Radiomanganese PET Detects Changes in Functional β-Cell Mass in Mouse Models of Diabetes

The noninvasive measurement of functional β-cell mass would be clinically valuable for monitoring the progression of type 1 and type 2 diabetes as well as the viability of transplanted insulin-producing cells. Although previous work using MRI has shown promise for functional β-cell mass determination through voltage-dependent Ca2+ channel (VDCC)–mediated internalization of Mn2+, the clinical utility of this technique is limited by the cytotoxic levels of the Mn2+ contrast agent. Here, we show that positron emission tomography (PET) is advantageous for determining functional β-cell mass using 52Mn2+ (t1/2: 5.6 days). We investigated the whole-body distribution of 52Mn2+ in healthy adult mice by dynamic and static PET imaging. Pancreatic VDCC uptake of 52Mn2+ was successfully manipulated pharmacologically in vitro and in vivo using glucose, nifedipine (VDCC blocker), the sulfonylureas tolbutamide and glibenclamide (KATP channel blockers), and diazoxide (KATP channel opener). In a mouse model of streptozotocin-induced type 1 diabetes, 52Mn2+ uptake in the pancreas was distinguished from healthy controls in parallel with classic histological quantification of β-cell mass from pancreatic sections. 52Mn2+-PET also reported the expected increase in functional β-cell mass in the ob/ob model of pretype 2 diabetes, a result corroborated by histological β-cell mass measurements and live-cell imaging of β-cell Ca2+ oscillations. These results indicate that 52Mn2+-PET is a sensitive new tool for the noninvasive assessment of functional β-cell mass.

The noninvasive measurement of functional b-cell mass would be clinically valuable for monitoring the progression of type 1 and type 2 diabetes as well as the viability of transplanted insulin-producing cells. Although previous work using MRI has shown promise for functional b-cell mass determination through voltage-dependent Ca 2+ channel (VDCC)-mediated internalization of Mn 2+ , the clinical utility of this technique is limited by the cytotoxic levels of the Mn 2+ contrast agent. Here, we show that positron emission tomography (PET) is advantageous for determining functional b-cell mass using 52 Mn 2+ (t 1/2 : 5.6 days). We investigated the whole-body distribution of 52 Mn 2+ in healthy adult mice by dynamic and static PET imaging. Pancreatic VDCC uptake of 52 Mn 2+ was successfully manipulated pharmacologically in vitro and in vivo using glucose, nifedipine (VDCC blocker), the sulfonylureas tolbutamide and glibenclamide (K ATP channel blockers), and diazoxide (K ATP channel opener). In a mouse model of streptozotocin-induced type 1 diabetes, 52 Mn 2+ uptake in the pancreas was distinguished from healthy controls in parallel with classic histological quantification of b-cell mass from pancreatic sections. 52 Mn 2+ -PET also reported the expected increase in functional b-cell mass in the ob/ob model of pretype 2 diabetes, a result corroborated by histological b-cell mass measurements and live-cell imaging of b-cell Ca 2+ oscillations. These results indicate that 52 Mn 2+ -PET is a sensitive new tool for the noninvasive assessment of functional b-cell mass.
Type 1 and type 2 diabetes, although arising from different etiologies, are each associated with the functional loss of insulin-secreting b-cells and can lead to life-threatening complications such as cardiovascular disease and neuropathy (1). As such, a variety of therapeutics are being pursued that aim to preserve, expand, or replace b-cells. An imaging modality capable of monitoring functional b-cell mass in vivo as well as the viability of islet-or stem cell-derived b-cell transplants would therefore be invaluable to future therapeutic investigations.
Human pancreatic islets occupy ;4.5% of the pancreas volume and are composed of a mixture of b-, a-, g-, d-, and e-cells (2,3). Because individual islets vary in size from 25 to 400 mm in diameter (3)(4)(5) and are nonuniformly distributed throughout the pancreas, quantification is challenging through noninvasive anatomical imaging techniques such as MRI or computed tomography (CT) (6). Alternatively, positron emission tomography (PET) is a technique that involves quantifying the in vivo distribution of a biologically relevant moiety by tracking a positron-emitting radioisotope. Compared with MRI and CT, PET has significantly greater imaging sensitivity and inherently probes physiology rather than anatomy (7), which may prove useful in the clinical quantification of functional b-cell mass.
