Tritium isotope effects in the measurement of the glycerol phosphate and dihydroxyacetone phosphate pathways of glycerolipid biosynthesis in rat liver

1973 ◽  
Vol 134 (4) ◽  
pp. 833.b1-833.b1
Keyword(s):  
Rat Liver ◽  
Isotope Effects ◽  
Dihydroxyacetone Phosphate ◽  
Glycerol Phosphate

scholarly journals Tritium isotope effects in the measurement of the glycerol phosphate and dihydroxyacetone phosphate pathways of glycerolipid biosynthesis in rat liver

1972 ◽  
Vol 130 (4) ◽  
pp. 1003-1012 ◽  
Author(s):  
Ray Manning ◽  
David N. Brindley
Keyword(s):  
Neutral Lipid ◽  
Rat Liver ◽  
Isotope Effects ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Dihydroxyacetone Phosphate ◽  
Glycerol Concentration ◽  
Glycerol Phosphate ◽  
Experimental Conditions ◽  
Liver Slices

1. Rat liver slices were employed to study the relative rates of incorporation of a mixture of [2-3H]- or [1,3-3H]-glycerol and [1-14C]glycerol into lipids. 2. With 0.1mm-glycerol approx. 82% of the newly synthesized lipid, calculated from 14C incorporation, was present as neutral lipid, 13% as phosphatidylcholine and 5% as phosphatidylethanolamine. Increasing the glycerol concentration to 40mm caused a decrease in the percentage of neutral lipid to 59% and a corresponding increase in the percentage of phosphatidylcholine to 36% of the newly synthesized lipid. 3. The (d.p.m. of 2-3H)/(d.p.m. of 1-14C) ratio in glycerolipid was considerably higher than that in precursor glycerol throughout the range of experimental conditions. In contrast the incorporation of a mixture of [1,3-3H]glycerol and [1-14C]glycerol into lipid occurred with little or no change in the 3H/14C ratio. 4. Respiring rat liver mitochondria were found to oxidize a mixture of sn-[2-3H]- and sn-[1-14C]-glycerol 3-phosphate with a resultant increase in the 3H/14C ratio of the remaining sn-glycerol 3-phosphate. This increase is due to a 3H isotope effect of the mitochondrial sn-glycerol 3-phosphate dehydrogenase (EC 1.1.99.5), which discriminates against sn-[2-3H]glycerol 3-phosphate during oxidation. 5. A method is described for the simultaneous determination of the relative contributions of the glycerol phosphate and dihydroxyacetone phosphate pathways of glycerolipid biosynthesis in rat liver slices. The method involves measurement of the (d.p.m. of 2-3H)/(d.p.m. of 1-14C) ratio in both sn-glycerol 3-phosphate and glycerolipid after incubation of rat liver slices with a mixture of [2-3H]glycerol and [1-14C]glycerol for various times. 6. By using this method it was shown that 40–50% of the glycerol incorporated into lipid by rat liver slices proceeded via the sn-glycerol 3-phosphate pathway and 50–60% was incorporated via dihydroxyacetone phosphate.

scholarly journals A study of the glycerol phosphate acyltransferase and dihydroxyacetone phosphate acyltransferase activities in rat liver mitochondrial and microsomal fractions. Relative distribution in parenchymal and non-parenchymal cells, effects of N-ethylmaleimide, palmitoyl-coenzyme A concentration, starvation, adrenalectomy and anti-insulin serum treatment

1979 ◽  
Vol 182 (3) ◽  
pp. 751-762 ◽  
Author(s):  
E J Bates ◽  
E D Saggerson
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Dihydroxyacetone Phosphate ◽  
Acute Administration ◽  
Relative Distribution ◽  
Glycerol Phosphate ◽  
Mitochondrial Fractions ◽  
Different Response ◽  
Parenchymal Cells ◽  
Post Mitochondrial Supernatant

