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The Features of Copper Metabolism in the Rat Liver during Development.

Zatulovskaia YA, Ilyechova EY, Puchkova LV - PLoS ONE (2015)

Bottom Line: In adults, serum copper concentration increased by about a factor of 3, while metallothionein-bound copper level decreased by a factor of 2.During development, the expression level of Cp, Sod1, Cox4i1, Atp7b, Ctr1, Ctr2, Cox17, and Ccs genes was significantly increased, and metallothionein was decreased.The copper routes in newborns are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics, Institute of Physics, Nanotechnology, and Telecommunications, Peter the Great St. Petersburg Polytechnic University, St. Petersburg, Russia.

ABSTRACT
Strong interest in copper homeostasis is due to the fact that copper is simultaneously a catalytic co-factor of the vital enzymes, a participant in signaling, and a toxic agent provoking oxidative stress. In mammals, during development copper metabolism is conformed to two types. In embryonic type copper metabolism (ETCM), newborns accumulate copper to high level in the liver because its excretion via bile is blocked; and serum copper concentration is low because ceruloplasmin (the main copper-containing protein of plasma) gene expression is repressed. In the late weaning, the ETCM switches to the adult type copper metabolism (ATCM), which is manifested by the unlocking of copper excretion and the induction of ceruloplasmin gene activity. The considerable progress has been made in the understanding of the molecular basis of copper metabolic turnover in the ATCM, but many aspects of the copper homeostasis in the ETCM remain unclear. The aim of this study was to investigate the copper metabolism during transition from the ETCM (up to 12-days-old) to the ATCM in the rats. It was shown that in the liver, copper was accumulated in the nuclei during the first 5 days of life, and then it was re-located to the mitochondria. In parallel with the mitochondria, copper bulk bound with cytosolic metallothionein was increased. All compartments of the liver cells rapidly lost most of their copper on the 13th day of life. In newborns, serum copper concentration was low, and its major fraction was associated with holo-Cp, however, a small portion of copper was bound to extracellular metallothionein and a substance that was slowly eluted during gel-filtration. In adults, serum copper concentration increased by about a factor of 3, while metallothionein-bound copper level decreased by a factor of 2. During development, the expression level of Cp, Sod1, Cox4i1, Atp7b, Ctr1, Ctr2, Cox17, and Ccs genes was significantly increased, and metallothionein was decreased. Atp7a gene's activity was fully repressed. The copper routes in newborns are discussed.

No MeSH data available.


