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ENDOCRINE AND REPRODUCTIVE

Mediation of -Endorphin by Isoferulic Acid to Lower Plasma Glucose in Streptozotocin-Induced Diabetic Rats

Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan City, Taiwan, Republic of China (I.-M.L., J.T.C.); Department of Pharmacy, Tajen Institute of Technology, Yen-Pou, Ping Tung Shien, Taiwan, Republic of China (I.-M.L.); and Department of Chinese Medicine, Jin-Ai Municipal Hospital, Taipei City, Taiwan, Republic of China (W.C.C.)

Received for publication May 4, 2003
Accepted July 29, 2003.

	Abstract

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We investigated the mechanism(s) by which isoferulic acid lowers plasma glucose levels in streptozotocin-induced diabetic rats (STZ-diabetic rats). In STZ-diabetic rats, isoferulic acid dose dependently lowered plasma glucose concentrations and increased plasma -endorphin-like immunoreactivity (BER). Both of these effects of isoferulic acid were abolished by pretreatment of rats with tamsulosin or 2-[2,6-dimethoxyphenoxyethyl]aminomethyl-1,4-benzodioxane hydrochloride (WB 4101) at doses sufficient to block ₁-adrenoceptors. Also, isoferulic acid enhanced BER release from isolated rat adrenal medulla in a concentration-dependent manner that could be abolished by treatment with ₁-adrenoceptor antagonists. Moreover, bilateral adrenalectomy in STZ-diabetic rats eliminated the activities of isoferulic acid, including the plasma glucose-lowering effect and the plasma BER-elevating effect. Naloxone and naloxonazine inhibited the plasma glucose-lowering activity of isoferulic acid at doses sufficient to block opioid µ-receptors. In contrast with the effect in wild-type diabetic mice, isoferulic acid failed to lower plasma glucose levels in opioid µ-receptor knockout diabetic mice. Treatment of STZ-diabetic rats with isoferulic acid three times in 1 day resulted in an increase in the expression of the glucose transporter subtype 4 form in soleus muscle. This effect was blocked by ₁-adrenoceptor or opioid µ-receptor antagonists. The reduction of elevated mRNA or protein level of hepatic phosphoenolpyruvate carboxykinase was also impeded in the same groups of STZ-diabetic rats. In conclusion, our results suggest that isoferulic acid may activate ₁-adrenoceptors to enhance the secretion of -endorphin, which can stimulate the opioid µ-receptors to increase glucose use or/and reduce hepatic gluconeogenesis, resulting in a decrease of plasma glucose in STZ-diabetic rats.

Recent studies (Liu et al., 1999a; Cheng et al., 2001b, 2002) have shown that activation of opioid µ-receptors by either exogenous -endorphin or chemical agents, such as loperamide or tramadol, might improve glucose homeostasis in diabetic rats in the absence of insulin. Also, activation of ₁-adrenoceptors in adrenal medulla by phenylephrine might enhance the secretion of -endorphin from the rat adrenal gland (Cheng et al., 2001c). Therefore, activation of ₁-adrenoceptors or opioid µ-receptors may lower plasma glucose levels in rats with type 1-like diabetes. Isoferulic acid can activate _1A-adrenoceptor, leading to increased glucose uptake into cultured mouse myoblast C₂C₁₂ cells (Liu et al., 2001b). However, the role of -endorphin in the plasma glucose-lowering action of isoferulic acid is still unclear. Thus, we investigated the mechanisms by which isoferulic acid reduces plasma glucose level in diabetic rats in the absence of insulin.

	Materials and Methods

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Animal Models. Male Wistar rats weighing 200 to 250 g were obtained from the Animal Center of National Cheng Kung University Medical College. Male wild-type (BDF1 mice) and opioid µ-receptor knockout mice (Loh et al., 1998), 8 to 10 weeks of age, were obtained from Professor H. H. Loh (Department of Pharmacology, University of Minnesota Medical School. Minneapolis, MN). STZ-diabetic rats, the model for type I-diabetes, were prepared by i.v. injecting STZ (60.0 mg/kg) into male Wistar rats 8 to 10 weeks of age. Mice with or without opioid µ-receptors received an i.p. injection of STZ at 50.0 mg/kg to induce diabetes according to the previous method (Liu et al., 2001a). Animals were considered to be diabetic if they had plasma glucose concentrations of 20.0 mM or greater in addition to polyuria and other diabetic features. All studies were carried out 2 weeks after the injection of STZ. 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.

Effect of Isoferulic Acid on Plasma -Endorphin-Like Immunoreactivity (BER) Level in STZ-Diabetic Rats. After an overnight fast, STZ-diabetic rats received an i.v. injection of isoferulic acid at the desired doses. Animals were anesthetized with sodium pentobarbital (30.0 mg/kg i.p.) and blood samples (0.1 ml) were collected from the tail vein for measurement of plasma glucose concentrations and BER. In a previous study (Liu et al., 2000a), a 5.0-mg/kg dose of isoferulic acid was found to produce the maximal plasma glucose-lowering effect in STZ-diabetic rats 30 min after single i.v. injection. Thus, the effect of isoferulic acid on plasma BER was determined using blood samples collected at 30 min after the i.v. injection. STZ-diabetic rats that received an injection of vehicle only (0.9% NaCl in distilled water) were used as controls. Further experiments were performed with the pharmacological inhibitors, such as the blockers of ₁-adrenoceptors (tamsulosin or WB 4101) and the antagonists of opioid µ-receptors (naloxone or naloxonazine). These inhibitors were injected intravenously into fasting rats 30 min before the intravenous injection of isoferulic acid.

