Control of pancreatic β-cell mass and function by gluco-incretin hormones : identification of novel regulatory mechanisms for the treatment of diabetes

Summary : Control of pancreatic ß-cell mass and function by gluco-incretin hormones: Identification of novel regulatory mechanisms for the treatment of diabetes The ß-cells of islets of Langerhans secrete insulin to reduce hyperglycemia. The number of pancreatic islet ß-cells and their capacity to secrete insulin is modulated in normal physiological conditions to respond to the metabolic demand of the organism. A failure of the endocrine pancreas to maintain an adequate insulin secretory capacity due to a reduced ß-cell number and function underlies the pathogenesis of both type 1 and type 2 diabetes. The molecular mechanisms controlling the glucose competence of mature ß-cells, i.e., the magnitude of their insulin secretion response to glucose, ß-cell replication, their differentiation from precursor cells and protection against apoptosis are poorly understood. To investigate these mechanisms, we studied the effects on ß-cells of the gluco-incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) which are secreted by intestinal endocrine cells after food intake. Besides acutely potentiating glucose-stimulated insulin secretion, these hormones induce ß-cell differentiation from precursor cells, stimulate mature ß-cell replication, and protect them against apoptosis. Therefore, understanding the molecular basis for gluco-incretin action may lead to the uncovering of novel ß-cell regulatory events with potential application for the treatment or prevention of diabetes. Islets from mice with inactivation of both GIP and GLP-1 receptor genes (dK0) present a defect in glucose-induced insulin secretion and are more sensitive than control islets to cytokine-induced apoptosis. To search for regulatory genes, that may control both glucose competence and protection against apoptosis, we performed comparative transcriptomic analysis of islets from control and dK0 mice. We found a strong down-regulation of the IGF1 Rexpression in dK0 islets. We demonstrated in both a mouse insulin-secreting cell line and primary islets, that GLP-1 stimulated IGF-1R expression and signaling. Importantly, GLP-1induced IGF-1R-dependent Akt phosphorylation required active secretion, indicating the presence of an autocrine activation mechanism. We further showed that activation of IGF-1R signaling was dependent on the secretion of IGF-2 and IGF-2 expression was regulated by nutrients. Finally, we demonstrated that the IGF-Z/IGF-1R autocrine loop was required for GLP-1 i) to protect ß-cells against cytokine-induced apoptosis, ii) to enhance their glucose competence and iii) to increase ß-cell proliferation. Résumé : Contrôle de la masse des cellules ß pancréatiques et de leur fonction par les hormones glucoincrétines: Identification de nouveaux mécanismes régulateurs pour le traitement du diabète Les cellules ß des îlots de Langerhans sécrètent l'insuline pour diminuer l'hyperglycémie. Le nombre de cellules ß et leur capacité à sécréter l'insuline sont modulés dans les conditions physiologiques normales pour répondre à la demande métabolique de l'organisme. Un échec du pancréas endocrine à maintenir sa capacité sécrétoire d'insuline dû à une diminution du nombre et de la fonction des cellules ß conduit au diabète de type 1 et de type 2. Les mécanismes moléculaires contrôlant la compétence au glucose des cellules ß matures, tels que, l'augmentation de la sécrétion d'insuline en réponse au glucose, la réplication des cellules ß, leur différentiation à partir de cellules précurseurs et la protection contre l'apoptose sont encore peu connus. Afin d'examiner ces mécanismes, nous avons étudié les effets sur les cellules ß des hormones gluco-incrétines, glucose-dépendent insulinotropic polypeptide (G1P) et glucagon-like peptide-1 (GLP-1) qui sont sécrétées par les cellules endocrines de l'intestin après la prise alimentaire. En plus de potentialiser la sécrétion d'insuline induite par le glucose, ces hormones induisent la différentiation de cellules ß à partir de cellules précurseurs, stimulent leur prolifération et les protègent contre l'apoptose. Par conséquent, comprendre les mécanismes d'action des gluco-incrétines