Related in: MedlinePlus

Ontogenetic changes of copper balance in the blood serum of rats.(A) Serum copper concentration and ceruloplasmin protein level increased coordinately during development. Ordinate axis: (left) copper concentration, μg/L (light bars), (right) ceruloplasmin protein concentration measured by rocket immunoelectrophoresis, mg/L (black bars), the means ± SD, n = 5. Inset: the representative protocol of immunoelectrophoresis. *Serum of adult rat was 2-fold diluted. Abscissa axis: age, days. (B) Blood serum oxidase and ferroxidase activities increased after ETCM→ATCM transition. Enzymatic activities were determined by gel-assay and expressed as a. u., the means ± SD, n = 3. Abscissa axis: age, days, *Р<0.05 in comparison with newborns. (C) In the serum of newborn and adult rats, the main copper portion was precipitated with ceruloplasmin. Ordinate axis: copper concentration, μg/L, light bars–actual serum copper concentration; black bars–copper concentration in ceruloplasmin precipitates. (D) Gel-filtration distribution of blood serum copper of P9 (red) and P60 (blue) rats. Ordinate axis: cooper concentration, μg/L; abscissa axis: gel-filtration fraction number. *—position of the maximum oxidase activity. Arrow show eluted position of cytochrome C. Inset: 12% SDS-PAGE of fractions from peak III of P9 and P60 rats. The samples were treated with SDS and 2-mercaptoethanol at 95°C, 5 min. Gel was stained with AgNO3. Right lane: molecular weight calibration markers from 3.4–100 kDa (ThermoScientific, cat. N26632, USA). (E) Metallothionein (MT) presents in the serum of newborn and adult rats. Upper: WB protocol of blood serum P9 and P60 rats with antibodies to MT (samples content 1.0 μl serum); below: relative level of MT in newborn and adult rats, the means ± SD (n = 3). (F) Serum copper and zinc are precipitated by antibodies to MT. Ordinate axis: Copper or zinc content in MT precipitates, % from atomic concentration copper or zinc in serum (n = 2).
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pone.0140797.g002: Ontogenetic changes of copper balance in the blood serum of rats.(A) Serum copper concentration and ceruloplasmin protein level increased coordinately during development. Ordinate axis: (left) copper concentration, μg/L (light bars), (right) ceruloplasmin protein concentration measured by rocket immunoelectrophoresis, mg/L (black bars), the means ± SD, n = 5. Inset: the representative protocol of immunoelectrophoresis. *Serum of adult rat was 2-fold diluted. Abscissa axis: age, days. (B) Blood serum oxidase and ferroxidase activities increased after ETCM→ATCM transition. Enzymatic activities were determined by gel-assay and expressed as a. u., the means ± SD, n = 3. Abscissa axis: age, days, *Р<0.05 in comparison with newborns. (C) In the serum of newborn and adult rats, the main copper portion was precipitated with ceruloplasmin. Ordinate axis: copper concentration, μg/L, light bars–actual serum copper concentration; black bars–copper concentration in ceruloplasmin precipitates. (D) Gel-filtration distribution of blood serum copper of P9 (red) and P60 (blue) rats. Ordinate axis: cooper concentration, μg/L; abscissa axis: gel-filtration fraction number. *—position of the maximum oxidase activity. Arrow show eluted position of cytochrome C. Inset: 12% SDS-PAGE of fractions from peak III of P9 and P60 rats. The samples were treated with SDS and 2-mercaptoethanol at 95°C, 5 min. Gel was stained with AgNO3. Right lane: molecular weight calibration markers from 3.4–100 kDa (ThermoScientific, cat. N26632, USA). (E) Metallothionein (MT) presents in the serum of newborn and adult rats. Upper: WB protocol of blood serum P9 and P60 rats with antibodies to MT (samples content 1.0 μl serum); below: relative level of MT in newborn and adult rats, the means ± SD (n = 3). (F) Serum copper and zinc are precipitated by antibodies to MT. Ordinate axis: Copper or zinc content in MT precipitates, % from atomic concentration copper or zinc in serum (n = 2).

Mentions: In P9 and P60 rats, the distribution of copper between cytosolic proteins was compared by gel-filtration. In the both samples copper was distributed as three major peaks (Fig 1C). The first copper peak is associated with high molecular weight protein fraction. In this fraction according to WB analysis serum protein Cp is present (Fig 1C, inset). Perhaps, it was blood Cp, which contaminated the cytosol during homogenization. In newborns, the copper content of this peak is less than in adults. This is consistent with the serum low Cp level at the ETCM (see Fig 2A). In the same fraction (N2 fraction) SOD activity was determined (Fig 1C, inset). The SOD-band had different mobility than SOD-bands from fractions N9-10. Perhaps, SOD3 was presented in this peak, because its native molecular weight (∼130 kDa) corresponded to position of peak I. In peak II, copper concentration was higher in adults. SOD activity was associated with it (Fig 1C, inset). The position of this peak in the chromatogram with regard to cytochrome C and Cp, as well as the presence of SOD-activity support that the copper of peak II is associated with Cu(II)/Zn(II)-SOD1. In newborns, the copper concentration in the peak III is more that in the adults. The position of this peak (approximate molecular weight 15–8 kDa) as well as co-localization of zinc with its allowed us identify peak III as MT’s. In P9 rats, but not in adults, minor copper peak IV (fractions N30-35) was present. In these fractions copper concentration was above background level approximately by a factor of 2. Electrophoresis in 15% PAG under denaturing conditions did not reveal any material to be stained with silver nitrate or Coomassie G250.


