K. V. Derkach

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

The third intracellular loop (ICL3) of G protein-coupled receptors has, as a rule, a key role in their interaction with heterotrimeric G proteins. We synthesized peptides corresponding to the C-terminal region of the ICL3 (C-ICL3) of 5-hydroxytryptamine receptors of the type 1B (5-HT1BR) and 6 (5-HT6R) and studied their influence on the functional activity of adenylyl cyclase signaling system (ACSS) in synaptosomal membranes isolated from the rat brain. The 5-HT1BR-peptide ARERKATKTL307–316K-amide mimicking agonist-activated 5-HT1BR reduced forskolin-stimulated adenylyl cyclase (AC) activity and activated pertussis toxin-sensitive G proteins. It lowered inhibitory effects of serotonin and 5-HT1BR-agonists on forskolin-stimulated AC activity and their stimulating effects on GTP binding. This was not the case in the presence of 5-HT1BR-antagonists. The 5-HT6R-peptides mimicking 5-HT6R activated both the basal AC activity and GTP binding of cholera toxin-sensitive G proteins. They lowered the stimulating effect of serotonin and 5-HT6R-agonists on AC and Gs proteins, but in the presence of 5-HT6R-antagonists their action was blocked. Of all the 5-HT6R-peptides with linear and dimeric structure we studied the palmitoylated peptide KHSRKALKASL258–268K(Pal)A-amide had a most pronounced effect both on the basal and 5-HT6R-agonist-stimulated ACSS. The data was obtained indicating that the peptides corresponding to C-ICL3 of 5-HT1BR and 5-HT6R selectively activate Gi and Gs proteins, respectively, and in a receptor-specific manner reduce signal transduction via serotonin-sensitive ACSS in the rat brain. The results of the study give strong evidence in favor of active participation of C-ICL3 of these 5-HTRs in their coupling with the G proteins.

On the tyrosine kinase mechanism of the novel effect of insulin and insulinlike growth factor I: Stimulation of the adenylyl cyclase system in muscle tissues

For the first time, insulinlike growth factor I (IGF-I), like insulin (Pertseva et al., Comp Biochem Physiol 112: 689-695, 1995), was shown to exercise a GTP-dependent stimulating action on adenylyl cyclase (AC; EC 4.6.1.1.) activity in the muscle tissues (membrane fraction) of mammal (rat) and mollusc (Anodonta cygnea). By studying the mechanism of the effect of peptides with selective inhibitors of tyrosine kinase activity, tyrphostin 47 (RG50864, 3,4-dihydroxy-alpha-cyanothiocinnamamide) and genistein (4,5,7-trihydroxyisoflavone), it was found that receptor tyrosine kinase is involved in this action. The data obtained suggest that the stimulating effect of insulin and IGF-1 is produced via the following signalling system: receptor tyrosine kinase --> stimulatory G-protein --> AC. Thus, the existence of a novel signalling pathway of transduction of signals generated by insulin and related peptides was hypothesised.

Intranasal insulin affects adenyl cyclase system in rat tissues in neonatal diabetes

The changes in hormone-regulated adenylyl cyclase (AC) signaling system implicated in control of the nervous, cardiovascular and reproductive systems may contribute to complications of diabetes mellitus (DM). We investigated the functional state of AC system in the brain, myocardium, ovary and uterus of rats with neonatal DM and examined the influence of intranasally administered insulin on the sensitivity of this system to biogenic amines and polypeptide hormones. The regulatory effects of somatostatin and 5-HT1BR-agonist 5-nonyloxytryptamine acting via Gi protein-coupled receptors were significantly decreased in DM and partially restored in insulin-treated rats. The effects of hormones, activators of AC, are changed in tissue- and receptorspecific manner, and intranasal insulin restored the effects rather close to the level in control. In insulin-treated non-diabetic rats, AC stimulating effects of isoproterenol and relaxin in the myocardium and of human chorionic gonadotropin in the ovaries were decreased, while the effects of hormones, inhibitors of AC, were increased. These data indicate that with intranasal insulin, Gi protein-mediated signaling pathways continue to gain strength. The obtained data on the influence of hormones on AC system in the brain, myocardium, ovary and uterus allow looking anew into the mechanisms of therapeutic effects of intranasal insulin.

