Experimental models of diabetes mellitus types 1 and 2 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 superfamily—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 normal state and in experimental diabetes mellitus types 1 and 2 (DM1, DM2). Normally, the peptides stimulated GS activity to the maximum at a concentration of 10⁻⁸ M in vitro. The efficiency ranking of the peptide action was as follows: insulin > IGF-1 > relaxin. In DM1 the basal GS activity did not change, while the effect of insulin in vitro decreased more sharply on the 30th day of diabetes as compared to IGF-1 and relaxin, i.e. the efficiency ranking was as follows: IGF-1 = relaxin > insulin. Administration of insulin in vivo did not recover the sensitivity of the enzyme to the action of the hormone in DM1. In DM2, GS activity (both in total and in the active form) decreased while the stimulatory effect of the peptides and EGF on the enzyme was absent. Insulin administered in vivo did not lead to the recovery of the enzyme activity. We conclude that it is insulin resistance pronounced in DM2 that mostly affects the basal GS activity as well as the enzyme regulation by peptides of insulin type and EGF in rat skeletal muscles, while insulin deficiency in DM1 is of lesser importance.     

Regulation of Adenylyl Cyclase Signaling System in Cell Cultures of Infusoria Dileptus anser and Tetrahymena pyriformis by Peptides of Insulin Superfamily

Hormone-sensitive adenylyl cyclase signaling system (ACS) provides transduction of a wide spectrum of hormonal signals in cells of the higher eucaryotes. At the same time, ACS in the lower eucaryotes at present is practically not studied. We studied regulatory effects on ACS of the infusoria Dileptus anser and Tetrahymena pyriformis of peptide hormones of the higher eukaryotes—insulin, IGF-1, and relaxin, whose action on ACS of the higher eucaryotes was the subject of our earlier studies. The action of these hormones at concentrations of 10^–10–10^–8 M on the AC activity in infusoria had clearly stimulating character, the dose–effect curves being of a bell-shaped form with a maximum of the stimulating effect of the hormones at concentrations of 10^–9–10^–8 M. the shape of the curves and the value of the stimulating effect of the peptide hormones depended substantially on the level of the AC basal activity in homogenates of infusorian cell cultures. All the hormones (10^–8 M) stimulated GTP-binding activity of G-proteins. It was shown by the example of relaxin that its stimulating effect on GTP-binding in infusorian cells was dose-dependent and increased in the range of hormone concentrations from 10^–10 to 10^–8 M to reach its maximum at concentrations of 10^–8–10^–7 M. In the presence of suramin, an inhibitor of heterotrimeric G-proteins, the stimulating effects of the hormones on the GTP-binding and the AC activity decreased essentially or were absent completely. This indicates that the heterotrimeric G-proteins are ones of components of the signaling cascade that mediates regulatory effects of the hormones of the insulin group on the AC activity in infusorian cell cultures. Based on the obtained data, it is suggested that the basic molecular mechanisms of regulation of ACS by insulin and the related peptides that are similar to those found in the higher vertebrates already begin to be formed as early as at the level of the lower eucaryotes.     

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 regulation of glucose-6-phosphate dehydrogenase and glycogen synthase activities by peptides of insulin superfamily in myometrium of pregnant women and its impairments in 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.     

