ISSN 0022-0930, Journal of Evolutionary Biochemistry and Physiology, 2013, Vol. 49, No. 2, pp. 153—164. © Pleiades Publishing, Ltd., 2013. Original Russian Text © A.O. Shpakov, K.V. Derkach, O.V. Chistyakova, I.V. Moiseyuk, I.B. Sukhov, V.M. Bondareva, 2013, published in Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, 2013, Vol. 49, No. 2, pp. 118—127.
COMPARATIVE AND ONTOGENIC BIOCHEMISTRY
Effect of Intranasal Insulin and Serotonin on Functional Activity of the Adenylyl Cyclase System in Myocardium, Ovary, and Uterus of Rats with Prolonged Neonatal Model of Diabetes Mellitus A. O. Shpakov, K. V. Derkach, O. V. Chistyakova, I. V. Moiseyuk, I. B. Sukhov, and V. M. Bondareva Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia E-mail:
[email protected] Received June 13, 2012
Abstract—The disturbances in hormonal signaling systems, adenylyl cyclase system (ACS) in particular, occur at the early stages of diabetes mellitus (DM) being one of the key causes of its complications. Since the correlation between the severity of DM and severity of disturbances in ACS is established, studying ACS activity can be used for monitoring DM and its complications and evaluating the effectiveness of their treatment. Recently, intranasal insulin (I–I) and the drugs increasing brain serotonin level, thus effectively restoring CNS functions, have begun to be used for the treatment of type 2 DM. However, the mechanisms of their action on peripheral tissues and organs at DM are not understood. The aim of this work was to study an influence of I–I and intranasal serotonin (I–S) on the functional activity of ACS in myocardium, ovary and uterus of rats with a neonatal model of type 2 DM. In the tissues of diabetic rats the changes in the regulation of adenylate cyclase (AC) by guanine nucleotides and hormones acting on enzyme in stimulatory and inhibitory manner were found, and these changes were characterized by receptor and tissue specificity. In diabetic rats I–I restored AC-stimulating effects of isoproterenol in the myocardium, that of guanine nucleotides and gonadotropin in the ovaries and relaxin in the uterus, as well as ACinhibiting effects of somatostatin in all tissues and norepinephrine in the myocardium. Treatment with I–S led to a partial recovery of AC-inhibiting effect of norepinephrine in the diabetic myocardium, but did not affect the regulation of AC by other hormones. These data indicate that I–I normalizes the functional activity of ACS in the myocardium and in the tissues of reproductive system of female rats with neonatal DM, whereas the effect of I–S on ACS in the studied tissues is less pronounced. These results should be considered for the design and optimization of the strategy of I–I and I–S application for the treatment of DM and its complications. DOI: 10.1134/S0022093013020047 Key words: adenylyl cyclase, adrenergic agonist, diabetes mellitus, gonadotropin, insulin, intranasal administration, myocardium, serotonin, somatostatin, ovaries.
Abbreviations used: AR—adrenergic receptor, AC—adenylyl cyclase, ACS—adenylyl cyclase system, GIDP—guanylyl imido diphosphate, I–I—intranasal insulin, I–S—intranasal sero-
tonin, DM—diabetes mellitus, SISR—selective inhibitors of serotonin reuptake, CHG—chorionic human gonadotropin, PACAP-38—pituitary adenylyl cyclase-activating polypeptide-38.
153
154
SHPAKOV et al.
INTRODUCTION We have recently shown that as early as before the appearance of diabetes mellitus (DM) complications in the nervous, cardiovascular, renal, and reproductive systems, multiple changes are revealed in the peripheral hormonal systems, including hormone-sensitive adenylyl cyclase system (ACS) [1–5]. These changes are supposed to induce disturbances in regulation of fundamental cellular processes by hormones and are among the major causes of DM complications [6–8]. The expression of the changes in the hormonal signaling systems in DM is established to be positively correlated with the severity and duration of disease, with the most pronounced changes generally revealed in the organs and tissues whose functions are disturbed to the greatest degree in DM [1, 3, 9, 10]. Therefore, study of the functional activity of hormonal systems is a quite perspective approach for highly sensitive monitoring of the state of the entire organism and its single organs and tissues under DM conditions, as well as for evaluation of efficiency and elucidation of mechanisms of therapeutic action of the preparations applied for treatment of DM and its complications. Traditionally, injections of peripheral insulin are used for treatment of the insulin-dependent type 1 DM and severe, decompensated forms of insulin-independent type 2 DM. However, such treatment induces a series of substantial complications, the major of which is a high risk of development of hypoglycemic conditions inducing severe complications in the CNS and cardiovascular system and in some cases leading to death due to development of hypoglycemic coma. For the recent years, other methods of hormone delivery have been applied for the search of alternative pathways of DM treatment, first of all, the intranasal one [11, 12]. Intranasal insulin (I–I) improves glycemic control, but does not significantly affect the level of peripheral glucose, which rules out a possibility of development of hypoglycemic conditions. I–I improves cognitive functions weakened under DM conditions, prevents development of retinopathy and decreases risk of cardiovascular diseases [13, 14]. However, the majority of works are focused on the clinical application of I–I, whereas the molecular mechanisms of its action
