Purification of liver cytosolic proteins that stimulate triacylglycerol synthesis

1981 ◽  
Vol 59 (11-12) ◽  
pp. 944-950 ◽  
Author(s):  
Daniel A. K. Roncari ◽  
Esther Y. W. Mack
Keyword(s):  
Molecular Weight ◽  
Adipose Tissue ◽  
Diacylglycerol Acyltransferase ◽  
Microsomal Enzymes ◽  
Acyltransferase Activity ◽  
Cytosolic Proteins ◽  
Heat Stable ◽  
Triacylglycerol Synthesis ◽  
Phosphatidate Phosphohydrolase ◽  
Liver Microsomal Enzymes

Heat-stable rat liver cytosolic proteins that stimulate triacylglycerol synthesis catalyzed by adipose tissue or liver microsomal enzymes have been isolated in a highly purified state. Their molecular weight ranges were found to be 35 000 – 45 000, 20 000 – 28 000, and 8000 – 12 000. The protein with molecular weight 20 000 – 28 000 and pI 7.3–7.7 was purified 4716-fold. The cytosolic proteins were stable at 85 °C for 15 min and were inactivated by proteases. They did not reveal any intrinsic phosphatidate phosphohydrolase or diacylglycerol acyltransferase activity, but led to enhanced conversion of phosphatidate to diacylgiycerol and triacylglycerol.

scholarly journals The activities of lipoprotein lipase and of enzymes involved in triacylglycerol synthesis in rat adipose tissue. Effects of starvation, dietary modification and of corticotropin injection

1981 ◽  
Vol 200 (2) ◽  
pp. 285-294 ◽  
Author(s):  
N Lawson ◽  
A D Pollard ◽  
R J Jennings ◽  
M I Gurr ◽  
D N Brindley
Keyword(s):  
Adipose Tissue ◽  
Lipoprotein Lipase ◽  
Corn Oil ◽  
Diacylglycerol Acyltransferase ◽  
Dietary Modification ◽  
Dihydroxyacetone Phosphate ◽  
Glycerol Phosphate ◽  
Triacylglycerol Synthesis ◽  
Phosphatidate Phosphohydrolase ◽  
Starch Diet

1. The effects of dietary modification, including starvation, and of corticotropin injection on the activities of acyl-CoA synthetase, glycerol phosphate acyltransferase, dihydroxyacetone phosphate acyltransferase, phosphatidate phosphohydrolase, diacylglycerol acyltransferase and lipoprotein lipase were measured in adipose tissue. 2. Lipoprotein lipase activities in heart were increased and those in adipose tissue were decreased when rats were fed on diets enriched with corn oil or beef tallow rather than with sucrose or starch. The lipoprotein lipase activity was lower in the adipose tissue of rats fed on the sucrose rather than on the starch diet. 3. Rats fed on the beef tallow diet had slightly higher activities of the total glycerol phosphate acyltransferase in adipose tissue than did rats fed on the sucrose or starch diet. The diacylglycerol acyltransferase and the mitochondrial glycerol phosphate acyltransferase activities were higher for the rats fed on the tallow diet than for those fed on the corn-oil diet. 4. Starvation significantly decreased the activities of lipoprotein lipase (after 24 and 48 h), acyl-CoA synthetase (after 24 h) and of the mitochondrial glycerol phosphate acyltransferase and the N-ethylmaleimide-insensitive dihydroxyacetone phosphate acyltransferase (after 48 h) in adipose tissue. The activities of the microsomal glycerol phosphate acyltransferase, diacylglycerol acyltransferase and the soluble phosphatidate phosphohydrolase were not significantly changed after 24 or 48 h of starvation. 5. The activities of lipoprotein lipase and phosphatidate phosphohydrolase in adipose tissue were decreased 15 min after corticotropin was injected into rats during November to December. No statistically significant differences were found when these experiments were performed during March to September. These differences may be related to the seasonal variation in acute lipolytic responses. 6. These results are discussed in relation to the control of triacylglycerol synthesis and lipoprotein metabolism.

