Statistical Mechanical Study of β-N-methylamino-L-alanine and its carbamate adducts as potential inhibitors of the AMPA glutamate receptor using Molecular Dynamics Simulations

To BMAA είναι ένα μη πρωτεϊνικό αμινοξύ που μπορεί να καταλήξει στον ανθρώπινο οργανισμό μέσω της τροφικής αλυσίδας. Όταν το ΒΜΑΑ αλληλεπιδρά με το διττανθρακικό ιόν στον άνθρωπο παράγονται καρβαμιδικά παράγωγα που έχουν παρόμοια δομή με το νευροδιαβιβαστή γλουταμινικό ιόν. Πιστεύεται ότι το BMAA και τα καρβαμιδικά παράγωγά του προσδένονται στο σημείο πρόσδεσης γλουταμινικών υποδοχέων προκαλώντας νευρικά συμπτώματα ασθενειών όπως το Πάρκινσον και η Αμυοτροφική Πλευρική Σκλήρυνση. Ο μηχανισμός δράσης του BMAA και των καρβαμιδικών παραγώγων του προσδεμένων σε γλουταμινικούς υποδοχείς δεν έχει μελετηθεί έως τώρα. Σε αυτήν την εργασία, ερευνούμε τη δεσμευτική συγγένεια του ΒΜΑΑ και των καρβαμιδικών παραγώγων του σε γλουταμινικούς υποδοχείς σε σύγκριση με τον φυσιολογικό αγωνιστή, το γλουταμινικό ιόν, προκειμένου να γίνει κατανοητό εάν αυτά μπορούν να δράσουν ως αναστολείς γλουταμινικών υποδοχέων. Αρχικά, εφαρμόζουμε προσομοιώσεις Μοριακής Δυναμικής του ΒΜΑΑ και των καρβαμιδικών παραγώγων του στον γλουταμινικό υποδοχέα ΑΜΡΑ, έτσι ώστε να μελετηθεί η σταθερότητα των μορίων στην S1/S2 περιοχή πρόσδεσης του υποδοχέα ΑΜΡΑ. Επιπλέον, χρησιμοποιούμε προσομοιώσεις Μοριακής Δυναμικής σε συνδυασμό με υπολογισμούς Διατάραξης Ελεύθερης Ενέργειας προκειμένου να υπολογιστεί η διαφορά της ελεύθερης ενέργειας πρόσδεσης του BMAA σε σχέση με το γλουταμινικό ιόν στον ΑΜΡΑ υποδοχέα. Τα αποτελέσματα δείχνουν ότι ένα από τα παράγωγα του ΒΜΑΑ, το β-καρβαμιδικό ιόν, παρουσιάζει ιδιαίτερη σταθερότητα στον υποδοχέα ΑΜΡΑ σε σύγκριση με τον φυσιολογικό αγωνιστή, το γλουταμινικό ιόν. Επιπλέον, τα αποτελέσματα από τους υπολογισμούς FEP υποδεικνύουν ότι το γλουταμινικό ιόν και το β-καρβαμιδικό παράγωγο του ΒΜΑΑ έχουν συγκρίσιμη συγγένεια πρόσδεσης στον υποδοχέα ΑΜΡΑ.BMAA is a neurotoxic non-protein amino acid, which may reach the human body through the food chain. When BMAA interacts with bicarbonate in the human body, carbamate adducts are produced, which share high structural similarity with the neurotransmitter glutamate. It is believed that BMAA and its carbamate adducts bind in the glutamate binding site of glutamate receptors (GluR) causing the neurodegenerative effects of diseases such as Amyotrophic Lateral Sclerosis and Parkinson’s Disease. The mechanism of BMAA action and its carbamate adducts bound to GluR has not been yet elucidated. In this work, we investigate the binding affinity of BMAA and its carbamate adducts to glutamate receptors in comparison to the natural agonist, glutamate, in order to understand whether these can act as glutamate inhibitors. Initially, we perform Molecular Dynamics (MD) simulations of BMAA and its carbamate adducts bound to the AMPA glutamate receptor in order to examine the stability of the ligands in the S1/S2 ligand-binding domain of the AMPA receptor. In addition, we perform MD simulations coupled with Free Energy Perturbation calculations to calculate the difference in the free energy of binding of BMAA to glutamate receptors compared to glutamate. Our findings indicate that one of the adducts of BMAA, β-carbamate adduct, has enhanced stability in the binding site of the AMPA receptor compared to the natural agonist, glutamate. In addition, the results from MD/FEP calculations reveal that glutamate and β-carbamate adduct of BMAA have comparable binding affinity in the AMPA glutamate receptor

Stimulation of GLP-1 secretion downstream of the ligand-gated ion channel TRPA1.

