β-Cell Receptor Tyrosine Kinases in Controlling Insulin Secretion and Exocytotic Machinery: c-Kit and Insulin Receptor

Abstract

Insulin secretion from pancreatic β-cells is initiated through channel-mediated depolarization, cytoskeletal remodeling, and vesicle tethering at the cell membrane, all of which can be regulated through cell surface receptors. Receptor tyrosine kinases (RTKs) promote β-cell development and postnatal signaling to improve β-cell mass and function, yet their activation has also been shown to initiate exocytotic events in β-cells. This review examines the role of RTK signaling in insulin secretion, with a focus on RTKs c-Kit and insulin receptor (IR). Pathways that control insulin release and the potential interplay between c-Kit and IR signaling are discussed, along with clinical implications of RTK therapy on insulin secretion.

The endocrine cells of the pancreatic islets of Langerhans secrete hormones that are responsible for the minute-to-minute regulation of glucose homeostasis, with the β-cells composing the majority of islet cell mass in mammals (1, 2). Upon stimulation, β-cells release insulin that binds to insulin receptors (IRs) in peripheral tissues (skeletal muscle, adipose tissue, liver) to stimulate glucose uptake and storage (3). Biphasic insulin release from β-cells is initiated with glucose-stimulated closure of ATP-sensitive K_ATP channels, increased intracellular calcium, and insulin granule exocytosis. The activation of select membrane receptors found on β-cells is an additional factor that can influence insulin secretion. The G protein–coupled receptor (GPCR) glucagon-like peptide-1 receptor (GLP-1R) is one of the most extensively examined β-cell receptors, with a well-established role increasing insulin secretion through increased levels of intracellular calcium and reduced voltage-gated potassium ion channel activity (4–6). Receptor tyrosine kinases (RTKs) are another class of membrane receptors present on cells throughout the islet. Although RTKs are known to maintain β-cell proliferation and survival, the activation of these receptors has also been reported to promote insulin release. This review will examine the role of β-cell RTKs, with a focus on c-Kit and IR, in regulating the trafficking and exocytosis of insulin granules.

Islet RTKs and Their Role in Islet Maintenance and Hormone Secretion

The islet microenvironment expresses various RTKs that regulate β-cell proliferation, function, and insulin synthesis and secretion (Fig. 1). Pancreatic development is one process that relies on the expression of multiple RTKs to achieve islet maturation. Epidermal growth factor receptor (EGFR) expression is necessary for embryonic β-cell maturation, islet migration, and maintenance of β-cell mass and proliferation (7, 8). Other members of the ErbB receptor family were also identified during murine pancreatic development studies (9). The receptor for nerve growth factor (NGF), Trk-A, follows a specific pattern of expression within endocrine cells that depends on the stage of embryonic development (10). Discoidin domain receptor tyrosine kinase 1 (DDR1) has recently been identified during early pancreatic development in emerging endocrine cells and in injury-induced ductal ligated pancreata (11). Platelet-derived growth factor receptor (PDGFR), which shares structural homology to c-Kit, regulates islet proliferation and has been found to decrease in islets with advancing age (12). Both EGFR and c-Met, the receptor for hepatocyte growth factor (HGF), promoted β-cell proliferation during pregnancy (13, 14). The contributions of c-Kit and IR to β-cell development and postnatal function are discussed in subsequent sections.

Figure 1.

RTKs in the islet microenvironment. The mature murine islet is composed of insulin-secreting β-cells in the core of the islet (blue) and other endocrine hormone–secreting cells along the islet’s periphery (red). β-cells express multiple RTKs that influence their growth and development. The direct effect of RTK activation on insulin secretion has been demonstrated to date in select RTKs. β-cells regulate and are influenced by RTKs expressed on the islet vasculature.

