Mitosox Red staining of cells exposed to normal and high glucose with or without sucralose
The presence of reactive oxygen species (ROS) was examined by Mitosox Red staining in the presence of normal glucose: no statistically significant changes in ROS accumulation were observed in any of the sucralose-exposed conditions (0.2 mM, 0.45 mM, 1 mM) (Fig. 1a, b).
A trend of reduction (1.4-fold, p = 0.09, between 0 and 1 mM) in MSC viability was observed with increasing doses of sucralose (Fig. 1c). A major reduction in cell viability was noted between 0 and 0.2 mM.
Significant upregulation of antioxidant gene such as extra-cellular glutathione peroxidase (GPX3) and gene associated with adipogenic differentiation such as CCAAT/enhancer-binding protein alpha (CEPBa) genes was observed in response to sucralose exposure with the same set of cells mentioned above in Fig. 1a. Here, MScs were exposed to normal glucose media containing sucralose concentrations of 0.2 to 1 mM.
When human mesenchymal stromal cells (hMSCs) were exposed to 0 mM and 1 mM sucralose (Fig. 1d), the main upregulated genes again were GPX3 and CEBPA (2.6- and 5.2-fold, p = 0.03 and 0.008, respectively). The gene upregulation was not noted at 0.2 mM but noted at 0.45 mM and 1 mM sucralose concentrations.
When the presence of reactive oxygen species (ROS) was examined in the presence of high glucose (DMEM 25 mM glucose) using Mitosox Red staining, elevated ROS accumulation was observed (Fig. 2a, b). ROS accumulation increased (1.4-, 1.4-, and 1.7-folds, p = 0.009, 0.001, and 0.0001, respectively) when cells were exposed to 1 mM sucralose in comparison to control (absence of sucralose) (by ImageJ analysis,).
Of note, cell florescence secondary to ROS accumulation was higher (above 60,000 CTFC units) in all conditions in the presence of high glucose, compared to normal glucose.
Cell viability analyses (by trypan blue exclusion method) showed a decrease of 1.5-fold and 1.4-fold respectively (p = 0.03 and 0.03, respectively) when cells were exposed to 1 mM sucralose in comparison to 0 mM sucralose and 0.2 mM (Fig. 2c). There was a decrease in viability between 0.2 and 1 mM indicating accumulating effect of cell toxicity in a dose-dependent fashion.
We also observed significant upregulation of antioxidant and adipogenic differentiation genes with same sets of cells mentioned previously in Fig. 2a when cells were exposed to high glucose (Fig. 2d). Genes including SOD3 (superoxide dismutase 3, an extra-cellular antioxidant), GPX1 (glutathione peroxidase 1, an cytosolic antioxidant), and GPX3 (1.6-, 1.6-, and 1.7-fold, p = 0.01, 0.03, and 0.02, respectively) were also upregulated in this experimental set when comparing human mesenchymal stromal cells (hMSCs) exposed to 0 mM and 1 mM sucralose.
Adipogenic genes such as CEBPa and PPARG were upregulated in an increasing dose-dependent fashion of sucralose concentration, in the presence of high glucose (HG).
Viability of hMSC was decreased in the presence of high glucose in comparison to normal glucose (Figs. 1c and 2c) in the same concentration of sucralose.
Effects of sucralose on adipogenic differentiation
hMSCs were exposed to adipogenic media (Lonza, Walkersville, MD, USA), to mimic an obesogenic environment. Zero millimolar, 0.2 mM, and 1 mM sucralose were added to the adipogenic media and cultured for 18 days (much longer period than 72 h, following standard differentiation times for MSCs). Gene expression analysis demonstrated a relative upregulation of genes associated with increased intracellular fat, such as CEBPA, FABP4 (fatty acid binding protein or adipocyte protein 2 is a carrier protein for fatty acid and primarily expressed in adipocytes), and ADIPOQ (adiponectin, a protein hormone produced in mature adipocytes), 2.05-fold, 3.45-fold, and 3.5-fold with p values less than 0.05, respectively (Fig. 3a).
We also stained hMSCs exposed to adipogenic media and sucralose with Oil Red O to identify oil droplet (Fig. 3b), as an indication of the adipogenesis process.