Hindawi Publishing Corporation
Biochemistry Research International
Volume 2010, Article ID 512056, 5 pages
Ovine6-Phosphogluconate Dehydrogenase(6PGD) in
Respectto DifferentMilk Yield
StamatinaTrivizaki,George P. Laliotis,Iosif Bizelis,MariaA.Charismiadou,
Department of Animal Science, Laboratory of Animal Breeding and Husbandry, Agricultural University of Athens,
Iera Odos 75, 118 55 Athens, Greece
Correspondence should be addressed to Iosif Bizelis, email@example.com
Received 21 June 2009; Accepted 1 September 2009
Academic Editor: Edna S. Kaneshiro
Copyright © 2010 Stamatina Trivizaki et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Ovine 6-phosphogluconate dehydrogenase (6PGD) is an enzyme of the pentose phosphate pathway, providing the necessary
compounds of NADPH for the synthesis of fatty acids. Much of research has been conducted both on enzymatic level and on
molecular level. However, toour knowledge, anycorrelation between enzymatic activity and6PGD gene expressionpattern related
to different physiological stages has not been yet reported. With this report, we tried to highlight if any correlation between
enzymatic activity and expression of ovine 6PGD gene exists, in respect to different milk yield. According to the determined
enzymatic activities and adipocytes characteristics, ewes with low milk production possessed a greater (P ≤ .001) 6PGD activity
and larger adipocytes than the highly productive ewes. Although 6PGD expression pattern was higher in low milk yield ewes than
in ewes with high milk production, this difference was not found statistically significant. Thus, 6PGD gene expression pattern was
not followed by so rapid and great/sizeable changes as it was observed for its respective enzymatic activity, suggesting that other
mechanisms such as post translation regulation may be involved in the regulation of the respective gene.
6-Phosphogluconate dehydrogenase (6PGD) is an oxidative
phosphogluconate into ribulose 5-phosphate in the presence
of NADP. This reaction is a component of the hexose mono-
phosphate shunt and pentose phosphate pathways.
The functional importance of the enzyme is generally
recognized in providing NADPH for fat synthesis and ribose
for nucleic acid synthesis . In farm animals, fat synthesis
affects the economic return of the producer . Excess
fat deposits influence negatively meat quality, grading of
carcasses, and in high milk yield animals, their health status
and future performance.
Prokaryotic and eukaryotic 6PGDs are proteins of about
470 amino acids whose sequences are highly conserved. The
amino sequences of almost 40 different 6PGDs have been
reported including human , mouse , rat , and pig
. The protein is a homodimer in which the monomers act
independently. Each contains a large, mainly alpha-helical,
domain and a smaller beta-alpha-beta domain, containing a
mixed parallel and antiparallel 6-stranded beta sheet. NADP
is bound in a cleft in the small domain, the substrate binding
in an adjacent pocket [7, 8].
In ruminants, extensive studies have been reported only
for sheep and its cDNA. Carnet and Walker  were the first
who determined the protein sequence of 6PGD reporting the
isolation and characterization of 466 amino acids. However,
of the peptides resulting during the protein determination.
Somers et al.  revised the amino acid sequence based
upon the isolation of the cDNA clones encoding the 6PGD
2 Biochemistry Research International
gene in sheep. Thus, the isolated cDNA encodes a protein of
482 aa with a molecular mass of 52kDa. The conservation
of the protein sequence is very high as it shares an over
50% similarity with the protein encoded by the E. coli 6PGD
It is known that in dairy ruminants major changes occur
during lactation in the metabolism of several tissues such
as adipose tissue, as part of the homeorhetic control of
the organism. In many cases, as the animals are unable to
consume sufficient energy during lactation, utilizes body
reserves. During the last decades a lot of research has
been conducted on enzymatic level showing the reaction
of 6PGD activity in respect to different stimuli [2, 11–16]
without, however, involving any study on molecular level
(i.e., mRNA transcripts). Herein, we report for the first time
the effectof different ovine milk yield of Chios breed ewes on
the characteristics of adipocytes, on enzymatic activity and
expression of 6PGD gene.
