| 1. |
- Bengtsson, Martin, et al.
(författare)
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Gene expression profiling in single cells from the pancreatic islets of Langerhans reveals lognormal distribution of mRNA levels.
- 2005
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Ingår i: Genome Research. - : Cold Spring Harbor Laboratory. - 1549-5469 .- 1088-9051. ; 15:10, s. 1388-1392
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Tidskriftsartikel (refereegranskat)abstract
- The transcriptional machinery in individual cells is controlled by a relatively small number of molecules, which may result in stochastic behavior in gene activity. Because of technical limitations in current collection and recording methods, most gene expression measurements are carried out on populations of cells and therefore reflect average mRNA levels. The variability of the transcript levels between different cells remains undefined, although it may have profound effects on cellular activities. Here we have measured gene expression levels of the five genes ActB, Ins1, Ins2, Abcc8, and Kcnj11 in individual cells from mouse pancreatic islets. Whereas Ins1 and Ins2 expression show a strong cell-cell correlation, this is not the case for the other genes. We further found that the transcript levels of the different genes are lognormally distributed. Hence, the geometric mean of expression levels provides a better estimate of gene activity of the typical cell than does the arithmetic mean measured on a cell population.
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| 2. |
- MacDonald, Patrick E., et al.
(författare)
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A K-ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of langerhans
- 2007
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Ingår i: PLoS Biology. - : Public Library of Science (PLoS). - 1545-7885. ; 5:6, s. 1236-1247
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Tidskriftsartikel (refereegranskat)abstract
- Glucagon, secreted from pancreatic islet a cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the a cells themselves. We examined hormone secretion and Ca2+ responses of a and b cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn (2+) signalling was blocked, but was reversed by low concentrations (1-20 mu M) of the ATP-sensitive K+ (K-ATP) channel opener diazoxide, which had no effect on insulin release or b cell responses. This effect was prevented by the K-ATP channel blocker tolbutamide (100 mu M). Higher diazoxide concentrations (>= 30 mu M) decreased glucagon and insulin secretion, and alpha-and beta-cell Ca2+ responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (< 1 mu M) stimulated glucagon secretion, whereas high concentrations (> 10 mu M) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the K-ATP channel, inhibition of voltage-gated Na+ (TTX) and N-type Ca2+ channels (omega-conotoxin), but not L-type Ca2+ channels (nifedipine), prevented glucagon secretion. Both the N-type Ca2+ channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an a-cell K-ATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.
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| 3. |
- Soni, Arvind, et al.
(författare)
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GPRC5B a putative glutamate-receptor candidate is negative modulator of insulin secretion
- 2013
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 441:3, s. 643-648
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Tidskriftsartikel (refereegranskat)abstract
- GPRC5B is an orphan receptor belonging to the group C family of G protein-coupled receptors (GPCRs). GPRC5B is abundantly expressed in both human and mouse pancreatic islets, and both GPRC5B mRNA and protein are up-regulated 2.5-fold in islets from organ donors with type 2 diabetes. Expression of Gprc5b is 50% lower in islets isolated from newborn (<3 weeks) than in adult (>36 weeks) mice. Lentiviral shRNA-mediated down-regulation of Gprc5b in intact islets from 12 to 16 week-old mice strongly (2.5-fold) increased basal (I mmol/l) and moderately (40%) potentiated glucose (20 mmol/l) stimulated insulin secretion and also enhanced the potentiating effect of glutamate on insulin secretion. Downregulation of Gprc5b protected murine insulin-secreting clonal MIN6 cells against cytokine-induced apoptosis. We propose that increased expression of GPRC5B contributes to the reduced insulin secretion and beta-cell viability observed in type-2 diabetes. Thus, pharmacological targeting of GPRC5B might provide a novel means therapy for the treatment and prevention of type-2 diabetes. (C) 2013 Elsevier Inc. All rights reserved.
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| 4. |
- 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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| 5. |
- Barg, Sebastian, et al.
(författare)
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Priming of insulin granules for exocytosis by granular Cl(-) uptake and acidification
- 2001
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Ingår i: Journal of Cell Science. - 0021-9533 .- 1477-9137. ; 114:Pt 11, s. 2145-54
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Tidskriftsartikel (refereegranskat)abstract
- ATP-dependent priming of the secretory granules precedes Ca(2+)-regulated neuroendocrine secretion, but the exact nature of this reaction is not fully established in all secretory cell types. We have further investigated this reaction in the insulin-secreting pancreatic B-cell and demonstrate that granular acidification driven by a V-type H(+)-ATPase in the granular membrane is a decisive step in priming. This requires simultaneous Cl(-) uptake through granular ClC-3 Cl(-) channels. Accordingly, granule acidification and priming are inhibited by agents that prevent transgranular Cl(-) fluxes, such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and an antibody against the ClC-3 channels, but accelerated by increases in the intracellular ATP:ADP ratio or addition of hypoglycemic sulfonylureas. We suggest that this might represent an important mechanism for metabolic regulation of Ca(2+)-dependent exocytosis that is also likely to be operational in other secretory cell types.
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| 6. |
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| 7. |
- Bokvist, K, et al.
