Recent advances in mathematical modeling and statistical analysis of exocytosis in endocrine cells

open5noMost endocrine cells secrete hormones as a result of Ca(2+)-regulated exocytosis, i.e., fusion of the membranes of hormone-containing secretory granules with the cell membrane, which allows the hormone molecules to escape to the extracellular space. As in neurons, electrical activity and cell depolarization open voltage-sensitive Ca(2+) channels, and the resulting Ca(2+) influx elevate the intracellular Ca(2+) concentration, which in turn causes exocytosis. Whereas the main molecular components involved in exocytosis are increasingly well understood, quantitative understanding of the dynamical aspects of exocytosis is still lacking. Due to the nontrivial spatiotemporal Ca(2+) dynamics, which depends on the particular pattern of electrical activity as well as Ca(2+) channel kinetics, exocytosis is dependent on the spatial arrangement of Ca(2+) channels and secretory granules. For example, the creation of local Ca(2+) microdomains, where the Ca(2+) concentration reaches tens of µM, are believed to be important for triggering exocytosis. Spatiotemporal simulations of buffered Ca(2+) diffusion have provided important insight into the interplay between electrical activity, Ca(2+) channel kinetics, and the location of granules and Ca(2+) channels. By confronting simulations with statistical time-to-event (or survival) regression analysis of single granule exocytosis monitored with TIRF microscopy, a direct connection between location and rate of exocytosis can be obtained at the local, single-granule level. To get insight into whole-cell secretion, simplifications of the full spatiotemporal dynamics have shown to be highly helpful. Here, we provide an overview of recent approaches and results for quantitative analysis of Ca(2+) regulated exocytosis of hormone-containing granules.openPedersen, Morten Gram; Tagliavini, Alessia; Cortese, Giuliana; Riz, Michela; Montefusco, FrancescoPedersen, MORTEN GRAM; Tagliavini, Alessia; Cortese, Giuliana; Riz, Michela; Montefusco, Francesc

Intraspecies Transmission of BASE Induces Clinical Dullness and Amyotrophic Changes

The disease phenotype of bovine spongiform encephalopathy (BSE) and the molecular/ biological properties of its prion strain, including the host range and the characteristics of BSE-related disorders, have been extensively studied since its discovery in 1986. In recent years, systematic testing of the brains of cattle coming to slaughter resulted in the identification of at least two atypical forms of BSE. These emerging disorders are characterized by novel conformers of the bovine pathological prion protein (PrPTSE), named high-type (BSE-H) and low-type (BSE-L). We recently reported two Italian atypical cases with a PrPTSE type identical to BSE-L, pathologically characterized by PrP amyloid plaques and known as bovine amyloidotic spongiform encephalopathy (BASE). Several lines of evidence suggest that BASE is highly virulent and easily transmissible to a wide host range. Experimental transmission to transgenic mice overexpressing bovine PrP (Tgbov XV) suggested that BASE is caused by a prion strain distinct from the BSE isolate. In the present study, we experimentally infected Friesian and Alpine brown cattle with Italian BSE and BASE isolates via the intracerebral route. BASE-infected cattle developed amyotrophic changes accompanied by mental dullness. The molecular and neuropathological profiles, including PrP deposition pattern, closely matched those observed in the original cases. This study provides clear evidence of BASE as a distinct prion isolate and discloses a novel disease phenotype in cattle

