Enzymatic deglycosylation of human thyroglobulin: fluorescence studies.

The interaction between the carbohydrate and the amino acid residues in human thyroglobulin has been studied. Previous reports showed that the removal of the two terminal carbohydrates of the complex chains leads to an increase in thyroglobulin binding to thyroid membranes. In our study, after enzymatic release with glycosidases of the sugar moieties from thyroglobulin, a time-dependent decrease in tryptophan fluorescence has been observed. This decrease was also associated with a shift in the emission peak from 335 to 340 nm. The strong quenching of tryptophan emission was also accompanied by a decrease in the exposure of tryptophan residues, as shown by a Stern-Volmer analysis with the neutral quencher acrylamide. These data, together with the increase in fluorescence of the dansylated deglycosylated thyroglobulin, strongly suggest that a significant conformational change of thyroglobulin follows the deglycosylation of the protein.

Modifications in platelet membrane transport functions in insulin-dependent diabetes mellitus and in gestational diabetes.

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.

Characterization of the serum from a patient with insulin resistance and hypoglycemia. Evidence for multiple populations of insulin receptor antibodies with different receptor binding and insulin-mimicking activities.

The serum from a patient with lupus nephritis, insulin resistance, and hypoglycemia was studied. This serum both inhibits the binding of 125I-insulin to its receptor and has insulin-like activity on fat cells (see refs. 1 and 2). The IgG fraction from this patient's serum one-half maximally inhibited 125I-insulin binding to IM-9 cells at 1 microM, but did not markedly inhibit 125I-monoclonal antibody binding even at concentrations as high as 4 microM. The IgG was then subjected to affinity chromatography on a protein A-Sepharose column. Four protein peaks were eluted from this column by a step pH gradient from 5.5 to 2.3. Three of the four peaks inhibited 125I-insulin binding to its receptors, but none was more potent than the unfractionated IgG itself. One IgG peak, however, was able to inhibit 125I-monoclonal antibody binding at tenfold lower concentrations than the unfractionated IgG. When the ability of the four IgG fractions to stimulate 2-deoxy[3H]-D-glucose transport in rat adipocytes was studied, two fractions showed stimulatory activity. Compared with unfractionated IgG, one had a weak ability to inhibit 125I-insulin binding, but tenfold more potency to mimic insulin action. The other had a strong ability to inhibit 125I-insulin binding but less potency to mimic insulin action. These studies indicate, therefore, that the serum contains multiple populations of antibodies to the insulin receptor, or portions of the plasma membrane adjacent to the receptor, which have different biologic effects.

Altered cellular Ca2+ and Na+ transport in diabetes mellitus.

Platelet intracellular Ca2+ concentration ([Ca2+]i) and its response to stimuli (ADP and thrombin) were studied in 15 insulin-dependent and 22 non-insulin-dependent diabetes mellitus patients with the fluorescent probe Fura 2. The activity of Ca2(+)-ATPase and Na(+)-K(+)-ATPase, membrane fluidity, and cholesterol and phospholipid content were also determined in platelet membranes. Compared with control subjects, diabetic patients showed 1) increased platelet [Ca2+]i in the resting state, 2) higher Ca2+ levels after stimulation with thrombin and ADP, due entirely to increased resting concentrations, 3) reduced activity of Na(+)-K(+)-ATPase, 4) increased activity of Ca2(+)-ATPase, 5) higher fluidity of the platelet membrane, and 6) increased membrane concentration of total phospholipids. Na(+)-K(+)-ATPase activity was inversely related to platelet [Ca2+]i in each group studied, whereas Ca2(+)-ATPase activity was positively correlated with intracellular Ca2+ levels. The data obtained in diabetic subjects suggest an abnormality in Ca2+ and Na+ transport across the platelet membrane that might be responsible for the reported platelet hyperreactivity to stimuli in diabetes.

Red blood cell age, pyruvate kinase activity, and insulin receptors. Evidence that monocytes and RBCs may behave differently.

Data emerging from insulin receptor studies performed on red blood cells (RBCs) and monocytes from the same subject are not always in agreement; dichotomy might occur since variations in mean RBC age are not taken into account or because insulin receptors on the two cell types behave differently. In the present investigation RBCs from normal male subjects were separated into five populations of different mean age by means of centrifugation of RBCs on a discontinuous gradient of buffered Percoll for 10 min at 1000 X g. Insulin binding varied significantly depending upon the RBC population tested and was closely correlated to the activity of pyruvate kinase (r2 = 0.86), a well-known marker of RBC age. These data suggested that pyruvate kinase assay might be helpful in studies of RBCs. To confirm this hypothesis, RBCs from 10 normal male subjects and 13 male patients with hemolytic anemia were studied; insulin binding was correlated to pyruvate kinase activity. By adjusting insulin binding to 2 X 10(9) RBCs/ml the range of data was abnormally high, but it became acceptable after adjusting insulin binding to pyruvate kinase activity (0.75 U/2 X 10(9) RBCs). The overall data indicated that insulin binding was highly correlated to pyruvate kinase activity (r2 = 0.82) but only slightly to reticulocyte number (r2 = 0.56) since not only reticulocytes but also erythrocytes lose receptors during maturation. Pyruvate kinase activity was measured in RBCs from normal men and from normally menstruating women at the seventh and twenty-fourth days of the cycle; results demonstrated that adjustment of data, according to mean RBC age, broadens dichotomy of monocyte and RBC data.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue-specific antibodies against the fibroblast insulin receptor in a patient with lupus nephritis and hypoglycemia.

