Insulin restores neuronal nitric oxide synthase expression in streptozotocin-induced diabetic rats

Life Sciences 68 (2000) 625–634

Insulin restores neuronal nitric oxide synthase expression in streptozotocin-induced diabetic rats Wen-Jen Yua, Show-Wen Juanga, Wan-Tai China, Tzong-Cherng Chia, Chih-Jen Changb, Juei-Tang Chenga,* a

Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan City, Taiwan 70101, R.O.C. b Department of Family Medicine, College of Medicine, National Cheng Kung University, Tainan City, Taiwan 70101, R.O.C. Received 5 November 1999; accepted 11 July 2000

Abstract Nitric oxide (NO) is known to play an important role in the pathophysiology of insulin-dependent diabetic mellitus (IDDM). In an attempt to investigate the relation between insulin and NO in IDDM, the present study employed male Wistar rats to induce IDDM by intravenous injection of streptozotocin (STZ). Four groups of rats were used; untreated normal control group, insulin treated STZ group, vehicle-treated STZ control, and one group of age-matched rats which were orally supplied with glucose to increase plasma glucose (glucose-challenged rats). Changes of the activity and gene expression of neuronal nitric oxide synthase (nNOS) were examined in cerebellum and kidney of these groups. The activity of nNOS in cerebellum, determined by conversion of [3H] L-arginine to [3H] L-citrulline, in STZ-induced diabetic rats was markedly lower than normal rats. Insulin treatment reversed the nNOS activity. Similar reversion by insulin treatment was also obtained in the gene expression of nNOS. However, the activity and gene expression of nNOS in glucose-challenged rats were not different from those in normal rats. The role of hyperglycemia can thus be ruled out. These findings indicated that an impairment of nNOS in the brain of rats with IDDM is mainly due to the absence of insulin. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Nitric oxide synthase; Insulin dependent diabetic mellitus; Streptozotocin; Insulin; Glucose-challenge; Wistar rat

Introduction Nitric oxide (NO), known as the endothelium derived relaxing factor (EDRF) [1], a vascular and neuronal messenger and/or a cytotoxic and cytostatic agent [2], is synthesized from * Corresponding author: Fax: 1 886-6-238-6548. E-mail address: [email protected] (J.-T. Cheng) 0024-3205/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 9 6 7 -X

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L-arginine by the nitric oxide synthase [3]. Isoforms of NO synthases were the continuously expressed neuronal (nNOS) and endothelial (eNOS) forms, which are calcium dependent, in addition to an another calcium independent form, the inducible NOS (iNOS)[4]. Streptozotocin (STZ)-treated rats are widely used as model for insulin dependent diabetic mellitus (IDDM) which are characterized by generated hyperglycemia, hyperuria, hypoinsulinemia, and a lower gain in weight [5]. The role of NOS in diabetic disorder varied considerably in different tissues. In the hypertensive STZ-diabetic rats, impaired NOS was observed in studies with the NOS stimulant and inhibitor, L-arginine and Nv-nitro-L-arginine methyl ester (L-NAME), respectively [6]. In isolated Langerhans islets and in total pancreas homogenate, less enzyme activity of NOS was observed in diabetic animal [7]. Moreover, the NOS activity was also decreased in the platelets of IDDM patient [8]. The role of NOS has also been implicated to form NO in infections and inflammation [9]; an increase in iNOS activity and/or gene up-regulation has been documented in the macrophage [10]. Cerebral NOS pathway was reported to regulate the peripheral insulin action and secretion [11], but the NOS gene expression in STZ-diabetic rats remains unknown. We reported that STZ-diabetic rats expressed a lower mRNA level for nNOS and a decreased activity of NOS in the cerebrocortex [12]. Hyperglycemia or hypoinsulinemia seems responsible for the change of NOS expression. In the present study, the role of insulin in the nNOS expression in cerebellum and kidney was investigated using the treatment with exogenous insulin in STZ-diabetic rats. The nNOS protein was well known to be expressed in central nervous system (CNS) tissues. Since NOS mediated malfunction of kidney was reported in the diabetic rats [13], the nNOS expression in kidney tissues was investigated to compare with that in the cerebellum. The role of hyperglycemia was also examined in rats manipulated with oral glucose intake.

