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Metformin, an oral antidiabetic drug, recently demonstrated a reducing effect on bile acids (BA) plasma concentrations in one patient with intrahepatic cholestasis of pregnancy (ICP) by unknown mechanism. Therefore, the aim of the present study was to examine the effect of metformin on BA homeostasis and related molecular pathways in the liver and intestine using a mouse model of ICP. The cholestasis was induced in female C57BL/6 mice by repeated administration of ethinylestradiol (10 mg/kg BW s.c.) and/or metformin (150 mg/kg BW orally) over 5 consecutive days with subsequent bile collection and molecular analysis of samples. We demonstrated that metformin significantly increased the rate of bile secretion in control mice. This increase was BA dependent and was produced both by increased liver BA synthesis via induced cholesterol 7α-hydroxylase (Cyp7a1) and by increased BA reabsorption in the ileum via induction of the apical sodium-dependent BA transporter (Asbt). In contrast, metformin further worsened ethinylestradiol-induced impairment of bile secretion. This reduction was also BA dependent and corresponded with significant downregulation of Bsep, and Ntcp, major excretory and uptake transporters for BA in hepatocytes, respectively. The plasma concentrations of BA were consequently significantly increased in the metformin-treated mice. Altogether, our data indicate positive stimulation of bile secretion by metformin in the intact liver, but this drug also induces serious impairment of BA biliary secretion, with a marked increase in plasma concentrations in estrogen-induced cholestasis. Our results imply that metformin should be used with caution in situations with hormone-dependent cholestasis, such as ICP.

Excessive iron accumulation in the liver, which accompanies certain genetic or metabolic diseases, impairs bile acids (BA) synthesis, but the influence of iron on the complex process of BA homeostasis is unknown. Thus, we evaluated the effect of iron overload (IO) on BA turnover in rats. Compared with control rats, IO (8 intraperitoneal doses of 100 mg/kg every other day) significantly decreased bile flow as a consequence of decreased biliary BA secretion. This decrease was associated with reduced expression of Cyp7a1, the rate limiting enzyme in the conversion of cholesterol to BA, and decreased expression of Bsep, the transporter responsible for BA efflux into bile. However, IO did not change net BA content in faeces in response to increased intestinal conversion of BA into hyodeoxycholic acid. In addition, IO increased plasma cholesterol concentrations, which corresponded with reduced Cyp7a1 expression and increased expression of Hmgcr, the rate-limiting enzyme in de novo cholesterol synthesis. In summary, this study describes the mechanisms impairing synthesis, biliary secretion and intestinal processing of BA during IO. Altered elimination pathways for BA and cholesterol may interfere with the pathophysiology of liver damage accompanying liver diseases with excessive iron deposition.

Aims Increased plasma soluble endoglin concentrations (sEng) are frequently detected in metabolic disorders accompanied with hypercholesterolemia in serum, but effect of sEng on the cholesterol biochemistry is unknown. Cholesterol and bile acids (BA) are important products of liver metabolism with numerous functions within the organism. Turnover of these substances requires precise regulation due to potential toxicities during their cumulation. In this study, we hypothesized that high sEng levels affect cholesterol homeostasis and BA turnover in mice liver. Main methods Nine-month-old transgenic male mice overexpressing human sEng and wild-type mice underwent plasma, bile, stool, and organ samples analysis by analytical, qRT-PCT and Western blot methods. Key findings sEng mice demonstrated decreased plasma total and LDL cholesterol concentrations due to upregulation of hepatic Sr-b1 and Ldlr receptors, increased liver cholesterol content, and increased Abcg8-mediated cholesterol efflux into bile. sEng also increased conversion of cholesterol into bile acids (BA) via upregulation of Cyp7a1 and increased Mdr1 expression. Plasma concentrations of BA were increased in sEng mice due to their enhanced reabsorption via ileum. Increased hepatic disposition of BA led to their increased biliary excretion coupled with choleretic activity. Significance For the first time, we have shown that high sEng plasma levels affect cholesterol and BA homeostasis on the basis of complex liver and intestinal effects. The significance of these findings for pathophysiology of diseases associated with increased sEng concentrations remains to be elucidated in prospective studies.