Stimulus-coupled incretin secretion from enteroendocrine cells plays a fundamental role in glucose homeostasis and could be targeted for the treatment of type 2 diabetes. Here, we investigated the expression and function of transient receptor potential (TRP) ion channels in enteroendocrine L cells producing GLP-1. By microarray and quantitative PCR analysis, we identified trpa1 as an L cell-enriched transcript in the small intestine. Calcium imaging of primary L cells and the model cell line GLUTag revealed responses triggered by the TRPA1 agonists allyl-isothiocyanate (mustard oil), carvacrol, and polyunsaturated fatty acids, which were blocked by TRPA1 antagonists. Electrophysiology in GLUTag cells showed that carvacrol induced a current with characteristics typical of TRPA1 and triggered the firing of action potentials. TRPA1 activation caused an increase in GLP-1 secretion from primary murine intestinal cultures and GLUTag cells, an effect that was abolished in cultures from trpa1(-/-) mice or by pharmacological TRPA1 inhibition. These findings present TRPA1 as a novel sensory mechanism in enteroendocrine L cells, coupled to the facilitation of GLP-1 release, which may be exploitable as a target for treating diabetes.This is an author-created, uncopyedited electronic version of an article accepted for publication in Diabetes. The American Diabetes Association (ADA), publisher of Diabetes, is not responsible for any errors or omissions in this version of the manuscript or any version derived from it by third parties. The definitive publisher-authenticated version will is available in Diabetes online at http://diabetes.diabetesjournals.org/content/early/2014/10/13/db14-0737.short?rss=1&patientinform-links=yes&legid=diabetes;db14-0737v1

Short-chain fatty acids stimulate glucagon-like peptide-1 secretion via the G-protein-coupled receptor FFAR2.

Interest in how the gut microbiome can influence the metabolic state of the host has recently heightened. One postulated link is bacterial fermentation of "indigestible" prebiotics to short-chain fatty acids (SCFAs), which in turn modulate the release of gut hormones controlling insulin release and appetite. We show here that SCFAs trigger secretion of the incretin hormone glucagon-like peptide (GLP)-1 from mixed colonic cultures in vitro. Quantitative PCR revealed enriched expression of the SCFA receptors ffar2 (grp43) and ffar3 (gpr41) in GLP-1-secreting L cells, and consistent with the reported coupling of GPR43 to Gq signaling pathways, SCFAs raised cytosolic Ca2+ in L cells in primary culture. Mice lacking ffar2 or ffar3 exhibited reduced SCFA-triggered GLP-1 secretion in vitro and in vivo and a parallel impairment of glucose tolerance. These results highlight SCFAs and their receptors as potential targets for the treatment of diabetes

Nifedipine Protects INS-1 β-Cell from High Glucose-Induced ER Stress and Apoptosis

Sustained high concentration of glucose has been verified toxic to β-cells. Glucose augments Ca2+-stimulated insulin release in pancreatic β-cells, but chronic high concentration of glucose could induce a sustained level of Ca2+ in β-cells, which leads to cell apoptosis. However, the mechanism of high glucose-induced β-cell apoptosis remains unclear. In this study, we use a calcium channel blocker, nifedipine, to investigate whether the inhibition of intracellular Ca2+ concentration could protect β-cells from chronic high glucose-induced apoptosis. It was found that in a concentration of 33.3 mM, chronic stimulation of glucose could induce INS-1 β-cells apoptosis at least through the endoplasmic reticulum stress pathway and 10 μM nifedipine inhibited Ca2+ release to protect β-cells from high glucose-induced endoplasmic reticulum stress and apoptosis. These results indicated that inhibition of Ca2+ over-accumulation might provide benefit to attenuate islet β-cell decompensation in a high glucose environment

Distinct Cytoplasmic and Nuclear Functions of the Stress Induced Protein DDIT3/CHOP/GADD153

DDIT3, also known as GADD153 or CHOP, encodes a basic leucine zipper transcription factor of the dimer forming C/EBP family. DDIT3 is known as a key regulator of cellular stress response, but its target genes and functions are not well characterized. Here, we applied a genome wide microarray based expression analysis to identify DDIT3 target genes and functions. By analyzing cells carrying tamoxifen inducible DDIT3 expression constructs we show distinct gene expression profiles for cells with cytoplasmic and nuclear localized DDIT3. Of 175 target genes identified only 3 were regulated by DDIT3 in both cellular localizations. More than two thirds of the genes were downregulated, supporting a role for DDIT3 as a dominant negative factor that could act by either cytoplasmic or nuclear sequestration of dimer forming transcription factor partners. Functional annotation of target genes showed cell migration, proliferation and apoptosis/survival as the most affected categories. Cytoplasmic DDIT3 affected more migration associated genes, while nuclear DDIT3 regulated more cell cycle controlling genes. Cell culture experiments confirmed that cytoplasmic DDIT3 inhibited migration, while nuclear DDIT3 caused a G1 cell cycle arrest. Promoters of target genes showed no common sequence motifs, reflecting that DDIT3 forms heterodimers with several alternative transcription factors that bind to different motifs. We conclude that expression of cytoplasmic DDIT3 regulated 94 genes. Nuclear translocation of DDIT3 regulated 81 additional genes linked to functions already affected by cytoplasmic DDIT3. Characterization of DDIT3 regulated functions helps understanding its role in stress response and involvement in cancer and degenerative disorders

