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Department of Molecular Medicine, Karolinska Institute (A.F.-M., N.S., P.T.-E., G.N.), 17176 Stockholm, Sweden; Department of Biotechnology, KTH Royal Institute of Technology (J.L.), Stockholm 10044, Sweden; and Institute for Genomic Research (R.L.M., J.Q., N.H.L.), Rockville, Maryland 20850
Address all correspondence and requests for reprints to: Dr. Amilcar Flores-Morales, Department of Molecular Medicine, CMM, L8:01, Karolinska Hospital, 17176 Stockholm, Sweden. E-mail: amilcar.flores{at}molmed.ki.se
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GH is primarily synthesized in the pituitary gland. It is the main regulator of longitudinal growth and exerts a multitude of effects on several tissues. In liver, GH regulates lipid, protein, and carbohydrate metabolism (4, 5). It is responsible for sex-specific regulation of the metabolism of xenobiotic compounds (6) and regulates the expression of multiple liver-specific secretory proteins, including insulin-like growth factor I (IGF-I), a key regulator of body growth (7). GH also regulates cardiovascular function through effects in both heart and peripheral vasculature, and it induces cardiac growth and increases contractility in GH-deficient individuals and animal models (8). In kidney, the GH/IGF-I axis may be a cause of the glomerular and proximal tubular hypertrophy that occurs in hypersomatotropic states and during the development of diabetic kidney disease (9). IGF-I has also been reported to be involved in compensatory renal hypertrophy, and renal regeneration after acute ischemic injury (10). Nevertheless, the molecular mechanisms responsible for these actions remain unknown in many cases.
Technological developments have made it possible to monitor changes in gene expression of multiple transcripts and thereby generate large gene expression profiles. DNA microarrays can be used to simultaneously monitor several thousand different transcripts (11). We believe that the identification of gene expression patterns induced by specific hormones will provide insights into the molecular events underlying their diverse and tissue-specific actions.
In the present study we have used DNA microarrays containing 3000 different rat complementary DNA (cDNA) clones to investigate the state of hypophysectomy in three rat tissues, namely liver, kidney, and heart. Common and tissue-specific changes in gene expression were analyzed. Pituitary-dependent transcripts were classified according to biochemical function. In the last part of this study we examined the effects of 3 weeks of continuous human GH (hGH) infusion into Hx animals. Attempts were made to correlate the newly described variations in the expression patterns to the physiological changes induced by hypophysectomy and GH treatment.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Total RNA was isolated using TRIzol (Life Technologies, Inc., Gaithersburg, MD), according to the protocol supplied by the manufacturer. The quality of the RNA samples was ascertained on a denaturing agarose gel. Equal amounts of total RNA from four animals in the same experimental group were pooled before mRNA purification. mRNA was purified from 1 mg total RNA using 35 mg oligo(deoxythymidine)-cellulose (Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated in a ribonuclease (RNase)-free binding buffer containing 20 mM Tris-HCl, 0.5 M NaCl, and 1 mM EDTA. Polyadenylated [poly(A)+] RNA was bound to the cellulose and washed four times using the same buffer, then washed once in a low salt buffer (0.1 M NaCl) and eluted twice in warm elution buffer (20 mM Tris-HCl and 1 mM EDTA).
Probe labeling and purification
Two to 4 µg poly(A)+ mRNA were used from
each group of animals for each experiment. Labeled cDNA was produced by
a random primed RT reaction in the presence of Cy-labeled nucleotides
(12). Mouse Cot1 DNA (10 µg; Life Technologies, Inc.) was added to each reaction, and the probes were
subsequently purified as previously described (12). The
hybridization buffer consisted of 5 x SSC (standard saline
citrate)/0.2% SDS, 10 µg poly(A) RNA, and 10 µg yeast transfer
RNA. The probe was added at a final volume of 15 µl to the array, a
plastic 22 x 22-mm coverslip (Grace Bio-Labs, Bend, OR) was
placed on top, and the array was put in a sealed hybridization
chamber (Corning, Inc., Corning, NY). Hybridization
took place at 65 C for 15–18 h. The array was then washed
(12) and immediately scanned using the GMS 418 scanner
(Affymetrix, Santa Clara, CA).