whose elucidation requires biochemical and physiological investigations on experimental models of DM have been not yet studied. Another approach started to be applied for treatment of the type 2 DM relatively recently is administration of drugs positively affecting the brain neurotransmitter systems regulated by biogenic amines, in particular by serotonin. These drugs acting through central mechanisms restore the tissues sensitivity to insulin and increase its secretion by pancreatic β-cells, thus improving glycemic control in diabetic patients [15–17]. At present, of major interest are selective inhibitors of serotonin reuptake (SISR), whose application leads to an increase in serotonin level in diabetic brain, restores the central serotoninergic system, and improves the severity and prognosis of the disease [15, 16]. In spite of the action mechanisms of the drugs increasing the serotonin level in the brain, in CNS under conditions of diabetic pathology are studied in detail, information on the mechanism of their action in peripheral systems, except for pancreas, is absent. It is to be noted that instead of SISR, widely used in clinical practice, serotonin itself at intranasal method of its administration might be used in experimental animals to increase the level of central serotonin that in this case comes directly to brain. The goal of this work was to study action of the intranasal insulin and serotonin administration on the functional activity and sensitivity of ACS to hormones in cardiac muscle and tissues of reproductive system of female rats with neonatal model of type 2 DM. The choice of neonatal model was dictated by the following circumstances. First, this model is close to human type 2 DM and is characterized by pronounced resistance of the nervous and peripheral tissues to insulin and moderate hyperglycemia typical of this DM form [18–20]. Second, earlier we identified the disturbances in regulation of ACS by hormones in the nervous and peripheral tissues of male and female rats with neonatal model of DM [2, 21, 22]. We used these and other disturbances in ACS as markers of the functional state of myocardium, uterus, and ovaries under conditions of neonatal DM. Third, we have shown earlier that I–I and intranasal serotonin (I–S) positively affect CNS under conditions of neonatal DM, which is indicated by
JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 49 No. 2 2013
EFFECT OF INTRANASAL INSULIN AND SEROTONIN ON FUNCTIONAL ACTIVITY
restoration of AC sensitivity to hormones in brain tissues, as well as by improvement of capability for learning and spatial memory in diabetic animals treated with these intranasal hormones [5, 20]. MATERIALS AND METHODS The female Wistar rats housed under the standard conditions and on the standard ration were used in experiments. Neonatal DM was induced by intraperitoneal injection to the 5-day-old rat pups of streptozotocin (Sigma-Aldrich, USA) diluted in 0.9% sodium chloride acidified with citric acid (pH 4.5) at a dose of 80 mg/kg of animal weight, as described in the work [19]. The animals from control group were given the acidified saline of the same volume. The test of glucose tolerance showed that streptozotocin-treated rats by the age of 3–4 months developed the pronounced resistance to insulin typical of the human type 2 DM. This was indicated by the results of the test of loading of animal with glucose (2 g/kg of weight), showed that 2 h after glucose administration its concentration in blood of diabetic animals was considerably higher than the normal level, whereas in the control animals it reached normal values. Measurement of glucose level was performed in whole blood obtained from tail vein of animals, using test-strips One Touch Ultra (USA) and glucometer from Life Scan Johnson & Johnson (Denmark). The intranasal hormone administration was performed by the method described earlier for I–I [23]. Crystal insulin (Lilly, USA) at a concentration of 24 IU/ml or serotonin (SigmaAldrich, USA) at a concentration of 1 mg/ml were dissolved in acidified saline (pH 4.5) and administered to diabetic and control rats intranasally once a day. The rat was turned to back and 10 μl of hormone solution (two drops of 5 μl volume) were administered to each nostril. Overall, each animal received 0.48 IU of insulin or 20 μg of serotonin. The treatment with intranasal hormones was carried out for 7 weeks. The control group was administered intranasally with acidified saline of the same volume and during the same period instead of hormone solutions. Thus, 6 groups of the 5-month-old rats were taken for investigation: control animals, not receiving hormones (group C, n = 10, body weight
155
277 ± 18 g, glucose level 4.8 ± 0.3 mМ), control animals administered I–I (Group CI, n = 9, 265 ± 16 g, 4.4 ± 0.3 mМ), control animals administered I–S (group IS, n = 9, 287 ± 17 g, 4.7 ± 0.2 mМ), animals with neonatal DM (group D, n = 8, 331 ± 16 g, 6.9 ± 0.4 mМ), diabetic animals administered I–I (group DI, n = 8, 298 ± 20 g, 6.1 ± 0.5 mМ), diabetic animals received I–S (group DS, n = 8, 318 ± 15 g, 6.6 ± 0.3 mМ). To perform biochemical experiments, there were used isoproterenol, noradrenalin, serotonin, chorionic human gonadotropin (CHG), somatostatin, pituitary adenylyl cyclase-activating polypeptide-38 (PACAP-38), forskolin and guanylyl imido diphosphate (GIDP) from SigmaAldrich (USA). Relaxin-2 was kindly provided by Prof. John Wade (Melbourne University, Australia). Radioactive [α-32P]-АTP (1000 Ci/μM) was obtained from the Public Corporation “Reaktornye materialy” (Isotop, Russia). The tissues were obtained 24 h after the last intranasal administration of hormone or physiological solution to the 6-month-old diabetic and control rats. The fraction of plasma membranes from the cardiac muscle was isolated as described earlier [24]. Myocardium separated from ventricles, fat, and heart valves, was rinsed with cold saline, minced and homogenized by using Polytron in 20 volumes of cold (4°C) 40 mM Tris-HCl buffer (pH 7.4) containing 5 mM MgCl2, 320 mM sucrose, and protease inhibitor cocktail (500 μM O-phenanthroline, 2 μM pepstatin, and 1 mM phenyl methyl sulfonyl fluoride) (buffer A). The homogenate was centrifuged at 480 g for 10 min, pellet was discarded, the supernatant was centrifuged at 27500 g for 20 min, the obtained pellet was resuspended in the buffer A without sucrose and centrifuged again at 27500 g for 20 min (all procedures were performed at 4°C). Isolation of fractions of ovaries and uterus plasma membrane was performed as described earlier [3]. The tissues of ovaries and uterus were minced and rinsed with cold A buffer and homogenized using Polytron in 20 volumes of the same buffer. The homogenate was centrifuged at 1500 g for 10 min, pellet was discarded, the supernatant was centrifuged at 20 000 g for 30 min, the obtained pellet was resuspended in 10 volumes of the buffer A without sucrose and centrifuged again at 20 000 g
JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 49 No. 2 2013
156
SHPAKOV et al.
