441 Enhancement of muscle glucose uptake by the vasopeptidase inhibitor, , is independent of insulin signaling and the AMP kinase pathway

Victor Wong, Linda Szeto, Kristine Uffelman, I George Fantus and Gary F Lewis Department of Medicine, Division of Endocrinology and Metabolism and the Department of Physiology, University of Toronto, Toronto, Ontario, Canada (Requests for offprints should be addressed to G F Lewis who is now at Toronto General Hospital, EN11-229, 200 Elizabeth Street, Toronto, Ontario M5G 2C4, Canada; Email: [email protected])

Abstract Omapatrilat (OMA), a vasopeptidase inhibitor (VPI), P!0$01). Immunoprecipitation and Western blot analysis of presently being tested in clinical trials for its anti- insulin receptor substrate 1 (IRS-1) revealed no changes in hypertensive properties, inhibits both -converting protein mass with OMA treatment. OMA did not enhance enzyme and neutral endopeptidase, and raises tissue basal or insulin-stimulated IRS-1 tyrosine phosphorylation bradykinin levels. Recent studies from our laboratory and or its subsequent association with the p85 regulatory subunit those of others have demonstrated that VPIs enhance muscle of phosphatidylinositol 3-kinase. Under basal and insulin- glucose uptake in animal models, and this effect is mediated stimulated conditions, OMA treatment did not alter the by the bradykinin–nitric oxide pathway. The mechanism of protein mass or the phosphorylation of Akt/protein kinase the effect of OMA on muscle glucose uptake, however, is B, p42/44 extracellular signal-regulated kinase or adenosine presently unknown. To investigate the effect of OMA on monophosphate-activated protein kinase, or GLUT4 protein insulin signaling, soleus muscle was isolated 2 or 5 min after expression. We conclude that the ability of OMA to enhance an i.v. bolus of insulin or saline from male Zucker fatty rats whole body and specifically muscle glucose uptake in Zucker (8–10 weeks of age), following a 5-day treatment period of fatty rats is not mediated by enhancing insulin or AMPK oral OMA (15 mg/kg per day) or drug vehicle (placebo). signaling. Future studies should examine whether hemo- OMA resulted in significantly lower systolic blood pressure dynamic effects of the drug, independent of insulin signaling, compared with the placebo-treated group (84$4G enhance glucose uptake in insulin-resistant skeletal muscle. 7$52 mmHg in OMA vs 112G2$18 mmHg in controls, Journal of Endocrinology (2006) 190, 441–450

Introduction both angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP; Floras 2002). The consequence of dual Insulin resistance is a major pathogenic factor for type-2 inhibition of NEP and ACE is a synergistic reduction in diabetes mellitus and is associated with a cluster of vasoconstriction and enhancement of vasodilation, thereby atherosclerotic cardiovascular disease risk factors (Reaven serving to reduce blood pressure (BP) more effectively than 1988). These risk factors include, but are not limited to, ACE inhibitors (ACEI). Of the VPIs, the most advanced in its hypertension, dyslipidemia, endothelial dysfunction, pro- development as a therapeutic agent is omapatrilat (OMA), coagulant and pro-inflammatory states, abdominal obesity, although its clinical development has been curtailed severely by and hepatic steatosis, a complex that has collectively been a high incidence of serious side effects, including angioedema termed the metabolic syndrome. The close link between (Rouleau et al. 2000, Guthrie & Reeves 2002, Lapointe & insulin resistance and the development of cardiovascular Rouleau 2002). We recently showed that OMA enhances complications suggests either a direct or an indirect causal muscle glucose uptake in Zucker fatty rats and these results mechanism and emphasizes the need to develop therapeutic have been confirmed by others using another VPI, mixanpril strategies that improve insulin sensitivity and its (Arbin et al. 2003, Wang et al. 2003). By using two independent co-morbidities. methods, euglycemic hyperinsulinemic clamp and insulin- Vasopeptidase inhibitors (VPIs) are new therapeutic agents bolus-mediated 2-deoxyglucose tissue uptake, our laboratory that are being developed for the treatment of hypertension and has previously demonstrated enhancement of whole body heart failure (Floras 2002). They act primarily by blocking and muscle glucose uptake by OMA (Wang et al. 2003).

Journal of Endocrinology (2006) 190, 441–450 DOI: 10.1677/joe.1.06396 0022–0795/06/0190–441 q 2006 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org

