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Department of Animal Sciences, University of Missouri, Columbia, Missouri 65211
Address all correspondence and requests for reprints to: Dr. M. C. Lucy, 164 Animal Sciences Research Center, University of Missouri, Columbia, Missouri 65211. E-mail: lucym{at}missouri.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Receptors for GH are found in many tissues; however, the greatest GHR concentrations are found within the liver (7, 8, 9). During early lactation, the liver is refractory to GH, such that blood IGF-I concentrations are diminished (10). The mechanisms that regulate changes in hepatic GH responsiveness and blood IGF-I during early lactation are not known. One possibility is that transcription of GHR messenger RNA (mRNA) changes within the liver during early lactation. The transcription of GHR mRNA is controlled by at least three discernible promoters that alternatively splice different exon 1 onto GHR mRNA (7, 8). The first promoter (GHR P1) is only active in liver, so that GHR mRNA transcripts from GHR P1 [GHR 1A (bovine and ovine), GHR1 (rat), GHR L1 (mouse), and GHR V1 (human)] are only found in liver (9, 11, 12, 13, 14, 15, 16, 17). GHR 1A mRNA is different from other GHR mRNA because of its liver specificity and regulation by GH (13, 14). A second promoter (GHR P2) is ubiquitously expressed, with its transcripts [GHR 1B (bovine and ovine), GHR2 (rat), GHR L2 (mouse), and GHR V2 (human)] found in liver as well as nonhepatic tissues, including adipose tissue and muscle (9, 11, 12, 13, 14, 15, 16, 17, 18, 19). A third promoter (GHR P3) was characterized in the bovine (20) and transcribes GHR 1C mRNA (analogous to rat GHR V4) (21). Within the bovine (b) GHR gene, GHR P3 is immediately downstream from GHR P2, and GHR P2 acts and an enhancer for GHR P3 (20). Therefore, GHR 1B and GHR 1C mRNA are found in similar tissues and are alike because both mRNA represent constitutive mRNA for the GHR (20). In each case (P1, P2, or P3 transcripts), the protein-coding region of the GHR is unchanged because the open reading frame for GHR begins in exon 2 (22). Alternative splicing of exon 1A, 1B, or 1C onto GHR mRNA (exons 2–10), therefore, leads to mRNA with alternative first exons (GHR 1A, 1B, or 1C) that translate a theoretically identical protein. The activities of GHR P1, P2, and P3 can be estimated by quantifying the amounts of GHR 1A, 1B, and 1C mRNA, respectively.
Changes in liver GHR may be caused by independent regulation of each of the GHR promoters. We examined this possibility by measuring the amount of liver GHR mRNA in dairy cattle as they transitioned from pregnancy to parturition and subsequent lactation. Changes in liver IGF-I mRNA and blood IGF-I concentrations during the same period were also measured. We report here that total GHR mRNA is diminished at parturition as the result of a diminished level of GHR 1A mRNA, but not alternative GHR mRNA. The loss of GHR 1A mRNA at parturition was associated with decreased IGF-I mRNA and decreased blood IGF-I. These data support the hypothesis that GHR P1 (transcribing GHR 1A) is regulated independently from alternative GHR promoters that transcribe constitutive GHR mRNA in the liver. Changes in total GHR mRNA caused by GHR P1 may contribute to decreased blood IGF-I concentrations during early lactation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Collection of liver tissue
Liver samples were collected by needle biopsy (24). In study 1,
samples were collected from cows (n = 5) 14 days before expected
parturition (day -14), at parturition (day 0), and 21 days postpartum.
For study 2, samples were collected from cows (n = 6) at -14, 0,
15, 30, 60, and 90 days postpartum. To collect the samples, a region on
the right rib cage at the 11th intercostal space (
225
cm2 in size) was clipped and cleansed with disinfectant
(Nolvasan solution, Fort Dodge Laboratory, Inc., Fort Dodge, IA) and
70% ethanol. Local anesthesia (5 ml 2% lidocane hydrochloride
solution) was then administered sc at the site of incision, and a
scalpel blade was used to penetrate the skin. Liver samples were
collected with a custom-made biopsy needle (16.5 cm length, 4 mm inside
diameter; study 1) or a commercially available 14-gauge biopsy needle
(15.2-cm length; Tru-Cut biopsy needle, Baxter Healthcare Corp.,
Deerfield, IL; study 2). Liver samples were placed in screw-cap
microcentrifuge tubes and frozen in liquid nitrogen immediately after
collection, then transported to the laboratory. Samples were stored at
-80 C until RNA extraction.
