The purpose of this study was to determine whether there are differences in intestinal Ca and phosphate transport in mice having different peak bone densities. Intestinal transport was measured in C57BL/6 (C57, low bone density) and C3H/He (C3H, high bone density) female mice. Unidirectional (mucosal to serosal) transport of Ca was 58% higher in C3H compared with C57 mice, as measured by everted duodenal sacs. The capacity of the duodenal mucosa to take up Ca was also higher in the C3H mice. This uptake highly correlated with Ca transport across the intestine. 1,25-Dihydroxyvitamin D3[1,25(OH)2D3], which stimulates intestinal Ca absorption, markedly stimulated unidirectional Ca transport and uptake to similar levels in both strains of mice. On the other hand, unidirectional phosphate transport in C3H mice was only 36% that of C57 mice. mRNA levels of the plasma membrane Ca pump were 90% higher in the duodenum of C3H mice. There was no difference between strains in duodenal calbindin or 24-hydroxylase mRNA levels. Regarding vitamin D metabolism, there was no difference in serum 1,25(OH)2D3 levels or in renal 1α-hydroxylase mRNA levels. The combination of high intestinal Ca transport and low phosphate transport may contribute to the high peak bone density seen in the C3H relative to the C57 mouse.
- 1,25-dihydroxyvitamin D3; calbindin
- intestinal 24-hydroxylase
- plasma membrane calcium pump
mice have been shown to have a wide range of bone densities (10). In particular, C57BL/6 (C57) mice have a low peak bone density, and C3H/He (C3H) mice have a high peak bone density. Peak bone density is an important determinant of bone mass later in life (1,31). Therefore, C57 and C3H strains have been used as model systems for determining important parameters regarding peak bone mass. Differences in a number of systemic and bone-related factors have been reported between these strains (13, 18, 23).
Absorption of dietary calcium (Ca) by the small intestine is an important component of Ca homeostasis (30). A major factor in attaining peak bone density in humans is the intestinal absorption of dietary Ca (1, 31). Ca absorption early in life may play a role in the attainment of peak bone mass and in prevention of low bone mass in older individuals. Intestinal Ca absorption may also be related to bone mass in mice. Ca balance studies have shown that Ca balance is decreased in C57 compared with C3H mice (11). In addition, Ca supplementation has been reported to improve peak bone mass in mice (25).
The purpose of this study was to determine whether there were differences in the intestinal absorption of Ca between the low bone density C57 and the high bone density C3H strains. Ca absorption was measured in vitro using everted intestinal sacs. A major regulator of intestinal Ca absorption is 1,25-dihydroxyvitamin D3[1,25(OH)2D3], which is the major biologically active metabolite of vitamin D3(30). Therefore, the responsiveness of the two strains to 1,25(OH)2D3 in terms of Ca absorption was determined. Finally, intestinal absorption of phosphorus was measured in vitro, because phosphorus is the other important mineral component of bone.
Expression of several key regulators of intestinal Ca absorption in C57 and C3H mice was compared. In the intestine, the mRNA levels for calbindin, the plasma membrane Ca pump, and the vitamin D 24-hydroxylase were determined. Calbindin, which is a soluble Ca-binding protein, and the Ca pump, which pumps Ca across the basolateral membrane of the absorptive cell, are two of the major components of the Ca absorptive pathway (29, 30). The vitamin D 24-hydroxylase is involved in the catabolism of 1,25(OH)2D3 in the intestine.
In the kidney, the mRNA levels of the 25(OH)D-1α-hydroxylase, the 1,25(OH)2D3-24-hydroxylase, and calbindin were determined. The 1α-hydroxylase synthesizes 1,25(OH)2D3 from 25(OH)D, which increases plasma 1,25(OH)2D3 levels. The 24-hydroxylase hydroxylates both 1,25(OH)2D3 and 25(OH)D, which decreases plasma 1,25(OH)2D3 levels. Renal calbindin may play a role in the tubular reabsorption of Ca.
