Lipid synthesis in lactating mammary gland

The mammary gland o f a high-yielding cow may produce as much as 1–2 kg o f fat in a day. Studies reveal unique mechanisms at work to produce typical milk lipid.     

Acyl specificity in triglyceride synthesis by lactating rat mammary gland.

We have studied the specificity of the acyl-CoA:diglyceride acyltransferase reaction in lactating rat mammary gland to provide a rational explanation at the enzyme level for the nonrandom distribution of fatty acids in milk fat triglycerides. Acyl-CoA:diglyceride acyltransferase activity was measured using various diglyceride and radioactive acyl-CoA substrates; products were identified as triglycerides by thin-layer and gas-liquid chromatography. Most of the enzymatic activity was located in the microsomal fraction and showed a broad specificity for the acyl donors tested C10, C12, C14, C16, C18, and C18:1 CoA esters). The acyltransferase activity was highly specific for sn-1,2-diglyceride enantiomers; rac-1,3- and sn-2,3-diglycerides were relatively inactive. The acyl-CoA specificity was not affected by the type of 1,2-diglyceride acceptor offered, although dilaurin was the best acceptor and sn-1,2-dilaurin greater than sn-1,2-dimyristin greater than sn-1,2-dipalmitin greater than sn-1,2-distearin. We have previously shown that in the microsomal fraction from lactating rat mammary gland, the acyltransferase activities concerned with the conversion of sn-glycero-3-phosphate to diacylglycerophosphate show a very marked specificity for long chain acyl-CoA's. Therefore, we conclude that the predominant localization of long chain fatty acids in the 1 and 2 positions, and of shorter chain fatty acids in the 3 position of the glycerol backbone, results at least in part from the specificities of the mammary gland acyltransferases.     

Proteomic analysis of microsomes from lactating bovine mammary gland

Mammary gland has multiple metabolic potential including for large-scale synthesis of milk proteins, carbohydrate, and lipids including nutrient triacylglycerols. We have carried out a proteomic analysis of mammary tissue to discover proteins that affect lipid metabolism. Unfractionated microsomes from lactating bovine mammary tissue were analyzed using one-dimensional SDS-PAGE with RPLC-ESI-MS/MS. This approach gave 703 proteins including 160 predicted transmembrane proteins. Proteins were classified according to their subcellular localizations and biological functions. Over 50 proteins were associated with cellular uptake, metabolism, and secretion of lipids, including some enzymes that have been previously associated with breast cancer and potential therapeutic targets. This database develops a proteomic view of the metabolic potential of mammary gland that can be expected to contribute to a greater understanding of gene expression and tissue remodeling associated with lactation, and to further dissection of normal and pathological processes in mammary tissue.     

Proteolytic modification of rat prolactin by subcellular fractions of the lactating rat mammary gland

The current study explored prolactin proteolysis by rat lactating mammary gland. 125I-labelled rat prolactin was incubated with tissue fractions of lactating mammary gland and the extent of prolactin degradation and fragment formation was visualized and densitometrically quantitated from autoradiographs derived from SDS-polyacrylamide gel electrophoresis under reducing conditions. At pH 4.5, the 25 000 X g pellet of mammary gland converted intact prolactin (23 kDa band) to proteolytic fragments (8-16 kDa bands) in a time- and tissue concentration-dependent fashion similar to that reported previously for rat ventral prostate. The prolactin-degrading and -fragmenting activity in lactating mammary gland was 5-10-times that observed for ventral prostate, the most active male tissue. This activity at acid pH was also demonstrable in other fractions of mammary gland but appeared to predominate in the cytosol. The above activities in mammary gland virtually disappeared at pH 7.4, appeared sensitive to aspartate and sulfhydryl proteinase inhibitors, and insensitive to serine and metalloenzyme proteinase inhibitors. The distribution of this activity could not be correlated with a particular enzyme marker. These characteristics of mammary gland activity differed significantly from those reported previously for prostate. When electrophoresis was conducted under non-reducing conditions, prolactin proteolysis in prostate and mammary gland was primarily associated with the formation of a more slowly migrating product (24 kDa band) with little spontaneous 8-16 kDa fragment formation. Re-electrophoresis of the 24 kDa band under reducing conditions resulted in the appearance of the 8 and 16 kDa fragments. In conclusion, prolactin is proteolytically modified by prostate and lactating mammary gland to a variant of intact hormone (24 kDa band) with a cleavage site in its large loop, by two or more widely distributed, acid-dependent proteinases. Lactating mammary gland, the principal target for prolactin, has the capacity to cleave the hormone in its loop at rates higher than any other tissue examined to date.     

