Formation of an Adduct between Insulin and the Toxic

Sep 6, 2007 - Laboratorio de Metabolismo Experimental, Centro de InVestigación Biomédica de Michoacán, Instituto. Mexicano del Seguro Social, Morelia,...
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Chem. Res. Toxicol. 2007, 20, 1477–1481

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Formation of an Adduct between Insulin and the Toxic Lipoperoxidation Product Acrolein Decreases Both the Hypoglycemic Effect of the Hormone in Rat and Glucose Uptake in 3T3 Adipocytes Rafael Medina-Navarro,† Alberto M. Guzmán-Grenfell,‡ Margarita Díaz-Flores,§ Genoveva Duran-Reyes,§ Clara Ortega-Camarillo,§ Ivonne M. Olivares-Corichi,| and Juan José Hicks*,‡ Laboratorio de Metabolismo Experimental, Centro de InVestigación Biomédica de Michoacán, Instituto Mexicano del Seguro Social, Morelia, Michoacán, México, Departamento de InVestigación en Bioquímica y Medicina Ambiental, Instituto Nacional de Enfermedades Respiratorias “Ismael Cosio Villegas”, México D. F., Unidad de InVestigación Médica en Bioquímica, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, México, D. F., and Sección de InVestigación y Posgrado, Escuela Superior de Medicina, Instituto Politécnico Nacional, México, D. F. ReceiVed April 26, 2007

Lipid peroxidation induced by reactive oxygen species might modify circulating biomolecules because of the formation of R,β-unsaturated or dicarbonylic aldehydes. In order to investigate the interaction between a lipoperoxidation product, acrolein, and a circulating protein, insulin, the acrolein–insulin adduct was obtained. To characterize the adduct, gel filtration chromatography, sodium dodecylsulfate–polyacrylamide gel electrophoresis and carbonyl determination were performed. Induction of hypoglycemia in the rat and stimulation of glucose uptake by 3T3 adipocytes were used to evaluate the biological efficiency of the adduct compared with that of native insulin (Mackness, B., Quarck, R., Verte, W., Mackness, M., and Holvoet, P. (2006) Arterioscler., Thromb. Vasc. Biol. 26, 1545–1550). Formation of the acrolein–insulin complex in vitro increased the carbonyl group concentration from 2.5 to 22.5 nmol/ mg of protein, and it formed without intermolecular aggregates (Halliwell, B., and Whiteman, M. (2004) Br. J. Pharmacol. 142, 231–255. The hypoglycaemic effect 18 min after administration to the rat is decreased by 25% (Robertson, R. P. (2004) J. Biol. Chem. 279, 42351–42354. An adduct concentration of 94 nM, compared to 10 nM for native insulin, was required to obtain the A50% (concentration needed to obtain 50% of maximum transport of glucose uptake by 3T3 adipocytes). In conclusion, formation of the acrolein–insulin adduct modifies the structure of insulin and decreases its hypoglycemic effect in rat and glucose uptake by 3T3 adipocytes. These results help explain how a toxic aldehyde prone to be produced in vivo can structurally modify insulin and change its biological action. Introduction The metabolic syndrome is a cluster of metabolic abnormalities in which insulin resistance is a major characteristic. Central pathophysiological features include atherogenic dyslipidemia, chiefly manifested by decreased high-density lipoprotein cholesterol (HDL-C1) together with increased triglycerides and small, dense, oxidized low-density lipoprotein (LDL) particles (1), hypertension, a proinflammatory state, increased acute phase reactants, and oxidative stress. This last dysfunction occurs when the balance between the production of reactive oxygen species * To whom correspondence should be addressed. Tel: 525-56658330. Fax: 525-56654623. E-mail: [email protected]. † Laboratorio de Metabolismo Experimental, Instituto Mexicano del Seguro Social. ‡ Instituto Nacional de Enfermedades Respiratorias “Ismael Cosio Villegas”. § Unidad de Investigación Médica en Bioquímica, Instituto Mexicano del Seguro Social. | Instituto Politécnico Nacional. 1 Abbreviations: HDL-C, high-density lipoprotein cholesterol; LDL, lowdensity lipoprotein; OLAARPs, oxidized lipid/amino acid reaction products; KRP, Kreb’s ringer phosphate; BSA, bovine serum albumin; SDS–PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; DNPH, 2,4dinitrophenylhydrazine; DMEM, Dulbecco’s modified Eagle’s medium.

