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Published in final edited form as: Nucl Med Biol. 2023 Jul 26;122-123:108370. doi: 10.1016/j.nucmedbio.2023.108370

Characterization of a S1PR2 specific 11C-labeled radiotracer in streptozotocin-induced diabetic murine model

Hao Jiang a, Tianyu Huang a, Yanbo Yu a, Charles Zhou a, Lin Qiu a, Hien Ngoc Mai a, Robert J Gropler a, Robyn S Klein b, Zhude Tu a,*
PMCID: PMC10949307  NIHMSID: NIHMS1965094  PMID: 37556928

Abstract

Background:

Diabetes mellitus is a chronic progressive metabolic disorder that affects millions of people worldwide. Emerging evidence suggests the important roles of sphingolipid metabolism in diabetes. In particular, sphingosine-1-phosphate (S1P) and S1P receptor 2 (S1PR2) have important metabolic functions and are involved in several metabolic diseases. In diabetes, S1PR2 can effectively preserve β cells and improve glucose/insulin tolerance in high-fat diet induced and streptozotocin (STZ)-induced diabetic mouse models. We previously developed a group of potent and selective S1PR2 ligands and radioligands.

Methods:

In this study, we continued our efforts and characterized our leading S1PR2 radioligand, [11C]TZ34125, in a STZ-induced diabetic mouse model. [11C]TZ34125 was radiosynthesized in an automated synthesis module and in vitro saturation binding assay was performed using recombinant human S1PR2 membrane. In vitro saturation autoradiography analysis was also performed to determine the binding affinity of [11C]TZ34125 against mouse tissues. Type-1 diabetic mouse model was developed following a single high dose of STZ in C57BL/6 mice. Ex vivo biodistribution was performed to evaluate the distribution and amount of [11C]TZ34125 in tissues. In vitro autoradiography analysis was performed to compare the uptake of [11C]TZ34125 between diabetic and control animals in mouse spleen and pancreas.

Results:

Our in vitro saturation binding assay using [11C]TZ34125 confirmed [11C]TZ34125 is a potent radioligand to recombinant human S1PR2 membrane with a Kd value of 0.9 nM. Saturation autoradiographic analysis showed [11C]TZ34125 has a Kd of 67.5, 45.9, and 25.0 nM to mouse kidney, spleen, and liver tissues respectively. Biodistribution study in STZ-induced diabetic mice showed the uptake of [11C]TZ34125 was significantly elevated in the spleen (~2 fold higher) and pancreas (~1.4 fold higher) compared to normal controls. The increased uptake of [11C]TZ34125 was further confirmed using autoradiographic analysis in the spleen and pancreases of STZ-induced diabetic mice, indicating S1PR2 can potentially act as a biomarker of diabetes in pancreases and inflammation in spleen. Future mechanistic analysis and in vivo quantitative assessment using non-invasive PET imaging in large animal model of diabetes is worthwhile.

Conclusions:

Overall, our data showed an increased uptake of our lead S1PR2-specific radioligand, [11C]TZ34125, in the spleen and pancreases of STZ-induced diabetic mice, and demonstrated [11C]TZ34125 has a great potential for preclinical and clinical usage for assessment of S1PR2 in diabetes and inflammation.

Keywords: S1PR2, [11C]TZ34125, Radiotracer, Diabetes, Streptozotocin

1. Introduction

Diabetes mellitus is a major health problem that affects >450 million people worldwide according to the data from World Health Organization. It is a chronic progressive metabolic disease characterized by inadequate insulin production or impaired response to insulin level, leading to hyperglycemia. It has a significant health impact on cardiovascular system, kidney, and nervous system and is the major cause of heart attacks, stroke, kidney failure, blindness, and others pathologies [1,2]. Sphingolipids are ubiquitous components of the cell membranes and their metabolite, sphingosine-1-phosphate (S1P), has important roles in physiological and pathophysiological conditions. S1P regulates cell growth and inhibits apoptosis and is involved in many pathophysiological processes such as atherosclerosis and inflammation [3]. Extracellular S1P signaling acts through a group of G protein-coupled receptors, sphingosine-1-phosphate receptor (S1PR) 1–5. These receptors have been associated with a variety of developmental and pathological processes [4]. In particular, recent studies demonstrate their roles in metabolic processes. For example, S1P plays a critical role in liver fibrogenesis through mediating homing of bone marrow mesenchymal stem cells via S1PR3 [5]. In human fibrotic liver, the expressions of S1PR1 and S1PR3 are heavily increased whereas the level of S1PR2 is dramatically decreased [6].