Mathematical Modeling of Cellular Mechanisms in Endocrine Secretory Cells

The endocrine secretory system is a small collection of cells with the specific aim to act like a sensor and to directly or indirectly control the critical functions of different other target cells. Their specific role is to release into circulation different kinds of hormones. Some endocrine cells release hormones that maintain background levels of the cell metabolism and the chemical "steady state" of the blood. In particular, keeping glucose concentration in the blood within a limited target range of euglycemia [80-120 mg/dl] is a very important task for the endocrine system. Although the endocrine cells involved in the glucose regulatory mechanism are various, it is well known that the pancreatic beta-cells secreting the insulin hormone are the direct and main responsible to maintain euglycemia. Indeed, insulin is secreted because of an increased glucose blood concentration in order to induce other cells to metabolize glucose, bringing its blood concentration back under the physiological limited range. There are also other hormones involved in the regulation of the blood glucose level, such as the insulinotropic gut hormones, i.e., glucagon-like peptide-1 (GLP-1) secreted by intestinal L-cells scattered in the intestinal epithelium among enterocytes and other secretory cells. GLP-1, together with other hormones, is responsible for the incretin effect, i.e., it potentates glucose-induced insulin secretion, and is dependent on the presence of nutrients in the lumen of the small intestine, which is the reason why insulin response when glucose is orally ingested is greater than when glucose is intravenously administrated. Unfortunately, there is still not a complete understanding about the molecular mechanisms underlying the stimulus-secretion coupling in L-cells and about the mechanisms underlying disturbances in pulsatile insulin secretion even though they are early markers of diabetes. Other endocrine cells respond to specific input and release potent, long acting hormones. A subgroup of this kind of cells are endocrine cells of the pituitary gland (i.e., melanotrophs, lactotrophs, somatotrophs, thyrotrophs, corticotrophs, and gonadotrophs). They secrete a number of hormones in response to input from the hypothalamus. These hormones act on other endocrine glands and other tissues including the brain to regulate, e.g., growth, reproduction, behavior, temperature, and water intake. Because of the extreme importance of the endocrine system and the need of a deeper understanding of the stimulus-secretion coupling in these cells, a combination of experimental data and mathematical modeling will be exploited to better investigate the cellular mechanisms involved. All the endocrine excitable cells share common critical features to induce exocytosis: they depolarize after exposure to the stimuli, as a consequence calcium channels open letting the calcium enter into the cell; the increase in calcium concentration triggers the fusion of the vesicles often docked to the membrane releasing their hormone outside the cell. In this work, for three main representative categories of excitable endocrine cells (beta-cells, L-cells and pituitary cells), different cellular mechanisms, often associated to the secretion machinery, will be investigated, taking advantage of a combination of mathematical modeling and experimental data. Regarding the beta-cells, the presented work focuses on the effect on the intracellular calcium oscillations, responsible of exocytosis, of reactive oxygen (ROS) and nitrogen species (NOS), which are common products of the mitochondrial electronic chain. In particular, ROS/NOS have been shown to lead disturbances in insulin secretion and beta-cell damage. However, ROS/NOS have also been shown to exert a stimulating short-term effect on insulin release. In order to investigate this twofold effect and the cellular mechanisms implied, a previous mathematical model of beta-cell Ca2+ handling is adapted to MIN6 cells, a pancreatic beta-cell line, and used to interpret experimental results. Our experiment and model results suggest a presumed link between ROS/RNS and disturbed pulsatile insulin secretion. Specifically, moderate ROS/RNS levels act on Endoplasmic Reticulum Ca2+ handling mechanisms, whereas higher levels of ROS/RNS also target other components involved in creating oscillatory behaviour. The Thesis moves to pituitary cells, where we analyze the effect on excitation-secretion coupling generated by the two typical electrical behaviors of pituitary cells: continuous spiking and so-called pseudo-plateau bursting. Experimental data show that a greater amplitude of Ca2+ fluctuations is associated with the bursting pattern than with spiking. This observation leads to the hypothesis that bursting cells release more hormone than spiking cells. Unfortunately, testing this hypothesis and measuring the secreted hormone level is difficult due to the small size of the cells (<10 μm diameter) and the necessity of using cell populations, which may include both spikers and bursters. Therefore, we use computer simulation to explore this hypothesis. We develop a stochastic mathematical model of single Ca2+ channel activity dependent on experimental electrophysiological recordings. The stochastic Ca2+ channel gating is then used to simulate the associated buffered Ca2+ diffusion with the modeling program CalC (Calcium Calculator). We use the computed submembrane Ca2+ profiles at varying distances from the calcium channels to drive an exocytosis model to get the resulting hormone release. We consider scenarios where Ca2+ channels were either spatially discrete or located in clusters and vary the distance of the channels from the