Cationic Amino Acid Transporter Research Articles

Augmented Endothelial Specific Cationic Amino Acid Transporter‐1 Expression Prevents Renal Fibrosis and Inflammation and Loss of Renal Function in Heart Failure

INTRODUCTIONIt is now well established that impaired renal function associated with heart failure (HF) increases morbidity and mortality in HF patients. There are no effective treatments to rescue renal function in HF and the precise mechanisms that drive renal injury in HF remain elusive. Transport of L‐arginine via cationic amino acid transporter‐1 (CAT‐1) influences renal nitric oxide bioavailability. We hypothesised that impaired L‐arginine transport via CAT‐1 is central to the development of renal dysfunction in HF.METHODSAdult male 18–26 week old wild‐type mice (WT), transgenic mice with dilated cardiomyopathy (DCM) and double transgenic mice with DCM and endothelial‐specific overexpression of CAT1 (HFCAT‐1) were used (n=8 per group). Transgenic mice with dilated cardiomyopathy (DCM) have cardiac specific overexpression of Mammalian sterile 20‐like kinase‐1 (Mst1) which leads to apoptosis and DCM. The double transgenic HFCAT‐1 mouse model has endothelial specific overexpression of CAT1 driven by the Tie‐2 promotor and cardiac specific overexpression of Mst1. Cardiac and renal function were assessed via echocardiography and via calculating the albumin to creatinine ratio, respectively. Fibrosis was assessed via Masson's trichrome staining. Renal inflammation was assessed via expression of pro‐ and anti‐inflammatory gene expression. Plasma nitrate and nitrite levels were assessed via ELISA. Isolated renal cells from WT mice (n=3) were incubated with CAT1 antisense (2.5 μM) or its scrambled oligonucleotide for a period of 4‐h and then CAT1, collagen III and collagen IV expression were measured by PCR.RESULTSExpression of renal CAT1 was less in DCM mice (by 21%) compared to WT mice (P=0.04) and this was restored in HFCAT1 mice. Urinary albumin/creatinine ratio was greater in DCM mice (by 72%) compared to WT (P=0.04). Urinary albumin/creatinine ratio was normalized in HFCAT1 mice. Renal mRNA expression of Il6 was less in HFCAT‐1 mice than DCM mice (P=0.03). Renal mRNA expression of Il10 was 57% less in DCM mice than in WT mice and this was restored in HFCAT‐1 mice (P<0.05). Renal mRNA expression of Pai1 was greater in DCM mice (by 42%), but not HFCAT‐1 mice, compared to WT mice (P=0.01). Tubulointerstitial and glomerular fibrosis were 89% and 76% greater respectively in DCM mice than in WT mice (P<0.05). Tubulointerstitial and glomerular fibrosis were normalized in HFCAT‐1 mice. CAT1 antisense reduced the expression of CAT1 mRNA in isolated renal cells by 46% and collagen III expression in these cells was 8 fold greater compared to control. Ejection fraction was 33% less in DCM mice than in WT (P<0.01) and this was not restored in HFCAT1 mice (P=0.24).CONCLUSIONRestoration of endothelial specific CAT1 expression normalizes renal inflammation and fibrosis and prevents the loss of GFR in experimental HF.Support or Funding InformationNHMRC Program grant (DK) and Baker Heart and Diabetes Institute Early Career Grant (NR)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Profound Decrease in Glomerular Arginine Transport by CAT (Cationic Amino Acid Transporter)-1 Contributes to the FLT-1 (FMS-Like Tyrosine Kinase 1) Induced Preeclampsia in the Pregnant Mice.

