Biological Trace Element Research

, Volume 143, Issue 2, pp 901–912 | Cite as

Dietary Supplementation of Boron Differentially Alters Expression of Borate Transporter (NaBCl) mRNA by Jejunum and Kidney of Growing Pigs

  • Shengfa F. Liao
  • James S. Monegue
  • Merlin D. Lindemann
  • Gary L. Cromwell
  • James C. Matthews
Article

Abstract

Inorganic boron (B), in the form of various borates, is readily absorbed across gastrointestinal epithelia. Although there is no stated B requirement, dietary B supplementation is thought to positively affect animal growth and metabolism, including promotion of bone strength and cell proliferation. Because of effective homeostatic control of plasma B levels, primarly by renal excretion, B toxicity in animals and humans is rare. The mechanisms responsible for improved animal performance and borate homeostasis are incompletely understood. Although a Na+-coupled borate transporter (NaBC1) has been identified, the effect of dietary B supplementation on expression of NaBCl has not been evaluated. An experiment was conducted with growing pigs to determine if NaBC1 mRNA was expressed by small intestinal epithelia and kidney of growing barrows and whether dietary B (as borate) supplementation would affect expression of NaBC1 mRNA. A concomitant objective was to test the hypothesis that B supplementation of a phosphorus (P)-deficient diet would improve calcium, phosphorus, or nitrogen retention. Twenty-four crossbred growing barrows (body weight = 74.0 ± 9.8 kg) were selected and used in a randomized complete block design experiment with a total of eight blocks and three B supplementation treatments (n = 8/treatment). A typical corn-soybean meal basal diet (calculated to contain 41 mg intrinsic B/kg) was formulated to meet or exceed nutrient requirements, except for P, and fed to all pigs for 12 days. The basal diet plus 0, 50, or 100 mg/kg of B (prilled sodium borate pentahydrate, \( {\hbox{N}}{{\hbox{a}}_2}{{\hbox{B}}_4}{{\hbox{O}}_7} \cdot 5{{\hbox{H}}_2}{\hbox{O}} \)) was then fed for 18 more days. Feces and urine were collected during days 6 to 16 of the B supplementation, and pigs were killed for collection of jejunal and ileal epithelia and kidney tissue. Real-time reverse transcription-PCR analysis revealed that NaBC1 mRNA was expressed by these tissues, a novel finding for jejunal and ileal epithelia. Boron supplementation increased jejunal, but decreased, renal NaBC1 mRNA expression, relative to the 0 mg/kg treatment. The finding that NaBC1 mRNA is regulatable by dietary B is novel. That B supplementation evoked opposite effects on jejunal and kidney NaBC1 mRNA expression indicates that transcriptional regulation of NaBC1 expression may constitute a part of the homeostatic control of B metabolism. In contrast to its effect on NaBC1 mRNA expression, B supplementation did not affect total tract digestibility or retention of phosphorus, calcium, or nitrogen.

Keywords

Boron supplementation Nutrient–gene interaction Kidney SLC4A11 Small intestine Swine 

Abbreviations

18S

18S ribosomal ribonucleic acid

B

Boron

Ca

Calcium

N

Nitrogen

NaBC1

Sodium-coupled borate carrier 1

P

Phosphorus

Introduction

Boron is an essential nutrient for all vascular plants and may be an essential nutrient for animals including humans [1, 2]. Although organic B may be inaccessible to animals, inorganic B such as borates, due to their high solubility, are readily absorbed across gastrointestinal epithelia and quickly distributed throughout body fluids and soft tissues [1, 3, 4]. Boron in plasma is primarily and rapidly excreted in urine; therefore, the concentration of urinary B can mirror the actual B intake and a higher B intake does not significantly increase the plasma B level [1, 5].

