Activation of μ-opioid receptors improves insulin sensitivity in obese Zucker rats

Life Sciences 80 (2007) 1508 – 1516 www.elsevier.com/locate/lifescie

Activation of μ-opioid receptors improves insulin sensitivity in obese Zucker rats Thing-Fong Tzeng a , Chia-Ying Lo b , Juei-Tang Cheng b , I-Min Liu c,⁎ a

b

Department of Internal Medicine, Pao Chien Hospital, Ping Tung City, Taiwan, ROC Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan City, Taiwan, ROC c Department of Pharmacy, Tajen University, Yanpu Shiang, Ping Tung Shien, Taiwan 90701, ROC Received 22 July 2006; accepted 12 January 2007

Abstract In the current study we investigated the effect of μ-opioid receptor activation on insulin sensitivity. In obese Zucker rats, an intravenous injection of loperamide (18 μg/kg, three times daily for 3 days) decreased plasma glucose levels and the glucose–insulin index. Both effects of loperamide were subsequently inhibited by the administration of 10 μg/kg of naloxone or 10 μg/kg of naloxonazine, doses sufficient to block μopioid receptors. Other metabolic defects characteristic of obese Zucker rats, such as defects in insulin signaling, the decreased expression of insulin receptor substrate (IRS)-1, the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3 kinase), and the glucose transporter subtype 4 (GLUT 4), and the reduction of phosphorylation in IRS-1 or Akt serine, were also studied. These defects were all reversed by loperamide treatment in a dose which overcame μ-opioid receptor blockade. Moreover, loss of tolbutamide-induced plasma glucose lowering action (10 mg/ kg) in wild-type mice given a fructose-rich diet was markedly delayed by repeated treatment with loperamide; however, this delay induced by loperamide did not occur in μ-opioid receptor knockout mice. These results indicate an important role of peripheral μ-opioid receptors in the loperamide-induced improvement of insulin sensitivity. Our results suggest that activation of peripheral μ-opioid receptors can ameliorate insulin resistance in animals, and provide a new target for therapy of insulin resistance. © 2007 Elsevier Inc. All rights reserved. Keywords: Insulin resistance; Loperamide; μ-Opioid receptors; Obese Zucker rats

Introduction Pain control is the best-known role for opioid use (Yaksh, 1997). In addition to modulation of immune system function (Smith, 2003), opioids also participate in the regulation of endocrine processes, including glucose metabolism. We have documented that the injection of β-endorphin into streptozotocin-induced diabetic rats (STZ-diabetic rats), a Type 1 diabeteslike rat model, increases glucose utilization in peripheral tissues, resulting in a larger reduction in plasma glucose (Cheng et al., 2002). Similarly, chemical agents such as loperamide and tramadol, possess the ability to decrease plasma glucose via activation of μ-opioid receptors in diabetic rats lacking insulin (Liu et al., 1999; Cheng et al., 2001). It seems that activation of ⁎ Corresponding author. Tel.: +886 7 346 0961; fax: +886 8 762 5308. E-mail address: [email protected] (I.-M. Liu). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.01.016

μ-opioid receptors plays an important role in the plasma glucoselowering response in the absence of insulin. The activation of μopioid receptors to improve insulin resistance thus seems plausible. It has been demonstrated that β-endorphin has the ability to reverse the impaired insulin-stimulated glucose disposal in rats rendered insulin resistant by a fructose-rich chow diet (Su et al., 2004). In addition, the induction of insulin resistance by a high intake of fructose is more rapid in μ-opioid receptor knockout mice than in wild-type mice (Cheng et al., 2003). Furthermore, it has been documented that activation of the μ-opioid receptors by loperamide reverses impaired insulinstimulated glucose uptake, with a marked recovery of the insulin signals damaged by cytokines in C2C12 cells (Tzeng et al., 2005; Ko et al., 2006). These results led to the idea that activation of μopioid receptors on the insulin-targeted organs may have a beneficial effect, reducing insulin resistance. However, the induction of insulin resistance by dietary carbohydrates or

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cytokines may be variable and cannot be entirely mimicked as it occurs in actual patients (Bessesen, 2001; Tzeng et al., 2005; Ko et al., 2006). Therefore, the role of μ-opioid receptors in the regulation of plasma glucose homeostasis under conditions of insulin resistance requires further clarification. Among the different animal models for Type 2 diabetes, genetically obese Zucker rats exhibit a range of metabolic aberrations, including hyperlipidemia, hyperglycemia, adipocyte hypertrophy, and hyperinsulinemia (Kasiske et al., 1992). These symptoms are similar to those observed in humans with Type 2 diabetes, making these rats a very useful animal model in which to study the disease. Although it has been documented that enhancement of insulin sensitivity via exercise training may be mediated by β-endorphin in genetically obese Zucker rats (Su et al., 2005), the direct role of μ-opioid receptors in the improvement of insulin resistance has not been thoroughly investigated. Due to the short half-life of opioid peptides, injection of exogenous β-endorphin may produce only a transient stimulation on μ-opioid receptors. Loperamide is widely used as a gastrointestinal anti-motility agent, exhibiting a higher affinity and selectivity for the μ-opioid receptors than δor κ-subtypes (Wuster and Herz, 1978; Nozaki-Taguchi and Yaksh, 1999). In fact, loperamide does not cross the blood–brain barrier and lacks the side effects of other centrally-acting opiates (DeHaven-Hudkins et al., 1999). In the present study, we therefore utilized obese Zucker rats and μ-opioid receptor knockout mice to determine whether the direct activation of peripheral μ-opioid receptors by loperamide could improve insulin action on glucose metabolism in the insulin resistant state. Materials and methods Materials Standard and fructose-rich (60% fructose) rat chows were obtained from Purina Mills, LLC (St. Louis, MO, USA). Loperamide (Imodium®) was kindly supplied from Yu-Shen Pharmaceutics (Taichung, Taiwan). Naloxone and naloxonazine were purchased from Research Biochemical, Inc. (Natick, MA, USA). Tolbutamide and protein A-Sepharose beads were obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Plasma glucose concentrations were measured by the glucose oxidase method using a commercial kit (Cat. #COD12503) from BioSystem (Costa Brava, Barcelona, Spain). Plasma insulin was determined using a rat insulin enzyme-linked immunosorbent assay (ELISA) kit (Cat. #EZRMI-13K) obtained from LINCO Research, Inc. (St. Charles, MO, USA). The BioRad protein assay kit (Bio-Rad Laboratories, CA, USA) was used for protein determination. Anti-insulin receptor (IR) β-subunit antibodies (Cat. #MS-634 for immunoprecipitation; Cat. #MS-636 for Western blotting), anti-insulin receptor substrate-1 (IRS-1) antibody (Cat. #MS-630), anti-PI3-kinase p85 subunit antibody (Cat. #RB1625), and anti-phosphotyrosine antibody (Cat. #MS445) were obtained from NeoMarkers (Fremont, CA, USA). Anti-phosphoserine (Ser473) Akt antibody was supplied by Cell Signaling Technology, Inc. (Cat. #9271; Beverly, MA, USA).