Blood sample collection
Blood samples were collected beginning at first biopsy (day
-14; study 1) or at weekly intervals (study 2) thereafter via
coccygeal venipuncture. Samples (20 ml) were collected into evacuated
glass tubes containing EDTA (Vacutainer, Becton Dickinson and Co., Franklin Lake, UT) and placed on ice immediately. Plasma
was harvested by centrifugation (3000 x g; 15 min) and
stored at -20 C until assay.
RNA isolation
Total cellular RNA was isolated from each liver sample using the
Trizol procedure (Life Technologies, Inc., Gaithersburg,
MD; study 1) or using the RNeasy Mini kit (QIAGEN,
Valencia, CA; study 2). After isolation, RNA was dissolved in sterile
water treated with 0.1% (vol/vol) diethylpyrocarbonate. Concentrations
of RNA were determined by measuring absorbance at 260 nm. The purity of
the RNA was determined by calculating the ratio of absorbencies at
260 and 280 nm and by electrophoresis of a RNA aliquot (2.5 µg)
from each sample through a 1% agarose gel in Tris-borate/EDTA buffer
(0.089 M Tris-borate and 0.002 M EDTA) with
ethidium bromide (0.5 µg/ml). Samples with intact 28S and 18S
ribosomal RNA were used for subsequent analyses. Isolated RNA was
stored at -80 C until analysis by ribonuclease protection assay
(RPA).
Complementary DNA (cDNA) synthesis and cloning
The cDNA for analysis of total IGF-I mRNA
(IGF-Itot; Fig. 1
and Table 1) was amplified by RT-PCR. The
oligonucleotide primers for the RT-PCR (forward,
5'-CTCCTCGCATCTCTTCTATCT-3'; reverse, 5'-CTCATCCACGATTCCTGTCT-3') were
designed from the ovine IGF-I cDNA sequence (25) that was identical to
the bovine (GenBank accession no. X15726). The 200-bp PCR fragment
contained the majority of exon 3 (137 bp) and part of exon 4 (63 bp) of
the IGF-I gene. The RT mixture contained 5 µg bovine liver RNA, 5
µl 5 x RT buffer, 25 nM deoxy-NTPs, 24 U
ribonuclease inhibitor (RNasin, Promega Corp., Madison,
WI), 15 U AMV reverse transcriptase (Promega Corp.), and
12.1 pM reverse primer. The mixture was heated at 65 C for
5 min, and RT was carried out at 42 C for 60 min. The mixture then was
heated at 65 C for 5 min and placed on ice immediately thereafter. Five
microliters of the RT mixture, 18 µl sterile water, 16 pM
forward primer, and 12.1 pM reverse primer were added to a
commercially available PCR bead (Ready-To-Go PCR Beads, Pharmacia Biotech, Piscataway, NJ). The thermal cycler program was 35
cycles at 94 C for 45 sec, 58 C for 45 sec, and 72 C for 60 sec. An
aliquot of the reaction and an appropriately sized DNA marker were
electrophoresed on a 1% agarose gel in Tris-borate/EDTA buffer with
ethidium bromide to verify the size of the product. The PCR product was
subcloned into pGEM-T Easy (Promega Corp.), and the DNA
insert was sequenced by automated DNA sequencing to verify the identity
and orientation.
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). The probe allowed
the simultaneous measurement of class 2 IGF-I mRNA that contained exons
2–4 (IGF-IC2; 314-bp protected fragment) as well as class
1 IGF-I transcripts that contained exons 1, 3, and 4
[IGF-I(C1), detected as an exon 3–4 protected fragment
(223 bp) in the IGF-IC2 RPA; the (C1) is inparentheses because the class 1 IGF-I mRNA is deduced from the
exon 3–4 fragment]. The forward oligonucleotide primer
(5'-GTCCTCAGTTTTCTATTCAC-3') was designed from the ovine IGF-I genomic
DNA sequence from GenBank (accession no. X69472). The reverse primer
was identical to the reverse primer used for the 200-bp IGF-I fragment.