Due to the effective homeostatic control of plasma B levels, which involves gastrointestinal absorption and renal excretion, animal tissue B concentrations are generally kept steady and B toxicity in animals and humans is rare [1, 4, 5]. Although molecular mechanisms responsible for this control are not clear [6], a previously cloned putative “bicarbonate” transporter [7] was recently shown [8] to be a Na+-coupled borate transporter (NaBC1, SLC4A11). In the presence of borate, NaBCl fuctions as a selective, electrogenic, voltage-regulated, and Na+-coupled B(OH)4 transporter [8]. In the absence of borate, NaBC1 cotransports Na+ and OH (H+). NaBCl protein has been identified in several polarized epithelial cell lines and in rat liver, pancreas, kidney, spleen, and parotid and submandibular salivary glands.

Previous research has shown that physiological amounts of B positively affect the metabolism of many biological compounds (glucose, amino acids, triglycerides, macrominerals, and estrogen) and, thus, the function of tissues (brain, skeleton, and immune system) [2]. Previous research [1, 2, 9] also indicates that B may be involved with bone mineralization and structure and suggests that dietary B may have a pronounced influence on Ca and P metabolism when animals are subjected to nutritional stressors, such as P or Ca deficiency. Moreover, dietary supplementation of B in pigs, rats, and chicks has been shown to increase animal feed intake, feed efficiency, bone strength, and/or growth performance [1, 9, 10]. However, the relationship between dietary B supplementation and expression of NaBCl is not known.

Understanding the substrate regulation of NaBC1 mRNA expression will facilitate the future study of tissue- and cell-specifc mechanisms responsible for B homeostasis. The primary objectives of this study were to (1) determine if NaBC1 mRNA is expressed by small intestinal epithelia and kidney tissue of growing pigs and, if so, (2) determine if the expression NaBC1 mRNA by these tissues is responsive to dietary B supplementation. The secondary objective was to test the hypothesis that increased B supplementation of the P-deficient diet would improve Ca, P, or N balance of these animals.

Materials and Methods

Animal Trial Procedure

The studies were conducted under protocols approved by the University of Kentucky Institutional Animal Care and Use Committee. Pigs were individually housed in stainless steel metabolism crates in an environmentally controlled room in an animal research facility on the University of Kentucky campus.

Based on overall body condition, 24 growing barrows [Hampshire or Duroc × (Yorkshire × Landrace), initial body weight (BW) = 74.0 ± 9.8 kg, eight sets of three littermates] were selected and used in a randomized complete bock design experiment involving three dietary treatments. Thus, there were eight pigs for each dietary treatment. A basal, corn-soybean-meal diet, which was not supplemented with any inorganic source of P, was prepared to meet or exceed the nutrient requirements estimated by NRC [11] with the exception of P (Table 1). This basal diet was used as a control diet (0 mg supplemental B/kg) and calculated to contain 41 mg intrinsic B/kg [12]. Two supplemental B treatment diets were fomulated by supplementing with inorganic borax (prilled sodium borate pentahydrate, \( {\hbox{N}}{{\hbox{a}}_2}{{\hbox{B}}_4}{{\hbox{O}}_7} \cdot 5{{\hbox{H}}_2}{\hbox{O}} \)) to provide additional B at 50 and 100 mg/kg diet. Thus, the final formulation of the 50 and 100 mg B/kg supplement diets was calculated to be 91 and 141 mg B/kg, respectively.
Table 1

Formulation and selected nutrient composition of the basal diet fed to growing barrows (As-Fed Basis)

Item

Composition

Ingredients (%)

 Corn

71.000

 Soybean meal (48% CP)

23.300

 Corn oil

4.000

 Limestone

1.150

 Salt

0.400

 Trace mineral premixa

0.075

 Vitamin premixb

0.075

 Total

100.000

Calculated composition

 CP, %

17.280

 Lys, %

0.900

 ME, kcal/kg

3,346

 B (mg/kg)

41.0

 Ca (%)

0.600

 P (total, %)

0.370

 P (available, %)

