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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 milk protein expression.Panel A: Western blot analysis of milk derived from wild-type [+/+], heterozygous [+/−] and homozygous [−/−] α-casein deficient mice. The α-casein protein was detected using a rabbit-anti α-casein antiserum. Panel B: Western blot analysis of milk derived from wild-type [+/+], heterozygous [+/−] and homozygous [−/−] α-casein deficient mice. The β-casein protein was detected using a goat-anti β-casein antiserum. Panel C: Western blot analysis of milk derived from wild-type [+/+], heterozygous [+/−] and homozygous [−/−] α-casein deficient mice. The grp78/BiP protein was detected using a goat-anti grp78 antiserum. Panel D: Densitometric analysis of milk protein abundance as detected by SDS-PAGE. Coomassie Blue stained gels with milk samples from wild-type [+/+] (n = 3), heterozygous [+/−] (n = 6) and homozygous [−/−] α-casein deficient mice (n = 5) were scanned in a Kodak densitometer and the net intensity of the milk proteins was compared with the intensities of molecular weight marker proteins of know concentration. Protein concentrations detected in the three genotypes for albumin, α-casein (acas), β-casein (bcas) and γ-casein protein (gcas) are presented. Comparisons of [−/−] vs [+/−] as analysed by one-way ANOVA are significant with p<0.001 (***); Comparisons of [+/−] vs [+/+] are significant with p<0.05 (*) where indicated. Panel E: The relative amounts of milk protein abundance were calculated for the ratios of α-casein to albumin (acas/alb.), α-casein to β-casein (acas/bcas), β-casein to γ-casein (bcas/gcas), β-casein to albumin (bcas/alb.) and γ-casein to albumin (gcas/alb.). For comparisons against wild-type mice in a one-way ANOVA p<0.01 is indicated by **, p<0.001 by ***. Exact P values are presented in table 5.
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pone-0021775-g003: Analysis of milk protein expression.Panel A: Western blot analysis of milk derived from wild-type [+/+], heterozygous [+/−] and homozygous [−/−] α-casein deficient mice. The α-casein protein was detected using a rabbit-anti α-casein antiserum. Panel B: Western blot analysis of milk derived from wild-type [+/+], heterozygous [+/−] and homozygous [−/−] α-casein deficient mice. The β-casein protein was detected using a goat-anti β-casein antiserum. Panel C: Western blot analysis of milk derived from wild-type [+/+], heterozygous [+/−] and homozygous [−/−] α-casein deficient mice. The grp78/BiP protein was detected using a goat-anti grp78 antiserum. Panel D: Densitometric analysis of milk protein abundance as detected by SDS-PAGE. Coomassie Blue stained gels with milk samples from wild-type [+/+] (n = 3), heterozygous [+/−] (n = 6) and homozygous [−/−] α-casein deficient mice (n = 5) were scanned in a Kodak densitometer and the net intensity of the milk proteins was compared with the intensities of molecular weight marker proteins of know concentration. Protein concentrations detected in the three genotypes for albumin, α-casein (acas), β-casein (bcas) and γ-casein protein (gcas) are presented. Comparisons of [−/−] vs [+/−] as analysed by one-way ANOVA are significant with p<0.001 (***); Comparisons of [+/−] vs [+/+] are significant with p<0.05 (*) where indicated. Panel E: The relative amounts of milk protein abundance were calculated for the ratios of α-casein to albumin (acas/alb.), α-casein to β-casein (acas/bcas), β-casein to γ-casein (bcas/gcas), β-casein to albumin (bcas/alb.) and γ-casein to albumin (gcas/alb.). For comparisons against wild-type mice in a one-way ANOVA p<0.01 is indicated by **, p<0.001 by ***. Exact P values are presented in table 5.

Mentions: Milk was isolated from lactating wild-type [+/+], heterozygous [+/−] and [−/−] animals, separated on a denaturing protein gel and analysed by Coomassie Blue staining. Two representative samples of milk derived from heterozygous [+/−] and [−/−] mice are shown are in Fig. 2a alongside a wild-type control milk sample. Milk from control animals displayed a series of protein bands, with the most prominent proteins corresponding to molecular weights previously observed for α-, β- and γ-casein (with apparent molecular weights of 42, 30 and 25 kDa, respectively) [28]. The identity of the protein bands was confirmed using mass-spectrometry. Table 4 details the data for the 8 most prominent protein bands indicated in Fig. 2c. These findings were also supported using Western blot analyses for α-casein and β-casein (Fig. 3a and 3b). Milk from α-casein deficient mice shows some characteristic protein bands which are absent from milk of wild-type or heterozygous mice (Fig. 2d). Mass-spectrometry analysis was used to identify some of these proteins. The major additional band with an apparent molecular weight of 78 kDa was identified as grp78/BiP. BiP is an ER resident protein which is involved in the assembly of multi-protein complexes like antibodies [29], [30]. The identity of the grp78/BiP protein was confirmed by Western blot analysis (Fig. 3c). In addition two further ER-resident proteins, grp94 and PDIA6 (protein disulfide-isomerase associated protein 6), were detected in milk of the α-casein deficient mice (Fig. 2d, table 4).


