Design Principles of Pancreatic Islets: Glucose-dependent Coordination of Hormone Pulses

Pancreatic islets are functional units involved in glucose homeostasis. The multicellular system comprises three main cell types; $\beta$ and $\alpha$ cells reciprocally decrease and increase blood glucose by producing insulin and glucagon pulses, while the role of $\delta$ cells is less clear. Although their spatial organization and the paracrine/autocrine interactions between them have been extensively studied, the functional implications of the design principles are still lacking. In this study, we formulated a mathematical model that integrates the pulsatility of hormone secretion and the interactions and organization of islet cells and examined the effects of different cellular compositions and organizations in mouse and human islets. A common feature of both species was that islet cells produced synchronous hormone pulses under low- and high- glucose conditions, while they produced asynchronous hormone pulses under normal glucose conditions. However, the synchronous coordination of insulin and glucagon pulses at low glucose was more pronounced in human islets that had more $\alpha$ cells. When $\beta$ cells were selectively removed to mimic diabetic conditions, the anti-synchronicity of insulin and glucagon pulses was deteriorated at high glucose, but it could be partially recovered when the re-aggregation of remaining cells was considered. Finally, the third cell type, $\delta$ cells, which introduced additional complexity in the multicellular system, prevented the excessive synchronization of hormone pulses. Our computational study suggests that controllable synchronization is a design principle of pancreatic islets.Comment: 24 pages, 7 figure

Design Principles of Pancreatic Islets: Glucose-Dependent Coordination of Hormone Pulses

Pancreatic islets are functional units involved in glucose homeostasis. The multicellular system comprises three main cell types; β and α cells reciprocally decrease and increase blood glucose by producing insulin and glucagon pulses, while the role of δ cells is less clear. Although their spatial organization and the paracrine/autocrine interactions between them have been extensively studied, the functional implications of the design principles are still lacking. In this study, we formulated a mathematical model that integrates the pulsatility of hormone secretion and the interactions and organization of islet cells and examined the effects of different cellular compositions and organizations in mouse and human islets. A common feature of both species was that islet cells produced synchronous hormone pulses under low- and high-glucose conditions, while they produced asynchronous hormone pulses under normal glucose conditions. However, the synchronous coordination of insulin and glucagon pulses at low glucose was more pronounced in human islets that had more α cells. When β cells were selectively removed to mimic diabetic conditions, the anti-synchronicity of insulin and glucagon pulses was deteriorated at high glucose, but it could be partially recovered when the re-aggregation of remaining cells was considered. Finally, the third cell type, δ cells, which introduced additional complexity in the multicellular system, prevented the excessive synchronization of hormone pulses. Our computational study suggests that controllable synchronization is a design principle of pancreatic islets

Beta Cell Hubs Dictate Pancreatic Islet Responses to Glucose

N.R.J. was supported by a Diabetes UK RW and JM Collins Studentship (12/0004601). J.B. was supported by a European Foundation for the Study of Diabetes (EFSD) Albert Renold Young Scientist Fellowship and a Studienstiftung des deutschen Volkes PhD Studentship. D.T. was supported by an Advanced Grant from the European Research Commission (268795). G.A.R. was supported by Wellcome Trust Senior Investigator (WT098424AIA) and Royal Society Wolfson Research Merit Awards, and by MRC Programme (MR/J0003042/1), Biological and Biotechnology Research Council (BB/J015873/1), and Diabetes UK Project (11/0004210) grants. G.A.R. and M.W. acknowledge COST Action TD1304 Zinc-Net. D.J.H. was supported by Diabetes UK R.D. Lawrence (12/0004431), EFSD/Novo Nordisk Rising Star and Birmingham Fellowships, a Wellcome Trust Institutional Support Award, and an MRC Project Grant (MR/N00275X/1) with G.A.R. D.J.H and G.A.R. were supported by Imperial Confidence in Concept (ICiC) Grants. J.F. was supported by an MRC Programme grant (MR/L02036X/1). L.P. provided human islets through collaboration with the Diabetes Research Institute, IRCCS San Raffaele Scientific Institute (Milan), within the European islet distribution program for basic research supported by JDRF (1-RSC-2014-90-I-X). P.M. and M.B. were supported by the Innovative Medicine Initiative Joint Undertaking under grant agreement no. 155005 (IMIDIA), resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies in kind contribution, and by the Italian Ministry of University and Research (PRIN 2010-2012). D.B. and E.B. provided human islets through the European Consortium for Islet Transplantation sponsored by JDRF (1-RSC-2014-100-I-X)

Are physiological oscillations 'physiological'?

