The Effects And Molecular Mechanisms Of VFA And Amino Acids On GH And PRL Synthesis And Secretion In DCAPCs

Low nutritional quality of cow’s milk has increasingly become a severechallenge faced by the healthy development of dairy industry in China. Milk fat andprotein are the main substances to determine the nutritional quality of cow’s milk.Cow’s milk quality could be affected by many factors, where neuroendocrine factorsplay an important regulatory role to fat and protein contents in cow’s milk. Growthhormone (GH) and prolactin (PRL) is the most important hormone for the regulationof milk protein and milk fat synthesis in dairy cows. The concentration of precursorsof milk components in serum may also regulate the GH and PRL synthesis orsecretion in dairy cow anterior pituitary cells (DCAPCs) with direct or indirectmanners. Therefore, the objective of the present experiment was to investigate theeffects and signaling mechanisms of short-chain fatty acids (SCFAs), β-hydroxy-butyric acid (BHBA), arginin, and tyrosine on the GH and PRL synthesis or secretionin DCAPCs cultured in vitro. In addition, the nutrient-hormone interaction networksregulating milk fat and milk protein synthesis were constructed to get a thoroughunderstanding about the effects and molecular mechanisms of neuroendocrine factorson fatty acid and protein synthesis of cow’s milk.Immunohistochemistry assay of GH in the anterior pituitary glands showed thatmost somatotrophs were located within the lateral wings inferior border of the anteriorpituitary. Immunohistochemistry assay of PRL in the anterior pituitary glands showedthat most lactotrophs were located within the lateral wings superior border of theanterior pituitary. Tissues where somatotrophs or lactotrophs were located were dicedinto small pieces at less than1mm3and incubated in CMF-HBSS containing0.3%Itype collagenase,0.1%hyaluronidase, and0.1‰Dnase. After6d in culture, theDCAPCs displayed a monolayer, cobblestone, epithelial-like morphology which arethe typical characteristics of the anterior pituitary cells. GH or PRL immunoreactivitywas present in DCAPCs. Therefore, this model can be used successfully for the study of the mechanisms of hypothalamic factors, peripheral hormones, cytokines, ordietary nutrients regulating GH or PRL synthesis and release.SCFAs bind to GPR41/43and lead to dissociation of heterotrimeric G proteincomplex into Gαiand βγ subunit, thereby inhibiting adenylyl cyclase(AC) activity.Inactivated AC results in a decrease of intracellular cAMP levels and a subsequentreduction in protein kinase A (PKA) activity. Inhibition of PKA activity inhibitscAMP response element binding protein (CREB) phosphorylation, thereby leading toa decrease of bovine GH gene transcription. The change of the phosphorylation levelsof CREB may decrease the phosphorylated CREB binding protein (CBP) or CBPcomplex interacts with pituitary specific transcription factor-1(Pit-1) resultantinhibition of transcription of the bovine GH and PRL gene. Consequently, SCFAsinhibit bovine GH and PRL gene transcription in DCAPCs. Moreover, SCFAsdecrease viability of DCAPCs, which may be an important cause for the inhibition ofGH and PRL synthesis or secretion.BHBA bind to GPR109A and lead to dissociation of heterotrimeric G proteincomplex into Gαiand βγ subunit, thereby inhibiting AC activity. Inactivated ACresults in a decrease of intracellular cAMP levels and a subsequent reduction in PKAactivity and CREB phosphorylation level, thereby leading to a decrease of bovine GHand Pit-1gene transcription. The proximal promoters of the GH and PRL gene includebinding sites for Pit-1, thus, the change of phosphorylation levels of CREB couldchange GH and PRL gene transcription level directly or indirectly. Otherwise, BHBAincreased the expression of monocarboxylate transporter1(MCT1) in DCAPCs. Andour finding demonstrates that BHBA triggers the AMPK pathway resulting in theinhibition of GH translation.NO is synthesized by nitric oxide synthase (NOS) enzyme group in DCAPCs,which utilizes the semiessential amino acid L-arginine as a substrate. Thearginine-NO pathway induced the increase of intracellular calcium concentration inDCAPCs, which may be an important cause for the increase of GH and PRL synthesisor secretion. Tyrosine was converted to dopamine(DA) by tyrosine hydroxylase (TH).The inhibitory action of tyrosine on PRL secretion is caused by DA biding to the D2receptors on the DCAPCs cell membrane. In addition, the nutrient-hormone interaction networks regulating milk fat andmilk protein synthesis were constructed. These results will provide scientific basis tofurther study the regulatory mechanisms of cow’s milk fat and protein synthesis andimprove the nutritional quality of cow’s milk.