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Involvement of Bradykinin and Nitric Oxide in Leptin-Mediated Glucose Uptake in Skeletal Muscle

Department of Medical Biochemistry, Ehime University School of Medicine, Shigenobu, Japan

Address all correspondence and requests for reprints to: Masatsugu Horiuchi, M.D., Ph.D., Department of Medical Biochemistry, Ehime University School of Medicine, Shigenobu, Onsen-gun, Ehime 791-0295, Japan. E-mail: horiuchi{at}m.ehime-u.ac.jp

	Abstract

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Regulation of glucose metabolism in peripheral tissues by leptin has been highlighted recently, although its mechanism is unclear. In this study, we postulated that bradykinin and nitric oxide (NO) are involved in the effect of leptin-mediated glucose uptake in peripheral tissues and examined these possibilities. Injection of leptin (200 pg/mouse) into the ventromedial hypothalamus-enhanced glucose uptake in skeletal muscle and brown adipose tissue, but not in white adipose tissue. Treatment with Hoe140 (0.1 mg/kg), bradykinin B2 receptor antagonist, or L-NAME (N^G-nitro-L-arginine methyl ester) (30 mg/kg), nitric oxide synthase inhibitor, did not influence the basal level of glucose uptake in skeletal muscle and the adipose tissue, whereas Hoe140 and L-NAME inhibited leptin-mediated glucose uptake in skeletal muscles, but had no effect in adipose tissue. However, Hoe140 and L-NAME did not inhibit insulin (1.0 U/kg)-mediated glucose uptake in all tissues examined. Taken together, these results suggest that leptin enhances bradykinin and/or the NO system, which contributes at least partially to the enhanced glucose uptake in skeletal muscles.

	Introduction

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LEPTIN, a hormone secreted by adipose tissue, and its receptor are integral components of the system to regulate fuel stores and energy balance at an optimum level. Leptin also signals nutritional status to several other physiological systems and modulates their functions. Leptin acts in the hypothalamus and also exerts direct effects in several peripheral tissues. Recently, Kamohara et al. (1) demonstrated that acute iv or intracerebroventricular (ICV) administrations of leptin resulted in increased glucose turnover and glucose uptake, but decreased hepatic glycogen content, whereas the plasma levels of insulin and glucose did not change. We also reported that microinjection of leptin into the ventromedial hypothalamus (VMH) significantly increased glucose uptake in several peripheral tissues in rats without a significant change of plasma insulin concentration (2, 3). This accumulating evidence suggests that leptin plays important roles in glucose homeostasis (1, 2, 3, 4, 5, 6, 7, 8, 9) while the mechanism is poorly understood.

The effects of bradykinin on glucose metabolism in peripheral tissues have been focused on. It has been suggested that bradykinin can potentiate insulin-induced glucose uptake in dog skeletal muscle and rat L6 myoblasts (10, 11). Very recently, Kishi et al. (12) proposed that bradykinin directly stimulates GLUT4 translocation and increases 2-deoxy-D-glucose (2-DG) uptake through an insulin-independent pathway in both 3T3-L1 adipocytes and L6 myotubes. Interestingly, nitric oxide synthase (NOS) is expressed in skeletal muscle (13, 14), and chronic treadmill training increased the expression of both type I and type III NOS (nNOS and eNOS) in soleus muscle, suggesting that NO is involved in the signal transduction mechanism to increase glucose transport during exercise (15, 16, 17). Moreover, it is well established that bradykinin increases eNOS activity, which consequently produces NO (18, 19). These results led us to postulate that the effect of leptin on glucose uptake in skeletal muscle is at least partially dependent on bradykinin and the NO system.

	Materials and Methods

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Animals
Male FVB/N mice (Nihon Clea, Tokyo, Japan) weighing 20–30 g were used. They were housed individually in plastic cages at 25 ± 1 C with lighting on from 0700 to 1900 h and given a laboratory diet and water ad libitum. Under pentobarbital anesthesia (50 mg/kg ip), mice were stereotaxically implanted with a chronic double-walled stainless steel cannula in the unilateral VMH according to the atlas of Franklin and Paxinos (20). The stereotaxic coordinates used were as follows: 1.5 mm anterior to the interaural line, 0.3 mm lateral to the sagittal suture, and 6.0 mm below the surface of the skull. The cannula was then anchored firmly to the skull with acrylic dental cement. Five days after implantation of the brain cannula, a silicone cardiac catheter was implanted into the right atrium through the external jugular vein. The mice were repeatedly handled during the recovery period to habituate them to the injection and blood sampling procedures. Correct placement of the tips of the cannulas was verified microscopically in brain sections stained with Cresyl violet when the experiments were completed. All experimental procedures were approved and carried out in the compliance with guidelines of Ehime University School of Medicine Committee on Animals.