Effect of Isoferulic Acid on Plasma Glucose Concentrations in Opioid µ-Receptor Knockout Diabetic Mice. Fasting STZ-diabetic mice with or without opioid µ-receptors were given an intravenous injection of 5.0 mg/kg isoferulic acid, the dose previously shown to have the maximal plasma glucose-lowering action in STZ-diabetic rats. After 30 min, blood samples (0.1 ml) were collected from the lower eye lid of mice under anesthesia with pentobarbital (30.0 mg/kg i.p.) using a chilled syringe containing 10 IU of heparin.

Isolation and Incubation of Adrenal Medulla. Adrenal glands were quickly removed from sacrificed STZ-diabetic rats and medullae were immediately dissected after removal of cortex as described previously (Vatta et al., 1997). The tissues were cut into approximately 1-mm-thick slices and transferred to a glass tube fitted with a mesh of nylon at the bottom to permit free interchange with the medium. The tissues were incubated for 15 min at 37°C, pH 7.4, and bubbled with a 95% O₂ and 5% CO₂ mixture under continued shaking with 2 ml of a modified Krebs' solution (118 mmol/l NaCl, 4.7 mmol/l KCl, 1.2 mmol/l MgCl₂, 1.0 mmol/l NaH₂PO₄, 2.5 mmol/l CaCl₂, 0.004 mmol/l EDTA-Na, 11.1 mmol/l dextrose, 25.0 mmol/l NaHCO₃, and 0.11 mmol/l ascorbic acid). Tissues were then transferred to fresh incubation tubes with or without ₁-adrenoceptor antagonists at the indicated concentrations for 15 min at 37°C (Cheng et al., 2001c). The tissues were then incubated with isoferulic acid at the indicated concentrations at 37°C for 30 min with continuous shaking at 40 cycles/min. Incubation was terminated by placing the tubes on ice. The medium from each incubation was collected and frozen at -70°C until the -endorphin assay was performed.

Adrenalectomized Rats. Bilateral adrenalectomy was performed in Wistar rats as described previously (Cheng et al., 2001a) using the dorsal approach under pentobarbital anesthesia (30.0 mg/kg i.p.). Sham-operated animals served as controls. Animals were allowed to recover for 2 weeks after the operation. After recovery, diabetes was induced by an injection of STZ as described above. The effect of isoferulic acid at 5.0 mg/kg was determined using blood samples collected at 30 min after the bolus injection.

Laboratory Determinations. The concentration of plasma glucose was measured by the glucose oxidase method using an analyzer (Quik-Lab, Ames; Miles Inc., Elkhart, IN). The determination of BER in samples was done using a commercially available enzyme-linked immunosorbent assay (Peninsula Laboratories, Belmont, CA)

Determination of Gene Expression. STZ-diabetic rats were injected every 8 h, three times daily, into the tail vein with vehicle, isoferulic acid (5.0 mg/kg), or isoferulic acid (5.0 mg/kg) plus pharmacological inhibitors, such as of ₁-adrenoceptor antagonists (tamsulosin or WB 4101) or opioid µ-receptor blockers (naloxone or naloxonazine). The inhibitors were injected intravenously into STZ-diabetic rats 30 min before the injection of isoferulic acid. In preliminary experiments (Liu et al., 2000a), isoferulic acid was found to significantly modify glucose transporters subtype 4 (GLUT 4) and hepatic phosphoenolpyruvate carboxykinase (PEPCK) mRNA and protein levels in STZ-diabetic rats after 1 day of treatment. Thus, animals were sacrificed after 1 day of treatment. Liver and soleus muscle were immediately removed, frozen in liquid nitrogen, and stored at -70°C for Northern and Western blot analysis. Blood samples were collected from the tail vein of these rats before they were sacrificed.

Northern Blot Analysis. Total RNA was extracted from liver or soleus muscle of experimental animals using the Ultraspec-II RNA extraction system. For Northern blot analysis, RNA (20 µg) was denatured in a solution containing 2.2 mM formaldehyde and 50% formamide (v/v) by heating at 55°C for 15 min. Aliquots of total RNA were size-fractionated in a 1.2% agarose/formaldehyde gel. Gels were stained with ethidium bromide staining to identify the position of the 18S and 28S rRNA subunits and to confirm that equivalent amounts of undegraded RNA had been loaded. The RNA was transferred to a Hybond-N membrane. GLUT 4 and PEPCK mRNA levels were detected using full-length cDNA probe radioactively labeled using the random primer method and hybridized under stringent conditions. The intensity of the mRNA bands on the blot was quantified by scanning densitometry (Hoefer, San Francisco, CA). Membranes were probed using -actin as an internal standard.

