Fatty Acid Synthetase Activity Research Articles

Dietary Regulation of Fatty Acid Synthetase and Microsomal Glycerophosphate Acyltransferase Activities in Rat Liver

Investigations were carried out to determine the response of rat hepatic soluble fatty acid synthetase (FAS) and microsomal glycerophosphate acyltransferase (GPAT) to qualitative and quantitative differences in dietary fat. The effects on these enzymes of fasting and feeding after a fast were also studied. FAS activities of livers of rats consuming either fat-free (FF), 2.5% or 5.0% cocoa butter (CB) diet for 1 week were equal. However, 2.5% and 5.0% safflower oil ( SO ) diets reduced FAS activities by 25% and 40%, respectively, and stock diet or fasting for 3 days reduced it 20% and 5%, respectively. Refeeding FF, 5.0% SO or 5.0% CB for 2 days following a 3-day fast resulted in a 50- to 60-fold increase in FAS activity. Maximum activities of FAS were reached between days 2 and 3 of refeeding regardless of the type of diet. Fat in the diet, saturated or unsaturated, did not inhibit this adaptive increase in FAS activity during the first few days. However, at day 7 of refeeding there was a highly significant drop in FAS activities of rats fed 5.0% SO while that of those fed either FF or 5.0% CB remained high. Standard GPAT assay conditions were developed using microsomes from rats maintained on a stock diet. Under these conditions the average specific activity of GPAT was 5.2 nmoles phosphatidic acid synthesized per minute per milligram microsomal protein. GPAT was not significantly different after fasting for 3 days, ingestion of diets containing 2.5%, 5.0%, 10%, or 15% SO or CB, nor after stock or FF diets. However, refeeding the FF, 5.0% SO, or 5.0% CB diets for 2 days following a 3-day fast increased its activity by 40% over the prefasting or fasting levels. It returned to the prefasting level between days 3 and 5 of the refeeding period. These studies show that the hepatic GPAT activity is relatively constant while the FAS activity varies with qualitative and quantitative dietary fat differences. J. Nutr. 103: 1414-1424, 197.3.

Regulation by dietary choline of hepatic fatty acid synthetase in the rat

Fatty acid synthetase activity was measured in the high-speed supernatant fraction of liver homogenates from rats fed a semisynthetic diet low in lipotropic factors. If choline was omitted from the diet, a significant increase of fatty acid synthetase activity was observed after two feedings of the deficient diet. Compared with controls, the increase of fatty acid synthetase activity was of a magnitude that could account for the amount of triglyceride accumulating in the hepatic floating lipid fraction. Gas-liquid chromatographic analysis of the floating lipid triglycerides showed an increased content of palmitic acid due to choline deficiency; this increase could be predicted from the increased fatty acid synthetase activity and its known characteristic yield of palmitic acid.

Metabolic enzymes in liver and kidney of the germfree rat

The effect of intestinal microflora on the activities of NADP-dependent dehydrogenases in liver and kidneys, and on fatty acid synthetase, ATP-citrate lyse, cytochrome oxidase and succinate dehydrogenase activities in liver of rats were studied. Germfree rats showed a decrease in liver succinate dehydrogenase activity, and no effect on liver cytochrome oxidase activity. The absence of an intestinal microflora resulted in a significant decrease of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities, and a substantial increase of ATP-citrate lyase and fatty acid synthetase activities in the liver. Liver malate dehydrogenase activity was comparable in both germfree and conventional animals. Germfree status had no effect on the activities of NADP-dependent dehydrogenases in the kidney. It is concluded that the absence of an actively metabolizing microflora was associated with quantitative shifts in the activity of hepatic enzymes involved in the metabolism of carbohydrates and fats.

Relative activities of acetyl-CoA carboxylase and fatty acid synthetase in chick liver: effects of dietary fat.

