Journal: Frontiers in Endocrinology

Article Title: Chenodeoxycholic Acid Modulates Bile Acid Synthesis Independent of Fibroblast Growth Factor 19 in Primary Human Hepatocytes

doi: 10.3389/fendo.2020.554922

Figure Lengend Snippet: Demographics.

Article Snippet: FGF19 concentration in cell supernatants was determined using ELISA (Human FGF19 Quantikine ELISA kit, R&D systems, Minneapolis, MN) according to the manufacturer’s instructions.

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, RNA Sequencing

Journal: Frontiers in Endocrinology

Article Title: Chenodeoxycholic Acid Modulates Bile Acid Synthesis Independent of Fibroblast Growth Factor 19 in Primary Human Hepatocytes

doi: 10.3389/fendo.2020.554922

Figure Lengend Snippet: Differentially expressed genes in primary human hepatocytes upon treatment with endogenously produced FGF19. Primary human hepatocytes were treated with endogenously produced FGF19 or vehicle control and total RNA was sequenced (n = 3). (A, B) Principal component analysis (PCA) showed that 97% of the variation in the samples could be explained by donor differences (PC1-2), while only 2% of the variation could be explained by the treatment (PC3-4). (C, D) A limited number of transcripts were differentially expressed upon treatment. Venn diagram (C) and bar plot (D) displaying all expressed transcripts (green) and differentially expressed transcripts after endogenously produced FGF19 treatment (yellow). The differentially expressed transcripts were not significantly enriched for any gene ontologies (GOs) or KEGG pathways. (E, F) Representative UCSC Genome Browser tracks of normalized FGF19 and CYP7A1 expression after endogenous FGF19 treatment. The tracks from top to bottom show the scale in the human genome, the location in the human genome, the gene including exons (black boxes) and introns (arrows), and the RNA-seq signal from the treatment. (G) Venn diagram displaying the overlap between differentially expressed transcripts after chenodeoxycholic acid (CDCA) (blue), recombinant FGF19 (red), or endogenously produced FGF19 (yellow) treatment compared to their respective vehicle controls. All three treatments resulted in mainly unique expressed transcripts, with little overlap among the treatments.

Article Snippet: FGF19 concentration in cell supernatants was determined using ELISA (Human FGF19 Quantikine ELISA kit, R&D systems, Minneapolis, MN) according to the manufacturer’s instructions.

Techniques: Produced, Control, Expressing, RNA Sequencing, Recombinant

Journal: Frontiers in Endocrinology

Article Title: Chenodeoxycholic Acid Modulates Bile Acid Synthesis Independent of Fibroblast Growth Factor 19 in Primary Human Hepatocytes

doi: 10.3389/fendo.2020.554922

Figure Lengend Snippet: Chenodeoxycholic acid (CDCA), but not recombinant FGF19, downregulated bile acids (BA) synthesis in primary human hepatocytes. (A, B) CYP7A1 mRNA expression (24 h, n = 13; 6 h, n = 10) and (C, D) cholic acid (CA) concentration (24 h, n = 5; 6 h, n = 5) in cell medium following treatment with FGF19 or CDCA at various concentrations for 24 h or 6 h. Data is presented as box-plot showing interquartile range (IQR) (box) and min-max (bars) with median marked with a line, red dots are the individual values, black dots in (A) represents RNA from donors that were also used for RNA sequencing. The plus sign represents mean value. Friedman test was used to assess differences between control and treatments and Dunn’s multiple comparison test was used post hoc . *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: FGF19 concentration in cell supernatants was determined using ELISA (Human FGF19 Quantikine ELISA kit, R&D systems, Minneapolis, MN) according to the manufacturer’s instructions.

Techniques: Recombinant, Expressing, Concentration Assay, RNA Sequencing, Control, Comparison

Journal: Frontiers in Endocrinology

Article Title: Chenodeoxycholic Acid Modulates Bile Acid Synthesis Independent of Fibroblast Growth Factor 19 in Primary Human Hepatocytes

doi: 10.3389/fendo.2020.554922

Figure Lengend Snippet: FGF19 was rapidly induced and secreted in a dose-dependent manner following chenodeoxycholic acid (CDCA) treatment. (A) FGF19 mRNA expression and (B) protein levels in cell medium after CDCA treatment. (C) Time course of FGF19 mRNA expression (relative to Ctrl) and secreted protein after treatment with a single dose of CDCA. Data in (A, B) is presented as box-plot showing interquartile range (IQR) (box) and min-max (bars) with median marked with a line, marked with red dots are the individual values and black dots in (A) represents RNA from donors that were also used for RNA sequencing. (24 h, n = 13; 6h, n = 10). The plus sign represents mean value. The time course is presented as median with IQR. Friedman test was used to assess differences between control and treatments and Dunn’s multiple comparison test was used post-hoc . *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: FGF19 concentration in cell supernatants was determined using ELISA (Human FGF19 Quantikine ELISA kit, R&D systems, Minneapolis, MN) according to the manufacturer’s instructions.