Mn 2+ , in a behavior that mimics Ca 2+ , is freely transported through voltage-dependent Ca 2+ channels (VDCC) (8,9). Because VDCC activation is required for the release of insulin from b-cells, Mn 2+ has been proposed as a molecular imaging agent for probing b-cell function and mass using Mn 2+ -enhanced MRI (10)(11)(12)(13)(14)(15). However, this technique is limited by significant cellular toxicity and the slow biological clearance of Mn 2+ , which prevents the possibility of repeated administration (9,16). Because the sensitivity of PET is several orders of magnitude higher than MRI, the objective of this work was to assess the feasibility of using PET to probe b-cell mass and function.
Herein, we take advantage of the exceptional sensitivity and quantitation capability of PET and the availability of radiomanganese ( 52 Mn 2+ ) to noninvasively image functional b-cell mass in vivo. When intravenously (i.v.) injected in mice, 52 Mn 2+ (t 1/2 : 5.6 days) showed a rapid accumulation in the mouse pancreas that was sensitive to glucose and pharmacological manipulation of VDCC. We also explored the potential of 52 Mn 2+ -PET to detect changes in functional b-cell mass in mouse models of type 1 and type 2 diabetes. Overall, our results point to the significant potential for 52 Mn 2+ to overcome several limitations of other b-cell imaging agents and potentially become a method of choice for studying b-cell physiology noninvasively.

52
Mn 2+ -PET Imaging Aliquots of the buffered 52 Mn 2+ solution (0.01 mol/L NaOAc, pH 6.5) were diluted to the desired injection volume (;200 mL) with PBS, typically 2-4 MBq (;50-100 mCi) per subject. The acquisition of PET images was performed with an Inveon mPET/mCT scanner (Siemens Preclinical Solutions). To study the biodistribution and clearance of 52 Mn 2+ in the mouse, 3.7 MBq (100 mCi) of 52 Mn 2+ was i.v. injected into female ICR mice. As a result of the long decay half-life of 52 Mn 2+ (t 1/2 : 5.6 days), multiple PET scans were recorded between 1 h and 13 days after injection of the radiotracer. Before each scan, mice were anesthetized with isoflurane (4% induction; 1% maintenance) and placed prone in the scanner, and 30-40 3 10 6 coincidence events per mouse static PET scans were acquired (time window, 3.432 ns; energy window, 3502650 keV).
The PET images were reconstructed in Inveon Acquisition Workplace (Siemens Preclinical Solutions) workstation using a nonscatter-corrected three-dimensional ordered subset expectation optimization/maximum a posteriori (OSEM3D/MAP) algorithm. Region-of-interest (ROI) analysis was performed after organs were manually delineated on the PET images. Tissue 52 Mn 2+ uptake values are reported as standardized uptake value (SUV), which is normalized to whole-body 52 Mn 2+ concentration to account for weight disparities between animal models. The percentage of injected dose per gram of tissue (%ID/g) is also reported in Supplementary Tables 6-9). To acquire dynamic PET scans, mice were anesthetized with isoflurane and the lateral tail vein was catheterized. Simultaneous with the administration of ;1.7 MBq (;50 mCi) of 52 Mn 2+ as a fast i.v. bolus, 1-h scans were recorded, and list-mode files were binned into 46 frames (12 3 5 s, 6 3 10 s, 6 3 30 s, 10 3 60 s, 6 3 150 s, 6 3 300 s) and the images reconstructed using the OSEM3D/MAP algorithm. For comparison, 1.7 MBq (50 mCi) 52 Mn 2+ was continuously infused with a syringe pump (Model 780100l; KD Scientific) during the first 30 min of a 60-min experiment, and list-mode files were binned into 30 frames (2 min each) and reconstructed using the OSEM3D/MAP algorithm.