The RT-PCR was performed as described above. The amount of reverse
primer used for the RT reaction was 12.1 pM, and 16
pM forward primer and 12.1 pM reverse primer
were used for the PCR. The RT-PCR product was subcloned into pGEM-T
Easy (Promega Corp.), and the DNA insert was sequenced by
automated DNA sequencing to verify the identity and orientation. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (91 bp) was also produced by RT-PCR using procedures similar to those described for the IGF-I cDNA. The cDNA sequence for bGAPDH was obtained from GenBank (Accession no. U85042). The primers (forward, 5'-ACACTGAGGACCAGGTTG-3'; reverse, 5'-TGGTCGTTGAGGGC-AATG-3') then were designed to amplify between bases 791–881 of the bGAPDH gene. The amount of reverse primer used for the RT reaction was 12.4 pM, and for the PCR, 16.6 pM forward primer and 12.4 pM reverse primer were used. The RT-PCR product was subcloned into pGEM-T Easy (Promega Corp.), and the DNA insert was sequenced by automated DNA sequencing to verify the identity and orientation. Ultimately, the cDNA insert was further subcloned into the EcoRI site of pGEM-3Z (Promega Corp.).
Three other plasmids used in these studies were described previously
(9, 27). The total amount of GHR mRNA (GHRtot; Fig. 1) was
measured using a GHR riboprobe antisense for exons 5–8 from the bGHR
cDNA (27). Additional GHR plasmids containing exons 1A, 2, and 3
(GHR1A/2/3) or exons 1B, 2, and 3
(GHR1B/2/3) were used to synthesize a riboprobe for GHR 1A
and GHR 1B mRNA, respectively. Production of the GHR 1A and GHR 1B
plasmids was described previously (9). Exons 2 and 3 proved identical
for GHR mRNA that contain different exon 1. Therefore, each RPA (GHR 1A
and GHR 1B) produced a full-length protected fragment as well as a
truncated fragment (121 bp, exons 2–3). The truncated fragment (exons
2–3) represented GHR mRNA that did not contain exon 1 of the
respective riboprobe. For the purpose of this paper, the exon 2–3
fragments from the GHR 1A and GHR 1B RPA were designated
GHR
E1A and GHRE1B, respectively.
Production of antisense riboprobe
Antisense ribonucleotide probes were produced as
previously described (9). Each plasmid was linearized with an
appropriate restriction enzyme and purified by phenol-chloroform
extraction. Riboprobes were synthesized with the appropriate RNA
polymerases using a RNA transcription kit (Stratagene, La
Jolla, CA). Transcription was allowed to proceed at 37 C for 90 min.
Plasmid templates were digested after transcription by using RQ1
deoxyribonuclease (Promega Corp.). Riboprobes were
purified after phenol-chloroform extraction, and free
[
-32P]CTP was then removed by Quick Spin (TE) Sephadex
G-50 columns (Roche Molecular Biochemicals, Indianapolis,
IN). The specific activities of the purified riboprobes were determined
by liquid scintillation counting. Riboprobes were stored at -80 C
until use.
RPAs
The RPAs were performed as described previously (9) using a
commercially available kit (RPA II, Ambion, Inc., Austin,
TX) with modifications. Twenty micrograms of total cellular RNA were
assayed in study 1. For study 2, 2.5 µg total cellular RNA were
assayed, and 15 µg yeast RNA were added to increase the visibility of
the pellet. Protected RNA fragments were identified by gel
electrophoresis in a 6% acrylamide-7 M urea gel
(Acryl-a-Mix 6, Promega Corp.). Electrophoresis was
performed at 200 V for 2 h. Gels were dried on a gel drier at 80 C
for 1 h. Autoradiography was performed using X-Omat-AR film
(Eastman Kodak Co., Rochester, NY) at -80 C for 18 h
(GHR) or 7 days (IGF-I) in study 1. In study 2, autoradiography was
performed at -80 C for 2 days (GHR) or 7 days (IGF-I). Protected
fragments were identified based on size by comparing them to the
undigested riboprobes as well as to a radiolabeled 100-bp DNA ladder.
