Metformin Increases Proliferative Activity and Viability of Multipotent Stromal Stem Cells Isolated from Adipose Tissue Derived from Horses with Equine Metabolic Syndrome

Abstract

In this study, we investigated the influence of metformin (MF) on proliferation and viability of adipose-derived stromal cells isolated from horses (EqASCs). We determined the effect of metformin on cell metabolism in terms of mitochondrial metabolism and oxidative status. Our purpose was to evaluate the metformin effect on cells derived from healthy horses (EqASC_HE) and individuals affected by equine metabolic syndrome (EqASC_EMS). The cells were treated with 0.5 μM MF for 72 h. The proliferative activity was evaluated based on the measurement of BrdU incorporation during DNA synthesis, as well as population doubling time rate (PDT) and distribution of EqASCs in the cell cycle. The influence of metformin on EqASC viability was determined in relation to apoptosis profile, mitochondrial membrane potential, oxidative stress markers and BAX/BCL-2 mRNA ratio. Further, we were interested in possibility of metformin affecting the Wnt3a signalling pathway and, thus, we determined mRNA and protein level of WNT3A and β-catenin. Finally, using a two-tailed RT-qPCR method, we investigated the expression of miR-16-5p, miR-21-5p, miR-29a-3p, miR-140-3p and miR-145-5p. Obtained results indicate pro-proliferative and anti-apoptotic effects of metformin on EqASCs. In this study, MF significantly improved proliferation of EqASCs, which manifested in increased synthesis of DNA and lowered PDT value. Additionally, metformin improved metabolism and viability of cells, which correlated with higher mitochondrial membrane potential, reduced apoptosis and increased WNT3A/β-catenin expression. Metformin modulates the miRNA expression differently in EqASC_HE and EqASC_EMS. Metformin may be used as a preconditioning agent which stimulates proliferative activity and viability of EqASCs.

Keywords: adipose-derived stromal cells; equine metabolic syndrome; metformin.

Figure 1

The influence of metformin on proliferative and metabolic activity of adipose-derived stromal cells isolated from horses (EqASCs). The proliferative activity was evaluated based on microphotographs obtained with epifluorescence microscope—scale bar 250 μm (a) population doubling time ratio (b,c) and metabolic activity (d,e). The statistically significant changes were indicated with asterisks; * p < 0.05; ** p < 0.01, while non-significant differences are marked as ns.

Figure 2

The influence of metformin on the distribution of EqASCs during the cell cycle, with representative histograms (a) and results of statistical analysis (b). The statistically significant changes are indicated with asterisks; * p < 0.05; ** p < 0.01 and *** p < 0.001. Non-significant differences are marked as ns.

Figure 3

The influence of metformin on mitochondrial membrane potential and oxidative. Representative dot plots indicating distribution of cells accordingly to the mitochondrial membrane potential (a). The ultrastructure of EqASC_HE and EqASC_EMS without metformin treatment and after metformin treatment. Mitochondria are indicated with red arrows, and nuclei with nu symbol, scale bar: 2 μm (b). Analysis of cell viability based on mitochondrial potential (c). Comparison of total depolarised cells in tested experiments (d). Evaluation of reactive oxygen species (e), nitric oxide (f) and superoxide dismutase activity (g). Significant changes are indicated with asterisks: * p < 0.05; ** p < 0.01 and *** p < 0.001, while non-significant differences are marked as ns.

Figure 3

The influence of metformin on mitochondrial membrane potential and oxidative. Representative dot plots indicating distribution of cells accordingly to the mitochondrial membrane potential (a). The ultrastructure of EqASC_HE and EqASC_EMS without metformin treatment and after metformin treatment. Mitochondria are indicated with red arrows, and nuclei with nu symbol, scale bar: 2 μm (b). Analysis of cell viability based on mitochondrial potential (c). Comparison of total depolarised cells in tested experiments (d). Evaluation of reactive oxygen species (e), nitric oxide (f) and superoxide dismutase activity (g). Significant changes are indicated with asterisks: * p < 0.05; ** p < 0.01 and *** p < 0.001, while non-significant differences are marked as ns.

Figure 4

The influence of metformin on the apoptosis profile. Representative dot plots indicating distribution of cells after annexin V/ propidium iodide staining (a) Representative images obtained after calcein/PI staining, scale bar—250 μm. (b) Analysis of cell viability (c) and apoptosis (d). Measured transcript levels for BAX (e) BCL-2 (f) and their ratio (g) Statistically significant changes are indicated with asterisks: * p < 0.05; ** p < 0.01 and *** p < 0.001, while non-significant differences are marked as ns.

Figure 4

The influence of metformin on the apoptosis profile. Representative dot plots indicating distribution of cells after annexin V/ propidium iodide staining (a) Representative images obtained after calcein/PI staining, scale bar—250 μm. (b) Analysis of cell viability (c) and apoptosis (d). Measured transcript levels for BAX (e) BCL-2 (f) and their ratio (g) Statistically significant changes are indicated with asterisks: * p < 0.05; ** p < 0.01 and *** p < 0.001, while non-significant differences are marked as ns.

Figure 5

Determination of Wnt-3/β-catenin expression (a,b) and corresponding intracellular accumulation of protein (c–e). Statistically significant changes are indicated with asterisks: * p < 0.05; ** p < 0.01 and *** p < 0.001, while non-significant differences are marked as ns.

Figure 6

miRNA expression measured with two-tailed RT-qPCR. The following miRNAs were tested: miR-16-5p (a), miR-21-5p (b), miR-29a-3p(c), miR-140-3p (d) and miR-145-5p (e). Data are normalised to U6 snRNA levels and expressed as fold changes compared to the sample with the lowest level. Statistically significant changes are indicated with asterisks: * p < 0.05; ** p < 0.01 and *** p < 0.001, while non-significant differences are marked as ns.