Administration of leptin, insulin, Hoe140, and L-NAME
The experiment was started at 1000 h. Food was removed at 0900 h. Recombinant murine leptin (200 pg) (Pero Tech EC, London, UK) dissolved in 0.2 µl saline solution was injected into the VMH in conscious mice through the implanted brain cannula using a Hamilton microsyringe. Control mice were injected with 0.2 µl saline into the VMH. Hoe140 (100 µg/kg) (Peptide Institute, Inc., Oosaka, Japan), L-NAME (30 mg/kg) (Funakoshi, Tokyo, Japan) or saline (0.1 ml) was injected into the right atrium through the cardiac catheter 15 min before and 45, 105, 165 min after the microinjection of leptin or saline. Insulin (1.0 U/kg) was injected into the right atrium through the implanted cardiac catheter before the injection of the 2-[³H] DG. Hoe140 (100 µg/kg), L-NAME (30 mg/kg), and saline (0.1 ml) were also injected into right atrium through the cardiac catheter 15 min before the injection of insulin or saline.

Measurement of rate constant of net tissue uptake of 2-deoxy-D-[³H]glucose
Three hours after the microinjection of leptin or 15 min after injection of insulin, 6.25 µCi 2-deoxy-D-[³H]glucose (2-[³H] DG) (10 Ci/mmol) and 1.25 µCi [¹⁴C]sucrose (4.5 mCi/mmol) (ICN Radiochemicals, Irvine, CA) dissolved in 0.05 ml saline solution were injected through the cardiac catheter. The catheter was then flushed immediately with 0.1 ml of saline. Blood was withdrawn just before the injection of Hoe140, L-NAME, or saline, and was also taken 0, 10, 15, and 20 min after injection of radioactive tracers. As soon as the final blood samples were obtained (20 min after injection of tracers), an overdose of sodium pentobarbital (100 mg/kg) was injected through the cardiac catheter and the mice were quickly decapitated. Interscapular brown adipose tissue (BAT), epididymal and retroperitoneal white adipose tissues (WAT), and skeletal muscles [extensor digitorum longus (EDL)], soleus, and red and white parts of gastrocnemius) were rapidly dissected and weighed. The rate constant of net tissue uptake of 2-[³H] DG was calculated as described previously (21). Plasma samples were analyzed for radioactivity of 2-[³H] DG and [¹⁴C] sucrose as well as glucose concentration, by the glucose oxidase method.

Statistical analysis
All values are expressed as mean ± SE. The effects of the different treatments on all data were evaluated with factorial ANOVA. When a significant effect was found, these results were further compared with Bonferroni’s multiple range test. The difference was considered significant if P < 0.05.

	Results

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Effects of leptin, bradykinin, and NO on rate constant of 2-[³H] DG uptake
First we examined the effect of leptin on glucose uptake in skeletal muscle and adipose tissue in mice. As shown in Fig. 1, injection of leptin into the VMH significantly increased the rate constant of 2-[³H] DG uptake in the white and red parts of gastrocnemius, soleus, and EDL muscles. Microinjection of leptin into the VMH also significantly increased the rate constant of 2-[³H] DG uptake in interscapular BAT, but not in epididymal and retroperitoneal WAT (Fig. 2). To investigate the possibility that bradykinin is involved in leptin-induced glucose uptake, we examined the effect of Hoe140 administration on the rate constant of 2-[³H] DG uptake. As shown in Fig. 1A, the increased rate constants of 2-[³H] DG uptake in the skeletal muscles by leptin were significantly reduced by Hoe140 administration. In contrast, Hoe140 did not influence the leptin-mediated increase in the rate constant of 2-[³H] DG uptake in BAT (Fig. 2A). Furthermore, to investigate the role of NO in leptin-induced glucose uptake in peripheral tissues, we examined the effect of L-NAME. We observed that the increases in glucose uptake in the skeletal muscles in response to leptin were significantly attenuated by L-NAME administration (Fig. 1B), whereas L-NAME did not affect the increase in glucose uptake in BAT (Fig. 2B).