Rat cDNA clone collection
We collected approximately 3000 rat cDNA clones, selected from
the TIGR Rat Gene Index (www.tigr.org) and from earlier subtractive
cloning experiments (13). Fifty percent of these clones
represent previously characterized rat genes or mouse/human orthologs,
and the rest are identified by expressed sequence tags and have
no significant homology to sequences contained in public databases.
Generation of cDNA microarrays
Bacterial colonies were grown overnight in 1.5 ml Luria Bertoni
medium in 96-well plates (Beckman Coulter, Inc., Palo
Alto, CA), and plasmid minipreparations were followed by a PCR
amplification of the inserts using vector-specific primers T3
(5'AATTAACCCTCACTAAAGGG-3') and T7 (5'-GTAATACGACTCACTATAGGGC-3'). The
amplified inserts, each produced by two pooled 100-µl PCR reactions,
were purified by ethanol precipitation and resuspended in 40 µl
3 x SSC, and purity was checked on an agarose gel. Approximately
10% of the cDNA clones rendered more than one band after PCR
amplification. CMT GAPS amino silane-coated slides
(Corning, Inc.) were used as adhesive surface for printing
using a GMS 417 arrayer (Affymetrix). The slides were postprocessed as
described previously (12) and stored in a dust-free dark
box until hybridization.
Chip design and interpretation
Individual cDNAs were spotted once on the glass surface, and
selected cDNAs were spotted twice at different locations on the chip to
serve as reproducibility controls. The hybridizations were repeated
using new sets of labeled targets, acting as an additional control of
reproducibility. The cut-off for selecting a gene as up- or
down-regulated was set at 1.5 based on comparison of the results from
the chip analysis with independent validation using RNase protection
assay (see Results). These results show that Cy5/Cy3 ratios
of approximately 1.5 correspond to higher ratios when calculated using
RNase protection analysis. This cut-off is also supported by
independent findings that show regulation by GH of the expression of
genes such as GLUT-1 (14), fibrinogen (15),
MHC-ß (16), CYP2C7, ferritin, transferrin
(17), and CYP3A2 (18). Furthermore, only
changes in gene expression that were reproducible and higher than the
cut-off in at least two independent experiments are presented. Data
from clones rendering more than one band after PCR amplification were
removed from the results because they represent more than one gene. All
genes mentioned here by name have been sequence verified by single pass
3'-sequencing.
Image analysis
Image analysis was performed using the ScanAlyze software
(available at http://rana.stanford.edu/software). The signal from each
spot was calculated as the average intensity for each channel.
Automatic and manual flagging were used to localize absent or very weak
spots (<1.6 times above background), which were excluded from
analysis. Normalization between the two fluorescent images was
performed, as previously described (19), by applying a
scaling factor to all intensities measured for the Cy5 channel so that
the mean log (Cy5/Cy3) for the subset of spots that were not flagged
was 0.
Solution hybridization/RNase protection analysis
Total nucleic acids (tNA) were isolated by homogenization of
tissue specimens, using a Polytron PT-2000 (Kinematica AG,
Lucerne, Switzerland). Digestion of samples with proteinase K
(Merck, Mannheim, Germany) and subsequent extraction with chloroform
and phenol were described previously (20). mRNA levels
corresponding to the expression of individual clones [stearyl coenzyme
A (CoA) desaturase 1 (SCD1), meprin A, and atrial natriuretic peptide]
were measured in tNA samples using a solution hybridization/RNase
protection assay as described previously (13).
Transcript-specific 35S-labeled complementary RNA
probes were transcribed in vitro from the respective cDNA
vector construct, according to the method of Melton et al.
(21). All probes were cloned into the pT3T7Pac vector.
