Category Archives: Minerals

Zinc Inhibits Stimulatory Effect of TGF-β on Mouse Osteoclasts

Abstract

Differential effects of transforming growth factor-beta on osteoclast-like cell formation in mouse marrow culture: relation to the effect of zinc-chelating dipeptides.

The effect of transforming growth factor-beta (TGF-beta) on osteoclast-like cell formation in mouse marrow culture in vitro was investigated. The bone marrow cells were cultured for 7 days in alpha-minimal essential medium containing a well-known bone resorbing agent. Osteoclast-like cell formation was estimated with staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of TGF-beta (10(-13)-10(-11) M) caused a significant increase in the number of osteoclast-like multinucleated cells (MNCs); the maximum effect was seen with 10(-12) MTGF-beta. With a higher concentration (10(-10) M) of TGF-beta, the growth factor dramatically inhibited the 1,25-dihydroxyvitamin D5 [1,25(OH)2D3; 10(-8) M]-induced formation of osteoclast-like MNCs. This inhibitory effect was also seen in the formation of osteoclast-like MNCs stimulated by parathyroid hormone (10(-8) M), prostaglandine E2 (10(-6) M), and interleukin-1 alpha (50 U/ml). The stimulatory effect of TGF-beta (10(-12) M) on osteoclast-like MNCs formation was inhibited by zinc sulfate (10(-6) M) or zinc-chelating dipeptide [beta-alanyl-L-histidinato zinc (AHZ), 10(-6) M]. The stimulating effect of TGF-beta was markedly weakened by the presence of EGTA (0.5 mM), a chelator of Ca2+. The inhibitory effect of zinc compounds was not seen in the presence of EGTA. Moreover, the inhibitory effect of TGF-beta (10(-10) M), zinc sulfate (10(-6) M), or AHZ (10(-6) M) on osteoclast-like MNCs formation was not demonstrated in mature osteoclastic cells, although calcitonin (3 x 10(-8) M) significantly inhibited the osteoclastic formation. The present study demonstrates that TGF-beta has a stimulating and an inhibiting effect on osteoclast-like cell formation in mouse marrow culture, and that zinc can inhibit the stimulatory effect of TGF-beta.

Yamaguchi M, Kishi S
Peptides 1995
PMID: 8745062

Zinc Restores Bone After Unloading in Rats

Abstract

Zinc decrease and bone metabolism in the femoral-metaphyseal tissues of rats with skeletal unloading.

Whether the decrease of zinc content in the femoral-metaphyseal tissues of rats with skeletal unloading is involved in the alteration of bone metabolism was investigated. Skeletal unloading was designed using the model of hindlimb suspension in rats. Animals were fed for 4 days with the unloading. The metaphyseal zinc content were significantly decreased by the unloading. Zinc accumulation in the metaphyseal tissues by a single oral administration of zinc sulfate (20 mg Zn/100 g body weight) was partially depressed by the unloading, although serum zinc concentration was higher than that in normal rats, suggesting an impaired movement of zinc from serum into bone tissues by the unloading. Skeletal unloading caused a significant decrease of alkaline phosphatase activity and deoxyribonucleic acid (DNA) content in the metaphyseal tissues. These decreases were completely restored by addition of zinc sulfate (10(-4) M) or beta-alanyl-L-histidinato zinc (AHZ; 10(-5) M) in a culture medium with the metaphyseal tissues in vitro. The effects of zinc compounds were abolished by the presence of cycloheximide (10(-8) M), suggesting that the zinc effect is based on a newly synthesized protein. Dipicolinate (10(-4) and 10(-5) M), a potent zinc-chelating agent, caused an appreciable decrease of zinc content and alkaline phosphatase activity in the metaphyseal tissues. This decrease was restored by zinc supplement. The present results suggest that the skeletal unloading-induced decrease of zinc content in the femoral-metaphyseal tissues plays a role in the deterioration of bone metabolism in the unloaded rats.

Yamaguchi M, Ehara Y
Calcif. Tissue Int. Sep 1995
PMID: 8574940

Review: Zinc-Carnosine and Bone Resorption

Abstract

beta-Alanyl-L-histidinato zinc and bone resorption.

