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Published December 4, 2014 Glycerol is a major substrate for glucose, glycogen, and nonessential amino acid synthesis in late-term chicken embryos1,2,3 N. E. Sunny4 and B. J. Bequette5 Department of Animal and Avian Sciences, University of Maryland, College Park 20742 ABSTRACT: The objective was to determine the contributions of glucose to glycogen synthesis and glycerol to glycogen, glucose, and nonessential AA (NEAA) synthesis on embryonic day (e) 14/15 and e19/20. Chicken embryos from small (56.6 ± 0.88 g) and large eggs (71.7 ± 1.09 g) were repeatedly dosed with either [13C3]glycerol (14 mg/d for 4 d) or [13C6]glucose (15 mg/d for 3 d) into the chorio-allantoic fluid before blood and tissue collection. 13C-Mass isotopomer enrichments in blood glucose, liver, and muscle glycogen, and blood and tissue NEAA were analyzed by mass spectrometry. Glucose metabolism did not differ between small- and large-egg embryos. Although glucose entry was 60% less for e20 compared with e15 embryos, e20 embryos conserved glucose more efficiently as a result of 2- to 3-fold greater (P < 0.001) rates of glucose carbon recycling. Importantly, the direct contribution of glucose to liver glycogen synthesis was minimal on e15, and on e20 direct incorporation of glucose into liver glycogen was only 17%. By comparison, [13C3]glycerol dosing led to the appearance of [M + 1], [M + 2], and [M + 3] isotopomers in blood glucose and in liver and muscle glycogen on e14 and e19. Here, the 13C-isotopomer enrichments in blood glucose were ~2-fold greater (P < 0.05) in small- than in large-egg embryos on e14 and e19. Furthermore, [13C3]glycerol dosing led to substantial labeling of [M + 1], [M + 2], and [M + 3] isotopomers of alanine, aspartate, and glutamate in blood and in tissues where 13C enrichments were greater (P < 0.05) in liver of small-egg embryos. In summary, this study provides unequivocal evidence that glycerol is a precursor for glucose and NEAA synthesis. Furthermore, glycerol, but not egg-derived glucose, is a major substrate for synthesis of liver and muscle glycogen and is an important anaplerotic substrate for the tricarboxylic acid cycle of embryos during later development. Key words: chicken embryo, glucose, glycerol, glycogen, metabolism, stable isotope ©2011 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2011. 89:3945–3953 doi:10.2527/jas.2011-3985 INTRODUCTION The limited carbohydrate content of the egg (<3%; Romanoff and Romanoff, 1967; Hu et al., 2011) necessitates maximal gluconeogenesis (Sunny and Bequette, 2010) and glycogen synthesis (Hazelwood, 1971) for embryonic survival and energy metabolism. Dynamic 1 Funded by a Maryland Agricultural Experiment Station grant to B. J. Bequette. 2 Presented in part at the annual meeting of the Federation of Animal Science Societies on July 7 to 11, 2008, Indianapolis, IN (N. E. Sunny, J. Moorefield, S. L. Owens, and B. J. Bequette. 2008. Use of glycerol for glucose, glycogen and non-essential amino acid synthesis by embryos from small and large chicken eggs.) 3 Author disclosures: N. E. Sunny and B. J. Bequette, no conflicts of interest. 4 Present address: Advanced Imaging Research Center, University of Texas Southwestern Medical Center, 2201 Inwood Rd., Dallas, TX. 5 Corresponding author: [email protected] Received February 18, 2011. Accepted June 30, 2011. changes in gluconeogenic flux and enzyme activities follow hormonal cues (Pearce, 1971, 1977; Lu et al., 2007; Sunny and Bequette, 2010), with a parallel increase in blood glucose from 6 to 8 mM in premature embryos to 10 to 12 mM by 2 to 3 wk posthatch (Hazelwood, 1971). Similarly, tissue glycogen begins to accumulate by embryonic day (e) 6 (via the uronic acid pathway), peaking on e12, declining 50% by e13, and then increasing >4-fold by e20 (Hazelwood, 1971). Even though several substrates can serve as precursors for glucose and glycogen synthesis (Langslow, 1978; Brady et al., 1979), their preference varies with availability, stage of embryonic development, and tissue localization of enzymes. Thus, lactate could be a contributor to gluconeogenesis during anaerobic respiration (Brady et al., 1979; Morgan, 2007), but because lactate ultimately derives from metabolism of other substrates (e.g., AA, glycerol), it would not make a net contribution. By contrast, AA and glycerol are the important substrates for gluconeogenesis in the kidney of posthatch chicks (Watford et al., 1981; Magnuson et al., 