Alterations of Hormone-Sensitive Adenylyl Cyclase System in the Tissues of Rats with Long-Term Streptozotocin Diabetes and the In�uence of Intranasal Insulin

One of the causes of complications in type 1 diabetes mellitus (T1DM) is the changes in adenylyl cyclase (AC) signaling system, identi�ed on the early stages of the disease. However, the most signi�cant disturbances in this system occur on the later stages of T1DM,which ultimately leads to severe complications, but functional state of theAC system in late T1DM is poorly understood.e aim of this work was to study alterations in AC system sensitive to biogenic amines and polypeptide hormones in the heart, brain, and testes of male rats with long-term, 7-month, streptozotocin T1DM and to assess the in�uence on them of 135-day therapy with intranasal insulin. It was shown that AC effects of ββ-adrenergic agonists in the heart, serotonin receptor agonists and PACAP-38 in the brain, chorionic gonadotropin and PACAP-38 in the testes, and somatostatin in all investigated tissues in long-term T1DM were drastically decreased. e treatment with intranasal insulin (0.48 IU/day) signi�cantly restored these effects. e results were obtained suggesting that long-term T1DM induces signi�cant alterations in hormone-sensitive AC system in the heart, brain, and testes that are much more pronounced, compared with short-term T1DM, and include a large number of hormonal regulations.


Introduction
e type 1 diabetes mellitus (T1DM), one of the most severe metabolic disorders in humans, characterized by hyperglycemia due to a relative or an absolute lack of insulin, leads to many complications, such as coronary heart diseases, hypertension, atherosclerosis, neurodegenerative diseases, cognitive de�cit, and dysfunctions of the reproductive system [1,2].One of the causes of these complications is the alterations in hormone-sensitive signaling systems, the adenylyl cyclase (AC) system in particular [3,4].It was shown that changes in the functional activity of AC system in the heart, brain, and reproductive tissues in experimental T1DM were tissue and hormone speci�c [5][6][7][8][9][10].Generally, the effects of hormones acting on AC via G proteins of the inhibitory type (G i ) were changed to a greater degree compared with those realized via G proteins of the stimulating type (G s ), likely due to a decrease of G i proteins expression and a reduction of their functional activity and coupling with upstream and downstream signal proteins.
e degree of alterations and abnormalities in hormonal signaling systems is well correlated with the severity of T1DM and its complications [11,12].As a result, at the later stages of the disease characterized by pronounced clinical symptoms, there are all reasons to expect the changes in these systems to be much expressed.However, the functional state of hormonal signaling systems in late T1DM has been studied rather poorly yet, which greatly complicates the treatment of this disease and the monitoring of diabetic patients.e aim of this work was to identify and study the alterations in AC system sensitive to biogenic amines and polypeptide hormones in the heart, brain, and testes of male rats with long-term, 7-month, streptozotocin (STZ) T1DM and to reveal the in�uence of 135-day therapy with intranasal insulin (I-I) on the functioning and sensitivity of this system to hormones.e intranasal route of insulin delivery was chosen proceeding from the fact that I-I has no signi�cant in�uence on the level of peripheral glucose and, therefore, does not provoke hypoglycemic episodes [13,14].I-I normalizes metabolic processes and improves cognitive functions, but the molecular mechanisms and targets of its action have not been well de�ned [15,16].It should be noted also that the information concerning the in�uence of I-I on human and experimental T1DM is scarce [17,18].