The current literature regarding RTK expression on the remaining islet endocrine cells (α-, δ-, PP-, and ε-cells) is limited. Unlike the receptor for insulin, the receptors for other islet hormones belong to the GPCR family (15, 16). The presence of IR has been confirmed on α-cells and is necessary for cell proliferation and the regulation of glucagon secretion through the Akt/P70S6K1 axis (17, 18). IR expression is also found in isolated single δ-cells from human islets and in the somatostatin-secreting TGP52 cell line (19, 20). Recent findings demonstrated that glucagon release from α-cells can be manipulated through the erythropoietin-producing hepatoma A (EphA) receptor, an RTK class that is unique due to forward and reverse signaling through β-cell/β-cell and β-cell/α-cell membrane receptor–membrane-bound ligand interactions (21, 22). However, the presence of RTKs and their activity in non–β-cell endocrine cells warrants further investigation.

RTKs are not only limited to hormone-secreting cells but are crucial for integrin–extracellular matrix (ECM) communication and islet vascularization. One study identified that β-cell fibroblast growth factor receptor 1 (FGFR1) promoted ERK phosphorylation, but its activation depended on the binding of α6β1 integrin to specific ECM ligands produced from endothelial cells (23), which increased both islet microvasculature remodeling and β-cell insulin secretion and survival (24). Vascular endothelial growth factor (VEGF) is secreted from β-cells and binds to VEGF receptors (VEGFRs) on islet endothelial cells to promote angiogenesis. Reduced islet vascularization, decreased β-cell mass, and impaired insulin and glucagon secretion were noted in mice with β-cell Vegf-a loss during embryonic development (25–27). Although inducing Vegf-a inactivation in the postnatal adult β-cell also lowered islet vascularization 3 months after its initial loss, these mice developed only mild glucose intolerance and unchanged β-cell mass (26), which indicates that islet endothelial cell regulation of β-cell mass occurs during select developmental time points. Hypervascularization of the islets through increased VEGFR expression can also detrimentally affect β-cell survival and lead to the development of hyperglycemia, stressing the importance of maintaining an optimal range for islet vascularization. Overexpression of Vegf-a during islet development or induced in adult β-cells led to increased endothelial cell density, but this expansion impaired the formation of islet clusters and decreased β-cell proliferation and mass in both mouse models (28, 29). Nonobese diabetic mice also had increased Vegf-a production from β-cells, which increased the expression of VEGFR on endothelial cells and subsequently led to islet inflammation and T-cell–mediated β-cell destruction (30).

In addition to controlling overall islet function, the activation of certain RTKs can directly regulate insulin granule release. NGF signaling through Trk-A maintained glucose-stimulated insulin secretion in mouse islets, and Trk-A internalization initiated F-actin reorganization and led to insulin exocytosis (31–33). Signaling through the c-Met receptor has also been observed to regulate secretion. Its disruption in murine β-cells resulted in impaired insulin release, with a minimal effect on islet morphology (34). IGF-1 receptor (IGF-1R), which shares structural homology with IR, was found to be necessary for glucose-stimulated insulin release and glucose sensing through the maintenance of Glut2 expression (35, 36). Fibroblast growth factor 21, through binding to its receptor, stimulated insulin release from isolated islets of diabetic rodent models under high-glucose conditions (37). RTKs have also been found to play a role in the negative feedback of insulin release, as observed with bidirectional signaling through EphA (21, 38). Although there are many RTKs in β-cells that contribute to their overall function, we will focus on the selected RTKs c-Kit and IR and their roles in insulin release.

