Triacylglycerol biosynthesis in the adipose tissue of the obese-hyperglycaemic mouse

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.

Section: Determination Ofprotein and Radioactivitymentioning

confidence: 99%

Problems encountered in measuring the activity of phosphatidate phosphohydrolase

Sturton¹,

Brindley²

1978

“…This is seen after feeding fructose (Lamb & Fallon, 1974;Fallon et al, 1977) and ethanol Vol. 168 and in genetic obesity (Jamdar et al, 1976;Fallon et al, 1977).…”

Section: Resultsmentioning

confidence: 99%

The effect of chronic diabetes, induced by streptozotocin, on the activities of some enzymes of glycerolipid synthesis in rat liver

1978

1. Rats were injected with a single dose of 35mg of streptozotocin/kg body wt. They exhibited a diabetes that was characterized by glycosuria, polyuria, polydipsia, hyperphagia, hyperglycaemia, increased concentrations of unesterified fatty acids, glycerol and triacylglycerols in the serum and an increased activity of glucose 6-phosphatase in the liver. 2. After 10 weeks the hepatic activities of the microsomal glycerol phosphate acyltransferase, phosphatidate phosphohydrolase, phosphatidate cytidylyltransferase, diacylglycerol acyltransferase, choline phosphotransferase, CDP-diacylglycerol-inositol phosphatidyltransferase and the soluble phosphatidate phosphohydrolase were measured. 3. The only significant changes were an increase in the activity of the soluble phosphatidate phosphohydrolase and a decrease in that of the CDP-diacylglycerolinositol phosphatidyltransferase in the diabetic rats. 4. These results are discussed in relation to the control of glycerolipid synthesis.Diabetes mellitus is characterized by hyperglycaemia, which is often accompanied by glycosuria, and by an increase in the consumption of food and water and in urine output. In severe cases there is ketonaemia, ketonuria and hypertriglyceridaemia. The last is usually caused by an accelerated secretion of very-low-density lipoprotein, and the severity of the hypertriglyceridaemia can be taken as an indication of the adequacy of the control of the diabetes (Nikkila & Kekki, 1973;Nikkila, 1974).In the present study rats were made chronically diabetic by a single injection of streptozotocin. These animals were then used to investigate whether the diabetic state is accompanied by changes in the relative activities of the enzymes responsible for the synthesis ofglycerolipids in the liver. It is hoped that these results will add to our understanding of how glycerolipid synthesis is controlled.

“…The extent of the Mg2+ stimulation in cytosolic fractions has normally been 4-8-fold [18,21,22,24]. The Mg2+-dependency of PAP in microsomal fractions is normally much lower, if it is observed at all [21,23,24].…”

Section: Effects Of Chlorpromazine On Pap Activitymentioning

confidence: 99%

“…In liver [18][19][20], adipose tissue [21,22], lung [23] and other tissues, the cytosolic PAP exhibits a fairly high stimulation with Mg2+. By contrast, PAP activity in microsomal and in other particulate fractions is increased to a smaller extent by Mg2+ [21,23,24]. This observation raises the questions: (a) Alternatively, the phosphatidate was dispersed in chloroform/methanol/water (5:4:1, by vol.)…”

Section: Introductionmentioning

confidence: 99%

A rapid assay for measuring the activity and the Mg2+ and Ca2+ requirements of phosphatidate phosphohydrolase in cytosolic and microsomal fractions of rat liver

Ltd¹

1987

1. A rapid extraction and purification scheme was designed for the recovery of [3H]diacylglycerol formed during the assay of phosphatidate phosphohydrolase. 2. The importance of removing polyvalent cations, particularly Ca2 , from the phosphatidate and other reagents used in the assay of the phosphohydrolase activity was demonstrated. This was achieved mainly by treating the phosphatidate with a chelating resin and by adding 1 mM-EGTA and 1 mM-EDTA to the assays. 3. The activity of the phosphohydrolase in dialysed samples of the soluble and microsomal fractions of rat liver was very low. 4. Addition of optimum concentrations of MgCl2 resulted in a 110-167-fold stimulation in activity. 5. CaCl2 was also able to stimulate phosphohydrolase activity, but to a much smaller extent than MgCl2. 6. Chlorpromazine, an amphiphilic cation, inhibited the reaction when it was measured in these experiments by using a mixed emulsion of phosphatidylcholine and phosphatidate at pH 7.4. 7. Microsomal fractions that were preincubated with albumin contained very low activities of the Mg2+-dependent phosphohydrolase. When these were then incubated with the soluble fraction in the presence of oleate, the soluble phosphohydrolase attached to the microsomal membranes, and it retained its high dependency on Mg2+.