β-hydroxybutyrate restricts glycolytic metabolism in bovine neutrophils during toll-like receptor 2/1 stimulation

Abstract

Ketosis commonly affects high-producing dairy cows during the peripartum and peak lactation periods and is associated with immunosuppression. Elevated blood levels of β-hydroxybutyrate (β-OHB), the primary ketone body, have been linked to impaired neutrophil bactericidal activity, potentially explaining the increased susceptibility to mammary and uterine infections. Since neutrophils primarily rely on glycolysis for energy, this study investigated the effects of high β-OHB concentration on the energy metabolism of bovine neutrophils stimulated with the toll-like receptor 2/1 (TLR2/1) agonist Pam3CSK4. At both subclinical (2.5 mM) and clinical (5.0 mM) ketosis concentrations, β-OHB strongly narrowed glycolytic flux and glycolytic ATP production in unstimulated cells, accompanied by reduced extracellular glucose uptake without changes in glycogen content. TLR2/1 stimulation robustly enhanced glycolysis and ATP production, upregulated glycolytic enzyme expression, promoted the translocation of glucose transporters (GLUTs) 1 and 3, boosted extracellular glucose uptake, and decreased glycogen concentration. These changes were accompanied by increased glycogen phosphorylase (PYGL) phosphorylation over glycogen synthase (GYS1) and the activation of protein kinase B (Akt), glycogen synthase kinase 3 beta (GSK3β), and protein kinase A (PKA) pathways. However, preincubation with 2.5 and 5.0 mM β-OHB attenuated these metabolic responses, reducing glycolytic flux, ATP production, glycolytic enzyme expression, GLUTs translocation, and extracellular glucose uptake, while glycogen stores remained stable. Notably, β-OHB did not impact the phosphorylation of PYGL, GYS1, GSK3β, or PKA substrate, but it did decrease Akt phosphorylation at 5.0 mM. These results suggest that elevated β-OHB concentrations restrict glycolytic metabolism in bovine neutrophils, compromising their bioenergetics and potentially contributing to impaired immune competence in ketotic dairy cows.