Table 1. Basal AC activity in the fractions of plasma membranes isolated from myocardium, ovaries and uterus of control and diabetic rats treated and not treated with intranasal hormones Group of animals
Myocardium
Ovaries
Uterus
Basal AC activity, pmol cAMP/min per mg of membrane protein Group C
26.7 ± 1.2
13.4 ± 1.1
19.7 ± 1.8
Group CI
25.5 ± 1.0
14.4 ± 1.1
18.4 ± 0.7
Group CS
24.9 ± 1.6
13.1 ± 0.7
20.8 ± 1.3
Group D
29.1 ± 0.7*
10.1 ± 1.1*
18.8 ± 1.5
Group DI
26.6 ± 1.2#
12.3 ± 1.0#
19.0 ± 1.6
Group DS
27.7 ± 1.9
10.8 ± 0.8
17.9 ± 1.2
Note: *—differences between the C and D groups are significant at p < 0.05; #—between the groups D and DI, p < 0.05.
for 30 min (4°C). The fractions of plasma membranes obtained from myocardium, ovaries, and uterus were resuspended in 50 mM Tris-HCl buffer (pH 7.4) to obtain the protein content in the sample of 1–3 mg/ml, and stored at –70°C. The protein concentration was determined by using Lowry method with bovine serum albumin as the standard. The activity of adenylyl cyclase (EC 4.6.1.1) was determined as described earlier [25]. The reaction mixture (the total volume of 50 μl) contained 50 mМ Tris-HCl (pH 7.5), 5 mМ MgCl2, 0.1 mМ cАMP, 1 mМ АTP, 1 μCi [α-32P]-АTP, 20 mМ creatine phosphate, 0.2 mg/ml creatine phosphokinase, and 50–100 μg of membrane protein. The reaction was carried our for 10 min at 37°C, started by addition of plasma membrane fraction, and stopped by addition of 100 μl of 0.5 M HCl. The samples were boiled for 6 min to destroy the complex between labeled cAMP and proteins, and the acid was neutralized with 100 μl of 1.5 M imidazole. [32P]-cAMP generated in the enzyme reaction was isolated on columns with aluminum oxide by using 8 ml of 10 mM imidazole-HCl buffer (pH 7.4) as eluent. The eluate was collected into scintillation flasks and the radioactivity was counted on scintillation counter LS 6500 (Beckman Instruments Inc., USA). Each measurement was performed in three independent experiments in three or four parallel samples. The results are
presented as pmol cAMP/min per mg of membrane protein. The basal AC activity was measured in the absence of hormonal and non-hormonal agents; the inhibiting effect of hormones was studied by their effect on activity of AC stimulated by forskolin (10–5 M). Statistical analysis of the obtained data was performed by using software ANOVA. The data are presented as M ± m of several independent experiments. The differences between values of AC activity in the fractions of plasma membranes isolated from tissues of different animal groups were considered significant at p < 0.05. RESULTS Basal AC activity in myocardium of the 6-months-old diabetic rats was higher, while in the ovaries, on the contrary, lower as compared with that in control animals (Table 1). In uterus, no considerable changes in the basal AC activity were revealed. the 7-week I–I treatment decreased the basal AC activity in myocardium to its control level and substantially increased the basal enzyme activity in ovaries. Treatment of diabetic rats with I–S in this respect was ineffective. In control rats, GIDP, a non-hydrolyzed analog of GTP-activating Gs-proteins, coupled with AC in stimulating manner increased the basal activity of enzyme in all studied tissues. In diabetic animals its effect decreased significantly, to the greatest degree in ovaries (Table 2). The I–I treatment partially restored the GIDP-stimulated activity of enzyme in ovaries, but weakly changed AC-effects of guanylyl nucleotide in myocardium and uterus. The AC-stimulating effects of forskolin that directly interacts with the catalytic site of enzyme were comparable in the tissues of diabetic and control rats (Table 2). Only in ovaries of diabetic rats the AC-effect of forskolin was significantly lower than in control. Treatment of diabetic and control rats with intranasal hormones weakly affected the AC-action of forskolin. Then we performed a comparative investigation of regulatory effects of hormones by acting on AC through the Gs-proteins-coupled receptors, as well an effect of I–I and I–S on them. In myocardium of the control animal adrenergic agonists— isoproterenol and noradrenalin, and peptide hor-
JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 49 No. 2 2013
EFFECT OF INTRANASAL INSULIN AND SEROTONIN ON FUNCTIONAL ACTIVITY
157
Table 2. The AC activity stimulated by GIDP and forskolin in membrane fractions isolated from tissues of control and diabetic rats and action on it of intranasal hormones—insulin and serotonin Group of animals
Myocardium
Ovaries
Uterus
Stimulation of AC by GIDP (10–5 М), pmol cAMP/min per mg of membrane protein Group C
86.2 ± 2.9 (+223)
32.7 ± 1.2 (+144)
59.9 ± 1.8 (+204)
Group CI
79.3 ± 2.2 (+211)
36.1 ± 2.0 (+151)
54.8 ± 2.0 (+198)
Group CS
84.1 ± 3.0 (+238)
31.3 ± 1.2 (+139)
59.3 ± 1.7 (+185)
Group D
76.2 ± 3.4* (+162)
21.4 ± 1.0* (+112)
50.8 ± 1.3* (+170)
76.1 ± 1.7 (+186)
29.8 ± 1.5#, & (+142)
53.6 ± 2.5 (+182)
77.3 ± 2.0 (+179)
23.3 ± 1.6& (+116)
47.6 ± 1.3& (+166)
Group DI Group DS
