Acyltransferase activities in the yolk sac ... - Wiley Online Library

Acyltransferase Activities in the Yolk Sac Membrane of the Chick Embryo Alison M.B. Murray*, Raphael Denis, and Brian K. Speake Department of Biochemistry and Nutrition, Scottish Agricultural College, Ayr, KA6 5HW, United Kingdom

ABSTRACT: The activities of some enzymes of glycerolipid synthesis and fatty acid oxidation were measured in subcellular fractions of the yolk sac membrane (YSM), an extra-embryonic tissue that mediates the transfer of lipid from the yolk to the circulation of the chick embryo. The activities of monoacylglycerol acyltransferase and carnitine palmitoyl transferase-1 in the YSM (respectively, 284.8 ± 13.2 nmol/min/mg microsomal protein and 145.6 ± 9.1 nmol/min/mg mitochondrial protein; mean ± SE; n = 4) at day 12 of development appear to be the highest yet reported for any animal tissue. Also, the carnitine palmitoyl transferase-1 of the YSM was very insensitive to inhibition by malonyl CoA. The maximal activities of glycerol-3-phosphate acyltransferase and diacylglycerol acyltransferase in the YSM (respectively, 26.7 ± 2.2 and 36.1 ± 2.1 nmol/min/mg microsomal protein) were also high compared with the reported values for various animal tissues. The very high enzymic capacity for glycerolipid synthesis supports the hypothesis that the yolk-derived lipids are subjected to hydrolysis followed by reesterification during transit across the YSM. The monoacylglycerol pathway appears to be the main route for glycerolipid resynthesis in the YSM. The results also suggest that the YSM has the capacity to perform simultaneously β-oxidation at a high rate in order to provide energy for the lipid transfer process. Paper no. L8246 in Lipids 34, 929–935 (September 1999).

The transfer of lipid from the yolk to the embryo and the utilization of the various lipid moieties by the developing tissues are the predominant metabolic features of avian embryonic development. This transfer is especially intense during the second half of the developmental period when essentially all the energy needs of the embryo are provided by the β-oxidation of yolk-derived fatty acids. The uptake of lipid from the yolk is performed by the yolk sac membrane (YSM), a highly vascularized extra-embryonic structure that grows outward from the body of the embryo during the early stages of development so as to completely surround the yolk by about the midpoint of the developmental period. Within the endo*To whom correspondence should be addressed at Department of Biochemistry and Nutrition, Scottish Agricultural College, Auchincruive, Ayr, KA6 5HW, United Kingdom. E-mail: [email protected] Abbreviations: CE, cholesteryl ester; CPT-1, carnitine palmitoyl transferase1; DAG, diacylglycerol; DGAT, diacylglycerol acyltransferase; GPAT, glycerol-3-phosphate acyltransferase; MGAT, monoacylglycerol acyltransferase; NEM, N-ethylmaleimide; PL, phospholipid; TAG, triacylglycerol; VLDL, very low density lipoprotein; YSM, yolk sac membrane. Copyright © 1999 by AOCS Press

dermal cells of the YSM, the yolk-derived lipids are assembled into particles of the very low density lipoprotein (VLDL) type, which are then secreted into the vascular system for delivery into the embryonic circulation (1–6). The question arises regarding the degree to which the yolk-derived lipids are modified during their transit across the endodermal cell layer of the YSM. Because the yolk essentially consists of a tightly packed mass of VLDL particles originating from synthesis in the maternal liver (7–9), the most parsimonious explanation would be the transcytosis, without alteration, of these lipoproteins across the YSM. Such a simplistic scenario is, however, very unlikely for several reasons. Most crucially, the yolk-precursor VLDL secreted by the maternal liver is a highly-specialized lipoprotein, uniquely adapted to fulfill its singular function of delivering lipid to the oocyte maturing in the ovary (1,7–11). By contrast, the primary fate of the VLDL that emerges from the YSM is to serve as a substrate for lipoprotein lipase in the capillaries which permeate the adipose tissue and muscle of the embryo, thus delivering fatty acids to the developing tissues (12). Such distinct roles must require differences in lipoprotein structure, and it is pertinent that the maternally derived VLDL particles that form the yolk are unusually small and regular (30 nm diameter ) (7–9) whereas those which are exocytosed from the basal face of the YSM endodermal cells are large and polydisperse (50–150 nm diameter) (1,3–5). In light of these considerations, the alternative situation in which the triacylglycerol (TAG), phospholipid (PL) and cholesteryl ester (CE) of the yolk are subjected to extensive hydrolysis following uptake into the YSM, thus releasing a range of products such as free fatty acids, glycerol, partial glycerides and free cholesterol into the endodermal cytoplasm, would appear to be more likely. The transfer of these hydrolytic products to the endoplasmic reticulum of the endodermal cells, followed by reesterification to re-form TAG, PL and CE in concert with the synthesis of apoproteins (13), would enable the assembly of VLDL particles with a composition suited to their functions in the embryo. A key prediction of this “lipid remodeling” hypothesis is that the YSM should express the enzymic capacity necessary for the reesterification process. Moreover, the activities of the acyltransferases involved in glycerolipid resynthesis in the

