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Genes to Cells (2009) 14, 445-456. doi:10.1111/j.1365-2443.2009.01285.x
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Rab11 and its effector Rip11 participate in regulation of insulin granule exocytosis

 Kenji Sugawara1, Tadao Shibasaki1, Akira Mizoguchi3, Tetsuya Saito1 and Susumu Seino1,2,4

1 Division of Cellular and Molecular Medicine, Department of Physiology and Cell Biology and
2 Division of Diabetes, Metabolism and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan
3 Department of Neural Regeneration and Cell Communication, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan
4 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corp., Kawaguchi, Saitama 332–0012, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Rab GTPases and their effectors play important roles in membrane trafficking between cellular compartments in eukaryotic cells. In the present study, we examined the roles of Rab11B and its effectors in insulin secretion in pancreatic β-cells. In the mouse insulin-secreting cell line MIN6, Rab11 was co-localized with insulin-containing granules, and over-expression of the GTP- or the GDP-bound form of Rab11B significantly inhibited regulated secretion, indicating involvement of Rab11B in regulated insulin secretion. To determine the downstream signal of Rab11-mediated insulin secretion, we examined the effects of various Rab11-interacting proteins on insulin secretion, and found that Rip11 is involved in cAMP-potentiated insulin secretion but not in glucose-induced insulin secretion. Analyses by immunocytochemistry and subcellular fractionation revealed Rip11 to be co-localized with insulin granules. The inhibitory effect of the Rip11 mutant was not altered in MIN6 cells lacking Epac2, which mediates protein kinase A (PKA)-independent potentiation of insulin secretion, compared with wild-type MIN6 cells. In addition, Rip11 was found to be phosphorylated by PKA in MIN6 cells. The present study shows that both Rab11 and its effector Rip11 participate in insulin granule exocytosis and that Rip11, as a substrate of PKA, regulates the potentiation of exocytosis by cAMP in pancreatic β-cells.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Insulin secretion from pancreatic β-cells is regulated by a variety of extracellular and intracellular signals (Ahren 2000; Henquin 2000; Wollheim 2000). Physiologically, glucose is the most important secretagogue of insulin secretion. Generation of metabolic signals such as ATP by glucose and subsequently a rise in the intracellular Ca2+ concentration elicits insulin granule exocytosis. cAMP is a critical intracellular signal in the mechanism of potentiation of insulin secretion. Gut hormones such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) activate cAMP signaling in pancreatic β-cells (Meier et al. 2002; Drucker 2006). The potentiation of insulin secretion by cAMP is now known to be mediated in both protein kinase A (PKA)-dependent and PKA-independent manners, the latter involving cAMP-GEFII/Epac2 (hereafter referred to as Epac2) (Renstrom et al. 1997; Ozaki et al. 2000; Kashima et al. 2001). However, the mechanism of cAMP action in insulin secretion is still unclear.

Rab proteins are members of the Ras superfamily of small GTPases that play key roles in the regulation of intracellular membrane trafficking; the actions of Rab proteins are mediated by their effectors (Novick & Zerial 1997; Takai et al. 2001), and more than 50 members have been identified in mammals (Takai et al. 2001). Among them, the Rab3 family and Rab27A have been reported to locate on the membrane of insulin granules and to be involved in insulin granule exocytosis in pancreatic β-cells (Regazzi et al. 1996; Iezzi et al. 1999; Yi et al. 2002). Rab11 is a ubiquitously expressed Rab protein, and is known to be involved in the endosomal recycling pathway in mammalian cells (Ullrich et al. 1996; Ren et al. 1998). In addition, Rab11 has been suggested to regulate the secretory processes, including regulated and constitutive secretions in the rat pheochromocytoma cell line PC12 (Urbé et al. 1993; Khvotchev et al. 2003), insulin-stimulated and constitutive secretion of the adipokine ACRP30 in adipocytes (Clarke et al. 2006), and secretion of cysteine proteases in enteric protozoan parasite (Mitra et al. 2007). The functions of Rab11 in vesicle trafficking are mediated by its effectors, which interact with Rab11 in a GTP-dependent manner. The Rab11 effectors identified to date are Rab11-FIPs (Rab11-family interacting proteins), which contain a conserved Rab11-binding domain at the C-terminus, including FIP1, FIP2, FIP3 (Hales et al. 2001), FIP4 (Wallace et al. 2002), Rip11/FIP5 (hereafter referred to as Rip11) (Prekeris et al. 2000), RCP (Lindsay et al. 2002), Rabphilin-11/Rab11BP (hereafter referred to as Rabphilin-11) (Mammoto et al. 1999; Zeng et al. 1999), Myosin Vb (Lapierre et al. 2001) and Sec15 (Zhang et al. 2004). Rip11 was originally identified as an effector of Rab11 co-localized with recycling endosomes and involved in protein trafficking from recycling endosome to the plasma membrane (Prekeris et al. 2000). However, the role of Rip11 in exocytosis is not known.

