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Published December 4, 2014 Gluconeogenesis differs in developing chick embryos derived from small compared with typical size broiler breeder eggs1,2 N. E. Sunny3 and B. J. Bequette4 Department of Animal and Avian Sciences, University of Maryland, College Park 20742 entry rate (g·d−1), and Cori cycling and glucose 13Crecycling (% of entry rate) increased (P < 0.05) with development. On e12 and e14, rates of glucose entry and Cori cycle flux were greater (P < 0.05) for embryos from small eggs. When standardized to BW (g·100 g of BW−1·d−1), glucose entry and Cori and non-Cori cycle fluxes were greater for embryos from small eggs. From e12 through e18, blood concentrations of gluconeogenic AA (threonine, glutamine, arginine, proline, isoleucine, and valine) were 25 to 48% less (P < 0.01) in embryos from small eggs. In conclusion, embryos from small eggs exhibit greater rates of GNG earlier in development compared with typical size eggs and, perhaps as a consequence, their reduced embryonic growth may result from diverting greater supplies of AA toward GNG. ABSTRACT: We hypothesized that, as the supply of preformed glucose diminishes during development, the embryo would transition to a greater rate of gluconeogenesis (GNG) and that GNG would be greater in embryos from small vs. typical size eggs. Gluconeogenesis by embryos from small (51.1 ± 3.46 g) and typical size (65 ± 4.35 g) broiler breeder eggs was measured by dosing [13C6]glucose (15 mg·egg−1) into the chorio-allantoic fluid for 3 consecutive days to achieve isotopic steadystate before blood collection on embryonic day (e) 12, e14, e16, and e18 (4 to 5 eggs·size−1·d−1). The 13C-Mass isotopomer enrichment of blood glucose was determined by gas chromatography-mass spectrometry. On e14, e16, and e18, but not on e12, embryos from small eggs weighed less (P < 0.05) than typical size eggs. For both sizes of eggs, blood glucose concentration, glucose Key words: amino acid, embryo, glucose, metabolism, poultry, stable isotope ©2010 American Society of Animal Science. All rights reserved. INTRODUCTION J. Anim. Sci. 2010. 88:912–921 doi:10.2527/jas.2009-2479 lized and embryos transition to a gluconeogenic state (Hazelwood, 1971; Watford et al., 1981; Savon et al., 1993). Blood glucose is less in embryos from small eggs (Latour et al., 1996), and embryos and hatchlings with reduced blood glucose and tissue glycogen have greater mortality and poor growth (Moran, 1989; Donaldson et al., 1992; Donaldson and Christensen, 1993; Ohta et al., 1999; Christensen et al., 2000, 2001). Clearly, adequate rates of gluconeogenesis (GNG) are essential, yet this process differs in embryos from small eggs. The quantitative changes in GNG and substrates metabolized for GNG that lead to reduced glucose status of embryos from smaller eggs are unknown. Our aim was to quantify GNG by embryos from small vs. typical size broiler breeder eggs, in particular glucose recycling and the contribution of nonglucose sources (e.g., AA, glycerol) to GNG. We hypothesized that the reduced supply of egg resources of small egg embryos (~51 g) vs. typical (~65 g) eggs necessitates that small egg embryos transition to greater rates of GNG and recycling to maintain normal blood glucose and tissue glycogen. To test this hypothesis, we developed a [13C6]glucose stable isotope approach in ovo to Nutrient supplies in avian embryos are finite. Thus, allocation of this supply must be timely, otherwise development of embryos will be compromised. At lay, breeder eggs are composed mainly of yolk lipids and albumen; there is little carbohydrate (0.2 to 0.3 g/65-g egg; Romanoff and Romanoff, 1967). However, glucose is required for basic metabolism, emergence, and glycogen synthesis (Picardo and Dickson, 1982; Noy and Sklan, 1998, 1999). Thus, once formed, glucose is uti- 1 Funded by a Maryland Agricultural Experiment Station (College Park) grant to B. J. Bequette. 2 Presented in part at the annual meetings of the Federation of American Societies for Experimental Biology: April 28 to May 2, 2007, Washington, DC (N. E. Sunny, J. Adamany, B. J. Bequette. 2007. Gluconeogenesis and carbon utilization in embryos from small and large chicken eggs). 3 Present address: Advanced Imaging Research Center, University of Texas Southwestern Medical Center, 2201 Inwood Rd., Dallas, TX. 4 Corresponding author: [email protected] Received September 11, 2009. Accepted November 24, 2009. 