Interestingly, mounting evidence suggests that sphingolipid metabolism plays an important role in obesity and diabetes. Numerous studies demonstrate the important role of sphingolipids in the development and progression of diabetes. For example, studies show ceramides can inhibit the function of the insulin signaling pathway and stimulate pancreatic β cell death [7–9]. In contrast, the sphingosine kinase 1 (SphK1)-S1P axis has a beneficial role on glucose homeostasis and positively mediates pancreatic β cell function and viability [10,11]. However, SphK2 can promote β cell lipotoxicity and has a negative impact on glucose tolerance [12]. In addition, the SphK-S1P axis can positively regulate the insulin response in the liver, for example, SphK1 overexpression improves glucose homeostasis in diabetic animal models [13–15]. Studies also report a positive role of the SphK1-S1P axis on insulin signaling in muscle; SphK1 overexpression reduces the concentration of ceramide in muscle and improves insulin sensitivity in high-fat diet mice [16–18]. Intriguingly, studies also show the intracellular SphK-S1P axis leans a pro-inflammatory response and negatively impacts insulin signaling on adipose tissue whereas the extracellular S1P- S1PRs axis shows a positive impact on adipose tissue insulin signaling [19–21]. In fact, in addition to SphK-S1P axis, recent studies illustrate the important role of S1P-S1PRs pathway in diabetes. Upregulation of S1PR can increase the number of β cells and insulin levels and thus has a hypoglycemic effect in diabetes. FTY720, a well-characterized S1PR agonist, can significantly promote pancreatic β cell proliferation and inhibit diabetes induced β cell apoptosis [22,23].

S1PR2 is one of five member G protein-coupled S1P receptors that is widely expressed in various types of organs. S1PR2 regulates apoptosis, proliferation, actin remodeling, and B cell positioning through coupling with Gi/o, Gq, and G12/13 proteins [24]. Emerging evidence shows important metabolic functions of S1PR2 in the liver and pancreas. It is recognized as the receptor for conjugated bile acids in the liver and can regulate glucose, bile acid synthesis, and lipid metabolism in liver [25]. S1PR2 knockout mice show less fibrotic tissue and increased regeneration in liver fibrosis mouse model [26]. In diabetes, S1PR2 can mediate early stages of pancreatic and system inflammatory response via NF-κB activation in acute pancreatitis [27]. Inhibition and knockout of S1PR2 can block the high-fat diet induced adipocyte hypertrophy and systemic glucose intolerance [28]. In addition, S1PR2 inhibition can regulate the morphology and function of mitochondria in human renal glomerular endothelial cell via RhoA/ROCK1/Drp1 pathway and contribute to high glucose milieu [29]. It is hypothesized that therapeutic interventions to inhibit S1PR2 expression and function potentially can modify the progression of diabetes. To date, the exact mechanisms of S1PR2 in diabetes remain unclear.

We have previously developed a group of S1PR2-specific ligands and radioligands and performed initial evaluations in rodent and non-human primate models [30–32]. The leading S1PR2 radioligand, [11C] TZ34125, contains similar core structures to the well-known S1PR2 selective antagonist JTE-013 (Fig. 1A) [33]. It is potent to S1PR2 with an IC50 of 9.52 nM and shows high selectivity over S1PR1 and S1PR3 (IC50 > 1000 nM) [30]. Herein, we report our efforts on characterizations of our promising S1PR2 radioligand, [11C]TZ34125, in a streptozotocin (STZ)-induced diabetic murine model. Our findings show that the uptake of [11C]TZ34125 increased in the spleen and pancreas of STZ-induced diabetic mice, suggesting the possibility of diabetes diagnosis through PET with S1PR2 radioligand.

Fig. 1.

Fig. 1.

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Characterization of S1PR2 specific radiotracer [11C]TZ34125 in STZ induced diabetic mouse model. (A) Chemical structures of S1PR2 specific antagonist JTE- 013; (B) Radiosynthesis of 11C labeled JTE-013 analog [11C]TZ34125; (C) Biodistribution study of [11C]TZ34125 in normal and STZ-induced diabetic mice.

2. Methods

2.1. Animals

All animal experiments were conducted following the Guidelines for the Care and Use of Research Animals under a research protocol approved by the Washing University Institutional Animal Care and Use Committee (IACUC) in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals. Ten week old male C57BL/7 mice (The Jackson Laboratory, Bar Harbor, ME) were used in this study. All animal studies were conducted in the Preclinical Imaging Facility at the Washington University School of Medicine in St. Louis.

