Effects of sodium, 1,25-dihydroxyvitamin D3 and parathyroid hormone fragment on inorganic P absorption and Type IIb sodium-phosphate cotransporter expression in ligated duodenal loops of broilers

Effects of sodium, 1,25-dihydroxyvitamin D3 and parathyroid hormone fragment on inorganic P absorption and Type IIb sodium-phosphate cotransporter expression in ligated duodenal loops of broilers X. D. Liao,1 H. Q. Suo,1 L. Lu, Y. X. Hu, L. Y. Zhang, and X. G. Luo2 Mineral Nutrition Research Division, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, P. R. China ABSTRACT Three experiments were conducted with 22-day-old Arbor Acres male broilers to study the effects of Na+ , 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ] and parathyroid hormone fragment [PTH (1– 34)] on inorganic P absorption and Type IIb sodiumphosphate cotransporter (NaP-IIb) mRNA and protein expression levels in ligated duodenal loops. The duodenal loops were perfused with solutions (pH = 6) containing zero, 50, or 150 mmol/L of Na+ as NaCl in Exp. 1, containing zero, 30, or 300 pmol/L of 1,25-(OH)2 D3 in Exp. 2, or containing zero, 65, or 650 pmol/L of PTH (1–34) in Exp. 3, respectively. Compared with the control, additions of 50 and 150 mmol/L of Na+ , 30 and 300 pmol/L of

1,25-(OH)2 D3 , or 65 and 650 pmol/L of PTH (1– 34) to the perfusates promoted (P < 0.02) the P absorption percentages and rates, respectively. Additions of the above-mentioned concentrations of Na+ or 1,25-(OH)2 D3 to the perfusates increased (P < 0.003) NaP-IIb mRNA level in the duodenum of broilers, and a similar trend (P = 0.08) was observed for PTH (1–34). The Na+ , 1,25-(OH)2 D3 , and PTH (1– 34) had no effects (P > 0.15) on NaP-IIb protein level in the duodenum of broilers. The results indicate that increased P absorption due to perfusions of Na+ , 1,25-(OH)2 D3 or PTH (1–34) might be attributed to enhanced NaP-IIb expression in the duodenum of broilers.

Key words: broilers, duodenal loops, phosphorus absorption, regulation factors, type IIb sodium-phosphate cotransporter 2017 Poultry Science 0:1–7 http://dx.doi.org/10.3382/ps/pex033

INTRODUCTION

Peterlik, 1979; Nemere, 1996; Nemere and Larsson, 2002; Zhao and Nemere, 2002). However, the mechanisms by which P absorption is regulated in the small intestine of broilers are not fully understood. Our laboratory using in situ small intestinal loops (Liu, 2012; Liu et al., 2016) demonstrated that the duodenum is the main site of P absorption, and P absorption is a saturated carrier mediated process in the duodenum of broilers. Besides, type IIb sodium-phosphate cotransporter (NaP-IIb) is a protein recently shown to play an important role in P transport in the small intestine of animals (Werner and Kinne, 2001; Xu et al., 2002; Murer et al., 2004; Giral et al., 2009; Liu, 2012). Whether additions of Na+ , 1,25-(OH)2 D3 , or PTH (1–34) could promote P absorption in the duodenum of broilers by up-regulating NaP-IIb expression has not been clarified. Therefore, the objective of the current study was to investigate the effects of Na+ , 1,25-(OH)2 D3 , and PTH (1–34) on P absorption and NaP-IIb mRNA and protein expression levels in the duodenum of chicks by using in situ ligated duodenal loops, so as to elucidate whether the above factors could promote P absorption by enhancing NaP-IIb expression.