Expression of the mRNA was quantified by measuring pixel density
(arbitrary units) of protected fragments using GP Tools 3.01
(Biophotonics Corp., Ann Arbor, MI).
Plasma concentrations of GH, IGF-I, and nonesterified fatty
acids
Plasma concentrations of IGF-I were measured in a single RIA
(28). The specific activity of the [125I]bIGF-I was 177
µCi/µg, and the assay sensitivity was 9.8 pg/tube. The intraassay
coefficient of variation (CV) was 11.9%. Plasma GH concentrations were
also measured in a single RIA. The RIA procedures described by Matteri
et al. (29) were modified and validated for bGH. Bovine
plasma (100 µl) was incubated with 300 µl primary antibody [AFP
11182B monkey anti-GH antibody, gift from the National Hormone and
Pituitary Program (A. F. Parlow, Scientific Director), final
dilution, 1:500,000], 50 µl [125I]bGH (66 µCi/µg;
20,000 cpm/tube), and 300 µl PBS plus 0.1% gelatin (PBSG; pH 7.0)
in glass tubes. The nonspecific binding tubes contained 400 µl PBSG,
50 µl [125I]bGH, and 300 µl 10% (vol/vol) bovine
plasma. The zero binding tubes contained 400 µl PBSG, 50 µl
[125I]bGH, and 300 µl primary antibody. The standard
curve contained 300 µl PBSG with 0.05, 0.1, 0.5, 1, 2, 5, or 10
ng/tube of recombinant bGH (Monsanto Co., St. Louis, MO), 50 µl
[125I]bGH, and 300 µl primary antibody. All tubes were
incubated for 20 h at 4 C. Secondary antibody (goat antimonkey
IgG; Antibodies, Inc., Davis, CA) was diluted to 1:20 in PBSG and
0.01% (wt/vol) sodium ethylmercurithiosalicylate (thimerosal,
Sigma Chemical Co., St. Louis, MO) and 2.6% (vol/vol)
normal monkey serum. One hundred microliters of secondary antibody and
1 ml 10% polyethylene glycol (Carbowax PEG 8000, Fisher Scientific, Fairlawn, NJ) were added to tubes after the
incubation (final volume, 1.85 ml). Tubes were vortexed for 1 min,
incubated at room temperature for 1 h, and centrifuged at
3000 x g at 4 C for 30 min. Supernatants were
discarded, and pellets were counted for 1 min. Increasing amounts of
bovine plasma (50, 100, or 200 µl in triplicate) produced a
displacement curve that was parallel to the standard curve. The
addition of 5, 10, or 20 ng bGH to 100 µl bovine plasma sample (10
ng/ml) yielded an average recovery of 80.6%. The intraassay CV for the
GH RIA was 12.2%, and the sensitivity of the assay was 50 pg/tube.
Plasma nonesterified fatty acid concentrations were measured in plasma samples collected on the day of biopsy (study 2). The assay was performed as described previously (30) using a commercially available kit (NEFA C, Wako Pure Chemical Industries, Ltd., Osaka, Japan). Intra- and interassay CVs were 6.9% and 5.1%, respectively.
Statistical analyses
The GAPDH control was used within each RPA. There was no effect
of day postpartum (P > 0.10) on GAPDH mRNA in any of
the analyses. Therefore, the pixel densities for the protected mRNA
fragments were divided by the pixel densities of the internal GAPDH
control to adjust for minor differences in mRNA precipitation and
loading. The adjusted mRNA pixel densities (arbitrary units) as well as
plasma concentrations of GH, IGF-I, and NEFA were analyzed by least
squares ANOVA using the General Linear Model Procedure of SAS (31). The
independent variables in the model were day postpartum and cow. The
effect of day postpartum was tested using the cow by day interaction.
