We have generated transgenic mice in which pancreatic β-cells express a fusion protein of three different imaging reporters. Expression of the trifusion reporter transgene had no apparent effect on mouse weight, islet morphology, fasting blood glucose, or glucose tolerance. We demonstrated that MIP-TF mouse β-cells were readily identified in pancreatic tissue sections using fluorescence microscopy, as in previous studies of MIP-EGFP mice (17
). Additionally, we showed that both BLI and microPET can noninvasively monitor MIP-TF mouse β-cells. Previous studies have generated transgenic mice expressing luciferase in their β-cells (3
). The β-cells of MIP-TF mice have especially high levels of luciferase activity, perhaps because the trifusion cDNA expression is enhanced by the long stretches of the insulin II gene genomic DNA that surround it and/or because its luciferase has been modified for greater enzymatic activity at 37°C.
Using microPET/CT, we could clearly delineate preferential accumulation of [18
F]FHBG in the pancreas of MIP-TF mice. Noninvasive microPET imaging of mouse pancreatic β-cells is technically challenging because of the small size of mice relative to small animal PET scanner spatial resolution, the small size of their pancreas, and its proximity to tracer excretion pathways (9
). Six hours postprobe injection, the vast majority of the probe had cleared through its excretion pathway, and there was sufficient probe retention in the pancreas such that the pancreas could be readily discerned from surrounding organs. After rendering mice diabetic by STZ administration, probe retention in the pancreas region was reduced by 91–95% in individual mice, confirming that the probe was originally retained by β-cells. The magnitude of pancreatic microPET signals showed a significant correlation with pancreatic bioluminescence from individual mice. Correlation between reporter proteins originating from the same fusion cDNA has been observed in previous in vitro and in vivo studies of tumors expressing linked reporter molecules (e.g., 15
) and allows flexibility in the modalities used to monitor biological processes.
After administering a high dose of STZ, bioluminescent signals from the MIP-TF mice dropped to essentially background levels, proving to be further evidence that the expression of luciferase was β-cell specific. Using the more slowly progressing multiple low-dose STZ model of type 1 diabetes, we observed a progressive loss of pancreatic bioluminescent signals, which preceded the appearance of hyperglycemia. By the time the mice developed severe hyperglycemia, only 8% of the bioluminescent signal remained. The timing and magnitude of the changes in pancreatic bioluminescence correspond well with the gradual loss of β-cells previously described in C57BL/6 mice after administration with low-dose STZ (27
). There was a linear correlation between β-cell area and bioluminescence for individual MIP-TF mice, suggesting that noninvasive monitoring of MIP-TF
gene expression by BLI can provide a biomarker of endogenous β-cell mass. In addition, we show that treatment with GCV induces hyperglycemia concomitant with a large decrease in bioluminescence from pancreas region. Because STZ administration is highly toxic to multiple organ systems, MIP-TF mice together with GCV treatment may provide a less toxic alternative to induce experimental type 1 diabetes. Potentially, this also provides a suicide gene system to specifically ablate β-cells arising from stem cell therapy using MIP-TF stem cells.
Hyperglycemia has been reported to induce proinsulin expression in the mouse liver and other organs (24
). Tg(RIP-luc) mice that were rendered diabetic by STZ administration robustly express luciferase in their liver, but the expression of proinsulin/insulin was not demonstrated at the protein level in their liver (23
). Although our MIP-TF mice express approximately 20-fold higher levels of pancreatic luciferase activity compared with Tg(RIP-luc) mice, we never detected bioluminescent signals from diabetic MIP-TF mouse livers or from other organs, indicating that if hyperglycemia activates transgene expression in liver cells or in other organs, it occurs in rare cells, or at extremely low levels, in these mice.
We generated the MIP-TF mice in C57BL/6 background because this mouse strain provides a very useful model of early type 2 diabetes when placed on a HFD (28
). MIP-TF mice displayed a rapid weight gain shortly after being placed on a HFD, as well as progressively impaired glucose tolerance and elevated fasting blood glucose levels, as expected. A prominent feature of this model is increased mesenteric adipose tissue (35
), and this overlaying tissue can attenuate pancreatic bioluminescence. Using a constant fluorescent standard to quantify this attenuation (as described in [5
]), we corrected the pancreatic bioluminescence based on the animal’s weight and found that the pancreatic signals in MIP-TF mice were about 3.6-fold greater than that from MIP-TF mice on a normal diet. The corrected bioluminescence showed a significant correlation with pancreatic β-cell area for individual MIP-TF mice, again suggesting that noninvasive monitoring of MIP-TF
gene expression by BLI can provide a biomarker of endogenous β-cell mass. The increased β-cell area in HFD-fed mice reflects, in large part, increased insulin resistance. Consistent with this, we observed that the corrected pancreatic bioluminescence signals from individual mice correlated with their AUC-IPGTT. Although CCD imaging in type 2 diabetes models may have to take into account changes in the animal’s body weight, microPET detection of reporter gene expression is unaffected by tissue depth since high-energy γ-photons are much more tissue penetrating (8
), and can be corrected for attenuation if needed, but microPET imaging is much more expensive.
In summary, we have generated transgenic MIP-TF C57BL/6 mice in which the β-cells express a fusion of three different imaging reporters. We have demonstrated imaging of MIP-TF pancreatic β-cells by fluorescent microscopy, BLI, and microPET. The MIP-TF mice enabled noninvasive monitoring of β-cells in models of type 1 diabetes and type 2 diabetes. We believe the MIP-TF mice can expedite studies in a broad range of diabetes research, including studies of β-cell development, diabetes pathogenesis, stem cell differentiation into β-cells, transdifferentiation, and islet survival after transplantation. We are currently breeding the TF gene into the NOD mouse background and hope to provide a new tool for studying β-cell loss and β-cell replication in the context of autoimmunity.