Effect of Pharmacological VDCC Manipulation on 52 Mn 2+ Uptake In Vivo Pancreatic 52 Mn 2+ uptake was stimulated using glucose and glibenclamide (Tocris Biosciences), which is known to promote insulin release in b-cells via blockade of ATP-sensitive K + channels (K ATP ). Mice were injected intraperitoneally (i.p.) with 100 mL of 1 g/kg glucose, or glucose plus 5 mg/kg glibenclamide in PBS, 15 min before the i.v. injection of 0.74-1.85 MBq (20 mCi) of 52 Mn 2+ . VDCC blockade was achieved via i.p. injection of 20 mg/kg nifedipine (MP Biomedicals) dissolved in DMSO. To activate K ATP channels, mice received an i.p. injection of 20 mg/kg diazoxide (Tocris Biosciences) in PBS, a clinically used K ATP agonist, 15 min before injection of 0.74 MBq (20 mCi) of 52 Mn 2+ . Wholebody PET scans were acquired 1 h after the injection of the radiotracer, after which ex vivo biodistribution analysis was performed.

52
Mn 2+ -PET Studies in a Type 1 Diabetes Model Type 1 diabetes was induced in female ICR mice via one i.p. injection of 180 mg/kg streptozotocin (STZ; MP Biomedical), a toxin that selectively destroys pancreatic b-cells (17). The injectable STZ solution (12.5 mg/mL) was prepared fresh in PBS. The weight of each mouse was measured daily, and blood glucose levels were recorded every other day with a TRUEresult glucometer (Trividia Health Inc.) using blood samples collected from the tail vein. Mice were considered diabetic after two consecutive blood glucose readings above 250 mg/dL and were used for 52 Mn 2+ -PET imaging studies 1 week after the STZ injection. To evaluate 52 Mn 2+ pancreatic uptake in diabetic mice, 0.74 MBq (20 mCi) of radioactivity was administered i.v., and static PET images were recorded 1 h after administration of the tracer. Ex vivo biodistribution was performed after PET acquisition. Pancreatic uptake of 52 Mn 2+ was measured in 10-week-old wild-type and ob/ob mice on the C57BL/6J background. For PET imaging, 0.74 MBq (20 mCi) of 52 Mn 2+ was i.v. injected 1 h before PET scan acquisition. Accumulation of 52 Mn 2+ in the pancreas and other organs of interest was also quantified by ex vivo biodistribution analysis.

Additional Materials and Methods
Animal studies were conducted under the approval of University of Wisconsin-Madison Institutional Animal Care and Use Committee. Detailed information on animal models, isotope production, ex vivo biodistribution, islet isolation and imaging, and b-cell mass measurement is provided in the Supplementary Data.

RESULTS
In Vivo Whole-Body PET and Biodistribution of 52

Mn 2+ in Normal Mice
The in vivo biodistribution of 52 Mn 2+ was investigated noninvasively with PET and ex vivo gamma counting. Figure 1A shows coronal planes intersecting the pancreas of ICR mice, in PET scans acquired between 1 h and 13 days after i.v. injection of 3.7 MBq (100 mCi) of 52 Mn 2+ . A rapid and prominent accumulation of 52 Mn 2+ was observed in the pancreas, kidneys, liver, heart, and salivary glands (5.  Table 1). Uptake of 52 Mn 2+ in the pancreas, which was highest at 1 h postinjection, was notably higher than that in the liver and kidneys at all time points. Such favorable pancreas-to-normal organ contrast ratios, which peaked at ;3 days after 52 Mn 2+ administration, facilitated an easy delineation of the pancreas (Supplementary Fig. 1). Uptake in the muscle was very low and had little variation during the study. Ex vivo biodistribution was performed after the last PET scan 13 days postinjection, confirming a marked accumulation of 52 Mn 2+ in the salivary gland, pancreas, kidneys, and to a lesser extent, the heart and liver ( Fig. 1C and Supplementary Table 2). Other organs, including the brain, lungs, bones, intestines, stomach, and spleen, displayed low 52 Mn 2+ uptake, typically less than 0.5 SUV.