Means were separated using Duncan’s multiple range test from SAS (31).
The data are presented as the least squares mean ±
SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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E1A,
GHR 1B, GHRE1B, IGF-Itot,
IGF-IC2, and IGF-I(C1) were quantified in liver
samples collected from dairy cattle on days -14, 0, and 21 relative to
parturition (day 0 = parturition; Fig. 2). There was an effect
of day on the amount of GHRtot mRNA (P <
0.01). The amount of GHRtot mRNA was lowest on day 0 and
greatest on days -14 and 21 postpartum (Fig. 2B). A similar response
profile was observed for the amount of GHR 1A mRNA (P
< 0.001). In contrast, the amount of GHRE1A mRNA (exon
2–3 transcripts in the GHR 1A RPA) did not differ across the days of
study (P > 0.10). This suggested that GHR transcripts
that do not contain exon 1A do not change during the periparturient
period. To examine this possibility, GHR 1B mRNA was analyzed because
GHR 1B is the primary alternative to GHR 1A mRNA (9). The amount of GHR
1B mRNA did not change during the periparturient period
(P > 0.10), whereas the amount of
GHRE1B (exon 2–3 transcripts in the GHR 1B RPA) changed
in parallel to that of GHR 1A mRNA (i.e. decreased
expression on day 0; P < 0.01). This was expected
because GHR 1A mRNA is the primary alternative to GHR 1B, and the
GHRE1B fragment should reflect the amount of GHR 1A
mRNA.
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IGF-Itot, P < 0.05; IGF-IC2,
P < 0.05; IGF-I(C1), P <
0.01). The amount of IGF-I mRNA [IGF-Itot,
IGF-IC2, or IGF-I(C1)] changed in parallel to
that of GHRtot and GHR 1A mRNA. In general, the IGF-I mRNA
decreased on day 0 and then increased to a level similar to that on day
-14 by day 21 postpartum. Plasma concentrations of IGF-I changed in
parallel to liver GHR 1A and IGF-I mRNA (P < 0.01;
decreased at parturition with subsequent increase at day 21; Fig. 2D).
Study 2
There was an effect of day postpartum (P < 0.001)
on the amount of GHR 1A mRNA because the amount of GHR 1A mRNA present
on day 0 was less than that observed on days -14 or 15 relative
to that on parturition (Fig. 3, A and C).
After day 15 postpartum, the amount of GHR 1A mRNA did not differ
across days. The amount of IGF-Itot mRNA paralleled the
changes observed for GHR 1A mRNA, because IGF-Itot mRNA was
lowest on day 0 (P < 0.05) relative to levels assessed
on days -14 and 15 (Fig. 3, B and C). After day 15 postpartum,
IGF-Itot mRNA did not differ across days.
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). An effect of day
postpartum was also detected for plasma concentrations of GH
(P < 0.001) and IGF-I (P < 0.001).
The plasma GH concentration increased on day 0 and peaked on day 15
postpartum (Fig. 4B). Subsequently, the GH concentration decreased
until day 30 postpartum and remained unchanged thereafter. Plasma
concentrations of IGF-I were opposite those of plasma nonesterified
fatty acids and GH. The IGF-I concentrations decreased on day 0 and
subsequently increased throughout the postpartum period, reaching
prepartum concentrations by 77 days postpartum (Fig. 4C). Changes in
plasma IGF-I concentrations paralleled the temporal relationship
observed for GHR 1A and IGF-Itot mRNA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present studies show that the decrease in liver GHR mRNA at
parturition was caused by a decrease in GHR 1A mRNA (Figs. 2 and 3).
During the parturition-induced decline in GHR, the amount of non-GHR 1A
mRNA (GHR 1B or GHRE1A) remained unchanged and was not
associated with the changes in the amount of total GHR mRNA in liver.