912 Gluconeogenesis in chicken embryos 913 quantify glucose entry and recycling and GNG from nonglucose substrates by chicken 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 any additional internal policy governing the use of embryonated eggs. Therefore, the current study was in compliance with both federal regulations and campus policy. Isotopic Tracer The d-[13C6]glucose was purchased from Cambridge Isotope Laboratories Inc. (Woburn, MA). Mass spectrometry analysis confirmed its chemical and isotopic purity (92.7% [13C6]glucose and 6.9% [13C5]glucose). Experimental Protocol Fertilized eggs from broiler breeders were obtained from Perdue Farms Inc. (Salisbury, MD). To create groups of small and typical size eggs, eggs were selected from a flock of 30-wk-old (51.1 ± 3.46 g eggs; n = 40) and 40-wk-old (65 ± 4.35 g eggs; n = 40) layers. Small and typical size eggs were incubated at standard temperature (37.5°C) and relative humidity (65%). On embryonic day (e) 9 (i.e., e9), all eggs were candled for viable embryos. The minimum sample size to detect a significant treatment effect with standard α (0.05) and power (80%) was determined to be 5 based on measured SD of various response variables from a similar study from our laboratory. To obtain a minimum sample size of 5 at each sampling time point (day of development) and to account for potential hatchability losses (35%) due to in ovo injection procedures, 8 viable eggs were randomly selected as a group. Four groups each from small and typical size eggs were incubated for collection on e12, e14, e16, and e18 (blocks) of development. Preliminary Study This study was conducted to determine the length of time after administering [13C6]glucose into the chorio-allantoic fluid of incubating eggs that the 13C-isotopomer enrichments of blood glucose reach isotopic (e.g., [M+2], [M+3], [M+6]) and isotopomer (e.g., [M+3]:[M+6]) steady-state. Starting on e14, a solution containing [13C6]glucose (15 mg in 75 µL of sterile water) was injected into the chorio-allantoic fluid of 8 eggs of similar weight. After 1, 2, 3, and 4 d of administering the [13C6]glucose, 2 eggs were removed and sampled for blood glucose enrichments. For tracer injection, the Figure 1. Fresh embryo weights from small (■; 51.1 ± 3.46 g at set) and typical (○; 65.0 ± 4.35 g at set) size eggs on embryonic day (e) 12, e14, e16, and e18 of development. Values are means ± SEM, n = 4 to 5 embryos. a–dWithin egg size group, means without a common letter differ, P < 0.05. *Within day of development, values were different between small and typical size egg embryos, P < 0.05. air space end of the egg was sterilized with ethanol [70% (vol/vol) with water] before piercing the eggshell. A 25-ga needle was inserted 2 to 3 mm beneath the eggshell membrane into the chorio-allantoic fluid where the [13C6]glucose solution was deposited. At this site of injection, the extensive chorio-allantoic blood capillary network allows for diffusion of nutrients into the embryo blood supply. Main Study Based on the preliminary study, isotopic and isotopomer steady-state labeling of glucose (Figure 1) was achieved after 3 consecutive days of administering the [13C6]glucose. Thus, for measurements on e12, e14, e16, and e18, [13C6]glucose (15 mg in 75 µL of sterile water) was administered into the chorio-allantoic fluid for 3 consecutive days beginning on e9, e11, e13, and e15, respectively. One day after the last [13C6]glucose injection, blood samples were collected. After removing the eggshell surrounding the air cell, the eggshell membrane was carefully peeled away to expose the extra embryonic membranes. Whole egg contents were carefully transferred to an ice-cold glass Petri-dish, taking care that the vitelline vessels (artery and vein) were clearly visible. Blood was sampled from embryos by making a small incision on the vitelline vessels and withdrawal of blood (0.5 mL) into a glass Pasteur pipette. Blood was processed as described below. Fresh weights of embryos (without residual yolk) were recorded before blood sampling. Blood Glucose and AA Concentrations The concentrations of glucose and AA were determined by isotope dilution with gas chromatographymass spectrometry (GC-MS) as described previously 914 Sunny and Bequette (El-Kadi et al., 2006). To a known weight (0.1 g) of fresh blood was added an equal known weight of a solution containing 0.05 mg of hydrolyzed [U-13C]algae protein powder (99 atom % 13C; Martek Biosciences Corp., Columbia, MD), 10 nmol of [indole-2H5]tryptophan, 5 nmol of [methyl-2H3]methionine, 20 nmol of [13C5]glutamate, 100 nmol of [13C5]glutamine, 50 nmol of [13C6]arginine, and 500 nmol of [13C6; 1, 2, 3, 4, 5, 6, 6- 2H7]glucose, and the samples stored frozen (−20°C). Thawed samples were deproteinized by addition (0.2 mL) of