Table 1.
Clinical characteristics of the obese patients.
Fig 1.
Expression of mARC1 and mARC2 in human adult and fetal liver.
A. mARC1 and mARC2 gene expression in human fetal (n = 14) and adult (n = 88) liver samples was determined by microarray analysis and presented as background corrected probe intensities. ***p<0.001. B. mARC1 and mARC2 protein expression in 8 fetal and 9 adult livers. Equal amounts of protein were analyzed for the expression of mARC1 (upper panel), mARC2 (middle panel). Specific bands for both proteins were identified at the level of approximately 35 kDa, an apparent molecular mass of mARC1 and 2. ERp29 was used as a loading control (lower panel).
Fig 2.
Expression of mARC1 and mARC2 in human subcutaneous and omental fat tissues from obese patients.
A. mARC1 and mARC2 gene expression in human subcutaneous and omental fat tissues (n = 6) and matched liver samples (n = 8) in obese patients as determined by qRT-PCR analysis. The Ct values were normalized to the housekeeping gene TBP. Data are expressed as mean ± S.D., *p<0.05. B. Expression of mARC1 and mARC2 proteins in the subcellular and omental fat tissue and matched liver samples in 8 obese patients (patient data are presented in the Table 1). The mitochondrial (P10) and post- mitochondrial (S10) fractions were isolated from the different tissues and analyzed by western blot for the presence of mARC1 and mARC2. Mitochondrial cytochrome b5 type B (CYB5B) is included as a mitochondrial marker protein.
Fig 3.
Nutritional status in both human and rats affects the mARC2 levels.
A. mARC2 protein levels in obese patients that were put on a caloric restriction diet (fasted) prior to surgery (n = 7) and control (non-fasted) individuals (n = 7) (patient data are presented in the Table 1). Equal amounts of protein from the hepatic mitochondrial fractions were analyzed by western blot for mARC2 and loading control, mitochondrial heat shock protein 70 (mHSP70) (lower panels). B. Both bands were quantified by densitometric analysis and mARC2 levels were normalized by mHSP70. The results represent the mean ±S.D. (n = 7 in each group), **p<0.01. C. mARC2 protein levels and the associated amidoxime reductase activity are decreased in the starvation treated rats. The mitochondrial fractions from the livers from control (n = 3) and starvation treated (n = 3) animals were analyzed for amidoxime reductase activity using benzamidoxime as a substrate (left panel). The results are presented as the mean ± S.D. values. ***, p<0.001. The mitochondrial fractions from these animals were also analyzed by western blot for the presence of mARC2 (right panel). mHSP70, mitochondrial chaperone was used as a loading control.
Table 2.
Statistical analysis of the mARC2 downregulation effect on specific lipids in the differentiated adipocytes (TG, triglycerides; DG, diglycerides).
Fig 4.
Basal and uncoupled mitochondrial respiration of adipocytes treated with mARC2 siRNA and ximelagatran.
A. mARC2 expression in the control and siRNA treated differentiated adipocytes in the presence and absence of 100 μM ximelagatran. B. Basal and CCCP-stimulated mitochondrial respiration in the control or mARC2 knocked down adipocytes in the presence or absence of ximelagatran. C. Remaining respiration after treatment with ximelagatran was expressed as a percentage of corresponding untreated samples. (n = 3). *, p<0.05.
Fig 5.
Redox state changes in primary human hepatocytes treated with ximelagatran.
A. Effect of ximelagatran treatment on the cell viability (cellular ATP content) and the cellular GSH (B) and GSH:GSSG ratio (C) in PHHs derived from three different donors. D. Analysis of the redox state of mitochondrial-specific Trx2 in control- and ximelagatran-treated PHHs by redox Western blotting. Densitometric analysis of the fractions of different Trx2 redox forms is shown on the right panel. Data are representative for one donor. *, p<0.05; **, p<0.01: ***, p<0.001.