The Functional State of Hormone-Sensitive Adenylyl Cyclase Signaling System in Diabetes Mellitus

Diabetes mellitus (DM) induces a large number of diseases of the nervous, cardiovascular, and some other systems of the organism. One of the main causes of the diseases is the changes in the functional activity of hormonal signaling systems which lead to the alterations and abnormalities of the cellular processes and contribute to triggering and developing many DM complications. The key role in the control of physiological and biochemical processes belongs to the adenylyl cyclase (AC) signaling system, sensitive to biogenic amines and polypeptide hormones. The review is devoted to the changes in the GPCR-G protein-AC system in the brain, heart, skeletal muscles, liver, and the adipose tissue in experimental and human DM of the types 1 and 2 and also to the role of the changes in AC signaling in the pathogenesis and etiology of DM and its complications. It is shown that the changes of the functional state of hormone-sensitive AC system are dependent to a large extent on the type and duration of DM and in experimental DM on the model of the disease. The degree of alterations and abnormalities of AC signaling pathways correlates very well with the severity of DM and its complications.

The Brain Adenylyl Cyclase Signaling System and Cognitive Functions in Rats with Neonatal Diabetes under the Influence of Intranasal Serotonin

Type 2 diabetes mellitus is often associated with the neurodegenerative changes, and this is regarded as being the cause of cognitive deficit and other brain dysfunctions. The data obtained by us and the other authors showed that alterations in the brain signaling systems regulated by different hormones and neurotransmitters contribute to triggering and development of neurodegenerative processes in diabetes. However, the activity of brain adenylyl cyclase system and the cognitive functions, their interrelation, and the influence of intranasal serotonin on them in type 2 diabetes are poorly understood yet. We investigated the hormonal sensitivity of adenylyl cyclase system in the brain of female rats with the neonatal model of type 2 diabetes and the influence of 8-weeks treatment with intranasal serotonin (20 μ g/rat daily) on the brain adenylyl cyclase system and cognition in diabetes. It was shown that in the diabetic brain the regulatory effects of hormones (relaxin, adrenergic agonists, dopamine, serotonin) activating adenylyl cyclase via Gs proteins changed in a receptor-specific manner and were restored by intranasal serotonin. The effects of hormones inhibiting adenylyl cyclase via Gi proteins were significantly decreased, especially in the case of agonists of type 1 serotonin receptors. The intranasal serotonin treatment led to their partial or complete restoration. Using Morris water maze test we showed that intranasal serotonin improves diabetes- associated impaired learning and spatial memory. Summing up, in the brain of diabetic rats the functional activity of hormone-sensitive adenylyl cyclase signaling system was altered, most dramatically in the G i -coupled cascades. The intranasal serotonin treatment improved both the signal transduction via the brain adenylyl cyclase system and cognitive functions in type 2 diabetes.

The Effect of Long-Term Intranasal Serotonin Treatment on Metabolic Parameters and Hormonal Signaling in Rats with High-Fat Diet/Low-Dose Streptozotocin-Induced Type 2 Diabetes.

In the last years the treatment of type 2 diabetes mellitus (DM2) was carried out using regulators of the brain signaling systems. In DM2 the level of the brain serotonin is reduced. So far, the effect of the increase of the brain serotonin level on DM2-induced metabolic and hormonal abnormalities has been studied scarcely. The present work was undertaken with the aim of filling this gap. DM2 was induced in male rats by 150-day high-fat diet and the treatment with low dose of streptozotocin (25 mg/kg) on the 70th day of experiment. From the 90th day, diabetic rats received for two months intranasal serotonin (IS) at a daily dose of 20 μg/rat. The IS treatment of diabetic rats decreased the body weight, and improved glucose tolerance, insulin-induced glucose utilization, and lipid metabolism. Besides, it restored hormonal regulation of adenylyl cyclase (AC) activity in the hypothalamus and normalized AC stimulation by β-adrenergic agonists in the myocardium. In nondiabetic rats the same treatment induced metabolic and hormonal alterations, some of which were similar to those in DM2 but expressed to a lesser extent. In conclusion, the elevation of the brain serotonin level may be regarded as an effective approach to treat DM2 and its complications.