Insulin action and signalling in fat and muscle from dexamethasone-treated rats

Glucocorticoids initiate whole body insulin resistance and the aim of the present study was to investigate effects of dexamethasone on protein expression and insulin signalling in muscle and fat tissue. Rats were injected with dexamethasone (1 mg/kg/day, i.p.) or placebo for 11 days before insulin sensitivity was evaluated in vitro in soleus and epitrochlearis muscles and in isolated epididymal adipocytes. Dexamethasone treatment reduced insulin-stimulated glucose uptake and glycogen synthesis by 30-70% in epitrochlearis and soleus, and insulin-stimulated glucose uptake by ∼40% in adipocytes. 8-bromo-cAMP-stimulated lipolysis was ∼2-fold higher in adipocytes from dexamethasone-treated rats and insulin was less effective to inhibit cAMP-stimulated lipolysis. A main finding was that dexamethasone decreased expression of PKB and insulin-stimulated Ser⁴⁷³ and Thr³⁰⁸ phosphorylation in both muscles and adipocytes. Expression of GSK-3 was not influenced by dexamethasone treatment in muscles or adipocytes and insulin-stimulated GSK-3β Ser⁹ phosphorylation was reduced in muscles only. A novel finding was that glycogen synthase (GS) Ser⁷ phosphorylation was higher in both muscles from dexamethasone-treated rats. GS expression decreased (by 50%) in adipocytes only. Basal and insulin-stimulated GS Ser⁶⁴¹ and GS Ser^{645,649,653,657} phosphorylation was elevated in epitrochlearis and soleus muscles and GS fractional activity was reduced correspondingly. In conclusion, dexamethasone treatment (1) decreases PKB expression and insulin-stimulated phosphorylation in both muscles and adipocytes, and (2) increases GS phosphorylation (reduces GS fractional activity) in muscles and decreases GS expression in adipocytes. We suggest PKB and GS as major targets for dexamethasone-induced insulin resistance.     

Histochemical studies on glycogen synthetase activity by autoradiographic method

Summary Glycogen synthetase (uridine diphosphate glucose-glycogen glucosyl transferase) was studied in different organs by a histoautoradiographic method and by usual staining methods. This activity was found to be present in muscles and liver of different animals. Human skin also showed some activity. Human liver and myocardium showed the highest activity.In the present study, it was found that the glucose-6-phosphate dependent form (D-form) of the glycogen synthetase predominates over the glucose-6-phosphate independent form (I-form) in all the organs except hamster liver where the I-form predominates.Addition of calcium chloride in the incubation medium, to prevent phosphorolytic breakdown of the newly synthesized glycogen, does not improve the reaction. No glucose is incorporated into glycogen from ¹⁴C-glucose-6-phosphate of the incubation medium for glycogen synthetase. Fixation in absolute alcohol at –20° is recommended for tissues where cytolysis is caused by the incubation medium.     

Insulin-Like Growth Factor I (IGF-I) Binding in Skeletal Muscle of Lamprey Lampetra fluviatilis Predominates over Insulin Binding and Increase in the Course of Pre-Spawning Migration

Binding of ¹²⁵I-insulin and ¹²⁵I-IGF-I to partially purified receptors of lamprey skeletal muscles was studied during pre-pawning migration. It has been shown that throughout this whole period the IGF-I binding to skeletal muscle predominates over the insulin binding. Besides, a certain time dynamics was observed: the insulin binding rose since October to reach maximum in February–March, then it decreased to a minimum level in May; the IGF-I binding also increased: it rose statistically significantly in March compared to October, became maximal in April, and then decreased to a minimum. The dynamics of the receptor IGF-I binding has been shown to depend on changes of receptor affinity, whereas the change of the insulin binding was determined by binding capacity (the number of binding sites). Highly specific IGF-I receptors of the lamprey skeletal muscle bound insulin with an affinity about 1% from that of IGF-I, while insulin receptors had identical affinity for the insulin and IGF-I binding. Both peptides, insulin and IGF-I, activated autophosphorylation of beta-subunits in their receptors. The increase of the IGF-I binding from October to April could be a factor that maintains a high functional activity of lamprey skeletal muscles in the course of the pre-pawning migration. It is suggested that IGF-I promotes maintaining this activity due to its property of inhibiting apoptosis.     

Stimulatory Effect of Insulin and Epidermal Growth Factor on Activity of Protein Kinase A, Glucose-6-Phosphate Dehydrogenase, and Glycogen Synthase in Skeletal Muscles of Chick Embryos and Chickens

