Mitochondrial glycerol phosphate acyltransferase directs the incorporation of exogenous fatty acids into triacylglycerol.

The mitochondrial isoform of glycerol-3-phosphate acyltransferase (GPAT), the first step in glycerolipid synthesis, is up-regulated by insulin and by high carbohydrate feeding via SREBP-1c, suggesting that it plays a role in triacylglycerol synthesis. To test this hypothesis, we overexpressed mitochondrial GPAT in Chinese hamster ovary (CHO) cells. When GPAT was overexpressed 3.8-fold, triacylglycerol mass was 2.7-fold higher than in control cells. After incubation with trace [(14)C]oleate ( approximately 3 microm), control cells incorporated 4.7-fold more label into phospholipid than triacylglycerol, but GPAT-overexpressing cells incorporated equal amounts of label into phospholipid and triacylglycerol. In GPAT-overexpressing cells, the incorporation of label into phospholipid, particularly phosphatidylcholine, decreased 30%, despite normal growth rate and phospholipid content, suggesting that exogenous oleate was directed primarily toward triacylglycerol synthesis. Transiently transfected HEK293 cells that expressed a 4.4-fold increase in GPAT activity incorporated 9.7-fold more [(14)C]oleate into triacylglycerol compared with control cells, showing that the effect of GPAT overexpression was similar in two different cell types that had been transfected by different methods. When the stable, GPAT-overexpressing CHO cells were incubated with 100 microm oleate to stimulate triacylglycerol synthesis, they incorporated 1.9-fold more fatty acid into triacylglycerol than did the control cells. Confocal microscopy of CHO and HEK293 cells transfected with the GPAT-FLAG construct showed that GPAT was located correctly in mitochondria and was not present elsewhere in the cell. These studies indicate that overexpressed mitochondrial GPAT directs incorporation of exogenous fatty acid into triacylglycerol rather than phospholipid and imply that (a) mitochondrial GPAT and lysophosphatidic acid acyltransferase produce a separate pool of lysophosphatidic acid and phosphatidic acid that must be transported to the endoplasmic reticulum where the terminal enzymes of triacylglycerol synthesis are located, and (b) this pool remains relatively separate from the pool of lysophosphatidic acid and phosphatidic acid that contributes to the synthesis of the major phospholipid species.

Diacylglycerol generated in CHO cell plasma membrane by phospholipase C is used for triacylglycerol synthesis.

The diacylglycerol (DAG) signal generated from membrane phospholipids by hormone-activated phospholipases is attenuated by mechanisms that include lipolysis or phospholipid resynthesis. To determine whether the DAG signal might also be terminated by incorporation of DAG into triacylglycerol (TAG), we studied the direct formation of TAG from endogenous DAG generated by bacterial phospholipase C (PLC). When Chinese hamster ovary (CHO) cells prelabeled with [(14)C]oleate were treated with PLC from Clostridium perfringens for 6 h, [(14)C]phospholipid decreased 15% and labeled TAG increased 60%. This transfer of (14)C label was even greater when the cells were simultaneously exposed to PLC and 100 microM oleic acid. PLC as well as oleate treatment concomitantly increased the TAG mass within the cell. Moreover, when phospholipids were prelabeled with [(3)H]glycerol, a subsequent increase in [(3)H]TAG indicated that an intact DAG moiety was channeled into the TAG structure. Incubating CHO cells with the diacylglycerol kinase inhibitor R59022 enhanced the formation of TAG from phospholipids hydrolyzed by PLC or by PLC in the presence of 100 microM oleate, but not by incubation with oleate alone, indicating that the DAG released from plasma membrane phospholipids does not require the formation of a phosphatidic acid precursor for TAG synthesis. Similarly, the diacylglycerol lipase inhibitor RHC 80267 did not alter TAG synthesis from plasma membrane DAG, further supporting direct incorporation of DAG into TAG. These studies indicate that DAG derived from plasma membrane phospholipid is largely used for TAG formation, and support the view that this mechanism can terminate DAG signals. The studies also suggest that a transport mechanism exists to move plasma membrane-derived DAG to the endoplasmic reticulum.-Igal, R. A., J. M. Caviglia, I. N. T. de Gómez Dumm, and R. A. Coleman. Diacylglycerol generated in CHO cell plasma membrane by phospholipase C is used for triacylglycerol synthesis. J. Lipid Res. 2001. 42: 88;-95.

