Effects of insulin, glucagon, dexamethasone, cyclic GMP and spermine on the stability of phosphatidate phosphohydrolase activity in cultured rat hepatocytes.
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ABSTRACT:
SUBMITTER: Pittner RA
PROVIDER: S-EPMC1147401 | biostudies-other | 1986 Nov
REPOSITORIES: biostudies-other
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Project description:Rat hepatocytes were incubated in monolayer culture for 8 h. Glucagon (10nM) increased the total phosphatidate phosphohydrolase activity by 1.7-fold. This effect was abolished by adding cycloheximide, actinomycin D or 500 pM-insulin to the incubations. The glucagon-induced increase was synergistic with that produced by an optimum concentration of 100 nM-dexamethasone. Theophylline (1mM) potentiated the effect of glucagon, but it did not affect the dexamethasone-induced increase in the phosphohydrolase activity. The relative proportion of the phosphohydrolase activity associated with membranes was decreased by glucagon when 0.15 mM-oleate was added 15 min before the end of the incubations to translocate the phosphohydrolase from the cytosol. This glucagon effect was not seen at 0.5 mM-oleate. Since glucagon also increased the total phosphohydrolase activity, the membrane-associated activity was maintained at 0.15 mM-oleate and was increased at 0.5 mM-oleate. This activity at both oleate concentrations was also increased in incubations that contained dexamethasone, particularly in the presence of glucagon. Insulin increased the relative proportion of phosphatidate phosphohydrolase that was associated with membranes at 0.15 mM-oleate, but not at 0.5 mM-oleate. It also decreased the absolute phosphohydrolase activity on the membranes at both oleate concentrations in incubations that also contained glucagon and dexamethasone. None of the hormonal combinations significantly altered the total glycerol phosphate acyltransferase activity. However, glucagon significantly increased the microsomal activities, and insulin had the opposite effect. Glucagon also decreased the mitochondrial acyltransferase activity. There was a highly significant correlation between the total phosphatidate phosphohydrolase activity and the synthesis of neutral lipids from glycerol phosphate and 0.5 mM-oleate in homogenates of cells from all of the hormonal combinations. Phosphatidate phosphohydrolase activity is increased in the long term by glucocorticoids and also by glucagon through cyclic AMP. In the short term, glucagon increases the concentration of fatty acid required to translocate the cytosolic reservoir of activity to the membranes on which phosphatidate is synthesized. Insulin opposes the combined actions of glucagon and glucocorticoids. The long-term events explain the large increases in the phosphohydrolase activity that occur in vivo in a variety of stress conditions. The expression of this activity depends on increases in the net availability of fatty acids and their CoA esters in the liver.
| S-EPMC1152646 | biostudies-other
Project description:ObjectiveThe physiologic significance of the nitric oxide (NO)/cGMP signaling pathway in islets is unclear. We hypothesized that cGMP-dependent protein kinase type I (cGKI) is directly involved in the secretion of islet hormones and glucose homeostasis.Research design and methodsGene-targeted mice that lack cGKI in islets (conventional cGKI mutants and cGKI? and I? rescue mice [?/?RM] that express cGKI only in smooth muscle) were studied in comparison to control (CTR) mice. cGKI expression was mapped in the endocrine pancreas by Western blot, immuno-histochemistry, and islet-specific recombination analysis. Insulin, glucagon secretion, and cytosolic Ca²(+) ([Ca²(+)](i)) were assayed by radioimmunoassay and FURA-2 measurements, respectively. Serum levels of islet hormones were analyzed at fasting and upon glucose challenge (2 g/kg) in vivo.ResultsImmunohistochemistry showed that cGKI is present in ?- but not in ?-cells in islets of Langerhans. Mice that lack ?-cell cGKI had significantly elevated fasting glucose and glucagon levels, whereas serum insulin levels were unchanged. High glucose concentrations strongly suppressed the glucagon release in CTR mice, but had only a moderate effect on islets that lacked cGKI. 8-Br-cGMP reduced stimulated [Ca²(+)](i) levels and glucagon release rates of CTR islets at 0.5 mmol/l glucose, but was without effect on [Ca²(+)](i) or hormone release in cGKI-deficient islets.ConclusionsWe propose that cGKI modulates glucagon release by suppression of [Ca²(+)](i) in ?-cells.
| S-EPMC3012166 | biostudies-literature
Project description:Rat hepatocytes were preincubated for 16 h with hormones or drugs and then for a further 8 h with 125I-human low-density lipoprotein (LDL). Glucagon (via cyclic AMP) and adrenaline (via cyclic AMP and alpha-effects) increased the binding of 125I-LDL to the LDL receptor, and the degradation of LDL to [125I]iodotyrosine. The effects on degradation were antagonized by dexamethasone, and the action of cyclic AMP on binding and degradation was inhibited by actinomycin D. The results are discussed in relation to the control of lipoprotein metabolism in diabetes.
