Chronic High Fructose Intake Reduces Serum 1,25 (OH)2D3 Levels in Calcium-Sufficient Rodents

Veronique Douard; Chirag Patel; Jacklyn Lee; Phuntila Tharabenjasin; Edek Williams; J. Christopher Fritton; Yves Sabbagh; Ronaldo P. Ferraris

doi:10.1371/journal.pone.0093611

Abstract

Excessive fructose consumption inhibits adaptive increases in intestinal Ca²⁺ transport in lactating and weanling rats with increased Ca²⁺ requirements by preventing the increase in serum levels of 1,25(OH)₂D₃. Here we tested the hypothesis that chronic fructose intake decreases 1,25(OH)₂D₃ levels independent of increases in Ca²⁺ requirements. Adult mice fed for five wk a high glucose-low Ca²⁺ diet displayed expected compensatory increases in intestinal and renal Ca²⁺ transporter expression and activity, in renal CYP27B1 (coding for 1α-hydroxylase) expression as well as in serum 1,25(OH)₂D₃ levels, compared with mice fed isocaloric glucose- or fructose-normal Ca²⁺ diets. Replacing glucose with fructose prevented these increases in Ca²⁺ transporter, CYP27B1, and 1,25(OH)₂D₃ levels induced by a low Ca²⁺ diet. In adult mice fed for three mo a normal Ca²⁺ diet, renal expression of CYP27B1 and of CYP24A1 (24-hydroxylase) decreased and increased, respectively, when the carbohydrate source was fructose instead of glucose or starch. Intestinal and renal Ca²⁺ transporter activity and expression did not vary with dietary carbohydrate. To determine the time course of fructose effects, a high fructose or glucose diet with normal Ca²⁺ levels was fed to adult rats for three mo. Serum levels of 1,25(OH)₂D₃ decreased and of FGF23 increased significantly over time. Renal expression of CYP27B1 and serum levels of 1,25(OH)₂D₃ still decreased in fructose- compared to those in glucose-fed rats after three mo. Serum parathyroid hormone, Ca²⁺ and phosphate levels were normal and independent of dietary sugar as well as time of feeding. Thus, chronically high fructose intakes can decrease serum levels of 1,25(OH)₂D₃ in adult rodents experiencing no Ca²⁺ stress and fed sufficient levels of dietary Ca²⁺. This finding is highly significant because fructose constitutes a substantial portion of the average diet of Americans already deficient in vitamin D.

Citation: Douard V, Patel C, Lee J, Tharabenjasin P, Williams E, Fritton JC, et al. (2014) Chronic High Fructose Intake Reduces Serum 1,25 (OH)₂D₃ Levels in Calcium-Sufficient Rodents. PLoS ONE 9(4): e93611. https://doi.org/10.1371/journal.pone.0093611

Editor: Daniel Tomé, Paris Institute of Technology for Life, Food and Environmental Sciences, France

Received: November 28, 2013; Accepted: March 5, 2014; Published: April 9, 2014

Copyright: © 2014 Douard et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the National Science Foundation (IOS-1121049 to RPF). The work also received support from the Foundation of UMDNJ (RPF), and the Benjamin Delessert Foundation (to V. Douard). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: Yves Sabbagh is a full time employee of Genzyme, a Sanofi company, and owns Sanofi stock. However, this does not alter in any way the authors’ adherence to PLOS ONE policies on sharing data and materials.

Introduction

Fructose is one of the key components of the American diet and represents more than 10% of daily calorie intake [1]. This remarkable and recent increase in fructose consumption coincides with an equally striking increase in prevalence of vitamin D deficiency or insufficiency in developed countries [2]. Since excessive fructose consumption and vitamin D deficiency are each associated with similar metabolic diseases (i.e., hypertension, chronic kidney disease, metabolic syndrome, obesity) [3], [4], [5], a better understanding of the interaction between fructose and vitamin D metabolism will contribute to better health recommendations. Fructose absorption across the intestinal apical membrane is mediated by the facilitative glucose transporter GLUT5 while basolateral exit is mediated by GLUT2 [6], [7]. Fructose is then metabolized in the liver and to a lesser extent in the kidney.

In the event of decreased Ca²⁺ levels in the serum due to low dietary intake or increased demand of Ca²⁺, the synthesis of the hormonally active form of vitamin D, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) is enhanced, leading to adaptive increases in intestinal Ca²⁺ absorption. If normal serum Ca²⁺ level cannot be maintained by intestinal absorption, then 1,25(OH)₂D₃ together with the parathyroid hormone (PTH) will mobilize bone Ca²⁺ and increase reabsorption of Ca²⁺ from the renal distal tubule [8]. Active intestinal and renal Ca²⁺ transport involves Ca²⁺ entry through the apical Ca²⁺ channel transient receptor potential vanilloid 6 and 5 (TRPV6 and TRPV5) respectively, its intracellular diffusion via Ca²⁺-binding proteins (CaBP9k and CaBP28k respectively), and its extrusion across the basolateral membrane mainly through the plasma membrane Ca²⁺-ATPase (PMCA1). 1,25(OH)₂D₃ stimulates the transcription, via the vitamin D receptor (VDR) [9], of TRPV6 and TRPV5, as well as CaBP9k and CaBP28k.

Our recent work has demonstrated that excessive fructose consumption affects 1,25(OH)₂D₃ and Ca²⁺ metabolism [10], [11]. In both lactating and rapidly growing weanling rats, fructose consumption inhibits adaptive increases in intestinal and renal Ca²⁺ transport, mainly by preventing the upregulation of TRPV6 and CaBP9k expression in the duodenum, and to a lesser extent, of TRPV5 and CaBP28k in the kidney. The mechanism underlying its inhibitory effect on Ca²⁺ transport is that chronic fructose intake also inhibits the adaptive increases in synthesis and blood levels of 1,25(OH)₂D₃ regulating intestinal and renal Ca²⁺ transport systems. Highlighting the key role of 1,25(OH)₂D₃, treatments with 1,25(OH)₂D₃ rescued the inhibitory effect of chronic fructose intake on intestinal Ca²⁺ absorption in weanling rats [11].

The precursor vitamin D₃ (originating from the skin or diet) is initially hydroxylated by 25-hydroxylase (encoded by CYP2R1) in the liver, producing calcidiol or 25(OH)D₃ that is further hydroxylated by 1α-hydroxylase (encoded by CYP27B1) to 1,25(OH)₂D₃ in the kidney. The circulating level of 1,25(OH)₂D₃ is balanced between synthesis by 1α-hydroxylase and degradation by 24-hydroxylase (encoded by CYP24A1), also mainly expressed in the kidney [12]. When serum levels of Ca²⁺ and 1,25(OH)₂D₃ are normal, 1,25(OH)₂D₃ inhibits its own synthesis by reducing CYP27B1 expression, while low serum levels of 1,25(OH)₂D₃ are often associated with a compensatory increase in CYP27B1 expression to restore homeostasis [13]. Chronic fructose-feeding seems to disrupt this balance in rats undergoing a Ca²⁺ challenge. In fructose-fed lactating rats and in fructose-fed rat models of chronic kidney disease, both 1,25(OH)₂D₃ levels and CYP27B1 expression are low [10], [11]. In rapidly growing, postweaning rats, fructose feeding not only reduced 1,25(OH)₂D₃ and CYP27B1 levels, but also increased CYP24A1 expression, suggesting that high fructose intakes enhance the catabolism and impair the synthesis of 1,25(OH)₂D₃ [11]. However, whether this occurred due to low Ca²⁺ or some other mechanism could not be determined.

The aim of this study is to test the hypothesis that chronic fructose consumption causes 1,25(OH)₂D₃ insufficiency independent of its effects on adaptive adjustments in Ca²⁺ transport. We first demonstrated that adult mice challenged with a low Ca²⁺ diet failed to upregulate Ca²⁺ transporter expression if fed a high 43% fructose diet for five wk, because fructose prevented compensatory increases in 1,25(OH)₂D₃ levels. To minimize potential diet-induced changes in Ca²⁺ transport, we then fed a second group of adult mice with normal Ca²⁺ but with a much higher (60%) fructose concentration for a longer duration of three mo, and eventually found the excessive fructose consumption did reduce levels of 1,25(OH)₂D₃ independent of Ca²⁺ transporter expression. Finally, we used rats to enable us to sample blood levels of 1,25(OH)₂D₃ and of other hormones, as well as of Ca²⁺ and Pi concentrations, each month and determine the time course of fructose effects on 1,25(OH)₂D₃.

