What Is the Metabolic Amplification of Insulin Secretion and Is It (Still) Relevant?

The pancreatic beta-cell transduces the availability of nutrients into the secretion of insulin. While this process is extensively modified by hormones and neurotransmitters, it is the availability of nutrients, above all glucose, which sets the process of insulin synthesis and secretion in motion. The central role of the mitochondria in this process was identified decades ago, but how changes in mitochondrial activity are coupled to the exocytosis of insulin granules is still incompletely understood. The identification of ATP-sensitive K+-channels provided the link between the level of adenine nucleotides and the electrical activity of the beta cell, but the depolarization-induced Ca2+-influx into the beta cells, although necessary for stimulated secretion, is not sufficient to generate the secretion pattern as produced by glucose and other nutrient secretagogues. The metabolic amplification of insulin secretion is thus the sequence of events that enables the secretory response to a nutrient secretagogue to exceed the secretory response to a purely depolarizing stimulus and is thus of prime importance. Since the cataplerotic export of mitochondrial metabolites is involved in this signaling, an orienting overview on the topic of nutrient secretagogues beyond glucose is included. Their judicious use may help to define better the nature of the signals and their mechanism of action

A Parallel Perifusion Slide From Glass for the Functional and Morphological Analysis of Pancreatic Islets.

An islet-on-chip system in the form of a completely transparent microscope slide optically accessible from both sides was developed. It is made from laser-structured borosilicate glass and enables the parallel perifusion of five microchannels, each containing one islet precisely immobilized in a pyramidal well. The islets can be in inserted via separate loading windows above each pyramidal well. This design enables a gentle, fast and targeted insertion of the islets and a reliable retention in the well while at the same time permitting a sufficiently fast exchange of the media. In addition to the measurement of the hormone content in the fractionated efflux, parallel live cell imaging of the islet is possible. By programmable movement of the microscopic stage imaging of five wells can be performed. The current chip design ensures sufficient time resolution to characterize typical parameters of stimulus-secretion coupling. This was demonstrated by measuring the reaction of the islets to stimulation by glucose and potassium depolarization. After the perifusion experiment islets can be removed for further analysis. The live-dead assay of the removed islets confirmed that the process of insertion and removal was not detrimental to islet structure and viability. In conclusion, the present islet-on-chip design permits the practical implementation of parallel perifusion experiments on a single and easy to load glass slide. For each immobilized islet the correlation between secretion, signal transduction and morphology is possible. The slide concept allows the scale-up to even higher degrees of parallelization

Microgrippers to handle Organoids and pancreatic Islets for Precision Measurements of biological Function

The model of the cultured single cell is considered insufficient to explain the physiological regulation taking place at the organ level. The same is true for the prediction of drug action at the organ level or at the level of the intact organism. For these reasons 3D cell culture models are in increasing demand. It is thus necessary to develop the instruments to handle such cell aggregates and organoids in a controlled, precise and gentle manner. Here, a microgripper is presented which is able to work in aqueous solutions and which is compatible with electrophysiological recordings of the cells immobilized by it. It was successfully employed to position isolated pancreatic islets and a 3D cell culture model of insulin-secreting cells, the so-called MIN6-pseudoislet. As required it was possible to measure the membrane potential of cells within these aggregates without any interference from the microgripper

Metabolic changes during pregnancy in glucose-intolerant NZO mice: A polygenic model with prediabetic metabolism