Materials and methods Animals The male Wistar rats aged 8-weeks old were obtained from the Animals Center of National Cheng Kung University Medical College. 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. They were housed in a climatecontrolled, light-regulated space with 12-hour light and dark cycles, water and food ad libitum. The IDDM state was induced, according to the previous method [5], by an intravenous injection of STZ at 60 mg/kg dissolved in 1% citrate buffer into the femoral vein of adult Wistar rats. Rats received a similar injection of vehicle at the same volume were used as control. Plasma samples taken from the femoral vein of rats was employed to determine the glucose level using glucose-oxidase method [14]. Rats with hyperglycemia in addition to hyperuria, hypoinsulinemia, and a lower gain in weight were used as the animal model of IDDM. Half of the IDDM rats were then subcutaneously injected with insulin (0.8 IU/kg) into femoral vein. The injection of drugs and blood sample collecting procedures were under anesthesia with pentobarbital (30 mg/kg, i.p.). The age-matched rats were orally administrated glucose (1 g/Kg body weight) every 2 hours for 1 week to induce hyperglycemia named as the glucose-challenged rats.

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NOS activity assays Cerebellum and kidneys tissues were used for determination of NOS. Rats were killed and cerebellum and kidneys tissues were immediately excised, cleaned with phosphate-buffer solution (PBS), frozen in liquid nitrogen, and stored at 2708C for Northern blotting and NOS activity assay. Homogenates were prepared in 10 mmol/L HEPES buffer, pH 7.4, containing 320 mmol/L sucrose, 1 mmol/L EDTA, 1mmol/L DL-dithiothreitol (DTT), 10 mg/mL leupeptin, and 2 mg/mL aprotinin at 08C to 48C with the aid of a tissue grinder fitted with a motordriven ground glass pestle. Homogenates were centrifuged at 12 000 3 g for 5 minutes under 48C to remove tissue debris without precipitation of plasma membrane fragments. The supernatant was used for determination of NOS activity and protein mass. Protein concentration was determined with a Bio-Rad kit. The activity of NOS was measured as our previous method [15]. In brief, enzyme reactions were conducted at 378C for 30 minutes in 40 mL of the supernatant and 100 mL of 40 mmol/L potassium phosphate buffer, pH 7, containing 4.8 mmol/L DL-valine, 1 mmol/L NADPH, 1 mmol/L MgCl2, 2 mmol/L CaCl2, 20 mmol/L L-arginine, 1 mg/mL calmodulin, and 1.25 mL/mL L-[3H]arginine (59 Ci per mmol/L, Amersham Life Science Inc). For each occasion, parallel measurements were obtained in the presence and absence of 1 mmol/L NG-methyl-L-arginine. The reactions were terminated by an addition of 0.86 mL ice-cold buffer containing 0.2 mmol/L EDTA. Dowex 50W-X8 resin (250 mg, Na1 form) was added to a 0.25-mL aliquot of the reaction mixture and shaken for at least 5 minutes to remove the remaining L-arginine. The Na1 form of Dowex 50W was prepared by washing the H1 form of the resin (100 to 200 mesh, Bio-Rad) with 1 mol/L NaOH four times and then washing with H2O until the pH fell below 7.5. The above mixture was then centrifuged, and a 100-mL aliquot of the supernatant containing L-citrulline was mixed with 10 mL of scintillation cocktail in a 20-mL scintillation vial and counted by a beta counter. Net radioactivity was determined by subtracting the counts per minute observed in the presence of NG-methyl-L-arginine from that observed in the absence of NG-methyl-Larginine. NOS activity was determined from the production of [3H]citrulline per minute per milligram of protein. The endotoxin lipopolysaccharide (LPS, 10 mg/ml) was used to stimulate the iNOS activity. Northern blotting The mRNA level was investigated using Northern blotting analysis as reported previously [15]. Tissues were homogenized and total RNA extraction was achieved with a commercial kit, Ultraspect II (Amersham), a modified guanidinium isothiocyanate-phenol-chloroform method. Thirty micrograms of total RNA was separated via an electrophoresis of agarose (1%) / formaldehyde (1 mol/L) gel for 2 hours using a mixture of 20 mmol/L 3-[N-morpholine]-propanesulfonic acid buffer. Then, RNA was transferred to nylon membranes in 20X saline-sodium citrate (SSC; 0.15 mol/L sodium citrate, pH 7.0). After the transfer, the RNA was covalently bound to nylon membrane using UV cross-linker (Stratagene) and dried at room temperature at least 2 hours for Northern blotting. The plasmid with cDNA of nNOS was obtained from Professor N. Taniguchi and the cDNA of b-actin was from Professor H. S. Liu. The cDNA probe of nNOS was labeled with [32-P]dCTP using a random primer kit from Amersham. The membrane was prehybridized in the Quickhyb buffer purchased from Amer-