Materials and Methods

All the procedures in this study were approved by the Institutional Animal Care and Use Committee, Rutgers, The State University of New Jersey.

Experimental Design

Experimental diets were modified from a standard American Institute of Nutrition (AIN)-93G formula by the manufacturer (Research Diets, New Brunswick, NJ). Previous work indicated that 4 wk is sufficient for fructose consumption to have significant effects on intestinal Ca²⁺ transport and serum 1,25(OH)₂D₃ levels [11]. Male mice and rats were used and were kept under standard conditions: 12-h light-dark cycle and 24°C. Study 1: mice fed fructose for five wk. Four-wk-old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Maine), of similar initial body weights were randomly divided into four groups (n = 5) and pair-fed a 43% glucose or 43% fructose diet each containing normal (0.5%) or deficient Ca²⁺ (0.02%) levels (body weights in Fig. S1). The levels of Ca²⁺ in the diet were based on previous studies [14], [15]. The rationale underlying dietary fructose concentrations is in the discussion, under “Limitations”. Study 2: mice fed fructose for three mo. Four-wk-old mice were randomly divided into three groups (n = 5) and fed ad libitum a 63% glucose, 63% fructose, or 63% starch diet containing normal Ca²⁺ levels for three mo (body weights in Fig. S2). The higher dietary fructose concentration and the longer feeding duration were designed to attempt to determine fructose effects on 1,25(OH)₂D₃ under conditions of Ca²⁺ sufficiency. Study 3: rats fed fructose for three mo. Four wk old rats were randomly divided into two groups (n = 6) fed a 43% glucose or 43% fructose diet containing normal Ca²⁺ levels ad libitum for three mo (body weights in Fig. S3). Rats were utilized to ensure a large enough volume of serum would be collected each month for monitoring of Ca²⁺, 1,25(OH)₂D₃, phosphate (Pi), as well as the putative Pi and 1,25(OH)₂D₃ regulators, fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH).

In vitro Intestinal Ca²⁺ Transport

Intestinal segments were everted immediately after isolation and prepared as sacs to determine Ca²⁺ transport rates at 37°C as described previously [3]. The everted gut sacs were made by using the first 4-cm of proximal duodenum where active transcellular transport of Ca²⁺ is localized [14] and then incubated in Ca²⁺ transport buffer [3]. The inner serosal and outer luminal compartments had equal initial concentrations (0.25 mM) of nonradioactive Ca²⁺, then a tracer concentration of ⁴⁵Ca²⁺ was added to the outer mucosal compartment. After 1 h, the active accumulation of ⁴⁵Ca²⁺ in the inner serosal compartment was calculated as a ratio of the final concentration of ⁴⁵Ca²⁺ in compartments, inside/outside.

Serum

Following earlier work [3], serum Pi concentrations were determined using QuantiChrom Pi Assay Kit (BioAssay Systems, Hayward, CA). The total serum Ca²⁺ concentrations were determined by previously described techniques using flame atomic absorption spectrophotometry (Perkin-Elmer Model 633, Norwalk, CT) [16].

1,25(OH)₂D₃, PTH and FGF23 Assays

Following earlier work [3], serum 1,25(OH)₂D₃ levels were measured by enzyme immunoassay (ImmunoDiagnosticSystems (IDS), Arizona). Serum samples were de-lipidated by adding a solution containing dextran sulphate and magnesium chloride, and then centrifuged at 10000×g for 10 min. The resulting supernatant containing the de-lipidated serum is then 1,25(OH)₂D₃ immuno-extracted from potential cross-reactants by incubation for 90 minutes with a highly specific solid phase monoclonal anti-1,25(OH)₂D₃ before assaying by intact enzyme-linked immuno-sorbent assay (ELISA) for 1,25(OH)₂D₃ levels according to the manufacturer’s instructions [17]. Intact FGF23 measurement was performed by ELISAs according to manufacturer’s instructions (Kainos, Tokyo, Japan). PTH was determined using a 2-site sandwich ELISA (Immunotopics, San Clemente, CA, USA).

Western Blot Analysis

Western blot analyses were performed using 50 μg of intestinal or renal protein extracts following earlier work [3]. For CaBP9k (anti-rabbit, Swant Swiss Antibodies, Switzerland), 4–20% Tris-HCl gels (BioRad, Hercules, CA) were used. For CYP27B1 (anti-rabbit, Santa Cruz Biotechnology Inc., CA) and CYP24A1 (Sigma Chemicals, MO), 12% Tris-HCl gels were used. All membranes were stripped and re-probed with β-actin antibody (anti-mouse, Chemicon International, MA). The membrane was blocked with 5% nonfat milk in Tris-buffered saline-Tween 20 (TBS-T) (0.1% Tween 20, 50 mmol/L Tris-HCL, 137 mmol/L NaCl, pH 7.4) buffer for 1 h. The blots were then incubated with the primary antibody, CaBP9k, CYP24A1 and CYP27B1 diluted 1∶1000, 1∶200 and 1∶1000, respectively, in 5% blocking agent in TBS-T buffer overnight at 4°C. Anti-β-actin antibody was diluted 1∶2000 in the same buffer but incubated with the membrane for one h at room temperature. The membranes were incubated with secondary antibodies, anti-rabbit (1∶20000, GE Healthcare Life Sciences, PA) or anti-mousse (1∶5000, GE Healthcare Life Sciences, PA), for one h at room temperature. The western blot was revealed by enhanced chemiluminescent (ECL) HRP substrate (Thermo Scientific, Rockford, IL USA).

Real Time RT-PCR

Total RNA from homogenized intestinal mucosa or kidney was isolated, reverse transcribed, and real-time RT-PCR performed using Mx3000P (Stratagene, La Jolla, CA) as previously described [18]. The reference gene was α-elongation factor1 (EF1α) whose expression is independent of age and dietary carbohydrate [19]. Previously published primer sequences and annealing temperatures [3] [19] are listed in Table S1. To examine the effects of animal age on expression of intestinal Ca²⁺ transporters and of renal 1,25(OH)₂D₃ metabolic enzymes, the glucose-fed mouse samples from Studies 1 and 2 were re-run together through real time RT-PCR with results normalized to the older, 4-mo-old, glucose-fed mice of study 2.

Statistical Analyses

Data are presented as means ± SEM. For studies one and three, a two-way ANOVA was used to determine the effect of fructose and Ca²⁺ level, and of fructose and feeding time course, respectively. If an initial two-way ANOVA indicated a significant effect, a one-way ANOVA followed by LSD test (STATVIEW, Abacus Concepts) determined if differences existed between groups. Differences were considered significant at P<0.05. In study two, a one-way ANOVA was used to test for fructose effects.

Results

Study 1: Mice Fed Ca²⁺-deficient Diets for Five Weeks

Differences in intestinal Ca²⁺ transport and transporter mRNA were associated with a low Ca²⁺ diet containing fructose as main carbohydrate source. Active transepithelial Ca²⁺ transport rate was modest in mice fed fructose-based, normal Ca²⁺ diet and was similar to that in mice fed a glucose-based, normal Ca²⁺ diet (Fig. 1A). When dietary Ca²⁺ level was made deficient, active transepithelial Ca²⁺ transport was enhanced by >2 fold when the carbohydrate source in the diet was glucose. However, no adaptive increases occurred on the fructose-based diet. We investigated the mRNA expression of the three transporters known to mediate active Ca²⁺ transport in the duodenum: TRPV6, CaBP9k and PMCA1 (Fig. 1B). mRNA levels of TRPV6 and CaBP9k increased significantly by ∼400- and 30-fold, respectively, in mice fed a glucose-based, low Ca²⁺ diet compared to the glucose-based, normal Ca²⁺ diet, paralleling the low Ca²⁺-induced increase in Ca²⁺ transport. In mice fed a fructose-based, low Ca²⁺ diet these mRNA levels increased by ∼150- and 10-fold, respectively, compared to those fed normal Ca²⁺ diet. Thus, for TRPV6 and CaBP9k, dietary fructose dampened adaptive increases of Ca²⁺ transporter expression by three-fold compared to glucose. Differences in CaBP9k protein levels clearly paralleled those of CaBP9k mRNA (Fig. S4). As we previously demonstrated, intestinal GLUT5 expression increased with dietary fructose, regardless of dietary Ca²⁺ level (Fig. S5), suggesting that the inhibitory effect of fructose on Ca²⁺ transporter expression is specific.

