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D L Severson

Publications and source records attributed to D L Severson.

At least 37 records · Page 2Linked to original sources
PubMed1995

Post-transcriptional mechanisms are responsible for the reduction in lipoprotein lipase activity in cardiomyocytes from diabetic rat hearts.

Lipoprotein lipase (LPL) activity is reduced in cardiomyocytes from rat hearts following the acute (4-5 day) induction of diabetes with 100 mg/kg streptozotocin. The molecular basis for this inhibitory effect of diabetes on LPL activity was investigated by measuring steady-state LPL mRNA content and the synthesis and turnover of LPL protein ([35S]methionine incorporation into immunoprecipitable LPL protein in pulse and pulse-chase experiments) in control and diabetic cardiomyocytes. LPL activity was reduced to approx. 50% of control in diabetic cardiomyocytes, but LPL mRNA levels and turnover (degradation) of newly synthesized LPL were unchanged. Synthesis of total protein and LPL were reduced to 72% and 71% of control respectively; therefore, relative rates of LPL synthesis were the same in control and diabetic cardiomyocytes. The diabetes-induced reduction in LPL synthesis was accompanied by a decrease in LPL mass to 78% of control, and a decrease in enzyme specific activity (0.48 to 0.33 m-unit/ng of LPL protein) since the decline in catalytic activity was greater than the decrease in LPL synthesis and mass. Thus, post-transcriptional mechanisms involving a reduction in LPL synthesis as part of a generalized decrease in total protein synthesis, together with a post-translational mechanism(s) that result in accumulation of inactive LPL protein, are responsible for the decreased LPL activity in cardiomyocytes from diabetic rat hearts.

Animals↗
PubMed1995

Characterization of a partially purified diacylglycerol lipase from bovine aorta.

A partially-purified diacylglycerol (DG) lipase from bovine aorta has been characterized with respect to the effects of lipid metabolites and two lipase inhibitors, phenylboronic acid and tetrahydrolipstatin (THL). DG lipase activity was determined by the hydrolysis of the sn-1 position of 1-[1-14C]palmitoyl-2-oleoyl-sn-glycerol. The products of the lipase reaction, 2-monoacylglycerol (2-monoolein) and non-esterified fatty acids (oleate, archidonate) produced a concentration-dependent (20-200 microM) inhibition of DG lipase activity. Oleoyl-CoA and dioleoylphosphatidic acid also inhibited aortic DG lipase activity, but lysophosphatidylcholine had little or no effect. The inhibition of aortic DG lipase by phenylboronic acid was competitive, with a Ki of approx. 4 mM. THL was a very potent inhibitor of aortic DG lipase; the concentration required for inhibition to 50% of control was 2-6 nM. THL inhibition was reduced when the concentration of substrate in the assay was increased. Attempts to identify the aortic DG lipase by covalent-labelling with [14C]THL were unsuccessful. Immunoblotting experiments revealed that hormone-sensitive triacylglycerol lipase (HSL) could not be detected in bovine aorta.

Animals↗
PubMed1995

Myocardial lipoprotein lipase activity: regulation by diabetes and fructose-induced hypertriglyceridemia.

The decrease in myocardial lipoprotein lipase (LPL) activity observed previously in acute, severe models of insulin-deficient diabetes may be a compensatory response to hypertriglyceridemia and a sustained increase in fatty acid delivery to cardiomyocytes. The administration of fructose (10% solution in the drinking water for 4 days) to rats produced hypertriglyceridemia, but heparin-releasable LPL activity from perfused hearts and total and heparin-releasable LPL activities in isolated cardiomyocytes were not reduced. The acute (4 day) induction of a mild diabetic state (60 mg/kg streptozotocin) resulted in modest hypertriglyceridemia, and a selective decrease in heparin-releasable LPL activity in perfused hearts; LPL activity in cardiomyocytes from diabetic rat hearts was not reduced. Therefore, the diabetes-induced fall in myocardial LPL activity is not secondary to hypertriglyceridemia, since fructose treatment did not change LPL activity. Perfusion of rat hearts with 100 microM lysophosphatidylcholine (LPC) released a small amount of LPL activity into the perfusate, but only if albumin was omitted from the perfusion solution. Thus, the selective reduction in heparin-releasable LPL activity in perfused diabetic hearts is probably not the consequence of displacement by LPC, a lipolytic product of the LPL-catalyzed degradation of triacylglycerol-rich lipoproteins. Circulating LPL activity in the plasma of diabetic rats was not decreased relative to control plasma enzyme activity; therefore, the reduction in heparin-releasable LPL activity is not because circulating LPL was less available for uptake by the endothelium in diabetic hearts.

