Published December 4, 2014
Expression and activity of key hepatic gluconeogenesis enzymes
in response to increasing intravenous infusions
of glucose in dairy cows1
B. Al-Trad,* T. Wittek,† G. B. Penner,‡ K. Reisberg,§ G. Gäbel,§ M. Fürll,#
and J. R. Aschenbach*2
*Institute of Physiology and Pathophysiology, University of Veterinary Medicine Vienna, 1210 Vienna, Austria;
†Division of Animal Production and Public Health, University of Glasgow, G61 1QH, United Kingdom;
‡Animal and Poultry Science, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5A8, Canada;
§Institute of Veterinary Physiology, and #Clinic for Large Animal Internal Medicine,
University of Leipzig, D-04103 Leipzig, Germany
ABSTRACT: The present study aimed at investigating whether increasing concentrations of glucose supply
have a depressive effect on the mRNA abundance and
activity of key gluconeogenic enzymes in dairy cows.
Twelve Holstein-Friesian dairy cows in mid-lactation
were intravenously infused with saline (SI; n = 6) or
a 40% glucose solution (GI; n = 6). For GI cows, the
infusion dose increased by 1.25%/d relative to the initial NEl requirement until a maximum dose equating to
surplus 30% NEl was reached on d 24. Cows receiving
SI received an equivalent volume of 0.9% saline solution. Blood samples were taken every 2 d, and liver
biopsies were collected every 8 d. A treatment × quadratic dose interaction (P < 0.01) was observed for the
concentration of plasma glucose and serum insulin. The
interactions were due to positive quadratic responses of
the concentrations of glucose and insulin for GI cows,
whereas the concentrations of glucose and insulin did
not change over time for SI cows. The concentration
of β-hydroxybutyric acid (BHBA) and serum urea nitrogen (BUN) responded in a treatment × quadratic
dose manner, such that greater decreases (P < 0.01) in
BHBA and BUN concentrations were observed for cows
receiving GI than SI as the dosage increased. Serum
NEFA concentration tended to follow a similar pattern
as serum BHBA and BUN; however, the interaction
was not significant (P = 0.07). The mRNA abundance
of gluconeogenesis enzymes followed a linear treatment
× dose interaction (P < 0.05) for only pyruvate carboxylase (PC), which was paralleled by a trend for a
linear treatment × dose interaction (P = 0.13) for PC
enzyme activity. The least PC expression and activity were observed at the largest glucose dosage. The
activity, but not mRNA abundance, of fructose 1,6-bisphosphatase (FBPase) showed treatment × quadratic
dose interactions (P < 0.05) with decreasing activity
at increasing glucose dose. Activities and expression
of phosphoenolpyruvate carboxykinase and glucose
6-phosphatase were not affected (P > 0.25) by treatment. In conclusion, hepatic gluconeogenic enzymes are
only moderately affected by slowly increasing glucose
supply, including a translational or posttranslational
downregulation of FBPase activity and a decrease in
the mRNA abundance of PC with possible consequences for PC enzyme activity.
Key words: dairy cow, enzyme activity, gene expression, glucose infusion, hepatic gluconeogenesis
©2010 American Society of Animal Science. All rights reserved.
J. Anim. Sci. 2010. 88:2998–3008
doi:10.2527/jas.2009-2463
INTRODUCTION
1
Experiments were performed at the University of Leipzig. The
authors thank C. Benson, A. Schmidt-Mähne, and I. Urbansky from
the University of Leipzig for technical help and G. F. Schusser (University of Leipzig) for infrastructural support. The work was supported by Pfizer Animal Health, Sandwich, UK.
2
Corresponding author: [email protected]
Received September 9, 2009.
Accepted May 1, 2010.
The majority of ingested carbohydrates are converted
to short-chain fatty acids in the forestomach of ruminants (Young, 1977; Seal and Reynolds, 1993). Under
many dietary settings, this implies limited availability
of glucose for absorption (Baird et al., 1980; Reynolds
et al., 1988), thereby generating a need for hepatic
gluconeogenesis (Young, 1977). Efficient gluconeogenesis is especially important in dairy cows because it is
the major pathway for maintaining adequate glucose
2998
Hepatic gluconeogenic enzymes in dairy cows
supply for the mammary gland (Reynolds et al., 1988;
Huntington et al., 2006). When cows approach peak
lactation, insufficient gluconeogenesis might contribute
to the occurrence of metabolic disorders, such as ketosis
and fatty liver (Mills et al., 1986; Rukkwamsuk et al.,
1999; Murondoti et al., 2004). Consequently, knowledge
on the regulation of hepatic gluconeogenesis has implications for the performance and health status of dairy
cows.
Although surplus supply of glucose has often been
shown to downregulate hepatic glucose production
(Bartley and Black, 1966; Thompson et al., 1975; Lomax et al., 1977; Rigout et al., 2002), it is a crucial
question whether this also involves a downregulation
of the involved enzymes. A downregulation of gluconeogenic enzymes could have consequences extending
beyond the period of surplus glucose or glucogenic substrate supply. For example, a downregulation of gluconeogenic enzymes in cows overfed prepartum could
exacerbate metabolic disturbances during the postpartum phase of the transition period (Rukkwamsuk et
al., 1999; Murondoti et al., 2004). Therefore, the objective of this study was to investigate the dose effect of
intravenous glucose infusion on the mRNA abundance
and activity of key enzymes of hepatic gluconeogenesis.
