Multifunctional Cyclic D,L‑α-Peptide Architectures Stimulate Non-Insulin Dependent Glucose Uptake in Skeletal Muscle Cells andProtect Them Against Oxidative StressRenana Shapira,† Safra Rudnick,† Bareket Daniel,† Olga Viskind,† Vered Aisha,† Michal Richman,†

Kamesh R. Ayasolla,†,§ Alex Perelman,†,‡ Jordan H. Chill,† Arie Gruzman,*,† and Shai Rahimipour*,†

†Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel

*S Supporting Information

ABSTRACT: Oxidative stress directly correlates with theearly onset of vascular complications and the progression ofperipheral insulin resistance in diabetes. Accordingly, exoge-nous antioxidants augment insulin sensitivity in type 2 diabeticpatients and ameliorate its clinical signs. Herein, we exploredthe unique structural and functional properties of the abioticcyclic D,L-α-peptide architecture as a new scaffold fordeveloping multifunctional agents to catalytically decomposeROS and stimulate glucose uptake. We showed that His-richcyclic D,L-α-peptide 1 is very stable under high H2O2concentrations, effectively self-assembles to peptide nanotubes,and increases the uptake of glucose by increasing thetranslocation of GLUT1 and GLUT4. It also penetrates cellsand protects them against oxidative stress induced under hyperglycemic conditions at a much lower concentration than α-lipoicacid (ALA). In vivo studies are now required to probe the mode of action and efficacy of these abiotic cyclic D,L-α-peptides as anovel class of antihyperglycemic compounds.

■ INTRODUCTION

Different aspects of oxidative cellular stress are associated withthe pathogenesis of several devastating human diseases,including diabetes, Alzheimer’s disease, Parkinson’s disease,and arteriosclerosis. It is well-known that the generation ofreactive oxygen species (ROS) in abnormal amounts or animpairment of the cells’ antioxidative protective systems canlead to cellular and tissue damage. For instance, in diabetes,oxidative stress directly correlates with the early onset ofvascular complications and the progression of peripheral insulinresistance. Thus, together with conventional antidiabetic drugtherapy, various antioxidant agents are usually incorporated intothe disease treatment regime.1 For example, α-lipoic acid(ALA) and vitamin C augment insulin sensitivity in type 2diabetic patients,2 while certain flavonoids decrease theconcentrations of thiobarbituric acid-reactive substances andof plasma glucose and improve insulin sensitivity in peripheraltissues (skeletal muscles and adipose tissue) in overweightdiabetic subjects.3 However, despite the involvement of ROS inthe pathogenesis of diabetes, existing antioxidants are not aseffective as mainstream antidiabetic drugs (biguanides,sulfonylureas, and thiazolidinediones) in the treatment ofdiabetes. Low bioactivity and bioavailability, reduced metabolicstability, and systemic toxicity are among the factors that limitthe efficacy of antioxidants in diabetes therapy.4 Therefore, itseems that an ideal antioxidant drug for diabetes treatment

should act through a combination of antioxidant mechanisms(such as scavenging ROS, chelating metals, and regenerating/de novo synthesizing endogenous antioxidants) and throughrepairing macromolecules damaged by ROS.5 Novel antioxidantmolecules exhibiting some of these properties with highpotency, efficacy, and bioavailability could become valuableagents not only for type 2 diabetes therapy but also for otherdiseases.A major pathophysiological defect in type 2 diabetes is

decreased glucose utilization in peripheral tissues as a result ofinsulin resistance leading to glucose accumulation in the bloodand to hyperglycemia.6,7 Thus, the discovery of drugs thatincrease the rate of glucose transport to skeletal muscles in anoninsulin dependent manner under hyperglycemic conditionswould constitute a very promising development in the searchfor novel antidiabetic therapeutic agents.8 Several antioxidantmolecules, such as ALA, exhibit a direct stimulatory effect onthe rate of glucose uptake in skeletal and cardiac muscles.9 Thiscombination of a “classical” antioxidative effect and an ability tostimulate glucose transport in skeletal muscles under hyper-glycemic conditions might explain why ALA exhibits the mostprominent antidiabetic therapeutic effect of all the antiox-idants.10 However, to be considered as potential antidiabetic

Received: April 10, 2013

Article

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therapeutics, new bioavailable antioxidants with high bioactivityand low systemic toxicity are required.Here, we demonstrate how an His-rich cyclic D,L-α-peptide

may be used as a basis for the development of a scaffold thatcan exhibit potent antioxidant activity and directly stimulateglucose uptake in rat L6 myotubes. Cyclic D,L-α-peptides withan even number of alternating D- and L-α amino acids can formflat and ring-shaped conformations, and depending on theirside chains and the external environment, they can stack on topof each other through an intermolecular hydrogen-bondingnetwork to form hollow and β-sheet-like tubular structures(Figure 1).11 In such multivalent structures, the side chains are

oriented perpendicularly to the plane of the nanotube that caninteract with their immediate environment. Moreover, unlikebiopolymers, in which the nature of the monomers and theirsequence dictate their three-dimensional structure and function,the structure of the cyclic D,L-α-peptide nanotubes is mainlydictated by hydrogen bonding in the backbone, which stemsfrom alternating D- and L-α-amino acid configuration.Cyclic D,L-α-peptides can exhibit diverse biological activities,

including antibacterial and antiviral properties.12 We haverecently shown that cyclic D,L-α-peptide hexamers can inducepotent antiamyloidogenic activity against β-amyloid protein(Aβ), which is responsible for the pathogenesis of Alzheimer’sdisease, and can protect cells against Aβ-induced toxicitywithout exhibiting any cytotoxicity.13

By using a wide range of chemical, biophysical, andbiochemical techniques, we demonstrate here that cyclic D,L-

α-peptides that are rich in His residues exhibit potentantioxidant activity by catalytically decomposing hydrogenperoxide and dramatically reducing the amount of hydroxylradicals even in the absence of transition metal ions such as ironor copper. Moreover, we show how active cyclic D,L-α-peptide 1increases the rate of glucose uptake in immortalized rat skeletalmuscle cells (L6) and protects the cells from oxidative stress-induced toxicity.

