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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

Hepatic copper concentration and copper distribution in the liver cells of newborn rats.(А) Copper is accumulated in the liver from embryonic stage development to 12thday of life. Ordinate axis: hepatic copper concentrations, μg/g wet weight (the means ± SD, n = 5); abscissa axis: age, days. (B) During accumulation copper is redistributed between subcellular compartments. Each dot represents the average from 3 experiments, in which the differences were not more than 10%. For each point, depending on the age of rats, 2–10 livers were combined to isolate subcellular fractions (nuclei–circle, mitochondria–rhomb, (endoplasmic reticulum + Golgi complex)–triangle, cytosol–rectangle). Ordinate axis: copper concentrations, μg/mg total protein; abscissa axis: age, days. (C) Gel-filtration distribution of copper in cytosol of the newborn (P9, red) and adult (P60, blue) rats. Cytosolic fraction was isolated from about 1 g liver tissue (a mixture of ∼350 mg of liver tissue from three rats) as described in Methods. Ordinate axis (left): copper (closed dots) or zinc (open dots) concentration, μg/L, Arrow shows eluted position of cytochrome C. Ordinate axis (right): optical density–D280 (dotted line) and D254 (dots). Abscissa axis: fraction number. Inset: WB with antibodies to ceruloplasmin (8% SDS-PAGE) and SOD activity (gel-test, 8% PAGE); the samples content 10 μl of the major copper fractions from peaks I, II, III, and IV of P9 cytosol.
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pone.0140797.g001: Hepatic copper concentration and copper distribution in the liver cells of newborn rats.(А) Copper is accumulated in the liver from embryonic stage development to 12thday of life. Ordinate axis: hepatic copper concentrations, μg/g wet weight (the means ± SD, n = 5); abscissa axis: age, days. (B) During accumulation copper is redistributed between subcellular compartments. Each dot represents the average from 3 experiments, in which the differences were not more than 10%. For each point, depending on the age of rats, 2–10 livers were combined to isolate subcellular fractions (nuclei–circle, mitochondria–rhomb, (endoplasmic reticulum + Golgi complex)–triangle, cytosol–rectangle). Ordinate axis: copper concentrations, μg/mg total protein; abscissa axis: age, days. (C) Gel-filtration distribution of copper in cytosol of the newborn (P9, red) and adult (P60, blue) rats. Cytosolic fraction was isolated from about 1 g liver tissue (a mixture of ∼350 mg of liver tissue from three rats) as described in Methods. Ordinate axis (left): copper (closed dots) or zinc (open dots) concentration, μg/L, Arrow shows eluted position of cytochrome C. Ordinate axis (right): optical density–D280 (dotted line) and D254 (dots). Abscissa axis: fraction number. Inset: WB with antibodies to ceruloplasmin (8% SDS-PAGE) and SOD activity (gel-test, 8% PAGE); the samples content 10 μl of the major copper fractions from peaks I, II, III, and IV of P9 cytosol.

Mentions: To test the ontogenetic-required changes of copper metabolism, copper concentration was determined in the rat liver tissue and subcellular organelles during the first 15 days of life and compared with adults. Results are summarized in Fig 1. They demonstrate that a progressive copper accumulation began at the embryonic stage of development (Fig 1A) and lasted until the 12th day of postnatal life (P12). Then copper concentration sharply dropped to the level that is normal for the adult rats. These results are entirely consistent with earlier data [14, 15], which formed the basis for a common concept that the rats to 12th day of life retain the ETCM, which is characterized by the accumulation of copper in liver. After the 13th day of life, the ETCM is switched to the ATCM, and copper is not accumulated in the liver anymore but it is excreted through the bile or included to Cp that was secreted to bloodstream. However an intracellular place copper accumulation is not defined precisely in earlier works. To obtain such data copper concentration in subcellular fractions from 1st to 20th days was measured (Fig 1B). It was shown that during the ETCM copper was re-distributed between the compartments of the hepatic cells. So, after birth copper was accumulated in the nuclei, but after 5th day of life copper concentration in the nuclei decreased. Simultaneously, the specific copper content increased in mitochondria, thereby reaching the peak value in P12 rats. As total mitochondrial fraction isolated by differential centrifugation comprises lysosomes and peroxisomes it did not confidently indicate the exact site of copper accumulation. So the mitochondrial fraction was additionally fractionated by equilibrium ultracentrifugation in a stepwise density gradient of sucrose. Isolated fractions were identified as described in Methods and recognized as mitochondria, lysosomes and peroxisomes, and copper concentration was separately measured in them. It was determined that the purified fraction of mitochondria contained about 90% of copper from the crude mitochondrial fraction. So, our study provides a strong evidence to consider mitochondria as a copper deposit organelle during ETCM. Furthermore, copper concentration increased in cytosolic and intracellular membrane fractions in P5 to P12 rats (Fig 1B). The shape of curves for the changes of copper content in these fractions practically coincides with the mitochondrial curve, but copper content in these organelles was strongly lower. After the P12, copper concentration decreased in all cell compartments (Fig 1B).