Download:

Figure 1. Fructose-fed mice demonstrate an inhibition of compensation for intestinal Ca²⁺transport rate and transporter expression induced by dietary Ca²⁺-deficiency.

(A) Ca²⁺ transport was measured using everted duodenal sacs from mice fed diets containing either 43% glucose or fructose, in combination with either normal or low Ca²⁺. Active transepithelial Ca²⁺ transport from the luminal to the basolateral compartment was expressed as a ratio of the final quantity of ⁴⁵Ca²⁺ inside/outside of the everted gut sacs. (B) mRNA expression levels of intestinal Ca²⁺ (TRPV6, CaBP9k, PMCA1) transporters. All expression data were analyzed by real-time PCR using EF1α as a reference and then normalized to levels in mice fed a normal Ca²⁺-glucose diet (Nor Ca²⁺-G). Data are means ± SEM (n = 6 per group). Differences (P<0.05) among means are indicated by differences in superscript letters, as analyzed by 1-way ANOVA LSD. Thus, within a gene of interest, bars with superscript “a” are > bars with “b” which in turn are > bars with “c”. Dietary fructose inhibits compensatory increases in Ca²⁺ transporter activity and expression induced by dietary Ca²⁺-deficiency.

https://doi.org/10.1371/journal.pone.0093611.g001

Differences in renal mRNA expression of synthesis and degradation enzymes for 1,25(OH)₂D₃ were associated with fructose and low Ca²⁺ diets. In the kidney of mice fed low Ca²⁺, mRNA levels of CYP27B1 increased significantly by 17- and 7-fold, respectively for glucose- and fructose-based diets, compared to a normal Ca²⁺ diet with glucose or fructose as carbohydrate source (Fig. 2A). Thus, dietary fructose lead to a significantly lower expression of CYP27B1 when associated with low Ca²⁺ levels, as compared to dietary glucose. CYP24A1 mRNA expression was significantly upregulated by ∼14-fold with low Ca²⁺, regardless of dietary sugar. Similar patterns of expression were found with protein levels (Fig. 2B).

Download:

Figure 2. Fructose inhibits compensatory increases in renal expression of the 1,25(OH)₂D₃ metabolizing enzyme CYP27B1 induced by dietary Ca²⁺deficiency in mice.

(A) mRNA expression levels of renal CYP27B1 (1α-hydroxylase) and CYP24A1 (24-hydroxylase). (B) The protein abundance of CYP27B1 and CYP24A1, using β-actin as a reference. (C) mRNA expression levels of renal TRPV6, CaBP28k, CaBP9k and PMCA1. Nor = normal; G = glucose; F = fructose. All mRNA expression level data were analyzed by real-time PCR using EF1α as a reference and normalized to levels in mice fed glucose and normal Ca²⁺ diet. Data are means ± SEM (n = 5–6 per group). Differences (P<0.05) among means are indicated by differences in superscript letters, as analyzed by 1-way ANOVA LSD. Thus, within a gene of interest, bars with superscript “a” are > bars with “b” which in turn > bars with “c”. Dietary fructose inhibits compensatory increases in renal expression of Ca²⁺ transporters and 1,25(OH)₂D₃ metabolizing enzymes induced by dietary Ca²⁺-deficiency.

https://doi.org/10.1371/journal.pone.0093611.g002

As expected, the circulating level of 1,25(OH)₂D₃ was significantly increased with low Ca²⁺ (Table 1) and paralleled the increases in renal mRNA and protein expression of CYP27B1 and CYP24A1 with low Ca²⁺. However, dietary fructose dampened the low Ca²⁺-induced increase in 1,25(OH)₂D₃ level which was not significantly different from those in mice fed normal Ca²⁺ diets (P>0.075 by one-way ANOVA). A low Ca²⁺ diet was associated with a significant 40% decrease in circulating level of FGF23, independent of dietary sugar concentrations. However, serum concentrations of Ca²⁺ and Pi which are primarily regulated by 1,25(OH)₂D₃ and FGF23, did not vary with diet.

Download:

Table 1. Blood chemistry of mice after consuming glucose- or fructose-based low and normal Ca²⁺ diets for five weeks.

https://doi.org/10.1371/journal.pone.0093611.t001

In the kidney, differences in mRNA expression of Ca²⁺ transporters were associated with low Ca²⁺ diets. Low dietary Ca²⁺ induced a ∼two-fold increase in renal TRVP5 and CaBP9k mRNA levels in combination with either glucose or fructose (Fig. 2C). In contrast, CaBP28k and PMCA1 mRNA expression did not vary with diet.

Study 2: Mice Fed Fructose for Three Months

In order to determine if chronic high fructose intake alters CYP27B1 and CYP24A1 expression independently of Ca²⁺demand and metabolism, mice were challenged for three mo with diets containing higher levels of fructose and sufficient levels of Ca²⁺. Similar rates of food intake were observed among mice fed glucose, fructose or starch diets (average of 0.09±0.01 g per g of bw per day for all mice). Duodenal Ca²⁺ transport rates did not vary with carbohydrate source and were similar in glucose-, fructose- and starch-fed mice (Fig. 3A). TRPV6, CaBP9k and PMCA1 mRNA expression did not vary among the three diets (Fig. 3B). CaBP9k protein expression was not detected by Western blot (data not shown), indicating a low expression level in the 4 mo old mice fed sufficient dietary Ca²⁺.

Download:

Figure 3. Chronic consumption of fructose has no significant effect on intestinal Ca²⁺ transport rate and transporter mRNA expression in mice fed for three mo a high fructose, starch, or glucose diet containing normal Ca²⁺ levels.

(A) Active transduodenal Ca²⁺ transport from the luminal to the basolateral compartment was expressed as a ratio of the final quantity of (⁴⁵Ca²⁺ inside/⁴⁵Ca²⁺ outside) of the everted sacs of mice fed diets containing 63% fructose, glucose or starch. (B) mRNA expression levels of intestinal Ca²⁺ (TRPV6, CaBP9k, PMCA1) transporters. All expression data were analyzed by real-time PCR using EF1α as a reference and normalized relative to levels seen in mice fed glucose diet. Data are means ± SEM (n = 5 per group). Chronic consumption of high fructose levels has no significant effect on intestinal Ca²⁺ transport rate and transporter mRNA expression.

https://doi.org/10.1371/journal.pone.0093611.g003

In the kidney, fructose feeding had remarkable effects on the mRNA expression of genes involved in regulating 1,25(OH)₂D₃ levels. CYP27B1 expression decreased and CYP24A1 expression increased when compared to starch or glucose feeding (Fig. 4A). However, fructose had no effect on TRPV5, CaBP9k, CaBP28k, or PMCA1 expression (Fig. 4B). Taken together, these findings from intestine and kidney indicate that excessive fructose consumption may regulate 1,25(OH)₂D₃ independent of any effect on Ca²⁺ transport.

Download:

Figure 4. Expression of 1,25(OH)₂D₃ metabolizing enzymes and of Ca²⁺transporters in the kidney of mice fed for three mo a high fructose, starch, or glucose diet containing normal Ca²⁺ levels.

