| 1. |
- Cheviet, Severine, et al.
(författare)
-
Tomosyn-1 is involved in a post-docking event required for pancreatic beta-cell exocytosis
- 2006
-
Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 119:14, s. 2912-2920
-
Tidskriftsartikel (refereegranskat)abstract
- Although the assembly of a ternary complex between the SNARE proteins syntaxin-1, SNAP25 and VAMP2 is known to be crucial for insulin exocytosis, the mechanisms controlling this key event are poorly understood. We found that pancreatic beta-cells express different isoforms of tomosyn-1, a syntaxin-1-binding protein possessing a SNARE-like motif. Using atomic force microscopy we show that the SNARE-like domain of tomosyn-1 can form a complex with syntaxin-1 and SNAP25 but displays binding forces that are weaker than those observed for VAMP2 (237 +/- 13 versus 279 +/- 3 pN). In pancreatic beta-cells tomosyn-1 was found to be concentrated in cellular compartments enriched in insulin-containing secretory granules. Silencing of tomosyn-1 in the rat beta-cell line INS-1E by RNA interference did not affect the number of secretory granules docked at the plasma membrane but led to a reduction in stimulus-induced exocytosis. Replacement of endogenous tomosyn-1 with mouse tomosyn-1, which differs in the nucleotide sequence from its rat homologue and escapes silencing, restored a normal secretory rate. Taken together, our data suggest that tomosyn-1 is involved in a post-docking event that prepares secretory granules for fusion and is necessary to sustain exocytosis of pancreatic beta-cells in response to insulin secretagogues.
|
|
| 2. |
- Härndahl, Linda, et al.
(författare)
-
Important role of phosphodiesterase 3B for the stimulatory action of cAMP on pancreatic beta -cell exocytosis and release of insulin.
- 2002
-
Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 277:40, s. 37446-37455
-
Tidskriftsartikel (refereegranskat)abstract
- Cyclic AMP potentiates glucose-stimulated insulin release and mediates the stimulatory effects of hormones such as glucagon-like peptide 1 (GLP-1) on pancreatic b-cells. By inhibition of cAMP-degrading phosphodiesterase (PDE) and, in particular, selective inhibition of PDE3 activity, stimulatory effects on insulin secretion have been observed. Molecular and functional information on b-cell PDE3 is, however, scarce. To provide such information, we have studied the specific effects of the PDE3B isoform by adenovirus-mediated overexpression. In rat islets and rat insulinoma cells, approximate 10-fold overexpression of PDE3B was accompanied by a 6-8-fold increase in membrane-associated PDE3B activity. The cAMP concentration was significantly lowered in transduced cells (INS-1(832/13), and insulin secretion in response to stimulation with high glucose (11.1 mM) was reduced by 40% (islets) and 50% (INS-1). Further, the ability of GLP-1 (100 nM) to augment glucose-stimulated insulin secretion was inhibited by approximately 30% (islets) and 70% (INS-1). Accordingly, when stimulating with cAMP, a substantial decrease (65%) in exocytotic capacity was demonstrated in patch-clamped single b-cells. In untransduced insulinoma cells, application of the PDE3-selective inhibitor OPC3911 (10 mM) was shown to increase glucose-stimulated insulin release as well as cAMP-enhanced exocytosis. The findings suggest a significant role of PDE3B as an important regulator of insulin secretory processes.
|
|
| 3. |
- Ivarsson, Rosita, et al.
(författare)
-
Myosin 5a controls insulin granule recruitment during late-phase secretion.
