Dietary protein level and stage of development affect expression of an intestinal peptide transporter (cPepT1) in chickens

The objective of this study was to evaluate the effect of dietary protein intake and stage of development on chicken intestinal peptide transporter (cPepT1) mRNA abundance. Chicks were sampled at embryonic d 16 (e 16) until the day of hatch (d 0). After hatch, mixed sex Cobb chicks were randomly assigned to diets containing 12, 18, or 24% crude protein (CP). Intakes of the 18 and 24% CP-fed birds were restricted to that consumed by the 12% CP-fed birds. Chickens were sampled on d 0, 1, 3, 5, 7, 10, 14, 21, 28, and 35. The experiment was repeated with the addition of a 4th group with free access to the 24% CP diet. PepT1 mRNA abundance in the duodenum, jejunum, and ileum was quantified by Northern blot. PepT1 mRNA was barely detectable between e 16 and 20 but increased 14- to 50-fold by d 0 (P < 0.001). In chickens fed 12% CP, cPepT1 mRNA abundance decreased throughout the experiment, whereas those fed the restricted 18 or 24% CP diets showed an increase in cPepT1 mRNA abundance (protein x time interaction, P < 0.01). In chicks with free access to the 24% CP diet, cPepT1 mRNA decreased until d 14 but returned to an intermediate level at d 35 (protein x time interaction, P < 0.01). The relative abundance of cPepT1 mRNA varied among intestinal segments with greater abundance occurring in the duodenal or jejunal sections (P < 0.05). Our results indicate that expression of cPepT1 mRNA is regulated by both dietary protein intake and developmental stage.     

促红细胞生成素对缺氧缺血性脑损伤保护作用机制的研究

目的 构建新生儿缺氧缺血性脑病的经典动物模型,并分析在缺氧缺血条件下,促红细胞生成素(EPO)治疗前后胶质细胞谷氨酸运载蛋白2(EAAT2)表达情况。方法 108只出生72 h的SD乳鼠随机分为六组:空白对照组、单纯缺氧组、单纯缺血组、缺氧缺血组、促红细胞生成素治疗组和生理盐水治疗组。缺氧缺血性脑病模型通过双侧结扎颈动脉并且缺氧处理2 h建立。观察并记录不同实验组中乳鼠行为的变化。利用蛋白质印迹技术从蛋白质水平上分别检测不同实验组中EAAT2蛋白的表达情况。利用实时荧光定量聚合酶链反应(PCR)技术从mRNA水平上分别检测不同实验组中新生乳鼠的海马区、大脑皮层EAAT1、EAAT2的基因表达情况。结果 模型呈现典型的缺氧缺血脑病特征。缺氧、缺血、缺氧缺血都可以导致海马组织、大脑皮层中EAAT1的mRNA水平及EAAT2表达量的降低。经过EPO治疗后,EAAT1及EAAT2的基因表达量明显上升。并且,大脑皮层中基因表达水平要高于海马组织中基因表达水平。结论 EAAT2表达量的下降可能是HIE发病的一个原因,EPO的疗效可能是通过上调EAAT2的表达量来实现。     

Dietary S-methylmethionine, a component of foods, has choline-sparing activity in chickens

Acid hydrolysis of dehulled soybean meal (SBM) and corn gluten meal (CGM) followed by chromatographic amino acid analysis (ninhydrin detection) revealed substantial quantities of S-methylmethionine (SMM) in both ingredients (1.65 g SMM/kg SBM; 0.5 g SMM/kg CGM). Young chicks were used to quantify the methionine- (Met) and choline-sparing bioactivity of crystalline L-SMM, relative to L-Met and choline chloride standards in 3 assays. A soy isolate basal diet was developed that could be made markedly deficient in Met, choline, or both. When singly deficient in choline or in both choline and Met, dietary SMM addition produced a significant (P < 0.01) growth response. In Assay 2, dietary SMM did not affect (P > 0.10) growth of chicks fed a Met-deficient, choline-adequate diet. A standard-curve growth assay revealed choline bioactivity values (wt:wt) of 14.2 +/- 0.8 and 25.9 +/- 5.1 g/100 g SMM based on weight gain and gain:food responses, respectively. A fourth assay, using standard-curve procedures, showed choline bioactivity values of 20.1 +/- 1.1 and 22.9 +/- 1.7 g/100 g SMM based on weight gain and gain:food responses, respectively. It is apparent that SMM in foods and feeds has methylation bioactivity, and this has implications for proper assessment of dietary Met and choline requirements as well as their bioavailability in foods and feeds.     