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Anti-GLUT 4 antibody was purchased from R&D Systems, Inc. (Cat. #BAM1262; Minneapolis, MN, USA). ECL™ Western Blotting Systems were obtained from Amersham Corp. (Braunschweig, Germany). All other reagents were from standard sources. Animals Lean (Fa/fa or Fa/Fa) and obese (fa/fa) strains of Zucker rats were obtained from Dr. Kajuro Komeda (Animal Research Center, Tokyo Medical College, Tokyo, Japan). Wild-type (BDF1 mice) and μ-opioid receptor knockout mice were obtained from Professor H.H. Loh (Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN, USA; Cheng et al., 2003). All animals were bred in the animal center of the National Cheng Kung University. They were maintained in a temperature-controlled room (25 ± 1 °C) under a 12 h light/12 h dark cycle, with lights on at 0600 daily. Obese Zucker male rats become insulin resistant within weeks of birth and suffer a subsequent decline in β-cell mass and function, such that diabetes ensues between 9 and 11 weeks of age. Obese Zucker female rats also become insulin resistant, but they remain normoglycemic unless placed on a specialized high-fat diet (Corsetti et al., 2000). Thus, only male rats and mice were used in the current study. Both lean and obese Zucker male rats (16 weeks old) were fed with standard chow during the experimental period; male mice were 8 weeks of age and maintained on standard chow for 2 weeks. One-half of the wildtype mice and one-half of the μ-opioid receptor knockout mice were then randomly assigned to receive fructose-rich chow for an additional 4 weeks to induce insulin resistance (Su et al., 2004). The remaining mice received standard chow during this same 4-week period. Food and water were available ad libitum. All animal procedures were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, as well as the guidelines of the Animal Welfare Act. Treatment of obese Zucker rats with loperamide Obese Zucker rats were used to assess whether loperamide could improve insulin action on glucose metabolism in an insulin resistant state. Thus, Zucker rats were given intravenous (iv) injections of loperamide, or loperamide plus a μ-opioid receptor antagonist (naloxone or naloxonazine) in the tail. The antagonist was injected 30 min before the injection of loperamide. Control rats received iv injections of an equivalent volume of the vehicle solution (i.e., saline). To measure the effect of loperamide on plasma glucose concentrations, blood samples were collected from the tail vein 30 min after the final loperamide injection while the rats were maintained under sodium pentobarbital (30 mg/kg, intraperitoneal [i.p.]) anesthesia. The concentration of plasma glucose was estimated with an analyzer using a commercial kit (Quik-Lab, Ames, Miles Inc., Elkhart, IN, USA). A commercially available ELISA kit was used to quantify the plasma levels of insulin; the obtained values were expressed as pmol of insulin-like immunoreactivity