Gestational diabetes mellitus (GDM) is a complex metabolic disease involving genetic and environmental factors. Recent studies have underlined its heterogeneity, so it is reasonable to divide patients into subpopulations depending on whether an insulin secretion or sensitivity defect is predominant. Since testing for GDM is usually performed in the second trimester, misinterpretation of prediabetes as gestational diabetes may occur. As with type 2 diabetes (T2DM), rodent models are needed for both GDM and prediabetes, but few do exist. Here, we compared the metabolic changes in pregnant normal NMRI mice with those in New Zealand obese (NZO) mice. Male animals of this strain are an established model of T2DM, whereas female mice of this strain are protected from hyperglycemia and β-cell death. We demonstrate that female NZO mice exhibited impaired glucose tolerance, preconceptional hyperinsulinemia, and hyperglucagonemia without any signs of manifest diabetes. The NZO model showed, compared with the NMRI control strain, a reduced proliferative response of the Langerhans islets during pregnancy (3.7 ± 0.4 vs. 7.2 ± 0.8% Ki-67-positive nuclei, p = .004). However, oral glucose tolerance tests revealed improved stimulation of insulin secretion in both strains. But this adaption was not sufficient to prevent impaired glucose tolerance in NZO mice compared with the NMRI control (p = .0002). Interestingly, glucose-stimulated insulin secretion was blunted in isolated primary NZO islets in perifusion experiments. In summary, the NZO mouse reflects important characteristics of human GDM and prediabetes in pregnancy and serves as a model for subpopulations with early alterations in glucose metabolism and primary insulin secretion defect

Nonmechanical parfocal and autofocus features based on wave propagation distribution in lensfree holographic microscopy

Performing long-term cell observations is a non-trivial task for conventional optical microscopy, since it is usually not compatible with environments of an incubator and its temperature and humidity requirements. Lensless holographic microscopy, being entirely based on semiconductor chips without lenses and without any moving parts, has proven to be a very interesting alternative to conventional microscopy. Here, we report on the integration of a computational parfocal feature, which operates based on wave propagation distribution analysis, to perform a fast autofocusing process. This unique non-mechanical focusing approach was implemented to keep the imaged object staying in-focus during continuous long-term and real-time recordings. A light-emitting diode (LED) combined with pinhole setup was used to realize a point light source, leading to a resolution down to 2.76 μm. Our approach delivers not only in-focus sharp images of dynamic cells, but also three-dimensional (3D) information on their (x, y, z)-positions. System reliability tests were conducted inside a sealed incubator to monitor cultures of three different biological living cells (i.e., MIN6, neuroblastoma (SH-SY5Y), and Prorocentrum minimum). Altogether, this autofocusing framework enables new opportunities for highly integrated microscopic imaging and dynamic tracking of moving objects in harsh environments with large sample areas

Changes in granule mobility and age contribute to changes in insulin secretion after desensitization or rest: incl. supplementary material

Introduction Functional impairment of the stimulus secretion coupling in pancreatic beta cells is an essential component of type 2 diabetes. It is known that prolonged stimulation desensitizes the secretion of insulin and thus contributes to beta cell dysfunction. Beta cell rest, in contrast, was shown to enhance the secretory response. Here, the underlying mechanisms were investigated. Research design and methods To characterize the consequences of desensitization or rest for the number and mobility of submembrane granules, insulin-secreting MIN6 cells were desensitized by 18-hour culture with 500 µM tolbutamide or rested by 18-hour culture with 1 µM clonidine. The granules were labeled by hIns-EGFP or hIns-DsRed E5, imaged by TIRF microscopy of the cell footprint area and analyzed with an observer-independent program. Additionally, the insulin content and secretion were measured. Results Concurrent with the insulin content, submembrane granules were only slightly reduced after desensitization but markedly increased after rest. Both types of pretreatment diminished arrivals and departures of granules in the submembrane space and increased the proportion of immobile long-term resident granules, but desensitization lowered and rest increased the number of exocytoses, in parallel with the effect on insulin secretion. Labeling with hIns-DsRed E5 (‘timer’) showed that desensitization did not affect the proportion of aged granules, whereas rest increased it. Aged granules showed a high mobility and made up only a minority of long-term residents. Long-term resident granules were more numerous after rest and had a lower lateral mobility, suggesting a firmer attachment to the membrane. Conclusion The number, mobility and age of submembrane granules reflect the preceding functional states of insulin-secreting cells. Representing the pool of releasable granules, their quantity and quality may thus form part of the beta cell memory on renewed stimulation