We recently reported that the serum from a patient with lupus nephritis, insulin resistance, and hypoglycemia contains multiple populations of antibodies directed at the human insulin receptor. In the present study, we found a subpopulation of antibodies (eluted from a protein A-Sepharose affinity column at pH 4.3) directed at the human fibroblast insulin receptor. When tested against human placental membranes, IM-9 lymphocytes, circulating monocytes and erythrocytes, and isolated adipocytes, the antibody subpopulation did not compete with 125I-insulin for binding to its receptor. In contrast, the antibody subpopulation competed with 125I-insulin for binding to the human fibroblast insulin receptor. This antibody subpopulation stimulated [3H]alpha-aminoisobutyric acid [( 3H]AIB) uptake to these cells. Unlike the effect of insulin, however, this regulation of transport was not antagonized by a mouse monoclonal antibody to the human insulin receptor that inhibits 125I-insulin binding. These studies indicate, therefore, that a tissue-specific antibody subpopulation can occur spontaneously in patients with antibodies to the human insulin receptor. Furthermore, they indicate the presence of anti-insulin receptor autoantibodies specifically directed against a tissue that is not primarily involved in glucose metabolism.

Evidence that two naturally occurring human insulin receptor alpha-subunit variants are immunologically distinct.

The IgG from a patient (Italy 2 [I2]) with hypoglycemia, due to autoantibodies to the insulin receptor, was purified on protein A Sepharose into two fractions that were tested in various human tissues and cells. The IgG fraction that bound protein A (absorbed IgG [IgGa]) nearly completely inhibited the binding of 125I-labeled insulin to various cells or tissues (placenta, IM-9, adipocytes, HEp-2-larynx cells, Epstein-Barr virus lymphocytes) but not greater than 50% of 125I-labeled insulin binding to human liver membranes. Conversely, both the IgG fraction from this patient, which did not bind protein A (flow-through IgG [IgGb]), and the IgGa fraction from a second similar patient (Italy 1 [I-1]) almost completely inhibited the binding of 125I-labeled insulin to liver membranes. The IgGa fraction from patient I-2 did not change receptor affinity because 50% inhibition of 125I-labeled insulin binding was not affected by either the presence or absence of these IgG fractions. Furthermore, liver binding data were not due to cross-reaction of 125I-labeled insulin to the insulinlike growth factor I receptor, and treatment of liver membranes with neuraminidase did not alter the inhibitory effect of the IgGa fraction from patient I-2 on 125I-labeled insulin binding to liver. Binding inhibition experiments performed with cells transfected with and overexpressing the -12 (human insulin receptor [HIR]-A) or the +12 (HIR-B) variant of HIR revealed that the IgGa fraction from patient I-2 inhibited 125I-labeled insulin binding to the HIR-A receptor but not to the HIR-B receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Species specificity of insulin binding and insulin receptor protein tyrosine kinase activity.

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.

Prednisone increases the number of insulin receptors on monocytes from normal subjects.

The effect of prednisone on insulin receptors on circulating monocytes was studied in 38 normal volunteers. Intake of prednisone in doses which are usually employed in clinical treatment (40 mg/day) was associated with a significant increase int the number of insulin receptors. The rise in insulin binding was maximal 7 h after the begining of the medication and it was dose related.

Insulin receptors during the menstrual cycle in normal women.

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.

Changes in insulin receptors during oral contraception.

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.

PIP: Combined estrogen/progestagen (OC) oral contraceptives have been reported to be associated with a deterioration of glucose tolerance and a decrease in insulin sensitivity. Since it has been suggested that steroids affect insulin receptor properties, the influence of OCs on insulin receptors was investigated. The study groups were composed of 9 normally-menstruating women (controls), 9 pill users, and 2 healthy women on OCs for the 1st 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 OCs. This effect occurs rapidly because it was also evident in the 2 women on OCs for the 1st 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.

Insulin receptors on monocytes and erythrocytes from obese patients.

Insulin receptors were studied in monocytes and erythrocytes [red blood cells (RBC)] isolated from 15 normal and 15 nondiabetic obese outpatients on an unrestricted diet. Insulin binding on both monocytes (P < 0.001) and RBC (P < 0.01) was higher in normal than in obese subjects due to different receptor concentrations. In some obese patients, binding to monocytes was decreased, while binding to RBC was normal. These data demonstrate that obese out-patients on an unrestricted diet have a reduced number of insulin receptors. It is suggested that interpretation of insulin binding based upon RBC should be used with caution, since a discrepancy exists in some subjects in the results obtained with these cells and monocytes.

Effect of dexamethasone and cortisone on insulin receptors in normal human male.

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.

Differences in insulin receptors between men and menstruating women and influence of sex hormones on insulin binding during the menstrual cycle.

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.

Human autoantibodies directed against the human, but not the rat, insulin receptor.

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.

Regulation of insulin receptor-associated tyrosine kinase by a polyclonal IgG.

The effect of a polyclonal anti-insulin receptor antibody (pIgG) on the insulin receptor tyrosine kinase (IRTK) activity toward poly-(Glu-Tyr) was examined using wheat germ agglutinin agarose-purified insulin receptors from rat liver membranes. The main effect of pIgG was a reduction of Vmax (from 60.8 to 31.8 pmol/min/mg), without changes of Km, when IRTK was activated by insulin. In contrast, when IRTK was activated by ATP preincubation, pIgG was unable to affect the reaction, suggesting that IRTK possesses at least two regulatory mechanisms, one of which can be affected by pIgG.

Androgens increase insulin receptor mRNA levels, insulin binding, and insulin responsiveness in HEp-2 larynx carcinoma cells.

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.

Diabetes mellitus induces red blood cell plasma membrane alterations possibly affecting the aging process.

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.

Aggregation-induced stability of dexamethasone receptors in rat kidney.

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.