Download Changes in gene expression of glucose transporters in lactating and

Changes in gene expression of glucose transporters in lactating
and nonlactating cows1
T. Komatsu*2, F. Itoh*, S. Kushibiki*, and K. Hodate†
*Department of Animal Physiology and Nutrition, National Institute of Livestock and Grassland Science,
Tsukuba, Ibaraki 305-0901, Japan; and †School of Veterinary Medicine and Animal Science,
Kitasato University, Towada, Aomori 034-8628, Japan
amount of GLUT1, whereas the adipose tissue of peaklactating cows did not (P < 0.05). There were no significant differences in the abundance of GLUT4 mRNA in
adipose tissue and muscle, whereas GLUT4 mRNA was
not detected in the mammary gland. The plasma insulin
concentration was greater (P < 0.05) in nonlactating
cows than in peak- and late-lactating cows. The results
of the present study indicate that in lactation, GLUT1
expression in the mammary gland and adipose tissue
is a major factor for insulin-independent glucose metabolism, and the expression of GLUT4 in muscle and adipose tissue is not an important factor in insulin resistance in lactation; however, the plasma insulin concentration may play a role in insulin-dependent glucose
metabolism. Factors other than GLUT4 may be involved in insulin resistance.
ABSTRACT: Glucose delivery and uptake by the
mammary gland are a rate-limiting step in milk synthesis. It is thought that insulin-independent glucose uptake decreases in tissues, except for the mammary
gland, and insulin resistance in the whole body increases following the onset of lactation. To study glucose
metabolism in peak-, late-, and nonlactating cows, the
expression of erythrocyte-type glucose transporter
(GLUT1) and the insulin-responsive glucose transporter (GLUT4) in the mammary gland, adipose tissue,
and muscle were assessed by Western blotting and realtime PCR. Our results demonstrated that the mammary gland of lactating cows expressed a large amount
of GLUT1, whereas the mammary gland of nonlactating
cows did not (P < 0.05). On the other hand, adipose
tissue of late and nonlactating cows expressed a large
Key Words: Adipose Tissue, Cows, Glucose Transporter, Lactation, Mammary Gland
2005 American Society of Animal Science. All rights reserved.
Introduction
J. Anim. Sci. 2005. 83:557–564
cose transporter in the lactating bovine mammary
gland (Zhao et al., 1996), but whether GLUT1 expression changes during the period of lactation is unknown.
Insulin-responsive glucose transporter (GLUT4) is
the transporter isoform primarily responsible for insulin-stimulated glucose transport and is therefore found
mainly in insulin-sensitive tissues, such as fat and muscle (James et al., 1989). In lactation, it is considered
that the increase in glucose supply to the mammary
gland is partially mediated by an increase in wholebody insulin resistance (McDowell et al., 1987), but the
change of expression of GLUT4 during lactation has
not been extensively investigated.
In ruminants, GH is a galactopoietic hormone (i.e.,
the systemic administration of GH to cows markedly
increases milk production). Growth hormone augments
peripheral insulin resistance in dairy cows following
the i.v. injection of insulin (Rose et al., 1997). It has
been reported that the plasma insulin concentration is
lower in lactating cows than in nonlactating cows (Peel
et al., 1983).
The objectives of this study were to investigate
changes in expression of GLUT1 and GLUT4 related
During lactation, lactose synthesis from glucose in
the mammary epithelial cells seems to be the rate-limiting step in milk synthesis. To supply glucose to the
mammary gland, insulin-independent glucose uptake
decreases in tissues, except for the mammary gland,
and insulin resistance in the whole body increases at
peak lactation (McDowell et al., 1987); however, the
mechanisms during lactation are still unclear.
Insulin-independent glucose transporter (GLUT1) is
expressed in many tissues, including the brain, kidney,
and mammary gland (Burant et al., 1991). It is reported
that GLUT1 may be the predominant facilitative glu-
1
The authors thank the staff of the Ruminants and Field Management Section, Natl. Inst. of Livest. and Grassl. Sci., for technical
assistance and animal management.
