[Effects of intranasal insulin and serotonin on functional activity of the adenylyl cyclase system in the myocardium, ovaries, and uterus of rats with prolonged neonatal model of diabetes mellitus].

Disturbances in hormonal signaling systems, in the adenylyl cyclase system (ACS) in particular, occur at early stages of diabetes mellitus (DM) and are one of the key causes of its complications. Since there is a correlation between the severity of DM and of disturbances in the ACS, the study of the ACS activity can be used to monitor DM and its complications and to evaluate effectiveness of their treatment. Comparatively recently, for treatment of the type 2 DM, there began to be used the intranasal insulin (I-I) and drugs increasing brain serotonin level, which effectively restore CNS functions. However, mechanisms of their action on peripheral tissues and organs with DM remain to be not understood. The goal of this work was to study effects. of I-I and intranasal serotonin (I-S) on the ACS functional activity in myocardium, ovary, and uterus of rats with a neonatal model of the type 2 DM. In tissues of diabetic rats there were revealed changes in regulation of adenylyl cyclase (AC) by guanine nucleotides and hormones that both stimulated and inhibited this enzyme, such changes being characterized by the receptor and tissue specificity. In diabetic rats, I-I restored the AC stimulating effects of isoproterenol in the myocardium, guanine nucleotides and gonadotropin in ovaries and relaxin in uterus, as well as the AC inhibitory effects of somatostatin in all tissues, and of noradrenaline in myocardium. Treatment with IS led to a partial restoration of the AC-inhibitory effect of noradrenalin in the diabetic myocardium, but did not affect regulation of AC by other hormones. These data indicate that I-I normalizes the ACS functional activity in myocardium and in tissues of the reproductive system of female rats with neonatal DM, whereas the effect of I-S on in the studied tissues is less pronounced. These results are necessary to be taken into account at development and optimization of strategy of use of I-I and I-S for treatment of DM and of its complications.

[The influence of long-term diabetes mellitus induced by streptozotocin treatment of six-week rats on the functional activity of adenylyl cyclase system].

The alterations occurring in diabetes mellitus (DM) of the type 1 in the adenylyl cyclase signaling system (ACSS) are one of the key causes of complications of the disease. As type 1 DM most often diagnosed in childhood and adolescence, the actual problem is the study of alterations in ACSS in the early development of the disease. For this we developed a prolonged model of type 1 DM, which was induced by treatment of six-week-old rats with moderate doses of streptozotocin (1 ½M-DM), and studied the functional state of ACSS in the brain, myocardium, and testes of rats with this model of the disease, seven months after its initiation. Model 1 ½-DM was compared with the seven-month model of type 1 DM, which was induced by streptozotocin treatment of adults, five-month-old, animals (5M-DM). It is shown that in 1 ½M-DM in the tissues of diabetic rats the functional activity of ACSS sensitive to biogenic amines and polypeptide hormones was significantly changed. In rats with 1 ½M-DM the adenylyl cyclase (AC) inhibiting effects of somatostatin (in all studied tissues), norepinephrine (in the myocardium and brain), and agonists of type 1 serotonin receptor (in the brain) were weakened the most. In the brain also decreased AC stimulating effects of relaxin, isoproterenol and agonists of G(s)-protein-coupled serotonin receptors, in the myocardium--corresponding effects of GppNHp, relaxin and ß-adrenergic agonists, and in the testes--AC effects of GppNHp and chorionic gonadotropin. When comparing the models 1 ½M-DM and 5M-DM, the most pronounced differences between them were found in the influence of DM on hormonal regulation of ACSS in the brain, and this refers both to AC stimulating effects of dopamine and PACAP-38, and to AC inhibiting effects of bromocryptine and somatostatin. These results indicate significant alterations in the hormonal regulation of the nervous, cardiovascular and reproductive systems in rats with early induction of type 1 DM, in some cases more severe compared with late model of 5M-DM. These alterations can be the basis for the development of diabetic cardiomyopathy, cognitive deficits and hypogonadotropic states, which are often detected in children and adolescents with type 1 DM.

[Experimental models of diabetes mellitus of the 1st and 2nd types in rats: regulation of activity of glycogen synthase by peptides of the insulin superfamily and by epidermal growth factor in skeletal muscles].

