Leaf apoplastic alkalization promotes transcription of the ABA-synthesizing enzyme Vp14 and stomatal closure in Zea mays.

The chloride component of NaCl salinity causes the leaf apoplast to transiently alkalinize. This transition in pH reduces stomatal aperture. However, whether this apoplastic pH (pHapo) transient initiates stomatal closure by interacting with other chloride stress-induced responses or whether the pH transient alone initiates stomatal closure is unknown. To clarify the problem, the transient alkalinization of the leaf apoplast was mimicked in intact maize (Zea mays L.) by infiltrating near-neutral pH buffers into the leaf apoplast. Effects of the pHapo transient could thus be investigated independently from other chloride stress-derived effects. Microscopy-based ratiometric live pHapo imaging was used to monitor pHapoin planta. LC-MS/MS and real-time quantitative reverse transcription-PCR leaf analyses showed that the artificially induced pHapo transient led to an increase in the concentrations of the stomata-regulating plant hormone abscisic acid (ABA) and in transcripts of the key ABA-synthesizing gene ZmVp14 in the leaf. Since stomatal aperture and stomatal conductance decreased according to pHapo, we conclude that the pHapo transient alone initiates stomatal closure. Therefore, the functionality does not depend on interactions with other compounds induced by chloride stress. Overall, our data indicate that the pH of the leaf apoplast links chloride salinity with the control of stomatal aperture via effects exerted on the transcription of ABA.

Early response to salt ions in maize (Zea mays L.).

Abscisic acid (ABA) regulates leaf growth and transpiration rate of plants exposed to salt stress. Despite the known fact that cell dehydration is instrumental for the modulation of ABA concentrations when NaCl is high in the external environment, it was never tested as to whether sodium (Na) or chlorine (Cl) also modulate ABA concentrations. To answer this question, a hydroponic study on maize (Zea mays) was established, by exposing plants to 50mM of sodium glucosamide or glucosamine chloride. The effect of both ions on ABA was investigated in an early stage before (i) the salt ions accumulated to toxic tissue concentrations and before (ii) cells dehydrated. This allowed studying early responses to Na and Cl separately, well before plants were stressed by these ions. Gas chromatography-mass spectrometry analysis was used to quantify ABA concentrations in roots and in leaves after a period of 2h after ion application. The transcript abundance of the key regulatory enzyme of the biosynthesis of ABA in maize, the 9-cis-epoxycarotenoid dioxygenase gene viviparous 14, was quantified via real-time quantitative-reverse-transcriptase-polymerase-chain-reaction. The results reveal that Cl and Na induce the increase of leaf tissue ABA concentrations at two hours after plants were exposed to 50mM of the ions. Surprisingly, this effect was more pronounced in response to the Cl component. The increase in the guard-cell regulating ABA concentration correlated with a reduced transpiration. Mainly because of this result we suggest that the early accumulation of ABA is useful in maintaining cell turgor.

Intestinal barrier analysis by assessment of mucins, tight junctions, and α-defensins in healthy C57BL/6J and BALB/cJ mice.

The intestinal barrier is gaining increasing attention because it is related to intestinal homeostasis and disease. Different parameters have been used in the past to assess intestinal barrier functions in experimental studies; however most of them are poorly defined in healthy mice. Here, we compared a number of barrier markers in healthy mice, established normal values and correlations. In 48 mice (24 C57BL/6J, 24 BALB/cJ background), we measured mucus thickness, and expression of mucin-2, α-defensin-1 and -4, zonula occludens-1, occludin, junctional adhesion molecule-A, claudin-1, 2 and -5. We also analyzed claudin-3 and fatty acid binding protein-2 in urine and plasma, respectively. A higher expression of mucin-2 protein was found in the colon compared to the ileum. In contrast, the α-defensins-1 and -4 were expressed almost exclusively in the ileum. The protein expression of the tight junction molecules claudin-1, occludin and zonula occludens-1 did not differ between colon and ileum, although some differences occurred at the mRNA level. No age- or gender-related differences were found. Differences between C57BL/6J and BALB/cJ mice were found for α-defensin-1 and -4 mRNA expression, and for urine and plasma marker concentrations. The α-defensin-1 mRNA correlated with claudin-5 mRNA, whereas α-defensin-4 mRNA correlated with claudin-3 concentrations in urine. In conclusion, we identified a number of murine intestinal barrier markers requiring tissue analyses or measurable in urine or plasma. We provide normal values for these markers in mice of different genetic background. Such data might be helpful for future animal studies in which the intestinal barrier is of interest.