Enhancement of microsomal phosphatidate phosphohydrolase and diacylglycerol acyltransferase activity by insulin during growth of rat adipocyte precursors in culture

1979 ◽  
Vol 57 (6) ◽  
pp. 573-577 ◽  
Author(s):  
Daniel A. K. Roncari ◽  
Esther Y. W. Mack ◽  
Dominic K. Yip
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Diacylglycerol Acyltransferase ◽  
Culture Conditions ◽  
Acyltransferase Activity ◽  
Intact Cells ◽  
Triacylglycerol Synthesis ◽  
Phosphatidate Phosphohydrolase ◽  
Morphological Maturation ◽  
Adipocyte Precursor

The availability of a propagating adipocyte precursor culture system has provided the opportunity to study biochemical processes under conditions in which any known interacting influences are controlled. We have studied the activity of various triacylglycerol-biosynthetic enzymes during maturation of rat epididymal adipocyte precursors and any possible effect of insulin on enzyme activity. At certain times in culture, the specific activity of microsomal phosphatidate phosphohydrolase (EC 3.1.3.4) and diacylglycerol acyltransferase (EC 2.3.1.20) is significantly enhanced in cells grown in the presence of added insulin. Under the culture conditions used in this study, the adipocyte precursors acquire several small lipid inclusions and become rounder, but do not assume the signet-ring appearance of mature fat cells during the first 2 weeks of monolayer confluence. Consequently, the effects of the hormone on enzyme activity become evident prior to complete morphological maturation. Phosphatidate phosphohydrolase is believed to be a rate-controlling enzyme in triacylglycerol synthesis in adipose tissue and liver. The fact that the adipocyte precursor microsomal, rather than cytosolic, phosphohydrolase is influenced by insulin suggests that the membrane-bound enzyme is the regulatory phosphohydrolase in intact cells. The enhancement of diacylglycerol acyltransferase activity may be of significance in the reesterification of fatty acids with diacylglycerols, a reaction that by passes the phosphohydrolase step. Thus, in addition to the well-known mechanisms by which insulin promotes triacylglycerol accretion in adipocytes and their precursors, the hormone significantly enhances the specific activity of critical enzymes of triacylglycerol synthesis.

scholarly journals Effects of streptozotocin-diabetes and insulin administration in vivo or in vitro on the activities of five enzymes in the adipose-tissue triacylglycerol-synthesis pathway

1987 ◽  
Vol 243 (1) ◽  
pp. 289-292 ◽  
Author(s):  
E D Saggerson ◽  
C A Carpenter
Keyword(s):  
Insulin Treatment ◽  
Streptozotocin Diabetes ◽  
Diacylglycerol Acyltransferase ◽  
Fatty Acyl ◽  
Diabetic Rats ◽  
Insulin Administration ◽  
Triacylglycerol Synthesis ◽  
Phosphatidate Phosphohydrolase

At 2 days after administration of streptozotocin (100 mg/kg), activities in rat epididymal fat-pads of the following enzymes were significantly decreased: fatty acyl-CoA synthetase (FAS), mitochondrial and microsomal forms of glycerolphosphate acyltransferase (GPAT), monoacylglycerolphosphate acyltransferase (MGPAT) and Mg2+-dependent phosphatidate phosphohydrolase (PPH). There were no significant changes in diacylglycerol acyltransferase or Mg2+-independent PPH. Insulin administration to diabetic rats over 2 days restored activities of FAS, both forms of GPAT, MGPAT and Mg2+-dependent PPH. Significant restoration of all five activities was also seen 2 h after a single administration of insulin, but was not observed 45 min after insulin treatment. Insulin significantly increased all five enzyme activities when adipocytes from diabetic rats were incubated for 2 h with a mixture of glucose, lactate, pyruvate and amino acids.