Methodology
e model of long-term streptozotocin T1DM in adult male rats and the intranasal insulin treatment were as follows.For experiments, adult male Wistar rats housed in plastic sawdust-covered cages with a normal light-dark cycle and free access to food and water were obtained.e experiments were carried out under the Bioethics Committee of Sechenov Institute of Evolutionary Physiology and Biochemistry, St. Petersburg, Russia (Institutional Guidelines, December 23, 2010) and "Guidelines for the treatment of animals in behavior research and teaching" [19].All efforts were made to minimize animal suffering and reduce the number of animals used.
Aer a one-week adaptation period, rats aged 17-18 weeks were randomly divided into diabetic and control groups.Experimental diabetes mellitus of the type 1 (T1DM) was induced by a three consecutive intraperitoneal injection of freshly prepared STZ (Sigma, St. Louis, Mo, USA) in 0.1 M citrate buffer (pH 4.5) on the �rst, tenth, and eightieth days of experiments at doses 40, 35, and 30 mg/kg of body weight, respectively.Daily serum glucose level was assessed using test strips One Touch Ultra (USA) and a glucometer (LifeScan Johnson & Johnson, Denmark) via puncture of the tail.Nine days aer each STZ administration, animals with fasting blood glucose level over 12 mM were considered diabetic and were used in further experiments.Diabetic rats had, in addition, a very pronounced glucosuria.e monitoring of glucose concentration in the urine was carried out using test strips (Combi-Screen Analyticon, Germany).e insulin concentration in rat serum was determined using Rat Insulin ELISA (Mercodia AB, Sweden).
At the seventy-�h day of experiment, 5 days before the last injection of STZ, diabetic rats were divided into two equal groups.One group of diabetic rats (Group DI) was treated with I-I and the other with citrate buffer without insulin (Group D).Control animals were also divided into two equal groups: one received I-I (Group CI) and the other buffer without it (Group C).Intranasal delivery of insulin to the rat brain was performed as described previously by orne and coworkers [20].Crystalline insulin at concentration 24 IU/mL was dissolved in 0.1 M citrate buffer, pH 4.5, and administered intranasally to both diabetic (Group DI) and control (Group CI) rats once a day.Each rat was placed in a supine position and then an average of 20 μL of insulin solution (0.48 IU) was administered by Eppendorf pipette as 5 μL drops in each nostril, in turn, every 1-2 min.Control animals were given the equal volume of saline, pH 4.5.e I-I treatment of rats from Groups DI and CI was carried out during 135 days.Twenty-four hours aer the last intranasal administration of insulin or saline (on the 210th day of the experiment), the animals were decapitated under anesthetics, and their heart, brain tissues (cerebral cortex, striatum, hypothalamus, and olfactory bulbs), and testes were rapidly dissected and frozen on dry ice.Finally, four groups of rats were investigated: control animals (Group C,   ), control animals treated with I-I (Group CI,   ), diabetic animals with 210-day streptozotocin-induced diabetes mellitus of the type 1 (T1DM) (Group D,   ), and diabetic animals treated with I-I (Group DI,   ).
To study the functional activity of hormone-sensitive AC system in long-term T1DM, we chose the hormones that regulate AC activity via G s -coupled (stimulation) and G icoupled receptors (inhibition) and play a key role in the functioning of the cardiovascular, nervous, and reproductive systems.e brain serotonin-regulated AC system was examined using 5-nonyloxytryptamine, a selective agonist of G i -coupled 5-hydroxytryptamine receptors (5-HTRs) of the subtype 1B (5-HT 1B R); EMD-386088, a selective agonist of G s -coupled 5-HT 6 R; and the brain dopamine-regulated AC system using nonselective dopamine and bromocriptine, a selective agonist of D 2 -dopamine receptors (D 2 -DARs).In all investigated tissues, the AC inhibiting the effect of somatostatin realized via G i -coupled somatostatin receptors was studied.e myocardial adrenergic signaling was studied using noradrenaline, an agonist of  1 -and  2 -adrenergic receptors ( 1 -AR and  2 -AR), and isoproterenol, a nonselective -AR agonist, and in the testes using hCG, a glycoprotein hormone produced during pregnancy, and PACAP-38 that stimulate AC via G s -coupled luteinizing hormone/hCG and PAC1 receptors, respectively.e preparation of cardiac membranes from the rat heart was performed according to Baker and Potter [21], with some modi�cations.e dissected hearts were placed in icecold 0.9% NaCl, and atria, fat, and valves were removed.e tissues were cut into small pieces; homogenized with a Polytron in 20 volumes of ice-cold 40 mM Tris-HCl buffer (pH 7.4) containing 5 mM MgCl 2 , 320 mM sucrose, and a cocktail of protease inhibitors 500 μM O-fenantrolin, 2 μM pepstatin, and 1 mM phenylmethylsulfonyl �uoride (Buffer A); and centrifuged at 480 ×g for 10 min at 4 ∘ C. e pellet was discarded, and the supernatant was centrifuged at 27 500 ×g for 20 min at 4 ∘ C. e pellet was resuspended in Buffer A (without sucrose) and then centrifuged at 27 500 ×g for 20 min.e preparation of synaptosomal membranes from the rat brain was performed as described earlier [22].e brain tissues were dissected on ice and homogenized with a Polytron in 30 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.4) containing 10 mM MgCl 2 , 2 mM EGTA, 10% (w/v) sucrose, and a cocktail of protease inhibitors (Buffer B). e obtained material underwent centrifuge procedures, each performed at 4 ∘ C. e crude homogenate was centrifuged at 1000 ×g for 10 min; the resulting pellet was discarded and the supernatant was centrifuged at 9000 ×g for 20 min.e pellet was resuspended in Buffer B (without sucrose) and centrifuged at 35 000 ×g for 10 min.e isolation of plasma membranes from testes was carried out as described earlier [23].e testes were placed in ice-cold Buffer A and homogenized with a Polytron.e homogenate was centrifuged at 1500 ×g for 10 min at 4 ∘ C. e supernatant was centrifuged at 20 000 ×g for 30 min at 4 ∘ C. e resulting pellet was washed in 10 volumes of Buffer A (without sucrose) and centrifuged again at 20 000 ×g for 30 min.e �nal pellet was resuspended in the 50 mM Tris-HCl buffer (pH 7.4) to produce the membrane fraction with a protein concentration range 1-3 mg/mL and stored at −70 ∘ C. e protein concentration of each membrane preparation in all experiments was measured by the method of Lowry and colleagues using BSA as a standard.
e adenylyl cyclase (AC, EC 4.6.1.1)activity was measured as described previously [22].e reaction mixture (�nal volume 50 μL) contained 50 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 , 1 mM ATP, 1 μCi [-32 P]-ATP, 0.1 mM cAMP, 20 mM creatine phosphate, 0.2 mg/mL creatine phosphokinase, and 15-45 μg of membrane protein.Incubation was carried out at 37 ∘ C for 10 min.e reaction was initiated by the addition of membrane protein and terminated by the addition of 100 μL of 0.5 M HCl, followed by immersing the tubes with mixture in a boiling water bath for 6 min; 100 μL of 1.5 M imidazole was added to each tube.In these conditions, the AC activity was linear.[ 32 P]-cAMP formed as a result of the enzyme reactions was separated using alumina for column chromatography.e samples were placed on neutral alumina columns, and cAMP was eluted with 10 mL of 10 mM imidazole-HCl buffer (pH 7.4).e eluates were collected in scintillation vials and counted using an LS 6500 scintillation counter (Beckman Instruments Inc., USA).Each assay was carried out in triplicate at least three times, and the results were expressed as picomole (pmol) cAMP/min per milligram (mg) of membrane protein.e basal activity was measured in the absence of hormones and forskolin.To measure AC inhibition by hormones, the enzyme was activated by forskolin (10 −5 M).
e data are presented as the weighted mean ± weighted standard deviation.e difference in the weight and in glucose and insulin plasma levels of control and diabetic animals and in the basal activity of AC in the tissue membrane fractions of control and diabetic animals as well as the difference in the AC activity in the membrane fractions treated by hormones and nonhormonal AC regulators (GppNHp and forskolin) in each case was statistically assessed using the one-way analysis of variance (ANOVA, t-test) and was considered signi�cant at   005.e assumption of normality was assessed using Shapiro-Wilk test.e results of the test con�rmed that the obtained data are a sample from a normal distribution at   005.