Female C57 and C3H mice were purchased from Harlan Industries (Indianapolis, IN) and were used between 8 and 12 wk of age. This age range was chosen because Ca balance studies suggested strain differences in Ca absorption at this age (11). Growth curves of the strains are very similar, and both strains increased in weight by ∼5% over this age range. Mice were fed a semisynthetic diet containing 1.2% Ca, 0.8% phosphorus, and 3.3 IU/g vitamin D3 (Purina Rodent Chow, Ralston-Purina, St. Louis, MO). To study the effect of 1,25(OH)2D3 on intestinal Ca absorption, mice were given intraperitoneal injections of 1,25(OH)2D3 (100 ng/100 g body wt) at 48, 24, and 6 h before death. This multidose protocol was used to study both the short-term and long-term effects of 1,25(OH)2D3 in rats (5). All studies were approved by the Animal Studies Committee of the St. Louis Veterans Administration Medical Center.
Measurement of intestinal Ca transport.
Intestinal Ca transport was measured in vitro using everted intestinal sacs as previously described for rats (5, 8, 9). On the day of the experiment, the mice were killed, and blood was collected into heparinized tubes. Plasma was frozen for later determination of plasma Ca, phosphorus, and 1,25(OH)2D3.
To measure intestinal transport, the abdominal cavity was exposed by midline incision, and a 5-cm segment of the intestine was removed. Intestinal segments were everted by first flushing them out with a fine, blunt-end needle. The segment end was then tied to the end of the needle, and the segment was carefully everted over the end of the needle. A small-gauge needle was then used to fill the segment with incubation buffer, and the ends were tied off. The incubation buffer consisted of (in mM) 125 NaCl, 10 fructose, 30 Tris, and 0.25 CaCl2 (pH 7.4 at 37°C). To study the duodenum, the region immediately distal to the pylorus was used, and to study the jejunum, the region at the midpoint of the small intestine was used. Leakage of the sacs was detected by a loss of internal fluid during the 1-h incubation. This occurred <5% of the time.
Everted, fluid-filled sacs were incubated in flasks containing 10 ml of the incubation buffer. To measure unidirectional Ca transport,45CaCl2 (ICN Radiochemicals, Costa Mesa, CA) was added to the buffer. Flasks were then gassed with 95% O2, stoppered, and incubated for 1 h at 37°C. At the end of the incubation period, sacs were removed from the flask and drained of internal fluid, and the amount of fluid in the sacs was determined. Samples of internal fluid from each sac were assayed in triplicate for 45Ca using a scintillation counter. The total amount of Ca transported was calculated by multiplying radioactive counts per microliter of fluid by volume of fluid in the sac and by specific activity. Ca transport measured in this way is sensitive to age, segment, 1,25(OH)2D3treatment, and dietary Ca in the rat (8, 5).
In addition to the amount of Ca that moved across the sac, the amount taken up by the intestinal segment was also determined in these experiments. After the internal fluid was drained, the intestinal segment was washed, solubilized, and counted for 45Ca. Tissue uptake was then expressed as Ca accumulation per milligram wet weight of tissue. Ca uptake by the small intestinal mucosa is sensitive to age, segment, 1,25(OH)2D3 treatment, and dietary Ca in the rat (2).
Measurement of intestinal phosphate transport.
Phosphate transport was measured in separate experiments using the same procedures described for Ca transport. During measurement of unidirectional phosphate transport, 1 mM NaH2PO4 and [32P]H2PO4 were present in the incubation buffer. The amount of phosphate transported was calculated by multiplying radioactive counts per microliter of fluid by volume of fluid in the sac and by specific activity. Tissue uptake of phosphate was expressed as phosphate accumulation per milligram wet weight of tissue. Phosphate transport and phosphate uptake is sensitive to age, segment, 1,25(OH)2D3 treatment, and dietary Ca in the rat (2).
Measurement of mRNA levels.
