WO2015091812A1 - Methods and pharmaceutical composition for the treatment of insulin resistance - Google Patents

Abstract

The present invention relates to methods and pharmaceutical composition for the treatment of insulin resistance. In particular, the present invention relates to a method for the treatment of insulin resistance in a subject in need thereof comprising administering the subject with a therapeutically effective amount of an agent selected from the group consisting of 2',5'-oligoadenylate (2-5 A) or derivatives compounds, TLR3 agonists, and IFN1 polypeptides.

Description

METHODS AND PHARMACEUTICAL COMPOSITION FOR THE TREATMENT

OF INSULIN RESISTANCE

FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical composition for the treatment of insulin resistance.

BACKGROUND OF THE INVENTION:

Obesity or high- fat diet conditions are associated with an increased risk to develop metabolic dysfunction such as insulin resistance (IR) , which is a central determinant of type 2 diabetes (T2D) pathogenesis \ Nevertheless, in about 30% of obese individuals, insulin sensitivity is preserved and they seem to be protected against metabolic complications ²'³. The mechanisms allowing these obese people to stay insulin-sensitive and metabolically healthy are not yet clearly understood.

Since the 50s, several studies have led to develop the idea that IR is a major consequence of the establishment of a chronic low-grade inflammatory state and oxidative stress ⁴'⁵. Excess of saturated free fatty acids (FFA) overly activates toll- like receptors (TLR), that leads to the local activation of innate immune response ⁶'⁷. It results in production and release of numerous cytokines as type I interferon (IFNI), interleukin (IL)-ip, IL-6 and tumor necrosis factor a (TNFa), and chemokines such as monocyte chemoattractant protein- 1 (MCP-1), which activate inflammation and oxidative stress. However, the specific mechanisms linking TLR activation, inflammation, oxidative stress and defect in insulin sensitivity have been only partially characterized.

Inflammation induces the expression and the activation of inflammatory kinases such as c-Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPK) and double-stranded RNA-dependent protein kinase (PKR) which both phosphorylate insulin receptor substrates (IRS) on serine residues, thus disrupting insulin signaling ⁸'⁹. Moreover, these two kinases activate a third one, the inhibitor of κΒ (ΙκΒ) kinase (IKK), or directly phosphorylate ΙκΒ, leading to activation of transcriptional nuclear factor κΒ (NF-κΒ) and expression of inflammatory genes ¹⁰.

In parallel, chronic FFA exposure is associated with increased reactive oxygen species (ROS) production (the main source being the mitochondrial electron transport chain) through a TLR4-dependent mechanism that also involves innate immunity and the release of pro- inflammatory cytokines ^{11 13}. Besides, ROS and in particular hydrogen peroxide (H₂O₂), are essential for many biological processes, including regulation of insulin signal transduction ⁴'¹⁴. Indeed, small amount of H₂0₂ is produced during insulin receptor stimulation and facilitates normal signal transduction of insulin to the different proteins of the cascade via tyrosine phosphorylation [e.g. insulin receptor, IRS-1] or serine phosphorylation (PKB/Akt) 15,16 jj_{ie S}t_eady-_State levels of tyrosine and serine phosphorylation are modulated by tyrosine (PTPases) or serine/threonine (PP2A) phosphatases that negatively regulate insulin action. These phosphatases are themselves directly sensitive to H₂0₂ oxidation, that causes their reversible inhibition and thus favors insulin signal transduction ^15~17. If ROS are indispensable for the proper insulin response, their excess may however cause its disruption and contribute to IR and T2D, in particular because they lead to JNK activation ¹⁸'¹⁹ . Consequently, ROS level must be tightly regulated and pro-oxidants are naturally neutralized or detoxified by enzymes such as superoxide dismutases (SOD), glutathione peroxidases (GPx) and catalase. "Oxidative stress" results from an imbalance between the generation of ROS and the anti- oxidative defenses.

Innate immunity, inflammation and oxidative stress processes are finely controlled by the induction of negative regulators to avoid unchecked immune reactions. IFNI plays an important role in the balance between activation and inhibition of immune responses, particularly through their anti- inflammatory property as inhibition of TNFa and regulation of IL10 production ²⁰. The mechanism of this positive effect of IFNI on insulin signaling has not been yet studied in depth.

The latent endoribonuclease (RNase L) is an essential actor of innate immunity and is induced by IFNI ²²'²³. During inflammation, its induction is essential to TLR4 signal transduction ²⁴ and to activation of dsRNA sensor as RIG-1 and MAD5²³. RNase L activity is strictly dependent on its binding by small oligoadenylates, the 2-5 A, which are synthesized by the 2-5 A oligoadenylate synthetase(s) (OAS), OAS1 and OAS2, also induced by IFNI during inflammation ²². RNase L, when activated by 2-5A, cleaves single-stranded RNA at UpNp sequences during their translation, inhibiting protein synthesis ²⁵. These cleaved mRNA can also themselves activate TLR3 pathway via their secondary structures ²³'²⁶. TLR3 activation leads to IFNI production and induction of interferon stimulated genes (ISG) such as OAS, thus to 2-5A synthesis ²¹. The 2-5A activate RNase L, leading to the amplification of TLR3 signaling. Moreover IFNI, in turn, induces TLR3 expression in a positive amplification loop ²⁸. RNase L activity is negatively regulated by RNase L inhibitor/ ATP-binding cassette subfamily E member 1 (RLI/ABCEl) ²⁹. The major role of RNase L and RLI in lipids accumulation, insulin response and cell fate during myogenesis and adipogenesis via regulation of MyoD and C/EBP (CCAAT-enhancer-binding protein)-homologous protein 10 (CHOP 10) mPvNA expression was demonstrated ^30~32. SUMMARY OF THE INVENTION:

The present invention relates to methods and pharmaceutical composition for the treatment of insulin resistance. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Skeletal muscle is an essential organ for insulin action and glucose metabolism, and the primary site of peripheral IR development ³³. It is well documented that elevation of plasma FFA is currently associated with obesity and causes inflammation, oxidative stress and IR in skeletal muscle ³⁴'³⁵. The current study was performed in order to investigate the implication of RNase L/RLI in FFA-induced IR in skeletal muscle cells during obesity. The results show that RNase L plays an important role in maintaining insulin response during inflammation in mouse and human muscle cells. Myotubes from obese insulin-resistant (OB- IR) individuals, when compared to insulin- sensitive (OB-IS), are characterized by a lower expression of the enzymes MnSOD and OAS. Cell transfection with 2-5A allows to restore insulin response as well as increased MnSOD expression in OB-IR myotubes. Moreover, investigating the mechanism by which RNase L regulates insulin response led us to demonstrate that reduced expression of MnSOD and OAS are linked to low level of TLR3. The inventors thus identify here for the first time a mechanism regulated by inflammation which allows maintenance of insulin response via TLR3 activation and account for the difference between insulin-sensitive and insulin-resistant obese people's muscle.