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YSM would need to be commensurate with the sheer intensity of lipid transfer during the second half of the embryonic period (1). Previous work by Sansbury and coworkers (14) delineated the ontogeny of acyltransferase expression in the liver of the chick embryo. In the present study we focused on the levels of acyltransferase activity exhibited by the YSM. The activities of glycerol-3-phosphate acyltransferase (GPAT) (EC 2.3.1.15) which catalyzes the first committed step of the glycerol phosphate pathway (15,16), monoacylglycerol acyltransferase (MGAT) (EC 2.3.1.22) which acylates sn-2-monoacylglycerol to form sn-1,2-diacylglycerol and thus catalyzes the defining step of the monoacylglycerol pathway (15), and diacylglycerol acyltransferase (DGAT) (EC 2.3.1.20) which catalyzes the conversion of sn-1,2-diacylglycerol to TAG, a step common to both the above pathways (15), were determined in the YSM throughout the second half of development. The levels of carnitine palmitoyl transferase-1 (CPT-1) (EC 2.3.1.21) expressed in mitochondria from the YSM of the embryo were also determined as an indication of the capacity of this tissue for β-oxidation (17,18). MATERIALS AND METHODS Embryos. Fertile eggs of the Ross 1 broiler–breeder strain were obtained from a commercial supplier (Ross Poultry, Thornhill, Scotland). The eggs were incubated at 37.8°C and 60% relative humidity in a bench-top incubator (Brinsea Products, Banwell, United Kingdom) with automatic egg turning. At various stages throughout development, the required number of embryos were sacrificed, and the YSM and liver were collected. The YSM was washed thoroughly in 0.85% (wt/vol) NaCl at 4°C to remove any adherent yolk. Hatching occurred after 21 d of incubation, and the required number of chicks were maintained for 1 d with the provision of drinking water but with no food prior to sacrifice. Preparation of subcellular fractions. Tissue samples were finely chopped with scissors and gently homogenized using a hand-held glass-Teflon homogenizer in 5 vol of 0.25 M sucrose containing 5 mM Tris-HCl buffer (pH 7.4) and 1 mM EGTA at 0°C. All subsequent centrifugations were performed at 4°C. The homogenate was centrifuged at 500 × g for 10 min, and the resultant supernatant was recentrifuged at 9,000 × g for 10 min to isolate mitochondria. The mitochondrial pellet was washed with homogenization medium and recentrifuged at 9,000 × g for 10 min. The washed mitochondria were then suspended in 5 mM Tris-HCl buffer (pH 7.4) containing 0.15 M KCl and 1 mM EGTA prior to enzyme assay. The 9,000 × g supernatant was then centrifuged at 100,000 × g for 60 min (Centrikon T-1170 Ultracentrifuge; Kontron Ltd., Watford, United Kingdom) to obtain the microsomal fraction. The microsomal pellet was resuspended in homogenization buffer and recentrifuged at 100,000 × g for 60 min. The washed microsomal pellet was then resuspended in the appropriate enzyme assay buffer. The purity of the mitochondrial and microsomal fractions was assessed by marker enzyme assays. Lactate dehydrogeLipids, Vol. 34, no. 9 (1999)