sulfosalicylic acid [15% (wt/vol) in water]. The supernatant was applied to 0.5 g of cation (AG-50, H+ form; BioRad, Hercules, CA) exchange resin, and the glucose-containing fraction collected after addition of 2 mL of water. This fraction was frozen and freeze-dried. Amino acids were eluted from the resin with 2 mL of 2 M NH4OH followed by water. This fraction was frozen and lyophilized to dryness. The di-O-isopropylidene acetate derivative of glucose was formed before separation by GC (HP-5; 30 m × 0.25 mm × 0.25 µm; Agilent Technologies, Palo Alto, CA) and selective ion monitoring with MS (5973N Mass Selective Detector coupled to a 6890 Series GC System; Agilent) under electron ionization conditions (Hachey et al., 1999). Ions with mass-to-charge (m/z) 287 (unlabelled) and 300 (internal glucose tracer) were monitored. Calibration curves were generated from gravimetric mixtures of labeled and unlabeled glucose, and the final concentration of glucose was corrected for the 13 C-isotopomer abundances of [M+1] to [M+6] glucose. Amino acids were converted to the t-butyldimethylsilyl derivative before separation by GC (HP-5ms, 30 m × 0.25 mm × 0.25 µm, Agilent Technologies) and selected ion monitoring with MS under electron ionization conditions. The following ions of m/z were monitored: alanine 260, 263; glycine 246, 248; valine 288, 293; leucine 302, 308; isoleucine 302, 308; proline 286, 291; methionine 292, 295; serine 390, 393; threonine 404, 408; phenylalanine 234, 242; aspartate 302, 304; glutamate 432, 437; lysine 300, 306; glutamine 431, 436; arginine 442, 448; tyrosine 302, 304; and tryptophan 244, 249. Calibration curves were generated from gravimetric mixtures of labeled and unlabeled AA. For all nonessential AA that had become 13C-labeled after catabolism of [13C6]glucose, a correction was made to account for isotopomer ([M+1], [M+2], and [M+3]) abundances (see below). 13 C-Mass Isotopomer Enrichments of Blood Glucose and AA For determination of blood glucose enrichments, samples (0.1 mL) were acidified with ice-cold sulphosalicylic acid [15% (wt/vol) in water] and centrifuged at 10,000 × g for 10 min at 25°C to precipitate proteins and other debris. Glucose was isolated from the acid supernatant and processed for GC-MS analysis as above. Ions with m/z of 287 to 293 were monitored. Amino acids were eluted from the resin with 2 mL of 2 M NH4OH followed by water. The eluate was lyopholized to dryness, and AA were 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 with MS was carried out under methane negative chemical ionization conditions. For enrichment of biochemically nonessential AA, the following ions of m/z were monitored: alanine 321 to 324, aspartate 421 to 425, glutamate 435 to 440, glutamine 361 to 366, and serine 533 to 536. All biochemically essential AA (including arginine, glycine, and proline) were also monitored, but as expected, there was no detection of 13C. Calculations The normalized crude ion abundances of glucose and AA were corrected for the natural abundance of stable isotopes present in the original molecule and that contributed by the derivative using 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 [13C6]glucose. Enrichments are reported as molar tracer:tracee ratios (TTR, mol of 13C-isotopomer [M+n] per 100 mol of 12C-isotopomer [M+0]), where n equals the number of 13C-atoms in the analyte (e.g., [M+1], [M+2], and [M+3] and [M+6]glucose). Entry of glucose into the blood of chick embryos derives from 4 sources: 1) GNG, 2) release of preexisting glucose from egg contents, 3) glycogenolysis, and 4) recycling of glucose molecules (Cori cycle). However, the net supply of glucose only derives from GNG and preexisting glucose in egg contents. Based on literature values (Romanoff and Romanoff, 1967), we estimated that the eggs used in the current study contained 0.2 to 0.3 g of preformed glucose at time of set, and so it is likely that by e12 this initial pool of preformed glucose had been largely metabolized, such that GNG contributed the majority of net glucose supplies up until the time of hatch. In the present study, after 3 consecutive days of dosing [13C6]glucose, blood glucose became a mixture of [M+6]glucose (derived from the administered tracer) and [M+1], [M+2], and [M+3]glucose (derived from 13 C-recycling from phosphoenolpyruvate and oxaloacetate). Metabolism of tracer [M+6]glucose via glycolysis leads to the synthesis of [M+3]pyruvate. Among the various fates of this [M+3]pyruvate is carboxylation to [M+3]oxaloacetate (via pyruvate carboxylase) in the tricarboxylic acid (TCA) cycle and, via operation of cytosolic or mitochondrial