The Dynamics of Calcium Signaling in Beta Cells—A Discussion on the Comparison of Experimental and Modelling Data

The stimulus–secretion coupling of the pancreatic beta cell is particularly complex, as it integrates the availability of glucose and other nutrients with the neuronal and hormonal input to generate rates of insulin secretion that are appropriate for the entire organism. It is beyond dispute however, that the cytosolic Ca2+ concentration plays a particularly prominent role in this process, as it not only triggers the fusion of insulin granules with the plasma membrane, but also regulates the metabolism of nutrient secretagogues and affects the function of ion channels and transporters. In order to obtain a better understanding of the interdependence of these processes and, ultimately, of the entire beta cell as a working system, models have been developed based on a set of nonlinear ordinary differential equations, and were tested and parametrized on a limited set of experiments. In the present investigation, we have used a recently published version of the beta cell model to test its ability to describe further measurements from our own experimentation and from the literature. The sensitivity of the parameters is quantified and discussed; furthermore, the possible influence of the measuring technique is taken into account. The model proved to be powerful in correctly describing the depolarization pattern in response to glucose and the reaction of the cytosolic Ca2+ concentration to stepwise increases of the extracellular K+ concentration. Additionally, the membrane potential during a KATP channel block combined with a high extracellular K+ concentration could be reproduced. In some cases, however, a slight change of a single parameter led to an abrupt change in the cellular response, such as the generation of a Ca2+ oscillation with high amplitude and high frequency. This raises the question as to whether the beta cell may be a partially unstable system or whether further developments in modeling are needed to achieve a generally valid description of the stimulus–secretion coupling of the beta cell

A Cellular Automaton Model as a First Model-Based Assessment of Interacting Mechanisms for Insulin Granule Transport in Beta Cells

In this paper a first model is derived and applied which describes the transport of insulin granules through the cell interior and at the membrane of a beta cell. A special role is assigned to the actin network, which significantly influences the transport. For this purpose, microscopically measured actin networks are characterized and then further ones are artificially generated. In a Cellular Automaton model, phenomenological laws for granule movement are formulated and implemented. Simulation results are compared with experiments, primarily using TIRF images and secretion rates. In this respect, good similarities are already apparent. The model is a first useful approach to describe complex granule transport processes in beta cells, and offers great potential for future extensions. Furthermore, the model can be used as a tool to validate hypotheses and associated mechanisms regarding their effect on exocytosis or other processes. For this purpose, the source code for the model is provided online

The changing view of insulin granule mobility: From conveyor belt to signaling hub

Before the advent of TIRF microscopy the fate of the insulin granule prior to secretion was deduced from biochemical investigations, electron microscopy and electrophysiological measurements. Since Calcium-triggered granule fusion is indisputably necessary to release insulin into the extracellular space, much effort was directed to the measure this event at the single granule level. This has also been the major application of the TIRF microscopy of the pancreatic beta cell when it became available about 20 years ago. To better understand the metabolic modulation of secretion, we were interested to characterize the entirety of the insulin granules which are localized in the vicinity of the plasma membrane to identify the characteristics which predispose to fusion. In this review we concentrate on how the description of granule mobility in the submembrane space has evolved as a result of progress in methodology. The granules are in a state of constant turnover with widely different periods of residence in this space. While granule fusion is associated +with prolonged residence and decreased lateral mobility, these characteristics may not only result from binding to the plasma membrane but also from binding to the cortical actin web, which is present in the immediate submembrane space. While granule age as such affects granule mobility and fusion probability, the preceding functional states of the beta cell leave their mark on these parameters, too. In summary, the submembrane granules form a highly dynamic heterogeneous population and contribute to the metabolic memory of the beta cells