Insulin inhibits the phosphorylation of alpha-Gi-2 in intact hepatocytes.
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ABSTRACT:
SUBMITTER: Morris NJ
PROVIDER: S-EPMC1136981 | biostudies-other | 1995 Jun
REPOSITORIES: biostudies-other
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Project description:Hepatocytes contain the Gi2 and Gi3 forms of the 'Gi-family' of guanine-nucleotide-binding proteins (G-proteins), but not Gi1. The anti-peptide antisera AS7 and I3B were shown to immunoprecipitate Gi2 and Gi3 selectively, and the antiserum CS1 immunoprecipitated the stimulatory G-protein Gs. Treatment of intact, 32P-labelled hepatocytes with one of glucagon, TH-glucagon ([1-N-alpha-trinitrophenylhistidine, 12-homoarginine]glucagon), Arg-vasopressin, angiotensin-II, the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) and 8-bromo-cyclic AMP elicited a time- and dose-dependent increase in the labelling of the alpha-subunit of immunoprecipitated Gi2 which paralleled the loss of ability of low concentrations of the non-hydrolysable GTP analogue guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) to inhibit forskolin-stimulated adenylate cyclase activity ('Gi'-function). The immunoprecipitation of phosphorylated Gi-2 alpha-subunit by the antiserum AS7 was blocked in a dose-dependent fashion by the inclusion of the C-terminal decapeptide of transducin, but not that of Gz (a 'Gi-like' G-protein which lacks the C-terminal cysteine group which is ADP-ribosylated by pertussis toxin in other members of the Gi family), in the immunoprecipitation assay. No labelling of the alpha-subunits of either Gi3 or Gs was observed. alpha-Gi2 was labelled in the basal state and this did not change over 15 min in the absence of ligand addition. In contrast to the monophasic dose-effect curves seen with vasopressin, angiotensin and TPA, the dose-effect curve for the glucagon-mediated increase in the labelling of alpha-Gi2 was markedly biphasic where the loss of Gi function paralleled the high-affinity component of the labelling of alpha-Gi2 caused by glucagon. TPA, TH-glucagon, angiotensin-II and vasopressin achieved similar maximal increases in the labelling of alpha-Gi2, which was approximately half that found after treatment of hepatocytes with either high glucagon concentrations (1 microM) or 8-bromocyclic AMP. Analysis of the phosphoamino acid content of immunoprecipitated alpha-Gi2 showed the presence of phosphoserine only. Incubation of hepatocyte membranes with [gamma-32P]ATP and purified protein kinase C, but not protein kinase A, led to the incorporation of label into immunoprecipitated alpha-Gi2. This labelling was abolished if membranes were obtained from cells which had received prior treatment with ligands shown to cause the phosphorylation of alpha-Gi2 in intact cells. We suggest that there are two possible sites for the phosphorylation of alpha-Gi2; one for C-kinase and the other for an unidentified kinase whose action is triggered by A-kinase activation.