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sham at 688C for 30 min, and hybridized in the same buffer for 3 hours. After hybridization , the membrane was washed twice in room temperature (RT) with buffer containing 23 SSC and 0.1% sodium dodecyl sulfate (SDS) at 408C for 20 min. Autoradiograms were obtained at 2708C using Kodak XAR film. The intensity of autoradiogram was scanned with a transmission densitometer. The blotted membrane was washed in 13 SSC plus 0.1% SDS buffer at 1008C for 1 min, and rehybridized with b-actin probe at 688C for 1 hour to obtain the internal standard. The washing process was described as above. Western blotting Western blotting analysis for NOS protein was carried out as described previously [3] with some modifications. Tissues were homogenized in the 20 % (weight/volume) 20 mmol/L Tris/EDTA buffer, pH 7.4, containing 100 mM pepstatin, 100 mg/mL aprotinin, 10 mmol/L, EDTA, 100 mg/mL leupeptin, 1 mmol/L phenanthroline, and 1 mmol/L E-64 (Sigma). The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on a 12.5 % gel, with a 3 % stacking gel. Gels were reach 2 mm thick and 30 mg of total protein/well was loaded. After SDS-PAGE, the protein was transferred into nitrocellulose paper with a transfer time of 1 hour and a voltage of 100 mV. Immunoblotting analysis was performed using a mouse monoclonal anti-nNOS or anti-nNOS antibody (1:1000) as primary antibody and a horse radish-peroxidase-conjugated goat anti-mouse IgG (1:2000) as secondary antibody. The bound antibody was detected using the Enhanced Chemi-Luminiscense (ECL) kit and quantified by densitometry. Statistical analysis Statistical analysis was made by the Student’s t-test for comparison of two means. P values less than 0.05 were considered statistically significant. Results In STZ-diabetic rats, plasma glucose became 479.9 6 10.4 mg/dL (mean 6 SEM, N5 10) which was markedly (P,0.001) different from the normal rats (87.7 6 10.4 mg/dL; mean 6 SEM, N510). Subcutaneously injection of insulin (0.8 IU/kg2/day) for three days reduced the plasma glucose to the normal value. In age-matched non-diabetic rats, oral administration of glucose (1 g/kg) every 2 hours for 1 day raised the plasma glucose to 116.2 6 8.7 mg/dL (mean 6 SEM, N5 9). After 1 week of glucose treatment, the plasma glucose in glucosechallenged rats increased to 145.0 6 26.6 mg/dL (mean 6 SEM, N59) which was significantly (P,0.05) higher than that of normal rats. The NOS activity determined by the conversion of [3H] L-arginine to [3H] L-citrulline was indicated as pmol/min/mg of tissue protein. Table 1 shows the basal activity of nNOS in cerebellum tissues and the level of iNOS activity stimulated with the endotoxin, lipopolysaccharide (LPS). The nNOS activity in cerebellum of STZ-diabetic rats was significantly lower than that of the normal control (P,0.05). After insulin treatment, the nNOS activity in diabetic rats was restored to the level similar to control (P.0.05). On the other hand, in the glucose-challenged rats, nNOS activity was higher than normal control (P,0.05).

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Table 1 Changes of neuronal nitric oxide synthase (nNOS) and inducible NOS (iNOS) both activity and enzyme (immunoblot) in the cerebellum of streptozotocin-induced diabetic rats (STZ) to compare with non-diabetic Wistar rats (normal), STZ-diabetic rats treated with insulin (STZ1insulin) and glucose-challenged rats (Hyperglycemic) Normal

STZ

STZ1insulin

Hyperglycemic

nNOS activity (pmol/min/mg) Immunoblot

3.3460.44 100

2.7160.10a 42.366.83a

3.0260.11 93.4669.56

4.3560.46a 96.4967.10

iNOS activity (pmol/min/mg) Immunoblot

2.9960.33 100

2.4860.05a 88.3962.68a

2.6260.26 97.6863.86

3.8260.44a 99.45614.07

iNOS1LPS activity (pmol/min/mg)

4.6860.61

3.9460.18a

5.4060.60

5.4160.16a

Values (mean 6 SEM) were obtained from the determination of 8–10 animals. a P,0.05 vs. value in Normal.