Neutral lipid storage disease: a genetic disorder with abnormalities in the regulation of phospholipid metabolism.

Neutral lipid storage disease (NLSD) is an autosomal recessive disorder characterized by the presence of numerous lipid droplets in virtually all tissues examined. The increased cellular triacylglycerol content results from defective recycling of triacylglycerol-derived diacylglycerol to phospholipids (Igal, R. A. and R. A. Coleman. 1996. J. Biol. Chem. 271: 16644-16651). In order to determine whether de novo glycerolipid synthesis is also altered in NLSD, we compared the ability of normal human skin fibroblasts and fibroblasts from a patient with NLSD to incorporate phospholipid precursors into cell lipids. NLSD cells had increased rates of incorporation of [14C]oleic acid and [3H]glycerol into triacylglycerol and all phospholipid species except phosphatidylethanolamine. However, the cell content of each phospholipid species was similar in control and NLSD cells, indicating a higher turnover rate in NLSD cells for phosphatidylcholine, phosphatidylinositol, phosphatidylserine, and sphingomyelin. Labeling with [14C]choline and [14C]ethanolamine confirmed the increase in the rate of phosphatidylcholine synthesis and the decreased rate of phosphatidylethanolamine synthesis through their respective CDP pathways. The activities of the major regulatory enzymes of triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine biosynthesis were similar in control and NLSD cells. Taken as a whole, this study provides strong evidence for an underlying regulatory defect in NLSD that alters the rates of synthesis and degradation of the major cellular phospholipids.

Early lipid alterations in spontaneously diabetic rats.

Human and experimental diabetes mellitus extensively alters lipid metabolism. The eSS is a rat strain that develops a spontaneous diabetes of slow evolution, resembling the non-insulin-dependent diabetes mellitus of young people. We report here disturbances in lipid metabolism of 5-month old eSS rats compared to age-matched alpha-controls. Normal plasmatic glucose levels were found in the fasted state, whereas a diabetic curve was evident for eSS rats after glucose load. Triglyceride content was elevated in plasma and in liver microsomal preparations of eSS animals, when compared to the controls. The diabetic strain revealed a significant fall in the amount of linoleic acid in liver and kidney microsomes and in erythrocyte membranes. In liver, an increase in 22:6 (n-3) was also noted. A depression in the content of linoleic acid as well as an enhancement of docosahexaenoic acid were detected in phosphatidylcholine and phosphatidylethanolamine fractions from liver microsomes of eSS rats. The fatty acid pattern of eSS rat testis showed a raise in the relative percentage of arachidonic and a decrease in 22:5 (n-6), 22:5 (n-3) and 22:6 (n-3) acids compared to their controls. Diabetic rats exhibited a significant increase in microsomal cholesterol content and cholesterol/phospholipid ratio in liver and testis. In the latter tissue, higher values of fluorescence anisotropy were also observed. The current observations indicate that in early stages of the diabetes onset, when eSS rats are still normoglycemic, severe alterations of lipid metabolism may contribute to the establishment and progression of the diabetic syndrome.

Modulation of rat liver lipid metabolism by prolactin.

The effect of chronic hyperprolactinemia on the delta6- and delta5-desaturation activity, total lipid and fatty acid composition, as well as fluorescence anisotropy, was studied in liver microsomes from anterior pituitary-grafted rats. We observed a depression in delta6-desaturation activity but no changes in the delta5-desaturation activity in the grafted animals. The microsomal fraction from the hyperprolactinemic rats contained significantly less amount of linoleic acid and a higher content of 20:4 n-6, 22:5 n-6 and 22:6 n-3 acids. Lipid rotational mobility was increased in microsomes as well as in liposomes obtained from the microsomes of transplanted animals. The fluidifying effect induced by PRL was located in the deepest zone of the membrane. The results obtained indicate that high levels of prolactin induce changes in polyunsaturated fatty acid distribution in liver microsomes, which regulates the lipid rotational mobility and hence membrane fluidity.