| S-EPMC1131453 | biostudies-other
Project description:A phosphorylated form of alpha-Gi-2 (the alpha-subunit of Gi-2), immunoprecipitated from hepatocytes under basal conditions, migrated as a single species of pI approximately 5.7, the labelling of which increased approximately 2-fold in cells challenged with either vasopressin or phorbol 12-myristate 13-acetate (PMA); agents which activate protein kinase C. In contrast, treatment of hepatocytes with 8-bromo-cyclic AMP produced a more acidic species of phosphorylated alpha-Gi-2 having a pI of approximately 5.4 and whose labelling was increased approximately 3-fold. Trypsin digestion of labelled alpha-Gi-2 isolated from hepatocytes under basal conditions identified, on two-dimensional peptide analyses, three positively charged phosphoserine-containing peptides (C1, C2 and C3), with only peptides C1 and C2 being evident upon less extensive digestion with trypsin. These are suggested to reflect a single site of phosphorylation, with proteolysis by trypsin being incomplete, and where C2 is larger than C1, which is larger than C3. An identical pattern of tryptic phosphopeptides was seen in hepatocytes treated with either vasopressin or PMA, although labelling of this group of peptides was increased by approximately 2-fold compared with the basal state. In contrast, treatment of hepatocytes with glucagon, 8-bromo-cyclic AMP or forskolin not only resulted in increased labelling of the 'basal' sites approximately 3-fold, but identified a novel positively charged tryptic phosphoserine-containing peptide (AN). All four tryptic peptides were susceptible to proteolysis by V8 protease. Treatment of labelled alpha-Gi-2 from basal and PMA-treated cells produced a pattern of peptides which was identical with those found when the tryptic phosphopeptide was treated with V8 protease. We tentatively suggest that, on alpha-Gi-2, Ser144 is phosphorylated through the action of protein kinase C and Ser207 is phosphorylated upon elevation of the intracellular concentrations of cyclic AMP.
| S-EPMC1137044 | biostudies-other
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.
| S-EPMC1147530 | biostudies-other
Project description:The molecular mechanism of hepatic cell growth and differentiation is ill defined. In the present study, we examined the putative role of tyrosine phosphorylation in normal rat liver development and in an in vitro model, the alpha-fetoprotein-producing (AFP+) and AFP-nonproducing (AFP-) clones of the McA-RH 7777 rat hepatoma. We demonstrated in vivo and in vitro that the AFP+ phenotype is clearly associated with enhanced tyrosine phosphorylation, as assessed by immunoblotting and flow cytometry. Moreover, immunoprecipitation of proteins with anti-phosphotyrosine antibody showed that normal fetal hepatocytes expressed the same phosphorylation pattern as stable AFP+ clones and likewise for adult hepatocytes and AFP- clones. The tyrosine phosphorylation of several proteins, including the beta-subunit of the insulin receptor, insulin receptor substrate-1, p85 regulatory subunit of phosphatidylinositol-3-kinase, and ras-guanosine triphosphatase-activating protein, was observed in AFP+ clones, whereas the same proteins were not phosphorylated in AFP- clones. We also observed that fetal hepatocytes and the AFP+ clones express 4 times more of the insulin receptor beta-subunit compared with adult hepatocytes and AFP- clones and, accordingly, that these AFP+ clones were more responsive to exogenous insulin in terms of protein tyrosine phosphorylation. Finally, growth rate in cells of AFP+ clones was higher than that measured in cells of AFP- clones, and inhibition of phosphatidylinositol-3-kinase by LY294002 and Wortmannin blocked insulin- and serum-stimulated DNA synthesis only in cells of AFP+ clones. These studies provide evidences in support of the hypothesis that signaling via insulin prevents hepatocyte differentiation by promoting fetal hepatocyte growth.
| S-EPMC25332 | biostudies-literature
Project description:Activation of arterial smooth muscle alpha(1)-adrenergic receptors results in vasoconstriction, as well as a secondary release of nitric oxide and slow vasodilation, presumably through gap junction communication from smooth muscle to endothelium. We hypothesized that this slow vasodilation is due to activation of eNOS through phosphorylation at Ser1179 and dephosphorylation at Thr495. Phosphorylation was measured by western blot using mouse mesenteric arteries that were cannulated and pressurized (75 mm Hg) and treated either by 1) 5 min of phenylephrine superfusion (10(-5)M) (PE5), 2) 15 min of phenylephrine (PE15), 3) 15 min phenylephrine followed by acetylcholine (10(-4)M) (PE+ACh), or 4) 20 min time control with no treatment (NT) [4-5 arteries pooled per treatment per blot; 5 blots performed]. These treatments allowed correlation between vasomotor changes, namely maximal constriction (PE5), slow vasodilation (PE15), and maximal dilation (PE+ACh), and relative phosphorylation changes. Phosphorylation of eNOS at Ser1179 was increased relative to NT by more than 2-fold at PE5 and remained similarly increased at PE15 and PE+ACh. Phosphorylation of eNOS at Thr495 was less in all treatments relative to NT, but not significantly. Treatment with L-NAME (10(-4)M) or endothelial denudation indicated that the slow dilation in response to phenylephrine was completely due to nitric oxide synthase and was endothelial dependent. These results indicate that eNOS phosphorylation at Ser1179 occurs before the slow dilation and is not actively involved in this vasodilation or dilation to acetylcholine, but may play a permissive role in eNOS activation by other mechanisms. It is not yet known what mechanism is responsible for Ser1179 phosphorylation with phenylephrine stimulation.
