A Comparative Review of the Extrinsic and Intrinsic Factors Regulating Lactose Synthesis.

Milk is critical for the survival of all mammalian offspring, where its production by a mammary gland is also positively associated with its lactose concentration. A clearer understanding of the factors that regulate lactose synthesis stands to direct strategies for improving neonatal health while also highlighting opportunities to manipulate and improve milk production and composition. In this review we draw a cross-species comparison of the extra- and intramammary factors that regulate lactose synthesis, with a special focus on humans, dairy animals, and rodents. We outline the various factors known to influence lactose synthesis including diet, hormones, and substrate supply, as well as the intracellular molecular and genetic mechanisms. We also discuss the strengths and limitations of various in vivo and in vitro systems for the study of lactose synthesis, which remains an important research gap.

A Comparative Review of the Cell Biology, Biochemistry, and Genetics of Lactose Synthesis.

Lactose is the primary carbohydrate in the milk of most mammals and is unique in that it is only synthesized by epithelial cells in the mammary glands. Lactose is also essential for the development and nutrition of infants. Across species, the concentration of lactose in milk holds a strong positive correlation with overall milk volume. Additionally, there is a range of examples where the onset of lactose synthesis as well as the content of lactose in milk varies between species and throughout a lactation. Despite this diversity, the precursors, genes, proteins and ions that regulate lactose synthesis have not received the depth of study they likely deserve relative to the significance of this simple and abundant molecule. Through this review, our objective is to highlight the requirements for lactose synthesis at the biochemical, cellular and temporal levels through a comparative approach. This overview also serves as the prelude to a companion review describing the dietary, hormonal, molecular, and genetic factors that regulate lactose synthesis.

Engineering endogenous fermentative routes in ethanologenic Escherichia coli W for bioethanol production from concentrated whey permeate.

Whey permeate (WP) is a lactose-rich waste effluent, generated during cheese manufacturing and further valorization steps, such as protein extraction. The production of ethanol by WP fermentation has been proposed to increase cost-competitiveness of dairy waste processing. In previous work, the Escherichia coli W strain was selected for its efficient growth in dairy waste and it was engineered to convert lactose into ethanol as the main fermentation product from WP and concentrated WP (CWP). To improve its performance, here the lactate dehydrogenase, fumarate reductase and pyruvate formate lyase fermentative routes were disrupted, obtaining new deletion strains. In test tubes, growth and fermentation profiles obtained in standard laboratory media and CWP showed large differences, and were affected by oxygen, medium and ethanologenic gene expression level. Among the tested strains, the one with triple deletion was superior in both high-oxygen and low-oxygen test tube fermentations, in terms of ethanol titer, rate and yield. The improved performance was due to a lower inhibition by medium acidification rather than an improved ethanol flux. The parent and triple deletion strains showed similar performance indexes in pH-controlled bioreactor experiments. However, the deletion strain showed lower base consumption and residual waste, in terms of both dry matter and chemical oxygen demand after distillation. It thus represents a step towards sustainable dairy wastewater valorization for bioenergy production by decreasing process operation costs.

AMP-activated protein kinase controls lipid and lactose synthesis in bovine mammary epithelial cells.

