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Biomedical subjects

G Grunberger

Publications and source records attributed to G Grunberger.

At least 73 records · Page 4Linked to original sources
PubMed1985

Structural difference of the insulin receptors from circulating monocytes and erythrocytes.

We compared insulin receptors obtained from cells widely used in human studies, the circulating monocytes and erythrocytes. Biochemically, these receptors possess both binding (alpha-subunit) and tyrosine kinase (beta-subunit) activities similar to insulin receptors from other sources. Subtle differences in molecular weight, however, were detected between the alpha-subunits of these two cell types when analyzed by NaDodSO4-PAGE. Crosslinked [125I]insulin-labeled alpha-subunit of the monocyte insulin receptor was of higher apparent molecular weight than the alpha-subunit derived from red cells. Neuraminidase treatment of the alpha-subunits from each cell type indicated more sialic acid residues were present on the monocyte than the red cell alpha-subunit. The structural properties of the insulin receptors of human circulating cells are similar but not identical to insulin receptors of other characterized systems.

Cell Transformation, Viral↗
PubMed1985

Use of tyrosine-containing polymers to characterize the substrate specificity of insulin and other hormone-stimulated tyrosine kinases.

Synthetic copolymers containing tyrosine residues were used to characterize the substrate specificity of the insulin receptor kinase and compare it to tyrosine kinases stimulated by epidermal growth factor, insulin-like growth factor-1 and phorbol ester. In partially purified receptor preparations from eight different tissues insulin best stimulated (highest V) phosphorylation of a random copolymer composed of glutamic and tyrosine residues at a 4:1 ratio (Glu/Tyr, 4:1). The insulin-stimulated phosphorylation of this polymer was highly significant also in receptor preparations from fresh human monocytes, where insulin binding and autophosphorylation were difficult to detect. Other tyrosine-containing polymers Ala/Glu/Lys/Tyr (6:2:5:1) and Glu/Ala/Tyr (6:3:1) were also phosphorylated by the insulin-stimulated kinase but to a lower extent. A tyrosine kinase stimulated by insulin-like growth factor-1, and one stimulated by phorbol ester also best phosphorylated the polymer Glu/Tyr (4:1). The three kinases differed only in their capability to phosphorylate Glu/Ala/Tyr (6:3:1) or Ala/Glu/Lys/Tyr (6:2:5:1). Glu/Tyr (4:1) was a poor substrate for the epidermal growth factor receptor kinase which best phosphorylated the polymer Glu/Ala/Tyr (6:3:1). Three additional polymers: Glu/Tyr (1:1), Glu/Ala/Tyr (1:1:1), and Lys/Tyr (1:1) failed to serve as substrates for all four tyrosine kinases tested. Taken together these findings suggest that. Hormone-sensitive tyrosine kinases have similar yet distinct substrate specificity and are likely to phosphorylate their native substrates on tyrosines adjacent to acidic (glutamic) residues. Tyrosine-containing polymer substrates are highly sensitive and convenient tools to study (hormone-sensitive) tyrosine kinases whose native substrates are unknown or present at low concentrations.

Animals↗
PubMed1985

Polypeptide hormone receptor phosphorylation: is there a role in receptor-mediated endocytosis of human growth hormone?

To determine whether receptor phosphorylation is a critical step in the internalization of polypeptide hormones and their receptors, we have studied a model system wherein insulin stimulates phosphorylation of its receptor and is also internalized. Using insulin as a positive control, we found that it stimulated a partially purified plasma membrane preparation of IM-9 lymphocytes to autophosphorylate its receptor and to catalyze the phosphorylation of a tyrosine-containing substrate. The human GH (hGH) receptor of the IM-9 lymphocytes, when coupled to [125I]iodo-hGH, migrated as a 140,000-dalton protein on polyacrylamide gel electrophoresis. This protein, in contrast to the insulin receptor, was not phosphorylated by the addition of hGH, nor did hGH stimulate this preparation to phosphorylate the tyrosine-containing substrate poly-(GluNa,Tyr)4:1, casein, or histone f2b under a variety of conditions. We conclude that receptor phosphorylation is not a critical intermediate in the receptor-mediated endocytosis of hGH and probably other polypeptide hormones and growth factors.