■ RESULTS AND DISCUSSION

Design and Synthesis. Amphiphilic cyclic D,L-α-peptideoctamers with at least two hydrophobic amino acids have beenshown to self-assemble into supramolecular structures and toinduce various biological activities with minimal toxicity.14 Ithas been shown that these structures adopt an orientationparallel to the plane of the membrane such that thehydrophobic side chains are inserted into the lipidiccomponents and the hydrophilic residues are exposed forinteraction with the immediate environment. Consequently,our design contained two adjacent Trp residues that inducesufficient hydrophobicity to ensure effective partitioning andself-assembly in lipid membranes. Moreover, His residues,which exhibit relatively low antioxidant activity,15 wereincorporated at the other four or five positions of the cyclicpeptides. Position one was fixed with Lys to allow the linearpeptides to be attached to a solid support via its free amine sidechain and to be cyclized through head-to-tail cyclization.Scheme 1 shows the structure of two cyclic D,L-α-peptides, 1and 2, that were synthesized by solid phase peptide synthesisusing the Fmoc methodology. The synthesis of the peptidesinvolved the preparation of linear peptides and their cyclizationon the solid support as described in Supporting InformationScheme S1. Cyclic D,L-α-peptide, 1, was also labeled with 4-chloro-7-nitrobenzofurazan (NBD-Cl) through the free ε-amine on Lys1 to follow its localization within cells.

Antioxidant Activity. The capability of the two cyclic D,L-α-peptides, 1 and 2, to reduce the amount of reactive oxygenspecies (ROS) in solution was evaluated and compared to thatof free His. Antioxidant activity was measured by the 2′,7′-dichlorofluorescin (DCFH) method. In this method, thenonfluorescent DCFH is oxidized by an ROS, such as H2O2,to form fluorescent 2,7-dichlorofluorescein (DCF; excitation485 nm, emission 535 nm).16 The antioxidant activity of thecyclic peptides was therefore estimated from their ability to

Figure 1. Schematic representation of nanotube assembly from cyclicD,L-peptides. For clarity, most of the side chains have been omitted.

Scheme 1. Chemical Structures of the Three His-Rich Cyclic D,L-α-Peptide Analogues Investigated in This Study

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prevent DCFH oxidation (Table 1). A solution of H2O2 (0.5mM) was incubated with DCFH in the absence or presence of

the cyclic peptides, and the amount of oxidized DCF wasdetermined fluorometrically.16 Free His and Trolox, a water-soluble derivative of Vitamin E with well-known antioxidantactivity, were used as the controls. Both cyclic peptidesdemonstrated significantly higher antioxidant activity than freeHis and Trolox. Indeed, cyclic peptides 2 and 1 were 30- and15-fold more active than His, respectively, and 7.5- and 3-foldmore active in respect to His residues concentrations. The highactivity of the cyclic peptides may originate from themultivalent presentation of His residues in the self-assembledarchitecture (Figure 1). The data also suggested thatincorporation of a fluorescent NBD group onto 1 did notchange its antioxidant activity.The antioxidant activity of the cyclic D,L-α-peptides was also

confirmed by electron paramagnetic resonance (EPR) spec-troscopy using the spin trapping method. N,N′-Dimethyl-pyrroline-N-oxide (DMPO) was utilized as the spin trap, andhydroxyl radicals were generated by the Fenton reaction, usingH2O2 and a catalytic amount of Fe2+.17 The amount of hydroxylradicals was estimated by integrating the corresponding EPRsignal (Figure 2). The active cyclic peptides dose-dependentlydecreased the amount of hydroxyl radicals, as the EPR signalsbecame smaller upon addition of the cyclic peptides (Figure 2;data are only shown for the more His-rich active peptide, 1).Table 1 compares the antioxidant activity (EC50) of 1 with thatof free His, using the EPR method. Notably, the DCFHexperiments together with the EPR measurements suggest thatthe cyclic peptides are catalytically active, as the amount ofpeptide required to decompose most of the H2O2 is less than0.1% (molar ratio).Next, we tried to elucidate the mode of action of the active

cyclic peptides. Many biologically interesting antioxidants andenzymes contain His residues in their active site, and these arefrequently involved in chelating transition metal ions.18 Freetransition metals, such as Fe2+ and Cu2+, play a major role ininducing oxidative damage to lipids, proteins, and nucleicacid.19 Through the Fenton reaction, these metal ions catalyzelipid peroxidation to form a variety of toxic byproducts, such astrans-4-hydroxy-2-nonenal (HNE) and trans-4-oxo-2-nonenal(ONE). The latter toxicants can potentially deplete intracellularlevels of glutathione and related cellular antioxidants. To ruleout the possibility that the activity of the His-rich cyclic D,L-α-peptides stemmed mainly from their metal chelating capabilityto inhibit the Fenton reaction, the antioxidant activity of 1 was

tested in the absence of Fe2+. In these experiments, hydroxylradicals were produced directly inside the EPR tube byhomolytic decomposition of H2O2 using UV irradiation(photolysis). The antioxidant activity of 1 in the absence ofFe2+ was very similar to that in the presence of Fe2+, suggestingthat the antioxidant activity of the His-rich peptide could alsostem from its direct interaction with H2O2, without involvingthe Fenton reaction.

