Structural and Functional Changes in Human Insulin Induced by the

ARTICLE pubs.acs.org/crt

Structural and Functional Changes in Human Insulin Induced by the Lipid Peroxidation Byproducts 4-Hydroxy-2-nonenal and 4-Hydroxy-2-hexenal Nicolas J. Pillon,*,†,‡,§,||,3 Roxane E. Vella,†,‡,§,||,3 Laurent Soul
ere,†,^,3 Michel Becchi,†,# Michel Lagarde,†,‡,§,|| and Christophe O. Soulage†,‡,§,|| †

Universite de Lyon, F-69600, Oullins, France INSERM UMR 1060, CarMeN, F-69621, Villeurbanne, France § INSA-Lyon, IMBL, F-69621, Villeurbanne, France INRA U1235, F-69600, Oullins, France ^ Laboratoire de Chimie Organique et Bioorganique, INSA-LYON, CNRS UMR 5246, ICBMS, F-69622, Villeurbanne, France # CNRS UMS 3444, CCMP, F-69367 Lyon, France

)

‡

ABSTRACT: Lipid peroxidation produces many reactive byproducts including 4-hydroxy-2-hexenal (HHE) and 4-hydroxy-2nonenal (HNE) derived from the peroxidation of n-3 and n-6 polyunsaturated fatty acids, respectively. HNE and HHE can modify circulating biomolecules through the formation of covalent adducts. It remains, however, unknown whether HHE and HNE could induce functional and structural changes in the insulin molecule, which may in turn be pivotal in the development of insulin resistance and diabetes. Recombinant human insulin was incubated in the presence of HHE or HNE, and the formation of covalent adducts on insulin was analyzed by mass spectrometry analysis. Insulin tolerance test in mice and stimulation of glucose uptake by 3T3 adipocytes and L6 muscle cells were used to evaluate the biological efficiency of adducted insulin compared with the native one. One to 5 adducts were formed on insulin through Michael adduction, involving histidine residues. Glucose uptake in 3T3-L1 and L6C5 cells as well as the hypoglycemic effect in mice was significantly reduced after treatment with adducted insulin compared to native insulin. The formation of HNE- and HHE-Michael adducts significantly disrupts the biological activity of insulin. These structural and functional abnormalities of the insulin molecule might contribute to the pathogenesis of insulin resistance.

’ INTRODUCTION Insulin is the main regulator of carbohydrate and fat metabolism. Impairment of insulin action on its target cells leads to insulin resistance, a stage in which increased concentrations of insulin are required to produce a given biological response. This common pathological stage is associated with several diseases such as diabetes mellitus, obesity, and atherosclerosis and leads to increased cardiovascular risk.1 To date, it is admitted that insulin resistance has both genetic and environmental factors, but the underlying mechanisms are not fully understood. There is, however, compelling evidence that oxidative stress is involved in the pathophysiology of metabolic syndrome and type 2 diabetes.2
5 During oxidative stress, cellular damages can result from the direct attack by radical species, and by any of the reactive oxidation byproducts from the breakdown of biomolecules. Polyunsaturated fatty acids (PUFA) are among the most susceptible and primary targets of oxidative stress, and their degradation generates a large variety of electrophilic compounds such as aldehydes and ketones.6 Peroxidation of n-6 and n-3 PUFA leads to the production of 4-hydroxy-2-nonenal (HNE) and 4-hydroxy2-hexenal (HHE), respectively. Reported plasma concentrations of HNE concentration ranges from 10 nM to 10 μM in humans.7,8 Plasma concentration of HHE is far less documented, but we recently reported that it ranges from 5
50 nM in healthy humans.9 Increased HNE levels have been reported in different r 2011 American Chemical Society

insulin resistance states, and serum 4-hydroxy-2-nonenalmodified proteins are elevated in type 2 diabetes mellitus patients and animals.10
14 A major determinant of the toxicity of lipid peroxidation byproducts is their huge ability to covalently modify protein reactive groups. HNE has been found to react with sulfhydryl and amino groups in proteins: mainly cysteine and histidine residues.15,16 HNE protein adducts have been detected in many proteins including low density lipoproteins, glucose-6phosphate dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase.17
20 Moreover, our group has recently shown that the toxicity of HHE, HNE, and several other aldehydes is largely due to their ability to form covalent adducts on proteins.21 These covalent modifications can potentially cause functional damage to proteins, and many enzymes have been reported to be inhibited or inactivated following covalent modification with the lipid peroxidation byproducts.17,18,22 Under conditions of oxidative stress, insulin, a polypeptide hormone composed of 51 amino acid residues, is exposed to modification by lipoperoxidation byproducts. Several amino acids are putative sites of adduction, and thus, covalent binding of aldehydes may affect the biological actions of this hormone. This applies to acrolein and methylglyoxal, whose fixation on insulin has been shown to Received: February 22, 2011 Published: April 04, 2011 752

dx.doi.org/10.1021/tx200084d | Chem. Res. Toxicol. 2011, 24, 752–762

Chemical Research in Toxicology

ARTICLE

reduce both hypoglycemic effects in rats and glucose uptake in 3T3-L1 adipocytes.23,24 The purpose of the present work was to provide new data on human insulin activity, particularly its biological efficiency, when adducts with the toxic lipid peroxidation byproducts HHE and HNE are formed. We used mass spectrometry to identify the amino acid residues at which the insulin molecule is modified and estimated its biological effects using an insulin tolerance test in mice and glucose transport in cultured 3T3-L1 adipocytes and L6C5 muscle cells.

