CDK4 is an essential insulin effector in adipocytes
Sylviane Lagarrigue, … , C. Ronald Kahn, Lluis Fajas
Sylviane Lagarrigue, … , C. Ronald Kahn, Lluis Fajas
Published December 14, 2015
Citation Information: J Clin Invest. 2016;126(1):335-348. https://doi.org/10.1172/JCI81480.
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Research Article Endocrinology Metabolism

CDK4 is an essential insulin effector in adipocytes

Abstract

Insulin resistance is a fundamental pathogenic factor that characterizes various metabolic disorders, including obesity and type 2 diabetes. Adipose tissue contributes to the development of obesity-related insulin resistance through increased release of fatty acids, altered adipokine secretion, and/or macrophage infiltration and cytokine release. Here, we aimed to analyze the participation of the cyclin-dependent kinase 4 (CDK4) in adipose tissue biology. We determined that white adipose tissue (WAT) from CDK4-deficient mice exhibits impaired lipogenesis and increased lipolysis. Conversely, lipolysis was decreased and lipogenesis was increased in mice expressing a mutant hyperactive form of CDK4 (CDK4R24C). We performed a global kinome analysis and found that mice lacking Cdk4 had impaired insulin signaling in the adipose tissue. Interestingly, our results demonstrated that insulin activates the cyclin D3-CDK4 complex, which, in turn, phosphorylates the insulin receptor substrate 2 (IRS2) at the Ser 388, likely creating a positive feedback loop to maintain adipocyte insulin signaling. Furthermore, we found that CCND3 expression and IRS2 serine 388 phosphorylation are increased in human obese subjects. Together, our results demonstrate that CDK4 is a major regulator of insulin signaling in WAT.

Authors

Sylviane Lagarrigue, Isabel C. Lopez-Mejia, Pierre-Damien Denechaud, Xavier Escoté, Judit Castillo-Armengol, Veronica Jimenez, Carine Chavey, Albert Giralt, Qiuwen Lai, Lianjun Zhang, Laia Martinez-Carreres, Brigitte Delacuisine, Jean-Sébastien Annicotte, Emilie Blanchet, Sébastien Huré, Anna Abella, Francisco J. Tinahones, Joan Vendrell, Pierre Dubus, Fatima Bosch, C. Ronald Kahn, Lluis Fajas

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Figure 6

CDK4 phosphorylates IRS2.

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CDK4 phosphorylates IRS2. (A) CCND3-CDK4 complex directly phosphorylates...
(A) CCND3-CDK4 complex directly phosphorylates full-length GST-IRS2 in vitro (n = 3). (B) In vitro phosphorylation of GST-IRS2 fragments (1–494aa, 495–744aa, 745–993aa, 994–1099aa, 1100–1321aa) by CCND3/CDK4. Left panel, SDS-PAGE stained with Coomassie blue for the loading control. Middle panels, autoradiography of the SDS-PAGE gels containing the different GST-IRS2 fragments, incubated with CCND3/CDK4. Right panel, RB1 recombinant protein was used as a positive control (n = 3). (C) Defective IRS2S388A and IRS2S1226A phosphorylation by CCND3-CDK4. Upper panel, autoradiography; lower panel, SDS-PAGE gel stained with Coomassie blue for the loading control. (n = 2). (D) Decrease in pAKT Ser473 phosphorylation in Flag-IRS2S388A electroporated Irs2–/– cells upon insulin stimulation, compared with the WT Flag-IRS2–transfected cells (n = 3). Original magnification, ×400. (E) Quantification of pAKT Ser473 fluorescence intensity for untransfected, Flag-IRS2–transfected, and Flag-IRS IRS2S388A electroporated Irs2–/– preadipocytes was performed with ImageJ software (http://imagej.nih.gov/ij/). At least 100 cells were quantified per condition. (F) Representative Western blot analysis showing impaired interaction between PIK3R1 and Flag-IRS2S388A mutant after insulin stimulation compared with cells transfected with Flag-IRS2 in 293T cells. (G) Quantification of the blot shown in F. A representative Western blot is shown. Data are expressed as mean ± SEM. Statistically significant differences were determined with 2-way ANOVA followed by Tukey’s multiple comparisons test (E–G). *P < 0.05.