Tag Archives: in vitro

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 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

Zinc-Carnosine Inhibits Bone Resorption In Viro

Abstract

Inhibitory effect of beta-alanyl-L-histidinato zinc on bone resorption in tissue culture.

The inhibitory effect of beta-alanyl-L-histidinato zinc (AHZ) on bone resorption in tissue culture was investigated. Calvaria were removed from weanling rats (3-week-old male) and cultured for periods up to 48 h in Dulbecco’s modified Eagle medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained 10(-7) to 10(-4) mol/l AHZ. The bone-resorbing factors, parathyroid hormone (1-34) (PTH; 10(-7) mol/l), prostaglandin E2 (10(-5) mol/l), interleukin-1 alpha (IL1 alpha; 50 U/ml), and lipopolysaccharide (10 micrograms/ml), caused a significant decrease in bone calcium content. The decreases in bone calcium content induced by bone-resorbing factors were completely inhibited by the coexistence of AHZ (10(-6) to 10(-4) mol/l). Also, AHZ (10(-5) mol/l) completely inhibited the PTH (10(-7) mol/l) or IL1 alpha (50 U/ml)-induced increase in medium glucose consumption and lactic acid production by bone tissue. Furthermore, AHZ (10(-5) mol/l) fairly blocked both PTH (10(-7) mol/l)-increased acid phosphatase and decreased alkaline phosphatase activities of bone tissue. The inhibitory effect of AHZ (10(-5) mol/l) on PTH (10(-7) mol/l)-stimulated bone resorption was clearly prevented by the presence of 10(-4) mol/l dipicolinate, a chelator of zinc. However, zinc sulfate (10(-7) to 10(-4) mol/l) did not inhibit the PTH (10(-7) mol/l)-stimulated bone resorption in tissue culture. These findings indicate that AHZ had a direct inhibitory effect on bone resorption in vitro, and the AHZ effect was found in the chemical form of zinc-chelated dipeptide.

Yamaguchi M, Segawa Y, Shimokawa N, Tsuzuike N…
Pharmacology 1992
PMID: 1465476

Zinc Increases IGF-1 Activity in Mouse Osteoblasts In Vitro

Abstract

Role of zinc in regulation of protein tyrosine phosphatase activity in osteoblastic MC3T3-E1 cells: zinc modulation of insulin-like growth factor-I’s effect.

Zinc, an essential trace element, has been demonstrated to stimulate bone growth in animal and human. The cellular mechanism by which zinc stimulates bone growth has not been fully clarified. The effect of hormone and zinc on protein tyrosine phosphatase activity in osteoblastic MC3T3-E1 cells was investigated. Cells were cultured for 72 h in medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers, and then exchanged to culture medium containing either vehicle, zinc sulfate or various hormones in the absence of 10% FBS. After medium change, cells were cultured for 48 h. Protein tyrosine phosphatase activity in the lysate of cells was significantly increased by culture with zinc (10(-6) – 10(-4) M). The effect of zinc in increasing the enzyme activity was completely blocked by culture with cycloheximide (10(-7 )M), an inhibitor of protein synthesis, or 5, 6-dichloro-l-beta-D- riboifuranosylbenzimidarzole (DRB) (10(-6) M), an inhibitor of translational activity. Addition of calcium chloride (10 microM) into the reaction mixture caused a significant increase in protein tyrosine phosphatase activity; this increase was completely blocked in the presence of trifluoperazine (50 microM), an antagonist of calmodulin. Culture with zinc caused a significant increase in Ca2+/calmodulin-dependent protein tyrosine phosphatase activity in osteoblastic cells. Protein tyrosine phosphatase activity was significantly raised by culture with parathyroid hormone (human PTH [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33]; 10(-7) M), 17beta-estradiol (10(-7) M), insulin-like growth factor-I (IGF-I; 10(-8) M) or insulin (10(-8) M). The enzyme activity was not significantly enhanced by the addition of calcium (10 microM) into the reaction mixture. The effect of PTH or IGF-I in increasing protein tyrosine phosphatase activity was completely blocked by culture with DRB. The IGF-I-induced increase in enzyme activity was significantly enhanced by culture with zinc. Such an effect was not seen in the case of PTH. Moreover, the effect of IGF-I in increasing proliferation of osteoblastic cells was significantly enhanced by culture with zinc. The effect of PTH was not enhanced by zinc. This study demonstrates that protein tyrosine phosphatase activity in osteoblastic cells is enhanced by various bone anabolic factors, and that zinc modulates the effect of IGF-I on protein tyrosine phosphatase activity and cell proliferation.