The hypothesis was that mRNA abundance and activity of hepatic gluconeogenic enzymes would decrease
with increasing dosage of glucose and that, at least, the
decreases of enzyme activity would persist beyond the
actual period of surplus glucose supply.
MATERIALS AND METHODS
Experimental procedures were preapproved by the
local authorities, Regierungspräsidium Leipzig (reference 24-9168.11, TVV 49/06).
Animals and Experimental Design
The use of animals, the general experimental design,
and the production traits during the experiment have
been described in detail in a previous report (Al-Trad
et al., 2009). In brief, experiments were carried out on
12 Holstein-Friesian cows from the dairy herd of the
University of Leipzig. Cows were confirmed to be in the
2nd to 4th month of gestation (193 ± 14 DIM) at the
start of the experiment and had an average BW and energy-corrected milk yield of 632 ± 33 kg and 30.5 ± 2.3
kg/d, respectively. Cows were housed in individual tie
stalls with straw bedding. Twice daily (0600 and 1500
h), cows received a diet based on grass haylage and
supplements containing a commercial energy concentrate for lactating dairy cows (Multilac, Leikra GmbH,
Leipzig, Germany) and soybean meal. The ingredient
and chemical composition of the diet has previously
been reported (Al-Trad et al., 2009). The diet was low
in starch (11.3% DM) and sugar content (4.4% DM) to
minimize the direct entry of glucose from the digestive
2999
tract. Water was available for ad libitum intake. Cows
were milked at 0630 and 1600 h.
Cows were assigned to a glucose infusion (GI; n =
6) or saline infusion (SI; n = 6) treatment balanced
for actual lactation performance and DIM. Infusions
were made via a 14-ga, 20-cm jugular catheter (Cavafix Certo Splittocan 338, Melsungen, Germany) that
was replaced every 8 d. Glucose infusion treatment consisted of continuous jugular infusions of 40% glucose
solution (Serumwerk Bernburg, Bernburg, Germany)
over a period of 28 d. The infusion dose was calculated
for each animal individually as a percentage of their
daily NEl requirement at the start of the study (see
Calculations and Statistical Analysis). Dosage was 0%
of the NEl requirement on d 0 and increased linearly
by 1.25% each day until a maximum dosage of surplus
30% of the NEl requirement was reached at d 24. This
was equivalent to 2.65 ± 0.19 kg of glucose per cow per
day. After maintaining the infusion dose at 30% of the
NEl requirement between d 24 and 28, responses to glucose withdrawal were assessed by withholding infusions
between d 29 and 32. Infusion canisters were loaded at
1500 h each day, starting in the afternoon of d 0. This
ensured that cows had been on the assigned dosage for
at least 19 h before the collection of biopsies (see below). The infusion protocol and the individual calculation of infusion volumes were identical for the SI treatment as the GI treatment except that the cows on the
SI treatment received a volume-equivalent dose of 0.9%
saline (Serumwerk Bernburg, Bernburg, Germany) as a
control treatment.
Sampling and Measurements
Liver biopsies were obtained between 1000 h and
1200 h on d 0, 8, 16, 24, and 32 with a 2.5-mm wide,
250-mm long biopsy needle (model Berlin, Walter Veterinär-Instrumente, Rietzneuendorf, Germany) under
ultrasonography control (Pie Medical Scanner 100 LC,
Pie Medical, Maastricht, the Netherlands) as described
by Gröhn and Lindberg (1982). After collection, liver
samples were washed immediately in ice-cold 0.9% saline solution. Samples for enzyme activity determinations (~200 mg) were snap-frozen in liquid nitrogen
and stored at −80°C. Samples (~300 mg) for real-time
reverse transcription-PCR (rt-PCR) were transferred
into tubes with 3 mL of RNAlater (Qiagen, Germantown, MD), placed in a refrigerator for 24 h, and stored
at −20°C thereafter.
Blood samples (20 mL for serum plus 20 mL for plasma) were taken from a coccygeal vein every 2 d at 1000
h and processed as described previously. Monovette
tubes with heparin (16 IU of lithium heparin/mL of
blood; Sarstedt, Nümbrecht, Germany) were used to
obtain plasma for glucose analysis. Kaolin-coated Monovette tubes (Sarstedt) were used for serum preparation to determine insulin, serum urea nitrogen (BUN),
NEFA, and β-hydroxybutyric acid (BHBA) concentra-
3000
Al-Trad et al.
tions. All plasma and serum samples were stored at
–20°C.
Blood Analyses
An automatic analyzer (Hitachi 912, Boehringer
Mannheim, Mannheim, Germany) was used to analyze the concentrations of plasma glucose (glucose/HK
kit, Roche Diagnostics GmbH, Mannheim, Germany;
Peterson and Young, 1968), serum BUN (urea/BUN
kit, Roche; Talke and Schubert, 1965), serum NEFA
(NEFA kit, Randox Laboratories Ltd., Crumlin, UK;
Matsubara et al., 1983) and serum BHBA (Ranbut/
kit, Randox; McMurray et al., 1984). A radiometric
immunoassay was used for analysis of serum insulin
(INS-IRMA Kit; BioSource Europe SA, Nivelles, Belgium), which has a high specificity for insulin and no
significant cross-reactivity to proinsulin (Temple et al.,
1990). The kit originally comes with a human calibration standard. However, we routinely prepare calibration standards by diluting bovine insulin in the human
insulin-free serum provided with the test to account for
the partial differences in the AA sequence between human and bovine insulin.
