By using p44tg mice with an ectopic p53
gene that encodes Δ40p53 (36
), we have identified a role for the tumor suppressor p53 in the maintenance of normal β-cell mass and glucose homeostasis. Although Δ40p53 has virtually no autonomous transcriptional activity, it can interact with full-length p53 to form tetramers that bind DNA and activate or suppress target gene expression (36
). Expression of ectopic Δ40p53 in the mouse alters the balance between the full-length and short isoforms that normally exists and hyperactivates p53 (22
). This increased activity of p53 can account for the higher levels of p21 transcripts, which we observed in β-cells from 10–12-month-old p44tg mice. The expression of Δ40p53 in p44tg mice can also account for the higher level of mRNA encoding the IGF-1R, whose transcription is differentially regulated by wild-type and mutant p53 (39
). An increased gene expression of IGF-1R
in liver and white adipose tissue from p44tg mice (Supplementary Fig. 5
) suggests a non–tissue-specific effect. Similar to embryonic fibroblasts derived from p44tg mice (22
), islets from young p44tg mice expressed significantly high levels of IGF-1R, indicating impaired trans
-suppression activity. Both the increased trans
-activation of p21 in old islets and the decreased trans
-suppression of the IGF-1R in the young can be explained by differences in expression of the transgene encoding Δ40p53 with age, as detected by primers that amplify sequences encoding the COOH-terminal domain of the p53 protein. Younger p44tg mice expressed control levels of Δ40p53 and slightly lower levels of p53, whereas older mice expressed normal levels of p53 and higher levels of Δ40p53. Biochemical experiments have demonstrated that the stability and activity of the p53 tetramer are exquisitely sensitive to the dose of Δ40p53, with low doses of Δ40p53 activating and high doses inactivating p53 function (40
). The differential effects of Δ40p53 on p53-dependent trans
-activation and trans
-repression with age could also explain how p21 levels increase in both age groups, but by different mechanisms. In old mice, the increase would be due to a direct effect of p53 on the p21 promoter, leading to increased transcription of the p21
gene. In young mice, on the other hand, increased p21 could be an indirect effect of higher IGF-1R expression and activation of IGF signaling due to impaired IGF-1R trans
-repression. Stimulation of the IGF-1 signal transduction pathway can increase p21 (42
), and further work is necessary to delineate the link among Δ40p53/p53, p21, and IGF-1R levels in β-cell proliferation.
β-Cell proliferation and glucose tolerance were impaired in 3-month-old p44tg animals and worsened to overt diabetes as the animals aged. Although random-fed blood glucose levels were normal in the transgenic mice at 3 and 10 months, the mutants displayed clear intolerance in response to a glucose challenge indicating deficiency in functional β-cells. Previous studies in rodents have reported normoglycemia even when β-cell mass is reduced. For example, Sreenan et al. (43
) report a reduced β-cell mass before the onset of diabetes in the nonobese diabetic mouse, and Tavana et al. (44
) describe the phenotypes of 1-month-old mice doubly mutant for p53 and nonhomologous end-joining deficiency that exhibit an ~50% decrease in β-cell mass and yet manifest blood glucose levels that are not significantly different from controls. Further, obese and nondiabetic humans have been reported to express a wide range of β-cell mass that is adequate to maintain euglycemia up to a specific threshold, and crossing the threshold correlates with fasting hyperglycemia and overt diabetes (45
). In our study, it is possible that p44tg mice that are significantly leaner than controls at all ages only cross this “threshold” at ~12 months of age when they begin to exhibit overt hyperglycemia. Additional longitudinal studies are necessary to investigate these observations. Thus, our data suggest that the balance between full-length and Δ40p53 isoforms plays a critical role in the maintenance of β-cell proliferation and glucose homeostasis, and may be important in regulating the cell cycle during aging. Further support for a role for p53 in glucose homeostasis is evident from recent studies. For example, Minamino et al. (46
) reported a role for adipose p53 in the regulation of insulin resistance. In our study, circulating proinflammatory cytokines, which are potential effectors in insulin resistance associated with diabetes, showed no significant differences between groups (Supplementary Fig. 4
). The cytokine expression profile in liver and white adipose tissue was also similar between groups. These data, along with the normal insulin sensitivity in the global p44tg mice, indicate that the glucose intolerance is due to intrinsic effects of p53 on β-cell mass rather than to secondary effects of transgene expression in other insulin sensitive tissues. Indeed, Tavana et al. (44
) reported that DNA double-strand breaks combined with an absence of p53-dependent apoptosis in mice leads to reduced β-cell replication and severe age-dependent diabetes, supporting our hypothesis that p53 plays a role in the regulation of β-cell proliferation.
