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

Agnieszka Smieszek,Katarzyna Kornicka,Krzysztof Marycz,Eva Rohlova,Mikael Kubista,Lucie Langerova,Lukas Valihrach,Peter Androvic,Jolanta Szłapka-Kosarzewska

doi:10.3390/cells8020080

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 (EqASCHE) and individuals affected by equine metabolic syndrome (EqASCEMS). 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 EqASCHE and EqASCEMS. Metformin may be used as a preconditioning agent which stimulates proliferative activity and viability of EqASCs.

Highlights

Equine metabolic syndrome (EMS) is considered as a burning issue in veterinary medicine, affecting more and more horses
Obtained results revealed that metformin may act as an agent that increases the proliferative activity of EqASCs derived both from healthy and equine metabolic syndrome (EMS) horses (Figure 1)
Microscopic evaluation of EqASCHE and EqASCEMS cultures showed that metformin does not affect the cells’ morphology—the cells maintain proper fibroblast-like morphotype

Summary

Introduction

Equine metabolic syndrome (EMS) is considered as a burning issue in veterinary medicine, affecting more and more horses. Obesity was excluded as a sine qua non condition in the course of the diagnostic procedure of EMS, specific local accumulation of adipose tissue, i.e., adiposity (cresty neck) is considered a diagnostic marker. It is thought that EMS horses that are not overweight might accumulate abdominal adipose tissue, which is not without physiological significance for the other organs, including liver. Adipose tissue produces a number of factors, including cytokines, adipokines, as well as hormones, all influencing the clinical picture of EMS horses. It was shown that adipocytes isolated from subcutaneous adipose tissue of EMS horses produce pro-inflammatory cytokines, i.e., tumour necrosis factor-alpha (TNF-α), interleukin

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Conclusion