Glutamine and cystine-enriched diets modulate aquaporins gene expression in the small intestine of piglets

Abstract

To understand the mechanism of action, we also determined the transcriptional profile of glycerol kinase (GK), the key enzyme in the regulation of glycerol uptake and metabolism [20] and the intracellular mediators involved in the regulation of AQPs expression, focusing on the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) and mammalian target of rapamycin (mTOR) transduction signals [21, 22]. [...]the impact of glutamine and cystine-enriched diets on intestinal permeability of piglets was evaluated using isolated membranes from the ileum portion of the small intestine. Twenty male piglets, weaned at 4 weeks, with an initial average weight of 8.8 ± 0.89 kg (mean ± SD) from a F1 cross between the Large White and Landrace breeds were allocated to four groups (5 pigs per diet) and received during a 4 week-period a basal diet with 27.5% of crude protein (NRC, 1998) and without supplementation (Control group) or supplemented with 8 kg/ton of glutamine (Gln group), cystine (Cys group) or the combination of the two amino acids in equal quantity (Gln + Cys group). Ingredients (g.kg-1) composition of the experimental diets. https://doi.org/10.1371/journal.pone.0245739.t001 Serum biochemical profile and redox status Glucose, urea, creatinine, total cholesterol, HDL-cholesterol, LDL-cholesterol, triacylglycerols (TAG), total protein, phospholipids, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and γ-glutamiltransferase (GGT) were analysed in serum using diagnostic kits (Roche Diagnostics, Mannheim, Germany) and a Modular Hitachi Analytical System (Roche Diagnostics).

About the Authors:

Inês Vieira da Silva

Roles Investigation, Methodology

Affiliations Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Lisbon, Portugal, Dept. Bioquímica e Biologia Humana, Faculdade de Farmácia, Universidade de Lisboa, Lisboa, Portugal

ORCID logo https://orcid.org/0000-0002-2916-4848

Bárbara P. Soares

Roles Methodology

Affiliations Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Lisbon, Portugal, Dept. Bioquímica e Biologia Humana, Faculdade de Farmácia, Universidade de Lisboa, Lisboa, Portugal

Catarina Pimpão

Roles Methodology

Affiliations Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Lisbon, Portugal, Dept. Bioquímica e Biologia Humana, Faculdade de Farmácia, Universidade de Lisboa, Lisboa, Portugal

ORCID logo https://orcid.org/0000-0002-2356-1105

Rui M. A. Pinto

Roles Methodology

Affiliations Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Lisbon, Portugal, JCS, Laboratório de Análises Clínicas Dr. Joaquim Chaves, Algés, Portugal

ORCID logo https://orcid.org/0000-0002-1667-7871

Teresa Costa

Roles Funding acquisition

Affiliation: Indukern Portugal, Lda., Centro Empresarial Sintra Estoril II, Sintra, Portugal

João P. B. Freire

Roles Funding acquisition, Resources

Affiliation: LEAF—Linking Engineering, Agriculture and Food, Departamento de Ciências e Engenharia de Biossistemas, Instituto Superior de Agronomia (ISA), Universidade de Lisboa, Lisboa, Portugal

Etienne Corrent

Roles Funding acquisition

Affiliation: Ajinomoto Animal Nutrition Europe, Paris, France

Tristan Chalvon-Demersay

Roles Funding acquisition

Affiliation: Ajinomoto Animal Nutrition Europe, Paris, France

José A. M. Prates

Roles Funding acquisition, Resources, Writing – review & editing

Affiliation: CIISA—Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Lisboa, Portugal

Paula A. Lopes

Roles Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Writing – original draft, Writing – review & editing

* E-mail: [email protected] (PAL); [email protected] (GS)

Affiliation: CIISA—Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Lisboa, Portugal

ORCID logo https://orcid.org/0000-0002-6755-4572

Graça Soveral

Roles Conceptualization, Formal analysis, Investigation, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing

* E-mail: [email protected] (PAL); [email protected] (GS)

Affiliations Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Lisbon, Portugal, Dept. Bioquímica e Biologia Humana, Faculdade de Farmácia, Universidade de Lisboa, Lisboa, Portugal

Introduction

Over the past decade, there has been increasing recognition of an association between disrupted intestinal barrier function and disease pathogenesis, in both animals and humans [1]. Recent studies have demonstrated that obesity, type 2 diabetes, inflammation and metabolic complications, such as dyslipidemia, are associated with intestinal barrier dysfunction and exacerbated by a lipid challenge [2, 3], suggesting that modulation of intestinal barrier permeability or personalized nutrition therapy may represent novel therapeutic approaches [4].

Glycerol concentration in plasma is determined primarily by adipose tissue lipolysis, but also by the variable amount reabsorbed in kidney tubules and gastrointestinal tract. In the interprandial state, starvation and exercise, glycerol from lipolysis represents an important substrate for gluconeogenesis together with lactate, pyruvate and some amino acids, as the case of glutamine [5]. Amino acids are building blocks of protein but also have many other important functions within the body, such as cell proliferation, immunity modulation, food intake control and energy provision [6]. While glutamine acts both as substrate and regulator of gluconeogenesis [5, 7, 8], cystine promotes adiposity by increasing visceral fat accumulation and lowering insulin sensitivity in conjunction with changes in genes expression involved in lipid and glucose metabolism [9]. In fasting conditions, triacylglycerol hydrolysis in the adipose tissue results in non-esterified fatty acids and glycerol, the latter being used for gluconeogenesis in the liver to assure normoglycemia. These metabolic events occurring exclusively in intracellular compartments depend on glycerol transport across different tissues [10]. Glycerol diffusion across cell membranes is facilitated by aquaglyceroporins, a subfamily of aquaporin water channels (AQPs) [11, 12] which are divided into three major sub-classes, according to their primary sequence and permeability features: (1) classical or orthodox aquaporins, considered primarily selective to water (AQP0, AQP1, AQP2, AQP4, AQP5, AQP6 and AQP8); (2) aquaglyceroporins, which have a less-constricted pore allowing permeation of small molecules like glycerol (AQP3, AQP7, AQP9 and AQP10); and (3) super-aquaporins or subcellular aquaporins, with intracellular localization and still undefined selectivity (AQP11 and AQP12) [13, 14]. Although the precise localization of AQPs in human intestine membranes is still unclear, their function as water channels in both healthy and pathological gastrointestinal related conditions, such as infectious diarrhea, inflammatory bowel disease and celiac disease, is well documented [15–18].