loaded onto a 5% concentration gel and then onto a 18% (w/v) polyacrylamide migration gel, and migration was performed for 30 min until the proteins were well penetrated in the concentration gel, then for 40 min under constant voltage (200 V). The gel was fixed overnight in a solution containing 30% (v/v) ethanol and 10% (v/v) acetic acid in water. The gel was washed for 10 min with 20% (v/v) ethanol in water and additionally for 10 min with distillated water. The gel was sensitized by incubation for 2 min in 0.1% (w/v) sodium thiosulfate and then rinsed two times for 1 min with distilled water. After rinsing, the gel was submerged by chilled 0.2% (w/v) silver nitrate solution and incubated for 30 min. After incubation, the silver nitrate was discarded, and the gel was rinsed twice with water and then developed with 0.02% (w/v) paraformaldehyde, 3% (w/v) K2CO3, and 0.01% (w/v) Na2S2O3 in water. After the desired intensity of staining was achieved, development was terminated by washing the gel with 5% (w/v) Tris base with 2.5% (v/v) acetic acid during 2 min. Silver-stained gels were stored in water at 4 C until analysis. Trypsin Digestion. Insulin and insulin adduct solutions were first reduced by 10 mM dithiothreitol in 50 mM NH4HCO3 for 30 min at 50 C and alkylated with 55 mM iodoacetamide in 50 mM NH4HCO3 for 30 min at room temperature in the dark. For proteolytic digestion, the solution was treated with 40 μL of trypsin (sequence grade, Promega, Madison, WI, USA) (0.1 μg/μL in 50 mM NH4HCO3) for 45 min at 50 C. The reaction was stopped by acidification with formic acid. Mass Spectrometry Analysis of Adducted Insulin. Insulin solutions were dialyzed, and mass spectrometry analyses were performed as previously described.15 For MALDI-TOF mass spectrometry analyses, a Voyager DE-PRO mass spectrometer (AB SCIEX, Courtaboeuf, France) was used. MALDI-TOF mass spectra were recorded in the 2.5
25 kDa mass range using linear mode and external calibration for insulin and insulin adduct solutions. The matrix was a sinapinic acid (Laser BioLabs, Sophia-Antipolis, France): solution at 1 mg/100 μL in CH3CN/H2O (50/50; v/v) containing 0.1% (v/v) trifluoroacetic acid (TFA). For peptide analysis after trypsin digestion, spectra were acquired in the 700
5000 Da mass range in positive ion reflector mode. A 1 mg/200 μL solution of R-cyano 4-hydroxycinnamic acid (CHCA) (LaserBioLab, Sophia-Antipolis, France) in H2O/CH3CN mixture (50/ 50) containing 0.1% (v/v) TFA was used as the matrix. Internal calibration was done using trypsin autolysis fragments at m/z 842.510 and 2211.105. Liquid chromatography coupled to Electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) was performed with a Q-Star XL (AB SCIEX, France) equipped with a nanospray ion source and a nano-LC system (Ultimate-Famos-Switchos, Dionex, Voisins Le Bretonneux, France). A 1 s TOF-MS survey scan was acquired over 400
1600 amu, followed by three 3 s production scans over a mass range of 65
2000 amu. The three most intense peptides with a charge state of 2 to 4 above a 30 count threshold were selected for fragmentation and were dynamically excluded for 60 s with 50 amu mass tolerance. The collision energy was set by the software according to the charge and mass of the precursor ion. The MS and MS/MS data were recalibrated by using internal reference ions from a trypsin autolysis peptide at m/z 842.51 [M þ H]þ and m/z 421.76 [M þ 2H]2þ. Chromatographic separation of peptides was obtained by using a C18 PepMap microprecolumn (0.3 mm  5 mm; Dionex) and a C18 PepMap nanocolumn (75 μm  150 mm; Dionex). After injection (1 μL injection volume, pick-up mode, in a 20 μL injection loop), samples were adsorbed and desalted on the precolumn with a H2O/CH3CN/ TFA (98:2:0.05; v/v/v) solvent mixture for 3 min at 25 μL/min flow rate. The peptide separation was developed using a linear 60 min gradient from 0 to 80%, where solvent A was 0.1% HCOOH in H2O/ CH3CN (95:5) and solvent B was 0.1% HCOOH in H2O/CH3CN (20:80) at approximately 250 nL/min flow rate.

