Rumen-protected methionine, histidine, and slow-release urea: Effects on plasma 3-methylhistidine and ubiquitin proteasome-related gene expression in skeletal muscle of dairy cows receiving a diet deficient in metabolizable protein
Skeletal muscle, the largest organ in vertebrates, plays a major role in homeostasis. The ubiquitin-proteasome system (UPS) is regarded as the main proteolytic pathway in muscle. It requires the coordinated reactions of 3 enzymes including ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligases (E3). We hypothesized that supplementation of diets deficient in metabolizable protein (MP) with slow-release urea or rumen-protected (RP) Met and His will affect the gene expression of UPS-related factors in skeletal muscle of dairy cows in support of decreased proteolysis. Sixty Holstein cows were blocked based on DIM and milk yield and randomly assigned to 1 of 5 diets: MP-adequate diet (AMP); MP-deficient diet [DMP; 5% below the requirements (NRC, 2001)]; DMP supplemented with slow-release urea as Optigen (Alltech Inc., Nicholasville, KY; DMPO); DMPO supplemented with RPMet (Mepron; Evonik Industries AG, Hanau, Germany; DMPOM); and DMPOM supplemented with RPHis (Balchem Corp., New Hampton, NY; DMPOMH). The experimental period was 10 wk, with first 2 wk as covariate period. Muscle biopsies were collected from M. longissimus dorsi during the last week of the experiment. The mRNA abundance of the following UPS-related target genes was quantified by qPCR: F-box protein 32 (FBXO32), muscle ring-finger protein 1 (MuRF-1), both being muscle-specific E3 ubiquitin ligases, ubiquitin-like modifier activating enzyme 1 (UBA1), and ubiquitin-conjugating enzymes (UBE2G1 and UBE2G2). Data were normalized based on the geometric mean of the 4 most stable reference genes: lipoprotein receptor-related protein 10, marvel domain containing 1, RNA polymerase II, and emerin. The concentration of 3-methylhistidine (3-MH) as marker of muscle catabolism was measured in plasma samples collected at the end of the experiment. Data were analyzed by the MIXED procedure of SAS. With the exception of MuRF-1, the mRNA abundance of the target genes was not affected by treatment. In DMP cows, about 2-fold more (P = 0.05) MuRF-1 mRNA than in DMPO was observed. Plasma 3-MH did not differ among treatments. In conclusion, the UPS seemed to be upregulated at the level of the mRNA during protein deficiency but this effect was apparently not sustained to increased 3-MH plasma concentrations.
Keywords: rumen-protected amino acid, ubiquitin-proteasome system, dairy cow