Endothelial dysfunction because of nitric oxide inactivation has been suggested to play a role in the pathogenesis of preeclampsia. During pregnancy, L-arginine transport by CAT-1 (cationic amino acid transporter 1), the only transporter for eNOS (endothelial nitric oxide synthase) is inhibited. We hypothesize that maternal arginine deficiency contributes to the development of preeclampsia. Adenovirus-mediated overexpression of sFlt-1 (soluble fms-like tyrosine kinase 1) in virgin and pregnant mice resulted in glomerular endotheliosis, hypertension, and albuminuria. L-arginine prevented the increase in blood pressure and albuminuria in Flt-1 pregnant but not in Flt-1 virgin mice. Flt-1 augmented arginine transport in pregnant but not in virgin dames. Ex vivo inhibition of CAT-2 leaving exclusively CAT-1 activity, decreased arginine transport velocities in Flt-1 animals more prominently in pregnant dames. Phosphorylated CAT-1/CAT-1 increased in pregnant, sFlt-1-pregnant, and sFlt-1 virgin mice. CAT-2 increased in Flt-1-pregnant and Flt-1-virgin dames. L-arginine augmented arginine transport in pregnant and Flt-pregnant mice and prevented the increase in pCAT-1 and CAT-2 expression. Glomerular cGMP (cyclic guanosine monophosphate) generation as a measure of eNOS activity was decreased in all Flt-1 treated animals. L-arginine abolished the decrease in cGMP levels only in Flt-1-pregnant mice. In conclusion, glomerular endothelial NO generation is compromised in Flt-1-pregnant mice because of CAT-1 inhibition induced by a combined effect of pregnancy and preeclampsia which involves: phosphorylation of CAT-1 and induction of CAT-2. These processes contribute to the clinical syndrome of preeclampsia in mice and are prevented by L-arginine.

Responses of broiler chickens to Eimeria challenge when fed a nucleotide-rich yeast extract

ABSTRACTNucleotide-rich yeast extract (YN) was investigated for effects on growth performance, jejunal physiology, and cecal microbial activity in Eimeria-challenged broiler chickens. A total of 360-day-old male chicks (Ross × Ross 708) were placed on floor pens and provided a corn–soybean meal-based diet without or with YN (500 g/MT; n = 12). On d 10, 6 replicates per diet were orally administered with 1 mL of E. acervulina and E. maxima sporulated oocysts and the rest (non-challenged control) were administered with 1 mL of distilled water. On d 15, 5 birds/pen were then necropsied for intestinal lesion scores, histomorphology and cecal digesta pH, short chain fatty acids (SCFA), and microbial community using Illumina Miseq platform. Supplemental YN improved (P = 0.01) Feed conversion ratio (FCR) during the prechallenge phase (d 0 to 10). In the postchallenge period (d 11 to 15), Eimeria depressed (P < 0.05) Body weight gain (BWG) relative to non-challenged birds, whereas YN-fed birds had a higher (P = 0.05) BWG compared to that of non-YN-fed birds. There was an interaction between YN and Eimeria on jejunal villi height (VH) (P = 0.001) and expression of cationic amino acid transporter 1(CAT1) (P = 0.04). Specifically, in the absence of Eimeria, YN-fed birds had a shorter VH (892 vs. 1,020 μm) relative to that of control but longer VH (533 vs. 447 μm) in the presence of Eimeria. With respect to CAT1, YN-fed birds had a higher (1.65 vs. 0.78) expression when subjected to Eimeria than when not challenged. Independently, Eimeria decreased (P < 0.01) the jejunal expression of maltase, Na glucose transporter 1 and occludin genes, ceca digesta abundance of genus Clostridium cluster XlVa and Oscillibacter but increased (P < 0.01) jejunal proliferating cell nuclear antigen and interleukin 10. Interaction between YN and Eimeria was observed for ceca digesta pH (P = 0.04) and total SCFA (P = 0.01) such that YN increased SCFA in the absence of Eimeria but reduced SCFA and increased pH in the presence of Eimeria. In summary, Eimeria impaired performance and gut function and shifted gut microbiome; YN improved performance independently, attenuated Eimeria damage on indices of gut function, and modulated cecal microbiome.

Cationic amino acid transporter PQLC2 is a potential therapeutic target in gastric cancer

Tumor cells overexpress amino acid transporters to meet the increased demand for amino acids. PQ loop repeat‐containing (PQLC)2 is a cationic amino acid transporter that might be involved in cancer progression. Here, we show that upregulation of PQLC2 is critical to gastric cancer (GC) development in vitro and in vivo. Both PQLC2 mRNA and protein were overexpressed in GC tissues, especially of the diffuse type. Overexpression of PQLC2 promoted cell growth, anchorage independence, and tumor formation in nude mice. This was due to activation of MEK/ERK1/2 and PI3K/AKT signaling. Conversely, PQLC2 knockdown caused growth arrest and cell death of cancer cells and suppressed tumor growth in a mouse xenograft model. These results suggest that targeting PQLC2 is an effective strategy for GC treatment.

