A short-term zinc-deficient diet decreases bone formation through down-regulated BMP2 in rat bone

Abstract

We investigated the effects of a short-term dietary zinc deficiency on bone metabolism. Zinc deficiency increased the mRNA expression of zinc uptake transporters such as Zip1, Zip13, and Zip14 in bone. However, zinc deficiency might not maintain zinc storage in bone, resulting in a decrease in bone formation through downregulation of the expression levels of osteoblastogenesis-related genes.

Key words:

• short-term zinc deficiency
• zinc transporter
• bone formation

Zinc, an essential trace element, is considered to play an important role in bone metabolism, because approximately 30% of the total body zinc content is stored in bone mass.¹⁾ In addition, it has been reported that bone loss was aggravated by dietary zinc deficiency, whereas it was prevented by zinc supplementation in ovariectomized rats, which suggested that there was a close relation between zinc and bone metabolism.^2,3) Furthermore, we previously observed that long-term (4 weeks) zinc deficiency decreases bone formation and increases bone resorption, resulting in bone loss.⁴⁾ Although several studies have proved that zinc deficiency leads to the development of osteoporosis^5,6), few studies have attempted to examine the effect of short-term dietary zinc deficiency on bone metabolism.

Zinc homeostasis is primarily maintained by regulation of its influx into and efflux out of cells. There are two families of zinc transporters in humans: the Slc39a (Zip) family with 14 members, which mediate the uptake of zinc, and the Slc30a (ZnT) family with at least 9 members, which mediate the export of zinc.⁷⁾ It was also reported that Zip1, Zip13, Zip14, ZnT5, and ZnT7 are expressed in a wide variety of tissues.⁷⁾ However, no information is available regarding the expression of zinc transporters in the bone tissue of rats fed a zinc deficient diet.

In the present study, to investigate whether a short-term dietary zinc deficiency induced an alteration in zinc transporters in bone and abnormal bone retardation, we fed control and zinc-deficient diets (30 and 1 mg Zn/kg, respectively) to male rats for 1 week.

Sixteen 4-week-old male Wistar rats (Clea Japan, Tokyo, Japan) were purchased from Clea Japan (Tokyo, Japan). After a 3-day adaptation period with a control diet, the rats were randomly divided into two groups: a control group (PF, n = 8) and a zinc deficiency group (ZD, n = 8). The rats in the ZD group were given ad libitum access to a zinc deficient diet. The rats in the PF group were pair-fed with the control diet to the mean intake of the ZD group. The experimental diets were prepared as described in a previous study.⁴⁾ After 1 week of treatment, the rats were fasted for 12 h, were then anesthetically sacrificed for dissection, and blood and femur samples were collected for analysis. These studies were approved by the Animal Studies Committee of Tokyo University of Agriculture, and all procedures involving rats were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals of Tokyo University of Agriculture. Serum zinc and calcium were measured as described previously.⁴⁾ The serum levels of osteocalcin and the C-terminal telopeptide of type I collagen (CTx) were measured as described previously.⁸⁾ Extraction of total RNA and synthesis of complementary DNA were performed as described previously.⁸⁾ For real-time PCR, the reaction mixture was prepared using the TaqMan Gene Expression Master Mix (Applied Biosystems, Foster, CA) with TaqMan gene expression assays (Applied Biosystems) for the following rat genes: Zip1, Zip13, Zip14, ZnT5, ZnT7, metallothionein (Mt)1a, Mt2A, bone morphogenetic protein 2 (BMP2), runt-related transcription factor 2 (Runx2), Osterix, alkaline phosphatase (ALP), Osteocalcin, Collagen typeⅠα 1(Col1a1), and β-actin. Respective Assay IDs were described previously.^8,9) Gene transcript levels in each sample were determined using the relative standard curve method. The level of gene transcripts is expressed as a ratio relative to β-actin mRNA, with the level in rats fed the control diet set to 1. The results are expressed as the means ± standard error of the mean (SEM). After conducting an F-test to determine the homogeneity of values, Student’s t-test was used to determine significant differences between the two groups. Significant differences were considered as a p < 0.05.

In this study, the serum zinc concentration was significantly lower in the ZD group compared with the PF group (Table 1). A study has reported that dietary zinc deficiency typically induces a decrease in serum zinc level in rodents within 5 days, which supports our results.¹⁰⁾

Table 1. Effects of short-term zinc deficiency on body weight, serum mineral levels, and markers of bone metabolism.

	PF	ZD
Body weight (g)
Initial	83.46 ± 1.14	83.37 ± 1.05
Final	110.92 ± 0.81	108.44 ± 1.29
Serum
Zinc (μM)	20.60 ± 0.67	8.57 ± 0.34*
Calcium (mM)	2.65 ± 0.05	2.63 ± 0.09
Osteocalcin (ng/ml)	137.52 ± 12.83	97.22 ± 7.81*
CTx (ng/ml)	135.22 ± 10.33	140.10 ± 7.22

Notes:

Values are expressed as means ± SEM for each group of eight rats.

* Significant differences from the PF group at p < 0.05.

Although the level of serum CTx, which is a bone resorption marker, was not significantly different between the PF and the ZD groups, the level of serum osteocalcin, which is a bone formation marker, was significantly decreased in the ZD group compared to the PF group. We previously reported that long-term (4 weeks) dietary zinc deficiency enhances bone resorption through low serum calcium level-induced secretion of PTH in rats.⁴⁾ Moreover, it has been demonstrated that long-term (6 weeks) dietary zinc deficiency decreases serum calcium level and calcium content of bone in ovariectomized rats.^3,11) In contrast, in the present study, we observed that short-term dietary zinc deficiency did not affect the serum calcium level. These data suggested that short-term dietary zinc deficiency may not induce bone resorption due to maintenance of the serum calcium level.