Diacylglycerol acyltransferase activity and triacylglycerol synthesis in germinating castor seed cotyledons

Lipids ◽  
2006 ◽  
Vol 41 (3) ◽  
pp. 281-285 ◽  
Author(s):  
Xiaohua He ◽  
Grace Q. Chen ◽  
Jiann-Tsyh Lin ◽  
Thomas A. McKeon
Keyword(s):  
Diacylglycerol Acyltransferase ◽  
Castor Seed ◽  
Acyltransferase Activity ◽  
Triacylglycerol Synthesis

Adipose tissue diacylglycerol acyltransferase activity and blood lipoprotein triglyceride enrichment in women with abdominal obesity

2014 ◽  
Vol 233 (1) ◽  
pp. 172-177 ◽  
Author(s):  
Julie Anne Côté ◽  
Mélanie Nadeau ◽  
Mathieu Leboeuf ◽  
Line Blackburn ◽  
André Tchernof
Keyword(s):  
Adipose Tissue ◽  
Abdominal Obesity ◽  
Diacylglycerol Acyltransferase ◽  
Acyltransferase Activity

scholarly journals Adipose-tissue Mg2+-dependent phosphatidate phosphohydrolase. Control of activity and subcellular distribution in vitro and in vivo

1986 ◽  
Vol 239 (2) ◽  
pp. 275-284 ◽  
Author(s):  
S J Taylor ◽  
E D Saggerson
Keyword(s):  
Adipose Tissue ◽  
Subcellular Distribution ◽  
Relative Proportion ◽  
Streptozotocin Diabetes ◽  
Filtration Method ◽  
Triacylglycerol Synthesis ◽  
Phosphatidate Phosphohydrolase

The subcellular distribution of Mg2+-dependent phosphatidate phosphohydrolase in rat adipocytes between a soluble and a membrane-bound fraction was measured by using both centrifugal fractionation and a novel Millipore-filtration method. The relative proportion of the phosphohydrolase associated with the particulate fraction was increased on incubation of cells with noradrenaline or palmitate. Insulin on its own decreased the proportion of the phosphohydrolase that was particulate and abolished the effect of noradrenaline, but not that of palmitate. The effect of noradrenaline on phosphohydrolase distribution was rapid, the effect being maximal within 10 min. Noradrenaline exerted this effect with a similar concentration-dependence to its lipolytic effect. Inclusion of albumin in homogenization buffers decreased the proportion of the phosphohydrolase that was particulate, but did not abolish the effect of noradrenaline. There was limited correlation between the proportion of the phosphohydrolase that was particulate and the measured rate of triacylglycerol synthesis in adipocytes incubated under a variety of conditions. Starvation, streptozotocin-diabetes and hypothyroidism decreased the specific activities of the phosphohydrolase and glycerolphosphate acyltransferase in homogenates from epididymal fat-pads. Restoration of these activities in the diabetic state was seen after administration of insulin over 2 days or, in the short term, within 2 h after a single administration of insulin. Administration of thyroxine over 3 days caused restoration of these activities in the hypothyroid state. Starvation and diabetes increased the proportion of the phosphohydrolase found in the microsomal fraction. This change was not seen when albumin was present in homogenization buffers. The possible role of fatty acids as regulators of the intracellular translocation of the phosphohydrolase, together with the role of this enzyme in the regulation of triacylglycerol synthesis in adipose tissue, is discussed.

Triacylglycerol synthesis and diacylglycerol acyltransferase activity during skeletal myogenesis

1990 ◽  
Vol 68 (12) ◽  
pp. 1393-1401 ◽  
Author(s):  
Victor S. Sauro ◽  
Kenneth P. Strickland
Keyword(s):  
Fatty Acid ◽  
Unsaturated Fatty Acids ◽  
Diacylglycerol Acyltransferase ◽  
Skeletal Myogenesis ◽  
Acyltransferase Activity ◽  
Triacylglycerol Synthesis ◽  
Tag Accumulation ◽  
Long Chain

