Download Glycerol is a major substrate for glucose, glycogen, and

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
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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-
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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.
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