Methionine supplementation during a hydrogen peroxide challenge alters components of insulin signaling and antioxidant proteins in subcutaneous adipose explants from dairy cows

Abstract

Enhanced postruminal supply of methionine (Met) during the peripartal period alters protein abundance of insulin, AA, and antioxidant signaling pathways in subcutaneous adipose tissue (SAT). Whether SAT is directly responsive to supply of Met and can induce molecular alterations is unknown. Our objective was to examine whether enhanced Met supply during an oxidative stress challenge in vitro alters insulin, AA, inflammation, and antioxidant signaling-related protein networks. Four late-lactation Holstein cows (average 27.0 kg of milk per day) were used for SAT collection. Tissue was incubated in duplicate for 4 h in a humidified incubator with 5% CO2 at 37 degrees C according to the following experimental design: control medium with an "ideal" profile of essential AA (CTR; Lys:Met 2.9:1), CTR plus 100 mu M H2O2 (HP), or CTR with greater Met supply plus 100 mu M H2O2 (HPMET; Lys:Met 2.5:1). Molecular targets associated with insulin signaling, lipolysis, antioxidant nuclear factor, erythroid 2 like 2 (NFE2L2), inflammation, and AA. metabolism were determined through reverse-transcription quantitative PCR and western blotting. Data were analyzed using the MIXED procedure of SAS 9.4 (SAS Institute Inc.). Among proteins associated with insulin signaling, compared with CTR, HP led to lower abundance of phosphorylated AKT serine/threonine kinase (p-AKT) and solute carrier family 2 member 4 (SLC2A4; insulin-induced glucose transporter). Although incubation with HPMET restored abundance of SLC2A4 to levels in the CTR and upregulated abundance of fatty acid synthase (FASN) and phosphorylated 5'-prime-AMP- activated protein kinase (p-AMPK), it did not alter p-AKT, which remained similar to HP. Among proteins associated with AA signaling, compared with CTR, challenge with HP led to lower abundance of phosphorylated mechanistic target of rapamycin (p-MTOR), and HPMET did not restore abundance to CTR levels. Among inflammation-related targets studied, incubartion with HPMET led to greater protein abundance of nuclear factor kappa B subunit p65 (NFKB-RELA). The response in NFKB observed with HPMET was associated with a marked upregulation of the antioxidant transcription regulator NFE2L2 and the antioxidant enzyme glutathione peroxidase 1 (GPX1). No effects of treatment were detected for mRNA abundance of proinflammatory cytokines or antioxidant enzymes, underscoring the importance of post-transcriptional regulation. Overall, data indicated that short-term challenge with H2O2 was particularly effective in reducing insulin and AA signaling. Although a greater supply of Met had little effect on those pathways, it seemed to restore the protein abundance of the insulin-induced glucose transporter. Overall, the concomitant upregulation of key inflammation and antioxidant signaling proteins when a greater level of Met was supplemented to oxidant-challenged SAT highlighted the potential role of this AA in regulating the inflammatory response and oxidant status. Further studies should be conducted to assess the role of postruminal supply of Met and other AA in the regulation of immune, antioxidant, and metabolic systems in peripartal cow adipose tissue.