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  • o-Phenanthroline br Materials and methods br Results br Disc


    Materials and methods
    Discussion Previously published in vitro and in vivo studies have proposed that PRMT3 is a coactivator for LXR that modulates LXR's lipogenic but not its function in cholesterol metabolism [9,10]. Here we show that chronic treatment of hyperlipidemic apoE knockout mice with the PRMT3 inhibitor SGC707 reduced the hepatic steatosis extent, while atherosclerotic lesion development was not negatively affected. This finding provides further evidence that through PRMT3 inhibition the LXR-driven (pathological) effects on fatty o-Phenanthroline and cholesterol metabolism can be uncoupled. As we expected based on previously published work [9,10] and the observation that plasma cholesterol levels, blood leukocyte numbers and the extent of in vivo foam cell formation are not changed by SGC707 treatment, atherosclerotic lesion susceptibility was not modified by pharmacological inhibition of PRMT3 activity. Interestingly, PRMT3 has recently emerged as a putative novel target in the context of atherosclerosis and cardiovascular disease in humans. Chen et al. detected a 2-fold higher mRNA expression of PRMT3 in myocardial tissue from patients suffering from cardiovascular disease as compared to that from subjects without atherosclerotic vessel disease [27]. In addition, human association studies by Shendre et al. have linked variation in the PRMT3 gene to a change in common carotid intima-media thickness (cCIMT), a measure of subclinical atherosclerosis [28]. Shendre et al. furthermore showed that the local European ancestry region in the PRMT3 gene is associated with a ~2 times higher stroke risk in African Americans [28]. This observational data suggest a role for PRMT3 in atherosclerosis. It is noteworthy that besides its proposed function as nuclear cofactor, PRMT3 belongs to the family of protein arginine N-methyltransferase enzymes that transfer methyl groups to the arginine residues of histones and other proteins to produce asymmetric dimethylarginine (ADMA) and, to a minor extent, symmetric dimethylarginine (SDMA). ADMA is a potent inhibitor of the formation of the anti-atherogenic agent nitric oxide [29]. Accordingly, ADMA increases atherosclerosis susceptibility in wild-type and apoE knockout mice [30,31]. In support of a negative impact of ADMA on atherosclerosis outcome in humans, high plasma ADMA levels serve as an independent predictor of high IMT levels in a variety of clinical settings [32,33]. Moreover, subjects in the highest quartile for plasma ADMA levels display a 3.9-fold increased risk for acute coronary events [34]. From our current study it can be concluded that complete inhibition of PRMT3 activity does not affect ADMA functionality in the cardiovascular context as we did not measure an effect on atherosclerosis outcome. It should however, be taken into account that we administered a dose of the PRMT3 inhibitor at which we aimed to be able to measure an effect on hepatic triglyceride accumulation. Although the hepatic steatosis extent was effectively lowered, it is currently unclear whether the dose of 10 mg kg−1 SGC707 is also sufficiently high to interfere with the ADMA/nitric oxide system. We did not detect an effect on systemic/plasma ADMA levels (data not shown). However, this does not necessarily exclude a potential (undetermined) impact of the chronic SGC707 treatment at an individual cellular level, i.e. in the aortic vessel wall. Further dedicated research, i.e. on vessel wall reactivity, may provide better insight in the potential of SGC707 to functionally modulate the body's ADMA/nitric oxide status and uncover the relevance in the cardiovascular disease setting, especially also since redundancy between the different PRMT family members has been suggested [35]. An interesting observation during our studies was that SGC707-treated mice, in marked contrast to control-treated mice, did not gain additional weight during the six week Western-type diet feeding period. Brown adipose tissue has emerged as a potential target to combat obesity due to its ability to burn fatty acids to produce heat, thereby eliminating storage of fat in white adipose tissue [36,37]. Since we did not primarily intend to investigate the effect of chronic SGC707 exposure on obesity development, a full metabolic profiling of the mice is unfortunately lacking. As such, we do not known the exact food intake nor the extent of heat produced by brown adipose tissue in the two groups of mice. Previous studies by Chen et al. have shown that exposure of C3H10T1/2 murine mesenchymal progenitor cells to the PRMT3 inhibitor compound 14u does not affect the ability of these cells to differentiate into brown adipocytes in vitro [38]. However, we observed a striking increase in browning of the subcutaneous white adipose tissue depot, which was paralleled by a marked increase in white adipose tissue UCP1 transcript levels. The decreased body weight gain can thus theoretically be the result of a higher heat production instead of fatty acid storage in white adipose tissue. However, dedicated in vivo studies are clearly warranted to decipher the exact function of PRMT3 in obesity development. The lower final body weight in SGC707-treated mice was also paralleled by a small change in the relative number of adipocytes in gonadal white adipose tissue. Strikingly, the change in gonadal white adipose tissue phenotype did not coincide with a decrease in the expression levels of the lipogenic genes SREBP-1c, FAS, ACC1, and SCD1, as we would have expected from our previous findings regarding PRMT3 functionality in liver [10]. The mRNA expression levels of LPL and CD36 were – however – lowered in gonadal white adipose tissue after SGC707 treatment. Adipose tissue-specific LPL deficiency does not change the obesogenic potential of mice [39]. In contrast, CD36 total body knockout mice as compared to their wild-type counterparts exhibit reduced adiposity in response to a high fat diet challenge, which appears to be driven by a reduction in the adipocyte uptake of lipoprotein-derived fatty acids and the associated reduction in white adipose tissue weight [40,41]. From these combined findings, it can be hypothesized that (1) the lipogenesis regulatory function of PRMT3 is perhaps restricted to hepatocytes and (2) that SGC707 treatment may actually decrease obesity development by lowering adipose tissue fatty acid uptake and switching white adipocytes to a more brown phenotype.