Reagents for in vitro transcription were obtained from
Promega Corp. (Madison, WI). The concentration of nucleic
acids in tNA samples was measured spectrophotometrically. To permit
accurate comparison of specific mRNA levels between different hormonal
statuses, the samples of interest were always analyzed at the same
assay occasion. The DNA content of the tNA samples was measured with
the DyNA Quant 200 system (Hoefer Scientific, San
Francisco, CA) according to the supplied protocol. The Hoechst dye was
purchased from Polysciences, Inc. (Warrington, PA). Samples were
analyzed in triplicate, and the results are expressed as counts per min
of specific mRNA per µg DNA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The microarrays were hybridized with fluorescent probes derived from poly(A)+ mRNA isolated from liver, heart, and kidney. Each hybridization compared Cy3-labeled cDNA, reverse transcribed from a pool of mRNA isolated from four 12-week-old, nontreated, male rats (normal), with Cy5-labeled cDNA produced from a pool of mRNA isolated from four age-matched animals that had been hypophysectomized for 6 weeks. In another set of experiments, Cy3-labeled cDNA derived from tissues of 12-week-old Hx animals was compared with Cy5-labeled cDNA from age-matched Hx animals that had been continuously treated with hGH for 3 weeks. Hybridizations were performed in duplicate, and only changes reproducible across independent replicates were considered. The hybridized fluorescent targets were detected, and signal intensities for the two fluorescent images were normalized, so that the mean log (Cy5/Cy3) of all detectable spots was zero (19).
An important consideration is that hypophysectomy causes a marked
decrease in RNA content in multiple tissues (22). In our
hands, and in agreement with previous results, the content of RNA per
cell is reduced by approximately 40% in liver by hypophysectomy, and
the fraction of mRNA in the total pool follows this pattern
(23). However, equal amounts of mRNA from tissues derived
from Hx and normal rats were used to guarantee proper array
quantification and data processing (12). By using this
normalization strategy, under- and overrepresented transcribed genes
could be identified in the mRNA pools (Tables 2
and 3), whereas the
ratios measured for the vast majority of genes, including housekeeping
genes such as ß-actin and histones, were approximately 1 in all three
tissues.
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. Of the 3000 arrayed clones, about 720
were detectable (signal 1.6-fold above background) in liver, 1600 in
heart, and 985 in kidney. Some of these were expressed in more than 1
tissue, whereas others could only be detected in 1 of the tissues
analyzed (Fig. 1A). Of the total detectable elements, 82 clones were
overexpressed (at least 1.5-fold increase), and 127 were underexpressed
(at least 1.5-fold decrease) in liver from Hx animals compared with
normal liver. This represents 29% of the detectable transcripts in
this tissue. In the kidney samples, 91 genes were overexpressed, and
136 were underexpressed in Hx animals. This number represents 23% of
the total number of detected transcripts. In heart, 125 genes were
overexpressed, and 141 were underexpressed in tissues from Hx rats
compared with normal tissues. The percentage of regulated genes thus
was lower in heart (16%) than in the other tissues. The identities of
the known genes and their putative functions are listed in Tables 2
and 3.
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shows the effects of chronic GH
treatment on gene expression (only genes with a putative identity are
listed) in kidney, liver, and heart of Hx animals. GH regulates the
expression of about 90 different genes in the tissues analyzed. In
liver, 28 genes were overexpressed after GH treatment, and 30 were
underexpressed. In kidney, 7 were overexpressed, and 9 were
underexpressed, whereas in heart 22 were overexpressed, and 10 were
underexpressed (Fig. 1B).
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confirmed the array information and showed that the atrial natriuretic
peptide, SCD1, and meprin A genes are indeed tissue enriched and that
GH reverses the negative effect of hypophysectomy on their respective
expression level. The analysis using RNase protection assay was then
further extended to include four additional genes assigned as
differentially regulated using DNA microarrays. As shown in Table 1, all seven selected transcripts were
confirmed to be differentially expressed when analyzed by RNase
protection assay. In terms of fold regulation, DNA chip analysis tended
to underestimate differences compared with RNase protection assay.
