Cooling-increased phospho-β-arrestin-1 and β-arrestin-1 expression levels in 3T3-L1 adipocytes

Cooling induces several responses that are modulated by molecular inhibitors and activators and receptor signaling. Information regarding potential targets involved in cold response mechanisms is still insufficient. We examined levels of the receptor-signaling mediator β-arrestin-1 and phospho-Ser-412 β-arrestin-1 in 3T3-L1 adipocytes exposed to 4-37 °C or treated with some molecular agents at 37°C. We also cooled cells with or without modification and signal-modulating agents. These conditions did not decrease cell viability, and western blot analysis revealed that exposure to 4 °C for 1.5h and to 28 and 32 °C for 24 and 48 h increased phospho-β-arrestin-1 and β-arrestin-1 levels and that exposure to 4 and 18 °C for 3 and 4.5h increased β-arrestin-1 level. Serum removal and rewarming abolished β-arrestin-1 alterations induced by cooling. Mithramycin A (a transcription inhibitor) treatment for 4 and 24h increased the level of β-arrestin-1 but not that of phospho-β-arrestin-1. The level of phospho-β-arrestin-1 was increased by okadaic acid (a phosphatase inhibitor), decreased by epinephrine and aluminum fluoride (receptor-signaling modulators), and unaffected by N-ethylmaleimide (an alkylating agent) at 37 °C. N-Ethylmaleimide and the receptor-signaling modulators did not alter β-arrestin-1 expression at 37 °C but impaired the induction of phospho-β-arrestin-1 at 28 and 32 °C without affecting the induction of β-arrestin-1. We show that cold-induced β-arrestin-1 alterations are partially mimicked by molecular agents and that the responsive machinery for β-arrestin-1 requires serum factors and N-ethylmaleimide-sensitive sites and is linked to rewarming- and receptor signaling-responsive machinery. Our findings provide helpful information for clarifying the cold-responsive machinery for β-arrestin-1 and elucidating low-temperature responses.