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

Diabetes is associated with an increased risk of atherosclerotic cardiovascular disease (CVD), and it is common in patients with known or at risk of CVD [1]. Various factors, including increased oxidative stress and subsequent activation of pro-inflammatory pathways, as well as increased cell proliferation in the arterial wall, contribute to accelerated atherosclerosis in diabetes [2]. Inflammation is a key factor in the pathogenesis of atherosclerosis and its complications [3‐5]. Positron emission tomography (PET) with a glucose analogue 2-deoxy-2-[¹⁸F]-fluoro-d-glucose (¹⁸F-FDG) is a feasible method for non-invasive imaging of atherosclerotic plaque inflammation in large arteries. Vascular uptake of ¹⁸F-FDG has also been observed in individuals without symptoms of CVD, in the presence of type 2 diabetes mellitus (T2DM) [6, 7], metabolic syndrome [8] and obesity [9]. Accumulation of ¹⁸F-FDG in atherosclerotic plaques has been attributed to high glucose uptake in macrophages [4, 10]. In addition to the number of macrophages, ¹⁸F-FDG uptake may reflect their polarization [11, 12] or augmentation of glucose uptake induced by hypoxia [13, 14]. Furthermore, high blood glucose levels in T2DM can diminish ¹⁸F-FDG uptake in atherosclerosis [7]. These factors may complicate the interpretation of ¹⁸F-FDG signal from arteries, especially in diabetic individuals, and therefore, alternative tracers for the quantification of inflammation in atherosclerosis have been investigated [15‐17].

Choline is taken up into cells by choline transporters, phosphorylated by choline kinase, further metabolized to phosphatidylcholine, and eventually incorporated into the cell membrane or used for lipoprotein assembly. Radiolabeled derivatives of choline have been utilized to detect increased choline uptake in tumor cells [18, 19] and in macrophages at the sites of inflammation [20, 21]. Previous studies have shown increased accumulation of radiolabeled choline derivatives in macrophage-rich atherosclerotic lesions [22, 23] or aortic aneurysms [24]. In asymptomatic patients, ¹¹C-choline or ¹⁸F-fluoromethylcholine (¹⁸F-FMCH) uptake has been detected in large vessels, predominantly in non-calcified atherosclerotic lesions [25, 26]. We hypothesized that the pro-inflammatory phenotype in T2DM is associated with increased choline uptake, thus making ¹⁸F-FMCH a potential tracer for monitoring the progression of atherosclerosis in diabetes.

For this study, we evaluated the uptake of ¹⁸F-FMCH and ¹⁸F-FDG in inflamed atherosclerotic plaques in non-diabetic and diabetic hypercholesterolemic mice [27]. We studied the kinetics and biodistribution of ¹⁸F-FMCH by PET/computed tomography (CT) imaging and in ex vivo tissue samples. Uptake of ¹⁸F-FMCH or ¹⁸F-FDG in atherosclerotic aorta was first quantified by gamma counting and then compared between plaques and adjacent normal vessel wall by autoradiography of aortic tissue sections. Furthermore, ¹⁸F-FMCH uptake was compared with the extent and phenotype of macrophages in atherosclerotic plaques, the plasma levels of inflammatory mediators and metabolic markers.

Total cholesterol levels were similar in non-diabetic (LDLR^−/−ApoB^100/100) and diabetic (IGF-II/LDLR^−/−ApoB^100/100) mice (31 ± 4.3 and 36 ± 12 mmol/l, respectively), as were also the fasting glucose levels (7.8 ± 1.0 and 9.1 ± 3.1 mmol/l, respectively). There was a tendency towards higher blood levels of insulin, C-peptide and IL-6 in diabetic mice (Additional file 1: Table S3). The total cholesterol was significantly lower in C57BL/6N control than in the hypercholesterolemic mice (1.8 ± 0.3, p < 0.001).

The aortas of both non-diabetic and diabetic hypercholesterolemic mice showed extensive atherosclerosis, mainly fibroatheroma-type lesions with large, confluent areas of Mac-3-positive macrophages, whereas no atherosclerosis was observed in control mice (Fig. 1). All plaques showed positivity for iNOS and MRC-1, indicating M1 and M2 polarized macrophages, respectively. The scavenger receptor CD36 co-localized with the macrophage-positive areas of plaques, while only occasional Ki-67 positive cells were observed. Occasional plaque calcifications were seen in 60 % of non-diabetic and 73 % of diabetic mice. The plaques of non-diabetic and diabetic mice showed no difference in any of the measured histological characteristics (Table 2).

The uptake of the PET tracer ¹⁸F-FMCH is increased in the atherosclerotic aorta of mice with T2DM. The uptake in atherosclerotic plaques was associated with the amount of Mac-3 positive macrophages in plaques, as well as the plasma levels of total cholesterol, certain metabolic markers and cytokines. In comparison with ¹⁸F-FDG, ¹⁸F-FMCH provided similar or higher target-to-background ratios between atherosclerotic plaques and the normal vessel wall, as well as aorta and blood or myocardium in diabetic mice. These results indicate that T2DM is associated with increased uptake of choline in atherosclerosis and that ¹⁸F-FMCH may be useful for the evaluation of vascular inflammation in diabetes.

Studies using ¹⁸F-FDG PET have revealed subclinical vascular inflammation in patients with abnormal glucose metabolism [6‐8]. More recent studies have indicated contribution of pericardial and visceral fat to vascular inflammation [9, 33]. Molecular imaging of vascular inflammation is a potential tool to study mechanisms of atherosclerosis, effects of therapies and eventually risk of CVD in high-risk individuals, such as those with diabetes [4].