c-Kit Activity in Islet Function and Insulin Secretion

c-Kit is the RTK for the ligand stem cell factor (SCF) and, similar to the islet RTKs discussed earlier, was observed in embryonic and newborn islets and become restricted to a small subpopulation of β-cells in the postnatal and adult rat pancreas (39–42). SCF treatment induced ERK phosphorylation in the INS-1 cell line and increased insulin⁺ cell expression in the PANC-1 cell line, highlighting its importance in β-cell development (41, 43). By using various mouse models, our research group has demonstrated that c-Kit activity is necessary in mature β-cells to maintain islet function and normoglycemia. Heterozygous mice (c-Kit^Wv/+) with an intracellular point mutation that disrupted receptor activation displayed reduced insulin content and glucose-stimulated insulin secretion (44). In contrast to the c-Kit^Wv/+ model, mice with a β-cell–specific overexpression of the human c-KIT receptor (c-KitβTg) had markedly greater islet mass, proliferation, and insulin release compared with wild-type controls (45). Increased vascularization was also noted in the islets of c-KitβTg mice through increased β-cell Vegf-a production, which promoted insulin secretion in mice on a normal diet but led to inflammation-induced β-cell apoptosis and islet dysfunction in mice on a long-term (22 weeks) high-fat diet (46). These in vivo studies have demonstrated that c-Kit signaling is necessary for β-cell development, survival, and function, yet the prolonged activation of the receptor may promote detrimental effects under metabolic stresses.

Although c-Kit activation on β-cells increased insulin production and release, the exact role of c-Kit signaling in the mechanism of insulin granule exocytosis is unknown. Secretory control through c-Kit activation has been extensively assessed in mast cells of the immune system, where histamine and IL-6 release were stimulated with SCF administration (47, 48). In mast cells, c-Kit activation via the phosphatidylinositol 3-kinase (PI3K) pathway and cytoskeletal remodeling were necessary for secretion (49, 50). Mice with a mutation that rendered c-Kit inactive exhibited reduced downstream PI3K/Akt signaling and decreased granule release response from mast cells (51). Although the direct role of c-Kit signaling on the regulation of β-cell insulin granule exocytosis has not been documented, we have found that c-KitβTg islets contained significantly elevated IR mRNA and protein expression (45). In light of these results, additional research is warranted to determine whether c-Kit directly regulates granule release in β-cells or requires interplay with other β-cell RTKs, including IR, for insulin exocytosis.

The Role of the β-Cell Autocrine Insulin/IR Axis in the Regulation of Insulin Secretion

In contrast to c-Kit, numerous studies have examined the capacity of IR activation to stimulate insulin release. There is little consensus on the role of insulin-stimulated insulin secretion because multiple studies have presented both positive and negative regulatory roles (summarized in Fig. 2). Findings from both in vitro cell models and in vivo rodent studies indicate that maintenance of the insulin/IR/insulin receptor substrate (IRS) signaling axis is important for biphasic insulin signaling and insulin granule exocytotic machinery. Transfection of βTC6-F7 cells with an overexpression of intracellular kinase mutated IR, which yielded reduced IR activity when compared with an overexpressing wild-type IR line, was shown to impair glucose-induced insulin secretion (52). The earliest study of mice with a β-cell–specific IR knockout demonstrated that reducing the presence of IR interfered with first phase insulin secretion (53). More recently, the adaptor protein APPL1, a regulator of Akt and an anchor between IR and IRS-1 (54), was identified as a potential upstream factor for the regulation of soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) Syntaxin 1a, Snap25, and Vamp2 in murine β-cells (55). Isolated single murine β-cells also demonstrated increased exocytotic events when stimulated with high doses of insulin (100 nM) and impaired secretion when pretreated with IR neutralization antibody (56).

Figure 2.

Effects of IR activation on insulin secretion. Insulin autocrine stimulation of β-cells can induce positive or negative feedback on insulin release. Positive feedback (right, green arrowheads): activation of the IR/IRS-1 pathway induces Akt phosphorylation, which increases intracellular Ca²⁺ release from the ER and SNARE protein levels and leads to increased insulin release from β-cells. Negative feedback (left, red arrowheads): insulin stimulation activates the IR/IRS/PI3K pathway, resulting in hyperpolarization of membrane K_ATP channel and a reduction in Ca²⁺-stimulated insulin release.