Phosphrous is an essential mineral for animals, and it plays an important role in nucleic acid synthesis, energy metabolism, muscle function, enzyme activity, lipid metabolism, and bone mineralization (Berndt and Kumar, 2009). In commercial broiler production, the amount of P routinely added to diets is to meet the requirements of fast-growing chicks (NRC, 1994). However, chicks utilize only about 60% of the dietary P, which could result in a large amount of P excretion and, thus, cause environment P pollution and the waste of P resources (Liu et al., 2012; 2013). Therefore, improving intestinal P absorption is a key point for this issue. Some studies have demonstrated that Na+ , 1,25-dihydroxyvitamin D3 [1,25-(OH)2 D3 ] and parathyroid hormone fragment [PTH (1–34)] could modulate P absorption in the small intestine of broilers (Taylor, 1974; Fuchs and  C 2017 Poultry Science Association Inc. Received June 14, 2016. Accepted January 18, 2017. 1 Both authors contributed equally to this study. 2 Corresponding author: [email protected]

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LIAO ET AL. Table 1. Composition of the basal diet for broilers (as-fed basis). Item

Percentage, % unless noted

Ingredients Ground yellow corn Soybean meal Soybean oil CaHPO4 ·2H2 O1 Limestone1 NaCl1 DL-Met1 Micronutrients1,2 Nutrient composition ME, MJ/kg CP3 Lys Met Met + Cys Ca3 Nonphytate P3

55.97 36.20 4.00 2.02 1.06 0.30 0.13 0.32 12.46 21.08 1.10 0.51 0.91 1.05 0.45

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Feed grade. Provided per kilogram of diet: vitamin A (all-trans retinol acetate), 15,000 IU; cholecalciferol, 4,500 IU; vitamin E (all-rac-α tocopherolacetate), 24 IU; vitamin K (menadione sodium bisulfate), 3.5 mg; thiamin (thiamin mononitrate), 3.0 mg; riboflavin, 9.6 mg; vitamin B6 , 3.0 mg; vitamin B12 , 0.018 mg; calcium pantothenate, 15.0 mg; niacin, 39.0 mg; folic acid, 1.5 mg; biotin, 0.15 mg; choline (choline chloride), 700 mg; Cu (CuSO4 ·5H2 O), 8 mg; Mn (MnSO4 ·H2 O), 110 mg; Fe (FeSO4 ·7H2 O), 80 mg; Zn (ZnSO4 ·7H2 O), 60 mg; I (KI), 0.35 mg; Se (Na2 SeO3 ), 0.15 mg. 3 Analyzed values and each value based on triplicate determinations. 2

MATERIALS AND METHODS Birds, Diets, and Experimental Design All experimental procedures were approved by the Office of Beijing Veterinarians. One-day-old Arbor Acres commercial male broiler chicks (Huadu Broiler Breeding Corp., Beijing, China) were used in 3 experiments. Chicks were housed in an electrically heated, thermostatically controlled room equipped with fiberglass feeders and stainless steel cages coated with plastics (100 × 50 × 45 cm), and maintained on a 24-hour constant light schedule from one to 21 d of age. Feed and tap water were available ad libitum. The basal cornsoybean meal diets were formulated to meet or exceed the NRC (1994) requirements of broilers for all nutrients (Table 1). Experiment 1 was conducted to investigate the effect of Na+ level on P absorption percentage and rate, and NaP-IIb mRNA and protein expressions in ligated duodenal loops of birds. The 3 treatments of different Na+ concentrations in the perfusates were zero, 50, and 150 mmol/L of Na+ as NaCl, respectively. Experiment 2 was conducted to investigate the effect of 1,25-(OH)2 D3 level on P absorption percentage and rate, and NaPIIb mRNA and protein expressions in ligated duodenal loops of birds. The 3 treatments of different 1,25(OH)2 D3 concentrations in the perfusates were zero, 30, and 300 pmol/L of 1,25-(OH)2 D3 , respectively. Experiment 3 was conducted to investigate the effect of PTH (1–34) level on P absorption percentage and rate, and

NaP-IIb mRNA and protein expressions in ligated duodenal loops of birds. The 3 treatments of different PTH (1–34) concentrations in the perfusates were zero, 65, and 650 pmol/L of PTH (1–34), respectively. The birds at 22 d of age with similar BW (900 ± 20 g) were assigned randomly to one of 10 replicate cages of one chick per cage for one treatment of each experiment. Each ligated duodenal loop of one bird was considered as one replicate.