Total RNA was isolated from intestinal mucosal scrapings using guanidinium isothiocyanate followed by cesium chloride centrifugation (12). mRNA levels of mouse intestinal calbindin, intestinal 24-hydroxylase, renal 1α-hydroxylase, and renal 24-hydroxylase were determined by ribonuclease protection assay (RPA). The RPA was performed as previously described, using specific probes (7). The RPA probe for the mouse intestinal calbindin D9k consisted of nucleotides 9–308 (27), the probe for the 24-hydroxylase consisted of nucleotides 945–1191 of the mouse renal cytochrome P-450 subunit CYP24 (17), and the probe for the 1α-hydroxylase consisted of nucleotides 127–610 of the mouse renal cytochrome P-450 subunit CYP1α (26). The RPA probe for the plasma membrane Ca (PMCA) pump was specific for the PMCA1b form of Ca pump. This is the major form found in the intestine and is the form thought to be involved in Ca absorption (16). The probe was generated by RT-PCR using total mouse intestinal RNA and primers based on rat PMCA1 sequence (forward primer: nucleotides 3557–3577; reverse primer: nucleotides 3869–3889) (24), as described by Varadi et al. (28). The 178-nucleotide PCR product was confirmed to be the PMCA1b form of the Ca pump by sequencing and comparison to the rat sequence (>98% homology). All probe sequences excluded substrate or cofactor binding sites and did not show homology to related proteins. The actin probe was rat β-actin (Ambion). RPA was performed using the RPAII kit from Ambion (Austin, TX). Probes were labeled with [32P]UTP (ICN Radiochemicals) using the Maxiscript T7 polymerase kit from Ambion. Bands were quantitated by scanning densitometry and normalized to actin mRNA.
Plasma Ca and phosphorus were measured colorimetrically using commercial kits (Sigma, St. Louis, MO). Plasma 1,25(OH)2D3 was measured using a commercial kit (ImmunoNuclear, Stillwater, MN).
Data from these experiments are reported as means ± SE of the number of animals indicated. Statistical analyses were performed using Student's two-tailed t-test. A confidence level of 95% or greater was considered significant.
Intestinal Ca transport was compared between C57 and C3H mice (Fig. 1 A). In the duodenum, Ca transport by C3H mice was 58% higher than the transport seen in C57 mice. In the jejunum, there was no difference between the two strains. In the same experiments, Ca uptake into the intestinal tissue was determined (Fig. 1 B). Ca uptake by the intestine paralleled Ca transport across the intestine. Ca uptake was 45% higher in the duodenum of the C3H mouse, but there was no difference in Ca uptake in the jejunum. In general, Ca transport and Ca uptake in both strains were less in the jejunum than in the duodenum. Ca transport was 7.07 ± 0.88 and 5.94 ± 0.92 pmol in the C57 mouse duodenum and jejunum, respectively. Ca uptake was 108 ± 6 and 65 ± 6 pmol/mg in the duodenum and jejunum, respectively. In the C3H mouse, Ca transport was 11.2 ± 1.6 and 5.52 ± 0.57 pmol in the duodenum and jejunum, respectively. Ca uptake was 157 ± 17 and 72 ± 9 pmol/mg in the duodenum and jejunum, respectively.
The effect of 1,25(OH)2D3 given in vivo on Ca transport and Ca uptake by the duodenum was then determined. 1,25(OH)2D3 significantly increased Ca transport to similar levels in both strains of mice (Fig.2 A). The component of Ca transport stimulated by 1,25(OH)2D3[1,25(OH)2D3 minus control] was calculated, and this also was not different between the two strains. The effect of 1,25(OH)2D3 on Ca uptake was similar (Fig.2 B). 1,25(OH)2D3 significantly increased Ca uptake in both strains of mice. Although the maximal level of stimulation was 29% less in the C57, this difference was not statistically significant. In addition, there was no difference in the 1,25(OH)2D3-stimulated component of Ca uptake.
Changes in Ca transport paralleled changes in Ca uptake in these experiments (Figs. 1 and 2). This suggested that these two physiological processes were correlated. To determine whether there was a correlation, Ca transport was plotted as a function of Ca uptake. Data from Fig. 2, A and B were used, because this experiment produced a wide range of Ca transport and uptake. There was a high degree of correlation between Ca uptake and Ca transport (Fig.3). This suggests that Ca transport across the intestine is a reflection of Ca uptake into the intestinal mucosa.
Intestinal phosphate transport was also measured in C57 and C3H mice (Fig. 4). Phosphate transport was measured in the duodenum, because this region showed significant differences regarding Ca transport between the two strains (Fig. 1). Duodenal phosphate transport in the C3H mouse was only 36% that of the C57 mouse (Fig. 4). Likewise, phosphate uptake by the C3H mouse was 55% that of the C57 mouse (Fig. 4). In absolute terms, phosphate transport was 88.6 ± 17.0 and 31.9 ± 3.5 nM in the C57 and C3H mice, respectively. Phosphate uptake was 1,534 ± 100 and 843 ± 46 pmol/mg, respectively.