Animals↗
PubMed1985

An alteration in apparent molecular weight of the insulin receptor from the human monocyte cell line U-937.

We have studied the structure of the insulin receptor from a human cultured monocyte cell line, U-937. The receptor is composed of alpha and beta subunits as seen in other insulin receptors, but these subunits are of greater apparent molecular weight (alpha 150,000 and beta 102,000) than in typical insulin receptors. Despite this, the U-937 insulin receptor appears to function normally. The alpha subunit binds insulin and the beta subunit is phosphorylated in response to insulin stimulation. Both subunits are expressed in the plasma membrane. Insulin binding isotherms are similar to those seen in IM-9 lymphocytes. Thus, the insulin receptor from U-937 monocytes appears functionally normal despite alterations in molecular weight of the subunits.

Cell Line↗
PubMed1985

Human circulating monocytes internalize 125I-insulin in a similar fashion to rat hepatocytes: relevance to receptor regulation in target and nontarget tissues.

Circulating monocytes bind 125I-insulin in a specific fashion and have been used to analyze the ambient receptor status in humans. When freshly isolated circulating monocytes are incubated with 125I-insulin and examined by electron microscopic autoradiography, approximately 18% of the labeled material is internalized after 15 minutes at 37 degrees C. By 2 hours at 37 degrees C, approximately one half of the 125I-insulin is internalized. Internalization occurs also at 15 degrees C but at a slower rate. Furthermore, the monocytes bind and internalize 125I-insulin in a manner that mirrors that of major target tissues, such as rat hepatocytes. These data suggest that the insulin receptor of the circulating monocyte might be regulated by adsorptive endocytosis in a manner analogous to that of target tissue, such as the liver.

Animals↗
PubMed1984

Defect in phosphorylation of insulin receptors in cells from an insulin-resistant patient with normal insulin binding.

Mononuclear blood cells were obtained from a patient with type A insulin resistance. The cells showed a normal ability to bind iodine 125-labeled insulin. Analysis of solubilized insulin receptors from the patient's cells revealed a defect in insulin-stimulated tyrosine kinase activity, which is closely associated with the receptor itself. The enzyme failed to phosphorylate the insulin receptor and showed a markedly reduced ability to phosphorylate exogenously added substrates. It appears that receptors from this insulin-resistant patient have a defect distal to the insulin-binding site (the alpha subunit of the receptor). The defect could be located in the beta subunit, which has an adenosine triphosphate-binding site, or in another receptor component that transfers a signal of insulin binding into kinase activity. This dissociation between the normal binding and the defective protein kinase component of the insulin receptor represents the first biochemical defect of the receptor distal to ligand binding.

Caseins↗
PubMed1984

Insulin-like growth factor-I (IGF-I) stimulates tyrosine kinase activity in purified receptors from a rat liver cell line.

Solubilized, lectin-purified receptor preparations from BRL 3A2 rat liver cells are rich in Type I and Type II IGF receptors, but possess few insulin receptors. High concentrations of IGF-I or insulin stimulate phosphorylation of a Mr congruent to 98K membrane protein in these preparations. Phosphorylation of a synthetic polymer of tyrosine and glutamic acid was stimulated by IGF-I greater than IGF-II congruent to insulin. These relative potencies, together with the results of immunodepletion experiments using an autoantibody to the insulin receptor, suggest that the effects of each of these hormones is mediated by the Type I IGF receptor. Our results are consistent with the Type I IGF receptor having intrinsic tyrosine kinase activity capable of phosphorylating the receptor itself and other substrates.

Animals↗
PubMed1984

Tumor-promoting phorbol ester stimulates tyrosine phosphorylation in U-937 monocytes.