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

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

Hepatic copper concentration and copper distribution in the liver cells of newborn rats.(А) Copper is accumulated in the liver from embryonic stage development to 12thday of life. Ordinate axis: hepatic copper concentrations, μg/g wet weight (the means ± SD, n = 5); abscissa axis: age, days. (B) During accumulation copper is redistributed between subcellular compartments. Each dot represents the average from 3 experiments, in which the differences were not more than 10%. For each point, depending on the age of rats, 2–10 livers were combined to isolate subcellular fractions (nuclei–circle, mitochondria–rhomb, (endoplasmic reticulum + Golgi complex)–triangle, cytosol–rectangle). Ordinate axis: copper concentrations, μg/mg total protein; abscissa axis: age, days. (C) Gel-filtration distribution of copper in cytosol of the newborn (P9, red) and adult (P60, blue) rats. Cytosolic fraction was isolated from about 1 g liver tissue (a mixture of ∼350 mg of liver tissue from three rats) as described in Methods. Ordinate axis (left): copper (closed dots) or zinc (open dots) concentration, μg/L, Arrow shows eluted position of cytochrome C. Ordinate axis (right): optical density–D280 (dotted line) and D254 (dots). Abscissa axis: fraction number. Inset: WB with antibodies to ceruloplasmin (8% SDS-PAGE) and SOD activity (gel-test, 8% PAGE); the samples content 10 μl of the major copper fractions from peaks I, II, III, and IV of P9 cytosol.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608700&req=5

pone.0140797.g001: Hepatic copper concentration and copper distribution in the liver cells of newborn rats.(А) Copper is accumulated in the liver from embryonic stage development to 12thday of life. Ordinate axis: hepatic copper concentrations, μg/g wet weight (the means ± SD, n = 5); abscissa axis: age, days. (B) During accumulation copper is redistributed between subcellular compartments. Each dot represents the average from 3 experiments, in which the differences were not more than 10%. For each point, depending on the age of rats, 2–10 livers were combined to isolate subcellular fractions (nuclei–circle, mitochondria–rhomb, (endoplasmic reticulum + Golgi complex)–triangle, cytosol–rectangle). Ordinate axis: copper concentrations, μg/mg total protein; abscissa axis: age, days. (C) Gel-filtration distribution of copper in cytosol of the newborn (P9, red) and adult (P60, blue) rats. Cytosolic fraction was isolated from about 1 g liver tissue (a mixture of ∼350 mg of liver tissue from three rats) as described in Methods. Ordinate axis (left): copper (closed dots) or zinc (open dots) concentration, μg/L, Arrow shows eluted position of cytochrome C. Ordinate axis (right): optical density–D280 (dotted line) and D254 (dots). Abscissa axis: fraction number. Inset: WB with antibodies to ceruloplasmin (8% SDS-PAGE) and SOD activity (gel-test, 8% PAGE); the samples content 10 μl of the major copper fractions from peaks I, II, III, and IV of P9 cytosol.
Mentions: To test the ontogenetic-required changes of copper metabolism, copper concentration was determined in the rat liver tissue and subcellular organelles during the first 15 days of life and compared with adults. Results are summarized in Fig 1. They demonstrate that a progressive copper accumulation began at the embryonic stage of development (Fig 1A) and lasted until the 12th day of postnatal life (P12). Then copper concentration sharply dropped to the level that is normal for the adult rats. These results are entirely consistent with earlier data [14, 15], which formed the basis for a common concept that the rats to 12th day of life retain the ETCM, which is characterized by the accumulation of copper in liver. After the 13th day of life, the ETCM is switched to the ATCM, and copper is not accumulated in the liver anymore but it is excreted through the bile or included to Cp that was secreted to bloodstream. However an intracellular place copper accumulation is not defined precisely in earlier works. To obtain such data copper concentration in subcellular fractions from 1st to 20th days was measured (Fig 1B). It was shown that during the ETCM copper was re-distributed between the compartments of the hepatic cells. So, after birth copper was accumulated in the nuclei, but after 5th day of life copper concentration in the nuclei decreased. Simultaneously, the specific copper content increased in mitochondria, thereby reaching the peak value in P12 rats. As total mitochondrial fraction isolated by differential centrifugation comprises lysosomes and peroxisomes it did not confidently indicate the exact site of copper accumulation. So the mitochondrial fraction was additionally fractionated by equilibrium ultracentrifugation in a stepwise density gradient of sucrose. Isolated fractions were identified as described in Methods and recognized as mitochondria, lysosomes and peroxisomes, and copper concentration was separately measured in them. It was determined that the purified fraction of mitochondria contained about 90% of copper from the crude mitochondrial fraction. So, our study provides a strong evidence to consider mitochondria as a copper deposit organelle during ETCM. Furthermore, copper concentration increased in cytosolic and intracellular membrane fractions in P5 to P12 rats (Fig 1B). The shape of curves for the changes of copper content in these fractions practically coincides with the mitochondrial curve, but copper content in these organelles was strongly lower. After the P12, copper concentration decreased in all cell compartments (Fig 1B).

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