Zinc deficiency has been demonstrated to decrease the mRNA expression of Runx2 and ALP, resulting in a decrease in bone formation in vitro.¹²⁾ We previously demonstrated that long-term (4 weeks) dietary zinc deficiency decreases serum osteocalcin, resulting in bone loss in rats.⁴⁾ In the present study, we observed that short-term dietary zinc deficiency decreased the gene expression of osteoblastogenesis-related genes, such as BMP2, Rnux2, Osterix, ALP, Osteocalcin, and Col1a1 in bone (Table 2). BMP2 target genes are transcription factors that have essential roles in skeletal development, including the regulation of Runx2 and osterix.¹³⁾ Therefore, the results of this study demonstrate that short-term dietary zinc deficiency decreased BMP2 expression, which reduced the gene expression of Runx2 and Osterix. Furthermore, the down-regulation of the expression of these osteoblast differentiation-related genes decreased ALP, Osteocalcin, and Col1a1 gene expression in zinc-deficient rats.

Table 2. Effect of short-term dietary zinc deficiency on the gene expression levels of osteoblastogenesis-related factors and of osteoblast-specific proteins in the femur.

	PF	ZD
BMP2	1.00 ± 0.04	0.57 ± 0.03*
Runx2	1.00 ± 0.03	0.75 ± 0.02*
Osterix	1.00 ± 0.06	0.51 ± 0.02*
ALP	1.00 ± 0.04	0.52 ± 0.01*
Osteocalcin	1.00 ± 0.04	0.71 ± 0.03*
Col1a1	1.00 ± 0.05	0.59 ± 0.04*

Notes:

The mRNA expression of BMP2, Runx2, Osterix, ALP, Osteocalcin, and Col1a1 was determined using qRT-PCR. The ordinate axis indicates the relative amount of mRNA compared with rats fed control diets. Gene expression levels were normalized with β-actin. Values are expressed as means ± SEM (n = 8).

* Significant differences from the PF group at p < 0.05.

Since we suspected that a short-term zinc deficiency led to a reduction in bone formation that was induced by depletion of zinc in bone, we measured the mRNA expression of zinc uptake transporters and Mt in bone. The mRNA expression of zinc uptake transporter genes such as Zip1, Zip13, and Zip14 was significantly increased in the ZD group compared with that in the PF group (Table 3). On the other hand, the mRNA expression of the zinc export transporter gene, ZnT5, was significantly decreased in the ZD group compared with that in the PF group, although there was no significant difference between the ZnT7 expression level in the PF and ZD groups. In addition, the mRNA expression of Mt-1a and 2A, which are zinc-dependent proteins that are closely related with zinc metabolism, was significantly decreased in the ZD group compared with that in the PF group. Onosaka et al. reported that Mt concentration linearly correlated with zinc content in the liver of rats subcutaneously injected with zinc.¹⁴⁾ Therefore, these results suggested that a short-term dietary zinc deficiency might decrease the zinc content in bone. Fong et al.¹⁵⁾ recently suggested that the lack of Mt would lead to a further dysregulated pool of the low available zinc, and thus exacerbate the negative effects of zinc deficiency on bone formation. These results suggested that the mRNA expression of zinc transporters such as Zip1, Zip13, Zip14, and ZnT5 in bone quickly respond to dietary zinc deficiency, which may be aimed at maintaining zinc content in bone, although these alterations could not maintain the zinc pool in bone. In addition, these results indicated that this reduction in zinc in bone may be a factor in decreased bone formation during a short-term dietary zinc deficiency.

Table 3. Effect of short-term dietary zinc deficiency on the gene expression levels of zinc transporters and metallothioneins in the femur.

	PF	ZD
Zip1	1.00 ± 0.03	1.31 ± 0.04*
Zip13	1.00 ± 0.03	1.38 ± 0.03*
Zip14	1.00 ± 0.03	1.44 ± 0.03*
ZnT5	1.00 ± 0.04	0.72 ± 0.02*
ZnT7	1.00 ± 0.03	0.98 ± 0.02
Mt1a	1.00 ± 0.06	0.53 ± 0.02*
Mt2A	1.00 ± 0.07	0.43 ± 0.02*

Notes:

The mRNA expression of Zip1, Zip13, Zip14, ZnT5, ZnT7, Mt1a, and Mt2A was determined using qRT-PCR. The ordinate axis indicates the relative amount of mRNA compared with rats fed control diets. Gene expression levels were normalized with β-actin. Values are expressed as means ± SEM (n = 8).

* Significant differences from the PF group at p < 0.05.

Author contributions

TS designed the experiments, carried out acquisition of data, statistical analysis, and wrote the manuscript. KS conceived of the research, supported the experiment, and assisted in the design and interpretation of experiments. HM and SK were involved in designing the experiment and interpreting the results. All authors contributed to the critical revision of the manuscript for important intellectual content. All authors read and approved the final manuscript.

Acknowledgments

We also thank assistant professor Tomoko Ishijima and research fellow Tsudoi Toyoda in the University of Tokyo for their assistance with the RNA extraction from bone.

Disclosure statement

No potential conflict of interest was reported by the authors.