| 1. |
- Godoy, Patricio, et al.
(författare)
-
Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME
- 2013
-
Ingår i: Archives of Toxicology. - : Springer Science and Business Media LLC. - 0340-5761 .- 1432-0738. ; 87:8, s. 1315-1530
-
Forskningsöversikt (refereegranskat)abstract
- This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4 alpha, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4 alpha), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.
|
|
| 2. |
- Krattinger, Regina, et al.
(författare)
-
Chenodeoxycholic acid significantly impacts the expression of miRNAs and genes involved in lipid, bile acid and drug metabolism in human hepatocytes
- 2016
-
Ingår i: Life Sciences. - : Elsevier BV. - 0024-3205 .- 1879-0631. ; 156, s. 47-56
-
Tidskriftsartikel (refereegranskat)abstract
- Aims: Bile acids (BAs) are important gut signaling hormones, influencing lipid, glucose, and energy homeostasis. The exact mechanisms behind these effects are not yet fully understood. Lately, they have come to the fore as putative therapeutics in metabolic diseases, such as e.g. nonalcoholic fatty liver disease (NAFLD). We elucidate to what extent BAs impacts on the mRNAome and microRNAome in hepatocytes to gather novel insights into the mechanisms behind metabolic and toxicologic effects of bile acids. Main methods: Five batches of primary human hepatocytes were treated with 50 mu mol/l chenodeoxycholic acid (CDCA) for 24 or 48 h. Total RNA was extracted, size fractionated and subjected to Next Generation Sequencing to generate mRNA and miRNA profiles. Key findings: Expression of 738 genes and 52 miRNAs were CDCA dependently decreased, whereas 1566 genes and 29 miRNAs were significantly increased in hepatocytes. Distinct gene clusters controlling BA and lipid homeostasis (FGF(R), APO and FABP family members, HMGCS2) and drug metabolism (CYP, UGT and SULT family members) were significantly modulated by CDCA. Importantly, CDCA affected distinct microRNAs, including miR-34a, -505, -885, -1260 and -552 that systematically correlated in expression with gene clusters responsible for bile acid, lipid and drug homeostasis incorporating genes, such as e.g. SLCO1B1, SLC22A7, FGF19, CYP2E1, CYP1A2, APO family members and FOXO3. Significance: Bile acids significantly modulate metabolic and drug associated gene networks that are connected to distinct shifts in the microRNAome These findings give novel insights on how BA enfold metabolic and system toxic effects.
|
|
| 3. |
- Krattinger, Regina, et al.
(författare)
-
microRNA-192 suppresses the expression of the farnesoid X receptor
- 2016
-
Ingår i: American Journal of Physiology - Gastrointestinal and Liver Physiology. - : American Physiological Society. - 0193-1857 .- 1522-1547. ; 310:11, s. G1044-G1051
-
Tidskriftsartikel (refereegranskat)abstract
- Farnesoid X receptor (FXR, NR1H4) plays an important role in the regulation of bile acid homeostasis in liver and intestine and may exert protective effects against certain forms of cancer such as colon carcinoma. However, the role of FXR in cell growth regulation, apoptosis, and carcinogenesis is still controversial. Similar to FXR, microRNA-192 (miR-192) is mainly expressed in the liver and colon and plays an important role in the pathogenesis of colon carcinoma. In this study, we investigated the extent to which FXR is regulated by miR-192. Two in silico-predicted binding sites for miR-192-3p within the NR1H4-3' untranslated region (UTR) were examined in vitro by luciferase reporter assays. Wild-type and mutated forms of the NR1H4-3' UTR were subcloned into a pmirGLO vector and cotransfected into Huh-7 cells with miR-192-3p. To study the effects of miR-192 on the expression of FXR, FXR target genes and cell proliferation, Huh-7 and Caco-2 cells were transfected with miR-192-5p and -3p mimics or antagomirs. In addition, the correlation between FXR and miR-192 expression was studied by linear regression analyses in colonic adenocarcinoma tissue from 27 patients. MiR-192-3p bound specifically to the NR1H4-3' UTR and significantly decreased luciferase activity. Transfection with miR-192 led to significant decreases in NR1H4 mRNA and protein levels as well as the mRNA levels of the FXR-inducible bile acid transporters OST alpha-OST beta and OATP1B3. Significant inverse correlations were detected in colonic adenocarcinoma between NR1H4 mRNA and miR-192-3p expression. In summary, microRNA-192 suppresses the expression of FXR and FXR target genes in vitro and in vivo.
|
|
| 4. |
|
|