Accordingly the present invention relates to a method for the treatment of insulin resistance in a subject in need thereof comprising administering the subject with a therapeutically effective amount of an agent selected from the group consisting of 2', 5'- oligoadenylate (2-5 A) or derivatives compounds, TLR3 agonists, TRIF agonists and IFN1 polypeptides.

In some embodiments, the subject is an overweight subject and more preferably an obese subject. "Overweight subject" refers herein to a subject preferably having a BMI of 25 to 30 kg/m². "Obesity" refers herein to a medical condition wherein the subject preferably has a BMI of >30 kg/m².

As used herein the term "insulin resistance" has its general meaning in the art and refers to the resistance of peripheral tissue (e.g. muscle) to the action of insulin to stimulate glucose uptake. Insulin resistance may be assessed in a subject by any assay well known in the art. In particular, insulin resitance may be assessed with HOMA_IR index (see EXAMPLE), which is frequently used in routine clinical medicine to estimate insulin sensitivity state. Typically an insulin sensitive subject has a HOMA_IR<1.5 and a subject whom the HOMA_IR index is superior to 3 is conventionally considered as insulin-resistant.

In some embodiments, the method of the present invention is particularly suitable for the treatment of muscle insulin resistance. As used herein the term "2',5'-oligoadenylate" or "2-5 A" has its general meaning in the art. Various 2',5'-oligoadenylate derivative compounds have been described in the prior art. Typically the derivatives compounds include but are not limited to those described in with EP0630249, W098/56385, US4464359, US4924624 and WO2012062847. As used herein the term "Toll like receptor (TLR)" has its general meaning in the art and describes a member of the Toll-like receptor family of proteins or a fragment thereof that senses a microbial product and/or initiates an innate or an adaptive immune response. Tolllike receptors (TLRs) are a family of germline-encoded transmembrane proteins that facilitate pathogen recognition and activation of the innate immune system. (Hoffmann J A et al, Science 284, 1313-1318 (1999); Rock F L et al, Proc Natl Acad Sci USA 95:588-593 (1998)). Toll-like Receptors include TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR 8, TLR9, TLR10, TR11 and TLR12.

The term "agonist" as used herein in referring to a TLR activating molecule, means a molecule that activates a TLR signaling pathway. As discussed above, a TLR signaling pathway is an intracellular signal transduction pathway employed by a particular TLR that can be activated by the TLR agonist. These pathways are MYD88 dependent and MYD88 independent pathways (T. Kawai, S. Akira / Seminars in Immunology 19 (2007) 24-32) that activate common intracellular mechanisms and include, for example, IRF3, NF-κΒ, Jun N- terminal kinase and mitogen-activated protein kinase. The TLR agonism for a particular compound may be assessed in any suitable manner. For example, assays for detecting TLR agonism of test compounds are described, for example, in U.S. Provisional Patent Application Ser. No. 60/432,650, filed Dec. 11, 2002, and recombinant cell lines suitable for use in such assays are described, for example, in U.S. Provisional Patent Application Ser. No. 60/432,651, filed Dec. 11, 2002.

TLR3 agonists are well known in the art. For example, TLR3 agonists include naturally-occurring double-stranded RNA (dsRNA); synthetic ds RNA; and synthetic dsRNA analogs; and the like (Alexopoulou et alal, 2001). An exemplary, non- limiting example of a synthetic dsRNA analog is Poly(I:C).

As used herein the term "TRIF" has its general meaning in the art and refers to TIR- domain-containing adapter-inducing interferon-β (TRIF) which is an adapter in responding to activation of to 11- like receptors (TLRs). Accordingly, a TRIF agonist refers to any compound that is able to activate TRIF. In some embodiments, the TRIF agonist is a TRIF polypeptide.

As used herein the term "Type I interferon" or "IFN1" has its general meaning in the art and refers collectively, to IFN-. alpha., IFN-.beta., IFN-omega, et al. or any mixture or combination thereof. Accordingly, the term "type 1 interferon" encompasses alpha interferons, beta interferons and other types of interferons classified as type 1 interferons. Particularly, this includes epsilon interferon, zeta interferon, and tau interferons such as tau 1 , 2, 3, 4, 5, 6, 7, 8, 9, and 10. Also, this includes variants thereof such as fragments, consensus interferons which mimic the structure of different type 1 interferon molecules such as alpha interferons, PEGylated versions thereof, type 1 interferons with altered glycosylation because of recombinant expression or mutagenesis, and the like.

Those skilled in the art are well aware of different type I interferons including those that are commercially available and in use as therapeutics. In particular the type 1 interferon will comprise a human a human alpha interferon and a human beta interferon. The polypeptide sequences for human interferon-alpha are deposited in database under accession numbers: AAA 52716, AAA 52724, and AAA 52713. The polypeptide sequences for human interferon-beta are deposited in database under accession numbers AAC41702, NP 002167, AAH 96152, AAH 96153, AAH 96150, AAH 96151, AAH 69314, and AAH 36040. The polypeptide sequences for human interferon-gamma are deposited in database under accession numbers AAB 59534, AAM 28885, CAA 44325, AAK 95388, CAA 00226, AAP 20100, AAP 20098, AAK 53058, and NP-000610. Interferon-alpha includes, but is not limited to, recombinant interferon-a2a (such as

ROFERON® interferon available from Hoffman-LaRoche, Nutley, N.J.), interferon-a2b (such as Intron-A interferon available from Schering Corp., Kenilworth, N.J., USA), a consensus interferon, and a purified interferon-a product. Use of interferon beta- la such as AVONEX™ (Biogen Idee MA Inc.) and REBIF™ (EMD Serono) and interferon-beta-lb, marketed in the United States as BETASERON™ (Berlex) and EXTAVIA™ (Novartis) are also encompassed in the present invention/

Suitable preparations, e.g., substantially pure preparations of the agent of the invention may be combined with pharmaceutically acceptable carriers, diluents, solvents, excipients, etc., to produce an appropriate pharmaceutical composition.

In some embodiments, the invention further provides a pharmaceutically acceptable composition comprising (i) at least one agent of the invention and (ii) a pharmaceutically acceptable carrier or excipient. This pharmaceutical composition is thus particularly suitable for the treatment of insulin resistance.

The term "pharmaceutically acceptable carrier, excipient, or vehicle" refers to a nontoxic carrier, excipient, or vehicle that does not destroy the pharmacological activity of the agent with which it is formulated. Pharmaceutically acceptable carriers, excipients, or vehicles that may be used in the compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose- based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration may be included. Pharmaceutically acceptable salts of the agents of this invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. Salts derived from appropriate bases include alkali metal {e.g., sodium and potassium), alkaline earth metal {e.g., magnesium), ammonium and NNo(Cl -4 alkyl)4 salts. This invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization.

It is to be understood that the pharmaceutical compositions of the invention, when administered to a subject, are preferably administered for a time and in an amount sufficient to treat the insulin resistance. In various embodiments of the invention an effective amount of the pharmaceutical composition is administered to a subject by any suitable route of administration including, but not limited to, intravenous, intramuscular, by inhalation (e.g. , as an aerosol), intraocularly, orally, rectally, intradermally, by application to the skin, etc. Accordingly the pharmaceutical composition is formulated to be compatible with its intended route of administration. Solutions or suspensions used for parenteral {e.g., intravenous), intramuscular, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use typically include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), phosphate buffered saline (PBS), or Ringer's solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium.

