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Endocrinology, doi:10.1210/en.2003-1010
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Endocrinology Vol. 145, No. 3 1367-1375
Copyright © 2004 by The Endocrine Society

Leptin Corrects Increased Gene Expression of Renal 25-Hydroxyvitamin D3-1{alpha}-Hydroxylase and -24-Hydroxylase in Leptin-Deficient, ob/ob Mice

Ayako Matsunuma, Tetsuya Kawane, Toyonobu Maeda, Setsuo Hamada and Noboru Horiuchi

Departments of Biochemistry (A.M., T.K., T.M., N.H.) and Pharmacy (S.H.), Ohu University School of Dentistry, Koriyama 963-8611, Japan

Address all correspondence and requests for reprints to: Noboru Horiuchi, Ph.D., Department of Biochemistry, Ohu University School of Dentistry, Koriyama 963-8611, Japan. E-mail: fwga4746{at}mb.infoweb.ne.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leptin, the ob gene product secreted by adipocytes, controls overall energy balance. We investigated leptin effects on bone metabolism using male leptin-deficient obese (ob/ob) mice, which had lower bone mineral density (BMD) and shorter femurs than lean (?/+) controls. Serum concentrations of calcium, phosphate, tartrate-resistant acid phosphatase (a bone resorption marker) and alkaline phosphatase, and urinary calcium and phosphate excretion were significantly elevated in ob/ob mice, whereas urinary concentrations of deoxypyridinoline did not differed between ob/ob and control mice. Because ob/ob mice develop severe hypogonadism, testosterone was administered to these mice for 10 wk (5 mg/kg, sc, twice weekly); this did not affect femoral BMD. Control and ob/ob mice showed similar vitamin D-receptor densities in bone and kidney; the obese mice had marked increases in serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and in mRNA expression and activities of renal 25-hydroxyvitamin D3-1{alpha}-hydroxylase (CYP27B1) and -24-hydroxylase (CYP24) compared with control mice. Leptin administration to ob/ob mice (4 mg/kg body weight, ip, every 12 h for 2 d) greatly reduced mRNAs and activities of 1{alpha}-hydroxylase and 24-hydroxylase. Elevated concentrations of serum calcium, phosphate, and 1,25-(OH)2D3 were normalized by leptin treatment. Thus, leptin suppresses renal gene overexpression for 1{alpha}-hydroxylase and 24-hydroxylase and corrects increased serum concentrations of calcium and phosphate in ob/ob mice. Therefore, low BMD in leptin-deficient mice appears to be related to stimulation of bone resorption by 1,25-(OH)2D3.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MUTATION OF THE obese gene (ob) results in obesity and increased body fat mass. The ob product, leptin, is a 16-kDa circulating hormone secreted by adipocytes and is thought to act primarily in the hypothalamus, where it influences body weight homeostasis by altering food intake and energy expenditure (1, 2, 3, 4, 5). In C57BL/6J mice, a single-base mutation of the ob gene at codon 105 results in production of a truncated, inactive form of leptin. In the homozygous state, the mutation provides a naturally occurring knockout model of leptin deficiency, the ob/ob mouse, which expresses a phenotype including obesity, increased body fat mass, hyperglycemia, hyperinsulinemia, hypothermia, sterility, and impaired thyroid function (6, 7, 8, 9, 10). Because leptin is produced almost exclusively by white adipose tissue, serum concentrations of leptin are increased in obesity and are strongly related to body fat mass (11). Leptin circulates in plasma and acts upon the hypothalamic nuclei after crossing the blood-brain barrier. The effects of leptin in the central nervous system include regulation of food intake, energy expenditure, growth, and sexual maturation (12, 13, 14). Leptin also acts in the periphery, where leptin receptors have been described (15, 16).

Integrity of the skeleton requires a dynamic balance between bone formation and bone resorption, which are controlled by cytokines and calcitropic hormones such as PTH and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. When bone resorption exceeds bone formation, diseases of bone metabolism, such as postmenopausal osteoporosis, can occur (17). Osteoporosis and obesity are inversely correlated because bone mineral density (BMD) increases with body weight and fat body mass. Thus, obesity is associated with increased BMD and has a protective effect against osteoporosis (18, 19). This protective effect of obesity has been partially explained by a combination of humoral and mechanical factors. Increased peripheral tissue mass may lead to a relative increase in serum concentration of estrogen, which elevates BMD. In addition, mechanical stress resulting from obesity has osteogenic effects on weight-bearing bone sites. However, Revilla et al. (20) suggested that the association between obesity and BMD is at least partially independent of sex steroids in postmenopausal women; this suggests a contribution from additional regulatory factors.