phosphoenolpyruvate carboxykinase, conversion of this [M+3]oxaloacetate to [M+3]phosphoneolpyruvate, the latter of which is the precursor for resynthesis (recycling) of glucose. Similarly, synthesis of [M+3]pyruvate in nonhepatic tissues 915 Gluconeogenesis in chicken embryos can lead to the synthesis of [M+3]lactate or [M+3]alanine, or both, which also contribute to the resynthesis of glucose by the liver and kidneys of avians. Thus, blood [M+6]glucose becomes diluted by glucose recycling, synthesis of glucose from unlabelled substrates, glycogenolysis in later development (e18 onward), and the release of preformed glucose from egg contents early in development. The sum of these processes, glucose entry rate (g/d), was calculated as [(92.7/ [M+6]glucose) – 1] 13 × [ C6]glucose injected (0.15 g/d), [1] where 92.7 is the isotopic purity (13C6) of the glucose tracer and [M+6]glucose is the TTR of blood glucose. Our aim was to estimate the proportion of glucose entry that derives from the metabolism of nonglucose sources (e.g., AA, glycerol) for GNG. Thus, it was necessary to quantify the proportion of GNG that arises from the recycling of glucose molecules (Cori cycle, i.e., all recycling via pyruvate and the TCA cycle). We used the following equation reported by Tayek and Katz (1996) to calculate glucose molecule recycling (Cori cycling, %): + [M+3] + [M+6])} × 100, [2] where [M+n] is moles of tracer per 100 moles of tracee for blood glucose. This Eq. [2] closely approximates the contribution of the Cori cycle to GNG (Tayek and Katz, 1996). Furthermore, this equation estimates the number of recycled glucose molecules, irrespective of the potential loss or exchange of 13C atoms with 12C in the TCA cycle (Tayek and Katz, 1996). Cori cycle flux (g of glucose·d−1) was calculated as the product of Eq. [1] and Eq. [2]. Non-Cori cycle flux (g of glucose·d−1) was calculated as the product of Eq. [1] and (100 – Eq. [2]). As an approximation of the extent of loss or exchange of 13C atoms with 12C in the TCA cycle, we calculated glucose 13C-recycling (%) according to Tayek and Katz (1996): {([M+1] + [M+2] × 2 + [M+3] × 3)/([M+1] + [M+2] × 2 + [M+3] × 3 + [M+6] × 6)} × 100. [M+3]alanine or asparate/ ([M+6]glucose + 0.5 × [M+3]glucose). [4] Formation of [M+3]glutamate and [M+3]glutamine from glucose catabolism occurs via the following pathway: pyruvate → oxaloacetate → α-ketoglutarate → glutamate → glutamine. However, the [M+3] isotopomer enrichments of glutamate and glutamine are 50% less as a consequence of the equilibrium reaction between oxaloacetate and fumarate. This metabolic cycle yields an equimolar mixture of 2 positional isotopomers of [M+3] oxaloacetate: one labeled in carbons 1 to 3 and the other in carbons 2 to 4. Subsequently, the decarboxylation step between citrate and α-ketoglutarate leads to the loss of carbon 1 of oxaloacetate (i.e., one-half of the [M+3]oxaloacetate enrichment). Thus, the contribution of glucose to the formation of glutamate and glutamine via pyruvate carboxylase is calculated as 2 × [M+3]glutamate or [M+3]glutamine/ {([M+1] + [M+2] + [M+3])/([M+1] + [M+2] only arise from catabolism of [M+3] and [M+6]glucose, the product:precursor relationship can be calculated to determine the contribution of glucose to alanine flux and to aspartate flux via pyruvate carboxylase: [3] This Eq. [3], when compared with Eq. [2], allows an assessment of the completeness of the recycling processes. Catabolism of glucose and glucose molecule recycling leads to the synthesis of [M+3] and [M+6]glucose. Metabolism of these 2 glucose isotopomers in the glycolytic pathway leads to the synthesis of [M+3]pyruvate and [M+3]alanine; [M+3]oxaloacetate and [M+3]aspartate; and [M+3]α-ketoglutarate, [M+3]glutamate, and [M+3]glutamine. Because these [M+3] isotopomers can ([M+6]glucose + 0.5 × [M+3]glucose). [5] Statistical Analysis After verifying the assumptions of normality and homogeneity of variance, results were analyzed by ANOVA using the MIXED procedure (SAS Inst. Inc., Cary, NC) with small and large eggs as treatment groups and days of incubation as the blocking factor. Treatment means were compared by Tukey-Kramer multiple comparison test. Data are presented as least squares means ± SEM, and the differences are considered significant at P < 0.05, whereas P < 0.1 was considered a trend. RESULTS Embryo Weight On e14, e16, and e18 (Figure 1), embryos from small eggs weighed less (P < 0.05) than those from typical size eggs. By e18, small egg embryos weighed 12.9% less (P < 0.01). Blood Glucose and AA Concentrations Blood glucose concentration was greatest (P < 0.001, Table 1) on