The Features of Copper Metabolism in the Rat Liver during Development.

Zatulovskaia YA, Ilyechova EY, Puchkova LV - PLoS ONE (2015)

Ontogenetic changes of copper balance in the blood serum of rats.(A) Serum copper concentration and ceruloplasmin protein level increased coordinately during development. Ordinate axis: (left) copper concentration, μg/L (light bars), (right) ceruloplasmin protein concentration measured by rocket immunoelectrophoresis, mg/L (black bars), the means ± SD, n = 5. Inset: the representative protocol of immunoelectrophoresis. *Serum of adult rat was 2-fold diluted. Abscissa axis: age, days. (B) Blood serum oxidase and ferroxidase activities increased after ETCM→ATCM transition. Enzymatic activities were determined by gel-assay and expressed as a. u., the means ± SD, n = 3. Abscissa axis: age, days, *Р<0.05 in comparison with newborns. (C) In the serum of newborn and adult rats, the main copper portion was precipitated with ceruloplasmin. Ordinate axis: copper concentration, μg/L, light bars–actual serum copper concentration; black bars–copper concentration in ceruloplasmin precipitates. (D) Gel-filtration distribution of blood serum copper of P9 (red) and P60 (blue) rats. Ordinate axis: cooper concentration, μg/L; abscissa axis: gel-filtration fraction number. *—position of the maximum oxidase activity. Arrow show eluted position of cytochrome C. Inset: 12% SDS-PAGE of fractions from peak III of P9 and P60 rats. The samples were treated with SDS and 2-mercaptoethanol at 95°C, 5 min. Gel was stained with AgNO3. Right lane: molecular weight calibration markers from 3.4–100 kDa (ThermoScientific, cat. N26632, USA). (E) Metallothionein (MT) presents in the serum of newborn and adult rats. Upper: WB protocol of blood serum P9 and P60 rats with antibodies to MT (samples content 1.0 μl serum); below: relative level of MT in newborn and adult rats, the means ± SD (n = 3). (F) Serum copper and zinc are precipitated by antibodies to MT. Ordinate axis: Copper or zinc content in MT precipitates, % from atomic concentration copper or zinc in serum (n = 2).
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Related In: Results  -  Collection

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Show All Figures
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pone.0140797.g002: Ontogenetic changes of copper balance in the blood serum of rats.(A) Serum copper concentration and ceruloplasmin protein level increased coordinately during development. Ordinate axis: (left) copper concentration, μg/L (light bars), (right) ceruloplasmin protein concentration measured by rocket immunoelectrophoresis, mg/L (black bars), the means ± SD, n = 5. Inset: the representative protocol of immunoelectrophoresis. *Serum of adult rat was 2-fold diluted. Abscissa axis: age, days. (B) Blood serum oxidase and ferroxidase activities increased after ETCM→ATCM transition. Enzymatic activities were determined by gel-assay and expressed as a. u., the means ± SD, n = 3. Abscissa axis: age, days, *Р<0.05 in comparison with newborns. (C) In the serum of newborn and adult rats, the main copper portion was precipitated with ceruloplasmin. Ordinate axis: copper concentration, μg/L, light bars–actual serum copper concentration; black bars–copper concentration in ceruloplasmin precipitates. (D) Gel-filtration distribution of blood serum copper of P9 (red) and P60 (blue) rats. Ordinate axis: cooper concentration, μg/L; abscissa axis: gel-filtration fraction number. *—position of the maximum oxidase activity. Arrow show eluted position of cytochrome C. Inset: 12% SDS-PAGE of fractions from peak III of P9 and P60 rats. The samples were treated with SDS and 2-mercaptoethanol at 95°C, 5 min. Gel was stained with AgNO3. Right lane: molecular weight calibration markers from 3.4–100 kDa (ThermoScientific, cat. N26632, USA). (E) Metallothionein (MT) presents in the serum of newborn and adult rats. Upper: WB protocol of blood serum P9 and P60 rats with antibodies to MT (samples content 1.0 μl serum); below: relative level of MT in newborn and adult rats, the means ± SD (n = 3). (F) Serum copper and zinc are precipitated by antibodies to MT. Ordinate axis: Copper or zinc content in MT precipitates, % from atomic concentration copper or zinc in serum (n = 2).
Mentions: In P9 and P60 rats, the distribution of copper between cytosolic proteins was compared by gel-filtration. In the both samples copper was distributed as three major peaks (Fig 1C). The first copper peak is associated with high molecular weight protein fraction. In this fraction according to WB analysis serum protein Cp is present (Fig 1C, inset). Perhaps, it was blood Cp, which contaminated the cytosol during homogenization. In newborns, the copper content of this peak is less than in adults. This is consistent with the serum low Cp level at the ETCM (see Fig 2A). In the same fraction (N2 fraction) SOD activity was determined (Fig 1C, inset). The SOD-band had different mobility than SOD-bands from fractions N9-10. Perhaps, SOD3 was presented in this peak, because its native molecular weight (∼130 kDa) corresponded to position of peak I. In peak II, copper concentration was higher in adults. SOD activity was associated with it (Fig 1C, inset). The position of this peak in the chromatogram with regard to cytochrome C and Cp, as well as the presence of SOD-activity support that the copper of peak II is associated with Cu(II)/Zn(II)-SOD1. In newborns, the copper concentration in the peak III is more that in the adults. The position of this peak (approximate molecular weight 15–8 kDa) as well as co-localization of zinc with its allowed us identify peak III as MT’s. In P9 rats, but not in adults, minor copper peak IV (fractions N30-35) was present. In these fractions copper concentration was above background level approximately by a factor of 2. Electrophoresis in 15% PAG under denaturing conditions did not reveal any material to be stained with silver nitrate or Coomassie G250.

