| 1. |
- Abadpour, Shadab, et al.
(författare)
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Glial cell-line derived neurotrophic factor protects human islets from nutrient deprivation and endoplasmic reticulum stress induced apoptosis
- 2017
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- One of the key limitations to successful human islet transplantation is loss of islets due to stress responses pre- and post-transplantation. Nutrient deprivation and ER stress have been identified as important mechanisms leading to apoptosis. Glial Cell-line Derived Neurotrophic Factor (GDNF) has recently been found to promote islet survival after isolation. However, whether GDNF could rescue human islets from nutrient deprivation and ER stress-mediated apoptosis is unknown. Herein, by mimicking those conditions in vitro, we have shown that GDNF significantly improved glucose stimulated insulin secretion, reduced apoptosis and proinsulin: insulin ratio in nutrient deprived human islets. Furthermore, GDNF alleviated thapsigargin-induced ER stress evidenced by reduced expressions of IRE1 alpha and BiP and consequently apoptosis. Importantly, this was associated with an increase in phosphorylation of PI3K/AKT and GSK3B signaling pathway. Transplantation of ER stressed human islets pre- treated with GDNF under kidney capsule of diabetic mice resulted in reduced expressions of IRE1 alpha and BiP in human islet grafts with improved grafts function shown by higher levels of human C-peptide post-transplantation. We suggest that GDNF has protective and anti-apoptotic effects on nutrient deprived and ER stress activated human islets and could play a significant role in rescuing human islets from stress responses.
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| 2. |
- Barg, Sebastian, et al.
(författare)
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Fast exocytosis with few Ca(2+) channels in insulin-secreting mouse pancreatic B cells
- 2001
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Ingår i: Biophysical Journal. - 1542-0086 .- 0006-3495. ; 81:6, s. 3308-3323
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Tidskriftsartikel (refereegranskat)abstract
- The association of L-type Ca(2+) channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with <500 alpha1(C) L-type Ca(2+) channels, corresponding to a Ca(2+) channel density of 0.9 channels per microm(2). Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs(-1) (approximately 650 granules/s) 25 ms after onset of the pulse and is completed within approximately 100 ms. This component represents a subset of approximately 60 granules situated in the immediate vicinity of the L-type Ca(2+) channels, corresponding to approximately 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca(2+) revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca(2+) concentration of 17 microM, and concentrations >25 microM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca(2+) buffer EGTA but abolished by addition of exogenous L(C753-893), the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca(2+) channel, which has been proposed to tether the Ca(2+) channels to the secretory granules. In keeping with the idea that secretion is determined by Ca(2+) influx through individual Ca(2+) channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca(2+) channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca(2+) from 2.6 to 10 mM. Recordings of single Ca(2+) channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca(2+) channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca(2+) channels as it ensures maximum usage of the Ca(2+) entering the cell while minimizing the influence of stochastic variations of the Ca(2+) channel activity.
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| 3. |
- Gromada, Jesper, et al.
(författare)
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CaM kinase II-dependent mobilization of secretory granules underlies acetylcholine-induced stimulation of exocytosis in mouse pancreatic B-cells
- 1999
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Ingår i: Journal of Physiology. - 1469-7793. ; 518:3, s. 745-759
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Tidskriftsartikel (refereegranskat)abstract
- 1. Measurements of cell capacitance were used to investigate the mechanisms by which acetylcholine (ACh) stimulates Ca2+-induced exocytosis in single insulin-secreting mouse pancreatic B-cells. 2. ACh (250 microM) increased exocytotic responses elicited by voltage-clamp depolarizations 2.3-fold. This effect was mediated by activation of muscarinic receptors and dependent on elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i) attributable to mobilization of Ca2+ from intracellular stores. The latter action involved interference with the buffering of [Ca2+]i and the time constant (tau) for the recovery of [Ca2+]i following a voltage-clamp depolarization increased 5-fold. As a result, Ca2+ was present at concentrations sufficient to promote the replenishment of the readily releasable pool of granules (RRP; > 0.2 microM) for much longer periods in the presence than in the absence of the agonist. 3. The effect of Ca2+ on exocytosis was mediated by activation of CaM kinase II, but not protein kinase C, and involved both an increased size of the RRP from 40 to 140 granules and a decrease in tau for the refilling of the RRP from 31 to 19 s. 4. Collectively, the effects of ACh on the RRP and tau result in a > 10-fold stimulation of the rate at which granules are supplied for release.
