Impact of culture media glucose levels on the intestinal uptake of organic cations

Abstract

There are several data concerning transporters expression and/or regulation in cell lines maintained in different conditions, such as medium glucose concentration. This work aimed to evaluate the influence of two different extracellular glucose concentrations, commonly used in culture media, on the intestinal absorption of organic cations. Thus, the effect of 5.5 mM glucose and 25 mM glucose (HG) in culture media, was studied on [(3)H]-MPP(+) (1-methyl-4-phenylpyridinium iodide) uptake in Caco-2 cells. Expression of human organic cation transporter type 1 (hOCT1) and human organic cation transporter type 3 (hOCT3) was investigated in cells cultured at both glucose concentrations. [(3)H]-MPP(+) uptake, as well as its affinity for the transporter, were significantly decreased in HG cells. Moreover, hOCT3 mRNA levels were reduced in HG cells. Functional confirmation of this result was made using hOCT3 inhibitors. In conclusion, maintenance of Caco-2 cells (commonly used in several in vitro studies on membrane transport) in HG conditions affects organic cation transport at the intestinal level. Hence, results obtained in these conditions must be analysed with great care, since extracellular glucose levels may originate changes in organic cation nutrient and drug bioavailability.

Fig. 1

The inhibitory effect on [³H]-MPP⁺ apical uptake by Caco-2 cells cultured in high glucose (HG cells) versus cultured under 5.5 mM (control cells). Osmolarity control was performed with mannitol. Confluent Caco-2 monolayers were preincubated at 37 °C for 60 min with Hanks’ buffer and then incubated with 200 nM [³H]-MPP⁺ for 5 min. Each value represents the mean ± SEM of the triplicates of two independent experiments (n = 6). * P < 0.05 in comparison to control cells

Fig. 2

(A) Representative bands of the effect of extracellular glucose concentration on organic cation transporter 1 (hOCT1) and organic cation transporter 3 (hOCT3) transcriptions. (B) Graphic representation of the quantification of mRNA expression for hOCT1 and hOCT3, normalized by comparison with the expression of GAPDH of each sample (R.E.L.—Relative Expression Level). hOCT3 expression was inhibited in Caco-2 cells cultured in high glucose (HG cells) versus cultured under 5.5 mM (control cells). Each value represents the mean ± SEM of three separate cultures.* P < 0.05

Fig. 3

Inhibitory effect of corticosterone (1 and 300 μM) and clonidine (50 μM) on [³H]-MPP⁺ apical uptake by control and HG Caco-2. Confluent Caco-2 monolayers were preincubated at 37 °C in the presence of H₂O (control) or the test compounds and then incubated with 200 nM [³H]-MPP⁺ for 5 min. Each value represents the mean ± SEM of the triplicates of two independent experiments (n = 6). * P < 0.05 (significant increase and decrease)