Insulin stimulates synthesis of soluble proteins in isolated rat hepatocytes.
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ABSTRACT:
SUBMITTER: Clark RL
PROVIDER: S-EPMC1162138 | biostudies-other | 1980 Sep
REPOSITORIES: biostudies-other
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Project description:The ability of the insulin-induced phospho-oligosaccharide to stimulate amino acid transport was studied in isolated rat hepatocytes. At low alpha-aminoisobutyric acid concentrations (0.1 mM), both 100 nM-insulin and 10 microM-phospho-oligosaccharide doubled amino acid uptake after 2 h of incubation. This stimulation was prevented by 0.1 mM-cycloheximide or 5 micrograms of actinomycin D/ml, indicating that the phospho-oligosaccharide, like insulin, was acting via the synthesis of a high-affinity transport component. The effects of the phospho-oligosaccharide and of insulin were blocked by Ins2P (2.5 mM), but not by myo-inositol, inositol hexaphosphoric acid or several monosaccharides such as mannose, glucosamine and galactose. Both the temporal effect on amino acid entry and the extent of stimulation of this process by the phospho-oligosaccharide indicate that this molecule mimics, and may mediate, some of the long-term actions of insulin. However, the effects of phospho-oligosaccharide and insulin were not exactly the same, since the effect of insulin, but not of the phospho-oligosaccharide, was additive with that of glucagon.
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Project description:There is increasing evidence for the existence of intrahepatic regulation of glucose metabolism by Kupffer cell products. Nitric oxide (NO) is known to inhibit gluconeogenic flux through pyruvate carboxylase and phosphoenolpyruvate carboxykinase. However, NO may also influence glucose metabolism at other levels. Using hepatocytes from fasted rats incubated with the NO-donor S-nitroso-N-acetylpenicillamine, we have now found that the synthesis of glycogen from glucose is even more sensitive to inhibition by NO than gluconeogenesis. Inhibition of glycogen production by NO was accompanied by a rise in intracellular glucose 6-phosphate and UDPglucose. Activity of glycogen synthase, as measured in extracts of hepatocytes after the cells had been exposed to NO, was decreased. Experiments with gel-filtered liver extracts revealed that inhibition of glycogen synthase was caused by an inhibitory effect of NO on the conversion of glycogen synthase b into glycogen synthase a.
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Project description: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.
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Project description:Previous work has shown that extracellular adenosine inhibits the incorporation of radiolabelled leucine into proteins in isolated rat hepatocytes [Tinton, Lefebvre, Cousin and Buc Calderon (1993) Biochim. Biophys. Acta 1176, 1-6]. In this study, we investigated whether its metabolism into adenine nucleotides, inosine or S-adenosylhomocysteine (AdoHcy) is required to induce such an impairment. Incubation of isolated hepatocytes in the presence of adenosine at 0.5 or 1 mM reduces the synthesis of proteins by about 45% after 120 min of incubation. Such an inhibition occurred without cell lysis and was not modified by adding the adenosine kinase inhibitor 5-iodotubercidin (15 microM) or the adenosine deaminase inhibitor coformycin (0.1 microM). It is therefore unlikely that the anabolic and catabolic pathways of adenosine are involved in the inhibition of protein synthesis. Adenosine (1 mM) increased the level of AdoHcy and S-adenosylmethionine by 20- and 5-fold respectively after 60 min of incubation and reduced the methylation index. These events as well as the inhibition of protein synthesis were strongly enhanced in the presence of L-homocysteine (2 mM). It is therefore concluded that the metabolism of adenosine into AdoHcy, which is known to be a potent inhibitor of cellular methylation reactions, may play an important role in the control of translation.
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Project description:Exposure of isolated rat hepatocytes to glucagon or chlorophenylthio cyclic AMP led to an inhibition of the incorporation of [1,2-14C]ethanolamine into phosphatidylethanolamine. Pulse-chase experiments and measurement of the activities of the enzymes involved in the CDP-ethanolamine pathway provided evidence that the inhibitory effect of glucagon on the synthesis de novo of phosphatidylethanolamine was not caused by a diminished conversion of ethanolamine phosphate into CDP-ethanolamine. The observations suggested that the glucagon-induced inhibition of the biosynthesis of phosphatidylethanolamine is probably due to a decreased supply of diacylglycerols, resulting in a decreased formation of phosphatidylethanolamine from CDP-ethanolamine and diacylglycerols.