Because the whole-body distribution of 52 Mn 2+ occurred largely within 1 h after i.v. administration (Fig. 1), a dynamic PET study was designed to investigate the 52 Mn 2+ kinetics during this time. Figure 2 shows the time-activity curves (TACs) resulting from ROI analysis of the dynamic PET data corresponding to the pancreas, liver, kidneys, heart, salivary gland, and muscle under two i.v. administration regimens: rapid bolus injection and 30-min continuous infusion. The analysis of the myocardial TAC revealed extremely fast blood extraction kinetics, with a blood circulation half-life of 10.7 6 3.5 s in mice administered a rapid i.v. 52 Mn 2+ bolus. Consequently, 52 Mn 2+ uptake was stabilized in the organs of interests within 5 min postinjection.  52 Mn 2+ (no anesthesia except during the PET scans). Coronal PET image slices were selected to best show pancreatic uptake. Arrows point to P, pancreas; H, heart; L, liver; I, intestines; and SG, salivary gland. B: ROI-based quantification of 52 Mn 2+ uptake in the heart, liver, kidneys, muscle, pancreas, and submandibular salivary gland. C: Ex vivo 52 Mn 2+ biodistribution of euthanized mice after the last PET scans, determined by gamma counting (n = 4).
A residual radioactivity of 2.60 6 0.41 SUV was observed in the heart at 1 h postinjection, which was consistent with the specific uptake of Mn 2+ ions by myocardial tissue. Compared with the static 1 h postinjection PET scans (Fig. 1), similar 52 Mn 2+ uptake values were observed after rapid bolus injection in the heart (2.11 6 0.20 vs. 2.60 6 0.41 SUV) and muscle (0.38 6 0.03 vs 0.16 6 0.02 SUV), whereas the liver (3.27 6 0.34 vs. 5.16 6 1.46 SUV) and kidneys (5.13 6 0.02 vs. 7.81 6 0.51 SUV) were much higher at the end of the dynamic scan after a rapid i.v. 52 Mn 2+ bolus. Interestingly, an ;50% reduction in pancreatic uptake, from 5.13 6 0.38 to 2.74 6 0.59 SUV, was observed in the dynamic studies.
As clearly depicted in Fig. 2 (red curves), continuous infusion of 52 Mn 2+ over a 30-min period resulted in a linear ramping in organ radioactivity, followed by an immediate plateau upon infusion termination that persisted for the remainder of the experiment. This method highlights the rapid distribution kinetics of 52 Mn 2+ and the comparable results obtained by a rapid bolus or continuous infusion of 52 Mn 2+ . Only the kidney displayed higher radioactivity at 1 h postinjection in mice administered the rapid bolus versus mice receiving the continuous 52 Mn 2+ infusion, 7.81 6 0.51 vs. 5.14 6 1.21 SUV, respectively (n = 4). During the continuous infusion regimen, the pancreas reached an uptake of 3.44 6 0.69 SUV, which, similar to the rapid bolus injection, was also significantly lower than that in the static PET scans at 1 h postinjection (5.13 6 0.38 SUV).
Previous reports have shown that many volatile anesthetics, including isoflurane, impair insulin secretion by inhibiting the glucose-dependent inactivation of K ATP channels (18,19). Plausibly, the observed decrease in pancreatic uptake of 52 Mn 2+ resulted from mice being anesthetized through the full extent of the studies during the dynamic PET scans. To investigate the effect of isoflurane anesthesia on pancreatic uptake of 52 Mn 2+ , 1 h postinjection biodistribution experiments were performed under different administration conditions in anesthetized (1% isoflurane) or awake mice. Supplementary Fig. 2 shows that isoflurane significantly inhibited the accumulation of 52 Mn 2+ in the pancreas regardless of the administration regimen (rapid bolus vs. infusion) or glucose stimulation, suggesting that isoflurane indeed acts as an indirect VDCC inhibitor upstream of K ATP channel closure.

Uptake of 52 Mn 2+ in Isolated Islets
To corroborate the mechanism of 52 Mn 2+ uptake in the pancreas and its dependence on VDCC, an ex vivo 52 Mn 2+ uptake study was performed in islets isolated from ob/ob mice to take advantage of the twofold greater islet yield versus wild-type mice. As a result of the similarities between Mn 2+ and Ca 2+ ions, Mn 2+ uptake by b-cells occurs via influx through VDCC (Fig. 3A) (20,21). Isolated islets were incubated with 0.37 MBq (10 mCi) of 52 Mn 2+ under several conditions that stimulate/inhibit VDCC (Fig. 3B). 52 Mn 2+ was readily taken up by islets, even in the presence of low (1 mmol/L) glucose, as expected for the high basal Ca 2+ and insulin secretion reported for ob/ob b-cells (22). 52 Mn 2+ uptake was significantly enhanced (P , 0.05) when the islets were stimulated with 10 mmol/L glucose. Importantly, 52 Mn 2+ uptake was completely blocked by the further application of diazoxide (50 mmol/L), which inhibits the opening of VDCC via activation of K ATP channels. As expected for the clearance of intracellular Ca 2+ in response to diazoxide (23), intracellular 52 Mn 2+ fell below the basal level. Conversely, glucose administered with the K ATP channel blocker tolbutamide (250 mmol/L) resulted in significantly higher 52 Mn 2+ retention than glucose alone. Taken together, these experiments demonstrate that pancreatic islet uptake of 52 Mn 2+ depends on the activity of b-cell VDCC.