release sites. We find that bursting is always at least as effective as spiking at evoking hormone release, and is often considerably more effective, even when normalizing to Ca2+ influx. From calcium diffusion and secretion in pituitary cells, we move our interest to the glucose-sensing mechanism and hormone secretion in intestinal L-cells. There are two main mechanisms implicated in glucose-triggered secretion of GLP-1. One mechanism involves glucose metabolism and closure of ATP-sensitive K+ channels, the other mechanism exploits the electrogenic nature of SGLTs. Glucose uptake is mostly dominated by GLUT2 facilitative glucose transport, and therefore this transporter is mostly involved in the glucose metabolism pathway. In order to elucidate the role of these two possible pathways in a physiological setting difficult to obtained experimentally, we develop a first mathematical time-spatial model of L-cell. In particular we model the electrical activity, the main currents, and other different mechanisms: glucose transportation inside the cell thought transporters SGLT and GLUT2 as well as its diffusion inside the cell; calcium influx and diffusion associated to calcium current; sodium influx and diffusion; ATP production and consumption dependent on glucose concentration. We exploit the whole cell spatial model developed to understand and have a deeper insight into the glucose-sensing mechanism and GLP-1 secretion. The model takes into account the spatial distribution of the cell, and therefore simulations permit to investigate the role of two transporters without the loss of cell polarization. Results suggest that SGLT1 transporter has a crucial role in triggering cell depolarization, and thus allowing Ca2+ influx and GLP-1 exocytosis. On the other hand, glucose metabolism alone is not sufficient to trigger cell depolarization, but it likely affects exocytosis machinery, still playing a minor role in GLP-1 secretion. In conclusion, in this work, we evaluated and investigated the main mechanisms which lead to hormone exocytosis in endocrine cells by means of mathematical models. We examined a subgroup of representative endocrine cells, beta-cells, pituitary cells and L-cells, and focused on different aspects linked to specific stimulus-secretion coupling. For each type of cell, we developed or built on mathematical models, by mean of them we explained and interpreted results observed in experiments. Indeed, experiment might have limitations due to different factors, such as the need of specific inhibiting substances; limitation in technologies and the impossibility to isolate the specific mechanism of interest. In all these cases, mathematical modeling results to be an essential tool to study biological mechanisms.Il sistema secretorio endocrino è formato da un insieme di cellule, che hanno l' obiettivo di agire da sensore e direttamente, o indirettamente, di controllare delle funzioni critiche nelle cellule target. Nello specifico, il loro ruolo è quello di rilasciare nella circolazione sanguigna diversi tipi di ormoni. Alcune cellule endocrine rilasciano ormoni che mantengono il metabolismo della cellula ad un livello basale e la così detta ”steady-state” chimica del sangue. In particolare, un compito importante del sistema endocrino è mantenere la concentrazione di glucosio nel sangue nel range di euglicemia [80-120 mg/dl]. Sebbene le cellule endocrine coinvolte nel meccanismo di regolazione del glucosio nel sangue siano diverse, è risaputo che le beta-cellule pancreatiche che secernano l'ormone insulina sono le principali responsabili nel mantenere l'euglicemia. Infatti, l'insulina viene secreta dopo un aumento della concentrazione di glucosio nel sangue e il suo effetto è quello di indurre le altre cellule a metabolizzare il glucosio presente nel sangue, riportando la sua concentrazione sotto il valore limite fisiologico. Oltre all'insulina, ci sono anche altri ormoni che agiscono per regolare il livello di glucosio nel sangue, come gli ormoni insulinotropici dell'intestino, per esempio glucagon-like peptide-1 (GLP-1) secreto dalle L-cellule intestinali, situate nell'epitelio intestinale tra gli enterociti e altre cellule secretorie. GLP-1, insieme ad altri ormoni, è responsabile dell'effetto incretina, effetto che potenzia la secrezione di insulina indotta dal glucosio stimolata dai nutrienti nel lumen dell'intestino tenue, motivo per cui la risposta dell'insulina quando il glucosio è assunto oralmente risulta essere maggiore rispetto a quando è somministrato per via intravenosa. Sfortunatamente, si conosce ancora poco sia riguardo i meccanismi molecolari alla base dell'associazione stimolo-secrezione delle L-cellule e sia riguardo i disturbi nella secrezione dell'insulina pulsatile, pur essendo questi i primi segnali del diabete. Altre cellule endocrine invece rispondono a specifici input e rilasciano ormoni potenti a lunga durata d'azione. Un sottogruppo di questo tipo di cellule è l'insieme delle cellule endocrine della ghiandola pituitaria (somatotrope, lactotrope, tirotrope, gonadotrope, corticotrope). Queste cellule secernono ormoni quando stimolate dall'ipotalamo. Gli ormoni così secreti agiscono su altre ghiandole endocrine e altri tessuti, incluso il cervello, per regolare ad esempio la crescita, la riproduzione, il comportamento, la temperatura, e l'assorbimento di acqua. Data l'estrema importanza del sistema endocrino e il bisogno di capire più in profondità l'associazione stimolo-secrezione in queste cellule, in questa tesi verrà utilizzata una combinazione di dati sperimentali e modelli matematici proprio con l'obiettivo di studiare i meccanismi alla base di questo meccanismo. Tutte le cellule endocrine eccitabili condividono delle caratteristiche comuni per indurre l'esocitosi: