This is the first study evaluating [11C]DTBZ for BCM imaging across rat models of diabetes and pre-diabetic conditions encompassing type 1 diabetes, type 2 diabetes, and non-diabetic β-cell hyperplasia and directly comparing the efficacy of BCM imaging with [11C]DTBZ vs. [18F]FP-(+)-DTBZ, which are currently the best-available imaging agents of BCM. The underlying strategy for imaging of pancreatic BCM with either [11C]DTBZ or [18F]FP-(+)-DTBZ is the specific uptake of these radiotracers into the insulin secretory granules of the β-cell mediated by VMAT2. It should be noted though that possible differences across rat strains of the association of VMAT2 levels with insulin content per β-cell is not known, and may impact the correlation of pancreatic uptake of DTBZ analogues with β-cell mass and its associated insulin content. Nevertheless, we observed an excellent correlation between pancreatic [18F]FP-(+)-DTBZ uptake as measured by pancreatic SUV and the indices of insulin content and percent β-cell area in the STZ-model of type 1 diabetes, and of [11C]DTBZ across the models of type 1 and type 2 diabetes and compensatory hyperplasia covering a ~10 fold range in BCM.
The results presented in this paper are based on total uptake of radiotracer in pancreas as measured by SUV. Although SUV is normalized by injected dose and body weight, a metric that quantitatively accounts for the availability of tracer (e.g., in plasma or a reference tissue lacking specific binding) would be a theoretically more accurate outcome. Previous studies investigating beta cell mass with VMAT2 radioligands have used the renal cortex as a reference region and the results of our displacement experiments suggested that the kidney lacks specific binding [13
]. We therefore analyzed the pancreas TACs by applying the multilinear reference tissue model using kidney data as input functions to estimate binding potential (BPND
), the ratio of specifically bound tracer to non-displaceable concentration at equilibrium [33
measurements from [11
C]DTBZ and [18
F]FP-(+)-DTBZ data correlated positively with pancreas insulin content (R2
=0.29 for [11
C]DTBZ and 0.26 for [18
F]FP-(+)-DTBZ), but less strongly than did pancreas SUV against insulin (R2
=0.32 for [11
C]DTBZ and 0.37 for [18
F]FP-(+)-DTBZ). As noted in Results, variability in the assignment of regions on the renal cortex and time-varying partial volume effects associated with imaging such a thin structure are likely sources of error, which may have added variability to the BPND
estimates. Fortunately, we anticipate that these factors will be less significant in human studies, due to a better match of structure size and spatial resolution.
Prior to the onset of type-2 diabetes, obesity often leads to peripheral insulin resistance. However, compensation by an enhanced insulin secretory response by the β-cell can maintain normoglycemic levels and minimize abnormal postpandrial glycemia. In part, increases in BCM can account for the hypersecretory response. In fact, type 2 DM is characterized by an increase
in BCM with time in response to peripheral insulin resistance. Indeed, type 2 DM is often associated with the “metabolic syndrome” characterized by obesity and hyperlipidemia. Zucker fatty (ZF) rats serve as a model of obesity and have demonstrated an increase in BCM with age and weight [34
]. With the increase in body fat mass of the ZF rat from 8 to 16 weeks, the changes in pancreatic β-cell were most evident in the increase in BCM, rather than pancreatic insulin. During this 8-week time period, BCM increased by ~2.6-fold and insulin by ~1.7 fold. In an earlier study of β-cell adaptation in Z-F rats, Liu et al. also found a modest 1.3-fold increase in the insulin content of 12-wk Z-F rats compared to 12-wk Zucker Lean rats, despite the 3.8-fold increase in BCM [26
]. An even greater disparity is observed in a comparison of the S-D and ZF rats in our study, where the 9.3-fold increase in BCM is associated only with a 1.5-fold increase in insulin content. These results indicate a marked decrease in the insulin content per β-cell in the ZF rats with the development of obesity. The positive correlation of the pancreatic SUV of the PET image with insulin content, but not with BCM in the ZF rat is consistent with an increase in the number of insulin secretory granules together with increased VMAT2 protein.