Dataset Description
e dataset associated with this Dataset Paper consists of 8 items, which are described as follows.
Dataset Item 1 (Table ).Body weight, plasma glucose, and plasma insulin levels in control rats without (Group C) and with (Group CI) I-I treatment and in rats with long-term, 7-month, STZ T1DM without (Group D) and with (Group DI) I-I treatment.e plasma glucose level in diabetic rats was considerably increased during the whole period aer the �rst treatment with STZ due to insulin de�ciency, and their body weight was signi�cantly lower as compared with control.In rats with 7-month STZ T1DM, glucose levels were approximately three times higher and the weight was reduced by 14% compared with the respective control (Table 1).In diabetic rats, the therapy with I-I led to an increase of the body weight and to a decrease of the elevated glucose level but had no effect on insulin de�cit.In control animals, I-I changed neither body weight nor glucose level and decreased a little insulin level.

Column 1: Group Name
Column 2: Body Weight (g) ).e basal AC activity in plasma membrane fractions isolated from the heart, brain, and testes of insulin-treated and -untreated diabetic and control rats (in picomole (pmol) cAMP/min per milligram (mg) of membrane protein).Group C is the control animals (  ); CI, the control animals treated with intranasal insulin (I-I) (  ); D, the diabetic animals with 210-day streptozotocininduced diabetes mellitus of the type 1 (T1DM) (  ); DI, the diabetic animals treated with I-I (  ).e basal AC activity in the heart and testes of diabetic rats, compared with control, was signi�cantly decreased (Table 2).e treatment with I-I did not in�uence on the basal AC activity in the heart and testes of control animals but restored it in diabetic rats.In the brain of diabetic rats, the basal AC activity did not change considerably compared with control, and I-I had little effect on it in both control and diabetic animals.is indicates weakening of catalytic function of AC in the diabetic heart and testes, but not in the brain.).AC stimulating effects of GppNHp activating G s proteins directly interacting with catalytic site T 1: Body weight, plasma glucose, and plasma insulin levels in control rats without (Group C) and with (Group CI) I-I treatment and in rats with long-term, 7-month, STZ T1DM without (Group D) and with (Group DI) I-I treatment.