si depolarizzano dopo l'esposizione allo stimolo, i canali di calcio voltaggio dipendenti si aprono, permettendo l'ingresso del calcio all'interno della cellula; l'aumento della concentrazione di calcio induce la fusione delle vescicole che sono ancorate alla membrana rilasciando il loro ormone all' esterno della cellula. In questa tesi verranno analizzati i meccanismi cellulari implicati nel processo di stimolazione della cellula associato all' esocitosi nelle tre categorie rappresentative di cellule endocrine eccitabili (beta-cellule pancreatiche, L-cellule intestinali e cellule pituitarie), utilizzando una combinazione di dati sperimentali e modelli matematici. Per quanto riguarda le beta-cellule, il lavoro presentato si focalizza sull'effetto delle sostanze ossidative sulle oscillazioni del calcio intracellulare, responsabile dell'esocitosi. Le sostanze ossidative e nitrogene, ROS e NOS, rispettivamente, sono prodotti cellulari che si formano durante la catena di trasporto degli elettroni nei mitocondri. In particolare, si è visto che ROS/NOS portano a disturbi nella secrezione di insulina e al danneggiamento delle beta-cellule. Tuttavia, è stata anche attribuita a queste sostanze un effetto a breve termine di stimolazione del rilascio di insulina. Per studiare questo duplice effetto e i meccanismi cellulari coinvolti, è stato adattato un modello matematico esistente relativo alle oscillazioni di calcio nelle beta-cellule alle cellule MIN6, una linea pancreatica di beta-cellule, ed è stato usato per interpretare i risultati sperimentali. I risultati sperimentai e del modello suggeriscono, presumibilmente, un collegamento tra ROS/NOS e i disturbi della secrezione di insulina pulsatile. Nello specifico, livelli moderati di NOS/ROS agiscono a livello di gestione del calcio del Reticolo Endoplasmatico, mentre livelli maggiori di ROS/NOS spesso hanno come target altre componenti che sono responsabili del comportamento oscillatorio. Il lavoro in seguito si sposta in direzione delle cellule pituitarie, per le quali analizziamo l'effetto di associazione stimolo-secrezione generato da due comportamenti tipici elettrici delle cellule pituitarie: spiking continuo e il così detto “pseudo-plateau” bursting. I dati sperimentali mostrano che al pattern del bursting è associata un'ampiezza maggiore delle fluttuazioni del Ca2+ rispetto alle fluttuazioni con spiking. Questa osservazione porta a formulare l'ipotesi che quando le cellule fanno bursting rilasciano una quantità maggiore di ormone. Sfortunatamente, testare questa ipotesi e misurare il livello di ormone secreto è difficile sia per le piccole dimensioni della cellula (dimetro <10 µm) che per la necessità di usare popolazione di cellule che può comprendere sia cellule che fanno bursting che cellule che fanno spiking. Per questo motivo, usiamo simulazioni per valutare questa ipotesi. I dati elettrofisiologici sperimentali dei due pattern misurati nella stessa cellula sono stati utilizzati come ingresso ad un modello matematico per l'attività stocastica del singolo canale di Ca2+. L'apertura stocastica del canale di calcio viene poi utilizzata per simulare la diffusione di Ca2+ associata utilizzando il programma CalC (Calcium Calculator). In seguito, i profili di Ca2+ ottenuti nella regione appena sotto la membrana a varie distanze dal canale di calcio vengono utilizzati per guidare un modello di esocitosi ed ottenere la quantità di ormone rilasciato corrispondente. Sono stati presi in considerazione sia uno scenario in cui i canali di Ca2+ sono distribuiti singolarmente tra la superficie della cellula, sia il caso in cui sono distribuiti suddivisi in cluster e variando la distanza dei canali dal sito di rilascio. Abbiamo trovato che bursting è sempre almeno tanto efficace ad indurre esocitosi quanto spiking e spesso è più efficace, anche quando viene normalizzato per la quantità di Ca2+ entrante. Dalla diffusione del calcio e la secrezione nelle cellule pituitarie, abbiamo spostato il nostro interesse al meccanismo di assorbimento del glucosio e secrezione ormonale nelle L-cellule intestinali. È stato sviluppato il primo modello spazio-temporale delle L-cellule, in particolare è stata modellata l'attività elettrica, le principali correnti e altri meccanismi quali il trasporto del glucosio all'interno della cellula attraverso i trasportatori SGLT1 e GLUT2 e la sua diffusione all'interno della cellula; il flusso di calcio associata alla corrente del calcio e diffusione; il flusso del Na+ e diffusione all'interno della cellula, e la produzione e l' utilizzo dell'ATP. Questo sistema di simulazione viene utilizzato per capire meglio i meccanismi delle L-cellule, focalizzandosi sul trasporto del glucosio all'interno della cellula sulla relazione tra glucosio e secrezione di GLP1. In conclusione, in questa tesi sono stati esaminati e studiati attraverso modelli matematici i principali meccanismi che portano all'esocitosi ormonale delle cellule endocrine. Un sottogruppo rappresentativo di cellule endocrine, beta-cellule, cellule pituitarie e L-cellule, è stato esaminato, ponendo l'attenzione sui diversi aspetti legati all'accoppiamento specifico stimolo-secrezione. Per ogni tipo di cellula, sono stati sviluppati o adattati dei modelli matematici che sono stati poi utilizzati per spiegare ed interpretare i risultati osservati sperimentalmente. Infatti, gli esperimenti possono avere dei limiti dovuti a diversi fattori come la necessità di dover utilizzare delle specifiche sostanze inibitorie o alla mancanza di una tecnologia appropriata, o all'impossibilità di isolare alcuni specifici meccanismi di interesse. È proprio in questi casi che i modelli matematici risultano essere uno strumento necessario e indispensabile per lo studio dei meccanismi biologici