The synthesis of milk components in bovine mammary epithelial cells (BMEC) requires an adequate supply of energy. The AMP-activated protein kinase (AMPK) is a cellular energy gauge that controls anabolic and catabolic processes to maintain a balance between energy supply and demand. The objectives of this study were to assess the role of AMPK on de novo lipid and lactose synthesis, as well as its regulation by glucose and acetate availability in BMEC. We isolated primary BMEC from the mammary tissue of 3 lactating Holstein cows and differentiated them with lactogenic hormones for 4 d. We measured protein abundance, site-specific phosphorylation, and proteolytic processing by immunoblotting. We quantified the expression of genes involved in lipid and lactose synthesis using real-time quantitative PCR. We measured de novo lipid and lactose synthesis by incorporation of radioactive substrates. We analyzed data by ANOVA using a randomized complete block design with PROC MIXED in SAS. To assess the effect of AMPK activation on milk component synthesis, we treated BMEC with 100 µM A-769662 (A76; an allosteric activator of AMPK) or vehicle control for 16 h. Consistent with activation of AMPK, A76 increased phosphorylation of its downstream targets ACC Ser79 and TSC2 Ser1387 by 144% and 26%, respectively. Activation of AMPK decreased lipid synthesis by 19%. This effect was accompanied by increased expression of FABP3. Activation of AMPK reduced the proportion of mature SREBP-1c. In addition, AMPK activation reduced lactose synthesis by 24% and lowered the expression of SLC2A1, the gene encoding GLUT1. To assess the regulation of AMPK by energy substrate availability, we incubated BMEC in a control medium containing 4 mM D-glucose and 1 mM sodium acetate, or medium lacking glucose or acetate, for 4 h. Compared with the control medium, deprivation of glucose or acetate promoted AMPKα phosphorylation at Thr172 by 84% or 58%, respectively. Activation of AMPK was significantly increased in BMEC only when the medium was devoid of glucose for at least 4 h. We concluded that activation of AMPK inhibits de novo lipid and lactose synthesis in BMEC. Further studies are needed to assess the physiological relevance of AMPK activation for milk composition in vivo and to identify the mechanisms mediating its effects on milk component synthesis.

Meta-analysis of mammary RNA seq datasets reveals the molecular understanding of bovine lactation biology.

A better understanding of the biology of lactation, both in terms of gene expression and the identification of candidate genes for the production of milk and its components, is made possible by recent advances in RNA seq technology. The purpose of this study was to understand the synthesis of milk components and the molecular pathways involved, as well as to identify candidate genes for milk production traits within whole mammary transcriptomic datasets. We performed a meta-analysis of publically available RNA seq transcriptome datasets of mammary tissue/milk somatic cells. In total, 11 562 genes were commonly identified from all RNA seq based mammary gland transcriptomes. Functional annotation of commonly expressed genes revealed the molecular processes that contribute to the synthesis of fats, proteins, and lactose in mammary secretory cells and the molecular pathways responsible for milk synthesis. In addition, we identified several candidate genes responsible for milk production traits and constructed a gene regulatory network for RNA seq data. In conclusion, this study provides a basic understanding of the lactation biology of cows at the gene expression level.

GLUT1 and GLUT8 support lactose synthesis in Golgi of murine mammary epithelial cells.

The mammary gland increases energy requirements during pregnancy and lactation to support epithelial proliferation and milk nutrients synthesis. Lactose, the principal carbohydrate of the milk, is synthetized in the Golgi of mammary epithelial cells by lactose synthase from glucose and UPD galactose. We studied the temporal changes in the expression of GLUT1 and GLUT8 in mammary gland and their association with lactose synthesis and proliferation in BALB/c mice. Six groups were used: virgin, pregnant at 2 and 17 days, lactating at 2 and 10 days, and weaning at 2 days. Temporal expression of GLUT1 and GLUT8 transporters by qPCR, western blot and immunohistochemistry, and its association with lactalbumin, Ki67, and cytokeratin 18 within mammary tissue was studied, along with subcellular localization. GLUT1 and GLUT8 transporters increased their expression during mammary gland progression, reaching 20-fold increasing in GLUT1 mRNA at lactation (p < 0.05) and 2-fold at protein level for GLUT1 and GLUT8 (p < 0.05 and 0.01, respectively). The temporal expression pattern was shared with cytokeratin 18 and Ki67 (p < 0.01). Endogenous GLUT8 partially co-localized with 58 K protein and α-lactalbumin in mammary tissue and with Golgi membrane-associated protein 130 in isolated epithelial cells. The spatial-temporal synchrony between expression of GLUT8/GLUT1 and alveolar cell proliferation, and its localization in cis-Golgi associated to lactose synthase complex, suggest that both transporters are involved in glucose uptake into this organelle, supporting lactose synthesis.

Microbial production of sialic acid and sialylated human milk oligosaccharides: Advances and perspectives.

