Initiation and elongation steps of mRNA translation are involved in the increase in milk protein yield caused by growth hormone administration during lactation (original) (raw)
Related papers
2013
The objective of this study was to determine if increased milk protein synthesis observed in lactating dairy cows treated with growth hormone (GH) was associated with mechanistic (or mammalian) target of rapamycin complex 1 (mTORC1) regulation of downstream factors controlling nucleocytoplasmic export and translation of mRNA. To address this objective, biochemical indices of mammary growth and secretory activity and the abundance and phosphorylation status of mTORC1 pathway factors were measured in mammary tissues harvested from nonpregnant lactating dairy cows 6 d after treatment with a slow-release formulation of GH or saline (n = 4/group). Treatment with GH increased mammary parenchymal weight and total protein content and tended to increase ribosome number and cell size, whereas protein synthetic efficiency, capacity, and cell number were unchanged. Cellular abundance of the mTORC1 components mTOR and (phosphorylated) mTOR Ser2448 increased, as did complex eukaryotic initiation factor 4E:eukaryotic initiation factor 4E binding protein 1 (eIF4E:4EBP1), whereas no change was observed for mTORC1-downstream targets 4EBP1, 4EBP1 Ser65 , p70/p85 S6K and p70 S6K Thre389/p85 S6K Thre412. Changes in activation were not observed for any of the targets measured. These results indicate that GH treatment influences signaling to mTORC1 but not downstream targets involved in the nucleocytoplasmic export and translation of mRNA. Increased eIF4E:4EBP1 complex formation indicates involvement of the mitogen-activated protein kinase (MAPK) pathway. Abundance of MAPK pathway components eIF4E, eIF4E Ser209 , eIF4E:eIF4G complex, MAP kinase-interacting serine/threonine-protein kinase 1 (MKNK1), MKNK1 Thr197202 , and ribosomal protein S6 kinase, 90kDa, polypeptide 1 (RPS6KA1) increased significantly in response to GH, whereas relative activation of the proteins was unchanged. Expres-sion of IGFBP3 and IGFBP5 increased, that of IGF1R decreased, and that of IGF1 remained unchanged in response to GH. PatSearch analysis of the milk caseins α S1 -casein, α S2 -casein, and β-casein, MAPK signaling target RPS6KA1, and proliferation gene IGFBP3 mRNA indicated that all contained putative eIF4E-sensitivity elements. In response to GH, these genes were all upregulated, suggesting that increased abundance of eIF4E and eIF4E Ser209 plays a role in mediating their nucleocytoplasmic export. We propose that, in response to GH, the IGF1-IGF1R-MAPK signaling cascade regulates eIF4E-mediated nucleocytoplasmic export and translation of mRNA, whereas mTOR controls cell renewal, cell turnover, and rRNA transcription through an alternative signaling cascade.
Administration of bovine growth hormone (GH) has been shown to increase milk production in dairy cows by coordinating the metabolism of various organs and tissues, so that nutrients are repartitioned to the mammary gland to support increased requirements for milk synthesis. Some of the targets for GH are adipose tissue, skeletal muscle, liver and the mammary gland. In muscle GH decreases glucose uptake and increases amino acid catabolism efficiency, whilst the liver responds by increasing the rate of basal gluconeogenesis through lypolysis of adipose reserves. We have previously shown that the increase in total milk protein yield following a six day treatment of dairy cows in mid-lactation with GH is associated with a change in protein translation initiation and elongation. However, whether these effects are tissue-specific has not been explored. The objective of this study was to determine if GH administration influences protein translation initiation and elongation in liver and sk...
Physiological Genomics, 2011
Growth hormone is one of few pharmacologic agents known to augment milk production in humans. We hypothesized that recombinant human GH (rhGH) increases the expression of cell proliferation and milk protein synthesis genes. Sequential milk and blood samples collected over four days were obtained from five normal lactating women. Following 24 h of baseline milk and blood sampling, rhGH (0.1 mg/kg/day) was administered subcutaneously once daily for 3 days. Gene expression changes were determined by microarray studies utilizing milk fat globule RNA isolated from each milk sample. Following rhGH administration, DNA synthesis and cell cycle genes were induced, while no significant changes were observed in the expression of milk synthesis genes. Expression of glycolysis and citric acid cycle genes were increased by day 4 compared with day 1, while lipid synthesis genes displayed a circadian-like pattern. Cell cycle gene upregulation occurred after a lag of ∼2 days, likely explaining the f...