2
Correspondence: Ikenodai 2 (phone: 81-29-838-8645; fax: 81-29838-8606; e-mail: [email protected]).
Received June 24, 2004.
Accepted November 29, 2004.
557
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Komatsu et al.
to glucose metabolism in peak-, late-, and nonlactating
cows, and to clarify the mechanisms of the decrease of
insulin-independent glucose uptake and the increase of
insulin resistance during lactation.
Materials and Methods
Animals and Tissues
Four peak-lactating (8 to 11 wk after parturition),
four late-lactating (40 to 50 wk after parturition), and
four nonlactating (dried off for 3 to 10 wk) Holstein
cows were used in this experiment. Cows were fed a
diet based on a total mixed ration, so as to maintain
BW (63.7% DM, 66.9% TDN, 13.5% CP, and 42.8% NDF;
DM basis) and milk yield (88.0% DM, 79.7% TDN,
18.75% CP, and 26.4% NDF; DM basis) according to
the Japanese Feeding Standard for Dairy Cattle (Agriculture, Forestry, and Fisheries Research Council Secretariat, 1999). The diets for lactating and nonlactating
cows were quartered and supplied at 0400, 1000, 1600,
and 2200 each day using an automatic feeder, and cows
were fasted for 12 h before slaughter. Lactating cows
were milked at 0830 and 1800. The mean milk yield of
the cows on the day before slaughter was 27.9 ± 2.51
kg/d in peak-lactating cows and 18.5 ± 2.16 kg/day in
late-lactating cows. The mean BW of the cows were 580
± 62.6 kg in peak-lactating cows, 665.8 ± 33.4kg in latelactating cows, and 657 ± 41.5 kg in nonlactating cows.
All animals received humane care as outlined in the
Guide for the Care and Use of Experimental Animals
(NILGS, 2002). Two 5-g of samples of intestinal adipose
tissue, mammary gland, and skeletal muscle (pectoralis
prefundis) were collected immediately after slaughter,
and frozen in liquid N. The biopsy site of mammary
gland selected was in the basal (upper) portion of the
left udder. Fat and large s.c. blood vessels were avoided
whenever possible during incision. Samples were stored
at −80°C before protein and RNA extraction.
RNA Isolation and cDNA Synthesis
Total RNA was extracted with TRIzol reagent (Invitrogen, Leek, The Netherlands) according to the manufacturer’s protocol, then treated with DNase I to remove residual genomic DNA, and quantified spectrophotometrically by light absorbance at a wavelength of
260 nM; the ratio of absorbance at 260 nM to that at
280 nM was always greater than 1.8. The method was
modified and optimized for extraction of RNA from adipose tissue. As opposed to the manufacturer’s protocol,
we used 10 mL of TRIzol/g of adipose tissue and added
4 mL of chloroform. The cDNA was synthesized from
100 ng of total RNA using a random hexamer (TaKaRa,
Tokyo, Japan) and Murine Moloney leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA).
Western Blotting
Tissue GLUT1 was analyzed by Western blotting as
reported previously (Abe et al., 2001). Tissues were ho-
mogenized in PBS containing 1 ␮g/mL of aprotinin and
1 mM phenylmethylsulfonyl fluoride using a Polytron
homogenizer, and centrifuged at 3,000 × g for 20 min at
4°C. Protein concentrations of the supernatant fraction
were measured using a BCA protein assay reagent kit
(Pierce, Rockford, IL) and BSA as a standard. The supernatant fraction was subjected to 10% SDS-PAGE,
electrophoretically transferred to a polyvinylidene
difluoride membrane, and blocked overnight at 4°C in
blocking buffer (Tris-buffered saline with 0.1% Triton
X-100 Sigma, St. Louis, MO] containing 5% nonfat dried
milk [Meiji, Tokyo, Japan]). Membranes were incubated with diluted (1:500) GLUT1 antiserum (Calbiochem, San Diego, CA) in blocking buffer at room temperature for 1 h. The membranes were then washed five
times at room temperature for 10 min in Tris-buffered
saline with 0.1% Triton X-100 and incubated for 1 h at
room temperature in blocking buffer, with a 1:3,000
dilution of goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase (Zymed, San Francisco CA). The luminescent signals on membranes were
visualized using an enhanced chemiluminescence detection kit (Amersham, Buckinghamshire, U.K.) according to the manufacturer’s instructions, followed by
exposure to x-ray film for 5 min. The resulting films
were analyzed by scanning densitometry. All samples
of mammary gland and adipose tissue were treated on
each blot.
