Changes with starvation in the rat of the lipoprotein lipase activity and hydrolysis of triacylglycerols from triacylglycerol-rich lipoproteins in adipose tissue preparations.

Lipoprotein lipase activity was higher in fat-pad pieces than in isolated adipocytes from the same fed rats, whereas hydrolysis of triacylglycerols from triacylglycerol-rich lipoproteins was similar in the two preparations when incubated either in basal conditions or in the presence of heparin. In both preparations there was a similar release of lipoprotein lipase activity into the medium during basal incubation, enhanced by the presence of heparin. In fat-pad pieces, but not in isolated adipocytes, incubation with heparin produced a decrease in the lipoprotein lipase activity measured in the tissue preparation. In fat-pad pieces from 24 h-starved rats, lipoprotein lipase activity was the same as in isolated adipocytes from the same animals and incubation with heparin did not affect the appearance of lipoprotein lipase in the medium or the utilization of triacylglycerols from triacylglycerol-rich lipoproteins. These results support the following conclusions. (1) The effectiveness of lipoprotein lipase in adipose tissue preparations in vitro depends more on its availability to the substrate than on its total activity. (2) Heparin acts on adipose tissue preparations from fed animals both by enhancing the release of pre-existing extracellular enzyme (which is absent in isolated adipocytes) and by enhancing the transfer outside the cells of the intracellular (and mainly undetectable) enzyme that is activated in the secretion process. (3) In adipose tissue from starved animals there is not only a decrease in the active extracellular form of lipoprotein lipase activity but also a reduction in the intracellular (and mainly undetectable) pool of the enzyme.     

Transfer of free and esterified cholesterol from low-density lipoproteins and high-density lipoproteins to human adipocytes

Cholesterol stored in human adipose tissue is derived from circulating lipoproteins. To delineate the cholesterol transport function of LDL and HDL, the movement of radiolabelled esterified cholesterol and free cholesterol from labelled LDL and HDL to human adipocytes was examined in the present study. LDL and HDL were enriched and labelled in esterified cholesterol with [14C]cholesterol by the action of plasma lipid transfer proteins and lecithin-cholesterol acyltransferase. Doubly labelled (3H,14C) LDL and HDL were prepared by exchanging free [3H]cholesterol into the 14C-labelled lipoproteins. 14C-labelled lipoprotein and 3H-labelled lipoprotein were also prepared separately and mixed to yield a mixed doubly labelled lipoprotein. Relative to the total amount added, proportionally more free than esterified cholesterol was transferred to the adipocytes upon incubation with any doubly labelled LDL and HDL. The calculated mass of free and esterified cholesterol transferred, however, varied with different labelled lipoproteins. 3H- and 14C-labelled LDL or HDL transferred 2-3-fold more esterified than free cholesterol while the reverse occurred with the mixed doubly labelled LDL or HDL. Thus, free cholesterol-depleted particles preferentially transferred cholesterol ester to the fat cells. In the presence of the homologous unlabelled native lipoprotein, the transfers of free and esterified cholesterol from labelled LDL or HDL were specifically inhibited. Selective transfer of esterified cholesterol relative to apoprotein was also observed when esterified cholesterol uptake from both LDL and HDL was assayed along with the binding of 125I-labelled lipoprotein. The cellular accumulation of cholesterol ether-labelled HDL (a non-hydrolyzable analogue of cholesterol ester) exceeded that of cholesterol ester consistent with significant hydrolysis of the latter physiological substrate. These results demonstrate preferential transfer of free cholesterol and esterified cholesterol over apoprotein for both LDL and HDL in human adipocytes. Furthermore, the data suggest that the cholesterol ester transport function of LDL and HDL can be enhanced by free cholesterol depletion and cholesterol ester enrichment of the particles, and affirms a role for adipose tissue in the metabolism of lipid-modified lipoproteins.     