(ROS) and the ability of cells or tissues to detoxify the free radicals produced during metabolic activity is tilted in favor of the former, leading to potential damage (2). Although hyperglycemia plays a key role in inducing oxidative stress in diabetic patients by several mechanisms (3), the contributions of other factors, such as transition metal imbalance conducing to a Fenton reaction, protein glycation (4), and overactivity of NADPH oxidase in phagocytes (5), might be considered. During oxidative stress, molecular and cell injury can result from the direct attack by reactive species on any or all of the major molecular targets, such as lipids, proteins, DNA, and carbohydrates (2), conducive to the generation of reactive oxidation products from the breakdown of these molecules. Lipid peroxidation (6) has been associated with important pathophysiological events in a variety of diseases, drug toxicities, and traumatic or ischemic injuries (7). This toxicity has been largely attributed to the Rβ-unsaturated or dicarbonylic aldehydes that are produced during lipid peroxidation (8, 9). Among these aldehydes, the most intensively studied have been malondialdehyde, 4-hydroxy-2-alkenals, 4,5-epoxy-2 alkenals, and more recently acrolein (10, 11). A common characteristic of all of these aldehydes is their ability to modify reactive groups in proteins, producing inter alia oxidized lipid/amino acid

10.1021/tx7001355 CCC: $37.00  2007 American Chemical Society Published on Web 09/06/2007

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reaction products (OLAARPs) (12). 4-Hydroxynonenal/protein adducts have been detected immunologically in modified LDL, glucose-6-phosphate dehydrogenase (13), and glyceraldehide3-phosphate dehydrogenase (14), affecting their chemical structure and biological function. During insulin circulation under conditions of oxidative stress linked to diabetes mellitus (15), lipoperoxidation products are present on oxidized plasma proteins, related to the modifications of insulin induced in vitro by the hydroxyl radical (16). As a result, insulin-dependent 14C-glucose utilization by human adipose tissue (17) is decreased. The purpose of the present work was (i) to provide new data on human insulin activity, particularly its biological efficiency, when an adduct with the toxic agent acrolein is formed; (ii) to evaluate the insulin tolerance test in rats in the presence of the adduct; and (iii) to investigate the decrease in glucose transport in cultured 3T3L1 adipocytes (18).

Materials and Methods Insulin Adduct Formation. Stock solutions of bovine insulin (Sigma. St. Louis, MO, USA) were prepared in Milli Q water, acidified with 1 N HCl until dissolution. Aliquots of insulin containing 855 µg/mL hormone (150 µM) were adjusted to pH 7.4 or pH 8.0 with 0.5 M Tris base and incubated for 2, 4, and 12 h at 37 °C with or without 300 µM acrolein (Sigma. St. Louis, MO, USA). At the end of the incubation, the pH of all of the samples was adjusted to 7.4, placed in micro-centrifuge filters (5 KDa cutoff) (Microcon, Millipore, Bedford, MA, USA), and centrifuged at 5000g for 45 min. The filter residues were washed and dialyzed three times and dissolved in Krebs Ringer Phosphate (KRP). Caution. The acrolein chemical is hazardous and should be handled carefully under a fume hood. Intraperitoneal Insulin Tolerance Test. Male Sprague–Dawley rats, weight 350 ( 5 g, were maintained under controlled light–dark conditions at 20 °C with food and water ad libitum. They were anesthetized with phenobarbital, and native bovine insulin or treated insulin (adduct) was administered intraperitoneally (0.35 mg in 100 µL/kg). Blood samples were taken from the tails at different times (0–90 min), and glucose concentration was measured by the glucose oxidase method using a glucose analyzer (Abbott Laboratories Inc., MediSense Products, Bedford, MA 01730 USA). Cell Culture. 3T3-L1 fibroblasts were cultured in Dulbecco’s Modified Eagle Medium (DMEM) medium with 10% bovine serum and differentiated in 6-well cell culture clusters, as previously reported (19). Before glucose transport measurements, the cell monolayers were treated with 1% bovine serum albumin (BSA) in KRP in order to remove bound insulin, according to the debinding procedure of Ronnett et al. (20). Glucose Transport. Glucose transport was measured in KRP buffer using 2-deoxy-[2,6-3H] glucose (Amersham Pharmacia Biotech., England) as described by Frost and Lane (19). Insulin adducts and untreated hormone (0, 0.1 nM, 1 nM, 10 nM, 100 nM, and 1 µM) were added to the cells in 1 mL of KRP. After 20 min of incubation at 37 °C in the dark and a second 20 min incubation with 50 µM 2-desoxy-[2,6-3H] glucose (1µCi/mL) under the same conditions, the culture medium was aspirated, and the cells were washed four times with cold 1% BSA in KRP and then lysed with 1% Triton. The mixture obtained (1 mL) was added to 10 mL of scintillation cocktail and counted on a Beckman LS 6000SE β counter. Sodium Dodecylsulfate–Polyacrylamide Gel Electrophoresis (SDS–PAGE). Aliquots containing bovine insulin (150 µM) were incubated at pH 7.4 or pH 8.0 for 0, 2, and 4 h at 37 °C in the presence or absence of 300 µM acrolein. Aliquots of 0.4 mL were filtered to separate protein. The filters were washed three times, and the protein was suspended in deionized water. Aliquots containing 100 µg of protein in 50 µL were boiled in a buffer containing 0.5 M Tris at pH 6.8, 1% SDS, and 10% β-mercapto-