In the present study, we have investigated the functional roles of Rab11 and its effectors in insulin granule exocytosis. We find that both Rab11 and its effector Rip11 participate in insulin granule exocytosis and that Rip11, as a substrate of PKA, regulates the potentiation of exocytosis by cAMP in pancreatic β-cells.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Rab11B is involved in regulated secretion in MIN6 cells

The Rab11 family consists of three members, Rab11A, Rab11B and Rab25. We first examined the expressions of members of the Rab11 family in the mouse insulin-secreting cell line MIN6 and mouse kidney by RT-PCR. All of the members were expressed in mouse kidney, whereas Rab11A and Rab11B, but not Rab25, were expressed in MIN6 cells (Fig. 1A). Both Rab11A and Rab11B were also expressed in mouse pancreatic islets (data not shown).


Figure 1
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  		Figure 1  Expressions of members of the Rab11 family in insulin-secreting cells and their effects on regulated exocytosis. (A) Expressions of the Rab11 family members. RT-PCR was carried out with cDNAs derived from mouse insulin-secreting MIN6 cells and mouse kidney using the gene-specific primers for members of the Rab11 family, including Rab11A, Rab11B and Rab25. (B) Subcellular localization of Rab11 in MIN6 cells. MIN6 cells were co-immunostained with mouse anti-Rab11 antibody, which recognizes both Rab11A and Rab11B, and guinea pig anti-insulin antibody, and visualized with Alexa488-conjugated donkey anti-mouse and Cy3-conjugated donkey anti-guinea pig IgG antibody, respectively. Bars indicate 5 µm. (C) Effects of Rab11 family proteins on regulated exocytosis in MIN6 cells. MIN6 cells transfected with hGH and various expression vectors; a control vector expressing GFP or vectors expressing Rab11A, Rab11B and Rab25, were incubated in KRBH containing 2.8 mM glucose (shaded columns), 16.8 mM glucose (open columns) or 16.8 mM glucose plus 10 µM forskolin/10 µM IBMX (filled columns) for 1 h hGH secretion is expressed as the amount released into the medium relative to the total cellular content (%). Similar results were obtained from five independent experiments (n = 3 for each) and expressed as means ± SE. **P < 0.005, ***P < 0.001 (Student's unpaired t-test). (D) Effects of a dominant negative form and a dominant active form of Rab11B on regulated exocytosis in MIN6 cells. MIN6 cells were transfected with hGH together with a control vector expressing GFP or vectors expressing a wild-type Rab11B (wt), and a dominant negative (DN) or a dominant active (DA) form of Rab11B. The transfected cells were subjected to hGH secretory experiments as shown in (C). Data are means ± SE. Similar results were obtained from five independent experiments (n = 3 for each). *P < 0.05, **P < 0.005, ***P < 0.001 (Student's unpaired t-test). Shaded, open and filled columns indicate hGH secretion incubated with 2.8 mM glucose, 16.8 mM glucose and 16.8 mM plus 10 µM forskolin/10 µM IBMX, respectively, as shown in (C).

 
We then examined the subcellular localization of Rab11 in MIN6 cells, using the antibody that recognizes both Rab11A and Rab11B. Endogenous Rab11 was co-localized with insulin granules (Fig. 1B).

We next examined Rab11 involvement in regulated exocytosis of insulin granules in MIN6 cells. For this purpose, we used MIN6 cells transfected with human growth hormone (hGH), and the direct effect of transfected Rab11 on secretion from MIN6 cells was determined by measuring hGH released from the cells. The secretion of hGH was stimulated with a high concentration of glucose (16.8 mM) or a high concentration of glucose plus 10 µM forskolin, an adenylate cyclase activator and 10 µM IBMX, a phosphodiesterase inhibitor (hereafter referred to as forskolin/IBMX).

In Rab11A- or Rab25-transfected cells, both glucose alone- and glucose plus forskolin/IBMX-induced hGH secretions were similar to those in green fluorescent protein (GFP)-transfected cells (control). In contrast, in Rab11B-transfected cells, both glucose alone- and glucose plus forskolin/IBMX-induced hGH secretions were significantly decreased by approximately 40% (P < 0.005) and approximately 50% (P < 0.001), respectively (Fig. 1C). To investigate the effects of active and inactive states of Rab11B on secretion, we used a dominant active or a negative mutant form of Rab11B. Over-expression of either mutant of Rab11B into MIN6 cells inhibited both glucose alone- and glucose plus forskolin/IBMX-induced hGH secretions (Fig. 1D). These results indicate that Rab11B is involved in regulated secretion of insulin in pancreatic β-cells.

Involvement of Rip11 in cAMP-potentiated insulin secretion

Rab proteins exert their actions by interacting with their effectors in a variety of cellular functions. To clarify the involvement of Rab11 effectors in insulin secretion, we first examined the expression of various Rab11 effectors in MIN6 cells. As shown in Fig. 2A, expressions of FIP2, FIP3, Rip11 and Rabphilin-11 were confirmed by RT-PCR analysis. We also confirmed expression of Myosin Vb (data not shown).