0.070

Intrinsic B composition was calculated using feedstuff values from Kerr et al. [12]. Boron premix (Rio Tinto Minerals, Greenwood Village, CO) was added to the basal diet at 0.033% and 0.067% to form Diets 2 and 3 with supplemental B concentrations of 50 and 100 mg/kg, respectively

aTrace mineral premix supplied the following ingredients per kilogram of diet: 120 mg of Fe (iron sulfate monohydrate), 150 mg of Zn (zinc oxide), 45 mg of Mn (manganous oxide), 12 mg of Cu (copper sulfate pentahydrate), 1.5 mg of I (calcium iodate), and 0.3 mg of Se (sodium selenite)

bVitamin premix supplied the following ingredients per kilogram of diet: 4,950 IU of vitamin A, 660 IU of vitamin D3, 33 IU of vitamin E, 4.8 mg of vitamin K (as menadione sodium bisulfite complex), 6.6 mg of riboflavin, 16.5 mg of pantothenic acid, 33.0 mg of niacin, 0.99 mg of folic acid, 0.165 mg of d-biotin, 24.5 μg of vitamin B12, and 3.3 mg of vitamin B6

Diets were fed in a gruel form to pigs at 3% of BW during the experiment with the feed being divided into two daily meals and placed in individual feeders attached to each metabolism crate. After consumption of each meal, water was added to the feeder to allow ad libitum access between meals.

Sample Collection

All pigs were fed the basal corn-soybean-meal diet (Table 1) for 12 days to standardize gastrointestinal tract physiologies. Pigs then were fed the basal diet with the appropriate B supplement for 18 days. Total feces and urine were collected from days 6 to 16 of this B supplement period. The beginning and end of the collection period was marked by the addition of 0.5% indigo carmine to the morning meal, and all the feces were collected daily, stored in plastic bags, and frozen at −20°C until the end of the collection period. The collection of urine was initiated 14 h after the feeding of the first marked meal and was completed 14 h after the feeding of the second marked meal at the end of the collection period. A total of 150 mL of 3 N HCl was added to the collection container at the beginning of each collection to prevent volatilization of urinary N. Urine was collected every 24 h and stored at −20°C.

At the end of the B supplement period, pigs were humanely killed by an overdose of sodium pentobarbital (Socumb, distributed by Butler Animal Health Supply, Dublin, OH) and the small intestine and right kidney taken for mRNA analysis. A 0.3-m jejunal section (0.5 m distal to the pyloric sphincter) and a 0.3-m ileal section (adjacent to the end of the ileum) were harvested, everted, and washed three times in ice-cold saline before epithelia were scraped from the underlying musculature with a glass slide on an ice-cold metal tray [13]. The scraped epithelial tissue was mixed and approximately 3 g placed in an aluminum foil pack and snap-frozen in liquid N2. For kidney tissue collection, a representative sample (3 g, containing proportional amounts of cortical and medulla tissues) was collected and snap-frozen as was for intestinal epithelial tissue [14]. The collected intestinal and kidney samples then were stored at −80°C.

Real-Time Reverse Transcription-PCR Analysis of NaBCl mRNA Expression

Total RNA was extracted from approximately 400 mg of the frozen jejunal, ileal, and kidney tissues using TRIzol reagent (Invitrogen, Carlsbad, CA) and a standard protocol [15]. The purity and concentration of the RNA samples was analyzed by a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE), which revealed that all samples were of high purity with 260/280 absorbance ratios greater than 1.98 and 260/230 absorbance ratios greater than 1.99. The integrity of the RNA was examined by gel electrophoresis using Agilent 2100 Bioanalyzer System (Agilent Technologies, Santa Clara, CA). Visualization of gel images and electropherograms showed that all RNA samples had high quality with RNA integrity numbers being greater than 7.6 and 28S/18S rRNA absorbance ratios greater than 1.9.