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Discussion |
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Our analysis of the effects of hypophysectomy demonstrates that the
expression of genes involved in virtually all cellular processes are
affected, including metabolism, cell structure, protein turnover, and
signal transduction (Fig. 1C). These wide effects are expected, as
multiple cellular processes are regulated by traditional hormones as
well as other trophic factors. Many of these changes in gene expression
reflect the physiological changes observed in the Hx rat.
Hypophysectomy causes a general decrease in the mRNA content of
different tissues (23). This was reflected in the present
study by the multiple genes underexpressed in tissues from Hx rats.
Table 2 shows the genes that were more
than 1.5-fold underexpressed after hypophysectomy.
Major effects were observed in genes involved in lipid and carbohydrate metabolism as well as in the mitochondrial respiratory chain. These findings reflect the metabolic changes observed in Hx animals, which have diminished metabolic rate, heat production, and oxygen consumption (3). Our observations that removal of the pituitary decreased the levels of several transcripts encoding various components of the electron transport chain, including cytochromes b and c and cytochrome c oxidase, are in agreement with previous studies of rat liver mitochondria. As early as 1972, Matsubara et al. reported that the respiratory cytochrome content of isolated mitochondria decreased in Hx animals in parallel with a decrease in respiratory activities (28). These changes have been attributed to an impaired function of the thyroid gland. Replacement therapy with thyroid hormone increases both respiration rates and mitochondrial mass (29).
Reduction in food intake, endogenous insulin secretion, and metabolic rate lead to diminished glucose utilization in multiple tissues in the Hx animal (26). This has been attributed to both a reduction in glucose transport and a progressive decrease in the activity of several enzymes involved in glucose oxidation (3, 26). When fed ad libitum, Hx rats maintain normal levels of blood glucose despite above normal rates of carbohydrate usage relative to other nutrients (27). The availability of fatty acids decreases, and the catabolism of amino acids increases (30). A striking difference was observed between heart and liver regarding the expression of genes encoding enzymes involved in fatty acid ß-oxidation and pyruvate usage. Our experiments showed that the expression of pyruvate dehydrogenase kinase (isoenzyme 4), a negative regulator of the oxidation of pyruvate to acetyl-CoA (31), was down-regulated in heart by hypophysectomy, but not in liver. This is in agreement with a preferential utilization of carbohydrates over lipids as an energy source in the former tissue. In addition, the genes involved in ß-oxidation of fatty acids (3-ketoacyl-CoA thiolase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 2,4-dienoyl-CoA reductase), an alternative source of acetyl-CoA for the Krebs cycle, were markedly reduced by hypophysectomy in heart. In liver, although pyruvate dehydrogenase kinase expression was less affected than in heart, the expression of lactate dehydrogenase was markedly reduced. As a result, there should be an increased availability of hepatic pyruvate for gluconeogenesis (27). In liver, the transcripts for the genes involved in ß-oxidation were less affected than those in heart, which is in line with previous reports that showed no difference in hepatic respiratory CO2 production after palmitate infusion in Hx vs. normal rats (32). Nevertheless, it is well known that hypophysectomy inhibits the hepatic production of ketone bodies during fasting. This has been attributed to decreased fatty acid mobilization rather than reduced capacity of the liver to produce ketone bodies (3). This decreased fatty acid mobilization from adipose tissue and liver may cause a reduced supply of fatty acids for ß-oxidation in heart and muscle (33). This is in agreement with the previous idea that carbohydrates are the favored fuel in these tissues in Hx animals (34).
The liver is responsible for the maintenance of normal blood glucose
levels (27). In fact, glycogen stores are reduced in
muscle and liver after hypophysectomy, and liver gluconeogenesis from
alanine, but not lactate, is increased, as is the amino acid transport
(27) and glutamic-pyruvate transaminase activity
(35) and expression (Table 3). This increase in gluconeogenesis
occurs despite a reduced expression of glucose-6-phosphatase and
fructose-1,6-biphosphatase (Table 2). It may be the case that this
reduced expression is sufficient for the requirements of the normally
fed rat, but not for the fasted animal. It is well known that Hx
animals are unable to maintain blood glucose levels during fasting.