Groups of Rats
Body Weight (g) Plasma Glucose (mM) Plasma Insulin (ng mL  of AC in the tissues of diabetic and nondiabetic rats.Group C is the control animals (  ); CI, the control animals treated with intranasal insulin (I-I) (  ); D, the diabetic animals with 210-day streptozotocin-induced diabetes mellitus of the type 1 (T1DM) (  ); DI, the diabetic animals treated with I-I (  ).In the heart, brain, and testes of control rats, AC stimulating effects of GppNHp (10 −5 M), nonhydrolysable analog of GTP, were 229, 204, and 105% over the basal activity of the enzyme, respectively.It is a common knowledge that GppNHp activates G s proteins and thus stimulates the basal AC activity.In the diabetic heart and testes, AC effects of GppNHp were decreased, most signi�cantly in the testes, and I-I restored the effects of GppNHp in the heart but did not in the testes (Figure 1(a) and Table 3(a)).In the diabetic brain, the AC effect of GppNHp decreased insigni�cantly.ese data indicate the attenuation of catalytic function of the membrane-bound AC in the heart and testes of rats with long-term T1DM, whereas in the diabetic brain the enzyme catalytic properties were not altered.Note that in the shortterm T1DM, we found no signi�cant changes in the basal and   GppNHp-stimulated AC activities in the heart and a small decrease of these activities in the testes [10,25].).AC stimulating effects of forskolin directly interacting with catalytic site of AC in the tissues of diabetic and nondiabetic rats.Group C is the control animals (  ); CI, the control animals treated with intranasal insulin (I-I) (  ); D, the diabetic animals with 210day streptozotocin-induced diabetes mellitus of the type 1 (T1DM) (  ); DI, the diabetic animals treated with I-I (  ).In the heart, brain, and testes of control rats, AC stimulating effects of diterpene forskolin (10 −5 M) were 661, 619, and 205%, respectively.It is a common knowledge that forskolin directly interacts with catalytic site of the enzyme.
In the diabetic heart and testes, AC effects of forskolin were decreased, most signi�cantly in the testes, and I-I restored the effects of forskolin in the heart but did not in the testes (Figure 1(b) and Table 3(b)).In the diabetic brain, the AC effect of forskolin did not change.ese data indicate the attenuation of catalytic function of the membrane-bound AC in the heart and testes of rats with long-term T1DM, whereas in the diabetic brain, the enzyme catalytic properties were not altered.Note that in the short-term T1DM, we found no signi�cant changes in the basal and forskolin-stimulated AC activities in the heart and a small decrease of these activities in the testes [10,25].).e AC effects of hormones activating the enzyme via G s protein-coupled receptors in the tissues of diabetic rats and under the in�uence of I-I.In the diabetic heart, the AC stimulating effects of isoproterenol and noradrenaline, the agonists of -adrenergic receptors (-AR), were decreased and partially restored by I-I.A most signi�cant decrease was observed with noradrenaline acting preferably via  1 -AR undergoing considerable downregulation in T1DM [26], whereas AC effect of isoproterenol, nonselective -AR agonist, was reduced to a lesser extent (Figure 2(a) and Table 4(a)).In our view, this may be due to the fact that the signaling pathways mediated via  2 -AR, the main target of isoproterenol, in the heart of rats with T1DM, even short-term, were preserved or enhanced, this being a compensatory mechanism triggered by reducing the functional activity of  1 -AR [11,27].us, in the heart of 14week STZ rats, the content of  1 -AR protein as well as  1 -AR mRNA was decreased to 35 and 45%, respectively, and the decrease of maximal chronotropic response to selective  1 -AR agonists was 70% of that in control, whereas the number   As we showed earlier, in the model of short-term T1DM and the neonatal model of type 2 diabetes mellitus (T2DM), although the stimulating effects of serotonin on AC activity and GTP binding of G s proteins in the diabetic brain were reduced, the corresponding effects of EMD-386088 and, as a result, 5-HT 6 R-mediated AC signaling did not change [16,25].ere was a decrease in the number of 5-HT 6 R and a reduced AC response to selective 5-HT 6 R agonists in the brain of patients with prolonged Alzheimer's disease due to the neurodegenerative alterations in neuronal and glial cells [28].e decrease in the sensitivity of AC to -AR-agonist isoproterenol points to a weakening of -AR signaling in the diabetic brain, which negatively in�uences the synaptic transmission provoked by impairment of the mechanism involving increase of intracellular cAMP concentration and protein synthesis [29] and induces disturbances in the cerebral microvessels, whose functions are controlled via different signaling systems, the adrenergic in particular [30].A signi�cant reduction of AC effect of PACAP-38 having an important role in protection of neuronal cells from damage and neurodegenerative changes [31] speaks in favor of abnormalities in PACAP-mediated neuroprotection in the case of long-term T1DM.It has been noticed that short-term T1DM and neonatal T2DM had no effect on the regulation of brain AC activity and G s protein binding by PACAP-38 [16,25].We showed that the 135-day therapy with I-I prevents the decline of AC stimulation induced by PACAP-38 in the diabetic brain.us, the ability of I-I to restore AC signaling pathways regulated by PACAP-38 and some biogenic amines may be one of the main mechanisms of positive in�uence of I-I on the impaired CNS functions in human and experimental DM [16,24,32].e AC effect of dopamine, contrary to the other investigated hormones, did not change in the diabetic brain.Earlier it was shown that dopamine-stimulated cAMP production in the brain of rats with 14-week alloxan DM or neonatal T2DM was markedly increased [16,33].is may be due to a compensatory increase in the expression of G s protein-coupled D 1 -DAR, as in the case of short-term STZ T1DM [34].
