Proteomic and Biological Analysis of the Effects of Metformin Senomorphics on the Mesenchymal Stromal Cells

Abstract

Senotherapeutics are new drugs that can modulate senescence phenomena within tissues and reduce the onset of age-related pathologies. Senotherapeutics are divided into senolytics and senomorphics. The senolytics selectively kill senescent cells, while the senomorphics delay or block the onset of senescence. Metformin has been used to treat diabetes for several decades. Recently, it has been proposed that metformin may have anti-aging properties as it prevents DNA damage and inflammation. We evaluated the senomorphic effect of 6 weeks of therapeutic metformin treatment on the biology of human adipose mesenchymal stromal cells (MSCs). The study was combined with a proteome analysis of changes occurring in MSCs' intracellular and secretome protein composition in order to identify molecular pathways associated with the observed biological phenomena. The metformin reduced the replicative senescence and cell death phenomena associated with prolonged in vitro cultivation. The continuous metformin supplementation delayed and/or reduced the impairment of MSC functions as evidenced by the presence of three specific pathways in metformin-treated samples: 1) the alpha-adrenergic signaling, which contributes to regulation of MSCs physiological secretory activity, 2) the signaling pathway associated with MSCs detoxification activity, and 3) the aspartate degradation pathway for optimal energy production. The senomorphic function of metformin seemed related to its reactive oxygen species (ROS) scavenging activity. In metformin-treated samples, the CEBPA, TP53 and USF1 transcription factors appeared to be involved in the regulation of several factors (SOD1, SOD2, CAT, GLRX, GSTP1) blocking ROS.

Keywords: aging; mesenchymal stem cells; senescence; senolytics; senomorphics.

FIGURE 1

Senescence levels in MSCs cultures. This histogram shows the percentage of senescent cells after three (3w), four (4w) and six (6w) weeks of in vitro culture, either in the absence (CTRL) or presence of metformin. Data are shown with a standard deviation (SD) n = 3 *p < 0.05, ***p < 0.001. For each time point, the symbol (*) indicates the statistical difference between the control culture and those treated with metformin. The pictures show representative images of senescent cells that tested positive for the beta-galactosidase activity (blue). The arrow heads indicate some typical senescent cells, which show flattened morphology and blue staining in the perinuclear area. The bar corresponds to 100 microns.

FIGURE 2

Apoptosis levels in MSCs cultures. This table shows the percentage of apoptotic cells after three, four and 6 weeks of in vitro culture, either in the absence (CTRL) or presence of metformin. Data are shown with standard deviation (SD) n = 3 *p < 0.05, **p < 0.01, ***p < 0.001. For each time point, the symbol (*) indicates the statistical difference between the control culture and those treated with metformin. The pictures show representative images of annexin-V detection by flow cytometry analysis. Apoptotic and non-apoptotic cells were identified by two separate dyes (Annexin V and 7AAD, respectively). The phosphatidylserine is bound by Annexin V (FITC-A labeled) on the external membrane of apoptotic cells, while 7AAD (PerCP-Cy5-5-A labeled) permeates and stains DNA in late-stage apoptotic and dead cells. Coloration enables the identification of three cell populations: non-apoptotic cells (Annexin V- and 7AAD+); early apoptotic cells (annexin V+ and 7AAD-); and late apoptotic or dead cells (Annexin V+ and 7AAD+). In our experimental conditions, both the early apoptotic and late apoptotic cells were grouped.

FIGURE 3

Venn analysis of proteins found in cell lysates and secretomes. These pictures show the proteins that occur in all the experimental conditions (CTRL, 3, 6, and 9 mM metformin treatment), and those that are only present in some.