Sialic acids (SAs) are important functional sugars, and monomers of sialylated human milk oligosaccharides (sialylated HMOs or sialyllactoses), which are crucial for improving infant development and can facilitate infant brain development, maintain brain health, and enhance immunity. The most common form of SA is N-acetylneuraminic acid (NeuAc), and the main forms of sialyllactoses are 6'-sialyllactose (6'-SL) and 3'-sialyllactose (3'-SL). As functional food additive, the demand for NeuAc and sialyllactoses will continuously increase due to their wide and important fields of application. However, NeuAc and sialyllactoses produced by traditional extraction methods are inefficient and may cause allergen contamination, and cannot keep up with the rapidly increasing market demand. Therefore, the production of NeuAc and sialyllactoses by sustainable biotechnological methods have attracted increasing attention. In particular, the development of metabolic engineering and synthetic biology techniques and strategies have promoted efficient biosynthesis of NeuAc and sialyllactoses. In this review, we first discussed the application of NeuAc and sialyllactoses. Secondly, metabolic engineering and protein engineering-fueled progress of whole-cell catalysis and de novo synthesis of NeuAc and sialyllactoses were systematically summarized and compared. Furthermore, challenges of efficient microbial production of NeuAc and sialyllactoses as well as strategies for overcoming the challenges were discussed, such as clustered regularly interspaced short palindromic repeats interference (CRISPRi)-aided identification of key precursor transport pathways, synergistically debottleneck of kinetic and thermodynamic limits in synthetic pathways, and dynamic regulation of metabolic pathways for balancing cell growth and production. We hope this review can further facilitate the understanding of limiting factors that hampered efficient production of sialic acid and sialyllactoses, as well as contribute to the development of strategies for the construction of efficient production hosts for high-level production of sialic acid and sialyllactose based on synthetic biology tools and strategies.

Engineering two species of yeast as cell factories for 2'-fucosyllactose.

Oligosaccharides present in human breast milk have been linked to beneficial effects on infant health. Inclusion of these human milk oligosaccharides (HMOs) in infant formula can recapitulate these health benefits. As a result, there is substantial commercial interest in a cost-effective source of HMOs as infant formula ingredients. Here we demonstrate that the yeast species Saccharomyces cerevisiae and Yarrowia lipolytica both can be engineered to produce 2'-fucosyllactose (2'FL), which is the most abundant oligosaccharide in human breast milk, at high titer and productivity. Both yeast species were modified to enable uptake of lactose and synthesis of GDP-fucose - the two precursors of 2'FL - by installing a lactose transporter and enzymes that convert GDP-mannose to GDP-fucose. Production of 2'FL was then enabled by expression of α-1,2-fucosyltransferases from various organisms. By screening candidate transporters from a variety of sources, we identified transporters capable of exporting 2'FL from yeast, which is a key consideration for any biocatalyst for 2'FL production. In particular, we identified CDT2 from Neurospora crassa as a promising target for further engineering to improve 2'FL efflux. Finally, we demonstrated production of 2'FL in fermenters at rates and titers that indicate the potential of engineered S. cerevisiae and Y. lipolytica strains for commercial 2'FL production.

Feed and nitrogen efficiency are affected differently but milk lactose production is stimulated equally when isoenergetic protein and fat is supplemented in lactating dairy cow diets.

