Characterization of mono-, di- and triacylglycerol lipase activities in the isolated rat heart

Stam, H.; Broekhoven-Schokker, S.; Hülsmann, W.C.

doi:10.1016/0005-2760(86)90013-5

Cited by 32 publications

(6 citation statements)

References 40 publications

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“…From diethyl-ether-anaesthetized rats, the hearts were quickly excised and perfused retrogradely in an open system according to Langendorff at a constant pressure of 80 cmH2O to remove the contained blood, as described elsewhere [31]. In a number of experiments low-flow ischaemia was induced by decreasing the coronary flow to 1 ml/min during 30 min, exactly as described elsewhere [5].…”

Section: Perfusion Protocolmentioning

confidence: 99%

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Properties of phosphatidate phosphohydrolase and diacylglycerol acyltransferase activities in the isolated rat heart. Effect of glucagon, ischaemia and diabetes

Schoonderwoerd

Broekhoven-Schokker

Hülsmann

et al. 1990

Biochemical Journal

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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.

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Section: Perfusion Protocolmentioning

confidence: 99%

“…Subcellular fractions were obtained as described in ref. [31]. Pellets of the subcellular fractions obtained were suspended in 0.25 M-sucrose/ mM-EDTA/1 mM-Tris/HCI, pH 7.4, and stored at -80°C until use.…”

Section: Perfusion Protocolmentioning

confidence: 99%

Properties of phosphatidate phosphohydrolase and diacylglycerol acyltransferase activities in the isolated rat heart. Effect of glucagon, ischaemia and diabetes

Schoonderwoerd

Broekhoven-Schokker

Hülsmann

et al. 1990

Biochemical Journal

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“…DG kinase has been purified to homogeneity from brain [20,21]. By comparison, most studies on DG lipase [6,10,[22][23][24] (2 mM) also selectively stimulated lipase activity (up to 10-13fold) 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.…”

Section: Introductionmentioning

confidence: 99%

Partial purification of a diacylglycerol lipase from bovine aorta

Lee

Severson

1994

Biochemical Journal

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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.

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“…At different time intervals the effluent samples were collected for measuring glycerol release. At the end of the experiments the hearts were removed from the apparatus and homogenized in 0.25 M sucrose, 10 mM Tris/HCl, 1 mM EDTA, pH 7.4 (38). In some experiments the hearts were perfused with 14C-glucose (1.66 mCi/mmol) during the ischemic period.…”

Section: Perfusion Protocolmentioning

confidence: 99%

“…TG lipase activity was measured using a [3H]-trioleirggum acacia emulsion as previously described (34,38). Protein content was determined by the biuret method (8), using bovine serum albumin as standard.…”

Section: Biochemical Assaysmentioning

confidence: 99%

Enhanced lipolysis of myocardial triglycerides during low-flow ischemia and anoxia in the isolated rat heart

et al. 1989

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We studied lipolysis in the isolated rat heart, measured as glycerol release during anoxia, low-flow ischemia and subsequent reperfusion. It was found that the rate of lipolysis was enhanced during ischemia/anoxia while the lipase activities in tissue extracts involved in the myocardial lipolysis and the amount of triglycerides were not affected. This indicates the dominant occurrence of a lipolysis-reesterification principle in ischemic and anoxic tissue. A common observation of ischemia/anoxia is an increase in the tissue NADH/NAD+ ratio. Therefore we investigated the effect of lactate and malate, both of which enhance the tissue redox state on myocardial lipolysis. Perfusion in the presence of lactate (10 mM) and malate (10 mM) both stimulated myocardial lipolysis by about five times. This suggests that the rate of reesterification of product fatty acids to triglycerides, which is determined by the NADH/NAD+ ratio, because of the increased formation of glycerol 3-phosphate from dihydroxy acetone phosphate, plays an important role in the regulation of lipolysis. The existence of triglyceride-fatty acid-triglyceride cycle is discussed.

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Characterization of mono-, di- and triacylglycerol lipase activities in the isolated rat heart

Cited by 32 publications

References 40 publications

Properties of phosphatidate phosphohydrolase and diacylglycerol acyltransferase activities in the isolated rat heart. Effect of glucagon, ischaemia and diabetes

Properties of phosphatidate phosphohydrolase and diacylglycerol acyltransferase activities in the isolated rat heart. Effect of glucagon, ischaemia and diabetes

Partial purification of a diacylglycerol lipase from bovine aorta

Enhanced lipolysis of myocardial triglycerides during low-flow ischemia and anoxia in the isolated rat heart

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