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Milk lacking α-casein leads to permanent reduction in body size in mice.

Kolb AF, Huber RC, Lillico SG, Carlisle A, Robinson CJ, Neil C, Petrie L, Sorensen DB, Olsson IA, Whitelaw CB - PLoS ONE (2011)

Bottom Line: In contrast, secretion of albumin, which is not synthesized in the mammary epithelium, into milk is not reduced.The absence of α-casein also significantly inhibits transcription of the other casein genes. α-Casein deficiency severely delays pup growth during lactation and results in a life-long body size reduction compared to control animals, but has only transient effects on physical and behavioural development of the pups.The results also confirm lactation as a critical window of metabolic programming and suggest milk protein concentration as a decisive factor in determining adult body weight.

View Article: PubMed Central - PubMed

Affiliation: Molecular Recognition Group, Hannah Research Institute, Ayr, United Kingdom. a.kolb@abdn.ac.uk

ABSTRACT
The major physiological function of milk is the transport of amino acids, carbohydrates, lipids and minerals to mammalian offspring. Caseins, the major milk proteins, are secreted in the form of a micelle consisting of protein and calcium-phosphate.We have analysed the role of the milk protein α-casein by inactivating the corresponding gene in mice. Absence of α-casein protein significantly curtails secretion of other milk proteins and calcium-phosphate, suggesting a role for α-casein in the establishment of casein micelles. In contrast, secretion of albumin, which is not synthesized in the mammary epithelium, into milk is not reduced. The absence of α-casein also significantly inhibits transcription of the other casein genes. α-Casein deficiency severely delays pup growth during lactation and results in a life-long body size reduction compared to control animals, but has only transient effects on physical and behavioural development of the pups. The data support a critical role for α-casein in casein micelle assembly. The results also confirm lactation as a critical window of metabolic programming and suggest milk protein concentration as a decisive factor in determining adult body weight.

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Analysis of markers of apoptosis in mammary tissue from α-casein deficient mice.Panel A: Western blot analysis of samples derived from two α-casein deficient mice and one heterozygous mouse (all taken at mid-lactation). The protein extracts were separated on a 10% (upper panel) and 15% (lower panel) polyacrylamide-gel blotted to nitrocellulose and detected using antisera against β-actin (upper panel) and the cleavage product of caspase 3 (lower panel). Extracts from RAW264 cells treated with 10 µM staurosporin (STS) for 6 h were used as positive control. The sizes of the protein molecular weight markers (Cell Signaling Technologies, biotinylated protein marker) are indicated as are the positions of the β-actin and caspase 3 proteins (arrows) Panel B: Analysis of caspase 3 and caspase 7 activity in cytoplasmic extracts of mammary gland tissue of control [+/+], heterozygous [+/−] and α-casein deficient mice [−/−] using a Caspase-Glo assay (Promega). Extracts derived from RAW264 cells treated with staurosporin were used as positive control. Panel C: Correlation of gene expression in wild type [+/+], heterozygous [+/−] and α-casein deficient mice [−/−] using quantitative PCR. Expression of the genes encoding the apoptosis related proteins nucleolar protein 3 (Nol3; up-regulated), Birc5 (up-regulated) and Traf1 (down-regulated) were correlated with the expression of the reference gene β-actin. Quantification was done in 3 [+/+], 6 [+/−] and 5 [−/−] mice. Statistical analysis using one-way ANOVA demonstrates that the expression changes for all three genes observed in α-casein deficient mice with respect to both wild-type and heterozygous mice occur with p<0.05. For comparisons against wild-type mice in a one-way ANOVA p<0.05 is indicated by *, and p<0.001 by ***.
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pone-0021775-g007: Analysis of markers of apoptosis in mammary tissue from α-casein deficient mice.Panel A: Western blot analysis of samples derived from two α-casein deficient mice and one heterozygous mouse (all taken at mid-lactation). The protein extracts were separated on a 10% (upper panel) and 15% (lower panel) polyacrylamide-gel blotted to nitrocellulose and detected using antisera against β-actin (upper panel) and the cleavage product of caspase 3 (lower panel). Extracts from RAW264 cells treated with 10 µM staurosporin (STS) for 6 h were used as positive control. The sizes of the protein molecular weight markers (Cell Signaling Technologies, biotinylated protein marker) are indicated as are the positions of the β-actin and caspase 3 proteins (arrows) Panel B: Analysis of caspase 3 and caspase 7 activity in cytoplasmic extracts of mammary gland tissue of control [+/+], heterozygous [+/−] and α-casein deficient mice [−/−] using a Caspase-Glo assay (Promega). Extracts derived from RAW264 cells treated with staurosporin were used as positive control. Panel C: Correlation of gene expression in wild type [+/+], heterozygous [+/−] and α-casein deficient mice [−/−] using quantitative PCR. Expression of the genes encoding the apoptosis related proteins nucleolar protein 3 (Nol3; up-regulated), Birc5 (up-regulated) and Traf1 (down-regulated) were correlated with the expression of the reference gene β-actin. Quantification was done in 3 [+/+], 6 [+/−] and 5 [−/−] mice. Statistical analysis using one-way ANOVA demonstrates that the expression changes for all three genes observed in α-casein deficient mice with respect to both wild-type and heterozygous mice occur with p<0.05. For comparisons against wild-type mice in a one-way ANOVA p<0.05 is indicated by *, and p<0.001 by ***.