Gluconeogenic Enzymes Activity
The activities of the following key hepatic gluconeogenic enzymes were measured: pyruvate carboxylase
(PC, EC 6.4.1.1), phosphoenolpyruvate carboxykinase
(PEPCK, EC 4.1.1.32), fructose 1,6-bisphosphatase
(FBPase, EC 3.1.3.11), and glucose 6-phospatase (G6Pase, EC 3.1.3.9). For PC and PEPCK assays, liver
tissues were homogenized with an Ultra-Turrax T25
homogenizer (IKA, Staufen, Germany). Thereafter,
mitochondria were disrupted by sonication (Bandelin
Sonoplus HD 2070; Bandelin Electronic, Germany) according to the protocol of Agca et al. (2002). Pyruvate
carboxylase activity was assayed spectrophotometrically at room temperature using the previously published
method of Crabtree et al. (1972). This procedure measures the reduction of 5,5′-dithiobis-2-nitrobenzoic acid
by CoA. The latter is released when citrate synthase
couples acetyl-CoA to oxaloacetate emerging from the
PC reaction. Activity of PEPCK was measured at 37°C
based on the 14CO2 incorporation assay of Ballard and
Hanson (1967) with the modifications described by
Agca et al. (2002). To avoid the release of 14CO2 into
the environment, the 14CO2 not incorporated into oxaloacetate was trapped in barium hydroxide (0.2 M) at
the end of the procedure.
For FBPase and G6-Pase assays, tissues were homogenized (1:10, wt/vol) in ice-cold homogenization buffer
containing 20 mM HEPES, 100 mM sucrose, and 0.25
mM EDTA (pH 7.4). The homogenate was centrifuged
for 5 min at 1,000 × g at 4°C to remove cell debris. The
supernatant was recentrifuged at 10,000 × g for 10 min
at 4°C. The resulting supernatant was used to determine FBPase and G6-Pase activities by the methods
of Marcus et al. (1973) and Swanson (1950), respectively. The latter methods spectrophotometrically measure the release of inorganic phosphate from fructose
1,6-bisphos­phate or glucose 6-phosphate.
RNA Isolation and Quantitative rt-PCR
Ribonucleic acid was extracted from liver tissues
stored in RNAlater using Trizol reagent (Invitrogen,
Carlsbad, CA) according to the manufacturer’s instructions. The resulting RNA pellets were dissolved
in RNase-free water, and the quantity and quality of
the isolated RNA were determined by absorbance at
260 and 280 nm. Total RNA (1 µg) was reverse-transcribed using oligo-(dT)15 primer in a 20-µL reaction
according to the manufacturer’s instructions (AMV-RT
kit, Roche Diagnostics, Mannheim, Germany). Reverse
transcription reactions were carried out at 25°C for
10 min followed by 42°C for 60 min and 95°C for 5 min.
The resulting first-strand cDNA was stored at −20°C
until use for rt-PCR.
Quantitative rt-PCR was carried out on a RotorGene 6000 (Corbett Research, Australia), using β2microglobulin (B2M) as a nonregulated reference gene.
Primers and dual-labeled fluorescent probes (Table 1)
for quantitative rt-PCR were designed using the Webbased quantitative rt-PCR probe design software provided by MWG Biotech AG (Ebersberg, Germany,
http://www.eurofinsdna.com/de/home.html) and synthesized by the same company. To ensure the specificity
of the primers and probes, a gel was run, in which a
single band of expected size was obtained. Primer and
probe concentrations were optimized to the concentration that provided the smallest fluorescence threshold
cycle (CT) values and the greatest increase of fluorescence compared with the background. For each sample,
the target gene and the control gene were run under
duplex reaction conditions in duplicate. The following reagents were used for amplification in 20 μL of
final volume: 1 μL of sample cDNA, 2 μL of Mg-free
10X buffer, 0.75 U of Dynazyme II DNA polymerase
(Finnzymes, Espoo, Finland), 5.5 mM MgCl2, 0.3 mM
dNTP, 900 nM of each primer, and 150 nM of each
probe for the genes of interests, and 200 nM of each
primer and 50 nM of the probe for the B2M. Amplification conditions for quantification were 95°C for 2 min
and 45 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C
for 30 s. After the amplification efficiency of each target and reference gene was validated, the relative gene
expression was determined by the 2−ΔΔCT method as
described by Livak and Schmittgen (2001). Gene expression was expressed as the normalized ratio of gene
expression relative to B2M mRNA using 1 sample from
the SI group as an interplate calibrator. The suitability
of B2M as a reference gene was checked by demonstrating that B2M CT values at the beginning of the experiment (i.e., d 0) were not different from those obtained
with later samples (d 8, 16, 24, 32) in both groups (P
> 0.05; data not shown).
