Two Forms of Microsomal Palmitoyl‐Coenzyme A Synthetase

1. Adipocytes were isolated from the interscapular brown fat and the epididymal white fat of normal, streptozotocin-diabetic and hypothyroid rats. 2. Measurements were made of the maximum rate of triacylglycerol synthesis by monitoring the incorporation of [U-14C]glucose into acylglycerol glycerol in the presence of palmitate (1 mM) and insulin (4 nM) and of the activities of the following triacylglycerol-synthesizing enzymes: fatty acyl-CoA synthetase (FAS), mitochondrial and microsomal forms of glycerolphosphate acyltransferase (GPAT), dihydroxyacetonephosphate acyltransferase (DHAPAT), monoacylglycerol phosphate acyltransferase (MGPAT), Mg2+-dependent phosphatidate phosphohydrolase (PPH) and diacylglycerol acyltransferase (DGAT). 3. FAS activity in brown adipocytes was predominantly localized in the mitochondrial fraction, whereas a microsomal localization of this enzyme predominated in white adipocytes. Subcellular distributions of the other enzyme activities in brown adipocytes were similar to those shown previously with white adipocytes [Saggerson, Carpenter, Cheng& Sooranna (1980) Biochem. J. 190, 183-189]. 4. Relative to cell DNA, brown adipocytes had lower activities of triacylglycerol-synthesizing enzymes and showed lower rates of metabolic flux into acylglycerols than did white adipocytes isolated from the same animals. 5. Diabetes decreased both metabolic flux into acylglycerols and the activities of triacylglycerol-synthesizing enzymes in white adipocytes. By contrast, although diabetes decreased metabolic flux into brown-adipocyte acylglycerols by 80%, there were no decreases in the activities of triacylglycerol-synthesizing enzymes, and the activity of PPH was significantly increased. 6. Hypothyroidism increased metabolic flux into acylglycerols in both cell types, and increased activities of all triacylglycerol-synthesizing enzymes in brown adipocytes. By contrast, in white adipocytes, although hypothyroidism increased the activities of FAS, microsomal GPAT and DGAT, this condition decreased the activities of mitochondrial GPAT and PPH. 7. It was calculated that the maximum capabilities for fatty acid oxidation and esterification are approximately equal in brown adipocytes. In white adipocytes esterification is predominant by approx. 100-fold. 8. Diabetes almost abolished incorporation of [U-14C]glucose into fatty acids in both adipocyte types. Hypothyroidism increased fatty acid synthesis in white and brown adipocytes by 50% and 1000% respectively.

Section: Enzyme Activities: Effects Of Diabetesmentioning

confidence: 99%

Comparison of triacylglycerol synthesis in rat brown and white adipocytes. Effects of hypothyroidism and streptozotocin-diabetes on enzyme activities and metabolic fluxes

Baht

Saggerson

1988

“…(a) Palmitoyl-CoA synthetase. This was assayed by two different methods, each of which was based on those described by Brandes et al (1973). In the first assay, which measures total palmitoyl-CoA synthetase activity, palmitoyl-CoA was generated from palmitate, CoA, ATP and Mg2+.…”

Section: Enzyme Assaysmentioning

confidence: 99%

“…of 0.2M-potassium phosphate buffer, pH17.4, and 1 vol. of a solution of 0.54guM-KOH in 0.4M-potassium phosphate buffer, pH6.8 (Brandes et al, 1973). Then 50,1 of 10mM-5,5'-dithiobis-(2-nitrobenzoic acid) in 0.2M-potassium phosphate buffer, pH7.4, was added and the A413 of the solution was measured immediately.…”

Section: Enzyme Assaysmentioning

confidence: 99%

Co-ordinate changes in enzymes of fatty acid synthesis, activation and esterification in rabbit mammary gland during pregnancy and lactation

Short¹,

Brindley²,

Dils³

1977

1. The activities of fatty acid synthetase, acyl-CoA synthetase, glycerol phosphate acyltransferase and phosphatidate phosphatase were measured in the mammary glands of rabbits from day 16 of pregnancy to day 15 of post partum. 2. There were significant correlations between the increases in activities of these enzymes during this period. This was the case whether the activities were expressed per mg of homogenate protein, per g wet wt. of tissue or per total wet weight of the whole glands. The only exception was the lack of correlation between the activities of fatty acid synthetase and of phosphatidate phosphatase per g wet wt. of tissue. 3. These co-ordinate increases are discussed in relation to the changes which occur in fatty acid metabolism in the mammary gland during pregnancy and lactation.

“…The activities of the total microsomal acyl-CoA synthetase (EC 6.2.1.3) and of glycerol phosphate acyltransferase do respond to some physiological stimuli, but the activities do not always correlate well with the overall rates of triacylglycerol synthesis, and the changes, when observed at all, are usually smaller than those for the soluble phosphatidate phosphohydrolase (Fallon & Kemp, 1968;Vavrecka et al, 1969;Aas & Daae, 1971;Mangiapane et al, 1973;Wiegand et al, 1973;Brandes et al, 1973;Lamb & Fallon, 1974;Fallon et al, 1977;; Table 2). The activity of the soluble phosphatidate phosphohydrolase can change rapidly in response to physiological stimuli and parallels the altered rate of triacylglycerol synthesis (Mangiapane et al, 1973;Lamb & Fallon, 1974;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.