Comparative utilization in vivo of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose and [1-C14] palmitate in the rat

Female rats were injected i.v. with comparable trace amounts of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose, or [1-14C] palmitate, and killed 30 min afterwards. The radioactivity remaining in plasma at that time was maximal in animals receiving [U-14C] glucose while the appearance of radioactive lipids was higher in the [U-14C] glycerol animals than in other groups receiving hydrosoluble substrates. The carcass, more than the liver, was the tissue where the greatest proportion of radioactivity was recovered, while the greatest percentage of radioactivity appeared in the liver in the form of lipids. The values of total radioactivity found in different tissues were very similar when using either labelled glucose or glycerol but the amount recovered as lipids was much greater in the latter. The maximal proportion of radioactive lipids appeared in the fatty-acid form in the liver, carcass, and lumbar fat pads when using [U-14C] glycerol as a hydrosoluble substrate, and the highest lipidic fraction appeared in adipose tissue as labelled, esterified fatty acids. In the spleen, heart, and kidney, most of the lipidic radioactivity from any of the hydrosoluble substrates appeared as glyceride glycerol. The highest proportion of radioactivity from [1-14C] palmitate appeared in the esterified fatty acid in adipose tissue, being followed in decreasing proportion by the heart, carcass, liver, kidney, and spleen. Thus at least in part, both labelled glucose and glycerol are used throughout different routes for their conversion in vivo to lipids. A certain proportion of glycerol is directly utilized by adipose tissue. The fatty acids esterification ability differs among the tissues and does not correspond directly with the reported activities of glycerokinase, suggesting that the alpha-glycerophosphate for esterification comes mainly from glucose and not from glycerol.     

Conversion of (U-14C)-glycerol, (2-3H)-glycerol and (1-14C)-palmitate into circulating lipoproteins in the rat.

The in vivo formation of labelled very low density lipoproteins (VLDL) from (U-14C)-glycerol, (2-3H)-glycerol and (1-14C)-palmitate was studied in fed female rats. The rate of disappearance of radioactivity from plasma after the i.v. injection with these tracers was similar for (U-14C)-glycerol and (1-14C)-palmitate. With (2-3H)-glycerol, plasma radioactivity at 10 min was lower than with the other substrates although it did not change thereafter. A certain proportion of radioactivity administered as glycerol appeared in plasma lipids, mainly in the VLDL glyceride glycerol fraction, although when (U-14C)-glycerol was the substrate, a considerable portion also appeared in the esterified fatty acids of these lipoproteins. When using (1-14C)-palmitate, practically all the circulating labelled esterified fatty acids appeared in the VLDL fraction, while the labelled free fatty acids appeared in lipoprotein of higher density, presumable free fatty acid-albumin complexes. This data is discussed in terms of the role of the liver in the rapid, continuous cycling of these substrates to yield VLDL-glycerides for their extrahepatic utilization.     

Differential incorporation of docosahexaenoic and arachidonic acids by the yolk sac membrane of the avian embryo

During avian development, lipoproteins derived from yolk lipid are assembled in the yolk sac membrane (YSM) for secretion into the embryonic circulation. To investigate how yolk polyunsaturated fatty acids, essential for the development of certain tissues, are distributed among the lipid classes of the lipoproteins, pieces of YSM were incubated in vitro with [14C]arachidonic and [14C]docosahexaenoic acids (DHA). There was a marked difference in the partitioning of these two precursors among the lipid classes of the tissue. Of the radioactivity incorporated into total lipid from [14C]-arachidonic acid during 1 h of incubation, 67.3% was esterified as phospholipid and 29.5% as triacylglycerol. In contrast, only 14.6% of the label incorporated from [14C]-DHA was esterified as phospholipid, whereas 73.2% was recovered in triacylglycerol. This pattern of differential partitioning was observed at all time points and across a 20-fold range of fatty acid concentrations. There was no evidence for conversion of the radioactive arachidonic and DHAs to other fatty acids prior to incorporation into tissue lipids. It is suggested that the selective incorporation of yolk-derived DHA into the triacylglycerol of secreted lipoproteins represents part of a mechanism for directing this polyunsaturate to particular embryonic tissues.     

Labelling studies in vivo on the metabolism of the acyl and glycerol moieties of the glycerolipids in the developing maize leaf.