In the kidney, nNOS in STZ-diabetic rats was also markedly lower than the control (Table 2). However, treatment with insulin failed to restore the nNOS activity in the kidney (Table 2). Glucose challenged rats exhibited a lower (P,0.05) nNOS activity compared to the control (Table 2). The trend of basal and stimulated level of inducible NOS (iNOS) activity was similar to that of nNOS in cerebellum (Table 1). In kidney homogenates incubated with LPS, the iNOS activity in each group was not statistical different (Table 2). Change of the immunoblot of NOS protein parallels the change of NOS activity. In the cerebellum region, the NOS protein was significantly (P,0.05) lower in STZ-diabetic rats than that in the normal control (Fig. 1, Table 1). In the glucose-challenged rats and insulin treated STZ-diabetic rats, the NOS protein in cerebellum remained the same as control. In the kidney of STZ-diabetic rats, the immunoblot of nNOS and iNOS proteins was significantly (P,0.05) decreased as shown in Fig. 2 and Table 2. In addition, treatment with insulin failed to restore the expression of nNOS protein (Fig. 2). Figure 3 showed the representative blot for the mRNA of nNOS expressed in cerebellum. In the cerebellum region, the STZ-diabetic rats expressed a lower mRNA level of nNOS than the normal control. Also, the decreased expression of nNOS mRNA was enhanced by the treatment of insulin (Fig. 3). However, expression of nNOS mRNA in glucose-challenged Table 2 Changes of neuronal nitric oxide synthase (nNOS) and inducible NOS (iNOS) both activity and enzyme (immunoblot) in the kidney of streptozotocin-induced diabetic rats (STZ) to compare with non-diabetic Wistar rats (normal), STZ-diabetic rats treated with insulin (STZ1insulin) and glucose-challenged rats (Hyperglycemic) Normal

STZ

STZ1insulin a

a

Hyperglycemic

nNOS activity (pmol/min/mg) Immunoblot

2.9260.21 100

2.4960.19 76.5169.00a

2.4360.25 86.1764.25a

2.5660.13a 99.5467.43

iNOS activity (pmol/min/mg) Immunoblot

2.4560.06 100

1.9060.23a 89.0566.60a

2.6460.26 104.47612.39

2.8860.14a 102.6563.88

iNOS1LPS activity (pmol/min/mg)

5.3760.27

5.0060.76

5.1060.50

Values (mean 6 SEM) were obtained from the determination of 8–10 animals. P,0.05 vs. value in Normal.

a

5.4460.22

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Fig. 1. Western blotting analysis of nitric oxide synthase including neuronal subtype (nNOS) and inducible subtype (iNOS) in the cerebellum of rats. Lane 1; Wistar rats, lane 2; streptozotocin-induced diabetic rats (STZ-rats), lane 3; insulin treated STZ-rats, and lane 4; glucose-challenged rats.

rats was the same as that in the normal control. The mRNA level of nNOS in the kidney showed a similar change as that in the cerebellum (Fig. 4). Discussion In the present study, we found a decrease of nNOS in the cerebellum and kidney of STZinduced diabetic rats. Because the medulla region of kidney contains nNOS [16], we compared kidney with cerebellum, which contains abundant nNOS [17]. In our previous study, lower NOS was observed in the cerebrocortex of STZ-diabetic rats [12]. Hyperglycemia and hypoinsulinemia were characterized as the major parameters in IDDM both in the experimental animal and patient. Thus, the decrease of NOS in IDDM may be also related to hyperglycemia and/or hypoinsulinemia. The role of hyperglycemia was investigated, but no significant change of the mRNA level of nNOS can be obtained in rats with hyperglycemia induced by oral administration of glucose. Furthermore, the activity of nNOS was increased in cerebellum, but decreased in kidney in these glucose-challenged rats. Mediation by hyperglycemia to lower nNOS in STZ-diabetic rats can thus be ruled out. In order to evaluate the effect of insulin on NOS pathway, the STZ-diabetic rats were subcutaneously injected with insulin. The presence of insulin receptor in brain for insulin that penetrates the blood-brain-barrier [18] made the CNS effect of peripheral injection of insulin possible. The mRNA level and protein production of nNOS was restored by insulin in addition to an enhanced enzymatic activity in the cerebellum. This is in agreement with previous studies that decrease of nNOS level may be an insulin-related effect [13,19].

Fig. 2. Western blotting analysis of nitric oxide synthase including neuronal subtype (nNOS) and inducible subtype (iNOS) in the kidney of rats. Lane 1; Wistar rats, lane 2; streptozotocin-induced diabetic rats (STZ-rats), lane 3; insulin treated STZ-rats, and lane 4; glucose-challenged rats.

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Fig. 3. Northern blotting analysis of the mRNA level for neuronal nitric oxide synthase (nNOS), and beta-actin (b-actin) in cerebellum of rat. Upper picture (A) shows the representative response and lower column (B) indicates the quantified data (n58). Lane 1; Wistar rats, lane 2; streptozotocin-induced diabetic rats (STZ-rats), lane 3; insulin treated STZ-rats, and lane 4; glucose-challenged rats. * P,0.05 vs. Wistar group.

