Proliferation rates of phogrin-downregulated MIN6 stable cells () and phogrin-overexpressed stable cells (S. Torii and T. Takeuchi, unpublished data) were reduced and elevated, respectively. Thus, phogrin appeared to be a positive regulator of β-cell growth. Because studies using stable cells seem inadequate for evaluation of primary functions of phogrin, we generated adenoviruses expressing shRNA. As expected, silencing of phogrin by Ad-shPhogrin caused a marked retardation of cell growth in both cultured cell lines and mouse islets (–). However, GSIS stayed unaffected by phogrin or IA-2 knockdown ( and ). Previous studies have shown that the deletion of IA-2 or phogrin gene or in combination in mice resulted in mild impairment of GSIS but did not affect β-cell mass (19
). However, more recent studies have suggested that islets from double knockout mice failed to show any secretion defect (45
), and β-cell regeneration was reduced in partially pancreatectomized ICA512 (IA-2) knockout mice (46
). Therefore, it is possible that the alteration in GSIS in knockout mice is indirect and that β-cell mass is recovered by compensatory function of other genes. Our present results support the notion that the primary function of phogrin is a regulation of β-cell growth but not of insulin secretion.
Glucose is a potent mitogen for β-cells and regulates various cellular dynamics, including insulin secretion and nutritional metabolism (1
). Although several signaling molecules such as PKC have been thought to be a mediator for glucose-induced β-cell growth (1
), recent studies have indicated a novel pathway in which the autocrine/paracrine function of secreted insulin promotes β-cell proliferation (3
). We demonstrated that β-cell growth retardation induced by phogrin knockdown was observed under the high-glucose culture (). Because phogrin was found to form a complex with insulin receptor () and because cell growth retardation by phogrin knockdown was not observed in insulin receptor–deficient cells (), insulin receptor is evidently a functional target of phogrin. Phogrin localizes to insulin-containing secretory granules and translocates to the plasma membrane whenever insulin exocytosis is induced () (36
). Thus, interaction of phogrin with insulin receptor is coordinately coupled with the autocrine action of insulin. Their interaction was strikingly promoted by the high glucose-induced insulin secretion (). In other words, phogrin regulates the glucose-induced β-cell growth through modulating the autocrine insulin signaling. Insulin secretion was never influenced by overexpression or repression of phogrin ( and ), thereby, modulation of autocrine effects by phogrin is not intervened by extracellular insulin dosage.
Our subcellular fractionation experiments indicate that phogrin interacts with insulin receptor on the plasma membrane (). However, EGFP-tagged phogrin in MIN6 or PC12 cells did not spread to whole plasma membrane under the evanescent microscopy observation (44
), suggesting that it remains on secretory granules during exocytotic events. On the other hand, the experimental result that phogrin antibody in culture medium accessed to the cell surface phogrin protein in MIN6 cells (49
) suggests that phogrin positively reaches the cell surface. Thus, the plasma membrane fraction in our assay may contain the attached granules that had been connected to the plasma membrane by lipid bilayers merger. Because insulin receptor is reportedly distributed to the methyl-β-cyclodextrin–sensitive microdomains of the plasma membrane in HIT-T15 cells (50
), it is possible that phogrin and insulin receptor colocalize at uncharacterized specific domains equivalent to the secretory granule targeting/fusion sites.
Our data indicate that phogrin interacts with insulin receptor and stabilizes IRS2 protein in β-cells. This hypothesis raises a question: What is the molecular mechanism of this interaction? Because phogrin and IA-2 have an inactive PTP domain in their cytoplasmic tail, it is possible that they bind to insulin receptor directly. In fact, the phogrin cytoplasmic fragment could bind to tyrosine-phosphorylated 95- to 100-kDa proteins in vitro (supplementary Fig. S2). Phogrin may regulate the insulin signaling through the tyrosine phosphorylation/dephosphorylation cycle. Insulin or IGF-I promotes degradation of IRS2 through PI 3-kinase, Akt, and mTOR signaling in adipocytes, hepatoma, and embryonic fibroblast cells (51
). Also, chronic exposure (>8 h) to high glucose and/or IGF-I induces proteasomal degradation of IRS2 in β-cells (43
). These proposed signaling pathways may be mediated by phogrin function for stabilization of IRS2.
Ablation of IRS2 in β-cells led to a decrease in β-cell mass and an increase in islet apoptosis (5
). In contrast, IRS2 expression in mice prevented diabetes by promoting β-cell growth (52
). Furthermore, IRS2 expression induced by glucose, GLP-1, and other signaling proteins has been shown to contribute to their regulatory functions for proliferation, indicating that IRS2 is a master regulator of β-cell growth (14
). Our data further indicate that IRS2 protein is stabilized under the control of phogrin and insulin receptor interaction for its cell growth regulation. This is because silencing of phogrin resulted in a marked reduction of IRS2 without change in other protein levels. Furthermore, adenoviral overexpression of phogrin caused a partial recovery of IRS2 level in MshP#33 and MshP#44 cells (). Thus, phogrin functions as a primary regulator of secreted insulin-mediated β-cell growth by stabilizing IRS2 protein.
To respond to hyperglycemia, β-cells proliferate and expand to compensate for increased insulin secretion demand (14
). The present observations suggested a novel mechanism in which phogrin contributes to glucose-induced proliferation of β-cells via insulin receptor and IRS2. Interestingly, insulin receptor and IRS2 mRNA levels reportedly decreased in islets isolated from human type 2 diabetes (53
). To prevent the decrease in insulin receptor and IRS2 functions, we suggest that phogrin and IA-2 in the β-cells are potential therapeutic targets for treating diabetes.