Fifty-six Holstein-Friesian cows were used in a randomized complete block design to test the effects of supplemental energy from protein (PT) and fat (FT) on lactation performance and nutrient digestibility in a 2 × 2 factorial arrangement. During the control period, cows were adapted for 28 d to a basal total mixed ration consisting of 34% grass silage, 33% corn silage, 5% grass hay, and 28% concentrate on a dry matter (DM) basis. Experimental rations were fed for 28 d immediately following the control period and consisted of (1) low protein, low fat (LP/LF), (2) high protein, low fat (HP/LF), (3) low protein, high fat (LP/HF), or (4) high protein and high fat (HP/HF). To obtain the HP and HF diets, intake of the basal ration was restricted and supplemented isoenergetically (net energy basis) with 2.0 kg/d of rumen-protected protein (soybean + rapeseed, 50:50 mixture on DM basis) and 0.68 kg/d of hydrogenated palm fatty acids (FA) on a DM basis. Milk production and composition, nutrient intake, and apparent digestibility were measured during the final 7 d of the control and experimental periods. No interaction was found between PT and FT on milk production and composition. Yields of milk, fat- and protein-corrected milk, and lactose increased in response to PT and FT and lactose concentration was unaffected by treatment. Milk protein concentration and yield increased in response to PT, and protein yield tended to increase in response to FT. Milk fat concentration and yield increased in response to FT and were unaffected by PT. Milk urea concentration increased and nitrogen efficiency decreased in response to PT. Feed and nitrogen efficiency were highest on the LP/HF diet and both parameters increased in response to FT, whereas milk urea concentration was not affected by FT. Energy from fat increased the concentration and yield of ≥16-carbon FA in milk and decreased the concentration of FA synthesized de novo, but had no effect on their yield. Concentration and yield of de novo-synthesized FA increased in response to PT. Concentration and yield of polyunsaturated FA increased and decreased in response to PT and FT, respectively. Apparent total-tract digestibility of crude fat decreased in response to PT, and FT increased crude protein digestibility. Energy supplementation through rumen-inert hydrogenated palm FA appears to be an efficient feeding strategy to stimulate milk production with regard to feed and nitrogen efficiency compared with supplementing an isoenergetic level of rumen-protected protein.

Peroxisome proliferator-activated receptor ß/δ does not regulate glucose uptake and lactose synthesis in bovine mammary epithelial cells cultivated in vitro.

The hypothesis of the study was that inhibition of PPARß/δ increases glucose uptake and lactose synthesis in bovine mammary epithelial cells by reducing the expression of the glucose transporter mRNA destabiliser calreticulin. Three experiments were conducted to test the hypothesis using immortalised bovine mammary alveolar (MACT) and primary bovine mammary (PBMC) cells. In Experiment 1, the most effective dose to inhibit PPARß/δ activity among two synthetic antagonists (GSK-3787 and PT-s58) was assessed using a gene reporter assay. In Experiment 2, the effect on glucose uptake and lactose synthesis was evaluated by measuring glucose and lactose in the media and expression of related key genes upon modulation of PPARß/δ using GSK-3787, the synthetic PPARß/δ agonist GW-501516, or a combination of the two in cells cultivated in plastic. In Experiment 3, the same treatments were applied to cells cultivated in Matrigel and glucose and lactose in media were measured. In Experiment 1 it was determined that a significant inhibition of PPARß/δ in the presence or absence of fetal bovine serum was achieved with ≥ 1000 nm GSK-3787 but no significant inhibition was observed with PT-s58. In Experiment 2, inhibition of PPARß/δ had no effect on glucose uptake and lactose synthesis but they were both increased by GW-501516 in PBMC. The mRNA abundance of PPARß/δ target gene pyruvate dehydrogenase kinase 4 was increased but transcription of calreticulin was decreased (only in MACT cells) by GW-501516. Treatment with GSK-3787 did not affect the transcription of measured genes. No effects on glucose uptake or lactose synthesis were detected by modulation of PPARß/δ activity on cells cultivated in Matrigel. The above data do not provide support for the original hypothesis and suggest that PPARß/δ does not play a major role in glucose uptake and lactose synthesis in bovine mammary epithelial cells.

Engineering of α-1,3-fucosyltransferases for production of 3-fucosyllactose in Escherichia coli.

Fucosyllactoses (FLs), present in human breast milk, have been reported to benefit human health immensely. Especially, 3-fucosyllactose (3-FL) has numerous benefits associated with a healthy gut ecosystem. Metabolic engineering of microorganisms is thought to be currently the only option to provide an economically feasible route for large-scale production of 3-FL. However, engineering principles for α-1,3-fucosyltransferases (1,3-FTs) are not well-known, resulting in the lower productivity of 3-FL than that of 2'-fucosyllactose (2'-FL), although both 2'-FL and 3-FL follow a common pathway to produce GDP-L-fucose. The C-terminus of 1,3-FTs is composed of heptad repeats, responsible for dimerization of the enzymes, and a peripheral membrane anchoring region. It has long been thought that truncation of most heptad repeats, retaining just 1 or 2, helps the soluble expression of 1,3-FTs. However, whether the introduction of truncated version of 1,3-FTs enhances the production of 3-FL in a metabolically engineered strain, is yet to be tested. In this study, the effect of these structural components on the production of 3-FL in Escherichia coli was evaluated through systematic truncation and elongation of the C-terminal regions of three 1,3-FTs from Helicobacter pylori. Although these three 1,3-FTs contained heptad repeats and membrane-anchoring regions of varying lengths, they commonly exhibited an optimal performance when the number of heptad repeats was increased, and membrane-binding region was removed. The production of 3-FL could be increased 10-20-fold through this simple strategy.

