Preclinical studies for measuring beta cell mass in vivo

本报告中描述的大部分工作来自哥伦比亚大学Paul Harris博士的观察，他使用差异基因表达研究，注意到β细胞（比其他胰腺细胞更大程度）表达水疱单胺转运蛋白-2 （VMAT-2）。Harris博士及其同事继续使用VMAT-2放射性标记配体11c -二氢四苯那嗪（11C-DTBZ）作为T1DM大鼠模型正电子发射断层扫描（PET）研究的显像剂进行研究，结果表明，在自身免疫过程中，随着β细胞质量下降，11C-DTBZ-PET胰腺信号也随之下降。的确，在那个模型中，PET信号似乎能够预测糖尿病的发病。为了将这项技术应用于T1DM风险人群的临床介入试验，我们启动了几项研究，利用来自人类尸体供体和各种小鼠和非人灵长类动物模型的胰岛来评估11C-DTBZ-PET。

Much of the work described in this report emanated from observations made by Dr. Paul Harris at Columbia University who, using differential gene expression studies, noted that beta cells (to a much greater degree than other pancreatic cells) express the vesicular monoamine transporter-2 (VMAT-2). Dr. Harris and colleagues went on to perform studies using a VMAT-2 radiolabeled ligand named 11C-dihydrotetrabenazine (11C-DTBZ) as an imaging agent for positron-emission tomography (PET) studies in a rat model for T1DM with results suggesting that as beta cell mass fell during the autoimmune process, so did the 11C-DTBZ-PET pancreatic signal. Indeed, in that model the PET signal seemed able to predict diabetes onset. With an eye toward developing the technique for application in clinical interventional trials enrolling humans at risk for T1DM, we initiated several studies to assess 11C-DTBZ-PET using islets from human cadaveric donors and various mouse and non-human primate models at our disposal. Consistent with the techniques promise, we found that: 1. Human islets immunostained with anti-VMAT-2 and with anti-insulin revealed nearly coincident staining, while other islet endocrine cells did not stain for VMAT-2. 2. Mixing variable human islet numbers with 3H-DTBZ generated radioactive counts directly correlated with the islet number. 3. Cold DTBZ could complete away 3H-DTBZ bound to human islets suggesting binding to specific receptors. 4. Labeling human islets with 3H-DTBZ, then injecting the labeled islets intravenously into mice promptly euthanized after that injection, generated radioactivity counts in the animals lungs consistent with the islet number injected. 5. Non-human primates injected with 11C-DTBZ then studied using PET revealed bright signals that co-registered with magnetic-resonance imaging (MRI) of the animals pancreas. Unfortunately, with further testing we found: 1. In our mouse models, native pancreatic DTBZ-PET signals did not reliably correlate with predicted beta cell mass. 2. Immunostaining of both mouse and non-human primate pancreas with anti-VMAT2 and anti-insulin (or other endocrine hormone-specific antibodies) was not consistent with beta cell specific VMAT2 expression. To test whether the apparent absence of beta cell VMAT2 expression might be secondary to species differences (i.e. the staining antibody was raised against human VMAT2), we immunostained other primate tissue (brain basal ganglia and adrenal medulla) known to express VMAT2 and found clear expression in those positive control tissues, but absent staining in appropriate negative control tissues. 3. Individual cells from isolated non-human primate islets studied using both flow cytometry and reverse-transcriptase-polymerase chain reaction (RT-PCR) revealed no evidence for beta cell specific VMAT2 expression. 4. 11C-DTBZ exists as both a positive enantiomer, and a mirror image negative enantiomer. Only the positive 11C-DTBZ enantiomer binds to VMAT2. We performed PET scans using both forms in the same primate and found that both generated similar PET signals. Taken together (in conjunction with data reported in work unit number DK055110-01), we concluded that while it showed considerable promise, 11C-DTBZ PET pancreatic signals are not sufficiently specific for pancreatic beta cells to serve as a useful technique for measuring beta cell mass in vivo and we have turned our attention to other techniques. A manuscript describing these results has been prepared for publication. We have moved on to assess, using the non-human primate model, whether pregnancy promotes beta cell proliferation as is reported in rodents. Under an approved animal use protocol, we have administered thymidine analogs to a pregnant non-human primate, then examined the animal's pancreas to assess rates of beta cell turnover. Another such study is planned in the next few weeks. Preliminary data suggests that as opposed to responses observed in rodents, adult primate beta cells display little if any capacity for proliferation, even during pregancy.