Hormonal Signaling Systems of the Brain in Diabetes Mellitus

Diabetes mellitus (DM) is nowadays a major global health problem affecting more than 200 million people worldwide. It is one of the most severe metabolic disorders in humans characterized by hyperglycemia due to a relative or an absolute lack of insulin or the action of insulin on its target tissue or both. Many neurodegenerative disorders, such as diabetic encephalopathy and Alzheimers disease (AD), are associated with the type 1, insulindependent, and the type 2, non-insulin-dependent, diabetes mellitus (DM1 and DM2). Manifestations of these disorders in diabetic patients include alterations in neurotransmission, electrophysiological abnormalities, structural changes and cognitive deficit (Biessels et al., 2001). In the recent time attention to the neurological consequences of DM in the CNS has increased considerably. Many approaches and tools have been used to study etiology and pathogenesis of DM and DM-associated neurodegenerative disorders, and their diagnostics and treatment. The most perspective approaches are based on a combined use of the methods of biochemistry, molecular biology and physiology, they include clinical investigations of diabetic patients and the experimental models of DM and their complications, such as the model of DM1 induced by streptozotocin (STZ) treatment of young or adult rodents, the neonatal model of DM2 induced by the STZ treatment of newborn rats, and also the models of spontaneous DM and nutritional background causing DM2, as well as the models produced by transgenic manipulations or gene knockout techniques are all successfully used to study the molecular, cellular and morphological changes in diabetic brain (Shafrir, 2010). A severe hyperglycemia in DM1, mild hyperglycemia typical of DM2, and recurrent hypoglycemia induced by inadequate insulin therapy are the major factors responsible for the development of CNS complications in DM. The brain is mainly a glucose-dependent organ, which can be damaged by hyperas well as by hypoglycemia (Scheen, 2010). Being a major problem in clinical practice, hypoglycemia unawareness is associated with an increased risk of coma. Note that low blood glucose level induces negative mood states, primarily self-reported “nervousness” (Boyle & Zrebiec, 2007). Moreover, patients with a history of severe hypoglycemia show a much higher level of anxiety compared to other DM patients (Wredling, 1992). The prolonged influence of mild hypoglycemia on the brain leads to deregulation of many processes in CNS, which underlines the importance of scrupulously avoiding even mild hypoglycemic episodes in patients with DM. Hypoglycemia induces

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

One of the causes of complications in type 1 diabetes mellitus (T1DM) is the changes in adenylyl cyclase (AC) signaling system, identified on the early stages of the disease. However, the most significant disturbances in this system occur on the later stages of T1DM, which ultimately leads to severe complications, but functional state of the AC system in late T1DM is poorly understood. The 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 influence on them of 135-day therapy with intranasal insulin. It was shown that AC effects of -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. The treatment with intranasal insulin (0.48 IU/day) significantly restored these effects. The results were obtained suggesting that long-term T1DM induces significant 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.

The Influence of Intranasal Insulin on Hypothalamic-Pituitary-Thyroid Axis in Normal and Diabetic Rats

The functions of hypothalamic-pituitary-thyroid axis are attenuated in type 1 diabetes mellitus due to insulin deficiency. The use of intranasally administered insulin is of considerable interest for treatment of diabetes and cognitive disorders, but its effect on the thyroid system has not been investigated yet. We studied the influence of long-term treatment with intranasal insulin on the hypothalamic-pituitary-thyroid axis of nondiabetic rats and diabetic animals with streptozotocin models of acute and mild type 1 diabetes mellitus. This treatment was carried out for 28 days in acute (daily does of 0.3, 0.6, and 1.5 IU of insulin per rat) and for 135 days in mild diabetes (daily dose of 0.45 IU/rat). Nondiabetic rats were treated in a similar manner. Intranasal insulin in both models of diabetes resulted in the improvement of thyroid status; manifested as increase of thyroid hormones levels and restoration of response to thyroliberin. In acute diabetes, a daily dose of 0.6 IU/rat was the most effective. Twenty eight days treatment of nondiabetic rats with intranasal insulin at a dose of 0.3 IU/rat resulted in a significant increase of free and total thyroxine levels. Longer treatment of rats with mild diabetes and nondiabetic animals significantly increased thyrotropin level. Thus, long-term intranasal insulin treatment restored the hypothalamic-pituitary-thyroid axis function in type 1 diabetes, but led to a significant increase in the thyrotropin level, which must be considered when designing a strategy for the use of intranasal insulin in clinical applications.

Brain signaling systems in the Type 2 diabetes and metabolic syndrome: promising target to treat and prevent these diseases

The changes in the brain signaling systems play an important role in etiology and pathogenesis of Type 2 diabetes mellitus (T2DM) and metabolic syndrome (MS), being a possible cause of these diseases. Therefore, their restoration at the early stages of T2DM and MS can be regarded as a promising way to treat and prevent these diseases and their complications. The data on the functional state of the brain signaling systems regulated by insulin, IGF-1, leptin, dopamine, serotonin, melanocortins and glucagon-like peptide-1, in T2DM and MS, are analyzed. The pharmacological approaches to restoration of these systems and improvement of insulin sensitivity, energy expenditure, lipid metabolism, and to prevent diabetic complications are discussed.