Developmental coordinate expression of triacylglycerol and small molecular weight apoB synthesis and secretion by rat hepatocytes.

Glycerolipid and apoB synthesis and secretion were examined in hepatocytes obtained from fetal, suckling (day 6), and adult rats in order to examine the developmental regulation of lipoprotein production. The capacity to synthesize [3H]triacylglycerol (from [3H]glycerol) followed the order: adult greater than day 6 greater than fetal. Addition of 1 mM oleic acid to the incubation media stimulated the incorporation of [3H]glycerol into triacylglycerol 6.7- and 3.6-fold by fetal and adult hepatocytes, respectively. After maximal stimulation by 1 mM oleic acid, triacylglycerol secretion by fetal cells was still only 39% of the amount secreted by adult cells that had been treated similarly. Fetal cells stimulated with 1 mM oleic acid synthesized the same amount of triacylglycerol as adult cells that had been treated with 0.1 mM oleic acid. However, the fetal cells secreted only one-third as much triacylglycerol, further demonstrating relatively impaired secretion of triacylglycerol. In order to determine whether low triacylglycerol secretion was associated with differences in apoB metabolism, cells were incubated with [35S]methionine and apoB was quantified after immunoprecipitation. Fetal cells synthesized and secreted nearly equal amounts of large molecular weight and small molecular weight apoB. In contrast, adult cells synthesized and secreted nearly twice as much small molecular weight apoB as large molecular weight apoB. Moreover, although fetal and adult cells secreted large molecular weight apoB at similar rates, adult cells synthesized and secreted small molecular weight apoB at rates that were nearly two times higher than fetal cells. These data suggest that the ability to assemble and secrete VLDL varies in parallel with the developmental expression of small molecular weight apoB. These studies also show the usefulness of the cultured rat hepatocyte model for examining the ontogeny and regulation of lipoprotein assembly/secretion.

Synthesis and release of fatty acids by human trophoblast cells in culture.

In order to determine whether placental cells can synthesize and release fatty acids, trophoblast cells from term human placentas were established in monolayer culture. The cells continued to secrete placental lactogen and progesterone and maintained specific activities of critical enzymes of triacylglycerol and phosphatidylcholine biosynthesis for 24 to 72 hr in culture. Fatty acid was rapidly synthesized from [14C]acetate and released by the cells. Palmitoleic, palmitic, and oleic acids were the major fatty acids synthesized from [14C]acetate and released. Small amounts of lauric, myristic, and stearic acids were also identified. [14C]acetate was also incorporated into cellular triacylglycerol, phospholipid, and cholesterol, but radiolabeled free fatty acid did not accumulate intracellularly. In a pulse-chase experiment, cellular glycerolipids were labeled with [1-14C]oleate; trophoblast cells then released 14C-labeled fatty acid into the media as the cellular content of labeled phospholipid and triacylglycerol decreased without intracellular accumulation of free fatty acid. Twenty percent of the 14C-label lost from cellular glycerolipid could not be recovered as a chloroform-extractable product, suggesting that some of the hydrolyzed fatty acid had been oxidized. These data indicate that cultured placenta trophoblast cells can release fatty acids that have either been synthesized de novo or that have been hydrolyzed from cellular glycerolipids. Trophoblast cells in monolayer culture should provide an excellent model for molecular studies of placental fatty acid metabolism and release.

Effect of dietary protein and food restriction on milk production and composition, maternal tissues and enzymes in lactating rats.