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(IRI) per liter of plasma. The test compounds used in the present study did not affect the binding of peptide with antibodies. All samples were analyzed in triplicate. Measurement of the glucose–insulin index After 3 days of loperamide treatment, an iv glucose tolerance test (IVGTT) was conducted for the assessment of insulin sensitivity (Godsland et al., 1994). Rats were food-restricted (4 g of chow given at 1800, which was immediately consumed) and given only water to drink the evening prior to performing the IVGTT. On the morning of the IVGTT, a blood sample (0.1 ml) was drawn from the tail vein of these rats for determination of baseline (12 h overnight fasting) plasma glucose and insulin concentrations. Next, each animal was injected iv with glucose (0.5 g/kg) and blood samples were drawn from the tail vein at 5, 10, 20, 30, 60, 90, and 120 min following glucose injection. Immediately after the completion of the IVGTT, all animals received 2 ml of sterile saline subcutaneously to compensate for plasma loss. Blood samples were thoroughly mixed with 10 IU heparin and centrifuged at 13,000×g to separate the plasma. Plasma concentrations of glucose and insulin were measured by the method described above. The glucose–insulin index was calculated as the product of the glucose and insulin areas under the curve (AUC), as described previously (Liu et al., 2005). Muscle processing Following the IVGTT, rats were sacrificed and the soleus muscle was immediately extirpated, washed with cold phosphate buffer, and cut into 200–300 mg portions, which were then stored separately at − 70 °C for subsequent immunoprecipitation and immunoblot analyses. Soleus muscle samples were weighed while still frozen and homogenized in ice-cold lysis buffer (1:10 wt/vol) containing 50 mmol/l HEPES (pH 7.6), 150 mmol/ l NaCl, 20 mmol/l sodium pyrophosphate, 20 mmol/l β-glycerophosphate, 10 mmol/l sodium fluoride, 2 mmol/l sodium orthovanadate (Na3VO4), 2 mmol/l EDTA (pH 8.0), 1% Nonidet P-40, 10% glycerol, 1 mmol/l phenylmethylsulfonyl fluoride, 1 mmol/l MgCl2, 1 mmol/l CaCl2, 10 μg/ml leupeptin, and 10 μg/ml aprotinin, as described previously (Christ et al., 2002). A Polytron homogenizer set to the maximum speed for 30 s was used for homogenization. Homogenates were incubated on ice for 20 min and then centrifuged at 15,000×g for 20 min at 4 °C. Muscle debris was removed, and the protein concentration in the crude extracts was estimated using the BioRad protein assay kit. The supernatant was stored at −80 °C until used. Immunoprecipitation and immunoblotting For immunoprecipitation, a 1-mg sample of total protein was incubated overnight at 4 °C with anti-IR β-subunit antibody or anti-IRS-1 antibody, followed by the addition of Protein ASepharose beads for 1 h. The bead–Protein A–antibody–antigen complexes were precipitated by brief centrifugation. The pellets were washed three times in ice-cold buffer (0.5% Triton X-100, 100 mmol/l Tris, pH 7.4, 10 mmol/l EDTA, and 2 mmol/l sodium

vanadate), resuspended in Laemmli sample buffer, and boiled for 5 min. The Sepharose beads were precipitated by brief centrifugation and the supernatant prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 10% acrylamide gel) using a Bio-Rad Mini-Protein II system (55 V and 130 V during the stacking and separation phases, respectively). Protein was transferred to a polyvinylidene difluoride (PVDF) membrane using the Bio-Rad Trans-Blot system (2 h at 20 V in 25 mmol/l Tris, 192 mmol/l glycine, and 20% MeOH). Following transfer, the membrane was probed with anti-IR β-subunit antibody, anti-IRS-1 antibody, or antiphosphotyrosine antibody, according to the manufacturer's instructions. For detection of the p85 regulatory subunit of PI3-kinase, Akt serine (Ser473) phosphorylation, and GLUT 4 content, equal amounts (50 μg) of protein were prepared from muscle homogenates, subjected to SDS-PAGE, transferred to a PVDF membrane as described above, and blotted with anti-PI3-kinase p85 subunit antibody, anti-phosphoserine (Ser473) Akt antibody, or anti-GLUT4 antibody, according to the manufacturer's instructions. After three, 5-min washes in TBST (20 mmol/ l Tris–HCl [pH 7.5], 150 mmol/l NaCl, and 0.05% Tween 20), membranes were incubated with the appropriate peroxidaseconjugated secondary antibodies. The membranes were then washed three times in TBST and visualized on X-ray film using the enhanced chemiluminescence detection system. Densities of the obtained immunoblots were quantified using a laser densitometer. The mean value for samples from vehicle-treated lean rats on each immunoblot, expressed in densitometry units, was adjusted to a value of 1.0. All experimental sample values were then expressed relative to this adjusted mean value. Insulin resistance induction in μ-opioid receptor knockout mice fed fructose-rich chow Wild-type or μ-opioid receptor knockout mice received iv injections of loperamide, at a dose of 18 μg/kg every 8 h, via the tail during the fructose-rich chow feeding period. Formation of insulin resistance in either wild-type mice or μ-opioid receptor knockout mice was characterized by a loss of the tolbutamideinduced plasma glucose-lowering effect (Chang et al., 1999). Briefly, mice received an i.p. injection of 10 mg/kg tolbutamide on the indicated date as described previously (Chang et al., 1999). To assess for development of insulin resistance, plasma glucose levels were determined using blood samples collected from the femoral vein of mice maintained under sodium pentobarbital (30 mg/kg, i.p.) anesthesia 1 h after tolbutamide injection. Concentrations of plasma glucose were measured by the method described above. Statistical analysis Data were expressed as the mean ± S.E.M. for the number (n) of animals in each group as indicated in the tables and figures. Statistical differences among groups were determined by using two-way repeated-measure ANOVA. Dunnett range post-hoc comparisons were used to determine the source of significant

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Table 1 Changes in plasma glucose and the glucose–insulin index during intravenous glucose tolerance test (IVGTT) in Zucker rats receiving repeated i.v. treatment with loperamide three times daily for 3 days Zucker rats

Plasma glucose (mg/dl)

Lean +Vehicle +Loperamide (18 μg/kg) Obese +Vehicle +Loperamide (μg/kg) 6 12 18

Glucose–insulin index (units × 106)

91.8 ± 5.8c 88.6 ± 5.2c

64.5 ± 17.2d 60.3 ± 16.8d

148.4 ± 6.8a

774.6 ± 62.4b

135.6 ± 7.3a 128.5 ± 6.1a,c 112.8 ± 5.4a,c

701.5 ± 59.3b 552.4 ± 69.5b,d 452.6 ± 61.4b,d

Values (mean ± S.E.M.) were obtained from each group of 8 animals. The vehicle of saline used to dissolve loperamide was given at the same volume. a P b 0.05 and bP b 0.01 versus vehicle-treated lean Zucker rats, respectively. c P b 0.05 and dP b 0.01 versus vehicle-treated obese Zucker rats, respectively.

