Effects of ethanol feeding on hepatic lipid synthesis

Rats were fed a high-fat, liquid diet containing either 36% of total calories as ethanol or an isocaloric amount of sucrose, for a period up to 35 days. At different time intervals we measured the effects of ethanol administration on the activities of a number of key enzymes involved in hepatic lipid synthesis. At the start of the experimental period the activities of acetyl-CoA carboxylase and fatty acid synthase, measured in liver homogenates, increased in the control as well as in the ethanol-fed group. After 35 days these enzyme activities were still elevated but there were no significant differences between the two groups. In hepatocytes isolated from controls as well as from ethanol-fed rats, short-term incubations with ethanol induced an increase in the rate of fatty acid synthesis and in the activities of acetyl-CoA carboxylase and fatty acid synthase. However, no alterations in the regulation of these enzymes by short-term modulators of lipogenesis were apparent in hepatocytes isolated from alcohol-treated animals. The results do not indicate a major role for the enzymes of de novo fatty acid synthesis in the development of the alcoholic fatty liver. The amount of liver triacylglycerols increased in ethanol-fed rats during the entire treatment period, whereas the hepatic levels of phosphatidylcholine and phosphatidylethanolamine were not affected by ethanol ingestion. Ethanol administration for less than 2 weeks increased the activities of phosphatidate phosphohydrolase, diacylglycerol acyltransferase, and microsomal phosphocholine cytidylyltransferase, whereas the cytosolic activity of phosphocholine cytidylyltransferase was slightly decreased. Upon prolonged ethanol administration the activities of these enzymes were slowly restored to control values after 35 days, suggesting development of some kind of adaptation. It is interesting that, although the activities of phosphatidate phosphohydrolase and diacylglycerol acyltransferase were restored to the levels found in the control rats, this effect was not accompanied by a stabilization or decrease of the concentration of hepatic triacylglycerols.     

Effects of vasopressin on the synthesis of phosphatidylethanolamines and phosphatidylcholines by isolated rat hepatocytes

The effect of vasopressin on the biosynthesis of phosphatidylcholines and phosphatidylethanolamines was investigated in freshly isolated rat hepatocytes in suspension. Treatment of hepatocytes with vasopressin inhibits the incorporation of [Me-14C]choline into phosphatidylcholines in a dose-dependent manner. The hormone does not affect the uptake, phosphorylation or oxidation of choline. Pulse-chase studies indicate that CTP:cholinephosphate cytidylyltransferase might be subject to hormonal regulation by vasopressin. In contrast with the inhibitory effect of vasopressin on the synthesis of phosphatidylcholines, this hormone stimulates the incorporation of [1,2-14C]ethanolamine into phosphatidylethanolamines in a dose-dependent manner. Pulse and pulse-chase studies with labelled ethanolamine show that the conversion of ethanolaminephosphate to CDPethanolamine as well as the formation of phosphatidylethanolamines from CDPethanolamine and diacylglycerol are enhanced. Determination of the effect of vasopressin on the activity of the enzymes of the synthesis de novo of phosphatidylethanolamines demonstrates an increase of the activity of ethanolaminephosphotransferase, probably as a result of the increased amount of diacylglycerol in vasopressin-treated cells.     

Cholinephosphotransferase and Diacylglycerol Acyltransferase (Substrate Specificities at a Key Branch Point in Seed Lipid Metabolism)

Many oilseed plants accumulate triacylglycerols that contain unusual fatty acyl structures rather than the common 16- and 18-carbon fatty acids found in membrane lipids of these plants. In vitro experiments demonstrate that triacylglycerols are synthesized via diacylglycerols in microsomal preparations and that this same sub-cellular fraction is the site for the synthesis of phosphatidylcholine, which in seeds is synthesized from diacylglycerol by CDP-choline: diacylglycerol cholinephosphotransferase. In microsomes from Cuphea lanceolata, a plant that accumulates fatty acids with 10 carbons and no double bonds (10:0) in its oil, the diacylglycerol acyltransferase exhibited 4-fold higher activity with 10:0/10:0 molecular species of diacylglycerol than with molecular species containing 18-carbon fatty acids. In castor bean (Ricinus communis), which accumulates oil containing ricinoleic acid, diricinoleoyldiacylglycerol was the favored substrate for triacylglycerol synthesis. In contrast to these modest specificities of the diacylglycerol acyltransferases, the cholinephosphotransferases from these plants and from safflower (Carthamus tinctorius) and rapeseed (Brassica napus) showed little or no specificity across a range of different diacylglycerol substrates. Consideration of these results and other data suggests that the targeting of unusual fatty acids to triacylglycerol synthesis and their exclusion from membrane lipids are not achieved on the basis of the diacylglycerol substrate specificities of the enzymes involved and may instead require the spatial separation of two different diacylglycerol pools.     