View larger version (32K): [in this window] [in a new window]   Figure 1. Rate constant of 2-[3H] DG uptake in skeletal muscle in response to intrahypothalamic injection of leptin with Hoe140 or L-NAME. Leptin was injected into VMH in conscious mice through the implanted cannula. Hoe140 (100 µg/kg) or L-NAME (30 mg/kg) was injected through the cardiac catheter 15 min before and 45, 105, and 165 after the injection of leptin. Three hours after microinjection of leptin or saline into the VMH, 6.25 µCi 2-[3H] DG and 1.25 µCi [14C]sucrose were injected. The Ki values of tissue 2-[3H] DG uptake were determined as described in Materials and Methods. Results are mean ± SE of 4–6 mice. * ,**, P < 0.05, 0.01 vs. control. #, ##, P < 0.05, 0.01 vs. leptin.

View larger version (27K): [in this window] [in a new window]   Figure 2. Rate constant of 2-[3H] DG uptake in WAT and BAT in response to intrahypothalamic injection of leptin with Hoe140 or with L-NAME. Leptin, Hoe140 and/or L-NAME were injected as described in the legend of Fig. 1. Results are mean ± SE of 4–6 mice. *, P < 0.05 vs. control.

View larger version (38K): [in this window] [in a new window]   Figure 3. Rate constant of 2-[3H] DG uptake in skeletal muscle in response to iv injection of insulin with Hoe140 or with L-NAME. Insulin (1.0 U/kg) was injected in conscious mice through the cardiac catheter with or without Hoe140 (100 µg/kg) or L-NAME (30 mg/kg). Hoe140 or L-NAME was injected 15 min before the injection of insulin. Then 15 min after iv injection of insulin or saline, each mouse was injected with 6.25 µCi 2-[3H] DG and 1.25 µCi [14C] sucrose. The Ki values of tissue 2-[3H] DG uptake were determined as described in Materials and Methods. Results are mean ± SE of 5–6 mice. *, **, P < 0.05, 0.01 vs. control.

View larger version (40K): [in this window] [in a new window]   Figure 4. Rate constant of 2-[3H] DG uptake in WAT and BAT in response to iv injection of insulin with Hoe140 or with L-NAME. Insulin, Hoe140 and/or L-NAME (30 mg/kg) were injected as described in the legend of Fig. 3. Results are mean ± SE of 5–6 mice. **, P < 0.01 vs. control.

	Discussion

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Recently, Kishi et al. demonstrated that bradykinin triggers GLUT4 translocation from the cytosol to the plasma membrane, and this mechanism is independent of the insulin signaling pathway (13). Consistent with these results, we observed that B2 receptor inhibition specifically attenuated the increased glucose uptake in skeletal muscle by leptin but not by insulin. In contrast, it is also reported that bradykinin enhanced the insulin sensitivity in the presence of insulin in an in vitro system (10, 11). The mechanism of the cross-talk between insulin and bradykinin needs to be addressed.

Bradykinin is a stimulator of NO synthase, and the produced NO is assumed to be involved in glucose uptake in skeletal muscle. There are bradykinin B2 receptors (11, 22, 23) and two different kinds of NO synthase, nNOS and eNOS, in skeletal muscle (18, 19). Indeed, there is some evidence suggesting the involvement of NO in glucose transport (15, 16, 17, 24, 25, 26). Therefore, we speculated that enhanced NO production via bradykinin activated by leptin plays some roles in leptin-mediated glucose uptake. Accordingly, we demonstrated in this study that L-NAME attenuated the increased glucose uptake by leptin in skeletal muscle. NO has a strong vasodilator function (27, 28) and one can argue that inhibition of the vasodilation by L-NAME may contribute to the decrease in leptin-mediated glucose uptake. However, administration of L-NAME did not affect the basal level of glucose uptake and did not inhibit the insulin-mediated glucose uptake. Taken together, our results suggest that leptin enhances bradykinin and/or the NO system and results in enhanced glucose uptake in skeletal muscle.

It has been reported that iv administration of leptin (1 g/kg BW) to anesthetized Wistar rats increased serum NO concentrations significantly in dose-dependent manner (28). Moreover, arterial relaxation by leptin appeared to be mediated by NO released from endothelium (29). Consistent with these observations, our results suggest that NO production would be increased by the administration of leptin in VMH at least partially due to bradykinin. In contrast, ICV leptin injection has been reported to be capable of inhibiting diencephalic nitric oxide synthase (NOS) activity and ICV leptin administration increases brain serotonin (5-HT) metabolism in mice (30, 31). To our knowledge, the effect of leptin on bradykinin is enigma, although here we provided some evidence that leptin may activate bradykinin production system. The mechanisms of these effects of leptin on bradykinin and NO have to be further explored.

Received August 10, 2000.

	References

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