Definition of a Skp2-c-Myc Pathway to Expand Human Beta-cells.

T2D (T2D) has traditionally been regarded to be the result of insulin resistance in liver, skeletal muscle and adipose tissue1,2,3. Recently, autopsy and genome-wide association studies (GWAS) suggest that it is also associated with β-cell deficiency and dysfunction4,5,6,7,8,9,10. The major factor associated with T2D is obesity, although not all obese subjects become diabetic1,3,10. In autopsy studies, patients with T2D display a reduced β-cell mass as compared to non-diabetic patients with comparable BMI. In contrast, β-cell mass is increased in non-diabetic obese subjects as compared to lean subjects8,11,12. In rodents, β-cell expansion in obesity models is associated with replication of endogenous β-cells1,3. However, there is little evidence for β-cell replication in human obesity or T2D. In humans, understanding how the β-cell mass evolves during insulin resistance and the development of T2D is challenging due to the limitations of autopsy studies. Studies in children and young adults suggest that it is possible that some people accrue lower than average β-cell mass during their first years of development13,14. These individuals would thus require greater expansion of β-cell mass in response to insulin resistance. Indeed, β-cell mass is primarily established during the first years after birth and is highly variable among children and young adults13,14. A second possibility is that if β-cell expansion can occur in adults, some individuals may not expand their β-cell mass as effectively as others in response to obesity and insulin resistance. A third possibility is that β-cell death and/or dedifferentiation may be more prevalent in some individuals, leading to the emergence of T2D. Finally, it is likely that combinations of the above occur. In any case, the failure of β-cells to adapt to insulin resistance seems to be central to the development of T2D, whether due to reduced β-cell proliferative response, and/or increased β-cell death, and/or loss of β-cell function and de-differentiation. A number of studies have linked the deregulation of cell cycle genes in β-cells with the development of T2D. In GWAS studies, T2D susceptibility loci have been identified in or near cell cycle genes6,7. In mouse genetic studies, the cell cycle inhibitor, p27kip1, has been linked to the development of T2D. For example, p27KIP1 progressively accumulates in the nuclei of pancreatic β-cells in T2D mouse models which lack either the insulin receptor substrate 2 (IRS2), or the leptin receptor15. In these two models of T2D, the genetic knockout of p27kip1 reduces the hyperglycemia, increases β-cell mass and maintains hyperinsulinemia, predominantly via β-cell proliferation. In addition, p27kip1 mRNA is increased in islets from human T2D donors as compared to non-diabetic donors16. p27kip1 may be either an activator or inhibitor of cell cycle progression. In rodent β-cells, p27kip1 has been shown to be a cell cycle inhibitor17,18,19. However, in other cell types, p27kip1 has also been shown to act as an activator of cell cycle. By facilitating the formation and stabilizing the complex formed between D-cyclins and cdk4 or cdk6, p27kip1 acts as a chaperone for the assembly and nuclear translocation of the complex20. This leads to an activation of cell cycle entry. With regards to human β-cells, p27kip1 is known to be expressed in whole human islets21 and in human β-cells, mostly in their cytoplasm22,23. […]. The precise role of p27kip1 in regulating β-cell mass and proliferation is not known in humans. p27kip1 expression is mostly regulated post-transcriptionaly by poly-ubiquitinylation and proteosomal degradation. The S-phase kinase-associated protein 2 (Skp2), a component of the SCF (Skp1-Cullin 1-F-box) E3 ubiquitin-ligase complex, has been shown to be the major p27kip1 -ubiquitin ligase. Although p27kip1 is a critical target of Skp2, many additional substrates of Skp2 has been identified. Many of these proteins, such as p21cip, p57kip2, E2F1, MEF, p130 Tob1, cyclin D1, cyclin E, Smad4 and c-myc are cell cycle regulators24. c-myc is unique among Skp2 targets, since Skp2 induces not only its ubiquitinylation, but also increases its transcriptional activity25,26,27. While several studies have focused on β-cell loss of function and/or de-differentiation and/or susceptibility to cell death in T2D, no prior study has examined the possibility that the proliferative response of human β-cell from T2D donors is impaired. We hypothesized that this is the case. We further hypothesized that abundance of p27kip1 may be elevated in the nuclei of human T2D β-cells, limiting their response to mitogens. Finally, we hypothesized that the downregulation of p27kip1 might lead to increased proliferative response of human β-cells.