nce in cells overloaded with glucose. 12 March 2011 | Volume 6 | Issue 3 | e17674 Supporting Information HK Localization and Glucose Fate of endogenous HKI and HKII decrease glucose clearance to similar EPA ethyl ester site extend. This quantitative analysis also shows that cell culture in the absence of glucose 
causes a slight increase in HK levels. This is important when considering the effect of glucose removal on glucose utilization. Indeed, we have shown that incubation in the absence of glucose decreases glucose clearance and we hypothesized that this effect was related to HK translocation away from mitochondria. Our quantitative analysis supports this hypothesis and shows that the effect on glucose clearance is not due to a decrease in HK level. Finally, we show, as expected, that over expression of HKs is accompanied by an increased level of intracellular HK. Thus, the decrease in glucose clearance observed with HKII over expression may be related to increased level of the enzyme in the cytoplasm, favoring glycogen synthesis. Author Contributions Conceived and designed the experiments: SJ JNW BR. Performed the experiments: SJ BR. Analyzed the data: SJ BR. Wrote the paper: SJ JNW BR. 13 March 2011 | Volume 6 | Issue 3 | e17674 Allele-Specific, Age-Dependent and BMI-Associated DNA Methylation of Human MCHR1 Stefanie Stepanow1, Kathrin Reichwald1, 10542155 Klaus Huse1, Ulrike Gausmann1, Almut Nebel2, Philip Rosenstiel2, Martin Wabitsch3, Pamela Fischer-Posovszky3, Matthias Platzer1 1 Genome Analysis, Leibniz Institute for Age Research Fritz Lipmann Institute, Jena, Germany, 2 Institute of Clinical Molecular Biology, Christian-Albrechts-University, Kiel, Germany, 3 Department 19296653 of Pediatrics and Adolescent Medicine, University of Ulm, Ulm, Germany Abstract Background: Melanin-concentrating hormone receptor 1 plays a significant role in regulation of energy balance, food intake, physical activity and body weight in humans and rodents. Several association studies for human obesity showed contrary results concerning the SNPs rs133072 and rs133073, which localize to the first exon of MCHR1. The variations constitute two main haplotypes. Both SNPs affect CpG dinucleotides, whereby each haplotype contains a potential methylation site at one of the two SNP positions. In addition, 15 CpGs in close vicinity of these SNPs constitute a weak CpG island. Here, we studied whether DNA methylation in this sequence context may contribute to population- and age-specific effects of MCHR1 alleles in obesity. Principal Findings: We analyzed DNA methylation of a 315 bp region of MCHR1 encompassing rs133072 and rs133073 and the CpG island in blood samples of 49 individuals by bisulfite sequencing. The AC haplotype shows a significantly higher methylation level than the GT haplotype. This allele-specific methylation is age-dependent. In young individuals the difference in DNA methylation between haplotypes is significant; whereas in individuals older than 60 years it is not detectable. Interestingly, the GT allele shows a decrease in methylation status with increasing BMI, whereas the methylation of the AC allele is not associated with this phenotype. Heterozygous lymphoblastoid cell lines show the same pattern of allele-specific DNA methylation. The cell line, which exhibits the highest difference in methylation levels between both haplotypes, also shows allele-specific transcription of MCHR1, which can be abolished by treatment with the DNA methylase inhibitor 5-aza-29-deoxycytidine. C