- 2005
-
Ingår i: Traffic: the International Journal of Intracellular Transport. - : Wiley. - 1398-9219. ; 6:11, s. 1027-1035
-
Tidskriftsartikel (refereegranskat)abstract
- We have examined the importance of the actin-based molecular motor myosin 5a for insulin granule transport and insulin secretion. Expression of myosin 5a was downregulated in clonal INS-1E cells using RNAinterference. Stimulated hormone secretion was reduced by 46% and single-cell exocytosis, measured by capacitance recordings, was inhibited by 42% after silencing. Silencing of Slac-2c/MYRIP, which links insulin granules to myosin 5a, resulted in similar inhibition of single-cell exocytosis. Antibody inhibition of the myosin 5a-Slac-2c/MYRIP interaction significantly reduced the recruitment of insulin granules for release. The pool of releasable granules independent of myosin 5a activity was estimated to approximately 550 granules. Total internal reflection microscopy was then applied to directly investigate granule recruitment to the plasma membrane. Silencing of myosin 5a inhibited granule recruitment during late phase of insulin secretion. In conclusion, we propose a model where insulin granules are transported through the actin network via both myosin 5a-mediated transport and via passive diffusion, with the former playing the major role during stimulatory conditions.
|
|
| 4. |
- Ivarsson, Rosita, et al.
(författare)
-
Redox control of exocytosis - Regulatory role of NADPH, thioredoxin, and glutaredoxin
- 2005
-
Ingår i: Diabetes. - 1939-327X. ; 54:7, s. 2132-2142
-
Tidskriftsartikel (refereegranskat)abstract
- Cellular redox state is an important metabolic variable, influencing many aspects of cell function like growth, apoptosis, and reductive biosynthesis. In this report, we identify NADPH as a candidate signaling molecule for exocytosis in neuroendocrine cells. In pancreatic beta-cells, glucose acutely raised the NADPH-to-NADP(+) ratio and stimulated insulin release in parallel. Furthermore, intracellular addition of NADPH directly stimulated exocytosis of insulin granules. Effects of NADPH on exocytosis are proposed to be mediated by the redox proteins glutaredoxin (GRX) and thioredoxin (TRX) on the basis of the following evidence: 1) Expression of GRX mRNA is very high in beta-cells compared with other studied tissues, and GRX protein expression is high in islets and in brain; 2) GRX and TRX are localized in distinct microdomains in the cytosol of beta-cells; and 3) microinjection of recombinant GRX potentiated effects of NADPH on exocytosis, whereas TRX antagonized the NADPH effect. We propose that the NADPEVGRX/ TRX redox regulation mediates a novel signaling pathway of nutrient-induced insulin secretion.
|
|
| 5. |
- Ivarsson, Rosita, et al.
(författare)
-
Temperature-Sensitive Random Insulin Granule Diffusion is a Prerequisite for Recruiting Granules for Release.
- 2004
-
Ingår i: Traffic: the International Journal of Intracellular Transport. - : Wiley. - 1398-9219. ; 5:10, s. 750-762
-
Tidskriftsartikel (refereegranskat)abstract
- Glucose-evoked insulin secretion exhibits a biphasic time course and is associated with accelerated intracellular granule movement. We combined live confocal imaging of EGFP-labelled insulin granules with capacitance measurements of exocytosis in clonal INS-1 cells to explore the relation between distinct random and directed modes of insulin granule movement, as well as exocytotic capacity. Reducing the temperature from 34 °C to 24 °C caused a dramatic 81% drop in the frequency of directed events, but reduced directed velocities by a mere 25%. The much stronger temperature sensitivity of the frequency of directed events (estimated energy of activation ~ 135 kJ/mol) than that of the granule velocities (~ 22 kJ/mol) suggests that cooling-induced suppression of insulin granule movement is attributable to factors other than reduced motor protein adenosine 5'-triphosphatase activity. Indeed, cooling suppresses random granule diffusion by ~ 50%. In the single cell, the number of directed events depends on the extent of granule diffusion. Finally, single-cell exocytosis exhibits a biphasic pattern corresponding to that observed in vivo, and only the component reflecting 2nd phase insulin secretion is affected by cooling. We conclude that random diffusive movement is a prerequisite for directed insulin granule transport and for the recruitment of insulin granules released during 2nd phase insulin secretion.