The regulatory effect of peptides of the insulin hyperfamily--insulin, insulin-like growth factor (IGF-1), and relaxin, as well as of epidermal growth factor (EGF) on activity of glycogen synthase (GS) in rat skeletal muscles was studied in norm and in experimental diabetes mellitus of the 1st and 2nd types (DM1, DM2). In norm, peptides in vitro stimulated maximally the GS activity at a concentration of 10-8 M. The row of efficiency of the peptide action was as follows: insulin > IGF-1 > relaxin. In DM1 the basal GS activity did not change, while effect of insulin in vitro was decreased more sharply as compared with action of IGF-1 and relaxin at the 30th day of development of diabetes, i. e., the efficiency row was as follows: IGF-1 = relaxin > insulin. Administration of insulin in vivo did not restore sensitivity of the enzyme to the action of hormone in DM1. In DM2, the GS activity (both the total and active form) decreased. while the stimulatory effect ofpeptides and EGF on the enzyme was absent. Insulin introduced in vitro did not lead to restoration of the enzyme reaction. The conclusion has been made that the insulin resistance affects the basal GS activity in rat skeletal muscles as well as the regulation of the enzyme by peptides of the insulin nature and by EGF, which is more obvious in DM2, than in DM1.

[The activity of receptor guanylyl cyclases in tissues of rats with neonatal streptozotocin diabetus mellitus and the influence of intranasal administration of insulin and serotonin].

Functional activity of hormanal signaling systems and their sensitivity to regulatory actions of hormones in diabetes mellitus (DM) and its complications are altered. The activity of receptor forms of guanylyl cyclases (rGC) sensitive to natriuretic peptides, ANP and CNP, in tissues of female rats with 240-days neonatal streptozotocin DM and the influence of intranasal administration of insulin and serotonin (6 weeks, daily dose is 0.48 IU of insulin or 20 microg of serotonin to rat) on this activity were studied. In diabetic animals, the increase of the basal activity of rGC in the myocardium and its decrease in the uterus and ovaries were found, whereas the brain showed no differences from control animals. The treatment of diabetic rats with insulin led to a decrease in the basal activity of rGC in the myocardium and to its restoration to normal level in the ovaries. Serotonin treatment induced a less pronounced compared with insulin decrease in the basal activity of the enzyme in the myocardium and also a slight increase the activity in the brain. In the myocardium of diabetic rats, the weakening of GC stimulating effect of ANP and, on the contrary, the strengthening of CNP effect were observed. In the ovaries, GC stimulating effect of CNP and, to a lesser degree, the corresponding effect of ANP were decreased. In the uterus and the brain, the sensitivity of rGC to hormones was practically not changed. The administration of insulin to diabetic rats induced an increase in GC effect of ANP in the myocardium to its values in control animals and a decrease in CNP effect as well as in partially restored GC effect of CNP in the ovaries. Serotonin treatment led to some increase in the effects of natriuretic peptides in the brain of both control and diabetic animals. Summing up, in neonatal model of type 2 DM in the myocardium and the tissues of reproductive system of rats the functioning of natriuretic peptide-sensitive rGC is altered in the myocardium and the tissues of reproductive system of model rats with neonatal type 2 DM. Treatment of animals with insulin substantially restores rGC activity while intranasal administration of serotonin has little effect.

[Functional state of adenylate cyclase signaling system in testis and ovary of fasted rats].

Sensitivity of the adenylate cyclase signaling system (ACSS) to polypeptide hormones and biogenic amines is studied in testis and ovary of rats after the 2- and 4-day fasting as compared with control animals. In tissues of the fasted rats there is shown a decrease in the basal activity of adenylate cyclase (AC) and of the basal level of the GTP binding of heterotrimeric G protein. An increase of duration of fasting from 2 to 4 days led to intensification of these changes. In the fasted rats, the stimulating effects of chorionic gonadotropin, PACAP-38. and isoproterenol on the AC activity realized via G protein of the stimulatory type are enhanced, whereas the inhibitory effects of somatostatin on the AC activity realized via G protein of the inhibitory type are reduced. In testis of the fasted rats the stimulating effect of serotonin acting on AC via both types of G proteins are increased, while the inhibitory effects of the hormone decrease. Thus, under conditions of fasting, in rat testis and ovary the ACSS sensitivity to regulatory effects of hormones is changing: its stimulatory effects are increased, while its inhibitory effects, on the contrary, are decreased. We suggest these changes is one of the key mechanisms of adaptation of organism to deficiency of nutritional resources to be aimed at intensifying the tissues catabolic processes, preferably, lypolysis.