1. GPAT (glycerol phosphate acyltransferase) and DHAPAT (dihydroxyacetone phosphate acyltransferase) activities were measured both in subcellular fractions prepared from fed rat liver and in whole homogenates prepared from freeze-stopped pieces of liver. 2. GPAT activity in mitochondria differed from the microsomal activity in that it was insensitive to N-ethylmaleimide, had a higher affinity towards the palmitoyl-CoA substrate and showed a different response to changes in hormonal and dietary status. 3. Starvation (48 h) significantly decreased mitochondrial GPAT activity. The ratio of mitochondrial to microsomal activities was also significantly decreased. The microsomal activity was unaffected by starvation, except after adrenalectomy, when it was significantly decreased. Mitochondrial GPAT activity was decreased by adrenalectomy in both fed and starved animals. 4. Acute administration of anti-insulin serum significantly decreased mitochondrial GPAT activity after 60 min without affecting the microsomal activity. 5. A new assay is described for DHAPAT. The subcellular distribution of this enzyme differed from that of GPAT. The highest specific activity of DHAPAT was found in a 23 000 gav. pellet obtained by centrifugation of a post-mitochondrial supernatant. This fraction also contained the highest specific activity of the peroxisomal marker uricase. DHAPAT activity in mitochondrial fractions or in the 23 000 gav. pellet was stimulated by N-ethylmaleimide, whereas that in microsomal fractions was slightly inhibited by this reagent. The GPAT and DHAPAT activities in mitochondrial fractions had a considerably higher affinity for the palmitoyl-CoA substrate. 6. Total liver DHAPAT activity was significantly decreased by starvation (48 h), but was unaffected by administration of anti-insulin serum. 7. The specific activities of GPAT and DHAPAT were lower in non-parenchymal cells compared with parenchymal cells, but the GPAT/DHAPAT ratio was 5–6-fold higher in the parenchymal cells.

scholarly journals The tritium isotope effect of sn-glycerol 3-phosphate oxidase and the effects of clofenapate and N-(2-benzoyloxyethyl)norfenfluramine on the esterification of glycerol phosphate and dihydroxyacetone phosphate by rat liver mitochondria

1973 ◽  
Vol 136 (2) ◽  
pp. 421-427 ◽  
Author(s):  
Mariana Bowley ◽  
Ray Manning ◽  
David N. Brindley
Keyword(s):  
Isotope Effect ◽  
Rat Liver ◽  
Lipid Synthesis ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Dihydroxyacetone Phosphate ◽  
Glycerol Phosphate ◽  
Simultaneous Synthesis

1. Owing to a 3H isotope effect, the mitochondrial sn-glycerol 3-phosphate oxidase (EC 1.1.99.5) had a mean activity which was 8.4 times less with sn-[2-3H]-rather than with sn-[1-14C]glycerol 3-phosphate as a substrate. 2. A method for measuring the simultaneous synthesis of lipid from glycerol phosphate and dihydroxyacetone phosphate in rat liver mitochondria is described. 3. The lipid synthesized by rat liver mitochondria from sn-[1-14C]glycerol 3-phosphate was mainly phosphatidate and lysophosphatidate, whereas that synthesized from dihydroxy[1-14C]acetone phosphate was mainly acyldihydroxyacetone phosphate. 4. Additions of NADPH facilitated the conversion of acyldihydroxyacetone phosphate into lysophosphatidate and phosphatidate. 5. Hydrazine (1.4mm) or KCN (1.4mm) inhibited the synthesis of lipids from dihydroxyacetone phosphate but not from glycerol phosphate. 6. Clofenapate (1–2.5mm) inhibited the synthesis of lipids from dihydroxyacetone phosphate but slightly stimulated synthesis from glycerol phosphate. 7. The methanesulphonate of N-(2-benzoyloxyethyl)norfenfluramine, at 0.25–0.75mm, inhibited lipid synthesis from both glycerol phosphate and dihydroxyacetone phosphate.

