Novel techniques for assessing beta cell turnover

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Background: Considerable data from rodent models has shown that, given time, pancreatic beta cell numbers can return to near normal following an insult that has destroyed most of animals insulin-producing beta cells. Even so, considerable controversy exists as to whether that beta cell recovery results exclusively from the proliferation of surviving beta cells, or whether other pancreatic stem cells can also differentiate into mature insulin producing islet beta cells. The question is even murkier in man since we cannot employ techniques used to study the question in rodents (e.g. transgenic mice with labeled beta cells, bromodeoxyuridine incorporation into DNA, etc). Even so, evidence strongly suggests that pancreatic beta cells are being replenished throughout life. For instance, despite the fact that individuals with even long standing type 1 diabetes mellitus (T1DM) appear to harbor anti-beta cell specific T cells capable of aggressively killing beta cell targets; autopsy based studies find pancreatic beta cells in most subjects dying with T1DM. Further, large case series that have evaluated patients with long-standing T1DM find that many if not most continue to make small amounts of C-peptide. For instance, as cited in our annual report for work unit number DK055111-01, we can demonstrate that C-peptide is produced by the pancreas in subjects with long-standing T1DM. These observations, and others, have led several investigators to ask whether pancreatic beta cell regeneration might prove a useful therapeutic approach to treat diabetes. As critical scientists, however, we must consider other data suggesting that beta cell mass does not significantly change in adult life. For instance, classic studies by Ogilvie from 1937 (Quarterly Journal of Medicine. 23: p287-300) suggest that beta cell mass is stable beyond age 25, though it is possible that observation is explained by beta cell loss rates being exactly equaled by regeneration rates in adults. As a consequence of this ongoing discussion, much interest has been focused on measuring a pancreatic beta cells regenerative capacity. Some scientists have approached the question by analyzing pancreatic biopsy specimens using immunohistochemical techniques, while we (and others) have attempted to promote beta cell functional recovery in clinical trials (see annual reports for work units numbered DK055104-03 and DK055111-01). A manuscript published by Spalding et al in 2005 (Cell. 122: p133-143) suggested to one of us (SP) a novel approach to assess pancreatic beta cell turnover rates by measuring the cells DNA 14C content. The principle underlying these studies is that earth's atmospheric 14C content has been tracked and is precisely known since the early 1900s. Further, we know that atmospheric 14C content sharply peaked in the early 1960s due to atmospheric nuclear tests then being performed. Since 14C decays with a half-life of nearly 6000 years, by measuring a cell's DNA 14C content, one can assess when that cells DNA was synthesized. Spalding et al validated the technique using DNA isolated from tree rings, and further used the technique to measure neuronal cell turnover rates in man. During the past 2 years, we have attempted to adapt the 14C technique to study pancreatic beta cell turnover rates. Such studies are difficult however, and for several reasons. One, the technique requires quite pure beta cells and large numbers of them; this can be difficult since the cells must be derived from a single donor. Two, one must scrupulously avoid contamination with carbon from any source (proteins, reagents used in the beta cell isolation process, etc) since that carbons 14C content could confound the analysis. Many techniques for isolating pure beta cells were attempted before we settled on immunostaining the cells for proinsulin and gaiting on the positive cells using flourscence activated cell sorting. With that technique, weve prepared pure beta cell DNA from two donors (born in 1926 and 1958), and in collaboration with Dr. Bruce Buchholz at the Lawrence Livermore Laboratories, have estimated that their beta cell DNA dated approximately 25 years younger than each donor. Our preliminary data suggests that the pancreatic islet beta cells from non-diabetic individuals do not turnover at a very rapid rate once individuals reach adulthood, and the work was recently cited in Science (Sept 12, 2008, page 1437). We have also obtained data employing pancreatic sections from subject autopsied days to months after receiving thymidine analogs for other clinical trials. Those sections were stained to look for insulin positive cells that had incorporated the analog, suggesting new DNA synthesis. These studies too suggest very limited beta cell turnover occurs during adult life. A manuscript reporting these results has been submitted but remains in the review process