1. |
- Brown, H, et al.
(författare)
-
Synaptotagmin III isoform is compartmentalized in pancreatic beta-cells and has a functional role in exocytosis
- 2000
-
Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 49:3, s. 383-391
-
Tidskriftsartikel (refereegranskat)abstract
- Synaptotagmin is involved in Ca2+-regulated secretion and has been suggested to serve as a general Ca2+ sensor on the membrane of secretory vesicles in neuronal cells. Insulin exocytosis from the pancreatic beta-cell is an example of a Ca2+-dependent secretory process. Previous studies of pancreatic beta-cells were unable to show presence of synaptotagmin I. We now present biochemical and immunohistochemical data showing that synaptotagmin III is present in pancreatic beta-cells as well as in the insulin-secreting cell line HIT-T15 and in rat insulinoma. By subcellular fractionation, we found synaptotagmin III in high-density fractions together with insulin and secretogranin I, indicating colocalization of synaptotagmin III and insulin in secretory granules. We could also show that blockade of synaptotagmin III by a specific antibody inhibited Ca2+-induced changes in beta-cell membrane capacitance, suggesting that synaptotagmin III is part of the functional protein complex regulating beta-cell exocytosis. The synaptotagmin III antibody did not affect the activity of the voltage-gated L-type Ca2+-channel. These findings are compatible with the view that synaptotagmin III, because of its distinct localization in the pancreatic beta-cell, functionally modulates insulin exocytosis. This indicates that synaptotagmin may have a general role in the regulation of exocytosis not only in neuronal cells but also in endocrine cells.
|
|
2. |
- Chandra, J, et al.
(författare)
-
Role of apoptosis in pancreatic beta-cell death in diabetes
- 2001
-
Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 5050 Suppl 1, s. S44-S47
-
Tidskriftsartikel (refereegranskat)abstract
- Apoptosis is a physiological form of cell death that occurs during normal development, and critical mediators of this process include caspases, reactive oxygen species, and Ca2+. Excessive apoptosis of the pancreatic beta-cell has been associated with diabetes. Consequently, apoptosis research has focused on how infiltrating macrophages or cytotoxic T-cells might kill pancreatic beta-cells using cytokines or death receptor triggering. Meanwhile, the intracellular events in the target beta-cell have been largely ignored. Elucidation of such targets might help develop improved treatment strategies for diabetes. This article will outline recent developments in apoptosis research, with emphasis on mechanisms that may be relevant to beta-cell death in type 1 and type 2 diabetes. Several of the models proposed in beta-cell killing converge on Ca2+ signaling, indicating that the pancreatic beta-cell may be an ideal system in which to carefully dissect the role of Ca2+ during apoptosis.
|
|
3. |
- Efanov, AM, et al.
(författare)
-
The novel imidazoline compound BL11282 potentiates glucose-induced insulin secretion in pancreatic beta-cells in the absence of modulation of K(ATP) channel activity
- 2001
-
Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 50:4, s. 797-802
-
Tidskriftsartikel (refereegranskat)abstract
- The insulinotropic activity of the novel imidazoline compound BL11282 was investigated. Intravenous administration of BL11282 (0.3 mg · kg–1 · min–1) to anesthetized rats did not change blood glucose and insulin levels under basal conditions, but produced a higher increase in blood insulin levels and a faster glucose removal from the blood after glucose infusion. Similarly, in isolated Wistar rat pancreatic islets, 0.1–100 μmol/l BL11282 potently stimulated glucose-induced insulin secretion but did not modulate basal insulin secretion. Unlike previously described imidazolines, BL11282 did not block ATP-dependent K+ channels. Furthermore, the compound stimulated insulin secretion in islets depolarized with high concentrations of KCl or permeabilized with electric shock. Insulinotropic activity of BL11282 was dependent on activity of protein kinases A and C. In pancreatic islets from spontaneously diabetic GK rats, the imidazoline compound restored the impaired insulin response to glucose. In conclusion, the imidazoline BL11282 constitutes a new class of insulinotropic compounds that exerts an exclusive glucose-dependent insulinotropic activity in pancreatic islets by stimulating insulin exocytosis.
|
|
4. |
- Gromada, J, et al.
