Date of Award
Doctor of Philosophy (PhD)
Klaus H. Kaestner
Diabetes mellitus is an increasingly prevalent metabolic disorder that is estimated to affect over 300 million people by 2025. Common to either type 1 or type 2 diabetes is a progressive inadequacy of functional β-cell mass. Recent studies have shown that during times of prolonged metabolic demand for insulin, the endocrine pancreas can respond by increasing β-cell mass, both by an increase in cell size and by changes in the balance of β-cell proliferation and apoptosis. Advances that further our knowledge of the molecular factors that control both β-cell proliferation and survival will be crucial for understanding the homeostasis of β-cell mass during adulthood, and are pivotal for any attempt to use instructive cues to induce the proliferation of terminally differentiated fully functional insulin-producing β-cells that are suitable for transplantation. However, no systematic study that investigates the expression profile of the islet’s response to pregnancy in vivo, a physiological state of insulin resistance, has been reported thus far.
In the first part of my thesis, I characterized the gene expression signature of pancreatic islets during pregnancy by performing large-scale expression profiling of islets isolated from 4- to 5-month-old non-pregnant and pregnant female mice at day 14.5 of gestation, the peak of β-cell proliferation. I identified a total of 1,907 genes as differentially expressed, and demonstrated the induction of both proliferative and survival pathways in the islet during pregnancy. A comparison of our pregnancy gene set with two additional models of islet expansion suggests that diverse mechanisms can be recruited to expand islet mass. One of the genes that is required for β-cell proliferation during pregnancy in mice is the transcription factor HNF4α.
In an attempt to translate knowledge gained using the pregnancy paradigm, I hypothesized that HNF4α is a human β-cell mitogen. To address this question, in the second part of my thesis, I employed adenoviral-mediated overexpression of a pancreas-specific isoform of HNF4α (HNF4α8) in primary human islets. HNF4α8 stimulated β-cells to enter the cell cycle, and led to a greater than 300-fold increase in the number of β-cells that entered S-phase, without detectable change in glucose stimulated insulin secretion. However, HNF4α8 overexpressing β-cells showed signs of cell cycle arrest, caused by activation of the DNA damage response associated with replication stress, ultimately resulting in a senescence-like phenotype independent of caspase-dependent apoptosis. Overexpression of HNF4α8 together with known β-cell mitogens, also further increased cell cycle entry of β-cells, strengthening the argument that HNF4α8 is a mitogenic signal in the human β-cell. Additionally, I observed a substantial proportion of β-cells stimulated to enter the cell cycle by CDK6 and CYCLIN D3 to also exhibit both markers of cell cycle arrest and double stranded DNA damage. In summary, the DNA damage response is a barrier to efficient human β-cell proliferation in vitro, and as such I suggest its evaluation in future attempts to stimulate β-cell replication.
Rieck, Sebastian, "The Generation of Fully Functional β-Cells by Proliferation: Lessons From Pregnancy and HNF4α" (2011). Publicly Accessible Penn Dissertations. 984.