permettrait de découvrir de nouveaux processus régulant les cellules ß avec d'éventuelles applications dans le traitement ou la prévention du diabète. Les îlots de souris ayant une double inactivation des gènes pour les récepteurs du GIP et du GLP-1 (dK0) présentent un défaut de sécrétion d'insuline stimulée par le glucose et une sensibilité accrue à l'apoptose induite par les cytokines. Afin de déterminer les gènes régulés, qui pourraient contrôler à la fois la compétence au glucose et la protection contre l'apoptose, nous avons effectué une analyse comparative transcriptomique sur des îlots de souris contrôles et dKO. Nous avons constaté une forte diminution de l'expression d'IGF-1R dans les îlots dKO. Nous avons démontré, à la fois dans une lignée cellulaire murine sécrétant l'insuline et dans îlots primaires, que le GLP-1 stimulait l'expression d'IGF-1R et sa voie de signalisation. Par ailleurs, la phosphorylation d'Akt dépendante d'IGF1-R induite parle GLP-1 nécessite une sécrétion active, indiquant la présence d'un mécanisme d'activation autocrine. Nous avons ensuite montré que l'activation de la voie de signalisation d'IGF-1R était dépendante de la sécrétion d'IGF-2, dont l'expression est régulée par les nutriments. Finalement, nous avons démontré que la boucle autocrine IGF-2/IGF-1R est nécessaire pour le GLP-1 i) pour protéger les cellules ß contre l'apoptose induite par les cytokines, ii) pour améliorer la compétence au glucose et iii) pour augmenter la prolifération des cellules ß. Résumé tout public : Contrôle de la masse des cellules ß pancréatiques et de leur fonction par les hormones gluco-incrétines: Identification de nouveaux mécanismes régulateurs pour le traitement du diabète Chez les mammifères, la concentration de glucose sanguine (glycémie) est régulée et maintenue à une valeur relativement constante d'environ 5 mM. Cette régulation est principalement contrôlée par 2 hormones produites par les îlots pancréatiques de Langerhans: l'insuline sécrétée par les cellules ß et le glucagon sécrété par les cellules a. A la suite d'un repas, l'augmentation de la glycémie entraîne la sécrétion d'insuline ce qui permet le stockage du glucose dans le foie, les muscles et le tissu adipeux afin de diminuer le taux de glucose circulant. Lors d'un jeûne, la diminution de la glycémie permet la sécrétion de glucagon favorisant alors la production de glucose par le foie, normalisant ainsi la glycémie. Le nombre de cellules ß et leur capacité sécrétoire s'adaptent aux variations de la demande métabolique pour assurer une normoglycémie. Une destruction complète ou partielle des cellules ß conduit respectivement au diabète de type 1 et de type 2. Bien que l'augmentation de la glycémie soit le facteur stimulant de la sécrétion d'insuline, des hormones gluco-incrétines, principalement le GLP-1 (glucagon-like peptide-1) et le GIP (glucose-dependent insulinotropic polypeptide) sont libérées par l'intestin en réponse aux nutriments (glucose, acides gras) et agissent au niveau des cellules ß, potentialisant la sécrétion d'insuline induite par le glucose, stimulant leur prolifération, induisant la différentiation de cellules précurseurs en cellules ß matures et les protègent contre la mort cellulaire (apoptose). Afin d'étudier plus en détail ces mécanismes, nous avons généré des souris déficientes pour les récepteurs du GIP et du GLP-l. Les îlots pancréatiques de ces souris présentent un défaut de sécrétion d'insuline stimulée par le glucose et une sensibilité accrue à l'apoptose par rapport aux îlots de souris contrôles. Nous avons donc cherché les gènes régulés pas ces hormones contrôlant la sécrétion d'insuline et la protection contre l'apoptose. Nous avons constaté une forte diminution de l'expression du récepteur à l'IGF-1 (IGF-1R) dans les îlots de souris déficientes pour les récepteurs des gluco-incrétines. Nous avons démontré dans un model de cellules ß en culture et d'îlots que le GLP-1 augmentait l'expression d'IGF-1R et la sécrétion de son ligand (IGF-2) permettant l'activation de la voie de signalisation. Finalement, nous avons montré que l'activation de la boucle IGF-2/IGF-1R induite par le GLP-1 était nécessaire pour la protection contre l'apoptose, l'augmentation de la sécrétion et la prolifération des cellules ß.