In this study, the expression and function of the water channel AQP1 and the glycerol channels AQP3, AQP7, AQP9 and AQP10 involved in volume regulation and energy homeostasis respectively, were investigated in the ileum portion of the small intestine of piglets. The pig is rapidly emerging as a biomedical model for energy metabolism and human obesity because of their similar metabolic features, cardiovascular systems, and proportional organ sizes [19]. To ascertain the effect of amino acids, glutamine and cystine-enriched diets, we firstly characterised the general metabolic state of piglets followed by the modulation of intestinal AQPs gene expression and activity. To understand the mechanism of action, we also determined the transcriptional profile of glycerol kinase (GK), the key enzyme in the regulation of glycerol uptake and metabolism [20] and the intracellular mediators involved in the regulation of AQPs expression, focusing on the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) and mammalian target of rapamycin (mTOR) transduction signals [21, 22]. Finally, the impact of glutamine and cystine-enriched diets on intestinal permeability of piglets was evaluated using isolated membranes from the ileum portion of the small intestine.

Material and methods

Animals and diets

All experimental procedures were reviewed and approved by the Animal Care Committee of Instituto Superior de Agronomia (ISA), Universidade de Lisboa (Lisbon, Portugal) and authorized by the National Veterinary Authority (Direcção Geral de Alimentação e Veterinária (Lisbon, Portugal), following the appropriate European Union guidelines (2010/63/EU Directive) to minimize animals suffering. Paula A. Lopes and José A. M. Prates hold a FELASA C grade, which enables them to design and conduct animal experimentation in the European Union.

This study was performed at the Animal Sector of the Engineer Bio-Systems, Instituto Superior de Agronomia, ISA, Universidade de Lisboa (Lisbon, Portugal). Before the beginning of the experiment, all piglets were subjected to the same management and received the conventional commercial starter diet (INICEAL PLASMA G), dosing 19.5% of crude protein (CP), 1.40% of lysine and 10.4 MJ/kg NE, manufactured by Eurocereal (Comercialização de Produtos Agro-Pecuários, SA, Portugal). Twenty male piglets, weaned at 4 weeks, with an initial average weight of 8.8 ± 0.89 kg (mean ± SD) from a F1 cross between the Large White and Landrace breeds were allocated to four groups (5 pigs per diet) and received during a 4 week-period a basal diet with 27.5% of crude protein (NRC, 1998) and without supplementation (Control group) or supplemented with 8 kg/ton of glutamine (Gln group), cystine (Cys group) or the combination of the two amino acids in equal quantity (Gln + Cys group). The ingredients of the experimental diets, together with their amino acid composition, are presented in Table 1. Throughout the experiment, piglets were weighed weekly before feeding. By the end of the experimental period, animals were sacrificed by cutting the carotid artery and external jugular veins after stunning by electronarcosis, according to EU legislation [23]. Blood samples were collected from the jugular vein and centrifuged at 1500 g for 15 min to obtain serum. Samples from the ileum portion of the small intestine (60 cm before the ileo-caecal valve) were removed, washed with saline, flash-frozen in liquid nitrogen and stored at -80°C for subsequent analysis.

[Figure omitted. See PDF.]

Table 1. Ingredients (g.kg-1) composition of the experimental diets.

https://doi.org/10.1371/journal.pone.0245739.t001

Serum biochemical profile and redox status

Glucose, urea, creatinine, total cholesterol, HDL-cholesterol, LDL-cholesterol, triacylglycerols (TAG), total protein, phospholipids, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and γ-glutamiltransferase (GGT) were analysed in serum using diagnostic kits (Roche Diagnostics, Mannheim, Germany) and a Modular Hitachi Analytical System (Roche Diagnostics). VLDL-cholesterol and total lipids were calculated as described by Friedewald et al. [24] and Covaci et al. [25], respectively. The quantitative colorimetric determinations of total antioxidant capacity (DTAC-100) and glutathione peroxidase activity (EGPX-100) were measured using commercial kits (BioAssay Systems, Hayward, CA, USA).

RNA isolation and cDNA synthesis

The RNA isolation was performed in the ileum samples by homogenization with a TissueLyser II (Qiagen, Hilden, Germany) using TRIzol® Reagent (Invitrogen, Barcelona, Spain) and extraction was performed according to the manufacturer’s instructions. For first strand cDNA synthesis, constant amounts of 1 μg of total RNA with quality ratios 260/280 and 260/230 between 1.8–2.2 (NanoDrop1 ND-2000c, ThermoFisher Scientific, Waltham, USA) were reverse transcribed in a 20 μl final volume using NZYFirst-strand cDNA synthesis Kit (NZYtech, Lisbon, Portugal), according to the manufacturer’s protocol.

