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Diabetic patients present alterations in the activity of a number of enzymes of the plasma membrane. The aim of this study was to verify if the modifications of the enzymatic activities in diabetes mellitus are associated with structural alterations of the cellular membrane. By means of the freeze-fracturing technique, we studied the structure of erythrocyte membranes from 15 insulin-dependent diabetic patients (24-43 years) and 15 age-matched healthy subjects (26-47 years). The kinetic properties of the Na+/K(+)-ATPase of the same membranes were also investigated. The Na+/K(+)-ATPase of the erythrocyte plasma membrane shows an uncompetitive inhibition in the diabetic subjects. As for the freeze-fracturing results, the intramembrane particles of the erythrocyte membranes from diabetic patients appear more clustered with respect to those obtained from controls. The uncompetitive inhibition of the enzyme suggests the presence of conformational modifications of the protein. This hypothesis is supported by the freeze-fracture results which indicate that the integral protein constituents of the membrane in diabetes tend to aggregate. Modifications of the interactions between the enzymatic subunits and the membrane lipid environment might be at the basis of the Na+/K(+)-ATPase alteration in diabetes.

Degenerate oligonucleotide primers complementary to the highly conserved subdomains III and VIII of subclass III tyrosine kinase receptors (TKr-III) were utilized to amplify rat aortic cDNA by polymerase chain reaction. Most of the cloned DNA products were rat platelet-derived growth factor receptor beta and macrophage-colony stimulating growth factor receptor cDNAs. Screening of the clones with probes coding for the receptor-specific kinase insert domain allowed the identification of a novel putative TKr-III cDNA, which hybridized with a approximately 6.1 kb mRNA with a distinctive tissue distribution. In situ hybridization on rat tissues and Northern analysis of cultured cells indicate that endothelial cells express a novel putative TKr-III mRNA.

Androgen receptors have been found in human larynx and androgens have been supposed to play an important role in promoting the growth of laryngeal carcinomas. The molecular mechanism underlaying this phenomenon is not at all understood. Aim of this work was to investigate the effects of two androgens (testosterone and dihydrotestosterone) on insulin receptor mRNA levels and insulin binding activity as well as on either metabolic or growth-promoting actions of insulin in a human larynx carcinoma cell line (HEp-2). We found that HEp-2 cells express a high affinity insulin receptor. Both androgens significantly increase insulin receptor mRNA levels and insulin receptor number in HEp-2 cells. Insulin action, evaluated either as total glucose utilization or as [3H]thymidine incorporation into DNA, significantly increased in HEp-2 treated with androgens in comparison to control cultures. Altogether, our data allow us to speculate that the increased insulin effectiveness we observed in the larynx carcinoma cell line HEp-2 after androgen treatment might be involved in the regulation of larynx cancer cells growth.

The human placenta plays an essential role in embryo development, in particular regulating the transport of ions, nutrients and immunoglobulins from the maternal to the fetal circulation. Trophoblast organization into a syncytial layer involves structural and functional steps that may be monitored and elucidated by in vitro studies. The structural stages by which the syncytial trophoblast is formed are not yet understood. In order to clarify the mechanism of trophoblast development, we studied the morphological characteristics of the syncytial trophoblast formation in culture and the functional changes (transport properties and membrane microviscosity) accompanying the structural modifications. By using both 5-nitroxystearate and 16-nitroxystearate as spin labels, we observed an initial increase in membrane order over 0-24 h of culture, which can be associated with two events: recovery of cell membranes from trypsin and initial aggregation of cytotrophoblasts. The similar behaviour of the order parameters determined with both probes indicates that membrane order changes both inside and in the outer part of the lipid bilayer. The subsequent decrease in membrane order observed at 36-48 h might be related to the process of cellular fusion. The increase in sodium/potassium pump activity in the first 24 h of culture might be an expression of cell recovery following trypsin treatment. The subsequent decrease might represent an adaptive mechanism by which metabolic energy is mainly used for morphogenetic changes.