Effect of 1-oleoyl-2-acetylglycerol and other lipids on phosphatidylcholine synthesis and cholinephosphate cytidylyltransferase activity in cultured type II pneumocytes

We previously reported that addition of phosphatidylglycerol to the culture medium stimulates phosphatidylcholine synthesis and cholinephosphate cytidylyltransferase activity in type II pneumocytes. In view of the known biological effects of diacylglycerols and since phosphatidylglycerol could be metabolized to diacylglycerol, we now examined the effects of diacylglycerols on the same parameters. The rate of choline incorporation into phosphatidylcholine was increased 30-60% by 10 microM phosphatidylglycerol, diolein, mixed diacylglycerols and 1-oleoyl-2-acetylglycerol (OAG). The effects of phosphatidylglycerol and OAG were not additive, suggesting a similar mechanism of action. The diacylglycerols and phosphatidylglycerol increased the activity of cholinephosphate cytidylyltransferase in type II cell sonicates by 35-50%, but had no effect on the activities of choline kinase, cholinephosphotransferase or 1-acylglycerophosphocholine acyltransferase. Again, the effects of OAG and phosphatidylglycerol on cytidylyltransferase were not additive. It is known that addition of lipids to the assay mixture increases the activity of cholinephosphate cytidylyltransferase in vitro and inclusion of the above lipids (1.1 mM) in the in vitro assay mixture increased cytidylyltransferase activity in type II cell sonicates. In addition, the stimulatory effects of OAG and of diolein, as well as of phosphatidylglycerol as reported previously, in the culture medium on cytidylyltransferase activity in type II cells were diminished or abolished when the assay was carried out in the presence of sufficient amounts of the same lipids to stimulate maximally the activity in vitro. These data show that lipids in the culture medium stimulate phosphatidylcholine biosynthesis in type II cells by direct activation of cholinephosphate cytidylyltransferase.     

Effect of eicosapentaenoic acid on triacylglycerol transport in CaCo-2 cells

The human intestinal cell line, CaCo-2, was used to study the effect of the n-3 fatty acid, eicosapentaenoic acid, on triacylglycerol secretion. In cells incubated with 250 microM eicosapentaenoic acid, the incorporation of [3H]glycerol into triacylglycerols secreted into the medium was decreased by 58% compared to cells incubated with 250 microM oleic acid. The incorporation of [3H]glycerol into cellular triacylglycerols was decreased 32% in cells incubated with eicosapentaenoic acid. In cells preincubated with [3H]glycerol to label existing triacylglycerols, the rates of secretion of preformed triacylglycerols were similar in response to the addition of either fatty acid. Initial uptake rates of the n-3 fatty acid were higher than for oleic acid. Both eicosapentaenoic acid and oleic acid were minimally oxidized to CO2. Oleic acid was predominantly incorporated into cellular triacylglycerols (62% vs. 47%), whereas more eicosapentaenoic acid was incorporated into cellular phospholipids (46% vs. 30%). Phospholipids of microsomes prepared from cells incubated with eicosapentaenoic acid were enriched in this fatty acid. The rate of synthesis of triacylglycerol and diacylglycerol acyltransferase activities were significantly less in microsomes prepared from cells incubated with eicosapentaenoic acid. Triacylglycerol mass secreted by CaCo-2 cells incubated with either fatty acid was similar. In CaCo-2 cells, eicosapentaenoic acid decreases the synthesis and secretion of newly synthesized triacylglycerol without decreasing the secretion of triacylglycerol mass. Modification of microsomal membrane phospholipid fatty acid composition is associated with a decrease in microsomal triacylglycerol synthesis and diacylglycerol acyltransferase activities.     