e16 and e18, and values did not differ between embryos from the 2 egg sizes. There was an effect of day of incubation (P < 0.05) on the concentrations of all AA measured except tyrosine (Tables 1 and 2). Concentrations of arginine, valine, leucine, isoleucine, 916 Sunny and Bequette Table 1. Glucose and nonessential AA concentrations in blood of embryos derived from small and typical size eggs on embryonic day (e) 12, e14, e16, and e18 of development1 Day of embryonic development Item Glucose, mM AA, µM Alanine Aspartate Glutamate Glutamine Serine Egg size e12 e14 b Small Typical 4.70 5.01b Small Typical Small Typical Small Typical Small Typical Small Typical 282.4b 350.6a 132.1a 120.0a 435.0a 392.8a 1,103.1a 1,354.3a 630.0ab 644.2b Main effect e16 2 — —2 293.3b 232.1b 83.2b 56.6c 155.2b 153.9c 770.7b 1,143.4b 533.1b 435.3d e18 SEM a a 6.26 6.58a 5.99 6.17a 391.5a 322.5a 123.0a 85.1b 176.5b 202.0b 1,021.1a 1,501.2a 587.8ab 574.7c 280.3b 324.7a 58.6b 79.4b 150.3b 161.0c 943.8ab 1,468.2a 699.6a 734.5a 0.21 0.19 24.0 29.7 13.9 6.5 28.8 11.5 74.6 61.7 42.2 23.2 Day of development Egg size <0.001 NS3 0.005 NS <0.001 NS <0.001 NS <0.001 <0.001 <0.001 NS a–d Within each row, means with different superscript letters differ from each other, P < 0.05. Values are means of 4 to 5 embryos. Small eggs weighed 51.1 (±3.46) g, and typical size eggs weighed 65.0 (±4.35) g at time of setting. 2 Samples for blood glucose concentration on e14 were destroyed during processing for gas chromatography-mass spectrometry. 3 NS = nonsignificant, P > 0.05. 1 threonine, glutamine, glycine, and tyrosine were less (P < 0.01) in blood of small egg embryos on e16 and e18, and those of proline, aspartate, and tryptophan were less (P < 0.05) on e18 compared with concentrations in typical size egg embyros. Glucose Entry, and Glucose Molecule and 13C-Recycling In the pilot study, we demonstrated that glucose isotopic ([M+3] and [M+6] enrichments) and isotopomer Table 2. Essential AA concentrations in blood of embryos derived from small and typical size eggs on embryonic day (e) 12, e14, e16, and e18 of development1 Day of embryonic development AA, µM Arginine Glycine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Threonine Tryptophan Tyrosine Valine a–c Egg size Small Typical Small Typical Small Typical Small Typical Small Typical Small Typical Small Typical Small Typical Small Typical Small Typical Small Typical Small Typical e12 e14 ab 358.4 346.3b 743.3b 733.6c 283.4a 282.1b 277.8a 299.2c 460.1b 457.0b 86.4a 67.7b 119.1ab 123.0a 241.2b 269.5c 427.1a 324.3b 153.7a 130.8a 412.1a 370.6b 458.5a 442.5b e16 ab 315.0 350.7b 949.3a 971.0b 279.2b 230.5b 228.0b 326.4bc 399.0b 482.4b 68.4b 58.8b 98.9b 103.2b 258.4b 307.9c 414.7a 525.8a 126.1b 129.3a 371.2ab 467.9a 356.7b 435.9b Main effect e18 a 367.9 491.4a 767.9b 1,179.7a 256.1ab 388.7a 282.2a 425.1a 844.6a 944.2a 83.5ab 67.5b 126.3a 117.9ab 342.2a 405.5b 361.5a 574.7a 126.2b 133.5a 296.6bc 406.0ab 363.7b 478.4b SEM b 287.3 382.3b 408.9c 595.8c 179.7c 297.9b 188.7b 359.2b 226.3c 267.2c 90.5a 119.7a 120.3ab 132.0a 375.5a 520.4a 464.4a 577.7a 106.7b 130.9a 246.3c 433.7ab 423.0ab 603.4a 23.4 24.1 43.0 55.7 15.3 18.5 19.6 17.7 41.0 45.7 6.8 6.5 9.4 5.6 24.4 29.2 36.6 44.0 8.3 2.4 38.3 27.0 23.2 25.5 Day of incubation Egg size <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 NS2 <0.001 NS 0.007 NS <0.001 <0.001 0.010 0.010 0.008 NS NS <0.001 <0.001 <0.001 Within each row, means with different superscript letters differ from each other, P < 0.05. Values are means of 4 to 5 embryos. Small eggs weighed 51.1 (±3.46) g, and typical size eggs weighed 65.0 (±4.35) g at time of setting. 2 NS = nonsignificant, P > 0.05. 1 917 Gluconeogenesis in chicken embryos 13 Table 3. Molar tracer:tracee ratios of blood glucose C-isotopomers in embryos derived from small and typical size eggs on embryonic day (e) 12, e14, e16, and e18 of development after 3 consecutive days of dosing [13C6]glucose into the chorio-allantoic fluid1 Glucose 13C-isotopomer,2 mol of isotopomer/100 mol of tracee Day of embryonic development e12 e14 e16 e18 Egg size M+1 M+2 M+3 M+6 Glucose molecule recycled,3 % Small Typical SEM Small Typical SEM Small Typical SEM Small Typical SEM 1.31 1.18 0.15 2.74 2.66 0.29 2.60* 1.26 0.30 3.48 3.64 0.65 1.21 1.02 0.13 1.10 1.08 0.16 0.72 0.53 0.13 2.06 1.54 0.54 0.38 0.38 0.05 0.29 0.30 0.05 0.17 0.15 0.04 0.51 0.38 0.14 6.95*** 18.67 1.21 1.17* 1.88 0.20 2.91 2.15 0.38 3.84 2.83 1.30 30.0** 12.2 3.4 77.7* 68.4 2.3 54.9 47.6 3.2 66.9 69.6 6.2 13 Glucose C-recycling,4 % 11.1* 3.8 1.6 45.7* 33.9 3.6 21.4 17.8 2.7 36.1 36.6 7.8 1 Values are means of 4 to 5 embryos. Small eggs weighed 51.1 (±3.46) g, and typical size eggs weighed 65.0 (±4.35) g at time of setting. Asterisks indicate difference from typical embryos at that time: *P < 0.05, **P < 0.01, ***P < 0.001. 