Hepatic gene expression in multiparous Holstein cows treated with bovine somatotropin and fed n-3 fatty acids in early lactation

Abstract

Multiparous cows were fed supplemental dietary fat and treated with bST to assess effects of n-3 fatty acid supply, bovine somatotropin (bST), and stage of lactation on hepatic gene expression. Cows were blocked by expected calving date and previous milk yield and assigned randomly to treatment. Supplemental dietary fat was provided from calving as either whole high-oil sunflower seeds (SS; 10% of dietary dry matter; n-6/n-3 ratio of 4.6) as a source of linoleic acid or a mixture of Alifet-High Energy and Alifet-Repro (AF; 3.5 and 1.5% of dietary dry matter, respectively; n-6/n-3 ratio of 2.6) as a source of protected n-3 fatty acids. Cows were treated with 0 (SSN, AFN) or 500 (SSY, AFY) mg of bST every 10 d from 12 to 70 d in milk (DIM) and at 14-d intervals thereafter. Liver biopsies were collected on -12, 10, 24, and 136 DIM for gene expression analysis. Growth hormone receptor (GHR), insulin-like growth factor-I (IGF-I), IGF-binding protein-3 (IGFBP3), hepatic nuclear factor 4alpha (HNF4alpha), fibroblast growth factor-21 (FGF-21), and peroxisome proliferator-activated receptor alpha (PPARalpha) were the target genes and hypoxanthine phosphoribosyltransferase (HPRT) was used as an endogenous control gene. Expression was measured by quantitative real-time reverse transcription-PCR analyses of 4 samples from each of 32 cows (8 complete blocks). Amounts of hepatic HPRT mRNA were not affected by bST or diet but were increased by approximately 3.8% in early lactation (3.42, 3.52, 3.54, and 3.41 x 10(4) message copies for -12, 10, 24, and 136 DIM, respectively). This small change had little detectable impact on the ability of HPRT to serve as an internal control gene. Amounts of hepatic GHR, IGF-I, and IGFBP3 mRNA were reduced by 1.5 to 2-fold after calving. Expression of GHR and IGF-I increased and IGFBP3 tended to increase within 12 d (by 24 DIM) of bST administration. These effects of bST persisted through 136 DIM. Hepatic HNF4alpha mRNA was not altered by DIM or any of the treatments. Abundance of PPARalpha mRNA was unchanged through 24 DIM but increased by 136 DIM. There was a trend for an interaction of bST, diet, and DIM on PPARalpha mRNA abundance from 24 to 136 DIM because the amount of PPARalpha mRNA increased in SSN, SSY, and AFN cows but was not altered in AFY cows. The amount of FGF-21 mRNA increased markedly in early lactation but, like HNF4alpha mRNA, was not affected by bST, diet, or their interactions. These results indicate 1) that bST induced increases in hepatic expression of GHR, IGF-I, and IGFBP3 when cows were in negative energy balance in early lactation, 2) there was no effect of reduced dietary n-6/n-3 content on hepatic gene expression, and 3) there was support for a potential homeorhetic role of hepatic FGF-21 via uncoupling the somatotropin-IGF-axis in early lactation.

Effects of herbage allowance of native grasslands in purebred and crossbred beef cows: metabolic, endocrine and hepatic gene expression profiles through the gestation–lactation cycle

“…Plasma and serum samples were stored at − 20°C until analysis. Liver biopsies Laporta, Astessiano, López-Mazz, Soca, Espasandin and Carriquiry (0.5 g of tissue approximately) were collected at − 165, − 75, − 45, and − 15 ± 10, +15 and +60 ± 3 days using a biopsy needle (Tru-Core ® -II Automatic Biopsy Instrument; Angiotech, Lausanne, Switzerland) according to Carriquiry et al (2009). Liver samples were immediately frozen in liquid nitrogen and stored at − 80°C until total RNA was isolated.…”

“…Quantitative real-time PCR Isolation of total RNA from the hepatic tissue and synthesis of cDNA by reverse transcription was performed according to Carriquiry et al (2009) (Supplementary Material S1). Primers (Supplementary Table S1) to specifically amplify cDNA of target genes: GHR, GHR-1A, IGF1, IGF-binding proteins-2 and -3 (IGFBP2, IGFBP3), acyl-CoA oxidase-1 (ACOX1), acyl-CoA dehydrogenase very long chain (ACADVL), pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase-1 (PCK1), fibroblast growth factor-21 (FGF21) and peroxisome proliferator-activated receptor-α (PPARA), and from endogenous controls: β-actin (ACTB) and hypoxanthine phosphoribosyltransferase (HPRT) were obtained from the literature (Loor et al, 2005;Carriquiry et al, 2009;Astessiano et al, 2012) or specifically designed using Primer3 (http://frodo.wi.mit.edu/) and bovine nucleotide sequences available from NCBI (http://www.ncbi.nlm.nih.gov/).…”

“…Primers (Supplementary Table S1) to specifically amplify cDNA of target genes: GHR, GHR-1A, IGF1, IGF-binding proteins-2 and -3 (IGFBP2, IGFBP3), acyl-CoA oxidase-1 (ACOX1), acyl-CoA dehydrogenase very long chain (ACADVL), pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase-1 (PCK1), fibroblast growth factor-21 (FGF21) and peroxisome proliferator-activated receptor-α (PPARA), and from endogenous controls: β-actin (ACTB) and hypoxanthine phosphoribosyltransferase (HPRT) were obtained from the literature (Loor et al, 2005;Carriquiry et al, 2009;Astessiano et al, 2012) or specifically designed using Primer3 (http://frodo.wi.mit.edu/) and bovine nucleotide sequences available from NCBI (http://www.ncbi.nlm.nih.gov/). Before use, primer product size (1% agarose gel separation) and sequence (Macrogen, Inc., Seoul, Korea) were determined to ensure that primers produced the desired amplicons (Supplementary Tables S2 and S3).…”

“…The expression of IGFBP3 or the IGFBP3 to IGFBP2 ratio could represent possible mechanisms, whereby insulin regulates plasma IGF-I concentrations (Mashek et al, 2001;Loor et al, 2005;Carriquiry et al, 2009). Circulating IGF-I forms a ternary complex (IGFBP3/IGF-I) that increases IGF-I half-life and potentiates its role, whereas IGFBP2 inhibits IGF-I action by preventing the binding to its receptors (Rajaram et al, 1997).…”

“…This was reflected in the high correlations determined in this study between plasma insulin and IGF-I and the IGFBP3 to IGFBP2 mRNA ratio. Reduced IGFBP3 and increased IGFBP2 have been associated with NEB (Rajaram et al, 1997;Loor et al, 2005;Carriquiry et al, 2009). The greater IGFBP2 mRNA and reduced IGFBP3 to IGFBP2 mRNA ratio observed in LO than in HI cows, indicated a more severe nutrient restriction in the former ones (Rajaram et al, 1997) and could explain, at least partly, their reduced circulating IGF-I concentrations (Mashek et al, 2001).…”