PS2-21. Effect of Ile, Leu, and Met and Insulinemic Energy Supplementation on Mammary Gland and Muscle Gene Expression.

Abstract We aimed to investigate the effect of insulinemic energy source (ES) with AA supplementation on mammary and muscle gene expression differences in lactating cows. Rumen-cannulated, multiparous Holstein cows (70±26 DIM, n = 16) were enrolled in a replicated 4x4 Latin square study with 21-d periods and treatments arranged as a 2x2 factorial. The two factors were ES: isoenergetic (2.86 Mcal/d) amounts of glucose (GLC) or acetate (ACE) as non-insulinemic control; and AA: Ile, Leu and Met (12, 50, and 20 g/d, respectively; ILM) or control (CON; 0 g/d), all dissolved in water and continuously infused into the abomasum, except during milkings. A diet formulated to meet 96% of the energy and 84% of metabolizable protein requirements was fed 6x/d. Ten coccygeal blood samples were collected on d 17-19 of each period, representing hourly diurnal sampling. Averaged cow-period results were analyzed with a mixed model containing fixed effects of ES, AA, their interaction, square, period, and random effect of cow within square. Mammary gland (MG) and muscle biopsies of one Latin square were performed on d 21 of each period for RNA sequencing (n = 16). Differentially expressed genes (DEG; P < 0.005) were obtained using edgeR package of R, and gene annotation was performed with DAVID v2023q4. Pathway enrichment analysis of DEG was done with IPA (Qiagen). Plasma glucose and insulin were increased by GLC vs ACE (71.5 vs 69.0 mg/dL and 0.58 vs 0.45 ug/L, respectively; P < 0.001). Insulin (+0.065 ug/L) and acetate (+0.29 mM) were increased by ILM vs CON (P < 0.004). Relative to CON, ILM increased plasma Met (46.8 vs 17.3 uM), and increased Leu to a greater extent for ACE than GLC (+52.4 vs + 27.4 uM; ES×AA: P = 0.009). In MG, GLC upregulated 62 genes and downregulated 40 genes relative to ACE (FDR< 0.1). Key upregulated genes (FDR≤0.05) included SLC27A2 (log fold change [logFC]= 3.1) involved in fatty acid metabolism; DIAPH3 (logFC= 2.3) involved in integrin-mediated signaling; and CENPE (logFC= 2.1), involved in cell mitosis. Key downregulated genes included SLC2A8 (logFC= -1.0) involved in glucose transmembrane transport; ATP2B2 (logFC= -1.2) involved in calcium ion transmembrane transport; and LOC520402 (logFC= -3.0), involved in immune response. No DEG were observed with AA infusion in MG (FDR >0.1). In muscle (FDR≤0.05), IGF1 (logFC= -1.7) and CHRNG (cholinergic receptor; logFC= -5.5) were downregulated by ES×AA interaction. GLC upregulated PPM1K (logFC= 1.0), a phosphatase involved in branched-chain AA catabolism, and downregulated ANGPTL4, involved in negative regulation of lipid biosynthesis (ES; FDR≤0.05). Canonical pathways at FDR< 0.4 included upregulation of cell structure, division, and migration processes, and downregulation of lipid and ATP synthesis for GLU vs ACE. Our results suggest ES and AA effects on gene expression are driven by the anabolic effects of insulin.