2.2. Chemistry and radiosynthesis of [11C]TZ34125

Commercially available chemicals including starting materials, reagents, and solvents were used directly unless otherwise stated. The radiosynthesis and quality control of S1PR2 specific radioligand [11C] TZ34125 from the in-house synthesized precursor, N-(2-Chloro-6-hydroxypyridin-4-yl)-2-(4-isopropyl-1,3-dimethyl-1H-pyrazolo[3,4-b] pyridin-6-yl)hydrazine-1-carboxamide, was accomplished as previously published (Fig. 1B) with minor modifications due to the use of a different cyclotron and hot cell [30].

Production of [11C]CH3I followed the reported method [34]. No-carrier added [11C]CO2 was produced through Washington University, Mallinckrodt Institute of Radiology TR19 cyclotron (Advanced Cyclotron Systems, Richmond, BC, Canada) from the 14N (p,α)11C nuclear reaction with proton bombardment of an aluminum target filled with 0.5 % O2 in nitrogen gas. The irradiation was achieved with a beam of 18.0 MeV at current of 40 μA for 25 min. After the irradiation was finished, the [11C]CO2 was shunted to the GE TracerLab FX MeI module to generate reactive [11C]methyl iodide ([11C]CH3I) for tracer production. Briefly, up to 88.8 GBq [11C]CO2 was trapped in molecular sieves and further reduced to [11C]CH4 in the presence of Ni catalyst and hydrogen at 350 °C. After removal of unreacted CO2 and H2O by passing through a chemical trap containing Ascarite and Sicapent in series, the [11C]CH4 was concentrated and purified by trapping in a carbosphere column. Once the [11C]CH4 was released, it reacted with iodine at 740 °C to produce [11C]CH3I in a recirculation loop. Approximately 10 min following the end-of-bombardment (EOB), 29.6–40.7 GBq of [11C] CH3I was delivered in the carrier gas to the hot cell where the radiosynthesis was accomplished.

[11C]CH3I was bubbled for a period of 2–3 min into a solution of precursor (1.0–1.3 mg) in DMF (0.2 mL) containing 3.0 μL of potassium hydroxide (5.0 M) at room temperature. When the trapping of radioactivity was complete, the sealed reaction vessel was heated at 85 °C for 5 min with stirring. Then the reaction was quenched with 1.3 mL of the HPLC mobile phase (45 % acetonitrile in 0.1 M ammonium formate, v/v, pH 6.5), and the mixture was loaded onto a reversed-phase HPLC system for purification (Agilent Zorbax SB-C18 column, 250 × 9.2 mm, mobile phase 45 % acetonitrile in 0.1 M ammonium formate, pH 6.5, flow rate 4.0 mL min− 1, detection wavelength 254 nm). Under these conditions, the desired product with a retention time at 15–17 min was collected into a vial that contained 50 mL Milli-Q water. Then the diluted product was passed through a C-18 Plus Sep-Pak® cartridge to concentrate the target component in the Sep-Pak cartridge. The Sep-Pak cartridge was rinsed using 20 mL of sterile water. Finally, the tracer trapped on the Sep-Pak® was eluted with 0.6 mL of ethanol, followed by 5.4 mL 0.9 % sodium chloride solution, passing through a 0.22 μm (Whatman Puradisc 13 mm syringe filter) sterile filter into a sterile pyrogen-free glass vial for delivery. For quality control, an aliquot of sample was assayed by an analytical HPLC system (Agilent Zorbax SB-C18 column, 250 × 4.6 mm, mobile phase 55 % acetonitrile in 0.1 M ammonium formate, pH 4.5, flow rate 1.2 mL min− 1, detection wavelength 254 nm). The sample was authenticated by co-injecting with the corresponding reference standard solution. The retention time was 5.5 min with radiochemical purity >99 %. The radiosynthesis typically took 55 min starting from the release of [11C]CH3I, with a yield at 16 ± 5 % and final amount at 740 ± 222 MBq (n > 5, decay corrected to the end of bombardment), specific activity >236 GBq/μmol (decayed corrected to EOB).

2.3. In vitro saturation binding assay

Saturation binding assay was carried out using [11C]TZ34125. In brief, 2 μg of recombinant human S1PR2 membrane protein was incubated with serial dilutions of [11C]TZ34125 ranging from 0.06 to 16 nM in 150 uL of 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM CaCl2, and 0.5 % bovine serum albumin (BSA) in a 96-well microplate for 1 h with gentle shaking, samples were then filtered and washed three times using a presoaked 96-well glass fiber filtration plate (Millipore, Billerica, MA). The filter was then dried and transferred to a scintillation vial with 2 mL of scintillation fluid and counted on a Beta liquid scintillation counter. Non-specific binding was determined by adding 5 μM of JTE-013.