For this purpose, any bland fixed oil may be employed including synthetic mono- or di- glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

In all cases, the composition should be sterile, if possible, and should be fluid to the extent that easy syringeability exists. Preferred pharmaceutical formulations are stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. In general, the relevant carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Prolonged absorption of oral compositions can be achieved by various means including encapsulation.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Preferably solutions for injection are free of endotoxin. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of oral solution (e.g. for pediatric purpose) tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. Formulations for oral delivery may advantageously incorporate agents to improve stability within the gastrointestinal tract and/or to enhance absorption.

A therapeutically effective amount of an agent of the invention typically ranges from about 0.001 to 100 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The pharmaceutical composition can be administered at various intervals and over different periods of time as required, e.g., multiple times per day, daily, every other day, once a week for between about 1 to 10 weeks, between 2 to 8 weeks, between about 3 to 7 weeks, about 4, 5, or 6 weeks, etc. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous and current treatments, the general health and/or age of the subject, and other diseases present. Generally, treatment of a subject with an inventive composition can include a single treatment or, in many cases, can include a series of treatments. It will be appreciated that a range of different dosage combinations (i.e., doses of the agent of the invention) can be used. Exemplary doses include milligram or microgram amounts or even nanogram amounts of the inventive compounds per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram.) It is furthermore understood that appropriate doses depend upon the potency of the agent, and may optionally be tailored to the particular recipient, for example, through administration of increasing doses until a preselected desired response is achieved. It is understood that the specific dose level for any particular subject may depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

The agents of the invention for use in accordance with the present invention may be administered alone, or as part of a combination therapy. If a combination of active agents is administered, then it may be administered simultaneously, separately or sequentially. In particular, the substances for use in connection with the treatment of insulin resistance is combined with one or more additional active substances selected from the group consisting of insulin, sulfonylureas, meglitinides, biguanides, thiazolidinediones, glilazones, a-glucosdase inhibitors, incretin mimetics such as e.g. GLP-1 analogues and GLP-1 agonists, DPP-4 inhibitors, amylin analogues, PPAR α/γ ligands, sodium-dependent glucose transporter 1 inhibitors, fructose 1,6-bisphosphatase inhibitors, glucagon inhibitors, and l lbeta-HSDl inhibitors. Non-limiting examples of the one or more additional active substance may be selected from the group consisting of insulin, glimepiride, glibenclamide, tolbutamide, gliciazide, glipzid, repaglinide, nateglinide, metformin, pioglitazones, rosiglitazones, acarbose, miglitol, liraglutide, exenafide, sitagliptin, vildagliptin saxagliptin, and alogliptin. In another embodiment of the present invention the one or more additional active substances are selected from the group consisting of thiazides, diuretics, ACE inhibitors, AT2 inhibitors, ARB, Ca2+ antagonists, a-blockers, β-blockers, cholesterol absorption inhibitors, hypolipidemic drugs, fibrates, anion exchangers, bile acid sequestrants, fish oils, HMG-CoA reductase inhibitors, and CBI cannabinoid receptor antagonists. Non-limiting examples of the one or more additional active substance may be selected from the group consisting of bendroflumetiazid, indapamid, hydrochlorothiazid, captopril, enalapril, lisinopril, fosinophil, perindopril, quinapril, ramipril, trandolapril, quinapril, fosinopril, candesartancilexefil, irbesarian, losartan, valsartan, telmisartan, eprosartan, olmesartanmedoxomil, nifedipin, amlodipin, nitrendipin, diltiazem, felodipin, verapamil, lacidipin, isradipin, tercanidipin, doxazosin, prazosin, terazosin, phentolamin, hydralazin, acebutolol, atenolol, bisoprolol, carvedilol, esmolol, labetalol, metoprolol, pindolol, propranolo, sotalol, tertatolol, timolol, melhyldopa, moxonidin, ezitimibe, gemfibrozil, bezafibrat, ienofibrate, nicotinic acid, acipimox, colestipol, colestyramin, fish oils, atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin, and rimonabant.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:

Figure 1: Insulin response in C2C12-derived mouse myogenic cells after palmitate treatment.

(A) RNase L expression was IPTG-induced (C2-RNaseL+) or not in differentiated C2- RNaseL cells. Total proteins were extracted from C2-RNaseL, C2-RNaseL+ and C2-RLI, and

RNase L and RLI expression was assessed by western-blot. A membrane scan obtained in each cell line is presented in the figure. RNase L and RLI levels in control C2-RNaseL were set at 100%. Error bars refer to the SEM obtained in three independent experiments. *P<0.05.

(B) Differentiated C2-RNaseL, C2-RNaseL+ and C2-RLI cells were incubated or not with palmitate then stimulated or not with insulin. To measure insulin response, P-Akt and

Akt proteins expression was assessed on total cellular extracts by western-blot. A membrane scan obtained in each cell line is presented in the figure. The control value, with neither palmitate treatment nor insulin stimulation, was set at 100% for each cell line. Error bars refer to the SEM obtained in three independent experiments. **P<0.01. Figure 2: Insulin response of normal-weight subjects' myotubes after siRNA- transfection and palmitate treatment.

(A) Human myotubes were transfected with control siRNA (Myo-siControl) or specific siRNA against RLI (Myo-siRLI) or RNase L (Myo-siRNaseL) and were then incubated or not with palmitate. Total RNA were then extracted from siRNA-transfected cells to measure expression of RNase L and RLI mRNA by q-PCR. For each mRNA, the mean level of mRNA expression in Myo-siControl was set at 1. Error bars refer to the SEM obtained from five samples issued from different donors. *P<0.05 and **P<0.01.

(B) Human myotubes were siRNA-transfected and were then incubated or not with palmitate. RNase L and RLI proteins expression was assessed on total cellular extracts by western-blot. Membrane scans obtained from two different samples are presented in the figure. RNase L and RLI levels in Myo-siControl without palmitate treatment were set at 100%. Error bars refer to the SEM obtained from five samples issued from different donors. *P<0.05.

(C) siRNA-transfected human myotubes were incubated or not with palmitate then stimulated or not with insulin. P-Akt and Akt proteins expression was assessed on total cellular extracts by western-blot. Membrane scans obtained from two different samples of each siRNA-transfected cell population are presented in the figure. Data are expressed relative to the fold induction of P-Akt/ Akt protein level in each cell population with neither palmitate treatment nor insulin stimulation (basal response), which was set at 1. Error bars refer to the SEM obtained from five samples issued from different donors. *P<0.05.

Figure 3: Insulin response and RNase L and RLI expression in insulin-sensitive and insulin-resistant obese subjects' myotubes.

(A) Differentiated myotubes from OB-IS (n=7) and OB-IR (n=8) subjects were incubated or not with insulin. P-Akt and Akt proteins expression was assessed on total cellular extracts by western-blot. Membrane scans obtained from four different subjects of each group are presented in the figure. Data are expressed relative to the fold induction of P- Akt/ Akt level in OB-IS subjects after insulin stimulation, which was set at 100%. **P<0.01.