Solubilized lectin-purified extracts from human monocyte-like cells (U-937) and freshly isolated human mononuclear cells preincubated in the presence of phorbol 12-myristate 13-acetate (PMA) stimulated phosphorylation of synthetic tyrosine-containing polymers and of casein. Tyrosine phosphorylation was confirmed by phospho amino acid analysis. PMA stimulated phosphorylation of exogenous substrates in a time- and concentration-dependent manner. This phosphorylation reaction did not require addition of phospholipid, diolein, or calcium. Biologically inactive phorbol compounds did not stimulate phosphorylation in this system. In addition, PMA enhanced phosphorylation of a Mr approximately equal to 140,000 protein as well as several other endogenous proteins in the U-937 extracts. PMA treatment stimulated predominantly phosphorylation on tyrosine residues of the Mr 140,000 protein. Tyrosine phosphorylation, typical of growth-promoting peptides such as insulin or epidermal growth factor, is believed to play a role in regulating normal and disordered cellular growth and proliferation. The demonstration of PMA-stimulated tyrosine phosphorylation might provide a clue to the mechanism of cellular differentiation and proliferation induced by the tumor promoter.

Cells, Cultured↗
PubMed1984

The insulin receptor of a human monocyte-like cell line: characterization and function.

Most clinical studies of the insulin receptor in man have been carried out in circulating cells, mainly monocytes. It is important to establish whether the function of the insulin receptor in blood cells reflects that of the major target tissues. Since peripheral monocytes do not divide, they cannot be studied under continuous cell culture conditions. We, therefore, studied the insulin receptor of a monocyte-like human cell line (U-937) to determine whether it could serve as a model for further investigation of the function of the insulin receptor. We found that the U-937 cells bind [125I]insulin specifically and in a time-, temperature-, concentration-, and pH-dependent fashion, similar to circulating monocytes. In addition, they internalize the hormone-receptor complex rapidly and extensively at 37 C in a fashion analogous to that of blood monocytes and hepatocytes. U-937 cells show a similar affinity for insulin as monocytes. Unlike hepatocytes, these cells do not appear to release extensive insulin-degrading activity and do not degrade cell surface-associated ligand during initial incubations. [125I] Insulin extracted or dissociated spontaneously from the cell surface shows full rebindability to fresh cells. The insulin receptor of the U-937 cells can be down-regulated as in the major target cells. As in cultured human lymphocytes, preincubation of U-937 cells with prednisolone results in significantly increased insulin binding. Incubation with a phorbol ester tumor promoter, in contrast, inhibits the extent of specific [125I]insulin binding to U-937 cells by altering the receptor affinity. The insulin receptor of the U-937 monocyte-like cell line mirrors the insulin receptor of blood monocytes and target cells; it is a useful tool for further in vitro studies of the insulin receptor.

Cell Line↗
PubMed1984

Tyrosine kinase activity of the insulin receptor of patients with type A extreme insulin resistance: studies with circulating mononuclear cells and cultured lymphocytes.

The syndrome of type A insulin resistance in nonobese women is characterized by hyperinsulinemia, resistance to exogenous insulin, acanthosis nigricans, polycystic ovaries, and masculinization. Insulin binding to intact circulating monocytes and cultured Epstein-Barr virus-transformed B-lymphocytes derived from these patients is decreased in some patients but normal in others. Insulin receptors consist of two subunits; the alpha-subunit contains the insulin-binding site, and the beta-subunit possesses an insulin-sensitive tyrosine-specific protein kinase activity. Insulin binding to circulating monocytes was decreased in five patients, suggesting a decreased number of alpha-subunits on the surface of cells from the patients with type A insulin resistance. In the present work, we demonstrated that there is a proportional decrease in the function of the beta-subunit (i.e. tyrosine kinase activity) in cells from these subjects. In one patient, insulin binding to circulating monocytes was normal, and the insulin-stimulated tyrosine kinase activity of the receptors was normal as well. In separate studies, using cultured Epstein-Barr virus-transformed lymphocytes from the same six patients with type A extreme insulin resistance, the results were similar, in that the functions of the alpha- and beta-subunits of the receptor from these cells correlated. Though heterogeneity among the six patients with type A extreme insulin resistance at the level of the kinase activity of their insulin receptors was demonstrated, it does not appear that a selective defect in beta-subunit phosphorylation per se can be implicated in the mechanisms of insulin resistance of these patients. These findings are distinct from our previously reported patient with normal binding and very low insulin-stimulated phosphorylation of the beta-subunit of the receptor of circulating monocytes, in whom it was speculated that selective reduction in beta-subunit phosphorylation was responsible for insulin resistance.