The pathogenesis of plasma membrane alterations present in diabetes mellitus is unclear. To add new insights to the question, platelet membrane properties were evaluated in 16 women presenting impaired glucose tolerance at the 28-29th week of gestation (GDM) and in 8 women with insulin-dependent diabetes mellitus (IDDM). 15 healthy pregnant women (HPW) and 21 healthy non-pregnant (HNPW) women were the control group for GDM and IDDM, respectively. Pregnancy (HPW vs. HNPW) provoked an increase in Ca(2+)-ATPase activity and a decrease in membrane fluidity; in contrast, Na+/K(+)-ATPase, intracellular free Ca2+ concentrations, membrane cholesterol and phospholipid content did not vary. Both GDM and IDDM showed lower Na+/K(+)-ATPase activity and higher Ca2+ concentration, compared to HPW and HNPW, respectively, whereas Ca(2+)-ATPase activity was higher only in IDDM; furthermore, membrane fluidity was lower in GDM and higher in IDDM. Finally, GDM showed higher membrane cholesterol content. Both GDM and IDDM showed a very good metabolic control so that variations reported cannot be due to hyperglycemia; it is tempting to suggest that membrane variations are present before the clinical metabolic alteration. Furthermore, both GDM and IDDM were on insulin therapy, therefore: (i) insulin may be the pathogenetic factor of higher intracellular free Ca2+ concentrations and lower Na+/K(+)-ATPase activity since they both varied accordingly in GDM and IDDM, but not of (ii) changes in Ca(2+)-ATPase, membrane fluidity and cholesterol content which did not vary accordingly in GDM and IDDM.

Various alterations of red blood cell (RBC) plasma membrane appear both in diabetes mellitus and during the physiological aging process. Diabetes mellitus decreases RBC life-span; therefore, it may change the plasma membrane by acting through its effect on the aging process. In order to clarify the issue, RBCs from normal subjects and insulin-dependent diabetic patients were fractionated in five subpopulations of different mean age (fraction 1: early young RBC, fraction 5: mature RBC). Thereafter, plasma membranes were prepared and enzymatic activities, membrane fluidity and lipid peroxidation were evaluated. NA+, K(+)-ATPase activity decreased during aging and it was higher in all RBC subpopulations from normal subjects in comparison to diabetic patients. Next, lipid peroxidation and fluidity increased during aging in both the study groups; in this case, however, in all subpopulations, except for that from fraction 1, RBCs from diabetic patients showed higher membrane fluidity and lipid peroxidation in comparison to normal subjects. Data herein reported suggest that diabetes mellitus affects the plasma membrane independently of (lipid peroxidation and fluidity) or dependently on (Na+, K(+)-ATPase) its effect on aging. In the case of lipid peroxidation and fluidity diabetes mellitus seems to affect the membrane by decreasing RBC life span, whereas in the case of Na+K(+)-ATPase it seems to alter this enzymatic activity which in turn might affect RBC aging. Acetylcholinesterase activity decreased during aging in RBCs from normal subjects, but it increased in RBCs from diabetic patients; RBC subpopulation from fraction 1, on the other hand, showed similar values in normal subjects and diabetic patients. In this case the effect of diabetes mellitus appears only during aging.

Maternal- fetal exchanges are mainly regulated by trophoblast, which displays an active role during embryo growth. Trophoblast organization into a syncytial layer involves structural and functional steps that may be monitored and better elucidated by "in vitro" studies. In light of this, we have carried out morphological and biochemical analyses in order to evaluate 1) the syncytiotrophoblast formation in culture (48 h, 5-30 days) the Na+/K+ATPase activity and 3) the plasmalemmal microviscosity changes occurring during "in vitro" trophoblast production. Morphological and biochemical modulations have been pointed out.