The specific activities of acetyl-CoA carboxylase and fatty acid synthetase were measured in the cytosol fraction of livers from chicks fed various levels of corn oil, cottonseed oil, corn-oil free fatty acids, or crude (79%) oleic acid. Activities of both enzymes were depressed by the addition of fat to a fat-free basal diet. The ratios of synthetase to carboxylase activity were greater than unity when up to 4% fat was fed, but less than unity when 8% or higher levels of fat were fed. The depressions of the activities of these enzymes appeared to be unrelated to the dietary level of linoleate. In in vitro experiments, 2 μM concentrations of palmityl-CoA or oleoyl-CoA depressed acetyl-CoA carboxylase activity. Concentrations of 20 μM of these acyl-CoA esters did not affect the activity of fatty acid synthetase.

Fatty acid synthetase activity in Euglena gracilis variety bacillarius. Characterization of an acyl carrier protein dependent system.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTFatty acid synthetase activity in Euglena gracilis var bacillarius. Characterization of an acyl carrier protein dependent systemMary L. Ernst-FonbergCite this: Biochemistry 1973, 12, 13, 2449–2455Publication Date (Print):June 1, 1973Publication History Published online1 May 2002Published inissue 1 June 1973https://doi.org/10.1021/bi00737a013RIGHTS & PERMISSIONSArticle Views52Altmetric-Citations25LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (885 KB) Get e-Alerts Get e-Alerts

On the Relationship between Fatty Acid Synthesis and the Total Activities of Acetyl Coenzyme A Carboxylase and Fatty Acid Synthetase in the Liver of Prenatal and Early Postnatal Chicks

Abstract Fructose and lactate markedly stimulated fatty acid synthesis from [1-14C]acetate in isolated cells prepared from the livers of unfed neonatal chicks (21 or 22 days of incubation). In liver cells obtained from unhatched chicks (19 days of incubation) fructose and lactate had very small stimulatory effects on fatty acid synthesis. The increase in fructose- and lactate-stimulated fatty acid synthesis during hatching was about 20-fold. Pulmonary respiration began on the 20th day and the chicks were hatched by the 21st day. Fatty acid synthesis from [1-14C]acetate (plus citrate) or [1,5-14C]-citrate in a cytosol fraction of liver increased about 20-fold during the hatching period. Concomitantly, there were 6.5- and 4-fold increases in the total activities of acetyl-CoA carboxylase and fatty acid synthetase, respectively. The effect of hatching on the total activity of fatty acid synthetase could be mimicked by incubating 18-day-old embryos in an atmosphere of 100% oxygen for 24 hours. The oxygen treatment did not affect the total activity of acetyl-CoA carboxylase or the rate of fatty acid synthesis in isolated hepatocytes (plus or minus fructose). Injecting glucose or fructose into 18-day-old embryos had no effect on the total activity of acetyl-CoA carboxylase or fatty acid synthetase. Feeding the neonatal chicks increased the total activities of both acetyl-CoA carboxylase and fatty acid synthetase. If the neonatal chicks were not fed, the total activity of acetyl-CoA carboxylase did not change for at least 3 days. In contrast, the total activity of fatty acid synthetase continued to increase during those 3 days of fasting. The total activities of both enzymes were increased when unfed neonatal chicks were given a single glucose or fructose injection. The effect was not observed until 6 hours after administration of the sugar despite the fact that the rate of fatty acid synthesis was stimulated by more than 5-fold at 1½ hours after the injection. The results suggest that acetyl-CoA carboxylase and fatty acid synthetase are not part of an operon or its eukaryote equivalent, and that changes in their total activities did not initiate changes in the rate of fatty acid synthesis.

Regulation of Fatty Acid Synthesis in the Liver of Prenatal and Early Postnatal Chicks: HEPATIC CONCENTRATIONS OF INDIVIDUAL FREE FATTY ACIDS AND OTHER METABOLITES