differences where appropriate. A P-value b 0.05 was considered statistically significant. Results General characteristics of obese Zucker rats repeatedly treated with loperamide In the preliminary experiments, loperamide was found to significantly improve the decrement of the insulin-stimulated glucose disposal rate in obese Zucker rats in a dose-dependent manner, from 6 to 18 μg/kg, after 3 days of treatment (Table 1), which was similar to the plasma glucose-lowering effect produced by loperamide in the same group of animals. The maximum reduction in plasma glucose (24.3 ± 3.5%) was achieved using 18 μg/kg of loperamide (Table 1). In fact, treatment of obese Zucker rats with loperamide at 18 μg/kg, Table 2 General characteristics of Zucker rats receiving repeated treatment with loperamide three times daily for 3 days Zucker rats

Lean +Vehicle +Loperamide (18 μg/kg) Obese +Vehicle +Loperamide (18 μg/kg) +Naloxone (10 μg/kg) +Naloxonazine (10 μg/kg)

Food intake (g/day)

Body weight Plasma glucose (g/rat) (mg/dl)

17.9 ± 5.1d 182.5 ± 9.1d 17.2 ± 6.3d 188.9 ± 10.2d

91.3 ± 5.9d 90.1 ± 6.4d

Plasma insulin (μU/ml) 17.9 ± 5.2d 18.2 ± 5.8d

45.2 ± 7.1b 285.6 ± 8.4b 148.6 ± 6.7b 224.9 ± 10.9b 42.8 ± 6.8b 278.3 ± 10.1b 112.2 ± 5.6b,d 161.6 ± 11.7b,d 43.7 ± 7.4b 290.7 ± 8.4b

142.7 ± 7.2b

44.8 ± 8.2b 289.8 ± 11.4b 144.9 ± 5.6b

220.9 ± 9.8b 219.8 ± 10.6b

Naloxone or naloxonazine was given by i.v. injection at 30 min before i.v. injection of loperamide. The vehicle of saline used to dissolve the test drugs was given at the same volume. Values (mean ± S.E.M.) were obtained from each group of 8 animals. bP b 0.01 versus vehicle-treated lean Zucker rats. dP b 0.01 versus vehicle-treated obese Zucker rats.

Fig. 1. (A) Glucose responses during 0.5 g/kg intravenous glucose tolerance test (IVGTT) in obese Zucker rats treated with loperamide (18 μg/kg, three times daily for 3 days; ▾), or with loperamide plus naloxone (10 μg/kg; ▿) or naloxonazine (10 μg/kg; ) (three times daily for 3 days). The lean (●) and obese (○) Zucker rats received the same volume of the vehicle (saline) used to dissolve the test drugs. (B) Insulin responses during IVGTT were also determined in these rats.

▪

three times daily for 1 day, had no significant influence on plasma glucose concentrations and the glucose disposal rate. Thus, in the present study, we used the most effective dose (i.e., 18 μg/kg) of loperamide to treat Zucker rats every 8 h, three times daily, for 3 days. There were no significant differences in plasma glucose concentrations in lean Zucker rats treated with loperamide or the vehicle (Table 1). The 3 day treatment regimen with loperamide did not influence feeding behavior and/or body weight of obese Zucker rats (Table 2). Nevertheless, fasting plasma glucose and insulin concentrations were significantly lower in loperamide-treated obese Zucker rats as compared to their vehicle-treated counterparts; these effects of loperamide were not observed in obese Zucker rats pretreated with naloxone (10 μg/kg) or naloxonzine (10 μg/kg; Table 2). Neither naloxone (10 μg/kg) nor naloxonazine (10 μg/kg) influenced the food intake or body weight in obese Zucker rats (Table 2). Effects of repeated loperamide treatment on the glucose disposal rate of obese Zucker rats During the IVGTT, obese Zucker rats exhibited significantly greater increases in plasma glucose and insulin concentrations

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compared to lean Zucker rats (Fig. 1A and B). Also, the AUC for plasma glucose and insulin concentrations during the IVGTT in obese Zucker rats was markedly higher than for the lean rats (Fig. 1A and B). Furthermore, the glucose–insulin index in obese Zucker rats following exogenous glucose loading was elevated 12-fold that of the lean group (Fig. 2C). Treatment with loperamide for 3 days reduced the plasma glucose concentration in obese Zucker rats during the IVGTT (Fig. 1A), and the total AUC for the glucose response was markedly lower than that for their vehicle-treated counterparts (Fig. 2A). In addition, treatment with loperamide for 3 days reduced plasma insulin concentrations in obese Zucker rats and the incremental area under the insulin curve during the IVGTT to 70% of the corresponding value obtained for the vehicletreated obese rats (Figs. 1B and 2B). Treatment with loperamide for 3 days also lowered the peak value of the glucose–insulin index in obese Zucker rats during the IVGTT to about 58% of the value for vehicle-treated obese rats (Fig. 2C). Strikingly, in obese rats pretreated with naloxone or naloxonazine, the loperamide-induced reduction of plasma levels in glucose and insulin, as well as the value of the glucose–insulin index during the IVGTT, was abolished (Fig. 1). Effect of repeated loperamide treatment on expression of insulin receptor-related signaling mediators in the soleus muscles of obese Zucker rats As shown in Fig. 3, the expression of IR protein in soleus muscle was similar between obese and lean Zucker rats. Similarly, the extent of tyrosine phosphorylation of the IR in soleus muscles was not significantly different between lean and obese rats. Additionally, both the protein expression and the degree of tyrosine phosphorylation of IRS-1 in the soleus muscle of obese Zucker rats after completion of the IVGTT were approximately 40% of the lean group. Furthermore, expression

Fig. 2. (A) The incremental areas under the curves (AUC) for glucose during 0.5 g/kg intravenous glucose tolerance test (IVGTT) in obese Zucker rats treated with loperamide (18 μg/kg, three times daily for 3 days), or with loperamide plus naloxone (10 μg/kg) or naloxonazine (10 μg/kg) (three times daily for 3 days). The lean and obese Zucker rats received the same volume of the vehicle (saline) used to dissolve the test drugs. (B) The insulin AUC during IVGTT was also determined in these rats. (C) The glucose-insulin index is calculated as the product of the glucose AUC and insulin AUC for each animal Values (mean ± S.E.M.) were obtained from each group of 6 animals. aP b 0.05 and bP b 0.01 versus vehicletreated lean Zucker rats, respectively. cP b 0.05 and dP b 0.01 versus vehicle-treated obese Zucker rats, respectively.