When fasting, the animals develop hypoglycemia despite a decrease in
the glycogen storages in liver, muscle, and heart (3, 26).
Seemingly, there is an uncoupling effect between glycogen degradation
and glucose secretion. Glycogen degradation is regulated by the enzyme
glycogen phosphorylase, which is posttranslationally activated through
adenylate cyclase-induced phosphorylation. Increased usage of glycogen
can be explained by the known hypersensitivity to glucagon observed in
the liver of Hx animals (36). On the other hand, the
secretion of glucose from liver requires its previous dephosphorylation
by glucose-6-phosphatase, an enzyme whose expression (Table 2) and
activity (37) are greatly reduced by hypophysectomy. The
other source of blood glucose during fasting is the gluconeogenic
pathway. The expression of the rate-limiting enzyme in this pathway,
fructose-1,6-biphosphatase, was also down-regulated by hypophysectomy
in liver and kidney (Table 2), presumably due to adrenal insufficiency
(38). It has been shown that the activity and expression
of this enzyme can be induced by treatment with glucocorticoids
(3).
The general decrease in metabolic rate is also reflected by a decrease
in the secretory function. In our study this was reflected by liver
expression of the mRNA encoding the main secreted liver protein, serum
albumin (39), which, together with some other genes
involved in the protein secretory pathway, showed reduced expression
after hypophysectomy (Table 2). For example, the expression of the
genes for endoplasmic reticulum-resident chaperone proteins such
as calreticulin, which is involved in protein folding of de
novo synthesized glycosylated proteins (40), was
reduced. The same was seen for the expression of ADP-ribosylation
factor 4, which is involved in vesicular trafficking
between the endoplasmic reticulum and the cis-Golgi
compartment (41). Moreover, farnesyl pyrophosphate
synthetase, a rate-limiting enzyme involved in the cholesterol
biosynthetic pathway (42), and stearyl-CoA desaturase
1 (SCD1), the key enzyme in the synthesis of unsaturated fatty acids
(43), were also down-regulated in liver. Cholesterol and
unsaturated fatty acids are essential for maintaining membrane fluidity
and are therefore subject to a coordinate regulation that is achieved
through the activation of common transcription factors, sterol
regulatory element-binding transcription factors
(44). Response elements for these factors are also
present in the promoters of
3-hydroxy-3-methylglutaryl-coenzyme A reductase and low
density lipoprotein (LDL) receptor genes. These genes are also involved
in lipid metabolism and are known to be down-regulated by
hypophysectomy (45). These findings may reflect the
known coordination between the synthesis of secreted proteins and the
biogenesis of the endoplasmic reticulum (46).
Many gene products involved in signal transduction were markedly
down-regulated by hypophysectomy in a tissue-specific nature. For
example, calbindin D was down-regulated in kidney, but it does not
appear to be expressed in heart or liver. The expression of this gene
together with osteonectin and matrix Gla protein (MGP) is regulated by
1,25-dihydroxycholecalciferol, the active hormone derived from vitamin
D (47, 48). 1,25-Dihydroxycholecalciferol synthesis by the
renal 25-hydroxyvitamin D3-1
-hydroxylase is
known to be reduced by hypophysectomy in rats (49, 50).
The low levels of this enzyme may explain why these genes are affected.
Osteonectin and MGP are matrix proteins involved in the remodeling of
the extracellular matrix and the regulation of bone formation. Mice
that are deficient in osteonectin develop severe osteopenia
(51). On the other hand, MGP knockout mice exhibit
inappropriate calcification of various cartilages and arteries, which
eventually lead to short stature and osteopenia (52).
Besides many other effects, hypophysectomy causes profound osteopenia,
primarily due to an inhibition of bone gain, and a decrease in bone
turnover (53). It will therefore be interesting to analyze
the expression of these genes in bone tissue and their involvement in
bone turnover during hypophysectomy. These genes are also expressed in
tissues others than bones. Osteonectin, for example, is also expressed
by myocardocytes (54), liver myofibroblasts
(55), and glomerular mesangial cells (56),
where it regulates the synthesis and deposition of extracellular matrix
proteins such as collagen.
