peroxidase homologue. METHODS AND RESULTS: We cloned the PXDNL cDNA from the human heart and identified its expression pattern by northern blot, in situ hybridization, and immunohistochemistry. PXDNL is expressed exclusively in the heart and it has evolved to lose its peroxidase activity. The protein is produced by cardiomyocytes and localizes to cell-cell junctions. We also demonstrate that PXDNL can form a complex with PXDN and antagonizes its peroxidase activity. Furthermore, we show an increased expression of PXDNL in the failing myocardium. CONCLUSION: PXDNL is a unique component of the heart with a recently evolved inactivation of peroxidase function. The elevation of PXDNL levels in the failing heart may contribute to ECM dysregulation due to its antagonism of PXDN function.

Irony comprehension and context processing in schizophrenia during remission--a functional MRI study.

Schizophrenic patients have Theory of Mind (ToM) deficits even during remission, but it is yet unknown whether this could be influenced. We examined the neural correlates of irony understanding in schizophrenic patients, as an indicator of ToM capacity, and evaluated how linguistic help inserted into the context phase could affect irony comprehension. Schizophrenic patients in remission and healthy controls were subjected to event-related functional MRI scanning while performing irony, 'irony with linguistic help', and control tasks. Patients understood irony significantly worse than healthy controls. The patients showed stronger brain activity in the parietal and frontal areas in the early phase of irony task, however the healthy controls exhibited higher activation in frontal, temporal and parietal regions in the latter phase of the irony task. Interestingly the linguistic help not only improved the patients' ToM performance, but it also evoked similar activation pattern to healthy controls.

Improvement in hepatic insulin sensitivity after Roux-en-Y gastric bypass in a rat model of obesity is partially mediated via hypothalamic insulin action.

AIMS/HYPOTHESIS: Roux-en-Y gastric bypass (RYGB) surgery, an effective treatment for morbid obesity, commonly leads to near complete resolution of type 2 diabetes. The underlying mechanisms, however, remain unclear and factors other than weight loss alone may be involved. METHODS: To determine whether increased hypothalamic insulin sensitivity after RYGB drives the rapid improvement in glucose metabolism, high-fat-fed rats received either an insulin receptor (IR) antisense vector or a control lentiviral vector that was microinjected into the ventromedial hypothalamus (VMH). Six weeks later, rats underwent RYGB or control gastrointestinal surgery. RESULTS: Four weeks after surgery, weight loss was comparable in RYGB and surgical controls. Nevertheless, only RYGB rats that received the control vector demonstrated both improved hepatic and peripheral insulin sensitivity. Insulin suppressed hepatic glucose production (HGP) by 50% (p < 0.05) with RYGB, whereas the effect of insulin on HGP was completely absent in VMH IR knockdown (IRkd) rats. By contrast, both RYGB groups displayed an identical twofold increase in insulin-stimulated peripheral glucose uptake. The animals that underwent control gastrointestinal surgery failed to show any improvement in either hepatic or peripheral insulin sensitivity; VMH IRkd did not influence the magnitude of insulin resistance. CONCLUSIONS/INTERPRETATION: Our findings demonstrate that RYGB surgery in high-fat-fed obese rats enhances hepatic and peripheral insulin sensitivity independently of weight loss. The improved hepatic, but not the peripheral, response to insulin is mediated centrally at the level of the VMH. These data provide direct evidence that the metabolic benefits of RYGB surgery are not simply a consequence of weight loss but likely in part involve the central nervous system.

Could the mechanisms of bariatric surgery hold the key for novel therapies? report from a Pennington Scientific Symposium.

Bariatric surgery is the most effective method for promoting dramatic and durable weight loss in morbidly obese subjects. Furthermore, type 2 diabetes is resolved in over 80% of patients. The mechanisms behind the amelioration in metabolic abnormalities are largely unknown but may be due to changes in energy metabolism, gut peptides and food preference. The goal of this meeting was to review the latest research to better understand the mechanisms behind the 'magic' of bariatric surgery. Replication of these effects in a non-surgical manner remains one of the ultimate challenges for the treatment of obesity and diabetes. Promising data on energy metabolism, gastrointestinal physiology, hedonic response and food intake were reviewed and discussed.