Seasonal changes in critical enzymes of lipogenesis and triacylglycerol synthesis in the marmot (Marmota flaviventris)

Fatty acid metabolism and triacylglycerol synthesis are critical processes for the survival of hibernating mammals that undergo a prolonged fasting period. Fatty acid synthase, fatty-acid-CoA ligase, diacylglycerol acyltransferase, and monoacylglycerol acyltransferase activities were measured in liver and in white and brown adipose tissue, in order to determine whether enzymes of lipogenesis and triacylglycerol synthesis vary seasonally during hibernation in the yellow-bellied marmot (Marmota flaviventris). Compared with mid-winter hibernation, fatty acid synthase activity was higher in all three tissues during early spring when marmots emerged from hibernation and in mid-summer when they were feeding, consistent with the synthesis of fatty acids from the carbohydrate-rich summer diet. Fatty-acid-CoA ligase and diacylglycerol acyltransferase activities were highest in summer in white adipose tissue when triacylglycerol synthesis would be expected to be high; diacylglycerol acyltransferase activity was also high in brown adipose tissue during spring and summer. In liver, however, diacylglycerol acyltransferase specific activity was highest during hibernation, suggesting that triacylglycerol synthesis may be prominent in liver in winter. Monoacylglycerol acyltransferase activity, which may aid in the retention of essential fatty-acids, was 80-fold higher in liver than in white or brown adipose tissue, but did not vary seasonally. Its dependence on palmitoyl-CoA suggests that a divalent cation might play a role in enzyme activation. The high hepatic diacylglycerol acyltransferase activity during hibernation suggests that the metabolism of very low density lipoprotein may be important in the movement of adipose fatty acids to brown adipose tissue and muscle during the rewarming that occurs periodically during hibernation. These studies suggest that enzymes of lipid metabolism vary seasonally in the marmot, consistent with requirements of this hibernator for triacylglycerol synthesis and metabolism.Abbreviations BAT brown adipose tissue - DGAT diacylglycerol acyltransferase - FAS fatty acid synthase - K _m Michaelis constant - MGAT monoacylglycerol acyltransferase - RQ respiratory quotiant - VLDL very low density lipoprotein - WAT white adipose tissue     

Regulation of triacylglycerol synthesis in the liver: a decrease in diacylglycerol acyltransferase activity after treatment of isolated rat hepatocytes with glucagon

Isolated rat hepatocytes were used to investigate the possibility of a short-term effect of glucagon on the synthesis of triacylglycerols in the liver. Incubation of hepatocytes in the presence of glucagon, followed by homogenization in a buffer containing F- (50 mM) and EDTA (2.5 mM), resulted in a 53% decrease in activity of microsomal diacylglycerol acyltransferase (EC 2.3.1.20), the only enzyme that is exclusively involved in the synthesis of triacylglycerols. The activity of cholinephosphotransferase (EC 2.7.8.2), which also uses diacylglycerols as substrate, was not decreased after exposure of the hepatocytes to glucagon. This may imply that triacylglycerol synthesis can be regulated independently of phosphatidylcholine synthesis. The activity of diacylglycerol acyltransferase in microsomes isolated from a homogenate of whole liver could be reduced by preincubating the microsomes with Mg2+ (5 mM), ATP (1 mM) and 105 000 X g supernatant. The enzyme could be reactivated by incubation of the washed microsomes with a 105 000 X g supernatant in the presence of dithiothreitol (5 mM). Fluoride (50 mM) inhibited this reactivation. It is concluded that the activity of diacylglycerol acyltransferase is subject to hormonal short-term control, possibly via a phosphorylation-dephosphorylation mechanism.     