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| 4. |
- Göpel, Sven, et al.
(författare)
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Activation of Ca(2+)-dependent K(+) channels contributes to rhythmic firing of action potentials in mouse pancreatic beta cells
- 1999
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Ingår i: Journal of General Physiology. - 0022-1295 .- 1540-7748. ; 114:6, s. 759-770
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Tidskriftsartikel (refereegranskat)abstract
- We have applied the perforated patch whole-cell technique to beta cells within intact pancreatic islets to identify the current underlying the glucose-induced rhythmic firing of action potentials. Trains of depolarizations (to simulate glucose-induced electrical activity) resulted in the gradual (time constant: 2.3 s) development of a small (<0.8 nS) K(+) conductance. The current was dependent on Ca(2+) influx but unaffected by apamin and charybdotoxin, two blockers of Ca(2+)-activated K(+) channels, and was insensitive to tolbutamide (a blocker of ATP-regulated K(+) channels) but partially (>60%) blocked by high (10-20 mM) concentrations of tetraethylammonium. Upon cessation of electrical stimulation, the current deactivated exponentially with a time constant of 6.5 s. This is similar to the interval between two successive bursts of action potentials. We propose that this Ca(2+)-activated K(+) current plays an important role in the generation of oscillatory electrical activity in the beta cell.
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| 5. |
- Göpel, Sven, et al.
(författare)
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Capacitance measurements of exocytosis in mouse pancreatic {alpha}-, {beta}- and {delta}-cells studied in intact islets of Langerhans.
- 2004
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Ingår i: Journal of Physiology. - : Wiley. - 1469-7793 .- 0022-3751. ; 556:3, s. 711-726
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Tidskriftsartikel (refereegranskat)abstract
- To determine the functions of fibromodulin (Fmod), a small leucine-rich keratan sulfate proteoglycan in tooth formation, we investigated the distribution of Fmod in dental tissues by immunohistochemistry and characterized the dental phenotype of 1-day-old Fmod-deficient mice using light and transmission electron microscopy. Immunohistochemistry was also used to compare the relative protein expression of dentin sialoprotein (DSP), dentin matrix protein-1 (DMP 1), bone sialoprotein (BSP), and osteopontin (OPN) between Fmod-deficient mice and wild-type mice. In normal mice and rats, Fmod immunostaining was mostly detected in the distal cell bodies of odontoblasts and in the stratum intermedium and was weaker in odontoblast processes and predentin. The absence of Fmod impaired dentin mineralization, increased the diameter of the collagen fibrils throughout the whole predentin, and delayed enamel formation. Immunohistochemistry provides evidence for compensatory mechanisms in Fmod-deficient mice. Staining for DSP and OPN was decreased in molars, whereas DMP 1 and BSP were enhanced. In the incisors, labeling for DSP, DMP 1, and BSP was strongly increased in the pulp and odontoblasts, whereas OPN staining was decreased. Positive staining was also seen for DMP 1 and BSP in secretory ameloblasts. Together these studies indicate that Fmod restricts collagen fibrillogenesis in predentin while promoting dentin mineralization and the early stages of enamel formation.