| S-EPMC1133285 | biostudies-other
Project description:The activity of many proteins orchestrating different biological processes is regulated by allostery, where ligand binding at one site alters the function of another site. Allosteric changes can be brought about by either a change in the dynamics of a protein, or alteration in its mean structure. We have investigated the mechanisms of allostery induced by chemically distinct ligands in the cGMP-binding, cGMP-specific phosphodiesterase, PDE5. PDE5 is the target for catalytic site inhibitors, such as sildenafil, that are used for the treatment of erectile dysfunction and pulmonary hypertension. PDE5 is a multidomain protein and contains two N-terminal cGMP-specific phosphodiesterase, bacterial adenylyl cyclase, FhLA transcriptional regulator (GAF) domains, and a C-terminal catalytic domain. Cyclic GMP binding to the GAFa domain and sildenafil binding to the catalytic domain result in conformational changes, which to date have been studied either with individual domains or with purified enzyme. Employing intramolecular bioluminescence resonance energy transfer, which can monitor conformational changes both in vitro and in intact cells, we show that binding of cGMP and sildenafil to PDE5 results in distinct conformations of the protein. Metal ions bound to the catalytic site also allosterically modulated cGMP- and sildenafil-induced conformational changes. The sildenafil-induced conformational change was temperature-sensitive, whereas cGMP-induced conformational change was independent of temperature. This indicates that different allosteric ligands can regulate the conformation of a multidomain protein by distinct mechanisms. Importantly, this novel PDE5 sensor has general physiological and clinical relevance because it allows the identification of regulators that can modulate PDE5 conformation in vivo.
| S-EPMC3048737 | biostudies-literature
Project description:During the perinatal period, the activity of the urea-cycle enzyme argininosuccinate lyase (ASL) is regulated by glucocorticoids, glucagon and insulin. In this study, the effects of glucagon and cyclic AMP (cAMP) analogues were examined on the synthesis of ASL and on the level of its corresponding mRNA in cultured foetal hepatocytes. Northern-blot analysis revealed that these agents only gave a transient induction of ASL mRNA amount, which reached a peak at 6 h and declined thereafter. This induction preceded the increase in enzyme activity and amount which could be observed for 2 or 3 days of culture. Stimulation of ASL mRNA accumulation by a combination of cAMP analogues and dexamethasone was additive, indicating that glucocorticoids and cAMP are both necessary to promote hepatocyte differentiation and that inductions could occur via independent pathways. Induction by cAMP analogues could be abolished by actinomycin D, suggesting a control mechanism at the transcriptional level. Puromycin was without effect on ASL mRNA induction by cAMP, indicating that no ongoing protein synthesis was required in the stimulation process.
| S-EPMC1132567 | biostudies-other
Project description:Myocardial triacylglycerol hydrolysis is subject to product inhibition. After hydrolysis of endogenous triacylglycerols, the main proportion of the liberated fatty acids is re-esterified to triacylglycerol, indicating the importance of fatty acid re-esterification in the regulation of myocardial triacylglycerol homoeostasis. Therefore, we characterized phosphatidate phosphohydrolase (PAP) and diacylglycerol acyltransferase (DGAT) activities, enzymes catalysing the final steps in the re-esterification of fatty acids to triacylglycerols in the isolated rat heart. The PAP activity was mainly recovered in the microsomal and soluble cell fractions, with an apparent Km of 0.14 mM for both the microsomal and the soluble enzyme. PAP was stimulated by Mg2+ and oleic acid. Oleic acid, like a high concentration of KCl, stimulated the translocation of PAP activity from the soluble to the particulate (microsomal) fraction. Myocardial DGAT had an apparent Km of 3.8 microM and was predominantly recovered in the particulate (microsomal) fraction. Both enzyme activities were significantly increased after acute streptozotocin-induced diabetes, PAP from 15.6 +/- 1.1 to 28.1 +/- 3.6 m-units/g wet wt. (P less than 0.01) and DGAT from 2.23 +/- 0.11 to 3.01 +/- 0.11 m-units/g wet wt. (P less than 0.01). In contrast with diabetes, low-flow ischaemia during 30 min did not affect PAP and DGAT activity in rat hearts. Perfusion with glucagon (0.1 microM) during 30 min did not affect total PAP activity, but changed the subcellular distribution. More PAP activity was recovered in the particulate fraction. DGAT activity was lowered by glucagon treatment from 0.37 +/- 0.03 to 0.23 +/- 0.02 m-unit/mg of microsomal protein (P less than 0.05). The role of PAP and DGAT activity and PAP distribution in the myocardial glucose/fatty acid cycle is discussed.