Activities of acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT) in microsomal preparations of developing sunflower and safflower seeds

The last step in triacylglycerols (TAG) biosynthesis in oil seeds, the acylation of diacylglycerols (DAG), is catalysed by two types of enzymes: the acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT). The relative contribution of these enzymes in the synthesis of TAG has not yet been defined in any plant tissue. In the presented work, microsomal preparations were obtained from sunflower and safflower seeds at different stages of development and used in DGAT and PDAT enzyme assays. The ratio between PDAT and DGAT activity differed dramatically between the two different species. DGAT activities were measured with two different acyl acceptors and assay methods using two different acyl-CoAs, and in all cases the ratio of PDAT to DGAT activity was significantly higher in safflower than sunflower. The sunflower DGAT, measured by both methods, showed significant higher activity with 18:2-CoA than with 18:1-CoA, whereas the opposite specificity was seen with the safflower enzyme. The specificities of PDAT on the other hand, were similar in both species with 18:2-phosphatidylcholine being a better acyl donor than 18:1-PC and with acyl groups at the sn-2 position utilised about fourfold the rate of the sn-1 position. No DAG:DAG transacylase activity could be detected in the microsomal preparations.     

Soybean phospholipid dependent reductions in triacylglycerol concentration and synthesis in the liver of fasted-refed rats.

The effect of dietary soybean phospholipid on the activities of hepatic triacylglycerol-synthesizing enzymes was compared with soybean oil in fasted-refed rats. Soybean oil at the dietary level corresponding to 20% but not at 5% fatty acid level (21.2 and 5.3% on weight bases, respectively) significantly decreased liver microsomal diacylglycerol acyltransferase activities measured with the endogenous diacylglycerol substrate. Dietary soybean phospholipid even at the dietary level corresponding to 2% fatty acids (3.4% on weight base) significantly decreased the acyltransferase activities measured with endogenous substrate. The dietary phospholipid further decreased the parameter as the dietary level increased, and at the 5% fatty acid level, it was lower than that obtained with soybean oil at 20% fatty acid level. Soybean oil and phospholipid decreased the diacylglycerol acyltransferase activities measured with the saturating concentration of exogenous dioleoylglycerol substrate only when the activities were expressed in terms of total activity (mumol/min per liver) but to much lesser extents. Dietary phospholipid compared to the oil profoundly decreased not only hepatic triacylglycerol but also microsomal diacylglycerol levels. It was indicated that the availability of microsomal diacylglycerol as the substrate for diacylglycerol transferase is the critical determinant in regulating hepatic triacylglycerol synthesis and concentration in this experimental situation. Alterations in the activities of microsomal glycerol 3-phosphate acyltransferase and of the enzymes in fatty acid synthesis could account for the phospholipid-dependent decrease in the microsomal concentration of this intermediate in triacylglycerol synthesis.     

Triacylglycerol synthesis in goat mammary gland. Factors influencing the esterification of fatty acids synthesized de novo.

ATP alone had no effect on incorporation of fatty acids synthesized de novo and membrane-bound diacylglycerol into triacylglycerol. Combined addition of ATP and Mg2+ totally inhibits incorporation of fatty acids synthesized de novo and stimulated incorporation of membrane-bound diacylglycerol. ATP, Mg2+ and glycerol 3-phosphate stimulate incorporation of fatty acids synthesized de novo into triacylglycerol, but inhibited the incorporation of membrane-bound diacylglycerol. Diacylglycerol generated in situ was shown to be superior to diacylglycerols preloaded on the membrane as substrate for the diacylglycerol acyltransferase. A model is proposed to explain the effect of absorbed exogenous fatty acid on fatty acid synthesis de novo in goat mammary gland.     

Fasting and insulin regulation of the utlization of acetoacetate for fatty acid synthesis.

The effects of fasting and of insulin on the incorporation of acetoacetate, β-OH-butyrate, or acetate into fatty acids of liver, adipose tissue, and carcass were studied in mice. Fasting decreases the incorporation of the three precursors, more so in liver than in the other tissues. Insulin totally restores lipogenesis in adipose tissue when the precursor is acetate or acetoacetate. Its effect is less marked on the incorporation of β-OH-butyrate and in the liver. The incorporation of acetate or acetoacetate into fatty acids by 100,000g supernatant protein of mouse liver was also studied. Fasting strongly decreases the incorporation of both compounds and insulin partially restores it. The activities of cytoplasmic acetyl-CoA and acetoacetyl-CoA synthetases were measured in the liver supernatant solution of fed or fasted mice. Fasting strongly decreases the activities of both enzymes; refeeding restores the activities; refeeding and insulin increase the activities above normal levels. Actinomycin suppresses the effect of insulin. The results strongly suggest that insulin is an inducer of the synthesis of cytoplasmic acetyl-CoA and acetoacetyl-CoA synthetases, and that both these synthetases are adaptative enzymes.     