2.1. Animal Treatment. The experiment was carried out in
the Experimental Station of the Agricultural University of
Athens. Twenty Chios breed ewes aged 2–4 years old and
with an average (±S.E) live weight of 55.4 ± 1.8kg were
randomly selected and divided in two groups according to
their milk production after the weaning (40 ± 3 days post
partum). Group A and Group B included animals with high
(each animal possessed >1700kg/day, n = 6) and low milk
production (each animal possessed <1100kg/day, n = 14),
The ewes were milked by machine and were fed ad
libitum twice daily, at 7:00 and 16:00 hour on an alfalfa
hay and concentrated feed in pellets, designed to meet
their maintenance and lactation requirements. Water was
freely available. Milk samples were collected and fat content
was determined according to Gerber method. Milk energy
content was estimated in Mj of Net Energy according to the
following equation : E = [91.17M(4.97 + f)]0.00418,
where: E = milk energy (Mj), M = milk yield (kg/day),
f = milk fat content (%).
Once per week samples of subcutaneous adipose tissue
from tail region were taken, by biopsy. A day before sampling
ewes fasted (24 hours) with free access in water, and in
the day of sampling ewes were anaesthetized with the use
of Zoletin 50 in 15–20mg/kg BW. One sample (4–5g)
from adipose tissue was immediately frozen at −20◦C for
the determination of enzyme activity and lipid extraction.
The second sample (0.5–1g) was placed in Krebs-Ringer
bicarbonate buffer (pH = 7.4, 37◦C) for the measurement
of adipocytes size and number, where as the third sample
(∼2.5–3.0g) was immediately frozen in liquid N2and stored
at −80◦C for further RNA extraction.
2.2. Biochemical Parameters. The diameter of 200 fat cells
from each sample was measured as described by Rodbell
. The mean fat cell volume (V) of the 200 cells and
mean adipocytes number/g adipose tissue were calculated
from the mean diameter (d) and the standard deviation (s).
The chemical fat content of adipose tissues was determined
as described by Folch et al. .
For enzyme assay the method described by Rogdakis 
was used. Enzyme activity was expressed as units per fat cell.
2.3. RNA Isolation and Reverse Transcription. Samples from
tail subcutaneous adipose tissue were taken by biopsy. Total
RNA from ovine adipose tissue was extracted using “Rneasy
Lipid Tissue Kit” (Qiagen Cat no. 74804). For first-strand
cDNA synthesis a two-step RT-PCR procedure was followed
using 1ug of the eluted total RNA, which was pre-treated
with DNase I and Omniscript Reversetranscriptase (Qiagen)
according to manufacturer’s recommendations.
2.4. Semiquantitative RT-PCR Analysis. Total RNA from
sheep adipose tissue was isolated as described above and
used as template for a semi-quantitative RT-PCR anal-
ysis. For analysis, Ambion’s QuantumRNA 18S Internal
Standards Kit was employed, resulting in a 324bp prod-
uct. For 6PGD gene amplification the specific primers
FP7: 5?-GGCCTACCACCTGATGAAGGACG-3?and RP8:
5?-GCCAAATTCAGTTGCTGCCTGTC-3?were used result-
ing in a 378bp product. Multiplex PCR conditions were:
94◦C for 3 minutes followed by 30 cycles each of 94◦C for
30 seconds, 60◦C for 30 seconds, 72◦C for 30 seconds, with
a final extension at 72◦C for 2 minutes (Taq polymerase,
New England Biolabs). The relative transcript amount
was determined using the Scion Image software v. 188.8.131.52
(http://www.scioncorp.com/). Each sample was measured 4
2.5. Statistical Analysis. Least Squares Procedures were
employed in statistical analysis . Fixed effects models
were used to describe each individual observation concern-
ing number and size of fat cells, enzymatic activity and
Table 1 shows the average daily net milk energy (Mj), fat
content (%), and milk production (kg). Group A possessed
higher milk production (2.083 ± 0.108kg) and higher net
milk energy (8.545 ± 0.454Mj, P ≤ .001) than Group
B (0.715 ± 0.073kg and 3.012 ± 0.289Mj, respectively).
Concerning the size of the observed adipocytes, the average
diameter of adipocytes of all ewes was 74.03 ± 3.14μm.
However, ewes of group A possessed statistically (P ≤ .01)
smaller adipocytes than group B (64.38 ± 4.49μm versus
83.67 ± 2.94μm, resp.).
