The presence of an adequate functional β-cell mass is critical for maintaining euglycemia in mammals. In states of altered metabolic demand, e.g., pregnancy or high-fat feeding, healthy β-cells maintain euglycemia, by increasing insulin secretion, through an elevated β-cell mass, or both (39
). Indeed, <20% of obese insulin-resistant individuals develop type 2 diabetes, and a majority of insulin-resistant humans are capable of maintaining euglycemia by β-cell compensation (9
). Recent studies focused on genomic analyses of type 2 diabetic patients have reported polymorphisms in genes that are close to those coding for key cell-cycle regulators (41
); these experiments suggest that expansion of β-cell mass are linked to proteins in cell-cycle progression. In this study, we provide direct genetic evidence that the cell cycle protein cyclin D2 is essential for compensatory β-cell expansion in response to insulin resistance.
We have reported earlier that cyclin D2 is essential for the physiological remodeling of β-cell mass in the postnatal period (13
). To address whether cyclin D2 is also required for β-cell expansion in states of insulin resistance, we crossed the cyclin D2 knockouts with two mouse models that exhibited varying degrees of insulin resistance. We chose the LIRKO model because it is tissue specific and is characterized by severe resistance and a dramatic β-cell hyperplastic response (22
). For the second model, we used mice lacking IRS1, which exhibited mild insulin resistance but still manifested islet hyperplasia (20
). The presence of a significantly increased number of proliferating β-cells in the islets from both models indicates the presence of hyperplasia—in part due to replication. Whereas imposition of cyclin D2 insufficiency led to diabetes in both models, severe hyperglycemia was evident in the D2KO/LIRKO compound knockouts at a much earlier age, suggesting that the severity of insulin resistance and the availability of cyclin D2 are important determinants of an appropriate islet hyperplastic response. On the other hand, in the IRS1KO mice, which develop mild-to-moderate postreceptor insulin resistance (20
), the superimposition of cyclin D2 insufficiency also led to the development of diabetes. However, the compound D2KO/IRS1KO mice continued to live until age 25 weeks and more closely mimicked the human progression of insulin resistance–mediated type 2 diabetes compared with the D2KO/LIRKO model. Further, the reduced expression of two key β-cell–specific markers, PDX1 and GLUT2, suggests that absence of cyclin D2, in the context of insulin resistance, can directly influence β-cell function and proliferation.
Upstream signaling pathways (e.g., insulin/IGF-I) are crucial in the regulation of β-cell replication (rev. in 9
) and may be linked with cyclin D2 in modulating β-cell mass. Indeed, we previously reported that increased β-cell replication in LIRKO mice correlates with increased insulin levels but not with glucose levels (23
). Furthermore, in this same study, we crossed LIRKO mice with β-cell–specific insulin receptor knockout (βIRKO) mice, a model that exhibits β-cell hypoplasia and manifests a phenotype resembling human type 2 diabetes (25
). Consistent with a role for the insulin receptor in modulating β-cell proliferation, βIRKO/LIRKO mice failed to develop islet hyperplasia and died as early as 8 weeks of age (23
). Interestingly, islets and β-cells derived from βIRKO mice show reduced cyclin D2 protein expression (C.H. and R.N.K., unpublished data), and it is possible that this absence of the cell-cycle protein contributes to poor islet growth and development of age-dependent diabetes in these mutants (43
). Recent experiments have also linked the cyclin/CDK4 complex with Akt in β-cell proliferation, indicating that Akt1 upregulates cyclin D1 and cyclin D2 levels and CDK4 activity (45
Cyclin/cyclin-dependent kinase complexes are key nodes in the regulation of the G1-to-S transition during cell-cycle progression. For example, global knockouts of CDK4 in mice develop β-cell hypoplasia and diabetes (16
). Previous work by our own group has shown that mice with a global knockout of p27, a cell-cycle inhibitor, regenerate β-cells more efficiently following streptozocin-induced diabetes (13
), while other investigators have reported that β-cell–specific overexpression of p27 leads to islet hypoplasia and diabetes (46
). Conversely, the cell-cycle inhibitor p21, which is the main target of the tumor suppressor p53, has recently been reported to be unessential for maintaining β-cell mass or function in vivo (47
). In a similar manner, β-cell–specific deletion of pRb, a protein reported to be a central regulator of the cell cycle, leads to minimal defects in β-cell replication, mass, and function (48
). Therefore, specific cell-cycle regulators play a critical role in β-cell proliferation regardless of the redundant expression of complementary family members. Intriguingly, Lavine et al. (49
) have reported an inability to detect cyclin D2 protein in proliferating human islets that overexpressed prepro-cholecystokinin. Whether human β-cells utilize cyclin D2 for proliferation in a context-dependent manner or are dependent on another key cyclin protein requires further investigation.
While prior studies support a role for PDX-1 in β-cell proliferation, it is unclear whether proteins in the cyclin/CDK pathway are direct targets of this transcription factor. On the other hand, in our study the decreased β-cell mass in the compound knockouts and reduced expression of PDX-1 suggest that cyclin D2 is upstream of the pancreas-duodenum homeodomain transcription factor. It is likely there are intermediates that link these two important proteins, and additional studies are warranted to define their roles in the regulation of β-cell proliferation.
It is worth noting that the cyclin D2 mutants were global knockouts; therefore, it is possible that lack of cyclin D2 in putative precursors contributing to neogenesis also depends on cyclin D2–mediated cell-cycle reentry to facilitate enhanced β-cell mass. Nevertheless, our conclusion that cyclin D2 is necessary for replication-based β-cell expansion in insulin-resistant states is supported by lineage trace studies, which provide firm evidence that self-duplication is the primary source of new β-cells in adult rodents (12
). Taken together, our studies support the hypothesis that cyclin D2, or its analog in humans, is a potential therapeutic target that can be harnessed to promote β-cell expansion in the treatment of type 1 diabetes and to delay the progression or prevent the development of type 2 diabetes.