Î±-Ketoglutarate Axis Regulates Intestinal ... - ACS Publications

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The AMPK/#-ketoglutarate axis regulates intestinal water and ions homeostasis in young pigs Liuqin He, niu huang, huan li, junquan tian, Xihong Zhou, tiejun li, kang yao, Guoyao Wu, and Yulong Yin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00324 • Publication Date (Web): 28 Feb 2017 Downloaded from http://pubs.acs.org on March 2, 2017

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Journal of Agricultural and Food Chemistry

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The AMPK/α-ketoglutarate axis regulates intestinal water and ions homeostasis

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in young pigs

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Liuqin He†,‡, , Niu Huang§, , Huan Li§, Junquan Tian †,‡, Xihong Zhou †, Tiejun Li †, KangYao†,§,*,

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Guoyao Wu# , and Yulong Yin†,*

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†

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Agriculture, Chinese Academy of Sciences; Scientific Observing and Experimental Station of

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Animal Nutrition and Feed Science in South-Central, Ministry of Agriculture; Hunan Provincial

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Engineering Research Center for Healthy Livestock and Poultry Production, Changsha, Hunan

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410125, China

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Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical

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‡

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§

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410128, China

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#

University of the Chinese Academy of Sciences, Beijing, 10008, China

College of Animal Science and Technology, Hunan Agricultural University, Changsha, Hunan,

Department of Animal Science, Texas A&M University, College Station, TX 77843, USA

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ACS Paragon Plus Environment


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ABSTRACT

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Water and ion absorption via sensitive aquaporins (AQPs) and ion channels are of critical

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importance in intestinal health. However, whether α-ketoglutarate (AKG) could improve intestinal

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water and ions homeostasis in LPS-challenged piglet and whether the AMP-activated protein

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kinase (AMPK) pathway was involved remains largely unknown.This study was conducted to

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investigate the effect of dietary AKG supplementation on the small intestinal water and ion

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homeostasis through modulating the AMPK pathway in piglet diarrhea model. A total of 32

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weaned piglets were used in a 2 × 2 factorial design; the major factors were diet (basal diet or 1%

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AKG diet) and challenge (Escherichia coli lipopolysaccharide (LPS) or saline). The results

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showed that LPS challenge increased the diarrhea index and affected the concentrations of serum

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Na+, K+, Cl-, glucose, AKG and its metabolites in piglets fed the basal or AKG diet. However, the

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addition of AKG attenuated diarrhea incidence and reversed these serum parameters

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concentrations. Most AQPs (e.g.,AQP1, AQP3, AQP4, AQP5, AQP8, AQP10, and AQP11) and

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ion transporters (NHE3, ENaC, DRA/PAT1) were widely distributed in the duodenum and

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jejunum of piglets. We also found that AKG up-regulated the expression of intestinal epithelial

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AQPs, while inhibiting the expression of ion transporters. LPS challenge decreased (P < 0.05) the

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gene and protein expression of AMPK pathway (AMPKα1, AMPKα2, SIRT1, PGC-1α, ACC,

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TORC2) in the jejunum and ileum. Notably, AKG supplementation enhanced the abundance of

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these proteins in the LPS-challenged piglets. Collectively, AKG plays an important role in

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increasing water and ion homeostasis through modulating the AMPK pathway. Our novel finding

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has important implications for the prevention and treatment of gut dysfunction in neonates.


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KEY WORDS: Aquaporins, ion transporters, α-ketoglutarate, AMPK, diarrhea

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INTRODUCTION

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Diarrhea, a major healthcare concern in both human medicine and animal production, is a

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critical intestinal illness characterized by various bacterial invasion, as well as imbalanced water

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and ion transfer across the intestinal mucosa.1 The main consequence of diarrhea is the

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disturbance of the gastrointestinal tract osmotic homeostasis, which would aggravate secondary

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injuries such as the infection of gastrointestinal tissues (e.g., infectious diarrhea)2, 3 and negatively

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affect the quality of life.4 However, the molecular mechanism for diarrhea formation/resolution

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are still poorly understood. Therefore, it is necessary to develop a new and effective strategy to

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prevent and treat diarrhea .

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In animals with diarrhea, the intestine exhibits an imbalance between transpot and secretion

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of ions and solutes,5 and the movement of water across the gut mucosa in an attempt to restore the

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appropriate intestinal permeability.6, 7 Recently, it has become clear that the discovery of intestinal

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aquaporins (AQPs) offers hope for the development of new therapies for diarrhea.8, 9 AQPs,

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including 13 AQPs (AQP0-12) in mammals, play a key role in the rapid transport of water across

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the gastrointestinal cell membrane.10, 11 Moreover, AQPs support bulk movements of fluid by

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giving high water permeability to membranes that secrete ions and solute (e.g., Na+, K+, Cl−, and

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HCO3−).12 This suggests that some AQPs may form macromolecular complexes with ion

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transporters (i.e., sodium channels, Cl−/ HCO3− exchanger, Na+/H+ exchanger).13 Thus the ion or

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water channels may function as the transport and secretion of both ions and water.