IMPACT OF L-CARNITINE SUPPLEMENTATION ON GROWTH OF BROILER CHICKEN THROUGH DETERMINATION OF CHANGES IN THE EXPRESSION OF CAT2, MYOD AND MYF5 GENES

This study explored the impact of supplementation of L- carnitine on the performance of broiler chicken, biochemical parameters and expression of cationic amino acid transporter (CAT2), myogenic determining factor (MYOD) and myogenic factor 5 (MYF5) genes. This study included two groups; the first one is the control group and the second one received L-carnitine at a dose 50 mg/kg/day in drinking water. After 35 day, the sera and muscle tissue were obtained for biochemical and real time polymerase chain reaction (qPCR) analysis. The obtained results revealed a significant increase in the body weight, total body weight gain and a significant decrease in food consumption, food conversion rate in L-carnitine-supplemented group as compared to the control group. Moreover, there was no significant changes in serum cholesterol, triacylglycerol, AST, ALT, total protein and uric acid levels which showed a slight decrease in L-carnitine-supplemented group in comparison with the control group. This study also revealed a significant up regulation for the expression of CAT2, MYOD and MYF5 genes in muscles of broiler chicken-supplemented with L- carnitine as compared to the control one. The present study concluded that supplementation of L- carnitine to broiler chicken was useful to increase the total body weight gain and muscle mass and this beneficial effect was associated with up regulation of CAT2, MYOD, and MYF5 genes.Key words: L-carnitine; broiler chicken; growth traits; CAT2; MYOD; MYF5

Analysis of LPI-causing mutations on y+LAT1 function and localization

Backgroundy+LAT1, encoded by SCL7A7, is the protein mutated in Lysinuric Protein Intolerance (LPI), a rare metabolic disease caused by a defective cationic amino acid (CAA, arginine, lysine, ornithine) transport at the basolateral membrane of intestinal and renal tubular cells. The disease is characterized by protein-rich food intolerance with secondary urea cycle disorder, but symptoms are heterogeneous with lung and immunological complications that are not explainable by the CAA transport defect. With the exception of the Finnish founder mutation (c.895-2A > T, LPIFin), LPI-causative mutations are heterogeneous and genotype-phenotype correlations have not been found. Here we addressed system y+L-mediated arginine uptake in monocytes from three LPI Italian patients and in lymphoblasts carrying the same mutations; in parallel, the genetic defects carried by the patients were reproduced as eGFP-tagged y+LAT1 mutants in transfected CHO cells to define the function and localization protein.ResultsSystem y+L activity is impaired in monocytes isolated from all LPI patients, and in CHO cells transfected with the three eGFP-y+LAT1 mutants, but not in lymphoblasts bearing the same mutations. The analysis of protein localization with confocal microscopy revealed that the eGFP-tagged mutants were retained inside the cytosol, with a pattern of expression quite heterogeneous among the mutants.ConclusionsThe three mutations studied of y+LAT1 transporter result in a defective arginine transport both in ex vivo (monocytes) and in vitro (CHO transfected cells) models, likely caused by the retention of the mutated proteins in the cytosol. The different effect of y+LAT1 mutation on arginine transport in monocytes and lymphoblasts is supposed to be due to the different expression of SLC7A7 mRNA in the two models, supporting the hypothesis that the impact of LPI defect largely depends on the relative abundance of LPI target gene in each cell type.

Macrophage-Derived IL1β and TNFα Regulate Arginine Metabolism in Neuroblastoma.

Neuroblastoma is the most common childhood solid tumor, yet the prognosis for high-risk disease remains poor. We demonstrate here that arginase 2 (ARG2) drives neuroblastoma cell proliferation via regulation of arginine metabolism. Targeting arginine metabolism, either by blocking cationic amino acid transporter 1 (CAT-1)-dependent arginine uptake in vitro or therapeutic depletion of arginine by pegylated recombinant arginase BCT-100, significantly delayed tumor development and prolonged murine survival. Tumor cells polarized infiltrating monocytes to an M1-macrophage phenotype, which released IL1β and TNFα in a RAC-alpha serine/threonine-protein kinase (AKT)-dependent manner. IL1β and TNFα established a feedback loop to upregulate ARG2 expression via p38 and extracellular regulated kinases 1/2 (ERK1/2) signaling in neuroblastoma and neural crest-derived cells. Proteomic analysis revealed that enrichment of IL1β and TNFα in stage IV human tumor microenvironments was associated with a worse prognosis. These data thus describe an immune-metabolic regulatory loop between tumor cells and infiltrating myeloid cells regulating ARG2, which can be clinically exploited. SIGNIFICANCE: These findings illustrate that cross-talk between myeloid cells and tumor cells creates a metabolic regulatory loop that promotes neuroblastoma progression.