Bottom Line: In adults, serum copper concentration increased by about a factor of 3, while metallothionein-bound copper level decreased by a factor of 2.During development, the expression level of Cp, Sod1, Cox4i1, Atp7b, Ctr1, Ctr2, Cox17, and Ccs genes was significantly increased, and metallothionein was decreased.The copper routes in newborns are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics, Institute of Physics, Nanotechnology, and Telecommunications, Peter the Great St. Petersburg Polytechnic University, St. Petersburg, Russia.

ABSTRACT
Strong interest in copper homeostasis is due to the fact that copper is simultaneously a catalytic co-factor of the vital enzymes, a participant in signaling, and a toxic agent provoking oxidative stress. In mammals, during development copper metabolism is conformed to two types. In embryonic type copper metabolism (ETCM), newborns accumulate copper to high level in the liver because its excretion via bile is blocked; and serum copper concentration is low because ceruloplasmin (the main copper-containing protein of plasma) gene expression is repressed. In the late weaning, the ETCM switches to the adult type copper metabolism (ATCM), which is manifested by the unlocking of copper excretion and the induction of ceruloplasmin gene activity. The considerable progress has been made in the understanding of the molecular basis of copper metabolic turnover in the ATCM, but many aspects of the copper homeostasis in the ETCM remain unclear. The aim of this study was to investigate the copper metabolism during transition from the ETCM (up to 12-days-old) to the ATCM in the rats. It was shown that in the liver, copper was accumulated in the nuclei during the first 5 days of life, and then it was re-located to the mitochondria. In parallel with the mitochondria, copper bulk bound with cytosolic metallothionein was increased. All compartments of the liver cells rapidly lost most of their copper on the 13th day of life. In newborns, serum copper concentration was low, and its major fraction was associated with holo-Cp, however, a small portion of copper was bound to extracellular metallothionein and a substance that was slowly eluted during gel-filtration. In adults, serum copper concentration increased by about a factor of 3, while metallothionein-bound copper level decreased by a factor of 2. During development, the expression level of Cp, Sod1, Cox4i1, Atp7b, Ctr1, Ctr2, Cox17, and Ccs genes was significantly increased, and metallothionein was decreased. Atp7a gene's activity was fully repressed. The copper routes in newborns are discussed.

No MeSH data available.


Related in: MedlinePlus