Limits...
Granuphilin molecularly docks insulin granules to the fusion machinery.

Gomi H, Mizutani S, Kasai K, Itohara S, Izumi T - J. Cell Biol. (2005)

Bottom Line: The Rab27a effector granuphilin is specifically localized on insulin granules and is involved in their exocytosis.Here we show that the number of insulin granules morphologically docked to the plasma membrane is markedly reduced in granuphilin-deficient beta cells.The enhanced secretion in mutant beta cells is correlated with a decrease in the formation of the fusion-incompetent syntaxin-1a-Munc18-1 complex, with which granuphilin normally interacts.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Endocrinology and Metabolism, Institute for Molecular and Cellular Regulation, Gunma University, Gunma 371-8512, Japan.

ABSTRACT
The Rab27a effector granuphilin is specifically localized on insulin granules and is involved in their exocytosis. Here we show that the number of insulin granules morphologically docked to the plasma membrane is markedly reduced in granuphilin-deficient beta cells. Surprisingly, despite the docking defect, the exocytosis of insulin granules in response to a physiological glucose stimulus is significantly augmented, which results in increased glucose tolerance in granuphilin- mice. The enhanced secretion in mutant beta cells is correlated with a decrease in the formation of the fusion-incompetent syntaxin-1a-Munc18-1 complex, with which granuphilin normally interacts. Furthermore, in contrast to wild-type granuphilin, its mutant that is defective in binding to syntaxin-1a fails to restore granule docking or the protein level of syntaxin-1a in granuphilin- beta cells. Thus, granuphilin not only is essential for the docking of insulin granules but simultaneously imposes a fusion constraint on them through an interaction with the syntaxin-1a fusion machinery. These findings provide a novel paradigm for the docking machinery in regulated exocytosis.

Show MeSH

Related in: MedlinePlus

Insulin secretion profiles in perifused islets. Islets isolated from age-matched (15- to 20-wk-old) male Grn+/Y (open squares) or Grn−/Y (closed circles) mice were perifused with standard LG (2.8 mM) Krebs-Ringer buffer for 30 min. Thereafter, the collection of each fraction (1 ml/min) was started, and an appropriate secretagogue was applied 10 min after the start. (A) Single stimulation by HG buffer. Islets were perifused with 16.7 mM glucose buffer for 30 min (horizontal black line) followed by 2.8 mM glucose buffer for 20 min (n = 5). (B) Repeated stimulation by 16.7 mM glucose buffer for 20 min (n = 4). (C) Stimulation by graded increase of glucose concentrations (dashed line) from 2.8 to 7.8, 16.7, and 33.4 mM each for 15 min (n = 6). (D–F) Depolarizing stimulation by high K+ buffer containing 20 mM KCl and 105 mM NaCl (D), 30 mM KCl and 95 mM NaCl (E), or 60 mM KCl and 65 mM NaCl (F) for 15 min (n = 5). (G and H) Application of 10 μM forskolin (G; n = 6) or 0.5 μM phorbol-12-myristate-13-acetate (H; n = 9). In the continuous presence of either drug (black and gray lines), islets were stimulated by 16.7 mM glucose buffer for 20 min (black line), with pre- and postincubation stimulation of 2.8 mM glucose buffer for 15 min (gray line). (I) Basal secretion. Data for the secretion at 2.8 mM glucose before the application of secretagogues in panels A–C, G, and H are combined (n = 30). Results are provided as mean ± SEM.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171228&req=5

fig4: Insulin secretion profiles in perifused islets. Islets isolated from age-matched (15- to 20-wk-old) male Grn+/Y (open squares) or Grn−/Y (closed circles) mice were perifused with standard LG (2.8 mM) Krebs-Ringer buffer for 30 min. Thereafter, the collection of each fraction (1 ml/min) was started, and an appropriate secretagogue was applied 10 min after the start. (A) Single stimulation by HG buffer. Islets were perifused with 16.7 mM glucose buffer for 30 min (horizontal black line) followed by 2.8 mM glucose buffer for 20 min (n = 5). (B) Repeated stimulation by 16.7 mM glucose buffer for 20 min (n = 4). (C) Stimulation by graded increase of glucose concentrations (dashed line) from 2.8 to 7.8, 16.7, and 33.4 mM each for 15 min (n = 6). (D–F) Depolarizing stimulation by high K+ buffer containing 20 mM KCl and 105 mM NaCl (D), 30 mM KCl and 95 mM NaCl (E), or 60 mM KCl and 65 mM NaCl (F) for 15 min (n = 5). (G and H) Application of 10 μM forskolin (G; n = 6) or 0.5 μM phorbol-12-myristate-13-acetate (H; n = 9). In the continuous presence of either drug (black and gray lines), islets were stimulated by 16.7 mM glucose buffer for 20 min (black line), with pre- and postincubation stimulation of 2.8 mM glucose buffer for 15 min (gray line). (I) Basal secretion. Data for the secretion at 2.8 mM glucose before the application of secretagogues in panels A–C, G, and H are combined (n = 30). Results are provided as mean ± SEM.