As a model of type 2 diabetes, ZDF rats exhibit a loss of glucose-stimulated insulin secretion prior to the loss of beta cell mass [35
]. The ZDF diabetic variant of the Zucker fatty rat colony [36
] is a model for prediabetes and type 2 DM as they manifest an increase in BCM till 18 weeks of age, followed by a progressive loss of β-cells [34
]. Despite this increase in BCM, ZDF rats manifest a progressive loss of insulin production from 10 weeks of age [37
]. In our study, there was significantly lower insulin content in 12 and 16 weeks old ZDF rats whereas the percent beta cell area remained unchanged. Indeed, we did not notice a change in [11
C]DTBZ SUV in ZDF rats over age consistent with the percent β-cell area changes. These results suggest that a constant number of insulin secretory vesicles as diabetes develops in the ZDF rat, but that an overall depletion of insulin content within the vesicles occurs. As a diagnostic tool, PET imaging of [11
C]DTBZ uptake in the pancreas may be useful for identifying those type 2 diabetic patients that have reduced β-cell function despite intact BCM, and guide the design of therapies to restore islet function.
Streptozotocin (STZ) induced diabetes in experimental animals serves as a model of Type 1 diabetes with the selective loss of beta cell mass. Compared to their healthy controls, we have noted a ~30% reduction of both the [11
C]DTBZ, and the [18
F]FP-(+)-DTBZ SUV in STZ treated rats, whereas Souza et al observed a 65% reduction in an alternative rat model of type 1 diabetes (Biobreeding Diabetic Prone: BB-DP) [16
]. A possible explanation for this difference may be accounted for by the distribution of VMAT2 in pancreatic islet innervations. It is known that VMAT2 is present on both sympathetic neurons and beta cells in pancreas [19
]. The concentration of VMAT2 in pancreatic sympathetic neurons is higher than in beta cells and can be distinguished based on appearance on immunohistochemical examination [19
]. The [11
C]DTBZ and [18
F]FP-(+)-DTBZ PET signal is a sum total of the DTBZ bound to VMAT2 on both sympathetic neurons and beta cells. It has been reported that STZ does not damage VMAT2 on sympathetic nerves [38
] but does damage VMAT2 in beta cells [39
]. Eight-fold reductions in VMAT2 transcript concentration were noted in beta cells following STZ treatments [39
]. On the other hand, there is a progressive damage to sympathetic nerves along with beta cells in BB-DP models [38
]. A combined damage to both sources of VMAT2 may be responsible for the lower pancreatic binding of DTBZ in BB-DP rats as compared to STZ-treated rats where only the VMAT2 in beta cells is damaged.
The displacement of ~60% of pancreatic [18
F]FP-(+)-DTBZ with unlabeled FP-(+)-DTBZ in our rats was similar to that seen with either pre-treatment or coinjection of the unlabeled compound [12
]. This displaceable pool of radioligand has been interpreted to represent FP-(+)-DTBZ binding sites on VMAT2. Not previously reported though, the fraction of displaceable [18
F]FP-(+)-DTBZ was dependent upon BCM, supporting the hypothesis that this represents VMAT2 binding sites within the pancreatic β-cells. However, even in those rats with little residual pancreatic insulin or BCM, a substantial pool of displaceable, and hence specifically bound radioligand remained in the pancreas. From a comparison of the healthy S-D rats and the STZ-treated rats, we estimate that ~65% of the specifically-bound [18
F]FP-(+)-DTBZ is binding to VMAT2 (or another saturable site) that is not associated with insulin-positive β-cells. When also including the nonspecifically bound tracer, we estimate that ~ 25% of the total uptake can be attributed to β-cell uptake. In addition to the probability that sympathetic nerves within the pancreas contribute to the pool of reversible binding sites, Saisho et al.
also identified VMAT-2 positive pancreatic polypeptide (PP) cells within the pancreatic islet [32
]. In our STZ-treated animals, the toxicity is limited largely to the β-cell, leaving the PP cells unaffected [40
]. Thus, the contribution of PP cells to the reversibly-bound radioligand pool may be significant in our studies, as well as in the PET imaging of humans. PP cells have been shown to constitute up to ~2% of the volume density in the head of the pancreas in healthy controls, as well as type 1 and type 2 diabetic patients [42
], and may have contributed to the [11
C]DTBZ specifically bound background signal present in the pancreas of C-peptide negative type 1 diabetic patients [17