Phosphoinositide 3-kinase as a novel functional target for the regulation of the insulin signaling pathway by SIRT1

https://doi.org/10.1016/j.mce.2011.01.008Get rights and content

Abstract

The protein deacetylase SIRT1, and its activator resveratrol, exert beneficial effects on glucose metabolism. Different SIRT1 targets have been identified, including PTP1B, AMPK, FOXO, PGC-1α and IRS2. The latter may underscore a tight link between SIRT1 and insulin signaling components. However, whether SIRT1 has a direct effect on insulin resistance and whether resveratrol acts directly or indirectly in this context is still a matter of controversy and this question has not been addressed in muscle cells.

Here, we show that SIRT1 protein expression is decreased in muscle biopsies and primary myotubes derived from type 2 diabetic patients, suggesting a contribution of diminished SIRT1 in the determination of muscle insulin resistance. To investigate the functional impact of SIRT1 on the insulin pathway, the activation of insulin downstream effector PKB was evaluated after SIRT1 inactivation by RNAi, SIRT1 overexpression, or resveratrol treatments. In muscle cells and HEK293 cells, downregulation of SIRT1 reduced, while overexpression increased, insulin-induced PKB activatory phosphorylation. Further molecular characterisation revealed that SIRT1 interacts in an insulin-independent manner with the PI3K adapter subunit p85. We then investigated whether resveratrol may improve insulin signaling in muscle cells via SIRT1, or alternative targets. Incubation of muscle cells with resveratrol reverted the insulin-resistant state induced by prolonged TNFα or insulin treatment. Resveratrol-dependent improvement of insulin-resistance occurred through inhibition of serine phosphorylation of IRS1/2, implicating resveratrol as a serine kinase inhibitor. Finally, a functional interaction between PI3K and SIRT1 was demonstrated in C. elegans, where constitutively active PI3K – mimicking increased IIS signaling – lead to shortened lifespan, while removal of sir-2.1 abolished PI3K-induced lifespan shortening.

Our data identify SIRT1 as a positive modulator of insulin signaling in muscle cells through PI3K, and this mechanism appears to be conserved from C. elegans through humans.

Introduction

The control of lifespan and glucose homeostasis are tightly linked processes in eukaryotes. Caloric restriction, an experimental condition that leads to lifespan prolongation, entails an amelioration of insulin signaling resulting in hypoinsulinaemia and improved glycaemic control.

Ample genetic evidence demonstrates that mild inhibition of insulin signaling components including the insulin receptor, insulin receptor substrate proteins (IRS), PI3K; or over-activation of FOXO transcription factors contribute to lifespan extension, which is paralleled by (or consequential to) improved metabolic profile (Holzenberger et al., 2004). PI3K catalyses the phosphorylation of PI(4,5)P2 to produce PIP3. The PIP3 phosphatase PTEN downregulates the insulin signaling pathway. Accordingly, overexpression of the PTEN homolog DAF-18, by downregulating IIS through termination of PI3K signaling, leads to lifespan extension in Caenorhabditis elegans (Mihaylova et al., 1999, Solari et al., 2005). Sir2, a NAD+ dependent protein deacetylase (Vaziri et al., 2001), modulates lifespan in lower eukaryotes including C. elegans (Tissenbaum and Guarente, 2001) and Drosophila melanogaster (Rogina and Helfand, 2004) by acting as calorie-restriction mimetic. It has also been shown that the small polyphenolic molecule resveratrol, acting via Sir2/SIRT1, induces lifespan extension (Baur et al., 2006, Howitz et al., 2003, Wood et al., 2004) and improves the glycaemic profile in rodent models (Baur et al., 2006).