Real-time quantitative PCR (RT-qPCR)

The transcript levels for AQP1, AQP3, AQP7, AQP9, AQP10, GK, mTOR and PIK3CG and housekeeping, β-actin (ACTB) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) genes were quantified in the ileum portion of small intestine samples by real-time quantitative PCR in a CFX96™ Real-Time PCR Detection System C1000 (BioRad, CA, USA). RT-qPCR was performed using TaqMan™ Universal Master Mix II with UNG (Life Technologies, CA, USA) and following Sus scrofa specific predesigned TaqMan™ Gene Expression Assays (primers FWD, REV and FAM-labelled probe; Life Technologies, CA, USA): AQP1, Ss03385017_u1; AQP3, Ss03389620_m1; AQP7, Ss03389863_m1; AQP9, Ss03389741_m1; AQP10, Ss03374224_m1; GK, Ss03818509_m1; mTOR, Ss03377427_u1; PIK3CG, Ss03392908_u1; ACTB, Ss03376081_u1 and HPRT1, Ss03388274_m1. The characterisation of the selected genes used in real-time quantitative PCR is described in Table 2. Probes were designed to hybridise between exons to ensure the detection of the corresponding transcript avoiding genomic DNA amplification. The cDNA was amplified at the following conditions: 95°C for 10 min, followed by 45 cycles of 15 seconds at 95°C and 1 min at 59°C, using the TaqMan® Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The reaction volume of 20 μL was prepared using 10 μL of TaqMan Master Mix, 1 μL of Taqman Assay and 9 μL of cDNA template diluted to 1:8 in molecular biology grade water. The reaction consisted of an initial denaturation step at 95°C for 3 min, 45 cycles of denaturation at 95°C for 10 seconds and annealing/extension at 62°C for 30 seconds. All results were normalised to the mean of the levels of both reference genes (ACTB and HPRT1) and relative quantification was calculated using a variation of the Livak method [26], described by Fleige and Pfaffl [27]. All samples were run in triplicate and the average values were calculated.

[Figure omitted. See PDF.]

Table 2. Gene specific primers used for RT-qPCR.

https://doi.org/10.1371/journal.pone.0245739.t002

Preparation of membrane vesicles from the ileum portion of small intestine

Membrane vesicles were prepared from the ileum portion of small intestine samples by differential centrifugation with buffer without detergents. Approximately 6 g of ileum from each piglet was chopped into small pieces, removing visible endothelial tissue, and homogenised in mannitol-HEPES buffer (100 mM mannitol, 2 mM Tris-HEPES, pH 7.4) in a Waring blender for 2 × 1 min and then with a Potter-Elvehjem homogeniser. The homogenate was diluted with 10 mM MgCl2, gently mixed for 20 min and centrifuged at 3000 g for 20 min at 4°C in a Beckman Coulter Allegra 6R centrifuge. The supernatant was centrifuged at 30000 g for 30 min at 4°C in a Beckman 12–21 MIE centrifuge with a JA-20 rotor to obtain a pellet of crude membranes that was resuspended in a mannitol buffer (300 mM mannitol, 0.1 mM MgSO4, 0.02% NaN3 and 20 mM Tris/HEPES pH 7.4), transferred to a syringe and sheared by vigorously passing it 10 times through a 21-gauge needle and immediately used for transport experiments of total membranes. This protocol was adapted from other authors [28–30].

The protein content was determined by the BCA assay (Pierce™ BCA Protein Assay Kit, Thermo Scientific, MA, USA). The enrichment in specific activity of the apical markers, leucine-aminopeptidase and alkaline phosphatase (membrane vesicles/crude homogenate), assayed as previously described [31] was 9.41 ± 3.53 and 4.20 ± 2.16 (n = 20, mean ± SEM), respectively [32]. To obtain vesicles enriched in basolateral membranes (BLMV), 1 mL of the membrane suspension was loaded on a continuous linear sucrose gradient (25–55% (w/v) sucrose in 10 mM Tris-HEPES, pH 7.4), following the protocol described in [33]. The gradient was centrifuged at 100000 g for 60 min, the upper band was collected and resuspended in mannitol buffer and centrifuged at 100000 g for 30 min to remove traces of sucrose. The pellet was then resuspended in mannitol buffer by repeated passage through a 21-gauge needle, as above. The enrichment in basolateral membranes of the final preparation determined by the specific activity of the basolateral marker K+-phosphatase [34] was 11.75 ± 5.23 (n = 20, mean ± SEM). Membrane preparations obtained were either immediately used for experiments or stored at -20°C for later use. The vesicle size of all membrane preparations determined by the Quasi-Elastic Light Scattering (QELS) technique (Brookhaven Instruments BI-90, Holtsville, NY, USA), as described by Soveral et al. [31] revealed homogenous vesicles populations with 300 ± 21 nm diameter (mean ± SEM), regardless the dietary treatment.

Membrane permeability of intestinal vesicles

Water and glycerol permeabilities of intestinal vesicles were evaluated by the stopped-flow technique (HI-TECH Scientific PQ/SF-53). Experiments were performed at 23°C for single measurements. Five replicates were analysed for each experimental condition. For the measurement of osmotic water permeability, membrane vesicles (0.2 mg protein/ml) resuspended in mannitol-HEPES buffer (300 mOsM) were mixed with an equal amount of isosmotic or hyperosmotic (500 mOsM) mannitol solutions to reach an inwardly directed gradient of the impermeant solute. The kinetics of vesicle shrinkage was measured from the time course of scattered light intensity at 400 nm until a stable light scatter signal was attained. The osmotic water permeability coefficient (Pf) was estimated by fitting the light scatter signal to a single exponential curve and using the linear relation between Pf and the exponential time constant k [35], Pf = k (V0/A)(1/Vw(osmout)∞), where Vw is the molar volume of water, V0/A is the initial volume to area ratio of the vesicle preparation, and (osmout)∞ is the final medium osmolarity after the applied osmotic gradient. For glycerol permeability, membrane vesicles equilibrated in 300 mOsM mannitol-HEPES buffer were challenged with a hyperosmotic glycerol solution (500 mOsM), creating an inwardly directed glycerol gradient. After the first fast vesicle shrinkage due to water outflow, glycerol influx in response to its chemical gradient was followed by water influx with subsequent vesicle reswelling. Glycerol permeability was calculated as Pgly = k (V0/A), where k is the single exponential time constant fitted to the light scattering signal of glycerol influx [36].

Statistical analysis

Statistics was carried out with the Generalised Linear Mixed (GLM) model of Statistical Analysis System (SAS) software, version 9.4 (SAS Institute, Cary, NC, USA). Once normality was tested by Kolmogorov-Smirnov test and variance homogeneity, significant multiple comparisons test was carried out using the PDIFF option, adjusted with Tukey-Kramer, to determine statistical differences among dietary treatments. All data were presented as mean and standard error of the mean (SEM). A P value lower than 0.05 was considered statistically significant.