1. When [2-3H]glycerol was supplied to developing maize-leaf laminae, label entered 3-sn-phosphatidycholine at a linear rate essentially from zero time, whereas other lipids were labelled at accelerating rates. On transfer of laminae from [3H]glycerol to unlabelled glycerol, radioactivity was rapidly lost from 3-sn-phosphatidylcholine and accumulated in other lipids, principally monogalactosyl diacyglycerol. 2. Degradation of these lipids showed that 3H was present only in the glycerol moiety of the lipids. 3. In double-labelling pulse-chase experiments with [14C]acetate, which labelled essentially only fatty acids and [3H]glycerol similar amounts of 14C and 3H radioactivity were lost from 3-sn-phosphatidylcholine and accumulated by monogalactosyl diacylglycerol. 4. The different molecular species of both lipids isolated from laminae during a double-labelled pulse-chase study were separated by argentation t.l.c., and the changes in the amount of radioactivity and the 14C/3H ratio in different species were compared. The greatest loss of radioactivity during the period in unlabelled substrates occurred from the 3-sn-phosphatidylcholine species containing oleate and from the dilinoleate species, and radioactivity accumulated by monogalactosyl diacyglycerol was mainly in the dilinolenate species. However, despite the considerable change in the radioactivity in these species during the chase, the 14C/3H ratio in each of them remained relatively unchanged. 5. It is proposed that 3-sn-phosphatidylcholine in the developing leaf may serve as a donor or linoleate-containing diacyl-glycerols which are incorporated into other lipids, principally monogalactosyl diacylglycerol.     

Lipid synthesis in the adult dog heartworm, Dirofilaria immitis.

1. Incorporation studies with three labelled substrates--[14C]2-glycerol, [14C]1-acetate and [14C]1-oleic acid--demonstrated that adult dog heartworms can synthesize all classes of complex lipids present, including free cholesterol. 2. Diacylglycerols and phosphoglycerides were most rapidly labelled regardless of the precursor employed. 3. 14C from glycerol was found in the aqueous phase of saponified lipids, whereas that from oleic acid was in the fatty acid portion. 4. Tag from acetate was predominantly in the fatty acid portion of saponified lipids and also occurred in the unesterified fatty acids. 5. Acetate and unesterified fatty acids, as represented by oleic acid, were more readily used for lipid synthesis than was glycerol.     

Microbial metabolism of amino alcohols. Biosynthetic utilization of ethanolamine for lipid synthesis by bacteria.

1. Ten bacteria utilizing [2-14C]ethanol-2-amine as the sole or major source of nitrogen for growth on glycerol + salts medium incorporated radioactivity into a variety of bacterial substances. A high proportion was commonly found in lipid fractions, particularly in the case of Erwinia carotovora. 2. Detailed studies of [14C]ethanolamine incorporation into lipids by five bacteria, including E. carotovora, showed that all detectable lipids were labelled. Even where phosphatidylethanolamine was the major lipid labelled, radioactivity was predominantly in the fatty acid rather than the base moiety. The labelled fatty acids were identified in each case. 3. The addition of acetate to growth media decreased the incorporation of radioactivity from ethanolamine into both fatty acid and phosphatidyl-base fragments of lipids from all the bacteria except Mycobacterium smegmatis. Experiments with [3H]ethanolamine and [14C]acetate confirmed that unlabelled acetate decreased the incorporation of both radioactive isotopes into lipids, except in the case of M. smegmatis. 4. Enzyme studies suggested one of two metabolic routes between ethanolamine and acetyl-CoA for each of four bacteria. A role for ethanolamine O-phosphate was not obligatory for the incorporation of [14C]ethanolamine into phospholipids, but correlated with CoA-independent aldehyde dehydrogenase activity.     

Hepatic lipase-mediated hydrolysis versus liver uptake of HDL phospholipids and triacylglycerols by the perfused rat liver