A review of the literature suggests that IR/IRS activity differentially affects intracellular Ca²⁺ influx from the cell membrane and endoplasmic reticulum (ER), which may explain the contrasting effects of autocrine insulin action during insulin release. It has been shown that IR signaling does not affect membrane depolarization–induced secretion but is necessary for mobilizing Ca²⁺ from intracellular stores (56). Insulin-stimulated isolated murine β-cells demonstrated hyperpolarization of the cell and mitochondrial membranes through activated K_ATP channels, which subsequently reduced the levels of intracellular Ca²⁺ necessary for insulin exocytosis (57). However, another report showed that treating murine β-cells with 100 nM of insulin heightened intracellular Ca²⁺ release from the ER, resulting in increased insulin secretion (58). This pathway relied on activation through the IR/IRS-1/Akt axis and is independent of K_ATP-induced depolarization. Irs-1 colocalized with the sarcoendoplasmic reticulum Ca²⁺ ATPase in βTC6-F7 cells, which inhibited Ca²⁺ reuptake into the ER and increased insulin secretion (59). Irs-1^−/− mice displayed reduced insulin granule exocytosis because of a shortened transient period of high intracellular Ca²⁺ levels after glucose stimulation (60), indicating that the IR/IRS-1 signaling axis is important for ER-regulated Ca²⁺ release. It has also been reported that isolated rat islets increased insulin release when Irs-1 was inhibited in a high-glucose environment (61), which suggests that IRS-1–induced insulin secretion can also be affected by glycemic levels. The data available at this time indicate that additional research is needed to determine the contrasting effects of insulin stimulation on intracellular Ca²⁺ levels and the downstream signaling pathways that connect receptor activation to granule exocytosis.

Interplay Between c-Kit and IR Signaling on Insulin Exocytosis

The role of Akt signaling through c-Kit and IR

Signaling through the PI3K/Akt pathway is well established in c-Kit– and IR-activated β-cells (45, 62). Importantly, Akt activation through the conversion of phosphatidylinositols via PI3K has been demonstrated in multiple experimental studies to promote insulin secretion. Loss of class IA PI3Ks in β-cells reduced intracellular Ca²⁺ levels and SNARE proteins, which inhibited insulin granule exocytosis (63). The α isoform of class II PI3Ks has also been linked to downstream Akt1 activation and insulin release under glucose-induced conditions (64). Akt is necessary for the phosphorylation of the Rab GTPase-activating protein AS160, responsible for glucose-stimulated insulin release, through IR/IRS-2 signaling (65). Focal adhesion kinase, which controls cytoskeletal remodeling and vesicle trafficking through integrin-initiated signaling, also phosphorylated the Akt/AS160 complex, indicating that this pathway may serve a role in focal adhesion kinase–mediated insulin release (66). In vitro studies have also determined that β-cell autocrine insulin signaling, in a glucose-induced environment, is necessary for activation of the PI3K/Akt pathway (67), which highlights the necessity of this signaling axis for insulin secretion. Our own studies have determined that c-Kit activation in mouse islets resulted in phosphorylation of serines 473 and serine 9 of Akt and GSK3β, respectively, leading to increased insulin secretion (45, 68). Recent work by others has shown that β-cell mTOR inactivation reduced Akt phosphorylation and inhibited insulin secretion without affecting islet mass (69). We also found that mTOR signaling in c-KitβTg mice is necessary for β-cell Vegf-a production and the subsequent promotion of islet vascularization and insulin secretion (46).

Interestingly, a temporal effect for PI3K suppression on rodent islets has been identified, where short-term inhibition of PI3K promoted increased newcomer granule exocytosis, whereas prolonged inhibition compromised insulin release (70). Additional support for an inhibitory role of the PI3K/Akt pathway was provided through experiments with p85α^−/− mice, which revealed that high-glucose treatments increased insulin release (71). It should also be noted that PI3K signaling through IR activation has been shown to inhibit insulin release in human islets (72). Although activation of the PI3K/Akt pathway has been reported to have differing effects on insulin secretion, it remains an important regulator of exocytosis in β-cells.