Mentions: In order to assess whether the reduction in protein and RNA expression is correlated with overall morphological changes, sections of mammary tissue were obtained from animals at peak lactation and analysed by haematoxylin/eosin (H&E) staining and immuno-histochemistry (Fig. 6). As expected α-casein expression was clearly detectable in tissue from wild-type control mice and heterozygous mice (Fig. 6a). In contrast no α-casein protein could be detected in sections of a-casein deficient mice (Fig. 6a). As expected sections analysed with pre-immune serum did not show any α-casein specific staining (Fig. 6a). No gross morphological alterations were detected in the sections of the different genotypes (Fig. 6b). This suggests that the absence of α-casein, although critical for overall milk composition, does not significantly impact on the survival of the mammary gland. To address this question further we measured the expression and activity of capase proteins in the mammary gland. Firstly we assessed the presence of cleaved caspase 3 in the protein extracts derived from control mice, heterozygous mice and α-casein deficient mice. No cleaved caspase 3 could be detected (Fig. 7a). In contrast cleaved caspase 3 protein with the expected molecular weight of 19 kDa was readily detected in the extracts of RAW264 cells treated with 10 µM staurosporin (Fig. 7a). Similarly, no specific signals for cleaved caspase 3 could be detected in immuno-histochemical analyses of tissue sections. In addition, caspase 3 and caspase 7 activities was measured in cytoplasmic protein extracts derived from mammary tissue of control, heterozygous and α-casein deficient mice. No significant differences in caspase 3 and 7 were detected (Fig. 7b). In contrast a significant increase in caspase activity could be detected in RAW cells treated with 10 µM staurosporin (Fig. 7b).


Milk lacking α-casein leads to permanent reduction in body size in mice.

Kolb AF, Huber RC, Lillico SG, Carlisle A, Robinson CJ, Neil C, Petrie L, Sorensen DB, Olsson IA, Whitelaw CB - PLoS ONE (2011)

Analysis of markers of apoptosis in mammary tissue from α-casein deficient mice.Panel A: Western blot analysis of samples derived from two α-casein deficient mice and one heterozygous mouse (all taken at mid-lactation). The protein extracts were separated on a 10% (upper panel) and 15% (lower panel) polyacrylamide-gel blotted to nitrocellulose and detected using antisera against β-actin (upper panel) and the cleavage product of caspase 3 (lower panel). Extracts from RAW264 cells treated with 10 µM staurosporin (STS) for 6 h were used as positive control. The sizes of the protein molecular weight markers (Cell Signaling Technologies, biotinylated protein marker) are indicated as are the positions of the β-actin and caspase 3 proteins (arrows) Panel B: Analysis of caspase 3 and caspase 7 activity in cytoplasmic extracts of mammary gland tissue of control [+/+], heterozygous [+/−] and α-casein deficient mice [−/−] using a Caspase-Glo assay (Promega). Extracts derived from RAW264 cells treated with staurosporin were used as positive control. Panel C: Correlation of gene expression in wild type [+/+], heterozygous [+/−] and α-casein deficient mice [−/−] using quantitative PCR. Expression of the genes encoding the apoptosis related proteins nucleolar protein 3 (Nol3; up-regulated), Birc5 (up-regulated) and Traf1 (down-regulated) were correlated with the expression of the reference gene β-actin. Quantification was done in 3 [+/+], 6 [+/−] and 5 [−/−] mice. Statistical analysis using one-way ANOVA demonstrates that the expression changes for all three genes observed in α-casein deficient mice with respect to both wild-type and heterozygous mice occur with p<0.05. For comparisons against wild-type mice in a one-way ANOVA p<0.05 is indicated by *, and p<0.001 by ***.
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Related In: Results  -  Collection