Altered orosensory sensitivity to oils in CCK-1 receptor deficient rats.

CCK-1 receptor deficient Otsuka Long Evans Tokushima Fatty (OLETF) rats are hyperphagic, which leads to subsequent obesity and diabetes. Additionally, they have increased sham intake and enhanced preference for sucrose solutions relative to control, Long Evans Tokushima Otsuka (LETO) rats. To determine the effects of oil on ingestion, we first measured real feeding of various concentrations of oil emulsions (12.5, 25, 50, 75, and 100%) in rats that were fed ad libitum. Secondly, to isolate the orosensory compontent of oils from post-ingestive consequences, as well as determine the contribution of energy status, we measured sham feeding in OLETF and LETO rats using one-bottle acceptance tests while non-deprived and overnight food deprived. Finally, to assess the orosensory effects of nutritive and non-nutritive oils, we used two-bottle preference tests in sham fed OLETF and LETO rats. We found that real feeding resulted in increased intake of high oil concentrations for OLETF rats relative to LETO rats. Similarly, OLETF rats consumed significantly more of higher concentration corn oils than LETO while non-deprived sham feeding. Conversely, OLETF rats overconsumed low concentration corn oil compared to LETO during overnight deprived sham-feeding tests. In two-bottle sham-feeding preference tests, both non-deprived OLETF and LETO rats preferred corn to mineral oil. Collectively, these results show that increased oil intake in OLETF rats is driven by both peripheral deficits to satiation and altered orosensory sensitivity.

Dopamine D2 receptors contribute to increased avidity for sucrose in obese rats lacking CCK-1 receptors.

Accumulating evidence has indicated a link between dopamine signaling and obesity in both animals and humans. We have recently demonstrated heightened avidity to sapid sweet solutions in the obese cholecystokinin (CCK)-1 receptor deficient Otsuka Long Evans Tokushima fatty (OLETF) rat. To investigate the dopamine dependence and the respective contribution of D1 and D2 receptor subtypes in this phenomenon, real and sham intake of 0.3 M sucrose solution was compared between prediabetic, obese OLETF and age-matched lean Long-Evans Tokushima Otsuka (LETO) cohorts following peripheral (i.p.) administration of equimolar doses (50-800 nmol/kg) of the D1 (R-(+) 7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine, SCH23390) and D2 (raclopride) selective receptor antagonists. Both antagonists were potent in reducing sucrose intake in both strains with both drugs suppressing sham intake starting at lower doses than real intake (200 nmol/kg vs. 400 nmol/kg for SCH23390, and 400 nmol/kg vs. 600 nmol/kg for raclopride, respectively). Furthermore, when percent suppression of intake, a measure that controlled for the higher baseline sucrose intake by obese rats was analyzed, OLETF rats expressed an increased sensitivity to raclopride in reducing ingestion of sucrose with a 1.7- and 2.9-fold lower inhibitory dose threshold (ID50) for real and sham intake conditions, respectively, compared with LETO controls. In contrast, SCH23390 caused no differential strain effect with respect to dosage whether sucrose was real or sham fed. These findings demonstrate that D2 receptors are involved in heightened increased consumption of sucrose observed in the OLETF obese rat.

Acute methylphenidate treatments reduce sucrose intake in restricted-fed bingeing rats.

