Chemical mechanism of insulin action via proteolytic formation of mediator peptides.
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Biomedical subjects
Publications and source records attributed to K Kikuchi.
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Four derivatives of the insuln B-chain COOH-terminal pentapeptide Arg-Gly-Phe-Phe-Tyr (B22-26) were synthesized and shown to be inactive alone. In the presence of submaximal concentrations of insulin or desoctapeptide insulin, peptides at concentrations of 10(-4) M and higher, markedly stimulated the actions of insulin on rat adipocytes including labeled glucose oxidation, activation of glycogen synthase, and stimulation of 2-deoxyglucose transport. The B-chain COOH-terminal heptapeptide, Gly-Phe-Phe-Tyr-Thr-Pro-Lys (B23-29) was inactive alone or in the presence of submaximal concentrations of insulin or desoctapeptide insulin, suggesting that argnine is required. Dose response curves of insulin, and desoctapeptide insulin for labeled glucose oxidation by rat adipocytes were shifted 1 log concentration unit to the left in the presence of peptide. Peptide shortened the lag time of labeled glucose oxidation and markedly enhanced the rate of 14CO2 production following the lag time. Peptides also enhanced insulin-like activities of concanavalin A and nonsuppressible insulin-like activity P.
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Brief treatment of rat adipocytes with low concentration of trypsin activated both cell membrane and intracellular insulin-sensitive functions in marked contrast H2O2 (1), increase in pH, and oxidized glutathione (papers I and II). Glucose oxidation was activated maximally by trypsin in 30 s and preceded maximal activation of glycogen synthase, which occurred in 60s. Trypsin action to activate glycogen synthase was further enhanced by insulin. Mitochondrial pyruvate dehydrogenase was also rapidly activated by trypsin. With both insulin and trypsin action, mediator generation was directly demonstrated by glycogen synthase phosphoprotein phosphatase activation. Trypsin is thus the most insulin-like of these four agents studied since it acts by the formation of chemical mediator peptide(s) which are similar but not identical to those produced by insulin.
Cell surface markers of 44 cases of non-Hodgkin's lymphoma (NHL) were studied with various surface markers, especially by using antihuman B lymphocyte serum (ABS), antihuman thymocyte serum (ATS-T), and antihuman peripheral T lymphocyte serum (ALS-T), which were rendered specific for human B lymphocytes, human thymocytes, and human peripheral T lymphocytes, respectively. An immunofluorescent study with ABS, ATS-T, and ALS-T enabled us to demonstrate the histologic localization of normal or neoplastic B and T cells in preserving the original structure of lymphoid organs or tumor tissues. The proportion of cell types in NHL was B cell type 59%, T1 (ATS-T reactive) type 7%, T2 (ALS-T reactive) type 23%, and Null (non T, non B) type 11%. The relationships among cell types, histologic findings, and clinical characteristics were also investigated. Patients with T1-NHL had mediastinal tumors, which were histologically classified into "Lymphoblastic lymphoma." These facts suggest that T1-NHL may have originated in the thymus. Patients with T2-NHL showed a high incidence of skin lesions. Median survival of ten patients with T1- and T2-NHL was seven months, which was much shorter than that of B- or Null-NHL.