2 M+n is moles of 13C-isotopomer per 100 mol of tracee, where n equals the number of 13C-atoms in glucose. 3 Calculated according to Eq. [2] as a proportion of glucose entry rate. 4 Calculated according to Eq. [3] as a proportion of glucose entry rate. ([M+3]:[M+6]) steady-states were attained after 3 consecutive days of [13C6]glucose administration (Figure 2). Similarly, isotopomer labeling kinetics of blood alanine, aspartate, and glutamate had also reached steady-state (data not shown). Thus, in the main study, glucose tracer was administered for 3 consecutive days with blood sampling on the next day. After 3 d of dosing [13C6]glucose, [M+1], [M+2], [M+3] and [M+6]glucose were the predominant isotopomers observed in blood, whereas [M+4] and [M+5] glucose isotopomers were absent. Furthermore, the TTR of [M+3]glucose did not exceed 1, and so the likelihood of 2 labeled triose molecules recombining to form [M+6]glucose was of low probability and should not affect calculations. Glucose molecule recycling (%) and glucose 13C-recycling (%) increased (P < 0.05) with day of development, and on e12 and e16 this recycling was greater (P < 0.05) in embryos from small eggs (Table 3). From e12 onward, glucose molecule recycling exceeded (P < 0.001) glucose 13C-recycling by at least 70%, indicating that there was considerable exchange and loss of 13C to fates other than GNG. On an absolute (g of glucose·embryo−1·d−1) and BW-adjusted basis (g of glucose·100 g of BW−1·d−1), glucose entry rate, Cori cycle flux, and non-Cori cycle flux on e12 and e14 were greater (P < 0.05) for embryos from small eggs, except on e14 when the absolute rate of non-Cori cycle flux did not differ (Figure 3). On e16 and e18, these fluxes did not differ between egg sizes, except for the absolute rate of non-Cori cycle flux, which on e16 was greater (P < 0.05) in embryos from typical size eggs. Transfer of [13C6]Glucose Carbon to Nonessential AA Figure 2. Molar tracer:tracee ratios (TTR, mol of isotopomer/100 mol of tracee) of blood [M+3] and [M+6]glucose [M is the mass of unlabeled glucose and M+n is the mass of unlabeled glucose plus the number (n) of 13C-atoms in glucose] after 1, 2, 3, or 4 consecutive days of injecting [13C6]glucose into the chorio-allantoic fluid of embryos beginning on embryonic day (e) 14 of development. Each time point represents the mean of 2 embryos. Values in brackets represent the ratio of [M+3]:[M+6]glucose. There was appreciable transfer of 13C to blood alanine, aspartate, glutamate, and glutamine (Table 4), whereas incorporation into serine, glycine, and proline was negligible (data not shown). For alanine, aspartate, glutamate, and glutamine, enrichments of the [M+1], [M+2] and [M+3] isotopomers increased with day of embryo development, this despite that fact that the same amount of [13C6]glucose was injected on each occasion. For alanine, the [M+1] and [M+3] isotopomers were more (P < 0.05) enriched compared with the [M+2] isotopomer. By contrast, the [M+1] and [M+2] isotopomers of aspartate, glutamate, and glutamine 918 Sunny and Bequette Figure 3. Glucose entry rate (A and B), Cori cycle flux (C and D), and non-Cori cycle flux (E and F) in embryos from small (■) and typical (○) size eggs on embryonic day (e) 12, e14, e16, and e18 of development. Values are means ± SEM, n = 4 to 5 embryos. a–cWithin egg size, means without a common letter differ, P < 0.05. *Within day of development, values were different for small and typical size egg embryos, P < 0.05. Divide values by the factor 180.18 to convert to moles. were more (P < 0.05) enriched compared with the [M+3] isotopomer. Based on the [M+3] isotopomer, which uniquely arises from metabolism of [13C6]glucose to pyruvate and thence via pyruvate carboxylase to oxaloaceate and α-ketoglutarate, the proportion of alanine, aspartate, glutamate, and glutamine synthesis (flux) from glucose increased with day of development. DISCUSSION To acquire quantitative data to describe adaptations in glucose metabolism by chick embryos from small (<55 g) and typical size (65 to 70 g) eggs, we developed an in ovo [13C6]glucose stable isotope injection approach. The [13C6]glucose was injected into the chorioallantoic fluid for several reasons. First, it proved to be technically challenging to cannulate the vitelline or other vessels of the embryo without causing hemorrhage and compromising the structural integrity of the surrounding membranes. Second, the