Insulin receptor gene expression in skeletal muscle and brown adipose tissue.
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Adipose Tissue, Brown↗
Biomedical subjects
J E Hesketh
Publications and source records attributed to J E Hesketh.
The cyclo-oxygenase inhibitors indomethacin and ibuprofen inhibit the insulin-induced stimulation of ribosomal RNA synthesis in L6 myoblasts.
Insulin stimulated total RNA accretion and the incorporation of [3H]uridine into RNA in L6 skeletal-muscle myoblasts. Incorporation of uridine into the rRNA was measured after either separation of 18 S and 28 S rRNA species by agarose-gel electrophoresis or separation of dissociated 40 S and 60 S ribosomal subunits on sucrose density gradients. Both methods showed a stimulation by insulin of uridine incorporation into the RNA of the two subunits. Two non-steroidal anti-inflammatory drugs, indomethacin and ibuprofen, which inhibit the metabolism of arachidonic acid by the cyclo-oxygenase pathway, inhibited the insulin-induced accretion of total cellular RNA and the incorporation of uridine into the RNA of both ribosomal subunits. The effect of insulin was observed both by using a tracer dose of [3H]uridine (5 microM) and in the presence of a high concentration (1 mM) of uridine to minimize possible changes in intracellular precursor pools. Neither insulin nor indomethacin was found to affect the incorporation of uridine into the total intracellular nucleotide pool, or the conversion of uridine into UTP. The ability of inhibitors of arachidonic acid metabolism to prevent insulin-induced increases in RNA metabolism suggests that a prostaglandin or other eicosanoid is involved in the signal mechanism whereby insulin stimulates RNA synthesis.
Animals↗
Sodium butyrate induces major morphological changes in C6 glioma cells that are correlated with increased synthesis of a spectrin-like protein.
Butyrate induced flattening and development of cell processes in rat glioma (C6) cells and this change was correlated with an increase in the synthesis of a polypeptide doublet with an apparent molecular weight of about 200 kDa. Blot analysis revealed that at least one of these polypeptides was a spectrin-like protein. Indirect immunofluorescence studies with the spectrin antiserum indicated that the antigen was present in the cell bodies, and also in the cell processes. Thus fodrin may be one the major targets for the action of butyrate on C6 cells.
Autoradiography↗
Effect of RNA synthesis inhibitors on insulin-induced protein synthesis by 3T3 cells.
1. The increased protein synthesis of quiescent 3T3 cells in response to insulin was separated into three distinct phases based on their response to various inhibitors of RNA synthesis. 2. The first increase in protein synthesis was insensitive to the inhibitors used, and probably resulted from activation of existing protein synthesizing mechanism. 3. The second phase was sensitive to a varying extent to alpha-amanitin and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, implying the need for new mRNA synthesis as well as the production of new ribosomes indicated by its further sensitivity to low concentration (10 ng/ml) of Actinomycin D. 4. The final phase was insensitive to inhibitors of new ribosome formation, but still depended on new mRNA. alpha-difluoromethylornithine, an inhibitor of de novo polyamine synthesis, partly inhibited the insulin induced stimulation of protein synthesis.
Amanitins↗
Increased protein synthesis response to insulin in fibroblasts treated with the diacylglycerol kinase inhibitor R59022.
Insulin stimulated protein synthesis in quiescent 3T3 fibroblasts. This effect of the hormone was greater in the presence of the diacylglycerol kinase inhibitor R59022 (10(-5) M) over a range of insulin concentrations from 1 microU to 1 mU/ml; R59022 increased the sensitivity of cells to insulin. The amount of radioactive diacylglycerol recovered from cells prelabelled with [3H]glycerol was increased transiently in response to insulin; the response was larger and prolonged in cells given the kinase inhibitor. The results (i) support the hypothesis that diacylglycerol production is part of the signal pathway by which insulin stimulates protein synthesis and (ii) suggest that inhibition of diacylglycerol breakdown leads to increased sensitivity to the hormone.
Animals↗
Evidence that insulin increases the proportion of polysomes that are bound to the cytoskeleton in 3T3 fibroblasts.
