J.P. Cant

Nutrient availability and lactogenic hormones regulate mammary protein synthesis through the mammalian target of rapamycin signaling pathway

The nutritional and endocrine factors affecting protein translation in the bovine mammary gland, and the molecular mechanisms mediating their effects, are not well understood. The objective of this study was to assess the role of the mammalian target of rapamycin (mTOR) signaling pathway in the regulation of mammary protein synthesis by nutrients and hormones. Mammary epithelial acini isolated from lactating dairy cows were treated in medium containing, alone or in combination, a mixture of AA or glucose and acetate (GA) as energy substrates, or a combination of the lactogenic hormones hydrocortisone, insulin, and prolactin (HIP). Changes in the rate of mammary protein synthesis and the phosphorylation state of components of the mTOR signaling pathway were measured. Mammary protein synthesis was 50% higher with increased availability of AA in medium. The presence of GA or treatment of mammary acini with HIP alone did not affect mammary protein synthesis. The stimulation of mammary protein synthesis by AA was enhanced by HIP treatment, but not by the presence of GA in medium. Treatment of mammary acini with HIP induced the phosphorylation of protein kinase B. This effect was augmented in the presence of either AA or GA in medium. The stimulation of mammary protein synthesis by AA and its enhancement by HIP were associated with increased phosphorylation of the mTOR substrates, p70 ribosomal protein S6 kinase-1, and eukaryotic initiation factor 4E (eIF4E)-binding protein-1 (4E-BP1), and dissociation of 4E-BP1 from eIF4E. The results suggest that nutrients and hormones may modulate mammary protein synthesis through the mTOR signaling pathway.

Nutritional Stimulation of Milk Protein Yield of Cows Is Associated with Changes in Phosphorylation of Mammary Eukaryotic Initiation Factor 2 and Ribosomal S6 Kinase 1

Production of protein by the lactating mammary gland is stimulated by intake of dietary energy and protein. Mass-action effects of essential amino acids (EAA) cannot explain all of the nutritional response. Protein synthesis in tissues of growing animals is regulated by nutrients through the mammalian target of rapamycin (mTOR) and integrated stress response (ISR) networks. To explore if nutrients signal through the mTOR and ISR networks in the mammary gland in vivo, lactating cows were feed-deprived for 22 h and then infused i.v. for 9 h with EAA+ glucose (Glc), Glc only, l-Met+l-Lys, l-His, or l-Leu. Milk protein yield was increased 33 and 27% by EAA+Glc and Glc infusions, respectively. Infusions of Met+Lys and His generated 35 and 41%, respectively, of the EAA+Glc response. Infusion of EAA+Glc reduced phosphorylation of the ISR target, eukaryotic initiation factor(eIF) 2, in mammary tissue and increased phosphorylation of the mTOR targets, ribosomal S6 kinase 1 (S6K1) and S6. Both responses are stimulatory to protein synthesis. Glucose did not significantly increase mammary S6K1 phosphorylation but reduced eIF2 phosphorylation by 62%, which implicates the ISR network in the stimulation of milk protein yield. In contrast, the EAA infusions increased (P < 0.05) or tended to increase (P < 0.1) mammary mTOR activity and only His, like Glc, decreased eIF2 phosphorylation by 62%. Despite activation of these protein synthesis signals to between 83 and 127% of the EAA+Glc response, EAA infusions produced less than one-half of the milk protein yield response generated by EAA+Glc, indicating that ISR and mTOR networks exert only a portion of the control over protein yield.

Mathematical analysis of the relationship between blood flow and uptake of nutrients in the mammary glands of a lactating cow

A dynamic mathematical model of blood flow regulation in the mammary glands of a lactating cow was constructed from a principle of local vasodilator release in response to changes in intracellular adenylate charge. An equation was derived to predict uptake of the milk precursors acetate, glucose, amino acids and fatty acids, as affected by mammary blood flow rate. Metabolism of the precursors to milk components and CO2 was simulated with a set of empirically derived equations. Relative rates of ATP production and utilization regulated both the number of perfused capillaries and the conductance of arteriolar segments in the mammary glands. The model simulated local control phenomena of functional and reactive hyperaemia, and simulation of autoregulation under changing arterial pressure suggested a predominance of precapillary sphincter regulation. It was predicted that an increase in blood flow without the mammary capacity to utilize blood metabolites efficiently would be detrimental to milk synthesis. Conversely, increased blood flow through changes in mammary activity resulted in predictions of higher milk production. It was proposed that the equation for uptake, [equation: see text] be used in analysis of mammary arteriovenous differences.

