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Patrik Rorsman

Publications and source records attributed to Patrik Rorsman.

At least 37 records · Page 2Linked to original sources
PubMed2004

Regulated exocytosis of GABA-containing synaptic-like microvesicles in pancreatic beta-cells.

We have explored whether gamma-aminobutyric acid (GABA) is released by regulated exocytosis of GABA-containing synaptic-like microvesicles (SLMVs) in insulin-releasing rat pancreatic beta-cells. To this end, beta-cells were engineered to express GABA(A)-receptor Cl(-)-channels at high density using adenoviral infection. Electron microscopy indicated that the average diameter of the SLMVs is 90 nm, that every beta-cell contains approximately 3,500 such vesicles, and that insulin-containing large dense core vesicles exclude GABA. Quantal release of GABA, seen as rapidly activating and deactivating Cl(-)-currents, was observed during membrane depolarizations from -70 mV to voltages beyond -40 mV or when Ca(2+) was dialysed into the cell interior. Depolarization-evoked GABA release was suppressed when Ca(2+) entry was inhibited using Cd(2+). Analysis of the kinetics of GABA release revealed that GABA-containing vesicles can be divided into a readily releasable pool and a reserve pool. Simultaneous measurements of GABA release and cell capacitance indicated that exocytosis of SLMVs contributes approximately 1% of the capacitance signal. Mathematical analysis of the release events suggests that every SLMV contains 0.36 amol of GABA. We conclude that there are two parallel pathways of exocytosis in pancreatic beta-cells and that release of GABA may accordingly be temporally and spatially separated from insulin secretion. This provides a basis for paracrine GABAergic signaling within the islet.

Animals↗
PubMed2004

Type 2 diabetes mellitus: not quite exciting enough?

Type 2 diabetes mellitus is a serious metabolic disease that afflicts around 5% of the population in Western societies and over 150 million people worldwide. It is characterized by elevation of the blood glucose concentration, usually presents in middle age, and is exacerbated by obesity. Both genetic and environmental factors contribute to the disease but in the vast majority of cases the aetiology is still not understood. Here we present a novel hypothesis for the aetiology of type 2 diabetes. We postulate that the electrical activity of the insulin-secreting beta-cells of the pancreas acts to integrate the genetic and environmental factors that predispose to disease risk. Our hypothesis is supported by a substantial amount of data gathered from a range of different disciplines and makes predictions that can be tested experimentally both in vitro and in man.

Age Factors↗
PubMed2004

Large dense-core vesicle exocytosis in pancreatic beta-cells monitored by capacitance measurements.

This article discusses the currently used methodologies for monitoring exocytosis as changes in cell capacitance. Details are given on composition of solutions, experimental protocols, and how the observed responses can be interpreted physiologically. The concepts are illustrated by examples from our own work on insulin-releasing pancreatic beta-cells. Finally, we consider the feasibility of applying capacitance measurements to endocrine cells in intact pancreatic islets, where the cells are electrically coupled to each other.

Animals↗
PubMed2004

Glucagon-like peptide-1: regulation of insulin secretion and therapeutic potential.

Glucagon-like peptide-1 (GLP-1) is an intestinally derived insulinotropic hormone currently under investigation for use as a novel therapeutic agent in the treatment of type 2 diabetes. One of several important effects of GLP-1 is on nutrient-induced pancreatic hormone release and is mediated by binding to a specific G-protein coupled receptor resulting in the activation of adenylate cyclase and an increase in cAMP generation. In the beta-cell, cAMP binds and modulates activities of both protein kinase A and cAMP-regulated guanine nucleotide exchange factor II, thereby enhancing glucose-dependent insulin secretion. The stimulatory action of GLP-1 on insulin secretion involves interaction with a plethora of signal transduction processes including ion channel activity, intracellular Ca(2+) handling and exocytosis of the insulin-containing granules. In this review we focus principally on recent advances in our understanding on the cellular mechanisms proposed to underlie GLP-1's insulinotropic effect and attempt to incorporate this knowledge into a working model for the control of insulin secretion. Lastly, this review discusses the applicability of GLP-1 as a therapeutic agent for the treatment of type 2 diabetes.

