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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 expression of the genes associated with copper metabolism during ETCM→ATCM transition.(A) RT-PCR analysis of the relative levels of mRNAs. Ordinate axis: the data expressed as a. u., the means ± SD (n = 4); *—P<0.05, **—P<0.01, ***—P<0.005. Light bars–P3, grey bars–P12, black bars–P60. (B) Western blot analysis of the relative content of proteins associated with copper metabolism. Upper: examples of WB and SOD activity protocols. The molecular weight of WB identified proteins corresponds to: Cp ∼130 kDa (8% SDS-PAGE), SOD1 ∼17 kDa (12% SDS-PAGE), COX4 ∼20 kDa (12% SDS-PAGE), MT ∼8 kDa (15% SDS-PAGE), COMMD1 ∼23 kDa (12% SDS-PAGE); SOD activity was identified by gel-assay (blue gel) as described in Methods. Below: densitometric quantification of WB and SOD1 activity data. Abscissa axis: relative protein content, a. u., the means ± SD (n = 4). Light bars–P9, black bars–P60 rats.
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pone.0140797.g003: Hepatic expression of the genes associated with copper metabolism during ETCM→ATCM transition.(A) RT-PCR analysis of the relative levels of mRNAs. Ordinate axis: the data expressed as a. u., the means ± SD (n = 4); *—P<0.05, **—P<0.01, ***—P<0.005. Light bars–P3, grey bars–P12, black bars–P60. (B) Western blot analysis of the relative content of proteins associated with copper metabolism. Upper: examples of WB and SOD activity protocols. The molecular weight of WB identified proteins corresponds to: Cp ∼130 kDa (8% SDS-PAGE), SOD1 ∼17 kDa (12% SDS-PAGE), COX4 ∼20 kDa (12% SDS-PAGE), MT ∼8 kDa (15% SDS-PAGE), COMMD1 ∼23 kDa (12% SDS-PAGE); SOD activity was identified by gel-assay (blue gel) as described in Methods. Below: densitometric quantification of WB and SOD1 activity data. Abscissa axis: relative protein content, a. u., the means ± SD (n = 4). Light bars–P9, black bars–P60 rats.

Mentions: We assessed the activity of listed above genes in the liver of the P3 (copper accumulates in nuclei), P12 (copper accumulates in mitochondria), and P60 (adult) rats. Processed results of the semi quantity RT-PCR analysis are presented Fig 3A. They show that Cp gene activity increased after ETCM→ATCM transition. There was a positive linear correlation between Cp-mRNA level, copper concentration and serum Cp protein contents as well as with its enzymatic activities (Figs 3A, 3B, 2A and 2B). In adult rats, the splice-isoform encoding GPI-Cp appeared. It is known that GPI-Cp mRNA did not form in the HepG2 liver cell culture [46] and, perhaps, GPI-Cp was produced by non-hepatocyte cells (e.g., Kupffer cells), whose amount increased during development. Adult rats had significantly higher COX4i1-mRNA level and COX4i1 protein concentration than the newborns (Fig 3A and 3B). The relative level of SOD1-mRNA, its protein concentration, and enzymatic activity significantly increased after ETCM→ATCM switching (Fig 3A and 3B). Also Cox4i1 and Sod1 gene activities were significantly increased between the 3rd and 12th days of life.


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

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

Hepatic expression of the genes associated with copper metabolism during ETCM→ATCM transition.(A) RT-PCR analysis of the relative levels of mRNAs. Ordinate axis: the data expressed as a. u., the means ± SD (n = 4); *—P<0.05, **—P<0.01, ***—P<0.005. Light bars–P3, grey bars–P12, black bars–P60. (B) Western blot analysis of the relative content of proteins associated with copper metabolism. Upper: examples of WB and SOD activity protocols. The molecular weight of WB identified proteins corresponds to: Cp ∼130 kDa (8% SDS-PAGE), SOD1 ∼17 kDa (12% SDS-PAGE), COX4 ∼20 kDa (12% SDS-PAGE), MT ∼8 kDa (15% SDS-PAGE), COMMD1 ∼23 kDa (12% SDS-PAGE); SOD activity was identified by gel-assay (blue gel) as described in Methods. Below: densitometric quantification of WB and SOD1 activity data. Abscissa axis: relative protein content, a. u., the means ± SD (n = 4). Light bars–P9, black bars–P60 rats.
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pone.0140797.g003: Hepatic expression of the genes associated with copper metabolism during ETCM→ATCM transition.(A) RT-PCR analysis of the relative levels of mRNAs. Ordinate axis: the data expressed as a. u., the means ± SD (n = 4); *—P<0.05, **—P<0.01, ***—P<0.005. Light bars–P3, grey bars–P12, black bars–P60. (B) Western blot analysis of the relative content of proteins associated with copper metabolism. Upper: examples of WB and SOD activity protocols. The molecular weight of WB identified proteins corresponds to: Cp ∼130 kDa (8% SDS-PAGE), SOD1 ∼17 kDa (12% SDS-PAGE), COX4 ∼20 kDa (12% SDS-PAGE), MT ∼8 kDa (15% SDS-PAGE), COMMD1 ∼23 kDa (12% SDS-PAGE); SOD activity was identified by gel-assay (blue gel) as described in Methods. Below: densitometric quantification of WB and SOD1 activity data. Abscissa axis: relative protein content, a. u., the means ± SD (n = 4). Light bars–P9, black bars–P60 rats.
Mentions: We assessed the activity of listed above genes in the liver of the P3 (copper accumulates in nuclei), P12 (copper accumulates in mitochondria), and P60 (adult) rats. Processed results of the semi quantity RT-PCR analysis are presented Fig 3A. They show that Cp gene activity increased after ETCM→ATCM transition. There was a positive linear correlation between Cp-mRNA level, copper concentration and serum Cp protein contents as well as with its enzymatic activities (Figs 3A, 3B, 2A and 2B). In adult rats, the splice-isoform encoding GPI-Cp appeared. It is known that GPI-Cp mRNA did not form in the HepG2 liver cell culture [46] and, perhaps, GPI-Cp was produced by non-hepatocyte cells (e.g., Kupffer cells), whose amount increased during development. Adult rats had significantly higher COX4i1-mRNA level and COX4i1 protein concentration than the newborns (Fig 3A and 3B). The relative level of SOD1-mRNA, its protein concentration, and enzymatic activity significantly increased after ETCM→ATCM switching (Fig 3A and 3B). Also Cox4i1 and Sod1 gene activities were significantly increased between the 3rd and 12th days of life.

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