Fig. 3. Representative immunoblots of protein expression and insulin-stimulated phosphorylation in isolated soleus muscles of Zucker rats following repeated treatment with loperamide or vehicle. Lanes: vehicle-treated lean Zucker rats (lane 1), vehicle-treated obese Zucker rats (lane 2), obese Zucker rats treated with loperamide (18 μg/kg, three times daily for 3 days) (lane 3), obese Zucker rats treated with loperamide (18 μg/kg) plus naloxonazine (10 μg/kg) (three times daily for 3 days) (lane 4). The vehicle (saline) used to dissolve the test drugs was given at the same volume. Similar results were obtained in another 4 determinations. Quantification of the data is shown in Table 3.

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of the p85 regulatory subunit of PI3-kinase and the degree of Akt serine (Ser473) phosphorylation in response to insulin in the soleus muscle of obese Zucker rats at the end of the IVGTT was nearly 30% of values in the lean rats. Under non-stimulated conditions, GLUT 4 protein expression in soleus muscles of obese Zucker rats was shown to be approximately 40% of that in lean controls. Repeated loperamide treatment for 3 days did not affect the protein expression and the extent of tyrosine phosphorylation of IR in obese Zucker rats, while both the protein level and the degree of tyrosine phosphorylation of IRS-1 were increased in soleus muscle of obese Zucker rats as compared to their vehicle treated counterparts (Fig. 3). Moreover, treatment with loperamide for 3 days significantly increased the expression of the p85 regulatory subunit of PI3-kinase as well as the degree of insulin-stimulated Akt serine phosphorylation in the soleus muscle of the obese rats (Fig. 3). The GLUT 4 protein expression in the soleus muscle of obese rats who received loperamide treatment also increased markedly (Fig. 3). However, naloxonazine (10 μg/kg) inhibited the loperamideinduced stimulatory effects on these signaling mediators in the soleus muscle of obese Zucker rats (Fig. 3). Quantification of the immunoblots is summarized in Table 3. Effect of loperamide on the induction of insulin resistance in mice with or without μ-opioid receptors After 28 days of a fructose-rich diet, the plasma glucose concentration in wild-type mice who did not receive loperamide injections was increased to 149.2 ± 4.8 mg/dl and the plasma glucose lowering activity of tolbutamide (10 mg/kg) in these mice was decreased to nearly 6.4 ± 2.1% (Table 4). In contrast, the plasma glucose-lowering activity of tolbutamide (10 mg/kg) was still about 33.9 ± 4.9% in wild-type mice fed with standard chow for 28 days; the plasma glucose concentration in this group at the end of the feeding period was 92.8 ± 5.2 mg/dl (Table 4). In

Table 3 Quantification of the changes in protein expression and insulin-stimulated phosphorylation in isolated soleus muscle of obese Zucker rats treated with loperamide three times daily for 3 days Arbitrary units

Lean Zucker rats Obese Zucker rats Vehicle

Vehicle

Loperamide

Loperamide + naloxonazine

IR IR-pTyr IRS-1 IRS-1-pTyr PI3K (p85α) pAkt (Ser473) GLUT 4

1.02 ± 0.02 1.01 ± 0.03 1.00 ± 0.04d 1.03 ± 0.02d 1.01 ± 0.02d 1.04 ± 0.02d 1.02 ± 0.02d

0.97 ± 0.03 0.99 ± 0.02 0.37 ± 0.02b 0.39 ± 0.02b 0.31 ± 0.02b 0.28 ± 0.03b 0.42 ± 0.02b

0.98 ± 0.02 1.01 ± 0.02 1.17 ± 0.02a,d 1.29 ± 0.02a,d 1.25 ± 0.03a,d 1.31 ± 0.02a,d 1.11 ± 0.03a,d

0.96 ± 0.02 0.98 ± 0.03 0.98 ± 0.02d 0.97 ± 0.04d 0.97 ± 0.02d 1.02 ± 0.03d 0.97 ± 0.02d

Values (mean ± S.E.M.) were obtained from each group of 5 different animals. Naloxonazine (10 μg/kg) was given by i.v. injection at 30 min before i.v. injection of loperamide (18 μg/kg). The vehicle of saline used to dissolve the test drugs was given at the same volume. Equal amounts of protein from each sample were loaded per lane during gel electrophoresis for comparison of signal intensity between samples. aP b 0.05 and bP b 0.01 versus vehicle-treated lean Zucker rats, respectively. dP b 0.01 versus vehicle-treated obese Zucker rats.