Stimulation of AC by forskolin (10–5 М), pmol cAMP/min per mg of membrane protein Group C
148 ± 4 (+454)
43 ± 2 (+221)
86 ± 4 (+337)
Group CI
142 ± 3 (+457)
41 ± 2 (+185)
89 ± 2 (+384)
Group CS
153 ± 5 (+514)
41 ± 4 (+213)
91 ± 2 (+338)
Group D
158 ± 7 (+443)
34 ± 2* (+237)
81 ± 4 (+331)
Group DI
155 ± 7 (+483)
37 ± 2 (+201)
79 ± 2& (+316)
Group DS
144 ± 4 (+420)
33 ± 3 (+206)
85 ± 3 (+375)
Note: Digits in brackets—the AC-stimulating effect of GIDP or forskolin related to the basal activity of enzyme (%). *— Differences between groups C and D are significant at p < 0.05; # D and DI, p < 0.05; &—between groups CI and DI or CS and DS, p < 0.05.
Fig. 1. The adenylyl cyclase-stimulating effects of hormones in myocardium of control and diabetic rats and effect on them of intranasal hormones. Isoproterenol and norepinephrine are taken at a concentration of 10–5 М, relaxin—of 10–8 М. *—Differences between the C and D groups are significant at p < 0.05; #—between D and DI, p < 0.05, &— between CS and DS, p < 0.05.
mone relaxin stimulated the basal AC activity, while treatment with I–I and I–S weakly affected their actions (Fig. 1). In myocardium of diabetic
rats the AC-stimulating effect of isoproterenol significantly increased, whereas treatment of the animals with I–I (but not with I–S) decreased it to
JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 49 No. 2 2013
158
SHPAKOV et al.
Fig. 2. Adenylyl cyclase-stimulating effects of hormones in ovaries (A) and uterus (B) of control and diabetic rats and influence on them of intranasal hormones. CHG and relaxin are taken at a concentration of 10–8 М, PACAP-38—of 10–6 М, isoproterenol and serotonin—of 10–5 М; *—differences between the C and D groups are significant at p < 0.05; #—between D and DI, p < 0.05, &—between CI and DI or CS and DS, p < 0.05.
the control level. The AC effect of noradrenalin in diabetic myocardium has changed weakly, whereas the corresponding effect of relaxin decreased significantly, with the I–I and I–S treatment producing no substantial action. The AC effect of noradrenalin in diabetic myocardium has changed weakly, whereas corre-
sponding effect of relaxin was significantly diminished, with I–I and I–S treatment not exerting considerable action on it. In ovaries of control rats the most pronounced AC-stimulating effect was observed for PACAP-38, in uterus—for relaxin (Fig. 2). Treatment with intranasal hormones did not substantially af-
JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 49 No. 2 2013
EFFECT OF INTRANASAL INSULIN AND SEROTONIN ON FUNCTIONAL ACTIVITY
159
Fig. 3. The AC-inhibiting effects of somatostatin and norepinephrine in tissues of control and diabetic rats and effect on them of intranasal hormones. Somatostatin is taken at a concentration of 10–6 М, norepinephrine—of 10–5 М; *— differences between the C and D groups are significant at p < 0.05; #—between D and DI or D and DS, p < 0.05; &— between CI and DI or CS and DS, p < 0.05.
fect the AC actions of hormones in ovaries and uterus of control animals. In ovaries of diabetic rats the effects of all studied hormones were considerably decreased, to the greatest degree—that of CHG. I–I restored the AC effect of gonadotropin, but did not change the AC effects of PACAP-38 and serotonin. The I–I treatment did not restore AC effects of hormone in ovaries of diabetic rats, with them remaining significantly lower that in the groups C, CI, and CS. In uterus of diabetic animals a significant decrease in AC effects was observed only for relaxin, with a partial restoration by treatment with I–I, but not with I–S (Fig. 2). These data indicate that both a decrease of the AC-stimulating effects in myocardium and tissues of reproductive system of female rats with neonatal DM and their restoration after I–I treatment are characterized by the receptor and tissue specificity. At the final stage, we compared the inhibitory AC effects of peptide hormone somatostatin in all
tissues and of noradrenalin in myocardium; for this, we studied effect of hormones on the AC activity stimulated by diterpene forskolin (10–5 М). Under conditions of DM, the AC-inhibiting effects of somatostatin were shown to be significantly decreased in all tissues, to the greatest degree in ovaries (Fig. 3). In diabetic myocardium the AC-inhibiting effect of noradrenalin, realized via α2-adrenetgic receptors coupled with Gi-proteins also was decreased. Treatment with I–I fully restored the AC effect of somatostatin in myocardium completely, while in uterus—partially, but almost did not affect it in ovaries. The intranasal treatment of diabetic rats with the both hormones resulted in recovery of the AC-inhibiting effect of noradrenalin, with the action of I–I being more effective in comparison with I–S (Fig. 3). I–S did not change significantly the AC-inhibiting somatostatin effects weakened under the DM conditions. Thus, in neonatal DM the AC-inhibiting effects of somatostatin and noradrenalin decreased,
JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 49 No. 2 2013
160
SHPAKOV et al.