1. beta-Alanyl-L-histidinato zinc (AHZ), in which zinc is chelated to beta-alanyl-L-histidine, is a new zinc compound. beta-Alanyl-L-histidine can uniquely chelated zinc ion in various essential trace metals. More recently, it has been demonstrated that this compound has more intensive effect than zinc sulfate on bone metabolism, suggesting a role as pharmacological tool in osteoporosis. This review describes mainly the action of AHZ on bone resorption as summarized in the following.
2. The prolonged oral administration of AHZ (10-100 mg/kg/day) can completely prevent bone loss in the femur of ovariectomized rats, indicating the preventive effect of AHZ on bone resorption in vivo.
3. The decrease in bone calcium content induced by various bone resorbing factors was completely inhibited by the presence of AHZ (10(-6)-10(-4) M) in bone tissue culture system in vitro.
4. Many bone resorbing agents can stimulate the formation (differentiation) of osteoclasts from marrow cells. AHZ (10(-6)-10(-4) M) clearly inhibited osteoclast-like cell formation in mouse marrow culture in vitro.
5. AHZ may act on the process of parathyroid hormone-induced protein kinase C activation which is involved in Ca(2+)-signaling in osteoclastic cells.

Yamaguchi M
Gen. Pharmacol. Oct 1995
PMID: 7590105

Zinc Inhibits Osteoclast-Like Cell Formation in Mouse Cells

Abstract

Inhibitory effect of zinc-chelating dipeptide on parathyroid hormone-stimulated osteoclast-like cell formation in mouse marrow cultures: involvement of calcium signaling.

A possible mechanism of zinc action inhibiting the PTH-induced osteoclast-like cell formation in mouse marrow culture system in vitro was investigated. Bone marrow cells were cultured for 7 days in alpha-minimal essential medium containing a well-known bone-resorbing agent parathyroid hormone (1-34) (PTH). Osteoclast-like cell formation was estimated with staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The effect of zinc sulfate (10(-6) M) or beta-alanyl-L-histidinato zinc (AHZ; 10(-6) M) inhibiting the PTH (10(-8) M)-induced osteoclast-like cell formation was clearly seen in the absence or presence of theophylline (10(-4) M). However, zinc compounds did not inhibit the stimulatory effect of dibutyryladenosine 3′,5′-cyclic monophosphate (DBcAMP; 10(-4) M) on osteoclast-like cell formation. The stimulating effect of PTH (10(-8) M) on osteoclast-like cell formation was clearly weakened (about 50%) in the presence of EGTA (1.0 mM) or dibucaine (10(-5) M). Phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator (10(-8) to 10(-6) M), clearly stimulated osteoclast-like cell formation. PMA effect was inhibited by the presence of AHZ (10(-6) M) or zinc sulfate (10(-8) M). However, the inhibitory effect of zinc compounds was not seen in the presence of both PTH (10(-8) M) and PMA (10(-6) M). The present findings suggest that zinc compounds inhibit PTH-stimulated osteoclast-like cell formation mediated through the Ca(2+)-dependent activation of protein kinase C.

Yamaguchi M, Kishi S
Peptides 1995
PMID: 7479295

Zinc-Carnosine > Zinc at Stimulating Bone Growth in Rats

Abstract

A new zinc compound, beta-alanyl-L-histidinato zinc, stimulates bone growth in weanling rats.

The effect of a new zinc compound. beta-alanyl-L-histidinato zinc (AHZ), on bone metabolism in weanling rats was investigated. Rats were orally administered AHZ (0.5-2.5 mg/100 g body weight) for 3 days, and 24h later they were killed. Administration of AHZ (1.0 and 2.5 mg/100 g) caused a significant increase of zinc content in the femoral diaphysis and a corresponding elevation of calcium content, alkaline phosphatase activity, and DNA content. A dose of 0.5 mg/100g AHZ did not produce an appreciable increase in bone components. When zinc sulfate (0.55 mg Zn/100g) was orally administered in rats for 3 days, the bone zinc content, calcium content, and alkaline phosphatase activity were raised significantly, but bone DNA content was not appreciably affected. Thus, the stimulation of AHZ (2.5 mg/100g), which corresponds to 0.55 mg Zn/100g, on bone metabolism was more intensive than that of zinc sulfate. These results suggest that AHZ can stimulate bone growth in weanling rats, and that the compound has a greater effect in comparison with zinc sulfate.

