Bioluminescence imaging in mouse models quantifies beta cell mass in the pancreas and after islet transplantation

Abstract

Purpose: We developed a mouse model that enables non-invasive assessment of changes in beta cell mass.

Procedures: We generated a transgenic mouse expressing luciferase under control of the mouse insulin I promoter [mouse insulin promoter-luciferase-Vanderbilt University (MIP-Luc-VU)] and characterized this model in mice with increased or decreased beta cell mass and after islet transplantation.

Results: Streptozotocin-induced, diabetic MIP-Luc-VU mice had a progressive decline in bioluminescence that correlated with a decrease in beta cell mass. MIP-Luc-VU animals fed a high-fat diet displayed a progressive increase in bioluminescence that reflected an increase in beta cell mass. MIP-Luc-VU islets transplanted beneath the renal capsule or into the liver emitted bioluminescence proportional to the number of islets transplanted and could be imaged for more than a year.

Conclusions: Bioluminescence in the MIP-Luc-VU mouse model is proportional to beta cell mass in the setting of increased and decreased beta cell mass and after transplantation.

Fig. 1

In vivo bioluminescence imaging of mice expressing luciferase under control of the mouse insulin I promoter detects light emission from the pancreas. a A MIP-Luc-VU transgenic mouse, but not control mouse, emitted light after administration of the luciferin substrate in an anatomical region consistent with the pancreas. b After injection of luciferin, the abdominal cavity was immediately opened to expose the pancreas and verify that all light emission was emanating from the pancreas; no other organs emitted light. c A MIP-Luc-VU mouse placed in a right lateral decubitis position displayed light emission from the pancreatic region. Regions of interest drawn over the bioluminescent region demonstrate the manner in which bioluminescence emission was quantified. d Three-dimensional reconstruction of bioluminescence from the pancreas of a MIP-Luc-VU mouse. Clusters of bioluminescent islets are reconstructed as red voxels within the volume of the mouse. The surface projection from the bioluminescent islets is displayed on the rainbow color scale. BLI in MIP-Luc-VU mice was similar in males and females and between mice of different generations and ages. There was no statistically significant difference between age-matched male and female control mice used in these studies (P=0.199, unpaired t test) used for subsequent STZ and high-fat diet experiments. The mice used in the STZ experiment and high-fat diet came from successive generations of MIP-Luc-VU mice. There was no significant difference between the STZ control cohort (four mice, approximately 60 islets/mouse) and high-fat control cohort (three mice) as determined by unpaired t test (P=0.5654). The control mice used in the high-fat diet were imaged at 1, 3, and 6 months of age and there was no significant difference in bioluminescence intensity as determined by ANOVA.

Fig. 2

MIP-Luc-VU mice have normal glucose tolerance, insulin secretion, and islet morphology. a Intraperitoneal glucose tolerance testing of MIP-Luc-VU animals (black squares) revealed similar response to a glucose bolus (1.5 g/kg body weight) as wild-type control animals (open circles; n=5 each group). b Isolated MIP-Luc-VU islets (black squares) displayed similar insulin release in response to islet secretagogues as islets isolated from wild-type animals (open circles) when tested in a cell perifusion apparatus (n=4 each group). The time of exposure to each islet secretagogue (16.8 mM glucose, 16.8 mM glucose+ 100 μM isobutyl methyl xanthine, and 2.8 mM glucose+300 μM tolbutamide) is indicated by the bar above the chart. Immunocytochemistry of MIP-Luc-VU islets stained for insulin (green), glucagon (red), and somatostatin (blue) (c); insulin (d); luciferase (e); and insulin (green) and luciferase (red) (f). This pattern of expression of luciferase was similar in different mice. There was not a significant difference between the number of luciferase-positive beta cells in the STZ control cohort (four mice, approximately 60 islets/mouse) and high-fat control cohort (three mice) as determined by unpaired t test (P=0.5654)—see Figures 4 and 5.

Fig. 3

Bioluminescence of MIP-Luc-VU islets correlates with the number of islets in vitro. Islets in quantities of 10, 25, 50, and 100 islets (left to right) were placed in a 24-well plate and in vitro bioluminescence imaging was performed after addition of luciferin (lower panel). Quantification of in vitro bioluminescence (left y-axis) reveals linear correlation between bioluminescence intensity and the number of islets (black squares; upper panel). Luciferase activity in islet lysates (right y-axis), as measured by luminometer, also correlates with the number of islets (open circles; upper panel).

Fig. 4

BLI declines after β cell destruction and correlates with β cell mass. a Treatment with streptozotocin (STZ, 175 mg/kg) leads to a progressive decline in bioluminescence (black squares) from MIP-Luc-VU mice (n=3) and is accompanied by an increase in blood glucose level (open circles; n=3). b The insulin content (white bar) and luciferase activity (black bar) of pancreata excised from untreated control MIP-Luc-VU mice is higher than in mice 8 days after STZ injection (n=4). Luciferase activity and insulin content measurements were performed on separate pancreatic extracts. c Immunocytochemistry of an islet from a MIP-Luc-VU mouse 8 days after STZ treatment was stained for insulin (green) and luciferase (red). The larger panel shows co-labeling of cells for insulin and luciferase. d The mass of β cells (white bar) and luciferase-expressing cells (black bar), as determined by morphometric analysis, of MIP-Luc-VU mice 8 days after STZ administration was significantly reduced compared with untreated control pancreata (n=4 mice, 30–40 islets per mouse; unpaired t test; *p<0.05; **p<0.01; ***p<0.005).

Fig. 5

BLI correlates with increased β cell mass induced by high-fat feeding. a MIP-Luc-VU mice fed a high-fat diet (black squares) displayed a progressive increase in body weight as compared with MIP-Luc-VU mice fed a regular diet (open circles). b Over the same time period MIP-Luc-VU mice fed the high-fat diet (black squares) had higher bioluminescence emission than MIP-Luc-VU mice fed a regular diet (open circles). c Light emission from a luminescent bead implanted at the pancreas was higher for mice fed a regular diet (left) than for a mouse fed a high-fat diet (right). d Correlation between the animal weight and the amount of light detected from a luminescent bead placed at the site of the pancreas. e Immunocytochemistry of an islet stained for insulin from mice fed a regular diet (left) or high-fat diet (right). f Morphometric analysis of insulin labeling of islets revealed greater β cell mass in mice fed the high-fat diet (white bars), reflected by increased BLI measurements (black bars), but more closely correlating with BLI measurements normalized for animal body habitus (hatched bars; unpaired t test; *p<0.05; **p<0.01; ***p<0.005).

Fig. 6

MIP-Luc-VU islets can be imaged and their mass assessed in vivo after transplantation. MIP-Luc-VU islets were transplanted beneath the kidney capsule (a) or into the liver via portal vein infusion (b) of NOD-scid mice. c Three-dimensional reconstruction of islets beneath the kidney capsule reveals a small, contiguous islet graft within the kidney. Islets are shown as red voxels within the volume of the mouse. d Three-dimensional reconstruction of islets infused via the portal vein reveals islets scattered throughout the liver. e The number of islets transplanted to the renal capsule correlates linearly with bioluminescence and the insulin content of the graft (n=4). f Islets transplanted to the liver display a similar correlation between the number of islets transplanted, bioluminescence, and hepatic insulin content (n=4). The relatively low R2 values in the liver reflect the variability in islet engraftment after portal vein infusion, as indicated by the variability in post-mortem insulin content measurements. The variability in BLI measurements mirrors this inconsistent engraftment of islets in the liver.