’ EXPERIMENTAL SECTION Reagents. Unless mentioned, all chemicals, reagents, and culture media were from Sigma Aldrich (Saint Quentin Fallavier, France). Insulin Adduct Formation. Human recombinant insulin (Actrapid, 100 UI/mL) was purchased from Novo Nordisk (La Defense, France). 4-Hydroxy-2-nonenal (HNE) and 4-hydroxy-2-hexenal (HHE) were synthesized as previously described.25 Briefly, insulin (2.5 μg/μL, 0.43 mM) was incubated for 2 to 16 h with HHE, HNE, or DMSO as control. The final concentration of DMSO was 10% (v/v) in each condition. Insulin solutions were then used for the different experimentations at the concentrations indicated below. Insulin was not purified from the free HHE or HNE before it was injected intraperitonally or applied on cells. However, suitable controls using equivalent concentrations of unreacted aldehydes were performed for each condition. For cell culture, insulin solution and therefore unreacted free aldehydes were diluted 0.43 mM to 100 nM (corresponding to a 4300-fold dilution) before addition into the cell culture media. Thus, the remnant concentration of HHE/HNE was below 1 μM, a concentration for which no effect was observed on glucose uptake in L6 cells (data not shown). For animal study, insulin was diluted from 0.43 mM to 23 μM (corresponding to 1800-fold dilution), and thus, the remnant HHE/HNE and DMSO concentrations were very low. Animals were injected 10 mL/kg of diluted insulin solution which would correspond to a maximal amount of 2.7 nmol/kg. Control experiments were performed in mice with injections of 2.7 nmol/kg HNE, HHE, or vehicle alone. Spectrophotometric DNPH Assay for Carbonyl Content Determination. Carbonyl groups on insulin were determined using 2,4-dinitrophenylhydrazine (2,4-DNPH) as previously described.26 Carbonyl content was determined from the absorbance at 370 nm using a molar absorption coefficient of 22,000 M
1 cm
1 and was normalized by the protein concentration measured at 280 nm. Dot Blot. Anti-HNE-Michael adduct (reference 393207) and antiHHE-Michael adduct (reference NOF-N213730-EX) antibodies were from Calbiochem (San Diego,USA) and Cosmobio (Tokyo, Japan), respectively. The antibody used to detect HHE-adducts (immunogen: HHE modified keyhole-lympet hemocyanine) was highly specific for HHE Michael adducts on histidine residues and therefore enables the specific detection of HHE-histidine in protein samples.27,28 Insulin (0.43 mM) was incubated for 16 h with 5 mM of HHE, HNE, or DMSO as the control. one hundred micrograms of insulin was loaded directly on a nitrocellulose membrane using the Bio-Dot apparatus (BioRad, Marne-la-Coquette, France). Following saturation with 5% BSA, membranes were probed overnight with primary antibodies, antiHHE-Michael adducts, or anti-HNE-Michael adducts. After incubation with HRP-coupled secondary antibodies, membranes were processed for chemiluminescence (ECL plus, GE Healthcare) and quantitated by densitometry using Quantity One software (BioRad). Electrophoresis and Silver Staining. Adducted insulins were mixed with a loading buffer containing glycerol and SDS (without β-mercaptoethanol to avoid reduction of disulfide bonds) and analyzed by Tricine-SDS
PAGE as described previously.29 Briefly, insulins were 753

dx.doi.org/10.1021/tx200084d |Chem. Res. Toxicol. 2011, 24, 752–762

Chemical Research in Toxicology

ARTICLE

Molecular Modeling. Molecular modeling studies were performed using Sybyl 7.2 software (TRIPOS, St. Louis, MO). Insulin monomers with R and T conformations were, respectively, obtained from pdb structure 1MSO30 and 3AIY.31 Molecular models of both insulin monomers with HHE Michael adducts were constructed from His B5 and His B10 by connecting the C3 to the most accessible nitrogen of imidazoles showing the lower steric hindrance with neighboring residues. Energy minimization of insulin monomers with HHE Michael adducts was then achieved using the conjugate gradient method with the TRIPOS force field and MMFF94 charges. HHE modified insulin monomers were then superimposed with native monomers using PyMol (http://www.pymol.org/).32 Figures 5 and 6 were created using YASARA (www.yasara.org)33 and PyMol, respectively. Intraperitonal Insulin Tolerance Test. All experiments were carried out according to the guidelines laid down by the French Minist
ere de l’Agriculture and E.U. Council Directive for the Care and Use of Laboratory Animals (No. 02889). Male CD1 mice (2
3 weeks) were purchased from Harlan (Gannat, France) and kept under controlled light
dark conditions at 20 C with food and water ad libitum. Before the experiment, animals were fasted overnight. Baseline glycemia was measured, and animals were then injected intraperitoneally with 0.5 UI/kg body weight of either native insulin or alkenal-adducted insulin as described above. Plasma glucose was measured from tail vein blood using a glucometer (Accu-Chek Performa, Roche, Meylan, France) at 0, 20, 40, 60, and 120 min following the injection. Glucose disappearance rate for ITT (KITT; %/min) was calculated as follows: KITT ¼

means final DMSO concentration was