Abstract A normal mash diet or a single glucose injection stimulates fatty acid synthesis and increases the total activities of acetyl coenzyme A carboxylase, fatty acid synthetase, and malic enzyme in the livers of neonatal chicks. Feeding and glucose injection caused a decrease in the concentration of free fatty acids and fatty acyl-CoA and an increase in α-glycerophosphate and free CoA. These concentration changes are appropriate for intermediates which may regulate both the rate of fatty acid synthesis and the concentration of the lipogenic enzymes. The hepatic concentrations of citrate and acetyl-CoA were unaffected by feeding or glucose injection. During the hatching process, total activities of acetyl-CoA carboxylase and fatty acid synthetase in the liver increased but the rate of fatty acid synthesis did not. Concomitantly, free stearate in the liver decreased, but none of the other metabolites indicated above were affected. Fatty acid synthetase increased and hepatic free stearate decreased when embryos were incubated in 100% oxygen. Stearate or a metabolite derived from stearate is a potential negative regulator of the concentration of fatty acid synthetase in chick liver. Injection of glucose into 18-day-old embryos markedly increased the α-glycerophosphate content of the liver but had no effect on the total activities of the lipogenic enzymes. Hence, α-glycerophosphate is not a direct regulator of the total activities of acetyl-CoA carboxylase or fatty acid synthetase in embryonic chicks.

Control of the synthesis of fatty-acid synthetase in rat liver by insulin, glucagon, and adenosine 3':5' cyclic monophosphate.

The usual increase in the activity of liver fatty-acid synthetase that occurs on refeeding of a fat-free diet to previously fasted rats is abolished in diabetic animals. Insulin specifically restores this increase by enhancement of the rate of synthesis of fatty-acid synthetase. However, glucagon and cyclic AMP inhibit the increase in the activity of fatty-acid synthetase. Therefore, the concentration of fatty-acid synthetase in rat liver is under the control of the relative concentrations of insulin and glucagon.

The Concentration of Malonyl-Coenzyme A and the Control of Fatty Acid Synthesis in Vivo

An enzymatic method has been developed for the measurement of malonyl-CoA in perchloric acid extracts of liver. With this method the relative activities of acetyl-CoA carboxylase and fatty acid synthetase have been studied in vivo by comparing the diet-induced changes in malonyl-CoA concentrations with rates of 3H2O incorporation into fatty acids. Malonyl-CoA concentrations were low in the fat-fed or 48-hours starved animal (0.004 to 0.006 µmole per g wet weight) but approached normal-fed levels (0.013 µmole per g wet weight) within 3 hours of refeeding the starved animal. The highest concentrations of malonyl-CoA (0.025 µmole per g wet weight) were found in the meal-fed animal. The de novo rates of fatty acid synthesis in vivo varied between 0.02 µmole of C2 units per min per g wet weight of liver in the starved or fat-fed animals to 0.45 µmole, of C2 units per min per g in the meal-fed group. Malonyl-CoA concentrations and the rate of tritium incorporation into fatty acids were increased above starved values in all fed groups except that group fed a high fat diet. A number of intermediary metabolites which have been proposed as controllers of the rates of fatty acid synthesis have been measured in freeze-clamped liver. There was no correlation between the rate of fatty acid synthesis and the liver content of citrate, ATP, ADP, glucose, glucose 6-phosphate, or α-glycerophosphate. In addition, short term control did not appear to be exerted by free mitochondrial [NAD+]:[NADH], free cytoplasmic [NAD+]:[NADH], or [NADP+]:[NADPH], energy charge or phosphorylation state. Increases of the malonyl-CoA content and the rate of fatty acid synthesis above the starved or fat-fed values occurred when the tissue content of long chain acyl-CoA decreased below starved values. It was concluded that short term control of fatty acid synthesis in vivo most likely results from an inhibition of acetyl-CoA carboxylase by long chain acyl-CoA. The variations in the rate of fatty acid synthesis which occurred in the carbohydrate-fed groups probably result from variations in the quantity of the enzyme fatty acid synthetase.

Fatty acid synthetase activity in the liver and adipose tissue of rats fed with various carbohydrates.