Early lipid alterations in spontaneously diabetic rats

Human and experimental diabetes mellitus extensively alters lipid metabolism. The eSS is a rat strain that develops a spontaneous diabetes of slow evolution, resembling the non-insulin-dependent diabetes mellitus of young people. We report here disturbances in lipid metabolism of 5-month old eSS rats compared to age-matched alpha-controls. Normal plasmatic glucose levels were found in the fasted state, whereas a diabetic curve was evident for eSS rats after glucose load. Triglyceride content was elevated in plasma and in liver microsomal preparations of eSS animals, when compared to the controls. The diabetic strain revealed a significant fall in the amount of linoleic acid in liver and kidney microsomes and in erythrocyte membranes. In liver, an increase in 22:6 (n-3) was also noted. A depression in the content of linoleic acid as well as an enhancement of docosahexaenoic acid were detected in phosphatidylcholine and phosphatidylethanolamine fractions from liver microsomes of eSS rats. The fatty acid pattern of eSS rat testis showed a raise in the relative percentage of arachidonic and a decrease in 22:5 (n-6), 22:5 (n-3) and 22:6 (n-3) acids compared to their controls. Diabetic rats exhibited a significant increase in microsomal cholesterol content and cholesterol/phospholipid ratio in liver and testis. In the latter tissue, higher values of fluorescence anisotropy were also observed. The current observations indicate that in early stages of the diabetes onset, when eSS rats are still normoglycemic, severe alterations of lipid metabolism may contribute to the establishment and progression of the diabetic syndrome.

Early lipid alterations in spontaneously diabetic rats

Human and experimental diabetes mellitus extensively alters lipid metabolism. The eSS is a rat strain that develops a spontaneous diabetes of slow evolution, resembling the non-insulin-dependent diabetes mellitus of young people. We report here disturbances in lipid metabolism of 5-month old eSS rats compared to age-matched alpha-controls. Normal plasmatic glucose levels were found in the fasted state, whereas a diabetic curve was evident for eSS rats after glucose load. Triglyceride content was elevated in plasma and in liver microsomal preparations of eSS animals, when compared to the controls. The diabetic strain revealed a significant fall in the amount of linoleic acid in liver and kidney microsomes and in erythrocyte membranes. In liver, an increase in 22:6 (n-3) was also noted. A depression in the content of linoleic acid as well as an enhancement of docosahexaenoic acid were detected in phosphatidylcholine and phosphatidylethanolamine fractions from liver microsomes of eSS rats. The fatty acid pattern of eSS rat testis showed a raise in the relative percentage of arachidonic and a decrease in 22:5 (n-6), 22:5 (n-3) and 22:6 (n-3) acids compared to their controls. Diabetic rats exhibited a significant increase in microsomal cholesterol content and cholesterol/phospholipid ratio in liver and testis. In the latter tissue, higher values of fluorescence anisotropy were also observed. The current observations indicate that in early stages of the diabetes onset, when eSS rats are still normoglycemic, severe alterations of lipid metabolism may contribute to the establishment and progression of the diabetic syndrome. (AU)

Triacsin C blocks de novo synthesis of glycerolipids and cholesterol esters but not recycling of fatty acid into phospholipid: evidence for functionally separate pools of acyl-CoA.

The trafficking of acyl-CoAs within cells is poorly understood. In order to determine whether newly synthesized acyl-CoAs are equally available for the synthesis of all glycerolipids and cholesterol esters, we incubated human fibroblasts with [14C]oleate, [3H]arachidonate or [3H]glycerol in the presence or absence of triacsin C, a fungal metabolite that is a competitive inhibitor of acyl-CoA synthetase. Triacsin C inhibited de novo synthesis from glycerol of triacylglycerol, diacylglycerol and cholesterol esters by more than 93%, and the synthesis of phospholipid by 83%. However, the incorporation of oleate or arachidonate into phospholipids appeared to be relatively unimpaired when triacsin was present. Diacylglycerol acyltransferase and lysophosphatidylcholine acyltransferase had similar dependences on palmitoyl-CoA in both liver and fibroblasts; thus it did not appear that acyl-CoAs, when present at low concentrations, would be preferentially used to acylate lysophospholipids. We interpret these data to mean that, when fatty acid is not limiting, triacsin blocks the acylation of glycerol 3-phosphate and diacylglycerol, but not the reacylation of lysophospholipids. Two explanations are possible: (1) different acyl-CoA synthetases exist that vary in their sensitivity to triacsin; (2) an independent mechanism channels acyl-CoA towards phospholipid synthesis when little acyl-CoA is available. In either case, the acyl-CoAs available to acylate cholesterol, glycerol 3-phosphate, lysophosphatidic acid and diacylglycerol and those acyl-CoAs that are used by lysophospholipid acyltransferases and by ceramide N-acyltransferase must reside in two non-mixing acyl-CoA pools or, when acyl-CoAs are limiting, they must be selectively channelled towards specific acyltransferase reactions.