Triacylglycerols are synthesised and utilized by transacylation reactions in microsomal preparations of developing safflower (Carthamus tinctorius L.) seeds

Microsomal membrane preparations from the immature cotyledons of safflower (Carthamus tinctorius) catalysed the interconversion of the neutral lipids, mono-, di-, and triacylglycerol. Membranes were incubated with neutral lipid substrates, ¹⁴C-labelled either in the acyl or glycerol moiety, and the incorporation of radioactivity into other complex lipids determined. It was clear that diacylglycerol gave rise to triacylglycerol and monoacylglycerol as well as phosphatidylcholine. Radioactivity from added [¹⁴C] triacylglycerol was to a small extent transferred to diacylglycerol whereas added [¹⁴C] monoacylglycerol was rapidly converted to diacylglycerols and triacylglycerols. The formation of triacylglycerol from diacylglycerol occurred in the absence of acyl-CoA and hence did not involve diacylglycerol acyltransferase (DAGAT) activity. Monoacylglycerol was not esterified by direct acylation from acyl-CoA. We propose that these reactions were catalyzed by a diacylglycerol: diacylglycerol transacylase which yielded triacylglycerol and monoacylglycerol, the reaction being freely reversible. The specific activity of the transacylase was some 25% of the diacylglycerol acyltransferase activity and, hence, during the net accumulation of oil, substantial newly formed triacylglycerol equilibrated with the diacylglycerol pool. In its turn the diacylglycerol rapidly interconverted with phosphatidylcholine, the major complex lipid substrate for Δ12 desaturation. Hence, the oleate from triacylglycerols entering phosphatidylcholine via this route could be further desaturated to linoleate. A model is presented which reconciles these observations with our current understanding of fatty acid desaturation in phosphatidylcholine and oil assembly in oleaceous seeds. Received: 8 November 1996 / Accepted: 5 February 1997     

Mammalian wax biosynthesis. II. Expression cloning of wax synthase cDNAs encoding a member of the acyltransferase enzyme family

Wax monoesters are synthesized by the esterification of fatty alcohols and fatty acids. A mammalian enzyme that catalyzes this reaction has not been isolated. We used expression cloning to identify cDNAs encoding a wax synthase in the mouse preputial gland. The wax synthase gene is located on the X chromosome and encodes a member of the acyltransferase family of enzymes that synthesize neutral lipids. Expression of wax synthase in cultured cells led to the formation of wax monoesters from straight chain saturated, unsaturated, and polyunsaturated fatty alcohols and acids. Polyisoprenols also were incorporated into wax monoesters by the enzyme. The wax synthase had little or no ability to synthesize cholesteryl esters, diacylglycerols, or triacylglycerols, whereas other acyltransferases, including the acyl-CoA:monoacylglycerol acyltransferase 1 and 2 enzymes and the acyl-CoA:diacylglycerol acyltransferase 1 and 2 enzymes, exhibited modest wax monoester synthesis activities. Confocal light microscopy indicated that the wax synthase was localized in membranes of the endoplasmic reticulum. Wax synthase mRNA was abundant in tissues rich in sebaceous glands such as the preputial gland and eyelid and was present at lower levels in other tissues. Coexpression of cDNAs specifying fatty acyl-CoA reductase 1 and wax synthase led to the synthesis of wax monoesters. The data suggest that wax monoester synthesis in mammals involves a two step biosynthetic pathway catalyzed by fatty acyl-CoA reductase and wax synthase enzymes.     