Hepatic triacylglycerol-lipase-mediated hydrolysis and liver uptake of high-density lipoprotein (HDL) lipid components were studied in a recirculating rat liver perfusion, a situation where the enzyme is physiologically expressed and active at the vascular bed. Human native HDL were labelled with tri-[3H]oleoylglycerol, [N-methyl-3H]dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl,2-[14C]linoleoylphosphatidylcholine (PLPC), 1-palmitoyl,2-[14C]linoleoylphosphatidyl-ethanolamine (PLPE) and 1-palmitoyl,2-[14C]palmitoylphosphatidylethanolamine (DPPE). (1) Relative degradation rates of phosphatidylethanolamine molecular species were 2- to 10-fold higher than those of phosphatidylcholine. Considering [14C] PLPC and [14C] PLPE as representative of HDL phosphatidylcholine and phosphatidylethanolamine, respectively, the amounts of lysophosphatidylcholine and lysophosphatidylethanolamine generated after a 60 min perfusion were comparable. The enzyme showed a clear preference for the molecular species bearing an unsaturated fatty acid at the 2 position of glycerol; this was the most pronounced in the case of phosphatidylethanolamine molecular species. (2) Relative liver uptake of HDL-phosphatidylethanolamine was 4- to 5-fold higher than that of HDL-phosphatidylcholine, irrespective of the constitutive fatty acids. Nevertheless, mass estimation indicated that 3 times more molecules of phosphatidylcholine than of phosphatidylethanolamine were transferred. No correlation could be found between the relative degradation rates of phospholipids and their relative liver uptake, indicating a dissociation between the two processes. (3) Perfusate decay and relative liver uptake of labelled HDL-triacylglycerol were higher than that of any phospholipid class. No circulating radiolabelled free fatty acids accumulated in the perfusate, but they were found acylated into liver cell phospholipids and triacylglycerols. (4) A prior 10-12-min washout of the liver vascular bed with heparin removed over 80% of the hepatic lipase activity, as assessed by specific immunoinhibition. Hepatic lipase-depleted liver displayed impaired phospholipid hydrolysis and triacyglycerol uptake, whereas the transfer of HDL phospholipids to liver tissue was unaffected.     

Studies on the mode of uptake of blood triglycerides by the mammary gland of the lactating goat. The uptake and incorporation into milk fat and mammary lymph of labelled glycerol, fatty acids and triglycerides

1. The mode of uptake of the precursors of milk fat by the mammary gland of the lactating goat has been examined by infusing radioactive fatty acids, glucerol or doubly labelled triglycerides into the mammary artery or jugular vein of animals surgically prepared to permit samples of arterial and venous blood to be withdrawn without disturbance to the animal. 2. Acetate was taken up by the mammary gland and incorporated into milk fat. The decrease in the specific radioactivity of blood acetate across the gland was evidence of acetate production, but there was no significant release of labelled lipid from the mammary gland. 3. When labelled long-chain fatty acids or glycerol were infused into the lactating goat, there was extensive transfer of radioactivity into milk in spite of the absence of net uptake of substrate by the mammary gland. The decrease in the specific radioactivity of each substrate across the mammary gland, however, showed that both fatty acids and glycerol were simultaneously taken up and released by mammary tissue. 4. The infusion of chylomicra and triglyceride emulsions labelled with (3)H and (14)C revealed that both glycerol and fatty acids were released during triglyceride uptake by mammary tissue. Changes in the (3)H/(14)C ratio during the transfer of triglyceride from blood into milk showed that at least 80% of the triglyceride was hydrolysed during uptake, but the potential re-utilization of both products of hydrolysis for triglyceride synthesis in mammary tissue implied that only a minimum value could be obtained from the change in the ratio. 5. The time-course of the transfer of (3)H and (14)C into milk and lymph were closely similar after the infusion of [2-(3)H]glycerol tri[1-(14)C]oleate or of a mixture of [2-(3)H]glycerol and [1-(14)C]oleate. 6. The results were consistent with the hypothesis that plasma triglycerides are extensively or completely hydrolysed during mammary uptake.     

Stimulation of hepatic triglyceride synthesis and secretion by clofibrate

Isolated hepatocytes prepared from rat and squirrel monkey livers were used to explore the mechanism of action of clofibrate, a hypolipidemic agent in current use. Addition of sodium clofibrate to cells suspended in Hanks medium stimulated the conversion of [1-¹⁴C]palmitate into esterified lipids and to ¹⁴CO₂. This agent also promoted the incorporation of [2-³H]glycerol into cellular lipids when fatty acids were present in the incubation medium. Triglycerides were the major lipid class increased by the drug. Sodium clofibrate enhanced the discharge of labeled lipids into the medium from liver cells prelabeled with [2-³H]glycerol. These data suggest that clofibrate does not lower plasma triglyceride levels by interference with hepatic triglyceride production or secretion.     