Isolation and characterization of mRNA from mammary gland of lactating cow
Agricultural and Biological Chemistry, 1981
mRNA was isolated from mammaryglands of lactating cow by affinity chromatography on poly(U)-Sepharose. The mRNAwas heterogeneous on 3% agarose gel electrophoresis in the presence of 6m urea. The molecular weight of the main peak was estimated to be 3.3 x 105. The mRNA was translated in a cell-free protein synthesizing system derived from wheat germ extract, and the translation products were analysed by the indirect immunoprecipitation method using specific antisera for casein components. About 50% of the total protein directed by this mRNAwas casein. The relative amounts of asl-, /?-, and fc-casein in the translation products were nearly the same as those in bovine milk. The immunoprecipitates were analysed on sodiumdodecyl sulfatepolyacrylamide gradient gel (15~20%) electrophoresis, and their mobilities were compared with those ofdephosphorylated and non-glycosylated casein as standard, asl-and /c-Casein synthesized in vitro migrated more slowly than standard caseins, while synthesized^-casein migrated slightly faster than the standard jS-casein. Casein is synthesized by the mammary epithelial cells of lactating animals under the multiple control by several hormones.1} After being synthesized on membrane bound polysomes, casein is transported to the Golgi apparatus,2) where it undergoes enzymic modification such as phosphorylation3) and glycosylation.4) During these processings, casein is assembled into casein micelle and then is secreted out of the cells.5) Therefore, mammary gland can be used as one of the model systems to study the mechanisms of the regulation of gene expression by hormones and post-translational modifications of secretory proteins. Isolation of mRNA is prerequisite to the studies of these mechanisms. Casein mRNA'shave been isolated from the mammary glands of ewe,6) rat,7>8) mouse,8) guinea pig,9) and rabbit.10
Essential Amino Acid Regulation of Cell Signaling and Casein Synthesis in Mammary Tissue
Specific AA have been demonstrated to activate signaling pathways that regulate translation initiation and to stimulate protein synthesis in mammary tissue. The objectives of this research were to determine the response to Ile, Leu, Met, and Thr in cellular signaling and α-S1 casein fractional synthesis rates (CFSR). An experiment was developed as a composite design. The experiment was replicated in tissue corresponding to 5 cows. Mammary tissue slices (0.12 ± 0.02 g) from lactating dairy cows were incubated 4 h in treatment media enriched with 2 H 5 Phe. Following incubation, slices were homogenized in lysis buffer and caseins were precipitated by acidification to pH 4.6. An aliquot of the pellet was trypsinized and 2 H 5 Phe enrichment in the 34-NLLRFFVAPFPE-45 peptide of α-S1 casein was measured by MALDI TOF-MS and used to determine CFSR (%/h). Western immunoblotting was performed to identify total and site-specific phosphorylated mammalian target of rapamycin (mTOR, Ser2448), eukaryotic elongation factor (eEF) 2 (Thr56), ribosomal protein (rp) S6 (Ser235/236), and eukaryotic initiation factor (eIF) 2α (Ser51). Addition of Ile, Leu, Met, or Thr had no effect on eIF2α phosphorylation. Isoleucine positively affected mTOR, and rpS6, and negatively affected eEF2 phosphorylation. Leu had a similar effect on eEF2, but not on mTOR or rpS6, and these two AA inhibited each other. Thr negatively interacted with Ile on mTOR and rpS6, and with Leu on eEF2. Increasing concentrations of Ile, Leu, Met, and Thr caused curvilinear increases in CFSR. The maximum response to Ile, Leu, iii Met, and Thr was at 71, 49, 60, and 65% of DMEM concentrations, respectively. All maximums were above plasma AA concentrations observed in lactating cows fed to meet NRC requirements. The CFSR estimated at those maximums were similar between AA (3.6 ± 0.6 %/h). Individual AA effects on CFSR did not correlate with mTOR signaling. Independent CFSR responses to individual essential AA observed in this study contradict the single-limiting AA theory assumed in current requirement systems. The saturable responses of CFSR to these 4 AA also demonstrate the deficiencies of a fixed postabsorptive AA efficiency approach for determining AA requirements for milk protein synthesis.