Bone mass is maintained by coordinated regulation of osteoblast and osteoclast numbers at the cellular level. Osteoblasts regulate recruitment and activity of osteoclasts through expression of receptor activator of nuclear-{kappa}B ligand (RANKL) and osteoprotegerin (OPG). RANKL, a member of the TNF family, is expressed on the osteoblast/stromal cell surface and binds to its receptor, receptor activator of nuclear-{kappa}B (RANK), on surfaces of hematopoietic precursor cells; in the presence of macrophage-colony stimulating factor, this binding stimulates osteoclastogenesis. OPG, a secreted glycoprotein of the TNF receptor superfamily, acts as a nonsignaling decoy receptor that binds to RANKL and prevents the activation of RANK. The RANK/RANKL/OPG axis couples osteoblast and osteoclast activity, controlling the balance between bone formation and resorption (21).

Recently, Ducy et al. and Takeda et al. (22, 23) demonstrated that leptin acts on hypothalamic neurons and inhibits bone formation by stimulating activity of sympathetic neurons whose axons penetrate the bone. In contrast, Steppan et al. (24) reported that leptin administration significantly increased femoral length, total body bone area, and BMD in male ob/ob mice. Administration of leptin to female rats was effective in reducing trabecular bone loss associated with estrogen deficiency but could not fully restore bone loss resulting from ovariectomy (25). Thus, leptin appears to have multiple central and peripheral effects on bone metabolism (26).

The active form of vitamin D, 1,25-(OH)2D3, is an important regulator of bone metabolism that elicits its biologic effects by binding to the vitamin D receptor (VDR) in target tissues such as bone, intestine, and kidney (27, 28). PTH and 1,25-(OH)2D3 increase serum calcium concentrations in humans and rodents. Major actions of 1,25-(OH)2D3 include augmentation of bone resorption and stimulation of intestinal calcium absorption. Metabolism of 25-hydroxyvitamin D3 (25-OHD3), a circulating form of vitamin D3, occurs mainly in the kidney where it is converted to 1,25-(OH)2D3 by 25-OHD3-1{alpha}-hydroxylase (CYP27B1) and also to 24,25-dihydroxyvitamin D3, a catabolic product, by 25-OHD3-24-hydroxylase (CYP24). The synthesis of these metabolites is reciprocally regulated by multiple factors, including 1,25-(OH)2D3 and PTH (27, 28).

Because PTH and vitamin D are the major regulators of mineral ion metabolism, suspicion has arisen that leptin deficiency may influence 25-OHD3 metabolism in the kidney and thus affect BMD in mice. The present study using male leptin-deficient (ob/ob) mice was undertaken to clarify involvement of leptin in calcium homeostasis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The animal studies were approved by the Institutional Animal Care and Use Committee of Ohu University and conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals. Eight-week-old heterozygous male and female mice (C57BL/6J ob/+) were purchased from Jackson Laboratory (Bar Harbor, ME) and were maintained on rodent chow (calcium, 1.42%; phosphorus, 1.16%; vitamin D3, 1.6 IU/g of diet). Five mice were housed to a cage in a temperature-regulated environment (22 ± 2C) with a cycle of 12 h of light and 12 h of darkness and with free access to food and water. Obese (ob/ob) and lean (?/+) mice were obtained by mating female (ob/+) mice with male (ob/+) mice. Mice were maintained on rodent chow for the periods indicated. In experiments involving leptin administration, 13-wk-old male C57BL/6J ob/ob mice that were ad libitum fed received ip injections of either vehicle alone (PBS) or recombinant murine leptin (Calbiochem, San Diego, CA; 4 mg/kg) at intervals of 12 h for 2 d. In experiments involving testosterone treatment, 13-wk-old male ob/ob mice received sc injections twice weekly of either vehicle (corn oil) or testosterone (Sigma, St. Louis, MO; 5 mg/kg) for 10 wk. Serum and urinary concentrations of calcium and phosphorus were measured colorimetrically, as was urinary creatinine. Serum concentrations of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) activities were measured as previously described (29). Serum 1,25-(OH)2D3 was measured by a RIA kit of 1,25-(OH)2D3 (ImmunoDiagnostic System, Boldon, UK), and serum mouse PTH was determined using an ELISA kit of Mouse Intact PTH (Immutopics, San Clemente, CA). Urinary excretion of deoxypyridinoline (Dpd) was measured with a PRIDIRINS-D ELISA kit (Quidel; Santa Clara, CA).