Recent evidence suggests that methylphenidate HCl may be effective at limiting the frequency and the amount of binge eating. The present study investigated if daily treatments with methylphenidate reduced the bingeing-like behavior observed in restricted-fed adult male rats. Three groups (n = 6) received peripheral injections of methylphenidate in doses of 1.5 or 0.75 mg/kg/day, or saline, 3 days prior and 7 days during a previously characterized intermittent feeding regimen that results in a gradual increase of sucrose and food intake. The higher, but not the lower, dose of methylphenidate reduced sucrose intake to an asymptotic level starting after 3 days of the feeding protocol and concurrently led to an increase in the intake of chow. The high dose methylphenidate group also had two-fold lower plasma insulin levels compared with the saline-treated animals at the time of sacrifice on the last day of the feeding regimen. Further histological assays revealed that the methylphenidate treatments, irrespective of the dose used, resulted in selectively higher dopamine transporter and D2-like receptor labeled bindings in the shell region of the nucleus accumbens. These results suggest that relatively low-dose methylphenidate treatments may be effective for the management of binge eating by reducing the intake of palatable foods and may not interfere with short-term regulation of energy balance. These findings further support the notion that the mesoaccumbens dopamine system plays an important role in restricted access-induced sucrose bingeing in this rat model.

Gustatory reward and the nucleus accumbens.

The concept of reward is central to psychology, but remains a cipher for neuroscience. Considerable evidence implicates dopamine in the process of reward and much of the data derives from the nucleus accumbens. Gustatory stimuli are widely used for animal studies of reward, but the connections between the taste and reward systems are unknown. In a series of experiments, our laboratory has addressed this issue using functional neurochemistry and neuroanatomy. First, using microdialysis probes, we demonstrated that sapid sucrose releases dopamine in the nucleus accumbens. The effect is dependent on oral stimulation and concentration. We subsequently determined that this response was independent of the thalamocortical gustatory system, but substantially blunted by damage to the parabrachial limbic taste projection. Further experiments using c-fos histochemistry confirmed that the limbic pathway was the prime carrier for the gustatory afferent activity that drives accumbens dopamine release.

NADPH-diaphorase positive neurons of the rat hippocampal formation: regional distribution, total number and colocalization with calcium binding proteins.

The present study aimed to asses the total number and distribution of the NADPH-diaphorase-positive non-pyramidal neurons in Ammon's horn and dentate gyrus of rat hippocampal formation. Cell bodies were counted according to the "disector" principle. The total numbers varied from 27 000 to 32 400. In all strains, approximately one third of the NADPH-diaphorase-reactive non-principal cells were found in the dentate gyrus and the remaining two thirds were within the Ammon's horn. Analysis of the dorsoventral differences revealed that approximately 70% of NADPH-diaphorase-positive cells were in the dorsal and 30% in the ventral hippocampus. Distribution of NADPH-diaphorase-reactive cells in the different layers of the dentate gyrus and Ammon's horn was similar in all strains. Double-labelling studies revealed colocalization of NADPH-diaphorase with calretinin, but none with calbindin or parvalbumin. NADPH-diaphorase-positive neurons appear to form the largest chemically identified subpopulation of the GABAergic inhibitory cell population of the hippocampal formation.

Accumbens dopamine mechanisms in sucrose intake.

Extracellular levels of dopamine (DA) and monoamine metabolites were measured in the nucleus accumbens (NAcc) during sucrose licking using microdialysis in freely moving rats. The converse relationship also was tested. Using bilateral reverse microdialysis, D1 and D2 receptor antagonists (SCH23390, sulpiride) and the DA uptake blocker nomifensine were introduced into NAcc while measuring both ingestive behavior and neurochemistry. Licking of 0.3 M sucrose caused a 305% (+/-69%) increase in NAcc DA compared with water intake. Reverse microdialysis of nomifensine at a dose that increased accumbens DA levels (1484+/-346%) led to an increase of sucrose intake (152.5+/-5.4%). Concurrent infusions of the D1 and D2 blockers with nomifensine brought sucrose ingestion back near to control levels (114.8+/-3.7%). The higher dose of the D2 antagonist sulpiride also increased DA levels and sucrose intake. In contrast, the lower dose of the D2, and both doses of the D1 antagonist had no chemical or behavioral effects. These results showed release of NAcc DA in response to sucrose licking and the converse, an augmentation of the behavior by uptake blockade. The same data, however, failed to prove that tonic, local accumbens D1 and D2 receptor activity influenced this ingestive behavior.