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The insulin-sensitizing effects of OMA are blocked by Animals bradykinin B receptor antagonist (ecatibant-140) and nitric 2 Obese (fa/fa) Zucker male rats (Charles River Laboratories, oxide synthase inhibitor (Danser et al. 2000, Wang et al. 2003). Saint Constant, Quebec, Canada) were studied at the age of Following on from our previous observations (Wang et al. 8–10 weeks, as this is the time when Zucker fa/farats progress 2003), the present study was designed to investigate whether from insulin-resistance to mild glucose intolerance. Limited the previously demonstrated OMA-induced improvement in studies were also performed in two leanstrains ( fa/K and K/K) muscle glucose uptake is mediated by enhanced insulin for comparison. The rats were housed in the Animal Care signaling via known pathways of insulin signaling in skeletal Facility of the Toronto General Hospital. They were exposed muscle. Our results demonstrate that OMA does not affect to a 12 h light:12 h darkness cycle and fed rat chow (Purina insulin-signaling molecules under basal or insulin-stimulated 5001, 4$5% fat, Ralston Purina Co., St Louis, MO, USA) and conditions. Furthermore, the enhancement of muscle water ad libitum. All procedures were approved by the Animal glucose uptake by OMA treatment cannot be attributed to Care Committee of the University Health Network, adenosine monophosphate-activated protein kinase a University of Toronto. (AMPKa) activation or to an increase in total GLUT4 protein expression. Surgery and animal handling After 3 days of adaptation to the facility, the rats were anesthetized by i.p. injection of ketamine:xylazine:acepro- Materials and Methods mazine (20:2:1 mg/ml, 1 ml/g body weight) and an indwel- ling catheter was inserted in the right internal jugular vein for Materials i.v. administration of the insulin or saline bolus. The catheter Polyethylene (PE-50) catheters were obtained from Cay Adams was tunneled subcutaneously and exteriorized to the back of (Aronson et al. 1997a), and each was extended with a segment of the neck. All surgeries (vessel cannulation) were performed silastic tubing (length 3 cm, internal diameter 0$05 cm) from under sterile conditions. The catheters were filled with a Dow Corning Co. (Midland, MI, USA). OMA was obtained mixture of 60% polyvinylpyrrolidone and heparin from Bristol-Myers Squibb (#186716-01, Lot9B10310 CA, (1000 USP/ml) to maintain patency for 5 days and closed at Princeton, NJ, USA). Human insulin was obtained from Eli the end with a metal pin. The catheters were flushed with Lilly and heparin from Organon (Toronto, Ont., Canada). saline once during the 5-day treatment of either OMA or Protease inhibitors used in this study such as aprotinin, placebo, on day 3. leupeptin, okadaic acid, phenylmethylsulfonyl fluoride (PMSF), E64, and pepstatin were obtained from Sigma. All Treatment of rats and muscle preparations electrophoresis and transfer processes were carried out in XCell The obese Zucker rats were randomized to receive oral SureLock Novex Mini-Cell and XCell II Blot module obtained gavage of either OMA (15 mg/kg per day) diluted in saline from Invitrogen. Nitrocellulose membranes were purchased (nZ12) or drug vehicle only (control, nZ12), once daily, for from Schleicher and Schuell Bioscience (Keene, NH, USA). a period of 5 days. This is the same dose of the drug used in Anti-phospho-tyrosine antibody was purchased from Santa our previous studies (Wang et al. 2003), in which we Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Agarose- demonstrated an insulin-sensitizing effect of OMA. All conjugated anti-insulin receptor substrate-1 (anti-IRS-1) and animals were given free access to regular chow and water anti-p85 subunit of phosphatidylinositol 3-kinase (PI3K) throughout the 5-day treatment period. On the third day of antibodies were purchased from Upstate Biotechnology, Inc. the study, systolic BP was measured by tail cuff in lightly (Lake Placid, NY, USA). Anti-AKT, anti-phospho-AKT anesthetized rats (approximately two-thirds of the anesthetic (Ser473), anti-phospho-AKT (Thr308), anti-AMPKa, anti- dosage for surgery as mentioned earlier), 6 h before their oral phospho AMPKa (Thr172), anti-extracellular signal-regulated treatment. Immediately afterwards, the catheter was flushed kinase 1 and 2 (anti-ERK1/2) mitogen-activated protein kinase with saline and filled with polyvinylpyrrolidone and heparin (MAPK; p44/p48) and anti-phospho-ERK1/2 MAPK admixture to maintain patency for the remaining days of the (Thr202/Tyr204), anti-acetyl CoA carboxylase (ACC), and treatment. Blood samples were obtained from the tail vein anti-phospho-ACC(Ser79) antibodieswere from Cell Signaling after an overnight fast (food was removed at 1800 h after the Technology, Inc. (Beverly, MA, USA). Rabbit polyclonal anti- final dose of treatments on day 5). Insulin (50 U/kg) was GLUT4 antibody was a generous gift from Dr Amira Klip administered as an i.v. bolus through the venous catheter to (Hospital for Sick Children, Toronto, Ont., Canada). Horse- nZ12 OMA-treated and nZ12 placebo-treated rats. Six radish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit animals in each of these two treatment groups had muscle immunoglobulin G (IgG) was purchased from Cell Signaling biopsies performed 2-min post-i.v. insulin bolus for Technology,Inc. Enhanced chemiluminescence (ECL) reagents assessment of IRS-1 activation, whereas six animals in each were obtained from Amersham. group had muscle biopsies performed at 5 min for assessment

Journal of Endocrinology (2006) 190, 441–450 www.endocrinology-journals.org

Downloaded from Bioscientifica.com at 09/27/2021 08:15:09PM via free access OMA and mechanism of insulin sensitization $ V WONG and others 443 of Akt phosphorylation and all other measures of insulin Tris (pH 6$8), 0$1% bromophenol blue, 5% signaling, as described below. We (unpublished observations) b-mercaptoethanol), boiled for 5 min, and separated by and others (Kim et al. 2000, Shiuchi et al. 2002) have found SDS-7$5% PAGE under reducing conditions. Proteins on the that peak stimulation of IRS-1 phosphorylation and p85 gels were transferred onto nitrocellulose membranes. association with IRS-1 occurs 2 min after the insulin bolus, Following blocking with Tris-buffered saline/0$1% Tween whereas downstream Akt peak phosphorylation occurs later, 20 (TBST) blocking buffer containing 0$1% Tween-20 and at approximately 5 min. A separate group of OMA- (nZ6) or 5% non-fat milk or BSA at room temperature for 1 h, the placebo-treated (nZ6) Zucker fatty rats received an i.v. bolus membranes were immunoblotted with anti-IRS-1, anti- of saline in order to assess basal (i.e. non-insulin stimulated) phosphotyrosine, or anti-PI3K p85 (1:1000) overnight at tissue levels of the parameters of interest. To determine 4 8C. After washing three times with TBST, the membranes whether the 12-h time lag following the last dose of OMA were incubated at room temperature for 1 h with HRP- could have influenced our results, an additional three Zucker conjugated secondary antibodies. Bands of interest were Z fatty rats (n 3) pre-treated with OMA received an additional visualized by ECL, scanned, and quantified by densitometry. dose of OMA 2 h prior to the insulin bolus. When necessary, some nitrocellulose membranes were Immediately after insulin administration, the animal was stripped subsequently at 50 8C for 30 min in stripping buffer placed under anesthesia with 5% isoflurane in 100% O2 and (62$5mM Tris–HCl (pH6$8), 2% SDS, and 100 mM $ $ maintained during surgery with 1 9–2 1% isoflurane in 100% b-mercaptoethanol) for a second round of immunoblotting. O2 through a nose cone to minimize stress and excitation. The To determine the protein levels of Akt, phospho-Akt depth of anesthesia was verified by noting the absence of (Ser473 and Thr308), p44/42 MAPK, phospho-p44/42 physical responses to firm paw pinch, and the right and left MAPK, AMPK, phospho-AMPK, ACC, and phospho- soleus skeletal muscles were removed from each rat 2 or 5 min ACC, equal amounts of protein (40 mg) were prepared from after insulin administration, as indicated earlier. We have muscle homogenates and subjected to SDS-PAGE and chosen the soleus muscle to assess insulin signaling, since our immunoblotting procedures as described above. previous studies showed a marked increase in glucose uptake in To determine the protein levels of GLUT4, 1 mg soleus this muscle group (Wang et al. 2003). Tissue samples were muscle was weighed and homogenized in ice-cold lysis buffer removed and snap frozen in liquid nitrogen and stored (20 mM NaHCO , 250 mM sucrose, 5 mM NaN ,0$2mM at K70 8C for later analysis. Approximately 50 mg of soleus 3 3 PMSF, 10 mM E64, 1 mM pepstatin, 1 mM leupeptin). The skeletal muscles were ground in a glass-on-glass homogenizer homogenates were centrifuged at 1300 g for 10 min. The containing ice-cold lysis buffer (50 mM Tris (pH 7$5), 1% pellets were resuspended in lysis buffer and re-centrifuged at Nonidet P-40, 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 2 mM ethylene glycol tetra , 1 mM Na VO , 1300 g for another 10 min. The supernatants from the first and 3 4 second centrifugations were pooled and spun at 229 000 g for 100 mM NaF, 10 mM Na4P2O4,1mM okadaic acid, 1 mM phenyl methyl sulfonyl fluoride, 10 mg/ml aprotinin, 10 mg/ 90 min. The resultant pellets containing the total or crude ml leupeptin). Insoluble materials were removed by centri- membranes were re-suspended in lysis buffer and protein fugation at 2000 r.p.m. for 10 min at 4 8C. Protein concen- concentrations of the extracts were estimated by the Lowry tration was determined by the Lowry method using BSA method as described previously. Samples were stored at K as standard. 70 8C until used. In order to determine whether the insulin bolus could have lowered blood glucose within the 5-min time-frame prior to Other laboratory methods performing the muscle biopsy, thereby eliciting a counter- regulatory response, blood glucose was measured by Sure Step Blood glucose was analyzed by Sure Step, One Touch One Touch glucometer (Lifescan, Inc., Johnson & Johnson, glucometer (Lifescan, Inc., Johnson & Johnson). Plasma Milpitas, CA, USA) in a subgroup of three animals prior to insulin was determined using a RIA kit from Linco Research insulin administration and again after 5 min. (St Charles, MO, USA). BP was monitored by Doppler probe with occlusive cuff on the tail artery manufactured by IITC, Inc., Life Science (Woodland Hills, CA, USA) and Immunoprecipitation and Western blot analysis measurements were acquired through Biopac AcqKnowledge For detection of tyrosine phosphorylation of IRS-1 and p85 Software (Goleta, CA, USA). regulatory subunit of PI3K association with IRS-1, muscle lysate containing equal amounts of protein (1 mg) were Statistical analysis subjected to immunoprecipitation overnight at 4 8C with agarose-conjugated rat polyclonal anti-IRS-1 antibodies. All data are presented as meansGS.E.M. Statistical significance Immune complexes were collected by brief centrifugation was determined for each parameter of interest by unpaired and washed three times with lysis buffer as mentioned above. Student’s t-test for OMA- vs placebo-treated animals. A value The equivalent amount of protein samples was resuspended in of P!0$05 was considered significant (as indicated by *) and 1!Laemmli sample buffer (2% SDS, 10% glycerol, 62$5mM P!0$01 was considered highly significant (as indicated by **). www.endocrinology-journals.org Journal of Endocrinology (2006) 190, 441–450