Bone mineral analysis, histology, and histomorphometry
After mice were killed, femurs and tibiae were removed. BMD of the right femurs was measured by dual-energy x-ray absorptiometry with a DCS-600 densitometer (Aloka, Tokyo, Japan), using software designated for small-animal measurements (29). Right tibiae were fixed in 10% buffered formalin, decalcified in 10% EDTA (pH 7.2), dehydrated, and embedded in paraffin. Sections were cut using a standard microtome, affixed to glass slides, and stained either with hematoxylin and eosin or by the Masson-trichrome method. Left tibiae were fixed with 70% ethanol and embedded in glycol methacrylate without decalcification. Sections (3-µm thick) were stained with the Villanueva Goldner stain. Histomorphometric analyses of bone sections were performed using a semiautomated system (Osteoplan II; Carl Zeiss, Thornwood, NY). Parameters for bone structure included bone volume per tissue volume (%), trabecular thickness (µm), trabecular number (per mm), and trabecular separation (µm). Parameters for bone resorption included number of osteoclasts per bone perimeter (per 100 mm), and osteoclast surface per bone surface (%) (30).

Probes for mRNA determination
The probe used for 24-hydroxylase in this study was a previously cloned 3.3-kb, full-length cDNA (31). The 1{alpha}-hydroxylase probe was a 2.4-kb full-length cDNA that we have cloned; the sequence was identical to the one reported by Takeyama et al. (32). Complementary DNA probes, such as a 561-bp length of mouse VDR (GenBank accession no. D31969), a 331-bp length of RANK (GenBank accession no. AF019046), a 295-bp length of RANKL (GenBank accession no. AF013170), and a 476-bp length of OPG (GenBank accession no. U94331), were synthesized by RT-PCR and cloned into a pCR2.1 vector (Invitrogen, San Diego, CA). The nucleotide sequences of the cDNAs corresponded to reported sequences. Probes were labeled with [{alpha}-32P]deoxycytidine triphosphate (110 Tbq/mmol; ICN Biochemical, Costa Mesa, CA) by the random oligopriming method using a Megaprime DNA labeling kit (Amersham, Buckinghamshire, UK). The cyclophilin cDNA probe was used as a control for loading of RNA on the gels and transfer to membranes.

Determination of mRNAs
Total RNA was extracted from femurs and renal cortex, and Northern blot analysis was performed as described previously (31). The membranes were hybridized with mouse 24-hydroxylase, VDR (renal preparation), or cyclophilin cDNA probes. Membrane signal intensity was quantified with a Molecular Imager FX (BIORAD, Hercules, CA); the resulting images were analyzed using Quantity One 4.1.1 (BIORAD) image analysis software. Amounts of the mRNA of interest were calculated relative to cyclophilin mRNA expression. RT-PCR was performed to determine 1{alpha}-hydroxylase, VDR (bone preparation), RANK, RANKL, and OPG mRNA expression using a Gene Amp RNA PCR kit (Applied Biosystems, Foster City, CA). One microgram of total RNA was reverse-transcribed at 42 C for 30 min to synthesize cDNA. Amplification reactions were then performed using the following primers and protocols: 1{alpha}-hydroxylase forward, 5'-TAACCCACTTCCTTTTTCGG-3', and reverse, 5'-GCTGGATTAAAAGAGTTGGG-3' (annealing at 55 C, 18 cycles, 412-bp product); VDR forward, 5'-GGAGCAACAGCACATTATCG-3', and reverse, 5'-CACCTGGAACTTTATGAGGG-3' (annealing at 55 C, 19 cycles, 561-bp product); RANK forward, 5'-TTATGAGCATCTCGGACGGT-3', and reverse, 5'-ACGTCGAGTTGTTCCTATGC-3' (annealing at 55 C, 26 cycles, 331-bp product); RANKL forward, 5'-CATTTGCACACCTCACCATC-3', and reverse, 5'-TACTTTCCTCCCTCGTGCTT-3' (annealing at 55 C, 26 cycles, 295-bp product); and OPG forward, 5'-TGTGTATTGCAGCCCAGTGT-3', and reverse, 5'-CTTACGGCTCTCACATCTCT-3' (annealing at 55 C, 22 cycles, 476-bp product). Relative amounts of these transcripts were corrected for the amount of cyclophilin or ß-actin mRNA that was present.

Western blot analysis of VDR
Kidneys and bone from male lean (?/+) and obese (ob/ob) mice were homogenized in a buffer. Polyclonal antibody raised against mouse VDR (Santa Cruz Biotechnology, Santa Cruz, CA) was used at a 1:1500 dilution. Incubation was conducted for 60 min at room temperature with a 1:2000 dilution of peroxidase-labeled antirabbit IgG (Amersham). Antigen-antibody complexes on membranes were visualized using an ECL-Plus Western blotting detection system (Amersham). The amounts of VDR protein were quantified by densitometric scanning of autoradiographs (33).