Preparations of Perfusion Solutions Engberg et al. (2002) reported that the pH value of chyme in the duodenum of 22-day-old broilers was 6.0, thus the perfusates injected into the duodenal loops were buffered with 15.5 mmol/L of morpholineoethanesulfonic (MES) acid based on the pH value (Ji et al., 2006; Bai et al., 2008; Yu et al., 2008; 2012). According to the results of a previous study from our laboratory (Liu, 2012; Liu et al., 2016), the duodenal loops were perfused with the solution containing 6.0 mmol P/L as KH2 PO4 , which was chosen according to the determined P concentration in the liquid chyme of the small intestine of chicks fed with a normal P diet. Considering the need for Na+ for the P uptake, the perfusates contained 150 mmol Na+ /L as NaCl in Exp. 2 and 3 (Wasserman and Taylor, 1973). The 3.0 mg/L of polyethylene glycol 3350 (Sigma, catalog number 202444, St. Louis, MO) was used as a nonabsorbable indicator (Schiller et al., 1997). Therefore, the basal perfusate was composed of 6.0 mmol P/L as KH2 PO4 , 150 mmol Na+ /L as NaCl (except Exp. 1), 3.0 mg/L of polyethylene glycol 3350, and 15.5 mmol/L of MES (pH = 6.0). In Exp. 1, the above-mentioned concentrations of Na+ as NaCl were added to the basal perfusate, and choline chloride was added to keep the perfusates of 3 treatments containing equal levels of Cl− ; in Exp. 2, the abovementioned concentrations of 1,25-(OH)2 D3 were added to the basal perfusate; in Exp. 3, the above-mentioned concentrations of PTH (1–34) were added to the basal perfusate.

Perfusion Protocol Perfusion protocol was adopted by previocus reports from our laboratory (Ji et al., 2006; Yu et al., 2008; Bai et al., 2012). Briefly, chicks were fasted overnight (12 h) and anesthetized by intravenous injection of sodium pentobarbital (40 mg/kg of BW). The abdomen was opened by midline incision. At one cm distal to the pylorus, an inlet polyethylene cannula (inner diameter, 1.0 mm; outer diameter, 1.6 mm) was inserted into a hole made with a 22-gauge needle through the intestinal wall into the lumen. The cannula was secured with suture tied around the intestine and tubing. A 10 cm section of the intestine was isolated and

REGULATION OF PHOSPHORUS ABSORPTION

an outlet cannula was inserted in a similar way at the opposite end of the loop. The loop was flushed with warm saline except for Exp. 1 (5% glucose solution) to remove any chyme that might be present, and then the distal loop was tied off to contain the perfusate. The duodenal loop was injected with 3 mL of perfusion solution with a calibrated syringe through the cannula, and then the cannula was covered by a removable stopper. The perfused loop was placed carefully back into the abdominal cavity for the duration of the experiment. All chicks were stable throughout anaesthesia, and there were no significant changes in heart rate, respiratory rate, or body temperature during the perfusion periods.

Sample Collections The perfusates in the ligated duodenal loops were harvested (2 mL) through the cannula at 40 min after perfusion, and this time was determined in the previous study from our laboratory (Liu, 2012; Liu et al., 2016). All chicks were killed by cervical dislocation. The ligated duodenal loops were excised, flushed with ice-cold saline, and slit lengthwise. The duodenal mucosa was scraped with an ice-cold microscope slide (Symolon et al., 2004), immediately frozen in liquid N2 , and then stored at −80◦ C for analyses of NaP-IIb mRNA and protein expressions.

Determinations of CP, Ca, and P Concentrations and Calculations− of P Absorption Concentrations of CP in feed ingredients and diet samples were determined according to the Kjeldahl method (Thiex et al., 2002). The Ca concentrations in feed ingredients and diet samples were measured using an inductively coupled plasma spectroscopy (Model IRIS Intrepid II, Thermal Jarrell Ash, Waltham, MA) after wet digestion with HNO3 and HClO4 (Huang et al., 2013). Phosphorus concentrations in the feed ingredients, diet samples, tap water, and fluid perfusates in the duodenal loops were determined colorimetrically using the vanadate-molybdate method (Davies et al., 1973). A nonabsorbable reference indicator (polyethylene glycol 3350) was used to correct the changes in P concentrations resulting from water absorption or duodenal secretion. The concentrations of polyethylene glycol in initial and final fluid contents were determined as described by Boulter and McMichael (1970). Final volumes of perfusates in duodenal loops were calculated by the change in concentration of the polyethylene glycol, and the P absorption percentage and rate were calculated according to these equations below:

VF =

CP(1) × VI CP(2)

UP =

CP(1) × VI − CP(2) × VF × 100 CP(1) × VI

UV =

CP(1) × VI − CP(2) × VF PT × PL

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where VF = final volume of perfusion solution (mL); CP(1) and CP(2) = initial and final concentrations (mg/L) of polyethylene glycol, respectively; VI = initial volume (mL) of injected dose; UP = absorption percentage of P (%); CP(1) and CP(2) = P concentrations (nmol/L) of initial and final perfusion solutions, respectively; UV = absorption rate of P (nmol·min−1 ·cm−1 ); PT = time (min) of perfusion; and PL = length (cm) of perfusion.

Isolations of Total RNA and Quantitative Real-time PCR The total RNA in duodenal mucosa was isolated using Trizol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The RNA concentration was measured by using the Nano Drop ND-1000 spectrofluorometer (Nano-Drop Technologies, Wilmington, DE), and the quality of total RNA was determined in agarose gels stained with ethidium bromide. One microgram of total RNA was subjected to reverse transcription by using the Super Script FirstStrand Synthesis System (Invitrogen, Carlsbad, CA). The PCR primers used to detect NaP-IIb, β -actin and GAPDH were as follows: 5 - CTG GAT GCA CTC CCT AGA GC-3 and 5 - TTA TCT TTG GCA CCC TCC TG -3 for NaP-IIb (NM 204474.1); 5 - GAG AAA TTG TGC GTG ACA TCA -3 and 5 - CCT GAA CCT CTC ATT GCC A -3 for β -actin (NM 205518.1); and 5 - CTT TGG CAT TGT GGA GGG TC -3 and 5 ACG CTG GGA TGA TGT TCT GG -3 for GAPDH (K01458.1). Real-time PCR reactions were performed on an ABI 7500 real-time PCR system using SYBRGreen PCR Master Mix (Applied Biosystems, Foster City, CA). The PCR reactions were performed for 10 min at 95◦ C, 40 cycles of 94◦ C for 15 s, and 60◦ C for one min. Relative mRNA expression of NaP-IIb gene was calculated using the 2−C T method reported by Livak and Schmittgen (2001). The geometric mean of internal reference genes, β -actin and GAPDH, was used to normalize the expression of the targeted gene. The run was performed in triplicate.

Tissue Preparations and Western Blotting The NaP-IIb protein level in duodenal mucosa was determined using the Western blot technique. Frozen duodenal mucosal samples (40 mg) were homogenized

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LIAO ET AL. Table 2. Effect of Na+ level on the percentages and rates of P absorption and NaP-IIb mRNA and protein levels in the duodenum of broliers1 (Exp. 1). Added Na+ level, mmol/L 0 50 150 Pooled SE P-value

P absorption percentage, %

P absorption rate, nmol·min−1 ·cm−1

NaP-IIb mRNA, RQ2

NaP-IIb protein, arbitrary unit3

32.2b 57.2a 61.0a 3.54 < 0.0001

12.8b 22.9a 25.0a 1.41 < 0.0001

0.82b 1.20a 1.11a 0.056 0.0003

0.48 0.64 0.65 0.067 0.16

Means with different superscripts with the same column differ significantly (P < 0.003). Data represent the means of 10 replicate duodenal loops (n = 10); NaP-IIb represents type IIb sodium-phosphate cotransporter; RQ represents relative quantity. 2 NaP-IIb mRNA was calculated as the ratio of NaP-IIb mRNA to the geometric mean of β -action mRNA and GAPDH, and RQ = 2−C T (CT = threshold cycle). 3 GAPDH protein was used to normalize the expression of the NaP-IIb protein, and the average expression of protein in the control was used as a calibrator. a,b 1