Insulin is able to produce two types of regulatory effects on muscles—metabolic and growth stimulating. Study of the effect of insulin and epidermal growth factor (EGF) on activity of cAMP-dependent protein kinase (PKA), glucose-6-phosphate dehydrogenase (G-6PDH), a starting enzyme of pentosephosphate pathway (PPP), and glycogen synthase (GS), a key enzyme of the glycogen synthesis, has shown that both types of the insulin effects do not arise simultaneously in the course of embryogenesis. The growth-stimulating effect mediated by adenylyl cyclase—cAMP is revealed since the 10th embryonal day. It was established for the first time that insulin could participate in growth stimulation by activating PKA in vivo and in vitro in muscles of the 10–14-day old embryos and the 8–10-day old chickens. The stimulating effect of insulin on G-6PDH activity is revealed since the same embryonal period and gradually increases. Insulin in vivo and in vitro simulates the glycogen synthase activity by increasing its conversion from non-active to active (GS-I) form only in the 15-day old embryos and in chickens. The stimulating effects of insulin and EGF on both G-6PDH activity (in embryos and chickens) and GS (in chickens) was shown to blocked by selective inhibitors of tyrosine kinases, thyrphostin 47 and genestein, in the dose-dependent manner, which indicates involvement of receptor of the tyrosine kinase type in these effects. The complex of the established facts permits concluding that insulin at early embryonal stages stimulates in the chicken muscles the PKA and G-6PDH activities involved in action of this hormone on growth, which is especially pronounced at the stage of myoblast proliferation. Meanwhile, the metabolic insulin effect (stimulation of the glycogen synthase system) appears in the second half of embryonal period and coincides in time with the period of muscle cell differentiation and active muscle contractures.     

The effect of insulin-like growth factor-1 on adult rat cardiac contractility

There is increasing evidence that insulin-like growth factor-1 (IGF-1) may play a role in both physiological and pathophysiological events in the mammalian myocardium. The present study investigated the acute effects of IGF-I on isometric force development in isolated rat cardiac muscle and on intracellular calcium (Ca²⁺) handling in isolated cardiac myocytes. IGF-I had a positive inotropic effect on rat ventricular papillary muscles increasing force development by 17.8 ± 4.6%, 18.5 ± 5.8% and 11.9 ± 4.9% (n = 12–20) at concentrations of 1, 10 and 100 ng/ml respectively. Isoprenaline increased tension in these papillary muscles by 56.7 ± 7.7% at a concentration of 100 nM (n = 22). In comparison, insulin increased papillary muscle force development by 11.6 ± 3.2%, 17.7 ± 4.1% and 19.7 ± 5.6% at concentrations of 1, 10 and 100 nM respectively (n = 16–20). In the single cardiac myocyte IGF-1 increased, the peak cytosolic free Ca²⁺ concentration, the amplitude of the Ca²⁺ transient and the time to peak Ca²⁺ as measured with the fluorescent bioprobe Indo-1 AM. The positive inotropic response to IGF-1 by rat ventricular muscle is therefore associated with a rise in free, peak cytosolic Ca²⁺ in isolated cardiac myocytes. Increasing insulin concentrations (1–1000 nM) elicited a progressive elevation in isometric force and free, cytosolic Ca²⁺. In contrast, in the presence of IGF-1, the maximal rise in isometric force and free cytosolic Ca²⁺ were both observed at 10 ng/ml. Recent reports have suggested that IGF-1 may act on the mammalian myocardium when administered chronically, but this study is amongst the first to demonstrate an acute effect of IGF-I on the mammalian heart. IGF-1 may prove then to be a novel cardioactive agent in both normal and pathophysiological states.     

Construction of a toxic insulin molecule: Selection and partial characterization of cells resistant to its killing effects