Regarding plasma levels, there was no significant difference in plasma Ca, phosphorus, or 1,25(OH)2D3 between the two strains (Table 1). Plasma 1,25(OH)2D3 levels were increased 12- to 14-fold in both strains by 1,25(OH)2D3treatment, and there was no significant difference in the final levels.
To characterize the mechanisms responsible for these strain differences in intestinal Ca absorption, basal expression was measured as well as expression in response to chronic 1,25(OH)2D3treatment. There was no difference in the basal (control) level of mRNA for calbindin in the duodenum of the two strains (Fig.5). 1,25(OH)2D3treatment had no significant effect on calbindin mRNA levels in these studies. Previous studies have shown that this chronic treatment with 1,25(OH)2D3 markedly increases calbindin mRNA levels in 1,25(OH)2D3-depleted rats (4). However, because these mice were not vitamin D deficient, they had high levels of calbindin mRNA in the duodenum at the beginning of the study. Thus it may be difficult for 1,25(OH)2D3 to further increase calbindin mRNA levels under these conditions.
In contrast to calbindin, the basal Ca pump mRNA levels were 90% higher in the C3H mice (Fig. 6). This strain difference was also seen in the 1,25(OH)2D3-treated groups. As with calbindin, 1,25(OH)2D3 treatment had no significant effect on Ca pump mRNA levels. This again may be due to the fact that these mice were not vitamin D-deficient.
In the same experiments, the mRNA levels of CYP24 were measured in the duodenum (Fig. 7). mRNA levels were virtually undetectable in the control animals, but they were markedly stimulated by 1,25(OH)2D3. This has been seen previously in the rat (3). There was no difference in the maximal level of stimulation by 1,25(OH)2D3between the two strains.
Finally, the mRNA levels of the vitamin D hydroxylases and calbindin D9k in the kidney were measured in these experiments (Table2). There was no significant difference in the basal (control) levels of CYP1α. This is consistent with the fact that there was no significant difference in plasma 1,25(OH)2D3 levels between strains (Table1). Treatment with 1,25(OH)2D3 significantly decreased the CYP1α mRNA in both strains. There was also no significant difference in the basal (control) levels CYP24. Treatment with 1,25(OH)2D3significantly increased CYP24 mRNA levels in both strains. Finally, there was no difference in renal calbindin D9k mRNA levels with either strain or 1,25(OH)2D3 treatment. This is similar to what was seen in the duodenum (Fig. 5).
These studies demonstrate that the Ca-transporting capacity of the duodenum of the C3H mouse is 58% greater than that of the C57 mouse (Fig. 1). There is no difference seen in the jejunum, suggesting that the duodenum is the major site of increased Ca transport by the C3H mouse. In the previously reported balance study, the apparent Ca absorption by C3H mice was ∼55% greater than that seen in C57 mice (11). However, differences in apparent Ca absorption may reflect differences in Ca secretion as well as Ca transport (6). The present in vitro studies strongly suggest the increase seen in the balance study is due to greater Ca transport rather than decreased Ca secretion by the C3H mouse.
The question arises as to why there is increased Ca transport by the duodenum of the C3H mouse. Intestinal Ca transport involves uptake of Ca by the absorptive cells, transport of Ca across the cells with the aid of calbindin, and extrusion of Ca out of the cells by an ATP-dependent Ca pump (29). Intestinal calbindin is a soluble, vitamin D-regulated, Ca-binding protein. Steady-state levels of calbindin correlate well with intestinal Ca transport under a variety of conditions in the rat (30). However, in these studies there were no differences in the calbindin mRNA levels between the two strains (Fig. 5).
In addition to calbindin, the ATP-dependent Ca pump plays a major role in Ca transport. A number of studies have shown a correlation between Ca pump mRNA levels and Ca transport regarding 1,25(OH)2D3 administration, dietary Ca, and dietary phosphorus (29). In the rat, Ca pump protein levels correlate with Ca transport in terms of age and 1,25(OH)2D3 administration (5). Interestingly, in the present study, Ca pump mRNA levels were 90% higher in C3H mice compared with C57 mice (Fig. 6). This was greater in magnitude than the increase in Ca transport seen in the C3H mice (58%). Thus the increase in duodenal Ca pump mRNA in C3H mice could account for the observed increase in Ca transport, assuming the increased mRNA levels resulted in increased Ca pump activity.