nase, citrate synthase, and NADPH-cytochrome c reductase were determined by published methods (19–21). Both fractions were free of cytosolic contamination as indicated by the absence of lactate dehydrogenase in the final washed suspensions. The mitochondrial preparation contained no detectable NADPH-cytochrome c reductase and the microsomal preparation contained no detectable citrate synthase, indicating a lack of cross-contamination between these subcellular fractions. Assay of MGAT. For each of the four enzymes investigated in this study, the reaction rate was linear with incubation time and with the concentration of microsomal or mitochondrial protein under the assay conditions described. Also, for each enzyme assay, the blank value obtained by stopping the reaction at time zero was subtracted from the measured incorporation. MGAT activity was determined by a previously reported method (22) with minor modification. The reaction mixture consisted of 24 mM Tris-HCl buffer (pH 7.5), 50 mM KCl, 8 mM MgSO4, 0.75 mM dithiothreitol, 0.625 mg bovine serum albumin (fatty acid-free), 15 µg each of phosphatidylcholine and phosphatidylserine, 0.25 mM sn-2-monoolein and 25 µM palmitoyl CoA, [14C] palmitoyl CoA (0.01 µCi), and 2 µg microsomal protein in a final volume of 0.5 mL. The sn-2-monoolein substrate, which was at least 99% pure as described by the suppliers (Sigma Chemical Co., Poole, Dorset, United Kingdom), was added as a dispersion in 0.1% (wt/vol) Tween 20; the final concentration of Tween 20 in the assay was 0.002% (wt/vol). The reaction was started by the addition of the microsomal protein, and the mixture was incubated at 37°C for 5 min. The reaction was terminated by the addition of 0.75 mL of 2-propanol/heptane/water (80:20:2, by vol). After 5 min, 0.5 mL heptane and 0.25 mL water were added, the samples were mixed and centrifuged at 600 × g for 5 min. The heptane layer was removed and washed twice with 1 mL of 0.5 M NaOH/ethanol/water (10:50:50, by vol). The radioactivity in a portion (0.25 mL) of the heptane layer was determined by scintillation counting. The reaction products in the heptane layer were identified as diacylglycerol (DAG) and TAG by thin-layer chromatography on silica gel G using a solvent system of heptane/isopropyl ether/acetic acid (60:40:4, by vol). The bands corresponding to DAG and TAG were scraped from the plates and the radioactivities were determined. The specific activity of MGAT was calculated from the radioactivity in DAG plus half that in TAG. Assay of GPAT. GPAT activity was determined by a previously described method (30) with slight modification (23). The reaction mixture consisted of 50 mM Tris-HCl buffer (pH 7.5), 0.12 M KCl, 1 mg bovine serum albumin (fatty acidfree), 100 µM palmitoyl CoA, 3 mM sn-glycerol-3-phosphate, [14C] glycerol-3-phosphate (0.2 µCi), and 50 µg of microsomal or mitochondrial protein in a final volume of 0.25 mL. The reaction was initiated by the addition of the microsomal or mitochondrial protein, and the mixture was incubated for 5 min (YSM) or 10 min (liver) at 30°C. The reaction was terminated by the addition of 2 mL water-saturated 1-butanol followed by 0.75 mL of butanol-saturated water.

