L. A. Kuznetsova

Functional defects in adenylyl cyclase signaling mechanisms of insulin and relaxin in skeletal muscles of rat with streptozotocin type 1 diabetes

Functional disturbance in the novel adenylyl cyclase signaling mechanism (ACSM) of insulin and relaxin action in rat streptozotocin (STZ) type I diabetes was studied on the basis of the authors’ conception of molecular defects in hormonal signaling systems as the main causes of endocrine diseases. Studying the functional state of molecular components of the ACSM and the mechanism as a whole, the following changes were found in the skeletal muscles of diabetic rats compared with control animals: 1) increase of insulin receptor binding due to an increase in the number of insulin binding sites with high and low affinity; 2) increase of the basal adenylyl cyclase (AC) activity and the reduction of AC-activating effect of non-hormonal agents (guanine nucleotides, sodium fluoride, forskolin); 3) reduction of ACSM response to stimulatory action of insulin and relaxin; 4) decrease of the insulin-activating effect on the key enzymes of carbohydrate metabolism, glycogen synthase and glucose-6-phosphate dehydrogenase. Hence, the functional activity of GTP-binding protein of stimulatory type, AC and their functional coupling are decreased during experimental type 1 diabetes that leads to the impairment of the transduction of insulin and relaxin signals via ACSM.

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.

Study of the Functional Organization of a Novel Adenylate Cyclase Signaling Mechanism of Insulin Action

In this study we continued decoding the adenylate cyclase signaling mechanism that underlies the effect of insulin and related peptides. We show for the first time that insulin signal transduction via an adenylate cyclase signaling mechanism, which is attended by adenylate cyclase activation, is blocked in the muscle tissues of the rat and the mollusk Anodonta cygnea in the presence of: 1) pertussis toxin, which impairs the action of the inhibitory GTP-binding protein (Gi); 2) wortmannin, a specific blocker of phosphatidylinositol 3-kinase; and 3) calphostin C, an inhibitor of different isoforms of protein kinase C. The treatment of sarcolemmal membrane fraction with cholera toxin increases basal adenylate cyclase activity and decreases the sensitivity of the enzyme to insulin. We suggest that the stimulating effect of insulin on adenylate cyclase involves the following stages of hormonal signal transduction cascade: receptor tyrosine kinase → Giprotein (βγ) → phosphatidylinositol 3-kinase → protein kinase C (ζ?) → Gsprotein → adenylate cyclase → cAMP.

Adenylyl cyclase signaling mechanisms of relaxin and insulin action: Similarities and differences

The adenylyl cyclase signaling mechanism (ACSM) of relaxin H2 action was discovered and deciphered in mammalian muscles. A study of signaling blocks involved in ACSM of relaxin in comparison with that of insulin previously detected showed a close similarity throughout the post‐receptor signaling chain of both hormones. The inhibitory action of tyrosine kinase blockers on the hormone AC activating effect indicates that the relaxin receptor involved in ACSM is likely to be of the tyrosine kinase type. However, a recent discovery of a relaxin receptor with serpentine architecture leaves open the question concerning the existence of receptor of the tyrosine kinase type. The structural‐functional organization of the ACSM due to the action of relaxin—shown here for the first time—can be presented as the following signaling sequence: relaxin receptor ⇒ Gi protein (βγ‐dimer) ⇒ phosphatidylinositol 3‐kinase ⇒ protein kinase Cζ ⇒ Gs protein ⇒ adenylyl cyclase. According to our hypothesis, the regulatory action of the insulin superfamily peptides on cell processes (proliferation, apoptosis, and metabolism) is mediated via ACSM.

A novel, adenylate cyclase, signaling mechanism of relaxin H2 action

Abstract: For the first time, the adenylate cyclase signaling mechanism (ACSM) of the action of relaxin H2 was revealed and deciphered in human and rat muscle tissues. The comparative study of signaling blocks forming the ACSM of relaxin and insulin (discovered earlier) showed that the postreceptor signaling chain of relaxin coincides with that of insulin. However, the type of relaxin receptor involved in ACSM remains obscure. Currently, the ACSM of relaxin may be represented as a signaling cascade: receptor ⇒ Gi protein (βγ‐dimer) ⇒ phosphatidylinositol 3‐kinase (PI3K) ⇒ protein kinase Cζ (PKCζ) ⇒ Gs protein ⇒ adenylate cyclase.

Relaxin Adenylyl Cyclase System of Pregnant Women with Diabetes: Functional Defects in Insulin and Relaxin Adenylyl Cyclase Signaling Systems in Myometrium of Pregnant Women with Type 1 Diabetes

Abstract: The study was conducted to reveal the functional disturbances in two novel insulin and relaxin adenylyl cyclase signaling mechanisms (ACSMs). It was shown for the first time that in myometrium of pregnant women with insulin insufficiency the functional defects of Gs‐protein‐AC coupling in insulin‐ and relaxin H2‐regulated AC systems were developed. As a result, the sensitivity of the signaling systems to both hormones and potentiation of their AC effects by guanine nucleotides were markedly decreased compared with that in control group. These functional defects in ACSM may lead to violation of the process of insulin and relaxin signal transduction.

