Synthesis of milk specific fatty acids and proteins by dispersed goat mammary-gland epithelial cells.
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ABSTRACT:
SUBMITTER: Hansen HO
PROVIDER: S-EPMC1147111 | biostudies-other | 1986 Aug
REPOSITORIES: biostudies-other
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Project description:1. Arteriovenous differences of plasma free amino acids across the lactating mammary glands of six goats have been measured. 2. In four experiments, measurements of blood flow, amino acid arteriovenous differences, milk yield and milk nitrogen showed that the uptake of nitrogen in the form of amino acids was sufficient to provide all the nitrogen of the milk proteins synthesized in the mammary gland. 3. In the same four experiments the uptake from the plasma and output into the milk of individual amino acids per unit time were compared. The uptakes of essential amino acids and glutamic acid were approximately equal to the corresponding output figures. The uptake of serine was consistently less than the output, and the uptake of other non-essential amino acids was very variable, in some experiments being approximately equal to the output figures and in others being considerably less. 4. As in cows, there was an uptake of ornithine in all experiments, though ornithine is absent from milk. In goats, though not in cows, the uptake of arginine was consistently greatly in excess of the requirement for arginine residues in milk protein. 5. The possible significance of the uptakes of arginine and ornithine for the synthesis of serine and other non-essential amino acids in the mammary gland is discussed. 6. The importance of clamping the external pudic vein, when sampling mammary venous blood from the caudal superficial epigastric vein, is indicated.
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Project description:ATP alone had no effect on incorporation of fatty acids synthesized de novo and membrane-bound diacylglycerol into triacylglycerol. Combined addition of ATP and Mg2+ totally inhibits incorporation of fatty acids synthesized de novo and stimulated incorporation of membrane-bound diacylglycerol. ATP, Mg2+ and glycerol 3-phosphate stimulate incorporation of fatty acids synthesized de novo into triacylglycerol, but inhibited the incorporation of membrane-bound diacylglycerol. Diacylglycerol generated in situ was shown to be superior to diacylglycerols preloaded on the membrane as substrate for the diacylglycerol acyltransferase. A model is proposed to explain the effect of absorbed exogenous fatty acid on fatty acid synthesis de novo in goat mammary gland.
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Project description:A key member of the nuclear receptor superfamily is the peroxisome proliferator-activated receptor alpha (PPARA) isoform, which in nonruminants is closely associated with fatty acid oxidation. Whether PPARA plays a role in milk fatty acid synthesis in ruminants is unknown. The main objective of the present study was to use primary goat mammary epithelial cells (GMEC) to activate PPARA via the agonist WY-14643 (WY) or to silence it via transfection of small-interfering RNA (siRNA). Three copies of the peroxisome proliferator-activated receptor response element (PPRE) contained in a luciferase reporter vector were transfected into GMEC followed by incubation with WY at 0, 10, 20, 30, 50, or 100 µM. A dose of 50 µM WY was most effective at activating PPRE without influencing PPARA mRNA abundance. Transfecting siRNA targeting PPARA decreased its mRNA abundance to 20% and protein level to 50% of basal levels. Use of WY upregulated FASN, SCD1, ACSL1, DGAT1, FABP4, and CD36 (1.1-, 1.5-, 2-, 1.4-, 1.5-, and 5-fold, respectively), but downregulated DGAT2 and PGC1A (-20% and -40%, respectively) abundance. In contrast, triacylglycerol concentration decreased and the content and desaturation index of C16:1 and C18:1 increased. Thus, activation of PPARA via WY appeared to channel fatty acids away from esterification. Knockdown of PPARA via siRNA downregulated ACACA, SCD1, AGPAT6, CD36, HSL, and SREBF1 (-43%, -67%, -16%, -56%, -26%, and -29%, respectively), but upregulated ACSL1, DGAT2, FABP3, and PGC1A (2-, 1.4-, 1.3-, and 2.5-fold, respectively) mRNA abundance. A decrease in the content and desaturation index of C16:1 and C18:1 coupled with an increase in triacylglycerol content accompanied those effects at the mRNA level. Overall, data suggest that PPARA could promote the synthesis of MUFA in GMEC through its effects on mRNA abundance of genes related to fatty acid synthesis, oxidation, transport, and triacylglycerol synthesis.