in 0.5 mL of ice-cold RIPA lysis buffer (Beyotime Institute of Biotechnology, catalog number P0013B, Haimen, China) supplemented with protease inhibitor (Biotool, catalog number B14001, Houston, TX). The homogenate was centrifuged at 10,000 × g for 10 min at 4◦ C, and the supernatant was collected for total protein determination using a BCA Protein Assay kit (Pierce, catalog number 23225, Rockford, IL). The extracted protein (30 ug) was subjected to electrophoresis on a 9% SDS-PAGE gel, and then electrotransfered onto the polyvinylidene fluoride membranes (MerckMillipore, catalog number IPVH00010, Billerica, MA). After the transfer, membranes were blocked for one h at room temperature in blocking buffer with 5% skimmed milk. In order to be able to investigate the proteins of different sizes on the same membrane, the membrane was cut into 2 horizontal strips (Heidebrecht et al., 2009). Then the strips were separately incubated overnight at 4◦ C with the following primary antibodies and dilution rates: NaP-IIb (Santa Cruz, CA, catalog number sc-160605, 1:250), and GAPDH (Huaxingbio, Beijing, China, catalog number HX1828, 1:5,000). After 4 washes for 10 min each in Tris-buffered saline with Tween, the strips with NaP-IIb were incubated with rabbit anti-goat horseradish peroxidase-conjugated antibody (Huaxingbio, Beijing, China, catalog number HX2030, 1:5,000) and the strips with GAPDH were incubated with goat anti-mouse horseradish peroxidaseconjugated antibody (Huaxingbio, Beijing, China, catalog number HX2032, 1:5,000). After one h of incubation at room temperature, the strips were washed 4 times for 10 min each, and bands were visualized by enhanced chemiluminescence using a High-sig ECL Western Blotting Substrate Kit (Tanon, Shanghai, China, catalog number 180–5001). The signals were recorded with a chemiluminescence image scanner (Tanon, Shanghai, China) and analyzed with Tanon Gis 1D software (Tanon, Shanghai, China). Data were presented as the ratio of NaP-IIb protein band intensity to GAPDH protein band intensity. The GAPDH protein was used to normalize the expression level of NaP-IIb, and the average expression of protein in the control was used as a calibrator.

Figure 1 Representative immunoblots of NaP-IIb protein expression in the duodenum of broilers from different perfused Na+ levels (Exp. 1). NaP-IIb represents type IIb sodium-phosphate cotransporter.

Statistical Analyses Data from Exp. 1, 2, and 3 were subjected to one-way ANOVA using the GLM procedures of SAS (version 9.2; SAS Inst. Inc., Cary, NC). Differences among means were tested by the LSD method. Each duodenal loop was the experimental unit. The P < 0.05 was considered to be statistically significant.

RESULTS Na+ Level on P Absorption and NaP-IIb Expression in Exp. 1 The Na+ level in the perfusates affected (P < 0.0004) P absorption percentage and rate as well as NaP-IIb mRNA level in the duodenum of broilers, but had no effect (P > 0.15) on NaP-IIb protein expression level (Table 2 and Figure 1). The addition of either 50 or 150 mmol/L of Na+ to the perfusates increased (P < 0.003) P absorption percentage and rate as well as NaP-IIb mRNA level in the duodenum of broilers compared with the control. However, no differences (P > 0.24) in these indices were observed between 50 and 150 mmol/L of Na+ levels. In addition, the addition of either 50 or 150 mmol/L of Na+ to the perfusates tended (P > 0.08) to increase NaP-IIb protein level in the duodenum of broilers.

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REGULATION OF PHOSPHORUS ABSORPTION Table 3. Effect of 1,25-(OH)2 D3 level on the percentages and rates of P absorption and NaP-IIb mRNA and protein levels in the duodenum of broilers1 (Exp. 2). Added 1,25-(OH)2 D3 level, pmol/L 0 30 300 Pooled SE P-value

