Journal: Scientific Reports

Article Title: Inhibition of mitochondrial function by metformin increases glucose uptake, glycolysis and GDF-15 release from intestinal cells

doi: 10.1038/s41598-021-81349-7

Figure Lengend Snippet: The effects of metformin on differentially expressed (DE) genes and metabolic pathways in mouse intestinal monolayer cultures. ( a ) Volcano plot displaying gene expression changes in duodenal organoid cells in 2D monolayer cultures treated with 1 mM metformin or no treatment control (n = 6 plates per condition from 2 different organoid lines). Fold-change (X-axis) compared to p-adjusted values (Y-axis) of individual genes altered by metformin treatment. Red dots represent DE genes (P < 0.05); black dots represent gene expression changes that were not statistically significant (Not Sig). Labelled genes represent the top 50 DE genes (sorted by P values). Analysis was performed using Bioconductor software packages in RStudio (v.1.2.5019), with gene annotation from the Ensembl dataset in BioMart (v2.40.5). Volcano plots were generated using the Geompoint function of the ggplot package. ( b , c ) KEGG enrichment analysis for changes in signalling ( b ) and metabolic ( c ) pathways affected by metformin treatment. Gene ratio is the number of DE genes in a KEGG pathway divided by total number of genes in the pathway. Count represents the numbers of DE genes. KEGG pathway analysis was performed using clusterprofiler. ( d ) Heatmaps showing DE genes (filtered by p-adjusted value < 0.05) involved in hexose metabolism. Relative expression is normalised to z-scores for each gene. Each box indicates a separate plate and the corresponding organoid line. Heatmaps were plotted using Pheatmap. ( e ) Schematic showing expression changes of genes involved in all hexose metabolism pathways. Red and blue show upregulated and downregulated genes, respectively.

Article Snippet: Analysis was performed using Bioconductor software packages in RStudio (v.1.2.5019), with gene annotation from the Ensembl dataset in BioMart (v2.40.5).

Techniques: Gene Expression, Control, Software, Generated, Expressing

Journal: Scientific Reports

Article Title: Inhibition of mitochondrial function by metformin increases glucose uptake, glycolysis and GDF-15 release from intestinal cells

doi: 10.1038/s41598-021-81349-7

Figure Lengend Snippet: Metformin causes mitochondrial dysfunction in intestinal cells. ( a ) Left: Changes in oxygen consumption rate (OCR) during the mitochondrial stress test. Cells were pre-treated with control (ENR media) ± metformin (1 mM) for 24 h, then incubated with glucose (10 mM) in XF basal media for 1 h prior to measurements. Dotted lines indicate compound addition. Oligomycin-A (OliA, 1 μM), FCCP (1 μM), Rotenone/Antimycin A (Rot/AA, 1 μM each). Results were normalised to the protein concentration measured by BCA assay. Right: Effect of metformin on measured mitochondrial respiration parameters. n = 5 wells from 3 independent experiments. ***P < 0.001. Two-way ANOVA and Bonferroni post-hoc test. ( b ) Effects of mitochondrial pyruvate carrier inhibitor UK-5099 compared to DMSO on OCR of control and metformin overnight pre-treated cells as measured by the Seahorse bioanalyser. Results were normalised to the protein concentration measured by BCA assay. Inset: Normalised OCR was calculated as differences after drug application and third time point measured before application ***P < 0.001 compared to DMSO in control cultures. n = 4–5 wells from 4 independent experiments. ( c ) Left: Example traces of Perceval fluorescence ratios from control and metformin treated cells. Glucose (Gluc, 10 mM), Rot/AA (1 μM) and 2-DG (50 mM) were applied as indicated. Right: Effect of 10 mM glucose (Gluc), Rot/AA and 2-DG on the Perceval fluorescence ratios in control (red) and metformin (blue) overnight pre-treated cells (***P < 0.001, Repeated-measures ANOVA and Bonferroni post-hoc test. Control: n = 14 from 5 dishes, metformin: n = 20 cells from 4 dishes). ( d ) Heatmaps showing differentially expressed genes (filtered by p-adjusted value of < 0.05) involved in mitochondrial function based from the MitoCarta 2.0 dataset. Relative expression is normalised to the z-scores for each gene. Each box indicates a separate plate and the corresponding organoid line. Analysis was performed using Bioconductor software packages in RStudio (v.1.2.5019), and the heatmap generated using Pheatmap. ( e ) Confocal images of intestinal cells treated with control or metformin (1 mM) stained with MitoTracker green. Scale bar: 50 µm. Magnified images of individual cells are shown. ( f ) Quantification of the percentage of total cells with fragmented, intermediate or elongated mitochondrial in control or metformin treated cells (***P < 0.001, Chi-square test. Control: n = 1660 cells and metformin: n = 2155 cells, from 3 organoid lines). ( g ) Quantification of mitochondrial morphology as the width/length ratio or roundness by imposition of spots to image regions using the Harmonizer software.

Article Snippet: Analysis was performed using Bioconductor software packages in RStudio (v.1.2.5019), with gene annotation from the Ensembl dataset in BioMart (v2.40.5).

Techniques: Control, Incubation, Protein Concentration, BIA-KA, Fluorescence, Expressing, Software, Generated, Staining