Deficiency of cationic amino acid transporter-2 protects mice from hyperoxia-induced lung injury.

The pathology of acute lung injury (ALI) involves inducible nitric oxide (NO) synthase (iNOS)-derived NO-induced apoptosis of pulmonary endothelial cells. In vitro, iNOS-derived NO production has been shown to depend on the uptake of l-arginine by the cationic amino acid transporters (CAT). To test the hypothesis that mice deficient in CAT-2 ( slc7a2-/- on a C57BL/6 background) would be protected from hyperoxia-induced ALI, mice ( slc7a2-/- or wild-type) were placed in >95% oxygen (hyperoxia) or 21% oxygen (control) for 60 h. In wild-type mice exposed to hyperoxia, the exhaled nitric oxide (exNO) was twofold greater than in wild-type mice exposed to normoxia ( P < 0.005), whereas in slc7a2-/- mice there was no significant difference between exNO in animals exposed to hyperoxia or normoxia ( P = 0.95). Hyperoxia-exposed wild-type mice had greater ( P < 0.05) lung resistance and a lower ( P < 0.05) lung compliance than did hyperoxia-exposed slc7a2-/- mice. The lung wet-to-dry weight ratio was greater ( P < 0.005) in the hyperoxia-exposed wild-type mice than in hyperoxia-exposed slc7a2-/- mice. Neutrophil infiltration was lower ( P < 0.05) in the hyperoxia-exposed slc7a2-/- mice than in the hyperoxia-exposed wild-type mice as measured by myeloperoxidase activity. The protein concentration in bronchoalveolar lavage fluid was lower ( P < 0.001) in the hyperoxia-exposed slc7a2-/- mice than in similarly exposed wild-type mice. The percent of TUNEL-positive cells in the lung following hyperoxia exposure was significantly lower ( P < 0.001) in the slc7a2-/- mice than in the wild-type mice. These results are consistent with our hypothesis that lack of CAT-2 protects mice from acute lung injury.

Rabbit SLC15A1, SLC7A1 and SLC1A1 genes are affected by site of digestion, stage of development and dietary protein content

Peptide transporter 1 (SLC15A1, PepT1), excitatory amino acid transporter 3 (SLC1A1, EAAT3) and cationic amino acid transporter 1 (SLC7A1, CAT1) were identified as genes responsible for the transport of small peptides and amino acids. The tissue expression pattern of rabbit (SLC15A1, SLC7A1 and SLC1A1) across the digestive tract remains unclear. The present study investigated SLC15A1, SLC7A1 and SLC1A1 gene expression patterns across the digestive tract at different stages of development and in response to dietary protein levels. Real time-PCR results indicated that SLC15A1, SLC7A1 and SLC1A1 genes throughout the rabbits’ entire development and were expressed in all tested rabbit digestive sites, including the stomach, duodenum, jejunum, ileum, colon and cecum. Furthermore, SLC7A1 and SLC1A1 mRNA expression occurred in a tissue-specific and time-associated manner, suggesting the distinct transport ability of amino acids in different tissues and at different developmental stages. The most highly expressed levels of all three genes were in the duodenum, ileum and jejunum in all developmental stages. All increased after lactation. With increased dietary protein levels, SLC7A1 mRNA levels in small intestine and SLC1A1 mRNA levels in duodenum and ileum exhibited a significant decreasing trend. Moreover, rabbits fed a normal level of protein had the highest levels of SLC15A1 mRNA in the duodenum and jejunum (P<0.05). In conclusion, gene mRNA differed across sites and with development suggesting time and sites related differences in peptide and amino acid absorption in rabbits. The effects of dietary protein on expression of the three genes were also site specific.

Dietary nutrient levels alter the metabolism of arginine family amino acids in the conceptus of Huanjiang mini-pigs.