Hepatic gluconeogenic enzymes in dairy cows
Calculations and Statistical Analysis
Net energy required for maintenance and lactation
(MJ/d) was calculated as 4.184 × {(BW0.75 × 0.08)
+ milk yield (kg) × [(0.0929 × fat %) + (0.0563 ×
protein %) + (0.0395 × lactose %)]} according to the
NRC (2001). Based on the energy requirement of the
individual cow, infusion dosage (kg of glucose/d) was
calculated as follows: designated dose × NEl/(15.6 MJ/
kg).
All data were analyzed using the PROC MIXED procedure (SAS Inst. Inc., Cary, NC) accounting for repeated measures. The model to test for global effects of
treatment, dosage, and the dosage × treatment interaction included the fixed effects of treatment (GI vs. SI),
dosage (representing dosage of 0, 10, 20, and 30% NEl
requirement), and their interaction. The NEl calculated
before the start of the experiment was used as a covariate. In addition, because cows were gradually exposed
to increasing dosage, dosage was included in the model
as a repeated measure. The covariate error structure
that yielded the smallest Akaike’s and Bayesian information criterion values for each dependent variable was
used. Because the first part of the statistical model
could not account for the steady increase in dosage over
time, we assessed the slope responses of the infusion
dosage and their interaction with treatment (i.e., linear
vs. quadratic effects) using the same model except that
dose was considered to be a continuous variable. The
biological significance of the statistical tests was finally
assessed based on the treatment effects derived from
the first model, the linear and quadratic responses of
dosage, and treatment × dosage-derived slope responses from the second model (Table 2). Priority was given
to treatment × dosage interactions because dosage effects can be partially confounded by time and infusion
volume given the long duration of the experiment and
the constant increase in infusion volume. Comparisons
of postinfusion samples (d 32) to preinfusion samples
(d 0) were performed using a Student’s paired t-test.
Differences were considered significant when P < 0.05,
and tendencies are discussed when P < 0.15.
RESULTS
Production Data
Production data from this study have been reported
previously (Al-Trad et al., 2009). Briefly, DMI was not
different between the GI and SI groups over the period
from d 0 to 24 (period mean ± pooled SEM for GI vs.
SI cows: 17.5 vs. 17.7 ± 0.9 kg/d). Correspondingly,
surplus glucose linearly improved the energy balance
of GI cows between d 0 and 24 (from −14.6 to 35.4
MJ/d), whereas it was unchanged in the SI group during the same period (from −10.4 to −1.8 MJ/d; pooled
SEM: 6.3 MJ/d). The improved energy balance was
coupled to a net BW gain of 32 kg in GI cows (from
651 to 670 kg) relative to the SI cows (from 614 to 601
3001
kg; pooled SEM: 28 kg), whereas the energy-corrected
milk yield was not affected by the treatment (period
mean between d 0 and 24 ± pooled SEM: 27.8 and 29.7
± 1.9 kg/d for GI and SI cows, respectively; Al-Trad
et al., 2009).
Plasma and Serum Metabolites
and Hormones
Plasma glucose and serum insulin concentrations
showed predominantly quadratic dosage effects and
treatment × quadratic dose interactions (P < 0.05; Figure 1A and 1B, respectively). Additionally, there was
a treatment effect on serum insulin concentration (P
= 0.005) and a trend for a treatment effect on plasma
glucose concentrations (P = 0.06). These effects were
due to increases in plasma glucose and serum insulin
concentrations in the GI group occurring mainly when
infusion dosages exceeded 20% NEl requirement, whereas cows receiving the SI treatment had no response to
increasing dosage (Figure 1).
Treatment did not affect the concentrations of BUN,
BHBA, and NEFA; however, BUN, BHBA, and NEFA
decreased in a quadratic manner in response to increasing dosage (P = 0.001; Figure 2A–2C). Additionally,
treatment × quadratic dosage interactions were detected for BUN and BHBA (P = 0.001) and tended to
be present for NEFA (P = 0.07), indicating that the
concentrations of these metabolites decreased specifically with increasing dosage of glucose. In contrast to
glucose and insulin concentrations, however, the major
part of the decrease in BUN, BHBA, and NEFA concentrations occurred at smaller infusion dosages <20%
NEl requirement.
Postinfusion values (d 32) were less (P = 0.01) in the
GI group for NEFA compared with pretreatment values (d 0). However, glucose, insulin, BHBA, and BUN
values were not different between d 0 and 32 in both
groups, indicating a quick reversal of the GI effect on
these other blood metabolites.