Mentions: To examine whether the reduced number of docked granules affects the insulin secretion ability, we performed perifusion analyses in isolated islets. In the granuphilin- islets, insulin secretion in response to 16.7 mM glucose was significantly enhanced in both the first and second phases, by ∼200% of the control levels (P < 0.05; Fig. 4 A), although the peak of first-phase secretion was delayed by ∼4 min. The increase of insulin secretion was observable after repeated exposure to high concentrations of glucose but became less evident with later stimuli (Fig. 4 B). Stimulation by graded increases of glucose concentrations again showed a higher insulin response (P < 0.003) but no significant change in glucose sensitivity (Fig. 4 C). Depolarization by a high KCl concentration (20 mM) also evoked stronger insulin release in the mutant islets (P < 0.005; Fig. 4 D). The application of a higher KCl concentration (30 and 60 mM) induced similar but not statistically significant increases (Fig. 4, E and F). Although the effect of forskolin (an activator of adenylate cyclase) in the presence of high glucose was indistinguishable between the mutant and control islets (Fig. 4 G), phorbol-12-myristate-13-acetate (a PKC activator) enhanced the release of insulin in the mutant islets (P < 0.002; Fig. 4 H). The differential effects of each secretagogue likely reflect differences in its mode of granule recruitment for fusion. Overall, in our experiments, the basal secretion was slightly but not significantly increased in the granuphilin-deficient islets (Fig. 4 I). The observed enhancement of insulin secretion in response to physiological or some nonphysiological stimuli in the granuphilin- β cells is consistent with previous findings that the overexpression of granuphilin in cultured β cell lines profoundly inhibits stimulus-induced secretion (Coppola et al., 2002; Torii et al., 2002). Therefore, granuphilin plays a negative regulatory role in secretagogue-evoked insulin secretion.


Granuphilin molecularly docks insulin granules to the fusion machinery.

Gomi H, Mizutani S, Kasai K, Itohara S, Izumi T - J. Cell Biol. (2005)

Insulin secretion profiles in perifused islets. Islets isolated from age-matched (15- to 20-wk-old) male Grn+/Y (open squares) or Grn−/Y (closed circles) mice were perifused with standard LG (2.8 mM) Krebs-Ringer buffer for 30 min. Thereafter, the collection of each fraction (1 ml/min) was started, and an appropriate secretagogue was applied 10 min after the start. (A) Single stimulation by HG buffer. Islets were perifused with 16.7 mM glucose buffer for 30 min (horizontal black line) followed by 2.8 mM glucose buffer for 20 min (n = 5). (B) Repeated stimulation by 16.7 mM glucose buffer for 20 min (n = 4). (C) Stimulation by graded increase of glucose concentrations (dashed line) from 2.8 to 7.8, 16.7, and 33.4 mM each for 15 min (n = 6). (D–F) Depolarizing stimulation by high K+ buffer containing 20 mM KCl and 105 mM NaCl (D), 30 mM KCl and 95 mM NaCl (E), or 60 mM KCl and 65 mM NaCl (F) for 15 min (n = 5). (G and H) Application of 10 μM forskolin (G; n = 6) or 0.5 μM phorbol-12-myristate-13-acetate (H; n = 9). In the continuous presence of either drug (black and gray lines), islets were stimulated by 16.7 mM glucose buffer for 20 min (black line), with pre- and postincubation stimulation of 2.8 mM glucose buffer for 15 min (gray line). (I) Basal secretion. Data for the secretion at 2.8 mM glucose before the application of secretagogues in panels A–C, G, and H are combined (n = 30). Results are provided as mean ± SEM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171228&req=5