Western Blot Analysis. After homogenization of liver and soleus muscle using a glass/Teflon homogenizer, the homogenates (50 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot analysis was performed using either an anti-rat GLUT 4 antibody (1:1000) in soleus muscle or an anti-rat PEPCK antibody (1:1000) in liver. The blots were probed with a goat polycolonal actin antibody (1:500) or a mouse monoclonal -tubulin antibody (1:500) to ensure that the amount of protein loaded into each lane of the gel was constant. Blots were incubated with the appropriate peroxidase-conjugated secondary antibodies. After removal of the secondary antibody, blots were washed and developed using the ECL-Western blotting system. Densities of the obtained immunoblots at 45 KDa for GLUT 4, 69.5 KDa for PEPCK, 43 KDa for actin, and 50 KDa for -tubulin were quantified using laser densitometer.

Drugs. Isoferulic acid was supplied by Professor F. L. Hsu (Department of Pharmacology, School of Pharmacy, Taipei Medical University, Taipei City, Taiwan). Streptozotocin and phenylephrine were obtained from Sigma-Aldrich (St. Louis, MO). WB 4101, naloxone, and naloxonazine were purchased from Sigma/RBI (Natick, MA). Tamsulosin was a generous gift from Dr. S. Yamada (Department of Biochemical Pharmacy, School of Pharmacy, Shizuoka KenRitsu University, Shizuoka City, Japan). A commercial kit for BER was purchased from Peninsula Laboratories. Ultraspec-II RNA extraction system was from Bioteck (Houston, TX). The plasmid containing cDNA for PEPCK was kindly supplied by Professor R. W. Hanson (Department of Biochemistry School of Medicine, Case Western Reserve University, Cleveland, OH). The plasmid containing cDNA of GLUT 4 was obtained from Professor C. Makepeace (Department of Cell Biology and Physiology, School of Medicine, Washington University, St. Louis, MO), and the -actin cDNA was from Professor S. S. Liu (Department of Microbiology and Immunology, National Cheng Kung University, Tainan City, Taiwan, Republic of China). Medaprime-labeling system kit, Hybond-N membrane, and ECL-Western blotting system were purchased from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). Monocolonal mouse anti-rat GLUT 4 antibody was from Genzyme (Cambridge, MA). Sheep anti-rat liver PEPCK was a gift from Professor D. K. Granner (Vanderbilt University, Nashville, TN). Goat polyclonal actin antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse monoclonal -tubulin antibody was from Zymed Laboratories (South San Francisco, CA). All other reagents were from standard sources.

Statistical Analysis. Data are expressed as the mean ± S.E.M. for the number (n) of animals in the group as indicated in tables and figures. Repeated measures analysis of variance was used to analyze the changes in plasma glucose and other parameters. The Dunnett range post hoc comparisons were used to determine the source of significant differences where appropriate. The concentration for 50% effect (ED₅₀) was obtained from nonlinear regression analysis. A p value < 0.05 was considered statistically significant.

	Results

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Effects of Isoferulic Acid on the Plasma Glucose Concentration and Plasma BER Level in STZ-Diabetic Rats. Thirty minutes after treatment, the plasma glucose concentration was decreased from a basal level of 27.5 ± 2.8 to 25.8 ± 3.1 mM, 24.1 ± 2.9, and 20.4 ± 3.4 mM in STZ-diabetic rats receiving i.v. injections of isoferulic acid at 0.5, 1.0, and 3.0 mg/kg, respectively (n = 8). Isoferulic acid at 5.0 mg/kg significantly decreased the plasma glucose concentration to 17.7 ± 2.5 mM (p < 0.01; n = 8), a 35.6 ± 3.8% decrease. Similar to our previous report (Liu et al., 2000a), the maximal plasma glucose-lowering activity in STZ-diabetic rats was achieved using isoferulic acid at 5.0 mg/kg and increasing the dose to 10.0 mg/kg had no further effect (Fig. 1A). A dose-dependent elevation of the plasma BER level was observed in the same group of STZ-diabetic rats after single i.v. injection of isoferulic acid at doses ranging from 1.0 to 10.0 mg/kg (Fig. 1B). Isoferulic acid at 5.0 mg/kg increased the plasma BER level from basal level of 48.2 ± 5.4 to 114.3 ± 6.4 pg/ml in STZ-diabetic rats; no additional increase of plasma BER was observed using higher concentrations of isoferulic acid. Intravenous injection of phenylephrine lowered plasma glucose levels in STZ-diabetic rats by 11.7 ± 2.5, 22.4 ± 2.1, and 29.3 ± 4.6% at the dose of 0.5, 1.0, and 5.0 µg/kg, respectively (n = 8). The plasma glucose-lowering activity if isoferulic acid at 5.0 mg/kg (35.6 ± 3.8%) was similar to that of 5.0 µg/kg phenylephrine (29.3 ± 4.6%). Intravenous injection of phenylephrine elevated the plasma BER level in STZ-diabetic rats to 60.3 ± 7.2 at 0.5 µg/kg, 82.3 ± 9.4 pg/ml at 1.0 µg/kg, and 101.3 ± 6.5 pg/ml at 5.0 µg/kg (n = 8). This was similar to the elevation of the BER level in STZ-diabetic rats treated intravenously with isoferulic acid at 5.0 mg/kg i.v. (114.3 ± 6.4 pg/ml). Thus, 5.0 mg/kg isoferulic acid was used in subsequent experiments.