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Results animals compared with saline treatment. Consistent with the tyrosine phosphorylation results, 5 days of OMA treatment Basal parameters in Zucker fatty rats alone did not stimulate basal or insulin-stimulated IRS-1/ PI3K association. Characteristics of the animals are shown in Table 1. Body weight The relative impairment of insulin signaling in obese was similar between control and OMA-treated groups after animals compared with Zucker lean rats was demonstrated by 5 days of treatment and there was no difference in fasting blood measuring insulin-stimulated IRS-1 tyrosine phosphorylation glucose, insulin, triglycerides (TGs), or free fatty acids (FFAs) and PI3K association in soleus muscles, 2 min after between OMA and control groups. BP was reduced signi- administration of the insulin bolus (data not illustrated). ficantly in the OMA-treated group compared with the placebo Zucker fatty rats (nZ3) had a clear impairment in IRS-1 group, verifying that the OMA-treated animals had a tyrosine phosphorylation and p85 association with IRS-1 biologically significant response to the drug. There was no compared with their lean counterparts (nZ2). Fatty rats difference in the blood glucose level before and 5 min after the demonstrated 155$7G29$5 and 168$6G27$6% enhancement insulin bolus in an additional three control and three of IRS-1 phosphorylation and p85 association respectively OMA-treated rats (glucose 4$9G0$5mmol/lpre-vs6$7G after an insulin bolus, when compared with rats administered 0$5 mmol/l 5-min post-insulin administration in the control a saline bolus. In contrast, lean rats demonstrated 430$0G50$7 group, PZnot significant (n.s.); 5$9G0$8 mmol/l pre- vs 5$5G and 581$1G30$2% enhancement of IRS-1 phosphorylation 0$8 mmol/l 5-min post-insulin bolus in the OMA-treated and p85 association respectively after an insulin bolus, when group, PZn.s.). Such an observation allows us to safely compared with rats administered a saline bolus. These data are eliminate the possibility that counter-regulatory hormonal consistent with previous studies (Hotamisligil et al. 1994, Anai changes may have occurred and impacted on insulin signaling. et al. 1998).

IRS-1 tyrosine phosphorylation and association with the p85 Akt/protein kinase B (PKB) and p42/44 MAP kinase regulatory subunit of PI3-kinase phosphorylation in Zucker fatty rat skeletal muscle Tyrosine phosphorylation of IRS-1 is shown in Fig. 1A. We next examined insulin-signaling proteins downstream of There were no changes in total IRS-1 protein expression in IRS-1 and the results are illustrated in Fig. 2. Akt protein the soleus muscles of control and OMA-treated rats after levels did not differ among the groups in this study. Insulin- 5 days. As expected, the insulin bolus significantly increased stimulated Akt serine phosphorylation was increased signi- IRS-1 tyrosine phosphorylation compared with saline in both ficantly compared with the saline-treated group. However, OMA- and placebo-treated rats, but the OMA treatment Akt phosphorylation in soleus muscle after OMA treatment neither induced any changes in basal tyrosine phosphoryl- was comparable to that seen in placebo-treated rats. OMA ation of IRS-1 proteins nor did it augment insulin-stimulated treatment did not augment insulin-stimulated Akt serine IRS-1 tyrosine phosphorylation. phosphorylation. Akt phosphorylation at threonine was also The association of IRS-1 with the p85 regulatory subunit examined and yielded similar observations (data not shown). of PI3K was also examined and is shown in Fig. 1B. In parallel In addition, we also examined Akt phosphorylation in other with an increase seen in IRS-1 tyrosine phosphorylation muscle groups, such as tibialis anterior, qastrocnemius, and induced by insulin, there was an increase in IRS-1 association quadriceps. Consistently, OMA treatment did not affect serine and threonine phosphorylation of Akt under basal or with the p85 regulatory subunit of PI3K in insulin-treated insulin-stimulated conditions in these other muscle groups (nZ5–6, data not shown). Table 1 Body weight, fasting plasma glucose, insulin, free fatty acid ERK1/2 (p42/44) MAPK protein expression was com- (FFA), triglyceride (TG) concentrations, and systolic blood pressure parable between the OMA and placebo groups (Fig. 2B). (BP) in control and OMA-treated Zucker fatty rats Insulin induced significant phosphorylation of MAPK compared with saline treatment. Five days of OMA treatment 15 mg/kg per day alone neither altered the phosphorylation status of ERK1/2 Placebo-treated OMA-treated Zucker fatty rats Zucker fatty rats nor did it augment insulin-stimulated ERK1/2 activation. (nZ12) (nZ12)

Weight (g) 382G13$2 386G12$1 AMPK protein expression and phosphorylation $ G $ $ G $ Glucose (mmol/l) 5 31 0 12 5 03 0 10 We examined whether chronic treatment of OMA could have Insulin (pmol/l) 1163G103 1112G118 FFA (mmol/l) 0$27G0$02 0$25G0$02 enhanced glucose uptake through AMPK phosphorylation in TG (mmol/l) 4$08G0$27 3$70G0$27 skeletal muscle of insulin-resistant rats. The results are shown Systolic BP (mmHg) 107G386G5* in Fig. 3. AMPK protein expression was not different between all groups examined. OMA treatment did not enhance *P!0$001 for differences between OMA- and placebo-treated rats. AMPK phosphorylation under basal or insulin-stimulated