The incorporation of radioactive fatty acids into the phospholipids of nerve-cell-body membranes in vivo. Evidence for highly labelled neuronal nuclei

1. Nerve cell bodies were isolated in bulk from cerebral cortices of 15 day-old rabbits after intrathecal injections of [³H]plamitate, [³H]oleate or [³H]arachidonate and [¹⁴C]glycerol. 2. Nuclear, microsomal and two mitochondrial fractions were isolated from homogenates of the radioactively labelled nerve cell bodies by using differential and discontinuous-gradient centrifugation. 3. After 7.5min in vivo, a high percentage (>80%) of the total ³H-labelled fatty acid radioactivity was found in the membrane fractions of the nerve cell bodies, whereas after 60min in vivo 50% of the total [¹⁴C]glycerol radioactivity was found in the high-speed supernatant. 4. The specific radioactivities of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, and the radioactivity in neutral lipid and non-esterified fatty acid fractions were determined in the four subfractions, as were the distributions of several marker enzymes and nucleates. 5. With respect of ³H-labelled fatty acid, the phospholipids of the nuclear fraction had the highest specific radioactivities of the four subfractions. However, for [¹⁴C]glycerol labelling, generally the ¹⁴C specific radioactivities for individual phospholipids were comparable in the four subfractions. This latter observation suggests transport of phospholipids synthesized de novo between membranes of the nerve cell body. 6. Double-labelling experiments demonstrated that individual phospholipids and the combined neutral lipids of the nuclear fraction had higher labelling ratios of ³H-labelled fatty acid/[¹⁴C]glycerol than did the corresponding lipids of the microsomal or mitochondrial fractions. 7. On the basis of the labelling results and the marker studies, it is proposed that it is indeed the nuclei of the nuclear fraction that have these lipids highly labelled with ³H-labelled fatty acid, and the existence of nuclear acyl transferases that are responsible for this fatty acid incorporation is suggested.     

In vitro lipid metabolism in the rat pancreas. I. Basal lipid metabolism.

1. The in vitro basal lipid metabolism of rat pancreatic fragments was compared with that in adipose tissue fragments and liver slices. 2. [1-14C]Acetate added to the media was mostly incorporated into palmitic acid and to a lesser extent into oleic acid. In addition, pancreatic tissue exhibited a marked capacity for elongation of polyunsaturated fatty acids by [1-14C]acetate and resulting desaturation when compared to adipose tissue and liver. 3. Data obtained in the presence of [U-14C]glucose, [1-14C]palmitate and 3H20 indicate that acetyl-CoA derived from glucose and from beta-oxidation of fatty acids contributed to de novo lipogenesis. 4. Oxidation of [1-14C]palmitic acid was 9-13 times higher in the pancreas than in adipose tissue or liver when expressed on a wet weight basis. 5. The fatty acid moiety of pancreatic glycerolipids could be derived from de novo synthesis, fatty acids added to the medium, or from fatty acids formed from the hydrolysis of endogenous lipids. The glycerol moiety could be derived either from glucose, or directly from glycerol through participation of glycerol kinase.     

Very low and low density lipoprotein synthesis and secretion by the human hepatoma cell line Hep-G2: effects of free fatty acid

The liver is a major source of the plasma lipoproteins; however, direct studies of the regulation of lipoprotein synthesis and secretion by human liver are lacking. Dense monolayers of Hep-G2 cells incorporated radiolabeled precursors into protein ([35S]methionine), cholesterol ([3H]mevalonate and [14C]acetate), triacylglycerol, and phospholipid ([3H]glycerol), and secreted them as lipoproteins. In the absence of free fatty acid in the media, the principal lipoprotein secretory product that accumulated had a density maximum of 1.039 g/ml, similar to serum low density lipoprotein (LDL). ApoB-100 represented greater than 95% of the radiolabeled apoprotein of these particles, with only traces of apoproteins A and E present. Inclusion of 0.8 mM oleic acid in the media resulted in a 54% reduction in radiolabeled triacylglycerol in the LDL fraction and a 324% increase in triacylglycerol in the very low density lipoprotein (VLDL) fraction. Similar changes occurred in the secretion of newly synthesized apoB-100. The VLDL contained apoB-100 as well as apoE. In the absence of exogenous free fatty acid, the radiolabeled cholesterol was recovered in both the LDL and the high density lipoprotein (HDL) regions. Oleic acid caused a 50% decrease in HDL radiolabeled cholesterol and increases of radiolabeled cholesterol in VLDL and LDL. In general, less than 15% of the radiolabeled cholesterol was esterified, despite the presence of cholesteryl ester in the cell. Incubation with oleic acid did not cause an increase in the total amount of radiolabeled lipid or protein secreted. We conclude that human liver-derived cells can secrete distinct VLDL and LDL-like particles, and the relative amounts of these lipoproteins are determined, at least in part, by the availability of free fatty acid.     

Fatty acids of Treponema pallidum and Borrelia burgdorferi lipoproteins.

