Martin Bengtsson

Voltage-Gated Ion Channels in Human Pancreatic β-Cells: Electrophysiological Characterization and Role in Insulin Secretion

OBJECTIVE— To characterize the voltage-gated ion channels in human β-cells from nondiabetic donors and their role in glucose-stimulated insulin release. RESEARCH DESIGN AND METHODS— Insulin release was measured from intact islets. Whole-cell patch-clamp experiments and measurements of cell capacitance were performed on isolated β-cells. The ion channel complement was determined by quantitative PCR. RESULTS— Human β-cells express two types of voltage-gated K+ currents that flow through delayed rectifying (KV2.1/2.2) and large-conductance Ca2+-activated K+ (BK) channels. Blockade of BK channels (using iberiotoxin) increased action potential amplitude and enhanced insulin secretion by 70%, whereas inhibition of KV2.1/2.2 (with stromatoxin) was without stimulatory effect on electrical activity and secretion. Voltage-gated tetrodotoxin (TTX)-sensitive Na+ currents (NaV1.6/1.7) contribute to the upstroke of action potentials. Inhibition of Na+ currents with TTX reduced glucose-stimulated (6–20 mmol/l) insulin secretion by 55–70%. Human β-cells are equipped with L- (CaV1.3), P/Q- (CaV2.1), and T- (CaV3.2), but not N- or R-type Ca2+ channels. Blockade of L-type channels abolished glucose-stimulated insulin release, while inhibition of T- and P/Q-type Ca2+ channels reduced glucose-induced (6 mmol/l) secretion by 60–70%. Membrane potential recordings suggest that L- and T-type Ca2+ channels participate in action potential generation. Blockade of P/Q-type Ca2+ channels suppressed exocytosis (measured as an increase in cell capacitance) by >80%, whereas inhibition of L-type Ca2+ channels only had a minor effect. CONCLUSIONS— Voltage-gated T-type and L-type Ca2+ channels as well as Na+ channels participate in glucose-stimulated electrical activity and insulin secretion. Ca2+-activated BK channels are required for rapid membrane repolarization. Exocytosis of insulin-containing granules is principally triggered by Ca2+ influx through P/Q-type Ca2+ channels.

γ-Aminobutyric Acid (GABA) Is an Autocrine Excitatory Transmitter in Human Pancreatic β-Cells

OBJECTIVE Paracrine signaling via γ-aminobutyric acid (GABA) and GABAA receptors (GABAARs) has been documented in rodent islets. Here we have studied the importance of GABAergic signaling in human pancreatic islets. RESEARCH DESIGN AND METHODS Expression of GABAARs in islet cells was investigated by quantitative PCR, immunohistochemistry, and patch-clamp experiments. Hormone release was measured from intact islets. GABA release was monitored by whole-cell patch-clamp measurements after adenoviral expression of α1β1 GABAAR subunits. The subcellular localization of GABA was explored by electron microscopy. The effects of GABA on electrical activity were determined by perforated patch whole-cell recordings. RESULTS PCR analysis detected relatively high levels of the mRNAs encoding GABAAR α2, β3, γ2, and π subunits in human islets. Patch-clamp experiments revealed expression of GABAAR Cl− channels in 52% of β-cells (current density 9 pA/pF), 91% of δ-cells (current density 148 pA/pF), and 6% of α-cells (current density 2 pA/pF). Expression of GABAAR subunits in islet cells was confirmed by immunohistochemistry. β-Cells secreted GABA both by glucose-dependent exocytosis of insulin-containing granules and by a glucose-independent mechanism. The GABAAR antagonist SR95531 inhibited insulin secretion elicited by 6 mmol/l glucose. Application of GABA depolarized β-cells and stimulated action potential firing in β-cells exposed to glucose. CONCLUSIONS Signaling via GABA and GABAAR constitutes an autocrine positive feedback loop in human β-cells. The presence of GABAAR in non–β-cells suggests that GABA may also be involved in the regulation of somatostatin and glucagon secretion.

Microfluidic enzyme immunosensors with immobilised protein A and G using chemiluminescence detection