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When water and ion channels are responsible for the transport of water and ions across cell

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membranes and intracellular osmotic homeostasis,14 the process also needs to consume large


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amounts of ATP. As a precursor of glutamine and glutamate,15 α-ketoglutarate (AKG) via the

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tricarboxylic acid (TCA) cycle generates a large amount of ATP, which is required for nutrient

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absorption and transport.16 In addition, AMP-activated protein kinase (AMPK) pathway

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stimulated by cellular functions serves in large part to refuel cellular ATP levels and plays a key

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role in energy metabolism.17-19 Lang et al.(2014) reported that AMPK was a powerful regulator of

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transport across cell membrane. Hence AMPK contributed to the regulation of channels, carriers,

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and pumps, such as diverse K+ channels, glucose carriers, Na+/Ca2+ exchanger, amino acid

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transporters, and Na+/K+ -ATPase.20 There is, however, currently no information regarding the

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effect of AKG on the regulation of the AMPK pathway associated with either water or ions

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transport in the intestinal epithelium under diarrhea conditions. Therefore, the present study was

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conducted to test the hypothesis that AKG improves the transport and secretion of intestinal water

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and ions through regulating the AMPK pathway in piglet diarrhea model.

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MATERIALS AND METHODS

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Animals and Experimental Design

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This study was approved by the animal welfare committee of the Institute of Subtropical

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Agriculture, Chinese Academy of Sciences (2013020; Changsha, China). Thirty-two healthy

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piglets of similar body weight (BW= 6.24 ± 0.11kg) (Landrace× Large White) (ZhengHong Co.,

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Changsha, Hunan, China) were weaned at 28 days, and were randomly assigned to either a basal

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(CON) or basal+1% AKG diet (n=16/diet). Then each group of weaned pigs was divided



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randomly into two sub-groups (n=8/treatment group, CON+LPS and AKG+LPS), which were fed

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their respective diets ad libitum. Of note, at 10:00 am on days 22, 25, 28 and 30, piglets in the

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CON+LPS and AKG+LPS groups were intraperitoneally injected with Escherichia coli

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lipopolysaccharide (E. coli LPS, 100µg/kg BW), respectively, whereas pigs in the CON and AKG

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groups were injected intraperitoneally with the same volume of sterile saline. The composition and

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nutrient levels of the diets met the nutrient specifications for 5 to 10 kg BW pig were described in

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our pervious study.21 The administration dosages of AKG (Wuhan Yuancheng Gongchuang

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Technology co., LTD, Wuhan, Hubei, China; purity ≥ 99.2%) and LPS (Escherichia coli serotype

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055:B5; Sigma Chemical, Inc., St Louis, MO, USA) were adopted according to the previous

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experiment. 22 All piglets had free access to water throughout the experimental period. On day 30,

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all pigs were anaesthetized according to our previous methods.21 Blood was obtained from the

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jugular vein after LPS challenge. Samples were collected from the proximal duodenum, middle

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jejunum, and distal ileum, then immediately frozen in liquid nitrogen and stored at -80 °C for

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subsequent analyses of gene and protein expression. One intestinal segment was fixed in 10 %

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neutral buffered formalin for examination of intestinal morphology.

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The determination of diarrhea index

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During the LPS challenge period, the defecation situation of every pig was observed and

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recorded, including the stool color and shape. Pig fecal samples were scored according to our

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observations, and then the diarrhea index of each pig was determined. The criterion for the

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diagnosis of diarrhea in pigs was described in previous studies.23, 24 Based on the criterion, a

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higher score means more severe diarrhea. Diarrhea index = the total scores of all the diarrhea


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piglets during the experiment period/(the total number of experiment days multiplied by pig total

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numbers).23

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Measurement of serum parameters and intestinal morphology

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Serum chlorine (Cl-), sodium (Na+), potassium (K+), calcium (Ca2+), and glucose were

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measured using spectrophotometric kits (Nanjing Jiangcheng Biotechnology Institute, Nanjing,

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Jiangsu, China) in accordance with the manufacturer’s instructions. Serum AKG, glutamate, and

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glutamine concentrations were determined by LC–MS/MS (HPLC Ultimate3000 and 3200 QTRAP

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LC–MS/MS; Applied Biosystems, Foster, CA, USA) as described previously.

