Type 2 diabetes enhances arterial uptake of choline in atherosclerotic mice: an imaging study with positron emission tomography tracer 18F-fluoromethylcholine

Background Diabetes is a risk factor for atherosclerosis associated with oxidative stress, inflammation and cell proliferation. The purpose of this study was to evaluate arterial choline uptake and its relationship to atherosclerotic inflammation in diabetic and non-diabetic hypercholesterolemic mice. Methods Low-density lipoprotein-receptor deficient mice expressing only apolipoprotein B100, with or without type 2 diabetes caused by pancreatic overexpression of insulin-like growth factor II (IGF-II/LDLR−/−ApoB100/100 and LDLR−/−ApoB100/100) were studied. Distribution kinetics of choline analogue 18F-fluoromethylcholine (18F-FMCH) was assessed in vivo by positron emission tomography (PET) imaging. Then, aortic uptakes of 18F-FMCH and glucose analogue 18F-fluorodeoxyglucose (18F-FDG), were assessed ex vivo by gamma counting and autoradiography of tissue sections. The 18F-FMCH uptake in atherosclerotic plaques was further compared with macrophage infiltration and the plasma levels of cytokines and metabolic markers. Results The aortas of all hypercholesterolemic mice showed large, macrophage-rich atherosclerotic plaques. The plaque burden and densities of macrophage subtypes were similar in diabetic and non-diabetic animals. The blood clearance of 18F-FMCH was rapid. Both the absolute 18F-FMCH uptake in the aorta and the aorta-to-blood uptake ratio were higher in diabetic than in non-diabetic mice. In autoradiography, the highest 18F-FMCH uptake co-localized with macrophage-rich atherosclerotic plaques. 18F-FMCH uptake in plaques correlated with levels of total cholesterol, insulin, C-peptide and leptin. In comparison with 18F-FDG, 18F-FMCH provided similar or higher plaque-to-background ratios in diabetic mice. Conclusions Type 2 diabetes enhances the uptake of choline that reflects inflammation in atherosclerotic plaques in mice. PET tracer 18F-FMCH is a potential tool to study vascular inflammation associated with diabetes. Electronic supplementary material The online version of this article (doi:10.1186/s12933-016-0340-6) contains supplementary material, which is available to authorized users.


Additional animal experiments: uptake of 18 F-FMCH in more advanced atherosclerosis and in fasting state
An additional group of older atherosclerotic mice with diabetes (IGF-II/LDLR -/-ApoB 100/100 ) that were fed with a high-fat diet of a longer duration (age 8-10 months, high-fat diet 6 months, n=9, Supplemental Table   1) were studied to evaluate the 18 F-FMCH uptake in more advanced stages of atherosclerosis. The mice were studied for 18 F-FMCH biodistribution, autoradiography and histology in a similar manner as the other mice in the study. The results were compared with 6-month-old IGF-II/LDLR -/-ApoB 100/100 mice and with C57BL/6N controls (where applicable).
The effects of fasting were studied in a small cohort of LDLR -/-ApoB 100/100 mice (age 6-7 months, high-fat diet 4 months, n=3, Supplemental Table 1). The mice were fasted for 4 hours and studied for ex vivo biodistribution and biomarkers in the plasma as described in the main article.

