Preclinical studies for measuring beta cell mass in vivo

本报告中描述的大部分工作源自哥伦比亚大学 Paul Harris 博士的观察，他利用差异基因表达研究指出，β 细胞（比其他胰腺细胞程度高得多）表达囊泡单胺转运蛋白 2 (VMAT-2)。 Harris 博士及其同事继续使用名为 11C-二氢丁苯那嗪 (11C-DTBZ) 的 VMAT-2 放射性标记配体作为显像剂，在 T1DM 大鼠模型中进行正电子发射断层扫描 (PET) 研究，结果表明，随着自身免疫过程中 β 细胞质量下降，11C-DTBZ-PET 胰腺信号也下降。 事实上，在该模型中，PET 信号似乎能够预测糖尿病的发病。 为了开发适用于招募有 T1DM 风险的人类的临床介入试验的技术，我们启动了几项研究，使用人类尸体捐献者的胰岛以及我们掌握的各种小鼠和非人类灵长类动物模型来评估 11C-DTBZ-PET。 与技术承诺一致，我们发现： 1.用抗VMAT-2和抗胰岛素免疫染色的人胰岛显示出几乎一致的染色，而其他胰岛内分泌细胞没有对VMAT-2进行染色。 2.将可变的人胰岛数量与 3H-DTBZ 混合产生与胰岛数量直接相关的放射性计数。 3.冷DTBZ可以消除与人胰岛结合的3H-DTBZ，表明与特定受体结合。 4.用3H-DTBZ标记人胰岛，然后将标记的胰岛静脉注射到注射后立即安乐死的小鼠中，在动物肺部产生与注射的胰岛数量一致的放射性计数。 5. 注射 11C-DTBZ 的非人类灵长类动物随后使用 PET 进行研究，发现与动物胰腺的磁共振成像 (MRI) 共同记录的明亮信号。 不幸的是，通过进一步测试我们发现： 1. 在我们的小鼠模型中，天然胰腺 DTBZ-PET 信号与预测的 β 细胞质量没有可靠的相关性。 2.用抗VMAT2和抗胰岛素（或其他内分泌激素特异性抗体）对小鼠和非人灵长类动物胰腺进行的免疫染色与β细胞特异性VMAT2表达不一致。 为了测试β细胞VMAT2表达的明显缺失是否可能继发于物种差异（即，针对人VMAT2产生染色抗体），我们对已知表达VMAT2的其他灵长类组织（脑基底神经节和肾上腺髓质）进行免疫染色，并发现在这些阳性对照组织中存在清晰的表达，但在适当的阴性对照组织中没有染色。 3. 使用流式细胞术和逆转录聚合酶链式反应 (RT-PCR) 对来自分离的非人灵长类胰岛的单个细胞进行研究，未发现 β 细胞特异性 VMAT2 表达的证据。 4. 11C-DTBZ 既有正对映体又有镜像负对映体。 仅阳性 11C-DTBZ 对映体与 VMAT2 结合。 我们对同一灵长类动物使用这两种形式进行 PET 扫描，发现两者都产生相似的 PET 信号。 综合起来（结合工作单位编号 DK055110-01 中报告的数据），我们得出的结论是，虽然 11C-DTBZ PET 胰腺信号显示出相当大的前景，但它对于胰腺 β 细胞的特异性不够，无法作为测量体内 β 细胞质量的有用技术，因此我们已将注意力转向其他技术。 描述这些结果的手稿已准备好出版。 我们继续使用非人类灵长类动物模型来评估怀孕是否会像啮齿类动物中报道的那样促进β细胞增殖。 根据批准的动物使用方案，我们对怀孕的非人类灵长类动物施用胸苷类似物，然后检查动物的胰腺以评估β细胞周转率。 计划在未来几周内进行另一项此类研究。 初步数据表明，与在啮齿类动物中观察到的反应相反，成年灵长类动物β细胞几乎没有表现出增殖能力，即使在怀孕期间也是如此。

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.