Adenosine Triphosphate↗
PubMed2004

Isoform-specific regulation of mood behavior and pancreatic beta cell and cardiovascular function by L-type Ca 2+ channels.

Ca(v)1.2 and Ca(v)1.3 L-type Ca(2+) channels (LTCCs) are believed to underlie Ca(2+) currents in brain, pancreatic beta cells, and the cardiovascular system. In the CNS, neuronal LTCCs control excitation-transcription coupling and neuronal plasticity. However, the pharmacotherapeutic implications of CNS LTCC modulation are difficult to study because LTCC modulators cause cardiovascular (activators and blockers) and neurotoxic (activators) effects. We selectively eliminated high dihydropyridine (DHP) sensitivity from Ca(v)1.2 alpha 1 subunits (Ca(v)1.2DHP-/-) without affecting function and expression. This allowed separation of the DHP effects of Ca(v)1.2 from those of Ca(v)1.3 and other LTCCs. DHP effects on pancreatic beta cell LTCC currents, insulin secretion, cardiac inotropy, and arterial smooth muscle contractility were lost in Ca(v)1.2DHP-/- mice, which rules out a direct role of Ca(v)1.3 for these physiological processes. Using Ca(v)1.2DHP-/- mice, we established DHPs as mood-modifying agents: LTCC activator-induced neurotoxicity was abolished and disclosed a depression-like behavioral effect without affecting spontaneous locomotor activity. LTCC activator BayK 8644 (BayK) activated only a specific set of brain areas. In the ventral striatum, BayK-induced release of glutamate and 5-HT, but not dopamine and noradrenaline, was abolished. This animal model provides a useful tool to elucidate whether Ca(v)1.3-selective channel modulation represents a novel pharmacological approach to modify CNS function without major peripheral effects.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2004

Palmitate stimulation of glucagon secretion in mouse pancreatic alpha-cells results from activation of L-type calcium channels and elevation of cytoplasmic calcium.

We have investigated the short-term effects of the saturated free fatty acid (FFA) palmitate on pancreatic alpha-cells. Palmitate (0.5 or 1 mmol/l bound to fatty acid-free albumin) stimulated glucagon secretion from intact mouse islets 1.5- to 2-fold when added in the presence of 1-15 mmol/l glucose. Palmitate remained stimulatory in islets depolarized with 30 mmol/l extracellular K(+) or exposed to forskolin, but it did not remain stimulatory after treatment with isradipine or triacsin C. The stimulatory action of palmitate on secretion correlated with a 3.5-fold elevation of intracellular free Ca(2+) when applied in the presence of 15 mmol/l glucose, a 40% stimulation of exocytosis (measured as increases in cell capacitance), and a 25% increase in whole-cell Ca(2+) current. The latter effect was abolished by isradipine, suggesting that palmitate selectively modulates l-type Ca(2+) channels. The effect of palmitate on exocytosis was not mediated by palmitoyl-CoA, and intracellular application of this FFA metabolite decreased rather than enhanced Ca(2+)-induced exocytosis. The stimulatory effects of palmitate on glucagon secretion were paralleled by a approximately 50% inhibition of somatostatin release. We conclude that palmitate increases alpha-cell exocytosis principally by enhanced Ca(2+) entry via l-type Ca(2+) channels and, possibly, relief from paracrine inhibition by somatostatin released by neighboring delta-cells.