In all of the tissues analyzed, hypophysectomy caused a marked
reduction of genes expressing -hemoglobin and ß-globin. The lack
of tissue specificity of this effect is expected, because the mRNA is
most likely to come from circulating erythrocytes trapped within the
tissues (nonperfused tissues were used). This may reflect the well
known physiological effects. After hypophysectomy, rats became anemic
with a decrease in hematocrit, hemoglobin concentration, red blood cell
count, and reticulocytes (57). GH can restore normal
levels of red blood cells independently of other hormones
(58).
Approximately 100 different genes were found to be overexpressed (>1.5-fold) in mRNA from Hx animals compared with similar amounts of mRNA from normal animals in the three tissues analyzed. Of the 62 genes of known function found to be overexpressed in mRNA of Hx rat tissues, 18 were related to the protein synthesis machinery, including the protein components of the ribosome and translation factors. Ribosomal proteins are stochiometrically assembled with ribosomal RNA to form the ribosomal subunits. Consequently, the expression of these genes is coregulated, and the existence of such a cluster of coregulated ribosomal genes was shown here as well as in previous studies using yeast and mammalian cell cultures (59). The genes belonging to this cluster were approximately 1.4–2 times overexpressed in the mRNA population from Hx compared with normal tissue. This result is in line with previous studies regarding the effect of hypophysectomy on protein synthesis. The severe inhibition of protein synthesis after removal of the pituitary gland is mainly due to a general reduction in mRNA content rather than a diminished number of ribosomes (23, 60, 61). The diminished protein synthesis rate by isolated hepatic ribosomes from Hx rats can be restored by adding exogenous RNA to these preparations (62).
In contrast to underexpressed genes, fewer genes were overexpressed across all three tissues of Hx rats beyond the levels of those belonging to the ribosomal cluster. As an example, ID-1, ID-3, and carbonic anhydrase III were found to be overexpressed in Hx kidney, but not in heart or liver. ID (inhibitors of DNA binding) proteins are helix-loop-helix proteins that form heterodimers with ubiquitous and/or tissue-specific basic helix-loop-helix proteins, and thereby inhibit their DNA-binding activity. The interaction between ID-1 or ID-3 and basic helix-loop-helix proteins, many of which are essential for cellular differentiation, has been proposed as a key regulatory event leading to negative regulation of cell differentiation and as positive regulators of G1 cell cycle control (63, 64). Carbonic anhydrase, a key enzyme in the regulation of acid-base balance by the kidney is involved in acidification of the urine and reabsorption of HCO3- (65). It may be the case that low food intake and diminished erythropoiesis (66) and albumin production (39) after hypophysectomy result in metabolic acidosis. Thus, a compensatory mechanism in kidney may be triggered by the induction of the expression of carbonic anhydrase (67).
In liver, the type IV cAMP-specific phosphodiesterase-4B was overexpressed beyond the levels of the ribosomal cluster. The type IV isoforms of phosphodiesterases are expressed in liver and regulated by glucagon at the level of enzymatic activity (68) and by cAMP at the level of gene expression (69). This finding may be related to the report of an increase in both basal and glucagon-stimulated adenylate cyclase activity in hepatocytes from Hx rats (70). An increased cAMP production may activate glycogen phosphorylase and account for the decrease in glycogen content found in Hx animals after fasting (3, 27).
Effects of chronic GH treatment on gene expression
Chronic hGH treatment of Hx animals results in clear restoration
of body growth among other physiological changes, which in our view
justify the selection of this mode of treatment for the study. The
discussion below has been focused on relating the variations seen in
gene expression to those physiological changes. No attempts have been
made to distinguish between the lactogenic and the somatogenic effects
of human GH in nonhepatic tissues, as such a study would require a
different experimental design. In the interpretation of our results it
is important to stress that the state of hypophysectomy creates a
physiological situation unique to the model itself (of multiple
hormonal deficiency), and this should be kept in mind when interpreting
the effects of GH.