(B) Glucose uptake was measured in myotubes of OB-IS (n=7) and OB-IR (n=8) subjects. Differentiated cells were incubated or not with insulin then with 6-NBDG in a glucose-free medium. Fluorescence intensity was measured at 540 nm wavelength. Each bar of the histogram represents the ratio of fluorescence measured in basal condition (without insulin) and after incubation with insulin. **P<0.01.

(C) Differentiated myotubes of OB-IS (n=7) and OB-IR (n=8) subjects were collected and expression of RNase L and RLI proteins was assessed on total cellular extracts by western-blot. Membrane scans obtained from four different subjects of each group are presented in the figure. The mean value of OB-IS group was set at 100%.

Figure 4: OAS and SOCS3 expression and insulin response in insulin-sensitive and insulin-resistant obese subjects' myotubes after 2-5 A transfection.

(A) Differentiated myotubes of OB-IS (n=7) and OB-IR (n=8) subjects were collected and expression of OAS2 protein was assessed on total cellular extracts by western-blot. Membrane scans obtained from four different subjects of each group are presented in the figure. The mean value of OB-IS group was set at 100%. *P<0.05.

(B) Expression of OAS 1 and OAS2 mRNA was measured by q-PCR in myotubes of OB-IS (n=7) and OB-IR (n=7) subjects. For each mRNA, the mean level of expression in OBIS group was set at 100%. *P<0.05.

(C) SOCS3 protein expression was assessed on total cellular extracts from myotubes of OB-IS (n=7) and OB-IR (n=8) subjects, by western-blot. Membrane scans obtained from four different subjects of each group are presented in the figure. The mean value of OB-IS group was set at 100%.

(D) Myotubes of OB-IS (n=7) and OB-IR (n=8) subjects were transfected or not with 2-5A and were then stimulated or not with insulin. P-Akt and Akt proteins expression was assessed on total cellular extracts by western-blot. Membrane scans obtained from four different subjects of each group are presented in the figure. Data are expressed relative to the percentage of induction of P-Akt/ Akt protein level in insulin-stimulated non-transfected OBIS myotubes, which was set at 100%. In the histogram, insulin-stimulated P-Akt/ Akt levels have been represented in 2-5A-transfected and non-transfected myotubes. *P<0.05 and **P<0.01.

(E) Glucose uptake was measured in myotubes of OB-IS (n=7) and OB-IR (n=8) subjects. Differentiated cells were transfected or not with 2-5A, then incubated or not with insulin and finally with 6-NBDG in a glucose-free medium. Fluorescence intensity was measured at 540 nm wavelength. Each bar of the histogram represents the ratio of fluorescence measured in basal condition (without insulin) and after incubation with insulin. **P<0.01. (F) Myotubes of OB-IS (n=7) and OB-IR (n=7) subjects were transfected or not with 2-5A and expression of OAS1 and OAS2 mRNA was then measured by q-PCR. For each mRNA, the mean level of expression in non-transfected OB-IS myotubes was set at 100%. *P<0.05.

Figure 5: Analysis of PKR and ΙκΒα expression and JNK and IRSl phosphorylation in obese insulin-sensitive and insulin-resistant subjects' myotubes.

Myotubes of OB-IS (n=7) and OB-IR (n=8) subjects were collected and expression of ΙκΒα, PKR, P-JNK, JNK, Ser312P-IRS l and IRS l proteins was assessed on total cellular extracts by western-blot. Membrane scans obtained from four different subjects of each group are presented in the figure. For representation of ΙκΒα and PKR expression levels and P- JNK/JNK and P-IRS 1/IRS 1 expression rates the mean value of OB-IS group was set at 100%.

Figure 6: Analysis of anti-oxidant enzymes and mitochondrial proteins expression in obese insulin-sensitive and insulin-resistant subjects' myotubes.

(A) Differentiated myotubes of OB-IS (n=7) and OB-IR (n=7) subjects were collected and expression of GPx 1 , 2 and 4, Cu/ZnSOD, MnSOD, catalase and NRF2 mRNA was measured by q-PCR. For each mRNA, the mean level of expression in OB-IS group was set at 100%. *P<0.05.

(B) Myotubes of OB-IS (n=8) and OB-IR (n=7) subjects were transfected or not with

2-5A and MnSOD protein expression was assessed on total cellular extracts, by western-blot. Membrane scans obtained from two different subjects of each group are presented in the figure. The mean level of MnSOD expression in OB-IS group, without 2-5A treatment, was set at 100%. *P<0.05 and **P<0.01.

(C) After 2-5 A transfection, expression of MnSOD mRNA was measured by q-PCR in myotubes of OB-IS (n=7) and OB-IR (n=8) subjects. The mean level of expression in non- transfected OB-IS myotubes was set at 100%. *P<0.05 and **P<0.01.

(D) COX IV and UCP3 proteins expression was assessed on total cellular extracts from myotubes of OB-IS (n=7) and OB-IR (n=8) subjects, by western-blot. Membrane scans obtained from four different subjects of each group are presented in the figure. The mean value of OB-IS group was set at 100%.

(E) Expression of TLR3 and TLR4 mRNA was measured by q-PCR in myotubes of OB-IS (n=7) and OB-IR (n=8) subjects. The mean level of expression in non-transfected OBIS myotubes was set at 100%. *P<0.05. (F) Myotubes of OB-IS (n=7) and OB-IR (n=7) subjects were transfected or not with 2-5 A and TLR3 mRNA expression was then measured by q-PCR. The mean level of expression in non-transfected OB-IS myotubes was set at 100%. *P<0.05. Figure 7: Role of RNase L and TLR3 activation during inflammation in insulin response of obese people's myotubes.

During obesity, chronic lipid overload triggers the development of global inflammation and oxidative stress, which are particularly deleterious for metabolic tissues such as skeletal muscle. RNase L, which is activated by the 2-5A synthesized by the OAS, cleaves mRNA, which in turn could activate TLR3 pathway. This activation allows the expression of MnSOD and of numerous ISG among which the OAS. The secondary activation of the OAS induced by cleaved mRNA leads to increased 2-5A production that all the more activates RNase L. It then creates an amplification loop that induces high levels of OAS and MnSOD expression in myotubes from insulin-sensitive obese people. By controlling H202 levels, high expression of MnSOD allows for maintaining Akt phosphorylation and glucose uptake, even when excessive ROS are produced. At the contrary, myotubes from obese insulin-resistant people express very low level of TLR3 which prevents this amplification loop to setting up. The consecutive under-expression of the OAS and MnSOD are thus responsible for oxidative stress development and inhibition of insulin response. IR: insulin receptor; MyD88: myeloid differentiation primary response gene 88.

