Using β-cells that completely lack either the insulin receptor or IRS-2, we found that the signaling effects of glucose require the presence of an intact insulin/IGF-I signaling pathway. Glucose stimulation of the mutant β-cells failed to activate key kinases that mediate important downstream effects, including proliferation, survival, and protein synthesis, indicating the significance of insulin signaling for the maintenance of β-cell mass.
Although previous studies, using β-cell lines with a partial knockdown of insulin receptors, have reported that the signaling effects of glucose in β-cells include the effects of secreted insulin, the presence of residual insulin signaling in these cells raised the confounding possibility that the effects of glucose were diluted by the ambient insulin that is continuously secreted by β-cells via the constitutive pathway. For example, in independent experiments Da Silva Xavier et al. (9
) and Ohsugi et al. (38
) achieved transient knockdown of insulin receptor expression levels in MIN6 cells that ranged between 80 and 90%. In these approaches the residual insulin signaling activity can potentially confound the interpretation of the effects of glucose in β-cells. Further, previous reports indicate potential nonspecific effects of siRNA (2
). Similarly, experiments designed to dissociate the effects of glucose from insulin secretion using somatostatin (57
) are unlikely to completely block regulated and constitutive insulin secretion that occurs in primary β-cells (19
). The use of β-cell lines from βIRKO and IRS-2-KO mice that lack expression of the insulin receptor or IRS-2, respectively, circumvents these limitations and allows for direct interpretation of the signaling effects of glucose.
Consistent with the signaling effects of insulin reported in the classical insulin target tissues (7
), insulin rapidly activated proteins in the insulin/IGF-1 signaling pathway in control β-cells, with peak effects occurring within a minute, in contrast to the delayed effects of glucose. While the delayed effects of glucose in control β-cells may be explained, in part, by the time required for the sugar to be transported into the cell and metabolized (43
), the failure of glucose to activate PI3 kinase or Akt kinase in the β-cells only when either the insulin receptor or IRS-2 is lacking clearly indicates the dependence of glucose on the two proteins for its signaling effects (Fig. ).
FIG. 9. Schematic of a link between glucose and insulin signaling in β-cells. (A) Potential direct effects of glucose and/or its metabolites on proteins in the insulin/IGF-1 signaling pathway. (B) Potential indirect effects of glucose and direct effects (more ...)
The defects in glucose-stimulated signaling also provide insights into the phenotypes of the βIRKO mice. For example, the activation of cleaved capsase-3, either by glucose stimulation of β-cells lacking insulin receptors (βIRKO β-cells) or by blocking insulin action in control β-cells by using an anti-insulin receptor antibody, indicates that functional insulin receptors activate antiapoptotic signaling in control β-cells. Indeed, βIRKO mice exhibit an age-dependent decrease in β-cell mass and a susceptibility to develop type 2 diabetes (25
). Thus, it is conceivable that ambient hyperglycemia promotes apoptosis of β-cells (12
) in insulin-resistant states due to poor insulin-stimulated activation of Akt (5
). Furthermore, the reduced stimulation of mTOR by glucose in βIRKO cells suggests that blunted protein synthesis also contributes to a reduced islet cell mass in βIRKO mice (35
We and others have previously reported that loss of insulin signaling in β-cells impairs acute-phase insulin secretion in response to glucose (9
). In agreement with these reports, expression of GLUT2 was stimulated by glucose and insulin, but not by IGF-1. This effect is diminished in βIRKO cells, suggesting that functional insulin receptors play a role in glucose-stimulated expression of GLUT2. It is possible that a reduced expression of GLUT2 in βIRKO cells plays some role in the ability of glucose to directly activate insulin signaling proteins. Our studies using the PI3 kinase blocker (LY294002) and the dominant negative p85 provide further evidence that the effect of glucose occurs in a PI3K-dependent manner (62
). In contrast, the effects of glucose on p-ERK and its potential downstream effects on cell proliferation and differentiation occur independently of insulin signaling (23
). While our studies provide a systematic characterization of alterations in the signaling proteins in the insulin/IGF-1 pathway in βIRKO versus βIRS2KO cell lines, our findings in the βIRKO cells are largely in agreement with those of Da Silva Xavier et al. (9
) and Ohsugi et al. (38
Insulin and glucose minimally activated IGF-1 receptors in controls, while phosphorylation of the IGF-1 receptor and activation of downstream proteins in the insulin/IGF-1 signaling cascade were clearly evident upon IGF-I stimulation of βIRKO cells. The upregulation of IGF-1 receptors in βIRKO cells indicates a potential long-term compensatory effect in the absence of functional insulin receptors. Thus, the significance of IGF-1 receptor activation in vivo for the maintenance of β-cell proliferation and survival, especially during states of β-cell insulin resistance, requires careful investigation (32
The similarities in signaling defects between βIRKO and IRS-2KO β-cells are consistent with the largely similar phenotypes of the mutant mouse models (24
). In contrast, IRS-1KO mice exhibit a compensatory increase in β-cell mass (3
) that is, in part, due to upregulation of IRS-2 (20
). The contrasting signaling effects due to loss of IRS-1 versus loss of IRS-2 indicate that these two substrates serve different downstream pathways in β-cells, with IRS-1 having a dominant effect in insulin secretion (28
) and IRS-2 being relevant for growth and/or apoptosis (8
). Cell-specific roles for the two substrates have also been reported in other cell types. For example, brown preadipocytes that lack IRS-1 fail to differentiate into mature adipocytes due to altered UCP-1 expression, compared to a mild defect in cells lacking IRS-2 (48
). In hepatocytes, IRS-1 and IRS-2 have been suggested to play complementary roles in regulating lipid and carbohydrate metabolism (13
). The availability of stable knockout β-cell lines created for the studies reported in this paper provides us with an opportunity to further dissect the differential signaling responses between IRS-1 and IRS-2 in β-cells.
In summary, our studies point to an important role for insulin/IGF-I signaling in the signaling responses activated by glucose stimulation with implications for a direct effect of insulin in the maintenance of β-cell mass and function (Fig. ).