The association of polysome redistribution with changes in protein synthesis was investigated in insulin-stimulated fibroblasts. Free polysomes were released by Nonidet-P40 and 25 mM KCl, cytoskeletal-bound polysomes were retained at 25 mM KCl but released at 130 mM, while membrane-bound polysomes were released by deoxycholate. Insulin increased the proportion of polysomes which were retained at 25 mM KCl but had no effect when extraction was carried out at 130 mM KCl, suggesting that more polysomes were associated with the cytoskeleton. Insulin also reduced the amount of actin released from the detergent-insoluble cytoskeleton indicating that the hormone affects microfilament organization.
Actins↗
The detection of a spectrin-like protein in Trypanosoma cruzi with a polyclonal antibody.
A rabbit serum raised against sheep erythrocyte spectrin stained mainly the flagella of epimastigote forms of the parasitic protozoan Trypanosoma cruzi. In Western blots of T. cruzi proteins solubilized with Nonidet P-40 it recognized mainly a polypeptide doublet with an apparent molecular weight of about 240 kDa, while a pre-immune rabbit serum and a tubulin monoclonal antibody did not. The results are consistent with the idea that a spectrin-like protein may be involved in cytoskeleton-membrane interactions in flagella of T. cruzi.
Animals↗
Effects of insulin, pertussis toxin and cholera toxin on protein synthesis and diacylglycerol production in 3T3 fibroblasts: evidence for a G-protein mediated activation of phospholipase C in the insulin signal mechanism.
The rapid increase in protein synthesis that occurs on addition of insulin (1 mU/ml) to stepped-down 3T3 cells was blocked by pre-incubation of the cells with pertussis toxin. Cholera toxin on the other hand stimulated protein synthesis and this effect was insensitive to actinomycin D and inhibited by pre-treatment of the cells with phorbol dibutyrate to deplete cell protein kinase C. Insulin was found to cause a rapid and transient increase in diacylglycerol (DAG) synthesis. The insulin-induced increase in diacylglycerol was blocked by pertussis toxin. Exogenous DAG (10 microM) stimulated protein synthesis within 1 hour. The results suggest that insulin stimulates ribosomal activity through a signal mechanism that involves a G-protein mediated activation of phospholipase C to increase DAG levels.
Animals↗
Cytochalasin B binding and actin content of endoplasmic reticulum subfractions isolated from L-929 cells.
Heavy rough (HR) endoplasmic reticulum (ER) membranes and a dense fraction of light rough (LR) membranes (LR I) of L-929 cells bind H-cytochalasin B extremely poorly in comparison to smooth (S) membranes and a fraction of LR membranes of low density (LR II). The LR and S subfractions of ER are apparently heterogeneous membrane populations with respect to cytochalasin B binding. The separation of proteins in HR and LR subfractions by electrophoresis followed by immunoblotting with monoclonal antibodies against actin showed that actin was not present in the former membranes while there were large amounts in the LR subfraction. It is concluded that membranes in the LR II fraction of ER are associated with actin-containing microfilaments of the cytoskeleton, but that HR membranes are not.
Actins↗
Assembly and characterisation of a multi-component cytoskeletal gel from adrenal medulla.
Incubation of bovine adrenal medullary cytoplasmic extracts results in the formation of three-dimensional supramolecular gels. Ultrastructurally, the gels display a network of fibres similar in appearance to the cytoskeleton within intact chromaffin cells. Analysis of the protein composition using both electrophoretic and immunoblotting techniques indicates that the gels are composed exclusively of cytoskeletal elements; microfilaments, microtubules and intermediate filament proteins have been identified as having a number of actin-associated proteins. Among the latter class of components the following polypeptides have been identified: filamin (300 kDa), fodrin (240 kDa), a 235 kDa polypeptide, myosin (200 kDa), caldesmon (70 kDa) and tropomyosins (39 kDa). All of these polypeptides co-sedimented with F-actin when gels were assembled in the absence of Ca2+. When gelation was performed in the presence of 10 microM Ca2+ actin, the 235 kDa polypeptide, 70 kDa caldesmon and tropomyosin were all absent from the gels. These results may suggest that the 235 kDa polypeptide, 70 kDa caldesmon and tropomyosins could act either individually or as a functional regulatory unit in controlling the Ca2+-activated reorganisation of the actin network in the cytoplasmic gels.
Actins↗
Immunohistochemical localization of a spectrin-like protein (fodrin) in nerve cells in culture.