Results

Baseline characterisation of the dietary groups

Table 3 presents data on growth performance parameters and serum metabolites of piglets fed on amino acid-enriched diets, Gln and Cys, individually or combined. No variations were found for animal’s final body weight (P>0.05) as well as feed intake (P>0.05). Piglets fed on the mixture of glutamine and cystine had decreased concentrations of glucose (P<0.001), urea (P<0.001), creatinine (P<0.001), cholesterol (P<0.001), HDL-cholesterol (P<0.001), LDL-cholesterol (P<0.001), total protein (P = 0.006), total lipids (P<0.001), and phospholipids (P<0.001), relative to the other dietary treatments. The same pattern of variation was found for hepatic function markers, AST (P = 0.003), ALT (P<0.001), ALP (P<0.001) and GGT (P = 0.000). Conversely, piglets fed glutamine plus cystine had higher concentrations of VLDL-cholesterol (P = 0.001) and triacylglycerols (P = 0.001) than piglets fed on cystine and control diets. The redox status evaluation, through the assessment of total antioxidant capacity and glutathione peroxidase enzyme activity, was unchanged across dietary treatments (P = 0.146 and P = 0.838, respectively).

[Figure omitted. See PDF.]

Table 3. Growth performance and serum biochemical metabolites of piglets fed on amino acid-enriched diets, glutamine and cystine, individually or combined.

https://doi.org/10.1371/journal.pone.0245739.t003

Glutamine and cystine, individually or combined, downregulate AQP1, AQP7 and AQP10 gene expression

Fig 1 presents the gene expression levels of the tested AQPs in the ileum portion of the small intestine of piglets fed on amino acid-enriched diets, glutamine and cystine, individually or combined. Comparing their transcriptional profile, AQP3 mRNA levels were found predominant over the others (AQP3a > AQP1b > AQP7c, AQP9c, AQP10c), regardless the addition of amino acids (P<0.001). Moreover, a clear pattern of downregulation of AQP1 (P<0.001) (Fig 1A), AQP7 (P = 0.001) (Fig 1B) and AQP10 (P<0.001) (Fig 1E) gene expression was found in piglets fed diets enriched in glutamine and cystine, individually or combined, relative to the control. The same trend was verified for AQP9 mRNA levels, although not reaching statistical significance (P = 0.090) (Fig 1D). Cystine-enriched diet upregulated AQP3 gene expression in relation to the other diets (P<0.001) (Fig 1C), but the combination of glutamine and cystine did not differ from the control (P>0.05) (Fig 1C).

[Figure omitted. See PDF.]

Fig 1.

mRNA expression levels of AQP1 (A), AQP7 (B), AQP3 (C), AQP9 (D) and AQP10 (E) in the ileum portion of the small intestine of piglets fed on amino acid-enriched diets, glutamine and cystine, individually or combined. Values are mean ± standard error of the mean (SEM) represented by vertical bars (n = 5). a,b,cMean values with unlike letters are significantly different (P<0.05).

https://doi.org/10.1371/journal.pone.0245739.g001

Cystine downregulates GK gene expression

Fig 2 presents the variation on gene expression levels of GK (A), mTOR (B) and PI3KCG (C) in the ileum portion of the small intestine of piglets fed glutamine and cystine, individually or combined. GK gene expression was downregulated by individually cystine relative to glutamine (P = 0.045) (Fig 2A). No further variations were found for GK mRNA levels among the other dietary treatments (P>0.05) (Fig 2A). The gene expression levels of mTOR (Fig 2B) and PI3KCG (Fig 2C) were kept unchanged by amino acid-enriched diets, glutamine and cystine, individually or combined (P = 0.206 and P = 0.119, respectively).

[Figure omitted. See PDF.]

Fig 2.

mRNA expression levels of GK (A), mTOR (B) and PI3KCG (C) in the ileum portion of the small intestine of piglets fed on amino acid-enriched diets, glutamine and cystine, individually or combined. Values are mean ± standard error of the mean (SEM) represented by vertical bars (n = 5). a,bMean values with unlike letters are significantly different (P<0.05).

https://doi.org/10.1371/journal.pone.0245739.g002

Cystine increases glycerol permeability in intestinal vesicles enriched in basolateral membranes

To evaluate if the detected AQPs gene modulation by glutamine and cystine dietary treatments would impact on intestinal water or glycerol permeability across intestine, the ileum portion of the small intestine of piglets’ groups under study was used to prepare vesicles enriched in apical and in basolateral membranes. The vesicles from all dietary groups were assayed for water (Pf) and glycerol (Pgly) permeability. Fig 3A and 3B depict typical stopped flow signals showing the time course of vesicle volume shrinkage due to water outflux (Fig 3A) and vesicle reswelling due to glycerol influx (Fig 3B) with their respective exponential fits used to calculate Pf and Pgly. Piglets fed on glutamine and cystine, individually or combined, displayed similar values of water permeability (P = 0.510) (Fig 3C) in both apical and basolateral membranes. Glycerol permeability was not different across dietary groups (P = 0.488) (Fig 3D) in vesicles enriched in apical membranes. However, in basolateral membranes, an increase in glycerol permeability was detected in piglets fed on cystine diet compared to the other dietary groups (P<0.001) (Fig 3E).

[Figure omitted. See PDF.]

Fig 3.