Animals↗
PubMed2004

Glucose inhibition of glucagon secretion from rat alpha-cells is mediated by GABA released from neighboring beta-cells.

gamma-Aminobutyric acid (GABA) has been proposed to function as a paracrine signaling molecule in islets of Langerhans. We have shown that rat beta-cells release GABA by Ca(2+)-dependent exocytosis of synaptic-like microvesicles. Here we demonstrate that GABA thus released can diffuse over sufficient distances within the islet interstitium to activate GABA(A) receptors in neighboring cells. Confocal immunocytochemistry revealed the presence of GABA(A) receptors in glucagon-secreting alpha-cells but not in beta- and delta-cells. RT-PCR analysis detected transcripts of alpha(1) and alpha(4) as well as beta(1-3) GABA(A) receptor subunits in purified alpha-cells but not in beta-cells. In whole-cell voltage-clamp recordings, exogenous application of GABA activated Cl(-) currents in alpha-cells. The GABA(A) receptor antagonist SR95531 was used to investigate the effects of endogenous GABA (released from beta-cells) on pancreatic islet hormone secretion. The antagonist increased glucagon secretion at 1 mmol/l glucose twofold and completely abolished the inhibitory action of 20 mmol/l glucose on glucagon release. Basal and glucose-stimulated secretion of insulin and somatostatin were unaffected by SR95531. The L-type Ca(2+) channel blocker isradipine evoked a paradoxical stimulation of glucagon secretion. This effect was not observed in the presence of SR95531, and we therefore conclude that isradipine stimulates glucagon secretion by inhibition of GABA release.

Animals↗
PubMed2004

ATP-sensitive K+ channel-dependent regulation of glucagon release and electrical activity by glucose in wild-type and SUR1-/- mouse alpha-cells.

Patch-clamp recordings and glucagon release measurements were combined to determine the role of plasma membrane ATP-sensitive K+ channels (KATP channels) in the control of glucagon secretion from mouse pancreatic alpha-cells. In wild-type mouse islets, glucose produced a concentration-dependent (half-maximal inhibitory concentration [IC50]=2.5 mmol/l) reduction of glucagon release. Maximum inhibition (approximately 50%) was attained at glucose concentrations >5 mmol/l. The sulfonylureas tolbutamide (100 micromol/l) and glibenclamide (100 nmol/l) inhibited glucagon secretion to the same extent as a maximally inhibitory concentration of glucose. In mice lacking functional KATP channels (SUR1-/-), glucagon secretion in the absence of glucose was lower than that observed in wild-type islets and both glucose (0-20 mmol/l) and the sulfonylureas failed to inhibit glucagon secretion. Membrane potential recordings revealed that alpha-cells generate action potentials in the absence of glucose. Addition of glucose depolarized the alpha-cell by approximately 7 mV and reduced spike height by 30% Application of tolbutamide likewise depolarized the alpha-cell (approximately 17 mV) and reduced action potential amplitude (43%). Whereas insulin secretion increased monotonically with increasing external K+ concentrations (threshold 25 mmol/l), glucagon secretion was paradoxically suppressed at intermediate concentrations (5.6-15 mmol/l), and stimulation was first detectable at >25 mmol/l K+. In alpha-cells isolated from SUR1-/- mice, both tolbutamide and glucose failed to produce membrane depolarization. These effects correlated with the presence of a small (0.13 nS) sulfonylurea-sensitive conductance in wild-type but not in SUR1-/- alpha-cells. Recordings of the free cytoplasmic Ca2+ concentration ([Ca2+]i) revealed that, whereas glucose lowered [Ca2+]i to the same extent as application of tolbutamide, the Na+ channel blocker tetrodotoxin, or the Ca2+ channel blocker Co2+ in wild-type alpha-cells, the sugar was far less effective on [Ca2+]i in SUR1-/- alpha-cells. We conclude that the KATP channel is involved in the control of glucagon secretion by regulating the membrane potential in the alpha-cell in a way reminiscent of that previously documented in insulin-releasing beta-cells. However, because alpha-cells possess a different complement of voltage-gated ion channels involved in action potential generation than the beta-cell, moderate membrane depolarization in alpha-cells is associated with reduced rather than increased electrical activity and secretion.

