Download Gluconeogenesis differs in developing chick embryos derived from

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. The observed changes in
gluconeogenic fluxes are consistent with previous measures of gluconeogenic enzyme activity and gene expression, as well as the patterns of hormonal regulation. Embryos from small eggs exhibited rates of GNG
that were greater than embryos from typical size eggs
earlier in development, and perhaps as a consequence,
the reduced embryonic growth of embryos from small
eggs may have resulted from the partition of greater
supplies of AA toward GNG. Future work should aim
to investigate the individual contribution of these AA
to GNG, the energy-sensing mechanisms that coordinate egg resource use, and application of approaches to
ensure adequate glucose status and improved survival
of embryos from small size eggs (<55 g) from broiler
breeders.
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