[Functional state of adenylyl cyclase signaling system in reproductive tissues of rats with experimental type 1 diabetes].

Diabetes mellitus (DM) of type 1 induces numerous disturbances in reproductive systems of males and females. We have shown earlier that the main cause of the complications in the case of DM is alteration of adenylyl cyclase signaling system (ACSS) sensitivity to hormones. The aim of the present work was identification of disturbances in hormone-regulated ACSS in reproductive tissues of rats with experimental type 1 DM (EDM1) induced by streptozotocin treatment. Testis of the rats with 5-days EDM1 showed significant decrease in the stimulatory effects of human chorionic gonadotropin (hCG) and PACAP-38 on adenyly] cyclase (AC) activity and G protein GTP-binding. Uterus of the rats with EDM1 exhibited decreased effects of relaxin, PACAP-38 and biogenic amines. In the ovaries, we showed the decrease in hCG effects only. Weakening of the inhibitory influence of somatostatin on ACSS activity was found in all studied tissues of rats with EDM1. Uterus displayed also decreased inhibitory effects of serotonin and adrenaline. Thus, regulatory effects of the hormones in ACSS sensitivity in reproductive tissues of the rats with EDM1 were decreased. The effects of hCG and AC inhibiting hormones were decreased to a greater extent. We suppose that the decrease in ACSS sensitivity to hormones in the case of EDM1 is responsible for pathological changes in reproductive systems of diabetic rats under condition of hyperglycemia and insulin deficiency which are typical for type 1 DM.

[Peptides derived from the third cytoplasmatic loop of serotonin 1B receptor subtype selectively inhibit serotonin signal transduction via a homologous receptor].

The third intracellular loops of hormonal receptors play the main role in the interaction of majority of the serpentine type receptors with heterotrimeric G-proteins. In recent years, it was shown that synthetic peptides corresponding to membrane-proximal regions of these loops could be selectively influenced with hormonal signal transduction via the receptors homologous to them and trigger signalling cascade in absence of the hormone. For the first time, we succeeded in synthesizing the peptides derived from C-terminal region of the third intracellular loop of the IB-subtype serotonin receptor and studied their influence on serotonin-sensitive adenylyl cyclase system in the rat brain. The peptides 300-316 and 306-316 (the numbers correspond to amino acid positions in the rat IB-subtype serotonin receptor) at micromolar concentrations in absence of hormone-stimulated GTP-binding of Gi,-proteins coupled with the IB-subtype serotonin receptors and inhibited forskolin-stimulated adenylyl cyclase activity. Using selective agonists and antagonists of serotonin receptors it was shown that the peptides 300-316 and 306--316 inhibited serotonin signal transduction via homologous to them receptor and weakly influenced other types of serotonin receptors. The peptide 300-316 is more active compared with its shorter analogue 306-316 in the selectivity and efficiency of action on adenylyl cyclase signalling system regulated via the IB-subtype serotonin receptors. These findings indicate that the regions 300-316 of the IB-subtype serotonin receptor are involved in interaction with Grproteins and consist of the main molecular determinants responsible for serotonin signal transduction to adenylyl cyclase.

[The regulation of glucose-6-phosphate dehydrogenase and glycogen synthase activities by insulin superfamily peptides in myometrium of pregnant women and its impairments under different types of diabetes mellitus].

