In T2DM patients, insulin signaling, especially PI3K/Akt signaling, is known to be diminished in muscle and liver owing to insulin resistance and/or decreased circulating insulin, leading to a reduction in glucose metabolism and subsequent hyperglycemia (Bandyopadhyay et al., 2005
). We and others have previously shown that β cells also possess insulin signaling systems that play a critical role in the regulation of β cell mass and function (Kulkarni et al., 1999
; Leibiger et al., 2001
; Ueki et al., 2006
). It is possible that decreased insulin signaling in β cells, resulting from either insulin resistance or decreased insulin secretion in these cells, may cause loss of β cell function and mass, leading to hypoinsulinemia and subsequent hyperglycemia.
We asked whether insulin signaling in the β cell in db/db
mice, an obese diabetic animal model, would play a role in the compensatory hyperinsulinemia in response to insulin resistance and the exacerbated hyperglycemia seen after decompensation. Indeed, as db/db
mice grow older and obese, hyperglycemia and defects in insulin secretion become evident, and phosphorylated Akt levels significantly decrease in β cells, indicating impaired insulin signaling within these cells. In accordance with the age-dependent impairment of insulin secretion and decreased β cell mass, expressions of insulin signaling molecules, such as class IA PI3K, decline. In addition to the reductions in expression of these molecules, posttranslational modulation of signaling molecules by increased oxidative stress, ER stress, and inflammation may further attenuate insulin signaling (Evans et al., 2003
; Hotamisligil, 2006
). These results suggest the possibility that insulin signaling in β cells plays a crucial role in the maintenance of β cell mass and functions in response to insulin resistance through the class IA PI3K pathway. Thus, we have tried to explore the roles of class IA PI3K in maintaining β cell functions and mass using the mice lacking all the major regulatory subunits of class IA PI3K specifically in β cells.
In β cells, unlike in hepatocytes, deletion of pik3r1
markedly suppresses downstream signaling, such as Akt activation, and results in impaired glucose tolerance with a defect in GSIS. Double knockout of pik3r1
in β cells (βDKO), which further shuts down the downstream signaling, causes an even greater defect in GSIS and decreased β cell mass. These defects are caused by severely impaired insulin secretion and a robust increase in apoptosis. Interestingly, βDKO mice do not develop overt diabetes, unlike insulin/IGF-1 receptor double knockout mice in which both PI3K and the Erk pathways are suppressed, even considering the fact that pik3r2
null mice exhibit a modest insulin-sensitive phenotype (Ueki et al., 2002b
The PI3K/Akt pathway can negatively affect IR/IGFR signaling via multiple mechanisms, such as serine phosphorylation of IRS1 and Raf (Um et al., 2004
). Abrogation of this signal may enhance tyrosine phosphorylation of IRS proteins and reduce serine phosphorylation of Raf, ultimately increasing the signaling to the Raf/Erk pathway. Indeed, βDKO islets exhibit significantly increased phosphorylation of Erk1/2 as well as increased BrdU incorporation. Thus, βDKO mice can preserve β cell mass presumably at a level necessary to maintain normoglycemia, despite a marked increase in apoptosis and impaired insulin secretion.
The current study has revealed that class IA PI3K plays a crucial role in maintaining β cell function through regulating cell-cell synchronization associated with Ca2+
influx and exocytosis machinery. Previous studies have reported that gap-junction channels composed of Connexin36 hexamer regulate the cell-cell synchronization in β cells through direct exchanges of metabolites and ions and that these communications are important for proper GSIS and oscillations (Nlend et al., 2006
; Ravier et al., 2005
). In βDKO islets, the expression of gjd2
is significantly reduced. Moreover, db/db
islets also show impaired cell-cell synchronization similar to that seen in the βDKO islets, with decreased expression of gjd2
in accordance with decreased insulin secretion. Thus, the impaired Ca2+
influx can be accounted for, at least in part, by the impaired cell-cell synchronization.