Real-Time PCR from cDNA
GLUT1 and GLUT4 mRNA. The abundance of
GLUT1 and GLUT4 mRNA was measured with the
use of gene-specific double-fluorescent-labeled probes,
TaqMan Universal PCR Mix and a 7700 Sequence Detector (Applied Biosystems, Foster, CA). Six-carboxyfluorescein and VIC (Applied Biosystems, Foster, CA)
were used as the 5′-fluorescent reporters, while 6-carboxy-tetramethyl-rhodamine (TAMRA) was added to
the 3′ end as a quencher. To compensate for variations
in input RNA amounts and the efficiency of reverse
transcription, the housekeeping gene’s glyceraldehyde3-phosphate dehydrogenase mRNA was quantified and
results were normalized to these values. The PCR amplification was performed using the primer and probe
(Applied Biosystems) sets outlined in Table 1. Primers
and TaqMan probes were designed using the computer
program Primer Express (Applied Biosystems). The
PCR was performed at 95°C for 10 min, followed by 45
cycles at 94°C for 30 s, 59°C for 30 s, and 72°C for 30
s. Use of the TaqMan probe resulted in reliable and
sensitive quantification of the real-time PCR (RT-PCR)
product with good linearity (Pearson correlation coefficient r > 0.99, not shown). All samples were all within
the standard curve.
Growth Hormone Receptor mRNA. Growth hormone
receptor (GHR) mRNA expression was measured by
RT-PCR using a LightCycler (Roche Diagnostics,
Mannheim, Germany) instrument with the QuantiTect
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Gene expression of GLUT in lactating cows
Table 1. Primers and probes for real-time polymerase chain reactiona
Item
Genbank
Accession No.
GLUT1
NM_174602
5′-GTGCTCCTGGTTCTGTTCTTCA-3′
5′-GCCAGAAGCAATCTCATCGAA-3′
5′-FAM-TCAAAGTTCCCGAGACAAAAGGCCG-TAMRA-3′
GLUT4
D63150
5′-GGACCGCGAATAGAAGAAAGAC-3′
5′-CAACTTCATCATCGGCATGG-3′
5′-FAM-TAGGGACCCATAGCATCCGCCACA-TAMRA-3′
GAPDH
BTU85042
5′-TGACCCCTTCATTGACCTTCA-3′
5′-GCCTTGACTGTGCCGTTGA-3′
5′-VIC-TTCCAGTATGATTCCACCCACGGCA-TAMRA-3′
GHR
NM_176608
5′-ACAACGCTTACTTCTGCGAGGTA-3′
5′-TTCCTGGTTAAAGCTTGGCTCTAC-3′
GAPDH
BTU85042
5′-CACCCTCAAGATTGTCAGCA-3′
5′-GGTCATAAGTCCCTCCACGA-3′
Primer
Probe
a
FAM = six-carboxy-fluorescein; TAMRA = 6-carboxy-tetramethyl-rhodamine; GAPDH = glyceraldehyde-3-phosphate dehydrogenase;
GLUT1 = insulin-independent glucose transporter; GLUT4 = insulin-responsive glucose transporter; GHR = growth hormone receptor.
SYBR Green PCR system (Qiagen K. K., Tokyo, Japan).
The PCR amplification was performed using the primer
sets outlined in Table 1. The primers were chosen with
an online software package (www-genome.wi.mit.edu/
cgi-bin/primer/primer3_www.cgi). The PCR was performed at 95°C for 15 min, followed by 50 cycles at 94°C
for 15 s, 55°C for 20 s, and 72°C for 10 s. All samples
were all within the standard curve.
