Elsevier

Metabolism

Volume 83, June 2018, Pages 81-91
Metabolism

Basic Science
Decreased RNF41 expression leads to insulin resistance in skeletal muscle of obese women

https://doi.org/10.1016/j.metabol.2018.01.014Get rights and content

Highlights

  • RNF41 expression is decreased in skeletal muscle of obese insulin resistant women.

  • RNF41 regulates positively skeletal muscle insulin sensitivity during human obesity.

  • RNF41 expression allows IFNI pathway activation downstream TLR4.

  • IFNI restores insulin response in skeletal muscle of obese insulin resistant women.

Abstract

Context

Toll-like receptor 4 (TLR4) activation contributes to obesity-associated insulin resistance in skeletal muscles (SM). TLR4 signaling involves two pathways: the myeloid differentiation primary response gene 88 (MyD88) leading to inflammatory cytokines production and the toll/interleukin-1 receptor domain-containing adapter-inducing interferon (IFN) I (TRIF)-dependent pathways leading to type 1 interferon (IFNI) and interferon stimulated genes (ISG) expression. The E3 ubiquitin ligase RNF41 allows the preferential activation of the TRIF-IFNI pathway; however, its role in insulin response has not been reported.

Methods

We measured RNF41 level and IFNI pathway activation (ISG expression) in SM biopsies of obese insulin sensitive (OIS) and obese insulin resistant (OIR) women. Then we isolated and differentiated in myotubes, primary human SM cell progenitors from OIS and OIR SM biopsies. We modulated RNF41 and ISG expression in these myotubes and investigated their effects on insulin response.

Results

RNF41 expression is down-regulated in vivo in OIR SM and myotubes compared to OIS SM and myotubes. TLR4 activation with palmitate induces TRIF-IFNI pathway and ISG in OIS myotubes but not in OIR myotubes. Inhibition of RNF41 expression with siRNF41 in OIS myotubes treated with palmitate attenuates insulin response, IFNI pathway activation and ISG induction, mimicking OIR phenotype. Further, overexpression of RNF41 in OIR myotubes increases insulin response and ISG expression. Exposure to IFNI or to its inducer polyinosinic-polycytidylic acid, restores ISG expression and insulin sensitivity in OIR myotubes and OIS myotubes transfected with siRNF41.

Conclusion

Our results identify RNF41 as essential to IFNI pathway activation in order to maintain muscle insulin sensitivity during human obesity.

Introduction

Obesity is a worldwide epidemic disease and a major risk factor for insulin resistance (IR) development in insulin-dependent organs such as skeletal muscles (SM) [1,2]. Ultimately, IR leads to the onset of type 2 diabetes [3,4]. In fact, SM is responsible for 80% of glucose uptake and metabolism in the postprandial state, and muscle failure in this function is often considered as the first defect causing IR [3,5]. These last years, it has been shown that during obesity, activation of the innate immunity receptor toll-like receptor (TLR) 4 by non-esterified fatty acids (NEFA) and/or lipopolysaccharides (LPS) from the gut microbiota plays a pivotal role in the development of IR [[6], [7], [8], [9]]. However, while the cellular and molecular mechanisms leading from TLR4 activation to IR are still not fully understood in humans, the regulation of its two downstream signaling pathways, the myeloid differentiation primary response gene 88 (MyD88)- and the toll/interleukin-1 receptor domain-containing adapter-inducing interferon (IFN) I/β (TRIF)-dependent pathways, has never been described in obesity nor in IR. The MyD88 pathway initiates the inflammatory cascade response via nuclear factor κB (NFκB) activation and further induction of inflammatory cytokine expression whereas the TRIF pathway leads to interferon regulatory factor 3 (IRF3) activation [[10], [11], [12]]. IRF3 binds the interferon-sensitive/stimulated response element (ISRE) and induces the transcription of IFNI, of interferon stimulated genes (ISG) such as the oligoadenylate synthetases (OAS) and of anti-inflammatory factors [13,14]. The OAS, themselves activated by double-stranded RNA, synthesize the 2′-5′ oligoadenylates (2-5A) which in turn activate the latent endoribonuclease L (RNase L) [15]. RNase L cleaves single-stranded RNA, giving rise to smaller RNA fragments able to activate TLR3 [16]. Of note, TLR3 signaling is, like TLR4, mediated via the TRIF pathway [10,11].

