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Updated: Jun 7, 2026
Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments
Published on: February 3, 2023
MDM2-related responses in 3T3-L1 adipocytes exposed to cooling and subsequent rewarming
Yasuhito Ohsaka1, Hoyoku Nishino
1Department of Pharmacology, Faculty of Pharmaceutical Sciences, Chiba Institute of Science, 15-8 Shiomi-cho, Choshi, Chiba 288-0025, Japan. y-ohsaka@cis.ac.jp
Abstract:
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.
Insights
Temperature changes, like cooling and rewarming, affect MDM2 protein levels and phosphorylation in adipocytes. These responses are linked to insulin signaling pathways and PI3K activity.
Area of Science:
- Cellular and Molecular Biology
- Metabolic Regulation
- Signal Transduction
Background:
- Insulin and Insulin-like Growth Factor-I stimulate phospho-Ser-166 MDM2 production via Akt.
- The impact of temperature fluctuations on 3T3-L1 adipocyte responses and MDM2 regulation is not well understood.
- Insulin's activation of phosphatidylinositol 3-kinase (PI3K)/Akt pathways is temperature-dependent.
Purpose of the Study:
- To investigate how temperature changes influence MDM2 phosphorylation and subcellular localization in 3T3-L1 adipocytes.
- To elucidate the role of PI3K/Akt signaling in mediating temperature-induced MDM2 responses.
- To understand the regulatory mechanisms of MDM2 complex formation under varying thermal conditions.
Main Methods:
- 3T3-L1 adipocytes were exposed to different temperatures (4 °C, 18 °C, 28 °C) followed by rewarming.
- Cells were treated with wortmannin (PI3K inhibitor) or mithramycin A (transcription inhibitor).
- Western blot analysis was used to examine phospho-Ser-166 MDM2, total MDM2 levels, and subcellular MDM2 complex formation.
Main Results:
- Cooling to 18 °C and 28 °C induced phospho-MDM2 and increased plasma membrane MDM2, without altering total MDM2.
- Cooling to 4 °C increased total MDM2 but did not induce phospho-MDM2; rewarming induced phospho-MDM2.
- Wortmannin partially inhibited phospho-molecule induction at 28/18 °C and completely blocked rewarming-induced phospho-MDM2 at 4 °C.
Conclusions:
- Temperature variations trigger MDM2-related cellular responses in adipocytes.
- These temperature-induced MDM2 responses are partly mediated by insulin signaling and PI3K-dependent pathways.
- Subcellular localization and phosphorylation of MDM2 are sensitive to thermal stress and rewarming in L1 adipocytes.