Column 1: Group Name  4(c)).e reduction in hCG-and PACAPinduced AC stimulation in the rat testes in long-term T1DM was more pronounced compared with that in short-term T1DM [10].ese results indicate that the abnormalities in testicular function in human and experimental T1DM can be ascribed to the changes in sensitivity of AC to gonadotropins and peptide hormones belonging to the vasoactive intestinal peptide/PACAP family.It should be mentioned that AC stimulation induced by vasoactive intestinal peptide in the seminal vesicle of rats with STZ T1DM was considerably reduced, which was associated with a decrease of G s subunit level [35,36].
Column 1: Group Name  Dataset Item 8 (Table ).e AC inhibitory effects of hormones acting via G i protein-coupled receptors in the tissues of diabetic and nondiabetic rats, which were estimated by the in�uence of the hormones on fors�olin-stimulated AC activity.AC inhibitory effects of somatostatin in all the investigated tissues, noradrenaline in the heart, and 5-HT 1B/1D R agonist 5-nonyloxytryptamine in the brain were decreased signi�cantly in T1DM compared with control (Figure 3 and Table 5).e corresponding effects of noradrenaline and D 2 -DAR agonist bromocriptine in the diabetic brain changed, but not very much.e therapy with I-I restored the inhibitory effects of noradrenaline in the heart and of 5-nonyloxytryptamine in the brain but in�uenced only a little the somatostatin effects in all the studied tissues.e signi�cant decrease of AC effect of 5-nonyloxytryptamine indicates attenuation of 5-HT 1 R-mediated AC signaling in the brain in the model of prolonged T1DM.ese �ndings are consistent with the data obtained by the other authors on the weakening of the behavioral response of rats with T1DM to 5-HT 1A R agonist 8-hydroxy-2-(dipropylamino) tetralin hydrobromide [37], and with our data concerning a decrease of regulatory effects of 5-nonyloxytryptamine on AC activity and G i proteins GTP binding in the brain of rats with neonatal T2DM [16].e abnormalities in 5-HT 1 R-mediated signaling pathways are able to induce the impairment of memory and cognitive functions and psychic disorders [38].e decrease of AC inhibitory effects of noradrenaline in the diabetic heart indicates alterations in cardiac signaling including G i -coupled  2 -AR, which may be caused by a decrease in the number of  2 -AR and by a reduction of expression and functional activity of G i proteins in the heart of rats with T1DM [5,6].As shown in our previous studies, the changes in  2 -AR signaling can be identi�ed at the early stages of STZ T1DM, being, however, less expressed compared with the later stages [8,25].e most dramatic changes were revealed in AC signaling cascades regulated by somatostatin.Somatostatin-induced AC inhibition was signi�cantly reduced in all tissues under study, and this decline surpassed that in short-term STZ T1DM and neonatal T2DM [16,25].ese data are the evidence for the degree of damage in somatostatin-regulated AC system enhanced with increasing duration and severity of the diabetic state.e reports are available describing the decrease of expression of some types of somatostatin receptors in the hypothalamus and pituitary in rats with T1DM [39].In the atria of 4week STZ rats, the suppressive effect of somatostatin on ANP secretion, realized via G i -coupled sst2-somatostatin receptor, and the level of mRNA and protein content for this receptor was markedly decreased [40].In the glucagon cells isolated from the pancreatic islets from patients with T2DM, there were found no sst1-and sst4-somatostatin receptors, and in the somatostatin cells, no sst1-, sst2-, sst3-, and sst-4somatostatin receptors were detected [41].is gave grounds for a suggestion that the decrease of the level and the function of somatostatin receptors in patients with T2DM and animals with T1DM may be due to the elevated level of circulating somatostatin inducing downregulation of the cognate receptors [42].Taking into consideration the importance and complexity of the physiological and biochemical effects of somatostatin and its involvement in the pathogenesis of a variety of diseases, it would be right to say that the disturbances in somatostatin-regulated AC system are likely to be responsible for the development of T1DM-associated dysfunctions in the cardiovascular, nervous, and reproductive systems.is indicates the attenuation of G i protein-coupled signaling cascades, especially somatostatin-regulated, in the heart, brain, and testes of rats with long-term STZ T1DM, and the partial restoration of these cascades with I-I treatment.