Adipocytes↗
PubMed1995

Thyroid status and potassium currents in rat ventricular myocytes.

The size and rate dependence of the transient (Ito) and steady-state (Iss) outward potassium currents were investigated in isolated rat ventricular myocytes obtained from euthyroid, hyperthyroid, and hypothyroid rats, using the whole cell, suction electrode voltage-clamp method. Under hypothyroid conditions Ito was reduced in size, with no significant change in Iss. In hypothyroid cells, the rate dependence of both currents was greatly enhanced, resulting in a much larger attenuation with increasing stimulation rates. A significant slowing of the recovery kinetics of Ito was observed. These effects of hypothyroidism were reversed by physiological triiodothyronine (T3) replacement. These current changes were reflected in an altered rate dependence of the action potential configuration in hypothyroid myocytes. Under hyperthyroid conditions no significant changes were observed in the amplitude or time course of recovery of Ito. Iss amplitude was increased, but no changes were found in its rate dependence. These results are discussed in terms of hormonal, long-term modulation of potassium currents, and in terms of cardiac pathology under conditions of altered thyroid status.

Animals↗
PubMed1994

Partial purification of a diacylglycerol lipase from bovine aorta.

A diacylglycerol (DG) lipase has been purified from a soluble subcellular fraction of bovine aorta by (NH4)2SO4 precipitation in the presence of 5.0% (w/v) Triton X-100, followed by chromatography on DEAE-Sephacel, heparin-Sepharose and octyl-Sepharose in the presence of either CHAPS or Triton X-100 detergents. Under basal conditions, the hydrolysis of a short-chain [3H]dioctanoylglycerol ([3H]diC8) substrate was much greater than that of a long-chain 1-[1-14C]palmitoyl-2-oleoyl-sn-glycerol (1-[14C]POG) substrate. Lipase activity measured with 1-[14C]POG was markedly enhanced by Triton X-100. In the presence of 0.1% Triton X-100, specific enzyme activities in the octyl-Sepharose fraction determined with 1-[14C]POG or 1-stearoyl-2-[1-14C]-arachidonoyl-sn-glycerol as substrates were the same as that measured with [3H]diC8. MgCl2 (5mM) or CaCl2 (2 mM) also selectively stimulated lipase activity (up to 10-13-fold) measured with the long-chain (1-[14C]POG) substrate only. The increase in relative specific activity in the octyl-Sepharose fraction was 60-fold and 155-fold, based on hydrolysis of [3H]diC8 and 1-[14C]POG (+ Triton X-100), respectively. Unlabelled diC8 was a competitive inhibitor of 1-[14C]POG hydrolysis, suggesting that a single lipase hydrolyses both the short-chain and long-chain DG substrates; selective stimulatory effects of non-ionic detergents and bivalent cations on the hydrolysis of 1-[14C]POG may be due to effects on the physical properties of the substrate preparation. Monoacylglycerol lipase, DG kinase and cholesterol esterase activities could not be detected in the partially purified lipase preparation.

Animals↗
PubMed1994

Regulation of myocardial lipoprotein lipase activity by diabetes and thyroid hormones.

Administration of streptozotocin (100 mg/kg) to adult Sprague-Dawley rats reduced both functional (heparin releasable) lipoprotein lipase activity in perfused hearts and total and heparin-releasable lipoprotein lipase activity in isolated cardiomyocytes, and produced a hypothyroid state (decreased plasma levels of triiodothyronine and thyroxine). Administration of replacement doses of triiodothyronine (3 or 10 micrograms/kg for 3 days) to diabetic rats normalized heparin-releasable lipoprotein lipase activity in perfused hearts, but the depressed lipoprotein lipase activity in cardiomyocytes from diabetic hearts was unchanged by in vivo thyroid hormone treatment. However, hypothyroidism in thyroidectomized rats did not alter lipoprotein lipase activity in either perfused hearts or isolated cardiomyocytes. Therefore, thyroid hormones may interact with some other factor(s) in this acute, insulin-deficient model of diabetes to selectively regulate functional, heparin-releasable lipoprotein lipase activity in perfused hearts.