Specific binding of [125I]insulin to circulating monocytes and erythrocytes from nine normal menstruating women and nine normal men was determined during a 28-day period (one sample every 7 days). In women, insulin binding was higher to both monocytes (P less than 0.001) and erythrocytes (P less than 0.02) in the follicular phase than in the luteal phase. In men, insulin binding to monocytes was similar to the follicular phase values for women; however, insulin binding to erythrocytes from men showed higher values than insulin binding to erythrocytes from women in both the follicular (P less than 0.001) and luteal (P less than 0.001) phases. These differences were due primarily to changes in receptor concentration rather than receptor affinity. An inverse relationship was found between insulin binding to monocytes and levels of 17 beta-estradiol, progesterone, and 17 alpha-hydroxyprogesterone; this relationship was not observed in insulin binding to erythrocytes. The present data, therefore, suggest that sex hormones may play a role in the control of insulin receptors. Furthermore, it appears that other factors exist during the follicular phase that lower insulin binding to erythrocyte insulin receptors. If insulin receptors on circulating cells reflect the behavior of the main insulin target tissues, the present data might in part explain the reduction in glucose tolerance reported by various authors in the second half of the menstrual cycle.

Glucose ingestion, food intake or acute exercise produced rapid variations in insulin binding on monocytes. Insulin seems to play a minor or any role in this phenomenon in contrast pancreatic glucagon might be involved since it usually rises when an increase in insulin receptor affinity has been observed. To investigate the role of pancreatic glucagon we have studied the effect of arginine infusion on monocyte insulin receptor in five normal subjects. Results indicate that the aminoacid employed does not induce any change in insulin receptor affinity and concentration suggesting that the simultaneous increase in plasma insulin and pancreatic glucagon levels does not exert any action.

[125I]-[D-Ala2]-beta EP*) binding to Epstein-Barr Virus (EBV) transformed B lymphocytes or to freshly isolated lymphocytes was characterized. The binding was time-, temperature- and pH-dependent; furthermore, it was reversible and cell concentration dependent. Maximum binding appeared at 15C in Tris buffer (pH 7.6) containing both BSA 0.5% and Bacitracin 0.1 mg/ml. beta EP inhibited BEP* binding to transformed lymphocytes and to freshly isolated lymphocytes by 50% at approximately 4 x 10(-8) M and 8 x 10(-9) M, respectively. Peptides representing amino-acid sequences 1-5, 1-16 and 1-17 of beta EP did not inhibit beta EP binding, neither did the opiates compounds Naloxone, Morphine, Bremazocine and Ethylketocyclazocine. On the contrary, beta EPd (6-31) inhibited beta EP* binding as effectively as beta EP, thus indicating that beta EP* binding to lymphocytes does not involve the N-terminal region of beta EP. 2 x 10(7)/ml freshly isolated lymphocytes bound 1.53 +/- 75%, whereas 2 x 10(6)/ml transformed lymphocytes bound 1.64 +/- 0.75% (mean +/- SD) beta EP* at tracer concentration. EBV transformed lymphocytes and freshly isolated lymphocytes may be useful to study beta EP receptors in humans.

Specific binding of 125I-insulin to circulating monocytes from eight normal menstruating women, four postmenopausal women and four men were studied four times during a 28-day period (one sample at 7-day intervals). Data indicate the presence of a higher specific cell binding fraction in the follicular phase compared to the luteal phase due to changes in insulin receptor concentration. No changes were observed in men or postmenopausal women during the same period of time suggesting that sex hormones should be included among the factors influencing insulin receptors.

Density gradient centrifugation analysis has been used to study rat kidney receptors for dexamethasone. Density gradients performed with cytosol incubated with 3H-dexamethasone in a Tris 10 mM, EDTA 1.5 mM, glycerol 5%, monothioglycerol 12 mM (pH 7.5) buffer never gave any good evidence of a 85 radioactivity peak probably due to instability of the complex. The stability of the complex for at least 30 h was achieved by homogenizing the tissue and incubating cytosol in a distilled water, glycerol 5% monothioglycerol 12 mM solution. Density gradient centrifugation analysis in this way showed radioactivity peak at the bottom of the tube probably due to the aggregation of the complex. Since this drawback is inherent in the technique it must be concluded that density gradient centrifugation analysis cannot be used to study the dexamethasone-receptor complex of the kidney.