1. The inclusion of sucrose in the diet of rats led to an increase in hepatic fatty acid synthetase activity compared with that of rats fed with starch as the sole carbohydrate. The higher activity occurred within 18h of the introduction of sucrose and persisted with fluctuations for the 30 days of the experiment. Reversal of the diets in some rats after 21 days led to changes in the enzyme activity to values appropriate to the second diet. The plasma triglyceride concentration followed a similar pattern. 2. A comparison of the effects of diets with starch, glucose, maltose, sucrose or fructose showed that fructose gave the highest values of triglyceride content and of fatty acid synthetase activity in liver, but the lowest values of the synthetase activity in adipose tissue and the lowest values of plasma insulin concentration. These effects may perhaps be attributed to the low insulin response to fructose and to the high affinity of the liver for this sugar. 3. When the diet contained fructose or sucrose there was a correlation between hepatic synthetase activity and plasma triglyceride concentration. Neither of these, however, was related to plasma insulin concentration. On the other hand, there was a correlation between plasma insulin concentration and fatty acid synthetase activity in adipose tissue. 4. When rats were starved and then re-fed the differences in enzyme activities induced by fructose or glucose were minimized. This, together with the varying degree of difference during the course of the experiments, may explain why other workers, using the starvation-re-feeding technique and making measurements on one day only, have failed to observe differences in the activities of lipogenic enzymes in animals fed with either fructose or glucose.

In vitro regulation by nadph of fatty acid biosynthesis with larval and pharate adult homogenates of the fly, Ceratitis capitata

Acetyl-CoA carboxylase and fatty acid synthetase activities have been studied in larval and pharate adult homogenates of Ceratitis capitata under the influence of various concentrations of NADPH. NADPH, 0.5–1.0 μmole per ml., activates acetyl-CoA carboxylase, whereas 1.0–5.0 μmoles per ml. produces a marked decrease, using either labelled acetate or acetyl-CoA as substrates. This NADPH influence cannot be interpreted through a concomitant activation of the synthesis of fatty acids from malonyl-CoA, since the incorporation of labelled acetate and acetyl-CoA into fatty acids becomes negatively regulated by NADPH in the same interval, 1.0–5.0 μmoles per ml. The NADPH effect is discussed through its competitive influence with respect to malonyl-CoA on the fatty acid synthetase.

Comparative effects of dietary regimens on the levels of enzymes regulating the synthesis of fatty acids and cholesterol in rat liver

The activities, in subcellular fractions of rat liver, for acetyl-CoA carboxylation, fatty acid synthesis, and β-hydroxy-β-methylglutaryl-CoA reduction and for the overall conversion of acetate to cholesterol were determined for animals in different nutritional states. Acetyl-CoA carboxylase and fatty acid synthetase activities increased or decreased together whenever the nutritional state was changed. A similar relationship was observed between β-hydroxy-β-methylglutaryl-CoA reductase activity and the synthesis of cholesterol from acetate. Feeding a fat-free diet after either a normal or fasted regimen increased the activity of acetyl-CoA carboxylase and fatty acid synthetase but suppressed the activities for β-hydroxy-β-methylglutaryl CoA reduction and cholesterol synthesis. Thus, the quantity of fat in the diet apparently regulates the levels of activity of the key enzymes of two divergent pathways in an opposite manner. The levels of these enzymes in turn control the proportion of acetyl-CoA used by each pathway. When a diet containing 2% cholesterol is fed, the levels of β-hydroxy-β-methylglutaryl-CoA reductase activity and cholesterol synthesis in liver are markedly depressed. In contrast, the levels of acetyl-CoA carboxylase and fatty acid synthetase activities are unaffected. These results suggest that cholesterol and fat exert their effects on the cholesterol-synthesizing and fatty acid-synthesizing pathways by different mechanisms.

Fatty Acid Synthetase from Chlamydomonas reinhardi: SITES OF TRANSCRIPTION AND TRANSLATION

Abstract Cell-free extracts of the unicellular green alga, Chlamydomonas reinhardi, catalyze the incorporation of acetyl-CoA and malonyl-CoA into long chain fatty acids. The fatty acid synthetase is dependent on added acyl carrier protein for activity, regardless if the cells are grown in the light or in the dark. The major products formed from acetyl-CoA and malonyl-CoA by cells in the light period of synchronous growth are palmitate, stearate, and arachidate, whereas in the dark period of synchronous growth more short fatty acids are formed. Rifampicin has no effect on the appearance of the fatty acid synthetase in synchronous cultures of C. reinhardi, thus suggesting that transcriptional steps necessary for formation of fatty acid synthetase occur in the nucleus. Cycloheximide has no effect on the activity of fatty acid synthetase in the synchronous cultures, whereas spectinomycin reduced the activity significantly. These findings strongly indicate that the chloroplast ribosomes are responsible for the de novo protein synthesis required for fatty acid synthetase in C. reinhardi.