| S-EPMC1131459 | biostudies-other
Project description:Lipin 1 is a bifunctional intracellular protein that regulates fatty acid metabolism in the nucleus via interactions with DNA-bound transcription factors and at the endoplasmic reticulum as a phosphatidic acid phosphohydrolase enzyme (PAP-1) to catalyze the penultimate step in triglyceride synthesis. However, livers of 8-day-old mice lacking lipin 1 (fld mice) exhibited normal PAP-1 activity and a 20-fold increase in triglyceride levels. We sought to further analyze the hepatic lipid profile of these mice by electrospray ionization mass spectrometry. Surprisingly, hepatic content of phosphatidate, the substrate of PAP-1 enzymes, was markedly diminished in fld mice. Similarly, other phospholipids derived from phosphatidate, phosphatidylglycerol and cardiolipin, were also depleted. Another member of the lipin family (lipin 2) is enriched in liver, and hepatic lipin 2 protein content was markedly increased by lipin 1 deficiency, food deprivation, and obesity, often independent of changes in steady-state mRNA levels. Importantly, RNAi against lipin 2 markedly reduced PAP-1 activity in hepatocytes from both wild type and fld mice and suppressed triglyceride synthesis under conditions of high fatty acid availability. Collectively, these data suggest that lipin 2 plays an important role as a hepatic PAP-1 enzyme.
| S-EPMC2652272 | biostudies-literature
Project description:When a particle-free supernatant fraction from rat liver was incubated at 37 degrees C with mitochondria and oleate, some of the enzyme phosphatidate phosphohydrolase (PAP), initially present in the particle-free supernatant, was recovered, after the incubation, bound to mitochondria. This translocation of PAP from cytosol to mitochondria was stimulated by oleate or palmitate in a similar fashion to the stimulation of translocation of PAP to endoplasmic reticulum [Martin-Sanz, Hopewell & Brindley (1984) FEBS Lett. 175, 284-288]. Translocation of PAP from particle-free supernatant to a partially purified mitochondrial-outer-membrane preparation was also stimulated by oleate. More PAP was bound to a mitochondrial-outer-membrane fraction washed in 0.5 M-NaCl before resuspension in sucrose than to a sucrose-washed mitochondrial-outer-membrane preparation. In contrast, washing of microsomal membranes in 0.5 M-NaCl did not enhance the binding of PAP to these membranes. PAP also binds to phosphatidate-loaded mitochondria or microsomes (microsomal fractions). In the experimental system employed, more PAP bound to mitochondria loaded with phosphatidate than to microsomes loaded with phosphatidate. The results are discussed in relation to the role of mitochondrial phosphatidate in liver lipid metabolism.
| S-EPMC1133467 | biostudies-other
Project description:Previously we have identified the presence of two different phosphatidate phosphohydrolase (PPH) activities in rat adipose tissue, based on Mg(2+)-dependency. In the present investigation, we have further characterized these isoenzymes, using both aqueous dispersed and membrane-bound phosphatidate as substrates and differentiated these activities on the basis of both Mg(2+)-dependency and N-ethylmaleimide (NEM)-sensitivity. These two distinguishing criteria gave identical estimates of PPH activities present in the different subcellular fractions. The microsomal and cytosol fractions contained mainly the Mg(2+)-dependent (NEM-sensitive) form, which was inhibited by various thiol reagents, was inactivated by heating at 55 degrees C for 20 min, and was decreased significantly within 2 h after intraperitoneal administration of cystamine (200 mg/kg). Such treatments had no effects on the Mg(2+)-independent (NEM-insensitive) form of PPH, which was mainly located in the plasma membranes, mitochondrial and microsomal fractions. Addition of Lipid A and guanosine 5'-[gamma-thio]triphosphate to the assay mixture had no effect on the PPH activities. The Mg(2+)-independent PPH form, which was thermostable in the intact subcellular fractions, became thermolabile when these fractions were disrupted in the presence of Triton X-100. The present studies demonstrate that: (1) the thermostability is not a satisfactory index to differentiate these isoenzymes; (2) the thiol/disulphide exchange may be involved in the regulation of Mg(2+)-dependent PPH activity; and (3) the PPH isoenzymes do not seem to be under G-protein control in adipose tissue, as reported previously in the mesangial cell line.
| S-EPMC1137057 | biostudies-other
Project description:The measurement of phosphate release from phosphatidate overestimates the microsomal activity of phosphatidate phosphohydrolase from rat liver, since phosphate is also produced via the glycerol phosphate that results from the deacylation of phosphatidate. The determination of phosphate production can be a reliable assay for the soluble phosphatidate phosphohydrolase in rat liver, because the glycerol phosphate formed is not hydrolysed under the conditions used.
| S-EPMC1184155 | biostudies-other