Non‐Nutritive Sweeteners Reduce Endothelial Cell Nitric Oxide Synthase Activity and Nitric Oxide Production

Abstract

Non-nutritive sweeteners (NNS), such as sucralose and acesulfame potassium (ace K), offer low-calorie alternatives to traditional sweeteners, such as glucose and fructose. Substituting NNSs for these traditional sweeteners, particularly in popular beverages such as soft drinks, has been shown to help mitigate some, not all, of sugar-associated cardiometabolic risk. However, the effect of NNSs on endothelial cell function are not fully understood. The experimental aim of this study was to determine if sucralose and ace k affects endothelial cell inflammation, apoptosis and nitric oxide (NO) production, key functional properties of vascular health. Human umbilical vein endothelial cells were cultured and treated with concentrations of sucralose (0.169mg/mL) and/or ace k (0.127mg/mL) that are equivalent to the amount in one 12 oz can of diet soda (i.e. Diet Coke with Splenda or Pepsi One) for 24 h. Neither sucralose, ace k, nor sucralose + ace k significantly altered markers of endothelial cell inflammation. Total cellular expression of the primary inflammation transcription factor, nuclear factor kappa B (NF-kB) was not different in endothelial cells treated with sucralose (46.0±10.6 AU), ace k (41.3±17.4 AU), sucralose + ace k (47.9±15.4 AU) or control (50.8±15.7 AU). Activated NF-kB was also similar in cells treated with sucralose (130.1±29.6 AU), ace k (122.0±32.4 AU), sucralose + ace k (118.4±33.5 AU) and control (116.7±16.3 AU). In addition, NNSs did not affect endothelial cell apoptosis. Total caspase 3 expression, a key apoptosis protein, was not significantly different between sucralose (65.1±14.0 AU), ace k (78.8±5.4 AU), sucralose + ace k (59.0±9.4 AU) and control (88.1±14.7 AU) conditions. Of note, the expression of active caspase 3 was also not significantly different between sucralose (13.0±3.0 AU), ace k (12.2±3.2 AU), sucralose + ace k (11.8±3.3 AU) and control (11.7±1.6 AU). Endothelial nitric oxide synthase (eNOS) is fundamental to endothelial cell function and the production of NO. Total eNOS expression was not significantly different in cells treated with sucralose (155.5±22.6 AU), ace k (260.9±53.5 AU), sucralose + ace k (175.3±36.3 AU) or control (245.3±27.0 AU); however, sucralose (180.9±36.9 AU), ace k (208.0±40.2 AU) and sucralose + ace k (115.1±17.5 AU) markedly lowered (P<0.05) eNOS activation (phosphorylated-(p)eNOS serine 1177) compared with the control condition (375.9±74.6 AU). Concordantly, NO production was significantly reduced in cells treated with sucralose (7.5±0.4 mmol/L), ace k (7.9±0.2 mmol/L), and sucralose + ace k (6.9±0.2 mmol/L) compared with control (9.1±0.5 mmol/L). The effect of NSSs on p-eNOS and NO production was similar across the conditions. In conclusion, NNSs commonly contained in artificially sweetened beverages do not affect specific endothelial cell inflammation or apoptotic proteins, however NNSs adversely affect endothelial cell nitric oxide production. Future studies are needed to determine whether NNS are associated with impaired endothelial vasodilator dysfunction and the potential vascular consquences of reduced NO bioavailabilty.