Pharmacological Manipulation of 52 Mn 2+ Pancreatic Uptake In Vivo
To verify the specificity of 52 Mn 2+ accretion in the pancreas noninvasively using PET imaging, 52 Mn 2+ pancreatic uptake was pharmacologically manipulated in vivo through the inhibition or stimulation of VDCC. In line with the in vitro results, inhibition of VDCC by direct blockade with nifedipine, or activation of K ATP with diazoxide, resulted in a significant (P , 0.0001) abrogation of the PET signal within the pancreas. Figure 4A shows coronal PET slices of the pancreas of ICR mice receiving 20 mg/kg i.p. injections of nifedipine or diazoxide 10-15 min before the administration of an i.v. 52 52 Mn 2+ uptake in groups administered diazoxide and nifedipine, respectively ( Fig. 4B and C). The remnant pancreatic signal suggests that i.p. drug injection resulted in the incomplete blockade of VDCC, or more likely, that some 52 Mn 2+ uptake is independent of VDCC (8). However, mice administered glibenclamide (5 mg/kg) exhibited a significant enhancement in pancreatic uptake of 52 Mn 2+ . The distribution of 52 Mn 2+ in other organs of interest, including the heart, liver, kidneys, spleen, and salivary gland, remained largely unaltered among the groups (Supplementary Tables 3 and  4). These results demonstrate the potential of 52 Mn 2+ PET imaging to noninvasively detect changes in functional b-cell mass. 52

Mn 2+ Uptake in Mouse Models of Type 1 and Type 2 Diabetes
We further investigated the correlation between pancreatic 52 Mn 2+ uptake and functional b-cell mass in a murine model of type 1 diabetes. Diabetes was induced in female ICR mice via a single STZ injection (180 mg/kg). Four days after injection (Fig. 5A), mice presented signs of hyperglycemia (blood glucose . 250 mg/dL) and weight loss that indicated a diabetic status. As seen in the PET images (Fig.  5B), 52 Mn 2+ accumulation within the pancreas of diabetic mice was reduced by ;60%, from 5.13 6 0.38 SUV (n = 3) in normal mice to 2.04 6 0.81 SUV (n = 3) in diabetic mice (P , 0.0001) (Fig. 5C, and Supplementary Tables 3 and 4). Ex vivo biodistribution analysis corroborated a very similar (58%) decrease in pancreatic accumulation of 52 Mn 2+ (Fig.  5D). To compare 52 Mn 2+ -PET imaging with a direct histological assessment of b-cell mass, three pancreata per treatment group were weighed, sectioned, and immunostained for insulin. On average, b-cell mass in the STZ-treated mice fell by ;70% compared with the sham-injected ICR mice (Fig. 5E), in good agreement with the 52 Mn 2+ -PET data. 52 Mn 2+ -PET imaging studies were also performed in C57BL/6J mice carrying the lep ob (ob/ob) spontaneous mutation that results in obesity and pretype 2 diabetes. In this case, 52 Mn 2+ accumulation in the pancreas of ob/ob mice was significantly (P , 0.001) higher than in the wild-type C57BL/6J mice (Fig. 6A). Pancreatic uptake 1 h after administration of 52 Mn 2+ (n = 3) was 4.89 6 0.68 SUV in wild-type C57BL/6J mice and 7.27 6 1.03 SUV in ob/ob mice (Fig. 6B). Ex vivo biodistribution analysis corroborated the statistically significant differences in pancreatic SUV between the groups (P , 0.001) ( Supplementary Fig. 3 6C). We also observed that ob/ob islet Ca 2+ levels, which oscillate in response to glucose stimulation (Fig. 6D), were strongly left shifted in their glucose dependence relative to controls (Figs. 6E and F), in agreement with the in vivo 52 Mn 2+ -PET results and prior functional studies (22). Notably, in contrast to the experiments in Fig. 4, where pharmacological VDCC manipulation was used to strictly define functional b-cell mass, the experiments in Figs. 5 and 6 show that basal 52 Mn 2+ uptake (measured in the absence of exogenous glucose or VDCC inhibitors) remains heavily dependent on changes in b-cell mass. Both approaches, when applied clinically, are likely to yield useful information.  52 Mn 2+ is observed in mice that received nifedipine and diazoxide before radiotracer administration. Mice that received glibenclamide (5 mg/kg) before radiotracer administration had significantly higher pancreatic uptake of 52 Mn 2+ than the control mice, based on both PET imaging (P = 0.02) and biodistribution (P = 0.047) studies. Data are presented as mean 6 SD (n = 3-4 mice per group). ***P < 0.001.