Development and evaluation of pid controllers for glucose control in people with type 1 diabetes mellitus

The aim of this research is to develop and evaluate a novel, model-based PID control design method to achieve normo-glycemia with an artificial pancreas. A key objective is to utilize a simple, control-relevant model that can be personalized for individual subjects based only on clinical information that is readily available, such as total daily insulin (TDI). The proposed PID design method is based on the Internal Model Control (IMC) approach and a simple dynamic model that has a single adjustable parameter that is personalized based on the subject’s TDI. The proposed controller design method was evaluated in an in silico study using the UVa/Padova metabolic simulator. Ten simulated subjects were used to determine a conservative value of the single IMC design parameter. This value provided good post-prandial responses and a reasonable degree of robustness for changes of+/- 50% in the insulin sensitivity. Then ten additional subjects were simulated in a validation study that included three meals (50, 40 and 50 g CHO) during a 30 hours. The PID controllers based on the personalized models performed better than controllers based on a fixed model. For the validation study, the average amount of time that the glucose concentration was in the desired range (70-180 mg/dL) was 88% for the personalized models and 83% for the fixed models. Similarly, the average values for the 180-250 mg/dL range were 10% and 16% for the personalized and fixed models, respectively. Neither design method resulted in hypoglycemia (<60 mg/dL). Simulation studies have demonstrated that the proposed controller design method based on personalized models is practical and superior to controllers based on a fixed mode