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Figure 1 Effects of omapatrilat (OMA) on basal and insulin-stimulated (A) IRS-1 tyrosine phosphorylation and (B) IRS-1 association with p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) in soleus muscle of Zucker fatty rats. Skeletal muscles were obtained 2 min after insulin (50 U/kg) or saline bolus. After immunoprecipitation (I.P.) with an anti-IRS-1 antibody, proteins were separated by SDS-PAGE and identified by Western blotting (I.B.)with (A) anti-phosphotyrosine (pY) antibody or (B) anti-p85 antibody as described in Materials and Methods. Control blots indicate that IRS-1 protein was no different between treatment groups. IRS-1 phosphorylation was stimulated by insulin administration, but OMA treatment did not alter IRS-1 phosphorylation or its association with PI3K under basal or insulin-stimulated conditions. Each group was expressed as a percent of control. Representative bands are shown. Values are meansGS.E.M. for six rats per treatment group. conditions. To confirm the absence of AMPK signaling in the differences in total ACC protein and serine phosphorylation skeletal muscle, we examined ACC, which is a downstream- of ACC were not observed (Fig. 4). signaling molecule of AMPK. Activated AMPK has been shown to induce Ser97 phosphorylation of ACC, thereby Total GLUT4 protein expression inhibiting ACC enzymatic activity (Ha et al. 1994, Park et al. OMA treatment did not change total cell GLUT4 protein 2002). In accordance with the above AMPK inactivity, under basal or insulin-stimulated conditions (Fig. 5).

Figure 2 Effect of OMA treatment on (A) Akt/protein kinase B (PKB) and (B) ERK1/2 MAPK phosphorylation. Zucker fatty rats were treated with either placebo or OMA for 5 days and soleus muscles were extracted 5 min after an i.v. bolus of insulin (50 U/kg) or saline. Treatment of OMA for 5 days did not affect basal or insulin- stimulated Akt/PKB or ERK1/2 MAPK phosphorylation. Each group was expressed as a percent of control. Representative bands are shown. Values are meansGS.E.M. for six soleus muscle samples per group. www.endocrinology-journals.org Journal of Endocrinology (2006) 190, 441–450

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Figure 3 Effect of 5 days OMA treatment on AMPK protein content and phosphorylation. Soleus muscles were taken 5 min after administration of saline or insulin bolus in placebo or OMA-treated Figure 5 Effect of OMA treatment on total cellular GLUT4 protein insulin-resistant animals. There were no changes in total AMPK expression. Zucker fatty animals were treated with placebo or OMA protein levels between any of the treatment groups, as illustrated in for 5 days and soleus muscles were taken 5 min after saline or the bar graph. In addition, treatment with OMA did not affect AMPK insulin bolus on the last day. There were no differences in total phosphorylation. Representative bands for AMPK phosphorylation GLUT4 protein levels between any of the treatment groups shown. are shown. Values are meansGS.E.M. for six rats per treatment group. Representative bands for GLUT4 expression are shown. Values are meansGS.E.M. for six animals per treatment group.

Discussion potential mechanisms underlying the improvement in insulin sensitivity by OMA. We were unable to demonstrate an effect In our previous study (Wang et al. 2003), we demonstrated that of OMA on insulin signaling in the skeletal muscle of Zucker OMA treatment, at the same dose as that used in the present fatty rats. Furthermore, OMA did not alter the activity of study (15 mg/kg per day for 5 days), induced profound whole- AMPK or GLUT4 protein expression in muscle tissue derived body insulin sensitization as measured by the euglycemic from treated animals. Our studies provide clear evidence that hyperinsulinemic clamp method, and enhanced insulin- the enhanced muscle glucose uptake by OMA cannot be stimulated muscle 2-deoxyglucose uptake in insulin-resistant attributed to the improvement in known insulin-signaling obese Zucker rats. In the present follow-up study,we examined pathways or stimulation of AMPK activity. Numerous studies in animals (Uehara et al. 1994, Henriksen & Jacob 1995, Jacob et al. 1996, Wa ng et al. 2003) and humans (Pollare et al. 1989, Lithell et al. 1990, To r l o n e et al. 1993, Falkner et al. 1995, Vuorinen-Markkola & Yki-Jarvinen 1995, Valensi et al. 1996, Tillmann et al. 1997, Fogari et al. 1998, Lender et al. 1999) have reported small improvements in insulin sensitivity by treatment with ACEI and their beneficial effect can be abolished completely by pre-treatment with HOE-140 (B2 kinin receptor blocker) in animal studies (To m i ya m a et al. 1994). Studies using , an ACEI, increased insulin sensitivity in normal rats but not in kininogen-deficient rats in which kinin formation is severely reduced (Damas et al. 1999). This observation strongly implicates the role of kinins in tissue glucose uptake. The contribution of bradykinin to glucose metabolism has been appreciated for some time, with a number of studies showing that bradykinin can increase glucose uptake in skeletal muscle (Leighton et al. 1996, Henriksen et al. 1998). In skeletal muscles, 64% of the kininase activity is attributed to Figure 4 Effect of 5 days OMA treatment on total soleus muscle ACE and the remaining 36% to NEP (Arbin et al. 2001). Since acetyl CoA carboxylase (ACC) and Ser97 phosphorylation of ACC. VPIs inhibit both ACE and NEP,they increase tissue bradykinin There were no changes in total ACC or Ser97 phosphorylation of levels to a greater extent than selective ACE inhibition alone ACC with OMA treatment, as illustrated in the bar graph. Representative bands for total ACC and serine phosphorylation are (Floras 2002), providing an explanation for the greater insulin- shown. Values are meansGS.E.M. for six rats per treatment group. sensitizing effect of VPIs than ACEI (Wan g et al. 2003).