Lactating rats have been fed either a protein-restricted diet (10 vs. 20% casein in the control diet) or the control diet at 80, 60 and 40% of the voluntary intake for 7 d from d 7 of lactation. Food consumption, changes in maternal live weight, litter live weight gain and the mass of several maternal tissues were determined together with the activity of several mammary and liver enzymes, including 10 that are essential for fatty acid and complex lipid synthesis. Milk production was estimated from the litter weight gain and litter weight. Lactating rats fed the 20% protein diet ad libitum consumed three times that of nonlactating rats; their liver and kidney masses were significantly higher and their adipose mass was lower. The livers of the lactating rats were fatty, containing 118 mg lipid/g compared with 42 mg/g for the nonlactating rats. Lactating rats fed either the protein-restricted diet or the control diet at 40 and 60% of the ad libitum intake of the control diet had lower mammary, liver and kidney masses than rats consuming the control diet ad libitum. Both protein and food restriction led to lower rates of milk production than those of ad libitum-fed control rats as evidenced by the decrease in litter live weight gains. The concentrations of total lipid, total protein and lactose in milk were not affected by these dietary treatments. The concentration of alpha-lactalbumin in milk of rats fed the low protein diet was, however, lower than that in the milk of all rats receiving the control diet, irrespective of intake. Consumption of the restricted diets resulted in only small changes in specific activities (mu/mg protein) of 15 mammary enzymes. In the livers, lactation led to higher specific activities of all four soluble lipogenic enzymes examined but did not affect the particulate enzymes involved in complex lipid synthesis. The dietary restrictions resulted in lower specific activities of the soluble enzymes compared with those of the lactating rats consuming the control diet ad libitum without affecting the particulate enzymes. Total activities of these enzymes were, however, lower than those for the control rats as a result of the smaller liver mass in the rats receiving the restricted diets.

Ontogeny of microsomal activities of triacylglycerol synthesis in guinea pig liver.

Because the onset of triacylglycerol-rich lipoprotein synthesis occurs in guinea pig liver during fetal life, we investigated the microsomal enzyme activities of triacylglycerol synthesis in fetal and postnatal guinea pig liver. Hepatic monoacylglycerol acyltransferase specific and total microsomal activities peaked by the 50th day of gestation and declined rapidly after birth to levels that were virtually unmeasurable in the adult. Peak fetal specific activity was more than 75-fold higher than observed in the adult. The specific activities of fatty acid CoA ligase and lysophosphatidic acid acyltransferase increased 2- to 3-fold before birth; lysophosphatidic acid acyltransferase increased a further 2.6-fold during the first week of life. Specific activities of phosphatidic acid phosphatase, microsomal glycerophosphate acyltransferase, and diacylglycerol acyltransferase varied minimally over the time course investigated. These data demonstrate that selective changes occur in guinea pig hepatic microsomal activities of triacylglycerol synthesis before birth. Because of an approximate 11-fold increase in hepatic microsomal protein between birth and the adult, however, major increases in total microsomal activity of all the triacylglycerol synthetic activities occurred after birth. The pattern of monoacylglycerol acyltransferase specific and total microsomal activities differs from that of the rat in occurring primarily during the last third of gestation instead of during the suckling period. This pattern provides evidence that hepatic monoacylglycerol acyltransferase activity probably does not function to acylate 2-monoacylglycerols derived from partial hydrolysis of diet-derived triacylglycerol.

Monoacylglycerol acyltransferase. Evidence that the activities from rat intestine and suckling liver are tissue-specific isoenzymes.

The monoglycerol acyltransferase (EC 2.3.1.22) (recommended name acylglycerol palmitoltransferase) activities from rat intestinal mucosa and suckling liver microsomes were compared in order to determine why substrate specificities differed in the two tissues. Suckling liver monoacylglycerol acyltransferase activity was highly specific for sn-2-mono-C18:1 glycerol and acylated rac-1-mono-C18:1 glycerol and 1- and 2-mono-C18:1 glycerol ethers poorly. In contrast, the substrate specificity of intestinal monoacylglycerol acyltransferase activity was broad. 1-Acyl- and 1- and 2-alkylglycerols were acylated at rates that were 45-78% of the rate observed with the preferred substrate sn-2-mono-C18:1 glycerol. Partial heat inactivation did not alter these relative specific activities, making it unlikely that intestinal microsomes contained a second acyltransferase capable of acylating the alternate substrates. The hypothesis that intestine and liver contain non-identical monoacylglycerol acyltransferase activities was further tested. Intestinal mucosa monoacylglycerol acyltransferase was much more thermolabile than the liver activity. Incubation with 50 microM diethylpyrocarbonate inactivated liver monoacylglycerol acyltransferase activity 84% but had little effect on the intestinal activity. Hydroxylamine completely reversed diethylpyrocarbonate inactivation, suggesting that critical histidine residues were more accessible in liver monoacylglycerol acyltransferase. 2,4,6-Trinitrobenzene sulfonic acid inactivated hepatic monoacylglycerol acyltransferase more than the intestinal activity, suggesting that critical lysine residues were more accessible. The intestinal and liver activities were also differently affected by acetone, detergents, MgCl2, phospholipids, and bovine serum albumin. Taken as a whole, the data strongly suggest that rat intestinal mucosa and suckling liver contain tissue-specific monoacylglycerol acyltransferase isoenzymes.