Animals↗
PubMed1994

Signal transduction in vascular smooth muscle: diacylglycerol second messengers and PKC action.

Agonist-stimulated phospholipid turnover can generate diacylglycerol (DAG), an intracellular second messenger that activates protein kinase C (PKC). DAG can be produced from the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by a phosphoinositide-specific phospholipase C and by the degradation of phosphatidylcholine (PC) by a phospholipase C or the concerted actions of phospholipase D and phosphatidate phosphohydrolase. In vascular smooth muscle, agonist-stimulated DAG accumulation is biphasic; PIP2 hydrolysis produces a transient increase in DAG, which is followed by a sustained phase of DAG accumulation from PC degradation. Metabolism of DAG attenuates PKC activation and thus results in signal termination. The metabolic fates for DAG include 1) ATP-dependent phosphorylation to form phosphatidic acid (DAG kinase), 2) hydrolysis to release fatty acids and glycerol (DAG and monoacylglycerol lipases), 3) synthesis of triacylglycerol (DAG acyltransferase), and 4) synthesis of PC (choline phosphotransferase). Hydrolysis through the lipase pathway is the predominant metabolic fate of DAG in vascular smooth muscle. Activation of PKC in vascular smooth muscle modulates agonist-stimulated phospholipid turnover, produces an increase in contractile force, and regulates cell growth and proliferation. Further research is required to investigate cross talk between signal transduction mechanisms involving lipid second messengers. In addition, spatial considerations such as nuclear PKC activation and the influence of diradylglycerol generation on the duration of PKC activation are important issues.

Animals↗
PubMed1993

Brain protein kinase C assay using MARCKS substrate reveals no translocation due to profound insulin-induced hypoglycemia.

Hypoglycemia sufficient to produce EEG isoelectricity or coma leads to neuronal death by an excitotoxic mechanism due to elevated extracellular levels of glutamate, aspartate and increased intracellular calcium. Since an elevated intracellular calcium concentration is known to translocate protein kinase C (PKC) from the cytosol to the membrane (a process thought to represent the in vivo activation of the enzyme), the objective of this investigation was to determine if calcium-dependent isoforms of PKC were translocated in specific brain regions of rats subjected to 40 min of insulin-induced hypoglycemic coma. The caudate nucleus and hippocampus (regions damaged by hypoglycemia showing extensive neuronal necrosis), and cerebellum (an undamaged, control region) of hypoglycemic rats were microdissected. Soluble and detergent (Triton X-100)-solubilized particulate fractions were partially purified by DEAE-Sephacel chromatography. PKC activity in both fractions was then measured using a novel assay based on the calcium- and lipid (phosphatidylserine and diolein)-dependent phosphorylation of the specific substrate myristoylated alanine rich C kinase substrate (MARCKS). The percentage distribution of PKC in the soluble and particulate (membrane-bound) fractions of all the brain regions from hypoglycemic rats was not significantly different from that in the control brains, indicating that 40 min of hypoglycemia does not result in PKC translocation as measured in subcellular fractions from brain tissue.

Animals↗
PubMed1993

Release of lipoprotein lipase from cardiac myocytes by low-molecular weight heparin.

Incubation of cardiac myocytes from rat heart with low-molecular weight heparin (LMWH; Mr approx. 3 kDa) for 30 min resulted in a concentration-dependent release of lipoprotein lipase (LPL) activity into the incubation medium. The release of lipoprotein lipase from cardiac myocytes isolated from both control and diabetic rat hearts induced by LMWH (10 micrograms/mL) following incubation times of 10 or 30 min was significantly greater than that produced by unfractionated heparin (10 and 30 micrograms/mL) or decavanadate (1 mM). Since LMWH released more LPL activity into the incubation medium than unfractionated heparin following a short (10 min) incubation time. LMWH is probably more effective in displacing LPL bound to heparan sulfate proteoglycan binding sites on the cell surface of cardiac myocytes.