ACYLTRANSFERASES OF THE CHICK EMBRYO

After shaking, the samples were centrifuged at 600 × g for 10 min, and the butanol layer was removed and washed three times with butanol-saturated water. The radioactivity in 1 mL of the washed butanol layer was determined by scintillation counting. To correct for any cross-contamination between subcellular organelles, we performed the assays after pre-incubation of the subcellular fractions at 37°C for 15 min in the presence or absence of 4.5 mM N-ethylmaleimide (NEM). The enzyme in the mitochondrial fraction showed no detectable inhibition by NEM indicating the absence of any contamination with the microsomal form. Up to 5% of the GPAT activity present in the microsomal fraction was resistant to inhibition by NEM, suggesting some contamination of the fraction by the mitochondrial form of the enzyme; the NEM-resistant activity was subtracted from the total microsomal activity in order to provide a corrected value for the activity of microsomal GPAT. Assay of DGAT. DGAT activity was assayed as previously described (22). The assay mixture consisted of 50 mM TrisHCl buffer (pH 7.5), 10 mM MgSO4, 0.25 mM dithiothreitol, 0.625 mg bovine serum albumin (fatty acid-free), 1.2 mM sn-1,2-DAG, 100 µM palmitoyl CoA, [14C]palmitoyl CoA (0.025 µCi), and 10 µg microsomal protein in a final volume of 0.5 mL. The sn-1,2-DAG was added as a dispersion in 0.1% (wt/vol) Tween 20; the final concentration of Tween 20 in the assay was 0.004% (wt/vol). The reaction was initiated by the addition of the microsomal protein, and the mixture was incubated at 37°C for 10 min. Termination of the reaction and extraction of the products were performed as described for the MGAT assay. Radioactivity associated with the TAG fraction isolated by thin-layer chromatography was used to calculate the enzyme activity. Assay of CPT-1. CPT-1 activity was measured in intact mitochondria as previously described (24). The assay mixture consisted of 5 mM Tris-HCl (pH 7.4), 0.15 M KCl, 5 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 5 mM ATP, 10 mg bovine serum albumin (fatty acid-free), 2 µg antimycin A, 4 µg rotenone, 250 µM palmitoyl CoA, 0.5 mM carnitine, [3H]carnitine (0.18 µCi), and 0.25 mg mitochondrial protein in a volume of 1 mL. The reaction was initiated by the addition of the mitochondrial protein, with the [3H]carnitine added 2 min later. The assay was for 2 min at 37°C and was terminated by the addition of 0.3 mL of 6 M HCl. The [3H]palmitoylcarnitine formed was quantified as described previously (25). Maximal CPT-1 activity was determined in the presence of 250 µM palmitoyl CoA whereas the sensitivity to inhibition by malonyl CoA was determined in the presence of 35 µM palmitoyl CoA. In the latter case, the enzyme activity in the presence of a range of malonyl CoA concentrations was determined and the sensitivity to this regulatory molecule was expressed as the ID50; i.e., the concentration (µM) of malonyl CoA which inhibited CPT-1 activity by 50%. In all experiments, the formation of palmitoylcarnitine was suppressed by over 90% by the highest malonyl CoA concentration (100 µM). This suggests that a high degree of membrane integrity was maintained in the mitochondrial

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preparations and that only CPT-1 activity was measured without any significant contribution from CPT-II. Protein determination. Mitochondrial and microsomal protein content was determined by the method of Lowry et al. (26) using bovine serum albumin as standard. Materials. L-[Methyl-3H]carnitine hydrochloride and 14 [1- C]palmitoyl CoA were obtained from Amersham International (Aylesbury, Buckinghamshire, United Kingdom). [U-14C]Glycerol-3-phosphate was obtained from ICN Biomedicals Ltd. (Thame, Oxfordshire, United Kingdom). Palmitoyl CoA was purchased from Pharmacia Biotech. (St. Albans, Hertfordshire, United Kingdom), and bovine serum albumin (fatty acid-free) was supplied by Advanced Protein Products (Brierley Hill, West Midlands, United Kingdom). All other biochemicals were obtained from Sigma Chemical Co. (Poole, Dorset, United Kingdom). Expression of results. Data are expressed as the mean ± SE of measurements on three to five replicate samples at each stage. In the case of the YSM, each replicate sample represents the subcellular fraction derived from an individual YSM. Because of the small size of the livers at days 10, 12, and 14, each replicate subcellular fraction was derived from four pooled livers. At the later developmental stages, the replicate samples were derived from individual livers. Statistical comparisons were performed using Student’s t-test. RESULTS MGAT activity in the YSM. The activity of MGAT in the microsomal fraction of the YSM was determined throughout the second half of the developmental period, from day 10 of embryonic life to 1 d after hatching (i.e., day 22). Very high levels of MGAT activity were maintained in the YSM between days 10 and 19 of embryo development, with a dramatic decrease (P < 0.001) in this activity then occurring betweeen days 19 and 20 (Fig. 1). GPAT activity in the YSM. The developmental changes in GPAT activity in the microsomal and mitochondrial fractions

FIG. 1. Activity of monoacylglycerol acyltransferase (MGAT) in the microsomal fraction of the yolk sac membrane during development. Values are means obtained from four yolk sac membranes at each stage, and vertical bars represent SE.