Stability of the Cyclic D,L-α-Peptide 1 towardOxidative Conditions. To show that the antioxidant cyclicD,L-α-peptides are stable under highly oxidative conditions themore His-rich, 1 was incubated for one month with H2O2 in thepresence of a catalytic amount of Fe2+ and analyzed by HPLC,mass spectroscopy, and NMR. Less than 5% of the cyclic D,L-α-peptide was damaged under these conditions (SupportingInformation Figure S1), suggesting that 1 is highly stabletoward H2O2. The high stability of the cyclic peptide is mostlikely related to the fast kinetics of its degradation of H2O2 or,alternatively, to the generation of self-assembled supra-molecular structures that are resistant to oxidative conditions.Previous studies have also shown that cyclic D,L-peptides can behighly resistant to proteolysis and stable in serum and otherbiological fluids.14

Evidence for the Self-Assembly. To show that cyclic D,L-α-peptides 1 and 2 can indeed self-assemble by stacking on topof each other, the fluorescence of 3 was followed over time. Ithas been shown that the self-assembly of cyclic D,L-α-peptidescontaining 1,4,5,8-naphthalenetetracarboxylic acid diimide(NDI) side chains could lead to excimer formation, whichcan be monitored by fluorescence spectroscopy.20 Figure 3shows that the fluorescent signal of 3 (1 μM) decreases overtime, most likely because of self-quenching of the NBD moeityinduced by the self-assembly of the cyclic D,L-α-peptide. Similarself-quenching of NBD has been observed in other self-assembling systems.21

Table 1. Antioxidant Activity of the Studied Cyclic D,L-α-Peptides

sequencea nameEC50 (DCFH)

(μM)bEC50 (EPR)

(μM)

[HwWhHhHk] 1 0.4 3[HwWhHhSk] 2 0.2 ND[HwWhHhHk(NBD)] 3 0.3 NDTrolox 0.35 pro-oxidantHistidine 6 100

aUpper and lower case letters represent L- and D-amino acid residues,respectively. Square brackets indicate a cyclic structure. bAntioxidantactivity was determined by the dichlorofluorescin (DCFH) method aswell as by electron paramagnetic resonance (EPR) spectroscopy usingthe Fenton reaction as the source of hydroxyl radicals and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the spin trap.

Figure 2. Cyclic D,L-α-peptide 1 dose dependently inhibited thegeneration of hydroxyl radicals from H2O2. To a solution of H2O2 (10mM), cyclic peptide 1 (1−1000 μM), DMPO (40 mM), and FeSO4·7H2O (10 mM) were added. The samples were diluted to 100 μL withphosphate buffer (15 mM, pH 7.4), transferred to a narrow Teflontube, and measured immediately by EPR. Amount of hydroxyl radicalswas estimated from integration of the second peak of the EPR signal(shown in the insert) and is shown in arbitrary units (au).

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The self-assembly of the cyclic D,L-α-peptide 1 to asupramolecular structure was also confirmed by transmissionelectron microscopy (TEM). A solution of 1 (100 μM) wasaged in phosphate buffer solution for 2 days, negatively stainedwith 2% uranyl acetate, and tested by TEM for nanotubeformation. These studies showed that two-day aged 1 couldgenerate a fibrillar network (Figure 3), which is in agreementwith our and other previous studies, suggesting that cyclic D,L-α-peptides can self-assemble into peptide nanotubes.11,13

Effect of the Cyclic D,L-α-Peptide 1 on GlucoseUptake. Having shown that cyclic D,L-α-peptide 1 exhibitspotent antioxidant activity, we next probed its capability toincrease the rate of glucose uptake in immortalized rat skeletalmuscle cells (L6). These cells spontaneously differentiate frommyoblasts to myotubes (multinuclear cellular clusters with highmorphological similarity to native muscle myofibrils) in theirlate growth stage in vitro, closely mimicking skeletal muscles.22

Consequently, L6 cells are a suitable model to probe the effectof the cyclic peptide on the intracellular translocation of glucosetransporters (GLUTs). Cells were incubated in a medium

containing a high concentration of glucose and with increasingamounts of 1 for 12 h and then tested for glucose uptake usinga standard [3H]-2-deoxy-D-glucose ([3H]dGlc) uptake assay.Cells incubated in a high glucose medium in the presence ofALA (2 mM) were used as positive controls. ALA was usedbecause it is a natural coenzyme that is known to stimulateglucose uptake and to exhibit antioxidant activity.23 L6 cellsincubated under the same conditions in the absence of anyadditive served as negative controls. Figure 4A shows that 1dose-dependently increases the rate of glucose uptake in L6myotubes as compared with the negative control. Thestimulatory effect of the cyclic peptide 1 is observed at aconcentration of 5 μM and exhibits its maximal effect at 25 μM.Similar glucose uptake was evident when L6 myotubes werestimulated with 2 mM of ALA, suggesting that 1 is at least 80times more effective than ALA in stimulating glucose uptake inskeletal muscle cells. No glucose uptake activity was observedwhen cells were incubated with 25 μM of ALA (data notshown).

Figure 3. His-rich cyclic D,L-α-peptides self-assemble to generate peptide nanotubes. (A) Reduction of the fluorescence of 3 (1 μM) over time and(B) TEM analysis of 1 (100 μM) aged for 2 days in PBS and negatively stained with uranyl acetate.

Figure 4. Effect of 1 on the rate of glucose uptake in L6 myotubes. (A) Dose response analysis. Myotube cultures were incubated with increasingconcentrations of 1 (open bars, for 12 h) or ALA (black bar, for 30 min) in a αMEM medium containing 23.5 mM D-glucose. Control myotubesreceived the vehicle only (0.1% DMSO (v/v)). After the indicated time interval, all cultures were washed and examined for glucose uptake using thestandard [3H]dGlc uptake assay. The basal rate of dGlc uptake of DMSO treated myotubes (3.48 ± 0.39 nmol mg−1 protein min−1) was taken as100% uptake. *, p < 0.05, in comparison with the vehicle-treated control. (B) Time-course analysis. The myotubes were incubated with 1 (○; 25μM) or ALA (■; 2 mM) for the indicated time periods. Control myotubes received the vehicle (0.1% DMSO) only (●). The cultures were thenanalyzed in a dGlc uptake assay. The basal rate of [3H]dGlc uptake at the beginning of the experiment (1.43 ± 0.12 nmol mg−1 protein min−1) wasassigned as 100%. *, p < 0.05, in comparison with the respective 23.5 mM glucose control.