Interestingly, we have previously shown that a defect in RNase L activity leads to IR development in myotubes obtained from obese individuals [17]. Besides, RNase L activation by 2-5A transfection enables this defective pathway to reactivate, restoring insulin response in human insulin-resistant myotubes. At the opposite, RNase L inhibition by RNase L inhibitor (RLI)/ATP binding cassette E1 (ABCE1) [18] induces IR in myotubes [17]. These results thus highlight the crucial role of the TRIF-IFNI pathway and particularly the activation of the OAS-RNase L pathway in maintaining insulin sensitivity in human myotubes.

Systemic insulin sensitivity is preserved in some obese individuals who do not display the typical metabolic disorders associated with obesity [5,19,20]. Being well aware of the existence of different subgroups of insulin sensitivity levels among obese individuals, we recently compared metabolic parameters and inflammation both at systemic and SM levels in obese insulin-sensitive (OIS) and obese insulin-resistant (OIR) post-menopausal women. In accordance with the literature [12], we observed a lower expression of IκBα in OIR SM compared to OIS SM, indicative of TLR4-MyD88 activation in OIR SM and not in OIS SM [5]. The aim of this study was to better understand the molecular mechanisms underlying IR development in SM during human obesity, in particular the regulation of TRIF-IFNI pathway. In this regard, we focused on RNF41, an E3 ubiquitin ligase ring finger protein 41 (RNF41) identified as an essential positive regulator of TRIF-IFNI pathway at the expense of the MyD88 pathway [21]. We thus compared TRIF-IFNI pathway activation through OAS-RNase L pathway and RNF41 expression in both SM biopsies and biopsy-derived cells from our cohort of OIS and OIR post-menopausal women, previously characterized for their systemic and SM insulin sensitivity [5]. Furthermore, we modulated RNF41 and ISG expression in myotubes differentiated from primary human SM cell progenitors and investigated the effects on insulin response.

Section snippets

Volunteers Recruitment

Twenty post-menopausal obese women were recruited at CHRU Montpellier. Eleven were insulin-sensitive (OIS: HOMAIR = 1.7 ± 0.6, BMI = 32.0 ± 1.5 kg/m2, age = 58.0 ± 4.4 years), and nine insulin-resistant (OIR; HOMAIR = 4.0 ± 0.8, BMI = 33.0 ± 1.8 kg/m2, age = 55 ± 3.8 years). A detailed characterization of these volunteers has been published previously [5]. Skeletal muscle (SM) biopsies were obtained from the left vastus lateralis [22] at rest and in fasting state, after local anesthesia with

TRIF-IFNI Pathway Activation is Altered in OIR SM

TRIF-IFNI pathway activation leads to increased expression of numerous ISG as ISG15, interferon responsive factor (IRF), Interferon-induced GTP-binding protein MX1, double stranded activated protein kinase (PKR), OAS [34]. However, as we previously showed that OAS-RNase L pathway allows maintaining insulin sensitivity in palmitate-treated human myotubes [17], we analyzed OAS and RNase L mRNA expression in OIS and OIR SM biopsies. RNase L mRNA expression was lower in OIR SM and OAS mRNA

Discussion

Several studies have shown the role of TLR4-mediated MyD88 activation in IR development [12]. However, TLR4 signaling involves a second pathway: the TRIF-IFNI-dependent pathway. In this study, we aimed to analyze the regulation by RNF41 of TRIF-IFNI pathway and insulin response in OIS and OIR SM. In agreement with other studies [12], we previously showed that IκB expression is lower in OIR compared to OIS SM, suggesting that MyD88 pathway is more activated in OIR SM [5]. Here, we show that

Fundings

This work was supported by grants from Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Montpellier (UM), Centre National de la Recherche Scientifique (CNRS) and CHRU Montpellier PROM8685. CA and OF were both recipient of a Ministère de l'Enseignement Supérieur et de la Recherche (MESR) fellowship.