while the treatment of diabetic rats with I–I resulted in their partial or complete restoration in uterus and myocardium, whereas that with I–S— only in a partial recovery of noradrenalin effect in myocardium. DISCUSSION Earlier, we have shown that in the brain and peripheral tissues of rats with neonatal model of DM with peculiarities typical of human type 2 DM— pronounced resistance of tissues to insulin and moderate hyperglycemia, as well as in the rats with experimental models of type 1 DM that has acute insulin deficit, the functional activity of ACS being disturbed [1–5, 22]. In the present work we have established that in myocardium, ovaries, and uterus tissues of the 6-months-old rats with neonatal DM the sensitivity of ACS to hormones acting on AC by both stimulating way through Gsproteins and inhibiting way through Gi-proteins is changed. The changes in activity of Gs-coupled signaling cascades are characterized by receptor and tissue specificity, which is indicated by the following facts. In myocardium of diabetic rats the AC-stimulating effect of isoproterenol, binding with β2-adrenergic receptors (β2-АР) with higher affinity in comparison to β1- и β3-АР, has significantly increased, whereas in ovaries this effect, in contrast, has decreased. The AC-effect of another adrenergic agonist noradrenalin, acting predominantly through β1-АР, in myocardium of rats with DM has changed insignificantly. The ACstimulating effect of PACAP-38 in diabetic animals clearly decreased in ovaries, but was weakly changed in uterus. In myocardium and uterus of diabetic rats the AC-stimulating effects of relaxin clearly decreased, whereas the effects of other studied hormones significantly increased (isoproterenol in myocardium) or changed insignificantly (noradrenalin in myocardium, PACAP-38 and serotonin in uterus). These results cannot be explained either by a decrease in functions of Gs-proteins, as the ability of GIDP, non-hydrolyzed GTP analog, to stimulate AC activity has decreased in all tissues, or by changes in catalytic properties of the AC molecule whose stimulation by forskolin, acting directly on catalytic site of en-
zyme, in neonatal DM has changed insignificantly. Therefore, alongside with a decrease in activity of Gs-proteins, an important role in alterations in the functioning Gs-coupled signaling cascades in peripheral tissues of diabetic rats might be played by activity and the number of hormonal receptors, as well as by efficiency of their coupling with downstream messengers of ACS. This is confirmed by the data on adrenergic system obtained by us and other authors. We have shown that in myocardium of diabetic rat the sensitivity of ACS to β2-АР-agonist isoproterenol increases, whereas that to noradrenalin, predominantly activating β1-АР, does not virtually changes. As we suppose, in the neonatal DM, functions of single β-АР types are redistributed in favor of intensification of β2-АР- and β3-АРdependent signaling pathways, as was established in the case of streptozotocin type 1 DM [10]. In rats with the type 1, the decrease in expression of β1-АР in myocardium with DM was found. In control, a part of β1-АР among all types of β-АР is 62 %, in DM—only 40%, but treatment of rats with peripheral insulin results in restoration of β1-АР expression in myocardium—their portion rises to 57%. At the same time, the part of β2-АР in diabetic myocardium, in contrast, considerably increases, which might be considered as a compensatory reaction to decrease in the number of β1-АР, and treatment with peripheral insulin decreases both the number of β2-АР and efficacy of their activation by β2-АР- agonists to control level [10]. In this respect, it is to be noted that treatment of diabetic rats with I–I performed by us induces partial recovery of adrenergic signaling pathways in myocardium. The AC effect of isoproterenol decreased to its values in control, while the diminished under DM condition AC-inhibiting effect of noradrenalin, realized through α2-АР, in contrast, rises during such treatment. In respect to Gi-coupled ACS we have not revealed the pronounced tissue and receptor specificity typical of Gs-coupled cascades. The AC-inhibiting effects of somatostatin acting through the Gi-proteins-coupled somatostatin receptors, were decreased in all studied tissues of diabetic rats, while in myocardium the AC-inhibiting effect of noradrenalin was weakened. Earlier, we have shown that disturbances in the Gi-coupled ACS
JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 49 No. 2 2013
EFFECT OF INTRANASAL INSULIN AND SEROTONIN ON FUNCTIONAL ACTIVITY
activity in the brain and peripheral tissues of rats with types 1 and 2 DM also are not characterized by receptor and tissue specificity, and expressed stronger than in the case of Gs-coupled cascades [2, 21]. The most probable cause of this is a change under the DM conditions of the functional activity of Gi-proteins, common components of ACinhibiting cascades. In favor of this there are the following facts. Expression and functional activity of Gαi-subunits in the tissues of patients suffering form DM and in the tissues of rats with experimental types 1 and 2 DM and acute hyperglycemia are strongly decreased, whereas the content of other types of α-subunits changes weakly or even increased [26–32]. Thus, expression of Gαi1- and Gαi2-subunits in the liver of patients with type 2 DM decreased by 40% [28]. Expression of Gαi2and Gαi3-subunits in the membranes isolated from liver of rats with the streptozotocin type 1 DM and mutant mice with type 2 DM also dramatically decreased [26]. In platelets of patients with the type 2 DM the content of Gαi2- and Gαi3-subunits has decreased by 51% and 25%, while Gαi1-subunits are absent altogether [27]. Diminished