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. In cultured islets the second phase of glucose stimulation was even enhanced. In fresh and in cultured islets CHX strongly reduced the content of proinsulin, but not of insulin, and moderately diminished the [Ca2+]i increase during stimulation. The oxygen consumption rate (OCR) of fresh islets was about 50% higher than that of cultured islets at basal glucose and was significantly increased by glucose but not tolbutamide. In fresh, but not in cultured, islets CHX diminished the glucose-induced OCR increase and changes in the NAD(P)H- and FAD-autofluorescence. It is concluded that short-term CHX exposure interferes with the signal function of the mitochondria, which have different working conditions in fresh and in cultured islets. The interference may not be an off-target effect but may result from the inhibited cytosolic synthesis of mitochondrial proteins.

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. However, few methods are available to characterize islet cell distribution in a statistically valid manner. Here we describe PyCreas, an open-source tool written in Python that allows semi-automated analysis of islet cell distribution based on images of pancreatic sections stained by immunohistochemistry or immunofluorescence. To ensure that the PyCreas tool is suitable for quantitative analysis of cell distribution in the islets at different metabolic states, we studied the localization and distribution of alpha, beta, and delta cells during gestation and prediabetes. We compared the islet cell distribution of pancreatic islets from metabolically healthy NMRI mice with that of New Zealand obese (NZO) mice, which exhibit impaired glucose tolerance (IGT) both preconceptionally and during gestation, and from C57BL/6 N (B6) mice, which acquire this IGT only during gestation. Since substrain(s) of the NZO mice are known to show a variant in the Abcc8 gene, we additionally examined preconceptional SUR1 knock-out (SUR1-KO) mice. PyCreas provided quantitative evidence that alterations in the Abcc8 gene are associated with an altered distribution pattern of islet cells. Moreover, our data indicate that this cannot be a consequence of prolonged hyperglycemia, as islet architecture is already altered in the prediabetic state. Furthermore, the quantitative analysis suggests that states of transient IGT, such as during common gestational diabetes mellitus (GDM), are not associated with changes in islet architecture as observed during long-term IGT. PyCreas provides the ability to systematically analyze the localization and distribution of islet cells at different stages of metabolic disease to better understand the underlying pathophysiology.

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.

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. While it is generally recognized that the amplifying signals are also derived from the mitochondrial metabolism, their exact nature is still unclear. The current study tests the hypothesis that the supply of cytosolic acetyl-CoA is a signal in the amplifying pathway. The contents of acetyl-CoA and acetyl-CoA plus CoA-SH were measured in isolated mouse islets. Insulin secretion was recorded in isolated perifused islets. In islets, the ATP-sensitive K+ channels of which were pharmacologically closed and which were preincubated without exogenous fuel, 10 mmol/L alpha-ketoisocaproate enhanced the acetyl-CoA content after 5 and 20 min incubations and decreased the acetyl-CoA plus CoA-SH within 5 min, but not after 20 min. In islets not exposed to drugs, the preincubation with 3 mmol/L glucose, a non-triggering concentration, elevated the acetyl-CoA content. This content was further increased after 5 min and 20 min incubations with 30 mmol/L glucose, concurrent with a strong increase in insulin secretion. Alpha-ketoisocaproate and glucose increase the supply of acetyl-CoA in the beta-cell cytosol during both phases of insulin secretion. Most likely, this increase provides a signal for the metabolic amplification.