We have constructed an insulin-diphtheria hormono-toxin which migrates as a single 29 kd band on 10% SDS polyacrylamide gel electrophoresis. This corresponds to a one to one molar ratio of the diphtheria A-chain (23 kDa) and insulin (6 kDa) molecules. The diphtheria A-chain: insulin (DTaI) hormono-toxin demonstrates cytotoxicity in V-79 Chinese hamster cells exhibiting an LD₅₀ of 1.1×10^–8M, which is 22 x more potent than whole diphtheria toxin. Also, DTaI can competitively displace [¹²⁵I]-insulin with an ED₅₀ of 1.1×10^–8 M, which is identical to the ED₅₀ of insulin (1.1×10^–8M) and showed limited cross-reactivity with the IGF-1 receptor (12% displacement of [¹²⁵I]-IGF-1 with a DTaI concentration of 1.1×10^–8 M). We have used DTaI to select conjugate-resistant clones from the V-79 Chinese hamster fibroblast parental cell line. Conjugate-resistant variants expressed insulin binding levels ranging from 8.0±2.0 fmoles/mg protein down to 3.6±0.5 fmoles/mg protein while insulin binding in the V-79 parental cell line was 11.2±0.2 fmoles/mg protein. Additionally, a number of conjugate resistant clones expressed variable ability to grow in medium containing 5% serum. The altered ability of these clones to grow in a serum-containing medium did not correlate directly with the changes observed for insulin binding. One mutant, IV-A1-j, did not grow in a serum-free defined medium containing insulin as the predominant mitogen. This IV-A1-j mutant had a lower number of insulin receptors, no change in insulin binding affinity, no change in the rate of internalization of [¹²⁵I]-insulin and no apparent difference in [¹²⁵I]-IGF-1 binding. Further, insulin-stimulated sugar transport was similar to that observed in the parental cell line. Based on these observations we suggest that 1) DTaI elicits its cytotoxicological effects through the insulin receptor trafficking pathway, 2) DTaI can be used to isolate cells altered at the level of insulin binding and/or action, and 3) signal transduction mechanisms responsible for mediating insulin-dependent cell growth can be pursued using mutants such as IV-A1-j.     

Effects of phorbol esters on insulin receptor function and insulin action in hepatocytes: evidence for heterogeneity

We investigated the effect of phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator on insulin receptors and insulin action in freshly isolated and primary cultures of rat hepatocytes. PMA (1 x 10^–7 M) did not alter insulin receptor numbers or affinity either acutely or chronically but within 60 minute inactivated insulin stimulated tyrosine kinase of the insulin receptor. PKC activation inhibitied insulin (1 x 10^–7M) stimulation of glycogen and lipid synthesis with a decrease or no change in basal glycogenesis and lipogenesis respectively. However, PKC activation did not alter insulin stimulated or basal amino acid transport even though PCK activation inhibited insulin stimulation of the insulin. receptor tyrosine kinase. Thus, within one tissue, PKC activation has differential effect on insulin action depending on which pathway is examined. Furthermore, insulin stimulation of the insulin receptor tyrosine kinase may not be a necessary step for all insulin signaling pathways.     

Regucalcin modulates hormonal effect on (Ca2+−Mg2+)-ATPase activity in rat liver plasma membranes

The interaction of various hormones and regucalcin on (Ca²⁺–Mg²⁺)-ATPase activity in rat liver plasma membranes was investigated. The presence of epinephrine (10^–6–10^–4 M), and insulin (10^–8–10^– M) in the reaction mixture produced a significant increase in (Ca²⁺–Mg²⁺)-ATPase activity, while the enzyme activity was decreased significantly by calcitonin, (3×10^–8–3×10^–6 M). These hormonal effects, except for calcitonin, were clearly inhibited by the presence of vanadate (10^–4 M) which can inhibit the Ca²⁺-dependent phosphorylation of enzyme. Meanwhile, regucalcin (0.25 and 0.50 M), isolated from rat liver cytosol, elevated significantly (Ca²⁺–Mg²⁺)-ATPase activity in the plasma membranes, although this elevation was not inhibited by vanadate (10^–4 M). the epinephrine (10^–5 M) or phenylephrine (10^–4 M)-induced increase in (Ca²⁺–Mg²⁺)-ATPase activity was disappeared in the presence of regucalcin; in this case the effect of regucalcin was also weakened. However, the inhibitory effect of calcitonin (3×10^–6 M) was not weakened by the presence of regucalcin (0.5 M). Moreover, GTP (10^–5 and 10^–4 M)-induced increase in (Ca²⁺–Mg²⁺)-ATPase activity was not seen in the presence of regucalcin (0.25 M). The present finding suggests that the activating mechanism of regucalcin on (Ca²⁺–Mg²⁺)-ATPase is not involved on GTP-binding protein which modulates the receptor-mediated hormonal effect in rat liver plasma membranes.     