Functional Coupling of Hormone Receptors with G Proteins in the Adenylate Cyclase System of the Rat Muscle Tissues and Brain under Conditions of Short-Term Hyperglycemia

The sensitivity of components of the adenylate cyclase signaling system (heterotrimer G proteins and adenylate cyclase enzyme) to the regulatory effects of hormones mediated through G proteins (stimulatory effect of isoproterenol and relaxin and inhibitory effects of somatostatin) was decreased in the myocardium of hyperglycemic rats under conditions of transitory hyperglycemia caused by intravenous glucose and in hyperglycemia associated with insulin insufficiency in 24-h type 1 streptozotocin-induced diabetes mellitus. Changes in hormone sensitivity of the adenylate cyclase system were tissue-specific: clearly manifest in the myocardium, minor in skeletal muscles, and virtually absent in the brain of hyperglycemic rats. The main disorders of this system in the myocardium were observed at the stage of hormone receptor coupling with G proteins, which was seen from reduced stimulatory effect of GppNHp on adenylate cyclase activity and attenuation of the regulatory effect of hormones on adenylate cyclase enzyme and G proteins functionally coupled with it.

Regulation of the activity of adenylate cyclase and protein kinase a of the infusorians Dileptus anser and Tetrahymena pyriformis by biogenic amines and peptide hormones.

The hormone-sensitive adenylate cyclase (AC) system is one of the key signal systems involved in the transduction of hormonal signals in eukaryotic cells. The structural and functional organization of the AC system of higher eukaryotes has been studied sufficiently well, whereas the structure and mechanisms of function of lower eukaryotes are poorly understood. At the same time, the AC system of lower eukaryotes is an evolutionally more ancient signal system, and its study is a promising method of studying the evolution of the hormonal signaling systems.

Regulatory calcium effect on adenylyl cyclase functional activity in the infusorian Dileptis anser

Earlier we have shown that some non-hormonal activators of adenylyl cyclase (AC) and hormones of higher vertebrate animals are able to affect functional activity of the AC system in the infusorian Dileptus anser. In the present work, sensitivity of this infusorian AC to Ca2+ was studied and it was found that calcium cations at concentrations of 0.5–10 μM stimulated significantly the enzyme activity in D. anser partially purified membranes. An increase of Ca2+ concentrations to 100 μM and higher led to the complete block of their stimulatory effect. In the EDTA-treated membranes the enzyme activity was reduced markedly, but it was restored significantly by addition of Ca2+. Calmodulin antagonists—chlorpromazine, W-7, and W-5—caused a dose-dependent decrease of the enzyme activity stimulated by 5 μM Ca2+ with IC50 values of 35, 137, and 174 M, respectively. The AC-stimulating effects of biogenic amines (serotonin and octopamine) were completely retained in the presence of 2.5 and 100 μM Ca2+, whereas effects of peptide hormones (relaxine and EGF) were hardly changed in the presence of 2.5 μM calcium ions, but were markedly inhibited by 100 μM Ca2+. In the EDTA-treated membranes, the AC effects of biogenic amines were reduced, while the effects of peptide hormones were not revealed. On addition of Ca2+, the AC effects of biogenic amines were completely restored, whereas the effects of peptide hormones were not detected or restored to a non-significant degree. Calmodulin antagonists slightly affected the AC effects of peptide hormones at concentrations efficient in the case of vertebrate AC, but decreased them markedly at higher concentrations. The AC effects of biogenic amines were little sensitive even to high antagonist concentrations. The obtained data show that targets of action of peptide hormones in the infusorian D. anser cell culture are the AC forms whose activity depends on calcium cations and possibly is regulated by Ca2+/calmodulin, whereas targets of action of biogenic amines are calcium-independent enzyme forms.

Comparative study of biological activity of insulins of lower vertebrates in the novel adenylyl cyclase test-system

The biological activity of insulins of lower vertebrates (teleosts-Oncorhynchus gorbuscha, Scorpaena porcus, chondrosteans-Acipenser guldenstaedti and cyclostomates-Lamperta fluviatilis) was studied and compared with that of standard pig insulin. The determination of biological activity was made using the novel adenylyl cyclase (AC) test-system based on the adenylyl cyclase signaling mechanism (ACSM) of insulin action discovered earlier by the authors. The biological activity of insulins was estimated as EC(50), i.e. concentration leading to half-maximal activating effect of the hormone (10(-11)-10(-7) M), in vitro, on adenylyl cyclase in two types of the target tissues: in membrane fractions of the muscles of rat and mollusc Anodonta cygnea. In rat, the efficiency of insulins was found to decrease in the following order: pig insulin>scorpaena insulin>gorbuscha insulin>sturgeon insulin>lamprey insulin. In the mollusc, the order was different: sturgeon insulin>scorpaena insulin>pig insulin>gorbuscha insulin. Lamprey insulin at the same concentrations did not apparently reach the maximal adenylyl cyclase activating effect. The suggestion was made that differences in the biological activity of insulins depend on the hormone structure and a number of indexes characteristic of the adenylyl cyclase test-system in the vertebrate and invertebrate tissues. The proposed adenylyl cyclase test-system is highly sensitive to insulin at physiological concentrations, has good reproduction and is easy to apply.