Transport mechanisms of the large neutral amino acid L-phenylalanine in the human intestinal epithelial caco-2 cell line

The transepithelial transport and the intracellular accumulation of the large neutral amino acid L-phenylalanine (L-Phe) were studied in monolayers of Caco-2 cells, cultivated in a bicameral insert system, to characterize the mechanisms involved in the absorption of this essential amino acid by the human intestinal mucosa. In our model, L-Phe was transported selectively in the apical (AP)-to-basolateral (BL) direction. AP-to-BL transport of L-Phe was temperature dependent and Na(+) independent, increased in the absence of protein synthesis and showed competition with large neutral and cationic amino acids. By contrast, transport in the BL-to-AP direction mainly resulted from passive movement (probably paracellular passage and transcellular diffusion). L-Phe accumulation into Caco-2 cells was higher from the BL pole than from the AP pole and characterized by the incorporation of most of the accumulated molecules into newly synthesized proteins. In addition, L-Phe accumulation was Na(+) dependent from both poles, whereas only accumulation from the AP pole was sensitive to inhibition by both large neutral and cationic amino acids. These results suggest that the processes involved in AP-to-BL transport and AP accumulation of this amino acid are very different from those involved in BL-to-AP transport and BL accumulation.     

严重创伤对肠上皮细胞中性氨基酸载体ASCT2表达的影响

目的:研究用肠上皮细胞系Caco-2定量分析在严重创伤情况下氨基酸的转运变化及其载体蛋白表达情况.方法:体外培养肠上皮细胞系Caco-2,首先分析其细胞膜表面Na -依赖性中性氨基酸转运及其转运载体功能特性.将细胞置于缺氧、营养剥夺和缺血等创伤条件下,继续培养1～6 h,观察其刷状缘膜囊氨基酸转运及其相应转运载体蛋白及mRNA表达情况.结果:Caco-2细胞膜表面Na -依赖性L-丙氨酸的转运速率,高于L-谷氨酰胺(876±67.5) pmol/mg 蛋白·min-1 vs (635±52.8) pmol/mg 蛋白·min-1,P＜0.01).在主要的中性氨基酸载体中,仅仅成功扩增出ASCT2的mRNA条带.与对照组相比,在营养剥夺及缺血条件下,L-丙氨酸和L-谷氨酰胺的转运速率明显下降(P＜0.01),而缺氧对氨基酸转运无明显影响.这种变化主要引起转运动力学Vmax值下降,Km值无明显变化.与对照组相比,营养剥夺及缺血条件下相关转运载体蛋白及mRNA表达下降.结论:严重创伤对Na -依赖性中性氨基酸转运及关键性转运载体调控特点不同.这对临床上危重症病人特殊的营养底物需求,可能提供积极的理论依据.     

肠道必需氨基酸代谢及其功能的研究进展

在肠道代谢过程中,食物必需氨基酸可不同程度地被肠道组织利用。这些必需氨基酸,除了用于合成肠黏膜蛋白质外,还可通过不同途径在肠上皮细胞内代谢。它们不仅是小肠黏膜的能量物质,同时还参与肠道内氨基酸、谷胱甘肽和多胺等多种生物活性物质的合成,对维持肠黏膜完整性和肠道功能有重要意义,对整个机体的代谢产生重要的影响。     

Zinc intestinal absorption in rats: specificity of amino acids as ligands

The luminal phase of zinc intestinal absorption may be mediated by low-molecular-weight substances originated in digestive, metabolic or secretory processes. Amino acids are considered primary candidates for this role through the formation of complexes with zinc. However, structural characteristics that may be indispensable for this physiological function have not been explored in vivo. We investigated the comparative effectiveness of four amino acids and their respective chemically related homologues on the absorption of zinc by the jejunum, ileum and colon of the rat using a perfusion procedure. L-Tryptophan (Trp) allowed for significantly greater zinc absorption than tryptophol (Tpl) in all areas of the gut. L-Histidine (His) and imidazole (Imd) had similar effects in both the jejunum and the ileum. Imd allowed for much greater zinc absorption from the colon than did His (His, 388 +/- 31; Imd, 937 +/- 107 pmol/min X cm, P less than 0.001). Proline (Pro) was a more effective ligand than pyroglutamate (Pyr) in the ileum (Pro, 559 +/- 19; Pyr, 352 +/- 22 pmol/min X cm, P less than 0.001), but not in the jejunum or the colon. L-Cysteine was superior to N-acetyl-L-cysteine only in the ileum (508 +/- 45 vs. 348 +/- 25 pmol/min X cm, P less than 0.01). The greater zinc absorption achieved by amino acids than by non-amino acid homologues in the small intestine appeared to be due to the presence of both mediated and nonmediated transport mechanisms for amino acids but of only nonmediated zinc uptake for the homologues. In the colon, where amino acid absorption does not take place, high structural affinity for zinc, such as that exhibited by Imd, allowed for considerable absorption of the trace element.     