Confirmation of natural gas explosion from methane quantification by headspace gas chromatography-mass spectrometry (HS-GC-MS) in postmortem samples: a case report.

A new analytical approach for measuring methane in tissues is presented. For the first time, the use of in situ-produced, stably labelled CDH(3) provides a reliable and precise methane quantification. This method was applied to postmortem samples obtained from two victims to help determine the explosion origin. There was evidence of methane in the adipose tissue (82 nmol/g) and cardiac blood (1.3 nmol/g) of one victim, which corresponded to a lethal methane outburst. These results are discussed in the context of the available literature to define an analysis protocol for application in the event of a gas explosion.

Response-to-comments about: "Is it really the method for carbon dioxide determination in human postmortem cardiac gas samples using Headspace-Gas Chromatography-Mass Spectrometry valid?" from T. Saffaj and B. Ihssane

Saffaj et al. recently criticized our method of monitoring carbon dioxide in human postmortem cardiac gas samples using Headspace-Gas Chromatography-Mass Spectrometry. According to the authors, their demonstration, based on the latest SFSTP guidelines (established after 2007 [1,2]) fitted for the validation of drug monitoring bioanalytical methods, has put in evidence potential errors. However, our validation approach was built using SFSTP guidelines established before 2007 [3-6]. We justify the use of these guidelines because of the post-mortem context of the study (and not clinical) and the gaseous state of the sample (and not solid or liquid). Using these guidelines, our validation remains correct.

A hierarchical approach to model parameter optimization for developmental systems.

In the context of Systems Biology, computer simulations of gene regulatory networks provide a powerful tool to validate hypotheses and to explore possible system behaviors. Nevertheless, modeling a system poses some challenges of its own: especially the step of model calibration is often difficult due to insufficient data. For example when considering developmental systems, mostly qualitative data describing the developmental trajectory is available while common calibration techniques rely on high-resolution quantitative data. Focusing on the calibration of differential equation models for developmental systems, this study investigates different approaches to utilize the available data to overcome these difficulties. More specifically, the fact that developmental processes are hierarchically organized is exploited to increase convergence rates of the calibration process as well as to save computation time. Using a gene regulatory network model for stem cell homeostasis in Arabidopsis thaliana the performance of the different investigated approaches is evaluated, documenting considerable gains provided by the proposed hierarchical approach.

Urinary analysis of four testosterone metabolites and pregnanediol by gas chromatography-combustion-isotope ratio mass spectrometry after oral administrations of testosterone.

The most frequently used method to demonstrate testosterone abuse is the determination of the testosterone and epitestosterone concentration ratio (T/E ratio) in urine. Nevertheless, it is known that factors other than testosterone administration may increase the T/E ratio. In the last years, the determination of the carbon isotope ratio has proven to be the most promising method to help discriminate between naturally elevated T/E ratios and those reflecting T use. In this paper, an excretion study following oral administration of 40 mg testosterone undecanoate initially and 13 h later is presented. Four testosterone metabolites (androsterone, etiocholanolone, 5 alpha-androstanediol, and 5 beta-androstanediol) together with an endogenous reference (5 beta-pregnanediol) were extracted from the urines and the delta(13)C/(12)C ratio of each compound was analyzed by gas chromatography-combustion-isotope ratio mass spectrometry. The results show similar maximum delta(13)C-value variations (parts per thousand difference of delta(13)C/(12)C ratio from the isotope ratio standard) for the T metabolites and concomitant changes of the T/E ratios after administration of the first and the second dose of T. Whereas the T/E ratios as well as the androsterone, etiocholanolone and 5 alpha-androstanediol delta(13)C-values returned to the baseline 15 h after the second T administration, a decrease of the 5 beta-androstanediol delta-values could be detected for over 40 h. This suggests that measurements of 5 beta-androstanediol delta-values allow the detection of a testosterone ingestion over a longer post-administration period than other T metabolites delta(13)C-values or than the usual T/E ratio approach.

Generic approach for the sensitive absolute quantification of large undigested peptides in plasma using a particular liquid chromatography-mass spectrometry setup.

