Ebina Y., Araki E., Taira M., Shimada F., Mori M., Craik C.S., Siddle K., Pierce ... Hara K., Yonezawa K., Sakaue H., Ando A., Koton K, Kitamura T., Kitamura Y., ...
Vol. 46, No. 2, October 1998 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL Pages259-266
ANGIOTENSIN-CONVERTING ENZYME INHIBITOR INCREASES INSULIN-INDUCED pp185 PHOSPHORYLATION IN LIVER AND MUSCLE OF OBESE RATS Daniela S. Carvalho, Veridiana Villa~a, Sigisfredo L. Brenelli, Carla R.O. Carvalho, Mario J.A. Saad Departamento de Clinica Mgdica, FCM, UNICAMP, Campinas, SP, Brasil Received March 30, 1998 Received after revision,June 15, 1998 SUMMARY: The clinical use of angiotensin-converting enzyme (ACE) inhibitors has been associated with increased insulin sensitivity. However, the molecular mechanism is unknown. The authors examined the early steps in insulin action, i.e., the phosphorylation status of the insulin receptor and of the pp185 in liver and muscle of obese rats treated acutely with captopril, using immunoblotting with antiphosphotyrosine antibodies. Following treatment with captopril there was an improvement in insulin-induced insulin receptor and pp185 phosphorylation in the liver and muscle of obese rats. This finding contribute to an explanation of the mechanism by which ACE inhibitors appear to improve insulin sensitivity. KEY WORDS: tyrosine phosphorylation, captopril, pp 185, insulin receptor, and insulin action INTRODUCTION Insulin initiates its metabolic and growth-promoting effects by binding to the ct subunit of its tetrameric receptor (1), leading to the autophosphorylation of specific tyrosine residues of the 13 subunit. This process enhances the tyrosine kinase activity of the receptor towards other protein substrates (1). Insulin receptor tyrosine kinase activity is essential for many, if not all, of the biological effects of insulin (1-3). In most cells, this primary event leads to the subsequent tyrosyl phosphorylation of a 165-185 kDa protein complex termed pp185 (1, 4-6). The pp185 complex contains at least two proteins that are substrates for insulin receptor, namely, insulin receptor substrate 1 (IRS-1) and insulin receptor substrate 2 (IKS-2) (7-9). Insulin resistance is a characteristic of many disease states, including type 2 diabetes, aging and obesity in both humans and animals. In animal models of obesity (10) and aging (11), a decrease in pp185 phosphorylation occurs which suggests molecular defects in insulin signaling that may contribute to insulin resistance. In aging rats, the ACE inhibitor, captopril, improves insulin sensitivity in parallel with increases in insulin receptor and IRS-1 tyro sine phosphorylations (12). In this study, the effects of captopril on insulin-induced insulin receptor and pp185 tyrosine phosphorylation in liver and muscle of obese rats were investigated. Correspondence to: MfirioJ.A Saad, M.D. Depto Clinica MedicaFCM-UNICAMP,Campinas, SP, Brasil, 13081970. Fax: + 512894107 1039-9712/98/140259~08505.00/0 259
Copyright 9 1998 by Academic Press A.~tralia All rights r reproduction in any form reserved.