2.4. In vitro saturation autoradiography analysis

For saturation autoradiography analysis, 14 μm fresh frozen tissue sections were prewarmed and pre-incubated with HBSS buffer containing 10 mM HEPES, 5 mM MgCl2, 1 mM CaCl2, and 0.5 % BSA, and 0.1 mM EDTA at pH 7.4 for 5 min. The sections were then incubated with a serial dilution of [11C]TZ34125 ranging from 0.06 to 50 nM for 30 min, and then washed, and air dried with a blower. Dried slides were incubated with a BAS-IP MS Storage Phosphor Screen (Cytiva, Marlborough, MA) overnight. Autoradiography signal was then visualized using a Typhoon FLA 7000 laser scanner (Fiji Photo Film, Tokyo, Japan). Images were then quantified using Multi Gauge v3.0 software (Fiji Photo Film, Tokyo, Japan). The non-specific binding was determined by incubation in the presence of 10 μM JTE-013.

2.5. STZ-induced type 1 diabetic mouse model

Type 1 diabetic mouse model was used as previously described [35]. In brief, induction of type 1 diabetic mice was achieved by intraperitoneal (i.p.) injection of a single high dose of STZ at 200 mg/kg at Day 1. STZ (Sigma-Aldrich, St Louis, MO) was freshly prepared by dissolving STZ in 50 mM sodium citrate buffer pH 4.5 for a final concentration of 10 mg/mL and was used immediately. 200 mg/kg of STZ in sodium citrate was injected for the study group, whereas the control group received an equal volume of sodium citrate buffer i.p. Mice were returned to the cages with normal food and 10 % sterilized sucrose water. On experimental day 3, 10 % sucrose water was switched back to regular water. On the day of STZ injection, and experimental day 3 and day 6 after STZ injection, the mouse body weight and fasting blood glucose levels were measured. All mice were fasted for 6 h (approximately from 7 a.m. to 1 p.m. each time), blood glucose level was tested from the tail vein with a One Touch Basic blood glucose monitoring system to monitor hyperglycemia in STZ-induced mice.

2.6. Ex vivo biodistribution study of [11C]TZ34125 in control and STZ-treated mice

To determine the expression of S1PR2 and evaluate the uptake of [11C]TZ34125 in response to STZ-induced diabetes, we performed biodistribution study in normal and STZ-treated C57BL/6 mice as previously described with minor modifications [36]. In brief, after STZ-induced diabetes was established, approximately 200 MBq/100 μL of [11C]TZ34125 was administrated to the mice intravenously via the tail vein. At 30 min post injection, animals were euthanized and tissues of interest including blood, lung, liver, spleen, kidney, muscle, heart, brain, thyroid, pancreas, and thymus were collected, weighted, and counted in a Beckman 8000 automated gamma counter (Beckman, Brea, CA). The uptake of [11C]TZ34125 in each tissue was expressed as background and decay-corrected percent injected dose per gram of tissue (%ID/g).

2.7. In vitro autoradiography study of [11C]TZ34125 in normal and STZ- induced diabetic mice

To compare the uptake of [11C]TZ34125 in spleen and pancreas of STZ-induced diabetic mice and control mice, we next performed an in vitro autoradiography study using the fresh-frozen tissue sections from normal and STZ-treated mice. Fourteen micron sections from snap-frozen spleen and pancreas were prepared using a Leica 1860 Cryostat and stored at − 80 °C until use. All sections were pre-warmed to room temperature briefly and pre-incubated in the HBSS buffer containing 10 mM HEPES, 5 mM MgCl2, 0.2 % BSA, and 0.1 mM EDTA at pH 7.4 for 5 min. All sections were then incubated with [11C]TZ34125 at ~370 kBq/ mL of buffer for 45 min with gentle shaking, washed with buffer for 5 min for three times, and dipped in ice-cold H2O for 1 min and dried gently with a blower. Dried slides were incubated with a BAS-IP MS Storage Phosphor Screen (Cytiva, Marlborough, MA) overnight at −20 °C in a Hypercassette autoradiography cassette. To determine non- specific binding, 5 μM of JTE-013 (TOCRIS, Bristol, UK) was added to the buffer during pre-incubation and incubation with [11C]TZ34125. Autoradiography signal was detected using a Typhoon FLA 7000 phosphor imaging system (GE Healthcare, Chicago, IL) and measured using the MultiGauge software program (Fujifilm, Tokyo, Japan). The data were background-corrected, and specific binding was determined by subtracting the signal of tissues with JTE-013 from the tissues with [11C] TZ34125 alone.