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Project description:In humans, fatty acid elongase 7 (ELOVL7) plays a role in synthesis of long-chain saturated fatty acids. Whether ELOVL7 protein plays a role in ruminants is unclear. The transcript abundance of ELOVL7 in goat mammary tissue was assessed at three stages of lactation. Results showed that ELOVL7 had the highest expression in the dry period compared with peak and late lactation period. Results revealed that ELOVL7 overexpression was correlated with lower expression in diacylglycerol O-acyltransferase 2 (DGAT2) and stearoyl-CoA desaturase 1 (SCD1), and had no significant effect on triacylglycerol concentration. Overexpression of ELOVL7 significantly decreased the concentration of palmitoleic (C16:1n7) and oleic (C18:1n9) acid, and increased the concentration of vaccenic (C18:1n7) and linoleic (C18:2) acid. Overexpression of ELOVL7 significantly upregulated the elongation index of C16:1 in goat epithelial mammary cells (GMEC), but had a minor effect on that of palmitate (C16:0). Knockdown of ELOVL7 decreased mRNA expression of fatty acid binding protein 3 (FABP3) and fatty acid desaturase 2 (FADS2) and had a minor effect on triacylglycerol concentration; however, it increased concentration of C18:1n9 in GMEC. The elongation indices of C16:0 and C16:1 did not differ due to knockdown of ELOVL7. The minor change for the C16:0 and stearate (C18:0) was observed after activation of ELOVL7, suggesting the two fatty acids are not the preferential substrates of ELOVL7 in cultured GMEC. However, changes in C18:1n9 and C18:2 after overexpression or knockdown of ELOVL7 indicated a biological functional role of ELOVL7. Collectively, our data highlighted a role of ELOVL7 in long-chain unsaturated fatty acid elongation in goat mammary epithelial cells.
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Project description:1. Highly purified rat mammary-gland acetyl-CoA carboxylase was inhibited by milk obtained from rats 12h after their young were weaned. 2. All the inhibitory activity was found in the particulate fraction (R(105)) obtained on centrifuging the milk. It could be extracted from milk fraction R(105) with acetone and identified as a complex mixture of non-esterified fatty acids, present in high concentration (nearly 10mm) in the milk. 3. Inhibition of acetyl-CoA carboxylase was observed at low concentrations (0.2-20mum) of several of these fatty acids when fresh fully active enzyme was used. Enzyme that had been partly inactivated by aging, or by storing in the absence of citrate, was stimulated by low concentrations but inhibited by high concentrations of fatty acids. 4. Various experiments suggested that fatty acids produce irreversible inactivation of acetyl-CoA carboxylase. 5. The effects of palmitoyl-CoA on mammary-gland acetyl-CoA carboxylase were found to resemble those of fatty acids, except that palmitoyl-CoA was effective at lower concentration. 6. The effect of milk fraction R(105) was tested on six other enzymes previously shown to decline to various extents after weaning. Although several of these enzymes were affected by unfractionated milk fraction R(105), none was significantly inhibited by the acetone extract or by low concentrations of lauric acid. 7. The findings are consistent, both qualitatively and quantitatively, with a regulatory mechanism whereby milk fatty acids shut off fatty acid synthesis in the mammary gland after weaning by inhibiting acetyl-CoA carboxylase.
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Project description:Synergistic regulation among microRNAs (miRNAs) is important to understand the mechanisms underlying the complex molecular regulatory networks in goats. Goat milk fat synthesis is driven by a gene network that involves many biological processes in the mammary gland. These biological processes are affected by several miRNAs rather than a single miRNA. Therefore, identifying synergistic miRNAs is necessary to further understand the functions of miRNAs and the metabolism of goat milk fat synthesis. Using qRT-PCR, we assessed the expression of 11 miRNAs that have the potential to regulate milk fat synthesis in the goat mammary gland. Six of these miRNAs exhibited expression during the lactation cycle. Additionally, we also found that prolactin, the key hormone that regulates lactation, promotes the expression of four miRNAs (miR-23a, miR-27b, miR-103, and miR-200a). Further functional analysis showed that overexpression of all four miRNAs by using recombinant adenovirus in goat mammary gland epithelial cells can affect gene mRNA expression associated with milk fat synthesis. Specifically, elevated miR-200a expression suppressed the mRNA expression of genes involved in fat droplet formation. To analyze the synergistic regulation among these four miRNAs (miR-23a, miR-27b, miR-103, and miR-200a), we used the Pearson correlation coefficient to evaluate the correlation between their expression levels in 30 lactating goats. As a result, we found a strong correlation and mutual regulation between three miRNA pairs (miR-23a and miR-27b, miR-103 and miR-200a, miR-27b and miR-200a). This study provides the first experimental evidence that miRNA expression is synergistically regulated in the goat mammary gland and has identified the potential biological role of miRNAs in goat milk fat synthesis. The identification of synergistic miRNAs is a crucial step for further understanding the molecular network of milk fat synthesis at a system-wide level.