Concluding Remarks
e study of the model of long-term T1DM is very important for understanding the molecular mechanisms responsible for the development of complications of this disease and for �nding the approaches to their therapy and diagnostics.We showed that in 7-month STZ T1DM the regulatory effects of a large number of hormones including both AC activators, such as ligands of -AR and 5-HT 6 R, PACAP-38, and gonadotropin, and AC inhibitors, such as noradrenaline, somatostatin, and selective agonist of 5-HT1B/1DR, change signi�cantly.e alterations in AC signaling may be regarded as a result of disturbances in the fundamental cellular processes in the tissues and organs of diabetic individuals and also as a mechanism compensating for the impairment of insulin-mediated signaling provoked by prolonged insulin de�ciency.e alterations in AC signaling we revealed in long-term T1DM are expressed much better compared with short-term T1DM studied in detail by us and the other authors earlier and cover a wide spectrum of hormonal regulations [5,[8][9][10][11].A prolonged, 135-day, treatment of diabetic rats with I-I resulted in restoration of the sensitivity of the cardiac AC system to nonhormonal regulators (GppNHp, forskolin) and adrenergic agonists of the brain AC system to selective agonists of 5-HTR and PACAP-38 and of the testicular AC system to hCG.Our results give strong evidence for the bene�t of I-I in the therapy of diabetic patients with prolonged T1DM aimed at improving functioning of the cardiovascular, nervous, and reproductive systems and preventing their impairments; they indicate that insulin signaling system, the key component of overall hormonal network in the brain, is an important target in the treatment of severe forms of T1DM.e increase in sensitivity of cardiac AC to adrenergic ligands and testicular AC to gonadotropin in diabetic rats receiving I-I suggests that intranasaly adminestered hormone acts on the cardiovascular system and on the testes, a distal component of the hypothalamic-pituitary-gonadal axis, via the central mechanisms due to the impovement of brain signaling impaired in long-term T1DM.