The arginine family amino acids (AFAAs) exert important roles in the metabolism, growth and development of the conceptus. However, to date, few studies have investigated the effects of maternal nutrient levels on the concentrations and metabolism of AFAAs in the conceptus. Compared to low nutrient diets, high nutrient diets increased (P<0.05) the concentrations of citrulline and proline (Pro) in plasma; the concentrations of arginine, glutamine, Pro and ornithine (Orn) in the amniotic fluid; and the concentrations of all detected AFAAs in the allantoic fluid, which were most pronounced on day 45 of pregnancy. High nutrient diets upregulated (P<0.05) mRNA expression of arginase I (Arg I), Pro oxidase and spermidine synthetase (SRM) in the fetal placenta, as well as Arg II, SRM and spermine synthetase (SMS) expression in the fetal liver (most pronounced on day 45 of pregnancy). The same effect was observed for mRNA expression of NO synthase and Orn aminotransferase (OAT), mainly on day 110 of pregnancy, and for mRNA expression of Arg I, Arg II, OAT, Orn decarboxylase and SMS throughout pregnancy. High nutrient diets upregulated (P<0.05) mRNA expression of Y+ L-type amino acid transporter (LAT) and cationic amino acid transporter 1 (CAT1) in the fetal jejunum throughout pregnancy. Dietary treatments did not affect (P>0.05) mRNA expression of Y+ LAT1, sodium-coupled neutral amino acid transporter 2 (SNAT2) and CAT1 in the fetal placenta, skeletal muscle and colon. High nutrient diets increased the concentration and transport of AFAAs in the mothers and conceptus, which likely improves growth and development of the conceptus. © 2018 Society of Chemical Industry.

The effect of dietary supplementation of low crude protein on intestinal morphology in pigs

To explore the effects of reducing the Cp levels on intestinal barrier function, low Cp (LP) and NRC standard Cp (NP) diets were fed to pigs from 45 to 160 days, and in vitro experiments were performed using monolayers of IPEC-J2 cells. The number of goblet cells, expression of proteins related to cell junction, amino acid transport, glucose transport, transepithelial electrical resistance (TEER), dextran permeability, and IL-6 secretion level were detected in pigs. The results demonstrated that a moderate reduction of Cp levels did not affect intestinal morphology, as demonstrated by a normal villi height, crypt depth and normal numbers of goblet cells. The maintenance of the intestinal structure obtained with LP was also confirmed by stable mRNA expression levels of muc2 and E-cadherin in the jejunum. We also found that LP did not affect the protein expression of cationic amino acid transporter 1 (CAT-1) and alanine serine cysteine transporter 1 (ASCT1) from 45 to 160 days. Moreover, the excitatory amino acid transporter 3 (EAAT3), sodium-glucose cotransporter 1 (SGLT1) and glucose transporter (GLUT2) protein expression levels in the jejunum were significantly increased at a certain age during the rearing period. Furthermore, we also demonstrated that a reduction in protein concentration up to 15% in the cultural medium of IPEC-J2 cells did not impact the mucosal barrier function. This study demonstrated that a moderate reduction of the protein level did not affect intestinal mucosal barrier function and morphology in the jejunum.

Transmissible gastroenteritis virus infection decreases arginine uptake by downregulating CAT-1 expression

Transmissible gastroenteritis virus (TGEV) is a coronavirus that causes severe diarrhea in suckling piglets. TGEV primarily targets and infects porcine intestinal epithelial cells, which play an important role in nutrient absorption. However, the effects of TGEV infection on nutrient absorption in swine have not yet been investigated. In this study, we evaluated the impact of TGEV infection on arginine uptake using the porcine small intestinal epithelial cell line IPEC-J2 as a model system. High performance liquid chromatography (HPLC) analyses showed that TGEV infection leads to reduced arginine uptake at 48 hours post-infection (hpi). Expression of cationic amino acid transporter 1 (CAT-1) was attenuated as well. TGEV infection induced activation of phospho-protein kinase C α (p-PKC α), phospho-epidermal growth factor receptor (p-EGFR), and enhanced the expression of caveolin-1, all of which appear to be involved in down-regulating arginine uptake and CAT-1 expression. These results illuminate the relationship between TGEV infection and nutrient absorption, and further our understanding of the mechanisms of TGEV infection.

Impact of mash and crumble diets on intestinal amino acids transporters, intestinal morphology and pancreatic enzyme activity of broilers.