| S-EPMC3534816 | biostudies-literature
Project description:Insulin and adrenergic stimulation are two divergent regulatory systems that may interact under certain pathophysiological circumstances. Here, we characterized a complex consisting of insulin receptor (IR) and ?2-adrenergic receptor (?2AR) in the heart. The IR/?2AR complex undergoes dynamic dissociation under diverse conditions such as Langendorff perfusions of hearts with insulin or after euglycemic-hyperinsulinemic clamps in vivo. Activation of IR with insulin induces protein kinase A (PKA) and G-protein receptor kinase 2 (GRK2) phosphorylation of the ?2AR, which promotes ?2AR coupling to the inhibitory G-protein, Gi. The insulin-induced phosphorylation of ?2AR is dependent on IRS1 and IRS2. After insulin pretreatment, the activated ?2AR-Gi signaling effectively attenuates cAMP/PKA activity after ?-adrenergic stimulation in cardiomyocytes and consequently inhibits PKA phosphorylation of phospholamban and contractile responses in myocytes in vitro and in Langendorff perfused hearts. These data indicate that increased IR signaling, as occurs in hyperinsulinemic states, may directly impair ?AR-regulated cardiac contractility. This ?2AR-dependent IR and ?AR signaling cross-talk offers a molecular basis for the broad interaction between these signaling cascades in the heart and other tissues or organs that may contribute to the pathophysiology of metabolic and cardiovascular dysfunction in insulin-resistant states.
| S-EPMC4113065 | biostudies-literature
Project description:IntroductionEpidemiological studies suggest that statins may promote the development or exacerbation of diabetes, but whether this occurs through inhibition of insulin secretion is unclear. This lack of understanding is partly due to the cellular models used to explore this phenomenon (cell lines or pooled islets), which are non-physiologic and have limited clinical transferability.MethodsHere, we study the effect of simvastatin on insulin secretion using single-islet cultures, an optimal compromise between biological observability and physiologic fidelity. We develop and validate a microfluidic device to study single-islet function ex vivo, which allows for switching between media of different compositions with a resolution of seconds. In parallel, fluorescence imaging provides real-time analysis of the membrane voltage potential, cytosolic Ca2+ dynamics, and insulin release during perfusion under 3 or 11 mM glucose.ResultsWe found that simvastatin reversibly inhibits insulin secretion, even in high-glucose. This phenomenon is very rapid (<60 s), occurs without affecting Ca2+ concentrations, and is likely unrelated to cholesterol biosynthesis and protein isoprenylation, which occur on a time span of hours.ConclusionsOur data provide the first real-time live demonstration that a statin inhibits insulin secretion in intact islets and that single islets respond differently from cell lines on a short time scale.FundingUniversity of Padova, EASD Foundation.