For each sample, reverse transcription of 3 μg of total RNA was conducted using SuperScript III First-Strand Synthesis System (Invitrogen) and a standard protocol [16]. The resulting cDNA products were stored at −80°C until used in real-time PCRs. Real-time PCR was performed as previously described [15] using an ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) and a Custom TaqMan Primer and Probe set designed in this laboratory and manufactured by Assays-by-Design service (Applied Biosystems 2004 [17]). Briefly, components of a 25-μL PCR were the Assays-by-Design Primer and Probe set (1.25 μL), cDNA template plus DNAse/RNAse free H2O (11.25 μL), and TaqMan Universal PCR Master Mix-No AmpErase UNG (12.5 μL). The PCR conditions used for the amplification and quantification were: an initial denaturing stage (95°C for 10 min), followed by 40 cycles of two amplification stages of denaturing (95°C for 15 s) and annealing/extention (60°C for 1 min), with a melting curve program (60°C to 95°C), a heating rate of 0.15°C/s, and continuous fluorescence measurements. Real-time PCR was performed in triplicate for each sample.

Development of NaBC1 mRNA Quantification Method

For real-time PCR primer-probe set design, a computationally predicted porcine NaBC1 mRNA sequence (GenBank accession number XM_001924562.1) and a reported porcine 18S rRNA sequence (GenBank accession number NR_002170.3) were retrieved from the GenBank database (National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD). To reduce the potential real-time PCR “noise” from genomic DNA contamination, NaBC1 primer-probe set was designed to produce an amplicon that bridges exons 14 and 15 junction. Each primer-probe set consisted of two unlabeled PCR primers and one TaqMan Minor Groove Binding probe labeled with a reporter dye, six-carboxy-fluorescein (FAM) at 5′ end. The sequences of these primers and probes, as well as the locations on their respective templates are presented in Table 2.
Table 2

Primers and probes used for real-time reverse transcription-PCR analysis of NaBC1 mRNA and 18S rRNA (18S)

Primer or Probea

Location on template, bp

Sequenceb

Amplicon size, bp

Real-time RT-PCR for NaBC1 (XM_001924562.1)

 Forward

309–327

5′-CCCCTACATGCGCGAGATC-3′

130

 Probe (fwd)

397–414

5′-FAM-ATTAAGATGGGCAAGTTC-3′

 Reverse

417–438

5′-CAGGCTCTCACTCGGGTTGTAG-3′

Real-time RT-PCR for 18S (NR_002170.3)

 Forward

1,328–1,350

5′-ACGCTATTGGAGCTGGAATTACC-3′

100

 Probe (fwd)

1,364–1,382

5′-FAM-CCAGACTTGCCCTCCAATG-3′

 Reverse

1,403–1,427

5′-CCCTGTAATTGGAATGAGTCCACTT-3′

aThe contents in parentheses are the accession numbers for the corresponding cDNA or gene sequences recorded in the GenBank database. These sequences were retrieved for the purpose of being used as templates to design primers and probes for real-time PCR analyses. The custom TaqMan probes were supplied in forward (fwd) orientation as indicated in the parentheses for each probe

bFAM = 6-carboxy-fluorescein, which is a reporter dye labeled at the 5′ end of TaqMan probe

Both NaBC1 and 18S real-time RT-PCR products were validated by sequence verification [16]. Briefly, the real-time PCR products were purified using the PureLink Quick Gel Extraction Kit (Invitrogen) before sequencing. The purified PCR products then were sequenced by the University of Florida DNA Sequencing Core Laboratory (Gainesville, FL), using appropriate forward and reverse primers (Table 2) for sequencing both sense and antisense DNA strands. The resulting sequences were compared to the GenBank template sequences that were used for primer-probe set design.