expression of Gαi-subunit is revealed in smooth muscle cells treated with high doses of glucose [30, 32]. These data indicate a decrease in the number and activity of Gαi-subunits at DM, which inevitably leads to weakened transduction of hormonal signals through Gi-coupled AC cascades and well agrees with our data on weakened AC-inhibiting effects of somatostatin and noradrenalin in peripheral tissues or rats with neonatal DM. We have shown that the I–I treatment restores AC effects of some hormones in peripheral tissues of diabetic rats measured under conditions of neonatal DM. Among them are AC-stimulating effects of isoproterenol in myocardium, CHG in ovaries and relaxin in uterus, as well as the ACinhibiting effects of somatostatin in all tissues and noradrenalin in myocardium. Alongside with this, I–I partially restored the AC-stimulating effect of GIDP in ovaries. This indicates that I–I, directly entering the brain, has pronounced recovery effect on not only brain signaling systems [20], but also on the ACS in peripheral organs and tissues. It is suggested that action of I–I on peripheral signaling systems are based on the mechanisms realized through the insulin signaling system of the brain
161
[7]. In this respect it is to be noted that two factors responsible for changes in hormonal signaling systems in DM are usually considered. The first of them—prolonged and weakly controlled hyperglycemia typical of the both DM types and the second—a decrease in activity of the central and peripheral insulin signaling systems, which might be associated with hormone deficit (DM of 1 type) or with disturbances in sensitivity of signaling cascades to insulin—insulin resistance (in the type 2 DM) [7]. The obtained data indicate that an increase in insulin level in the brain after its intranasal administration leads to recovery of the insulin signaling system in brain and functionally connected with it central and peripheral hormonal signaling systems under conditions of insulin resistance and insignificant decrease in the insulin level typical of the neonatal DM. However, it is to be noted that I–I restores not all hormonal signaling systems weakened under DM. Thus, treatment of diabetic animals with I–I restored the AC-inhibiting effect of somatostatin and AC effects of adrenergic agonists in myocardium, but did not affect them in ovaries. At the same time, in ovaries of diabetic rats, I–I restored effect of CHG. All this indicate receptor and tissue specificity in I–I action, what should be taken into account for development of treatment strategy of the patients with DM using the intranasal hormone administration. After intranasal administration, serotonin directly enters the brain, which leads to an increase in its level in CNS and restores the brain serotoninergic system weakened under DM conditions [33]. According to our data, the I–S treatment improves cognitive functions in rats with neonatal DM, which is expressed as an increase of long-term spatial memory and the capability for training in Morris water test, as well as leads to partial recovery of ACS in brain tissues [5]. Other authors have found that the long-term treatment of diabetic patients by using SISR, that induces an increase in the brain serotonin level not only prevents depression typical of types 1 and 2 DM, but also improves glycemic control, and in the case of type 2 DM increases sensitivity of peripheral tissues to insulin [15, 16]. Based on this, we assumed that I–S may directly affect the functional activity of not only central, but also of peripheral
JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 49 No. 2 2013
162
SHPAKOV et al.
signaling systems. However, we have not revealed pronounced effect of long-term, for 7 weeks, I–S treatment, effective in respect with brain ACS on regulation of AC by hormones in peripheral tissues. An exception is the AC-inhibiting effect of norepinephrine in myocardium, which was partially restored after the I–S treatment. Hence, the action of I–S on the functional state of animals is mainly associated with recovery of the central signaling systems, which differs it from I–I restoring functional activity of both central and peripheral Ac cascades. Thus, our obtained data on studying the effect of the 7-weeks intranasal administration of insulin and serotonin on the functional activity of ACS in peripheral tissues of rats with the 6-months neonatal DM allow us to make the following conclusions. The target of the therapeutic I–I action is a wide net of hormonal systems including both CNS and some peripheral tissues, which indicates perspectives of its application for not only treatment of neurodegenerative disorders induced by DM, but also of complications from the cardiovascular and reproductive systems. The I–S treatment that, as we showed it earlier, considerably improves cognitive functions weakened under conditions of neonatal DM is less effective in respect to recovery of peripheral AC cascades, which indirectly indicates the lower efficacy of I–S for treatment of dysfunctions in cardiovascular and reproductive systems in comparison with I–I. The restoring I–I action on ACS is characterized by tissue and receptor specificity, which indicates its selectivity in restoration of peripheral hormonal systems. Deciphering of the molecular basis of action of intranasal hormones may help in near future to develop and optimize new approaches for monitoring and treatment of DM and its complications on the basis of combined application of intranasal and peripheral hormones. ACKNOWLEDGMENTS The study was supported by the Russian Federation Ministry of Education and Science (contract no. 8486) and by the Russian Foundation for Basic Research (projects nos. 12-04-00434 and 12-04-32034).