(författare)
-
ATP-sensitive K+ channel-dependent regulation of glucagon release and electrical activity by glucose in wild-type and SUR1-/- mouse alpha-cells
- 2004
-
Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 5353 Suppl 3, s. S181-S189
-
Tidskriftsartikel (refereegranskat)abstract
- 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 α-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 (∼50%) was attained at glucose concentrations >5 mmol/l. The sulfonylureas tolbutamide (100 μmol/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 α-cells generate action potentials in the absence of glucose. Addition of glucose depolarized the α-cell by ∼7 mV and reduced spike height by 30% Application of tolbutamide likewise depolarized the α-cell (∼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 α-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−/− α-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 α-cells, the sugar was far less effective on [Ca2+]i in SUR1−/− α-cells. We conclude that the KATP channel is involved in the control of glucagon secretion by regulating the membrane potential in the α-cell in a way reminiscent of that previously documented in insulin-releasing β-cells. However, because α-cells possess a different complement of voltage-gated ion channels involved in action potential generation than the β-cell, moderate membrane depolarization in α-cells is associated with reduced rather than increased electrical activity and secretion.
|
|
5. |
- Ji, JZ, et al.
(författare)
-
Modulation of L-type Ca(2+) channels by distinct domains within SNAP-25
- 2002
-
Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 51:5, s. 1425-1436
-
Tidskriftsartikel (refereegranskat)abstract
- Cognate soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins are now known to associate the secretory vesicle with both the target plasma membrane and Ca2+ channels in order to mediate the sequence of events leading to exocytosis in neurons and neuroendocrine cells. Neuroendocrine cells, particularly insulin-secreting islet β-cells, t-SNARE proteins, 25-kDa synaptosomal-associated protein (SNAP-25), and syntaxin 1A, independently inhibit the L-type Ca2+ channel (LCa). However, when both are present, they actually exhibit stimulatory actions on the LCa. This suggests that the positive regulation of the LCa is conferred by a multi-SNARE protein complex. We hypothesized an alternate explanation, which is that each of these SNARE proteins possess distinct inhibitory and stimulatory domains that act on the LCa. These SNARE proteins were recently shown to bind the Lc753–893 domain corresponding to the II and III intracellular loop of the α1C subunit of the LCa. In this study, using patch-clamp methods on primary pancreatic β-cells and insulinoma HIT-T15 cells, we examined the functional interactions of the botulinum neurotoxin A (BoNT/A) cleavage products of SNAP-25, including NH2-terminal (1–197 amino acids) and COOH-terminal (amino acid 198–206) domains, on the LCa, particularly at the Lc753–893 domain. Intracellular application of SNAP-251–206 in primary β-cells decreased LCa currents by ∼15%. The reduction in LCa currents was counteracted by coapplication of Lc753–893. Overexpression or injection of wild-type SNAP-25 in HIT cells reduced LCa currents by ∼30%, and this inhibition was also blocked by the recombinant Lc753–893 peptide. Expression of BoNT/A surprisingly caused an even greater reduction of LCa currents (by 41%), suggesting that the BoNT/A cleavage products of SNAP-25 might possess distinct inhibitory and positive regulatory domains. Indeed, expression of SNAP-251–197 increased LCa currents (by 19% at 10 mV), and these effects were blocked by the Lc753–893 peptide. In contrast, injection of SNAP-25198–206 peptide into untransfected cells inhibited LCa currents (by 47%), and more remarkably, these inhibitory effects dominated over the stimulatory effects of SNAP-251–197 overexpression (by 34%). Therefore, the SNARE protein SNAP-25 possesses distinct inhibitory and stimulatory domains that act on the LCa. The COOH-terminal 197–206 domain of SNAP-25, whose inhibitory actions dominate over the opposing stimulatory NH2-terminal domain, likely confers the inhibitory actions of SNAP-25 on the LCa. We postulate that the eventual accelerated proteolysis of SNAP-25 brought about by BoNT/A cleavage allows the relatively intact NH2-terminal SNAP-25 domain to assert its stimulatory action on the LCa to increase Ca2+ influx, and this could in part explain the observed weak or inconsistent inhibitory effects of BoNT/A on insulin secretion. The present study suggests that distinct domains within SNAP-25 modulate LC subtype Ca2+ channel activity in both primary β-cells and insulinoma HIT-T15 cells.
|
|
6. |
- Kamp, F, et al.