Measurement of renal hydroxylase activities
Activities of renal 1{alpha}-hydroxylase and 24-hydroxylase were determined using kidney homogenates and substrate as described previously (34). The renal cortex of male mice was minced and washed in ice-cold homogenization buffer (0.19 M sucrose, 25 mM sodium succinate, 2 mM MgCl2, 1 mM EDTA, and 20 mM Tris-HEPES, pH 7.4) and then homogenized in the same solution (20 ml/g tissue). For measurement of 1{alpha}-hydroxylase activity, 5 µg of 25-OHD3 (Phillips Duphar, Amsterdam, Netherlands), dissolved in 10 µl of ethanol, was added to 1 ml of 5% homogenate, and the mixture was incubated at 37 C for 45 min. Reactions were stopped by the addition of 1 ml of acetonitrile. Synthesis of 1,25-(OH)2D3 was quantified by a radioreceptor assay for 1,25-(OH)2D3. Data are expressed as femtomoles per milligram of protein per minute. Because 1{alpha}-hydroxylase activity was very low, we used the sensitive method of Lobaugh et al. (35), which can accurately measure in vitro production of 1,25-(OH)2D3 at low enzyme concentrations in mouse kidney homogenates. This method indicated the maximum velocity of 1{alpha}-hydroxylase activity. For the 24-hydroxylase activity assay, tritiated 1,25-(OH)2D3 (250 pmol, 50,000 cpm) from DuPont NEN Life Science Products (Boston, MA) was used as the substrate in an incubation carried out at 37 C for 15 min using 1 ml of 5% kidney homogenate. Reactions were stopped by the addition of 1 ml of acetonitrile. Vitamin D metabolites were extracted with a C18/Sep-Pak (Waters, Milford, MA) and separated by high-performance liquid chromatography. Activity of 24-hydroxylase is expressed in femtomoles per milligram of protein per minute.

Statistical analyses
Data are described as the means ± SEM. Differences between treated and untreated groups were assessed using the Student’s t test. Multiple comparisons were evaluated by one-way ANOVA followed by Scheffé’s F test. Statistical analysis was performed with the Statview 4.02 software package (Abacus Concepts, Berkeley, CA). A P value < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Biochemical markers in lean control and ob/ob mice
Serum markers of bone metabolism were compared between lean control and obese (ob/ob) mice (Table 1Go). Obese mice that were 24-wk-old had significantly elevated serum calcium, phosphorus, and 1,25-(OH)2D3 concentrations compared with control mice. Because serum ALP activity is the most commonly used marker of bone formation, we measured total ALP activity in serum; this was significantly greater in both 13- and 24-wk-old ob/ob mice compared with control mice. Serum TRAP activity, a bone resorption marker, significantly increased in ob/ob mice (Table 1Go). Urinary excretion of calcium and phosphorus was significantly greater in ob/ob mice that were both 14 wk and 20 wk old compared with control mice (Table 2Go). Dpd and pyridinoline are nonreducible cross-links that stabilize collagen chains within the extracellular matrix. Because significant amounts of Dpd are found only in bone collagen and urinary Dpd is released from bone matrix during bone resorption, urinary excretion of Dpd was monitored as a bone resorption marker in obese and control mice. The leptin deficiency present in ob/ob mice did not affect the urinary excretion of Dpd (Table 2Go).


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  		TABLE 1. Serum calcium, phosphorus, 1,25-(OH)2D3, ALP, and TRAP activity in obese (ob/ob) and lean control mice

 

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  		TABLE 2. Urinary excretion of calcium, phosphorus, and Dpd in ob/ob and control mice

 
BMD and histologic analysis of long bones
We assessed BMD and femoral length in ob/ob and control mice. Femoral BMD was significantly less in both 13- and 24-wk-old ob/ob mice compared with lean control mice, whereas femoral length of mutant mice was decreased at both ages (Fig. 1Go). Figure 2Go compares representative histologic sections of proximal tibia from 13-wk-old ob/ob and control mice. Sections from control mice had a normal appearance with adequate compact bone thickness and trabecular bone volume predominating over relatively scant marrow space (Fig. 2Go, A and C). In contrast, obese (ob/ob) mice showed relatively thin compact bone and less trabecular bone than sections from control mice (Fig. 2Go, B and D). In the sections stained by the Masson-trichrome technique (Fig. 2Go, C and D), collagen fibers abundant in bone and cartilage were stained blue. In these sections, leptin-deficient (ob/ob) mice again showed lower trabecular bone volume and thinner compact bone than control mice. Thus, histologic analysis of the tibia confirmed a decrease in BMD in femurs from ob/ob mice. Bone histomorphometric analysis showed no significant differences in trabecular bone of tibiae between ob/ob and control mice (data not shown). Because ob/ob mice manifest hypogonadism and testosterone increases bone mass, the effect on BMD of testosterone administration to the mutant mice was studied. Long-term administration of testosterone (10 wk) did not influence femoral BMD in ob/ob mice (data not shown). This established that low BMD in ob/ob mice is not a result of reduced serum testosterone concentrations.