(A) Marked effects of excessive fructose intake on mRNA expression levels of renal 1,25(OH)₂D₃ metabolic enzymes, CYP27B1 and CYP24A1. (B) mRNA expression levels of renal TRPV6, CaBP28k, CaBP9k and PMCA1. All expression data were analyzed by real-time PCR using EF1α as a reference and normalized relative to levels seen in mice fed glucose diet. Data are means ± SEM (n = 5 per group). Differences (P<0.05) among means are indicated by differences in superscript letters, as analyzed by 1-way ANOVA LSD. Thus, within a gene of interest, bars with superscript “a” are > bars with “b”. Chronic consumption of high fructose levels has dramatic effects on mRNA expression of renal 1,25(OH)₂D₃ metabolizing enzymes but not on Ca²⁺ transporter mRNA expression.

https://doi.org/10.1371/journal.pone.0093611.g004

To examine possible mechanisms behind the difference in Ca²⁺ transport rates between Figs. 1A and 3A, we reanalyzed the intestinal transporter and renal 1,25(OH)₂D₃ regulating enzymes in a direct comparison of mRNA expression levels in mice fed normal Ca²⁺ and glucose from study 1 (those mice were 2.5 mo old at sacrifice) and mice fed normal Ca²⁺ and glucose from study 2 (those mice were 4 mo old at sacrifice). Intestinal mRNA expression levels of TRPV6 and CaBP9k were both ∼100-fold lower at 4 than at 2.5 mo of age (Fig. 5A). Low CaBP9k expression at 4 mo-old mice fed normal Ca²⁺ explains the undetectable CaBP9k protein levels in these mice. Despite the large age-related differences in Ca²⁺ transport and transporter expression, expression of renal CYP27B1 and CYP24A1 was the same (Fig. 5B).

Download:

Figure 5. Effects of age on expression of intestinal Ca²⁺ transporters and of renal 1,25(OH)₂D₃ metabolic enzymes in mice.

A comparison of the expression levels of Ca²⁺ transporters (A) and 1,25(OH)₂D₃ metabolic enzymes (B) in four mo old mice after three mo of feeding on normal Ca²⁺ diet containing 63% glucose (from study 2) and in two mo old mice fed a normal Ca²⁺ diet containing 43% glucose for five wk (from study 1). All expression data were analyzed by real-time PCR using EF1α as a reference and normalized relative to levels seen in four mo old mice. Data are means ± SEM (n = 5–6 per group). Differences (P<0.05) between means are indicated by asterisks. Expression of intestinal Ca²⁺ transporters decreases with age.

https://doi.org/10.1371/journal.pone.0093611.g005

Study 3: Rats Fed Fructose for Three Months

Having demonstrated that prolonged feeding of a fructose diet altered renal expression of 1,25(OH)₂D₃ metabolizing enzymes independent of changes in expression of Ca²⁺ transporters and binding proteins, we proceeded to evaluate the time course of the fructose effect on 1,25(OH)₂D₃ levels. Rats fed a fructose or glucose diet displayed similar rates of food intake as well as similar final body weights (Fig. S3A, B). Similar to Study 2, the expression levels of intestinal and renal transporters with normal Ca²⁺ were also the same on fructose- and glucose-based diets (Figs. 6, 7). While it might have been possible to get a statistically significant fructose effect with a greater n, another reason we failed to get significance was the known age-related reductions in expression of Ca transporters that would also reduce the magnitude of the fructose effect. However, fructose feeding did lead to a two-fold decrease in mRNA expression of the 1,25(OH)₂D₃ synthesizing renal enzyme, CYP27B1 (Fig. 7A). Meanwhile, CYP24A1 expression did not change. Protein levels demonstrated similar results (Fig. 7B).

Download:

Figure 6. Effect of chronic fructose feeding on Ca²⁺ transporter expression in rat intestine.

Rats were fed for three mo a normal Ca²⁺ diet containing either 43% glucose or fructose. mRNA expression levels of intestinal Ca²⁺ (TRPV6, CaBP9k, PMCA1) transporters remained similar between diets.

https://doi.org/10.1371/journal.pone.0093611.g006

Download:

Figure 7. Effect of chronic fructose feeding on renal expression of 1,25(OH)₂D₃ metabolizing enzymes and of Ca²⁺transporters.

Rats were fed for three mo a normal Ca²⁺ diet containing either 43% glucose or fructose. (A) mRNA expression level of renal 1,25(OH)₂D₃ metabolic enzymes, CYP27B1 and CYP24A1 (B) The protein abundance of CYP27B1 and CYP24B1 using β-actin as a reference. (C) mRNA expression levels of renal TRPV6, CaBP28k, CaBP9k and PMCA1. All expression data were analyzed by real-time PCR using EF1α as a reference and normalized relative to levels seen in rat fed glucose diet. Data are means ± SEM (n = 6 per group). Differences (P<0.05) between means are indicated by asterisks. Chronic consumption of high fructose diets reduces mRNA and protein expression of CYP27B1.

https://doi.org/10.1371/journal.pone.0093611.g007

As expected, the decreased CYP27B1 expression after three mo of consuming a high fructose diet was associated with a significantly lower circulating level of 1,25(OH)₂D₃ versus the glucose diet group (Table 2). The most pronounced effect was with duration as in both diet groups, 1,25(OH)₂D₃ serum concentrations fell by over 40% from two to three and four mo of age. Conversely, circulating levels of FGF23 were not affected by the dietary sugar, and increased about 20% between three and four mo of age, suggesting that age-related reductions in 1,25(OH)₂D₃ levels occur prior to increases in FGF23. Serum PTH, Ca²⁺ and Pi did not vary with duration or dietary sugar.

Download:

Table 2. Time course of the fructose-induced reduction in serum levels of 1,25-(OH)₂D₃ in adult rats fed for three mo a normal Ca²⁺ diet with either glucose or fructose.

https://doi.org/10.1371/journal.pone.0093611.t002

Discussion

We show in mice that excessive fructose intake inhibits adaptive increases in intestinal Ca²⁺ absorption and in 1,25(OH)₂D₃ levels, in response to dietary Ca²⁺ restriction. Thus, regardless of the source of Ca²⁺ challenge (growth [11], lactation [10], renal disease [3], dietary insufficiency (this paper)), excessive fructose consumption will diminish, if not completely abolish, the main physiological responses: increased intestinal absorption and renal reabsorption of Ca²⁺. Very importantly, we also demonstrate in both rats and mice that chronic fructose intake can decrease 1,25(OH)₂D₃ levels independent of Ca²⁺ demand, and can alter CYP27B1 or CYP24A1 expression in the absence of physiological and nutritional Ca²⁺ challenge.

Excessive Fructose Intake Decreases 1,25(OH)₂D₃

Results from these current studies and our previous work [10], [11] have consistently shown that fructose feeding prevents adaptive increases in serum levels of 1,25(OH)₂D_3. This fructose-induced decrease in serum 1,25(OH)₂D₃ concentrations was not associated with a reduction in serum levels of its precursor 25(OH)D₃, suggesting fructose had little effect on the initial steps of vitamin D synthesis. Instead, these findings indicate a specific effect of fructose at the renal step of either 1,25(OH)₂D₃ synthesis or degradation [10]. The fructose-induced decrease in 1,25(OH)₂D₃ levels was always associated with a fructose-induced decrease in CYP27B1 expression and less consistently with increased CYP24A1 expression. So far the effect of fructose on 1,25(OH)₂D₃ had been observed only when Ca²⁺ had been challenged. We demonstrate in the current study that chronic fructose intake reduces over time normal circulating levels of 1,25(OH)₂D₃ independently of any increase in Ca²⁺ requirement.

Chronic Fructose Intake and Intestinal Ca²⁺ Transport

Total intestinal Ca²⁺ absorption consists of a passive paracellular and a 1,25(OH)₂D₃-dependent active transcellular pathway [20]. Since the ultimate source of Ca²⁺ is intestinal absorption, vertebrates undergoing Ca²⁺ stress adaptively increase active Ca²⁺ transcellular transport, achieved by augmenting levels of 1,25(OH)₂D₃. Here we confirmed in mouse our previous findings in rat models showing that marked increases in active absorption of Ca²⁺ are associated with equally remarkable increases in TRPV6 and CaBP9K expression, and that high fructose intakes dramatically compromise these adaptive increases in Ca²⁺ transport rates as well as in TRPV6 and CaBP9k mRNA or protein levels [10], [11].