2003); thus these substrates are also likely to be im- 3945 3946 Sunny and Bequette portant contributors to gluconeogenesis by the embryo. However, glutamate and glutamine, which form ~14% of egg protein, were found to be insignificant contributors to glucose synthesis in e19 embryos (Sunny et al., 2007). Extensive metabolism of yolk triglyceride in the liver of late-term embryos releases fatty acids for energy (Deeming and Ferguson, 1991; Sato et al., 2006) and an abundant supply of glycerol, a potential substrate for glucose, glycogen, and nonessential AA [NEAA; via tricarboxylic acid (TCA) cycle metabolism] synthesis. Employing an in ovo [13C3]glycerol injection approach, we determined if glycerol is a precursor for glucose, glycogen (liver and breast muscle), and NEAA synthesis in chicken embryos. We hypothesized that the contribution of glycerol to gluconeogenesis, glycogen, and NEAA synthesis will be less in small- compared with large-egg embryos because of the decreased initial yolk content of the small egg (Lourens et al., 2006). We confirmed that glycerol is a major substrate for glucose, glycogen, and NEAA synthesis in developing embryos, but that glycerol is a greater contributor to glucose synthesis in small- compared with large-egg embryos. MATERIALS AND METHODS The University of Maryland Institutional Animal Care and Use Committee follows the guidelines mandated by the Animal Welfare Act, Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the Guidelines for the Care and Use of Laboratory Animals, which do not regulate the use of embryonated avian eggs in research. Further, the University of Maryland Institutional Animal Care and Use Committee does not have an additional internal policy governing the use of embryonated eggs at this time. Therefore, the current study was in compliance with both federal regulations and campus policy. Egg Incubation and Experimental Protocol Fertilized small (56.6 ± 0.24 g) and large (71.7 ± 0.29 g) eggs were obtained from Perdue Farms Inc. (Salisbury, MD) from a broiler flock of the same age (40 wk old). All eggs were incubated at standard temperature and relative humidity of 37°C and 65%, respectively. On e9, all the eggs were candled for viable embryos. Based on our previous experience (Sunny and Bequette, 2010), the minimum sample size to detect a significant treatment effect with standard α (0.05) and power (80%) was determined to be 5. Thus, to account for potential hatchability losses (35%) due to in ovo injection procedures, 8 viable eggs were randomly selected for incubation in each treatment group. Study 1: In Ovo [13C6]Glucose Injection One group (n = 8) each from small and large eggs was randomly selected on e12 or e17 for injection of [13C6]glucose (15 mg in 75 μL of sterile water) into the chorio-allantoic fluid for 3 consecutive days. Previously, we established that isotopic and isotopomer steadystate labeling of glucose was achieved after 3 consecutive days of administering [13C6]glucose (Sunny and Bequette, 2010). For tracer injections, the air-space end of the egg was sterilized with 70% ethanol before piercing the eggshell. A 25-gauge needle was used to inject the tracer solution, which was deposited 2 to 3 mm beneath the eggshell membrane into the chorio-allantoic fluid. In this compartment of injection, the tracer will mix uniformly with the fluid compartment and be absorbed by the developing embryo through the extensive chorio-allantoic capillary network. The day after the last injection of [13C6]glucose, blood, and tissue samples were collected and stored at −80°C until further analysis to determine parameters of glucose metabolism and incorporation of glucose into liver and breast muscle glycogen. Study 2: In Ovo [13C3]Glycerol, Preliminary Study Similar to the in ovo [13C6]glucose delivery method, [ C3]glycerol (99 atom % 13C, Cambridge Isotope Laboratories Inc., Andover, MA) was utilized as a metabolic tracer to determine whether egg glycerol is a substrate for gluconeogenesis, glycogenesis, and NEAA synthesis. To confirm accurate tracer delivery and achievement of steady-state labeling of the metabolites of interest, [13C3]glycerol (14 mg in 75 µL of sterile water) was injected as above into the chorio-allantoic fluid of 4 eggs of similar weight starting on e14. One egg was sampled after 1, 2, 3, or 4 d of administering the [13C6]glycerol, and 13C-isotopomer enrichments in blood glucose were measured (see below). 