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morphology was defined as in the previous study. 25

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Intestinal

cDNA synthesis and quantification mRNA by real-time polymerase chain reactions (PCR) analysis

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Extraction of total RNA and its reverse transcription were performed according to our previous

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study.26 Primers were designed with the use of Primer 5.0 according to the gene sequence of pigs

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(http://www.ncbi.nlm.nih.gov/pubmed/) to produce amplification products (Table 1). β-actin and

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GAPDH were used as a housekeeping gene to normalize target gene transcript levels. Real-time

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PCR was performed as described previously.27 Relative gene expression was normalized and

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expressed as a ratio of the target gene to the control gene using the formula 2- ( ∆∆Ct), where ∆∆ Ct =

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(Ct Target – Ct β-actin/GADPH )treatment - (Ct Target – Ct β-actin/GADPH)control.

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Western bolt analysis

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Jejunum and ileum were extracted with cytomembrane protein and total protein extraction

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reagents (Thermo Fisher Scientific Inc., New York, NK, USA) in accordance with the

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manufacturer’s instructions. The relative expression of protein was determined by western blot

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technique as described previously.28 1)The cytomembrane protein antibodies used in this study



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were as followed: AQP3 (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), AQP4 (1:1000;

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Abcam, Cambridge, LON, UK), AQP8 (1:200; Santa Cruz Biotechnology, Dallas, TX, USA),

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α-ENaC (1:1000; Abcam, UK), NHE3 (1:200; Santa Cruz Biotechnology, Dallas, TX, USA),

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DRA/PAT1 (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), and β-ENaC (1:500;

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Proteintech group.,Inc, Los Angeles, CA,USA). Mouse β-ATPase Na+/K+ antibody (1:2000;

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Abcam, Cambridge, LON, UK ) was used as the cytomembrane protein loading control. 2)The

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total protein antibodies used in this study were as followed: AMPKα1 (1:500; Proteintech group.,

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Inc, Los Angeles, CA,USA), AMPKα2 (1:2000; Proteintech group.,Inc, Los Angeles, CA,USA),

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PGC-1α (1:1000; Abcam, Cambridge, LON, UK), ACC (1:1000; Cell Signaling Technology,

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Danvers, MA, USA), TORC2 (1:1000; Proteintech group.,Inc, Los Angeles, CA,USA), SIRT1

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(1:1000; Proteintech group., Inc, Los Angeles, CA,USA). All total protein measurements were

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normalized to β-actin (1:4000; Proteintech group.,Inc, Los Angeles, CA,USA) and data are

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expressed relative to the values in control piglets.

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Statistical analysis

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All statistical analyses were performed by one-way ANOVA or factorial ANOVA using a

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mixed procedure (PROC MIXED) of SAS software version 9.2 (SAS Institute Inc., Cary, NC,

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USA). The statistical model included the effects of challenge (saline or LPS), diet (basal or AKG),

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and their interactions. All data were presented as Least Squares means plus pooled SEM. The

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Tukey multiple comparison test was used to evaluate the differences among the treatments.

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Probability values ≤ 0.05 were taken to indicate statistical significance.

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RESULTS

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Diarrhea index, intestinal morphology, and serum parameters

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To simulate in vivo diarrhea conditions, we induced piglet diarrhea using the intraperitoneal

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administration of Escherichia coli LPS. Therefore, the primary focus was to monitor the diarrhea

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index (Figure 1a), intestinal morphology(Figure 1b), and serum parameters of weaned piglets. The

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data showed that LPS challenge remarkably increased (P < 0.05) the diarrhea index in piglets fed

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the basal or AKG diet. Compared with the basal diet, AKG supplementation decreased (P < 0.05)

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the diarrhea incidence in piglets. An improvement in the intestinal morphology of both saline- and

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LPS-challenged piglets fed the AKG diet was observed ( Direct data in supplementary material).

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The administration of AKG reduced (P < 0.05) serum sodium concentration (Figure 2B) while

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increasing the concentrations of serum potassium (Figure 2C), chloride (Figure 2A), AKG (Figure

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2E), glutamate(Figure 2G), and glutamine(Figure 2H) (P < 0.05). Meanwhile, LPS also affected

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(P < 0.05) the concentrations of these serum ions, glucose (Figure 2F), and amino acids(glutamate

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and glutamine) in piglets fed the basal or AKG diet. Neither diets nor LPS challenge affected

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serum calcium concentration (Figure 2D). There is no LPS challenge × diet interaction effect (P

Î±-Ketoglutarate Axis Regulates Intestinal ... - ACS Publications

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