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pone-0021775-g007: Analysis of markers of apoptosis in mammary tissue from α-casein deficient mice.Panel A: Western blot analysis of samples derived from two α-casein deficient mice and one heterozygous mouse (all taken at mid-lactation). The protein extracts were separated on a 10% (upper panel) and 15% (lower panel) polyacrylamide-gel blotted to nitrocellulose and detected using antisera against β-actin (upper panel) and the cleavage product of caspase 3 (lower panel). Extracts from RAW264 cells treated with 10 µM staurosporin (STS) for 6 h were used as positive control. The sizes of the protein molecular weight markers (Cell Signaling Technologies, biotinylated protein marker) are indicated as are the positions of the β-actin and caspase 3 proteins (arrows) Panel B: Analysis of caspase 3 and caspase 7 activity in cytoplasmic extracts of mammary gland tissue of control [+/+], heterozygous [+/−] and α-casein deficient mice [−/−] using a Caspase-Glo assay (Promega). Extracts derived from RAW264 cells treated with staurosporin were used as positive control. Panel C: Correlation of gene expression in wild type [+/+], heterozygous [+/−] and α-casein deficient mice [−/−] using quantitative PCR. Expression of the genes encoding the apoptosis related proteins nucleolar protein 3 (Nol3; up-regulated), Birc5 (up-regulated) and Traf1 (down-regulated) were correlated with the expression of the reference gene β-actin. Quantification was done in 3 [+/+], 6 [+/−] and 5 [−/−] mice. Statistical analysis using one-way ANOVA demonstrates that the expression changes for all three genes observed in α-casein deficient mice with respect to both wild-type and heterozygous mice occur with p<0.05. For comparisons against wild-type mice in a one-way ANOVA p<0.05 is indicated by *, and p<0.001 by ***.
Mentions: In order to assess whether the reduction in protein and RNA expression is correlated with overall morphological changes, sections of mammary tissue were obtained from animals at peak lactation and analysed by haematoxylin/eosin (H&E) staining and immuno-histochemistry (Fig. 6). As expected α-casein expression was clearly detectable in tissue from wild-type control mice and heterozygous mice (Fig. 6a). In contrast no α-casein protein could be detected in sections of a-casein deficient mice (Fig. 6a). As expected sections analysed with pre-immune serum did not show any α-casein specific staining (Fig. 6a). No gross morphological alterations were detected in the sections of the different genotypes (Fig. 6b). This suggests that the absence of α-casein, although critical for overall milk composition, does not significantly impact on the survival of the mammary gland. To address this question further we measured the expression and activity of capase proteins in the mammary gland. Firstly we assessed the presence of cleaved caspase 3 in the protein extracts derived from control mice, heterozygous mice and α-casein deficient mice. No cleaved caspase 3 could be detected (Fig. 7a). In contrast cleaved caspase 3 protein with the expected molecular weight of 19 kDa was readily detected in the extracts of RAW264 cells treated with 10 µM staurosporin (Fig. 7a). Similarly, no specific signals for cleaved caspase 3 could be detected in immuno-histochemical analyses of tissue sections. In addition, caspase 3 and caspase 7 activities was measured in cytoplasmic protein extracts derived from mammary tissue of control, heterozygous and α-casein deficient mice. No significant differences in caspase 3 and 7 were detected (Fig. 7b). In contrast a significant increase in caspase activity could be detected in RAW cells treated with 10 µM staurosporin (Fig. 7b).

Bottom Line: In contrast, secretion of albumin, which is not synthesized in the mammary epithelium, into milk is not reduced.The absence of α-casein also significantly inhibits transcription of the other casein genes. α-Casein deficiency severely delays pup growth during lactation and results in a life-long body size reduction compared to control animals, but has only transient effects on physical and behavioural development of the pups.The results also confirm lactation as a critical window of metabolic programming and suggest milk protein concentration as a decisive factor in determining adult body weight.

View Article: PubMed Central - PubMed

Affiliation: Molecular Recognition Group, Hannah Research Institute, Ayr, United Kingdom. a.kolb@abdn.ac.uk

ABSTRACT
The major physiological function of milk is the transport of amino acids, carbohydrates, lipids and minerals to mammalian offspring. Caseins, the major milk proteins, are secreted in the form of a micelle consisting of protein and calcium-phosphate.We have analysed the role of the milk protein α-casein by inactivating the corresponding gene in mice. Absence of α-casein protein significantly curtails secretion of other milk proteins and calcium-phosphate, suggesting a role for α-casein in the establishment of casein micelles. In contrast, secretion of albumin, which is not synthesized in the mammary epithelium, into milk is not reduced. The absence of α-casein also significantly inhibits transcription of the other casein genes. α-Casein deficiency severely delays pup growth during lactation and results in a life-long body size reduction compared to control animals, but has only transient effects on physical and behavioural development of the pups. The data support a critical role for α-casein in casein micelle assembly. The results also confirm lactation as a critical window of metabolic programming and suggest milk protein concentration as a decisive factor in determining adult body weight.

Show MeSH
Related in: MedlinePlus