The Proton Leak of the Inner Mitochondrial Membrane Is Enlarged in Freshly Isolated Pancreatic Islets.

In a number of investigations on the mechanism of the metabolic amplification of insulin secretion, differences between the response of freshly isolated islets and of islets cultured for one day have been observed. Since no trivial explanation like insufficient numbers of viable cells after cell culture could be found, a more thorough investigation into the mechanisms responsible for the difference was made, concentrating on the function of the mitochondria as the site where the metabolism of nutrient stimulators of secretion forms the signals impacting on the transport and fusion of insulin granules. Using combinations of inhibitors of oxidative phosphorylation, we come to the conclusion that the mitochondrial membrane potential is lower and the exchange of mitochondrial reducing equivalents is faster in freshly isolated islets than in cultured islets.

Short-Term Inhibition of Translation by Cycloheximide Concurrently Affects Mitochondrial Function and Insulin Secretion in Islets from Female Mice.

Since glucose stimulates protein biosynthesis in beta cells concomitantly with the stimulation of insulin release, the possible interaction of both processes was explored. The protein biosynthesis was inhibited by 10 μM cycloheximide (CHX) 60 min prior to the stimulation of perifused, freshly isolated or 22 h-cultured NMRI mouse islets. CHX reduced the insulinotropic effect of 25 mM glucose or 500 μM tolbutamide in fresh but not in cultured islets.

PyCreas: a tool for quantification of localization and distribution of endocrine cell types in the islets of Langerhans.

Manifest diabetes, but also conditions of increased insulin resistance such as pregnancy or obesity can lead to islet architecture remodeling. The contributing mechanisms are as poorly understood as the consequences of altered cell arrangement. For the quantification of the different cell types but also the frequency of different cell-cell contacts within the islets, different approaches exist.

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 Ca 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.

Regulation of insulin secretion in mouse islets: metabolic amplification by alpha-ketoisocaproate coincides with rapid and sustained increase in acetyl-CoA content.

Glucose and alpha-ketoisocaproate, the keto acid analogue of leucine, stimulate insulin secretion in the absence of other exogenous fuels. Their mitochondrial metabolism in the beta-cell raises the cytosolic ATP/ADP ratio, thereby providing the triggering signal for the exocytosis of the insulin granules. However, additional amplifying signals are required for the full extent of insulin secretion stimulated by these fuels.

Differences in lipid metabolism in acquired versus preexisting glucose intolerance during gestation: role of free fatty acids and sphingosine-1-phosphate.

Background: The prevalence of gestational diabetes mellitus (GDM) is increasing worldwide. There is increasing evidence that GDM is a heterogeneous disease with different subtypes. An important question in this context is whether impaired glucose tolerance (IGT), which is a typical feature of the disease, may already be present before pregnancy and manifestation of the disease.

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.

Metabolic Regulation of Hormone Secretion in Beta-Cells and Alpha-Cells of Female Mice: Fundamental Differences.

It is unclear whether the secretion of glucagon is regulated by an alpha-cell-intrinsic mechanism and whether signal recognition by the mitochondrial metabolism plays a role in it. To measure changes of the cytosolic ATP/ADP ratio, single alpha-cells and beta-cells from NMRI mice were adenovirally transduced with the fluorescent indicator PercevalHR. The cytosolic Ca2+ concentration ([Ca2+]i) was measured by use of Fura2 and the mitochondrial membrane potential by use of TMRE.

An Immersible Microgripper for Pancreatic Islet and Organoid Research.

To improve the predictive value of in vitro experimentation, the use of 3D cell culture models, or organoids, is becoming increasingly popular. However, the current equipment of life science laboratories has been developed to deal with cell monolayers or cell suspensions. To handle 3D cell aggregates and organoids in a well-controlled manner, without causing structural damage or disturbing the function of interest, new instrumentation is needed.

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

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.

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.

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.

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.

Pharmacological inhibition of thioredoxin reductase increases insulin secretion and diminishes beta cell viability.

Apparently, both a decrease in beta cell function and in beta cell mass contribute to the progressive worsening of type 2 diabetes. So, it is of particular interest to define factors which are relevant for the regulation of insulin secretion and at the same time for the maintenance of beta cell mass. The NADPH-thioredoxin system has a candidate role for such a dual function.