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The mechanism of enhanced muscle glucose uptake by 1998), we postulated that activation of this pathway may bradykinin remains controversial, with some studies showing provide an alternative mechanism to explain VPI-induced that bradykinin mediates its insulin-sensitizing effects via the insulin-sensitizing effects in insulin-resistant skeletal muscle. insulin receptor-signaling pathway (Henriksen et al. 1996, OMA, however, had no effect on AMPKa protein content or Carvalho et al. 1997, Nawano et al. 1999), and other studies its phosphorylation using antibodies that recognize both demonstrating that the effects are independent of the insulin a-isoforms. Therefore, it is unlikely that the increase in receptor (Shiuchi et al. 2002). We found that under basal or glucose uptake induced by OMA treatment in Zucker fatty insulin-stimulated conditions, OMA treatment has no effect rats is dependent on changes in AMPK activity. on Akt serine and threonine phosphorylation (data not We also examined one component of the MAPK (Au et al. shown). Our finding is in agreement with the results of a 2003) signaling. Recent evidence has suggested that MAPK- study that demonstrated that treatment of type-2 diabetic signaling cascades contribute to the regulation of insulin action KK-Ay mice with , an ACEI, significantly and protein expression in skeletal muscle (Goodyear et al. 1996, enhanced 2-deoxyglucose uptake in skeletal muscle in a Aronson et al. 1997a,b). Furthermore, muscle contractions bradykinin–nitric oxide-dependent fashion without altera- have been demonstrated to activate ERK1/2, isoforms of tions in IRS-1 phosphorylation (Shiuchi et al. 2002). In MAPK, and exercise training by obese Zucker rats leads to the contrast, Carvalho et al. (1997) reported that injection of upregulation of ERK2 protein expression (Osman et al. 2001). captopril or bradykinin enhanced insulin-induced increase in This raises an interesting possibility that enhanced signal insulin receptor phosphorylation, IRS-1 phosphorylation, transduction via the MAPK pathway may play an important and PI3K association with IRS-1 in aged rats. Similarly, role in the increased expression of glucose catabolic enzymes, Nawano et al. (1999) also reported that the administration of GLUT4, and other insulin signaling proteins. We were unable , another ACEI, also improved insulin sensitivity to demonstrate an effect of OMA treatment on basal or insulin- which was associated with increased insulin-induced IRS-1 stimulated ERK1/2 phosphorylation. tyrosine phosphorylation and PI3K activity in female obese There was no difference in fasting blood glucose, insulin, Zucker rats (Nawano et al. 1999). It is possible that differences TGs, or FFAs between OMA-treated and control groups. in therapeutic agents and the animal models may account for These parameters are insensitive indicators of differences in the contrasting results. insulin sensitivity in the highly insulin-resistant Zucker fatty We found no effect of OMA on total muscle tissue GLUT4 rat. In our previous study (Wang et al. 2003), there were also expression. Technically, it is extremely difficult and requires no significant differences in these parameters, whereas OMA large amounts of muscle tissue to examine GLUT4 treatment was associated with marked improvement in insulin translocation from intracellular vesicles to the plasma sensitivity as measured by the gold standard method, the membrane ex vivo in muscle. In the present study, therefore, euglycemic hyperinsulinemic clamp technique. we were only able to measure total cellular GLUT4 and found How do we reconcile our previous findings, confirmed by no increase after 5-days OMA treatment. Our findings do not others, of a significant increase in insulin-stimulated glucose preclude an increase in GLUT4 translocation from intra- uptake induced by VPIs, and the present ‘negative’ findings cellular organelles to the plasma membrane with OMA regarding some of the molecular mechanisms known to treatment. Indeed, Kishi et al. (1998) examined bradykinin mediate glucose uptake in myocytes? We used the same animal effects on GLUT4 translocation in vitro in L6 myoblasts stably model in the present and our previous study,as well as the same expressing c-myc epitope-tagged GLUT4 (GLUT4myc) and dose and duration of OMA treatment. The significantly lower found that bradykinin directly triggers GLUT4 translocation, BP in the OMA- vs placebo-treated rats in the present study, thereby increasing the rate of glucose uptake through an similar to the BP changes noted in our previous study,indicates insulin-independent pathway. It remains an intriguing that the drug was indeed ingested and was biologically active. possibility that OMA can increase glucose uptake through Although we cannot definitively exclude an effect of BP on direct stimulation of GLUT4 translocation. Future studies insulin signaling, indirect evidence suggests that it does not using very different experimental model systems to the account for the insulin-sensitizing properties of OMA. In our present study will be required to address this possibility. previous publication (Wang et al. 2003), BP was precisely In addition to insulin, skeletal muscle glucose transport is matched between different pharmacological treatment experi- also stimulated by exercise. AMPK is a known regulator of mental groups and yet insulin sensitization was markedly glucose metabolism in skeletal muscle in response to exercise increased only by OMA. Changes in BP,therefore, cannot fully (Winder & Hardie 1996, Hutber et al. 1997, Vavvas et al. explain the insulin-sensitizing effect of OMA. In addition, in 1997). Activation of AMPK in muscle leads to an increase the present study, we detected no differences in the cellular in glucose transport, accompanied by increased translocation insulin-signaling cascade in muscle tissue, despite a significant of GLUT4 to the plasma membrane (Merrill et al. 1997, difference in BP between OMA- and placebo-treated animals. Hayashi et al. 1998, Kurth-Kraczek et al. 1999). Since The dose of insulin used in the present study was in the mid- AMPK appears to increase glucose metabolism through an range of doses that have been used by others and indeed insulin insulin-independent mechanism and is possibly nitric administration effectively stimulated phosphorylation of IRS- oxide-dependent (Lund et al. 1995, Hayashi et al. 1997, 1, Akt/PKB and association of the p85 subunit of PI3K with www.endocrinology-journals.org Journal of Endocrinology (2006) 190, 441–450