GH and Insulin Assays
Venous blood samples were collected at 0800 on the
day of slaughter. Plasma was harvested from the blood
samples and stored at −20°C for later determination
of GH and insulin. Plasma GH concentrations were
determined by RIA as described by Johke (1978).
Plasma insulin concentrations were determined using
a commercially available RIA kit (Eiken Chemical,
Tokyo, Japan).
Statistical Analyses
All values were expressed as means ± SEM. The data
were analyzed by one-way ANOVA with the StatView
5 (SAS Inst., Inc., Cary, NC) software package. When
the ANOVA showed a significant effect for the phase,
phases were compared by the Fisher’s protected LSD
as a multiple comparison test. A P-value less than 0.05
denoted the presence of a statistically significant difference.
Results
The expression of GLUT1 protein and mRNA was
assessed in the bovine mammary gland and adipose
tissue at various stages of lactation. It has been reported that the antibody used in this study recognized
GLUT1 protein in bovine tissues (Abe et al., 2001). In
the mammary glands of peak- and late-lactating cows,
Western blotting analysis showed that GLUT1 was detected with similar intensity, whereas in nonlactating
cows, it was barely detectable (Figure 1A). The antibody
of GLUT1 could specifically recognize separate 42- and
45-kDa proteins. In the mammary gland, RT-PCR analysis also showed that the mRNA level of bovine GLUT1
in peak- and late-lactating cows was three times greater
(P < 0.05) than that in nonlactating cows (Figure 1B).
In adipose tissue, GLUT1 protein expression was detected in late- and nonlactating cows, whereas it was
barely detectable in peak-lactating cows (Figure 2A).
Unlike in the mammary gland, it was detected as a
single band in adipose tissue. Use of the TaqMan probe
resulted in reliable and sensitive quantification of the
RT-PCR product, with good linearity (Person correlation coefficient r > 0.99, not shown). Values were standardized to glyceraldehyde-3-phosphate dehydrogenase and expressed as a percentage of the control. Realtime PCR analysis also showed that the mRNA level
of GLUT1 in late and nonlactating cows was six times
greater (P < 0.05) than that in peak-lactating cows (Figure 2B). In muscle, GLUT1 protein and mRNA were
not detected in at any stage cows (data not shown).
There were no significant differences in the abundance of GLUT4 mRNA among the peak-, late-, and
nonlactating cows in the adipose tissue (Figure 3A) or
muscle (Figure 3B), and GLUT4 mRNA was not detected in the mammary gland of the cows in any lactating stage (data not shown).
There were no significant differences in the abundance of GHR mRNA among cows in the various lactating stages in the mammary gland or adipose tissue
(Figures 4A and B).
The insulin concentration was greater (P < 0.05) in
nonlactating than in peak- and late-lactating cows (Figure 5A). In contrast, there were no significant differences in the concentrations of GH among the cows in
the various stages (Figure 5B).
Discussion
Glucose is the major precursor for lactose synthesis.
Therefore, the supply of glucose to the mammary gland
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Komatsu et al.
Figure 1. Western blot analysis of erythrocyte-type glucose transporter (GLUT1) protein (A) and GLUT1 mRNA
(B) abundance in the mammary gland in peak-, late-, and nonlactating cows. Data are means ± SEM for four cows
per group and are expressed as the ratio of GLUT1 mRNA relative to glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) mRNA (peak lactating = 100%). Means with different letters (a, b) differ, P < 0.05.
is an important factor in the control of milk yield. The
mammary gland mainly expresses the glucose transporter GLUT1, the sodium glucose-linked transporter
SGLT (Zhao et al., 1999), and in mouse, GLUT12
(Macheda et al., 2003). The GLUT1 is the major glucose
transporter species in lactating cows (Zhao et al., 1996).
In the present study, no difference between GLUT1
protein and mRNA abundance in the mammary gland
was detected at peak vs. late lactation, whereas GLUT1
protein and mRNA were barely detectable in dry cows.