Galactolipid synthesis in chromoplasts in vitro

Isolated chromoplasts from Narcissus pseudonarcissus flowers contain: a fatty acid synthesizing system; acyl-CoA synthetase (EC 6.2.1.3); glycero-phosphate acyltransferase (EC 2.3.1.15); acylglycero-phosphate acyltransferase; phosphatidate phosphatase (EC 3.1.3.4); diacylglycerol galactosyltransferase (EC 2.4.1.46); and diacylgalactosylglycerol galactosyltransferase, i.e. all enzymatic activities necessary for the synthesis of diacylgalactosylglycerol and diacylgalabiosylglycerol from acetate, HCO _- ³ , sn-glycerol 3-phosphate, and UDP-d-galactose. Diacylgalactosylglycerol and diacylgalabiosylglycerol, however, are synthesized from these precursors to only a very low extent in an in vitro system. This is attributed to a specificity of diacylglycerol galactosyltransferase for highly unsaturated diacylglycerols. Specificities of acyltransferase reactions were also found.     

Stimulation of phosphatidylethanolamine synthesis in isolated rat hepatocytes by phorbol 12-myristate 13-acetate

Incubation of freshly isolated rat hepatocytes in the presence of phorbol 12-myristate 13-acetate stimulates the incorporation of [1,2-14C]ethanolamine into phosphatidylethanolamines. This stimulation is strongly dependent on the ethanolamine concentration in the medium and becomes apparent at ethanolamine concentrations above 25 microM. Treatment of hepatocytes with phorbol 12-myristate 13-acetate results in a decreased labelling of intracellular ethanolamine, ethanolaminephosphate and CDPethanolamine. Exposure of cells to phorbol 12-myristate 13-acetate induces an increase of the activity of the enzymes CTP: ethanolaminephosphate cytidylyltransferase and ethanolaminephosphotransferase. These effects are accompanied by a decrease of the pool size of ethanolaminephosphate and CDPethanolamine and an increase of the level of diacylglycerols after 30 min of incubation in the presence of phorbol 12-myristate 13-acetate. Upon prolonged incubation, the CDPethanolamine and diacylglycerol pools are restored to the level found in untreated cells. These results indicate that stimulation of phosphatidylethanolamine synthesis by phorbol 12-myristate 13-acetate is probably exerted at the level of CTP : ethanolaminephosphate cytidylytransferase, although there may be an additional effect on the subsequent step of phosphatidylethanolamine synthesis, the formation of phosphatidylethanolamines from CDPethanolamine and diacylglycerols.     

Secretion of lipoprotein lipid and synthesis of fatty acids, cholesterol and triacylglycerol by hepatocytes of fasted-refed rats

Synthetic rates of fatty acid, cholesterol and triacylglycerols, and contents and secretion of lipoprotein lipids, were determined in hepatocytes of rats fed ad libitum a fat-containing stock diet or of rats fasted for 48 h and then refed for 24 or 48 h with stock diet or with a glucose-rich fat-free diet. When compared with the values for the ad libitum-fed rats, fatty acid synthesis was lower in fasted rats, slightly increased in rats refed with the stock diet, but several-fold elevated after refeeding the glucose-rich fat-free diet. Cholesterol synthesis was decreased in the fasted cells, and restored to the control level upon refeeding either diet. Triacylglycerol synthesis from exogenous oleate was greatly stimulated in the cells of fasted-refed rats above the rate in cells of the ad libitum-fed rats, the increase being considerably higher after refeeding the glucose-rich fat-free diet than the stock diet. The amount of triacylglycerol secreted by the cells was also elevated by the fasting-refeeding treatment, but the difference between the two diets was much less pronounced than seen for the lipids' synthetic rates. This imbalance may underlie the huge accumulation of this lipid observed in the heptatocytes after refeeding the rats for 48 h with the glucose-rich fat-free diet.     

Glycerolipid synthetic capacity of rat liver peroxisomes

Investigations on rat liver peroxisomal glycerolipid synthetic capability were performed. Highly purified peroxisomal preparations contained dihydroxyacetone-phosphate acyltransferase, acyldihydroxyacetone-phosphate reductase, and fatty acid-CoA ligase activities. Glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, diacylglycerol cholinephosphotransferase, diacylglycerol ethanolaminephosphotransferase and ethanol acyltransferase activities were low in activity or not detected. These results suggest that the peroxisomes are specialized to contribute to the synthesis of ether-linked glycerolipids. If peroxisomes contribute towards the synthesis of non-ether-linked glycerolipids (i.e., ester-linked) then translocation of acyl glycerophosphatide (acyl dihydroxyacetone phosphatide) from peroxisomes to endoplasmic reticulum would be expected to occur.     