Enzymatic Synthesis of 6'-Sialyllactose, a Dominant Sialylated Human Milk Oligosaccharide, by a Novel exo-α-Sialidase from Bacteroides fragilis NCTC9343.

Gut bacteria provide a rich source of glycosidases that can recognize and/or hydrolyze glycans for nutrition. Interestingly, some glycosidases have also been found to catalyze transglycosylation reactions in vitro and thus can be used for oligosaccharide synthesis. In this work, six putative and one known exo-α-sialidase genes-three from Bacteroides fragilis NCTC9343, three from Clostridium perfringens ATCC 13124, and one known from Bifidobacterium bifidum JCM1254-were subjected to gene cloning and heterogeneous expression in Escherichia coli The recombinant enzymes were purified, characterized for substrate specificity, and screened for transglycosylation activity. A sialidase, named BfGH33C, from B. fragilis NCTC9343 was found to possess excellent transglycosylation activity for the synthesis of sialylated human milk oligosaccharide. The native BfGH33C was a homodimer with a molecular weight of 113.6 kDa. The Km and kcat values for 4-methylumbelliferyl N-acetyl-α-d-neuraminic acid and sialic acid dimer were determined to be 0.06 mM and 283.2 s-1, and 0.75 mM and 329.6 s-1, respectively. The enzyme was able to transfer sialyl from sialic acid dimer or oligomer to lactose with high efficiency and strict α2-6 regioselectivity. The influences of the initial substrate concentration, pH, temperature, and reaction time on transglycosylation were investigated in detail. Using 40 mM sialic acid dimer (or 40 mg/ml oligomer) and 1 M lactose (pH 6.5) at 50°C for 10 min, BfGH33C could specifically produce 6'-sialyllactose, a dominant sialylated human milk oligosaccharide, at a maximal conversion ratio above 20%. It provides a promising alternative to the current chemical and enzymatic methods for obtaining sialylated oligosaccharides.IMPORTANCE Sialylated human milk oligosaccharides are significantly beneficial to the neonate, as they play important roles in supporting resistance to pathogens, gut maturation, immune function, and brain and cognitive development. Therefore, access to the sialylated oligosaccharides has attracted increasing attention both for the study of saccharide functions and for the development of infant formulas that could mimic the nutritional value of human milk. Nevertheless, nine-carbon sialic acids are rather complicated for the traditional chemical modifications, which require multiple protection and deprotection steps to achieve a specific glycosidic bond. Here, the exo-α-sialidase BfGH33C synthesized 6'-sialyllactose in a simple step with high transglycosylation activity and strict regioselectivity. Additionally, it could utilize oligosialic acid, which was newly prepared in an easy, economical way to reduce the substrate cost, as a glycosyl donor. All the studies laid a foundation for the practical use of BfGH33C in large-scale synthesis of sialylated oligosaccharides in the future.

Quercetin improves postpartum hypogalactia in milk-deficient mice via stimulating prolactin production in pituitary gland.