Immunohistochemical stainings
Aortic root sections were immunostained for macrophages (Mac-3), M1 and M2 polarization markers (iNOS and MRC-1), and CD36. For the Mac-3 staining, the paraffin sections were first de-paraffinized and rehydrated. After washes and bovine serum albumin (BSA) blocking, the sections were incubated with primary antibody (Anti-Mac-3 M3/84, 1:500, BD Pharmingen) for one hour followed by peroxidase treatment and washes. Rat-on-Mouse HRP-polymer RT 517 kit (Biocare Medical, Concord, CA, USA) was utilized according to the manufacturer's instructions, followed by chromogen (DAB, Dako K3468) and counterstaining with Mayer's hematoxylin. Consecutive sections were stained for iNOS and MRC-1. The paraffin sections were de-paraffinized, rehydrated and pre-incubated in 10 mM citric acid. After washes and BSA blocking, the primary antibody (anti-iNOS ab15323, 1:200, or anti-MRC-1 ab64693, 1:500, Abcam, Cambridge, UK) was added and incubated for one hour. Secondary antibody (Dako EnVision anti-rabbit K4003) was added and incubated for 30 min after washing and H 2 O 2 treatment. The sections were again washed, followed by the addition of chromogen and counterstaining as described above. For CD36, the protocol was similar, except for the pre-incubation, which was performed by boiling in 10 mM tris-EDTA buffer. The primary antibody was anti-CD36 ab 80978, 1:500, (Abcam). The Ki-67 stainings were performed on longitudinally cut aortic cryosections. The sections were first fixed in 10 % formalin and pre-incubated in 10 mM boiling citric acid. After washes and blocking with 5 % goat serum in 3 % BSA, the primary antibody (monoclonal rat anti-mouse clone TEC-3, M7249, 1:1000, Dako) was incubated overnight. Secondary antibody (polyclonal rabbit anti-rat E0468, 1:200, Dako) was incubated for 30 minutes after washes and H 2 O 2 treatment, followed by tertiary antibody (EnVision anti-rabbit, K4003, Dako). The detection and counterstaining was performed similarly as described above.

In vivo stability of 18 F-FMCH
Intravenously administered 18 F-FMCH showed rapid in vivo radiotracer metabolism with no differences between the mouse strains. Twenty minutes after injection, 7.9 ± 0.78 % of plasma total radioactivity originated from the intact tracer (radio-HPLC retention time 4.0 min). In the urine, 40 ± 2.4 % of the radioactivity was excreted as intact tracer. The main radioactive metabolite in mice plasma was 18 F-betaine (retention time 2.9 min). Representative radio-HPLC chromatograms are presented in Supplemental Figure   1.

Fasting has no significant effect on 18 F-FMCH uptake or plasma markers
The mice that fasted for 4 hours before 18 F-FMCH injection showed very similar characteristics as the nonfasted mice of the same strain (Supplemental Table 1 and Table 1 of the main article). There were no differences in 18 F-FMCH uptake between different tissues (Supplemental Table 2 and Table 3 of the main article) or in the measured markers in the plasma, except for a subtle change in PLTP activity (Supplemental Table 3).

Lipoprotein fraction measurements
The lipoprotein distributions were determined from pooled plasma samples in IGF-II/LDLR -/-ApoB 100/100 , LDLR -/-ApoB 100/100 and C57BL/6N mice. The levels of cholesterol, triglycerides and phospholipids in very lowdensity lipoproteins (VLDL) and intermediate-and low-density lipoproteins (IDL-LDL) were higher in both of the atherosclerotic mouse models than in controls (Supplemental Figure 3). The lipoprotein profile was shifted towards higher levels of VLDL-associated cholesterol and phospholipids, and lower levels of these same lipids associated with IDL-LDL in IGF-II/LDLR -/-ApoB 100/100 mice as compared with LDLR -/-ApoB 100/100 mice. The high-density lipoprotein (HDL) associated cholesterol levels had no differences between strains, whereas the HDL-associated phospholipid levels tended to be lower in the IGF-II/LDLR -/-ApoB 100/100 mice.
The differences, however, were modest and measured from only one pool of plasma from each strain, so no direct conclusions can be made. No statistically significant differences were observed between fasted and non-fasted mice (results in the main article). 18 F-FMCH uptake in lungs was lower in aged IGF-II/LDLR -/-ApoB 100/100 mice as compared to 6month old mice of the same strain (results in the main article). NA=not analyzed. Total cholesterol (mmol/l) 31 ± 1.3 36 ± 3.7 1.8 ± 0.11 a 31 ± 3.9