Spatiotemporal Modeling of Triggering and Amplifying Pathways in GLP-1 Secreting Intestinal L Cells

none2nononeTagliavini, Alessia; Pedersen, Morten GramTagliavini, Alessia; Pedersen, MORTEN GRA

Reactive oxygen and nitrogen species disturb Ca2+ oscillations in insulin-secreting MIN6 \u3b2-cells

Disturbances in pulsatile insulin secretion and Ca2+ oscillations in pancreatic -cells are early markers of diabetes, but the underlying mechanisms are still incompletely understood. Reactive oxygen/nitrogen species (ROS/RNS) are implicated in reduced -cell function, and ROS/RNS target several Ca2+ pumps and channels. Thus, we hypothesized that ROS/RNS could disturb Ca2+ oscillations and downstream insulin pulsatility. We show that ROS/RNS production by photoactivation of aluminum phthalocyanine chloride (AlClPc) abolish or accelerate Ca2+ oscillations in the MIN6 -cell line, depending on the amount of ROS/RNS. Application of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor thapsigargin modifies the Ca2+ response to high concentrations of ROS/RNS. Further, thapsigargin produces effects that resemble those elicited by moderate ROS/RNS production. These results indicate that ROS/RNS interfere with endoplasmic reticulum Ca2+ handling. This idea is supported by theoretical studies using a mathematical model of Ca2+ handling adapted to MIN6 cells. Our results suggest a putative link between ROS/RNS and disturbed pulsatile insulin secretion

Calcium signaling and secretory granule pool dynamics underlie biphasic insulin secretion and its amplification by glucose: experiments and modeling.

Glucose-stimulated insulin secretion from pancreatic β-cells is controlled by a triggering pathway that culminates in calcium influx and regulated exocytosis of secretory granules, and by a less understood amplifying pathway that augments calcium-induced exocytosis. In response to an abrupt increase in glucose concentration, insulin secretion exhibits a first peak followed by a lower sustained second phase. This biphasic secretion pattern is disturbed in diabetes. It has been attributed to depletion and subsequent refilling of a readily releasable pool of granules or to the phasic cytosolic calcium dynamics induced by glucose. Here, we apply mathematical modeling to experimental data from mouse islets to investigate how calcium and granule pool dynamics interact to control dynamic insulin secretion. Experimental calcium traces are used as inputs in three increasingly complex models of pool dynamics, which are fitted to insulin secretory patterns obtained using a set of protocols of glucose and tolbutamide stimulation. New calcium and secretion data for so-called staircase protocols, in which the glucose concentration is progressively increased, are presented. These data can be reproduced without assuming any heterogeneity in the model, in contrast to previous modeling, because of nontrivial calcium dynamics. We find that amplification by glucose can be explained by increased mobilization and priming of granules. Overall, our results indicate that calcium dynamics contribute substantially to shaping insulin secretion kinetics, which implies that better insight into the events creating phasic calcium changes in human β-cells is needed to understand the cellular mechanisms that disturb biphasic insulin secretion in diabetes

Concise Whole-Cell Modeling of BKCa-CaV Activity Controlled by Local Coupling and Stoichiometry

none4nononeMontefusco, Francesco; Tagliavini, Alessia; Ferrante, Marco; Pedersen, Morten GramMontefusco, Francesco; Tagliavini, Alessia; Ferrante, Marco; Pedersen, Morten Gra