Postpartum dysgalactia is a common clinical problem for lactating women. Seeking out the safe and efficient phytoestrogens will be a promising strategy for postpartum dysgalactia therapy. In this study, the postpartum mice within four groups, including control group, the model group, and the treatment groups intragastrically administrated with normal saline, bromocriptine, bromocriptine plus 17α-ethinyl estradiol, and bromocriptine plus quercetin, respectively, were used. The results showed that quercetin, a kind of natural phytoestrogen, could efficiently promote lactation yield and mammary gland development in the agalactosis mice produced by bromocriptine administration. Mechanically, quercetin, such as 17α-ethinyl estradiol, significantly stimulated prolactin (PRL) production and deposition in the mammary gland in the agalactosis mice determined by western blotting, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay, respectively. Furthermore, quercetin could increase the expression of ß-casein, stearoyl-CoA desaturase, fatty acid synthase, and α-lactalbumin in the breast tissues that are responsible for the production of fatty acid, lactose, and galactose in the milk at the transcriptional level determined by quantitative polymerase chain reaction. Specifically, quercetin promoted primary mammary epithelial cell proliferation and stimulated prolactin receptor (PRLR) expression probably via AKT activation in vitro. In conclusion, this study indicates that estrogen-like quercetin promotes mammary gland development and lactation yield in milk-deficient mice, probably via stimulating PRL expression and release from the pituitary gland, as well as induces PRLR expression in primary mammary epithelial cells.

Effects of graded removal of lysine from an intravenously infused amino acid mixture on lactation performance and mammary amino acid metabolism in lactating goats.

To investigate responses of milk protein synthesis and mammary AA metabolism to a graded decrease of postruminal Lys supply, 4 lactating goats fitted with jugular vein, mammary vein, and carotid artery catheters and transonic blood flow detectors on the external pudic artery were used in a 4 × 4 Latin square experiment. Goats were fasted for 24 h and then received a 9-h intravenous infusion of an AA mixture plus glucose. Milk yield was recorded and samples were taken in h 2 to 8 of the infusion period; a mammary biopsy was performed in the last hour. Treatments were graded decrease of lysine content in the infusate to 100 (complete), 60, 30, or 0% as in casein. Lysine-removed infusions linearly decreased milk yield, tended to decrease lactose yield, and tended to increase milk fat to protein ratio. Milk protein content and yield were linearly decreased by graded Lys deficiency. Mammary Lys uptake was concomitantly decreased, but linear regression analysis found no significant relationship between mammary Lys uptake and milk protein yield. Treatments had no effects on phosphorylation levels of the downstream proteins measured in the mammalian target or rapamycin pathway except for a tended quadratic effect on that of eukaryotic initiation factor 2, which was increased and then decreased by graded Lys deficiency. Removal of Lys from the infusate linearly increased circulating glucagon and glucose. Removal of Lys from the infusate linearly decreased arterial and venous concentrations of Lys. Treatments also had a significant quadratic effect on venous Lys, suggesting mechanisms to stabilize circulating Lys at a certain range. The 2 infusions partially removing Lys resulted in a similar 20% decrease, whereas the 0% Lys infusion resulted in an abrupt 70% decrease in mammary Lys uptake compared with that of the full-AA mixture infusion. Consistent with the abrupt decrease, mammary Lys uptake-to-output ratio decreased from 2.2 to 0.92, suggesting catabolism of Lys in the mammary gland could be completely prevented when the animal faced severe Lys deficiency. Mammary blood flow was linearly increased, consistent with the linearly increased circulating nitric oxide by graded Lys deficiency, indicating mechanisms to ensure the priority of the mammary gland in acquiring AA for milk protein synthesis. Infusions with Lys removed increased mammary clearance rate of Lys numerically by 2 to 3 fold. In conclusion, the decreased milk protein yield by graded Lys deficiency was mainly a result of the varied physiological status, as indicated by the elevated circulating glucagon and glucose, rather than a result of the decreased mammary Lys uptake or depressed signals in the mTOR pathway. Mechanisms of Lys deficiency to promote glucagon secretion and mammary blood flow and glucagon to depress milk protein synthesis need to be clarified by future studies.

Metabolic Transition of Milk Lactose Synthesis and Up-regulation by AKT1 in Sows from Late Pregnancy to Lactation.