Representative stopped flow light scatter signals for evaluation of water permeability (Pf) (A) and glycerol permeability (Pgly) (B) of intestinal membrane vesicles, showing the time course of vesicle shrinkage (water outflux) or reswelling (glycerol influx) and the respective exponential fit used to calculate permeability. Effect of amino acid-enriched diets in glutamine, cystine and the combination of glutamine and cystine on Pf (C) and Pgly from intestinal vesicles (D) and from vesicles enriched in basolateral membranes (BLMV) (E) from the ileum portion of small intestine of piglets. Values are mean ± standard error of the mean (SEM) represented by vertical bars (n = 5). a,bMean values with unlike letters are significantly different (P<0.001).

https://doi.org/10.1371/journal.pone.0245739.g003

Discussion

Ever since aquaporins were discovered, research on these transmembrane proteins has evolved providing knowledge on their pathophysiological importance [37]. AQPs were initially thought as bidirectional water-selective channels but they also play a central role in glycerol trafficking across the cell membrane barrier [11, 12] bridging tissues and organs, and contributing to energy homeostasis. The regulation of glycerol membrane permeation in the gastrointestinal tract is crucial to control fat deposition, lipolysis and gluconeogenesis. AQPs act as mediators of glycerol delivery underlying current investigation on molecular composition and regulation of intestinal barrier function. Therefore, targeting AQPs in the small intestine might turn into a novel approach to control metabolic disorders, such as insulin resistance, diabetes and obesity [38]. However, at present, scarce information is available, about the effects of nutrients, such as individual amino acids, on AQPs regulation in animal cells [39].

In this study, we used piglets fed glutamine and cystine-enriched diets to investigate the effects of supplemented amino acids on the general metabolic state of animals, through the assessment of growth performance and serum biochemical metabolites, followed by the modulation of AQPs gene expression and activity. The growth performance parameters of piglets, including daily feed intake, were not changed by amino acids supplementation. In terms of palatability, glutamine and cystine have been considered highly attractive in what taste properties concerns [40]. In fact, previous research has revealed that the supplementation of cystine and theanine (an analogue of glutamine) can increase appetite in rodents and humans [41, 42], contradicting our results and also, that the structure and function of the gut are beneficially preserved by glutamine [43].

For the biochemical profile, a consistent decreased pattern was found for glucose, cholesterol, HDL-cholesterol, LDL-cholesterol and total protein when pigs were fed the mixture of glutamine plus cystine. The impairment of total cholesterol and glucose at levels within normal range stands out as a promising result, thus suggesting the maintenance of glucose [44] and lipid metabolism [45] homeostasis. The same applies for total protein [46]. Urea and creatinine variations were indicative of unaffected renal function [47]. The systemic redox status was kept under balance in piglets from all dietary treatments. In fact, Wang et al. [48] reported that glutamine improves intestinal oxidative defence capacity. Cysteine (cystine is the oxidized dimer form of cysteine [49]) is generally the limiting amino acid for glutathione synthesis in humans [49–51], as in pigs [50], with recognised antioxidant properties [49] preventing many diseases, such as cancer, heart attack, stroke and diabetes [52]. In a larger scale trial using the same protocol, we revealed that the supplementation of glutamine and cystine improved total antioxidant capacity [53].

Regarding the gene expression of AQPs in piglets, the most expressed aquaporin isoform was AQP3, closely followed by AQP1 and the remaining isoforms tested, regardless the dietary treatment. The magnitude and localization of the transcriptional profile found for these AQPs are in line with the literature [18, 38, 54–57]. Both amino acids, individually or combined, were responsible for a dramatic reduction on the transcriptional profile of AQP1 (at least by 61.8%), AQP7 (at least by 71.0%) and AQP10 (at least by 88.8%). The same trend applies to AQP9, although not reaching statistical significance. However, the expected outcomes on reduction of water permeability were not verified in the ileum membranes from any of the piglets fed glutamine and cystine, individually or combined. In fact, our data shows that Pf was unaffected by diets. This finding contradicts other authors, who reported decreased permeability given by the integrity of the barrier, promoted by glutamine in an experimental mouse model [58]. Disparate results might be a consequence on the use of different animal models (piglets versus rodents), or the different technical approach used here to measure isolated membranes permeability. In fact, here we used a biophysical method to evaluate permeability of isolated membranes, while other studies have looked at the permeability of the whole tissue, which is totally dependent on the integrity of the epithelia. Although the relationship between gene, protein and function is not always directly proportional [59], we may also speculate that the higher intestinal permeability to water might occur upstream of the small intestine, at the level of duodenum or jejunum. Most importantly, the measured water permeability is the end result of the sum of two diffusional pathways, water channels and membrane phospholipid bilayer. Thus, the observed partial reduction at the transcriptional profile of AQP1 promoted by glutamine and cystine is difficult to evaluate under the experimental conditions of this study and may have a minor impact on transepithelial permeability.

AQPs display differential distribution in apical and basolateral epithelial membranes, although their precise localization in intestinal epithelia is still controversial [18]. For instance, AQP3 was found predominantly expressed in basolateral membranes of the small intestine [56, 60]. However, in other study comparing normal subjects vs. celiac disease patients, AQP3 was abundantly expressed but the protein localized at enterocyte apical membranes [16]. Discrepancies in results were attributed to differences in used antibodies, tissue sampling procedures and conservation, patient condition (fasted or fed), and/or other methodological details. Moreover, in pathological conditions, AQPs localization may shift from apical to basolateral membranes, as observed for AQP7 in colon inflammatory bowel disease patients [15]. In this study, we found AQP3 as the most abundantly expressed in piglets’ ileum, in agreement with rodents and humans [18, 56] where AQP3 is predominantly located in basolateral membranes. Using intestinal membrane vesicles enriched in basolateral membranes, we were able to ascertain the impact of AQP3 transcript upregulation by dietary cystine supplementation on intestinal glycerol permeability. Inversely, although AQP7 and AQP10 are described as predominantly located in apical membranes [56, 61], significant changes in their much lower transcript level compared to AQP3 may result in undetectable changes on glycerol permeability induced by these amino acid-enriched diets.