|
|
| 6. |
- Ivarsson, Rosita
(författare)
-
Transport and release of insulin granules during biphasic insulin secretion
- 2005
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Glucose-evoked insulin secretion exhibits two phases: The first phase represents exocytosis of insulin granules situated at the plasma membrane, whereas the second phase reflects insulin release from granules continuously recruited to the plasma membrane. This thesis investigates different aspects of phasic insulin secretion, with the specific aims to elucidate the possible contribution of insulin granule transport, as well as to study the influence of the nucleotides cAMP and NADPH to this process. Time-lapse confocal imaging revealed that insulin granules move by an interplay of directed and random movements. The directed movements, representing kinesin-mediated transport of insulin granules along microtubules, constitute an efficient mechanism to transfer granules, whereas random movements are slow but essential for facilitating the interaction between granules and motorproteins. Glucose accelerated granule velocities by ~50%, but had only modest effects on the frequency of directed events. In contrast, cAMP elevated the number of translocations by ~90%, but had no effect on average granule velocity. This indicates that granule mobility is a regulated process and that different stimulators of insulin secretion influence granule mobility via distinct mechanisms. Near the plasma membrane granules are transported through the actin web via myosin 5a-mediated translocations. Silencing of myosin 5a expression using RNAinterference inhibited hormone secretion by 46% under stimulatory conditions. Myosin 5a-mediated transport was accelerated by glucose during late phase of insulin secretion, supporting the view that granules released at this stage are recruited from the inner side of the actin network. The possible role of NADPH and cAMP for granule transport and phasic insulin secretion was also investigated. Capacitance recordings of insulin exocytosis revealed that physiological concentrations of NADPH stimulated insulin release by 84% and 102% in mouse and rat beta-cells, respectively. NADPH was also found to increase in response to glucose, which suggests NADPH as a possible candidate metabolite for the amplifying pathway of glucose-evoked insulin secretion. Furthermore, infusion of NADPH in combination with the redox protein glutaredoxin stimulated insulin secretion by additional 54%, indicating that glutaredoxin acts as an NADPH effector. Finally, we demonstrated that the nature of cAMP-signals are transient (~40 s) but in spite of this are able to exert a time-dependent potentiation, or memory effect, on insulin secretion and granule mobility. We speculate that this potentiation might be due to elevated recruitment of new granules to the release sites at the plasma membrane. In conclusion: (1) Physical translocation of insulin granules is a regulated process that is stimulated during second phase insulin secretion. (2) Kinesin 1- and myosin 5a-mediated translocations in combination with diffusional movements are essential for efficient recruitment of new granules to the plasma membrane. (3) The potentiating effect of cAMP on insulin secretion might at least in part be a result of elevated granule transport. And finally (4) NADPH is a candidate for mediating the stimulatory effect of glucose on second phase insulin secretion.
|
|
| 7. |
- Jing, Xingjun, et al.
(författare)
-
CaV2.3 calcium channels control second-phase insulin release.
- 2005
-
Ingår i: Journal of Clinical Investigation. - 0021-9738. ; 115:1, s. 146-154
-
Tidskriftsartikel (refereegranskat)abstract
- Concerted activation of different voltage-gated Ca2+ channel isoforms may determine the kinetics of insulin release from pancreatic islets. Here we have elucidated the role of R-type CaV2.3 channels in that process. A 20% reduction in glucose-evoked insulin secretion was observed in CaV2.3-knockout (CaV2.3–/–) islets, close to the 17% inhibition by the R-type blocker SNX482 but much less than the 77% inhibition produced by the L-type Ca2+ channel antagonist isradipine. Dynamic insulin-release measurements revealed that genetic or pharmacological CaV2.3 ablation strongly suppressed second-phase secretion, whereas first-phase secretion was unaffected, a result also observed in vivo. Suppression of the second phase coincided with an 18% reduction in oscillatory Ca2+ signaling and a 25% reduction in granule recruitment after completion of the initial exocytotic burst in single CaV2.3–/– ß cells. CaV2.3 ablation also impaired glucose-mediated suppression of glucagon secretion in isolated islets (27% versus 58% in WT), an effect associated with coexpression of insulin and glucagon in a fraction of the islet cells in the CaV2.3–/– mouse. We propose a specific role for CaV2.3 Ca2+ channels in second-phase insulin release, that of mediating the Ca2+ entry needed for replenishment of the releasable pool of granules as well as islet cell differentiation.
|
|
| 8. |
- Reinbothe, Thomas, 1981, et al.