DISCUSSION
The loss of functional b-cell mass is a cornerstone of both type 1 and type 2 diabetes (24). Here, we leveraged the ability of b-cells to sequester divalent metal ions (e.g., Ca 2+ , Mn 2+ , Zn 2+ , and Co 2+ ), which is essential to the production and release of insulin. Although b-cell function has been widely investigated based on the measurement of Ca 2+ currents in vitro (25), little progress has been achieved in exploring divalent metal intake for measurement of functional b-cell mass in vivo. Part of this is a result of the lack of effective methods to track the distribution of most of these metals. Fortunately, 52 Mn 2+ has both magnetic and nuclear properties that facilitate its noninvasive detection by MRI, and with greater sensitivity, PET. In the present work, we report the use of 52 Mn 2+ for noninvasive PET imaging of the mouse pancreas. When injected i.v., 52 Mn 2+ accumulated significantly into pancreatic tissue with exceptionally fast blood extraction kinetics. 52 Mn 2+ uptake in the  52 Mn 2+ administration in ob/ob mice and C57BL/6J controls. The pancreas (P) is demarcated by white dashed contours. B: Image-derived quantification expressed as SUV indicated a significant difference in 52 Mn 2+ pancreatic uptake between groups (mean 6 SD; n = 3). C: Quantification of b-cell mass for wildtype and ob/ob mice (n = 3 mice/group). Scale bars 5 400 mm. D: Recordings of islet Ca 2+ in response to glucose (10 mmol/L and 7 mmol/L) from wild-type and ob/ob C57BL/6J mice. E: Contingency plot shows the range of islet behaviors at each glucose level. F: The oscillatory plateau fraction, reflecting the plasma membrane glucose sensitivity, was calculated as the fraction of time spent in the active state during each oscillation at 10 mmol/L glucose. C57BL/6J, n = 141 islets from 4 mice; ob/ob, n = 126 islets from 4 mice. Results reflect mean 6 SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. pancreas, which peaked within minutes after administration in healthy mice, was on the order of 5-6 SUV, with a slow clearance half-life of ;8 days. Our results demonstrate that in vivo measurements of functional b-cell mass by 52 Mn 2+ -PET offers complementary information to existing b-cell function assays that lack spatial information (e.g., measurement of c-peptide release), as well as measures of b-cell mass that cannot predict b-cell function (e.g., PET imaging with radiolabeled exendin-4).
A key finding of our work was that 50-60% of the observed pancreatic radioactivity resulted from the specific uptake of 52 Mn 2+ through VDCC, implicating b-cells. By pharmacologically manipulating the mechanism of VDCC in isolated islets, we were able to correlate the uptake of radioactive 52 Mn 2+ with Ca 2+ uptake. This agrees with other in vitro studies using nonradioactive Mn 2+ that reported a correlation between Mn 2+ uptake and changes in b-cell functional capacity arising from the triggering pathway (14,15,26). Similarly, in vivo studies demonstrated that chemical inhibition of VDCC, using nifedipine or diazoxide, resulted in a drastic decline in the accumulation of 52 Mn 2+ within the pancreas. In addition, the stimulation of VDCC with glucose and glibenclamide led to an increase in 52 Mn 2+ uptake in the pancreas. Overall, our results indicated that the 52 Mn 2+ uptake observed in the pancreas was largely mediated by and dependent on the functional b-cell mass, keeping in mind that 52 Mn 2+ -PET would be blind to defects in the neurohormonal amplifying pathways of insulin secretion, which are largely VDCC independent (27).