Subcellular location and topography of rat hepatic monoacylglycerol acyltransferase activity.

Monoacylglycerol acyltransferase activity from suckling rat liver was localized to the microsomal subcellular fraction by differential centrifugation and comparison with the partitioning of selected marker enzymes. Chymotrypsin, pronase, and proteinase K inactivated the monoacylglycerol acyltransferase activity in detergent-disrupted microsomes, but not in intact microsomes, falsely suggesting a lumenal location for the enzyme. The impermeant inhibitors mercury-dextran and 4,4'-diisothiocyano,-2,2'-disulfonic acid stilbene inhibited monoacylglycerol acyltransferase in intact microsomes. These data, as well as the lack of latency and the inability of the substrate palmitoyl-CoA to readily permeate hepatic microsomes from suckling rats, strongly suggest that the enzyme's active site faces the cytosolic surface of the endoplasmic reticulum.

Hepatic monoacylglycerol acyltransferase. Characterization of an activity associated with the suckling period in rats.

Hepatic monoacylglycerol acyltransferase activity is 700-fold higher during the suckling period than in the adult rat. Specific activity in total particulate preparations rose from 9.4 nmol/min/mg before birth to a peak of 78 nmol/min/mg on the 6th to 8th postnatal days. Monoacylglycerol acyltransferase activity fell sharply after day 8 and was 1.6 and 0.1 nmol/min/mg on day 28 and in adult rats, respectively. The activity had a pH optimum at 8.0 and was activated by albumin and by phospholipids. With [3H]palmitoyl-CoA and sn-2-monooleoylglycerol, more than 96% of the products were di- and triacylglycerols. More than 92% of the diacylglycerol product was the 1,2 isomer. The activity was stable at 43 degrees C for 50 min. Thermal inactivation showed t 1/2 values of 8 min and 4.5 min at 53.5 and 55 degrees C, respectively. In suckling rats, monoacylglycerol acyltransferase activities in liver and intestinal mucosa were 150- to 800-fold higher than in other tissues. Monoacylglycerol acyltransferase activity was 12.5-fold greater with palmitoyl-CoA than with octanoyl-CoA. Acetyl-CoA was not a substrate. The sn-2-monoacylglycerols were strongly preferred over the sn-1 isomers. No direct relationship was noted between 2-monoacylglycerol chain length and apparent Km value. The presence of high levels of monoacylglycerol acyltransferase activity in suckling rat liver suggests that the monoacylglycerol pathway functions as a major route of hepatic glycerolipid synthesis during the suckling period but not in adult animals.

Microsomal and lysosomal enzymes of triacylglycerol metabolism in rat placenta.

The placenta plays a major role in transporting lipid to the developing foetus. Since previous studies have suggested that placental lipid transport involves intermediate esterification steps, we investigated selected microsomal and lysosomal enzymes of triacylglycerol metabolism in rat placenta. Between gestational days 10 and 14, microsomal phosphatidic acid phosphatase specific activity was 6-fold greater than the activity in adult rat liver. Phosphatidic acid phosphatase activity decreased 50% on day 15. Studies employing several different phosphorylated substrates indicated a high degree of substrate specificity. Lysosomal triacylglycerol lipase and cholesterol esterase activities decreased about 50% between days 15 and 18, then rose late in gestation. No changes were observed in the specific activities of fatty acid: CoA ligase, glycerolphosphate acyltransferase, lysophosphatidate acyltransferase, diacylglycerol acyltransferase or diacylglycerol cholinephosphotransferase during the final 12 days of gestation. Kinetic observations (competitive inhibition by alternative substrates, pH-dependence and thermal inactivation) were consistent with the hypothesis that glycerol phosphate and dihydroxyacetone phosphate can be acylated by a single microsomal enzyme in placenta. Except for fatty acid: CoA ligase, the activities of microsomal and lysosomal enzymes of triacylglycerol metabolism were comparable with those in adult rat liver. These observations are consistent with physiological studies suggesting that triacylglycerol synthetic and degradative pathways are very active in rat placenta.