13 Study 2: In Ovo [13C3]Glycerol, Main Study Based on the pilot study, we established that isotopic and isotopomer steady states had been achieved after 4 consecutive days of administering [13C3]glycerol (Figure 1). Thus, 1 group (n = 8) each from small and large eggs was randomly selected on e10 or e15 for injection of [13C3]glycerol, followed by collection of blood and tissues on either e14 or e19. Sample Collection and Analysis After either [13C6]glucose (study 1) or [13C3]glycerol (study 2) injections, each group of eggs was dissected to collect blood and tissue samples. After removing the eggshell surrounding the air cell, the eggshell membrane was carefully peeled away to expose the extraembryonic membranes. Whole egg contents were then carefully transferred to a Petri dish, taking care that the vitelline vessels (artery and vein) were clearly exposed. Blood from embryos was sampled by making a small incision in a vitelline vessel and blood withdrawn Glucose, glycogen, and glycerol metabolism in chicken embryos 3947 lated glycogen pellet was washed twice with 0.5 mL of ice-cold ethanol (95%) to remove residual-free glucose followed by centrifugation for 10 min (10,000 × g at 4°C). The glycogen pellet was dried for 2 to 3 h at room temperature and free glucose liberated by incubation (1 h at 55°C) in 250 μL of buffer (0.3 M acetic acid and 2 M acetate; 1:1; pH 4.5) containing 0.5 mg of amyloglucosidase (31.2 units/mg of solid; Sigma-Aldrich, St. Louis, MO). The incubation mixture was lyophilized to dryness and the di-O-isopropylidene acetate derivative of glucose formed before GC-MS as described. 13 C-Enrichments in AA 13 Figure 1. Molar tracer:tracee ratios (moles of C-isotopomer per 100 mol of tracee) of blood [M + 2]- and [M + 3]-glucose (M + n is moles of 13C-isotopomer per 100 mol of tracee, where n equals the number of 13C atoms in the molecule) and [M + 2]:[M + 3] after 1, 2, 3, or 4 consecutive days of injecting [13C3]glycerol into the chorioallantoic fluid of embryos beginning on embryonic d 14. into a glass Pasteur pipette. Blood was transferred to a 2-mL tube and frozen immediately at −20°C for later analysis. Liver, intestine, muscle, and kidney tissues were dissected, rinsed with ice-cold normal saline to remove excess blood and other debris, and transferred to 2-mL plastic tubes for storage at −80°C. 13 For blood (100 μL) and tissue (50 mg) samples, NEAA were isolated by cation-exchange (AG 50W-X8 resin, 100 to 200 mesh; Bio-Rad Laboratories, Hercules, CA), and AA were eluted from the resin with 2 volumes of 2 M NH4OH, followed by 1 vol of water. The elute was lyophilized to dryness, reconstituted in 250 µL of double-distilled water, dried under a stream of N2 gas, and AA converted to their heptafluoro-butyryl isobutyl derivatives (MacKenzie and Tenaschuk, 1979a,b) before separation by GC (Heliflex AT-Amino acid, 25 m × 0.53 mm × 1.20 µm, Alltech, Deerfield, IL). Selected ion monitoring was carried out by MS under methanenegative chemical ionization conditions. The following ions of m/z were monitored: alanine 321 to 324, aspartate 421 to 425, and glutamate 435 to 440. C-Enrichments in Glucose and Tissue Glycogen Calculations For determination of blood glucose enrichments, samples (100 μL) were acidified with ice-cold 15% sulphosalicylic acid (wt/vol) and centrifuged for 10 min (10,000 × g at 25°C) to precipitate proteins and other debris. The elute containing free glucose was collected after passing the supernatant over 0.5 g of cation exchange resin. The solution was concentrated by lyophilization and analyzed by gas chromatography mass spectrometry (GC-MS) for glucose 13C-isotopomer enrichments after formation of the di-O-isopropylidene acetate derivative of glucose. After separation on a fused silica capillary column (HP-5; 30 m × 0.25 mm i.d., 1 µm Hewlett-Packard, Palo Alto, CA) with helium as carrier gas, selective ion monitoring of ions with mass-to-charge (m/z) of 287 to 293 was recorded by MS (5973N Mass Selective Detector coupled to a 6890 Series GC System, Agilent, Palo Alto, CA) under electrical ionization conditions (Hannestad and Lundblad, 1997). For determination of glucose enrichments in liver (100 mg) and breast muscle (200 mg) glycogen, tissues were homogenized in 0.5 to 1.0 mL of ice-cold 30% sulphosalicylic acid (wt/vol) and centrifuged for 10 min (10,000 × g at 25°C) to precipitate proteins and other debris. The glycogen was extracted after the addition of ice-cold ethanol (95%) to the supernatant (2:1) and centrifugation for 20 min (10,000 × g at 4°C). The