| 1. |
- Barg, Sebastian, et al.
(författare)
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A Subset of 50 Secretory Granules in Close Contact With L-Type Ca(2+) Channels Accounts for First-Phase Insulin Secretion in Mouse beta-Cells.
- 2002
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Ingår i: Diabetes. - 1939-327X .- 0012-1797. ; 51 Suppl 1, s. 74-82
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Tidskriftsartikel (refereegranskat)abstract
- Capacitance measurements were applied to mouse pancreatic beta-cells to elucidate the cellular mechanisms underlying biphasic insulin secretion. We report here that only <50 of the beta-cell's >10,000 granules are immediately available for release. The releasable granules tightly associate with the voltage-gated alpha(1C) Ca(2+) channels, and it is proposed that the release of these granules accounts for first-phase insulin secretion. Subsequent replenishment of the releasable pool by priming of previously nonreleasable granules is required for second-phase insulin secretion. The latter reaction depends on intragranular acidification due to the concerted action of granular bafilomycin-sensitive v-type H(+)-ATPase and 4,4-diisothiocyanostilbene-2,2-disulfonate--blockable ClC-3 Cl(-) channels. Lowering the cytoplasmic ATP/ADP ratio prevents granule acidification, granule priming, and refilling of the releasable pool. The latter finding provides an explanation to the transient nature of insulin secretion elicited by, for example, high extracellular K(+) in the absence of metabolizable fuels.
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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. |
- 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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| 4. |
- Barg, Sebastian, et al.
(författare)
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The stimulatory action of tolbutamide on Ca2+-dependent exocytosis in pancreatic beta cells is mediated by a 65-kDa mdr-like P-glycoprotein
- 1999
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 96:10, s. 5539-5544
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Tidskriftsartikel (refereegranskat)abstract
- Intracellular application of the sulfonylurea tolbutamide during whole-cell patch-clamp recordings stimulated exocytosis >5-fold when applied at a cytoplasmic Ca2+ concentration of 0.17 microM. This effect was not detectable in the complete absence of cytoplasmic Ca2+ and when exocytosis was elicited by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). The stimulatory action could be antagonized by the sulfonamide diazoxide, by the Cl--channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), by intracellular application of the antibody JSB1 [originally raised against a 170-kDa multidrug resistance (mdr) protein], and by tamoxifen (an inhibitor of the mdr- and volume-regulated Cl- channels). Immunocytochemistry and Western blot analyses revealed that JSB1 recognizes a 65-kDa protein in the secretory granules. This protein exhibited no detectable binding of sulfonylureas and is distinct from the 140-kDa sulfonylurea high-affinity sulfonylurea receptors also present in the granules. We conclude that (i) tolbutamide stimulates Ca2+-dependent exocytosis secondary to its binding to a 140-kDa high-affinity sulfonylurea receptor in the secretory granules; and (ii) a granular 65-kDa mdr-like protein mediates the action. The processes thus initiated culminate in the activation of a granular Cl- conductance. We speculate that the activation of granular Cl- fluxes promotes exocytosis (possibly by providing the energy required for membrane fusion) by inducing water uptake and an increased intragranular hydrostatic pressure.
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| 5. |
- Barghouth, Mohammad, et al.
(författare)
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The structure of insulin granule core determines secretory capacity being reduced in type-2 diabetes
- 2022
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Exocytosis in excitable cells is essential for their physiological functions. Although the exocytotic machinery controlling cellular secretion has been well investigated, the function of the vesicular cargo, i.e. secretory granular content remains obscure. Here we combine dSTORM imaging and single-domain insulin antibody, to dissect the in situ structure of insulin granule cores (IGCs) at nano level. We demonstrate that the size and shape of the IGCs can be regulated by the juxta-granular molecules Nucleobindin-2 and Enolase-1, that further contribute to the stimulated insulin secretion. IGCs located at the plasma membrane are larger than those in the cytosol. The IGCs size is decreased by ∼20% after glucose stimulation due to the release of the peripheral part of IGCs through incomplete granule fusion. Importantly, the reduction of the IGCs size is also observed in non-stimulatory pancreatic β-cells from diabetic db/db mice, Akita (Ins2+/-) mice and human Type-2 diabetic donors, in accordance with impaired secretion. These findings overall highlight the structure of exocytotic insulin cores as a novel modality amenable to targeting in the stimulated exocytosis in β-cells with impaired insulin secretion.Competing Interest StatementThe authors have declared no competing interest.