Oxidant stress-induced loss of IRS-1 and IRS-2 proteins in rat skeletal muscle: Role of p38 MAPK

Oxidative stress is characterized as an imbalance between the cellular production of oxidants and the cellular antioxidant defenses and contributes to the development of numerous cardiovascular and metabolic disorders, including hypertension and insulin resistance. The effects of prolonged oxidant stress in vitro on the insulin-dependent glucose transport system in mammalian skeletal muscle are not well understood. This study examined the in vitro effects of low-level oxidant stress (60–90 μM, H₂O₂) for 4 h on insulin-stimulated (5 mU/ml) glucose transport activity (2-deoxyglucose uptake) and on protein expression of critical insulin signaling factors (insulin receptor (IR), IR substrates IRS-1 and IRS-2, phosphatidylinositol 3-kinase, Akt, and glycogen synthase kinase-3 (GSK-3)) in isolated soleus muscle of lean Zucker rats. This oxidant stress exposure caused significant (50%, p < 0.05) decreases in insulin-stimulated glucose transport activity that were associated with selective loss of IRS-1 (59%) and IRS-2 (33%) proteins, increased (64%) relative IRS-1 Ser³⁰⁷ phosphorylation, and decreased phosphorylation of Akt Ser⁴⁷³ (50%) and GSK-3β Ser⁹ (43%). Moreover, enhanced (37%) phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was observed. Selective inhibition of p38 MAPK (10 μM A304000) prevented a significant portion (29%) of the oxidant stress-induced loss of IRS-1 (but not IRS-2) protein and allowed partial recovery of the impaired insulin-stimulated glucose transport activity. These results indicate that in vitro oxidative stress in mammalian skeletal muscle leads to substantial insulin resistance of distal insulin signaling and glucose transport activity, associated with a selective loss of IRS-1 protein, in part due to a p38 MAPK-dependent mechanism.     

Effects of insulin,triiodothyronine, and serotonin on plant seed development

Summary Treatment with insulin, triiodothyronine or serotonin resulted in increases of root length, root weight, coleoptile weight and mitotic index of germlings from barley seeds at concentration of 10^–8 M. All three hormones were superior in activity to the natural and synthetic plant hormones (3-indoleacetic acid, naphthylacetic acid, trichlorophenoxyacetic acid) tested for comparison. The experimental observations suggest that plant cells also have receptors to which hormones of vertebrates can bind, and that plants cells also respond to such hormones.Supported by the Scientific Research Council, Ministry of Health, Hungary 1-01-0302-02-1/Cs.     

Interaction between aflatoxin B1 and oxytetracycline in isolated rat hepatocytes

Isolated rat hepatocytes were used as an in vitro model to investigate A possible interaction between oxytetracycline (OXT) and aflatoxin B₁ (AFB₁). LDH leakage, RNA and protein synthesis and glycogen accumulation were measured in the presence of both drugs, either separately or in combination. The evolution of LDH leakage during the incubation was identical in untreated and treated cells. AFB₁ inhibited RNA and protein synthesis at a concentration of 10^–7 M and 10^–6 M, respectively, and higher, whereas OXT did not influence RNA synthesis but inhibited protein synthesis at the highest tested concentration, 10^–3 M. As far as glycogen is concerned, rats were injected with glucagon before sacrifice in order to obtain a constant synthesis rate in isolated hepatocytes. AFB₁ inhibited the accumulation of glycogen from 10^–6 M upward. This effect was never observed before 90 min of incubation. OXT had no effect on glycogen synthesis. In the presence of both drugs, no interaction was demonstrated as far as RNA and protein synthesis were concerned, but OXT opposed the inhibition induced by AFB₁ on glycogen accumulation. If the in vivo protection, provided by OXT against AFBI-induced toxicity, is due to a direct interference in the toxic mechanisms of the mycotoxin, these results show that OXT does not influence the AFB₁-inhibition of RNA and protein synthesis. The latter are early and sensitive parameters inhibited by AFB₁. On the contrary, taking into consideration the results on glycogen accumulation, it seems more interesting to investigate further this metabolism.Abbreviations AFB₁ Aflatoxin B₁ - OXT Oxytetracycline - DMEM Dulbecco's Modified Eagle's Medium - LDH Lactate Dehydrogenase - DMSO Dimethyl Sulfoxide - BSA Bovine Serum Albumin     