iso- For both studies, the normalized crude ion abundances for glucose and AA were corrected for the natural abundance of stable isotopes present in the original molecule and that from the derivative by employing the matrix approach (Fernandez et al., 1996). Natural isotopomer distributions in unlabelled glucose and AA were quantified from blood samples taken from embryos that had not received isotopic tracers. Corrected enrichments are reported as molar tracer:tracee ratios (mol of 13C-isotopomer [M + n] per 100 mol of 12 C-isotopomer [M + 0]), where n equals the number of 13C atoms in the analyte (e.g., [M + 1], [M + 2], [M + 3], and [M + 6]-glucose). For study 1, glucose entry rate, and glucose molecule, and 13C-recycling were calculated as described previously (Sunny and Bequette, 2010). The proportion of glycogen synthesized directly from blood glucose was calculated from the ratio [M + 6]-glycogen:[M + 6]-blood glucose. In the liver, glycogen synthesis can also be derived via gluconeogenesis, termed the indirect pathway. The precursors that can contribute to the indirect pathway include recycled glucose molecules (lactate and alanine), glycerol, and AA. The contribution of recycled glucose molecules to hepatic glycogen synthesis can be calculated from blood [M + 3]- and [M + 6]-glucose relative to [M + 3]-glycogen. Anaerobic and partial metabolism of [M + 3]- and [M + 6]-glucose in peripheral tissues leads to release of 3948 Sunny and Bequette [M + 3]-lactate and [M + 3]-alanine. These 3-carbon skeletons carry the same enrichment as the [M + 6]-glucose but only one-half of the enrichment of the [M + 3]-glucose. Extraction of these 3-carbon skeletons by the liver and via activity of the Cori and alanine cycles, the [M + 3]-lactate and [M + 3]-alanine are incorporated into glucose. Thus, synthesis of heptatic glycogen from recycled glucose molecules (%) is calculated by the equation and isotopomer steady state (based on the ratio [M + 2]:[M + 3]) was attained by d 4. Thus, the rates of [13C3]glycerol entry into the gluconeogenic pathway and into the TCA cycle (i.e., 13C-recycling) have attained a metabolic steady-state, an important criterion of tracer flux analyses. Based on these results and our experience with the in ovo [13C6]glucose tracer approach (Sunny and Bequette, 2010), in the main study (Study 2) [13C3] glycerol was administered in ovo for 4 consecutive days before blood and tissue sampling on d 5. [M + 3]-glycogen/{(0.5 × [M + 3]-glucose) + [M + 6]-glucose} × 100. Synthesis of hepatic glycogen from nonglucose sources (i.e., glycerol and AA) is that proportion not derived directly from glucose and from recycled glucose molecules. Statistical Analysis After verifying for assumptions of normality and homogeneity of variance, results were analyzed by ANOVA using mixed procedure (SAS Inst. Inc., Cary, NC) with small and large eggs as treatment groups and days of incubation as blocks. Treatment means were compared by Tukey-Kramer multiple-comparison test. Data are presented as least squares means ± SED, and the differences were considered significant at P < 0.05. RESULTS Embryo weights were heavier (P < 0.05) from the large compared with small eggs on e14/15 (11.4 ± 0.53 g vs. 13.3 ± 0.47 g) and e19/20 (31.0 ± 0.58 g vs. 36.7 ± 0.34 g). The time course of enrichments of blood [M + 2]- and [M + 3]-glucose isotopomers and the ratio of [M + 2]/[M + 3] after 1, 2, 3, or 4 d of injecting [13C3]glycerol are shown in Figure 1. Glucose isotopic Glucose Metabolism Glucose metabolism differed (P < 0.05) between e15 and e20 embryos (Table 1), but remained similar between small- and large-egg embryos. Therefore, data in Table 1 are means of small- and large-egg embryos. Glucose entry rate on e15 was greater than on e20 (1.50 ± 0.146 vs. 0.61 ± 0.240 g/d). Glucose molecule recycling (%; 49.9 ± 2.94 vs. 83.1 ± 2.96) and glucose 13 C-recycling (%; 23.4 ± 2.14 vs. 61.3 ± 5.14) were substantially greater (P < 0.001) on e20, suggesting that e20 embryos are more efficiently conserving 3-carbon skeletons after anaerobic metabolism of glucose. Glycogen Synthesis from Glucose Although there was appreciable enrichment of blood [M + 6]-glucose in e15 embryos after 3 d of [13C6]glucose administration, there was negligible enrichment of the [M + 6]-glucose isotopomer in liver glycogen, suggesting that there was essentially no direct contribution of blood glucose to liver glycogen synthesis. However, there was detectable contribution of blood glucose to muscle