Cyclic AMP stimulates fructose transport in neonatal rat small intestine

Intestinal fructose transporter (GLUT5) expression normally increases significantly after completion of weaning in neonatal rats. Increases in GLUT5 mRNA, protein, and activity can be induced in early weaning pups by precocious consumption of dietary fructose or by perfusion of the small intestine with fructose solutions. Little is known about the signal transduction pathway of the dietary fructose-mediated increase in GLUT5 expression during early intestinal development. Recent microarray results indicate that key gluconeogenic enzymes modulated by cAMP are markedly upregulated by fructose perfusion; hence, we tested the hypothesis that cAMP plays an important role in regulating intestinal fructose absorption by simultaneously perfusing adenylyl cyclase, phosphodiesterase, or protein kinase A (PKA) inhibitors along with fructose. Intestinal fructose uptake rates increased by 100% in rat pups perfused with 8-bromo-cAMP. Simultaneous fructose and dideoxyadenosine (DDA; inhibitor of adenylyl cyclase) perfusion completely inhibited increases in fructose uptake rate induced by perfusion with fructose alone. Fructose perfusion increased intestinal mucosal cAMP concentrations by 27%, but simultaneous perfusion of fructose and DDA inhibited the fructose-induced increase in cAMP. However, GLUT5 and sodium-glucose cotransporter (SGLT1) mRNA abundance and glucose transport rates were each not significantly affected by 8-bromo-cAMP and DDA. Moreover, simultaneous perfusion of the small intestine with fructose and PKA inhibitor or N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamid. 2HCl, both inhibitors of PKA, did not prevent the fructose-induced increases in GLUT5 mRNA abundance and fructose uptake rate. Cyclic AMP appears to modulate fructose transport without affecting GLUT5 mRNA abundance, and without involving PKA.     

Microbial phytase does not improve protein-amino acid utilization in soybean meal fed to young chickens

Three growth trials were conducted with young chicks to evaluate crude protein (CP) utilization in soybean meal (SBM) as affected by dietary addition of microbial phytase. In assay 1, chicks were fed two CP-deficient (50 or 150 g CP/kg) levels of dehulled SBM, and each SBM level was then supplemented with equimolar amounts of cystine or methionine (Met) or with 1200 U phytase/kg. At 50 g CP/kg, cystine or Met supplementation improved (P < 0.05) measures of growth performance, but when 150 g CP/kg from SBM was fed, only Met addition improved (P < 0.05) weight gain, food efficiency and protein efficiency ratio (PER). Thus, Cys was more limiting than Met in the diet that contained 50 g CP/kg, but Met was clearly first-limiting in the diet that contained 150 g CP/kg. Phytase supplementation did not improve (P > 0.10) chick performance at either level of CP. Chicks in assay 2 were fed 100 g CP/kg furnished by SBM, casein or corn gluten meal in the absence and presence of 1200 U phytase/kg. Weight gain, gain/food and PER values were greater (P < 0.05) in chicks fed SBM than in those fed casein, and greater (P < 0.05) in chicks fed casein than in those fed corn gluten meal. Phytase supplementation had no effect (P > 0.10) on any measure of chick performance, regardless of the protein source fed. In assay 3, three deficient levels of CP (50, 100 and 150 g/kg) from SBM were fed in the absence and presence of 1200 U dietary phytase/kg. Weight gain, food efficiency and protein accretion increased linearly (P < 0.05) as a function of protein intake, but phytase supplementation had no effect (P > 0.10) on slopes of the body weight and protein accretion curves. Likewise, phytase addition did not affect (P > 0.10) measures of protein utilization, i.e., weight gain/protein intake and protein gain/protein intake at any of the CP levels that were fed. Because sulfur amino acids are the growth-limiting factors when protein-deficient levels of SBM are fed to young chicks, we conclude that dietary addition of phytase does not improve sulfur amino acid utilization in SBM.     