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| 6. |
- Barghouth, Mohammad, et al.
(författare)
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The T-type calcium channel CaV3.2 regulates insulin secretion in the pancreatic β-cell
- 2022
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Ingår i: Cell Calcium. - : Elsevier BV. - 0143-4160. ; 108
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Tidskriftsartikel (refereegranskat)abstract
- Voltage-gated Ca2+ (CaV) channel dysfunction leads to impaired glucose-stimulated insulin secretion in pancreatic β-cells and contributes to the development of type-2 diabetes (T2D). The role of the low-voltage gated T-type CaV channels in β-cells remains obscure. Here we have measured the global expression of T-type CaV3.2 channels in human islets and found that gene expression of CACNA1H, encoding CaV3.2, is negatively correlated with HbA1c in human donors, and positively correlated with islet insulin gene expression as well as secretion capacity in isolated human islets. Silencing or pharmacological blockade of CaV3.2 attenuates glucose-stimulated cytosolic Ca2+ signaling, membrane potential, and insulin release. Moreover, the endoplasmic reticulum (ER) Ca2+ store depletion is also impaired in CaV3.2-silenced β-cells. The linkage between T-type (CaV3.2) and L-type CaV channels is further identified by the finding that the intracellular Ca2+ signaling conducted by CaV3.2 is highly dependent on the activation of L-type CaV channels. In addition, CACNA1H expression is significantly associated with the islet predominant L-type CACNA1C (CaV1.2) and CACNA1D (CaV1.3) genes in human pancreatic islets. In conclusion, our data suggest the essential functions of the T-type CaV3.2 subunit as a mediator of β-cell Ca2+ signaling and membrane potential needed for insulin secretion, and in connection with L-type CaV channels.
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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. |
- Cataldo, Rodrigo, et al.
(författare)
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MAFA and MAFB regulate exocytosis-related genes in human β-cells
- 2022
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Ingår i: Acta Physiologica. - : Wiley. - 1748-1716 .- 1748-1708. ; 234:2
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Tidskriftsartikel (refereegranskat)abstract
- AIMS: Reduced expression of exocytotic genes is associated with functional defects in insulin exocytosis contributing to impaired insulin secretion and type 2 diabetes (T2D) development. MAFA and MAFB transcription factors regulate β-cell physiology, and their gene expression is reduced in T2D β cells. We investigate if loss of MAFA and MAFB in human β cells contributes to T2D progression by regulating genes required for insulin exocytosis.METHODS: Three approaches were performed: (1) RNAseq analysis with the focus on exocytosis-related genes in MafA-/- mouse islets, (2) correlational analysis between MAFA, MAFB and exocytosis-related genes in human islets and (3) MAFA and MAFB silencing in human islets and EndoC-βH1 cells followed by functional in vitro studies.RESULTS: The expression of 30 exocytosis-related genes was significantly downregulated in MafA-/- mouse islets. In human islets, the expression of 29 exocytosis-related genes correlated positively with MAFA and MAFB. Eight exocytosis-related genes were downregulated in MafA-/- mouse islets and positively correlated with MAFA and MAFB in human islets. From this analysis, the expression of RAB3A, STXBP1, UNC13A, VAMP2, NAPA, NSF, STX1A and SYT7 was quantified after acute MAFA or MAFB silencing in EndoC-βH1 cells and human islets. MAFA and MAFB silencing resulted in impaired insulin secretion and reduced STX1A, SYT7 and STXBP1 (EndoC-βH1) and STX1A (human islets) mRNA expression. STX1A and STXBP1 protein expression was also impaired in islets from T2D donors which lack MAFA expression.CONCLUSION: Our data indicate that STXBP1 and STX1A are important MAFA/B-regulated exocytosis genes which may contribute to insulin exocytosis defects observed in MAFA-deficient human T2D β cells.