EXAMPLE: Material & Methods

Mouse myogenic cells

For conditional expression of RNase L, human cDNA was cloned in the LacSwitch II inducible mammalian expression system (Stratagene, Massy, France). The C2C12 clone in which RNase L is conditionally expressed (C2-RNaseL) and C2-RLI cells which over-express RLI were previously described ³⁰'³². For induction of RNase L (C2-RNaseL⁺), differentiated C2-RNaseL cells were treated with isopropyl-P-D-thiogalactopyranoside (IPTG) (2 mM) during six hours.

Human cells Primary human skeletal muscle cells from three normal-weight (19<BMI<25 kg/m²) subjects were provided by the Agence Francaise contre les Myopathies (AFM), in accordance with the French and European legislations.

The present study involved fifteen non-diabetic obese (BMI>30 kg/m²) subjects, all with no personal or familial history of diabetes. Their HOMA_IR index, which is frequently used in routine clinical medicine to estimate insulin sensitivity state, was evaluated to separate them into insulin-sensitive (OB-IS: HOMA_IR<1.5) and insulin-resistant (OB-IR: HOMA_IR>3.5) (a subject whom the HOMA_IR index is superior to 3 is conventionally considered as insulin-resistant). The characteristics of these subjects are presented in Table 1.

Group OB-IS (n=7) OB-IR (n=8)

Gender Male Male

Age (years) 50.4 ± 2.9 51.6 ± 1.7

Body mass index

32.6 ± 1.2 33.7 ± 0.9

(kg/m²)

Body fat (%) 30.1 ± 1.4 32.0 ± 1.7

Fasting blood glucose

5.32 ± 0.20 5.50 ± 0.15

[G] (mM)

Fasting plasma insulin

6.89 ± 0.68 16.66 ± 1.84**

[I] (pU/ml)

HOMA_IR 1.64 ± 0.19 4.08 ± 0.61 *

Table 1. Clinical characteristics of obese subjects.

Values are means ± SEM.

**P<0.001 fasting plasma insulin level in OB-IR subjects compared to OB-IS.

**P=0.002 Homeostasis model assessment of insulin resistance (HOMA_IR =

[I]x[G]/22.5) index in OB-IR subjects compared to OB-IS.

Skeletal muscle cells were obtained from biopsies in the vastus lateralis ⁵⁵. This experimental protocol was approved by the local Ethic Committee (03/10/GESE, Montpellier, France) and informed and written consent was obtained from all participants. NCTO 1644942 ClinicalTrials.gov.

Myoblastes isolation, purification and culture were performed as described by Barro et al. ⁵⁶. Cells between passages 2 and 6 were used for all the experiments. Cell differentiation

At confluence, C2C12-derived cells and human myoblastes were switched to muscle differentiation medium [Dulbecco's modified eagle medium (DMEM, Lonza, Basel, Switzerland) supplemented with 2% (V/V) FCS (PAN Biotech, Dutscher, Brumath, France)] for five days.

Cell transfection

Human cells were transfected four days after induction of muscle differentiation. siRNA were transfected with HiPerFect (Qiagen, Courtaboeuf, France) following manufacturer instructions. Cells were incubated with siRNA for eight hours at 37°C.

A non-stabilized non-modified 2-5A₃ was enzymatically produced with dsRNA- activated OAS from IFNP-treated Hela cells (500 U/ml) ⁵⁷ and was transfected at 10 nM final concentration with the Calcium Phosphate Transfection Kit (Sigma-Aldrich, Saint-Quentin Fallavier, France) following manufacturer instructions. Cells were incubated for eight hours at 37°C with 2-5A then total RNA were extracted as described in following paragraph. RNase L activation by 2-5A was checked by controlling 28S and 18S specific cleavage as previously described ⁵⁸'⁵⁹ (data not shown).

Induction of inflammation and insulin treatment

Lipid-containing media were prepared by conjugation of palmitate with FFA-free BSA

(molar ratio palmitate/BSA ~2.5) ⁶⁰ Cells were incubated for 16 hours at 37°C with 750 μΜ palmitate.

For insulin response, cells were treated or not with human insulin (1 nM) for 10 minutes at 37°C.

Western-blot assay

Differentiated cells were washed and lysed in SDS-PAGE sample buffer [300 mM Tris (pH 8.9), 5% (W/V) SDS, 750 mM β-mercaptoethanol, 20% (V/V) glycerol, bromophenol blue]. Then, proteins were fractionated and transferred electrophoretically to nitrocellulose membranes to be incubated with the different antibodies and analyzed with the Odyssey Infrared Imaging System LI-COR (Biosciences, ScienceTec, Courtaboeuf, France) as previously described ³¹. We used the primary antibodies against the following proteins: Phospho-Akt (Ser473), Akt, ΙκΒα, PKR, Phospho-SAPK JNK (Thrl83/Tyrl85), SAPK/JNK, IRS1, SOCS3 and COX IV (nine from Cell Signaling, Ozyme, Saint-Quentin Yvelines, France), RNase L and Phospho-IRSl (Ser312) (two from Abeam, Paris, France), RNase L (H300), ABCE1 (mouse) and OAS2 (three from Santa Cruz, Tebu-Bio, Le Perray en Yvelines, France), ABCE1 (human) (Abnova, Tebu-Bio), (Pierce, Thermo Scientific, Fermentas GmbH, Sankt Leon-Rot, Germany), a-tubulin and UCP3 (two from Sigma- Aldrich). The secondary antibodies were conjugated to IRDye800® (Rockland, Tebu-Bio). a- tubulin protein level was measured in each sample as an indicator of proteins quantity loading. Proteins levels were then quantified from membranes scans with the ImageJ software and corrected with corresponding a-tubulin levels. Glucose uptake

After 10 minutes incubation at 37°C with or without 1 nM insulin, differentiated human cells were incubated 30 minutes at 37°C with 300 μΜ 6-[N-(7-nitrobenz-2-oxa-l,3- diazol-4-yl) amino] -2-deoxy-glucose (6-NBDG, Molecular Probes, Fisher Scientific), in a glucose-free medium (InVitrogen, Life Technologies, Saint Aubin, France) ³¹'⁶¹. Fluorescence intensity of 6-NBDG was measured at 540 nm wavelength (465 nm excitation wavelength) with an Infinite 200PRO TECAN (Lyon, France). Fluorescence values were normalized by cell number.

Reverse transcription and real-time quantitative PCR (q-PCR)

Total RNA were isolated using TRIzol (InVitrogen). Complementary DNA (cDNA) were generated by reverse transcription with the Verso cDNA Synthesis Kit (Thermo Scientific) following manufacturer instructions. To quantify genes expression, complementary DNA were used as templates in SYBR Green I Master real-time q-PCR assays on a LightCycler®480 (Roche Diagnostics, Meylan, France), in a total volume of 10 μΐ. Cycle number was 40. Sample data were analyzed according to the comparative cycle threshold method and were normalized by stable reference gene of GAPDH. Gene sequences for primer design were obtained from the NCBI Reference Sequences database. Primers were chosen using the Primer3 and LightCycler Probe Design (Roche) softwares. Forward and reverse primers were designed on different exon sequences when possible.