On the other hand, ATP production and the expressions of slc2l2
and mitochondria-related genes are slightly decreased in βDKO islets, consistent with the recent report that the expression of slc2l2
and mitochondrial functions are regulated by insulin signaling in β cells (Assmann et al., 2009
; Liu et al., 2009
). Although electrophysiological experiments fail to indicate significant impairment of the steps upstream of Ca2+
influx, at least at the single β cell level, these defects might also contribute to the impairment of GSIS by deletion of class IA PI3K. Meanwhile, the mRNA expression of gck
shows no reduction in βDKO islets, although gck
expression is reduced both in βIRKO and in βIGFRKO islets. Recently, it has been reported that class II PI3K (PI3K-C2α) may play an important role in GSIS in pancreatic β cells downstream of the IR (Leibiger et al., 2010
). In this report, impairment of class II PI3K signaling has been shown to result in the reduction of gck
expression. Thus, the expression of gck
might be regulated through class II PI3K signaling, not through class IA PI3K signaling downstream of IR.
Insulin secretion occurs to a much lesser extent in βDKO islets compared to Pik3r2KO islets, even when using caged-Ca2+
stimulation, suggesting another defect downstream of Ca2+
influx, such as in the exocytosis machinery (Takahashi et al., 2004
). Indeed, expressions of SNARE complex genes are markedly reduced in the islets of βDKO mice, and these reductions can contribute to defects in insulin secretion downstream of Ca2+
Are these insulin secretion defects directly caused by ablating PI3K signaling? Expression of FoxO1-3A in the Pik3r2KO islets reduces the expression of snap25 and gjd2, both of which have the putative FoxO1 binding sites conserved across species in their promoter region, suggesting that FoxO1 binds to the promoter regions and inhibits the transcription of these genes. Thus, activation of PI3K/Akt appears to promote nuclear excursion of FoxO1, thereby increasing the expression levels of these genes. Indeed, expression of GagAkt normalizes the expression of these genes with other SNARE complex genes, such as vamp2, stx1a, and rab27a. These data also suggest that other transcription factor(s) may regulate the expression of other SNARE complex genes through Akt activation, independently of FoxO1. Importantly, restoration of the expression of these genes by Akt activation almost completely normalizes GSIS in vitro.
T2DM patients show decreased islet mRNA and protein expressions of SNAP25, Syntaxin 1a, and VAMP2 compared to nondiabetic subjects (Ostenson et al., 2006
). Moreover, Connexin36 is also expressed in human islets, and it has been suggested that this may play a role in insulin secretion (Serre-Beinier et al., 2009
). Taken together, these findings suggest that these SNARE complex proteins and Connexin36, whose expression is regulated by the PI3K/Akt pathway downstream of insulin signaling, may control the function of the human β cell. In addition, downregulation of these proteins by impaired PI3K/Akt activity may play a crucial role in the deteriorated GSIS, one of the characteristics of T2DM.
Similar to db/db
mice in the current study, human obese T2DM patients show hyperinsulinemia with a defect in early-phase GSIS at the early stage of T2DM and then develop severe diabetes with hypoinsulinemia and reduced β cell mass in the advanced stage (Butler et al., 2003
; Rhodes, 2005
). It is possible that insulin resistance in β cells inhibits PI3K activity by some mechanisms, such as decreased expressions of insulin signaling molecules, including class IA PI3K. As shown in βDKO mice, decreased PI3K activity in β cells can impair insulin secretion by suppressing glucose metabolism, cell-cell communication, and SNARE proteins and by increasing apoptosis. However, β cell mass can be maintained by enhanced Erk signaling induced by decreased PI3K activity as long as input of the insulin signal to activate IRS/Erk pathway is preserved by hyperinsulinemia in response to peripheral insulin resistance. Meanwhile, at an advanced stage of diabetes, severely impaired IR activation due to further reductions in expression levels of IR/IRS2/PI3K and relative hypoinsulinemia may abolish both PI3K/Akt and Erk activity. This leads to exacerbation of impaired insulin secretion, decreased β cell mass, and subsequent hypoinsulinemia, making a vicious cycle.
Taken together, class IA PI3K in β cells is indispensable for normal insulin secretion mainly through controlling intracellular Ca2+ levels, maintaining cell-cell synchronization, and regulating SNARE complex proteins. It is also important for antiapoptosis and regulates β cell mass, in cooperation with cell proliferation controlled by the Erk pathway. Thus, enhancing the class IA PI3K pathway, not only in peripheral tissues but also in β cells, may provide a therapeutic strategy for T2DM.