(författare)
-
Sulfonylureas rapidly cross phospholipid bilayer membranes by a free-diffusion mechanism
- 2003
-
Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 52:10, s. 2526-2531
-
Tidskriftsartikel (refereegranskat)abstract
- Because sulfonylureas directly activate the exocytotic machinery, we were interested in the extent to which these compounds penetrate the β-cell plasma membrane and the underlying molecular mechanism(s). We now provide evidence that sulfonylureas cross phospholipid bilayer membranes rapidly and effectively by a free-diffusion mechanism. Two sulfonylurea compounds investigated by 1H nuclear magnetic resonance spectroscopy, glibenclamide and tolbutamide, were found to incorporate into phospholipid bilayers, with the ionizable sulfonamide exposed to the aqueous interface and its apparent dissociation constant (pKa) increased to ∼7.0. Diffusion of weak amphiphilic acids across membranes is associated with a measurable change in pH. Thus, by using a fluorescence-based pH assay, we could investigate the diffusion of sulfonylurea compounds across phospholipid bilayer membranes. A fluorescent pH indicator (pyranin or [2′,7′-bis (2-carboxyethyl)-5(6)-carboxyfluorescein] [BCECF]) was trapped in egg phosphatidylcholine vesicles. Addition of glibenclamide decreased internal pH (pHin), and addition of albumin reversed this drop by 50%. With the same amount of tolbutamide, the decrease in pHin was much smaller, primarily because of the lower partitioning of tolbutamide into phospholipid bilayers. Using similar protocols, we also demonstrated diffusion by the same mechanism across the β-cell plasma membrane. Thus, we now provide a molecular mechanism by which sulfonylureas can penetrate the plasma membrane and reach intracellular sites regulating exocytosis.
|
|
7. |
- Kohler, M, et al.
(författare)
-
On-line monitoring of apoptosis in insulin-secreting cells
- 2003
-
Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 52:12, s. 2943-2950
-
Tidskriftsartikel (refereegranskat)abstract
- Apoptosis was monitored in intact insulin-producing cells both with microfluorometry and with two-photon laser scanning microscopy (TPLSM), using a fluorescent protein based on fluorescence resonance energy transfer (FRET). TPLSM offers three-dimensional spatial information that can be obtained relatively deep in tissues. This provides a potential for future in vivo studies of apoptosis. The cells expressed a fluorescent protein (C-DEVD-Y) consisting of two fluorophores, enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), linked by the amino acid sequence DEVD selectively cleaved by caspase-3–like proteases. FRET between ECFP and EYFP in C-DEVD-Y could therefore be monitored on-line as a sensor of caspase-3 activation. The relevance of using caspase-3 activation to indicate β-cell apoptosis was demonstrated by inhibiting caspase-3–like proteases with Z-DEVD-fmk and thereby showing that caspase-3 activation was needed for high-glucose–and cytokine-induced apoptosis in the β-cell and for staurosporine-induced apoptosis in RINm5F cells. In intact RINm5F cells expressing C-DEVD-Y and in MIN6 cells expressing the variant C-DEVD-Y2, FRET was lost at 155 ± 23 min (n = 9) and 257 ± 59 min (n = 4; mean ± SE) after activation of apoptosis with staurosporine (6 μmol/l), showing that this method worked in insulin-producing cells.
|
|
8. |
- Larsson, O, et al.