矢车菊素-3-葡萄糖苷在Caco-2细胞模型的吸收机制研究

目的研究花色苷矢车菊素-3-葡萄糖苷(Cy-3-G)在Caco-2细胞模型的吸收机制。方法建立Caco-2单层细胞模型,观察Cy-3-G的转运情况。加入不同浓度的维拉帕米(P-gp抑制剂)、MK571(MRP2抑制剂)、根皮苷(SGLT1抑制剂)及根皮素(GLUT2抑制剂),观察P-gp、MRP2、SGLT1及GLUT2等转运蛋白在Cy-3-G肠道吸收中的作用。结果 Cy-3-G在Caco-2细胞模型的吸收率随着时间延长逐渐升高,2 h时吸收率在0.76%~2.41%之间,但随着花色苷浓度的升高而降低。维拉帕米和MK571对Cy-3-G在Caco-2细胞模型的吸收无影响(P>0.05),根皮苷和根皮素可显著抑制Cy-3-G的吸收(P<0.05)。结论 P-gp和MRP2对Cy-3-G在肠道的吸收无影响,SGLT1和GLUT2均参与了Cy-3-G在小肠的吸收。[营养学报,2013,35(2):191-194,198]     

Acute administration of cefepime lowers L-carnitine concentrations in early lactation stage rat milk

Our study investigated the potential for important in vivo drug-nutrient transport interactions at the lactating mammary gland using the L-carnitine transporter substrates, cefepime and L-carnitine, as proof-of-concept. On d 4 (n = 6/treatment) and d 10 (n = 6/treatment) of lactation, rats were administered cefepime (250 mg/h) or saline by continuous i.v. infusion (4 h). Serum and milk L-carnitine and cefepime concentrations were quantified by HPLC-UV. In whole mammary gland, organic cation/carnitine transporter (OCTN)1, OCTN2, OCTN3, amino acid transporter B(0,+) (ATB(0,+)), and L-carnitine transporter 2 expression were determined by quantitative RT-PCR and by western blot and immunohistochemistry when possible. Cefepime caused a 56% decrease in milk L-carnitine concentrations on lactation d 4 (P = 0.0048) but did not affect milk L-carnitine at lactation d 10 or serum L-carnitine concentrations at either time. The mean L-carnitine and cefepime milk:serum ratios (M/S) decreased from 9.1 +/- 0.4 to 4.9 +/- 0.6 (P < 0.0001) and 0.89 +/- 0.3 to 0.12 +/- 0.02 (P = 0.0473), respectively, between d 4 and d 10 of lactation. In both groups, OCTN2 (P < 0.0001), OCTN3 (P = 0.0039), and ATB(0,+) (P = 0.004) mRNA expression and OCTN2 protein (P < 0.0001) were higher in mammary glands at d 4 of lactation compared with d 10. Immunohistochemistry revealed OCTN1 and OCTN2 localization in the mammary alveolar epithelium and OCTN3 expression in the interstitial space and blood vessel endothelium. In conclusion, cefepime significantly decreased milk L-carnitine concentrations only at d 4 of lactation. Relative to d 10, enhanced expression of OCTN2 and ATB(0,+) in mammary glands at d 4 of lactation and higher M/S (L-carnitine and cefepime) suggests cefepime competes with L-carnitine for L-carnitine transporters expressed in the lactating mammary gland to adversely affect L-carnitine milk concentrations and these effects depend upon lactation stage.     

ATB0/ASCT2 expression in residual rabbit bowel is decreased after massive enterectomy and is restored by growth hormone treatment