Fatty acid synthetase of brain: development, influence of nutritional and hormonal factors and comparison with liver enzyme.

Abstract— The activity of fatty acid synthetase was studied in the brain and liver of the developing rat. Synthetase activity in brain was considerably higher in foetal and suckling rats than in older animals However, except for a small transient rise in the perinatal period, activity in liver was low until weaning when a dramatic rise occurred. Activity in brain varied according to the quantity of dietary fat only in long‐term experiments, whereas in liver nutritional influences clearly predominated in determining the rapid developmental changes of synthetase activity. Administration of hydrocortisone diminished hepatic activity but did not change brain synthetase. In the hypothyroid state activity in brain and liver was consistently decreased. However, in the hyperthyroid state hepatic activity increased but activity in brain did not change. The relatively high activity of fatty acid synthetase during brain development has been discussed in relation to the critical role of this enzyme system in brain metabolism. The effect of the hypothyroid state on the activity of brain synthetase suggests the possibility of hormonal control of this enzyme activity. The responses of hepatic synthetase to the hormonal influences delineate a specific step by which these compounds may exert their effect on fatty acid biosynthesis.

Effect of hydrogenated soybean fat on rat adipose tissue and on liver acetyl-CoA carboxylase and fatty acid synthetase activities

Effect of hydrogenated soybean fat on rat adipose tissue and on liver acetyl-CoA carboxylase and fatty acid synthetase activities

Fat metabolism in higher plants: LI. Palmitic and stearic synthesis by an avocado supernatant system

The synthesis of palmitic and stearic acids by soluble proteins from avocado mesocarp has been examined. Evidence suggests that palmitate is made de novo and stearate by elongation of a C 16 precursor. While the synthesis of palmitate from malonyl CoA is arsenite insensitive, the elongation reaction from palmitate to stearate is arsenite sensitive. The pattern of synthesis of the two acids is neither dependent on the malonyl CoA/acetyl CoA ratio nor is altered by the addition of exogenous lipids and detergents. The reaction products are 20% thiol esters and 80% free fatty acids. When the incubation system was separated by gel filtration on Sephadex G-75 or G-150 columns two protein peaks were rapidly eluted. Over 60% of the 14C fatty acids were eluted with the protein peaks, the majority of the 14C fatty acids being in the first peak which contained only 10% of the total protein. The predominant acid of the first peak was stearate with 85% as the free acid. The second peak contained 30% thiol esters, including shorter-chain acids. It also contained fatty acid synthetase activity and [ 3H]ACP. When [ 14C-malonyl] CoA is added to a reaction mixture containing protein from this peak, palmitate is found exclusively. Thus, the de novo system is associated with this peak. The elongation system is no longer demonstrable with peaks 1 or 2. Unspecific binding of free fatty acids was negligible. [ 14C]Palmityl-acyl carrier protein was elongated to stearate by an acetone powder of avocado supernatant fraction. It was a more effective precursor than [ 14C]palmitic acid or [ 14C-palmityl] CoA. From these results we conclude that avocado supernatant fraction contains a fatty acid synthetase-forming palmitate and an elongation system which forms stearate by malonyl CoA addition to a C 16 precursor, possibly palmityl-ACP. The latter system is arsenite sensitive.

Mode d'action de l'insuline et du jeûne sur la synthèse des acides gras du foie et du tissu adipeux

Summary Homogenates and particle-free (100 000 g) supernatants of the liver or epididymal adipose tissue from fed or 24 hours fasted rats were incubated in the presence of either acetyl-CoA 14 C or malonyl-CoA 14 C. The animals were treated or not with insulin/or actinomycin D. Unlabeled glucose was, or not, added to the incubation medium. The radioactivity of the fatty acids synthesized from these labeled precursors, as well as the activities of acetyl-CoA carboxylase and fatty acid synthetase, were measured. In the liver tissue of fasted rats, insulin administrated i.p. 3 hours before their killing, stimulated the activity of both acetyl-CoA carboxylase and fatty acid synthetase. Actinomycin D removed this effect of insulin. In adipose tissue, no inhibition of fatty acid synthesis either from acetyl-CoA 14 C or from malonyl-CoA 14 C was observed. Insulin was without effect on the incorporation of these precursors, whether or not glucose was added to the incubation medium. In vivo lack of NADPH and not an enzymatic deficiency or a decrease in the tissue ability to generate acetyl-CoA from glucose seems to be the limiting factor of the fatty acid synthesis in adipose tissue of 24 hours fasted rats and the action of insulin may be simply explained by the increase of glucose uptake.