Despite widespread and striking examples of physiological oscillations, their functional role is often unclear. Even glycolysis, the paradigm example of oscillatory biochemistry, has seen questions about its oscillatory function. Here, we take a systems approach to summarize evidence that oscillations play critical physiological roles. Oscillatory behavior enables systems to avoid desensitization, to avoid chronically high and therefore toxic levels of chemicals, and to become more resistant to noise. Oscillation also enables complex physiological systems to reconcile incompatible conditions such as oxidation and reduction, by cycling between them, and to synchronize the oscillations of many small units into one large effect. In pancreatic beta cells, glycolytic oscillations are in synchrony with calcium and mitochondrial oscillations to drive pulsatile insulin release, which is pivotal for the liver to regulate blood glucose dynamics. In addition, oscillation can keep biological time, essential for embryonic development in promoting cell diversity and pattern formation. The functional importance of oscillatory processes requires a rethinking of the traditional doctrine of homeostasis, holding that physiological quantities are maintained at constant equilibrium values, a view that has largely failed us in the clinic. A more dynamic approach will enable us to view health and disease through a new light and initiate a paradigm shift in treating diseases, including depression and cancer. This modern synthesis also takes a deeper look into the mechanisms that create, sustain and abolish oscillatory processes, which requires the language of nonlinear dynamics, well beyond the linearization techniques of equilibrium control theory

New Understanding of β-Cell Heterogeneity and In Situ Islet Function

Insulin-secreting β-cells are heterogeneous in their regulation of hormone release. While long known, recent technological advances and new markers have allowed the identification of novel subpopulations, improving our understanding of the molecular basis for heterogeneity. This includes specific subpopulations with distinct functional characteristics, developmental programs, abilities to proliferate in response to metabolic or developmental cues, and resistance to immune-mediated damage. Importantly, these subpopulations change in disease or aging, including in human disease. Although discovering new β-cell subpopulations has substantially advanced our understanding of islet biology, a point of caution is that these characteristics have often necessarily been identified in single β-cells dissociated from the islet. β-Cells in the islet show extensive communication with each other via gap junctions and with other cell types via diffusible chemical messengers. As such, how these different subpopulations contribute to in situ islet function, including during plasticity, is not well understood. We will discuss recent findings revealing functional β-cell subpopulations in the intact islet, the underlying basis for these identified subpopulations, and how these subpopulations may influence in situ islet function. Furthermore, we will discuss the outlook for emerging technologies to gain further insight into the role of subpopulations in in situ islet function.</jats:p

Molecular Mechanisms Orchestrating the Dynamics of Secretory Vesicle Pools.

The secretion of chemical messengers via Ca2+-dependent exocytosis of vesicles is fundamental to a wide-range of physiological events. Rab GTPases and SNARE proteins govern the temporal and spatial precision of transmitter release. Yet, little is known about their role in specifying the size and filling kinetics of functionally defined vesicle pools, which impact the strength and efficiency of exocytosis. We first sought to delineate the distinct vs. overlapping roles of highly homologous Rab GTPase proteins, Rab3 and Rab27, which display high sequence homology, share protein-effectors, and may functionally compensate. To define their actions, we overexpressed Rab3GAP and/or EPI64A GTPase-activating protein in wild-type or Rab27-null cells to transit the Rab3 family or Rab27A to a GDP-bound inactive state. We found Rab27A is essential for generation of the functionally defined immediately releasable pool, Rab3 is essential for a kinetically rapid filling of the RRP, and both cooperate in populating the readily releasable granule pool (RRP). We conclude that while Rab3 and Rab27A cooperate to generate release-ready vesicles in β-cells, they also direct unique kinetic and functional properties of the exocytotic pathway. We also investigated how the SNARE Tomosyn1 (Tomo1) regulates the partitioning of synaptic vesicle (SV) pools in hippocampal neurons. Tomo1 inhibits SV priming at the plasma membrane. Yet, its localization to SVs and cytosol uniquely positions it to coordinate SV pool partitioning. We that find that Tomo1 controls SV transition between the Resting Pool and Total Recycling Pool (TRP), and modulates the RRP size. Tomo1’s regulation of SV distribution between pools is sensitive to neural activity and requires Cdk5. We provide novel evidence for an interaction between Tomo1 and Rab3A-GTP, and through this with Synapsin1 proteins, known regulators of SV recruitment. In addition, Tomo1 regulatory control over the TRP occurred independent of its C-terminal SNARE domain. Hence, Tomo1 actions on neurotransmission extend beyond its known inhibition of SV priming into the RRP and may involve other effector proteins. Altogether, our results advance the understanding of how Rab and Tomosyn proteins coordinate steps of the vesicle cycle that lead to functional heterogeneity among vesicles and thus may determine modes of transmitter release.PHDNeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/116755/1/vcazares_1.pd