A generic LC-MS approach for the absolute quantification of undigested peptides in plasma at mid-picomolar levels is described. Nine human peptides namely, brain natriuretic peptide (BNP), substance P (SubP), parathyroid hormone 1-34 (PTH), C-peptide, orexines A and B (Orex-A and -B), oxytocin (Oxy), gonadoliberin-1 (gonadothropin releasing-hormone or luteinizing hormone-releasing hormone, LHRH) and α-melanotropin (α-MSH) were targeted. Plasma samples were extracted via a 2-step procedure: protein precipitation using 1vol of acetonitrile followed by ultrafiltration of supernatants on membranes with a MW cut-off of 30 kDa. By applying a specific LC-MS setup, large volumes of filtrates (e.g., 2×750 μL) were injected and the peptides were trapped on a 1mm i.d.×10 mm length C8 column using a 10× on-line dilution. Then, the peptides were back-flushed and a second on-line dilution (2×) was applied during the transfer step. The refocalized peptides were resolved on a 0.3mm i.d. C18 analytical column. Extraction recovery, matrix effect and limits of detection were evaluated. Our comprehensive protocol demonstrates a simple and efficient sample preparation procedure followed by the analysis of peptides with limits of detection in the mid-picomolar range. This generic approach can be applied for the determination of most therapeutic peptides and possibly for endogenous peptides with latest state-of-the-art instruments.

NCoR1 is a conserved physiological modulator of muscle mass and oxidative function.

Transcriptional coregulators control the activity of many transcription factors and are thought to have wide-ranging effects on gene expression patterns. We show here that muscle-specific loss of nuclear receptor corepressor 1 (NCoR1) in mice leads to enhanced exercise endurance due to an increase of both muscle mass and of mitochondrial number and activity. The activation of selected transcription factors that control muscle function, such as MEF2, PPARβ/δ, and ERRs, underpins these phenotypic alterations. NCoR1 levels are decreased in conditions that require fat oxidation, resetting transcriptional programs to boost oxidative metabolism. Knockdown of gei-8, the sole C. elegans NCoR homolog, also robustly increased muscle mitochondria and respiration, suggesting conservation of NCoR1 function. Collectively, our data suggest that NCoR1 plays an adaptive role in muscle physiology and that interference with NCoR1 action could be used to improve muscle function.

Quantification of typical antipsychotics in human plasma by ultra-high performance liquid chromatography tandem mass spectrometry for therapeutic drug monitoring.

A selective and sensitive method was developed for the simultaneous quantification of seven typical antipsychotic drugs (cis-chlorprothixene, flupentixol, haloperidol, levomepromazine, pipamperone, promazine and zuclopenthixol) in human plasma. Ultra-high performance liquid chromatography (UHPLC) was used for complete separation of the compounds in less than 4.5min on an Acquity UPLC BEH C18 column (2.1mm×50mm; 1.7μm), with a gradient elution of ammonium formate buffer pH 4.0 and acetonitrile at a flow rate of 400μl/min. Detection was performed on a tandem quadrupole mass spectrometer (MS/MS) equipped with an electrospray ionization interface. A simple protein precipitation procedure with acetonitrile was used for sample preparation. Thanks to the use of stable isotope-labeled internal standards for all analytes, internal standard-normalized matrix effects were in the range of 92-108%. The method was fully validated to cover large concentration ranges of 0.2-90ng/ml for haloperidol, 0.5-90ng/ml for flupentixol, 1-450ng/ml for levomepromazine, promazine and zuclopenthixol and 2-900ng/ml for cis-chlorprothixene and pipamperone. Trueness (89.1-114.8%), repeatability (1.8-9.9%), intermediate precision (1.9-16.3%) and accuracy profiles (<30%) were in accordance with the latest international recommendations. The method was successfully used in our laboratory for routine quantification of more than 500 patient plasma samples for therapeutic drug monitoring. To the best of our knowledge, this is the first UHPLC-MS/MS method for the quantification of the studied drugs with a sample preparation based on protein precipitation.

Probabilistic evidential assessment of gunshot residue particle evidence (Part II) : Bayesian parameter estimation for experimental count data.

Part I of this series of articles focused on the construction of graphical probabilistic inference procedures, at various levels of detail, for assessing the evidential value of gunshot residue (GSR) particle evidence. The proposed models - in the form of Bayesian networks - address the issues of background presence of GSR particles, analytical performance (i.e., the efficiency of evidence searching and analysis procedures) and contamination. The use and practical implementation of Bayesian networks for case pre-assessment is also discussed. This paper, Part II, concentrates on Bayesian parameter estimation. This topic complements Part I in that it offers means for producing estimates useable for the numerical specification of the proposed probabilistic graphical models. Bayesian estimation procedures are given a primary focus of attention because they allow the scientist to combine (his/her) prior knowledge about the problem of interest with newly acquired experimental data. The present paper also considers further topics such as the sensitivity of the likelihood ratio due to uncertainty in parameters and the study of likelihood ratio values obtained for members of particular populations (e.g., individuals with or without exposure to GSR).