Assessment of the Intestinal Barrier with Five Different Permeability Tests in Healthy C57BL/6J and BALB/cJ Mice.

BACKGROUND: Intestinal permeability is thought to be of major relevance for digestive and nutrition-related diseases, and therefore has been studied in numerous mouse models of disease. However, it is unclear which tools are the preferable ones, and how normal values should be defined. AIMS: To compare different in vivo permeability tests in healthy mice of commonly used genetic backgrounds. METHODS: We assessed the intestinal barrier in male and female C57BL/6J and BALB/cJ mice of different ages, using four orally administered permeability markers, FITC-dextran 4000 (FITC-D4000) and ovalbumin (OVA) measured in plasma, and polyethylene glycol (PEG) and lactulose/mannitol (Lac/Man) measured in urine, and by assessing lipopolysaccharide (LPS) in portal vein plasma. RESULTS: After gavage, FITC-D4000, OVA, Lac/Man, and PEG400, but not PEG4000, were detectable in plasma or urine. Female mice tended to have a higher permeability according to the FITC-D4000, OVA, and PEG400 tests, but the Lac/Man ratio was higher in males. No significant differences between the two mouse strains of young and old mice were observed except for mannitol recovery, which was higher in BALB/cJ mice compared to C57BL/6J mice (p < 0.05). Virtually no LPS was detected in healthy mice. For all markers, normal values have been defined based on 5th-95th percentile ranges of our data. CONCLUSION: Selected oral permeability tests, such as FITC-D4000, OVA, PEG400, and Lac/Man, as well as LPS measurements in portal vein plasma, could be suitable for the evaluation of the intestinal barrier in mice, if used in a standardized way.

Altered intestinal neuroendocrine gene expression in humans with obesity.

OBJECTIVE: Gastrointestinal hormones are critically involved in the regulation of food intake and body weight. Previous studies support an interplay between gastrointestinal hormones and the serotonergic system. This study explored intestinal neuroendocrine expression patterns in humans with obesity versus nonobese humans. METHODS: Jejunum samples were collected from 164 humans with obesity (120 women; BMI (mean ± SD): 43.5 ± 6.6 kg/m(2) ) while they underwent Roux-en-Y gastric bypass surgery and from 18 nonobese humans (7 women; BMI: 23.5 ± 3.0 kg/m(2) ) undergoing distinct intestinal surgeries. mRNA expression of cholecystokinin (CCK), peptide YY3-36 (PYY), nesfatin1, ghrelin, ghrelin O-acyltransferase (GOAT), leptin, leptin receptor (leptinR), glucagon-like-peptide 1 receptor (GLP1R), serotonin transporter (SERT), tryptophan hydroxylase 1 (TPH1), and serotonin receptor 3A (5HT3A R) was determined with qRT-PCR. Ghrelin and GOAT protein expression was quantified using immunohistological stainings. Statistical analyses were performed with SPSS. RESULTS: Jejunum samples from humans with obesity showed a higher expression of GOAT (mRNA and protein), TPH1, and SERT mRNA compared with the nonobese humans (all P < 0.05). Positive correlations were observed between TPH1, CCK, PYY, and nesfatin1 in nonobese and GOAT, ghrelin, TPH1, SERT, CCK, and PYY in humans with obesity (all P < 0.01). CONCLUSIONS: Our top-down approach substantiates the dysregulation of jejunal neuroendocrine hormones in obesity.

Effect of high sugar intake on glucose transporter and weight regulating hormones in mice and humans.