The interplay between AQPs has been reported previously in human obesity. AQP3 and AQP7 have been described to modulate glycerol efflux from adipose tissue, thus controlling the glycerol influx into hepatocytes, via AQP9 to prevent the excessive lipid accumulation and the subsequent aggravation of hyperglycemia [21]. While AQP3 upregulation by cystine supplementation may suggest facilitated glycerol absorption from the intestinal lumen favoring triacylglycerols synthesis and lipid accumulation in the adipose tissue, the downregulation of AQP3 by glutamine-enriched diet shown here (at least by 69.4%) might be a promising strategy to limit the excess dietary glycerol entrance into the bloodstream. Therefore, these data suggest that intestinal AQP3 might also participate in the interplay between adipose AQP7 and hepatic AQP9 to maintain body energy homeostasis.

Glycerol kinase plays a central role in adipogenesis and gluconeogenesis promoting the modification of glycerol in its biological active form, glycerol-3-phosphate [62]. Herein, the low mRNA levels of GK detected in the ileum of piglets are justified by tissue specificity, as glycerol kinase is highly expressed in liver, kidney and testis [63], rather than in the small intestine. However, it is interesting to notice that glutamine-enriched diet increased GK expression (at least by 77.0%) in relation to cystine-enriched diet leaving this molecule available to be used in the enterocyte metabolism, simultaneously with the impairment of AQP3 expression, the main route for dietary glycerol to reach the bloodstream. In turn, PI3K/Akt/mTOR is a signaling pathway regulating AQP3 expression in different metabolic events [21, 39, 64]. Our data presented no variations on gene expression levels of mTOR and PI3KCG by diets and consequently, no association between glycerol channels and these intracellular mediators was accomplished in the small intestine of piglets, under these experimental conditions.

Conclusions

The integrative methodological approach applied in this study using the ileum portion of the small intestine of piglets unveils AQPs gene regulation by dietary amino acids, thus providing a novel insight into the molecular players involved in gut physiology. A clear influence on the transcriptional profile of the water channel AQP1 and aquaglyceroporins AQP7 and AQP10 by glutamine and cystine, individually or combined, was observed, although their impact on the total water movements might be masked by a larger contribution of the lipid bilayer diffusional pathway. However, upregulation of AQP3 gene together with increased glycerol permeability of intestinal basolateral membranes, suggest that glycerol intestinal reabsorption via intestinal AQP3 might be favored in piglets fed cystine diet, revealing AQP3 as a new player in fat accumulation. Altogether, our data show that amino acids dietary supplementation can modulate intestinal AQPs expression and unveil AQP3 as a promising target in adipogenesis regulation. Due to the similarities between pigs and humans, this study allows the translation of porcine data to humans and warrants further additional research to validate its findings.

Acknowledgments

The authors acknowledge Clínica Médica e Diagnóstico Dr. Joaquim Chaves (Algés, Portugal) for the technical assistance regarding the serum metabolites determination.

Citation: Vieira da Silva I, P. Soares B, Pimpão C, M. A. Pinto R, Costa T, Freire JPB, et al. (2021) Glutamine and cystine-enriched diets modulate aquaporins gene expression in the small intestine of piglets. PLoS ONE 16(1): e0245739. https://doi.org/10.1371/journal.pone.0245739

References

1. Groschwitz KR, Hogan SP. Intestinal barrier function: molecular regulation and disease pathogenesis. J Allergy Clin Immunol. 2009; 124(1): 3–20. pmid:19560575

2. Cox AJ, Zhang P, Bowden DW, Devereaux B, Davoren PM, Cripps AW, et al. Increased intestinal permeability as a risk factor for type 2 diabetes. Diabetes Metab. 2017; 43(2): 163–6. pmid:27745826

3. Genser L, Aguanno D, Soula HA, Dong L, Trystram L, Assmann K, et al. (2018) Increased jejunal permeability in human obesity is revealed by a lipid challenge and is linked to inflammation and type 2 diabetes. J Pathol. 2018; 246(2): 217–30. pmid:29984492

4. Camilleri M, Lyle BJ, Madsen KL, Jonnenburg J, Verbeke K, Wu GD. Role for diet in normal gut barrier function: developing guidance within the framework of food-labeling regulations. Am J Physiol Gastrointest Liver Physiol. 2019; 317(1): G17–39. pmid:31125257

5. Stumvoll M, Perriello G, Meyer C, Gerich J (1999) Role of glutamine in human carbohydrate metabolism in kidney and other tissues. Kidney Int. 1999; 55(3): 778–92. pmid:10027916

6. Wu G. Amino acids: metabolism, functions, and nutrition. Amino Acids. 2009; 37(1): 1–17. pmid:19301095

7. Perriello G, Nurjhan N, Stumvoll M, Buccci A, Welle S, Dailey G, et al. Regulation of gluconeogenesis by glutamine in normal postabsorptive humans. Am J Physiol. 1997; 272(3 Pt 1): E437–E45. pmid:9124550

8. Gerich JE, Meyer C, Stumvoll MW. Hormonal control of renal and systemic glutamine metabolism. J Nutr. 2000; 130(4S Suppl): 995S–1001S. pmid:10736369

9. Elshorbagy AK, Church C, Valdivia-Garcia M, Smith AD, Refsum H, Cox R. Dietary cystine level affects metabolic rate and glycaemic control in adult mice. J Nutr Biochem. 2012; 23(4): 332–40. pmid:21543215

10. da Silva IV, Soveral, G. Aquaporins in obesity. Adv Exp Med Biol. 2017; 969: 227–38. pmid:28258577

11. Agre P. Aquaporin water channels (Nobel Lecture). Angew Chem Int Ed Engl. 2004; 43(33): 4278–90. pmid:15368374

12. Carbrey JM, Agre P. Discovery of the aquaporins and development in the field. In Handbook of Experimental Pharmacology, vol. 190, pp. 3–28 [Beitz E, editors]. Berlin, Heidelberg: Springer. 2009. https://doi.org/10.1007/978-3-540-79885-9_1 pmid:19096770

13. Verkman AS. Aquaporins: translating bench research to human disease. J Exp Biol. 2009; 212(Pt 11): 1707–15. pmid:19448080

14. Ishibashi K, Tanaka Y, Morishita Y. The role of mammalian superaquaporins inside the cell. Biochim Biophys Acta. 2014; 1840: 1507–12. pmid:24189537