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Figure 1. Intraperitoneal insulin tolerance test. Insulin samples were incubated with and without acrolein for 4 h at pH 7.4 and 8.0. After incubation, the pH was adjusted to 7.4 and processed as described. The samples (0.3 mg/Kg) were then injected into rats after an overnight fast. At time 0 and every 15 mm thereafter, blood glucose concentrations were measured by the glucose oxidase method. Data are the mean ( SD; (n ) 5) *p < 0.01 vs insulin, pH 7.4; **p < O 001 vs insulin, pH 7.4; p < 0.05 vs insulin, pH 7.4.

ethanol. Separation was achieved in 20% T and 0.5% C (200:1 acrylamide/bisacrylamide), and when using a separating gel buffer at pH 9.3 (21). Gel Filtration Chromatography. A solution containing 1 mg of insulin/mL of deionized water was incubated for 12 h at 37 °C in the presence or absence of 300 µM acrolein, and the pH was adjusted to 7.4 or 8.0 using 0.5 M Tris. After the incubation and before dialysis, samples containing 50 µg of protein were dissolved in a buffer containing 0.5 M Tris at pH 6.8, 1% SDS, and 15 mM dithiothreitol to preclude spontaneous aggregation. Under these conditions, the protein was applied to a Sephadex G-50 column (10 × 3.0 cm), equilibrated, and eluted with 150 mM phosphate buffer (pH 7) at 20 °C. Cytochrome c (1 mg/mL) was applied to the column under the same conditions. The protein concentrations in the 1 mL fractions collected were measured by the method of Bradford (22). Determination of Carbonyls in Protein. The insulin–acrolein adduct and untreated insulin (control) were resuspended in 1 mL of 10 mM 2,4-dinitrophenylhydrazine (DNPH) in 2 M HCl. The samples were incubated at laboratory temperature in the dark for 60 min, stirring at 15 min intervals. After several centrifuge-washing steps, the protein carbonyl content was determined (23) by measuring the absorbance of the protein-2,4-dinitrophenylhydrazone derivative at 375 nm. The molar extinction coefficient for DPNH was taken as ε ) 22,000 M-1 cm-1. The protein concentration was taken as the reference parameter. Data Analyses. The means and SE were calculated using standard procedures. The statistical significance of differences between group means was evaluated by Student’s t test, taking a probability of 0.05 % as the criterion of significance. All values reported are the mean ( SD.

Results Insulin Tolerance. The effect of protein modification due to adduct formation with acrolein on the intraperitoneal insulin tolerance test in rats is shown in Figure 1. A rapid reduction of blood glucose level from 120 to 55 mg/dL was obtained 18 min after insulin administration, irrespective of previous incubation at pH 7.4 or pH 8.0. The response to the adduct was less than that to the control. The adduct formed at pH 7.4 decreased the biological activity of insulin by approximately 25% 18 min after administration. The effect was more marked when the adduct formed at pH 8.0 was administered; the biological effect