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We also determined the effect of 1 on the rate of glucoseuptake. L6 myotubes were incubated with the peptide or ALAfor different time periods, and the rate of glucose uptake wasdetermined. Treatment of the cells with ALA (2 mM) caused arapid increase in glucose uptake, which reached a maximumvalue after 30 min of incubation. In contrast, 1 (25 μM) beganto influence glucose uptake only after 6 h and achieved itsmaximal effect at 12 h (Figure 4B).Effect of the Cyclic Peptide 1 on the Translocation of

GLUT1 and GLUT4. We also tested the effect of 1 on GLUT1and GLUT4 translocation in cells that overexpress GLUT1myc

and GLUT4myc. L6 cells overexpressing glucose transporterGLUT1myc (found in many types of human cells) orGLUT4myc (the major insulin-responsive transporter inhumans, found primarily in striated muscle and adipose tissue)were created to enable detailed investigation of the regulationof GLUT intracellular translocation.24 One of the mostimportant regulatory mechanisms by which cells control theirrate of glucose uptake is by varying the glucose transportercontent of their plasma membrane.25 Figure 5A shows thattreatment of the cells with ALA (2 mM, used as a knowninducer of GLUT1 and GLUT4 plasma membrane trans-

Figure 5. Cyclic D,L-α-peptide 1 and ALA induce GLUT1 and GLUT4 translocation to the plasma membrane of L6 myotubes overexpressingGLUT1myc or GLUT4myc. (A) L6 myotube cultures were washed, received serum free-αMEM with 0.5% bovine serum albumin (BSA) containing23.5 mM D-glucose, and were incubated for 9 h. During the last 8 h of incubation, the myotubes received 1 (25 μM) or vehicle containing 0.1%DMSO (v/v). Insulin (100 nM) and ALA (2 mM) were introduced during the last 30 min of incubation. At the end of the incubation, the cultureswere taken for immunodetection of surface GLUT1myc (open bars) or GLUT4myc (closed bars), as described in the Experimental Section. TheWestern blot in (B) depicts the total myotube content of the corresponding glucose transporters in whole cell lysates. #, p < 0.05 in comparison withvehicle treated L6 myotubes for GLUT1myc. *, p < 0.05, in comparison with vehicle treated L6 myotubes for GLUT4myc.

Figure 6. (A) Effect of 1 on cell survival under oxidative stress conditions. L6 myotubes were grown in 23.5 mM glucose medium for 7 days (tomimic hyperglycemic conditions in vivo) and exposed to 1 (25 μM for 12 h) or to ALA (2 mM for 30 min). Control myotubes received the vehicleonly (DMSO, 0.1% v/v). Cells were then incubated for 5 h with glucose oxidase (GO; 50 mU mL−1) to induce oxidative stress. Cell viability wasdetermined by a standard MTT assay. *, p < 0.05 in comparison with the absorption level of samples without GO, #, p < 0.05 in comparison with theabsorption level of samples treated with GO. (B) Effect of 1 on TEAC in L6 myotubes. Myotube cultures were treated with 1 (25 μM for 12 h) orALA (2 mM, 2 h) in αMEM supplemented with 2% (v/v) FCS and 5.5 or 23.5 mM D-glucose. Control cells received 0.1% DMSO. Followingincubation of the myotubes, the cells were lysed and then TEAC measurements were performed as described in the Experimental Section. *, p < 0.05in comparison with the absorption level of a sample containing 0.1% DMSO in 5.5 mM D-glucose.

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location)23 or 1 (25 μM) increased the surface abundance ofGLUT1myc in the L6 myotubes as measured by theimmunocolorimetric method. The treatment increased theGLUT4myc content of the plasma membrane to an evengreater extent, with 1 achieving ∼60% the efficacy of thepositive control, insulin, which only induces GLUT4 plasmamembrane translocation.26 These treatments did not alter thetotal content of GLUT1 or GLUT4 in the cells, as determinedin whole cell lysates of myc epitope-tagged L6 myotubes(Figure 5B) or in wild-type L6 myotubes (data not shown).Insulin and ALA affected the abundance of GLUT1 andGLUT4 in the plasma membrane of myotubes very rapidly(within 30−40 min), whereas 8 h were required for the cyclicD,L-α-peptide. On the basis of these results, we anticipate thatthe mechanism by which 1 increases intracellular translocationof GLUTs differs from the mechanisms utilized by insulin andALA.Cytotoxicity Assay. We also investigated the protective

effect of 1 on oxidative stress-induced toxicity in L6 myotubesusing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) cell viability assay. Oxidative stress conditionswere induced by adding glucose oxidase (GO, 50 mU mL−1) tothe culture medium of L6 myotubes.27 The myotubes weregrown in a medium in which a high glucose concentration (23.5mM) was maintained chronically for the 7 days prior to theexperiment to mimic hyperglycemic conditions in vivo.28 Underthese conditions, the presence of GO results in continuousproduction of H2O2, which reaches a plateau concentration of29.0 ± 9.6 μM at 5 h of incubation.27 Figure 6A displays theeffect of 1 on GO-mediated L6 myotube toxicity. ALA (2 mM)was used in place of 1 as a positive control. The GO-treatedcells show a nearly 50% decrease in cell viability because of theoxidative stress induced under these conditions. The presenceof a low concentration of 1 (25 μM) significantly (P < 0.05)increased the survival of L6 cells. Similar protective activity wasobserved only when cells were treated with 2 mM of ALA.29

These results suggest that 1 is considerably more potent thanALA in protecting L6 cells against the oxidative stressconditions induced by a high glucose concentration.

Intracellular Antioxidant Activity Assay. To obtainmore detailed information about the intracellular activity of 1and, specifically, how it reduces oxidative stress in L6 myotubes,the total antioxidant capacity of the cell lysates was measuredby the Trolox equivalent antioxidant capacity (TEAC) assay kit(Cayman, USA). The assay measures the formation of the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) radical cation(ABTS•+) that is induced by metmyoglobin and hydrogenperoxide from ABTS.30 Trolox, a water-soluble vitamin Eanalogue, serves as a standard that inhibits the formation ofABTS•+. L6 myotubes were incubated in the absence orpresence of 1 (25 μM for 8 h) or ALA (2 mM, for 30 min) in ahigh glucose medium, and the resulting cell lysates weresubjected to the TEAC measurement assay. Figure 6B showsthe intracellular antioxidant capacity of 1 in comparison withthat of ALA. While 1 increased the TEAC in L6 myotubeslysates at a concentration of 25 μM, similar activity wasobserved with ALA only at a concentration of 2 mM, suggestingthat the cyclic peptide exhibits greater intracellular antioxidantactivity in L6 myotubes under hyperglycaemic conditions.