The LDLR^−/−ApoB^100/100 mouse model is a well characterized and widely utilized model of hypercholesterolemia and atherosclerosis. Abnormalities in apolipoprotein E (ApoE) rarely contribute to human hypercholesterolemia, and therefore, the LDLR^−/− mouse model better mimics the human disease. The addition of the gene modification leading to the expression of only apolipoprotein B100 on the LDLR^−/− background further raises the cholesterol level and amplifies the progression of atherosclerosis [28]. Despite the only mildly elevated fasting glucose levels, the IGF-II/LDLR^−/−ApoB^100/100 mice overexpressing IGF-II in pancreatic beta cells represent the insulin resistance and impaired glucose tolerance that is consistent with the T2DM phenotype. Although their lipid profile is similar to that of the LDLR^−/−ApoB^100/100, the diabetic mice represent accelerated development of macrovascular complications, including more calcified, less organized and more complex lesions with higher IL-6 expression [27, 34]. Our findings of elevated plasma insulin, C-peptide and IL-6 levels are in line with the T2DM phenotype. Taken together, these data suggest a more pro-inflammatory milieu in the diabetic IGF-II/LDLR^−/−ApoB^100/100 mice as compared with the non-diabetic LDLR^−/−ApoB^100/100 mice.

The ¹⁸F-FMCH uptake was increased in the atherosclerotic aorta of diabetic mice as compared with non-diabetic mice. In addition, the correlations between the ¹⁸F-FMCH plaque uptake and the plasma C-peptide, insulin and leptin levels support the association of the uptake to diabetic phenotype. In both diabetic and non-diabetic mice, the plaques represented similar amounts of M1 and M2 polarized macrophages and also scavenger receptor CD36. Therefore, the higher ¹⁸F-FMCH uptake in the inflamed plaques may reflect increased choline transport [23] and metabolism in the macrophages. Interestingly, ¹⁸F-FMCH seems to have an enhanced cellular intake in diabetic mice, since the tracer tends to be more cleared from the blood and less excreted in the urine at the 20-minute time point as compared with non-diabetic mice. The absolute uptake of ¹⁸F-FMCH was higher in the aortas of diabetic mice as compared with non-diabetic mice, as was also the aorta-to-blood uptake ratio due to lower remaining tracer in circulation. Because blood will always be included in the ROI due to the limited resolution of PET images, high aorta-to-blood ratio is a good property for the in vivo imaging of tracer uptake in atherosclerotic plaques.

In the current study, the plaque-to-wall uptake ratio of ¹⁸F-FMCH (on average 2.6) was within the same range as that of ¹¹C-choline (2.3) or ¹⁸F-fluorocholine (3.5) in previous studies in mice [22, 23]. In line with previous observations, we discovered that ¹⁸F-FMCH accumulated in the most inflamed plaques, as shown by high macrophage density. The plaque uptake of ¹⁸F-FMCH also showed correlation with different metabolic and inflammatory markers. Elevated levels of total cholesterol, PLTP, RANTES, IL-6, insulin and leptin, which showed correlation with ¹⁸F-FMCH uptake, have been linked with increased atherosclerosis [35‐42] thus lending support to the association between ¹⁸F-FMCH uptake and the risk of atherosclerosis. Recent reports, however, suggest that PLTP activity is inversely correlated with carotid artery disease and linked with PON-1 [43], and that PON-1 is regarded as an atheroprotective enzyme [44, 45].

In previous studies, arterial uptake of ¹⁸F-FDG or ¹¹C-choline did not typically co-localize with calcifications seen on CT supporting the concept that inflammation and calcification occur at different stages of atherosclerosis [4, 25]. Our findings with ¹⁸F-FMCH suggest the same pattern since vascular tracer uptake was lower in 9-month-old mice (% IA/g 1.7, plaque-to-wall ratio 2.2, see Additional file 1) than in 6-month-old mice (% IA/g 2.0, plaque-to-wall ratio 2.6), despite more extensive vascular calcification (calcifications in all animals at the age of 9 months vs. in 73 % of animals at the age of 6 months), but less macrophages.

Although ¹⁸F-FDG is the most commonly used tracer for atherosclerotic plaque imaging, the high physiological tracer uptake in the myocardium has limited its application for imaging coronary artery inflammation, although it is possible to reduce the uptake by means of metabolic interventions [4]. In this study, the myocardial uptake was lower for ¹⁸F-FMCH than for ¹⁸F-FDG, and, moreover, the myocardial uptake of ¹¹C-choline has been negligible in patients [25], thus promoting the feasibility of ¹⁸F-FMCH in coronary artery imaging. As compared with ¹⁸F-FDG, ¹⁸F-FMCH showed superior plaque-to-wall ratio, equal aorta-to-blood ratio and higher aorta-to-myocardium ratio in the diabetic mice, suggesting potential for monitoring atherosclerosis progression in diabetes.

There are some limitations in our study. The reasons for insulin resistance in our mouse model are not fully understood, the model does not represent the full phenotypic spectrum of T2DM in patients, and our findings need to be verified in clinical studies [46]. The feasibility of ¹⁸F-FMCH for detecting atherosclerotic plaque inflammation by in vivo PET imaging remains to be investigated because, in our study, only young IGF-II/LDLR^−/−ApoB^100/100 mice were imaged with PET/CT and it was not possible to visualize the small plaques existing at the young age. Taken together, our findings support the concept of utilizing choline-based radiopharmaceuticals in the imaging of inflammation in diabetic atherosclerosis, but further in vivo imaging studies in patients are needed to proof this.

The uptake of the radiolabeled choline derivative ¹⁸F-FMCH was increased by type 2 diabetes in the aortas of atherosclerotic mice and reflected inflammation in atherosclerotic plaques. Comparison with ¹⁸F-FDG indicates that ¹⁸F-FMCH is a potential tracer for monitoring the atherosclerotic vascular inflammation in diabetes.