2.8. Immunohistochemistry of S1PR2 in fresh frozen tissues

To confirm the distribution of [11C]TZ34125 in mouse tissues, immunohistochemistry of S1PR2 was performed in fresh-frozen mouse spleen and pancreas tissues. Fourteen micron sections from fresh frozen tissues were prepared and used. All sections were pre-warmed at room temperature and fixed with 4 % paraformaldehyde in PBS for 10 min and then washed with PBS three times. Sections were then incubated with ReadyProbes Endogenous HRP and AP Blocking Solution (ThermoFisher, Waltham, MA) for 10 min followed by blocking with 5 % horse serum for 2 h. All sections were then incubated with anti-S1PR2 antibody (Novus Biologicals, Littleton, CO) at 4 °C overnight and then washed in PBS three times. Sections were then incubated with HRP conjugated goat anti-rabbit IgG antibody for 1 h and developed using ImmPACT DAB (Vector Laboratories, Burlingame, CA). Slides were scanned automatically using a Hamamatsu NanoZoomer slide scanning system (Hamamatsu, Shizuoka, Japan).

2.9. Statistical analysis

All data were analyzed with Prism 9.1 (GraphPad Software, San Diego, CA). For body weight and blood glucose level, multiple t-tests were performed. For biodistribution study, two-way ANOVA followed by Fisher’s LSD multiple comparisons was used. For autoradiography study, the student t-test was used. A P value ≤0.05 was considered to be statistically significant.

3. Result

3.1. Radioligand preparation

Radiosynthesis of [11C]TZ34125 was carried out as described before with minor modifications (Fig. 1B) [30]. For each batch, the radiochemical yield was ~16 % (decay corrected to the end of bombardment, EOB). The radiochemical purity, chemical purity, and specific activity were > 99 %, > 95 %, and > 236 GBq/μmol (EOB) respectively.

3.2. In vitro characterization of [11C]TZ34125

We have previously reported that TZ34125 was a potent and selective S1PR2 ligand with an IC50 of 9.52 nM for S1PR2 and > 1000 nM for S1PR1 and S1PR3 using competitive binding assay with fresh-made [32P]S1P [30]. In this study, we performed a direct in vitro characterization of [11C]TZ34125 using a saturation binding assay with recombinant human S1PR2 membranes. Our data showed [11C]TZ34125 had a Kd of 0.9 nM, further indicating [11C]TZ34125 is highly potent to S1PR2 (Fig. 2A). Furthermore, we also performed a saturation autoradiographic analysis in tissue sections from mouse kidney, spleen, and liver. [11C]TZ34125 had Kd values of 67.5 nM, 45.9 nM, and 25.0 nM in mouse kidney, spleen, and liver respectively, which was slightly higher than ligand-membrane binding assay possibly due to the species difference. Our data indicated [11C]TZ34125 is a potent S1PR2 radioligand. Interestingly, the uptake of [11C]TZ34125 was much higher in the liver than kidney and spleen, indicating the expression of S1PR2 was higher in the mouse liver than kidney and spleen (Fig. 2B-E). We then determined the specificity of [11C]TZ34125 by blocking with well-validated S1PR2 specific antagonist JTE-013 and comparing the autoradiography analysis with immunohistochemistry analysis using anti-S1PR2 antibody. Our results showed [11C]TZ34125 was able to be blocked by JTE-013 in mouse tissues and the distribution of [11C]TZ34125 matched well with anti-S1PR2 antibody in the mouse spleen and pancreas. In particular, [11C]TZ34125 and S1PR2 were mainly distributed in the red pulp of spleen (Fig. 2F). Overall, these data demonstrate [11C]TZ34125 is a potent and S1PR2 specific radioligand.

Fig. 2.

Fig. 2.

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In vitro saturation binding and autoradiography characterization of [11C]TZ34125. (A) Saturation binding assay showed [11C]TZ34125 was potent for recombinant human S1PR2 membrane with a Kd of 0.9 nM; (B–D) Saturation autoradiographic analysis showed Kd values of 67.5 nM, 58.9 and 25.0 nM of [11C] TZ34125 in the mouse kidney (B), spleen (C), and liver (D); (E) Representative images of saturation autoradiography of [11C]TZ34125 in kidney, spleen, and liver; (F) [11C]TZ34125 was able to be blocked by S1PR2 specific antagonist JTE-013 and the distrubtion of [11C]TZ34125 matched well with immunohistology analysis in mouse spleen and pancreases.