Figure 2
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  		Figure 2  Effect of Rab11 effectors on regulated exocytosis in MIN6 cells. (A) Expressions of Rab11 effectors in MIN6 cells. RT-PCR was carried out using the gene-specific primers for the various Rab11 effectors, including FIP1, FIP2, FIP3, FIP4, Rip11 and Rabphilin-11. RT-PCR was carried out using cDNAs of MIN6 cells from three independent preparations. (B) Schematic representation of FIP2, FIP3, Rip11, Rabphilin-11, Myosin Vb and their mutants. Green, red, yellow and blue colors indicate C2 domain, Rab11-binding domain (RBD), EF-hand and proline-rich region, respectively. (C) Effects of FIP3 and Rabphilin-11 mutants on regulated exocytosis in MIN6 cells. MIN6 cells were transfected with hGH together with GFP (control), FIP3{Delta}EF-hand, FIP3{Delta}RBD, Rph11-N or Rph11-C. The transfected cells were incubated in KRBH containing 2.8 mM glucose (shaded columns), 16.8 mM glucose (open columns) or 16.8 mM glucose plus 10 µM forskolin/10 µM IBMX (filled columns) for 1 h hGH secretion is expressed as the amount released into the medium relative to the total cellular content (%). Similar results were obtained from two independent experiments (n = 3 for each) and expressed as means ± SE. NS, no significant difference (Student's unpaired t-test). (D) Effects of Myosin Vb mutant on regulated exocytosis in MIN6 cells. MIN6 cells were transfected with hGH and Myosin Vb-tail and subjected to hGH secretory experiments as shown in (C). Similar results were obtained from two independent experiments (n = 3 for each) and expressed as means ± SE. (E) Effects of FIP2 and Rip11 mutants on regulated exocytosis in MIN6 cells. MIN6 cells were transfected with hGH together with FIP2{Delta}RBD or Rip11{Delta}RBD and subjected to hGH secretory experiments as shown in (C). Similar results were obtained from four independent experiments (n = 3 for each) and expressed as means ± SE. ***P < 0.001 (Student's unpaired t-test).

 
We then attempted to determine the functional roles of these effectors in insulin secretion. For this purpose, we prepared respective dominant negative mutants of the various effectors (Fig. 2B). These mutants were transfected into MIN6 cells, and hormone secretion was examined. FIP3{Delta}EF-hand, FIP3{Delta}RBD, Rph11-N, Rph11-C or Myosin Vb-tail had almost no effect on hGH secretion stimulated with a high concentration of glucose (16.8 mM) or a high concentration of glucose plus forskolin/IBMX (Fig. 2C,D). In contrast, in MIN6 cells transfected with Rip11{Delta}RBD, secretion stimulated with glucose alone was not changed, whereas secretion stimulated with glucose plus forskolin/IBMX was significantly inhibited, compared to control (P < 0.001). Secretion stimulated with glucose plus forskolin/IBMX was also inhibited in FIP2{Delta}RBD-transfected cells significantly, compared to control (Fig. 2E). These results indicate that Rip11 and FIP2 are involved in cAMP-potentiated insulin secretion in pancreatic β-cells. In this study, we investigated the role of Rip11 in insulin granule exocytosis in further detail.

Subcellular localization of Rip11 in MIN6 cells

We examined subcellular localization of Rip11 in MIN6 cells. As shown in Fig. 3A,B, GFP-Rip11 was co-localized with Rab11A and Rab11B. We then examined co-localization of Rip11 with organelles in MIN6 cells, using well-characterized organellar markers, Early Endosomal Antigen 1 (EEA1) for early endosomes, Golgi58k for Golgi apparatus, and transferrin receptor for recycling endosomes. GFP-Rip11 was partially co-localized with EEA1, Golgi58k and transferrin receptor, suggesting that Rip11 is partially localized in early endosomes, Golgi apparatus, and recycling endosomes in MIN6 cells (Fig. 3C–E).


Figure 3
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  		Figure 3  Subcellular localization of Rip11 in MIN6 cells. MIN6 cells were transfected with vectors expressing GFP-Rip11. The transfected cells were fixed, permeabilized and immunostained with rabbit anti-Rab11A (A), rabbit anti-Rab11B (B), rabbit anti-EEA1 (early endosome marker) (C), mouse anti-Golgi58k (Golgi apparatus marker) (D) and mouse anti-transferrin receptor (TfR) (recycling endosome marker) (E) antibodies, which were visualized with Cy3-conjugated donkey anti-mouse or anti-rabbit IgG antibodies. Each row represents GFP-Rip11 (green), organelle markers (red), and merged images, respectively. Bars indicate 5 µm.

 
We next examined co-localization of Rip11 with insulin-containing large dense-core granules. Confocal microscopic analysis showed that GFP-Rip11 was partially co-localized with insulin granules (Fig. 4A). To further confirm co-localization of Rip11 with insulin granules, subcellular fractionation analysis was carried out by sucrose gradient centrifugation. Immunoblot analysis revealed that Rip11 was co-localized with transferrin receptor, a marker for recycling endosome at light density fractions and Chromogranin A, a marker for large dense-core granules at heavy density fractions (Fig. 4B). We also carried out immunogold electron microscopic analysis. As shown in Fig. 4C, Rip11 was present on large dense-core granules. These results indicate that Rip11 is partially co-localized with insulin granules.