For each tissue, standard curves for relative quantitation of NaBC1 and 18S cDNA content were constructed [17, 18, 19] using a single cDNA sample that was prepared from equal volumes of cDNA samples generated from all 24 pigs. The 18S cDNA quantities were used as the endogenous control to normalize the variations in mRNA inputs and RT reaction efficiencies [20]. Specifically, each cDNA sample was serially diluted by 2.5×, 5×, 25×, 125×, 625×, 3,125×, 15,625×, 78,125×, and 390,625×, and the linear ranges for cDNA quantification were established to ascertain appropriate amounts of cDNA to be used for the real-time PCRs. As a result, the dilutions of cDNA samples for NaBC1 and 18S were defined to be 1:5 and 1:15,625, respectively, and the minimal threshold (CT) values detected at these dilutions were 27 to 33 and 25 to 30 for NaBC1 and 18S, respectively.

The relative quantities of NaBC1 cDNA were normalized to 18S cDNA content by calculating the quantity ratios of NaBC1/18S. The relative expression of NaBC1 mRNA was normalized to 18S rRNA content because numerous studies have revealed that 18S content is very stable and can be used as an endogenous control to normalize the expression of other genes in response to various stimuli [16, 17, 20, 21]. Consistent with this understanding, the potential effect of B supplementation on the expression levels of 18S was evaluated and no B supplementation effect (P ≥ 0.37) on 18S rRNA expression was found (data not shown). Thus, the use of 18S rRNA content to normalize the relative expression of NaBC1 mRNA was validated.

Nutrient Balance Sample Preparation and Analysis

Diet, feces, and urine samples were prepared and analyzed as previously described [22]. Briefly, to obtain representative urine samples for nutrient analysis, the collected urine was thawed at room temperature and proportionally composited by sample weight for each pig according to the recorded daily excretion. Composited samples remained frozen at all times until analysis. All frozen feces were dried in a forced-air oven at 55°C for 1 week, then air-equilibrated, weighed, and ground through a 1-mm screen using a laboratory mill. The ground feces for each pig from each collection period were thoroughly mixed in a single bag. From this bag, a sample was obtained and reground using a smaller high-speed grinder (type 4041, model KSM 2-4, Braun Inc., Woburn, MA). After being composited, feces were stored at 4°C to 8°C.

The DM content of feed and feces was assessed according to an adaptation of the AOAC [23] method involving overnight drying (105°C) of the samples in a convection oven and then calculating moisture contents as the difference between weighings. The total contents of nutrients found in feces, urine, and feed were calculated as the product of nutrient concentration and total amount of material. Individual samples were analyzed in duplicate, and analysis was repeated when abnormal variation was observed (generally when the CV was >5%). The N concentration was measured using Dumas methodology in an automatic N analyzer (model FP-2000, Leco Corp., Saint Joseph, MI). Ignition of blanks and EDTA samples with known N contents was conducted daily to calibrate the equipment and to check for drift in the readings. The Ca concentration was assessed by flame atomic absorption spectrophotometry (Thermoelemental, SOLAAR M5, Thermo Electron Corp., Verona, WI) according to a modification of the AOAC [24] procedure (method 927.02). The P concentration in feed and feces was assessed by a gravimetric method (modification of method 968.08) [25], in which samples were weighed, ashed, acid-digested, diluted to 250 mL and then reacting 50 mL of the liquid with Quimociac Solution [25], filtered, and the precipitate obtained was weighed to calculate the P concentration. The P concentration in urine was assessed as inorganic P by a colorimetric procedure (Number 360-UVP, Sigma Diagnostics, St. Louis, MO) using a spectrophotometer (model Ultrospec IIE, 4057 UV/visible, LKB Biochrom Ltd., Cambridge, UK). The concentration was measured under UV light at 340 nm. A commercial reagent (ammonium molybdate, 0.40 mmol/L in sulfuric acid with surfactant; catalog number 360-3, Sigma Diagnostics) was used along with a set of three standards (catalog number 360-5, Sigma Diagnostics) containing 1, 5, and 15 mg of P/dL.