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FIG. 2. Activity of glycerol-3-phosphate acyltransferase (GPAT) in the microsomal (●) and mitochondrial (■ ■ ) fractions of the yolk sac membrane during development. Values are means obtained from four yolk sac membranes at each stage and vertical bars represent SE.

of the YSM are shown in Figure 2. The activity of the microsomal enzyme increased between days 10 and 12 (P < 0.05) and decreased continuously thereafter so that the activity at day 22 was only 25% (P < 0.001) of the peak value at day 12. Only a small proportion of the total YSM GPAT activity was accounted for by the mitochondrial form of the enzyme at all stages. For example, at day 12 the microsomes contained 92% of the total activity with only 8% present in the mitochondria. By day 19, the mitochondrial enzyme was undetectable. DGAT activity in the YSM. Substantial levels of DGAT activity were expressed in the microsomes of the YSM throughout the developmental period studied (Fig. 3). Although this activity appeared to reach a peak value around days 12 to 14 with a decrease thereafter, these changes were not statistically significant. CPT-1 activity in the YSM. Very high activities of CPT-1 were expressed in the mitochondria of the YSM at days 10 and 12 (Fig. 4). After this stage the activity decreased continuously with the result that the level of this enzyme at day 22 was only 27% of the peak value at day 12 (P < 0.001). The

FIG. 4. Activity of carnitine palmitoyl transferase-1 (CPT-1) in the mitochondrial fraction of the yolk sac membrane during development. Values are means obtained from three to five yolk sac membranes at each stage and vertical bars represent SE.

sensitivity of CPT-1 from the YSM of the embryo to inhibition by malonyl CoA did not change to any major extent during the embryonic period. The ID50 (µM) values for this inhibitory effect were 18.5 ± 2.4, 20.0 ± 2.0, 16.0 ± 1.1, 21.0 ± 3.4, and 15.0 ± 2.2 at days 10, 12, 14, 16, and 18, respectively. Enzyme activities in the liver of the embryo. The activity of MGAT in the embryonic liver increased almost threefold (P < 0.001) between days 12 and 19 of development and then decreased by about 50% (P < 0.001) over the hatching period (Table 1). These hepatic activities were, however, far lower than the activities of MGAT determined in the YSM. At day 12, for example, the level of MGAT in the liver was only 9% of the activity expressed in the YSM (P < 0.001). The activity of GPAT in the liver microsomes showed little change from day 12 to day 22 of development, was far lower than the activity of MGAT in the liver, and was also far lower than the activity of GPAT in the YSM. About 16 to 25% of the total GPAT expressed in the liver was due to the mitochondrial form of the enzyme. The hepatic DGAT activity increased by 2.3-fold (P < 0.05) from day 12 to day 19, and this elevated activity was maintained over the hatching period. The activities of DGAT in the liver at days 19 and 22 were not significantly different from the concurrent activities of this enzyme in the YSM. The activity of CPT-1 in the liver mitochondria TABLE 1 Acyltransferase Activities in the Liver of the Chick Embryoa Enzyme MGAT (n = 4) GPATmic (n = 6) GPATmit (n = 6) DGAT (n = 4) CPT-1 (n = 4)

FIG. 3. Activity of diacylglycerol acyltransferase (DGAT) in the microsomal fraction of the yolk sac membrane during development. Values are means obtained from four yolk sac membranes at each stage and vertical bars represent SE.

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Day 12 24.9 ± 4.3 2.7 ± 0.1 0.5 ± 0.1 9.8 ± 2.2 80.3 ± 13.7

Day 19

Day 22b

70.0 ± 2.7 2.5 ± 0.3 0.6 ± 0.1 22.5 ± 4.1 53.7 ± 6.2

38.0 ± 3.1 3.6 ± 0.3 1.2 ± 0.1 26.4 ± 4.7 52.0 ± 2.1

a nmol/min/mg microsomal (for MGAT, GPATmic , DGAT) or mitochondrial (for GPATmit, CPT-1) protein (mean ± SE). b Represents 1 d after hatching. MGAT, monoacylglycerol acyltransferase; GPATmic and GPATmit, glycerol-3-phosphate acyltransferase (microsomal and mitochondrial forms, respectively); DGAT, diacylglycerol acyltransferase; CPT-1, carnitine palmitoyl transferase-1.