Affinity proteins were covalently immobilised on silicon microchips with overall dimensions of 13.1 x 3.2 mm, comprising 42 porous flow channels of 235 microm depth and 25 microm width, and used to develop microfluidic immunosensors based on horseradish peroxidase (HRP), catalysing the chemiluminescent oxidation of luminol/p-iodophenol (PIP). Different hydrophilic polymers with long flexible chains (polyethylenimine (PEI), dextran (DEX), polyvinyl alcohol, aminodextran) and 3-aminopropyltriethoxysilane (APTS) were employed for modification of the silica surfaces followed by attachment of protein A or G. The resulting immunosensors were compared in an affinity capture assay format, where the competition between the labelled antigen and the analyte for antibody-binding sites took place in the bulk of the solution. The formed immunocomplexes were then trapped by the microchip affinity capture support and the amount of bound tracer was monitored by injection of luminol, PIP and H2O2. All immunosensors were capable of detecting atrazine at the sub-microg l(-1) level. The most sensitive assays were obtained with PEI and DEX polymer modified supports and immobilised protein G, with limits of detection of 0.006 and 0.010 microg l(-1), and IC50 values of 0.096 and 0.130 microg l(-1), respectively. The protein G based immunosensors were regenerated with 0.4 M glycine-HCl buffer pH 2.2, with no loss of activity observed for a storage and operating period of over 8 months. To estimate the applicability of the immunosensors to the analysis of real samples, PEI and DEX based protein G microchips were used to detect atrazine in surface water and fruit juice, spiked with known amounts of the atrazine, giving recovery values of 87-102 and 88-124% at atrazine fortification levels of 0.5-3 and 80-240 microg l(-1), respectively.

GABA, a natural immunomodulator of T lymphocytes

gamma-aminobutyric acid (GABA) is the main neuroinhibitory transmitter in the brain. Here we show that GABA in the extracellular space may affect the fate of pathogenic T lymphocytes entering the brain. We examined in encephalitogenic T cells if they expressed functional GABA channels that could be activated by the low (nM-1 microM), physiological concentrations of GABA present around neurons in the brain. The cells expressed the alpha1, alpha4, beta2, beta3, gamma1 and delta GABAA channel subunits and formed functional, extrasynaptic-like GABA channels that were activated by 1 microM GABA. 100 nM and higher GABA concentrations decreased T cell proliferation. The results are consistent with GABA being immunomodulatory.

A dominant mutation in Snap25 causes impaired vesicle trafficking, sensorimotor gating, and ataxia in the blind-drunk mouse.

The neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is essential for synaptic vesicle exocytosis, but its study has been limited by the neonatal lethality of murine SNARE knockouts. Here, we describe a viable mouse line carrying a mutation in the b-isoform of neuronal SNARE synaptosomal-associated protein of 25 kDa (SNAP-25). The causative I67T missense mutation results in increased binding affinities within the SNARE complex, impaired exocytotic vesicle recycling and granule exocytosis in pancreatic β-cells, and a reduction in the amplitude of evoked cortical excitatory postsynaptic potentials. The mice also display ataxia and impaired sensorimotor gating, a phenotype which has been associated with psychiatric disorders in humans. These studies therefore provide insights into the role of the SNARE complex in both diabetes and psychiatric disease.

Single-cell gene expression profiling using reverse transcription quantitative real-time PCR

Even in an apparently homogeneous population of cells there are considerable differences between individual cells. A response to a stimulus of a cell population or tissue may be consistent and gradual while the single-cell response might be binary and apparently irregular. The origin of this variability may be preprogrammed or stochastic and a study of this phenomenon will require quantitative measurements of individual cells. Here, we describe a method to collect dispersed single cells either by glass capillaries or flow cytometry, followed by quantitative mRNA profiling using reverse transcription and real-time PCR. We present a single cell lysis protocol and optimized priming conditions for reverse transcription. The large cell-to-cell variability in single-cell gene expression measurements excludes it from standard data analysis. Correlation studies can be used to find common regulatory elements that are indistinguishable at the population level. Single-cell gene expression profiling has the potential to become common practice in many laboratories and a powerful research tool for deeper understanding of molecular mechanisms.

Improved performance in silicon enzyme microreactors obtained by homogeneous porous silicon carrier matrix

The catalytic performance of porous silicon (PS) micro enzyme reactors (muIMER) is strongly dependent on the PS matrix morphology for enzyme immobilisation. PS was achieved in the muIMER by anodisation in a HF-ethanol mixture. PS etching of structured silicon surfaces commonly results in an inhomogeneous pore formation. The deep channel microreactors described herein have previously suffered from these phenomena, yielding non-optimised muIMERs. In order to obtain a homogeneous PS layer on the deep microreactor channel walls, different reactor geometries (channel wall thicknesses of 50 and 75 mum) were anodised at 10 and 50 mA cm(-2) for anodisation times ranging between 0 and 50 min. The muIMERs were evaluated by immobilising two types of enzymes, glucose oxidase (GOx) and trypsin, and the resulting catalytic turnover was monitored by a colorimetric assay. It was found that reactors with a homogeneous PS matrix displayed improved performance. The trypsin muIMERs were used to digest a protein, beta-casein, in an on-line format and the digest was analysed by MALDI-TOF MS. The importance of tailoring the muIMER geometry and the PS-matrix is crucial for the protein digestion. Successful protein identification after only 12 s. digestion was demonstrated for the best reactor, 75 mum channel wall, 25 mum channel width, anodised at 50 mA cm(-2) for 10 min.