Statistical Analysis

Gene expression was analyzed by using Mixed procedure of SAS (SAS Inst., Inc., Cary, NC, USA) with dietary B level as a fixed effect, block as a random effect, and individual pigs as experimental units. For gene expression data, an individual value was considered an outlier if it was more than two standard deviations away from the mean. Means (from balanced observations) or least squares means (from unbalanced observations) were compared using orthogonal contrasts. Specifically, a single degree of freedom contrast comparing the unsupplemented controls to the mean of the supplemented treatments was made and, when significant at P < 0.15, was followed by another single degree of freedom contrast comparing the two B supplementation levels.

Nutrient digestibility and retention data were analyzed by ANOVA using the GLM procedure (SAS Inst. Inc., Cary, NC). The model for analysis included the effects of littermate set (i.e., replicate) and diet (i.e., treatment). In all analyses, the experimental unit was the pig. The α level used for determination of statistical significance was 0.05, with ≤0.15 used to declare a tendency for significance. The level of 0.15 was used for declaration of a tendency due to the more limited statistical power of gene expression work in relation to the variation associated with those assays. However, the level of 0.05 was retained for actual declaration of significance.

Results and Discussion

NaBCl mRNA Was Expressed by Both Pig Intestinal Epithelia and Kidney Tissues

Real-time RT-PCR products were generated for NaBC1 mRNA and 18S rRNA from each tissue. Bi-directional sequencing revealed that both NaBCl (130 bp) and 18S (100 bp) cDNA shared 100% identity with their corresponding template sequences (Fig. 1) and were identical for all three tissues. These validated pig NaBCl mRNA and 18S rRNA sequences now reside in GenBank with accession numbers, HQ127317 and HQ127318, respectively.
Fig. 1

Comparison of the NaBC1 and 18S real-time RT-PCR products (Product) to their respective GenBank sequences (accession numbers indicated) which were used as templates for primer and probe design. Underlines indicate the positions of forward and reverse primers, highlights mark the positions of probes, and vertical lines indicate identical base pairs between Product and template sequences. These products now reside in GenBank with accession number HQ127317 for NaBC1 and accession number HQ127318 for 18S rRNA

To our knowledge, this report of NaBCl mRNA expression by jejunal and ileal epithelia, and kidney tissue, by pigs is the first for mammals outside of humans [26] and rodents including rats [8, 26] and mice [27, 28, 29]. With regard to intestinal expression of NaBC1 mRNA, NaBC1 expression has been reported for human duodenum, but not found in human or rat jejunum, ileum, or colon [26]. In contrast, NaBC1 mRNA is expressed by mouse colon [27], and our study shows that at least jejunal and ileal epithelia of pigs express NaBC1. Regarding the renal expression of NaBC1, the kidney of all studied species express mRNA for NaBC1. Moreover, in humans and rats, NaBC1 protein has been localized to the brush border membrane of proximal tubule cells and the apical membrane of intercalcated cells, and the basolateral domain of collecting ducts [26].

As noted above, NaBC1 (SLC4A11) is a Na+-coupled borate transporter. Although the kinetics of NaBC1-mediated coupled borate and Na absorption have not been well described, recent studies with slc4a11 knock-out mice [28, 29] and the seminal siRNA-induced cellular NaBC1 knockout model [8] have revealed the unambiguous importance of NaBC1 expression to cellular and whole-body physiology. For example, the loss of NaBC1 expression in the cornea leads to altered Na and Cl concentrations, whereas in the inner ear, slc4a11 knock-out in fibrocytes leads to deafness [29]. Furthermore, in terms of its importance to cell proliferation and growth, silencing RNA-mediated the knock-down of NaBC1 expression reduces thymidine incorporation by about 50%, presumably through a loss of borate-stimulated activation of the mitogen-activated protein kinase pathway [6, 8].