Adenosine Triphosphate↗
PubMed2003

Impaired insulin secretion and glucose tolerance in beta cell-selective Ca(v)1.2 Ca2+ channel null mice.

Insulin is secreted from pancreatic beta cells in response to an elevation of cytoplasmic Ca(2+) resulting from enhanced Ca(2+) influx through voltage-gated Ca(2+) channels. Mouse beta cells express several types of Ca(2+) channel (L-, R- and possibly P/Q-type). beta cell-selective ablation of the gene encoding the L-type Ca(2+) channel subtype Ca(v)1.2 (betaCa(v)1.2(-/-) mouse) decreased the whole-cell Ca(2+) current by only approximately 45%, but almost abolished first-phase insulin secretion and resulted in systemic glucose intolerance. These effects did not correlate with any major effects on intracellular Ca(2+) handling and glucose-induced electrical activity. However, high-resolution capacitance measurements of exocytosis in single beta cells revealed that the loss of first-phase insulin secretion in the betaCa(v)1.2(-/-) mouse was associated with the disappearance of a rapid component of exocytosis reflecting fusion of secretory granules physically attached to the Ca(v)1.2 channel. Thus, the conduit of Ca(2+) entry determines the ability of the cation to elicit secretion.

Animals↗
PubMed2003

Phosphatidylinositol 4-kinase serves as a metabolic sensor and regulates priming of secretory granules in pancreatic beta cells.

Insulin secretion is controlled by the beta cell's metabolic state, and the ability of the secretory granules to undergo exocytosis increases during glucose stimulation in a membrane potential-independent fashion. Here, we demonstrate that exocytosis of insulin-containing secretory granules depends on phosphatidylinositol 4-kinase (PI 4-kinase) activity and that inhibition of this enzyme suppresses glucose-stimulated insulin secretion. Intracellular application of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] stimulated exocytosis by promoting the priming of secretory granules for release and increasing the number of granules residing in a readily releasable pool. Reducing the cytoplasmic ADP concentration in a way mimicking the effects of glucose stimulation activated PI 4-kinase and increased exocytosis whereas changes of the ATP concentration in the physiological range had little effect. The PI(4,5)P(2)-binding protein Ca(2+)-dependent activator protein for secretion (CAPS) is present in beta cells, and neutralization of the protein abolished both Ca(2+)- and PI(4,5)P(2)-induced exocytosis. We conclude that ADP-induced changes in PI 4-kinase activity, via generation of PI(4,5)P(2), represents a metabolic sensor in the beta cell by virtue of its capacity to regulate the release competence of the secretory granules.

1-Phosphatidylinositol 4-Kinase↗
PubMed2003

Imidazoline NNC77-0074 stimulates insulin secretion and inhibits glucagon release by control of Ca(2+)-dependent exocytosis in pancreatic alpha- and beta-cells.