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Table 4 Effects of loperamide on the insulin resistance induction in wild-type mice or in μ-opioid receptor knockout mice during the fructose-rich chow feeding period Plasma glucose (mg/dl)

Plasma glucose lowering activity of tolbutamide (%)

Wild-type mice Knockout mice Wild-type mice Knockout mice Standard chow feeding + Vehicle Day 0 93.1 ± 4.7 94.1 ± 5.1 Day 7 93.7 ± 6.2 94.5 ± 5.6 Day 14 93.9 ± 5.6 94.8 ± 4.7c Day 21 93.3 ± 4.9c 94.2 ± 5.2d Day 28 92.8 ± 5.3 95.1 ± 6.4d Fructose-rich chow feeding + Vehicle Day 0 93.8 ± 5.5 93.6 ± 4.9 Day 7 95.2 ± 5.2 98.7 ± 6.4 Day 14 98.2 ± 4.9 122.8 ± 4.3a Day 21 117.6 ± 6.1 143.2 ± 5.8b Day 28 149.2 ± 4.8b 182.5 ± 4.5b Fructose-rich chow feeding + Loperamide Day 0 92.5 ± 3.7 94.2 ± 4.3 Day 7 93.2 ± 4.3 98.4 ± 5.2 Day 14 95.8 ± 6.2 128.6 ± 4.8a Day 21 108.4 ± 5.3 145.6 ± 6.5b Day 28 123.8 ± 5.6a,c 188.3 ± 5.9b

34.3 ± 6.4 33.8 ± 6.1 34.1 ± 5.7c 34.2 ± 5.8d 33.9 ± 4.9d

32.9 ± 4.6 32.6 ± 6.5 31.8 ± 6.3c 31.9 ± 4.7d 32.1 ± 5.6d

34.1 ± 4.4 30.4 ± 5.7 23.3 ± 5.4a 15.1 ± 3.6b 6.4 ± 2.1b

33.2 ± 6.1 27.3 ± 6.3 15.2 ± 3.8a 7.8 ± 4.3b 0.9 ± 1.5b

33.8 ± 5.4 32.1 ± 4.5 28.1 ± 6.4 21.4 ± 5.7a 10.1 ± 1.8b

32.1 ± 4.2 26.8 ± 4.9 13.2 ± 5.3b 5.2 ± 6.1b 1.1 ± 1.7b

Mice fed with fructose-rich chow received loperamide (18 μg/kg) injections into the tail vein every 8 h during the feeding period. The vehicle of saline used to dissolve loperamide was given at the same volume. Mice were received an i.p. injection of 10 mg/kg tolbutamide on the indicated date. Values (mean ± S.E.M.) were obtained from 8 experiments. aP b 0.05 and b P b 0.01 versus vehicle-treated mice fed with standard chow at the indicated date in each group, respectively. cP b 0.05 and dP b 0.01 versus vehicletreated mice fed with fructose-rich chow at the indicated date in each group, respectively.

the presence of loperamide, the plasma glucose levels in wildtype mice fed fructose-rich chow for 28 days were changed to 123.8 ± 5.6 mg/dl and the plasma glucose-lowering activity of tolbutamide in these mice remained at 10.1 ± 1.8% (Table 4). In μ-opioid receptor knockout mice who were not treated with loperamide, the plasma glucose concentration was markedly elevated to 182.5 ± 4.5 mg/dl on the 28th day of the fructose-rich chow diet; in addition, the plasma glucose lowering activity of tolbutamide (10 mg/kg) in the same group of mice was markedly reduced to 0.9 ± 1.5% (Table 4). Similarly, the plasma glucose concentration in μ-opioid receptor knockout mice receiving loperamide at 18 μg/kg on the fructose-rich chow diet was significantly elevated to 188.3 ± 5.9 mg/dl by the 28th day; the lowering activity of tolbutamide (10 mg/kg) in these mice was only 1.1 ± 1.7% (Table 4). In fact, the plasma glucoselowering activity of tolbutamide (10 mg/kg) in μ-opioid receptor knockout mice receiving a 28-day standard chow diet remained at 32.1 ± 5.6% (Table 4). Discussion We found that loperamide can lower plasma glucose and raise insulin-mediated glucose utilization in obese Zucker rats in a dose-dependent fashion. Loperamide exhibited potent affinity and selectivity for the cloned human μ-opioid receptors (Ki = 3 nmol/l) as compared with the δ- (Ki = 48 nmol/l) and

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κ-subtypes (Ki = 1156 nmol/l; DeHaven-Hudkins et al., 1999). It is plausible that the affinity and selectivity of loperamide at the higher concentration might shift from μ-opioid receptors to other subtypes of opioid receptor (DeHaven-Hudkins et al., 1999). The concentration of loperamide used in the present study was actually b 3 nmol/l, suggesting that the action of loperamide was mediated largely by μ-opioid receptors. Furthermore, the action of loperamide was abolished by naloxonazine at doses sufficient to result in μ-opioid receptor blockade (Mhatre and Holloway, 2003). Clearly, these results support the notion that peripheral μ-opioid receptor activation is beneficial for the improvement of insulin resistance. The generally accepted dogma regarding insulin action places insulin at the point of multiple organ adaptations to ingested nutrients, in particular, dietary carbohydrates (Bessesen, 2001). Studies in rats have demonstrated that a high intake of fructose produces a decline of insulin sensitivity in the liver and peripheral tissues (Elliott et al., 2002). Importantly, the pathophysiology of Type 2 diabetes mellitus involves defects in both insulin secretion and insulin action. Individuals with insulin resistance, reduced insulin secretion, or impaired glucose tolerance are known to have an increased risk of developing diabetes (Giorgino et al., 2005). In order to further support the idea that peripheral μ-opioid receptor activation is beneficial for the improvement of insulin sensitivity, μ-opioid receptor knockout mice were then fed with a diet containing 60% fructose to further examine the role of this receptor in the action of loperamide on the induction of insulin resistance. In the present study, the plasma glucose-lowering activity of tolbutamide was extinguished rapidly in μ-opioid receptor knockout mice receiving a fructose-rich diet, whereas the response to tolbutamide was retained in wild-type mice receiving a similar fructose-rich diet for the same time period of time. Loss of the plasma glucose-lowering response to tolbutamide-stimulated endogenous insulin release has been interpreted as the development of insulin-resistance (Chang et al., 1999). These results suggest that a decrease in insulin action was more marked in μ-opioid receptor knockout mice and is consistent with a previous report that insulin resistance is more easily induced in the absence of μ-opioid receptors (Cheng et al., 2003). Thus, μopioid receptors has been shown to have a beneficial role in the amelioration of impaired insulin action. Although it has been reported that opioid agonists increase food intake (Bodnar, 1998), loperamide did not modify the food consumption or body weight of obese Zucker rats. This is sensible because loperamide primarily exerts its effect on μ-opioid receptors located in the peripheral tissues (DeHaven-Hudkins et al., 1999). Taken together, loperamide reversed the responsiveness to insulin in animals with insulin resistance, highlighting the coincident changes between peripheral μ-opioid receptor activation and insulin sensitivity. Insulin resistance is caused by the decreased ability of peripheral target tissues, especially the skeletal muscle, to respond properly to normal circulating insulin, although insulin resistance is known to be associated with cardiovascular complications (Petersen and Shulman, 2002). On the basis of histochemical staining and enzymatic analysis, skeletal muscle