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Project description:Previous work demonstrates that proteases present in human milk release hundreds of peptides derived from milk proteins. However, the question of whether human milk protein digestion begins within the mammary gland remains incompletely answered. The primary objective of this study was to determine whether proteolytic degradation of human milk proteins into peptides begins within the mammary gland. The secondary objectives were to determine which milk proteases participate in the proteolysis and to predict which released peptides have bioactivity. Lactating mothers (n = 4) expressed their milk directly into a mixture of antiproteases on ice followed by immediate freezing of the milk to limit post-expression protease activity. Samples were analyzed for their peptide profiles via mass spectrometry and database searching. Peptidomics-based protease prediction and bioactivity prediction were each performed with several different approaches. The findings demonstrate that human milk contains more than 1,100 unique peptides derived from milk protein hydrolysis within the mammary gland. These peptides derived from 42 milk proteins and included 306 potential bioactive peptides. Based on the peptidomics data, plasmin was predicted to be the milk protease most active in the hydrolysis of human milk proteins within the mammary gland. Milk proteases actively cleave milk proteins within the mammary gland, initiating the release of functional peptides. Thus, the directly breastfed infant receives partially pre-digested proteins and numerous bioactive peptides.
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Project description:Milk is the primary source of nutrition for young mammals including humans. The nutritional value of milk is mainly attributable to fats and proteins fractions. In comparison to cow milk, goat milk contains greater amounts of total fat, including much higher levels of the beneficial unsaturated fatty acids. MicroRNAs (miRNAs), a well-defined group of small RNAs containing about 22 nucleotides (nt), participate in various metabolic processes across species. However, little is known regarding the role of miRNAs in regulating goat milk composition. In the present study, we performed high-throughput sequencing to identify mammary gland-enriched miRNAs in lactating goats. We identified 30 highly expressed miRNAs in the mammary gland, including miR-103. Further studies revealed that miR-103 expression correlates with the lactation. Further functional analysis showed that over-expression of miR-103 in mammary gland epithelial cells increases transcription of genes associated with milk fat synthesis, resulting in an up-regulation of fat droplet formation, triglyceride accumulation, and the proportion of unsaturated fatty acids. This study provides new insight into the functions of miR-103, as well as the molecular mechanisms that regulate milk fat synthesis.
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Project description:Fatty acid synthetase from goat mammary gland was subjected to limited proteolysis by trypsin and elastase. Both proteolytic enzymes selectively cleaved the chain-terminating thioester hydrolase component from the enzyme complex, leaving all other partial activities intact in the core peptides. Trypsin, but not elastase, caused extensive degradation of the released thioester hydrolase. The released thioester hydrolase could be purified to homogeneity by gel filtration. The molecular weight was estimated as 29 000 and the enzyme showed only significant hydrolytic activity toward long-chain acyl-CoA esters. The core peptides retained the ability to synthesize medium-chain acyl-CoA esters in the presence of 2,6-di-O-methyl-alpha-cyclodextrin. The results conclusively show that the terminating thioester hydrolase of goat mammary-gland fatty acid synthetase is not involved in termination of medium-chain-length fatty acid synthesis by this enzyme.
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Project description:This study aimed to examine the effects of increasing levels of three 18-carbon fatty acids (stearate, oleate and linoleate) on mammary lipogenesis, and to evaluate their effects on the milk lipogenic pathway in porcine mammary epithelial cells (pMECs). We found that increasing the three of 18-carbon fatty acids enhanced the cellular lipid synthesis in a dose-dependent manner, as reflected by the increased (triacylglycerol) TAG content and cytosolic lipid droplets in pMECs. The increased lipid synthesis by the three 18-carbon fatty acids was probably caused by the up-regulated expression of major genes associated with milk fat biosynthesis, including CD36 (long chain fatty acid uptake); GPAM, AGPAT6, DGAT1 (TAG synthesis); PLIN2 (lipid droplet formation); and PPARγ (regulation of transcription). Western blot analysis of CD36, DGAT1 and PPARγ proteins confirmed this increase with the increasing incubation of 18-carbon fatty acids. Interestingly, the mRNA expressions of ACSL3 and FABP3 (fatty acids intracellular activation and transport) were differentially affected by the three 18-carbon fatty acids. The cellular mRNA expressions of ACSL3 and FABP3 were increased by stearate, but were decreased by oleate or linoleate. However, the genes involved in fatty acid de novo synthesis (ACACA and FASN) and the regulation of transcription (SREBP1) were decreased by incubation with increasing concentrations of 18-carbon fatty acids. In conclusion, our findings provided evidence that 18-carbon fatty acids (stearate, oleate and linoleate) significantly increased cytosolic TAG accumulation in a dose-dependent manner, probably by promoting lipogenic genes and proteins that regulate the channeling of fatty acids towards milk TAG synthesis in pMECs.
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