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| 6. |
- Göpel, Sven
(författare)
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Electrophysiology of pancreatic A-, B- and D-cells studied in intact mouse islets of Langerhans
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Abstract The electrophysiological properties of insulin-secreting b-cells, glucagon-releasing a-cells and somatostation-producing d-cells in intact mouse pancreatic islets were investigated using the perforated patch whole-cell technique. It is demonstrated that it is possible to functionally identify the different cell types in situ. The ?-cells have a cell capacitance of 7.4 pF and differ from the ?- and ?-cells in lacking TTX-sensitive Na+-channels. Patch-clamp analysis of ?-cells in intact islets was applied to explain the grouping of action potentials to bursts in b-cells exposed to intermediate glucose concentrations. Electrical stimulation resulted in the gradual development of an outward K+-current, which deactivated slowly upon cessation of stimulation with a time course broadly consistent with a role in bursting (Kslow-current). A similar current was seen in isolated ?-cells but the amplitude was reduced by >80%, possibly explaining the lack of bursting in these cells. Inhibition of Ca2+-influx abolished the Kslow- current but it was resistant to apamin and charybdotoxin, two blockers of Ca2+-activated K+-channels. Ca2+-entry may instead lead to activation of ATP-regulated K+-channels by accelerated ATP-consumption as suggested by the observation that the Kslow-current was partially blocked by tolbutamide. The ?-cells are smaller than ?-cells as witnessed by a lower value of the cell capacitance (4.4 pF). They contain an L-type Ca2+-current, a TTX-sensitive Na+-current and a delayed outward K+-current. They also contained KATP-channels but the channel density was <50% of that seen in ?-cells. Glucagon-secreting ?-cells were occasionally seen to generate overshooting action potentials in the absence of glucose. An increased glucose concentration (to inhibit glucagon release) suppressed electrical activity but the membrane potential was paradoxically more depolarised than in the absence of the sugar. The size of the ?-cells was comparable to that of the ?-cell (5 pF) and like the ?-cells they contained a Na+-current that remained activatable at physiological membrane potentials. However, the steady-state inactivation of the Na+-current in the ?-cells occurred at voltages 20 mV more negative than seen in ?-cells (-47 mV instead of –28 mV). The a-cells are equipped with KATP-channels but the density was only »15% of that in the ?-cell. It is proposed that glucose inhibits glucagon release by closing KATP-channels. The associated depolarization leads to inactivation of the Na+- and T-type Ca2+-channels culminating in suppression of ?-cell electrical activity and glucagon release.
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| 7. |
- Göpel, Sven, et al.
(författare)
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Patch-clamp characterisation of somatostatin-secreting -cells in intact mouse pancreatic islets
- 2000
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Ingår i: Journal of Physiology. - 1469-7793 .- 0022-3751. ; 528:3, s. 497-507
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Tidskriftsartikel (refereegranskat)abstract
- The perforated patch whole-cell configuration of the patch-clamp technique was applied to superficial cells in intact mouse pancreatic islets. Three types of electrical activity were observed corresponding to alpha-, beta- and delta-cells. The delta-cells were electrically active in the presence of glucose but lacked the oscillatory pattern seen in the beta-cells. By contrast, the alpha-cells were electrically silent at high glucose concentrations but action potentials could be elicited by removal of the sugar. Both alpha- and beta-cells contained transient voltage-activated K+ currents. In the delta-cells, the K+ currents activated above -20 mV and were completely blocked by TEA (20 mM). The alpha-cells differed from the delta-cells in possessing a TEA-resistant K+ current activating already at -40 mV. Immunocytochemistry revealed the presence of Kv3.4 channels in delta-cells and TEA-resistant Kv4.3 channels in alpha-cells. Thus the presence of a transient TEA-resistant current can be used to functionally separate the delta- and alpha-cells. A TTX-sensitive Na+ current developed in delta-cells during depolarisations beyond -30 mV and reached a peak amplitude of 350 pA. Steady-state inactivation of this current was half-maximal at -28 mV. The delta-cells were also equipped with a sustained Ca2+ current that activated above -30 mV and reached a peak of 60 pA when measured at 2.6 mM extracellular Ca2+. A tolbutamide-sensitive KATP channel conductance was observed in delta-cells exposed to glucose-free medium. Addition of tolbutamide (0.1 mM) depolarised the delta-cell and evoked electrical activity. We propose that the KATP channels in delta-cells serve the same function as in the beta-cell and couple an elevation of the blood glucose concentration to stimulation of hormone release.
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| 8. |
- Göpel, Sven, et al.