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  		FIG. 1. BMD (A) and length of the femur (B) in obese (ob/ob) and lean control mice. Control data (open columns) and ob/ob data (gray columns) obtained in 13-wk-old and 24-wk-old mice are shown. At these time points, mice were killed under anesthesia, and femurs were excised. BMD was determined by dual-energy x-ray absorptiometry using software designed for small animal measurements. Values are expressed as the mean ± SEM for six animals. *, P < 0.01 compared with lean control mice at the same age.

 


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  		FIG. 2. Histologic analysis of the proximal tibia in control (A, C) and ob/ob (B, D) mice. Tibiae from 13-wk-old mice were excised, and proximal portions were fixed, decalcified, and embedded. Sections were stained with hematoxylin and eosin (A, B) or by the Masson-trichrome method (C, D).

 
Effect of leptin on serum biochemical markers
Leptin-deficient (ob/ob) mice were injected with murine leptin 4 mg/kg ip every 12 h for 2 d (i.e. four times). Serum biochemical parameters were measured 12 h after the last injection. Leptin administration abolished serum calcium and phosphorus elevations in ob/ob mice, returning these serum concentrations to the values seen in lean control mice (Table 3Go). Surprisingly, serum concentrations of immunoreactive mouse PTH were greatly elevated by murine leptin administration in ob/ob mice, which had PTH concentrations similar to those in control mice before treatment. Although ob/ob mice had markedly increased serum concentrations of 1,25-(OH)2D3, leptin administration at the above dose clearly decreased the elevated serum 1,25-(OH)2D3 concentrations, alleviating hypervitaminosis (Table 3Go).


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  		TABLE 3. Effect of leptin on serum parameters in ob/ob mice

 
Effects of leptin in bone
Because 1,25-(OH)2D3 acts via binding to VDR on the target tissues, VDR gene expression was examined in bone from these mice. Expression of VDR mRNA and protein in mouse bone was not affected by the presence or absence of leptin (data not shown), indicating that, in mice, the effect of 1,25-(OH)2D3 in stimulating bone resorption depended on circulating quantities of 1,25-(OH)2D3. Next, whole-bone expression for RANK, RANKL, and OPG mRNA was determined in ob/ob and control mice because bone resorption and formation are linked through expression of these proteins. Expression of the RANK, RANKL, and OPG genes in bone was significantly increased in ob/ob mice, whereas short-term administration of leptin (2 d) to these mice did not affect expression of these mRNAs (Fig. 3Go).



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  		FIG. 3. Effect of leptin on mRNA expression of RANK (A), RANKL (B), and OPG (C) in bone from 13-wk-old control and ob/ob mice. Mice were injected with either vehicle or murine leptin (4 mg/kg, ip) every 12 h for a total of four injections. Mice were killed 12 h after the last injections. Control mice were killed for total RNA extraction of femurs. The mRNAs were determined by RT-PCR and normalized to cyclophilin mRNA. Data are expressed as the mean ± SEM for six measurements. *, P < 0.01 compared with control mice.

 
Effect of leptin on renal 25-OHD3 metabolism
When we assessed the effect of leptin on renal VDR expression, control and ob/ob mice treated with vehicle or leptin showed similar amounts of renal VDR mRNA and protein expression (data not shown). The high serum concentrations of 1,25-(OH)2D3 in ob/ob mice prompted us to examine expression of 1{alpha}-hydroxylase and 24-hydroxylase, which are enzymes required for regulation of the final rate-limiting steps of vitamin D metabolism. Renal expression of the 1{alpha}-hydroxylase mRNA and its enzyme activity was markedly enhanced in leptin-deficient (ob/ob) mice; leptin administration of 4 mg/kg ip per 12 h for 2 d clearly reduced 1{alpha}-hydroxylase mRNA expression and enzyme activity (Fig. 4Go). The ob/ob mice showed highly increased renal 24-hydroxylase mRNA expression, and administration of leptin attenuated the excessive enzyme gene expression (Fig. 5AGo). The 24-hydroxylase activity of kidney increased in ob/ob mice, but leptin administration to the ob/ob mice markedly decreased the enzyme activity (Fig. 5BGo).