We have investigated the effects of the novel imidazoline compound (+)-2-(2-(4,5-dihydro-1H-imidazol-2-yl)-thiopene-2-yl-ethyl)-pyridine (NNC77-0074) on stimulus-secretion coupling in isolated pancreatic alpha- and beta-cells. NNC77-0074 stimulated glucose-dependent insulin secretion in intact mouse pancreatic islets. No effect was observed at </=2.5 mM glucose and maximal stimulation occurred at 10-15 mM glucose. NNC77-0074 produced a concentration-dependent stimulation of insulin secretion. Half-maximal (EC(50)) stimulation was observed at 24 microM and at maximally stimulatory concentrations insulin release was doubled. The stimulatory action of NNC77-0074 on insulin secretion was not associated with membrane depolarisation or a change in the activity of ATP-sensitive K(+) channels. Using capacitance measurements, we found that NNC77-0074 stimulated depolarisation-induced exocytosis 2.6-fold without affecting the whole-cell Ca(2+) current when applied via the extracellular medium. The concentration dependence of the stimulatory action was determined by intracellular application of NNC77-0074 through the recording pipette. NNC77-0074 stimulated exocytosis half-maximal at 44 nM and at maximally stimulatory concentrations the rate of exocytosis was increased twofold. NNC77-0074 stimulated depolarised-induced insulin secretion from islets exposed to diazoxide and high external KCl (EC(50)=0.45 microM). The stimulatory action of NNC77-0074 was dependent on protein kinase C activity. NNC77-0074 potently inhibited glucagon secretion from rat islets (EC(50)=11 nM). This was not associated with a change in spontaneous electrical activity and ATP-sensitive K(+) channel activity but resulted from a reduction of the rate of Ca(2+)-dependent exocytosis in single rat alpha-cells (EC(50)=9 nM). Inhibition of exocytosis by NNC77-0074 was pertussis toxin-sensitive and mediated by activation of the protein phosphatase calcineurin. In rat somatotrophs, PC12 cells and mouse cortical neurons NNC77-0074 did not stimulate Ca(2+)-evoked exocytosis, whereas the other imidazoline compounds phentolamine and efaroxan produced 2.5-fold stimulation of exocytosis. Our data suggest that the imidazoline compound NNC77-0074 constitutes a novel class of antidiabetic compounds that stimulates glucose-dependent insulin release while inhibiting glucagon secretion. These actions are exclusively exerted by modulation of exocytosis of the insulin- and glucagon-containing granules.

Animals↗
PubMed2003

SUR1 regulates PKA-independent cAMP-induced granule priming in mouse pancreatic B-cells.

Measurements of membrane capacitance were applied to dissect the cellular mechanisms underlying PKA-dependent and -independent stimulation of insulin secretion by cyclic AMP. Whereas the PKA-independent (Rp-cAMPS-insensitive) component correlated with a rapid increase in membrane capacitance of approximately 80 fF that plateaued within approximately 200 ms, the PKA-dependent component became prominent during depolarizations >450 ms. The PKA-dependent and -independent components of cAMP-stimulated exocytosis differed with regard to cAMP concentration dependence; the K(d) values were 6 and 29 micro M for the PKA-dependent and -independent mechanisms, respectively. The ability of cAMP to elicit exocytosis independently of PKA activation was mimicked by the selective cAMP-GEFII agonist 8CPT-2Me-cAMP. Moreover, treatment of B-cells with antisense oligodeoxynucleotides against cAMP-GEFII resulted in partial (50%) suppression of PKA-independent exocytosis. Surprisingly, B-cells in islets isolated from SUR1-deficient mice (SUR1(-/-) mice) lacked the PKA-independent component of exocytosis. Measurements of insulin release in response to GLP-1 stimulation in isolated islets from SUR1(-/-) mice confirmed the complete loss of the PKA-independent component. This was not attributable to a reduced capacity of GLP-1 to elevate intracellular cAMP but instead associated with the inability of cAMP to stimulate influx of Cl(-) into the granules, a step important for granule priming. We conclude that the role of SUR1 in the B cell extends beyond being a subunit of the plasma membrane K(ATP)-channel and that it also plays an unexpected but important role in the cAMP-dependent regulation of Ca(2+)-induced exocytosis.

ATP-Binding Cassette Transporters↗
PubMed2002

Fast insulin secretion reflects exocytosis of docked granules in mouse pancreatic B-cells.

A readily releasable pool (RRP) of granules has been proposed to underlie the first phase of insulin secretion. In the present study we combined electron microscopy, insulin secretion measurements and recordings of cell capacitance in an attempt to define this pool ultrastructurally. Mouse pancreatic B-cells contain approximately 9,000 granules, of which 7% are docked below the plasma membrane. The number of docked granules was reduced by 30% (200 granules) during 10 min stimulation with high K+. This stimulus depolarized the cell to -10 mV, elevated cytosolic [Ca2+] ([Ca2+](i)) from a basal concentration of 130 nM to a peak of 1.3 microM and released 0.5 ng insulin/islet, corresponding to 200-300 granules/cell. The Ca2+ transient decayed towards the prestimulatory concentration within approximately 200 s, presumably reflecting Ca2+ channel inactivation. Renewed stimulation with high K+ failed to stimulate insulin secretion when applied in the absence of glucose. The size of the RRP, derived from the insulin measurements, is similar to that estimated from the increase in cell capacitance elicited by photolytic release of caged Ca2+. We propose that the RRP represents a subset of the docked pool of granules and that replenishment of RRP can be accounted for largely by chemical modification of granules already in place or situated close to the plasma membrane.