Western blot analysis showed two bands corresponding
to GLUT1 in the mammary gland, as reported by Zhao
et al. (1996). Tracer studies have shown that there is
a linear relationship between the rate of glucose transport and milk yield in cows (Kronfeld, 1982). In the
mammary gland, we found no change in the expression
of GLUT1 between peak and late lactation, implying
that a decrease of the glucose supply to the mammary
Gene expression of GLUT in lactating cows
561
Figure 2. Western blot analysis of insulin-independent glucose transporter (GLUT1) protein (A), and GLUT1 mRNA
(B) abundance in adipose tissue in peak-, late-, and nonlactating cows. Data are means ± SEM for four cows per group
and are expressed as the ratio of GLUT1 mRNA relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
mRNA (peak lactating = 100%). Means with different letters differ, P < 0.05.
gland and apoptosis of mammary epithelial cells (Wilde
et al., 1997) may cause the decrease of milk yield at
late lactation. In addition, it has been reported that
resistin mRNA abundance decreases in the lactating
mammary gland (Komatsu et al., 2003). Therefore, resistin may serve to make the regulation of glucose uptake by insulin effective. Because GLUT4 was not detected in the mammary gland, a novel insulin-depen-
dent glucose transporter (e.g., GLUT12; Macheda et al.,
2003) may be involved in glucose uptake.
In ruminants, triacylglycerol is synthesized from
glycerol-3 phosphate, a metabolite derived from glucose, and adipose tissue mainly expresses GLUT1 and
GLUT4. Although we also showed GLUT1 protein and
mRNA abundance in adipose tissue, no difference of
expression was observed between non- and late-lactat-
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Komatsu et al.
Figure 3. Insulin-responsive glucose transporter
(GLUT4) mRNA abundance in adipose tissue (A) and
muscle (B) in peak-, late-, and nonlactating cows. Data
are means ± SEM for four cows per group and are expressed as the ratio of GLUT4 mRNA relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA
(peak lactating = 100%). The GLUT4 mRNA was not detected in the mammary gland.
ing cows, whereas hardly any expression was detected
in peak-lactating cows. Western blot analysis showed
a single band, in contrast with the case of mammary
glands. The cause of this difference is unknown. The
Figure 4. Growth hormone receptor (GHR) gene abundance in mammary gland (A) and adipose tissue (B) in
peak-, late, and nonlactating cows. Data are means ± SEM
for four cows per group and are expressed as the ratio
of GHR mRNA relative to glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) mRNA (peak lactating =
100%).
decrease of GLUT1 expression in adipose tissue in peak
lactation may contribute to the increased supply of
plasma glucose to the mammary gland. The GLUT4
mRNA levels detected in the adipose tissue and muscle
did not vary significantly among the various stages of
lactation in cows. It is considered that insulin resistance
563
Gene expression of GLUT in lactating cows
GHR mRNA levels detected in the mammary gland and
adipose tissue did not significantly vary in the various
stages of lactating cows. It has been reported that administration of GH to dairy cows increases milk yield
(Pocius et al., 1986), so the effect may be caused via
GHR activation in addition to the GH concentration,
not by regulation of the abundance of GHR mRNA
and protein.
In conclusion, GLUT1 seems to be the major glucose
transporter isoform in the bovine mammary gland and
adipose tissue during lactation. The change in insulinindependent glucose uptake during lactation was associated with a change in the expression of GLUT1. We
did not detect any change of GLUT4 expression in adipose tissue or muscle, which suggests that the change
of insulin-dependent glucose uptake in lactation is not
regulated by GLUT4 expression. Insulin concentration
and GLUT4 activation may be involved in regulating
lactation. Plasma insulin concentration may play a role
in regulating insulin-dependent glucose metabolism,
but the expression of GLUT4 in the mammary gland
and adipose tissue is not an important factor of insulin
resistance in lactation.
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Figure 5. Plasma insulin (A) and GH (B) concentrations
in peak-, late-, and nonlactating cows. Data are means ±
SEM for four cows per group. Means with different letters
(a, b) differ, P < 0.05.
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