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  		FIG. 4. Effect of leptin administration on renal 1{alpha}-hydroxylase mRNA expression (A) and enzyme activity (B) in 13-wk-old control and ob/ob mice. Mice were injected with either vehicle or murine leptin (4 mg/kg, ip) every 12 h for a total of four injections. Mice were killed 12 h after the last injection. Control mice were killed for analysis of renal 1{alpha}-hydroxylase expression. Amounts of 1{alpha}-hydroxylase mRNA were determined by RT-PCR and normalized to those of ß- actin mRNA. The enzyme activity was measured in kidney homogenates using excess amounts of substrate. Data are presented as the mean ± SEM for six measurements. *, P < 0.01 compared with control mice. #, P < 0.01 compared with vehicle-treated ob/ob mice.

 


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  		FIG. 5. Effect of leptin administration on renal 24-hydroxylase mRNA expression (A) and enzyme activity (B) in 13-wk-old control and ob/ob mice. The animal protocol was the same as for Fig. 4Go. Control mice were killed for analysis of renal 24-hydroxylase expression. Amounts of 24-hydroxylase mRNA were determined by Northern blotting and normalized to those of cyclophilin mRNA. The enzyme activity was measured in kidney homogenates using tritiated 1,25-(OH)2D3 as a substrate. Data are presented as the mean ± SEM for six measurements. *, P < 0.01 compared with control mice. #, P < 0.01 compared with vehicle-treated ob/ob mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using leptin-deficient (ob/ob) and leptin-resistant (db/db) mice, Ducy et al. (22) first demonstrated that intracerebroventricular infusion of leptin resulted in bone loss. High BMD of these animals reflected accelerated bone formation that appeared to result from increased osteoblastic activity as opposed to an increase in osteoblast number. Recently, Takeda et al. (23) demonstrated a leptin-dependent antiosteogenic network in the hypothalamus. Leptin-deficient mice had reduced sympathetic tone, and administration of a ß- adrenergic agonist reduced bone mass in these mice. Thus, leptin acts on the central nervous system, especially the hypothalamus, causing sympathetic nerve-mediated inhibition of bone formation.

On the other hand, a number of studies have examined the peripheral effects of leptin on regulation of bone metabolism. Clinical studies have demonstrated that circulating leptin concentrations were positively associated with bone mass at various sites, particularly in postmenopausal women (36). Experimentally, leptin administration increased femoral length, total bone area, and BMD in ob/ob mice (24) and counteracted reduction of bone volume induced by ovariectomy in rats (25). A histomorphometric experiment demonstrated that leptin treatment of ob/ob mice stimulated endocortical bone formation by increasing osteoblast numbers and dynamic indices of bone formation (37). The db/db mice with lack of leptin signaling had decreased BMD compared with normal mice and showed osteopenia (38). Recently, Tamasi et al. (39) indicated the trend toward decreasing structural and bone formation parameters in the obese (fa/fa) leptin receptor-deficient Zucker rats, suggesting that leptin exerts a positive effect on bone. Furthermore, Thomas et al. (40) showed that leptin acted on human marrow stromal cells to promote osteoblastic differentiation and to inhibit adipocyte differentiation. Osteoblasts were shown to express the long form of the leptin receptor (OB-Rb), which conveys the hormone signal (41, 42). In recent investigations, leptin was produced and secreted by osteoblasts in vitro and promoted mineralization by these cells (42, 43). Leptin acted as a bone growth factor on the chondrocytes of skeletal growth centers (44). According to Holloway et al. (45), leptin inhibited osteoclast generation in peripheral blood mononuclear cells and splenic cells. We found that leptin deficiency clearly attenuated BMD and growth in mouse femurs. However, histomorphometric analysis showed that there were no differences in trabecular bone of tibiae between ob/ob and control mice. Low BMD in ob/ob mice may be involved in a larger space of bone marrow than in the bone marrow space of control mice. Collectively, our findings are consistent with previously reported results (24, 25, 37, 38, 39, 40, 42, 43, 44, 45). Although the basis of the apparent discrepancy between our findings and other findings (22) is unknown, there are several differences of experimental condition between both studies. We used male ob/ob mice to measure BMD of femurs, whereas bone metabolism of vertebrae was mainly analyzed in female ob/ob mice in the other study (22). Further investigations will be necessary to clarify the cause of divergent results in terms of bone structure of ob/ob mice.

Hypogonadism in humans and rodents leads to accelerated bone loss, whereas androgen replacement therapy has been found to prevent bone loss after orchidectomy and to increase BMD in osteoporosis and hypogonadism in men (46). Androgen continues to be important in the maintenance of bone mass in male humans and animals (47). Obese (ob/ob) mice manifest sterility associated with reduced testicular secretion of testosterone (48, 49). In the present study, however, we were unable to demonstrate any effects of testosterone on BMD in ob/ob mice, which indicated that low BMD in obese mice was not caused by decreases in testosterone. Considering the results of studies as a whole, leptin has both central and peripheral effects on bone metabolism, as usefully summarized by Khosla (26). Conceivably, more actions of leptin on calcium homeostasis may exist.