15. Hardin JA, Wallace LE, Wong JFK, O'Loughlin EV, Urbanski SJ, Gall DG, et al. Aquaporin expression is downregulated in a murine model of colitis and in patients with ulcerative colitis, Crohn's disease and infectious colitis. Cell Tissue Res. 2004; 318(2): 313–23. pmid:15338270

16. Laforenza U, Miceli E, Gastaldi G, Scaffino MF, Ventura U, Fontana JM, et al. Solute transporters and aquaporins are impaired in celiac disease. Biol Cell. 2010; 102(8): 457–67. pmid:20415666

17. Ikarashi N, Kon R, Iizasa T, Suzuki N, Hiruma R, Suenaga K, et al. (2012) Inhibition of aquaporin-3 water channel in the colon induces diarrhea. Biol Pharm Bull. 35(6), 957–62. pmid:22687538

18. Laforenza U. Water channel proteins in the gastrointestinal tract. Mol Asp Med. 2012; 33(5–6): 642–50. pmid:22465691

19. Spurlock ME, Gabler NK. The development of porcine models of obesity and the metabolic syndrome. J Nutr. 2008; 138(2): 397–402. pmid:18203910

20. Engelking LR. Gluconeogenesis. In Textbook of Veterinary Physiological Chemistry, 3rd ed, chapter 37, pp. 225–30 [Engelking LR, editors]. Academic Press. 2015.

21. Rodríguez A, Catalán V, Gómez-Ambrosi J, García-Navarro S, Rotellar F, Valentí V, et al. Insulin- and leptin-mediated control of aquaglyceroporins in human adipocytes and hepatocytes is mediated via the PI3K/Akt/mTOR signaling cascade. J Clin Endocrinol Metab. 2011; 96(4): E586–E97. pmid:21289260

22. Gu L-Y, Qiu L-W, Chen X-F, Lv L, Mei Z-C. Expression of aquaporin 3 and aquaporin 9 is regulated by oleic acid through the PI3K/Akt and p38 MAPK signaling pathways. Zhonghua Gan Zang Bing Za Zhi. 2013; 21(10): 753–8. pmid:24331633

23. Council Regulation (EC). Official Journal of the European Union. No 1/2005 of 22 December 2004. 2005.

24. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972; 18(6): 499–502. https://doi.org/10.1093/clinchem/18.6.499 pmid:4337382

25. Covaci A, Voorspoels S, Thomsen C, van Bavel B, Neels H. Evaluation of total lipids using enzymatic methods for the normalization of persistent organic pollutant levels in serum. Sci Total Environ. 2006; 366(1): 361–6. pmid:16624383

26. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001; 25(4): 402–8. pmid:11846609

27. Fleige S, Pfaffl MW. RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med. 2006; 27(2–3): 126–39. pmid:16469371

28. Fujita M, Ota H, Kawai K, Matsui H, Nakao M. Differential isolation of microvillous and basolateral plasma membranes from intestinal mucosa: mutually exclusive distribution of digestive enzymes and ouabain-sensitive ATPase. Biochim Biophys Acta. 1972, 274(2): 336–47. pmid:4340259

29. Shirazi-Beechey SP, Davies AG, Tebbutt K, Dyer J, Ellis A, Taylor CJ, et al. Preparation and properties of brush-border membrane vesicles from human small intestine. Gastroenterol. 1990; 98(3): 676–85. pmid:2298371

30. Tacnet F, Lauthier F, Ripoche P. Mechanisms of zinc transport into pig small intestine brush-border membrane vesicles. J Physiol. 1993; 465: 57–72. pmid:8229851

31. Soveral G, Macey R, Moura T. Water permeability of brush border membrane vesicles from kidney proximal tubule. J Membrane Biol. 1997; 158(3): 219–28. pmid:9263884

32. Danielsen EM, Hansen GH, Niels-Christiansen LL. Localization and biosynthesis of aminopeptidase N in pig fetal small intestine. Gastroenterol. 1995; 109(4): 1039–50. pmid:7557068

33. Pinches SA, Gribble SM, Beechey RB, Ellis A, Shaw JM, Shirazi-Beechey SP. Preparation and characterization of basolateral membrane vesicles from pig and human colonocytes: the mechanism of glucose transport. Biochem J. 1993; 294 (Pt 2): 529–34. pmid:8396917

34. Colas B, Maroux S. Simultaneous isolation of brush border and basolateral membrane from rabbit enterocytes. Presence of brush border hydrolases in the basolateral membrane of rabbit enterocytes. Biochim Biophys Acta 1980; 600(2): 406–20. pmid:7407121

35. van Heeswijk MP, van Os CH. Osmotic water permeabilities of brush border and basolateral membrane vesicles from rat renal cortex and small intestine. J Membr Biol. 1986; 92(2): 183–93. pmid:3761362

36. Dix JA, Ausiello DA, Jung CY, Verkman AS. Target analysis studies of red cell water and urea transport. Biochim Biophys Acta. 1985; 821(2): 243–52. pmid:2998469

37. Agre P, Sasaki S, Chrispeels MJ. Aquaporins: a family of water channel proteins. Am J Physiol. 1993; 265(3 Pt 2): F461. pmid:7692747

38. Pelagalli A, Squillacioti C, Mirabella N, Meli R. Aquaporins in health and disease: an overview focusing on the gut of different species. Int J Mol Sci. 2016; 17(8): 1213. pmid:27472320

39. Zhu C, Jiang Z, Bazer FW, Johnson GA, Burghardt RC, Wu G. Aquaporins in the female reproductive system of mammals. Front Biosci. 2015; 20: 838–71. pmid:25553483

40. Kasumyan AO, Morsi AMK. Taste sensitivity of common carp Cyprinus Carpio to free amino acids and classical taste substances. J Ichthyol. 1996; 36(5): 391–403.