Effect of acidosis on skeletal muscle metabolism with and without propranolol

1990 ◽  
Vol 68 (7) ◽  
pp. 870-876
Author(s):  
J. K. Barclay ◽  
T. E. Graham ◽  
B. R. Wolfe ◽  
J. Van Duk ◽  
B. A. Wilson
Keyword(s):  
Skeletal Muscle ◽  
Muscle Metabolism ◽  
Inhibitory Effect ◽  
Metabolic Effects ◽  
Dihydroxyacetone Phosphate ◽  
Glycerol Phosphate ◽  
Muscle Lactate ◽  
Skeletal Muscle Metabolism ◽  
Glycogen Concentration ◽  
Glycolytic Intermediates

Does the stimulatory effect of circulating catecholamines counteract the inhibitory effect of acidosis on skeletal muscle metabolism? To investigate this possibility, we studied gastrocnemii in dogs breathing either air (n = 10) or 4% carbon dioxide in air (n = 10) at rest and during contractions. In five dogs from each group, we infused propranolol into the arterial supply of the right and left muscles for 40 min. After 30 min of infusion, the left muscle was stimulated at 3 Hz for 10 min. During the 10th min of contractions, we removed and froze both muscles in liquid nitrogen. Oxygen uptake and blood flow to the left muscle prior to or during stimulation was not affected by acidosis either with or without propranolol. Glycogen concentration in resting muscle was unaffected by acidosis with or without propranolol. There was an acidosis related decrease of approximately 50% in the glycolytic intermediates (glucose 6-phosphate, fructose 1,6-diphosphate, α-glycerol phosphate, and dihydroxyacetone phosphate) in unstimulated muscles without β-blockade. At rest, acidosis decreased muscle lactate by 50% with and 64% without propranolol, but lactate release was decreased only with acidosis without propranolol (1.4–0.1 μmol/kg∙s). Acidosis without propranolol had no effect on the changes in glycogen concentration or the change in the concentration of glycolytic intermediates resulting from contractions. In β-blocked muscle, the difference between stimulated and unstimulated concentrations of glycogen and glycolytic intermediates including lactate was 20–50% smaller with acidosis. Thus, with β-blockade, the acidotic effects at rest disappeared and an inhibition of the metabolic adjustment to contractions appeared, indicating that circulating catecholamines do modify some metabolic effects of acidosis.Key words: oxidative muscle, glycogen, lactate efflux, glycolytic intermediates, β-blockade.

scholarly journals The effects of diet on the esterification of glycerol phosphate, dihydroxyacetone phosphate and 2-hexadecylglycerol by homogenates of rat adipose tissue

1976 ◽  
Vol 160 (3) ◽  
pp. 701-706 ◽  
Author(s):  
P F Dodds ◽  
D N Brindley ◽  
M I Gurr
Keyword(s):  
Adipose Tissue ◽  
Beef Tallow ◽  
Corn Oil ◽  
Specific Activity ◽  
Body Weight Gain ◽  
Male Rats ◽  
Dihydroxyacetone Phosphate ◽  
Glycerol Phosphate ◽  
Triacylglycerol Content ◽  
Rat Adipose Tissue

1. Male rats were fed for 5 weeks after weaning on a diet containing (by weight) 59% of starch or on diets that contained 39% of starch and 20% of either sucrose, beef tallow or corn oil. 2. The rats fed on the beef tallow consumed more energy than did the rats fed on the starch and sucrose diets. The rats fed on the corn oil drank less water than did the other groups of rats. 3. There were no significant differences between the four groups in terms of body-weight gain, epididymal-fat-pad weight and in the size, number and triacylglycerol content of the adipocytes in the fat-pads. 4. There was a significant correlation (P < 0.001) between the activities of glycerol phosphate acyltransferase and monoacylglycerol acyltransferase in individual rats. Both of these activities were highest in the group fed on the high-starch diet and both correlated with the consumption of glucose by individual rats in the four groups. 5. The percentage of glycerol phosphate converted into diacylglycerol and triacylglycerol was positively correlated with the mean diameters, surface area and triacylglycerol content of the adipocytes for individual rats and was greates in the sucrose-fed rats. 6. The specific activity of dihydroxyacetone phosphate acyltransferase was highest in the rats fed on beef tallow. This activity was positively correlated with the energy intake for all dietary groups over the 5-week feeding period. 7. The results are discussed in terms of the functions of the three routes of glycerolipid synthesis in adipose tissue.

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