Increased concentrations of serum calcium and phosphate in leptin-deficient (ob/ob) mice may significantly contribute to the phenotypes seen in mutants, such as reduced femoral BMD. In the present study, we examined the serum concentrations of calcitropic factors and their functional interactions to determine the causes of the phenotypes. We showed that ob/ob mice had normal concentrations of PTH, confirming the findings of Ducy et al. (22). Furthermore, we found a marked increase in serum 1,25-(OH)2D3 concentrations in these mice, resulting in elevation of serum calcium and phosphate concentrations via 1,25-(OH)2D3 actions on bone (27, 28). Administration of leptin to ob/ob mice markedly suppressed excessive renal 25-OHD3-1{alpha}-hydroxylase gene expression and enzyme activity, which ultimately corrected the elevation of serum calcium and phosphate concentrations. Expression of 1{alpha}-hydroxylase, the 1,25-(OH)2D3-synthesizing enzyme, and 24-hydroxylase, a catabolic enzyme for vitamin D metabolites, is regulated reciprocally in normal humans and animals (27, 28, 31). However, absence of leptin stimulates renal expression of both enzymes. Leptin deficiency generally promotes 1{alpha}-hydroxylase expression, overcoming inhibition of enzyme synthesis that normally would result from increased serum concentrations of 1,25-(OH)2D3. These excesses presumably induce exaggerated renal production of 24-hydroxylase in ob/ob mice (31, 34).

Short-term injection of leptin strikingly increases serum concentrations of immunoreactive mouse PTH in ob/ob mice. Although PTH is a potent stimulator of renal 1{alpha}-hydroxylase (50, 51), leptin-treated ob/ob mice with high serum concentrations of PTH manifested lower 1{alpha}-hydroxylase expression in the kidney and lower 1,25-(OH)2D3 in serum compared with vehicle-treated mutant mice. Thus, PTH may not be a major regulator of renal 1{alpha}-hydroxylase in these mice. In contrast, PTH inhibits renal 24-hydroxylase expression in rodents (52). Suppression of 24-hydroxylase mRNA expression and enzyme activity by leptin administration to ob/ob mice may result, at least in part, from elevation of the serum PTH concentration. Increases in serum PTH induced by leptin administration strongly suggest that leptin acts on the parathyroid glands to stimulate PTH synthesis. Further investigation is required to define regulation of PTH synthesis and secretion by leptin in ob/ob mutant mice.

The biologic effects of leptin are mediated by interactions with the leptin receptor, especially OB-Rb, in target cells (53). Importantly, the mouse kidney has been found to express more OB-Rb mRNA than other mouse tissues except for the lung (12, 54); this finding implies that leptin directly affects renal function. Jackson and Li (55) reported that short-term injection or infusion of large doses of leptin results in natriuresis and diuresis. In a kinetic study (56), leptin was actively incorporated and metabolized by renal cells. Thus, leptin is likely to act directly on the kidney to attenuate mRNA expression of 1{alpha}-hydroxylase and 24-hydroxylase in ob/ob mice. To better understand the effects of leptin administration in ob/ob mice, we determined whether exogenous leptin altered VDR expression in bone and kidney in mutant mice. No such change was demonstrated, indicating that actions of leptin upon regulation of vitamin D metabolism do not involve changes in VDR in target cells.

In this study, the serum concentration of 1,25-(OH)2D3 was elevated in ob/ob mice as a consequence of increased expression of renal 1{alpha}-hydroxylase because these mice were released from constitutive suppression of 1{alpha}-hydroxylase expression by leptin. The 1,25-(OH)2D3 hormone is the main in vivo stimulator of mRNA and protein expression of 24- hydroxylase, which catalyzes 24,25-dihydroxyvitamin D3 synthesis (31, 34, 57). Indeed, increased expression of 24- hydroxylase would be expected in kidneys of ob/ob mice in response to the high serum concentration of 1,25-(OH)2D3. In these mice, exogenous leptin markedly inhibited renal expression of both 1{alpha}-hydroxylase and 24-hydroxylase and was very effective in normalizing elevated serum concentrations of calcium and phosphate. The major biologic function of 1,25-(OH)2D3 in bone is to stimulate bone calcium and phosphate resorption when dietary calcium and phosphate are inadequate to maintain serum concentrations of these ions within the normal range (27, 28). Because serum TRAP activity, a resorption marker, increased in ob/ob mice, it was suggested that the mutant mice had stimulated bone resorption. Bone resorption as a response to elevation of serum 1,25-(OH)2D3 was at least partly responsive for lower BMD of femurs in ob/ob mice compared with lean (control) mice. Our present histologic analysis demonstrated that ob/ob mice had impaired mineralization and attenuated bone volume in the tibial epiphysis and metaphysis, which is consistent with the observation of significant reduction of femoral BMD in mutant mice. Moreover, the efficiency of intestinal calcium and phosphate absorption increased in ob/ob mice whose concentration of serum 1,25-(OH)2D3 was markedly elevated. Augmented production of 1,25-(OH)2D3 in ob/ob mouse kidney stimulates the intestinal absorption of mineral ions and the bone resorption, resulting in elevated serum concentrations of calcium and phosphate in the mutant mice.