REFERENCES 1. Shpakov, A.O., Kuznetsova, L.A., Plesneva, S.A., Gur’yan, I.A., Vlasov, G.P., and Pertseva, M.N., Identification of Disturbances in Hormone-Sensitive AC-System in Rat Tissues with Types 1 and 2 Diabetes Using Functional Proves and Synthetic Nanosize Peptides, Tekhnologii Zhivykh System, 2007, vol. 4, no. 5–6, pp. 96–108. 2. Shpakov, A.O., Derkach, K.V., and Bondareva, V.M., Changes in Hormone Sensitivity of the Adenylate Cyclase Signaling System in the Testicular Tissue of Rats with Neonatal StreptozotocinInduced Diabetes, Byull. Eksper. Biol. Med., 2009, vol. 148, no. 9, pp. 282–286. 3. Shpakov, A.O., Bondareva, V.M., and Chistyakova, O.V., Functional State of Adenylyl Cyclase Signaling System in Reproductive Tissues of Rats with Experimental Type 1 Diabetes, Tsitologiya, 2010, vol. 52, no. 2, pp. 177–183. 4. Shpakov, A.O., Kuznetsova, L.A., Plesneva, S.A., Kolychev, A.P., Bondareva, V.M., Chistyakova, O.V., and Pertseva, M.N., Functional Defects in Adenylyl Cyclase Signaling Mechanisms of Insulin and Relaxin Action in Skeletal Muscles of Rat with Streptozotocin Type 1 Diabetes, Central Eur. J. Biol., 2006, vol. 1, pp. 530–544. 5. Shpakov, A.O., Derkach, K.V., Chistyakova, O.V., Sukhov, V.B., Shipilov, V.N., and Bondareva, V.M., The Brain Adenylyl Cyclase Signaling System in Cognitive Functions in Rat with Neonatal Diabetes under the Effect of Intranasal Serotonin, J. Metab. Syndr., 2012, vol. 1, no. 2, http:// dx.doi.org/10.4172/jms.1000104. 6. Pertseva, M.N. and Shpakov, A.O., On Conception of Molecular Defects in Hormonal Signaling Systems as the Cause of Endocrine Disorders, Ross. Fiziol. Zh. im. I.M. Sechenova, 2004, vol. 90, no. 8, pp. 446–447. 7. Shpakov, A.O., Alterations in Hormonal Signaling Systems in Diabetes Mellitus: Origin, Causality and Specificity, Endocrinol. Metab. Syndr., 2012, vol. 1, no. 2, DOI: 2161–n1017-1-e106. 8. Shpakov, A.O., The Functional State of Biogenic Amines and Acetylcholine-Regulated Signaling Systems of the Brain in Diabetes Mellitus, Tsitologiya, 2012, vol. 54, no. 6, pp. 459–468. 9. Shima, S., Fukase, H., and Akamatsu, N., Adrenergic Receptors and Adenylate Cyclase Activity in Hepatocytes of the Streptozotocin-Diabetic Rats, Endocrinol. Jpn., 1992, vol. 39, pp. 157–163. 10. Dinçer, U.D., Bidasee, K.R., Güner, S., Tay, A., Ozçelikay, A.T., and Altan, V.M., The Effect of Diabetes on Expression of β1-, β2-, and β3-Adre-
JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 49 No. 2 2013
EFFECT OF INTRANASAL INSULIN AND SEROTONIN ON FUNCTIONAL ACTIVITY
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
noreceptors in Rat Hearts, Diabetes, 2001, vol. 50, pp. 455–461. Vig, P.J., Subramony, S.H., D’Souza, D.R., Wei, J., and Lopez, M.E., Intranasal Administration of IGF-1 Improves Behavior and Purkinje Cell Pathology in SCA1 Mice, Brain Res. Bull., 2006, vol. 69, pp. 573–579. Benedict, C., Hallschmid, M., Schmitz, K., Schultes, B., Ratter, F., Fehm, H.L., Born, J., and Kern, W., Intranasal Insulin Improves Memory in Humans: Superiority of Insulin Aspart, Neuropsychopharmacol., 2007, vol. 32, pp. 239–243. Stockhorst, U., de Fries, D., Steingrueber, H.J., and Scherbaum, W.A., Insulin and the CNS: Effects on Food Intake, Memory, and Endocrine Parameters and the Role of Intranasal Insulin Administration in Humans, Physiol. Behav., 2004, vol. 83, pp. 47–54. Benedict, C., Frey, W.H., 2nd, Schiöth, H.B., Schultes, B., Born, J., and Hallschmid, M., Intranasal Insulin as a Therapeutic Option in the Treatment of Cognitive Impairments, Exp. Gerontol., 2011, vol. 46, pp. 112–115. Lustman, P., Freedland, K., Griffith, L., and Clouse, R., Fluoxetine for Depression in Diabetes: a Randomized Double-Blind Placebo-Controlled Trial, Diabetes Care, 2000, vol. 23, pp. 618–623. Zhou, L., Sutton, G., Rochford, J., Semple, R., Lam, D., Oksanen, L.J., Thornton-Jones, Z.D., Clifton, P.G., Yueh, C.Y., Evans, M.L., McCrimmon, R.J., Elmquist, J.K., Butler, A.A., and Heisler, L.K., Serotonin 2C Receptor Agonists Improve Type 2 Diabetes via Melanocortin-4 Receptor Signaling Pathways, Cell. Metab., 2007, vol. 6, pp. 398–405. Kerr, J., Timpe, E., and Petkewicz, K., Bromocriptine Mesylate for Glycemic Management in Type 2 Diabetes Mellitus, Ann. Pharmacother., 2010, vol. 44, pp. 1777–1785. Blondel, O., Bailbé, D., and Portha, B., Relation of Insulin Deficiency to Impaired Insulin Action in NIDDM Adult Rats Given Streptozotocin as Neonates, Diabetes, 1989, vol. 38, pp. 610–617. Hemmings, S. and Spafford, D., Neonatal STZ Model of Type II Diabetes Mellitus in the Fischer 344 Rat: Characteristics and Assessment of the Status of the Hepatic Adrenergic Receptors, Int. J. Biochem. Cell. Biol., 2000, vol. 32, pp. 905–919. Chistyakova, O.V., Bondareva, V.M., Shipilov, V.N., Sukhov, I.B., and Shpakov, A.O., A Positive Effect of Intranasal Insulin on Spatial Memory in Rats with Neonatal Diabetes Mellitus, Endocrinol. Studies, 2011, vol. 1, e16, pp. 67–75. Shpakov, A.O., Kuznetsova, L.A., Plesneva, S.A.,
22.