Two weeks after 70% enterectomy, glutamine (Gln) transport is downregulated in rabbit residual bowel due to a decrease in system B(0) activity. Providing epidermal growth factor (EGF) and growth hormone (GH) restores Gln transport by increasing systems A and B(0,+) activities. We hypothesized that changes in Na(+)-dependent broad-spectrum neutral amino acid transporter (ATB(0)/ASCT2) protein and mRNA expression correlate with system B(0) activity. New Zealand White rabbits underwent 70% jejunoileal resection or no resection. Resected rabbits immediately received parenteral EGF, GH, both, or neither agent for 2 wk. Tissues harvested from jejunum, ileum, and colon were subjected to Western and Northern blot analyses for ATB(0)/ASCT2 protein and mRNA. In all tissues, ATB(0)/ASCT2 mRNA was reduced by approximately 50% in resected rabbits compared with nonresected controls. Similar reductions in protein amount occurred in the ileum and cecum. None of the growth factor treatments restored ATB(0)/ASCT2 protein, but GH treatment increased ATB(0)/ASCT2 mRNA abundance 250% in the residual ileum. Because changes in the ATB(0)/ASCT2 protein amount paralleled those in the system B(0) activity in this model, it is likely that this is the protein responsible for this transport system. The increase in mRNA abundance in rabbits treated with GH for 2 wk may be a harbinger of subsequent increases in transporter protein and activity. Unlike reported upregulation of transporters in human colon after small bowel resection, ATB(0)/ASCT2 protein and mRNA expression in rabbit colon are decreased, suggesting different regulatory pathways.     

Glucose transporters (GLUT and SGLT): expanded families of sugar transport proteins

The number of known glucose transporters has expanded considerably over the past 2 years. At least three, and up to six, Na+-dependent glucose transporters (SGLT1-SGLT6; gene name SLC5A) have been identified. Similarly, thirteen members of the family of facilitative sugar transporters (GLUT1-GLUT12 and HMIT; gene name SLC2A) are now recognised. These various transporters exhibit different substrate specificities, kinetic properties and tissue expression profiles. The number of distinct gene products, together with the presence of several different transporters in certain tissues and cells (for example, GLUT1, GLUT4, GLUT5, GLUT8, GLUT12 and HMIT in white adipose tissue), indicates that glucose delivery into cells is a process of considerable complexity.     

Protein digestion in weanling pigs: effect of dietary protein source

The digestion and absorption of dietary protein was measured in weanling pigs (5 wk of age). Diets containing corn and either dried skim milk (DSM), soybean meal (SBM) or corn gluten meal (CGM) were fed for 7 d, and the contents of the stomach and six segments of the small intestine were collected. Nitrogen digestibility increased linearly from the proximal duodenum to the distal ileum, and was highest for DSM, intermediate for SBM and lowest for CGM diets at the distal ileum. The content of free amino acids in digesta increased 8-fold (SBM and CGM) to 12-fold (DSM) between the stomach and the proximal duodenum and reached a maximum concentration in the distal jejunum. Digestion of DSM was more proximal than was that of SBM. Although there was some accumulation of small peptides in the duodenum with a subsequent decrease in the jejunum, the molecular weight profiles of the soluble proteins were relatively constant throughout the small intestine. Protein solubility and the rate of proteolysis in the stomach and upper small intestine were the primary factors that limited the digestion of SBM and CGM.     

Lactobacillus plantarum consumption increases PepT1-mediated amino acid absorption by enhancing protein kinase C activity in spontaneously colitic mice

Although probiotic consumption has generally been shown to have many beneficial effects for the prevention and treatment of inflammatory bowel disease, the effects of Lactobacillus plantarum (LP) on intestinal nutrient absorption, particularly oligopeptide transporter 1 (PepT1)-mediated absorption of dietary protein under inflammatory conditions, has not yet been characterized. In this study, we first investigated the effects of LP consumption on plasma amino acid concentrations and PepT1-mediated absorption of cephalexin in the small intestine of wild-type (WT) mice and interleukin-10 knockout (IL-10(-/-)) mice, a model of spontaneous colitis. We then analyzed expression and distribution of PepT1 and protein kinase C (PKC) activity in the jejunum of these mice. LP consumption (10(9) colony-forming units/0.5 mL) delivered by gavage once per day for 4 wk increased the total plasma amino acid concentration and the concentration of plasma cephalexin through enhancement of PepT1-mediated uptake in LP treated IL-10(-/-) mice compared with IL-10(-/-) mice. However, Western blotting and quantitative PCR analysis revealed no significant differences in PepT1 protein and mRNA expression between LP-treated and untreated mice. Additionally, immunofluorescence analysis showed that PepT1 did not appear to be mislocalized in IL-10(-/-) mice. Interestingly, IL-10(-/-) mice had significantly lower PKC activity and expression of phosphorylated PKC compared with WT mice, and these decreases could be prevented by LP treatment. These data suggest that consumption of LP enhances PepT1-mediated amino acid absorption, likely through alterations in PKC activity, as opposed to changes in expression or distribution of PepT1 in the small intestine of IL-10(-/-) mice.     