Figure 4
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  		Figure 4  Co-localization of Rip11 with insulin granules in MIN6 cells. (A) Co-immunostaining of GFP-Rip11 with insulin granules. MIN6 cells were transfected with vectors expressing GFP-Rip11. The transfected cells were fixed, permeabilized and immunostained with guinea pig anti-insulin antibody, which was visualized with Cy3-conjugated donkey anti-guinea pig IgG antibody. Each image represents GFP-Rip11 (green), insulin granules (red) and merged images, respectively. Bars indicate 5 µm. (B) Subcellular fractionation of MIN6 cells. MIN6 cells were homogenized, and organelles in the supernatants were separated on discontinuous sucrose gradients. Fractions were subjected to immunoblot analysis with anti-Rip11, anti-transferrin receptor (TfR) (recycling endosome marker), Chromogranin A (large dense-core granule marker), Synaptophysin (synaptic-like microvesicle marker) and Na+–K+-ATPase {alpha}-1 (plasma membrane marker). (C) Detection of localization of endogenous Rip11 in MIN6 cells by immunoelectron microscopy. Rip11 was immunostained with anti-Rip11 antibody and visualized with gold-labeled secondary antibody, followed by silver enhancement. Red arrows indicate co-localization of Rip11 with large dense-core granules. Bar indicates 1 µm. N indicates nucleus.

 
Rip11 contributes to both first and second phases of insulin secretion

To determine the functional role of Rip11 in insulin secretion, we introduced GFP-Rip11{Delta}RBD, a dominant negative Rip11, into MIN6 cells. Three days after infection of MIN6 cells with adenovirus carrying GFP-Rip11{Delta}RBD, the cells were stimulated with 16.8 mM glucose alone or 16.8 mM glucose plus forskolin/IBMX for 1 h. Similar to the results of hGH secretion in MIN6 cells transfected with Rip11{Delta}RBD (Fig. 2E), over-expression of GFP-Rip11{Delta}RBD had no effect on glucose-induced insulin secretion, although it significantly inhibited potentiation of glucose-induced insulin secretion by forskolin/IBMX (Fig. 5A).


Figure 5
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  		Figure 5  Effects of a dominant negative form of Rip11 on cAMP-potentiated insulin secretion in MIN6 cells. (A) MIN6 cells infected with adenoviruses carrying GFP or GFP-Rip11{Delta}RBD at a multiplicity of infection of 10 were preincubated in KRBH containing 2.8 mM glucose for 30 min. After preincubation, the cells were incubated with in KRBH containing 2.8 mM glucose (shaded columns), 16.8 mM glucose (open columns) or 16.8 mM glucose plus 10 µM forskolin/10 µM IBMX (filled columns) for 1 h. Similar results were obtained from three independent experiments (n = 3 for each) and expressed as means ± SE. ***P < 0.001 (Student's unpaired t-test). (B) Perifusion experiment of MIN6 cells over-expressing Rip11{Delta}RBD. MIN6 cells seeded on cover slides were infected with adenoviruses carrying GFP (open circles) or GFP-Rip11{Delta}RBD (filled circles) at a multiplicity of infection of 10. After preincubation, cover slides were mounted in a perifusion chamber. The cells were perifused in KRBH containing 2.8 mM glucose for 10 min, when the perfusate was switched to KRBH containing 16.8 mM glucose plus 10 µM forskolin/10 µM IBMX. Eluted fractions were collected at 1-min intervals, and insulin in each fraction was measured by insulin assay kit. Data were obtained from three independent experiments and expressed as means ± SE. *P < 0.05 (Student's unpaired t-test).

 
Glucose-induced insulin secretion occurs in a biphasic manner, the first phase of a prompt, marked, and transient increase followed by the second phase of a moderate and sustained increase. cAMP potentiates both phases of glucose-induced insulin secretion in isolated perifused pancreatic β-cells (Prentki & Matschinsky 1987). We thus examined the involvement of Rip11 in the insulin secretory phases using perifused MIN6 cells. No significant difference was observed at basal state. However, when MIN6 cells were infected with Rip11{Delta}RBD, both the first phase (the first 7 min after stimulation) and the second phase potentiations (8–19 min after stimulation) by glucose plus forskolin/IBMX were significantly suppressed (Fig. 5B). These results indicate that Rip11 is involved in both first and second phases of cAMP-potentiated insulin secretion.

Rip11 is involved in PKA-dependent potentiation of insulin secretion

cAMP is now known to potentiate insulin secretion in both PKA-dependent and PKA-independent mechanisms, the latter involving Epac2 (Seino & Shibasaki 2005). We used pancreatic β-cells lacking Epac2 (Shibasaki et al. 2007) to determine which mechanism involves Rip11. Over-expression of Rip11{Delta}RBD in Epac2-lacking β-cells inhibited hGH secretion potentiated by forskolin/IBMX (Fig. 6A), indicating that Rip11 is involved in PKA-dependent potentiation of insulin secretion.