Statistical analysis

Values represented in the graphs are means ± standard errors of the mean (SEM). Statistical analyses were performed using SigmaStat software. Statistical significance was determined by using the Holm-Sidak method. Two groups were considered statistically significantly different for <0.05.

Results

RNase L limits deleterious effect of palmitate on insulin signaling in mouse myogenic cells.

To determine the impact of RNase L/RLI expression on insulin signaling during inflammation, we evaluated insulin response in C2C12-derived mouse myogenic cells expressing different levels of RNase L or RLI, after FFA treatment. The saturated FFA palmitate was used here in order to mimic the effect of elevated circulating FFA, known to induce an acute pro -inflammatory response via TLR pathway in obesity 34. As previously described 28,30 and shown in figure 1A, RNase L and RLI expression was significantly increased in C2-RNaseL+ (*P=0.028) and C2-RLI (*P=0.024) respectively, compared to C2- RNaseL control cells.

Without induction of inflammation, insulin response was totally abolished in cells over-expressing RLI (C2-RLI), as shown by no increase of P-Akt/Akt expression rate following insulin stimulation, while Akt phosphorylation was induced in the two other C2C12-derived cell lines (figure IB). In basal conditions, we observed no any difference in insulin response between cells over-expressing RNase L (C2-RNaseL+) and C2-RNaseL. This could be explained on account of the fact that, without inflammation induction, insulin response was already high in C2-RNaseL. On the other hand, palmitate treatment blunted insulin response in C2-RNaseL and C2-RNaseL+, C2-RNaseL+ however maintaining a significant higher level of P-Akt/Akt expression rate than C2-RNaseL (figure IB). As expected, there was no insulin-stimulated Akt phosphorylation in C2-RLI after palmitate exposure (figure IB).

Down-regulation of RLI expression impacts palmitate-induced impairment of insulin response in myotubes from normal- weight subjects.

Human primary myotubes, obtained from skeletal muscle of normal- weight subjects were transfected with specific RNase L (Myo-siRNaseL) or RLI (Myo-siRLI) siRNA or with a non-specific siRNA (Myo-siControl) before palmitate treatment and/or insulin stimulation.

In Myo-siControl, palmitate treatment induced RNase L and RLI proteins expression without increasing RNase L or RLI mRNA levels (figures 2 A and 2B). This indicates that the induction of these proteins would be the result of a post-transcriptional regulatory mechanism such as increased protein translation, as yet demonstrated for RNase L35.

Both RNase L and RLI mRNA levels were significantly lower in Myo-siRNaseL (*P=0.039) and Myo-siRLI (*P=0.029) respectively, compared to Myo-siControl, demonstrating the effectiveness of siRNA transfection i.e. specific mRNA degradation (figure 2A). However, these significant differences were not confirmed at protein level, suggesting a low RNase L and RLI proteins turnover and/or a stable expression of these proteins, as it has already been observed by others for RNase L 36. On the other way, induction of RNase L and RLI proteins expression following palmitate treatment was not observed in Myo-siRNase L and Myo-siRLI, respectively. Indeed, in palmitate-treated cells, RNase L and RLI expression was significantly lower in Myo-siRNaseL (*P=0.014) and Myo-siRLI (*P=0.05) respectively, compared to Myo-siControl (figure 2B). These results, which show an inhibition by siRNA of palmitate-induced RNase L and RLI proteins translation would be explained by the fact that siRNA can regulate gene expression through both mRNA stability and mRNA translation37.

Palmitate treatment similarly impaired insulin signaling in Myo-siControl and Myo- siRNaseL, as shown by a relative fold expression of insulin- stimulated P-Akt/Akt rate inferior to 1 (0.75±0.07 and 0.81±0.10 respectively), while it was significantly maintained and even increased in Myo-siRLI (1.41±0.30) (figure 2C; *P=0.032). The absence of RNase L siRNA effect on insulin response in Myo-siRNaseL could be explained by the fact that, at these concentrations of palmitate, inhibition of insulin response is yet maximal (compare insulin- stimulated P-Akt/Akt expression rate in cells transfected with Control and RNase L siRNA in figure 2C).

OAS expression is impaired in human myotubes from OB-IR subjects and activation of RNase L with exogenous 2-5A allows to restore insulin response in OB-IR myotubes.

As stated in the introduction, not all obese people develop IR. Our results indicating that RNase L/RLI could impact insulin response in muscle cells, prompted us to analyze their expression in primary myotubes from obese subjects divided into two groups, insulin- sensitive (OB-IS) and insulin-resistant (OB-IR), on the basis of their homeostasis model assessment of IR (HOMAIR) index (table 1). Accordingly with previous studies 38,39, insulin-stimulated P-Akt/Akt expression rate and glucose uptake level were significantly reduced, by 56% (figure 3 A; **P=0.005) and 31% (figure 3B; **P=0.003) respectively, in OB-IR group compared to OB-IS. In an unexpected way, RNase L and RLI levels were similar in the two groups of obese subjects (figure 3C).

However, OAS2 protein level was decreased by 69% in myotubes of OB-IR group compared to OB-IS (figure 4A; *P=0.045). We also evaluated OASl protein expression but failed to detect it. Moreover, OAS mRNA levels were also diminished in OB-IR group (figure 4B; *P=0.05). However, contrary to OAS2 mRNA, the inter-subject variability did not allow for demonstrating any significant difference in OASl mRNA expression between the two groups. We hypothesized that the decreased levels of OAS mRNA in OB-IR myotubes could be due to an increase of the suppressor of cytokine signaling 3 (SOCS3) expression, which is induced during inflammation and is known as an important negative regulator of OAS expression 40 and insulin response in muscle 41 ,42. However, we found no difference in SOCS3 expression between OB-IS and OB-IR groups (figure 4C).

As OAS are responsible for 2-5A synthesis which is necessary for RNase L activation, we transfected OB-IS and OB-IR myotubes with an exogenous 2-5A. In presence of 2-5A, insulin-stimulated P-Akt/Akt expression rate and glucose uptake level were significantly increased in OB-IR group, by 119% (figure 4D; *P=0.013) and 38% (figure 4E; **P=0.006) respectively, whereas it had no effect in OB-IS. Moreover, insulin- stimulated P-Akt/Akt and glucose uptake levels, which were significantly different between OB-IS and OB-IR groups, were no more significantly different between non-transfected OB-IS and 2-5A-transfected OB-IR myotubes, indicating that RNase L activation by exogenous 2-5A restored insulin response in OB-IR myotubes (figures 4D and 4E). Important to note, 2-5A transfection also induced an increase of OASl and OAS2 mRNA expression in OB-IR myotubes, by 560 and 575%) respectively (figure 4F; *P=0.047), that cancelled out the significance of the difference in OAS2 mRNA expression between OB-IS and OB-IR groups.

RNase L regulates neither PKR and ΙκΒ expression, nor JNK and IRS1 (serine 312) phosphorylation.