Lactose plays a crucial role in controlling milk volume by inducing water toward into the mammary secretory vesicles from the mammary epithelial cell cytoplasm, thereby maintaining osmolality. In current study, we determined the expression of several lactose synthesis related genes, including glucose transporters (glucose transporter 1, glucose transporter 8, sodium-glucose cotransporter 1, sodium-glucose cotransporter 3, and sodium-glucose cotransporter 5), lactose synthases (α-lactalbumin and ß1,4-galactosyltransferase), and hexokinases (hexokinase-1 and hexokinase-2) in sow mammary gland tissue at day 17 before delivery, on the 1st day of lactation and at peak lactation. The data showed that glucose transporter 1 was the dominant glucose transporter within sow mammary gland and that expression of each glucose transporter 1, sodium-glucose cotransporter 1, hexokinase-1, hexokinase-2, α-lactalbumin, and ß1,4-galactosyltransferase were increased (p < 0.05) when the sows transited from late pregnancy to peak lactation. AKT1 over-expressed mammary epithelial cells were then constructed, and the results indicated that AKT1 increases (p < 0.01) the expression of hexokinase-1 and glucose transporter 1. In summary, lactose synthesis was significantly elevated with the increase of milk production and AKT1 could positively regulate lactose synthesis.

An insufficient glucose supply causes reduced lactose synthesis in lactating dairy cows fed rice straw instead of alfalfa hay.

The objective of the present study was to investigate the nutrient availability for milk production in the mammary gland of lactating cows fed different forage-based diets. The 3 diets contained 30% corn stover (CS), 30% rice straw (RS), or 23% alfalfa hay plus 7% Chinese wild rye hay (AH) as a forage source. All diets contained 15% of DM as corn silage and 55% of DM as concentrate. The percentage of milk lactose was always lower in the RS-fed cows than in the cows fed AH or CS during the 12-wk feeding trial ( < 0.01). Ruminal propionate concentrations were lower in the RS group than in the AH group ( = 0.03). The ratio of insulin to glucagon in the mammary venous plasma was greater in the AH group than in the CS or RS group ( = 0.04). The abundance of the pyruvate carboxylase mRNA in the liver was lower in the RS group than in the AH or CS group ( = 0.04), and the abundance of mitochondrial phosphoenolpyruvate carboxykinase, IGF-1 receptor, and phosphofructokinase-liver, phosphofructokinase-muscle, and phosphofructokinase-platelet mRNA in the liver were lower in the RS group than in the AH group ( < 0.05). The mammary glucose uptake was greater in the AH-fed cows than in the CS- or RS-fed cows ( = 0.02). The mRNA abundance of the glucose transporters in the mammary gland was similar among the 3 treatments. The mRNA abundance of α-lactalbumin in the mammary gland of the cows fed RS tended to be greater compared with that of the cows fed AH or CS. The milk potassium concentration was greater in the cows fed RS than those fed AH or CS ( < 0.01). In summary, the insufficient ruminal propionate concentrations in the cows fed RS were associated with lower gluconeogenesis in the liver, resulting in the shortage of glucose supply for mammary utilization.

Effects of glucose on lactose synthesis in mammary epithelial cells from dairy cow.

BACKGROUND: Lactose, as the primary osmotic component in milk, is the major determinant of milk volume. Glucose is the primary precursor of lactose. However, the effect of glucose on lactose synthesis in dairy cow mammary glands and the mechanism governing this process are poorly understood. RESULTS: Here we showed that glucose has the ability to induce lactose synthesis in dairy cow mammary epithelial cells, as well as increase cell viability and proliferation. A concentration of 12 mM glucose was the optimum concentration to induce cell growth and lactose synthesis in cultured dairy cow mammary epithelial cells. In vitro, 12 mM glucose enhanced lactose content, along with the expression of genes involved in glucose transportation and the lactose biosynthesis pathway, including GLUT1, SLC35A2, SLC35B1, HK2, ß4GalT-I, and AKT1. In addition, we found that AKT1 knockdown inhibited cell growth and lactose synthesis as well as expression of GLUT1, SLC35A2, SLC35B1, HK2, and ß4GalT-I. CONCLUSIONS: Glucose induces cell growth and lactose synthesis in dairy cow mammary epithelial cells. Protein kinase B alpha acts as a regulator of metabolism in dairy cow mammary gland to mediate the effects of glucose on lactose synthesis.