Proposed model of c-Kit and IR interplay for insulin release

c-KitβTg islets exhibited increased expression of Ir and Irs-1 (45), which establishes a link between the c-Kit and IR/IRS pathways that can contribute to the insulin release axis. Increased insulin release was initially observed in 8-week c-KitβTg mice and continued to 28 weeks of age (46). These results suggest that transient c-Kit signaling in β-cells can lead to IR/IRS upregulation through the following: (i) a direct interaction that affects the activity of the IR/IRS pathway [previously proposed in Feng et al. (73)]; and (ii) indirect IR activation through increased insulin secretion from c-Kit signaling (Fig. 3A). However, recent data from our group indicated that the prolonged expression of c-Kit on β-cells resulted in negative feedback through serine phosphorylation of Irs-1^Ser612, a known mechanism that downregulates IR/IRS signaling (75). This led to reduced signaling through the Akt pathway and subsequent defective insulin release. Decreasing the expression of IR on the β-cells of aging c-KitβTg mice improved their glucose tolerance. Interestingly, a recent study identified that IGF-1R was increased in aged β-cells that have poor insulin release (76). Activation of PDGFR has also been shown to promote IRS serine phosphorylation in adipocyte and vascular smooth muscle cells through PI3K/Akt/mTOR signaling (77, 78). The effects of c-Kit overexpression on β-cells can therefore influence the serine phosphorylation of IRS and promote dysregulated signaling of this axis through direct feedback from c-Kit–activated Akt signaling or from prolonged c-Kit–initiated insulin release (Fig. 3B). This evidence suggests that the IR axis may have a detrimental effect on insulin secretion in aged β-cells and that regulation of c-Kit–IR interplay must be considered to maintain appropriate insulin levels that contribute to the alleviation of hyperglycemia.

Figure 3.

Proposed c-Kit–IR interplay within the β-cell. (A) Transient receptor activation: c-Kit and IR both promote PI3K/Akt intracellular signaling, which leads to maintained insulin secretion. c-Kit receptor activation is proposed to promote upregulated IR/IRS signaling (via phosphorylation of tyrosine resides, green) through (i) direct activation and (ii) indirect activation through insulin secretion. Phosphorylation of IRS-1 tyrosine 608 in rodents and 612 in humans is linked to PI3K binding (74). (B) Chronic receptor activation: Sustained signaling through the PI3K/Akt pathway can lead to negative feedback through IRS-1 phosphorylation of serine residues (red) and impair insulin release. Multiple serine sites (e.g., Ser612) have been reported as phosphorylation targets from PI3K/Akt/mTOR/S6K1 signaling [for an extensive review, please see Copps and White (74)].

Potential Research Targets and Clinical RTK Therapeutic Applications for c-Kit–IR Pathway Activation

One of the major challenges for islet transplantation is preserving the insulin secretory efficacy of islets in patients. Inducing isolated islet proliferation and survival through ligand stimulation of RTKs has been examined as a potential therapy for improving islet transplantation. Increased HGF production from rodent islets via adenoviral transduction resulted in improved islet survival after transplantation and prevented the loss of intraportally transplanted islet function from immunosuppressive drug treatment (79, 80). Nonhuman primate islets transduced with murine HGF were also able to achieve euglycemic levels through increased islet survival when transplanted to streptozotocin (STZ)-induced diabetic mice (81). Similar improvements in islet survival and glucose tolerance in STZ-diabetic mice were noted when transplanted islets were pretreated with NGF (82). Improving the vascularization of rodent and human islets through their transfection with VEGF-A mRNA has been reported to increase β-cell volume (83) and restored glucose control in diabetic mouse models (83–85). The cotransplant of islet-containing scaffold grafts with a Vegf-a–releasing alginate sphere in STZ-diabetic mice also improved islet survival, vascularization, and the time interval for mice to reach restored normoglycemia (86). Additional studies have shown that delivering RTK ligands through transplanted islet-rich scaffolds or gels improved glucose homeostasis in diabetic mice (87, 88), which presents the effective role of RTK signaling as a promising strategy for extending the function of transplanted islets.