SIRT1 also participates in the control of glucose homeostasis by regulating gluconeogenesis (Rodgers et al., 2005), insulin secretion (Bordone et al., 2006), lipid mobilization from adipocytes (Picard et al., 2004) and fatty acid oxidation in skeletal muscle (Gerhart-Hines et al., 2007). By virtue of these multiple effects on glucose homeostasis, and its capability to protect pancreatic β-cells against cytokine toxicity (Lee et al., 2009), SIRT1 is a promising pharmacological therapeutic target for the treatment of insulin-resistance and subsequent type 2 diabetes (Liang et al., 2009). Several molecular mechanisms account for metabolic control by SIRT1, including (i) deacetylation-dependent activation of PGC1α (Nemoto et al., 2005), leading to increased mitochondrial function (Rodgers et al., 2008), (ii) SIRT1-mediated histone deacetylation on the PTP1B promoter, resulting in PTP1B gene repression (Sun et al., 2007) and (iii) deacetylation-dependent nuclear retention of FoxO1, promoting expression of gluconeogenesis-related genes (Frescas et al., 2005). Besides these transcriptionally related mechanisms, recent research indicates that SIRT1 modulates IRS2 tyrosine phosphorylation (Zhang, 2007) and improves insulin sensitivity in adipocytes (Yoshizaki et al., 2009). In view of these findings, we investigated the effects of SIRT1, and its activator resveratrol, on insulin signaling in human skeletal myotubes, skeletal muscle being the most relevant tissue for glucose disposal. Furthermore, we studied the interplay of IIS – elicited by overexpression of a constitutively active PI3K – and the SIRT1 homolog SIR-2.1 in C. elegans, monitoring lifespan as readout, to determine whether the interaction between SIRT1 and insulin signaling is a highly conserved phenomenon.

Here we show that (i) SIRT1 protein expression levels are decreased in myotubes obtained from patients with type 2 diabetes, (ii) SIRT1 interacts in an insulin-independent manner with the PI3K adapter subunit p85, and modulates insulin signaling at physiological insulin concentrations in skeletal muscle cells, (iii) the SIRT1 activator resveratrol protects muscle cells – including human primary myotubes – from insulin-resistance induced by TNFα or prolonged hyperinsulinaemia and (iv) lifespan shortening in C. elegans, caused by amplification of IIS signaling through expression of a constitutively active PI3K, is reverted by ablation of the sir-2.1 gene.

Section snippets

Materials and cell cultures

Resveratrol and recombinant human insulin were from Sigma. Recombinant human TNFα was from PeproTech (London). The following antibodies were used: anti-phospho-Ser-473 PKB, (Cell Signaling Technology,), anti-PKB, IRS1, IRS2, SIRT1, and anti-pY (Santa Cruz Biotechnology), anti-p85α (Upstate Biotechnology). Flag-SIRT1 and MYC-SIRT1 plasmids were from Drs. E. Verdin and B.M. Burgering (North et al., 2003, van der Horst et al., 2004). MYC-SIRT1, Flag-SIRT1 adenovirus were produced as described (

SIRT1 protein expression is decreased in muscle biopsies and primary myotubes derived from type 2 diabetic subjects

Research on cultured cell lines indicated that SIRT1 is required to achieve full insulin-induced IRS2 tyrosine phosphorylation in hepatocytes (Zhang, 2007). As peripheral insulin-resistance in type 2 diabetes is characterised by decreased insulin action in skeletal muscle (Fig. 1A and Cozzone et al., 2008), the tissue prominently contributing to glucose disposal, we investigated whether alterations of SIRT1 expression occur in muscle biopsies and primary myotubes derived from patients with type

Discussion

As SIRT1 exerts positive metabolic effects through several mechanisms (reviewed in Chaudhary and Pfluger, 2009), and in view of the decreased SIRT1 protein expression observed by us (Fig. 1) and others (de Kreutzenberg et al., 2010) in muscle and peripheral blood mononuclear cells from insulin resistant patients, we sought to investigate the role of SIRT1, and its activator resveratrol, on the insulin signaling cascade in muscle cells, since in this cell type insulin signaling is quantitatively

Acknowledgements

We thank Drs. B.M. Burgering (University Medical Center Utrecht, Utrecht, The Netherlands) and E. Verdin (Gladstone Institute of Virology and Immunology, University of California,) for donation of SIRT1 plasmids. We acknowledge Cyrille Debard and Graeme Lancaster for preparation and maintenance of human myotubes and discussion, respectively. Nematode strains were provided by the Caenorhabditis Genetic Center. This work was supported by INSERM (To LP, Programme National de Recherche sur le

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