chorio-allantoic route was chosen because of the extensive capillary network that surrounds it and that would facilitate direct uptake into the blood supply of the embryo. Therefore, daily injection of tracer into the chorio-allantoic compartment had the effect of a semicontinuous delivery of tracer. To verify this assumption, we injected [13C6] glucose into the chorio-allantoic fluid of e14 embryos for 1 to 4 consecutive days. The results indicated that 3 consecutive days of dosing [13C6]glucose was sufficient to achieve isotopic ([M+3] and [M+6]glucose) and isotopomer ([M+3]:[M+6]glucose) steady-states in blood glucose. The achievement of isotopomer steady-state was an important criterion to satisfy when calculating GNG and glucose molecule and 13C-recycling. Last, the [13C6]glucose injected per day (15 mg) approximates to be 6% of the blood glucose pool (~250 mg) of a 60-g broiler breeder egg embryo on e16 (Romanoff and Romanoff, 1967), which we believe is unlikely to have a significant metabolic (mass) effect when administered in ovo. The ability of the chick embryo to maintain elevated rates of GNG and glycogen metabolism is critical to ensure vital supplies of glucose during pipping and hatching (Freeman, 1969) and to maintain supplies of glucose for proper development of the immune, nervous, and muscular systems (Moran, 2007; Humphrey and Rudrappa, 2008). Plasma glucose can be detected as early as e4 of development, when gluconeogenic enzyme activity begins to increase up until time of hatch (Hazelwood, 1971; Watford et al., 1981; Savon et al., 1993). This increase in gluconeogenic activity raises plasma glucose from 3.33 mM on e4 to 8.33 mM at hatch (Hazelwood, 1971). Increased rates of GNG and glycogenesis occur after e13 (Pearce, 1977; Picardo and Dickson, 1982). Consistent with these observations, we observed increasing blood glucose concentrations and 919 Gluconeogenesis in chicken embryos 13 Table 4. Molar tracer:tracee ratios of blood alanine, aspartate, glutamate, and glutamine C-isotopomers and the proportion of their flux derived from blood glucose via pyruvate carboxylase on embryonic day (e) 12, e14, e16, and e18 of development1 13 C-Isotopomer,2 moles of isotopomer/100 mol of tracee AA Day of embryonic development Alanine e12 e14 e16 e18 Aspartate e12 e14 e16 e18 Glutamate e12 e14 e16 e18 Glutamine e12 e14 e16 e18 SEM SEM SEM SEM M+1 M+2 M+3 % of flux from glucose3 0.55a 0.81b 0.52a 0.93b 0.07 0.76b 1.26a 0.67b 0.50b 0.08 0.53c 1.76b 1.60b 3.51a 0.14 0.38c 1.47b 1.45b 2.98a 0.24 0.27b 0.24b 0.09c 0.44a 0.04 0.00 0.10 0.14 0.33 0.04 0.23c 1.02b 1.33b 3.45a 0.17 1.93c 3.02b 3.54b 5.97a 0.30 0.58ab 0.42b 0.20c 0.85a 0.06 0.02b 0.04b 0.06b 0.14a 0.02 0.05b 0.10b 0.13b 0.38a 0.02 0.09b 0.14b 0.17b 0.41a 0.04 4.9c 27.3a 6.4c 19.3b 2.3 0.2b 2.2ab 2.1ab 3.5a 1.4 0.9b 13.8a 9.2a 13.2a 2.6 1.7b 20.0a 12.5a 14.7a 2.6 a–c For each AA, within each column, means with different superscript letters differ from each other, P < 0.05. Differences were not significant between small and typical size egg embryos; thus, values represent the combined means of the 2 groups. SEM = SE of the difference between means. 2 M+n is moles of 13C-isotopomer per 100 mol of tracee, where n equals the number of 13C-atoms in the AA. 3 The proportion of each AA derived from glucose increased with day of development, P < 0.05. 1 fluctuations in glucose metabolism for both egg sizes between e12 and e18. Glucose entry rate, Cori cycle flux (glucose molecule recycling), and non-Cori cycle flux (i.e., GNG) increased dramatically by e14. About e13 to 14, hypophyseal and adrenocortical activity develops leading to increased release of epinephrine and a concomitant breakdown of glycogen in the liver (Hazelwood, 1971; Jenkins and Porter, 2004). Around the same time, the insulin:glucagon ratio increases, along with corticosterone, favoring deposition of glucose into glycogen (Lu et al., 2007). These changes are associated with increased blood glucose concentrations, as activities of gluconeogenic enzymes increase and attain maxima by e16 or 17, thereafter decreasing around hatch (Pearce, 1977). Thus, blood entry of glucose from glycogen breakdown in the liver and the rapid increase in activities of gluconeogenic enzymes will be responsible for the increased rates of glucose entry we observed on e14. Further, with the negative feedback exerted by corticosterone on hypophyseal and adrenocortical activity (Jenkins and Porter, 2004), glucose metabolism stabilizes across e16 and e18. We hypothesized that embryos from small eggs, due to their reduced supplies of preformed glucose from egg components, would