fibers can be classified into one of three distinct categories: type I (slow twitch, oxidative), type IIa (fast twitch, oxidative and glycolytic), and type IIb (fast twitch, glycolytic; Essén et al., 1975). It has been documented that insulin action on glucose uptake and metabolism is much greater in skeletal muscle composed primarily of oxidative fibers (e.g., the soleus) as compared to glycolytic fibers (e.g., the epitrochlearis and extensor digitorum longus), even though the soleus muscle represents a small portion of the total muscle mass (Song et al., 1999). The increase in insulin action in the soleus muscle is likely to be related to increased protein expression and/or functional activity of several key components of the insulin signal transduction cascade. Thus, insulin resistance mainly occurs in oxidative muscles rather than glycolytic muscles (Song et al., 1999). We have demonstrated that naloxonazine blocks the effect of β-endorphin to enhance the uptake of radioactive glucose into the soleus muscle of STZ-diabetic rats, the Type 1 diabetes-like animal model (Cheng et al., 2002). The presence of μ-opioid receptors in soleus muscle can thus be considered, although confirmatory histological evidence is lacking. Therefore, the soleus muscle, but not glycolytic fibers, seems to be an appropriate tissue for investigating the role of μ-opioid receptor activation on changes in insulin signals. Defects in the insulin signaling cascade leading to impaired glucose utilization have been proposed as the pathogenesis of insulin resistance (Shulman, 2000). Insulin receptor substrate (IRS) molecules are key mediators in insulin signaling and four members of the IRS family have been identified (Sesti et al., 2001). Although IRS-1 and IRS-2 play a central role in tissues that are responsible for glucose metabolism, neither IRS-1 nor IRS-2 are functionally interchangeable. IRS-1 appears to have its major role in skeletal muscle, whereas IRS-2 appears to regulate hepatic insulin action as well as pancreatic β cell development and survival (Sesti et al., 2001). Therefore, defects in IRS-1 signaling in skeletal muscle cannot be entirely compensated for by IRS-2 (Sesti et al., 2001). In contrast, IRS-3 and IRS-4 genes appear to play a redundant role in the IRS signaling system (Sesti et al., 2001). Insulin responsiveness in skeletal muscle is known to be determined by GLUT 4 protein concentrations, and any impairment in GLUT 4 expression may result in insulin resistance (Kern et al., 1999). It has been documented that the reduced glucose clearance in obese Zucker rats is mainly related to the decrease of GLUT 4 function in skeletal muscle cells, probably due to abnormalities of insulin signals, including IRS-1 and PI3-kinase (King et al., 1992). Thus, evaluation the changes in IRS-1-related signals, but not other IRS molecules in the soleus muscle of obese Zucker rats, seems to be a more reliable means to demonstrate the beneficial effects of peripheral μopioid receptor activation on improvement of insulin resistance. Current data showed that serine/threonine kinase protein B, also known as Akt, is a potential link between insulinstimulated PI3-kinase and IRS-1 (Klippel et al., 1997). Impairment of Akt serine phosphorylation in cells from animals exhibiting insulin resistance has been documented (Anai et al., 1998). We found that loperamide increased the expression and phosphorylation of IRS-1 in the soleus muscle of obese Zucker rats. Also, the expression of p85, Akt serine phosphorylation,

