Sucralose Stimulates Mitochondrial Bioenergetics in Caco-2 Cells

Abstract

Sucralose is a non-caloric artificial sweetener widely used in processed foods that reportedly affects energy homeostasis through partially understood mechanisms. Mitochondria are organelles fundamental for cellular bioenergetics that are closely related to the development of metabolic diseases. Here, we addressed whether sucralose alters mitochondrial bioenergetics in the enterocyte cell line Caco-2. Sucralose exposure (0.5-50 mM for 3-24 h) increased cellular reductive power assessed through MTT assay, suggesting enhanced bioenergetics. Low doses of sucralose (0.5 and 5 mM) for 3 h stimulated mitochondrial respiration, measured through oxygraphy, and elevated mitochondrial transmembrane potential and cytoplasmic Ca²⁺, evaluated by fluorescence microscopy. Contrary to other cell types, the increase in mitochondrial respiration was insensitive to inhibition of mitochondrial Ca²⁺ uptake. These findings suggest that sucralose alters enterocyte energy homeostasis, contributing to its effects on organismal metabolism.

Keywords: Ca2+; artificial sweetener; metabolism; mitochondria; sucralose.

Figure 1

Sucralose increases cellular reductive power. Caco-2 cells (p.95-105) were cultured in control condition or treated with 0.5, 1, 5, 15, or 50 mM sucralose (Suc) for 3, 6, 18, and 24 h Then, their MTT reductase activity was measured through MTT assay (n = 4–5). Data are shown as mean ± SEM. *P < 0.05 compared with control cells of the respective time using one-way ANOVA followed by a Holm-Sidak post-test.

Figure 2

Sucralose stimulates mitochondrial bioenergetics in Caco-2 cells. (A) Sucralose increases cell respiration. Caco-2 cells (p.95-105) were cultured in control condition or treated with 0.5 or 5 mM sucralose (Suc) for 3 h. Their oxygen consumption rate was measured through oxygraphy using Clark's electrode (n = 4–5). Baseline, non-ATP-associated (400 nM oligomycin) and uncoupled (12 μM CCCP) respiration conditions were analyzed. *P < 0.05 compared with control respiration in the same condition. (B) Sucralose increases mitochondrial transmembrane potential. Caco 2 cells (p95-105) were cultured in control condition or treated for 3 h with 0.5 or 5 mM sucralose. Then, their mitochondrial transmembrane potential was measured as the incorporation of the voltage-sensitive fluorescent mitochondrial dye TMRM by live-cell epifluorescence microscopy. Upper panel: Representative images of TMRM-stained Caco-2. Lower panel: Quantification of TMRM fluorescence, arbitrary units (n = 4). *P < 0.05 compared with control cells. (C) Sucralose does not change ATP levels. Caco-2 cells (p95-105) were cultured in control condition or treated with 0.5 or 5 mM sucralose for 3 h. ATP levels were measured through luciferin/luciferase-based ATP assay (n = 5–6). All data are shown as mean ± SEM and were analyzed using one-way ANOVA followed by a Holm-Sidak post-test.

Figure 3

Sucralose did not change AMPK protein levels in Caco-2 cells. Caco-2 cells (p.95-105) were cultured in control condition or treated with 0.5 or 5 mM sucralose (Suc) for 3 h. Thr172 p-AMPK, total AMPK and GAPDH protein levels were determined through western blot (n = 6). Data are shown as means ± SEM (n = 6) and groups were compared using one-way ANOVA followed by a Holm-Sidak post-test.

Figure 4

Sucralose increases mitochondrial bioenergetics in Caco-2 cells independently of mitochondrial Ca²⁺ entry. (A) Sucralose acutely increases cytosolic Ca²⁺. Caco-2 cells (p.95-105) were cultured in the control condition, loaded with Fura-2 AM, mounted in an open recording chamber and 350 and 380 nm fluorescence was imaged through epifluorescence microscopy. Baseline fluorescence was measured for 25 min and then for 125 min after the addition of KRH (Control) or sucralose (Suc) at a final concentration of 0.5 or 5 mM (n = 4). The graph shows representative kinetics of the Fura-2 AM 350/380 nm fluorescence ratio. (B) Changes in Fura-2 AM 350/380 nm fluorescence ratio after sucralose addition. *P < 0.05 compared to baseline. (C) Sucralose-induced boost of mitochondrial respiration is independent of the mitochondrial Ca⁺² uniporter. Caco-2 cells (p.95-105) were cultured in control condition or treated with 0.5 or 5 mM sucralose for 3 h in the presence or absence of 10 μM ruthenium red (RuRed). Then, their oxygen consumption rate was measured by oxygraphy using Clark's electrode (n = 6). Baseline, non-ATP-associated (400 nM oligomycin) and uncoupled (12 μM CCCP) respiration conditions were analyzed. *P < 0.05 compared to the baseline of control cells in the same condition of presence or absence of RuRed. All data are shown as mean ± SEM and were analyzed using one-way ANOVA followed by a Holm-Sidak post-test.