Function of arginase in lactating mammary gland

The potential for a considerable formation of ornithine exists in lactating mammary gland because of its arginase content. Late in lactation arginase reaches an activity in the gland higher than that present in any rat tissue except liver. Occurrence of the urea cycle can be excluded since two enzymes for the further reaction of ornithine in the cycle, carbamoyl phosphate synthetase I and ornithine carbamoyltransferase, are both absent from this tissue. Instead, carbamoyl phosphate synthetase II appears early in lactation, associated with accumulation of aspartate carbamoyltransferase and DNA, consistent with the proposed role of these enzymes in pyrimidine synthesis. The facts require another physiological role for arginase apart from its known function in the urea cycle. Significant activity of ornithine aminotransferase develops in mammary gland in close parallel with the arginase. By this reaction, ornithine can be converted into glutamic semialdehyde and subsequently into proline. The enzymic composition of the lactating mammary gland is therefore appropriate for the major conversion of arginine into proline that is known to occur in the intact gland.     

The study of spermidine-stimulated polypeptide synthesis in cell-free translation of mRNA from lactating mouse mammary gland

The stimulatory effect of spermidine on the translation of poly(A)⁺ mRNA from lactating mouse mammary glands in a wheat germ system was studied. Spermidine stimulated total polypeptide synthesis about 2.5-fold relative to that occurring in the presence of an optimal concentration of Mg²⁺ alone. The size and the number of polysomes were about 1.6-times larger in the presence of spermidine than in its absence. A similar magnitude of increase in peptide chain initiation, 1.4-fold, was found when the extent of peptide chain initiation was measured by determining the residual polypeptide synthesis subsequent to the addition of inhibitor(s) of peptide chain initiation to the in vitro translation system with or without spermidine at various times of the incubation. Time-course study of the release of polypeptide from polysomes showed that spermidine stimulated this process to a much greater extent than peptide chain initiation, indicating that the polyamine also increases the rate of peptide chain elongation. The extent of stimulation of peptide chain elongation by spermidine was estimated to be about 1.5-fold when the disappearance of isotope-labeled nascent peptides from polysomes was measured by pulse-chase experiments. These results indicate that spermidine stimulates the cell-free translation of mammary mRNA by increasing the rates of both initiation and elongation of polypeptide synthesis to almost the same extent. The polyamine also reduced the relative amount of incomplete polypeptides, thereby increasing the yield of full-length translational products.     

Properties of ornithine decarboxylase-antizyme from mammary gland of lactating rats

Ornithine decarboxylase-antizyme was induced in mammary gland of fasted lactating rats by administration of 1,3-diaminopropan-2-ol. Antizyme from mammary gland showed similar chemical and kinetic behavior to that previously reported by Canellakis and co-workers for antizyme from liver [J. S. Heller, W. F. Fong, and E. S. Canellakis (1976) Proc. Natl. Acad. Sci. USA 72, 1858-1862]; specifically the inhibitor was nondialyzable, heat labile, and ribonuclease insensitive, and the inhibition was time independent, proportional to the concentration of antizyme present, and noncompetitive with respect to the substrate, ornithine. However, ornithine decarboxylase-antizyme from mammary gland eluted from Sephadex G-75 with an apparent molecular mass of 55 kDa, compared with 27 kDa, for antizyme from liver under identical conditions. The elution pattern was unaffected by the presence of high salt concentrations, indicating that the larger size was not due to macromolecular complexes. The presence of antizyme-ornithine decarboxylase complex was detected in mammary gland of untreated lactating rats fasted for 6 or 24 h, thus indicating that antizyme plays a role in the regulation of ornithine decarboxylase in mammary gland under physiological conditions.     