Stimulation of neutral amino acid transport by fructose in epithelial cells isolated from rat intestine.

Interest in the nutritional and physiological implications of the high dietary intakes of fructose from sucrose and isomerized corn sweeteners has directed attention to the specific metabolic properties of this monosaccharide. Epithelial cells isolated from the small intestine of Wistar rats fed a stock diet showed 24% to 57% higher transport of 1 mM leucine, isoleucine, valine, alanine, phenylalanine tryptophan and histidine in the presence of 10 mM fructose than in its absence. In contrast, 10mM glucose generally inhibited the transport of these amino acids and 10mM sorbose had no effect on leucine transport. Fructose failed to consistently stimulate the transport of basic amino acids and generally inhibited the transport of glucose and galactose, indicating that the stimulation was relatively specific for neutral amino acids. Cells preloaded with fructose optimally stimulated leucine transport in the absence of extracellular fructose. Ouabain and dinitrophenol inhibited the stimulation of leucine transport by intracellular fructose after 2 minutes. A stimulation mediated by an exchange transport mechanism was rejected on the basis of the failure of 1 mM neutral amino acids either to inhibit the transport of 10 mM fructose or to accelerate the movement of fructose out of fructose-loaded cells. Although the mechanism remains unknown, these results demonstrate a specific effect of fructose at the intestinal level that produces a stimulation of neutral amino acid transport.     

肠黏膜刷状缘中性氨基酸载体ASCT2的结构和功能特点

ASCT2是肠黏膜刷状缘的一种Na 依赖性中性氨基酸载体,它的功能主要是转运中性氨基酸,如丙氨酸、丝氨酸、半胱氨酸、苏氨酸、谷氨酰胺和天冬酰胺等.对ASCT2分子结构、功能特点和调控特性等的研究,有助于对肠道功能的认识,并为临床短肠综合征(SBS)及其他肠功能障碍疾病提供新的治疗对策.     

Oral bioavailability of glyphosate: studies using two intestinal cell lines

Glyphosate is a commonly used nonselective herbicide that inhibits plant growth through interference with the production of essential aromatic amino acids. In vivo studies in mammals with radiolabeled glyphosate have shown that 34% of radioactivity was associated with intestinal tissue 2 h after oral administration. The aim of our research was to investigate the transport, binding, and toxicity of glyphosate to the cultured human intestinal epithelial cell line, Caco-2, and the rat small intestinal crypt-derived cell line, ileum epithelial cells-18 (IEC-18). An in vitro analysis of the transport kinetics of [14C]-glyphosate showed that 4 h after exposure, approximately 8% of radiolabeled glyphosate moved through the Caco-2 monolayer in a dose-dependent manner. Binding of glyphosate to cells was saturable and approximately 4 x 10(11) binding sites/cell were estimated from bound [14C]. Exposure of Caco-2 cells to > or =10 mg/ml glyphosate reduced transmembrane electrical resistance (TEER) by 82 to 96% and increased permeability to [3H]-mannitol, indicating that paracellular permeability increased in glyphosate-treated cells. At 10-mg/ml glyphosate, both IEC-18 and Caco-2 cells showed disruption in the actin cytoskeleton. In Caco-2 cells, significant lactate dehydrogenase leakage was observed when cells were exposed to 15 mg/ml of glyphosate. These data indicate that at doses >10 mg/ml, glyphosate significantly disrupts the barrier properties of cultured intestinal cells.     

The indicator amino acid oxidation method identified limiting amino acids in two parenteral nutrition solutions in neonatal piglets

Recent studies using the indicator amino acid oxidation (IAAO) technique in TPN-fed piglets and infants have been instrumental in defining parenteral amino acid requirements. None of the commercial products in use are ideal when assessed against these new data. Our objectives were to determine whether the oxidation of an indicator amino acid would decline with the addition of amino acids that were limiting in the diets of TPN-fed piglets, and to use this technique to identify limiting amino acids in a new amino acid profile. Piglets (n = 26) were randomized to receive TPN with amino acids provided by Vaminolact (VM) or by a new profile (NP). After 5 d of TPN administration, lysine oxidation was measured using a constant infusion of L- [1-(14)C]-lysine. Immediately following the first IAAO study, the piglets were further randomized within diet group to receive either 1) supplemental aromatic amino acids (AAA), 2) sulfur amino acids (SAA) or 3) both (AAA+SAA) (n = 4-5 per treatment group). A second IAAO study was carried out 18 h later. In the first IAAO study, lysine oxidation was high for both groups (18 vs. 21% for VM and NP, respectively, P = 0.055). The addition of AAA to VM induced a 30% decline in lysine oxidation compared with baseline (P < 0.01). Similarly, SAA added to NP lowered lysine oxidation by approximately 30% (P < 0.01). The application of the IAAO technique facilitates rapid evaluation of the amino acids that are limiting to protein synthesis in parenteral solutions.     