Figure 6
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  		Figure 6  Involvement of Rip11 in PKA-dependent regulated exocytosis in MIN6 cells and identification of PKA phosphorylation of Rip11. (A) Effects of Rip11{Delta}RBD on regulated exocytosis in pancreatic β-cells lacking Epac2. Pancreatic β-cells lacking Epac2 were transfected with hGH and Rip11{Delta}RBD and incubated in KRBH containing 2.8 mM glucose (shaded columns), 16.8 mM glucose (open columns) or 16.8 mM glucose plus 10 µM forskolin/10 µM IBMX (filled columns) for 1 h hGH secretion is expressed as the amount released into the medium relative to the total cellular content (%). Similar results were obtained from two independent experiments (n = 3 for each) and expressed as means ± SE. **P < 0.005 (Student's unpaired t-test). (B) Phosphorylation of Rip11 by PKA. MIN6 cells were transfected with vectors expressing Myc-wild-type Rip11. The transfected cells were incubated in KRBH containing 2.8 mM glucose, 16.8 mM glucose or 16.8 mM glucose plus 10 µM forskolin/10 µM IBMX, with or without 1 µM okadaic acid (Calbiochem, San Diego, CA), a phosphatase inhibitor, for 30 min. The cells were then lysed in KRBH containing 0.1% TritonX-100, protease inhibitor cocktail, and 1 µM okadaic acid. The homogenates were centrifuged at 15 000 g for 10 min. The proteins in the supernatants were subjected to immunoblot analysis with anti-Rip11 antibody. Arrowhead indicates the second phosphorylation of Rip11. (C) Identification of the serine residues of Rip11 responsible for phosphorylation by PKA. MIN6 cells were transfected with vectors expressing Myc-wild-type Rip11 or its mutant, in which serine at position 14, 35, 243, 244, 307, 335, 356, 357, 474 or 539 was substituted with alanine. The cells were incubated in KRBH containing 16.8 mM glucose plus 10 µM forskolin/10 µM IBMX, and PKA inhibitor H-89 in the absence or presence of 1 µM okadaic acid for 30 min. The cells were then lysed in KRBH containing 0.1% TritonX-100, protease inhibitor cocktail and 1 µM okadaic acid, and subjected to immunoblot analysis with anti-Rip11 antibody. Arrowhead indicates the second phosphorylation of Rip11.

 
Phosphorylation of Rip11 by forskolin/IBMX in MIN6 cells and identification of the phosphorylated residues

When human intestinal epithelial cell line Caco-2 was treated with okadaic acid, a phosphatase inhibitor, a slight decrease in the mobility of Rip11 on SDS-PAGE was observed, indicating the phosphorylation of Rip11 (Prekeris et al. 2000). Phosphorylation of Rip11 has been proposed to play an important role in the regulation of its trafficking and translocation (Prekeris et al. 2000).

We then examined phosphorylation of Rip11 in MIN6 cells. MIN6 cells transfected with Myc-Rip11 were treated with okadaic acid (1 µM) in the presence or absence of glucose alone or glucose plus forskolin/IBMX. As shown in Fig. 6B, the electrophoretic mobility of Rip11 was slightly decreased in okadaic acid-treated cells, indicating phosphorylation of Rip11 in MIN6 cells. Interestingly, when the cells were stimulated with a high concentration of glucose (16.8 mM) plus forskolin/IBMX, the mobility of Myc-Rip11 was further decreased, indicating the second phosphorylation (arrowhead in Fig. 6B). However, the second phosphorylation of Rip11 was not detected when MIN6 cells were treated with H-89, a PKA inhibitor (Fig. 6B). These results indicate that Rip11 is a direct substrate of phosphorylation by PKA.

To identify the PKA phosphorylation sites in Rip11 induced by forskolin/IBMX stimulation, sequence analysis was carried out using NETPHOSK program (Blom et al. 2004), which predicts the consensus sites of PKA phosphorylation. We predicted nine potential serine residues (14, 35, 243, 244, 335, 356, 357, 474 and 539) for PKA phosphorylation, based on NETPHOSK program. In addition, it has been reported that serine residue at position 307 of Rip11 can be phosphorylated (Collins et al. 2005). We thus constructed 10 different Myc-Rip11 mutants, in which the serine at positions 14, 35, 243, 244, 307, 335, 356, 357, 474 or 539 was substituted with alanine. MIN6 cells transfected with these mutants were stimulated with a high concentration of glucose (16.8 mM) plus forskolin/IBMX in the presence of okadaic acid. In MIN6 cells transfected with S357A mutant of Rip11, the second phosphorylation was not detected (Fig. 6C). We conclude that Rip11 is a direct substrate of PKA in insulin-secreting cells and that it is phosphorylated at serine 357.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Rab11 is known to be localized to the endocytotic recycling endosomes and the trans-Golgi network, and to be involved in the endosomal recycling pathway (Ullrich et al. 1996; Ren et al. 1998). In addition to their roles in the recycling pathway, Rab11B has recently been suggested to regulate Ca2+-triggered exocytosis in PC12 cells (Khvotchev et al. 2003). In this study, over-expression of wild type, GDP-bound form, or GTP-bound form of Rab11B all inhibited both glucose alone- and glucose plus forskolin/IBMX-induced secretion in MIN6 cells (Fig. 1D). Generally, the impairment of the GTP/GDP cycle of Rab protein causes inhibition of membrane traffic (Takai et al. 2001). The inhibitory effect of Rab11 mutants indicates the involvement of GTP/GDP cycle of Rab11 in insulin secretion. These results are consistent with studies indicating that over-expression of wild type, GDP-bound form, and GTP-bound form of Rab11B or Rab3 in PC12 cells also inhibits Ca2+-triggered exocytosis (Schluter et al. 2002; Khvotchev et al. 2003). Together, these results provide the first evidence that Rab11B is involved in regulated exocytosis of insulin granules in pancreatic β-cells.

