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Summary Incubation of intact mouse diaphragms with insulin in the absence of glucose resulted in a rapid inhibition of the subsequent cell-free phosphorylation of endogenous protein substrates in tissue extracts. The phosphorylation of added histone was inhibited to a lesser extent. The inhibition was observed both in the absence and in the presence of added cyclic 35 adenosine monophosphate. Acrylamide gel electrophoresis of phosphorylation products revealed a number of major phosphorylated polypeptides. The phosphorylation of several polypeptides was inhibited following short treatment with insulin. These results represent a novel experimental approach to the elucidation of the mechanism of the action of insulin and are consistent with our hypothesis that the inhibition of protein kinase activities in the tissue may be the first step in this mechanism.Abbreviations Tricine
N-tris(hydroxymethyl)methyl glycine
- EDTA
ethylenediamine tetraacetic acid
- EGTA
ethyleneglycol-bis-(-amino ethyl ether) N,N1-tetraacetic acid
- Mes
2(N-morpholino)ethane sulfonic acid
J. Larner is an established investigator of the American Diabetes Association. 相似文献
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Early uptake curves of L-lactate and of mannitol were measured in quartered, incubated mouse diaphragms. Uptake was determined at 15, 30, and 45 s for various concentrations of lactate in the external solution as well as in the presence and absence of the competitive inhibitor of lactate transport, alpha-cyano-4-hydroxycinnimate. In normal preparations, when the external lactate concentration was 10 mM or less, the ratio of lactate-to mannitol space in the tissue was 1.7. This value was nearly independent of time and of external concentration. In normal preparations, when the external lactate concentration was greater than 10 mM, the ratio of lactate-to-mannitol space rose with time. At a fixed time, however, this ratio fell with increasing lactate concentration. In the inhibited preparations, the ratio of lactate-to-mannitol space rose with time at all concentrations. When lactate concentration was greater than 5 mM, this ratio was independent of the external concentration. The results suggest that there are two modes of lactate entry into these muscle cells. Entry can occur by means of a saturable system. When external lactate concentration is low, entry rates for this process are rapid compared with diffusional rates. This system probably saturates at concentrations near 10 mM and can facilitate transport in either direction. In addition, an appreciable passive leak is present. This leak accounts for about one fourth of the membrane transfer when external lactate is low, but is equal to the carrier transfer when lactate concentration is 30 mM. A model was developed to describe the entry of a permeating solute, such as lactate, into an isolated tissue. 相似文献
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Growth factors have an important role in the regulation of cell growth, division and differentiation. They are also involved in the regulation of embryonic growth and differentiation. Insulin and insulin-like growth factor I (IGF I) play an important part in these events in the later stages of embryogenesis, when organogenesis is completed. In this study, we are presenting evidence that insulin and IGF I are also secreted by embryonic tissues during the prepancreatic stage of mouse development. We found measurable amounts of insulin and IGF I in 8- to 12-day-old mouse embryos. We also showed that embryonic cells derived from 8-, 9- and 10-day-old mouse embryos secrete insulin, IGF I and/or related molecules. Furthermore, the same growth factors, when added to the culture of 9-day-old mouse embryonic cells, stimulate their proliferation. These results lead to the conclusion that insulin can stimulate the growth of embryonic cells during the period when pancreas is not yet formed, which is indirect evidence for a paracrine (or autocrine) type of action. 相似文献
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1. A cytosol serine-protein kinase was isolated and partially purified from lactating mouse mammary gland which is cAMP-independent, inhibited by the bioflavonoid quercetin and probably connected to the growth of mammary cells. 2. After supra-physiological doses of insulin (0.4 IU daily) given in vivo the activity of this kinase rises 2.4 times, but at 5 times higher doses of insulin the activity was completely inhibited. 3. The biphasic effect of insulin suggests that this protein kinase might be connected to the growth effect of the hormone. 相似文献
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M Korc H Sankaran K Y Wong J A Williams I D Goldfine 《Biochemical and biophysical research communications》1978,84(2):293-299