fig4: Insulin secretion profiles in perifused islets. Islets isolated from age-matched (15- to 20-wk-old) male Grn+/Y (open squares) or Grn−/Y (closed circles) mice were perifused with standard LG (2.8 mM) Krebs-Ringer buffer for 30 min. Thereafter, the collection of each fraction (1 ml/min) was started, and an appropriate secretagogue was applied 10 min after the start. (A) Single stimulation by HG buffer. Islets were perifused with 16.7 mM glucose buffer for 30 min (horizontal black line) followed by 2.8 mM glucose buffer for 20 min (n = 5). (B) Repeated stimulation by 16.7 mM glucose buffer for 20 min (n = 4). (C) Stimulation by graded increase of glucose concentrations (dashed line) from 2.8 to 7.8, 16.7, and 33.4 mM each for 15 min (n = 6). (D–F) Depolarizing stimulation by high K+ buffer containing 20 mM KCl and 105 mM NaCl (D), 30 mM KCl and 95 mM NaCl (E), or 60 mM KCl and 65 mM NaCl (F) for 15 min (n = 5). (G and H) Application of 10 μM forskolin (G; n = 6) or 0.5 μM phorbol-12-myristate-13-acetate (H; n = 9). In the continuous presence of either drug (black and gray lines), islets were stimulated by 16.7 mM glucose buffer for 20 min (black line), with pre- and postincubation stimulation of 2.8 mM glucose buffer for 15 min (gray line). (I) Basal secretion. Data for the secretion at 2.8 mM glucose before the application of secretagogues in panels A–C, G, and H are combined (n = 30). Results are provided as mean ± SEM.
Mentions: To examine whether the reduced number of docked granules affects the insulin secretion ability, we performed perifusion analyses in isolated islets. In the granuphilin- islets, insulin secretion in response to 16.7 mM glucose was significantly enhanced in both the first and second phases, by ∼200% of the control levels (P < 0.05; Fig. 4 A), although the peak of first-phase secretion was delayed by ∼4 min. The increase of insulin secretion was observable after repeated exposure to high concentrations of glucose but became less evident with later stimuli (Fig. 4 B). Stimulation by graded increases of glucose concentrations again showed a higher insulin response (P < 0.003) but no significant change in glucose sensitivity (Fig. 4 C). Depolarization by a high KCl concentration (20 mM) also evoked stronger insulin release in the mutant islets (P < 0.005; Fig. 4 D). The application of a higher KCl concentration (30 and 60 mM) induced similar but not statistically significant increases (Fig. 4, E and F). Although the effect of forskolin (an activator of adenylate cyclase) in the presence of high glucose was indistinguishable between the mutant and control islets (Fig. 4 G), phorbol-12-myristate-13-acetate (a PKC activator) enhanced the release of insulin in the mutant islets (P < 0.002; Fig. 4 H). The differential effects of each secretagogue likely reflect differences in its mode of granule recruitment for fusion. Overall, in our experiments, the basal secretion was slightly but not significantly increased in the granuphilin-deficient islets (Fig. 4 I). The observed enhancement of insulin secretion in response to physiological or some nonphysiological stimuli in the granuphilin- β cells is consistent with previous findings that the overexpression of granuphilin in cultured β cell lines profoundly inhibits stimulus-induced secretion (Coppola et al., 2002; Torii et al., 2002). Therefore, granuphilin plays a negative regulatory role in secretagogue-evoked insulin secretion.

Bottom Line: The Rab27a effector granuphilin is specifically localized on insulin granules and is involved in their exocytosis.Here we show that the number of insulin granules morphologically docked to the plasma membrane is markedly reduced in granuphilin-deficient beta cells.The enhanced secretion in mutant beta cells is correlated with a decrease in the formation of the fusion-incompetent syntaxin-1a-Munc18-1 complex, with which granuphilin normally interacts.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Endocrinology and Metabolism, Institute for Molecular and Cellular Regulation, Gunma University, Gunma 371-8512, Japan.

ABSTRACT
The Rab27a effector granuphilin is specifically localized on insulin granules and is involved in their exocytosis. Here we show that the number of insulin granules morphologically docked to the plasma membrane is markedly reduced in granuphilin-deficient beta cells. Surprisingly, despite the docking defect, the exocytosis of insulin granules in response to a physiological glucose stimulus is significantly augmented, which results in increased glucose tolerance in granuphilin- mice. The enhanced secretion in mutant beta cells is correlated with a decrease in the formation of the fusion-incompetent syntaxin-1a-Munc18-1 complex, with which granuphilin normally interacts. Furthermore, in contrast to wild-type granuphilin, its mutant that is defective in binding to syntaxin-1a fails to restore granule docking or the protein level of syntaxin-1a in granuphilin- beta cells. Thus, granuphilin not only is essential for the docking of insulin granules but simultaneously imposes a fusion constraint on them through an interaction with the syntaxin-1a fusion machinery. These findings provide a novel paradigm for the docking machinery in regulated exocytosis.

Show MeSH
Related in: MedlinePlus