PKR plays an important role in blunting insulin signaling during inflammation via IRS1 phosphorylation on serine residues, directly or indirectly by phosphorylating and activating JNK 8,9. Moreover, PKR also regulates IKK activation, leading to ΙκΒ degradation and NF-KB activation 10. On the other hand, it has been demonstrated that RNase L modulates PKR expression 43. However, we observed no difference in ΙκΒ, PKR, P- JNK/JNK and Ser312P-IRSl/IRSl expression levels between OB-IS and OB-IR groups (figure 5). RNase L regulates MnSOD expression.

During obesity, lipid overload causes oxidative stress by increasing ROS production and/or altering anti-oxidant defenses, that can lead to muscle IR 32. We thus checked in OB- IS and OB-IR myotubes mRNA expression of several major anti-oxidant enzymes and of the transcriptional nuclear factor erythroid 2-related factor 2 (NRF2) which regulates their expression. Only MnSOD mRNA was significantly under-expressed, by 77%, in myotubes of OB-IR subjects compared to OB-IS (figure 6A; *P=0.021), which was confirmed at the protein level with 66% under-expression (figure 6B, see white bars; **P=0.005). Furthermore, RNase L activation with exogenous 2-5 A significantly increased MnSOD mRNA expression, by 285%) (figure 6C; **P=0.004), as well as MnSOD protein expression, by 176%o (figure 6B; *P=0.033), in OB-IR myotubes, allowing to cancel out the significances between OB-IS and OB-IR groups. As we observed for OAS1 and OAS2 mRNA (figure 4F), 2-5A transfection increased MnSOD mRNA level in OB-IS myotubes, this effect being here statistically significant. We assume that, even when MnSOD mRNA is highly expressed, over-activation of RNase L by exogenous 2-5 A could lead to its induction. However, the level of MnSOD protein was not modified after 2-5A transfection in OB-IS myotubes as it was in OB-IR myotubes (figure 6B), which could be indicative of a regulation of MnSOD expression by a post-transcriptional mechanism.

MnSOD is the mitochondrial superoxide dismutase; however its low expression in

OB-IR myotubes does not seem due to a different mitochondrial mass between OB-IS and OB-IR myotubes, as we observed no variation in the expression of two other mitochondrial proteins: the mitochondrial uncoupling protein 3 (UCP3) and the subunit IV of cytochrome c oxidase (COX IV) (figure 6D).

TLR3 expression is decreased in OB-IR myotubes.

As RNase L activation by exogenous 2-5A restored the expression of MnSOD and OAS in OB-IR myotubes, we concluded that a common regulator of these proteins was affected in OB-IR myotubes contrary to OB-IS. An essential regulatory mechanism of MnSOD and OAS expression during inflammation and oxidative stress involves TLR3 activation. Indeed, previous studies have shown that TLR3 activation i) protects cells against oxidative stress through the up-regulation of MnSOD 44,45 and ii) induces IFNI production and OAS expression after TLR3 recognition of small mRNA fragments. We thus measured TLR mRNA expression in OB-IS and OB-IR myotubes: unlike TLR4, TLR3 mRNA was significantly under-expressed, by 72%, in OB-IR myotubes compared to OB-IS (figure 6E; *P=0.048). Finally, as OAS2 and MnSOD mRNA, 2-5A transfection induced a significant increase, by 195%, of TLR3 mRNA expression in OB-IR myotubes (figure 6F; *P=0.031), allowing to cancel out the significance between OB-IS and OB-IR groups.

Discussion:

Obesity has an undeniable impact on health by promoting the occurrence of chronic diseases including T2D 1. Although it is known that IR appears several decades before the onset of T2D, the pathophysiological mechanisms involved in the development of the disease are still not fully understood. Importantly, it is difficult to explain why a subset (30%>) of obese individuals seems preserved from IR and other metabolic disorders during weight gain 2,3. The identification of the pathways implicated in the regulation of insulin response in these two groups of obese people is a key issue to understand the pathogenesis of IR.

Skeletal muscle, due to its mass, is an essential organ in maintenance of glycemic homeostasis and plays a key role in IR and T2D pathogenesis 31. So, understanding the cellular mechanisms that regulate insulin response in muscle of obese people is of primary importance and could help to understand the differences between insulin-sensitive and insulin-resistant subjects.

During the last decade, the central role played by innate immunity and TLR activation in the pathogenesis of IR has been demonstrated. RNase L, which is an essential component of the innate immune response, is activated through IFN production following TLR 3/4 activation and could amplify TLR activation 20-22. RNase L activity is regulated by its binding to 2-5 A, which is synthesized by OAS, and by RLI. We recently demonstrated that insulin response was impaired in RNase L-/~MEFs and partially restored after re-introduction of RNase L expression in these cells 29. These findings incited us to study the potential role of RNase L/RLI in regulation of insulin response, at muscle level.

Here, our results reveal that RNase L activity allows for maintaining an insulin response during FFA-induced inflammation in mouse and human myotubes. Indeed, in our two models, the impairment of insulin response induced by palmitate treatment, which mimics inflammation observed during obesity, was limited in cells expressing higher level of RNase L/RLI ratio (C2-RNaseL+ and Myo-siRLI) compared to respective control cells. Insulin-stimulated phosphorylation of Akt was even improved by RLI siRNA transfection in palmitate-treated human myotubes, showing the importance of RNase L activity to limit FFA deleterious effect on insulin response.

As stated above, one intriguing and still not explained fact is the existence of insulin- sensitive individuals among obese people. If RNase L and RLI levels were similar in two groups of obese individuals with different insulin sensitivity states, the levels of OAS2 protein and OAS1 and OAS2 mRNA were lower in OB-IR myotubes. 2-5 A level could thus be the limiting factor for a correct insulin response. Indeed, 2-5A transfection restored insulin- stimulated P-Akt/Akt and glucose uptake levels in OB-IR myotubes to comparable levels of those in OB-IS myotubes. These results confirm that low 2-5A production, due to decreased expression of OAS, leads to under-activation of RNase L and impaired insulin response in OB-IR myotubes. Our findings underline the importance of RNase L activity in the development of IR in skeletal muscle during obesity.

The next objectives of this study were to determine how RNase L can regulate insulin response and why OAS levels are low in OB-IR myotubes. One of the known targets of RNase L is another mediator of innate immunity, the enzyme PKR 43. PKR is activated in tissues of obese mice and, consequently, can directly or indirectly (through JNK activation) induce negative phosphorylation of IRS 1 as well as activation of IKK. As a result, these two activated pathways lead to the disruption of insulin response combined with the synthesis of inflammatory cytokines 8,9. However, we neither found any significant variation of PKR and IKB expression levels, nor of P-JNK JNK and Ser312P-IRSl/IRSl expression rates between OB-IS and OB-IR myotubes. According to these observations, the mechanism by which RNase L activity allows for maintaining insulin response in myotubes during obesity does not depend on PKR regulation.