| S-EPMC1135636 | biostudies-other
Project description:Insulin resistance is a hallmark of type 2 diabetes. Although multiple genetic and physiological factors interact to cause insulin resistance, deregulated signaling by phosphorylation is a common underlying mechanism. In particular, the specific phosphorylation-dependent regulatory mechanisms and signaling outputs of insulin are poorly understood in hepatocytes, which represents one of the most important insulin-responsive cell types. Using primary rat hepatocytes as a model system, we performed reductive dimethylation (ReDi)-based quantitative mass spectrometric analysis and characterized the phosphoproteome that is regulated by insulin as well as its key downstream kinases including Akt, mTORC1, and S6K. We identified a total of 12 294 unique, confidently localized phosphorylation sites and 3805 phosphorylated proteins in this single cell type. Detailed bioinformatic analysis on each individual data set identified both known and previously unrecognized targets of this key insulin downstream effector pathway. Furthermore, integrated analysis of the hepatic Akt/mTORC1/S6K signaling axis allowed the delineation of the substrate specificity of several close-related kinases within the insulin signaling pathway. We expect that the data sets will serve as an invaluable resource, providing the foundation for future hypothesis-driven research that helps delineate the molecular mechanisms that underlie the pathogenesis of type 2 diabetes and related metabolic syndrome.
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Project description:We studied internalization of 125I-labelled insulin in isolated rat hepatocytes. Using the acidification technique, we were able to dissociate the ligand from its cell-surface receptors, and thus to separate internalized from surface-bound insulin. Because during the first 5 min of incubation of 125I-labelled insulin with freshly isolated hepatocytes there is no loss of internalized label, the ratio of the amount of internalized ligand to the amount of cell-surface-bound ligand may serve as an index of insulin internalization. Within the first 10 min of insulin's interaction with hepatocytes, the plot of the above ratio as a function of time yields a straight line. The slope of this line is referred to as the endocytic rate constant (Ke) for insulin and denotes the probability with which the insulin-receptor complex is internalized in 1 min. At the insulin concentration of 0.295 ng/ml, the Ke is 0.049 min-1. It is independent of insulin concentration until the latter exceeds 1 ng/ml. At the insulin concentration of 3.2 ng/ml, the Ke accelerates to 0.131 min-1. With the Ke being the probability of insulin-receptor-complex internalization, 4.9% of occupied insulin receptors will be internalized in 1 min at an insulin concentration of 0.295 ng/ml, and 13.1% of occupied insulin receptors will be internalized in 1 min at 3.2 ng/ml. When the insulin concentration decreases from 3.2 to 0.3 ng/ml, the Ke decreases accordingly. The half-time of occupied receptor internalization is 15.4 min at the lower insulin concentration and 5.3 min at the higher insulin concentration.
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Project description:Extracellular Ca2+ stimulated fatty acid synthesis in isolated rat hepatocytes. Orthovanadate (0.2--2.0 mM), an inhibitor of Ca2+-dependent ATPases, stimulated fatty acid synthesis in both the presence and the absence of extracellular Ca2+. Insulin stimulated fatty acid synthesis only in the presence of extracellular Ca2+. The contribution of extracellular Ca2+ to insulin stimulation of fatty acid synthesis is discussed.
| S-EPMC1158177 | biostudies-other
Project description:Glutamine stimulated glycogen synthesis and lactate production in hepatocytes from overnight-fasted normal and diabetic rats. The effect, which was half-maximal with about 3 mM-glutamine, depended on glucose concentration and was maximal below 10 mM-glucose. beta-2-Aminobicyclo[2.2.1.]heptane-2-carboxylic acid, an analogue of leucine, stimulated glutaminase flux, but inhibited the stimulation of glycogen synthesis by glutamine. Various purine analogues and inhibitors of purine synthesis were found to inhibit glycogen synthesis from glucose, but they did not abolish the stimulatory effect of glutamine on glycogen synthesis. The correlation between the rate of glycogen synthesis and synthase activity suggested that the stimulation of glycogen synthesis by glutamine depended solely on the activation of glycogen synthase. This activation of synthase was not due to a change in total synthase, nor was it caused by a faster inactivation of glycogen phosphorylase, as was the case after glucose. It could, however, result from a stimulation of synthase phosphatase, since, after the addition of 1 nM-glucagon or 10 nM-vasopressin, glutamine did not interfere with the inactivation of synthase, but did promote its subsequent re-activation. Glutamine was also found to inhibit ketone-body production and to stimulate lipogenesis.
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Project description:The effect of microcystin-LR, an inhibitor of protein phosphatases PP1 and PP2A, was studied on protein synthesis by measuring the incorporation of labelled amino acid into protein in isolated rat hepatocytes. Microcystin-LR inhibited protein synthesis in the first minutes of the incubation period, and half-maximum effect was obtained at about 60 nM. Such an inhibition was also observed in the presence of different protein phosphatase inhibitors, i.e. okadaic acid, calyculin A and microcystin-RR. This effect was observed in whole hepatocytes, in the supernatant of the post-mitochondrial fraction and in the microsomal fraction. It was independent of a substrate supply and of the labelled amino acid used. Furthermore, this inhibition preceded the previously reported glucose-6-phosphatase activation induced by microcystin-LR [Claeyssens, Chédeville and Lavoinne (1993) FEBS Lett. 315, 7-10].
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