Domestic Animal Endocrinology, 2004
In ruminants, both milking frequency and exogenous GH treatment affect milk production. In a previous report, we showed that the modulation of milk yield due to variations in milking frequency and GH treatment was associated with variations in mammary cell numbers. The aim of this study was to clarify the different mechanisms governing the effects of GH treatment and milking frequency on signal transducer and activator of transcription (Stat) expression and activation, and on the expression of genes involved in mammary cell differentiation. Six Saanen goats in late lactation were milked once daily from one half-udder and thrice daily from the other half-udder for 23 days. At the same time, the goats were divided into two groups: GH-treated versus control group. After slaughter of the goats, soluble mammary proteins and RNA were extracted from half-udder samples. Levels of Stat5, Stat3 and Stat1 proteins and the Stat activation by phosphorylation were analysed by Western blot. The amounts of Stat5 protein and mRNA were significantly elevated by GH treatment in all half-udders (milked once or thrice daily). Positive Stat5 immunoreactivity was principally localised in the nuclei of epithelial cells, with heterogeneous intensity between cells. No significant changes in Stat5 protein phosphorylation levels were observed. Furthermore, GH significantly increased Stat1 protein levels, without modifying the level of Stat1 tyrosine phosphorylation, and tended to reduce the abundance of Stat3 protein. In contrast, milking frequency failed to modify Stat gene expression, protein level and phosphorylation. Using Northern blot, we showed that levels of kappa casein and prolactin receptor mRNA were not affected by the treatments. These observations suggest that GH probably acts specifically on mammary cells by regulating the expression of Stat1, 3 and 5. In contrast, milking frequency does not act through this regulatory pathway.
PloS one, 2015
The objectives of this study were to (1) identify changes in plasma and mammary intracellular amino acid (AA) profiles in dairy cows treated with growth hormone (GH), and (2) evaluate the expression of mammary gland genes involved in the transport of AA identified in (1). Eight non-pregnant (n = 4 per group) lactating dairy cows were treated with a single subcutaneous injection of either a slow-release formulation of commercially available GH (Lactotropin 500 mg) or physiological saline solution. Six days after treatment, cows were milked and blood collected from the jugular vein for the analysis of free AA in the plasma. Cows were euthanized and mammary tissue harvested. Treatment with GH increased milk, protein, fat and lactose yields, with no effect on dry matter intake. Plasma concentrations of lysine and group I AA decreased significantly, and arginine, methionine, tyrosine and arginine-family AA tended to decrease in GH-treated cows. Concentrations of intracellular glycine, se...
Functional & Integrative Genomics, 2009
Murine milk protein gene expression requires insulin, hydrocortisone, and prolactin; however, the role of insulin is not well understood. This study, therefore, examined the requirement of insulin for milk protein synthesis. Mammary explants were cultured in various combinations of the lactogenic hormones and global changes in gene expression analysed using Affymetrix microarray. The expression of 164 genes was responsive to insulin, and 18 were involved in protein synthesis at the level of transcription and posttranscription, as well as amino acid uptake and metabolism. The folate receptor gene was increased by fivefold, highlighting a potentially important role for the hormone in folate metabolism, a process that is emerging to be central for protein synthesis. Interestingly, gene expression of two milk protein transcription factors, Stat5a and Elf5, previously identified as key components of prolactin signalling, both showed an essential requirement for insulin. Subsequent experiments in HCll cells confirmed that Stat5a and Elf5 gene expression could be induced in the absence of prolactin but in the presence of insulin. Whereas prolactin plays an essential role in phosphorylating and activating Stat5a, gene expression is only induced when insulin is present. This indicates insulin plays a crucial role in the transcription of the milk protein genes.
Growth Hormone and Milking Frequency Act Differently on Goat Mammary Gland in Late Lactation
Journal of Dairy Science, 2003
In ruminants, milk yield can be affected by treatment with growth hormone (rbGH) and/or changes in frequency of milking. Frequent milkings encourage the maintenance of lactation, whereas infrequent milkings result in mammary involution. Our objective was to evaluate the influence of rbGH treatment and milking frequency on mammary gland morphology and milk composition. After adaptation to twice-daily milkings, six Saanen goats in late lactation were milked once daily from one udder-half and thrice-daily from the other udder-half. Concurrently, three of the six goats received daily injections of rbGH. After 23 d of treatment, milking frequency significantly affected milk yield (+8% vs. −26% for thrice-vs. once-daily milking). Additionally, treatments of rbGH increased milk yield from thrice-daily milked udder-halves (+19%), but failed to abate the reduction in milk yield from once-daily milked udderhalves (−31%). Mammary glands were heavier in the frequently milked udder-halves and in GH-treated goats. Based on histological and DNA analysis of mammary tissues, it was determined that milking frequency clearly affected epithelial cell numbers and alveolar diameter, whereas rbGH induced a potential cell hypertrophy and only a tendency to increase and/or maintain the mammary cell number. RNA concentration and kappa casein gene expression were not affected by treatments. In udder-halves milked once-daily, low casein:whey protein ratios, high Na + :K + ratios, and high somatic cell counts (SCC) were indicative of changes in epithelial permeability, which rbGH treatment facilitated. The present data suggest that milking frequency and exogenous treatments of rbGH use different cellular mechanisms to influence mammary gland morphology and milk production. (