3.3. STZ-induced diabetic mouse models

A single high dose of STZ at 200 mg/kg in sodium citrate buffer was administrated in 10 week old C57BL/6 mice (Fig. 1C). On day 0, right before the injection of STZ, the body weight of control mice (22.10 ± 1.26 g) was almost identical to the STZ-treated mice (21.63 ± 1.14 g) with a P-value of 0.559; the fasting blood glucose level of the control group (288.25 ± 44.37 mg/dL) was also comparable to the STZ-treated group (251.50 ± 36.77 mg/dL) with a P-value of 0.190. At day 3 after the injection of STZ, a decrease in the body weight of STZ-treated mice (18.75 ± 1.20 g) was identified compared with control mice (22.10 ± 1.11 g) with a P-value of 0.002; whereas a dramatic increase in the fasting blood glucose level of the STZ group (612.83 ± 119.04 mg/dL) was observed compared with the control group (269.00 ± 59.20 mg/dL) with a P-value of <0.001. On day 6 after injection of STZ, the body weight of STZ-treated mice (17.52 ± 1.46 g) kept decreasing whereas the body weight of control mice remain identical (22.25 ± 0.91 g) to the beginning of the study with a P-value of <0.001 between the two groups; the fasting blood glucose level in the STZ treated group (639.17 ± 60.23 mg/dL) was even higher compared the value at day 3, while the control group showed a slight decrease (190.75 ± 28.09 mg/dL) with a P-value <0.001 between the two groups (Fig. 3). Overall, a slight decrease in body weight and a dramatic increase in fasting blood glucose level in the STZ-treated group indicate the STZ-induced diabetic mouse model was successfully established.

Fig. 3.

Fig. 3.

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Whole body weight and blood glucose level of normal and STZ-induced diabetic mice. (A) STZ treated mice showed an identical body weight at beginning of the study and 15.2 % and 21.3 % reduction on Day 3 (P = 0.002) and Day 6 (P = 0.0004); (B) The blood glucose level in STZ treated mice was identical to normal control beginning of the study, and then increased to 2.28 and 3.35 fold to control mice at Day 3 (P = 0.001) and Day 6 (P < 0.000). Data represent mean ± SD, n = 4– 6, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

3.4. Biodistribution study of [11C]TZ34125 in normal and STZ-induced diabetic mice

After the STZ-induced diabetic mouse model was established, biodistribution of [11C]TZ34125 was performed in normal control mice and STZ-induced diabetic mice (Fig. 4). In general, at 30 min post injection, the uptake of [11C]TZ34125 was high in lung, liver, spleen, kidney, and pancreas with %ID/g values of 3.79, 10.97, 2.57, 6.73, and 4.28 respectively; whereas the uptake of [11C]TZ34125 was low in muscle, brain, thyroid, and thymus with %ID/g value of 1.32, 0.33, 1.67, and 1.87 respectively. Interestingly, the uptake of [11C]TZ34125 was significantly different in STZ-induced diabetic mice compared with normal mice (Two-way ANOVA: F(1, 77) = 11.38, P = 0.0012). In particular, the uptake of [11C]TZ34125 significantly increased in the spleen and pancreases of STZ-treated mice with %ID/g values of 5.01 ± 0.77 and 2.57 ± 0.33 compared with %ID values of 5.84 ± 0.09 and 4.28 ± 0.46 in the normal control mice. The increased [11C]TZ34125 uptake in the STZ-treated mice was ~2 fold higher in the spleen and ~ 1.4 fold higher in the pancreas with a P-value of <0.0001 and 0.0078 respectively. An increase of [11C]TZ34125 was also found in the muscle of diabetic mice (~1.8 fold increase) but with no statistical difference. In addition, for practical imaging of pancreatic β cells, the accumulation ratio to the surrounding pancreatic organs is important. A high radioactive uptake ratio of endocrine-to-exocrine pancreas and a high radioactive uptake ratio of pancreas-to-surrounding organs are necessary to detec and quantify specific signal in the pancreas [37]. Based on the uptake of [11C]TZ34125 in our biodistribution study, an increase of pancreas to surrounding organs was observed. Student t-test showed a significant increase of pancreas to liver ratio in STZ-treated mice compare to normal control mice(Fig. 4B).

Fig. 4.

Fig. 4.

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Ex vivo biodistribution study of [11C] TZ34125 in normal and STZ-induced diabetic mice. (A) Two-way ANOVA analysis showed the uptake of [11C]TZ34125 in STZ-treated mice was significantly different from normal control mice (F(1,77) = 11.38, P = 0.001). In particular, Fisher’s LSD test followed by ANOVA showed the uptake of [11C]TZ34125 was significantly increased in the spleen (~ 2-fold, P ≤ 0.0001) and pancreases (~ 1.4 fold, P = 0.0078) of STZ-treated mice compared with normal control mice. (B) Ratio of [11C] TZ34125 in pancreas to liver showed a statistically significant increase in STZ-treated mice. Data represent mean ± SEM, n = 4– 6, * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001.