In the present work, under Ca²⁺ restricted conditions, the inhibition of adaptive increases in TRPV6 and CaBP9k expression by fructose likely explains the limited upregulation of active intestinal Ca²⁺transport in rodents with high fructose intakes. CaBP9k and TRPV6 had been shown to play a major role in the adaptation of intestinal Ca²⁺ transport under low Ca²⁺ conditions, and after induction by 1,25(OH)₂D₃ [14]. When dietary supply of Ca²⁺ is normal, TRPV6 null mice or CaBP9k null mice display a normal Ca²⁺ transport. However, in double KO TRPV6^−/−/CaBP9k^−/− mice, the adaptive increase in intestinal active Ca²⁺ transport in response to low Ca²⁺ diet or 1,25(OH)₂D₃ injection was reduced by 50% compared to that of wild-type mice, indicating that both CaBP9k and TRPV6 are needed for optimal Ca²⁺ transport under a Ca²⁺ challenge condition [14]. The same adaptive increase was also seriously impaired in the single KO TRPV6^−/− mice [14], suggesting that TRPV6 plays a major role in active intestinal Ca²⁺ absorption.

We [10], [11] have not found rodent PMCA1 levels to vary with Ca²⁺ stress and other workers have even used this gene for reference or “housekeeping” purposes [21] because of its stable expression even as TRPV6 levels change markedly. This difference in regulation between TRPV’s and PMCA’s is understandable because Ca²⁺ transport across the apical membrane is the rate-limiting step [22].

Effect of Age

In four mo old mice and rats, fructose did not affect the active Ca²⁺ transport or Ca²⁺ transporter expression likely because active Ca²⁺ transport constitutes a very small component of total intestinal transport in older, Ca²⁺-sufficient animals. Comparing Fig. 3A and 1A, Ca²⁺ transport rate was also lower in older mice (study 2) which were fed normal Ca²⁺ for three mo than in younger mice fed the same sufficient level of dietary Ca²⁺ in Study 1. Compared to the 2.5 mo old mice, the modest rates of active Ca²⁺ transport rates exhibited by four mo old mice were also associated with reduced expression of TRPV6 and CaBP9K, as previously demonstrated in rats [23]. In turn, age-related decreases in Ca²⁺ transport and transporter expression occurring in adult rodents between two and four mo of age may be linked to the age-related decrease in serum 1,25(OH)₂D₃ levels occurring simultaneously, as shown in this study and as previously demonstrated in rats [24]. The role of age in modulating 1,25(OH)₂D₃ levels should be further investigated in mice on the same diets as a limitation of our age comparison is that different (43% carbohydrate, Study 1 and 63%, Study 2) diets were utilized.

In this and in previous work, adult rats and mice fed sufficient Ca²⁺ and Pi levels and not under Ca²⁺ stress have normal serum Ca²⁺ and Pi levels even when fed high fructose diets. The active, NaPi2b-mediated component of intestinal Pi transport also decreases dramatically in postweaning rodents as growth rate decreases markedly with age [25]. These findings suggest that paracellular transport of Ca²⁺ and Pi may be sufficient to maintain serum levels as demand for these minerals in older rodents is low.

Interactions among Blood Levels of 1,25(OH)₂D₃, FGF23 and PTH

FGF23 is a hormone synthesized mainly in the osteocytes and osteoblasts of bone and regulates blood levels of Pi via removal of Pi transporters from the apical membrane of kidney cells, thus reducing Pi reabsorption and increasing urinary excretion [10], [26]. FGF23 also regulates 1,25(OH)₂D₃ by inhibiting CYP27B1 expression and increasing CYP24A1 expression [27]. Moreover, FGF23 synthesis is positively regulated by increased serum levels of 1,25(OH)₂D₃ or of Pi.

In a previous study using growing rats, we found that chronic consumption of fructose was associated with a significant increase in circulating levels of FGF23, which could potentially be the mechanism by which fructose reduces 1,25(OH)₂D₃ levels [11]. However, in the present study, in mice fed for five wk a fructose-based, low Ca²⁺ diet and in rats fed for three mo a fructose-based, normal Ca²⁺ diet (high-fructose conditions that reduce circulating levels of 1,25(OH)₂D₃), FGF23 production did not increase with fructose feeding. In mice, the failure of fructose to induce FGF23 production may be due to the confounding effects of hypocalcaemia that acutely reduces circulating level of FGF23 [28]. Our data confirm this since in Ca²⁺ deficiency, circulating levels of FGF23 are low, thus allowing 1,25(OH)₂D₃ circulating levels to remain elevated and prevent decreases in blood Ca²⁺. In adult rats, the mechanism of the fructose-induced decrease in 1,25(OH)₂D₃ levels remains unclear, as fructose-induced increases in FGF23 levels were not significant perhaps due to a statistically insufficient number of samples. Fructose, however, is known to injure the kidneys [3], [10], [29] which are the site of 1,25(OH)₂D₃ synthesis. PTH levels were not affected by fructose as shown in this as well as in our previous studies [10], [11].

Limitations

There are limitations to this study, both in conduct and extrapolation of the results to the human condition. Despite similar food intakes, fructose feeding was associated with a significantly greater kidney mass index (kidney weight/body weight) in both the longer-term studies (two and three) in both mice and rats (Figs. S2 and S3). Additionally, rodents and humans have different diets. The AIN recommends either 73% (AIN-93M (maintenance) diet) or 63% (AIN-93G (growth) diet) carbohydrate, levels higher than the average for humans (54%) consuming a high carbohydrate diet [30]. Thus, rodents consuming 43 or 63% sugars, though consuming more % carbohydrate than humans, are eating less than the carbohydrate amount and concentration in a standard AIN-93M laboratory rodent diet. The popularity of>60% fructose diets is demonstrated in the fact that >130 published studies in the last five years specifically used pellets containing ∼60% fructose, because such a level typically causes pathological effects in a mouse within 4 wk. Here we used high concentrations of fructose to allow us to detect any potential effect it may have on expected outcomes. While our previous studies found 63% fructose diets to cause a significant decrease in serum 1,25(OH)₂D₃ levels [10], [11] after 4–6 wk of feeding, the present paper demonstrated that lowering the amount of fructose in the diet could lead to similar decreases in serum 1,25(OH)₂D₃, but only after a longer period of feeding (3 mo). Humans consume lower concentrations of fructose that are often mixed with other carbohydrates, but studies have shown that mixing with glucose may accelerate fructose effects. Because of the cumulative effects of fructose demonstrated here, lower fructose concentrations will likely cause similar deleterious effects as higher fructose concentrations if consumed for a longer time period [31], [32].

Conclusion and Perspective

Despite these limitations, we have clearly demonstrated that a chronic intake of high levels of dietary fructose can lead to a decrease in circulating levels of 1,25(OH)₂D₃ independent of dietary Ca²⁺ levels and of physiological increases in Ca²⁺ requirement. This work is highly relevant since fructose, a sugar contained in many types of foods that are being consumed at high levels, may contribute to the increasing prevalence of vitamin D insufficiency, especially in sensitive populations with high, processed sugar intakes such as in children and teenagers [33], [34]. Indeed, when consumption is normalized to body weight, these age groups are the highest consumers of fructose, with an average total ingestion reaching ∼2 g kg⁻¹ day⁻¹ occurring at a time when there is a very high demand for vitamin D during growth [35].

Supporting Information

Figure S1.

The body and kidney weight of mice pair-fed diets containing either 43% glucose or fructose, in combination with either normal or low Ca²⁺. A) Initial and final body weight after 5 wk of feeding on the experimental diets. B) Kidney weight after 5 wk. C) Kidney somatic index. Bars are means ± SEM; n = 5–8. Nor = normal; G = glucose; F = fructose. Differences (P<0.05) among means are indicated by differences in superscript letters, as analyzed by 1-way ANOVA LSD. Thus, bars with superscript “a” are > bars with “b” and similar to those with “ab”.

https://doi.org/10.1371/journal.pone.0093611.s001

(TIF)

Figure S2.