The interaction of Rab protein and its effectors requires the activation of Rab protein (Takai et al. 2001). As Rab protein is activated by its specific guanine nucleotide exchange factor (GEF), the regulation of GEF activity by intracellular signals is important for Rab-mediated membrane traffic (Takai et al. 2001). Although GEF toward Rab11 has not yet been identified, the GEF may convert Rab11 from the GDP to GTP-bound form in a glucose-dependent manner and thus facilitate the regulation of insulin secretion in pancreatic β-cells.

Sec15, a component of the exocyst complex, has recently been identified as an effector of Rab11 in mammalian cells (Zhang et al. 2004). Sec15 is co-localized with Rab11 in COS cells, and interacts with Rab11 in a GTP-dependent manner. Over-expression of the respective truncated mutant of Sec6, Sec8 or Exoc3l, an isoform of Sec6, all of which are components of the exocyst, inhibits glucose-induced secretion in MIN6 cells (Tsuboi et al. 2005; Saito et al. 2008), suggesting that the exocyst is involved in glucose-induced insulin secretion in pancreatic β-cells. Thus, taken together with our present finding, Rab11 participates in glucose-induced insulin secretion and cAMP-potentiated insulin secretion by interacting with Sec15 and Rip11, respectively.

Various Rab11 effectors have been identified to date, including FIP1, FIP2, FIP3, FIP4, Rip11, Rabphilin-11, Myosin Vb and Sec15. In this study, we found that Rip11 is expressed in both insulin-secreting cells (Fig. 2A) and mouse pancreatic islets (data not shown), and that over-expression of Rip11 lacking a Rab11-binding domain inhibits glucose-induced insulin secretion by cAMP, but not glucose-induced insulin secretion (Fig. 2E). In addition, Rip11 is partially co-localized with insulin granules in MIN6 cells (Fig. 4). These findings suggest that Rip11 directly interacts with insulin granules and regulates the recruitment of the granules to plasma membrane in a cAMP-dependent manner. In the course of identification of the Rab11 effector protein involved in insulin secretion, over-expression of FIP2 mutant lacking the Rab11-binding domain also inhibited forskolin/IBMX-potentiated insulin secretion, although the inhibitory level was lower than that in Rip11 mutant lacking the Rab11-binding domain (Fig. 2E). FIP2 may also have functions similar to Rip11 in cAMP-induced potentiation of insulin secretion in pancreatic β-cells.

Although a rise in the intracellular Ca2+ concentration is the primary signal in the regulation of exocytosis, other intracellular signals are also critical, cAMP being especially important in many secretory cells (Seino & Shibasaki 2005). cAMP is now known to potentiate insulin secretion in both a PKA-dependent pathway, followed by phosphorylation of the relevant proteins, and a PKA-independent pathway, involving Epac2/Rap1 signaling (Seino & Shibasaki 2005; Shibasaki et al. 2007). Although PKA is a major cAMP target, the precise mechanisms of the PKA-dependent pathway in insulin granule exocytosis are not fully understood. Our present study demonstrates that Rip11 is involved in the PKA-dependent mechanism, based on the following evidence. In the hGH secretory experiment, over-expression of Rip11 mutant lacking the Rab11-binding domain inhibited the potentiation of secretion by forskolin/IBMX in pancreatic β-cells lacking Epac2 (Fig. 6A). Moreover, in the phosphorylation experiment, Rip11 was additionally phosphorylated by forskolin/IBMX in MIN6 cells. The first step in the phosphorylation process occurred without any stimulation. In a previous study, the first step phosphorylation was shown to be involved in translocation of Rip11 and regulation of the apical recycling system in polarized MDCK cells (Prekeris et al. 2000). The present study has clarified the second phosphorylation of Rip11, which occurs at serine residue 357 in MIN6 cells stimulated with forskolin/IBMX (Fig. 6B,C). Serine residue 357 constitutes an Arg-His-Arg-X-Ser-Ile motif (X: any amino acid), possessing basic residues at –2 to –4 positions and hydrophobic residue at +1 position. The motif is compatible with the recognition amino acid sequence for phosphorylation by PKA. Furthermore, the second phosphorylation was not detected when MIN6 cells were treated with H-89, a PKA inhibitor. Considering this evidence together, we conclude that Rip11 is a direct substrate of PKA in PKA-dependent potentiation of insulin secretion. Phosphorylation of Rip11 by PKA may promote the recruitment of insulin granules to the plasma membrane in both phases (first and second phase) of the potentiation of insulin secretion.

In this study, we have found a novel function of Rip11 in pancreatic β-cells. This finding suggests the possibility that insulin granule exocytosis potentiated by cAMP is coupled with recycling mechanisms mediated by Rip11. Phogrin, an integral membrane protein of dense-core granules in endocrine and neuroendocrine cells, has been reported to be internalized and recycled to immature insulin granules after exocytotic stimulation in MIN6 cells (Vo et al. 2004). Using a similar mechanism, Rip11 might recycle proteins involved in exocytotic processes back to immature insulin granules and then accelerate the maturation of the granules upon cAMP stimulation.