View larger version (10K): [in this window] [in a new window]   Fig. 1. A, plasma glucose-lowering activity produced by an i.v. injection of isoferulic acid into STZ-diabetic rats. B, plasma BER level in STZ-diabetic rats receiving i.v. injections of isoferulic acid. Values of mean and bar of S.E.M. were obtained from each group of eight animals. Vehicle (0.9% NaCl in distilled water) only was given at the same volume. *, p < 0.05 and **, p < 0.01 versus data from animals treated with vehicle (0).

Effects of ₁-Adrenoceptor Antagonists on the Actions of Isoferulic Acid in STZ-Diabetic Rats. Table 1 shows that the increase of plasma BER levels induced by isoferulic acid in STZ-diabetic rats was attenuated in a dosedependent manner by treatment of rats with tamsulosin or WB 4101 30 min after the injection of isoferulic acid (5.0 mg/kg). Pretreatment with tamsulosin at 1.0 mg/kg or WB 4101 at 1.0 mg/kg completely reversed the action of isoferulic acid. Moreover, tamsulosin or WB 4101 at 1.0 mg/kg did not influence basal levels of plasma BER in STZ-diabetic rats.

View this table: [in this window] [in a new window]   TABLE 1 Effects of 1-adrenoceptor antagonists on the isoferulic acid-induced changes of BER and glucose concentrations in plasma of STZ-diabetic rats The antagonists were given by i.v. injection 30 min before the injection of isoferulic acid. Vehicle (0.9% NaCl in distilled water) was given at the same volume. Values (mean ± S.E.M.) were obtained from each group of eight animals. Basal level shows the value from fasted animals treated with vehicle.

Similar to the effect on the plasma BER level, the plasma glucose-lowering activity of isoferulic acid at 5.0 mg/kg was reduced in a dose-dependent manner by pretreatment of rats with tamsulosin. Tamsulosin at 1.0 mg/kg completely blocked the plasma glucose-lowering activity of isoferulic acid. Similarly, the plasma glucose-lowering activity of isoferulic acid (5.0 mg/kg) in STZ-diabetic rats was inhibited by pretreatment of rats with WB 4101 at 1.0 mg/kg, the same dose that blocked the effects of isoferulic acid on plasma BER levels. However, treatment of STZ-diabetic rats with tamsulosin or WB 4101 alone at 1.0 mg/kg had no effect on plasma glucose concentrations (Table 1).

Effect of Isoferulic Acid on the Secretion of BER from Adrenal Medulla Isolated from STZ-Diabetic Rats. Fig. 2 shows the effect of isoferulic acid on the secretion of BER from adrenal medulla isolated from STZ-diabetic rats. Isoferulic acid increased the levels of BER in the culture medium in a concentration-dependent manner from 0.001 to 10.0 µM. The effective concentration (EC₅₀) of isoferulic acid was approximately 52.2 nM. The stimulatory effect of isoferulic acid on BER secretion was not further increased even using a concentration of 10.0 µM. Thus, 1.0 µM isoferulic acid was used in subsequent experiments.

View larger version (10K): [in this window] [in a new window]   Fig. 2. Effect of isoferulic acid on BER secretion from isolated adrenal medulla from STZ-diabetic rats. Results are expressed as picogram per milligram protein of adrenal medulla and represent the mean ± S.E.M. of seven independent experiments.

Effects of ₁-Adrenoceptor Antagonists on Isoferulic Acid-Stimulated -Endorphin Secretion from Adrenal Medulla Isolated from STZ-Diabetic Rats. Table 2 shows that tamsulosin and WB 4101 blocked isoferulic acid-stimulated BER secretion from rat adrenal medulla in a concentration-dependent manner. Preincubation with tamsulosin at 100.0 nM reduced BER secretion in response to 1.0 µM isoferulic acid from 252.7 ± 13.1 to 105.7 ± 10.7 pg/mg protein of adrenal medulla, a level close to the basal value (100.2 ± 9.8 pg/mg protein of adrenal medulla) obtained from samples incubating with vehicle only. WB 4101 at 1.0 µM also blocked the increase of BER secretion stimulated by isoferulic acid (1.0 µM) to near the basal level. Neither tamsulosin nor WB 4101 affected the spontaneous secretion of BER, even at concentrations sufficient to completely block ₁-adrenoceptors (Table 2).

View this table: [in this window] [in a new window]   TABLE 2 Effects of 1-adrenoceptor antagonists on isoferulic acid-induced changes of BER in isolated adrenal medulla of STZ-diabetic rats After preincubation for 15 min with inhibitors or vehicle (modified Krebs' solution), the adrenal medulla was exposed to isoferulic acid (1.0 mmol/l) for another 30 min. Results are the means ± S.E.M. of six determinations.

Bilateral Adrenalectomy in STZ-Diabetic Rats Abolishes the Effect of Isoferulic Acid on Plasma Glucose and BER Levels. Bilateral adrenalectomy was performed in STZ-diabetic rats. Two weeks after adrenalectomy, there were no significant differences in the basal plasma levels of glucose and BER between adrenalectomized STZ-diabetic rats and sham-operated control (Table 3). However, the ability of isoferulic acid to decrease plasma glucose levels and to increase plasma BER levels was abolished in STZ-diabetic rats with bilateral adrenalectomy but was unaltered in the sham-operated STZ-diabetic rats (Table 3).