Specific insulin receptors were measured in isolated mouse pancreatic acini. Scatchard analyses revealed a high affinity binding site with a Kd of 1.67 nM and a lower affinity site with a Kd of 83 nM. Binding of insulin to these receptors was rapid, one-half maximal binding occurring at 2 min and maximal binding at 30 min. Insulin stimulated the uptake of the glucose analogue 2-deoxy-D-glucose; maximum effects were detected at 1.67 μM. Insulin, in contrast, had no direct effects on alpha-aminoisobutyric acid uptake. The finding of high affinity insulin receptors in pancreatic acinar cells supports the hypothesis that insulin may directly regulate specific functions in the exocrine pancreas. 相似文献
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The mouse diaphragm muscle fiber was studied using the loose patch clamp technique. The voltage gated sodium currents were evoked by step pulses from a holding potential of about −70 mV. Following the activation of the sodium current, a very large and fast outward current was evoked. The sensitivity of this current to 4-aminopyridine and tetraethylammonium indicates the potassium ion as the possible carrier for the channel. Furthermore, the sensitivity to tetrodotoxin and extracellular sodium demonstrated the sodium dependence of this current. 相似文献
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ADP-ribosylation of ADPR-transferase and topoisomerase I in intact mouse epidermal cells JB6 总被引:5,自引:0,他引:5
Poly(ADP-ribosylation) [poly(ADPR)] is a posttranslational modification of chromosomal proteins that affects the structural and functional properties of chromatin. We have studied poly(ADPR) of ADPR-transferase and topoisomerase I in intact mouse epidermal cells JB6 (clone 41) by a combination of affinity chromatography on phenylboronate and immunoblotting with monoclonal antibodies against poly(ADPR) chains and polyclonal antibodies against ADPR-transferase and topoisomerase I, respectively. Constitutive, steady-state poly(ADPR) substitution of ADPR-transferase was estimated at 4% and that of topoisomerase I at 0.1%. Active oxygen produced extracellularly by xanthine-xanthine oxidase and the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine transiently increased the level of poly(ADPR) substitution of these enzymes by a factor of 6-10. While the poly(ADPR) substitution of ADPR-transferase remained elevated after 60 min of incubation, the poly(ADPR) substitution of topoisomerase I had returned to control values within this time. Benzamide (100 microM) partially prevented the stimulation of poly(ADPR) synthesis by these agents. We speculate that self-inactivation of ADPR-transferase by poly(ADPR) represents a feedback mechanism that has the function to avoid excessive poly(ADPR) synthesis and concomitant NAD and ATP depletion. Inactivation of topoisomerase I in the neighborhood of DNA breakage may temporarily shut down DNA replication and allow DNA repair to occur. 相似文献
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Insulin stimulated the accumulation of alpha-aminoisobutyric acid by the rat diaphragm in vivo. This stimulation, but not the accumulation in the absence of insulin, was reduced by 30-40% by insulin inhibitor present in extracts of liver. 相似文献
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In order to find out how insulin acts on airway smooth muscle and which mechanisms could be involved, we studied the effect of insulin on contraction induced, first, by KCl and, second, by Acetylcholine (Ach), before and after epithelium removal, and finally in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor. Tracheal smooth muscle strips from 24 rabbits, 6 being used for each experiment. Each muscle strip was pretreated with a solution containing either 80 mM KCl or 10(-5) Ach and increasing doses of insulin (range 10(-10)--10(-5) M) in the presence or absence of 10(-4) M L-NAME. A reference curve for contraction evoked by 80 mM KCl or 10(-5) M Ach in the presence or absence of 10(-4) M L-NAME was recorded each time before the pretreatment mentioned above. Insulin evoked a concentration-dependent inhibition of tracheal smooth muscle contraction, induced by 80 mM KCl or 10(-5) M Ach. After epithelium removal, insulin (10(-8), 10(-7) M) evoked statistically significant increases to the contractions induced by 10(-5) M Ach compared to the contractions induced by 10(-5) M Ach and insulin in the presence of epithelium (P < 0.05). These increases were higher when 10(-4) M l-NAME was added to the bath (P < 0.05). In conclusion, these results indicate that insulin inhibits tracheal smooth muscle contraction by acting on epithelium and releasing NO. 相似文献