Acanthosis Nigricans↗
PubMed1983

The insulin-stimulated receptor kinase is a tyrosine-specific casein kinase.

Insulin stimulates a kinase that phosphorylates tyrosines in the insulin receptor; this kinase is tightly associated with the insulin receptor itself. We now show that the insulin-stimulated casein kinase, present in solubilized, lectin-purified receptor preparations from rat liver, is indistinguishable from the insulin receptor kinase. As with phosphorylation of the insulin receptor, insulin selectively enhanced by 2-3-fold the phosphorylation of tyrosines in casein. The insulin-stimulated activities of both kinases were inactivated at 37 degrees C with the same t0.5 of 5 min and were identically affected by alkylating agents. Both receptor and casein kinase activities were specifically coprecipitated by anti-receptor antibodies or by insulin and anti-insulin antibodies. When the latter type of immune complexes were incubated with an excess of insulin, both kinase activities were quantitatively recovered. We therefore conclude that insulin-stimulated receptor and casein phosphorylations are probably catalyzed by a single enzyme which is tightly associated with the receptor itself. Now, by replacing casein for receptor as substrate, it is possible to measure the enzymatic activity of this receptor-related kinase itself, i.e. independent of the receptor as substrate. Detection of this activity is improved in the presence of certain alkylating agents. Use of artificial substrates (in combination with alkylating agents) is particularly important to dissect the functional components of the receptor complex, to study mechanisms of enzyme regulation and especially in situations where the available receptor for study is limited, e.g. fresh or cultured cells from patients.

Amino Acids↗
PubMed1983

Insulin stimulates phosphorylation of serine residues in soluble insulin receptors.

Using lectin affinity-purified receptor preparations from human hepatoma cells, insulin (10(-7)M) specifically stimulated phosphorylation of the 95,000 dalton (beta) subunit of its own receptor. Phospho-amino acid analysis of the receptor subunit revealed that insulin increased at least 2.5-fold the content of phosphoserine and of phosphotyrosine. In intact cells, the major effect of insulin is to increase the phosphoserine content of its receptor. These findings are the first demonstration of an insulin-stimulated serine kinase in a cell-free system.

Carcinoma, Hepatocellular↗
PubMed1983

Protein kinase activity of the insulin receptor in human circulating and cultured mononuclear cells.

In lectin-purified receptor preparations from human monocyte-like cell (U-937), insulin (10(-7)M) stimulated phosphorylation of the 95,000 dalton subunit of its own receptor. In addition, insulin stimulated phosphorylation of exogenously added substrates like casein, (T,G)-A--L, and histones. Phosphorylation of the synthetic peptide (T,G)-A--L indicates the presence of at least one insulin-dependent tyrosine kinase in these cell extracts. Insulin receptor preparations from freshly isolated human mononuclear blood cells were also shown to possess insulin-dependent casein and (T,G)-A--L kinase activity. Phosphorylations in these systems are specific for insulin and dependent on insulin concentration. A simple and rapid method is described that is relevant for clinical investigations of early postbinding events.

Cells, Cultured↗
PubMed1983

Auditory brain-stem responses in adrenomyeloneuropathy.