Effects of Opioid µ-Receptor Antagonists on Isoferulic Acid-Induced Plasma Glucose-Lowering Activity in STZ-Diabetic Rats. Table 4 shows the dose-dependent effect of naloxone and naloxonazine on the activity of isoferulic acid in STZ-diabetic rats. In the presence of 10.0 µg/kg naloxone, the plasma glucose concentration in treated with 5.0 mg/kg isoferulic acid was 28.4 ± 3.7 mM, which was not statistically different from the basal level (28.2 ± 4.1 mM). The effects of naloxonazine (10.0 µg/kg) were similar to those of naloxone; the plasma glucose level in naloxonazine (10.0 µg/kg)-pretreated STZ-diabetic rats after treatment with 5.0 mg/kg isoferulic acid was 29.0 ± 4.5 mM, a level near the basal level. Neither naloxone nor naloxonazine alone had any effect on basal plasma glucose levels in STZ-diabetic rats, even at the highest dose.

Effect of Isoferulic Acid on Plasma Glucose Levels in Opioid µ-Receptor Knockout Diabetic Mice. Fig. 3 shows that isoferulic acid at the maximal dose (5.0 mg/kg) had no effect in diabetic mice lacking opioid µ-receptors. The concentration of plasma glucose in opioid µ-receptor knockout diabetic mice was not significantly influenced by isoferulic acid (from 28.4 ± 2.5 to 27.1 ± 2.6 mM; p > 0.05). In contrast, in the presence of opioid µ-receptors in diabetic mice, isoferulic acid (5.0 mg/kg) decreased the plasma glucose concentration from 27.2 ± 2.0 to 17.6 ± 1.9 mM. The plasma glucose-lowering activity of isoferulic acid in these wild-type diabetic mice was approximately 35.2 ± 3.8%, similar to the effect of isoferulic acid in STZ-diabetic rats (Fig. 3).

View larger version (11K): [in this window] [in a new window]   Fig. 3. Effects of isoferulic acid (5.0 mg/kg i.v.) on plasma glucose concentrations in opioid µ-receptor knockout mice and wild-type control. Values of mean and S.E.M. bar were obtained from each group of six animals. **, p < 0.01 versus data obtained from animals before treatment in each group.

Effects of ₁-Adrenoceptor Antagonists on Isoferulic Acid-Induced Changes of mRNA and Protein Levels of GLUT 4 in Soleus Muscle of STZ-Diabetic Rats. Similar to our previous report (Liu et al., 2000a), treatment of STZ-diabetic rats with isoferulic acid (5.0 mg/kg) three times for 1 day resulted in an elevation of GLUT 4 mRNA level in soleus muscle (Figs. 4 and 5, top). Western blot analysis showed a similar effect of isoferulic acid (5.0 mg/kg) on levels of GLUT 4 protein in soleus muscle (Figs. 4 and 5, bottom). Pretreatment of rats with tamsulosin (1.0 mg/kg) or WB 4101 (1.0 mg/kg) completely abolished the activity of isoferulic acid on GLUT 4 mRNA (Fig. 4, top). Also, isoferulic acid did result in an elevation of GLUT 4 protein levels in diabetic rats pretreated with ₁-adrenoceptor antagonists (Fig. 4, bottom). Table 5 presents a quantification of these data regarding the mRNA and protein levels of GLUT 4.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Top, representative response of mRNA level for GLUT 4 or -actin in soleus muscle isolated from STZ-diabetic rats receiving treatment with isoferulic acid or isoferulic acid and 1-adrenoceptor antagonists in combination, three times daily for 1 day. Bottom, identification of GLUT 4 protein level using immunoblot analysis. Lane 1, vehicle (0.9% NaCl in distilled water)-treated STZ-diabetic rats; lane 2, isoferulic acid (5.0 mg/kg)-treated STZ-diabetic rats; lane 3, isoferulic acid (5.0 mg/kg) plus tamsulosin (1.0 mg/kg)-treated STZ-diabetic rats; and lane 4, isoferulic acid (5.0 mg/kg) plus WB 4101 (1.0 mg/kg)-treated STZ-diabetic rats.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Top, representative response of mRNA level for GLUT 4 or -actin in soleus muscle isolated from STZ-diabetic rats receiving treatment with isoferulic acid or isoferulic acid and opioid µ-receptor antagonists in combination, three times daily for 1 day. Bottom, identification of GLUT 4 protein level using immunoblot analysis. Lane 1, vehicle (0.9% NaCl in distilled water)-treated STZ-diabetic rats; lane 2, isoferulic acid (5.0 mg/kg)-treated STZ-diabetic rats; lane 3, isoferulic acid (5.0 mg/kg) plus naloxone (10.0 µg/kg)-treated STZ-diabetic rats; and lane 4, isoferulic acid (5.0 mg/kg) plus naloxonazine (10.0 µg/kg)-treated STZ-diabetic rats.

View this table: [in this window] [in a new window]   TABLE 5 Changes of plasma glucose concentrations and quantification of the mRNA and protein levels for GLUT 4 in isolated soleus muscle and hepatic PEPCK from STZ-diabetic rats treated with isoferulic acid and 1-adrenoceptor antagonists in combination Vehicle (0.9% NaCl in distilled water) was given at the same volume. Basal level shows the value from animals treated with vehicle. Values (mean ± S.E.M.) were obtained from each group of six animals.

