MDM2-related responses in 3T3-L1 adipocytes exposed to cooling and subsequent rewarming

Insulin-like growth factor-I and insulin induce the production of phospho-Ser-166 MDM2, a target of Akt, and influence the formation of the MDM2 complex. The glycolipid hormone insulin differentially activates phosphatidylinositol 3-kinase (PI3K)/Akt pathways in 3T3-L1 (L1) adipocytes incubated at 19 °C. Responses of L1 adipocytes to different temperature changes and their regulatory mechanisms are poorly understood. We exposed L1 adipocytes to cooling and subsequent rewarming in the presence or absence of wortmannin, a PI3K inhibitor, or mithramycin A, a transcription inhibitor, and examined the induction of phospho-Ser-166 MDM2 and MDM2 and the subcellular formation of the MDM2 complex using western blot analysis. Exposure to 28 and 18 °C induced phospho-MDM2 in cells and increased the level of MDM2 in the plasma membrane of cells. These temperatures did not affect the total MDM2 level. Similar results were obtained when the cells were treated with insulin. Exposure to 4 °C increased the total MDM2 level and did not induce phospho-MDM2, which was induced by rewarming at 37 °C after cooling at 4 °C without any alteration in the protein level. Mithramycin A (10 μM) did not alter the increase in protein level induced at 4 °C. The induction of phospho-molecules at 28 and 18 °C was impaired slightly by 1 μM of wortmannin but not by 0.1 μM of wortmannin. This low concentration of wortmannin completely blocked the induction of phospho-MDM2 by rewarming. Our results indicate that temperature changes induce MDM2-related responses, including those that are stimulated by receptor responses and dependent on a kinase inhibitor, in L1 adipocytes.