Elsevier

Cellular Signalling

Volume 26, Issue 2, February 2014, Pages 233-239
Cellular Signalling

Evidence for a regulatory role of Cullin-RING E3 ubiquitin ligase 7 in insulin signaling

https://doi.org/10.1016/j.cellsig.2013.11.005Get rights and content

Highlights

  • Dysfunctional insulin signaling is a major cause of type 2 diabetes.

  • Loss CRL7 resulted in enhanced insulin signaling in vivo and in vitro.

  • We report CRL7 as a novel regulator of the insulin signaling pathway.

  • CRL7 might constitute a novel therapeutical target for type 2 diabetes.

Abstract

Dysfunctional regulation of signaling pathways downstream of the insulin receptor plays a pivotal role in the pathogenesis of insulin resistance and type 2 diabetes. In this study we report both in vitro and in vivo experimental evidence for a role of Cullin-RING E3 ubiquitin ligase 7 (CRL7) in the regulation of insulin signaling and glucose homeostasis. We show that Cul7−/− mouse embryonic fibroblasts displayed enhanced AKT and Erk MAP kinase phosphorylation upon insulin stimulation. Depletion of CUL7 by RNA interference in C2C12 myotubes led to increased activation of insulin signaling pathways and cellular glucose uptake, as well as a reduced capacity of these cells to execute insulin-induced degradation of insulin receptor substrate 1 (IRS1). In vivo, heterozygosity of either Cul7 or Fbxw8, both key components of CRL7, resulted in elevated PI3 kinase/AKT activation in skeletal muscle tissue upon insulin stimulation when compared to wild-type controls. Finally, Cul7+/− or Fbxw8+/− mice exhibited enhanced insulin sensitivity and plasma glucose clearance. Collectively, our findings point to a yet unrecognized role of CRL7 in insulin-mediated control of glucose homeostasis by restraining PI3 kinase/AKT activities in skeletal muscle cells.

Introduction

Diabetes mellitus is a major metabolic disorder affecting more than 285 million people worldwide [1]. 90% of the patients suffer from type 2 diabetes, which is characterized by glucose intolerance, insulin resistance and defects in pancreatic insulin secretion [2]. Insulin exerts its effects in target tissues by binding to insulin tyrosine kinase receptors, resulting in the recruitment and phosphorylation of insulin receptor substrate (IRS) proteins. In particular IRS1 and IRS2 were shown to play critical roles in insulin signaling by transmitting receptor stimulation to the activation of PI3 kinase/AKT and Erk MAPK pathways, which mediate the metabolic and mitogenic effects of insulin, respectively [3]. In addition, activation of PI3 kinase also triggers the translocation of vesicles containing glucose transporter 4 (GLUT4) from intracellular storage pools to the cell membrane, thereby enabling uptake of glucose into the cell [4].

A major cause of insulin resistance and type 2 diabetes is thought to be dysfunctional regulation of cellular signal transduction downstream of the insulin receptor [5], [6]. Several studies provided evidence for dysregulation of key signaling molecules, such as IRS1, IRS2 and AKT1, in insulin resistant cells and tissues due to altered transcriptional or posttranslational (such as ubiquitin-mediated proteasomal degradation) activities [7]. Timely degradation of intracellular proteins by the ubiquitin-proteasome system (UPS) is a precisely controlled process that regulates a broad spectrum of fundamental cellular functions, ranging from cell cycle progression to signal transduction. Central to this process is the recognition of a substrate protein by an E3 ubiquitin ligase, a pivotal step for initiating the ubiquitination reaction that joins the target protein covalently with polyubiquitin chains, thereby leading to its degradation by the 26S proteasome [8].

We have previously identified IRS1 as a proteolytic target of Cullin-RING E3 ubiquitin ligase 7 (CRL7), a multimeric enzyme composed of the scaffold protein Cullin7 (CUL7), the RING finger protein ROC1 and the SKP1-FBXW8 substrate targeting subunit [9], [10], [11]. It was shown that CRL7 induced degradation of IRS1 is part of a negative feedback loop via mammalian target of rapamycin complex 1 (mTORC1) and p70 S6 kinase (S6K) activities to restrain PI3 kinase/AKT signaling upon activation of the insulin-like growth factor 1 (IGF-1) receptor [9], [10]. Collectively, these studies provided experimental evidence for a novel role of CRL7 in IGF-1 signaling in vitro. Based on these observations, we sought to investigate whether CRL7 also contributes to the regulation of insulin signaling and evaluate its biological relevance in vivo.

Section snippets

Animals

Cul7+/− mice were generated by replacing exons 2–4 of the Cul7 gene with a PGK-neo cassette as described in detail previously [9]. Fbxw8+/− mice were generated by injecting embryonic stem cells harboring a gene trap insertion in intron 3 of the Fbxw8 gene (Clone RRT057, BayGenomics gene-trap resource) into C57BL/6J blastocysts [12]. The injected blastocysts were then transplanted into pseudopregnant females using standard protocols [13]. A similar approach was used by DeCaprio and colleagues

Loss of CUL7 is associated with hyper-activation of AKT and Erk in response to insulin

We previously identified IRS1 as a substrate of CRL7 and demonstrated that mouse embryonic fibroblasts (MEFs) deficient of Cul7 display hyper-activation of IRS1 downstream PI3K/AKT and Erk MAPK pathways upon IGF-1 stimulation [9]. As both IGF-1 and insulin receptors utilize IRS1 for signal transduction [3], we sought to further investigate the effect of insulin receptor activation in Cul7+/+, Cul7+/− and Cul7−/− MEFs. When compared to the Cul7+/+ cells (Fig. 1A, lanes 1–4), insulin stimulation

Discussion

In this study we provide both in vitro and in vivo evidence for a regulatory role of CRL7 in regulation of insulin signaling and glucose homeostasis. We show that Cul7−/− mouse embryonic fibroblasts displayed an increase in the activation of AKT and Erk phosphorylation upon insulin stimulation. Depletion of CUL7 by siRNA in C2C12 myotubes led to enhanced activation of downstream pathways of the insulin receptor, cellular glucose uptake upon insulin stimulation, and impaired insulin-triggered

Competing interest statement

The authors declare no competing financial interests.

Acknowledgments

This work was supported by research grant SA 1706/3-1 from the German Research Foundation (to A.S.) and Marie Curie International Reintegration grant 256584 (to A.S.). Z.-Q. P. was supported by the US Public Health Service grants GM61051 and CA095634. We thank Milena Dürrbaum for optimizing the IRS1 degradation assay in C2C12 myotubes.

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