Effects of Opioid µ-Receptor Antagonists on Isoferulic Acid-Induced Changes of the mRNA and Protein Levels of GLUT 4 in Soleus Muscle of STZ-Diabetic Rats. The elevation of GLUT 4 mRNA level in STZ-diabetic rats was no more seen after 1-day treatment with naloxone (10.0 µg/kg) and isoferulic acid (5.0 mg/kg) in combination (Fig. 5, top). The effect of isoferulic acid (5.0 mg/kg) on the GLUT 4 mRNA in diabetic rats was also inhibited by pretreatment with naloxonazine (10.0 µg/kg) compared with the group treated with isoferulic acid (5.0 mg/kg) only (Fig. 5, top). A similar antagonism by naloxone (10.0 µg/kg) or naloxonazine (10.0 µg/kg) on the enhancement of the GLUT 4 protein level induced by isoferulic acid was also obtained (Fig. 5, bottom). Table 6 shows the changes of mRNA and protein levels of GLUT 4 induced by these treatments.

Effects of ₁-Adrenoceptor Antagonists on Isoferulic Acid-Induced Changes of mRNA and Protein Levels of Hepatic PEPCK in STZ-Diabetic Rats. Similar to our previous report (Liu et al., 2000a), the level of PEPCK mRNA from livers of STZ-diabetic rats repeatedly treated with isoferulic acid (5.0 mg/kg) was markedly reduced (Figs. 6 and 7, top). The representative results of the immunoblot of liver proteins are shown in Figs. 6 and 7, bottom. Repeated treatment of STZ-diabetic rats with isoferulic acid also resulted in a marked reduction of the PEPCK protein level to about 45% of the level in vehicle-treated STZ-diabetic rats. However, pretreatment with tamsulosin (1.0 mg/kg) or WB 4101 (1.0 mg/kg) reversed the effect of isoferulic acid (Fig. 6, top). In contrast, the hepatic PEPCK protein levels in STZ-diabetic rats treated with ₁-adrenoceptors antagonists were not different from those in the vehicle-treated group (Fig. 6, bottom). Table 5 presents the quantification of the data from these experiments examining mRNA and protein levels of PEPCK.

View larger version (23K): [in this window] [in a new window]   Fig. 6. Top, representative response of mRNA level for PEPCK or -actin in liver isolated from STZ-diabetic rats receiving treatment with isoferulic acid or isoferulic acid and 1-adrenoceptor antagonists in combination, three times daily for 1 day. Bottom, identification of hepatic PEPCK protein level using immunoblot analysis. Lane 1, vehicle (0.9% NaCl in distilled water)-treated STZ-diabetic rats; lane 2, isoferulic acid (5.0 mg/kg)-treated STZ-diabetic rats; lane 3, isoferulic acid (5.0 mg/kg) plus tamsulosin (1.0 mg/kg)-treated STZ-diabetic rats; and lane 4, isoferulic acid (5.0 mg/kg) plus WB 4101 (1.0 mg/kg)-treated STZ-diabetic rats.

View larger version (37K): [in this window] [in a new window]   Fig. 7. Top, representative response of mRNA level for PEPCK or -actin in liver isolated from STZ-diabetic rats receiving treatment with isoferulic acid or isoferulic acid and opioid µ-receptor antagonists in combination, three times daily for 1 day. Bottom, identification of PEPCK protein level using immunoblot analysis. Lane 1, vehicle (0.9% NaCl in distilled water)-treated STZ-diabetic rats; lane 2, isoferulic acid (5.0 mg/kg)-treated STZ-diabetic rats; lane 3, isoferulic acid (5.0 mg/kg) plus naloxone (10.0 µg/kg)-treated STZ-diabetic rats; and lane 4, isoferulic acid (5.0 mg/kg) plus naloxonazine (10.0 µg/kg)-treated STZ-diabetic rats.

Effects of Opioid µ-Receptor Antagonists on Isoferulic Acid-Induced Changes of mRNA and Protein Levels of Hepatic PEPCK in STZ-Diabetic Rats. The effect of isoferulic acid on the PEPCK mRNA level in STZ-diabetic rats was not reversed by naloxone (10.0 µg/kg) to the level in the vehicle-treated group (Fig. 7, top). However, the reduction of PEPCK mRNA levels in response to treatment of rats with isoferulic acid (5.0 mg/kg) after pretreatment of the animals with naloxone (10.0 µg/kg) was approximately 2-fold the level achieved in animals treated with isoferulic acid (5.0 mg/kg) only. A similar effect was seen in the naloxonazine (10.0 µg/kg)-pretreated group (Fig. 7, top). The protein level of PEPCK was modified upon blockade of opioid µ-receptors by naloxone (10.0 µg/kg) or naloxonazine (10.0 µg/kg) (Fig. 7, bottom). Table 6 presents the quantification of all data from these experiments examining mRNA and protein levels of PEPCK.