Cross-Species Comparison of Genes Related to Nutrient Sensing Mechanisms Expressed along the Intestine

Introduction Intestinal chemosensory receptors and transporters are able to detect food-derived molecules and are involved in the modulation of gut hormone release. Gut hormones play an important role in the regulation of food intake and the control of gastrointestinal functioning. This mechanism is often referred to as “nutrient sensing”. Knowledge of the distribution of chemosensors along the intestinal tract is important to gain insight in nutrient detection and sensing, both pivotal processes for the regulation of food intake. However, most knowledge is derived from rodents, whereas studies in man and pig are limited, and cross-species comparisons are lacking. Aim To characterize and compare intestinal expression patterns of genes related to nutrient sensing in mice, pigs and humans. Methods Mucosal biopsy samples taken at six locations in human intestine (n = 40) were analyzed by qPCR. Intestinal scrapings from 14 locations in pigs (n = 6) and from 10 locations in mice (n = 4) were analyzed by qPCR and microarray, respectively. The gene expression of glucagon, cholecystokinin, peptide YY, glucagon-like peptide-1 receptor, taste receptor T1R3, sodium/glucose cotransporter, peptide transporter-1, GPR120, taste receptor T1R1, GPR119 and GPR93 was investigated. Partial least squares (PLS) modeling was used to compare the intestinal expression pattern between the three species. Results and conclusion The studied genes were found to display specific expression patterns along the intestinal tract. PLS analysis showed a high similarity between human, pig and mouse in the expression of genes related to nutrient sensing in the distal ileum, and between human and pig in the colon. The gene expression pattern was most deviating between the species in the proximal intestine. Our results give new insights in interspecies similarities and provide new leads for translational research and models aiming to modulate food intake processes in man

Susceptibility of Pancreatic Beta Cells to Fatty Acids Is Regulated by LXR/PPARα-Dependent Stearoyl-Coenzyme A Desaturase

Chronically elevated levels of fatty acids-FA can cause beta cell death in vitro. Beta cells vary in their individual susceptibility to FA-toxicity. Rat beta cells were previously shown to better resist FA-toxicity in conditions that increased triglyceride formation or mitochondrial and peroxisomal FA-oxidation, possibly reducing cytoplasmic levels of toxic FA-moieties. We now show that stearoyl-CoA desaturase-SCD is involved in this cytoprotective mechanism through its ability to transfer saturated FA into monounsaturated FA that are incorporated in lipids. In purified beta cells, SCD expression was induced by LXR- and PPARα-agonists, which were found to protect rat, mouse and human beta cells against palmitate toxicity. When their SCD was inhibited or silenced, the agonist-induced protection was also suppressed. A correlation between beta cell-SCD expression and susceptibility to palmitate was also found in beta cell preparations isolated from different rodent models. In mice with LXR-deletion (LXRβ-/- and LXRαβ-/-), beta cells presented a reduced SCD-expression as well as an increased susceptibility to palmitate-toxicity, which could not be counteracted by LXR or PPARα agonists. In Zucker fatty rats and in rats treated with the LXR-agonist TO1317, beta cells show an increased SCD-expression and lower palmitate-toxicity. In the normal rat beta cell population, the subpopulation with lower metabolic responsiveness to glucose exhibits a lower SCD1 expression and a higher susceptibility to palmitate toxicity. These data demonstrate that the beta cell susceptibility to saturated fatty acids can be reduced by stearoyl-coA desaturase, which upon stimulation by LXR and PPARα agonists favors their desaturation and subsequent incorporation in neutral lipids

ER stress in rodent islets of langerhans is concomitant with obesity and β-cell compensation but not with β-cell dysfunction and diabetes

Objective: The objective of this study was to determine whether ER stress correlates with β-cell dysfunction in obesity-associated diabetes. Methods: Quantitative RT-PCR and western blot analysis were used to investigate changes in the expression of markers of ER stress, the unfolded protein response (UPR) and β-cell function in islets isolated from (1) non-diabetic Zucker obese (ZO) and obese female Zucker diabetic fatty (fZDF) rats compared with their lean littermates and from (2) high-fat-diet-fed fZDF rats (HF-fZDF), to induce diabetes, compared with age-matched non-diabetic obese fZDF rats. Results: Markers of an adaptive ER stress/UPR and β-cell function are elevated in islets isolated from ZO and fZDF rats compared with their lean littermates. In islets isolated from HF-fZDF rats, there was no significant change in the expression of markers of ER stress compared with age matched, obese, non-diabetic fZDF rats. Conclusions: These results provide evidence that obesity-induced activation of the UPR is an adaptive response for increasing the ER folding capacity to meet the increased demand for insulin. As ER stress is not exacerbated in high-fat-diet-induced diabetes, we suggest that failure of the islet to mount an effective adaptive UPR in response to an additional increase in insulin demand, rather than chronic ER stress, may ultimately lead to β-cell failure and hence diabetes