Insulin–Acrolein Adduct Decreases Hormonal ActiVity

Figure 2. Effect of modified insulin on glucose transport. Regular insulin and insulin incubated with acrolein were processed as described. The dotted lines intercepting the x-axis indicate the insulin concentration where the transport activation reaches 50% (A50%) and corresponds to the followings values: 4.5, 12.3, and 94 nM (nonmodified pH 7.4, modified pH 7.4, and modified pH 8.0). Each point represents the mean ( SD of 5 experiments. *p < 005 vs insulin at pH 7.4; **p < 0.001 vs insulin at pH 7.4.

of insulin on induction of hypoglycemia in the rat was inhibited by almost 100% (p < 0.001; Figure 1). The activation of glucose uptake measured in 3T3-L1 cells was defined by the rate of glucose transport in the absence and presence of sufficient insulin to achieve the maximal response. Under our experimental conditions, the concentration of unmodified insulin at which glucose transport activation was basal was 0.1 nM, and the insulin concentrations necessary to achieve maximum and 50% maximum (A50%) glucose transport activation were 1000 nM and 4.5 nM, respectively (Figure 2). The concentration of adduct obtained by incubation with acrolein at pH 7.4 necessary to reach the A50% was 12.3 nM, which is more than twice the A50% value for native insulin. For the acrolein-modified protein incubated at pH 8, the concentration required to achieve the A50% value (94 nM) was more than 20 times that of unmodified insulin. In this case, the adduct concentration required to produce basal glucose uptake was approximately 10 nM, almost the concentration at which unmodified insulin reaches 50% maximal activity (Figure 2). Carbonyl Group Formation. Figure 3 shows carbonyl group formation resulting from the incorporation of acrolein into insulin, measured as carbonyl-derived hydrazone formation/mg protein (abscissa). The protein carbonyl content of the acrolein– insulin complex was highly dependent on the pH at which the adduct was formed. Up to 1 h of incubation at pH 8, the carbonyl concentration of insulin increased rapidly and then remained high throughout the incubation period (from 23 to 26 nmol/mg protein on average). At pH 7.4, the protein carbonyl content increased slowly and reached a maximum of 8.4 ( 1.3 nmol/ mg protein in 4 h. It was possible to observe spontaneous carbonyl formation in unmodified insulin (2.9 ( 1.0 nmol/mg protein after 4 h of incubation) (Figure 3). Each point represents the mean ( SE of four experiments. Molecular Weight Modification. The possibility of molecular aggregate formation by cross-linking of the insulin–acrolein adduct was considered. The molecular sizes of the compounds resulting from incubation of insulin with acrolein at different pH values were determined by SDS–PAGE and gel filtration chromatography. Figure 5 shows the elution patterns of insulin

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Figure 3. Carbonyl introduction to insulin exposed to acrolein. Insulin (1 mg/mL) was incubated with and without acrolein at 37 °C for a 4-h period, in de-ionized water at pH 7.4 and 8. At time 0 and before each hour, aliquots of the samples were collected, alt pH values adjusted (7.4), and processed as described in the Materials and Methods section. The insulin carbonyl content was determined using 2,4- dinitrophenylhydrazine. Each point is the mean ( SD from 3 experiments. *p < 0.01 vs insulin at pH 7.4; **p < 0.001 vs insulin at pH 7.4.

Figure 4. Scarce insulin aggregates in the SDS–PAGE (20% and separation buffer at pH 9.3) by the reaction with acrolein from 0, 2, and 4 h. Before the incubation period, insulin was processed as described. 1, insulin at pH 7.4; 2, insulin + acrolein at pH 7.4; 3, insulin + acrolein at pH 8.0; c, cytochrome c (Mol. Wt. 12.3 kDa).

Figure 5. Gel filtration elution profile of insulin and insulin exposed to acrolein. Before incubation (12 h), insulin and treated insulin were applied to a Sephadex G-50 column (10 × 3.0 cm) equilibrated and eluted using 150 mM phosphate buffer at pH 7. The profile of cytochrome c corresponds approximately to the void volume. Each fraction volume corresponds to 1 mL.

and the adducts on Sephadex G-50 gel filtration chromatography. The insulin–acrolein adducts showed the highest protein concentration in fraction 7, while the concentration of unmodified insulin peaked in fraction 8. The elution of the adduct might suggest an increase in molecular weight compared with that of insulin. However, this observation was not considered evidence for stable dimer or polymer formation because the SDS–PAGE results (Figure 4) showed only one band, corresponding to insulin, for both unmodified insulin and adducts. This indicates that polymers are not formed during the interaction between insulin and acrolein under the experimental conditions used here.