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Continuous exposure of follicles/oocytes to elevated levels of insulin compromises embryonic developmental competence, although the underlying cellular mechanisms are unknown. The objectives of the present study were to determine whether mouse oocytes have insulin receptors and a functional insulin signaling cascade, and whether insulin exposure during oocyte growth or maturation influences meiotic progression and chromatin remodeling. Immunoblot and immunocytochemical analyses of germinal vesicle-intact (GVI) oocytes demonstrated the presence of insulin receptor-beta. Insulin receptor expression in oocytes was increased by gonadotropin stimulation, and remained elevated throughout meiotic maturation. Fully grown GVI oocytes contained 3-phosphoinositide-dependent protein kinase-1 (PDPK1), thymoma viral proto-oncogene 1 (AKT1), and glycogen synthase kinase 3 (GSK3). In vitro maturation of GVI oocytes in 5 microg/ml insulin had no influence on meiotic progression or the incidence of normal metaphase II (MII) chromosome condensation. Treatment of oocytes during maturation had no effect on GSK3A/B protein expression or phosphorylation of S21/9. However, the culturing of preantral follicles for 10 days with 5 microg/ml insulin increased the phosphorylation of oocyte GSK3B, indicating GSK3 inactivation. The rates of development to metaphase I (MI) were similar for oocytes obtained from insulin-treated follicles and controls, whereas the incidence of abnormal MI chromatin condensation was significantly higher in oocytes obtained from follicles cultured with insulin compared to those cultured without insulin. These results demonstrate that oocytes contain a functional insulin signaling pathway, and that insulin exposure during oocyte growth results in chromatin remodeling aberrations. These findings begin to elucidate the mechanisms by which chronic elevated insulin influences oocyte meiosis, chromatin remodeling, and embryonic developmental competence. 相似文献
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Insulin stimulates tyrosine phosphorylation of its receptor beta-subunit in intact rat hepatocytes. 总被引:1,自引:1,他引:0
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R Ballotti A Kowalski M F White Y Le Marchand-Brustel E Van Obberghen 《The Biochemical journal》1987,241(1):99-104
We studied the phosphorylation of the beta subunit of the insulin receptor in intact freshly isolated rat hepatocytes, labelled with [32P]Pi. Insulin receptors partially purified by wheat-germ agglutinin chromatography were immunoprecipitated with either antibodies to insulin receptor or antibodies to phosphotyrosine. Receptors derived from cells incubated in the absence of insulin contained only phosphoserine. Addition of insulin to hepatocytes led to a dose-dependent increase in receptor beta-subunit phosphorylation, with half-maximal stimulation being observed at 2 nM-insulin. Incubation of cells with 100 nM-insulin showed that, within 1 min of exposure to the hormone, maximal receptor phosphorylation occurred, which was followed by a slight decrease and then a plateau. This insulin-induced stimulation of its receptor phosphorylation was largely accounted for by phosphorylation on tyrosine residues. Sequential immunoprecipitation of receptor with anti-phosphotyrosine antibodies and with anti-receptor antibodies, and phosphoamino acid analysis of the immunoprecipitated receptors, revealed that receptors that failed to undergo tyrosine phosphorylation were phosphorylated on serine residues. The demonstration of a functional hormone-sensitive insulin-receptor kinase in normal cells strongly supports a role for this receptor enzymic activity in mediating biological effects of insulin. 相似文献
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Cross- protein structures. I. Insulin fibrils 总被引:5,自引:0,他引:5
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Lipidic mitochondrial inclusions in experimentally-induced in vitro myopathy of mouse diaphragm. 总被引:1,自引:0,他引:1
J D Howl S J Publicover 《Comparative biochemistry and physiology. B, Comparative biochemistry》1990,95(4):833-837
1. Treatment of murine skeletal muscle with the Ca2(+)-channel agonist, Bay K 8644, induces myopathic changes including configurational changes of the mitochondria. 2. During the progression of the myopathy the mitochondria apparently engulf neighboring lipid droplets. 3. Mitochondrial changes are most marked when damage to the muscle is most severe, are prevented under conditions that should reduce Bay K 8644-induced Ca2(+)-influx, and are not modified by various maneuvers designed to prevent the generation of free radical species. 相似文献
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Calcium/phospholipid-dependent protein kinase activity (protein kinase C) was identified in rat diaphragm membrane and cytosol fractions by means of in vitro phosphorylation either of histones or of a specific 87 kDa protein substrate, combined with phosphopeptide-mapping techniques. Both insulin and tumor-promoting phorbol ester treatment of the diaphragm preparations led to increased protein kinase C activity in the membrane fractions. In contrast to the phorbol ester, however, insulin did not induce a concomitant decrease in cytosolic activity, indicating that translocation of the enzyme had not taken place. Thus, insulin appears to increase specifically membrane protein kinase C activity in rat skeletal muscle, possibly through a mechanism not identical to that induced by phorbol esters. 相似文献