(författare)
-
Glutaredoxin-1 mediates NADPH-dependent stimulation of calcium-dependent insulin secretion
- 2009
-
Ingår i: Mol Endocrinol. - : The Endocrine Society. - 1944-9917 .- 0888-8809. ; 23:6, s. 893-900
-
Tidskriftsartikel (refereegranskat)abstract
- Nicotinamide adenine dinucleotide phosphate (NADPH) enhances Ca(2+)-induced exocytosis in pancreatic beta-cells, an effect suggested to involve the cytosolic redox protein glutaredoxin-1 (GRX-1). We here detail the role of GRX-1 in NADPH-stimulated beta-cell exocytosis and glucose-stimulated insulin secretion. Silencing of GRX-1 by RNA interference reduced glucose-stimulated insulin secretion in both clonal INS-1 832/13 cells and primary rat islets. GRX-1 silencing did not affect cell viability or the intracellular redox environment, suggesting that GRX-1 regulates the exocytotic machinery by a local action. By contrast, knockdown of the related protein thioredoxin-1 (TRX-1) was ineffective. Confocal immunocytochemistry revealed that GRX-1 locates to the cell periphery, whereas TRX-1 expression is uniform. These data suggest that the distinct subcellular localizations of TRX-1 and GRX-1 result in differences in substrate specificities and actions on insulin secretion. Single-cell exocytosis was likewise suppressed by GRX-1 knockdown in both rat beta-cells and clonal 832/13 cells, whereas after overexpression exocytosis increased by approximately 40%. Intracellular addition of NADPH (0.1 mm) stimulated Ca(2+)-evoked exocytosis in both cell types. Interestingly, the stimulatory action of NADPH on the exocytotic machinery coincided with an approximately 30% inhibition in whole-cell Ca(2+) currents. After GRX-1 silencing, NADPH failed to amplify insulin release but still inhibited Ca(2+) currents in 832/13 cells. In conclusion, NADPH stimulates the exocytotic machinery in pancreatic beta-cells. This effect is mediated by the NADPH acceptor protein GRX-1 by a local redox reaction that accelerates beta-cell exocytosis and, in turn, insulin secretion.
|
|
| 9. |
- Renström, Erik, et al.
(författare)
-
Inositol 3,4,5,6-tetrakisphosphate inhibits insulin granule acidification and fusogenic potential.
- 2002
-
Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 277:30, s. 26717-26720
-
Tidskriftsartikel (refereegranskat)abstract
- ClC Cl(-) channels in endosomes, synaptosomes, lysosomes, and beta-cell insulin granules provide charge neutralization support for the functionally indispensable acidification of the luminal interior by electrogenic H(+)-ATPases (Jentsch, T. J., Stein, V., Weinreich, F., and Zdebik, A. A. (2002) Physiol. Rev. 82, 503-568). Regulation of ClC activity is, therefore, of widespread biological significance (Forgac, M. (1999) J. Biol. Chem. 274, 12951-12954). We now ascribe just such a regulatory function to the increases in cellular levels of inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P(4)) that inevitably accompany activation of the ubiquitous Ins(1,4,5)P(3) signaling pathway. We used confocal imaging to record insulin granule acidification in single mouse pancreatic beta-cells. Granule acidification was reduced by perfusion of single cells with 10 microm Ins(3,4,5,6)P(4) (the concentration following receptor activation), whereas at 1 microm ("resting" levels), Ins(3,4,5,6)P(4) was ineffective. This response to Ins(3,4,5,6)P(4) was not mimicked by 100 microm Ins(1,4,5,6)P(4) or by 100 microm Ins(1,3,4,5,6)P(5). Ins(3,4,5,6)P(4) did not affect granular H(+)-ATPase activity or H(+) leak, indicating that Ins(3,4,5,6)P(4) instead inhibited charge neutralization by ClC. The Ins(3,4,5,6)P(4)-mediated inhibition of vesicle acidification reduced exocytic release of insulin as determined by whole-cell capacitance recordings. This may impinge upon type 2 diabetes etiology. Regulatory control over vesicle acidification by this negative signaling pathway in other cell types should be considered.
|
|