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IRS-1 in the present study. In order to determine whether the vascular function (Laakso et al. 1992, Steinberg et al. 1996, 12-h time-period following administration of the last dose of Baron 2002) and insulin itself is vasodilatory, thereby OMA accounted for the absence of effect of OMA on insulin enhancing its own and/or glucose access to muscle tissues signaling in the present study, additional experiments were (Steinberg et al. 1996, Baron et al. 2000). Clark and colleagues performed in three OMA-treated and three placebo-treated have championed the concept that increased nutrient capillary rats in which an additional dose of OMA or saline respectively recruitment in muscle beds plays an important role in were administered 2 h prior to the insulin bolus. There was no mediating glucose delivery to myocytes, and thus it is a critical significant difference in the activation of IRS-1 or Akt/PKB in determinant of whole-body insulin sensitivity (Vincent et al. these animals, suggesting that the time-period following the 2002, 2004, Zhang et al. 2004). Recent studies have last dose of OMA was not the reason for the negative results of demonstrated that insulin’s ability to enhance capillary this study. In the present study, we used only the soleus muscle recruitment is nitric oxide dependent (Shankar et al. 2000, to examine insulin signaling, since we found that soleus had the Vincent et al. 2003). Furthermore, Wallis et al. (2002) have greatest enhancement of glucose uptake. In addition, we found shown that the insulin-resistant obese Zucker rat has markedly no effect of OMA on insulin-stimulated Akt/PKB in three impaired insulin-stimulated capillary recruitment. Our other skeletal muscle groups: tibilias anterior, gastrocnemius, previous observation that the nitric oxide synthase inhibitor and quadriceps (data not shown). We (unpublished obser- significantly blocked the insulin-sensitizing effects of OMA is vations) and others (Kim et al. 2000, Shiuchi et al. 2002)have consistent with this concept (Wang et al. 2003). It remains an found that peak stimulation of IRS-1 phosphorylation and p85 interesting possibility that capillary recruitment can play a vital association with IRS-1 occurs 2 min after the insulin bolus, role in the enhancement of insulin-stimulated glucose whereas downstream Akt peak phosphorylation occurs later, at transport, brought about by administration of VPIs. approximately 5 min. Therefore, the negative results of the In conclusion, enhancement of muscle and whole-body present study cannot be explained on the basis of down- glucose uptake with the VPI, OMA, cannot be attributed to regulation of insulin signaling due to delayed taking of muscle enhanced insulin signaling, activation of AMPK, or increase in biopsies. The only cavegt to consider that may account for a total GLUT4 protein content. We speculate that OMA lack of difference is that OMA and ACE inhibitors may alter the timing or extent of insulin induced feedback inhibition of mediates its effect on glucose uptake by muscle capillary bed signaling, which occurs following the peak of IRS phosphory- recruitment, which increases glucose delivery to myocytes. lation and Akt activation. Further work delineating a larger Future investigations should focus on the link between the time course will answer this question. hemodynamic changes induced by vasoactive agents, such as It remains for future studies to investigate alternative VPIs, and their ability to enhance glucose uptake. Although pathways such as atypical protein kinase C z (aPKCz)(Farese these agents are presently being developed primarily for the 2002). Studies suggest that the aPKCz is a downstream treatment of hypertension and heart failure, given the mediator of PI3K and that its activation is required for insulin association between insulin-resistant states and cardiovascular stimulation of GLUT4 translocation and ultimately glucose disease, this pleotropic effect of VPIs could make them uptake (Bandyopadhyay et al. 1997, Standaert et al. 1997, extremelyattractive therapeutic tools for the treatment of those Kotani et al. 1998). aPKCz is an attractive candidate as several with the metabolic syndrome. in vitro studies using overexpression of dominant-negative and wild-type kinase can abrogate and enhance insulin-stimulated glucose uptake respectively (Bandyopadhyay et al. 1997, 2000, Acknowledgements Etgen et al. 1999). Interestingly, aPKCz has been implicated in insulin resistance and this is supported by studies in vivo This work was supported by an operating grant from the demonstrating impaired insulin-stimulated aPKCz activity in Heart and Stroke Foundation of Ontario (G E L) and from skeletal muscle tissue of animal models of insulin resistance and the Canadian Institutes of Health Research (I G F). G F L is diabetes, such as Goto–Kakizaki, high-fat fed rats, and obese the recipient of a Career Investigator Award from the Heart diabetic monkeys (Kanoh et al. 2001, 2003, Standaert et al. and Stroke Foundation of Canada and holds a Canada 2002). Similarly, defective aPKCz activity in skeletal muscle of Research Chair in Diabetes. V W is the recipient of a obese humans with impaired glucose tolerance and in type-2 studentship from the Banting and Best Diabetes Centre, diabetes humans has also been reported (Vollenweider et al. University of Toronto. We would like to acknowledge the 2002, Kim et al. 2003). Since our data do not exclude the technical assistance of Laura Stavar, Alan Prendergast, Dr possibility that the activity or membrane localization of aPKCz Zhiqiang Jia, and Mikim Patel, University of Toronto. We could also be altered by OMA treatment, it will be interesting would also like to thank Dr Amira Klip, University of to investigate this aspect of insulin signaling. Toronto, for her kind advice and GLUT4 antibodies. Last, but Another intriguing possibility, muscle capillary bed recruit- not least, we would like to thank Dr P Brubaker, University of ment, could explain an increase in muscle glucose uptake by Toronto, for her suggestions and input. The authors declare OMA in vivo that is not mediated by enhanced insulin that there is no conflict of interest that would prejudice the signaling. Insulin resistance is closely linked to alterations in impartiality of this scientific work.