Antiapoptotic Effect of Cyclic Peptide 1. Next, wetested the effect of 1 on the activation and phosphorylation ofthe stress-activated protein kinase, c-Jun N-terminal kinase(JNK), as a modulator of cell apoptosis.31 L6 myotubes wereincubated chronically in a medium containing a highconcentration of glucose for 7 days, to mimic the chronichyperglycemia of diabetes,32 and then exposed to 1 for 8 h or toALA for 3 h. The levels of total and phosphorylated JNK werethen determined by Western blotting. Chronic incubation ofthe cells in a high glucose medium significantly increased thephosphorylation of both isoforms of JNK (54 and 46 kDa).33

Co-incubation of the cells with 1 or with ALA considerablysuppressed the high glucose-induced JNK phosphorylation(Figure 7), providing direct evidence that 1 can effectivelyreduce oxidative stress and associated apoptosis in L6myotubes.

Cell Localization Assay. Having shown that 1 can potentlyincrease glucose uptake and reduce the intracellular oxidativestress induced by a chronically hyperglycemic environmentwhile suppressing high glucose-induced JNK phosphorylation,

Figure 7. (A) Western blot analyses of cells treated with 1 or ALA. Whole cell lysates were prepared from myotube cultures that were treated with 1or ALA as described in the legend of Figure 6. Western blot analyses were performed with antibodies against total JNK and phosphorylated JNK (p-54, p-46, and α-tubulin), as described in the Experimental Section. (B) Density of the resulting signals was calculated using the ImageJ program. Thegraph represents the relative densities of a 54 kDa JNK protein (open bars) and a 46 kDa JNK protein (closed bars), respectively. The band densityis normalized according to total JNK. *, p < 0.05 in comparison with suitable control (23.5 mM glucose for ALA and 23.5 mM glucose treated withDMSO for 1).

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we next studied the uptake of 3 by L6 myotubes. Cells wereincubated for 4 h with 3 (10 μM), washed, and observed undera confocal fluorescent microscope. To verify that the cyclicpeptide is localized to the intracellular space, the outermembrane and the nucleus of the live cells were stained withAlexa-red wheat-germ agglutinin (WGA) and Hoechst dye,respectively (Figure 8). Previous studies have shown that

antiviral cyclic D,L-α-peptides prevent the development of lowpH in endocytic vesicles by their possible penetration into theendosomal compartments of the cells and by arresting theescape of virions from the endosome, without exhibiting anytoxicity to the cells.12b Our microscopic studies show directlythat cyclic D,L-α-peptide 3 can effectively penetrate into thecells, most likely into its intracellular vesicles (Figure 8). Theeffective penetration of 3 may be partly enabled by thepermeation of the self-assembled nanoarchitecture of 3 by theL6 myotubes. Further studies are required to probe the exactmechanism by which 3 penetrates the cells and the cellcompartment within which it is localized.

■ CONCLUSIONSOur findings emphasize the great potential of 1 in thedevelopment of novel, potent, and long-acting antihyperglyce-mic compounds. This study shows that antioxidative cyclic D,L-α-peptides may represent a new class of compounds for thedevelopment of novel antidiabetic drugs because of their effectof augmenting the rate of glucose uptake at pharmacologicallyrelevant concentrations (5−25 μM). This compound alsosignificantly increased the abundance of GLUT-1 and -4 in themyotube plasma membrane and protected L6 myotubes fromoxidative stress. All these effects were obtained at a much lowerconcentration than that required for the canonic antidiabeticantioxidant compound ALA. The high in vitro activity of 1 maybe originated from its self-assembly capability in cellular milieubecause the concentration required for the biological activity ishigher than the concentration that self-assembly occurs.Moreover, it is expected that hydrogen-bond-assisted self-assembly of the cyclic D,L-α-peptides will be more efficient inhydrophobic environment, such as cell’s membrane. Furtherexperiments including toxicity, pharmacokinetic, and efficacyexperiments in animal models are required to probe the utilityof these abiotic peptides as a novel class of antihyperglycemiccompounds.

■ EXPERIMENTAL SECTIONGeneral. All chemicals and reagents were of analytical grade. 2-

Chlorotrityl-chloride, 9-fluorenylmethoxycarbonyl (Fmoc)-protectedamino acid derivatives, and all other reagents for solid-phase peptidesynthesis were purchased either from Novabiochem (San Diego, CA)or GL Biochem (Shanghai, China) and used as received. ALA,Bradford reagent, bovine serum albumin (BSA), MTT, GO, D-glucose,o-phenylenediamine dihydrochloride (OPD), and a protease inhibitorcocktail were purchased from Sigma-Aldrich Chemicals (Rehovot,Israel). Mercaptoethanol, phenylmethylsulfonyl fluoride (PMSF),sodium orthovanadate, sodium-β-glycerophosphate, sodium pyrophos-phate, and sodium dodecyl sulfate (SDS) were purchased from AlfaAesar (Ward Hill, MA). [3H]-2-Deoxy-D-glucose ([3H]dGlc) [2.22TBq mmol−1 (60 Ci mmol−1)] was supplied by American Radio-labeled Chemicals (St. Louis, MO). Anti-α-tubulin antibody waspurchased from Millipore (Billerica, MA), while rabbit phospho-SAPK/JNK (Thr183/Tyr185) and total rabbit SAP/JNK antibodieswere purchased from Cell Signaling Technology (Beverly, MA). Anti-C-Myc (A-14) and rabbit polyclonal antiglucose transporter GLUT1and GLUT4 antibodies were obtained from Santa Cruz Biotechnology(Santa Cruz, CA) and Abcam (Cambridge, MA), respectively.Horseradish peroxidase-conjugated antirabbit IgG and the EZ-ECLchemoluminescence detection kit were obtained from JacksonImmunoResearch (West Grove, PA). Goat serum, fetal calf serum(FCS), L-glutamine, α-MEM, and antibiotics were purchased fromBiological Industries (Beth-Haemek, Israel), while the total antioxidantmeasurement kit was purchased from Cayman Chemical Company(Ann Arbor, MI). Liquid scintillator aquasafe 300-plus solution waspurchased from Zinsser Analytical (Frankfurt, Germany).