was high at day 12, decreasing by about 33% at the later stages. The activity of CPT-1 in the liver at day 12 was, however, only about half the activity expressed in the YSM at this time (P < 0.01). As a measure of the sensitivity of hepatic CPT-1 to inhibition by malonyl CoA, the ID50 values were 11.0 ± 1.1 and 7.3 ± 0.8 µM at days 12 and 19, respectively. DISCUSSION A degree of caution is required in extrapolating the activities of the enzymes of glycerolipid synthesis as determined in vitro to the actual levels of expression of these activities in vivo, particularly since these assays involve membranebound enzymes and hydrophobic substrates/products. However, with this proviso, a major finding of the present study is that the YSM of the chick embryo displays an exceptionally high enzymic capacity for glycerolipid synthesis. This is particularly evident in the case of MGAT, which is expressed in the YSM at an unprecedented level compared with the reported activities for other avian or mammalian tissues at various developmental stages (22,27–29). Even the intestinal mucosa of adult mammals, regarded as the classic tissue for the operation of the monoacylglycerol pathway, exhibits much lower MGAT activities than those reported here for the YSM. For example, the activity (nmol/min/mg protein) of this enzyme in the intestinal mucosa of the neonatal (28) and adult (22) rat is approximately 100 compared with a peak of almost 300 in the YSM. Apart from its expression in the intestine, the tissue-specific distribution of MGAT in the adult mammal is very restricted (15). In particular, the adult liver is normally almost devoid of MGAT activity although this enzyme is transiently expressed in neonatal rat liver, attaining a level that is 30–40% of that achieved by the YSM (28). Thus the YSM of the avian embryo should be added to the select list of animal tissues possessing high MGAT activity. The activity of GPAT in the YSM is also very high in comparison with the reported levels of this enzyme in tissues of the adult mammal. For instance, the activities of this enzyme (nmol/min/mg protein) in the liver and intestine of the adult rat are approximately 6.0 and 0.5, respectively (22), compared with 26.7 in the YSM at day 12. The activity of DGAT, the only enzyme concerned exclusively with TAG synthesis, was also relatively high in the YSM. For example, the DGAT activities (nmol/min/mg protein) in the liver and intestine of the adult rat are approximately 3.0 and 5.0, respectively (22), compared with a maximum of 36.1 in the YSM. An additional point is that the activities of MGAT, GPAT, and DGAT are far higher in the YSM than in the developing liver, or indeed in any other tissue, of the chick embryo. These activities have previously been reported for the liver and certain other chick embryo tissues (14), and our present values obtained for the liver are consistent with this earlier study. The overall conclusion is that the YSM possesses an almost unprecedented enzymic potential for the synthesis of glycerolipids via the acylation of partial glycerides or glycerol-3phosphate.

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This conclusion clearly lends support to the hypothesis that, during their transit across the YSM, the lipids of the yolk are hydrolyzed to their constituent free fatty acids, glycerol, partial glycerides, free cholesterol, and the like, which are then reesterified to resynthesize TAG, PL, and CE in a process coupled to the assembly of VLDL particles. The fact that MGAT activity in the YSM was greater than that of GPAT by an order of magnitude may be taken to indicate that the monoacylglycerol pathway forms the predominant means of reesterification in this tissue, with the glycerol phosphate pathway performing a supporting role. Previous observations that the YSM also displays very high levels of acyl-CoA:cholesterol acyltransferase activity (ACAT) (30) as well as a high expression of the mRNA for apoprotein B (31) are also consistent with this view. Complementary to these biochemical studies is the evidence from electron microscopy (1–5). In brief, this indicates that yolk droplets and granules are engulfed by the apical surface of the YSM endodermal cells and that this uptake is followed by the fusion of the phagocytotic vesicles within the cell to form large lipid-rich vacuoles. During the second half of the embryonic period, this uptake is so intense that these lipid-rich vacuoles occupy a large proportion of the cytoplasmic space and totally dominate the appearance of the cells. Meanwhile, the opposite side of the cell is observed to be extremely active in the exocytosis of lipoproteins into the vascular system of the YSM. Lipid-rich spherules corresponding to VLDL particles are packed into the cisternae of the endoplasmic reticulum and Golgi. Moreover, secretory vesicles containing VLDL particles can be observed in the process of fusion with the basal plasma menbrane, thus releasing these lipoproteins into the circulation. The results described in the present study may help to define the biochemical processes that link the absorptive events at the apical surface with the subsequent secretory events at the basal surface of the endodermal cells. There is evidence that, as a result of fusion with lysosomes, the large lipid-rich vacuoles formed by the phagocytotic process are converted to socalled lipolysosomes in which the hydrolysis of the vacuolar contents proceeds at a high rate, catalyzed by lysosomal lipases and proteases (32). We propose that the products of this hydrolysis (free fatty acids, partial glycerides, glycerol, free cholesterol, lyso-PL, and the like) diffuse out of the large vacuoles and are transported to the endoplasmic reticulum where reesterification to synthesize TAG, PL, and CE is rapidly achieved by the very high activities of MGAT, GPAT, DGAT, acylCoA:acyltransferase, and presumably of the other enzymes required for complex lipid synthesis. The coordination of this reesterification process with the translation of the mRNA for apoprotein B (31) will promote the assembly of VLDL particles in the lumen of the endoplasmic reticulum (13). The exceptional activity of MGAT in the YSM implies that sn-2monoacylglycerol is a major product of yolk lipid hydrolysis in the large vacuoles with the further inference that the lysosomal lipase which acts on the yolk-derived TAG preferentially hydrolyzes the ester bonds at the sn-1 and -3 positions. Lipids, Vol. 34, no. 9 (1999)