An almost two fold greater number (P
adipocytes per g of adipose tissue, was observed in ewes of
group A in contrast to that of group B. The average number
of adipocytes of tail adipose tissue of group A ewes was
4.58 ± 0.51 ∗ 106/g adipose tissue while group B possessed
2.20 ±0.34 ∗ 106adipocytes/g adipose tissue.
≤ .01) of
Biochemistry Research International3
Table 1: Average milk yield (kg), milk fat content (%) and net milk
energy (Mj) of experimental ewes.
Means with different superscript letters differ significantly (∗∗∗P ≤ .001.)
7 89 10 11 12 13 14 15 1617 1819 20
High milk yield
Low milk yield
Figure 1: Expression analysis of 6PGD gene in tail adipose tissue of
high and low milk yield ewes. All samples were normalized to 18S
RNA gene. 10μL of each sample were loaded in the agarose gel for
electrophoresis. As the experimental conditions were the same for
all the samples, the observed differences in the low milk yield group
may be due to differences concerns the sample itself.
The determined average enzymatic activity was signif-
icantly (P ≤ .05) greater in ewes of group B than that
observed in group A (364.80 ± 49.73nmol NADPH ∗
min−1/106adipocytes and 76.80 ± 13.50nmol NADPH
∗ min−1/106adipocytes, resp.). Concerning 6PGD gene
expression in the two groups during lactation (Figure 1)
an increased transcript expression was observed in ewes of
group B compared with the ewes of group A (0.693 ± 0.060
versus 0.595 ± 0.085, resp.). However, this difference was
not statistically significant. Moreover, 6PGD expression did
not follow the acute changes of enzymatic activity and milk
energy level concerning the two groups (Figure 2).
During lactation, udder is the tissue with the highest
metabolic activity. In this study, the metabolic adaptations
of tail adipose tissue of Chios ewes during milk production
were of particular interest.
Factors such as genotype, feed availability, body condi-
tion, number and weight of lambs at birth, environmental
stimuli and their interaction may influence the daily milk
production. According to Chilliard et al. , the mobiliza-
tion of body fat depends on the initial body weight, as has
been observed in sheep and dairy cows. As no differentiation
of the body weight was observed during the study, the
comparability of parameters between the examined groups
Regarding the number of adipocytes observed in this
of the observed number of fat cells/gadipose tissue in respect
to that of group B ewes. The observed range of values
Group AGroup B
NADPH/106adipocytes), gene expression of 6PGD and milk
energy (Mj) of lactating ewes.
2: Correlation amongenzymatic activity (nmol
regarding the number of fat cells observed in this experiment
was significantly higher than the values recorded by previous
researchers [11, 23, 24]. The difference is probably due to
different productivity breeds of ewes (meat type breeds) and
to the fact that the lambs in the experiments of Vernon et al.
 and Travers et al.  have not been weaned.
Comparing the size of fat cells in tail adipose tissue of
the ewes it was observed that the fat cells were on average
significantly smaller in ewes with high milk production
than in ewes with low milk production. This result was
requirements than that of ewes with low milk production
and, thus, the pathway of lipolysis was more intensive in tail
adipose tissue in order to cover the energy requirements of
udder . It is obvious that the catabolic activity of tail
adipose tissue of ewes of group A in this study was higher
as the quantity and the milk energy were increased. This
observation is consistent with observations of Vernon et al.
Diversification of lipogenic dehydrogenases activity such
as 6PGD between animals with different milk productivity
indicates the dependence of lipogenesis of ewes during
the activityof 6PGD is reduced asthe energyof the produced
milk is increased (Figure 2). This observation was expected,
as the ewes in group B due to the low milk production
had lower energy requirements in order to meet the energy
needs of the udder. In group A, a significantly less 6PGD
activity was observed (Figure 2). In this case, the metabolic
pathway of liposynthesis is limited, reducing thus the activity
of lipogenic enzymes in order energy to be utilized for the
maintenance of homeostasis of the body.
As shown in Figure 2, 6PGD activity in tail adipose tissue
is reduced with the increase of milk energy. However, the
difference in the values of 6PGD gene expression between
the two groups was not statistically significant, although
a decrease of expression was observed with the increase
of milk production. The fact that the number of 6PGD
gene transcripts between the two groups did not change
significantly indicates that the control of 6PGD in adipose
tissue during lactation may take place in translation and/or
4 Biochemistry Research International
post-translation level, as previous authors have noted for
important lipogenic genes during lactation, such as ACC
[24, 26]. Moreover, in the low yield ewes group there seems
to be an increasing rate of transcription of the 6PGD gene.