Animals↗
PubMed1993

Acute diabetes does not reduce heparin-releasable lipoprotein lipase activity in perfused hearts from Wistar-Kyoto rats.

The induction of diabetes (3-5 days duration) in Wistar-Kyoto rats by the administration of streptozotocin (100 mg/kg) did not increase plasma concentrations of triacylglycerols or free fatty acids, and did not reduce heparin-releasable (functional) lipoprotein lipase activity in perfused hearts. By comparison, diabetic Sprague-Dawley rats were characterized as having hypertriglyceridemia and decreased heparin-releasable lipoprotein lipase activity in perfused hearts. Therefore, the diabetes-induced reduction in myocardial lipoprotein lipase activity in Sprague-Dawley rat hearts may, at least in part, be a compensatory response to the hypertriglyceridemia and increased fatty acid delivery to the myocardial cell, which is a characteristic feature of most severe, insulin-deficient models of diabetes mellitus. Although functional, endothelium-bound lipoprotein lipase activity was not reduced in diabetic perfused hearts from Wistar-Kyoto rats, cellular and heparin-releasable lipoprotein lipase activity was reduced in cardiac myocyte preparations, suggesting that other mechanisms in addition to plasma triacylglycerol must regulate lipoprotein lipase activity in the whole diabetic Wistar-Kyoto rat heart and that cardiac myocytes may not be the exclusive source of functional lipoprotein lipase in the diabetic myocardium.

Acute Disease↗
PubMed1993

Metabolism of a long-chain diacylglycerol by permeabilized A10 smooth muscle cells.

The regulatory effects of diacylglycerol (DAG) second messengers will be terminated by metabolism. A long-chain DAG, 1-palmitoyl-2-[1-14C]oleoyl-sn-glycerol (2-[14C]POG), was metabolized by cultured A10 smooth muscle cells after permeabilization by preincubation with 340 U/ml alpha-toxin from Staphylococcus aureus. In contrast to results with the cell-permeable DAG analogue, dioctanoyl-glycerol ([3H]diC8), no appreciable 2-[14C]POG degradation could be detected in control A10 cells not treated with alpha-toxin. With permeabilized A10 cells, 2-[14C]POG was mainly converted into lipolytic products of a lipase pathway, monoacylglycerol (MG) and fatty acid (FA); very little radioactivity was incorporated into triacylglycerol (TG) or phospholipid (PL) via reactions catalyzed by either DAG acyltransferase, cholinephosphotransferase, or DAG kinase. Similar results were obtained in experiments with 1-stearoyl-2-[1-14C]arachidonoyl-sn-glycerol. The conversion of 2-[14C]POG into PL and TG was not enhanced by the addition of 1 mM ATP-MgCl2, 1 mM CDP-choline, or 1 mM oleoyl-CoA to the alpha-toxin-treated A10 cells. The formation of FA and MG by permeabilized A10 cells was inhibited by DAG lipase inhibitors, U-57,908 (50 microM) and tetrahydrolipstatin (1-25 nM). The predominant contribution of the lipase pathway to the metabolism of a long-chain DAG, 2-[14C]POG, by alpha-toxin-treated A10 cells is similar to results for the degradation of [3H]diC8 by intact A10 cells.

Animals↗
PubMed1992

Comparison of an endogenous protein kinase C substrate in rat aorta with rat brain MARCKS.

We have compared the properties of a rat aorta-derived protein kinase C substrate (p75) with those of 80 kDa kinase C substrates from rat brain (MARCKS) and rabbit aorta (p80). Rat aortic p75 appeared to be closely related to rat brain MARCKS on the basis of: solubility in perchloric acid and trichloroacetic acid, heat stability, isoelectric point (pI approximately 4.2), overall V8 protease phosphopeptide map, and immunocrossreactivity with an antibody directed against the N-terminal domain of MARCKS. However, p75 could be distinguished from rat brain MARCKS and from the rabbit aorta-derived p80 on the basis of its consistently more rapid electrophoretic mobility in SDS-containing gels, and in terms of a unique proteolytic phosphopeptide found in MARCKS but not in aortic p75. We conclude that p75 probably belongs to the family of protein kinase C substrates represented by MARCKS, and that differences in post-translational processing (glycosylation) or mRNA processing may account for the unique properties of the p75 protein in rat aortic tissue.