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 milk protein expression.Panel A: Western blot analysis of milk derived from wild-type [+/+], heterozygous [+/−] and homozygous [−/−] α-casein deficient mice. The α-casein protein was detected using a rabbit-anti α-casein antiserum. Panel B: Western blot analysis of milk derived from wild-type [+/+], heterozygous [+/−] and homozygous [−/−] α-casein deficient mice. The β-casein protein was detected using a goat-anti β-casein antiserum. Panel C: Western blot analysis of milk derived from wild-type [+/+], heterozygous [+/−] and homozygous [−/−] α-casein deficient mice. The grp78/BiP protein was detected using a goat-anti grp78 antiserum. Panel D: Densitometric analysis of milk protein abundance as detected by SDS-PAGE. Coomassie Blue stained gels with milk samples from wild-type [+/+] (n = 3), heterozygous [+/−] (n = 6) and homozygous [−/−] α-casein deficient mice (n = 5) were scanned in a Kodak densitometer and the net intensity of the milk proteins was compared with the intensities of molecular weight marker proteins of know concentration. Protein concentrations detected in the three genotypes for albumin, α-casein (acas), β-casein (bcas) and γ-casein protein (gcas) are presented. Comparisons of [−/−] vs [+/−] as analysed by one-way ANOVA are significant with p<0.001 (***); Comparisons of [+/−] vs [+/+] are significant with p<0.05 (*) where indicated. Panel E: The relative amounts of milk protein abundance were calculated for the ratios of α-casein to albumin (acas/alb.), α-casein to β-casein (acas/bcas), β-casein to γ-casein (bcas/gcas), β-casein to albumin (bcas/alb.) and γ-casein to albumin (gcas/alb.). For comparisons against wild-type mice in a one-way ANOVA p<0.01 is indicated by **, p<0.001 by ***. Exact P values are presented in table 5.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3138747&req=5

pone-0021775-g003: Analysis of milk protein expression.Panel A: Western blot analysis of milk derived from wild-type [+/+], heterozygous [+/−] and homozygous [−/−] α-casein deficient mice. The α-casein protein was detected using a rabbit-anti α-casein antiserum. Panel B: Western blot analysis of milk derived from wild-type [+/+], heterozygous [+/−] and homozygous [−/−] α-casein deficient mice. The β-casein protein was detected using a goat-anti β-casein antiserum. Panel C: Western blot analysis of milk derived from wild-type [+/+], heterozygous [+/−] and homozygous [−/−] α-casein deficient mice. The grp78/BiP protein was detected using a goat-anti grp78 antiserum. Panel D: Densitometric analysis of milk protein abundance as detected by SDS-PAGE. Coomassie Blue stained gels with milk samples from wild-type [+/+] (n = 3), heterozygous [+/−] (n = 6) and homozygous [−/−] α-casein deficient mice (n = 5) were scanned in a Kodak densitometer and the net intensity of the milk proteins was compared with the intensities of molecular weight marker proteins of know concentration. Protein concentrations detected in the three genotypes for albumin, α-casein (acas), β-casein (bcas) and γ-casein protein (gcas) are presented. Comparisons of [−/−] vs [+/−] as analysed by one-way ANOVA are significant with p<0.001 (***); Comparisons of [+/−] vs [+/+] are significant with p<0.05 (*) where indicated. Panel E: The relative amounts of milk protein abundance were calculated for the ratios of α-casein to albumin (acas/alb.), α-casein to β-casein (acas/bcas), β-casein to γ-casein (bcas/gcas), β-casein to albumin (bcas/alb.) and γ-casein to albumin (gcas/alb.). For comparisons against wild-type mice in a one-way ANOVA p<0.01 is indicated by **, p<0.001 by ***. Exact P values are presented in table 5.
Mentions: Milk was isolated from lactating wild-type [+/+], heterozygous [+/−] and [−/−] animals, separated on a denaturing protein gel and analysed by Coomassie Blue staining. Two representative samples of milk derived from heterozygous [+/−] and [−/−] mice are shown are in Fig. 2a alongside a wild-type control milk sample. Milk from control animals displayed a series of protein bands, with the most prominent proteins corresponding to molecular weights previously observed for α-, β- and γ-casein (with apparent molecular weights of 42, 30 and 25 kDa, respectively) [28]. The identity of the protein bands was confirmed using mass-spectrometry. Table 4 details the data for the 8 most prominent protein bands indicated in Fig. 2c. These findings were also supported using Western blot analyses for α-casein and β-casein (Fig. 3a and 3b). Milk from α-casein deficient mice shows some characteristic protein bands which are absent from milk of wild-type or heterozygous mice (Fig. 2d). Mass-spectrometry analysis was used to identify some of these proteins. The major additional band with an apparent molecular weight of 78 kDa was identified as grp78/BiP. BiP is an ER resident protein which is involved in the assembly of multi-protein complexes like antibodies [29], [30]. The identity of the grp78/BiP protein was confirmed by Western blot analysis (Fig. 3c). In addition two further ER-resident proteins, grp94 and PDIA6 (protein disulfide-isomerase associated protein 6), were detected in milk of the α-casein deficient mice (Fig. 2d, table 4).

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