41. Kurihara S, Shibahara S, Arisaka H, Akiyama Y. Enhancement of antigen-specific immunoglobulin G production in mice by co-administration of L-cystine and L-theanine. J Vet Med Sci. 2007; 69(12): 1263–70. pmid:18176023

42. Tsuchiya T, Honda H, Oikawa M, Kakita T, Oyama A, Oishi H, et al. Oral administration of the amino acids cystine and theanine attenuates the adverse events of S-1 adjuvant chemotherapy in gastrointestinal cancer patients. Int J Clin Oncol. 2016; 21(6): 1085–90. pmid:27306219

43. van der Hulst RR, van Kreel BK, von Meyenfeldt MF, Brummer RJ, Arends JW, Deutz NE, et al. Glutamine and the preservation of gut integrity. The Lancet. 1993; 341(8857): 1363–5. pmid:8098788

44. Madeira MS, Pires VMR, Alfaia CM, Lopes PA, Martins SV, Pinto RMA, et al. Restriction of dietary protein does not promote hepatic lipogenesis in lean or fatty pigs. Br J Nutr. 2016; 115(8): 1339–51. pmid:26927728

45. Jackson PGG, Cockcroft PD. Laboratory reference values: biochemistry. In Clinical Examination of Farm Animals, Appendix 3, pp. 303–5. Oxford: Blackwell Science. 2002. https://doi.org/10.1002/9780470752425.app3

46. Madeira MS, Rolo EA, Lopes PA, Ramos DA, Alfaia CM, Pires VMR, et al. Betaine and arginine supplementation of low protein diets improves plasma lipids but does not affect hepatic fatty acid composition and related gene expression profiling in pigs. J Sci Food Agric. 2018; 98(2): 598–608. pmid:28664986

47. Kaneko JJ, Harvey JW, Bruss ML. Clinical Biochemistry of Domestic Animals, 5th ed, pp. 895–900. San Diego, CA: Academic Press. 1997. https://doi.org/10.1016/B978-012396305-5/50032-4

48. Wang J, Chen L, Li P, Li X, Zhou H, Wang F, et al. Gene expression is altered in piglet small intestine by weaning and dietary glutamine supplementation. J Nutr. 2008; 138(6): 1025–32. pmid:18492829

49. Wu G, Fang Y-Z, Yang S, Lupton JR, Turner ND. Glutathione metabolism and its implications for health. J Nutr. 2004; 134(3): 489–92. pmid:14988435

50. Jahoor F, Jackson A, Gazzard B, Philips G, Sharpstone D, Frazer ME, et al. Erythrocyte glutathione deficiency in symptom-free HIV infection is associated with decreased synthesis rate. Am J Physiol. 276(1): E205–11. pmid:9886968

51. Lyons J, Rauh-Pfeiffer A, Yu YM, Lu XM, Zurakowski D, Tompkins RG, et al. Blood glutathione synthesis rates in healthy adults receiving a sulfur amino acid-free diet. Proc Natl Acad Sci. USA 2000; 97(10): 5071–6. pmid:10792033

52. Townsend DM, Tew KD, Tapiero H. The importance of glutathione in human disease. Biomed Pharmacother. 2003; 57(3–4): 145–55. pmid:12818476

53. Freire J, Prates J, Pinho M, Lopes PA, Madeira M, Alfaia C, et al. Effect of glutamine and/or cystine on gut health, immunity and redox status in post-weaning piglets. In Energy and protein metabolism and nutrition. EAAP Scientific Series, 138, pp. 307–8. Wageningen Academic Publishers. 2019. https://doi.org/10.3920/978-90-8686-891-9

54. Kitchen P, Day RE, Salman MM, Conner MT, Bill RM, Conner AC. Beyond water homeostasis: diverse functional roles of mammalian aquaporins. Biochim Biophys Acta 2015; 1850(12): 2410–21. pmid:26365508

55. Kreida S, Tornroth-Horsefield S. Structural insights into aquaporin selectivity and regulation. Curr Opin Struct Biol. 2015; 33, 126–34. pmid:26342685

56. Laforenza U, Bottino C, Gastaldi G. Mammalian aquaglyceroporin function in metabolism. Biochim Biophys Acta Biomembr. 2016; 1858(1): 1–11. pmid:26456554

57. Zhu C, Chen Z, Jiang Z. Expression, distribution and role of aquaporin water channels in human and animal stomach and intestines. Int J Mol Sci. 2016; 17(9): 1399. pmid:27589719

58. dos Santos RGC, Viana ML, Generoso SV, Arantes RE, Correia MITD, Cardoso VN. Glutamine supplementation decreases intestinal permeability and preserves gut mucosa integrity in an experimental mouse model. J Parenter Enteral Nutr. 2010; 34(4): 408–13. pmid:20631386

59. Joaquim LM, El-Hani CN. A genética em transformação: crise e revisão do conceito de gene. Scientiae Studia 2010; 8(1): 93–128. https://doi.org/10.1590/S1678-31662010000100005

60. Mobasheri A, Wray S, Marples D. Distribution of AQP2 and AQP3 water channels in human tissue microarrays. J Mol Histol. 2005; 36(1–2): 1–14. pmid:15703994

61. Ishii M, Ohta K, Katano T, Urano K, Watanabe J, Miyamoto A, et al. Dual functional characteristic of human aquaporin 10 for solute transport. Cell Physiol Biochem. 2011; 27(6): 749–56. pmid:21691092

62. Parr LS, Sriram G, Nazarian R, Rahib L, Dipple KM. The ATP-stimulated translocation promoter (ASTP) activity of glycerol kinase plays central role in adipogenesis. Mol Genet Metab. 2018; 124(4): 254–65. pmid:29960856

63. Lin EC. Glycerol utilization and its regulation in mammals. Annu Rev Biochem. 1977; 46: 765–95. pmid:197882

64. Huang X, Huang L, Shao M. Aquaporin 3 facilitates tumor growth in pancreatic cancer by modulating mTOR signaling. Biochem Biophys Res Commun. 2017; 486(4): 1097–1102. pmid:28377226