Glucagonotropic and Glucagonostatic Effects of KATP Channel Closure and Potassium Depolarization.

The role of depolarization in the inverse glucose-dependence of glucagon secretion was investigated by comparing the effects of KATP channel block and of high potassium. The secretion of glucagon and insulin by perifused mouse islets was simultaneously measured. Lowering glucose raised glucagon secretion before it decreased insulin secretion, suggesting an alpha cell-intrinsic signal recognition.

Fresh and cultured mouse islets differ in their response to nutrient stimulation.

Observing different kinetics of nutrient-induced insulin secretion in fresh and cultured islets under the same condition we compared parameters of stimulus secretion coupling in freshly isolated and 22-h-cultured NMRI mouse islets. Stimulation of fresh islets with 30 mM glucose after perifusion without nutrient gave a continuously ascending secretion rate. In 22-h-cultured islets the same protocol produced a brisk first phase followed by a moderately elevated plateau, a pattern regarded to be typical for mouse islets.

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.

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.

Deletion of the RabGAP TBC1D1 Leads to Enhanced Insulin Secretion and Fatty Acid Oxidation in Islets From Male Mice.

The Rab guanosine triphosphatase-activating protein (RabGAP) TBC1D1 has been shown to be a key regulator of glucose and lipid metabolism in skeletal muscle. Its function in pancreatic islets, however, is not yet fully understood. Here, we aimed to clarify the specific impact of TBC1D1 on insulin secretion and substrate use in pancreatic islets.

The inhibitor of connexin Cx36 channels, mefloquine, inhibits voltage-dependent Ca channels and insulin secretion.

The antimalarial agent, mefloquine, inhibits the function of connexin Cx36 gap junctions and hemichannels and has thus become a tool to investigate their physiological relevance in pancreatic islets. In view of earlier reports on a K channel-block by mefloquine, the specificity of mefloquine as a pharmacological tool was investigated. Mouse pancreatic islets and single beta cells were used to measure membrane potential, whole cell currents, Ca channel activity, cytosolic Ca concentration ([Ca]) and insulin secretion.

Selective manipulation of superparamagnetic nanoparticles for product purification and microfluidic diagnostics.

The needs of scalable product purification as well as the demand for sensitive diagnostics for highly dilute entities can be addressed with the utilization of tailored superparamagnetic nanoparticles. Recent developments have led to more efficient fluidic systems at different scales with suspended nanoparticles or nanoparticle aggregates. However, magnetic nanoparticle systems differ widely in properties and their applications are characterized by very specific challenges.

Glucose but not KCl diminishes submembrane granule turnover in mouse beta-cells.

KCl depolarization is widely used to mimic the depolarization during glucose-stimulated insulin secretion. Consequently, the insulin secretion elicited by KCl is often regarded as the equivalent of the first phase of glucose-induced insulin secretion. Here, the effects of both stimuli were compared by measuring the secretion of perifused mouse islets, the cytosolic Ca concentration of single beta-cells and the mobility of submembrane insulin granules by TIRF microscopy of primary mouse beta-cells.

A 3D microfluidic perfusion system made from glass for multiparametric analysis of stimulus-secretioncoupling in pancreatic islets.

Microfluidic perfusion systems (MPS) are well suited to perform multiparametric measurements with small amounts of tissue to function as an Organ on Chip device (OOC). Such microphysiolgical characterization is particularly valuable in research on the stimulus-secretion-coupling of pancreatic islets. Pancreatic islets are fully functional competent mini-organs, which serve as fuel sensors and transduce metabolic activity into rates of hormone secretion.

Metabolic amplification of insulin secretion is differentially desensitized by depolarization in the absence of exogenous fuels.

Objective: The metabolic amplification of insulin secretion is the sequence of events which enables the secretory response to a fuel secretagogue to exceed the secretory response to a purely depolarizing stimulus. The signals in this pathway are incompletely understood. Here, we have characterized an experimental procedure by which the amplifying response to glucose is reversibly desensitized, while the response to α-ketoisocaproic acid (KIC) is unchanged.