Our laboratory’s research proposed that RTK therapy targeting c-Kit and IR will increase the long-term function and survival of transplanted islets. Evidence from our previous work involving c-Kit signaling in murine islets has repeatedly shown that receptor activation increased insulin release under normal and short-term high-fat diet feeding (45) and promoted β-cell survival by downregulating the proapoptotic Fas receptor (89). c-Kit signaling also improved islet vascularization through increased β-cell Vegf-a secretion (46), which has been established as one strategy that can increase the survival and function of transplanted islets. The potential translation of c-Kit and SCF therapies in human islets has been demonstrated through our laboratory’s previous work on the human fetal pancreas. Phosphorylation of c-Kit in human islet-epithelial cell clusters in vitro, via either exogenous SCF treatment or antibody-targeted receptor activation, increased the proliferation and survival of insulin⁺ cells, increased the percentage of PDX-1⁺ and insulin⁺ cells found within the culture, and led to Akt phosphorylation (39, 90). Insulin treatment of isolated human and murine islets demonstrated a similar outcome, where islets had increased survival through Akt phosphorylation and Pdx1 nuclear translocation (62, 91). The activation of c-Kit and IR through exogenous SCF and insulin treatment, respectively, can therefore be used to improve long-term outcomes in recipients of transplanted islets.

Although activation of c-Kit–IR signaling pathways increases the capacity for β-cells to proliferate, diminishes cellular apoptosis, and increases insulin release to improve glucose tolerance, there is the potential for negative effects to emerge from this RTK therapy. Because of its prosurvival and replicative effects, activation of c-Kit signaling through SCF stimulation has been identified in a variety of different tumor types, and SCF treatment in pancreatic cell lines positive for c-Kit expression has increased their proliferation and invasion (92, 93). High levels of circulating insulin have been shown to induce inflammation in mice fed a high-fat diet and reduce the lifespan and insulin sensitivity in aged mice, and subsequently induced a diabetic phenotype in these rodent models (94, 95). Inhibitors that target RTKs have been examined for their potential to alleviate diabetic complications that emerge from the negative effects of prolonged intracellular activation, such as the promotion of islet inflammation and eventual β-cell apoptosis (96). The development of RTK therapies for transplanted islets must identify an optimal window where transient receptor activation will alleviate hyperglycemia by improving insulin secretory function while minimizing the cellular dysfunction observed with prolonged activation.

Summary and Perspectives

The secretion of insulin from β-cells is a controlled process that can be stimulated through many different means. RTKs are essential for the maintenance of normal β-cell development and function, and they play an important role in the regulation of granule exocytosis. Activation and signaling through the receptor c-Kit can increase the release of insulin and improve glycemia. Although the role of insulin activation of IR is controversial, a number of studies that focused on the expression and activation of IR in β-cells have found that this receptor is positively associated with insulin exocytosis. Intracellular signaling through the PI3K/Akt pathway shared by both c-Kit and IR activation is necessary for regulating insulin granule release. Interplay between β-cell membrane receptors can modulate granule release, but sustained activation of c-Kit and IR in β-cells can blunt insulin secretion, leading to insufficient control of blood glucose. Understanding the mechanisms through which c-Kit and IR control the release of insulin is essential for the potential application of RTK-based therapies because determining appropriate temporal or dosage levels will be necessary to optimize β-cell function in diabetes treatment.

Glossary

Abbreviations:

DDR1

discoidin domain receptor tyrosine kinase 1

ECM

extracellular matrix

EGFR

epidermal growth factor receptor

EphA

erythropoietin-producing hepatoma A

ER

endoplasmic reticulum

FGFR1

fibroblast growth factor receptor 1

GLP-1R

glucagon-like peptide-1 receptor

GPCR

G protein–coupled receptor

HGF

hepatocyte growth factor

IGF-1R

IGF-1 receptor

IR

insulin receptor

IRS

insulin receptor substrate

NGF

nerve growth factor

PDGFR

platelet-derived growth factor receptor

PI3K

phosphatidylinositol 3-kinase

RTK

receptor tyrosine kinase

SCF

stem cell factor

SNARE

soluble N-ethylmaleimide–sensitive factor attachment protein receptor

STZ

streptozotocin

VEGF

vascular endothelial growth factor

VEGFR

vascular endothelial growth factor receptor