R-type Ca 2+ -channel-evoked CICR regulates glucose-induced somatostatin secretion

Pancreatic islets have a central role in blood glucose homeostasis. In addition to insulin-producing β-cells and glucagon-secreting α-cells, the islets contain somatostatin-releasing δ-cells. Somatostatin is a powerful inhibitor of insulin and glucagon secretion. It is normally secreted in response to glucose and there is evidence suggesting its release becomes perturbed in diabetes. Little is known about the control of somatostatin release. Closure of ATP-regulated K+-channels (KATP-channels) and a depolarization-evoked increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) have been proposed to be essential. Here, we report that somatostatin release evoked by high glucose (≥10 mM) is unaffected by the KATP-channel activator diazoxide and proceeds normally in KATP-channel-deficient islets. Glucose-induced somatostatin secretion is instead primarily dependent on Ca2+-induced Ca2+-release (CICR). This constitutes a novel mechanism for KATP-channel-independent metabolic control of pancreatic hormone secretion.

Long-Term Exposure to Glucose and Lipids Inhibits Glucose-Induced Insulin Secretion Downstream of Granule Fusion With Plasma Membrane

Mouse β-cells cultured at 15 mmol/l glucose for 72 h had reduced ATP-sensitive K+ (KATP) channel activity (−30%), increased voltage-gated Ca2+ currents, higher intracellular free Ca2+ concentration ([Ca2+]i; +160%), more exocytosis (monitored by capacitance measurements, +100%), and greater insulin content (+230%) than those cultured at 4.5 mmol/l glucose. However, they released 20% less insulin when challenged with 20 mmol/l glucose. Glucose-induced (20 mmol/l) insulin secretion was reduced by 60–90% in islets cocultured at 4.5 or 15 mmol/l glucose and either oleate or palmitate (0.5 mmol/l). Free fatty acid (FFA)-induced inhibition of secretion was not associated with any major changes in [Ca2+]i or islet ATP content. Palmitate stimulated exocytosis by twofold or more but reduced K+-induced secretion by up to 60%. Basal (1 mmol/l glucose) KATP channel activity was 40% lower in islets cultured at 4.5 mmol/l glucose plus palmitate and 60% lower in islets cultured at 15 mmol/l glucose plus either of the FFAs. Insulin content decreased by 75% in islets exposed to FFAs in the presence of high (15 mmol/l), but not low (4.5 mmol/l), glucose concentrations, but the number of secretory granules was unchanged. FFA-induced inhibition of insulin secretion was not associated with increased transcript levels of the apoptosis markers Bax (BclII-associated X protein) and caspase-3. We conclude that glucose and FFAs reduce insulin secretion by interference with the exit of insulin via the fusion pore.

Progression of Diet-Induced Diabetes in C57BL6J Mice Involves Functional Dissociation of Ca2+ Channels From Secretory Vesicles

OBJECTIVE The aim of the study was to elucidate the cellular mechanism underlying the suppression of glucose-induced insulin secretion in mice fed a high-fat diet (HFD) for 15 weeks. RESEARCH DESIGN AND METHODS C57BL6J mice were fed a HFD or a normal diet (ND) for 3 or 15 weeks. Plasma insulin and glucose levels in vivo were assessed by intraperitoneal glucose tolerance test. Insulin secretion in vitro was studied using static incubations and a perfused pancreas preparation. Membrane currents, electrical activity, and exocytosis were examined by patch-clamp technique measurements. Intracellular calcium concentration ([Ca2+]i) was measured by microfluorimetry. Total internal reflection fluorescence microscope (TIRFM) was used for optical imaging of exocytosis and submembrane depolarization-evoked [Ca2+]i. The functional data were complemented by analyses of histology and gene transcription. RESULTS After 15 weeks, but not 3 weeks, mice on HFD exhibited hyperglycemia and hypoinsulinemia. Pancreatic islet content and β-cell area increased 2- and 1.5-fold, respectively. These changes correlated with a 20–50% reduction of glucose-induced insulin secretion (normalized to insulin content). The latter effect was not associated with impaired electrical activity or [Ca2+]i signaling. Single-cell capacitance and TIRFM measurements of exocytosis revealed a selective suppression (>70%) of exocytosis elicited by short (50 ms) depolarization, whereas the responses to longer depolarizations were (500 ms) less affected. The loss of rapid exocytosis correlated with dispersion of Ca2+ entry in HFD β-cells. No changes in gene transcription of key exocytotic protein were observed. CONCLUSIONS HFD results in reduced insulin secretion by causing the functional dissociation of voltage-gated Ca2+ entry from exocytosis. These observations suggest a novel explanation to the well-established link between obesity and diabetes.