The body weight, feeding rate, and kidney weight of mice fed diets containing normal Ca²⁺ and 63% glucose, fructose or starch. A) Body weight after 3 mo of feeding on the special diets. B) Feeding rate per day normalized to body weight. C) Kidney weight after 3 mo of feeding on the special diets. D) Kidney somatic index. Bars are means ± SEM; n = 5–8. Differences (P<0.05) among means are indicated by differences in superscript letters, as analyzed by 1-way ANOVA LSD. Thus, bars with superscript “a” are > bars with “b”.

https://doi.org/10.1371/journal.pone.0093611.s002

(TIF)

Figure S3.

The body and kidney weight of rat fed diets containing either 43% glucose or fructose. A) Body weight after 3 mo of feeding of the special diets. B) Feeding rate per day normalized to body weight. C) Kidney weight after 3 mo of feeding of the special diets. D) Kidney somatic index. Bars are means ± SEM; n = 5–8. Differences (P<0.05) among means are indicated by differences in superscript letters, as analyzed by 1-way ANOVA LSD. Thus, bars with superscript “a” are > bars with “b”.

https://doi.org/10.1371/journal.pone.0093611.s003

(TIF)

Figure S4.

CaBP9k protein abundance. The protein abundance of CaBP9k was determined in the small intestine of mice fed diets containing either 43% glucose or fructose, in combination with either normal or low Ca²⁺. β-actin was used as a reference. Nor = normal; G = glucose; F = fructose.

https://doi.org/10.1371/journal.pone.0093611.s004

(TIF)

Figure S5.

mRNA levels of GLUT5. Expression level of GLUT5 in the intestine of A) mice fed diets containing either 43% glucose or fructose, in combination with either normal or low Ca²⁺ for 5 wk; B) mice fed diets containing normal Ca²⁺ in combination with 63% glucose, fructose or starch for 3 mo; and C) rats fed diets containing either 43% glucose or fructose for 3 mo. All expression data were analyzed by real-time PCR using EF1α as a reference and normalized relative to levels seen in rat fed glucose diet. Nor = normal; G = glucose; F = fructose. Data are means ± SEM (n = 6 per group). Differences (P<0.05) among means are indicated by differences in superscript letters, as analyzed by 1-way ANOVA LSD. Thus, for each study, bars with superscript “a” are > bars with “b”.

https://doi.org/10.1371/journal.pone.0093611.s005

(TIF)

Table S1.

Primer sequences.

https://doi.org/10.1371/journal.pone.0093611.s006

(DOC)

Acknowledgments

We are deeply indebted to Frank Kemp for help in analyzing serum Ca²⁺ and Pi levels.

Author Contributions

Conceived and designed the experiments: VD RPF. Performed the experiments: VD CP JL PT EW YS. Analyzed the data: VD. Contributed reagents/materials/analysis tools: YS. Wrote the paper: VD RPF JCF.

References

1. Bray GA (2013) Energy and fructose from beverages sweetened with sugar or high-fructose corn syrup pose a health risk for some people. Adv Nutr 4: 220–225.
- View Article
- Google Scholar
2. Forrest KY, Stuhldreher WL (2011) Prevalence and correlates of vitamin D deficiency in US adults. Nutr Res 31: 48–54.
- View Article
- Google Scholar
3. Douard V, Asgerally A, Sabbagh Y, Sugiura S, Shapses SA, et al. (2010) Dietary Fructose Inhibits Intestinal Calcium Absorption and Induces Vitamin D Insufficiency in CKD. J Am Soc Nephrol 21: 261–271.
- View Article
- Google Scholar
4. vinh quoc Lu‘o’ng K, Nguyen LT (2013) The beneficial role of vitamin D in obesity: possible genetic and cell signaling mechanisms. Nutrition journal 12: 89.
- View Article
- Google Scholar
5. Dekker MJ, Su Q, Baker C, Rutledge AC, Adeli K (2010) Fructose: a highly lipogenic nutrient implicated in insulin resistance, hepatic steatosis, and the metabolic syndrome. Am J Physiol Endocrinol Metab 299: E685–694.
- View Article
- Google Scholar
6. Manolescu AR, Witkowska K, Kinnaird A, Cessford T, Cheeseman C (2007) Facilitated hexose transporters: new perspectives on form and function. Physiology (Bethesda) 22: 234–240.
- View Article
- Google Scholar
7. Thorens B, Mueckler M (2010) Glucose transporters in the 21st Century. Am J Physiol Endocrinol Metab 298: E141–145.
- View Article
- Google Scholar
8. Hoenderop JGJ, Dardenne O, Van Abel M, Van Der Kemp AWCM, Van Os CH, et al. (2002) Modulation of renal Ca2+ transport protein genes by dietary Ca2+ and 1,25-dihydroxyvitamin D3 in 25-hydroxyvitamin D3-1alpha-hydroxylase knockout mice. FASEB J 16: 1398–1406.
- View Article
- Google Scholar
9. Haussler MR, Whitfield GK, Haussler CA, Hsieh JC, Thompson PD, et al. (1998) The nuclear vitamin D receptor: biological and molecular regulatory properties revealed. J Bone Miner Res 13: 325–349.
- View Article
- Google Scholar
10. Douard V, Suzuki T, Sabbagh Y, Lee J, Shapses S, et al. (2012) Dietary fructose inhibits lactation-induced adaptations in rat 1,25-(OH)(2)D(3) synthesis and calcium transport. FASEB J 26: 707–721.
- View Article
- Google Scholar
11. Douard V, Sabbagh Y, Lee J, Patel C, Kemp FW, et al. (2013) Excessive fructose intake causes 1,25-(OH)2D3-dependent inhibition of intestinal and renal calcium transport in growing rats. Am J Physiol Endocrinol Metab 304: 1303–1313.
- View Article
- Google Scholar
12. Christakos S, Ajibade DV, Dhawan P, Fechner AJ, Mady LJ (2012) Vitamin D: metabolism. Rheum Dis Clin North Am 38: 1–11.
- View Article
- Google Scholar
13. Sakaki T, Kagawa N, Yamamoto K, Inouye K (2005) Metabolism of vitamin D3 by cytochromes P450. Front Biosci 10: 119–134.
- View Article
- Google Scholar
14. Benn BS, Ajibade D, Porta A, Dhawan P, Hediger M, et al. (2008) Active intestinal calcium transport in the absence of transient receptor potential vanilloid type 6 and calbindin-D9k. Endocrinology 149: 3196–3205.
- View Article
- Google Scholar
15. Lieben L, Benn BS, Ajibade D, Stockmans I, Moermans K, et al. (2010) Trpv6 mediates intestinal calcium absorption during calcium restriction and contributes to bone homeostasis. Bone 47: 301–308.
- View Article
- Google Scholar
16. Bogden JD, Kemp FW, Huang AE, Shapses SA, Ambia-Sobhan H, et al. (2008) Bone mineral density and content during weight cycling in female rats: effects of dietary amylase-resistant starch. Nutr Metab (Lond) 5: 34.
- View Article
- Google Scholar
17. Yu X, Sabbagh Y, Davis SI, Demay MB, White KE (2005) Genetic dissection of phosphate- and vitamin D-mediated regulation of circulating Fgf23 concentrations. Bone 36: 971–977.
- View Article
- Google Scholar
18. Douard V, Cui XL, Soteropoulos P, Ferraris RP (2008) Dexamethasone sensitizes the neonatal intestine to fructose induction of intestinal fructose transporter (Slc2A5) function. Endocrinology 149: 409–423.
- View Article
- Google Scholar
19. Douard V, Choi HI, Elshenawy S, Lagunoff D, Ferraris RP (2008) Developmental reprogramming of rat GLUT5 requires glucocorticoid receptor translocation to the nucleus. J Physiol 586: 3657–3673.
- View Article
- Google Scholar
20. Hoenderop JGJ, Nilius B, Bindels RJM (2005) Calcium absorption across epithelia. Physiol Rev 85: 373–422.
- View Article
- Google Scholar
21. Brini M (2009) Plasma membrane Ca(2+)-ATPase: from a housekeeping function to a versatile signaling role. Pflugers Archiv : European journal of physiology 457: 657–664.
- View Article
- Google Scholar
22. Schedl HP, Wilson HD (1985) Calcium uptake by intestinal brush border membrane vesicles. Comparison with in vivo calcium transport. The Journal of clinical investigation 76: 1871–1878.
- View Article
- Google Scholar
23. Brown AJ, Krits I, Armbrecht HJ (2005) Effect of age, vitamin D, and calcium on the regulation of rat intestinal epithelial calcium channels. Arch Biochem Biophys 437: 51–58.
- View Article
- Google Scholar
24. Wood RJ, Fleet JC, Cashman K, Bruns ME, Deluca HF (1998) Intestinal calcium absorption in the aged rat: evidence of intestinal resistance to 1,25(OH)2 vitamin D. Endocrinology. 139: 3843–3848.
- View Article
- Google Scholar
25. Kirchner S, Muduli A, Casirola D, Prum K, Douard V, et al. (2008) Luminal fructose inhibits rat intestinal sodium-phosphate cotransporter gene expression and phosphate uptake. Am J Clin Nutr 87: 1028–1038.
- View Article
- Google Scholar
26. Shimada T, Urakawa I, Yamazaki Y, Hasegawa H, Hino R, et al. (2004) FGF-23 transgenic mice demonstrate hypophosphatemic rickets with reduced expression of sodium phosphate cotransporter type IIa. Biochem Biophys Res Comm 314: 409–414.
- View Article
- Google Scholar
27. Shimada T, Hasegawa H, Yamazaki Y, Muto T, Hino R, et al. (2004) FGF-23 Is a potent regulator of vitamin D metabolism and phosphate homeostasis. J Bone Miner Res 19: 429–435.
- View Article
- Google Scholar
28. Rodriguez-Ortiz ME, Lopez I, Munoz-Castaneda JR, Martinez-Moreno JM, Ramirez AP, et al. (2012) Calcium deficiency reduces circulating levels of FGF23. J Am Soc Nephrol 23: 1190–1197.
- View Article
- Google Scholar
29. Nakayama T, Kosugi T, Gersch M, Connor T, Sanchez-Lozada LG, et al. (2010) Dietary fructose causes tubulointerstitial injury in the normal rat kidney. Am J Physiol Renal Physiol 298: F712–720.
- View Article
- Google Scholar
30. Krauss RM, Blanche PJ, Rawlings RS, Fernstrom HS, Williams PT (2006) Separate effects of reduced carbohydrate intake and weight loss on atherogenic dyslipidemia. Am J Clin Nutr 83: 1025–1031.
- View Article
- Google Scholar
31. Johnson RJ, Perez-Pozo SE, Sautin YY, Manitius J, Sanchez-Lozada LG, et al. (2009) Hypothesis: could excessive fructose intake and uric acid cause type 2 diabetes? Endocr Rev 30: 96–116.
- View Article
- Google Scholar
32. Douard V, Sabbagh Y, Suzuki T, Ferraris R (2012) A high fructose intake prevents compensatory increases in intestinal calcium transport in rodent models physiologically and nutritionally deficient in calcium Gastroenterology. 142: S557.
- View Article
- Google Scholar
33. Marriott BP, Cole N, Lee E (2009) National estimates of dietary fructose intake increased from 1977 to 2004 in the United States. J Nutr 139: 1228S–1235S.
- View Article
- Google Scholar
34. Douard V, Ferraris RP (2013) The role of fructose transporters in diseases linked to excessive fructose intake. J Physiol 591: 401–414.
- View Article
- Google Scholar
35. Bischoff-Ferrari HA (2011) Vitamin D: role in pregnancy and early childhood. Ann Nutr Metab 59: 17–21.
- View Article
- Google Scholar

