Category Archives: Zinc-Carnosine

Zinc Restores Protein Synthesis in Unloaded Rats

Abstract

Zinc stimulates protein synthesis in the femoral-metaphyseal tissues of normal and skeletally unloaded rats.

The effect of zinc on protein synthesis in the femoral-metaphyseal tissues of normal and skeletally unloaded rats was investigated. Skeletal unloading was designed using the model of hindlimb suspension in rats. Animals were fed for 2 or 4 days during the unloading. [3H]Leucine was added to the reaction mixture containing the 5500 g supernatant fraction of the homogenate prepared from the femoral-metaphyseal tissues. In vitro protein synthesis was significantly decreased in the bone tissues from the rats which had undergone unloading for 2 or 4 days. When the metaphyseal tissues were cultured for 24 h in the presence of zinc sulfate (10(-5) M) or beta-alanyl-L-histidinato zinc (AHZ, 10(-5) M), zinc compounds clearly stimulated protein synthesis in the metaphyseal tissues from the 4-day unloaded rats. The zinc effect was also seen in the metaphyseal tissues from normal rats. The addition of zinc sulfate (10(-5) M) or AHZ (10(-7) to 10(-5) M) into the reaction mixture containing the 5500 g supernatant fraction of metaphyseal homogenate from normal or unloaded rats produced a significant increase in protein synthesis. This increase was clearly inhibited in the presence of cycloheximide (10(-7) M). The present result demonstrates that protein synthesis is impaired in the femoral-metaphyseal tissues of rats with skeletal unloading, and that this impairment is clearly restored by zinc supplementation.

Ehara Y, Yamaguchi M
Res Exp Med (Berl) 1997
PMID: 9089885

Zinc-Carnosine Has Proliferative Effect on Mouse Osteoblasts In Vitro

Abstract

Stimulatory effect of beta-alanyl-L-histidinato zinc on cell proliferation is dependent on protein synthesis in osteoblastic MC3T3-E1 cells.

The effect of beta-alanyl-L-histidinato zinc (AHZ) on bone metabolism was investigated in osteoblastic MC3T3-E1 cells. Cells were cultured for 3 days at 37 degrees C in a 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 for appropriate periods of time. The presence of AHZ (10(-7)-10(-5) M) stimulated the proliferation of cells. AHZ (10(-6) and 10(-5) M) increased deoxyribonucleic acid (DNA) content in the cells with 48 hr-culture. This increase was completely blocked by the presence of cycloheximide (10(-6) M) or hydroxyurea (10(-3) M). Also, the presence of cycloheximide (10(-6) M) completely inhibited the AHZ (10(-5) M)-induced increase in the proliferation of cells. Meanwhile, parathyroid hormone (10(-7) M), estrogen (10(-9) M) and insulin (10(-8) M) significantly increased cellular DNA content. However, these hormonal effects clearly lowered in comparison with that of AHZ (10(-5) M). Dibutyryl cyclic AMP (10(-4) M) and zinc sulfate (10(-5) M) did not cause a significant increase in cellular DNA content. The present results support the view that AHZ has a direct specific proliferative effect on osteoblastic cells in vitro and that this effect is dependent on protein synthesis.

Hashizume M, Yamaguchi M
Mol. Cell. Biochem. May 1993
PMID: 8350864

Zinc-Carnosine > Zine at Stimulating DNS Synthesis in Mouse Osteoblast Cells

Abstract

Stimulatory effect of zinc-chelating dipeptide on deoxyribonucleic acid synthesis in osteoblastic MC3T3-E1 cells.

Whether deoxyribonucleic acid (DNA) synthesis in osteoblastic MC3T3-E1 cells is stimulated by zinc, an activator of bone formation, was investigated in vitro. After subculture for 3 days, the cells were cultured for up to 3 days (72 h) with zinc sulfate or zinc-chelated dipeptide (beta-alanyl-L-histidinato zinc; AHZ) in the range of 10(-7) to 10(-5) M. The culture with zinc compounds (10(-5) M) produced a significant increase of cell number, DNA content, and protein concentration in the cells, as reported previously. The culture with zinc compounds (10(-6) and 10(-5) M) clearly stimulated DNA synthesis in the homogenate, when it was estimated by the incorporation of [3H]deoxythymidine 5′-triphosphate into the DNA in the homogenate of cells. The AHZ effect was greater than that of zinc sulfate. The culture together with cycloheximide (19(-6) M) completely abolished the zinc compounds (10(-5) M)-induced increase of DNA synthesis in the cells, suggesting that the zinc compound effect is based on a newly synthesized protein component. Moreover, when zinc sulfate (10(-7) and 10(-6) M) or AHZ (10(-8) to 10(-5) M) was added into the reaction mixture with the homogenate of cells cultured without zinc compounds, the DNA synthesis was clearly increased. The effect of addition of zinc compounds (10(-6) M) on the DNA synthesis was completely inhibited by the presence of staurosporine (10(-8) M), an inhibitor of protein kinase C, or okadaic acid (10(-7) M), an inhibitor of protein phosphatase. The present study demonstrates that zinc compounds have a stimulatory effect on DNA synthesis in osteoblastic cells.