This, may be due to more possible involvement of other
mechanisms than the regulation of translation for example,
the presence of a SNP, which might facilitate the rate of
transcription (group B) or affect the post transcriptional
events (group A).
To sum up, the level of milk production significantly
affects the metabolic role of tail subcutaneous adipose
tissue. In ewes with high milk production, the pathway
of lipogenesis is limited by reducing the size of fat cells
and reducing the activity of 6PGD. In ewes with low milk
production this restriction is less intense, as these animals
have lower energy requirements compared to ewes with high
milk production. The changes in the number of 6PGD gene
transcripts in tail adipose tissue are not analogous either to
the changes of 6PGD activity or to the level of milk pro-
duction. 6PGD expression in the tail adipose tissue during
dairy production is likely to be controlled through post-
further research should be conducted in order to detect
any potential relationship between gene expression, 6PGD
activity and milk yield in sheep.
NADPH: Reduced nicotinamide adenine dinucleotide
PCR: Polymerase chain reaction
RT: Reverse transcription.
Acetyl coenzyme A carboxylase
DNA complementary to RNA
Nicotinamide adenine dinucleotide phosphate
The experiment was approved by the Bioethical Committee
of the Agricultural University of Athens.
 C. Phillips, S. Gover, and M. J. Adams, “Structure of 6-
phosphogluconate dehydrogenase refined at 2˚ A resolution,”
Acta Crystallographica Section D, vol. 51, part 3, pp. 290–304,
 K. E. Belk, J. W. Savell, S. K. Davis, J. F. Taylor, J. E. Womack,
and S. B. Smith, “Tissue-specific activity of pentose cycle
oxidative enzymes during feeder lamb development,” Journal
of Animal Science, vol. 71, no. 7, pp. 1796–1804, 1993.
 S. K. W. Tsui, J. Y. W. Chan, M. M. Y. Waye, K. P.
Fung, and C. Y. Lee, “Identification of a cDNA encoding 6-
phosphogluconate dehydrogenase from a human heart cDNA
library,” Biochemical Genetics, vol. 34, no. 9-10, pp. 367–373,
 J. Mitoma, S. Furuya, M. Shimizu, et al., “Mouse 3-
phosphoglycerate dehydrogenase gene: genomic organization,
chromosomal localization, and promoter analysis,” Gene, vol.
334, no. 1-2, pp. 15–22, 2004.
map and comparative maps for mouse or human homologous
rat genes,” Mammalian Genome, vol. 5, no. 2, pp. 63–83, 1994.
 I. Harbitz, B. Chowdhary, R. Chowdhary, et al., “Isolation,
characterization and chromosomal assignment of a par-
tial cDNA for porcine 6-phosphogluconate dehydrogenase,”
Hereditas, vol. 112, no. 1, pp. 83–88, 1990.
 M. A. Rosemeyer, “The biochemistry of glucose-6-phosphate
dehydrogenase, 6-phosphogluconate dehydrogenase and glu-
tathione reductase,” Cell Biochemistry and Function, vol. 5, no.
2, pp. 79–95, 1987.
 M. J. Adams, S. Gover, R. Leaback, C. Phillips, and D. O.
Somers, “The structure of 6-phosphogluconate dehydroge-
B, vol. 47, part 5, pp. 817–820, 1991.
 A. Carne and J. E. Walker, “Amino acid sequence of ovine 6-
phosphogluconate dehydrogenase,” The Journal of Biological
Chemistry, vol. 258, no. 21, pp. 12895–12906, 1983.
 D. O. Somers, S. M. Medd, J. E. Walker, and M. J. Adams,
“Sheep 6-phosphogluconate dehydrogenase. Revised protein
sequence based upon the sequences of cDNA clones obtained
with the polymerase chain reaction,” Biochemical Journal, vol.
288, part 3, pp. 1061–1067, 1992.