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| 9. |
- De Marinis, Yang, et al.
(författare)
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Enhancement of glucagon secretion in mouse and human pancreatic alpha cells by protein kinase C (PKC) involves intracellular trafficking of PKCalpha and PKCdelta.
- 2010
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Ingår i: Diabetologia. - : Springer Science and Business Media LLC. - 1432-0428 .- 0012-186X. ; 53:4, s. 717-729
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Tidskriftsartikel (refereegranskat)abstract
- AIMS/HYPOTHESIS: Protein kinase C (PKC) regulates exocytosis in various secretory cells. Here we studied intracellular translocation of the PKC isoenzymes PKCalpha and PKCdelta, and investigated how activation of PKC influences glucagon secretion in mouse and human pancreatic alpha cells. METHODS: Glucagon release from intact islets was measured in static incubations, and the amounts released were determined by RIA. Exocytosis was monitored as increases in membrane capacitance using the patch-clamp technique. The expression of genes encoding PKC isoforms was analysed by real-time PCR. Intracellular PKC distribution was assessed by confocal microscopy. RESULTS: The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated glucagon secretion from mouse and human islets about fivefold (p < 0.01). This stimulation was abolished by the PKC inhibitor bisindolylmaleimide (BIM). Whereas PMA potentiated exocytosis more than threefold (p < 0.001), BIM inhibited alpha cell exocytosis by 60% (p < 0.05). In mouse islets, the PKC isoenzymes, PKCalpha and PKCbeta1, were highly abundant, while in human islets PKCeta, PKCepsilon and PKCzeta were the dominant variants. PMA stimulation of human alpha cells correlated with the translocation of PKCalpha and PKCdelta from the cytosol to the cell periphery. In the mouse alpha cells, PKCdelta was similarly affected by PMA, whereas PKCalpha was already present at the cell membrane in the absence of PMA. This association of PKCalpha in alpha cells was principally dependent on Ca(2+) influx through the L-type Ca(2+) channel. CONCLUSIONS/INTERPRETATION: PKC activation augments glucagon secretion in mouse and human alpha cells. This effect involves translocation of PKCalpha and PKCdelta to the plasma membrane, culminating in increased Ca(2+)-dependent exocytosis. In addition, we demonstrated that PKCalpha translocation and exocytosis exhibit differential Ca(2+) channel dependence.
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| 10. |
- De Marinis, Yang, et al.
(författare)
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GLP-1 inhibits and adrenaline stimulates glucagon release by differential modulation of N- and L-type Ca2+ channel-dependent exocytosis.
- 2010
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Ingår i: Cell Metabolism. - : Elsevier BV. - 1550-4131. ; 11:6, s. 543-553
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Tidskriftsartikel (refereegranskat)abstract
- Glucagon secretion is inhibited by glucagon-like peptide-1 (GLP-1) and stimulated by adrenaline. These opposing effects on glucagon secretion are mimicked by low (1-10 nM) and high (10 muM) concentrations of forskolin, respectively. The expression of GLP-1 receptors in alpha cells is <0.2% of that in beta cells. The GLP-1-induced suppression of glucagon secretion is PKA dependent, is glucose independent, and does not involve paracrine effects mediated by insulin or somatostatin. GLP-1 is without much effect on alpha cell electrical activity but selectively inhibits N-type Ca(2+) channels and exocytosis. Adrenaline stimulates alpha cell electrical activity, increases [Ca(2+)](i), enhances L-type Ca(2+) channel activity, and accelerates exocytosis. The stimulatory effect is partially PKA independent and reduced in Epac2-deficient islets. We propose that GLP-1 inhibits glucagon secretion by PKA-dependent inhibition of the N-type Ca(2+) channels via a small increase in intracellular cAMP ([cAMP](i)). Adrenaline stimulates L-type Ca(2+) channel-dependent exocytosis by activation of the low-affinity cAMP sensor Epac2 via a large increase in [cAMP](i).
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