Acylglycerol recycling from triacylglycerol to phospholipid, not lipase activity, is defective in neutral lipid storage disease fibroblasts.

Neutral lipid storage disease (NLSD) is an autosomal recessive disorder in which excess triacylglycerol (TG) accumulates in most cells. Although it has been hypothesized that the TG accumulation is caused by a functional defect in cytosolic lipase activity, we were able to expose TG hydrolysis in NLSD cells by using triacsin C, an inhibitor of acyl-CoA synthetase that blocks the reincorporation of hydrolyzed fatty acids into glycerolipids. Our data suggest that TG lipolysis in NLSD cells is masked by rapid TG resynthesis, occurring because released acylglycerols cannot be used for phospholipid synthesis. In uptake studies, triacsin C blocked the incorporation of [3H]glycerol into glycerolipids, incorporation of [14C]oleate into TG, but not incorporation of [14C]oleate into phospholipid. Thus, the drug inhibited both de novo synthesis of glycerolipids via the glycerol-3-phosphate pathway and the synthesis of TG from diacylglycerol. The drug did not appear to block reacylation of lysophospholipids. Triacsin C caused a loss of about 60% of the TG mass from both NLSD and oleate-loaded control cells. Rates of TG lipolysis were similar in NLSD cells and oleate-loaded control cells labeled with [6-(7-nitro-2,1,3-benzoxadiazol-4-yl)-amino]hexanoic acid or labeled with [14C]oleate or [3H]glycerol and chased in the presence of triacsin C. During a 96-h chase, [14C]oleate reincorporation into the different phospholipid species increased only in control cells. Similar results were observed when NLSD, and control cells were chased after labeling with [3H]glycerol. These data strongly suggest that normal human fibroblasts mobilize stored TG for phospholipid synthesis and that recycling to PC occurs via a TG-derived mono- or diacylglycerol intermediate. Normal recycling to phosphatidylethanolamine may primarily involve TG-derived acyl groups rather than an acylglycerol precursor. NLSD cells appear to have a block in this recycling pathway with the result that both hydrolyzed fatty acids and the acylglycerol backbone are re-esterified to form TG. Because the NLSD phenotype includes ichthyosis, fatty liver, myopathy, cardiomyopathy, and mental retardation, the recycling pathway appears to be critical for the normal function of skin, liver, muscle, heart, and the central nervous system.

Abnormal metabolism of polyunsaturated fatty acids in adrenal glands of diabetic rats.

Studies carried out on the adrenal glands of experimental diabetic rats have shown an important inhibition in polyenoic fatty acid biosynthesis. This effect was demonstrated by testing the activities of long-chain fatty acyl-CoA synthetase, the delta 5- and delta 6-desaturases of the (n-6) essential fatty-acid series and the delta 6-desaturase of the (n-3) series in liver and adrenal microsomes. The depression in desaturating activity in the insulin-deprived animals was independent of that produced on acyl-CoA-thioester biosynthesis. Experiments measuring the incorporation and transformation of [1-14C]eicosa-8,11,14-trienoic acid in adrenocortical cells isolated from streptozotocin-diabetic animals demonstrated a significant inhibition of arachidonic acid biosynthesis compared to controls. Insulin injections in diabetic rats partially restored the delta 5- and delta 6-desaturase activities. This effect could result from direct action by the hormone since the restoration was reproduced when arachidonic acid biosynthesis was measured after insulin was added to the incubation medium of adrenocortical cells isolated from diabetic animals. The results of the present study provide new information about the implication of this abnormal metabolism in the adrenal gland of diabetic rats.