Dataset Availability
e dataset associated with this Dataset Paper is dedicated to the public domain using the CC0 waiver and is available at http://dx.doi.org/10.7167/2013/698435/dataset.

Con�ict of �nterests
e authors declare that they have no con�ict of interests.

T 3 :
e stimulating effects of GppNHp (a) and forskolin (b) on the basal AC activity in the heart, brain, and testes in diabetic and control rats treated with I-I or saline.C: control animals; CI: control animals treated with intranasal insulin (I-I); D: diabetic animals with 210-day streptozotocin-induced diabetes mellitus of the type 1 (T1DM); DI: diabetic animals treated with I-I.GppNHp and forskolin were taken at 10 −5 M. Values are the weighted mean ± weighted standard deviation of three individual experiments, each performed in triplicate.D, P = 10 −9 ; C-CI, P = 0.3460) 54.6 ± 0.6 (C-D, P = 0.0322; C-CI, P = 0.2613) 20.9 ± 0.3 (C-D, P = 10 −6 ; C-CI, P = 0.3268) Average of the �rst series 68

F 1 :
D, P = 10 −8 ; C-CI, P = 0.1781) 166 ± 4 (C-D, P = 0.3585; C-CI, P = 0.2458) 41 ± 2 (C-D, P = 10 −9 ; C-CI, P = 0.3261) Average of the �rst series 191 e stimulating effects of (a) GppNHp and (b) forskolin on the basal AC activity in the heart, brain, and testes in diabetic and control rats treated with I-I or saline.C: control animals; CI: control animals treated with intranasal insulin (I-I); D: diabetic animals with 210-day streptozotocin-induced diabetes mellitus of the type 1 (T1DM); DI: diabetic animals treated with I-I.GppNHp and forskolin were taken at 10 −5 M. Values are the weighted mean ± weighted standard deviation of three individual experiments, each performed in triplicate.* and * * * denote the statistically signi�cant difference between Groups C and D at   005 and 0.001; ###, the same between Groups D and DI at   0001; &, &&, and &&&, the same between Groups CI and DI at   005, 0.01, and 0.001, respectively.