Mathematical Modeling of Electrical Activity and Exocytosis in Intestinal L-cells and Pancreatic Beta-cells

In healthy subjects, glucose concentration is tightly kept in a limited range around its basal value thanks to complex regulatory mechanisms. Impairment of this regulatory system is the cause of several metabolic disorders, such as diabetes, characterized by chronic hyperglycemia, which leads to severe micro- and macrovascular complications. Different hormones are involved in this regulation, with insulin being one of the most important and well studied. It is physiologically secreted at every meal by pancreatic beta-cells in response to increased blood glucose levels, in order to lower glucose concentration. Other substances can stimulate insulin secretion, for example glucagon-like peptide-1 (GLP-1) is an insulinotropic hormone released from intestinal L-cells in response to food ingestion. It is, together with other hormones, responsible for the so-called incretin effect, i.e., the fact that glucose ingested orally elicits a greater insulin response than glucose administered intravenously, even when glucose concentrations in plasma are matched. In type-2 diabetes, both insulin and GLP-1 secretion is impaired. In this context, a combination of experimental data and mathematical modeling could help in getting a deeper insight into the cellular mechanisms leading to the secretion of both hormones. In most of the excitable cells, the steps leading to secretion are quite similar: a trigger initiates the electrical activity in the cell, which leads to the opening of voltage gated calcium channels and a subsequent calcium influx inside the cell; the increase in calcium levels allows the vesicles to fuse with the plasma membrane and to release their content outside the cell. In this work, the different steps leading to secretion will be analyzed by means of a combination of both experimental data and mathematical modeling, with reference to the intestinal L-cells and the pancreatic beta-cells. Regarding the intestinal L-cells, a mathematical model of electrical activity was built to investigate the stimulus-secretion pathway, which is still poorly understood. However, two glucose-sensing mechanisms are known to contribute in the sensing of luminal glucose: the sodium-glucose cotransporters (SGLT) and ATP-sensitive K+-channels (K(ATP)-channels). The results showed how the two glucose-sensing mechanisms interact, and suggested that the depolarizing effect of SGLT currents is modulated by K(ATP)-channels activity. On the other hand, the stimulus-secretion pathway in pancreatic beta-cells is well established. SK-channels and calcium dynamics were included in a previous mathematical model of electrical activity in human beta-cells to investigate the heterogeneous and non-intuitive electrophysiological responses to ion channel antagonists. By using our model we also studied paracrine signals, and simulated slow oscillations by adding a glycolytic oscillatory component to the electrophysiological model. The model was further developed by including Kir channels, which play a critical role in the cardiac cells, by determining the shape of cardiac action potential. The inclusion of the Kir2.1 current in the model resulted in a clear improvement of the model behavior, by slowing down the spiking dynamics, thanks to the small outward Kir2.1 current, which tends to stabilize the inter-spike membrane potential. As a result of the beta-cell electrical depolarization, calcium channels open, leading to an influx and a subsequent diffusion of calcium inside the cell, which in turn triggers exocytosis. Hence, from the electrical activity analysis, we moved to the investigation of the relationship between insulin granule exocytosis, calcium levels, distance from calcium channels and channel clustering in beta-cells. This subproject is based on Total Internal Reflection Fluorescence (TIRF) microscopy, consisting in simultaneous visualization of two different fluorophores. The first fluorophore is used to label insulin-containing vesicles, and to differentiate them into two groups: the ones that undergo exocytosis in response to depolarization, and the ones that do not. The second fluorophore, a genetically encoded calcium indicator (R-GECO), is attached to the plasma membrane and permits visualizing calcium levels during the stimulus. Simulations were performed using the modeling program CalC, which implements calcium diffusion and buffering. Simulated calcium levels and the corresponding R-GECO signal were evaluated at different distances from the channel. The comparison of the simulations to the TIRF microscopy data allowed estimating the average distance from the channel of the granules that undergo exocytosis. Calcium diffusion simulations were coupled to a simple model for insulin granule exocytosis to investigate different pools of granules, in terms of vicinity to the calcium channel and calcium affinity. Furthermore, the fusion probability was evaluated both in a single channel, and in a cluster-of-channels context. Simulations confirmed that the hypothesis of a cluster significantly increases the fusion probability and a certain dependence between the channels in the cluster is functional advantageous. So far we analyzed cellular mechanisms, which translate in insulin secretion. Hence, the natural step was to move from a cellular point of view to a bigger scale considering the whole pancreas. In this context, the so called minimal model approach might become useful, by allowing the determination of indexes to assess beta-cell function in different experimental groups. A minimal model specific for the perfused pancreas experimental setting was built adapting the C-peptide minimal model previously applied to the intravenous glucose tolerance test. The model was initially applied to untreated pancreata and afterward used for the assessment of pharmacologically relevant agents (GLP-1, the GLP-1 receptor agonist lixisenatide, and a GPR40/FFAR1 agonist, SAR1) to quantify and differentiate their effect on insulin secretion. Model application showed that lixisenatide reaches improvement of beta-cell function similarly to GLP-1 and demonstrated that SAR1 leads to an additional improvement of beta-cell function in the presence of postprandial GLP-1 levels. In conclusion, in this work different aspects of GLP-1 and insulin secretion were investigated by means of a combination of experimental data and mathematical models. Starting from the modeling of electrical activity in both L-cells and beta-cells, we moved to the calcium diffusion and exocytosis of insulin vesicles, concluding with a minimal model of insulin secretion