Dietary Boron Supplementation Increased Jejunal But Decreases Renal, NaBC1 mRNA Content

Despite the importance of NaBC1 presence to various cell functions, the regulation of NaBC1 expression by borate has not been reported. Because feed intake was dictated by body weight, which was equalized across dietary B supplementation treatment, pigs consumed the same amount of diet (Table 3, DM intake of 1.73 to 1.74 kg/day; P = 0.94) but either 71, 157, or 202 mg B/day, depending on whether they received 0, 50, or 100 mg B/kg as a supplement to the basal diet, which was calculated to contain 41 mg intrinsic B/kg. B supplementation tended to increase (P = 0.12) jejunal NaBCl mRNA content by 213% compared to the basal diet treatment (Fig. 2), but NaBC1 mRNA content between the two levels of supplementation did not differ (P = 0.65).
Table 3

Effect of dietary B supplementation on apparent total tract digestibility and retention of dry matter, Ca, P, and N

 

Supplemental B (mg/kg)

SE

P valuea

0

50

100

Dry matter

 Intake (kg/d)

1.73

1.73

1.74

0.013

0.94

 Digestibility (%)

90.95

91.18

90.80

0.249

0.56

Calcium

 Intake (g/day)

9.59

9.60

9.63

0.074

0.94

 Excreted in feces (g/day)

4.05

3.96

4.04

0.133

0.86

 Digestibility (%)

57.67

58.64

58.03

1.350

0.88

 Excreted in urine (g/day)

2.12

2.75

2.58

0.252

0.23

 Retention (% of intake)

35.46

30.10

31.23

3.362

0.51

Phosphorous

 Intake, (g/day)

6.70

6.70

6.72

0.047

0.94

 Excreted in feces (g/day)

4.53

4.61

4.76

0.094

0.26

 Digestibility (%)

30.75

30.48

28.48

1.499

0.47

 Excreted in urine (g/day)

0.00

0.01

0.01

0.002

0.45

 Retention (% of intake)

30.69

30.37

28.38

1.500

0.47

Nitrogen

 Intake (g/day)

46.34

46.38

46.51

0.358

0.94

 Excreted in feces (g/day)

4.99

4.93

5.16

0.212

0.73

 Digestibility (%)

89.16

89.36

88.89

0.433

0.75

 Excreted in urine (g/day)

29.85

31.84

30.21

1.686

0.68

 Retention (% of intake)

24.50

20.23

23.93

3.720

0.69

Each value represents a mean calculated from eight individually penned pigs for each dietary treatment

aP values were obtained from ANOVA F test

Fig. 2

Effect of dietary B supplementation on NaBC1 mRNA expression by jejunal epithelium. Bars represent the least squares means of the relative NaBC1 mRNA quantities normalized to the relative 18S rRNA quantities. Error bars represent the standard error (SE, n = 7, 7, and 6 for 0, 50, and 100 mg B/kg, respectively). Dietary supplementation of B resulted in a greater (P = 0.12) expression (the mean response of the 50 and 100 mg B/kg supplemented diets) of NaBC1 mRNA

For ileal epithelia, dietary B supplementation did not (P = 0.55) affect NaBC1 mRNA content (Fig. 3). For kidney tissue, in contrast to jejunal epithlia, dietary B supplementation decreased (P = 0.04) NaBCl mRNA content by 35% compared to the non-supplemented basal treatment group (Fig. 4). As for jejunal epithelia, NaBCl mRNA content of pigs fed the 100 mg B/kg did not differ (P = 0.19) from those pigs fed the 50 mg B/kg supplements.
Fig. 3

Effect of dietary B supplementation on NaBC1 mRNA expression by ileal epithelium. Bars represent the least squares means of the relative NaBC1 mRNA quantities normalized to the relative 18S rRNA quantities. Error bars represent the standard error (SE, n = 7, 7, and 6 for 0, 50, and 100 mg B/kg, respectively). There was no mRNA expression response to supplementation of the diet with B (P = 0.55)