Yamaguchi M, Ozaki K
Res Exp Med (Berl) 1990
PMID: 2349394

Zinc-Carnosine > Zinc at Stimulating Osteoblasts In Vitro

Abstract

Effect of zinc-chelating dipeptides on osteoblastic MC3T3-E1 cells: activation of aminoacyl-tRNA synthetase.

The effect of zinc-chelating dipeptides on osteoblastic MC3T3-E1 cells was investigated. As zinc compounds, we used zinc sulfate, AHZ, di(N-acetyl-beta-alanyl-L-histidinato)zinc (AAHZ), and di(histidino)zinc (HZ). Cells were cultured for 72 h in the presence of zinc compounds (10(-8)-10(-5) M). The effect of AHZ (10(-7) and 10(-6) M) to increase protein and deoxyribonucleic acid (DNA) contents in the cells was the greatest in comparison with those of other zinc compounds. Zinc sulfate and HZ at 10(-7) M did not have an effect on the cellular protein content. AHZ (10(-6) M) had a potent effect on cell proliferation, although zinc sulfate (10(-6) M) had no effect. beta-Alanyl-L-histidine (10(-6) and 10(-5) M) did not have an appreciable effect on the cells. Those effects of AHZ (10(-6) M) on osteoblastic cells were completely abolished by the presence of cycloheximide (10(-6) M). AHZ (10(-8)-10(-5) M) directly activated [3H]leucyl-tRNA synthetase in the cell homogenate, whereas the effect of zinc sulfate was seen at 10(-6) and 10(-5) M. The present study suggests that the chemical form of zinc-chelating beta-alanyl-L-histidine (AHZ) can reveal a potent anabolic effect on osteoblastic cells, and that AHZ directly stimulates protein synthesis.

Yamaguchi M, Kishi S, Hashizume M
Peptides 1994
PMID: 7700838

Zinc-Carnosine Increases Proteins Involved in Bone Formation In Vitro

Abstract

Effect of beta-alanyl-L-histidinato zinc on protein components in osteoblastic MC3T3-El cells: increase in osteocalcin, insulin-like growth factor-I and transforming growth factor-beta.

The effect of beta-alanyl-L-histidinato zinc (AHZ) on protein components in osteoblastic MC3T3-E1 cells was investigated. Cells were cultured for 3 days at 37 degrees C in CO2 incubator in plastic dishes containing alpha-modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus various concentrations of AHZ or other reagents, and the cells were cultured further 3 or 6 days. The homgenate of cells was analyzed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The presence of AHZ (10(-7) to 10(-5) M) caused an appreciable increase of many protein components in cells. Especially, the 67 killo-dalton (kDa) and 44 kDa proteins which are the major components from control cells were clearly increased by the presence of AHZ. Furthermore, the concentrations of osteocalcin, insulin-like growth factor-I and transforming growth factor-beta in the culture medium secreted from osteoblastic cells were markedly increased by the presence of AHZ (10(-6) and 10(-5) M). The effect of AHZ was a greater than that of zinc sulfate (10(-6) and 10(-5) M). The present findings suggest that AHZ can increase many proteins which are involved in the stimulation of bone formation and cell proliferation in osteoblastic cells.

Yamaguchi M, Hashizume M
Mol. Cell. Biochem. Jul 1994
PMID: 7845370

Zinc-Carnosine Resorption Inhibition is Not Through Osteoblasts in Mouse Cells

Abstract

Effect of parathyroid hormone and interleukin-1 alpha in osteoblastic MC3T3-E1 cells: interaction with beta-alanyl-L-histidinato zinc.