Effect of insulin on the incorporation of3H-inositol into the inositol phospholipids (PI,PIP, PIP2) and Glycosyl-phoshatidylinositols (GPIs) of Tetrahymena pyriformis

The unicellular tetrahymena contains inositol phospholipids (PI, PIP, PIP₂) and GPIs. Treatment with 10^–5M insulin decreases the total³H-inositol incorporation and incorporation into PI. 24 h after 10^–6M insulin treatment there is an elevation of these parameters. Second treatment with 10^–6M insulin doubles and 10^–5M decreases these levels. This means that the effect on phosphoinositide turnover by insulin in Tetrahymena is rather concentration dependent. Inositol incorporation into GPIs is also influenced by insulin.     

Adenylyl Cyclase Signaling Mechanism of Relaxin Action

In connection with our discovery of the adenylyl cyclase signaling mechanism (ACSM) of action of some peptides belonging to the insulin superfamily, a possibility of its involvement in action of another insulin superfamily peptide, relaxin, was studied. It was shown for the first time that human relaxin-2 (10^–12–10^–8 M) activated adenylyl cyclase (AC) in a dose-dependent manner. The maximal peptide effect was revealed at a concentration of 10^–8 M. Under condition of the hormonal action the basal enzyme activity increased by +310% in human myometrium, by +117%, in rat skeletal muscles, and by +49%, in foot smooth muscles of the bivalve mollusc Anodonta cygnea. Insulin and mammalian insulin-like growth factor-I (IGF-I) also produced the AC activating effect in these muscles. The order of efficiency of these peptides, based on their ability to induce the maximal AC stimulating effect, was as follows: relaxin > IGF-I > insulin (human myometrium); IGF-I > relaxin > insulin (rat skeletal muscle); insulin-like peptide of Anodonta (ILPA) > IGF-I > insulin > relaxin (molluscan muscle). The relaxin activating effect on AC was potentiated by a guanine nucleotide, the non-hydrolyzed analog of GTP, guanylylimidodiphosphate (Gpp[NH]p), which indicates participation of G_s-protein in realization of this effect. This effect was inhibited by a tyrosine kinase selective blocker, tyrphostin 47, and a phosphatidylinositol-3-kinase (PI-3-K) selective blocker, wortmannin. Thus, for the first time, participation of ACSM in the relaxin action has been established. This mechanism, as suggested at the present time state of its study, includes the following signal pathway: receptor-tyrosine kinase PI-3-K G_s-protein AC.     

Chronic and endogenous regulation of insulin receptors by catecholamines in adipocytes from patients with a phaeochromocytoma

Insulin binding in adipocytes from patients with a phaeochromocytoma (PH) approached that of the controls (C) at low and higher concentrations of unlabeled insulin. The apparent receptor affinity was unchanged (ED50: PH 0.50×10^–9M and C0.60×10^–9M). Scatchard analysis of the binding data using the negative cooperative model revealed a 46% decrease in the total number of receptors together with no changes in both K^–e (PH 0.55×10⁹M^–1 and C 0.36×10⁹M^–1) and K^–f (PH 0.13×10⁹ M^–1 and C 0.07×10⁹ M^–1). According to the two site model, an altered proportion in the two classes of insulin binding sites was detected. This was accompanied by a catecholamine-desensitization of the adipocytes to the antilipolytic action of insulin. These events could represent a final situation of a chronic and endogeneous regulation by high levels of catecholamines of insulin receptors in human adipose tissue.     

Effect of urethane (ethyl carbamate) on the mitosis of phytohaemagglutinin stimulated human lymphocytesin vitro

Summary The action of urethane at concentrations between 10^–1 and 10^–8 M on the rate of mitosis of phytohaemagglutinin-stimulated human lymphocytes in culture has been investigated. At 10^–1M urethane is cytotoxic, at all other concentrations tested it has some inhibitory activity except at 10^–5M and 10^–6M where it stimulates mitosis. These results are discussed in relation to previously reported data and compared with the action of hydroxyurea in the same system.I wish to thank the Smith Kline and French Foundation for financial support and Mrs. B. Haynes and Miss C. Watson for technical assistance.