Our next question was whether this method was sensitive enough to detect changes in functional b-cell mass that occur during diabetes progression and disease. First, we used an experimental animal model of STZ-induced type 1 diabetes, which showed an ;60% reduction in the pancreatic accumulation of 52 Mn 2+ , while uptake in other organs remained unaltered. Direct measurements of b-cell mass by histology showed a comparable decline (;70%), indicating the capability of 52 Mn 2+ -PET imaging for revealing extreme cases of b-cell loss. Equally relevant results were observed in the obese mouse (ob/ob) model of pretype 2 diabetes, where significant (P , 0.0001) enhancement in pancreatic uptake of 52 Mn 2+ was observed. These results agree with previous reports showing that ob/ob mice do not progress to type 2 diabetes (28,29) but rather present markedly higher functional b-cell mass compared with wild-type mice at 10 weeks of age, as assessed by histology and live-cell Ca 2+ imaging. Probing the gradual gain/loss of functional b-cell mass using 52 Mn 2+ (t 1/2 = 5.6 days) was not feasible as a result of its long radioactive half-life. Rather than trying to account for the remnant 52 Mn 2+ radioactivity in the pancreas, longitudinal studies would benefit from the use of the shorter-lived 51 Mn 2+ (t 1/2 : 45.6 min), which would facilitate regular monitoring of variations in functional b-cell mass without the interference of remnant radioactivity. This is the subject of our future work.
Because significant changes can occur in b-cell function long before changes in anatomical mass are observed (30)(31)(32), it is advantageous that the relative uptake of 52/51 Mn 2+ in vivo depends on the functional b-cell mass. This ability has the potential to shed light on the rate of functional b-cell mass decline in asymptomatic patients with type 1 diabetes as well as the survival of b-cell transplants, including stem cell-derived b-cells. Whether 51/52 Mn 2+ -PET is capable of measuring very subtle changes in this parameter is the pivotal question to its clinical significance. The diffusion of Mn 2+ through b-cell VDCCs depends on their activation by glucose or drug-based stimulation. This has been shown here through in vitro and in vivo functional suppression and enhancement studies. For a given state of glucose activation, the amount of Mn 2+ cellular internalization by the pancreas is partly controlled by the number of available VDCCs, which should be proportional to functional b-cell mass. However, accurate quantification of functional b-cell mass will require subtraction of nonspecific exocrine pancreas uptake of the radiotracer. For this reason, the 52 Mn 2+ -PET measurements in ob/ob mice underestimate the predicted effect on functional b-cell mass. Calibration may be accomplished through pharmacological manipulation to acutely suppress VDCCmediated uptake of 52 Mn 2+ using nifedipine, diazoxide, or other suppressing agents. However, same-subject calibration will require the implementation of back-to-back PET scans using the shorter-lived 51 Mn 2+ or the performance of dynamic PET scanning under a 52 Mn 2+ infusion regimen that allows for the deconvolution of the endocrine signal from the exocrine background.
Aside from VDCC, there are several endogenous mechanisms for Mn 2+ uptake, including facilitated diffusion, divalent metal transporter-1 (DMT1), Zrt-and Irt-related protein-8 (ZIP-8), transferrin receptor (TfR), and ionotropic glutamate receptor Ca 2+ channels (8). The relative role that these alternative pathways play in 52 Mn 2+ transport remains unknown, but a lack of effect by nifedipine suggests that VDCC may not be a primary transport route in many normal tissues. In this regard, our studies also provided valuable information on the kinetics of the whole-body distribution of 52 Mn 2+ . Besides the pancreas, 52 Mn 2+ also accumulated in the liver, heart, kidneys, and salivary gland. No clear sign of renal excretion was observed despite the significant kidney uptake, and most of the 52 Mn 2+ clearance occurred through the hepatobiliary system. From the analysis of the image-derived dynamic TAC of the heart, we estimated a 52 Mn 2+ circulation half-life of 10.7 6 3.5 s after a rapid bolus i.v. injection, which aligned with previous studies showing an extremely fast (;0.8 min) blood clearance of 54 Mn in dogs (33). Such fast extraction kinetics indicate that compartmental modeling could be used to describe 52 Mn 2+ uptake in a more quantitative manner. A stable residual myocardial uptake was observed after complete blood clearance, ;1 min after injection. Of note was the marked underestimation of myocardial uptake by PET compared with biodistribution results, with an average recovery coefficient of 0.54. This is an inherent limitation of the PET imaging-based quantification of small volumes such as the myocardial wall or adrenal glands, which in mice are affected by significant partial volume effects (34,35).
