Introduction
Type 2 diabetes mellitus is known to be caused by peripheral insulin resistance and pancreatic beta cell failure, and a number of important findings have been made for the molecular mechanism of impaired insulin secretion. The cytoskeleton and cell adhesion of pancreatic beta cells are reported to be involved in insulin secretion, and low-molecular-weight G proteins also play a major role in insulin secretion. About 40 years ago, an electron microscopy study showed that F-actin functions as a barrier for insulin granules [
1]. This was the first report to show a relation between the beta cell cytoskeleton and insulin secretion.
Recent studies have found that adjacent beta cells influence each other’s insulin secretion by modulating the F-actin barrier through ephrin receptor (Eph)–ephrin signalling [
2]. Such findings suggest that the molecules that regulate the cytoskeleton are important for insulin secretion. In addition, cell culture experiments have also revealed that the amount of insulin secretion is reduced when there is a decrease in the production of cell division cycle 42 (CDC42), which is a low-molecular-weight G protein [
3]. Furthermore, RAB27A, member RAS oncogene family (RAB27A) has been shown to play an important role in the docking of insulin granules to the cell membrane [
4]. The various roles of small G proteins, including the above examples, in pancreatic beta cells have been well described [
5]. In this study, we examined the G-protein ras-related C3 botulinum toxin substrate 1 (RAC1), which plays major roles in various phenomena in the cytoskeleton, in oxidative stress and in secretion. Because of its diverse functions, RAC1 sometimes plays both positive and negative roles in pancreatic beta cells [
6]. Previous studies using dominant-negative mutations have shown that inhibiting the functions of RAC1 in cultured beta cells results in decreased insulin secretion [
7,
8], suggesting that RAC1 could be an important molecule for insulin secretion; however, no in vivo analyses or studies of the mechanism have been reported to date, and the role of RAC1 at the individual level remains unclear. Here, we conducted a functional analysis of RAC1 in beta cells, using genetically modified mice and cultured cell lines.
Results
Next, we observed the cytoskeleton of glucose-stimulated INS-1 cells. When stimulated with low concentrations of glucose, the F-actin in the INS-1 cells stained well, but when stimulated with high concentrations of glucose, the amount of stained F-actin decreased significantly. However, when RAC1 was knocked down by siRNA in INS-1 cells, significantly reduced RAC1 production was confirmed by immunostaining (Fig.
4f–i), and F-actin was fully stained, even during high-glucose stimulation, indicating that the F-actin remained significantly more intact in RAC1-deficient cells than in control cells during high-glucose stimulation (Fig.
4b–e). For further confirmation of the results, western blotting was performed to quantify the polymer F-actin and the depolymerised monomer G-actin in the glucose-stimulated INS-1 cells. Under normal conditions, glucose stimulation causes a depolymerisation of F-actin, and consequently the monomer G-actin increases in quantity. However, in the INS-1 cells knocked down for RAC1 by siRNA, the polymer F-actin was present in larger quantities even under glucose stimulation, and no increase in the quantity of G-actin was found (Fig.
4j). Although the proportion of F-actin of the total actin in normal INS-1 cells is reported not to exceed 55% [
23,
24], in the present study we found this ratio to be around 70%. This may have been because of the 2.8 mmol/l glucose challenge, which increased the intensity of F-actin polymerisation. Additionally, the toxicity of DMSO [
23] and differences in the F-actin assay [
24] might have affected the proportion of F-actin. These results suggest that glucose stimulation causes depolymerisation of the F-actin into G-actin in INS-1 cells, but when
Rac1 expression is reduced, depolymerisation is inhibited and F-actin remains intact.
Discussion
RAC1 is known to contribute to various disorders, including cancer and neurological disorders [
25‐
27]. In addition, RAC1 is associated with the translocation of GLUT4 via the non-phosphatidylinositol 3-kinase pathway in the muscle of patients with diabetes mellitus [
28,
29]. It is also known that many low-molecular-weight G proteins, including RAC1, influence insulin secretion in pancreatic beta cells. Although the importance of the low-molecular-weight G protein in relation to insulin secretion is recognised, RAC1-specific functional analysis has never been performed in pancreatic beta cells. Therefore, we generated beta
Rac1
−/− mice and analysed them.
It was expected that ablation of RAC1, which mediates cell adhesion and migration, would show morphological abnormalities. Epithelial hyperplasia and a reduced basal cell layer are actually found in epithelial-specific
Rac1-knockout mice [
30]. However, unexpectedly, no major influence was observed in the pancreatic beta cell mass or in pancreatic islet density in the present study, possibly because other ras-homologous (Rho)-GTPases) compensated to form the pancreatic beta cell mass.