	Discussion

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The STZ-diabetic rat, an animal model for type 1-like diabetic mellitus, was used to confirm that isoferulic acid has plasma glucose-lowering activity as was reported previously (Liu et al., 1999b, 2000a). In the present study, a dose-dependent increase of plasma BER was also obtained in STZ-diabetic rats who received a bolus injection of isoferulic acid. The increase of plasma BER level seemed to be associated with the lowering of plasma glucose concentration in STZ-diabetic rats. Elevation of -endorphins seemed to be related to the plasma glucose-lowering activity of isoferulic acid in the absence of insulin. In fact, plasma glucose-lowering activities are not entirely dependent on insulin regulation; for example, physical exercise is traditionally considered beneficial in insulin-dependent diabetes mellitus (Wasserman and Zinman, 1994). In addition, endogenous -endorphin or opioid receptor activation has been reported to positively regulate glucose homeostasis in an insulin-deficient state (Cheng et al., 2001b, 2002). In this report, we investigated the role of endogenous -endorphin in the insulin-independent plasma glucose-lowering activity of isoferulic acid in diabetic rats with insulin deficiency.

Phenylephrine was used to activate ₁-adrenoceptors in STZ-diabetic rats and produced a dose-dependent plasma glucose-lowering activity accompanying plasma BER elevation in STZ-diabetic rats. A positive relationship between phenylephrine and isoferulic acid in the activation of ₁-adrenoceptors was obtained. Thus, the elevation of -endorphin via activation of ₁-adrenoceptors may be involved in the activity of isoferulic acid. Pretreatment with the ₁-adrenoceptor antagonist tamsulosin (Muramatsu et al., 1998) and WB 4101 (Minneman et al., 1988) blocked the plasma glucose-lowering activity of isoferulic acid in a dose-dependent manner. The isoferulic acid-induced increase of plasma BER was also reduced in the presence of ₁-adrenoceptor antagonists. Although tamsulosin acts on both _1A-adrenoceptor and _1D-adrenoceptor, tamsulosin is more selective for the _1A-subtype (Muramatsu et al., 1998). Recently, we demonstrated that the _1A-adrenoceptor is the major subtype of ₁-adrenoceptors in C₂C₁₂ cells (Liu et al., 2000b). Activation of _1A-adrenoceptor mediates the isoferulic acid increase of glucose uptake into C₂C₁₂ cells (Liu et al., 2001b). Therefore, an increase of endogenous -endorphin secretion via activation of ₁-adrenoceptors by isoferulic acid is probably primarily due to the _1A-adrenoceptor subtype. However, further investigations are necessary to verify this finding.

Although -endorphin is released along with adrenocorticotrophic hormone from the pituitary gland (Vargo et al., 1977), the adrenal gland is also a source of -endorphin (Arefolov et al., 1986; Mitsuma et al., 1987). In an attempt to determine whether the adrenal gland is involved in the isoferulic acid-induced release of -endorphin, adrenalectomy was carried out in STZ-diabetic rats. The plasma glucose-lowering activity of isoferulic acid was abolished in STZ-diabetic rats after bilateral adrenalectomy. Similar to previous reports (Koenig et al., 1986; Cheng et al., 2001a), the basal plasma level of BER in STZ-diabetic rats that received bilateral adrenalectomy was not different from that in shamoperated controls. Moreover, no increase in the plasma BER level was obtained in these adrenalectomized diabetic rats after treatment with isoferulic acid at the effective doses. Thus, secretion of endogenous -endorphin from the adrenal gland is likely to be involved in the activity of isoferulic acid.

To confirm this hypothesis, we used an enzyme-linked immunosorbent assay to investigate the direct effect of isoferulic acid on the secretion of -endorphin in isolated adrenal medulla. We found that isoferulic acid-enhanced BER secretion from adrenal medulla isolated from STZ-diabetic rat in a concentration-related manner. It has been documented that the increase of -endorphin release induced by phenylephrine in rat adrenal medulla is blocked by prazosin and tamsulosin (Cheng et al., 2001c). Because tamsulosin or WB 4101 abolished isoferulic acid-stimulated secretion of -endorphin, activation of ₁-adrenoceptors in the adrenal medulla by isoferulic acid resulting in enhanced -endorphin secretion would be a plausible mechanism. Furthermore, our data support reports that pituitary gland-independent release of endogenous opioids is operative in other organs (Arefolov et al., 1986; Mitsuma et al., 1987).

Our previous reports (Liu et al., 1999a; Cheng et al., 2001b,2002) indicated that opioid µ-receptors participated in the regulation of glucose metabolism in an insulin deficient state. Endogenous -endorphin has physiological activities that are mediated by opioid µ-receptors (Goldstein, 1987; Pasternak, 1993). Both naloxone (Martin, 1967) and naloxonazine (Ling et al., 1986) at doses sufficient to block opioid µ-receptors (Cheng et al., 2001b) suppressed the plasma glucose-lowering activity of isoferulic acid in STZ-diabetic rats. This suggests that the plasma glucose-lowering activity of isoferulic acid in STZ-diabetic rats may be mediated by opioid µ-receptors. However, these antagonists may have nonspecific effects in addition to the blockade of opioid µ-receptors. Therefore, we used opioid µ-receptor knockout mice (Loh et al., 1998) to further examine the role of opioid µ-receptor activation in the activity of isoferulic acid. In contrast with the effect in mice with opioid µ-receptors, isoferulic acid did not have plasma glucose-lowering activity in opioid µ-receptor knockout diabetic mice. These data suggest that opioid µ-receptors play an essential role in the hypoglycemic activity of isoferulic acid in the insulin-deficient state.