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 latter type resembles in its clinical manifestation prediabetes that has not yet manifested as type 2 diabetes (T2DM). Altered lipid metabolism plays a crucial role in the disorder's pathophysiology. The aim was to investigate the role of lipids which are relevant in diabetes-like phenotypes in these both models with different time of initial onset of IGT. METHODS: Two rodent models reflecting different characteristics of human GDM were used to characterize changes in lipid metabolism occurring during gestation. Since the New Zealand obese (NZO)-mice already exhibit IGT before and during gestation, they served as a subtype model for GDM with preexisting IGT (preIGT) and were compared with C57BL/6 N mice with transient IGT acquired during gestation (aqIGT). While the latter model does not develop manifest diabetes even under metabolic stress conditions, the NZO mouse is prone to severe disease progression later in life. Metabolically healthy Naval Medical Research Institute (NMRI) mice served as controls. RESULTS: In contrast to the aqIGT model, preIGT mice showed hyperlipidemia during gestation with elevated free fatty acids (FFA), triglycerides (TG), and increased atherogenic index. Interestingly, sphingomyelin (SM) concentrations in the liver decreased during gestation concomitantly with an increase in the sphingosine-1-phosphate (S1P) concentration in plasma. Further, preIGT mice showed impaired hepatic weight adjustment and alterations in hepatic FFA metabolism during gestation. This was accompanied by decreased expression of peroxisome proliferator-activated receptor alpha (PPARα) and lack of translocation of fatty acid translocase (FAT/CD36) to the hepatocellular plasma membrane. CONCLUSION: The preIGT model showed impaired lipid metabolism both in plasma and liver, as well as features of insulin resistance consistent with increased S1P concentrations, and in these characteristics, the preIGT model differs from the common GDM subtype with aqIGT. Thus, concomitantly elevated plasma FFA and S1P concentrations, in addition to general shifts in sphingolipid fractions, could be an interesting signal that the metabolic disorder existed before gestation and that future pregnancies require more intensive monitoring to avoid complications. This graphical abstract was created with BioRender.com .

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.

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. Perifused islets were used to measure the secretion of glucagon and insulin. At 5 mM glucose, the PercevalHR ratio in beta-cells was significantly lower than in alpha-cells. Lowering glucose to 1 mM decreased the ratio to 69% within 10 minutes in beta-cells, but only to 94% in alpha-cells. In this situation, 30 mM glucose, 10 mM alpha-ketoisocaproic acid, and 10 mM glutamine plus 10 mM BCH (a nonmetabolizable leucine analogue) markedly increased the PercevalHR ratio in beta-cells. In alpha-cells, only glucose was slightly effective. However, none of the nutrients increased the mitochondrial membrane potential in alpha-cells, whereas all did so in beta-cells. The kinetics of the PercevalHR increase were reflected by the kinetics of [Ca2+]i. increase in the beta-cells and insulin secretion. Glucagon secretion was markedly increased by washing out the nutrients with 1 mM glucose, but not by reducing glucose from 5 mM to 1 mM. This pattern was still recognizable when the insulin secretion was strongly inhibited by clonidine. It is concluded that mitochondrial energy metabolism is a signal generator in pancreatic beta-cells, but not in alpha-cells.

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. In particular, the precise and stable positioning in a cell bath with flow rates sufficient to characterize the kinetic responses to physiological or pharmacological stimuli can be a demanding task. Here, we present data that demonstrate that microgrippers are well suited to this task. The current version is able to work in aqueous solutions and was shown to position isolated pancreatic islets and 3D aggregates of insulin-secreting MIN6-cells. A stable hold required a gripping force of less than 30 µN and did not affect the cellular integrity. It was maintained even with high flow rates of the bath perfusion, and it was precise enough to permit the simultaneous microfluorimetric measurements and membrane potential measurements of the single cells within the islet through the use of patch-clamp electrodes.

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. 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.

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.

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.

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. Here, we have characterized the effects of a highly specific inhibitor of thioredoxin reductase, AM12, on the viability and function of insulin-secreting MIN6 cells and isolated NMRI mouse islets. Viability was checked by MTT testing and the fluorescent live-dead assay. Apoptosis was assessed by annexin V assay. Insulin secretion of perifused islets was measured by ELISA. The cytosolic Ca2+ concentration was measured by the Fura technique. Acute exposure of perifused pancreatic islets to 5 µM AM12 was without significant effect on insulin secretion. Islets cultured for 24 h in 0.5 or 5 µM AM12 showed unchanged basal secretion during perifusion, but the response to 30 mM glucose was significantly enhanced by 5 µM. Twenty-four-hour exposure to 5 µM AM12 proved to be without effect on the viability of MIN6 cells, whereas longer exposure was clearly toxic. Islets were more susceptible, showing initial signs of apoptosis after 24-h exposure to 5 µM AM12. The activity of the NADPH-thioredoxin system is indispensable for beta cell viability but may have a limiting effect on glucose-induced insulin secretion.