As already mentioned, the primary characteristic of the Hx animal is growth failure consequent to the loss of GH. Accompanying this are multiple alterations in the intermediary metabolism. Thus, the Hx animal has a higher proportion of fat and a lower proportion of protein in its carcass. Chronic hGH administration restores the growth rate, improves the nitrogen balance, and reverses the changes in body composition found in Hx animals (3, 30). The protein anabolic effects of GH include increased uptake of amino acids in peripheral tissues, decreased protein breakdown and usage of amino acids for energy expenditure, and increased protein synthesis (71, 72). These effects are partly accomplished by increasing the utilization of FFA in peripheral tissues. Thus, GH treatment promotes lipolysis and prevents lipogenesis in adipose tissue, which increases the availability of FFA for energy expenditure (4, 33). The liver plays a key role in the regulation of lipid metabolism by its production and uptake of lipoproteins. GH affects hepatic lipid metabolism on many levels, including increased lipoprotein and triglyceride production, increased secretion of very low density lipoprotein, and increased expression of LDL receptors (73).
In the present study the strong effects of hypophysectomy on the expression of genes involved in the metabolism of carbohydrates and lipids and in ATP production reflect the importance of pituitary hormones in cellular homeostasis. Furthermore, the modest number of genes that showed normalized expression levels in Hx rats upon chronic hGH administration substantiates the importance of other pituitary hormones in the control of metabolic pathways. The loss of TSH and ACTH leads to secondary thyroid and adrenal insufficiency, whereas FSH and LH depletion lead to a reduced production of sex steroids. The gene encoding glucose-6-phosphatase, the final enzyme in the gluconeogenic pathway, which showed a decreased expression in liver and kidney from our Hx rats, could exemplify this. The activity of this enzyme is known to be induced by GH in Hx rats substituted with thyroid hormone and ACTH (37). The GH-mediated effect on this gene product was recently shown to include induced mRNA expression in the liver of aged rats, which are not as deficient in thyroid and adrenal hormones as the Hx rats (our unpublished results). Similarly, hepatic glutamate dehydrogenase mRNA expression is induced by GH in aged rats (our unpublished results), but only showed decreased expression in our Hx rats and no major changes in mRNA levels after GH administration. In contrast, the hepatic expression of glutamate pyruvate transaminase was induced in Hx rats compared with normal rats and was repressed upon hGH treatment. This finding confirms results obtained measuring the enzymatic activity and is in line with previous reports on GH-mediated down-regulation of urea synthesis and increased nitrogen balance (74).
Other genes may also contribute to the anabolic effects of GH. For
example, transketolase expression (together with other enzymes in the
pentose phosphate pathway) was reduced after hypophysectomy and was
induced after GH treatment (Table 4).
This enzyme participates in the synthesis of ribose phosphate, which is
needed for the production of RNA. In line with this is the finding that
the expression of xanthine dehydrogenase, which is responsible for the
degradation of nucleotides, was down-regulated in liver by GH. A
reduced level of mRNA expression was observed for peroxisome
proliferator-activated receptor (PPAR) in GH-treated
Hx rats together with gene products previously shown to be regulated by
PPAR, including 2,4-dienoyl-CoA reductase and 3-hydroxyacyl-CoA
dehydrogenase (75, 76). The increased expression of
PPAR observed in Hx rats might therefore be a consequence of GH
deficiency.
Analysis of GH-induced changes in gene expression showed that most of
the effects were highly tissue specific. Liver was the most responsive
tissue, which agrees with previous findings about GH receptor
expression and GH sensitivity of this tissue (77). The
expression of SCD1 was greatly reduced in liver after hypophysectomy
and was induced by GH treatment (Fig. 2). SCD catalyzes the oxidation
of stearyl CoA to form the monounsaturated fatty acid oleyl-CoA. Oleic
acid is the major unsaturated fatty acid in lipid stores, fat tissue,
and membrane phospholipids. The ratio of stearic/oleic acid has been
implicated in the regulation of cell growth and differentiation through
effects in cell membrane fluidity and signal transduction. An increased
ratio of stearic to oleic acid, for example, induces apoptosis in rat
ventricular myocytes, whereas oleate-enriched LDL is resistant to
oxidation (78, 79). Enzymes involved in detoxification and
xenobiotic metabolism were also GH regulated in liver
(Table 4), which is in agreement with previous reports
(6).