Leptin administration to ob/ob mice alters serum concentrations of IGF-I, glucocorticoids, and GH (58). Because leptin increases production of bone anabolic factors such as GH and IGF-I and has been found to stimulate osteoblastic differentiation in an immortalized human stromal cell line (40), ob/ob mice would be likely to show retarded femoral growth, in agreement with prior observations (24). We also showed decreased BMD in ob/ob mice compared with lean controls, confirming the findings of Steppan at al. (24). Studies in vitro (25, 45) have demonstrated effects of leptin on the RANK/RANKL/OPG system, which regulates differentiation and activation of osteoclasts in bone; leptin was found to decrease mRNA expression of RANK and RANKL but increase gene expression of OPG, suggesting that leptin inhibited osteoclastic function. In contrast, our in vivo study of mouse bone demonstrated that leptin deficiency elevated mRNA expression for RANK, RANKL, and OPG. This disagreement between the present results and those of other studies (25, 45) remains to be explained and may involve differences between in vivo and in vitro systems. However, increased gene expression for RANK, RANKL, and OPG in bone of ob/ob mice may be related to augmented concentration of serum 1,25-(OH)2D3 because 1,25-(OH)2D3 increased expression of these mRNAs in osteoblasts (59).

Bone-specific ALP is an osteoblastic product that plays an important role in mineralization. Leptin was reported to increase ALP mRNA expression and stimulate mineralization in cultured osteoblasts (43). In contrast, our in vivo study demonstrated increased total ALP activity from serum of leptin-deficient mice. Circulating ALP activity is derived from several tissues including intestine, spleen, kidney, liver, and bone. The two most common sources of ALP activity in circulation are liver and bone, with total activity being strongly dependent upon the hepatic isozyme. Therefore, simple measurement of total ALP activity does not provide specific information concerning bone formation (60), and synthesis and secretion of hepatic ALP in the mutant mice might be abnormally high. Determinations of bone-specific ALP are needed in ob/ob mice.

In summary, we demonstrated that leptin-deficient (ob/ob) mice have low BMD and retarded femoral growth. We also found that serum concentrations of calcium, phosphate, and 1,25-(OH)2D3 are significantly elevated in ob/ob mice compared with lean (control) mice, although leptin replacement in ob/ob mice markedly reduced these excesses. Furthermore, we show for the first time that mRNA expression of renal 1{alpha}-hydroxylase and 24-hydroxylase are greatly increased in ob/ob mice, with the excesses again reduced by leptin administration. Moreover, leptin suppresses renal 1{alpha}-hydroxylase and 24-hydroxylase activity in ob/ob mice. These findings indicate that loss of leptin function contributes to aberrant regulation of renal 25-OHD3 metabolism. As a result, enhanced renal production of 1,25-(OH)2D3 would promote bone resorption in ob/ob mice, leading to mild elevation of serum calcium and phosphate concentrations and low BMD.


   Acknowledgments
 
We thank Dr. H. Itoh and Dr. J. Sugiura (Department of Pathology, Ohu University) for their valuable advice of histologic analysis, and Dr. H. Yamato and Dr. H. Murayama (Kureha Chemical Co., Tokyo, Japan) for bone histomorphometry.


   Footnotes
 
This work was supported in part by grants from the Japan Society for the Promotion of Science.

Abbreviations: ALP, Alkaline phosphatase; BMD, bone mineral density; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; 25-OHD3, 25-hydroxyvitamin D3; Dpd, deoxypyridinoline; OB-Rb, long form of the leptin receptor; OPG, osteoprotegerin; RANK, receptor activator of nuclear-{kappa}B; RANKL, receptor activator of nuclear-{kappa}B ligand; TRAP, tartrate-resistant acid phosphatase; VDR, vitamin D receptor.

Received August 6, 2003.

Accepted for publication November 24, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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