A fundamental ultrastructural feature shared by the spirochetal pathogens Treponema pallidum subsp. pallidum (T. pallidum) and Borrelia burgdorferi, the etiological agents of venereal syphilis and Lyme disease, respectively, is that their most abundant membrane proteins contain covalently attached fatty acids. In this study, we identified the fatty acids covalently bound to lipoproteins of B. burgdorferi and T. pallidum and examined potential acyl donors to these molecules. Palmitate was the predominant fatty acid of both B. burgdorferi and T. pallidum lipoproteins. T. pallidum lipoproteins also contained substantial amounts of stearate, a fatty acid not typically prevalent in prokaryotic lipoproteins. In both spirochetes, the fatty acids of cellular lipids differed from those of their respective lipoproteins. To characterize phospholipids in these organisms, spirochetes were metabolically labeled with [3H]palmitate or [3H]oleate; B. burgdorferi contained only phosphatidylglycerol and phosphatidylcholine, while T. pallidum contained phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and cardiolipin. Although palmitate predominated in the lipoproteins, there were no apparent differences in the incorporation of these two fatty acids into phospholipids (putative acyl donors). Phospholipase A1 and A2 digestion of phosphatidylcholine from B. burgdorferi and T. pallidum labeled with either [3H]palmitate or [3H]oleate also revealed that neither fatty acid was incorporated preferentially into the 1 and 2 positions (potential acyl donor sites) of the glycerol backbone. The combined findings suggest that fatty acid utilization during lipoprotein synthesis is determined largely by the fatty acid specificities of the lipoprotein acyl transferases. These findings also provide the basis for ongoing efforts to elucidate the relationship between lipoprotein acylation and the physiological functions and inflammatory activities of these molecules.     

Glyceride metabolism in cultured cells dissociated from rat cerebral cortex

Abstract— [1-¹⁴C]stearic acid and [2-³H]glycerol were rapidly taken up and esterified into triacylglycerol and phospholipids by rat brain cells cultivated in monolayers. Expressed in terms of pool size, the incorporation of glycerol and stearate into triacylglycerol was 6- and 8-fold, respectively, higher than the incorporation into the choline phosphoglycerides. Tritium-labelled glycerol in both triacylglycerol and glycerophosphatides was diluted more rapidly than the [¹⁴C] labelled fatty acids. Chase experiments indicated a transfer of fatty acid from one lipid class to another, mainly from triacylglycerol to phospholipids, with no apparent loss of radioactivity. The accumulation of triacylglycerol in the brain cells was a function of both the presence of exogenous fatty acids in the culture medium and the metabolic needs of the cells; as long as the cells were involved in active formation of membranes the proportion of triacylglycerol was relatively small; its concentration increased while cell division slowed down in older, fully monolayered cultures.     

Effect of rat serum on glycerol metabolism in adipose tissue in vitro

It has been proposed that the lipolytic effect of serum is based on the presence of either lipoproteins or catecholamines. To test these hypotheses, pieces of epididymal fat pads from fed rats were incubated in the presence of albumin and glucose for 120 min. The addition of rat serum (5 mul/vial) enhanced the rates of both glycerol release to the media and [U-14C] glycerol utilization by the tissue. Heparin did not alter these parameters or the response produced by serum. VLDL from rat plasma also enhanced glycerol release and utilization for the formation of CO2 and lipids, and heparin significantly augmented these effects. Neither of the conditions studied affected the percentual distribution of 14C-lipid fractions in the tissues. It is known that in similar conditions to those used in the present study, adrenaline produces a decrease in the utilization of glycerol. Thus our findings do not support the proposed hypothesis explaining the fat-mobilizing action of serum, the mechanism of which remains to be determined.     