beta-Alanyl-L-histidinato zinc (AHZ), which is an activator of bone formation, has an inhibitory effect of bone resorption. Whether AHZ can inhibit the effect of parathyroid hormone (PTH) or interleukin-1 alpha (IL-1 alpha), which is a bone resorbing factor, on osteoblastic MC3T3-E1 cells was investigated. After subculture for 3 days, the cells were cultured for 48 h with peptides. Parathyroid hormone (10(-9)-10(-7) M) or IL-1 alpha (50 U/ml) caused a significant decrease in the cellular alkaline phosphatase activity and a remarkable increase of prostaglandin E2 (PGE2) production in the cells. Parathyroid hormone (10(-7) M) or IL-1 alpha (50 U/ml) did not have an appreciable effect on the protein content of the cells. beta-Alanyl-L-histidinato zinc (10(-5) M) significantly increased the cellular alkaline phosphatase activity and protein content, whereas it had no effect on PGE2 production. This increasing effect of AHZ was also seen in the presence of PTH (10(-7) M) or IL-1 alpha (50 U/ml), although the effect of PTH and IL-1 alpha to stimulate PGE2 production was not modulated by AHZ treatment. The present finding suggests that the inhibitory effect of AHZ on bone resorption is not through osteoblasts.

Yamaguchi M, Hashizume M
Peptides 1994
PMID: 7937338

Zinc-Carnosine Prevents Effects of Aluminium on Bone in Rats

Abstract

Beta-alanyl-L-histidinato zinc prevents the toxic effect of aluminium on bone metabolism in weanling rats.

The preventive effect of beta-alanyl-L-histidinato zinc (AHZ) on the toxic action of aluminium on bone metabolism was investigated in the femoral diaphysis of weanling rats. Aluminium chloride (5.0, 10.0 and 20.0 mumol A1/100 g body weight) was orally administered for 3 days. The dose of 10.0 and 20.0 mumol A1/100 g caused a significant increase in serum calcium concentration and bone acid phosphatase activity, while bone alkaline phosphatase activity and calcium content were not altered significantly. Moreover, the bone DNA content was significantly decreased by the doses of 10.0 and 20.0 mumol A1/100 g. Meanwhile, the increase in serum calcium concentration caused by the administration of aluminium (20 mumol/100 g) was completely prevented by the simultaneous administration of AHZ (1.0 and 2.5 mg/100 g) for 3 days, although AHZ alone did not have any effect. Also, the effects of aluminium (20.0 mumol/100 g) to increase bone acid phosphatase activity and to decrease the bone DNA content were completely blocked by the simultaneous administration of AHZ (1.0 and 2.5 mg/100 g). AHZ (1.0 and 2.5 mg/100 g) alone had the effect to increase bone DNA content but not bone acid phosphatase activity. The present study indicates that AHZ can prevent the revelation of the toxic effect of aluminium on bone metabolism in rats.

Yamaguchi M, Ozaki K
Pharmacology 1990
PMID: 2096394

Zinc-Carnosine Stimulates Bone In Vitro

Abstract

Stimulatory effect of beta-alanyl-L-histidinato zinc on bone formation in tissue culture.

The present investigation was undertaken to clarify the in vitro effect of beta-alanyl-L-histidinato zinc (AHZ) on bone metabolism in tissue culture. Calvaria were removed from weanling rats (3-week-old male) and cultured for periods up to 96 h in Dulbecco’s modified Eagle medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained 10(-8) to 10(-4) mol/l AHZ. The bone cellular zinc content was significantly increased in cultures with concentrations of AHZ greater than 10(-6) mol/l. With 10(-5) mol/l zinc sulfate, the bone cellular zinc content was significantly elevated. Bone calcium content was significantly increased by the presence of 10(-7) to 10(-4) mol/l AHZ. This increase was blocked by the presence of 10(-7) mol/l cycloheximide. Bone alkaline phosphatase activity was elevated in the presence of AHZ (10(-7) to 10(-4) mol/l), whereas it did not significantly alter acid phosphatase activity Bone collagen and DNA contents were significantly increased by 10(-7) to 10(-5) mol/l AHZ, while they were not significantly elevated by zinc sulfate (10(-7) and 10(-6) mol/l). The AHZ (10(-5) mol/l)-induced increase in bone alkaline phosphatase activity and DNA content were prevented by 10(-4) mol/l dipicolinate, a chelator of zinc. Furthermore, the AHZ (10(-5) mol/l)-induced increase in bone alkaline phosphatase activity, collagen and DNA contents were blocked by 10(-7) mol/l cycloheximide. These findings indicate that AHZ had a direct stimulatory effect on bone mineralization in vitro, and that bone protein synthesis was a necessary component of this response. The AHZ effect was more intensive than that of zinc sulfate.

Yamaguchi M, Miwa H
Pharmacology 1991
PMID: 1852783