Although some reports have shown that RAC1 contributes to insulin secretion in the pancreatic beta cell line [
7,
8], no functional analysis of RAC1 in animals has been performed. This is the first report to evaluate blood glucose levels and serum insulin levels of the beta
Rac1
−/− mouse. A significant reduction in insulin secretion was found in beta
Rac1
−/− mice in the OGTT compared with controls. Moreover, significantly reduced insulin secretion was observed in beta
Rac1
−/− mice when isolated islets were stimulated with glucose. However, no differences in insulin secretion between groups were observed after high KCl or tolbutamide stimulation. Previous reports found no activated RAC1 after high KCl stimulation and showed that when the mutant of inactivated RAC1 was transfected into the cells, glucose-responsive insulin secretion was inhibited without any influence on insulin secretion after high KCl stimulation [
7]. In other words, RAC1 in pancreatic beta cells appeared to mediate glucose-specific insulin secretion. Recently, isoprenylcysteine carboxyl methyltransferase (ICMT) was shown to play an important role in glucose-induced RAC1 activation, suggesting that ICMT is a pivotal molecule in glucose-stimulated insulin secretion in pancreatic beta cells [
31].
There are many reports that only the first phase of insulin secretion is found after non-nutrient stimulation [
32‐
34]. However, it was not clear which type of molecule and which type of mechanism led to the differences in insulin secretion after glucose stimulation. Our results suggest that the key molecule distinguishing the second phase from the first phase of insulin secretion might have been RAC1. Actually, we demonstrated that only the second phase of insulin secretion was significantly reduced in beta
Rac1
−/− mice. Previous reports on cultured cell revealed that RAC1 controlled the second phase of insulin secretion [
8], which was demonstrated in vivo for the first time in the present study.
In terms of factors regulating the first and second phases of insulin secretion, previous studies have suggested that the readily releasable pool, comprising docked insulin granules constitutes the first phase, and the reserve pool (RP), which is located further away, constitutes the second phase [
35]. However, a recently proposed model has the second phase of insulin secretion controlled by an RP formed out of the actin network, irrespective of the distance from the cell membrane or of docking onto the membrane [
18]. It is already well known that the actin cytoskeleton plays an important role in insulin secretion [
1,
36], and reports have shown that glucose stimulates the recruitment of insulin granules to the cell membrane through actin remodelling [
37,
38]. It was recently reported that the Rho–Rho-associated coiled-coil containing protein kinase (ROCK) signalling pathway, in which Rho, another low-molecular-weight G protein, plays an important role, regulates insulin secretion via actin reorganisation in pancreatic beta cells [
39]. In our study, with high KCl stimulation and circumstances promoting decreased
Rac1 expression, no actin remodelling occurred, suggesting that glucose-stimulated RAC1 activation contributes to F-actin depolymerisation.
In 2007, Konstantinova et al demonstrated that EphA–ephrin-A signalling mediates communication between adjacent pancreatic beta cells, thereby regulating insulin secretion [
2]. They showed that EphA–ephrin-A-mediated cell communication is bidirectional, and that EphA forward signalling inhibits insulin secretion while ephrin-A reverse signalling stimulates insulin secretion. They also investigated the downstream targets of EphA–ephrin-A signalling and found that the EphA forward signalling-induced decrease in insulin secretion is accompanied by inhibition of RAC1 activity and an increase in F-actin intensity, while the ephrin-A-reverse-signalling-induced increase in insulin secretion is accompanied by enhanced RAC1 activity with decreased F-actin intensity. However, it remains unclear whether RAC1 activation is linked directly with F-actin intensity, and also with insulin secretion. Here, we demonstrated that activation of RAC1 promotes insulin secretion via depolymerisation of F-actin, suggesting a pivotal role of RAC1 in insulin secretion regulated by communication between adjacent pancreatic beta cells.
Recently, synapses of amphids defective (SAD-A) kinase was identified as a kinase that phosphorylates PAK1, an effector of RAC1 [
40]. Although evidence for molecules that act downstream of RAC1 has been slowly accumulating, it is currently not clear how RAC1 regulates the depolymerisation of F-actin. CDC42 was reported to regulate F-actin remodelling in pancreatic beta cell lines [
41,
42], and RAC1 acts downstream of CDC42. Both RAC1 and CDC42 regulate the second phase of insulin secretion, and adequate ‘cycling’ of these proteins between the active state (GTP-bound) and the inactive state (GDP-bound) is important in insulin secretion [
41,
42]. Considering that inhibition of CDC42 cycling between GTP- and GDP-bound forms impairs insulin secretion and F-actin regulation [
41], it is likely that the absence of RAC1 cycling resulted in inhibition of F-actin depolymerisation in the RAC-deficient cells used in the present study. Thus, this study provides evidence for a crucial role of RAC1 cycling in insulin secretion via F-actin depolymerisation in vivo.
GLP-1 secretion in L cells of the small intestine was recently reported to be controlled by the actin barrier, suggesting that low-molecular-weight G proteins such as RAC1 and CDC42 contribute to the molecular mechanism [
43]. There are also reports of reduced incretin secretion being found in patients with type 2 diabetes mellitus [
44,
45], but the secretion mechanism of incretin remains largely unclear. Interestingly, not only insulin secretion but also incretin secretion might be controlled by RAC1 and CDC42, but further studies are required. Moreover, application of RAC1 to early diagnosis and treatment of diabetes mellitus warrants further investigation.
Acknowledgements
We thank S. Seino and T. Shibasaki for helpful discussions, and M. Nagano, M. Oya and A. Tanida for technical assistance.