(författare)
-
Phosphatidylinositol 4,5-bisphosphate and ATP-sensitive potassium channel regulation: a word of caution
- 2000
-
Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 49:9, s. 1409-1412
-
Tidskriftsartikel (refereegranskat)abstract
- Phosphatidylinositol 4,5-bisphosphate (PIP2) has been suggested to play an important role as an endogenous regulator of ATP-sensitive potassium (KATP) channels consisting of Kir6.2 as a pore-forming subunit. These studies show the ability of PIP2 to activate KATP channel activity and to counteract the inhibitory effect of ATP, implying that PIP2 could serve the function of modulating the sensitivity of KATP channels to the cytoplasmic free ATP concentration. Careful examination of the literature reveals that the definitive physiologically relevant experiments to establish efficacy of PIP2 on this channel may still have to be performed. Our reservations are based on the handling of PIP2 in cell-free experiments and in various strategies designed to modulate PIP2 concentrations in intact cells. Furthermore, a potent stimulatory effect of phosphatidylinositol 3,4,5trisphosphate, a downstream metabolite of PIP2, on KATP channel activity raises the possibility that the effects on the KATP channel may not be directly related to PIP2.
|
|
9. |
- Zaitsev, SV, et al.
(författare)
-
Imidazoline compounds protect against interleukin 1beta-induced beta-cell apoptosis
- 2001
-
Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 5050 Suppl 1, s. S70-S76
-
Tidskriftsartikel (refereegranskat)abstract
- Imidazoline compounds have been considered for the treatment of type 2 diabetes. We have now investigated the effects of imidazolines on interleukin (IL)-1beta-induced beta-cell apoptosis and the signal transduction pathways involved. Inhibition of Ca2+ influx into beta-cells by D-600, a blocker of voltage-gated L-type Ca2+ channels, suppressed IL-1beta-induced apoptosis. Our data show that calcineurin, Ca2+/calmodulin-dependent serine/threonine protein phosphatase 2B, is responsible for the effect of Ca2+ on beta-cell apoptosis. We also demonstrate that IL-1beta-mediated apoptosis correlates with expression of inducible nitric oxide synthase (iNOS) and the increase in intracellular production of nitric oxide. An inhibitor of cGMP-dependent protein kinase (PKG), KT5823, suppressed IL-1beta-induced apoptosis, suggesting the involvement of a PKG-dependent pathway in the apoptotic process. One of the major findings in this study is that imidazoline compounds RX871024 and efaroxan, suggested as prototypes of a new generation of drugs against type 2 diabetes, can protect against IL-1beta-induced apoptosis in pancreatic beta-cells, possibly by their inhibition of the expression of iNOS, a key element in the IL-1beta-induced apoptotic pathway in pancreatic beta-cells. These data suggest that imidazoline compounds should be explored as a potential therapeutic agent for the treatment of both type 1 and type 2 diabetes.
|
|
10. |
- Zhuang, H, et al.
(författare)
-
Cloning of a T-type Ca2+ channel isoform in insulin-secreting cells
- 2000
-
Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 49:1, s. 59-64
-
Tidskriftsartikel (refereegranskat)abstract
- The T-type Ca2+ channel is an important determinant of electrical activity and of Ca2+ influx in rat and human pancreatic beta-cells. We have identified and sequenced a cDNA encoding a T-type Ca2+ channel alpha1-subunit derived from INS-1, the rat insulin-secreting cell line. The sequence of the cDNA indicates a protein composed of 2,288 amino acids that shares 96.3% identity to alpha1G, the neuronal T-type Ca2+ channel subunit. The transmembrane domains of the protein are highly conserved, but the isoform contains three distinct regions and 10 single amino acid substitutions in other regions. Sequencing rat genomic DNA revealed that the alpha1-subunit we cloned is an alternative splice isoform of alpha1G. By using specific primers and reverse transcription-polymerase chain reaction, we demonstrated that both splice variants are expressed in rat islets. The isoform deduced from INS-1 was also expressed in brain, neonatal heart, and kidney. Functional expression of this alpha1G isoform in Xenopus oocytes generated low voltage-activated Ba2+ currents. These results provide the molecular biological basis for studies of function of T-type Ca2+ channels in beta-cells, which is where these channels may play critical roles in diabetes.
|
|