Esults in a more significant CPT-1 mRNA abundance in the mammary gland, (Fig. 4a). In addition, the in silico analysis of the rat CPT-1 promoter region that we performed with the MatInspector program for transcription factor binding sites (unpublished observations) showed several putative estrogen response elements (ERE), suggesting that estrogens may directly regulate the transcription of CPT-1. During lactation, there is a decrease in the expression of CPT-1 and these changes are related to the sharp increase in mammary gland lipogenesis, which is needed to synthesize large amounts of triglycerides for milkFigure 4. The expression of genes involved in lipid oxidation and lipolysis in the mammary gland of dams fed different proportions (10/73, 20/63 or 30/53 ) of dietary protein/ dietary carbohydrates (DP/DCH) during gestation and lactation. (A) The relative mRNA levels of carnitine palmitoyl transferase 1 (CPT-1) and (B) hormone 11967625 sensitive lipase (HSL). Values are the mean 6 SEM. n = 5. *p,0.05, **p,0.01, ***p,0.001. doi:10.1371/journal.pone.0069338.gassociated with the development of fatty liver during lactation (Fig. 5d). S6K phosphorylation that is one of the target proteins of mTOR1 was largely unchanged during gestation and lactation by the different proportions of DP/DCH (Fig. 5b, e). The energy status of the liver cells, as represented by the phosphorylated AMPK (P-AMPK)/total AMPK ratio, was slightly increased during lactation (Fig. 5b, f). During gestation, but not 1315463 during lactation, the expression of the amino acid degrading enzymes, such as serine dehydratase (SDH), was significantly increased only when rats consumed a high protein diet in the group of 30/53 DP/DCH diet (Fig. 5g). The metabolic adaptations that occur during gestation and lactation in adipose tissue were very similar to those observed in the liver. The expression of lipogenic genes, as well as that of HSL, only increased when rats consumed a low-protein/high-carbohydrate diet (Fig. 6a). In fact, FAS abundance decreased with the progression of gestation and lactation (Fig. 6b, c). During gestation, S6K is fully active after phosphorylation at Thr389. At delivery, the phosphorylation of S6K almost disappeared but rapidly increased again during the lactation period, reaching values similar to those observed during gestation (Fig. 6b, d). During the gestation period, AMPK is partially activated by phosphorylation at Thr172, but this phosphorylation decreased rapidly during delivery and fully Methionine enkephalin chemical information restored during lactation (Fig. 6b, e).Dietary Protein and Mammary Gland MetabolismFigure 5. The expression of metabolic genes in the liver of dams fed different proportions (10/73, 20/63 or 30/53 ) of dietary protein/dietary carbohydrates (DP/DCH) during gestation and lactation. (A) The relative mRNA levels of sterol regulatory element binding protein 1 (SREBP1), fatty acid synthase (FAS), and pyruvate kinase (PK). (B) A representative Mirin biological activity immunoblot of AMP-activated protein kinase (AMPK), threonine phosphorylation of AMP-activated protein kinase (P-AMPK), p70 S6 kinase (S6K), threonine phosphorylation of S6 kinase (P-S6K), fatty acid synthase (FAS) and actin. (C) Western blot densitometric analysis of FAS/ACTIN. (D) A representative histology picture of the liver of rats fed 10/73, 20/ 63 or 30/53 DP/DCH at day 5 of lactation. Western blot densitometric analysis of (E) P-S6K/S6K, (F) P-AMPK/AMPK. (G) The relative mRNA levels of serine dehydratase (SDH). Values are the mean 6 S.Esults in a more significant CPT-1 mRNA abundance in the mammary gland, (Fig. 4a). In addition, the in silico analysis of the rat CPT-1 promoter region that we performed with the MatInspector program for transcription factor binding sites (unpublished observations) showed several putative estrogen response elements (ERE), suggesting that estrogens may directly regulate the transcription of CPT-1. During lactation, there is a decrease in the expression of CPT-1 and these changes are related to the sharp increase in mammary gland lipogenesis, which is needed to synthesize large amounts of triglycerides for milkFigure 4. The expression of genes involved in lipid oxidation and lipolysis in the mammary gland of dams fed different proportions (10/73, 20/63 or 30/53 ) of dietary protein/ dietary carbohydrates (DP/DCH) during gestation and lactation. (A) The relative mRNA levels of carnitine palmitoyl transferase 1 (CPT-1) and (B) hormone 11967625 sensitive lipase (HSL). Values are the mean 6 SEM. n = 5. *p,0.05, **p,0.01, ***p,0.001. doi:10.1371/journal.pone.0069338.gassociated with the development of fatty liver during lactation (Fig. 5d). S6K phosphorylation that is one of the target proteins of mTOR1 was largely unchanged during gestation and lactation by the different proportions of DP/DCH (Fig. 5b, e). The energy status of the liver cells, as represented by the phosphorylated AMPK (P-AMPK)/total AMPK ratio, was slightly increased during lactation (Fig. 5b, f). During gestation, but not 1315463 during lactation, the expression of the amino acid degrading enzymes, such as serine dehydratase (SDH), was significantly increased only when rats consumed a high protein diet in the group of 30/53 DP/DCH diet (Fig. 5g). The metabolic adaptations that occur during gestation and lactation in adipose tissue were very similar to those observed in the liver. The expression of lipogenic genes, as well as that of HSL, only increased when rats consumed a low-protein/high-carbohydrate diet (Fig. 6a). In fact, FAS abundance decreased with the progression of gestation and lactation (Fig. 6b, c). During gestation, S6K is fully active after phosphorylation at Thr389. At delivery, the phosphorylation of S6K almost disappeared but rapidly increased again during the lactation period, reaching values similar to those observed during gestation (Fig. 6b, d). During the gestation period, AMPK is partially activated by phosphorylation at Thr172, but this phosphorylation decreased rapidly during delivery and fully restored during lactation (Fig. 6b, e).Dietary Protein and Mammary Gland MetabolismFigure 5. The expression of metabolic genes in the liver of dams fed different proportions (10/73, 20/63 or 30/53 ) of dietary protein/dietary carbohydrates (DP/DCH) during gestation and lactation. (A) The relative mRNA levels of sterol regulatory element binding protein 1 (SREBP1), fatty acid synthase (FAS), and pyruvate kinase (PK). (B) A representative immunoblot of AMP-activated protein kinase (AMPK), threonine phosphorylation of AMP-activated protein kinase (P-AMPK), p70 S6 kinase (S6K), threonine phosphorylation of S6 kinase (P-S6K), fatty acid synthase (FAS) and actin. (C) Western blot densitometric analysis of FAS/ACTIN. (D) A representative histology picture of the liver of rats fed 10/73, 20/ 63 or 30/53 DP/DCH at day 5 of lactation. Western blot densitometric analysis of (E) P-S6K/S6K, (F) P-AMPK/AMPK. (G) The relative mRNA levels of serine dehydratase (SDH). Values are the mean 6 S.