P absorption percentage,%

P absorption rate, nmol·min−1 ·cm−1

NaP-IIb mRNA, RQ2

NaP-IIb protein, arbitrary unit3

44.0b 63.3a 63.3a 2.72 < 0.0001

17.9b 25.9a 26.7a 1.13 < 0.0001

0.77b 1.20a 1.10a 0.06 0.0001

0.57 0.61 0.63 0.05 0.75

Means with different superscripts with the same column differ significantly (P < 0.0008). Data represent the means of 10 replicate duodenal loops (n = 10); NaP-IIb represents type IIb sodium-phosphate cotransporter; RQ represents relative quantity. 2 NaP-IIb mRNA was calculated as the ratio of NaP-IIb mRNA to the geometric mean of β -action mRNA and GAPDH, and RQ = 2−C T (CT = threshold cycle). 3 GAPDH protein was used to normalize the expression of the NaP-IIb protein, and the average expression of protein in the control was used as a calibrator. a,b 1

dition of either 65 or 650 pmol/L of PTH (1–34) to the perfusates tended to have higher (P > 0.03) NaP-IIb mRNA and protein expression levels in the duodenum of broilers than those in the control.

DISCUSSION Figure 2. Representative immunoblots of NaP-IIb protein expression in the duodenum of broilers from different perfused 1,25-(OH)2 D3 levels (Exp. 2). NaP-IIb represents type IIb sodium-phosphate cotransporter; 1,25-(OH)2 D3 represents 1,25-dihydroxyvitamin D3 .

1,25-(OH)2 D3 Level on P Absorption and NaP-IIb Expression in Exp. 2 The 1,25-(OH)2 D3 level in the perfusates affected (P < 0.0002) P absorption percentage and rate as well as NaP-IIb mRNA expression level in the duodenum of broilers, but had no effect (P > 0.74) on NaP-IIb protein expression level (Table 3 and Figure 2). The addition of either 30 or 300 pmol/L of 1,25-(OH)2 D3 to the perfusates increased (P < 0.0008) P absorption percentage and rate as well as NaP-IIb mRNA level in the duodenum of broilers compared with the control, whereas no differences (P > 0.26) in these indices were observed between 30 and 300 pmol/L of 1,25-(OH)2 D3 levels.

PTH (1–34) Level on P Absorption and NaP-IIb Expression in Exp. 3 The PTH (1–34) level in the perfusates affected (P < 0.02) P absorption percentage and rate in the duodenum of broilers, but had no effect (P > 0.07) on NaP-IIb mRNA or protein expression levels (Table 4 and Figure 3). The addition of either 65 or 650 pmol/L of PTH (1–34) to the perfusates increased (P < 0.02) P absorption percentage and rate in the duodenum of broilers compared with the control, but no differences (P > 0.62) in these indices were observed between 65 and 650 pmol/L of PTH (1–34) levels. Besides, the ad-

As P is an expensive mineral ingredient, dietary supplements with excess amounts of P would significantly increase the feed cost of the broiler production (Woyengo and Nyachoti, 2010). Besides, excess P that is not utilized by broilers may enter the environment and can lead to eutrophication of fresh waters, and, thus, cause environmental P pollution (Knowlton et al., 2004; Liu et al., 2012; 2013). One way of reducing P excretion is to improve intestinal P absorption of broilers. The present study showed that additions of Na+ , 1,25-(OH)2 D3 or PTH (1–34) to the perfusates could increase P absorption percentages and rates in the major site of P absorption, the duodenum of broilers. Intestinal phosphate absorption depends on the presence of extracellular Na+ . Its concentrations could affect the affinity of epithelial cells, and reduction of Na+ level could reduce maximal absorption velocity (Fuchs and Peterlik, 1979). Taylor (1974) found that gut sacs needed Na+ for the phosphate uptake and storage steps; Matsumoto et al. (1980) also reported that phosphate uptake into the brush border vesicles was a Na+ -dependent saturated process. Furthermore, it was found that Na+ could stimulate P absorption in Caco-2 cells. The P absorption increased as Na+ (20 to 137 mmol/L) level increased (Ingvild and Matthlas, 1986). In the present study, we found that Na+ promoted P absorption in the duodenum of broilers, which was consistent with the study of Berner et al. (1976), who reported that Na+ stimulated P uptake by brush border vesicles from rat duodenum. We also found that additions of Na+ up-regulated NaP-IIb mRNA level and had a trend to enhance the NaP-IIb protein level in the duodenum of broilers, suggesting that the increased P absorption might be attributed to enhanced duodenal NaP-IIb expression. This has been not reported before,