The regulatory effects of insulin, insulin-like growth factor 1 (IGF-1), and relaxin on glucose-6-phosphate dehydrogenase (G6PDH) and glycogen synthase (GS) activities have been studied in myometrium of pregnant women of control group and with diabetes mellitus of different etiology. In patients with type 1 diabetes G6PDH activity did not differ from the control group, but the enzyme activity was sharply decreased in pregnant women with type 2 diabetes and gestational diabetes. In the control group maximal stimulation of G6PDH activity was observed at 10(-9) M of peptides and their stimulating effect decreased in the following order: insulin > relaxin > IGF-1. In pregnant women with types 1 diabetes insulin effect on the enzyme activity was lower than in the control, and the effects of IGF-1 and relaxin were absent. In the group of pregnant women with type 2 diabetes and gestational diabetes the effects of insulin and IGF-1 were decreased, but the effect of relaxin was somewhat higher thus giving the following order in their efficiency relaxin > IGF-1 = insulin. At 10(-9) M peptides exhibited similar stimulating effects on the active form of GS-I, but had no influence on the total enzyme activity in the control group of pregnant women. In patients with type 1 diabetes GS activity remained unchanged (versus control), and peptides did not stimulate the enzyme activity. In patients with type 2 diabetes a significant decrease in GS activity was accompanied by the decrease in the effect of peptides, giving the following order of their efficiency: insulin = IGF-1 > relaxin. In myometrium of pregnant women with gestational (treated and untreated) diabetes GS activity decreased, the effect of insulin was weaker, whereas the effects of relaxin and IGF-1 increased thus giving the following order of their efficiency: relaxin > IGF-1 > insulin. Insulin therapy of type 1 diabetes incompletely restored sensitivity of the enzymes to the peptide actions. At the same time, in women with gestational diabetes and subjected to insulin therapy the stimulating effect of relaxin on the enzyme activities increased. This fact suggests that relaxin exhibits replacement functions under conditions of attenuated insulin action.

[Signal pathway of insulin and insulin-like growth factor 1 (IGF-1) as a potential regulator of lifespan].

The experimental material accumulated for two decades allows concluding that regulation of lifespan has hormonal control based on the evolutionary conservative insulin/IGF-1 receptor signal pathway. Data obtained on the commonly accepted models of longevity - nematode Caenorhabditis elegans, Drosophila Drosophila melanogaster, and rodents - demonstrate that reduction of the insulin/IGF- 1 signal pathway leads to an increase of the lifespan. There is shown involvement of the longevity mechanism of a large group of genes whose products perform control of metabolism, alimentary behavior, reproduction, resistance to oxidative stress. Discussed in this review are current concepts of the insulin/IGF-1 signal system as a regulatory "longevity module" and of its possible role in prolongation of life in the higher vertebrates, including human.

[Streptozocin model of diabetes mellitus in the mollusc Anodonta cygnea: functional state of the adenylyl cyclase mechanisms of action of peptides of the insulin superfamily and their effect on carbohydrate metabolism enzymes].

In terms of development of evolutionary biomedicine using invertebrate animals as models for study of molecular grounds of various human diseases, for the first time the streptozocin (ST) model of insulin-dependent diabetes in the mollusc Anodonta cygnea has been developed. This model is based on the following authors' data: (1) redetection of insulin-related peptides (IRP) in mollusk tissues: (2) discovery of the adenylyl cyclase signal mechanism (ACSM) of action of insulin and other peptides of the insulin superfamily in tissues of mammals, human, and mollusc. A. cygnea; (3) concept of molecular defects in hormonal signal systems as causes of endocrine diseases. Studies on the ST model have revealed in mollusc smooth muscle on the background of hyperglycemia at the 2nd, 4th, and 8th day after the ST administration a decrease of the ACSM response to activating action of insulin, IGF-1, and relaxin. These functional disturbances were the most pronounced at the 2nd day of development and rather less marked at the 4th and 8th day. Analysis of data on effect of hormonal and non-hormonal (NaF, GIDP, and forskolin) ACSM activators has shown that the causes of impair of signal-transducing function of this mechanism are (1) a hyperglycemia-induced increase of the basal AC activity and as a consequence--a decrease of the enzyme catalytic potentials in response to hormone; (2) a decrease of functions of Gs-protein and of its coupling with AC. Besides, administration of ST produced in the mollusc muscles an attenuation of regulation by insulin of carbohydrate metabolism enzyme (glucose-6-phosphate dehydrogenase, glycogensynthase). The pattern of disturbances in the studied parameters in the mollusc is very similar to that revealed by the authors in rat and human muscle tissues in type 1 diabetes.

[Evaluation of the diagnostic significance of cytologic studies in pancreatic tumors].