We suggest that changes in the amount of liver GHR mRNA are primarily
determined by the expression of GHR 1A mRNA during this period. These
data provide evidence that the liver-specific GHR P1 is responding to
physiological changes in periparturient cattle. The activity of GHR P1
was independent from that of other promoters (i.e. GHR P2 or
GHR P3) that control constitutive liver GHR mRNA expression because we
did not detect a decrease in either GHR 1B or GHRE1A
during the period of decreased GHR 1A mRNA. The relationship between
the amounts of GHR P1 activity and GHR 1A mRNA assumes that the
stability of GHR 1A mRNA is unchanged in periparturient cow liver. We
know of no evidence to suggest altered stability of GHR 1A mRNA
relative to that of other GHR mRNA. Nevertheless, the relative
stabilities of GHR 1A, 1B, and 1C mRNA should be tested in controlled
experiments.
The paradigm that we observed for GHR regulation agrees with previous studies of GHR mRNA in rats, where liver-specific GHR1 mRNA was regulated independently from other GHR transcripts (14, 15). Indeed, GHR1 mRNA was shown to be sexually dimorphic and developmentally regulated (14, 15). The observations made during the present study support the concept of independent regulation of GHR mRNA by at least two promoters. Furthermore, our observations extend the physiological evidence for GHR promoter regulation to postpartum animals.
A decrease in GHR1 after parturition was also observed in the postpartum rat (14). Some aspects of GHR1 and GHR 1A mRNA regulation in rat and cattle, however, were different, because GHR 1A increased to a prepartum level by day 15 postpartum. GHR1 was increased by pregnancy in the rat and did not achieve expression equivalent to that in prepartum (pregnant) animals during the postpartum period (14). The increase in GHR 1A mRNA in the postpartum bovine reflects a slightly different paradigm for GHR 1A. In the ovine, GHR 1A expression is initiated in fetal liver during late gestation (11, 32), and an adult level of GHR 1A expression in the bovine and ovine neonate occurs shortly after birth (11, 32, 33). GHR1 in the rat is also developmentally regulated (increased expression with the shift toward the female pattern of GH secretion in the rat), but the greatest increase in GHR1 occurs in pregnant animals (14). Therefore, the failure of GHR1 to achieve prepartum levels in the postpartum rat probably reflects the strong influence of pregnancy on GHR1 expression. The effects of pregnancy on bovine GHR 1A have not been tested, but our observation that nonpregnant postpartum cattle have similar GHR 1A mRNA expression as prepartum pregnant cattle (day -14) suggests that pregnancy does not strongly up-regulate GHR 1A in the bovine.
The transcription of IGF-I mRNA is also controlled by at least two different promoters (6, 26, 34, 35, 36). The activity of IGF-I promoters leads to the alternative splicing of different leader exons onto exon 3, where the coding for fully processed IGF-I begins (6, 26). The amount of IGF-I mRNA was lowest at parturition, and class 1 and class 2 IGF-I mRNA were equally down-regulated. Therefore, unlike GHR mRNA, the IGF-I mRNA were temporally regulated in periparturient cows. The mechanisms that control IGF-I mRNA within this experimental model, therefore, were conserved across both IGF-I promoters that regulate IGF-I synthesis and secretion. This may suggest that DNA sequences that are common to both IGF-I promoters ultimately determine IGF-I mRNA expression. In sheep, both class 1 and class 2 IGF-I mRNA declined in response to undernutrition, but class 2 IGF-I mRNA was more sensitive to decreased nutrient levels than class 1 IGF-I mRNA (26). In the present study, however, we did not observe significant differences in the relative changes in class 1 and class 2 IGF-I mRNA expression. The mechanisms for control of IGF-I mRNA expression in periparturient cattle, therefore, may not be the same as those in animals undergoing acute nutritional deprivation (26).