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Discussion In this work, we have demonstrated the structural chemical change induced in human insulin by interaction with acrolein, an R,β unsaturated aldehyde produced during lipid peroxidation. The formation of an insulin adduct with acrolein affects its biological activity, decreasing its hypoglycemic activity in vivo and attenuating insulin-dependent glucose uptake in cultured cells. Insulin activity is particularly sensitive to intramolecular cross-linking since conformational changes at the receptor binding site are a prerequisite for biological function; therefore, any interference with the conformation and functional group structure of the hormone might affect its activity. A completely inactive analogue (24) called mini-proinsulin contains a peptide bond between the A and B chains (GlyA1–LysB29), which displaces the C-terminal region of the B chain and increases the rigidity of the molecule (25). In contrast, the loss of this constraint induces a higher bioactivity, as seen in analogues in which the R-NH2 group of GlyA1 and the ε-NH2 of LysB29 are cross-linked by chemical reagents (26, 27). When the data obtained from these experiments were considered in light of the enhancement of conformational stability and decrease in flexibility caused by the addition of an intramolecular crosslink to the insulin molecule, a 6-carbon GlyA1–LysB29 link was found to be optimal for enhanced stability and decreased flexibility (28). This leads to a plausible explanation for the decrease in activity subsequent to the interaction between acrolein and insulin (Figures 1 and 2) since the first aldolic condensation product of acrolein is precisely six carbons long. Aldehydes such as acrolein and malondialdehyde, which contain an R-H, can undergo aldol-type self-condensation (29) and form dimers, trimers, or polymers in aqueous solution. The formation of an acrolein dimer or polymer and then of condensation products was suggested by Uchida et al. (10). The loss of insulin activity shown in Figures 1 and 2 was greater when the adduct was obtained at alkaline pH, which favors aldol condensation (Figure 3). This would allow the formation of more carbonyls at pH 8.0 than at pH 7.4, which can be detected during osazone formation during acid hydrolysis, and would lead to more molecules of acrolein being cross-linked to insulin in the adduct. The effect of pH on adduct formation therefore supports the possibility that the acrolein–insulin interaction might be effected by an aldolic product, rather than a single interaction with the N-terminal group LysB29 and the terminal GlyA1 and PheB30 as reported for glycated insulin. Monoglycated insulin was approximately 20% less effective (30) and diglycated insulin more than 43% less effective (31) than native insulin in stimulating glucose uptake by isolated abdominal muscles of mice; these modifications also decrease hormone activity. The concentrations of acrolein and other 2-alkenals in plasma and tissues must depend on the characteristics and intensity of the lipoperoxidation process. The concentration of 4-hydroxy2-nonenal (HNE) in plasma or tissues under basal physiological conditions is considered to lie in the range 0.1–0.3 µM (32), increasing in pathological states to 2.0 and 5.4 µM in plasma and liver, respectively (33). In the ventricular fluid of patients with Alzheimer’s disease, HNE concentrations reach 120 µM (34). Tissue aldehyde concentrations in particular are higher under the oxidative stress accompanying diabetes mellitus. In the pancreatic islet, the HNE concentration has been reported to be 23–35 µM (35). In summary, we have demonstrated in the present article that acrolein, a toxic lipoperoxidation product generated as a consequence of ROS action during oxidative stress, is capable

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of forming an adduct with insulin that shows the following characteristics: (I) Optimal formation of the acrolein–insulin complex is pH dependent and is favored at pH 8. (II) This adduct might be a product of intramolecular aldolic condensation and not due to Schiff base formation with the N-terminal amine groups of insulin. (III) There is no evidence for intermolecular interactions forming insulin–acrolein aggregates (Figures 4 and 5). (IV) The adduct formation affects the biological activity of insulin in vivo, decreasing (a) its hypoglycemic effect in the rat and (b) the stimulation of glucose transport in cultured cells. Finally, we considered whether the adduct can form in vivo, taking into account the insulin concentration (0.08–0.1 µM) and the acrolein/insulin molar ratio (2/1) used in the present work. The acrolein concentration required to produce a similar response in vivo is estimated to be at around 0.1–0.2 µM. Acknowledgment. This study was supported in part by an award from FUNSALUD (Fundación Mexicana para la Salud) and SILANES. We gratefully acknowledge Antonio Lopez de Silanes for the sponsorship.

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