The objective of this study was to evaluate effects of mash and crumble pre-starter diets on pancreatic enzyme activity, intestinal morphology, gene expression of intestinal peptide and amino acid (AA) transporters of broilers. Broilers in battery cages were assigned to different feed forms of pre-starter diet from 1 to 10days of age. Significantly increased body weight gain (BWG), feed intake (FI) and lowered FCR were observed in birds fed crumble pre-starter diet (CPD, p<0.05). Feed forms had no effect on whole and small intestine length, but relative intestinal length and relative small intestinal length significantly increased in the broilers fed a mash pre-starter diet (MPD, p<0.05). Feeding CPD increased the weight of pancreas (p<0.05), but relative weight of the pancreas was not influenced by treatments. Pancreatic protease and amylase activities significantly increased in the broilers fed CPD (p<0.05) but the activity of lipase was not influenced. Crypt depth (CD) and villus height (VH) were higher in broilers fed CPD (p<0.05) but villus width (VW), villus surface area (VSA) and villus height-to-crypt depth ratio (VCR) were not influenced by treatments. mRNA levels for peptide transporter 1 (PepT1), Na+ -independent cationic AA transporter1 (CAT1), Na+ -independent cationic and Na+ -dependent neutral AA transporter 1 (y+ LAT1) and Na+ -dependent neutral AA transporter (B0 AT) were lower in birds fed CPD (p<0.05). There were no differences in mRNA abundance of Na+ -independent cationic and zwitterionic AA transporter (b0,+ AT) among treatments. Overall, the present data showed that feeding crumble diet during first 10days of age, through higher FI, enhanced intestinal histomorphology, increased digestive enzyme activity is beneficial to growth performance of broilers. Indeed, dietary form can be an important factor in the expression of jejunal transporters.

Structural and functional studies on a prokaryotic homologue of the mammalian SLC7 cationic amino acid transporters

The Amino acid/Polyamine/Cationic transporter superfamily is among the largest of the sec-ondary active transporters. APC family members use either a proton or sodium gradient to drive substrate translocation across the membrane. Members of the APC superfamily are responsible for the uptake of amino acids and are found in all kingdoms of life. Amino acids, and their derivatives, are crucial for both prokaryotic and eukaryotic cell biology, being the building blocks of a variety of macromolecules and play important roles in metabolism and cell signalling. Therefore, the systems responsible for their transport are important targets of further study, in particular to gain greater insight into how these proteins recognise and discriminate between different types of amino acids. The transport mechanism of several APC members has been elucidated using a variety of structural, biophysical and functional approaches. However, these studies have focussed on sodium coupled transporters due to their function as neurotransmitter transporters. Here, the crystal structure of the first pro-ton-coupled amino acid transporter of the APC superfamily, from Geobacillus kaustophilus, GkApcT, is presented. GkApcT closely related to the mammalian cationic amino acid trans-porters (CATs) of the SLC7 family, responsible for the transport arginine and lysine into the cell. The mechanism of transport was further investigated using liposome-based functional assays to characterise the substrate specificity and kinetics. The crystal structure was used to guide a detailed site-directed mutagenesis study, which identified several conserved res-idues involved in substrate binding and proton-coupling. The current structure serves as a template to further understand proton coupled amino acid transport in bacteria and to un-derstand mammalian amino acid transport via the SLC7 and SLC36 families.

Mineralocorticoid receptor blockade improves arginine transport and nitric oxide generation through modulation of cationic amino acid transporter-1 in endothelial cells

Blockade of the mineralocorticoid receptor (MCR) has been shown to improve endothelial function far beyond blood pressure control. In the current studies we have looked at the effect of MCR antagonists on cationic amino acid transporter-1 (CAT-1), a major modulator of endothelial nitric oxide (NO) generation. Using radio-labeled arginine, {[3H] l-arginine} uptake was determined in human umbilical vein endothelial cells (HUVEC) following incubation with either spironolactone or eplerenone with or without silencing of MCR. Western blotting for CAT-1, PKCα and their phosphorylated forms were performed. NO generation was measured by using Griess reaction assay. Both Spironolactone and eplerenone significantly increased endothelial arginine transport, an effect which was further augmented by co-incubation with aldosterone, and blunted by either silencing of MCR or co-administration of amiloride. Following MCR blockade, we identified two bands for CAT-1. The addition of tunicamycin (an inhibitor of protein glycosylation) or MCR silencing resulted in disappearance of the extra band and prevented the increase in arginine transport. Only spironolactone decreased CAT-1 phosphorylation through inhibition of PKCα (CAT-1 inhibitor). Subsequently, incubation with either MCR antagonists significantly augmented NO2/NO3 levels (stable NO metabolites) and this was attenuated by silencing of MCR or tunicamycin. GO 6076 (PKCα inhibitor) intensified the increase of NO metabolites only in eplerenone treated cells. In conclusion spironolactone and eplerenone augment arginine transport and NO generation through modulation of CAT-1 in endothelial cells. Both MCR antagonists activate CAT-1 by inducing its glycosylation while only spironolactone inhibits PKCα.