The decrease in liver IGF-I on day 0 may have been caused by a decline
in GHR mRNA that led to a loss of GHR protein that precluded
GH-dependent IGF-I synthesis (5). If true, the decrease in liver IGF-I
mRNA should have occurred after the reduction in GHR mRNA. This
possibility will need to be examined in future experiments in which a
greater number of time points are sampled. It will also be necessary to
measure GHR protein and correlate GHR 1A mRNA with GH binding to liver
tissue, because the rate of translation of GHR 1A relative to that of
other GHR variants has not been tested. A second possibility is that
GHR 1A and IGF-I mRNA are decreased in a synchronized manner through a
common pathway or through two independent pathways acting at the same
time. Expression of liver-specific genes, including GHR and IGF-I, is
regulated by several transcription factors, such as hepatocyte nuclear
factor-1 (HNF-1), -3, -4, and -6 as well as CAAT/enhancer-binding
proteins (C/EBP) (37, 38). The 5'-untranslated region of the IGF-I gene
contains binding sites for HNF-1, HNF-3, and C/EBP (6), and the IGF-I
promoter was activated in vitro by cotransfection of
HNF-1 (36) or C/EBP (35) expression plasmids with an IGF-I
promoter/reporter construct. The activity of GHR P1 may also be
regulated by liver-specific transcription factors, because GHR P1
contains binding sites for HNF and C/EBP (13). Therefore, the GHR 1A
and IGF-I promoters may be controlled by a common pathway that
ultimately coordinates their expression in liver.
The nutritional and physiological mechanisms that cause the specific down-regulation of GHR 1A at calving are unknown. During the periparturient period, cows undergo metabolic changes to accommodate the nutrient demands for lactation (1, 2). There is a transient period of lipid mobilization (measured as increased plasma concentrations of nonesterified fatty acids) that is associated with negative energy balance in postpartum cows (1, 2). The nutritional constraints during this period were associated with the specific loss of GHR 1A and suggest that nutritional effects on the GHR within liver (39) may be acting directly on GHR P1. It is also possible that insulin or glucocorticoids are mediating the responses that we observed. Insulin concentrations in blood are low during early lactation (40), and in diabetic models, IGF-I as well as GHR mRNA expressions were dependent on insulin (6, 38, 41, 42). Glucocorticoids initiate parturition as well as lactation and begin the process of nutrient partitioning in lactating animals (1). In general, glucocorticoids decrease the ability of GH to increase IGF-I release (43, 44). The effects of glucocorticoids on GHR mRNA in ruminants, however, were stimulatory, particularly in the fetal liver during late gestation (45, 46).
In summary, the loss of GHR mRNA at parturition was associated with a reduction in the amount of GHR 1A mRNA. The amount of alternative GHR mRNA was unchanged and was not associated with changes in total GHR mRNA during this period. The amount of IGF-I mRNA also decreased at parturition. The decrease in IGF-I mRNA was caused by a reduced amount of both class 1 and class 2 IGF-I mRNA. Unlike IGF-I mRNA, the regulation of total GHR mRNA depended on the independent control of two distinct GHR mRNA. The constitutive GHR (i.e. GHR 1B) did not undergo a decline at parturition. The inducible GHR (GHR 1A) underwent a dramatic decrease that was associated with a decrease in total GHR mRNA as well as a decrease in liver IGF-I mRNA and plasma IGF-I concentrations. These data support the concept of independent regulation of GHR mRNA by GHR P1 (transcribes GHR 1A) and other constitutive GHR promoters (GHR P2 and GHR P3). The mechanisms through which each promoter is controlled will require additional investigation.
