Luminal leptin inhibits l-glutamine transport in rat small intestine: involvement of ASCT2 and B⁰AT1

Abstract

is the primary metabolic fuel for enterocytes. Glutamine from the diet is transported into the absorptive cells by two sodium-dependent neutral amino acid transporters present at the apical membrane: ASCT2/SLC1A5 and B 0 AT1/ SLC6A19. We have demonstrated that leptin is secreted into the stomach lumen after a meal and modulates the transport of sugars after binding to its receptors located at the brush border of the enterocytes. The present study was designed to address the effect of luminal leptin on Na ϩ -dependent glutamine (Gln) transport in rat intestine and identify the transporters involved. We found that 0.2 nM leptin inhibited uptake of Gln and phenylalanine (Phe) (substrate of B 0 AT1) using everted intestinal rings. In Ussing chambers, 10 mM Gln absorption followed as Na ϩ -induced short-circuit current was inhibited by leptin in a dose-dependent manner (maximum inhibition at 10 nM; IC50 ϭ ϳ0.1 nM). Phe absorption was also decreased by leptin. Western blot analysis after 3-min incubation of the intestinal loops with 10 mM Gln, showed marked increase of ASCT2 and B 0 AT1 protein in the brush-border membrane that was reduced by rapid preincubation of the intestinal lumen with 1 nM leptin. Similarly, the increase in ASCT2 and B 0 AT1 gene expression induced by 60-min incubation of the intestine with 10 mM Gln was strongly reduced after a short preincubation period with leptin. Altogether these data demonstrate that, in rat, leptin controls the active Gln entry through reduction of both B 0 AT1 and ASCT2 proteins traffic to the apical plasma membrane and modulation of their gene expression.Ussing chamber; intestinal rings; leptin receptor; phenylalanine; qPCR GLUTAMINE (Gln) is the most abundant amino acid in the plasma. It is absorbed by the intestine, making this organ a key player in the whole body Gln homeostasis. As Gln is a precursor of the nucleosides and glucose synthesis and is involved in the acid-base balance in the kidney, it is crucial for the interorgan nitrogen flux. Gln is also the most important energy source for the enterocytes, lymphocytes, and fibroblasts and is necessary for the growth and viability of cells maintained in culture (5). Gln from the diet is transported into the enterocyte by two sodium-dependent neutral amino acid transporters present in the apical membrane: ASCT2 (System ASC) and B 0 AT1 (System B) (6). ASCT2/SLC1A5 shows high affinity for alanine (Ala), serine, cysteine, threonine, and Gln (K 0.5 ϭ ϳ20 M), whereas B 0 AT1/ SLC6A19 is a low-affinity transporter (K 0.5 ranging from 1.4 to 4 mM) with preference for large neutral amino acids, including those with bulky lateral chain as phenylalanine (Phe), a specific substrate for this transporter (6). In the cell, most of the Gln is metabolized to cover its energetic requirements, and the rest is transported to the blood by the basolateral sodium-independent exchanger LAT-2/4F2hc, SLC7A8/SLC3A2 (system L) (6).Leptin was initially described as an adipostatic signal controlling food intake and energy expenditure (32). Toda...

In vivo regulation of intestinal absorption of amino acids by leptin

“…These results described here confirm the findings from our previous in vitro studies in rat and in Caco-2 cells, where leptin rapidly inhibited Gln uptake by decreasing the expression in the brush border membrane of the enterocytes of the main Gln transporters present in the intestine, ASCT2 and B 0 AT1 (Ducroc et al 2010, Fanjul et al 2012. We could, therefore, expect that the traffic of these two transporters from the plasma membrane to the intracellular compartments would also be directly regulated by leptin in vivo.…”

“…Similar results were obtained with 5 mM b-Ala (data not shown). Gln absorption experiments were performed at 5 mM and pH 7.4 consistent with the functional characteristics of the intestinal transporters involved, ASCT2 and B 0 AT1 (Bröer 2008, Ducroc et al 2010). …”

“…During the last 15 years, our group and the group of A Bado have been investigating the physiological role of leptin in absorption of nutrients using different physiological and biochemical approaches (Lostao et al 1998, Barrenetxe et al 2001, Ducroc et al 2005, 2010, Iñigo et al 2007, Sakar et al 2009, Fanjul et al 2012. All these results lead to the conclusion that leptin can be considered to be a new gastrointestinal hormone, which is secreted by the stomach (Cammisotto & Bendayan 2012), and rapidly regulates intestinal absorption of nutrients acting from the apical membrane of the enterocytes (Lostao et al 1998, Ducroc et al 2005, 2010, Iñigo et al 2007, Fanjul et al 2012.…”

“…All these results supported our initial discovery about the role of luminal leptin as regulator of intestinal sugar absorption, and were followed by further studies on leptin modulation of sugar transport (Barrenetxe et al 2001, Iñigo et al 2004, 2007, Ducroc et al 2005, Sakar et al 2009). With regard to amino acid absorption, we have recently shown in vitro using rat intestine and Caco-2 cells that leptin also inhibits glutamine (Gln) and phenylalanine transport by the regulation of gene expression and protein activity of the implicated transporters, ASCT2 and B 0 AT1 (Ducroc et al 2010, Fanjul et al 2012. Also, we have reported that in Caco-2 cells, leptin inhibits uptake of proline (Pro) and b-alanine (b-Ala) by PAT1 (Fanjul et al 2012).…”

Leptin regulates sugar and amino acids transport in the human intestinal cell line Caco‐2

“…Uptake of 0.1 mM Phe, which is mainly mediated by B 0 AT1 (Broer 2008), was also inhibited by leptin, which decreased B 0 AT1 abundance in BBMV as well. As it has been reported in rat intestine (Ducroc et al 2010), the lower control uptake values for 0.1 mM Phe compared with the control uptake values for 0.1 mM Gln, would be explained by the lower affinity of B 0 AT1 compared with ASCT2 [Broer et al 2004, Broer 2008, Utsunomiya-Tate et al 1996.…”