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 5

CDK4 is activated by insulin and translates insulin signaling in adipocytes.

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CDK4 is activated by insulin and translates insulin signaling in adipocy...
(A) CDK4 activity in vivo. SDS-PAGE autoradiography showing RB1 phosphorylation by CDK4 immunoprecipitated from 3T3-L1 mature adipocytes after insulin stimulation. The left panel shows RB1 phosphorylation by recombinant CDK4 used as a positive control. (B) Western blot analysis showing the inhibition of insulin-induced RB1 phosphorylation on Ser780 by CDK4 knockdown in mature 3T3-L1 adipocytes. (C) CCND3 and CDK4 association is increased upon insulin stimulation, but decreased upon cotreatment with insulin and AKT inhibitor in mature 3T3-L1 adipocytes. (D) Volcano plot showing differences in putative kinase activities between 22-week-old control and Cdk4nc mice injected (portal vein) with insulin for 3 minutes (n = 4). Kinases with a positive kinase statistic show higher activity in Cdk4nc samples compared with control samples, whereas kinases with negative kinase statistic show lower activity in Cdk4nc samples compared with control samples. (E) Based on the upstream kinase activity (D) results and on the GeneGO analysis of the PamGene experiment, the putative role of CDK4 upstream of AKT in the insulin signaling pathway is represented. (F) Immunoblot showing AKT phosphorylation on Thr308 and Ser473 in response to insulin injection (3 minutes) in 22-week-old fasted control and Cdk4nc mice (n = 2 for NaCl and n = 4 for insulin treatment). (G and H) Coimmunoprecipitation experiments showing the interaction between endogenous IRS2 and PIK3R1. IRS2 (G) and PI3Kp85A (H) were immunoprecipitated and the presence of PI3K p85A (G) and IRS2 (H) was detected by Western blot analysis from 23-week-old Cdk4+/+ and Cdk4nc mice treated with insulin for 3 minutes. (n = 2 for NaCl and n = 3 for insulin treatment). (I and J) Coimmunoprecipitation experiments showing the interaction between endogenous PIK3R1 or IRS2 and CDK4. CDK4 was immunoprecipitated and the presence of PIK3R1 (n = 3) (I) and IRS2 (n = 1) (J) was detected by Western blot analysis in 3T3-L1 mature adipocytes treated with insulin. A representative Western blot is shown. Unless specified otherwise, all experiments are representative of 3 independent experiments.