Thus, Rab11/Rip11 participates in various membrane-trafficking events as well as in the recycling pathway. Further investigation is required to clarify the upstream and downstream signals of Rab11/Rip11 in the regulation of insulin secretion.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Antibodies

Rabbit anti-Rip11 antibody was kindly provided by Dr Rytis Prekeris (University of Colorado School of Medicine). Rabbit anti-Rab11A, mouse anti-human transferrin receptor and guinea pig anti-insulin antibodies were obtained from Zymed (San Francisco, CA). Rabbit anti-Rab11B antibody was obtained from Cell Signaling Technology (Beverly, MA). Rabbit anti-Early Endosomal Antigen 1 (EEA1) antibody was obtained from Affinity Bioreagents (Golden, CO). Mouse anti-Golgi58k antibody was obtained from Sigma-Aldrich (Poole, UK). Mouse anti-Rab11 antibody and mouse anti-Chromogranin A antibody were obtained from BD Transduction Laboratories (Lexington, KY). Mouse anti-Synaptophysin antibody was obtained from Roche (Mannheim, Germany). Mouse anti-Na+-K+-ATPase {alpha}-1 antibody was obtained from Upstate Biotechnology (Lake Placid, NY). Alexa488-conjugated donkey anti-rabbit and anti-mouse IgG antibodies were obtained from Invitrogen (Carlsbad, CA). Cy3-conjugated donkey anti-guinea pig, anti-mouse and anti-rabbit IgG antibodies were obtained from Chemicon (Temecula, CA).

Cell culture and transfection

MIN6 cells were cultured in Dulbecco's modified Eagle's medium containing 25 mM glucose and 10% (v/v) fetal bovine serum under a humidified condition of 95% air and 5% CO2. Transfections were carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

RT-PCR analysis

Total RNA from MIN6 cells and mouse pancreatic islets was isolated using the RNAeasy Kit (Qiagen, Hilden, Germany). For reverse transcription, ReverTra Ace-{alpha}-Kit (TOYOBO, Osaka, Japan) was used. A mouse multiple tissue cDNA panel was also used (Clontech, Palo Alto, CA). RT-PCR was carried out with the primers for mouse Rab11A (forward 5'-ATGGGCACCCGCGACGACG and reverse 5'-TTAGATGTTCTGACAGCACTGCACC), mouse Rab11B (forward 5'-ATGGGGACCCGGGACGACG and reverse 5'-TCACA GGCTCTGGCAGCACTG), mouse Rab25 (forward 5'-ATGGGGAATCGAACAGATGAAG and reverse 5'-TCAGA GGCTGATGCAACAGGC), FIP1 (forward 5'-CAGGT GGGCAAGGAGAAGTAC and reverse 5'-CTGGCCGT CATGTTATTCCTC), FIP2 (forward 5'-CAGGTGGCAAT CAATCTCAATG and reverse 5'-AACTCAGGATTGGCAT CAGG), FIP3 (forward 5'-GGGGTCTGAGAGCACCTACAG and reverse 5'-GTGAGAAAAGCAGGGTCCTC), FIP4 (forward 5'-TGTTTGGATCCCAATGACCTG and reverse 5'-AGCTCAGGCTCCTTCTCCTC), Rip11 (forward 5'-GGCCGAGAGAAGTACAGCAC and reverse 5'-CTGGAT GGTCACCTGGATCTC), Rabphilin-11 (forward 5'-CCG GCTATCCTGTAGGGTCTC and reverse 5'-GCCTTTT CGGAATCTTGTTG) and Myosin Vb (forward 5'-AGCTC CCCAGACAGCTACAGC and reverse 5'-GCCTCGG AAACACGCATCCTC).

Constructions of plasmids and adenoviral vectors

Mouse Rab11A, Rab11B, Rab25, FIP2 lacking Rab11-binding domain (RBD) (residues 1–439, FIP2{Delta}RBD), FIP3 lacking EF-hand (residues 604–1047, FIP3{Delta}EF-hand), FIP3 lacking RBD (residues 604–1005, FIP3{Delta}RBD), wild-type Rip11, Rip11 lacking RBD (residues 1–578, Rip11{Delta}RBD), amino-terminus of Rabphilin-11 (residues 1–638, Rph11-N), carboxyl-terminus of Rabphilin-11 (residues 612–740, Rph11-C) or carboxyl-terminus of Myosin Vb (residues 1231–1818, Myosin Vb-tail) was obtained from a MIN6 cDNA library by using RT-PCR and subcloned into pEGFP-C (Clontech) or pCMV-Tag3 (Stratagene, La Jolla, CA). Site-directed mutagenesis was carried out using a KOD-Plus-Mutagenesis kit (TOYOBO) according to the manufacturer's instructions. In Rab11B, serine at position 25 was substituted with asparagine for a dominant negative mutant, and glutamine at position 70 was substituted with leucine for a dominant active mutant. Adenoviruses carrying GFP cDNA (Clontech) and GFP-Rip11{Delta}RBD cDNA were constructed using ViraPower Adenoviral Expression System (Invitrogen).