The role that diacylglycerol acyltransferase (DAGAT) may play in the switch in lipid metabolism from predominantly triacylglycerol- and phospholipid-synthesizing myoblasts to predominantly phospholipid-synthesizing myotubes has been studied during L6 skeletal myogenesis. Fatty acid induced triacylglycerol (TAG) accumulation in vivo was found to be optimal with long-chain, unsaturated fatty acids. The fatty acid induced TAG accumulation was significantly greater in myoblasts than that in myotubes. DAGAT activity in vitro was found to be associated with the particulate (membrane) fraction only. The inhibition by many thiol-specific reagents (N-ethylmaleimide, p-chloromercuribenzoate, iodoacetate, 5,5′-dithiobis(2-nitrobenzoic acid)) suggest that a thiol group is at or near the active site. In general, optimal DAGAT activity in vitro was observed when long-chain unsaturated acyl-CoAs and diacylglycerols (DAGs) containing long acyl chains were used as substrates for in vitro TAG synthesis (although 1,2-didecanoin was also very effective). DAGAT activity (expressed relative to DNA) was shown to decline over twofold during skeletal myogenesis when measured in the absence of exogenous DAG. However, in the presence of exogenous (1 mM) DAG, there was no significant change in DAGAT activity, suggesting that the levels of this enzyme are not altered during skeletal myogenesis. These results indicate that endogenous DAG levels are limiting TAG synthesis in L6 myotubes. However, DAG content of myotubes was significantly greater than that of myoblasts, suggesting that there may be an increased competition for DAG (perhaps owing to enhanced phospholipid synthesis) during skeletal myogenesis. The combined effects of decreased synthesis and increased degradation (reported earlier) of TAG may account for the decrease in endogenous TAG contents observed during skeletal myogenesis.Key words: diacylglycerol acyltransferase, TAG synthesis, skeletal myogenesis.

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

1976 ◽  
Vol 158 (2) ◽  
pp. 327-334 ◽  
Author(s):  
S C Jamdar ◽  
D Shapiro ◽  
H J Fallon
Keyword(s):  
Adipose Tissue ◽  
Microsomal Fraction ◽  
Soluble Fraction ◽  
Diacylglycerol Acyltransferase ◽  
Obese Mouse ◽  
Obese Mice ◽  
Triacylglycerol Biosynthesis ◽  
Phosphatidate Phosphohydrolase ◽  
Membrane Bound

Obesity in obese-hyperglycaemic mouse is associated with an increase in number and size of adipocytes. Adipocytes from the obese mouse showed increased incorporation of [14C]acetate and[14C]glucose into triacylglycerol. This increased capacity of triacylglycerol formation was correlated with increased activities of various triacylglycerol-forming enzymes measured in the microsomal fraction of adipose tissue from obese mice. Microsomal fractions from lean and obese mice contained sn-glycerol 3-phosphate acyltransferase, phosphatidate phosphohydrolase and diacylglycerol acyltransferase. Phosphatidate phosphohydrolase was also detected in the soluble fraction. In the presence of Mg2+, the phosphatidate phsophohydrolase from the soluble and the microsomal fractions was active towards membrane-bound phosphatidate. Among the three enzymes studied here, the increase in Mg2+-dependent phosphatidate phosphohydrolase was most prominent in adipose tissue of obese mice.

scholarly journals Glycerolipid biosynthesis in rat adipose tissue. Influence of adipocyte size

1981 ◽  
Vol 194 (1) ◽  
pp. 293-298 ◽  
Author(s):  
S C Jamdar ◽  
L J Osborne ◽  
J A Zeigler
Keyword(s):  
Adipose Tissue ◽  
Subcutaneous Fat ◽  
Diacylglycerol Acyltransferase ◽  
Adipocyte Size ◽  
Filtration Method ◽  
Fat Depots ◽  
Lipid Formation ◽  
Phosphatidate Phosphohydrolase ◽  
One Step ◽  
Rat Adipose Tissue

A simple one-step filtration method is described to separate larger adipocytes from the smaller ones by using nylon screen (52 microM pore size). Adipocytes retained on the screen were larger (60-90 micrometers) compared with those that passed through the screen. By using this separation technique, activities of various enzymes involved in triacylglycerol formation from sn-glycerol 3-phosphate were measured in the larger and smaller adipocytes isolated from gonadal fat-depots. The homogenates from larger adipocytes were more active in lipid formation compared with those derived from small adipocytes. This was evident from the increased activities of sn-glycerol 3-phosphate acyltransferase. Mg2+-dependent phosphatidate phosphohydrolase and diacylglycerol acyltransferase in the larger adipocytes. The activities of these enzymes were also measured in the adipocytes isolated from gonadal, perirenal and subcutaneous fat-depots. Subcutaneous adipocytes were smaller and were less active in lipid formation than gonadal and perirenal adipocytes. These measurements in the activities of individual enzymes provide evidence that the entire pathway of esterification via sn-glycerol 3-phosphate is accelerated in the larger adipocytes.

Sign in / Sign up

Export Citation Format

Share Document