The distribution of fodrin in neurones in culture was demonstrated by immunohistochemistry with anti-spectrin antibodies. There was strong staining of the plasma membrane and cell extensions and also labelling of a cytoskeletal network throughout the cytoplasm.
Animals↗
Rapid response of protein synthesis to insulin in 3T3 cells: effects of protein kinase C depletion and differences from the response to serum repletion.
Quiescent 3T3 cells grown in media containing 4% foetal calf serum showed different responses to insulin and to serum repletion (to 12%). Insulin stimulated protein synthesis within 1 h and this early response was insensitive to actinomycin D. The later insulin response showed progressive sensitivity to actinomycin D. The serum response was slower, not occurring until 1 h, and was inhibited by actinomycin D. Depletion of cell protein kinase C by pre-treatment with phorbol ester caused a total block of the immediate response to insulin but had little effect on the response to serum or the later response to insulin. Acute phorbol ester treatment stimulated protein synthesis.
Animals↗
Immunolocalization of a spectrin-like protein (fodrin) in pancreatic acinar cells.
A spectrin-like protein (fodrin) was localized in porcine pancreas using an immunoperoxidase procedure with antibodies raised against erythrocyte spectrin. Fodrin was primarily associated with the cell plasma membrane although some was also detectable in the cytoplasm of the acinar cells. The membrane labelling of the acinar cells was uneven such that the lateral and basal membranes were strongly labelled by anti-spectrin antibodies whereas the apical membranes were poorly labelled. The implications of the results to secretion and to the occurrence of specific membrane domains are discussed.
Animals↗
Insulin inhibits the phosphorylation of the membrane cytoskeletal protein spectrin in pig erythrocytes.
Incubation of either ghost membranes with 32P- -ATP or intact erythrocytes with 32P-inorganic phosphate led to phosphorylation of the beta-chain of the major membrane-associated protein spectrin. This phosphorylation was reduced by 30% by insulin (10-100 microU/ml) both in membranes and in intact cells. The results show that the membrane-cytoskeleton is responsive to extracellular signals such as hormone receptor activation.
Animals↗
Regulation of microtubule assembly.
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Animals↗
Cold-stable microtubules and microtubule-organizing centres in astrocytes in primary culture.
Immunocytochemical staining with anti-tubulin antibodies showed cold-stable microtubules in astrocytes in culture. On rewarming, microtubule-organizing centre (MTOC) could be visualized. MTOCs showed structural complexity, from 2-3 fibres to a mass of interweaving fibres. Only one MTOC per cell was found in cultures of both polygonal and stellate-bearing cells.
Animals↗
Differences in polypeptide composition and enzyme activity between cold-stable and cold-labile microtubules and study of microtubule alkaline phosphatase activity.
Cold-stable and cold-labile microtubules were prepared by two cycles of assembly and disassembly and two periods of exposure to cold. The cold-labile preparations were shown to contain a higher proportion of a high molecular mass microtubule-associated protein (MAP 2) than cold-stable preparations. The cold-stable preparations showed a much higher alkaline phosphatase activity. Stimulation of microtubule assembly by zinc led to increases in both cold stability and alkaline phosphatase activity.
Alkaline Phosphatase↗
Microtubule assembly in rat brain extracts. Further characterization of the effects of zinc on assembly and cold stability.
Microtubule assembly has been studied turbidometrically in supernatant fluids prepared from rat brain by high-speed centrifugation. It was confirmed that assembly occurred in the absence of added GTP. Zinc ions (500 microM, but in the presence of 1 mM EGTA) stimulated assembly under these conditions. Zinc-stimulated assembly produced microtubules with normal characteristics, as judged by electron microscopy, SDS-polyacrylamide gel electrophoresis and inhibition of assembly by fructose-6-phosphate or colchicine. However, microtubules formed in the presence of such zinc concentrations were more stable to cold than controls, although the rate constant for the disassembly reaction was unchanged. Neither the stimulation of assembly by zinc nor the effect on cold stability was affected by trifluoperazine suggesting that a calmodulin-related mechanism is not involved. Microtubule "seeds" had little effect in the presence of zinc, suggesting that it may be acting on the nucleation phase of the assembly reaction. This was supported by the findings that zinc reduced the critical concentration of brain supernatant necessary for assembly and that zinc did not affect the rate constant for assembly. The results suggest zinc can in some way stabilize microtubules; possible mechanisms are discussed.
Animals↗