Non-Invasive Bioluminescence Imaging of β-Cell Function in Obese-Hyperglycemic [<i>ob/ob</i>] Mice

<div><p>Background</p><p>Type 2 diabetes results from failure of the β-cells to compensate for increased insulin demand due to abnormal levels of metabolic factors. The <i>ob/ob</i>(lep-/-) mouse has been extensively studied as an animal model of type 2 diabetes. Previous studies have shown a correlation between β-cell function and bioluminescent imaging in lean genetically engineered mice. The ability to noninvasively monitor β-cell function in <i>ob/ob</i> mice could provide new information on β-cell regulation in type 2 diabetes.</p><p>Methods</p><p>To create the B6 Albino <i>ob/ob</i> MIP-luc mice (ob/ob-luc), the <i>ob/ob</i> mouse was crossed with the CD1 MIP-luc mouse. All mice were backcrossed over multiple generations to ensure the genetic background of the transgenic mice was over 96% similar to the background of the original <i>ob/ob</i> mouse. Animal weight, blood glucose levels, insulin in plasma, and in vivo bioluminescence (BLI) were monitored weekly or biweekly for up to 70 weeks of age. BL imaging was performed using IVIS Spectrum (Perkin Elmer) and calculated by integrating the bioluminescence signal between 5 and 10 min after i.v. injection of D-luciferin. Insulin immunohistochemistry determined islet beta cell count and insulin secretion assay determined islet insulin function.</p><p>Results</p><p>There were significant increases in BLI and insulin levels as the <i>ob/ob</i>-luc mice aged while glucose levels gradually decreased. <i>Ob/ob</i>-luc were sacrificed at different time points to determine ex vivo BLI, islet function and total β-cell numbers using a cell counting training algorithm developed for the Vectra image analysis system (Perkin Elmer). The number of β-cells increased as the mice aged and all three ex vivo measurements correlated with BLI.</p><p>Conclusions</p><p>The <i>ob/ob</i>-luc mice can serve as a model of metabolic stress, similar to human type 2 diabetes using BLI as a surrogate marker for β-cell function.</p></div

Characterization of <i>ob/ob</i>-luc mice.

<p>A) Weight increases significantly with age in <i>ob/ob</i>-luc mice consistent with the original <i>ob/ob</i> mouse model on the B6 black background. As early as week 8 the <i>ob/ob</i>-luc mice are 2.5 times the weight of their lean control counterparts. Blood was taken from fasting mice following imaging to measure B) glucose and C) insulin. Glucose levels in <i>ob/ob</i>-luc mice (closed symbols) are significantly higher than lean controls (open symbols) at week 8 (p<0.001, t-test). As the <i>ob/ob</i>-luc mice age the glucose levels decrease and reach similar levels as lean controls by week 45. Insulin levels in <i>ob/ob</i>-luc mice increase with age while insulin levels in lean mice remain stable with age. Both glucose and insulin levels follow similar trends as normal <i>ob/ob</i> mice, indicating that the additional genetic manipulation (albino and MIP-luc) did not affect the physical characteristics of the <i>ob/ob</i> model. D) There is a good correlation (R² = 0.79) between plasma insulin levels and <i>in vivo</i> BLI measurements.</p

Correlation of <i>ex vivo</i> bioluminescence with quantitative histology.

<p>A) Total number of β-cells was counted from sections of pancreas from <i>ob/ob</i>-luc mice (N = 3–8/group). Mice were grouped to minimize variation since individual mice reach disease stages at different time points. B) Total bioluminescence was measured from <i>ex vivo</i> images of pancreata from <i>ob/ob</i>-luc mice. When the data are grouped similar to β-cell number there is an upwards trend in β-cell number as the mice age. (The horizontal lines in figures A and B indicate a statistically significant difference between the two groups, *p<0.001, One way ANOVA) C) There is a strong correlation between the number of β-cells counted by histology and <i>ex vivo</i> BLI measurements.</p

Luciferase expression is restricted to the pancreas.

<p>A) An example of BLI on <i>ob/ob</i>-luc mice. The animals of the same age were injected i.p. with D-luciferin and imaged 5 min later. Signal is present in the midsection of the animals only. B) Biodistribution of luciferase in the organs of <i>ob/ob</i>-luc mice. Three animals with similar intensities in the midsection were dissected and the organs were imaged with an IVIS Spectrum. ROI analysis was performed on the organs. The background bar is highlighted to show all organs except pancreas are at background levels while pancreas is significantly higher than other organs (*p<0.001, One way ANOVA). C) Zoomed in BLI of a pancreas. D) The pancreas was sectioned and stained with anti-insulin antibody. Both BLI and insulin staining have similar punctate patterns.</p

Insulin measurements on individual islets confirms <i>in vivo</i> BLI and insulin measurements.

<p>A) Bioluminescence imaging of islets isolated from <i>ob/ob</i> luciferase mice at 10, 22, 40, and 64 weeks of age displayed increased bioluminescence with increasing age up to 40 weeks of age, followed by decreased BLI at 64 weeks of age. B) Isolated islets displayed increased total insulin content with increasing age up to 40 weeks of age. Insulin content of islets from 64 week old mice was not significantly different than at 40 weeks. C) Isolated <i>ob/ob</i>-luc islets displayed insulin release in response to insulin secretagogues. The response from 64 week old islets is represented by open symbols to distinguish it from the other groups. High glucose (16.7 mM) and IBMX induced higher insulin release in islets from 40 week old mice and lower insulin release in islets from 64 week old mice (*p = 0.015, One way ANOVA).</p

<b>Coefficient of Variation for Area Under the Curve measurements of i.p. vs. i.v. administration of D-luciferin in </b><b><i>ob/ob</i></b><b>-luc mice.</b>

<p>Test-retest scans were performed every other day for three days on a total of 12 animals. I.P. administration was tested the first week and i.v. administration was tested the following week and dynamic profiles were generated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106693#pone.0106693.s002" target="_blank">Figure S2</a>. AUC for the entire time course and for just 5–10 min interval was measured for each day and the Coefficient of Variation (CV)  =  ((STD/AUC)*100) was calculated for all three days for each mouse. The CV was then averaged for all 12 mice. I.V. is more consistent than i.p. and there is no statistical significant difference in performing a 5 min acquisition 5 min after D-luciferin administration vs. measuring for the entire time course.</p><p><b>Coefficient of Variation for Area Under the Curve measurements of i.p. vs. i.v. administration of D-luciferin in </b><b><i>ob/ob</i></b><b>-luc mice.</b></p

Discovery of MK-3168: A PET Tracer for Imaging Brain Fatty Acid Amide Hydrolase

We report herein the discovery of a fatty acid amide hydrolase (FAAH) positron emission tomography (PET) tracer. Starting from a pyrazole lead, medicinal chemistry efforts directed toward reducing lipophilicity led to the synthesis of a series of imidazole analogues. Compound <b>6</b> was chosen for further profiling due to its appropriate physical chemical properties and excellent FAAH inhibition potency across species. [¹¹C]-<b>6</b> (MK-3168) exhibited good brain uptake and FAAH-specific signal in rhesus monkeys and is a suitable PET tracer for imaging FAAH in the brain