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References Guthrie R & Reeves RA 2002 Omapatrilat provides long-term control of hypertension: a randomized trial of treatment withdrawal. Journal of Clinical Anai M, Funaki M, Ogihara T, Terasaki J, Inukai K, Katagiri H, Fukushima Y, Hypertension 4 169–172. Yazaki Y, Kikuchi M, Oka Y et al. 1998 Altered expression levels and Ha J, Daniel S, Broyles SS & Kim KH 1994 Critical phosphorylation sites for impaired steps in the pathway to phosphatidylinositol 3-kinase activation acetyl-CoA carboxylase activity. Journal of Biological Chemistry 269 via insulin receptor substrates 1 and 2 in Zucker fatty rats. Diabetes 47 22162–22168. 13–23. Hayashi T, Wojtaszewski JF & Goodyear LJ 1997 Exercise regulation of Arbin V, Claperon N, Fournie-Zaluski MC, Roques BP & Peyroux J 2001 glucose transport in skeletal muscle. American Journal of Physiology 273 Acute effect of the dual angiotensin-converting enzyme and neutral E1039–E1051. Hayashi T, Hirshman MF, Kurth EJ, Winder WW & Goodyear LJ 1998 endopeptidase 24-11 inhibitor mixanpril on insulin sensitivity in obese 0 Zucker rat. British Journal of Pharmacology 133 495–502. Evidence for 5 AMP-activated protein kinase mediation of the effect of Arbin V, Claperon N, Fournie-Zaluski MC, Roques BP & Peyroux J 2003 muscle contraction on glucose transport. Diabetes 47 1369–1373. Effects of dual angiotensin-converting enzyme and neutral endopeptidase Henriksen EJ & Jacob S 1995 Effects of captopril on glucose transport activity 24-11 chronic inhibition by mixanpril on insulin sensitivity in lean and in skeletal muscle of obese Zucker rats. Metabolism 44 267–272. obese Zucker rats. Journal of Cardiovascular Pharmacology 41 254–264. Henriksen EJ, Jacob S, Augustin HJ & Dietze GJ 1996 Glucose transport Aronson D, Dufresne SD & Goodyear LJ 1997a Contractile activity stimulates activity in insulin-resistant rat muscle. Effects of angiotensin-converting the c-Jun NH2-terminal kinase pathway in rat skeletal muscle. Journal of enzyme inhibitors and bradykinin antagonism. Diabetes 45 S125–S128. Biological Chemistry 272 25636–25640. Henriksen EJ, Jacob S, Fogt DL & Dietze GJ 1998 Effect of chronic Aronson D, Violan MA, Dufresne SD, Zangen D, Fielding RA & Goodyear bradykinin administration on insulin action in an animal model of insulin LJ 1997b Exercise stimulates the mitogen-activated protein kinase resistance. American Journal of Physiology 275 R40–R45. pathway in human skeletal muscle. Journal of Clinical Investigation 99 Hotamisligil GS, Budavari A, Murray D & Spiegelman BM 1994 Reduced 1251–1257. tyrosine kinase activity of the insulin receptor in obesity-diabetes. Central Au WS, Kung HF & Lin MC 2003 Regulation of microsomal triglyceride role of tumor necrosis factor-alpha. Journal of Clinical Investigation 94 transfer protein gene by insulin in HepG2 cells: roles of MAPKerk and 1543–1549. MAPKp38. Diabetes 52 1073–1080. Hutber CA, Hardie DG & Winder WW 1997 Electrical stimulation Bandyopadhyay G, Standaert ML, Galloway L, Moscat J & Farese RV 1997 inactivates muscle acetyl-CoA carboxylase and increases AMP-activated Evidence for involvement of protein kinase C (PKC)-zeta and protein kinase. American Journal of Physiology 272 E262–E266. noninvolvement of diacylglycerol-sensitive PKCs in insulin-stimulated Jacob S, Henriksen EJ, Fogt DL & Dietze GJ 1996 Effects of and glucose transport in L6 myotubes. Endocrinology 138 4721–4731. verapamil on glucose transport in insulin-resistant rat skeletal muscle. Bandyopadhyay G, Kanoh Y, Sajan MP, Standaert ML & Farese RV 2000 Metabolism 45 535–541. Effects of adenoviral gene transfer of wild-type, constitutively active, and Kanoh Y, Bandyopadhyay G, Sajan MP, Standaert ML & Farese RV 2001 kinase-defective protein kinase C-lambda on insulin-stimulated glucose Rosiglitazone, insulin treatment, and fasting correct defective activation of transport in L6 myotubes. Endocrinology 141 4120–4127. protein kinase C-zeta/lambda by insulin in vastus lateralis muscles and Baron AD 2002 Insulin resistance and vascular function. Journal of Diabetes and adipocytes of diabetic rats. Endocrinology 142 1595–1605. its Complications 16 92–102. Kanoh Y, Sajan MP, Bandyopadhyay G, Miura A, Standaert ML & Farese RV Baron AD, Tarshoby M, Hook G, Lazaridis EN, Cronin J, Johnson A & 2003 Defective activation of atypical protein kinase C zeta and lambda by Steinberg HO 2000 Interaction between insulin sensitivity and muscle insulin and phosphatidylinositol-3,4,5-(PO4)(3) in skeletal muscle of rats perfusion on glucose uptake in human skeletal muscle: evidence for following high-fat feeding and streptozotocin-induced diabetes. Endo- capillary recruitment. Diabetes 49 768–774. crinology 144 947–954. Carvalho CR, Thirone AC, Gontijo JA, Velloso LA & Saad MJ 1997 Effect of Kim YB, Peroni OD, Franke TF & Kahn BB 2000 Divergent regulation of captopril, , and bradykinin on early steps of insulin action. Diabetes Akt1 and Akt2 isoforms in insulin target tissues of obese Zucker rats. 46 1950–1957. Diabetes 49 847–856. Damas J, Bourdon V & Lefebvre PJ 1999 Insulin sensitivity, clearance and Kim YB, Kotani K, Ciaraldi TP, Henry RR & Kahn BB 2003 Insulin- release in kininogen-deficient rats. Experimental Physiology 84 549–557. stimulated protein kinase C lambda/zeta activity is reduced in skeletal Danser AH, Tom B, de Vries R & Saxena PR 2000 L-NAME-resistant muscle of humans with obesity and type 2 diabetes: reversal with weight bradykinin-induced relaxation in porcine coronary arteries is NO-depen- reduction. Diabetes 52 1935–1942. dent: effect of ACE inhibition. British Journal of Pharmacology 131 195–202. Kishi K, Muromoto N, Nakaya Y, Miyata I, Hagi A, Hayashi H & Ebina Y Etgen GJ, Valasek KM, Broderick CL & Miller AR 1999 In vivo adenoviral 1998 Bradykinin directly triggers GLUT4 translocation via an insulin- delivery of recombinant human protein kinase C-zeta stimulates glucose independent pathway. Diabetes 47 550–558. transport activity in rat skeletal muscle. Journal of Biological Chemistry 274 Kotani K, Ogawa W, Matsumoto M, Kitamura T, Sakaue H, Hino Y, Miyake 22139–22142. K, Sano W, Akimoto K, Ohno S et al. 1998 Requirement of atypical Falkner B, Canessa M & Anzalone D 1995 Effect of angiotensin converting protein kinase C lambda for insulin stimulation of glucose uptake but not enzyme inhibitor () on insulin sensitivity and sodium transport in for Akt activation in 3T3-L1 adipocytes. Molecular and Cell Biology 18 mild hypertension. American Journal of Hypertension 8 454–460. 6971–6982. Farese RV 2002 Function and dysfunction of aPKC isoforms for glucose Kurth-Kraczek EJ, Hirshman MF, Goodyear LJ & Winder WW 1999 50 transport in insulin-sensitive and insulin-resistant states. American Journal of AMP-activated protein kinase activation causes GLUT4 translocation in Physiology. Endocrinology and Metabolism 283 E1–E11. skeletal muscle. Diabetes 48 1667–1671. Floras JS 2002 Vasopeptidase inhibition: a novel approach to cardiovascular Laakso M, Edelman SV, Brechtel G & Baron AD 1992 Impaired insulin- therapy. Canadian Journal of Cardiology 18 177–182. mediated skeletal muscle blood flow in patients with NIDDM. Diabetes 41 Fogari R, Zoppi A, Corradi L, Lazzari P, Mugellini A & Lusardi P 1998 1076–1083. Comparative effects of lisinopril and losartan on insulin sensitivity in the Lapointe N & Rouleau JL 2002 Cardioprotective effects of vasopeptidase treatment of non diabetic hypertensive patients. British Journal of Clinical inhibitors. Canadian Journal of Cardiology 18 415–420. Pharmacology 46 467–471. Leighton B, Sanderson AL, Young ME, Radda GK, Boehm EA & Clark JF Goodyear LJ, Chang PY, Sherwood DJ, Dufresne SD & Moller DE 1996 1996 Effects of treatment of spontaneously hypertensive rats with the Effects of exercise and insulin on mitogen-activated protein kinase signaling angiotensin-converting enzyme inhibitor trandolapril and the calcium pathways in rat skeletal muscle. American Journal of Physiology 271 antagonist verapamil on the sensitivity of glucose metabolism to insulin in E403–E408. rat soleus muscle in vitro. Diabetes 45 S120–S124. www.endocrinology-journals.org Journal of Endocrinology (2006) 190, 441–450

Downloaded from Bioscientifica.com at 09/27/2021 08:15:09PM via free access 450 V WONG and others $ OMA and mechanism of insulin sensitization

