A Novel Antagonist Peptide Reveals a Physiological Role of Insulin-Like Peptide 5 in Control of Colorectal Function.

Insulin-like peptide 5 (INSL5), the natural ligand for the relaxin family peptide receptor 4 (RXFP4), is a gut hormone that is exclusively produced by colonic L-cells. We have recently developed an analogue of INSL5, INSL5-A13, that acts as an RXFP4 agonist in vitro and stimulates colorectal propulsion in wild-type mice but not in RXFP4-knockout mice. These results suggest that INSL5 may have a physiological role in the control of colorectal motility. To investigate this possibility, in this study we designed and developed a novel INSL5 analogue, INSL5-A13NR. This compound is a potent antagonist, without significant agonist activity, in two in vitro assays. We report here for the first time that this novel antagonist peptide blocks agonist-induced increase in colon motility in mice that express RXFP4. Our data also show that colorectal propulsion induced by intracolonic administration of bacterial products (short-chain fatty acids, SCFAs) is antagonized by INSL5-A13NR. Therefore, INSL5-A13NR is an important research tool and potential drug lead for the treatment of colon motility disorders, such as bacterial diarrheas.

The identification of neuronal control pathways supplying effector tissues in the stomach.

The stomach acts as a buffer between the ingestion of food and its processing in the small intestine. It signals to the brain to modulate food intake and it in turn regulates the passage of a nutrient-rich fluid, containing partly digested food, into the duodenum. These processes need to be finely controlled, for example to restrict reflux into the esophagus and to transfer digesta to the duodenum at an appropriate rate. Thus, the efferent pathways that control gastric volume, gastric peristalsis and digestive juice production are critically important. We review these pathways with an emphasis on the identities of the final motor neurons and comparisons between species. The major types of motor neurons arising from gastric enteric ganglia are as follows: immunohistochemically distinguishable excitatory and inhibitory muscle motor neurons; four neuron types innervating mucosal effectors (parietal cells, chief cells, gastrin cells and somatostatin cells); and vasodilator neurons. Sympathetic efferent neurons innervate intramural arteries, myenteric ganglia and gastric muscle. Vagal efferent neurons with cell bodies in the brain stem do not directly innervate gastric effector tissues; they are pre-enteric neurons that innervate each type of gastric enteric motor neuron. The principal transmitters and co-transmitters of gastric motor neurons, as well as key immunohistochemical markers, are the same in rat, pig, human and other species.

Tissue-specific deletion of mouse basolateral uniporter LAT4 (Slc43a2) reveals its crucial role in small intestine and kidney amino acid transport.

KEY POINTS: LAT4 is a broadly expressed uniporter selective for essential branched chain amino acids, methionine and phenylalanine, which are involved in epithelial transport. Its global deletion leads to an early malnutrition-like phenotype and death within 10 days after birth. Here, we tested the impact of deleting LAT4 selectively in the mouse intestine. This affected slightly the absorption of amino acids (AAs) and delayed gastrointestinal motility; however, it had no major phenotypic effect, even when combined with aromatic AA uniporter TAT1 knockout (KO). Conversely, kidney tubule-selective deletion of LAT4 led to a substantial aminoaciduria that strongly increased under a high protein diet. Combining a partial tubular LAT4 deletion with TAT1 KO implicated their synergistic action on AA reabsorption. These results show that LAT4 plays an important role for kidney AA reabsorption, but that its functional role in intestinal AA absorption is largely dispensable. ABSTRACT: Amino acid (AA) transporter LAT4 (Slc43a2) functions as facilitated diffusion uniporter for essential neutral AAs and is highly expressed at the basolateral membrane of small intestine (SI) and kidney tubule epithelia. Previously, we showed that LAT4 global knockout (KO) mice were born at the expected Mendelian ratio but died within 10 days. Their failure to gain weight and a severe malnutrition-like phenotype contrasted with apparently normal feeding, suggesting a severe intestinal AA absorption defect. In the present study, using conditional global and tissue-specific LAT4 KO mouse models, we nullified this hypothesis, demonstrating that the selective lack of intestinal LAT4 does not impair postnatal development, although it leads to an absorption defect accompanied by delayed gastrointestinal motility. Kidney tubule-specific LAT4 KO led to a substantial aminoaciduria as a result of a reabsorption defect of AAs transported by LAT4 and of other AAs that are substrates of the antiporter LAT2, demonstrating, in vivo, the functional co-operation of these two transporters. The major role played by basolateral uniporters in the kidney was further supported by the observation that, in mice lacking TAT1, another neutral AA uniporter, a partial LAT4 KO led to a synergistic increase of urinary AA loss. Surprisingly in the SI, the same combined KO induced no major effect, suggesting yet unknown compensatory mechanisms. Taken together, the lethal malnutrition-like phenotype observed previously in LAT4 global KO pups is suggested to be the consequence of a combinatorial effect of LAT4 deletion in the SI, kidney and presumably other tissues.

Phosphorylation of mouse intestinal basolateral amino acid uniporter LAT4 is controlled by food-entrained diurnal rhythm and dietary proteins.

Adaptive regulation of epithelial transporters to nutrient intake is essential to decrease energy costs of their synthesis and maintenance, however such regulation is understudied. Previously we demonstrated that the transport function of the basolateral amino acid uniporter LAT4 (Slc43a2) is increased by dephosphorylation of serine 274 (S274) and nearly abolished by dephosphorylation of serine 297 (S297) when expressed in Xenopus oocytes. Phosphorylation changes in the jejunum of food-entrained mice suggested an increase in LAT4 transport function during food expectation. Thus, we investigated further how phosphorylation, expression and localization of mouse intestinal LAT4 respond to food-entrained diurnal rhythm and dietary protein content. In mice entrained with 18% protein diet, LAT4 mRNA was not submitted to diurnal regulation, unlike mRNAs of luminal symporters and antiporters. Only in duodenum, LAT4 protein expression increased during food intake. Concurrently, S274 phosphorylation was decreased in all three small intestinal segments, whereas S297 phosphorylation was increased only in jejunum. Interestingly, during food intake, S274 phosphorylation was nearly absent in ileum and accompanied by strong phosphorylation of mTORC1 target S6. Entraining mice with 8% protein diet provoked a shift in jejunal LAT4 localization from the cell surface to intracellular stores and increased S274 phosphorylation in both jejunum and ileum during food anticipation, suggesting decreased transport function. In contrast, 40% dietary protein content led to increased LAT4 expression in jejunum and its internalization in ileum. Ex vivo treatments of isolated intestinal villi fraction demonstrated that S274 phosphorylation was stimulated by protein kinase A. Rapamycin-sensitive insulin treatment and amino acids increased S297 phosphorylation, suggesting that the response to food intake might be regulated via the insulin-mTORC1 pathway. Ghrelin, an oscillating orexigenic hormone, did not affect phosphorylation of intestinal LAT4. Overall, we show that phosphorylation, expression and localization of intestinal mouse LAT4 responds to diurnal and dietary stimuli in location-specific manner.