Hepatic mRNA Abundance and Activity
of Gluconeogenic Enzymes
Hepatic gluconeogenic enzyme activity and relative
mRNA abundance are listed in Table 2. Treatment did
not affect the mRNA abundance or enzyme activity
measured in this study. However, the relative mRNA
abundance of PC showed a linear treatment × dosage interaction (P = 0.048) and a tendency for a quadratic dosage effect (P = 0.06), indicating decreased
abundance of PC mRNA with increasing glucose dosage. The decreased abundance of PC mRNA coincided
with a numerical decrease in PC activity only at the
greatest dosage of glucose (linear treatment × dosage
interaction; P = 0.13). The relative mRNA abundance
of the mitochondrial isoform of PEPCK (PEPCKM) decreased already at smaller infusion dosage (10%
NEl requirement) in a quadratic manner (P = 0.01),
BC118352.1
NM_177946.3
NM_174737.2
XM_583200.3
NM_001034447.1
NM_001076124.1
B2M
PC
PEPCK-C
PEPCK-M
FBPase
G6-Pase
102–123
138–168
170–192
2,042–2,058
2,066–2,095
2,102–2,122
2,184–2,203
2,234–2,260
2,268–2,288
2,000–2,019
2,069–2,096
2,101–2,120
975–994
997–1,023
1,055–1,073
458–478
495–517
529–547
Nucleotide
range
Forward: 5′-AGCGTCCTCCAAAGATTCAAGT-3′
Probe: 5′-FAM-CACCAGAAGATGGAAAGCCAAATTACCTGAA-BHQ1-3′
Reverse: 5′-GGATGGAACCCATACACATAGCA-3′
Forward: 5′-CAACGCCGTGGGCTACA-3′
Probe: 5′-ROX-CCCCGACAATGTGGTCTTCAAGTTCTGCGA-BHQ2-3′
Reverse: 5′-ATGTCCATGCCATTCTCCTTG-3′
Forward: 5′-TTGGGGAGGGAAATAGCAGG-3′
Probe: 5′-JOE-ATGCACCTTTGTTCAACTTAGGGACAC-TAMRA-3′
Reverse: 5′-AGGGGGAAAGACAAAGAAGAC-3′
Forward: 5′-ACACCACCCAGTTGTTCTCC-3′
Probe: 5′-JOE-TGACAGAACAGGTCAACCAGGATCTGCC-TAMRA-3′
Reverse: 5′-TCCAGTTCAGCCAGCACTTC-3′
Forward: 5′-AGAAGGCAGGAGGAATGGCT-3′
Probe: 5′-ROX-CACCGGGAAGGAAACTGTGCTGGACAT-BHQ2-3′
Reverse: 5′-CAGGAGACCCCAAGATGAT-3′
Forward: 5′-GTCACATCCACCCTCTCTATC-3′
Probe: 5′-JOE-AGCCAACCTACAGATTTCGGTGC-TAMRA-3′
Reverse: 5′-CCAGAATCCCAACCACAAC-3′
Primers and probes sequences3
1
B2M = β2-microglobulin; PEPCK-C = cytosolic phosphoenolpyruvate carboxykinase; PEPCK-M = mitochondrial phosphoenolpyruvate carboxykinase; PC = pyruvate carboxylase; FBPase =
fructose 1,6-bisphosphatase; G6-Pase = glucose 6-phosphatase, catalytic subunit.
2
From the database of the National Center for Biotechnology Information (NCBI); all sequences were derived from cattle.
3
FAM = 6-carboxy-fluorescein; TAMRA = 6-carboxy-tetramethylrhodamine; JOE = 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein; ROX = 6-carboxy-X-rhodamine; BHQ1,2 = blackhole
quencher-1,2.
GenBank
accession2
Gene1
Table 1. Sequences of primers and probes used for quantitative real-time reverse transcription-PCR
3002
Al-Trad et al.
SI
GI
SI
GI
SI
GI
SI
GI
SI
GI
SI
GI
SI
GI
SI
GI
SI
GI
Treatment
2.68
2.84
31.2
26.9
36.2
36.4
26.8
30.3
1.09
1.15
0.96
0.56
0.43
0.30
1.14
0.96
0.62
0.62
0
2.63
2.16
27.4
31.1
32.7
36.7
27.8
29.8
0.69
0.72
0.48
0.68
0.32
0.23
0.80
0.77
0.39
0.43
10
2.94
2.97
28.7
23.6
36.4
38.7
25.1
31.4
0.87
0.72
0.76
0.41
0.26
0.24
0.95
0.61
0.41
0.40
20
2.84
1.46
26.2
23.5
38.4
28.6
24.8
27.8
1.06
0.56
0.54
0.40
0.39
0.22
0.74
0.63
0.56
0.38
30
3.5
3.7
2.1
0.38
0.08
0.08
0.05
0.14
0.20
SEM
0.51
0.91
0.56
0.49
0.65
0.06
0.14
0.17
0.42
Treatment
0.40
0.46
0.03
0.35
0.13
0.01
0.16
0.07
0.14
Linear
0.57
0.47
0.65
0.47
0.02
0.41
0.01
0.74
0.06
Quadratic
Dosage
0.87
0.04
0.77
0.13
0.25
0.73
0.96
0.81
0.04
Linear
0.81
0.02
0.37
0.55
0.34
0.87
0.54
0.36
0.51
Quadratic
Treatment × dosage
interaction
2.64
2.43
25.5
25.8
35.3
36.9
26.0
22.6
1.29
0.94
0.80
0.65
0.44
0.37
0.86
1.17
0.59
0.63
Mean2
0.37
0.46
3.0
1.9
2.4
4.3
3.0
3.6
0.19
0.21
0.08
0.10
0.04
0.08
0.01
0.08
0.07
0.07
SEM
0.94
0.63
0.02
0.77
0.57
0.82
0.87
0.02
0.40
0.33
0.09
0.86
0.94
0.26
0.16
0.18
0.87
0.89
P-value3
Postinfusion
2
Data are from 6 observations in 6 animals.
Postinfusion samples were collected 4 d after the end of the infusion period at an infusion dosage of 0%.
3
The values at the infusion dosage of 0% NEl listed in column 3 (i.e., preinfusion values) were compared with the postinfusion values by paired Student’s t-test.