glycogen (%; 7.03 ± 1.12) of e15 embryos (Table 2), which was expected because muscle lacks the necessary gluconeogenic enzymes. For both e15 and e20 embryos, the lesser isotopomers ([M + 1], [M + 2], and [M + 3]) in blood glucose and glucose units in liver and muscle Table 1. Embryonic weights and metabolism of glucose in embryonic day (e) 15 and 20 embryos1 Day of development Item e15 2 e20 P-value ± ± ± ± ± ± ± ± <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 13 Glucose enrichment, mol of C-isotopomer/ 100 mol of tracee M + 1 M + 2 M + 3 M + 6 Embryo weights, g Glucose entry, g/d Glucose molecule recycling, % Glucose carbon recycling, % 0.46 0.46 0.17 1.08 12.4 1.50 49.9 23.4 ± ± ± ± ± ± ± ± 0.06 0.05 0.02 0.14 0.50 0.15 2.94 2.14 8.71 9.48 3.64 4.92 33.9 0.61 83.1 61.3 0.74 1.07 0.46 1.07 0.46 0.24 2.96 5.14 1 Except for embryo weights, differences between small and large embryos were not significant (P > 0.05). Therefore, values are pooled means of 10 to 15 embryos at each stage. Data are means ± SE with P < 0.05 between e15 and e20 embryos. 2 M + n is moles of 13C-isotopomer per 100 mol of tracee, where n equals the number of 13C atoms in the glucose molecule. 3949 Glucose, glycogen, and glycerol metabolism in chicken embryos 13 Table 2. Tissue glycogen C-isotopomers and synthesis in embryonic day (e) 15 and 20 embryos determined after 3 consecutive days of dosing [13C6]glucose1 e15 Item Liver 13 e20 Muscle Liver 2 Glucose C-isotopomer, mol of isotopomer/ 100 mol of tracee in glycogen M + 1 M + 2 M + 3 M + 6 Glycogen synthesis Direct pathway, % Indirect via glucose recycling, % Indirect from other sources, % 0.50 ± 0.07 0.59 ± 0.07 0.23 ± 0.02 NS 0.20 0.32 0.15 0.06 ± ± ± ± 0.04 0.04 0.02 0.01 0 21.1 ± 2.47 78.8 ± 2.45 7.0 ± 1.12 14.4 ± 1.72 78.6 ± 2.49 1.54 2.08 0.98 1.09 ± ± ± ± 0.18 0.38 0.14 0.30 16.5 ± 6.06 10.8 ± 2.20 72.7 ± 8.18 1 Except for embryo weights, differences between small and large embryos were not significant. Therefore values are pooled means of 10 to 15 embryos at each stage. Data are means ± SE; NS = not significantly different from 0. 2 M + n is moles of 13C-isotopomer per 100 mol of tracee, where n equals the number of 13C atoms in the glucose molecule. glycogen were significantly enriched from [13C6]glucose administration (Table 2; e20 muscle glycogen enrichment not determined). These lesser isotopomers arise due to recycling of glucose 13C-skeletons in the TCA cycle and their return for gluconeogenesis, and via the indirect pathway for glycogen synthesis. In contrast to e15 embryos, [M + 6]-glucose units in liver glycogen of e20 embryos were significantly labeled, and there were also substantial enrichments of [M + 1], [M + 2], and [M + 3] isotopomers. This suggests that there is direct synthesis of glycogen from blood glucose (17%) in the liver on e20 but that the indirect pathway of glycogen synthesis predominates. We further calculated that on e15 and e20, 12% of hepatic glycogen was synthesized via the indirect route from glucose molecule recycling, which does not represent a net supply to glycogen synthesis. The remaining 73% was estimated to be derived via the indirect pathway from AA and glycerol, both of which represent a net supply of precursors. Glucose and Glycogen Synthesis from Glycerol In ovo injection of [13C3]glycerol ([M + 3]glycerol) resulted in significant enrichments of [M + 1], [M + 2], and [M + 3] in blood glucose, and liver and muscle glycogen (Figure 2). On e14 and e19, all the blood glucose isotopomers were more enriched (P < 0.05) in smallcompared with large-egg embryos (Figure 2A). Blood glucose isotopomer enrichments ([M + 1], [M + 2], and [M + 3]) were also greater (P < 0.05) in e19 than e14 embryos (Figure 2A). Enrichments of [M + 1], [M + 2], and [M + 3] isotopomers in glucose units of liver glycogen were similar between small- and large-egg embryos, and also between e14 and e19 of development (Figure 2B). On e14, enrichments of [M + 1], [M + 2], and [M + 3] isotopomers in glucose units of muscle glycogen of small-egg embryos were greater (P < 0.05) compared with their larger counterparts and with those of small- and largeegg embryos on e19 (Figure 2C). NEAA Enrichments from [13C3]Glycerol In ovo injection of [13C3]glycerol resulted in significant enrichment of [M + 1], [M + 2], and [M + 3] isotopomers in alanine, aspartate, and glutamate in blood and in tissues (Table 3; and Supplemental Tables 1, 2, and 3, in the online version of this paper). On e14 and e19, liver