Effects of AMP-activated protein kinase (AMPK) signaling and essential amino acids on mammalian target of rapamycin (mTOR) signaling and protein synthesis rates in mammary cells.

Regulation of mammary protein synthesis potentially changes the relationships between AA supply and milk protein output represented in current nutrient requirement models. Glucose and AA regulate muscle protein synthesis via cellular signaling pathways involving mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK). The objective of this study was to investigate the effects of essential AA (EAA) and acetate or glucose on mTOR and AMPK signaling pathways and milk protein synthesis rates. A bovine mammary epithelial cell line, MAC-T, was subjected to different media containing 0 or 3.5 mmol/L EAA concentrations with 0 or 5 mmol/L acetate or 0 or 17.5 mmol/L glucose in 2 separate 2 × 2 factorial studies. In a separate set of experiments, lactogenic bovine mammary tissue slices were subjected to the same treatments except that the low EAA treatment contained a low level of EAA (0.18 mmol/L). Supplementation of EAA enhanced phosphorylation of mTOR (Ser2448) and eukaryotic initiation factor 4E binding protein 1 (4EBP1, Thr37/46), and reduced phosphorylation of eukaryotic elongation factor 2 (eEF2, Thr56) in MAC-T cells. Concentration of ATP and phosphorylation of AMPK increased and decreased, respectively, in the presence of EAA in MAC-T cells. Acetate, EAA, or glucose numerically reduced AMPK phosphorylation by about 16% in mammary tissue slices. Provision of EAA increased phosphorylation of mTOR and 4EBP1, intracellular total EAA concentration, and casein synthesis rates in mammary tissue slices, irrespective of the presence of acetate or glucose in the medium. Phosphorylation of mTOR had a marginally negative association with AMPK phosphorylation, which was positively related to eEF2 phosphorylation. Casein synthesis rates were positively and more strongly linked to mTOR phosphorylation than the negative link between eEF2 phosphorylation and casein synthesis rates. A 100% increase in mTOR phosphorylation was associated with an increase in the casein synthesis rate of 0.74%·h(-1), whereas a 100% increase in eEF2 phosphorylation was related to a decline in the casein synthesis rate of 0.33%·h(-1). Although AMPK phosphorylation was responsive to cellular energy status and had a negative effect on mTOR-mediated signals in bovine mammary epithelial cells, its effect on milk protein synthesis rates appeared to be marginal compared with the mTOR-mediated regulation of milk protein synthesis by EAA.

Effects of milk replacer composition on growth, body composition, and nutrient excretion in preweaned Holstein heifers.

Twenty-four newborn Holstein heifer calves were fed 1 of 4 milk replacers (MR): control (20% CP, 21% fat; MR fed at 441 g/d); high protein/low fat (HPLF; 28% CP, 20% fat; MR fed at 951 g/d); high protein/high fat (HPHF; 27% CP, 28% fat; MR fed at 951 g/d); and HPHF MR fed at a higher rate (HPHF+; 27% CP, 28% fat; MR fed at 1,431 g/d). Dry calf starter (20% CP, 1.43% fat) composed of ground corn (44.4%), 48% CP soybean meal (44.4%), cottonseed hulls (11.2%), and molasses (1.0%) was offered free choice. Heifers were obtained from a commercial dairy, blocked by groups of 8 in the order acquired, and randomly assigned to treatments within group. Upon arrival at the research farm, heifers were fed the control for 2 feedings. Treatments were imposed when heifers were 4 +/- 1 d of age. Heifers were on study for 61 +/- 1 d. Body weight and body size measures were taken weekly. Four-day total collection of feed refusals, feces, and urine was initiated at 57 +/- 1 d of age. Heifers were slaughtered at the end of the collection period to evaluate body composition. Preplanned contrasts were used to compare control to all, HPLF to HPHF, and HPHF to HPHF+. Heifers fed the control diet consumed more starter than those fed other treatment diets, but their total dry matter intake and apparent dry matter digestibility were lowest. Fecal output was highest in heifers fed the control diet, whereas urine output and urine N excretion were lowest. Nitrogen intake and urine N excretion were greater for heifers fed HPHF+ compared with HPHF but were not affected by MR fat content (HPLF vs. HPHF). Retention (g/d) of N and P was greater in heifers fed all nutrient-dense diets compared with those fed the control diet, but was not improved by increasing fat in the milk replacer (HPLF vs. HPHF) or by increasing the amount fed. Addition of fat to the milk replacer (HPLF vs. HPHF) increased empty body weight fat content without improving average daily gain or frame measures. Increasing the volume fed (HPHF vs. HPHF+) increased growth rate and empty body weight, but HPHF+ heifers were neither taller nor longer and their carcasses contained more fat. Clear improvements in growth and nutrient retention were observed with more nutrient-dense diets, but most of the improvements were seen with the increased protein intake relative to the control MR; adding fat to the high protein MR did not further improve lean tissue gain.

Casein secretion and cytological differentiation in mammary tissue from bulls of high or low genetic merit.