| S-EPMC5118248 | biostudies-literature
Project description:The 52 kDa subunit of the peripheral-plasma-membrane insulin-stimulated high-affinity cyclic AMP phosphodiesterase can be specifically detected by the antibody PM1 by Western-blotting procedures and also can be immunoprecipitated from a hepatocyte extract. PM1-mediated immunoprecipitation from hepatocyte extracts showed that insulin treatment of intact 32P-labelled hepatocytes caused the rapid phosphorylation of the peripheral-plasma-membrane cyclic AMP phosphodiesterase. Phosphoamino acid analysis and the use of a phosphotyrosine-specific antibody indicated that phosphorylation occurred on tyrosyl residue(s) of this phosphodiesterase. Prior treatment of hepatocytes with glucagon (10 nM) completely blocked the insulin-mediated tyrosyl phosphorylation of this 52 kDa protein, as detected with both the PM1 and the anti-phosphotyrosine antibodies. Treatment of hepatocytes with glucagon alone did not increase the phosphorylation state of the peripheral-plasma-membrane cyclic AMP phosphodiesterase. The specific anti-phosphotyrosine antibody also detected the insulin-stimulated phosphorylation of proteins of 180 kDa, 95 kDa and 39 kDa. Prior treatment of hepatocytes with glucagon decreased the ability of insulin to phosphorylate the 180 kDa and 39 kDa species, but not the 95 kDa species.
| S-EPMC1138914 | biostudies-other
Project description:G protein-coupled receptors transduce diverse extracellular signals, such as neurotransmitters, hormones, chemokines, and sensory stimuli, into intracellular responses through activation of heterotrimeric G proteins. G proteins play critical roles in determining specificity and kinetics of subsequent biological responses by modulation of effector proteins. We have developed a fluorescence resonance energy transfer (FRET)-based assay to directly measure mammalian G protein activation in intact cells and found that Gi proteins activate within 1-2 s, which is considerably slower than activation kinetics of the receptors themselves. More importantly, FRET measurements demonstrated that Galphai- and Gbetagamma-subunits do not dissociate during activation, as has been previously postulated. Based on FRET measurements between Galphai-yellow fluorescent protein and Gbetagamma-subunits that were fused to cyan fluorescent protein at various positions, we conclude that, instead, G protein subunits undergo a molecular rearrangement during activation. The detection of a persistent heterotrimeric composition during G protein activation will impact the understanding of how G proteins achieve subtype-selective coupling to effectors. This finding will be of particular interest for unraveling Gbetagamma-induced signaling pathways.
| S-EPMC307695 | biostudies-literature
Project description:BackgroundIn several neurodegenerative diseases, hyperphosphorylation at position Ser129 is found in fibrillar deposits of alpha-synuclein (asyn), implying a pathophysiological role of asyn phosphorylation in neurodegeneration. However, recent animal models applying asyn phosphorylation mimics demonstrated a protective effect of phosphorylation. Since metal-ion induced asyn oligomers were identified as a potential neurotoxic aggregate species with membrane pore-forming abilities, the current study was undertaken to determine effects of asyn phosphorylation on oligomer membrane binding.MethodsWe investigated the influence of S129 phosphorylation on interactions of metal-ion induced asyn oligomers with small unilamellar lipid vesicles (SUV) composed of POPC and DPPC applying the phosphorylation mimic asyn129E. Confocal single-particle fluorescence techniques were used to monitor membrane binding at the single-particle level.ResultsBinding of asyn129E monomers to gel-state membranes (DPPC-SUV) is slightly reduced compared to wild-type asyn, while no interactions with membranes in the liquid-crystalline state (POPC-SUV) are seen for both asyn and asyn129E. Conversely, metal-ion induced oligomer formation is markedly increased in asyn129E. Surprisingly, membrane binding to POPC-SUV is nearly absent in Fe(3+) induced asyn129E oligomers and markedly reduced in Al(3+) induced oligomers.ConclusionThe protective effect of pseudophosphorylation seen in animal models may be due to impeded oligomer membrane binding. Phosphorylation at Ser129 may thus have a protective effect against neurotoxic asyn oligomers by preventing oligomer membrane binding and disruption of the cellular electrophysiological equilibrium. Importantly, these findings put a new complexion on experimental pharmaceutical interventions against POLO-2 kinase.
| S-EPMC4049638 | biostudies-literature