Dynamic modeling of subcellular phenylpropanoid metabolism in Arabidopsis lignifying cells

Lignin is a polymer that significantly inhibits saccharification of plant feedstocks. Adjusting the composition or reducing the total lignin content have both been demonstrated to result in an increase in sugar yield from biomass. However, because lignin is essential for plant growth, it cannot be manipulated with impunity. Thus, it is important to understand the control of carbon flux towards lignin biosynthesis such that optimal modifications to it can be made precisely. Phenylalanine (Phe) is the common precursor for all lignin subunits and it is commonly accepted that all biosynthetic steps, spanning multiple subcellular compartments, are known, yet an in vivo model of how flux towards lignin is controlled is lacking. To address this deficiency, we formulated and parameterized a kinetic model based on data from feeding Arabidopsis thaliana basal lignifying stems with ring labeled [13C6]-Phe. Several candidate models were compared by an information theoretic approach to select the one that best matched the experimental observations. Here we present a dynamic model of phenylpropanoid metabolism across several subcellular compartments that describes the allocation of carbon towards lignin biosynthesis in wild-type Arabidopsis stems. Flux control coefficients for the enzymes in the pathway starting from arogenate dehydratase through 4-coumarate: CoA ligase were calculated and show that the plastidial cationic amino-acid transporter has the highest impact on flux.

Pre-pregnancy maternal obesity associates with endoplasmic reticulum stress in human umbilical vein endothelium

Obesity associates with the endoplasmic reticulum (ER) stress-induced endothelial dysfunction. Pregnant women with pre-pregnancy maternal obesity (PGMO) may transfer this potential risk to their offspring; however, whether ER stress occurs and associates with foetoplacental endothelial dysfunction in PGMO is unknown. We studied the l-arginine transport and nitric oxide (NO) synthesis in human umbilical vein endothelial cells (HUVECs) from women with PGMO or with a normal pre-pregnancy weight. We analysed the expression and activation of the ER stress sensors protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6). PGMO associated with lower endothelial NO synthase activity due to increased Thr495-inhibitor and decreased Ser1177-stimulator phosphorylation. However, higher expression and activity of the human cationic amino acid transporter 1 was found. PGMO caused activation of PERK and its downstream targets eukaryotic initiation factor 2 (eIF2α), C/EBP homologous protein 10 (CHOP), and tribbles-like protein 3 (TRB3). Increased IRE1α protein abundance (but not its phosphorylation or X-box binding protein 1-mRNA splicing) and increased c-Jun N-terminal kinase 1 phosphorylation was seen in PGMO. A preferential nuclear location of the activating transcription factor 6 (ATF6) was found in HUVECs from PGMO. All the changes seen in PGMO were blocked by TUDCA but unaltered by tunicamycin. Thus, PGMO may determine a state of ER stress via upregulation of the PERK–eIF2α–CHOP–TRB3 axis signalling in HUVECs. This phenomenon results in foetoplacental vascular endothelial dysfunction at birth.

Fetoplacental endothelial exosomes modulate high d-glucose-induced endothelial dysfunction

INTRODUCTIONGestational diabetes mellitus (GDM) is associated with fetoplacental endothelial dysfunction, which may be induced by hyperglycemia. We hypothesized that endothelial exosomes, which are extracellular nanovesicles affecting endothelial function, play a role in the high glucose (HG)-induced endothelial dysfunction. METHODSExosomes were isolated from HUVECs incubated with basal glucose (5.5 mmol/L; HUVEC- BG; exo-BG) and from HUVECs incubated with HG for 24 h (25 mmol/L; HUVEC-HG; exo-HG) in exosome-free medium. Exosomes were isolated and characterized by ultracentrifugation, sucrose gradient, electron microscopy, nanotracking analysis and Western blotting. HUVEC-BG and HUVEC-HG were exposed to exo-BG and exo-HG in two different concentrations: 5 μg and 1 μg exosome protein/mL. The exosomal effect on endothelial cell function was determined by wound healing assay, expression of endothelial nitric oxide synthase (eNOS), human cationic amino acid transporter type 1 (hCAT-1), vascular endothelial growth factor (VEGF) and intracellular adhesion molecule type 1 (ICAM-1) by Western blotting, qPCR or flow cytometry. RESULTSHG increased the exosomal release from HUVECs, endothelial wound healing and expression of phosphorylated (P∼Ser1177)-eNOS, hCAT-1, VEGF and ICAM-1. Exo-HG also increased endothelial cell wound healing, P∼Ser1177-eNOS, hCAT-1 and ICAM-1 expression in HUVEC-BG. Exo-BG reverted the effect of HG on endothelial cell wound healing and hCAT-1 mRNA expression to normal values. DISCUSSIONOur results show that HG may induce endothelial dysfunction in HUVECs and that exosomes from HUVEC-HG mimicked some of the effects of HG. This study contributes to the unraveling of the mechanism by which hyperglycemia affects the fetoplacental vasculature in GDM.