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LIAO ET AL. Table 4. Effect of PTH (1–34) level on the percentages and rates of P absorption and NaP-IIb mRNA and protein levels in the duodenum of broilers1 (Exp. 3). Added PTH (1–34) level, pmol/L 0 65 650 Pooled SE P-value

P absorption percentage, %

P absorption rate, nmol·min−1 ·cm−1

NaP-IIb mRNA, RQ2

NaP-IIb protein, arbitrary unit3

44.0b 58.7a 57.7a 3.31 0.01

17.9b 24.9a 23.9a 1.38 0.004

0.77 1.17 1.07 0.12 0.08

0.50 0.63 0.59 0.05 0.16

Means with different superscripts with the same column differ significantly (P < 0.02). Data represent the means of 10 replicate duodenal loops (n = 10); NaP-IIb represents type IIb sodium-phosphate cotransporter; RQ represents relative quantity. 2 NaP-IIb mRNA was calculated as the ratio of NaP-IIb mRNA to the geometric mean of β -action mRNA and GAPDH, and RQ = 2−C T (CT = threshold cycle). 3 GAPDH protein was used to normalize the expression of the NaP-IIb protein, and the average expression of protein in the control was used as a calibrator. a,b 1

Figure 3. Representative immunoblots of NaP-IIb protein expression in the duodenum of broilers from different perfused PTH (1–34) levels (Exp. 3). NaP-IIb represents type IIb sodium-phosphate cotransporter; PTH (1–34) represents parathyroid hormone fragment.

and, thus, provides scientific experimental bases and methods for how to improve P absorption in broilers. Some studies have demonstrated that 1,25(OH)2 D3 could stimulate phosphate uptake and transport in the perfused duodenal loops, intestinal brush border membrane vesicles, and isolated intestinal cells of chicks (Matsumoto et al., 1980; Nemere, 1996; Zhao and Nemere, 2002). However, the mechanisms by which 1,25(OH)2 D3 modulates P absorption are not fully understood. The results from the present study demonstrated that 1,25-(OH)2 D3 could stimulate P absorption in the duodenal loops of broilers by up-regulating NaPi-IIb mRNA expression. Similar results were reported by Han et al. (2009), who found that 1α-OH D3 could improve intestinal NaP-IIb mRNA expression and increase phosphate utilization in naturally growing broilers, and together with the present study, indicating that the increased P absorption observed due to vitamin D3 perfusions might result from the enhancements of NaPi-IIb mRNA expression in the duodenum of broilers. Dogma for the past decades has dictated that PTH has no direct effect on intestinal P absorption; however, some previous studies have indicated that PTH had a direct effect on intestinal P absorption (Nemere, 1996; Nemere and Larsson, 2002). Nemere and Larsson (2002) found the presence of receptors of PTH in the intestine, and proved that PTH could stimulate P absorption not only through signal transduction mechanisms, but also by promoting nuclear effects that might include altered gene transcription and cell proliferation. The present

data demonstrate that PTH (1–34) promoted P absorption in the duodenum of broilers, which might be associated with the trend of improved NaP-IIb mRNA expression. However, the Na+ , 1,25-(OH)2 D3 and PTH (1–34) had no significant effects on NaP-IIb protein expression in the duodenum of broilers, which might be due to the shorter perfusion duration in this study. In conclusion, the results from the current study indicate that additions of Na+ , 1,25-(OH)2 D3 or PTH (1–34) to the perfusates promoted P absorption percentages and rates as well as up-regulated NaP-IIb expression in the duodenum of broilers. The increased P absorption due to Na+ , 1,25-(OH)2 D3 or PTH (1–34) perfusions might be attributed to enhanced NaP-IIb expression in the duodenum of broilers.

ACKNOWLEDGMENTS This work was supported by the National Nonprofit Institute Research Grant (2016ywf-yb-8; Beijing, P. R. China), the National Natural Science Foundation of China (project no. 31472116; Beijing, P. R. China), the Key Program of the National Natural Science Foundation of China (project no. 31630073; Beijing, P. R. China), the China Agriculture Research System (project no. CARS-42; Beijing, P. R. China), and the Agricultural Science and Technology Innovation Program (ASTIP-IAS08; Beijing, P. R. China).

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