Activity of lipogenic enzymes and plasma triglyceride levels in the rat and the chicken as determined by the nature of the dietary fat and dietary carbohydrate.

The plasma triglyceride levels of rats fed diets containing sucrose were higher than those of rats fed starch. The presence of saturated fat in the diet further elevated these levels. No significant differences were found in the activities of hepatic lipogenic enzymes in groups fed saturated or polyunsaturated fat, although these activities were much higher after feeding sucrose. The activities of lipogenic enzymes in the adipose tissue were very low. It is suggested that sucrose elevates plasma triglycerides by stimulating hepatic lipogenesis, whereas saturated fat may produce the same effect by reducing plasma clearance. In cockerels, no significant difference was found in the plasma triglyceride levels of those fed either sucrose or starch or with saturated or polyunsaturated fat. Sucrose increased hepatic fatty acid synthetase activity, while polyunsaturated fat decreased it. It was presumed that efficient plasma clearance prevented any rise in plasma triglyceride concentrations.

Fatty Acid Synthetase from Lactating Rat Mammary Gland: III. DISSOCIATION AND REASSOCIATION

Abstract Fatty acid synthetase from lactating rat mammary gland is shown to be a cold-labile multienzyme complex. The native (13 S) form slowly dissociates into half-molecular weight (9 S) subunits on aging in the cold. Fatty acid synthetase activity observed on addition of the 9 S subunits to the assay system between 20 and 30°, is shown to result from rapid reassociation of the subunits to the 13 S form. Dissociation of the enzyme into subunits is accompanied by a change in the number of protein sulfhydryl groups accessible to 5,5'-dithiobis(2-nitrobenzoate), but reassociation to the parent form is not dependent on the presence of a reduced thiol. The dissociation does not involve oxidation of protein sulfhydryl groups, as the total number of sulfhydryl groups titratable with 5,5'-dithiobis(2-nitrobenzoate) in the presence of 6 m urea was the same in the native enzyme and its subunits. Blocking more than two of the 28 subunit sulfhydryl groups with p-chloromercuribenzoate restricted the ability of the subunits to undergo heat-induced reassociation. Evidence is presented which suggests that although protein sulfhydryl groups may be involved in the dissociation-reassociation phenomenon, hydrophobic bonding is probably a more critical factor. Similar studies carried out with the fatty acid synthetase purified from rat liver indicate that this enzyme too exhibits cold lability.

Intracelluar localizaion of fatty acid synthesis in chick-embryo liver and stimulation of synthesis by exogenous glucose

In homogenates of chick-embryo liver and in the subcellular fractions isolated from those homogenates the mitochondrial system of fatty acid elongation and fatty acid synthetase can be measured independently. The assay medium for measuring the microsomal system of fatty acid elongation also measures some mitochondrial elongation activity. Acetyl-CoA carboxylase (acetyl-CoA: carbon dioxide ligase (ADP), EC 6.4.1.2) can be measured independently in the cytosol but not in the crude homogenates, presumably because of the activity of the mitochondrial enzymes pyruvate carboxylase (pyruvate: carbon dioxide ligase (ADP), EC 6.4.1.1) and propionyl-CoA carboxylase (propionyl-CoA: carbon dioxide ligase (ADP), EC 6.4.1.3). 1. 2. The predominant system of fatty acid synthesis in chick-embryo liver is mitochondrial elongation ; microsomal elongation and fatty acid synthetase activities are low in comparison. 2. 3. The activities of fatty acid synthetase and acetyl-CoA carboxylase in livers of 15-day embryos can be stimulated by injection of 180 mg of glucose into eggs at 4.5 days of incubation.