Splanchnic exchange of amino acids after amino acid ingestion in patients with chronic renal insufficiency

Splanchnic exchange (net uptake or release) of amino acids (AAs) was evaluated by measuring arterial-hepatic venous differences for AAs and hepatic blood flow in patients with chronic renal insufficiency (CRI) and control subjects before and for 70 min after the ingestion of an AA mixture simulating an animal protein meal. In CRI after AA ingestion, splanchnic exchange area for total nonessential AAs (NEAAs) is increased 135% over control subjects because of an augmented escape of proline, glutamate, serine, glycine, alanine, and cyst(e)ine; contrarily, glutamine shows an increased splanchnic uptake. Splanchnic exchange area for total essential AAs (EAAs) is increased only by 67% over controls because of a higher escape of threonine, isoleucine, phenylalanine, and histidine. Abnormalities in arterial areas for AAs parallel those in splanchnic areas except for glutamine and isoleucine. Data indicate that in CRI, at least for 70 min after an AA meal, splanchnic organs metabolize abnormally ingested AAs and export an increased and unbalanced bulk of AAs, severely affecting postprandial arterial profile of AAs.     

Evidence for multiple signaling pathways in the regulation of gene expression by amino acids in human cell lines

In mammals, plasma concentrations of amino acids (AA) are affected by nutritional or pathologic conditions. Alterations in AA profiles have been reported as a result of a deficiency of any one of the essential AA, a dietary imbalance of AA or an insufficient intake of protein. In recent years, evidence has accumulated that AA availability regulates the expression of several genes involved in the regulation of a number of cellular functions or AA metabolism. Nevertheless, the molecular mechanisms involved in the AA regulation of mammalian gene expression are limited, particularly the signaling pathways mediating the AA response. This work provides a better understanding of the signaling pathways involved in the AA control of gene expression. We studied the expression of C/EBP homologous protein (CHOP) and asparagine synthetase (AS) in response to deprivation of a single AA and investigated the possible link between protein synthesis inhibition due to amino acid limitation and gene expression. We have shown the following: 1) several mechanisms are involved in the AA control of gene expression. When omitted from the culture medium, each AA can activate one (or several) specific signaling pathways leading to the regulation of one specific pattern of genes. 2) AA limitation by itself can induce gene expression independently of a cellular stress due to protein synthesis inhibition. Together, these results suggest that AA control of gene expression involves several specific mechanisms by which one AA (or one group of AA) can activate one signaling pathway and thus alter one specific pattern of gene expression.     

肠上皮细胞中性氨基酸载体B0AT1的真核表达及稳定转染体系的建立

目的: 构建可表达人中性氨基酸载体B0AT1基因的真核表达载体,建立重组质粒转染的Hela细胞系,筛选出稳定表达人B0AT1的细胞株. 方法: 提取人正常小肠上皮细胞总RNA,采用RT-PCR方法扩增出B0AT1基因片段,EcoRⅠ和XbaⅠ双酶切后,将其插入至真核表达载体pcDNA3.1中,构建重组表达质粒pcDNA3.1-B0AT1,转化E.coli DH5α菌株感受态细胞,将阳性克隆脂质体法转染Hela细胞,经G418筛选获得稳定表达株,用RT-PCR法检测B0AT1基因的mRNA表达. 结果: 从小肠上皮细胞中成功克隆到人B0AT1基因.酶切和序列测定表明已成功构建真核表达载体pcDNA3.1-B0AT1,将此重组质粒转染的Hela细胞,可稳定表达人B0AT1基因,并筛选出稳定表达B0AT1的Hela细胞系.氨基酸摄取实验证实,转染pcDNA3.1-B0AT1的Hela细胞具有B0AT1基因的生物学活性. 结论: 重组人中性氨基酸载体B0AT1克隆成功,并在Hela细胞中获得稳定表达.     

