Short-chain fatty acids inhibit growth hormone and prolactin gene transcription via cAMP/PKA/CREB signaling pathway in dairy cow anterior pituitary cells

Abstract

Short-chain fatty acids (SCFAs) play a key role in altering carbohydrate and lipid metabolism, influence endocrine pancreas activity, and as a precursor of ruminant milk fat. However, the effect and detailed mechanisms by which SCFAs mediate bovine growth hormone (GH) and prolactin (PRL) gene transcription remain unclear. In this study, we detected the effects of SCFAs (acetate, propionate, and butyrate) on the activity of the cAMP/PKA/CREB signaling pathway, GH, PRL, and Pit-1 gene transcription in dairy cow anterior pituitary cells (DCAPCs). The results showed that SCFAs decreased intracellular cAMP levels and a subsequent reduction in PKA activity. Inhibition of PKA activity decreased CREB phosphorylation, thereby inhibiting GH and PRL gene transcription. Furthermore, PTX blocked SCFAs- inhibited cAMP/PKA/CREB signaling pathway. These data showed that the inhibition of GH and PRL gene transcription induced by SCFAs is mediated by Gi activation and that propionate is more potent than acetate and butyrate in inhibiting GH and PRL gene transcription. In conclusion, this study identifies a biochemical mechanism for the regulation of SCFAs on bovine GH and PRL gene transcription in DCAPCs, which may serve as one of the factors that regulate pituitary function in accordance with dietary intake.

Figure 1

The effect of short-chain fatty acids (SCFAs) on the mRNA levels of GH, PRL and Pit-1 in dairy cow anterior pituitary cells (DCAPCs). DCAPCs were treated with 0, 0.1, 0.5, 1.0, 2.5 and 5.0 mmol/L acetate (A); propionate (B); and butyrate (C) for 24 h, respectively. The cells were also treated with or without prior PTX incubation (100 ng/L) for 2 h, and then stimulated with 1.0 mmol/L acetate (D); propionate (E); or butyrate (F) for 24 h, respectively. Each treatment replicated 12 times. * p < 0.05, ** p < 0.01 vs. the control group.

Figure 2

The effect of SCFAs on the intracellular cAMP levels in DCAPCs. DCAPCs were treated with 1.0 mmol/L acetate, propionate, and butyrate for 0, 0.5, 1.0, 2.0, and 3.0 h, respectively (A); The cells were also treated with or without prior PTX incubation (100 ng/L) for 2 h, and then stimulated with 1.0 mmol/L acetate, propionate, or butyrate for 3 h, respectively (B). Each treatment replicated 12 times. * p < 0.05, ** p < 0.01 vs. the control group.

Figure 3

The effect of SCFAs on the PKA activity in DCAPCs. The cells were treated with or without prior PTX incubation (100 ng/L) for 2 h, and then stimulated with 1.0 mmol/L acetate, propionate, or butyrate for 3 h, respectively. Each treatment replicated 12 times. *p < 0.05, **p < 0.01 vs. the control group; # p < 0.05 vs. the non PTX group.

Figure 4

The effect of SCFAs on the CREB phosphorylation in DCAPCs. The cells were treated with or without prior PTX incubation (100 ng/L) for 2 h, and then stimulated with 1.0 mmol/L acetate, propionate, or butyrate for 6 h, respectively. Each treatment replicated 12 times. * p < 0.05, ** p < 0.01 vs. the control group; ## p < 0.01 vs. the non PTX group.

Figure 5

SCFAs inhibit the cAMP/PKA/CREB signaling pathway to mediate GH and PRL gene transcription in DCAPCs. SCFAs bind to GPCR and lead to dissociation of heterotrimeric G protein complex into G_αi and G_βγ subunit. The exchange of GTP from GDP results in activation of the G_αi, thereby inhibiting adenylyl cyclase (AC) activity. This process results in a decrease of intracellular cAMP levels and a subsequent reduction in PKA activity. Inhibition of PKA activity inhibits CREB phosphorylation, thereby decreasing GH and PRL gene transcription directly or indirectly. The A-protomer of PTX penetrates into the host cells result in inactivation of G_αi, hence, inhibits SCFAs mediated signaling pathway. In addition, inhibition of PKA activity may result in a decreased of Ca²⁺ influx through decreased l-type Ca²⁺ channel mean open time. Therefore, SCFAs may also inhibit bovine GH and PRL secretion via G_αi-mediated inhibition of Ca²⁺ channel.