3.5. Autoradiography study of [11C]TZ34125 in normal and STZ-induced diabetic mice

To further compare the uptake of [11C]TZ34125 in the spleen and pancreas of STZ-induced diabetic mice and control mice, we next performed in vitro autoradiography in fresh frozen mouse spleen and pancreas sections (Fig. 5). In general, [11C]TZ34125 was mainly distributed in the white pulp of the spleen with a relatively lower level in the red pulp of spleen. Blocking study with S1PR2 specific antagonist JTE-013 significantly reduced the signal of [11C]TZ34125 indicating [11C]TZ34125 is specific to S1PR2. In addition, the distribution of [11C] TZ34125 matched well with immunohistochemistry analysis of S1PR2, further confirming the specificity of [11C]TZ34125 toward S1PR2. Similar to the biodistribution study, compared to the normal control, the uptake of [11C]TZ34125 in the spleen and pancreas of STZ-induced diabetic mice showed a significant increase, indicating the active S1PR2 was elevated by STZ induction. The level of [11C]TZ34125 increased ~1.4 fold and ~ 1.6 fold in the spleen and pancreas of STZ- treated mice with a P value of 0.042 and 0.029 respectively.

Fig. 5.

Fig. 5.

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In vitro autoradiography study of [11C]TZ34125 in the spleen and pancreas of normal and STZ-induced diabetic mice. (A) The binding of [11C]TZ34125 was significantly higher in the spleen and pancreas of STZ-induced diabetic mice compared to the normal control; (B) student t-test showed a significant increase in the spleen (~1.4 fold, P = 0.0420) and pancreases (~1.6 fold, P = 0.0285) of STZ mice. Data represent mean ± SD, n = 5, * P ≤ 0.05.

4. Discussion

S1P-S1PR2 pathways crucial role in metabolic processes. For example, S1P-S1PR2 can induce epithelial-mesenchymal transition changes through Rho kinase activation in renal tubule [38]. Similarly, S1P-S1PR2 can also activate Rho kinase and lead portal hypertension [39]. Conjugated bile acids can activate the protein kinase B and extracellular signal-regulated kinase 1 and 2 (ERK1/2) via S1PR2 in hepatocytes and cholangiocarcinoma cells and mediate obstructive cholestasis in liver [40]. S1PR2 expression is also increased in advanced fibrotic human liver [41]. In the present study, we confirm the important role of S1PR2 in metabolic disorder by characterization of S1PR2 specific radioligand, [11C]TZ34125, in a STZ-induced diabetic mouse model. Our results showed increased uptake of [11C]TZ34125 in the spleen and pancreas of STZ-induced diabetic mice. These results are consistent with previous studies [42] and show that S1PR2 is a promising diagnostic and therapeutic target for diabetes.

We previously reported our efforts on S1PR2 ligand development and radiosynthesis of potent and selective S1PR2 radiotracers and in vivo evaluation for S1PR2 expression using noninvasive PET imaging [30–32]. Our lead radioligand [11C]TZ34125 shares similar core structures with the well-known S1PR2 antagonist, JTE-013. We previously used competitive binding assay against [32P]S1P in human S1PR membrane and demonstrated TZ34125 has high potency (IC50 = 9.52 nM) and specificity to S1PR2 (IC50 > 1000 nM to other S1PRs) [30]. In the present study, we performed saturation binding assay using [11C] TZ34125 on recombinant human S1PR2 membrane and mouse kidney and liver. Similar to competitive binding assay in human S1PR2 membrane, the saturation binding assay showed [11C]TZ34125 is very potent with a Kd of 0.9 nM. Additionally, autoradiographic saturation binding analysis in mouse tissues also confirmed [11C]TZ34125 is a potent S1PR2 radioligand, though the Kd obtained from autoradiographic analysis of the direct binding in mouse tissues was slightly higher than in recombinant human S1PR2 membrane, possibly due to the different sensitivities between the two assays and/or differential binding affinities between species. Overall, our in vitro evaluation showed [11C] TZ34125 is a potent radioligand toward S1PR2.

