Medical College of Wisconsin
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Non-nutritive sweeteners competitively inhibit P-glycoprotein in liver. FASEB J 2022 May;36 Suppl 1

Date

05/14/2022

Pubmed ID

35554226

DOI

10.1096/fasebj.2022.36.S1.R4915

Scopus ID

2-s2.0-85130031224

Abstract

Non-nutritive sweeteners (NNS) are popular sugar replacements that provide sweet taste with little to no calories and are used in a wide range of products, including foods, beverages, and medications. Although NNS are recognized as safe for the general population at the Acceptable Daily Intake (ADI) by the Food and Drug Administration, the effects of NNS on specific patient populations and on human development are not completely understood. Previously, our lab has shown that in mice, pups exposed to NNS during early development present alterations in liver detoxification and metabolic enzymes. This led us to question whether NNS may disrupt detoxification in the liver, including transmembrane efflux transporters such as P-glycoprotein (PGP). PGP plays a major role in the distribution of common xenobiotics and drugs, such as antibiotics, antidepressants, and antivirals, by excreting their conjugated metabolites from liver cells to the general circulation (blood and bile). Mutations in PGP, along with administration of pharmacological inhibitors lead to intracellular accumulation of its substrates and toxicity. We hypothesize that two common NNS, acesulfame potassium (AceK) and sucralose (Sucr), inhibit PGP's efflux function in liver cells and alter PGP expression, suggesting reduced detoxification capacity in the liver. To investigate the effects of NNS on hepatic PGP, we first exposed the HepG2 liver carcinoma cell line to AceK and Sucr and assessed PGP mRNA expression by qPCR. Cells were treated with varying concentrations of AceK, Sucr, or combined NNS (AceK+Sucr) that spanned levels below and above the ADI for each sweetener for either 24h acute exposure or 72h chronic exposure. In parallel, PGP protein levels were assessed by Western blot. 24h combined exposure of AceK and Sucr, at concentrations above the ADI, increased PGP transcript expression, but had no effect at concentrations below or equal to the ADI. Only levels above the ADI of AceK and Sucr treatment at 72h led to increased PGP mRNA levels. Combined NNS treatment for 72h, however, increased PGP transcript levels at lower concentrations than acute exposure. In western blot analysis of protein expression, concentrations of combined NNS below the ADI increased PGP levels, in contrast to the qPCR data. As PGP inhibitor drugs have been shown to similarly affect its expression, we then wished to understand whether NNS impact PGP's efflux action. Therefore, a cell-free assay measuring PGP's ATPase activity was used. Complementarily, in vitro assays of substrate retention and substrate efflux in HepG2 were performed to measure cellular efflux. We found that physiologically relevant NNS concentrations led to significant stimulation of ATPase activity, similar to potent PGP competitive inhibitors such as Verapamil. Similarly, low concentrations of AceK, Sucr, or combined NNS, comparable to 1/8th to 1 times the ADI for each, significantly retain PGP substrate Calcein-AM intracellularly, or reduce efflux of PGP substrate Rhodamine 123, confirming PGP inhibition by NNS. Our results provide evidence that two common NNS, AceK and Sucr, are both substrates and inhibitors of PGP, and that both acute and chronic exposure to those NNS impacts PGP expression. Most importantly, altered PGP efflux function was observed after exposure of hepatic cells to sub-ADI concentrations of NNS within expected exposure to common foods and beverages, suggesting risks for NNS consumers when taking medications that utilize the PGP as a main detoxification transporter.

Author List

Danner L, Valdes R, Olivier-Van Stichelen S

Author

Stephanie Olivier-Van Stichelen PhD Assistant Professor in the Biochemistry department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Adenosine Triphosphatases
Animals
Humans
Liver
Mice
Non-Nutritive Sweeteners
RNA, Messenger