Type 2 diabetes is characterized by peripheral insulin resistance followed by a failure of compensation by the pancreatic β cell and, ultimately, frank hyperglycemia (21
). Traditionally, these have been discrete pathophysiological lesions; however, it is increasingly clear that the insulin-signaling cascade may play an important role in the functioning of the islet/β cell (9
). Previous work (9
) and the results of the present study indicate that the IRS proteins are present in pancreatic islets and that the β-cells lacking IRS-1 or IRS-2 show altered function and/or growth. In addition, our recent demonstration of altered glucose sensing in a β-cell tissue-specific knockout of the insulin receptor suggests that insulin resistance at the level of the β cell itself may contribute to the failure of pancreatic compensation, thereby suggesting a unifying theory for the pathogenesis of type 2 diabetes (11
In the present study, we have demonstrated that although IRS-1 knockout mice appear to have a normal β-cell hyperplastic response to insulin resistance (7
), IRS-1 deficiency leads to decreased insulin content of β cells and a reduced acute phase secretion to both glucose and L
-arginine. The diminished glucose-stimulated insulin secretion likely contributes to the glucose intolerance observed in the IRS-1–/–
mice. When arginine is used as a stimulant, there is also a reduced insulin response in the IRS-1–/–
mice. Although the precise mechanism of action of arginine is poorly understood, the reduced response to both glucose and arginine suggests that the lack of IRS-1 in the β cell impairs a final step in the insulin secretory process. This phenotype is different from those both in the β-cell–specific insulin receptor knockout that influences glucose-stimulated insulin release (11
), and in the IRS-2 knockout that manifests no secretory defects but altered β-cell growth (28
). It is notable that in addition to the insulin receptor, IRS-1 acts as a substrate for IGF-1, prolactin, and growth hormone receptors (29
). The presence of these receptors in insulin-producing cells (2
) indicates they may potentially activate IRS-1 in islets. Similarly, as several cytokines and growth factors activate IRS-2 (35
), it is possible that this protein is a substrate for the growth-promoting IGF-1 receptor in the β cells (25
). The reduced number of insulin-positive cells in islets isolated from mice heterozygous for the IGF-1 receptor and homozygous for IRS-2 suggests that IGF-1 receptor–mediated β-cell growth is likely mediated by IRS-2 (36
). Taken together, these data suggest that insulin receptors and IGF-1 receptors, as well as IRS-1 and IRS-2, each have distinct functions in the islet/β-cells.
Although it is clear from our in vivo experiments that the acute insulin secretory pattern is blunted, basal circulating insulin levels are higher in the IRS-1–/–
mice than in controls. This mild hyperinsulinemia is due to increased secretion, as evidenced by increased C-peptide levels, and may be due to 2 factors. First, IRS-1–/–
mice are insulin resistant (6
), and this by itself may cause hyperinsulinemia due to normal feedback mechanisms (11
). In addition, the circulating leptin levels are 50% lower in the growth-retarded IRS-1–/–
mice, similar to obese individuals carrying a variant of IRS-1 (37
). Leptin has been shown to suppress insulin secretion in vivo and in vitro (15
) and may result in the removal of a tonic insulin inhibitory effect resulting in unrestrained insulin secretion.
To define the nature of the insulin secretory defect in the absence of this and other in vivo feedback mechanisms, functional studies were conducted on isolated islets. This also enabled us to compare directly islet function in islets of similar size. Although small and large islets could be detected in the IRS-1–/–
mice, the insulin content in both of these types of islets was lower than in WT islets. Because the IRS-1–/–
mice are growth retarded, the islet size/body mass ratio is actually greater in these mice when compared with WT controls. This factor and a greater proportion of large islets and a smaller total blood volume contribute in part to the mildly higher circulating insulin in the knockout mice. When incubated in vitro, IRS-1–deficient islets secrete lower insulin in the basal state. In vitro glucose stimulation showed a diminished release in islets isolated from IRS-1+/–
and even more so with IRS-1–/–
mice, indicating a genetic dose response. These secretory responses were lower even after correcting for the lower insulin content, indicating a secretory defect in the islets lacking IRS-1. An earlier study by Terauchi et al. (7
) also reported a reduced basal insulin release and consistently lower secretory responses to increasing concentration of glucose in batch-incubated and perifused islets isolated from IRS-1–/–
mice compared with WT controls. However, no reference is made as to whether islet size is taken into consideration in these experiments as considered in our study.
Similar results were obtained with cultured β-cell lines derived from IRS-1–/–
mice. Furthermore, after transfection of the IRS-1–/–
cells to restore IRS-1 protein, there is a significant increase in insulin content consistent with an important role for IRS-1 in the regulation of insulin stores in the β cells. Interestingly, expression of a mutant IRS-1 (glycine for arginine in codon 972) in rat insulinoma cells has been reported to result in a lower insulin content and a lower secretory response to glucose and glybenclamide compared with cells expressing WT IRS-1 (39
). Indeed, polymorphisms in IRS-1 occur at a 2-fold higher frequency in patients with type 2 diabetes compared with unaffected individuals (40
). It is interesting that carriers of this mutation manifest low serum insulin and C-peptide levels (41
). The results presented in this study suggest that the presence of altered IRS-1 molecules contribute to the insulin deficiency in these patients.
The exact molecular consequences of IRS-1 deficiency that lead to the altered insulin content and secretion need further investigation. We and others (24
) have shown that glucose stimulates phosphorylation of IRS-1 in islets. In addition, this promotes IRS-1 association with the regulatory subunit of PI 3-kinase (24
) and activates pathways involved in β-cell proliferation (25
). Expression of the mutant IRS-1 in rat insulinoma cells showed a decrease in binding of the p85 subunit of PI 3- kinase (39
). PI 3-kinase activity has been implicated in multiple vesicle trafficking roles and in insulin and growth factor–regulated gene expression (44
). We are currently performing single-cell amperometric experiments to dissect the mechanism of the loss of glucose-stimulated insulin secretion in IRS-1–/–
cells. Preliminary data show a delayed rise in Ca+
in response to glucose consistent with the data in IRS-1–deficient islets and cultured β-cell lines (data not shown). It is therefore conceivable that the insulin receptor/IRS-1/PI 3-kinase pathway is involved in islet secretory function and insulin gene expression.
The presence of a β-cell defect is thought to be one of the prerequisites for full development of type 2 diabetes. However, defining the temporal onset of islet decompensation and localizing the functional defects in humans has been difficult. The IRS-1 knockout mouse provides an excellent genetic model in that it exhibits early insulin resistance but manifests only impaired glucose tolerance and does not become overtly diabetic. The demonstrable detectable defect in islet function, especially clear in vitro, therefore implicates confounding systemic factors that mask the islet dysfunction. One interesting line of approach would be to investigate the IRS-1 knockout mice on other genetic backgrounds, as polymorphic modifier genes could well alter the phenotypes and produce frank diabetes. In summary, the present data provide novel evidence of a role for IRS-1 in an insulin-signaling cascade that links it to insulin content and secretion in the islet β cell, a linkage that may be disrupted in patients with type 2 diabetes manifesting insulin resistance.