A human skin multifunctional O-acyltransferase that catalyzes the synthesis of acylglycerols, waxes, and retinyl esters

Acyl-CoA-dependent O-acyltransferases catalyze reactions in which fatty acyl-CoAs are joined to acyl acceptors containing free hydroxyl groups to produce neutral lipids. In this report, we characterize a human multifunctional O-acyltransferase (designated MFAT) that belongs to the acyl-CoA:diacylglycerol acyltransferase 2/acyl-CoA:monoacylglycerol acyltransferase (MGAT) gene family and is highly expressed in the skin. Membranes of insect cells and homogenates of mammalian cells overexpressing MFAT exhibited significantly increased MGAT, acyl-CoA:fatty acyl alcohol acyltransferase (wax synthase), and acyl-CoA:retinol acyltransferase (ARAT) activities, which catalyze the synthesis of diacylglycerols, wax monoesters, and retinyl esters, respectively. Furthermore, when provided with the appropriate substrates, intact mammalian cells overexpressing MFAT accumulated more waxes and retinyl esters than control cells. We conclude that MFAT is a multifunctional acyltransferase that likely plays an important role in lipid metabolism in human skin.     

Precursor supply and hepatic enzyme activities as regulators of triacylglycerol synthesis in isolated hepatocytes and perfused liver

The relative significance of alterations in precursor supply and enzyme activities for the rate of triacylglycerol synthesis was studied in isolated hepatocytes and perfused livers. Precursor availability was varied in vitro by changing the fatty acid concentration in the incubation medium or adding ethanol to the perfusion medium in order to increase the cellular glycerol 3-phosphate concentration. The rate of glycerolipid synthesis in hepatocytes, measured in terms of the label incorporated into the various lipid classes from tritiated glycerol, was strongly dependent on the fatty acid concentration up to 2 mm of oleate (fatty acid/albumin molar ratio $\text{7}\text{1}$). Ethanol in vitro increased the incorporation of labeled oleate into phosphatidic acid and diacylglycerol in the isolated perfused liver, but its effect on the incorporation into triacylglycerol was small. Ethanol in vitro increased the label incorporation into both diacylglycerol and triacylglycerol in the livers from cortisol-treated rats. Although cortisol treatment increased the soluble phosphatidate phosphohydrolase activity 4.4-fold in the hepatocytes, it had no effect on the rate of triacylglycerol synthesis, whereas fasting increased this rate about 3-fold, although only a moderate concomitant increase in soluble phosphatidate phosphohydrolase activity was observed. Neither cortisol treatment nor fasting affected the microsomal glycerol-3-phoshate acyltransferase activity. The results demonstrate that substrate availability can override enzyme modulations in the regulation of triacylglycerol synthesis and that phosphatidate phosphohydrolase is not the main regulator of triacylglycerol synthesis.     

Acyl-coenzyme A: cholesterol acyltransferase family

The enzymes of the acyl-coenzyme A: cholesterol acyltransferase (ACAT) family are responsible for the in vivo synthesis of neutral lipids. They are potential drug targets for the intervention of atherosclerosis, hyperlipidemia, obesity, type II diabetes and even Alzheimer’s disease. ACAT family enzymes are integral endoplasmic reticulum (ER) membrane proteins and can be divided into ACAT branch and acyl-coenzyme A: diacylglycerol acyltransferase 1 (DGAT1) branch according to their substrate specificity. The ACAT branch catalyzes synthesis of cholesteryl esters using long-chain fatty acyl-coenzyme A and cholesterol as substrates, while the DGAT1 branch catalyzes synthesis of triacylglycerols using fatty acylcoenzyme A and diacylglycerol as substrates. In this review, we mainly focus on the recent progress in the structural research of ACAT family enzymes, including their disulfide linkage, membrane topology, subunit interaction and catalysis mechanism.