Animals↗
PubMed2002

Delay between fusion pore opening and peptide release from large dense-core vesicles in neuroendocrine cells.

Peptidergic neurotransmission is slow compared to that mediated by classical neurotransmitters. We have studied exocytotic membrane fusion and cargo release by simultaneous capacitance measurements and confocal imaging of single secretory vesicles in neuroendocrine cells. Depletion of the readily releasable pool (RRP) correlated with exocytosis of 10%-20% of the docked vesicles. Some remaining vesicles became releasable after recovery of RRP. Expansion of the fusion pore, seen as an increase in luminal pH, occurred after approximately 0.3 s, and peptide release was delayed by another 1-10 s. We conclude that (1) RRP refilling involves chemical modification of vesicles already in place, (2) the release of large neuropeptides via the fusion pore is negligible and only proceeds after complete fusion, and (3) sluggish peptidergic transmission reflects the time course of vesicle emptying.

Animals↗
PubMed2002

Cellular function in multicellular system for hormone-secretion: electrophysiological aspect of studies on alpha-, beta- and delta-cells of the pancreatic islet.

We review a new 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. By using low-resistance patch electrodes, rapid current responses can be monitored under 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 Ca(2+)-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 (approximately 1 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 have implications for neuroscience research such as the mechanism of oscillatory electrical activity in general and processes involved in the glucose-sensing in some neurons, which response to changes of blood glucose concentration.

Animals↗
PubMed2002

Nateglinide, but not repaglinide, stimulates growth hormone release in rat pituitary cells by inhibition of K channels and stimulation of cyclic AMP-dependent exocytosis.

OBJECTIVE: GH causes insulin resistance, impairs glycemic control and increases the risk of vascular diabetic complications. Sulphonylureas stimulate GH secretion and this study was undertaken to investigate the possible stimulatory effect of repaglinide and nateglinide, two novel oral glucose regulators, on critical steps of the stimulus-secretion coupling in single rat somatotrophs. METHODS: Patch-clamp techniques were used to record whole-cell ATP-sensitive K(+) (K(ATP)) and delayed outward K(+) currents, membrane potential and Ca(2+)-dependent exocytosis. GH release was measured from perifused rat somatotrophs. RESULTS: Both nateglinide and repaglinide dose-dependently suppressed K(ATP) channel activity with half-maximal inhibition being observed at 413 nM and 13 nM respectively. Both compounds induced action potential firing in the somatotrophs irrespective of whether GH-releasing hormone was present or not. The stimulation of electrical activity by nateglinide, but not repaglinide, was associated with an increased mean duration of the action potentials. The latter effect correlated with a reduction of the delayed outward K(+) current, which accounts for action potential repolarization. The latter effect had a K(d) of 19 microM but was limited to 38% inhibition. When applied at concentrations similar to those required to block K(ATP) channels, nateglinide in addition potentiated Ca(2+)-evoked exocytosis 3.3-fold (K(d)=3 microM) and stimulated GH release 4.5-fold. The latter effect was not shared by repaglinide. The stimulation of exocytosis by nateglinide was mimicked by cAMP and antagonized by the protein kinase A inhibitor Rp-cAMPS. CONCLUSION: Nateglinide stimulates GH release by inhibition of plasma membrane K(+) channels, elevation of cytoplasmic cAMP levels and stimulation of Ca(2+)-dependent exocytosis. By contrast, the effect of repaglinide was confined to inhibition of the K(ATP) channels.

Animals↗