Three monoclonal anti-insulin receptor antibodies have been labelled with 125I according to various methods (Cloramine T, Lactoperoxidase and IODO-GEN). The effect of labelling on antibody structure and function has been characterized using the following parameters: a) specific activity obtained in four different labelling procedures, at least; b) TCA labelled antibody precipitable 90 days after labelling; c) interaction between labelled antibodies and the insulin receptor; d) ability of antibodies to inhibit insulin-stimulated receptor auto-phosphorylation. Cloramine T method produced labelled antibody with constant specific activity; however, some preparations were unstable and showed reduced capacity to recognize the insulin receptor. Lactoperoxidase method produced stable antibodies; however, specific activity was highly variable and antibodies had low capacity to interact with the insulin receptor. The IODO-GEN method produced antibodies with constant specific activity, stable, high capacity to interact with the insulin receptor, and, moreover, maintaining in full the capacity to inhibit the insulin-stimulated auto-phosphorylation of the insulin receptor, since it does not induce antibody alterations which in turn affect antibody-receptor interaction biological action.

Prior studies with monoclonal antibodies produced against the human insulin receptor in mice revealed that these antibodies may be species specific. Whether species-specific antibodies to the insulin receptor occur spontaneously in patients, however, has not been previously investigated. Recently, we found that the serum immunoglobulin G from a patient with lupus nephritis, insulin resistance, and hypoglycemia contained multiple subpopulations of antibodies directed at the human insulin receptor. We report herein that one such subpopulation has a high affinity for the human insulin receptor. This antibody subpopulation at 10 nM half-maximally inhibited [125I]insulin binding to human IM-9 lymphocytes, circulating erythrocytes and monocytes, isolated adipocytes, and placenta membranes. In contrast, this antibody subpopulation did not inhibit [125I]insulin binding to isolated rat adipocytes and hepatocytes, even at concentrations as high as 100 nM. These studies indicate that species-specific antibodies can occur spontaneously in patients with antiinsulin receptor antibodies.

Studies have been carried out with labeled substrates on isolated adipocytes of male rats injected with sulpho-conjugated estrogens. Three days treatment did not affect insulin receptor concentration or affinity in the cells. Conversely glucose uptake is reduced in the absence of insulin, or by reduced responsiveness in the presence of it. After ten days treatment, insulin binding impaired on account of reduced receptor concentration, whereas glucose uptake was normal in the absence of insulin, increased at low and decreased at high insulin concentration, on account of both decreased responsiveness and increased sensibility to insulin. Thus it is suggested that estrogens acts on glucose uptake prior to act on the insulin receptor in the adipocytes.

Combined estrogen/progestagen oral contraceptives (OC) have been reported to be associated with a deterioration of glucose tolerance and a decrease in insulin sensitivity; thus, since it has been suggested that steroids affect insulin receptor properties, the influence of OC on insulin receptors was investigated. The study groups were composed of nine normal menstruating women (controls), nine pill users, and two healthy women on OC for the first time. Insulin receptors on monocytes were evaluated at 7-day intervals during the 28 days between menses. Insulin receptor concentration and/or affinity did not show any variation in pill users during the test period and did not differ from values observed in controls in the luteal phase; consequently, the insulin receptor concentration in pill users is lower than that during the follicular phase or in men. The physiological variation of insulin receptor concentration and the increase of receptor affinity in the midfollicular phase, which characterize the normal menstrual cycle, are therefore abolished by OC. This effect occurs rapidly because it was also evident in the two women on OC for the first time. No difference was observed in fasting blood glucose and serum immunoreactive insulin concentrations between control subjects and pill users. The present data appear to confirm that sex steroids affect the insulin receptor and lend further support to the concept that caution must be used in clinical studies of insulin receptors when women are included. In addition, the results suggest that insulin receptors may play a role in the glucose intolerance and insulin insensitivity which have been described in pill users.