Several PET tracers have been studied as potential b-cell imaging agents. [ 11 C]-dihydrotetrabenazine (DTBZ) and the 18 F-labeled DTBZ analog FP-(+)-DTBZ have been shown to have high affinity for the type 2 vesicular acetylcholine transporter (VMAT2), which is expressed on the surface of rodent b-cells but appears to be entirely absent from pancreatic exocrine tissue (36)(37)(38)(39). Unfortunately, primate models have shown very low pancreatic uptake of [ 11 C]-DTBZ, which suggests that the degree of VMAT2 expression is species dependent (40)(41)(42). More work is needed in quantifying human b-cell expression of VMAT2 and how expression density is related to b-cell function. Another promising PET approach for monitoring in vivo b-cell mass involves compartmental kinetic analysis of [ 11 C]5-hydroxytryptophan (HTP), a compound used clinically for detection of neuroendocrine tumors (43). This compound has shown differential clearance from endocrine versus exocrine pancreatic tissues based on differences in the presence of serotonin biosynthesis machinery, which effect [ 11 C]5-HTP retention in endocrine cells. With this approach, [ 11 C]5-HTP tracer uptake is observed to decrease both longitudinally in patients with type 2 diabetes and cross-sectionally in patients with type 1 diabetes compared with healthy control subjects (44,45). Another category of tracers includes radiolabeled (e.g., 18 F, 68 Ga, 64 Cu, 111 In) derivatives of exendin-4, a glucagon-like protein-1 receptor agonist (46)(47)(48)(49)(50). Fluorescence microscopy has shown that glucagonlike protein-1 receptor is only located on b-cells within the human pancreas (51), making it an attractive molecular imaging target as a result of its specificity. In animal models, exendin-4-PET suffers from low pancreatic uptake relative to the surrounding organs (,0.3%ID/g in Sprague-Dawley rats at 1 h postinjection, whereas proximal kidney uptake is .25%ID/g) (52). Although the kidney uptake of 52 Mn 2+ is also significant, the pancreatic uptake of 52 Mn 2+ is comparatively high, such that improved pancreas-tobackground ratios are attained by 52 Mn 2+ -PET. In general, the implementation of tracers targeting surface receptors of b-cells is extremely challenging, considering the low total mass of b-cells, diffuse pancreatic distribution, and heterogeneous receptor expression.
Aside from following the gain/loss of b-cell mass in patients with prediabetes/diabetes, there is a pressing need for a noninvasive method for longitudinal imaging of b-cell transplants. Islet transplantation has been shown to lead to temporary insulin independence in patients with type 1 diabetes (53,54) but has not been widely adopted because of the need for donor tissues. Recent advances in selective stem cell differentiation techniques will likely lead to wider clinical adoption of b-cell transplantation therapies (55). The ability to noninvasively track the survival and function of transplanted b-cells would enable research into the patient-specific efficacy assessment of immune-modulating therapies and the development of new therapeutic strategies (56). In this case, the use of 51 Mn 2+ -PET for monitoring islet transplant survival is compelling because it can be performed repeatedly and safely over time.
In conclusion, the prominent pancreatic uptake, rapid localization kinetics, and well-characterized metabolic pathways of 52 Mn 2+ make it a promising agent for noninvasive PET imaging of functional b-cell mass and was corroborated by various post hoc experiments such as histological b-cell mass measurements and live-cell imaging of b-cell Ca 2+ oscillations. With further development, 51 Mn 2+ -PET may also find broad applications in venues ranging from basic research to clinical patient care.