In diabetes, elevation of blood glucose is a consequence of increased hepatic glucose output in concert with reduced peripheral glucose use (Consoli et al., 1989). PEPCK, which catalyzes a regulatory step in gluconeogenesis, is one of the key enzymes in hepatic carbohydrate metabolism (Consoli et al., 1989). Insulin deficiency is clearly associated with a change in hepatic metabolism (Consoli et al., 1989). Moreover, reduction in insulin-mediated glucose uptake caused by decreasing gene expression of GLUT 4 has been reported in diabetes skeletal muscle, a major site for glucose disposal (Berger et al., 1989; Sivitz et al., 1989). In STZ-diabetic rats, we found that the plasma glucose-lowering activity of isoferulic acid was associated with attenuation of the increased hepatic PEPCK gene expression (Liu et al., 2000a). Meanwhile, an increase in the gene expression of GLUT 4 may contribute to the enhancement of glucose use in isoferulic acid-treated STZ-diabetic rats (Liu et al., 2000a). In the present study, ₁-adrenoceptor antagonists were used to elucidate the role of this receptor in isoferulic acid-induced changes in glucose metabolism-related gene expression. We observed that the mRNA and protein levels of GLUT 4 increased by isoferulic acid were reversed by pretreatment with ₁-adrenoceptor antagonists at the dose sufficient to inhibit -endorphin secretion. Phospholipase C and protein kinase C also participate in the regulation of the glucose transporter system (Ishizuka et al., 1990; Van Epps-Fung et al., 1997). This is consistent with the report that activation of ₁-adrenoceptors by isoferulic acid leading to enhanced glucose uptake into myoblast C₂C₁₂ cells was mediated via the phospholipase C-protein kinase C-related pathway (Liu et al., 2001b). Therefore, an increase of GLUT 4 gene expression by isoferulic acid after activation of ₁-adrenoceptors may use the same signaling pathway. Moreover, we found that pretreatment with ₁-adrenoceptor antagonists significantly affected hepatic PEPCK gene expression in isoferulic acid-treated STZ-diabetic rats. Thus, the effect of isoferulic acid on glucose homeostasis, including PEPCK gene expression, was also related to the activation of ₁-adrenoceptors.

We have previously demonstrated that endogenous -endorphin via the activation of opioid receptor is a positive regulator in the glucose use and a negative modulator in hepatic gluconeogenesis in the insulin-deficient state (Liu et al., 1999a; Cheng et al., 2001b, 2002). Thus, we used opioid µ-receptor antagonists to clarify the role of this receptor in isoferulic acid-induced changes in gene expression associated with glucose metabolism. In the presence of opioid µ-receptor antagonists, isoferulic acid failed to elevate GLUT 4 mRNA and protein levels in the soleus muscle of diabetic rats. Activation of opioid µ-receptors by isoferulic acid mediated the increase of GLUT 4 gene expression in soleus muscle of diabetic rats lacking insulin. Also, the suppression of PEPCK gene expression in STZ-diabetic rat by isoferulic acid was blocked by opioid µ-receptor antagonists. It is likely that normalization of hepatic gluconeogenesis by isoferulic acid is also mediated through activation of opioid µ-receptors. Together, we demonstrated that both ₁-adrenoceptors and opioid µ-receptors were involved in the regulation of glucose by isoferulic acid in an insulin deficiency state. In adrenal glands, ₁-adrenoceptors were stimulated by isoferulic acid resulting in increased secretion of -endorphin, which activated opioid µ-receptors to modify gene expression of GLUT 4 and PEPCK, gene products that are associated with glucose homeostasis and involved in lowering the high plasma glucose levels in STZ-diabetic rats.

In conclusion, our results suggest that activation of ₁-adrenoceptors, especially the _1A-subtype, by isoferulic acid in the adrenal medulla may enhance the secretion of endogenous -endorphin from the adrenal gland of STZ-diabetic rats. The plasma glucose-lowering activity of isoferulic acid was obtained by the released -endorphin via an activation of opioid µ-receptors to achieve the enhancement of GLUT 4 gene expression and/or the amelioration of the elevated hepatic PEPCK gene expression. Thus, the improvement of glucose use by isoferulic acid in skeletal muscle and the decline of hepatic gluconeogenesis in liver leading to lowering of the plasma glucose concentration in STZ-diabetic rats may be mediated by endogenous -endorphin.

	Acknowledgements

 
We thank Professor F. L. Hsu for the donation of isoferulic acid and Professor H. H. Loh for the kind supply of opioid µ-receptor knockout mice and wild-type mice. We are grateful to Professors R. W. Hanson, C. Makepeace, and S. S. Liu for kindly providing plasmid-containing cDNAs. We also thank Professor D. K. Granner for the supply of antibodies specific to PEPCK and Professor Y. C. Tong for editing.

	Footnotes

 
ABBREVIATIONS: STZ-diabetic rats, streptozotocin-induced diabetic rats; BER, -endorphin-like immunoreactivity, GLUT 4, glucose transporters subtype 4; PEPCK, phosphoenolpyruvate carboxykinase; WB 4101, 2-[2,6-dimethoxyphenoxyethyl]aminomethyl-1,4-benzodioxane hydrochloride.

The present study is supported in part by a grant from National Science Council (NSC90-2320-B006-039) of the Republic of China.

DOI: 10.1124/jpet.103.053900.

Address correspondence to: Professor Juei-Tang Cheng, Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan City, Taiwan 70101, Republic of China. E-mail: jtcheng{at}mail.ncku.edu.tw

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