4
Data are expressed as the normalized ratio of gene abundance relative to β2-microglobulin mRNA abundance using 1 sample from SI group as an interplate calibrator (CT = cycle number at the
threshold level of log-based fluorescence).
5
PC = pyurvate carboxylase; PEPCK-C = cytosolic phosphoenolpyruvate carboxykinase; PEPCK-M = mitochondrial phosphoenolpyruvate carboxykinase; FBPase = fructose 1,6-bisphosphatase;
G6-Pase = glucose 6-phosphatase, catalytic subunit.
1
G6-Pase
FBPase
PEPCK
Activity,
nmol/(mg∙min)
PC
G6-Pase
FBPase
PEPCK-M
PEPCK-C
Relative mRNA
abundance
(normalized
2−ΔΔCT)4,5
PC
Item
Infusion dosage, % of NEl
P-value for contrasts
Table 2. Relative mRNA abundance and activity of hepatic gluconeogenic enzymes for cows on saline infusion (SI) or glucose infusion (GI)1
Hepatic gluconeogenic enzymes in dairy cows
3003
3004
Al-Trad et al.
and the expression of the cytosolic isoform of PEPCK
tended to decrease linearly (P = 0.07) with increasing
dosage. Corresponding to the decreases in the mRNA
abundance for PEPCK, the enzyme assay showed a linear decrease in PEPCK activity with increasing glucose
dosage (P = 0.03). However, the decreases in PEPCK
mRNA abundance and enzyme activity occurred in the
SI and GI groups with no significant treatment × dosage interactions, indicating time-dependent or infusion
volume-dependent changes with no relationship to glucose treatment. A linear decrease (P = 0.01) in both
groups with no treatment × dosage interaction was also
observed for FBPase mRNA abundance. However, FBPase activity remained stable in the SI group despite
the decreasing mRNA abundance but was decreased in
the GI group at the largest dosage of 30% NEl requirement (treatment × quadratic dosage, P = 0.02). Treatment did not affect the mRNA abundance of G6-Pase,
although a quadratic decrease with increasing dose (P
= 0.02) was observed for both treatments. Similarly to
PEPCK-M, the steepest decrease in the mRNA abundance of G6-Pase occurred already at smaller infusion
dosages of 10% NEl requirement. However, the activity of G6-Pase was not affected by treatment, dosage,
or their interactions. As such, the changes of mRNA
abundance and enzyme activity were uncoordinated
for FBPase and G6-Pase, indicating that mRNA abundance had no measurable influence on the activity of
these enzymes.
The significantly or numerically decreased activities
of FBPase and PC and the decreased transcription of
PC in the GI group were restored to preinfusion values by d 32 [i.e., at 4 d after suspending the infusion
(Table 2)]. Similarly, PEPCK mRNA abundance and
activity was restored to preinfusion values except for
a slight remaining reduction in PEPCK activity in the
SI group (P = 0.02). However, the activity of G6-Pase
was decreased in the GI but not the SI group after
stopping infusion (P = 0.02), although G6-Pase mRNA
abundance increased to preinfusion values at the same
time.
DISCUSSION
Hepatic gluconeogenesis is a key component of total
glucose entry in cattle (Young, 1977; Huntington et al.,
2006). Approximately 60 to 85% of the glucose entry is
utilized by the mammary gland in lactating dairy cows
(Annison et al., 1974; Bickerstaffe et al., 1974). Consequently, the kinetics and regulation of gluconeogenesis
are an area of great interest with regard to milk production. The fact that several previous studies failed to
detect a positive influence of surplus glucose supply on
milk production (Amaral et al., 1990; Hurtaud et al.,
1998; Al-Trad et al., 2009) supports the concept that
gluconeogenesis is mostly functioning at a very appropriate amount with regard to lactation requirements
(Al-Trad et al., 2009).
Figure 1. Plasma glucose (A) and serum insulin concentrations (B)
of cows treated with glucose (■) or saline (●). Open symbols depict
pre- and postinfusion values that were not included in statistical trend
modeling. Data are expressed as least squares means; pooled SEM:
0.31 mmol/L (A), 120 pmol/L (B). Probability levels for statistical
contrasts were as follows: treatment: 0.06 (A), 0.005 (B); dose linear:
0.03 (A), 0.10 (B); dose quadratic: 0.02 (A), 0.001 (B); treatment ×
dose linear: 0.61 (A), 0.44 (B); treatment × dose quadratic: 0.001 (A),
0.006 (B). Postinfusion values (d 32) were not different from preinfusion values (d 0; P > 0.05).
As with any biochemical pathway, the regulation of
gluconeogenesis may occur at one or more of the following levels: 1) regulation of substrate supply, 2) regulation of activity of catalytic enzymes, and 3) regulation
of end product utilization. Of these, glucogenic substrate supply is considered the control point best amenable to feeding management (Veenhuizen et al., 1988;
Overton et al., 1999). Increasing the entry rate of propionate and glucogenic AA from the portal-drained viscera has become a successful strategy to support cattle
in periods of glucose shortage, especially in the critical
period postpartum. Such strategies include measures to
increase DMI, to modify fermentation patterns, or to
directly add glucogenic precursors to the diet (Overton
and Waldron, 2004). On the other hand, upregulation
of glucose oxidation or anabolism are primary adaptations of ruminants themselves in times of excessive sup-
Hepatic gluconeogenic enzymes in dairy cows
ply of glucogenic precursors (Judson and Leng, 1973b;
Veenhuizen et al., 1988) or glucose (Amaral et al., 1990;
Rigout et al., 2002). However, data are limiting on how
the regulation of catalytic activity is utilized to meet
imbalances between glucogenic precursor/glucose availability and glucose demand.