23.
24.
25.
26.
27.
28.
163
and Pertseva, M.N., The Disturbance of the Transduction of Adenylyl Cyclase Inhibiting Hormonal Signal in Myocardium and Brain of Rats with Experimental Type 2 Diabetes, Tsitologiya, 2007, vol. 49, no. 6, pp. 442–450. Shpakov, A.O., Chistyakova, O.V., Derkach, K.V., Moiseyuk, I.V., and Bondareva, V.M., Intranasal Insulin Affects Adenylyl Cyclase System in Rat Tissues in Neonatal Diabetes, Central Eur. J. Biol., 2012, vol. 7, pp. 33–47. Thorne, R., Pronk, G., Padmanabhan, V., and Frey, W., Delivery of Insulin-Like Growth FactorI to the Rat Brain and Spinal Cord along Olfactory and Trigeminal Pathways following Intranasal Administration, Neurosci., 2004, vol. 127, pp. 481– 496. Shpakov, A.O., Shpakova, E.A., Tarasenko, I.I., Derkach, K.V, Chistyakova, O.V, Avdeeva, E.A., and Vlasov, G.P., The Influence of Peptides Corresponding to the Third Intracellular Loop of Luteinizing Hormone Receptor on Basal and Hormone-Stimulated Activity of the Adenylyl Cyclase Signaling System, Global J. Biochem., 2011, vol. 2, no. 1, pp. 59–73. Shpakov, A.O., Shpakova, E.A., Tarasenko, I.I., Derkach, K.V, and Vlasov, G.P., The Peptides Mimicking the Third Intracellular Loop of 5-Hydroxytryptamine Receptors of the Types 1B and 6 Selectively Activate G Proteins and Receptor-Specifically Inhibit Serotonin Signaling via the Adenylyl Cyclase System, Int. J. Pept. Res. Ther., 2010, vol. 16, pp. 95–105. Bushfield, M., Griffiths, S.L., Murphy, G.J., Pyne, N.J., Knowler, J.T., Milligan, G., Parker, P.J., Mollner, S., and Houslay, M.D., Diabetes-Induced Alterations in the Expression, Functioning and Phosphorylation State of the Inhibitory Guanine Nucleotide Regulatory Protein Gi-2 in Hepatocytes, Biochem. J., 1990, vol. 271, pp. 365–372. Livingstone, C., McLellan, A.R., McGregor, M.A., Wilson, A., Connell, J.M., Small, M., Milligan, G., Paterson, K.R., and Houslay, M.D., Altered GProtein Expression and Adenylate Cyclase Activity in Platelets of Non-Insulin-Dependent Diabetic (NIDDM) Male Subjects, Biochim. Biophys. Acta, 1991, vol. 1096, pp. 127–133. Caro, J.F., Raju, M.S., Caro, M., Lynch, C.J., Poulos, J., Exton, J.H., and Thakkar, J.K., Guanine Nucleotide Binding Regulatory Proteins in Liver from Obese Humans with and without Type II Diabetes: Evidence for Altered “Cross-Talk” between the Insulin Receptor and Gi-Proteins, J. Cell. Biochem., 1994, vol. 54, pp. 309–319.
JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 49 No. 2 2013
164
SHPAKOV et al.
29. Gettys, T.W., Ramkumar, V., Surwit, R.S., and Taylor, I.L., Tissue-Specific Alterations in G Protein Expression in Genetic versus Diet-Induced Models of Non-Insulin-Dependent Diabetes Mellitus in the Mouse, Metab. Clin. Exp., 1995, vol. 44, pp. 771–778. 30. Hashim, S., Li, Y., Nagakura, A., Takeo, S., and Anand-Srivastava, M.B., Modulation of G-Protein Expression and Adenylyl Cyclase Signaling by High Glucose in Vascular Smooth Muscle, Cardiovasc. Res., 2004, vol. 63, pp. 709–718. 31. Rodgers, B.D., Bernier, M., and Levine, M.A., Endocrine Regulation of G-Protein Subunit Produc-
tion in an Animal Model of Type 2 Diabetes Mellitus, J. Endocrinol., 2001, vol. 168, pp. 509–515. 32. Li, Y., Descorbeth, M., and Anand-Srivastava, M.B., Role of Oxidative Stress in High Glucose-Induced Decreased Expression of Giα Proteins and Adenylyl Cyclase Signaling in Vascular Smooth Muscle Cells, Am. J. Physiol. Heart Circ. Physiol., 2008, vol. 294, pp. 2845–2854. 33. Shpakov, A., Chistyakova, O., Derkach, K., and Bondareva, V., Hormonal Signaling Systems of the Brain in Diabetes Mellitus, In: Neurodegenerative Diseases (Chang, R.C., Ed.), Intech Open Access Publisher, Rijeka, Croatia, 2011, pp. 349–386.
JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 49 No. 2 2013