alanine, aspartate, and glutamate 13Cisotopomers were all more enriched in small- compared with large-egg embryos (P < 0.05), and for aspartate and glutamate isotopomers their enrichment in small egg embryos was greater (P < 0.05) on e19 compared with e14. DISCUSSION Maintaining increased rates of gluconeogenesis and synthesis of glycogen in liver and muscle are critical processes for survival and emergence of chicken embryos during development and at the time of hatch. These metabolic processes, which follow hormonal and nutrient cues (Jenkins and Porter, 2004; Lu et al., 2007; Sunny and Bequette, 2010), are highly active, especially during the latter half of embryonic development and extending into the immediate posthatch period (Bequette et al., 2010). However, the egg-nutrient substrates that provide the necessary carbon skeletons for gluconeogenesis and glycogen synthesis in ovo are not known with certainty. Knowledge of which egg nutrients are the major gluconeogenic precursors is fundamental to hen feeding, breeding, and management practices to optimize egg components for survival and growth of broilers. We utilized the in ovo stable isotope tracer delivery approach (Sunny and Bequette, 2010) previously developed in our laboratory to investigate whether glucose and glycerol serve as substrates for gly- 3950 Sunny and Bequette Table 3. Molar tracer:tracee ratios (mol of 13C-isotopomer per 100 mol of tracee) of AA isotopomers [M + 1], [M + 2], and [M + 3] in the liver of embryonic day (e) 14 and 19 chicken embryos after in ovo [13C3]glycerol injection for 4 consecutive days1,2 Moles of 13C-isotopomer per 100 mol of tracee Item Egg type3 Alanine e14 Small Large Small Large Small Large Small Large Small Large Small Large e19 Aspartate e14 e19 Glutamate e14 e19 [M + 1] [M + 2] [M + 3] 1.37 0.66 1.53 0.97 ±0.22 ±0.14* ±0.27 ±0.33* 1.31 0.75 1.57 1.04 ±0.16 ±0.11* ±0.24 ±0.29* 0.97 0.62 1.02 0.55 ±0.11 ±0.12* ±0.17 ±0.14* 2.43 1.47 5.23 3.30 ±0.29 ±0.23* ±0.72† ±0.93* 1.56 0.93 3.16 1.90 ±0.18 ±0.15* ±0.45† ±0.56* 0.26 0.15 0.46 0.36 ±0.03 ±0.02* ±0.08† ±0.13* 1.54 1.05 3.03 1.86 ±0.17 ±0.12* ±0.43† ±0.41* 1.49 0.94 3.24 1.90 ±0.18 ±0.17* ±0.47† ±0.54* 0.19 0.13 0.34 0.25 ±0.03 ±0.03* ±0.06† ±0.09* 1 Values are means ± SE of means of 6 to 7 embryos with significance declared at P < 0.05. *P < 0.05 between small- and large-egg embryos of each embryonic stage. †P < 0.05 between e14 and e19 of either small- or large-egg embryos. 2 M + n is moles of 13C-isotopomer per 100 mol of tracee, where n equals the number of 13C atoms in the AA molecule. 3 Small = small-egg embryos; large = large-egg embryos. Figure 2. Molar tracer:tracee ratios (moles of 13C isotopomer per 100 mol of tracee) of [M + 1], [M + 2], and [M + 3] isotopomers (M + n is moles of 13C-isotopomer per 100 mol of tracee, where n equals the number of 13C atoms in the AA molecule) in (A) blood glucose, (B) liver glycogen, and (C) muscle glycogen of embryonic day (e) 14 and 19 embryos from small or large eggs after injection of [13C3]glycerol into the chorioallantoic fluid of embryos. Each bar represents the mean of 6 to 7 embryos. a–cWithin each graph, bars without common letters differ (P < 0.05). Glucose, glycogen, and glycerol metabolism in chicken embryos cogen and glycogen/glucose synthesis, respectively. The results demonstrated that glycerol is a more significant substrate than glucose for hepatic glycogen synthesis during embryonic development. Thus, glycerol derived from yolk lipids appears to serve as a major precursor for gluconeogenesis and glycogen synthesis, as well as for NEAA synthesis via metabolism in the TCA cycle. Developing chicken embryos maintain increased rates of glucose entry (turnover) to maintain a high glucose status and embryonic energy metabolism in support of the developing immune, nervous, and muscular systems (Freeman, 1969; Moran, 2007; Humphrey and Rudrappa, 2008). Consistent with our previous report (Sunny and Bequette, 2010), glucose entry was significantly greater on e15 compared with e20, in line with the hypophyseal and adrenocortical development of the embryo at e15 (Hazelwood, 1971; Jenkins and Porter, 2004). The decrease in glucose entry on e20 could be attributed to the increase in the insulin:glucagon ratio at this later stage of embryonic development (Lu et al., 2007), which is consistent with the reported patterns of gluconeogenic enzyme gene expression that wanes and stabilizes toward the time of hatch (Pearce, 1977). Further, glycogen synthesis