Peptide Purification and Analysis. Peptides were purified byRP-HPLC on a RP-18 semipreparative column (DENALI; 250 mm ×22 mm; 10 μm, GraceVydac), using a linear gradient of 0.1%trifluoroacetic acid (TFA) in water as buffer A and 0.1% TFA inacetonitrile as buffer B. Eluent composition was 20−100% B over 45min with flow rate of 10 mL/min provided by a ProStar 210 VarianHPLC system. Characterization of the purified peptides was carriedout by an Agilent 1200 HPLC system using either a GraceVydac RP-18 column (DENALI; 150 mm × 2.1 mm; 3 μm) and a gradient of10−100%B over 25 min or a GraceVydac RP-8 column (150 mm ×2.1 mm; 5 μm) using a gradient of 0−100%B over 35 min with a flowrate of 0.3 mL/min. The purity of the peptides was always over 95%based on analytical HPLC. High-resolution mass spectroscopy analysisof the purified peptides was performed on a Bruker Autoflex III TOF/TOF MALDI mass spectrometer (Bruker, Germany) in positive ionreflector mode.

NMR Spectroscopy. NMR measurements were conducted on asample containing 3 mg/mL (3 mM) of the cyclic peptides 1, 2, and 3dissolved in neat 2H6-DMSO and placed in a Wilmad (WilmadLabglass, Vineland, NJ, USA) NMR tube. All NMR measurementswere conducted at 298 K on a DRX700 Bruker spectrometer using acryogenic triple-resonance TCI probe head equipped with z-axispulsed field gradients. 2D homonuclear spectra acquired and utilizedfor assignment of 1H resonances included total correlation spectros-copy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY),and rotating-frame Overhauser effect spectroscopy (ROESY) experi-ments. Experiments (2−4 h each) were acquired with 4−8 scans per t1transient, and 4096 (512) complex points and an acquisition time of324 (56.2) ms in the direct (indirect) dimensions, respectively. Mixingtimes for the correlation spectra were 200 ms for the TOCSY andROESY experiments, and 250−500 ms for the NOESY experiment.

2D heteronuclear spectra acquired and utilized for assignment of13C resonances included heteronuclear multiple quantum correlation(HMQC) and heteronuclear multiple bond correlation (HMBC)experiments, optimized for the detection of 1JHC and 2−3JHCcorrelations, respectively. Experiment (8−16 h each) were acquiredwith 32−64 scans per t1 transient and 2048−4096 (200−400) complexpoints and an acquisition time of 162−324 (18−36) ms in the 1H(13C) dimensions, respectively. All spectra were processed using theTopSpin 2.1 package (Bruker BioSpin, Karlsruhe, Germany). 1H

Figure 8. Intracellular accumulation of the cyclic D,L-α-peptide, 3, byL6 myotubes. (A) Confocal image of live L6 cells incubated with 3(green spots) and (B) the corresponding close-up image. Cells wereincubated for 4 h with 3 (10 μM). The membranes and the nuclei ofthe live cells were stained with Alexa-red WGA (red) and Hoechst dye(blue), respectively. Cells were then visualized with an Olympus FV-1000 confocal microscope.

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chemical shifts were referenced to the DMSO peak (2.51 against 4,4-dimethyl-4-silapentane-1-sulfonic acid, DSS). 13C chemical shifts wereindirectly referenced to DSS.Assignment of 1H resonances was performed by utilizing the typical

TOCSY intraresidual correlations between the 1HN proton andaliphatic protons (1Hα, 1Hβ etc.) and NOESY/ROESY interresidualcorrelations between these protons in adjacent residues. Particularlyuseful in this context were the 1Hα(i − 1)/1HN(i) correlations, whichare expected to be strong for the cyclic D,L-alternating peptide.Assignment of aromatic 1H resonances was performed by utilizing thetypical TOCSY and NOESY/ROESY intra-aromatic correlations andconnecting them to the backbone resonances by detecting theintraresidual ROESY correlation between the 1Hβ and 1Hδ protons.The majority of 13C resonances could be assigned using theheteronuclear spectra by interpreting cross-peaks using one- andtwo/three-bond scalar couplings and comparing chemical shifts totables of typical aromatic 13C values.Synthesis of Cyclic D,L-α-Peptides. Cyclic D,L-α-peptides were

synthesized on 2-Cl-trityl resin as described previously,12b with minormodifications. Briefly, Fmoc-Lys-ODmab (0.78 g, 1 mmol; whereDmab is 2-(ethylhexyl)-4-(dimethylamino)benzoate))34 and diisopro-pylethylamine (DIEA; 2 mL) in CH2Cl2 (8 mL) were added to 2-Cl-trityl chloride polystyrene resin (1 g, 1.6 mmol g−1), and the mixturewas agitated for 4 h. The resin was then washed with CH2Cl2 (×3) andmethanol and dried. The loading of the resin was then determined byquantification of Fmoc release following treatment of the resin with20% piperidine in dimethylformamide (DMF). Peptides were thensynthesized on an AAPPTec automated peptide synthesizer (Vantage)in a 40 μmol scale. Coupling reactions were mediated by 5 mol excessof HBTU/DIEA (where HBTU is O-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate). Following completion oflinear peptides, the resin was treated with 2% hydrazine hydrate inDMF to remove the Dmab protecting group. The resin was thenwashed with 5% DIEA in DMF and the linear peptide was cyclized,while still on the resin, using an N-methyl-2-pyrrolidone (NMP)solution of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluor-ophosphate (PyBOP), hydroxybenzotriazole (HOBt), and DIEA (5, 5,and 15 equiv, respectively) for 6 h. The peptides were cleaved from theresin by treatment with a mixture of 95:2.5:2.5% trifluoroaceticacid:triisopropylsilane:water (2 mL) for 2 h. Crude peptides were thenpurified to homogeneity (>95% purity, based on HPLC) bypreparative RP-HPLC using a C18 column and identified by analyticalHPLC, high-resolution matrix-assisted laser desorption/ionization(MALDI) mass spectroscopy, and NMR (see Supporting Informationfor detailed analyses).Synthesis of 3. NBD-Cl was dissolved in methanol (1 mg/mL) to