Studies on the positional integrity of glyceride fatty acids during digestion and absorption in rats

1. Rats previously starved for 24hr. were separately given by intraduodenal injections 0.5ml. of a dispersion containing 10mg. of sodium taurocholate, with 50mg. of glycerol 1,3-dioleate 2[1-(14)C]-palmitate, glycerol 1,2-dioleate 3[1-(14)C]-palmitate, a mixture of [1-(14)C]palmitic acid and triolein, or a mixture of [1-(14)C]-palmitic acid and oleic acid. 2. At the end of 30min., the net amounts, and the radioactivity, of the neutral-lipid components recovered from the intestinal lumen and mucosa, and the position of the labelled palmitic acid in the mucosal triglycerides, were determined. 3. When glycerol 1,3-dioleate 2[1-(14)C]-palmitate was administered, most of the labelled acid was retained in the di- and monoglycerides of the lumen; the triglycerides were the major components containing the radioactivity in the mucosa and 75-80% of the labelled acid was located at the beta-position of these triglycerides. 4. When glycerol 1,2-dioleate 3[1-(14)C]-palmitate was administered, the labelled acid was readily split off in the lumen and virtually no radioactivity could be traced in the monoglyceride fraction; in the intestinal mucosa, triglycerides were again the chief components containing most of the radioactivity, and 80-85% of the labelled acid was esterified at the outer positions of the glycerol. 5. When [1-(14)C]palmitic acid mixed with triolein was administered, the concentrations of free fatty acids increased markedly in the intestinal lumen and mucosa, and 80-88% of the radioactivity of the mucosal triglycerides was located at the outer positions of the glycerol. 6. When [1-(14)C]palmitic acid mixed with oleic acid was administered, the labelled acid accumulated in the lumen as well as in the cell, and it was randomly incorporated into all three positions of the mucosal triglycerides.     

Metabolism of 15-hydroxyeicosatetraenoic acid by Caco-2 cells

Monolayers of Caco-2 cells, a human enterocyte cell line, were incubated with [1-14C]15-hydroxyeicosatetraenoic acid (15-HETE), a lipid mediator of inflammation, and [1-14C]arachidonic acid. Both fatty acids were taken up readily and metabolized by Caco-2 cells. [1-14C]Arachidonic acid was directly esterified in cellular phospholipids and, to a lesser extent, in triglycerides. When [1-14C]15-hydroxyeicosatetraenoic acid was incubated with Caco-2 cells, about 10% was directly esterified into cellular lipids but most (55%) was beta-oxidized to ketone bodies, CO2, and acetate, with very little accumulation of shorter carbon chain products of partial beta-oxidation. The radiolabeled acetate generated from beta-oxidation of [1-14C]15-hydroxyeicosatetraenoic acid was incorporated into the synthesis of new fatty acids, primarily [14C]palmitate, which in turn was esterified into cellular phospholipids, with lesser amounts in triglycerides. Caco-2 cells were also incubated with [5,6,8,9,11,12,14,15-3H]15-hydroxyeicosatetraenoic acid; most of the radiolabel was recovered either in ketone bodies or in [3H]palmitate esterified in phospholipids and triglycerides, demonstrating that most of the [3H]15-hydroxyeicosatetraenoic acid underwent several cycles of beta-oxidation. The binding of both 15-hydroxyeicosatetraenoic acid and arachidonic acid to hepatic fatty acid binding protein, the only fatty acid binding protein in Caco-2 cells, was measured. The Kd (6.0 microM) for 15-HETE was three-fold higher than that for arachidonate (2.1 microM).     

Alteration of adipocyte metabolism in omega3 fatty acid-depleted rats.

Presently an insufficient supply of long-chain polyunsaturated omega3 fatty acid is prevalent in Western populations leading to potential metabolic consequences. Based on this fact, this study deals mainly with various aspects of lipid metabolism in second generation female omega3-depleted rats. The parametrial fat and body weights were higher in omega3-depleted than control animals. This coincided with liver steatosis but did not alter heart triglyceride/phospholipid ratio. The net uptake of [U-14C] palmitate by adipocytes was also higher in omega3-depleted rats than in control animals. The uptake of D-[U- 4C] glucose or [1,2 (-14)C] acetate by adipocytes was lower, however in omega3-depleted than control animals and was unaffected by insulin in the former as distinct from latter animals. Despite comparable basal lipolysis, the increase in glycerol output from adipocytes provoked by theophylline was higher in omega3-depleted than control rats. The fatty acid pattern of lipids in adipose tissue was characterized in the omega3-depleted rats by a much lower omega3 content, higher apparent Delta 9-saturase and elongase activities, lower efficiency for the conversion of C18:2omega6 to C20:4omega6 and higher efficiency for the conversion of C18:3omega3 to C20:5omega3. These features were compared to those prevailing in liver and plasma lipids. The present study thus extends knowledge on the alteration of lipid metabolism resulting from a deficiency in long-chain polyunsaturated omega3 fatty acids.