Notch inhibition of RAS signaling through MAP kinase phosphatase LIP-1 during C. elegans vulval development.

During Caenorhabditis elegans vulval development, a signal from the anchor cell stimulates the RTK/RAS/MAPK (receptor tyrosine kinase/RAS/mitogen-activated protein kinase) signaling pathway in the closest vulval precursor cell P6.p to induce the primary fate. A lateral signal from P6.p then activates the Notch signaling pathway in the neighboring cells P5.p and P7.p to prevent them from adopting the primary fate and to specify the secondary fate. The MAP kinase phosphatase LIP-1 mediates this lateral inhibition of the primary fate. LIN-12/NOTCH up-regulates lip-1 transcription in P5.p and P7.p where LIP-1 inactivates the MAP kinase to inhibit primary fate specification. LIP-1 thus links the two signaling pathways to generate a pattern.

The C. elegans Mi-2 chromatin-remodelling proteins function in vulval cell fate determination.

The Mi-2 protein is the central component of the recently isolated NuRD nucleosome remodelling and histone deacetylase complex. Although the NuRD complex has been the subject of extensive biochemical analyses, little is known about its biological function. Here we show that the two C. elegans Mi-2 homologues, LET-418 and CHD-3, play essential roles during development. The two proteins possess both shared and unique functions during vulval cell fate determination, including antagonism of the Ras signalling pathway required for vulval cell fate induction and the proper execution of the 2 degrees cell fate of vulval precursor cells, a process under the control of LIN-12 Notch signalling.

Accumbens cholinergic interneurons play a role in the regulation of body weight and metabolism.

The aims of the present study were (1) to determine whether selective lesions of the accumbens cholinergic interneurons impair feeding and body weight regulation, and (2) to characterize the nature of disturbances using motivational and metabolic challenges. Rats with bilateral cholinotoxic (AF64A) lesions in the nucleus accumbens showed a significant and lasting lag in body weight gain in comparison to the sham-operated controls. This failure to gain weight was not due to a decrease in feeding because lesioned rats actually ate more food and drank more water than controls under basal conditions. Lesion-induced deficits were also exposed when the rats were challenged with food deprivation or cold exposure. Lesioned rats ate less than controls when 24 h food deprived and maintained both a higher core temperature and a higher metabolic rate than controls following either 24-h food deprivation or exposure to cold. Thyroid hormones, insulin, and blood glucose levels were, however, within the physiological range, and no sensory and motor disturbances were observed. The results suggest that the altered body weight regulation is partly due to the enhanced metabolic responsiveness to stress. Possible explanations for the effects of the lesions are also discussed in the context of motivational alterations, including possible dopamine-acetylcholine interactions.

The Caenorhabditis elegans APC-related gene apr-1 is required for epithelial cell migration and Hox gene expression.

Inactivation of the Caenorhabditis elegans APC-related gene (apr-1) has pointed at two separate functions of apr-1. First, apr-1 is required for the migration of epithelial cells during morphogenesis of the embryo. In this process, APR-1 may act in a Cadherin/alpha-Catenin/beta-Catenin complex as a component of adherens junctions. Second, apr-1 is required for Hox gene expression, most likely by positively regulating the activity of the Wingless signaling pathway. During embryogenesis, apr-1 is required for the expression of ceh-13 labial in anterior seam and muscle cells and during larval development, apr-1 is necessary for the expression of lin-39 deformed in the vulval precursor cells. Thus, APR-1 may positively regulate the activity of the beta-Catenin/Armadillo-related proteins HMP-2 in migrating epithelial cells and BAR-1 in the vulval precursor cells.