Immunofluorescence microscopy

MIN6 cells were plated on cover slides and transfected with Rip11. After 3 days, the cells were fixed in 3% formaldehyde, permeabilized with 0.1% TritonX-100, and blocked with PBS containing 3% BSA and 0.1% TritonX-100, and then incubated with appropriate primary antibodies overnight at 4 °C in a humidified chamber. Cells were then incubated with Alexa488 or Cy3-conjugated secondary antibodies. The sections were mounted on cover slides using Perma Fluor Mounting Medium (Thermo, Pittsburgh, PA) and observed by confocal laser scanning microscopy (Olympus Fluoview FV1000, Tokyo, Japan).

Subcellular fractionation

Discontinuous sucrose gradient fractionation of MIN6 cells was carried out as previously described (Kotake et al. 1997). Briefly, the cells were washed twice with PBS and scraped. The cells were pelleted by centrifuging at 200 g for 3 min. The cell pellet was resuspended in 3 mL of homogenate buffer containing 5 mM HEPES (pH 7.4), 1 mM EDTA, 0.25 M sucrose and protease inhibitor cocktail (Roche). The suspension was homogenized in a Dounce Tissue Grinder (Wheaton, Melville, NJ) with four strokes. The homogenate was then centrifuged at 3000 g for 10 min. The supernatant was loaded on to 9 mL of discontinuous sucrose gradient containing 1 mL of each sucrose concentration of 0.45, 0.65, 0.85, 1.05, 1.25, 1.45, 1.65, 1.85 and 2.05 M with 5 mM HEPES and 0.5 mM EDTA, and the gradient was ultracentrifuged at 4 °C at 110 000 g for 18 h. Fractions (0.5 mL) were collected from the top of gradients, and proteins in each fraction were precipitated with 10% trichloro acetic acid (TCA), and then resuspended in SDS-PAGE-loading buffer and subjected to immunoblot analysis.

Immunoelectron microscopy

Immunoelectron microscopy using the silver-enhanced immunogold method was carried out as previously described (Mizoguchi et al. 2002). MIN6 cells were seeded on Lab-Tek tissue culture chamber slides (Nunc Inc., Naperville, IL). After 3 days, cells were fixed in 2% parafomaldehyde in 0.1 M phosphate buffer (pH 7.4) at room temperature for 4 h, rinsed once with 0.1 M phosphate buffer, and blocked with 20% Block Ace (Dainihonseiyaku, Osaka, Japan) in PBS containing 0.05% saponin for 10 min. The cells were then incubated with the rabbit anti-Rip11 antibody, followed by incubation with a secondary antibody coupled with 1.4-nm gold particles (Nanoprobes Inc., Yaphank, NY). The sample-bound gold particles were silver-enhanced by the HQ-silver kit (Nanoprobes Inc.) at 18 °C for 12 min in the dark. The samples were post-fixed with 0.5% osmium oxide in phosphate buffer for 1 h, dehydrated by passage through a graded series of ethanol, and embedded in Epon 812. Ultrathin sections were then made and analyzed using an electron microscope (JEM-1011EX; JEOL, Tokyo, Japan).

Measurements of regulated exocytosis

One day after transfection of the expression plasmids for hGH, MIN6 cells were harvested and replated in 24-well plates (2 x 105 cells/well). Forty-eight hours after replating, the cells were washed twice and preincubated for 20 min in HEPES-balanced Krebs–Ringer bicarbonate buffer (KRBH: 119 mM NaCl, 4.74 mM KCl, 2.54 mM CaCl2, 1.19 mM MgCl2, 1.19 mM KH2PO4, 25 mM NaHCO3 and 10 mM HEPES, pH 7.4) containing 0.1% BSA with 2.8 mM glucose. After preincubation, the cells were incubated for 60 min with KRBH containing 2.8 mM glucose, 16.8 mM glucose or 16.8 mM glucose plus 10 µM forskolin/10 µM IBMX. Released hGH was measured by ELISA kit (Roche). The amount of hGH secretion in each experiment was normalized by the cellular hGH content. To measure cellular hGH content, cellular lysates were obtained using KRBH containing 0.1% TritonX-100. Data were evaluated for statistical significance using Student's unpaired t-test.

Perifusion experiments

For the perifusion experiments on insulin secretion, MIN6 cells were seeded at a density of 2.0 x 105 cells on cover slides. The following day, the cells were infected with recombinant adenoviruses for GFP alone or GFP-Rip11{Delta}RBD (multiplicity of infection of 10) and maintained for 48 h. The cells were then incubated in KRBH containing 2.8 mM glucose for 50 min and mounted in a perifusion chamber. The cells were perifused in KRBH containing 2.8 mM glucose for 10 min, when perfusate was switched to KRBH containing 16.8 mM glucose plus 10 µM forskolin/10 µM IBMX. Eluted fractions were collected at 1-min intervals, and released insulin in each fraction was measured by insulin assay kit (Medical Biological Laboratories, Nagoya, Japan).


   Acknowledgements
 
We thank Dr Rytis Prekeris (University of Colorado School of Medicine) for providing us with anti-Rip11 antibody. This work was supported by a Core Research for Evolutional Science and Technology grant from the Japan Science and Technology Agency and a Grant-in-Aid for Specially Promoted Research and Scientific Research Grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Yoshimi Takai

* Correspondence: seino{at}med.kobe-u.ac.jp


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Accepted: 24 December 2008





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