Animals↗
PubMed1992

Long term incubation of cardiac myocytes with oleic acid and very-low density lipoprotein reduces heparin-releasable lipoprotein lipase activity.

An exogenous [3H]triolein emulsion was hydrolyzed by intact cardiac myocytes with functional LPL located on the cell surface. This surface-bound LPL could be released into the medium when cardiac myocytes were incubated with heparin. Incubation of cardiac myocytes with VLDL, or the products of TG breakdown, oleic acid or 2-monoolein, did not increase LPL activity in the medium. However, incubation of cardiac myocytes with either VLDL or oleic acid for > 60 min did reduce heparin-releasable LPL activity. In the heart, this inhibitory effect of FFA could regulate the translocation of LPL from its site of synthesis in the cardiac myocyte to its functional site at the capillary endothelium.

Animals↗
PubMed1992

Activation of lipoprotein lipase in cardiac myocytes by glycosylation requires trimming of glucose residues in the endoplasmic reticulum.

Incubation of cycloheximide-treated cardiac myocytes results in a time-dependent increase in cellular and heparin-releasable lipoprotein lipase (LPL) activities. N-Methyldeoxynojirimycin (1 mM) and castanospermine (100 micrograms/ml), inhibitors of glucosidases in the endoplasmic reticulum (ER), prevented the increase in cellular LPL activity. The glucosidase inhibitors did not influence the synthesis or turnover of LPL protein. Therefore activation of LPL by glycosylation in cardiac myocytes requires the trimming of glucose residues in oligosaccharide chains by glucosidases of the ER.

1-Deoxynojirimycin↗
PubMed1992

Synergistic actions of epidermal growth factor-urogastrone and vasopressin in cultured aortic A-10 smooth muscle cells.

In cultured rat aorta-derived A-10 cells, epidermal growth factor-urogastrone (EGF-URO) acts synergistically with arginine vasopressin (AVP) to augment the AVP-mediated release of 3H-arachidonate (3H-AA) from 3H-AA prelabeled cells. On its own, EGF-URO had no effect on AA release and had no effect on calcium influx or efflux either in the absence or presence of AVP. The synergistic action of EGF-URO was not affected by actinomycin D, cycloheximide, indomethacin, by the diacylglycerol lipase inhibitor U-57,908, or by the tyrosine kinase inhibitors genistein (GS) and tyrphostin (TP). TP did, nonetheless, completely abrogate 3H-thymidine incorporation triggered in the presence of EGF-URO. Although EGF-URO stimulated an increase in calpactin-II (lipocortin-I) phosphorylation in permeabilized cells, no such increase was detected in intact cells exposed to EGF-URO either alone or in combination with AVP, under conditions where EGF-URO augmented the action of AVP. The phospholipase A2 inhibitor, mepacrine, had no effect on AVP-mediated AA release, but abolished the synergistic action of EGF-URO. We conclude that in contrast with our previous results with gastric smooth muscle strips, wherein EGF-URO acts via the diacylglycerol lipase-mediated metabolism of diacylglycerol, and in keeping with observations with cultured mesangial cells, EGF-URO acts synergistically with AVP in A-10 cells via the activation of phospholipase A2. This synergistic action of EGF-URO does not appear to be due to increased levels of cyclooxygenase and would appear not to require increased tyrosine kinase activity.

Animals↗
PubMed1992

Inhibition of myocardial lipoprotein lipase by U-57,908 (RHC 80267).

U-57,908 (RHC 80267) was shown to inhibit lipoprotein lipase (LPL) activity in cardiac myocytes from rat hearts; the concentrations required for inhibition to 50% of control activity were 1.1 microM and 2.5 microM for myocyte homogenates and a post-heparin medium preparation, respectively. The inhibition of LPL activity by U-57,908 was not changed when the concentration of the triolein substrate and apolipoprotein CII activator in the assay was reduced. The availability of U-57,908 as a potent and selective LPL inhibitor may provide a useful experimental approach in studies on lipoprotein metabolism.

Animals↗