Yamaguchi M, Fukagawa M
Calcif. Tissue Int. Jan 2005
PMID: 15477998

Zinc, but not Zinc-Carnosine, Enhances Anabolic Effect of IGF-1 in Mouse Osteoblasts In Vitro

Abstract

Zinc modulation of insulin-like growth factor’s effect in osteoblastic MC3T3-E1 cells.

Whether the anabolic effect of insulin-like growth factor-I (IGF-I) in osteoblastic MC3T3-E1 cells is modulated by zinc, an activator of bone formation, was investigated in vitro. After subculture for 3 days, the cells were cultured for 72 h with IGF-I (10(-8) M). The peptide produced a significant increase of protein concentration, deoxyribonucleic acid (DNA) content, and cell number in the cells. These increases were markedly enhanced by the presence of zinc sulfate (10(-5) M), but not zinc-chelating dipeptide (beta-alanyl-L-histidinato zinc; 10(-5) M). Also, the cellular alkaline phosphatase activity was synergistically increased by the presence of both IGF-I and zinc sulfate. Thus, effect was not seen in the presence of both insulin (10(-8) M) and zinc sulfate (10(-5) M). The effect of zinc sulfate to enhance the IGF-I-increased alkaline phosphatase activity and protein concentration in the cells was clearly prevented by the presence of cycloheximide (10(-6) M), staurosporin (10(-8) M), or okadaic acid (10(-7) M) with an effective concentration. However, staurosporin had a partial inhibiting effect on the IGF-I or the IGF-I plus zinc-induced increases in cellular protein, although okadaic acid entirely blocked the IGF-I or the IGF-I plus zinc effect. The present study demonstrates that the anabolic effect of IGF-I in osteoblastic cells is enhanced by zinc ion. The enhancement by zinc may be mediated through the signaling pathway of protein kinase C and protein phosphatase in the cells.

Matsui T, Yamaguchi M
Peptides 1995
PMID: 8532589

Zinc-Carnosine > Zinc at Enhancing Estrogen’s Anabolic Effect on Osteoblasts In Vitro

Abstract

Zinc enhancement of 17beta-estradiol’s anabolic effect in osteoblastic MC3T3-E1 cells.

The anabolic effect of 17beta-estradiol in osteoblastic MC3T3-E1 cells was investigated. The cells were cultured for 3 days in the medium containing either vehicle or 17beta-estradiol (10(-11)-10(-9) M). 17beta-Estradiol significantly increased alkaline phosphatase activity and protein concentration in the cells. The steroid (10(-9) M) also significantly elevated the cell numbers and the cellular DNA content. The anabolic effect by 17beta-estradiol was blocked by the presence of dipicolinate (10(-3) M), a chelator of zinc ion, suggesting a role of cellular zinc in osteoblastic cell function. The presence of zinc sulfate (10(-5) M) or beta-alanyl-L-histidinato zinc (AHZ) (10(-5) M) significantly enhanced the 17beta-estradiol (10(-10) or 10(-9) M)-induced increase of alkaline phosphatase activity and protein concentration in the cells; the effect of AHZ was greater than that of zinc sulfate. The enhancement by zinc compounds was not based on the augmentation of osteoblastic cell numbers. The co-addition of cycloheximide (10(-6) M), an inhibitor of protein synthesis, completely blocked the zinc compound (10(-5) M)-induced enhancement of 17beta-estradiol’s (10(-9) M) effect to increase alkaline phosphatase activity and protein concentration in the cells. Moreover, the anabolic effect of 17beta-estradiol together with or without zinc compounds was abolished by the presence of staurosporine (10(-8) M), an inhibitor of protein kinase C, or of okadaic acid (10(-7) M), an inhibitor of protein phosphatase. The present study demonstrates that the anabolic effect of 17beta-estradiol is enhanced by zinc-chelating dipeptide in osteoblastic MC3T3-E1 cells, and that the enhancing effect may involve protein synthesis and protein kinase activity.

