The mechanism of glycerol transport by human aquaporin 9 (hAQP9), which is a liver-specific AQP water channel and can also transport glycerol, was investigated by using the Xenopus laevis oocyte expression system. It was found that specific glycerol uptake by hAQP9 was concentration-dependent (saturable) at 25 degrees C, conforming to the Michaelis-Menten kinetics with the maximum transport rate (J(max)) of 0.84 pmol/min/oocyte and the Michaelis constant (K(m)) of 9.2 microM, and temperature-dependent, being reduced by about 70% when temperature was lowered from 25 degrees C to 4 degrees C. Such dependences on concentration and temperature are characteristic of a carrier-mediated type of mechanism rather than a channel type, which is expected not to depend on them. Furthermore, several glycerol-related compounds, such as monoacetin, were found to specifically inhibit hAQP9-mediated glycerol uptake, indicating a possibility of competition with glycerol. hAQP9-mediated glycerol uptake was, however, found not to require Na+. All these results suggest that hAQP9 functions as a facilitative carrier for glycerol, although it had been believed to function as a channel. Findings in the present study provide novel insight into its glycerol-transporting mechanism and would help exploring a possibility that hAQP9 inhibitors might help lower blood glucose level by reducing gluconeogenesis by limiting hepatic glycerol uptake.
The HCT-15 human colon cancer cell line has a Na(+)-dependent carrier-mediated transport system for the uptake of glycerol. A similar transport system has been suggested to be present also in the small intestine and is of interest with regard to its role in the absorption of glycerol and possibly some structurally related compounds. To help clarifying functional characteristics of such glycerol transport systems, we examined the effect of butyrate, an agent known to facilitate the differentiation of cells, on glycerol uptake in HCT-15 cells. The uptake of glycerol (0.4 microM) was found to be about 5-fold greater in HCT-15 cells pretreated with butyrate (2 mM) for 24 h than in those untreated. The increase in the uptake by the butyrate treatment was due to an increase in the maximum transport rate. The effect of butyrate was almost completely suppressed when actinomycin D, an inhibitor of gene transcription, and cycloheximide, an inhibitor of protein synthesis, were added to the medium during the butyrate treatment. These results support the suggestion that a specific carrier protein is involved in glycerol uptake by HCT-15 cells and the carrier protein is one of those inducible by butyrate-induced cell differentiation.
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