Acknowledgments

The authors gratefully acknowledge Marie Hokayem for the critical reading of the manuscript.

Authors' contribution

Conceived and designed the experiments: CBi and TS. Experimental investigations: CA, CBi, CB, OF, KL, PS, AB. Recruitment of the volunteers and SM biopsies: AS and JM. Data analysis: CBi, CB, CA. Wrote the manuscript: CBi and OF. Review of the manuscript: all authors.

Conflicts of interest

None.

References (58)

  • C. Wang et al.

    Protein kinase C theta (PKCtheta)-dependent phosphorylation of PDK1 at Ser504 and Ser532 contributes to palmitate-induced insulin resistance

    J Biol Chem

    (2009)
  • C.J. Vlahos et al.

    A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002)

    J Biol Chem

    (1994)
  • D.E. Dostal et al.

    An improved method for absolute quantification of mRNA using multiplex polymerase chain reaction: determination of renin and angiotensinogen mRNA levels in various tissues

    Anal Biochem

    (1994)
  • E.T. Kase et al.

    Primary defects in lipolysis and insulin action in skeletal muscle cells from type 2 diabetic individuals

    Biochim Biophys Acta

    (2015)
  • J.M. Dos Santos et al.

    The effect of exercise on skeletal muscle glucose uptake in type 2 diabetes: an epigenetic perspective

    Metabolism

    (2015)
  • Y.T. Wang et al.

    Human serum RNase-L level is inversely associated with metabolic syndrome and age

    Cardiovasc Diabetol

    (2017)
  • G. Schreiber

    The molecular basis for differential type I interferon signaling

    J Biol Chem

    (2017)
  • B.B. Kahn et al.

    Obesity and insulin resistance

    J Clin Invest

    (2000)
  • R.A. DeFronzo et al.

    Skeletal muscle insulin resistance is the primary defect in type 2 diabetes

    Diabetes Care

    (2009)
  • C. Amouzou et al.

    Skeletal muscle insulin resistance and absence of inflammation characterize insulin-resistant grade I obese women

    PLoS One

    (2016)
  • M.R. Dasu et al.

    Free fatty acids in the presence of high glucose amplify monocyte inflammation via toll-like receptors

    Am J Physiol Endocrinol Metab

    (2011)
  • I. Jialal et al.

    Toll-like receptor status in obesity and metabolic syndrome: a translational perspective

    J Clin Endocrinol Metab

    (2014)
  • L.A. Velloso et al.

    TLR4 at the crossroads of nutrients, gut microbiota and metabolic inflammation

    Endocr Rev

    (2015)
  • L.B. Ivashkiv et al.

    Regulation of type I interferon responses

    Nat Rev Immunol

    (2014)
  • K. Honda et al.

    IRFs: master regulators of signalling by toll-like receptors and cytosolic pattern-recognition receptors

    Nat Rev Immunol

    (2006)
  • O. Fabre et al.

    Defects in TLR3 expression and RNase L activation lead to decreased MnSOD expression and insulin resistance in muscle cells of obese people

    Cell Death Dis

    (2014)
  • M. Bluher

    Are there still healthy obese patients?

    Curr Opin Endocrinol Diabetes Obes

    (2012)
  • C. Wang et al.

    The E3 ubiquitin ligase Nrdp1 ‘preferentially’ promotes TLR-mediated production of type I interferon

    Nat Immunol

    (2009)
  • C. Thomas et al.

    Relationships between maximal muscle oxidative capacity and blood lactate removal after supramaximal exercise and fatigue indexes in humans

    J Appl Physiol

    (2004)
  • Cited by (0)

    View full text