Although the mechanisms that regulate the dynamics of β-cell turnover during aging are still being unraveled, several studies including our own support a role for replication as the major mechanism that underlies the compensatory growth response to insulin resistance (1
). Two proteins that are important for this compensatory growth response are cyclin D2 (5
) and PDX-1, the pancreatic duodenal homeobox domain protein (3
). The lower expression of cyclin D2 in islets would potentially exacerbate the loss of β-cells and lead to glucose intolerance and overt diabetes. In this study, the expression of PDX-1 was also altered in p44tg β-cells. Although the gene expression of PDX-1
was increased in both young and old p44tg mice, the protein expression in islets was low, suggesting compensatory effects in gene expression or more dominant regulation of the transcription factor at the protein level. In old p44tg mice, however, there were fewer β-cell nuclei in which PDX-1 could be detected. Immunohistochemistry (data not shown) also revealed that the localization of PDX-1 correlated inversely with that of FoxO1, a transcription factor that regulates expression of this homeodomain protein (34
). Because FoxO1 transactivates cell cycle inhibitors (p21 and p27) (35
) and represses cell cycle activators (cyclin D1 and D2), nuclear localization of FoxO1 could have a major impact on cell cycle progression in aging β-cells. Further subcellular fractionation approaches are necessary to directly address this question. It is also possible that FoxO1 and p53 interact with deacetylase sirtuin 1 and function in a cooperative manner in an aging environment (48
). Thus, the alterations in both PDX-1 and cyclin D2, in our model of accelerated aging, underscore the concept that these two proteins are involved in β-cell replication during aging.
Two CDKIs, namely, p16 and p19, have been reported to be involved in β-cell regenerative failure in diabetes (49
). Our observations of enhanced expression of p16
genes confirm the premature aging phenotype of p44tg mice (Supplementary Fig. 6
). However, the regulatory link between p53 and the two aging markers in the maintenance or amplification of the phenotype requires further investigation.
Maier et al. (22
) previously described that young p44tg mice displayed signs of aging in bone. Further, the osteoblast-secreted molecule, osteocalcin, has recently been reported to modulate insulin secretion and insulin gene expression that correlated with expression of CREB
). In our study, we observed a mild but significant decrease in osteocalcin levels only in old p44tg mice; however, gene expression of insulin, CREB
, or NeuroD
was unaltered (Supplementary Fig. 7
Finally, the significance of the presence of p53 in non–β-cell fractions obtained from dispersed islet cells (29
) remains unclear. Although the relative increase in α-cells in p44tg islets was not associated with an increase in glucagon gene expression or circulating glucagon levels, we observed a significant increase in the number of somatostatin-secreting δ-cells and gene expression in the mutant islets. The presence of a p53 response element in the somatostatin promoter (Genomatix) warrants further studies to evaluate whether the increase in somatostatin actually decreases insulin secretion or limits β-cell growth in a paracrine manner in the p44tg mice.
In summary, increased dosage of Δ40p53 in mice promotes hypoinsulinemia and glucose intolerance, ultimately leading to overt diabetes with age and early death. The suppression of β-cell proliferation secondary to changes in the expression of p21, cyclin D2, and PDX-1 in this mouse model of accelerated aging implicates impaired p53 function in the development of type 2 diabetes in the elderly.