Thyroglobulin biosynthesis was studied in thyroid glands of rats treated during 30 days with a daily dose of propranolol, a non-selective beta-adrenergic blocking drug. Studies were carried out by extraction of soluble proteins from homogenates after incubation of the glands in presence of [3H]-4,5-L-leucine and [3H]-D-1-galactose. Thyroglobulin 19S, 12S and 4-8S soluble proteins were separated and identified by ultracentrifugation in a saccharose gradient. The glands of rats treated with propranolol showed a decreased amount of soluble proteins as well as a decreased incorporation of [3H] labeled markers. 19S thyroglobulin is poorly represented, but very large amounts of the 12S monomer are present; this suggests an impairment in the dimerization of the 12S subunit, absent in normal controls. This impairment could be due to a structure modification. Propranolol reduces the biosynthesis of thyroglobulin in thyroid gland as well as the formation of T3 from T4 in peripheral tissues; its therapeutical use against thyrotoxicosis is thus justified. The study of its action on the dimerisation of the 12S subunit could be of interest to clear the mechanism of thyroglobulin biosynthesis.

The effect of monoclonal anti-insulin receptor antibody MA 10 on [125I]insulin binding and on insulin receptor protein tyrosine kinase activity was investigated in human and rat tissues. It was observed that MA 10 inhibits insulin binding to human, but not rat, tissues while inhibiting insulin-stimulated receptor autophosphorylation and protein tyrosine kinase activity in both human and rat tissues. These data suggest that MA 10 is directed against a region of the insulin receptor that is in between the insulin-binding domain and the beta-subunit and that in human, but not rat, tissues, this region is involved in insulin binding.

Results of a recent study suggested that depending upon the glucocorticoid or the model used, opposite changes occur in insulin binding; in fact, the increase in insulin receptor number on monocytes after prednisone ingestion in man appears to contrast with previous reports in animals in which a decrease was shown after dexamethasone. To establish whether this apparent discrepancy depends upon the model used in human studies (i.e. monocytes), the effect of dexamethasone and corticone intake on normal men was evaluated. A significant decrease was observed in insulin binding on circulating monocytes 24, 48, and 72 h after both steroids, mainly due to reduced receptor affinity. Furthermore, steroid treatment increased insulinemia which did not appear to be related to insulin binding. These data are in agreement with results in animal studies and appear to suggest that previous data on prednisone do not depend upon the model used (i.e. monocytes) but upon the hormone itself, thus indicating that glucocorticoids, depending upon their chemical structure, may produce opposite changes in membrane insulin binding sites. Furthermore, since dexamethasone and cortisone affect plasma insulin levels in the same fashion as previously reported with prednisone, it is suggested that the variation in insulin binding observed after glucocorticoid treatment is not due to variations in insulinemia.

Insulin binding to circulating monocytes was studied in 22 normal volunteers before and 1, 3 and 5 h after a 1400 Kcal meal. Results indicate that 3 h after food intake there is an increase in the specific cell binding fraction (P less than 0.001) with a change in receptor affinity. Data emerging from the present study demonstrate that there are rapid changes in insulin receptor properties during the day. These changes probably play a role in the regulation of the hormonal and metabolic pattern in normal subjects.

The stability of the dexamethasone-receptor complex of rat kidney cytosol was studied using homogenizing media of varying pH and composition. It was found that the complex is less stable with buffers usually employed for the study of steroid receptors such as Tris, EDTA etc., whereas the stability increases considerably with a solution composed of monothioglycerol 12 mmol/l and glycerol 5% in distilled water. Sepharose 4-B column chromatography revealed that the increased stability was associated with a larger form (probably an aggregate) of the receptor-steroid complex.

Carbohydrate chains formation into thyroglobulin (Tg) is a prerequisite for thyroid hormones formation and completeness of carbohydrates chains is necessary for secretion of Tg into the follicles. Tg biosynthesis has been investigated by in vitro experiments, incubating rat thyroid glands with labeled amino-acid and carbohydrate in the presence of tunicamycin, a specific inhibitor of protein glycosylation. Tunicamycin inhibit Tg biosynthesis which is impaired in carbohydrate chains addition but slightly in the polypeptide synthesis, as shown by inhibition of 3H-glucosamine incorporation. Thus tunicamycin inhibits carbohydrate incorporation into Tg without affecting the polypeptide chain growth and decreases its secretion into the follicles.