In heart, GH induced the expression of atrial natriuretic peptide in Hx rats. These animals otherwise show low levels of this factor and attenuated diuresis and natriuresis in response to blood volume expansion (80). The expression levels of the hemoglobin genes were also restored, a finding in line with the known positive actions of GH on hemopoiesis (58). The hormonal regulation of vasculogenesis deserves more attention, keeping in mind the known positive effects of GH (81). The present study shows that GH down-regulates the liver expression of collagen type XVIII, the precursor of endostatin, a newly described inhibitor of angiogenesis. In the same tissue hypophysectomy reduces and GH induces the expression of the hepatic fibrinogen/angiopoietin-related protein, a recently described secreted protein that prevents endothelial cell apoptosis (82). These findings may constitute a novel mechanism used by GH to regulate the cardiovascular system.
In kidney, GH stimulated the expression of stanniocalcin I, a newly characterized hormone in mammals that stimulates phosphate reabsorption at proximal tubules (83). This finding may explain previous observations describing a positive effect of GH on phosphate transport in Hx rats fed a low phosphorous diet as well as in GH-deficient children (84). Regulation of stanniocalcin I by GH may be an important mechanism for positive calcium balance and the induction of longitudinal bone growth that is unique to GH action (85, 86). Several genes involved in kidney damage, such as fibronectin, collagen, and meprin A, were induced by GH in kidney (87, 88). Meprin A is the major matrix-degrading metalloproteinase in rat kidney. Its expression is polarized, as it is expressed in the brush border membrane of renal proximal tubular epithelial cells (89). It has been suggested that meprin A plays a role in the pathophysiology of acute renal failure after a variety of injuries to the kidney. Redistribution of this metalloendopeptidase to the basolateral membrane domain during acute renal failure results in degradation of the extracellular matrix and damage to adjacent peritubular structures (89, 90). It is a well known phenomenon that chronic GH treatment may result in kidney damage, and that hypophysectomy protects against glomerulosclerosis induced by diabetes (9). It will be interesting to further explore the role of GH-regulated genes such as meprin A in the development of diabetic nephropathy as well as the role of GH in this process.
The cDNA array used here contains a small sampling of rat cDNA transcripts and is far from a complete representation of all rat cDNAs. The present study therefore only represents a partial survey of genes regulated by hypophysectomy or genes responsive to GH treatment. Furthermore, the experimental models used, i.e. hypophysectomy and chronic hGH infusion, provide only partial information. The effects of hypophysectomy are dependent on developmental stage, and the effects of GH are dose dependent, significantly depend on the mode of administration (6), and if the GH treatment is long term would include actions of induced IGF-I. In extended studies the use of our cDNA microarray to study acute effects of GH on Hx animals would provide further insights into the tissue-specific changes, as the present study focused on a more stabilized condition after weeks of treatment. A study of the acute effects of GH on liver gene expression in another model of GH deficiency using a filter array of 588 rat cDNAs has recently been published (91). However, direct comparison to the present study cannot readily be made because a different experimental design was employed, and the two studies used a nonoverlapping set of GH-regulated genes.
In the present study cDNA arrays were used to characterize the state of hypophysectomy. Our data support earlier observations and can be put in a context of previous physiological knowledge. We also identified several genes that were previously not known to be regulated by either Hx or hGH treatment. In addition, a number of genes of unknown function (expressed sequence tags) responded to hypophysectomy and hGH in this study. In future experiments the value of DNA arrays to annotate such genes in terms of regulation will be studied. Further work is therefore required to extend our knowledge, and DNA array technology will serve as a valuable tool.
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2 A.F-M. and N.S. contributed equally to this work.
3 Supported by the Swedish Medical Research Council.
Received December 15, 2000.
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