(författare)
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Co-localization of L-type Ca2+ channels and insulin-containing secretory granules and its significance for the initiation of exocytosis in mouse pancreatic B-cells
- 1995
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Ingår i: EMBO Journal. - 1460-2075. ; 14:1, s. 50-57
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Tidskriftsartikel (refereegranskat)abstract
- We have monitored L-type Ca2+ channel activity, local cytoplasmic Ca2+ transients, the distribution of insulin-containing secretory granules and exocytosis in individual mouse pancreatic B-cells. Subsequent to the opening of the Ca2+ channels, exocytosis is initiated with a latency < 100 ms. The entry of Ca2+ that precedes exocytosis is unevenly distributed over the cell and is concentrated to the region with the highest density of secretory granules. In this region, the cytoplasmic Ca2+ concentration is 5- to 10-fold higher than in the remainder of the cell reaching concentrations of several micromolar. Single-channel recordings confirm that the L-type Ca2+ channels are clustered in the part of the cell containing the secretory granules. This arrangement, which is obviously reminiscent of the 'active zones' in nerve terminals, can be envisaged as being favourable to the B-cell as it ensures that the Ca2+ transient is maximal and restricted to the part of the cell where it is required to rapidly initiate exocytosis whilst at the same time minimizing the expenditure of metabolic energy to subsequently restore the resting Ca2+ concentration.
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| 8. |
- Galvanovskis, Juris, et al.
(författare)
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Probability of Exocytosis in Pancreatic β-Cells : Dependence on Ca2+ Sensing Latency Times, Ca2+ Channel Kinetic Parameters, and Channel Clustering
- 2008
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Ingår i: Biosimulation in Drug Development. - Weinheim, Germany : Wiley-VCH Verlag GmbH & Co. KGaA. - 9783527316991 ; , s. 299-311
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Bokkapitel (refereegranskat)abstract
- The fusion of secretory vesicles and granules with the cell membrane prior to the release of their content into the extracellular space requires a transient increase of free Ca2+ concentration in the vicinity of the fusion site. Usually there is a short temporal delay in the onset of the actual fusion of membranes with reference to the rising free Ca2+ levels. This delay is described as a latency time of the Ca2+-sensing system of the secretory machinery and has been observed in several cell types, including pancreatic β-cells. The presence of a delay time of a finite length inherent to the secretory machinery of the cell has an essential effect on the probability for a certain granule to fuse with the cell membrane and to release its contents into the extracellular space during the action potential. We investigate here, theoretically and by numerical simulations, the extent of this influence and its dependence on the parameters of Ca2+ channels, channel clustering, the Ca2+-sensing system, and the length of depolarizing pulses.We use a linear probabilistic model for a random opening and closing of channels that yields an explicit expression for the Laplace transforms of the waiting time distributions for an event that at least one channel is open during the latency time. This allows one in principle to calculate the probability that a vesicle will fuse with the cell membrane during the action potential. We compare our theoretical results with numerical simulatio
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| 9. |
- Hatamie, Amir, et al.
(författare)
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Nanoscale Amperometry Reveals that Only a Fraction of Vesicular Serotonin Content is Released During Exocytosis from Beta Cells
- 2021
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Ingår i: Angewandte Chemie-International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 60:14, s. 7593-7596
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Tidskriftsartikel (refereegranskat)abstract
- Recent work has shown that chemical release during the fundamental cellular process of exocytosis in model cell lines is not all-or-none. We tested this theory for vesicular release from single pancreatic beta cells. The vesicles in these cells release insulin, but also serotonin, which is detectible with amperometric methods. Traditionally, it is assumed that exocytosis in beta cells is all-or-none. Here, we use a multidisciplinary approach involving nanoscale amperometric chemical methods to explore the chemical nature of insulin exocytosis. We amperometrically quantified the number of serotonin molecules stored inside of individual nanoscale vesicles (39 317 +/- 1611) in the cell cytoplasm before exocytosis and the number of serotonin molecules released from single cells (13 310 +/- 1127) for each stimulated exocytosis event. Thus, beta cells release only one-third of their granule content, clearly supporting partial release in this system. We discuss these observations in the context of type-2 diabetes.
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| 10. |
- Muratore, Massimo, et al.
(författare)
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The vascular architecture of the pancreatic islets: A homage to August Krogh
- 2021
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Ingår i: Comparative Biochemistry and Physiology -Part A : Molecular and Integrative Physiology. - : Elsevier BV. - 1095-6433. ; 252
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Tidskriftsartikel (refereegranskat)abstract
- The vascular network supporting the islets of Langerhans represents a highly specialised system of arterioles, capillaries and venules. Several features of the islet vasculature (density and fenestration of the capillaries) ensure rapid exchange of nutrients and hormones, which is central to the islets' capacity to control of systemic metabolism via reciprocal changes of insulin and glucagon secretion. Here we discuss how changes in islet blood flow may underlie pulsatile insulin secretion, which becomes impaired in type-2 diabetes. Improved understanding of the architecture and regulation of pancreas/islet blood flow may therefore illuminate the causes underlying this common metabolic disorder. The pioneering work of August Krogh on blood flow, oxygen diffusion and capillary anatomy (that was awarded with the Nobel Prize in 1920) is a cornerstone in these efforts and remains relevant to today's research. © 2020 The Authors
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