Techniques: Expressing, RNA Sequencing, Control, Comparison

Journal: Frontiers in Endocrinology

Article Title: Chenodeoxycholic Acid Modulates Bile Acid Synthesis Independent of Fibroblast Growth Factor 19 in Primary Human Hepatocytes

doi: 10.3389/fendo.2020.554922

Figure Lengend Snippet: Medium containing FGF19 produced by primary human hepatocytes downregulated CYP7A1 mRNA expression. (A) Layout of the experiment showing how chenodeoxycholic acid (CDCA) was used to induce FGF19 synthesis in primary human hepatocytes. Endogenously produced FGF19 was then applied to naïve hepatocytes for 24 h. (B) Levels of FGF19 in control and conditioned medium. (C) CYP7A1 mRNA expression and (D) cholic acid (CA) concentration in control and conditioned medium. Data is presented as box-plot showing interquartile range (IQR) (box) and min-max (bars) with median marked with a line, marked with red dots are the individual values, black dots in (C) represents RNA from donors that were also used for RNA sequencing. (n = 10). The plus sign represents mean value. Wilcoxon matched-pairs signed rank test was used to assess differences between control and conditioned medium. *p < 0.05, ns: non significant.

Article Snippet: FGF19 concentration in cell supernatants was determined using ELISA (Human FGF19 Quantikine ELISA kit, R&D systems, Minneapolis, MN) according to the manufacturer’s instructions.

Techniques: Produced, Expressing, Control, Concentration Assay, RNA Sequencing

Journal: Frontiers in Endocrinology

Article Title: Chenodeoxycholic Acid Modulates Bile Acid Synthesis Independent of Fibroblast Growth Factor 19 in Primary Human Hepatocytes

doi: 10.3389/fendo.2020.554922

Figure Lengend Snippet: Differentially expressed genes in primary human hepatocytes upon treatment with chenodeoxycholic acid (CDCA) or recombinant FGF19. Total RNA from primary human hepatocytes treated with recombinant FGF19, CDCA, or vehicle control was sequenced (n = 3). (A, B) Principal component analysis (PCA) showed that 88% of the variation in the samples could be explained by donor differences (PC1-2), while 11% of the variation could be explained by the treatment (PC3-4). (C, D) A limited number of transcripts were differentially expressed upon treatment. Venn diagram (C) and bar plot (D) displaying all expressed transcripts (black) and differentially expressed ones after recombinant FGF19 treatment (red) or CDCA treatment (blue). (E, F) Representative UCSC Genome Browser tracks of normalized FGF19 and CYP7A1 expression after recombinant FGF19 or CDCA treatment. The tracks from top to bottom show the scale in the human genome, the location in the human genome, the gene including exons (black boxes) and introns (arrows), and the RNA-seq signal from each treatment. (G) Unique chenodeoxycholic acid (CDCA)-DE transcripts were used for gene ontology (GO) biological processes (BP), molecular function (MF), and KEGG pathway analysis. Displayed are all significant ontologies/pathways in each ontology. The size of the bubble indicates number of genes in each category and the color represents the significance. (H) GO-term interaction network of the 7 most significant GO-BP terms in (G) . GO terms are in green circles, and gene names are in grey circles. Unique DE transcripts after recombinant FGF19 gave no significant enrichment in GO or KEGG pathway-related terms.

Article Snippet: FGF19 concentration in cell supernatants was determined using ELISA (Human FGF19 Quantikine ELISA kit, R&D systems, Minneapolis, MN) according to the manufacturer’s instructions.

Techniques: Recombinant, Control, Expressing, RNA Sequencing

Journal: Frontiers in Endocrinology

Article Title: Chenodeoxycholic Acid Modulates Bile Acid Synthesis Independent of Fibroblast Growth Factor 19 in Primary Human Hepatocytes

doi: 10.3389/fendo.2020.554922

Figure Lengend Snippet: Knockdown of FGF19 did not alter downregulation of CYP7A1 mRNA expression by chenodeoxycholic acid (CDCA). Primary human hepatocytes were treated with siRNA targeting FGF19 or non-targeting control. (A) FGF19 mRNA expression, (B) protein levels in medium in control cultures, after induction by 10 µM CDCA. (C) CYP7A1 mRNA expression, (D) cholic acid (CA) concentration in cell medium. Data is presented as median with interquartile range (IQR). Friedman test was used to assess differences between control and treatments and Dunn’s multiple comparison test was used post-hoc . Each colored dot represents one individual case (n = 3). *p < 0.05.

Article Snippet: FGF19 concentration in cell supernatants was determined using ELISA (Human FGF19 Quantikine ELISA kit, R&D systems, Minneapolis, MN) according to the manufacturer’s instructions.

Techniques: Knockdown, Expressing, Control, Concentration Assay, Comparison