 S. B. Smith and R. L. Prior, “Comparisons of lipogenesis
and glucose metabolism between ovine and bovine adipose
tissues,” Journal of Nutrition, vol. 116, no. 7, pp. 1279–1286,
 J. M. Thompson and R. M. Butterfield, “Changes in body
composition relative to weight and maturity of Australian
Dorset Horn rams and wethers. 4. Adipocyte number and
volume in dissected fat partitions,” Animal Production, vol. 46,
pp. 387–393, 1988.
 E. Panopoulou, S. Deligeorgis, T. Papadimitriou, and E.
Rogdakis, “Carcass composition, size of fat cells and NADP
generating dehydrogenases activity in adipose tissue of the fat-
tailed Chios and thin-taled Karagouniko sheep breed,” Journal
of Animal Breeding and Genetics, vol. 106, pp. 51–58, 1989.
 A. Ayala, I. Fabregat, and A. Machado, “The role of
NADPH in the regulation of glucose-6-phosphate and 6-
phosphogluconate dehydrogenases in rat adipose tissue,”
Molecular and Cellular Biochemistry, vol. 105, no. 1, pp. 1–5,
 E. Rogdakis, M. Charismiadou, S. Orphanos, E. Panopoulou,
and I. Bizelis, “Cellularity and enzymatic activity of adipose
tissue in the Karagouniko dairy breed of sheep from birth to
maturity,” Journal of Animal Breeding and Genetics, vol. 114,
no. 5, pp. 385–396, 1997.
 M. Rippa, P. P. Giovannini, M. P. Barrett, F. Dallocchio, and S.
Hanau, “6-phosphogluconate dehydrogenase: the mechanism
of action investigated by a comparison of the enzyme from
different species,” Biochimica et Biophysica Acta, vol. 1429, no.
1, pp. 83–92, 1998.
 A. P. Mavrogenis and C. Papachristoforou, “Estimation of the
energy value of milk and prediction of fat-corrected milk yield
Biochemistry Research International5 Download full-text
in sheep and goats,” Small RuminantResearch, vol. 1, no. 3, pp.
adipose tissue,” The Journal of Biological Chemistry, vol. 239,
pp. 753–755, 1964.
 J. Folch, M. Lees, and G. Sloane-Stanley, “A simple method
for the isolation and purification of total lipids from animal
tissues,” The Journal of Biological Chemistry, vol. 226, pp. 497–
 E. Rogdakis, “Untersuchungen ueber die Aktivitaet NADPH-
liefernder Enzyme im Fettgewebe des Schweines. I. Mitt:
biopsietechnik und enzymteste sowie enzymatische Unterschiede
zwischen verschiedenen anatomischen Stellen des Fettgewebes,”
Zeitschrift f¨ ur Tierphysiologie, Tierern¨ ahrung und Futtermit-
telkunde, vol. 33, pp. 329–338, 1974.
 SAS/STAT User’s Guide, Version 6. SAS Inst., Inc. Cary, NC,
 Y. Chilliard, A. Ferlay, Y. Faulconnier, M. Bonnet, J. Rouel,
and F. Bocquier, “Adipose tissue metabolism and its role in
adaptations to undernutrition in ruminants,” Proceedings of
the Nutrition Society, vol. 59, no. 1, pp. 127–134, 2000.
 R. G. Vernon, A. Faulkner, E. Finley, H. Pollock, and E.
kidney, skeletal muscle, adipose tissue and mammary gland of
lactating and non-lactating sheep,” Journal of Animal Science,
vol. 64, no. 5, pp. 1395–1411, 1987.
 M. T. Travers, R. G. Vernon, and M. C. Barber, “Repression
of the acetyl-CoA carboxylase gene in ovine adipose tissue
during lactation: the role of insulin responsiveness,” Journal
of Molecular Endocrinology, vol. 19, no. 2, pp. 99–107, 1997.
 J. P. McNamara and J. K. Hillers, “Regulation of bovine
adipose tissue metabolism during lactation. 1. Lipid synthesis
in response to increased milk production and decreased
energy intake,” Journal of Dairy Science, vol. 69, no. 12, pp.
 R. G. Vernon, M. C. Barber, and E. Finley, “Modulation of
the activity of acetyl-CoA carboxylase and other lipogenic
enzymes by growth hormone, insulin and dexamethasone
in sheep adipose tissue and relationship to adaptations to
lactation,” Biochemical Journal, vol. 274, no. 2, pp. 543–548,