Dietary and Endogenous Amino Acids Are the Main Contributors to Microbial Protein in the Upper Gut of Normally Nourished Pigs

Although amino acids (AA) synthesized by enteric microbiota in the upper gut of nonruminants can be absorbed, they do not necessarily make a net contribution to the hosts AA supply. That depends on whether protein or nonprotein nitrogen sources are used for microbial protein production. We determined the contributions of urea, endogenous protein (EP), and dietary protein (DP) to microbial valine (M.VAL) at the distal ileum of growing pigs, based on isotope dilutions after a 4-d continuous infusion of l-[1-(13)C]valine to label EP and of [(15)N(15)N]urea. Eight barrows were assigned to either a cornstarch and soybean meal-based diet with or without 12% added fermentable fiber from pectin. Dietary pectin did not affect (P > 0.10) the contributions of the endogenous and DP to M.VAL. More than 92% of valine in microbial protein in the upper gut was derived from preformed AA from endogenous and DP, suggesting that de novo synthesis makes only a small contribution to microbial AA.

Responses of the bovine mammary glands to absorptive supply of single amino acids

In this review, we discuss the mechanismsof responses of various tissues of the lactating dairy cow, particularly the mammary glands, to perturbations in supply of single amino acids that result in observed milk protein yields. Additions of methionine, lysine, histidine or leucine to the absorptive supply cause arterial concentrations of these amino acids to increase, mammary extractions to drop and mammary blood flow to decrease. Single subtractions of essential amino acids have the opposite effect. Changes in mammary blood flow that have been recorded can be explained as attempts by the mammary glands to restore intracellular ATP balance in the face of altered concentrations of energy metabolites in the general circulation. In a quantitative sense, milk protein yield is relatively insensitive to fluctuations in arterial amino acid concentrations but can be stimulated by any one of a number of amino acids. In this context, it is suggested that the designation of a limiting amino acid is not appropriate t...

Effects of supplementing glycerol and soybean oil in drinking water on feed and water intake, energy balance, and production performance of periparturient dairy cows

The objective of this study was to determine the effects of supplementing glycerol and soybean oil in drinking water on feed and water intake, calculated energy balance, and production performance of periparturient dairy cows. Ninety multiparous Holstein dairy cows were randomly assigned to 1 of 3 treatments: 1) no nutrients supplemented in the drinking water (control); 2) 20 g/L of glycerin supplemented in the drinking water (glycerol); and 3) 10 g/L of soybean oil supplemented in the drinking water (SBO). The trial lasted from 7 d prepartum to 7 d postpartum. Cows were offered a close-up and milking cow TMR for ad libitum intake, pre- and postpartum, respectively. The dry matter intake of cows supplemented with glycerol and SBO was lower than for the control cows throughout the experimental period but not different from each other. Water intake for the control cows was greater than the average for the glycerol and SBO cows prepartum, and greater than for SBO cows but similar to that of glycerol cows postpartum. Glycerol cows consumed more water than SBO cows. There were no differences in energy intake and energy balance of the cows pre- and postpartum. Serum triacylglycerol concentration for glycerol cows was lower than for the control and SBO cows prepartum and was lower than for the SBO cows postpartum. There were no differences in the serum nonesterified fatty acids and glucose concentrations throughout the experiment. There were no differences in the serum beta-hydroxybutyrate (BHBA) concentrations at parturition, but serum BHBA concentration of the glycerol cows was greater than for control and SBO cows during the prepartum period. However, during the postpartum period, serum BHBA concentrations of the control cows were greater than for glycerol and SBO cows. There were no differences in calf birth weights or milk yield and composition. Although the glucogenic property of glycerol supplemented in the drinking water at 20 g/L may not have been sufficient to elicit a milk yield response, it did reduce the concentration of BHBA postpartum.

Selenomethionine increases proliferation and reduces apoptosis in bovine mammary epithelial cells under oxidative stress.

The decline in mammary epithelial cell number as lactation progresses may be due, in part, to oxidative stress. Selenium is an integral component of several antioxidant enzymes. The present study was conducted to examine the effect of oxidative stress and selenomethionine (SeMet) on morphology, viability, apoptosis, and proliferation of bovine mammary epithelial cells (BMEC) in primary culture. Cells were isolated from mammary glands of lactating dairy cows and grown for 3 d in a low-serum gel system containing lactogenic hormones and 0 or 100 μM H2O2 with 0, 10, 20, or 50 nM SeMet. Hydrogen peroxide stress increased intracellular H2O2 to 3 times control concentrations and induced a loss of cuboidal morphology, cell-cell contact, and viability of BMEC by 25%. Apoptotic cell number more than doubled during oxidative stress, but proliferating cell number was not affected. Supplementation with SeMet increased glutathione peroxidase activity 2-fold and restored intracellular H2O2 to control levels with a concomitant return of morphology and viability to normal. Apoptotic BMEC number was decreased 76% below control levels by SeMet and proliferating cell number was increased 4.2-fold. These findings suggest that SeMet modulated apoptosis and proliferation independently of a selenoprotein-mediated reduction of H2O2. In conclusion, SeMet supplementation protects BMEC from H2O2-induced apoptosis and increased proliferation and cell viability under conditions of oxidative stress.