Transport mechanisms of the imino acid L-proline in the human intestinal epithelial caco-2 cell line

The intestinal transport of L-proline (L-Pro) has been investigated in various animal species with the use of different tissue preparations. Because major qualitative differences have been observed among the species, it is difficult to extent the results obtained with animal models to humans. In addition, studies on human tissue are lacking because of difficulties in obtaining material for experiments. To characterize the mechanisms involved in the intestinal absorption of L-Pro in humans, the transport of this nonessential imino acid was studied in monolayers of human intestinal Caco-2 cells that were cultivated on microporous membranes. In this model, L-Pro was transported selectively in the apical (AP)-to-basolateral (BL) direction. This transport was significantly reduced by metabolic inhibitors and by an incubation at 4 degrees C; it was Na(+) dependent and showed competition with (methylamino)-alpha-isobutyric acid and L-hydroxyproline. By contrast, transport in the BL-to-AP direction resulted to a large extent from passive movement (paracellular passage and transcellular diffusion). L-Pro accumulation by Caco-2 cells was significantly greater from the AP pole than from the BL pole. About 30-50% of the accumulated molecules were incorporated into newly synthesized proteins in a process inhibited by cycloheximide, whereas the remainder were extensively metabolized into non-amino acid compounds. L-Pro accumulations from the AP and BL poles were both Na(+) dependent, but they exhibited different characteristics. AP accumulation was inhibited by competition with (methylamino)-alpha-isobutyric acid, L-hydroxyproline and, to a lesser extent, D-Pro, whereas BL accumulation was inhibited by competition with L-hydroxyproline, (methylamino)-alpha-isobutyric acid, alpha-aminoisobutyric acid, L-histidine and small neutral amino acids. The results indicate that AP-to-BL transport and AP accumulation of L-Pro exhibited very different characteristics than BL-to-AP transport and BL accumulation.     

Renal metabolism of amino acids: its role in interorgan amino acid exchange

The kidneys play a role in the synthesis and interorgan exchange of several amino acids. The quantitative importance of renal amino acid metabolism in the body is not, however, clear. We review here the role of the kidney in the interorgan exchange of amino acids, with emphasis on quantitative aspects. We reviewed relevant literature by using a computerized literature search (PubMed) and checking relevant references from the identified articles. Our own data are discussed in the context of the literature. The kidney takes up glutamine and metabolizes it to ammonia. This process is sensitive to pH and serves to maintain acid-base homeostasis and to excrete nitrogen. In this way, the metabolism of renal glutamine and ammonia is complementary to hepatic urea synthesis. Citrulline, derived from intestinal glutamine breakdown, is converted to arginine by the kidney. Renal phenylalanine uptake is followed by stoichiometric tyrosine release, and glycine uptake is accompanied by serine release. Certain administered oligopeptides (eg, glutamine dipeptides) are converted by the kidneys to their constituent components before they can be used in metabolic processes. The kidneys play an important role in the interorgan exchange of amino acids. Quantitatively, for several important amino acids, the kidneys are as important as the gut in intermediary metabolism. The kidneys may be crucial "mediators" of the beneficial effects of specialized, disease-specific feeding solutions such as those enriched in glutamine dipeptides.     

Treatment of acid maltase deficiency with a diet high in branched-chain amino acids

Acid maltase deficiency in adults is associated with progressive muscle weakness and may effect respiratory muscles resulting in respiratory failure. The biochemical and clinical manifestations of acid maltase deficiency arise from a marked deficiency of the lysosomal enzyme alpha-glucosidase (acid maltase), which normally degrades glycogen to free glucose. In the past few years, high-protein diets have provided an alternative energy source for these patients and resulted in improved muscle strength. Recently, we treated a ventilator-dependent acid maltase-deficient patient with a general diet supplemented with branched-chain amino acids. Branched-chain amino acids are the principal amino acids involved in muscle protein synthesis and utilization. While on this diet, the patient had improvement of respiratory function and muscle strength and was able to be weaned from the ventilator during the day. In addition to his nutritional status, levels of serum branched-chain amino acids, showed improvement within 2 months after the diet started. This diet shows potential advantages over a high-protein diet without supplemented branched-chain amino acids for the treatment of acid maltase deficiency. These include theoretical sparing of amino acids required for muscle protein synthesis by providing higher concentrations of postprandial branched-chain amino acids in the circulation. Also, the liquid formula would be better tolerated by a ventilator-dependent or debilitated patient rather than a high-protein general diet. Further experience with branched-chain amino acid formulas will be needed to substantiate their efficacy in the treatment of acid maltase deficiency.     

淋巴小结相关上皮细胞在肠黏膜屏障中的作用

淋巴小结相关上皮细胞(M细胞)是覆盖在器官相关淋巴组织(OALT)上的滤泡相关上皮(FAE)中特殊分化出来的一种细胞.在胃肠道,M细胞主要承担着向黏膜下转运肠腔内的抗原,进而诱发免疫反应的功能.作为胃肠道黏膜免疫屏障的门户,M细胞被认为是肠黏膜免疫屏障中重要的功能性通道.以下就M细胞的特殊结构、生物学特性及其在肠黏膜屏障中的特殊作用作一综述.