Fig. 4

Effect of dietary B supplementation on NaBC1 mRNA expression by kidney tissue. Bars represent the least squares means of the relative NaBC1 mRNA quantities normalized to the relative 18S rRNA quantities. Error bars represent the standard error (SE, n = 8, 8, and 8 for 0, 50, and 100 mg B/kg, respectively). Dietary supplementation of B resulted in a lower (P = 0.04) expression (the mean response of the 50 and 100 mg B/kg supplemented diets) of NaBC1 mRNA

To our knowledge, this is the first study demonstrating that dietary B levels can affect the expression of small intestinal (jejunal) and kidney NaBC1 mRNA. Previous studies using cell culture and laboratory animal have suggested that homeostatic control of B in animals mainly involves unrestricted gastrointestinal absorption of B or borate and modulated renal excretion of B or borate [6, 29, 30], albeit by unknown molecular mechanisms. In the present study, essentially doubling (157 mg B/kg) the B intake of the zero B supplement group (71 mg B/kg) resulted in an increased potential for jejnual B uptake capacity but a decreased renal resorption potential, if the observed changes in NaBC1 mRNA paralleled actual changes in NaBC1 transporter capacity. Essentially tripling (202 mg B/kg) the intrinsic B content of the basal diet gave no indication of any further change in expression of NaBC1 mRNA.

These findings indicate the importance and senstivity of NaBC1 expression by the jejnum and kidney to whole-body B homeostasis, especially when considered in context of the known pattern of NaBC1 localization to the brush border of proximal tublues, apical membranes of intercalated ducts, and basolateral membranes of inner medullary ducts of human and rat kidneys [26]. However, whether the decreased renal NaBC1 mRNA expression found in this study specifically indicates decreased re-absorption of borate from urine to blood or the decreased excretion of borate from blood to urine remains to be elucidated.

Boron Supplementation Did Not Affect Ca, P, or N Digestibility and Retention

Consideration of previous research using human and animal models [1, 2, 9, 31] indicates that B may be involved with bone mineralization and structure and suggests that dietary B may have a pronounced influence on Ca and P metabolism when animals are subjected to nutritional stressors, such as P or Ca deficiency. In the kidney, loss of NaBC1 expression due to slc4a11 knock-out results in polyuria, with an accompanying increase in urine of Na (43%), Cl (23%), K (21%), and Mg (37%) [29]. The purpose of the nutrient balance component of the present trial was to test the hypothesis that increased B supplementation of a P-deficient diet would improve Ca, P, or N balance of growing barrows fed a P-deficient diet. As seen in Table 1, available P constituted 0.07% of the diet. Therefore, the diet was deficient in P as it provided only 37% of the P requirement [11].

As noted above, pigs consumed the same amount of diet but either a calculated amount of 71, 157, or 202 mg B/day, depending on whether they received the 0, 50, or 100 mg B/kg of diet supplement. However, despite these differences in B intake at a constant low level of dietary P, no differences were found in the fecal excretion, digestibility, urinary excretion, or retention of P (P ≥ 0.26), Ca (P ≥ 0.23), or N (P ≥ 0.68; Table 3).

Summary

NaBC1 mRNA is expressed by at least jejunal epithelia, ileal epithlia, and kidney tissue of growing barrows. NaBC1 mRNA content was increased in jejunal but decreased in kidney tissues by supplementation of B as borate. Supplementation at thrice the intrinsic dietary B level for 18 days gave no greater response than supplementation at twice the intrinsic dietary B level. These novel findings indicate that a transcriptional regulation of NaBC1 expression may constitute a part of the homeostatic control of B metabolism and that NaBC1 protein expression may be responsive to borate levels in digesta, blood, or both. In contrast, B supplementation did not affect the measured digestibility of the P-deficient diet, nor retention of Ca, P, and N.

Notes

Disclosures

None

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Shengfa F. Liao
    • 1
  • James S. Monegue
    • 1
  • Merlin D. Lindemann
    • 1
  • Gary L. Cromwell
    • 1
  • James C. Matthews
    • 1
  1. 1.Department of Animal and Food SciencesUniversity of KentuckyLexingtonUSA

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