and storage, vital for pipping and hatching, has been shown to increase up to 4-fold from ~e13 onward (Hazelwood, 1971). This rapid rate of glycogen synthesis and storage may also be a contributing factor to the observed decreased glucose entry rate on e20 (i.e., reduced glycogen turnover). Even though glucose entry was lesser on e20, these e20 embryos conserved glucose carbon more efficiently than their e15 counterparts due to a 2- and 3-fold increase in glucose molecule and glucose 13C recycling, respectively. Such increased rates of glucose molecule recycling (>80%) in the e20 embryos is demonstration of the switch to anaerobic glycolysis and that glucose is a vital commodity to the evolving metabolism of the embryo. This naturally leads to the question: what are the precursor sources for gluconeogenesis and glycogenesis during embryonic development? Potential endogenous egg resources include preformed glucose, triglycerideglycerol, and gluconeogenic AA. Recently, we measured yolk and albumen for carbohydrate contents throughout development of smaller- (53 g) and larger- (69 g) sized egg embryos (Hu et al., 2011). Even though the initial total supply of glucose plus mannose at the time of set was 250 to 300 mg, by e11 very little glucose remained and the rate of mannose utilization decreased in parallel with the continually dwindling supply of the egg components. Thus, from e11 onward, the embryo begins to rely more on gluconeogenesis from substrates present in the yolk and albumen. We have previously investigated whether glutamine and glutamate serve as gluconeogenic precursors (Sunny et al., 2007). Both AA account for a large proportion of albumen (~14%; Ohta et al., 1999), and both are in increased concentrations in embryo plasma (~22%; Sunny and Bequette, 2010). However, neither of these AA was found to be metabolized for gluconeogenesis 3951 on e19. Even though these AA could potentially contribute to gluconeogenesis during early stages of embryonic development, it prompted us to investigate the role of the glycerol moiety of triglyceride; an abundant substrate for gluconeogenesis. Egg yolk contains 60 to 70% triglyceride that is extensively metabolized during the latter half of embryo development, providing >90% of the energy requirements of the embryo (Sato et al., 2006). Hydrolysis of triglycerides releases glycerol, a 3-carbon skeleton that enters the glycolytic pathway at the triose phosphate level. Consequently, the glycerol moiety can contribute via reverse glycolysis to the synthesis of glucose or via forward glycolysis to the synthesis of pyruvate. Further metabolism of pyruvate by the TCA cycle can subsequently lead to the synthesis of NEAA and energy generation via flux through acetylCoA. In consequence, glycerol carbon has the potential to contribute not only to gluconeogenesis, but also to TCA cycle energy generation and to NEAA synthesis (Bequette et al., 2010). Indeed, there was appreciable enrichment of 13C-isotopomers in alanine, aspartate, and glutamate in blood and in the free pool of tissues after in ovo administration of [13C3]glycerol. Alanine, aspartate, and glutamate are in rapid equilibrium with their respective TCA cycle intermediates pyruvate, oxaloacetate, and α-ketoglutarate (Katz et al., 1989; Wykes et al., 1998; Bequette et al., 2006); thus, their 13C-labeling patterns mirror the activity and substrate fluxes through the TCA cycle. The TCA cycle is the primary energy generating pathway in the embryo, requiring large inputs of anaplerotic substrates to maintain continuous flux through the TCA cycle and to replenish metabolic intermediates lost through cataplerotic (synthetic) pathways. The present results demonstrate that glycerol serves both of these latter functions. First, the large enrichments of the [M + 1] and [M + 2] 13C-isotopomers in alanine, aspartate, and glutamate are a clear demonstration that the glycerol carbon skeleton enters into and completes several turns of the TCA cycle, thus providing anaplerotic carbon to replenish the TCA cycle and mitochondrial energy generation. Second, the appearance of the [M + 3] 13C-isotopomer in these 3 NEAA in blood further confirms that glycerol is an important source of carbon skeletons for cataplerotic reactions of the TCA cycle (i.e., pyruvate, oxaloacetate, and α-ketoglutarate) from which NEAA synthesis originates. In ovo injection of [13C3]glycerol (i.e., [M + 3]glycerol) into the chorio-allantoic fluid resulted in the appearance of [M + 3]-glucose