Regulation of glucose transporter SGLT1 by ubiquitin ligase Nedd4-2 and kinases SGK1, SGK3, and PKB

OBJECTIVE: Serum- and glucocorticoid-inducible kinase 1 (SGK1) inhibits the ubiquitin ligase neuronal cell expressed developmentally downregulated 4-2 (Nedd4-2), which retards the retrieval of the epithelial Na+ channel ENaC. Accordingly, SGK1 enhances ENaC abundance in the cell membrane. The significance of this effect is shown by an association of an E8CC/CT;I6CC polymorphism in the SGK1 gene with increased blood pressure. However, strong expression of SGK1 in enterocytes not expressing ENaC points to further functions of SGK1. This study was performed to test for regulation of Na+-coupled glucose transporter 1 (SGLT1) by Nedd4-2, SGK1, and/or the related kinases SGK3 and PKB. Additional studies searched for an association of the SGK1 gene with BMI. RESEARCH METHODS AND PROCEDURES: mRNA encoding SGLT1, wild-type Nedd4-2, inactive (C938S)Nedd4-2, wild type SGK1, constitutively active (S422D)SGK1 or inactive (K127N)SGK1, wild-type SGK3, and constitutively active (T308DS473D)PKB or inactive (T308AS473A)PKB were injected into Xenopus oocytes, and glucose transport was quantified from glucose-induced current (I(glc)). BMI was determined in individuals with or without the E8CC/CT;I6CC polymorphism. RESULTS: I(glc) was significantly decreased by coexpression of Nedd4-2 but not of (C938S)Nedd4-2. Coexpression of SGK1, (S422D)SGK1, SGK3, or (T308DS473D)PKB, but not of (K127N)SGK1 or (T308AS473A)PKB, enhanced I(glc) and reversed the effect of Nedd4-2. SGK1 and SGK3 phosphorylated Nedd4-2. Deletion of the SGK/PKB phosphorylation sites in Nedd4-2 blunted the kinase effects. BMI was significantly (p < 0.008) greater in individuals with the E8CC/CT;I6CC polymorphism than in individuals without. DISCUSSION: Overactivity of SGK1 may lead not only to excessive ENaC activity and hypertension but also to enhanced SGLT1 activity and obesity.     

Expression of a cloned ovine gastrointestinal peptide transporter (oPepT1) in Xenopus oocytes induces uptake of oligopeptides in vitro

We determined the primary structure, tissue distribution and in vitro functional characterization of a peptide transporter, oPepT1, from ovine intestine. Ovine PepT1 (oPepT1) cDNA was 2829-bp long, encoding a protein of 707 amino acid residues with an estimated molecular size of 78 kDa and an isoelectric point (pI) of 6.57. Transport function of oPepT1 was assessed by expressing oPepT1 in Xenopus oocytes using a two-electrode voltage-clamp technique. The transport process was electrogenic and pH dependent, but independent of Na+, Cl- and Ca2+. The oPepT1 displayed a broad substrate specificity for transport of neutral and charged dipeptides and tripeptides. All dipeptides and tripeptides examined evoked inward currents in a saturable manner, with an affinity constant (Kt) ranging from 27 micromol/L to 3.0 mmol/L. No responses were detected from tetrapeptides or free amino acids. Northern blot analysis demonstrated that oPepT1 was expressed in the small intestine, omasum and rumen, but was not expressed in liver and kidney. The presence of the peptide transporter in the forestomach at such levels could provide nutritionally important amino acid nitrogen to ruminants.     

Distribution of the H+/peptide transporter PepT1 in human intestine: up-regulated expression in the colonic mucosa of patients with short-bowel syndrome