Secretory membranes of the lactating mammary gland

Summary The lactating mammary gland is one of the most highly differentiated and metabolically active organs in the body. Membranes of the lactating mammary cell have important roles in transmitting from one membrane to another of hormonal information and in milk secretion, which is the final event. During milk secretion, the projection of the surface membrane into the alveolar lumen by enveloping intracellular lipid droplets with the apical plasma membrane is one of the most remarkable aspects of biological membrane action throughout nature.This review focuses on current knowledge about membranes in the lactating mammary gland. (1) Advances in the isolation and properties of membranes, especially the plasma membrane and Golgi-derived secretory vesicles, concerned with milk secretion from the lactating mammary gland are described. (2) Milk serum components are secreted by fusing the membranes of secretory vesicles that condense milk secretions with the plasma membrane in the apical regions. This occurs through the formation of a tubular-shaped projection and vesicular depression in a ball-and-socket configuration, as well as by simple fusion. (3) Intracellular lipid droplets are directly extruded from the mammary epithelial cells by progressive envelopment of the plasma membranes in the apical regions. (4) The balance between the surface volume lost in enveloping lipid droplets and that provided by fusion of the secretory vesicle and other vesicles with the apical plasma membrane is discussed. (5) The membrane surrounding a milk fat globule, which is referred to as the milk fat globule membrane (MFGM), is composed of at least the coating membrane of an intracellular lipid droplet, of the apical plasma membrane and secretory vesicle membrane, and of a coat material. Consequently, MFGM is molecularly different from the plasma membrane in composition. (6) MFGM of bovine milk is structurally composed of an inner coating membrane and outer plasma membrane just after segregation. These two membranes are fused and reorganized through a process of vesiculation and fragmentation to stabilize the fat globules. Hypothetical structural models for MFGM from bovine milk fat globules just after secretion and after rearrangement are proposed.Abbrevations MFGM milk fat globule membrane - HEPES N-2-hydroxylpiperazine-N-2-ethanesulfonic acid - INT 2-(p-indophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate - Sph sphingomyelin - PC phosphatidyl choline - PE phosphatidyl ethanolamine - PS phosphatidyl serine - PI phosphatidyl inositol - PAS periodic acid-Schiff reagent - CB Coomassie brilliant blue R-250 Dedicated to Professor Stuart Patton on the occasion of his 70th birthday.     

The pattern of fatty acid synthesis in lactating rabbit mammary gland studied in vivo

The biosynthesis of fatty acids has been studied in lactating rabbits at 6h after intravenous injection of sodium [1-(14)C]acetate. The specific radioactivities of the individual fatty acids (C(6:0) to C(14:0)) and the proportions of these fatty acids synthesized were similar in mammary tissue and milk. Hexanoic acid had the highest specific radioactivity, and the C(8:0)-C(14:0) fatty acids had similar specific radioactivities, which were about five times those of C(16) and C(18) acids. No radioactivity was detected in fatty acids of chain length C(14) in these tissues were similar to those of the long-chain fatty acids in the milk and mammary gland. The results show that the C(4:0)-C(14:0) fatty acids are synthesized within the mammary gland rather than by fatty acid uptake from circulating blood or by oxidation of long-chain fatty acids within the gland. We conclude that de novo synthesis of esterified fatty acids in vivo by this tissue has a high degree of chain-length specificity.     

Triacylglycerol hydrolysis by cells isolated from lactating rat mammary gland

The hydrolysis of exogenous trioleoylglycerol emulsions by suspensions of cells prepared from lactating rat mammary gland has been investigated.Cell integrity remains high throughout short (at least 30 min) incubations, during which extracellular hydrolysis of trioleoylglycerol proceeds at a mean rate (11 preparations) of 1.9 nmol oleate (and 0.6 nmol glycerol) released/min per mg protein. This hydrolysis shows partial dependence upon added serum and partial inhibition by protamine sulphate—both characteristic properties of lipoprotein lipase-catalyzed lipolysis. One or more monoacylglycerol hydrolase enzymes may also contribute to the measured lipolysis. Evidence is presented consistent with the hypothesis that a surface-located lipoprotein lipase is responsible for the observed lipolysis. Very little lipoprotein lipase activity is released from the cell surface by heparin.During trioleoylglycerol hydrolysis, non-esterified oleate does not accumulate in the cells or in the medium in quantities stoicheiometric with glycerol release. Analyses indicate that it passes into the cells without prior equilibration with the extracellular oleate pool(s). Once inside the cells, oleate is rapidly re-esterified into the triacylglycerol fraction.The possible relevance of these findings to the physiological mechanism of fatty acid uptake from triacylglycerol at the capillary endothelium is discussed.