OBJECTIVE: Sugar consumption has increased dramatically over the last decades in Western societies. Especially the intake of sugar-sweetened beverages seems to be a major risk for the development of obesity. Thus, we compared liquid versus solid high-sugar diets with regard to dietary intake, intestinal uptake and metabolic parameters in mice and partly in humans. METHODS: Five iso-caloric diets, enriched with liquid (in water 30% vol/vol) or solid (in diet 65% g/g) fructose or sucrose or a control diet were fed for eight weeks to C57bl/6 mice. Sugar, liquid and caloric intake, small intestinal sugar transporters (GLUT2/5) and weight regulating hormone mRNA expression, as well as hepatic fat accumulation were measured. In obese versus lean humans that underwent either bariatric surgery or small bowel resection, we analyzed small intestinal GLUT2, GLUT5, and cholecystokinin expression. RESULTS: In mice, the liquid high-sucrose diet caused an enhancement of total caloric intake compared to the solid high-sucrose diet and the control diet. In addition, the liquid high-sucrose diet increased expression of GLUT2, GLUT5, and cholecystokinin expression in the ileum (P<0.001). Enhanced liver triglyceride accumulation was observed in mice being fed the liquid high-sucrose or -fructose, and the solid high-sucrose diet compared to controls. In obese, GLUT2 and GLUT5 mRNA expression was enhanced in comparison to lean individuals. CONCLUSIONS: We show that the form of sugar intake (liquid versus solid) is presumably more important than the type of sugar, with regard to feeding behavior, intestinal sugar uptake and liver fat accumulation in mice. Interestingly, in obese individuals, an intestinal sugar transporter modulation also occurred when compared to lean individuals.

Effect of tryptophan supplementation on diet-induced non-alcoholic fatty liver disease in mice.

Intestinal serotonin (5-hydroxytrypamine, 5-HT) metabolism is thought to play a role in gut functions by regulating motility, permeability and other functions of the intestine. In the present study, we investigated the effect of tryptophan (TRP), the precursor of 5-HT, supplementation on intestinal barrier functions and non-alcoholic fatty liver disease (NAFLD). An established mouse model of NAFLD induced by feeding a fructose-rich diet (N group) was used in the present study. TRP was administered orally for 8 weeks to C57BL/6J control or NAFLD mice. NAFLD-related liver parameters (hepatic TAG and Oil Red O staining), intestinal barrier parameters (tight-junction protein occludin and portal plasma lipopolysaccharides (LPS)) and 5-HT-related parameters (5-HT, 5-HT transporter (SERT) and motility) were measured. We observed reduced duodenal occludin protein concentrations (P= 0·0007), high portal plasma LPS concentrations (P= 0·005) and an elevated liver weight:body weight ratio (P= 0·01) in the N group compared with the parameters in the control group. TRP supplementation led to an increase in occludin concentrations (P= 0·0009) and consecutively reduced liver weight:body weight ratio (P= 0·009) as well as overall hepatic fat accumulation in the N group (P= 0·05). In addition, the N group exhibited reduced SERT protein expression (P= 0·002), which was normalised by TRP supplementation (P= 0·02). For the first time, our data indicate that oral TRP supplementation attenuates experimental NAFLD in mice. The underlying mechanisms are not clear, but probably involve stabilisation of the intestinal barrier in the upper small intestine and amelioration of the dysregulated intestinal serotonergic system.

Lactobacillus rhamnosus GG protects against non-alcoholic fatty liver disease in mice.

OBJECTIVE: Experimental evidence revealed that obesity-associated non-alcoholic fatty liver disease (NAFLD) is linked to changes in intestinal permeability and translocation of bacterial products to the liver. Hitherto, no reliable therapy is available except for weight reduction. Within this study, we examined the possible effect of the probiotic bacterial strain Lactobacillus rhamnosus GG (LGG) as protective agent against experimental NAFLD in a mouse model. METHODS: Experimental NAFLD was induced by a high-fructose diet over eight weeks in C57BL/J6 mice. Fructose was administered via the drinking water containing 30% fructose with or without LGG at a concentration resulting in approximately 5×10(7) colony forming units/g body weight. Mice were examined for changes in small intestinal microbiota, gut barrier function, lipopolysaccharide (LPS) concentrations in the portal vein, liver inflammation and fat accumulation in the liver. RESULTS: LGG increased beneficial bacteria in the distal small intestine. Moreover, LGG reduced duodenal IκB protein levels and restored the duodenal tight junction protein concentration. Portal LPS (P≤0.05) was reduced and tended to attenuate TNF-α, IL-8R and IL-1ß mRNA expression in the liver feeding a high-fructose diet supplemented with LGG. Furthermore liver fat accumulation and portal alanine-aminotransferase concentrations (P≤0.05) were attenuated in mice fed the high-fructose diet and LGG. CONCLUSIONS: We show for the first time that LGG protects mice from NAFLD induced by a high-fructose diet. The underlying mechanisms of protection likely involve an increase of beneficial bacteria, restoration of gut barrier function and subsequent attenuation of liver inflammation and steatosis.