The study was undertaken to specify cytomorphological criteria for prostatic duct adenocarcinoma (PDAC), by assessing fine-needle aspiration biopsy (FNAB) specimens. Histologically verified cases were selected and the archival cytological material that constituted Leishman stained smears obtained by percutaneous FNAB under ultrasound guidance from 72 patients with PDAC (n = 50) or ductal hyperplasia (DP) (n = 22) was revised. For statistical analysis, the authors selected the following cytological signs of malignancy: anisonucleosis, increased nuclei, hyperchromia, uneven chromatin distribution, enucleation, uneven nuclear outline, apocytes; increased nucleoli, nucleolar polymorphism, multiple nucleoli, increased cells, anisocytosis (polymorphism of the size and shape of a cell, mitoses, piled nuclei, papillary structures, slightly glandular structures. The statistical analysis identified three most important cytological criteria for PDAC: anisocytosis, anisonucleosis, and piled nuclei. Keeping in mind all three signs at once increases the probability of detecting of PDAC up with 90%. The detection of only anisocytosis in the cytological specimen showed a 40% probability of the presence of PDAC. Three additional cytological signs of PDAC were also identified: increased nuclei, uneven nuclear outline, and slightly adhesive structures. The probability of PDAC was 100% if the specimen contained three most significant signs and any of the additional signs. By taking into account the indicators of diagnostic efficiency (DE), the best criteria were as follows: anisonucleosis, uneven nuclear outline, anisocytosis. The optimal indicators of DE for anisonucleosis were 95.83% diagnostic sensitivity (DSen), 95.83% diagnostic specificity (DSp), and 95.83% DE. When two indicators (anisonucleosis and uneven nuclear outline) were simultaneously used, DSen was 93.42; DSp, 98.53% (the highest); DE, 95.83%. Thus, the following cytological signs are defined as important for differentiation of PH and PDAC: anisocytosis, anisonucleosis, piled nuclei, increased nuclei, uneven nuclear outline, slightly adhesive structures.

[Involvement of adenylyn cyclase signaling mechanisms in relaxing effect of relaxin and insulin on the rat uterus, trachea and human myometrium].

Participation of adenylyl cyclase signaling mechanisms of relaxin and insulin action in their regulating influence on the process of relaxation of the rat uterine and tracheal smooth muscles and human myometrium was shown. The study was based on the discovery of novel adenylyl cyclase signaling mechanisms of relaxin and insulin action in the muscle of vertebrates which involve: receptor --> Gi protein (betagamma dimer) --> phosphatidylinositol-3-kinase --> protein kinase Csigma (zeta) --> Gs protein --> adenylyl cyclase --> cAMP. In the rat uterus, trachea and human myometrium, relaxin, insulin and isoproterenol induced relaxation of KCl-contraction. The order of efficiency of the agents based upon their ability to induce the inhibiting effect on the KCl-contraction was as follows: relaxin = insulin > isoproterenol. The hormones induce activating effect on adenylyl cyclase leading to production of cAMP in the rat uterine and tracheal smooth muscles and human myometrium. It is shown that cAMP reproduces relaxing effect of the hormones under study. Thus, the involvement of novel adenylyl cyclase signaling mechanisms of relaxin and insulin action in realization of their relaxation effect on rat uterus, trachea and human myometrium was revealed for the first time.

[Liver cancer: immunohistochemical and electron microscopic study].

Primary liver tumors, including 7 low-grade hepatocellular carcinomas (HC), 12 average-grade HC (including 2 mixed tumors and 2 cholangiocellular carcinomas (ChC)), obtained from 23 patients, were histologically, immunohistochemically, and electron microscopically. Certain markers were immunohistochemically studied to identify HC and ChC and differentiate liver carcinoma from metastatic tumors of the same organ.

[Angiomyolipoma of the kidneys].

59 angiomyolipomas were studied: immunohistochemistry (IHC) was used in 24 cases, electron microscopy (EM) in 6 cases. 57 tumors had a typical structure, 2 did not contain fat component. Premelanosomes were found by EM in one tumor. Smooth muscle actin was found in all tumor cells. Involvement of lymph nodes was observed in 2 patients. Histogenesis of this tumor is discussed.