need to transition to greater rates of GNG earlier in development compared with embryos from typical size eggs. Furthermore, to support these greater rates of GNG, we expected embryos from small eggs to have a greater reliance on AA and other ≥3-carbon chain substrates (e.g., triglyceride glycerol) earlier in development to support net synthesis of glucose for storage of glycogen in liver and muscle in preparation for hatch. Indeed, glucose entry rate and non-Cori cycle flux (i.e., net glucose synthesis from nonglucose substrates) were greatest on e12 for embryos from small eggs, both on an absolute (per egg) and weight-adjusted basis. Interestingly, these greater rates of glucose metabolism on e12 occurred when the weights of the embryos were not different between the egg sizes. Supporting the greater rates of non-Cori cycle flux by embryos from small eggs necessitates metabolism of egg resources that will provide at least a 3-carbon chain. Assuming that the preformed glucose content of eggs is 300 mg/65-g egg (Romanoff and Romanoff, 1967), the small eggs (51 g) would have 235 mg of preformed glucose and the typical size eggs (65 g) 300 mg available at time set. Given that fresh embryo weights did not differ between egg sizes on e12, in turn suggesting that metabolic demands for glucose are similar, the small egg embryos would need to synthesize an additional 65 mg of glucose to make up for the deficit. Correspondingly, small egg embryos synthesized an additional 70 mg of glucose from nonglucose sources (non-Cori cycle flux) on e12 alone compared with embryos from typical size eggs. Further, because AA can only contribute at most one 3-carbon triosphosphate to glucose synthesis, 920 Sunny and Bequette the equivalent amount of AA needed to synthesize 70 mg of glucose is 100 mg. Although we cannot rule out metabolism of yolk triglyceride glycerol for GNG, we believe albumen-derived glucogenic AA make a larger contribution to GNG in embryos from small compared with typical size eggs. In support of this view, blood concentrations of the glucogenic AA threonine, isoleucine, valine, glutamine, and tyrosine, as well as concentrations of the nonglucogenic AA arginine, leucine, glycine, and proline, were all less in embryos from small eggs throughout development. We believe that this reduced availability of AA partly explains the reduced embryonic weights of the embryos from small eggs. Furthermore, although the absolute rate of non-Cori cycle flux reached a maximum in embryos from typical size eggs on e16, a time that coincides with the greatest rates of glycogen synthesis and storage (Hazelwood, 1971), non-Cori cycle flux was significantly less and continued to decline in embryos from small eggs from e16 onward. Although we did not observe reduced blood glucose concentrations in embryos from small eggs, our observation that these embryos had reduced rates of non-Cori cycle flux is consistent with previous observations that blood glucose is less in embryos from small compared with larger eggs (Latour et al., 1996). Potential consequences of reduced glucose status are greater rates of mortality, reduced BW gain, and suboptimal performance of hatchlings (Moran, 1989; Donaldson et al., 1992; Donaldson and Christensen, 1993; Christensen et al., 2000, 2001). Furthermore, our conclusion would be consistent with observations that in ovo injection of glucose, AA, or both in e7 or e16 embryos from typical size eggs improves chick BW gains (4 to 7%) by d 7 and 56 posthatch and increases liver glycogen at hatch (Ohta et al., 1999; Ferket and Zehava, 2002; Uni et al., 2005). As a general observation, fractional glucose molecule recycling exceeded glucose 13C-recycling throughout development, and there was no notable difference between the eggs sizes for this relationship. If glucose carbon recycling is complete, then we would expect fractional glucose molecule and 13C-recycling to be similar. That they were not similar indicates that there is considerable transfer of glucose carbon to other metabolites, namely AA, bicarbonate, glycerol, and fatty acids. Of the nonessential AA, alanine, aspartate, glutamate, and glutamine became 13C-labeled, whereas there was no or negligible transfer of 13C to serine, glycine, proline, and arginine. With respect to the latter 4 AA, our observations indicate that these should be considered essential AA for the developing embryo, relying solely upon albumen for their supply. In conclusion, the present study is the first to quantify rates of glucose metabolism by chick embryos from e12 to e18 of development. 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