Effect of supplemental yeast cell walls on growth performance, gut mucosal glutathione pathway, proteolytic enzymes and transporters in growing broiler chickens.

This study aimed to investigate the effect of supplemental yeast cell walls (YCW) on growth performance, gut mucosal glutathione pathway, proteolytic enzymes, and transporters in broiler chickens from 1 to 21 d of age. One-day-old broiler chickens (n = 480) were randomly allocated to 4 treatments with 6 replicates of 20 chicks each for diets containing YCW at 0, 0.5, 1.0, and 1.5 g/kg. The results showed that YCW supplementation increased (P < 0.05) ADFI and ADG by 15.3% and 16.0%, respectively, and the 2 higher doses of YCW had greater effects (P < 0.05) than the lower dose. For the glutathione pathway in the duodenal mucosa, the levels of reduced glutathione and glutathione reductase in the YCW treatments were increased (P < 0.05) by 15.6% and 17.4%, respectively, but glutathione S-transferases was not affected. Similarly, the YCW increased (P < 0.05) the mucosal activities of trypsin, dipeptidyl, and leucine aminopeptidase by 12.4%, 5.5%, and 17.3%, respectively, and the mRNA profiles of neutral, cationic, and oligopeptide transporters by 62.5%, 69.1%, and 11.5%, respectively. The YCW also increased (P < 0.05) the concentrations of Thr, Met, Ile, Leu, Lys, Arg, and Tyr in the blood by 8.8% to 39.2%. Additionally, the ADFI, ADG, reduced glutathione, trypsin, dipeptidyl aminopeptidase, leucine aminopeptidase, and cationic amino acid transporter increased linearly and quadratically (P < 0.05) with YCW supplementation. The results suggest that YCW supplementation can beneficially modulate intestinal glutathione pathway, proteolytic enzyme activity, and nutrient transport in growing animals.

Effects of catecholamines on secretion of interferon tau and expression of genes for synthesis of polyamines and apoptosis by ovine trophectoderm.

Ovine trophectoderm (oTr1) cells were used to investigate effects of epinephrine (EP), norepinephrine (NE), and dopamine (DA) on their proliferation, migration and adhesion, secretion of interferon tau (IFNT), and expression of genes for synthesis of polyamines and apoptosis. Expression of mRNAs for agmatinase (AGMAT), arginine decarboxylase (ADC), ornithine decarboxylase (ODC1), and solute carrier family 7 (SLC7A1) (cationic amino acid transporter, Y+system), member 1 increased (P<0.05) in oTr1 cells in response to EP and DA. However, expression of SLC7A1 decreased at high doses of EP and expression of ADC mRNA by oTr1 cells decreased in response to 20 and 40ng/ml NE, and 40ng/ml DA. Migration of oTr1 cells increased in response to EP, DA, and NE after 48 h of treatment. However, proliferation of oTr1 cells was inhibited by 300pg/ml EP after 96h and DA at 20 and 100ng/ml. EP increased adhesion of oTr1 cells. The secretion of IFNT increased in response to 300pg/ml EP, 100ng/ml NE and DA after 48 h and at 96 h, and both DA (40ng/ml) and NE (100ng/ml). Expression of mRNAs for apoptotic genes (caspase 3, cathpsin B, BCL2 associated X protein "bax," B-cell lymphoma 2 "bcl2," and proto-oncogene "cmyc") decreased (P<0.05) in response to catecholamines, but DA did not affect (P<0.05) expression of cMYC mRNA. These results indicate that catecholamines play important roles in conceptus development during the peri-implantation period of pregnancy through effects on synthesis of polyamines, secretion of IFNT, and expression of apoptotic genes by oTr1 cells.