Yamaguchi M, Matsui T
Peptides 1996
PMID: 8959758

 

Zinc-Carnosine Prevents Deterioration of Bone in Ovariectomized Rats

Abstract

Preventive effect of beta-alanyl-L-histidinato zinc on the deterioration of bone metabolism in ovariectomized rats.

The preventive effect of beta-alanyl-L-histidinato zinc (AHZ) on the deterioration of bone metabolism was investigated in the femoral diaphysis of ovariectomized rats. AHZ (10, 30 and 100 mg/kg body weight/d) was orally administered to ovariectomized rats for 6 weeks. Ovariectomy produced a significant decrease in estradiol, calcitonin, calcium and inorganic phosphorus concentrations in the serum as compared with those from sham-operated rats. The dose of 30 and 100 mg AHZ/kg prevented any decrease in serum inorganic phosphorus concentration caused by ovariectomy. Alkaline phosphatase activity, deoxyribonucleic acid (DNA) and calcium contents in the femoral diaphysis of ovariectomized rats significantly decreased in comparison with those from sham-operated rats. These decreases were completely prevented by the dose of AHZ (10, 30 and 100 mg/kg). Electron microscopical analysis showed a rough alteration of bone matrix in the femoral diaphysis of ovariectomized rats. This alteration was clearly modified by the doses of AHZ (10, 30 and 100 mg/kg). Also, dosages of AHZ (30 and 100 mg/kg) restored the atrophy of osteoblasts and cartilage cells caused by ovariectomy. The present study suggests that oral administration of AHZ can prevent the deterioration of bone metabolism by ovariectomy. AHZ may have a therapeutic role in the treatment of osteoporosis.

Segawa Y, Tsuzuike N, Tagashira E, Yamaguchi M
Biol. Pharm. Bull. May 1993
PMID: 8364496

Zinc Inhibits Osteoclast-Like Cell In Rat Cells

Abstract

Zinc compounds inhibit osteoclast-like cell formation at the earlier stage of rat marrow culture but not osteoclast function.

The effect of zinc compounds on osteoclast-like cell formation in rat 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 hormone (1, 25-dihydroxyvitamin D3 and parathyroid hormone [1-34]). Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of 1, 25-dihydroxyvitamin D3 (10(-8) M) or parathyroid hormone (PTH; 10(-8) M) induced a remarkable increase in osteoclast-like multinucleated cells (MNC). These increases were clearly inhibited by the presence of zinc sulfate or zinc-chelating dipeptide (beta-alanyl-L-histidinato zinc; AHZ) in the concentration range of 10(-7) to 10(-5) M. The inhibitory effect was seen at the earlier stage of osteoclast-like MNC formation. However, zinc compounds (10(-6) M) did not have an effect on PTH (10(-8) M)-induced osteoclast-like cell formation in the presence of EGTA (5 x 10(-4) M), dibucaine (10(-5) M) or staurosporine (10(-9) M). Moreover, when osteoclasts isolated from rat femoral-diaphyseal tissues were cultured for 24 h in the presence of zinc compounds (10(-7) to 10(-5) M), the compounds did not have an effect on cell numbers or lysosomal enzymes activity (acid phosphatase and beta-glucuronidase) in the cells. The present study clearly demonstrates that zinc compounds inhibit osteoclast-like cell formation at the earlier stage with differentiation of marrow cells.

Yamaguchi M, Kishi S
Mol. Cell. Biochem. May 1996
PMID: 8817479

Zinc-Carnosine Stimulates Bone Formation in Arthritic Rats

Abstract

Effect of beta-alanyl-L-histidinato zinc on bone metabolism in rats with adjuvant arthritis.

The effect of a new zinc compound, beta-alanyl-L-histidinato zinc (AHZ), on osteopenia was investigated in rats with adjuvant arthritis. Arthritis was induced in female rats by administering 1% Mycobacterium butyricum (MB) into the subplantar surface of the right hind paw. AHZ (10, 30 and 100 mg/kg body weight) was orally administered to MB-treated rats 28 times at 24-h intervals, and the rats were bled 24 h after the last administration. Treatment with MB caused a remarkable increase in paw volume and a corresponding decrease in the ratio of albumin per globulin in serum, indicating that the treatment induces inflammation. These alterations were not significantly changed by the administration of AHZ (10, 30 and 100 mg/kg). Serum calcium and zinc concentrations are significantly decreased in rats with adjuvant arthritis. These decreases were completely restored by the administration of AHZ (30 and 100 mg/kg). Furthermore, the inflammation-induced decreases in alkaline phosphatase activity and calcium content in the femoral diaphysis were clearly blocked by the administration of AHZ (30 and 100 mg/kg). Also, the larger doses of AHZ (30 and 100 mg/kg) produced a significant increase in femoral-diaphyseal deoxyribonucleic acid and in the zinc content in rats with adjuvant arthritis. These results suggest that AHZ has a stimulating effect on bone formation in the femoral diaphysis of rats with adjuvant arthritis, although the compound did not have an anti-arthritic effect.

Segawa Y, Tsuzuike N, Itokazu Y, Tagashira E…
Biol. Pharm. Bull. Jul 1993
PMID: 8401397

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