We evaluated tissue content and secretion of milk proteins by mammary parenchymal explants prepared from tissue of mature Holstein bulls as a possible index for genetic merit. Explants from eight selection-line and eight control-line bulls were cultured for 48 or 96 h in medium with 5% fetal bovine serum with or without the addition of lactogenic hormones. Four selection-line and four control-line bulls were also treated for 7 d with estrogen and progesterone or placebo before tissue was removed on d 15 of the experiment. Overall, tissue content and secretion of casein were increased approximately twofold in selection-line bulls. Differences between genetic lines were evident only with the addition of lactogenic hormones. However, treatment of bulls with steroids was not necessary for casein secretion or detection of differences between genetic lines. Averaged for both lines, 96-h media concentrations of casein were 89-fold greater for explants cultured with added lactogenic hormones. Epithelial cells were classified as nonsecretory, droplet-containing, or degenerated. The appearance of droplet-containing cells was markedly increased in cultures supplemented with lactogenic hormones, and the cells were most often located within epithelial folds or pockets. Only occasionally did we observe areas of alveolar-like tissue. The data demonstrate that it is possible to induce the secretion of casein in mammary explants prepared from sexually mature bulls, that lactogenic hormones markedly stimulate secretion, and that differences in genetic merit may be expressed.

Effects of plane of nutrition, growth hormone and unsaturated fat on mammary growth in prepubertal lambs.

Effects of plane of nutrition, growth hormone treatment and dietary polyunsaturated fat on mammary development were assessed in prepubertal ewe lambs. Ten lambs (15.6 kg initial body weight) were assigned to each of four treatment groups. Treatments included: (A) lambs given ad libitum access to a high-energy ration; (G) fed as group A but injected s.c. daily with bovine growth hormone (.08 mg/kg BW); (R) feed intake restricted to achieve a growth rate of about 120 g/d; and (S) given ad libitum access to a ration including a formaldehyde-protected sunflower seed supplement. Diets were isonitrogenous and isocaloric and were fed from about 7 to 22 wk of age. At slaughter, the udder was removed and divided into halves. One udder half was trimmed and the parenchyma was dissected from surrounding stroma for compositional analyses. Final body weight and rate of gain averaged 41.2, 42.8, 27.3 and 46.0 kg, and 251, 267, 112 and 311 g/d for groups A, G, R and S, respectively. In the same treatment order, mammary parenchymal weight averaged 15.3, 20.3, 14.2 and 25.6 g. Parenchymal dry, fat-free tissue and DNA content were 1.7, 2.5, 1.3 g and 12.6, 24.1, 10.4 and 18.8 mg, respectively. Mammary parenchymal development was stimulated by polyunsaturated fat but was not affected by plane of nutrition. Supplemental dietary lipid may promote mammogenesis in ruminants.

Effects of plane of nutrition, growth hormone and unsaturated fat on growth hormone, insulin and prolactin receptors in prepubertal lambs.

The influence of plane of nutrition, growth hormone (GH) treatment and dietary polyunsaturated fat on serum concentrations of GH and insulin (INS) and binding capacities of GH, INS and prolactin (PRL) in liver, mammary parenchyma and adipose tissue was assessed in prepubertal ewe lambs. Ten lambs were assigned to each of four treatment groups. Treatments included: (A) lambs with ad libitum access to a high-energy ration; (G) lambs fed as group A and treated with bovine GH (.08 mg/kg/d); (R) lambs with feed intake restricted to limit ADG to about 120 g; and (S) lambs with ad libitum access to a ration including formaldehyde-protected sunflower seed. Diets, all approximately isonitrogenous and isocaloric, were fed from about 7 to 22 wk of age. Weekly blood samples were collected during wk 6 to 14 of the trial. Averaged across sampling dates, mean serum concentrations of GH were elevated in G lambs (P less than .05) and INS concentrations differed in the order G greater than A greater than R = S (P less than .05). Crude membranes for binding assays were prepared from liver, mammary parenchyma and adipose tissue. Mean concentrations of GH receptors in liver and PRL receptors in mammary parenchyma were elevated in group S lambs (P less than .01). Dietary polyunsaturated fat increased the number of GH receptors in liver and PRL receptors in mammary parenchyma. Increased availability of receptors may mediate the stimulation of mammary growth observed in lambs fed polyunsaturated fat.

Effect of dose of bovine growth hormone on milk composition: alpha-lactalbumin, fatty acids, and mineral elements.

Tissue-specific effects of bovine growth hormone on lactating dairy cows were examined by analysis of milk composition. Milk samples were from 6 cows that received subcutaneous injections of 0, 5, 10, 25, 50, and 100 IU/d of growth hormone in a Latin-square design. Samples from the last 5 d of each 10-d treatment period were pooled for analyses of milk components. Concentration of alpha-lactalbumin in milk increased progressively across the treatment range up to 32% above controls (1.30 mg/nl) at the 100 IU dose. Specific alpha-lactalbumin synthesis (expressed as a percent of total milk protein) was also increased. Secretion of de novo synthesized fatty acids (short and medium chain length) in milk was increased, but response plateaued between the 50 and 100 IU/d. Secretion of preformed (long-chain) fatty acids progressively increased across the entire dose range. Thus, the percentage of long-chain fatty acids in milk increased at the highest doses of hormone. Changes in fatty acid composition of milk were apparently related to energy status; the milk response to 50 and 100 IU/d of growth hormone caused cows to be in or near negative energy balance. Exogenous growth hormone did not affect the concentration of calcium, phosphorus, sodium, iron, copper, and manganese in milk. Results are consistent with growth hormone functioning in homeorhesis to coordinate the partitioning of all nutrients to support the increased secretion of milk and milk components.