BACKGROUND: Intestinal adaptation after massive bowel resection in animal models is characterized by increased gut-mucosal growth and expression of nutrient transporters. Few data about these indexes exist in humans with short-bowel syndrome (SBS). OBJECTIVE: The objective was to compare small-bowel and colonic mucosal growth and expression of the peptide transporter PepT1 in adults with or without SBS. DESIGN: Mucosal biopsy specimens were obtained from the small bowel and colon of 33 control subjects with intact intestine and from 13 SBS patients dependent on parenteral nutrition because of chronic malabsorption. Gut-mucosal crypt depth, villus height, and villus width were measured, and expression of PepT1 was determined by Northern blotting, in situ hybridization, and immunohistochemistry. RESULTS: The indexes of small-bowel and colonic mucosal growth were not significantly different between the 2 groups. PepT1 expression was high in the apical region of duodenal, jejunal, and ileal villus epithelial cells; low in absorptive colonocytes; and not significantly different in the distal small intestine of the 2 groups. However, the abundance of PepT1 mRNA in the colon of SBS patients was more than 5-fold that in control subjects (P < 0.01). CONCLUSIONS: Gut adaptation in SBS patients does not appear to involve an increase in gut-mucosal crypt depth or villus size. PepT1 is abundant along the small-bowel brush border in humans; expression in the colon indicates that the large intestine has a mechanism for luminal di- and tripeptide transport. Up-regulation of colonic PepT1 in SBS may adaptively improve accrual of malabsorbed di- and tripeptides, independent of changes in the mucosal surface area.     

Growth hormone and epidermal growth factor upregulate specific sodium-dependent glutamine uptake systems in human intestinal C2BBe1 cells

Glutamine (Gln) is one of the major oxidative fuels of the enterocyte and enters from the lumen via Na(+)-dependent transport mechanisms. When given parenterally, growth hormone (GH) + epidermal growth factor (EGF) increase apical Gln uptake after massive enterectomy in rabbits. Although both receptors are basolateral, GH and EGF are present in luminal contents. We hypothesized that short-term luminal growth factor exposure to enterocytes increases apical Gln uptake by selective upregulation of systems A, B(0,+), or ASC+B(0). A monolayer of C2(BBe)1 cells was exposed for 10 or 60 min to GH (500 microg/L), EGF (100 microg/L), both, or neither. Initial uptake of [(3)H]Gln (50 micromol/L) was measured in the presence of Na(+) or choline. The contributions of systems A, B(0,+), and ASC+B(0) were determined by competitive inhibition with arginine and/or alpha-(methylamino)butyric acid. Gln uptake was linear for up to 8 min. Na(+)-independent transport was negligible. Under control conditions the relative contributions of systems A, B(0,+), and ASC+B(0) were 0, 19 +/- 6, and 80 +/- 4%, respectively. GH alone had no effect on Gln transport. After 10 min of EGF exposure, Na(+)-dependent Gln uptake increased by 50% (P < 0.001) with no change in individual transport systems. Combined EGF and GH for 60 min increased Gln transport by system B(0,+) nearly 250% (P < 0.001) and system A from undetectable levels to 16% of total transport (P < 0.01). Thus, short-term luminal exposure to EGF+GH increases Na(+)-dependent Gln transport mainly by upregulating system B(0+).     

The glucose transporter families SGLT and GLUT: molecular basis of normal and aberrant function

Glucose enters eucaryotic cells via 2 different types of membrane associated carrier proteins, the Na+-coupled glucose transporters (SGLT) and glucose transporter facilitators (GLUT). Three members of the SGLT family function as sugar transporters (SGLT1 and SGLT2) or sensors (SGLT3). The human GLUT family consists of 14 members, of which 11 have been shown to catalyze sugar transport. The individual isotypes exhibit different substrate specificity, kinetic characteristics, and expression profiles, thereby allowing a tissue-specific adaptation of glucose uptake through regulation of their gene expression. Furthermore, some transporters (eg, GLUT4 and GLUT8) are regulated by their subcellular distribution. In addition to catalyzing glucose entry into cells, some isotypes (eg, GLUT2) seem to be involved in the mechanisms of glucosensing of pancreatic beta-cells, neuronal, or other cells, thereby playing a major role in the hormonal and neural control. Targeted disruption in mice has helped to elucidate the physiologic function of some isotypes (GLUT1, GLUT2, GLUT4). Furthermore, several congenital defects of sugar metabolism are caused by aberrant transporter genes (eg, the glucose-galactose malabsorption syndrome, SGLT1; the glucose transporter 1 deficiency syndrome; and the Fanconi-Bickel syndrome, GLUT2). In addition, a malfunction of glucose transporter expression or regulation (GLUT4) appears to contribute to the insulin resistance syndrome.