give a light-yellow solution. Cyclic D,L-α-peptide 1 (5 mg, 4.21 μmol)in methanol (400 μL) was then added to 900 μL of NBD-Cl solution(4.5 μmol) and the pH was adjusted to 8.5 by adding DIEA (3.5equiv). The reaction was left overnight at room temperature, purifiedto homogeneity by preparative HPLC, and analyzed by analyticalHPLC, NMR, and MS. HRMS (Na-adduct ion) m/z: calcd forC64H68N24NaQ11 1371.5397, found 1371.5392 (see SupportingInformation for detailed analyses).Self-Assembly of the Cyclic D,L-α-Peptides. A 1:1 (v/v)

solution of 3 (180 μL, 1 μM) in dimethylsulfoxide (DMSO) and 2-(N-morpholino)ethanesulfonic acid (MES; 25 mM, pH 6.6) wasadded to the wells of a black 96-well plate. The florescence of the wellswas then followed over time at an excitation of 470 nm and emissionof 500−600 nm. The self-assembly of 1 was also studied by TEM. Asolution of 1 (100 μM) was aged for 2 days at room temperature andthen analyzed by TEM. Samples (5 μL) were placed on the TEMgrids, blotted after 30 s with a filter paper, and dried for 20 min. Thesamples were negatively stained with 2% uranyl acetate in water (5 μL)for 30 s, blotted with filter paper, and dried for an additional 20 min.Samples were then analyzed by a Tecnai G2 TEM (FEI TecnaiTMG2, Hillsboro, OR) operated at 120 kV.Cell Culture Experiments. L6 skeletal myocytes were grown and

allowed to differentiate into multinuclear myotubes (85−90% yield) inαMEM supplemented with 2% (v/v) FCS, as described elsewhere.35

All experiments were conducted on fully differentiated myotubes. Toevaluate the effect of the cyclic D,L-α-peptide, 1, on GO-mediatedtoxicity, cells were incubated in the absence or presence of either 1 (25μM) or ALA (2 mM) in a high glucose medium (23.5 mM). Afterincubation at 37 °C for the indicated time, GO (50 mU mL−1) wasadded to the cell medium for 5 h to generate oxidative stress, aspreviously described.27 The protecting activity of the tested agents wasthen determined by the MTT assay.

[3H]dGlc Uptake Assay. The rate of [3H]dGlc uptake inmyotubes was determined as previously described.35 Briefly, L6myotube cultures were preincubated in α-MEM supplemented with2% FCS (v/v) and 5.5 or 23.5 mM D-glucose for 24 h. Four hoursbefore the experiment, the medium was changed to FCS-free mediumwith 0.5% BSA. The myotubes were then treated with increasingconcentrations of 1 or ALA for 12 h or 30 min, respectively. Thecultures were then rinsed with PBS (×3) and incubated with a solutionof dGlc (0.1 mM) and [3H]dGlc (1.3 μCi mL−1) in PBS (pH 7.4) for5 min at room temperature. At the end of the assay, the myotubeswere washed with ice cold PBS (×3) and lysed in 0.1% (w/v) SDS,and the level of radioactivity was determined by liquid scintillationmeasurement (Tri-Carb 2810TR, PerkinElmer, Waltham, MA).

Preparation of Cell Lysates for Western Blot Analyses.Wholecell lysates were prepared as previously described with some minormodifications.36 Myotube cultures were washed with ice-cold PBS andlysed for 40 min in an ice-cold lysis buffer (Tris-HCl; 50 mM, pH 7.5,ethylenediaminetetraacetic acid (EDTA; 1 mM), ethyleneglycoltetra-acetic acid (EGTA; 1 mM), Na3VO4 (1 mM), NaCl (150 mM), NaF(50 mM), sodium-glycerophosphate (10 mM), sodium pyrophosphate(5 mM), and phenylmethylsulfonyl fluoride (PMSF; 1 mM)),supplemented with nonyl phenoxypolyethoxylethanol (NP-40; 0.1%(v/v), 2-β-mercaptoethanol (0.1%, v/v), and protease inhibitorcocktail (1:100 dilution). The resulting cell lysates were centrifugedat 8,700g for 30 min at 4 °C, and the supernatant fractions wereseparated and kept at −20 °C until used. Protein content in thesupernatant was determined according to the Bradford assay, using aBSA standard dissolved in the same buffer. Aliquots (5−60 μg ofprotein) were then mixed with the sample buffer (Tris-HCl (62.5 mM,pH 6.8), 2% SDS (w/v), 10% glycerol (v/v), dithiothreitol (DTT; 50mM), and 0.01% bromophenol blue (w/v)), heated at 95 °C for 5min, and separated on 10−12% SDS-PAGE. Western blot analyseswere then performed using commercially available antibodies andaccording to the antibody supplier’s protocol.