The present study was designed to evaluate the effect
of increasing glucose supply on 4 key hepatic gluconeogenesis enzymes in mid-lactating dairy cows. The intention was to explore the whole range of nutritionally
relevant surplus glucose. When assuming postruminal
starch digestion as the major way to provide glucose
directly to dairy cows and proceeding from maximum
rates of postruminal starch digestion in the order of ~5
kg/d (McCarthy et al., 1989; Taylor and Allen, 2005),
a functional maximum uptake capacity (fMUC) of the
small intestine can be calculated in the range of ~2.8
kg of glucose/d according to the formula of Cant et al.
(1999). In the cows of the present study, this fMUC was
modeled by the infusion dosage of 30% NEl requirement
(i.e., 2.65 kg of glucose/d). It became clear that this
infusion dosage may not only be representative for the
intestinal limit of glucose absorption. It is also a dose at
which cows show dysregulation of glucose homeostasis
as evidenced by increased concentrations of blood glucose and insulin. Obviously, the limit of intestinal absorption of glucose and the ability to use the absorbed
glucose appear very coordinated in dairy cows.
The present study additionally modeled the whole
range of gluconeogenic requirement to meet the glucose demand. Diets were formulated to be low in starch
and sugar to provide basal conditions with minimized
direct entry of glucose from the digestive tract. Consequently, hepatic gluconeogenesis had to meet the complete demand for glucose entry at the beginning of the
experiment. By contrast, the infusion dosage of 2.65 kg
of glucose/d at the end of the GI period completely accounted for the glucose demand, which would be 2.27
kg/d at 27.8 kg of energy-corrected milk yield/d according to the formula by Danfær (1994). The latter
means that gluconeogenesis was, in theory, completely
dispensable at an infusion dosage of 30% NEl requirement.
The main finding was that the effect of GI on gluconeogenic enzyme activity was very moderate. Enzymatic
activity decreased (or tended to decrease) for only FBPase and PC, and only at extremely large surpluses of
glucose supply (i.e., infusion of 30% NEl requirement).
Moreover, both decreases were rapidly reversible within
4 d after the end of infusion. We further elucidated that
the glucose-induced decreases in enzyme activity were
not linearly related to decreased mRNA abundance because mRNA abundance and enzyme activity changed
in a coordinated manner only for PEPCK with a tendency for PC, indicating that the activities of FBPase
and G6-Pase are predominantly regulated by translational and posttranslational events.
Three of the investigated enzymes, PEPCK, FBPase,
and G6-Pase, belong to the common gluconeogenic
3005
Figure 2. Serum urea nitrogen (BUN; panel A), β-hydroxybutyric
acid (BHBA; panel B), and NEFA (panel C) concentrations for blood
samples taken every 2 d for cows treated with glucose (■) or saline
(●). Open symbols depict pre- and postinfusion values that were not
included in statistical trend modeling. Data are expressed as least
squares means; pooled SEM, 1.6 mg/dL (A), 0.15 mmol/L (B), 29
µmol/L (C). Probability levels for statistical contrasts were as follows:
treatment: 0.22 (A), 0.98 (B), 0.56 (C); dosage linear: 0.001 (A), 0.06
(B), 0.001 (C); dosage quadratic: 0.001 (A), 0.001 (B), 0.001 (C);
treatment × dosage linear: 0.12 (A), 0.76 (B), 0.19 (C); treatment ×
dosage quadratic: 0.001 (A), 0.001 (B), 0.07 (C). Postinfusion values
(d 32) were different from preinfusion values (d 0) for only the glucose
infusion group in panel C (P = 0.01).
3006
Al-Trad et al.
pathway, whereas PC serves to shuttle lactate and
glucogenic AA into this pathway (Pilkis and Granner,
1992; Jitrapakdee and Wallace, 1999). Unlike in nonruminant species, lactate and glucogenic AA are not the
dominating glucogenic precursors in ruminants where
propionate is quantitatively most important (Amaral
et al., 1990; Huntington et al., 2006). Propionate enters the common gluconeogenic pathway at PEPCK
after initial conversion by propionyl-CoA carboxylase
to oxaloacetate (Halarnkar and Blomquist, 1989). Accordingly, PEPCK has been identified as a main ratelimiting enzyme involved in glucose production from
propionate in dairy cows (Greenfield et al., 2000). Although the rate of propionate utilization for hepatic
gluconeogenesis was not measured in the current study,
the nonsignificant treatment × dosage interaction for
the expression and activity of PEPCK provides indirect
support that infused glucose did not specifically compromise gluconeogenesis from propionate at PEPCK.