There has been some debate as to whether calbindin or the Ca pump is most important as the rate-limiting step of Ca transport. In many studies, there are parallel changes in the expression of these two proteins in response to vitamin D, dietary Ca, and age (5). However, in the present study, only Ca pump expression was significantly higher in the C3H mice, suggesting that changes in the Ca pump alone may alter overall Ca transport. However, there may also be strain differences in other components of the Ca transport system, such as the newly discovered Ca channel (15). This channel may play a role in the uptake of Ca into absorptive cells.
In these studies, the strain differences in Ca absorption were not due to differences in the production and action of 1,25(OH)2D3. First, plasma 1,25(OH)2D3 levels were not significantly different in the two strains (Table 1). Second, the basal mRNA levels for CYP1α and CYP24 in the kidney were not different (Table 2). Finally, the capacity of 1,25(OH)2D3 to increase duodenal Ca transport was the same in both strains (Fig. 2). Also, 1,25(OH)2D3 increased duodenal CYP24 mRNA to the same level (Fig. 6). This is consistent with the fact that vitamin D receptor levels in the duodenum have been reported to be the same in both strains (11).
In addition to differences in Ca absorption, there are also differences in duodenal phosphate transport between the two species (Fig. 4). Interestingly, phosphate transport is higher in the C57 mouse. This argues against some kind of generalized defect in transepithelial ion transport in the C57 mouse. Because phosphate, along with Ca, is a major component of bone, this increased phosphate transport may have physiological significance. There is evidence that the ratio of dietary Ca to phosphate is important for optimal bone mineralization. In adult and aged mice, high dietary phosphate in relation to dietary Ca may result in reduced bone mineralization (22). In vitamin D receptor knockout mice, dietary phosphorus must be low relative to dietary Ca to prevent the low bone density associated with these animals (19). Thus it is possible that reduced Ca transport combined with increased phosphate transport may contribute to the reduced peak bone mass seen in the C57 mouse.
This is not to suggest that differences in mineral absorption alone account for all of the increased peak bone mass seen in the C3H mice. It is likely that several factors are involved, some of which may be related to Ca absorption. For example, C3H mice have higher levels of insulin-like growth factors (IGF-I) in serum and bone than C57 mice (21). Higher levels of serum IGF-I may increase bone formation directly. In addition, they could contribute to the increased Ca absorption seen in the C3H strain. It has been shown that growth hormone and IGF-I stimulate intestinal Ca absorption (14). Regarding bone itself, histomorphometric studies have shown that bone formation rate is greater in the C3H mice compared with the C57 mice (23). This is consistent with the fact that C3H mice have higher levels of alkaline phosphatase and higher osteoprogenitor cell numbers (13). There is also evidence that bone resorption may be slowed in the C3H mice, because they produce fewer osteoclasts (18).
Finally, these studies demonstrate that the everted intestinal sac is a useful in vitro technique for characterizing Ca absorption in the mouse. Previous studies of Ca absorption in the mouse have used in vivo techniques such as balance studies (11) and the ligated intestinal loop (20). In the present study, everted intestinal sacs detected the expected differences between intestinal segments (Fig. 1) and the marked stimulation by 1,25(OH)2D3 (Fig. 2). They have also been used to study the effect of age and dietary Ca in mice (unpublished studies). With the large number of transgenic mice now available, it may be possible to use this technique to dissect out the important components and regulators of the intestinal Ca transport system itself.
This work was supported by the St. Louis Geriatric Research, Education, and Clinical Center, the Medical Research Service of the Department of Veterans Affairs, and National Institute on Aging Grant AG-12587. The probe for the mouse cytochrome P-450 subunit of the 1α-hydroxylase was kindly provided by Dr. Anthony Portale, University of California, San Francisco, CA.
Address for reprint requests and other correspondence: H. J. Armbrecht, Geriatric Center (11G-JB), St. Louis Veterans Administration Medical Center, St. Louis, MO 63125 (E-mail:).
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