Colorimetric Determination of Surface GLUT1myc andGLUT4myc in L6 Myotubes. The colorimetric detection of surfaceGLUT4myc or GLUT1myc in L6 myotubes was performed asdescribed.37 Briefly, GLUT1myc and GLUT4myc L6 cell cultures(provided by Dr. A. Klip, Hospital for Sick Children (Toronto,Canada)) were incubated with rabbit anti-C-Myc antibody (1:200dilution), washed, and fixed with 3% formaldehyde and further reactedwith goat HRP-conjugated antirabbit IgG (1:2000 dilution). Asolution of o-phenylenediamine dihydrochloride (OPD) was addedto the washed cells, and the absorbance of the samples was measuredat 492 nm to estimate the relative abundance of GLUT1myc orGLUT4myc on the plasma membrane of the myotubes.

Determination of H2O2 Concentration in the Medium. Theconcentration of H2O2 generated by the glucose oxidase/glucosesystem was determined as described by Thurman et al.38

TEAC Measurement. TEAC measurements were conducted usinga total antioxidant capacity testing kit. Myotube cultures were washedand received fresh αMEM supplemented with 2% (v/v) FCS and 5.5or 23.5 mM D-glucose. 1 (25 μM for 12 h) or ALA (2 mM, 2 h) wasthen added to the myotubes (control cells received 0.1% DMSO).Following incubation of the myotubes, the myotubes were washedwith cold PBS three times to remove any remnants of the medium.The last portion of PBS was aspirated by suction. Cells were harvestedto cold lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, and 16.2 mMNP-40) and collected into centrifuge tubes. Cells underwent threefreezing-defrosting cycles in liquid nitrogen and were then intensivelytreated three times by Vortex, with an intertreatment interval of 10min. After centrifugation (8000g for 30 min at 4 °C), the formation of

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ABTS•+ in the cell lysates was determined (λ = 750 nm) using anELISA reader. The TEAC equivalent was calculated according to theamount of Trolox equivalents per 1 mg of total protein, as described inthe kit manual. A standard Trolox calibration curve obtained fromknown concentrations of dose dependent inhibition of ABSToxidation was used for this calculation. The total protein amountwas determined by the Bradford method.Cellular Localization of 3 by Confocal Microscope. L6

myotubes were grown on 35 mm disposable glass-bottomed tissueculture plates (MatTek) and treated with 3 (10 μM) for 4 h inmedium containing 1% FBS. Cells were washed with PBS (×3) andfresh medium was added to each sample. The nuclei of the live cellswere then stained with Hoechst, while the membranes were stainedwith Alexa Fluor 680-conjugated wheat germ agglutinin (WGA,Invitrogen). Live cell imaging was performed on an Olympus FV-1000confocal microscope operated at 37 °C.Statistical Analysis. Results are given as mean ± SEM. Statistical

significance (p < 0.05) was calculated among experimental groupsusing the two-tailed Student’s t test.

■ ASSOCIATED CONTENT*S Supporting InformationReaction sequence for the synthesis of cyclic D,L-α-peptideoctamers, cyclic D,L-α-peptide 1 is highly stable toward highconcentrations of hydrogen peroxide, analytical HPLC analyses,matrix-assisted laser desorption/ionization (MALDI) massspectroscopy, and NMR analyses of the cyclic D,L-α-peptides.This material is available free of charge via the Internet athttp://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*For A.G.: phone, 972 3 7384597; E-mail, gruzmaa@biu.ac.il.For S.R.: phone, 972 3 5317412; E-mail, rahimis@biu.ac.il.Present Addresses‡Rhenium Ltd., Modi’in 7171101, Israel§Department of Nephrology, Feinstein Institute for MedicalResearch, Hofstra North Shore-LIJ School of Medicine, GreatNeck, Long Island, NY 11021NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis study was supported in part by a Bar-Ilan University newfaculty grant (A.G.) and grants from the United States−IsraelBinational Science Foundation and the Chief Scientist of theIsrael Ministry of Health and Israel Ministry for Senior Citizens(S.R.).

■ ABBREVIATIONS USEDAβ, β-amyloid protein; ABTS•+, 2,2′-azinobis-(3-ethylbenzo-thiazoline-6-sulfonate) radical cation; ALA, α-lipoic acid; BSA,bovine serum albumin; DCF, 2,7-dichlorofluorescein; DCFH,2′,7′-dichlorofluorescin; DCM, dichloromethane; dGlc, 2-deoxy-D-glucose; DMF, dimethylformamide; DIEA, diisopro-pylethylamine; Dmab, 2-(ethylhexyl)-4-(dimethylamino)-benzoate; DMPO, N,N′-dimethyl-pyrroline-N-oxide; DMSO,dimethylsulfoxide; EPR, electron paramagnetic resonance; FCS,fetal calf serum; Fmoc, fluorenylmethyloxycarbonyl; GLUTs,glucose transporters; GO, glucose oxidase; HBTU, O-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phos-phate; [3H]dGlc, [3H]-2-deoxy-D-glucose; HMBC, heteronu-clear multiple bond correlation; HMQC, heteronuclear multi-ple quantum correlation; HNE, trans-4-hydroxy-2-nonenal;

HOBt, 1-hydroxy-benzotriazole; JNK, c-Jun N-terminal kinase;L6 cells, immortalized rat skeletal muscle cells; MALDI, matrix-assisted laser desorption/ionization; MES, 2-(N-morpholino)-ethanesulfonic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NBD, 4-chloro-7-nitrobenzofur-azan; NDI, 1,4,5,8-naphthalenetetracarboxylic acid diimide;NMP, N-methyl-2-pyrrolidone; NOESY, nuclear Overhausereffect spectroscopy; ONE, trans-4-oxo-2-nonenal; OPD, o-phenylenediamine dihydrochloride; PMSF, phenylmethylsul-fonyl fluoride; PyBOP, benzotriazol-1-yl-oxytripyrrolidinophos-phonium hexafluorophosphate; ROS, reactive oxygen species;ROESY, rotating-frame Overhauser effect spectroscopy; SDS,sodium dodecyl sulfate; TEAC, Trolox equivalent antioxidantcapacity; TEM, transmission electron microscopy; TFA,trifluoroacetic acid; TIS, triisopropylsilane; TOCSY, totalcorrelation spectroscopy; WGA, wheat-germ agglutinin

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