in liver and muscle glycogen on e14 and e19. The appearance of [M + 3]-glucose units in glycogen can only arise from the direct incorporation of [M + 3]glycerol via gluconeogenesis, suggesting that glycerol is potentially a major precursor for hepatic glycogen synthesis. This was particularly noteworthy considering that in ovo injection of [13C6]glucose (i.e., [M + 6]-glucose) resulted in very low enrichment of [M + 6]-glucose units in liver and muscle glycogen of e15 embryos. Glycogenesis can occur via phosphorylation of 3952 Sunny and Bequette glucose and incorporation of glucose-6-phosphate into glycogen (direct pathway) or through the incorporation of 3-carbon intermediates entering via reverse glycolysis (indirect pathway). In humans and rats, only a small proportion of dietary glucose is incorporated directly into liver glycogen, with the majority of hepatic glycogen synthesis occurring via the indirect pathway (Katz and McGarry, 1984; Huang and Veech, 1988). We observed that >85% of hepatic glycogen synthesis in the chicken embryo occurred via the indirect pathway, which is consistent with humans and rats. We further estimated that the contribution of glucose-carbon recycling via the indirect pathway accounted for 15% of glycogen synthesis, with the remaining 76% derived from metabolism of AA and glycerol (average of e15 and e20 liver). Indeed, the large [M + 3]-glycogen enrichments when [13C3]glycerol was administered further confirm the role of glycerol as a major contributor to the indirect pathway of glycogen synthesis. Even though the direct contribution of glucose to glycogen synthesis was minimal on e15, 17% of liver glycogen derived from direct synthesis from glucose on e20. This may reflect the fact that both direct and indirect pathways of glycogen synthesis function in unison during later stages of embryonic development to support the necessary increased rates of glycogen synthesis and breakdown, which when coupled with increased rates of glucose carbon recycling, serve to maintain euglycemia and energy reserves for hatching. Blood [M + 3]-glucose was more enriched in smallcompared with large-egg embryos after daily dosing with [13C3]glycerol. Unfortunately, technical difficulties encountered when measuring the isotopic enrichment of blood glycerol (i.e., the precursor pool) precluded calculation of the proportional contribution of glycerol to glucose (and glycogen) synthesis. However, there are several possibilities that can account for these differences in [M + 3]-glucose enrichment, and thus, identification of potential metabolic differences between egg sizes. First, the rate of gluconeogenesis could have been greater in small-egg embryos, resulting in greater incorporation of the [13C3]glycerol. However, glucose entry and glucose recycling were not different between small- and large-egg embryos on e15 and e20. Second, the plasma pool size and turnover rate of glycerol may have been less in small-egg embryos, leading to less dilution of the dosed [13C3]glycerol. This possibility is also less likely because glycerol concentrations on e14 and e18 are similar in broiler and layer embryos (Sato et al., 2006). The rate of glycerol entry rate is also related to the size of the yolk compartment. We have observed decreased dry yolk weights on e20 for smallcompared with large-egg embryos (4.95 vs. 7.47 g; Hu et al., 2011), derived from eggs of similar initial size (53 vs. 69 g) as in the current study. However, this difference in yolk-glycerol supply (50%), and thus dilution of blood glycerol, does not fully account for the difference in blood [M + 3]-glucose enrichment (78 to 148%) in small- compared with large-egg embryos on e15 and e19. The last possibility is that small-egg embryos derive a larger proportion of glucose precursor supply from glycerol compared with large-egg embryos. This will need to be confirmed, however, with direct evidence from precursor:product tracer studies. In summary, this study provides clear evidence in chicken embryos that 1) hepatic glycogen synthesis occurs predominantly via the indirect pathway with AA and glycerol supplying the majority of the precursor supply; 2) glycerol, but not blood glucose, is the predominant precursor for hepatic glycogen synthesis; and 3) glycerol is a significant contributor to glucose and NEAA synthesis and to anaplerotic fluxes in the TCA cycle. LITERATURE CITED Bequette, B. J., S. W. El-Kadi, and N. E. Sunny. 2010. 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