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. Raising glucose affected glucagon and insulin secretion at the same time. However, depolarization by tolbutamide, gliclazide, or 15 mM KCl increased insulin secretion before the glucagon secretion receded. In contrast to the robust depolarizing effect of arginine and KCl (15 and 40 mM) on single alpha cells, tolbutamide was of variable efficacy. Only when applied before other depolarizing agents had tolbutamide a consistent depolarizing effect and regularly increased the cytosolic Ca2+ concentration. When tested on inside-out patches tolbutamide was as effective on alpha cells as on beta cells. In the presence of 1 µM clonidine, to separate insulinotropic from glucagonotropic effects, both 500 µM tolbutamide and 30 µM gliclazide increased glucagon secretion significantly, but transiently. The additional presence of 15 or 40 mM KCl in contrast led to a marked and lasting increase of the glucagon secretion. The glucagon secretion by SUR1 knockout islets was not increased by tolbutamide, whereas 40 mM KCl was of unchanged efficiency. In conclusion a strong and sustained depolarization is compatible with a marked and lasting glucagon secretion. KATP channel closure in alpha cells is less readily achieved than in beta cells, which may explain the moderate and transient glucagonotropic effect.

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. This was also the response of cultured islets to the nutrient secretagogue alpha-ketoisocaproic acid, whereas the secretion of fresh islets increased similarly fast but remained strongly elevated. The responses of fresh and cultured islets to purely depolarizing stimuli (tolbutamide or KCl), however, were closely similar. Signs of apoptosis and necrosis were rare in both preparations. In cultured islets, the glucose-induced rise of the cytosolic Ca2+ concentration started from a lower value and was larger as was the increase of the ATP/ADP ratio. The prestimulatory level of mitochondrial reducing equivalents, expressed as the NAD(P)H/FAD fluorescence ratio, was lower in cultured islets, but increased more strongly than in fresh islets. When culture conditions were modified by replacing RPMI with Krebs-Ringer medium and FCS with BSA, the amount of released insulin varied widely, but the kinetics always showed a predominant first phase. In conclusion, the secretion kinetics of fresh mouse islets is more responsive to variations of nutrient stimulation than cultured islets. The more uniform kinetics of the latter may be caused by a different use of endogenous metabolites.

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.

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 ß-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.

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. We analyzed the dynamics of glucose-stimulated insulin secretion (GSIS) and lipid metabolism in isolated islets from Tbc1d1-deficient (D1KO) mice. To further investigate the underlying cellular mechanisms, we conducted pharmacological studies in these islets. In addition, we determined morphology and number of both pancreatic islets and insulin vesicles in ß-cells using light and transmission electron microscopy. Isolated pancreatic islets from D1KO mice exhibited substantially increased GSIS compared with wild-type (WT) controls. This was attributed to both enhanced first and second phase of insulin secretion, and this enhanced secretion persisted during repetitive glucose stimuli. Studies with sulfonylureas or KCl in isolated islets demonstrated that TBC1D1 exerts its function via a signaling pathway at the level of membrane depolarization. In line, ultrastructural analysis of isolated pancreatic islets revealed both higher insulin-granule density and number of docked granules in ß-cells from D1KO mice compared with WT controls. Like in skeletal muscle, lipid use in isolated islets was enhanced upon D1KO, presumably as a result of a higher mitochondrial fission rate and/or higher mitochondrial activity. Our results clearly demonstrate a dual role of TBC1D1 in controlling substrate metabolism of the pancreatic islet.

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. This review summarizes advances in the synthesis of superparamagnetic particles and displays states and trends in research making use of these particles in biotechnological downstream processing and in biosensing.