Mechanistic investigation of coordinated conformational changes in multisubunit ion channels and enzymes

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2008.Vita.Includes bibliographical references.Many enzymes and ion channels consist of multiple subunits and/or multiple distinct functional components. Coordinated conformational changes through allosteric interactions between subunits and/or between functional units can efficiently regulate protein activity. This dissertation describes investigations of coordinated conformational changes in two systems: the ATP-sensitive potassium (KATP) channel and the ATP-dependent bacterial protease, ClpAP. KATP channels consist of two protein subunits: a pore-forming subunit, Kir6.2 and a regulatory subunit, SUR1. Kir6.2 is an inwardly rectifying potassium channel, and SUR1 belongs to the ATP-binding cassette (ABC) superfamily. Using patch clamp techniques, KATP channel activity was observed directly with single-channel resolution. The results indicate that noise from stochastic channel gating is significantly reduced compared to what would be observed for identical and independent channels, and provide evidence that negatively cooperative interactions between neighboring KATP channels are the source of the noise reduction. Simulations further suggest that negative coupling among KATP channels in pancreatic beta cells could be important for reliable signal transduction. Energetic coupling between Kir6.2 and SUR1 subunits was also investigated. Single-channel records were analyzed to detect the violations of microscopic reversibility in channel gating that would occur if Kir6.2 conformational transitions were driven by the energy from ATP hydrolysis by SUR1. Although no violations of detailed balance in channel gating are detected on the time scale where ATP hydrolysis takes place, unexpected non-equilibrium gating is observed on longer time scales. These results imply that channel gating is coupled to non-equilibrium processes other than ATP hydrolysis by SUR1. The second system studied for coordinated conformational change was ClpAP.(cont.) ClpAP is composed of an ATPase, ClpA and a serine peptidase, ClpP. ClpA uses the free energy of ATP hydrolysis to unfold protein substrates and translocate them to ClpP, which proteolyzes them. To investigate how protein translocation by ClpA is coupled to proteolysis by ClpP, size distributions of peptide products were measured. The observation of non-exponential size distributions, in combination with simulations predicting how different mechanisms would influence the size distribution, supports the hypothesis that peptide product sizes are controlled by coordinated conformational changes of ClpA and ClpP.by Kee-Hyun Choi.Ph.D

Neuronal oscillations on an ultra-slow timescale: daily rhythms in electrical activity and gene expression in the mammalian master circadian clockwork

This is the author accepted manuscript. The final version is available from Wiley via the DOI in this record.Neuronal oscillations of the brain, such as those observed in the cortices and hippocampi of behaving animals and humans, span across wide frequency bands, from slow delta waves (0.1 Hz) to ultra-fast ripples (600 Hz). Here, we focus on ultra-slow neuronal oscillators in the hypothalamic suprachiasmatic nuclei (SCN), the master daily clock that operates on interlocking transcription-translation feedback loops to produce circadian rhythms in clock gene expression with a period of near 24 h (< 0.001 Hz). This intracellular molecular clock interacts with the cell's membrane through poorly understood mechanisms to drive the daily pattern in the electrical excitability of SCN neurons, exhibiting an up-state during the day and a down-state at night. In turn, the membrane activity feeds back to regulate the oscillatory activity of clock gene programs. In this review, we emphasise the circadian processes that drive daily electrical oscillations in SCN neurons, and highlight how mathematical modelling contributes to our increasing understanding of circadian rhythm generation, synchronisation and communication within this hypothalamic region and across other brain circuits.M.D.C.B is supported by the University ofExeter Medical School (UEMS). C.O.D’s work was partially supported bythe National Science Foundation under grant nos. DMS-1412877 and DMS-155237, and the U.S. Army Research Laboratory and the U.S. ArmyResearch Office under Grant No. W911NF-16-1-0584