As oxidative stress is induced during obesity and plays an important role in IR development, we checked the expression levels of several enzymes implicated in the antioxidant defense. Only MnSOD was weakly expressed in OB-IR myotubes, that does not seem related to a lower mitochondria mass, as attested by the similar levels of two other mitochondrial proteins (UCP3 and COX IV) in OB-IS and OB-IR myotubes. MnSOD expression is necessary to maintain insulin sensitivity during obesity. Indeed, not only its expression is up-regulated by treatments that improve insulin sensitivity 46,47, but muscle transfection with plasmid allowing for increased MnSOD expression in vivo preserves insulin response during high-fat diet-induced obesity 48. We hypothesize that the high MnSOD expression observed in OB-IS myotubes may i) counteract the highly toxic superoxide anion (02·-) produced in excess by mitochondria during lipid overload 32 and ii) rise H202 production which is essential to maintain insulin sensitivity 49,50. As an explanation of MnSOD under-expression in OB-IR myotubes, we identified a defect in TLR3 expression, which is known to protect against inflammation and oxidative stress in different models 44,51-54. The concomitant significant increase in MnSOD and TLR3 expression following 2- 5A transfection would be in favor of our hypothesis.

We propose that, during FFA-induced inflammation, RNase L is activated by the OAS-synthesized 2-5A. RNase L then cleaves mRNA in smaller RNA, which in turn could activate TLR3 pathway. This activation induces the expression of the anti-oxidant enzyme MnSOD and numerous ISG among which the OAS. This secondary activation of OAS by cleaved mRNA entails increased 2-5A production that all the more activates RNase L. It then creates an amplification loop that leads to higher activation of TLR3 and higher levels of OAS and MnSOD, as observed in myotubes from OB-IS subjects. By controlling H202 level, MnSOD allows for maintaining Akt phosphorylation and glucose uptake, even when excessive ROS are produced. At the contrary, myotubes of OB-IR subjects express very low level of TLR3 which prevents this amplification loop to setting up. The consecutive under- expression of the OAS and MnSOD thus causes the inhibition of insulin response in myotubes of OB-IR subjects. The global mechanism we identified here is recapitulated by a scheme, in figure 7.

Our results highlight that TLR3 signaling and RNase L/RLI play a critical role in the pathogenesis of obesity-associated IR. Importantly, for the first time, we identify a molecular mechanism induced by inflammation which could regulate anti-oxidant defenses and be responsible for the different insulin- sensitivity states observed among obese people, at skeletal muscle level. Going further into this pathway, in particular through the identification of regulatory factors of TLR3 expression, could lead to the determination of central mechanisms regulating insulin response during obesity and the discovery of new therapeutic targets.

LIST OF ABBREVATIONS: ABCE1 : ATP -binding cassette sub-family E member 1

CHOP10: C/EBP-homologous protein 10

COX IV: cytochrome c oxidase

Cu/ZnSOD: copper/zinc superoxide dismutase

C/EBP: CCAAT-enhancer-binding protein DMEM: Dulbecco's modified eagle medium

FFA: free fatty acids

GPx: glutathione peroxidase

IF I: type I interferon

IKB: inhibitor of κΒ

IKK: IKB kinase

IL: inter leukin

IPTG: isopropyl-P-D-thiogalactopyranoside

IRS: insulin receptor substrate

Η₂0₂ : hydrogen peroxide

HOM A_IR: homeostasis model assessment of insulin resistance

J K: c-Jun N-terminal kinase

PP2A: protein phosphatase 2 A

MCP-1 : monocyte chemoattractant protein- 1

MnSOD: manganese superoxide dismutase

6-NBDG: 6-[N-(7-nitrobenz-2-oxa-l ,3-diazol-4-yl) amino]-2-deoxy-glucose

NF-KB: nuclear factor κΒ

Nrf2: nuclear factor erythroid 2 -related factor 2

OAS: oligoadenylate synthetase

PKB: protein kinase B

PKR: double-stranded RNA-dependent protein kinase

PTP-1B: phosphotyrosine phosphatase- IB

q-PCR: real-time quantitative PCR

RLI: RNase L inhibitor

RNase L: latent endoribonuclease

ROS: reactive oxygen species

SAPK: stress-activated protein kinase

SEM: standard error of the mean

SOCS3: suppressor of cytokine signaling 3

TLR: toll-like receptor

TNFa: tumor necrosis factor a

UCP3 : uncoupling protein 3

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Claims

CLAIMS:

A method for the treatment of insulin resistance in a subject in need thereof comprising administering the subject with a therapeutically effective amount of an agent selected from the group consisting of 2',5'-oligoadenylate (2-5A) or derivatives compounds, TLR3 agonists, and IFN1 polypeptides.

The method of claim 1 herein the subject is an overweight subject and more preferably an obese subject.

The method of claim 1 wherein the TLR3 agonist is selected from the group consisting of naturally-occurring double-stranded RNA (dsRNA) synthetic ds RNA; and synthetic dsRNA analogs.

The method of claim 1 wherein the IFN1 polypeptide is selected from the group consisting of IFN-. alpha, IFN-.beta, IFN-omega, or any mixture or combination thereof.

The method of claim 1 wherein the agent is combined with one or more additional active substances selected from the group consisting of insulin, sulfonylureas, meglitinides, biguanides, thiazolidinediones, glilazones, a-glucosdase inhibitors, incretin mimetics such as e.g. GLP-1 analogues and GLP-1 agonists, DPP-4 inhibitors, amylin analogues, PPAR α/γ ligands, sodium-dependent glucose transporter 1 inhibitors, fructose 1,6-bisphosphatase inhibitors, glucagon inhibitors, and l lbeta- HSD1 inhibitors. Non- limiting examples of the one or more additional active substance may be selected from the group consisting of insulin, glimepiride, glibenclamide, tolbutamide, gliciazide, glipzid, repaglinide, nateglinide, metformin, pioglitazones, rosiglitazones, acarbose, miglitol, liraglutide, exenafide, sitagliptin, vildagliptin saxagliptin, and alogliptin. In another embodiment of the present invention the one or more additional active substances are selected from the group consisting of thiazides, diuretics, ACE inhibitors, AT2 inhibitors, ARB, Ca2+ antagonists, a-blockers, β-blockers, cholesterol absorption inhibitors, hypolipidemic drugs, fibrates, anion exchangers, bile acid sequestrants, fish oils, HMG-CoA reductase inhibitors, and CBI cannabinoid receptor antagonists. Non-limiting examples of the one or more additional active substance may be selected from the group consisting of bendroflumetiazid, indapamid, hydrochlorothiazid, captopril, enalapril, lisinopril, fosinophil, perindopril, quinapril, ramipril, trandolapril, quinapril, fosinopril, candesartancilexefil, irbesarian, losartan, valsartan, telmisartan, eprosartan, olmesartanmedoxomil, nifedipin, amlodipin, nitrendipin, diltiazem, felodipin, verapamil, lacidipin, isradipin, tercanidipin, doxazosin, prazosin, terazosin, phentolamin, hydralazin, acebutolol, atenolol, bisoprolol, carvedilol, esmolol, labetalol, metoprolol, pindolol, propranolo, sotalol, tertatolol, timolol, melhyldopa, moxonidin, ezitimibe, gemfibrozil, bezafibrat, ienofibrate, nicotinic acid, acipimox, colestipol, colestyramin, fish oils, atorvastatin, f uvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin, and rimonabant.