Genetic variation at CHRNA5-CHRNA3-CHRNB4 interacts with smoking status to influence body mass index.

BACKGROUND: Cigarette smoking is associated with lower body mass index (BMI), and a commonly cited reason for unwillingness to quit smoking is a concern about weight gain. Common variation in the CHRNA5-CHRNA3-CHRNB4 gene region (chromosome 15q25) is robustly associated with smoking quantity in smokers, but its association with BMI is unknown. We hypothesized that genotype would accurately reflect smoking exposure and that, if smoking were causally related to weight, it would be associated with BMI in smokers, but not in never smokers. METHODS: We stratified nine European study samples by smoking status and, in each stratum, analysed the association between genotype of the 15q25 SNP, rs1051730, and BMI. We meta-analysed the results (n = 24 198) and then tested for a genotype × smoking status interaction. RESULTS: There was no evidence of association between BMI and genotype in the never smokers {difference per T-allele: 0.05 kg/m(2) [95% confidence interval (95% CI): -0.05 to 0.18]; P = 0.25}. However, in ever smokers, each additional smoking-related T-allele was associated with a 0.23 kg/m(2) (95% CI: 0.13-0.31) lower BMI (P = 8 × 10(-6)). The effect size was larger in current [0.33 kg/m(2) lower BMI per T-allele (95% CI: 0.18-0.48); P = 6 × 10(-5)], than in former smokers [0.16 kg/m(2) (95% CI: 0.03-0.29); P = 0.01]. There was strong evidence of genotype × smoking interaction (P = 0.0001). CONCLUSIONS: Smoking status modifies the association between the 15q25 variant and BMI, which strengthens evidence that smoking exposure is causally associated with reduced BMI. Smoking cessation initiatives might be more successful if they include support to maintain a healthy BMI.

S-system parameter estimation for noisy metabolic profiles using newton-flow analysis.

Biochemical systems are commonly modelled by systems of ordinary differential equations (ODEs). A particular class of such models called S-systems have recently gained popularity in biochemical system modelling. The parameters of an S-system are usually estimated from time-course profiles. However, finding these estimates is a difficult computational problem. Moreover, although several methods have been recently proposed to solve this problem for ideal profiles, relatively little progress has been reported for noisy profiles. We describe a special feature of a Newton-flow optimisation problem associated with S-system parameter estimation. This enables us to significantly reduce the search space, and also lends itself to parameter estimation for noisy data. We illustrate the applicability of our method by applying it to noisy time-course data synthetically produced from previously published 4- and 30-dimensional S-systems. In addition, we propose an extension of our method that allows the detection of network topologies for small S-systems. We introduce a new method for estimating S-system parameters from time-course profiles. We show that the performance of this method compares favorably with competing methods for ideal profiles, and that it also allows the determination of parameters for noisy profiles.

The differentiation of fibre- and drug type Cannabis seedlings by gas chromatography/mass spectrometry and chemometric tools

Cannabis cultivation in order to produce drugs is forbidden in Switzerland. Thus, law enforcement authorities regularly ask forensic laboratories to determinate cannabis plant's chemotype from seized material in order to ascertain that the plantation is legal or not. As required by the EU official analysis protocol the THC rate of cannabis is measured from the flowers at maturity. When laboratories are confronted to seedlings, they have to lead the plant to maturity, meaning a time consuming and costly procedure. This study investigated the discrimination of fibre type from drug type Cannabis seedlings by analysing the compounds found in their leaves and using chemometrics tools. 11 legal varieties allowed by the Swiss Federal Office for Agriculture and 13 illegal ones were greenhouse grown and analysed using a gas chromatograph interfaced with a mass spectrometer. Compounds that show high discrimination capabilities in the seedlings have been identified and a support vector machines (SVMs) analysis was used to classify the cannabis samples. The overall set of samples shows a classification rate above 99% with false positive rates less than 2%. This model allows then discrimination between fibre and drug type Cannabis at an early stage of growth. Therefore it is not necessary to wait plants' maturity to quantify their amount of THC in order to determine their chemotype. This procedure could be used for the control of legal (fibre type) and illegal (drug type) Cannabis production.