(författare)
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Regulation of glucagon release in mouse -cells by KATP channels and inactivation of TTX-sensitive Na+ channels
- 2000
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Ingår i: Journal of Physiology. - 1469-7793 .- 0022-3751. ; 528:3, s. 509-520
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Tidskriftsartikel (refereegranskat)abstract
- The perforated patch whole-cell configuration of the patch-clamp technique was applied to superficial glucagon-secreting alpha-cells in intact mouse pancreatic islets. alpha-cells were distinguished from the beta- and delta-cells by the presence of a large TTX-blockable Na+ current, a TEA-resistant transient K+ current sensitive to 4-AP (A-current) and the presence of two kinetically separable Ca2+ current components corresponding to low- (T-type) and high-threshold (L-type) Ca2+ channels. The T-type Ca2+, Na+ and A-currents were subject to steady-state voltage-dependent inactivation, which was half-maximal at -45, -47 and -68 mV, respectively. Pancreatic alpha-cells were equipped with tolbutamide-sensitive, ATP-regulated K+ (KATP) channels. Addition of tolbutamide (0.1 mM) evoked a brief period of electrical activity followed by a depolarisation to a plateau of -30 mV with no regenerative electrical activity. Glucagon secretion in the absence of glucose was strongly inhibited by TTX, nifedipine and tolbutamide. When diazoxide was added in the presence of 10 mM glucose, concentrations up to 2 microM stimulated glucagon secretion to the same extent as removal of glucose. We conclude that electrical activity and secretion in the alpha-cells is dependent on the generation of Na+-dependent action potentials. Glucagon secretion depends on low activity of KATP channels to keep the membrane potential sufficiently negative to prevent voltage-dependent inactivation of voltage-gated membrane currents. Glucose may inhibit glucagon release by depolarising the alpha-cell with resultant inactivation of the ion channels participating in action potential generation.
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| 9. |
- Göpel, Sven, et al.
(författare)
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Voltage-gated and resting membrane currents recorded from B-cells in intact mouse pancreatic islets
- 1999
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Ingår i: Journal of Physiology. - 1469-7793 .- 0022-3751. ; 521:3, s. 717-728
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Tidskriftsartikel (refereegranskat)abstract
- 1. The perforated patch whole-cell configuration of the patch-clamp technique was applied to superficial cells in intact pancreatic islets. Immunostaining in combination with confocal microscopy revealed that the superficial cells consisted of 35 % insulin-secreting B-cells and 65 % non-B-cells (A- and D-cells). 2. Two types of cell, with distinct electrophysiological properties, could be functionally identified. One of these generated oscillatory electrical activity when the islet was exposed to 10 mM glucose and had the electrophysiological characteristics of isolated B-cells maintained in tissue culture. 3. The Ca2+ current recorded from B-cells in situ was 80 % larger than that of isolated B-cells. It exhibited significant (70 %) inactivation during 100 ms depolarisations. The inactivation was voltage dependent and particularly prominent during depolarisations evoking the largest Ca2+ currents. 4. Voltage-dependent K+ currents were observed during depolarisations to membrane potentials above -20 mV. These currents inactivated little during a 200 ms depolarisation and were unaffected by varying the holding potential between -90 and -30 mV. 5. The maximum resting conductance in the absence of glucose, which reflects the conductance of ATP-regulated K+ (KATP) channels, amounted to approximately 4 nS. Glucose produced a concentration-dependent reduction of KATP channel conductance with half-maximal inhibition observed with 5 mM glucose. 6. Combining voltage- and current-clamp recording allowed the estimation of the gap junction conductance between different B-cells. These experiments indicated that the input conductance of the B-cell at stimulatory glucose concentrations ( approximately 1 nS) is almost entirely accounted for by coupling to neighbouring B-cells.
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| 10. |
- Kanno, T, et al.
(författare)
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Cellular function in multicellular system for hormone-secretion: electrophysiological aspect of studies on alpha-, beta- and delta-cells of the pancreatic islet
- 2002
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Ingår i: Neuroscience Research. - 0168-0102. ; 42:2, s. 79-90
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Forskningsöversikt (refereegranskat)abstract
- We review a neck method to explore the cellular functions in multicellular system by application of the perforated patch-clamp technique to intact pancreatic islet of Langerhans. Using this approach, the integrity of the islet is preserved and intercellular communication via gap junctions and paracrine processes are maintained. 13 using low-resistance patch electrodes, rapid current responses can be monitored wider voltage-clamp control. We have applied this methodology to answer questions not resolved by patch-clamp experiments on isolated single insulin-secreting, beta-cells. First, the role of a K+-current dependent on Ca2+-influx for the termination of burst of action potentials in beta-cells could be documented. Neither the current, nor the bursting pattern of electrical activity is preserved in isolated beta-cells. Second. the conductance of gap junctions (similar to1 nS) between beta-cells was determined. Third, electrical properties of glucagon-producing alpha- and somatostatin-secreting delta-cells and the different mechanisms for glucose-sensing in these cells could be explored. The findings emanating from these experiments may hake implications for neuroscience research such as the mechanism of oscillatory electrical activity in general anti processes involved in the glucose-sensing in some neurons, which response to changes of blood glucose concentration. (C) 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
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