In order to explore the potential biological application of [11C] TZ34125, we next evaluated the uptake of [11C]TZ34125 in a STZ-induced diabetic mouse model using ex vivo biodistribution, and in vitro autoradiographic study. As stated above, S1PR2 has been associated with several metabolic diseases and is recognized as a promising diagnostic and therapeutic target in these diseases. In diabetes, administration of JTE-013 or knockout of S1PR2 can effectively preserve pancreatic β cells and improve glucose/insulin tolerance in high-fat diet induced and STZ-induced diabetic mouse models [28,43]. Additionally, the expression of S1PR2 is directly elevated in the spleen of STZ-induced diabetic mice [42]. S1P can activate JNK and inhibit Akt signaling through S1PR2 in pancreatic β cells and counteracts insulin signaling [44]. Consistent with these studies, we observed an increase of [11C] TZ34125 uptake in the pancreas of STZ-induced diabetic mice. It is notable that β cells could be heavily destroyed in our model, the increase of S1PR2 in response to STZ treatment and as a result of β cell loss may indicate a protective or compensatory role of S1PR2 in response to β cell damage and disruption of insulin signaling, further investigating the molecular mechanisms of S1PR2 in insulin signaling will advance our understanding the role of S1PR2 in β cell damage and insulin signaling. Nevertheless, our data indicates [11C]TZ34125 is a promising S1PR2 radiotracer and is suitable for quantitative assessment of S1PR2 expression in the pancreas of the diabetic mouse. Although the high expression of S1PR2 in abdominal tissues may pose challenges for PET measurement of S1PR2 expression in mouse pancreas due to the small size of the mouse pancreas and the sensitivy of microPET imaging, PET with [11C]TZ34125 for the detection of S1PR2 elevation in diabetes is possible in large animal models such as nonhuman primates and in human subjects. One of the limitations of this study is we only tested the uptake of [11C]TZ34125 at the late stage of β cell damage following treatment of mice with a high dose of STZ. Multiple low doses of STZ, high-fat diet combined with STZ treatment, and early events following STZ treatment were not tested. S1PR2 is likely involved in various aspects of insulin signaling and pancreatic β cell function, thus changes of S1PR2 in response to β cell damage is expected. Future studies using different diabetic models and evaluation of changes of [11C]TZ34125 and S1PR2 expression over time in response to the STZ treatment will help to understand the precise role of S1PR2 in insulin signaling in pancreas.

In addition to the pancreas, an elevated uptake of [11C]TZ34125 was also observed in the spleen of diabetic mice compared to control mice. In fact, previous studies demonstrate S1PR2 plays important roles in inflammation. During inflammation, the endothelial S1PR2 becomes activated with an increase in endothelial permeability to promote the innate immune response [45]. Others have reported that S1PR2 knockout mice or administration of JTE-013 exhibited a reduced vascular permeability in spleen and reduced LPS-induced inflammation [45]. In STZ-induced diabetic mice, though the cytotoxic effects of STZ are quite selective to the destruction of pancreatic islet β cells, recent studies showed STZ can cause lymphopenia and can be toxic to other organs [46,47]. STZ can increase oxidative stress, inflammation, and endothelial dysfunction [48]. In particular, STZ treatment led to a significant loss of spleen weight, diffused white pulp structure, reduced mature lymphocytes, downregulated circulatory lymphocytes, and upregulation of reactive oxygen species [49]. In our case, a significant increase of [11C]TZ34125 uptake was observed in the STZ induced diabetic mice indicating an upregulation of S1PR2 in spleen in response to STZ treatment. This is likely due to the acute inflammatory response caused by STZ treatment which further induced endothelial S1PR2 expression in the spleen. While the exact mechanism remains to be illustrated with further investigation, our current finding confirmed the critical role of S1PR2 in inflammation. Our S1PR2 specific radioligand [11C]TZ34125 has great potential for the quantitative assessment of inflammatory S1PR2 induction.

5. Conclusion

We characterized the expression of S1PR2 in a STZ-induced diabetic mouse model using our well-validated S1PR2 specific radioligand [11C] TZ34125. Our data demonstrated that STZ-induced S1PR2 elevation in mouse spleen and pancreas can be detected by [11C]TZ34125. Pancreatic S1PR2 is a potential marker for the diagnosis of diabetes, and splenic S1PR2 could play an important role for inflammatory response. Future in vivo evaluation using non-invasive PET imaging of [11C] TZ34125 in larger animal models is worthwhile. [11C]TZ34125 is a promising S1PR2 radioligand and has great potential for preclinical and clinical usage for the assessment of S1PR2 in vivo.

Acknowledgment

This work is supported by the National Institutes of Health under projects NS103988 and EB025815. We would like to acknowledge Robert Dennett and Michael Nickels at the Washington University Cyclotron Facility. We especially would like to acknowledge Lynne Jones at Mallinckrodt Institute of Radiology and Nicole Fettig and Margaret Morris at the Mallinckrodt Institute of Radiology Preclinical Imaging Facility for their technical assistance.

Footnotes

Ethics statement

The animal experiments were carried out in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals under a protocol reviewed and approved by the Washing University Institutional Animal Care and Use Committee (IACUC).

Declaration of competing interest

The authors declare no competing financial interest.

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