Previous glucose metabolism studies in ruminants confirm this suggestion by showing that hepatic propionate
extraction efficiency and capacity for glucose synthesis
from propionate are very resistant to changes in glucose
supply (Judson and Leng, 1973a; Baird et al., 1980) and
insulin concentrations (Brockman, 1990; Eisemann and
Huntington, 1994; Huntington et al., 2006). Therefore,
our enzyme measurements in context with previous investigations on glucose metabolism support the view
that the liver of ruminants has an increased metabolic
priority to utilize propionate for gluconeogenesis (Brockman, 1990; Huntington et al., 2006). This conclusion is
not surprising because over 90% of portal propionate is
cleared by the liver and most of this amount is used for
hepatic glucose synthesis (Armentano, 1992). Efficient
propionate clearance and metabolism might not only
be important for the glucose balance in lactating dairy
cows but may also prevent adverse consequences of propionate accumulation in the blood (e.g., decreased feed
intake; Allen et al., 2005).
In contrast to PEPCK, mRNA abundance of PC
tended to be specifically suppressed by GI. This confirmed earlier findings that PC mRNA abundance is
most consistently changed with varying concentrations
of glucose or energy supply in ruminants (Bradford and
Allen, 2005; Velez and Donkin, 2005; Loor et al., 2006).
Coincidence of a decrease in the mRNA abundance of
PC (by 51% at 30% NEl requirement) with a numerical decrease in PC activity at the greatest dosage of
glucose (by 49% at 30% NEl requirement) might suggest some contribution of decreased PC transcription
to the decreased enzyme activity. It may be speculated
that the observed decrease in PC enzyme activity, although not statistically significant, might have contributed to a decreased deamination of AA because PC is
required for hepatic glucose synthesis from glucogenic
AA (Greenfield et al., 2000; Velez and Donkin 2005).
A decreased deamination of AA, in turn, was suggested
by the decreased serum BUN concentration in the GI
group and has the unique metabolic advantage of spar-
ing AA for protein synthesis pathways (e.g., muscle or
milk protein synthesis).
An enzyme activity significantly affected by GI was
FBPase but only at the greatest dosage of infused glucose (i.e., 30% of the daily NEl requirement, equating
to 2.65 kg of surplus glucose/d). The linear treatment
× dosage interaction was only evident at the enzyme
activity level and not at the mRNA level, indicating
posttranscriptional regulation of enzyme activity. Fructose 1,6-bisphosphatase is the enzyme that releases
fructose 6-phosphate from the gluconeogenic pathway
(Pilkis and Granner, 1992), which, after conversion to
glucose 6-phosphate, can release glucose by the action
of G6-Pase (see below). Fructose 1,6-bisphosphatase
thus controls the overall output of gluconeogenesis regardless of the precursors utilized. Consequently, the
decrease in FBPase activity might be seen as an effective measure to counteract hyperglycemia occurring at
very large infusion dosages. The decrease in FBPase
activity could be directly related to the hyperglycemia
because excess glucose, together with insulin, stimulates the intracellular accumulation of fructose 2,6-bisphosphate, which is a potent competitive inhibitor of
FBPase (Pilkis et al., 1988; Pilkis and Granner, 1992).
Additionally, a rapid proteasomal degradation of FBPase has been demonstrated in yeast upon exposure to
glucose (Gancedo, 1971; Brown and Chiang, 2009). The
rapid translocation of FBPase to the nucleus observed
in cultured rat hepatocytes after exposure to glucose or
insulin (Yáñez et al., 2004) could indicate that similar
proteasomal degradation of FBPase is possible in the
mammalian liver during hyperglycemia.
Apart from investigating the dosage effects of glucose,
the second intention of the present study was to test the
response in gluconeogenic capacity after withdrawing
an extremely large glucose load. It became evident that
PC mRNA abundance and FBPase activity were fully
restored within only 4 d after withdrawing GI. This
supports the view that gluconeogenic enzymes in cattle
liver adapt rapidly to changes in glucose supply. On the
other hand, it indicates that the dysregulation or maladaptation of gluconeogenic enzymes described in postparturient dairy cows are not a primary insufficiency
of carbohydrate metabolism but are likely secondary
to disturbances of lipid metabolism (Rukkwamsuk et
al., 1999; Murondoti et al., 2004). The latter conclusion
does not change when considering that withdrawing
large-dose GI led to posttranscriptional downregulation
of G6-Pase in the present study because the physiological activity of G6-Pase is regulated by the intracellular concentration of its substrate (glucose 6-phosphate;
van Schaftingen and Gerin, 2002). The decrease in
the activity of G6-Pase after stopping glucose infusion
could thus be related to an increased availability of glucose 6-phosphate if glucose 6-phosphate does not only
emerge from gluconeogenesis but also from glycogenolysis (Nordlie et al., 1999; van Schaftingen and Gerin,
2002). Because the liver accumulates excess amounts of
glycogen during increasing GI (Al-Trad et al., 2009),
Hepatic gluconeogenic enzymes in dairy cows
the increased utilization of this glycogen after stopping
the infusion could lead to increased availability of glucose 6-phosphate and, subsequently, to a reduction in
G6-Pase activity.
In conclusion, our study demonstrated that increases of glucose supply have, in general, no negative effect on the activity of key gluconeogenesis enzymes in
mid-lactating dairy cows. Only very large dosages selectively suppress PC mRNA abundance and FBPase
activity. Both effects were fully reversed within only 4
d after the end of large-dose GI. The latter indicates
that glucose metabolism is rather robust with regard to
changes in glucose supply in either direction, at least in
the absence of other metabolic disturbances.
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