Aims To evaluate iron biochemistry and contributing liver mechanisms during obstructive cholestasis and pravastatin treatment in rats. Main methods A rat model of cholestasis induced by bile duct ligation (BDL) was used for the study. The detection of iron and the expression of relevant molecules were performed one week after surgery in the control, and cholestatic animals after treatment with either saline or pravastatin (1 mg/kg/day). Key findings Saline-administered BDL rats showed, in comparison to sham-operated animals, a significant increase in plasma iron concentration, increased liver protein content of heme oxygenase-1 (HO-1) and a decline in the expression of hepcidin. Ferroportin 1 expression was increased with a simultaneous reduction in intrahepatic iron concentration. The administration of pravastatin to BDL animals attenuated proliferation changes in liver parenchyma, prevented HO-1 induction, restored hepatic mRNA hepcidin expression to control levels and induced the expression of ferritin, transferrin receptors (TfR1/2) and divalent metal transporter-1. This was accompanied by an increased content of intrahepatic iron when compared to the BDL animals, and a reduction of hyperbilirubinemia. Significance Cholestasis-induced increase in plasma and decrease in hepatic iron levels were associated with up-regulation of liver HO-1 and ferroportin 1. Pravastatin alleviated cholestatic liver impairment and raised liver iron content by modulation of heme catabolism and an increase of hepatic iron uptake and storage capacity.

Background: Epigenetic de novo methylation of CpG-rich areas located in the promotors of gene p15INK4b (CDKN2B) as it is capable to silence tumor suppressor genes and knowledge of telomere-telomerase complex may bring an important sign into molecular backround of malignant transformation in patients with myelodysplastic syndromes (MDS). Aims: Intensity of gene methylation, the variability of telomere length (TRF) and telomerase activity (TA) were studied in bone marrow (BM) or peripheral blood (PB) cells of patients with MDS before treatment and during the course of the disease to evaluate its prognostic significance and association with clinical outcomes and disease progression. Methods: 110 patients with MDS were stratiffied according to the WHO classification: 18 × RA, 3 × RARS, 32 × RCMD, 12 × 5q-syndrome, 17 × RAEB1, 19 × RAEB2 and 9 × MDS/MPS. BM cells from 30 age matched healthy donors served as controls, 5 leukemic cell lines were examined as positive controls. The level of DNA methylation was quantified by methylation specific PCR after sodium bisulphate modification of DNA, CpGenom DNA Modification kit from Scintila. The methylation status was dedicated as Methylation Indices (MI) in the range 0.0–1.0. The telomere length was determined as terminal restriction fragment (TRF index in kbp) after Southern analysis of genomic DNA, TeloTAGGG Telomere Length Assay kit from Roche. The activity of telomerase was assessed by Quantitative TRAP assay based on a photometric enzyme immunoassay on solid phase, Telo TAGGG Telomerase PCR Elisa plus kit from Roche. We postulated TRF shorter than 7.5 kbp as reduced telomeres and values of TA higher than 0.0263 as positive telomerase activity. Results: Aberrant methylation of CDKN2B gene was present in 64% of MDS untreated patients showing significant heterogeneity in WHO subgroups. Mean values of MI increased as follows: from 0.18 ± 0.13 in RCMD, 0.21 ± 0.11 in RA/RARS, 0.26 ± 0.19 in 5q- syndrome, 0.33 ± 0.17 in RAEB1, 0.34 ± 0.17 in RAEB2, up to 0.41 ± 0.14 in MDS/MPS. A significant difference was found between early forms of MDS (RA/RARS, RCMD, 5q-syndrome) and advanced MDS with excess of blasts (RAEB1, RAEB2, MDS/MPS). High level of methylation CDKN2B gene was observed in 70% (33/47) of patients with advances forms of MDS (MI=0.36 ± 0.17) in comparison with early phases of MDS (MI=0.20 ± 0.14) in 60% (39/65) of patients. In one patient with RAEB2, a significant decrease in MI from 0.44 in BM and 0.37 in PB to 0,25 to 0,04, respectively was observed after 3 months of treatment with decitabine together with simultaneous prolongation of TRF from 7,62 kbp to 10.5 kbp. The patient achieved partial remission of the disease after 6 months of treatment with corresponding values of MI=0.20 and TRF=9.63kbp. In another patient, a progression of the disease from RCMD towards RAEB was connected vith increase in MI from 0.00 to 0.39 that correlated with elevation of telomerase activity from 0 to 0.0534. Conclusions: The results confirm association of aberrant gene methylation with progression of early MDS to advanced forms with excess of blasts. Our findings also showed a certain degree of correlation between molecular changes in gene methylation, telomere length and telomerase activity in different phases of disease. Thus, determination of these markers may contribute to more precise estimation of an individual MDS patient’s risk and for decision of an optimal treatment strategy.