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and GLUT 4 in obese Zucker rats was restored by repeated treatment with loperamide. Indeed, the effects of loperamide on IRS-1-associated insulin signaling appear to depend on the activation of μ-opioid receptors, as demonstrated by naloxonazine antagonism. IRS-1 tyrosine phosphorylation in response to insulin stimulation generally increases the association of IRS-1 with the p85 subunit of PI3-kinase, resulting in increased PI3-kinase activity, which in turn leads to activation of Akt and, ultimately, to an enhancement in insulin-stimulated glucose disposal (Carvalho et al., 2000). Although the effects of loperamide on PI3-kinase activity and glucose transport into soleus muscles of obese Zucker rats were not evaluated in the present study, it is conceivable that activation of μ-opioid receptors located on the peripheral tissues enhance PI3-kinase activity by insulin, thereby leading to a stimulation of Akt serine phosphorylation to increase GLUT 4 activity. The significance of whole-muscle expression of GLUT 4 protein for insulinmediated glucose transport activity has been recognized (King et al., 1992). Indeed, the essential need for an increase in GLUT4 protein for improvement in muscle insulin resistance has been documented (Ivy, 2004). Thus, although the change involving glucose translocation in soleus muscle was not evaluated, the findings of this report supporting the amelioration by peripheral μ-opioid receptor activation of defects in the IRS1-associated PI 3-kinase step of the insulin signal transduction pathway for glucose transport into skeletal muscle of obese Zucker rats were above suspicion. Protein kinase C (PKC) activation by a phospholipase C (PLC) dependent-mechanism is the main signal linked to μ-opioid receptors. Indeed, we have observed that activation of μ-opioid receptors in myoblast C2C12 cells by loperamide may increase glucose uptake via the PLC–PKC pathway (Liu et al., 2004)., Atypical PKCs have been shown to be essential in insulin stimulation of glucose transport in muscle tissue (Heled et al., 2003). Although atypical PKCs are located downstream from IRS-1 and PI 3-kinase in the established insulin signal pathways, IRS-1 is also a novel physiological substrate for atypical PKCs (Heled et al., 2003). Thus, the relationship in insulin signals between post-receptor in the IRS-1-associated PI3-kinase step and activation of peripheral μ-opioid receptors to improve insulin resistance is worthy of investigation in the future. Nevertheless, improvements in insulin sensitivity through the activation of peripheral μ-opioid receptors overcoming defects in insulin signal transduction related to the post-receptor in IRS-1-associated PI3kinase step have been defined in soleus muscle from obese Zucker rats. The rate of gastric emptying is a key determinant of postprandial glucose concentration. If gastric emptying is accelerated, the presentation of meal-derived glucose to the circulation is poorly timed with insulin delivery. In individuals with diabetes, the absent or delayed secretion of insulin further exacerbates postprandial hyperglycemia (Kruger et al., 1999). Recently, a decrease in gastrointestinal motility by glucoregulatory hormones leading to the improvement of glucose tolerance has been documented (Kendall et al., 2006). Although a major action of loperamide is to slow gastrointestinal motility, improvement of insulin resistance by an activation of peripheral μ-opioid receptors

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was observed in obese Zucker rats in the present study. Therefore, development of the stimulant for peripheral μ-opioid receptors devoid of central opiate-like effects seems useful as an adjuvant for the prevention and/or management of insulin resistance. In conclusion, the present study demonstrated that activation of μ-opioid receptors located on the soleus muscle by loperamide reversed the impairment of insulin-stimulated glucose disposal in obese Zucker rats. This improvement of insulin resistance was associated with the amelioration of the post-receptor insulin signaling, including an increase in protein expression and tyrosine phosphorylation of IRS-1, the expression of p85 regulatory subunit of PI3-kinase, and Akt Ser473 phosphorylation, resulting in enhanced insulin-stimulated GLUT 4 protein. These data strengthen the basis for recommending peripheral μ-opioid receptors as a new target for development of agonists to improve insulin action for patients with insulin resistance. Acknowledgements We thank Yu-Shen Pharmaceutics (Taichung, Taiwan) for the kind supply of loperamide and Dr. Kajuro Komeda (Animal Research Center, Tokyo Medical College, Tokyo, Japan) for the kind supply of Zucker rats. Thanks are also due to Professor H. H. Loh (Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN, USA) for the donation of μopioid receptor knockout and wild-type mice. The present study was supported in part by a grant from the National Science Council (NSC-90-2320-B006-013) of Taiwan, the Republic of China. References Anai, M., Funaki, M., Ogihara, T., Terasaki, J., Inukai, K., Katagiri, H., Fukushima, Y., Yazaki, Y., Kikuchi, M., Oka, Y., Asano, T., 1998. Altered expression levels and impaired steps in the pathway to phosphatidylinositol 3-kinase activation via insulin receptor substrates 1 and 2 in Zucker fatty rats. Diabetes 47 (1), 13–23. Bessesen, D.H., 2001. The role of carbohydrates in insulin resistance. The Journal of Nutrition 131 (10), 2782S–2786S. Bodnar, R.J., 1998. Recent advances in the understanding of the effects of opioid agents on feeding and appetite. Expert Opinion on Investigational Drugs 7 (4), 485–497. Carvalho, E., Rondinone, C., Smith, U., 2000. Insulin resistance in fat cells from obese Zucker rats—evidence for an impaired activation and translocation of protein kinase B and glucose transporter 4. Molecular and Cellular Biochemistry 206 (1–2), 7–16. Chang, S.L., Lin, J.G., Chi, T.C., Liu, I.M., Cheng, J.T., 1999. An insulindependent hypoglycaemia induced by electroacupuncture at the Zhongwan (CV12) acupoint in diabetic rats. Diabetologia 42 (2), 250–255. Cheng, J.T., Liu, I.M., Chi, T.C., Tzeng, T.F., Lu, F.H., Chang, C.J., 2001. Plasma glucose lowering effect of tramadol in streptozotocin-induced diabetic rats. Diabetes 50 (12), 2815–2821. Cheng, J.T., Liu, I.M., Tzeng, T.F., Tsai, C.C., Lai, T.Y., 2002. Plasma glucose lowering effect of β-endorphin in streptozotocin-induced diabetic rats. Hormone and Metabolic Research 34 (10), 570–576. Cheng, J.T., Liu, I.M., Hsu, C.F., 2003. Rapid induction of insulin resistance in opioid mu-receptor knock-out mice. Neuroscience Letters 339 (2), 139–142. Christ, C.Y., Hunt, D., Hancock, J., Garcia-Macedo, R., Mandarino, L.J., Ivy, J.L., 2002. Exercise training improves muscle insulin resistance but not insulin receptor signaling in obese Zucker rats. Journal of Applied Physiology 92 (2), 736–744.

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