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°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 μM H2O2 (HP), or CTR with greater Met supply plus 100 μM 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, incubation 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.