Lender D, Arauz-Pacheco C, Breen L, Mora-Mora P, Ramirez LC & Torlone E, Britta M, Rambotti AM, Perriello G, Santeusanio F, Brunetti P & Raskin P 1999 A double blind comparison of the effects of amlodipine Bolli GB 1993 Improved insulin action and glycemic control after long- and on insulin sensitivity in hypertensive patients. American term angiotensin-converting enzyme inhibition in subjects with arterial Journal of Hypertension 12 298–303. hypertension and type II diabetes. Diabetes Care 16 1347–1355. Lithell HO, Pollare T & Berne C 1990 Insulin sensitivity in newly detected Uehara M, Kishikawa H, Isami S, Kisanuki K, Ohkubo Y, Miyamura N, hypertensive patients: influence of captopril and other antihypertensive Miyata T, Yano T & Shichiri M 1994 Effect on insulin sensitivity of agents on insulin sensitivity and related biological parameters. Journal of angiotensin converting enzyme inhibitors with or without a sulphydryl Cardiovascular Pharmacology 15 S46–S52. group: bradykinin may improve insulin resistance in dogs and humans. Lund S, Holman GD, Schmitz O & Pedersen O 1995 Contraction stimulates Diabetologia 37 300–307. translocation of glucose transporter GLUT4 in skeletal muscle through a Valensi P, Derobert E, Genthon R & Riou JP 1996 Effect of on mechanism distinct from that of insulin. PNAS 92 5817–5821. insulin sensitivity in obese patients. Time-course study of glucose infusion Merrill GF, Kurth EJ, Hardie DG & Winder WW 1997 AICA riboside rate during euglycaemic hyperinsulinaemic clamp. Diabetes Metabolism 22 increases AMP-activated protein kinase, fatty acid oxidation, and glucose 197–200. uptake in rat muscle. American Journal of Physiology 273 E1107–E1112. Vavvas D, Apazidis A, Saha AK, Gamble J, Patel A, Kemp BE, Witters LA & Nawano M, Anai M, Funaki M, Kobayashi H, Kanda A, Fukushima Y, Ruderman NB 1997 Contraction-induced changes in acetyl-CoA Inukai K, Ogihara T, Sakoda H, Onishi Y et al. 1999 Imidapril, an carboxylase and 50-AMP-activated kinase in skeletal muscle. Journal of angiotensin-converting enzyme inhibitor, improves insulin sensitivity by enhancing signal transduction via insulin receptor substrate proteins and Biological Chemistry 272 13255–13261. improving vascular resistance in the Zucker fatty rat. Metabolism 48 Vincent MA, Dawson D, Clark AD, Lindner JR, Rattigan S, Clark MG & 1248–1255. Barrett EJ 2002 Skeletal muscle microvascular recruitment by physiological Osman AA, Hancock J, Hunt DG, Ivy JL & Mandarino LJ 2001 Exercise hyperinsulinemia precedes increases in total blood flow. Diabetes 51 42–48. training increases ERK2 activity in skeletal muscle of obese Zucker rats. Vincent MA, Barrett EJ, Lindner JR, Clark MG & Rattigan S 2003 Inhibiting Journal of Applied Physiology 90 454–460. NOS blocks microvascular recruitment and blunts muscle glucose uptake in Park SH, Gammon SR, Knippers JD, Paulsen SR, Rubink DS & Winder response to insulin. American Journal of Physiology. Endocrinology and WW 2002 Phosphorylation-activity relationships of AMPK and acetyl- Metabolism 285 E123–E129. CoA carboxylase in muscle. Journal of Applied Physiology 92 2475–2482. Vincent MA, Clerk LH, Lindner JR, Klibanov AL, Clark MG, Rattigan S & Pollare T, Lithell H & Berne C 1989 A comparison of the effects of Barrett EJ 2004 Microvascular recruitment is an early insulin effect that hydrochlorothiazide and captopril on glucose and lipid metabolism in regulates skeletal muscle glucose uptake in vivo. Diabetes 53 1418–1423. patients with hypertension. New England Journal of Medicine 321 868–873. Vollenweider P,Menard B & Nicod P 2002 Insulin resistance, defective insulin Reaven GM 1988 Banting lecture 1988. Role of insulin resistance in human receptor substrate 2-associated phosphatidylinositol-30 kinase activation, disease. Diabetes 37 1595–1607. and impaired atypical protein kinase C (zeta/lambda) activation in Rouleau JL, Pfeffer MA, Stewart DJ, Isaac D, Sestier F, Kerut EK, Porter CB, myotubes from obese patients with impaired glucose tolerance. Diabetes 51 Proulx G, Qian C & Block AJ 2000 Comparison of vasopeptidase inhibitor, 1052–1059. omapatrilat, and lisinopril on exercise tolerance and morbidity in patients Vuorinen-Markkola H & Yki-Jarvinen H 1995 Antihypertensive therapy with with heart failure: IMPRESS randomised trial. Lancet 356 615–620. enalapril improves glucose storage and insulin sensitivity in hypertensive Shankar RR, Wu Y, Shen HQ, Zhu JS & Baron AD 2000 Mice with gene patients with non-insulin-dependent diabetes mellitus. Metabolism 44 disruption of both endothelial and neuronal nitric oxide synthase exhibit 85–89. insulin resistance. Diabetes 49 684–687. Wallis MG, Wheatley CM, Rattigan S, Barrett EJ, Clark AD & Clark MG Shiuchi T, Cui TX, Wu L, Nakagami H, Takeda-Matsubara Y, Iwai M & 2002 Insulin-mediated hemodynamic changes are impaired in muscle of Horiuchi M 2002 ACE inhibitor improves insulin resistance in diabetic Zucker obese rats. Diabetes 51 3492–3498. mouse via bradykinin and NO. Hypertension 40 329–334. Wang CH, Leung N, Lapointe N, Szeto L, Uffelman KD, Giacca A, Standaert ML, Galloway L, Karnam P,Bandyopadhyay G, Moscat J & Farese RV Rouleau JL & Lewis GF 2003 Vasopeptidase inhibitor omapatrilat 1997 Protein kinase C-zeta as a downstream effector of phosphatidylinositol 3-kinase during insulin stimulation in rat adipocytes. Potential role in glucose induces profound insulin sensitization and increases myocardial glucose transport. Journal of Biological Chemistry 272 30075–30082. uptake in Zucker fatty rats: Studies comparing a vasopeptidase inhibitor, Standaert ML, Ortmeyer HK, Sajan MP,Kanoh Y,Bandyopadhyay G, Hansen angiotensin-converting enzyme inhibitor, and angiotensin II type I BC & Farese RV 2002 Skeletal muscle insulin resistance in obesity- receptor blocker. Circulation 107 1923–1929. associated type 2 diabetes in monkeys is linked to a defect in insulin Winder WW & Hardie DG 1996 Inactivation of acetyl-CoA carboxylase and activation of protein kinase C-zeta/lambda/iota. Diabetes 51 2936–2943. activation of AMP-activated protein kinase in muscle during exercise. Steinberg HO, Chaker H, Leaming R, Johnson A, Brechtel G & Baron AD American Journal of Physiology 270 E299–E304. 1996 Obesity/insulin resistance is associated with endothelial dysfunction. Zhang L, Vincent MA, Richards SM, Clerk LH, Rattigan S, Clark MG & Implications for the syndrome of insulin resistance. Journal of Clinical Barrett EJ 2004 Insulin sensitivity of muscle capillary recruitment in vivo. Investigation 97 2601–2610. Diabetes 53 447–453. Tillmann HC, Walker RJ, Lewis-Barned NJ, Edwards EA & Robertson MC 1997 A long-term comparison between enalapril and captopril on insulin sensitivity in normotensive non-insulin dependent diabetic volunteers. Journal of Clinical Pharmacy and Therapeutics 22 273–278. Received in final form 7 April 2006 Tomiyama H, Kushiro T,Abeta H, Ishii T,Takahashi A, Furukawa L, Asagami T, Accepted 12 April 2006 Hino T, Saito F & Otsuka Y 1994 Kinins contribute to the improvement of insulin sensitivity during treatment with angiotensin converting enzyme Made available online as an Accepted Preprint inhibitor. Hypertension 23 450–455. 27 April 2006

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