Disorder in Milk Proteins: α-Lactalbumin. Part B. A Multifunctional Whey Protein Acting as an Oligomeric Molten Globular "Oil Container" in the Anti-Tumorigenic Drugs, Liprotides.

This is a second part of the three-part article from a series of reviews on the abundance and roles of intrinsic disorder in milk proteins. We continue to describe α-lactalbumin, a small globular Ca2+-binding protein, which besides being one of the two components of lactose synthase that catalyzes the final step of the lactose biosynthesis in the lactating mammary gland, possesses a multitude of other functions. In fact, recent studies indicated that some partially folded forms of this protein possess noticeable bactericidal activity and other forms might be related to induction of the apoptosis of tumor cells. In its anti-tumorigenic function, oligomeric α-lactalbumin serves as a founding member of a new family of anticancer drugs termed liprotides (for lipids and partially denatured proteins), where an oligomeric molten globular protein acts as an "oil container" or cargo for the delivery of oleic acid to the cell membranes.

Pro-inflammatory cytokine TNF-α is a key inhibitory factor for lactose synthesis pathway in lactating mammary epithelial cells.

Lactose is a milk-specific carbohydrate synthesized by mammary epithelial cells (MECs) in mammary glands during lactation. Lactose synthesis is downregulated under conditions causing inflammation such as mastitis, in which MECs are exposed to high concentrations of inflammatory cytokines. In this study, we investigated whether inflammatory cytokines (TNF-α, IL-1ß, and IL-6) directly influence the lactose synthesis pathway by using two types of murine MEC culture models: the monolayer culture of MECs to induce lactogenesis; and the three-dimensional culture of MECs surrounded by Matrigel to induce reconstitution of the alveolar structure in vitro. TNF-α caused severe down-regulation of lactose synthesis-related genes concurrently with the degradation of glucose transporter 1 (GLUT1) from the basolateral membranes in MECs. IL-1ß also caused degradation of GLUT1 along with a decrease in the expression level of ß-1,4-galactosylransferase 3. IL-6 caused both up-regulation and down-regulation of the expression levels of lactose synthesis-related genes in MECs. These results indicate that TNF-α, IL-1ß, and IL-6 have different effects on the lactose synthesis pathway in MECs. Furthermore, TNF-α triggered activation of NFκB and inactivation of STAT5, suggesting that NFκB and STAT5 signaling pathways are involved in the multiple adverse effects of TNF-α on the lactose synthesis pathway.

XBP1 Regulates the Biosynthetic Capacity of the Mammary Gland During Lactation by Controlling Epithelial Expansion and Endoplasmic Reticulum Formation.

Cells composing the mammary secretory compartment have evolved a high capacity to secrete not only proteins but also triglycerides and carbohydrates. This feature is illustrated by the mouse, which can secrete nearly twice its own weight in milk proteins, triglycerides and lactose over a short 20-day lactation. The coordination of synthesis and export of products in other secretory cells is orchestrated in part by the transcription factor X-box binding protein 1 (XBP1). To assess the role of XBP1 in mammary epithelial cells (MEC), we studied floxed XBP1 female mice lacking (wild type; WT) or expressing the Cre recombinase under the control of the ovine ß-lactoglobulin promoter (ΔXBP1(MEC)). Pregnant ΔXBP1(MEC) females had morphologically normal mammary development and gave birth to the same number of pups as WT mice. Their litters, however, suffered a weight gain deficit by lactation day 3 (L3)3 that grew to 80% by L14. ΔXBP1(MEC) dams had only modest changes in milk composition (-21% protein, +24% triglyceride) and in the expression of associated genes in isolated MEC. By L5, WT glands were fully occupied by dilated alveoli, whereas ΔXBP1(MEC) glands contained fewer, mostly unfilled alveoli and retained a prominent adipocyte population. The smaller epithelial compartment in ΔXBP1(MEC) glands was explained by lower MEC proliferation and increased apoptosis. Finally, endoplasmic reticulum ribbons were less abundant in ΔXBP1(MEC) at pregnancy day 18 and failed to increase in abundance by L5. Collectively, these results show that XBP1 is required for MEC population expansion during lactation and its ability to develop an elaborate endoplasmic reticulum compartment.