[ref1] 1. Bray GA (2013) Energy and fructose from beverages sweetened with sugar or high-fructose corn syrup pose a health risk for some people. Adv Nutr 4: 220–225.
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Forrest KY, Stuhldreher WL (2011) Prevalence and correlates of vitamin D deficiency in US adults. Nutr Res 31: 48–54.
View Article
Google Scholar

[5] View Article

[6] Google Scholar

[ref3] 3. Douard V, Asgerally A, Sabbagh Y, Sugiura S, Shapses SA, et al. (2010) Dietary Fructose Inhibits Intestinal Calcium Absorption and Induces Vitamin D Insufficiency in CKD. J Am Soc Nephrol 21: 261–271.
View Article
Google Scholar

[8] View Article

[9] Google Scholar

[ref4] 4. vinh quoc Lu‘o’ng K, Nguyen LT (2013) The beneficial role of vitamin D in obesity: possible genetic and cell signaling mechanisms. Nutrition journal 12: 89.
View Article
Google Scholar

[11] View Article

[12] Google Scholar

[ref5] 5. Dekker MJ, Su Q, Baker C, Rutledge AC, Adeli K (2010) Fructose: a highly lipogenic nutrient implicated in insulin resistance, hepatic steatosis, and the metabolic syndrome. Am J Physiol Endocrinol Metab 299: E685–694.
View Article
Google Scholar

[14] View Article

[15] Google Scholar

[ref6] 6. Manolescu AR, Witkowska K, Kinnaird A, Cessford T, Cheeseman C (2007) Facilitated hexose transporters: new perspectives on form and function. Physiology (Bethesda) 22: 234–240.
View Article
Google Scholar

[17] View Article

[18] Google Scholar

[ref7] 7. Thorens B, Mueckler M (2010) Glucose transporters in the 21st Century. Am J Physiol Endocrinol Metab 298: E141–145.
View Article
Google Scholar

[20] View Article

[21] Google Scholar

[ref8] 8. Hoenderop JGJ, Dardenne O, Van Abel M, Van Der Kemp AWCM, Van Os CH, et al. (2002) Modulation of renal Ca2+ transport protein genes by dietary Ca2+ and 1,25-dihydroxyvitamin D3 in 25-hydroxyvitamin D3-1alpha-hydroxylase knockout mice. FASEB J 16: 1398–1406.
View Article
Google Scholar

[23] View Article

[24] Google Scholar

[ref9] 9. Haussler MR, Whitfield GK, Haussler CA, Hsieh JC, Thompson PD, et al. (1998) The nuclear vitamin D receptor: biological and molecular regulatory properties revealed. J Bone Miner Res 13: 325–349.
View Article
Google Scholar

[26] View Article

[27] Google Scholar

[ref10] 10. Douard V, Suzuki T, Sabbagh Y, Lee J, Shapses S, et al. (2012) Dietary fructose inhibits lactation-induced adaptations in rat 1,25-(OH)(2)D(3) synthesis and calcium transport. FASEB J 26: 707–721.
View Article
Google Scholar

[29] View Article

[30] Google Scholar

[ref11] 11. Douard V, Sabbagh Y, Lee J, Patel C, Kemp FW, et al. (2013) Excessive fructose intake causes 1,25-(OH)2D3-dependent inhibition of intestinal and renal calcium transport in growing rats. Am J Physiol Endocrinol Metab 304: 1303–1313.
View Article
Google Scholar

[32] View Article

[33] Google Scholar

[ref12] 12. Christakos S, Ajibade DV, Dhawan P, Fechner AJ, Mady LJ (2012) Vitamin D: metabolism. Rheum Dis Clin North Am 38: 1–11.
View Article
Google Scholar

[35] View Article

[36] Google Scholar

[ref13] 13. Sakaki T, Kagawa N, Yamamoto K, Inouye K (2005) Metabolism of vitamin D3 by cytochromes P450. Front Biosci 10: 119–134.
View Article
Google Scholar