Vol. 46, No. 2, 1998
BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL
MATERIALS AND METHODS
The reagents for SDS-PAGE and immunoblotting were from Bio-Rad (Richmond, CA, USA). Tris, phenylmethylsulfonylfluoride(PMSF), dithiothreitol, Tween 20, and glycerol were from Sigma Co. (St. Louis, MO, USA). [t25I]Protein A and nitrocellulose paper (HYBOND ECL) were from Amersham (Aylesbury, UK). Sodium thiopental and human recombinant insulin (Humulin R) were from Elli Lilly Co. (Indianapolis, USA). Male Wistar rats were from the UNICAMP Central Animal Breeding Center. Monoclonal antiphosphotyrosine antibodies and anti-insulin receptor and antiIRS-1 were from Santa Cruz Technology (Santa Cruz, CA). Animals Twelve month-old, obese (450-520 g) male Wistar rats were provided with standard rodent chow and water ad libitum. Food was withdrawn 12-14 h before the experiments. Experimental procedures Rats received captopril (2 mg/kg BW) orally 6 h and 1 h before the experiment. The animals were subsequently anesthetized with sodium thiopental (100mg/kg BW, i.p.) and used 10-15 min later, i.e. as soon as anesthesia was assured by the loss of pedal and corneal reflexes. The abdominal cavity was opened, the portal vein exposed, and 0.5 ml of normal saline (0.9% NaC1) with or without 6 gg insulin was injected. After 30 s, the liver was removed, minced coarsely and homogenized immediately in approximately ten volumes of solubilization buffer (100 mM Tris-HC1 pH 7.4, 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium vanadate, 10% SDS) in a boiling bath water using a polytron PTA 20S generator (model PT 10/35, Brinkmann Instruments, Westbury, NY) operated at maximum speed for 20 s. After centrifugation, the resulting supernatant fractions were treated with Laemmli sample buffer (13) containing 100 mM dithiothreitol, and equal amounts of protein (150lag) were then resolved by SDS-PAGE 6.5% using a Bio-Rad miniature lab gel apparatus (Mini-Protean, Bio-Rad Laboratories, Richmond, CA). Mr standards were myosin (194 kDa), B galactosidase (116 kDa), bovine serum albumin (85 kDa), and ovalbumin (49.5 kDa). Approximately 90 s after the injections described above, hindlimb muscle was quickly excised and homogenized as described for liver. Protein determinations were performed by the Bradford dye binding method using the Bio-Rad reagent and BSA as the standard. Electrotransfer of proteins from the gel to nitrocellulose was performed for 2 hours at 120 V (constant) in a Bio-Rad miniature transfer apparatus (Mini-Protean) as described by Towbin et al. (14) except for the addition of 0.02% SDS to the transfer buffer to enhance the elution of high Mr proteins. Non-specific protein binding to the nitrocellulose was reduced by pre-incubating the filter 2 hours at room temperature in blocking buffer (5% nonfat dry milk, 10 mM Tris, 150 mM NaC1, and 0.02% Tween 20). The nitrocellulose blot was incubated with antiphosphotyrosine antibodies (ll,tg/mi) overnight at 4~ C and then washed for 30 min with the blocking buffer without milk. The blots were subsequently incubated with 2 ~tCi of [125I] protein A (30 laCi/~g) in 10 ml of blocking buffer for 2 h at room temperature and then washed again for 30 minutes as described above. [125I] Protein A bound to the antiphosphotyrosine and antipeptide antibodies was detected by autoradiography using prefiashed Kodak XAR film (Eastman Kodak, Rochester, NY) with Cronex Lightning Plus intensifying screens (DuPont, Wilmington, DE) at -80~ for 12-48 h. Band intensities were quantitated by optical densitometry (model GS 300, Hoefer Scientific Instruments, San Francisco, CA) of the developed autoradiographs. Estimation of in vivo insulin action using the 15-rain insulin tolerance test (ITT) Some animals from both groups underwent an intravenous ITT (0.5 ml of 61,tg of insulin i.v.), and samples for blood glucose determination were collected at 0 (basal), 4, 8, 12 and 16 min after injection. Rats were anesthetized, 40 ~tl of blood were collected from the tail, and the blood glucose
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BIOCHEMISTRY ond MOLECULAR BIOLOGY INTERNATIONAL
level was measured by the glucose oxidase method. Thereafter, the rate constant for plasma glucose disappearance (Kin) was calculated from the formula 0.693/tvz. The plasma glucose tvz was calculated from the slope of the least square analysis of the plasma glucose concentration during the linear phase of decline (15).
Statistical analysis Experiments were performed by parallel study of the different animal groups. For comparisons, Student's unpaired t test was used. The level of significance was set at p