Yamaguchi M, Matsui T
Calcif. Tissue Int. Jun 1997
PMID: 9164827

Zinc Induces Bone Formation in Rat Cells

Abstract

Zinc stimulation of bone protein synthesis in tissue culture. Activation of aminoacyl-tRNA synthetase.

The present investigation was undertaken to clarify the effect of zinc on bone protein synthesis in tissue culture. Calvaria were removed from 3-week-old male rats and cultured for periods up to 96 hr in Dulbecco’s Modified Eagle Medium (high glucose, 4500 mg/dl) supplemented with antibiotics and bovine serum albumin. The calvaria were incubated at 37 degrees in 5% CO2/95% air in the medium containing 10(-6)-10(-4) M zinc. Zinc content in bone cells was increased when the culture was treated with 10(-5) and 10(-4) M zinc for 48 hr. When calvaria cultured in the presence of 10(-4) M zinc were pulsed with [14C]uridine, the incorporation of [14C]uridine into the bone RNA was not increased significantly. In the pulse with [3H]leucine, the presence of 10(-5) to 10(-4) M zinc in the medium caused a significant increase in the incorporation of [3H]leucine into the acid-insoluble residues of bone tissue. This increase was blocked completely by treatment with 10(-7) M cycloheximide, an inhibitor of protein synthesis. When [3H]leucine was added into the reaction mixture containing the 5500 g supernatant fraction of the homogenate prepared from calvaria cultured in the presence of 10(-4) M zinc, the in vitro protein synthesis was increased about 2-fold. The activity of [3H]leucyl-tRNA synthetase in the 105,000 g supernatant fraction (cytosol) of the bone homogenate was increased about 2-fold by the culture with 10(-4) M zinc. The presence of 10(-4) M dipicolinate, a specific chelator of zinc, in the culture medium negated the effect of zinc on [3H]leucyl-tRNA synthetase activity. The addition of 10(-7) to 10(-6) M zinc into the reaction mixture containing enzyme extracts obtained from uncultured rat calvaria caused a 2-fold increase of [3H]leucyl-tRNA synthetase activity. These results clearly indicate that zinc induces the stimulation of protein synthesis at the translational level in bone cells. The present study further supports the view that zinc increases protein synthesis in bone cells and that the metal induces bone formation.

Yamaguchi M, Oishi H, Suketa Y
Biochem. Pharmacol. Nov 1988
PMID: 2461201

Korean Black Raspberry Enhances Osteoblast Function In Vitro

Abstract

Rubus coreanus Miq. extract promotes osteoblast differentiation and inhibits bone-resorbing mediators in MC3T3-E1 cells.

To prevent bone loss that occurs with increasing age, certain nutritional and pharmacological factors are needed. In the present study, the ethanol extract from the fruit of Rubus coreanus Miq. (RCE) was investigated for its effect on the function of osteoblastic MC3T3-E1 cells. RCE (10approximately50 microg/ml) caused a significant elevation in cell viability, alkaline phosphatase (ALP) activity, collagen content, and osteocalcin secretion in the cells. The effect of RCE (50 microg/ml) in increasing cell viability, ALP activity, and collagen content was prevented by the presence of 10(-6) M cycloheximide and 10(-6) M tamoxifen, suggesting that RCE’s effect results from a newly synthesized protein component and might be partly involved in estrogen action. We then examined the effect of RCE on the H(2)O(2)-induced apoptosis and production of local factors in osteoblasts. Treatment with RCE (10approximately50 microg/ml) decreased the 0.2 mM H(2)O(2)-induced apoptosis and production of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and nitric oxide (NO) in osteoblasts. Our data indicate that the enhancement of osteoblast function by Rubus coreanus Miq. may result in the prevention of osteoporosis and inflammatory bone diseases.

Lee KH, Choi EM
Am. J. Chin. Med. 2006
PMID: 16883635