Although iNOS expression in DM remained controversial [20,21], we found that immunoblot of iNOS protein was decreased in STZ-diabetic rats and insulin restored the iNOS protein production. However, we failed to detect the mRNA of iNOS in both tissues using Northern blotting analysis probably due to limited expression of iNOS in non-stimulated condition. Otherwise, the basal level of iNOS activity was decreased in STZ-diabetic rats while increased in glucose-challenged rats in both tissues. Actually, insulin restored the iNOS activity indicating the lowering of basal iNOS activity was insulin-related. Effect of insulin on iNOS expression in kidney tissue may be via cytokine/endotoxin mediated pathway [22]. An increase of basal iNOS activity in glucose-challenged rats seems also insulin-related since insulin secretion was raised in rats receiving glucose [23].

Fig. 4. Northern blotting analysis of the mRNA level for neuronal nitric oxide synthase (nNOS), and beta-actin (b-actin) in kidney of rat. Upper picture (A) shows the representative response and lower column (B) indicates the quantified data (n58). Lane 1; Wistar rats, lane 2; streptozotocin-induced diabetic rats (STZ-rats), lane 3; insulin treated STZ-rats, and lane 4; glucose-challenged rats. * P,0.05 vs. Wistar group.

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The relationship between NO and DM has been widely documented [24–26]. Due to the vascular relaxing activity, increase of NO should reduce the blood pressure to some extent [27]. In fact, cardiovascular diseases (CVD) are very common in DM patients [28]. The expression of NOS seems important in the DM-induced CVD. An impairment of NOS expression, especially constitutive expressed NOS (cNOS), in vascular system has been documented [6]. Actually, in IDDM patients, a reduction of NOS was found in platelet [8]. Thus, a lower level of NOS possibly accelerated the development of CVD in DM patient [29]. There are two isoforms of cNOS, endothelial NOS (eNOS) and neuronal NOS (nNOS). In the vascular system, eNOS is the major form of cNOS in keeping with in the homeostasis of blood pressure [30]. Neuronal NOS was mainly expressed in the nervous system, however, limit information of nNOS in IDDM was available. Studies of chronic inhibition of NOS indicated that central NOS pathway may be involved in the regulation of the peripheral insulin action and secretion [11]. Induction of iNOS is known to be activated by endotoxin and/or cytokines [22] and in some circumstances concurrently inhibited the function of cNOS [31]. In this study, we provided the evidence that NOS expression in CNS can be restored by exogenous insulin. Failure of insulin to restore the nNOS expression in kidney of STZ-diabetic rats may be due to the defect in insulin binding to the receptors. Although the amount of insulin receptor in the kidney was increased, impaired renal function was found with a reduced insulin binding activity in the kidney of IDDM rats [32]. Furthermore, the number of insulin receptor in cerebellum of IDDM rats was the same as normal control [33] and exogenous insulin can restore the NOS expression in this area. Therefore, decrease of insulin binding activity in cerebellum of IDDM rats can be ruled out. Since oral glucose uptake was reported to induce an increase of plasma insulin level [34] and hyperinsulinemia in turn caused a decrease of the insulin binding activity in kidney [35], a decrease of nNOS activity in the kidney of glucosechallenged rats seems reasonable. Acknowledgments We thank Professor Taniguchi, N. and Professor Liu. H. S. for the kindly supply of plasmid, and Professor Kwan, D.C. for editing. The present study was supported in part by a grant from National Science Council (NSC88-2314-B006-043) of the Republic of China. References 1. Forstermann U, Pollock JS, Nakane M. Nitric oxide synthase in the cardiovascular system. Trends in Cardiovascular Medicine 1993;3(1) :104–110. 2. Nathan C, Xie QW. Nitric oxide synthases: roles, tolls, and controls. Cell 1994;78(6):915–918. 3. Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proceedings of the National Academy of Science of the United States of American 1987;84(24):9265–9269. 4. Fosterman U, Shmidt HH, Pollock JS, Sheng H, Mitchell JA, Warner TD, Nakane M, Murad F. Isoforms of nitric oxide synthase. Characterization and purification from different cell types. Biochemical Pharmacology 1991;42(10): 1849–1857. 5. O’Donnell MP, Kasiske BL, Keane WF. Glomerular hemodynamic and structural alterations in experimental diabetes mellitus. FASEB Journal 1988;2(8): 2339–2347.

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