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Footnotes |
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Received December 31, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
LAP. Mol Endocrinol 8:1636–1645 activates promoter 1 of the human
insulin-like growth factor I gene via two distinct binding sites. Mol
Endocrinol 9:1488–1499This article has been cited by other articles:
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R. P. Radcliff, B. L. McCormack, D. H. Keisler, B. A. Crooker, and M. C. Lucy Partial Feed Restriction Decreases Growth Hormone Receptor 1A mRNA Expression in Postpartum Dairy Cows J Dairy Sci, February 1, 2006; 89(2): 611 - 619. [Abstract] [Full Text] [PDF]
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H. Jiang, M. C. Lucy, B. A. Crooker, and W. E. Beal Expression of Growth Hormone Receptor 1A mRNA is Decreased in Dairy Cows but not in Beef Cows at Parturition J Dairy Sci, April 1, 2005; 88(4): 1370 - 1377. [Abstract] [Full Text] [PDF]
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J. R. Roche, E. S. Kolver, and J. K. Kay Influence of Precalving Feed Allowance on Periparturient Metabolic and Hormonal Responses and Milk Production in Grazing Dairy Cows J Dairy Sci, February 1, 2005; 88(2): 677 - 689. [Abstract] [Full Text] [PDF]
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R. P. Radcliff, M. J. VandeHaar, Y. Kobayashi, B. K. Sharma, H. A. Tucker, and M. C. Lucy Effect of Dietary Energy and Somatotropin on Components of the Somatotropic Axis in Holstein Heifers J Dairy Sci, May 1, 2004; 87(5): 1229 - 1235. [Abstract] [Full Text] [PDF]
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J. C. Velez and S. S. Donkin Bovine Somatotropin Increases Hepatic Phosphoenolpyruvate Carboxykinase mRNA in Lactating Dairy Cows J Dairy Sci, May 1, 2004; 87(5): 1325 - 1335. [Abstract] [Full Text] [PDF]
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 R. P. Rhoads, J. W. Kim, B. J. Leury, L. H. Baumgard, N. Segoale, S. J. Frank, D. E. Bauman, and Y. R. Boisclair Insulin Increases the Abundance of the Growth Hormone Receptor in Liver and Adipose Tissue of Periparturient Dairy Cows J. Nutr., May 1, 2004; 134(5): 1020 - 1027. [Abstract] [Full Text] [PDF]
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 R. Webb, P. C. Garnsworthy, J.-G. Gong, and D. G. Armstrong Control of follicular growth: Local interactions and nutritional influences J Anim Sci, January 1, 2004; 82(13_suppl): E63 - 74. [Abstract] [Full Text] [PDF]
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R. P. Radcliff, B. L. McCormack, B. A. Crooker, and M. C. Lucy Plasma Hormones and Expression of Growth Hormone Receptor and Insulin-Like Growth Factor-I mRNA in Hepatic Tissue of Periparturient Dairy Cows J Dairy Sci, December 1, 2003; 86(12): 3920 - 3926. [Abstract] [Full Text] [PDF]
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R. P. Radcliff, B. L. McCormack, B. A. Crooker, and M. C. Lucy Growth Hormone (GH) Binding and Expression of GH Receptor 1A mRNA in Hepatic Tissue of Periparturient Dairy Cows J Dairy Sci, December 1, 2003; 86(12): 3933 - 3940. [Abstract] [Full Text] [PDF]
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N. H. Ciccioli, R. P. Wettemann, L. J. Spicer, C. A. Lents, F. J. White, and D. H. Keisler Influence of body condition at calving and postpartum nutrition on endocrine function and reproductive performance of primiparous beef cows J Anim Sci, December 1, 2003; 81(12): 3107 - 3120. [Abstract] [Full Text] [PDF]
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R. P. Rhoads, C. McManaman, K. L. Ingvartsen, and Y. R. Boisclair The Housekeeping Genes GAPDH and Cyclophilin Are Regulated by Metabolic State in the Liver of Dairy Cows J Dairy Sci, November 1, 2003; 86(11): 3423 - 3429. [Abstract] [Full Text] [PDF]
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Y. Wang, S. Eleswarapu, W. E. Beal, W. S. Swecker Jr., R. M. Akers, and H. Jiang Reduced Serum Insulin-Like Growth Factor (IGF) I Is Associated with Reduced Liver IGF-I mRNA and Liver Growth Hormone Receptor mRNA in Food-Deprived Cattle J. Nutr., August 1, 2003; 133(8): 2555 - 2560. [Abstract] [Full Text] [PDF]
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P. E. Kendall, T. L. Auchtung, K. S. Swanson, R. P. Radcliff, M. C. Lucy, J. K. Drackley, and G. E. Dahl Effect of photoperiod on hepatic growth hormone receptor 1A expression in steer calves J Anim Sci, June 1, 2003; 81(6): 1440 - 1446. [Abstract] [Full Text] [PDF]
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 H. Jiang and M. C. Lucy Involvement of Hepatocyte Nuclear Factor-4 in the Expression of the Growth Hormone Receptor 1A Messenger Ribonucleic Acid in Bovine Liver Mol. Endocrinol., June 1, 2001; 15(6): 1023 - 1034. [Abstract] [Full Text] [PDF]
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