Specific anti-human thymus xenoantiserum (ATS) was utilized for characterizing a human thymus antigen (HTA) preferentially expressed on human thymocytes. Binding of ATS with different cell types was studied by immunofluorescence and immunoperoxidase techniques, as well as by radioimmunoprecipitation (RIP) followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). ATS reacted selectively with the majority (87%) of human thymocytes among normal lymphoid cell populations tested. In the thymus, HTA was expressed largely on cortical thymocytes but not on those cells located in the thymic medulla. When 20 cases of different types of lymphatic leukemias were studied with ATS, HTA was found on acute lymphoblastic leukemia cells rosetting with sheep erythrocytes (T-ALL) but not on other lymphatic leukemia cases, including chronic lymphocytic leukemia cells rosetting with sheep red blood cells (T-CLL). SDS-PAGE analysis of immunoprecipitates formed between ATS and radiolabeled cell-surface components of human thymocytes demonstrated that ATS bound a 48,000-molecular-weight component that could be adsorbed to Sepharose 4B coupled with Les culinaris hemagglutinin and could be labeled by periodate-tritiated borohydride, indicating that HTA is a sialoglycoprotein. Similar antigen molecules were also precipitated by ATS from T-ALL cells but not from other lymphatic leukemia cells. Five T-ALL-derived cell lines were tested with ATS by immunofluorescence and by RIP and SDS-PAGE, but only two cell lines (MOLT-3 and P12/Ich) possessed HTA molecules on their cell membrane.
Morphologic and immunohistochemical studies were performed to examine the existence of lymphocytes in the brain of rats. Special attention was paid to the time course of the appearance of lymphocytes in and around the pineal gland. Rabbit anti-rat T cell and anti-rat immunoglobulin sera were used for identification of T and B cells in tissue sections. Immunoperoxidase and immunofluorescence techniques were employed to identify cells reacting with anti-T and anti-immunoglobulin sera. No lymphocytes were found in the brain of rats until 20 days after birth. Small clusters of lymphocytes appeared in the pineal region by 30 days of age, after which they gradually increased in number, forming massive clusters in the pineal region by 120 days. Along with an increase in the number of lymphocytic cells, there was a gradual increase of cells reacting with anti-T cell serum. These T cells were only a minority of pineal lymphocytes in younger animals, but 90% or more cells were stained by anti-T cell serum at 120 days after birth. The remaining cells did not react with anti-immunoglobulin sera either. These findings suggest that the gradual increase of T lymphocytes in the rat pineal region is a simple reflection of the normal course of maturation of T cells, and the pineal gland in the rat may have some role in immune responses within the brain.
Using immunological techniques urinary hCG, pregnancy-specific beta 1-glycoprotein (SP1) and beta-subunit of hCG (beta-hCG) levels were measured in the serum and cerebrospinal fluid of patients with trophoblastic tumours. After removal of hydatidiform moles, urinary hCG, beta-hCG and SP1 levels were similar, but SP1 levels tended to exceed serum beta-hCG levels. SP1 usually disappeared first. In patients with metastatic choriocarcinoma, SP1 levels appeared to be lower than beta-hCG values in serum and cerebrospinal fluid, but urinary hCG, beta-hCG and SP1 concentrations all decreased during chemotherapy. Measurements of SP1 levels may well be useful in the monitoring of patients being treated for trophoblastic tumours.
A case of immunoglobulin E (IgE) myeloma with clinical features of "classic" myeloma is presented. Skeletal roentgenograms showed osteoporosis and compression fractures of the vertebrae but no osteosclerosis. Protein analyses revealed an M component of the IgE kappa type with a concentration of 3.1 g/dl. Although morphologic examination revealed that the plasma cells were not so differentiated, well-developed Golgi apparatus and abundant rough-surfaced endoplasmic reticulum were observed. An indirect immunofluorescence technique showed characteristic apple green fluorescence. The E myeloma protein of our patient had no antibody activity. Treatment with melphalan or cyclophosphamide resulted in a decrease in the serum IgE level and in the level of Bence Jones protein in the urine. The clinical and laboratory features of IgE myeloma were summarized and compared with those of other classes of myeloma.