7 T 4 :
e stimulating effects of hormones on AC activity in (a) the heart, (b) brain, and (c) testes of diabetic and control rats with and without therapy by I-I.C: control animals; CI: control animals treated with intranasal insulin (I-I); D: diabetic animals with 210-day streptozotocin-induced diabetes mellitus of the type 1 (T1DM); DI: diabetic animals treated with I-I.Isoproterenol, noradrenaline, serotonin, EMD-386088, and dopamine were taken at 10 −5 M, PACAP-38 at 10 −7 M, and hCG at 10 −8 M. Values are the weighted mean ± weighted standard deviation of three individual experiments, each performed in triplicate.
(a) In picomole (pmol) of cAMP/min per milligram (mg) of membrane protein

F 2 :Column 5 :- 386088 Column 6 :Column 7 :- 38 Dataset Item 7 (
e stimulating effects of hormones on AC activity in (a) the heart, (b) brain, and (c) testes of diabetic and control rats with and without therapy by I-I.C: control animals; CI: control animals treated with intranasal insulin (I-I); D: diabetic animals with 210-day streptozotocin-induced diabetes mellitus of the type 1 (T1DM); DI: diabetic animals treated with I-I.Isoproterenol, noradrenaline, serotonin, EMD-386088, and dopamine were taken at 10 −5 M, PACAP-38 at 10 −7 M, and hCG at 10 −8 M. Values are the weighted mean ± weighted standard deviation of three individual experiments, each performed in triplicate.* * and * * * denote the statistically signi�cant difference between Groups C and D at    and 0.001; #, ##, and ###, the same between Groups D and DI at   5, 0.01, and 0.001; &, &&, and &&&, the same between Groups CI and DI at   5, 0.01, and 0.001, respectively.EMDDopamine PACAPTable).In the diabetic testes, the AC effects of hCG, structural and functional homologue of luteinizing hormone, and PACAP-38 were decreased signi�cantly, and the effect of hCG was partially restored by I-I treatment (Figure 2(c) and Table

F 3 :
e inhibitory effects of hormones on forskolin-stimulated AC activity in the tissues of diabetic and control rats with and without I-I treatment.C: control animals; CI: control animals treated with intranasal insulin (I-I); D: diabetic animals with 210-day streptozotocininduced diabetes mellitus of the type 1 (T1DM); DI: diabetic animals treated with I-I; 1, 3, and 7: somatostatin (10 −6 M) in the heart, brain, and testes, respectively; 2 and 4: noradrenaline (10 −5 M) in the heart and brain, respectively; 5: bromocriptine (10 −5 M) in the brain; 6: 5nonyloxytryptamine (10 −5 M) in the brain.e stimulating effect of forskolin (10 −5 M) on the basal AC activity is taken as 100%.Values are the weighted mean ± weighted standard deviation of three individual experiments, each performed in triplicate.* and * * * denote the statistically signi�cant difference between Groups C and D at   5 and 0.001; #, ##, and ###, the same between Groups D and DI at   5, 0.01, and 0.001; &, &&, and &&&, the same between Groups CI and DI at   5, 0.01, and 0.001, respectively.

Column 5 : 7 :Column 9 :
Percentage in HeartColumn 6: Brain Column Percentage in Brain Percentage in Testes HT 6 R-mediated pathways in the diabetic brain but does not exclude attenuation of the functional activity of the other types of 5-HTR that are also coupled with AC via G s proteins.