We studied three patients with adrenomyeloneuropathy. Complete audiologic assessment was obtained: two patients showed unimpaired peripheral hearing and one showed a mild high-frequency hearing loss. Auditory brain-stem responses were abnormal in both ears of all subjects, with one subject showing no response above wave I, and the other two having significant wave I to III and wave III to V interval prolongations. We concluded that auditory brain-stem response testing provides a simple, valid, reliable method for demonstrating neurologic abnormality in adrenomyeloneuropathy even prior to evidence of clinical signs.

Adolescent↗
PubMed1983

Insulin receptors in normal and disease states.

The binding of insulin to its receptor has been studied under various physiological and pathological conditions. Quantitative studies have involved human circulating cells such as monocytes and erythrocytes, adipocytes, placental cells, and cultured cells such as fibroblasts and transformed lymphocytes. In animals, other target tissues such as liver and muscle have been studied and correlated with the human studies. Various physiological conditions such as diurnal rhythm, diet, age, exercise and the menstrual cycle affect insulin binding; in addition, many drugs perturb the receptor interaction. Disease affecting the insulin receptor can be divided into five general categories: (1) Receptor regulation--this involves diseases characterized by hyper- or hypoinsulinaemia. Hyperinsulinaemia in the basal state usually leads to receptor 'down' regulation as seen in obesity, type II diabetes, acromegaly and islet cell tumours. Hypoinsulinaemia such as seen in anorexia nervosa or type I diabetes may lead to elevated binding. (2) Antireceptor antibodies--these immunoglobulins bind to the receptor and competitively inhibit insulin binding. They may act as agonists, antagonists or partial agonists. (3) Genetic diseases which produce fixed alterations in both freshly isolated and cultured cells. (4) Diseases of receptor specificity where insulin may bind with different affinity to its own receptor or related receptors such as receptors for insulin-like growth factors. (5) Disease of affinity modulation where physical factors such as pH, temperature, ions, etc. may modify binding. In this review, we have considered primarily abnormality in insulin receptor binding. There are numerous other functions of the receptor such as coupling and transmission of the biological signal. These mechanisms are frequently referred to as postreceptor events, but more properly should be referred to as postbinding events since the receptor subserves other functions in addition to recognition and binding of insulin.

Acromegaly↗
PubMed1982

Hypoglycemia associated with antibodies to the insulin receptor.

Antibodies to the insulin receptor are insulinomimetic in vitro, although they generally induce insulin resistance in vivo. We report the novel case of a patient who presented with fasting hypoglycemia as the sole manifestation of autoantibodies to the insulin receptor. Prednisone therapy (120 mg per day) produced a rise in fasting glucose to more than 100 mg per deciliter (6 mmol per liter) within 48 hours, although there was no detectable change in the titer of antireceptor antibodies. After 10 weeks of therapy, the titer of antireceptor antibodies had fallen approximately 100-fold, and prednisone could be discontinued without recurrence of hypoglycemia. This case demonstrates that antireceptor antibodies must be considered in the differential diagnosis of hypoglycemia, especially in patients with other manifestations of autoimmunity.

Adipose Tissue↗
PubMed1982

Affinity alteration of insulin receptor induced by a phorbol ester.

The effect of a phorbol ester tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA) on 125I-insulin binding to human cells was examined. TPA markedly inhibits insulin binding to cultured human lymphocytes and macrophages but has a minimal effect on human fibroblasts. This inhibition is temperature, time, and concentration dependent. The inhibition of insulin binding to the cells at 37 degrees C occurs within minutes and diminishes by 6 h of incubation. Insulin binding is decreased by TPA whether the phorbol ester is added before, after, or simultaneously with 125I-insulin to the cell suspension. Scatchard analysis of binding to IM-9 lymphocytes indicates that TPA affects the affinity rather than the number of insulin receptors. The phorbol ester has only a small effect on 125I-human growth hormone binding in cultured human lymphocytes. TPA perturbs the insulin receptor of cultured human lymphocytes in a fashion similar to its effect on the epidermal growth factor receptor of several other cell types. The specific mechanism of TPA action that affects the receptor of these two potent growth factors (i.e., insulin and epidermal growth factor), however, is unknown.

Cell Line↗