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The very high activity of CPT-1 in the mitochondria of the YSM, as reported in the present study, is indicative of an exceptionally high capacity for β-oxidation in this structure. The maximal CPT-1 activity measured in the YSM was about three times greater than the highest levels of this enzyme reported for the rat liver under any physiological state (17,18,24). Furthermore, the YSM enzyme was relatively insensitive to the physiological inhibitor, malonyl CoA. In fact, the CPT-1 activity of the adult rat liver is almost 10 times more sensitive to malonyl CoA inhibition compared with the YSM enzyme (17,18). These results suggest that a proportion of the yolk-derived lipid is oxidized in the mitochondria of the YSM in order to provide the energy for the translocation of the bulk of lipid across the endodermal layer. The present paper also reports for the first time the developmental expression of CPT-1 activity in the liver of the chick embryo; at day 12, this hepatic activity was only about half that concurrently expressed in the YSM but was nevertheless about 50% greater than the highest activities reported for the liver of the adult rat (17,18,24). CPT-1 in the mitochondria of the chick embryo liver was more sensitive than the YSM enzyme to inhibition by malonyl CoA, but was still relatively resistant to this inhibitor when compared with the enzyme from the liver of the adult rat (17,18). In the present study, we also determined the activity of CPT-1 in mitochondria isolated from the liver of the adult chicken (female, fed state). The activity of this enzyme in the adult was 20.8 ± 1.4 nmol/min/mg mitochondrial protein (n = 4 livers), far lower than the values obtained from the YSM and liver of the embryo at all stages. The aim of the present work was to determine the activities of the acyltransferases in the YSM during the second half of the developmental period when the transfer of lipid from the yolk to the embryo is most intensive. The results indicate that the maximal or near-maximal activities of these enzymes have already been attained by day 10 of development, the earliest time-point studied. In future work, it would be of interest to measure these enzyme activities in the YSM during the first half of the developmental period in order to delineate the ontogeny of their expression and to determine the timing of their induction. ACKNOWLEDGMENTS We are grateful to the Scottish Office Agriculture Environment and Fisheries Department for financial support.

REFERENCES 1. Speake, B.K., Murray, A.M.B., and Noble, R.C. (1998) Transport and Transformations of Yolk Lipids During Development of the Avian Embryo, Prog. Lipid Res. 37, 1–32. 2. Noble, R.C., and Cocchi, M. (1990) Lipid Metabolism and the Neonatal Chicken, Prog. Lipid Res. 29, 107–140. 3. Lambson, R.O. (1970) An Electron Microscopic Study of the Endodermal Cells of the Yolk Sac of the Chick During Incubation and After Hatching, Am. J. Anat. 129, 1–20. 4. Mobbs, I.G., and McMillan, D.B. (1979) Structure of the Endodermal Epithelium of the Chick Yolk Sac During Early Stages of Development, Am. J. Anat. 155, 287–310.

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[Received April 26, 1999, and in revised form July 26, 1999; revision accepted August 10, 1999]

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