[38] View Article

[39] Google Scholar

[ref14] 14. Benn BS, Ajibade D, Porta A, Dhawan P, Hediger M, et al. (2008) Active intestinal calcium transport in the absence of transient receptor potential vanilloid type 6 and calbindin-D9k. Endocrinology 149: 3196–3205.
View Article
Google Scholar

[41] View Article

[42] Google Scholar

[ref15] 15. Lieben L, Benn BS, Ajibade D, Stockmans I, Moermans K, et al. (2010) Trpv6 mediates intestinal calcium absorption during calcium restriction and contributes to bone homeostasis. Bone 47: 301–308.
View Article
Google Scholar

[44] View Article

[45] Google Scholar

[ref16] 16. Bogden JD, Kemp FW, Huang AE, Shapses SA, Ambia-Sobhan H, et al. (2008) Bone mineral density and content during weight cycling in female rats: effects of dietary amylase-resistant starch. Nutr Metab (Lond) 5: 34.
View Article
Google Scholar

[47] View Article

[48] Google Scholar

[ref17] 17. Yu X, Sabbagh Y, Davis SI, Demay MB, White KE (2005) Genetic dissection of phosphate- and vitamin D-mediated regulation of circulating Fgf23 concentrations. Bone 36: 971–977.
View Article
Google Scholar

[50] View Article

[51] Google Scholar

[ref18] 18. Douard V, Cui XL, Soteropoulos P, Ferraris RP (2008) Dexamethasone sensitizes the neonatal intestine to fructose induction of intestinal fructose transporter (Slc2A5) function. Endocrinology 149: 409–423.
View Article
Google Scholar

[53] View Article

[54] Google Scholar

[ref19] 19. Douard V, Choi HI, Elshenawy S, Lagunoff D, Ferraris RP (2008) Developmental reprogramming of rat GLUT5 requires glucocorticoid receptor translocation to the nucleus. J Physiol 586: 3657–3673.
View Article
Google Scholar

[56] View Article

[57] Google Scholar

[ref20] 20. Hoenderop JGJ, Nilius B, Bindels RJM (2005) Calcium absorption across epithelia. Physiol Rev 85: 373–422.
View Article
Google Scholar

[59] View Article

[60] Google Scholar

[ref21] 21. Brini M (2009) Plasma membrane Ca(2+)-ATPase: from a housekeeping function to a versatile signaling role. Pflugers Archiv : European journal of physiology 457: 657–664.
View Article
Google Scholar

[62] View Article

[63] Google Scholar

[ref22] 22. Schedl HP, Wilson HD (1985) Calcium uptake by intestinal brush border membrane vesicles. Comparison with in vivo calcium transport. The Journal of clinical investigation 76: 1871–1878.
View Article
Google Scholar

[65] View Article

[66] Google Scholar

[ref23] 23. Brown AJ, Krits I, Armbrecht HJ (2005) Effect of age, vitamin D, and calcium on the regulation of rat intestinal epithelial calcium channels. Arch Biochem Biophys 437: 51–58.
View Article
Google Scholar

[68] View Article

[69] Google Scholar

[ref24] 24. Wood RJ, Fleet JC, Cashman K, Bruns ME, Deluca HF (1998) Intestinal calcium absorption in the aged rat: evidence of intestinal resistance to 1,25(OH)2 vitamin D. Endocrinology. 139: 3843–3848.
View Article
Google Scholar

[71] View Article

[72] Google Scholar

[ref25] 25. Kirchner S, Muduli A, Casirola D, Prum K, Douard V, et al. (2008) Luminal fructose inhibits rat intestinal sodium-phosphate cotransporter gene expression and phosphate uptake. Am J Clin Nutr 87: 1028–1038.
View Article
Google Scholar

[74] View Article

[75] Google Scholar

[ref26] 26. Shimada T, Urakawa I, Yamazaki Y, Hasegawa H, Hino R, et al. (2004) FGF-23 transgenic mice demonstrate hypophosphatemic rickets with reduced expression of sodium phosphate cotransporter type IIa. Biochem Biophys Res Comm 314: 409–414.
View Article
Google Scholar

[77] View Article

[78] Google Scholar

[ref27] 27. Shimada T, Hasegawa H, Yamazaki Y, Muto T, Hino R, et al. (2004) FGF-23 Is a potent regulator of vitamin D metabolism and phosphate homeostasis. J Bone Miner Res 19: 429–435.
View Article
Google Scholar

[80] View Article

[81] Google Scholar

[ref28] 28. Rodriguez-Ortiz ME, Lopez I, Munoz-Castaneda JR, Martinez-Moreno JM, Ramirez AP, et al. (2012) Calcium deficiency reduces circulating levels of FGF23. J Am Soc Nephrol 23: 1190–1197.
View Article
Google Scholar

[83] View Article

[84] Google Scholar

[ref29] 29. Nakayama T, Kosugi T, Gersch M, Connor T, Sanchez-Lozada LG, et al. (2010) Dietary fructose causes tubulointerstitial injury in the normal rat kidney. Am J Physiol Renal Physiol 298: F712–720.
View Article
Google Scholar

[86] View Article

[87] Google Scholar

[ref30] 30. Krauss RM, Blanche PJ, Rawlings RS, Fernstrom HS, Williams PT (2006) Separate effects of reduced carbohydrate intake and weight loss on atherogenic dyslipidemia. Am J Clin Nutr 83: 1025–1031.
View Article
Google Scholar

[89] View Article

[90] Google Scholar

[ref31] 31. Johnson RJ, Perez-Pozo SE, Sautin YY, Manitius J, Sanchez-Lozada LG, et al. (2009) Hypothesis: could excessive fructose intake and uric acid cause type 2 diabetes? Endocr Rev 30: 96–116.
View Article
Google Scholar

[92] View Article

[93] Google Scholar

[ref32] 32. Douard V, Sabbagh Y, Suzuki T, Ferraris R (2012) A high fructose intake prevents compensatory increases in intestinal calcium transport in rodent models physiologically and nutritionally deficient in calcium Gastroenterology. 142: S557.
View Article
Google Scholar

[95] View Article

[96] Google Scholar

[ref33] 33. Marriott BP, Cole N, Lee E (2009) National estimates of dietary fructose intake increased from 1977 to 2004 in the United States. J Nutr 139: 1228S–1235S.
View Article
Google Scholar

[98] View Article

[99] Google Scholar

[ref34] 34. Douard V, Ferraris RP (2013) The role of fructose transporters in diseases linked to excessive fructose intake. J Physiol 591: 401–414.
View Article
Google Scholar

[101] View Article

[102] Google Scholar

[ref35] 35. Bischoff-Ferrari HA (2011) Vitamin D: role in pregnancy and early childhood. Ann Nutr Metab 59: 17–21.
View Article
Google Scholar

[104] View Article

[105] Google Scholar

Figures

Abstract

Introduction

Materials and Methods

Experimental Design

In vitro Intestinal Ca2+ Transport

Serum

1,25(OH)2D3, PTH and FGF23 Assays

Western Blot Analysis

Real Time RT-PCR

Statistical Analyses

Results

Study 1: Mice Fed Ca2+-deficient Diets for Five Weeks

Study 2: Mice Fed Fructose for Three Months

Study 3: Rats Fed Fructose for Three Months

Discussion

Excessive Fructose Intake Decreases 1,25(OH)2D3

Chronic Fructose Intake and Intestinal Ca2+ Transport

Effect of Age

Interactions among Blood Levels of 1,25(OH)2D3, FGF23 and PTH

Limitations

Conclusion and Perspective

Supporting Information

Figure S1.

Figure S2.

Figure S3.

Figure S4.

Figure S5.

Table S1.

Acknowledgments

Author Contributions

References

In vitro Intestinal Ca²⁺ Transport

1,25(OH)₂D₃, PTH and FGF23 Assays

Study 1: Mice Fed Ca²⁺-deficient Diets for Five Weeks

Excessive Fructose Intake Decreases 1,25(OH)₂D₃

Chronic Fructose Intake and Intestinal Ca²⁺ Transport

Interactions among Blood Levels of 1,25(OH)₂D₃, FGF23 and PTH