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Peripheral blood lymphocytes from some of patients with mycosis fungoides disease showed decreased ability to form rosettes with sheep erythrocytes. This decreased percentage of E-rosette forming cells could be normalized when those cells were incubated in culture for 20 hr. Since these data led us to considering a possible inhibitory factor present in patients' sera, we tested their ability to inhibit E-rosetting by T lymphocytes from normal donors, and found that sera from mycosis fungoides patients with low levels of E-rosetting blood lymphocytes showed greater inhibitory effect on E-rosette formation by normal T cells when compared to those either from normal donors or from mycosis patients who had almost normal levels of E-rosetting blood lymphocyte number. The E-rosette inhibitory factor was sensitive to 2-mercaptoethanol treatment and was copurified with serum IgM by ammonium sulfate precipitation and by sequential gel filtrations, suggesting that it might be an anti-T lymphocyte antibody naturally occurring during the disease process.
One- and two-dimensional polyacrylamide gel electrophoresis (PAGE) was performed on immunoprecipitates formed between anti-human T-cell xeno-antiserum (ATS) and cell-surface glycoproteins of human lymphocytes, that had been radioiodinated by lactoperoxidase and purified on a Lentil lectin-coupled Sepharose 4B column. In some experiments, the cells were 3H-labeled by periodate-tritiated borohydride. ATS that was absorbed with B cells recognized a number of cell-surface antigens expressed preferentially on human thymus and T cells, with molecular weights of 150K (T150), 94K (T94), 72K (T72), and 65K (T65) daltons. Whereas T150 appeared to consist of multiple components of heavily sialylated glycoproteins and to be expressed largely on thymus and T cells, and to a much lesser extent on B cells, the remaining T94, T72, and T65 glycoproteins seemed to be present on thymus and T cells but absent from B cells. Two-dimensional PAGE analysis of these T-cell glycoproteins precipitated by ATS demonstrated that T94 was an acidic glycoprotein with pI of 4, while T72 and T65, the latter being found on thymus and T cells but not on T cell-type leukemic cells, exhibited marked electric charge heterogeneity with pI ranging from 4 to 7. These data clearly suggest that human thymus and T cells possess a complex antigenic make-up on their cell surfaces, comparable to that of mouse T cells with a variety of Ly antigen systems.
Phosphoprotein phosphatase IA, which represents the major glycogen synthase phosphatase activity in rat liver cytosol, has been purified to apparent homogeneity by chromatography on DEAE-cellulose, histone - Sepharose-4B and Sephadex G-100. The molecular weight of the purified enzyme was 40 000 by gel filtration and 48 000 by sodium dodecyl sulfate gel electrophoresis, Phosphatase IA is therefore a monomeric protein. When treated with 80% ethanol at room temperature, phosphatase IA underwent an inactivation which was totally prevented by 2 mM MgCl2. Catalytically, phosphatase IA has a preference for glycogen synthase D compared with phosphatases IB and II and obligatorily requires Mg2+ or Mn2+ for activity. Maximum activity was attained at 5 mM MgCl2. Since Mg2+ does not activate other phosphoprotein phosphatases in rat liver cytosol, we propose the term 'Mg2+-dependent glycogen synthase phosphatase' for phosphatase IA.
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To study the mechanism of the potentiating effect of lithium chloride (LiCl) on methamphetamine (MA)-induced stereotypy in mice, effects of various drugs on the action of LiCl on the stereotypy and pharmacokinetics of MA in different brain regions and liver were examined. The potentiating effect of LiCl disappeared in mice pretreated with atropine or scopolamine whereas LiCl potentiated the stereotypy in mice pretreated with p-chlorophenylalanine, alpha-methyl-p-tyrosine, nialamide, physostigmine or butylscopolamine. The concentrations of MA in the striatum, brainstem and liver were increased and the half-life of MA in these tissues was prolonged by LiCl. The effect of LiCl on the kinetics of MA in the brain, but not that in the liver, was blocked by scopolamine. LiCl prolonged the half-life of MA in the brain and liver in mice treated with physostigmine or butylscopolamine. The inhibition of MA elimination from the brain and its blockade by scopolamine seem to explain the potentiating action of LiCl on the stereotypy and the antagonism by scopolamine of the behavioural action of LiCl, respectively.