Decline in mammary translational capacity during intravenous glucose infusion into lactating dairy cows

The objective of this study was to determine effects of glucose on milk protein yield and mammary mammalian target of rapamycin (mTOR) activity in dairy cattle in early lactation. Eight multiparous cows at 73 ± 8 d in milk were randomly assigned to 2 treatments in a crossover design for two 6-d periods. Treatments were jugular infusion of either saline (Sal) or 896 g/d glucose (Glc). All cows were fed a total mixed ration with 42% neutral detergent fiber, had free access to water, and were milked twice a day. Within each period, blood samples were taken (d 5) and mammary tissue was collected by biopsy (d 6) from each hindquarter for Western blot analysis. In addition to Sal and Glc treatments, on d 6, rapamycin dissolved in 50% dimethyl sulfoxide was administered via the teat canals into the left quarters, with a control solution administered into the right quarters. Rapamycin had no effect on milk protein yields or phosphorylation state of mTOR signaling proteins. Infusions of Glc significantly increased milk yield but only tended to increase milk protein yields. Milk fat tended to be decreased in cows infused with Glc, whereas lactose yields were significantly increased. Glucose infusion did not increase plasma glucose levels, but insulin and nonessential AA concentrations increased by 21 and 16%, respectively, branched-chain AA concentrations decreased 24%, and essential AA concentrations tended to decrease by 14%. Infusion of Glc significantly decreased abundances of both phosphorylated and total ribosomal S6 kinase 1 (S6K1) in mammary tissue by 27 and 11%, respectively. Abundance of phosphorylated eukaryotic initiation factor 4E-binding protein 1 (4EBP1) decreased significantly by 25%, whereas total 4EBP1 exhibited a tendency to decrease by 16%. We conclude that the mTOR signaling pathway is not the only regulator of milk protein synthesis. Decreases in essential AA concentrations in plasma suggest that protein synthesis was stimulated in nonmammary tissues of the body, presumably skeletal muscle.

Effect of enhanced whole-milk feeding in calves on subsequent first-lactation performance.

The objective of this study was to determine the effect of enhanced whole-milk (WM) feeding systems in calves from birth to 8wk of age on subsequent first-lactation performance. The experiment was conducted as a completely randomized design consisting of 2 treatment groups. At birth, 152 Holstein heifer calves were randomly assigned to 1 of 2 treatments: (i) 4L of WM/d or (ii) 8L of WM/d. The calves were bucket fed 2 or 4L of WM twice daily at 0700 and 1600h. Each calf was housed individually in temperature-controlled nurseries and had ad libitum access to water and textured calf starter daily. Calves consumed greater volumes of textured calf starter when fed 4 versus 8L of WM/d. Water intakes mirrored starter intakes, leading to greater water consumption at weaning. Calves reared on 8L of WM/d were heavier at d 56 than calves reared on 4L of WM/d. The average daily gain of the calves offered 8L of WM/d from d 0 to 56 was greater than that of calves offered 4L of WM/d. Structural measurements were significantly greater for calves that consumed 8L of WM/d. The differences observed in withers height and live BW due to WM feeding level were not apparent by 3 and 12mo of age, respectively. Rumen pH was higher in calves that consumed 8L of WM/d than in calves that consumed 4L of WM/d. Whole-milk feeding level did not affect age at first calving or milk-production parameters. These results suggest that enhanced WM feeding improved growth performance until 3mo of age. However, first-lactation results indicated no lactation-performance benefits of increased nutrition and growth performance during the milk-fed period in dairy calves.

Simulation analysis of substrate utilization in the mammary gland of lactating cows.

A kinetic modelling approach was developed and investigated with the aim of predicting the utilization of major substrates in the mammary gland and milk secretion rates in the lactating cow at varying concentrations of substrate in arterial blood. The model includes kinetic equations of transport and metabolism of glucose, acetate, free amino acids and free fatty acids in secretory cells and a phenomenological description of autoregulation of local blood flow, in which an energy criterion of control has been used. The predicted relationships between the rate of milk secretion and glucose levels in the blood are consistent with experimental results. Differential stimulation of alpha-lactalbumin synthesis causes increments in local blood flow and milk secretion rate in the model. The results of the study suggest that there is no simple relationship between the level of substrates in the blood and milk yield and contents of fat and protein in milk. This is because the effect on production of varying patterns of substrate concentrations in the blood is mediated by network interactions at the level of secretory cell metabolism and microcirculation. However, dynamic modelling provides a rational framework for developing such predictive tools.